Thermodynamics of DNA target site recognition by homing endonucleases

PubMed Central

Eastberg, Jennifer H.; Smith, Audrey McConnell; Zhao, Lei; Ashworth, Justin; Shen, Betty W.; Stoddard, Barry L.

2007-01-01

The thermodynamic profiles of target site recognition have been surveyed for homing endonucleases from various structural families. Similar to DNA-binding proteins that recognize shorter target sites, homing endonucleases display a narrow range of binding free energies and affinities, mediated by structural interactions that balance the magnitude of enthalpic and entropic forces. While the balance of ΔH and TΔS are not strongly correlated with the overall extent of DNA bending, unfavorable ΔHbinding is associated with unstacking of individual base steps in the target site. The effects of deleterious basepair substitutions in the optimal target sites of two LAGLIDADG homing endonucleases, and the subsequent effect of redesigning one of those endonucleases to accommodate that DNA sequence change, were also measured. The substitution of base-specific hydrogen bonds in a wild-type endonuclease/DNA complex with hydrophobic van der Waals contacts in a redesigned complex reduced the ability to discriminate between sites, due to nonspecific ΔSbinding. PMID:17947319

Prostate Cancer Clinical Consortium Clinical Research Site:Targeted Therapies

DTIC Science & Technology

2015-10-01

AWARD NUMBER: W81XWH-14-2-0159 TITLE: Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies PRINCIPAL INVESTIGATOR...Sep 2015 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies 5b. GRANT NUMBER... therapy resistance/sensitivity, identification of new therapeutic targets through high quality genomic analyses, providing access to the highest quality

Catalog of MicroRNA Seed Polymorphisms in Vertebrates

PubMed Central

Calin, George Adrian; Horvat, Simon; Jiang, Zhihua; Dovc, Peter; Kunej, Tanja

2012-01-01

MicroRNAs (miRNAs) are a class of non-coding RNA that plays an important role in posttranscriptional regulation of mRNA. Evidence has shown that miRNA gene variability might interfere with its function resulting in phenotypic variation and disease susceptibility. A major role in miRNA target recognition is ascribed to complementarity with the miRNA seed region that can be affected by polymorphisms. In the present study, we developed an online tool for the detection of miRNA polymorphisms (miRNA SNiPer) in vertebrates (http://www.integratomics-time.com/miRNA-SNiPer) and generated a catalog of miRNA seed region polymorphisms (miR-seed-SNPs) consisting of 149 SNPs in six species. Although a majority of detected polymorphisms were due to point mutations, two consecutive nucleotide substitutions (double nucleotide polymorphisms, DNPs) were also identified in nine miRNAs. We determined that miR-SNPs are frequently located within the quantitative trait loci (QTL), chromosome fragile sites, and cancer susceptibility loci, indicating their potential role in the genetic control of various complex traits. To test this further, we performed an association analysis between the mmu-miR-717 seed SNP rs30372501, which is polymorphic in a large number of standard inbred strains, and all phenotypic traits in these strains deposited in the Mouse Phenome Database. Analysis showed a significant association between the mmu-miR-717 seed SNP and a diverse array of traits including behavior, blood-clinical chemistry, body weight size and growth, and immune system suggesting that seed SNPs can indeed have major pleiotropic effects. The bioinformatics analyses, data and tools developed in the present study can serve researchers as a starting point in testing more targeted hypotheses and designing experiments using optimal species or strains for further mechanistic studies. PMID:22303453

TarPmiR: a new approach for microRNA target site prediction.

PubMed

Ding, Jun; Li, Xiaoman; Hu, Haiyan

2016-09-15

The identification of microRNA (miRNA) target sites is fundamentally important for studying gene regulation. There are dozens of computational methods available for miRNA target site prediction. Despite their existence, we still cannot reliably identify miRNA target sites, partially due to our limited understanding of the characteristics of miRNA target sites. The recently published CLASH (crosslinking ligation and sequencing of hybrids) data provide an unprecedented opportunity to study the characteristics of miRNA target sites and improve miRNA target site prediction methods. Applying four different machine learning approaches to the CLASH data, we identified seven new features of miRNA target sites. Combining these new features with those commonly used by existing miRNA target prediction algorithms, we developed an approach called TarPmiR for miRNA target site prediction. Testing on two human and one mouse non-CLASH datasets, we showed that TarPmiR predicted more than 74.2% of true miRNA target sites in each dataset. Compared with three existing approaches, we demonstrated that TarPmiR is superior to these existing approaches in terms of better recall and better precision. The TarPmiR software is freely available at http://hulab.ucf.edu/research/projects/miRNA/TarPmiR/ CONTACTS: haihu@cs.ucf.edu or xiaoman@mail.ucf.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

A novel, non-functional, COL1A1 polymorphism is not associated with lumbar disk disease in young male Greek subjects unlike that of the Sp1 site

PubMed Central

Bei, Thalia; Tilkeridis, Constantinos; Garantziotis, Stavros; Boikos, Sosipatros A.; Kazakos, Konstantinos; Simopoulos, Constantinos; Stratakis, Constantine A.

2011-01-01

OBJECTIVE We recently reported the association of the Sp1 site polymorphism of the COL1A1 gene with lumbar disk disease (LDD). In the present study we searched for a different polymorphism of the COL1A1 gene (which is usually not in linkage disequilibrium with the Sp1 site) in subjects with LDD. DESIGN Blood was collected from 24 Greek army recruits, aged 29±7.6 years, with LDD, and 66 healthy men, aged 26±4.38 years, matched for body mass index (BMI) and age, with normal BMD and with no history of trauma or fractures, who served as controls. DNA was extracted and the COL1A1 gene was sequenced. Of the control subjects, 12 were army recruits and 54 were selected from the general population. RESULTS The four base-pair insertion polymorphism in the COL1A1 gene analyzed by polymerase chain reaction amplification of DNA produces two different fragments (alleles A1 and A2): 14 patients (58.3%) were homozygous for A2A2, versus 35 controls (53%), while 3 patients (12.5%) were A1A1, and 8 of the control subjects (12%) had this genotype. There were no statistically significant differences in the presence of the two alleles of this polymorphism between patients with LDD and control subjects. CONCLUSIONS A four base-pair insertion polymorphism of the COL1A1 gene is not associated with the presence of LDD in young males, unlike the Sp1 site polymorphism of the same gene. These data reinforce the association between LDD and the functional polymorphisms of the Sp1 site by showing that other polymorphic sites of the of the COL1A1 gene in the same population of patients are not linked to the disease. PMID:18694864

Polymorphisms A387P in thrombospondin-4 and N700S in thrombospondin-1 perturb calcium binding sites.

PubMed

Stenina, Olga I; Ustinov, Valentin; Krukovets, Irene; Marinic, Tina; Topol, Eric J; Plow, Edward F

2005-11-01

Recent genetic studies have associated members of the thrombospondin (TSP) gene family with premature cardiovascular disease. The disease-associated polymorphisms lead to single amino acid changes in TSP-4 (A387P) and TSP-1 (N700S). These substitutions reside in adjacent domains of these highly homologous proteins. Secondary structural predictive programs and the homology of the domains harboring these amino acid substitutions to those in other proteins pointed to potential alterations of putative Ca2+ binding sites that reside in close proximity to the polymorphic amino acids. Since Ca2+ binding is critical for the structure and function of TSP family members, direct evidence for differences in Ca2+ binding by the polymorphic forms was sought. Using synthetic peptides and purified recombinant variant fragments bearing the amino acid substitutions, we measured differences in Tb3+ luminescence as an index of Ca2+ binding. The Tb3+ binding constants placed the TSP-1 region affected by N700S polymorphism among other high-affinity Ca2+ binding sites. The affinity of Ca2+ binding was lower for peptides (3.5-fold) and recombinant fragments (10-fold) containing the S700 vs. the N700 form. In TSP-4, the P387 form acquired an additional Ca2+ binding site absent in the A387 form. The results of our study suggest that both substitutions (A387P in TSP-4 and N700S in TSP-1) alter Ca2+ binding properties. Since these substitutions exert the opposite effects on Ca2+ binding, a decrease in TSP-1 and an increase in TSP-4, the two TSP variants are likely to influence cardiovascular functions in distinct but yet pathogenic ways.

Retention of ferrofluid aggregates at the target site during magnetic drug targeting

NASA Astrophysics Data System (ADS)

Asfer, Mohammed; Saroj, Sunil Kumar; Panigrahi, Pradipta Kumar

2017-08-01

The present study reports the retention dynamics of a ferrofluid aggregate localized at the target site inside a glass capillary (500 × 500 μm2 square cross section) against a bulk flow of DI water (Re = 0.16 and 0.016) during the process of magnetic drug targeting (MDT). The dispersion dynamics of iron oxide nanoparticles (IONPs) into bulk flow for different initial size of aggregate at the target site is reported using the brightfield visualization technique. The flow field around the aggregate during the retention is evaluated using the μPIV technique. IONPs at the outer boundary experience a higher shear force as compared to the magnetic force, resulting in dispersion of IONPs into the bulk flow downstream to the aggregate. The blockage effect and the roughness of the outer boundary of the aggregate resulting from chain like clustering of IONPs contribute to the flow recirculation at the downstream region of the aggregate. The entrapment of seeding particles inside the chain like clusters of IONPs at the outer boundary of the aggregate reduces the degree of roughness resulting in a streamlined aggregate at the target site at later time. The effect of blockage, structure of the aggregate, and disturbed flow such as recirculation around the aggregate are the primary factors, which must be investigated for the effectiveness of the MDT process for in vivo applications.

Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies

DTIC Science & Technology

2017-10-01

AWARD NUMBER: W81XWH-14-2-0159 TITLE: Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies PRINCIPAL INVESTIGATOR...Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for...AND SUBTITLE Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT

Infraspecific DNA methylation polymorphism in cotton (Gossypium hirsutum L.).

PubMed

Keyte, Anna L; Percifield, Ryan; Liu, Bao; Wendel, Jonathan F

2006-01-01

Cytosine methylation is important in the epigenetic regulation of gene expression and development in plants and has been implicated in silencing duplicate genes after polyploid formation in several plant groups. Relatively little information exists, however, on levels and patterns of methylation polymorphism (MP) at homologous loci within species. Here we explored the levels and patterns of methylation-polymorphism diversity at CCGG sites within allotetraploid cotton, Gossypium hirsutum, using a methylation-sensitive amplified fragment length polymorphism screen and a selected set of 20 G. hirsutum accessions for which we have information on genetic polymorphism levels and relationships. Methylation and MP exist at high levels within G. hirsutum: of 150 HpaII/MspI sites surveyed, 48 were methylated at the inner cytosine (32%) and 32 of these were polymorphic (67%). Both these values are higher than comparable measures of genetic diversity using restriction fragment length polymorphisms. The high percentage of methylation-polymorphic sites and potential relationship to gene expression underscore the potential significance of MP within and among populations. We speculate that biased correlation of methylation-polymorphic sites and genes in cotton may be a consequence of polyploidy and the attendant doubling of all genes.

Target-mediated drug disposition model for drugs with two binding sites that bind to a target with one binding site.

PubMed

Gibiansky, Leonid; Gibiansky, Ekaterina

2017-10-01

The paper extended the TMDD model to drugs with two identical binding sites (2-1 TMDD). The quasi-steady-state (2-1 QSS), quasi-equilibrium (2-1 QE), irreversible binding (2-1 IB), and Michaelis-Menten (2-1 MM) approximations of the model were derived. Using simulations, the 2-1 QSS approximation was compared with the full 2-1 TMDD model. As expected and similarly to the standard TMDD for monoclonal antibodies (mAb), 2-1 QSS predictions were nearly identical to 2-1 TMDD predictions, except for times of fast changes following initiation of dosing, when equilibrium has not yet been reached. To illustrate properties of new equations and approximations, several variations of population PK data for mAbs with soluble (slow elimination of the complex) or membrane-bound (fast elimination of the complex) targets were simulated from a full 2-1 TMDD model and fitted to 2-1 TMDD models, to its approximations, and to the standard (1-1) QSS model. For a mAb with a soluble target, it was demonstrated that the 2-1 QSS model provided nearly identical description of the observed (simulated) free drug and total target concentrations, although there was some minor bias in predictions of unobserved free target concentrations. The standard QSS approximation also provided a good description of the observed data, but was not able to distinguish between free drug concentrations (with no target attached and both binding site free) and partially bound drug concentrations (with one of the binding sites occupied by the target). For a mAb with a membrane-bound target, the 2-1 MM approximation adequately described the data. The 2-1 QSS approximation converged 10 times faster than the full 2-1 TMDD, and its run time was comparable with the standard QSS model.

Metabolic and Target-Site Mechanisms Combine to Confer Strong DDT Resistance in Anopheles gambiae

PubMed Central

Mitchell, Sara N.; Rigden, Daniel J.; Dowd, Andrew J.; Lu, Fang; Wilding, Craig S.; Weetman, David; Dadzie, Samuel; Jenkins, Adam M.; Regna, Kimberly; Boko, Pelagie; Djogbenou, Luc; Muskavitch, Marc A. T.; Ranson, Hilary; Paine, Mark J. I.; Mayans, Olga; Donnelly, Martin J.

2014-01-01

The development of resistance to insecticides has become a classic exemplar of evolution occurring within human time scales. In this study we demonstrate how resistance to DDT in the major African malaria vector Anopheles gambiae is a result of both target-site resistance mechanisms that have introgressed between incipient species (the M- and S-molecular forms) and allelic variants in a DDT-detoxifying enzyme. Sequencing of the detoxification enzyme, Gste2, from DDT resistant and susceptible strains of An. gambiae, revealed a non-synonymous polymorphism (I114T), proximal to the DDT binding domain, which segregated with strain phenotype. Recombinant protein expression and DDT metabolism analysis revealed that the proteins from the susceptible strain lost activity at higher DDT concentrations, characteristic of substrate inhibition. The effect of I114T on GSTE2 protein structure was explored through X-ray crystallography. The amino acid exchange in the DDT-resistant strain introduced a hydroxyl group nearby the hydrophobic DDT-binding region. The exchange does not result in structural alterations but is predicted to facilitate local dynamics and enzyme activity. Expression of both wild-type and 114T alleles the allele in Drosophila conferred an increase in DDT tolerance. The 114T mutation was significantly associated with DDT resistance in wild caught M-form populations and acts in concert with target-site mutations in the voltage gated sodium channel (Vgsc-1575Y and Vgsc-1014F) to confer extreme levels of DDT resistance in wild caught An. gambiae. PMID:24675797

Polymorphism at codon 36 of the p53 gene.

PubMed

Felix, C A; Brown, D L; Mitsudomi, T; Ikagaki, N; Wong, A; Wasserman, R; Womer, R B; Biegel, J A

1994-01-01

A polymorphism at codon 36 in exon 4 of the p53 gene was identified by single strand conformation polymorphism (SSCP) analysis and direct sequencing of genomic DNA PCR products. The polymorphic allele, present in the heterozygous state in genomic DNAs of four of 100 individuals (4%), changes the codon 36 CCG to CCA, eliminates a FinI restriction site and creates a BccI site. Including this polymorphism there are four known polymorphisms in the p53 coding sequence.

A complex dominance hierarchy is controlled by polymorphism of small RNAs and their targets.

PubMed

Yasuda, Shinsuke; Wada, Yuko; Kakizaki, Tomohiro; Tarutani, Yoshiaki; Miura-Uno, Eiko; Murase, Kohji; Fujii, Sota; Hioki, Tomoya; Shimoda, Taiki; Takada, Yoshinobu; Shiba, Hiroshi; Takasaki-Yasuda, Takeshi; Suzuki, Go; Watanabe, Masao; Takayama, Seiji

2016-12-22

In diploid organisms, phenotypic traits are often biased by effects known as Mendelian dominant-recessive interactions between inherited alleles. Phenotypic expression of SP11 alleles, which encodes the male determinants of self-incompatibility in Brassica rapa, is governed by a complex dominance hierarchy 1-3 . Here, we show that a single polymorphic 24 nucleotide small RNA, named SP11 methylation inducer 2 (Smi2), controls the linear dominance hierarchy of the four SP11 alleles (S 44 > S 60 > S 40 > S 29 ). In all dominant-recessive interactions, small RNA variants derived from the linked region of dominant SP11 alleles exhibited high sequence similarity to the promoter regions of recessive SP11 alleles and acted in trans to epigenetically silence their expression. Together with our previous study 4 , we propose a new model: sequence similarity between polymorphic small RNAs and their target regulates mono-allelic gene expression, which explains the entire five-phased linear dominance hierarchy of the SP11 phenotypic expression in Brassica.

Polymorphic amplified typing sequences (PATS) and pulsed-field gel electrophoresis (PFGE) yield comparable results in the strain typing of a diverse set of bovine Escherichia coli O157 isolates

USDA-ARS?s Scientific Manuscript database

The PCR-based Escherichia coli O157 (O157) strain typing system, Polymorphic Amplified Typing Sequences (PATS), targets insertions-deletions (Indels) and single nucleotide polymorphisms (SNPs) at the XbaI and AvrII(BlnI) restriction enzyme sites, respectively, besides amplifying four known virulenc...

Templated sequence insertion polymorphisms in the human genome

NASA Astrophysics Data System (ADS)

Onozawa, Masahiro; Aplan, Peter

2016-11-01

Templated Sequence Insertion Polymorphism (TSIP) is a recently described form of polymorphism recognized in the human genome, in which a sequence that is templated from a distant genomic region is inserted into the genome, seemingly at random. TSIPs can be grouped into two classes based on nucleotide sequence features at the insertion junctions; Class 1 TSIPs show features of insertions that are mediated via the LINE-1 ORF2 protein, including 1) target-site duplication (TSD), 2) polyadenylation 10-30 nucleotides downstream of a “cryptic” polyadenylation signal, and 3) preference for insertion at a 5’-TTTT/A-3’ sequence. In contrast, class 2 TSIPs show features consistent with repair of a DNA double-strand break via insertion of a DNA “patch” that is derived from a distant genomic region. Survey of a large number of normal human volunteers demonstrates that most individuals have 25-30 TSIPs, and that these TSIPs track with specific geographic regions. Similar to other forms of human polymorphism, we suspect that these TSIPs may be important for the generation of human diversity and genetic diseases.

Using information content and base frequencies to distinguish mutations from genetic polymorphisms in splice junction recognition sites.

PubMed

Rogan, P K; Schneider, T D

1995-01-01

Predicting the effects of nucleotide substitutions in human splice sites has been based on analysis of consensus sequences. We used a graphic representation of sequence conservation and base frequency, the sequence logo, to demonstrate that a change in a splice acceptor of hMSH2 (a gene associated with familial nonpolyposis colon cancer) probably does not reduce splicing efficiency. This confirms a population genetic study that suggested that this substitution is a genetic polymorphism. The information theory-based sequence logo is quantitative and more sensitive than the corresponding splice acceptor consensus sequence for detection of true mutations. Information analysis may potentially be used to distinguish polymorphisms from mutations in other types of transcriptional, translational, or protein-coding motifs.

Polymorphisms in O-methyltransferase genes are associated with stover cell wall digestibility in European maize (Zea mays L.).

PubMed

Brenner, Everton A; Zein, Imad; Chen, Yongsheng; Andersen, Jeppe R; Wenzel, Gerhard; Ouzunova, Milena; Eder, Joachim; Darnhofer, Birte; Frei, Uschi; Barrière, Yves; Lübberstedt, Thomas

2010-02-12

OMT (O-methyltransferase) genes are involved in lignin biosynthesis, which relates to stover cell wall digestibility. Reduced lignin content is an important determinant of both forage quality and ethanol conversion efficiency of maize stover. Variation in genomic sequences coding for COMT, CCoAOMT1, and CCoAOMT2 was analyzed in relation to stover cell wall digestibility for a panel of 40 European forage maize inbred lines, and re-analyzed for a panel of 34 lines from a published French study. Different methodologies for association analysis were performed and compared. Across association methodologies, a total number of 25, 12, 1, 6 COMT polymorphic sites were significantly associated with DNDF, OMD, NDF, and WSC, respectively. Association analysis for CCoAOMT1 and CCoAOMT2 identified substantially fewer polymorphic sites (3 and 2, respectively) associated with the investigated traits. Our re-analysis on the 34 lines from a published French dataset identified 14 polymorphic sites significantly associated with cell wall digestibility, two of them were consistent with our study. Promising polymorphisms putatively causally associated with variability of cell wall digestibility were inferred from the total number of significantly associated SNPs/Indels. Several polymorphic sites for three O-methyltransferase loci were associated with stover cell wall digestibility. All three tested genes seem to be involved in controlling DNDF, in particular COMT. Thus, considerable variation among Bm3 wildtype alleles can be exploited for improving cell-wall digestibility. Target sites for functional markers were identified enabling development of efficient marker-based selection strategies.

Polymorphisms in O-methyltransferase genes are associated with stover cell wall digestibility in European maize (Zea mays L.)

PubMed Central

2010-01-01

Background OMT (O-methyltransferase) genes are involved in lignin biosynthesis, which relates to stover cell wall digestibility. Reduced lignin content is an important determinant of both forage quality and ethanol conversion efficiency of maize stover. Results Variation in genomic sequences coding for COMT, CCoAOMT1, and CCoAOMT2 was analyzed in relation to stover cell wall digestibility for a panel of 40 European forage maize inbred lines, and re-analyzed for a panel of 34 lines from a published French study. Different methodologies for association analysis were performed and compared. Across association methodologies, a total number of 25, 12, 1, 6 COMT polymorphic sites were significantly associated with DNDF, OMD, NDF, and WSC, respectively. Association analysis for CCoAOMT1 and CCoAOMT2 identified substantially fewer polymorphic sites (3 and 2, respectively) associated with the investigated traits. Our re-analysis on the 34 lines from a published French dataset identified 14 polymorphic sites significantly associated with cell wall digestibility, two of them were consistent with our study. Promising polymorphisms putatively causally associated with variability of cell wall digestibility were inferred from the total number of significantly associated SNPs/Indels. Conclusions Several polymorphic sites for three O-methyltransferase loci were associated with stover cell wall digestibility. All three tested genes seem to be involved in controlling DNDF, in particular COMT. Thus, considerable variation among Bm3 wildtype alleles can be exploited for improving cell-wall digestibility. Target sites for functional markers were identified enabling development of efficient marker-based selection strategies. PMID:20152036

Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing

PubMed Central

Lee, Ciaran M; Cradick, Thomas J; Fine, Eli J; Bao, Gang

2016-01-01

The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. PMID:26750397

Restriction site polymorphism-based candidate gene mapping for seedling drought tolerance in cowpea [Vigna unguiculata (L.) Walp.].

PubMed

Muchero, Wellington; Ehlers, Jeffrey D; Roberts, Philip A

2010-02-01

Quantitative trait loci (QTL) studies provide insight into the complexity of drought tolerance mechanisms. Molecular markers used in these studies also allow for marker-assisted selection (MAS) in breeding programs, enabling transfer of genetic factors between breeding lines without complete knowledge of their exact nature. However, potential for recombination between markers and target genes limit the utility of MAS-based strategies. Candidate gene mapping offers an alternative solution to identify trait determinants underlying QTL of interest. Here, we used restriction site polymorphisms to investigate co-location of candidate genes with QTL for seedling drought stress-induced premature senescence identified previously in cowpea. Genomic DNA isolated from 113 F(2:8) RILs of drought-tolerant IT93K503-1 and drought susceptible CB46 genotypes was digested with combinations of EcoR1 and HpaII, Mse1, or Msp1 restriction enzymes and amplified with primers designed from 13 drought-responsive cDNAs. JoinMap 3.0 and MapQTL 4.0 software were used to incorporate polymorphic markers onto the AFLP map and to analyze their association with the drought response QTL. Seven markers co-located with peaks of previously identified QTL. Isolation, sequencing, and blast analysis of these markers confirmed their significant homology with drought or other abiotic stress-induced expressed sequence tags (EST) from cowpea and other plant systems. Further, homology with coding sequences for a multidrug resistance protein 3 and a photosystem I assembly protein ycf3 was revealed in two of these candidates. These results provide a platform for the identification and characterization of genetic trait determinants underlying seedling drought tolerance in cowpea.

Engineering synthetic TAL effectors with orthogonal target sites

PubMed Central

Garg, Abhishek; Lohmueller, Jason J.; Silver, Pamela A.; Armel, Thomas Z.

2012-01-01

The ability to engineer biological circuits that process and respond to complex cellular signals has the potential to impact many areas of biology and medicine. Transcriptional activator-like effectors (TALEs) have emerged as an attractive component for engineering these circuits, as TALEs can be designed de novo to target a given DNA sequence. Currently, however, the use of TALEs is limited by degeneracy in the site-specific manner by which they recognize DNA. Here, we propose an algorithm to computationally address this problem. We apply our algorithm to design 180 TALEs targeting 20 bp cognate binding sites that are at least 3 nt mismatches away from all 20 bp sequences in putative 2 kb human promoter regions. We generated eight of these synthetic TALE activators and showed that each is able to activate transcription from a targeted reporter. Importantly, we show that these proteins do not activate synthetic reporters containing mismatches similar to those present in the genome nor a set of endogenous genes predicted to be the most likely targets in vivo. Finally, we generated and characterized TALE repressors comprised of our orthogonal DNA binding domains and further combined them with shRNAs to accomplish near complete repression of target gene expression. PMID:22581776

Ty1-copia elements reveal diverse insertion sites linked to polymorphisms among flax (Linum usitatissimum L.) accessions.

PubMed

Galindo-González, Leonardo; Mhiri, Corinne; Grandbastien, Marie-Angèle; Deyholos, Michael K

2016-12-07

Initial characterization of the flax genome showed that Ty1-copia retrotransposons are abundant, with several members being recently inserted, and in close association with genes. Recent insertions indicate a potential for ongoing transpositional activity that can create genomic diversity among accessions, cultivars or varieties. The polymorphisms generated constitute a good source of molecular markers that may be associated with phenotype if the insertions alter gene activity. Flax, where accessions are bred mainly for seed nutritional properties or for fibers, constitutes a good model for studying the relationship of transpositional activity with diversification and breeding. In this study, we estimated copy number and used a type of transposon display known as Sequence-Specific Amplification Polymorphisms (SSAPs), to characterize six families of Ty1-copia elements across 14 flax accessions. Polymorphic insertion sites were sequenced to find insertions that could potentially alter gene expression, and a preliminary test was performed with selected genes bearing transposable element (TE) insertions. Quantification of six families of Ty1-copia elements indicated different abundances among TE families and between flax accessions, which suggested diverse transpositional histories. SSAPs showed a high level of polymorphism in most of the evaluated retrotransposon families, with a trend towards higher levels of polymorphism in low-copy number families. Ty1-copia insertion polymorphisms among cultivars allowed a general distinction between oil and fiber types, and between spring and winter types, demonstrating their utility in diversity studies. Characterization of polymorphic insertions revealed an overwhelming association with genes, with insertions disrupting exons, introns or within 1 kb of coding regions. A preliminary test on the potential transcriptional disruption by TEs of four selected genes evaluated in three different tissues, showed one case of significant

Evolutionary transitions to new DNA methyltransferases through target site expansion and shrinkage.

PubMed

Rockah-Shmuel, Liat; Tawfik, Dan S

2012-12-01

DNA-binding and modifying proteins show high specificity but also exhibit a certain level of promiscuity. Such latent promiscuous activities comprise the starting points for new protein functions, but this hypothesis presents a paradox: a new activity can only evolve if it already exists. How then, do novel activities evolve? DNA methyltransferases, for example, are highly divergent in their target sites, but how transitions toward novel sites occur remains unknown. We performed laboratory evolution of the DNA methyltransferase M.HaeIII. We found that new target sites emerged primarily through expansion of the original site, GGCC, and the subsequent shrinkage of evolved expanded sites. Variants evolved for sites that are promiscuously methylated by M.HaeIII [GG((A)/(T))CC and GGCGCC] carried mutations in 'gate-keeper' residues. They could thereby methylate novel target sites such as GCGC and GGATCC that were neither selected for nor present in M.HaeIII. These 'generalist' intermediates were further evolved to obtain variants with novel target specificities. Our results demonstrate the ease by which new DNA-binding and modifying specificities evolve and the mechanism by which they occur at both the protein and DNA levels.

Multilocus patterns of polymorphism and selection across the X chromosome of Caenorhabditis remanei.

PubMed

Cutter, Asher D

2008-03-01

Natural selection and neutral processes such as demography, mutation, and gene conversion all contribute to patterns of polymorphism within genomes. Identifying the relative importance of these varied components in evolution provides the principal challenge for population genetics. To address this issue in the nematode Caenorhabditis remanei, I sampled nucleotide polymorphism at 40 loci across the X chromosome. The site-frequency spectrum for these loci provides no evidence for population size change, and one locus presents a candidate for linkage to a target of balancing selection. Selection for codon usage bias leads to the non-neutrality of synonymous sites, and despite its weak magnitude of effect (N(e)s approximately 0.1), is responsible for profound patterns of diversity and divergence in the C. remanei genome. Although gene conversion is evident for many loci, biased gene conversion is not identified as a significant evolutionary process in this sample. No consistent association is observed between synonymous-site diversity and linkage-disequilibrium-based estimators of the population recombination parameter, despite theoretical predictions about background selection or widespread genetic hitchhiking, but genetic map-based estimates of recombination are needed to rigorously test for a diversity-recombination relationship. Coalescent simulations also illustrate how a spurious correlation between diversity and linkage-disequilibrium-based estimators of recombination can occur, due in part to the presence of unbiased gene conversion. These results illustrate the influence that subtle natural selection can exert on polymorphism and divergence, in the form of codon usage bias, and demonstrate the potential of C. remanei for detecting natural selection from genomic scans of polymorphism.

Computational Predictions Provide Insights into the Biology of TAL Effector Target Sites

PubMed Central

Grau, Jan; Wolf, Annett; Reschke, Maik; Bonas, Ulla; Posch, Stefan; Boch, Jens

2013-01-01

Transcription activator-like (TAL) effectors are injected into host plant cells by Xanthomonas bacteria to function as transcriptional activators for the benefit of the pathogen. The DNA binding domain of TAL effectors is composed of conserved amino acid repeat structures containing repeat-variable diresidues (RVDs) that determine DNA binding specificity. In this paper, we present TALgetter, a new approach for predicting TAL effector target sites based on a statistical model. In contrast to previous approaches, the parameters of TALgetter are estimated from training data computationally. We demonstrate that TALgetter successfully predicts known TAL effector target sites and often yields a greater number of predictions that are consistent with up-regulation in gene expression microarrays than an existing approach, Target Finder of the TALE-NT suite. We study the binding specificities estimated by TALgetter and approve that different RVDs are differently important for transcriptional activation. In subsequent studies, the predictions of TALgetter indicate a previously unreported positional preference of TAL effector target sites relative to the transcription start site. In addition, several TAL effectors are predicted to bind to the TATA-box, which might constitute one general mode of transcriptional activation by TAL effectors. Scrutinizing the predicted target sites of TALgetter, we propose several novel TAL effector virulence targets in rice and sweet orange. TAL-mediated induction of the candidates is supported by gene expression microarrays. Validity of these targets is also supported by functional analogy to known TAL effector targets, by an over-representation of TAL effector targets with similar function, or by a biological function related to pathogen infection. Hence, these predicted TAL effector virulence targets are promising candidates for studying the virulence function of TAL effectors. TALgetter is implemented as part of the open-source Java library

[Genetic diversity and genetic structure of endangered wild Sinopodophyllum emodi by start codon targeted polymorphism].

PubMed

Chen, Da-Xia; Zhao, Ji-Feng; Liu, Xiang; Wang, Chang-Hua; Zhang, Zhi-Wei; Qin, Song-Yun; Zhong, Guo-Yue

2013-01-01

Revealed the genetic diversity level and genetic structure characteristics in Sinopodophyllum emodi, a rare and endangered species in China. We detected the genetic polymorphism within and among six wild populations (45 individuals) by the approach of Start Codon Targeted (SCoT) Polymorphism. The associated genetic parameters were calculated by POP-GENE1.31 and the relationship was constructed based on UPGMA method. A total of 350 bands were scored by 27 primers and 284 bands of them were polymorphic. The average polymorphic bands of each primer were 10.52. At species level, there was a high level of genetic diversity among six populations (PPB = 79.27%, N(e) = 1.332 7, H = 0.210 9 and H(sp) = 0.328 6). At population level, the genetic diversity level was low (PPB = 10.48% (4.00% -23.71%), N(e) = 1.048 7 (1.020 7-1.103 7), H = 0.029 7 (0.012 9-0.063 1), H(pop) = 0.046 2 (0.019 9-0.098 6). The Nei's coefficient of genetic differentiation was 0.841 1, which was consistent with the Shannon's coefficient of genetic differentiation (0.849 4). Two calculated methods all showed that most of the genetic variation existed among populations. The gene flow (N(m) = 0.094 4) was less among populations, indicating that the degree of genetic differentiation was higher. Genetic similarity coefficient were changed from 0.570 8 to 0.978 7. By clustering analysis, the tested populations were divided into two classes and had a tendency that the same geographical origin or material of similar habitats clustered into one group. The genetic diversity of samples of S. emodi is high,which laid a certain foundation for effective protection and improvement of germplasm resources.

Characteristics of Food Industry Web Sites and "Advergames" Targeting Children

ERIC Educational Resources Information Center

Culp, Jennifer; Bell, Robert A.; Cassady, Diana

2010-01-01

Objective: To assess the content of food industry Web sites targeting children by describing strategies used to prolong their visits and foster brand loyalty; and to document health-promoting messages on these Web sites. Design: A content analysis was conducted of Web sites advertised on 2 children's networks, Cartoon Network and Nickelodeon. A…

Target-classification approach applied to active UXO sites

NASA Astrophysics Data System (ADS)

Shubitidze, F.; Fernández, J. P.; Shamatava, Irma; Barrowes, B. E.; O'Neill, K.

2013-06-01

This study is designed to illustrate the discrimination performance at two UXO active sites (Oklahoma's Fort Sill and the Massachusetts Military Reservation) of a set of advanced electromagnetic induction (EMI) inversion/discrimination models which include the orthonormalized volume magnetic source (ONVMS), joint diagonalization (JD), and differential evolution (DE) approaches and whose power and flexibility greatly exceed those of the simple dipole model. The Fort Sill site is highly contaminated by a mix of the following types of munitions: 37-mm target practice tracers, 60-mm illumination mortars, 75-mm and 4.5'' projectiles, 3.5'', 2.36'', and LAAW rockets, antitank mine fuzes with and without hex nuts, practice MK2 and M67 grenades, 2.5'' ballistic windshields, M2A1-mines with/without bases, M19-14 time fuzes, and 40-mm practice grenades with/without cartridges. The site at the MMR site contains targets of yet different sizes. In this work we apply our models to EMI data collected using the MetalMapper (MM) and 2 × 2 TEMTADS sensors. The data for each anomaly are inverted to extract estimates of the extrinsic and intrinsic parameters associated with each buried target. (The latter include the total volume magnetic source or NVMS, which relates to size, shape, and material properties; the former includes location, depth, and orientation). The estimated intrinsic parameters are then used for classification performed via library matching and the use of statistical classification algorithms; this process yielded prioritized dig-lists that were submitted to the Institute for Defense Analyses (IDA) for independent scoring. The models' classification performance is illustrated and assessed based on these independent evaluations.

Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strauch, Eva-Maria; Bernard, Steffen M.; La, David

Many viral surface glycoproteins and cell surface receptors are homo-oligomers1, 2, 3, 4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5, 6, 7, 8. In the first step, a small protein ismore » designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.« less

Photoaffinity labeling in target- and binding-site identification

PubMed Central

Smith, Ewan; Collins, Ian

2015-01-01

Photoaffinity labeling (PAL) using a chemical probe to covalently bind its target in response to activation by light has become a frequently used tool in drug discovery for identifying new drug targets and molecular interactions, and for probing the location and structure of binding sites. Methods to identify the specific target proteins of hit molecules from phenotypic screens are highly valuable in early drug discovery. In this review, we summarize the principles of PAL including probe design and experimental techniques for in vitro and live cell investigations. We emphasize the need to optimize and validate probes and highlight examples of the successful application of PAL across multiple disease areas. PMID:25686004

Restriction Site Tiling Analysis: accurate discovery and quantitative genotyping of genome-wide polymorphisms using nucleotide arrays

PubMed Central

2010-01-01

High-throughput genotype data can be used to identify genes important for local adaptation in wild populations, phenotypes in lab stocks, or disease-related traits in human medicine. Here we advance microarray-based genotyping for population genomics with Restriction Site Tiling Analysis. The approach simultaneously discovers polymorphisms and provides quantitative genotype data at 10,000s of loci. It is highly accurate and free from ascertainment bias. We apply the approach to uncover genomic differentiation in the purple sea urchin. PMID:20403197

Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites.

PubMed

Betel, Doron; Koppal, Anjali; Agius, Phaedra; Sander, Chris; Leslie, Christina

2010-01-01

mirSVR is a new machine learning method for ranking microRNA target sites by a down-regulation score. The algorithm trains a regression model on sequence and contextual features extracted from miRanda-predicted target sites. In a large-scale evaluation, miRanda-mirSVR is competitive with other target prediction methods in identifying target genes and predicting the extent of their downregulation at the mRNA or protein levels. Importantly, the method identifies a significant number of experimentally determined non-canonical and non-conserved sites.

Sexual selection and genetic colour polymorphisms in animals.

PubMed

Wellenreuther, Maren; Svensson, Erik I; Hansson, Bengt

2014-11-01

Genetic colour polymorphisms are widespread across animals and often subjected to complex selection regimes. Traditionally, colour morphs were used as simple visual markers to measure allele frequency changes in nature, selection, population divergence and speciation. With advances in sequencing technology and analysis methods, several model systems are emerging where the molecular targets of selection are being described. Here, we discuss recent studies on the genetics of sexually selected colour polymorphisms, aiming at (i) reviewing the evidence of sexual selection on colour polymorphisms, (ii) highlighting the genetic architecture, molecular and developmental basis underlying phenotypic colour diversification and (iii) discuss how the maintenance of such polymorphisms might be facilitated or constrained by these. Studies of the genetic architecture of colour polymorphism point towards the importance of tight clustering of colour loci with other trait loci, such as in the case of inversions and supergene structures. Other interesting findings include linkage between colour loci and mate preferences or sex determination, and the role of introgression and regulatory variation in fuelling polymorphisms. We highlight that more studies are needed that explicitly integrate fitness consequences of sexual selection on colour with the underlying molecular targets of colour to gain insights into the evolutionary consequences of sexual selection on polymorphism maintenance. © 2014 John Wiley & Sons Ltd.

Single nucleotide polymorphisms at miR-146a/196a2 and their primary ovarian insufficiency-related target gene regulation in granulosa cells.

PubMed

Cho, Sung Hwan; An, Hui Jeong; Kim, Kyung Ah; Ko, Jung Jae; Kim, Ji Hyang; Kim, Young Ran; Ahn, Eun Hee; Rah, HyungChul; Lee, Woo Sik; Kim, Nam Keun

2017-01-01

MicroRNAs post-transcriptionally regulate gene expression in animals and plants. The aim of this study was to identify new target genes for microRNA polymorphisms (miR-146aC>G and miR-196a2T>C) in primary ovarian insufficiency (POI). We cloned and transfected miR-146aC>G and miR-196a2T>C into human granulosa cells and used microarrays and qPCR-arrays to examine the changes in the messenger RNA expression profile. We show miR-146aC>G and miR-196a2T>C change the mRNA expression patterns in granulosa cell. In each case, mRNAs were up or down-regulated after treatments with miR-146a C or G and miR-196a2 T or C. We found that miR-146a led to a significantly altered regulation of the mRNA levels of FOXO3, FOXL2 and CCND2 compared to controls. We also found that the polymorphisms of miR-146a led to a significantly altered regulation of CCND2 and FOXO3. Our results suggest that miR-146aC>G and miR-196a2T>C can regulate the levels of many of their target transcripts. In addition, specific target genes of miR-146aC>G polymorphisms may be involved in granulosa cell regulation.

Single nucleotide polymorphisms at miR-146a/196a2 and their primary ovarian insufficiency-related target gene regulation in granulosa cells

PubMed Central

Cho, Sung Hwan; An, Hui Jeong; Kim, Kyung Ah; Ko, Jung Jae; Kim, Ji Hyang; Kim, Young Ran; Ahn, Eun Hee; Rah, HyungChul; Lee, Woo Sik

2017-01-01

MicroRNAs post-transcriptionally regulate gene expression in animals and plants. The aim of this study was to identify new target genes for microRNA polymorphisms (miR-146aC>G and miR-196a2T>C) in primary ovarian insufficiency (POI). We cloned and transfected miR-146aC>G and miR-196a2T>C into human granulosa cells and used microarrays and qPCR-arrays to examine the changes in the messenger RNA expression profile. We show miR-146aC>G and miR-196a2T>C change the mRNA expression patterns in granulosa cell. In each case, mRNAs were up or down-regulated after treatments with miR-146a C or G and miR-196a2 T or C. We found that miR-146a led to a significantly altered regulation of the mRNA levels of FOXO3, FOXL2 and CCND2 compared to controls. We also found that the polymorphisms of miR-146a led to a significantly altered regulation of CCND2 and FOXO3. Our results suggest that miR-146aC>G and miR-196a2T>C can regulate the levels of many of their target transcripts. In addition, specific target genes of miR-146aC>G polymorphisms may be involved in granulosa cell regulation. PMID:28841705

Color polymorphic lures target different visual channels in prey.

PubMed

White, Thomas E; Kemp, Darrell J

2016-06-01

Selection for signal efficacy in variable environments may favor color polymorphism, but little is known about this possibility outside of sexual systems. Here we used the color polymorphic orb-web spider Gasteracantha fornicata, whose yellow- or white-banded dorsal signal attracts dipteran prey, to test the hypothesis that morphs may be tuned to optimize either chromatic or achromatic conspicuousness in their visually noisy forest environments. We used data from extensive observations of naturally existing spiders and precise assessments of visual environments to model signal conspicuousness according to dipteran vision. Modeling supported a distinct bias in the chromatic (yellow morph) or achromatic (white morph) contrast presented by spiders at the times when they caught prey, as opposed to all other times at which they may be viewed. Hence, yellow spiders were most successful when their signal produced maximum color contrast against viewing backgrounds, whereas white spiders were most successful when they presented relatively greatest luminance contrast. Further modeling across a hypothetical range of lure variation confirmed that yellow versus white signals should, respectively, enhance chromatic versus achromatic conspicuousness to flies, in G. fornicata's visual environments. These findings suggest that color polymorphism may be adaptively maintained by selection for conspicuousness within different visual channels in receivers. © 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.

Target Site Recognition by a Diversity-Generating Retroelement

PubMed Central

Guo, Huatao; Tse, Longping V.; Nieh, Angela W.; Czornyj, Elizabeth; Williams, Steven; Oukil, Sabrina; Liu, Vincent B.; Miller, Jeff F.

2011-01-01

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification. PMID:22194701

Near Surface Swimming of Salmonella Typhimurium Explains Target-Site Selection and Cooperative Invasion

PubMed Central

Kreibich, Saskia; Vonaesch, Pascale; Andritschke, Daniel; Rout, Samuel; Weidner, Kerstin; Sormaz, Milos; Songhet, Pascal; Horvath, Peter; Chabria, Mamta; Vogel, Viola; Spori, Doris M.; Jenny, Patrick; Hardt, Wolf-Dietrich

2012-01-01

Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by S. Typhimurium. This enteropathogenic bacterium employs adhesins (e.g. fim) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically, S. Typhimurium invasion is focused on a subset of cells and multiple bacteria invade via the same ruffle. It has remained unclear how this is achieved. We have studied target-site selection in tissue culture by time lapse microscopy, movement pattern analysis and modeling. Flagellar motility (but not chemotaxis) was required for reaching the host cell surface in vitro. Subsequently, physical forces trapped the pathogen for ∼1.5–3 s in “near surface swimming”. This increased the local pathogen density and facilitated “scanning” of the host surface topology. We observed transient TTSS-1 and fim-independent “stopping” and irreversible TTSS-1-mediated docking, in particular at sites of prominent topology, i.e. the base of rounded-up cells and membrane ruffles. Our data indicate that target site selection and the cooperative infection of membrane ruffles are attributable to near surface swimming. This mechanism might be of general importance for understanding infection by flagellated bacteria. PMID:22911370

Initial basalt target site selection evaluation for the Mars penetrator drop test

NASA Technical Reports Server (NTRS)

Bunch, T. E.; Quaide, W. L.; Polkowski, G.

1976-01-01

Potential basalt target sites for an air drop penetrator test were described and the criteria involved in site selection were discussed. A summary of the background field geology and recommendations for optimum sites are also presented.

Mathematical description of drug-target interactions: application to biologics that bind to targets with two binding sites.

PubMed

Gibiansky, Leonid; Gibiansky, Ekaterina

2018-02-01

The emerging discipline of mathematical pharmacology occupies the space between advanced pharmacometrics and systems biology. A characteristic feature of the approach is application of advance mathematical methods to study the behavior of biological systems as described by mathematical (most often differential) equations. One of the early application of mathematical pharmacology (that was not called this name at the time) was formulation and investigation of the target-mediated drug disposition (TMDD) model and its approximations. The model was shown to be remarkably successful, not only in describing the observed data for drug-target interactions, but also in advancing the qualitative and quantitative understanding of those interactions and their role in pharmacokinetic and pharmacodynamic properties of biologics. The TMDD model in its original formulation describes the interaction of the drug that has one binding site with the target that also has only one binding site. Following the framework developed earlier for drugs with one-to-one binding, this work aims to describe a rigorous approach for working with similar systems and to apply it to drugs that bind to targets with two binding sites. The quasi-steady-state, quasi-equilibrium, irreversible binding, and Michaelis-Menten approximations of the model are also derived. These equations can be used, in particular, to predict concentrations of the partially bound target (RC). This could be clinically important if RC remains active and has slow internalization rate. In this case, introduction of the drug aimed to suppress target activity may lead to the opposite effect due to RC accumulation.

Voltage-gated sodium channel polymorphism and metabolic resistance in pyrethroid-resistant Aedes aegypti from Brazil.

PubMed

Martins, Ademir Jesus; Lins, Rachel Mazzei Moura de Andrade; Linss, Jutta Gerlinde Birgitt; Peixoto, Alexandre Afranio; Valle, Denise

2009-07-01

The nature of pyrethroid resistance in Aedes aegypti Brazilian populations was investigated. Quantification of enzymes related to metabolic resistance in two distinct populations, located in the Northeast and Southeast regions, revealed increases in Glutathione-S-transferase (GST) and Esterase levels. Additionally, polymorphism was found in the IIS6 region of Ae. aegypti voltage-gated sodium channel (AaNa(V)), the pyrethroid target site. Sequences were classified in two haplotype groups, A and B, according to the size of the intron in that region. Rockefeller, a susceptible control lineage, contains only B sequences. In field populations, some A sequences present a substitution in the 1011 site (Ile/Met). When resistant and susceptible individuals were compared, the frequency of both A (with the Met mutation) and B sequences were slightly increased in resistant specimens. The involvement of the AaNa(V) polymorphism in pyrethroid resistance and the metabolic mechanisms that lead to potential cross-resistance between organophosphate and pyrethroids are discussed.

Polymorphisms at the Ligand Binding Site of the Vitamin D Receptor Gene and Osteomalacia

PubMed Central

Ak, Duygu Gezen; Kahraman, Hakkí; Dursun, Erdinç; Duman, Belgin Süsleyici; Erensoy, Nevin; Alagöl, Faruk; Tanakol, Refik; Yılmazer, Selma

2005-01-01

Vitamin D receptor (VDR) gene polymorphisms have been suggested as possible determinants of bone mineral density (BMD) and calcium metabolism. In this study, our aim was to determine whether there is an association between VDR gene polymorphism and osteomalacia or not. We determined ApaI and TaqI polymorphisms in the vitamin D receptor gene in 24 patients with osteomalacia and 25 age-matched healthy controls. Serum calcium, phosphorus, ALP, PTH, 25OHD levels were also examined. We used PCR and RFLP methods to test for an association between osteomalacia and polymorphisms within, intron 8 and exon 9 of the VDR gene. When the control and patients were compared for their ApaI and TaqI genotypes there was no relationship between VDR gene allelic polymorphisms and osteomalacia. Whereas a nearly significant difference for A allele was found in the allellic distribution of the patients (p = 0.08). Also no association between biochemical data and VDR gene polymorphisms was observed. PMID:16403954

Detection of the Single Nucleotide Polymorphism at Position rs2735940 in the Human Telomerase Reverse Transcriptase Gene by the Introduction of a New Restriction Enzyme Site for the PCR-RFLP Assay.

PubMed

Wang, Sihua; Ding, Mingcui; Duan, Xiaoran; Wang, Tuanwei; Feng, Xiaolei; Wang, Pengpeng; Yao, Wu; Wu, Yongjun; Yan, Zhen; Feng, Feifei; Yu, Songcheng; Wang, Wei

2017-09-01

It has been shown that the single nucleotide polymorphism (SNP) of the rs2735940 site in the human telomerase reverse transcriptase ( hTERT ) gene is associated with increased cancer risk. The traditional method to detect SNP genotypes is polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, there is a limitation to utilizing PCR-RFLP due to a lack of proper restriction enzyme sites at many polymorphic loci. This study used an improved PCR-RFLP method with a mismatched base for detection of the SNP rs2735940. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and in addition, the restriction enzyme Msp I for CRS-PCR was cheaper than other enzymes. We used this novel assay to determine the allele frequencies in 552 healthy Chinese Han individuals, and found the allele frequencies to be 63% for allele C and 37% for allele T In summary, the modified PCR-RFLP can be used to detect the SNP of rs2735940 with low cost and high efficiency. © 2017 by the Association of Clinical Scientists, Inc.

The impact of target site accessibility on the design of effective siRNAs.

PubMed

Tafer, Hakim; Ameres, Stefan L; Obernosterer, Gregor; Gebeshuber, Christoph A; Schroeder, Renée; Martinez, Javier; Hofacker, Ivo L

2008-05-01

Small-interfering RNAs (siRNAs) assemble into RISC, the RNA-induced silencing complex, which cleaves complementary mRNAs. Despite their fluctuating efficacy, siRNAs are widely used to assess gene function. Although this limitation could be ascribed, in part, to variations in the assembly and activation of RISC, downstream events in the RNA interference (RNAi) pathway, such as target site accessibility, have so far not been investigated extensively. In this study we present a comprehensive analysis of target RNA structure effects on RNAi by computing the accessibility of the target site for interaction with the siRNA. Based on our observations, we developed a novel siRNA design tool, RNAxs, by combining known siRNA functionality criteria with target site accessibility. We calibrated our method on two data sets comprising 573 siRNAs for 38 genes, and tested it on an independent set of 360 siRNAs targeting four additional genes. Overall, RNAxs proves to be a robust siRNA selection tool that substantially improves the prediction of highly efficient siRNAs.

Recovery of perennial vegetation in military target sites in the eastern Mohave Desert, Arizona

USGS Publications Warehouse

Steiger, John W.; Webb, Robert H.

2000-01-01

The effect of the age of geomorphic surfaces on the recovery of desert vegetation in military target sites was studied in the Mohave and Cerbat Mountains of northwestern Arizona. The target sites were cleared of all vegetation during military exercises in 1942-1943 and have not been subsequently disturbed. The degree of recovery was measured by calculating percentage-similarity (PS) and correlation-coefficient indices on the basis of differences in cover, density, and volume of species growing in and out of each target site. PS values, ranging from 22.7 to 95.1 percent (100 percent = identical composition), indicate a wide range of recovery that is partially controlled by the edaphic properties of the geomorphic surfaces. Statistical analyses show a strong pattern that indicates a greater variability in the degree of recovery for sites on older surfaces than on younger surfaces and a weak pattern that indicates an inverse relation between the degree of recovery and geomorphic age. Comparisons of the different effects of target site construction on the edaphic characteristics of each target site provides an explanation for these patterns and suggests the soil properties critical to the recovery process. Statistically significant negative or positive response to disturbance for most species are independent of the age of the geomorphic surfaces; however, there is strong evidence for a shift in response for the common perennial species Acamptopappus sphaerocephalus, and to a lesser extent, Salazaria mexicana, Encelia farinosa, and Coldenia canescens, among different geomorphic surfaces.

Species-specific markers for the differential diagnosis of Trypanosoma cruzi and Trypanosoma rangeli and polymorphisms detection in Trypanosoma rangeli.

PubMed

Ferreira, Keila Adriana Magalhães; Fajardo, Emanuella Francisco; Baptista, Rodrigo P; Macedo, Andrea Mara; Lages-Silva, Eliane; Ramírez, Luis Eduardo; Pedrosa, André Luiz

2014-06-01

Trypanosoma cruzi and Trypanosoma rangeli are kinetoplastid parasites which are able to infect humans in Central and South America. Misdiagnosis between these trypanosomes can be avoided by targeting barcoding sequences or genes of each organism. This work aims to analyze the feasibility of using species-specific markers for identification of intraspecific polymorphisms and as target for diagnostic methods by PCR. Accordingly, primers which are able to specifically detect T. cruzi or T. rangeli genomic DNA were characterized. The use of intergenic regions, generally divergent in the trypanosomatids, and the serine carboxypeptidase gene were successful. Using T. rangeli genomic sequences for the identification of group-specific polymorphisms and a polymorphic AT(n) dinucleotide repeat permitted the classification of the strains into two groups, which are entirely coincident with T. rangeli main lineages, KP1 (+) and KP1 (-), previously determined by kinetoplast DNA (kDNA) characterization. The sequences analyzed totalize 622 bp (382 bp represent a hypothetical protein sequence, and 240 bp represent an anonymous sequence), and of these, 581 (93.3%) are conserved sites and 41 bp (6.7%) are polymorphic, with 9 transitions (21.9%), 2 transversions (4.9%), and 30 (73.2%) insertion/deletion events. Taken together, the species-specific markers analyzed may be useful for the development of new strategies for the accurate diagnosis of infections. Furthermore, the identification of T. rangeli polymorphisms has a direct impact in the understanding of the population structure of this parasite.

DNA methylation and methylation polymorphism in ecotypes of Jatropha curcas L. using methylation-sensitive AFLP markers.

PubMed

Mastan, Shaik G; Rathore, Mangal S; Bhatt, Vacha D; Chikara, J; Ghosh, A

2014-12-01

We investigated DNA methylation and polymorphism in the methylated DNA using AFLP based methylation-sensitive amplification polymorphism (MS-AFLP) markers in ecotypes of Jatropha curcas L. growing in similar and different geo-ecological conditions. Three ecotypes growing in different geo-ecological conditions with environmental heterogeneity (Group-1) and five ecotypes growing in similar environmental conditions (Group-2) were assessed. In ecotypes growing in group-1, 44.32 % DNA was methylated and of which 93.59 % DNA was polymorphic. While in group-2, 32.27 % DNA was methylated, of which 51.64 % DNA was polymorphic. In site 1 and site 2 of group-1, overall methylation was 18.94 and 22.44 % respectively with difference of 3.5 %, while overall polymorphism was 41.14 and 39.23 % with a difference of 1.91 %. In site 1 and site 2 of group-2, overall methylation was 24.68 and 24.18 % respectively with difference of 0.5 %, while overall polymorphism was 12.19 and 12.65 % with a difference of 0.46 %. The difference of methylation percentage and percentage of methylation polymorphism throughout the genome of J. curcas at site 1 and 2 of group-1 is higher than that of J. curcas at site 1 and 2 of group-2. These results correlated the physico-chemical properties of soil at these sites. The variations of physico-chemical properties of soil at Chorwadla (site 1 in group-1 and site 2 in group-2) compared to the soil at Brahmapur (site 2 in group-1) is higher than that of soil at Neswad (site 1 in group-2). The study suggests that these homologous nucleotide sequences probably play important role in ecotype adaptation to environmental heterogeneity by creating epiallelic variations hence in evolution of ecotypes/clines or forms of species showing phenotypic/genotypic differences in different geographical areas.

Search for methylation-sensitive amplification polymorphisms associated with the mantled variant phenotype in oil palm (Elaeis guineensis Jacq).

PubMed

Jaligot, E; Beulé, T; Baurens, F-C; Billotte, N; Rival, A

2004-02-01

The methylation-sensitive amplification polymorphism (MSAP) technique has been employed on somatic embryo-derived oil palms (Elaeis guineensis Jacq.) to identify methylation polymorphisms correlated with the "mantled" somaclonal variation. The variant phenotype displays an unstable feminization of male organs in both male and female flowers. Using MSAP, the methylation status of CCGG sites was compared in three normal versus three mantled regenerants sampled in clonal populations obtained through somatic embryogenesis from four genotypically distinct mother palms. Overall, 64 selective primer combinations were used and they have amplified 23 markers exhibiting a differential methylation pattern between the two phenotypes. Our results indicate that CCGG sites are poorly affected by the considerable decrease in global DNA methylation that has been previously associated with the mantled phenotype. Each of the 23 markers isolated in the present study could discriminate between the two phenotypes only when they were from the same genetic origin. This result hampers at the moment the direct use of MSAP markers for the early detection of variants, even though valuable information on putative target sequences will be obtained from a further characterization of these polymorphic markers.

Toll-like receptor polymorphisms in malaria-endemic populations

PubMed Central

Greene, Jennifer A; Moormann, Ann M; Vulule, John; Bockarie, Moses J; Zimmerman, Peter A; Kazura, James W

2009-01-01

Background Toll-like receptors (TLR) and related downstream signaling pathways of innate immunity have been implicated in the pathogenesis of Plasmodium falciparum malaria. Because of their potential role in malaria pathogenesis, polymorphisms in these genes may be under selective pressure in populations where this infectious disease is endemic. Methods A post-PCR Ligation Detection Reaction-Fluorescent Microsphere Assay (LDR-FMA) was developed to determine the frequencies of TLR2, TLR4, TLR9, MyD88-Adaptor Like Protein (MAL) single nucleotide polymorphisms (SNPs), and TLR2 length polymorphisms in 170 residents of two regions of Kenya where malaria transmission is stable and high (holoendemic) or episodic and low, 346 residents of a malaria holoendemic region of Papua New Guinea, and 261 residents of North America of self-identified ethnicity. Results The difference in historical malaria exposure between the two Kenyan sites has significantly increased the frequency of malaria protective alleles glucose-6-phoshpate dehydrogenase (G6PD) and Hemoglobin S (HbS) in the holoendemic site compared to the episodic transmission site. However, this study detected no such difference in the TLR2, TLR4, TLR9, and MAL allele frequencies between the two study sites. All polymorphisms were in Hardy Weinberg Equilibrium in the Kenyan and Papua New Guinean populations. TLR9 SNPs and length polymorphisms within the TLR2 5' untranslated region were the only mutant alleles present at a frequency greater than 10% in all populations. Conclusion Similar frequencies of TLR2, TLR4, TLR9, and MAL genetic polymorphisms in populations with different histories of malaria exposure suggest that these innate immune pathways have not been under strong selective pressure by malaria. Genotype frequencies are consistent with Hardy-Weinberg Equilibrium and the Neutral Theory, suggesting that genetic drift has influenced allele frequencies to a greater extent than selective pressure from malaria or any

Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

NASA Astrophysics Data System (ADS)

Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

2016-07-01

Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

Characteristics of food industry web sites and "advergames" targeting children.

PubMed

Culp, Jennifer; Bell, Robert A; Cassady, Diana

2010-01-01

To assess the content of food industry Web sites targeting children by describing strategies used to prolong their visits and foster brand loyalty; and to document health-promoting messages on these Web sites. A content analysis was conducted of Web sites advertised on 2 children's networks, Cartoon Network and Nickelodeon. A total of 290 Web pages and 247 unique games on 19 Internet sites were examined. Games, found on 81% of Web sites, were the most predominant promotion strategy used. All games had at least 1 brand identifier, with logos being most frequently used. On average Web sites contained 1 "healthful" message for every 45 exposures to brand identifiers. Food companies use Web sites to extend their television advertising to promote brand loyalty among children. These sites almost exclusively promoted food items high in sugar and fat. Health professionals need to monitor food industry marketing practices used in "new media." Published by Elsevier Inc.

Widespread occurrence of both metabolic and target-site herbicide resistance mechanisms in Lolium rigidum populations.

PubMed

Han, Heping; Yu, Qin; Owen, Mechelle J; Cawthray, Gregory R; Powles, Stephen B

2016-02-01

Lolium rigidum populations in Australia and globally have demonstrated rapid and widespread evolution of resistance to acetyl coenzyme A carboxylase (ACCase)-inhibiting and acetolactate synthase (ALS)-inhibiting herbicides. Thirty-three resistant L. rigidum populations, randomly collected from crop fields in a most recent resistance survey, were analysed for non-target-site diclofop metabolism and all known target-site ACCase gene resistance-endowing mutations. The HPLC profile of [(14) C]-diclofop-methyl in vivo metabolism revealed that 79% of these resistant L. rigidum populations showed enhanced capacity for diclofop acid metabolism (metabolic resistance). ACCase gene sequencing identified that 91% of the populations contain plants with ACCase resistance mutation(s). Importantly, 70% of the populations exhibit both non-target-site metabolic resistance and target-site ACCase mutations. This work demonstrates that metabolic herbicide resistance is commonly occurring in L. rigidum, and coevolution of both metabolic resistance and target-site resistance is an evolutionary reality. Metabolic herbicide resistance can potentially endow resistance to many herbicides and poses a threat to herbicide sustainability and thus crop production, calling for major research and management efforts. © 2015 Society of Chemical Industry.

Widespread Site-Dependent Buffering of Human Regulatory Polymorphism

PubMed Central

Kutyavin, Tanya; Stamatoyannopoulos, John A.

2012-01-01

The average individual is expected to harbor thousands of variants within non-coding genomic regions involved in gene regulation. However, it is currently not possible to interpret reliably the functional consequences of genetic variation within any given transcription factor recognition sequence. To address this, we comprehensively analyzed heritable genome-wide binding patterns of a major sequence-specific regulator (CTCF) in relation to genetic variability in binding site sequences across a multi-generational pedigree. We localized and quantified CTCF occupancy by ChIP-seq in 12 related and unrelated individuals spanning three generations, followed by comprehensive targeted resequencing of the entire CTCF–binding landscape across all individuals. We identified hundreds of variants with reproducible quantitative effects on CTCF occupancy (both positive and negative). While these effects paralleled protein–DNA recognition energetics when averaged, they were extensively buffered by striking local context dependencies. In the significant majority of cases buffering was complete, resulting in silent variants spanning every position within the DNA recognition interface irrespective of level of binding energy or evolutionary constraint. The prevalence of complex partial or complete buffering effects severely constrained the ability to predict reliably the impact of variation within any given binding site instance. Surprisingly, 40% of variants that increased CTCF occupancy occurred at positions of human–chimp divergence, challenging the expectation that the vast majority of functional regulatory variants should be deleterious. Our results suggest that, even in the presence of “perfect” genetic information afforded by resequencing and parallel studies in multiple related individuals, genomic site-specific prediction of the consequences of individual variation in regulatory DNA will require systematic coupling with empirical functional genomic measurements

Terminal Restriction Fragment Length Polymorphism Analysis Program, a Web-Based Research Tool for Microbial Community Analysis

PubMed Central

Marsh, Terence L.; Saxman, Paul; Cole, James; Tiedje, James

2000-01-01

Rapid analysis of microbial communities has proven to be a difficult task. This is due, in part, to both the tremendous diversity of the microbial world and the high complexity of many microbial communities. Several techniques for community analysis have emerged over the past decade, and most take advantage of the molecular phylogeny derived from 16S rRNA comparative sequence analysis. We describe a web-based research tool located at the Ribosomal Database Project web site (http://www.cme.msu.edu/RDP/html/analyses.html) that facilitates microbial community analysis using terminal restriction fragment length polymorphism of 16S ribosomal DNA. The analysis function (designated TAP T-RFLP) permits the user to perform in silico restriction digestions of the entire 16S sequence database and derive terminal restriction fragment sizes, measured in base pairs, from the 5′ terminus of the user-specified primer to the 3′ terminus of the restriction endonuclease target site. The output can be sorted and viewed either phylogenetically or by size. It is anticipated that the site will guide experimental design as well as provide insight into interpreting results of community analysis with terminal restriction fragment length polymorphisms. PMID:10919828

Association of the Serotonin Receptor 3E Gene as a Functional Variant in the MicroRNA-510 Target Site with Diarrhea Predominant Irritable Bowel Syndrome in Chinese Women.

PubMed

Zhang, Yu; Li, Yaoyao; Hao, Zhenfeng; Li, Xiangming; Bo, Ping; Gong, Weijuan

2016-04-30

The functional variant (rs56109847) in the 3'-untranslated regions (3'-UTR) of the serotonin receptor 3E (HTR3E) gene is associated with female diarrhea predominant irritable bowel syndrome (IBS-D) in British populations. However, the relationship of the polymorphism both to HTR3E expression in the intestine and to the occurrence of Chinese functional gastrointestinal disorders has yet to be examined. Polymerase chain reaction amplification and restriction fragment length polymorphism analyses were employed to detect polymorphisms among Chinese Han women, particularly 107 patients with IBS-D, 99 patients with functional dyspepsia (FD), 115 patients with mixed IBS and 69 patients with IBS-D + FD. We also assessed microRNA-510 (miR-510) and HTR3Eexpression in human colonic mucosal tissues with immunohistochemistry and other methods. Dual-luciferase reporter assays were conducted to examine the binding ability of miR-510 and HTR3E 3'-UTR. Genotyping data showed the variant rs56109847 was significantly associated with IBS-D, but not with FD, mixed-IBS, or FD + IBS-D. HTR3E was abundantly expressed around the colonic mucosal glands but less expressed in the stroma. miR-510 expression decreased, whereas HTR3E expression increased in the colonic mucosal tissue of patients with IBS-D compared with those in controls. HTR3E expression was significantly higher in patients with the GA genotype than that in patients with the GG genotype. The single-nucleotide polymorphisms disrupted the binding site of miR-510 and significantly upregulated luciferase expression in HEK293 and HT-29 cells. The single-nucleotide polymorphisms rs56109847 led to reduced microRNA binding and overexpression of the target gene in intestinal cells, thereby increasing IBS-D risk in the Chinese Han population. The decreased expression of miR-510 might contribute to IBS-D.

Target sites for the transposition of rat long interspersed repeated DNA elements (LINEs) are not random.

PubMed Central

Furano, A V; Somerville, C C; Tsichlis, P N; D'Ambrosio, E

1986-01-01

The long interspersed repeated DNA family of rats (LINE or L1Rn family) contains about 40,000 6.7-kilobase (kb) long members (1). LINE members may be currently mobile since their presence or absence causes allelic variation at three single copy loci (2, 3): insulin 1, Moloney leukemia virus integration 2 (Mlvi-2) (4), and immunoglobulin heavy chain (Igh). To characterize target sites for LINE insertion, we compared the DNA sequences of the unoccupied Mlvi-2 target site, its LINE-containing allele, and several other LINE-containing sites. Although not homologous overall, the target sites share three characteristics: First, depending on the site, they are from 68% to 86% (A+T) compared to 58% (A+T) for total rat DNA (5). Depending on the site, a 7- to 15-bp target site sequence becomes duplicated and flanks the inserted LINE member. The second is a version (0 or 1 mismatch) of the hexanucleotide, TACTCA, which is also present in the LINE member, in a highly conserved region located just before the A-rich right end of the LINE member. The third is a stretch of alternating purine/pyrimidine (PQ). The A-rich right ends of different LINE members vary in length and composition, and the sequence of a particularly long one suggests that it contains the A-rich target site from a previous transposition. PMID:3012480

RNase L targets distinct sites in influenza A virus RNAs.

PubMed

Cooper, Daphne A; Banerjee, Shuvojit; Chakrabarti, Arindam; García-Sastre, Adolfo; Hesselberth, Jay R; Silverman, Robert H; Barton, David J

2015-03-01

Influenza A virus (IAV) infections are influenced by type 1 interferon-mediated antiviral defenses and by viral countermeasures to these defenses. When IAV NS1 protein is disabled, RNase L restricts virus replication; however, the RNAs targeted for cleavage by RNase L under these conditions have not been defined. In this study, we used deep-sequencing methods to identify RNase L cleavage sites within host and viral RNAs from IAV PR8ΔNS1-infected A549 cells. Short hairpin RNA knockdown of RNase L allowed us to distinguish between RNase L-dependent and RNase L-independent cleavage sites. RNase L-dependent cleavage sites were evident at discrete locations in IAV RNA segments (both positive and negative strands). Cleavage in PB2, PB1, and PA genomic RNAs suggests that viral RNPs are susceptible to cleavage by RNase L. Prominent amounts of cleavage mapped to specific regions within IAV RNAs, including some areas of increased synonymous-site conservation. Among cellular RNAs, RNase L-dependent cleavage was most frequent at precise locations in rRNAs. Our data show that RNase L targets specific sites in both host and viral RNAs to restrict influenza virus replication when NS1 protein is disabled. RNase L is a critical component of interferon-regulated and double-stranded-RNA-activated antiviral host responses. We sought to determine how RNase L exerts its antiviral activity during influenza virus infection. We enhanced the antiviral activity of RNase L by disabling a viral protein, NS1, that inhibits the activation of RNase L. Then, using deep-sequencing methods, we identified the host and viral RNAs targeted by RNase L. We found that RNase L cleaved viral RNAs and rRNAs at very precise locations. The direct cleavage of IAV RNAs by RNase L highlights an intimate battle between viral RNAs and an antiviral endonuclease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

PAM multiplicity marks genomic target sites as inhibitory to CRISPR-Cas9 editing.

PubMed

Malina, Abba; Cameron, Christopher J F; Robert, Francis; Blanchette, Mathieu; Dostie, Josée; Pelletier, Jerry

2015-12-08

In CRISPR-Cas9 genome editing, the underlying principles for selecting guide RNA (gRNA) sequences that would ensure for efficient target site modification remain poorly understood. Here we show that target sites harbouring multiple protospacer adjacent motifs (PAMs) are refractory to Cas9-mediated repair in situ. Thus we refine which substrates should be avoided in gRNA design, implicating PAM density as a novel sequence-specific feature that inhibits in vivo Cas9-driven DNA modification.

A novel W1999S mutation and non-target site resistance impact on acetyl-CoA carboxylase inhibiting herbicides to varying degrees in a UK Lolium multiflorum population.

PubMed

Kaundun, Shiv Shankhar; Bailly, Geraldine C; Dale, Richard P; Hutchings, Sarah-Jane; McIndoe, Eddie

2013-01-01

Acetyl-CoA carboxylase (ACCase) inhibiting herbicides are important products for the post-emergence control of grass weed species in small grain cereal crops. However, the appearance of resistance to ACCase herbicides over time has resulted in limited options for effective weed control of key species such as Lolium spp. In this study, we have used an integrated biological and molecular biology approach to investigate the mechanism of resistance to ACCase herbicides in a Lolium multiflorum Lam. from the UK (UK21). The study revealed a novel tryptophan to serine mutation at ACCase codon position 1999 impacting on ACCase inhibiting herbicides to varying degrees. The W1999S mutation confers dominant resistance to pinoxaden and partially recessive resistance to cycloxydim and sethoxydim. On the other hand, plants containing the W1999S mutation were sensitive to clethodim and tepraloxydim. Additionally population UK21 is characterised by other resistance mechanisms, very likely non non-target site based, affecting several aryloxyphenoxyproprionate (FOP) herbicides but not the practical field rate of pinoxaden. The positive identification of wild type tryptophan and mutant serine alleles at ACCase position 1999 could be readily achieved with an original DNA based derived cleaved amplified polymorphic sequence (dCAPS) assay that uses the same PCR product but two different enzymes for positively identifying the wild type tryptophan and mutant serine alleles identified here. This paper highlights intrinsic differences between ACCase inhibiting herbicides that could be exploited for controlling ryegrass populations such as UK21 characterised by compound-specific target site and non-target site resistance.

DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance.

PubMed

Zhao, Jie-hong; Zhang, Ji-shun; Wang, Yi; Wang, Ren-gang; Wu, Chun; Fan, Long-jiang; Ren, Xue-liang

2011-11-01

DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth, development, and polyploidization. However, there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics. We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco, Nicotiana tabacum, using a methylation-sensitive amplified polymorphism (MSAP) technique. The results showed that methylation existed at a high level among tobacco accessions, among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic. A cluster analysis revealed distinct patterns of geography-specific groups. In addition, three polymorphic sites significantly related to tobacco mosaic virus (TMV) resistance were explored. This suggests that tobacco breeders should pay more attention to epigenetic traits.

DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance*

PubMed Central

Zhao, Jie-hong; Zhang, Ji-shun; Wang, Yi; Wang, Ren-gang; Wu, Chun; Fan, Long-jiang; Ren, Xue-liang

2011-01-01

DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth, development, and polyploidization. However, there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics. We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco, Nicotiana tabacum, using a methylation-sensitive amplified polymorphism (MSAP) technique. The results showed that methylation existed at a high level among tobacco accessions, among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic. A cluster analysis revealed distinct patterns of geography-specific groups. In addition, three polymorphic sites significantly related to tobacco mosaic virus (TMV) resistance were explored. This suggests that tobacco breeders should pay more attention to epigenetic traits. PMID:22042659

Simultaneous human platelet antigen genotyping and detection of novel single nucleotide polymorphisms by targeted next-generation sequencing.

PubMed

Davey, Sue; Navarrete, Cristina; Brown, Colin

2017-06-01

Twenty-nine human platelet antigen systems have been described to date, but the majority of current genotyping methods are restricted to the identification of those most commonly associated with alloantibody production in a clinical context. This can result in a protracted investigation if causative human platelet antigens are rare or novel. A targeted next-generation sequencing approach was designed to detect all known human platelet antigens with the additional capability of identifying novel mutations in the encoding genes. A targeted enrichment, high-sensitivity HaloPlex assay was designed to sequence all exons and flanking regions of the six genes known to encode human platelet antigens. Indexed DNA libraries were prepared from 47 previously human platelet antigen-genotyped samples and subsequently combined into one of three pools for sequencing on an Illumina MiSeq platform. The generated FASTQ files were aligned and scrutinized for each human platelet antigen polymorphism using SureCall data analysis software. Forty-six samples were successfully genotyped for human platelet antigens 1 through 29bw, with an average per base coverage depth of 1144. Concordance with historical human platelet antigen genotypes was 100%. A putative novel mutation in Exon 10 of the integrin β-3 (ITGB3) gene from an unsolved case of fetal neonatal alloimmune thrombocytopenia was also detected. A next-generation sequencing-based method that can accurately define all known human platelet antigen polymorphisms was developed. With the ability to sequence up to 96 samples simultaneously, our HaloPlex design could be used for high-throughput human platelet antigen genotyping. This method is also applicable for investigating fetal neonatal alloimmune thrombocytopenia when rare or novel human platelet antigens are suspected. © 2017 AABB.

PAM multiplicity marks genomic target sites as inhibitory to CRISPR-Cas9 editing

PubMed Central

Malina, Abba; Cameron, Christopher J. F.; Robert, Francis; Blanchette, Mathieu; Dostie, Josée; Pelletier, Jerry

2015-01-01

In CRISPR-Cas9 genome editing, the underlying principles for selecting guide RNA (gRNA) sequences that would ensure for efficient target site modification remain poorly understood. Here we show that target sites harbouring multiple protospacer adjacent motifs (PAMs) are refractory to Cas9-mediated repair in situ. Thus we refine which substrates should be avoided in gRNA design, implicating PAM density as a novel sequence-specific feature that inhibits in vivo Cas9-driven DNA modification. PMID:26644285

Kappa-casein polymorphisms among cattle breeds and bison herds

USGS Publications Warehouse

Cronin, M.A.; Cockett, N.

1993-01-01

We identified the HindIII restriction site polymorphism Of kappa-casein in cattle reported by Pinder et al. (Animal Genetics 22, 11, 1991) and found an additonal polymorphism (RsaI) in cattle and bison. The Hin dIII and Rsa I restriction sites were mapped and three haplotypes (alleles) were identified. Preliminary screening of 39 cattle and 71 bison revealed one allele restricted to cattle, one restricted to bison, and one shared by the species. No fixed allelic differences were observed among cattle breeds or among bison herds or subspecies.

Construction of a directed hammerhead ribozyme library: towards the identification of optimal target sites for antisense-mediated gene inhibition.

PubMed Central

Pierce, M L; Ruffner, D E

1998-01-01

Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA. Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict. Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA. The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention. PMID:9801305

An integrated CRISPR Bombyx mori genome editing system with improved efficiency and expanded target sites.

PubMed

Ma, Sanyuan; Liu, Yue; Liu, Yuanyuan; Chang, Jiasong; Zhang, Tong; Wang, Xiaogang; Shi, Run; Lu, Wei; Xia, Xiaojuan; Zhao, Ping; Xia, Qingyou

2017-04-01

Genome editing enabled unprecedented new opportunities for targeted genomic engineering of a wide variety of organisms ranging from microbes, plants, animals and even human embryos. The serial establishing and rapid applications of genome editing tools significantly accelerated Bombyx mori (B. mori) research during the past years. However, the only CRISPR system in B. mori was the commonly used SpCas9, which only recognize target sites containing NGG PAM sequence. In the present study, we first improve the efficiency of our previous established SpCas9 system by 3.5 folds. The improved high efficiency was also observed at several loci in both BmNs cells and B. mori embryos. Then to expand the target sites, we showed that two newly discovered CRISPR system, SaCas9 and AsCpf1, could also induce highly efficient site-specific genome editing in BmNs cells, and constructed an integrated CRISPR system. Genome-wide analysis of targetable sites was further conducted and showed that the integrated system cover 69,144,399 sites in B. mori genome, and one site could be found in every 6.5 bp. The efficiency and resolution of this CRISPR platform will probably accelerate both fundamental researches and applicable studies in B. mori, and perhaps other insects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Search for methylation-sensitive amplification polymorphisms in mutant figs.

PubMed

Rodrigues, M G F; Martins, A B G; Bertoni, B W; Figueira, A; Giuliatti, S

2013-07-08

Fig (Ficus carica) breeding programs that use conventional approaches to develop new cultivars are rare, owing to limited genetic variability and the difficulty in obtaining plants via gamete fusion. Cytosine methylation in plants leads to gene repression, thereby affecting transcription without changing the DNA sequence. Previous studies using random amplification of polymorphic DNA and amplified fragment length polymorphism markers revealed no polymorphisms among select fig mutants that originated from gamma-irradiated buds. Therefore, we conducted methylation-sensitive amplified polymorphism analysis to verify the existence of variability due to epigenetic DNA methylation among these mutant selections compared to the main cultivar 'Roxo-de-Valinhos'. Samples of genomic DNA were double-digested with either HpaII (methylation sensitive) or MspI (methylation insensitive) and with EcoRI. Fourteen primer combinations were tested, and on an average, non-methylated CCGG, symmetrically methylated CmCGG, and hemimethylated hmCCGG sites accounted for 87.9, 10.1, and 2.0%, respectively. MSAP analysis was effective in detecting differentially methylated sites in the genomic DNA of fig mutants, and methylation may be responsible for the phenotypic variation between treatments. Further analyses such as polymorphic DNA sequencing are necessary to validate these differences, standardize the regions of methylation, and analyze reads using bioinformatic tools.

Diverse Actions and Target-Site Selectivity of Neonicotinoids: Structural Insights

PubMed Central

Matsuda, Kazuhiko; Kanaoka, Satoshi; Akamatsu, Miki; Sattelle, David B.

2009-01-01

The nicotinic acetylcholine receptors (nAChRs) are targets for human and veterinary medicines as well as insecticides. Subtype-selectivity among the diverse nAChR family members is important for medicines targeting particular disorders, and pest-insect selectivity is essential for the development of safer, environmentally acceptable insecticides. Neonicotinoid insecticides selectively targeting insect nAChRs have important applications in crop protection and animal health. Members of this class exhibit strikingly diverse actions on their nAChR targets. Here we review the chemistry and diverse actions of neonicotinoids on insect and mammalian nAChRs. Electrophysiological studies on native nAChRs and on wild-type and mutagenized recombinant nAChRs have shown that basic residues particular to loop D of insect nAChRs are likely to interact electrostatically with the nitro group of neonicotinoids. In 2008, the crystal structures were published showing neonicotinoids docking into the acetylcholine binding site of molluscan acetylcholine binding proteins with homology to the ligand binding domain (LBD) of nAChRs. The crystal structures showed that 1) glutamine in loop D, corresponding to the basic residues of insect nAChRs, hydrogen bonds with the NO2 group of imidacloprid and 2) neonicotinoid-unique stacking and CH-π bonds at the LBD. A neonicotinoid-resistant strain obtained by laboratory-screening has been found to result from target site mutations, and possible reasons for this are also suggested by the crystal structures. The prospects of designing neonicotinoids that are safe not only for mammals but also for beneficial insects such as honey bees (Apis mellifera) are discussed in terms of interactions with non-α nAChR subunits. PMID:19321668

Computational Approach for Epitaxial Polymorph Stabilization through Substrate Selection.

PubMed

Ding, Hong; Dwaraknath, Shyam S; Garten, Lauren; Ndione, Paul; Ginley, David; Persson, Kristin A

2016-05-25

With the ultimate goal of finding new polymorphs through targeted synthesis conditions and techniques, we outline a computational framework to select optimal substrates for epitaxial growth using first principle calculations of formation energies, elastic strain energy, and topological information. To demonstrate the approach, we study the stabilization of metastable VO2 compounds which provides a rich chemical and structural polymorph space. We find that common polymorph statistics, lattice matching, and energy above hull considerations recommends homostructural growth on TiO2 substrates, where the VO2 brookite phase would be preferentially grown on the a-c TiO2 brookite plane while the columbite and anatase structures favor the a-b plane on the respective TiO2 phases. Overall, we find that a model which incorporates a geometric unit cell area matching between the substrate and the target film as well as the resulting strain energy density of the film provide qualitative agreement with experimental observations for the heterostructural growth of known VO2 polymorphs: rutile, A and B phases. The minimal interfacial geometry matching and estimated strain energy criteria provide several suggestions for substrates and substrate-film orientations for the heterostructural growth of the hitherto hypothetical anatase, brookite, and columbite polymorphs. These criteria serve as a preliminary guidance for the experimental efforts stabilizing new materials and/or polymorphs through epitaxy. The current screening algorithm is being integrated within the Materials Project online framework and data and hence publicly available.

Computational Approach for Epitaxial Polymorph Stabilization through Substrate Selection

DOE PAGES

Ding, Hong; Dwaraknath, Shyam S.; Garten, Lauren; ...

2016-05-04

With the ultimate goal of finding new polymorphs through targeted synthesis conditions and techniques, we outline a computational framework to select optimal substrates for epitaxial growth using first principle calculations of formation energies, elastic strain energy, and topological information. To demonstrate the approach, we study the stabilization of metastable VO 2 compounds which provides a rich chemical and structural polymorph space. Here, we find that common polymorph statistics, lattice matching, and energy above hull considerations recommends homostructural growth on TiO 2 substrates, where the VO 2 brookite phase would be preferentially grown on the a-c TiO 2 brookite plane whilemore » the columbite and anatase structures favor the a-b plane on the respective TiO 2 phases. Overall, we find that a model which incorporates a geometric unit cell area matching between the substrate and the target film as well as the resulting strain energy density of the film provide qualitative agreement with experimental observations for the heterostructural growth of known VO 2 polymorphs: rutile, A and B phases. The minimal interfacial geometry matching and estimated strain energy criteria provide several suggestions for substrates and substrate-film orientations for the heterostructural growth of the hitherto hypothetical anatase, brookite, and columbite polymorphs. Our criteria serve as a preliminary guidance for the experimental efforts stabilizing new materials and/or polymorphs through epitaxy. The current screening algorithm is being integrated within the Materials Project online framework and data and hence publicly available.« less

Computational Approach for Epitaxial Polymorph Stabilization through Substrate Selection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, Hong; Dwaraknath, Shyam S.; Garten, Lauren

With the ultimate goal of finding new polymorphs through targeted synthesis conditions and techniques, we outline a computational framework to select optimal substrates for epitaxial growth using first principle calculations of formation energies, elastic strain energy, and topological information. To demonstrate the approach, we study the stabilization of metastable VO2 compounds which provides a rich chemical and structural polymorph space. We find that common polymorph statistics, lattice matching, and energy above hull considerations recommends homostructural growth on TiO2 substrates, where the VO2 brookite phase would be preferentially grown on the a-c TiO2 brookite plane while the columbite and anatase structuresmore » favor the a-b plane on the respective TiO2 phases. Overall, we find that a model which incorporates a geometric unit cell area matching between the substrate and the target film as well as the resulting strain energy density of the film provide qualitative agreement with experimental observations for the heterostructural growth of known VO2 polymorphs: rutile, A and B phases. The minimal interfacial geometry matching and estimated strain energy criteria provide several suggestions for substrates and substrate-film orientations for the heterostructural growth of the hitherto hypothetical anatase, brookite, and columbite polymorphs. These criteria serve as a preliminary guidance for the experimental efforts stabilizing new materials and/or polymorphs through epitaxy. The current screening algorithm is being integrated within the Materials Project online framework and data and hence publicly available.« less

Computational Approach for Epitaxial Polymorph Stabilization through Substrate Selection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, Hong; Dwaraknath, Shyam S.; Garten, Lauren

With the ultimate goal of finding new polymorphs through targeted synthesis conditions and techniques, we outline a computational framework to select optimal substrates for epitaxial growth using first principle calculations of formation energies, elastic strain energy, and topological information. To demonstrate the approach, we study the stabilization of metastable VO 2 compounds which provides a rich chemical and structural polymorph space. Here, we find that common polymorph statistics, lattice matching, and energy above hull considerations recommends homostructural growth on TiO 2 substrates, where the VO 2 brookite phase would be preferentially grown on the a-c TiO 2 brookite plane whilemore » the columbite and anatase structures favor the a-b plane on the respective TiO 2 phases. Overall, we find that a model which incorporates a geometric unit cell area matching between the substrate and the target film as well as the resulting strain energy density of the film provide qualitative agreement with experimental observations for the heterostructural growth of known VO 2 polymorphs: rutile, A and B phases. The minimal interfacial geometry matching and estimated strain energy criteria provide several suggestions for substrates and substrate-film orientations for the heterostructural growth of the hitherto hypothetical anatase, brookite, and columbite polymorphs. Our criteria serve as a preliminary guidance for the experimental efforts stabilizing new materials and/or polymorphs through epitaxy. The current screening algorithm is being integrated within the Materials Project online framework and data and hence publicly available.« less

E2F1 somatic mutation within miRNA target site impairs gene regulation in colorectal cancer.

PubMed

Lopes-Ramos, Camila M; Barros, Bruna P; Koyama, Fernanda C; Carpinetti, Paola A; Pezuk, Julia; Doimo, Nayara T S; Habr-Gama, Angelita; Perez, Rodrigo O; Parmigiani, Raphael B

2017-01-01

Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.

Landscape of Insertion Polymorphisms in the Human Genome

PubMed Central

Onozawa, Masahiro; Goldberg, Liat; Aplan, Peter D.

2015-01-01

Nucleotide substitutions, small (<50 bp) insertions or deletions (indels), and large (>50 bp) deletions are well-known causes of genetic variation within the human genome. We recently reported a previously unrecognized form of polymorphic insertions, termed templated sequence insertion polymorphism (TSIP), in which the inserted sequence was templated from a distant genomic region, and was inserted in the genome through reverse transcription of an RNA intermediate. TSIPs can be grouped into two classes based on nucleotide sequence features at the insertion junctions; class 1 TSIPs show target site duplication, polyadenylation, and preference for insertion at a 5′-TTTT/A-3′ sequence, suggesting a LINE-1 based insertion mechanism, whereas class 2 TSIPs show features consistent with repair of a DNA double strand break by nonhomologous end joining. To gain a more complete picture of TSIPs throughout the human population, we evaluated whole-genome sequence from 52 individuals, and identified 171 TSIPs. Most individuals had 25–30 TSIPs, and common (present in >20% of individuals) TSIPs were found in individuals throughout the world, whereas rare TSIPs tended to cluster in specific geographic regions. The number of rare TSIPs was greater than the number of common TSIPs, suggesting that TSIP generation is an ongoing process. Intriguingly, mitochondrial sequences were a frequent template for class 2 insertions, used more commonly than any nuclear chromosome. Similar to single nucleotide polymorphisms and indels, we suspect that these TSIPs may be important for the generation of human diversity and genetic diseases, and can be useful in tracking historical migration of populations. PMID:25745018

A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine site of tubulin.

PubMed

Menchon, Grégory; Prota, Andrea E; Lucena-Agell, Daniel; Bucher, Pascal; Jansen, Rolf; Irschik, Herbert; Müller, Rolf; Paterson, Ian; Díaz, J Fernando; Altmann, Karl-Heinz; Steinmetz, Michel O

2018-05-29

Microtubule-targeting agents (MTAs) like taxol and vinblastine are among the most successful chemotherapeutic drugs against cancer. Here, we describe a fluorescence anisotropy-based assay that specifically probes for ligands targeting the recently discovered maytansine site of tubulin. Using this assay, we have determined the dissociation constants of known maytansine site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine site on β-tubulin. The high-resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of an additional sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next-generation MTAs for the treatment of cancer.

Using PCR-RFLP technology to teach single nucleotide polymorphism for undergraduates.

PubMed

Zhang, Bo; Wang, Yan; Xu, Xiaofeng; Guan, Xingying; Bai, Yun

2013-01-01

Recent studies indicated that the aberrant gene expression of peroxiredoxin-6 (prdx6) was found in various kinds of cancers. Because of its biochemical function and gene expression pattern in cancer cells, the association between genetic polymorphism of Prdx6 and cancer onset is interesting. In this report, we have developed and implemented a serial experiment in molecular biology laboratory course to teach single nucleotide polymorphism (SNP) to undergraduate students majoring in molecular biology or genetics. The flanking sequence of rs4382766 was located in Prdx6 gene, which contained a restriction site of SspI, and was used as a target in this lab course. The students could mimic real research by integrating different techniques, such as database retrieving, genomic DNA isolation, PCR, and restriction enzyme assay. This serial experiment of PCR-RFLP helps students set up intact idea of molecular biology and understand the relation among individual experiments. Students were found to be more enthusiastic during the laboratory classes than those in the former curriculum. Copyright © 2013 Wiley Periodicals, Inc.

Association of VAV2 and VAV3 polymorphisms with cardiovascular risk factors

PubMed Central

Perretta-Tejedor, Nuria; Fernández-Mateos, Javier; García-Ortiz, Luis; Gómez-Marcos, Manuel A.; Recio-Rodríguez, José I.; Agudo-Conde, Cristina; Rodriguez-Sánchez, Emiliano; Morales, Ana I.; López-Hernández, Francisco J.; López-Novoa, José M.; González-Sarmiento, Rogelio; Martínez-Salgado, Carlos

2017-01-01

Hypertension, diabetes and obesity are cardiovascular risk factors closely associated to the development of renal and cardiovascular target organ damage. VAV2 and VAV3, members of the VAV family proto-oncogenes, are guanosine nucleotide exchange factors for the Rho and Rac GTPase family, which is related with cardiovascular homeostasis. We have analyzed the relationship between the presence of VAV2 rs602990 and VAV3 rs7528153 polymorphisms with cardiovascular risk factors and target organ damage (heart, vessels and kidney) in 411 subjects. Our results show that being carrier of the T allele in VAV2 rs602990 polymorphism is associated with an increased risk of obesity, reduced levels of ankle-brachial index and diastolic blood pressure and reduced retinal artery caliber. In addition, being carrier of T allele is associated with increased risk of target organ damage in males. On the other hand, being carrier of the T allele in VAV3 rs7528153 polymorphism is associated with a decreased susceptibility of developing a pathologic state composed by the presence of hypertension, diabetes, obesity or cardiovascular damage, and with an increased risk of developing altered basal glycaemia. This is the first report showing an association between VAV2 and VAV3 polymorphisms with cardiovascular risk factors and target organ damage. PMID:28157227

Novel and viable acetylcholinesterase target site for developing effective and environmentally safe insecticides.

PubMed

Pang, Yuan-Ping; Brimijoin, Stephen; Ragsdale, David W; Zhu, Kun Yan; Suranyi, Robert

2012-04-01

Insect pests are responsible for human suffering and financial losses worldwide. New and environmentally safe insecticides are urgently needed to cope with these serious problems. Resistance to current insecticides has resulted in a resurgence of insect pests, and growing concerns about insecticide toxicity to humans discourage the use of insecticides for pest control. The small market for insecticides has hampered insecticide development; however, advances in genomics and structural genomics offer new opportunities to develop insecticides that are less dependent on the insecticide market. This review summarizes the literature data that support the hypothesis that an insect-specific cysteine residue located at the opening of the acetylcholinesterase active site is a promising target site for developing new insecticides with reduced off-target toxicity and low propensity for insect resistance. These data are used to discuss the differences between targeting the insect-specific cysteine residue and targeting the ubiquitous catalytic serine residue of acetylcholinesterase from the perspective of reducing off-target toxicity and insect resistance. Also discussed is the prospect of developing cysteine-targeting anticholinesterases as effective and environmentally safe insecticides for control of disease vectors, crop damage, and residential insect pests within the financial confines of the present insecticide market.

Influence of quasi-specific sites on kinetics of target DNA search by a sequence-specific DNA-binding protein.

PubMed

Kemme, Catherine A; Esadze, Alexandre; Iwahara, Junji

2015-11-10

Functions of transcription factors require formation of specific complexes at particular sites in cis-regulatory elements of genes. However, chromosomal DNA contains numerous sites that are similar to the target sequences recognized by transcription factors. The influence of such "quasi-specific" sites on functions of the transcription factors is not well understood at present by experimental means. In this work, using fluorescence methods, we have investigated the influence of quasi-specific DNA sites on the efficiency of target location by the zinc finger DNA-binding domain of the inducible transcription factor Egr-1, which recognizes a 9 bp sequence. By stopped-flow assays, we measured the kinetics of Egr-1's association with a target site on 143 bp DNA in the presence of various competitor DNAs, including nonspecific and quasi-specific sites. The presence of quasi-specific sites on competitor DNA significantly decelerated the target association by the Egr-1 protein. The impact of the quasi-specific sites depended strongly on their affinity, their concentration, and the degree of their binding to the protein. To quantitatively describe the kinetic impact of the quasi-specific sites, we derived an analytical form of the apparent kinetic rate constant for the target association and used it for fitting to the experimental data. Our kinetic data with calf thymus DNA as a competitor suggested that there are millions of high-affinity quasi-specific sites for Egr-1 among the 3 billion bp of genomic DNA. This study quantitatively demonstrates that naturally abundant quasi-specific sites on DNA can considerably impede the target search processes of sequence-specific DNA-binding proteins.

Influence of Quasi-Specific Sites on Kinetics of Target DNA Search by a Sequence-Specific DNA-Binding Protein

PubMed Central

2015-01-01

Functions of transcription factors require formation of specific complexes at particular sites in cis-regulatory elements of genes. However, chromosomal DNA contains numerous sites that are similar to the target sequences recognized by transcription factors. The influence of such “quasi-specific” sites on functions of the transcription factors is not well understood at present by experimental means. In this work, using fluorescence methods, we have investigated the influence of quasi-specific DNA sites on the efficiency of target location by the zinc finger DNA-binding domain of the inducible transcription factor Egr-1, which recognizes a 9 bp sequence. By stopped-flow assays, we measured the kinetics of Egr-1’s association with a target site on 143 bp DNA in the presence of various competitor DNAs, including nonspecific and quasi-specific sites. The presence of quasi-specific sites on competitor DNA significantly decelerated the target association by the Egr-1 protein. The impact of the quasi-specific sites depended strongly on their affinity, their concentration, and the degree of their binding to the protein. To quantitatively describe the kinetic impact of the quasi-specific sites, we derived an analytical form of the apparent kinetic rate constant for the target association and used it for fitting to the experimental data. Our kinetic data with calf thymus DNA as a competitor suggested that there are millions of high-affinity quasi-specific sites for Egr-1 among the 3 billion bp of genomic DNA. This study quantitatively demonstrates that naturally abundant quasi-specific sites on DNA can considerably impede the target search processes of sequence-specific DNA-binding proteins. PMID:26502071

Transcription Factor Binding Site Polymorphism in the Motilin Gene Associated with Left-Sided Displacement of the Abomasum in German Holstein Cattle

PubMed Central

Mömke, Stefanie; Sickinger, Marlene; Rehage, Jürgen; Doll, Klaus; Distl, Ottmar

2012-01-01

Left-sided displacement of the abomasum (LDA) is a common disease in many dairy cattle breeds. A genome-wide screen for QTL for LDA in German Holstein (GH) cows indicated motilin (MLN) as a candidate gene on bovine chromosome 23. Genomic DNA sequence analysis of MLN revealed a total of 32 polymorphisms. All informative polymorphisms used for association analyses in a random sample of 1,136 GH cows confirmed MLN as a candidate for LDA. A single nucleotide polymorphism (FN298674:g.90T>C) located within the first non-coding exon of bovine MLN affects a NKX2-5 transcription factor binding site and showed significant associations (ORallele = 0.64; −log10Pallele = 6.8, −log10Pgenotype = 7.0) with LDA. An expression study gave evidence of a significantly decreased MLN expression in cows carrying the mutant allele (C). In individuals heterozygous or homozygous for the mutation, MLN expression was decreased by 89% relative to the wildtype. FN298674:g.90T>C may therefore play a role in bovine LDA via the motility of the abomasum. This MLN SNP appears useful to reduce the incidence of LDA in German Holstein cattle and provides a first step towards a deeper understanding of the genetics of LDA. PMID:22536407

A Novel W1999S Mutation and Non-Target Site Resistance Impact on Acetyl-CoA Carboxylase Inhibiting Herbicides to Varying Degrees in a UK Lolium multiflorum Population

PubMed Central

Kaundun, Shiv Shankhar; Bailly, Geraldine C.; Dale, Richard P.; Hutchings, Sarah-Jane; McIndoe, Eddie

2013-01-01

Background Acetyl-CoA carboxylase (ACCase) inhibiting herbicides are important products for the post-emergence control of grass weed species in small grain cereal crops. However, the appearance of resistance to ACCase herbicides over time has resulted in limited options for effective weed control of key species such as Lolium spp. In this study, we have used an integrated biological and molecular biology approach to investigate the mechanism of resistance to ACCase herbicides in a Lolium multiflorum Lam. from the UK (UK21). Methodology/Principal Findings The study revealed a novel tryptophan to serine mutation at ACCase codon position 1999 impacting on ACCase inhibiting herbicides to varying degrees. The W1999S mutation confers dominant resistance to pinoxaden and partially recessive resistance to cycloxydim and sethoxydim. On the other hand, plants containing the W1999S mutation were sensitive to clethodim and tepraloxydim. Additionally population UK21 is characterised by other resistance mechanisms, very likely non non-target site based, affecting several aryloxyphenoxyproprionate (FOP) herbicides but not the practical field rate of pinoxaden. The positive identification of wild type tryptophan and mutant serine alleles at ACCase position 1999 could be readily achieved with an original DNA based derived cleaved amplified polymorphic sequence (dCAPS) assay that uses the same PCR product but two different enzymes for positively identifying the wild type tryptophan and mutant serine alleles identified here. Conclusion/Significance This paper highlights intrinsic differences between ACCase inhibiting herbicides that could be exploited for controlling ryegrass populations such as UK21 characterised by compound-specific target site and non-target site resistance. PMID:23469130

Renton's Quendall Terminals on List of EPA Superfund Sites Targeted for Immediate, Intense Attention

EPA Pesticide Factsheets

EPA released the list of Superfund sites that Administrator Pruitt has targeted for intense and immediate attention, including the Quendall Terminals Site, a former creosote facility on the shore of Lake Washington in Renton, Washington.

Association of the Serotonin Receptor 3E Gene as a Functional Variant in the MicroRNA-510 Target Site with Diarrhea Predominant Irritable Bowel Syndrome in Chinese Women

PubMed Central

Zhang, Yu; Li, Yaoyao; Hao, Zhenfeng; Li, Xiangming; Bo, Ping; Gong, Weijuan

2016-01-01

Background/Aims The functional variant (rs56109847) in the 3′-untranslated regions (3′-UTR) of the serotonin receptor 3E (HTR3E) gene is associated with female diarrhea predominant irritable bowel syndrome (IBS-D) in British populations. However, the relationship of the polymorphism both to HTR3E expression in the intestine and to the occurrence of Chinese functional gastrointestinal disorders has yet to be examined. Methods Polymerase chain reaction amplification and restriction fragment length polymorphism analyses were employed to detect polymorphisms among Chinese Han women, particularly 107 patients with IBS-D, 99 patients with functional dyspepsia (FD), 115 patients with mixed IBS and 69 patients with IBS-D + FD. We also assessed microRNA-510 (miR-510) and HTR3E expression in human colonic mucosal tissues with immunohistochemistry and other methods. Dual-luciferase reporter assays were conducted to examine the binding ability of miR-510 and HTR3E 3′-UTR. Results Genotyping data showed the variant rs56109847 was significantly associated with IBS-D, but not with FD, mixed-IBS, or FD + IBS-D. HTR3E was abundantly expressed around the colonic mucosal glands but less expressed in the stroma. miR-510 expression decreased, whereas HTR3E expression increased in the colonic mucosal tissue of patients with IBS-D compared with those in controls. HTR3E expression was significantly higher in patients with the GA genotype than that in patients with the GG genotype. The single-nucleotide polymorphisms disrupted the binding site of miR-510 and significantly upregulated luciferase expression in HEK293 and HT-29 cells. Conclusions The single-nucleotide polymorphisms rs56109847 led to reduced microRNA binding and overexpression of the target gene in intestinal cells, thereby increasing IBS-D risk in the Chinese Han population. The decreased expression of miR-510 might contribute to IBS-D. PMID:26787495

Detection of DNA methylation changes in micropropagated banana plants using methylation-sensitive amplification polymorphism (MSAP).

PubMed

Peraza-Echeverria, S; Herrera-Valencia, V A.; Kay, A -J.

2001-07-01

The extent of DNA methylation polymorphisms was evaluated in micropropagated banana (Musa AAA cv. 'Grand Naine') derived from either the vegetative apex of the sucker or the floral apex of the male inflorescence using the methylation-sensitive amplification polymorphism (MSAP) technique. In all, 465 fragments, each representing a recognition site cleaved by either or both of the isoschizomers were amplified using eight combinations of primers. A total of 107 sites (23%) were found to be methylated at cytosine in the genome of micropropagated banana plants. In plants micropropagated from the male inflorescence explant 14 (3%) DNA methylation events were polymorphic, while plants micropropagated from the sucker explant produced 8 (1.7%) polymorphisms. No DNA methylation polymorphisms were detected in conventionally propagated banana plants. These results demonstrated the usefulness of MSAP to detect DNA methylation events in micropropagated banana plants and indicate that DNA methylation polymorphisms are associated with micropropagation.

Sulfadiazine resistance in Toxoplasma gondii: no involvement of overexpression or polymorphisms in genes of therapeutic targets and ABC transporters

PubMed Central

Doliwa, Christelle; Escotte-Binet, Sandie; Aubert, Dominique; Sauvage, Virginie; Velard, Frédéric; Schmid, Aline; Villena, Isabelle

2013-01-01

Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondii strains naturally resistant to sulfadiazine and we developed in vitro two sulfadiazine resistant strains, RH-RSDZ and ME-49-RSDZ, by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhps and dhfr and/or the ABC transporter genes family. To identify genotypic and/or phenotypic markers of resistance in T. gondii, we sequenced and analyzed the expression levels of therapeutic targets dhps and dhfr, three ABC genes, two Pgp, TgABC.B1 and TgABC.B2, and one MRP, TgABC.C1, on sensitive strains compared to sulfadiazine resistant strains. Neither polymorphism nor overexpression was identified. Contrary to Plasmodium, in which mutations and/or overexpression within gene targets and ABC transporters are involved in antimalarial resistance, T. gondii sulfadiazine resistance is not related to these toxoplasmic genes studied. PMID:23707894

Susceptibility to breast cancer and three polymorphisms of GSTZ1.

PubMed

Saadat, Iraj; Khalili, Maryam; Nafissi, Samane; Omidvari, Shahpour; Saadat, Mostafa

2012-03-01

Glutathione S-transferases class zeta (GSTζ) is involved in the detoxification of xenobiotic compounds and catalyzes the biotransformation of a variety of α-haloacids including dichloroacetic acid and chlorofluoroacetic acid. It has been reported that, in mice, deficiency of Gstz1 (a member of GSTζ) resulted in the generation of a constant level of oxidative stress. The present study was carried out to investigate the association between genetic polymorphisms of GSTZ1 (in promoter site G-1002A and in coding sites Glu32Lys and Gly42Arg) and risk of breast cancer. We included 106 females with breast cancer and 106 healthy females frequency matched for age. The study polymorphisms were not associated with risk of breast cancer (p>0.05). The polymorphisms of GSTZ1 showed strong linkage disequilibrium among cancer patients and control subjects (p<0.0001). There was no significant difference between cancer patients and controls for frequencies of the GSTZ1 haplotypes (p>0.05). It seems there is no meaningful relationship between the genetic polymorphisms of GSTZ1 and risk of breast cancer.

Structural insight of dopamine β-hydroxylase, a drug target for complex traits, and functional significance of exonic single nucleotide polymorphisms.

PubMed

Kapoor, Abhijeet; Shandilya, Manish; Kundu, Suman

2011-01-01

Human dopamine β-hydroxylase (DBH) is an important therapeutic target for complex traits. Several single nucleotide polymorphisms (SNPs) have also been identified in DBH with potential adverse physiological effect. However, difficulty in obtaining diffractable crystals and lack of a suitable template for modeling the protein has ensured that neither crystallographic three-dimensional structure nor computational model for the enzyme is available to aid rational drug design, prediction of functional significance of SNPs or analytical protein engineering. Adequate biochemical information regarding human DBH, structural coordinates for peptidylglycine alpha-hydroxylating monooxygenase and computational data from a partial model of rat DBH were used along with logical manual intervention in a novel way to build an in silico model of human DBH. The model provides structural insight into the active site, metal coordination, subunit interface, substrate recognition and inhibitor binding. It reveals that DOMON domain potentially promotes tetramerization, while substrate dopamine and a potential therapeutic inhibitor nepicastat are stabilized in the active site through multiple hydrogen bonding. Functional significance of several exonic SNPs could be described from a structural analysis of the model. The model confirms that SNP resulting in Ala318Ser or Leu317Pro mutation may not influence enzyme activity, while Gly482Arg might actually do so being in the proximity of the active site. Arg549Cys may cause abnormal oligomerization through non-native disulfide bond formation. Other SNPs like Glu181, Glu250, Lys239 and Asp290 could potentially inhibit tetramerization thus affecting function. The first three-dimensional model of full-length human DBH protein was obtained in a novel manner with a set of experimental data as guideline for consistency of in silico prediction. Preliminary physicochemical tests validated the model. The model confirms, rationalizes and provides

Novel and Viable Acetylcholinesterase Target Site for Developing Effective and Environmentally Safe Insecticides

PubMed Central

Pang, Yuan-Ping; Brimijoin, Stephen; Ragsdale, David W; Zhu, Kun Yan; Suranyi, Robert

2012-01-01

Insect pests are responsible for human suffering and financial losses worldwide. New and environmentally safe insecticides are urgently needed to cope with these serious problems. Resistance to current insecticides has resulted in a resurgence of insect pests, and growing concerns about insecticide toxicity to humans discourage the use of insecticides for pest control. The small market for insecticides has hampered insecticide development; however, advances in genomics and structural genomics offer new opportunities to develop insecticides that are less dependent on the insecticide market. This review summarizes the literature data that support the hypothesis that an insect-specific cysteine residue located at the opening of the acetylcholinesterase active site is a promising target site for developing new insecticides with reduced off-target toxicity and low propensity for insect resistance. These data are used to discuss the differences between targeting the insect-specific cysteine residue and targeting the ubiquitous catalytic serine residue of acetylcholinesterase from the perspective of reducing off-target toxicity and insect resistance. Also discussed is the prospect of developing cysteine-targeting anticholinesterases as effective and environmentally safe insecticides for control of disease vectors, crop damage, and residential insect pests within the financial confines of the present insecticide market. PMID:22280344

Balance between transmitted HLA preadapted and nonassociated polymorphisms is a major determinant of HIV-1 disease progression.

PubMed

Mónaco, Daniela C; Dilernia, Dario A; Fiore-Gartland, Andrew; Yu, Tianwei; Prince, Jessica L; Dennis, Kristine K; Qin, Kai; Schaefer, Malinda; Claiborne, Daniel T; Kilembe, William; Tang, Jianming; Price, Matt A; Farmer, Paul; Gilmour, Jill; Bansal, Anju; Allen, Susan; Goepfert, Paul; Hunter, Eric

2016-09-19

HIV-1 adapts to a new host through mutations that facilitate immune escape. Here, we evaluate the impact on viral control and disease progression of transmitted polymorphisms that were either preadapted to or nonassociated with the new host's HLA. In a cohort of 169 Zambian heterosexual transmission pairs, we found that almost one-third of possible HLA-linked target sites in the transmitted virus Gag protein are already adapted, and that this transmitted preadaptation significantly reduced early immune recognition of epitopes. Transmitted preadapted and nonassociated polymorphisms showed opposing effects on set-point VL and the balance between the two was significantly associated with higher set-point VLs in a multivariable model including other risk factors. Transmitted preadaptation was also significantly associated with faster CD4 decline (<350 cells/µl) and this association was stronger after accounting for nonassociated polymorphisms, which were linked with slower CD4 decline. Overall, the relative ratio of the two classes of polymorphisms was found to be the major determinant of CD4 decline in a multivariable model including other risk factors. This study reveals that, even before an immune response is mounted in the new host, the balance of these opposing factors can significantly influence the outcome of HIV-1 infection. © 2016 Mónaco et al.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)-A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes.

PubMed

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare . However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

Comprehensive analysis of Salmonella sequence polymorphisms and development of a LDR-UA assay for the detection and characterization of selected serotypes.

PubMed

Lauri, Andrea; Castiglioni, Bianca; Mariani, Paola

2011-07-01

Salmonella is a major cause of food-borne disease, and Salmonella enterica subspecies I includes the most clinically relevant serotypes. Salmonella serotype determination is important for the disease etiology assessment and contamination source tracking. This task will be facilitated by the disclosure of Salmonella serotype sequence polymorphisms, here annotated in seven genes (sefA, safA, safC, bigA, invA, fimA, and phsB) from 139 S. enterica strains, of which 109 belonging to 44 serotypes of subsp. I. One hundred nineteen polymorphic sites were scored and associated to single serotypes or to serotype groups belonging to S. enterica subsp. I. A diagnostic tool was constructed based on the Ligation Detection Reaction-Universal Array (LDR-UA) for the detection of polymorphic sites uniquely associated to serotypes of primary interest (Salmonella Hadar, Salmonella Infantis, Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum, Salmonella Virchow, and Salmonella Paratyphi B). The implementation of promiscuous probes allowed the diagnosis of ten further serotypes that could be associated to a unique hybridization pattern. Finally, the sensitivity and applicability of the tool was tested on target DNA dilutions and with controlled meat contamination, allowing the detection of one Salmonella CFU in 25 g of meat.

Rotifer rDNA-specific R9 retrotransposable elements generate an exceptionally long target site duplication upon insertion.

PubMed

Gladyshev, Eugene A; Arkhipova, Irina R

2009-12-15

Ribosomal DNA genes in many eukaryotes contain insertions of non-LTR retrotransposable elements belonging to the R2 clade. These elements persist in the host genomes by inserting site-specifically into multicopy target sites, thereby avoiding random disruption of single-copy host genes. Here we describe R9 retrotransposons from the R2 clade in the 28S RNA genes of bdelloid rotifers, small freshwater invertebrate animals best known for their long-term asexuality and for their ability to survive repeated cycles of desiccation and rehydration. While the structural organization of R9 elements is highly similar to that of other members of the R2 clade, they are characterized by two distinct features: site-specific insertion into a previously unreported target sequence within the 28S gene, and an unusually long target site duplication of 126 bp. We discuss the implications of these findings in the context of bdelloid genome organization and the mechanisms of target-primed reverse transcription.

Impact of M36I polymorphism on the interaction of HIV-1 protease with its substrates: insights from molecular dynamics

PubMed Central

2014-01-01

Background Over the last decades, a vast structural knowledge has been gathered on the HIV-1 protease (PR). Noticeably, most of the studies focused the B-subtype, which has the highest prevalence in developed countries. Accordingly, currently available anti-HIV drugs target this subtype, with considerable benefits for the corresponding patients. However, in developing countries, there is a wide variety of HIV-1 subtypes carrying PR polymorphisms related to reduced drug susceptibility. The non-active site mutation, M36I, is the most frequent polymorphism, and is considered as a non-B subtype marker. Yet, the structural impact of this substitution on the PR structure and on the interaction with natural substrates remains poorly documented. Results Herein, we used molecular dynamics simulations to investigate the role of this polymorphism on the interaction of PR with six of its natural cleavage-sites substrates. Free energy analyses by MMPB/SA calculations showed an affinity decrease of M36I-PR for the majority of its substrates. The only exceptions were the RT-RH, with equivalent affinity, and the RH-IN, for which an increased affinity was found. Furthermore, molecular simulations suggest that, unlike other peptides, RH-IN induced larger structural fluctuations in the wild-type enzyme than in the M36I variant. Conclusions With multiple approaches and analyses we identified structural and dynamical determinants associated with the changes found in the binding affinity of the M36I variant. This mutation influences the flexibility of both PR and its complexed substrate. The observed impact of M36I, suggest that combination with other non-B subtype polymorphisms, could lead to major effects on the interaction with the 12 known cleavage sites, which should impact the virion maturation. PMID:25573486

A Polymorphic p53 Response Element in KIT Ligand Influences Cancer Risk and Has Undergone Natural Selection

PubMed Central

Zeron-Medina, Jorge; Wang, Xuting; Repapi, Emmanouela; Campbell, Michelle R.; Su, Dan; Castro-Giner, Francesc; Davies, Benjamin; Peterse, Elisabeth F.P.; Sacilotto, Natalia; Walker, Graeme J.; Terzian, Tamara; Tomlinson, Ian P.; Box, Neil F.; Meinshausen, Nicolai; De Val, Sarah; Bell, Douglas A.; Bond, Gareth L.

2014-01-01

SUMMARY The ability of p53 to regulate transcription is crucial for tumor suppression and implies that inherited polymorphisms in functional p53-binding sites could influence cancer. Here, we identify a polymorphic p53 responsive element and demonstrate its influence on cancer risk using genome-wide data sets of cancer susceptibility loci, genetic variation, p53 occupancy, and p53-binding sites. We uncover a single-nucleotide polymorphism (SNP) in a functional p53-binding site and establish its influence on the ability of p53 to bind to and regulate transcription of the KITLG gene. The SNP resides in KITLG and associates with one of the largest risks identified among cancer genome-wide association studies. We establish that the SNP has undergone positive selection throughout evolution, signifying a selective benefit, but go on to show that similar SNPs are rare in the genome due to negative selection, indicating that polymorphisms in p53-binding sites are primarily detrimental to humans. PMID:24120139

Sex, Drugs, and Rock ‘N’ Roll: Hypothesizing Common Mesolimbic Activation as a Function of Reward Gene Polymorphisms

PubMed Central

Blum, Kenneth; Werner, Tonia; Carnes, Stefanie; Carnes, Patrick; Bowirrat, Abdalla; Giordano, John; Marlene-Oscar-Berman; Gold, Mark

2014-01-01

The nucleus accumbens, a site within the ventral striatum, plays a prominent role in mediating the reinforcing effects of drugs of abuse, food, sex, and other addictions. Indeed, it is generally believed that this structure mandates motivated behaviors such as eating, drinking, and sexual activity, which are elicited by natural rewards and other strong incentive stimuli. This article focuses on sex addiction, but we hypothesize that there is a common underlying mechanism of action for the powerful effects that all addictions have on human motivation. That is, biological drives may have common molecular genetic antecedents, which if impaired, lead to aberrant behaviors. Based on abundant scientific support, we further hypothesize that dopaminergic genes, and possibly other candidate neurotransmitter-related gene polymorphisms, affect both hedonic and anhedonic behavioral outcomes. Genotyping studies already have linked gene polymorphic associations with alcohol and drug addictions and obesity, and we anticipate that future genotyping studies of sex addicts will provide evidence for polymorphic associations with specific clustering of sexual typologies based on clinical instrument assessments. We recommend that scientists and clinicians embark on research coupling the use of neuroimaging tools with dopaminergic agonistic agents to target specific gene polymorphisms systematically for normalizing hyper- or hypo-sexual behaviors. PMID:22641964

[Clustered regularly interspaced short palindromic repeats (CRISPR) site in Bacillus anthracis].

PubMed

Gao, Zhiqi; Wang, Dongshu; Feng, Erling; Wang, Bingxiang; Hui, Yiming; Han, Shaobo; Jiao, Lei; Liu, Xiankai; Wang, Hengliang

2014-11-04

To investigate the polymorphism of clustered regularly interspaced short palindromic repeats (CRISPR) in Bacillu santhracis and the application to molecular typing based on the polymorphism of CRISPR in B. anthracis. We downloaded the whole genome sequence of 6 B. anthracis strains and extracted the CRISPR sites. We designed the primers of CRISPR sites and amplified the CRISPR fragments in 193 B. anthracis strains by PCR and sequenced these fragments. In order to reveal the polymorphism of CRISPR in B. anthracis, wealigned all the extracted sequences and sequenced results by local blasting. At the same time, we also analyzed the CRISPR sites in B. cereus and B. thuringiensis. We did not find any polymorphism of CRISPR in B. anthracis. The molecular typing approach based on CRISPR polymorphism is not suitable for B. anthracis, but it is possible for us to distinguish B. anthracis from B. cereus and B. thuringiensis.

A mathematical model of single target site location by Brownian movement in subcellular compartments.

PubMed

Kuthan, Hartmut

2003-03-07

The location of distinct sites is mandatory for many cellular processes. In the subcompartments of the cell nucleus, only very small numbers of diffusing macromolecules and specific target sites of some types may be present. In this case, we are faced with the Brownian movement of individual macromolecules and their "random search" for single/few specific target sites, rather than bulk-averaged diffusion and multiple sites. In this article, I consider the location of a distant central target site, e.g. a globular protein, by individual macromolecules executing unbiased (i.e. drift-free) random walks in a spherical compartment. For this walk-and-capture model, the closed-form analytic solution of the first passage time probability density function (p.d.f.) has been obtained as well as the first and second moment. In the limit of a large ratio of the radii of the spherical diffusion space and central target, well-known relations for the variance and the first two moments for the exponential p.d.f. were found to hold with high accuracy. These calculations reinforce earlier numerical results and Monte Carlo simulations. A major implication derivable from the model is that non-directed random movement is an effective means for locating single sites in submicron-sized compartments, even when the diffusion coefficients are comparatively small and the diffusing species are present in one copy only. These theoretical conclusions are underscored numerically for effective diffusion constants ranging from 0.5 to 10.0 microm(2) s(-1), which have been reported for a couple of nuclear proteins in their physiological environment. Spherical compartments of submicron size are, for example, the Cajal bodies (size: 0.1-1.0 microm), which are present in 1-5 copies in the cell nucleus. Within a small Cajal body of radius 0.1 microm a single diffusing protein molecule (with D=0.5 microm(2) s(-1)) would encounter a medium-sized protein of radius 2.5 nm within 1 s with a probability near

Engineered Proteins Program Mammalian Cells to Target Inflammatory Disease Sites.

PubMed

Qudrat, Anam; Mosabbir, Abdullah Al; Truong, Kevin

2017-06-22

Disease sites in atherosclerosis and cancer feature cell masses (e.g., plaques/tumors), a low pH extracellular microenvironment, and various pro-inflammatory cytokines such as tumor necrosis factor α (TNFα). The ability to engineer a cell to seek TNFα sources allows for targeted therapeutic delivery. To accomplish this, here we introduced a system of proteins: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca 2+ -activated RhoA (named CaRQ), vesicular stomatitis virus glycoprotein G (VSVG), and thymidine kinase. Upon binding TNFα, TNFR1chi generates a Ca 2+ signal that in turn activates CaRQ-mediated non-apoptotic blebs that allow migration toward the TNFα source. Next, the addition of VSVG, upon low pH induction, causes membrane fusion of the engineered and TNFα source cells. Finally, after ganciclovir treatment cells undergo death via the thymidine kinase suicide mechanism. Hence, we assembled a system of proteins that forms the basis of engineering a cell to target inflammatory disease sites characterized by TNFα secretion and a low-pH microenvironment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Centredale Manor Superfund Site in Rhode Island included on EPA List of Targeted for Immediate Attention

EPA Pesticide Factsheets

Today, the U.S. Environmental Protection Agency released the list of Superfund sites that Administrator Pruitt has targeted for immediate and intense attention. The Centredale Manor Restoration Project superfund site is one of the 21 sites on the list.

Regulatory polymorphisms modulate the expression of HLA class II molecules and promote autoimmunity

PubMed Central

Raj, Prithvi; Rai, Ekta; Song, Ran; Khan, Shaheen; Wakeland, Benjamin E; Viswanathan, Kasthuribai; Arana, Carlos; Liang, Chaoying; Zhang, Bo; Dozmorov, Igor; Carr-Johnson, Ferdicia; Mitrovic, Mitja; Wiley, Graham B; Kelly, Jennifer A; Lauwerys, Bernard R; Olsen, Nancy J; Cotsapas, Chris; Garcia, Christine K; Wise, Carol A; Harley, John B; Nath, Swapan K; James, Judith A; Jacob, Chaim O; Tsao, Betty P; Pasare, Chandrashekhar; Karp, David R; Li, Quan Zhen; Gaffney, Patrick M; Wakeland, Edward K

2016-01-01

Targeted sequencing of sixteen SLE risk loci among 1349 Caucasian cases and controls produced a comprehensive dataset of the variations causing susceptibility to systemic lupus erythematosus (SLE). Two independent disease association signals in the HLA-D region identified two regulatory regions containing 3562 polymorphisms that modified thirty-seven transcription factor binding sites. These extensive functional variations are a new and potent facet of HLA polymorphism. Variations modifying the consensus binding motifs of IRF4 and CTCF in the XL9 regulatory complex modified the transcription of HLA-DRB1, HLA-DQA1 and HLA-DQB1 in a chromosome-specific manner, resulting in a 2.5-fold increase in the surface expression of HLA-DR and DQ molecules on dendritic cells with SLE risk genotypes, which increases to over 4-fold after stimulation. Similar analyses of fifteen other SLE risk loci identified 1206 functional variants tightly linked with disease-associated SNPs and demonstrated that common disease alleles contain multiple causal variants modulating multiple immune system genes. DOI: http://dx.doi.org/10.7554/eLife.12089.001 PMID:26880555

A simple repeat polymorphism in the MITF-M promoter is a key regulator of white spotting in dogs.

PubMed

Baranowska Körberg, Izabella; Sundström, Elisabeth; Meadows, Jennifer R S; Rosengren Pielberg, Gerli; Gustafson, Ulla; Hedhammar, Åke; Karlsson, Elinor K; Seddon, Jennifer; Söderberg, Arne; Vilà, Carles; Zhang, Xiaolan; Åkesson, Mikael; Lindblad-Toh, Kerstin; Andersson, Göran; Andersson, Leif

2014-01-01

The white spotting locus (S) in dogs is colocalized with the MITF (microphtalmia-associated transcription factor) gene. The phenotypic effects of the four S alleles range from solid colour (S) to extreme white spotting (s(w)). We have investigated four candidate mutations associated with the s(w) allele, a SINE insertion, a SNP at a conserved site and a simple repeat polymorphism all associated with the MITF-M promoter as well as a 12 base pair deletion in exon 1B. The variants associated with white spotting at all four loci were also found among wolves and we conclude that none of these could be a sole causal mutation, at least not for extreme white spotting. We propose that the three canine white spotting alleles are not caused by three independent mutations but represent haplotype effects due to different combinations of causal polymorphisms. The simple repeat polymorphism showed extensive diversity both in dogs and wolves, and allele-sharing was common between wolves and white spotted dogs but was non-existent between solid and spotted dogs as well as between wolves and solid dogs. This finding was unexpected as Solid is assumed to be the wild-type allele. The data indicate that the simple repeat polymorphism has been a target for selection during dog domestication and breed formation. We also evaluated the significance of the three MITF-M associated polymorphisms with a Luciferase assay, and found conclusive evidence that the simple repeat polymorphism affects promoter activity. Three alleles associated with white spotting gave consistently lower promoter activity compared with the allele associated with solid colour. We propose that the simple repeat polymorphism affects cooperativity between transcription factors binding on either flanking sides of the repeat. Thus, both genetic and functional evidence show that the simple repeat polymorphism is a key regulator of white spotting in dogs.

A Simple Repeat Polymorphism in the MITF-M Promoter Is a Key Regulator of White Spotting in Dogs

PubMed Central

Meadows, Jennifer R. S.; Rosengren Pielberg, Gerli; Gustafson, Ulla; Hedhammar, Åke; Karlsson, Elinor K.; Seddon, Jennifer; Söderberg, Arne; Vilà, Carles; Zhang, Xiaolan; Åkesson, Mikael; Lindblad-Toh, Kerstin; Andersson, Göran; Andersson, Leif

2014-01-01

The white spotting locus (S) in dogs is colocalized with the MITF (microphtalmia-associated transcription factor) gene. The phenotypic effects of the four S alleles range from solid colour (S) to extreme white spotting (sw). We have investigated four candidate mutations associated with the sw allele, a SINE insertion, a SNP at a conserved site and a simple repeat polymorphism all associated with the MITF-M promoter as well as a 12 base pair deletion in exon 1B. The variants associated with white spotting at all four loci were also found among wolves and we conclude that none of these could be a sole causal mutation, at least not for extreme white spotting. We propose that the three canine white spotting alleles are not caused by three independent mutations but represent haplotype effects due to different combinations of causal polymorphisms. The simple repeat polymorphism showed extensive diversity both in dogs and wolves, and allele-sharing was common between wolves and white spotted dogs but was non-existent between solid and spotted dogs as well as between wolves and solid dogs. This finding was unexpected as Solid is assumed to be the wild-type allele. The data indicate that the simple repeat polymorphism has been a target for selection during dog domestication and breed formation. We also evaluated the significance of the three MITF-M associated polymorphisms with a Luciferase assay, and found conclusive evidence that the simple repeat polymorphism affects promoter activity. Three alleles associated with white spotting gave consistently lower promoter activity compared with the allele associated with solid colour. We propose that the simple repeat polymorphism affects cooperativity between transcription factors binding on either flanking sides of the repeat. Thus, both genetic and functional evidence show that the simple repeat polymorphism is a key regulator of white spotting in dogs. PMID:25116146

Spy: a new group of eukaryotic DNA transposons without target site duplications.

PubMed

Han, Min-Jin; Xu, Hong-En; Zhang, Hua-Hao; Feschotte, Cédric; Zhang, Ze

2014-06-24

Class 2 or DNA transposons populate the genomes of most eukaryotes and like other mobile genetic elements have a profound impact on genome evolution. Most DNA transposons belong to the cut-and-paste types, which are relatively simple elements characterized by terminal-inverted repeats (TIRs) flanking a single gene encoding a transposase. All eukaryotic cut-and-paste transposons so far described are also characterized by target site duplications (TSDs) of host DNA generated upon chromosomal insertion. Here, we report a new group of evolutionarily related DNA transposons called Spy, which also include TIRs and DDE motif-containing transposase but surprisingly do not create TSDs upon insertion. Instead, Spy transposons appear to transpose precisely between 5'-AAA and TTT-3' host nucleotides, without duplication or modification of the AAATTT target sites. Spy transposons were identified in the genomes of diverse invertebrate species based on transposase homology searches and structure-based approaches. Phylogenetic analyses indicate that Spy transposases are distantly related to IS5, ISL2EU, and PIF/Harbinger transposases. However, Spy transposons are distinct from these and other DNA transposon superfamilies by their lack of TSD and their target site preference. Our findings expand the known diversity of DNA transposons and reveal a new group of eukaryotic DDE transposases with unusual catalytic properties. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Unusual target site disruption by the rare-cutting HNH restriction endonuclease PacI

PubMed Central

Shen, Betty; Heiter, Daniel F.; Chan, Siu-Hong; Wang, Hua; Xu, Shuang-Yong; Morgan, Richard D.; Wilson, Geoffrey G.; Stoddard, Barry L.

2010-01-01

The crystal structure of the rare-cutting HNH restriction endonuclease PacI in complex with its eight base pair target recognition sequence 5'-TTAATTAA-3' has been determined to 1.9 Å resolution. The enzyme forms an extended homodimer, with each subunit containing two zinc-bound motifs surrounding a ββα-metal catalytic site. The latter is unusual in that a tyrosine residue likely initiates strand-cleavage. PacI dramatically distorts its target sequence from Watson-Crick duplex DNA basepairing, with every base separated from its original partner. Two bases on each strand are unpaired, four are engaged in non-canonical A:A and T:T base pairs, and the remaining two bases are matched with new Watson-Crick partners. This represents a highly unusual DNA binding mechanism for a restriction endonuclease, and implies that initial recognition of the target site might involve significantly different contacts from those visualized in the DNA-bound cocrystal structures. PMID:20541511

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

PubMed Central

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

Compositions and methods for detecting single nucleotide polymorphisms

DOEpatents

Yeh, Hsin-Chih; Werner, James; Martinez, Jennifer S.

2016-11-22

Described herein are nucleic acid based probes and methods for discriminating and detecting single nucleotide variants in nucleic acid molecules (e.g., DNA). The methods include use of a pair of probes can be used to detect and identify polymorphisms, for example single nucleotide polymorphism in DNA. The pair of probes emit a different fluorescent wavelength of light depending on the association and alignment of the probes when hybridized to a target nucleic acid molecule. Each pair of probes is capable of discriminating at least two different nucleic acid molecules that differ by at least a single nucleotide difference. The methods can probes can be used, for example, for detection of DNA polymorphisms that are indicative of a particular disease or condition.

Analysis on establishing Chang'E-3 landing site as a reflectance calibration target

NASA Astrophysics Data System (ADS)

Liu, Bin; Fu, Xiaohui; Zeng, Xingguo; Yao, Meijuan; Zhang, Hongbo; Su, Yan; Zhao, Shu; Xue, Xiping; Li, Chunlai; Zou, Yongliao

2015-04-01

Recent lunar orbital observations suggested that the surface reflectance calculated based on the Apollo 16 standard area and Apollo 16 sample laboratory measurement is significantly different from its true value [1-3], one reason is the composition and maturity differences between the 62231 sampling site and the Apollo 16 standard site existed, the other reason is the physical properties of the returned lunar sample, such as porosity, have been changed during the sampling operations. So more new standard targets on the Moon, besides the widely used Apollo 16 area, are needed for imaging spectrometers on lunar missions to improve their reflectance calibration accuracies. The Chang'E-3 VIS/NIR Imaging Spectrometer (VNIS), which is just fixed at the front of the Yutu rover [4], equipped with a white spectralon panel as reflectance calibration standard, can perform in situ multispectral observations around the Chang'E-3 landing site without altering the physical and mineralogical natures of lunar soils. Therefore, it provides an opportunity to establish a new reliable standard target for in-flight reflectance calibration. The reflectance calibration target should be compositional homogeneous, the topography of which must be flat, and the reflectance should be identical with no nearby units of other different materials. As we have known, Chang'e-3 probe landed on the Mare Imbrium basin in the east part of Sinus Iridum, the landing site is relatively flat at a spatial coverage of ~660km2, and this region belongs to Eratosthenian low-Ti/high-Ti mare basalts [5-6]. According to much higher resolution topography data, elemental data and reflectance data of Chang'E-2 and Chang'E-3[7-8], we preliminary analyse the possibility on establishing Chang'E-3 landing site as a reflectance calibration target. Firstly, the overall terrain of the 4 km×4 km area around the landing site is flat, but there are still three bigger craters existed. Secondly, the composition on Chang'E-3

Footprints of ancient-balanced polymorphisms in genetic variation data from closely related species

PubMed Central

Gao, Ziyue; Przeworski, Molly; Sella, Guy

2015-01-01

When long-lasting, balancing selection can lead to “trans-species” polymorphisms that are shared by two or more species identical by descent. In such cases, the gene genealogy at the selected site clusters by allele instead of by species, and nearby neutral sites also have unusual genealogies because of linkage. While this scenario is expected to leave discernible footprints in genetic variation data, the specific patterns remain poorly characterized. Motivated by recent findings in primates, we focus on the case of a biallelic polymorphism under ancient balancing selection and derive approximations for summaries of the polymorphism data from two species. Specifically, we characterize the length of the segment that carries most of the footprints, the expected number of shared neutral single nucleotide polymorphisms (SNPs), and the patterns of allelic associations among them. We confirm the accuracy of our approximations by coalescent simulations. We further show that for humans and chimpanzees—more generally, for pairs of species with low genetic diversity levels—these patterns are highly unlikely to be generated by neutral recurrent mutations. We discuss the implications for the design and interpretation of genome scans for ancient balanced polymorphisms in primates and other taxa. PMID:25403856

Study of the HFE gene common polymorphisms in French patients with sporadic amyotrophic lateral sclerosis.

PubMed

Praline, Julien; Blasco, Hélène; Vourc'h, Patrick; Rat, Valérian; Gendrot, Chantal; Camu, William; Andres, Christian R

2012-06-15

Our objective was to investigate whether the C282Y (p.Cys 282 Tyr) and H63D (p. His 63 Asp) HFE polymorphisms were associated with sporadic amyotrophic lateral sclerosis (SALS) in the French population. We searched for a relation of HFE polymorphisms with the clinical characteristics of the disease. The HFE polymorphisms were studied in 824 patients with SALS and 583 controls. We compared the frequency of the polymorphisms between SALS and controls groups by univariate and multivariate statistics, taking into account gender, site, age-at-onset and survival. We did not observe significant difference in the frequency of H63D polymorphism between SALS and control group. We observed a significant difference for C282Y between patients and controls with a low frequency of the Y allele in patients (3.2%) compared to our control group (5.9%). Disease duration, distribution of gender, site-of-onset, age-at-onset did not differ between groups taking into account genotypes of each polymorphism. Our results in this large cohort of ALS patients indicate that H63D polymorphism is not associated with SALS in the French population. This conclusion does not exclude a weak effect of the HFE gene polymorphisms in certain ALS populations, or an effect of other rare HFE gene variants. Copyright © 2012 Elsevier B.V. All rights reserved.

Immunological Reactivity Using Monoclonal and Polyclonal Antibodies of Autoimmune Thyroid Target Sites with Dietary Proteins

PubMed Central

Herbert, Martha

2017-01-01

Many hypothyroid and autoimmune thyroid patients experience reactions with specific foods. Additionally, food interactions may play a role in a subset of individuals who have difficulty finding a suitable thyroid hormone dosage. Our study was designed to investigate the potential role of dietary protein immune reactivity with thyroid hormones and thyroid axis target sites. We identified immune reactivity between dietary proteins and target sites on the thyroid axis that includes thyroid hormones, thyroid receptors, enzymes, and transport proteins. We also measured immune reactivity of either target specific monoclonal or polyclonal antibodies for thyroid-stimulating hormone (TSH) receptor, 5′deiodinase, thyroid peroxidase, thyroglobulin, thyroxine-binding globulin, thyroxine, and triiodothyronine against 204 purified dietary proteins commonly consumed in cooked and raw forms. Dietary protein determinants included unmodified (raw) and modified (cooked and roasted) foods, herbs, spices, food gums, brewed beverages, and additives. There were no dietary protein immune reactions with TSH receptor, thyroid peroxidase, and thyroxine-binding globulin. However, specific antigen-antibody immune reactivity was identified with several purified food proteins with triiodothyronine, thyroxine, thyroglobulin, and 5′deiodinase. Laboratory analysis of immunological cross-reactivity between thyroid target sites and dietary proteins is the initial step necessary in determining whether dietary proteins may play a potential immunoreactive role in autoimmune thyroid disease. PMID:28894619

Searching target sites on DNA by proteins: Role of DNA dynamics under confinement

PubMed Central

Mondal, Anupam; Bhattacherjee, Arnab

2015-01-01

DNA-binding proteins (DBPs) rapidly search and specifically bind to their target sites on genomic DNA in order to trigger many cellular regulatory processes. It has been suggested that the facilitation of search dynamics is achieved by combining 3D diffusion with one-dimensional sliding and hopping dynamics of interacting proteins. Although, recent studies have advanced the knowledge of molecular determinants that affect one-dimensional search efficiency, the role of DNA molecule is poorly understood. In this study, by using coarse-grained simulations, we propose that dynamics of DNA molecule and its degree of confinement due to cellular crowding concertedly regulate its groove geometry and modulate the inter-communication with DBPs. Under weak confinement, DNA dynamics promotes many short, rotation-decoupled sliding events interspersed by hopping dynamics. While this results in faster 1D diffusion, associated probability of missing targets by jumping over them increases. In contrast, strong confinement favours rotation-coupled sliding to locate targets but lacks structural flexibility to achieve desired specificity. By testing under physiological crowding, our study provides a plausible mechanism on how DNA molecule may help in maintaining an optimal balance between fast hopping and rotation-coupled sliding dynamics, to locate target sites rapidly and form specific complexes precisely. PMID:26400158

Fragment length polymorphisms among independent isolates of Epstein-Barr virus from immunocompromised and normal hosts.

PubMed

Katz, B Z; Niederman, J C; Olson, B A; Miller, G

1988-02-01

DNA restriction fragment length polymorphisms of Epstein-Barr virus (EBV) DNA were used as a molecular epidemiological tool to study multiple isolates of virus from the same and different individuals. We studied 35 EBV isolates: 19 from seven immunocompromised children and 16 from seven college students with mononucleosis. Analysis of the fragment length polymorphisms in this collection of isolates permitted several conclusions. Sites of polymorphism were most often encountered in regions with repetitive DNA. Epidemiologically unrelated patients harbored viruses that could be readily distinguished; by contrast, two infants and their mothers harbored similar viruses. Isolates from different sites in the same patient were similar. Variations between different clinical isolates of EBV mimic those found between different laboratory strains of the virus. Fragment length polymorphisms thus provide a useful marker for studying transmission and pathogenesis of EBV infections.

CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites

PubMed Central

Naito, Yuki; Hino, Kimihiro; Bono, Hidemasa; Ui-Tei, Kumiko

2015-01-01

Summary: CRISPRdirect is a simple and functional web server for selecting rational CRISPR/Cas targets from an input sequence. The CRISPR/Cas system is a promising technique for genome engineering which allows target-specific cleavage of genomic DNA guided by Cas9 nuclease in complex with a guide RNA (gRNA), that complementarily binds to a ∼20 nt targeted sequence. The target sequence requirements are twofold. First, the 5′-NGG protospacer adjacent motif (PAM) sequence must be located adjacent to the target sequence. Second, the target sequence should be specific within the entire genome in order to avoid off-target editing. CRISPRdirect enables users to easily select rational target sequences with minimized off-target sites by performing exhaustive searches against genomic sequences. The server currently incorporates the genomic sequences of human, mouse, rat, marmoset, pig, chicken, frog, zebrafish, Ciona, fruit fly, silkworm, Caenorhabditis elegans, Arabidopsis, rice, Sorghum and budding yeast. Availability: Freely available at http://crispr.dbcls.jp/. Contact: y-naito@dbcls.rois.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25414360

Coupling Neurogenetics (GARS™) and a Nutrigenomic Based Dopaminergic Agonist to Treat Reward Deficiency Syndrome (RDS): Targeting Polymorphic Reward Genes for Carbohydrate Addiction Algorithms

PubMed Central

Blum, Kenneth; Simpatico, Thomas; Badgaiyan, Rajendra D.; Demetrovics, Zsolt; Fratantonio, James; Agan, Gozde; Febo, Marcelo; Gold, Mark S.

2016-01-01

Earlier work from our laboratory, showing anti-addiction activity of a nutraceutical consisting of amino-acid precursors and enkephalinase inhibition properties and our discovery of the first polymorphic gene (Dopamine D2 Receptor Gene [DRD2]) to associate with severe alcoholism serves as a blue-print for the development of “Personalized Medicine” in addiction. Prior to the later genetic finding, we developed the concept of Brain Reward Cascade, which continues to act as an important component for stratification of addiction risk through neurogenetics. In 1996 our laboratory also coined the term “Reward Deficiency Syndrome (RDS)” to define a common genetic rubric for both substance and non-substance related addictive behaviors. Following many reiterations we utilized polymorphic targets of a number of reward genes (serotonergic, Opioidergic, GABAergic and Dopaminergic) to customize KB220 [Neuroadaptogen- amino-acid therapy (NAAT)] by specific algorithms. Identifying 1,000 obese subjects in the Netherlands a subsequent small subset was administered various KB220Z formulae customized according to respective DNA polymorphisms individualized that translated to significant decreases in both Body Mass Index (BMI) and weight in pounds. Following these experiments, we have been successfully developing a panel of genes known as “Genetic Addiction Risk Score” (GARSpDX)™. Selection of 10 genes with appropriate variants, a statistically significant association between the ASI-Media Version-alcohol and drug severity scores and GARSpDx was found A variant of KB220Z in abstinent heroin addicts increased resting state functional connectivity in a putative network including: dorsal anterior cingulate, medial frontal gyrus, nucleus accumbens, posterior cingulate, occipital cortical areas, and cerebellum. In addition, we show that KB220Z significantly activates, above placebo, seed regions of interest including the left nucleus accumbens, cingulate gyrus, anterior

Vegetation and the importance of insecticide-treated target siting for control of Glossina fuscipes fuscipes.

PubMed

Esterhuizen, Johan; Njiru, Basilio; Vale, Glyn A; Lehane, Michael J; Torr, Stephen J

2011-09-01

Control of tsetse flies using insecticide-treated targets is often hampered by vegetation re-growth and encroachment which obscures a target and renders it less effective. Potentially this is of particular concern for the newly developed small targets (0.25 high × 0.5 m wide) which show promise for cost-efficient control of Palpalis group tsetse flies. Consequently the performance of a small target was investigated for Glossina fuscipes fuscipes in Kenya, when the target was obscured following the placement of vegetation to simulate various degrees of natural bush encroachment. Catches decreased significantly only when the target was obscured by more than 80%. Even if a small target is underneath a very low overhanging bush (0.5 m above ground), the numbers of G. f. fuscipes decreased by only about 30% compared to a target in the open. We show that the efficiency of the small targets, even in small (1 m diameter) clearings, is largely uncompromised by vegetation re-growth because G. f. fuscipes readily enter between and under vegetation. The essential characteristic is that there should be some openings between vegetation. This implies that for this important vector of HAT, and possibly other Palpalis group flies, a smaller initial clearance zone around targets can be made and longer interval between site maintenance visits is possible both of which will result in cost savings for large scale operations. We also investigated and discuss other site features e.g. large solid objects and position in relation to the water's edge in terms of the efficacy of the small targets.

Lack of association between the APEX1 Asp148Glu polymorphism and sporadic amyotrophic lateral sclerosis.

PubMed

Coppedè, Fabio; Lo Gerfo, Annalisa; Carlesi, Cecilia; Piazza, Selina; Mancuso, Michelangelo; Pasquali, Livia; Murri, Luigi; Migliore, Lucia; Siciliano, Gabriele

2010-02-01

Impairments in DNA repair enzymes have been observed in amyotrophic lateral sclerosis (ALS) tissues, particularly in the activity of the apurinic/apyrimidinic endonuclease 1 (APEX1). Moreover, it was suggested that the common APEX1 Asp148Glu polymorphism might be associated with ALS risk. To further address this question we performed the present study aimed at evaluating the contribution of the APEX1 Asp148Glu polymorphism in sporadic ALS (sALS) risk and clinical presentation, including age and site of onset and disease progression. We screened 134 sALS Italian patients and 129 matched controls for the presence of the APEX1 Asp148Glu polymorphism. No difference in APEX1 Asp148Glu allele and genotype frequencies was found between the groups, nor was the polymorphism associated with age and site of onset or disease progression. Present results do not support a role for the APEX1 Asp148Glu polymorphism in sALS pathogenesis in the Italian population.

[Correlation between polymorphisms in the coagulation factor VII gene hypervariable region 4 site and the risk of coronary heart disease in population with different ethnic backgrounds: a Meta-analysis].

PubMed

Wang, Li-li; Ma, Bin; Qian, Dun; Pang, Jun; Yao, Ya-li

2013-12-01

To assess the correlation between polymorphisms in the coagulation factor VII (F VII)gene hypervariable region 4 (HVR4)site and risk related to coronary heart disease (CHD)in different ethnic populations, especially the Asian populations. Publications up to April 2013, from CBM, CNKI, Wanfang Database,VIP, PubMed, Cochrane Library and Embase were searched to collect data from case-control studies related to F VII gene HVR4 site and CHD in populations from different ethnicities. Quality of studies was evaluated, available data extracted and both RevMan 5.1 and Stata 11.0 softwares were used for Meta-analysis. Fifteen case-control studies were included, involving 3167 cases with CHD group and 3168 cases in the control group. on this Meta-analysis showed that:a)polymorphism of the F VII gene HVR4 site H7/H6+H5 and CHD, b)H7H7/H6H6 + H7H6 and CHD were both slightly correlated between people with different ethnic backgrounds. However, the H6 allele versus H7+H5 allele and CHD showed different results-a high correlation seen in different ethnic groups. H5 allele versus H6+H7 allele and CHD did not appear significant difference(OR = 1.20, 95%CI:0.76-1.90, P = 0.43). Both F VII gene HVR4 polymorphisms H7 allele and the H7H7 genotype might have served as protective factors for CHD in different ethnic groups, H6 allele might serve as a risk factor for CHD, but H5 allele was likely not to be associated with CHD in different ethnic groups.

In silico dissection of miRNA targetome polymorphisms and their role in regulating miRNA-mediated gene expression in esophageal cancer.

PubMed

Nariman-Saleh-Fam, Ziba; Bastami, Milad; Somi, Mohammad Hossein; Samadi, Naser; Abbaszadegan, Mohammad Reza; Behjati, Farkhondeh; Ghaedi, Hamid; Tavakkoly-Bazzaz, Javad; Masotti, Andrea

2016-12-01

Esophageal cancer is the eighth most common cancer worldwide. Also middle-aged obese adults with higher body mass index during childhood have a greater risk to develop esophageal cancer. The contribution of microRNAs to esophageal cancer has been extensively studied and it became clear that these noncoding RNAs may play crucial roles in pathogenesis, diagnosis and prognosis of the disease. Increasing evidences have suggested that polymorphisms perturbing microRNA targetome (i.e., the compendium of all microRNA target sites) are associated with cancers including esophageal cancer. However, the extent to which such variants contribute to esophageal cancer is still unclear. In this study, we applied an in silico approach to systematically identify polymorphisms perturbing microRNA targetome in esophageal cancer and performed various analyses to predict the functional consequences of the occurrence of these variants. The computational results were integrated to provide a prioritized list of the most potentially disrupting esophageal cancer-implicated microRNA targetome polymorphisms along with the in silico insight into the mechanisms with which such variations may modulate microRNA-mediated regulation. The results of this study will be valuable for future functional experiments aimed at dissecting the roles of microRNA targetome polymorphisms in the onset and progression of esophageal cancer.

Polymorphism discovery and allele frequency estimation using high-throughput DNA sequencing of target-enriched pooled DNA samples

PubMed Central

2012-01-01

Background The central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility. Results In total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952) of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612) were intronic and 9% (n = 464) were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS). Significant (P < 0.01) mean allele frequency differentials between the low and high fertility groups were observed for 720 SNPs (58 NSS). Allele frequencies for 43 of the SNPs were also determined by genotyping the 150 individual animals (Sequenom® MassARRAY). No significant differences (P > 0.1) were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total). Conclusions The results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post

Protecting Important Sites for Biodiversity Contributes to Meeting Global Conservation Targets

PubMed Central

Butchart, Stuart H. M.; Scharlemann, Jörn P. W.; Evans, Mike I.; Quader, Suhel; Aricò, Salvatore; Arinaitwe, Julius; Balman, Mark; Bennun, Leon A.; Bertzky, Bastian; Besançon, Charles; Boucher, Timothy M.; Brooks, Thomas M.; Burfield, Ian J.; Burgess, Neil D.; Chan, Simba; Clay, Rob P.; Crosby, Mike J.; Davidson, Nicholas C.; De Silva, Naamal; Devenish, Christian; Dutson, Guy C. L.; Fernández, David F. Día z; Fishpool, Lincoln D. C.; Fitzgerald, Claire; Foster, Matt; Heath, Melanie F.; Hockings, Marc; Hoffmann, Michael; Knox, David; Larsen, Frank W.; Lamoreux, John F.; Loucks, Colby; May, Ian; Millett, James; Molloy, Dominic; Morling, Paul; Parr, Mike; Ricketts, Taylor H.; Seddon, Nathalie; Skolnik, Benjamin; Stuart, Simon N.; Upgren, Amy; Woodley, Stephen

2012-01-01

Protected areas (PAs) are a cornerstone of conservation efforts and now cover nearly 13% of the world's land surface, with the world's governments committed to expand this to 17%. However, as biodiversity continues to decline, the effectiveness of PAs in reducing the extinction risk of species remains largely untested. We analyzed PA coverage and trends in species' extinction risk at globally significant sites for conserving birds (10,993 Important Bird Areas, IBAs) and highly threatened vertebrates and conifers (588 Alliance for Zero Extinction sites, AZEs) (referred to collectively hereafter as ‘important sites’). Species occurring in important sites with greater PA coverage experienced smaller increases in extinction risk over recent decades: the increase was half as large for bird species with>50% of the IBAs at which they occur completely covered by PAs, and a third lower for birds, mammals and amphibians restricted to protected AZEs (compared with unprotected or partially protected sites). Globally, half of the important sites for biodiversity conservation remain unprotected (49% of IBAs, 51% of AZEs). While PA coverage of important sites has increased over time, the proportion of PA area covering important sites, as opposed to less important land, has declined (by 0.45–1.14% annually since 1950 for IBAs and 0.79–1.49% annually for AZEs). Thus, while appropriately located PAs may slow the rate at which species are driven towards extinction, recent PA network expansion has under-represented important sites. We conclude that better targeted expansion of PA networks would help to improve biodiversity trends. PMID:22457717

Polymorphisms in MicroRNA Genes And Genes Involving in NMDAR Signaling and Schizophrenia: A Case-Control Study in Chinese Han Population.

PubMed

Zhang, Yanxia; Fan, Mei; Wang, Qingzhong; He, Guang; Fu, Yingmei; Li, Huafang; Yu, Shunying

2015-08-10

Disturbances in glutamate signaling caused by disruption of N-methyl-D-aspartate-type glutamate receptor (NMDAR) have been implicated in schizophrenia. Findings suggested that miR-219, miR-132 and miR-107 could involve in NMDAR signaling by influencing the expression of pathway genes or the signaling transmission and single nucleotide polymorphisms (SNPs) within miRNA genes or miRNA target sites could result in their functional changes. Therefore, we hypothesized that SNPs in miRNAs and/or their target sites were associated with schizophrenia. 3 SNPs in hsa-pri-miR-219/132/107 and 6 SNPs in 3'UTRs of GRIN2A/2B/3A and CAMK2G were selected and genotyped in a case-control study of 1041 schizophrenia cases and 953 healthy controls in Chinese Han population. In the present study, GRIN2B rs890 showed significant associations with schizophrenia. Further functional analyses showed that the rs890 variant C allele led to significantly lower luciferase activity, compared with the A allele. MDR analysis showed that a 4-locus model including rs107822, rs2306327, rs890 and rs12342026 was the best model. These findings suggest that GRIN2B may be associated with schizophrenia and interaction effects of the polymorphisms in hsa-miR-219, CAKM2G, GRIN2B and GRIN3A may confer susceptibility to schizophrenia in the Chinese Han population.

Repopulation of calibrations with samples from the target site: effect of the size of the calibration.

NASA Astrophysics Data System (ADS)

Guerrero, C.; Zornoza, R.; Gómez, I.; Mataix-Solera, J.; Navarro-Pedreño, J.; Mataix-Beneyto, J.; García-Orenes, F.

2009-04-01

Near infrared (NIR) reflectance spectroscopy offers important advantages because is a non-destructive technique, the pre-treatments needed in samples are minimal, and the spectrum of the sample is obtained in less than 1 minute without the needs of chemical reagents. For these reasons, NIR is a fast and cost-effective method. Moreover, NIR allows the analysis of several constituents or parameters simultaneously from the same spectrum once it is obtained. For this, a needed steep is the development of soil spectral libraries (set of samples analysed and scanned) and calibrations (using multivariate techniques). The calibrations should contain the variability of the target site soils in which the calibration is to be used. Many times this premise is not easy to fulfil, especially in libraries recently developed. A classical way to solve this problem is through the repopulation of libraries and the subsequent recalibration of the models. In this work we studied the changes in the accuracy of the predictions as a consequence of the successive addition of samples to repopulation. In general, calibrations with high number of samples and high diversity are desired. But we hypothesized that calibrations with lower quantities of samples (lower size) will absorb more easily the spectral characteristics of the target site. Thus, we suspect that the size of the calibration (model) that will be repopulated could be important. For this reason we also studied this effect in the accuracy of predictions of the repopulated models. In this study we used those spectra of our library which contained data of soil Kjeldahl Nitrogen (NKj) content (near to 1500 samples). First, those spectra from the target site were removed from the spectral library. Then, different quantities of samples of the library were selected (representing the 5, 10, 25, 50, 75 and 100% of the total library). These samples were used to develop calibrations with different sizes (%) of samples. We used partial least

Cross-species amplification of mitochondrial DNA sequence-tagged-site markers in conifers: the nature of polymorphism and variation within and among species in Picea.

PubMed

Jaramillo-Correa, J P; Bousquet, J; Beaulieu, J; Isabel, N; Perron, M; Bouillé, M

2003-05-01

Primers previously developed to amplify specific non-coding regions of the mitochondrial genome in Angiosperms, and new primers for additional non-coding mtDNA regions, were tested for their ability to direct DNA amplification in 12 conifer taxa and to detect sequence-tagged-site (STS) polymorphisms within and among eight species in Picea. Out of 12 primer pairs, nine were successful at amplifying mtDNA in most of the taxa surveyed. In conifers, indels and substitutions were observed for several loci, allowing them to distinguish between families, genera and, in some cases, between species within genera. In Picea, interspecific polymorphism was detected for four loci, while intraspecific variation was observed for three of the mtDNA regions studied. One of these (SSU rRNA V1 region) exhibited indel polymorphisms, and the two others ( nad1 intron b/c and nad5 intron1) revealed restriction differences after digestion with Sau3AI (PCR-RFLP). A fourth locus, the nad4L- orf25 intergenic region, showed a multibanding pattern for most of the spruce species, suggesting a possible gene duplication. Maternal inheritance, expected for mtDNA in conifers, was observed for all polymorphic markers except the intergenic region nad4L- orf25. Pooling of the variation observed with the remaining three markers resulted in two to six different mtDNA haplotypes within the different species of Picea. Evidence for intra-genomic recombination was observed in at least two taxa. Thus, these mitotypes are likely to be more informative than single-locus haplotypes. They should be particularly useful for the study of biogeography and the dynamics of hybrid zones.

Prospects and limitations for p-type doping in boron nitride polymorphs

NASA Astrophysics Data System (ADS)

Weston, Leigh; van de Walle, Chris G.

Using first-principles calculations, we examine the potential for p-type doping of BN polymorphs via substitutional impurities. Based on density functional theory with a hybrid functional, our calculations reveal that group-IV elements (C, Si) substituting at the N site result in acceptor levels that are more than 1 eV above the valence-band maximum in all of the BN polymorphs, and hence far too deep to allow for p-type doping. On the other hand, group-II elements (Be, Mg) substituting at the B site lead to shallower acceptor levels. However, for the ground-state hexagonal phase (h-BN), we show that p-type doping at the B site is inhibited by the formation of hole polarons. Our calculations reveal that hole localization is intrinsic to sp2 bonded h-BN, and this places fundamental limits on hole conduction in this material. In contrast, the sp3 bonded wurtzite (w-BN) and cubic (c-BN) polymorphs are capable of forming shallow acceptor levels. For Be dopants, the acceptor ionization energies are 0.31 eV and 0.24 eV for w-BN and c-BN, respectively; these values are only slightly larger than the ionization energy of the Mg acceptor in GaN. This work was supported by NSF.

Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site Definition and Plasma Stability

DTIC Science & Technology

2015-11-01

increased PhScN potency as a result of preventing endoproteolytic degradation. Finally, the in vivo lung extravasation and colonization data, as well as...successful colonization are late stages in breast cancer progression that are ultimately fatal. Hence, prevention of extravasation which leads to colony...Award Number: TITLE: “Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site Definition and Plasma

The Science and Issues of Human DNA Polymorphisms: A Training Workshop for High School Biology Teachers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Micklos, David A.

2006-10-30

of human polymorphism kits by Carolina Biological rose from 700 units in 1999 to 1,132 in 2000 â a 62% increase. Competing kits using the Alu system, and based substantially on our earlier work, are also marketed by Biorad and Edvotek. In parallel with the lab experiments, we developed a suite of database/statistical applications and easy-to-use interfaces that allow students to use their own DNA data to explore human population genetics and to test theories of human evolution. Database searches and statistical analyses are launched from a centralized workspace. Workshop participants were introduced to these and other resources available at the DNALC WWW site (http://vector.cshl.org/bioserver/): 1) Allele Server tests Hardy-Weinberg equilibrium and statistically compares PV92 data from world populations. 2) Sequence Server uses DNA sequence data to search Genbank using BLASTN, compare sequences using CLUSTALW, and create phylogenetic trees using PHYLIP. 3) Simulation Server uses a Monte Carlo generator to model the long-term effects of drift, selection, and population bottlenecks. By targeting motivated and innovative biology faculty, we believe that this project offered a cost-effective means to bring high school biology education up-to-the-minute with genomic biology. The workshop reached a target audience of highly professional faculty who have already implemented hands-on labs in molecular genetics and many of whom offer laboratory electives in biotechnology. Many attend professional meetings, develop curriculum, collaborate with scientists, teach faculty workshops, and manage equipment-sharing programs. These individuals are life-long learners, anxious for deeper insight and additional training to further extend their leadership. This contention was supported by data from a mail survey, conducted in February-March 2000 and 2001, of 256 faculty who participated in workshops conducted during the current term of DOE support. Seventy percent of participants

Impact of vitamin D receptor gene polymorphisms in pathogenesis of Type-1 diabetes mellitus

PubMed Central

Kamel, Mahmoud M; Fouad, Shawky A; Salaheldin, Omina; El-Razek, Abd El-Rahman A Abd; El-Fatah, Abeer I Abd

2014-01-01

Background: Type 1 diabetes mellitus (TIDM) results from an immune-mediated destruction of insulin-producing-cells in the pancreatic islets of Langerhans. There are clear differences in immunogenetic predisposition to type1 diabetes among countries. Studies have indicated that vitamin D supplementation in early childhood decreases the risk of TIDM. Vitamin D exerts its action via the nuclear vitamin D receptor (VDR), which shows an extensive polymorphism. VDR gene polymorphisms have been associated with altered gene expression or gene function. Four single nucleotide polymorphisms (SNPs) in the VDR gene produce variation in four recognition sites. These recognition sites variants include Fok I, Bsm I, Apa I and Taq I. Aim of the study: TO investigate the relationship between VDR gene polymorphisms (at positions Taq I and Apa I) and the incidence of TIDM in Egyptian peoples. Subjects and methods: This study included 74 patients with type 1 DM in addition to 28 healthy age and sex matched control subjects. All of them were subjected to full history taking and clinical examination. Three ml of venous blood were withdrawn from each patient at fasting and postprandial times and used for genomic DNA extraction, estimation of Hb A1C, as well as, fasting and postprandial C-peptide and blood glucose levels. Results: Apa I recognition site was found in low frequency in diabetic patients (14/74) 18.9% while, its frequency was high (16/28) 57.1% among normal subjects. Taq I has two recognition sites. The first was found at nucleotide number 293 that was found in a frequency of (2/28) 7.1% in normal non-diabetic individuals while it was detected in (14/74) 18.9% in diabetic patients. The second Taq I recognition site was found at nucleotide number 494 without any differences between diabetic and normal individuals. Conclusion: This study indicates that there is an association between VDR genetic polymorphism and incidence of TIDM in Egyptian patients. PMID:25664062

Intragenomic polymorphisms among high-copy loci: a genus-wide study of nuclear ribosomal DNA in Asclepias (Apocynaceae).

PubMed

Weitemier, Kevin; Straub, Shannon C K; Fishbein, Mark; Liston, Aaron

2015-01-01

Despite knowledge that concerted evolution of high-copy loci is often imperfect, studies that investigate the extent of intragenomic polymorphisms and comparisons across a large number of species are rarely made. We present a bioinformatic pipeline for characterizing polymorphisms within an individual among copies of a high-copy locus. Results are presented for nuclear ribosomal DNA (nrDNA) across the milkweed genus, Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual's consensus and at each position reads differing from the consensus were tallied using a custom perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%). Most nrDNA positions (91%) were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the "noncoding" ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided here is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming).

Outreach for Outreach: Targeting social media audiences to promote a NASA kids’ web site

NASA Astrophysics Data System (ADS)

Pham, C. C.

2009-12-01

The Space Place is a successful NASA web site that benefits upper elementary school students and educators by providing games, activities, and resources to stimulate interest in science, technology, engineering, and mathematics, as well as to inform the audience of NASA’s contributions. As online social networking grows to be a central component of modern communication, The Space Place has explored the benefits of integrating social networks with the web site to increase awareness of materials the web site offers. This study analyzes the capabilities of social networks, and specifically the demographics of Twitter and Facebook. It then compares these results with the content, audience, and perceived demographics of The Space Place web site. Based upon the demographic results, we identified a target constituency that would benefit from the integration of social networks into The Space Place web site. As a result of this study, a Twitter feed has been established that releases a daily tweet from The Space Place. In addition, a Facebook page has been created to showcase new content and prompt interaction among fans of The Space Place. Currently, plans are under way to populate the Space Place Facebook page. Each social network has been utilized in an effort to spark excitement about the content on The Space Place, as well as to attract followers to the main NASA Space Place web site. To pursue this idea further, a plan has been developed to promote NASA Space Place’s social media tools among the target audience.

Analysis of ploidy and the patterns of amplified fragment length polymorphism and methylation sensitive amplified polymorphism in strawberry plants recovered from cryopreservation.

PubMed

Hao, Yu-Jin; You, Chun-Xiang; Deng, Xui-Xin

2002-01-01

Shoot-tips of 10 strawberry genotypes were successfully cryopreserved using a modified encapsulation-dehydration method. All genotypes survived cryopreservation with high survival and regeneration rates. Eight Joho single-bud sibling lines were established as a model system for genetic analysis. Although cytological examination found chromosomal variation in both non-cryopreserved and cryopreserved samples, the ploidy constitution remained relatively stable after cryopreservation. DNA samples digested with MseI and PstI were used for amplified fragmentation length polymorphism (AFLP) assay. In 16 primer combinations, only one, namely, PCCA-MCAG, detected one site where band pattern changed after cryopreservation, which might be contributed to the change in DNA methylation status at PstI recognition site. Methylation sensitive amplified polymorphism (MSAP) assay was carried out for further investigation on the influence of cryopreservation on DNA methylation status. It was found that cryopreservation induced a significant change in DNA methylation status.

Molecular Characterization of Monoclonal Antibodies that Inhibit Acetylcholinesterase by Targeting the Peripheral Site and Backdoor Region

PubMed Central

Essono, Sosthène; Mondielli, Grégoire; Lamourette, Patricia; Boquet, Didier; Grassi, Jacques; Marchot, Pascale

2013-01-01

The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis. PMID:24146971

Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site Definition and Plasma Stability

DTIC Science & Technology

2015-11-01

systemic therapy to prevent breast cancer bone colony progression. Figure 6. Colocalization of Ac-PhscNGGK-Bio with DiI in lung– extravasated SUM149PT cells...breast cancer progression that are ultimately fatal. Hence, prevention of extravasation which leads to colony formation would increase life...1 Award Number: W81XWH-12-1-0097 TITLE: “Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site

Polymorphs and polymorphic cocrystals of temozolomide.

PubMed

Babu, N Jagadeesh; Reddy, L Sreenivas; Aitipamula, Srinivasulu; Nangia, Ashwini

2008-07-07

Crystal polymorphism in the antitumor drug temozolomide (TMZ), cocrystals of TMZ with 4,4'-bipyridine-N,N'-dioxide (BPNO), and solid-state stability were studied. Apart from a known X-ray crystal structure of TMZ (form 1), two new crystalline modifications, forms 2 and 3, were obtained during attempted cocrystallization with carbamazepine and 3-hydroxypyridine-N-oxide. Conformers A and B of the drug molecule are stabilized by intramolecular amide N--HN(imidazole) and N--HN(tetrazine) interactions. The stable conformer A is present in forms 1 and 2, whereas both conformers crystallized in form 3. Preparation of polymorphic cocrystals I and II (TMZBPNO 1:0.5 and 2:1) were optimized by using solution crystallization and grinding methods. The metastable nature of polymorph 2 and cocrystal II is ascribed to unused hydrogen-bond donors/acceptors in the crystal structure. The intramolecularly bonded amide N-H donor in the less stable structure makes additional intermolecular bonds with the tetrazine C==O group and the imidazole N atom in stable polymorph 1 and cocrystal I, respectively. All available hydrogen-bond donors and acceptors are used to make intermolecular hydrogen bonds in the stable crystalline form. Synthon polymorphism and crystal stability are discussed in terms of hydrogen-bond reorganization.

HFE p.H63D polymorphism does not influence ALS phenotype and survival.

PubMed

Chiò, Adriano; Mora, Gabriele; Sabatelli, Mario; Caponnetto, Claudia; Lunetta, Christian; Traynor, Bryan J; Johnson, Janel O; Nalls, Mike A; Calvo, Andrea; Moglia, Cristina; Borghero, Giuseppe; Monsurrò, Maria Rosaria; La Bella, Vincenzo; Volanti, Paolo; Simone, Isabella; Salvi, Fabrizio; Logullo, Francesco O; Nilo, Riva; Giannini, Fabio; Mandrioli, Jessica; Tanel, Raffaella; Murru, Maria Rita; Mandich, Paola; Zollino, Marcella; Conforti, Francesca L; Penco, Silvana; Brunetti, Maura; Barberis, Marco; Restagno, Gabriella

2015-10-01

It has been recently reported that the p.His63Asp polymorphism of the HFE gene accelerates disease progression both in the SOD1 transgenic mouse and in amyotrophic lateral sclerosis (ALS) patients. We have evaluated the effect of HFE p.His63Asp polymorphism on the phenotype in 1351 Italian ALS patients (232 of Sardinian ancestry). Patients were genotyped for the HFE p.His63Asp polymorphism (CC, GC, and GG). All patients were also assessed for C9ORF72, TARDBP, SOD1, and FUS mutations. Of the 1351 ALS patients, 363 (29.2%) were heterozygous (GC) for the p.His63Asp polymorphism and 30 (2.2%) were homozygous for the minor allele (GG). Patients with CC, GC, and GG polymorphisms did not significantly differ by age at onset, site of onset of symptoms, and survival; however, in SOD1 patients with CG or GG polymorphism had a significantly longer survival than those with a CC polymorphism. Differently from what observed in the mouse model of ALS, the HFE p.His63Asp polymorphism has no effect on ALS phenotype in this large series of Italian ALS patients. Copyright © 2015 Elsevier Inc. All rights reserved.

Gene polymorphisms associated with functional dyspepsia.

PubMed

Kourikou, Anastasia; Karamanolis, George P; Dimitriadis, George D; Triantafyllou, Konstantinos

2015-07-07

Functional dyspepsia (FD) is a constellation of functional upper abdominal complaints with poorly elucidated pathophysiology. However, there is increasing evidence that susceptibility to FD is influenced by hereditary factors. Genetic association studies in FD have examined genotypes related to gastrointestinal motility or sensation, as well as those related to inflammation or immune response. G-protein b3 subunit gene polymorphisms were first reported as being associated with FD. Thereafter, several gene polymorphisms including serotonin transporter promoter, interlukin-17F, migration inhibitory factor, cholecystocynine-1 intron 1, cyclooxygenase-1, catechol-o-methyltransferase, transient receptor potential vanilloid 1 receptor, regulated upon activation normal T cell expressed and secreted, p22PHOX, Toll like receptor 2, SCN10A, CD14 and adrenoreceptors have been investigated in relation to FD; however, the results are contradictory. Several limitations underscore the value of current studies. Among others, inconsistencies in the definitions of FD and controls, subject composition differences regarding FD subtypes, inadequate samples, geographical and ethnical differences, as well as unadjusted environmental factors. Further well-designed studies are necessary to determine how targeted genes polymorphisms, influence the clinical manifestations and potentially the therapeutic response in FD.

The ATP-binding site of type II topoisomerases as a target for antibacterial drugs.

PubMed

Maxwell, Anthony; Lawson, David M

2003-01-01

DNA topoisomerases are essential enzymes in all cell types and have been found to be valuable drug targets both for antibacterial and anti-cancer chemotherapy. Type II topoisomerases possess a binding site for ATP, which can be exploited as a target for chemo-therapeutic agents. High-resolution structures of protein fragments containing this site complexed with antibiotics or an ATP analogue have provided vital information for the understanding of the action of existing drugs and for the potential development of novel anti-bacterial agents. In this article we have reviewed the structure and function of the ATPase domain of DNA gyrase (bacterial topoisomerase II), particularly highlighting novel information that has been revealed by structural studies. We discuss the efficacy and mode of action of existing drugs and consider the prospects for the development of novel agents.

Occurrence Prospect of HDR and Target Site Selection Study in Southeastern of China

NASA Astrophysics Data System (ADS)

Lin, W.; Gan, H.

2017-12-01

Hot dry rock (HDR) geothermal resource is one of the most important clean energy in future. Site selection a HDR resource is a fundamental work to explore the HDR resources. This paper compiled all the HDR development projects domestic and abroad, and summarized the location of HDR geothermal geological index. After comparing the geological background of HDR in the southeast coastal area of China, Yangjiang Xinzhou in Guangdong province, Leizhou Peninsula area, Lingshui in Hainan province and Huangshadong in Guangzhou were selected from some key potential target area along the southeast coast of China. Deep geothermal field model of the study area is established based on the comprehensive analysis of the target area of deep geothermal geological background and deep thermal anomalies. This paper also compared the hot dry rock resources target locations, and proposed suggestions for the priority exploration target area and exploration scheme.

Intragenomic polymorphisms among high-copy loci: a genus-wide study of nuclear ribosomal DNA in Asclepias (Apocynaceae)

PubMed Central

Straub, Shannon C.K.; Fishbein, Mark; Liston, Aaron

2015-01-01

Despite knowledge that concerted evolution of high-copy loci is often imperfect, studies that investigate the extent of intragenomic polymorphisms and comparisons across a large number of species are rarely made. We present a bioinformatic pipeline for characterizing polymorphisms within an individual among copies of a high-copy locus. Results are presented for nuclear ribosomal DNA (nrDNA) across the milkweed genus, Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual’s consensus and at each position reads differing from the consensus were tallied using a custom perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%). Most nrDNA positions (91%) were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the “noncoding” ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided here is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming). PMID:25653903

Microscopic origin of entropy-driven polymorphism in hybrid organic-inorganic perovskite materials

NASA Astrophysics Data System (ADS)

Butler, Keith T.; Svane, Katrine; Kieslich, Gregor; Cheetham, Anthony K.; Walsh, Aron

2016-11-01

Entropy is a critical, but often overlooked, factor in determining the relative stabilities of crystal phases. The importance of entropy is most pronounced in softer materials, where small changes in free energy can drive phase transitions, which has recently been demonstrated in the case of organic-inorganic hybrid-formate perovskites. In this Rapid Communication we demonstrate the interplay between composition and crystal structure that is responsible for the particularly pronounced role of entropy in determining polymorphism in hybrid organic-inorganic materials. Using ab initio based lattice dynamics, we probe the origins and effects of vibrational entropy of four archetype perovskite (A B X3 ) structures. We consider an inorganic material (SrTiO3), an A -site hybrid-halide material (CH3NH3) PbI3 , a X -site hybrid material KSr (BH4)3 , and a mixed A - and X -site hybrid-formate material (N2H5) Zn (HCO2)3 , comparing the differences in entropy between two common polymorphs. The results demonstrate the importance of low-frequency intermolecular modes in determining the phase stability in these materials. The understanding gained allows us to propose a general principle for the relative stability of different polymorphs of hybrid materials as temperature is increased.

Mohawk Tannery Hazardous Waste Site in New Hampshire included on EPA List of Targeted for Immediate Attention

EPA Pesticide Factsheets

Today, the U.S. Environmental Protection Agency released the list of Superfund sites that Administrator Pruitt has targeted for immediate and intense attention. The former Mohawk Tannery facility (a.k.a. Granite State Leathers) is one of the 21 sites on th

TERT promoter mutations in bladder cancer affect patient survival and disease recurrence through modification by a common polymorphism.

PubMed

Rachakonda, P Sivaramakrishna; Hosen, Ismail; de Verdier, Petra J; Fallah, Mahdi; Heidenreich, Barbara; Ryk, Charlotta; Wiklund, N Peter; Steineck, Gunnar; Schadendorf, Dirk; Hemminki, Kari; Kumar, Rajiv

2013-10-22

The telomerase reverse transcriptase (TERT) promoter, an important element of telomerase expression, has emerged as a target of cancer-specific mutations. Originally described in melanoma, the mutations in TERT promoter have been shown to be common in certain other tumor types that include glioblastoma, hepatocellular carcinoma, and bladder cancer. To fully define the occurrence and effect of the TERT promoter mutations, we investigated tumors from a well-characterized series of 327 patients with urothelial cell carcinoma of bladder. The somatic mutations, mainly at positions -124 and -146 bp from ATG start site that create binding motifs for E-twenty six/ternary complex factors (Ets/TCF), affected 65.4% of the tumors, with even distribution across different stages and grades. Our data showed that a common polymorphism rs2853669, within a preexisting Ets2 binding site in the TERT promoter, acts as a modifier of the effect of the mutations on survival and tumor recurrence. The patients with the mutations showed poor survival in the absence [hazard ratio (HR) 2.19, 95% confidence interval (CI) 1.02-4.70] but not in the presence (HR 0.42, 95% CI 0.18-1.01) of the variant allele of the polymorphism. The mutations in the absence of the variant allele were highly associated with the disease recurrence in patients with Tis, Ta, and T1 tumors (HR 1.85, 95% CI 1.11-3.08). The TERT promoter mutations are the most common somatic lesions in bladder cancer with clinical implications. The association of the mutations with patient survival and disease recurrence, subject to modification by a common polymorphism, can be a unique putative marker with individualized prognostic potential.

Target and Non-target Site Mechanisms Developed by Glyphosate-Resistant Hairy beggarticks (Bidens pilosa L.) Populations from Mexico

PubMed Central

Alcántara-de la Cruz, Ricardo; Fernández-Moreno, Pablo T.; Ozuna, Carmen V.; Rojano-Delgado, Antonia M.; Cruz-Hipolito, Hugo E.; Domínguez-Valenzuela, José A.; Barro, Francisco; De Prado, Rafael

2016-01-01

In 2014 hairy beggarticks (Bidens pilosa L.) has been identified as being glyphosate-resistant in citrus orchards from Mexico. The target and non-target site mechanisms involved in the response to glyphosate of two resistant populations (R1 and R2) and one susceptible (S) were studied. Experiments of dose-response, shikimic acid accumulation, uptake-translocation, enzyme activity and 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene sequencing were carried out in each population. The R1 and R2 populations were 20.4 and 2.8-fold less glyphosate sensitive, respectively, than the S population. The resistant populations showed a lesser shikimic acid accumulation than the S population. In the latter one, 24.9% of 14C-glyphosate was translocated to the roots at 96 h after treatment; in the R1 and R2 populations only 12.9 and 15.5%, respectively, was translocated. Qualitative results confirmed the reduced 14C-glyphosate translocation in the resistant populations. The EPSPS enzyme activity of the S population was 128.4 and 8.5-fold higher than the R1 and R2 populations of glyphosate-treated plants, respectively. A single (Pro-106-Ser), and a double (Thr-102-Ile followed by Pro-106-Ser) mutations were identified in the EPSPS2 gene conferred high resistance in R1 population. Target-site mutations associated with a reduced translocation were responsible for the higher glyphosate resistance in the R1 population. The low-intermediate resistance of the R2 population was mediated by reduced translocation. This is the first glyphosate resistance case confirmed in hairy beggarticks in the world. PMID:27752259

Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes.

PubMed Central

Schmidt, R J; Ketudat, M; Aukerman, M J; Hoschek, G

1992-01-01

opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the -300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the O2 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family. PMID:1392590

Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti.

PubMed

Häcker, Irina; Harrell Ii, Robert A; Eichner, Gerrit; Pilitt, Kristina L; O'Brochta, David A; Handler, Alfred M; Schetelig, Marc F

2017-03-07

Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting.

A polymorphism in the promoter region of PD-L1 serves as a binding-site for SP1 and is associated with PD-L1 overexpression and increased occurrence of gastric cancer.

PubMed

Tao, Li-Hua; Zhou, Xin-Ru; Li, Fu-Chao; Chen, Qi; Meng, Fan-Yi; Mao, Yong; Li, Rui; Hua, Dong; Zhang, Hong-Jian; Wang, Wei-Peng; Chen, Wei-Chang

2017-03-01

PD-L1 is a member of the B7 family co-inhibitory molecules and plays a critical role in tumor immune escape. In this study, we found a polymorphism rs10815225 in the PD-L1 promoter region was significantly associated with the occurrence of gastric cancer. The GG homozygous frequency was higher in the cancer patients than that in the precancerous lesions, which was higher than that in the health controls. This polymorphism locates in the binding-site of Sp1 transcription factor (SP1). The expression level of PD-L1 mRNA in the GG homozygous cancer patients was apparently higher than that in the GC heterozygotes. Luciferase reporter results showed that SP1 bonded to rs10815225 G-allelic PD-L1 promoter instead of C-allelic. Upregulation and knockdown of SP1 resulted in elevation and attenuation of PD-L1 in SGC-7901 cells, respectively. The chromatin immunoprecipitation results further confirmed the binding of SP1 to the promoter of PD-L1. Additionally, rs10815225 was found to be in disequilibrium with a functional polymorphism rs4143815 in the PD-L1 3'-UTR, and the haplotypes of these two polymorphisms were also markedly related to gastric cancer risk. These results revealed a novel mechanism underlying genetic polymorphisms influencing PD-L1 expression modify gastric cancer susceptibility.

A genetic variant of miR-148a binding site in the SCRN1 3'-UTR is associated with susceptibility and prognosis of gastric cancer.

PubMed

Song, Peng; Zhu, Haixia; Zhang, Dong; Chu, Haiyan; Wu, Dongmei; Kang, Meiyun; Wang, Meilin; Gong, Weida; Zhou, Jianwei; Zhang, Zhengdong; Zhao, Qinghong

2014-11-17

Single nucleotide polymorphisms (SNPs) in the 3'-untranslated regions targeted by putative mircoRNA can change its binding strength, affecting the susceptibility and prognosis of cancer. We aimed to investigate the associations between SNPs within miR-148a binding sites and gastric cancer (GC) risk and prognosis. Using bioinformatics tools, we selected two SNPs (SCRN1 rs6976789 and PDYN rs2235749) located in miR-148a target sites. We genotyped the two SNPs in a case-control study comprising 753 GC patients and 949 cancer-free subjects. We found a significantly increased risk of GC associated with the SCRN1 rs6976789 C>T polymorphism [adjusted OR = 1.25, 95% confidence interval (CI) = 1.02-1.53; CT/TT vs. CC]. However, no significant association was found between the PDYN rs2235749 and GC risk in all genetic models. Furthermore, we evaluated whether SCRN1 rs6976789 affected the survival of GC patients. Results showed that individuals with SCRN1 rs6976789 TT genotype had poorer overall survival compared with those carried CC/CT genotypes in intestinal-type GC (adjusted HR = 2.47, 95% CI = 1.21-5.05). Luciferase report assay showed that the rs6976789 variant T allele influenced the binding ability of miR-148a. Our results suggested that the SCRN1 rs6976789 polymorphism may play an important role in the GC development and progression.

Association study between XRCC1 gene polymorphisms and sporadic amyotrophic lateral sclerosis.

PubMed

Coppedè, Fabio; Migheli, Francesca; Lo Gerfo, Annalisa; Fabbrizi, Maria Rita; Carlesi, Cecilia; Mancuso, Michelangelo; Corti, Stefania; Mezzina, Nicoletta; del Bo, Roberto; Comi, Giacomo P; Siciliano, Gabriele; Migliore, Lucia

2010-01-01

The aim of the present study was to investigate the possible contribution of three common functional polymorphisms in the DNA repair protein X-ray repair cross-complementing group 1 (XRCC1), namely Arg194Trp (rs1799782), Arg280His (rs25489) and Arg399Gln (rs25487), to sporadic amyotrophic lateral sclerosis (SALS). We genotyped 206 Italian SALS patients and 203 matched controls for XRCC1 Arg194Trp, Arg280His and Arg399Gln polymorphisms by means of PCR/RFLP technique, searching for association between any of the studied polymorphisms and disease risk, age and site of onset. We observed a statistically significant difference in XRCC1 Gln399 allele frequencies between SALS cases and controls (0.39/0.28; p=0.001). The present study suggests that the XRCC1 Arg399Gln polymorphism might contribute to SALS risk.

Candidate gene study of genetic thrombophilic polymorphisms in pre-eclampsia and recurrent pregnancy loss in Sinhalese women.

PubMed

Dissanayake, Vajira H W; Sirisena, Nirmala D; Weerasekera, Lakshini Y; Gammulla, Chumithri G; Seneviratne, Harshalal R; Jayasekara, Rohan W

2012-09-01

Genetic thrombophilias are known to contribute to adverse pregnancy outcomes. Studies in Western populations show that 5, 10-methylenetetrahydrofolate reductase (MTHFR) 677C>T and Factor V (F5) 1691G>A (Leiden) polymorphisms are commonly associated with pre-eclampsia and recurrent spontaneous pregnancy loss. The objective of this study was to investigate the association of MTHFR 677C>T (rs1801133); 1298A>C (rs1801131) and F5 1691G>A (rs6025); 4070A>G (rs1800595) polymorphisms with pre-eclampsia and recurrent pregnancy loss among Sinhalese women in Sri Lanka. Genotype and allele frequencies at each polymorphic site in the MTHFR and F5 genes and the haplotypes defined by them were determined in 175 Sinhalese women with pre-eclampsia, 171 normotensive controls, 200 Sinhalese women with two or more recurrent pregnancy losses and 200 controls with two or more living children and no pregnancy losses. Genotyping was done by polymerase chain reaction/restriction fragment length polymorphism. Odds ratios and χ(2) -testing were performed to compare genotype/haplotype frequencies at each polymorphic site for both cases and controls. The genotype frequencies at each polymorphic site in the MTHFR 677C>T; 1298A>C; F5 1691G>A and 4070A>G genes and the haplotypes defined by them were not significantly associated with either pre-eclampsia or recurrent pregnancy loss. There was no significant association of genetic thrombophilia with either early or late pregnancy losses. The MTHFR and F5 polymorphisms and the haplotypes defined by them were not significantly associated with either pre-eclampsia or recurrent pregnancy loss in this group of Sinhalese women. © 2012 The Authors. Journal of Obstetrics and Gynaecology Research © 2012 Japan Society of Obstetrics and Gynecology.

SdsA polymorph isolation and improvement of their crystal quality using nonconventional crystallization techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

De la Mora, Eugenio; Flores-Hernández, Edith; Jakoncic, Jean

SdsA, a sodium dodecyl sulfate hydrolase, from Pseudomonas aeruginosa was crystallized in three different crystal polymorphs and their three-dimensional structure was determined. The different polymorphs present different crystal packing habits. One of the polymorphs suggests the existence of a tetramer, an oligomeric state not observed previously, while the crystal packing of the remaining two polymorphs obstructs the active site entrance but stabilizes flexible regions of the protein. Nonconventional crystallization methods that minimize convection, such as counterdiffusion in polyvinyl alcohol gel coupled with the influence of a 500 MHz (10.2 T) magnetic field, were necessary to isolate the poorest diffracting polymorphmore » and increase its internal order to determine its structure by X-ray diffraction. In conclusion, the results obtained show the effectiveness of nonconventional crystallographic methods to isolate different crystal polymorphs.« less

Genetic Polymorphism of SUMO-Specific Cysteine Proteases - SENP1 and SENP2 in Breast Cancer.

PubMed

Mirecka, Alicja; Morawiec, Zbigniew; Wozniak, Katarzyna

2016-10-01

SENP proteases take part in post-translational modification of proteins known as sumoylation. They catalyze three distinct processes during sumoylation: processing of SUMO protein, deconjugation of SUMO from the target protein, and chain editing which mentions to the dismantling of SUMO chain. Many proteins that are involved in the basic processes of cells, such as regulation of transcription, DNA repair or cell cycle control, are sumoylated. The aim of these studies was to investigate an association between polymorphic variants (SNPs) of the SENP1 gene (c.1691 + 36C > T, rs12297820) and SENP2 gene (c.902C > A, p.Thr301Lys, rs6762208) and a risk of breast cancer occurrence. We performed a case-control study in 324 breast cancer cases and 335 controls using PCR-RLFP. In the case of the SENP1 gene polymorphism we did not find any association between this polymorphism and breast cancer risk. In the case of SENP2 gene polymorphism we observed higher risk of breast cancer for carriers of the A allele (OR =1.33; 95 % CI 1.04-1.69). Our analysis also showed the genotype C/C (OR =0.67, 95 % CI 0.48-0.93) and the allele C (OR =0.75, 95 % CI 0.59-0.69) of this polymorphism decrease a risk of breast cancer. We also checked the distribution of genotypes and frequency of alleles of the SENP1 and SENP2 genes polymorphisms in groups of patients with different hormone receptor status, patients with positive and negative lymph node status and patients with different tumor grade. Odds ratio analysis showed a higher risk of metastases in women with the genotype C/C (OR =2.07, 95 % CI 1.06-4.05) and allele C (OR =2.10 95 % CI 1.10-4.01) of the c.1691 + 36C > T SENP1 gene polymorphism. Moreover, we observed reduced risk in women with the allele T (OR =0.48, 95 % CI 0.25-0.91) in this polymorphic site. In the case of SENP2 gene polymorphism we observed that the A/A genotype correlated with the lack of estrogen receptor (OR =1.94, 95 % CI 1.04-3.62). Our results suggest

Chloroplast DNA polymorphism and evolutional relationships between Asian cultivated rice (Oryza sativa) and its wild relatives (O. rufipogon).

PubMed

Li, W J; Zhang, B; Huang, G W; Kang, G P; Liang, M Z; Chen, L B

2012-12-17

We analyzed chloroplast DNA (cpDNA) polymorphism and phylogenic relationships between 6 typical indica rice, 4 japonica rice, 8 javanica rice, and 12 Asian common wild rice (Oryza rufipogon) strains collected from different latitudes in China by comparing polymorphism at 9 highly variable regions. One hundred and forty-four polymorphic bases were detected. The O. rufipogon samples had 117 polymorphic bases, showing rich genetic diversity. One hundred and thirty-one bases at 13 sites were identified with indica/japonica characteristics; they showed differences between the indica and japonica subspecies at these sites. The javanica strains and japonica shared similar bases at these 131 polymorphic sites, suggesting that javanica is closely related to japonica. On the basis of length analyses of the open reading frame (ORF)100 and (ORF)29-tRNA-Cys(GCA) (TrnC(GCA)) fragments, the O. rufipogon strains were classified into indica/japonica subgroups, which was consistent with the results of the phylogenic tree assay based on concatenated datasets. These results indicated that differences in indica and japonica also exist in the cpDNA genome of the O. rufipogon strains. However, these differences demonstrated a certain degree of primitiveness and incompleteness, as an O. rufipogon line may show different indica/ japonica attributes at different sites. Consequently, O. rufipogon cannot be simply classified into the indica/japonica types as O. sativa. Our data support the hypothesis that Asian cultivated rice, O. indica and O. japonica, separately evolved from Asian common wild rice (O. rufipogon) strains, which have different indica-japonica differentiation trends.

Association of I-FABP gene polymorphism and the risk of coronary heart disease.

PubMed

Yuan, Dong; Yu, Changqing; Zeng, Chunyu

2015-01-01

The study aimed to investigate the association between polymorphism of I-FABP gene and coronary heart disease (CHD). 225 patients with CHD were randomly recruited into the case group, and 196 healthy elderly volunteers were recruited from Medical Examination Center of our hospital as control. General clinical data were collected and plasma TC, TG, LDL-C, HDL-C levels were measured. Besides, polymerase chain reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) technology were used to detect the polymorphism of Hha-I enzyme cleavage sites in I-FABP gene in the study population. Hha-I cleavage sites occurred at codon 54 in exon in the coding sequencing of I-FABP gene in all participants. After cleavage with Hha-I enzyme, the genotypes were identified as wild-type A/A, heterozygous mutant A/T and homozygous mutant T/T. In case group, A/T and T/T genetic carriers had significantly higher levels of TC, TG and LDL-C than A/A carriers (P<0.05). However, in control group, similar differences were not observed (P>0.05). BMI, dietary habits and I-FABP alleles were independent risk factors of CHD. The polymorphism of I-FABP gene existed in the study population. And this genetic variation had influence on lipid metabolism, which was associated with the risk of developing CHD. I-FABP gene polymorphism may contribute to the increased genetic susceptibility to CHD.

Electron petrography of silica polymorphs associated with pseudotachylite, Vredefort structure, South Africa

NASA Technical Reports Server (NTRS)

White, J. C.

1992-01-01

High-pressure silica polymorphs (coesite and stishovite) were described from the Vredefort structure in association with pseudotachylite veinlets. In addition to the fundamental significance of the polymorphs to genetic interpretations of the structure, it was additionally argued that the type of pseudotachylite with which they occur forms during the compressional phase of the shock process, while the larger, classic pseudotachylite occurrences are barren of polymorphs and formed during passage of the rarefaction wave. This identification of temporal relationships among transient shock features at a regional scale is similar to observations from the Manicouagan structure, Quebec, where texturally distinct diaplectic plagioclase glasses formed during both compressional and decompressional phases of the shock process. The clarification of such relationships impinges directly on interpretations of natural shock processes and the identification of high probability targets for polymorph searches. Detailed analytical scanning (SEM) and transmission electron microscopy (TEM) were utilized to further establish the nature of both the pseudotachylite and the silica polymorph occurrences in the Vredefort rocks. The results of this investigation are discussed.

HOTAIR gene polymorphisms contribute to increased neuroblastoma susceptibility in Chinese children.

PubMed

Yang, Xu; He, Jing; Chang, Yitian; Luo, Annie; Luo, Ailing; Zhang, Jiao; Zhang, Ruizhong; Xia, Huimin; Xu, Ling

2018-06-15

Neuroblastoma is the most frequently diagnosed extracranial solid tumor in children. Previous studies have shown that single-nucleotide polymorphisms in some genes are associated with the risk of multiple cancers, including neuroblastoma. Although Hox transcript antisense intergenic RNA (HOTAIR) gene polymorphisms have been investigated in a variety of cancers, to the authors' knowledge the relationships between HOTAIR gene polymorphisms and neuroblastoma susceptibility have not been reported to date. The objective of the current study was to evaluate the correlation between HOTAIR gene polymorphisms and neuroblastoma risk in Chinese children. The authors genotyped 6 polymorphisms (rs920778 A>G, rs12826786 C>T, rs4759314 A>G, rs7958904 G>C, rs874945 C>T, and rs1899663 C>A) of the HOTAIR gene in 2 Chinese populations including 393 neuroblastoma cases and 812 healthy controls. The strength of the associations was evaluated using odds ratios and 95% confidence intervals. Further stratification analyses were conducted to explore the association between the HOTAIR gene polymorphisms rs12826786 C>T, rs874945 C>T, and rs1899663 C>A with neuroblastoma susceptibility in terms of age, sex, clinical stage of disease, and sites of origin. The authors found that the rs12826786 C>T (P =.013), rs874945 C>T (P =.020), and rs1899663 C>A (P =.029) polymorphisms were significantly associated with increased neuroblastoma risk. In stratification analyses, these associations were more predominant in females and among patients with tumor in the retroperitoneal region or mediastinum. The remaining 3 polymorphisms were not found to be related to neuroblastoma susceptibility. The results of the current study verified that HOTAIR gene polymorphisms are associated with increased neuroblastoma risk and suggest that HOTAIR gene polymorphisms might be a potential biomarker for neuroblastoma susceptibility. Cancer 2018;124:2599-606. © 2018 American Cancer Society. © 2018 American Cancer Society.

Restriction fragment length polymorphism of the human c-fms gene.

PubMed Central

Xu, D Q; Guilhot, S; Galibert, F

1985-01-01

By using blot hybridization with a v-fms probe, a polymorphism for EcoRI, HindIII, and BamHI restriction endonuclease sites associated with the human c-fms locus was observed in a random adult population. This restriction fragment length polymorphism can be explained on the basis of the existence of two alleles, a and b, and is due to a short (congruent to 500 base pairs) deletion characteristic of allele a. The distribution in the analyzed population (48 unrelated individuals) is 23% heterozygotes ab, 75% homozygotes bb, and 2% homozygotes aa. Though the inheritance of this polymorphism follows a Mendelian pattern, the children from couples ab X bb are of the following genotype: 74% ab and 26% bb. These deviations from the expected frequencies of 50% suggest a selective pressure in favor of heterozygotes. Images PMID:2986142

Aryl-substituted aminobenzimidazoles targeting the hepatitis C virus internal ribosome entry site

PubMed Central

Ding, Kejia; Wang, Annie; Boerneke, Mark A.; Dibrov, Sergey M.; Hermann, Thomas

2014-01-01

We describe the exploration of N1-aryl-substituted benzimidazoles as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The design of the compounds was guided by the co-crystal structure of a benzimidazole viral translation inhibitor in complex with the RNA target. Structure-binding activity relationships of aryl-substituted benzimidazole ligands were established that were consistent with the crystal structure of the translation inhibitor complex. PMID:24856063

Visualizing multiple inter-organelle contact sites using the organelle-targeted split-GFP system.

PubMed

Kakimoto, Yuriko; Tashiro, Shinya; Kojima, Rieko; Morozumi, Yuki; Endo, Toshiya; Tamura, Yasushi

2018-04-18

Functional integrity of eukaryotic organelles relies on direct physical contacts between distinct organelles. However, the entity of organelle-tethering factors is not well understood due to lack of means to analyze inter-organelle interactions in living cells. Here we evaluate the split-GFP system for visualizing organelle contact sites in vivo and show its advantages and disadvantages. We observed punctate GFP signals from the split-GFP fragments targeted to any pairs of organelles among the ER, mitochondria, peroxisomes, vacuole and lipid droplets in yeast cells, which suggests that these organelles form contact sites with multiple organelles simultaneously although it is difficult to rule out the possibilities that these organelle contacts sites are artificially formed by the irreversible associations of the split-GFP probes. Importantly, split-GFP signals in the overlapped regions of the ER and mitochondria were mainly co-localized with ERMES, an authentic ER-mitochondria tethering structure, suggesting that split-GFP assembly depends on the preexisting inter-organelle contact sites. We also confirmed that the split-GFP system can be applied to detection of the ER-mitochondria contact sites in HeLa cells. We thus propose that the split-GFP system is a potential tool to observe and analyze inter-organelle contact sites in living yeast and mammalian cells.

Binding site and affinity prediction of general anesthetics to protein targets using docking.

PubMed

Liu, Renyu; Perez-Aguilar, Jose Manuel; Liang, David; Saven, Jeffery G

2012-05-01

The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explored whether a computational method, AutoDock, could serve as such a tool. High-resolution crystal data of water-soluble proteins (cytochrome C, apoferritin, and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus [GLIC]) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (http://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants were compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent cocrystallization data. Docking calculations for 6 general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known 50% effective concentration (EC(50)) values were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC(50) values and octanol/water partition coefficients for the 6 general anesthetics. All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (P = 0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site

Binding Site and Affinity Prediction of General Anesthetics to Protein Targets Using Docking

PubMed Central

Liu, Renyu; Perez-Aguilar, Jose Manuel; Liang, David; Saven, Jeffery G.

2012-01-01

Background The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explore whether a computational method, AutoDock, could serve as such a tool. Methods High-resolution crystal data of water soluble proteins (cytochrome C, apoferritin and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus, GLIC) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (https://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants are compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent co-crystallization data. Docking calculations for six general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known EC50 were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC50s and octanol/water partition coefficients for the six general anesthetics. Results All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (p=0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site located in the

The Role of Dopamine in Anticipatory Pursuit Eye Movements: Insights from Genetic Polymorphisms in Healthy Adults

PubMed Central

Hennig, Jürgen

2016-01-01

Abstract There is a long history of eye movement research in patients with psychiatric diseases for which dysfunctions of neurotransmission are considered to be the major pathologic mechanism. However, neuromodulation of oculomotor control is still hardly understood. We aimed to investigate in particular the impact of dopamine on smooth pursuit eye movements. Systematic variability in dopaminergic transmission due to genetic polymorphisms in healthy subjects offers a noninvasive opportunity to determine functional associations. We measured smooth pursuit in 110 healthy subjects genotyped for two well-documented polymorphisms, the COMT Val158Met polymorphism and the SLC6A3 3′-UTR-VNTR polymorphism. Pursuit paradigms were chosen to particularly assess the ability of the pursuit system to initiate tracking when target motion onset is blanked, reflecting the impact of extraretinal signals. In contrast, when following a fully visible target sensory, retinal signals are available. Our results highlight the crucial functional role of dopamine for anticipatory, but not for sensory-driven, pursuit processes. We found the COMT Val158Met polymorphism specifically associated with anticipatory pursuit parameters, emphasizing the dominant impact of prefrontal dopamine activity on complex oculomotor control. In contrast, modulation of striatal dopamine activity by the SLC6A3 3′-UTR-VNTR polymorphism had no significant functional effect. Though often neglected so far, individual differences in healthy subjects provide a promising approach to uncovering functional mechanisms and can be used as a bridge to understanding deficits in patients. PMID:28101524

The Role of Dopamine in Anticipatory Pursuit Eye Movements: Insights from Genetic Polymorphisms in Healthy Adults.

PubMed

Billino, Jutta; Hennig, Jürgen; Gegenfurtner, Karl R

2016-01-01

There is a long history of eye movement research in patients with psychiatric diseases for which dysfunctions of neurotransmission are considered to be the major pathologic mechanism. However, neuromodulation of oculomotor control is still hardly understood. We aimed to investigate in particular the impact of dopamine on smooth pursuit eye movements. Systematic variability in dopaminergic transmission due to genetic polymorphisms in healthy subjects offers a noninvasive opportunity to determine functional associations. We measured smooth pursuit in 110 healthy subjects genotyped for two well-documented polymorphisms, the COMT Val 158 Met polymorphism and the SLC6A3 3'-UTR-VNTR polymorphism. Pursuit paradigms were chosen to particularly assess the ability of the pursuit system to initiate tracking when target motion onset is blanked, reflecting the impact of extraretinal signals. In contrast, when following a fully visible target sensory, retinal signals are available. Our results highlight the crucial functional role of dopamine for anticipatory, but not for sensory-driven, pursuit processes. We found the COMT Val 158 Met polymorphism specifically associated with anticipatory pursuit parameters, emphasizing the dominant impact of prefrontal dopamine activity on complex oculomotor control. In contrast, modulation of striatal dopamine activity by the SLC6A3 3'-UTR-VNTR polymorphism had no significant functional effect. Though often neglected so far, individual differences in healthy subjects provide a promising approach to uncovering functional mechanisms and can be used as a bridge to understanding deficits in patients.

LuciPHOr: Algorithm for Phosphorylation Site Localization with False Localization Rate Estimation Using Modified Target-Decoy Approach*

PubMed Central

Fermin, Damian; Walmsley, Scott J.; Gingras, Anne-Claude; Choi, Hyungwon; Nesvizhskii, Alexey I.

2013-01-01

The localization of phosphorylation sites in peptide sequences is a challenging problem in large-scale phosphoproteomics analysis. The intense neutral loss peaks and the coexistence of multiple serine/threonine and/or tyrosine residues are limiting factors for objectively scoring site patterns across thousands of peptides. Various computational approaches for phosphorylation site localization have been proposed, including Ascore, Mascot Delta score, and ProteinProspector, yet few address direct estimation of the false localization rate (FLR) in each experiment. Here we propose LuciPHOr, a modified target-decoy-based approach that uses mass accuracy and peak intensities for site localization scoring and FLR estimation. Accurate estimation of the FLR is a difficult task at the individual-site level because the degree of uncertainty in localization varies significantly across different peptides. LuciPHOr carries out simultaneous localization on all candidate sites in each peptide and estimates the FLR based on the target-decoy framework, where decoy phosphopeptides generated by placing artificial phosphorylation(s) on non-candidate residues compete with the non-decoy phosphopeptides. LuciPHOr also reports approximate site-level confidence scores for all candidate sites as a means to localize additional sites from multiphosphorylated peptides in which localization can be partially achieved. Unlike the existing tools, LuciPHOr is compatible with any search engine output processed through the Trans-Proteomic Pipeline. We evaluated the performance of LuciPHOr in terms of the sensitivity and accuracy of FLR estimates using two synthetic phosphopeptide libraries and a phosphoproteomic dataset generated from complex mouse brain samples. PMID:23918812

Site-Specific Targeting of Platelet-Rich Plasma via Superparamagnetic Nanoparticles

PubMed Central

Talaie, Tara; Pratt, Stephen J.P.; Vanegas, Camilo; Xu, Su; Henn, R. Frank; Yarowsky, Paul; Lovering, Richard M.

2015-01-01

Background: Muscle strains are one of the most common injuries treated by physicians. Standard conservative therapy for acute muscle strains usually involves short-term rest, ice, and nonsteroidal anti-inflammatory medications, but there is no clear consensus regarding treatments to accelerate recovery. Recently, clinical use of platelet-rich plasma (PRP) has gained momentum as an option for therapy and is appealing for many reasons, most notably because it provides growth factors in physiological proportions and it is autologous, safe, easily accessible, and potentially beneficial. Local delivery of PRP to injured muscles can hasten recovery of function. However, specific targeting of PRP to sites of tissue damage in vivo is a major challenge that can limit its efficacy. Hypothesis: Location of PRP delivery can be monitored and controlled in vivo with noninvasive tools. Study Design: Controlled laboratory study. Methods: Superparamagnetic iron oxide nanoparticles (SPIONs) can be visualized by both magnetic resonance imaging (MRI) (in vivo) and fluorescence microscopy (after tissue harvesting). PRP was labeled with SPIONs and administered by intramuscular injections of SPION-containing platelets. MRI was used to monitor the ability to manipulate and retain the location of PRP in vivo by placement of an external magnet. Platelets were isolated from whole blood and incubated with SPIONs. Following SPION incubation with PRP, a magnetic field was used to manipulate platelet location in culture dishes. In vivo, the tibialis anterior (TA) muscles of anesthetized Sprague-Dawley rats were injected with SPION-containing platelets, and MRI was used to track platelet position with and without a magnet worn over the TA muscles for 4 days. Results: The method used to isolate PRP yielded a high concentration (almost 4-fold increase) of platelets. In vitro experiments showed that the platelets successfully took up SPIONs and then rapidly responded to an applied magnetic field

[Correlation analysis between single nucleotide polymorphism of FGF5 gene and wool yield in rabbits].

PubMed

Li, Chun-Xiao; Jiang, Mei-Shan; Chen, Shi-Yi; Lai, Song-Jia

2008-07-01

Single nucleotide polymorphism (SNP) in exon 1 and 3 of fibroblast growth factor (FGF5) gene was studied by DNA sequencing in Yingjing angora rabbit, Tianfu black rabbit and California rabbit. A frameshift mutation (TCT insert) at base position 217 (site A) of exon 1 and a T/C missense mutation at base position 59 (site B) of exon 3 were found in Yingjing angora rabbit with a high frequency; a T/C same-sense mutation at base position 3 (site C) of exon 3 was found with similar frequency in three rabbit breeds. Least square analysis showed that different genotypes had no significant association with wool yield in site A, and had high significant association with wool yield in site B (P<0.01) and significant association with wool yield in site C (P<0.05). It was concluded from the results that FGF5 gene could be the potential major gene affecting wool yield or link with the major gene, and polymorphic loci B and C may be used as molecular markers for im-proving wool yield in angora rabbits.

Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

PubMed Central

Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

2016-01-01

This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

Cooperation between a hierarchical set of recruitment sites targets the X chromosome for dosage compensation

PubMed Central

Albritton, Sarah Elizabeth; Kranz, Anna-Lena; Winterkorn, Lara Heermans; Street, Lena Annika; Ercan, Sevinc

2017-01-01

In many organisms, it remains unclear how X chromosomes are specified for dosage compensation, since DNA sequence motifs shown to be important for dosage compensation complex (DCC) recruitment are themselves not X-specific. Here, we addressed this problem in C. elegans. We found that the DCC recruiter, SDC-2, is required to maintain open chromatin at a small number of primary DCC recruitment sites, whose sequence and genomic context are X-specific. Along the X, primary recruitment sites are interspersed with secondary sites, whose function is X-dependent. A secondary site can ectopically recruit the DCC when additional recruitment sites are inserted either in tandem or at a distance (>30 kb). Deletion of a recruitment site on the X results in reduced DCC binding across several megabases surrounded by topologically associating domain (TAD) boundaries. Our work elucidates that hierarchy and long-distance cooperativity between gene-regulatory elements target a single chromosome for regulation. DOI: http://dx.doi.org/10.7554/eLife.23645.001 PMID:28562241

Cloning of polymorphisms (COP): enrichment of polymorphic sequences from complex genomes

PubMed Central

Li, Jingfeng; Wang, Fuli; Zabarovska, Veronika; Wahlestedt, Claes; Zabarovsky, Eugene R.

2000-01-01

Here we describe a new procedure (cloning of polymorphisms, COP) for enrichment of single nucleotide polymorphisms (SNPs) that represent restriction fragment length polymorphisms (RFLPs). COP would be applicable to the isolation of SNPs from particular regions of the genome, e.g. CpG islands, chromosomal bands, YACs or PAC contigs. A combination of digestion with restriction enzymes, treatment with uracil-DNA glycosylase and mung bean nuclease, PCR amplification and purification with streptavidin magnetic beads was used to isolate polymorphic sequences from the genomes of two human samples. After only two cycles of enrichment, 80% of the isolated clones were found to contain RFLPs. A simple method for the PCR detection of these polymorphisms was also developed. PMID:10606669

Sparse networks of directly coupled, polymorphic, and functional side chains in allosteric proteins.

PubMed

Soltan Ghoraie, Laleh; Burkowski, Forbes; Zhu, Mu

2015-03-01

Recent studies have highlighted the role of coupled side-chain fluctuations alone in the allosteric behavior of proteins. Moreover, examination of X-ray crystallography data has recently revealed new information about the prevalence of alternate side-chain conformations (conformational polymorphism), and attempts have been made to uncover the hidden alternate conformations from X-ray data. Hence, new computational approaches are required that consider the polymorphic nature of the side chains, and incorporate the effects of this phenomenon in the study of information transmission and functional interactions of residues in a molecule. These studies can provide a more accurate understanding of the allosteric behavior. In this article, we first present a novel approach to generate an ensemble of conformations and an efficient computational method to extract direct couplings of side chains in allosteric proteins, and provide sparse network representations of the couplings. We take the side-chain conformational polymorphism into account, and show that by studying the intrinsic dynamics of an inactive structure, we are able to construct a network of functionally crucial residues. Second, we show that the proposed method is capable of providing a magnified view of the coupled and conformationally polymorphic residues. This model reveals couplings between the alternate conformations of a coupled residue pair. To the best of our knowledge, this is the first computational method for extracting networks of side chains' alternate conformations. Such networks help in providing a detailed image of side-chain dynamics in functionally important and conformationally polymorphic sites, such as binding and/or allosteric sites. © 2014 Wiley Periodicals, Inc.

Examination of CRISPR/Cas9 design tools and the effect of target site accessibility on Cas9 activity.

PubMed

Lee, Ciaran M; Davis, Timothy H; Bao, Gang

2018-04-01

What is the topic of this review? In this review, we analyse the performance of recently described tools for CRISPR/Cas9 guide RNA design, in particular, design tools that predict CRISPR/Cas9 activity. What advances does it highlight? Recently, many tools designed to predict CRISPR/Cas9 activity have been reported. However, the majority of these tools lack experimental validation. Our analyses indicate that these tools have poor predictive power. Our preliminary results suggest that target site accessibility should be considered in order to develop better guide RNA design tools with improved predictive power. The recent adaptation of the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system for targeted genome engineering has led to its widespread application in many fields worldwide. In order to gain a better understanding of the design rules of CRISPR/Cas9 systems, several groups have carried out large library-based screens leading to some insight into sequence preferences among highly active target sites. To facilitate CRISPR/Cas9 design, these studies have spawned a plethora of guide RNA (gRNA) design tools with algorithms based solely on direct or indirect sequence features. Here, we demonstrate that the predictive power of these tools is poor, suggesting that sequence features alone cannot accurately inform the cutting efficiency of a particular CRISPR/Cas9 gRNA design. Furthermore, we demonstrate that DNA target site accessibility influences the activity of CRISPR/Cas9. With further optimization, we hypothesize that it will be possible to increase the predictive power of gRNA design tools by including both sequence and target site accessibility metrics. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability

PubMed Central

Kim, Min Jung; Chia, Ian V.; Costantini, Frank

2008-01-01

Axin is a scaffold protein for the β-catenin destruction complex, and a negative regulator of canonical Wnt signaling. Previous studies implicated the six C-terminal amino acids (C6 motif) in the ability of Axin to activate c-Jun N-terminal kinase, and identified them as a SUMOylation target. Deletion of the C6 motif of mouse Axin in vivo reduced the steady-state protein level, which caused embryonic lethality. Here, we report that this deletion (Axin-ΔC6) causes a reduced half-life in mouse embryonic fibroblasts and an increased susceptibility to ubiquitination in HEK 293T cells. We confirmed the C6 motif as a SUMOylation target in vitro, and found that mutating the C-terminal SUMOylation target residues increased the susceptibility of Axin to polyubiquitination and reduced its steady-state level. Heterologous SUMOylation target sites could replace C6 in providing this protective effect. These findings suggest that SUMOylation of the C6 motif may prevent polyubiquitination, thus increasing the stability of Axin. Although C6 deletion also caused increased association of Axin with Dvl-1, this interaction was not altered by mutating the lysine residues in C6, nor could heterologous SUMOylation motifs replace the C6 motif in this assay. Therefore, some other specific property of the C6 motif seems to reduce the interaction of Axin with Dvl-1.—Kim, M. J., Chia, I. V., Costantini, F. SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability. PMID:18632848

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

PubMed Central

Agúndez, Leticia; González-Prieto, Coral; Machón, Cristina; Llosa, Matxalen

2012-01-01

Background Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering. Methodology/Principal Findings We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome. Conclusions/Significance The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells. PMID:22292089

Association between DNA methyltransferase gene polymorphism and Parkinson's disease.

PubMed

Pezzi, Julio Carlos; de Bem, Cintia Monique Boschmann Ens; da Rocha, Tatiane Jacobsen; Schumacher-Schuh, Artur F; Chaves, Marcia Lorena Fagundes; Rieder, Carlos Roberto; Hutz, Mara H; Fiegenbaum, Marilu; Camozzato, Ana Luiza

2017-02-03

Parkinson's disease (PD) is a common and complex neurodegenerative disorder, the second most prevalent, only behind Alzheimer's disease. Recent studies suggest that environmental factors may contribute for neurodegeneration through induction of epigenetic modifications, such as DNA methylation, that is carried out by enzymes, such as DNMT1 and DNMT3B. This present study targeted to investigate the association among DNMT1 and DNMT3B polymorphisms with PD. Five hundred and twenty-two participants (214 PD patients following UK Brain Bank criteria and 308 healthy individuals) were evaluated. DNA was obtained from whole blood and genotypes were detected by an allelic discrimination assay using TaqMan ® MGB probes on a real-time PCR system. The polymorphisms studied were rs2162560 and rs759920 (DNMT1) and rs2424913, rs998382 and rs2424932 (DNMT3B). Was found association between DNMT3B rs2424913 in T allele carriers with PD. The presence of the T allele was associated with PD (OR=1.80, 95% CI 1.16-2.81, p=0.009). No significant difference was observed for others DNMT3B SNPs. Also, no association between PD and the control group were observed for DNMT1 polymorphisms. This is the first study addressing an association between DNMT3B polymorphism and PD. The polymorphism may play a role in the pathogenesis of PD. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

mRNA-engineered mesenchymal stem cells for targeted delivery of interleukin-10 to sites of inflammation.

PubMed

Levy, Oren; Zhao, Weian; Mortensen, Luke J; Leblanc, Sarah; Tsang, Kyle; Fu, Moyu; Phillips, Joseph A; Sagar, Vinay; Anandakumaran, Priya; Ngai, Jessica; Cui, Cheryl H; Eimon, Peter; Angel, Matthew; Lin, Charles P; Yanik, Mehmet Fatih; Karp, Jeffrey M

2013-10-03

Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapy to treat several diseases and are compelling to consider as vehicles for delivery of biological agents. However, MSCs appear to act through a seemingly limited "hit-and-run" mode to quickly exert their therapeutic impact, mediated by several mechanisms, including a potent immunomodulatory secretome. Furthermore, MSC immunomodulatory properties are highly variable and the secretome composition following infusion is uncertain. To determine whether a transiently controlled antiinflammatory MSC secretome could be achieved at target sites of inflammation, we harnessed mRNA transfection to generate MSCs that simultaneously express functional rolling machinery (P-selectin glycoprotein ligand-1 [PSGL-1] and Sialyl-Lewis(x) [SLeX]) to rapidly target inflamed tissues and that express the potent immunosuppressive cytokine interleukin-10 (IL-10), which is not inherently produced by MSCs. Indeed, triple-transfected PSGL-1/SLeX/IL-10 MSCs transiently increased levels of IL-10 in the inflamed ear and showed a superior antiinflammatory effect in vivo, significantly reducing local inflammation following systemic administration. This was dependent on rapid localization of MSCs to the inflamed site. Overall, this study demonstrates that despite the rapid clearance of MSCs in vivo, engineered MSCs can be harnessed via a "hit-and-run" action for the targeted delivery of potent immunomodulatory factors to treat distant sites of inflammation.

siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs.

PubMed

Vickers, Timothy A; Crooke, Stanley T

2012-07-01

While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

Polymorphous computing fabric

DOEpatents

Wolinski, Christophe Czeslaw [Los Alamos, NM; Gokhale, Maya B [Los Alamos, NM; McCabe, Kevin Peter [Los Alamos, NM

2011-01-18

Fabric-based computing systems and methods are disclosed. A fabric-based computing system can include a polymorphous computing fabric that can be customized on a per application basis and a host processor in communication with said polymorphous computing fabric. The polymorphous computing fabric includes a cellular architecture that can be highly parameterized to enable a customized synthesis of fabric instances for a variety of enhanced application performances thereof. A global memory concept can also be included that provides the host processor random access to all variables and instructions associated with the polymorphous computing fabric.

Regulation of CCL2 expression by an upstream TALE homeodomain protein-binding site that synergizes with the site created by the A-2578G SNP.

PubMed

Page, Stephen H; Wright, Edward K; Gama, Lucio; Clements, Janice E

2011-01-01

CC Chemokine Ligand 2 (CCL2) is a potent chemoattractant produced by macrophages and activated astrocytes during periods of inflammation within the central nervous system. Increased CCL2 expression is correlated with disease progression and severity, as observed in pulmonary tuberculosis, HCV-related liver disease, and HIV-associated dementia. The CCL2 distal promoter contains an A/G polymorphism at position -2578 and the homozygous -2578 G/G genotype is associated with increased CCL2 production and inflammation. However, the mechanisms that contribute to the phenotypic differences in CCL2 expression are poorly understood. We previously demonstrated that the -2578 G polymorphism creates a TALE homeodomain protein binding site (TALE binding site) for PREP1/PBX2 transcription factors. In this study, we identified the presence of an additional TALE binding site 22 bp upstream of the site created by the -2578 G polymorphism and demonstrated the synergistic effects of the two sites on the activation of the CCL2 promoter. Using chromatin immunoprecipitation (ChIP) assays, we demonstrated increased binding of the TALE proteins PREP1 and PBX2 to the -2578 G allele, and binding of IRF1 to both the A and G alleles. The presence of TALE binding sites that form inverted repeats within the -2578 G allele results in increased transcriptional activation of the CCL2 distal promoter while the presence of only the upstream TALE binding site within the -2578 A allele exerts repression of promoter activity.

Fitness cost implications of phiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae)

USDA-ARS?s Scientific Manuscript database

Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understandin...

Targeted Delivery of LXR Agonist Using a Site-Specific Antibody-Drug Conjugate.

PubMed

Lim, Reyna K V; Yu, Shan; Cheng, Bo; Li, Sijia; Kim, Nam-Jung; Cao, Yu; Chi, Victor; Kim, Ji Young; Chatterjee, Arnab K; Schultz, Peter G; Tremblay, Matthew S; Kazane, Stephanie A

2015-11-18

Liver X receptor (LXR) agonists have been explored as potential treatments for atherosclerosis and other diseases based on their ability to induce reverse cholesterol transport and suppress inflammation. However, this therapeutic potential has been hindered by on-target adverse effects in the liver mediated by excessive lipogenesis. Herein, we report a novel site-specific antibody-drug conjugate (ADC) that selectively delivers a LXR agonist to monocytes/macrophages while sparing hepatocytes. The unnatural amino acid para-acetylphenylalanine (pAcF) was site-specifically incorporated into anti-CD11a IgG, which binds the α-chain component of the lymphocyte function-associated antigen 1 (LFA-1) expressed on nearly all monocytes and macrophages. An aminooxy-modified LXR agonist was conjugated to anti-CD11a IgG through a stable, cathepsin B cleavable oxime linkage to afford a chemically defined ADC. The anti-CD11a IgG-LXR agonist ADC induced LXR activation specifically in human THP-1 monocyte/macrophage cells in vitro (EC50-27 nM), but had no significant effect in hepatocytes, indicating that payload delivery is CD11a-mediated. Moreover, the ADC exhibited higher-fold activation compared to a conventional synthetic LXR agonist T0901317 (Tularik) (3-fold). This novel ADC represents a fundamentally different strategy that uses tissue targeting to overcome the limitations of LXR agonists for potential use in treating atherosclerosis.

Analysis of the relationship between interleukin polymorphisms within miRNA-binding regions and alcoholic liver disease.

PubMed

Novo-Veleiro, I; Cieza-Borrella, C; Pastor, I; González-Sarmiento, R; Laso, F-J; Marcos, M

2018-05-01

Alcohol consumption promotes inflammation through the Toll-like receptor 4 (TLR4)/nuclear factor (NF)-?B pathway, leading to organic damage. Some micro-RNA (miRNA) molecules modulate this inflammatory response by downregulating TLR4/NF-?B pathway mediators, like interleukins (ILs). Thus, polymorphisms within IL genes located near miRNA binding sites could modify the risk of ethanol-induced damage. The present study analyzed potential relationships between alcoholism or alcoholic liver disease (ALD) and IL12B 2124 G>T (rs1368439), IL16 5000 C>T (rs1131445), IL1R1 3114 C>T (rs3917328), and NFKB1 3400 A>G (rs4648143) polymorphisms. The study included 301 male alcoholic patients and 156 male healthy volunteers. Polymorphisms were genotyped using TaqMan ® PCR assays for allelic discrimination. Allele and genotype frequencies were compared between groups. Logistic regression analysis was performed to analyze the inheritance model. Analysis of the IL1R1 (rs3917328) polymorphism showed that the proportion of alleleT carriers (CT and TT genotypes) was higher in healthy controls (9.7%) than in alcoholic patients (6.5%; P=.042). However, multivariable logistic regression analyses did not yield a significant result. No differences between groups were found for other analyzed polymorphisms. Our study describes, for the first time, the expected frequencies of certain polymorphisms within miRNA-binding sites in alcoholic patients with and without ALD. Further studies should be developed to clarify the potential relevance of these polymorphisms in alcoholism and ALD development. Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Medicina Interna (SEMI). All rights reserved.

Correlation between the development of calcium oxalate stones and polymorphisms in the fibronectin gene in the Uighur population of the Xinjiang region of China.

PubMed

Murat, M; Aekeper, A; Yuan, L Y; Alim, T; Du, G J; Abdusamat, A; Wu, G W; Aniwer, Y

2015-10-29

Here, we have investigated the correlation between calcium oxalate stone formation and Fn gene polymorphisms in urinary calculi patients among the Uighur population (Xinjiang region). In this case control study, genomic DNA extracted from the peripheral blood of 129 patients with calcium oxalate stones (patient group) and 94 normal people (control group) was used to genotype polymorphisms in the rs6725958, rs10202709, and rs35343655 sites of the Fn gene by polymerase chain reaction-restriction fragment length polymorphism. Subsequently, the association between different genotypes and susceptibility to calcium oxalate stone formation was compared among the patient and control groups. Single nucleotide polymorphisms (SNPs) were detected in the rs6725958, rs10202709, and rs35343655 sites of the Fn gene among the patient and control groups. The genotype distributions of the three loci complied with the Hardy-Weinberg equilibrium. The results of allele frequencies of the patient/control group for polymorphisms in the rs6725958 site of the Fn gene were C = 179 (69.92%)/119 (63.30%) and A = 77 (30.08%)/69 (36.70%), in the rs10202709 site were C = 245 (95.70%)/176 (93.63%) and T = 11 (4.30%)/12 (6.38%), and in the rs35343655 site of the Fn gene were A = 139 (54.30%)/87 (46.28%) and G = 117 (45.70%)/101 (53.72%). We observed no significant differences between the three SNPs and development of calcium oxalate stones. Polymorphisms in rs6725958, rs10202709, and rs35343655 of the Fn gene had no obvious effect on the susceptibility to the development of calcium oxalate stones in the Uighur population, residing in the Xinjiang region of China.

Characterization of natural polymorphic sites of the HIV-1 integrase before the introduction of HIV-1 integrase inhibitors in Germany

PubMed Central

Meixenberger, Karolin; Pouran Yousef, Kaveh; Somogyi, Sybille; Fiedler, Stefan; Bartmeyer, Barbara; von Kleist, Max; Kücherer, Claudia

2014-01-01

Introduction The aim of our study was to analyze the occurrence and evolution of HIV-1 integrase polymorphisms during the HIV-1 epidemic in Germany prior to the introduction of the first integrase inhibitor raltegravir in 2007. Materials and Methods Plasma samples from drug-naïve HIV-1 infected individuals newly diagnosed between 1986 and 2006 were used to determine PCR-based population sequences of the HIV-1 integrase (amino acids 1–278). The HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. We calculated the frequency of amino acids at each position of the HIV-1 integrase in 337 subtype B strains for the time periods 1986–1989, 1991–1994, 1995–1998, 1999–2002, and 2003–2006. Positions were defined as polymorphic if amino acid variation was >1% in any period. Logistic regression was used to identify trends in amino acid variation over time. Resistance-associated mutations were identified according to the IAS 2013 list and the HIVdb, ANRS and GRADE algorithms. Results Overall, 56.8% (158/278) amino acid positions were polymorphic and 15.8% (25/158) of these positions exhibited a significant trend in amino acid variation over time. Proportionately, most polymorphic positions (63.3%, 31/49) were detected in the N-terminal zinc finger domain of the HIV-1 integrase. Motifs and residues essential for HIV-1 integrase activity were little polymorphic, but within the minimal non-specific DNA binding region I220-D270 up to 18.1% amino acid variation was noticed, including four positions with significant amino acid variation over time (S230, D232, D256, A265). No major resistance mutations were identified, and minor resistance mutations were rarely observed without trend over time. E157Q considered by HIVdb, ANRS, and GRADE algorithms was the most frequent resistance-associated polymorphism with an overall prevalence of 2.4%. Conclusions Detailed knowledge of the evolutionary variation of HIV-1 integrase polymorphisms is important to understand

Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies

DTIC Science & Technology

2016-10-01

2016 4 . TITLE AND SUBTITLE 5a. CONTRACT NUMBER Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies 5b. GRANT NUMBER 5c...new biomarker driven trials directly to patients W81XWH-14-2-0159 None listed 20 Table of Contents Page 1. Introduction…………………………………………………………. 4 2...Keywords……………………………………………………………. 4 3. Accomplishments ..……..…………………………………………... 4 4 . Impact…………………………...…………………………………… 8 5. Changes/Problems

Polymorphism of heat shock protein 70-2 and enterocutaneous fistula in Chinese population

PubMed Central

Chen, Jun; Ren, Jian-An; Han, Gang; Gu, Guo-Sheng; Wang, Ge-Fei; Wu, Xiu-Wen; Zhou, Bo; Hu, Dong; Wu, Yin; Zhao, Yun-Zhao; Li, Jie-Shou

2014-01-01

AIM: To investigate whether the heat shock protein 70-2 (HSP70-2) polymorphism is associated with enterocutaneous fistulas in a Chinese population. METHODS: This study included 131 patients with enterocutaneous/enteroatmospheric fistulas. Patients with inflammatory bowel disease or other autoimmune diseases were excluded from this study. All patients with enterocutaneous/enteroatmospheric fistulas were followed up for three months to observe disease recurrence. In addition, a total of 140 healthy controls were also recruited from the Jinling Hospital, matched according to the sex and age of the patient population. Genomic DNA was extracted from peripheral blood from each participant. The HSP70-2 restriction fragment length polymorphism related to the polymorphic PstI site at position 1267 was characterized by polymerase chain reaction (PCR). First PCR amplification was carried out, and then PCR products were digested with PstI restriction enzyme. The DNA lacking the polymorphic PstI site within HSP70-2 generates a product of 1117 bp in size (allele A), whereas the HSP70-2 PstI polymorphism produces two fragments of 936 bp and 181 bp in size (allele B). RESULTS: The frequency of the HSP70-2 PstI polymorphism did not differ between patients and controls; however, the A allele was more predominant in patients with enterocutaneous fistulas than in controls (60.7% vs 51.4%, P = 0.038, OR = 1.425, 95%CI: 1.019-1.994). Sixty-one patients were cured by a definitive operation, drainage operation, or percutaneous drainage while 52 patients were cured by nonsurgical treatment. There was no significant difference in the frequency of the HSP70-2 PstI polymorphism between the patients who had surgery compared to those who did not (P = 0.437, OR = 1.237, 95%CI: 0.723-2.117). Moreover, 11 patients refused any treatment for economic reasons or tumor burden, and 7 patients with enterocutaneous fistulas (5.8%) died during the follow-up period. However, there was no significant

Rivastigmine hydrogen tartrate polymorphs: Solid-state characterisation of transition and polymorphic conversion via milling

NASA Astrophysics Data System (ADS)

Amaro, Maria Inês; Simon, Alice; Cabral, Lúcio Mendes; de Sousa, Valéria Pereira; Healy, Anne Marie

2015-11-01

Rivastigmine (RHT) is an active pharmaceutical ingredient that is used for the treatment of mild to moderately severe dementia in Alzheimer's disease, and is known to present two polymorphic forms and to amorphise upon granulation. To date there is no information in the scientific or patent literature on polymorphic transition and stability. Hence, the aim of the current study was to gain a fundamental understanding of the polymorphic forms by (1) evaluating RHT thermodynamic stability (monotropy or enantiotropy) and (2) investigating the potential for polymorphic transformation upon milling. The two polymorphic and amorphous forms were characterised using X-ray powder diffractometry, thermal analyses, infra-red spectroscopy and water sorption analysis. The polymorphic transition was found to be spontaneous (ΔG0 < 0) and exothermic (ΔH0 < 0), indicative of a monotropic polymorph pair. The kinetic studies showed a fast initial polymorphic transition characterised by a heterogeneous nucleation, followed by a slow crystal growth. Ball milling can be used to promote the polymorphic transition and for the production of RHT amorphous form.

Adaptive Role of Inversion Polymorphism of Drosophila subobscura in Lead Stressed Environment

PubMed Central

Kenig, Bojan; Kurbalija Novičić, Zorana; Patenković, Aleksandra; Stamenković-Radak, Marina; Anđelković, Marko

2015-01-01

Local adaptation to environmental stress at different levels of genetic polymorphism in various plants and animals has been documented through evolution of heavy metal tolerance. We used samples of Drosophila subobscura populations from two differently polluted environments to analyze the change of chromosomal inversion polymorphism as genetic marker during laboratory exposure to lead. Exposure to environmental contamination can affect the genetic content within a particular inversion and produce targets for selection in populations from different environments. The aims were to discover whether the inversion polymorphism is shaped by the local natural environments, and if lead as a selection pressure would cause adaptive divergence of two populations during the multigenerational laboratory experiment. The results showed that populations retain signatures from past contamination events, and that heavy metal pollution can cause adaptive changes in population. Differences in inversion polymorphism between the two populations increased over generations under lead contamination in the laboratory. The inversion polymorphism of population originating from the more polluted natural environment was more stable during the experiment, both under conditions with and without lead. Therefore, results showed that inversion polymorphism as a genetic marker reflects a strong signature of adaptation to the local environment, and that historical demographic events and selection are important for both prediction of evolutionary potential and long-term viability of natural populations. PMID:26102201

Adaptive Role of Inversion Polymorphism of Drosophila subobscura in Lead Stressed Environment.

PubMed

Kenig, Bojan; Kurbalija Novičić, Zorana; Patenković, Aleksandra; Stamenković-Radak, Marina; Anđelković, Marko

2015-01-01

Local adaptation to environmental stress at different levels of genetic polymorphism in various plants and animals has been documented through evolution of heavy metal tolerance. We used samples of Drosophila subobscura populations from two differently polluted environments to analyze the change of chromosomal inversion polymorphism as genetic marker during laboratory exposure to lead. Exposure to environmental contamination can affect the genetic content within a particular inversion and produce targets for selection in populations from different environments. The aims were to discover whether the inversion polymorphism is shaped by the local natural environments, and if lead as a selection pressure would cause adaptive divergence of two populations during the multigenerational laboratory experiment. The results showed that populations retain signatures from past contamination events, and that heavy metal pollution can cause adaptive changes in population. Differences in inversion polymorphism between the two populations increased over generations under lead contamination in the laboratory. The inversion polymorphism of population originating from the more polluted natural environment was more stable during the experiment, both under conditions with and without lead. Therefore, results showed that inversion polymorphism as a genetic marker reflects a strong signature of adaptation to the local environment, and that historical demographic events and selection are important for both prediction of evolutionary potential and long-term viability of natural populations.

Polymorphism analysis of prion protein gene in 11 Pakistani goat breeds

PubMed Central

Hassan, Mohammad Farooque; Khan, Sher Hayat; Babar, Masroor Ellahi; Yang, Lifeng; Ali, Tariq; Khan, Jamal Muhammad; Shah, Syed Zahid Ali; Zhou, Xiangmei; Hussain, Tanveer; Zhu, Ting; Hussain, Tariq; Zhao, Deming

2016-01-01

ABSTRACT The association between caprine PrP gene polymorphisms and its susceptibility to scrapie has been investigated in current years. As the ORF of the PrP gene is extremely erratic in different breeds of goats, we studied the PrP gene polymorphisms in 80 goats which belong to 11 Pakistani indigenous goat breeds from all provinces of Pakistan. A total of 6 distinct polymorphic sites (one novel) with amino acid substitutions were identified in the PrP gene which includes 126 (A -> G), 304 (G -> T), 379 (A -> G), 414 (C -> T), 428 (A -> G) and 718 (C -> T). The locus c.428 was found highly polymorphic in all breeds as compare to other loci. On the basis of these PrP variants NJ phylogenetic tree was constructed through MEGA6.1 which showed that all goat breeds along with domestic sheep and Mauflon sheep appeared as in one clade and sharing its most recent common ancestors (MRCA) with deer species while Protein analysis has shown that these polymorphisms can lead to varied primary, secondary and tertiary structure of protein. Based on these polymorphic variants, genetic distance, multidimensional scaling plot and principal component analyses revealed the clear picture regarding greater number of substitutions in cattle PrP regions as compared to the small ruminant species. In particular these findings may pinpoint the fundamental control over the scrapie in Capra hircus on genetic basis. PMID:27388702

49 CFR 325.77 - Computation of open site requirements-nonstandard sites.

Code of Federal Regulations, 2010 CFR

2010-10-01

... microphone target point is other than 50 feet (15.2 m), the test site must be an open site within a radius... microphone target point. (b) Plan view diagrams of nonstandard test sites are shown in Figures 3 and 4... (18.3 m) distance between the microphone location point and the microphone target point. (See § 325.79...

49 CFR 325.77 - Computation of open site requirements-nonstandard sites.

Code of Federal Regulations, 2011 CFR

2011-10-01

... microphone target point is other than 50 feet (15.2 m), the test site must be an open site within a radius... microphone target point. (b) Plan view diagrams of nonstandard test sites are shown in Figures 3 and 4... (18.3 m) distance between the microphone location point and the microphone target point. (See § 325.79...

TargetM6A: Identifying N6-Methyladenosine Sites From RNA Sequences via Position-Specific Nucleotide Propensities and a Support Vector Machine.

PubMed

Li, Guang-Qing; Liu, Zi; Shen, Hong-Bin; Yu, Dong-Jun

2016-10-01

As one of the most ubiquitous post-transcriptional modifications of RNA, N 6 -methyladenosine ( [Formula: see text]) plays an essential role in many vital biological processes. The identification of [Formula: see text] sites in RNAs is significantly important for both basic biomedical research and practical drug development. In this study, we designed a computational-based method, called TargetM6A, to rapidly and accurately target [Formula: see text] sites solely from the primary RNA sequences. Two new features, i.e., position-specific nucleotide/dinucleotide propensities (PSNP/PSDP), are introduced and combined with the traditional nucleotide composition (NC) feature to formulate RNA sequences. The extracted features are further optimized to obtain a much more compact and discriminative feature subset by applying an incremental feature selection (IFS) procedure. Based on the optimized feature subset, we trained TargetM6A on the training dataset with a support vector machine (SVM) as the prediction engine. We compared the proposed TargetM6A method with existing methods for predicting [Formula: see text] sites by performing stringent jackknife tests and independent validation tests on benchmark datasets. The experimental results show that the proposed TargetM6A method outperformed the existing methods for predicting [Formula: see text] sites and remarkably improved the prediction performances, with MCC = 0.526 and AUC = 0.818. We also provided a user-friendly web server for TargetM6A, which is publicly accessible for academic use at http://csbio.njust.edu.cn/bioinf/TargetM6A.

Decaleside: a new class of natural insecticide targeting tarsal gustatory sites

NASA Astrophysics Data System (ADS)

Rajashekar, Yallappa; Rao, Lingamallu J. M.; Shivanandappa, Thimmappa

2012-10-01

Natural sources for novel insecticide molecules hold promise in view of their eco-friendly nature, selectivity, and mammalian safety. Recent progress in understanding the biology of insect olfaction and taste offers new strategies for developing selective pest control agents. We have isolated two natural insecticidal molecules from edible roots of Decalepis hamiltonii named Decalesides I and II, which are novel trisaccharides, highly toxic to household insect pests and stored-product insects. We have experimentally shown that insecticidal activity requires contact with tarsi on the legs but is not toxic orally. The insecticidal activity of molecules is lost by hydrolysis, and various sugars modify toxic response, showing that the insecticidal activity is via gustatory sites on the tarsi. Selective toxicity to insects by virtue of their gustatory site of action and the mammalian safety of the new insecticides is inherent in their chemical structure with 1-4 or 1-1 α linkage that is easily hydrolyzed by digestive enzymes of mammals. Decalesides represent a new chemical class of natural insecticides with a unique mode of action targeting tarsal chemosensory/gustatory system of insects.

Polymorphisms of Interlukin-1β rs16944 confer susceptibility to myelodysplastic syndromes.

PubMed

Yin, Congcong; He, Na; Li, Peng; Zhang, Chen; Yu, Jie; Hua, Mingqiang; Ji, Chunyan; Ma, Daoxin

2016-11-15

Genetic factors have been shown to be associated with Myelodysplastic syndromes (MDS) susceptibility. In recent years, the role of inflammation in the promotion of tumor growth is supported by a broad range of experimental and clinical evidence. But the relationship between polymorphisms in NOD-like receptor protein 3 (NLRP3) inflammasome and MDS is rarely reported. Thus, we conducted a case-control study, and genotyped five single nucleotide polymorphisms (SNPs) (NLRP3, IL-1β, IL-18, CARD8, and NF-κB) in MDS patients and healthy controls. The association of different genotypes with patient characteristics was analyzed. Comparing MDS patients with controls, GG genotype of IL-1β (rs16944) was observed to be associated with a significantly increased risk of MDS 78/166 (48.8%) vs 26/96 (27.0%), OR=2.1, CI (1.0-4.4). No significant association was identified regarding the rest of investigated polymorphisms and MDS susceptibility. Complex karyotypes were more frequent in patients with GG genotype of IL-1β (rs16944). Patients with IL-1β polymorphisms (rs16944) GG and GA had lower hemoglobin than those without. Patients with IL-1β polymorphisms (rs16944) GG had higher IPSS scores than those without IL-1β polymorphisms. In conclusion, our present data shows that the IL-1β polymorphisms (rs16944) GG were frequently occurred in MDS. IL-1β (rs16944) GG genotype might serve as a novel biomarker and potential targets for MDS. Copyright © 2016 Elsevier Inc. All rights reserved.

Theory-Guided Synthesis of a Metastable Lead-Free Piezoelectric Polymorph

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garten, Lauren M; Ndione, Paul F; Beaton, Daniel A

Many technologically critical materials are metastable under ambient conditions, yet the understanding of how to rationally design and guide the synthesis of these materials is limited. This work presents an integrated approach that targets a metastable lead-free piezoelectric polymorph of SrHfO3. First-principles calculations predict that the previous experimentally unrealized, metastable P4mm phase of SrHfO3 should exhibit a direct piezoelectric response (d33) of 36.9 pC N-1 (compared to d33 = 0 for the ground state). Combining computationally optimized substrate selection and synthesis conditions lead to the epitaxial stabilization of the polar P4mm phase of SrHfO3 on SrTiO3. The films are structurallymore » consistent with the theory predictions. A ferroelectric-induced large signal effective converse piezoelectric response of 5.2 pm V-1 for a 35 nm film is observed, indicating the ability to predict and target multifunctionality. This illustrates a coupled theory-experimental approach to the discovery and realization of new multifunctional polymorphs.« less

Polymorphism of BMP4 gene in Indian goat breeds differing in prolificacy.

PubMed

Sharma, Rekha; Ahlawat, Sonika; Maitra, A; Roy, Manoranjan; Mandakmale, S; Tantia, M S

2013-12-10

Bone morphogenetic proteins (BMPs) are members of the TGF-β (transforming growth factor-beta) superfamily, of which BMP4 is the most important due to its crucial role in follicular growth and differentiation, cumulus expansion and ovulation. Reproduction is a crucial trait in goat breeding and based on the important role of BMP4 gene in reproduction it was considered as a possible candidate gene for the prolificacy of goats. The objective of the present study was to detect polymorphism in intronic, exonic and 3' un-translated regions of BMP4 gene in Indian goats. Nine different goat breeds (Barbari, Beetal, Black Bengal, Malabari, Jakhrana (Twinning>40%), Osmanabadi, Sangamneri (Twinning 20-30%), Sirohi and Ganjam (Twinning<10%)) differing in prolificacy and geographic distribution were employed for polymorphism scanning. Cattle sequence (AC_000167.1) was used to design primers for the amplification of a targeted region followed by direct DNA sequencing to identify the genetic variations. Single nucleotide polymorphisms (SNPs) were not detected in exon 3, the intronic region and the 3' flanking region. A SNP (G1534A) was identified in exon 2. It was a non-synonymous mutation resulting in an arginine to lysine change in a corresponding protein sequence. G to A transition at the 1534 locus revealed two genotypes GG and GA in the nine investigated goat breeds. The GG genotype was predominant with a genotype frequency of 0.98. The GA genotype was present in the Black Bengal as well as Jakhrana breed with a genotype frequency of 0.02. A microsatellite was identified in the 3' flanking region, only 20 nucleotides downstream from the termination site of the coding region, as a short sequence with more than nineteen continuous and repeated CA dinucleotides. Since the gene is highly evolutionarily conserved, identification of a non-synonymous SNP (G1534A) in the coding region gains further importance. To our knowledge, this is the first report of a mutation in the coding

Polymorphic variants of neurotransmitter receptor genes may affect sexual function in aging males: data from the HALS study.

PubMed

Jóźków, Paweł; Słowińska-Lisowska, Małgorzata; Łaczmański, Łukasz; Mędraś, Marek

2013-01-01

Human behavior is influenced by a number of brain neurotransmitters. Central dopamine, serotonin and melanocortin systems have special importance for male sexual function. We searched for associations between male aging symptoms and polymorphic sites of serotonin (5-HTR1B), melanocortin (MC4R) and dopamine (DRD2, DRD4) receptors. In a population-based sample, genotyping of 5-HTR1B (polymorphism: G861C), MC4R (polymorphisms: C-2745T, Val103Ile), DRD2 (polymorphism: C313T) and DRD4 (polymorphism: 48-bp VNTR) was performed in 387 healthy men. The Aging Males' Symptoms (AMS) scale was used to evaluate specific ailments of aging men. We analyzed answers to questions from the AMS scale. Five points of the questionnaire addressed sexual symptoms of the aging male: feeling of passing one's peak, decrease in beard growth, decrease in ability/frequency to perform sexually, decrease in the number of morning erections, and decrease in sexual desire/libido (lacking pleasure in sex, lacking desire for sexual intercourse). Relations between reported symptoms and variants of the polymorphic sites of the studied genes were assessed. After adjusting for confounding factors (education, arterial hypertension, physical activity, weight, waist circumference) an association between the sexual dimension of AMS and genetic variants of 5-HTR1B G861C (p = 0.04) was observed. Variability of neurotransmitter receptor genes may be associated with sexual symptoms of aging in men. Copyright © 2013 S. Karger AG, Basel.

Mitochondrial DNA polymorphism in a maternal lineage of Holstein cows.

PubMed Central

Hauswirth, W W; Laipis, P J

1982-01-01

Two mitochondrial genotypes are shown to exist within one Holstein cow maternal lineage. They were detected by the appearance of an extra Hae III recognition site in one genotype. The nucleotide sequence of this region has been determined and the genotypes are distinguished by an adenine/guanine base transition which creates the new Hae III site. This point mutation occurs within an open reading frame at the third position of a glycine codon and therefore does not alter the amino acid sequence. The present pattern of genotypes within the lineage demands that multiple shifts between genotypes must have occurred within the past 20 years with the most rapid shift taking place in no more than 4 years and indicates that mitochondrial DNA polymorphism can occur between maternally related mammals. The process that gave rise to different genotypes in one lineage is clearly of fundamental importance in understanding intraspecific mitochondrial polymorphism and evolution in mammals. Several potential mechanisms for rapid mitochondrial DNA variation are discussed in light of these results. Images PMID:6289312

Development of EST Intron-Targeting SNP Markers for Panax ginseng and Their Application to Cultivar Authentication.

PubMed

Wang, Hongtao; Li, Guisheng; Kwon, Woo-Saeng; Yang, Deok-Chun

2016-06-04

Panax ginseng is one of the most valuable medicinal plants in the Orient. The low level of genetic variation has limited the application of molecular markers for cultivar authentication and marker-assisted selection in cultivated ginseng. To exploit DNA polymorphism within ginseng cultivars, ginseng expressed sequence tags (ESTs) were searched against the potential intron polymorphism (PIP) database to predict the positions of introns. Intron-flanking primers were then designed in conserved exon regions and used to amplify across the more variable introns. Sequencing results showed that single nucleotide polymorphisms (SNPs), as well as indels, were detected in four EST-derived introns, and SNP markers specific to "Gopoong" and "K-1" were first reported in this study. Based on cultivar-specific SNP sites, allele-specific polymerase chain reaction (PCR) was conducted and proved to be effective for the authentication of ginseng cultivars. Additionally, the combination of a simple NaOH-Tris DNA isolation method and real-time allele-specific PCR assay enabled the high throughput selection of cultivars from ginseng fields. The established real-time allele-specific PCR assay should be applied to molecular authentication and marker assisted selection of P. ginseng cultivars, and the EST intron-targeting strategy will provide a potential approach for marker development in species without whole genomic DNA sequence information.

microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3' untranslated region: function in replication and influence of RNA secondary structure.

PubMed

Gerresheim, Gesche K; Dünnes, Nadia; Nieder-Röhrmann, Anika; Shalamova, Lyudmila A; Fricke, Markus; Hofacker, Ivo; Höner Zu Siederdissen, Christian; Marz, Manja; Niepmann, Michael

2017-02-01

We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3' untranslated region (3'UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3'UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3'UTR target site inhibits translation directed by the HCV 5'UTR. Thus, the miR-122 target sites in the 3'-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.

The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

PubMed

Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

2017-07-15

Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach

PubMed Central

Abi-Ghanem, Josephine; Chusainow, Janet; Karimova, Madina; Spiegel, Christopher; Hofmann-Sieber, Helga; Hauber, Joachim; Buchholz, Frank; Pisabarro, M. Teresa

2013-01-01

Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre’s enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine. PMID:23275541

Alpha-tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II.

PubMed

Dong, L-F; Low, P; Dyason, J C; Wang, X-F; Prochazka, L; Witting, P K; Freeman, R; Swettenham, E; Valis, K; Liu, J; Zobalova, R; Turanek, J; Spitz, D R; Domann, F E; Scheffler, I E; Ralph, S J; Neuzil, J

2008-07-17

Alpha-tocopheryl succinate (alpha-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of alpha-TOS has not been identified. Here, we show that alpha-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (Q(P) and Q(D), respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of alpha-TOS compared to that of UbQ for the Q(P) and Q(D) sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of alpha-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to alpha-TOS. We propose that alpha-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.

α-Tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II

PubMed Central

Dong, Lan-Feng; Low, Pauline; Dyason, Jeffrey C.; Wang, Xiu-Fang; Prochazka, Lubomir; Witting, Paul K.; Freeman, Ruth; Swettenham, Emma; Valis, Karel; Liu, Ji; Zobalova, Renata; Turanek, Jaroslav; Spitz, Doug R.; Domann, Frederick E.; Scheffler, Immo E.; Ralph, Stephen J.; Neuzil, Jiri

2009-01-01

α-Tocopheryl succinate (α-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of α-TOS has not been identified. Here we show that α-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ) binding site (QP and QD, respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of α-TOS compared to that of UbQ for the QP and QD sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and undergo apoptosis in the presence of α-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to α-TOS. We propose that α-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy. PMID:18372923

Vitamin D receptor gene polymorphisms and the risk of rickets among Asians: a meta-analysis.

PubMed

Mao, Song; Huang, Songming

2014-03-01

To evaluate the association between vitamin D receptor (VDR) gene polymorphisms and the risk of rickets among Asians. Eligible studies were included in our meta-analysis by searching PubMed, Embase, Cochrane and China National Knowledge Infrastructure databases according to a predefined criteria. A random effects model was used to calculate the combined ORs and its corresponding 95% CI. 16 studies were recruited for the analysis of the association between VDR BsmI (rs1544410), TaqI (rs731236), FokI (rs2228570) and ApaI (rs7975232) gene polymorphisms and the risk of rickets among Asians, most of whom were from China. B allele/BB genotype was associated with the susceptibility of rickets (p=0.017 and 0.044, respectively), and bb genotype was associated with lower risk of rickets (p=0.033). F allele/FF genotype was associated with the susceptibility of rickets (p<10(-4)), and ff genotype was associated with lower risk of rickets (p<10(-4)). AA genotype was associated with the onset of rickets (p=0.044). No significant association was observed between TaqI polymorphism the risk of rickets. A allele/aa genotype was not associated with the risk of rickets. No evidence of publication bias was observed. B allele/BB genotype at the BsmI site, F allele/FF genotype at the FokI site and AA genotype at the ApaI site may be risk factors for the onset of rickets among Asians; bb genotype at the BsmI site and ff genotype at the FokI site may be protective factors against the risk of rickets among Asians.

An innovative way to highlight the power of each polymorphism on elite athletes phenotype expression.

PubMed

Contrò, Valentina; Schiera, Gabriella; Abbruzzo, Antonino; Bianco, Antonino; Amato, Alessandra; Sacco, Alessia; Macchiarella, Alessandra; Palma, Antonio; Proia, Patrizia

2018-01-12

The purpose of this study was to determine the probability of soccer players having the best genetic background that could increase performance, evaluating the polymorphism that are considered Performance Enhancing Polymorphism (PEPs) distributed on five genes: PPARα, PPARGC1A, NRF2, ACE e CKMM. Particularly, we investigated how each polymorphism works directly or through another polymorphism to distinguish elite athletes from non-athletic population. Sixty professional soccer players (age 22.5 ± 2.2) and sixty healthy volunteers (age 21.2± 2.3) were enrolled. Samples of venous blood was used to prepare genomic DNA. The polymorphic sites were scanned using PCR-RFLP protocols with different enzyme. We used a multivariate logistic regression analysis to demonstrate an association between the five PEPs and elite phenotype. We found statistical significance in NRF2 (AG/GG genotype) polymorphism/soccer players association (p < 0.05) as well as a stronger association in ACE polymorphism (p =0.02). Particularly, we noticed that the ACE ID genotype and even more the II genotype are associated with soccer player phenotype. Although the other PEPs had no statistical significance, we proved that some of these may work indirectly, amplifying the effect of another polymorphism; for example, seems that PPARα could acts on NRF2 (GG) enhancing the effect of the latter, notwithstanding it had not shown a statistical significance. In conclusion, to establish if a polymorphism can influence the performance, it is necessary to understand how they act and interact, directly and indirectly, on each other.

A single nucleotide polymorphism in osteonectin 3’ untranslated region regulates bone volume and is targeted by miR-433

PubMed Central

Dole, Neha S.; Kapinas, Kristina; Kessler, Catherine B.; Yee, Siu-Pok; Adams, Douglas J.; Pereira, Renata C.; Delany, Anne M.

2014-01-01

Osteonectin/SPARC is one of the most abundant non-collagenous extracellular matrix proteins in bone, regulating collagen fiber assembly and promoting osteoblast differentiation. Osteonectin-null and –haploinsufficient mice have low turnover osteopenia, indicating that osteonectin contributes to normal bone formation. In male idiopathic osteoporosis patients, osteonectin 3’ UTR single nucleotide polymorphism (SNP) haplotypes that differed only at SNP1599 (rs1054204) were previously associated with bone mass. Haplotype A (containing SNP1599G) was more frequent in severely affected patients, whereas haplotype B (containing SNP1599C) was more frequent in less affected patients and healthy controls. We hypothesized that SNP1599 contributes to variability in bone mass by modulating osteonectin levels. Osteonectin 3’UTR reporter constructs demonstrated that haplotype A has a repressive effect on gene expression compared to B. We found that SNP1599G contributed to a miR-433 binding site and miR-433 inhibitor relieved repression of the haplotype A, but not B, 3’ UTR reporter construct. We tested our hypothesis in vivo, using a knock-in approach to replace the mouse osteonectin 3’ UTR with human haplotype A or B 3’ UTR. Compared to haplotype A mice, bone osteonectin levels were higher in haplotype B mice. B mice displayed higher bone formation rate and gained more trabecular bone with age. When parathyroid hormone was administered intermittently, haplotype B mice gained more cortical bone area than A mice. Cultured marrow stromal cells from B mice deposited more mineralized matrix and had higher osteocalcin mRNA compared with A mice, demonstrating a cell-autonomous effect on differentiation. Altogether, SNP1599 differentially regulates osteonectin expression and contributes to variability in bone mass, by a mechanism that may involve differential targeting by miR-433. This work validates the findings of the previous candidate gene study, and it assigns a

Genetic polymorphisms and expression of minisatellite mutations in a 3-generation population around the Semipalatinsk nuclear explosion test-site, Kazakhstan.

PubMed

Bolegenova, N K; Bekmanov, B O; Djansugurova, L B; Bersimbaev, R I; Salama, S A; Au, W W

2009-11-01

We have reported previously that a population near the Semipalatinsk nuclear explosion test site had significantly increased minisatellite mutations (MM), suggesting increased germ-line mutation rates from the exposure in 3 generations. We hypothesize that the MM can be used as a surrogate biomarker for functional genetic alterations, e.g. gene mutations and chromosome aberrations. Therefore, we have investigated the influence of polymorphisms in genes on the expression of MM in the same two populations (247 and 172 individuals, for exposed and control, respectively, in 3 generations), and their relationships with radiation exposure. We have chosen the analyses of three polymorphic DNA - repair genes (XRCC1, XRCC1 and XRCC3) and two xenobiotic detoxification genes (GSTT1 and GSTM1). Among the exposed and in comparison with the wild-type gene, the functionally active XRCC1 Arg194Trp was significantly associated with low MM and over-represented in the exposed compared with the control populations. In a similar analysis, the functionally deficient XRCC1 Arg399Glu and XRCC3 Trp241Met were associated with increased and significantly reduced MM, respectively, but these variant genes were under-represented in the exposed population. Both GSTT1 and GSTM1 nulls were significantly associated with increased MM. The former was under-represented but the latter was significantly over-represented in the exposed compared with the control populations. In summary, the data indicate that the expected enzymatic functions of the polymorphic genes are consistent with the MM expression, except the XRCC1 Arg399Glu variant gene. In addition, the variant genes were retained in the three generations in association with their useful function, except for the GSTM1 null. However, the MM frequencies in the exposed were not consistently and significantly higher than those in the control populations, radiation exposure may therefore not have been the only cause for the high MM frequency among the

Population genetics of polymorphism and divergence for diploid selection models with arbitrary dominance.

PubMed

Williamson, Scott; Fledel-Alon, Adi; Bustamante, Carlos D

2004-09-01

We develop a Poisson random-field model of polymorphism and divergence that allows arbitrary dominance relations in a diploid context. This model provides a maximum-likelihood framework for estimating both selection and dominance parameters of new mutations using information on the frequency spectrum of sequence polymorphisms. This is the first DNA sequence-based estimator of the dominance parameter. Our model also leads to a likelihood-ratio test for distinguishing nongenic from genic selection; simulations indicate that this test is quite powerful when a large number of segregating sites are available. We also use simulations to explore the bias in selection parameter estimates caused by unacknowledged dominance relations. When inference is based on the frequency spectrum of polymorphisms, genic selection estimates of the selection parameter can be very strongly biased even for minor deviations from the genic selection model. Surprisingly, however, when inference is based on polymorphism and divergence (McDonald-Kreitman) data, genic selection estimates of the selection parameter are nearly unbiased, even for completely dominant or recessive mutations. Further, we find that weak overdominant selection can increase, rather than decrease, the substitution rate relative to levels of polymorphism. This nonintuitive result has major implications for the interpretation of several popular tests of neutrality.

Are "functionally related polymorphisms" of renin-angiotensin-aldosterone system gene polymorphisms associated with hypertension?

PubMed

Hahntow, Ines N; Mairuhu, Gideon; van Valkengoed, Irene Gm; Koopmans, Richard P; Michel, Martin C

2010-06-02

Genotype-phenotype association studies are typically based upon polymorphisms or haplotypes comprised of multiple polymorphisms within a single gene. It has been proposed that combinations of polymorphisms in distinct genes, which functionally impact the same phenotype, may have stronger phenotype associations than those within a single gene. We have tested this hypothesis using genes encoding components of the renin-angiotensin-aldosterone system and the high blood pressure phenotype. Our analysis is based on 1379 participants of the cross-sectional SUNSET study randomly selected from the population register of Amsterdam. Each subject was genotyped for the angiotensinogen M235T, the angiotensin-converting enzyme insertion/deletion and the angiotensin II type 1 receptor A1166C polymorphism. The phenotype high blood pressure was defined either as a categorical variable comparing hypertension versus normotension as in most previous studies or as a continuous variable using systolic, diastolic and mean blood pressure in a multiple regression analysis with gender, ethnicity, age, body-mass-index and antihypertensive medication as covariates. Genotype-phenotype relationships were explored for each polymorphism in isolation and for double and triple polymorphism combinations. At the single polymorphism level, only the A allele of the angiotensin II type 1 receptor was associated with a high blood pressure phenotype. Using combinations of polymorphisms of two or all three genes did not yield stronger/more consistent associations. We conclude that combinations of physiologically related polymorphisms of multiple genes, at least with regard to the renin-angiotensin-aldosterone system and the hypertensive phenotype, do not necessarily offer additional benefit in analyzing genotype/phenotype associations.

Identification of single nucleotide polymorphism in ginger using expressed sequence tags

PubMed Central

Chandrasekar, Arumugam; Riju, Aikkal; Sithara, Kandiyl; Anoop, Sahadevan; Eapen, Santhosh J

2009-01-01

Ginger (Zingiber officinale Rosc) (Family: Zingiberaceae) is a herbaceous perennial, the rhizomes of which are used as a spice. Ginger is a plant which is well known for its medicinal applications. Recently EST-derived SNPs are a free by-product of the currently expanding EST (Expressed Sequence Tag) databases. The development of high-throughput methods for the detection of SNPs (Single Nucleotide Polymorphism) and small indels (insertion/deletion) has led to a revolution in their use as molecular markers. Available (38139) Ginger EST sequences were mined from dbEST of NCBI. CAP3 program was used to assemble EST sequences into contigs. Candidate SNPs and Indel polymorphisms were detected using the perl script AutoSNP version 1.0 which has used 31905 ESTs for detecting SNPs and Indel sites. We found 64026 SNP sites and 7034 indel polymorphisms with frequency of 0.84 SNPs / 100 bp. Among the three tissues from which the EST libraries had been generated, Rhizomes had high frequency of 1.08 SNPs/indels per 100 bp whereas the leaves had lowest frequency of 0.63 per 100 bp and root is showing relative frequency 0.82/100bp. Transitions and transversion ratio is 0.90. In overall detected SNP, transversion is high when compare to transition. These detected SNPs can be used as markers for genetic studies. Availability The results of the present study hosted in our webserver www.spices.res.in/spicesnip PMID:20198184

Site index curves for black, white, scarlet, and chestnut oaks in the Central States.

Treesearch

Willard H. Carmean

1971-01-01

Stem analyses showed polymorphic patterns of height growth for each species and for different levels of site quality. New site index curves are presented that show better height growth in later years than predicted by older harmonized site index curves.

DNA abasic site-directed formation of fluorescent silver nanoclusters for selective nucleobase recognition

NASA Astrophysics Data System (ADS)

Ma, Kun; Cui, Qinghua; Liu, Guiying; Wu, Fei; Xu, Shujuan; Shao, Yong

2011-07-01

DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation related diseases. Various methods for SNP detection have been proposed and many are already in use. Here, we find that the abasic site (AP site) in the DNA duplex can be developed as a capping scaffold for the generation of fluorescent silver nanoclusters (Ag NCs). As a proof of concept, the DNA sequences from fragments near codon 177 of cancer supression gene p53 were used as a model for SNP detection by in situ formed Ag NCs. The formation of fluorescent Ag NCs in the AP site-containing DNA duplex is highly selective for cytosine facing the AP site and guanines flanking the site and can be employed in situ as readout for SNP detection. The fluorescent signal-on sensing for SNP based on this inorganic fluorophore is substantially advantageous over the previously reported signal-off responses using low-molecular-weight organic ligands. The strong dependence of fluorescent Ag NC formation on the sequences surrounding the AP site was successfully used to identify mutations in codon 177 of cancer supression gene p53. We anticipate that this approach will be employed to develop a practical SNP detection method by locating an AP site toward the midway cytosine in a target strand containing more than three consecutive cytosines.

Theory-Guided Synthesis of a Metastable Lead-Free Piezoelectric Polymorph.

PubMed

Garten, Lauren M; Dwaraknath, Shyam; Walker, Julian; Mangum, John S; Ndione, Paul F; Park, Yoonsang; Beaton, Daniel A; Gopalan, Venkatraman; Gorman, Brian P; Schelhas, Laura T; Toney, Michael F; Trolier-McKinstry, Susan; Persson, Kristin A; Ginley, David S

2018-05-10

Many technologically critical materials are metastable under ambient conditions, yet the understanding of how to rationally design and guide the synthesis of these materials is limited. This work presents an integrated approach that targets a metastable lead-free piezoelectric polymorph of SrHfO 3 . First-principles calculations predict that the previous experimentally unrealized, metastable P4mm phase of SrHfO 3 should exhibit a direct piezoelectric response (d 33 ) of 36.9 pC N -1 (compared to d 33 = 0 for the ground state). Combining computationally optimized substrate selection and synthesis conditions lead to the epitaxial stabilization of the polar P4mm phase of SrHfO 3 on SrTiO 3 . The films are structurally consistent with the theory predictions. A ferroelectric-induced large signal effective converse piezoelectric response of 5.2 pm V -1 for a 35 nm film is observed, indicating the ability to predict and target multifunctionality. This illustrates a coupled theory-experimental approach to the discovery and realization of new multifunctional polymorphs. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Canine olfactory receptor gene polymorphism and its relation to odor detection performance by sniffer dogs.

PubMed

Lesniak, Anna; Walczak, Marta; Jezierski, Tadeusz; Sacharczuk, Mariusz; Gawkowski, Maciej; Jaszczak, Kazimierz

2008-01-01

The outstanding sensitivity of the canine olfactory system has been acknowledged by using sniffer dogs in military and civilian service for detection of a variety of odors. It is hypothesized that the canine olfactory ability is determined by polymorphisms in olfactory receptor (OR) genes. We investigated 5 OR genes for polymorphic sites which might affect the olfactory ability of service dogs in different fields of specific substance detection. All investigated OR DNA sequences proved to have allelic variants, the majority of which lead to protein sequence alteration. Homozygous individuals at 2 gene loci significantly differed in their detection skills from other genotypes. This suggests a role of specific alleles in odor detection and a linkage between single-nucleotide polymorphism and odor recognition efficiency.

Identification of single-nucleotide polymorphisms of the prion protein gene in sika deer (Cervus nippon laiouanus)

PubMed Central

Jeong, Hyun-Jeong; Lee, Joong-Bok; Park, Seung-Yong; Song, Chang-Seon; Kim, Bo-Sook; Rho, Jung-Rae; Yoo, Mi-Hyun; Jeong, Byung-Hoon; Kim, Yong-Sun

2007-01-01

Polymorphisms of the prion protein gene (PRNP) have been detected in several cervid species. In order to confirm the genetic variations, this study examined the DNA sequences of the PRNP obtained from 33 captive sika deer (Cervus nippon laiouanus) in Korea. A total of three single-nucleotide polymorphisms (SNPs) at codons 100, 136 and 226 in the PRNP of the sika deer were identified. The polymorphic site located at codon 100 has not been reported. The SNPs detected at codons 100 and 226 induced amino acid substitutions. The SNP at codon 136 was a silent mutation that does not induce any amino acid change. The genotype and allele frequencies were determined for each of the SNPs. PMID:17679779

Polymorphisms in miRNA genes and their involvement in autoimmune diseases susceptibility.

PubMed

Latini, Andrea; Ciccacci, Cinzia; Novelli, Giuseppe; Borgiani, Paola

2017-08-01

MicroRNAs (miRNAs) are small non-coding RNA molecules that negatively regulate the expression of multiple protein-encoding genes at the post-transcriptional level. MicroRNAs are involved in different pathways, such as cellular proliferation and differentiation, signal transduction and inflammation, and play crucial roles in the development of several diseases, such as cancer, diabetes, and cardiovascular diseases. They have recently been recognized to play a role also in the pathogenesis of autoimmune diseases. Although the majority of studies are focused on miRNA expression profiles investigation, a growing number of studies have been investigating the role of polymorphisms in miRNA genes in the autoimmune diseases development. Indeed, polymorphisms affecting the miRNA genes can modify the set of targets they regulate or the maturation efficiency. This review is aimed to give an overview about the available studies that have investigated the association of miRNA gene polymorphisms with the susceptibility to various autoimmune diseases and to their clinical phenotypes.

The association of RANK gene C421T and C575T polymorphisms with bone mineral density in postmenopausal Turkish women.

PubMed

Işleten, Banu; Durmaz, Burak; Durmaz, Berrin; Onay, Hüseyin; Ozkınay, Ferda; Durmaz, Asude; Turan, Volkan; Oztekin, Kemal

2013-10-01

To investigate the association between C421T polymorphism within exon 4, C575T polymorphism within exon 6 of the RANK gene and bone mineral density (BMD) variations in postmenopausal Turkish women. One hundred seventy-eight postmenopausal women (patients = 100 and controls = 78) who applied to Ege University Faculty of Medicine, Department of Physical Medicine and Rehabilitation, for osteoporosis examination were analyzed. BMDs of the lumbar spine and femoral sites were measured. Patient and control groups were established based on their T-score values being above and/or below -1. After venous blood sampling, C421T and C575T polymorphisms of the RANK gene were assessed through PCR process following DNA extraction. Genotype frequencies for the C421T and C575T polymorphisms were compared between the control group and the patient group. No significant difference was detected between the two groups for both polymorphisms. There was also no significant difference between the control and patient groups in terms of the combined genotype (p = 0.752) and the combined haplotype analysis of the C421T and C575T polymorphisms (p = 0.723). In the control and patient groups separately, no significant differences in BMD values either at the femoral sites or at the lumbar spine were detected between the combined genotypes of the two polymorphisms. The genotypes, combined genotypes and allele frequencies of C421T and C575T polymorphisms of the RANK gene have not been found to be associated with BMD in Turkish women. Further studies including both sexes and more cases are required.

Targeting the Allosteric Site of Oncoprotein BCR-ABL as an Alternative Strategy for Effective Target Protein Degradation.

PubMed

Shimokawa, Kenichiro; Shibata, Norihito; Sameshima, Tomoya; Miyamoto, Naoki; Ujikawa, Osamu; Nara, Hiroshi; Ohoka, Nobumichi; Hattori, Takayuki; Cho, Nobuo; Naito, Mikihiko

2017-10-12

Protein degradation technology based on hybrid small molecules is an emerging drug modality that has significant potential in drug discovery and as a unique method of post-translational protein knockdown in the field of chemical biology. Here, we report the first example of a novel and potent protein degradation inducer that binds to an allosteric site of the oncogenic BCR-ABL protein. BCR-ABL allosteric ligands were incorporated into the SNIPER (Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers) platform, and a series of in vitro biological assays of binding affinity, target protein modulation, signal transduction, and growth inhibition were carried out. One of the designed compounds, 6 (SNIPER(ABL)-062), showed desirable binding affinities against ABL1, cIAP1/2, and XIAP and consequently caused potent BCR-ABL degradation.

The effect of colour polymorphism on thermoregulation in an orb web spider.

PubMed

Rao, Dinesh; Mendoza-Cuenca, Luis

2016-08-01

Spiders that build aerial webs in open areas face the risk of overheating due to incident solar radiation. These spiders can counteract overheating by either moving the web to another site or by adopting behavioural thermoregulation within the web. Since moving can be costly, studies have suggested that a passive but effective method of reducing heat load is by light reflectance through body colouration. We explored the interaction between colour and thermoregulation in a colour polymorphic species, under both field and laboratory conditions. We show that in natural conditions, the spiders maintain their body temperature below that of the ambient, but with no difference in surface temperature between colour morphs. In laboratory experiments with internal temperature measurements, white morphs bore the risk of overheating better than the yellow morphs since they heated up slower and cooled faster. We suggest that the thermoregulatory properties of colour polymorphism in Verrucosa arenata have physiological consequences and may play an important role in the maintenance of colour polymorphism in this species.

The effect of colour polymorphism on thermoregulation in an orb web spider

NASA Astrophysics Data System (ADS)

Rao, Dinesh; Mendoza-Cuenca, Luis

2016-08-01

Spiders that build aerial webs in open areas face the risk of overheating due to incident solar radiation. These spiders can counteract overheating by either moving the web to another site or by adopting behavioural thermoregulation within the web. Since moving can be costly, studies have suggested that a passive but effective method of reducing heat load is by light reflectance through body colouration. We explored the interaction between colour and thermoregulation in a colour polymorphic species, under both field and laboratory conditions. We show that in natural conditions, the spiders maintain their body temperature below that of the ambient, but with no difference in surface temperature between colour morphs. In laboratory experiments with internal temperature measurements, white morphs bore the risk of overheating better than the yellow morphs since they heated up slower and cooled faster. We suggest that the thermoregulatory properties of colour polymorphism in Verrucosa arenata have physiological consequences and may play an important role in the maintenance of colour polymorphism in this species.

White Pine Site Index for the Southern Forest Survey

Treesearch

Bernard R. Parresol; John S. Vissage

1998-01-01

Second-growth white pine age-height data a A base-ageinvariant polymorphic site index equation was used to model the white pine (Pinus strobus L.) site-quality data provided by Frothingham (1914). These data are the accepted standard used by the Southern Forest Inventory and Analysis unit of the U.S. Department of Agriculture, Forest Service. An all...

Floral polymorphism and the fitness implications of attracting pollinating and florivorous insects.

PubMed

de Jager, Marinus L; Ellis, Allan G

2014-01-01

Floral polymorphism is frequently attributed to pollinator-mediated selection. Multiple studies, however, have revealed the importance of non-pollinating visitors in floral evolution. Using the polymorphic annual daisy Ursinia calenduliflora, this study investigated the importance of different insect visitors, and their effects on fitness, in the maintenance of floral polymorphism. The spatial structure of a discrete floral polymorphism was characterized based on the presence/absence of anthocyanin floret spots in U. calenduliflora. A 3-year observational study was then conducted in polymorphic populations to investigate differences in visitation rates of dominant visitors to floral morphs. Experiments were performed to explore the floral preference of male and female Megapalpus capensis (the dominant insect visitor) and their effectiveness as pollinators. Next, floral damage by antagonistic florivores and the reproductive success of the two floral morphs were surveyed in multiple populations and years. Floral polymorphism in U. calenduliflora was structured spatially, as were insect visitation patterns. Megapalpus capensis males were the dominant visitors and exhibited strong preference for the spotted morph in natural and experimental observations. While this may indicate potential fitness benefits for the spotted morph, female fitness did not differ between floral morphs. However, as M. capensis males are very efficient at exporting U. calenduliflora pollen, their preference may likely increase the reproductive fitness of the spotted morph through male fitness components. The spotted morph, however, also suffered significantly greater costs due to ovule predation by florivores than the spotless morph. The results suggest that pollinators and florivores may potentially exert antagonistic selection that could contribute to the maintenance of floral polymorphism across the range of U. calenduliflora. The relative strength of selection imposed by each agent is

Androgen Receptor Gene Polymorphisms and Alterations in Prostate Cancer: Of Humanized Mice and Men

PubMed Central

Robins, Diane M.

2011-01-01

Germline polymorphisms and somatic mutations of the androgen receptor (AR) have been intensely investigated in prostate cancer but even with genomic approaches their impact remains controversial. To assess the functional significance of AR genetic variation, we converted the mouse gene to the human sequence by germline recombination and engineered alleles to query the role of a polymorphic glutamine (Q) tract implicated in cancer risk. In a prostate cancer model, AR Q tract length influences progression and castration response. Mutation profiling in mice provides direct evidence that somatic AR variants are selected by therapy, a finding validated in human metastases from distinct treatment groups. Mutant ARs exploit multiple mechanisms to resist hormone ablation, including alterations in ligand specificity, target gene selectivity, chaperone interaction and nuclear localization. Regardless of their frequency, these variants permute normal function to reveal novel means to target wild type AR and its key interacting partners. PMID:21689727

Associations of Polymorphisms in the Apolipoprotein APOA1-C3-A5 Gene Cluster with Acute Coronary Syndrome

PubMed Central

Ding, Yan; Zhu, Ming An; Wang, Zhi Xiao; Zhu, Jing; Feng, Jing Bo; Li, Dong Sheng

2012-01-01

Background. Acute coronary syndromes (ACSs) are clinically cardiovascular events associated with dyslipidemia in common. Single nucleotide polymorphisms (SNPs) and haplotypes in the APOA1/C3/A5 gene cluster are associated with diabetes and familial combined hyperlipidaemia (FCH). Little is known about whether the polymorphisms in these genes affect lipid homeostasis in patients with ACSs. The present paper aimed to examine these associations with 4 SNPs in the APOA1 −75G > A, the APOC3 −455T > C, and APOA5 −1131T > C, c.553G > T variant to ACSs in Chinese Han. Methods. Chinese Han of 229 patients with ACSs and 254 unrelated controls were analyzed. Four SNPs in APOA1/C3/A5 cluster were genotyped and lipid was determined. Results. Our data show that minor allelic frequencies of APOC3 −455T > C, APOA5 −1131T > C, and c.553G > T polymorphisms in patients with ACSs were significantly higher than control group (P < 0.05). Furthermore, the 3 polymorphic sites were strongly of linkage disequilibrium, and minor alleles of 3 SNP sites had higher TG level than wild alleles (P < 0.05), APOC3 −455C and APOA5 c.553T allele carriers also had lower level of HDL-C. Conclusions. The minor alleles of APOC3 −455T > C, APOA5 −1131T > C, and c.553G > T polymorphisms are closely associated with ACSs. PMID:22675253

Association study of interleukin-4 polymorphisms with paranoid schizophrenia in the Polish population: a critical approach.

PubMed

Fila-Danilow, Anna; Kucia, Krzysztof; Kowalczyk, Malgorzata; Owczarek, Aleksander; Paul-Samojedny, Monika; Borkowska, Paulina; Suchanek, Renata; Kowalski, Jan

2012-08-01

Changes in immunological system are one of dysfunctions reported in schizophrenia. Some changes based on an imbalance between Th1 and Th2 cytokines results from cytokine gene polymorphisms. Interleukin-4 gene (IL4) is considered as a potential candidate gene in schizophrenia association studies. The aim of the current case-control study was to examine whether the -590C/T (rs2243250) and -33C/T (rs2070874) IL4 gene polymorphisms are implicated in paranoid schizophrenia development in the Polish population. Genotyping of polymorphisms was performed by using PCR-RFLP technique. The genotypes and alleles distribution of both SNPs were analysed in patients (n = 182) and healthy individuals constituted the control group (n = 215). The connection between some clinical variables and studied polymorphisms has been examined as well. We did not revealed any association between the -590C/T and -33C/T polymorphisms and paranoid schizophrenia. In case of both SNPs the homozygous TT genotype was extremely rare. Both polymorphic sites of the IL4 gene were found to be in a very strong linkage disequilibrium. However we did not identify a haplotype predispose to paranoid schizophrenia. No associations were also observed between the clinical course and psychopathology of the disease and the genotypes of both analysed polymorphisms. Our results suggest that the polymorphisms -590C/T in IL4 gene promoter region and -33C/T in the 5'-UTR are not involved in the pathophysiology of paranoid schizophrenia in Polish residents.

Simultaneous quantification of protein phosphorylation sites using liquid chromatography-tandem mass spectrometry-based targeted proteomics: a linear algebra approach for isobaric phosphopeptides.

PubMed

Xu, Feifei; Yang, Ting; Sheng, Yuan; Zhong, Ting; Yang, Mi; Chen, Yun

2014-12-05

As one of the most studied post-translational modifications (PTM), protein phosphorylation plays an essential role in almost all cellular processes. Current methods are able to predict and determine thousands of phosphorylation sites, whereas stoichiometric quantification of these sites is still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted proteomics is emerging as a promising technique for site-specific quantification of protein phosphorylation using proteolytic peptides as surrogates of proteins. However, several issues may limit its application, one of which relates to the phosphopeptides with different phosphorylation sites and the same mass (i.e., isobaric phosphopeptides). While employment of site-specific product ions allows for these isobaric phosphopeptides to be distinguished and quantified, site-specific product ions are often absent or weak in tandem mass spectra. In this study, linear algebra algorithms were employed as an add-on to targeted proteomics to retrieve information on individual phosphopeptides from their common spectra. To achieve this simultaneous quantification, a LC-MS/MS-based targeted proteomics assay was first developed and validated for each phosphopeptide. Given the slope and intercept of calibration curves of phosphopeptides in each transition, linear algebraic equations were developed. Using a series of mock mixtures prepared with varying concentrations of each phosphopeptide, the reliability of the approach to quantify isobaric phosphopeptides containing multiple phosphorylation sites (≥ 2) was discussed. Finally, we applied this approach to determine the phosphorylation stoichiometry of heat shock protein 27 (HSP27) at Ser78 and Ser82 in breast cancer cells and tissue samples.

Development of EST Intron-Targeting SNP Markers for Panax ginseng and Their Application to Cultivar Authentication

PubMed Central

Wang, Hongtao; Li, Guisheng; Kwon, Woo-Saeng; Yang, Deok-Chun

2016-01-01

Panax ginseng is one of the most valuable medicinal plants in the Orient. The low level of genetic variation has limited the application of molecular markers for cultivar authentication and marker-assisted selection in cultivated ginseng. To exploit DNA polymorphism within ginseng cultivars, ginseng expressed sequence tags (ESTs) were searched against the potential intron polymorphism (PIP) database to predict the positions of introns. Intron-flanking primers were then designed in conserved exon regions and used to amplify across the more variable introns. Sequencing results showed that single nucleotide polymorphisms (SNPs), as well as indels, were detected in four EST-derived introns, and SNP markers specific to “Gopoong” and “K-1” were first reported in this study. Based on cultivar-specific SNP sites, allele-specific polymerase chain reaction (PCR) was conducted and proved to be effective for the authentication of ginseng cultivars. Additionally, the combination of a simple NaOH-Tris DNA isolation method and real-time allele-specific PCR assay enabled the high throughput selection of cultivars from ginseng fields. The established real-time allele-specific PCR assay should be applied to molecular authentication and marker assisted selection of P. ginseng cultivars, and the EST intron-targeting strategy will provide a potential approach for marker development in species without whole genomic DNA sequence information. PMID:27271615

Methylation of avpr1a in the cortex of wild prairie voles: effects of CpG position and polymorphism

PubMed Central

Maguire, S. M.; Phelps, S. M.

2017-01-01

DNA methylation can cause stable changes in neuronal gene expression, but we know little about its role in individual differences in the wild. In this study, we focus on the vasopressin 1a receptor (avpr1a), a gene extensively implicated in vertebrate social behaviour, and explore natural variation in DNA methylation, genetic polymorphism and neuronal gene expression among 30 wild prairie voles (Microtus ochrogaster). Examination of CpG density across 8 kb of the locus revealed two distinct CpG islands overlapping promoter and first exon, characterized by few CpG polymorphisms. We used a targeted bisulfite sequencing approach to measure DNA methylation across approximately 3 kb of avpr1a in the retrosplenial cortex, a brain region implicated in male space use and sexual fidelity. We find dramatic variation in methylation across the avrp1a locus, with pronounced diversity near the exon–intron boundary and in a genetically variable putative enhancer within the intron. Among our wild voles, differences in cortical avpr1a expression correlate with DNA methylation in this putative enhancer, but not with the methylation status of the promoter. We also find an unusually high number of polymorphic CpG sites (polyCpGs) in this focal enhancer. One polyCpG within this enhancer (polyCpG 2170) may drive variation in expression either by disrupting transcription factor binding motifs or by changing local DNA methylation and chromatin silencing. Our results contradict some assumptions made within behavioural epigenetics, but are remarkably concordant with genome-wide studies of gene regulation. PMID:28280564

Rational modification of protein stability by targeting surface sites leads to complicated results

PubMed Central

Xiao, Shifeng; Patsalo, Vadim; Shan, Bing; Bi, Yuan; Green, David F.; Raleigh, Daniel P.

2013-01-01

The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions. PMID:23798426

Epitope-based peptide vaccine design and target site depiction against Ebola viruses: an immunoinformatics study.

PubMed

Khan, M A; Hossain, M U; Rakib-Uz-Zaman, S M; Morshed, M N

2015-07-01

Ebola viruses (EBOVs) have been identified as an emerging threat in recent year as it causes severe haemorrhagic fever in human. Epitope-based vaccine design for EBOVs remains a top priority because a mere progress has been made in this regard. Another reason is the lack of antiviral drug and licensed vaccine although there is a severe outbreak in Central Africa. In this study, we aimed to design an epitope-based vaccine that can trigger a significant immune response as well as to prognosticate inhibitor that can bind with potential drug target sites using various immunoinformatics and docking simulation tools. The capacity to induce both humoral and cell-mediated immunity by T cell and B cell was checked for the selected protein. The peptide region spanning 9 amino acids from 42 to 50 and the sequence TLASIGTAF were found as the most potential B and T cell epitopes, respectively. This peptide could interact with 12 HLAs and showed high population coverage up to 80.99%. Using molecular docking, the epitope was further appraised for binding against HLA molecules to verify the binding cleft interaction. In addition with this, the allergenicity of the epitopes was also evaluated. In the post-therapeutic strategy, docking study of predicted 3D structure identified suitable therapeutic inhibitor against targeted protein. However, this computational epitope-based peptide vaccine designing and target site prediction against EBOVs open up a new horizon which may be the prospective way in Ebola viruses research; the results require validation by in vitro and in vivo experiments. © 2015 John Wiley & Sons Ltd.

TargetSpy: a supervised machine learning approach for microRNA target prediction.

PubMed

Sturm, Martin; Hackenberg, Michael; Langenberger, David; Frishman, Dmitrij

2010-05-28

Virtually all currently available microRNA target site prediction algorithms require the presence of a (conserved) seed match to the 5' end of the microRNA. Recently however, it has been shown that this requirement might be too stringent, leading to a substantial number of missed target sites. We developed TargetSpy, a novel computational approach for predicting target sites regardless of the presence of a seed match. It is based on machine learning and automatic feature selection using a wide spectrum of compositional, structural, and base pairing features covering current biological knowledge. Our model does not rely on evolutionary conservation, which allows the detection of species-specific interactions and makes TargetSpy suitable for analyzing unconserved genomic sequences.In order to allow for an unbiased comparison of TargetSpy to other methods, we classified all algorithms into three groups: I) no seed match requirement, II) seed match requirement, and III) conserved seed match requirement. TargetSpy predictions for classes II and III are generated by appropriate postfiltering. On a human dataset revealing fold-change in protein production for five selected microRNAs our method shows superior performance in all classes. In Drosophila melanogaster not only our class II and III predictions are on par with other algorithms, but notably the class I (no-seed) predictions are just marginally less accurate. We estimate that TargetSpy predicts between 26 and 112 functional target sites without a seed match per microRNA that are missed by all other currently available algorithms. Only a few algorithms can predict target sites without demanding a seed match and TargetSpy demonstrates a substantial improvement in prediction accuracy in that class. Furthermore, when conservation and the presence of a seed match are required, the performance is comparable with state-of-the-art algorithms. TargetSpy was trained on mouse and performs well in human and drosophila

TargetSpy: a supervised machine learning approach for microRNA target prediction

PubMed Central

2010-01-01

Background Virtually all currently available microRNA target site prediction algorithms require the presence of a (conserved) seed match to the 5' end of the microRNA. Recently however, it has been shown that this requirement might be too stringent, leading to a substantial number of missed target sites. Results We developed TargetSpy, a novel computational approach for predicting target sites regardless of the presence of a seed match. It is based on machine learning and automatic feature selection using a wide spectrum of compositional, structural, and base pairing features covering current biological knowledge. Our model does not rely on evolutionary conservation, which allows the detection of species-specific interactions and makes TargetSpy suitable for analyzing unconserved genomic sequences. In order to allow for an unbiased comparison of TargetSpy to other methods, we classified all algorithms into three groups: I) no seed match requirement, II) seed match requirement, and III) conserved seed match requirement. TargetSpy predictions for classes II and III are generated by appropriate postfiltering. On a human dataset revealing fold-change in protein production for five selected microRNAs our method shows superior performance in all classes. In Drosophila melanogaster not only our class II and III predictions are on par with other algorithms, but notably the class I (no-seed) predictions are just marginally less accurate. We estimate that TargetSpy predicts between 26 and 112 functional target sites without a seed match per microRNA that are missed by all other currently available algorithms. Conclusion Only a few algorithms can predict target sites without demanding a seed match and TargetSpy demonstrates a substantial improvement in prediction accuracy in that class. Furthermore, when conservation and the presence of a seed match are required, the performance is comparable with state-of-the-art algorithms. TargetSpy was trained on mouse and performs well

RNA interference as a method for target-site screening in the Western Corn Rootworm, Diabrotica virgifera virgifera

USDA-ARS?s Scientific Manuscript database

RNA interference (RNAi) is one of the most powerful and extraordinarily-specific means by which to silence genes. The ability of RNAi to silence genes makes it possible to ascertain function from genomic data, thereby making it an excellent choice for target-site screening. To test the efficacy of...

A 40-bp VNTR polymorphism in the 3'-untranslated region of DAT1/SLC6A3 is associated with ADHD but not with alcoholism.

PubMed

Šerý, Omar; Paclt, Ivo; Drtílková, Ivana; Theiner, Pavel; Kopečková, Marta; Zvolský, Petr; Balcar, Vladimir J

2015-06-11

ADHD and alcoholism are psychiatric diseases with pathophysiology related to dopamine system. DAT1 belongs to the SLC6 family of transporters and is involved in the regulation of extracellular dopamine levels. A 40 bp variable number tandem repeat (VNTR) polymorphism in the 3'-untranslated region of DAT1/SLC6A3 gene was previously reported to be associated with various phenotypes involving disturbed regulation of dopaminergic neurotransmission. A total of 1312 subjects were included and genotyped for 40 bp VNTR polymorphism of DAT1/SLC6A3 gene in this study (441 alcoholics, 400 non-alcoholic controls, 218 ADHD children and 253 non ADHD children). Using miRBase software, we have performed a computer analysis of VNTR part of DAT1 gene for presence of miRNA binding sites. We have found significant relationships between ADHD and the 40 bp VNTR polymorphisms of DAT1/SLC6A3 gene (P < 0.01). The 9/9 genotype appeared to reduce the risk of ADHD about 0.4-fold (p < 0.04). We also noted an occurrence of rare genotypes in ADHD (frequency different from controls at p < 0.01). No association between alcoholism and genotype frequencies of 40 bp VNTR polymorphism of DAT1/SLC6A3 gene has been detected. We have found an association between 40 bp VNTR polymorphism of DAT1/SLC6A3 gene and ADHD in the Czech population; in a broad agreement with studies in other population samples. Furthermore, we detected rare genotypes 8/10, 7/10 and 10/11 present in ADHD boys only and identified miRNAs that should be looked at as potential novel targets in the research on ADHD.

Validation of microRNA pathway polymorphisms in esophageal adenocarcinoma survival.

PubMed

Faluyi, Olusola O; Eng, Lawson; Qiu, Xin; Che, Jiahua; Zhang, Qihuang; Cheng, Dangxiao; Ying, Nanjiao; Tse, Alvina; Kuang, Qin; Dodbiba, Lorin; Renouf, Daniel J; Marsh, Sharon; Savas, Sevtap; Mackay, Helen J; Knox, Jennifer J; Darling, Gail E; Wong, Rebecca K S; Xu, Wei; Azad, Abul Kalam; Liu, Geoffrey

2017-02-01

Polymorphisms in miRNA and miRNA pathway genes have been previously associated with cancer risk and outcome, but have not been studied in esophageal adenocarcinoma outcomes. Here, we evaluate candidate miRNA pathway polymorphisms in esophageal adenocarcinoma prognosis and attempt to validate them in an independent cohort of esophageal adenocarcinoma patients. Among 231 esophageal adenocarcinoma patients of all stages/treatment plans, 38 candidate genetic polymorphisms (17 biogenesis, 9 miRNA targets, 5 pri-miRNA, 7 pre-miRNA) were genotyped and analyzed. Cox proportional hazard models adjusted for sociodemographic and clinicopathological covariates helped assess the association of genetic polymorphisms with overall survival (OS) and progression-free survival (PFS). Significantly associated polymorphisms were then evaluated in an independent cohort of 137 esophageal adenocarcinoma patients. Among the 231 discovery cohort patients, 86% were male, median diagnosis age was 64 years, 34% were metastatic at diagnosis, and median OS and PFS were 20 and 12 months, respectively. GEMIN3 rs197412 (aHR = 1.37, 95%CI: [1.04-1.80]; P = 0.02), hsa-mir-124-1 rs531564 (aHR = 0.60, 95% CI: [0.53-0.90]; P = 0.05), and KIAA0423 rs1053667 (aHR = 0.51, 95% CI: [0.28-0.96]; P = 0.04) were found associated with OS. Furthermore, GEMIN3 rs197412 (aHR = 1.33, 95% CI: [1.03-1.74]; P = 0.03) and KRT81 rs3660 (aHR = 1.29, 95% CI: [1.01-1.64]; P = 0.04) were found associated with PFS. Although none of these polymorphisms were significant in the second cohort, hsa-mir-124-1 rs531564 and KIAA0423 rs1053667 had trends in the same direction; when both cohorts were combined together, GEMIN3 rs197412, hsa-mir-124-1 rs531564, and KIAA0423 rs1053667 remained significantly associated with OS. We demonstrate the association of multiple miRNA pathway polymorphisms with esophageal adenocarcinoma prognosis in a discovery cohort of patients, which did not validate in a separate cohort

Glioma Dual-Targeting Nanohybrid Protein Toxin Constructed by Intein-Mediated Site-Specific Ligation for Multistage Booster Delivery

PubMed Central

Chen, Yingzhi; Zhang, Meng; Jin, Hongyue; Li, Dongdong; Xu, Fan; Wu, Aihua; Wang, Jinyu; Huang, Yongzhuo

2017-01-01

Malignant glioma is one of the most untreatable cancers because of the formidable blood-brain barrier (BBB), through which few therapeutics can penetrate and reach the tumors. Biologics have been booming in cancer therapy in the past two decades, but their application in brain tumor has long been ignored due to the impermeable nature of BBB against effective delivery of biologics. Indeed, it is a long unsolved problem for brain delivery of macromolecular drugs, which becomes the Holy Grail in medical and pharmaceutical sciences. Even assisting by targeting ligands, protein brain delivery still remains challenging because of the synthesis difficulties of ligand-modified proteins. Herein, we propose a rocket-like, multistage booster delivery system of a protein toxin, trichosanthin (TCS), for antiglioma treatment. TCS is a ribosome-inactivating protein with the potent activity against various solid tumors but lack of specific action and cell penetration ability. To overcome the challenge of its poor druggability and site-specific modification, intein-mediated ligation was applied, by which a gelatinase-cleavable peptide and cell-penetrating peptide (CPP)-fused recombinant TCS toxin can be site-specifically conjugated to lactoferrin (LF), thus constructing a BBB-penetrating, gelatinase-activatable cell-penetrating nanohybrid TCS toxin. This nanohybrid TCS system is featured by the multistage booster strategy for glioma dual-targeting delivery. First, LF can target to the BBB-overexpressing low-density lipoprotein receptor-related protein-1 (LRP-1), and assist with BBB penetration. Second, once reaching the tumor site, the gelatinase-cleavable peptide acts as a separator responsive to the glioma-associated matrix metalloproteinases (MMPs), thus releasing to the CPP-fused toxin. Third, CPP mediates intratumoral and intracellular penetration of TCS toxin, thereby enhancing its antitumor activity. The BBB penetration and MMP-2-activability of this delivery system were

Site Targeted Press Coated Delivery of Methylprednisolone Using Eudragit RS 100 and Chitosan for Treatment of Colitis.

PubMed

Jagdale, Swati; Chandekar, Apoorva

2016-01-01

Inflammatory bowel disease (IBD) is one of the five most prevalent gastrointestinal disease burdens which commonly require lifetime care. Worldwide incidence rate of ulcerative colitis and Crohn's disease is about 16.8% and 13.4% respectively. Colitis is an inflammation of the colon. Colon targeted drug delivery will direct the drug to the colon. The drug will reach at the site of action and hence its side effects as well as dose can be reduced. Recent patent describes treatment of ulcerative colitis using anti CD3 antibodies, with nicotine and anti-depressant drugs, budesonide foam etc. Present study deals with optimization of site targeted methylprednisolone delivery for treatment of colitis. Chitosan and Eudragit RS 100 were used as coating polymers. Tablets were prepared by press coated technology. The core tablets contain drug, avicel as binder, croscarmellose sodium as super disintegrant and dicalcium phosphate as diluent. Drug excipient compatibility was carried out using FTIR, UV and DSC. Design of experiment was used to optimize the formulation. Tablets were evaluated for thickness, weight variation, hardness, swelling index, in-vitro drug release and release of drug in simulated media. Optimized batch (B2) contained chitosan 40% and eudragit RS 100 17.5%. B2 showed in-vitro drug release 85.65 ± 7.6% in 6.8 pH phosphate buffer and 96.7 ±9.1% in simulated media after 7.5 hours. In-vivo x-ray placebo study for formulation B2 had shown that the tablet reached to the ascending colon after 5 hours. This indicated a potential site targeted delivery of optimized batch B2.

Polymorphisms in the leptin gene promoter in Brazilian beef herds.

PubMed

Guimarães, R C; Azevedo, J S N; Corrêa, S C; Campelo, J E G; Barbosa, E M; Gonçalves, E C; Silva Filho, E

2016-12-02

Brazil is the world's largest producer of beef cattle; however, the quality of its herds needs to be improved. The use of molecular markers as auxiliary tools in selecting animals for reproduction with high pattern for beef production would significantly improve the quality of the final beef product in Brazil. The leptin gene has been demonstrated to be an excellent candidate gene for bovine breeding. The objective of this study was to sequence and compare the leptin gene promoter of Brazil's important cattle breeds in order to identify polymorphisms in it. Blood samples of the Nellore, Guzerat, Tabapuã, and Senepol breeds were collected for genomic DNA extraction. The genomic DNA was used as a template for polymerase chain reaction (PCR) to amplify a 1575-bp fragment, which in turn was sequenced, aligned, and compared between animals of different breeds. Twenty-three single nucleotide polymorphic sites, including transitions and transversions, were detected at positions -1457, -1452, -1446, -1397, -1392, -1361, -1238, -963,-901, -578, -516, -483, -478, -470, -432, -430, -292, -282, -272, -211, -202, -170, and -147. Additionally, two insertion sites at positions -680 and -416 and two deletion sites at positions -1255 and -1059 were detected. As the promoter region of the leptin gene has been demonstrated to vary among breeds, these variations must be tested for their use as potential molecular markers for artificial selection of animals for enhanced beef production in different systems of bovine production in Brazil.

Apolipoprotein B and E genetic polymorphisms in the Cayapa Indians of Ecuador.

PubMed

Scacchi, R; Corbo, R M; Rickards, O; Mantuano, E; Guevara, A; De Stefano, G F

1997-06-01

Three DNA polymorphisms (XbaI, EcoRI, and Ins/Del) of the apolipoprotein B (APOB) gene and the CfoI polymorphism of the APOE gene were investigated in a sample of 96 Cayapa Indians from Ecuador. The frequencies of the X+ (0.182), R+ (1.000), and Del alleles (0.432) at the three APOB sites were found to be higher than and to differ significantly from those reported for East Asians. No comparisons could be made between the Cayapa and other native Americans because of the lack of data on these sites. We observed in our sample that, like native American populations but unlike East Asians, the APOE allele frequencies were characterized by the absence of the APOE*2 allele and by a high frequency of the APOE*4 allele (0.280). Besides a probable drift effect, the high APOE*4 value was tentatively attributed to an effect of selection. Because this allele enhances the absorption of cholesterol by the intestine, it could confer an advantage to carriers in an unfavorable environment (i.e., diet poor in cholesterol).

A new single-nucleotide polymorphism database for rainbow trout generated through whole genome re-sequencing

USDA-ARS?s Scientific Manuscript database

Single-nucleotide polymorphisms (SNPs) are highly abundant markers, which are broadly distributed in animal genomes. For rainbow trout, SNP discovery has been done through sequencing of restriction-site associated DNA (RAD) libraries, reduced representation libraries (RRL), RNA sequencing, and whole...

Single nucleotide polymorphism analysis using different colored dye dimer probes

NASA Astrophysics Data System (ADS)

Marmé, Nicole; Friedrich, Achim; Denapaite, Dalia; Hakenbeck, Regine; Knemeyer, Jens-Peter

2006-09-01

Fluorescence quenching by dye dimer formation has been utilized to develop hairpin-structured DNA probes for the detection of a single nucleotide polymorphism (SNP) in the penicillin target gene pbp2x, which is implicated in the penicillin resistance of Streptococcus pneumoniae. We designed two specific DNA probes for the identification of the pbp2x genes from a penicillin susceptible strain R6 and a resistant strain Streptococcus mitis 661 using green-fluorescent tetramethylrhodamine (TMR) and red-fluorescent DY-636, respectively. Hybridization of each of the probes to its respective target DNA sequence opened the DNA hairpin probes, consequently breaking the nonfluorescent dye dimers into fluorescent species. This hybridization of the target with the hairpin probe achieved single nucleotide specific detection at nanomolar concentrations via increased fluorescence.

MAPK Target Sites of Eyes Absent Are Not Required for Eye Development or Survival in Drosophila

PubMed Central

Jusiak, Barbara; Abulimiti, Abuduaini; Haelterman, Nele; Chen, Rui; Mardon, Graeme

2012-01-01

Eyes absent (Eya) is a highly conserved transcription cofactor and protein phosphatase that plays an essential role in eye development and survival in Drosophila. Ectopic eye induction assays using cDNA transgenes have suggested that mitogen activated protein kinase (MAPK) activates Eya by phosphorylating it on two consensus target sites, S402 and S407, and that this activation potentiates the ability of Eya to drive eye formation. However, this mechanism has never been tested in normal eye development. In the current study, we generated a series of genomic rescue transgenes to investigate how loss- and gain-of-function mutations at these two MAPK target sites within Eya affect Drosophila survival and normal eye formation: eya+GR, the wild-type control; eyaSAGR, which lacks phosphorylation at the two target residues; and eyaSDEGR, which contains phosphomimetic amino acids at the same two residues. Contrary to the previous studies in ectopic eye development, all eya genomic transgenes tested rescue both eye formation and survival equally effectively. We conclude that, in contrast to ectopic eye formation, MAPK-mediated phosphorylation of Eya on S402 and S407 does not play a role in normal development. This is the first study in Drosophila to evaluate the difference in outcomes between genomic rescue and ectopic cDNA-based overexpression of the same gene. These findings indicate similar genomic rescue strategies may prove useful for re-evaluating other long-standing Drosophila developmental models. PMID:23251383

[Polymorphic loci and polymorphism analysis of short tandem repeats within XNP gene].

PubMed

Liu, Qi-Ji; Gong, Yao-Qin; Guo, Chen-Hong; Chen, Bing-Xi; Li, Jiang-Xia; Guo, Yi-Shou

2002-01-01

To select polymorphic short tandem repeat markers within X-linked nuclear protein (XNP) gene, genomic clones which contain XNP gene were recognized by homologous analysis with XNP cDNA. By comparing the cDNA with genomic DNA, non-exonic sequences were identified, and short tandem repeats were selected from non-exonic sequences by using BCM search Launcher. Polymorphisms of the short tandem repeats in Chinese population were evaluated by PCR amplification and PAGE. Five short tandem repeats were identified from XNP gene, two of which were polymorphic. Four and 11 alleles were observed in Chinese population for XNPSTR1 and XNPSTR4, respectively. Heterozygosities were 47% for XNPSTR1 and 70% for XNPSTR4. XNPSTR1 and XNPSTR4 localized within 3' end and intron 10, respectively. Two polymorphic short tandem repeats have been identified within XNP gene and will be useful for linkage analysis and gene diagnosis of XNP gene.

Molecular nucleation mechanisms and control strategies for crystal polymorph selection.

PubMed

Van Driessche, Alexander E S; Van Gerven, Nani; Bomans, Paul H H; Joosten, Rick R M; Friedrich, Heiner; Gil-Carton, David; Sommerdijk, Nico A J M; Sleutel, Mike

2018-04-04

The formation of condensed (compacted) protein phases is associated with a wide range of human disorders, such as eye cataracts, amyotrophic lateral sclerosis, sickle cell anaemia and Alzheimer's disease. However, condensed protein phases have their uses: as crystals, they are harnessed by structural biologists to elucidate protein structures, or are used as delivery vehicles for pharmaceutical applications. The physiochemical properties of crystals can vary substantially between different forms or structures ('polymorphs') of the same macromolecule, and dictate their usability in a scientific or industrial context. To gain control over an emerging polymorph, one needs a molecular-level understanding of the pathways that lead to the various macroscopic states and of the mechanisms that govern pathway selection. However, it is still not clear how the embryonic seeds of a macromolecular phase are formed, or how these nuclei affect polymorph selection. Here we use time-resolved cryo-transmission electron microscopy to image the nucleation of crystals of the protein glucose isomerase, and to uncover at molecular resolution the nucleation pathways that lead to two crystalline states and one gelled state. We show that polymorph selection takes place at the earliest stages of structure formation and is based on specific building blocks for each space group. Moreover, we demonstrate control over the system by selectively forming desired polymorphs through site-directed mutagenesis, specifically tuning intermolecular bonding or gel seeding. Our results differ from the present picture of protein nucleation, in that we do not identify a metastable dense liquid as the precursor to the crystalline state. Rather, we observe nucleation events that are driven by oriented attachments between subcritical clusters that already exhibit a degree of crystallinity. These insights suggest ways of controlling macromolecular phase transitions, aiding the development of protein-based drug

Polymorphism of antibiotic-inactivating enzyme driven by ecology expands the environmental resistome.

PubMed

Kim, Dae-Wi; Thawng, Cung Nawl; Choi, Jung-Hye; Lee, Kihyun; Cha, Chang-Jun

2018-01-01

The environmental resistome has been recognized as the origin and reservoir of antibiotic resistance genes and considered to be dynamic and ever expanding. In this study, a targeted gene sequencing approach revealed that the polymorphic diversity of the aminoglycoside-inactivating enzyme AAC(6')-Ib was ecological niche-specific. AAC(6')-Ib-cr, previously known as a clinical variant, was prevalent in various soils and the intestines of chickens and humans, suggesting that this variant might not have arisen from adaptive mutations in the clinic but instead originated from the environment. Furthermore, ecologically dominant polymorphic variants of AAC(6')-Ib were characterized and found to display different substrate specificities for quinolones and aminoglycosides, conferring the altered resistance spectra. Interestingly, a novel variant with the D179Y substitution showed an extended resistance spectrum to the recently developed fluoroquinolone gemifloxacin. Our results suggest that soil and animal microbiomes could be major reservoirs of antibiotic resistance; polymorphic diversity expands the antibiotic resistome in the environment, resulting in the potential emergence of novel resistance.

Site classification of ponderosa pine stands under stocking control in California

Treesearch

Robert F. Powers; William W. Oliver

1978-01-01

Existing systems for estimating site index of ponderosa pine (Pinus ponderosa Laws.) do not apply well to California stands where stocking is controlled. A more suitable system has been developed using trends in natural height growth, derived from stem analysis of dominant trees in California. This site index system produces polymorphic patterns of...

Diversifying Selection Underlies the Origin of Allozyme Polymorphism at the Phosphoglucose Isomerase Locus in Tigriopus californicus

PubMed Central

Schoville, Sean D.; Flowers, Jonathan M.; Burton, Ronald S.

2012-01-01

The marine copepod Tigriopus californicus lives in intertidal rock pools along the Pacific coast, where it exhibits strong, temporally stable population genetic structure. Previous allozyme surveys have found high frequency private alleles among neighboring subpopulations, indicating that there is limited genetic exchange between populations. Here we evaluate the factors responsible for the diversification and maintenance of alleles at the phosphoglucose isomerase (Pgi) locus by evaluating patterns of nucleotide variation underlying previously identified allozyme polymorphism. Copepods were sampled from eleven sites throughout California and Baja California, revealing deep genetic structure among populations as well as genetic variability within populations. Evidence of recombination is limited to the sample from Pescadero and there is no support for linkage disequilibrium across the Pgi locus. Neutrality tests and codon-based models of substitution suggest the action of natural selection due to elevated non-synonymous substitutions at a small number of sites in Pgi. Two sites are identified as the charge-changing residues underlying allozyme polymorphisms in T. californicus. A reanalysis of allozyme variation at several focal populations, spanning a period of 26 years and over 200 generations, shows that Pgi alleles are maintained without notable frequency changes. Our data suggest that diversifying selection accounted for the origin of Pgi allozymes, while McDonald-Kreitman tests and the temporal stability of private allozyme alleles suggests that balancing selection may be involved in the maintenance of amino acid polymorphisms within populations. PMID:22768211

Molecular and functional characterization of polymorphisms in the secreted phospholipase A2 group X gene: relevance to coronary artery disease.

PubMed

Gora, Sarah; Perret, Claire; Jemel, Ikram; Nicaud, Viviane; Lambeau, Gérard; Cambien, François; Ninio, Ewa; Blankenberg, Stefan; Tiret, Laurence; Karabina, Sonia-Athina

2009-07-01

Among secreted phospholipases A2 (sPLA2s), human group X sPLA2 (hGX sPLA2) is emerging as a novel attractive therapeutic target due to its implication in inflammatory diseases. To elucidate whether hGX sPLA2 plays a causative role in coronary artery disease (CAD), we screened the human PLA2G10 gene to identify polymorphisms and possible associations with CAD end-points in a prospective study, AtheroGene. We identified eight polymorphisms, among which, one non-synonymous polymorphism R38C in the propeptide region of the sPLA2. The T-512C polymorphism located in the 5' untranslated region was associated with a decreased risk of recurrent cardiovascular events during follow-up. The functional analysis of the R38C polymorphism showed that it leads to a profound change in expression and activity of hGX sPLA2, although there was no detectable impact on CAD risk. Due to the potential role of hGX sPLA2 in inflammatory processes, these polymorphisms should be investigated in other inflammatory diseases.

Novel therapeutic approaches for pulmonary arterial hypertension: Unique molecular targets to site-specific drug delivery.

PubMed

Vaidya, Bhuvaneshwar; Gupta, Vivek

2015-08-10

Pulmonary arterial hypertension (PAH) is a cardiopulmonary disorder characterized by increased blood pressure in the small arterioles supplying blood to lungs for oxygenation. Advances in understanding of molecular and cellular biology techniques have led to the findings that PAH is indeed a cascade of diseases exploiting multi-faceted complex pathophysiology, with cellular proliferation and vascular remodeling being the key pathogenic events along with several cellular pathways involved. While current therapies for PAH do provide for amelioration of disease symptoms and acute survival benefits, their full therapeutic potential is hindered by patient incompliance and off-target side effects. To overcome the issues related with current therapy and to devise a more selective therapy, various novel pathways are being investigated for PAH treatment. In addition, inability to deliver anti-PAH drugs to the disease site i.e., distal pulmonary arterioles has been one of the major challenges in achieving improved patient outcomes and improved therapeutic efficacy. Several novel carriers have been explored to increase the selectivity of currently approved anti-PAH drugs and to act as suitable carriers for the delivery of investigational drugs. In the present review, we have discussed potential of various novel molecular pathways/targets including RhoA/Rho kinase, tyrosine kinase, endothelial progenitor cells, vasoactive intestinal peptide, and miRNA in PAH therapeutics. We have also discussed various techniques for site-specific drug delivery of anti-PAH therapeutics so as to improve the efficacy of approved and investigational drugs. This review will provide gainful insights into current advances in PAH therapeutics with an emphasis on site-specific drug payload delivery. Copyright © 2015 Elsevier B.V. All rights reserved.

Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing

PubMed Central

Fukuda, Masatora; Kurihara, Kei; Yamaguchi, Shota; Oyama, Yui; Deshimaru, Masanobu

2014-01-01

Adenosine-to-inosine (A-to-I) RNA editing is an endogenous regulatory mechanism involved in various biological processes. Site-specific, editing-state–dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. Previously, we designed an artificial hammerhead ribozyme (HHR) for selective, site-specific RNA cleavage dependent on the A-to-I RNA editing state. In the present work, we developed an improved strategy for constructing a trans-acting HHR that specifically cleaves target editing sites in the adenosine but not the inosine state. Specificity for unedited sites was achieved by utilizing a sequence encoding the intrinsic cleavage specificity of a natural HHR. We used in vitro selection methods in an HHR library to select for an extended HHR containing a tertiary stabilization motif that facilitates HHR folding into an active conformation. By using this method, we successfully constructed highly active HHRs with unedited-specific cleavage. Moreover, using HHR cleavage followed by direct sequencing, we demonstrated that this ribozyme could cleave serotonin 2C receptor (HTR2C) mRNA extracted from mouse brain, depending on the site-specific editing state. This unedited-specific cleavage also enabled us to analyze the effect of editing state at the E and C sites on editing at other sites by using direct sequencing for the simultaneous quantification of the editing ratio at multiple sites. Our approach has the potential to elucidate the mechanism underlying the interdependencies of different editing states in substrate RNA with multiple editing sites. PMID:24448449

Glutathione enzyme and selenoprotein polymorphisms associate with mercury biomarker levels in Michigan dental professionals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goodrich, Jaclyn M.; Wang, Yi; Gillespie, Brenda

Mercury is a potent toxicant of concern to both the general public and occupationally exposed workers (e.g., dentists). Recent studies suggest that several genes mediating the toxicokinetics of mercury are polymorphic in humans and may influence inter-individual variability in mercury accumulation. This work hypothesizes that polymorphisms in key glutathione synthesizing enzyme, glutathione s-transferase, and selenoprotein genes underlie inter-individual differences in mercury body burden as assessed by analytical mercury measurement in urine and hair, biomarkers of elemental mercury and methylmercury, respectively. Urine and hair samples were collected from a population of dental professionals (n = 515), and total mercury content wasmore » measured. Average urine (1.06 {+-} 1.24 ug/L) and hair mercury levels (0.49 {+-} 0.63 ug/g) were similar to national U.S. population averages. Taqman assays were used to genotype DNA from buccal swab samples at 15 polymorphic sites in genes implicated in mercury metabolism. Linear regression modeling assessed the ability of polymorphisms to modify the relationship between mercury biomarker levels and exposure sources (e.g., amalgams, fish consumption). Five polymorphisms were significantly associated with urine mercury levels (GSTT1 deletion), hair mercury levels (GSTP1-105, GSTP1-114, GSS 5 Prime ), or both (SEPP1 3 Prime UTR). Overall, this study suggests that polymorphisms in selenoproteins and glutathione-related genes may influence elimination of mercury in the urine and hair or mercury retention following exposures to elemental mercury (via dental amalgams) and methylmercury (via fish consumption). -- Highlights: Black-Right-Pointing-Pointer We explore the influence of 15 polymorphisms on urine and hair Hg levels. Black-Right-Pointing-Pointer Urine and hair Hg levels in dental professionals were similar to the US population. Black-Right-Pointing-Pointer GSTT1 and SEPP1 polymorphisms associated with urine Hg levels. Black

Association between SLC11A1 (NRAMP1) polymorphisms and susceptibility to tuberculosis in Chinese Holstein cattle.

PubMed

Liu, Kaihua; Zhang, Bin; Teng, Zhaochun; Wang, Youtao; Dong, Guodong; Xu, Cong; Qin, Bo; Song, Chunlian; Chai, Jun; Li, Yang; Shi, Xianwei; Shu, Xianghua; Zhang, Yifang

2017-03-01

We investigated the associations between SLC11A1 polymorphisms and susceptibility to tuberculosis (TB) in Chinese Holstein cattle, using a case-control study of 136 animals that had positive reactions to TB tests and showed symptoms and 96 animals that had negative reactions to tests and showed no symptoms. Polymerase chain reaction (PCR) sequencing and the restriction fragment length polymorphism (RFLP) technique were used to detect and determine SLC11A1 polymorphisms. Association analysis identified significant correlations between SLC11A1 polymorphisms and susceptibility/resistance to TB, and two genetic markers for SLC11A1 were established using PCR-RFLP. Sequence alignment of SLC11A1 revealed seven single-nucleotide polymorphisms (SNPs). This is the first report of MaeII PCR-RFLP markers for the SLC11A1-SNP3 site and PstI PCR-RFLP markers for the SLC11A1-SNP5 and SLC11A1-SNP6 sites in Chinese Holstein cattle. Logistic regression analysis indicated that SLC11A1-SNP1, SLC11A1-SNP3, and SLC11A1-SNP5 were significantly associated with susceptibility/resistance to TB. Two genotypes of SLC11A1-SNP3 were susceptible to TB, whereas one genotype of SLC11A1-SNP1 and two genotypes of SLC11A1-SNP5 were resistant. Haplotype analysis showed that nine haplotypes were potentially resistant to TB. After Bonferroni correction, three of the haplotypes remained significantly associated with TB resistance. SLC11A1 is a useful candidate gene related to TB in Chinese Holstein cattle. Copyright © 2016 Elsevier Ltd. All rights reserved.

Single nucleotide polymorphisms in the mitochondrial displacement loop and outcome of esophageal squamous cell carcinoma.

PubMed

Zhang, Ruixing; Wang, Rui; Zhang, Fengbin; Wu, Chensi; Fan, Haiyan; Li, Yan; Wang, Cuiju; Guo, Zhanjun

2010-11-26

Accumulation of single nucleotide polymorphisms (SNPs) in the displacement loop (D-loop) of mitochondrial DNA (mtDNA) has been described for different types of cancers and might be associated with cancer risk and disease outcome. We used a population-based series of esophageal squamous cell carcinoma (ESCC) patients for investigating the prediction power of SNPs in mitochondrial D-loop. The D-loop region of mtDNA was sequenced for 60 ESCC patients recorded in the Fourth Hospital of Hebei Medical University between 2003 and 2004. The 5 year survival curve were calculated with the Kaplan-Meier method and compared by the log-rank test at each SNP site, a multivariate survival analysis was also performed with the Cox proportional hazards method. The SNP sites of nucleotides 16274G/A, 16278C/T and 16399A/G were identified for prediction of post-operational survival by the log-rank test. In an overall multivariate analysis, the 16278 and 16399 alleles were identified as independent predictors of ESCC outcome. The length of survival of patients with the minor allele 16278T genotype was significantly shorter than that of patients with 16278C at the 16278 site (relative risk, 3.001; 95% CI, 1.029 - 8.756; p = 0.044). The length of survival of patients with the minor allele 16399G genotype was significantly shorter than that of patients with the more frequent allele 16399A at the 16399 site in ESCC patients (relative risk, 3.483; 95% CI, 1.068 - 11.359; p = 0.039). Genetic polymorphisms in the D-loop are independent prognostic markers for patients with ESCC. Accordingly, the analysis of genetic polymorphisms in the mitochondrial D-loop can help identify patient subgroups at high risk of a poor disease outcome.

Association between XRCC1 polymorphisms and laryngeal cancer susceptibility in a Chinese sample population.

PubMed

Wu, W Q; Zhang, L S; Liao, S P; Lin, X L; Zeng, J; Du, D

2016-10-05

Laryngeal cancer is the major malignant tumor affecting the upper respiratory tract. Previous studies have reported on the association between XRCC1 genetic polymorphisms and risk of laryngeal cancer, but with conflicting results. In this study, we attempted to assess the association between XRCC1 Arg194Trp, Arg280His and Arg399Gln polymorphisms and risk of laryngeal cancer in a Chinese population. A total of 126 laryngeal cancer patients and 254 control subjects were recruited to this study from the Second Medical College of Jinan University between December 2013 and May 2015. The XRCC1 Arg194Trp, Arg280His, and Arg399Gln polymorphic sites were genotyped by polymerase chain reaction-restriction fragment length polymorphism. Our results revealed a significant association between the AA genotype of XRCC1 Arg280His [odds ratio (OR) = 2.51, 95% confidence interval (CI) = 1.29-4.87, P = 0.01] and an increased risk of laryngeal cancer susceptibility compared to the GG genotype. Moreover, the A allele showed a higher risk of laryngeal cancer susceptibility compared to the G allele (OR = 1.63, 95%CI = 1.19-2.50, P = 0.002). In conclusion, the results of our study suggest that the AA genotype and A allele of the XRCC1 Arg280His polymorphism are associated with an increased laryngeal cancer risk in a Chinese population.

Onco-Regulon: an integrated database and software suite for site specific targeting of transcription factors of cancer genes

PubMed Central

Tomar, Navneet; Mishra, Akhilesh; Mrinal, Nirotpal; Jayaram, B.

2016-01-01

Transcription factors (TFs) bind at multiple sites in the genome and regulate expression of many genes. Regulating TF binding in a gene specific manner remains a formidable challenge in drug discovery because the same binding motif may be present at multiple locations in the genome. Here, we present Onco-Regulon (http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm), an integrated database of regulatory motifs of cancer genes clubbed with Unique Sequence-Predictor (USP) a software suite that identifies unique sequences for each of these regulatory DNA motifs at the specified position in the genome. USP works by extending a given DNA motif, in 5′→3′, 3′ →5′ or both directions by adding one nucleotide at each step, and calculates the frequency of each extended motif in the genome by Frequency Counter programme. This step is iterated till the frequency of the extended motif becomes unity in the genome. Thus, for each given motif, we get three possible unique sequences. Closest Sequence Finder program predicts off-target drug binding in the genome. Inclusion of DNA-Protein structural information further makes Onco-Regulon a highly informative repository for gene specific drug development. We believe that Onco-Regulon will help researchers to design drugs which will bind to an exclusive site in the genome with no off-target effects, theoretically. Database URL: http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm PMID:27515825

Evolutionary Determinants of Morphological Polymorphism in Colonial Animals.

PubMed

Simpson, Carl; Jackson, Jeremy B C; Herrera-Cubilla, Amalia

2017-07-01

Colonial animals commonly exhibit morphologically polymorphic modular units that are phenotypically distinct and specialize in specific functional tasks. But how and why these polymorphic modules have evolved is poorly understood. Across colonial invertebrates, there is wide variation in the degree of polymorphism, from none in colonial ascidians to extreme polymorphism in siphonophores, such as the Portuguese man-of-war. Bryozoa are a phylum of exclusively colonial invertebrates that uniquely exhibit almost the entire range of polymorphism, from monomorphic species to others that rival siphonophores in their polymorphic complexity. Previous approaches to understanding the evolution of polymorphism have been based on analyses of (1) the functional role of polymorphs or (2) presumed evolutionary costs and benefits based on evolutionary theory that postulates polymorphism should be evolutionarily sustainable only in more stable environments because polymorphism commonly leads to the loss of feeding and sexual competence. Here we use bryozoans from opposite shores of the Isthmus of Panama to revisit the environmental hypothesis by comparison of faunas from distinct oceanographic provinces that differ greatly in environmental variability, and we then examine the correlations between the extent of polymorphism in relation to patterns of ecological succession and variation in life histories. We find no support for the environmental hypothesis. Distributions of the incidence of polymorphism in the oceanographically unstable Eastern Pacific are indistinguishable from those in the more stable Caribbean. In contrast, the temporal position of species in a successional sequence is collinear with the degree of polymorphism because species with fewer types of polymorphs are competitively replaced by species with higher numbers of polymorphs on the same substrata. Competitively dominant species also exhibit patterns of growth that increase their competitive ability. The

A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep.

PubMed

Clop, Alex; Marcq, Fabienne; Takeda, Haruko; Pirottin, Dimitri; Tordoir, Xavier; Bibé, Bernard; Bouix, Jacques; Caiment, Florian; Elsen, Jean-Michel; Eychenne, Francis; Larzul, Catherine; Laville, Elisabeth; Meish, Françoise; Milenkovic, Dragan; Tobin, James; Charlier, Carole; Georges, Michel

2006-07-01

Texel sheep are renowned for their exceptional meatiness. To identify the genes underlying this economically important feature, we performed a whole-genome scan in a Romanov x Texel F2 population. We mapped a quantitative trait locus with a major effect on muscle mass to chromosome 2 and subsequently fine-mapped it to a chromosome interval encompassing the myostatin (GDF8) gene. We herein demonstrate that the GDF8 allele of Texel sheep is characterized by a G to A transition in the 3' UTR that creates a target site for mir1 and mir206, microRNAs (miRNAs) that are highly expressed in skeletal muscle. This causes translational inhibition of the myostatin gene and hence contributes to the muscular hypertrophy of Texel sheep. Analysis of SNP databases for humans and mice demonstrates that mutations creating or destroying putative miRNA target sites are abundant and might be important effectors of phenotypic variation.

Abundant off-target edits from site-directed RNA editing can be reduced by nuclear localization of the editing enzyme.

PubMed

Vallecillo-Viejo, Isabel C; Liscovitch-Brauer, Noa; Montiel-Gonzalez, Maria Fernanda; Eisenberg, Eli; Rosenthal, Joshua J C

2018-01-02

Site-directed RNA editing (SDRE) is a general strategy for making targeted base changes in RNA molecules. Although the approach is relatively new, several groups, including our own, have been working on its development. The basic strategy has been to couple the catalytic domain of an adenosine (A) to inosine (I) RNA editing enzyme to a guide RNA that is used for targeting. Although highly efficient on-target editing has been reported, off-target events have not been rigorously quantified. In this report we target premature termination codons (PTCs) in messages encoding both a fluorescent reporter protein and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein transiently transfected into human epithelial cells. We demonstrate that while on-target editing is efficient, off-target editing is extensive, both within the targeted message and across the entire transcriptome of the transfected cells. By redirecting the editing enzymes from the cytoplasm to the nucleus, off-target editing is reduced without compromising the on-target editing efficiency. The addition of the E488Q mutation to the editing enzymes, a common strategy for increasing on-target editing efficiency, causes a tremendous increase in off-target editing. These results underscore the need to reduce promiscuity in current approaches to SDRE.

4G/5G Plasminogen Activator Inhibitor-1 Polymorphisms and Haplotypes Are Associated with Pneumonia

PubMed Central

Yende, Sachin; Angus, Derek C.; Ding, Jingzhong; Newman, Anne B.; Kellum, John A.; Li, Rongling; Ferrell, Robert E.; Zmuda, Joseph; Kritchevsky, Stephen B.; Harris, Tamara B.; Garcia, Melissa; Yaffe, Kristine; Wunderink, Richard G.

2007-01-01

Rationale: Plasminogen activator inhibitor (PAI)-1 inhibits urokinase and tissue plasminogen activator, required for host response to infection. Whether variation within the PAI-1 gene is associated with increased susceptibility to infection is unknown. Objectives: To ascertain the role of the 4G/5G polymorphism and other genetic variants within the PAI-1 gene. We hypothesized that variants associated with increased PAI-1 expression would be associated with an increased occurrence of community-acquired pneumonia (CAP). Methods: Longitudinal analysis (>12 yr) of the Health, Aging, and Body Composition cohort, aged 65–74 years at start of analysis. Measurements and Main Results: We genotyped the 4G/5G PAI-1 polymorphism and six additional single nucleotide polymorphisms. Of the 3,075 subjects, 272 (8.8%) had at least one hospitalization for CAP. Among whites, variants at the PAI4G,5G, PAI2846, and PAI7343 sites had higher risk of CAP (P = 0.018, 0.021, and 0.021, respectively). At these sites, variants associated with higher PAI-1 expression were associated with increased CAP susceptibility. Compared with the 5G/5G genotypes at PAI4G,5G site, the 4G/4G and 4G/5G genotypes were associated with a 1.98-fold increased risk of CAP (95% confidence interval, 1.2–3.2; P = 0.006). In whole blood stimulation assay, subjects with a 4G allele had 3.3- and 1.9-fold increased PAI-1 expression (P = 0.043 and 0.034, respectively). In haplotype analysis, the 4G/G/C/A haplotype at the PAI4G,5G, PAI2846, PAI4588, and PAI7343 single nucleotide polymorphisms was associated with higher CAP susceptibility, whereas the 5G/G/C/A haplotype was associated with lower CAP susceptibility. No associations were seen among blacks. Conclusions: Genotypes associated with increased expression of PAI-1 were associated with increased susceptibility to CAP in elderly whites. PMID:17761618

Max-E47, a Designed Minimalist Protein that Targets the E-Box DNA Site In Vivo and In Vitro

PubMed Central

Xu, Jing; Chen, Gang; De Jong, Antonia T.; Shahravan, S. Hesam; Shin, Jumi A.

2009-01-01

Max-E47 is a designed hybrid protein comprising the Max DNA-binding basic region and E47 HLH dimerization subdomain. In the yeast one-hybrid system (Y1H), Max-E47 shows strong transcriptional activation from the E-box site, 5'-CACGTG, targeted by the Myc/Max/Mad network of transcription factors; two mutants, Max-E47Y and Max-E47YF, activate more weakly from the E-box in the Y1H. Quantitative fluorescence anisotropy titrations to gain free energies of protein:DNA binding gave low nM Kd values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14 nM, 15 nM, 9 nM, and 6 nM, respectively), with no detectable binding to a nonspecific control duplex. Because these minimalist, E-box-binding hybrids have no activation domain and no interactions with the c-MycbHLHZ, as shown by the yeast two-hybrid assay, they can potentially serve as dominant-negative inhibitors that suppress activation of E-box-responsive genes targeted by transcription factors including the c-Myc/Max complex. As proof-of-principle, we used our modified Y1H, which allows direct competition between two proteins vying for a DNA target, to show that Max-E47 effectively outcompetes the native MaxbHLHZ for the E-box; weaker competition is observed from the two mutants, consistent with Y1H results. These hybrids provide a minimalist scaffold for further exploration of the relationship between protein structure and DNA-binding function and may have applications as protein therapeutics or biochemical probes capable of targeting the E-box site. PMID:19449889

Lack of association between JAK3 gene polymorphisms and cardiovascular disease in Spanish patients with rheumatoid arthritis.

PubMed

García-Bermúdez, Mercedes; López-Mejías, Raquel; Genre, Fernanda; Castañeda, Santos; Corrales, Alfonso; Llorca, Javier; González-Juanatey, Carlos; Ubilla, Begoña; Miranda-Filloy, José A; Pina, Trinitario; Gómez-Vaquero, Carmen; Rodríguez-Rodríguez, Luis; Fernández-Gutiérrez, Benjamín; Balsa, Alejandro; Pascual-Salcedo, Dora; López-Longo, Francisco J; Carreira, Patricia; Blanco, Ricardo; Martín, Javier; González-Gay, Miguel A

2015-01-01

Rheumatoid arthritis (RA) is a polygenic disease associated with accelerated atherosclerosis and increased cardiovascular (CV) mortality. JAK/STAT signalling pathway is involved in autoimmune diseases and in the atherosclerotic process. JAK3 is a highly promising target for immunomodulatory drugs and polymorphisms in JAK3 gene have been associated with CV events in incident dialysis patients. Therefore, the aim of this study was to assess the potential role of JAK3 polymorphisms in the development of CV disease in patients with RA. 2136 Spanish RA patients were genotyped for the rs3212780 and rs3212752 JAK3 gene polymorphisms by TaqMan assays. Subclinical atherosclerosis was evaluated in 539 of these patients by carotid ultrasonography (US). No statistically significant differences were found when each polymorphism was assessed according to carotid intima-media thickness values and presence/absence of carotid plaques in RA, after adjusting the results for potential confounders. Moreover, no significant differences were obtained when RA patients were stratified according to the presence/absence of CV events after adjusting for potential confounders. In conclusion, our results do not confirm association between JAK3 polymorphisms and CV disease in RA.

Lack of Association between JAK3 Gene Polymorphisms and Cardiovascular Disease in Spanish Patients with Rheumatoid Arthritis

PubMed Central

García-Bermúdez, Mercedes; López-Mejías, Raquel; Castañeda, Santos; Corrales, Alfonso; González-Juanatey, Carlos; Ubilla, Begoña; Miranda-Filloy, José A.; Pina, Trinitario; Gómez-Vaquero, Carmen; Rodríguez-Rodríguez, Luis; Fernández-Gutiérrez, Benjamín; Balsa, Alejandro; Pascual-Salcedo, Dora; López-Longo, Francisco J.; Carreira, Patricia; Blanco, Ricardo; Martín, Javier; González-Gay, Miguel A.

2015-01-01

Rheumatoid arthritis (RA) is a polygenic disease associated with accelerated atherosclerosis and increased cardiovascular (CV) mortality. JAK/STAT signalling pathway is involved in autoimmune diseases and in the atherosclerotic process. JAK3 is a highly promising target for immunomodulatory drugs and polymorphisms in JAK3 gene have been associated with CV events in incident dialysis patients. Therefore, the aim of this study was to assess the potential role of JAK3 polymorphisms in the development of CV disease in patients with RA. 2136 Spanish RA patients were genotyped for the rs3212780 and rs3212752 JAK3 gene polymorphisms by TaqMan assays. Subclinical atherosclerosis was evaluated in 539 of these patients by carotid ultrasonography (US). No statistically significant differences were found when each polymorphism was assessed according to carotid intima-media thickness values and presence/absence of carotid plaques in RA, after adjusting the results for potential confounders. Moreover, no significant differences were obtained when RA patients were stratified according to the presence/absence of CV events after adjusting for potential confounders. In conclusion, our results do not confirm association between JAK3 polymorphisms and CV disease in RA. PMID:25815310

Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR.

PubMed

Kamau, Everlyn; Agoti, Charles N; Lewa, Clement S; Oketch, John; Owor, Betty E; Otieno, Grieven P; Bett, Anne; Cane, Patricia A; Nokes, D James

2017-03-01

Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples. N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses. An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Multianalyte, dipstick-type, nanoparticle-based DNA biosensor for visual genotyping of single-nucleotide polymorphisms.

PubMed

Litos, Ioannis K; Ioannou, Penelope C; Christopoulos, Theodore K; Traeger-Synodinos, Jan; Kanavakis, Emmanuel

2009-06-15

DNA biosensors involve molecular recognition of the target sequence by hybridization with specific probes and detection by electrochemical, optical or gravimetric transduction. Disposable, dipstick-type biosensors have been developed recently, which enable visual detection of DNA without using instruments. In this context, we report a multianalyte DNA biosensor for visual genotyping of two single-nucleotide polymorphisms (SNPs). As a model, the biosensor was applied to the simultaneous genotyping of two SNPs, entailing the detection of four alleles. A PCR product that flanks both polymorphic sites is subjected to a single primer extension (PEXT) reaction employing four allele-specific primers, each containing a region complementary to an allele and a characteristic segment that enables subsequent capture on a test zone of the biosensor. The primers are extended with dNTPs and biotin-dUTP only if there is perfect complementarity with the interrogated sequence. The PEXT mixture is applied to the biosensor. As the developing buffer migrates along the strip, all the allele-specific primers are captured by immobilized oligonucleotides at the four test zones of the biosensor and detected by antibiotin-functionalized gold nanoparticles. As a result, the test zones are colored red if extension has occurred denoting the presence of the corresponding allele in the original sample. The excess nanoparticles are captured by immobilized biotinylated albumin at the control zone of the sensor forming another red zone that indicates the proper performance of the system. The assay was applied successfully to the genotyping of twenty clinical samples for two common SNPs of MBL2 gene.

Glutathione enzyme and selenoprotein polymorphisms associate with mercury biomarker levels in Michigan dental professionals

PubMed Central

Goodrich, Jaclyn M.; Wang, Yi; Gillespie, Brenda; Werner, Robert; Franzblau, Alfred; Basu, Niladri

2012-01-01

Mercury is a potent toxicant of concern to both the general public and occupationally exposed workers (e.g., dentists). Recent studies suggest that several genes mediating the toxicokinetics of mercury are polymorphic in humans and may influence inter-individual variability in mercury accumulation. This work hypothesizes that polymorphisms in key glutathione synthesizing enzyme, glutathione s-transferase, and selenoprotein genes underlie inter-individual differences in mercury body burden as assessed by analytical mercury measurement in urine and hair, biomarkers of elemental mercury and methylmercury, respectively. Urine and hair samples were collected from a population of dental professionals (n=515), and total mercury content was measured. Average urine (1.06±1.24 ug/L) and hair mercury levels (0.49±0.63 ug/g) were similar to national U.S. population averages. Taqman assays were used to genotype DNA from buccal swab samples at 15 polymorphic sites in genes implicated in mercury metabolism. Linear regression modeling assessed the ability of polymorphisms to modify the relationship between mercury biomarker levels and exposure sources (e.g., amalgams, fish consumption). Five polymorphisms were significantly associated with urine mercury levels (GSTT1 deletion), hair mercury levels (GSTP1-105, GSTP1-114, GSS 5’), or both (SEPP1 3’UTR). Overall, this study suggests that polymorphisms in selenoproteins and glutathione-related genes may influence elimination of mercury in the urine and hair or mercury retention following exposures to elemental mercury (via dental amalgams) and methylmercury (via fish consumption). PMID:21967774

Correlation between the NPPB gene promoter c.-1298 G/T polymorphism site and pulse pressure in the Chinese Han population.

PubMed

Zeng, K; Wu, X D; Cai, H D; Gao, Y G; Li, G; Liu, Q C; Gao, F; Chen, J H; Lin, C Z

2014-04-29

The aim of this study was to investigate the correlation between the natriuretic peptide precursor B (NPPB) gene single nucleotide polymorphism (SNP) c.-1298 G/T and pulse pressure (PP) of the Chinese Han population and the association between genotype and clinical indicators of hypertension. Peripheral blood was collected from 180 unrelated patients with hypertension and 540 healthy volunteers (control group), and DNA was extracted to amplify the 5'-flanking region and 2 exons of the NPPB gene by polymerase chain reaction; the fragment was sequenced after purification. The clinical data of all subjects were recorded, the distribution of the NPPB gene c.-1298 G/T polymorphism was determined, and differences in clinical indicators between the two groups were evaluated. The mean arterial pressure PP, and creatinine levels were significantly higher in the hypertension group than in the control group (P<0.05), but no other clinical indicators differed between the groups. There were no significant differences in genotype frequency and distribution of the NPPB gene c.-1298 G/T polymorphism between the hypertension group and the control group (P>0.05); in the control group, the mean PP of individuals with the SNP c.-1298 GG genotype was greater than that of individuals with the GT+TT genotype (P<0.05). In conclusion, there was no significant correlation between the NPPB gene c.-1298 G/T polymorphism and the incidence of essential hypertension in the Han population; however, the PP of the SNP c.-1298 GG genotype was greater than that of the GT+TT genotype in the control group.

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans.

PubMed

Rand, D M; Kann, L M

1996-07-01

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.

Targeting of Magnetic Nanoparticle-coated Microbubbles to the Vascular Wall Empowers Site-specific Lentiviral Gene Delivery in vivo.

PubMed

Heun, Yvonn; Hildebrand, Staffan; Heidsieck, Alexandra; Gleich, Bernhard; Anton, Martina; Pircher, Joachim; Ribeiro, Andrea; Mykhaylyk, Olga; Eberbeck, Dietmar; Wenzel, Daniela; Pfeifer, Alexander; Woernle, Markus; Krötz, Florian; Pohl, Ulrich; Mannell, Hanna

2017-01-01

In the field of vascular gene therapy, targeting systems are promising advancements to improve site-specificity of gene delivery. Here, we studied whether incorporation of magnetic nanoparticles (MNP) with different magnetic properties into ultrasound sensitive microbubbles may represent an efficient way to enable gene targeting in the vascular system after systemic application. Thus, we associated novel silicon oxide-coated magnetic nanoparticle containing microbubbles (SO-Mag MMB) with lentiviral particles carrying therapeutic genes and determined their physico-chemical as well as biological properties compared to MMB coated with polyethylenimine-coated magnetic nanoparticles (PEI-Mag MMB). While there were no differences between both MMB types concerning size and lentivirus binding, SO-Mag MMB exhibited superior characteristics regarding magnetic moment, magnetizability as well as transduction efficiency under static and flow conditions in vitro . Focal disruption of lentiviral SO-Mag MMB by ultrasound within isolated vessels exposed to an external magnetic field decisively improved localized VEGF expression in aortic endothelium ex vivo and enhanced the angiogenic response. Using the same system in vivo , we achieved a highly effective, site-specific lentiviral transgene expression in microvessels of the mouse dorsal skin after arterial injection. Thus, we established a novel lentiviral MMB technique, which has great potential towards site-directed vascular gene therapy.

Genetic Association Study of KCNQ5 Polymorphisms with High Myopia.

PubMed

Liao, Xuan; Yap, Maurice K H; Leung, Kim Hung; Kao, Patrick Y P; Liu, Long Qian; Yip, Shea Ping

2017-01-01

Identification of genetic variations related to high myopia may advance our knowledge of the etiopathogenesis of refractive error. This study investigated the role of potassium channel gene (KCNQ5) polymorphisms in high myopia. We performed a case-control study of 1563 unrelated Han Chinese subjects (809 cases of high myopia and 754 emmetropic controls). Five tag single-nucleotide polymorphisms (SNPs) of KCNQ5 were genotyped, and association testing with high myopia was conducted using logistic regression analysis adjusted for sex and age to give P asym values, and multiple comparisons were corrected by permutation test to give P emp values. All five noncoding SNPs were associated with high myopia. The SNP rs7744813, previously shown to be associated with refractive error and myopia in two GWAS, showed an odds ratio of 0.75 (95% CI 0.63-0.90; P emp = 0.0058) for the minor allele. The top SNP rs9342979 showed an odds ratio of 0.75 (95% CI 0.64-0.89; P emp = 0.0045) for the minor allele. Both SNPs are located within enhancer histone marks and DNase-hypersensitive sites. Our data support the involvement of KCNQ5 gene polymorphisms in the genetic susceptibility to high myopia and further exploration of KCNQ5 as a risk factor for high myopia.

[Study on association of CTLA4 gene polymorphism with Grave's disease in Guangxi Zhuang nationality population].

PubMed

Liang, Xing-huan; Qin, Ying-fen; Ma, Yan; Xie, Xin-rong; Xie, Kai-qing; Luo, Zuo-jie

2006-06-01

To investigate the relationship between the polymorphic (AT)n repeats in 3ountranslated region of exon 4 of CTLA4 gene [CTLA4(AT)n] and Graveso disease (GD) in Zhuang nationality population of Guangxi province. The studied groups comprised 48 patients with GD and 44 normal controls. Amplification of target DNA was carried out by polymerase chain reaction (PCR). The amplified products were run by 8% polyacrylamide gel electrophoresis, and then followed by 0.1% silver staining. Some of amplified products were sequenced directly. Nineteen alleles of CTLA4 gene microsatellite polymorphism were found in Guangxi Zhuang nationality individuals. The 106 bp long allele was apparently increased in patients with GD of Zhuang nationality but not in healthy controls (P< 0.05). CTLA4 gene microsatellite polymorphism is strongly associated with Graveso disease in Zhuang nationality population of Guangxi province. CTLA4(AT)n 106 bp may be the susceptible gene in GD patients of Zhuang nationality in Guangxi; 19 alleles of CTLA4 gene microsatellite polymorphism were found in Guangxi Zhuang nationality individuals.

The Third Ambient Aspirin Polymorph

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shtukenberg, Alexander G.; Hu, Chunhua T.; Zhu, Qiang

Polymorphism in aspirin (acetylsalicylic acid), one of the most widely consumed medications, was equivocal until the structure of a second polymorph II, similar in structure to the original form I, was reported in 2005. Here, the third ambient polymorph of aspirin is described. Lastly, it was crystallized from the melt and its structure was determined using a combination of X-ray powder diffraction analysis and crystal structure prediction algorithms.

The Third Ambient Aspirin Polymorph

DOE PAGES

Shtukenberg, Alexander G.; Hu, Chunhua T.; Zhu, Qiang; ...

2017-05-17

Polymorphism in aspirin (acetylsalicylic acid), one of the most widely consumed medications, was equivocal until the structure of a second polymorph II, similar in structure to the original form I, was reported in 2005. Here, the third ambient polymorph of aspirin is described. Lastly, it was crystallized from the melt and its structure was determined using a combination of X-ray powder diffraction analysis and crystal structure prediction algorithms.

Genotype imputation in a coalescent model with infinitely-many-sites mutation

PubMed Central

Huang, Lucy; Buzbas, Erkan O.; Rosenberg, Noah A.

2012-01-01

Empirical studies have identified population-genetic factors as important determinants of the properties of genotype-imputation accuracy in imputation-based disease association studies. Here, we develop a simple coalescent model of three sequences that we use to explore the theoretical basis for the influence of these factors on genotype-imputation accuracy, under the assumption of infinitely-many-sites mutation. Employing a demographic model in which two populations diverged at a given time in the past, we derive the approximate expectation and variance of imputation accuracy in a study sequence sampled from one of the two populations, choosing between two reference sequences, one sampled from the same population as the study sequence and the other sampled from the other population. We show that under this model, imputation accuracy—as measured by the proportion of polymorphic sites that are imputed correctly in the study sequence—increases in expectation with the mutation rate, the proportion of the markers in a chromosomal region that are genotyped, and the time to divergence between the study and reference populations. Each of these effects derives largely from an increase in information available for determining the reference sequence that is genetically most similar to the sequence targeted for imputation. We analyze as a function of divergence time the expected gain in imputation accuracy in the target using a reference sequence from the same population as the target rather than from the other population. Together with a growing body of empirical investigations of genotype imputation in diverse human populations, our modeling framework lays a foundation for extending imputation techniques to novel populations that have not yet been extensively examined. PMID:23079542

Broadly neutralizing antibodies from human survivors target a conserved site in the Ebola virus glycoprotein HR2-MPER region.

PubMed

Flyak, Andrew I; Kuzmina, Natalia; Murin, Charles D; Bryan, Christopher; Davidson, Edgar; Gilchuk, Pavlo; Gulka, Christopher P; Ilinykh, Philipp A; Shen, Xiaoli; Huang, Kai; Ramanathan, Palaniappan; Turner, Hannah; Fusco, Marnie L; Lampley, Rebecca; Kose, Nurgun; King, Hannah; Sapparapu, Gopal; Doranz, Benjamin J; Ksiazek, Thomas G; Wright, David W; Saphire, Erica Ollmann; Ward, Andrew B; Bukreyev, Alexander; Crowe, James E

2018-05-07

Ebola virus (EBOV) in humans causes a severe illness with high mortality rates. Several strategies have been developed in the past to treat EBOV infection, including the antibody cocktail ZMapp, which has been shown to be effective in nonhuman primate models of infection 1 and has been used under compassionate-treatment protocols in humans 2 . ZMapp is a mixture of three chimerized murine monoclonal antibodies (mAbs) 3-6 that target EBOV-specific epitopes on the surface glycoprotein 7,8 . However, ZMapp mAbs do not neutralize other species from the genus Ebolavirus, such as Bundibugyo virus (BDBV), Reston virus (RESTV) or Sudan virus (SUDV). Here, we describe three naturally occurring human cross-neutralizing mAbs, from BDBV survivors, that target an antigenic site in the canonical heptad repeat 2 (HR2) region near the membrane-proximal external region (MPER) of the glycoprotein. The identification of a conserved neutralizing antigenic site in the glycoprotein suggests that these mAbs could be used to design universal antibody therapeutics against diverse ebolavirus species. Furthermore, we found that immunization with a peptide comprising the HR2-MPER antigenic site elicits neutralizing antibodies in rabbits. Structural features determined by conserved residues in the antigenic site described here could inform an epitope-based vaccine design against infection caused by diverse ebolavirus species.

p62 Targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding.

PubMed

Itakura, Eisuke; Mizushima, Noboru

2011-01-10

Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)-associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway.

Finding the target sites of RNA-binding proteins

PubMed Central

Li, Xiao; Kazan, Hilal; Lipshitz, Howard D; Morris, Quaid D

2014-01-01

RNA–protein interactions differ from DNA–protein interactions because of the central role of RNA secondary structure. Some RNA-binding domains (RBDs) recognize their target sites mainly by their shape and geometry and others are sequence-specific but are sensitive to secondary structure context. A number of small- and large-scale experimental approaches have been developed to measure RNAs associated in vitro and in vivo with RNA-binding proteins (RBPs). Generalizing outside of the experimental conditions tested by these assays requires computational motif finding. Often RBP motif finding is done by adapting DNA motif finding methods; but modeling secondary structure context leads to better recovery of RBP-binding preferences. Genome-wide assessment of mRNA secondary structure has recently become possible, but these data must be combined with computational predictions of secondary structure before they add value in predicting in vivo binding. There are two main approaches to incorporating structural information into motif models: supplementing primary sequence motif models with preferred secondary structure contexts (e.g., MEMERIS and RNAcontext) and directly modeling secondary structure recognized by the RBP using stochastic context-free grammars (e.g., CMfinder and RNApromo). The former better reconstruct known binding preferences for sequence-specific RBPs but are not suitable for modeling RBPs that recognize shape and geometry of RNAs. Future work in RBP motif finding should incorporate interactions between multiple RBDs and multiple RBPs in binding to RNA. WIREs RNA 2014, 5:111–130. doi: 10.1002/wrna.1201 PMID:24217996

Recent Progress in the Design and Discovery of RXR Modulators Targeting Alternate Binding Sites of the Receptor.

PubMed

Su, Ying; Zeng, Zhiping; Chen, Ziwen; Xu, Dan; Zhang, Weidong; Zhang, Xiao-Kun

2017-01-01

Retinoid X receptors (RXRs) occupy a central position within the nuclear receptor superfamily. They not only function as important transcriptional factors but also exhibit diverse nongenomic biological activities. The pleiotropic actions of RXRs under both physiological and pathophysiological conditions confer RXRs important drug targets for the treatment of cancer, and metabolic and neurodegenerative diseases. RXR modulators have been studied for the purpose of developing both drug molecules and chemical tools for biological investigation of RXR. Development of RXR modulators has focused on small molecules targeting the canonical ligand-binding pocket. However, accumulating results have demonstrated that there are other binding mechanisms by which small molecules interact with RXR to act as RXR modulators. This review discusses the recent development in the design and discovery of RXR modulators with a focus on those targeting novel binding sites on RXR.

Design and Synthesis of Irreversible Analogues of Bardoxolone Methyl for the Identification of Pharmacologically Relevant Targets and Interaction Sites.

PubMed

Wong, Michael H L; Bryan, Holly K; Copple, Ian M; Jenkins, Rosalind E; Chiu, Pak Him; Bibby, Jaclyn; Berry, Neil G; Kitteringham, Neil R; Goldring, Christopher E; O'Neill, Paul M; Park, B Kevin

2016-03-24

Semisynthetic triterpenoids such as bardoxolone methyl (methyl-2-cyano 3,12-dioxooleano-1,9-dien-28-oate; CDDO-Me) (4) are potent inducers of antioxidant and anti-inflammatory signaling pathways, including those regulated by the transcription factor Nrf2. However, the reversible nature of the interaction between triterpenoids and thiols has hindered attempts to identify pharmacologically relevant targets and characterize the sites of interaction. Here, we report a shortened synthesis and SAR profiling of 4, enabling the design of analogues that react irreversibly with model thiols, as well as the model protein glutathione S-transferase P1, in vitro. We show that one of these analogues, CDDO-epoxide (13), is comparable to 4 in terms of cytotoxicity and potency toward Nrf2 in rat hepatoma cells and stably modifies specific cysteine residues (namely, Cys-257, -273, -288, -434, -489, and -613) within Keap1, the major repressor of Nrf2, both in vitro and in living cells. Supported by molecular modeling, these data demonstrate the value of 13 for identifying site(s) of interaction with pharmacologically relevant targets and informing the continuing development of triterpenoids as novel drug candidates.

Association analysis of a polymorphism of the monoamine oxidase B gene with Parkinson`s disease in a Japanese population

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morimoto, Yuji; Murayama, Nobuhiro; Kuwano, Akira

1995-12-18

The polymorphic allele of the monoamine oxidase B (MAO-B) gene detected by polymerase chain reaction (PCR) and single-stranded conformation polymorphism (SSCP) was associated with Parkinson`s disease (PD) in Caucasians. We characterized this polymorphic allele, allele 1, of the MAO-B gene using direct sequencing of PCR products. A single DNA substitution (G-A), resulting gain of Mae III restriction site was detected in intron 13 of the MAO-B gene. The allele associated with PD in Caucasians was twice as frequent as in healthy Japanese, but the association of the allele of the MAO-B gene was not observed in Japanese patients with PD.more » 7 refs., 2 figs., 1 tab.« less

The relative contribution of target-site mutations in complex acaricide resistant phenotypes as assessed by marker assisted backcrossing in Tetranychus urticae.

PubMed

Riga, Maria; Bajda, Sabina; Themistokleous, Christos; Papadaki, Stavrini; Palzewicz, Maria; Dermauw, Wannes; Vontas, John; Leeuwen, Thomas Van

2017-08-23

The mechanisms underlying insecticide and acaricide resistance in insects and mites are often complex, including additive effects of target-site insensitivity, increased metabolism and transport. The extent to which target-site resistance mutations contribute to the resistance phenotype is, however, not well studied. Here, we used marker-assisted backcrossing to create 30 congenic lines carrying nine mutations (alone, or in combination in a few cases) associated with resistance to avermectins, pyrethroids, mite growth inhibitors and mitochondrial complex III inhibitors (QoI) in a polyphagous arthropod pest, the spider mite Tetranychus urticae. Toxicity tests revealed that mutations in the voltage-gated sodium channel, chitin synthase 1 and cytochrome b confer high levels of resistance and, when fixed in a population, these mutations alone can result in field failure of acaricide treatment. In contrast, although we confirmed the implication of mutations in glutamate-gated chloride channels in abamectin and milbemectin insensitivity, these mutations do not lead to the high resistance levels that are often reported in abamectin resistant strains of T. urticae. Overall, this study functionally validates reported target-site resistance mutations in T. urticae, by uncoupling them from additional mechanisms, allowing to finally investigate the strength of the conferred phenotype in vivo.

TNF-alpha -308G/A and -238G/A polymorphisms and its protein network associated with type 2 diabetes mellitus.

PubMed

Jamil, Kaiser; Jayaraman, Archana; Ahmad, Javeed; Joshi, Sindhu; Yerra, Shiva Kumar

2017-09-01

Several reports document the role of tumor necrosis factor alpha ( TNF-α ) and lipid metabolism in the context of acute inflammation as a causative factor in obesity-associated insulin resistance and as one of the causative parameter of type 2 diabetes mellitus (T2DM). Our aim was to investigate the association between -308G/A and -238G/A polymorphisms located in the promoter region of the TNF-α gene in T2DM in the Indian population with bioinformatics analysis of TNF-α protein networking with an aim to find new target sites for the treatment of T2DM. Demographics of 100 diabetes patients and 100 healthy volunteers were collected in a structured proforma and 3 ml blood samples were obtained from the study group, after approval of Institutional Ethics Committee of the hospital (IEC). The information on clinical parameters was obtained from medical records. Genomic DNA was extracted; PCR-RFLP was performed using TNF-α primers specific to detect the presence of SNPs. Various bioinformatics tools such as STRING software were used to determine its network with other associated genes. The PCR-RFLP studies showed that among the -238G/A types the GG genotype was 87%, GA genotype was 12% and AA genotype was 1%. Almost a similar pattern of results was obtained with TNF-α -308G/A polymorphism. The results obtained were evaluated statistically to determine the significance. By constructing TNF-α protein interaction network we could analyze ontology and hubness of the network to identify the networking of this gene which may influence the functioning of other genes in promoting T2DM. We could identify new targets in T2DM which may function in association with TNF-α . Through hub analysis of TNF-α protein network we have identified three novel proteins RIPK1, BIRC2 and BIRC3 which may contribute to TNF- mediated T2DM pathogenesis. In conclusion, our study indicated that some of the genotypes of TNF-α -308G/A, -238G/A were not significantly associated to type 2 diabetes

Identification of thyroid hormone receptor binding sites and target genes using ChIP-on-chip in developing mouse cerebellum.

PubMed

Dong, Hongyan; Yauk, Carole L; Rowan-Carroll, Andrea; You, Seo-Hee; Zoeller, R Thomas; Lambert, Iain; Wade, Michael G

2009-01-01

Thyroid hormone (TH) is critical to normal brain development, but the mechanisms operating in this process are poorly understood. We used chromatin immunoprecipitation to enrich regions of DNA bound to thyroid receptor beta (TRbeta) of mouse cerebellum sampled on post natal day 15. Enriched target was hybridized to promoter microarrays (ChIP-on-chip) spanning -8 kb to +2 kb of the transcription start site (TSS) of 5000 genes. We identified 91 genes with TR binding sites. Roughly half of the sites were located in introns, while 30% were located within 1 kb upstream (5') of the TSS. Of these genes, 83 with known function included genes involved in apoptosis, neurodevelopment, metabolism and signal transduction. Two genes, MBP and CD44, are known to contain TREs, providing validation of the system. This is the first report of TR binding for 81 of these genes. ChIP-on-chip results were confirmed for 10 of the 13 binding fragments using ChIP-PCR. The expression of 4 novel TH target genes was found to be correlated with TH levels in hyper/hypothyroid animals providing further support for TR binding. A TRbeta binding site upstream of the coding region of myelin associated glycoprotein was demonstrated to be TH-responsive using a luciferase expression system. Motif searches did not identify any classic binding elements, indicating that not all TR binding sites conform to variations of the classic form. These findings provide mechanistic insight into impaired neurodevelopment resulting from TH deficiency and a rich bioinformatics resource for developing a better understanding of TR binding.

Discovery and validation of vascular endothelial growth factor (VEGF) pathway polymorphisms in esophageal adenocarcinoma outcome.

PubMed

Eng, Lawson; Azad, Abul Kalam; Qiu, Xin; Kong, Qin Quinn; Cheng, Dangxiao; Ying, Nanjiao; Tse, Alvina; Kuang, Qin; Dodbiba, Lorin; Renouf, Daniel J; Marsh, Sharon; Savas, Sevtap; Mackay, Helen J; Knox, Jennifer J; Darling, Gail E; Wong, Rebecca K S; Xu, Wei; Liu, Geoffrey; Faluyi, Olusola O

2015-09-01

Polymorphisms in the vascular endothelial growth factor (VEGF)/angiogenesis pathway have been implicated previously in cancer risk, prognosis and response to therapy including in esophageal adenocarcinoma. Prior esophageal adenocarcinoma studies focused on using candidate polymorphisms, limiting the discovery of novel polymorphisms. Here, we applied the tagSNP (single nucleotide polymorphism) approach to identify new VEGF pathway polymorphisms associated with esophageal adenocarcinoma prognosis and validated them in an independent cohort of esophageal adenocarcinoma patients. In 231 esophageal adenocarcinoma patients of all stages/treatment plans, 58 genetic polymorphisms (18 KDR, 7 VEGFA and 33 FLT1) selected through tagging and assessment of predicted function were genotyped. Cox-proportional hazard models adjusted for important socio-demographic and clinico-pathological factors were applied to assess the association of genetic polymorphisms with overall survival (OS) and progression-free survival (PFS). Significantly associated polymorphisms were then validated in an independent cohort of 137 esophageal adenocarcinoma patients. Among the 231 discovery cohort patients, 86% were male, median diagnosis age was 64 years, 34% were metastatic at diagnosis and median OS and PFS were 20 and 12 months, respectively. KDR rs17709898 was found significantly associated with PFS (adjusted hazard ratio, aHR = 0.69, 95% confidence interval (CI): 0.53-0.90; P = 5.9E-3). FLT1 rs3794405 and rs678714 were significantly associated with OS (aHR = 1.44, 95% CI: 1.04-1.99; P = 0.03 and aHR = 1.50, 95% CI: 1.01-2.24; P = 0.045, respectively). No VEGFA polymorphisms were found significantly associated with either outcome. Upon validation, FLT1 rs3794405 remained strongly associated with OS (aHR = 1.59, 95% CI: 1.04-2.44; P = 0.03). FLT1 rs3794405 is significantly associated with OS in esophageal adenocarcinoma, whereby each variant allele confers a 45-60% increased risk of mortality

Genetic basis of interindividual susceptibility to cancer cachexia: selection of potential candidate gene polymorphisms for association studies.

PubMed

Johns, N; Tan, B H; MacMillan, M; Solheim, T S; Ross, J A; Baracos, V E; Damaraju, S; Fearon, K C H

2014-12-01

Cancer cachexia is a complex and multifactorial disease. Evolving definitions highlight the fact that a diverse range of biological processes contribute to cancer cachexia. Part of the variation in who will and who will not develop cancer cachexia may be genetically determined. As new definitions, classifications and biological targets continue to evolve, there is a need for reappraisal of the literature for future candidate association studies. This review summarizes genes identified or implicated as well as putative candidate genes contributing to cachexia, identified through diverse technology platforms and model systems to further guide association studies. A systematic search covering 1986-2012 was performed for potential candidate genes / genetic polymorphisms relating to cancer cachexia. All candidate genes were reviewed for functional polymorphisms or clinically significant polymorphisms associated with cachexia using the OMIM and GeneRIF databases. Pathway analysis software was used to reveal possible network associations between genes. Functionality of SNPs/genes was explored based on published literature, algorithms for detecting putative deleterious SNPs and interrogating the database for expression of quantitative trait loci (eQTLs). A total of 154 genes associated with cancer cachexia were identified and explored for functional polymorphisms. Of these 154 genes, 119 had a combined total of 281 polymorphisms with functional and/or clinical significance in terms of cachexia associated with them. Of these, 80 polymorphisms (in 51 genes) were replicated in more than one study with 24 polymorphisms found to influence two or more hallmarks of cachexia (i.e., inflammation, loss of fat mass and/or lean mass and reduced survival). Selection of candidate genes and polymorphisms is a key element of multigene study design. The present study provides a contemporary basis to select genes and/or polymorphisms for further association studies in cancer cachexia, and

Association of Interleukin-10 gene promoter polymorphisms with obstructive sleep apnea.

PubMed

Özdaş, Sibel; Özdaş, Talih; Acar, Mustafa; Erbek, Selim S; Köseoğlu, Sabri; Göktürk, Gökhan; Izbirak, Afife

2016-05-01

Interleukin-10 (IL) is an anti-inflammatory cytokine that regulates normal sleep patterns, and recent studies have reported that it is a potential useful biomarker to identify presence and severity of sleep apnea syndrome (OSAS). Promoter polymorphisms of IL-10 gene have been associated with altered expression levels, which contributes to OSAS. The aim of this study was to determine the prevalence of -1082 G/A, -819 C/T, and -592 C/A promoter polymorphisms of IL-10 gene in individuals with OSAS and controls. An open-label study was performed in the Otorhinolaryngology and Sleep Disorders Outpatient Clinics. One hundred four cases with OSAS were included as the study group, and 78 individuals without OSAS were included as the controls. DNAs were extracted from peripheral blood leukocytes, and the sites that encompassed those polymorphisms were identified by DNA sequencing analyses. Data were analyzed with SNPStats and multifactor dimensionality reduction (MDR) software. The prevalence of OSAS was higher in males in the study group when compared to controls (P = 0.0003). The IL-10-1082 G/A, -819 C/T, and -592 C/A SNPs, and their minor alleles were associated with a significantly increased risk for OSAS compared to the controls (P ˂ 0.05 for all). Furthermore, ATA haplotype frequency was significantly higher in the study group compared to the control group, but the GCC haplotype frequency was lower (P = 0.0001 and P = 0.0001). As indicated in MDR analysis, combinations of IL-10 gene were associated with OSAS in single-, double-, and triple-locus analyses. The prevalences of the IL-10 gene promoter polymorphisms were different in OSAS patients and the controls in Turkish population. IL-10 gene polymorphisms may lead to altered inflammatory cascade, which might contribute to OSAS. Further studies on larger cohorts are needed to validate our findings.

Prostate-Specific Membrane Antigen-Targeted Site-Directed Antibody-Conjugated Apoferritin Nanovehicle Favorably Influences In Vivo Side Effects of Doxorubicin.

PubMed

Dostalova, Simona; Polanska, Hana; Svobodova, Marketa; Balvan, Jan; Krystofova, Olga; Haddad, Yazan; Krizkova, Sona; Masarik, Michal; Eckschlager, Tomas; Stiborova, Marie; Heger, Zbynek; Adam, Vojtech

2018-06-11

Herein, we describe the in vivo effects of doxorubicin (DOX) encapsulated in ubiquitous protein apoferritin (APO) and its efficiency and safety in anti-tumor treatment. APODOX is both passively (through Enhanced Permeability and Retention effect) and actively targeted to tumors through prostate-specific membrane antigen (PSMA) via mouse antibodies conjugated to the surface of horse spleen APO. To achieve site-directed conjugation of the antibodies, a HWRGWVC heptapeptide linker was used. The prostate cancer-targeted and non-targeted nanocarriers were tested using subcutaneously implanted LNCaP cells in athymic mice models, and compared to free DOX. Prostate cancer-targeted APODOX retained the high potency of DOX in attenuation of tumors (with 55% decrease in tumor volume after 3 weeks of treatment). DOX and non-targeted APODOX treatment caused damage to liver, kidney and heart tissues. In contrast, no elevation in liver or kidney enzymes and negligible changes were revealed by histological assessment in prostate cancer-targeted APODOX-treated mice. Overall, we show that the APO nanocarrier provides an easy encapsulation protocol, reliable targeting, high therapeutic efficiency and very low off-target toxicity, and is thus a promising delivery system for translation into clinical use.

Detection of Epidemic USA300 Community-Associated Methicillin-Resistant Staphylococcus aureus Strains by Use of a Single Allele-Specific PCR Assay Targeting a Novel Polymorphism of Staphylococcus aureus pbp3

PubMed Central

Chadwick, Sean G.; Prasad, Aditya; Smith, W. Lamar; Mordechai, Eli; Adelson, Martin E.

2013-01-01

In recent years, the dramatic increase in community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections has become a significant health care challenge. Early detection of CA-MRSA is important because of its increased virulence associated with the arginine catabolic mobile element (ACME), Panton-Valentine leukocidin (PVL), and other toxins that may contribute to disease severity. In particular, the USA300 epidemic clone has emerged and now represents the cause of as much as 98% of CA-MRSA skin and soft tissue infections in the United States. Current diagnostic assays used to identify CA-MRSA strains are based on complex multiplex PCRs targeting the staphylococcal cassette chromosome mec (SCCmec) DNA junction, a multitude of genes, and noncoding DNA fragments or on a number of lengthy sequence-typing methods. Here, two nucleotide polymorphisms, G88A and G2047A, that were found to be in strict linkage disequilibrium in the S. aureus penicillin-binding protein 3 (pbp3) gene were also found to be highly associated with the USA300 clone of CA-MRSA. Clinical isolates that contained this pbp3 allele were also positive for the presence of SCCmec type IV, the ACME, and the PVL toxin gene and matched the t008 or t121 molecular spa types, which are associated specifically with the USA300 CA-MRSA clone. A single allele-specific PCR targeting the G88A polymorphism was developed and was found to be 100% sensitive and specific for the detection of USA300 CA-MRSA and 91.5% sensitive and 100% specific for the detection of all CA-MRSA isolates in this study. PMID:23698534

HapMap-based study on the association between MPO and GSTP1 gene polymorphisms and lung cancer susceptibility in Chinese Han population

PubMed Central

Gu, Jun-dong; Hua, Feng; Mei, Chao-rong; Zheng, De-jie; Wang, Guo-fan; Zhou, Qing-hua

2014-01-01

Aim: Myeloperoxidase (MPO) and glutathione S-transferase pi 1 (GSTP1) are important carcinogen-metabolizing enzymes. The aim of this study was to investigate the association between the common polymorphisms of MPO and GSTP1 genes and lung cancer risk in Chinese Han population. Methods: A total of 266 subjects with lung cancer and 307 controls without personal history of the disease were recruited in this case control study. The tagSNPs approach was used to assess the common polymorphisms of MOP and GSTP1 genes and lung cancer risk according to the disequilibrium information from the HapMap project. The tagSNP rs7208693 was selected as the polymorphism site for MPO, while the haplotype-tagging SNPs rs1695, rs4891, rs762803 and rs749174 were selected as the polymorphism sites for GSTP1. The gene polymorphisms were confirmed using real-time PCR, cloning and sequencing. Results: The four GSTP1 haplotype-tagging SNPs rs1695, rs4891, rs762803 and rs749174, but not the MPO tagSNP rs7208693, exhibited an association with lung cancer susceptibility in smokers in the overall population and in the studied subgroups. When Phase 2 software was used to reconstruct the haplotype for GSTP1, the haplotype CACA (rs749174+rs1695 + rs762803+rs4891) exhibited an increased risk of lung cancer among smokers (adjust odds ratio 1.53; 95%CI 1.04–2.25, P=0.033). Furthermore, diplotype analyses demonstrated that the significant association between the risk haplotype and lung cancer. The risk haplotypes co-segregated with one or more biologically functional polymorphisms and corresponded to a recessive inheritance model. Conclusion: The common polymorphisms of the GSTP1 gene may be the candidates for SNP markers for lung cancer susceptibility in Chinese Han population. PMID:24786234

A comparative genomics strategy for targeted discovery of single-nucleotide polymorphisms and conserved-noncoding sequences in orphan crops.

PubMed

Feltus, F A; Singh, H P; Lohithaswa, H C; Schulze, S R; Silva, T D; Paterson, A H

2006-04-01

Completed genome sequences provide templates for the design of genome analysis tools in orphan species lacking sequence information. To demonstrate this principle, we designed 384 PCR primer pairs to conserved exonic regions flanking introns, using Sorghum/Pennisetum expressed sequence tag alignments to the Oryza genome. Conserved-intron scanning primers (CISPs) amplified single-copy loci at 37% to 80% success rates in taxa that sample much of the approximately 50-million years of Poaceae divergence. While the conserved nature of exons fostered cross-taxon amplification, the lesser evolutionary constraints on introns enhanced single-nucleotide polymorphism detection. For example, in eight rice (Oryza sativa) genotypes, polymorphism averaged 12.1 per kb in introns but only 3.6 per kb in exons. Curiously, among 124 CISPs evaluated across Oryza, Sorghum, Pennisetum, Cynodon, Eragrostis, Zea, Triticum, and Hordeum, 23 (18.5%) seemed to be subject to rigid intron size constraints that were independent of per-nucleotide DNA sequence variation. Furthermore, we identified 487 conserved-noncoding sequence motifs in 129 CISP loci. A large CISP set (6,062 primer pairs, amplifying introns from 1,676 genes) designed using an automated pipeline showed generally higher abundance in recombinogenic than in nonrecombinogenic regions of the rice genome, thus providing relatively even distribution along genetic maps. CISPs are an effective means to explore poorly characterized genomes for both DNA polymorphism and noncoding sequence conservation on a genome-wide or candidate gene basis, and also provide anchor points for comparative genomics across a diverse range of species.

Intein-mediated site-specific synthesis of tumor-targeting protein delivery system: Turning PEG dilemma into prodrug-like feature

PubMed Central

Chen, Yingzhi; Zhang, Meng; Jin, Hongyue; Tang, Yisi; Wang, Huiyuan; Xu, Qin; Li, Yaping; Li, Feng; Huang, Yongzhuo

2017-01-01

Poor tumor-targeted and cytoplasmic delivery is a bottleneck for protein toxin-based cancer therapy. Ideally, a protein toxin drug should remain stealthy in circulation for prolonged half-life and reduced side toxicity, but turn activated at tumor. PEGylation is a solution to achieve the first goal, but creates a hurdle for the second because PEG rejects interaction between the drugs and tumor cells therein. Such PEG dilemma is an unsolved problem in protein delivery. Herein proposed is a concept of turning PEG dilemma into prodrug-like feature. A site-selectively PEGylated, gelatinase-triggered cell-penetrating trichosanthin protein delivery system is developed with three specific aims. The first is to develop an intein-based ligation method for achieving site-specific modification of protein toxins. The second is to develop a prodrug feature that renders protein toxins remaining stealthy in blood for reduced side toxicity and improved EPR effect. The third is to develop a gelatinase activatable cell-penetration strategy for enhanced tumor targeting and cytoplasmic delivery. Of note, site-specific modification is a big challenge in protein drug research, especially for such a complicated, multifunctional protein delivery system. We successfully develop a protocol for constructing a macromolecular prodrug system with intein-mediated ligation synthesis. With an on-column process of purification and intein-mediated cleavage, the site-specific PEGylation then can be readily achieved by conjugation with the activated C-terminus, thus constructing a PEG-capped, cell-penetrating trichosanthin system with a gelatinase-cleavable linker that enables tumor-specific activation of cytoplasmic delivery. It provides a promising method to address the PEG dilemma for enhanced protein drug delivery, and importantly, a facile protocol for site-specific modification of such a class of protein drugs for improving their druggability and industrial translation. PMID:27914267

Thiopurine methyltransferase polymorphisms and mercaptopurine tolerance in Turkish children with acute lymphoblastic leukemia.

PubMed

Albayrak, Meryem; Konyssova, Uljan; Kaya, Zuhre; Gursel, Turkiz; Guntekin, Sezen; Percin, E Ferda; Kocak, Ulker

2011-11-01

Thiopurine methyltransferase (TPMT) enzyme is involved in the metabolism of 6-mercaptopurine (6-MP), a key component of acute lymphoblastic leukemia (ALL) treatment protocols in children. The aims of this study were to investigate the frequency of common genetic polymorphisms associated with low TPMT activity and correlations of polymorphic variants with 6-MP tolerance in a group of Turkish children with ALL. Genotyping for G238C, A719G, and G460A mutations were performed by using NanoChip Technology. Adverse reactions during the first 6 months of maintenance therapy with oral 6-MP and methotrexate were retrospectively analyzed from patient's files. Five (8.6%) of 58 children with ALL had a polymorphic TPMT allele: 4 (3.4%) were heterozygous for TPMT*3A (G460A and A719G), and one (0.9%) was heterozygous for TPMT*3C (A719G). No cases with TPMT*3B (G460A) or TPMT*2 (G238C) variants were identified. Children with TPMT*3A and *3C had significantly lower leukocyte and neutrophil counts and percentage of target 6-MP dosage, and longer periods with ≥grade 2 infections, ≥grade 2 liver toxicity, and chemotherapy interruptions than the children with wild-type TPMT during the first 24 weeks of maintenance therapy. The frequency and distribution of common TPMT polymorphisms in Turkish children with ALL is similar to other Caucasian populations. Polymorphic variants were associated with excessive 6-MP toxicity supporting the suggestion that TPMT genotyping should be performed before institution of 6-MP therapy.

Assessment of genetic diversity in Vigna unguiculata L. (Walp) accessions using inter-simple sequence repeat (ISSR) and start codon targeted (SCoT) polymorphic markers.

PubMed

Igwe, David Okeh; Afiukwa, Celestine Azubike; Ubi, Benjamin Ewa; Ogbu, Kenneth Idika; Ojuederie, Omena Bernard; Ude, George Nkem

2017-11-17

Assessment of genetic diversity of Vigna unguiculata (L.) Walp (cowpea) accessions using informative molecular markers is imperative for their genetic improvement and conservation. Use of efficacious molecular markers to obtain the required knowledge of the genetic diversity within the local and regional germplasm collections can enhance the overall effectiveness of cowpea improvement programs, hence, the comparative assessment of Inter-simple sequence repeat (ISSR) and Start codon targeted (SCoT) markers in genetic diversity of V. unguiculata accessions from different regions in Nigeria. Comparative analysis of the genetic diversity of eighteen accessions from different locations in Nigeria was investigated using ISSR and SCoT markers. DNA extraction was done using Zymogen Kit according to its manufacturer's instructions followed by amplifications with ISSR and SCoT and agarose gel electrophoresis. The reproducible bands were scored for analyses of dendrograms, principal component analysis, genetic diversity, allele frequency, polymorphic information content, and population structure. Both ISSR and SCoT markers resolved the accessions into five major clusters based on dendrogram and principal component analyses. Alleles of 32 and 52 were obtained with ISSR and SCoT, respectively. Numbers of alleles, gene diversity and polymorphic information content detected with ISSR were 9.4000, 0.7358 and 0.7192, while SCoT yielded 11.1667, 0.8158 and 0.8009, respectively. Polymorphic loci were 70 and 80 in ISSR and SCoT, respectively. Both markers produced high polymorphism (94.44-100%). The ranges of effective number of alleles (Ne) were 1.2887 ± 0.1797-1.7831 ± 0.2944 and 1.7416 ± 0.0776-1.9181 ± 0.2426 in ISSR and SCoT, respectively. The Nei's genetic diversity (H) ranged from 0.2112 ± 0.0600-0.4335 ± 0.1371 and 0.4111 ± 0.0226-0.4778 ± 0.1168 in ISSR and SCoT, respectively. Shannon's information index (I) from ISSR and SCoT were 0

Influence of genetic polymorphisms of FPGS, GGH, and MTHFR on serum methotrexate levels in Chinese children with acute lymphoblastic leukemia.

PubMed

Wang, Shu-mei; Sun, Lu-lu; Zeng, Wei-xin; Wu, Wan-shui; Zhang, Guo-liang

2014-08-01

To investigate the correlation between common genetic polymorphisms of folylpolyglutamate synthase (FPGS), gamma-glutamyl hydrolase (GGH), and methylenetetrahydrofolate reductase (MTHFR) and serum levels of methotrexate (MTX) in Chinese children with acute lymphoblastic leukemia (ALL). Ninety-one children with ALL who received high-dose MTX were recruited. The polymorphisms FPGS (rs1544105 G>A), GGH (rs3758149 C>T), and MTHFR (rs1801133 C>T) were genotyped through polymerase chain reaction-restriction fragment length polymorphism analysis. Serum MTX was measured by fluorescence polarization immunoassay. The association between targeted polymorphisms and MTX concentration-to-dose (C/D) ratios was assessed, and between targeted polymorphisms and the percent of MTX above the therapeutic threshold (40 µmol/L). The minor allele frequencies of rs1544105 G (34.1%), rs3758149 T (19.2%), and rs1801133 C (48.4%) observed in our population were significantly lower than those reported for European populations (64.2, 30.8, and 69.0%, respectively). The association between the GGH rs3758149 polymorphism and MTX C/D was gender-specific; in girls, the MTX C/D at 24 h of GGH rs3758149 CC carriers (12.09 μmol/L per g/m(2)) was significantly lower than that of CT or TT carriers (16.80 μmol/L per g/m(2)). The percent of serum MTX above the therapeutic threshold in GGH rs3758149 CC carriers (18.3%) was significantly lower than that of CT and TT carriers (38.7%). The MTX C/D ratios at 24 h and the percent of MTX >40 µmol/L for the A-T-T (three variant alleles) haplotype were significantly higher than those for other haplotypes combined (P < 0.05). These data indicate that FPGS rs1544105, GGH rs3758149, and MTHFR rs1801133 polymorphisms contribute to the variability of MTX pharmacokinetics, and their genotyping may be useful to reduce toxicities associated with MTX therapy.

A new single-nucleotide polymorphisms database for rainbow trout generated through whole genome resequencing of selected samples

USDA-ARS?s Scientific Manuscript database

Single-nucleotide polymorphisms (SNPs) are highly abundant markers, which are broadly distributed in animal genomes. For rainbow trout, SNP discovery has been done through sequencing of restriction-site associated DNA (RAD) libraries, reduced representation libraries (RRL), RNA sequencing, and whole...

Distinguishing functional polymorphism from random variation in the sequences of >10,000 HLA-A, -B and -C alleles.

PubMed

Robinson, James; Guethlein, Lisbeth A; Cereb, Nezih; Yang, Soo Young; Norman, Paul J; Marsh, Steven G E; Parham, Peter

2017-06-01

HLA class I glycoproteins contain the functional sites that bind peptide antigens and engage lymphocyte receptors. Recently, clinical application of sequence-based HLA typing has uncovered an unprecedented number of novel HLA class I alleles. Here we define the nature and extent of the variation in 3,489 HLA-A, 4,356 HLA-B and 3,111 HLA-C alleles. This analysis required development of suites of methods, having general applicability, for comparing and analyzing large numbers of homologous sequences. At least three amino-acid substitutions are present at every position in the polymorphic α1 and α2 domains of HLA-A, -B and -C. A minority of positions have an incidence >1% for the 'second' most frequent nucleotide, comprising 70 positions in HLA-A, 85 in HLA-B and 54 in HLA-C. The majority of these positions have three or four alternative nucleotides. These positions were subject to positive selection and correspond to binding sites for peptides and receptors. Most alleles of HLA class I (>80%) are very rare, often identified in one person or family, and they differ by point mutation from older, more common alleles. These alleles with single nucleotide polymorphisms reflect the germ-line mutation rate. Their frequency predicts the human population harbors 8-9 million HLA class I variants. The common alleles of human populations comprise 42 core alleles, which represent all selected polymorphism, and recombinants that have assorted this polymorphism.

Distinguishing functional polymorphism from random variation in the sequences of >10,000 HLA-A, -B and -C alleles

PubMed Central

Cereb, Nezih; Yang, Soo Young; Marsh, Steven G. E.; Parham, Peter

2017-01-01

HLA class I glycoproteins contain the functional sites that bind peptide antigens and engage lymphocyte receptors. Recently, clinical application of sequence-based HLA typing has uncovered an unprecedented number of novel HLA class I alleles. Here we define the nature and extent of the variation in 3,489 HLA-A, 4,356 HLA-B and 3,111 HLA-C alleles. This analysis required development of suites of methods, having general applicability, for comparing and analyzing large numbers of homologous sequences. At least three amino-acid substitutions are present at every position in the polymorphic α1 and α2 domains of HLA-A, -B and -C. A minority of positions have an incidence >1% for the ‘second’ most frequent nucleotide, comprising 70 positions in HLA-A, 85 in HLA-B and 54 in HLA-C. The majority of these positions have three or four alternative nucleotides. These positions were subject to positive selection and correspond to binding sites for peptides and receptors. Most alleles of HLA class I (>80%) are very rare, often identified in one person or family, and they differ by point mutation from older, more common alleles. These alleles with single nucleotide polymorphisms reflect the germ-line mutation rate. Their frequency predicts the human population harbors 8–9 million HLA class I variants. The common alleles of human populations comprise 42 core alleles, which represent all selected polymorphism, and recombinants that have assorted this polymorphism. PMID:28650991

Inter-seasonal maintenance of individual nest site preferences in hawksbill sea turtles.

PubMed

Kamel, Stephanie J; Mrosovsky, N

2006-11-01

Within a single population of hawksbill sea turtles (Eretmochelys imbricata), we found a behavioral polymorphism for maternal nest site choice with respect to beach microhabitat characteristics. Some females preferred to nest in littoral forest and in places with overstory vegetation cover, and others preferred to nest in more open, deforested areas. Nest site choice was consistent within and between nesting seasons two years apart. This was not a result of females simply returning to the same location along the shoreline; beach sections used by individual turtles varied between seasons. Nest site choice was not influenced by changes in beach environment (e.g., beach width and foliage cover) or by changes in females' reproductive output (e.g., clutch size), suggesting that fidelity to particular microhabitats is a major determinant of the observed nesting patterns. Because hawksbills exhibit temperature-dependent sex determination, if the behavioral polymorphism in nest site choice has a genetic basis, as is plausible, then this would have implications for sex ratio evolution and offspring survival. By taking an individual-based approach to the study of maternal behavior we reveal previously overlooked individual variation and hope to provide some impetus for more detailed studies of nest site choice.

Identification of Splice Variants, Targeted MicroRNAs and Functional Single Nucleotide Polymorphisms of the BOLA-DQA2 Gene in Dairy Cattle

PubMed Central

Hou, Qinlei; Huang, Jinming; Ju, Zhihua; Li, Qiuling; Li, Liming; Wang, Changfa; Sun, Tao; Wang, Lingling; Hou, Minghai

2012-01-01

Major histocompatibility complex, class II, DQ alpha 2, also named BOLA-DQA2, belongs to the Bovine Leukocyte Antigen (BOLA) class II genes which are involved in the immune response. To explore the variability of the BOLA-DQA2 gene and resistance to mastitis in cows, the splice variants (SV), targeted microRNAs (miRNAs), and single nucleotide polymorphisms (SNPs) were identified in this study. A new SV (BOLA-DQA2-SV1) lacking part of exon 3 (195 bp) and two 3′-untranslated regions (UTR) (52 bp+167 bp) of the BOLA-DQA2 gene was found in the healthy and mastitis-infected mammary gland tissues. Four of 13 new SNPs and multiple nucleotide polymorphisms resulted in amino acid changes in the protein and SNP (c. +1283 C>T) may affect the binding to the seed sequence of bta-miR-2318. Further, we detected the relative expressions of two BOLA-DQA2 transcripts and five candidated microRNAs binding to the 3′-UTR of two transcripts in the mammary gland tissues in dairy cattle by using the quantitative real-time polymerase chain reaction. The result showed that expression of the BOLA-DQA2-SV1 mRNA was significantly upregulated 2.67-fold (p<0.05) in mastitis-infected mammary tissues (n=5) compared with the healthy mammary gland mammary tissues (n=5). Except for bta-miR-1777a, miRNA expression (bta-miR-296, miR-2430, and miR-671) was upregulated 1.75 to 2.59-fold (p<0.05), whereas miR-2318 was downregulated in the mastitis cows. Our findings reveal that BOLA-DQA2-SV1 may play an important role in the mastitis resistance in dairy cattle. Whether the SNPs affect the structure of the BOLA-DQA2 gene or association with mastitis resistance is unknown and warrants further investigation. PMID:22084936

Microsatellites as targets of natural selection.

PubMed

Haasl, Ryan J; Payseur, Bret A

2013-02-01

The ability to survey polymorphism on a genomic scale has enabled genome-wide scans for the targets of natural selection. Theory that connects patterns of genetic variation to evidence of natural selection most often assumes a diallelic locus and no recurrent mutation. Although these assumptions are suitable to selection that targets single nucleotide variants, fundamentally different types of mutation generate abundant polymorphism in genomes. Moreover, recent empirical results suggest that mutationally complex, multiallelic loci including microsatellites and copy number variants are sometimes targeted by natural selection. Given their abundance, the lack of inference methods tailored to the mutational peculiarities of these types of loci represents a notable gap in our ability to interrogate genomes for signatures of natural selection. Previous theoretical investigations of mutation-selection balance at multiallelic loci include assumptions that limit their application to inference from empirical data. Focusing on microsatellites, we assess the dynamics and population-level consequences of selection targeting mutationally complex variants. We develop general models of a multiallelic fitness surface, a realistic model of microsatellite mutation, and an efficient simulation algorithm. Using these tools, we explore mutation-selection-drift equilibrium at microsatellites and investigate the mutational history and selective regime of the microsatellite that causes Friedreich's ataxia. We characterize microsatellite selective events by their duration and cost, note similarities to sweeps from standing point variation, and conclude that it is premature to label microsatellites as ubiquitous agents of efficient adaptive change. Together, our models and simulation algorithm provide a powerful framework for statistical inference, which can be used to test the neutrality of microsatellites and other multiallelic variants.

Microsatellites as Targets of Natural Selection

PubMed Central

Haasl, Ryan J.; Payseur, Bret A.

2013-01-01

The ability to survey polymorphism on a genomic scale has enabled genome-wide scans for the targets of natural selection. Theory that connects patterns of genetic variation to evidence of natural selection most often assumes a diallelic locus and no recurrent mutation. Although these assumptions are suitable to selection that targets single nucleotide variants, fundamentally different types of mutation generate abundant polymorphism in genomes. Moreover, recent empirical results suggest that mutationally complex, multiallelic loci including microsatellites and copy number variants are sometimes targeted by natural selection. Given their abundance, the lack of inference methods tailored to the mutational peculiarities of these types of loci represents a notable gap in our ability to interrogate genomes for signatures of natural selection. Previous theoretical investigations of mutation-selection balance at multiallelic loci include assumptions that limit their application to inference from empirical data. Focusing on microsatellites, we assess the dynamics and population-level consequences of selection targeting mutationally complex variants. We develop general models of a multiallelic fitness surface, a realistic model of microsatellite mutation, and an efficient simulation algorithm. Using these tools, we explore mutation-selection-drift equilibrium at microsatellites and investigate the mutational history and selective regime of the microsatellite that causes Friedreich’s ataxia. We characterize microsatellite selective events by their duration and cost, note similarities to sweeps from standing point variation, and conclude that it is premature to label microsatellites as ubiquitous agents of efficient adaptive change. Together, our models and simulation algorithm provide a powerful framework for statistical inference, which can be used to test the neutrality of microsatellites and other multiallelic variants. PMID:23104080

Application of industrial scale genomics to discovery of therapeutic targets in heart failure.

PubMed

Mehraban, F; Tomlinson, J E

2001-12-01

In recent years intense activity in both academic and industrial sectors has provided a wealth of information on the human genome with an associated impressive increase in the number of novel gene sequences deposited in sequence data repositories and patent applications. This genomic industrial revolution has transformed the way in which drug target discovery is now approached. In this article we discuss how various differential gene expression (DGE) technologies are being utilized for cardiovascular disease (CVD) drug target discovery. Other approaches such as sequencing cDNA from cardiovascular derived tissues and cells coupled with bioinformatic sequence analysis are used with the aim of identifying novel gene sequences that may be exploited towards target discovery. Additional leverage from gene sequence information is obtained through identification of polymorphisms that may confer disease susceptibility and/or affect drug responsiveness. Pharmacogenomic studies are described wherein gene expression-based techniques are used to evaluate drug response and/or efficacy. Industrial-scale genomics supports and addresses not only novel target gene discovery but also the burgeoning issues in pharmaceutical and clinical cardiovascular medicine relative to polymorphic gene responses.

Barriers to Liposomal Gene Delivery: from Application Site to the Target.

PubMed

Saffari, Mostafa; Moghimi, Hamid Reza; Dass, Crispin R

2016-01-01

Gene therapy is a therapeutic approach to deliver genetic material into cells to alter their function in entire organism. One promising form of gene delivery system (DDS) is liposomes. The success of liposome-mediated gene delivery is a multifactorial issue and well-designed liposomal systems might lead to optimized gene transfection particularly in vivo. Liposomal gene delivery systems face different barriers from their site of application to their target, which is inside the cells. These barriers include presystemic obstacles (epithelial barriers), systemic barriers in blood circulation and cellular barriers. Epithelial barriers differ depending on the route of administration. Systemic barriers include enzymatic degradation, binding and opsonisation. Both of these barriers can act as limiting hurdles that genetic material and their vector should overcome before reaching the cells. Finally liposomes should overcome cellular barriers that include cell entrance, endosomal escape and nuclear uptake. These barriers and their impact on liposomal gene delivery will be discussed in this review.

The chromodomain of Tf1 integrase promotes binding to cDNA and mediates target site selection.

PubMed

Chatterjee, Atreyi Ghatak; Leem, Young Eun; Kelly, Felice D; Levin, Henry L

2009-03-01

The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDDelta, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDDelta caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting.

Role of Plasmodium vivax Dihydropteroate Synthase Polymorphisms in Sulfa Drug Resistance

PubMed Central

Riangrungroj, Pinpunya; Chitnumsub, Penchit; Ittarat, Wanwipa; Kongkasuriyachai, Darin; Uthaipibull, Chairat; Yuthavong, Yongyuth

2016-01-01

Dihydropteroate synthase (DHPS) is a known sulfa drug target in malaria treatment, existing as a bifunctional enzyme together with hydroxymethyldihydropterin pyrophosphokinase (HPPK). Polymorphisms in key residues of Plasmodium falciparum DHPS (PfDHPS) have been characterized and linked to sulfa drug resistance in malaria. Genetic sequencing of P. vivax dhps (Pvdhps) from clinical isolates has shown several polymorphisms at the positions equivalent to those in the Pfdhps genes conferring sulfa drug resistance, suggesting a mechanism for sulfa drug resistance in P. vivax similar to that seen in P. falciparum. To characterize the role of polymorphisms in the PvDHPS in sulfa drug resistance, various mutants of recombinant PvHPPK-DHPS enzymes were expressed and characterized. Moreover, due to the lack of a continuous in vitro culture system for P. vivax parasites, a surrogate P. berghei model expressing Pvhppk-dhps genes was established to demonstrate the relationship between sequence polymorphisms and sulfa drug susceptibility and to test the activities of PvDHPS inhibitors on the transgenic parasites. Both enzyme activity and transgenic parasite growth were sensitive to sulfadoxine to different degrees, depending on the number of mutations that accumulated in DHPS. Ki values and 50% effective doses were higher for mutant PvDHPS enzymes than the wild-type enzymes. Altogether, the study provides the first evidence of sulfa drug resistance at the molecular level in P. vivax. Furthermore, the enzyme inhibition assay and the in vivo screening system can be useful tools for screening new compounds for their activities against PvDHPS. PMID:27161627

Does epigenetic polymorphism contribute to phenotypic variances in Jatropha curcas L.?

PubMed

Yi, Chengxin; Zhang, Shilu; Liu, Xiaokun; Bui, Ha T N; Hong, Yan

2010-11-23

There is a growing interest in Jatropha curcas L. (jatropha) as a biodiesel feedstock plant. Variations in its morphology and seed productivity have been well documented. However, there is the lack of systematic comparative evaluation of distinct collections under same climate and agronomic practices. With the several reports on low genetic diversity in jatropha collections, there is uncertainty on genetic contribution to jatropha morphology. In this study, five populations of jatropha plants collected from China (CN), Indonesia (MD), Suriname (SU), Tanzania (AF) and India (TN) were planted in one farm under the same agronomic practices. Their agronomic traits (branching pattern, height, diameter of canopy, time to first flowering, dormancy, accumulated seed yield and oil content) were observed and tracked for two years. Significant variations were found for all the agronomic traits studied. Genetic diversity and epigenetic diversity were evaluated using florescence Amplified Fragment Length Polymorphism (fAFLP) and methylation sensitive florescence AFLP (MfAFLP) methods. Very low level of genetic diversity was detected (polymorphic band <0.1%) within and among populations. In contrast, intermediate but significant epigenetic diversity was detected (25.3% of bands were polymorphic) within and among populations. More than half of CCGG sites surveyed by MfAFLP were methylated with significant difference in inner cytosine and double cytosine methylation among populations. Principal coordinates analysis (PCoA) based on Nei's epigenetic distance showed Tanzania/India group distinct from China/Indonesia/Suriname group. Inheritance of epigenetic markers was assessed in one F1 hybrid population between two morphologically distinct parent plants and one selfed population. 30 out of 39 polymorphic markers (77%) were found heritable and followed Mendelian segregation. One epiallele was further confirmed by bisulphite sequencing of its corresponding genomic region. Our study

Does epigenetic polymorphism contribute to phenotypic variances in Jatropha curcas L.?

PubMed Central

2010-01-01

Background There is a growing interest in Jatropha curcas L. (jatropha) as a biodiesel feedstock plant. Variations in its morphology and seed productivity have been well documented. However, there is the lack of systematic comparative evaluation of distinct collections under same climate and agronomic practices. With the several reports on low genetic diversity in jatropha collections, there is uncertainty on genetic contribution to jatropha morphology. Result In this study, five populations of jatropha plants collected from China (CN), Indonesia (MD), Suriname (SU), Tanzania (AF) and India (TN) were planted in one farm under the same agronomic practices. Their agronomic traits (branching pattern, height, diameter of canopy, time to first flowering, dormancy, accumulated seed yield and oil content) were observed and tracked for two years. Significant variations were found for all the agronomic traits studied. Genetic diversity and epigenetic diversity were evaluated using florescence Amplified Fragment Length Polymorphism (fAFLP) and methylation sensitive florescence AFLP (MfAFLP) methods. Very low level of genetic diversity was detected (polymorphic band <0.1%) within and among populations. In contrast, intermediate but significant epigenetic diversity was detected (25.3% of bands were polymorphic) within and among populations. More than half of CCGG sites surveyed by MfAFLP were methylated with significant difference in inner cytosine and double cytosine methylation among populations. Principal coordinates analysis (PCoA) based on Nei's epigenetic distance showed Tanzania/India group distinct from China/Indonesia/Suriname group. Inheritance of epigenetic markers was assessed in one F1 hybrid population between two morphologically distinct parent plants and one selfed population. 30 out of 39 polymorphic markers (77%) were found heritable and followed Mendelian segregation. One epiallele was further confirmed by bisulphite sequencing of its corresponding genomic

Generation of Hypertension-Associated STK39 Polymorphism Knockin Cell Lines With the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 System.

PubMed

Mandai, Shintaro; Mori, Takayasu; Sohara, Eisei; Rai, Tatemitsu; Uchida, Shinichi

2015-12-01

Previous genome-wide association studies identified serine threonine kinase 39 (STK39), encoding STE20/SPS1-related proline/alanine-rich kinase, as one of a limited number of hypertension susceptibility genes. A recent meta-analysis confirmed the association of STK39 intronic polymorphism rs3754777 with essential hypertension, among previously reported hypertension-associated STK39 polymorphisms. However, the biochemical function of this polymorphism in the mechanism responsible for hypertension is yet to be clarified. We generated rs3754777G>A knockin human cell lines with clustered regularly interspaced short palindromic repeats-mediated genome engineering. Homozygous (A/A) and heterozygous (G/A) knockin human embryonic kidney cell lines were generated using a double nickase, single-guide RNAs targeting STK39 intron 5 around single-nucleotide polymorphism, and a 100-bp donor single-stranded DNA oligonucleotide. Reverse transcription polymerase chain reaction with sequencing analyses revealed the identical STK39 transcripts among the wild-type and both knockin cell lines. Quantitative reverse transcription polymerase chain reaction showed increased STK39 mRNA expression, and immunoblot analysis revealed increases in total and phosphorylated STE20/SPS1-related proline/alanine-rich kinase with increased phosphorylated Na-K-Cl cotransporter isoform 1 in both knockin cell lines. The largest increases in these molecules were observed in the homozygous cell line. These findings indicated that this intronic polymorphism increases STK39 transcription, leading to activation of the STE20/SPS1-related proline/alanine-rich kinase-solute carrier family 12A signaling cascade. Increased interactions between STE20/SPS1-related proline/alanine-rich kinase and the target cation-chloride cotransporters may be responsible for hypertension susceptibility in individuals with this polymorphism. © 2015 American Heart Association, Inc.

p62 targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding

PubMed Central

Itakura, Eisuke

2011-01-01

Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)–associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway. PMID:21220506

Eccentric muscle challenge shows osteopontin polymorphism modulation of muscle damage.

PubMed

Barfield, Whitney L; Uaesoontrachoon, Kitipong; Wu, Chung-Sheih; Lin, Stephen; Chen, Yue; Wang, Paul C; Kanaan, Yasmine; Bond, Vernon; Hoffman, Eric P

2014-08-01

A promoter polymorphism of the osteopontin (OPN) gene (rs28357094) has been associated with multiple inflammatory states, severity of Duchenne muscular dystrophy (DMD) and muscle size in healthy young adults. We sought to define the mechanism of action of the polymorphism, using allele-specific in vitro reporter assays in muscle cells, and a genotype-stratified intervention in healthy controls. In vitro reporter constructs showed the G allele to respond to estrogen treatment, whereas the T allele showed no transcriptional response. Young adult volunteers (n = 187) were enrolled into a baseline study, and subjects with specific rs28357094 genotypes enrolled into an eccentric muscle challenge intervention [n = 3 TT; n = 3 GG/GT (dominant inheritance model)]. Female volunteers carrying the G allele showed significantly greater inflammation and increased muscle volume change as determined by magnetic resonance imaging T1- and T2-weighted images after eccentric challenge, as well as greater decrement in biceps muscle force. Our data suggest a model where the G allele enables enhanced activities of upstream enhancer elements due to loss of Sp1 binding at the polymorphic site. This results in significantly greater expression of the pro-inflammatory OPN cytokine during tissue remodeling in response to challenge in G allele carriers, promoting muscle hypertrophy in normal females, but increased damage in DMD patients. © The Author 2014. Published by Oxford University Press.

The novel cyst nematode effector protein 30D08 targets host nuclear functions to alter gene expression in feeding sites.

PubMed

Verma, Anju; Lee, Chris; Morriss, Stephanie; Odu, Fiona; Kenning, Charlotte; Rizzo, Nancy; Spollen, William G; Lin, Marriam; McRae, Amanda G; Givan, Scott A; Hewezi, Tarek; Hussey, Richard; Davis, Eric L; Baum, Thomas J; Mitchum, Melissa G

2018-05-04

Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Microfluidic droplet enrichment for targeted sequencing

PubMed Central

Eastburn, Dennis J.; Huang, Yong; Pellegrino, Maurizio; Sciambi, Adam; Ptáček, Louis J.; Abate, Adam R.

2015-01-01

Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications. PMID:25873629

Deficient prepulse inhibition in schizophrenia detected by the multi-site COGS.

PubMed

Swerdlow, Neal R; Light, Gregory A; Sprock, Joyce; Calkins, Monica E; Green, Michael F; Greenwood, Tiffany A; Gur, Raquel E; Gur, Ruben C; Lazzeroni, Laura C; Nuechterlein, Keith H; Radant, Allen D; Ray, Amrita; Seidman, Larry J; Siever, Larry J; Silverman, Jeremy M; Stone, William S; Sugar, Catherine A; Tsuang, Debby W; Tsuang, Ming T; Turetsky, Bruce I; Braff, David L

2014-02-01

Startle inhibition by weak prepulses (PPI) is studied to understand the biology of information processing in schizophrenia patients and healthy comparison subjects (HCS). The Consortium on the Genetics of Schizophrenia (COGS) identified associations between PPI and single nucleotide polymorphisms in schizophrenia probands and unaffected relatives, and linkage analyses extended evidence for the genetics of PPI deficits in schizophrenia in the COGS-1 family study. These findings are being extended in a 5-site "COGS-2" study of 1800 patients and 1200 unrelated HCS to facilitate genetic analyses. We describe a planned interim analysis of COGS-2 PPI data. Eyeblink startle was measured in carefully screened HCS and schizophrenia patients (n=1402). Planned analyses of PPI (60 ms intervals) assessed effects of diagnosis, sex and test site, PPI-modifying effects of medications and smoking, and relationships between PPI and neurocognitive measures. 884 subjects met strict inclusion criteria. ANOVA of PPI revealed significant effects of diagnosis (p=0.0005) and sex (p<0.002), and a significant diagnosis×test site interaction. HCS>schizophrenia PPI differences were greatest among patients not taking 2nd generation antipsychotics, and were independent of smoking status. Modest but significant relationships were detected between PPI and performance in specific neurocognitive measures. The COGS-2 multi-site study detects schizophrenia-related PPI deficits reported in single-site studies, including patterns related to diagnosis, prepulse interval, sex, medication and other neurocognitive measures. Site differences were detected and explored. The target COGS-2 schizophrenia "endophenotype" of reduced PPI should prove valuable for identifying and confirming schizophrenia risk genes in future analyses. Copyright © 2013 Elsevier B.V. All rights reserved.

Deficient prepulse inhibition in schizophrenia detected by the multi-site COGS

PubMed Central

Swerdlow, Neal R.; Light, Gregory A.; Sprock, Joyce; Calkins, Monica E.; Green, Michael F.; Greenwood, Tiffany A.; Gur, Raquel E.; Gur, Ruben C.; Lazzeroni, Laura C.; Nuechterlein, Keith H.; Radant, Allen D.; Ray, Amrita; Seidman, Larry J.; Siever, Larry J.; Silverman, Jeremy M.; Stone, William S.; Sugar, Catherine A.; Tsuang, Debby W.; Tsuang, Ming T.; Turetsky, Bruce I.; Braff, David L.

2014-01-01

Background Startle inhibition by weak prepulses (PPI) is studied to understand the biology of information processing in schizophrenia patients and healthy comparison subjects (HCS). The Consortium on the Genetics of Schizophrenia (COGS) identified associations between PPI and single nucleotide polymorphisms in schizophrenia probands and unaffected relatives, and linkage analyses extended evidence for the genetics of PPI deficits in schizophrenia in the COGS-1 family study. These findings are being extended in a 5-site “COGS-2” study of 1800 patients and 1200 unrelated HCS to facilitate genetic analyses. We describe a planned interim analysis of COGS-2 PPI data. Methods Eyeblink startle was measured in carefully screened HCS and schizophrenia patients (n=1402). Planned analyses of PPI (60 ms intervals) assessed effects of diagnosis, sex and test site, PPI-modifying effects of medications and smoking, and relationships between PPI and neurocognitive measures. Results 884 subjects met strict inclusion criteria. ANOVA of PPI revealed significant effects of diagnosis (p=0.0005) and sex (p<0.002), and a significant diagnosis × test site interaction. HCS > schizophrenia PPI differences were greatest among patients not taking 2nd generation antipsychotics, and were independent of smoking status. Modest but significant relationships were detected between PPI and performance in specific neurocognitive measures. Discussion The COGS-2 multi-site study detects schizophrenia-related PPI deficits reported in single-site studies, including patterns related to diagnosis, prepulse interval, sex, medication and other neurocognitive measures. Site differences were detected and explored. The target COGS-2 schizophrenia “endophenotype” of reduced PPI should prove valuable for identifying and confirming schizophrenia risk genes in future analyses. PMID:24405980

The effects of genetic polymorphism on treatment response of recombinant human growth hormone.

PubMed

Chen, Shi; You, Hanxiao; Pan, Hui; Zhu, Huijuan; Yang, Hongbo; Gong, Fengying; Wang, Linjie; Jiang, Yu; Yan, Chengsheng

2017-12-06

Recombinant human growth hormone (rhGH) has been widely used in clinical treatment of growth hormone deficiency (GHD) or non GHD since 1985 and technology have achieved a great development in different long-acting formulations. Although the mathematical models for predicting the growth hormone response could help clinicians get to an individual personalized growth dose, many patients just can't reach the target height and the growth hormone responses differed.Genetic polymorphisms may play a role in the varies of individual responses in this treatment process.This article gives an overview of the genetic polymorphisms research of growth hormone in recent years, in order to give some potential suggestion and guide for the dose titration during treatment. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites

PubMed Central

Ji, Wei-ke; Hatch, Anna L; Merrill, Ronald A; Strack, Stefan; Higgs, Henry N

2015-01-01

While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites. DOI: http://dx.doi.org/10.7554/eLife.11553.001 PMID:26609810

Detection of canonical A-to-G editing events at 3' UTRs and microRNA target sites in human lungs using next-generation sequencing.

PubMed

Soundararajan, Ramani; Stearns, Timothy M; Griswold, Anthony L; Mehta, Arpit; Czachor, Alexander; Fukumoto, Jutaro; Lockey, Richard F; King, Benjamin L; Kolliputi, Narasaiah

2015-11-03

RNA editing is a post-transcriptional modification of RNA. The majority of these changes result from adenosine deaminase acting on RNA (ADARs) catalyzing the conversion of adenosine residues to inosine in double-stranded RNAs (dsRNAs). Massively parallel sequencing has enabled the identification of RNA editing sites in human transcriptomes. In this study, we sequenced DNA and RNA from human lungs and identified RNA editing sites with high confidence via a computational pipeline utilizing stringent analysis thresholds. We identified a total of 3,447 editing sites that overlapped in three human lung samples, and with 50% of these sites having canonical A-to-G base changes. Approximately 27% of the edited sites overlapped with Alu repeats, and showed A-to-G clustering (>3 clusters in 100 bp). The majority of edited sites mapped to either 3' untranslated regions (UTRs) or introns close to splice sites; whereas, only few sites were in exons resulting in non-synonymous amino acid changes. Interestingly, we identified 652 A-to-G editing events in the 3' UTR of 205 target genes that mapped to 932 potential miRNA target binding sites. Several of these miRNA edited sites were validated in silico. Additionally, we validated several A-to-G edited sites by Sanger sequencing. Altogether, our study suggests a role for RNA editing in miRNA-mediated gene regulation and splicing in human lungs. In this study, we have generated a RNA editome of human lung tissue that can be compared with other RNA editomes across different lung tissues to delineate a role for RNA editing in normal and diseased states.

Distribution of virulence factors and association with emm polymorphism or isolation site among beta-hemolytic group G Streptococcus dysgalactiae subspecies equisimilis.

PubMed

Lo, Hsueh-Hsia; Cheng, Wei-Shan

2015-01-01

Distribution of virulence factors and association with emm polymorphism or isolation site among beta-hemolytic group G Streptococcus dysgalactiae subspecies equisimilis. Streptococcus dysgalactiae subspecies equisimilis (SDSE), the dominant human pathogenic species among group G streptococci, is the causative agent of several invasive and non-invasive diseases worldwide. However, limited information is available about the distribution of virulence factors among SDSE isolates, or their association with emm types and the isolation sites. In this study, 246 beta-hemolytic group G SDSE isolates collected in central Taiwan between February 2007 and August 2011 were under investigation. Of these, 66 isolates were obtained from normally sterile sites and 180 from non-sterile sites. emm typing revealed 32 types, with the most prevalent one being stG10.0 (39.8%), followed by stG245.0 (15.4%), stG840.0 (12.2%), stG6.1 (7.7%), and stG652.0 (4.1%). The virulence genes lmb (encoding laminin-binding protein), gapC (glyceraldehyde 3-phosphate dehydrogenase), sagA (streptolysin S), and hylB (hyaluronidase) existed in all isolates. Also, 99.2% of the isolates possessed slo (streptolysin O) and scpA (C5a peptidase) genes. In addition, 72.8%, 14.6%, 9.4%, and 2.4% of the isolates possessed the genes ska (streptokinase), cbp (putative collagen-binding protein, SDEG_1781), fbp (putative fibronectin-binding protein, SDEG_0161), and sicG (streptococcal inhibitor of complement), respectively. The only superantigen gene detected was spegg (streptococcus pyrogenic exotoxin G(dys) ), which was possessed by 74.4% of the isolates; these isolates correlated with non-sterile sites. Positive correlations were observed between the following emm types and virulence genes: stG10.0 and stG840.0 with spegg, stG6.1 and stG652.0 with ska, and stG840.0 with cbp. On the other hand, negative correlations were observed between the following: stG245.0, stG6.1, and stG652.0 types with spegg, stG10.0 with ska

Human Alu insertion polymorphisms in North African populations.

PubMed

Cherni, Loth; Frigi, Sabeh; Ennafaa, Hajer; Mtiraoui, Nabil; Mahjoub, Touhami; Benammar-Elgaaied, Amel

2011-10-01

Several features make Alu insertions a powerful tool used in population genetic studies: the polymorphic nature of many Alu insertions, the stability of an Alu insertion event and, furthermore, the ancestral state of an Alu insertion is known to be the absence of the Alu element at a particular locus and the presence of an Alu insertion at the site that forward mutational change. This study analyses seven Alu insertion polymorphisms in a sample of 297 individuals from the autochthonous population of Tunisia (Thala, Smar, Zarzis, and Bou Salem) and Libya with the aim of studying their genetic structure with respect to the populations of North Africa, Western, Eastern and Central Europe. The comparative analyses carried out using the MDS and AMOVA methods reveal the existence of spatial heterogeneity, and identify four population groups. Study populations (Libya, Smar, Zarzis, and Bou Salem) are closest to North African populations whereas Thala is isolated and is closest to Western European populations. In conclusion, Results of the present study support the important role that migratory movements have played in the North African gene pool, at least since the Neolithic period.

PPARγ gene polymorphism, C-reactive protein level, BMI and periodontitis in post-menopausal Japanese women.

PubMed

Wang, Yangming; Sugita, Noriko; Yoshihara, Akihiro; Iwasaki, Masanori; Miyazaki, Hideo; Nakamura, Kazutoshi; Yoshie, Hiromasa

2016-03-01

Several studies have reported inconsistent results regarding the association between the PPARγPro12Ala polymorphism and obesity. Obese individuals had higher C-reactive protein (CRP) levels compared with those of normal weight, and PPARγ activation could significantly reduce serum high-sensitive CRP level. We have previously suggested that the Pro12Ala polymorphism represents a susceptibility factor for periodontitis, which is a known risk factor for increased CRP level. The aim was to investigate associations between PPARγ gene polymorphism, serum CRP level, BMI and/or periodontitis among post-menopausal Japanese women. The final sample in this study comprised 359 post-menopausal Japanese women. Periodontal parameters, including PD, CAL and BOP, were measured per tooth. PPARγPro12Ala genotype was determined by PCR-RFLP. Hs-CRP value was measured by a latex nephelometry assay. No significant differences in age, BMI or periodontal parameters were found between the genotypes. The percentages of sites with PD ≥ 4 mm were significantly higher among the hsCRP ≥ 1 mg/l group than the hsCRP < 1 mg/l group (p = 0.003). Positive correlations were found between serum hsCRP levels and the percentages of sites with PD ≥ 4 mm (p = 0.043) in PPARγ Ala allele carriers, and BMI (p = 0.033) in non-carriers. After adjustment for model covariates, BMI was significantly associated with serum hsCRP level. The PPARγPro12Ala polymorphism was not independently associated with periodontitis, serum CRP level or BMI in post-menopausal Japanese women. However, serum hsCRP level correlated with periodontitis in Ala allele carriers, and with BMI in non-carriers. © 2014 John Wiley & Sons A/S and The Gerodontology Society. Published by John Wiley & Sons Ltd.

Identifying disease polymorphisms from case-control genetic association data.

PubMed

Park, L

2010-12-01

In case-control association studies, it is typical to observe several associated polymorphisms in a gene region. Often the most significantly associated polymorphism is considered to be the disease polymorphism; however, it is not clear whether it is the disease polymorphism or there is more than one disease polymorphism in the gene region. Currently, there is no method that can handle these problems based on the linkage disequilibrium (LD) relationship between polymorphisms. To distinguish real disease polymorphisms from markers in LD, a method that can detect disease polymorphisms in a gene region has been developed. Relying on the LD between polymorphisms in controls, the proposed method utilizes model-based likelihood ratio tests to find disease polymorphisms. This method shows reliable Type I and Type II error rates when sample sizes are large enough, and works better with re-sequenced data. Applying this method to fine mapping using re-sequencing or dense genotyping data would provide important information regarding the genetic architecture of complex traits.

Target-site resistance to neonicotinoids.

PubMed

Crossthwaite, Andrew J; Rendine, Stefano; Stenta, Marco; Slater, Russell

2014-10-01

Neonicotinoid insecticides selectively target the invertebrate nicotinic acetylcholine receptor and disrupt excitatory cholinergic neurotransmission. First launched over 20 years ago, their broad pest spectrum, variety of application methods and relatively low risk to nontarget organisms have resulted in this class dominating the insecticide market with global annual sales in excess of $3.5 bn. This remarkable commercial success brings with it conditions in the field that favour selection of resistant phenotypes. A number of important pest species have been identified with mutations at the nicotinic acetylcholine receptor associated with insensitivity to neonicotinoids. The detailed characterization of these mutations has facilitated a greater understanding of the invertebrate nicotinic acetylcholine receptor.

Linking Oviposition Site Choice to Offspring Fitness in Aedes aegypti: Consequences for Targeted Larval Control of Dengue Vectors

PubMed Central

Wong, Jacklyn; Morrison, Amy C.; Stoddard, Steven T.; Astete, Helvio; Chu, Yui Yin; Baseer, Imaan; Scott, Thomas W.

2012-01-01

Background Current Aedes aegypti larval control methods are often insufficient for preventing dengue epidemics. To improve control efficiency and cost-effectiveness, some advocate eliminating or treating only highly productive containers. The population-level outcome of this strategy, however, will depend on details of Ae. aegypti oviposition behavior. Methodology/Principal Findings We simultaneously monitored female oviposition and juvenile development in 80 experimental containers located across 20 houses in Iquitos, Peru, to test the hypothesis that Ae. aegypti oviposit preferentially in sites with the greatest potential for maximizing offspring fitness. Females consistently laid more eggs in large vs. small containers (β = 9.18, p<0.001), and in unmanaged vs. manually filled containers (β = 5.33, p<0.001). Using microsatellites to track the development of immature Ae. aegypti, we found a negative correlation between oviposition preference and pupation probability (β = −3.37, p<0.001). Body size of emerging adults was also negatively associated with the preferred oviposition site characteristics of large size (females: β = −0.19, p<0.001; males: β = −0.11, p = 0.002) and non-management (females: β = −0.17, p<0.001; males: β = −0.11, p<0.001). Inside a semi-field enclosure, we simulated a container elimination campaign targeting the most productive oviposition sites. Compared to the two post-intervention trials, egg batches were more clumped during the first pre-intervention trial (β = −0.17, P<0.001), but not the second (β = 0.01, p = 0.900). Overall, when preferred containers were unavailable, the probability that any given container received eggs increased (β = 1.36, p<0.001). Conclusions/Significance Ae. aegypti oviposition site choice can contribute to population regulation by limiting the production and size of adults. Targeted larval control strategies may unintentionally lead to

Targeted Nanotechnology for Cancer Imaging

PubMed Central

Toy, Randall; Bauer, Lisa; Hoimes, Christopher; Ghaghada, Ketan B.; Karathanasis, Efstathios

2014-01-01

Targeted nanoparticle imaging agents provide many benefits and new opportunities to facilitate accurate diagnosis of cancer and significantly impact patient outcome. Due to the highly engineerable nature of nanotechnology, targeted nanoparticles exhibit significant advantages including increased contrast sensitivity, binding avidity and targeting specificity. Considering the various nanoparticle designs and their adjustable ability to target a specific site and generate detectable signals, nanoparticles can be optimally designed in terms of biophysical interactions (i.e., intravascular and interstitial transport) and biochemical interactions (i.e., targeting avidity towards cancer-related biomarkers) for site-specific detection of very distinct microenvironments. This review seeks to illustrate that the design of a nanoparticle dictates its in vivo journey and targeting of hard-to-reach cancer sites, facilitating early and accurate diagnosis and interrogation of the most aggressive forms of cancer. We will report various targeted nanoparticles for cancer imaging using X-ray computed tomography, ultrasound, magnetic resonance imaging, nuclear imaging and optical imaging. Finally, to realize the full potential of targeted nanotechnology for cancer imaging, we will describe the challenges and opportunities for the clinical translation and widespread adaptation of targeted nanoparticles imaging agents. PMID:25116445

Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

PubMed Central

Atherton, Richard; Bhavnani, Darlene; Calvopiña, Manuel; Vicuña, Yosselin; Cevallos, William; Eisenberg, Joseph

2013-01-01

The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay) were analysed using a semi-nested polymerase chain reaction-restriction fragment length polymorphism assay that targets the glutamate dehydrogenase (gdh) gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61%) were classified as assemblage B (26 as BIII and 16 as BIV), 22 (32%) as assemblage A (3 as AI and 19 as AII) and five (7%) as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A. PMID:23827993

Involvement of human histamine N-methyltransferase gene polymorphisms in susceptibility to atopic dermatitis in korean children.

PubMed

Lee, Hee Seon; Kim, Seung-Hyun; Kim, Kyung Won; Baek, Ji Young; Park, Hae-Sim; Lee, Kyung Eun; Hong, Jung Yeon; Kim, Mi Na; Heo, Won Il; Sohn, Myung Hyun; Kim, Kyu-Earn

2012-01-01

Histamine N-methyltransferase (HNMT) catalyzes one of two major histamine metabolic pathways. Histamine is a mediator of pruritus in atopic dermatitis (AD). The aim of this study was to evaluate the association between HNMT polymorphisms and AD in children. We genotyped 763 Korean children for allelic determinants at four polymorphic sites in the HNMT gene: -465T>C, -413C>T, 314C>T, and 939A>G. Genotyping was performed using a TaqMan fluorogenic 5' nuclease assay. The functional effect of the 939A>G polymorphism was analyzed. Of the 763 children, 520 had eczema and 542 had atopy. Distributions of the genotype and allele frequencies of the HNMT 314C>T polymorphism were significantly associated with non-atopic eczema (P=0.004), and those of HNMT 939A>G were significantly associated with eczema in the atopy groups (P=0.048). Frequency distributions of HNMT -465T>C and -413C>T were not associated with eczema. Subjects who were AA homozygous or AG heterozygous for 939A>G showed significantly higher immunoglobulin E levels than subjects who were GG homozygous (P=0.009). In U937 cells, the variant genotype reporter construct had significantly higher mRNA stability (P<0.001) and HNMT enzyme activity (P<0.001) than the common genotype. Polymorphisms in HNMT appear to confer susceptibility to AD in Korean children.

Thiamethoxam acts as a target-site synergist of spinosad in resistant strains of Frankliniella occidentalis.

PubMed

Guillén, Juan; Bielza, Pablo

2013-02-01

Previous studies have suggested that the resistance mechanism towards spinosad in Frankliniella occidentalis (Pergande) is an altered target site. Like the neonicotinoids, the spinosyns act on nicotinic acetylcholine receptors (nAChRs) in insects, but at a distinct site. The changes in nAChRs related to spinosad resistance in thrips might involve interaction with neonicotinoids. In this study, the efficacy of spinosad and neonicotinoids, alone and in combination, was evaluated in susceptible and spinosad-resistant thrips strains. The neonicotinoids tested were imidacloprid, thiacloprid, acetamiprid, thiamethoxam and clothianidin. No cross-resistance was shown between spinosad and any of the neonicotinoids. However, an increased toxicity was observed when a mixture of spinosad with thiamethoxam or clothianidin was tested. No synergism was found in the susceptible strains. The more spinosad-resistant the thrips strain, the stronger was the synergism. Data suggest that spinosad and thiamethoxam may interact at the nAChRs in spinosad-resistant thrips, facilitating enhanced insecticidal action. Copyright © 2012 Society of Chemical Industry.

Polymorphisms in Gag spacer peptide 1 confer varying levels of resistance to the HIV- 1 maturation inhibitor bevirimat.

PubMed

Adamson, Catherine S; Sakalian, Michael; Salzwedel, Karl; Freed, Eric O

2010-04-20

The maturation inhibitor bevirimat (BVM) potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking capsid-spacer peptide 1 (CA-SP1) cleavage. Recent clinical trials demonstrated that a significant proportion of HIV-1-infected patients do not respond to BVM. A patient's failure to respond correlated with baseline polymorphisms at SP1 residues 6-8. In this study, we demonstrate that varying levels of BVM resistance are associated with point mutations at these residues. BVM susceptibility was maintained by SP1-Q6A, -Q6H and -T8A mutations. However, an SP1-V7A mutation conferred high-level BVM resistance, and SP1-V7M and T8Delta mutations conferred intermediate levels of BVM resistance. Future exploitation of the CA-SP1 cleavage site as an antiretroviral drug target will need to overcome the baseline variability in the SP1 region of Gag.

MiR-149 Compromises the Reactions of Liver Cells to Fatty Acid via its Polymorphism and Increases Non-Alcoholic Fatty Liver Disease (NAFLD) Risk by Targeting Methylene Tetrahydrofolate Reductase (MTHFR).

PubMed

An, Xianchao; Yang, Zonglin; An, Zhengzhuang

2017-05-16

BACKGROUND Non-alcoholic fatty liver disease (NAFLD) is a worldwide health problem, and microRNA (miRNA) has been reported to be involved in NAFLD. The objective of our study was to explore the effect of polymorphism in miR-149 on the pathogenesis of NAFLD. MATERIAL AND METHODS Real-time PCR was performed to explore the effect of long-chain fatty acid (FFA) on the level of miR-149 and methylene tetrahydrofolate reductase (MTHFR). Then in-silicon analysis and luciferase assay were investigated to verify MTHFR was the target gene of miR-149. Finally, Western-blot analysis and real-time PCR were performed to confirm the control of MTHFR by miR-149. RESULTS In this study, we found that miR-149 was apparently upregulated in hepatocytes genotyped as TT treated with FFA; and MTHFR in hepatocytes genotyped as TT treated with FFA was evidently downregulated compared to control. Whereas, FFA had no obvious effect on MTHFR level in hepatocytes genotyped as CC. We searched an online miRNA database and found that miR-149 was a regulator of MTHFR expression, which was confirmed by luciferase assay. In hepatocytes genotyped as TT and treated with or without FFA, miR-149 mimic dose-dependently decreased the level of MTHFR, and miR-149 inhibitor dose-dependently increased the level of MTHFR. And in hepatocytes genotyped as CC treated with or without FFA exhibited a similar inhibition effect of miR-149 on expression of MTHFR. CONCLUSIONS The data suggested that the polymorphism in miR-149 played an important role in the development of NAFLD via altering the expression of miR-149 as well as its target, MTHFR.

MiR-149 Compromises the Reactions of Liver Cells to Fatty Acid via its Polymorphism and Increases Non-Alcoholic Fatty Liver Disease (NAFLD) Risk by Targeting Methylene Tetrahydrofolate Reductase (MTHFR)

PubMed Central

An, Xianchao; Yang, Zonglin; An, Zhengzhuang

2017-01-01

Background Non-alcoholic fatty liver disease (NAFLD) is a worldwide health problem, and microRNA (miRNA) has been reported to be involved in NAFLD. The objective of our study was to explore the effect of polymorphism in miR-149 on the pathogenesis of NAFLD. Material/Methods Real-time PCR was performed to explore the effect of long-chain fatty acid (FFA) on the level of miR-149 and methylene tetrahydrofolate reductase (MTHFR). Then in-silicon analysis and luciferase assay were investigated to verify MTHFR was the target gene of miR-149. Finally, Western-blot analysis and real-time PCR were performed to confirm the control of MTHFR by miR-149. Results In this study, we found that miR-149 was apparently upregulated in hepatocytes genotyped as TT treated with FFA; and MTHFR in hepatocytes genotyped as TT treated with FFA was evidently downregulated compared to control. Whereas, FFA had no obvious effect on MTHFR level in hepatocytes genotyped as CC. We searched an online miRNA database and found that miR-149 was a regulator of MTHFR expression, which was confirmed by luciferase assay. In hepatocytes genotyped as TT and treated with or without FFA, miR-149 mimic dose-dependently decreased the level of MTHFR, and miR-149 inhibitor dose-dependently increased the level of MTHFR. And in hepatocytes genotyped as CC treated with or without FFA exhibited a similar inhibition effect of miR-149 on expression of MTHFR. Conclusions The data suggested that the polymorphism in miR-149 played an important role in the development of NAFLD via altering the expression of miR-149 as well as its target, MTHFR. PMID:28507283

Regionally clustered ABCC8 polymorphisms in a prospective cohort predict cerebral oedema and outcome in severe traumatic brain injury.

PubMed

Jha, Ruchira Menka; Koleck, Theresa A; Puccio, Ava M; Okonkwo, David O; Park, Seo-Young; Zusman, Benjamin E; Clark, Robert S B; Shutter, Lori A; Wallisch, Jessica S; Empey, Philip E; Kochanek, Patrick M; Conley, Yvette P

2018-04-19

ABCC8 encodes sulfonylurea receptor 1, a key regulatory protein of cerebral oedema in many neurological disorders including traumatic brain injury (TBI). Sulfonylurea-receptor-1 inhibition has been promising in ameliorating cerebral oedema in clinical trials. We evaluated whether ABCC8 tag single-nucleotide polymorphisms predicted oedema and outcome in TBI. DNA was extracted from 485 prospectively enrolled patients with severe TBI. 410 were analysed after quality control. ABCC8 tag single-nucleotide polymorphisms (SNPs) were identified (Hapmap, r 2 >0.8, minor-allele frequency >0.20) and sequenced (iPlex-Gold, MassArray). Outcomes included radiographic oedema, intracranial pressure (ICP) and 3-month Glasgow Outcome Scale (GOS) score. Proxy SNPs, spatial modelling, amino acid topology and functional predictions were determined using established software programs. Wild-type rs7105832 and rs2237982 alleles and genotypes were associated with lower average ICP (β=-2.91, p=0.001; β=-2.28, p=0.003) and decreased radiographic oedema (OR 0.42, p=0.012; OR 0.52, p=0.017). Wild-type rs2237982 also increased favourable 3-month GOS (OR 2.45, p=0.006); this was partially mediated by oedema (p=0.03). Different polymorphisms predicted 3-month outcome: variant rs11024286 increased (OR 1.84, p=0.006) and wild-type rs4148622 decreased (OR 0.40, p=0.01) the odds of favourable outcome. Significant tag and concordant proxy SNPs regionally span introns/exons 2-15 of the 39-exon gene. This study identifies four ABCC8 tag SNPs associated with cerebral oedema and/or outcome in TBI, tagging a region including 33 polymorphisms. In polymorphisms predictive of oedema, variant alleles/genotypes confer increased risk. Different variant polymorphisms were associated with favourable outcome, potentially suggesting distinct mechanisms. Significant polymorphisms spatially clustered flanking exons encoding the sulfonylurea receptor site and transmembrane domain 0/loop 0 (juxtaposing the channel pore

Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

NASA Astrophysics Data System (ADS)

Czulak, J.; Guerreiro, A.; Metran, K.; Canfarotta, F.; Goddard, A.; Cowan, R. H.; Trochimczuk, A. W.; Piletsky, S.

2016-05-01

Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike

Association of interleukin-1 beta (-511C/T) polymorphisms with osteoporosis in postmenopausal women.

PubMed

Chao, Tai-Hung; Yu, Hsing-Ning; Huang, Chi-Chuan; Liu, Wen-Shen; Tsai, Ya-Wen; Wu, Wen-Tung

2010-01-01

Osteoporosis is a common disease of the elderly, in which genetic and clinical factors contribute to the disease phenotype. Since the production of interleukin-1 (IL-1) has been implicated in the bone mass and skeletal disorders, we investigated whether IL-1 system gene polymorphisms are associated with the pathogenesis of osteoporosis in postmenopausal Taiwanese women. Osteoporosis is diagnosed by dual-energy x-ray absorptiometry, which measures bone mineral density (BMD) at multiple skeletal sites. We studied the IL-1α (-889C/T), IL-1β (-511C/T) and the 86 base pair variable number tandem repeat (VNTR) in intron 2 of the IL-1 receptor antagonist (IL-1ra) gene in 117 postmenopausal women with osteoporosis and 135 control subjects without a history of symptomatic osteoporosis. These gene polymorphisms were analyzed by polymerase chain reaction and restriction fragment length polymerase. Blood sugar and other risk factors were also determined. The frequencies of IL-1β (-511C/T) genotypes (P=.022, odds ratio=1.972) and alleles (P=.02, odds ratio=2.909) showed a statistically significant difference between the two groups. However, we did not find any statistically significant difference in IL-1β and IL-1ra polymorphisms (P>.05). We also observed a positive relationship between osteoporosis and cholesterol and a weak inverse relationship between blood sugar and osteoporosis in postmenopausal women. These experimental results suggest that the pathogenesis of osteoporosis is associated with IL-1β (-511C/T) polymorphism in postmenopausal women. This polymorphism is an independent risk factor for osteoporosis.

Controlling the selective formation of calcium sulfate polymorphs at room temperature.

PubMed

Tritschler, Ulrich; Van Driessche, Alexander E S; Kempter, Andreas; Kellermeier, Matthias; Cölfen, Helmut

2015-03-23

Calcium sulfate is a naturally abundant and technologically important mineral with a broad scope of applications. However, controlling CaSO4 polymorphism and, with it, its final material properties still represents a major challenge, and to date there is no universal method for the selective production of the different hydrated and anhydrous forms under mild conditions. Herein we report the first successful synthesis of pure anhydrite from solution at room temperature. We precipitated calcium sulfate in alcoholic media at low water contents. Moreover, by adjusting the amount of water in the syntheses, we can switch between the distinct polymorphs and fine-tune the outcome of the reaction, yielding either any desired CaSO4 phase in pure state or binary mixtures with predefined compositions. This concept provides full control over phase selection in CaSO4 mineralization and may allow for the targeted fabrication of corresponding materials for use in various areas. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chromosomal polymorphism in mammals: an evolutionary perspective.

PubMed

Dobigny, Gauthier; Britton-Davidian, Janice; Robinson, Terence J

2017-02-01

Although chromosome rearrangements (CRs) are central to studies of genome evolution, our understanding of the evolutionary consequences of the early stages of karyotypic differentiation (i.e. polymorphism), especially the non-meiotic impacts, is surprisingly limited. We review the available data on chromosomal polymorphisms in mammals so as to identify taxa that hold promise for developing a more comprehensive understanding of chromosomal change. In doing so, we address several key questions: (i) to what extent are mammalian karyotypes polymorphic, and what types of rearrangements are principally involved? (ii) Are some mammalian lineages more prone to chromosomal polymorphism than others? More specifically, do (karyotypically) polymorphic mammalian species belong to lineages that are also characterized by past, extensive karyotype repatterning? (iii) How long can chromosomal polymorphisms persist in mammals? We discuss the evolutionary implications of these questions and propose several research avenues that may shed light on the role of chromosome change in the diversification of mammalian populations and species. © 2015 Cambridge Philosophical Society.

Rhodium(II) proximity-labeling identifies a novel target site on STAT3 for inhibitors with potent anti-leukemia activity

PubMed Central

Minus, Matthew B.; Liu, Wei; Vohidov, Farrukh; Kasembeli, Moses M.; Long, Xin; Krueger, Michael; Stevens, Alexandra; Kolosov, Mikhail I.; Sison, Edward Allen R.; Ball, Zachary T.

2015-01-01

Nearly 40% of children with acute myeloid leukemia (AML) suffer relapse due to chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). In this paper, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML. PMID:26480340

Dengue vector management using insecticide treated materials and targeted interventions on productive breeding-sites in Guatemala.

PubMed

Rizzo, Nidia; Gramajo, Rodrigo; Escobar, Maria Cabrera; Arana, Byron; Kroeger, Axel; Manrique-Saide, Pablo; Petzold, Max

2012-10-30

In view of the epidemiological expansion of dengue worldwide and the availability of new tools and strategies particularly for controlling the primary dengue vector Aedes aegypti, an intervention study was set up to test the efficacy, cost and feasibility of a combined approach of insecticide treated materials (ITMs) alone and in combination with appropriate targeted interventions of the most productive vector breeding-sites. The study was conducted as a cluster randomized community trial using "reduction of the vector population" as the main outcome variable. The trial had two arms: 10 intervention clusters (neighborhoods) and 10 control clusters in the town of Poptun Guatemala. Activities included entomological assessments (characteristics of breeding-sites, pupal productivity, Stegomyia indices) at baseline, 6 weeks after the first intervention (coverage of window and exterior doorways made of PermaNet 2.0 netting, factory treated with deltamethrin at 55 mg/m2, and of 200 L drums with similar treated material) and 6 weeks after the second intervention (combination of treated materials and other suitable interventions targeting productive breeding-sites i.e larviciding with Temephos, elimination etc.). The second intervention took place 17 months after the first intervention. The insecticide residual activity and the insecticidal content were also studied at different intervals. Additionally, information about demographic characteristics, cost of the intervention, coverage of houses protected and satisfaction in the population with the interventions was collected. At baseline (during the dry season) a variety of productive container types for Aedes pupae were identified: various container types holding >20 L, 200 L drums, washbasins and buckets (producing 83.7% of all pupae). After covering 100% of windows and exterior doorways and a small number of drums (where the commercial cover could be fixed) in 970 study households, tropical rains occurred in the area and

Dengue vector management using insecticide treated materials and targeted interventions on productive breeding-sites in Guatemala

PubMed Central

2012-01-01

Background In view of the epidemiological expansion of dengue worldwide and the availability of new tools and strategies particularly for controlling the primary dengue vector Aedes aegypti, an intervention study was set up to test the efficacy, cost and feasibility of a combined approach of insecticide treated materials (ITMs) alone and in combination with appropriate targeted interventions of the most productive vector breeding-sites. Methods The study was conducted as a cluster randomized community trial using “reduction of the vector population” as the main outcome variable. The trial had two arms: 10 intervention clusters (neighborhoods) and 10 control clusters in the town of Poptun Guatemala. Activities included entomological assessments (characteristics of breeding-sites, pupal productivity, Stegomyia indices) at baseline, 6 weeks after the first intervention (coverage of window and exterior doorways made of PermaNet 2.0 netting, factory treated with deltamethrin at 55 mg/m2, and of 200 L drums with similar treated material) and 6 weeks after the second intervention (combination of treated materials and other suitable interventions targeting productive breeding-sites i.e larviciding with Temephos, elimination etc.). The second intervention took place 17 months after the first intervention. The insecticide residual activity and the insecticidal content were also studied at different intervals. Additionally, information about demographic characteristics, cost of the intervention, coverage of houses protected and satisfaction in the population with the interventions was collected. Results At baseline (during the dry season) a variety of productive container types for Aedes pupae were identified: various container types holding >20 L, 200 L drums, washbasins and buckets (producing 83.7% of all pupae). After covering 100% of windows and exterior doorways and a small number of drums (where the commercial cover could be fixed) in 970 study households, tropical

Glyphosate resistance in Ambrosia trifida: Part 2. Rapid response physiology and non-target-site resistance.

PubMed

Moretti, Marcelo L; Van Horn, Christopher R; Robertson, Renae; Segobye, Kabelo; Weller, Stephen C; Young, Bryan G; Johnson, William G; Douglas Sammons, R; Wang, Dafu; Ge, Xia; d' Avignon, André; Gaines, Todd A; Westra, Philip; Green, Amanda C; Jeffery, Taylor; Lespérance, Mackenzie A; Tardif, François J; Sikkema, Peter H; Christopher Hall, J; McLean, Michael D; Lawton, Mark B; Schulz, Burkhard

2018-05-01

The glyphosate-resistant rapid response (GR RR) resistance mechanism in Ambrosia trifida is not due to target-site resistance (TSR) mechanisms. This study explores the physiology of the rapid response and the possibility of reduced translocation and vacuolar sequestration as non-target-site resistance (NTSR) mechanisms. GR RR leaf discs accumulated hydrogen peroxide within minutes of glyphosate exposure, but only in mature leaf tissue. The rapid response required energy either as light or exogenous sucrose. The combination of phenylalanine and tyrosine inhibited the rapid response in a dose-dependent manner. Reduced glyphosate translocation was observed in GR RR, but only when associated with tissue death caused by the rapid response. Nuclear magnetic resonance studies indicated that glyphosate enters the cytoplasm and reaches chloroplasts, and it is not moved into the vacuole of GR RR, GR non-rapid response or glyphosate-susceptible A. trifida. The GR RR mechanism of resistance is not associated with vacuole sequestration of glyphosate, and the observed reduced translocation is likely a consequence of rapid tissue death. Rapid cell death was inhibited by exogenous application of aromatic amino acids phenylalanine and tyrosine. The mechanism by which these amino acids inhibit rapid cell death in the GR RR phenotype remains unknown, and it could involve glyphosate phytotoxicity or other agents generating reactive oxygen species. Implications of these findings are discussed. The GR RR mechanism is distinct from the currently described glyphosate TSR or NTSR mechanisms in other species. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Relationship between target organ damage and blood pressure, retinal vessel calibre, oxidative stress and polymorphisms in VAV-2 and VAV-3 genes in patients with hypertension: a case-control study protocol (LOD-Hipertension).

PubMed

Gomez-Marcos, Manuel A; Gonzalez-Sarmiento, Rogelio; Recio-Rodríguez, José I; Agudo-Conde, Cristina; Gamella-Pozuelo, Luis; Perretta-Tejedor, Nuria; Martínez-Salgado, Carlos; García-Ortiz, Luis

2014-04-03

Target organ damage (TOD) is associated with increased cardiovascular risk. The study objectives were to analyse the relationship of TOD to blood pressure, size of retinal arteries and veins, oxidative stress and different polymorphisms in the VAV-2 and VAV-3 genes in participants with hypertension. A case-control study to analyse the relationship between clinical, biochemical and genetic parameters and presence of cardiac, vascular and renal TOD in 486 patients with hypertension. Participants with TOD will be considered as cases, and those without TOD will be enrolled as controls. This will be a collaborative study conducted by the groups of Primary Care, Cardiovascular and Metabolic and Degenerative Diseases of the Instituto de Investigación Biomédica of Salamanca (IBSAL). Assessment of cardiac, renal and vascular TOD. Measurement of peripheral and central blood pressure, size of eye fundus arteries and veins, and oxidative stress, and polymorphisms in the VAV-2 and VAV-3 genes. The study will be conducted after approval is obtained from the Ethics Committee of Hospital Clínico Universitario of Salamanca. All study participants will sign an informed consent to agree to participate in the study, and another consent to agree on the genetic study, in compliance with the Declaration of Helsinki and the WHO standards for observational studies. The results of this study will allow for an understanding of the relationship of the different TODs with blood pressure, retinal artery and vein diameters, oxidative stress and polymorphisms in VAV-2 and VAV-3 genes. Clinical Trials. gov Identifier: NCT02022618.

Molecular nucleation mechanisms and control strategies for crystal polymorph selection

NASA Astrophysics Data System (ADS)

van Driessche, Alexander E. S.; van Gerven, Nani; Bomans, Paul H. H.; Joosten, Rick R. M.; Friedrich, Heiner; Gil-Carton, David; Sommerdijk, Nico A. J. M.; Sleutel, Mike

2018-04-01

The formation of condensed (compacted) protein phases is associated with a wide range of human disorders, such as eye cataracts, amyotrophic lateral sclerosis, sickle cell anaemia and Alzheimer’s disease. However, condensed protein phases have their uses: as crystals, they are harnessed by structural biologists to elucidate protein structures, or are used as delivery vehicles for pharmaceutical applications. The physiochemical properties of crystals can vary substantially between different forms or structures (‘polymorphs’) of the same macromolecule, and dictate their usability in a scientific or industrial context. To gain control over an emerging polymorph, one needs a molecular-level understanding of the pathways that lead to the various macroscopic states and of the mechanisms that govern pathway selection. However, it is still not clear how the embryonic seeds of a macromolecular phase are formed, or how these nuclei affect polymorph selection. Here we use time-resolved cryo-transmission electron microscopy to image the nucleation of crystals of the protein glucose isomerase, and to uncover at molecular resolution the nucleation pathways that lead to two crystalline states and one gelled state. We show that polymorph selection takes place at the earliest stages of structure formation and is based on specific building blocks for each space group. Moreover, we demonstrate control over the system by selectively forming desired polymorphs through site-directed mutagenesis, specifically tuning intermolecular bonding or gel seeding. Our results differ from the present picture of protein nucleation, in that we do not identify a metastable dense liquid as the precursor to the crystalline state. Rather, we observe nucleation events that are driven by oriented attachments between subcritical clusters that already exhibit a degree of crystallinity. These insights suggest ways of controlling macromolecular phase transitions, aiding the development of protein

CRISPR/Cas9-Mediated Insertion of loxP Sites in the Mouse Dock7 Gene Provides an Effective Alternative to Use of Targeted Embryonic Stem Cells.

PubMed

Bishop, Kathleen A; Harrington, Anne; Kouranova, Evguenia; Weinstein, Edward J; Rosen, Clifford J; Cui, Xiaoxia; Liaw, Lucy

2016-07-07

Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful ∼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation. Copyright © 2016 Bishop et al.

Associations of the eNOS G894T gene polymorphism with target organ damage in children with newly diagnosed primary hypertension.

PubMed

Śladowska-Kozłowska, Joanna; Litwin, Mieczysław; Niemirska, Anna; Wierzbicka, Aldona; Roszczynko, Marta; Szperl, Małgorzata

2015-12-01

The endothelial nitric oxide synthase (eNOS) G894T gene polymorphism is associated with the risk of primary hypertension (PH) and vascular complications in adults with PH. We explored the associations of the G894T polymorphism with 24-h ambulatory blood pressure, left ventricular mass (LVM), carotid intima media thickness (cIMT), urinary albumin excretion, oxidative stress and inflammatory parameters in 126 children with newly diagnosed PH and in 83 healthy children. Among the 126 children with PH 92 (73%) had ambulatory hypertension and 34 (27%) had severe ambulatory hypertension. Left ventricular hypertrophy (LVH) was detected in 39 (31%) patients, cIMT of >2 standard deviation scores in 21 (16.6%) patients, albuminuria of >30 mg/24 h in 18 (14.3%) patients and metabolic syndrome (MS) in 22 (17.5%) patients. The frequency of the T allele was 52.4% in the PH group and 54.2% in the control group (not significant), and in both groups the frequency of the T allele was consistent with the Hardy-Weinberg equilibrium. Compared with G allele carriers, hypertensive T allele carriers had increased cIMT (p < 0.05) and more severe albuminuria (not significant, p = 0.1); there was no difference between the groups in hypertension severity and LVM. T and G allele distribution did not differ between patients with and without metabolic syndrome. No significant correlations between the assessed parameters and the eNOS G894T gene polymorphism were found in the controls, although T allele carriers tended to have an increased cIMT (p = 0.09). The eNOS T allele is not more prevalent among hypertensive children than among healthy ones, but it is associated with early vascular damage in children with PH, independent of metabolic abnormalities. No associations between the eNOS G894T polymorphism and metabolic abnormalities were found.

Genetic polymorphisms related to efficacy and overuse of triptans in chronic migraine.

PubMed

Gentile, Giovanna; Borro, Marina; Lala, Noemi; Missori, Serena; Simmaco, Maurizio; Martelletti, Paolo

2010-10-01

Migraine is a common type of headache and its most severe attacks are usually treated with triptans, the efficacy of which is extremely variable. Several SNPs in genes involved in metabolism and target mechanisms of triptans have been described. To define an association between genetic profile and triptan response, we classified a migrainous population on the basis of triptan response and characterized it for polymorphisms in the genes coding for monoamine oxidase A, G protein β3 and the cytochrome CYP1A2. Analysis of the association between genotypic and allelic frequencies of the analyzed SNPs and the grade of response to triptan administration showed a significant correlation for MAOA uVNTR polymorphism. Further stratification of patients in abuser and non-abuser groups revealed a significant association with triptan overuse and, within the abusers, with drug response to the CYP1A2*1F variant.

Assessment of Azorean ancestry by Alu insertion polymorphisms.

PubMed

Branco, Claudia C; Palla, Raquel; Lino, Sílvia; Pacheco, Paula R; Cabral, Rita; De Fez, Laura; Peixoto, Bernardo R; Mota-Vieira, Luisa

2006-01-01

Knowledge of population ancestry from genetic markers is essential, for example, to understand the history of human migration and to carry out admixture and association studies. Here we assess the genome ancestry of the Azorean population through analysis of six Alu polymorphic sites (TPA-25, ACE, APO, B65, PV92, and D1) in 65 Azoreans and 30 Portuguese unrelated blood donors and compare data for the Y-chromosome and mtDNA. Allele frequencies were calculated by direct counting. Statistical analysis was performed using Arlequin 2.0. Nei's genetic distance was calculated with DISPAN software, and trees were constructed by neighbor joining (NJ) using PHYLIP 3.63. The results show that all Alu insertions were polymorphic. APO is the closest to fixation. The less frequent insertions are PV92 and D1 in the Azores and Portugal, respectively. ACE and TPA-25 show the highest values of heterozygosity in both populations. Allele frequencies are very similar to those obtained in European populations. These results are validated by the Y-chromosome and mtDNA data, where the majority of the maternal and paternal lineages are European. Overall, these data are reflected in the phylogenetic tree, in which the Azoreans and the Portuguese branch with Catalans, Andalusians, Moroccans, and Algerians. We conclude that the population of the Azores shows no significant genetic differences from that of mainland Portugal and that it is an outbred population. Moreover, the data validate the use of Alu insertion polymorphisms to assess the origin and history of human populations. Am. J. Hum. Biol. 18:223-226, 2006. (c) 2006 Wiley-Liss, Inc.

Specter: linear deconvolution for targeted analysis of data-independent acquisition mass spectrometry proteomics.

PubMed

Peckner, Ryan; Myers, Samuel A; Jacome, Alvaro Sebastian Vaca; Egertson, Jarrett D; Abelin, Jennifer G; MacCoss, Michael J; Carr, Steven A; Jaffe, Jacob D

2018-05-01

Mass spectrometry with data-independent acquisition (DIA) is a promising method to improve the comprehensiveness and reproducibility of targeted and discovery proteomics, in theory by systematically measuring all peptide precursors in a biological sample. However, the analytical challenges involved in discriminating between peptides with similar sequences in convoluted spectra have limited its applicability in important cases, such as the detection of single-nucleotide polymorphisms (SNPs) and alternative site localizations in phosphoproteomics data. We report Specter (https://github.com/rpeckner-broad/Specter), an open-source software tool that uses linear algebra to deconvolute DIA mixture spectra directly through comparison to a spectral library, thus circumventing the problems associated with typical fragment-correlation-based approaches. We validate the sensitivity of Specter and its performance relative to that of other methods, and show that Specter is able to successfully analyze cases involving highly similar peptides that are typically challenging for DIA analysis methods.

Survivin -31 G/C polymorphism might contribute to colorectal cancer (CRC) risk: a meta-analysis.

PubMed

Yao, Linhua; Hu, Yi; Deng, Zhongmin; Li, Jingjing

2015-01-01

Published data has shown inconsistent findings about the association of survivin -31 G/C polymorphism with the risk of colorectal cancer (CRC). This meta-analysis quantitatively assesses the results from published studies to provide a more precise estimate of the association between survivin -31 G/C polymorphism as a possible predictor of the risk of CRC. We conducted a literature search in the PubMed, Web of Science, and Cochrane Library databases. Stata 12 software was used to calculate the pooled odds ratios (ORs) with 95% confidence intervals (CIs) based on the available data from each article. Six studies including 1840 cases with CRC and 1804 controls were included in this study. Survivin -31 G/C polymorphism was associated with a significantly increased risk of CRC (OR = 1.78; 95% CI, 1.53-2.07; I(2) = 0%). In the race subgroup analysis, both Asians (OR = 1.72; 95% CI, 1.44-2.05; I(2) = 0%) and Caucasians (OR = 1.93; 95% CI, 1.46-2.55; I(2) = 0%) with survivin -31 G/C polymorphism had increased CRC risk. In the subgroup analysis according to site of CRC, survivin -31 G/C polymorphism was not associated with colon cancer risk (OR = 2.02; 95% CI, 0.79-5.22; I(2) = 82%). However, this polymorphism was significantly associated with rectum cancer risk (OR = 1.98; 95% CI, 1.42-2.74; I(2) = 0%). In the subgroup analysis by clinical stage, both early stage (I+II) and advanced stage (III+IV) were associated with survivin -31 G/C polymorphism (OR = 1.61; 95% CI, 1.20-2.16; I(2) = 0% and OR = 2.30; 95% CI, 1.70-3.13; I(2) = 0%, respectively). In the subgroup analysis by smoke status, both smokers and non-smokers with survivin -31 G/C polymorphism showed increased CRC risk (OR = 1.47; 95% CI, 1.01-2.13; I(2) = 60% and OR = 1.71; 95% CI, 1.28-2.30; I(2) = 0%, respectively). In the subgroup analysis by drink status, both drinkers and non-drinkers with survivin -31 G/C polymorphism showed increased CRC risk (OR = 1.58; 95% CI, 1.06-2.37; I(2) = 8% and OR = 1.61; 95% CI, 1

Structural basis for microRNA targeting

DOE PAGES

Schirle, Nicole T.; Sheu-Gruttadauria, Jessica; MacRae, Ian J.

2014-10-31

MicroRNAs (miRNAs) control expression of thousands of genes in plants and animals. miRNAs function by guiding Argonaute proteins to complementary sites in messenger RNAs (mRNAs) targeted for repression. In this paper, we determined crystal structures of human Argonaute-2 (Ago2) bound to a defined guide RNA with and without target RNAs representing miRNA recognition sites. These structures suggest a stepwise mechanism, in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing. Pairing to nt 2 to 5 promotes conformational changes that expose nt 2 to 8 and 13 to 16 for further target recognition. Interactions withmore » the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner, whereas an adenosine binding-pocket opposite guide nt 1 further facilitates target recognition. Spurious slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion. Finally, these results explain the conserved nucleotide-pairing patterns in animal miRNA target sites first observed over two decades ago.« less

Preferential nucleation during polymorphic transformations

DOE PAGES

Sharma, H.; Sietsma, J.; Offerman, S. E.

2016-08-03

Polymorphism is the ability of a solid material to exist in more than one phase or crystal structure. Polymorphism may occur in metals, alloys, ceramics, minerals, polymers, and pharmaceutical substances. Unresolved are the conditions for preferential nucleation during polymorphic transformations in which structural relationships or special crystallographic orientation relationships (OR’s) form between the nucleus and surrounding matrix grains. We measured in-situ and simultaneously the nucleation rates of grains that have zero, one, two, three and four special OR’s with the surrounding parent grains. These experiments show a trend in which the activation energy for nucleation becomes smaller – and thereforemore » nucleation more probable - with increasing number of special OR’s. As a result, these insights contribute to steering the processing of polymorphic materials with tailored properties, since preferential nucleation affects which crystal structure forms, the average grain size and texture of the material, and thereby - to a large extent - the final properties of the material.« less

A resource of single-nucleotide polymorphisms for rainbow trout generated by restriction-site associated DNA sequencing of doubled haploids

USDA-ARS?s Scientific Manuscript database

Salmonid genomes are considered to be in a pseudo-tetraploid state as a result of an evolutionarily recent genome duplication event. This situation complicates single nucleotide polymorphism (SNP) discovery in rainbow trout as many putative SNPs are actually paralogous sequence variants (PSVs) and ...

Gene duplication in the major insecticide target site, Rdl, in Drosophila melanogaster

PubMed Central

Remnant, Emily J.; Good, Robert T.; Schmidt, Joshua M.; Lumb, Christopher; Robin, Charles; Daborn, Phillip J.; Batterham, Philip

2013-01-01

The Resistance to Dieldrin gene, Rdl, encodes a GABA-gated chloride channel subunit that is targeted by cyclodiene and phenylpyrazole insecticides. The gene was first characterized in Drosophila melanogaster by genetic mapping of resistance to the cyclodiene dieldrin. The 4,000-fold resistance observed was due to a single amino acid replacement, Ala301 to Ser. The equivalent change was subsequently identified in Rdl orthologs of a large range of resistant insect species. Here, we report identification of a duplication at the Rdl locus in D. melanogaster. The 113-kb duplication contains one WT copy of Rdl and a second copy with two point mutations: an Ala301 to Ser resistance mutation and Met360 to Ile replacement. Individuals with this duplication exhibit intermediate dieldrin resistance compared with single copy Ser301 homozygotes, reduced temperature sensitivity, and altered RNA editing associated with the resistant allele. Ectopic recombination between Roo transposable elements is involved in generating this genomic rearrangement. The duplication phenotypes were confirmed by construction of a transgenic, artificial duplication integrating the 55.7-kb Rdl locus with a Ser301 change into an Ala301 background. Gene duplications can contribute significantly to the evolution of insecticide resistance, most commonly by increasing the amount of gene product produced. Here however, duplication of the Rdl target site creates permanent heterozygosity, providing unique potential for adaptive mutations to accrue in one copy, without abolishing the endogenous role of an essential gene. PMID:23959864

Genetic Architectures of Quantitative Variation in RNA Editing Pathways

PubMed Central

Gu, Tongjun; Gatti, Daniel M.; Srivastava, Anuj; Snyder, Elizabeth M.; Raghupathy, Narayanan; Simecek, Petr; Svenson, Karen L.; Dotu, Ivan; Chuang, Jeffrey H.; Keller, Mark P.; Attie, Alan D.; Braun, Robert E.; Churchill, Gary A.

2016-01-01

RNA editing refers to post-transcriptional processes that alter the base sequence of RNA. Recently, hundreds of new RNA editing targets have been reported. However, the mechanisms that determine the specificity and degree of editing are not well understood. We examined quantitative variation of site-specific editing in a genetically diverse multiparent population, Diversity Outbred mice, and mapped polymorphic loci that alter editing ratios globally for C-to-U editing and at specific sites for A-to-I editing. An allelic series in the C-to-U editing enzyme Apobec1 influences the editing efficiency of Apob and 58 additional C-to-U editing targets. We identified 49 A-to-I editing sites with polymorphisms in the edited transcript that alter editing efficiency. In contrast to the shared genetic control of C-to-U editing, most of the variable A-to-I editing sites were determined by local nucleotide polymorphisms in proximity to the editing site in the RNA secondary structure. Our results indicate that RNA editing is a quantitative trait subject to genetic variation and that evolutionary constraints have given rise to distinct genetic architectures in the two canonical types of RNA editing. PMID:26614740

Effect of P450 Oxidoreductase Polymorphisms on the Metabolic Activities of Ten Cytochrome P450s Varied by Polymorphic CYP Genotypes in Human Liver Microsomes.

PubMed

Fang, Yan; Gao, Na; Tian, Xin; Zhou, Jun; Zhang, Hai-Feng; Gao, Jie; He, Xiao-Pei; Wen, Qiang; Jia, Lin-Jing; Jin, Han; Qiao, Hai-Ling

2018-06-27

Background/ Aims: Little is known about the effect of P450 oxidoreductase (POR) gene polymorphisms on the activities of CYPs with multiple genotypes. We genotyped 102 human livers for 18 known POR single nucleotide polymorphisms (SNPs) with allelic frequencies greater than 1% as well as for 27 known SNPs in 10 CYPs. CYP enzyme activities in microsomes prepared from these livers were determined by measuring probe substrate metabolism by high performance liquid chromatograph. We found that the effects of the 18 POR SNPs on 10 CYP activities were CYP genotype-dependent. The POR mutations were significantly associated with decreased overall Km for CYP2B6 and 2E1, and specific genotypes within CYP1A2, 2A6, 2B6, 2C8, 2D6 and 2E1 were identified as being affected by these POR SNPs. Notably, the effect of a specific POR mutation on the activity of a CYP genotype could not be predicted from other CYP genotypes of even the same CYP. When combining one POR SNP with other POR SNPs, a hitherto unrecognized effect of multiple-site POR gene polymorphisms (MSGP) on CYP activity was uncovered, which was not necessarily consistent with the effect of either single POR SNP. The effects of POR SNPs on CYP activities were not only CYP-dependent, but more importantly, CYP genotype-dependent. Moreover, the effect of a POR SNP alone and in combination with other POR SNPs (MSGP) was not always consistent, nor predictable. Understanding the impact of POR gene polymorphisms on drug metabolism necessitates knowing the complete SNP complement of POR and the genotype of the relevant CYPs. © 2018 The Author(s). Published by S. Karger AG, Basel.

Genetic polymorphisms in the microRNA binding-sites of the thymidylate synthase gene predict risk and survival in gastric cancer.

PubMed

Shen, Rong; Liu, Hongliang; Wen, Juyi; Liu, Zhensheng; Wang, Li-E; Wang, Qiming; Tan, Dongfeng; Ajani, Jaffer A; Wei, Qingyi

2015-09-01

Thymidylate synthase (TYMS) plays a crucial role in folate metabolism as well as DNA synthesis and repair. We hypothesized that functional polymorphisms in the 3' UTR of TYMS are associated with gastric cancer risk and survival. In the present study, we tested our hypothesis by genotyping three potentially functional (at miRNA binding sites) TYMS SNPs (rs16430 6bp del/ins, rs2790 A>G and rs1059394 C>T) in 379 gastric cancer patients and 431 cancer-free controls. Compared with the rs16430 6bp/6bp + 6bp/0bp genotypes, the 0bp/0bp genotype was associated with significantly increased gastric cancer risk (adjusted OR = 1.72, 95% CI = 1.15-2.58). Similarly, rs2790 GG and rs1059394 TT genotypes were also associated with significantly increased risk (adjusted OR = 2.52, 95% CI = 1.25-5.10 and adjusted OR = 1.57, 95% CI = 1.04-2.35, respectively), compared with AA + AG and CC + CT genotypes, respectively. In the haplotype analysis, the T-G-0bp haplotype was associated with significantly increased gastric cancer risk, compared with the C-A-6bp haplotype (adjusted OR = 1.34, 95% CI = 1.05-1.72). Survival analysis revealed that rs16430 0bp/0bp and rs1059394 TT genotypes were also associated with poor survival in gastric cancer patients who received chemotherapy treatment (adjusted HR = 1.61, 95% CI = 1.05-2.48 and adjusted HR = 1.59, 95% CI = 1.02-2.48, respectively). These results suggest that these three variants in the miRNA binding sites of TYMS may be associated with cancer risk and survival of gastric cancer patients. Larger population studies are warranted to verify these findings. © 2014 Wiley Periodicals, Inc.

The Chromodomain of Tf1 Integrase Promotes Binding to cDNA and Mediates Target Site Selection▿ †

PubMed Central

Chatterjee, Atreyi Ghatak; Leem, Young Eun; Kelly, Felice D.; Levin, Henry L.

2009-01-01

The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDΔ, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDΔ caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting. PMID:19109383

Putative Prostate Cancer Risk SNP in an Androgen Receptor‐Binding Site of the Melanophilin Gene Illustrates Enrichment of Risk SNPs in Androgen Receptor Target Sites

PubMed Central

Bu, Huajie; Narisu, Narisu; Schlick, Bettina; Rainer, Johannes; Manke, Thomas; Schäfer, Georg; Pasqualini, Lorenza; Chines, Peter; Schweiger, Michal R.; Fuchsberger, Christian

2015-01-01

ABSTRACT Genome‐wide association studies have identified genomic loci, whose single‐nucleotide polymorphisms (SNPs) predispose to prostate cancer (PCa). However, the mechanisms of most of these variants are largely unknown. We integrated chromatin‐immunoprecipitation‐coupled sequencing and microarray expression profiling in TMPRSS2‐ERG gene rearrangement positive DUCaP cells with the GWAS PCa risk SNPs catalog to identify disease susceptibility SNPs localized within functional androgen receptor‐binding sites (ARBSs). Among the 48 GWAS index risk SNPs and 3,917 linked SNPs, 80 were found located in ARBSs. Of these, rs11891426:T>G in an intron of the melanophilin gene (MLPH) was within a novel putative auxiliary AR‐binding motif, which is enriched in the neighborhood of canonical androgen‐responsive elements. T→G exchange attenuated the transcriptional activity of the ARBS in an AR reporter gene assay. The expression of MLPH in primary prostate tumors was significantly lower in those with the G compared with the T allele and correlated significantly with AR protein. Higher melanophilin level in prostate tissue of patients with a favorable PCa risk profile points out a tumor‐suppressive effect. These results unravel a hidden link between AR and a functional putative PCa risk SNP, whose allele alteration affects androgen regulation of its host gene MLPH. PMID:26411452

Chemical structure determines target organ carcinogenesis in rats

PubMed Central

Carrasquer, C. A.; Malik, N.; States, G.; Qamar, S.; Cunningham, S.L.; Cunningham, A.R.

2012-01-01

SAR models were developed for 12 rat tumour sites using data derived from the Carcinogenic Potency Database. Essentially, the models fall into two categories: Target Site Carcinogen – Non-Carcinogen (TSC-NC) and Target Site Carcinogen – Non-Target Site Carcinogen (TSC-NTSC). The TSC-NC models were composed of active chemicals that were carcinogenic to a specific target site and inactive ones that were whole animal non-carcinogens. On the other hand, the TSC-NTSC models used an inactive category also composed of carcinogens but to any/all other sites but the target site. Leave one out validations produced an overall average concordance value for all 12 models of 0.77 for the TSC-NC models and 0.73 for the TSC-NTSC models. Overall, these findings suggest that while the TSC-NC models are able to distinguish between carcinogens and non-carcinogens, the TSC-NTSC models are identifying structural attributes that associate carcinogens to specific tumour sites. Since the TSC-NTSC models are composed of active and inactive compounds that are genotoxic and non-genotoxic carcinogens, the TSC-NTSC models may be capable of deciphering non-genotoxic mechanisms of carcinogenesis. Together, models of this type may also prove useful in anticancer drug development since they essentially contain chemicals moieties that target specific tumour site. PMID:23066888

Mutations and polymorphisms in FSH receptor: functional implications in human reproduction.

PubMed

Desai, Swapna S; Roy, Binita Sur; Mahale, Smita D

2013-12-01

FSH brings about its physiological actions by activating a specific receptor located on target cells. Normal functioning of the FSH receptor (FSHR) is crucial for follicular development and estradiol production in females and for the regulation of Sertoli cell function and spermatogenesis in males. In the last two decades, the number of inactivating and activating mutations, single nucleotide polymorphisms, and spliced variants of FSHR gene has been identified in selected infertile cases. Information on genotype-phenotype correlation and in vitro functional characterization of the mutants has helped in understanding the possible genetic cause for female infertility in affected individuals. The information is also being used to dissect various extracellular and intracellular events involved in hormone-receptor interaction by studying the differences in the properties of the mutant receptor when compared with WT receptor. Studies on polymorphisms in the FSHR gene have shown variability in clinical outcome among women treated with FSH. These observations are being explored to develop molecular markers to predict the optimum dose of FSH required for controlled ovarian hyperstimulation. Pharmacogenetics is an emerging field in this area that aims at designing individual treatment protocols for reproductive abnormalities based on FSHR gene polymorphisms. The present review discusses the current knowledge of various genetic alterations in FSHR and their impact on receptor function in the female reproductive system.

Is colour polymorphism advantageous to populations and species?

PubMed

Forsman, Anders

2016-06-01

I am writing in response to an article by Bolton, Rollins and Griffith (2015) entitled 'The danger within: the role of genetic, behavioural and ecological factors in population persistence of colour polymorphic species' that was recently published as an Opinion under the NEWS AND VIEWS section in Molecular Ecology. Bolton et al. (Molecular Ecology, 2015, 24, 2907) argue that colour polymorphism may reduce population fitness and increase extinction risk and emphasize that this is contrary to predictions put forward by Forsman et al. (Ecology, 89, 2008, 34) and Wennersten & Forsman (Biological Reviews 87, 2012, 756) that the existence of multiple colour morphs with co-adapted gene complexes and associated trait values may increase the ecological and evolutionary success of polymorphic populations and species. Bolton et al. (Molecular Ecology, 2015, 24, 2907) further state that there is no clear evidence from studies of 'true polymorphic species' that polymorphism promotes population persistence. In response, I (i) challenge their classifications of polymorphisms and revisit the traditional definitions recognizing the dynamic nature of polymorphisms, (ii) review empirical studies that have examined whether and how polymorphism is associated with extinction risk, (iii) discuss the roles of trait correlations between colour pattern and other phenotypic dimensions for population fitness and (iv) highlight that the causes and mechanisms that influence the composition and maintenance of polymorphisms are different from the consequences of the polymorphic condition and how it may impact on aspects of ecological success and long-term persistence of populations and species. © 2016 John Wiley & Sons Ltd.

Genetic polymorphisms in lung disease: bandwagon or breakthrough?

PubMed Central

Iannuzzi, Michael C; Maliarik, Mary; Rybicki, Benjamin

2002-01-01

The study of genetic polymorphisms has touched every aspect of pulmonary and critical care medicine. We review recent progress made using genetic polymorphisms to define pathophysiology, to identify persons at risk for pulmonary disease and to predict treatment response. Several pitfalls are commonly encountered in studying genetic polymorphisms, and this article points out criteria that should be applied to design high-quality genetic polymorphism studies. PMID:11980584

Detection of canonical A-to-G editing events at 3′ UTRs and microRNA target sites in human lungs using next-generation sequencing

PubMed Central

Soundararajan, Ramani; Stearns, Timothy M.; Griswold, Anthony J.; Mehta, Arpit; Czachor, Alexander; Fukumoto, Jutaro; Lockey, Richard F.; King, Benjamin L.; Kolliputi, Narasaiah

2015-01-01

RNA editing is a post-transcriptional modification of RNA. The majority of these changes result from adenosine deaminase acting on RNA (ADARs) catalyzing the conversion of adenosine residues to inosine in double-stranded RNAs (dsRNAs). Massively parallel sequencing has enabled the identification of RNA editing sites in human transcriptomes. In this study, we sequenced DNA and RNA from human lungs and identified RNA editing sites with high confidence via a computational pipeline utilizing stringent analysis thresholds. We identified a total of 3,447 editing sites that overlapped in three human lung samples, and with 50% of these sites having canonical A-to-G base changes. Approximately 27% of the edited sites overlapped with Alu repeats, and showed A-to-G clustering (>3 clusters in 100 bp). The majority of edited sites mapped to either 3′ untranslated regions (UTRs) or introns close to splice sites; whereas, only few sites were in exons resulting in non-synonymous amino acid changes. Interestingly, we identified 652 A-to-G editing events in the 3′ UTR of 205 target genes that mapped to 932 potential miRNA target binding sites. Several of these miRNA edited sites were validated in silico. Additionally, we validated several A-to-G edited sites by Sanger sequencing. Altogether, our study suggests a role for RNA editing in miRNA-mediated gene regulation and splicing in human lungs. In this study, we have generated a RNA editome of human lung tissue that can be compared with other RNA editomes across different lung tissues to delineate a role for RNA editing in normal and diseased states. PMID:26486088

Mutation analysis of BRCA1/2 mutations with special reference to polymorphic SNPs in Indian breast cancer patients.

PubMed

Shah, Nidhi D; Shah, Parth S; Panchal, Yash Y; Katudia, Kalpesh H; Khatri, Nikunj B; Ray, Hari Shankar P; Bhatiya, Upti R; Shah, Sandip C; Shah, Bhavini S; Rao, Mandava V

2018-01-01

Germline mutations BRCA1 and BRCA2 contribute almost equally in the causation of breast cancer (BC). The type of mutations in the Indian population that cause this condition is largely unknown. In this cohort, 79 randomized BC patients were screened for various types of BRCA1 and BRCA2 mutations including frameshift, nonsense, missense, in-frame and splice site types. The purified extracted DNA of each referral patient was subjected to Sanger gene sequencing using Codon Code Analyzer and Mutation Surveyor and next-generation sequencing (NGS) methods with Ion torrent software, after appropriate care. The data revealed that 35 cases were positive for BRCA1 or BRCA2 (35/79: 44.3%). BRCA2 mutations were higher (52.4%) than BRCA1 mutations (47.6%). Five novel mutations detected in this study were p.pro163 frameshift, p.asn997 frameshift, p.ser148 frameshift and two splice site single-nucleotide polymorphisms (SNPs). Additionally, four nonsense and one in-frame deletion were identified, which all seemed to be pathogenic. Polymorphic SNPs contributed the highest percentage of mutations (72/82: 87.8%) and contributed to pathogenic, likely pathogenic, likely benign, benign and variant of unknown significance (VUS). Young age groups (20-60 years) had a high frequency of germline mutations (62/82;75.6%) in the Indian population. This study suggested that polymorphic SNPs contributed a high percentage of mutations along with five novel types. Younger age groups are prone to having BC with a higher mutational rate. Furthermore, the SNPs detected in exons 10, 11 and 16 of BRCA1 and BRCA2 were higher than those in other exons 2, 3 and 9 polymorphic sites in two germline genes. These may be contributory for BC although missense types are known to be susceptible for cancer depending on the type of amino acid replaced in the protein and associated with pathologic events. Accordingly, appropriate counseling and treatment may be suggested.

The relationship between the target effective site concentration of rocuronium and the degree of recovery from neuromuscular blockade in elderly patients

PubMed Central

Fan, Xiaochong; Ma, Minyu; Li, Zhisong; Gong, Shengkai; Zhang, Wei; Wen, Yuanyuan

2015-01-01

Objective: To study the relationship between the target effective site concentration (Ce) of rocuronium and the degree of recovery from neuromuscular blockade in elderly patients. Methods: 50 elderly patients (ASA grade II) scheduled for selective surgical procedure under general anaesthesia were randomly divided into two groups, A and B, with 25 cases in each group. The Ce of rocuronium for intubation was 3 μg·ml-1 in both groups, and the Ce during operation were 0.8 and 1.0 μg·ml-1 in group A and B, respectively. When target controlled infusion of rocuronium was stopped, without the administration of reversal agents for neuromuscular blockade, the relationship between Ce and the first twitch height (T1) was studied by regression analysis. Results: There was a significant linear relationship between Ce and T1, and there was no statistical difference in regression coefficient and interception between group A and B (P>0.05). Conclusion: The degree of recovery from neuromuscular blockade could be judged by the target effective site concentration of rocuronium at the time of reversal from neuromuscular blockade in the elderly patients. PMID:26629159

Target-site mutations conferring resistance to glyphosate in feathertop Rhodes grass (Chloris virgata) populations in Australia.

PubMed

Ngo, The D; Krishnan, Mahima; Boutsalis, Peter; Gill, Gurjeet; Preston, Christopher

2018-05-01

Chloris virgata is a warm-season, C 4 , annual grass weed affecting field crops in northern Australia that has become an emerging weed in southern Australia. Four populations with suspected resistance to glyphosate were collected in South Australia, Queensland and New South Wales, Australia, and compared with one susceptible (S) population to confirm glyphosate resistance and elucidate possible mechanisms of resistance. Based on the rate of glyphosate required to kill 50% of treated plants (LD 50 ), glyphosate resistance (GR) was confirmed in four populations of C. virgata (V12, V14.2, V14.16 and V15). GR plants were 2-9.7-fold more resistant and accumulated less shikimate after glyphosate treatment than S plants. GR and S plants did not differ in glyphosate absorption and translocation. Target-site EPSPS mutations corresponding to Pro-106-Leu (V14.2) and Pro-106-Ser (V15, V14.16 and V12) substitutions were found in GR populations. The population with Pro-106-Leu substitution was 2.9-4.9-fold more resistant than the three other populations with Pro-106-Ser substitution. This report confirms glyphosate resistance in C. virgata and shows that target-site EPSPS mutations confer resistance to glyphosate in this species. The evolution of glyphosate resistance in C. virgata highlights the need to identify alternative control tactics. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Targeting hunter distribution based on host resource selection and kill sites to manage disease risk.

PubMed

Dugal, Cherie J; van Beest, Floris M; Vander Wal, Eric; Brook, Ryan K

2013-10-01

Endemic and emerging diseases are rarely uniform in their spatial distribution or prevalence among cohorts of wildlife. Spatial models that quantify risk-driven differences in resource selection and hunter mortality of animals at fine spatial scales can assist disease management by identifying high-risk areas and individuals. We used resource selection functions (RSFs) and selection ratios (SRs) to quantify sex- and age-specific resource selection patterns of collared (n = 67) and hunter-killed (n = 796) nonmigratory elk (Cervus canadensis manitobensis) during the hunting season between 2002 and 2012, in southwestern Manitoba, Canada. Distance to protected area was the most important covariate influencing resource selection and hunter-kill sites of elk (AICw = 1.00). Collared adult males (which are most likely to be infected with bovine tuberculosis (Mycobacterium bovis) and chronic wasting disease) rarely selected for sites outside of parks during the hunting season in contrast to adult females and juvenile males. The RSFs showed selection by adult females and juvenile males to be negatively associated with landscape-level forest cover, high road density, and water cover, whereas hunter-kill sites of these cohorts were positively associated with landscape-level forest cover and increasing distance to streams and negatively associated with high road density. Local-level forest was positively associated with collared animal locations and hunter-kill sites; however, selection was stronger for collared juvenile males and hunter-killed adult females. In instances where disease infects a metapopulation and eradication is infeasible, a principle goal of management is to limit the spread of disease among infected animals. We map high-risk areas that are regularly used by potentially infectious hosts but currently underrepresented in the distribution of kill sites. We present a novel application of widely available data to target hunter distribution based on host resource

A 3'UTR polymorphism marks differential KLRG1 mRNA levels through disruption of a miR-584-5p binding site and associates with pemphigus foliaceus susceptibility.

PubMed

Cipolla, Gabriel A; Park, Jong Kook; de Oliveira, Liana A; Lobo-Alves, Sara Cristina; de Almeida, Rodrigo C; Farias, Ticiana D J; Lemos, Débora de S; Malheiros, Danielle; Lavker, Robert M; Petzl-Erler, Maria Luiza

2016-10-01

Genetic variations mapping to 3' untranslated regions (3'UTRs) may overlap with microRNA (miRNA) binding sites, therefore potentially interfering with translation inhibition or messenger RNA (mRNA) degradation. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) located within the 3'UTRs of six candidate genes and predicted to interfere with miRNA ligation could account for disease-relevant differential mRNA levels. Focusing on pemphigus foliaceus (PF) - an autoimmune blistering skin condition with unique endemic patterns - we investigated whether nine 3'UTR SNPs from the CD1D, CTLA4, KLRD1, KLRG1, NKG7, and TNFSF13B genes differentially expressed in PF were disease-associated. The heterozygous genotype of the KLRG1 rs1805672 polymorphism was associated with increased predisposition to PF (A/G vs. A/A: P=0.038; OR=1.60), and a trend for augmented susceptibility was observed for carriers of the G allele (P=0.094; OR=1.44). In silico analyses suggested that rs1805672 G allele could disrupt binding of miR-584-5p, and indicated rs1805672 as an expression Quantitative Trait Locus (eQTL), with an effect on KLRG1 gene expression. Dual-luciferase assay showed that miR-584-5p mediated approximately 50% downregulation of the reporter gene's activity through the 3'UTR of KLRG1 harboring rs1805672 A allele (vs. miRNA-negative condition, P=0.006). This silencing relationship was lost after site-directed mutation to G allele (vs. miRNA-negative condition, P=0.391; vs. rs1805672 A allele, P=0.005). Collectively, these results suggest that a disease-associated SNP located within the 3'UTR of KLRG1 directly interferes with miR-584-5p binding, allowing for KLRG1 mRNA differential accumulation, which in turn may contribute to pathogenesis of autoimmune diseases, such as pemphigus. Copyright © 2016 Elsevier B.V. All rights reserved.

A Malaria Vaccine Based on the Polymorphic Block 2 Region of MSP-1 that Elicits a Broad Serotype-Spanning Immune Response

PubMed Central

Cowan, Graeme J. M.; Creasey, Alison M.; Dhanasarnsombut, Kelwalin; Thomas, Alan W.; Remarque, Edmond J.; Cavanagh, David R.

2011-01-01

Polymorphic parasite antigens are known targets of protective immunity to malaria, but this antigenic variation poses challenges to vaccine development. A synthetic MSP-1 Block 2 construct, based on all polymorphic variants found in natural Plasmodium falciparum isolates has been designed, combined with the relatively conserved Block 1 sequence of MSP-1 and expressed in E.coli. The MSP-1 Hybrid antigen has been produced with high yield by fed-batch fermentation and purified without the aid of affinity tags resulting in a pure and extremely thermostable antigen preparation. MSP-1 hybrid is immunogenic in experimental animals using adjuvants suitable for human use, eliciting antibodies against epitopes from all three Block 2 serotypes. Human serum antibodies from Africans naturally exposed to malaria reacted to the MSP-1 hybrid as strongly as, or better than the same serum reactivities to individual MSP-1 Block 2 antigens, and these antibody responses showed clear associations with reduced incidence of malaria episodes. The MSP-1 hybrid is designed to induce a protective antibody response to the highly polymorphic Block 2 region of MSP-1, enhancing the repertoire of MSP-1 Block 2 antibody responses found among immune and semi-immune individuals in malaria endemic areas. The target population for such a vaccine is young children and vulnerable adults, to accelerate the acquisition of a full range of malaria protective antibodies against this polymorphic parasite antigen. PMID:22073118

IL-1 polymorphism and periimplantitis. A literature review.

PubMed

Bormann, Kai-Hendrik; Stühmer, Constantin; Z'Graggen, Marcel; Kokemöller, Horst; Rücker, Martin; Gellrich, Nils-Claudius

2010-01-01

The most important factor leading to periimplantitis with bone loss appears to be an inflammatory process due to plaque accumulation. The object of this article was to present a review of the literature on a possible correlation between IL-1 polymorphism and periimplantitis. Research was carried out in the PUBMED and WEB OF KNOWLEDGE literature databases and 27 relevant articles were found. Of these articles, 4 groups of authors came to the conclusion that no correlation exists between IL-1 polymorphism and periimplantitis. In 5 articles by 4 groups of authors, the influence of IL-1 polymorphism on periimplantitis is unclear. 9 studies prove a correlation between IL-1 polymorphism and periimplantitis, and 6 studies also document a direct linkage between gene polymorphism and periimplantitis, if certain cofactors are present. IL-1 polymorphism is frequently connected with "noninfectious periimplant bone loss". Other studies prove that the inflammatory mediators and IL-1beta were significantly elevated in the gingival crevicular fluid (GCF) of infected implants. Many studies document that IL-1 polymorphism alone cannot be considered a risk factor for bone loss, but in combination with smoking, it is closely associated with periimplant bone loss. More studies are needed to discover possible correlations between IL-1 polymorphism and periimplantitis.

Rhodium(II) Proximity-Labeling Identifies a Novel Target Site on STAT3 for Inhibitors with Potent Anti-Leukemia Activity.

PubMed

Minus, Matthew B; Liu, Wei; Vohidov, Farrukh; Kasembeli, Moses M; Long, Xin; Krueger, Michael J; Stevens, Alexandra; Kolosov, Mikhail I; Tweardy, David J; Sison, Edward Allan R; Redell, Michele S; Ball, Zachary T

2015-10-26

Nearly 40 % of children with acute myeloid leukemia (AML) suffer relapse arising from chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). Herein, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Conformational flexibility and packing plausibility of repaglinide polymorphs

NASA Astrophysics Data System (ADS)

Rani, Dimpy; Goyal, Parnika; Chadha, Renu

2018-04-01

The present manuscript highlights the structural insight into the repaglinide polymorphs. The experimental screening for the possible crystal forms were carried out using various solvents, which generated three forms. The crystal structure of Form II and III was determined using PXRD pattern whereas structural analysis of Form I has already been reported. Form I, II and II was found to exist in P212121, PNA21 and P21/c space groups respectively. Conformational analysis was performed to account the conformational flexibility of RPG. The obtained conformers were further utilized to obtain the information about the crystal packing pattern of RPG polymorphs by polymorph prediction module. The lattice energy landscape, depicting the relationship between lattice energy and density of the polymorphs has been obtained for various possible polymorphs. The experimentally isolated polymorphs were successfully fitted into lattice energy landscape.

Confirming therapeutic target of protopine using immobilized β2 -adrenoceptor coupled with site-directed molecular docking and the target-drug interaction by frontal analysis and injection amount-dependent method.

PubMed

Liu, Guangxin; Wang, Pei; Li, Chan; Wang, Jing; Sun, Zhenyu; Zhao, Xinfeng; Zheng, Xiaohui

2017-07-01

Drug-protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β 2 -adrenoceptor (β 2 -AR) by linkage of the receptor on macroporous silica gel surface through N,N'-carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site-directed molecular docking. Subsequent application of immobilized β 2 -AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount-dependent method. The association constants of protopine to β 2 -AR by the 2 methods were (1.00 ± 0.06) × 10 5 M -1 and (1.52 ± 0.14) × 10 4 M -1 . The numbers of binding sites were (1.23 ± 0.07) × 10 -7 M and (9.09 ± 0.06) × 10 -7 M, respectively. These results indicated that β 2 -AR is the specific target for therapeutic action of protopine in vivo. The target-drug binding occurred on Ser 169 in crystal structure of the receptor. Compared with frontal analysis, injection amount-dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high-throughput drug-receptor interaction analysis. Copyright © 2017 John Wiley & Sons, Ltd.

TGF{beta}1 polymorphisms and late clinical radiosensitivity in patients treated for gynecologic tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruyck, Kim de; Van Eijkeren, Marc; Claes, Kathleen

2006-07-15

Purpose: To investigate the association between six transforming growth factor {beta}1 gene (TGF{beta}1) polymorphisms (-1.552delAGG, -800G>A, -509C>T, Leu10Pro, Arg25Pro, Thr263Ile) and the occurrence of late normal tissue reactions after gynecologic radiotherapy (RT). Methods and Materials: Seventy-eight women with cervical or endometrial cancer and 140 control individuals were included in the study. According to the Common Terminology Criteria for Adverse Events version 3.0 (CTCAEv3.0) scale, 25 patients showed late adverse RT reactions (CTC2+), of whom 11 had severe complications (CTC3+). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), single base extension and genotyping assays were performed to examine the polymorphic sites inmore » TGF{beta}1. Results: Homozygous variant -1.552delAGG, -509TT, and 10Pro genotypes were associated with the risk of developing late severe RT reactions. Triple (variant) homozygous patients had a 3.6 times increased risk to develop severe RT reactions (p = 0.26). Neither the -800A allele, nor the 25Pro allele or the 263Ile allele were associated with clinical radiosensitivity. There was perfect linkage disequilibrium (LD) between the -1.552delAGG and the -509C>T polymorphisms, and tight LD between the -1.552/-509 and the Leu10Pro polymorphisms. Haplotype analysis revealed two major haplotypes but could not distinguish radiosensitive from nonradiosensitive patients. Conclusions: The present study shows that homozygous variant TGF{beta}1 -1.552delAGG, -509TT, and 10Pro genotypes may be associated with severe clinical radiosensitivity after gynecologic RT.« less

Novel GABA receptor pesticide targets.

PubMed

Casida, John E; Durkin, Kathleen A

2015-06-01

The γ-aminobutyric acid (GABA) receptor has four distinct but overlapping and coupled targets of pesticide action importantly associated with little or no cross-resistance. The target sites are differentiated by binding assays with specific radioligands, resistant strains, site-directed mutagenesis and molecular modeling. Three of the targets are for non-competitive antagonists (NCAs) or channel blockers of widely varied chemotypes. The target of the first generation (20th century) NCAs differs between the larger or elongated compounds (NCA-IA) including many important insecticides of the past (cyclodienes and polychlorocycloalkanes) or present (fiproles) and the smaller or compact compounds (NCA-IB) highly toxic to mammals and known as cage convulsants, rodenticides or chemical threat agents. The target of greatest current interest is designated NCA-II for the second generation (21st century) of NCAs consisting for now of isoxazolines and meta-diamides. This new and uniquely different NCA-II site apparently differs enough between insects and mammals to confer selective toxicity. The fourth target is the avermectin site (AVE) for allosteric modulators of the chloride channel. NCA pesticides vary in molecular surface area and solvent accessible volume relative to avermectin with NCA-IBs at 20-22%, NCA-IAs at 40-45% and NCA-IIs at 57-60%. The same type of relationship relative to ligand-docked length is 27-43% for NCA-IBs, 63-71% for NCA-IAs and 85-105% for NCA-IIs. The four targets are compared by molecular modeling for the Drosophila melanogaster GABA-R. The principal sites of interaction are proposed to be: pore V1' and A2' for NCA-IB compounds; pore A2', L6' and T9' for NCA-IA compounds; pore T9' to S15' in proximity to M1/M3 subunit interface (or alternatively an interstitial site) for NCA-II compounds; and M1/M3, M2 interfaces for AVE. Understanding the relationships of these four binding sites is important in resistance management and in the discovery and use

Target capture enrichment of nuclear SNP markers for massively parallel sequencing of degraded and mixed samples.

PubMed

Bose, Nikhil; Carlberg, Katie; Sensabaugh, George; Erlich, Henry; Calloway, Cassandra

2018-05-01

DNA from biological forensic samples can be highly fragmented and present in limited quantity. When DNA is highly fragmented, conventional PCR based Short Tandem Repeat (STR) analysis may fail as primer binding sites may not be present on a single template molecule. Single Nucleotide Polymorphisms (SNPs) can serve as an alternative type of genetic marker for analysis of degraded samples because the targeted variation is a single base. However, conventional PCR based SNP analysis methods still require intact primer binding sites for target amplification. Recently, probe capture methods for targeted enrichment have shown success in recovering degraded DNA as well as DNA from ancient bone samples using next-generation sequencing (NGS) technologies. The goal of this study was to design and test a probe capture assay targeting forensically relevant nuclear SNP markers for clonal and massively parallel sequencing (MPS) of degraded and limited DNA samples as well as mixtures. A set of 411 polymorphic markers totaling 451 nuclear SNPs (375 SNPs and 36 microhaplotype markers) was selected for the custom probe capture panel. The SNP markers were selected for a broad range of forensic applications including human individual identification, kinship, and lineage analysis as well as for mixture analysis. Performance of the custom SNP probe capture NGS assay was characterized by analyzing read depth and heterozygote allele balance across 15 samples at 25 ng input DNA. Performance thresholds were established based on read depth ≥500X and heterozygote allele balance within ±10% deviation from 50:50, which was observed for 426 out of 451 SNPs. These 426 SNPs were analyzed in size selected samples (at ≤75 bp, ≤100 bp, ≤150 bp, ≤200 bp, and ≤250 bp) as well as mock degraded samples fragmented to an average of 150 bp. Samples selected for ≤75 bp exhibited 99-100% reportable SNPs across varied DNA amounts and as low as 0.5 ng. Mock degraded samples at 1�

Infectious polymorphic toxins delivered by outer membrane exchange discriminate kin in myxobacteria.

PubMed

Vassallo, Christopher N; Cao, Pengbo; Conklin, Austin; Finkelstein, Hayley; Hayes, Christopher S; Wall, Daniel

2017-08-18

Myxobacteria are known for complex social behaviors including outer membrane exchange (OME), in which cells exchange large amounts of outer membrane lipids and proteins upon contact. The TraA cell surface receptor selects OME partners based on a variable domain. However, traA polymorphism alone is not sufficient to precisely discriminate kin. Here, we report a novel family of OME-delivered toxins that promote kin discrimination of OME partners. These SitA lipoprotein toxins are polymorphic and widespread in myxobacteria. Each sitA is associated with a cognate sitI immunity gene, and in some cases a sitB accessory gene. Remarkably, we show that SitA is transferred serially between target cells, allowing the toxins to move cell-to-cell like an infectious agent. Consequently, SitA toxins define strong identity barriers between strains and likely contribute to population structure, maintenance of cooperation, and strain diversification. Moreover, these results highlight the diversity of systems evolved to deliver toxins between bacteria.

PhosphoSitePlus, 2014: mutations, PTMs and recalibrations

PubMed Central

Hornbeck, Peter V.; Zhang, Bin; Murray, Beth; Kornhauser, Jon M.; Latham, Vaughan; Skrzypek, Elzbieta

2015-01-01

PhosphoSitePlus® (PSP, http://www.phosphosite.org/), a knowledgebase dedicated to mammalian post-translational modifications (PTMs), contains over 330 000 non-redundant PTMs, including phospho, acetyl, ubiquityl and methyl groups. Over 95% of the sites are from mass spectrometry (MS) experiments. In order to improve data reliability, early MS data have been reanalyzed, applying a common standard of analysis across over 1 000 000 spectra. Site assignments with P > 0.05 were filtered out. Two new downloads are available from PSP. The ‘Regulatory sites’ dataset includes curated information about modification sites that regulate downstream cellular processes, molecular functions and protein-protein interactions. The ‘PTMVar’ dataset, an intersect of missense mutations and PTMs from PSP, identifies over 25 000 PTMVars (PTMs Impacted by Variants) that can rewire signaling pathways. The PTMVar data include missense mutations from UniPROTKB, TCGA and other sources that cause over 2000 diseases or syndromes (MIM) and polymorphisms, or are associated with hundreds of cancers. PTMVars include 18 548 phosphorlyation sites, 3412 ubiquitylation sites, 2316 acetylation sites, 685 methylation sites and 245 succinylation sites. PMID:25514926

Prediction of TF target sites based on atomistic models of protein-DNA complexes

PubMed Central

Angarica, Vladimir Espinosa; Pérez, Abel González; Vasconcelos, Ana T; Collado-Vides, Julio; Contreras-Moreira, Bruno

2008-01-01

Background The specific recognition of genomic cis-regulatory elements by transcription factors (TFs) plays an essential role in the regulation of coordinated gene expression. Studying the mechanisms determining binding specificity in protein-DNA interactions is thus an important goal. Most current approaches for modeling TF specific recognition rely on the knowledge of large sets of cognate target sites and consider only the information contained in their primary sequence. Results Here we describe a structure-based methodology for predicting sequence motifs starting from the coordinates of a TF-DNA complex. Our algorithm combines information regarding the direct and indirect readout of DNA into an atomistic statistical model, which is used to estimate the interaction potential. We first measure the ability of our method to correctly estimate the binding specificities of eight prokaryotic and eukaryotic TFs that belong to different structural superfamilies. Secondly, the method is applied to two homology models, finding that sampling of interface side-chain rotamers remarkably improves the results. Thirdly, the algorithm is compared with a reference structural method based on contact counts, obtaining comparable predictions for the experimental complexes and more accurate sequence motifs for the homology models. Conclusion Our results demonstrate that atomic-detail structural information can be feasibly used to predict TF binding sites. The computational method presented here is universal and might be applied to other systems involving protein-DNA recognition. PMID:18922190

Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity.

PubMed

Ritter, Dustin W; Roberts, Jason R; McShane, Michael J

2013-04-10

Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0-400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay. Copyright © 2013 Elsevier Inc. All rights reserved.

Environmental exposure to arsenic, AS3MT polymorphism and prevalence of diabetes in Mexico

PubMed Central

Drobná, Zuzana; Del Razo, Luz M.; García-Vargas, Gonzalo G.; Sánchez-Peña, Luz C.; Barrera-Hernández, Angel; Stýblo, Miroslav; Loomis, Dana

2014-01-01

Exposure to arsenic in drinking water is associated with increased prevalence of diabetes. We previously reported an association of diabetes and urinary concentration of dimethylarsinite (DMAsIII), a toxic product of arsenic methylation by arsenic ( +3 oxidation state) methyltransferase (AS3MT). Here we examine associations between AS3MT polymorphism, arsenic metabolism and diabetes. Fasting blood glucose, oral glucose tolerance and self-reported diagnoses were used to identify diabetic individuals. Inorganic arsenic and its metabolites were measured in urine. Genotyping analysis focused on six polymorphic sites of AS3MT. Individuals with M287T and G4965C polymorphisms had higher levels of urinary DMAsIII and were more frequently diabetic than the respective wild-type carriers, although the excess was not statistically significant. Odds ratios were 11.4 (95% confidence interval (CI) 2.2–58.8) and 8.8 (95% CI 1.6–47.3) for the combined effects of arsenic exposure >75th percentile and 287T and 4965C genotypes, respectively. Carriers of 287T and 4965C may produce more DMAsIII and be more likely to develop diabetes when exposed to arsenic. PMID:23093101

Polymorphisms of the IL-1beta and IL-1beta-inducible genes in ulcerative colitis.

PubMed

Nohara, Hiroaki; Saito, Yuki; Higaki, Singo; Okayama, Naoko; Hamanaka, Yuichiro; Okita, Kiwamu; Hinoda, Yuji

2002-11-01

Ulcerative colitis (UC) is a chronic disorder of undetermined etiology, but a genetic predisposition to UC is well recognized. Among cytokines induced in UC, interleukin 1 (IL-1) appears to have a central role because of its immunological upregulatory and proinflammatory activities. The aim of this study was to assess whether UC is associated with polymorphisms of the IL-1beta gene and three additional genes inducible with IL-1beta in Japanese subjects. A total of 96 patients with UC and 106 ethnically matched controls were genotyped at polymorphic sites in IL-1beta, matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 3 (MMP-3), and inducible nitric oxide synthase (iNOS) genes, using polymerase chain reaction (PCR)-based methods. There was no significant difference in genotype distributions of IL-1beta, MMP-1, MMP-3, and iNOS genes between controls and UC patients in a Japanese population. Also, no significant association of those polymorphisms with various clinical parameters of the patients was found. However, concerning association of age at onset with clinical factors in UC, the frequency of pancolitis was significantly higher in UC patients with age at onset being less than 30 years than in those more than 30 years of age (P = 0.049). No association of the IL-1beta and three IL-1beta-inducible gene polymorphisms with UC was observed in a Japanese population.

The Groucho Co-repressor Is Primarily Recruited to Local Target Sites in Active Chromatin to Attenuate Transcription

PubMed Central

Jennings, Barbara H.

2014-01-01

Gene expression is regulated by the complex interaction between transcriptional activators and repressors, which function in part by recruiting histone-modifying enzymes to control accessibility of DNA to RNA polymerase. The evolutionarily conserved family of Groucho/Transducin-Like Enhancer of split (Gro/TLE) proteins act as co-repressors for numerous transcription factors. Gro/TLE proteins act in several key pathways during development (including Notch and Wnt signaling), and are implicated in the pathogenesis of several human cancers. Gro/TLE proteins form oligomers and it has been proposed that their ability to exert long-range repression on target genes involves oligomerization over broad regions of chromatin. However, analysis of an endogenous gro mutation in Drosophila revealed that oligomerization of Gro is not always obligatory for repression in vivo. We have used chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) to profile Gro recruitment in two Drosophila cell lines. We find that Gro predominantly binds at discrete peaks (<1 kilobase). We also demonstrate that blocking Gro oligomerization does not reduce peak width as would be expected if Gro oligomerization induced spreading along the chromatin from the site of recruitment. Gro recruitment is enriched in “active” chromatin containing developmentally regulated genes. However, Gro binding is associated with local regions containing hypoacetylated histones H3 and H4, which is indicative of chromatin that is not fully open for efficient transcription. We also find that peaks of Gro binding frequently overlap the transcription start sites of expressed genes that exhibit strong RNA polymerase pausing and that depletion of Gro leads to release of polymerase pausing and increased transcription at a bona fide target gene. Our results demonstrate that Gro is recruited to local sites by transcription factors to attenuate rather than silence gene expression by promoting histone deacetylation

New Polymorph Form of Dexamethasone Acetate.

PubMed

Silva, Ronaldo Pedro da; Ambrósio, Mateus Felipe Schuchter; Piovesan, Luciana Almeida; Freitas, Maria Clara Ramalho; Aguiar, Daniel Lima Marques de; Horta, Bruno Araújo Cautiero; Epprecht, Eugenio Kahn; San Gil, Rosane Aguiar da Silva; Visentin, Lorenzo do Canto

2018-02-01

A new monohydrated polymorph of dexamethasone acetate was crystallized and its crystal structure characterized. The different analytical techniques used for describing its structural and vibrational properties were: single crystal and polycrystal X-ray diffraction, solid state nuclear magnetic resonance, infrared spectroscopy. A Hirshfeld surface analysis was carried out through self-arrangement cemented by H-bonds observed in this new polymorph. This new polymorph form appeared because of self-arrangement via classical hydrogen bonds around the water molecule. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Assessment of nevirapine bioavailability from targeted sites in the human gastrointestinal tract.

PubMed

Macha, Sreeraj; Yong, Chan-Loi; MacGregor, Thomas R; Castles, Mark; Quinson, Anne-Marie; Rouyrre, Nicolas; Wilding, Ian

2009-12-01

This study investigated absorption of nevirapine (NVP) from targeted sites of the gastrointestinal tract using remotely activated capsules and gamma scintigraphy. A total of 24 participants were randomized to receive 50 mg NVP orally as a suspension or via remotely activated capsules for release into the ascending colon. The 24 participants were then rerandomized into parallel groups of n = 8 for drug release into the ileum, jejunum, or descending colon. The mean gastric emptying time of capsules ranged from 0.88 to 3.35 hours. The small intestinal and colon transit time ranged from 4.08 to 7.76 hours and 17.6 to 21.2 hours, respectively, and capsule recovery time ranged from 27.6 to 34.4 hours. The relative bioavailability ratio of NVP in the jejunum was 1.06 (90% confidence interval [CI]: 1.00-1.12) compared to suspension. In the ileum, ascending colon, and descending colon, bioavailability decreased to 0.89 (0.80-0.99), 0.82 (0.71-0.95), and 0.58 (0.22-1.53), respectively. The absorption rate decreased by approximately 10-fold from the jejunum (3.83 h(-1)) to the descending colon (0.338 h(-1)), and t(max) increased from 2.42 hours (jejunum) to 16.3 hours (descending colon). Overall, NVP is absorbed from all 4 sites of the gastrointestinal tract, and the rate of absorption decreased from the jejunum to the descending colon. Relative bioavailability of NVP was in the order of jejunum > ileum > ascending colon > descending colon.

Disappearing Polymorphs Revisited

PubMed Central

Bučar, Dejan-Krešimir; Lancaster, Robert W; Bernstein, Joel

2015-01-01

Nearly twenty years ago, Dunitz and Bernstein described a selection of intriguing cases of polymorphs that disappear. The inability to obtain a crystal form that has previously been prepared is indeed a frustrating and potentially serious problem for solid-state scientists. This Review discusses recent occurrences and examples of disappearing polymorphs (as well as the emergence of elusive crystal forms) to demonstrate the enduring relevance of this troublesome, but always captivating, phenomenon in solid-state research. A number of these instances have been central issues in patent litigations. This Review, therefore, also highlights the complex relationship between crystal chemistry and the law. PMID:26031248

The GLUT-1 XbaI gene polymorphism is associated with vascular calcifications in nondiabetic uremic patients.

PubMed

Rufino, Margarita; Hernández, Domingo; Barrios, Ysamar; Salido, Eduardo

2008-01-01

Glucose transporters mediate the facilitative uptake of glucose into cells, with GLUT-1 being the predominant isoform in vascular smooth muscle cell (VSMC). Clones of human cells overexpressing the GLUT-1 transporter showed a high increase in intracellular glucose concentrations, mimicking the diabetic milieu. It is possible that high intracellular glucose together with uremic factors may play an important role in vascular calcification by transforming VSMC into osteoblast-like cells. The XbaI polymorphism in the GLUT-1 gene has been linked to variations in GLUT-1 expression, with consequent changes in intracellular glucose concentration. To assess the association between the GLUT-1 XbaI gene polymorphism and the presence of VC in nondiabetic uremic patients, a total of 105 nondiabetic patients on hemodialysis were studied. VC were evaluated by conventional simple X-ray. Mean values of serum calcium, phosphorous, cholesterol, triglycerides, HbA1c, PTH and insulin were measured. Height, weight, BMI and waist circumference were also determined. The GLUT-1 XbaI polymorphism in the second intron of the gene was ascertained by means of the polymerase chain reaction and restriction fragment length polymorphism analysis on DNA isolated from peripheral blood DNA. In the absence of an XbaI site, a fragment of 305 bp was seen (so-called x allele), whereas fragments of 232 and 73 bp were generated if the XbaI site was present (X allele). Genotype distribution in all patients was similar to the Caucasian population. However, when the patients were grouped according to the presence or absence of VC, there were marked differences in the frequency of the GLUT1 genotypes: the xx GLUT-1 genotype was more prevalent in the group with VC (30.7 vs. 4.5%, p = 0.001). Stepwise logistic regression demonstrated that the xx GLUT-1 genotype was independently associated with the presence of VC after adjusting for other variables such as age, calcium x phosphrus product, BMI and time on

LPA gene: interaction between the apolipoprotein(a) size ('kringle IV' repeat) polymorphism and a pentanucleotide repeat polymorphism influences Lp(a) lipoprotein level.

PubMed

Røsby, O; Berg, K

2000-01-01

In order to search for factors influencing the Lp(a) lipoprotein level, we have examined the apolipoprotein(a) (apo(a)) size polymorphism as well as a pentanucleotide (TTTTA) repeat polymorphism in the 5' control region of the LPA gene. Lp(a) lipoprotein levels were compared between individuals with different genotypes as defined by pulsed field gel electrophoresis of DNA plugs, and PCR of DNA samples followed by polyacrylamide gel electrophoresis. DNA plugs and DNA were prepared from blood samples collected from blood donors. Twenty-seven different K IV repeat alleles were observed in the 71 women and 92 men from which apo(a) size polymorphism results were obtained. Alleles encoding 26-32 Kringle IV repeats were the most frequent. Alleles encoding seven to 11 TTTTA repeats were detected in the 84 women and 122 men included in the pentanucleotide polymorphism study, and homozygosity for eight TTTTA repeats was the most common genotype. The eight TTTTA repeat allele occurred with almost any apo(a) allele. An inverse relationship between number of K IV repeats and Lp(a) concentration was confirmed. The contributions of the apo(a) size polymorphism and the pentanucleotide repeat polymorphism to the interindividual variance of Lp(a) lipoprotein concentrations were 9.7 and 3.5%, respectively (type IV sum of squares). Nineteen per cent of the variance in Lp(a) lipoprotein level appeared to be the result of the multiplication product (interaction) between the apo(a) size polymorphism and the pentanucleotide repeat polymorphism. The contribution of the apo(a) size polymorphism alone to the variation in Lp(a) lipoprotein level was lower than previously reported. However, the multiplicative interaction effect between the K IV repeat polymorphism and the pentanucleotide repeat polymorphism may be an important factor explaining the variation in Lp(a) lipoprotein levels among the populations.

Molecular dynamics simulations and statistical coupling analysis of GPI12 in L. major: functional co-evolution and conservedness reveals potential drug-target sites.

PubMed

Singh, Shailza; Mandlik, Vineetha; Shinde, Sonali

2015-03-01

GPI12 represents an important enzyme in the GPI biosynthetic pathway of several parasites like 'Leishmania'. GPI activity is generally regulated through either the hindrance in GPI complex assembly formation or the modulation of the lipophosphoglycan (LPG) flux to either reduce or enhance the pathogenicity in an organism. Of the various GPI molecules known, GPI12 is an important enzyme in the GPI biosynthetic pathway which can be exploited as a target due to the substrate specificity difference in parasites and humans. In the present study, the functional importance of the co-evolving residues of the GPI12 protein of Leishmania has been highlighted using the GPI proteins belonging to the GlcNAC-deacetylase family. Exploring the active site of the GPI12 protein and designing inhibitors against the functional residues provide ways and means to change the efficiency of deacetylation activity of the enzyme. The activity of de-N-acetylase is low in the absence of metal ions like zinc. Hence we designed eight small molecules in order to modulate the activity of GPI12. Compound 8 was found to be an appropriate choice to target the agonist (GPI12) active site thereby targeting the residues which were essential in the Zn binding and chelation activity. Inhibition of these sites offered a strong constraint to block the protein activity and in turn GPI biosynthesis.

Human papillomavirus type 56 polymorphism in Canadian women with and without cervical lesions.

PubMed

Rodrigues-Coutlée, Catherine; Archambault, Jacques; Money, Deborah; Ramanakumar, Agnihotram V; Raboud, Janet; Hankins, Catherine; Koushik, Anita; Richardson, Harriet; Brassard, Paul; Franco, Eduardo L; Coutlée, Francois

2013-12-01

The genomic diversity of high-risk human papillomaviruses (HPV) has been associated with viral persistence and HPV-induced lesions. Studies on HPV56 persistence are still pending. To assess the association between HPV56 polymorphism and HPV56 persistence and presence of high-grade cervical intraepithelial neoplasia (CIN2,3) or cancer. HPV56-positive cervical specimens from 204 women selected from a total of 4669 participants recruited in 5 epidemiological studies (parent studies) were further analyzed by PCR-sequencing of the long control region (LCR). Of the 81 women followed prospectively in cohort studies who could be classified, 34 had persistent and 47 had transient HPV56 infections. Variant HPV56-LCR-MTL-21 was detected more frequently in persistent infections (52.9%, 95% CI: 36.7-68.6%) than in transient infections (25.5%, 95% CI: 15.1-39.4). Considering only women recruited in a cohort of women infected or at high risk for HIV infection, infection with variant HPV56-LCR-MTL-21 (OR=4.4, 95% CI: 1.3-14.5) was significantly associated with HPV56 persistence controlling in multivariate analysis for high risk HPV detection and HIV infection. A variation at nucleotide 7800 in HPV56-LCR-MTL-21 resulted in the loss of a binding site for Elf-1 embedded in one of the E2 binding sites, a potential activator or repressor of expression of the HPV genome. HPV56 polymorphism was not associated with CIN2,3 or cancer in women enrolled in cross-sectional and case-control studies. Polymorphism in HPV56 may influence the risk that infections with this type will persist. Copyright © 2013 Elsevier B.V. All rights reserved.

A Comparative Genomics Strategy for Targeted Discovery of Single-Nucleotide Polymorphisms and Conserved-Noncoding Sequences in Orphan Crops1[W

PubMed Central

Feltus, F.A.; Singh, H.P.; Lohithaswa, H.C.; Schulze, S.R.; Silva, T.D.; Paterson, A.H.

2006-01-01

Completed genome sequences provide templates for the design of genome analysis tools in orphan species lacking sequence information. To demonstrate this principle, we designed 384 PCR primer pairs to conserved exonic regions flanking introns, using Sorghum/Pennisetum expressed sequence tag alignments to the Oryza genome. Conserved-intron scanning primers (CISPs) amplified single-copy loci at 37% to 80% success rates in taxa that sample much of the approximately 50-million years of Poaceae divergence. While the conserved nature of exons fostered cross-taxon amplification, the lesser evolutionary constraints on introns enhanced single-nucleotide polymorphism detection. For example, in eight rice (Oryza sativa) genotypes, polymorphism averaged 12.1 per kb in introns but only 3.6 per kb in exons. Curiously, among 124 CISPs evaluated across Oryza, Sorghum, Pennisetum, Cynodon, Eragrostis, Zea, Triticum, and Hordeum, 23 (18.5%) seemed to be subject to rigid intron size constraints that were independent of per-nucleotide DNA sequence variation. Furthermore, we identified 487 conserved-noncoding sequence motifs in 129 CISP loci. A large CISP set (6,062 primer pairs, amplifying introns from 1,676 genes) designed using an automated pipeline showed generally higher abundance in recombinogenic than in nonrecombinogenic regions of the rice genome, thus providing relatively even distribution along genetic maps. CISPs are an effective means to explore poorly characterized genomes for both DNA polymorphism and noncoding sequence conservation on a genome-wide or candidate gene basis, and also provide anchor points for comparative genomics across a diverse range of species. PMID:16607031

High-density polymorphisms analysis of 23 candidate genes for association with bone mineral density.

PubMed

Giroux, Sylvie; Elfassihi, Latifa; Clément, Valérie; Bussières, Johanne; Bureau, Alexandre; Cole, David E C; Rousseau, François

2010-11-01

Osteoporosis is a bone disease characterized by low bone mineral density (BMD), a highly heritable and polygenic trait. Women are more prone than men to develop osteoporosis due to a lower peak bone mass and accelerated bone loss at menopause. Peak bone mass has been convincingly shown to be due to genetic factors with heritability up to 80%. Menopausal bone loss has been shown to have around 38% to 49% heritability depending on the site studied. To have more statistical power to detect small genetic effects we focused on premenopausal women. We studied 23 candidate genes, some involved in calcium and vitamin-D regulation and others because estrogens strongly induced their gene expression in mice where it was correlated with humerus trabecular bone density. High-density polymorphisms were selected to cover the entire gene variability and 231 polymorphisms were genotyped in a first sample of 709 premenopausal women. Positive associations were retested in a second, independent, sample of 673 premenopausal women. Ten polymorphisms remained associated with BMD in the combined samples and one was further associated in a large sample of postmenopausal women (1401 women). This associated polymorphism was located in the gene CSF3R (granulocyte colony stimulating factor receptor) that had never been associated with BMD before. The results reported in this study suggest a role for CSF3R in the determination of bone density in women. Copyright © 2010 Elsevier Inc. All rights reserved.

Thermal, spectroscopic, and ab initio structural characterization of carprofen polymorphs.

PubMed

Bruni, Giovanna; Gozzo, Fabia; Capsoni, Doretta; Bini, Marcella; Macchi, Piero; Simoncic, Petra; Berbenni, Vittorio; Milanese, Chiara; Girella, Alessandro; Ferrari, Stefania; Marini, Amedeo

2011-06-01

Commercial and recrystallized polycrystalline samples of carprofen, a nonsteroidal anti-inflammatory drug, were studied by thermal, spectroscopic, and structural techniques. Our investigations demonstrated that recrystallized sample, stable at room temperature (RT), is a single polymorphic form of carprofen (polymorph I) that undergoes an isostructural polymorphic transformation by heating (polymorph II). Polymorph II remains then metastable at ambient conditions. Commercial sample is instead a mixture of polymorphs I and II. The thermodynamic relationships between the two polymorphs were determined through the construction of an energy/temperature diagram. The ab initio structural determination performed on synchrotron X-Ray powder diffraction patterns recorded at RT on both polymorphs allowed us to elucidate, for the first time, their crystal structure. Both crystallize in the monoclinic space group type P2(1) /c, and the unit cell similarity index and the volumetric isostructurality index indicate that the temperature-induced polymorphic transformation I → II is isostructural. Polymorphs I and II are conformational polymorphs, sharing a very similar hydrogen bond network, but with different conformation of the propanoic skeleton, which produces two different packing. The small conformational change agrees with the low value of transition enthalpy obtained by differential scanning calorimetry measurements and the small internal energy computed with density functional methods. Copyright © 2011 Wiley-Liss, Inc.

MT119, a new planar-structured compound, targets the colchicine site of tubulin arresting mitosis and inhibiting tumor cell proliferation.

PubMed

Zhang, Zhixiang; Meng, Tao; Yang, Na; Wang, Wei; Xiong, Bing; Chen, Yi; Ma, Lanping; Shen, Jingkang; Miao, Ze-Hong; Ding, Jian

2011-07-01

Microtubule-targeted drugs are now indispensable for the therapy of various cancer types worldwide. In this article, we report MT119 [6-[2-(4-methoxyphenyl) -ethyl]-9-[(pyridine-3-ylmethyl)amino]pyrido[2',1':2,3]imida-zo[4,5-c]isoquinolin-5(6H)-one] as a new microtubule-targeted agent. MT119 inhibited tubulin polymerization significantly both in tumor cells and in cell-free systems, which was followed by the disruption of mitotic spindle assembly. Surface plasmon resonance-based analyses showed that MT119 bound to purified tubulin directly, with the K(D) value of 10.6 μM. The binding of MT119 in turn caused tubulin conformational changes as evidenced by the quenched tryptophan fluorescence, the reduction of the bis-ANS reactivity and the decreased DTNB-sulfhydryl reaction rate. Competitive binding assays further revealed that MT119 bound to tubulin at its colchicine site. Consequently, by inhibiting tubulin polymerization, MT119 arrested different tumor cells at mitotic phase, which contributed to its potent antitumor activity in vitro. MT119 was also similarly cytotoxic to vincristine-, adriamycin- or mitoxantrone-resistant cancer cells and to their corresponding parental cells. Together, these data indicate that MT119 represents a new class of colchicine-site-targeted inhibitors against tubulin polymerization, which might be a promising starting point for future cancer therapeutics. Copyright © 2010 UICC.

Polymorphisms in the presumptive promoter region of the SLC2A9 gene are associated with gout in a Chinese male population.

PubMed

Li, Changgui; Chu, Nan; Wang, Binbin; Wang, Jing; Luan, Jian; Han, Lin; Meng, Dongmei; Wang, Yunlong; Suo, Peisu; Cheng, Longfei; Ma, Xu; Miao, Zhimin; Liu, Shiguo

2012-01-01

Glucose transporter 9 (GLUT9) is a high-capacity/low-affinity urate transporter. To date, several recent genome-wide association studies (GWAS) and follow-up studies have identified genetic variants of SLC2A9 associated with urate concentrations and susceptibility to gout. We therefore investigated associations between gout and polymorphisms and haplotypes in the presumptive promoter region of GLUT9 in Chinese males. The approximately 2000 bp presumptive promoter region upstream of the start site of exon 1 of GLUT9 was sequenced and subjected to genetic analysis. A genotype-phenotype correlation was performed and polymorphisms-induced changes in transcription factor binding sites were predicted. Of 21 SNPs identified in GLUT9, five had not been previously reported. Two of the SNPs (rs13124007 and rs6850166) were associated with susceptibility to gout (p = 0.009 and p = 0.042, respectively). The C allele of rs13124007 appeared to be the risk allele for predisposition to gout (p = 0.006, OR 1.709 [95% CI 1.162-2.514]). For rs6850166, an increased risk of gout was associated with the A allele (p = 0.029, OR 1.645 [95% CI 1.050-2.577]). After Bonferroni correction, there was statistically difference in rs13124007 allele frequencies between gout cases and controls (P = 0.042). Haplotype analyses showed that haplotype GG was a protective haplotype (p = 0.0053) and haplotype CA was associated with increased risk of gout (p = 0.0326). Genotype-phenotype analysis among gout patients revealed an association of rs13124007 with serum triglycerides levels (P = 0.001). The C to G substitution in polymorphism rs13124007 resulted in a loss of a binding site for transcription factor interferon regulatory factor 1 (IRF-1). Polymorphisms rs13124007 and rs6850166 are associated with susceptibility to gout in Chinese males.

Inheritance of restriction fragment length polymorphisms, random amplified polymorphic DNAs and isozymes in coastal Douglas-fir

Treesearch

K.D. Jermstad; A.M. Reem; J.R. Henifin; N.C. Wheeler; D.B Neale

1994-01-01

A total of 225 new genetic loci [151 restriction fragment length polymorphisms (RFLP) and 74 random amplified polymorphic DNAs (RAPD)] in coastal Douglas- fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] have been identified using a three-generation outbred pedigree. The Mendelian inheritance of 16 RFLP loci and 29...

Targeting smooth emergence: the effect site concentration of remifentanil for preventing cough during emergence during propofol-remifentanil anaesthesia for thyroid surgery.

PubMed

Lee, B; Lee, J-R; Na, S

2009-06-01

The administration of short-acting opioids can be a reliable and safe method to prevent coughing during emergence from anaesthesia but the proper dose or effect site concentration of remifentanil for this purpose has not been reported. We therefore investigated the effect site concentration (Ce) of remifentanil for preventing cough during emergence from anaesthesia with propofol-remifentanil target-controlled infusion. Twenty-three ASA I-II grade female patients, aged 23-66 yr undergoing elective thyroidectomy were enrolled in this study. EC(50) and EC(95) of remifentanil for preventing cough were determined using Dixon's up-and-down method and probit analysis. Propofol effect site concentration at extubation, mean arterial pressure, and heart rate (HR) were compared in patients with smooth emergence and without smooth emergence. Three out of 11 patients with remifentanil Ce of 1.5 ng ml(-1) and all seven patients with Ce of 2.0 ng ml(-1) did not cough during emergence; the EC(50) of remifentanil that suppressed coughing was 1.46 ng ml(-1) by Dixon's up-and-down method, and EC(95) was 2.14 ng ml(-1) by probit analysis. Effect site concentration of propofol at awakening was similar in patients with a smooth emergence and those without smooth emergence, but HR and arterial pressure were higher in those who coughed during emergence. Clinically significant hypoventilation was not seen in any patient. We found that the EC(95) of effect site concentration of remifentanil to suppress coughing at emergence from anaesthesia was 2.14 ng ml(-1). Maintaining an established Ce of remifentanil is a reliable method of abolishing cough and thereby targeting smooth emergence from anaesthesia.

Endometriosis and RAS system gene polymorphisms: the association of ACE A2350G polymorphism with endometriosis in Polish individuals.

PubMed

Kowalczyńska, Liliana J; Ferenc, Tomasz; Wojciechowski, Michał; Mordalska, Anna; Pogoda, Krzysztof; Malinowski, Andrzej

2014-05-01

To analyze the polymorphisms of angiotensin I converting enzyme (ACE) gene (insertion/deletion [I/D], A2350G) and angiotensin II type 1 receptor gene (A1166C) in women with endometriosis and to determine the correlation of the identified genotypes with the severity of the disease. Additionally, to estimate the prognostic value of the polymorphisms in patients with endometriosis treated due to infertility. The study group included 241 women, the control group (without endometriosis)-127. The molecular analysis was performed by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism technique. For I/D ACE and A1166C AT1 polymorphisms no significant differences were observed between the study and control groups and between the severity grades of the disease (p>0.05). For A2350G ACE polymorphism the frequency of genotypes for the study and control groups respectively was the following: AA-31.54%, AG-54.36%, GG-14.11% and AA-55.12%, AG-36.22%, GG-8.66% (x(2)=19.36, p<0.0001). Statistically significant differences were found between the frequency of A and G alleles between both groups (x(2)=15.16, p=0.0001), but not when individual grades of the disease severity were compared. There was no association between the investigated polymorphisms and the effect of infertility treatment. A2350G polymorphism (allele G, AG genotype) of ACE gene seems to be associated with the development of endometriosis.

Association of ACE and MDR1 Gene Polymorphisms with Steroid Resistance in Children with Idiopathic Nephrotic Syndrome.

PubMed

Dhandapani, Mohanapriya Chinambedu; Venkatesan, Vettriselvi; Rengaswamy, Nammalwar Bollam; Gowrishankar, Kalpana; Nageswaran, Prahlad; Perumal, Venkatachalam

2015-08-01

The purpose of the study was to investigate the distribution of insertion/deletion (I/D) polymorphisms of the angiotensin-converting enzyme (ACE) gene and three exonic polymorphisms of the multidrug resistance 1 (MDR1) gene (C3435T, C1236T, and G2677T) in children diagnosed with idiopathic nephrotic syndrome (INS). The study group consisted of 100 healthy controls and 150 INS patients, of which 50 were steroid resistant. Genomic DNA from blood samples was isolated from both of these groups and genotyping of the ACE and MDR1 genes was performed by polymerase chain reaction (PCR) using specific primers. There was no significant difference observed in the genotypic distribution and D allele frequency of the ACE gene. The two single-nucleotide polymorphisms (SNPs), C1236T and C3435T, of the MDR1 gene showed no significance, whereas the SNP G2677T/A was significantly associated with the genotypes GT and GA of the MDR1 gene, indicating it may be a potential marker to detect drug resistance. Screening these polymorphisms will pave the way to better understand the molecular mechanisms of the disease, which may be useful in developing targeted therapies for INS patients.

The Tubulin-Based-Polymorphism Method Provides a Simple and Effective Alternative to the Genomic Profiling of Grape

PubMed Central

Mastromauro, Francesco; Gianì, Silvia; Morello, Laura

2016-01-01

The TBP (Tubulin-Based-Polymorphism) method, based on a nuclear ILP (Intron-Length-Polymorphism) molecular marker, has been used for genotyping 37 accessions of the genus Vitis inclusive of different species, rootstocks, wild and cultivated subspecies. A distinct DNA barcode made up by a different number of amplicons, was attributed to each of the different accessions. TBP data were compared with those obtained, with the use of an internationally validated set of six SSR markers. Genetic relationships among the different accessions, dendrogram distributions, correlation values and polymorphic index values (PICs) were definitively comparable when not in favor of TBP. Such an experimental consistency is based upon a genomic organization of the multiple members of the β-tubulin gene family, the targets of TBP-mediated amplification, that is conserved in Vitis as in any other plant species. The TBP amplicons can actually be used as a useful source of sequence polymorphisms for generating primer pairs capable of identifying specific cultivars in a simple assay. An example for the identification of the ‘Sangiovese’ cv. is reported. More generally, these data are discussed in terms of the actual advantages that the introduction of the TBP method in the field of grape characterization and genotyping can provide. PMID:27643687

Altering the Polymorphic Accessibility of Polycyclic Aromatic Hydrocarbons with Fluorination

DOE PAGES

Hiszpanski, Anna M.; Woll, Arthur R.; Kim, Bumjung; ...

2017-04-25

Here, substituting hydrogen with fluorine is an extensively employed strategy to improve the macroscopic properties of compounds for use in fields as diverse as pharmaceutics and optoelectronics. The role fluorine substitution plays on polymorphism—the ability of a compound to adopt more than one crystal structure—has not been previously studied. Yet, this understanding is important as different polymorphs of the same compound can result in drastically different bulk properties (e.g., solubility, absorptivity, and conductivity). Strategies to either promote or suppress the crystallization of particular polymorphs are thus desired. Here, we show that substituting hydrogen with fluorine affects the polymorphic behavior ofmore » contorted hexabenzocoronene (cHBC). A polycyclic aromatic hydrocarbon and molecular semiconductor, cHBC exhibits two polymorphs (i.e., P2 1/c crystal structure which we refer to as polymorph I and a triclinic crystal structure which we refer to as polymorph II) that are accessible through postdeposition processing of amorphous films. While the same two polymorphs remain accessible in fluorinated derivatives of cHBC, fluorination appears to favor the formation of polymorph I, with progressively smaller energy barrier for transformation from polymorph II to polymorph I with fluorination.« less

Altering the Polymorphic Accessibility of Polycyclic Aromatic Hydrocarbons with Fluorination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hiszpanski, Anna M.; Woll, Arthur R.; Kim, Bumjung

Here, substituting hydrogen with fluorine is an extensively employed strategy to improve the macroscopic properties of compounds for use in fields as diverse as pharmaceutics and optoelectronics. The role fluorine substitution plays on polymorphism—the ability of a compound to adopt more than one crystal structure—has not been previously studied. Yet, this understanding is important as different polymorphs of the same compound can result in drastically different bulk properties (e.g., solubility, absorptivity, and conductivity). Strategies to either promote or suppress the crystallization of particular polymorphs are thus desired. Here, we show that substituting hydrogen with fluorine affects the polymorphic behavior ofmore » contorted hexabenzocoronene (cHBC). A polycyclic aromatic hydrocarbon and molecular semiconductor, cHBC exhibits two polymorphs (i.e., P2 1/c crystal structure which we refer to as polymorph I and a triclinic crystal structure which we refer to as polymorph II) that are accessible through postdeposition processing of amorphous films. While the same two polymorphs remain accessible in fluorinated derivatives of cHBC, fluorination appears to favor the formation of polymorph I, with progressively smaller energy barrier for transformation from polymorph II to polymorph I with fluorination.« less

Serum amyloid A1: Structure, function and gene polymorphism

PubMed Central

Sun, Lei; Ye, Richard D.

2017-01-01

Inducible expression of serum amyloid A (SAA) is a hallmark of the acute-phase response, which is a conserved reaction of vertebrates to environmental challenges such as tissue injury, infection and surgery. Human SAA1 is encoded by one of the four SAA genes and is the best-characterized SAA protein. Initially known as a major precursor of amyloid A (AA), SAA1 has been found to play an important role in lipid metabolism and contributes to bacterial clearance, the regulation of inflammation and tumor pathogenesis. SAA1 has five polymorphic coding alleles (SAA1.1 – SAA1.5) that encode distinct proteins with minor amino acid substitutions. Single nucleotide polymorphism (SNP) has been identified in both the coding and non-coding regions of human SAA1. Despite high levels of sequence homology among these variants, SAA1 polymorphisms have been reported as risk factors of cardiovascular diseases and several types of cancer. A recently solved crystal structure of SAA1.1 reveals a hexameric bundle with each of the SAA1 subunits assuming a 4-helix structure stabilized by the C-terminal tail. Analysis of the native SAA1.1 structure has led to the identification of a competing site for high-density lipoprotein (HDL) and heparin, thus providing the structural basis for a role of heparin and heparan sulfate in the conversion of SAA1 to AA. In this brief review, we compares human SAA1 with other forms of human and mouse SAAs, and discuss how structural and genetic studies of SAA1 have advanced our understanding of the physiological functions of the SAA proteins. PMID:26945629