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Sample records for vh chain gene

  1. Altered phenotypic expression of immunoglobulin heavy-chain variable-region (VH) genes in Alicia rabbits probably reflects a small deletion in the VH genes closest to the joining region.

    Science.gov (United States)

    Allegrucci, M; Newman, B A; Young-Cooper, G O; Alexander, C B; Meier, D; Kelus, A S; Mage, R G

    1990-07-01

    Rabbits of the Alicia strain have a mutation (ali) that segregates with the immunoglobulin heavy-chain (lgh) locus and has a cis effect upon the expression of heavy-chain variable-region (VH) genes encoding the a2 allotype. In heterozygous a1/ali or a3/ali rabbits, serum immunoglobulins are almost entirely the products of the normal a1 or a3 allele and only traces of a2 immunoglobulin are detectable. Adult homozygous ali/ali rabbits likewise have normal immunoglobulin levels resulting from increased production of a-negative immunoglobulins and some residual ability to produce the a2 allotype. By contrast, the majority of the immunoglobulins of wild-type a2 rabbits are a2-positive and only a small percentage are a-negative. Genomic DNAs from homozygous mutant and wild-type animals were indistinguishable by Southern analyses using a variety of restriction enzyme digests and lgh probes. However, when digests with infrequently cutting enzymes were analyzed by transverse alternating-field electrophoresis, the ali DNA fragments were 10-15 kilobases smaller than the wild type. These fragments hybridized to probes both for VH and for a region of DNA a few kilobases downstream of the VH genes nearest the joining region. We suggest that this relatively small deletion affects a segment containing 3' VH genes with important regulatory functions, the loss of which leads to the ali phenotype. These results, and the fact that the 3' VH genes rearrange early in B-cell development, indicate that the 3' end of the VH locus probably plays a key role in regulation of VH gene expression.

  2. VH gene expression and regulation in the mutant Alicia rabbit. Rescue of VHa2 allotype expression.

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    Chen, H T; Alexander, C B; Young-Cooper, G O; Mage, R G

    1993-04-01

    Rabbits of the Alicia strain, derived from rabbits expressing the VHa2 allotype, have a mutation in the H chain locus that has a cis effect upon the expression of VHa2 and VHa- genes. A small deletion at the most J-proximal (3') end of the VH locus leads to low expression of all the genes on the entire chromosome in heterozygous ali mutants and altered relative expression of VH genes in homozygotes. To study VH gene expression and regulation, we used the polymerase chain reaction to amplify the VH genes expressed in spleens of young and adult wild-type and mutant Alicia rabbits. The cDNA from reverse transcription of splenic mRNA was amplified and polymerase chain reaction libraries were constructed and screened with oligonucleotides from framework regions 1 and 3, as well as JH. Thirty-three VH-positive clones were sequenced and analyzed. We found that in mutant Alicia rabbits, products of the first functional VH gene (VH4a2), (or VH4a2-like genes) were expressed in 2- to 8-wk-olds. Expression of both the VHx and VHy types of VHa- genes was also elevated but the relative proportions of VHx and VHy, especially VHx, decreased whereas the relative levels of expression of VH4a2 or VH4a2-like genes increased with age. Our results suggest that the appearance of sequences resembling that of the VH1a2, which is deleted in the mutant ali rabbits, could be caused by alterations of the sequences of the rearranged VH4a2 genes by gene conversions and/or rearrangement of upstream VH1a2-like genes later in development.

  3. Primary cutaneous B-cell lymphoma is associated with somatically hypermutated immunoglobulin variable genes and frequent use of VH1-69 and VH4-59 segments.

    Science.gov (United States)

    Perez, M; Pacchiarotti, A; Frontani, M; Pescarmona, E; Caprini, E; Lombardo, G A; Russo, G; Faraggiana, T

    2010-03-01

    Accurate assessment of the somatic mutational status of clonal immunoglobulin variable region (IgV) genes is relevant in elucidating tumour cell origin in B-cell lymphoma; virgin B cells bear unmutated IgV genes, while germinal centre and postfollicular B cells carry mutated IgV genes. Furthermore, biases in the IgV repertoire and distribution pattern of somatic mutations indicate a possible antigen role in the pathogenesis of B-cell malignancies. This work investigates the cellular origin and antigenic selection in primary cutaneous B-cell lymphoma (PCBCL). We analysed the nucleotide sequence of clonal IgV heavy-chain gene (IgVH) rearrangements in 51 cases of PCBCL (25 follicle centre, 19 marginal zone and seven diffuse large B-cell lymphoma, leg-type) and compared IgVH sequences with their closest germline segment in the GenBank database. Molecular data were then correlated with histopathological features. We showed that all but one of the 51 IgVH sequences analysed exhibited extensive somatic hypermutations. The detected mutation rate ranged from 1.6% to 21%, with a median rate of 9.8% and was independent of PCBCL histotype. Calculation of antigen-selection pressure showed that 39% of the mutated IgVH genes displayed a number of replacement mutations and silent mutations in a pattern consistent with antigenic selection. Furthermore, two segments, VH1-69 (12%) and VH4-59 (14%), were preferentially used in our case series. Data indicate that neoplastic B cells of PBCBL have experienced germinal centre reaction and also suggest that the involvement of IgVH genes is not entirely random in PCBCL and that common antigen epitopes could be pathologically relevant in cutaneous lymphomagenesis.

  4. Characterization of a rabbit germ-line VH gene that is a candidate donor for VH gene conversion in mutant Alicia rabbits.

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    Chen, H T; Alexander, C B; Mage, R G

    1995-06-15

    Normal rabbits preferentially rearrange the 3'-most VH gene, VH1, to encode Igs with VHa allotypes, which constitute the majority of rabbit serum Igs. A gene conversion-like mechanism is employed to diversify the primary Ab repertoire. In mutant Alicia rabbits that derived from a rabbit with VHa2 allotype, the VH1 gene was deleted. Our previous studies showed that the first functional gene (VH4) or VH4-like genes were rearranged in 2- to 8-wk-old homozygous Alicia. The VH1a2-like sequences that were found in splenic mRNA from 6-wk and older Alicia rabbits still had some residues that were typical of VH4. The appearances of sequences resembling that of VH1a2 may have been caused by gene conversions that altered the sequences of the rearranged VH or there may have been rearrangement of upstream VH1a2-like genes later in development. To investigate this further, we constructed a cosmid library and isolated a VH1a2-like gene, VH12-1-6, with a sequence almost identical to VH1a2. This gene had a deleted base in the heptamer of its recombination signal sequence. However, even if this defect diminished or eliminated its ability to rearrange, the a2-like gene could have acted as a donor for gene-conversion-like alteration of rearranged VH genes. Sequence comparisons suggested that this gene or a gene like it could have acted as a donor for gene conversion in mutant Alicia and in normal rabbits.

  5. B lymphocyte selection and age-related changes in VH gene usage in mutant Alicia rabbits.

    Science.gov (United States)

    Zhu, X; Boonthum, A; Zhai, S K; Knight, K L

    1999-09-15

    Young Alicia rabbits use VHa-negative genes, VHx and VHy, in most VDJ genes, and their serum Ig is VHa negative. However, as Alicia rabbits age, VHa2 allotype Ig is produced at high levels. We investigated which VH gene segments are used in the VDJ genes of a2 Ig-secreting hybridomas and of a2 Ig+ B cells from adult Alicia rabbits. We found that 21 of the 25 VDJ genes used the a2-encoding genes, VH4 or VH7; the other four VDJ genes used four unknown VH gene segments. Because VH4 and VH7 are rarely found in VDJ genes of normal or young Alicia rabbits, we investigated the timing of rearrangement of these genes in Alicia rabbits. During fetal development, VH4 was used in 60-80% of nonproductively rearranged VDJ genes, and VHx and VHy together were used in 10-26%. These data indicate that during B lymphopoiesis VH4 is preferentially rearranged. However, the percentage of productive VHx- and VHy-utilizing VDJ genes increased from 38% at day 21 of gestation to 89% at birth (gestation day 31), whereas the percentage of VH4-utilizing VDJ genes remained at 15%. These data suggest that during fetal development, either VH4-utilizing B-lineage cells are selectively eliminated, or B cells with VHx- and VHy-utilizing VDJ genes are selectively expanded, or both. The accumulation of peripheral VH4-utilizing a2 B cells with age indicates that these B cells might be selectively expanded in the periphery. We discuss the possible selection mechanisms that regulate VH gene segment usage in rabbit B cells during lymphopoiesis and in the periphery.

  6. Similarities in the immunoglobulin response and VH gene usage in rhesus monkeys and humans exposed to porcine hepatocytes

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    Borie Dominic C

    2006-03-01

    Full Text Available Abstract Background The use of porcine cells and organs as a source of xenografts for human patients would vastly increase the donor pool; however, both humans and Old World primates vigorously reject pig tissues due to xenoantibodies that react with the polysaccharide galactose α (1,3 galactose (αGal present on the surface of many porcine cells. We previously examined the xenoantibody response in patients exposed to porcine hepatocytes via treatment(s with bioartficial liver devices (BALs, composed of porcine cells in a support matrix. We determined that xenoantibodies in BAL-treated patients are predominantly directed at porcine αGal carbohydrate epitopes, and are encoded by a small number of germline heavy chain variable region (VH immunoglobulin genes. The studies described in this manuscript were designed to identify whether the xenoantibody responses and the IgVH genes encoding antibodies to porcine hepatocytes in non-human primates used as preclinical models are similar to those in humans. Adult non-immunosuppressed rhesus monkeys (Macaca mulatta were injected intra-portally with porcine hepatocytes or heterotopically transplanted with a porcine liver lobe. Peripheral blood leukocytes and serum were obtained prior to and at multiple time points after exposure, and the immune response was characterized, using ELISA to evaluate the levels and specificities of circulating xenoantibodies, and the production of cDNA libraries to determine the genes used by B cells to encode those antibodies. Results Xenoantibodies produced following exposure to isolated hepatocytes and solid organ liver grafts were predominantly encoded by genes in the VH3 family, with a minor contribution from the VH4 family. Immunoglobulin heavy-chain gene (VH cDNA library screening and gene sequencing of IgM libraries identified the genes as most closely-related to the IGHV3-11 and IGHV4-59 germline progenitors. One of the genes most similar to IGHV3-11, VH3-11cyno, has

  7. [Cloning of VH and VL Gene of Human anti-IL1RAP McAb and Construction of Recombinant Chimeric Receptor].

    Science.gov (United States)

    Yin, Ling-Ling; Ruan, Su-Hong; Tian, Yu; Zhao, Kai; Xu, Kai Lin

    2015-10-01

    To clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs). The VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells. The VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired. Human anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.

  8. Sequences of the joining region genes for immunoglobulin heavy chains and their role in generation of antibody diversity.

    OpenAIRE

    Gough, N M; Bernard, O

    1981-01-01

    To assess the contribution to immunoglobulin heavy chain diversity made by recombination between variable region (VH) genes and joining region (JH) genes, we have determined the sequence of about 2000 nucleotides spanning the rearranged JH gene cluster associated with the VH gene expressed in plasmacytoma HPC76. The active VH76 gene has recombined with the second germ-line JH gene. The region we have studied contains two other JH genes, designated JH3 and JH4. No other JH gene was found withi...

  9. Side chain requirements for affinity and specificity in D5, an HIV-1 antibody derived from the VH1-69 germline segment.

    Science.gov (United States)

    Stewart, Alex; Harrison, Joseph S; Regula, Lauren K; Lai, Jonathan R

    2013-04-08

    Analysis of factors contributing to high affinity antibody-protein interactions provides insight into natural antibody evolution, and guides the design of antibodies with new or enhanced function. We previously studied the interaction between antibody D5 and its target, a designed protein based on HIV-1 gp41 known as 5-Helix, as a model system [Da Silva, G. F.; Harrison, J. S.; Lai, J. R., Biochemistry, 2010, 49, 5464-5472]. Antibody D5 represents an interesting case study because it is derived from the VH1-69 germline segment; this germline segment is characterized by a hydrophobic second heavy chain complementarity determining region (HCDR2) that constitutes the major functional paratope in D5 and several antibodies derived from the same progenitor. Here we explore side chain requirements for affinity and specificity in D5 using phage display. Two D5-based libraries were prepared that contained diversity in all three light chain complementarity determining regions (LCDRs 1-3), and in the third HCDR (HCDR3). The first library allowed residues to vary among a restricted set of six amino acids (Tyr/Ala/Asp/Ser/His/Pro; D5-Lib-I). The second library was designed based on a survey of existing VH1-69 antibody structures (D5-Lib-II). Both libraries were subjected to multiple rounds of selection against 5-Helix, and individual clones characterized. We found that selectants from D5-Lib-I generally had moderate affinity and specificity, while many clones from D5-Lib-II exhibited D5-like properties. Additional analysis of the D5-Lib-II functional population revealed position-specific biases for particular amino acids, many that differed from the identity of those side chains in D5. Together these results suggest that there is some permissiveness for alternative side chains in the LCDRs and HCDR3 of D5, but that replacement with a minimal set of residues is not tolerated in this scaffold for 5-Helix recognition. This work provides novel information about this high

  10. Contribution of V(H replacement products in mouse antibody repertoire.

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    Lin Huang

    Full Text Available VH replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS near the 3' end of a rearranged VH gene and the 23-bp RSS from an upstream unrearranged VH gene. Due to the location of the cRSS, VH replacement leaves a short stretch of nucleotides from the previously rearranged VH gene at the newly formed V-D junction, which can be used as a marker to identify VH replacement products. To determine the contribution of VH replacement products to mouse antibody repertoire, we developed a Java-based VH Replacement Footprint Analyzer (VHRFA program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify VH replacement products. The overall frequency of VH replacement products in these IgH genes is 5.29% based on the identification of pentameric VH replacement footprints at their V-D junctions. The identified VH replacement products are distributed similarly in IgH genes using most families of VH genes, although different families of VH genes are used differentially. The frequencies of VH replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified VH replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.

  11. Detection of clonal immunoglobulin heavy chain gene rearrangements by the polymerase chain reaction and capillary gel electrophoresis.

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    Fan, Hongxin; Robetorye, Ryan S

    2013-01-01

    Although well-established diagnostic criteria exist for mature B-cell neoplasms, a definitive diagnosis of a B-cell lymphoproliferative disorder cannot always be obtained using more conventional techniques such as flow cytometric immunophenotyping, conventional cytogenetics, fluorescence in situ hybridization, or immunohistochemistry. However, because B-cell malignancies contain identically rearranged immunoglobulin heavy chain genes, the polymerase chain reaction (PCR) can be a fast, convenient, and dependable option to identify clonal B-cell processes. This chapter describes the use of PCR and capillary electrophoresis to identify clonal immunoglobulin heavy chain (IGH) variable and joining region (VH-JH) gene rearrangements (IGH VH-JH PCR) using a commercially available method employing multiple multiplex PCR tubes that was originally developed as the result of a large European BIOMED-2 collaborative study (Invivoscribe Technologies). The core protocol involves the use of three separate master mix tubes that target the conserved framework (FR1, FR2, and FR3) and joining (J) regions of the IGH gene. Analysis of these three framework regions can detect approximately 88% of clonal IGH gene rearrangements.

  12. Genomic analysis of a 1 Mb region near the telomere of Hessian fly chromosome X2 and avirulence gene vH13

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    Chen Ming-Shun

    2006-01-01

    Full Text Available Abstract Background To have an insight into the Mayetiola destructor (Hessian fly genome, we performed an in silico comparative genomic analysis utilizing genetic mapping, genomic sequence and EST sequence data along with data available from public databases. Results Chromosome walking and FISH were utilized to identify a contig of 50 BAC clones near the telomere of the short arm of Hessian fly chromosome X2 and near the avirulence gene vH13. These clones enabled us to correlate physical and genetic distance in this region of the Hessian fly genome. Sequence data from these BAC ends encompassing a 760 kb region, and a fully sequenced and assembled 42.6 kb BAC clone, was utilized to perform a comparative genomic study. In silico gene prediction combined with BLAST analyses was used to determine putative orthology to the sequenced dipteran genomes of the fruit fly, Drosophila melanogaster, and the malaria mosquito, Anopheles gambiae, and to infer evolutionary relationships. Conclusion This initial effort enables us to advance our understanding of the structure, composition and evolution of the genome of this important agricultural pest and is an invaluable tool for a whole genome sequencing effort.

  13. Preferrential rearrangement in normal rabbits of the 3' VHa allotype gene that is deleted in Alicia mutants; somatic hypermutation/conversion may play a major role in generating the heterogeneity of rabbit heavy chain variable region sequences.

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    Allegrucci, M; Young-Cooper, G O; Alexander, C B; Newman, B A; Mage, R G

    1991-02-01

    The rabbit is unique in having well-defined allotypes in the variable region of the heavy chain. Products of the VHa locus, (with alleles a1, a2, and a3), account for the majority of the serum immunoglobulins. A small percentage of the serum immunoglobulins are a-negative. In 1986, Kelus and Weiss described a mutation that depressed the expression of the Ig VH a2 genes in an a1/a2 rabbit. From this animal the Alicia rabbit strain was developed and the mutation was termed ali. We previously showed, using Southern analysis and the transverse alternating field electrophoresis technique, that the difference between the ali rabbit and normal is a relatively small deletion including some of the most 3' VH genes. The most JH proximal 3' VH1 genes in DNA from normal rabbits of a1, a2 and a3 haplotypes encode a1, a2 and a3 molecules respectively, and it has been suggested that these genes are responsible for allelic inheritance of VHa allotypes. The present study suggests that the 3' end of the VH locus probably plays a key role in regulation of VH gene expression in rabbits because VH gene(s) in this region are the target(s) of preferential VDJ rearrangements. This raises the possibility that mechanisms such as somatic gene conversion and hypermutation are at work to generate the antibody repertoire in this species. Our data support the view that the 3' VH1 gene may be the preferred target for rearrangement in normal rabbits, and for the normal chromosome in heterozygous ali animals. However, homozygous ali rabbits with a deletion that removed the a2-encoding VH1 on both chromosomes do survive, rearrange other VH genes and produce normal levels of immunoglobulins as well as a significant percentage of B cells which bear the a2 allotype. This challenges the view that one VH gene, VH1, is solely responsible for the inheritance pattern of VHa allotypes.

  14. No evidence for the use of DIR, D-D fusions, chromosome 15 open reading frames or VH replacement in the peripheral repertoire was found on application of an improved algorithm, JointML, to 6329 human immunoglobulin H rearrangements

    DEFF Research Database (Denmark)

    Ohm-Laursen, Line; Nielsen, Morten; Larsen, Stine R

    2006-01-01

    gene (VH) replacement. Safe conclusions require large, well-defined sequence samples and algorithms minimizing stochastic assignment of segments. Two computer programs were developed for analysis of heavy chain joints. JointHMM is a profile hidden Markow model, while JointML is a maximum...

  15. VH repertoire in progeny of long term lymphoid-cultured cells used to reconstitute immunodeficient mice

    International Nuclear Information System (INIS)

    Denis, K.A.; Timson, L.K.; Witte, O.N.

    1989-01-01

    VH gene utilization in the progeny of long term lymphoid-cultured cells used for reconstitution of severe combined immunodeficient mice under varying conditions was determined. Hybridomas made from the spleens of these animals were evaluated for clonality and donor origin and a panel of 146 independent hybridomas were subsequently examined for VH expression. Hybridomas derived from the spleens of SCID mice reconstituted with fresh cells, used as a control, utilized VH families in proportion to their numerical representation in the genome. However, hybridomas from the spleens of mice reconstituted with long term cultured cells utilized a predominance of the two VH gene families most proximal to JH, characteristic of cells early in B lymphocyte development. Coinjection of thymocytes with cultured fetal liver cells, to provide good levels of T lymphocytes, did not alter this pattern of VH utilization. Irradiation (3 Gy) of the mice before cultured cell injection, which leads to more complete reconstitution of the B cell compartment, was effective in removing this bias in the VH repertoire. Hybridomas derived from these mice expressed their VH genes more in proportion to family size, characteristic of cells later in B lymphocyte development. In this manner, long term lymphoid-cultured cells can be used to study the transitions that occur in VH repertoire expression which appear to be mediated by either B lymphocyte developmental microenvironment or population size

  16. Diversity and repertoire of IgW and IgM VH families in the newborn nurse shark.

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    Rumfelt, Lynn L; Lohr, Rebecca L; Dooley, Helen; Flajnik, Martin F

    2004-05-06

    Adult cartilaginous fish express three immunoglobulin (Ig) isotypes, IgM, IgNAR and IgW. Newborn nurse sharks, Ginglymostoma cirratum, produce 19S (multimeric) IgM and monomeric/dimeric IgM1gj, a germline-joined, IgM-related VH, and very low amounts of 7S (monomeric) IgM and IgNAR proteins. Newborn IgNAR VH mRNAs are diverse in the complementarity-determining region 3 (CDR3) with non-templated nucleotide (N-region) addition, which suggests that, unlike in many other vertebrates, terminal deoxynucleotidyl transferase (TdT) expressed at birth is functional. IgW is present in the lungfish, a bony fish sharing a common ancestor with sharks 460 million years ago, implying that the IgW VH family is as old as the IgM VH family. This nurse shark study examined the IgM and IgW VH repertoire from birth through adult life, and analyzed the phylogenetic relationships of these gene families. IgM and IgW VH cDNA clones isolated from newborn nurse shark primary and secondary lymphoid tissues had highly diverse and unique CDR3 with N-region addition and VDJ gene rearrangement, implicating functional TdT and RAG gene activity. Despite the clear presence of N-region additions, newborn CDR3 were significantly shorter than those of adults. The IgM clones are all included in a conventional VH family that can be classified into five discrete groups, none of which is orthologous to IgM VH genes in other elasmobranchs. In addition, a novel divergent VH family was orthologous to a published monotypic VH horn shark family. IgW VH genes have diverged sufficiently to form three families. IgM and IgW VH serine codons using the potential somatic hypermutation hotspot sequence occur mainly in VH framework 1 (FR1) and CDR1. Phylogenetic analysis of cartilaginous fish and lungfish IgM and IgW demonstrated they form two major ancient gene groups; furthermore, these VH genes generally diversify (duplicate and diverge) within a species. As in ratfish, sandbar and horn sharks, most nurse shark IgM VH

  17. Diversity and repertoire of IgW and IgM VH families in the newborn nurse shark

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    Dooley Helen

    2004-05-01

    Full Text Available Abstract Background Adult cartilaginous fish express three immunoglobulin (Ig isotypes, IgM, IgNAR and IgW. Newborn nurse sharks, Ginglymostoma cirratum, produce 19S (multimeric IgM and monomeric/dimeric IgM1gj, a germline-joined, IgM-related VH, and very low amounts of 7S (monomeric IgM and IgNAR proteins. Newborn IgNAR VH mRNAs are diverse in the complementarity-determining region 3 (CDR3 with non-templated nucleotide (N-region addition, which suggests that, unlike in many other vertebrates, terminal deoxynucleotidyl transferase (TdT expressed at birth is functional. IgW is present in the lungfish, a bony fish sharing a common ancestor with sharks 460 million years ago, implying that the IgW VH family is as old as the IgM VH family. This nurse shark study examined the IgM and IgW VH repertoire from birth through adult life, and analyzed the phylogenetic relationships of these gene families. Results IgM and IgW VH cDNA clones isolated from newborn nurse shark primary and secondary lymphoid tissues had highly diverse and unique CDR3 with N-region addition and VDJ gene rearrangement, implicating functional TdT and RAG gene activity. Despite the clear presence of N-region additions, newborn CDR3 were significantly shorter than those of adults. The IgM clones are all included in a conventional VH family that can be classified into five discrete groups, none of which is orthologous to IgM VH genes in other elasmobranchs. In addition, a novel divergent VH family was orthologous to a published monotypic VH horn shark family. IgW VH genes have diverged sufficiently to form three families. IgM and IgW VH serine codons using the potential somatic hypermutation hotspot sequence occur mainly in VH framework 1 (FR1 and CDR1. Phylogenetic analysis of cartilaginous fish and lungfish IgM and IgW demonstrated they form two major ancient gene groups; furthermore, these VH genes generally diversify (duplicate and diverge within a species. Conclusion As in ratfish

  18. The evolution of multiple isotypic IgM heavy chain genes in the shark.

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    Lee, Victor; Huang, Jing Li; Lui, Ming Fai; Malecek, Karolina; Ohta, Yuko; Mooers, Arne; Hsu, Ellen

    2008-06-01

    The IgM H chain gene organization of cartilaginous fishes consists of 15-200 miniloci, each with a few gene segments (V(H)-D1-D2-J(H)) and one C gene. This is a gene arrangement ancestral to the complex IgH locus that exists in all other vertebrate classes. To understand the molecular evolution of this system, we studied the nurse shark, which has relatively fewer loci, and characterized the IgH isotypes for organization, functionality, and the somatic diversification mechanisms that act upon them. Gene numbers differ slightly between individuals ( approximately 15), but five active IgM subclasses are always present. Each gene undergoes rearrangement that is strictly confined within the minilocus; in B cells there is no interaction between adjacent loci located > or =120 kb apart. Without combinatorial events, the shark IgM H chain repertoire is based on junctional diversity and, subsequently, somatic hypermutation. We suggest that the significant contribution by junctional diversification reflects the selected novelty introduced by RAG in the early vertebrate ancestor, whereas combinatorial diversity coevolved with the complex translocon organization. Moreover, unlike other cartilaginous fishes, there are no germline-joined VDJ at any nurse shark mu locus, and we suggest that such genes, when functional, are species-specific and may have specialized roles. With an entire complement of IgM genes available for the first time, phylogenetic analyses were performed to examine how the multiple Ig loci evolved. We found that all domains changed at comparable rates, but V(H) appears to be under strong positive selection for increased amino acid sequence diversity, and surprisingly, so does Cmicro2.

  19. Limited number of immunoglobulin VH regions expressed in the mutant rabbit "Alicia".

    Science.gov (United States)

    DiPietro, L A; Short, J A; Zhai, S K; Kelus, A S; Meier, D; Knight, K L

    1990-06-01

    A unique feature of rabbit Ig is the presence of VH region allotypic specificities. In normal rabbits, more than 80% of circulating immunoglobulin molecules bear the VHa allotypic specificities, al, a2 or a3; the remaining 10% to 20% of immunoglobulin molecules lack VHa allotypic specificities and are designated VHa-. A mutant rabbit designated Alicia, in contrast, has predominantly serum immunoglobulin molecules that lack the VHa allotypic specificities (Kelus and Weiss, Proc. Natl. Acad. Sci. USA 1986. 83: 4883). To study the nature and molecular complexity of VHa- molecules, we cloned and determined the nucleotide sequence of seven cDNA prepared from splenic RNA of an Alicia rabbit. Six of the clones appeared to encode VHa- molecules; the framework regions encoded by these clones were remarkably similar to each other, each having an unusual insertion of four amino acids at position 10. This insertion of four amino acids has been seen in only 2 of 54 sequenced rabbit VH genes. The similarity of the sequences of the six VHa- clones to each other and their dissimilarity to most other VH genes leads us to suggest that the VHa- molecules in Alicia rabbits are derived predominantly from one or a small number of very similar VH genes. Such preferential utilization of a small number of VH genes may explain the allelic inheritance of VH allotypes.

  20. Gene Expression Profiling to Predict Clinical Outcome of Breast Cancer: reproducing, analyzing and extending the Nature publication by vhVeer et al

    NARCIS (Netherlands)

    Li R.; Visser, H.M.

    2010-01-01

    Chemotherapy and hormonal therapy as adjuvant systemic therapies to inhibit breast cancer recurrence are not necessary for each patient. In Veer's paper "Gene expression profiling predicts clinical outcome of breast cancer" (Nature 2002, PMID: 11823860), they introduced a method based on DNA

  1. Sterile DJH rearrangements reveal that distance between gene segments on the human Ig H chain locus influences their ability to rearrange

    DEFF Research Database (Denmark)

    Hansen, Tina Østergaard; Lange, Anders Blaabjerg; Barington, Torben

    2015-01-01

    Rearrangement of the Ig locus occurs in two steps. First, a JH gene is rearranged to a D gene followed by a VH gene rearranging to the DJH rearrangement. By next generation sequencing, we analyzed 9969 unique DJH rearrangements and 5919 unique VHDJH rearrangements obtained from peripheral blood B...... frequently than JH locus distal D genes, whereas VH locus proximal D genes were observed more frequently in nonproductive VHDJH rearrangements. We further demonstrate that the distance between VH, D, and JH gene segments influence their ability to rearrange within the human Ig locus....

  2. Diversity Analysis of the Immunoglobulin M Heavy Chain Gene in ...

    African Journals Online (AJOL)

    A full-length cDNA encoding the immunoglobulin (IgM) heavy chain gene of Nile tilapia was successfully cloned using the 5' and 3' RACE techniques. The complete cDNA of the Nile tilapia IgM heavy chain gene is 1,921 bp in length and has an open reading frame (ORF) of 1,740 bp, which corresponds to 580 amino acid ...

  3. Draft Genome Sequences of the Fish Pathogen Vibrio harveyi Strains VH2 and VH5

    DEFF Research Database (Denmark)

    Castillo, Daniel; D'Alvise, Paul; Middelboe, Mathias

    2015-01-01

    Vibrio harveyi is an important marine pathogen that is responsible for vibriosis outbreaks in cultured fish and invertebrates worldwide. Here, we announce the draft genome sequences of V. harveyi strains VH2 and VH5, isolated from farmed juvenile Seriola dumerili during outbreaks of vibriosis...... in Crete, Greece....

  4. Biased immunoglobulin light chain gene usage in the shark1

    Science.gov (United States)

    Iacoangeli, Anna; Lui, Anita; Naik, Ushma; Ohta, Yuko; Flajnik, Martin; Hsu, Ellen

    2015-01-01

    This study of a large family of kappa light (L) chain clusters in nurse shark completes the characterization of its classical immunoglobulin (Ig) gene content (two heavy chain classes, mu and omega, and four L chain isotopes, kappa, lambda, sigma, and sigma-2). The shark kappa clusters are minigenes consisting of a simple VL-JL-CL array, where V to J recombination occurs over a ~500 bp interval, and functional clusters are widely separated by at least 100 kb. Six out of ca. 39 kappa clusters are pre-rearranged in the germline (GL-joined). Unlike the complex gene organization and multistep assembly process of Ig in mammals, each shark Ig rearrangement, somatic or in the germline, appears to be an independent event localized to the minigene. This study examined the expression of functional, non-productive, and sterile transcripts of the kappa clusters compared to the other three L chain isotypes. Kappa cluster usage was investigated in young sharks, and a skewed pattern of split gene expression was observed, one similar in functional and non-productive rearrangements. These results show that the individual activation of the spatially distant kappa clusters is non-random. Although both split and GL-joined kappa genes are expressed, the latter are prominent in young animals and wane with age. We speculate that, in the shark, the differential activation of the multiple isotypes can be advantageously used in receptor editing. PMID:26342033

  5. Biased Immunoglobulin Light Chain Gene Usage in the Shark.

    Science.gov (United States)

    Iacoangeli, Anna; Lui, Anita; Naik, Ushma; Ohta, Yuko; Flajnik, Martin; Hsu, Ellen

    2015-10-15

    This study of a large family of κ L chain clusters in nurse shark completes the characterization of its classical Ig gene content (two H chain isotypes, μ and ω, and four L chain isotypes, κ, λ, σ, and σ-2). The shark κ clusters are minigenes consisting of a simple VL-JL-CL array, where V to J recombination occurs over an ~500-bp interval, and functional clusters are widely separated by at least 100 kb. Six out of ~39 κ clusters are prerearranged in the germline (germline joined). Unlike the complex gene organization and multistep assembly process of Ig in mammals, each shark Ig rearrangement, somatic or in the germline, appears to be an independent event localized to the minigene. This study examined the expression of functional, nonproductive, and sterile transcripts of the κ clusters compared with the other three L chain isotypes. κ cluster usage was investigated in young sharks, and a skewed pattern of split gene expression was observed, one similar in functional and nonproductive rearrangements. These results show that the individual activation of the spatially distant κ clusters is nonrandom. Although both split and germline-joined κ genes are expressed, the latter are prominent in young animals and wane with age. We speculate that, in the shark, the differential activation of the multiple isotypes can be advantageously used in receptor editing. Copyright © 2015 by The American Association of Immunologists, Inc.

  6. Immunoglobulin diversification in B cell malignancies: internal splicing of heavy chain variable region as a by-product of somatic hypermutation

    NARCIS (Netherlands)

    Bende, R. J.; Aarts, W. M.; Pals, S. T.; van Noesel, C. J. M.

    2002-01-01

    In this study we describe alternative splicing of somatically mutated immunoglobulin (Ig) variable heavy chain (V-H) genes in three distinct primary B cell non-Hodgkin's lymphomas (B-NHL). In two V4-34 expressing lymphomas, ie a post-germinal center type B cell chronic lymphocytic leukemia (B-CLL)

  7. Characterization of additional rabbit IgM allotypes and the effect of suppression of a VH locus allotypes on the expression of n Cμ locus allotype

    International Nuclear Information System (INIS)

    Gilman-Sachs, A.; Roux, K.H.; Horing, W.J.; Dray, S.

    1982-01-01

    Anti-allotype antisera were produced that identified eight rabbit IgM allotypic specificities, n80, n81, n82, n83, n84, n85, n86, and n87. The n locus Cμ genes controlling these IgM allotypic specificities are closely linked to the a (VH subgroup) locus. The genes controlling these allotypic specificities were found to be in the heavy chain chromosomal region and were assigned to 11 haplotypes present in our rabbit colony. The n locus and a locus genes appeared in the haplotypes in six combinations: a 1 n 81 , a 2 n/sup 81,n87/, a 1 n/sup 80,83/, a 2 n/sup 80,82,87/, a 3 n/sup 81,84,85/ and a 3 n/sup 80,84,86,87/. By radioprecipitation analysis, 70 to 80% of serum IgM reacts with the antiserum directed to each n locus allotypic specificity found encoded in one haplotype; thus, each allotypic specificity of the haplotype is present on the same IgM molecule. When sera from a locus allotype-suppressed homozygous rabbits were tested for expression of each n locus allotypic specificity, n80, n81, and n87 were still expressed, whereas n82, n83, n84, n85, and n86 were not. These data provide direct evidence that some IgM specificities are expressed independently of the a locus (i.e., ''true''), and other s are dependent on the expression of an a locus specificity (i.e., conformational). The expression of the ''true'' allotypic specificities probably reflects genetic control of the germline Cμ gene, and the expression of ''conformationally dependent'' allotypic specificities probably reflects the interaction of VH and Cμ gene segments. This distinction is important and must be recognized when evaluating the genetics and structure of the IgM molecule

  8. Data of evolutionary structure change: 1JOEA-1VH2A [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available ID>1VH2 A 1VH2A RDHLE----GVVDV ...>H ---- EEe> ATOM 480 CA ARG A 68 6.679 -2...entryIDChain> RDHLNGDSIEIIDI >H EE> 1VH2 A 1VH2A GHDQP--IPGVS ...e> -- > ATOM 806 CA GLY A 112 3.574 -8

  9. Platelet-derived growth factor (PDGF) B-chain gene expression by activated blood monocytes precedes the expression of the PDGF A-chain gene

    International Nuclear Information System (INIS)

    Martinet, Y.; Jaffe, H.A.; Yamauchi, K.; Betsholtz, C.; Westermark, B.; Heldin, C.H.; Crystal, R.G.

    1987-01-01

    When activated, normal human blood monocytes are known to express the c-sis proto-oncogene coding for PDGF B-chain. Since normal human platelet PDGF molecules are dimers of A and B chains and platelets and monocytes are derived from the same marrow precursors, activated blood monocytes were simultaneously evaluated for their expression of PDGF A and B chain genes. Human blood monocytes were purified by adherence, cultured with or without activation by lipopolysaccharide and poly(A)+ RNA evaluated using Northern analysis and 32 P-labeled A-chain and B-chain (human c-sis) probes. Unstimulated blood monocytes did not express either A-chain or B-chain genes. In contrast, activated monocytes expressed a 4.2 kb mRNA B-chain transcript at 4 hr, but the B-chain mRNA levels declined significantly over the next 18 hr. In comparison, activated monocytes expressed very little A-chain mRNA at 4 hr, but at 12 hr 1.9, 2.3, and 2.8 kb transcripts were observed and persisted through 24 hr. Thus, activation of blood monocytes is followed by PDGF B-chain gene expression preceding PDGF A-chain gene expression, suggesting a difference in the regulation of the expression of the genes for these two chains by these cells

  10. Characterization of human cardiac myosin heavy chain genes

    International Nuclear Information System (INIS)

    Yamauchi-Takihara, K.; Sole, M.J.; Liew, J.; Ing, D.; Liew, C.C.

    1989-01-01

    The authors have isolated and analyzed the structure of the genes coding for the α and β forms of the human cardiac myosin heavy chain (MYHC). Detailed analysis of four overlapping MYHC genomic clones shows that the α-MYHC and β-MYHC genes constitute a total length of 51 kilobases and are tandemly linked. The β-MYHC-encoding gene, predominantly expressed in the normal human ventricle and also in slow-twitch skeletal muscle, is located 4.5 kilobases upstream of the α-MYHC-encoding gene, which is predominantly expressed in normal human atrium. The authors have determined the nucleotide sequences of the β form of the MYHC gene, which is 100% homologous to the cardiac MYHC cDNA clone (pHMC3). It is unlikely that the divergence of a few nucleotide sequences from the cardiac β-MYHC cDNA clone (pHMC3) reported in a MYHC cDNA clone (PSMHCZ) from skeletal muscle is due to a splicing mechanism. This finding suggests that the same β form of the cardiac MYHC gene is expressed in both ventricular and slow-twitch skeletal muscle. The promoter regions of both α- and β-MYHC genes, as well as the first four coding regions in the respective genes, have also been sequenced. The sequences in the 5'-flanking region of the α- and β-MYHC-encoding genes diverge extensively from one another, suggesting that expression of the α- and β-MYHC genes is independently regulated

  11. Local Chromatin Features Including PU.1 and IKAROS Binding and H3K4 Methylation Shape the Repertoire of Immunoglobulin Kappa Genes Chosen for V(D)J Recombination

    OpenAIRE

    Louise S. Matheson; Daniel J. Bolland; Peter Chovanec; Felix Krueger; Simon Andrews; Hashem Koohy; Anne E. Corcoran

    2017-01-01

    V(D)J recombination is essential for the generation of diverse antigen receptor (AgR) repertoires. In B cells, immunoglobulin kappa (Igκ) light chain recombination follows immunoglobulin heavy chain (Igh) recombination. We recently developed the DNA-based VDJ-seq assay for the unbiased quantitation of Igh VH and DH repertoires. Integration of VDJ-seq data with genome-wide datasets revealed that two chromatin states at the recombination signal sequence (RSS) of VH genes are highly predictive o...

  12. vh@nnlo-v2: new physics in Higgs Strahlung

    Science.gov (United States)

    Harlander, Robert V.; Klappert, Jonas; Liebler, Stefan; Simon, Lukas

    2018-05-01

    Introducing version 2 of the code vh@nnlo [1], we study the effects of a number of new-physics scenarios on the Higgs-Strahlung process. In particular, the cross section is evaluated within a general 2HDM and the MSSM. While the Drell-Yan-like contributions are consistently taken into account by a simple rescaling of the SM result, the gluon-initiated contribution is supplemented by squark-loop mediated amplitudes, and by the s-channel exchange of additional scalars which may lead to conspicuous interference effects. The latter holds as well for bottom-quark initiated Higgs Strahlung, which is also included in the new version of vh@nnlo. Using an orthogonal rotation of the three Higgs CP eigenstates in the 2HDM and the MSSM, vh@nnlo incorporates a simple means of CP mixing in these models. Moreover, the effect of vector-like quarks in the SM on the gluon-initiated contribution can be studied. Beyond concrete models, vh@nnlo allows to include the effect of higher-dimensional operators on the production of CP-even Higgs bosons. Transverse momentum distributions of the final state Higgs boson and invariant mass distributions of the Vϕ final state for the gluon- and bottom-quark initiated contributions can be studied. Distributions for the Drell-Yan-like component of Higgs Strahlung can be included through a link to MCFM. vh@nnlo can also be linked to FeynHiggs and 2HDMC for the calculation of Higgs masses and mixing angles. It can also read these parameters from an SLHA-file as produced by standard spectrum generators. Throughout the manuscript, we highlight new-physics effects in various numerical examples, both at the inclusive level and for distributions.

  13. Regulation of laminin beta2 chain gene expression in human cancer cell lines

    DEFF Research Database (Denmark)

    Durkin, M E; Nielsen, F C; Loechel, F

    2001-01-01

    of the human laminin beta2 chain gene generates two isoforms of the 5' untranslated region of the beta2 chain mRNA. The translational efficiencies of the two laminin beta2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell beta2 chain m......RNA, transient transfection of chimeric beta2 chain leader/luciferase expression plasmids in clone A cells, and translation of in vitro synthesized RNAs in rabbit reticulocyte lysates....

  14. A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties

    Science.gov (United States)

    Tiller, Thomas; Schuster, Ingrid; Deppe, Dorothée; Siegers, Katja; Strohner, Ralf; Herrmann, Tanja; Berenguer, Marion; Poujol, Dominique; Stehle, Jennifer; Stark, Yvonne; Heßling, Martin; Daubert, Daniela; Felderer, Karin; Kaden, Stefan; Kölln, Johanna; Enzelberger, Markus; Urlinger, Stefanie

    2013-01-01

    This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds. PMID:23571156

  15. Using gene co-expression network analysis to predict biomarkers for chronic lymphocytic leukemia

    Directory of Open Access Journals (Sweden)

    Borlawsky Tara B

    2010-10-01

    Full Text Available Abstract Background Chronic lymphocytic leukemia (CLL is the most common adult leukemia. It is a highly heterogeneous disease, and can be divided roughly into indolent and progressive stages based on classic clinical markers. Immunoglobin heavy chain variable region (IgVH mutational status was found to be associated with patient survival outcome, and biomarkers linked to the IgVH status has been a focus in the CLL prognosis research field. However, biomarkers highly correlated with IgVH mutational status which can accurately predict the survival outcome are yet to be discovered. Results In this paper, we investigate the use of gene co-expression network analysis to identify potential biomarkers for CLL. Specifically we focused on the co-expression network involving ZAP70, a well characterized biomarker for CLL. We selected 23 microarray datasets corresponding to multiple types of cancer from the Gene Expression Omnibus (GEO and used the frequent network mining algorithm CODENSE to identify highly connected gene co-expression networks spanning the entire genome, then evaluated the genes in the co-expression network in which ZAP70 is involved. We then applied a set of feature selection methods to further select genes which are capable of predicting IgVH mutation status from the ZAP70 co-expression network. Conclusions We have identified a set of genes that are potential CLL prognostic biomarkers IL2RB, CD8A, CD247, LAG3 and KLRK1, which can predict CLL patient IgVH mutational status with high accuracies. Their prognostic capabilities were cross-validated by applying these biomarker candidates to classify patients into different outcome groups using a CLL microarray datasets with clinical information.

  16. Poloidal rotation and the evolution of H-mode and VH-mode profiles

    International Nuclear Information System (INIS)

    Hinton, F.L.; Staebler, G.M.; Kim, Y.B.

    1993-12-01

    The physics which determines poloidal rotation, and its role in the development of profiles during H- and VH-modes, is discussed. A simple phenomenological transport model, which incorporates the rvec E x rvec B flow shear suppression of turbulence, is shown to predict profile evolution similar to that observed experimentally during H-mode and VH-mode

  17. Characterization of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) in Shanghai, China: molecular and cytogenetic characteristics, IgV gene restriction and hypermutation patterns.

    Science.gov (United States)

    Irons, Richard D; Le, Anh; Bao, Liming; Zhu, Xiongzeng; Ryder, John; Wang, Xiao Qin; Ji, Meirong; Chen, Yan; Wu, Xichun; Lin, Guowei

    2009-12-01

    The clinical, cytogenetic and molecular features of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), a disease previously considered to be rare in Asia, were examined in consecutive series of 70 cases diagnosed by our laboratory over a 30-month period. Clonal abnormalities were observed in 80% of CLL/SLL cases using a combination of conventional cytogenetic and fluorescence in situ hybridization (FISH) analysis. Those involving 14q32/IGH were the most frequent (24 cases), followed by trisomy 12 and 11q abnormalities. IgV(H) gene usage was non-random with over-representation of V(H)4-34, V(H)3-23 and a previously unreported increase in V(H)3-48 gene use. Somatic hypermutation (SHM) of IgV(H) germline sequences was observed in 56.5% of cases with stereotyped patterns of SHM observed in V(H)4-34 heavy chain complimentary-determining (HCDR1) and framework region CFR2 sequences. These findings in a Chinese population suggest subtle geographical differences in IgV(H) gene usage while the remarkably specific pattern of SHM suggest that a relatively limited set of antigens may be involved in the development of this disease worldwide. IgV(H) gene mutation status was a significant predictor of initial survival in CLL/SLL. However, an influence of karyotype on prognosis was not observed.

  18. Gm typing by immunoglobulin heavy-chain gene RFLP analysis.

    OpenAIRE

    Jazwinska, E C; Dunckley, H; Propert, D N; Gatenby, P A; Serjeantson, S W

    1988-01-01

    This study was undertaken to investigate a means of assigning Gm allotypes to Caucasians by RFLP analysis. A single immunoglobulin heavy-chain gamma-4 cDNA probe (HU gamma 4) was hybridized with genomic DNA digested separately with two restriction enzymes, TaqI and PvuII. Results showed excellent correlation (P less than .001) between serologically defined Gm allotypes G1m(1), G1m(2), G2m(23), and G1m;G3m (3;5,10) and RFLPs identified with the (HU gamma 4) probe. We conclude that it is now po...

  19. Increased expression of electron transport chain genes in uterine leiomyoma.

    Science.gov (United States)

    Tuncal, Akile; Aydin, Hikmet Hakan; Askar, Niyazi; Ozkaya, Ali Burak; Ergenoglu, Ahmet Mete; Yeniel, Ahmet Ozgur; Akdemir, Ali; Ak, Handan

    2014-01-01

    The etiology and pathophysiology of uterine leiomyomas, benign smooth muscle tumors of the uterus, are not well understood. To evaluate the role of mitochondria in uterine leiomyoma, we compared electron transport gene expressions of uterine leiomyoma tissue with myometrium tissue in six uterine leiomyoma patients by RT-PCR array. Our results showed an average of 1.562 (±0.445) fold increase in nuclear-encoded electron transport genes. These results might suggest an increase in size, number, or activity of mitochondria in uterine leiomyoma that, to our knowledge, has not been previously reported. © 2014 by the Association of Clinical Scientists, Inc.

  20. Diversity of immunoglobulin lambda light chain gene usage over developmental stages in the horse.

    Science.gov (United States)

    Tallmadge, Rebecca L; Tseng, Chia T; Felippe, M Julia B

    2014-10-01

    To further studies of neonatal immune responses to pathogens and vaccination, we investigated the dynamics of B lymphocyte development and immunoglobulin (Ig) gene diversity. Previously we demonstrated that equine fetal Ig VDJ sequences exhibit combinatorial and junctional diversity levels comparable to those of adult Ig VDJ sequences. Herein, RACE clones from fetal, neonatal, foal, and adult lymphoid tissue were assessed for Ig lambda light chain combinatorial, junctional, and sequence diversity. Remarkably, more lambda variable genes (IGLV) were used during fetal life than later stages and IGLV gene usage differed significantly with time, in contrast to the Ig heavy chain. Junctional diversity measured by CDR3L length was constant over time. Comparison of Ig lambda transcripts to germline revealed significant increases in nucleotide diversity over time, even during fetal life. These results suggest that the Ig lambda light chain provides an additional dimension of diversity to the equine Ig repertoire. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Reference genes for gene expression analysis by real-time reverse transcription polymerase chain reaction of renal cell carcinoma.

    Science.gov (United States)

    Bjerregaard, Henriette; Pedersen, Shona; Kristensen, Søren Risom; Marcussen, Niels

    2011-12-01

    Differentiation between malignant renal cell carcinoma and benign oncocytoma is of great importance to choose the optimal treatment. Accurate preoperative diagnosis of renal tumor is therefore crucial; however, existing imaging techniques and histologic examinations are incapable of providing an optimal differentiation profile. Analysis of gene expression of molecular markers is a new possibility but relies on appropriate standardization to compare different samples. The aim of this study was to identify stably expressed reference genes suitable for the normalization of results extracted from gene expression analysis of renal tumors. Expression levels of 8 potential reference genes (ATP5J, HMBS, HPRT1, PPIA, TBP, 18S, GAPDH, and POLR2A) were examined by real-time reverse transcription polymerase chain reaction in tumor and normal tissue from removed kidneys from 13 patients with renal cell carcinoma and 5 patients with oncocytoma. The expression levels of genes were compared by gene stability value M, average gene stability M, pairwise variation V, and coefficient of variation CV. More candidates were not suitable for the purpose, but a combination of HMBS, PPIA, ATP5J, and TBP was found to be the best combination with an average gene stability value M of 0.9 and a CV of 0.4 in the 18 tumors and normal tissues. A combination of 4 genes, HMBS, PPIA, ATP5J, and TBP, is a possible reference in renal tumor gene expression analysis by reverse transcription polymerase chain reaction. A combination of four genes, HMBS, PPIA, ATP5J and TBP, being stably expressed in tissues from RCC is possible reference genes for gene expression analysis.

  2. VH mode accessibility and global H-mode properties in previous and present JET configurations

    Energy Technology Data Exchange (ETDEWEB)

    Jones, T T.C.; Ali-Arshad, S; Bures, M; Christiansen, J P; Esch, H P.L. de; Fishpool, G; Jarvis, O N; Koenig, R; Lawson, K D; Lomas, P J; Marcus, F B; Sartori, R; Schunke, B; Smeulders, P; Stork, D; Taroni, A; Thomas, P R; Thomsen, K [Commission of the European Communities, Abingdon (United Kingdom). JET Joint Undertaking

    1994-07-01

    In JET VH modes, there is a distinct confinement transition following the cessation of ELMs, observed in a wide variety of tokamak operating conditions, using both NBI and ICRF heating methods. Important factors which influence VH mode accessibility such as magnetic configuration and vessel conditions have been identified. The new JET pumped divertor configuration has much improved plasma shaping control and power and particle exhaust capability and should permit exploitation of plasmas with VH confinement properties over an even wider range of operating regimes, particularly at high plasma current; first H-modes have been obtained in the 1994 JET operating period and initial results are reported. (authors). 7 refs., 6 figs.

  3. Fundamental characteristics of the immunoglobulin VH repertoire of chickens in comparison with those of humans, mice, and camelids.

    Science.gov (United States)

    Wu, Leeying; Oficjalska, Katarzyna; Lambert, Matthew; Fennell, Brian J; Darmanin-Sheehan, Alfredo; Ní Shúilleabháin, Deirdre; Autin, Bénédicte; Cummins, Emma; Tchistiakova, Lioudmila; Bloom, Laird; Paulsen, Janet; Gill, Davinder; Cunningham, Orla; Finlay, William J J

    2012-01-01

    Examination of 1269 unique naive chicken V(H) sequences showed that the majority of positions in the framework (FW) regions were maintained as germline, with high mutation rates observed in the CDRs. Many FW mutations could be clearly related to the modulation of CDR structure or the V(H)-V(L) interface. CDRs 1 and 2 of the V(H) exhibited frequent mutation in solvent-exposed positions, but conservation of common structural residues also found in human CDRs at the same positions. In comparison with humans and mice, the chicken CDR3 repertoire was skewed toward longer sequences, was dominated by small amino acids (G/S/A/C/T), and had higher cysteine (chicken, 9.4%; human, 1.6%; and mouse, 0.25%) but lower tyrosine content (chicken, 9.2%; human, 16.8%; and mouse 26.4%). A strong correlation (R(2) = 0.97) was observed between increasing CDR3 length and higher cysteine content. This suggests that noncanonical disulfides are strongly favored in chickens, potentially increasing CDR stability and complexity in the topology of the combining site. The probable formation of disulfide bonds between CDR3 and CDR1, FW2, or CDR2 was also observed, as described in camelids. All features of the naive repertoire were fully replicated in the target-selected, phage-displayed repertoire. The isolation of a chicken Fab with four noncanonical cysteines in the V(H) that exhibits 64 nM (K(D)) binding affinity for its target proved these constituents to be part of the humoral response, not artifacts. This study supports the hypothesis that disulfide bond-constrained CDR3s are a structural diversification strategy in the restricted germline v-gene repertoire of chickens.

  4. Jet substructure and probes of CP violation in Vh production

    Energy Technology Data Exchange (ETDEWEB)

    Godbole, R.M. [Centre for High Energy Physics, Indian Institute of Science,Sir C.V. Raman Road, Bangalore 560012 (India); Miller, D.J. [School of Physics and Astronomy, Scottish Universities Physics Alliance, University of Glasgow,University Avenue, Glasgow G12 8QQ, Scotland (United Kingdom); Mohan, K.A. [Centre for High Energy Physics, Indian Institute of Science,Sir C.V. Raman Road, Bangalore 560012 (India); White, C.D. [School of Physics and Astronomy, Scottish Universities Physics Alliance, University of Glasgow,University Avenue, Glasgow G12 8QQ, Scotland (United Kingdom)

    2015-04-20

    We analyse the hVV (V=W,Z) vertex in a model independent way using Vh production. To that end, we consider possible corrections to the Standard Model Higgs Lagrangian, in the form of higher dimensional operators which parametrise the effects of new physics. In our analysis, we pay special attention to linear observables that can be used to probe CP violation in the same. By considering the associated production of a Higgs boson with a vector boson (W or Z), we use jet substructure methods to define angular observables which are sensitive to new physics effects, including an asymmetry which is linearly sensitive to the presence of CP odd effects. We demonstrate how to use these observables to place bounds on the presence of higher dimensional operators, and quantify these statements using a log likelihood analysis. Our approach allows one to probe separately the hZZ and hWW vertices, involving arbitrary combinations of BSM operators, at the Large Hadron Collider.

  5. Cas9-triggered chain ablation of cas9 as a gene drive brake

    OpenAIRE

    Wu, Bing; Luo, Liqun; Gao, Xiaojing J.

    2016-01-01

    With the advent of clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) technology, researchers can construct gene drives that can bias the inheritance of edited alleles to alter entire populations. As demonstrated with the mutagenic chain reaction in Drosophila4, the CRISPR-Cas9 system can propagate genomic modification together with the genome-editing machinery itself. Although gene drives might have the potential to control insect-borne di...

  6. Rapid and Sensitive Assessment of Globin Chains for Gene and Cell Therapy of Hemoglobinopathies

    NARCIS (Netherlands)

    Loucari, C.C. (Constantinos C.); Patsali, P. (Petros); T.B. van Dijk (Thamar); Stephanou, C. (Coralea); Papasavva, P. (Panayiota); Zanti, M. (Maria); Kurita, R. (Ryo); Nakamura, Y. (Yukio); S. Christou (Soteroula); Sitarou, M. (Maria); J.N.J. Philipsen (Sjaak); C.W. Lederer (Carsten); M. Kleanthous (Marina)

    2018-01-01

    textabstractThe β-hemoglobinopathies sickle cell anemia and β-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of

  7. Expression of porcine myosin heavy chain 1 gene in Berkshire loins ...

    African Journals Online (AJOL)

    Expression of porcine myosin heavy chain 1 gene in Berkshire loins with a high pH24 value. Jin Hun Kang, Woo Young Bang, Eun Jung Kwon, Yong Hwa Lee, Da Hye Park, Eun Seok Cho, Min Ji Kim, Jong-Soon Choi, Hwa Chun Park, Beom Young Park, Chul Wook Kim ...

  8. Histidine at Position 195 is Essential for Association of Heme- b in Lcp1VH2

    Science.gov (United States)

    Oetermann, Sylvia; Vivod, Robin; Hiessl, Sebastian; Hogeback, Jens; Holtkamp, Michael; Karst, Uwe; Steinbüchel, Alexander

    2018-05-01

    The latex clearing protein (Lcp) is the key enzyme of polyisoprene degradation in actinomycetes (Yikmis and Steinbüchel in Appl Environ Microbiol 78:4543-4551, https://doi.org/10.1128/AEM.00001-12 , 2012). In this study it was shown that Lcp from Gordonia polyisoprenivorans VH2 (Lcp1VH2) harbors a non-covalently bound heme b as cofactor, which was identified by pyridine hemochrome spectra and confirmed by LC/ESI-ToF-MS. It contains iron, most likely in the Fe3+ state. We focused on the characterization of the heme-cofactor, its accessibility with respect to the conformation of Lcp1VH2, and the identification of putative histidine residues involved in the coordination of heme. A change was detectable in UV/Vis-spectra of reduced Lcp1VH2 when imidazole was added, showing that Lcp1VH2 "as isolated" occurs in an open state, directly being accessible for external ligands. In addition, three highly conserved histidines (H195, H200 and H228), presumably acting as ligands coordinating the heme within the heme pocket, were replaced with alanines by site-directed mutagenesis. The effect of these changes on in vivo rubber-mineralization was investigated. The lcp- deletion mutant complemented with the H195A variant of lcp1 VH2 was unable to mineralize poly( cis-1,4-isoprene). In vitro analyses of purified, recombinant Lcp1VH2H195A confirmed the loss of enzyme activity, which could be ascribed to the loss of heme. Hence, H195 is essential for the association of heme- b in the central region of Lcp1VH2.

  9. Oestrogen influences on mitochondrial gene expression and respiratory chain activity in cortical and mesencephalic astrocytes.

    Science.gov (United States)

    Araújo, G W; Beyer, C; Arnold, S

    2008-07-01

    The regulation of mitochondrial energy metabolism plays an essential role in the central nervous system (CNS). Abnormalities of the mitochondrial respiratory chain often accompany neurodegenerative diseases. This makes mitochondria a perfect target for strategies of cellular protection against toxic compounds and pathological conditions. Steroid hormones, such as oestrogen, are well-known to fulfil a protective role in the brain during ischaemic and degenerative processes. Because astrocytes function as the major energy supplier in the CNS, we have analysed oestrogen effects on the mitochondrial respiratory chain of this cell type. In our studies, we applied semi- and quantitative polymerase chain reaction analysis of gene expression and polarographic measurements of the respiratory chain activity of mitochondria. We observed that structural and functional properties were regulated dependent on the oestrogen exposure time and the brain region, but independent of the nuclear oestrogen receptors. We could demonstrate that long-term oestrogen exposure increases the subunit gene expression of respiratory chain complexes and the mitochondrial DNA content, thereby indicating an up-regulation of the amount of mitochondria per cell together with an increase of mitochondrial energy production. This could represent an important indirect mechanism by which long-term oestrogen exposure protects neurones from cell death under neurotoxic conditions. On the other hand, we observed short-term effects of oestrogen on the activity of mitochondrial, proton-pumping respiratory chain complexes. In astrocytes from the cortex, respiratory chain activity was decreased, whereas it was increased in astrocytes from the mesencephalon. An increased production of reactive oxygen species would be the consequence of an increased respiratory chain activity in mesencephalic astrocytes. This could explain the different efficiencies of oestrogen-mediated short-term protection in distinct brain

  10. Rapid analysis of rearranged kappa light chain genes of circulating polysaccharide-specific B lymphocytes by means of immunomagnetic beads and the polymerase chain reaction

    DEFF Research Database (Denmark)

    Hougs, L; Barington, T; Madsen, HO

    1993-01-01

    reaction (PCR) using in addition a degenerate kappa light chain signal peptide region primer. The PCR product was cloned into the M13mp18 phage. The cloning efficiency was 100-600 clones/ml of blood. Of the 86 clones sequenced, 90% represented rearranged kappa light chain genes from different antibody...... of the B lymphocytes activated in vivo. Here, we present a method for rapid analysis of the rearranged kappa light chain genes used by human circulating antigen-specific B lymphocytes. After vaccination with Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugated with protein, the Hib...

  11. Metabolism of very long-chain Fatty acids: genes and pathophysiology.

    Science.gov (United States)

    Sassa, Takayuki; Kihara, Akio

    2014-02-01

    Fatty acids (FAs) are highly diverse in terms of carbon (C) chain-length and number of double bonds. FAs with C>20 are called very long-chain fatty acids (VLCFAs). VLCFAs are found not only as constituents of cellular lipids such as sphingolipids and glycerophospholipids but also as precursors of lipid mediators. Our understanding on the function of VLCFAs is growing in parallel with the identification of enzymes involved in VLCFA synthesis or degradation. A variety of inherited diseases, such as ichthyosis, macular degeneration, myopathy, mental retardation, and demyelination, are caused by mutations in the genes encoding VLCFA metabolizing enzymes. In this review, we describe mammalian VLCFAs by highlighting their tissue distribution and metabolic pathways, and we discuss responsible genes and enzymes with reference to their roles in pathophysiology.

  12. Structural organization of the human short-chain acyl-CoA dehydrogenase gene

    DEFF Research Database (Denmark)

    Corydon, M J; Andresen, B S; Bross, P

    1997-01-01

    Short-chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric mitochondrial flavoenzyme that catalyzes the initial reaction in short-chain fatty acid beta-oxidation. Defects in the SCAD enzyme are associated with failure to thrive, often with neuromuscular dysfunction and elevated urinary excretion...... shown to be associated with ethylmalonic aciduria. From analysis of 18 unrelated Danish families, we show that the four SCAD gene polymorphisms constitute five allelic variants of the SCAD gene, and that the 625A variant together with the less frequent variant form of the three other polymorphisms (321C....... The evolutionary relationship between SCAD and five other members of the acyl-CoA dehydrogenase family was investigated by two independent approaches that gave similar phylogenetic trees....

  13. Human heavy-chain variable region gene family nonrandomly rearranged in familial chronic lymphocytic leukemia

    International Nuclear Information System (INIS)

    Shen, A.; Humphries, C.; Tucker, P.; Blattner, F.

    1987-01-01

    The authors have identified a family of human immunoglobulin heavy-chain variable-region (V/sub H/) genes, one member of which is rearranged in two affected members of a family in which the father and four of five siblings developed chronic lymphocytic leukemia. Cloning and sequencing of the rearranged V/sub H/ genes from leukemic lymphocytes of three affected siblings showed that two siblings had rearranged V/sub H/ genes (V/sub H/TS1 and V/sub H/WS1) that were 90% homologous. The corresponding germ-line gene, V/sub H/251, was found to part of a small (four gene) V/sub H/ gene family, which they term V/sub H/V. The DNA sequence homology to V/sub H/WS1 (95%) and V/sub H/TS1 (88%) and identical restriction sites on the 5' side of V/sub H/ confirm that rearrangement of V/sub H/251 followed by somatic mutation produced the identical V/sub H/ gene rearrangements in the two siblings. V/sub H/TS1 is not a functional V/sub H/ gene; a functional V/sub H/ rearrangement was found on the other chromosome of this patient. The other two siblings had different V/sub H/ gene rearrangements. All used different diversity genes. Mechanisms proposed for nonrandom selection of a single V/sub H/ gene include developmental regulation of this V/sub H/ gene rearrangement or selection of a subpopulation of B cells in which this V/sub H/ has been rearranged

  14. Antibody Heavy Chain Variable Domains of Different Germline Gene Origins Diversify through Different Paths

    Directory of Open Access Journals (Sweden)

    Ufuk Kirik

    2017-11-01

    Full Text Available B cells produce antibodies, key effector molecules in health and disease. They mature their properties, including their affinity for antigen, through hypermutation events; processes that involve, e.g., base substitution, codon insertion and deletion, often in association with an isotype switch. Investigations of antibody evolution define modes whereby particular antibody responses are able to form, and such studies provide insight important for instance for development of efficient vaccines. Antibody evolution is also used in vitro for the design of antibodies with improved properties. To better understand the basic concepts of antibody evolution, we analyzed the mutational paths, both in terms of amino acid substitution and insertions and deletions, taken by antibodies of the IgG isotype. The analysis focused on the evolution of the heavy chain variable domain of sets of antibodies, each with an origin in 1 of 11 different germline genes representing six human heavy chain germline gene subgroups. Investigated genes were isolated from cells of human bone marrow, a major site of antibody production, and characterized by next-generation sequencing and an in-house bioinformatics pipeline. Apart from substitutions within the complementarity determining regions, multiple framework residues including those in protein cores were targets of extensive diversification. Diversity, both in terms of substitutions, and insertions and deletions, in antibodies is focused to different positions in the sequence in a germline gene-unique manner. Altogether, our findings create a framework for understanding patterns of evolution of antibodies from defined germline genes.

  15. Expression and developmental control of platelet-derived growth factor A-chain and B-chain/Sis genes in rat aortic smooth muscle cells

    International Nuclear Information System (INIS)

    Majesky, M.W.; Benditt, E.P.; Schwartz, S.M.

    1988-01-01

    Cultured arterial smooth muscle cells (SMC) can produce platelet-derived growth factor (PDGF)-like molecules. This property raises the possibility that SMC-derived PDGFs function as autocrine/paracrine regulators in the formation and maintenance of the artery wall. In this study the authors have asked if levels of mRNAs directing synthesis of PDFG are modulated in aortic SMC during postnatal development. The authors report here that genes encoding PDGF A- and B-chain precursors are expressed at similar low levels in intact aortas from newborn and adult rats. Marked differences in regulation of transcript abundance of these genes were revealed when aortic SMC were grown in cell culture. PDGF B-chain transcripts accumulated in passaged newborn rat SMC but not adult rat SMC, whereas PDGF A-chain RNA was found in comparable amounts in SMC from both age groups. Similarly, SMC from newborn rats secreted at least 60-fold more PDGF-like activity into conditioned medium than did adult rat SMC. These results show that PDGF A- and B-chain genes are transcribed in the normal rat aorta and provide evidence for age-related change in the control of PDGF B-chain gene expression in aortic SMC. Independent regulation of transcript levels in cultured SMC leaves open the possibility that PDGFs of different composition (AA, AB, BB) play different roles in normal function of the artery wall

  16. Rapid and Sensitive Assessment of Globin Chains for Gene and Cell Therapy of Hemoglobinopathies

    Science.gov (United States)

    Loucari, Constantinos C.; Patsali, Petros; van Dijk, Thamar B.; Stephanou, Coralea; Papasavva, Panayiota; Zanti, Maria; Kurita, Ryo; Nakamura, Yukio; Christou, Soteroulla; Sitarou, Maria; Philipsen, Sjaak; Lederer, Carsten W.; Kleanthous, Marina

    2018-01-01

    The β-hemoglobinopathies sickle cell anemia and β-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of induced or exogenous β-like globins. Reversed-phase high-performance liquid chromatography (HPLC) allows versatile and inexpensive globin quantification, but commonly applied protocols suffer from long run times, high sample requirements, or inability to separate murine from human β-globin chains. The latter point is problematic for in vivo studies with gene-addition vectors in murine disease models and mouse/human chimeras. This study demonstrates HPLC-based measurements of globin expression (1) after differentiation of the commonly applied human umbilical cord blood–derived erythroid progenitor-2 cell line, (2) in erythroid progeny of CD34+ cells for the analysis of clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of the globin regulator BCL11A, and (3) of transgenic mice holding the human β-globin locus. At run times of 8 min for separation of murine and human β-globin chains as well as of human γ-globin chains, and with routine measurement of globin-chain ratios for 12 nL of blood (tested for down to 0.75 nL) or of 300,000 in vitro differentiated cells, the methods presented here and any variant-specific adaptations thereof will greatly facilitate evaluation of novel therapy applications for β-hemoglobinopathies. PMID:29325430

  17. Cell surface expression of single chain antibodies with applications to imaging of gene expression in vivo

    International Nuclear Information System (INIS)

    Northrop, Jeffrey P.; Bednarski, Mark; Li, King C.; Barbieri, Susan O.; Lu, Amy T.; Nguyen, Dee; Varadarajan, John; Osen, Maureen; Star-Lack, Josh

    2003-01-01

    Imaging of gene expression in vivo has many potential uses for biomedical research and drug discovery, ranging from the study of gene regulation and cancer to the non-invasive assessment of gene therapies. To streamline the development of imaging marker gene technologies for nuclear medicine, we propose a new approach to the design of reporter/probe pairs wherein the reporter is a cell surface-expressed single chain antibody variable fragment that has been raised against a low molecular weight imaging probe with optimized pharmacokinetic properties. Proof of concept of the approach was achieved using a single chain antibody variable fragment that binds with high affinity to fluorescein and an imaging probe consisting of fluorescein isothiocyanate coupled to the chelator diethylene triamine penta-acetic acid labeled with the gamma-emitter 111 In. We demonstrate specific high-affinity binding of this probe to the cell surface-expressed reporter in vitro and assess the in vivo biodistribution of the probe both in wild-type mice and in mice harboring tumor xenografts expressing the reporter. Specific uptake of the probe by, and in vivo imaging of, tumors expressing the reporter are shown. Since ScFvs with high affinities can be raised to almost any protein or small molecule, the proposed methodology may offer a new flexibility in the design of imaging tracer/reporter pairs wherein both probe pharmacokinetics and binding affinities can be readily optimized. (orig.)

  18. Hypermutation in shark immunoglobulin light chain genes results in contiguous substitutions.

    Science.gov (United States)

    Lee, Susan S; Tranchina, Daniel; Ohta, Yuko; Flajnik, Martin F; Hsu, Ellen

    2002-04-01

    Among 631 substitutions present in 90 nurse shark immunoglobulin light chain somatic mutants, 338 constitute 2-4 bp stretches of adjacent changes. An absence of mutations in perinatal sequences and the bias for one mutating V gene in adults suggest that the diversification is antigen dependent. The substitutions shared no patterns, and the absence of donor sequences, including from family members, supports the idea that most changes arose from nontemplated mutation. The tandem mutations as a group are distinguished by consistently fewer transition changes and an A bias. We suggest this is one of several pathways of hypermutation diversifying shark antigen-receptor genes--point mutations, tandem mutations, and mutations with a G-C preference--that coevolved with or preceded gene rearrangement.

  19. Diagnostic value of immunoglobulin κ light chain gene rearrangement analysis in B-cell lymphomas.

    Science.gov (United States)

    Kokovic, Ira; Jezersek Novakovic, Barbara; Novakovic, Srdjan

    2015-03-01

    Analysis of the immunoglobulin κ light chain (IGK) gene is an alternative method for B-cell clonality assessment in the diagnosis of mature B-cell proliferations in which the detection of clonal immunoglobulin heavy chain (IGH) gene rearrangements fails. The aim of the present study was to evaluate the added value of standardized BIOMED-2 assay for the detection of clonal IGK gene rearrangements in the diagnostic setting of suspected B-cell lymphomas. With this purpose, 92 specimens from 80 patients with the final diagnosis of mature B-cell lymphoma (37 specimens), mature T-cell lymphoma (26 specimens) and reactive lymphoid proliferation (29 specimens) were analyzed for B-cell clonality. B-cell clonality analysis was performed using the BIOMED-2 IGH and IGK gene clonality assays. The determined sensitivity of the IGK assay was 67.6%, while the determined sensitivity of the IGH assay was 75.7%. The sensitivity of combined IGH+IGK assay was 81.1%. The determined specificity of the IGK assay was 96.2% in the group of T-cell lymphomas and 96.6% in the group of reactive lesions. The determined specificity of the IGH assay was 84.6% in the group of lymphomas and 86.2% in the group of reactive lesions. The comparison of GeneScan (GS) and heteroduplex pretreatment-polyacrylamide gel electrophoresis (HD-PAGE) methods for the analysis of IGK gene rearrangements showed a higher efficacy of GS analysis in a series of 27 B-cell lymphomas analyzed by both methods. In the present study, we demonstrated that by applying the combined IGH+IGK clonality assay the overall detection rate of B-cell clonality was increased by 5.4%. Thus, we confirmed the added value of the standardized BIOMED-2 IGK assay for assessment of B-cell clonality in suspected B-cell lymphomas with inconclusive clinical and cyto/histological diagnosis.

  20. Genetic recombination within the human T-cell receptor α-chain gene complex

    International Nuclear Information System (INIS)

    Robinson, M.A.; Kindt, T.J.

    1987-01-01

    Genetic analyses of the human T-cell receptor (TCR) α-chain genes indicate that recombination events may occur frequently within this gene complex. Examination of the inheritance of restriction fragment length polymorphisms (RFLP) detected by using probes for constant or variable region gene segments made it possible to assign TCRα haplotypes to the 16 parents and 43 offspring of eight families studied. A total of six RFLP, three for the constant region and three for variable region segments, were examined in the present studies. Most enzyme and probe combinations tested revealed no polymorphism and those finally selected for the study showed limited polymorphism in that only two or, in one case, three allelic forms of the gene were seen. In spite of limited variability at this level, extensive heterogeneity was observed for the combinations of markers present in haplotypes, suggesting that frequent recombination events have occurred. Most strikingly, multiple combinations of RFLP occurring in close proximity of the TCRα constant region gene were observed in this study. A high recombination frequency for the TCRα gene complex is further supported by the observation that two children, one in each of two families, inherited recombinant TCRα haplotypes

  1. Genes involved in long-chain alkene biosynthesis in Micrococcus luteus

    Energy Technology Data Exchange (ETDEWEB)

    Beller, Harry R.; Goh, Ee-Been; Keasling, Jay D.

    2010-01-07

    Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which four decades ago was reported to biosynthesize iso- and anteiso branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty-acid overproducing E. coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-CoA produced the same C27 monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or -ACP) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (?-ketoacyl-ACP synthase III), which catalyzes decarboxylative Claisen condensation during

  2. Cardiac-Specific Gene Expression Facilitated by an Enhanced Myosin Light Chain Promoter

    Directory of Open Access Journals (Sweden)

    Wolfgang Boecker

    2004-04-01

    Full Text Available Background: Adenoviral gene transfer has been shown to be effective in cardiac myocytes in vitro and in vivo. A major limitation of myocardial gene therapy is the extracardiac transgene expression. Methods: To minimize extracardiac gene expression, we have constructed a tissue-specific promoter for cardiac gene transfer, namely, the 250-bp fragment of the myosin light chain-2v (MLC-2v gene, which is known to be expressed in a tissue-specific manner in ventricular myocardium followed by a luciferase (luc reporter gene (Ad.4 × MLC250.Luc. Rat cardiomyocytes, liver and kidney cells were infected with Ad.4 × MLC.Luc or control vectors. For in vivo testing, Ad.4 × MLC250.Luc was injected into the myocardium or in the liver of rats. Kinetics of promoter activity were monitored over 8 days using a cooled CCD camera. Results: In vitro: By infecting hepatic versus cardiomyocyte cells, we found that the promoter specificity ratio (luc activity in cardiomyocytes per liver cells was 20.4 versus 0.9 (Ad.4 × MLC250.Luc vs. Ad.CMV. In vivo: Ad.4 × MLC250.Luc significantly reduced luc activity in liver (38.4-fold, lung (16.1-fold, and kidney (21.8-fold versus Ad.CMV (p = .01; whereas activity in the heart was only 3.8-fold decreased. The gene expression rate of cardiomyocytes versus hepatocytes was 7:1 (Ad.4 × MLC.Luc versus 1:1.4 (Ad.CMV.Luc. Discussion: This new vector may be useful to validate therapeutic approaches in animal disease models and offers the perspective for selective expression of therapeutic genes in the diseased heart.

  3. A polymerase chain reaction-based methodology to detect gene doping.

    Science.gov (United States)

    Carter, Adam; Flueck, Martin

    2012-04-01

    The non-therapeutic use of genes to enhance athletic performance (gene doping) is a novel threat to the world of sports. Skeletal muscle is a prime target of gene therapy and we asked whether we can develop a test system to produce and detect gene doping. Towards this end, we introduced a plasmid (pCMV-FAK, 3.8 kb, 50 μg) for constitutive expression of the chicken homologue for the regulator of muscle growth, focal adhesion kinase (FAK), via gene electro transfer in the anti-gravitational muscle, m. soleus, or gastrocnemius medialis of rats. Activation of hypertrophy signalling was monitored by assessing the ribosomal kinase p70S6K and muscle fibre cross section. Detectability of the introduced plasmid was monitored with polymerase chain reaction in deoxyribonucleic acids (DNA) from transfected muscle and serum. Muscle transfection with pCMV-FAK elevated FAK expression 7- and 73-fold, respectively, and increased mean cross section by 52 and 16% in targeted muscle fibres of soleus and gastrocnemius muscle 7 days after gene electro transfer. Concomitantly p70S6K content was increased in transfected soleus muscle (+110%). Detection of the exogenous plasmid sequence was possible in DNA and cDNA of muscle until 7 days after transfection, but not in serum except close to the site of plasmid deposition, 1 h after injection and surgery. The findings suggest that the reliable detection of gene doping in the immoral athlete is not possible unless a change in the current practice of tissue sampling is applied involving the collection of muscle biopsy close to the site of gene injection.

  4. Genetic stability of gene targeted immunoglobulin loci. I. Heavy chain isotype exchange induced by a universal gene replacement vector.

    Science.gov (United States)

    Kardinal, C; Selmayr, M; Mocikat, R

    1996-11-01

    Gene targeting at the immunoglobulin loci of B cells is an efficient tool for studying immunoglobulin expression or generating chimeric antibodies. We have shown that vector integration induced by human immunoglobulin G1 (IgG1) insertion vectors results in subsequent vector excision mediated by the duplicated target sequence, whereas replacement events which could be induced by the same constructs remain stable. We could demonstrate that the distribution of the vector homology strongly influences the genetic stability obtained. To this end we developed a novel type of a heavy chain replacement vector making use of the heavy chain class switch recombination sequence. Despite the presence of a two-sided homology this construct is universally applicable irrespective of the constant gene region utilized by the B cell. In comparison to an integration vector the frequency of stable incorporation was strongly increased, but we still observed vector excision, although at a markedly reduced rate. The latter events even occurred with circular constructs. Linearization of the construct at various sites and the comparison with an integration vector that carries the identical homology sequence, but differs in the distribution of homology, revealed the following features of homologous recombination of immunoglobulin genes: (i) the integration frequency is only determined by the length of the homology flank where the cross-over takes place; (ii) a 5' flank that does not meet the minimum requirement of homology length cannot be complemented by a sufficient 3' flank; (iii) free vector ends play a role for integration as well as for replacement targeting; (iv) truncating recombination events are suppressed in the presence of two flanks. Furthermore, we show that the switch region that was used as 3' flank is non-functional in an inverted orientation.

  5. Comparative genomic analysis of the arthropod muscle myosin heavy chain genes allows ancestral gene reconstruction and reveals a new type of 'partially' processed pseudogene

    Directory of Open Access Journals (Sweden)

    Kollmar Martin

    2008-02-01

    Full Text Available Abstract Background Alternative splicing of mutually exclusive exons is an important mechanism for increasing protein diversity in eukaryotes. The insect Mhc (myosin heavy chain gene produces all different muscle myosins as a result of alternative splicing in contrast to most other organisms of the Metazoa lineage, that have a family of muscle genes with each gene coding for a protein specialized for a functional niche. Results The muscle myosin heavy chain genes of 22 species of the Arthropoda ranging from the waterflea to wasp and Drosophila have been annotated. The analysis of the gene structures allowed the reconstruction of an ancient muscle myosin heavy chain gene and showed that during evolution of the arthropods introns have mainly been lost in these genes although intron gain might have happened in a few cases. Surprisingly, the genome of Aedes aegypti contains another and that of Culex pipiens quinquefasciatus two further muscle myosin heavy chain genes, called Mhc3 and Mhc4, that contain only one variant of the corresponding alternative exons of the Mhc1 gene. Mhc3 transcription in Aedes aegypti is documented by EST data. Mhc3 and Mhc4 inserted in the Aedes and Culex genomes either by gene duplication followed by the loss of all but one variant of the alternative exons, or by incorporation of a transcript of which all other variants have been spliced out retaining the exon-intron structure. The second and more likely possibility represents a new type of a 'partially' processed pseudogene. Conclusion Based on the comparative genomic analysis of the alternatively spliced arthropod muscle myosin heavy chain genes we propose that the splicing process operates sequentially on the transcript. The process consists of the splicing of the mutually exclusive exons until one exon out of the cluster remains while retaining surrounding intronic sequence. In a second step splicing of introns takes place. A related mechanism could be responsible for

  6. Selection of suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using quantitative real-time polymerase chain reaction

    DEFF Research Database (Denmark)

    Zornhagen, K. W.; Kristensen, A. T.; Hansen, Anders Elias

    2015-01-01

    Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a sensitive technique for quantifying gene expression. Stably expressed reference genes are necessary for normalization of RT-qPCR data. Only a few articles have been published on reference genes in canine tumours....... The objective of this study was to demonstrate how to identify suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using RT-qPCR. Primer pairs for 17 potential reference genes were designed and tested in archival tumour biopsies from six dogs. The geNorm algorithm...

  7. Muscle fiber type specific induction of slow myosin heavy chain 2 gene expression by electrical stimulation

    International Nuclear Information System (INIS)

    Crew, Jennifer R.; Falzari, Kanakeshwari; DiMario, Joseph X.

    2010-01-01

    Vertebrate skeletal muscle fiber types are defined by a broad array of differentially expressed contractile and metabolic protein genes. The mechanisms that establish and maintain these different fiber types vary throughout development and with changing functional demand. Chicken skeletal muscle fibers can be generally categorized as fast and fast/slow based on expression of the slow myosin heavy chain 2 (MyHC2) gene in fast/slow muscle fibers. To investigate the cellular and molecular mechanisms that control fiber type formation in secondary or fetal muscle fibers, myoblasts from the fast pectoralis major (PM) and fast/slow medial adductor (MA) muscles were isolated, allowed to differentiate in vitro, and electrically stimulated. MA muscle fibers were induced to express the slow MyHC2 gene by electrical stimulation, whereas PM muscle fibers did not express the slow MyHC2 gene under identical stimulation conditions. However, PM muscle fibers did express the slow MyHC2 gene when electrical stimulation was combined with inhibition of inositol triphosphate receptor (IP3R) activity. Electrical stimulation was sufficient to increase nuclear localization of expressed nuclear-factor-of-activated-T-cells (NFAT), NFAT-mediated transcription, and slow MyHC2 promoter activity in MA muscle fibers. In contrast, both electrical stimulation and inhibitors of IP3R activity were required for these effects in PM muscle fibers. Electrical stimulation also increased levels of peroxisome-proliferator-activated receptor-γ co-activator-1 (PGC-1α) protein in PM and MA muscle fibers. These results indicate that MA muscle fibers can be induced by electrical stimulation to express the slow MyHC2 gene and that fast PM muscle fibers are refractory to stimulation-induced slow MyHC2 gene expression due to fast PM muscle fiber specific cellular mechanisms involving IP3R activity.

  8. Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction

    International Nuclear Information System (INIS)

    Noonan, K.E.; Beck, C.; Holzmayer, T.A.; Chin, J.E.; Roninson, I.B.; Wunder, J.S.; Andrulis, I.L.; Gazdar, A.F.; Willman, C.L.; Griffith, B.; Von Hoff, D.D.

    1990-01-01

    The resistance of tumor cells ot chemotheraprutic drugs is a major obstacle to successful cancer chemotherapy. In human cells, expression of the MDR1 gene, encoding a transmembrane efflux pump (P-glycoprotein), leads to decreased intracellular accumulation and resistance to a variety of lipophilic drugs (multidrug resistance; MDR). The levels of MDR in cell lines selected in bitro have been shown to correlate with the steady-state levels of MDR1 mRNA and P-glycoprotein. In cells with a severalfold increase in cellular drug resistance, MDR1 expression levels are close to the limits of detection by conventional assays. MDR1 expression has been frequently observed in human tumors after chemotherapy and in some but not all types of clinically refactory tumors untreated with chemotherapeutic drugs. The authors have devised a highly sensitive, specific, and quantitative protocol for measuring the levels of MDR1 mRNA in clincal samples, based on the polymerase chain reaction. They have used this assay to measure MDR1 gene expression in MDR cell lines and >300 normal tissues, tumor-derived cell lines, and clinical specimens of untreated tumors of the types in which MDR1 expression was rarely observed by standard assays. Low levels of MDR1 expression were found by polymerase chain reaction in most solid tumors and leukemias tested. The frequency of samples without detectable MDR1 expression varied among different types of tumors; MDR1-negative samples were ost common among tumor types known to be relatively responsive to chemotherapy

  9. Effects of Long Chain Fatty Acid Synthesis and Associated Gene Expression in Microalga Tetraselmis sp.

    Directory of Open Access Journals (Sweden)

    T. Catalina Adarme-Vega

    2014-06-01

    Full Text Available With the depletion of global fish stocks, caused by high demand and effective fishing techniques, alternative sources for long chain omega-3 fatty acids are required for human nutrition and aquaculture feeds. Recent research has focused on land-based cultivation of microalgae, the primary producers of omega-3 fatty acids in the marine food web. The effect of salinity on fatty acids and related gene expression was studied in the model marine microalga, Tetraselmis sp. M8. Correlations were found for specific fatty acid biosynthesis and gene expression according to salinity and the growth phase. Low salinity was found to increase the conversion of C18:4 stearidonic acid (SDA to C20:4 eicosatetraenoic acid (ETA, correlating with increased transcript abundance of the Δ-6-elongase-encoding gene in salinities of 5 and 10 ppt compared to higher salinity levels. The expression of the gene encoding β-ketoacyl-coenzyme was also found to increase at lower salinities during the nutrient deprivation phase (Day 4, but decreased with further nutrient stress. Nutrient deprivation also triggered fatty acids synthesis at all salinities, and C20:5 eicosapentaenoic acid (EPA increased relative to total fatty acids, with nutrient starvation achieving a maximum of 7% EPA at Day 6 at a salinity of 40 ppt.

  10. Polymerase chain reaction detection of retinoblastoma gene deletions in paraffin-embedded mouse lung adenocarcinomas

    International Nuclear Information System (INIS)

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1991-01-01

    A Polymerase chain reaction (PCR) technique was used to detect deletions in the mouse retinoblastoma (mRb) gene using microtomed sections from paraffin-embedded radiation-induced and spontaneous tumors as the DNA source. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. Absence of any of these fragments relative to control PCR products on a Southern blot indicated a deletion of that portion of the mRb gene. Tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death. Spontaneous tumors as well as those from irradiated mice (569 cGy of 60 Co γ rays or 60 cGy of JANUS neutrons) were analyzed. Tumors in six neutron-irradiated mice also had no mRb deletions. However, one of six tumors from γ-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice showed a deletion in one or both mRb alleles. All deletions detected were in the 5' region of the mRb gene

  11. Genes involved in long-chain alkene biosynthesis in Micrococcus luteus.

    Science.gov (United States)

    Beller, Harry R; Goh, Ee-Been; Keasling, Jay D

    2010-02-01

    Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which 4 decades ago was reported to biosynthesize iso- and anteiso-branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty acid-overproducing Escherichia coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-coenzyme A (CoA) produced the same C(27) monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or-ACP [acyl carrier protein]) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (beta-ketoacyl-ACP synthase III), which

  12. Evolutionary analysis of the kinesin light chain genes in the yellow fever mosquito Aedes aegypti: gene duplication as a source for novel early zygotic genes.

    Science.gov (United States)

    Biedler, James K; Tu, Zhijian

    2010-07-08

    The maternal zygotic transition marks the time at which transcription from the zygotic genome is initiated and a subset of maternal RNAs are progressively degraded in the developing embryo. A number of early zygotic genes have been identified in Drosophila melanogaster and comparisons to sequenced mosquito genomes suggest that some of these early zygotic genes such as bottleneck are fast-evolving or subject to turnover in dipteran insects. One objective of this study is to identify early zygotic genes from the yellow fever mosquito Aedes aegypti to study their evolution. We are also interested in obtaining early zygotic promoters that will direct transgene expression in the early embryo as part of a Medea gene drive system. Two novel early zygotic kinesin light chain genes we call AaKLC2.1 and AaKLC2.2 were identified by transcriptome sequencing of Aedes aegypti embryos at various time points. These two genes have 98% nucleotide and amino acid identity in their coding regions and show transcription confined to the early zygotic stage according to gene-specific RT-PCR analysis. These AaKLC2 genes have a paralogous gene (AaKLC1) in Ae. aegypti. Phylogenetic inference shows that an ortholog to the AaKLC2 genes is only found in the sequenced genome of Culex quinquefasciatus. In contrast, AaKLC1 gene orthologs are found in all three sequenced mosquito species including Anopheles gambiae. There is only one KLC gene in D. melanogaster and other sequenced holometabolous insects that appears to be similar to AaKLC1. Unlike AaKLC2, AaKLC1 is expressed in all life stages and tissues tested, which is consistent with the expression pattern of the An. gambiae and D. melanogaster KLC genes. Phylogenetic inference also suggests that AaKLC2 genes and their likely C. quinquefasciatus ortholog are fast-evolving genes relative to the highly conserved AaKLC1-like paralogs. Embryonic injection of a luciferase reporter under the control of a 1 kb fragment upstream of the AaKLC2.1 start

  13. Evolutionary analysis of the kinesin light chain genes in the yellow fever mosquito Aedes aegypti: gene duplication as a source for novel early zygotic genes

    Directory of Open Access Journals (Sweden)

    Tu Zhijian

    2010-07-01

    Full Text Available Abstract Background The maternal zygotic transition marks the time at which transcription from the zygotic genome is initiated and a subset of maternal RNAs are progressively degraded in the developing embryo. A number of early zygotic genes have been identified in Drosophila melanogaster and comparisons to sequenced mosquito genomes suggest that some of these early zygotic genes such as bottleneck are fast-evolving or subject to turnover in dipteran insects. One objective of this study is to identify early zygotic genes from the yellow fever mosquito Aedes aegypti to study their evolution. We are also interested in obtaining early zygotic promoters that will direct transgene expression in the early embryo as part of a Medea gene drive system. Results Two novel early zygotic kinesin light chain genes we call AaKLC2.1 and AaKLC2.2 were identified by transcriptome sequencing of Aedes aegypti embryos at various time points. These two genes have 98% nucleotide and amino acid identity in their coding regions and show transcription confined to the early zygotic stage according to gene-specific RT-PCR analysis. These AaKLC2 genes have a paralogous gene (AaKLC1 in Ae. aegypti. Phylogenetic inference shows that an ortholog to the AaKLC2 genes is only found in the sequenced genome of Culex quinquefasciatus. In contrast, AaKLC1 gene orthologs are found in all three sequenced mosquito species including Anopheles gambiae. There is only one KLC gene in D. melanogaster and other sequenced holometabolous insects that appears to be similar to AaKLC1. Unlike AaKLC2, AaKLC1 is expressed in all life stages and tissues tested, which is consistent with the expression pattern of the An. gambiae and D. melanogaster KLC genes. Phylogenetic inference also suggests that AaKLC2 genes and their likely C. quinquefasciatus ortholog are fast-evolving genes relative to the highly conserved AaKLC1-like paralogs. Embryonic injection of a luciferase reporter under the control of a

  14. Deletion Analysis Of The Duchenne/Becker Muscular Dystrophy Gene Using Multiplex Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Dastur R

    2003-01-01

    Full Text Available The diagnosis of Duchenne Muscular Dystrophy (DMD and Becker Muscular Dystrophy (BMD is mainly based on clinical profile, serum CPK values, muscle biopsy and immunostaining for dystrophin. Most recent and accurate method for diagnosing DMD/BMD is by detection of mutations in the DMD gene. This was done in 100 unrelated patients using 19 exons including the promoter region in two sets of multiplex polymerase chain reaction (PCR. These primers amplify most of the exons in the deletion prone ′hotspot′ regions allowing determination of deletion end point. Intragenic deletions were detected in 74 patients indicating that the use of PCR-based assays will allow deletion detection help in prenatal diagnosis for most of the DMD/BMD patients. The frequency of deletions observed in the present study was 74%.

  15. Beta limits in H-modes and VH-modes in JET

    Energy Technology Data Exchange (ETDEWEB)

    Smeulders, P; Hender, T C; Huysmans, G; Marcus, F; Ali-Arshad, S; Alper, B; Balet, B; Bures, M; Deliyanakis, N; Esch, H de; Fshpool, G; Jarvis, O N; Jones, T T.C.; Ketner, W; Koenig, R; Lawson, K; Lomas, P; O` Brien, D; Sadler, G; Stok, D; Stubberfield, P; Thomas, P; Thomen, K; Wesson, J [Commission of the European Communities, Abingdon (United Kingdom). JET Joint Undertaking; Nave, M F [Universidade Tecnica, Lisbon (Portugal). Inst. Superior Tecnico

    1994-07-01

    In Hot-ion H- and VH-modes, the highest achieved beta was about 10% below the Troyon value in the best case of discharge 26087. The operational space of the high beta discharges obtained before March 1992 has been explored as function of the parameters H{sub ITER89P}, {beta}{sub n}, q{sub 95}, I{sub p}. Also, a limiting envelope on the fusion reactivity as a function of the average plasma pressure and beta has been observed with R{sub DD} related to {beta}{sub {phi}}{sup 2}.B{sub {phi}}{sup 4}. MHD stability analysis shows that the JET VH modes at the edge are in the second region for ballooning mode stability. The dependence of ballooning stability and the n=1 external kink on the edge current density is analyzed. (authors). 6 figs., 6 refs.

  16. Impurity penetration and transport during VH-mode on DIII-D

    International Nuclear Information System (INIS)

    Lippmann, S.I.; Evans, T.E.; Jackson, G.L.; West, W.P.

    1992-05-01

    A new modeling effort is made in order to understand the observed relatively low levels of impurity contamination during the VH-mode phase on DIII-D, as compared to those observed during the H-mode phase of selected discharges. The key element is the inclusion of the real 2-D flux surface geometry in the prediction of impurity penetration of sputtered atoms through the scrape-off layer into the core plasma. Of the elements which determine the impurity content in the plasma: sputtering yield, penetration, and core transport, the penetration through the scrape-off layer is found to be the most determinative factor. The low impurity content in VH-mode is attributed to the development of a scrape-off layer with higher density and temperature properties than those normally obtained in H-mode

  17. Rapid screening for nuclear genes mutations in isolated respiratory chain complex I defects.

    Science.gov (United States)

    Pagniez-Mammeri, Hélène; Lombes, Anne; Brivet, Michèle; Ogier-de Baulny, Hélène; Landrieu, Pierre; Legrand, Alain; Slama, Abdelhamid

    2009-04-01

    Complex I or reduced nicotinamide adenine dinucleotide (NADH): ubiquinone oxydoreductase deficiency is the most common cause of respiratory chain defects. Molecular bases of complex I deficiencies are rarely identified because of the dual genetic origin of this multi-enzymatic complex (nuclear DNA and mitochondrial DNA) and the lack of phenotype-genotype correlation. We used a rapid method to screen patients with isolated complex I deficiencies for nuclear genes mutations by Surveyor nuclease digestion of cDNAs. Eight complex I nuclear genes, among the most frequently mutated (NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1 and NDUFV2), were studied in 22 cDNA fragments spanning their coding sequences in 8 patients with a biochemically proved complex I deficiency. Single nucleotide polymorphisms and missense mutations were detected in 18.7% of the cDNA fragments by Surveyor nuclease treatment. Molecular defects were detected in 3 patients. Surveyor nuclease screening is a reliable method for genotyping nuclear complex I deficiencies, easy to interpret, and limits the number of sequence reactions. Its use will enhance the possibility of prenatal diagnosis and help us for a better understanding of complex I molecular defects.

  18. Poly(Amido Amine)s Containing Agmatine and Butanol Side Chains as Efficient Gene Carriers.

    Science.gov (United States)

    Won, Young-Wook; Ankoné, Marc; Engbersen, Johan F J; Feijen, Jan; Kim, Sung Wan

    2016-04-01

    A new type of bioreducible poly(amido amine) copolymer is synthesized by the Michael addition polymerization of cystamine bisacrylamide (CBA) with 4-aminobutylguanidine (agmatine, AGM) and 4-aminobutanol (ABOL). Since the positively charged guanidinium groups of AGM and the hydroxybutyl groups of ABOL in the side chains have shown to improve the overall transfection efficiency of poly(amido amine)s, it is hypothesized that poly(CBA-ABOL/AGM) synthesized at the optimal ratio of both components would result in high transfection efficiency and minimal toxicity. In this study, a series of the poly(CBA-ABOL/AGM) copolymers is synthesized as gene carriers. The polymers are characterized and luciferase transfection efficiencies of the polymers in various cell lines are investigated to select the ideal ratio between AGM and ABOL. The poly(CBA-ABOL/AGM) containing 80% AGM and 20% ABOL has shown the best transfection efficiency with the lowest cytotoxicity, indicating that this polymer is very promising as a potent and nontoxic gene carrier. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Detection of canine cytokine gene expression by reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Pinelli, E; van der Kaaij, S Y; Slappendel, R; Fragio, C; Ruitenberg, E J; Bernadina, W; Rutten, V P

    1999-08-02

    Further characterization of the canine immune system will greatly benefit from the availability of tools to detect canine cytokines. Our interest concerns the study on the role of cytokines in canine visceral leishmaniasis. For this purpose, we have designed specific primers using previously published sequences for the detection of canine IL-2, IFN-gamma and IL10 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). For IL-4, we have cloned and sequenced this cytokine gene, and developed canine-specific primers. To control for sample-to-sample variation in the quantity of mRNA and variation in the RT and PCR reactions, the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a housekeeping gene, were determined in parallel. Primers to amplify G3PDH were designed from consensus sequences obtained from the Genbank database. The mRNA levels of the cytokines mentioned here were detected from ConA-stimulated peripheral mononuclear cells derived from Leishmania-infected dogs. A different pattern of cytokine production among infected animals was found.

  20. Histidine at Position 195 is Essential for Association of Heme-b in Lcp1VH2

    Science.gov (United States)

    Oetermann, Sylvia; Vivod, Robin; Hiessl, Sebastian; Hogeback, Jens; Holtkamp, Michael; Karst, Uwe; Steinbüchel, Alexander

    2018-03-01

    The latex clearing protein (Lcp) is the key enzyme of polyisoprene degradation in actinomycetes (Yikmis and Steinbüchel in Appl Environ Microbiol 78:4543-4551, https://doi.org/10.1128/AEM.00001-12, 2012). In this study it was shown that Lcp from Gordonia polyisoprenivorans VH2 (Lcp1VH2) harbors a non-covalently bound heme b as cofactor, which was identified by pyridine hemochrome spectra and confirmed by LC/ESI-ToF-MS. It contains iron, most likely in the Fe3+ state. We focused on the characterization of the heme-cofactor, its accessibility with respect to the conformation of Lcp1VH2, and the identification of putative histidine residues involved in the coordination of heme. A change was detectable in UV/Vis-spectra of reduced Lcp1VH2 when imidazole was added, showing that Lcp1VH2 "as isolated" occurs in an open state, directly being accessible for external ligands. In addition, three highly conserved histidines (H195, H200 and H228), presumably acting as ligands coordinating the heme within the heme pocket, were replaced with alanines by site-directed mutagenesis. The effect of these changes on in vivo rubber-mineralization was investigated. The lcp- deletion mutant complemented with the H195A variant of lcp1 VH2 was unable to mineralize poly(cis-1,4-isoprene). In vitro analyses of purified, recombinant Lcp1VH2H195A confirmed the loss of enzyme activity, which could be ascribed to the loss of heme. Hence, H195 is essential for the association of heme-b in the central region of Lcp1VH2.

  1. Detection of clonal T-cell receptor beta and gamma chain gene rearrangement by polymerase chain reaction and capillary gel electrophoresis.

    Science.gov (United States)

    Fan, Hongxin; Robetorye, Ryan S

    2013-01-01

    Although established diagnostic criteria exist for mature T-cell neoplasms, a definitive diagnosis of a T-cell lymphoproliferative disorder cannot always be obtained using more conventional techniques such as flow cytometric immunophenotyping, conventional cytogenetics, fluorescence in situ hybridization, or immunohistochemistry. However, because T-cell malignancies contain identically rearranged T-cell receptor gamma (TCRG) and/or beta (TCRB) genes, the polymerase chain reaction (PCR) can be a fast, convenient, and dependable option to identify clonal T-cell processes. This chapter describes the use of PCR and capillary electrophoresis to identify clonal TCRB and TCRG gene rearrangements (TCRB and TCRG PCR) using a commercially available method employing multiple multiplex PCR tubes that was originally developed as the result of a large European BIOMED-2 collaborative study (Invivoscribe Technologies). The core protocol for the TCRB assay involves the use of three separate multiplex master mix tubes. Tubes A and B target the framework regions within the variable and joining regions of the TCRB gene, and Tube C targets the diversity and joining regions of the TCRB gene. The core protocol for the TCRG assay utilizes two multiplex master mix tubes (Tubes A and B) that target the variable and joining regions of the TCRG gene. Use of the five BIOMED-2 TCRB and TCRG PCR multiplex tubes in parallel can detect approximately 94% of clonal TCR gene rearrangements.

  2. Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors.

    Directory of Open Access Journals (Sweden)

    Radhika Thokala

    Full Text Available Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML. CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL, and has been an effective target for T cells expressing chimeric antigen receptors (CARs. The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb, coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR's in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies.

  3. Isolation of salt stress gene(s) from some haloterant streptomyces strains using polymerase chain reaction (abstract)

    International Nuclear Information System (INIS)

    Mohammad, S.H.

    2005-01-01

    We studied salt tolerance range in sixteen halotolerant streptomyces strains to isolate salt regulated genes using polymerase chain reaction (PCR) technology. A group of these strains was isolated from Sedi-creer (S. niveus Sc-2 and S. sendenensis Sc-II); El-Malahat (Alexndria) (S. graminofaciens Ma-13): Qaroon's lake (S. albovinaceus QA-44, S. luteofluorescens Qa-51, S. albidoflavous Qa-53 and S. erthaeus QA-84). The other group represents the strains isolated from different soils from Damaaita (S. violans Da-3). Ismailia (S. alboflavus-Is-10). Port said (S. bobili Ps-12) and Sinai sandy soil (streptomyces species Si-1, S. truirus Si-4, S. lateritius Si-6, S. hawaiiensis Si-8, S. muavecolor Si-9 and S. melanogenes Si-11). These strains were varied in their salt tolerance range in particular, with increasing NaCl concentration in the growth medium up to 14%. It was also noted that all the applied Streptomyces strains appeared abundant growth at NaCl concentrations of 0.05, 3.5 and 7.0%. When NaCl was added at concentration of 10.5%, all of them except S. melanogenes Si-II strain gave moderate growth. On the contrary, NaCl at concentration of 14% inhibited the growth of 50% of strains under investigation. But the other 50% of these strains gave moderate growth at the same NaCl concentration. At the molecular level, the PCR was successfully used for isolating the mtlD and P5CS genes from 3 (S. alboinaceus Qa-44, S. albidoflavus Qa-53, S. erthraeus QA-84) and 4 (S. albovunaecaus Qa-44, Streptomyces species Si-I, S. luteofluorescens Qa-51, S. latritius Si-6) strains, respectively. As PCR fragments with a size of about 1095 and 2100 bp were amplified from the DNA genome of these strains using the primer pairs (P1 and P2) and (P3 and P4), respectively. These results confirmed the ability to use PCR for isolation or detection of any gene based on its nucleotide sequencing in any microorganism. Furthermore, one can recommended the use of the applied halotolerant

  4. Association mapping of starch chain length distribution and amylose content in pea (Pisum sativum L.) using carbohydrate metabolism candidate genes.

    Science.gov (United States)

    Carpenter, Margaret A; Shaw, Martin; Cooper, Rebecca D; Frew, Tonya J; Butler, Ruth C; Murray, Sarah R; Moya, Leire; Coyne, Clarice J; Timmerman-Vaughan, Gail M

    2017-08-01

    Although starch consists of large macromolecules composed of glucose units linked by α-1,4-glycosidic linkages with α-1,6-glycosidic branchpoints, variation in starch structural and functional properties is found both within and between species. Interest in starch genetics is based on the importance of starch in food and industrial processes, with the potential of genetics to provide novel starches. The starch metabolic pathway is complex but has been characterized in diverse plant species, including pea. To understand how allelic variation in the pea starch metabolic pathway affects starch structure and percent amylose, partial sequences of 25 candidate genes were characterized for polymorphisms using a panel of 92 diverse pea lines. Variation in the percent amylose composition of extracted seed starch and (amylopectin) chain length distribution, one measure of starch structure, were characterized for these lines. Association mapping was undertaken to identify polymorphisms associated with the variation in starch chain length distribution and percent amylose, using a mixed linear model that incorporated population structure and kinship. Associations were found for polymorphisms in seven candidate genes plus Mendel's r locus (which conditions the round versus wrinkled seed phenotype). The genes with associated polymorphisms are involved in the substrate supply, chain elongation and branching stages of the pea carbohydrate and starch metabolic pathways. The association of polymorphisms in carbohydrate and starch metabolic genes with variation in amylopectin chain length distribution and percent amylose may help to guide manipulation of pea seed starch structural and functional properties through plant breeding.

  5. Physical linkage of a human immunoglobulin heavy chain variable region gene segment to diversity and joining region elements

    International Nuclear Information System (INIS)

    Schroeder, H.W. Jr.; Walter, M.A.; Hofker, M.H.; Ebens, A.; Van Dijk, K.W.; Liao, L.C.; Cox, D.W.; Milner, E.C.B.; Perlmutter, R.M.

    1988-01-01

    Antibody genes are assembled from a series of germ-line gene segments that are juxtaposed during the maturation of B lymphocytes. Although diversification of the adult antibody repertoire results in large part from the combinatorial joining of these gene segments, a restricted set of antibody heavy chain variable (V H ), diversity (D H ), and joining (J H ) region gene segments appears preferentially in the human fetal repertoire. The authors report here that one of these early-expressed V H elements (termed V H 6) is the most 3' V H gene segment, positioned 77 kilobases on the 5' side of the J H locus and immediately adjacent to a set of previously described D H sequences. In addition to providing a physical map linking human V H , D H , and J H elements, these results support the view that the programmed development of the antibody V H repertoire is determined in part by the chromosomal position of these gene segments

  6. Diversity analysis of the immunoglobulin M heavy chain gene in Nile ...

    African Journals Online (AJOL)

    nu tom

    2015-07-22

    Jul 22, 2015 ... related industries and supply chains, such as hatcheries, feed manufacturers ... system has a high risk of disease outbreaks. The bulk of ...... immunoglobulin heavy-chain locus in zebrafish: identification and expression of a ...

  7. Evaluation of reference gene suitability for quantitative expression analysis by quantitative polymerase chain reaction in the mandibular condyle of sheep.

    Science.gov (United States)

    Jiang, Xin; Xue, Yang; Zhou, Hongzhi; Li, Shouhong; Zhang, Zongmin; Hou, Rui; Ding, Yuxiang; Hu, Kaijin

    2015-10-01

    Reference genes are commonly used as a reliable approach to normalize the results of quantitative polymerase chain reaction (qPCR), and to reduce errors in the relative quantification of gene expression. Suitable reference genes belonging to numerous functional classes have been identified for various types of species and tissue. However, little is currently known regarding the most suitable reference genes for bone, specifically for the sheep mandibular condyle. Sheep are important for the study of human bone diseases, particularly for temporomandibular diseases. The present study aimed to identify a set of reference genes suitable for the normalization of qPCR data from the mandibular condyle of sheep. A total of 12 reference genes belonging to various functional classes were selected, and the expression stability of the reference genes was determined in both the normal and fractured area of the sheep mandibular condyle. RefFinder, which integrates the following currently available computational algorithms: geNorm, NormFinder, BestKeeper, and the comparative ΔCt method, was used to compare and rank the candidate reference genes. The results obtained from the four methods demonstrated a similar trend: RPL19, ACTB, and PGK1 were the most stably expressed reference genes in the sheep mandibular condyle. As determined by RefFinder comprehensive analysis, the results of the present study suggested that RPL19 is the most suitable reference gene for studies associated with the sheep mandibular condyle. In addition, ACTB and PGK1 may be considered suitable alternatives.

  8. Selection of suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using quantitative real-time polymerase chain reaction.

    Science.gov (United States)

    Zornhagen, K W; Kristensen, A T; Hansen, A E; Oxboel, J; Kjaer, A

    2015-12-01

    Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a sensitive technique for quantifying gene expression. Stably expressed reference genes are necessary for normalization of RT-qPCR data. Only a few articles have been published on reference genes in canine tumours. The objective of this study was to demonstrate how to identify suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using RT-qPCR. Primer pairs for 17 potential reference genes were designed and tested in archival tumour biopsies from six dogs. The geNorm algorithm was used to analyse the most suitable reference genes. Eight potential reference genes were excluded from this final analysis because of their dissociation curves. β-Glucuronidase (GUSB) and proteasome subunit, beta type, 6 (PSMB6) were most stably expressed with an M value of 0.154 and a CV of 0.053 describing their average stability. We suggest that choice of reference genes should be based on specific testing in every new experimental set-up. © 2014 John Wiley & Sons Ltd.

  9. Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle

    Science.gov (United States)

    Baldwin, K. M.; Haddad, F.

    2001-01-01

    The goal of this mini-review is to summarize findings concerning the role that different models of muscular activity and inactivity play in altering gene expression of the myosin heavy chain (MHC) family of motor proteins in mammalian cardiac and skeletal muscle. This was done in the context of examining parallel findings concerning the role that thyroid hormone (T(3), 3,5,3'-triiodothyronine) plays in MHC expression. Findings show that both cardiac and skeletal muscles of experimental animals are initially undifferentiated at birth and then undergo a marked level of growth and differentiation in attaining the adult MHC phenotype in a T(3)/activity level-dependent fashion. Cardiac MHC expression in small mammals is highly sensitive to thyroid deficiency, diabetes, energy deprivation, and hypertension; each of these interventions induces upregulation of the beta-MHC isoform, which functions to economize circulatory function in the face of altered energy demand. In skeletal muscle, hyperthyroidism, as well as interventions that unload or reduce the weight-bearing activity of the muscle, causes slow to fast MHC conversions. Fast to slow conversions, however, are seen under hypothyroidism or when the muscles either become chronically overloaded or subjected to intermittent loading as occurs during resistance training and endurance exercise. The regulation of MHC gene expression by T(3) or mechanical stimuli appears to be strongly regulated by transcriptional events, based on recent findings on transgenic models and animals transfected with promoter-reporter constructs. However, the mechanisms by which T(3) and mechanical stimuli exert their control on transcriptional processes appear to be different. Additional findings show that individual skeletal muscle fibers have the genetic machinery to express simultaneously all of the adult MHCs, e.g., slow type I and fast IIa, IIx, and IIb, in unique combinations under certain experimental conditions. This degree of

  10. Detection of Chloramphenicol Resistance Genes (cat in Clinical Isolates of Pseudomonas aeruginosa with Polymerase Chain Reaction Method

    Directory of Open Access Journals (Sweden)

    Tiana Milanda

    2014-12-01

    Full Text Available Pseudomonas aeruginosa is an opportunistic Gram negative bacteria, which may cause infection in eyes, ears, skin, bones, central nervous system, gastrointestinal tract, circulatory system, heart, respiratory system, and urinary tract. Recently, chloramphenicol is no longer used as the main option of the therapy due of its resistance case. The aim of this research was to detect the presence of gene which is responsible to chloramphenicol resistance in clinical isolates of P.aeruginosa. These bacteria isolated from pus of external otitis patients in Hasan Sadikin Hospital in Bandung City. Polymerase Chain Reaction (PCR method (colony-PCR and DNA-PCR were performed to detect this resistance gene. Electropherogram from PCR products showed that the chloramphenicol resistance in clinical isolates of P. aeruginosa was caused by cat gene (317 bp. Based on this research, cat gene may be used to detect the chloramphenicol resistance in patients with external ostitis.

  11. Knocking out the MFE-2 gene of Candida bombicola leads to improved medium-chain sophorolipid production.

    Science.gov (United States)

    Van Bogaert, Inge N A; Sabirova, Julia; Develter, Dirk; Soetaert, Wim; Vandamme, Erick J

    2009-06-01

    The nonpathogenic yeast Candida bombicola synthesizes sophorolipids. These biosurfactants are composed of the disaccharide sophorose linked to a long-chain hydroxy fatty acid and have potential applications in the food, pharmaceutical, cosmetic and cleaning industries. In order to expand the range of application, a shift of the fatty acid moiety towards medium-chain lengths would be recommendable. However, the synthesis of medium-chain sophorolipids by C. bombicola is a challenging objective. First of all, these sophorolipids can only be obtained by fermentations on unconventional carbon sources, which often have a toxic effect on the cells. Furthermore, medium-chain substrates are partially metabolized in the beta-oxidation pathway. In order to redirect unconventional substrates towards sophorolipid synthesis, the beta-oxidation pathway was blocked on the genome level by knocking out the multifunctional enzyme type 2 (MFE-2) gene. The total gene sequence of the C. bombicola MFE-2 (6033 bp) was cloned (GenBank accession number EU371724), and the obtained nucleotide sequence was used to construct a knock-out cassette. Several knock-out mutants with the correct geno- and phenotype were evaluated in a fermentation on 1-dodecanol. All mutants showed a 1.7-2.9 times higher production of sophorolipids, indicating that in those strains the substrate is redirected towards the sophorolipid synthesis.

  12. Detection of Brazilian hantavirus by reverse transcription polymerase chain reaction amplification of N gene in patients with hantavirus cardiopulmonary syndrome

    OpenAIRE

    Marcos Lázaro Moreli; Ricardo Luiz Moro de Sousa; Luiz Tadeu Moraes Figueiredo

    2004-01-01

    We report a nested reverse transcription-polymerase chain reaction (RT-PCR) assay for hantavirus using primers selected to match high homology regions of hantavirus genomes detected from the whole blood of hantavirus cardiopulmonary syndrome (HCPS) patients from Brazil, also including the N gene nucleotide sequence of Araraquara virus. Hantavirus genomes were detected in eight out of nine blood samples from the HCPS patients by RT-PCR (88.9% positivity) and in all 9 blood samples (100% positi...

  13. SNPs within the beta myosin heavy chain (MYH7 and the pyruvate kinase muscle (PKM2 genes in horse

    Directory of Open Access Journals (Sweden)

    Vincenzo Russo

    2010-01-01

    Full Text Available Two highly expressed skeletal muscle genes (the MYH7 gene encoding the myosin heavy chain slow/β-cardiac isoform and the PKM2 gene encoding the pyruvate kinase muscle isoforms were investigated with the objective to identify DNA markers in horses. A panel of DNA samples from different horse breeds was analysed using a PCR-single strand conformation polymorphism (SSCP approach. Four and two alleles were identified for the MYH7 and PKM2 loci, respectively. Mendelian inheritance of alleles of the two investigated genes was confirmed analysing horse families. Sequencing of PCR products obtained from the MYH7 and PKM2 genes made it possible to characterise two SSCP alleles for each gene. The polymorphisms found in the MYH7 and PKM2 genes were further studied in 61 and 68 horses of three (Italian Heavy Draught Horse, Italian Saddler and Murgese and five (Franches-Montagnes, Haflinger, Italian Heavy Draught Horse, Murgese and Standardbred breeds, respectively. Allele frequencies of the two loci varied among the considered breeds. The SNPs discovery in MYH7 and PKM2 genes makes it possible to locate new molecular markers to ECA1. The identified markers could be used in association analysis with performance traits in horses.

  14. The Natural Product Osthole Attenuates Yeast Growth by Extensively Suppressing the Gene Expressions of Mitochondrial Respiration Chain.

    Science.gov (United States)

    Wang, Zhe; Shen, Yan

    2017-03-01

    The fast growing evidences have indicated that the natural product osthole is a promising drug candidate for fighting several serious human diseases, for example, cancer and inflammation. However, the mode-of-action (MoA) of osthole remains largely incomplete. In this study, we investigated the growth inhibition activity of osthole using fission yeast as a model, with the goal of understanding the osthole's mechanism of action, especially from the molecular level. Microarray analysis indicated that osthole has significant impacts on gene transcription levels (In total, 214 genes are up-regulated, and 97 genes are down-regulated). Gene set enrichment analysis (GSEA) indicated that 11 genes belong to the "Respiration module" category, especially including the components of complex III and V of mitochondrial respiration chain. Based on GSEA and network analysis, we also found that 54 up-regulated genes belong to the "Core Environmental Stress Responses" category, particularly including many transporter genes, which suggests that the rapidly activated nutrient exchange between cell and environment is part of the MoA of osthole. In summary, osthole can greatly impact on fission yeast transcriptome, and it primarily represses the expression levels of the genes in respiration chain, which next causes the inefficiency of ATP production and thus largely explains osthole's growth inhibition activity in Schizosaccharomyces pombe (S. pombe). The complexity of the osthole's MoA shown in previous studies and our current research demonstrates that the omics approach and bioinformatics tools should be applied together to acquire the complete landscape of osthole's growth inhibition activity.

  15. Simplification of Markov chains with infinite state space and the mathematical theory of random gene expression bursts

    Science.gov (United States)

    Jia, Chen

    2017-09-01

    Here we develop an effective approach to simplify two-time-scale Markov chains with infinite state spaces by removal of states with fast leaving rates, which improves the simplification method of finite Markov chains. We introduce the concept of fast transition paths and show that the effective transitions of the reduced chain can be represented as the superposition of the direct transitions and the indirect transitions via all the fast transition paths. Furthermore, we apply our simplification approach to the standard Markov model of single-cell stochastic gene expression and provide a mathematical theory of random gene expression bursts. We give the precise mathematical conditions for the bursting kinetics of both mRNAs and proteins. It turns out that random bursts exactly correspond to the fast transition paths of the Markov model. This helps us gain a better understanding of the physics behind the bursting kinetics as an emergent behavior from the fundamental multiscale biochemical reaction kinetics of stochastic gene expression.

  16. Well-defined block copolymers for gene delivery to dendritic cells: probing the effect of polycation chain-length.

    Science.gov (United States)

    Tang, Rupei; Palumbo, R Noelle; Nagarajan, Lakshmi; Krogstad, Emily; Wang, Chun

    2010-03-03

    The development of safe and efficient polymer carriers for DNA vaccine delivery requires mechanistic understanding of structure-function relationship of the polymer carriers and their interaction with antigen-presenting cells. Here we have synthesized a series of diblock copolymers with well-defined chain-length using atom transfer radical polymerization and characterized the influence of polycation chain-length on the physico-chemical properties of the polymer/DNA complexes as well as the interaction with dendritic cells. The copolymers consist of a hydrophilic poly(ethylene glycol) block and a cationic poly(aminoethyl methacrylate) (PAEM) block. The average degree of polymerization (DP) of the PAEM block was varied among 19, 39, and 75, with nearly uniform distribution. With increasing PAEM chain-length, polyplexes formed by the diblock copolymers and plasmid DNA had smaller average particle size and showed higher stability against electrostatic destabilization by salt and heparin. The polymers were not toxic to mouse dendritic cells (DCs) and only displayed chain-length-dependent toxicity at a high concentration (1mg/mL). In vitro gene transfection efficiency and polyplex uptake in DCs were also found to correlate with chain-length of the PAEM block with the longer polymer chain favoring transfection and cellular uptake. The polyplexes induced a modest up-regulation of surface markers for DC maturation that was not significantly dependent on PAEM chain-length. Finally, the polyplex prepared from the longest PAEM block (DP of 75) achieved an average of 20% enhancement over non-condensed anionic dextran in terms of uptake by DCs in the draining lymph nodes 24h after subcutaneous injection into mice. Insights gained from studying such structurally well-defined polymer carriers and their interaction with dendritic cells may contribute to improved design of practically useful DNA vaccine delivery systems. Copyright 2009 Elsevier B.V. All rights reserved.

  17. Immunoglobulin heavy chain exclusion in the shark.

    Directory of Open Access Journals (Sweden)

    Karolina Malecek

    2008-06-01

    Full Text Available The adaptive immune system depends on specific antigen receptors, immunoglobulins (Ig in B lymphocytes and T cell receptors (TCR in T lymphocytes. Adaptive responses to immune challenge are based on the expression of a single species of antigen receptor per cell; and in B cells, this is mediated in part by allelic exclusion at the Ig heavy (H chain locus. How allelic exclusion is regulated is unclear; we considered that sharks, the oldest vertebrates possessing the Ig/TCR-based immune system, would yield insights not previously approachable and reveal the primordial basis of the regulation of allelic exclusion. Sharks have an IgH locus organization consisting of 15-200 independently rearranging miniloci (VH-D1-D2-JH-Cmu, a gene organization that is considered ancestral to the tetrapod and bony fish IgH locus. We found that rearrangement takes place only within a minilocus, and the recombining gene segments are assembled simultaneously and randomly. Only one or few H chain genes were fully rearranged in each shark B cell, whereas the other loci retained their germline configuration. In contrast, most IgH were partially rearranged in every thymocyte (developing T cell examined, but no IgH transcripts were detected. The distinction between B and T cells in their IgH configurations and transcription reveals a heretofore unsuspected chromatin state permissive for rearrangement in precursor lymphocytes, and suggests that controlled limitation of B cell lineage-specific factors mediate regulated rearrangement and allelic exclusion. This regulation may be shared by higher vertebrates in which additional mechanistic and regulatory elements have evolved with their structurally complex IgH locus.

  18. The normally expressed kappa immunoglobulin light chain gene repertoire and somatic mutations studied by single-sided specific polymerase chain reaction (PCR); frequent occurrence of features often assigned to autoimmunity

    DEFF Research Database (Denmark)

    Juul, L; Hougs, L; Andersen, V

    1997-01-01

    The expressed human kappa light chain gene repertoire utilized by healthy individuals was studied by two different single-sided specific PCR techniques to avoid bias for certain V genes. A total of 103 rearranged kappa sequences from peripheral blood mononuclear cells from healthy individuals were...

  19. Metabolism of Very Long-Chain Fatty Acids: Genes and Pathophysiology

    OpenAIRE

    Sassa, Takayuki; Kihara, Akio

    2014-01-01

    Fatty acids (FAs) are highly diverse in terms of carbon (C) chain-length and number of double bonds. FAs with C>20 are called very long-chain fatty acids (VLCFAs). VLCFAs are found not only as constituents of cellular lipids such as sphingolipids and glycerophospholipids but also as precursors of lipid mediators. Our understanding on the function of VLCFAs is growing in parallel with the identification of enzymes involved in VLCFA synthesis or degradation. A variety of inherited diseases, suc...

  20. A Select Subset of Electron Transport Chain Genes Associated with Optic Atrophy Link Mitochondria to Axon Regeneration in Caenorhabditis elegans.

    Science.gov (United States)

    Knowlton, Wendy M; Hubert, Thomas; Wu, Zilu; Chisholm, Andrew D; Jin, Yishi

    2017-01-01

    The role of mitochondria within injured neurons is an area of active interest since these organelles are vital for the production of cellular energy in the form of ATP. Using mechanosensory neurons of the nematode Caenorhabditis elegans to test regeneration after neuronal injury in vivo , we surveyed genes related to mitochondrial function for effects on axon regrowth after laser axotomy. Genes involved in mitochondrial transport, calcium uptake, mitophagy, or fission and fusion were largely dispensable for axon regrowth, with the exception of eat-3/Opa1 . Surprisingly, many genes encoding components of the electron transport chain were dispensable for regrowth, except for the iron-sulfur proteins gas-1, nduf-2.2, nduf-7 , and isp-1 , and the putative oxidoreductase rad-8 . In these mutants, axonal development was essentially normal and axons responded normally to injury by forming regenerative growth cones, but were impaired in subsequent axon extension. Overexpression of nduf-2.2 or isp-1 was sufficient to enhance regrowth, suggesting that mitochondrial function is rate-limiting in axon regeneration. Moreover, loss of function in isp-1 reduced the enhanced regeneration caused by either a gain-of-function mutation in the calcium channel EGL-19 or overexpression of the MAP kinase DLK-1. While the cellular function of RAD-8 remains unclear, our genetic analyses place rad-8 in the same pathway as other electron transport genes in axon regeneration. Unexpectedly, rad-8 regrowth defects were suppressed by altered function in the ubiquinone biosynthesis gene clk-1 . Furthermore, we found that inhibition of the mitochondrial unfolded protein response via deletion of atfs-1 suppressed the defective regrowth in nduf-2.2 mutants. Together, our data indicate that while axon regeneration is not significantly affected by general dysfunction of cellular respiration, it is sensitive to the proper functioning of a select subset of electron transport chain genes, or to the

  1. ATLAS searches for VH, HH, VV, V+$\\gamma$/$\\gamma\\gamma$ resonances

    CERN Document Server

    AUTHOR|(INSPIRE)INSPIRE-00441490; The ATLAS collaboration

    2017-01-01

    The discovery of a Higgs boson at the Large Hadron Collider motivates searches for physics beyond the Standard Model in channels involving coupling to the Higgs boson. A search for massive resonances decaying into couples of bosons is described. The considered final states are: $HH$, $VH$, $VV$, $V\\gamma$ and $\\gamma\\gamma$ with $V$ indicating either the $W$ or the $Z$ boson. Final states with different number of leptons or photons and where, in many cases, at least one Higgs decays into a b-quark pair are studied using different jet reconstruction techniques which allow to optimize the signal acceptance for low or high Higgs boson transverse momentum. The most recent diboson resonance searches using LHC Run 2 data are described.

  2. Collaborative ring trial of the papaya endogenous reference gene and its polymerase chain reaction assays for genetically modified organism analysis.

    Science.gov (United States)

    Wei, Jiaojun; Li, Feiwu; Guo, Jinchao; Li, Xiang; Xu, Junfeng; Wu, Gang; Zhang, Dabing; Yang, Litao

    2013-11-27

    The papaya (Carica papaya L.) Chymopapain (CHY) gene has been reported as a suitable endogenous reference gene for genetically modified (GM) papaya detection in previous studies. Herein, we further validated the use of the CHY gene and its qualitative and quantitative polymerase chain reaction (PCR) assays through an interlaboratory collaborative ring trial. A total of 12 laboratories working on detection of genetically modified organisms participated in the ring trial and returned test results. Statistical analysis of the returned results confirmed the species specificity, low heterogeneity, and single-copy number of the CHY gene among different papaya varieties. The limit of detection of the CHY qualitative PCR assay was 0.1%, while the limit of quantification of the quantitative PCR assay was ∼25 copies of haploid papaya genome with acceptable PCR efficiency and linearity. The differences between the tested and true values of papaya content in 10 blind samples ranged from 0.84 to 6.58%. These results indicated that the CHY gene was suitable as an endogenous reference gene for the identification and quantification of GM papaya.

  3. Disruption of KEX1 gene reduces the proteolytic degradation of secreted two-chain Insulin glargine in Pichia pastoris.

    Science.gov (United States)

    Sreenivas, Suma; Krishnaiah, Sateesh M; Shyam Mohan, Anil H; Mallikarjun, Niveditha; Govindappa, Nagaraja; Chatterjee, Amarnath; Sastry, Kedarnath N

    2016-02-01

    Insulin glargine is a slow acting analog of insulin used in diabetes therapy. It is produced by recombinant DNA technology in different hosts namely E. coli and Pichia pastoris. In our previous study, we have described the secretion of fully folded two-chain Insulin glargine into the medium by over-expression of Kex2 protease. The enhanced levels of the Kex2 protease was responsible for the processing of the glargine precursor with in the host. Apart from the two-chain glargine product we observed a small proportion of arginine clipped species. This might be due to the clipping of arginine present at the C-terminus of the B-chain as it is exposed upon Kex2 cleavage. The carboxypeptidase precursor Kex1 is known to be responsible for clipping of C-terminal lysine or arginine of the proteins or peptides. In order to address this issue we created a Kex1 knock out in the host using Cre/loxP mechanism of targeted gene deletion. When two-chain glargine was expressed in the Kex1 knock out host of P. pastoris GS115 the C-terminal clipped species reduced by ∼80%. This modification further improved the process by reducing the levels of product related impurities. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Structural organization of the human and mouse laminin beta2 chain genes, and alternative splicing at the 5' end of the human transcript

    DEFF Research Database (Denmark)

    Durkin, M E; Gautam, M; Loechel, F

    1996-01-01

    We have determined the structural organization of the human and mouse genes that encode the laminin beta2 chain (s-laminin), an essential component of the basement membranes of the neuromuscular synapse and the kidney glomerulus. The human and mouse genes have a nearly identical exon-intron organ......We have determined the structural organization of the human and mouse genes that encode the laminin beta2 chain (s-laminin), an essential component of the basement membranes of the neuromuscular synapse and the kidney glomerulus. The human and mouse genes have a nearly identical exon...

  5. [Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant III single chain antibody].

    Science.gov (United States)

    Han, Dong-gang; Duan, Xiao-yi; Guo, You-min; Zhou, Qi; Wang, Quan-ying; Yang, Guang-xiao

    2010-01-01

    To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system. The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody. The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII. An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.

  6. Draft Genome Sequence of Vibrio parahaemolyticus VH3, Isolated from an Aquaculture Environment in Greece

    DEFF Research Database (Denmark)

    Castillo, Daniel; Jun, Jin Woo; D'Alvise, Paul

    2015-01-01

    Vibrio parahaemolyticus is an important foodborne pathogen responsible for gastroenteritis outbreaks globally. It has also been identified as an important pathogen in aquatic organisms. Here, we report a draft genome sequence of V. parahaemolyticus, strain VH3, isolated from farmed juvenile greater...

  7. A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription.

    Science.gov (United States)

    Zhou, Xin X; Zou, Xinzhi; Chung, Hokyung K; Gao, Yuchen; Liu, Yanxia; Qi, Lei S; Lin, Michael Z

    2018-02-16

    Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

  8. Low-intensity laser irradiation at 660 nm stimulates transcription of genes involved in the electron transport chain.

    Science.gov (United States)

    Masha, Roland T; Houreld, Nicolette N; Abrahamse, Heidi

    2013-02-01

    Low-intensity laser irradiation (LILI) has been shown to stimulate cellular functions leading to increased adenosine triphosphate (ATP) synthesis. This study was undertaken to evaluate the effect of LILI on genes involved in the mitochondrial electron transport chain (ETC, complexes I-IV) and oxidative phosphorylation (ATP synthase). Four human skin fibroblast cell models were used in this study: normal non-irradiated cells were used as controls while wounded, diabetic wounded, and ischemic cells were irradiated. Cells were irradiated with a 660 nm diode laser with a fluence of 5 J/cm(2) and gene expression determined by quantitative real-time reverse transcription (RT) polymerase chain reaction (PCR). LILI upregulated cytochrome c oxidase subunit VIb polypeptide 2 (COX6B2), cytochrome c oxidase subunit VIc (COX6C), and pyrophosphatase (inorganic) 1 (PPA1) in diabetic wounded cells; COX6C, ATP synthase, H+transporting, mitochondrial Fo complex, subunit B1 (ATP5F1), nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) 1 alpha subcomplex, 11 (NDUFA11), and NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) in wounded cells; and ATPase, H+/K+ exchanging, beta polypeptide (ATP4B), and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C2 (subunit 9) (ATP5G2) in ischemic cells. LILI at 660 nm stimulates the upregulation of genes coding for subunits of enzymes involved in complexes I and IV and ATP synthase.

  9. Fundamental characteristics of the expressed immunoglobulin VH and VL repertoire in different canine breeds in comparison with those of humans and mice.

    Science.gov (United States)

    Steiniger, Sebastian C J; Dunkle, William E; Bammert, Gary F; Wilson, Thomas L; Krishnan, Abhiram; Dunham, Steven A; Ippolito, Gregory C; Bainbridge, Graeme

    2014-05-01

    Complementarity determining regions (CDR) are responsible for binding antigen and provide substantial diversity to the antibody repertoire, with VH CDR3 of the immunoglobulin variable heavy (VH) domain playing a dominant role. In this study, we examined 1200 unique canine VH and 500 unique variable light (VL) sequences of large and small canine breeds derived from peripheral B cells. Unlike the human and murine repertoire, the canine repertoire is heavily dominated by the Canis lupus familiaris IGHV1 subgroup, evolutionarily closest to the human IGHV3 subgroup. Our studies clearly show that the productive canine repertoire of all analyzed breeds shows similarities to both human and mouse; however, there are distinct differences in terms of VH CDR3 length and amino acid paratope composition. In comparison with the human and murine antibody repertoire, canine VH CDR3 regions are shorter in length than the human counterparts, but longer than the murine VH CDR3. Similar to corresponding human and mouse VH CDR3, the amino acids at the base of the VH CDR3 loop are strictly conserved. For identical CDR positions, there were significant changes in chemical paratope composition. Similar to human and mouse repertoires, the neutral amino acids tyrosine, glycine and serine dominate the canine VH CDR3 interval (comprising 35%) although the interval is nonetheless relatively depleted of tyrosine when compared to human and mouse. Furthermore, canine VH CDR3 displays an overrepresentation of the neutral amino acid threonine and the negatively charged aspartic acid while proline content is similar to that in the human repertoire. In general, the canine repertoire shows a bias towards small, negatively charged amino acids. Overall, this analysis suggests that functional canine therapeutic antibodies can be obtained from human and mouse sequences by methods of speciation and affinity maturation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Characterization of vitellogenin gene expression in round goby (Neogobius melanostomus) using a quantitative polymerase chain reaction assay.

    Science.gov (United States)

    Bowley, Lucas A; Alam, Farhana; Marentette, Julie R; Balshine, Sigal; Wilson, Joanna Y

    2010-12-01

    A growing concern over endocrine disruption in aquatic species has prompted the development of molecular assays to monitor environmental impacts. This study describes the development of quantitative polymerase chain reaction (qPCR) assays to characterize the expression of two vitellogenin (Vtg) genes in the invasive round goby (Neogobius melanostomus). Fragments from the 18SrRNA (housekeeping gene), Vtg II, and Vtg III genes were cloned and sequenced. The qPCR assays were developed to detect hepatic Vtg expression in goby. The assays detected induction of both Vtg genes in nonreproductive males following a two-week laboratory exposure to 17β-estradiol (≥1 mg/kg i.p. injection). The assays were applied to goby from Hamilton Harbour, Lake Ontario (Canada), including those from sites where feminization and intersex of goby has been documented. Both Vtg genes had significantly higher expression in females compared to males. Male reproductive goby adopt either parental or sneaker tactics; Vtg II expression was higher in sneaker than in parental males but parental and nonreproductive males did not differ from each other. The Vtg III expression was significantly higher in sneaker males followed by parental males and nonreproductive males, respectively. The Vtg II and III expression in nonreproductive males was elevated in the contaminated site with documented intersex. This assay provides an important tool for the use of an invasive species in monitoring endocrine disruption in the Great Lakes region. Copyright © 2010 SETAC.

  11. Candidate gene biodosimeters of mice and human exposure to ionizing radiation by quantitative reverse transcription polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Hamed Rezaeejam

    2015-01-01

    Full Text Available Understanding of cellular responses to ionizing radiation (IR is essential for the development of predictive markers useful for assessing human exposure. Biological markers of exposure to IR in human populations are of great interest for assessing normal tissue injury in radiation oncology and for biodosimetry in nuclear incidents and accidental radiation exposures. Traditional radiation exposure biomarkers based on cytogenetic assays (biodosimetry, are time-consuming and do not provide results fast enough and requires highly trained personnel for scoring. Hence, the development of rapid biodosimetry methods is one of the highest priorities. Exposure of cells to IR activates multiple signal transduction pathways, which result in complex alterations in gene-expression. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR has become the benchmark for the detection and quantification of RNA targets and is being utilized increasingly in monitoring the specific genes with more accurately and sensitively. This review evaluates the RT-qPCR as a biodosimetry method and we investigated the papers from 2000 up to now, which identified the genes-expression related the DNA repair, cell cycle checkpoint, and apoptosis induced by ionization radiation in peripheral blood and determined as biodosimeters. In conclusion, it could be say that RT-qPCR technique for determining the specific genes as biodosimeters could be a fully quantitative reliable and sensitive method. Furthermore, the results of the current review will help the researchers to recognize the most expressed genes induced by ionization radiation.

  12. MR molecular imaging of tumours using ferritin heavy chain reporter gene expression mediated by the hTERT promoter

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yan [Third Military Medical University, Department of Radiology, XinQiao Hospital, ChongQing (China); The First Affiliated Hospital of ChengDu Medical College, Department of Radiology, ChengDu (China); Gong, Ming-fu; Yang, Hua; Zhang, Song; Wang, Guang-xian; Su, Tong-sheng; Wen, Li; Zhang, Dong [Third Military Medical University, Department of Radiology, XinQiao Hospital, ChongQing (China)

    2016-11-15

    Using the human telomerase reverse transcriptase (hTERT) promoter and the modified ferritin heavy chain (Fth) reporter gene, reporter gene expression for MRI was examined in telomerase positive and negative tumour cells and xenografts. Activity of the reporter gene expression vector Lenti-hTERT-Fth1-3FLAG-Puro was compared to constitutive CMV-driven expression and to the untransfected parental control in five tumour cell lines: A549, SKOV3, 293T, U2OS and HPDLF. In vitro, transfected cells were evaluated for FLAG-tagged protein expression, iron accumulation and transverse relaxation. In vivo, tumours transduced by lentiviral vector injection were imaged using T2*WI. Changes in tumour signal intensity were validated by histology. Only telomerase positive tumour cells expressed FLAG-tagged Fth and displayed an increase in R2* above the parental control, with a corresponding change in T2*WI. In addition, only telomerase positive tumours, transduced by injection of the reporter gene expression construct, exhibited a change in signal intensity on T2*WI. Tumour histology verified the expression of FLAG-tagged Fth and iron accumulation in telomerase positive tissue. Reporter gene expression for MRI, using the Fth reporter and the hTERT promoter, may be a useful strategy for the non-invasive diagnosis of many types of cancer. (orig.)

  13. Haplotyping the human T-cell receptor β-chain gene complex by use of restriction fragment length polymorphisms

    International Nuclear Information System (INIS)

    Charmley, P.; Chao, A.; Gatti, R.A.; Concannon, P.; Hood, L.

    1990-01-01

    The authors have studied the genetic segregation of human T-cell receptor β-chain (TCRβ) genes on chromosome 7q in 40 CEPH (Centre d'Etude du Polymorphisme Humain) families by using restriction fragment length polymorphisms (RFLPs). They constructed haplotypes from eight RFLPs by using variable- and constant-region cDNA probes, which detect polymorphisms that span more than 600 kilobases of the TCRβ gene complex. Analysis of allele distributions between TCRβ genes revealed significant linkage disequilibrium between only 6 of the 28 different pairs of RFLPs. This linkage disequilibrium strongly influences the most efficient order to proceed for typing of these RFLPs in order to achieve maximum genetic informativeness, which in this study revealed a 97.3% level of heterozygosity within the TCRβ gene complex. The results should provide new insight into recent reports of disease associations with the TCRβ gene complex and should assist in designing future experiments to detect or confirm the existence of disease-susceptibility loci in this region of the human genome

  14. Homologous recombination in hybridoma cells: heavy chain chimeric antibody produced by gene targeting.

    OpenAIRE

    Fell, H P; Yarnold, S; Hellström, I; Hellström, K E; Folger, K R

    1989-01-01

    We demonstrate that murine myeloma cells can efficiently mediate homologous recombination. The murine myeloma cell line J558L was shown to appropriately recombine two transfected DNA molecules in approximately 30% of cells that received and integrated intact copies of both molecules. This activity was then exploited to direct major reconstructions of an endogenous locus within a hybridoma cell line. Production of antigen-specific chimeric heavy chain was achieved by targeting the human IgG1 h...

  15. Dynein Heavy Chain, Encoded by Two Genes in Agaricomycetes, Is Required for Nuclear Migration in Schizophyllum commune.

    Directory of Open Access Journals (Sweden)

    Melanie Brunsch

    Full Text Available The white-rot fungus Schizophyllum commune (Agaricomycetes was used to study the cell biology of microtubular trafficking during mating interactions, when the two partners exchange nuclei, which are transported along microtubule tracks. For this transport activity, the motor protein dynein is required. In S. commune, the dynein heavy chain is encoded in two parts by two separate genes, dhc1 and dhc2. The N-terminal protein Dhc1 supplies the dimerization domain, while Dhc2 encodes the motor machinery and the microtubule binding domain. This split motor protein is unique to Basidiomycota, where three different sequence patterns suggest independent split events during evolution. To investigate the function of the dynein heavy chain, the gene dhc1 and the motor domain in dhc2 were deleted. Both resulting mutants were viable, but revealed phenotypes in hyphal growth morphology and mating behavior as well as in sexual development. Viability of strain Δdhc2 is due to the higher expression of kinesin-2 and kinesin-14, which was proven via RNA sequencing.

  16. Adaptation of respiratory chain biogenesis to cytochrome c oxidase deficiency caused by SURF1 gene mutations

    Czech Academy of Sciences Publication Activity Database

    Kovářová, Nikola; Vrbacká-Čížková, Alena; Pecina, Petr; Stránecký, V.; Pronicka, E.; Kmoch, S.; Houštěk, Josef

    2012-01-01

    Roč. 1822, č. 7 (2012), s. 1114-1124 ISSN 0925-4439 R&D Projects: GA MZd(CZ) NS9759; GA MZd(CZ) NT12370; GA ČR(CZ) GD305/08/H037 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : mitochondrial disorder * SURF1 gene * Leigh syndrome * gene expression * oxidative phosphorylation * cytochrome c oxidase Subject RIV: FG - Pediatrics Impact factor: 4.910, year: 2012

  17. Detection of retinoblastoma gene deletions in spontaneous and radiation-induced mouse lung adenocarcinomas by polymerase chain reaction

    International Nuclear Information System (INIS)

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1994-01-01

    A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma gene using histological sections from radiation-induced and spontaneous tumors as the DNA source. Six mouse Rb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. The absence of any of these fragments relative to control PCR products on a Southern blot indicated a deletion of that portion of the mouse Rb gene. Tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death. Spontaneous tumors as well as those from irradiated mice (5.69 Gy 60 Co γ rays or 0.6 Gy JANUS neutrons, which have been found to have approximately equal radiobiological effectiveness) were analyzed for mouse Rb deletions. Tumors in 6 neutron-irradiated mice had no mouse Rb deletions. However, 1 of 6 tumors from γ-irradiated mice (17%) and 6 of 18 spontaneous tumors from unirradiated mice (33%) showed a deletion in one or both mouse Rb alleles. All deletions detected were in the 5' region of the mouse Rb gene. 36 refs., 2 figs., 2 tabs

  18. Isolation and Characterization of a Gene Specific to Lager Brewing Yeast That Encodes a Branched-Chain Amino Acid Permease

    Science.gov (United States)

    Kodama, Yukiko; Omura, Fumihiko; Ashikari, Toshihiko

    2001-01-01

    We found two types of branched-chain amino acid permease gene (BAP2) in the lager brewing yeast Saccharomyces pastorianus BH-225 and cloned one type of BAP2 gene (Lg-BAP2), which is identical to that of Saccharomyces bayanus (by-BAP2-1). The other BAP2 gene of the lager brewing yeast (cer-BAP2) is very similar to the Saccharomyces cerevisiae BAP2 gene. This result substantiates the notion that lager brewing yeast is a hybrid of S. cerevisiae and S. bayanus. The amino acid sequence homology between S. cerevisiae Bap2p and Lg-Bap2p was 88%. The transcription of Lg-BAP2 was not induced by the addition of leucine to the growth medium, while that of cer-BAP2 was induced. The transcription of Lg-BAP2 was repressed by the presence of ethanol and weak organic acid, while that of cer-BAP2 was not affected by these compounds. Furthermore, Northern analysis during beer fermentation revealed that the transcription of Lg-BAP2 was repressed at the beginning of the fermentation, while cer-BAP2 was highly expressed throughout the fermentation. These results suggest that the transcription of Lg-BAP2 is regulated differently from that of cer-BAP2 in lager brewing yeasts. PMID:11472919

  19. Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription.

    Science.gov (United States)

    Johnston, Stephen; Gallaher, Zachary; Czaja, Krzysztof

    2012-05-15

    Quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2(-∆∆Ct) normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxin selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference β-III tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.

  20. Multiplex reverse transcription polymerase chain reaction to study the expression of virulence and stress response genes in Staphylococcus aureus.

    Science.gov (United States)

    Shrihari, Rohinishree Yadahalli; Singh, Negi Pradeep

    2012-02-01

    Staphylococcus aureus survives well in different stress conditions. The ability of this organism to adapt to various stresses is the result of a complex regulatory response, which is attributed to regulation of multiple genes. The aims of the present study were (1) to develop a multiplex PCR for the detection of genes which are involved in stress adaptation (asp23, dnaK, and groEL); alternative sigma factor (sigB) and virulence determination (entB and spa) and (2) to study the expression of these genes during stress conditions for S. aureus culture collection strains (FRI 722 and ATCC 6538) and S. aureus food isolates at mRNA level using multiplex reverse transcription polymerase chain reaction (RT-PCR). During heat shock treatment groEL, dnaK, asp23, sodA, entB, spa, and sigB genes were up regulated up to 2.58, 2.07, 2.76, 2.55, 3.55, 2.71, and 2.62- folds, respectively, whereas in acid shock treatment, sodA and groEL were up regulated; dnaK was downregulated; and entB and sigB genes were not expressed in food isolates. Multiplex PCR assay standardized in this study offers an inexpensive alternative to uniplex PCR for detection of various virulence and stress response genes. This study is relevant to rapid and accurate detection of potential pathogenic S. aureus in foods. © 2012 Institute of Food Technologists®

  1. Sampling the genomic pool of protein tyrosine kinase genes using the polymerase chain reaction with genomic DNA.

    Science.gov (United States)

    Oates, A C; Wollberg, P; Achen, M G; Wilks, A F

    1998-08-28

    The polymerase chain reaction (PCR), with cDNA as template, has been widely used to identify members of protein families from many species. A major limitation of using cDNA in PCR is that detection of a family member is dependent on temporal and spatial patterns of gene expression. To circumvent this restriction, and in order to develop a technique that is broadly applicable we have tested the use of genomic DNA as PCR template to identify members of protein families in an expression-independent manner. This test involved amplification of DNA encoding protein tyrosine kinase (PTK) genes from the genomes of three animal species that are well known development models; namely, the mouse Mus musculus, the fruit fly Drosophila melanogaster, and the nematode worm Caenorhabditis elegans. Ten PTK genes were identified from the mouse, 13 from the fruit fly, and 13 from the nematode worm. Among these kinases were 13 members of the PTK family that had not been reported previously. Selected PTKs from this screen were shown to be expressed during development, demonstrating that the amplified fragments did not arise from pseudogenes. This approach will be useful for the identification of many novel members of gene families in organisms of agricultural, medical, developmental and evolutionary significance and for analysis of gene families from any species, or biological sample whose habitat precludes the isolation of mRNA. Furthermore, as a tool to hasten the discovery of members of gene families that are of particular interest, this method offers an opportunity to sample the genome for new members irrespective of their expression pattern.

  2. Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay.

    Science.gov (United States)

    Cronin, Maureen; Pho, Mylan; Dutta, Debjani; Stephans, James C; Shak, Steven; Kiefer, Michael C; Esteban, Jose M; Baker, Joffre B

    2004-01-01

    Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.

  3. BstXI RFLP in the human inter-alpha-trypsin inhibitor light chain gene

    Energy Technology Data Exchange (ETDEWEB)

    Leveillard, T; Bourguignon, J; Sesbouee, R; Hanauer, A; Salier, J P; Diarra-Mehrpour, M; Martin, J P

    1988-03-25

    The 1.2 kb EcoRI/SmaI fragment of lambdaHuLITI2 was used as probe. lambdaHuLITI2 is a full length cDNA clone coding for human inter-alpha-trypsin inhibitor light chain isolated from immunochemical screening of a lambdagt11 library. Its sequence coding for HI-30 and alpha-1-microglobulin is in agreement. BstXI identifies five invariant bands at 5.0 kb, 2.3 kb, 1.5 kb, 1.1 kb, and 0.7 kb and a diallelic polymorphism with DNA fragments at 2.0 kb or 1.7 kb.

  4. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.

    Science.gov (United States)

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John

    2015-10-01

    Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine. Published by Elsevier B.V.

  5. [Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble expression of single chain Fv].

    Science.gov (United States)

    Sheng, Wei-Jin; Miao, Qing-Fang; Zhen, Yong-Su

    2009-06-01

    Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy. The present study prepared single chain Fv (scFv) directed against EGFR. Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted. VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E. The phagemid containing scFv were transformed into electro-competent E. coli TG1 cells. The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA. A total of 48 clones from the library were selected randomly and 45 clones were identified positive. After infecting E. coli HB2151 cells with one positive clone, soluble recombinant antibodies about 27 kD were produced and located in the periplasm and the supernatant. The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues. VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig. The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane. The successful preparation of anti-EGFR scFv will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.

  6. Genes influenced by the non-muscle isoform of Myosin light chain kinase impact human cancer prognosis.

    Directory of Open Access Journals (Sweden)

    Tong Zhou

    Full Text Available The multifunctional non-muscle isoform of myosin light chain kinase (nmMLCK is critical to the rapid dynamic coordination of the cytoskeleton involved in cancer cell proliferation and migration. We identified 45 nmMLCK-influenced genes by bioinformatic filtering of genome-wide expression in wild type and nmMLCK knockout (KO mice exposed to preclinical models of murine acute inflammatory lung injury, pathologies that are well established to include nmMLCK as an essential participant. To determine whether these nmMLCK-influenced genes were relevant to human cancers, the 45 mouse genes were matched to 38 distinct human orthologs (M38 signature (GeneCards definition and underwent Kaplan-Meier survival analysis in training and validation cohorts. These studies revealed that in training cohorts, the M38 signature successfully identified cancer patients with poor overall survival in breast cancer (P<0.001, colon cancer (P<0.001, glioma (P<0.001, and lung cancer (P<0.001. In validation cohorts, the M38 signature demonstrated significantly reduced overall survival for high-score patients of breast cancer (P = 0.002, colon cancer (P = 0.035, glioma (P = 0.023, and lung cancer (P = 0.023. The association between M38 risk score and overall survival was confirmed by univariate Cox proportional hazard analysis of overall survival in the both training and validation cohorts. This study, providing a novel prognostic cancer gene signature derived from a murine model of nmMLCK-associated lung inflammation, strongly supports nmMLCK-involved pathways in tumor growth and progression in human cancers and nmMLCK as an attractive candidate molecular target in both inflammatory and neoplastic processes.

  7. Responses of Myosin Heavy Chain Phenotypes and Gene Expressions in Neck Muscle to Micro- an Hyper-Gravity in Mice

    Science.gov (United States)

    Ohira, Tomotaka; Ohira, Takashi; Kawano, F.; Shibaguchi, T.; Okabe, H.; Ohno, Y.; Nakai, N.; Ochiai, T.; Goto, K.; Ohira, Y.

    2013-02-01

    Neck muscles are known to play important roles in the maintenance of head posture against gravity. However, it is not known how the properties of neck muscle are influenced by gravity. Therefore, the current study was performed to investigate the responses of neck muscle (rhomboideus capitis) in mice to inhibition of gravity and/or increase to 2-G for 3 months to test the hypothesis that the properties of neck muscles are regulated in response to the level of mechanical load applied by the gravitational load. Three male wild type C57BL/10J mice (8 weeks old) were launched by space shuttle Discovery (STS-128) and housed in Japanese Experimental Module “KIBO” on the International Space Station in mouse drawer system (MDS) project, which was organized by Italian Space Agency. Only 1 mouse returned to the Earth alive after 3 months by space shuttle Atlantis (STS-129). Neck muscles were sampled from both sides within 3 hours after landing. Cage and laboratory control experiments were also performed on the ground. Further, 3-month ground-based control experiments were performed with 6 groups, i.e. pre-experiment, 3-month hindlimb suspension, 2-G exposure by using animal centrifuge, and vivarium control (n=5 each group). Five mice were allowed to recover from hindlimb suspension (including 5 cage control) for 3 months in the cage. Neck muscles were sampled bilaterally before and after 3-month suspension and 2-G exposure, and at the end of 3-month ambulation recovery. Spaceflight-associated shift of myosin heavy chain phenotype from type I to II and atrophy of type I fibers were observed. In response to spaceflight, 17 genes were up-regulated and 13 genes were down-regulated vs. those in the laboratory control. Expression of 6 genes were up-regulated and that of 88 genes were down-regulated by 3-month exposure to 2-G vs. the age-matched cage control. In response to chronic hindlimb suspension, 4 and 20 genes were up- or down-regulated. Further, 98 genes responded

  8. Identification of nickel response genes in abnormal early developments of sea urchin by differential display polymerase chain reaction.

    Science.gov (United States)

    Ryu, Tae Kwon; Lee, Gunsup; Rhee, Yong; Park, Heung-Sik; Chang, Man; Lee, Sukchan; Lee, Jaean; Lee, Taek-Kyun

    2012-10-01

    Bioassays and biomarkers have been previously developed to assess the effects of heavy metal contaminants on the early life stages of the sea urchin. In this study, malformation in the early developmental processes was observed in sea urchin (Strongylocentrotus intermedius) larvae exposed to 10 ppm Ni for over 30 h. The most critical stage at which the triggering of nickel effects takes place is thought to be the blastula stage, which occurs after fertilization in larval development. To investigate the molecular-level responses of sea urchin exposed to heavy metal stress and to explore the differentially expressed genes that are induced or repressed by nickel, differential display polymerase chain reaction (DD-PCR) was used with sea urchin mRNAs. The malformation-related genes expressed in the early life stages of the sea urchin were cloned from larvae exposed to 10 ppm of nickel for 15 h, and accessed via DD-PCR. Sequence analysis results revealed that each of the genes evidenced high homology with EGF2, PCSK9, serine/threonine protein kinase, apolipophorin precursor protein, and MGC80921 protein/transcript variant 2. This result may prove useful in the development of novel biomarkers for the assessment of heavy metal stresses on sea urchin embryos. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis.

    Science.gov (United States)

    Fox, Bridget C; Devonshire, Alison S; Baradez, Marc-Olivier; Marshall, Damian; Foy, Carole A

    2012-08-15

    Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT-qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Eh-pH diagrams of the V-H2O system at 25,60 and 1000C

    International Nuclear Information System (INIS)

    Silva, F.T. da; Ogasawara, T.; Cassa, J.C.S.

    1987-01-01

    Free energies of the formation of vanadium ions in aqueous solutions at elevated temperatures were determined. Eh-pH diagrams at 25 0 C, 60 0 C and 100 0 C at different vanadium concentrations, using microcomputers were calculated and obtained. The effects of temperature and vanadium concentration on the predominance of vanadium species were examined. The Eh-pH diagrams for the V-H 2 O system were evaluated by comparisons with experimental data. (Author) [pt

  11. Wheat-specific gene, ribosomal protein l21, used as the endogenous reference gene for qualitative and real-time quantitative polymerase chain reaction detection of transgenes.

    Science.gov (United States)

    Liu, Yi-Ke; Li, He-Ping; Huang, Tao; Cheng, Wei; Gao, Chun-Sheng; Zuo, Dong-Yun; Zhao, Zheng-Xi; Liao, Yu-Cai

    2014-10-29

    Wheat-specific ribosomal protein L21 (RPL21) is an endogenous reference gene suitable for genetically modified (GM) wheat identification. This taxon-specific RPL21 sequence displayed high homogeneity in different wheat varieties. Southern blots revealed 1 or 3 copies, and sequence analyses showed one amplicon in common wheat. Combined analyses with sequences from common wheat (AABBDD) and three diploid ancestral species, Triticum urartu (AA), Aegilops speltoides (BB), and Aegilops tauschii (DD), demonstrated the presence of this amplicon in the AA genome. Using conventional qualitative polymerase chain reaction (PCR), the limit of detection was 2 copies of wheat haploid genome per reaction. In the quantitative real-time PCR assay, limits of detection and quantification were about 2 and 8 haploid genome copies, respectively, the latter of which is 2.5-4-fold lower than other reported wheat endogenous reference genes. Construct-specific PCR assays were developed using RPL21 as an endogenous reference gene, and as little as 0.5% of GM wheat contents containing Arabidopsis NPR1 were properly quantified.

  12. Phylogenetic grouping and distribution of virulence genes in Escherichia coli along the production and supply chain of pork around Hubei, China.

    Science.gov (United States)

    Khan, Sher Bahadar; Zou, Geng; Cheng, Yu-Ting; Xiao, Ran; Li, Lu; Wu, Bin; Zhou, Rui

    2017-06-01

    Escherichia coli is an important foodborne zoonotic pathogen. A total of 285 strains of E. coli were isolated from the production and supply chain of pork in Hubei, China and characterized. Their phylogroups (A, B1, B2, and D) and virulence genes of public health importance become more and more diverse along the production and supply chain. Copyright © 2016. Published by Elsevier B.V.

  13. Deletion Analysis Of The Duchenne/Becker Muscular Dystrophy Gene Using Multiplex Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Dastur P

    2004-01-01

    Full Text Available The diagnosis of Duchenna Muscular Dystrophy (DMD and Becker Muscular Dystorphy (BMD is mainly based on clinical profile, serum CPK values, muscle biopsy and immunostaining for dystrophin. This was done in 100 unrelated patients using 19 exons including the promoter region in two sets of multiplex polymerase chain reaction (PCR. These primers amplify most of the exons in the deletion prone ′hot spot′ regions allowing determinations of deletion end points. Intragenic deletions were detected in 74 patients indicating that the use of PCR- based assays will allow deletion detection help in prenatal diagnosis for most of the DMD/BMD patients. The frequency of deletions observed in the present study was 74%.

  14. Local Chromatin Features Including PU.1 and IKAROS Binding and H3K4 Methylation Shape the Repertoire of Immunoglobulin Kappa Genes Chosen for V(DJ Recombination

    Directory of Open Access Journals (Sweden)

    Louise S. Matheson

    2017-11-01

    Full Text Available V(DJ recombination is essential for the generation of diverse antigen receptor (AgR repertoires. In B cells, immunoglobulin kappa (Igκ light chain recombination follows immunoglobulin heavy chain (Igh recombination. We recently developed the DNA-based VDJ-seq assay for the unbiased quantitation of Igh VH and DH repertoires. Integration of VDJ-seq data with genome-wide datasets revealed that two chromatin states at the recombination signal sequence (RSS of VH genes are highly predictive of recombination in mouse pro-B cells. It is unknown whether local chromatin states contribute to Vκ gene choice during Igκ recombination. Here we adapt VDJ-seq to profile the Igκ VκJκ repertoire and present a comprehensive readout in mouse pre-B cells, revealing highly variable Vκ gene usage. Integration with genome-wide datasets for histone modifications, DNase hypersensitivity, transcription factor binding and germline transcription identified PU.1 binding at the RSS, which was unimportant for Igh, as highly predictive of whether a Vκ gene will recombine or not, suggesting that it plays a binary, all-or-nothing role, priming genes for recombination. Thereafter, the frequency with which these genes recombine was shaped both by the presence and level of enrichment of several other chromatin features, including H3K4 methylation and IKAROS binding. Moreover, in contrast to the Igh locus, the chromatin landscape of the promoter, as well as of the RSS, contributes to Vκ gene recombination. Thus, multiple facets of local chromatin features explain much of the variation in Vκ gene usage. Together, these findings reveal shared and divergent roles for epigenetic features and transcription factors in AgR V(DJ recombination and provide avenues for further investigation of chromatin signatures that may underpin V(DJ-mediated chromosomal translocations.

  15. Polymerase chain reaction assay targeting cytochrome b gene for the detection of dog meat adulteration in meatball formulation.

    Science.gov (United States)

    Rahman, Md Mahfujur; Ali, Md Eaqub; Hamid, Sharifah Bee Abd; Mustafa, Shuhaimi; Hashim, Uda; Hanapi, Ummi Kalthum

    2014-08-01

    A polymerase chain reaction (PCR) assay for the assessment of dog meat adulteration in meatballs was developed. The assay selectively amplified a 100-bp region of canine mitochondrial cytochrome b gene from pure, raw, processed and mixed backgrounds. The specificity of the assay was tested against 11 animals and 3 plants species, commonly available for meatball formulation. The stability of the assay was proven under extensively autoclaving conditions that breakdown target DNA. A blind test from ready to eat chicken and beef meatballs showed that the assay can repeatedly detect 0.2% canine meat tissues under complex matrices using 0.04 ng of dog DNA extracted from differentially treated meatballs. The simplicity, stability and sensitivity of the assay suggested that it could be used in halal food industry for the authentication of canine derivatives in processed foods. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Molecular detection of field isolates of Turkey Eimeria by polymerase chain reaction amplification of the cytochrome c oxidase I gene.

    Science.gov (United States)

    Rathinam, T; Gadde, U; Chapman, H D

    2015-07-01

    Oocysts of Eimeria spp. were isolated from litter samples obtained from 30 commercial turkey farms. Genomic DNA was extracted from clean oocysts, and polymerase chain amplification of the species-specific cytochrome c oxidase subunit I (COI) gene was performed for five species of turkey Eimeria. The species tested were Eimeria adenoeides, Eimeria meleagrimitis, Eimeria meleagridis, Eimeria dispersa, and Eimeria gallopavonis. All DNA samples were positive for E. meleagrimitis, nine were positive for E. adenoeides, two were positive for E. dispersa, and none for E. meleagridis and E. gallopavonis. E. meleagrimitis occurred as a single species in 21 (70 %) of the farms while 9 (30 %) farms had a mixed species with E. meleagrimitis and E. adenoeides and 2 (7 %) were triple positive with E. meleagrimitis, E. adenoeides, and E. dispersa. This is the first account of the field prevalence of turkey Eimeria species using molecular methods.

  17. Induction of porcine host defense peptide gene expression by short-chain fatty acids and their analogs.

    Directory of Open Access Journals (Sweden)

    Xiangfang Zeng

    Full Text Available Dietary modulation of the synthesis of endogenous host defense peptides (HDPs represents a novel antimicrobial approach for disease control and prevention, particularly against antibiotic-resistant infections. However, HDP regulation by dietary compounds such as butyrate is species-dependent. To examine whether butyrate could induce HDP expression in pigs, we evaluated the expressions of a panel of porcine HDPs in IPEC-J2 intestinal epithelial cells, 3D4/31 macrophages, and primary monocytes in response to sodium butyrate treatment by real-time PCR. We revealed that butyrate is a potent inducer of multiple, but not all, HDP genes. Porcine β-defensin 2 (pBD2, pBD3, epididymis protein 2 splicing variant C (pEP2C, and protegrins were induced markedly in response to butyrate, whereas pBD1 expression remained largely unaltered in any cell type. Additionally, a comparison of the HDP-inducing efficacy among saturated free fatty acids of different aliphatic chain lengths revealed that fatty acids containing 3-8 carbons showed an obvious induction of HDP expression in IPEC-J2 cells, with butyrate being the most potent and long-chain fatty acids having only a marginal effect. We further investigated a panel of butyrate analogs for their efficacy in HDP induction, and found glyceryl tributyrate, benzyl butyrate, and 4-phenylbutyrate to be comparable with butyrate. Identification of butyrate and several analogs with a strong capacity to induce HDP gene expression in pigs provides attractive candidates for further evaluation of their potential as novel alternatives to antibiotics in augmenting innate immunity and disease resistance of pigs.

  18. Detection of clonal B cells in microdissected reactive lymphoproliferations: possible diagnostic pitfalls in PCR analysis of immunoglobulin heavy chain gene rearrangement

    DEFF Research Database (Denmark)

    Zhou, X.G.; Sandvej, K.; Gregersen, Niels

    1999-01-01

    Aims-To evaluate the specificity of standard and fluorescence based (GENESCAN) polymerase chain reaction (PCR) immunoglobulin heavy chain (IgH) gene rearrangement analysis in complete and microdissected paraffin wax embedded sections from lymphoid proliferations. Methods-PCR IgH gene rearrangement...... because of preferential priming or detection of local B cell clones. Data from clonal analysis of small, microdissected or lymphocyte poor samples must be evaluated critically. It is recommended that analyses should be run in parallel on at least two tissue specimens. Only reproducible bands present...

  19. PCR-based clonality analysis of B-cell lymphomas in paraffin-embedded tissues: diagnostic value of immunoglobulin kappa and lambda light chain gene rearrangement investigation.

    Science.gov (United States)

    Amara, Khaled; Trimeche, Mounir; Ziadi, Sonia; Sriha, Badreddine; Mokni, Moncef; Korbi, Sadok

    2006-01-01

    Polymerase chain reaction (PCR)-based analysis, employed for detecting immunoglobulin heavy chain (IgH) gene rearrangements, has become a diagnostic tool widely used in the investigation of B-cell lymphomas, but the overall sensitivity of these methods does not exceed 80%, notably in germinal center (GC) and post-GC B-cell origin lymphomas. Many PCR strategies devised for detecting immunoglobulin light chain (IgL) gene rearrangements have been developed to enhance the clonality detection rates. However, the feasibility of these methods in routine clinical diagnosis using paraffin-embedded tissues has not yet been investigated sufficiently. We studied a large series of 108 cases of B-cell lymphomas, as well as 20 reactive lymphoid tissues using degenerate primers to amplify immunoglobulin kappa (Igkappa) and lambda (Iglambda) light chain genes. B-cell clonality was further investigated using semi-nested PCR for IgH gene rearrangements. B-cell clonality was detected in 74%, 56.5%, and 43.5% of cases using IgH, Igkappa, and Iglambda PCR, respectively. By combining these methods, the clonality detection rate increased to 93.5%. Only polyclonal patterns were noted in reactive lymphoid samples. We concluded that in addition to the established methods for IgH analysis, a PCR-based approach for IgL gene rearrangements analysis improves the clonality detection rate in over 90% of B-cell lymphoma cases using routine histological specimens with poor preservation of the genomic DNA.

  20. Identification of four new mutations in the short-chain acyl-CoA dehydrogenase (SCAD) gene in two patients

    DEFF Research Database (Denmark)

    Gregersen, N; Winter, V S; Corydon, M J

    1998-01-01

    We have shown previously that a variant allele of the short-chain acyl-CoA dehydrogenase ( SCAD ) gene, 625G-->A, is present in homozygous form in 7% of control individuals and in 60% of 135 patients with elevated urinary excretion of ethylmalonic acid (EMA). We have now characterized three disease......-causing mutations (confirmed by lack of enzyme activity after expression in COS-7 cells) and a new susceptibility variant in the SCAD gene of two patients with SCAD deficiency, and investigated their frequency in patients with elevated EMA excretion. The first SCAD-deficient patient was a compound heterozygote...... for two mutations, 274G-->T and 529T-->C. These mutations were not present in 98 normal control alleles, but the 529T-->C mutation was found in one allele among 133 patients with elevated EMA excretion. The second patient carried a 1147C-->T mutation and the 625G-->A polymorphism in one allele...

  1. Detection of Ampicillin Resistance Genes (bla in Clinical Isolates of Escherichia coli with Polymerase Chain Reaction Method

    Directory of Open Access Journals (Sweden)

    Tiana Milanda

    2014-09-01

    Full Text Available Escherichia coli is a rod negative Gram which could be pathogenic, if its value increases or located in outer gastrointestinal tract. Pathogenic E. coli will produce enterotoxin which will cause diarrhoea or infection in urine tract. Ampicilin was one of particular antibiotics to overcome infection. Ampicilin nowadays is no longer used as primary medicine, because of its resistance case. The aim of this research is to detect the presence of gene which is responsible to ampicilin resistant E. coli. We used isolated midstream urine from cystitis object in Hasan Sadikin Hospital (RSHS as samples. Polymerase Chain Reaction (PCR method (colony-PCR and DNA-PCR were done to invenstigate the antibiotic resistency. Based on the result of antibiotic susceptibility testing to ampicillin, E. coli samples were resistant to ampicilin. Elektroforegram products of colony-PCR and DNA-PCR showed that the resistance case of ampicilin caused by bla gene (199 bp. Selective and rational antibiotic treatment is required to prevent ampicillin resistance in patients with symptoms

  2. Detection of Brazilian hantavirus by reverse transcription polymerase chain reaction amplification of N gene in patients with hantavirus cardiopulmonary syndrome

    Directory of Open Access Journals (Sweden)

    Marcos Lázaro Moreli

    2004-10-01

    Full Text Available We report a nested reverse transcription-polymerase chain reaction (RT-PCR assay for hantavirus using primers selected to match high homology regions of hantavirus genomes detected from the whole blood of hantavirus cardiopulmonary syndrome (HCPS patients from Brazil, also including the N gene nucleotide sequence of Araraquara virus. Hantavirus genomes were detected in eight out of nine blood samples from the HCPS patients by RT-PCR (88.9% positivity and in all 9 blood samples (100% positivity by nested-PCR. The eight amplicons obtained by RT-PCR (P1, P3-P9, including one obtained by nested-PCR (P-2 and not obtained by RT-PCR, were sequenced and showed high homology (94.8% to 99.1% with the N gene of Araraquara hantavirus. Although the serologic method ELISA is the most appropriate test for HCPS diagnosis, the use of nested RT-PCR for hantavirus in Brazil would contribute to the diagnosis of acute hantavirus disease detecting viral genomes in patient specimens as well as initial genomic characterization of circulating hantaviruses.

  3. Detection of giardine gene in local isolates of Giardia duodenalis by polymerase chain reaction (PCR).

    Science.gov (United States)

    Latifah, I; Teoh, K Y; Wan, K L; Rahmah, M; Normaznah, Y; Rohani, A

    2005-12-01

    Giardia duodenalis is an intestinal parasite that causes diarrhoea and malabsorption in children. The parasite also infects AIDS patients with a weak immune system. A study was carried out on six local isolates of Giardia duodenalis (110, 7304, 6304, M007, 2002 and 6307) from faeces of Orang Asli patients admitted to the Gombak Hospital. WB, a reference pathogenic strain from human and G. muris from a wild mouse, were commercially obtained from the American Type Culture Collection (ATCC). All the isolates were cultured axenically in TYI-S-33 medium. Two sets of primers were used for the techniques: primers LP1 and RP1 and primers LP2 and RP2. The sets of primers amplified giardine gene of 171 bp and 218 bp in sizes respectively. The study showed that the two sets of primers could detect G. duodenalis to the genus and species level specifically.

  4. Characterization of the human laminin beta2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS)

    DEFF Research Database (Denmark)

    Durkin, M E; Jäger, A C; Khurana, T S

    1999-01-01

    The laminin beta2 chain is an important constituent of certain kidney and muscle basement membranes. We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin beta2 chain gene (LAMB2) on chromosome 3p21.3-->p21.2, a region paralogous with the chromosome...... 7q22-->q31 region that contains the laminin beta1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3' end of LAMB2 lies 16 kb from the 5' end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we...... found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3' end of LAMB2L was similar to exons 8-12 of the laminin beta2 chain gene. The LAMB2L-LAMB2-QARS cluster lies telomeric to the gene encoding the laminin-binding protein...

  5. Multiplex reverse transcription-polymerase chain reaction combined with on-chip electrophoresis as a rapid screening tool for candidate gene sets

    DEFF Research Database (Denmark)

    Wittig, Rainer; Salowsky, Rüdiger; Blaich, Stephanie

    2005-01-01

    Combining multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with microfluidic amplicon analysis, we developed an assay for the rapid and reliable semiquantitative expression screening of 11 candidate genes for drug resistance in human malignant melanoma. The functionality of thi...

  6. Characterisation of Toxoplasma gondii isolates using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of the non-coding Toxoplasma gondii (TGR)-gene sequences

    DEFF Research Database (Denmark)

    Høgdall, Estrid; Vuust, Jens; Lind, Peter

    2000-01-01

    of using TGR gene variants as markers to distinguish among T. gondii isolates from different animals and different geographical sources. Based on the band patterns obtained by restriction fragment length polymorphism (RFLP) analysis of the polymerase chain reaction (PCR) amplified TGR sequences, the T...

  7. Clinical significance of productive immunoglobulin heavy chain gene rearrangements in childhood acute lymphoblastic leukemia.

    Science.gov (United States)

    Katsibardi, Katerina; Braoudaki, Maria; Papathanasiou, Chrissa; Karamolegou, Kalliopi; Tzortzatou-Stathopoulou, Fotini

    2011-09-01

    We analyzed the CDR3 region of 80 children with B-cell acute lymphoblastic leukemia (B-ALL) using the ImMunoGeneTics Information System and JOINSOLVER. In total, 108 IGH@ rearrangements were analyzed. Most of them (75.3%) were non-productive. IGHV@ segments proximal to IGHD-IGHJ@ were preferentially rearranged (45.3%). Increased utilization of IGHV3 segments IGHV3-13 (11.3%) and IGHV3-15 (9.3%), IGHD3 (30.5%), and IGHJ4 (34%) was noted. In pro-B ALL more frequent were IGHV3-11 (33.3%) and IGHV6-1 (33.3%), IGHD2-21 (50%), IGHJ4 (50%), and IGHJ6 (50%) segments. Shorter CDR3 length was observed in IGHV@6, IGHD7, and IGHJ1 segments, whereas increased CDR3 length was related to IGHV3, IGHD2, and IGHJ4 segments. Increased risk of relapse was found in patients with productive sequences. Specifically, the relapse-free survival rate at 5 years in patients with productive sequences at diagnosis was 75% (standard error [SE] ±9%), whereas in patients with non-productive sequences it was 97% (SE ±1.92%) (p-value =0.0264). Monoclonality and oligoclonality were identified in 81.2% and 18.75% cases at diagnosis, respectively. Sequence analysis revealed IGHV@ to IGHDJ joining only in 6.6% cases with oligoclonality. The majority (75%) of relapsed patients had monoclonal IGH@ rearrangements. The preferential utilization of IGHV@ segments proximal to IGHDJ depended on their location on the IGHV@ locus. Molecular mechanisms occurring during IGH@ rearrangement might play an essential role in childhood ALL prognosis. In our study, the productivity of the rearranged sequences at diagnosis proved to be a significant prognostic factor.

  8. Local Chromatin Features Including PU.1 and IKAROS Binding and H3K4 Methylation Shape the Repertoire of Immunoglobulin Kappa Genes Chosen for V(D)J Recombination.

    Science.gov (United States)

    Matheson, Louise S; Bolland, Daniel J; Chovanec, Peter; Krueger, Felix; Andrews, Simon; Koohy, Hashem; Corcoran, Anne E

    2017-01-01

    V(D)J recombination is essential for the generation of diverse antigen receptor (AgR) repertoires. In B cells, immunoglobulin kappa ( Igκ ) light chain recombination follows immunoglobulin heavy chain ( Igh ) recombination. We recently developed the DNA-based VDJ-seq assay for the unbiased quantitation of Igh VH and DH repertoires. Integration of VDJ-seq data with genome-wide datasets revealed that two chromatin states at the recombination signal sequence (RSS) of VH genes are highly predictive of recombination in mouse pro-B cells. It is unknown whether local chromatin states contribute to Vκ gene choice during Igκ recombination. Here we adapt VDJ-seq to profile the Igκ VκJκ repertoire and present a comprehensive readout in mouse pre-B cells, revealing highly variable Vκ gene usage. Integration with genome-wide datasets for histone modifications, DNase hypersensitivity, transcription factor binding and germline transcription identified PU.1 binding at the RSS, which was unimportant for Igh , as highly predictive of whether a Vκ gene will recombine or not, suggesting that it plays a binary, all-or-nothing role, priming genes for recombination. Thereafter, the frequency with which these genes recombine was shaped both by the presence and level of enrichment of several other chromatin features, including H3K4 methylation and IKAROS binding. Moreover, in contrast to the Igh locus, the chromatin landscape of the promoter, as well as of the RSS, contributes to Vκ gene recombination. Thus, multiple facets of local chromatin features explain much of the variation in Vκ gene usage. Together, these findings reveal shared and divergent roles for epigenetic features and transcription factors in AgR V(D)J recombination and provide avenues for further investigation of chromatin signatures that may underpin V(D)J-mediated chromosomal translocations.

  9. Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

    Science.gov (United States)

    Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

    2016-10-01

    IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

  10. Identification of a distinct type IV collagen α chain with restricted kidney distribution and assignment of its gene to the locus of X chromosome-linked Alport syndrome

    International Nuclear Information System (INIS)

    Hostikka, S.L.; Hoeyhtyae, M.; Tryggvason, K.; Eddy, R.L.; Byers, M.G.; Shows, T.B.

    1990-01-01

    The authors have identified and extensively characterized a type IV collagen α chain, referred to as α5(IV). Four overlapping cDNA clones isolated contain an open reading frame for 543 amino acid residues of the carboxyl-terminal end of a collagenous domain, a 229-residue carboxyl-terminal noncollagenous domain, and 1201 base pairs coding for a 3' untranslated region. The collagenous Gly-Xaa-Yaa repeat sequence has five imperfections that coincide with those in the corresponding region of the α1(IV) chain. The noncollagenous domain has 12 conserved cysteine residues and 83% and 63% sequence identity with the noncollagenous domains of the α1(IV) and α2(IV) chains, respectively. The α5(IV) chain has less sequence identity with the putative bovine α3(IV) and α4(IV) chains. Antiserum against an α5(IV) synthetic peptide stained a polypeptide chain of about 185 kDa by immunoblot analysis and immunolocalization of the chain in human kidney was almost completely restricted to the glomerulus. The gene was assigned to the Xq22 locus by somatic cell hybrids and in situ hybridization. This may be identical or close to the locus of the X chromosome-linked Alport syndrome that is believed to be a type IV collagen disease

  11. [Overexpression of four fatty acid synthase genes elevated the efficiency of long-chain polyunsaturated fatty acids biosynthesis in mammalian cells].

    Science.gov (United States)

    Zhu, Guiming; Saleh, Abdulmomen Ali Mohammed; Bahwal, Said Ahmed; Wang, Kunfu; Wang, Mingfu; Wang, Didi; Ge, Tangdong; Sun, Jie

    2014-09-01

    Three long-chain polyunsaturated fatty acids, docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), are the most biologically active polyunsaturated fatty acids in the body. They are important in developing and maintaining the brain function, and in preventing and treating many diseases such as cardiovascular disease, inflammation and cancer. Although mammals can biosynthesize these long-chain polyunsaturated fatty acids, the efficiency is very low and dietary intake is needed to meet the requirement. In this study, a multiple-genes expression vector carrying mammalian A6/A5 fatty acid desaturases and multiple-genes expression vector carrying mammalian Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases coding genes was used to transfect HEK293T cells, then the overexpression of the target genes was detected. GC-MS analysis shows that the biosynthesis efficiency and level of DHA, EPA and ARA were significantly increased in cells transfected with the multiple-genes expression vector. Particularly, DHA level in these cells was 2.5 times higher than in the control cells. This study indicates mammal possess a certain mechanism for suppression of high level of biosynthesis of long chain polyunsaturated fatty acids, and the overexpression of Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases broke this suppression mechanism so that the level of DHA, EPA and ARA was significantly increased. This study also provides a basis for potential applications of this gene construct in transgenic animal to produce high level of these long-chain polyunsaturated fatty acid.

  12. Differences in mitochondrial gene expression profiles, enzyme activities and myosin heavy chain types in yak versus bovine skeletal muscles.

    Science.gov (United States)

    Lin, Y Q; Xu, Y O; Yue, Y; Jin, S Y; Qu, Y; Dong, F; Li, Y P; Zheng, Y C

    2012-08-29

    Hypoxia can affect energy metabolism. We examined gene expression and enzyme activity related to mitochondrial energy metabolism, as well as myosin heavy chain (MyHC) types in yaks (Bos grunniens) living at high altitudes. Real-time quantitative PCR assays indicated that the yak has significantly lower levels of carnitine palmitoyltransferase (CPT) mRNA in the biceps femoris and lower levels of uncoupling protein 3 (UCP3) mRNA in both biceps femoris and longissimus dorsi than in Yellow cattle. No significant differences between yak and Yellow cattle were observed in the activities of mitochondrial β-hydroxyacyl-CoA dehydrogenase, isocitrate dehydrogenase and cytochrome oxidase in the same muscles. Semi-quantitative RT-PCR analysis showed that the MyHC 1 mRNA levels in yak biceps femoris was lower than in Yellow cattle. We conclude that the yak has significantly lower mRNA levels of CPT, UCP3, and MyHC 1 in biceps femoris than in Yellow cattle, suggesting that the yak biceps femoris has lower fatty acid oxidation capacity and greater glycolytic metabolic potential.

  13. Characterisation of myosin heavy chain gene variants in the fast and slow muscle fibres of gammarid amphipods.

    Science.gov (United States)

    Whiteley, N M; Magnay, J L; McCleary, S J; Nia, S Khazraee; El Haj, A J; Rock, J

    2010-10-01

    Recent molecular work has revealed a large diversity of myosin heavy chain (MyHC) gene variants in the abdominal musculature of gammarid amphipods. An unusual truncated MyHC transcript from the loop 1 region (Variant A(3)) was consistently observed in multiple species and populations. The current study aimed to determine whether this MyHC variant is specific to a particular muscle fibre type, as a change in net charge to the loop 1 region of Variant A(3) could be functionally significant. The localisation of different fibre types within the abdominal musculature of several gammarid species revealed that the deep flexor and extensor muscles are fast-twitch muscle fibres. The dorsal superficial muscles were identified as slow fibres and the muscles extrinsic to the pleopods were identified as intermediate fibres. Amplification of loop 1 region mRNA from isolated superficial extensor and deep flexor muscles, and subsequent liquid chromatography and sequence analysis revealed that Variant A(3) was the primary MyHC variant in slow muscles, and the conserved A(1) sequence was the primary variant in fast muscles. The specific role of Variant A(3) in the slow muscles remains to be investigated. 2010 Elsevier Inc. All rights reserved.

  14. Quantitative fucK gene polymerase chain reaction on sputum and nasopharyngeal secretions to detect Haemophilus influenzae pneumonia.

    Science.gov (United States)

    Abdeldaim, Guma M K; Strålin, Kristoffer; Olcén, Per; Blomberg, Jonas; Mölling, Paula; Herrmann, Björn

    2013-06-01

    A quantitative polymerase chain reaction (PCR) for the fucK gene was developed for specific detection of Haemophilus influenzae. The method was tested on sputum and nasopharyngeal aspirate (NPA) from 78 patients with community-acquired pneumonia (CAP). With a reference standard of sputum culture and/or serology against the patient's own nasopharyngeal isolate, H. influenzae etiology was detected in 20 patients. Compared with the reference standard, fucK PCR (using the detection limit 10(5) DNA copies/mL) on sputum and NPA showed a sensitivity of 95.0% (19/20) in both cases, and specificities of 87.9% (51/58) and 89.5% (52/58), respectively. In a receiver operating characteristic curve analysis, sputum fucK PCR was found to be significantly superior to sputum P6 PCR for detection of H. influenzae CAP. NPA fucK PCR was positive in 3 of 54 adult controls without respiratory symptoms. In conclusion, quantitative fucK real-time PCR provides a sensitive and specific identification of H. influenzae in respiratory secretions. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Modular Construction of Large Non-Immune Human Antibody Phage-Display Libraries from Variable Heavy and Light Chain Gene Cassettes.

    Science.gov (United States)

    Lee, Nam-Kyung; Bidlingmaier, Scott; Su, Yang; Liu, Bin

    2018-01-01

    Monoclonal antibodies and antibody-derived therapeutics have emerged as a rapidly growing class of biological drugs for the treatment of cancer, autoimmunity, infection, and neurological diseases. To support the development of human antibodies, various display techniques based on antibody gene repertoires have been constructed over the last two decades. In particular, scFv-antibody phage display has been extensively utilized to select lead antibodies against a variety of target antigens. To construct a scFv phage display that enables efficient antibody discovery, and optimization, it is desirable to develop a system that allows modular assembly of highly diverse variable heavy chain and light chain (Vκ and Vλ) repertoires. Here, we describe modular construction of large non-immune human antibody phage-display libraries built on variable gene cassettes from heavy chain and light chain repertoires (Vκ- and Vλ-light can be made into independent cassettes). We describe utility of such libraries in antibody discovery and optimization through chain shuffling.

  16. Heterologous co-expression of accA, fabD, and thioesterase genes for improving long-chain fatty acid production in Pseudomonas aeruginosa and Escherichia coli.

    Science.gov (United States)

    Lee, Sunhee; Jeon, Eunyoung; Jung, Yeontae; Lee, Jinwon

    2012-05-01

    The goal of the present study was to increase the content of intracellular long-chain fatty acids in two bacterial strains, Pseudomonas aeruginosa PA14 and Escherichia coli K-12 MG1655, by co-overexpressing essential enzymes that are involved in the fatty acid synthesis metabolic pathway. Recently, microbial fatty acids and their derivatives have been receiving increasing attention as an alternative source of fuel. By introducing two genes (accA and fabD) of P. aeruginosa into the two bacterial strains and by co-expressing with them the fatty acyl-acyl carrier protein thioesterase gene of Streptococcus pyogenes (strain MGAS10270), we have engineered recombinant strains that are efficient producers of long-chain fatty acids (C16 and C18). The recombinant strains exhibit a 1.3-1.7-fold increase in the production of long-chain fatty acids over the wild-type strains. To enhance the production of total long-chain fatty acids, we researched the carbon sources for optimized culture conditions and results were used for post-culture incubation period. E. coli SGJS17 (containing the accA, fabD, and thioesterase genes) produced the highest content of intracellular total fatty acids; in particular, the unsaturated fatty acid content was about 20-fold higher than that in the wild-type E. coli.

  17. Magnetovoltage Measurements and Field Sweep Rate Dependence of V-H curves in Superconducting Polycrystalline Y1Ba2Cu3O7-x

    International Nuclear Information System (INIS)

    Yetis, H; Altinkok, A; Olutas, M; Kilic, A; Kilic, K; Cetin, O

    2006-01-01

    Magnetovoltage measurements (V-H curves) were carried out in superconducting polycrystalline bulk Y 1 Ba 2 Cu 3 O 7-x (YBCO) material as a function of current (I), temperature (T), field sweep rate (dH/dt) and field orientation with respect to the transport current. A relative decrease in the dissipation measured in V-H curves was observed as dH/dt is increased, which implies that the time spent to plot the whole cycle has an importance on the evolution of the V-H curves. Thus, it could be possible to observe the relaxation effects in magnetovoltage measurements. In addition, the several significant steps and plateaus in V-H curves evolve depending on the magnitude of the transport current and also dH/dt. These observations were attributed to locking of the flux lines to decrease or increase in size of the easy motion flow channels. The strong hysteresis effects in V-H curves were discussed mainly by means of the flux trapping within the granularity of sample and the different degree of the inhomogeneous flux motion with respect to the sweeping of the external magnetic field up and down

  18. A Comprehensive Analysis of the Phylogeny, Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis, an Endangered Reptile Species

    Science.gov (United States)

    Lu, Yan; Zhang, Chenglin; Wu, Xiaobing; Han, Haitang; Zhao, Yaofeng; Ren, Liming

    2016-01-01

    Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 Cλ genes, each preceded by a Jλ gene, and 86 potentially functional Vλ genes upstream of (Jλ-Cλ)n. The Igκ locus contains a single Cκ gene, 6 Jκs and 62 functional Vκs. All VL genes are classified into a total of 31 families: 19 Vλ families and 12 Vκ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed Vλ and Vκ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates. PMID:26901135

  19. A Comprehensive Analysis of the Phylogeny, Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis, an Endangered Reptile Species.

    Directory of Open Access Journals (Sweden)

    Xifeng Wang

    Full Text Available Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 Cλ genes, each preceded by a Jλ gene, and 86 potentially functional Vλ genes upstream of (Jλ-Cλn. The Igκ locus contains a single Cκ gene, 6 Jκs and 62 functional Vκs. All VL genes are classified into a total of 31 families: 19 Vλ families and 12 Vκ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed Vλ and Vκ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates.

  20. Effects of Leucine Supplementation and Serum Withdrawal on Branched-Chain Amino Acid Pathway Gene and Protein Expression in Mouse Adipocytes

    Science.gov (United States)

    Vivar, Juan C.; Knight, Megan S.; Pointer, Mildred A.; Gwathmey, Judith K.; Ghosh, Sujoy

    2014-01-01

    The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of

  1. Effects of leucine supplementation and serum withdrawal on branched-chain amino acid pathway gene and protein expression in mouse adipocytes.

    Directory of Open Access Journals (Sweden)

    Abderrazak Kitsy

    Full Text Available The essential branched-chain amino acids (BCAA, leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2 and branched-chain alpha keto acid dehydrogenase (Bckdha was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4 compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our

  2. The frequency of a disease-causing point mutation in the gene coding for medium-chain acyl-CoA dehydrogenase in sudden infant death syndrome

    DEFF Research Database (Denmark)

    Banner, Jytte; Gregersen, N; Kølvraa, S

    1993-01-01

    A number of rare inherited metabolic disorders are known to lead to death in infancy. Deficiency of medium-chain acyl CoA dehydrogenase has, on clinical grounds, been related particularly to sudden infant death syndrome. The contribution of this disorder to the etiology of sudden infant death...... syndrome is still a matter of controversy. The present study investigated 120 well-defined cases of sudden infant death syndrome in order to detect the frequency of the most common disease-causing point mutation in the gene coding for medium-chain acyl-CoA dehydrogenase (G985) compared with the frequency...... in the general population. A highly specific polymerase chain reaction assay was applied on dried blood spots. No over-representation of homo- or heterozygosity for G985 appears to exist in such a strictly defined population, for which reason it may be more relevant to look at a broader spectrum of clinical...

  3. Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill.

    Science.gov (United States)

    de Almeida, Márcia R; Ruedell, Carolina M; Ricachenevsky, Felipe K; Sperotto, Raul A; Pasquali, Giancarlo; Fett-Neto, Arthur G

    2010-09-20

    Eucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in E. globulus microcuttings. By the use of two distinct algorithms, geNorm and NormFinder, we have assessed gene expression stability of eleven candidate reference genes in E. globulus: 18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI and 33380. The candidate reference genes were evaluated in microccuttings rooted in vitro, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: IDH and SAND for geNorm, and H2B and TUA for NormFinder. Both programs identified UBI and 18S as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the ARGONAUTE1 gene was evaluated in relation to the most stable candidate genes indicated by each algorithm. Our study showed that expression stability varied between putative reference genes tested in E. globulus. Based on the AGO1 relative expression

  4. Depth-dependent Vertical-to-Horizontal (V/H) Ratios of Free-Field Ground Motion Response Spectra for Deeply Embedded Nuclear Structures

    Energy Technology Data Exchange (ETDEWEB)

    Wei, X. [Brookhaven National Lab. (BNL), Upton, NY (United States); Braverman, J. [Brookhaven National Lab. (BNL), Upton, NY (United States); Miranda, M. [Brookhaven National Lab. (BNL), Upton, NY (United States); Rosario, M. E. [Brookhaven National Lab. (BNL), Upton, NY (United States); Costantino, C. J. [Brookhaven National Lab. (BNL), Upton, NY (United States)

    2015-02-01

    This report documents the results of a study to determine the depth-dependent V/H ratios of ground motion response spectra in the free field. The V/H ratios reported herein were developed from a worldwide database of surface and downhole acceleration recordings obtained from 45 vertical array stations. This database was specifically compiled for this project, and includes information from a diversity of active tectonic regions (California, Alaska, Taiwan, Japan), site conditions (rock to soft soil), ground motion intensity levels (PGAs between 0.01 g and 0.50 g), magnitudes (between ML 2.78 and JMA 8.1), epicentral distances (between 3.2 km and 812 km), and source depths (between 1.2 km and 112 km), as well as sensors at surface and at a wide range of depths relevant to the project. To study the significance of the depth effect, V/H ratios from all the records were sorted into a number of depth bins relevant to the project, and statistics (average, standard deviation, coefficient of variation, 16th, 50th, and 84th percentiles) of the V/H ratios within each bin were computed. Similar analyses were repeated, controlling for different site conditions, ground motion intensity levels, array locations, and source depths, to study their relative effect on the V/H ratios. Our findings confirm the importance of the depth effect on the V/H ratios. The research findings in this report can be used to provide guidance on the significance of the depth effect, and the extent to which this effect should be considered in the seismic design of deeply embedded SMR structures and NPP structures in general.

  5. Cloning of skeletal myosin heavy chain gene family from adult pleopod muscle and whole larvae of shrimps.

    Science.gov (United States)

    Koyama, Hiroki; Piyapattanakorn, Sanit; Watabe, Shugo

    2013-06-01

    The physiological and biological properties of skeletal muscle in crustacea have not been well understood compared with those of vertebrates. The present study focused on myosin, the major protein in skeletal muscle, from shrimps. In our previous study, two full-length genes encoding myosin heavy chain (MHC), a large subunit of the myosin molecule, were cloned from abdominal fast skeletal muscle of kuruma Marsupenaeus japonicus, black tiger Penaeus monodon and Pacific white Penaeus vannamei shrimps, and named as MHCa and MHCb. In this study, we renamed these as MHC1 and MHC2, respectively, due to the presence of various isoforms newly identified. Partial MHC sequences were identified from pleopod muscle of these shrimps. Two MHCs, named MHC3 and MHC4, were identified from pleopod muscle of kuruma shrimp, whereas two MHCs, named MHC4 and MHC5, were cloned from Pacific white shrimp pleopod. MHC3 was cloned only from black tiger shrimp pleopod. Partial MHC sequences from zoea, mysis, and postlarvae of black tiger and Pacific white shrimps were also determined. The phylogenetic tree demonstrated that most MHCs from pleopod muscle and larval MHCs formed clades with MHC1 and MHC2, respectively. These MHCs were considered to be of fast type, since MHC1 and MHC2 are fast-type MHCs according to our previous study. MHC5 obtained from pleopod muscle of Pacific white shrimp in this study was monophyletic with American lobster Homarus americanus S2 slow tonic MHC previously reported, indicating that MHC5 from Pacific white shrimp is of slow type. Copyright © 2013 Wiley Periodicals, Inc.

  6. Antimicrobial medium- and long-chain free fatty acids prevent PrfA-dependent activation of virulence genes in Listeria monocytogenes

    DEFF Research Database (Denmark)

    Sternkopf Lillebæk, Eva Maria; Lambert Nielsen, Stine; Scheel Thomasen, Rikke

    2017-01-01

    of virulence factors required for bacterial entry, intracellular replication and cell-to-cell spread. PrfA-dependent activation of virulence genes occurs primarily in the blood and during intracellular infection. In contrast, PrfA does not play a significant role in regulation of virulence gene expression...... antimicrobial free fatty acids act to downregulate transcription of PrfA-activated virulence genes. Interestingly, the inhibitory effect is also evident in cells encoding a constitutively active variant of PrfA. Collectively, our data suggest that antimicrobial medium- and long-chain free fatty acids may act...... as signals to prevent PrfA-mediated activation of virulence genes in environments where PrfA activation is not required, such as in food and the gastrointestinal tract....

  7. Close linkage of the mouse and human CD3 γ- and δ-chain genes suggests that their transcription is controlled by common regulatory elements

    International Nuclear Information System (INIS)

    Saito, H.; Koyama, T.; Georgopoulos, K.; Clevers, H.; Haser, W.G.; LeBien, T.; Tonegawa, S.; Terhorst, C.

    1987-01-01

    Antigen receptors on the T-cell surface are noncovalently associated with at least four invariant polypeptide chains, CD3-γ, -δ, -epsilon, and -zeta. The mouse CD3-γ gene, consisting of seven exons, was found to be highly homologous to the CD3-γ described earlier. Both the high level of sequence homology and the exon/intron organization indicate that the CD3-γ and -δ genes arose by gene duplication. Surprisingly, murine and human genomic DNA clones could be isolated that contained elements of both the CD3-γ and CD3-δ genes. In fact, the putative transcription start site of the mouse CD3-γ gene is less than 1.4 kilobases from the transcription initiation site of the mouse CD3-δ gene. Common elements that regulate the divergent transcription of the two genes are therefore proposed to be located in the intervening 1.4-kilobase DNA segment. This might contribute to the coordinate expression of the CD3-γ and -δ genes during intrathymic maturation of T lymphocytes

  8. Genetic polymorphism of toll-like receptors 4 gene by polymerase chain reaction-restriction fragment length polymorphisms, polymerase chain reaction-single-strand conformational polymorphism to correlate with mastitic cows

    Directory of Open Access Journals (Sweden)

    Pooja H. Gupta

    2015-05-01

    Full Text Available Aim: An attempt has been made to study the toll-like receptors 4 (TLR4 gene polymorphism from cattle DNA to correlate with mastitis cows. Materials and Methods: In present investigation, two fragments of TLR4 gene named T4CRBR1 and T4CRBR2 of a 316 bp and 382 bp were amplified by polymerase chain reaction (PCR, respectively from Kankrej (22 and Triple cross (24 cattle. The genetic polymorphisms in the two populations were detected by a single-strand conformational polymorphism in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results: Results showed that both alleles (A and B of two loci were found in all the two populations and the value of polymorphism information content indicated that these were highly polymorphic. Statistical results of χ2 test indicated that two polymorphism sites in the two populations fit with Hardy–Weinberg equilibrium (p˂0.05. Meanwhile, the effect of polymorphism of TLR4 gene on the somatic cell score (SCS indicated the cattle with allele a in T4CRBR1 showed lower SCS than that of allele B (p<0.05. Thus, the allele A might play an important role in mastitis resistance in cows. Conclusion: The relationship between the bovine mastitis trait and the polymorphism of TLR4 gene indicated that the bovine TLR4 gene may play an important role in mastitis resistance.

  9. Antimicrobial medium- and long-chain free fatty acids prevent PrfA-dependent activation of virulence genes in Listeria monocytogenes.

    Science.gov (United States)

    Sternkopf Lillebæk, Eva Maria; Lambert Nielsen, Stine; Scheel Thomasen, Rikke; Færgeman, Nils J; Kallipolitis, Birgitte H

    The foodborne pathogen Listeria monocytogenes is the causative agent of the invasive disease listeriosis. Infection by L. monocytogenes involves bacterial crossing of the intestinal barrier and intracellular replication in a variety of host cells. The PrfA protein is the master regulator of virulence factors required for bacterial entry, intracellular replication and cell-to-cell spread. PrfA-dependent activation of virulence genes occurs primarily in the blood and during intracellular infection. In contrast, PrfA does not play a significant role in regulation of virulence gene expression in the intestinal environment. In the gastrointestinal phase of infection, the bacterium encounters a variety of antimicrobial agents, including medium- and long-chain free fatty acids that are commonly found in our diet and as active components of bile. Here we show that subinhibitory concentrations of specific antimicrobial free fatty acids act to downregulate transcription of PrfA-activated virulence genes. Interestingly, the inhibitory effect is also evident in cells encoding a constitutively active variant of PrfA. Collectively, our data suggest that antimicrobial medium- and long-chain free fatty acids may act as signals to prevent PrfA-mediated activation of virulence genes in environments where PrfA activation is not required, such as in food and the gastrointestinal tract. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  10. Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search.

    Science.gov (United States)

    Piller, Nicolas; Decosterd, Isabelle; Suter, Marc R

    2013-07-10

    The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated.We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system - specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model. We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process

  11. Rapid screening of spontaneous and radiation-induced structural changes at the vestigial gene of Drosophila melanogaster by polymerase chain reaction

    International Nuclear Information System (INIS)

    Aleksandrov, I.D.; Lapidus, I.L.; Aleksandrova, M.V.; Karpovskij, A.L.; Korablinova, S.V.; Levkovich, N.V.

    1998-01-01

    A total of 27 independent isolated spontaneous and gamma-ray-induced heritable mutations at the vestigial gene of Drosophila melanogaster were analysed by a rapid deletion screening method with polymerase chain reaction (PCR) amplification. According to the results obtained 36.4% (4 of 11) of spontaneous mutants and 62.5% (10 of 16) of gamma-ray-induced ones have revealed deficiency of one or more fragments studied. The rest of spontaneous and radiation mutants showed no alterations in the PCR patterns, indicating possible small scale changes (point mutations) inside the gene region studied or, probably, the gross lesions situated elsewhere. The distribution of the mutation damages in the gene region studied are discussed

  12. Apparent polyploidization after gamma irradiation: pitfalls in the use of quantitative polymerase chain reaction (qPCR) for the estimation of mitochondrial and nuclear DNA gene copy numbers.

    Science.gov (United States)

    Kam, Winnie W Y; Lake, Vanessa; Banos, Connie; Davies, Justin; Banati, Richard

    2013-05-30

    Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization.

  13. Linkage of the gene that encodes the alpha 1 chain of type V collagen (COL5A1) to type II Ehlers-Danlos syndrome (EDS II).

    Science.gov (United States)

    Loughlin, J; Irven, C; Hardwick, L J; Butcher, S; Walsh, S; Wordsworth, P; Sykes, B

    1995-09-01

    Ehlers-Danlos syndrome (EDS) is a group of heritable disorders of connective tissue with skin, ligaments and blood vessels being the main sites affected. The commonest variant (EDS II) exhibits an autosomal dominant mode of inheritance and is characterized by joint hypermobility, cigarette paper scars, lax skin and excessive bruising. As yet no gene has been linked to EDS II, nor has linkage been established to a specific region of the genome. However, several candidate genes encoding proteins of the extracellular matrix have been excluded. Using an intragenic simple sequence repeat polymorphism, we report linkage of the COL5A1 gene, which encodes the alpha 1(V) chain of type V collagen, to EDS II. A maximum LOD score (Zmax) for linkage of 8.3 at theta = 0.00 was generated for a single large pedigree.

  14. Doxorubicin in vivo rapidly alters expression and translation of myocardial electron transport chain genes, leads to ATP loss and caspase 3 activation.

    Directory of Open Access Journals (Sweden)

    Amy V Pointon

    2010-09-01

    Full Text Available Doxorubicin is one of the most effective anti-cancer drugs but its use is limited by cumulative cardiotoxicity that restricts lifetime dose. Redox damage is one of the most accepted mechanisms of toxicity, but not fully substantiated. Moreover doxorubicin is not an efficient redox cycling compound due to its low redox potential. Here we used genomic and chemical systems approaches in vivo to investigate the mechanisms of doxorubicin cardiotoxicity, and specifically test the hypothesis of redox cycling mediated cardiotoxicity.Mice were treated with an acute dose of either doxorubicin (DOX (15 mg/kg or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ (25 mg/kg. DMNQ is a more efficient redox cycling agent than DOX but unlike DOX has limited ability to inhibit gene transcription and DNA replication. This allowed specific testing of the redox hypothesis for cardiotoxicity. An acute dose was used to avoid pathophysiological effects in the genomic analysis. However similar data were obtained with a chronic model, but are not specifically presented. All data are deposited in the Gene Expression Omnibus (GEO. Pathway and biochemical analysis of cardiac global gene transcription and mRNA translation data derived at time points from 5 min after an acute exposure in vivo showed a pronounced effect on electron transport chain activity. This led to loss of ATP, increased AMPK expression, mitochondrial genome amplification and activation of caspase 3. No data gathered with either compound indicated general redox damage, though site specific redox damage in mitochondria cannot be entirely discounted.These data indicate the major mechanism of doxorubicin cardiotoxicity is via damage or inhibition of the electron transport chain and not general redox stress. There is a rapid response at transcriptional and translational level of many of the genes coding for proteins of the electron transport chain complexes. Still though ATP loss occurs with activation caspase 3 and these

  15. Frequent expression loss of Inter-alpha-trypsin inhibitor heavy chain (ITIH) genes in multiple human solid tumors: A systematic expression analysis

    International Nuclear Information System (INIS)

    Hamm, Alexander; Knuechel, Ruth; Dahl, Edgar; Veeck, Juergen; Bektas, Nuran; Wild, Peter J; Hartmann, Arndt; Heindrichs, Uwe; Kristiansen, Glen; Werbowetski-Ogilvie, Tamra; Del Maestro, Rolando

    2008-01-01

    The inter-alpha-trypsin inhibitors (ITI) are a family of plasma protease inhibitors, assembled from a light chain – bikunin, encoded by AMBP – and five homologous heavy chains (encoded by ITIH1, ITIH2, ITIH3, ITIH4, and ITIH5), contributing to extracellular matrix stability by covalent linkage to hyaluronan. So far, ITIH molecules have been shown to play a particularly important role in inflammation and carcinogenesis. We systematically investigated differential gene expression of the ITIH gene family, as well as AMBP and the interacting partner TNFAIP6 in 13 different human tumor entities (of breast, endometrium, ovary, cervix, stomach, small intestine, colon, rectum, lung, thyroid, prostate, kidney, and pancreas) using cDNA dot blot analysis (Cancer Profiling Array, CPA), semiquantitative RT-PCR and immunohistochemistry. We found that ITIH genes are clearly downregulated in multiple human solid tumors, including breast, colon and lung cancer. Thus, ITIH genes may represent a family of putative tumor suppressor genes that should be analyzed in greater detail in the future. For an initial detailed analysis we chose ITIH2 expression in human breast cancer. Loss of ITIH2 expression in 70% of cases (n = 50, CPA) could be confirmed by real-time PCR in an additional set of breast cancers (n = 36). Next we studied ITIH2 expression on the protein level by analyzing a comprehensive tissue micro array including 185 invasive breast cancer specimens. We found a strong correlation (p < 0.001) between ITIH2 expression and estrogen receptor (ER) expression indicating that ER may be involved in the regulation of this ECM molecule. Altogether, this is the first systematic analysis on the differential expression of ITIH genes in human cancer, showing frequent downregulation that may be associated with initiation and/or progression of these malignancies

  16. Frequent expression loss of Inter-alpha-trypsin inhibitor heavy chain (ITIH genes in multiple human solid tumors: A systematic expression analysis

    Directory of Open Access Journals (Sweden)

    Werbowetski-Ogilvie Tamra

    2008-01-01

    Full Text Available Abstract Background The inter-alpha-trypsin inhibitors (ITI are a family of plasma protease inhibitors, assembled from a light chain – bikunin, encoded by AMBP – and five homologous heavy chains (encoded by ITIH1, ITIH2, ITIH3, ITIH4, and ITIH5, contributing to extracellular matrix stability by covalent linkage to hyaluronan. So far, ITIH molecules have been shown to play a particularly important role in inflammation and carcinogenesis. Methods We systematically investigated differential gene expression of the ITIH gene family, as well as AMBP and the interacting partner TNFAIP6 in 13 different human tumor entities (of breast, endometrium, ovary, cervix, stomach, small intestine, colon, rectum, lung, thyroid, prostate, kidney, and pancreas using cDNA dot blot analysis (Cancer Profiling Array, CPA, semiquantitative RT-PCR and immunohistochemistry. Results We found that ITIH genes are clearly downregulated in multiple human solid tumors, including breast, colon and lung cancer. Thus, ITIH genes may represent a family of putative tumor suppressor genes that should be analyzed in greater detail in the future. For an initial detailed analysis we chose ITIH2 expression in human breast cancer. Loss of ITIH2 expression in 70% of cases (n = 50, CPA could be confirmed by real-time PCR in an additional set of breast cancers (n = 36. Next we studied ITIH2 expression on the protein level by analyzing a comprehensive tissue micro array including 185 invasive breast cancer specimens. We found a strong correlation (p Conclusion Altogether, this is the first systematic analysis on the differential expression of ITIH genes in human cancer, showing frequent downregulation that may be associated with initiation and/or progression of these malignancies.

  17. A novel variable antibody fragment dimerized by leucine zippers with enhanced neutralizing potency against rabies virus G protein compared to its corresponding single-chain variable antibody fragment.

    Science.gov (United States)

    Li, Zhuang; Cheng, Yue; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2015-12-01

    Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency. Copyright © 2015. Published by Elsevier Ltd.

  18. Changes in Hepatic TRβ Protein Expression, Lipogenic Gene Expression, and Long-Chain Acylcarnitine Levels During Chronic Hyperthyroidism and Triiodothyronine Withdrawal in a Mouse Model.

    Science.gov (United States)

    Ohba, Kenji; Sinha, Rohit Anthony; Singh, Brijesh Kumar; Iannucci, Liliana Felicia; Zhou, Jin; Kovalik, Jean-Paul; Liao, Xiao-Hui; Refetoff, Samuel; Sng, Judy Chia Ghee; Leow, Melvin Khee-Shing; Yen, Paul Michael

    2017-06-01

    Thyroid hormone (TH) has important roles in regulating hepatic metabolism. It was previously reported that most hepatic genes activated by a single triiodothyronine (T3) injection became desensitized after multiple injections, and that approximately 10% of target genes did not return to basal expression levels after T3 withdrawal, despite normalization of serum TH and thyrotropin (TSH) levels. To determine the possible mechanism(s) for desensitization and incomplete recovery of hepatic target gene transcription and their effects on metabolism, mRNA and/or protein expression levels of key regulators of TH action were measured, as well as metabolomic changes after chronic T3 treatment and withdrawal. Adult male mice were treated with daily injections of T3 (20 μg/100 g body weight) for 14 days followed by the cessation of T3 for 10 days. Livers were harvested at 6 hours, 24 hours, and 14 days after the first T3 injection, and at 10 days after withdrawal, and then analyzed by quantitative reverse transcription polymerase chain reaction, Western blotting, and metabolomics. Although TH receptor (TRα and TRβ) mRNAs decreased slightly after chronic T3 treatment, only TRβ protein decreased before returning to basal expression level after withdrawal. The expression of other regulators of TH action was unchanged. TRβ protein expression was also decreased in adult male monocarboxylate transporter-8 (Mct8)-knockout mice, an in vivo model of chronic intrahepatic hyperthyroidism. Previously, increased hepatic long-chain acylcarnitine levels were found after acute TH treatment. However, in this study, long-chain acylcarnitine levels were unchanged after chronic T3, and paradoxically increased after T3 withdrawal. Pathway analyses of the previous microarray results showed upregulation of lipogenic genes after acute T3 treatment and withdrawal. Phosphorylation of acetyl-CoA carboxylase also decreased after T3 withdrawal. Decreased hepatic TRβ protein expression occurred

  19. MicroRNA-124-3p expression and its prospective functional pathways in hepatocellular carcinoma: A quantitative polymerase chain reaction, gene expression omnibus and bioinformatics study.

    Science.gov (United States)

    He, Rong-Quan; Yang, Xia; Liang, Liang; Chen, Gang; Ma, Jie

    2018-04-01

    The present study aimed to explore the potential clinical significance of microRNA (miR)-124-3p expression in the hepatocarcinogenesis and development of hepatocellular carcinoma (HCC), as well as the potential target genes of functional HCC pathways. Reverse transcription-quantitative polymerase chain reaction was performed to evaluate the expression of miR-124-3p in 101 HCC and adjacent non-cancerous tissue samples. Additionally, the association between miR-124-3p expression and clinical parameters was also analyzed. Differentially expressed genes identified following miR-124-3p transfection, the prospective target genes predicted in silico and the key genes of HCC obtained from Natural Language Processing (NLP) were integrated to obtain potential target genes of miR-124-3p in HCC. Relevant signaling pathways were assessed with protein-protein interaction (PPI) networks, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Protein Annotation Through Evolutionary Relationships (PANTHER) pathway enrichment analysis. miR-124-3p expression was significantly reduced in HCC tissues compared with expression in adjacent non-cancerous liver tissues. In HCC, miR-124-3p was demonstrated to be associated with clinical stage. The mean survival time of the low miR-124-3p expression group was reduced compared with that of the high expression group. A total of 132 genes overlapped from differentially expressed genes, miR-124-3p predicted target genes and NLP identified genes. PPI network construction revealed a total of 109 nodes and 386 edges, and 20 key genes were identified. The major enriched terms of three GO categories included regulation of cell proliferation, positive regulation of cellular biosynthetic processes, cell leading edge, cytosol and cell projection, protein kinase activity, transcription activator activity and enzyme binding. KEGG analysis revealed pancreatic cancer, prostate cancer and non-small cell lung cancer as the

  20. Side-chain amino-acid-based pH-responsive self-assembled block copolymers for drug delivery and gene transfer.

    Science.gov (United States)

    Kumar, Sonu; Acharya, Rituparna; Chatterji, Urmi; De, Priyadarsi

    2013-12-10

    Developing safe and effective nanocarriers for multitype of delivery system is advantageous for several kinds of successful biomedicinal therapy with the same carrier. In the present study, we have designed amino acid biomolecules derived hybrid block copolymers which can act as a promising vehicle for both drug delivery and gene transfer. Two representative natural chiral amino acid-containing (l-phenylalanine and l-alanine) vinyl monomers were polymerized via reversible addition-fragmentation chain transfer (RAFT) process in the presence of monomethoxy poly(ethylene glycol) based macro-chain transfer agents (mPEGn-CTA) for the synthesis of well-defined side-chain amino-acid-based amphiphilic block copolymers, monomethoxy poly(ethylene glycol)-b-poly(Boc-amino acid methacryloyloxyethyl ester) (mPEGn-b-P(Boc-AA-EMA)). The self-assembled micellar aggregation of these amphiphilic block copolymers were studied by fluorescence spectroscopy, atomic force microscopy (AFM) and scanning electron microscopy (SEM). Potential applications of these hybrid polymers as drug carrier have been demonstrated in vitro by encapsulation of nile red dye or doxorubicin drug into the core of the micellar nanoaggregates. Deprotection of side-chain Boc- groups in the amphiphilic block copolymers subsequently transformed them into double hydrophilic pH-responsive cationic block copolymers having primary amino groups in the side-chain terminal. The DNA binding ability of these cationic block copolymers were further investigated by using agarose gel retardation assay and AFM. The in vitro cytotoxicity assay demonstrated their biocompatible nature and these polymers can serve as "smart" materials for promising bioapplications.

  1. Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in the rumen epithelium.

    Science.gov (United States)

    Die, Jose V; Baldwin, Ransom L; Rowland, Lisa J; Li, Robert; Oh, Sunghee; Li, Congjun; Connor, Erin E; Ranilla, Maria-Jose

    2017-01-01

    The rumen is lined on the luminal side by a stratified squamous epithelium that is responsible for not only absorption, but also transport, extensive short-chain fatty acid (SCFA) metabolism and protection. Butyrate has been demonstrated to initiate the differentiation of the tissue following introduction of solid feed to the weaning neonate as well as affecting the metabolism of other nutrients and absorption of nutrients in in vitro experiments. The objective of the present study was to validate expression stability of eight putative reference genes bovine rumen, considering the intrinsic heterogeneity of bovine rumen with regard to different luminal characteristics due to direct infusion of butyrate to double the intra-ruminal content of the rumen liquor. Our focus was on identifying stable reference genes which are suitable to normalize real-time RT-qPCR experiments from rumen samples collected from clinical assays, irrespective of localization within the organ and the across physiological state. The most stably expressed genes included: ACTB, UXT, DBNDD2, RPS9, DDX54 and HMBS. Their high stability values suggest these reference genes will facilitate better evaluation of variation of across an array of conditions including: localization within the rumen, differences among cattle fed an array of rations, as well as response to development in the weaning animal. Moreover, we anticipate these reference genes may be useful for expression studies in other ruminants.

  2. Determination of haemolytic and non haemolytic genes profiles of Bacillus cereus strains isolated from food samples by polymerase chain reaction (pcr) technique

    Science.gov (United States)

    Jawad, Nisreen; Ahemd, Asmat; Abdullah, Aminah

    2018-04-01

    The aim of this study was to investigate the presence of Bacillus cereus and detection of enterotoxigenic genes in food samples by utilizing a Polymerase Chain Reaction technique (PCR). In this study the providence of B. cereus was carried out to food samples. The B. cereus isolates were investigated for enterotoxigenic gene. The cooked seafood, and raw milk samples were purchased from several restaurants and market in the area of (Bangi, Kajang, Serdang and UKM) Selangor, Malaysia. A total of 60 samples have been analyzed. B. cereus contamination has been formed between 1.4×105 - 3×105 cfu/mL of cooked seafood and raw milk samples. Five colonies have been detected as B. cereus using biochemical test. All B. cereus isolates named BC1 to BC27, were characterized for haemolytic enterotoxin (HBL) complex encoding genes (hblA), non-haemolytic enterotoxin encoding gene (NheA). 10 isolates have been reported to be positive towards hblA and 12 isolates were positive towards NheA. The presence of B. cereus and their enterotoxigenic genes in cooked seafood and raw milk from to food samples obtained may pose a potential risk for public health.

  3. Glycosyltransferases as marker genes for the quantitative polymerase chain reaction-based detection of circulating tumour cells from blood samples of patients with breast cancer undergoing adjuvant therapy.

    Science.gov (United States)

    Kölbl, Alexandra C; Hiller, Roman A; Ilmer, Mathias; Liesche, Friederike; Heublein, Sabine; Schröder, Lennard; Hutter, Stefan; Friese, Klaus; Jeschke, Udo; Andergassen, Ulrich

    2015-08-01

    Altered glycosylation is a predominant feature of tumour cells; it serves for cell adhesion and detachment, respectively, and facilitates the immune escape of these cells. Therefore changes in the expression of glycosyltransferase genes could help to identify circulating tumour cells (CTCs) in the blood samples of cancer patients using a quantitative polymerase chain reaction (PCR) approach. Blood samples of healthy donors were inoculated with certain numbers of established breast cancer cell line cells, thus creating a model system. These samples were analysed by quantitative PCR for the expression of six different glycosyltransferase genes. The three genes with the best results in the model system were consecutively applied to samples from adjuvant breast cancer patients and of healthy donors. FUT3 and GALNT6 showed the highest increase in relative expression, while GALNT6 and ST3GAL3 were the first to reach statistically significant different ∆CT-values comparing the sample with and without addition of tumour cells. These three genes were applied to patient samples, but did not show any significant results that may suggest the presence of CTCs in the blood. Although the relative expression of some of the glycosyltransferase genes exhibited reasonable results in the model system, their application to breast cancer patient samples will have to be further improved, e.g. by co-analysis of patient blood samples by gold-standard methods.

  4. Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR analysis in the rumen epithelium.

    Directory of Open Access Journals (Sweden)

    Jose V Die

    Full Text Available The rumen is lined on the luminal side by a stratified squamous epithelium that is responsible for not only absorption, but also transport, extensive short-chain fatty acid (SCFA metabolism and protection. Butyrate has been demonstrated to initiate the differentiation of the tissue following introduction of solid feed to the weaning neonate as well as affecting the metabolism of other nutrients and absorption of nutrients in in vitro experiments. The objective of the present study was to validate expression stability of eight putative reference genes bovine rumen, considering the intrinsic heterogeneity of bovine rumen with regard to different luminal characteristics due to direct infusion of butyrate to double the intra-ruminal content of the rumen liquor. Our focus was on identifying stable reference genes which are suitable to normalize real-time RT-qPCR experiments from rumen samples collected from clinical assays, irrespective of localization within the organ and the across physiological state. The most stably expressed genes included: ACTB, UXT, DBNDD2, RPS9, DDX54 and HMBS. Their high stability values suggest these reference genes will facilitate better evaluation of variation of across an array of conditions including: localization within the rumen, differences among cattle fed an array of rations, as well as response to development in the weaning animal. Moreover, we anticipate these reference genes may be useful for expression studies in other ruminants.

  5. Polymerase chain reaction amplification and cloning of immunogenic protein NAD-dependent beta hydroxybutyryl CoA dehydrogenase gene of Clostridium chauvoei

    Directory of Open Access Journals (Sweden)

    Saroj K. Dangi

    2014-10-01

    Full Text Available Aim: The present study was aimed at polymerase chain reaction (PCR amplification and cloning of NAD-dependent betahydroxybutyryl coenzyme A dehydrogenase (BHBD gene of Clostridium chauvoei. Materials and Methods: C. chauvoei was cultured and confirmed by 16-23S rDNA spacer region primers. The primers for nad-bhbd gene of C. chauvoei were designed to aid in cloning into pRham-N-His SUMO-Kan vector, and nad-bhbd gene was amplified by PCR. The amplified nad-bhbd gene was purified and cloned into pRham-N-His SUMO-Kan expression vector. The recombinant plasmid was transformed into E. cloni 10 G cells and the clone was confirmed by colony PCR using the pRham-SUMO-NAD-For and pRham-SUMO-NAD-Rev primers and also by sequencing. Results: PCR amplification of nad-bhbd gene yielded a product length of 844 base pairs which was cloned into pRham-NHis SUMO-Kan vector followed by transformation into E. cloni 10G chemically competent cells. The recombinant clones were characterized by colony PCR, sequencing, followed by basic local alignment search tool (BLAST analysis to confirm the insert. Conclusions: Immunogenic protein NAD- dependent BHBD of C. chauvoei was cloned and the recombinant clones were confirmed by colony PCR and sequencing analysis.

  6. Identification of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) by using polymerase chain reaction amplification and restriction analysis of the mitochondrial cytochrome b gene.

    Science.gov (United States)

    Carrera, E; García, T; Céspedes, A; González, I; Sanz, B; Hernández, P E; Martín, R

    1998-04-01

    Restriction site analysis of polymerase chain reaction (PCR) products from a conserved region of the cytochrome b gene has been used for the identification of fresh and smoked samples of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Digestion of the 359-bp PCR product with the endonucleases EcoRV and TaqI yielded specific banding patterns for salmon and trout. This genetic marker can be very useful for detecting fraudulent substitution of the cheaper smoked trout for the more expensive smoked salmon.

  7. Identification of human rotavirus serotype by hybridization to polymerase chain reaction-generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7

    International Nuclear Information System (INIS)

    Flores, J.; Sears, J.; Schael, I.P.; White, L.; Garcia, D.; Lanata, C.; Kapikian, A.Z.

    1990-01-01

    We have synthesized 32 P-labeled hybridization probes from a hyperdivergent region (nucleotides 51 to 392) of the rotavirus gene encoding the VP7 glycoprotein by using the polymerase chain reaction method. Both RNA (after an initial reverse transcription step) and cloned cDNA from human rotavirus serotypes 1 through 4 could be used as templates to amplify this region. High-stringency hybridization of each of the four probes to rotavirus RNAs dotted on nylon membranes allowed the specific detection of corresponding sequences and thus permitted identification of the serotype of the strains dotted. The procedure was useful when applied to rotaviruses isolated from field studies

  8. Identification of human rotavirus serotype by hybridization to polymerase chain reaction-generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7

    Energy Technology Data Exchange (ETDEWEB)

    Flores, J.; Sears, J.; Schael, I.P.; White, L.; Garcia, D.; Lanata, C.; Kapikian, A.Z. (National Institutes of Health, Bethesda, MD (USA))

    1990-08-01

    We have synthesized {sup 32}P-labeled hybridization probes from a hyperdivergent region (nucleotides 51 to 392) of the rotavirus gene encoding the VP7 glycoprotein by using the polymerase chain reaction method. Both RNA (after an initial reverse transcription step) and cloned cDNA from human rotavirus serotypes 1 through 4 could be used as templates to amplify this region. High-stringency hybridization of each of the four probes to rotavirus RNAs dotted on nylon membranes allowed the specific detection of corresponding sequences and thus permitted identification of the serotype of the strains dotted. The procedure was useful when applied to rotaviruses isolated from field studies.

  9. Revisit complexation between DNA and polyethylenimine — Effect of length of free polycationic chains on gene transfection

    DEFF Research Database (Denmark)

    Yue, Yanan; Jin, Fan; Deng, Rui

    2011-01-01

    Our revisit of the complexation between DNA and polyethylenimine (PEI) by using a combination of laser light scattering and gel electrophoresis confirms that nearly all the DNA chains are complexed with PEI to form polyplexes when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) r...

  10. Long-chain acyl-CoA-dependent regulation of gene expression in bacteria, yeast and mammals

    DEFF Research Database (Denmark)

    Black, P N; Færgeman, Nils J.; DiRusso, C C

    2000-01-01

    ). Both repression and activation are dependent upon the function of either of the acyl-CoA synthetases Faa1p or Faa4p. In mammals, purified hepatocyte nuclear transcription factor 4alpha (HNF-4alpha) like E. coli FadR, binds long chain acyl-CoA directly. Coexpression of HNF-4alpha and acyl-CoA synthetase...

  11. Cloning and inactivation of a branched-chain-amino-acid aminotransferase gene from Staphylococcus carnosus and characterization of the enzyme

    DEFF Research Database (Denmark)

    Madsen, Søren M; Beck, Hans Christian; Ravn, Peter

    2002-01-01

    Staphylococcus carnosus and Staphylococcus xylosus are widely used as aroma producers in the manufacture of dried fermented sausages. Catabolism of branched-chain amino acids (BCAAs) by these strains contributes to aroma formation by production of methyl-branched aldehydes and carboxy acids. The ...

  12. Erythroid Kruppel-like factor (EKLF) is recruited to the γ-globin gene promoter as a co-activator and is required for γ-globin gene induction by short-chain fatty acid derivatives

    Science.gov (United States)

    Perrine, Susan P.; Mankidy, Rishikesh; Boosalis, Michael S.; Bieker, James J.; Faller, Douglas V.

    2011-01-01

    Objectives The erythroid Kruppel-like factor (EKLF) is an essential transcription factor for β-type globin gene switching, and specifically activates transcription of the adult β-globin gene promoter. We sought to determine if EKLF is also required for activation of the γ-globin gene by short-chain fatty acid (SCFA) derivatives, which are now entering clinical trials. Methods The functional and physical interaction of EKLF and co-regulatory molecules with the endogenous human globin gene promoters was studied in primary human erythroid progenitors and cell lines, using chromatin immunoprecipitation (ChIP) assays and genetic manipulation of the levels of EKLF and co-regulators. Results and conclusions Knockdown of EKLF prevents SCFA-induced expression of the γ-globin promoter in a stably expressed μLCRβprRlucAγprFluc cassette, and prevents induction of the endogenous γ-globin gene in primary human erythroid progenitors. EKLF is actively recruited to endogenous γ-globin gene promoters after exposure of primary human erythroid progenitors, and murine hematopoietic cell lines, to SCFA derivatives. The core ATPase BRG1 subunit of the human SWI/WNF complex, a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure, is also required for γ-globin gene induction by SCFA derivatives. BRG1 is actively recruited to the endogenous γ-globin promoter of primary human erythroid progenitors by exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes. PMID:19220418

  13. Detection and analysis of hemolysin genes in Aeromonas hydrophila isolated from Gouramy (Osphronemus gouramy) by polymerase chain reaction (PCR)

    Science.gov (United States)

    Rozi; Rahayu, K.; Daruti, D. N.

    2018-04-01

    The goal of this study was to detect of Aeromonas hydrophila carrying the hlyA gene in guramy by PCR assay. A total of 5 A. hydrophila strains were isolated from gouramy with different location and furthermore genotypic of all A. hydrophila strains havedetected by PCR assay for 16S rRNA gene. The primers used in the PCR targeted a 592-bp fragment of the hlyA gene coding for the hemolysin gene. Particularly hlyA genes are responsible for haemolysin toxins production in this genus. After gel electrophoresis, the amplicons from representative strains of the A. hydrophila were purified using extraction kit and were subjected to the DNA sequencing analysis. The results showed that: (i) the 592bp amplicon of the hlyA gene was detected in 5/6 of the A. hydrophila; (ii) the nucleotide blast results of hemolysin gene sequences of the strains of A. hydrophila revealed a high homology of 90-97 % with published sequences, and;(iii) the protein blast showed 95-98 % homology when compared to the published sequences. The PCR clearly identified the haemolysin-producing strains of A. hydrophila by detection in hlyA genes and may have application as a rapid species-specific virulence test.

  14. Use of Hybridization Chain Reaction-Fluorescent In Situ Hybridization To Track Gene Expression by Both Partners during Initiation of Symbiosis.

    Science.gov (United States)

    Nikolakakis, K; Lehnert, E; McFall-Ngai, M J; Ruby, E G

    2015-07-01

    The establishment of a productive symbiosis between Euprymna scolopes, the Hawaiian bobtail squid, and its luminous bacterial symbiont, Vibrio fischeri, is mediated by transcriptional changes in both partners. A key challenge to unraveling the steps required to successfully initiate this and many other symbiotic associations is characterization of the timing and location of these changes. We report on the adaptation of hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) to simultaneously probe the spatiotemporal regulation of targeted genes in both E. scolopes and V. fischeri. This method revealed localized, transcriptionally coregulated epithelial cells within the light organ that responded directly to the presence of bacterial cells while, at the same time, provided a sensitive means to directly show regulated gene expression within the symbiont population. Thus, HCR-FISH provides a new approach for characterizing habitat transition in bacteria and for discovering host tissue responses to colonization. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. Genotyping of major histocompatibility complex Class II DRB gene in Rohilkhandi goats by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing

    Directory of Open Access Journals (Sweden)

    Kush Shrivastava

    2015-10-01

    Full Text Available Aim: To study the major histocompatibility complex (MHC Class II DRB1 gene polymorphism in Rohilkhandi goat using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP and nucleotide sequencing techniques. Materials and Methods: DNA was isolated from 127 Rohilkhandi goats maintained at sheep and goat farm, Indian Veterinary Research Institute, Izatnagar, Bareilly. A 284 bp fragment of exon 2 of DRB1 gene was amplified and digested using BsaI and TaqI restriction enzymes. Population genetic parameters were calculated using Popgene v 1.32 and SAS 9.0. The genotypes were then sequenced using Sanger dideoxy chain termination method and were compared with related breeds/species using MEGA 6.0 and Megalign (DNASTAR software. Results: TaqI locus showed three and BsaI locus showed two genotypes. Both the loci were found to be in Hardy–Weinberg equilibrium (HWE, however, population genetic parameters suggest that heterozygosity is still maintained in the population at both loci. Percent diversity and divergence matrix, as well as phylogenetic analysis revealed that the MHC Class II DRB1 gene of Rohilkhandi goats was found to be in close cluster with Garole and Scottish blackface sheep breeds as compared to other goat breeds included in the sequence comparison. Conclusion: The PCR-RFLP patterns showed population to be in HWE and absence of one genotype at one locus (BsaI, both the loci showed excess of one or the other homozygote genotype, however, effective number of alleles showed that allelic diversity is present in the population. Sequence comparison of DRB1 gene of Rohilkhandi goat with other sheep and goat breed assigned Rohilkhandi goat in divergence with Jamanupari and Angora goats.

  16. Branched-chain alpha-keto acid catabolism via the gene products of the bkd operon in Enterococcus faecalis: a ne, secreted metabolite serving as a temporary redox sink.

    NARCIS (Netherlands)

    Ward, D.E.; van der Weijden, C.C.; van der Merwe, M.J.; Westerhoff, H.V.; Claiborne, A.; Snoep, J.L.

    2000-01-01

    Recently the bkd gene cluster from Enterococcus faecalis was sequenced, and it was shown that the gene products constitute a pathway for the catabolism of branched-chain α-keto acids. We have now investigated the regulation and physiological role of this pathway. Primer extension analysis identified

  17. Tissue-specific expression of the human laminin alpha5-chain, and mapping of the gene to human chromosome 20q13.2-13.3 and to distal mouse chromosome 2 near the locus for the ragged (Ra) mutation

    DEFF Research Database (Denmark)

    Durkin, M E; Loechel, F; Mattei, M G

    1997-01-01

    , heart, lung, skeletal muscle, kidney, and pancreas. The human laminin alpha5-chain gene (LAMA5) was assigned to chromosome 20q13.2-q13.3 by in situ hybridization, and the mouse gene (Lama5) was mapped by linkage analysis to a syntonic region of distal chromosome 2, close to the locus for the ragged (Ra...

  18. Structural and functional characterization of the exonuclease I (sbcB) gene and gene product from Escherichia coli and a Markov chain analysis of DNA sequences

    International Nuclear Information System (INIS)

    Phillips, G.J.

    1987-01-01

    The nucleotide sequence for the structural gene for exonuclease I (sbcB) from Escherichia coli was determined. Two putative promotes for this gene were identified and were predicted to have weak transcription initiation activity. In addition, the sbcB coding region contains many non-optimal codons. These observations are consistent with the suggestions that sbcB is a poorly expressed gene. Several mutant exonuclease I genes were cloned onto pBR322 plasmids. These genes represented both sbcB and xonA mutation. One of the xonA mutation (xonA6) was associated with a 1.2-kb insertion of an IS-30 related mobile genetic element in the 3'-region of the gene. Two of the mutations (xonA2 and xonA6) encode unstable polypeptides. Determination of exonucleolytic activity on single-stranded DNA from cell extracts containing each of the cloned mutant genes revealed no correlation between residual exonucleolytic activity and the pheno-types of sbcB and xonA mutants. A proposal that the exonuclease I protein contains an additional activity besides its ability to degrade single-stranded DNA is presented. Characterization of E. coli strains which overproduce exonuclease I showed increased sensitivity to UV irradiation

  19. Real-time polymerase chain reaction assay for endogenous reference gene for specific detection and quantification of common wheat-derived DNA (Triticum aestivum L.).

    Science.gov (United States)

    Vautrin, Sonia; Zhang, David

    2007-01-01

    A species-specific endogenous reference gene system was developed for polymerase chain reaction (PCR)-based analysis in common wheat (Triticum aestivum L.) by targeting the ALMT1 gene, an aluminium-activated malate transporter. The primers and probe were elaborated for real-time PCR-based qualitative and quantitative assay. The size of amplified product is 95 base pairs. The specificity was assessed on 17 monocot and dicot plant species. The established real-time PCR assay amplified only T. aestivum-derived DNA; no amplification occurred on other phylogenetically related species, including durum wheat (T. durum). The robustness of the system was tested on the DNA of 15 common wheat cultivars using 20 000 genomic copies per PCR the mean cycle threshold (Ct) values of 24.02 +/- 0.251 were obtained. The absolute limits of detection and quantification of the real-time PCR assay were estimated to 2 and 20 haploid genome copies of common wheat, respectively. The linearity was experimentally validated on 2-fold serial dilutions of DNA from 650 to 20 000 haploid genome copies. All these results show that the real-time PCR assay developed on the ALMT1 gene is suitable to be used as an endogenous reference gene for PCR-based specific detection and quantification of T. aestivum-derived DNA in various applications, in particular for the detection and quantification of genetically modified materials in common wheat.

  20. Ginger extract mitigates ethanol-induced changes of alpha and beta - myosin heavy chain isoforms gene expression and oxidative stress in the heart of male wistar rats.

    Science.gov (United States)

    Shirpoor, Alireza; Zerehpoosh, Mitra; Ansari, Mohammad Hasan Khadem; Kheradmand, Fatemeh; Rasmi, Yousef

    2017-09-01

    The association between ethanol consumption and heart abnormalities, such as chamber dilation, myocyte damage, ventricular hypertrophy, and hypertension is well known. However, underlying molecular mediators involved in ethanol-induced heart abnormalities remain elusive. The aim of this study was to investigate the effect of chronic ethanol exposure on alpha and beta - myosin heavy chain (MHC) isoforms gene expression transition and oxidative stress in rats' heart. It was also planned to find out whether ginger extract mitigated the abnormalities induced by ethanol in rats' heart. Male wistar rats were divided into three groups of eight animals as follows: Control, ethanol, and ginger extract treated ethanolic (GETE) groups. After six weeks of treatment, the results revealed a significant increase in the β-MHC gene expression, 8- OHdG amount, and NADPH oxidase level. Furthermore, a significant decrease in the ratio of α-MHC/β-MHC gene expression to the amount of paraoxonase enzyme in the ethanol group compared to the control group was found. The consumption of Ginger extract along with ethanol ameliorated the changes in MHC isoforms gene expression and reduced the elevated amount of 8-OHdG and NADPH oxidase. Moreover, compared to the consumption of ethanol alone, it increased the paraoxonase level significantly. These findings indicate that ethanol-induced heart abnormalities may in part be associated with MHC isoforms changes mediated by oxidative stress, and that these effects can be alleviated by using ginger extract as an antioxidant molecule. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Search for $VH\\rightarrow$ leptons + $b\\bar{b}$ with the ATLAS experiment at the LHC

    CERN Document Server

    AUTHOR|(CDS)2079589

    The search for a Higgs boson decaying to a $b\\bar{b}$ pair is one of the key analyses ongoing at the ATLAS experiment. Despite being the largest branching ratio decay for a Standard Model Higgs boson, a large dataset is necessary to perform this analysis because of the very large backgrounds affecting the measurement. To discriminate the electroweak $H\\rightarrow b\\bar{b}$ signal from the large QCD backgrounds, the associated production of the Higgs with a $W$ or a $Z$ boson decaying leptonically is used. Different techniques have been proposed to enhance the signal over background ratio in the $VH(b\\bar{b})$ channel, from dedicated kinematic cuts, to a single large radius jet to identify the two collimated $b$'s in the Higgs high transverse momentum regime, to multivariate techniques. The high-$p_{\\mathrm{T}}$ approach, using a large radius jet to identify the $b$'s coming from the Higgs decay, has been tested against an analysis based on kinematic cuts for a dataset of $4.7$ fb$^{-1}$ luminosity at $\\sqrt{s...

  2. Development of a quantitative competitive reverse transcriptase polymerase chain reaction for the quantification of growth hormone gene expression in pigs

    Directory of Open Access Journals (Sweden)

    Maurício Machaim Franco

    2003-01-01

    Full Text Available After the advent of the genome projects, followed by the discovery of DNA polymorphisms, basic understanding of gene expression is the next focus to explain the association between polymorphisms and the level of gene expression, as well as to demonstrate the interaction among genes. Among the various techniques for the investigation of transcriptional profiling involving patterns of gene expression, quantitative PCR is the simplest analytical laboratory technique. The objective of this work was to analyze two strategies of a competitive PCR technique for the quantification of the pig growth hormone (GH gene expression. A pair of primers was designed targeting exons 3 and 5, and two competitive PCR strategies were performed, one utilizing a specific amplicon as a competitor, and the other utilizing a low-stringency PCR amplicon as a competitor. The latter strategy proved to be easier and more efficient, offering an accessible tool that can be used in any kind of competitive reaction, facilitating the study of gene expression patterns for both genetics and diagnostics of infectious diseases.

  3. MHC Class I Chain-Related Gene A Polymorphisms and Linkage Disequilibrium with HLA-B and HLA-C Alleles in Ocular Toxoplasmosis

    Science.gov (United States)

    Ayo, Christiane Maria; Camargo, Ana Vitória da Silveira; Frederico, Fábio Batista; Siqueira, Rubens Camargo; Previato, Mariana; Murata, Fernando Henrique Antunes; Silveira-Carvalho, Aparecida Perpétuo; Barbosa, Amanda Pires; Brandão de Mattos, Cinara de Cássia; de Mattos, Luiz Carlos

    2015-01-01

    This study investigated whether polymorphisms of the MICA (major histocompatibility complex class I chain-related gene A) gene are associated with eye lesions due to Toxoplasma gondii infection in a group of immunocompetent patients from southeastern Brazil. The study enrolled 297 patients with serological diagnosis of toxoplasmosis. Participants were classified into two distinct groups after conducting fundoscopic exams according to the presence (n = 148) or absence (n = 149) of ocular scars/lesions due to toxoplasmosis. The group of patients with scars/lesions was further subdivided into two groups according to the type of the ocular manifestation observed: primary (n = 120) or recurrent (n = 28). Genotyping of the MICA and HLA alleles was performed by the polymerase chain reaction-sequence specific oligonucleotide technique (PCR-SSO; One Lambda®) and the MICA-129 polymorphism (rs1051792) was identified by nested polymerase chain reaction (PCR-RFLP). Significant associations involving MICA polymorphisms were not found. Although the MICA*002~HLA-B*35 haplotype was associated with increased risk of developing ocular toxoplasmosis (P-value = 0.04; OR = 2.20; 95% CI = 1.05–4.60), and the MICA*008~HLA-C*07 haplotype was associated with protection against the development of manifestations of ocular toxoplasmosis (P-value = 0.009; OR: 0.44; 95% CI: 0.22–0.76), these associations were not statistically significant after adjusting for multiple comparisons. MICA polymorphisms do not appear to influence the development of ocular lesions in patients diagnosed with toxoplasmosis in this study population. PMID:26672749

  4. Pre-silencing of genes involved in the electron transport chain (ETC) pathway is associated with responsiveness to abatacept in rheumatoid arthritis.

    Science.gov (United States)

    Derambure, C; Dzangue-Tchoupou, G; Berard, C; Vergne, N; Hiron, M; D'Agostino, M A; Musette, P; Vittecoq, O; Lequerré, T

    2017-05-25

    In the current context of personalized medicine, one of the major challenges in the management of rheumatoid arthritis (RA) is to identify biomarkers that predict drug responsiveness. From the European APPRAISE trial, our main objective was to identify a gene expression profile associated with responsiveness to abatacept (ABA) + methotrexate (MTX) and to understand the involvement of this signature in the pathophysiology of RA. Whole human genome microarrays (4 × 44 K) were performed from a first subset of 36 patients with RA. Data validation by quantitative reverse-transcription (qRT)-PCR was performed from a second independent subset of 32 patients with RA. Gene Ontology and WikiPathways database allowed us to highlight the specific biological mechanisms involved in predicting response to ABA/MTX. From the first subset of 36 patients with RA, a combination including 87 transcripts allowed almost perfect separation between responders and non-responders to ABA/MTX. Next, the second subset of patients 32 with RA allowed validation by qRT-PCR of a minimal signature with only four genes. This latter signature categorized 81% of patients with RA with 75% sensitivity, 85% specificity and 85% negative predictive value. This combination showed a significant enrichment of genes involved in electron transport chain (ETC) pathways. Seven transcripts from ETC pathways (NDUFA6, NDUFA4, UQCRQ, ATP5J, COX7A2, COX7B, COX6A1) were significantly downregulated in responders versus non-responders to ABA/MTX. Moreover, dysregulation of these genes was independent of inflammation and was specific to ABA response. Pre-silencing of ETC genes is associated with future response to ABA/MTX and might be a crucial key to susceptibility to ABA response.

  5. Metagenomes of complex microbial consortia derived from different soils as sources for novel genes conferring formation of carbonyls from short-chain polyols on Escherichia coli.

    Science.gov (United States)

    Knietsch, Anja; Waschkowitz, Tanja; Bowien, Susanne; Henne, Anke; Daniel, Rolf

    2003-01-01

    Metagenomic DNA libraries from three different soil samples (meadow, sugar beet field, cropland) were constructed. The three unamplified libraries comprised approximately 1267000 independent clones and harbored approximately 4.05 Gbp of environmental DNA. Approximately 300000 recombinant Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from short-chain (C2 to C4) polyols such as 1,2-ethanediol, 2,3-butanediol, and a mixture of glycerol and 1,2-propanediol on indicator agar. Twenty-four positive E. COLI clones were obtained during the initial screen. Fifteen of them contained recombinant plasmids, designated pAK201-215, which conferred a stable carbonyl-forming phenotype on E. coli Sequencing revealed that the inserts of pAK201-215 encoded 26 complete and 14 incomplete predicted protein-encoding genes. Most of these genes were similar to genes with unknown functions from other microorganisms or unrelated to any other known gene. The further analysis was focused on the 7 plasmids (pAK204, pAK206, pAK208, and pAK210-213) recovered from the positive clones, which exhibited an NAD(H)-dependent alcohol oxidoreductase activity with polyols or the correlating carbonyls as substrates in crude extracts. Three genes (ORF6, ORF24, and ORF25) conferring this activity were identified during subcloning of the inserts of pAK204, pAK211, and pAK212. The sequences of the three deduced gene products revealed no significant similarities to known alcohol oxidoreductases, but contained putative glycine-rich regions, which are characteristic for binding of nicotinamide cofactors. Copyright 2003 S. Karger AG, Basel

  6. Expression of the Long Intergenic Non-Protein Coding RNA 665 (LINC00665) Gene and the Cell Cycle in Hepatocellular Carcinoma Using The Cancer Genome Atlas, the Gene Expression Omnibus, and Quantitative Real-Time Polymerase Chain Reaction.

    Science.gov (United States)

    Wen, Dong-Yue; Lin, Peng; Pang, Yu-Yan; Chen, Gang; He, Yun; Dang, Yi-Wu; Yang, Hong

    2018-05-05

    BACKGROUND Long non-coding RNAs (lncRNAs) have a role in physiological and pathological processes, including cancer. The aim of this study was to investigate the expression of the long intergenic non-protein coding RNA 665 (LINC00665) gene and the cell cycle in hepatocellular carcinoma (HCC) using database analysis including The Cancer Genome Atlas (TCGA), the Gene Expression Omnibus (GEO), and quantitative real-time polymerase chain reaction (qPCR). MATERIAL AND METHODS Expression levels of LINC00665 were compared between human tissue samples of HCC and adjacent normal liver, clinicopathological correlations were made using TCGA and the GEO, and qPCR was performed to validate the findings. Other public databases were searched for other genes associated with LINC00665 expression, including The Atlas of Noncoding RNAs in Cancer (TANRIC), the Multi Experiment Matrix (MEM), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) networks. RESULTS Overexpression of LINC00665 in patients with HCC was significantly associated with gender, tumor grade, stage, and tumor cell type. Overexpression of LINC00665 in patients with HCC was significantly associated with overall survival (OS) (HR=1.47795%; CI: 1.046-2.086). Bioinformatics analysis identified 469 related genes and further analysis supported a hypothesis that LINC00665 regulates pathways in the cell cycle to facilitate the development and progression of HCC through ten identified core genes: CDK1, BUB1B, BUB1, PLK1, CCNB2, CCNB1, CDC20, ESPL1, MAD2L1, and CCNA2. CONCLUSIONS Overexpression of the lncRNA, LINC00665 may be involved in the regulation of cell cycle pathways in HCC through ten identified hub genes.

  7. Quantitation of apolipoprotein epsilon gene expression by competitive polymerase chain reaction in a patient with familial apolipoprotein E deficiency.

    Science.gov (United States)

    Dobmeyer, J M; Rexin, M; Dobmeyer, T S; Klein, S A; Rossol, R; Feussner, G

    1998-06-22

    A simple method of obtaining semiquantitative and reliable data on apolipoprotein (apo) sigma gene expression is described. We detected apo sigma specific sequences by reverse transcription (rT)-PCR. For quantitative measurement, an apo sigma DNA standard was produced allowing the development of a competitive PCR-method. The efficiency of RNA extraction and cDNA synthesis was controlled by quantitation of a housekeeping gene (glyceraldehyde-3-phosphatedehydrogenase, G3PDH) in separate reactions. To imitate a defined induction of apo sigma gene expression, serial twofold dilutions of total RNA were reversely transcribed and the respective cDNAs used to perform a competitive apo sigma and G3PDH PCR. The change in apo sigma cDNA and G3PDH cDNA was 1.7-2.3-fold with an expected value of 2.0-fold. Standard deviations in three independently performed experiments were within a range of < 15% of the mean, indicating low intra-assay variation and high reproducibility. To illustrate this method, apo sigma gene expression was measured in a patient with complete lack of functional active apo E in comparison to healthy controls. The method presented here might be valuable in assessment of apo sigma gene expression in human disease.

  8. The Present and Future of Whole Genome Sequencing (WGS and Whole Metagenome Sequencing (WMS for Surveillance of Antimicrobial Resistant Microorganisms and Antimicrobial Resistance Genes across the Food Chain

    Directory of Open Access Journals (Sweden)

    Elena A. Oniciuc

    2018-05-01

    Full Text Available Antimicrobial resistance (AMR surveillance is a critical step within risk assessment schemes, as it is the basis for informing global strategies, monitoring the effectiveness of public health interventions, and detecting new trends and emerging threats linked to food. Surveillance of AMR is currently based on the isolation of indicator microorganisms and the phenotypic characterization of clinical, environmental and food strains isolated. However, this approach provides very limited information on the mechanisms driving AMR or on the presence or spread of AMR genes throughout the food chain. Whole-genome sequencing (WGS of bacterial pathogens has shown potential for epidemiological surveillance, outbreak detection, and infection control. In addition, whole metagenome sequencing (WMS allows for the culture-independent analysis of complex microbial communities, providing useful information on AMR genes occurrence. Both technologies can assist the tracking of AMR genes and mobile genetic elements, providing the necessary information for the implementation of quantitative risk assessments and allowing for the identification of hotspots and routes of transmission of AMR across the food chain. This review article summarizes the information currently available on the use of WGS and WMS for surveillance of AMR in foodborne pathogenic bacteria and food-related samples and discusses future needs that will have to be considered for the routine implementation of these next-generation sequencing methodologies with this aim. In particular, methodological constraints that impede the use at a global scale of these high-throughput sequencing (HTS technologies are identified, and the standardization of methods and protocols is suggested as a measure to upgrade HTS-based AMR surveillance schemes.

  9. Molecular detection of β-lactamase and integron genes in clinical strains of Klebsiella pneumoniae by multiplex polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Mansour Sedighi

    Full Text Available Abstract INTRODUCTION: Infections caused by β-lactamase-producing gram-negative bacteria, such as Klebsiella pneumoniae, are increasing globally with high morbidity and mortality. The aim of the current study was to determine antimicrobial susceptibility patterns and the prevalence of antibiotic resistance genes (β-lactamase and integron genes using multiplex PCR. METHODS One-hundred K. pneumoniae isolates were collected from different clinical samples. Antibiotic susceptibility testing was performed with thirteen different antibiotics. Multiplex-PCR was used to detect β-lactamase (bla TEM, bla CTX-M, bla SHV , bla VEB, bla PER, bla GES, bla VIM, bla IMP, bla OXA, and bla KPC and integron genes (int I, int II, and int III. RESULTS: The highest and lowest rate of resistance was exhibited against amikacin (93% and imipenem (8%, respectively. The frequency of β-lactamase-positive K. pneumoniae was 37%, and the prevalence of the bla TEM, bla CTX-M, bla SHV , bla VEB, bla PER, bla GES, bla VIM, bla IMP, bla OXA, and bla KPC genes was 38%, 24%, 19%, 12%, 6%, 11%, 33%, 0%, 28%, and 23%, respectively. Of the 100 isolates, eight (8% were positive for class I integrons; however, class II and III integrons were not detected in any of the strains. CONCLUSIONS: These results indicate co-carriage of a number of β-lactamase genes and antibiotic resistance integrons on the same plasmids harboring multi-drug resistance genes. It seems that these properties help to decrease treatment complications due to resistant bacterial infections by rapid detection, infection-control programs and prevention of transmission of drug resistance.

  10. Design and Generation of Humanized Single-chain Fv Derived from Mouse Hybridoma for Potential Targeting Application.

    Science.gov (United States)

    Khantasup, Kannika; Chantima, Warangkana; Sangma, Chak; Poomputsa, Kanokwan; Dharakul, Tararaj

    2015-12-01

    Single-chain variable antibody fragments (scFvs) are attractive candidates for targeted immunotherapy in several human diseases. In this study, a concise humanization strategy combined with an optimized production method for humanizing scFvs was successfully employed. Two antibody clones, one directed against the hemagglutinin of H5N1 influenza virus, the other against EpCAM, a cancer biomarker, were used to demonstrate the validity of the method. Heavy chain (VH) and light chain (VL) variable regions of immunoglobulin genes from mouse hybridoma cells were sequenced and subjected to the construction of mouse scFv 3-D structure. Based on in silico modeling, the humanized version of the scFv was designed via complementarity-determining region (CDR) grafting with the retention of mouse framework region (FR) residues identified by primary sequence analysis. Root-mean-square deviation (RMSD) value between mouse and humanized scFv structures was calculated to evaluate the preservation of CDR conformation. Mouse and humanized scFv genes were then constructed and expressed in Escherichia coli. Using this method, we successfully generated humanized scFvs that retained the targeting activity of their respective mouse scFv counterparts. In addition, the humanized scFvs were engineered with a C-terminal cysteine residue (hscFv-C) for site-directed conjugation for use in future targeting applications. The hscFv-C expression was extensively optimized to improve protein production yield. The protocol yielded a 20-fold increase in production of hscFv-Cs in E. coli periplasm. The strategy described in this study may be applicable in the humanization of other antibodies derived from mouse hybridoma.

  11. Identification of a Key Gene Involved in Branched-Chain Short Fatty Acids Formation in Natto by Transcriptional Analysis and Enzymatic Characterization in Bacillus subtilis.

    Science.gov (United States)

    Hong, Chenlu; Chen, Yangyang; Li, Lu; Chen, Shouwen; Wei, Xuetuan

    2017-03-01

    Natto as a fermented soybean product has many health benefits for human due to its rich nutritional and functional components. However, the unpleasant odor of natto, caused by the formation of branched-chain short fatty acids (BCFAs), prohibits the wide acceptance of natto products. This work is to identify the key gene of BCFAs formation and develop the guidance to reduce natto odor. Transcriptional analysis of BCFAs synthesis pathway genes was conducted in two Bacillus subtilis strains with obvious different BCFAs synthesis abilities. The transcriptional levels of bcd, bkdAA, and ptb in B. subtilis H-9 were 2.7-fold, 0.7-fold, and 8.9-fold higher than that of B. subtilis H-4, respectively. Therefore, the ptb gene with the highest transcriptional change was considered as the key gene in BCFAs synthesis. The ptb encoded enzyme Ptb was further characterized by inducible expression in Escherichia coli. The recombinant Ptb protein (about 32 kDa) was verified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis. The catalysis functions of Ptb were confirmed on substrates of isovaleryl-CoA and isobutyryl-CoA, and the higher catalysis efficiency of Ptb on isovaleryl-CoA explained the higher level of isovaleric acid in natto. The optimal activities of Ptb were observed at 50 °C and pH 8.0, and the enzymatic activity was inhibited by Ca 2+ , Zn 2+ , Ba 2+ , Mn 2+ , Cu 2+ , SDS, and EDTA. Collectively, this study reports a key gene responsible for BCFAs formation in natto fermentation and provides potential strategies to solve the odor problem.

  12. Cloning and inactivation of a branched-chain-amino-acid aminotransferase gene from Staphylococcus carnosus and characterization of the enzyme

    DEFF Research Database (Denmark)

    Madsen, Søren M; Beck, Hans Christian; Ravn, Peter

    2002-01-01

    . The first step in the catabolism is most likely a transamination reaction catalyzed by BCAA aminotransferases (IlvE proteins). In this study, we cloned the ilvE gene from S. carnosus by using degenerate oligonucleotides and PCR. We found that the deduced amino acid sequence was 80% identical...... were essential for optimal cell growth....

  13. Novel X-Linked Genes Revealed by Quantitative Polymerase Chain Reaction in the Green Anole, Anolis carolinensis

    Czech Academy of Sciences Publication Activity Database

    Rovatsos, M.; Altmanová, M.; Johnson Pokorná, Martina; Kratochvíl, L.

    2014-01-01

    Roč. 4, č. 11 (2014), s. 2107-2113 ISSN 2160-1836 R&D Projects: GA ČR GAP506/10/0718 Institutional support: RVO:67985904 Keywords : gene dosage * lizard * qPCR Subject RIV: EG - Zoology Impact factor: 3.198, year: 2014

  14. Evaluation of multiplex ligation-dependent probe amplification analysis versus multiplex polymerase chain reaction assays in the detection of dystrophin gene rearrangements in an Iranian population subset

    Directory of Open Access Journals (Sweden)

    Nayereh Nouri

    2014-01-01

    Full Text Available Background: The Duchenne muscular dystrophy (DMD gene is located in the short arm of the X chromosome (Xp21. It spans 2.4 Mb of the human genomic DNA and is composed of 79 exons. Mutations in the Dystrophin gene result in DMD and Becker muscular dystrophy. In this study, the efficiency of multiplex ligation-dependent probe amplification (MLPA over multiplex polymerase chain reaction (PCR assays in an Iranian population was investigated. Materials and Methods: Multiplex PCR assays and MLPA analysis were carried out in 74 patients affected with DMD. Results: Multiplex PCR detected deletions in 51% of the patients with DMD. MLPA analysis could determine all the deletions detected by the multiplex PCR. Additionally, MLPA was able to identify one more deletion and duplication in patients without detectable mutations by multiplex PCR. Moreover, MLPA precisely determined the exact size of the deletions. Conclusion: Although MLPA analysis is more sensitive for detection of deletions and duplications in the dystrophin gene, multiplex PCR might be used for the initial analysis of the boys affected with DMD in the Iranian population as it was able to detect 95% of the rearrangements in patients with DMD.

  15. Participation of different genes in the ruptures repair of double chain in Escherichia coli stumps exposed to gamma radiation

    International Nuclear Information System (INIS)

    Serment G, J. H.; Martinez M, E.; Alcantara D, D.

    2013-01-01

    All living organisms are naturally exposed to radiation from different sources. Ionizing radiation produces a plethora of lesions upon DNA that can be categorized as single and double strand breaks and base damage. Among them, unrepaired double strand breaks (Dbs) have the greatest biological significance, since they are responsible of cell death. In Escherichia coli this kind of lesions are repaired mostly by homologous recombination. In this work the participation of some recombination genes in the repair of Dbs is evaluated. Escherichia coli defective strains were exposed to gamma radiation and incubated for different periods in ideal conditions. Both micro electrophoresis and pulse field gel electrophoresis techniques were used to evaluate the kinetics of repair of such lesions, reflecting the importance of each defective gene in the process. (Author)

  16. Whey protein hydrolysate and branched-chain amino acids downregulate inflammation-related genes in vascular endothelial cells.

    Science.gov (United States)

    Da Silva, Marine S; Bigo, Cyril; Barbier, Olivier; Rudkowska, Iwona

    2017-02-01

    A recent review of clinical studies reports that dairy products may improve inflammation, a key etiologic cardiovascular disease risk factor. Yet the impact of dairy proteins on inflammatory markers is controversial and could be mediated by a differential impact of whey proteins and caseins. In this study, we hypothesized that whey proteins may have a greater anti-inflammatory effect than caseins. A model of human umbilical vein endothelial cells, with or without TNF-α stimulation, was used to investigate the effect of several dairy protein compounds on inflammation. Specifically, the impact of whey proteins either isolate or hydrolysate, caseins, and their amino acids on expression of TNF, VCAM-1, SOD2, and eNOS was examined. After a 24-hour incubation period, whey protein hydrolysate, leucine, isoleucine, and valine attenuated the TNF-α-induced endothelial inflammation by normalizing TNF and eNOS gene expression. This effect was not observed in unstimulated cells. Oppositely, caseins, a whey protein/casein mixture (1:4 w/w), and glutamine aggravated the TNF-α-induced TNF and SOD2 gene expression. Yet caseins and whey protein/casein mixture decreased VCAM-1 expression in both unstimulated and stimulated human umbilical vein endothelial cells. Measurement of TNF-α in cell supernatants by immunoassay substantiates gene expression data without reaching statistical significance. Taken together, this study showed that whey proteins and their major amino acids normalize TNF-α-induced proinflammatory gene expression in endothelial cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Identification of planorbids from Venezuela by polymerase chain reaction amplification and restriction fragment length polymorphism of internal transcriber spacer of the RNA ribosomal gene

    Directory of Open Access Journals (Sweden)

    Caldeira Roberta L

    2000-01-01

    Full Text Available Snails of the genus Biomphalaria from Venezuela were subjected to morphological assessment as well as polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP analysis. Morphological identification was carried out by comparison of characters of the shell and the male and female reproductive apparatus. The PCR-RFLP involved amplification of the internal spacer region ITS1 and ITS2 of the RNA ribosomal gene and subsequent digestion of this fragment by the restriction enzymes DdeI, MnlI, HaeIII and MspI. The planorbids were compared with snails of the same species and others reported from Venezuela and present in Brazil, Cuba and Mexico. All the enzymes showed a specific profile for each species, that of DdeI being the clearest. The snails were identified as B. glabrata, B. prona and B. kuhniana.

  18. The most common mutation causing medium-chain acyl-CoA dehydrogenase deficiency is strongly associated with a particular haplotype in the region of the gene

    DEFF Research Database (Denmark)

    Kølvraa, S; Gregersen, N; Blakemore, A I

    1991-01-01

    RFLP haplotypes in the region containing the medium-chain acyl-CoA dehydrogenase (MCAD) gene on chromosome 1 have been determined in patients with MCAD deficiency. The RFLPs were detected after digestion of patient DNA with the enzymes BanII. PstI and TaqI and with an MCAD cDNA-clone as a probe....... Of 32 disease-causing alleles studied, 31 possessed the previously published A----G point-mutation at position 985 of the cDNA. This mutation has been shown to result in inactivity of the MCAD enzyme. In at least 30 of the 31 alleles carrying this G985 mutation a specific RFLP haplotype was present...

  19. Fluxomic evidence for impaired contribution of short-chain acyl-CoA dehydrogenase to mitochondrial palmitate β-oxidation in symptomatic patients with ACADS gene susceptibility variants.

    Science.gov (United States)

    Dessein, Anne-Frédérique; Fontaine, Monique; Joncquel-Chevalier Curt, Marie; Briand, Gilbert; Sechter, Claire; Mention-Mulliez, Karine; Dobbelaere, Dries; Douillard, Claire; Lacour, Arnaud; Redonnet-Vernhet, Isabelle; Lamireau, Delphine; Barth, Magalie; Minot-Myhié, Marie-Christine; Kuster, Alice; de Lonlay, Pascale; Gregersen, Niels; Acquaviva, Cécile; Vianey-Saban, Christine; Vamecq, Joseph

    2017-08-01

    Despite ACADS (acyl-CoA dehydrogenase, short-chain) gene susceptibility variants (c.511C>T and c.625G>A) are considered to be non-pathogenic, encoded proteins are known to exhibit altered kinetics. Whether or not, they might affect overall fatty acid β-oxidation still remains, however, unclear. De novo biosynthesis of acylcarnitines by whole blood samples incubated with deuterated palmitate (16- 2 H 3 ,15- 2 H 2 -palmitate) is suitable as a fluxomic exploration to distinguish between normal and disrupted β-oxidation, abnormal profiles and ratios of acylcarnitines with different chain-lengths being indicative of the site for enzymatic blockade. Determinations in 301 control subjects of ratios between deuterated butyrylcarnitine and sum of deuterated C2 to C14 acylcarnitines served here as reference values to state specifically functional SCAD impairment in patients addressed for clinical and/or biological suspicion of a β-oxidation disorder. Functional SCAD impairment was found in 39 patients. The 27 patients accepting subsequent gene studies were all positive for ACADS mutations. Twenty-six of 27 patients were positive for c.625G>A variant. Twenty-three of 27 patients harbored susceptibility variants as sole ACADS alterations (18 homozygous and 3 heterozygous for c.625G>A, 2 compound heterozygous for c.625G>A/c.511C>T). Our present fluxomic assessment of SCAD suggests a link between ACADS susceptibility variants and abnormal β-oxidation consistent with known altered kinetics of these variants. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Immunoglobulin Heavy Chain Variable Region and Major Histocompatibility Region Genes Are Linked to Induced Graves' Disease in Females From Two Very Large Families of Recombinant Inbred Mice

    Science.gov (United States)

    Aliesky, Holly; Banuelos, Bianca; Magana, Jessica; Williams, Robert W.; Rapoport, Basil

    2014-01-01

    Graves' hyperthyroidism is caused by antibodies to the TSH receptor (TSHR) that mimic thyroid stimulation by TSH. Stimulating TSHR antibodies and hyperthyroidism can be induced by immunizing mice with adenovirus expressing the human TSHR A-subunit. Prior analysis of induced Graves' disease in small families of recombinant inbred (RI) female mice demonstrated strong genetic control but did not resolve trait loci for TSHR antibodies or elevated serum T4. We investigated the genetic basis for induced Graves' disease in female mice of two large RI families and combined data with earlier findings to provide phenotypes for 178 genotypes. TSHR antibodies measured by inhibition of TSH binding to its receptor were highly significantly linked in the BXD set to the major histocompatibility region (chromosome 17), consistent with observations in 3 other RI families. In the LXS family, we detected linkage between T4 levels after TSHR-adenovirus immunization and the Ig heavy chain variable region (Igvh, chromosome 12). This observation is a key finding because components of the antigen binding region of Igs determine antibody specificity and have been previously linked to induced thyroid-stimulating antibodies. Data from the LXS family provide the first evidence in mice of a direct link between induced hyperthyroidism and Igvh genes. A role for major histocompatibility genes has now been established for genetic susceptibility to Graves' disease in both humans and mice. Future studies using arrays incorporating variation in the complex human Ig gene locus will be necessary to determine whether Igvh genes are also linked to Graves' disease in humans. PMID:25051451

  1. 16S Ribosomal Ribonucleic Acid Gene Polymerase Chain Reaction in the Diagnosis of Bloodstream Infections: A Systematic Review and Meta-Analysis.

    Science.gov (United States)

    Su, Guoming; Fu, Zhuqing; Hu, Liren; Wang, Yueying; Zhao, Zuguo; Yang, Weiqing

    2015-01-01

    We aim to evaluate the accuracy of the 16S ribosomal ribonucleic acid (rRNA) gene polymerase chain reaction (PCR) test in the diagnosis of bloodstream infections through a systematic review and meta-analysis. A computerized literature search was conducted to identify studies that assessed the diagnostic value of 16S rRNA gene PCR test for bloodstream infections. Study quality was assessed using the revised Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. We calculated the sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR) and their 95% confidence intervals (95% CI) for each study. Summary receiver operating characteristic (SROC) curve was used to summarize overall test performance. Statistical analysis was performed in Meta-DiSc 1.4 and Stata/SE 12.0 software. Twenty-eight studies were included in our meta-analysis. Using random-effect model analysis, the pooled sensitivity, specificity, PLR, NLR, and DOR were 0.87 (95% CI, 0.85-0.89), 0.94 (95% CI, 0.93-0.95), 12.65 (95% CI, 8.04-19.90), 0.14 (95% CI, 0.08-0.24), and 116.76 (95% CI, 52.02-262.05), respectively. The SROC curve indicated that the area under the curve (AUC) was 0.9690 and the maximum joint sensitivity and specificity (Q*) was 0.9183. In addition, heterogeneity was statistically significant but was not caused by the threshold effect. Existing data suggest that 16S rRNA gene PCR test is a practical tool for the rapid screening of sepsis. Further prospective studies are needed to assess the diagnostic value of PCR amplification and DNA microarray hybridization of 16S rRNA gene in the future.

  2. Chain inequivalence in bovine methemoglobin. Progress report, December 1, 1979-November 30, 1980

    International Nuclear Information System (INIS)

    Ilan, Y.A.; Ilan, Y.; Chevion, M.; Czapski, G.

    1980-01-01

    Using pulse-radiolysis, a single-heme in the tetramer of bovine methemoglobin was reduced to the ferro state, producing a valence hybrid (VH). The kinetics of oxygen binding to the VH as well as the re-oxidation of the ferro-heme to the ferric state were studied as a function of pH. The kinetics of the oxygenation revealed the existence of two species, characterized by high and low affinities for oxygen that are associated with two quaternary structures (R and T, respectivey). Above pH 7.7 only the R state could be observed, while below pH 6.5 the T state was dominant. The reaction between the VH and ferricyanide at pH 7.75 (R state) consisted of two equal contributions attributed to the β and α subunits within the tetramer, respectively. At pH 6.3 (T state) a similar phenomenon was observed indicating chain inequivalences both in the T and the R states of methemoglobin. In the presence of inositol hexaphosphate, the T → R transition was shifted up by about 0.35 pH units. Yet similar rate constants exhibiting similar chain inequivalences have been measured

  3. Effects of dietary supplementation of lipid-coated zinc oxide on intestinal mucosal morphology and expression of the genes associated with growth and immune function in weanling pigs

    Directory of Open Access Journals (Sweden)

    Young Min Song

    2018-03-01

    Full Text Available Objective The present study was conducted to investigate the effects of a lipid-coated zinc oxide (ZnO supplement Shield Zn (SZ at the sub-pharmacological concentration on intestinal morphology and gene expression in weanling pigs, with an aim to gain insights into the mechanism of actions for SZ. Methods Forty 22-day-old weanling pigs were fed a nursery diet supplemented with 100 or 2,500 mg Zn/kg with uncoated ZnO (negative control [NC] or positive control [PC], respectively, 100, 200, or 400 mg Zn/kg with SZ for 14 days and their intestinal tissues were taken for histological and molecular biological examinations. The villus height (VH and crypt depth (CD of the intestinal mucosa were measured microscopically following preparation of the tissue specimen; expression of the genes associated with growth and immune function was determined using the real-time quantitative polymerase chain reaction. Results There was no difference in daily gain, gain:feed, and diarrhea score between the SZ group and either of NC and PC. The VH and VH:CD ratio were less for the SZ group vs NC in the jejunum and duodenum, respectively (p<0.05. The jejunal mucosal mRNA levels of insulin-like growth factor (IGF-I and interleukin (IL-10 regressed and tended to regress (p = 0.053 on the SZ concentration with a positive coefficient, respectively, whereas the IL-6 mRNA level regressed on the SZ concentration with a negative coefficient. The mRNA levels of IGF-I, zonula occludens protein-1, tumor necrosis factor-α, IL-6, and IL-10 did not differ between the SZ group and either of NC and PC; the occludin and transforming growth factor-β1 mRNA levels were lower for the SZ group than for PC. Conclusion The present results are interpreted to suggest that dietary ZnO provided by SZ may play a role in intestinal mucosal growth and immune function by modulating the expression of IGF-I, IL-6, and IL-10 genes.

  4. Basic Helix-Loop-Helix Transcription Factor Bmsage Is Involved in Regulation of fibroin H-chain Gene via Interaction with SGF1 in Bombyx mori

    Science.gov (United States)

    Li, Qiong-Yan; Hu, Wen-Bo; Zhou, Meng-Ting; Nie, Hong-Yi; Zhang, Yin-Xia; Peng, Zhang-Chuan; Zhao, Ping; Xia, Qing-You

    2014-01-01

    Silk glands are specialized in the synthesis of several secretory proteins. Expression of genes encoding the silk proteins in Bombyx mori silk glands with strict territorial and developmental specificities is regulated by many transcription factors. In this study, we have characterized B. mori sage, which is closely related to sage in the fruitfly Drosophila melanogaster. It is termed Bmsage; it encodes transcription factor Bmsage, which belongs to the Mesp subfamily, containing a basic helix–loop–helix motif. Bmsage transcripts were detected specifically in the silk glands of B. mori larvae through RT-PCR analysis. Immunoblotting analysis confirmed the Bmsage protein existed exclusively in B. mori middle and posterior silk gland cells. Bmsage has a low level of expression in the 4th instar molting stages, which increases gradually in the 5th instar feeding stages and then declines from the wandering to the pupation stages. Quantitative PCR analysis suggested the expression level of Bmsage in a high silk strain was higher compared to a lower silk strain on day 3 of the larval 5th instar. Furthermore, far western blotting and co-immunoprecipitation assays showed the Bmsage protein interacted with the fork head transcription factor silk gland factor 1 (SGF1). An electrophoretic mobility shift assay showed the complex of Bmsage and SGF1 proteins bound to the A and B elements in the promoter of fibroin H-chain gene(fib-H), respectively. Luciferase reporter gene assays confirmed the complex of Bmsage and SGF1 proteins increased the expression of fib-H. Together, these results suggest Bmsage is involved in the regulation of the expression of fib-H by being together with SGF1 in B. mori PSG cells. PMID:24740008

  5. Identification of clonally rearranged T-cell receptor beta chain genes in HTLV-I carriers as a potential instrument for early detection of neoplasia

    Directory of Open Access Journals (Sweden)

    M.M. Sales

    2005-05-01

    Full Text Available We analyzed the genetic recombination pattern of the T-cell receptor beta-chain gene (TCR-beta in order to identify clonal expansion of T-lymphocytes in 17 human T-lymphotropic virus type I (HTLV-I-positive healthy carriers, 7 of them with abnormal features in the peripheral blood lymphocytes. Monoclonal or oligoclonal expansion of T-cells was detected in 5 of 7 HTLV-I-positive patients with abnormal lymphocytes and unconfirmed diagnosis by using PCR amplification of segments of TCR-beta gene, in a set of reactions that target 102 different variable (V segments, covering all members of the 24 V families available in the gene bank, including the more recently identified segments of the Vbeta-5 and Vbeta-8 family and the two diversity beta segments. Southern blots, the gold standard method to detect T-lymphocyte clonality, were negative for all of these 7 patients, what highlights the low sensitivity of this method that requires a large amount of very high quality DNA. To evaluate the performance of PCR in the detection of clonality we also analyzed 18 leukemia patients, all of whom tested positive. Clonal expansion was not detected in any of the negative controls or healthy carriers without abnormal lymphocytes. In conclusion, PCR amplification of segments of rearranged TCR-beta is reliable and highly suitable for the detection of small populations of clonal T-cells in asymptomatic HTLV-I carriers who present abnormal peripheral blood lymphocytes providing an additional instrument for following up these patients with potentially higher risk of leukemia.

  6. Anatomical and Physiological Changes after Paclitaxel-Coated Balloon for Atherosclerotic De Novo Coronary Lesions: Serial IVUS-VH and FFR Study.

    Directory of Open Access Journals (Sweden)

    Soe Hee Ann

    Full Text Available To assess the serial changes of de novo coronary lesions treated with paclitaxel-coated balloon (PCB using intravascular ultrasound virtual histology (IVUS-VH and fractional flow reserve (FFR.This prospective observational study enrolled 27 patients with coronary artery disease treated with PCB who underwent coronary angiography, IVUS-VH and FFR before, immediately after intervention and at 9 months. 28 de novo lesions were successfully treated with PCB. Angiographic late luminal loss was 0.02 ± 0.27 mm. Mean vessel and lumen areas showed increase at 9 months (12.0 ± 3.5 mm(2 to 13.2 ± 3.9 mm(2, p <0.001; and 5.4 ± 1.2 mm(2 to 6.5 ± 1.8 mm(2, p <0.001, respectively. Although mean plaque area was unchanged (6.6 ± 2.6 mm2 to 6.6 ± 2.4 mm(2, p = 0.269, percent atheroma volume decreased significantly (53.4 ± 7.9% to 49.5 ± 6.4%, p = 0.002. The proportion of plaque compositions including fibrous, fibrofatty, dense calcium and necrotic core by IVUS-VH was unchanged at 9 months. The FFR of the treated lesion was 0.71 ± 0.13 pre-procedure, 0.87 ± 0.06 post-procedure and 0.84 ± 0.06 at follow-up.De novo coronary lesions treated with PCB showed persistent anatomical and physiological patency with plaque redistribution and vessel remodeling without chronic elastic recoil or plaque compositional change during follow-up.

  7. Novel real-time polymerase chain reaction assay for simultaneous detection of recurrent fusion genes in acute myeloid leukemia.

    Science.gov (United States)

    Dolz, Sandra; Barragán, Eva; Fuster, Óscar; Llop, Marta; Cervera, José; Such, Esperanza; De Juan, Inmaculada; Palanca, Sarai; Murria, Rosa; Bolufer, Pascual; Luna, Irene; Gómez, Inés; López, María; Ibáñez, Mariam; Sanz, Miguel A

    2013-09-01

    The recent World Health Organization classification recognizes different subtypes of acute myeloid leukemia (AML) according to the presence of several recurrent genetic abnormalities. Detection of these abnormalities and other molecular changes is of increasing interest because it contributes to a refined diagnosis and prognostic assessment in AML and enables monitoring of minimal residual disease. These genetic abnormalities can be detected using single RT-PCR, although the screening is still labor intensive and costly. We have developed a novel real-time RT-PCR assay to simultaneously detect 15 AML-associated rearrangements that is a simple and easily applicable method for use in clinical diagnostic laboratories. This method showed 100% specificity and sensitivity (95% confidence interval, 91% to 100% and 92% to 100%, respectively). The procedure was validated in a series of 105 patients with AML. The method confirmed all translocations detected using standard cytogenetics and fluorescence in situ hybridization and some additional undetected rearrangements. Two patients demonstrated two molecular rearrangements simultaneously, with BCR-ABL1 implicated in both, in addition to RUNX1-MECOM in one patient and PML-RARA in another. In conclusion, this novel real-time RT-PCR assay for simultaneous detection of multiple AML-associated fusion genes is a versatile and sensitive method for reliable screening of recurrent rearrangements in AML. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  8. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics

    Directory of Open Access Journals (Sweden)

    Peter Bannas

    2017-11-01

    Full Text Available Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL. The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL. VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv. scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa and nanobody-based human heavy chain antibodies (75 kDa can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb. Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo. Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo. Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody

  9. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics.

    Science.gov (United States)

    Bannas, Peter; Hambach, Julia; Koch-Nolte, Friedrich

    2017-01-01

    Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL). The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL). VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv). scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa) and nanobody-based human heavy chain antibodies (75 kDa) can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb). Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo . Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo . Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody-based human heavy

  10. Two very long chain fatty acid acyl-CoA synthetase genes, acs-20 and acs-22, have roles in the cuticle surface barrier in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Eriko Kage-Nakadai

    Full Text Available In multicellular organisms, the surface barrier is essential for maintaining the internal environment. In mammals, the barrier is the stratum corneum. Fatty acid transport protein 4 (FATP4 is a key factor involved in forming the stratum corneum barrier. Mice lacking Fatp4 display early neonatal lethality with features such as tight, thick, and shiny skin, and a defective skin barrier. These symptoms are strikingly similar to those of a human skin disease called restrictive dermopathy. FATP4 is a member of the FATP family that possesses acyl-CoA synthetase activity for very long chain fatty acids. How Fatp4 contributes to skin barrier function, however, remains to be elucidated. In the present study, we characterized two Caenorhabditis elegans genes, acs-20 and acs-22, that are homologous to mammalian FATPs. Animals with mutant acs-20 exhibited defects in the cuticle barrier, which normally prevents the penetration of small molecules. acs-20 mutant animals also exhibited abnormalities in the cuticle structure, but not in epidermal cell fate or cell integrity. The acs-22 mutants rarely showed a barrier defect, whereas acs-20;acs-22 double mutants had severely disrupted barrier function. Moreover, the barrier defects of acs-20 and acs-20;acs-22 mutants were rescued by acs-20, acs-22, or human Fatp4 transgenes. We further demonstrated that the incorporation of exogenous very long chain fatty acids into sphingomyelin was reduced in acs-20 and acs-22 mutants. These findings indicate that C. elegans Fatp4 homologue(s have a crucial role in the surface barrier function and this model might be useful for studying the fundamental molecular mechanisms underlying human skin barrier and relevant diseases.

  11. Two novel mutations in the BCKDK (branched-chain keto-acid dehydrogenase kinase) gene are responsible for a neurobehavioral deficit in two pediatric unrelated patients.

    Science.gov (United States)

    García-Cazorla, Angels; Oyarzabal, Alfonso; Fort, Joana; Robles, Concepción; Castejón, Esperanza; Ruiz-Sala, Pedro; Bodoy, Susanna; Merinero, Begoña; Lopez-Sala, Anna; Dopazo, Joaquín; Nunes, Virginia; Ugarte, Magdalena; Artuch, Rafael; Palacín, Manuel; Rodríguez-Pombo, Pilar; Alcaide, Patricia; Navarrete, Rosa; Sanz, Paloma; Font-Llitjós, Mariona; Vilaseca, Ma Antonia; Ormaizabal, Aida; Pristoupilova, Anna; Agulló, Sergi Beltran

    2014-04-01

    Inactivating mutations in the BCKDK gene, which codes for the kinase responsible for the negative regulation of the branched-chain α-keto acid dehydrogenase complex (BCKD), have recently been associated with a form of autism in three families. In this work, two novel exonic BCKDK mutations, c.520C>G/p.R174G and c.1166T>C/p.L389P, were identified at the homozygous state in two unrelated children with persistently reduced body fluid levels of branched-chain amino acids (BCAAs), developmental delay, microcephaly, and neurobehavioral abnormalities. Functional analysis of the mutations confirmed the missense character of the c.1166T>C change and showed a splicing defect r.[520c>g;521_543del]/p.R174Gfs1*, for c.520C>G due to the presence of a new donor splice site. Mutation p.L389P showed total loss of kinase activity. Moreover, patient-derived fibroblasts showed undetectable (p.R174Gfs1*) or barely detectable (p.L389P) levels of BCKDK protein and its phosphorylated substrate (phospho-E1α), resulting in increased BCKD activity and the very rapid BCAA catabolism manifested by the patients' clinical phenotype. Based on these results, a protein-rich diet plus oral BCAA supplementation was implemented in the patient homozygous for p.R174Gfs1*. This treatment normalized plasma BCAA levels and improved growth, developmental and behavioral variables. Our results demonstrate that BCKDK mutations can result in neurobehavioral deficits in humans and support the rationale for dietary intervention. © 2014 WILEY PERIODICALS, INC.

  12. Molecular characterization of elongase of very long-chain fatty acids 6 (elovl6) genes in Misgurnus anguillicaudatus and their potential roles in adaptation to cold temperature.

    Science.gov (United States)

    Chen, Jingwen; Cui, Yun; Yan, Jie; Jiang, Jimin; Cao, Xiaojuan; Gao, Jian

    2018-08-05

    Elongase of very long-chain fatty acids 6 (ELOVL6) is a rate-limiting enzyme catalyzing elongation of saturated and monounsaturated long-chain fatty acid. Although functional characteristics of Elovl6 have been demonstrated in mammal, the role of elovl6 in fish remains unclear. In this study, we firstly cloned three isoforms of elovl6 (elovl6a, elovl6b and elovl6-like) from loach (Misgurnus anguillicaudatus). Molecular characterizations of the three elovl6 isoforms in loach and their expressions of early life stages and different tissues were then determined. We also functionally characterized the three elovl6 isoforms using heterologous expression in baker's yeast. Results obtained here showed the three elovl6 proteins in loach can elongate C16:0 and C16:1 to C18:0 and C18:1, respectively. At last, to confirm the role of three loach elovl6 isoforms for elongation of fatty acids in adaption to cold stress, differences in skin histological structures, body fatty acid compositions, expressions of four hepatic lipogenesis or lipolysis related genes, and expressions of the three elovl6 isoforms and their related gene uncoupling protein 1 (ucp1) in different tissues were investigated in the loach reared in two different water temperatures (28 °C and 4 °C) for ten days. Cold stress increased ratios of C18/C16 and C20:5n-3/C18:3n-3 in loach body, and induced expressions of hepatic acyl-CoA delta-9 desaturase 1 (scd1), sterol-regulator element-binding protein 1 (srebp1), carnitine palmitoyltransferase 1 (cpt1) and fatty acid synthase (fas). Meanwhile, significant differences were found in expressions of the three elovl6 isoforms in different tissues between 28 °C and 4 °C groups. Overall, this study suggests that the three elovl6 isoforms in loach have ability to elongate C16 to C18, and elovl6 proteins in loach may play a role in adaptation to cold stress. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Falling chains

    OpenAIRE

    Wong, Chun Wa; Yasui, Kosuke

    2005-01-01

    The one-dimensional fall of a folded chain with one end suspended from a rigid support and a chain falling from a resting heap on a table is studied. Because their Lagrangians contain no explicit time dependence, the falling chains are conservative systems. Their equations of motion are shown to contain a term that enforces energy conservation when masses are transferred between subchains. We show that Cayley's 1857 energy nonconserving solution for a chain falling from a resting heap is inco...

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

    Science.gov (United States)

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

    2004-01-01

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

  15. The Ku Protein Complex Interacts with YY1, Is Up-Regulated in Human Heart Failure, and Represses α Myosin Heavy-Chain Gene Expression

    Science.gov (United States)

    Sucharov, Carmen C.; Helmke, Steve M.; Langer, Stephen J.; Perryman, M. Benjamin; Bristow, Michael; Leinwand, Leslie

    2004-01-01

    Human heart failure is accompanied by repression of genes such as α myosin heavy chain (αMyHC) and SERCA2A and the induction of fetal genes such as βMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human αMyHC promoter. We have now identified a region of the αMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human αMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases αMyHC mRNA expression and increases skeletal α-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the αMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human αMyHC promoter during heart failure. PMID:15367688

  16. A mutation in the dynein heavy chain gene compensates for energy deficit of mutant SOD1 mice and increases potentially neuroprotective IGF-1

    Directory of Open Access Journals (Sweden)

    Larmet Yves

    2011-04-01

    Full Text Available Abstract Background Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. ALS patients, as well as animal models such as mice overexpressing mutant SOD1s, are characterized by increased energy expenditure. In mice, this hypermetabolism leads to energy deficit and precipitates motor neuron degeneration. Recent studies have shown that mutations in the gene encoding the dynein heavy chain protein are able to extend lifespan of mutant SOD1 mice. It remains unknown whether the protection offered by these dynein mutations relies on a compensation of energy metabolism defects. Results SOD1(G93A mice were crossbred with mice harboring the dynein mutant Cramping allele (Cra/+ mice. Dynein mutation increased adipose stores in compound transgenic mice through increasing carbohydrate oxidation and sparing lipids. Metabolic changes that occurred in double transgenic mice were accompanied by the normalization of the expression of key mRNAs in the white adipose tissue and liver. Furthermore, Dynein Cra mutation rescued decreased post-prandial plasma triglycerides and decreased non esterified fatty acids upon fasting. In SOD1(G93A mice, the dynein Cra mutation led to increased expression of IGF-1 in the liver, increased systemic IGF-1 and, most importantly, to increased spinal IGF-1 levels that are potentially neuroprotective. Conclusions These findings suggest that the protection against SOD1(G93A offered by the Cramping mutation in the dynein gene is, at least partially, mediated by a reversal in energy deficit and increased IGF-1 availability to motor neurons.

  17. Rapid switch-off of the human myosin heavy chain IIX gene after heavy load muscle contractions is sustained for at least four days.

    Science.gov (United States)

    Andersen, J L; Gruschy-Knudsen, T

    2018-02-01

    Long-term heavy load contractions decrease the relative amount of the myosin heavy chain (MHC) IIX isoform in human skeletal muscle, but the timing of the down-regulation in the short term is unknown. Untrained subjects performed two resistance bouts, in two consecutive days, with one leg, the other leg serving as a control (age 24±1, n=5). Muscle biopsies were obtained in both legs before, immediately after, and 24, 54, and 96 hours after exercise. Serial cryosection analysis combined immunohistochemistry and ATPase histochemistry with In Situ hybridization to identify the distribution of MHC isoforms and their corresponding transcripts, enabling identification of transitional fibers. Fibers positive solely for MHC IIX mRNA decreased in the exercised leg throughout the study period. At 96 hours post-exercise, no fibers solely expressed MHC IIX mRNA. In contrast, the number of fibers expressing MHC IIA mRNA increased throughout the study period. The percentage of fibers expressing mRNA for MHC I was unchanged in both legs at all time points. Pronounced depletion of glycogen in the MHC IIX fibers of the exercised leg verifies that the type IIX fibers were active during the heavy load contractions. Major mismatch between MHC at the mRNA and protein levels was only found in the fibers of the exercised leg. These data provide unequivocal in situ evidence of an immediate shutdown of the MHC IIX gene after resistance exercise. A further novel finding was that the silencing of the MHC IIX gene is sustained at least 4 days after removal of the stimulus. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Methylmethane-sulphonate and X-ray-induced mutations in the Chinese hamster hprt gene: mRNA phenotyping using polymerase chain reactions

    International Nuclear Information System (INIS)

    Chaudhry, M.A.; Fox, Margaret

    1990-01-01

    Alterations in the hprt gene of Chinese hamster cells were determined in 71 spontaneous, methylmethane sulphonate (MMS)-and X-ray induced mutants, using the Southern blot hybridization technique. To investigate the possibility of small deletions, MMS-induced mutants were studied with proves derived from exons 3 and 9 but no evidence of specific deletion of these two exons was found. The polymerase chain reaction (PCR) was used to phenotype hprt transcripts in 48 MMS, X-ray and spontaneous Chinese hamster mutants by amplifying the coding region of their cDNA. Among 22 MMS-induced mutants the message was present in 16 instances. An analysis of 20 X-ray-induced mutants showed the presence of hprt mRNA in 11 of them with five having low levels of transcription. Among six spontaneous mutants, four were negative for mRNA on standard Northern blots and in one the message was only detected after PCR amplification. Direct DNA sequencing of 10 mutants revealed the presence of base substitutions in five of them while a 7 bp deletion was found in another. No mutations were found in another four mutants, suggesting the presence of mutation outside the coding region. (author)

  19. Development and validation of a SYBR Green I-based real-time polymerase chain reaction method for detection of haptoglobin gene deletion in clinical materials.

    Science.gov (United States)

    Soejima, Mikiko; Tsuchiya, Yuji; Egashira, Kouichi; Kawano, Hiroyuki; Sagawa, Kimitaka; Koda, Yoshiro

    2010-06-01

    Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin (Hp) antibodies. Being homozygous for the Hp gene deletion (HP(del)) is the only known cause of congenital anhaptoglobinemia, and clinical diagnosis of HP(del) before transfusion is important to prevent anaphylactic shock. We recently developed a 5'-nuclease (TaqMan) real-time polymerase chain reaction (PCR) method. A SYBR Green I-based duplex real-time PCR assay using two forward primers and a common reverse primer followed by melting curve analysis was developed to determine HP(del) zygosity in a single tube. In addition, to obviate initial DNA extraction, we examined serially diluted blood samples as PCR templates. Allelic discrimination of HP(del) yielded optimal results at blood sample dilutions of 1:64 to 1:1024. The results from 2231 blood samples were fully concordant with those obtained by the TaqMan-based real-time PCR method. The detection rate of the HP(del) allele by the SYBR Green I-based method is comparable with that using the TaqMan-based method. This method is readily applicable due to its low initial cost and analyzability using economical real-time PCR machines and is suitable for high-throughput analysis as an alternative method for allelic discrimination of HP(del).

  20. Polymerase chain reaction-hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation.

    Science.gov (United States)

    Xu, Chen; Zhang, Nan; Huo, Qianyu; Chen, Minghui; Wang, Rengfeng; Liu, Zhili; Li, Xue; Liu, Yunde; Bao, Huijing

    2016-04-15

    In this article, we discuss the polymerase chain reaction (PCR)-hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA-BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase-streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR-hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR-hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Increased Pathogen Identification in Vascular Graft Infections by the Combined Use of Tissue Cultures and 16S rRNA Gene Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Evelyne Ajdler-Schaeffler

    2018-06-01

    Full Text Available Background: Vascular graft infections (VGI are difficult to diagnose and treat, and despite redo surgery combined with antimicrobial treatment, outcomes are often poor. VGI diagnosis is based on a combination of clinical, radiological, laboratory and microbiological criteria. However, as many of the VGI patients are already under antimicrobial treatment at the time of redo surgery, microbiological identification is often difficult and bacterial cultures often remain negative rendering targeted treatment impossible. We aimed to assess the benefit of 16S rRNA gene polymerase chain reaction (broad-range PCR for better microbiological identification in patients with VGI.Methods: We prospectively analyzed the clinical, microbiological, and treatment data of patients enrolled in the observational Vascular Graft Cohort Study (VASGRA, University Hospital Zurich, Switzerland. The routine diagnostic work-up involved microbiological cultures of minced tissue samples, and the use of molecular techniques in parallel. Patient-related and microbiological data were assessed in descriptive analyses, and we calculated sensitivity, specificity, negative and positive predictive value for broad-range 16S rRNA gene PCR versus culture (considered as gold standard.Results: We investigated 60 patients (median age 66 years (Interquartile range [IQR] 59–75 with confirmed VGI between May 2013 and July 2017. The prevalence of antimicrobial pretreatment at the time of sampling was high [91%; median days of antibiotics 7 days (IQR 1–18]. We investigated 226 microbiological specimens. Thereof, 176 (78% were culture-negative and 50 (22% were culture-positive. There was a concordance of 70% (158/226 between conventional culture and broad-range PCR (sensitivity 58% (95% CI 43–72; specificity 74% (67–80%. Among the group of 176 culture-negative specimens, 46 specimens were broad-range PCR-positive resulting in identification of overall 69 species. Among the culture and

  2. Three monoclonal antibodies to the VHS virus glycoprotein: comparison of reactivity in relation to differences in immunoglobulin variable domain gene sequences

    DEFF Research Database (Denmark)

    Lorenzen, Niels; Cupit, P.M.; Secombes, C.J.

    2000-01-01

    and their neutralising activity was evident. Binding kinetic analyses by plasmon resonance identified differences in the dissociation rate constant (kd) as a possible explanation for the different reactivity levels of the MAbs. The Ig variable heavy (VH) and light (V kappa) domain gene sequences of the three hybridomas...... were compared. The inferred amino acid sequence of the two neutralising antibody VH domains differed by three amino acid residues (97% identity) and only one residue difference was evident in the Vk. domains. In contrast, IP1H3 shared only 38 and 39% identity with the 3F1A2 and 3F1H10 VH domains...... respectively and 49 and 50% identity with the 3F1A2 and 3F1H10 VK domains respectively. The neutralising antibodies were produced by hybridomas originating from the same fusion and the high nucleotide sequence homology of the variable Ig gene regions indicated that the plasma cell partners of the hybridomas...

  3. The properties of the single chicken MHC classical class II alpha chain (B-LA) gene indicate an ancient origin for the DR/E-like isotype of class II molecules

    DEFF Research Database (Denmark)

    Salomonsen, Jan; Marston, Denise; Avila, David

    2003-01-01

    for the cloning and sequencing of the cDNA. We found only one class II alpha chain transcript, which bears the major features of a classical class II alpha sequence, including the critical peptide-binding residues. The chicken sequence is more similar to human DR than to the DQ, DP, DO or DM isotypes, most...... the mammalian DR and E isotypes in three properties: the presence of the critical peptide-binding residues, the low level of polymorphism and sequence diversity, and the recombinational separation from the class II beta chain genes. These results indicate that the sequence features of this lineage are both......In mammals, there are MHC class II molecules with distinctive sequence features, such as the classical isotypes DR, DQ and DP. These particular isotypes have not been reported in non-mammalian vertebrates. We have isolated the class II (B-L) alpha chain from outbred chickens as the basis...

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

    Science.gov (United States)

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

    2015-04-01

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

  5. Relevance of the immunoglobulin VH somatic mutation status in patients with chronic lymphocytic leukemia treated with fludarabine, cyclophosphamide, and rituximab (FCR) or related chemoimmunotherapy regimens.

    Science.gov (United States)

    Lin, Katherine I; Tam, Constantine S; Keating, Michael J; Wierda, William G; O'Brien, Susan; Lerner, Susan; Coombes, Kevin R; Schlette, Ellen; Ferrajoli, Alessandra; Barron, Lynn L; Kipps, Thomas J; Rassenti, Laura; Faderl, Stefan; Kantarjian, Hagop; Abruzzo, Lynne V

    2009-04-02

    Although immunoglobulin V(H) mutation status (IgV(H) MS) is prognostic in patients with chronic lymphocytic leukemia (CLL) who are treated with alkylating agents or single-agent fludarabine, its significance in the era of chemoimmunotherapy is not known. We determined the IgV(H) somatic mutation status (MS) in 177 patients enrolled in a phase 2 study of fludarabine, cyclophosphamide, and rituximab (FCR) and in 127 patients treated with subsequent chemoimmunotherapy protocols. IgV(H) MS did not impact significantly on the complete remission (CR) rate of patients receiving FCR or related regimens. However, CR duration was significantly shorter in patients with CLL that used unmutated IgV(H) than those whose CLL used mutated IgV(H) (TTP 47% vs 82% at 6 years, P IgV(H) MS emerged as the only determinant of remission duration (hazard ratio 3.8, P IgV(H) status.

  6. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  7. The role of electric field shear stabilization of turbulence in the H-mode to VH-mode transition in DIII-D

    International Nuclear Information System (INIS)

    Burrell, K.H.; Osborne, T.H.; Groebner, R.J.; Rettig, C.L.

    1993-01-01

    VH-mode plasma exhibit energy confinement times up to 2.4 times the DIII-D/JET H-mode scaling relation and up to 3.9 times the value given by ITER89-P L-mode scaling. If this confinement improvement can be exploited in reactor plasmas, smaller prototype reactors with significantly lower unit cost can be produced. Accordingly, understanding and optimizing the confinement improvement is of significant interest. One of the possible explanations for this bulk confinement improvement is stabilization of turbulence by shear in the radial electric field, similar to the present explanation for the confinement improvement at the extreme plasma edge at the L to H transition. Preliminary measurements have shown that the region of the plasma where the electric field gradient is steepest broadens when the plasma goes from H-mode to VH-mode. More recent measurements have confirmed this broadening and have shown that the change in the electric field gradient occurs prior to the change in the thermal transport. In addition, transport analysis shows that the electric field shear increases in the same region between magnetic flux coordinate p=0.6 and 0.9 where the local thermal transport decreases. Furthermore, far infra-red (FIR) scattering measurements have detected density fluctuations in the region around p=0.8 which could be responsible for enhanced transport and which disappear at the time that the electric shear increases. These fluctuations appear as bursts of density fluctuations in the 0.5 to 1.5 MHz range. The time between bursts increases as the electric field shear increases. Once these bursts disappear, the major change in confinement takes place in most discharges. When isolated bursts occur, the heat and angular momentum pulse connected with the burst are detectable on the plasma profile diagnostics. (author) 13 refs., 4 figs

  8. The role of the radial electric field in confinement and transport in H-mode and VH-mode discharges in the DIII-D tokamak

    International Nuclear Information System (INIS)

    Gohil, P.; Burrell, K.H.; Groebner, R.J.; Osborne, T.H.; Doyle, E.J.; Rettig, C.L.

    1993-08-01

    Measurements of the radial electric field, E r , with high spatial and high time resolution in H-mode and VH-mode discharges in the DIII-D tokamak have revealed the significant influence of the shear in E r on confinement and transport in these discharges. These measurements are made using the DIII-D Charge Exchange Recombination (CER) System. At the L-H transition in DIII-D plasmas, a negative well-like E r profile develops just within the magnetic separatrix. A region of shear in E r results, which extends 1 to 2 cm into the plasma from the separatrix. At the transition, this region of sheared E r exhibits the greatest increase in impurity ion poloidal rotation velocity and the greatest reduction in plasma fluctuations. A transport barrier is formed in this same region of E x B velocity shear as is signified by large increases in the observed gradients of the ion temperature, the carbon density, the electron temperature and electron density. The development of the region of sheared E r , the increase in impurity ion poloidal rotation, the reduction in plasma turbulence, and the transport barrier all occur simultaneously at the L-H transition. Measurements of the radial electric field, plasma turbulence, thermal transport, and energy confinement have been performed for a wide range of plasma conditions and configurations. The results support the supposition that the progression of improving confinement at the L-H transition, into the H-mode and then into the VH-mode can be explained by the hypothesis of the suppression of plasma turbulence by the increasing penetration of the region of sheared E x B velocity into the plasma interior

  9. Light Scattering Study of Mixed Micelles Made from Elastin-Like Polypeptide Linear Chains and Trimers

    Science.gov (United States)

    Terrano, Daniel; Tsuper, Ilona; Maraschky, Adam; Holland, Nolan; Streletzky, Kiril

    Temperature sensitive nanoparticles were generated from a construct (H20F) of three chains of elastin-like polypeptides (ELP) linked to a negatively charged foldon domain. This ELP system was mixed at different ratios with linear chains of ELP (H40L) which lacks the foldon domain. The mixed system is soluble at room temperature and at a transition temperature (Tt) will form swollen micelles with the hydrophobic linear chains hidden inside. This system was studied using depolarized dynamic light scattering (DDLS) and static light scattering (SLS) to determine the size, shape, and internal structure of the mixed micelles. The mixed micelle in equal parts of H20F and H40L show a constant apparent hydrodynamic radius of 40-45 nm at the concentration window from 25:25 to 60:60 uM (1:1 ratio). At a fixed 50 uM concentration of the H20F, varying H40L concentration from 5 to 80 uM resulted in a linear growth in the hydrodynamic radius from about 11 to about 62 nm, along with a 1000-fold increase in VH signal. A possible simple model explaining the growth of the swollen micelles is considered. Lastly, the VH signal can indicate elongation in the geometry of the particle or could possibly be a result from anisotropic properties from the core of the micelle. SLS was used to study the molecular weight, and the radius of gyration of the micelle to help identify the structure and morphology of mixed micelles and the tangible cause of the VH signal.

  10. Systematic Analysis of Gene Expression Alterations and Clinical Outcomes for Long-Chain Acyl-Coenzyme A Synthetase Family in Cancer.

    Directory of Open Access Journals (Sweden)

    Wei-Ching Chen

    Full Text Available Dysregulated lipid metabolism contributes to cancer progression. Our previous study indicates that long-chain fatty acyl-Co A synthetase (ACSL 3 is essential for lipid upregulation induced by endoplasmic reticulum stress. In this report, we aimed to identify the role of ACSL family in cancer with systematic analysis and in vitro experiment. We explored the ACSL expression using Oncomine database to determine the gene alteration during carcinogenesis and identified the association between ACSL expression and the survival of cancer patient using PrognoScan database. ACSL1 may play a potential oncogenic role in colorectal and breast cancer and play a potential tumor suppressor role in lung cancer. Co-expression analysis revealed that ACSL1 was coexpressed with MYBPH, PTPRE, PFKFB3, SOCS3 in colon cancer and with LRRFIP1, TSC22D1 in lung cancer. In accordance with PrognoScan analysis, downregulation of ACSL1 in colon and breast cancer cell line inhibited proliferation, migration, and anchorage-independent growth. In contrast, increase of oncogenic property was observed in lung cancer cell line by attenuating ACSL1. High ACSL3 expression predicted a better prognosis in ovarian cancer; in contrast, high ACSL3 predicted a worse prognosis in melanoma. ACSL3 was coexpressed with SNUPN, TRIP13, and SEMA5A in melanoma. High expression of ACSL4 predicted a worse prognosis in colorectal cancer, but predicted better prognosis in breast, brain and lung cancer. ACSL4 was coexpressed with SERPIN2, HNRNPCL1, ITIH2, PROCR, LRRFIP1. High expression of ACSL5 predicted good prognosis in breast, ovarian, and lung cancers. ACSL5 was coexpressed with TMEM140, TAPBPL, BIRC3, PTPRE, and SERPINB1. Low ACSL6 predicted a worse prognosis in acute myeloid leukemia. ACSL6 was coexpressed with SOX6 and DARC. Altogether, different members of ACSLs are implicated in diverse types of cancer development. ACSL-coexpressed molecules may be used to further investigate the role of ACSL

  11. Ku70 is required for late B cell development and immunoglobulin heavy chain class switching.

    Science.gov (United States)

    Manis, J P; Gu, Y; Lansford, R; Sonoda, E; Ferrini, R; Davidson, L; Rajewsky, K; Alt, F W

    1998-06-15

    Immunoglobulin (Ig) heavy chain (HC) class switch recombination (CSR) is a late B cell process that involves intrachromosomal DNA rearrangement. Ku70 and Ku80 form a DNA end-binding complex required for DNA double strand break repair and V(D)J recombination. Ku70(-/-) (K70T) mice, like recombination activating gene (RAG)-1- or RAG-2-deficient (R1T or R2T) mice, have impaired B and T cell development at an early progenitor stage, which is thought to result at least in part from defective V(D)J recombination (Gu, Y., K.J. Seidl, G.A. Rathbun, C. Zhu, J.P. Manis, N. van der Stoep, L. Davidson, H.L. Cheng, J.M. Sekiguchi, K. Frank, et al. 1997. Immunity. 7:653-665; Ouyang, H., A. Nussenzweig, A. Kurimasa, V.C. Soares, X. Li, C. Cordon-Cardo, W. Li, N. Cheong, M. Nussenzweig, G. Iliakis, et al. 1997. J. Exp. Med. 186:921-929). Therefore, to examine the potential role of Ku70 in CSR, we generated K70T mice that carry a germline Ig HC locus in which the JH region was replaced with a functionally rearranged VH(D)JH and Ig lambda light chain transgene (referred to as K70T/HL mice). Previously, we have shown that B cells from R1T or R2T mice carrying these rearranged Ig genes (R1T/HL or R2T/HL mice) can undergo CSR to IgG isotypes (Lansford, R., J. Manis, E. Sonoda, K. Rajewsky, and F. Alt. 1998. Int. Immunol. 10:325-332). K70T/HL mice had significant numbers of peripheral surface IgM+ B cells, which generated serum IgM levels similar to those of R2T/HL mice. However, in contrast to R2T/HL mice, K70T/HL mice had no detectable serum IgG isotypes. In vitro culture of K70T/HL B cells with agents that induce CSR in normal or R2T/HL B cells did lead to the induction of germline CH transcripts, indicating that initial signaling pathways for CSR were intact in K70T/HL cells. However, treatment with such agents did not lead to detectable CSR by K70T/HL B cells, and instead, led to cell death within 72 h. We conclude that Ku70 is required for the generation of B cells that have

  12. Measurement of indicator genes using global complementary DNA (cDNA) amplification, by polyadenylic acid reverse transcriptase polymerase chain reaction (poly A RT-PCR): A feasibility study using paired samples from tissue and ductal juice in patients undergoing pancreatoduodenectomy.

    Science.gov (United States)

    Sanyal, Sudip; Siriwardena, Ajith K; Byers, Richard

    2018-06-01

    The aim of this study is to compare gene expression profiles in RNA isolated from pancreatic ductal juice with the RNA expression profiles of the same genes from matched intra-operative tissue samples from pancreatic tumours. Intra-operative sampling of pancreatic juice and collection of matched tissue samples was undertaken in patients undergoing pancreatoduodenectomy for clinically suspected pancreatic cancer and a precursor lesion, main-duct intraductal papillary mucinous neoplasm. RNA was isolated and Poly A PCR was used to globally amplify the RNA. Real-time polymerase chain reaction (RT-PCR) was used to measure expression levels of 17 genes selected from microarray studies. Spearman's rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue. The study was approved by Regional Ethics Committee. Mesothelin (MSLN) showed significant correlation (p cDNA using poly A PCR is technically feasible. Application of the technique to non-invasively obtained pancreatic juice during endoscopic assessment of tumours and the use of gene arrays of cancer indicator genes are the next steps in development of this technique. Copyright © 2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.

  13. CHAIN 2

    International Nuclear Information System (INIS)

    Bailey, D.

    1998-04-01

    The Second Processing Chain (CHAIN2) consists of a suite of ten programs which together provide a full local analysis of the bulk plasma physics within the JET Tokamak. In discussing these ten computational models this report is intended to fulfil two broad purposes. Firstly it is meant to be used as a reference source for any user of CHAIN2 data, and secondly it provides a basic User Manual sufficient to instruct anyone in running the CHAIN2 suite of codes. In the main report text each module is described in terms of its underlying physics and any associated assumptions or limitations, whilst deliberate emphasis is put on highlighting the physics and mathematics of the calculations required in deriving each individual datatype in the standard module PPF output. In fact each datatype of the CHAIN2 PPF output listed in Appendix D is cross referenced to the point in the main text where its evaluation is discussed. An effort is made not only to give the equation used to derive a particular data profile but also to explicitly define which external data sources are involved in the computational calculation

  14. Cloning and characterization of human very-long-chain acyl-CoA dehydrogenase cDNA, chromosomal assignment of the gene and identification in four patients of nine different mutations within the VLCAD gene

    DEFF Research Database (Denmark)

    Andresen, B S; Bross, P; Vianey-Saban, C

    1996-01-01

    Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of four straight-chain acyl-CoA dehydrogenase (ACD) enzymes, which are all nuclear encoded mitochondrial flavoproteins catalyzing the initial step in fatty acid beta-oxidation. We have used the very fast, Rapid Amplification of cDNA Ends (RACE...

  15. A single-chain fusion molecule consisting of peptide, major histocompatibility gene complex class I heavy chain and beta2-microglobulin can fold partially correctly, but binds peptide inefficiently

    DEFF Research Database (Denmark)

    Sylvester-Hvid, C; Buus, S

    1999-01-01

    of a recombinant murine MHC-I molecule, which could be produced in large amounts in bacteria. The recombinant MHC-I protein was expressed as a single molecule (PepSc) consisting of the antigenic peptide linked to the MHC-I heavy chain and further linked to human beta2-microglobulin (hbeta2m). The PepSc molecule...... electrophoresis (SDS-PAGE). Serological analysis revealed the presence of some, but not all, MHC-I-specific epitopes. Biochemically, PepSc could bind peptide, however, rather ineffectively. We suggest that a partially correctly refolded MHC-I has been obtained....

  16. Amplicon based RNA interference targeting V2 gene of cotton leaf curl Kokhran virus-Burewala strain can provide resistance in transgenic cotton plants

    Science.gov (United States)

    An RNAi based gene construct designated “C2” was used to target the V2 region of the cotton leaf curl virus (CLCuV) genome which is responsible for virus movement. The construct was transformed into two elite cotton varieties MNH-786 and VH-289. A shoot apex method of plant transformation using Agr...

  17. Single base mutation in the proα2(I) collagen gene that causes efficient splicing of RNA from exon 27 to exon 29 and synthesis of a shortened but in-frame proα2(I) chain

    International Nuclear Information System (INIS)

    Tromp, G.; Prockop, D.J.

    1988-01-01

    Previous observations demonstrated that a lethal variant of osteogenesis imperfecta had two altered alleles for proα2(I) chains of type I procollagen. One mutation produced a nonfunctioning allele in that there was synthesis of mRNA but no detectable synthesis of proα2(I) chains from the allele. The mutation in the other allele caused synthesis of shortened proα2(I) chains that lacked most or all of the 18 amino acids encoded by exon 28. Subclones of the proα2(I) gene were prepared from the proband's DNA and the DNA sequence was determined for a 582-base-pair (bp) region that extended from the last 30 bp of intervening sequence 26 to the first 26 bp of intervening sequence 29. Data from six independent subclones demonstrated that all had the same sequence as a previously isolated normal clone for the proα2(I) gene except that four subclones had a single base mutation at the 3' end of intervening sequence 27. The mutation was a substitution of guanine for adenine that changed the universal consensus sequence for the 3' splicing site of RNA from -AG- to -GG-. S1 nuclease experiments demonstrated that about half the proα2(I) mRNA in the proband's fibroblasts was abnormally spliced and that the major species of abnormal proα2(I) mRNA was completely spliced from the last codon of exon 27 to the first codon of exon 29. The mutation is apparently unique among RNA splicing mutations of mammalian systems in producing a shortened polypeptide chain that is in-frame in terms of coding sequences, that is used in the subunit assembly of a protein, and that contributes to a lethal phenotype

  18. Next-Generation DNA Sequencing of VH/VL Repertoires: A Primer and Guide to Applications in Single-Domain Antibody Discovery.

    Science.gov (United States)

    Henry, Kevin A

    2018-01-01

    Immunogenetic analyses of expressed antibody repertoires are becoming increasingly common experimental investigations and are critical to furthering our understanding of autoimmunity, infectious disease, and cancer. Next-generation DNA sequencing (NGS) technologies have now made it possible to interrogate antibody repertoires to unprecedented depths, typically by sequencing of cDNAs encoding immunoglobulin variable domains. In this chapter, we describe simple, fast, and reliable methods for producing and sequencing multiplex PCR amplicons derived from the variable regions (V H , V H H or V L ) of rearranged immunoglobulin heavy and light chain genes using the Illumina MiSeq platform. We include complete protocols and primer sets for amplicon sequencing of V H /V H H/V L repertoires directly from human, mouse, and llama lymphocytes as well as from phage-displayed V H /V H H/V L libraries; these can be easily be adapted to other types of amplicons with little modification. The resulting amplicons are diverse and representative, even using as few as 10 3 input B cells, and their generation is relatively inexpensive, requiring no special equipment and only a limited set of primers. In the absence of heavy-light chain pairing, single-domain antibodies are uniquely amenable to NGS analyses. We present a number of applications of NGS technology useful in discovery of single-domain antibodies from phage display libraries, including: (i) assessment of library functionality; (ii) confirmation of desired library randomization; (iii) estimation of library diversity; and (iv) monitoring the progress of panning experiments. While the case studies presented here are of phage-displayed single-domain antibody libraries, the principles extend to other types of in vitro display libraries.

  19. A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent

    DEFF Research Database (Denmark)

    Alifrangis, Michael; Enosse, Sonia; Pearce, Richard

    2005-01-01

    Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum dihydrofolate reductase (dhfr), and dihydropteroate synthetase (dhps), and chloroquine resistance transporter (Pfcrt) genes are used as molecular markers of P. falciparum resistance to sulfadoxine/pyrimethamine and chloroquine....... However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)- and enzyme-linked immunosorbent assay (ELISA...

  20. Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in the rumen epithelium

    Science.gov (United States)

    The rumen is lined on the luminal side by a stratified squamous epithelium that is responsible for not only absorption, but also transport, extensive short-chain fatty acid (SCFA) metabolism and protection. Butyrate has been demonstrated to initiate the differentiation of the tissue following intro...

  1. Dietary medium-chain saturated fatty acids induce gene expression of energy metabolism-related pathways in adipose tissue of abdominally obese subjects

    NARCIS (Netherlands)

    Matualatupauw, J.C.; Bohl, Mette; Gregersen, Søren; Hermansen, K.; Afman, L.A.

    2017-01-01

    Background:Dietary medium-chain saturated fatty acids (MC-SFAs) have been shown to reduce total body fat. Previously, we showed that MC-SFAs prevent body fat accumulation, despite weight gain. Here, we aim to explore potential molecular mechanisms underlying the protective effect of MC-SFAs on body

  2. Heavy Chain Diseases

    Science.gov (United States)

    ... of heavy chain produced: Alpha Gamma Mu Alpha Heavy Chain Disease Alpha heavy chain disease (IgA heavy ... the disease or lead to a remission. Gamma Heavy Chain Disease Gamma heavy chain disease (IgG heavy ...

  3. Recurrent vomiting and ethylmalonic aciduria associated with rare mutations in the short-chain acyl-CoA dehydrogenase (SCAD) gene

    DEFF Research Database (Denmark)

    Seidel, J.; Streck, S.; Bellstedt, K.

    2003-01-01

    blood spots. Neither of the frequent SCAD gene variants 625G>A and 511C>T was present, but direct sequencing of the promoter and coding regions of the SCAD gene revealed that the patient had mutations on both alleles: 417G>C (Trpl15Cys) and 1095G>T (Gln341His). Neither mutation has been described before...

  4. Postnatal changes of gene expression for tissue inhibitors of metalloproteinase-1 and -2 and cystatins S and C, in rat submandibular gland demonstrated by quantitative reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Nishiura, T; Abe, K

    1999-01-01

    The rat submandibular gland is not fully developed at birth and definitive differentiation takes place postnatally. The steady-state mRNA expression for the four proteinase inhibitor molecules, tissue inhibitors of metalloproteinase (TIMP)-1 and -2, and cystatins S and C, and for a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), in rat submandibular glands was measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) at different stages of postnatal development. The gene-expression patterns of TIMP-1 and -2 relative to G3PDH were similar to each other. The TIMP-2 and cystatin C genes were more highly expressed than those of TIMP-1 and cystatin S at all stages. Moreover, the gene expressions of TIMP-1 and -2, and of cystatins S and C, were predominant between 1 and 7, and 7 and 12 weeks of age, respectively, and coincided developmentally with the regression of terminal tubule cells and the differentiation of granular convoluted tubule cells, respectively. Quantitative competitive RT-PCR allowed accurate measurement of small changes in the steady-state concentrations of these proteinase-inhibitor mRNA molecules.

  5. A G {r_arrow} A transition at position IVS-11 +1 of the HEX A {alpha}-chain gene in a non-Ashkenazic Mexican Tay-Sachs infant

    Energy Technology Data Exchange (ETDEWEB)

    Miranda, S.R.P.; Gwon, S.; DeGasperi, R. [New York Univ. Medical Center, NY (United States)] [and others

    1994-09-01

    Tay-Sachs disease (TSD) is an autosomal recessive storage disorder caused by a deficiency of the lysosomal enzyme, {beta}-N-acetylhexosaminidase A (Hex A), a heteropolymer composed of two polypeptides, {alpha} and {beta}. Mutations in the {alpha}-chain gene render the enzyme defective, resulting in the accumulation of g{sub m2} ganglioside in the nervous system. Deficiency of Hex A was detected in a non-Ashkenazic girl of Mexican origin indicating infantile onset of TSD. Molecular investigation of the {alpha}-chain gene excluded the typical Ashkenazic 4 bp insertion in the exon 11 and the intron 12 splice-junction mutations by Hae III and Dde I restriction analysis, respectively. Single strand conformation polymorphism (SSCP) analysis showed a different pattern in the sample where exon 11 and flanking regions were amplified in the patient DNA as compared to the migration of control DNA. Sequencing of PCR amplified genomic DNA containing exon 11 and flanking intronic regions showed a single base substitution (G {r_arrow} A) at position IVS-11 +1. This mutation creates a recognition site for the restriction enzyme Mbo II. Digestion of exon 11 and flanking regions with Mbo II demonstrated homozygosity of the patient for this mutation and heterozygosity in the mother. mRNA from cultured fibroblasts obtained from a normal control and from the propositus was reverse transcribed. The cDNAs coding for Hex A {alpha}-chain were amplified in four overlapping fragments. In the patient sample it was not possible to amplify the fragment containing the exon 11/intron 11 junction, indicating that this mutation alters normal RNA processing of the Hex A pre-mRNA resulting in the deficiency of Hex A activity.

  6. Evaluation of RNA extraction methods and identification of putative reference genes for real-time quantitative polymerase chain reaction expression studies on olive (Olea europaea L.) fruits.

    Science.gov (United States)

    Nonis, Alberto; Vezzaro, Alice; Ruperti, Benedetto

    2012-07-11

    Genome wide transcriptomic surveys together with targeted molecular studies are uncovering an ever increasing number of differentially expressed genes in relation to agriculturally relevant processes in olive (Olea europaea L). These data need to be supported by quantitative approaches enabling the precise estimation of transcript abundance. qPCR being the most widely adopted technique for mRNA quantification, preliminary work needs to be done to set up robust methods for extraction of fully functional RNA and for the identification of the best reference genes to obtain reliable quantification of transcripts. In this work, we have assessed different methods for their suitability for RNA extraction from olive fruits and leaves and we have evaluated thirteen potential candidate reference genes on 21 RNA samples belonging to fruit developmental/ripening series and to leaves subjected to wounding. By using two different algorithms, GAPDH2 and PP2A1 were identified as the best reference genes for olive fruit development and ripening, and their effectiveness for normalization of expression of two ripening marker genes was demonstrated.

  7. Fusion of a viral antigen to invariant chain leads to augmented T-cell immunity and improved protection in gene-gun DNA-vaccinated mice

    DEFF Research Database (Denmark)

    Grujic, Mirjana; Holst, Peter J; Christensen, Jan P

    2009-01-01

    It has recently been demonstrated that a recombinant replication-deficient human adenovirus 5 (Ad5) vector expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) fused to the p31 invariant (Ii) chain confers broad, long-lasting T-cell immunity that completely protects C57BL/6 mice...... with the unlinked construct. In contrast, substantial protection against peripheral challenge was not observed. Additional experiments with T-cell subset-depleted or perforin-deficient mice revealed that virus control in vaccinated mice depends critically on cytotoxic CD8(+) T cells. Finally, priming with the naked...

  8. Transcript Quantification of Genes Involved in Steviol Glycoside Biosynthesis in Stevia rebaudiana Bertoni by Real-Time Polymerase Chain Reaction (RT-PCR).

    Science.gov (United States)

    Modi, Arpan; Kumar, Nitish; Narayanan, Subhash

    2016-01-01

    Stevia (Stevia rebaudiana Bertoni) is a medicinal plant having sweet, diterpenoid glycosides known as steviol glycosides which are 200-300 times sweeter than sucrose (0.4 % solution). They are synthesized mainly in the leaves via plastid localized 2-C-methyl-D-erythrose-4-phosphate pathway (MEP pathway). Fifteen genes are involved in the formation of these glycosides. In the present protocol, a method for the quantification of transcripts of these genes is shown. The work involves RNA extraction and cDNA preparation, and therefore, procedures for the confirmation of DNA-free cDNA preparation have also been illustrated. Moreover, details of plant treatments are not mentioned as this protocol may apply to relative gene expression profile in any medicinal plant with any treatment. The treatments are numbered as T0 (Control), T1, T2, T3, and T4.

  9. VH-92A Presidential Helicopter (VH-92A)

    Science.gov (United States)

    2015-12-01

    provide safe, reliable, and timely transportation for the President, Vice President, Foreign Heads of State, and other official parties as directed by...the Director of the White House Military Office. Presidential helicopter transportation requirements are executed by Marine Helicopter Squadron One...Review Jul 2016 Jul 2016 Jan 2017 Jul 2016 Milestone C Jan 2019 Jan 2019 Jul 2019 Jan 2019 IOT &E Complete Mar 2020 Mar 2020 Sep 2020 Mar 2020 IOC Jul

  10. In situ analysis of the variable heavy chain gene of an IgM/IgG-expressing follicular lymphoma - Evidence for interfollicular trafficking of tumor cells

    NARCIS (Netherlands)

    Aarts, Wilhelmina M.; Bende, Richard J.; Vaandrager, Jan-Willem; Kluin, Philip M.; Langerak, Anton W.; Pals, Steven T.; van Noesel, Carel J. M.

    2002-01-01

    is generally assumed that follicular lymphomas (FL) not only morphologically resemble normal germinal centers but have retained some functional characteristics of their non-neoplastic counterparts as well. Recent IgV gene analyses on a panel of FLs however, strongly suggested that FLs do not retain

  11. Expression of multiple slow myosin heavy chain genes reveals a diversity of zebrafish slow twitch muscle fibres with differing requirements for Hedgehog and Prdm1 activity.

    Science.gov (United States)

    Elworthy, Stone; Hargrave, Murray; Knight, Robert; Mebus, Katharina; Ingham, Philip W

    2008-06-01

    The zebrafish embryo develops a series of anatomically distinct slow twitch muscle fibres that characteristically express genes encoding lineage-specific isoforms of sarcomeric proteins such as MyHC and troponin. We show here that different subsets of these slow fibres express distinct members of a tandem array of slow MyHC genes. The first slow twitch muscle fibres to differentiate, which are specified by the activity of the transcription factor Prdm1 (also called Ubo or Blimp1) in response to Hedgehog (Hh) signalling, express the smyhc1 gene. Subsequently, secondary slow twitch fibres differentiate in most cases independently of Hh activity. We find that although some of these later-forming fibres also express smyhc1, others express smyhc2 or smyhc3. We show that the smyhc1-positive fibres express the ubo (prdm1) gene and adopt fast twitch fibre characteristics in the absence of Prdm1 activity, whereas those that do not express smyhc1 can differentiate independently of Prdm1 function. Conversely, some smyhc2-expressing fibres, although independent of Prdm1 function, require Hh activity to form. The adult trunk slow fibres express smyhc2 and smyhc3, but lack smyhc1 expression. The different slow fibres in the craniofacial muscles variously express smyhc1, smyhc2 and smyhc3, and all differentiate independently of Prdm1.

  12. Detection of a Bacteriophage Gene Encoding a Mu-like Portal Protein in Haemophilus parasuis Reference Strains and Field Isolates by Nested Polymerase Chain Reaction

    Science.gov (United States)

    A nested PCR assay was developed to determine the presence of a gene encoding a bacteriophage Mu-like portal protein, gp29, in 15 reference strains and 31 field isolates of Haemophilus parasuis. Specific primers, based on the gene’s sequence, were utilized. A majority of the virulent reference strai...

  13. Dynein light chain family in Tetrahymena thermophila.

    Science.gov (United States)

    Wilkes, David E; Rajagopalan, Vidyalakshmi; Chan, Clarence W C; Kniazeva, Ekaterina; Wiedeman, Alice E; Asai, David J

    2007-02-01

    Dyneins are large protein complexes that produce directed movement on microtubules. In situ, dyneins comprise combinations of heavy, intermediate, light-intermediate, and light chains. The light chains regulate the locations and activities of dyneins but their functions are not completely understood. We have searched the recently sequenced Tetrahymena thermophila macronuclear genome to describe the entire family of dynein light chains expressed in this organism. We identified fourteen genes encoding putative dynein light chains and seven genes encoding light chain-like proteins. RNA-directed PCR revealed that all 21 genes were expressed. Quantitative real time reverse transcription PCR showed that many of these genes were upregulated after deciliation, indicating that these proteins are present in cilia. Using the nomenclature developed in Chlamydomonas, Tetrahymena expresses two isoforms each of LC2, LC4, LC7, and Tctex1, three isoforms of p28, and six LC8/LC8-like isoforms. Tetrahymena also expresses two LC3-like genes. No Tetrahymena orthologue was found for Chlamydomonas LC5 or LC6. This study provides a complete description of the different genes and isoforms of the dynein light chains that are expressed in Tetrahymena, a model organism in which the targeted manipulation of genes is straightforward. (c) 2006 Wiley-Liss, Inc.

  14. Characterization of Biomphalaria orbignyi, Biomphalaria peregrina and Biomphalaria oligoza by polymerase chain reaction and restriction enzyme digestion of the internal transcribed spacer region of the RNA ribosomal gene

    Directory of Open Access Journals (Sweden)

    Spatz Linus

    2000-01-01

    Full Text Available The correct identification of Biomphalaria oligoza, B. orbignyi and B. peregrina species is difficult due to the morphological similarities among them. B. peregrina is widely distributed in South America and is considered a potential intermediate host of Schistosoma mansoni. We have reported the use of the polymerase chain reaction and restriction fragment length polymorphism analysis of the internal transcribed spacer region of the ribosomal DNA for the molecular identification of these snails. The snails were obtained from different localities of Argentina, Brazil and Uruguay. The restriction patterns obtained with MvaI enzyme presented the best profile to identify the three species. The profiles obtained with all enzymes were used to estimate genetic similarities among B. oligoza, B. peregrina and B. orbignyi. This is also the first report of B. orbignyi in Uruguay.

  15. X-linked Alport syndrome associated with a synonymous p.Gly292Gly mutation alters the splicing donor site of the type IV collagen alpha chain 5 gene.

    Science.gov (United States)

    Fu, Xue Jun; Nozu, Kandai; Eguchi, Aya; Nozu, Yoshimi; Morisada, Naoya; Shono, Akemi; Taniguchi-Ikeda, Mariko; Shima, Yuko; Nakanishi, Koichi; Vorechovsky, Igor; Iijima, Kazumoto

    2016-10-01

    X-linked Alport syndrome (XLAS) is a progressive hereditary nephropathy caused by mutations in the type IV collagen alpha chain 5 gene (COL4A5). Although many COL4A5 mutations have previously been identified, pathogenic synonymous mutations have not yet been described. A family with XLAS underwent mutational analyses of COL4A5 by PCR and direct sequencing, as well as transcript analysis of potential splice site mutations. In silico analysis was also conducted to predict the disruption of splicing factor binding sites. Immunohistochemistry (IHC) of kidney biopsies was used to detect α2 and α5 chain expression. We identified a hemizygous point mutation, c.876A>T, in exon 15 of COL4A5 in the proband and his brother, which is predicted to result in a synonymous amino acid change, p.(Gly292Gly). Transcript analysis showed that this mutation potentially altered splicing because it disrupted the splicing factor binding site. The kidney biopsy of the proband showed lamellation of the glomerular basement membrane (GBM), while IHC revealed negative α5(IV) staining in the GBM and Bowman's capsule, which is typical of XLAS. This is the first report of a synonymous COL4A5 substitution being responsible for XLAS. Our findings suggest that transcript analysis should be conducted for the future correct assessment of silent mutations.

  16. Chain reaction

    International Nuclear Information System (INIS)

    Balogh, Brian.

    1991-01-01

    Chain Reaction is a work of recent American political history. It seeks to explain how and why America came to depend so heavily on its experts after World War II, how those experts translated that authority into political clout, and why that authority and political discretion declined in the 1970s. The author's research into the internal memoranda of the Atomic Energy Commission substantiates his argument in historical detail. It was not the ravages of American anti-intellectualism, as so many scholars have argued, that brought the experts back down to earth. Rather, their decline can be traced to the very roots of their success after World War II. The need to over-state anticipated results in order to garner public support, incessant professional and bureaucratic specialization, and the sheer proliferation of expertise pushed arcane and insulated debates between experts into public forums at the same time that a broad cross section of political participants found it easier to gain access to their own expertise. These tendencies ultimately undermined the political influence of all experts. (author)

  17. Mutations in the Caenorhabditis elegans orthologs of human genes required for mitochondrial tRNA modification cause similar electron transport chain defects but different nuclear responses.

    Science.gov (United States)

    Navarro-González, Carmen; Moukadiri, Ismaïl; Villarroya, Magda; López-Pascual, Ernesto; Tuck, Simon; Armengod, M-Eugenia

    2017-07-01

    Several oxidative phosphorylation (OXPHOS) diseases are caused by defects in the post-transcriptional modification of mitochondrial tRNAs (mt-tRNAs). Mutations in MTO1 or GTPBP3 impair the modification of the wobble uridine at position 5 of the pyrimidine ring and cause heart failure. Mutations in TRMU affect modification at position 2 and cause liver disease. Presently, the molecular basis of the diseases and why mutations in the different genes lead to such different clinical symptoms is poorly understood. Here we use Caenorhabditis elegans as a model organism to investigate how defects in the TRMU, GTPBP3 and MTO1 orthologues (designated as mttu-1, mtcu-1, and mtcu-2, respectively) exert their effects. We found that whereas the inactivation of each C. elegans gene is associated with a mild OXPHOS dysfunction, mutations in mtcu-1 or mtcu-2 cause changes in the expression of metabolic and mitochondrial stress response genes that are quite different from those caused by mttu-1 mutations. Our data suggest that retrograde signaling promotes defect-specific metabolic reprogramming, which is able to rescue the OXPHOS dysfunction in the single mutants by stimulating the oxidative tricarboxylic acid cycle flux through complex II. This adaptive response, however, appears to be associated with a biological cost since the single mutant worms exhibit thermosensitivity and decreased fertility and, in the case of mttu-1, longer reproductive cycle. Notably, mttu-1 worms also exhibit increased lifespan. We further show that mtcu-1; mttu-1 and mtcu-2; mttu-1 double mutants display severe growth defects and sterility. The animal models presented here support the idea that the pathological states in humans may initially develop not as a direct consequence of a bioenergetic defect, but from the cell's maladaptive response to the hypomodification status of mt-tRNAs. Our work highlights the important association of the defect-specific metabolic rewiring with the pathological phenotype

  18. Detection of methicillin-resistance gene in Staphylococcus epidermidis strains isolated from patients in Al-Zahra Hospital using polymerase chain reaction and minimum inhibitory concentration methods

    Directory of Open Access Journals (Sweden)

    Ebtehaj Pishva

    2013-01-01

    Conclusion: The frequency of mecA gene in S. epidermidis isolates was 75.34%. Among the various applied tests used for determining methicillin resistance, sensitivity and specificity of PCR were the highest and reached 100%. Sensitivity and specificity were found to be 95.3% and 94.7%, respectively, for phenotypic test (E-test and 86.5% and 80.9%, respectively, for disk diffusion method. Based on the above results, it seems that resistance of S. epidermidis to methicillin is on the rise, and therefore more research is warranted.

  19. Analysis of the relationship between end-to-end distance and activity of single-chain antibody against colorectal carcinoma.

    Science.gov (United States)

    Zhang, Jianhua; Liu, Shanhong; Shang, Zhigang; Shi, Li; Yun, Jun

    2012-08-22

    We investigated the relationship of End-to-end distance between VH and VL with different peptide linkers and the activity of single-chain antibodies by computer-aided simulation. First, we developed (G4S)n (where n = 1-9) as the linker to connect VH and VL, and estimated the 3D structure of single-chain Fv antibody (scFv) by homologous modeling. After molecular models were evaluated and optimized, the coordinate system of every protein was built and unified into one coordinate system, and End-to-end distances calculated using 3D space coordinates. After expression and purification of scFv-n with (G4S)n as n = 1, 3, 5, 7 or 9, the immunoreactivity of purified ND-1 scFv-n was determined by ELISA. A multi-factorial relationship model was employed to analyze the structural factors affecting scFv: rn=ABn-ABO2+CDn-CDO2+BCn-BCst2. The relationship between immunoreactivity and r-values revealed that fusion protein structure approached the desired state when the r-value = 3. The immunoreactivity declined as the r-value increased, but when the r-value exceeded a certain threshold, it stabilized. We used a linear relationship to analyze structural factors affecting scFv immunoreactivity.

  20. Myocardial gene expression of microRNA-133a and myosin heavy and light chains, in conjunction with clinical parameters, predict regression of left ventricular hypertrophy after valve replacement in patients with aortic stenosis.

    Science.gov (United States)

    Villar, Ana V; Merino, David; Wenner, Mareike; Llano, Miguel; Cobo, Manuel; Montalvo, Cecilia; García, Raquel; Martín-Durán, Rafael; Hurlé, Juan M; Hurlé, María A; Nistal, J Francisco

    2011-07-01

    Left ventricular (LV) reverse remodelling after valve replacement in aortic stenosis (AS) has been classically linked to the hydraulic performance of the replacement device, but myocardial status at the time of surgery has received little attention. To establish predictors of LV mass (LVM) regression 1 year after valve replacement in a surgical cohort of patients with AS based on preoperative clinical and echocardiographic parameters and the myocardial gene expression profile at surgery. Transcript levels of remodelling-related proteins and regulators were determined in LV intraoperative biopsies from 46 patients with AS by RT-PCR. Using multiple linear regression analysis, an equation was developed (adjusted R²=0.73; pregression analysis identified microRNA-133a as a significant positive predictor of LVM normalisation, whereas β-myosin heavy chain and BMI constituted negative predictors. Hypertrophy regression 1 year after pressure overload release is related to the preoperative myocardial expression of remodelling-related genes, in conjunction with the patient's clinical background. In this scenario, miR-133 emerges as a key element of the reverse remodelling process. Postoperative improvement of valve haemodynamics does not predict the degree of hypertrophy regression or LVM normalisation. These results led us to reconsider the current reverse remodelling paradigm and (1) to include criteria of hypertrophy reversibility in the decision algorithm used to decide timing for the operation; and (2) to modify other prevailing factors (overweight, diabetes, etc) known to maintain LV hypertrophy.

  1. Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers' diarrhea samples.

    Science.gov (United States)

    Youmans, Bonnie P; Ajami, Nadim J; Jiang, Zhi-Dong; Petrosino, Joseph F; DuPont, Herbert L; Highlander, Sarah K

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

  2. Development of a nested polymerase chain reaction for amplification of a sequence of the p57 gene of Renibacterium salmoninarum that provides a highly sensitive method for detection of the bacterium in salmonid kidney

    Science.gov (United States)

    Chase, D.M.; Pascho, R.J.

    1998-01-01

    Nucleic acid-based assays have shown promise for diagnosing Renibacterium salmoninarum in tissues and body fluids of salmonids. DeVelopment of a nested polymerase chain reaction (PCR) method to detect a 320 bp DNA segment of the gene encoding the p57 protein of R. salmoninarum is described. Whereas a conventional PCR for a 383 bp segment of the p57 gene reliably detected 1000 R. salmoninarum cells per reaction in kidney tissue, the nested PCR detected as few as 10 R. salmoninarum per reaction in kidney tissue. Two DNA extraction methods for the nested PCR were compared and the correlation between replicate samples was generally higher in samples extracted by the QIAamp system compared with those extracted by the phenol/chloroform method. The specificity of the nested PCR was confirmed by testing DNA extracts of common bacterial fish pathogens and a panel of bacterial species reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) for R. salmoninarum. Kidney samples from 74 naturally infected chinook Salmon were examined by the nested PCR, the ELISA, and the FAT, and the detected prevalences of R. salmoninarum were 61, 47, and 43%, respectively.

  3. Bacterial genomes lacking long-range correlations may not be modeled by low-order Markov chains: the role of mixing statistics and frame shift of neighboring genes.

    Science.gov (United States)

    Cocho, Germinal; Miramontes, Pedro; Mansilla, Ricardo; Li, Wentian

    2014-12-01

    We examine the relationship between exponential correlation functions and Markov models in a bacterial genome in detail. Despite the well known fact that Markov models generate sequences with correlation function that decays exponentially, simply constructed Markov models based on nearest-neighbor dimer (first-order), trimer (second-order), up to hexamer (fifth-order), and treating the DNA sequence as being homogeneous all fail to predict the value of exponential decay rate. Even reading-frame-specific Markov models (both first- and fifth-order) could not explain the fact that the exponential decay is very slow. Starting with the in-phase coding-DNA-sequence (CDS), we investigated correlation within a fixed-codon-position subsequence, and in artificially constructed sequences by packing CDSs with out-of-phase spacers, as well as altering CDS length distribution by imposing an upper limit. From these targeted analyses, we conclude that the correlation in the bacterial genomic sequence is mainly due to a mixing of heterogeneous statistics at different codon positions, and the decay of correlation is due to the possible out-of-phase between neighboring CDSs. There are also small contributions to the correlation from bases at the same codon position, as well as by non-coding sequences. These show that the seemingly simple exponential correlation functions in bacterial genome hide a complexity in correlation structure which is not suitable for a modeling by Markov chain in a homogeneous sequence. Other results include: use of the (absolute value) second largest eigenvalue to represent the 16 correlation functions and the prediction of a 10-11 base periodicity from the hexamer frequencies. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Enhancement of antitumor activity by using a fully human gene encoding a single-chain fragmented antibody specific for carcinoembryonic antigen

    Directory of Open Access Journals (Sweden)

    Shibaguchi H

    2017-08-01

    Full Text Available Hirotomo Shibaguchi,1,* Naixiang Luo,1,* Naoto Shirasu,1,* Motomu Kuroki,2 Masahide Kuroki1 1Department of Biochemistry, Faculty of Medicine, Fukuoka University, Fukuoka, Japan; 2School of Nursing, Faculty of Medicine, Fukuoka University, Fukuoka, Japan *These authors equally contributed to this work Abstract: Human leukocyte antigen and/or costimulatory molecules are frequently lacking in metastatic tumor cells, and thus tumor cells are able to escape from the immune system. Although lymphocytes with a chimeric antigen receptor (CAR is a promising approach for overcoming this challenge in cancer immunotherapy, administration of modified T cells alone often demonstrates little efficacy in patients. Therefore, in order to enhance the antitumor activity of immune cells in the cancer microenvironment, we used lymphocytes expressing CAR in combination with a fusion protein of IL-2 that contained the single-chain fragmented antibody (scFv specific for the carcinoembryonic antigen. Among a series of CAR constructs, with or without a spacer and the intracellular domain of CD28, the CAR construct containing CD8α, CD28, and CD3ζ most effectively activated and expressed INF-γ in CAR-bearing T cells. Furthermore, in comparison with free IL-2, the combination of peripheral blood mononuclear cells expressing CAR and the fusion protein containing IL-2 significantly enhanced the antitumor activity against MKN-45 cells, a human gastric cancer cell line. In conclusion, this novel combination therapy of CAR and a fusion protein consisting of a functional cytokine and a fully human scFv may be a promising approach for adoptive cancer immunotherapy. Keywords: chimeric antigen receptor, fusion protein, human scFv, CEA, combination therapy

  5. The relationship between quantitative human epidermal growth factor receptor 2 gene expression by the 21-gene reverse transcriptase polymerase chain reaction assay and adjuvant trastuzumab benefit in Alliance N9831.

    Science.gov (United States)

    Perez, Edith A; Baehner, Frederick L; Butler, Steven M; Thompson, E Aubrey; Dueck, Amylou C; Jamshidian, Farid; Cherbavaz, Diana; Yoshizawa, Carl; Shak, Steven; Kaufman, Peter A; Davidson, Nancy E; Gralow, Julie; Asmann, Yan W; Ballman, Karla V

    2015-10-01

    The N9831 trial demonstrated the efficacy of adjuvant trastuzumab for patients with human epidermal growth factor receptor 2 (HER2) locally positive tumors by protein or gene analysis. We used the 21-gene assay to examine the association of quantitative HER2 messenger RNA (mRNA) gene expression and benefit from trastuzumab. N9831 tested the addition of trastuzumab to chemotherapy in stage I-III HER2-positive breast cancer. For two of the arms of the trial, doxorubicin and cyclophosphamide followed by paclitaxel (AC-T) and doxorubicin and cyclophosphamide followed by paclitaxel and trastuzumab concurrent chemotherapy-trastuzumab (AC-TH), recurrence score (RS) and HER2 mRNA expression were determined by the 21-gene assay (Oncotype DX®) (negative 10 % positive cells = positive), 91 % for RT-PCR versus central fluorescence in situ hybridization (FISH) (≥2.0 = positive) and 94 % for central IHC versus central FISH. In the primary analysis, the association of HER2 expression by 21-gene assay with trastuzumab benefit was marginally nonsignificant (nonlinear p = 0.057). In hormone receptor-positive patients (local IHC) the association was significant (p = 0.002). The association was nonlinear with the greatest estimated benefit at lower and higher HER2 expression levels. Concordance among HER2 assessments by central IHC, FISH, and RT-PCR were similar and high. Association of HER2 mRNA expression with trastuzumab benefit as measured by time to distant recurrence was nonsignificant. A consistent benefit of trastuzumab irrespective of mHER2 levels was observed in patients with either IHC-positive or FISH-positive tumors. Trend for benefit was observed also for the small groups of patients with negative results by any or all of the central assays. Clinicaltrials.gov NCT00005970 . Registered 5 July 2000.

  6. Mycobacterium leprae specific genomic target in the promoter region of probable 4-alpha-glucanotransferase (ML1545) gene with potential sensitivity for polymerase chain reaction based diagnosis of leprosy.

    Science.gov (United States)

    Sundeep Chaitanya, V; Das, Madhusmita; Eisenbach, Tiffany L; Amoako, Angela; Rajan, Lakshmi; Horo, Ilse; Ebenezer, Mannam

    2016-06-01

    With the absence of an effective diagnostic tool for leprosy, cases with negative bacteriological index and limited clinical manifestations often pose diagnostic challenges. In this study, we investigated the utility of a novel Mycobacterium leprae specific 112-bp DNA sequence in the promoter region of probable 4-alpha-glucanotransferase (pseudogene, ML1545) for polymerase chain reaction (PCR) based diagnosis of leprosy in comparison to that of the RLEP gene. DNA was extracted from slit skin scrapings of 180 newly diagnosed untreated leprosy cases that were classified as per Ridley Jopling classifications and bacteriological index (BI). Primers were designed using Primer Blast 3.0 and PCR was performed with annealing temperatures of 61°C for ML1545 and 58°C for the RLEP gene using conventional gradient PCR. The results indicated a significant increase in PCR positivity of ML1545 when compared to RLEP across the study groups (164/180 [91.11%] were positive for ML1545 whereas 114/180 (63.33%) were positive for RLEP [pleprosy cases with negative BI, 28 (48.28%) were positive for RLEP and 48 (82.76%) were positive for ML1545 (p=.0001, z=3.8). Of the 42 borderline tuberculoid leprosy cases, 23 (54.76%) were positive for RLEP whereas 37 (88.09%) were positive for ML1545 (pleprosy and BI-positive groups. ML1545 can be a potential gene target for PCR-based diagnosis of leprosy especially in cases where clinical manifestations were minimal. Copyright © 2016 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

  7. Expansion of B-1a cells with germline heavy chain sequence in lupus mice

    Directory of Open Access Journals (Sweden)

    Nichol E Holodick

    2016-03-01

    Full Text Available B6.Sle1.Sle2.Sle3 (B6.TC lupus-prone mice carrying the NZB allele of Cdkn2c, encoding for the cyclin-dependent kinase inhibitor P18INK4, accumulate B-1a cells due to a higher rate of proliferative self-renewal. However, it is unclear whether this affects primarily early appearing B-1a cells of fetal origin or later appearing B-1a cells that emerge from bone marrow. B-1a cells are the major source of natural autoantibodies, and it has been shown that their protective nature is associated with a germline-like sequence, which is characterized by few N-nucleotide insertions and a repertoire skewed towards rearrangements predominated during fetal life, VH11 and VH12. To determine the nature of B-1a cells expanded in B6.TC mice, we amplified immunoglobulin genes by PCR from single cells in mice. Sequencing showed a significantly higher proportion of B-1a cell antibodies display fewer N-additions in B6.TC mice than in B6 control mice. Following this lower number of N-insertions within the CDR-H3 region, the B6.TC B-1a cells display shorter CDR-H3 length than B6 B-1a cells. The absence of N-additions is a surrogate for fetal origin, as TdT expression starts after birth in mice. Therefore, our results suggest that the B-1a cell population is not only expanded in autoimmune B6.TC mice but also qualitatively different with the majority of cells from fetal origin. Accordingly, our sequencing results also demonstrated overuse of VH11 and VH12 in autoimmune B6.TC mice as compared to B6 controls. These results suggest that the development of lupus autoantibodies in these mice is coupled with skewing of the B-1a cell repertoire and possible retention of protective natural antibodies.

  8. Direct interactions of OCA-B and TFII-I regulate immunoglobulin heavy-chain gene transcription by facilitating enhancer-promoter communication.

    Science.gov (United States)

    Ren, Xiaodi; Siegel, Rachael; Kim, Unkyu; Roeder, Robert G

    2011-05-06

    B cell-specific coactivator OCA-B, together with Oct-1/2, binds to octamer sites in promoters and enhancers to activate transcription of immunoglobulin (Ig) genes, although the mechanisms underlying their roles in enhancer-promoter communication are unknown. Here, we demonstrate a direct interaction of OCA-B with transcription factor TFII-I, which binds to DICE elements in Igh promoters, that affects transcription at two levels. First, OCA-B relieves HDAC3-mediated Igh promoter repression by competing with HDAC3 for binding to promoter-bound TFII-I. Second, and most importantly, Igh 3' enhancer-bound OCA-B and promoter-bound TFII-I mediate promoter-enhancer interactions, in both cis and trans, that are important for Igh transcription. These and other results reveal an important function for OCA-B in Igh 3' enhancer function in vivo and strongly favor an enhancer mechanism involving looping and facilitated factor recruitment rather than a tracking mechanism. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Pregnancy after preimplantation diagnosis for a deletion in the dystrophin gene by polymerase chain reaction in embryos obtained after intracytoplasmic sperm injection

    Energy Technology Data Exchange (ETDEWEB)

    Lissens, W.; Liu, J.; Van Broeckhoven, C. [University Hospital, Brussels (Belgium)] [and others

    1994-09-01

    Duchenne muscular dystrophy (DMD) is one of the most common X-linked recessive diseases. In order to be able to perform a DMD-specific preimplantation diagnosis (PID) in a female carrier of a deletion of exons 3 to 18 in the dystrophin gene, we have developed a PCR assay to detect the deletion based on sequences of exon 17. The efficiency of this PCR was evaluated on 50 single blastomeres from 12 normal control embryos and on 41 blastomeres for 9 male and 3 female embryos from the female DMD carrier, obtained after a first preimplantation diagnosis by sexing. The exon 17 region was amplified with 100% efficiency, except in all 21 blastomeres from 6 male embryos from the carrier where no PCR signals were observed. The negative results in these blastomeres were interpreted as being found only in male embryos carrying the deletion. Intracytoplasmic sperm injection was carried out on the carrier`s metaphase II oocytes retrieved after ovarian stimulation. Embryos were analyzed for the presence of exon 17 and 2 male embryos were found to be deleted, while 4 embryos showed normal amplification signals. Three of the latter embryos were replaced, resulting in a singleton pregnancy. Amniotic cell analysis showed a normal female karyotype and DNA analysis indicated a non-carrier.

  10. Participation of different genes in the ruptures repair of double chain in Escherichia coli stumps exposed to gamma radiation; Participacion de diferentes genes en la reparacion de rupturas de doble cadena en cepas de Escherichia coli expuestas a radiacion gamma

    Energy Technology Data Exchange (ETDEWEB)

    Serment G, J. H.; Martinez M, E.; Alcantara D, D., E-mail: jorge.serment@inin.gob.mx [ININ, Departamento de Biologia, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

    2013-05-01

    All living organisms are naturally exposed to radiation from different sources. Ionizing radiation produces a plethora of lesions upon DNA that can be categorized as single and double strand breaks and base damage. Among them, unrepaired double strand breaks (Dbs) have the greatest biological significance, since they are responsible of cell death. In Escherichia coli this kind of lesions are repaired mostly by homologous recombination. In this work the participation of some recombination genes in the repair of Dbs is evaluated. Escherichia coli defective strains were exposed to gamma radiation and incubated for different periods in ideal conditions. Both micro electrophoresis and pulse field gel electrophoresis techniques were used to evaluate the kinetics of repair of such lesions, reflecting the importance of each defective gene in the process. (Author)

  11. Statistical test of reproducibility and operator variance in thin-section modal analysis of textures and phenocrysts in the Topopah Spring member, drill hole USW VH-2, Crater Flat, Nye County, Nevada

    International Nuclear Information System (INIS)

    Moore, L.M.; Byers, F.M. Jr.; Broxton, D.E.

    1989-06-01

    A thin-section operator-variance test was given to the 2 junior authors, petrographers, by the senior author, a statistician, using 16 thin sections cut from core plugs drilled by the US Geological Survey from drill hole USW VH-2 standard (HCQ) drill core. The thin sections are samples of Topopah Spring devitrified rhyolite tuff from four textural zones, in ascending order: (1) lower nonlithophysal, (2) lower lithopysal, (3) middle nonlithophysal, and (4) upper lithophysal. Drill hole USW-VH-2 is near the center of the Crater Flat, about 6 miles WSW of the Yucca Mountain in Exploration Block. The original thin-section labels were opaqued out with removable enamel and renumbered with alpha-numeric labels. The sliders were then given to the petrographer operators for quantitative thin-section modal (point-count) analysis of cryptocrystalline, spherulitic, granophyric, and void textures, as well as phenocryst minerals. Between operator variance was tested by giving the two petrographers the same slide, and within-operator variance was tested by the same operator the same slide to count in a second test set, administered at least three months after the first set. Both operators were unaware that they were receiving the same slide to recount. 14 figs., 6 tabs

  12. Polymorphisms in the non-muscle myosin heavy chain gene (MYH9 are associated with lower glomerular filtration rate in mixed ancestry diabetic subjects from South Africa.

    Directory of Open Access Journals (Sweden)

    Tandi Edith Matsha

    Full Text Available OBJECTIVE: Though single nucleotide polymorphisms (SNPs in the non-muscle myosin gene (MYH9 have been reported to explain most of the excess risk of nondiabetic chronic kidney disease (CKD, in African-Americans, some studies have also shown associations with diabetic end-stage renal disease. We investigated the association of MYH9 SNPs with renal traits in a mixed-ancestry South African population prone to diabetes. RESEARCH DESIGN AND METHODS: Three SNPs known to be associated with CKD (rs4821480, rs5756152 and rs12107 were genotyped using Taqman assay in 716 adults (198 with diabetes from the Bellville-South community, Cape Town. Glomerular filtration rate was estimated (eGFR and urinary albumin/creatinine ratio (ACR assessed. Multivariable regressions were used to relate the SNPs with renal traits. RESULTS: Mean age was 53.6 years, with the expected differences observed in characteristics by diabetic status. Significant associations were found between rs575152 and serum creatinine, and eGFR in the total population, and in diabetic participants (all p≤0.003, but not in non-diabetics (all p≥0.16, with significant interactions by diabetes status (interaction-p≤0.009. The association with ACR was borderline in diabetic participants (p = 0.05 and non-significant in non-diabetics (p = 0.85, with significant interaction (interaction p = 0.02. rs12107 was associated with fasting-, 2-hour glucose and HbA1c in diabetic participants only (interaction-p≤0.003, but not with renal traits. CONCLUSION: MYH9 SNPs were associated with renal traits only in diabetic participants in this population. Our findings and other studies suggest that MYH9 may have a broader genetic risk effect on kidney diseases.

  13. Prognostic value of ZAP-70 expression in chronic lymphocytic leukemia as assessed by quantitative polymerase chain reaction and flow cytometry.

    Science.gov (United States)

    Adams, Rebecca L C; Cheung, Catherine; Banh, Raymond; Saal, Russell; Cross, Donna; Gill, Devinder; Self, Marlene; Klein, Kerenaftali; Mollee, Peter

    2014-03-01

    Chronic lymphocytic leukemia (CLL) is a disorder in which the tempo of disease progression is highly variable, and prognostic markers that can be utilized at diagnosis are regarded as clinically important. Currently, there are several prognostic factors, such as immunoglobulin heavy chain (IgVH) mutational status, and ZAP-70 protein expression in neoplastic B-cells, that have demonstrated significant discriminative power in the prognostication of CLL. They are, however, largely unavailable in the routine diagnostic laboratory setting. In this study, we characterized the IgVH status and ZAP-70 expression by molecular techniques in a cohort of 108 patients with CLL, and correlated these results with three different methods of ZAP-70 expression by flow cytometry. We then assessed the results of these methods in terms of prognostic power as characterized by time to first treatment (TTFT). By comparing three different flow cytometry methods using receiver–operator curve (ROC) analysis, we identified that by utilizing a corrected mean fluorescence intensity (CorrMFI) algorithm for assessing ZAP-70 expression, there was good correlation with both IgVH mutational status, and ZAP-70 expression as assessed by qPCR. We were also able to show that ZAP-70 expression, as assessed by both qPCR and the CorrMFI method, was prognostic of TTFT. While confirmation in a larger patient cohort, with longer follow-up is required, we believe that the CorrMFI represents the most promising method currently available in a routine diagnostic setting for the assessment of ZAP-70 expression in CLL patients. © 2013 International Clinical Cytometry Society.

  14. CHAINS-PC, Decay Chain Atomic Densities

    International Nuclear Information System (INIS)

    1994-01-01

    1 - Description of program or function: CHAINS computes the atom density of members of a single radioactive decay chain. The linearity of the Bateman equations allows tracing of interconnecting chains by manually accumulating results from separate calculations of single chains. Re-entrant loops can be treated as extensions of a single chain. Losses from the chain are also tallied. 2 - Method of solution: The Bateman equations are solved analytically using double-precision arithmetic. Poles are avoided by small alterations of the loss terms. Multigroup fluxes, cross sections, and self-shielding factors entered as input are used to compute the effective specific reaction rates. The atom densities are computed at any specified times. 3 - Restrictions on the complexity of the problem: Maxima of 100 energy groups, 100 time values, 50 members in a chain

  15. Comparison between Radioisotopic and Non-radioisotopic Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) Procedures in the Detection of Mutations at the rpoB Gene Associated with Rifampicin Resistance in Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Lee, H.; Bang, H.E.; Johnson, R.; Jordaan, A.M.; Victor, T.C. . E-mail : tv@sun.ac.za; Dar, L.; Khan, B.K.; Cho, S.N. . E-mail : raycho@yonsei.ac.kr

    2006-01-01

    Rapid and sensitive detection of mutations at the rpoB gene of Mycobacterium tuberculosis would be of great importance for proper management of tuberculosis (TB) patients and control of multi-drug resistant TB. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) using both radioisotopic and non-radioisotopic methods have been widely used for detecting such mutations. However, the silver staining method, which is the most frequently employed in PCR-SSCP, has been reported to be producing results of varying sensitivity. Radioisotope-based methods have shown greater sensitivity in detecting the rpoB mutations than the silver staining method. The primary objective of this study was therefore to compare the radioisotopic method with the silver staining method detection of mutations of rpoB gene by PCR-SSCP in the same laboratory. Purified DNAs from M. tuberculosis H37Rv were serially diluted and used for PCR amplification with and without radionuclides. The PCR products were then detected by silver staining and autoradiography methods. In addition, clinical isolates were analyzed by PCR-SSCP. The radioisotopic method showed about four-fold increase in the detection of PCR products over ethidium bromide staining in agarose gel. When compared with silver staining, the radioisotopic method gave a sensitivity of more than 10-fold in detecting PCR products and about 8-fold in PCR-SSCP. Radioisotope-based detection methods provided a clearer resolution in PCR-SSCP than the silver staining method when applied to clinical isolates of M. tuberculosis. Radioisotope-based detection method was shown to be more sensitive than non-isotope-based method in detecting PCR products and mutations at the rpoB gene of M. tuberculosis by PCR-SSCP. It may be noted that mutations in the rpoB gene as a marker have significant clinical importance because of the increasing number of MDR-TB cases in the world. It is especially relevant to MDR and Extreme Drug Resistance TB

  16. Markov Chain Ontology Analysis (MCOA).

    Science.gov (United States)

    Frost, H Robert; McCray, Alexa T

    2012-02-03

    Biomedical ontologies have become an increasingly critical lens through which researchers analyze the genomic, clinical and bibliographic data that fuels scientific research. Of particular relevance are methods, such as enrichment analysis, that quantify the importance of ontology classes relative to a collection of domain data. Current analytical techniques, however, remain limited in their ability to handle many important types of structural complexity encountered in real biological systems including class overlaps, continuously valued data, inter-instance relationships, non-hierarchical relationships between classes, semantic distance and sparse data. In this paper, we describe a methodology called Markov Chain Ontology Analysis (MCOA) and illustrate its use through a MCOA-based enrichment analysis application based on a generative model of gene activation. MCOA models the classes in an ontology, the instances from an associated dataset and all directional inter-class, class-to-instance and inter-instance relationships as a single finite ergodic Markov chain. The adjusted transition probability matrix for this Markov chain enables the calculation of eigenvector values that quantify the importance of each ontology class relative to other classes and the associated data set members. On both controlled Gene Ontology (GO) data sets created with Escherichia coli, Drosophila melanogaster and Homo sapiens annotations and real gene expression data extracted from the Gene Expression Omnibus (GEO), the MCOA enrichment analysis approach provides the best performance of comparable state-of-the-art methods. A methodology based on Markov chain models and network analytic metrics can help detect the relevant signal within large, highly interdependent and noisy data sets and, for applications such as enrichment analysis, has been shown to generate superior performance on both real and simulated data relative to existing state-of-the-art approaches.

  17. Reverse transcriptase-quantitative polymerase chain reaction (RT ...

    African Journals Online (AJOL)

    The reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a highly specific polymerase chain reaction (PCR) method that allows one to detect very low transcription levels of functional gene(s) in soil. RT-qPCR helps us to know the active members of the microbial community, and their activities can be ...

  18. Stimulatory and inhibitory mechanisms of slow muscle-specific myosin heavy chain gene expression in fish: Transient and transgenic analysis of torafugu MYHM86-2 promoter in zebrafish embryos

    International Nuclear Information System (INIS)

    Asaduzzaman, Md.; Kinoshita, Shigeharu; Bhuiyan, Sharmin Siddique; Asakawa, Shuichi; Watabe, Shugo

    2013-01-01

    The myosin heavy chain gene, MYH M86-2 , exhibited restricted expression in slow muscle fibers of torafugu embryos and larvae, suggesting its functional roles for embryonic and larval muscle development. However, the transcriptional mechanisms involved in its expression are still ambiguous. The present study is the first extensive analysis of slow muscle-specific MYH M86-2 promoter in fish for identifying the cis-elements that are crucial for its expression. Combining both transient transfection and transgenic approaches, we demonstrated that the 2614 bp 5′-flanking sequences of MYH M86-2 contain a sufficient promoter activity to drive gene expression specific to superficial slow muscle fibers. By cyclopamine treatment, we also demonstrated that the differentiation of such superficial slow muscle fibers depends on hedgehog signaling activity. The deletion analyses defined an upstream fragment necessary for repressing ectopic MYH M86-2 expression in the fast muscle fibers. The transcriptional mechanism that prevents MYH M86-2 expression in the fast muscle fibers is mediated through Sox6 binding elements. We also demonstrated that Sox6 may function as a transcriptional repressor of MYH M86-2 expression. We further discovered that nuclear factor of activated T cells (NFAT) binding elements plays a key role and myocyte enhancer factor-2 (MEF2) binding elements participate in the transcriptional regulation of MYH M86-2 expression. - Highlights: ► MYH M86-2 is highly expressed in slow muscle fibers of torafugu embryos and larvae. ► MYH M86-2 promoter activity depends on the hedgehog signaling. ► Sox6 binding elements inhibits MYH M86-2 expression in fast muscle fibers. ► Sox6 elements function as transcriptional repressor of MYH M86-2 promoter activity. ► NFAT and MEF2 binding elements play a key role for directing MYH M86-2 expression

  19. Complete deficiency of mitochondrial trifunctional protein due to a novel mutation within the beta-subunit of the mitochondrial trifunctional protein gene leads to failure of long-chain fatty acid beta-oxidation with fatal outcome

    NARCIS (Netherlands)

    Schwab, Karl Otfried; Ensenauer, Regina; Matern, Dietrich; Uyanik, Gökhan; Schnieders, Birgit; Wanders, Ronald A.; Lehnert, Willy

    2003-01-01

    The mitochondrial trifunctional protein (MTP) is a multienzyme complex which catalyses three of the four chain-shortening reactions in the beta-oxidation of long-chain fatty acids. Clinically, failure of long-chain fatty acid beta-oxidation leads to hypoketotic hypoglycaemia associated with coma,

  20. Logistic chain modelling

    NARCIS (Netherlands)

    Slats, P.A.; Bhola, B.; Evers, J.J.M.; Dijkhuizen, G.

    1995-01-01

    Logistic chain modelling is very important in improving the overall performance of the total logistic chain. Logistic models provide support for a large range of applications, such as analysing bottlenecks, improving customer service, configuring new logistic chains and adapting existing chains to

  1. Detection of Cryptosporidium species in feces or gastric contents from snakes and lizards as determined by polymerase chain reaction analysis and partial sequencing of the 18S ribosomal RNA gene.

    Science.gov (United States)

    Richter, Barbara; Nedorost, Nora; Maderner, Anton; Weissenböck, Herbert

    2011-05-01

    Cryptosporidiosis is a well-known gastrointestinal disease of snakes and lizards. In the current study, 672 samples (feces and/or gastric contents or regurgitated food items) of various snakes and lizards were examined for the presence of cryptosporidia by polymerase chain reaction (PCR) assay targeting a part of the 18S ribosomal RNA gene. A consecutive sequencing reaction was used to identify the cryptosporidian species present in PCR-positive samples. Cryptosporidium varanii (saurophilum) was detected in 17 out of 106 (16%) samples from corn snakes (Pantherophis guttatus) and in 32 out of 462 (7%) samples from leopard geckos (Eublepharis macularius). Cryptosporidium serpentis was found in 8 out of 462 (2%) leopard gecko samples, but in no other reptile. The Cryptosporidium sp. "lizard genotype" was present in 1 leopard gecko sample, and 1 sample from a corn snake showed a single nucleotide mismatch to this genotype. Pseudoparasitic cryptosporidian species were identified in 5 out of 174 (3%) ophidian samples, but not in lizards. Other sequences did not show complete similarity to previously published Cryptosporidium sequences. The results stress the importance for diagnostic methods to be specific for Cryptosporidium species especially in snakes and show a relatively high prevalence of C. varanii in leopard geckos and corn snakes. © 2011 The Author(s)

  2. Development of nested polymerase chain reaction-based diagnosis of duck enteritis virus and detection of DNA polymerase gene from non-descriptive duck breeds of West Bengal, India

    Directory of Open Access Journals (Sweden)

    Partha Sarathi Mandal

    2017-03-01

    Full Text Available Aim: The study was undertaken to detect the clinical signs, postmortem lesions of embryonated duck plague (DP infected eggs, and histopathological changes of chorioallantoic membrane (CAM in non-descriptive ducks of West Bengal with special reference to standardize nested polymerase chain reaction (PCR. Materials and Methods: After postmortem of suspected carcasses, samples were collected for virus isolation and identification through specific pathogen free (Khaki Campbell embryonated duck eggs. PCR was also done as confirmatory test after doing postmortem of duck embryos. DP specific nested PCR was standardized for better confirmation of the disease. Sensitivity of nested primers was also tested for DP virus. Results: Gross, postmortem and histopathological changes were prominent in dead embryos. First set of primer was able to detect 602 bp fragments of DNA polymerase gene of duck enteritis virus from infected CAM. Subsequently, a DP specific nested PCR which was very much sensitive for very small amount of viral genome was successfully standardized. After NCBI blast nucleotide sequence of nested PCR product (Accession No. HG425076 showed homology with the sequences data available in GenBank. Conclusion: The study concludes that PCR assay is very much helpful to diagnose DP disease and developed nested PCR is a double confirmatory diagnostic tool for DP.

  3. Clinical value of miR-452-5p expression in lung adenocarcinoma: A retrospective quantitative real-time polymerase chain reaction study and verification based on The Cancer Genome Atlas and Gene Expression Omnibus databases.

    Science.gov (United States)

    Gan, Xiao-Ning; Luo, Jie; Tang, Rui-Xue; Wang, Han-Lin; Zhou, Hong; Qin, Hui; Gan, Ting-Qing; Chen, Gang

    2017-05-01

    The role and mechanism of miR-452-5p in lung adenocarcinoma remain unclear. In this study, we performed a systematic study to investigate the clinical value of miR-452-5p expression in lung adenocarcinoma. The expression of miR-452-5p in 101 lung adenocarcinoma patients was detected by quantitative real-time polymerase chain reaction. The Cancer Genome Atlas and Gene Expression Omnibus databases were joined to verify the expression level of miR-452-5p in lung adenocarcinoma. Via several online prediction databases and bioinformatics software, pathway and network analyses of miR-452-5p target genes were performed to explore its prospective molecular mechanism. The expression of miR-452-5p in lung adenocarcinoma in house was significantly lower than that in adjacent tissues (p < 0.001). Additionally, the expression level of miR-452-5p was negatively correlated with several clinicopathological parameters including the tumor size (p = 0.014), lymph node metastasis (p = 0.032), and tumor-node-metastasis stage (p = 0.036). Data from The Cancer Genome Atlas also confirmed the low expression of miR-452 in lung adenocarcinoma (p < 0.001). Furthermore, reduced expression of miR-452-5p in lung adenocarcinoma (standard mean deviations = -0.393, 95% confidence interval: -0.774 to -0.011, p = 0.044) was validated by a meta-analysis. Five hub genes targeted by miR-452-5p, including SMAD family member 4, SMAD family member 2, cyclin-dependent kinase inhibitor 1B, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta, were significantly enriched in the cell-cycle pathway. In conclusion, low expression of miR-452-5p tends to play an essential role in lung adenocarcinoma. Bioinformatics analysis might be beneficial to reveal the potential mechanism of miR-452-5p in lung adenocarcinoma.

  4. Sustainable Supply Chain Design

    DEFF Research Database (Denmark)

    Bals, Lydia; Tate, Wendy

    A significant conceptual and practical challenge is how to integrate triple bottom line (TBL; including economic, social and environmental) sustainability into global supply chains. Although this integration is necessary to slow down global resource depletion, understanding is limited of how...... to implement TBL goals across the supply chain. In supply chain design, the classic economic perspective still dominates, although the idea of the TBL is more widely disseminated. The purpose of this research is to add to the sustainable supply chain management literature (SSCM) research agenda...... by incorporating the physical chain, and the (information and financial) support chains into supply chain design. This manuscript tackles issues of what the chains are designed for and how they are designed structurally. Four sustainable businesses are used as illustrative case examples of innovative supply chain...

  5. Construction of a Single Chain Variable Fragment Antibody (scFv) against Carbaryl and Its Interaction with Carbaryl.

    Science.gov (United States)

    Xiuyuan, Zhang; Zhihong, Huang; Lixia, Wang; Xiaonan, Liu

    2015-05-01

    Carbaryl is a low molecular weight insecticide that inhibits cholinesterase. Residues of carbaryl in food and the environment have damaged human health. A high-specificity scFv that can identify carbaryl is still lacking. In the present study, an anti-carbaryl scFv gene was prepared by cloning VL and VH genes from hybridoma cells secreting monoclonal antibody, then VH and VL were fused together using splicing by overlap extension (SOE) PCR with a flexible polypeptide linker connector (Gly4Ser)3, and then the scFv-pET-26b recombinant plasmid was constructed and transformed into E. coli BL21 for expression using IPTG as an inducer. The expressed recombinant protein was identified by SDS-PAGE and ELISA. The three-dimensional structure of the anti-carbaryl scFv was constructed by computer modeling, and carbaryl was docked to the scFv model to obtain the structure of the binding complex. The binding site was composed of Ala51, Ser52, Ile51, Gly54, Ser56, Arg98, and Gly100. This helps to understand the mechanism of interaction between anti-carbaryl antibody and antigen. Furthermore, it provides guidance for in vitro affinity maturation of anti-carbaryl antibody.

  6. Casscf/ci Calculations for First Row Transition Metal Hydrides - the TIH(4-PHI), VH(5-DELTA), CRH(6-SIGMA-PLUS), MNH(7-SIGMA-PLUS), FEH(4,6-DELTA) and NIH(2-DELTA) States

    Science.gov (United States)

    Walch, S. P.; Bauschlicher, C. W., Jr.

    1983-04-01

    Calculations are performed for the predicted ground states of TiH(4-phi), VH(5-delta), CrH(6-sigma-plus), MnH(7-sigma-plus), Fett(4,6-delta) and NiH(2-delta). For FeH both the 6-delta and 4-delta states are studied, since both are likely candidates for the ground state. The ground state symmetries are predicted based on a combination of atomic coupling arguments and coupling of 4s(2)3d(n) and 4s(1)3d(n+1) terms in the molecular system. Electron correlation is included by a CASSCF/CI (SD) treatment. The CASSCF includes near-degeneracy effects, while correlation of the 3d electrons in included at the CI level.

  7. Mining of biomarker genes from expressed sequence tags and differential display reverse transcriptase-polymerase chain reaction in the self-fertilizing fish, Kryptolebias marmoratus and their expression patterns in response to exposure to an endocrine-disrupting alkylphenol, bisphenol A.

    Science.gov (United States)

    Lee, Young-Mi; Rhee, Jae-Sung; Hwang, Dae-Sik; Kim, Il-Chan; Raisuddin, Sheikh; Lee, Jae-Seong

    2007-06-30

    Expressed sequence tags (ESTs) and differentially expressed cDNAs from the self-fertilizing fish, Kryptolebias marmoratus were mined to develop alternative biomarkers for endocrine-disrupting chemicals (EDCs). 1,577 K. marmoratus cDNA clones were randomly sequenced from the 5'-end. These clones corresponded to 1,518 and 1,519 genes in medaka dbEST and zebrafish dbEST, respectively. Of the matched genes, 197 and 115 genes obtained Unigene IDs in medaka dbEST and zebrafish dbEST, respectively. Many of the annotated genes are potential biomarkers for environmental stresses. In a differential display reverse transcriptase-polymerase chain reaction (DD RT-PCR) study, 56 differential expressed genes were obtained from fish liver exposed to bisphenol A. Of these, 16 genes were identified after BLAST search to GenBank, and the annotated genes were mainly involved in catalytic activity and binding. The expression patterns of these 16 genes were validated by real-time RT-PCR of liver tissue from fish exposed to bisphenol A. Our findings suggest that expression of these 16 genes is modulated by endocrine disrupting chemicals, and therefore that they are potential biomarkers for environmental stress including EDCs exposure.

  8. Effects of branched-chain volatile fatty acids on lactation performance and mRNA expression of genes related to fatty acid synthesis in mammary gland of dairy cows.

    Science.gov (United States)

    Liu, Q; Wang, C; Guo, G; Huo, W J; Zhang, S L; Pei, C X; Zhang, Y L; Wang, H

    2018-02-12

    Branched-chain volatile fatty acids (BCVFA) supplements could promote lactation performance and milk quality by improving ruminal fermentation and milk fatty acid synthesis. This study was conducted to evaluate the effects of BCVFA supplementation on milk performance, ruminal fermentation, nutrient digestibility and mRNA expression of genes related to fatty acid synthesis in mammary gland of dairy cows. A total of 36 multiparous Chinese Holstein cows averaging 606±4.7 kg of BW, 65±5.2 day in milk (DIM) with daily milk production of 30.6±0.72 kg were assigned to one of four groups blocked by lactation number, milk yield and DIM. The treatments were control, low-BCVFA (LBCVFA), medium-BCVFA (MBCVFA) and high-BCVFA (HBCVFA) with 0, 30, 60 and 90 g BCVFA per cow per day, respectively. Experimental periods were 105 days with 15 days of adaptation and 90 days of data collection. Dry matter (DM) intake tended to increase, but BW changes were similar among treatments. Yields of actual milk, 4% fat corrected milk, milk fat and true protein linearly increased, but feed conversion ratio (FCR) linearly decreased with increasing BCVFA supplementation. Milk fat content linearly increased, but true protein content tended to increase. Contents of C4:0, C6:0, C8:0, C10:0, C12:0, C14:0 and C15:0 fatty acids in milk fat linearly increased, whereas other fatty acids were not affected with increasing BCVFA supplementation. Ruminal pH, ammonia N concentration and propionate molar proportion linearly decreased, but total VFA production and molar proportions of acetate and butyrate linearly increased with increasing BCVFA supplementation. Consequently, acetate to propionate ratios linearly increased. Digestibilities of DM, organic matter, CP, NDF and ADF also linearly increased. In addition, mRNA expressions of peroxisome proliferator-activated receptor γ, sterol regulatory element-binding factor 1 and fatty acid-binding protein 3 linearly increased, mRNA expressions of acetyl

  9. The Global Value Chain

    DEFF Research Database (Denmark)

    Sørensen, Olav Jull

    The conference paper aims to develop the global value chain concept by including corporate internal value adding activities and competition to the basic framework in order to turn the global value chain into a strategic management tool......The conference paper aims to develop the global value chain concept by including corporate internal value adding activities and competition to the basic framework in order to turn the global value chain into a strategic management tool...

  10. Pesquisa de marcadores para os genes da cadeia pesada da beta-miosina cardíaca e da proteína C de ligação à miosina em familiares de pacientes com cardiomiopatia hipertrófica Research of markers for the genes of the heavy chain of cardiac beta-myosin and myosin binding protein C in relatives of patients with hypertrophic cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Adriana Paula Tirone

    2005-06-01

    Full Text Available OBJETIVO: Estudar os marcadores moleculares para os genes da cadeia pesada da beta-miosina cardíaca e da proteína-C de ligação à miosina em familiares de portadores de cardiomiopatia hipertrófica. MÉTODOS: Foram estudadas 12 famílias que realizaram anamnese, exame físico, eletrocardiograma, ecocardiograma e coleta de sangue para o estudo genético através da reação em cadeia da polimerasse. RESULTADOS: Dos 227 familiares 25% eram acometidos, sendo 51% do sexo masculino com idade média de 35±19 (2 a 95 anos. A análise genética mostrou ligação com o gene da b-miosina cardíaca em uma família e, em outra, ligação com o gene da proteína C de ligação à miosina. Em cinco famílias foram excluídas ligações com os dois genes; em duas, a ligação com o gene da proteína C de ligação à miosina, porém para o gene da b-miosina os resultados foram inconclusivos; em duas famílias os resultados foram inconclusivos para os dois genes e em uma foi excluída ligação para o gene da b-miosina mas ficou inconclusivo para o gene da proteína C de ligação à miosina. CONCLUSÃO: Em nosso meio, talvez predominem outros genes que não aqueles descritos na literatura, ou que existam outras diferenças genéticas relacionadas com a origem de nossa população e/ou fatores ambientais.OBJECTIVE: To study the molecular markers for the genes of the heavy chain of cardiac beta-myosin and the myosin binding protein C in relatives of carriers of hypertrophic cardiomyopathy. METHODS: Twelve families who had anamnesis, physical exam, electrocardiogram, echocardiogram and blood collection for the genetic study through the chain reaction of polymerase. RESULTS: From the 227 relatives, 25% were ill-taken, with 51% men, with an average age of 35±19 (2 to 95 years old. The genetic analysis showed a connection with the gene of the cardiac b-myosin in a family and, in another, a connection with the gene of the myosin-binding protein C. In five

  11. Chain transitivity in hyperspaces

    International Nuclear Information System (INIS)

    Fernández, Leobardo; Good, Chris; Puljiz, Mate; Ramírez, Ártico

    2015-01-01

    Given a non-empty compact metric space X and a continuous function f: X → X, we study the dynamics of the induced maps on the hyperspace of non-empty compact subsets of X and on various other invariant subspaces thereof, in particular symmetric products. We show how some important dynamical properties transfer across induced systems. These amongst others include, chain transitivity, chain (weakly) mixing, chain recurrence, exactness by chains. From our main theorem we derive an ε-chain version of Furstenberg’s celebrated 2 implies n Theorem. We also show the implications our results have for dynamics on continua.

  12. Decisive Markov Chains

    OpenAIRE

    Abdulla, Parosh Aziz; Henda, Noomene Ben; Mayr, Richard

    2007-01-01

    We consider qualitative and quantitative verification problems for infinite-state Markov chains. We call a Markov chain decisive w.r.t. a given set of target states F if it almost certainly eventually reaches either F or a state from which F can no longer be reached. While all finite Markov chains are trivially decisive (for every set F), this also holds for many classes of infinite Markov chains. Infinite Markov chains which contain a finite attractor are decisive w.r.t. every set F. In part...

  13. Gushing metal chain

    Science.gov (United States)

    Belyaev, Alexander; Sukhanov, Alexander; Tsvetkov, Alexander

    2016-03-01

    This article addresses the problem in which a chain falls from a glass from some height. This phenomenon demonstrates a paradoxical rise of the chain over the glass. To explain this effect, an initial hypothesis and an appropriate theory are proposed for calculating the steady fall parameters of the chain. For this purpose, the modified Cayley's problem of falling chain given its rise due to the centrifugal force of upward inertia is solved. Results show that the lift caused by an increase in linear density at the part of chain where it is being bent (the upper part) is due to the convergence of the chain balls to one another. The experiments confirm the obtained estimates of the lifting chain.

  14. Characterization of myosin light chain in shrimp hemocytic phagocytosis.

    Science.gov (United States)

    Han, Fang; Wang, Zhiyong; Wang, Xiaoqing

    2010-11-01

    Myosin light chain, a well-known cytoskeleton gene, regulates multiple processes that are involved in material transport, muscle shrink and cell division. However, its function in phagocytosis against invading pathogens in crustacean remains unknown. In this investigation, a myosin light chain gene was obtained from Marsupenaeus japonicus shrimp. The full-length cDNA of this gene was of 766 bp and an open reading frame (ORF) of 462 bp encoding a polypeptide of 153 amino acids. The myosin light chain protein was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified GST fusion protein. As revealed by immuno-electron microscopy, the myosin light chain protein was only expressed in the dark bands of muscle. In the present study, the myosin light chain gene was up-regulated in the WSSV-resistant shrimp as revealed by real-time PCR and western blot. And the phagocytic percentage and phagocytic index using FITC-labeled Vibrio parahemolyticus were remarkably increased in the WSSV-resistant shrimp, suggesting that the myosin light chain protein was essential in hemocytic phagocytosis. On the other hand, RNAi assays indicated that the phagocytic percentage and phagocytic index were significantly decreased when the myosin light chain gene was silenced by sequence-specific siRNA. These findings suggested that myosin light chain protein was involved in the regulation of hemocytic phagocytosis of shrimp. Copyright 2010 Elsevier Ltd. All rights reserved.

  15. DNA segment containing C/sub β1/, a gene for the constant region of the β chain of the T-cell antigen receptor, was inserted into chromosome 6 in cells from one patients with human T-cell leukemia

    International Nuclear Information System (INIS)

    Ino, T.; Kurosawa, Y.; Yoshida, M.C.; Hirano, M.

    1987-01-01

    DNA rearrangements that occurred in the vicinity of T-cell antigen receptor β-chain gene clusters residing on chromosome 7 were examined in human T-cell acute lymphoblastic leukemia cells. In one patient, it was observed that, for the T-cell receptor β-chain genes, a D/sub β 1/-J/sub β2.3/ (where D is diversity and J is joining) junction was found on one chromosome, while the other chromosome kept the germ-line configuration. If this D/sub β/-J/sub β/ junction was formed by the customary deletion mechanism, the C/sub β1/ gene (where C is constant) located between the D/sub β1/ and J/sub Β2.3/ loci should have disappeared from this chromosome. The C/sub β1/ gene indeed was absent from the rearranged chromosome 7, but it was found on chromosome 6 as an inserted segment. The implications of the observations are discussed

  16. The Search for VH → VWW Standard Model Higgs Production in the Trilepton Signature 5.9fb-1 of Data from p(bar p) Collisions at √s = 1.96 GeV

    International Nuclear Information System (INIS)

    Nett, Jason Michael

    2010-01-01

    We present here the search for Standard Model VH → VWW → lll + E T (missing energy due to neutrinos) production, where V is a W or Z weak vector boson, which uses up to 5.9 fb -1 of integrated luminosity. This analysis has recently added to the CDF high-mass Higgs group three new signal topologies characterized by a tri-lepton signature, which are chosen to isolate the VH → VWW associated production signals in the three-lepton signature. As such, we define three new regions for a WH analysis, a ZH 1-jet analysis, and a ZH (ge) 2-jet analysis with which we expect to contribute an additional ∼ 5.8% (for m H = 165 GeV) acceptance to the current H → WW dilepton analysis. The ZH trilepton regions are defined by events passing a Z-boson selection: events having at least one lepton pairing (among three possible pairings) with opposite sign, same flavor, and a dilepton invariant mass within (76.0, 106.0) GeV - a ± 15 GeV window around the Z-boson mass. The WH trilepton region is then defined as the set of trilepton events that are complement to those chosen by the Z-boson selection. These three new event topologies make a substantial contribution to the H → WW group result. As a measure of the sensitivity of this search, we compute the median expected limit on the at 95% confidence level ('C.L.') on the production cross section (effectively the rate of production) for a Standard Model Higgs boson and report the result as a ratio to the theoretical production cross section. An observed limit ratio of one or less at a given mass would rule out the production of a Standard Model Higgs boson at that mass with 95% confidence. At m H = 165 GeV, the WH analysis expected limits reach 7.2 times the standard model cross section; the ZH 1-jet analysis is set at 29 times the expected standard model cross section; the ZH (ge) 2-jet analysis is set at 9.9 times the expected standard model cross section; and the combined trilepton analysis is set at 4.9 times the expected

  17. Editorial: Supply Chain Management

    Directory of Open Access Journals (Sweden)

    Dimitrios Aidonis

    2017-05-01

    Full Text Available This special issue has followed up the 3rd Olympus International Conference on Supply Chains held on Athens Metropolitan Expo, November 7 & 8 2015, Greece. The Conference was organized by the Department of Logistics Technological Educational Institute of Central Macedonia, in collaboration with the: a Laboratory of Quantitative Analysis, Logistics and Supply Chain Management of the Department of Mechanical Engineering, Aristotle University of Thessaloniki (AUTH, b Greek Association of Supply Chain Management (EEL of Northern Greece and the c Supply Chain & Logistics Journal. During the 2-Days Conference more than 60 research papers were presented covering the following thematic areas: (i Transportation, (ii Best Practices in Logistics, (iii Information and Communication Technologies in Supply Chain Management, (iv Food Logistics, (v New Trends in Business Logistics, and (vi Green Supply Chain Management. Three keynote invited speakers addressed interesting issues for the Operational Research, the Opportunities and Prospects of Greek Ports chaired Round Tables with other Greek and Foreign Scientists and Specialists.

  18. Supply Chain Management og Supply Chain costing

    DEFF Research Database (Denmark)

    Nielsen, Steen; Mortensen, Ole

    2002-01-01

    Formålet med denne artikel er at belyse de muligheder som ligger i at integrere virksomhedens økonomiske styring med begrebet Supply Chain Management (SCM). Dette søges belyst ved først at beskrive den teoretiske ramme, hvori SCM indgår. Herefter analyseres begrebet Supply Chain Costing (SCC) som...... Århus. Et resultat er, at via begrebet Supply Chain Costing skabes der mulighed for at måle logistikkædens aktiviteter i kr./øre. Anvendelsen af denne information har også strategisk betydning for at kunne vælge kunde og leverandør. Ved hjælp af integrationen skabes der også helt nye mulighed...

  19. Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle

    OpenAIRE

    Stull, James T.; Kamm, Kristine E.; Vandenboom, Rene

    2011-01-01

    Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometic bindin...

  20. Supply chain components

    OpenAIRE

    Vieraşu, T.; Bălăşescu, M.

    2011-01-01

    In this article I will go through three main logistics components, which are represented by: transportation, inventory and facilities, and the three secondary logistical components: information, production location, price and how they determine performance of any supply chain. I will discuss then how these components are used in the design, planning and operation of a supply chain. I will also talk about some obstacles a supply chain manager may encounter.

  1. Supply chain components

    Directory of Open Access Journals (Sweden)

    Vieraşu, T.

    2011-01-01

    Full Text Available In this article I will go through three main logistics components, which are represented by: transportation, inventory and facilities, and the three secondary logistical components: information, production location, price and how they determine performance of any supply chain. I will discuss then how these components are used in the design, planning and operation of a supply chain. I will also talk about some obstacles a supply chain manager may encounter.

  2. Markov Tail Chains

    OpenAIRE

    janssen, Anja; Segers, Johan

    2013-01-01

    The extremes of a univariate Markov chain with regularly varying stationary marginal distribution and asymptotically linear behavior are known to exhibit a multiplicative random walk structure called the tail chain. In this paper we extend this fact to Markov chains with multivariate regularly varying marginal distributions in Rd. We analyze both the forward and the backward tail process and show that they mutually determine each other through a kind of adjoint relation. In ...

  3. Economy, market and chain

    OpenAIRE

    Sukkel, W.; Hommes, M.

    2009-01-01

    In their pursuit of growth and professionalisation, the Dutch organic sector focuses primarily on market development. But how do you stimulate the market for organic foods? This is the subject of many research projects concerning market, consumer preferences and the supply chain. These projects focus specifically at consumer purchasing behaviour, product development, supply chain formation and minimising cost price. As a rule, this research takes place in close cooperation with chain actors

  4. Supply chain planning classification

    Science.gov (United States)

    Hvolby, Hans-Henrik; Trienekens, Jacques; Bonde, Hans

    2001-10-01

    Industry experience a need to shift in focus from internal production planning towards planning in the supply network. In this respect customer oriented thinking becomes almost a common good amongst companies in the supply network. An increase in the use of information technology is needed to enable companies to better tune their production planning with customers and suppliers. Information technology opportunities and supply chain planning systems facilitate companies to monitor and control their supplier network. In spite if these developments, most links in today's supply chains make individual plans, because the real demand information is not available throughout the chain. The current systems and processes of the supply chains are not designed to meet the requirements now placed upon them. For long term relationships with suppliers and customers, an integrated decision-making process is needed in order to obtain a satisfactory result for all parties. Especially when customized production and short lead-time is in focus. An effective value chain makes inventory available and visible among the value chain members, minimizes response time and optimizes total inventory value held throughout the chain. In this paper a supply chain planning classification grid is presented based current manufacturing classifications and supply chain planning initiatives.

  5. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

    Science.gov (United States)

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An

    2015-01-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. PMID:26209665

  6. Shark Ig light chain junctions are as diverse as in heavy chains.

    Science.gov (United States)

    Fleurant, Marshall; Changchien, Lily; Chen, Chin-Tung; Flajnik, Martin F; Hsu, Ellen

    2004-11-01

    We have characterized a small family of four genes encoding one of the three nurse shark Ig L chain isotypes, called NS5. All NS5 cDNA sequences are encoded by three loci, of which two are organized as conventional clusters, each consisting of a V and J gene segment that can recombine and one C region exon; the third contains a germline-joined VJ in-frame and the fourth locus is a pseudogene. This is the second nurse shark L chain type where both germline-joined and split V-J organizations have been found. Since there are only two rearranging Ig loci, it was possible for the first time to examine junctional diversity in defined fish Ig genes, comparing productive vs nonproductive rearrangements. N region addition was found to be considerably more extensive in length and in frequency than any other vertebrate L chain so far reported and rivals that in H chain. We put forth the speculation that the unprecedented efficiency of N region addition (87-93% of NS5 sequences) may be a result not only of simultaneous H and L chain rearrangement in the shark but also of processing events that afford greater accessibility of the V or J gene coding ends to terminal deoxynucleotidyltransferase.

  7. Use of molecular modeling and site-directed mutagenesis to define the structural basis for the immune response to carbohydrate xenoantigens

    Directory of Open Access Journals (Sweden)

    Yew Margaret

    2007-03-01

    Full Text Available Abstract Background Natural antibodies directed at carbohydrates reject porcine xenografts. They are initially expressed in germline configuration and are encoded by a small number of structurally-related germline progenitors. The transplantation of genetically-modified pig organs prevents hyperacute rejection, but delayed graft rejection still occurs, partly due to humoral responses. IgVH genes encoding induced xenoantibodies are predominantly, not exclusively, derived from germline progenitors in the VH3 family. We have previously identified the immunoglobulin heavy chain genes encoding VH3 xenoantibodies in patients and primates. In this manuscript, we complete the structural analysis of induced xenoantibodies by identifying the IgVH genes encoding the small proportion of VH4 xenoantibodies and the germline progenitors encoding xenoantibody light chains. This information has been used to define the xenoantibody/carbohydrate binding site using computer-simulated modeling. Results The VH4-59 gene encodes antibodies in the VH4 family that are induced in human patients mounting active xenoantibody responses. The light chain of xenoantibodies is encoded by DPK5 and HSIGKV134. The structural information obtained by sequencing analysis was used to create computer-simulated models. Key contact sites for xenoantibody/carbohydrate interaction for VH3 family xenoantibodies include amino acids in sites 31, 33, 50, 57, 58 and the CDR3 region of the IgVH gene. Site-directed mutagenesis indicates that mutations in predicted contact sites alter binding to carbohydrate xenoantigens. Computer-simulated modeling suggests that the CDR3 region directly influences binding. Conclusion Xenoantibodies induced during early and delayed xenograft responses are predominantly encoded by genes in the VH3 family, with a small proportion encoded by VH4 germline progenitors. This restricted group can be identified by the unique canonical structure of the light chain, heavy

  8. Supply Chain Management

    DEFF Research Database (Denmark)

    Wieland, Andreas; Handfield, Robert B.

    Supply chain management has made great strides in becoming a discipline with a standalone body of theories. As part of this evolution, researchers have sought to embed and integrate observed supply chain management phenomena into theoretical statements. In our review, we explore where we have been...

  9. Critical Chain Exercises

    Science.gov (United States)

    Doyle, John Kevin

    2010-01-01

    Critical Chains project management focuses on holding buffers at the project level vs. task level, and managing buffers as a project resource. A number of studies have shown that Critical Chain project management can significantly improve organizational schedule fidelity (i.e., improve the proportion of projects delivered on time) and reduce…

  10. Value Chain Engineering

    DEFF Research Database (Denmark)

    Wæhrens, Brian Vejrum; Slepniov, Dmitrij

    2015-01-01

    This workbook is recommended for the attention of students of and managers in Danish small and medium sized enterprises (SMEs). Danish SMEs are currently facing a number of key challenges related to their position in global value chains. This book provides an insight into value chain management t...

  11. Fields From Markov Chains

    DEFF Research Database (Denmark)

    Justesen, Jørn

    2005-01-01

    A simple construction of two-dimensional (2-D) fields is presented. Rows and columns are outcomes of the same Markov chain. The entropy can be calculated explicitly.......A simple construction of two-dimensional (2-D) fields is presented. Rows and columns are outcomes of the same Markov chain. The entropy can be calculated explicitly....

  12. Integrated supply chain risk management

    OpenAIRE

    Riaan Bredell; Jackie Walters

    2007-01-01

    Integrated supply chain risk management (ISCRM) has become indispensable to the theory and practice of supply chain management. The economic and political realities of the modern world require not only a different approach to supply chain management, but also bold steps to secure supply chain performance and sustainable wealth creation. Integrated supply chain risk management provides supply chain organisations with a level of insight into their supply chains yet to be achieved. If correctly ...

  13. An Application of the Direct Coulomb Electron Pair Production Process to the Energy Measurement of the "VH-Group" in the "Knee" Region of the "All-Particle" Energy Spectrum

    Science.gov (United States)

    Derrickson, J. H.; Wu, J.; Christl, M. J.; Fountain, W. F.; Parnell, T. A.

    1999-01-01

    The "all-particle" cosmic ray energy spectrum appears to be exhibiting a significant change in the spectral index just above approximately 3000 TeV. This could indicate (1) a change in the propagation of the cosmic rays in the galactic medium, and/or (2) the upper limit of the supernova shock wave acceleration mechanism, and/or (3) a new source of high-energy cosmic rays. Air shower and JACEE data indicate the spectral change is associated with a composition change to a heavier element mixture whereas DICE does not indicate this. A detector concept will be presented that utilizes the energy dependence of the production of direct Coulomb electron-positron pairs by energetic heavy ions. Monte Carlo simulations of a direct electron pair detector consisting of Pb target foils interleaved with planes of 1-mm square scintillating optical fibers will be discussed. The goal is to design a large area, non-saturating instrument to measure the energy spectrum of the individual cosmic ray elements in the "VH-group" for energies greater than 10 TeV/nucleon.

  14. Supply Chain Connectivity: Enhancing Participation in the Global Supply Chain

    OpenAIRE

    Patalinghug, Epictetus E.

    2015-01-01

    Supply chain connectivity is vital for the efficient flow of trade among APEC economies. This paper reviews the literature and supply chain management, describes the barriers to enhancing participation in global supply chain, analyzes the various measures of supply chain performance, and suggests steps for the Philippines to fully reap the benefits of the global value chain.

  15. Understanding the supply chain

    Directory of Open Access Journals (Sweden)

    Aćimović Slobodan

    2006-01-01

    Full Text Available Supply chain management represents new business philosophy and includes strategically positioned and much wider scope of activity in comparison with its "older brother" - management of logistics. Philosophy of the concept of supply chain is directed to more coordination of key business functions of every link in distribution chain in the process of organization of the flow of both goods and information, while logistic managing instruments are focused on internal optimum of flows of goods and information within one company. Applying the concept of integrated supply chain among several companies makes the importance of operative logistics activity even greater on the level of one company, thus advancing processes of optimum and coordination within and between different companies and confirms the importance of logistics performances for the company’s profitability. Besides the fact that the borders between companies are being deleted, this concept of supply chain in one distribution channel influences increasing of importance of functional, i.e. traditional business managing approaches but instead it points out the importance of process managing approaches. Although the author is aware that "there is nothing harder, more dangerous and with uncertain success, but to find a way for introducing some novelties (Machiavelli, it would be even his additional stimulation for trying to bring closer the concept and goals of supply chain implementation that are identified in key, relevant, modern, theoretical and consulting approaches in order to achieve better understanding of the subject and faster implementation of the concept of supply chain management by domestic companies.

  16. Plastic value chains

    DEFF Research Database (Denmark)

    Baxter, John; Wahlstrom, Margareta; Zu Castell-Rüdenhausen, Malin

    2014-01-01

    Optimizing plastic value chains is regarded as an important measure in order to increase recycling of plastics in an efficient way. This can also lead to improved awareness of the hazardous substances contained in plastic waste, and how to avoid that these substances are recycled. As an example......, plastics from WEEE is chosen as a Nordic case study. The project aims to propose a number of improvements for this value chain together with representatives from Nordic stakeholders. Based on the experiences made, a guide for other plastic value chains shall be developed....

  17. Project Decision Chain

    DEFF Research Database (Denmark)

    Rolstadås, Asbjørn; Pinto, Jeffrey K.; Falster, Peter

    2015-01-01

    To add value to project performance and help obtain project success, a new framework for decision making in projects is defined. It introduces the project decision chain inspired by the supply chain thinking in the manufacturing sector and uses three types of decisions: authorization, selection......, and plan decision. A primitive decision element is defined where all the three decision types can be accommodated. Each task in the primitive element can in itself contain subtasks that in turn will comprise new primitive elements. The primitive elements are nested together in a project decision chain....

  18. Myeloma-Derived Light Chain Paired with a Diagnostic Monoclonal Antibody Hinders Immunoassay Performance.

    Science.gov (United States)

    Tu, Bailin; Tieman, Bryan; Moore, Jeffrey; Pan, You; Muerhoff, A Scott

    2017-06-01

    Monoclonal antibodies are widely used as the capture and detection reagents in diagnostic immunoassays. In the past, myeloma fusion partners expressing endogenous heavy and/or light chains were often used to generate hybridoma cell lines. As a result, mixed populations of antibodies were produced that can cause inaccurate test results, poor antibody stability, and significant lot-to-lot variability. We describe one such scenario where the P3U1 (P3X63Ag8U.1) myeloma fusion partner was used in the generation of a hybridoma producing protein induced vitamin K absence/antagonist-II (PIVKA II) antibody. The hybridoma produces three subpopulations of immunoglobulin as determined by ion exchange (IEx) chromatography that exhibit varying degrees of immunoreactivity (0%, 50%, or 100%) to the target antigen as determined by Surface Plasmon Resonance. To produce an antibody with the highest possible sensitivity and specificity, the antigen-specific heavy and light chain variable domains (VH and VL) were cloned from the hybridoma and tethered to murine IgG1 and kappa scaffolds. The resulting recombinant antibody was expressed in Chinese hamster ovary cells and is compatible for use in a diagnostic immunoassay.

  19. Tobacco rattle virus (TRV) based silencing of cotton enoyl-CoA reductase (ECR) gene and the role of very long chain fatty acids in normal leaf development and resistance to wilt disease

    Science.gov (United States)

    A Tobacco rattle virus (TRV) based virus-induced gene silencing (VIGS) assay was employed as a reverse genetic approach to study gene function in cotton (Gossypium hirsutum). This approach was used to investigate the function of Enoyl-CoA reductase (GhECR) in pathogen defense. Amino acid sequence al...

  20. Editorial: Supply Chain Management

    Directory of Open Access Journals (Sweden)

    Aidonis, D.

    2012-01-01

    Full Text Available This special issue has followed up the 2nd Olympus International Conference on Supply Chains held on October 5-6, 2012, in Katerini, Greece. The Conference was organized by the Department of Logistics of Alexander Technological Educational Institution (ATEI of Thessaloniki, in collaboration with the Laboratory of Quantitative Analysis, Logistics and Supply Chain Management of the Department of Mechanical Engineering, Aristotle University of Thessaloniki (AUTH. During the 2-Days Conference more than 50 research papers were presented covering the following thematic areas: (i Business Logistics, (ii Transportation, Telematics and Distribution Networks, (iii Green Logistics, (iv Information and Communication Technologies in Supply Chain Management, and (v Services and Quality. Three keynote invited speakers addressed interesting issues for the Humanitarian Logistics, Green Supply Chains of the Agrifood Sector and the Opportunities and Prospects of Greek Ports chaired Round Tables with other Greek and Foreign Scientists and Specialists.

  1. Characterizing Oregon's supply chains.

    Science.gov (United States)

    2013-03-01

    In many regions throughout the world, freight models are used to aid infrastructure investment and : policy decisions. Since freight is such an integral part of efficient supply chains, more realistic : transportation models can be of greater assista...

  2. Moldova - Value Chain Training

    Data.gov (United States)

    Millennium Challenge Corporation — The evaluation of the GHS value chain training subactivity wwas designed to measure the extent, if any, to which the training activities improved the productivity...

  3. Innovation Across the Supply Chain

    DEFF Research Database (Denmark)

    Druehl, Cheryl; Carrillo, Janice; Hsuan, Juliana

    Innovation is an integral part of every firm’s ongoing operations. Beyond product innovation, supply chain innovations offer a unique source of competitive advantage. We synthesize recent research on innovation in the supply chain, specifically, innovative supply chain processes...

  4. Supply chain risk management

    OpenAIRE

    Christian Hollstein; Frank Himpel

    2013-01-01

    Background: Supply chain risk management increasingly gains prominence in many international industries. In order to strengthen supply chain structures, processes, and networks, adequate potentials for risk management need to be built (focus on effective logistics) and to be utilized (focus on efficient logistics). Natural-based disasters, such as the case of Fukushima, illustrate how crucial risk management is. Method: By aligning a theoretical-conceptual framework with empirical-induct...

  5. Genes and Gene Therapy

    Science.gov (United States)

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  6. MASTERING SUPPLY CHAIN RISKS

    Directory of Open Access Journals (Sweden)

    Borut Jereb

    2012-11-01

    Full Text Available Risks in supply chains represent one of the major business issues today. Since every organizationstrives for success and uninterrupted operations, efficient supply chain risk management is crucial.During supply chain risk research at the Faculty of Logistics in Maribor (Slovenia some keyissues in the field were identified, the major being the lack of instruments which can make riskmanagement in an organization easier and more efficient. Consequently, a model which captures anddescribes risks in an organization and its supply chain was developed. It is in accordance with thegeneral risk management and supply chain security standards, the ISO 31000 and ISO 28000families. It also incorporates recent finding from the risk management field, especially from theviewpoint of segmenting of the public.The model described in this paper focuses on the risks itself by defining them by different keydimensions, so that risk management is simplified and can be undertaken in every supply chain andorganizations within them. Based on our mode and consequent practical research in actualorganizations, a freely accessible risk catalog has been assembled and published online from the risksthat have been identified so far. This catalog can serve as a checklist and a starting point in supplychain risk management in organizations. It also incorporates experts from the field into a community,in order to assemble an ever growing list of possible risks and to provide insight into the model andits value in practice.

  7. Electronic structure of homoleptic transition metal hydrides: TiH4, VH4, CrH4, MnH4, FeH4, CoH4, and NiH4

    International Nuclear Information System (INIS)

    Hood, D.M.; Pitzer, R.M.; Schaefer III, H.F.

    1979-01-01

    Ab initio molecular electronic structure theory has been applied to the family of transition metal tetrahydrides TiH 4 through NiH 4 . For the TiH 4 molecule a wide range of contracted Gaussian basis sets has been tested at the self-consistent-field (SCF) level of theory. The largest basis, labeled M(14s 11p 6d/10s 8p 3d), H(5s 1p/3s 1p), was used for all members of the series and should yield wave functions approaching true Hartree-Fock quality. Predicted SCF dissociation energies (relative to M+4H) and M--H bond distances are TiH 4 132 kcal, 1.70 A; VH 4 86 kcal, 1.64 A; CrH 4 65 kcal, 1.59 A; MnH 4 -- 36 kcal, 1.58 A; FeH 4 0 kcal, 1.58 A; CoH 4 27 kcal, 1.61 A; and NiH 4 18 kcal, 1.75 A. It should be noted immediately that each of these SCF dissociation energies will be increased by electron correlation effects by perhaps as much as 90 kcal. For all of these molecules except TiH 4 excited states have also been studied. One of the most interesting trends seen for these excited states is the shortening of the M--H bond as electrons are transferred from the antibonding 4t 2 orbital to the nonbonding 1e orbitals

  8. Phasic Triplet Markov Chains.

    Science.gov (United States)

    El Yazid Boudaren, Mohamed; Monfrini, Emmanuel; Pieczynski, Wojciech; Aïssani, Amar

    2014-11-01

    Hidden Markov chains have been shown to be inadequate for data modeling under some complex conditions. In this work, we address the problem of statistical modeling of phenomena involving two heterogeneous system states. Such phenomena may arise in biology or communications, among other fields. Namely, we consider that a sequence of meaningful words is to be searched within a whole observation that also contains arbitrary one-by-one symbols. Moreover, a word may be interrupted at some site to be carried on later. Applying plain hidden Markov chains to such data, while ignoring their specificity, yields unsatisfactory results. The Phasic triplet Markov chain, proposed in this paper, overcomes this difficulty by means of an auxiliary underlying process in accordance with the triplet Markov chains theory. Related Bayesian restoration techniques and parameters estimation procedures according to the new model are then described. Finally, to assess the performance of the proposed model against the conventional hidden Markov chain model, experiments are conducted on synthetic and real data.

  9. Nodal-chain metals.

    Science.gov (United States)

    Bzdušek, Tomáš; Wu, QuanSheng; Rüegg, Andreas; Sigrist, Manfred; Soluyanov, Alexey A

    2016-10-06

    The band theory of solids is arguably the most successful theory of condensed-matter physics, providing a description of the electronic energy levels in various materials. Electronic wavefunctions obtained from the band theory enable a topological characterization of metals for which the electronic spectrum may host robust, topologically protected, fermionic quasiparticles. Many of these quasiparticles are analogues of the elementary particles of the Standard Model, but others do not have a counterpart in relativistic high-energy theories. A complete list of possible quasiparticles in solids is lacking, even in the non-interacting case. Here we describe the possible existence of a hitherto unrecognized type of fermionic excitation in metals. This excitation forms a nodal chain-a chain of connected loops in momentum space-along which conduction and valence bands touch. We prove that the nodal chain is topologically distinct from previously reported excitations. We discuss the symmetry requirements for the appearance of this excitation and predict that it is realized in an existing material, iridium tetrafluoride (IrF 4 ), as well as in other compounds of this class of materials. Using IrF 4 as an example, we provide a discussion of the topological surface states associated with the nodal chain. We argue that the presence of the nodal-chain fermions will result in anomalous magnetotransport properties, distinct from those of materials exhibiting previously known excitations.

  10. Vortex-homogenized matrix solid-phase dispersion for the extraction of short chain chlorinated paraffins from indoor dust samples.

    Science.gov (United States)

    Chen, Yu-Hsuan; Chang, Chia-Yu; Ding, Wang-Hsien

    2016-11-11

    A simple and effective method for determining short chain chlorinated paraffins (SCCPs) in indoor dust is presented. The method employed a modified vortex-homogenized matrix solid-phase dispersion (VH-MSPD) prior to its detection by gas chromatography - electron-capture negative-ion mass spectrometry (GC-ECNI-MS) operating in the selected-ion-monitoring (SIM) mode. Under the best extraction conditions, 0.1-g of dust sample was dispersed with 0.1-g of silica gel by using vortex (2min) instead of using a mortar and pestle (3min). After that step, the blend was transferred to a glass column containing 3-g acidic silica gel, 2-g basic silica gel, and 2-g of deactivated silica gel, used as clean-up co-sorbents. Then, target analytes were eluted with 5mL of n-hexane/dichloromethane (2:1, v/v) mixture. The extract was evaporated to dryness under a gentle stream of nitrogen. The residue was then re-dissolved in n-hexane (10μL), and subjected to GC-ECNI-MS analysis. The limits of quantitation (LOQs) ranged from 0.06 to 0.25μg/g for each SCCP congener. Precision was less than 7% for both intra- and inter-day analysis. Trueness was above 89%, which was calculated by mean extraction recovery. The VH-MSPD combined with GC-ECNI-MS was successfully applied to quantitatively detect SCCPs from various indoor dust samples, and the concentrations ranged from 1.2 to 31.2μg/g. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Supply chain quality management

    Directory of Open Access Journals (Sweden)

    Hannan Amoozad Mahdiraji

    2012-10-01

    Full Text Available In recent years, there are several methods introduced for the improvement of operational performances. Total quality management and supply chain management are two methods recommended for this purpose. These two approaches have been studied in most researches separately, while they have objectives in common, and this makes them a strategic means, which can be used, simultaneously. Total quality management and supply chain management play significant roles to increase the organizational competitiveness power. Moreover, they have only one purpose that is customer satisfaction, and they are different only on their approaches to reach their objectives. In this research, we aim to study both approaches of quality management and supply chain, their positive increasing effects that may be generated after their integration. For this purpose, the concept and definitions of each approach is studied, independently, their similarities and differences are recognized, and finally, the advantages of their integration are introduced.

  12. Chain inflation revisited

    International Nuclear Information System (INIS)

    Chialva, Diego; Danielsson, Ulf H

    2008-01-01

    This paper represents an in-depth treatment of the chain inflation scenario. We fully determine the evolution of the universe in the model, the conditions necessary in order to have a successful inflationary period, and the matching with the observational results regarding the cosmological perturbations. We study in great detail, and in general, the dynamics of the background, as well as the mechanism of generation of the perturbations. We also find an explicit formula for the spectrum of adiabatic perturbations. Our results prove that chain inflation is a viable model for solving the horizon, entropy and flatness problems of standard cosmology and for generating the right amount of adiabatic cosmological perturbations. The results are radically different from those found in previous works on the subject. Finally, we argue that there is a natural way to embed chain inflation into flux compactified string theory. We discuss the details of the implementation and how to fit observations

  13. Supply-Chain Optimization Template

    Science.gov (United States)

    Quiett, William F.; Sealing, Scott L.

    2009-01-01

    The Supply-Chain Optimization Template (SCOT) is an instructional guide for identifying, evaluating, and optimizing (including re-engineering) aerospace- oriented supply chains. The SCOT was derived from the Supply Chain Council s Supply-Chain Operations Reference (SCC SCOR) Model, which is more generic and more oriented toward achieving a competitive advantage in business.

  14. Essentials of supply chain management

    CERN Document Server

    Hugos, Michael H

    2011-01-01

    The latest thinking, strategies, developments, and technologies to stay current in supply chain management Presenting the core concepts and techniques of supply chain management in a clear, concise and easily readable style, the Third Edition of Essentials of Supply Chain Management outlines the most crucial tenets and concepts of supply chain management.

  15. Integrated supply chain risk management

    Directory of Open Access Journals (Sweden)

    Riaan Bredell

    2007-11-01

    Full Text Available Integrated supply chain risk management (ISCRM has become indispensable to the theory and practice of supply chain management. The economic and political realities of the modern world require not only a different approach to supply chain management, but also bold steps to secure supply chain performance and sustainable wealth creation. Integrated supply chain risk management provides supply chain organisations with a level of insight into their supply chains yet to be achieved. If correctly applied, this process may optimise management decision-making and assist in the protection and enhancement of shareholder value.

  16. Solitons in Granular Chains

    International Nuclear Information System (INIS)

    Manciu, M.; Sen, S.; Hurd, A.J.

    1999-01-01

    The authors consider a chain of elastic (Hertzian) grains that repel upon contact according to the potential V = adelta u , u > 2, where delta is the overlap between the grains. They present numerical and analytical results to show that an impulse initiated at an end of a chain of Hertzian grains in contact eventually propagates as a soliton for all n > 2 and that no solitons are possible for n le 2. Unlike continuous, they find that colliding solitons in discrete media initiative multiple weak solitons at the point of crossing

  17. Nuclear safety chains

    International Nuclear Information System (INIS)

    Robbins, M.C.; Eames, G.F.; Mayell, J.R.

    1981-01-01

    An original scheme has been developed for expressing the complex interrelationships associated with the engineered safeguards provided for a nuclear power station. This management tool, based upon network diagrams called Nuclear Safety Chains, looks at the function required of a particular item of safety plant, defines all of the vital supplies and support features necessary for successful operation, and expresses them in visual form, to facilitate analysis and optimisation for operations and maintenance staff. The safety chains are confined to manual schemes at present, although they are designed to be compatible with modern computer techniques. Their usefulness with any routine maintenance planning application on high technology plant is already being appreciated. (author)

  18. Markov set-chains

    CERN Document Server

    Hartfiel, Darald J

    1998-01-01

    In this study extending classical Markov chain theory to handle fluctuating transition matrices, the author develops a theory of Markov set-chains and provides numerous examples showing how that theory can be applied. Chapters are concluded with a discussion of related research. Readers who can benefit from this monograph are those interested in, or involved with, systems whose data is imprecise or that fluctuate with time. A background equivalent to a course in linear algebra and one in probability theory should be sufficient.

  19. The design of supply chains

    OpenAIRE

    Bøge Sørensen, Lars

    2004-01-01

    Keywords Supply Chain Management, Supply Chain Design, Literature study Abstract Argues stability is a design objective for supply chain design alongside cost, lead time and responsiveness. Performs an extensive literature study on supply chain design, identifies methods, theories and objectives in the existing literature. Describes the concept external specificity and how it’s used to design supply chains. Using the concept upstream, archetypes of risk minimal and maximal desi...

  20. Standard handbook of chains chains for power transmission and material handling

    CERN Document Server

    2005-01-01

    A BRIEF HISTORY OF THE DEVELOPMENT OF CHAINEarly DevelopmentsCog ChainCast Detachable ChainCast Pintle ChainPrecision Roller ChainEngineering Steel ChainSilent ChainFlat-Top ChainTerminologyA CHAIN OVERVIEW: USES AND ADVANTAGESGeneralTypes of ChainScope of Chains CoveredStyles and Forms of ChainsStraight and Offset Link ChainsChains With and Without RollersUses of ChainStandard Chains and Their UsesThe Advantages of Chains in ApplicationsAdvantages of Roller Chains in DrivesAdvantages of Silent Chain Drives

  1. Development Value Chains meet Business Supply Chains : The concept of Global Value Chains unraveled

    NARCIS (Netherlands)

    S. Drost (Sarah); J.C.A.C. van Wijk (Jeroen); S.R. Vellema (Sietze)

    2011-01-01

    textabstractValue chain promotion is considered a key element of private sector development strategies and pro-poor growth. However, (value) chain concepts are rather complex and unclear. This paper unravels the concept of global value chains and studies the diversity of key value chain-related

  2. Innovation in Supply Chains

    DEFF Research Database (Denmark)

    Maier, Maximilian; Korbel, Jakob; Brem, Alexander

    2015-01-01

    . Moreover, along with the fourth industrial revolution – industry 4.0 – new technologies such as cyber physical systems are fast gaining popularity. Hence, based on the analysis of relevant literature, we further develop the supply chain committee model, developed by Kaluza et al. (2003) to demonstrate how...

  3. Markov Chain Monte Carlo

    Indian Academy of Sciences (India)

    be obtained as a limiting value of a sample path of a suitable ... makes a mathematical model of chance and deals with the problem by .... Is the Markov chain aperiodic? It is! Here is how you can see it. Suppose that after you do the cut, you hold the top half in your right hand, and the bottom half in your left. Then there.

  4. Serum Free Light Chains

    Science.gov (United States)

    ... lab's website in order to provide you with background information about the test(s) you had performed. You will need to return ... the free light chains have a much shorter half-life (3-5 hours) than ... cell disorders, the test is also used for assessing response and minimal ...

  5. Galileo Chain Thermometer

    Science.gov (United States)

    Ucke, C.; Schlichting, H. J.

    2017-01-01

    This relatively rare thermometer has a rather unusual display: lower temperatures are located at the top of the scale, higher ones at the bottom. A sphere on a chain floats in a suitable liquid, sinking at high temperatures when the density of the liquid decreases and rising in the increased density at low temperatures. With reasonable effort and…

  6. Supply Chain Costing

    DEFF Research Database (Denmark)

    Asmussen, Jesper Normann; Kristensen, Jesper; Wæhrens, Brian Vejrum

    Based Costing (ABC) på et forsyningskædeniveau – heri benævnt Supply Chain Costing (SCC). Udoverdefordelederfindesved ABCtilføjerSCCogså et økonomisk grundlag til det strategiske rationale, der ofte ligger bag opbygningen af virksomhedens forsyningskæde, og kan dermed medvirke til konkret...

  7. Global Value Chain Configuration

    DEFF Research Database (Denmark)

    Hernandez, Virginia; Pedersen, Torben

    2017-01-01

    modes chosen and the different ways of coordinating them. We also examine the outcomes of a global value chain configuration in terms of performance and upgrading. Our aim is to review the state of the art of these issues, identify research gaps and suggest new lines for future research that would...

  8. Exploration Supply Chain Simulation

    Science.gov (United States)

    2008-01-01

    The Exploration Supply Chain Simulation project was chartered by the NASA Exploration Systems Mission Directorate to develop a software tool, with proper data, to quantitatively analyze supply chains for future program planning. This tool is a discrete-event simulation that uses the basic supply chain concepts of planning, sourcing, making, delivering, and returning. This supply chain perspective is combined with other discrete or continuous simulation factors. Discrete resource events (such as launch or delivery reviews) are represented as organizational functional units. Continuous resources (such as civil service or contractor program functions) are defined as enabling functional units. Concepts of fixed and variable costs are included in the model to allow the discrete events to interact with cost calculations. The definition file is intrinsic to the model, but a blank start can be initiated at any time. The current definition file is an Orion Ares I crew launch vehicle. Parameters stretch from Kennedy Space Center across and into other program entities (Michaud Assembly Facility, Aliant Techsystems, Stennis Space Center, Johnson Space Center, etc.) though these will only gain detail as the file continues to evolve. The Orion Ares I file definition in the tool continues to evolve, and analysis from this tool is expected in 2008. This is the first application of such business-driven modeling to a NASA/government-- aerospace contractor endeavor.

  9. Perturbed Markov chains

    OpenAIRE

    Solan, Eilon; Vieille, Nicolas

    2015-01-01

    We study irreducible time-homogenous Markov chains with finite state space in discrete time. We obtain results on the sensitivity of the stationary distribution and other statistical quantities with respect to perturbations of the transition matrix. We define a new closeness relation between transition matrices, and use graph-theoretic techniques, in contrast with the matrix analysis techniques previously used.

  10. Economy, market and chain

    NARCIS (Netherlands)

    Sukkel, W.; Hommes, M.

    2009-01-01

    In their pursuit of growth and professionalisation, the Dutch organic sector focuses primarily on market development. But how do you stimulate the market for organic foods? This is the subject of many research projects concerning market, consumer preferences and the supply chain. These projects

  11. Heavy chain only antibodies

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud

    2013-01-01

    Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen...

  12. Markov Chain Monte Carlo

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 7; Issue 3. Markov Chain Monte Carlo - Examples. Arnab Chakraborty. General Article Volume 7 Issue 3 March 2002 pp 25-34. Fulltext. Click here to view fulltext PDF. Permanent link: https://www.ias.ac.in/article/fulltext/reso/007/03/0025-0034. Keywords.

  13. Supply chain reliability modelling

    Directory of Open Access Journals (Sweden)

    Eugen Zaitsev

    2012-03-01

    Full Text Available Background: Today it is virtually impossible to operate alone on the international level in the logistics business. This promotes the establishment and development of new integrated business entities - logistic operators. However, such cooperation within a supply chain creates also many problems related to the supply chain reliability as well as the optimization of the supplies planning. The aim of this paper was to develop and formulate the mathematical model and algorithms to find the optimum plan of supplies by using economic criterion and the model for the probability evaluating of non-failure operation of supply chain. Methods: The mathematical model and algorithms to find the optimum plan of supplies were developed and formulated by using economic criterion and the model for the probability evaluating of non-failure operation of supply chain. Results and conclusions: The problem of ensuring failure-free performance of goods supply channel analyzed in the paper is characteristic of distributed network systems that make active use of business process outsourcing technologies. The complex planning problem occurring in such systems that requires taking into account the consumer's requirements for failure-free performance in terms of supply volumes and correctness can be reduced to a relatively simple linear programming problem through logical analysis of the structures. The sequence of the operations, which should be taken into account during the process of the supply planning with the supplier's functional reliability, was presented.

  14. METRODOS: Meteorological preprocessor chain

    DEFF Research Database (Denmark)

    Astrup, P.; Mikkelsen, T.; Deme, S.

    2001-01-01

    The METRODOS meteorological preprocessor chain combines measured tower data and coarse grid numerical weather prediction (NWP) data with local scale flow models and similarity scaling to give high resolution approximations of the meteorological situation. Based on available wind velocity and dire...

  15. Co-regulation of the atrial natriuretic factor and cardiac myosin light chain-2 genes during alpha-adrenergic stimulation of neonatal rat ventricular cells. Identification of cis sequences within an embryonic and a constitutive contractile protein gene which mediate inducible expression.

    Science.gov (United States)

    Knowlton, K U; Baracchini, E; Ross, R S; Harris, A N; Henderson, S A; Evans, S M; Glembotski, C C; Chien, K R

    1991-04-25

    To study the mechanisms which mediate the transcriptional activation of cardiac genes during alpha adrenergic stimulation, the present study examined the regulated expression of three cardiac genes, a ventricular embryonic gene (atrial natriuretic factor, ANF), a constitutively expressed contractile protein gene (cardiac MLC-2), and a cardiac sodium channel gene. alpha 1-Adrenergic stimulation activates the expression and release of ANF from neonatal ventricular cells. As assessed by RNase protection analyses, treatment with alpha-adrenergic agonists increases the steady-state levels of ANF mRNA by greater than 15-fold. However, a rat cardiac sodium channel gene mRNA is not induced, indicating that alpha-adrenergic stimulation does not lead to an increase in the expression of all cardiac genes. Studies employing a series of rat ANF luciferase and rat MLC-2 luciferase fusion genes identify 315- and 92-base pair cis regulatory sequences within an embryonic gene (ANF) and a constitutively expressed contractile protein gene (MLC-2), respectively, which mediate alpha-adrenergic-inducible gene expression. Transfection of various ANF luciferase reporters into neonatal rat ventricular cells demonstrated that upstream sequences which mediate tissue-specific expression (-3003 to -638) can be segregated from those responsible for inducibility. The lack of inducibility of a cardiac Na+ channel gene, and the segregation of ANF gene sequences which mediate cardiac specific from those which mediate inducible expression, provides further insight into the relationship between muscle-specific and inducible expression during cardiac myocyte hypertrophy. Based on these results, a testable model is proposed for the induction of embryonic cardiac genes and constitutively expressed contractile protein genes and the noninducibility of a subset of cardiac genes during alpha-adrenergic stimulation of neonatal rat ventricular cells.

  16. Effect of praziquantel on the differential expression of mouse hepatic genes and parasite ATP binding cassette transporter gene family members during Schistosoma mansoni infection.

    Directory of Open Access Journals (Sweden)

    Melissa C Sanchez

    2017-06-01

    Full Text Available Schistosomiasis is a chronic parasitic disease caused by sexually dimorphic blood flukes of the genus Schistosoma. Praziquantel (PZQ is the only drug widely available to treat the disease but does not kill juvenile parasites. Here we report the use of next generation sequencing to study the transcriptional effect of PZQ on murine hepatic inflammatory, immune and fibrotic responses to Schistosoma mansoni worms and eggs. An initial T helper cell 1 (Th1 response is induced against schistosomes in mice treated with drug vehicle (Vh around the time egg laying begins, followed by a T helper cell 2 (Th2 response and the induction of genes whose action leads to granuloma formation and fibrosis. When PZQ is administered at this time, there is a significant reduction in egg burden yet the hepatic Th1, Th2 and fibrotic responses are still observed in the absence of granuloma formation suggesting some degree of gene regulation may be induced by antigens released from the dying adult worms. Quantitative real-time PCR was used to examine the relative expression of 16 juvenile and adult S. mansoni genes during infection and their response to Vh and PZQ treatment in vivo. While the response of stress genes in adult parasites suggests the worms were alive immediately following exposure to PZQ, they were unable to induce transcription of any of the 9 genes encoding ATP-binding cassette (ABC transporters tested. In contrast, juvenile schistosomes were able to significantly induce the activities of ABCB, C and G family members, underscoring the possibility that these efflux systems play a major role in drug resistance.

  17. The design of supply chains

    DEFF Research Database (Denmark)

    Bøge Sørensen, Lars

    2004-01-01

    Keywords Supply Chain Management, Supply Chain Design, Literature studyAbstract Argues stability is a design objective for supply chain design alongside cost, leadtime and responsiveness. Performs an extensive literature study on supply chain design,identifies methods, theories and objectives...... in the existing literature. Describes the conceptexternal specificity and how it's used to design supply chains. Using the concept upstream,archetypes of risk minimal and maximal design are identified. Downstream the conceptdescribes two viable scenarios, one minimizing the impact, the other minimizing...... theprobability of (intended) departure of a supply chain partner. Finally, principles for supplychain design are described and managerial outlined....

  18. Decision-Making for Supply Chain Integration Supply Chain Integration

    CERN Document Server

    Lettice, Fiona; Durowoju, Olatunde

    2012-01-01

    Effective supply chain integration, and the tight co-ordination it creates, is an essential pre-requisite for successful supply chain management.  Decision-Making for Supply Chain Integration is a practical reference on recent research in the area of supply chain integration focusing on distributed decision-making problems. Recent applications of various decision-making tools for integrating supply chains are covered including chapters focusing on: •Supplier selection, pricing strategy and inventory decisions in multi-level supply chains, •RFID-enabled distributed decision-making, •Operational risk issues and time-critical decision-making for sensitive logistics nodes, Modelling end to end processes to improve supply chain integration, and •Integrated systems to improve service delivery and optimize resource use. Decision-Making for Supply Chain Integration provides an insight into the tools and methodologies of this field with support from real-life case studies demonstrating successful application ...

  19. Effects of dietary valine:lysine ratio on the performance, amino acid composition of tissues and mRNA expression of genes involved in branched-chain amino acid metabolism of weaned piglets

    Directory of Open Access Journals (Sweden)

    Ye Tong Xu

    2018-01-01

    Full Text Available Objective The goal of this study was to investigate the effects of dietary standard ileal digestible (SID valine:lysine ratios on performance, intestinal morphology, amino acids of liver and muscle, plasma indices and mRNA expression of branched-chain amino acid (BCAA metabolism enzymes. Methods A total of 144 crossbred pigs (Duroc×Landrace×Large White weaned at 28±4 days of age (8.79±0.02 kg body weight were randomly allotted to 1 of 4 diets formulated to provide SID valine:lysine ratios of 50%, 60%, 70%, or 80%. Each diet was fed to 6 pens of pigs with 6 pigs per pen (3 gilts and 3 barrows for 28 days. Results Average daily gain increased quadratically (p<0.05, the villous height of the duodenum, jejunum and ileum increased linearly (p<0.05 as the SID valine:lysine ratio increased. The concentrations of plasma α-keto isovaleric and valine increased linearly (p<0.05, plasma aspartate, asparagine and cysteine decreased (p<0.05 as the SID valine:lysine ratio increased. An increase in SID lysine:valine levels increased mRNA expression levels of mitochondrial BCAA transaminase and branched-chain α-keto acid dehydrogenase in the longissimus dorsi muscle (p<0.05. Conclusion Using a quadratic model, a SID valine:lysine ratio of 68% was shown to maximize the growth of weaned pigs which is slightly higher than the level recommended by the National Research Council [6].

  20. The IASI detection chain

    Science.gov (United States)

    Nicol, Patrick; Fleury, Joel; Le Naour, Claire; Bernard, Frédéric

    2017-11-01

    IASI (Infrared Atmospheric Sounding Interferometer) is an infrared atmospheric sounder. It will provide meteorologist and scientific community with atmospheric spectra. The instrument is composed of a Fourier transform spectrometer and an associated infrared imager. The presentation will describe the spectrometer detection chain architecture, composed by three different detectors cooled in a passive cryo-cooler (so called CBS : Cold Box Subsystem) and associated analog electronics up to digital conversion. It will mainly focus on design choices with regards to environment constraints, implemented technologies, and associated performances. CNES is leading the IASI program in collaboration with EUMETSAT. The instrument Prime is ALCATEL SPACE responsible, notably, of the detection chain architecture. SAGEM SA provides the detector package (so called CAU : Cold Acquisition Unit).

  1. Sympathetic chain Schwannoma

    International Nuclear Information System (INIS)

    Al-Mashat, Faisal M.

    2009-01-01

    Schwannomas are rare, benign, slowly growing tumors arising from Schwann cells that line nerve sheaths. Schwannomas arising from the cervical sympathetic chain are extremely rare. Here, we report a case of a 70-year-old man who presented with only an asymptomatic neck mass. Physical examination revealed a left sided Horner syndrome and a neck mass with transmitted pulsation and anterior displacement of the carotid artery. Computed tomography (CT) showed a well-defined non-enhancing mass with vascular displacement. The nerve of origin of this encapsulated tumor was the sympathetic chain. The tumor was excised completely intact. The pathologic diagnosis was Schwannoma (Antoni type A and Antoni type B). The patient has been well and free of tumor recurrence for 14 months with persistence of asymptomatic left sided Horner syndrome. The clinical, radiological and pathological evaluations, therapy and postoperative complications of this tumor are discussed. (author)

  2. Extended linear chain compounds

    CERN Document Server

    Linear chain substances span a large cross section of contemporary chemistry ranging from covalent polymers, to organic charge transfer com­ plexes to nonstoichiometric transition metal coordination complexes. Their commonality, which coalesced intense interest in the theoretical and exper­ imental solid state physics/chemistry communities, was based on the obser­ vation that these inorganic and organic polymeric substrates exhibit striking metal-like electrical and optical properties. Exploitation and extension of these systems has led to the systematic study of both the chemistry and physics of highly and poorly conducting linear chain substances. To gain a salient understanding of these complex materials rich in anomalous aniso­ tropic electrical, optical, magnetic, and mechanical properties, the conver­ gence of diverse skills and talents was required. The constructive blending of traditionally segregated disciplines such as synthetic and physical organic, inorganic, and polymer chemistry, crystallog...

  3. Environmental Retail Supply Chains

    DEFF Research Database (Denmark)

    Kotzab, Herbert; Munch, Hilde; de Faultrier, Birgitte

    2011-01-01

    which were grouped into eight categories; they refer to “fundamental environmental attitude”, “use of energy”, “use of input material”, “product”, “packaging”, “transport”, “consumption” and “waste”. The level of environmental supply chain management can be characterised as very operational and very...... short-term oriented (green operations). Long-term oriented green design initiatives were hardly observed. Furthermore, the specific environmental activities of three retailers from Denmark, France and the UK were compared. Research limitations/implications – The empirical study investigates supply chain...... operations of retailers and excludes other areas of retail management. The results are based on material that is published by the respective companies and thus do not include internal reports. Originality/value – The main contribution of this paper is to test the proposition that global retailers follow...

  4. Towards Intelligent Supply Chains

    DEFF Research Database (Denmark)

    Siurdyban, Artur; Møller, Charles

    2012-01-01

    applied to the context of organizational processes can increase the success rate of business operations. The framework is created using a set of theoretical based constructs grounded in a discussion across several streams of research including psychology, pedagogy, artificial intelligence, learning...... of deploying inapt operations leading to deterioration of profits. To address this problem, we propose a unified business process design framework based on the paradigm of intelligence. Intelligence allows humans and human-designed systems cope with environmental volatility, and we argue that its principles......, business process management and supply chain management. It outlines a number of system tasks combined in four integrated management perspectives: build, execute, grow and innovate, put forward as business process design propositions for Intelligent Supply Chains....

  5. Port supply chain integration : analyzing biofuel supply chains

    NARCIS (Netherlands)

    Stevens, Leonie C. E.; Vis, Iris F. A.

    2016-01-01

    This paper focuses on port supply chain integration to strengthen operational and business performance. We provide a structured and comprehensive method to enable port supply chain integration and demonstrate its applicability to the biofuel supply chain. We define the value proposition, role,

  6. The innovation value chain.

    Science.gov (United States)

    Hansen, Morten T; Birkinshaw, Julian

    2007-06-01

    The challenges of coming up with fresh ideas and realizing profits from them are different for every company. One firm may excel at finding good ideas but may have weak systems for bringing them to market. Another organization may have a terrific process for funding and rolling out new products and services but a shortage of concepts to develop. In this article, Hansen and Birkinshaw caution executives against using the latest and greatest innovation approaches and tools without understanding the unique deficiencies in their companies' innovation systems. They offer a framework for evaluating innovation performance: the innovation value chain. It comprises the three main phases of innovation (idea generation, conversion, and diffusion) as well as the critical activities performed during those phases (looking for ideas inside your unit; looking for them in other units; looking for them externally; selecting ideas; funding them; and promoting and spreading ideas companywide). Using this framework, managers get an end-to-end view of their innovation efforts. They can pinpoint their weakest links and tailor innovation best practices appropriately to strengthen those links. Companies typically succumb to one of three broad "weakest-link" scenarios. They are idea poor, conversion poor, or diffusion poor. The article looks at the ways smart companies - including Intuit, P&G, Sara Lee, Shell, and Siemens- modify the best innovation practices and apply them to address those organizations' individual needs and flaws. The authors warn that adopting the chain-based view of innovation requires new measures of what can be delivered by each link in the chain. The approach also entails new roles for employees "external scouts" and "internal evangelists," for example. Indeed, in their search for new hires, companies should seek out those candidates who can help address particular weaknesses in the innovation value chain.

  7. Supply Chain Initiatives Database

    Energy Technology Data Exchange (ETDEWEB)

    None

    2012-11-01

    The Supply Chain Initiatives Database (SCID) presents innovative approaches to engaging industrial suppliers in efforts to save energy, increase productivity and improve environmental performance. This comprehensive and freely-accessible database was developed by the Institute for Industrial Productivity (IIP). IIP acknowledges Ecofys for their valuable contributions. The database contains case studies searchable according to the types of activities buyers are undertaking to motivate suppliers, target sector, organization leading the initiative, and program or partnership linkages.

  8. Supply chain finance

    Directory of Open Access Journals (Sweden)

    Kasavica Petar

    2014-01-01

    Full Text Available The concept of supply chain finance is a response to global illiquidity, intensified through the global economic crisis and globalization of commercial and financial flows. The growing illiquidity undermines credit ratings of economic entities, thereby reducing the potential for achieving the projected goals (profitability and portfolio quality. In order to overcome this, banks have introduced certain products flexible to the requirements of specific transactions. The concerned products redirect the focus from a client's credit rating and risk to the credit rating and risk of a business partner (buyer, resulting in benefits for all transaction participants ('win-win-win'. Moreover, the activities are targeted at transaction analysis, i.e. the isolation and protection of the cash flow as the source of financial instrument's repayment. On the other hand, there has been an increasing number of transactions based on the risk of the commercial bank of the client's business partner, or on the risk of collateral (inventory. The focus is actually placed on the financing of adequate supply chain stages, given that counterparty relationship management has been proven to be crucial for efficient management of one's own business. The tensions existing in the relations between partners (increasingly long payment deadlines are in the basis of the supply chain finance concept. Decisions made by banks are based on the entire supply chain (wide information basis, thereby shifting the focus from the product (as was the case before the crisis to the client's needs. Thus, decisions become increasingly comprehensive, quicker, and more precise, and portfolios less risky. Through the individual portfolio of banks, the market of national economies also becomes safer and more liquid. These are rather profitable transactions, because, due to the risk transfer, financing is enabled to companies to whom classic crediting in most cases is not available.

  9. Pairwise Choice Markov Chains

    OpenAIRE

    Ragain, Stephen; Ugander, Johan

    2016-01-01

    As datasets capturing human choices grow in richness and scale---particularly in online domains---there is an increasing need for choice models that escape traditional choice-theoretic axioms such as regularity, stochastic transitivity, and Luce's choice axiom. In this work we introduce the Pairwise Choice Markov Chain (PCMC) model of discrete choice, an inferentially tractable model that does not assume any of the above axioms while still satisfying the foundational axiom of uniform expansio...

  10. Distinguishing Hidden Markov Chains

    OpenAIRE

    Kiefer, Stefan; Sistla, A. Prasad

    2015-01-01

    Hidden Markov Chains (HMCs) are commonly used mathematical models of probabilistic systems. They are employed in various fields such as speech recognition, signal processing, and biological sequence analysis. We consider the problem of distinguishing two given HMCs based on an observation sequence that one of the HMCs generates. More precisely, given two HMCs and an observation sequence, a distinguishing algorithm is expected to identify the HMC that generates the observation sequence. Two HM...

  11. Approximate quantum Markov chains

    CERN Document Server

    Sutter, David

    2018-01-01

    This book is an introduction to quantum Markov chains and explains how this concept is connected to the question of how well a lost quantum mechanical system can be recovered from a correlated subsystem. To achieve this goal, we strengthen the data-processing inequality such that it reveals a statement about the reconstruction of lost information. The main difficulty in order to understand the behavior of quantum Markov chains arises from the fact that quantum mechanical operators do not commute in general. As a result we start by explaining two techniques of how to deal with non-commuting matrices: the spectral pinching method and complex interpolation theory. Once the reader is familiar with these techniques a novel inequality is presented that extends the celebrated Golden-Thompson inequality to arbitrarily many matrices. This inequality is the key ingredient in understanding approximate quantum Markov chains and it answers a question from matrix analysis that was open since 1973, i.e., if Lieb's triple ma...

  12. The coprime quantum chain

    Science.gov (United States)

    Mussardo, G.; Giudici, G.; Viti, J.

    2017-03-01

    In this paper we introduce and study the coprime quantum chain, i.e. a strongly correlated quantum system defined in terms of the integer eigenvalues n i of the occupation number operators at each site of a chain of length M. The n i ’s take value in the interval [2,q] and may be regarded as S z eigenvalues in the spin representation j  =  (q  -  2)/2. The distinctive interaction of the model is based on the coprimality matrix \\boldsymbolΦ : for the ferromagnetic case, this matrix assigns lower energy to configurations where occupation numbers n i and n i+1 of neighbouring sites share a common divisor, while for the anti-ferromagnetic case it assigns a lower energy to configurations where n i and n i+1 are coprime. The coprime chain, both in the ferro and anti-ferromagnetic cases, may present an exponential number of ground states whose values can be exactly computed by means of graph theoretical tools. In the ferromagnetic case there are generally also frustration phenomena. A fine tuning of local operators may lift the exponential ground state degeneracy and, according to which operators are switched on, the system may be driven into different classes of universality, among which the Ising or Potts universality class. The paper also contains an appendix by Don Zagier on the exact eigenvalues and eigenvectors of the coprimality matrix in the limit q\\to ∞ .

  13. Adjustable chain trees for proteins

    DEFF Research Database (Denmark)

    Winter, Pawel; Fonseca, Rasmus

    2012-01-01

    A chain tree is a data structure for changing protein conformations. It enables very fast detection of clashes and free energy potential calculations. A modified version of chain trees that adjust themselves to the changing conformations of folding proteins is introduced. This results in much...... tighter bounding volume hierarchies and therefore fewer intersection checks. Computational results indicate that the efficiency of the adjustable chain trees is significantly improved compared to the traditional chain trees....

  14. A chain of FPU cells

    NARCIS (Netherlands)

    Verhulst, F.

    2015-01-01

    In contrast to the classical Fermi-Pasta-Ulam (FPU) chain, the inhomogeneous FPU chain shows nearly all the principal resonances. Using this fact, we can construct a periodic FPU chain of low dimension, for instance a FPU cell of four degrees-of-freedom, that can be used as a building block for a

  15. Designing structural supply chain flexibility

    NARCIS (Netherlands)

    Mulinski, Ksawery Jan

    2012-01-01

    In a continuously changing business environment the role of supply chain flexibility is constantly increasing. A flexible supply chain can ensure survival in quickly changing market conditions as well as enable sustainable growth. This thesis explores the topic of supply chain flexibility with focus

  16. Process Algebra and Markov Chains

    NARCIS (Netherlands)

    Brinksma, Hendrik; Hermanns, H.; Brinksma, Hendrik; Hermanns, H.; Katoen, Joost P.

    This paper surveys and relates the basic concepts of process algebra and the modelling of continuous time Markov chains. It provides basic introductions to both fields, where we also study the Markov chains from an algebraic perspective, viz. that of Markov chain algebra. We then proceed to study

  17. Process algebra and Markov chains

    NARCIS (Netherlands)

    Brinksma, E.; Hermanns, H.; Brinksma, E.; Hermanns, H.; Katoen, J.P.

    2001-01-01

    This paper surveys and relates the basic concepts of process algebra and the modelling of continuous time Markov chains. It provides basic introductions to both fields, where we also study the Markov chains from an algebraic perspective, viz. that of Markov chain algebra. We then proceed to study

  18. Risk Management in Logystics Chains

    OpenAIRE

    Butrin, Andrey; Vikulov, Vladimir

    2013-01-01

    Article is devoted to risk management of supply chain. The authors considered indicators of supply chain risks, including risks caused by supplier. Authors formed a method of optimizing the level of supply chain risk in the integration with suppliers and customers.

  19. Academic Education Chain Operation Model

    OpenAIRE

    Ruskov, Petko; Ruskov, Andrey

    2007-01-01

    This paper presents an approach for modelling the educational processes as a value added chain. It is an attempt to use a business approach to interpret and compile existing business and educational processes towards reference models and suggest an Academic Education Chain Operation Model. The model can be used to develop an Academic Chain Operation Reference Model.

  20. Branched-Chain Amino Acids

    Directory of Open Access Journals (Sweden)

    Matteo Ghiringhelli

    2015-07-01

    Full Text Available Our study is focused on evaluation and use of the most effective and correct nutrients. In particular, our attention is directed to the role of certain amino acids in cachectic patients. During parenteral nutrition in humans, physician already associates in the PN-bags different formulations including amino acids, lipids and glucose solutions or essential amino acids solution alone or exclusively branched-chain amino acids (BCAA. Studies investigated the effects of dietary BCAA ingestion on different diseases and conditions such as obesity and metabolic disorders, liver disease, muscle atrophy, cancer, impaired immunity or injuries (surgery, trauma, burns, and sepsis. BCAAs have been shown to affect gene expression, protein metabolism, apoptosis and regeneration of hepatocytes, and insulin resistance. They have also been shown to inhibit the proliferation of liver cancer cells in vitro, and are essential for lymphocyte proliferation and dendritic cell maturation. Oral or parenteral administration of these three amino acids will allow us to evaluate the real efficacy of these compounds during a therapy to treat malnutrition in subjects unable to feed themselves.

  1. Radiology's value chain.

    Science.gov (United States)

    Enzmann, Dieter R

    2012-04-01

    A diagnostic radiology value chain is constructed to define its main components, all of which are vulnerable to change, because digitization has caused disaggregation of the chain. Some components afford opportunities to improve productivity, some add value, while some face outsourcing to lower labor cost and to information technology substitutes, raising commoditization risks. Digital image information, because it can be competitive at smaller economies of scale, allows faster, differential rates of technological innovation of components, initiating a centralization-to-decentralization technology trend. Digitization, having triggered disaggregation of radiology's professional service model, may soon usher in an information business model. This means moving from a mind-set of "reading images" to an orientation of creating and organizing information for greater accuracy, faster speed, and lower cost in medical decision making. Information businesses view value chain investments differently than do small professional services. In the former model, producing a better business product will extend image interpretation beyond a radiologist's personal fund of knowledge to encompass expanding external imaging databases. A follow-on expansion with integration of image and molecular information into a report will offer new value in medical decision making. Improved interpretation plus new integration will enrich and diversify radiology's key service products, the report and consultation. A more robust, information-rich report derived from a "systems" and "computational" radiology approach will be facilitated by a transition from a professional service to an information business. Under health care reform, radiology will transition its emphasis from volume to greater value. Radiology's future brightens with the adoption of a philosophy of offering information rather than "reads" for decision making. Staunchly defending the status quo via turf wars is unlikely to constitute a

  2. Musical Markov Chains

    Science.gov (United States)

    Volchenkov, Dima; Dawin, Jean René

    A system for using dice to compose music randomly is known as the musical dice game. The discrete time MIDI models of 804 pieces of classical music written by 29 composers have been encoded into the transition matrices and studied by Markov chains. Contrary to human languages, entropy dominates over redundancy, in the musical dice games based on the compositions of classical music. The maximum complexity is achieved on the blocks consisting of just a few notes (8 notes, for the musical dice games generated over Bach's compositions). First passage times to notes can be used to resolve tonality and feature a composer.

  3. Chains of securitization

    Directory of Open Access Journals (Sweden)

    Marieke de Goede

    2017-12-01

    Full Text Available Securitization has a dual rationality that twins a financial sense to one of modern statecraft and national security. This forum contribution advocates a focus on material practices as a means of further exploring the entanglement of finance and security. In particular, it advances a notion of ‘chains of securitization’, arguing that such a concept provides researchers with a concrete way to analyze how ‘financial’ objects, such as derivatives, are assembled to generate (insecurities, as well as how ‘security’ objects, such as suspicious transactions, are assembled across public/private domains.

  4. Detox fashion supply chain

    CERN Document Server

    2017-01-01

    This first volume on detox fashion discusses various interesting topics including a Toxic-Free Supply Chain for Textiles and Clothing; Environmental Issues in Textiles; Global Regulations, Restrictions & Research; Making the Change: Consumer Adoption of Sustainable Fashion; and Strategies for Detoxing Your Wardrobe. It provides an overview of the chemical-related issues confronting the fashion sector, summarizes global regulations, and discusses how to make the change by changing consumers’ attitude towards adopting sustainable fashion, as well as the best strategies for detoxing our wardrobes.

  5. 6d string chains

    Science.gov (United States)

    Gadde, Abhijit; Haghighat, Babak; Kim, Joonho; Kim, Seok; Lockhart, Guglielmo; Vafa, Cumrun

    2018-02-01

    We consider bound states of strings which arise in 6d (1,0) SCFTs that are realized in F-theory in terms of linear chains of spheres with negative self-intersections 1,2, and 4. These include the strings associated to N small E 8 instantons, as well as the ones associated to M5 branes probing A and D type singularities in M-theory or D5 branes probing ADE singularities in Type IIB string theory. We find that these bound states of strings admit (0,4) supersymmetric quiver descriptions and show how one can compute their elliptic genera.

  6. Shared Value Chain Design

    DEFF Research Database (Denmark)

    Bals, Lydia; Tate, Wendy L.

    In Sustainable Supply Chain Management (SSCM) research still the classic economic perspective is the dominating perspective, although the triple bottom line (including economic, social and ecological) is well accepted. The theoretical foundation for the paper is Stakeholder Theory. Case studies...... of social businesses funded by the Germany-based company Yunus Social Business (YSB; http://www.yunussb.com) are presented, extracting the basic model types and how they involve and affect the stakeholders. These models reveal innovative approaches to enable positive economic, social and ecological impacts...

  7. Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells

    NARCIS (Netherlands)

    D.D. Drabek (Dubravka); R. Janssens (Rick); Boer, E. (Ernie de); Rademaker, R. (Rik); Kloess, J. (Johannes); J.J. Skehel (John ); Grosveld, F. (Frank)

    2016-01-01

    textabstractSeveral technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and

  8. Concurrent Product & Supply Chain Creation

    DEFF Research Database (Denmark)

    Gubi, Ebbe

    it is a structural premise. We also know that logistics costs generally are estimated 15-20% of total product costs. Accordingly, it stands to reason that a company can reduce costs, and thereby gain an edge on its competitors, by tailoring the supply chain in question to an individual product or product family; i.......e. by creating Focused Supply Chains. At the same time, customer satisfaction can be increased. As a second means to achieving a better fit between product and supply chain, the firm can deploy Design for Logistics, the discipline of considering the supply chain during product creation. The thesis sets out...... and supply chains should be created concurrently and integrated. The concept of Concurrent Product & Supply Chain Creation is introduced, and the two main components Focused Supply Chains and Design For Logistics are explained and exemplified by use of Bang & Olufsen....

  9. Self-lacing atom chains

    International Nuclear Information System (INIS)

    Zandvliet, Harold J W; Van Houselt, Arie; Poelsema, Bene

    2009-01-01

    The structural and electronic properties of self-lacing atomic chains on Pt modified Ge(001) surfaces have been studied using low-temperature scanning tunnelling microscopy and spectroscopy. The self-lacing chains have a cross section of only one atom, are perfectly straight, thousands of atoms long and virtually defect free. The atomic chains are composed of dimers that have their bonds aligned in a direction parallel to the chain direction. At low temperatures the atomic chains undergo a Peierls transition: the periodicity of the chains doubles from a 2 x to a 4 x periodicity and an energy gap opens up. Furthermore, at low temperatures (T<80 K) novel quasi-one-dimensional electronic states are found. These quasi-one-dimensional electronic states originate from an electronic state of the underlying terrace that is confined between the atomic chains.

  10. Evaluation of the impact of genetic polymorphisms in glutathione-related genes on the association between methylmercury or n-3 polyunsaturated long chain fatty acids and risk of myocardial infarction: a case-control study

    Directory of Open Access Journals (Sweden)

    Norberg Margareta

    2011-04-01

    Full Text Available Abstract Background The n-3 polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid, which are present in fish, are protective against myocardial infarction. However, fish also contains methylmercury, which influences the risk of myocardial infarction, possibly by generating oxidative stress. Methylmercury is metabolized by conjugation to glutathione, which facilitates elimination. Glutathione is also an antioxidant. Individuals with certain polymorphisms in glutathione-related genes may tolerate higher exposures to methylmercury, due to faster metabolism and elimination and/or better glutathione-associated antioxidative capacity. They would thus benefit more from the protective agents in fish, such as eicosapentaenoic+docosahexaenoic acid and selenium. The objective for this study was to elucidate whether genetic polymorphisms in glutathione-related genes modify the association between eicosapentaenoic+docosahexaenoic acid or methylmercury and risk of first ever myocardial infarction. Methods Polymorphisms in glutathione-synthesizing (glutamyl-cysteine ligase catalytic subunit, GCLC and glutamyl-cysteine ligase modifier subunit, GCLM or glutathione-conjugating (glutathione S-transferase P, GSTP1 genes were genotyped in 1027 individuals from northern Sweden (458 cases of first-ever myocardial infarction and 569 matched controls. The impact of these polymorphisms on the association between erythrocyte-mercury (proxy for methylmercury and risk of myocardial infarction, as well as between plasma eicosapentaenoic+docosahexaenoic acid and risk of myocardial infarction, was evaluated by conditional logistic regression. The effect of erythrocyte-selenium on risk of myocardial infarction was also taken into consideration. Results There were no strong genetic modifying effects on the association between plasma eicosapentaenoic+docosahexaenoic acid or erythrocyte-mercury and risk of myocardial infarction risk. When eicosapentaenoic

  11. Human beta 2 chain of laminin (formerly S chain): cDNA cloning, chromosomal localization, and expression in carcinomas

    DEFF Research Database (Denmark)

    Wewer, U M; Gerecke, D R; Durkin, M E

    1994-01-01

    or other known laminin genes. Immunostaining showed that the beta 2 chain is localized to the smooth muscle basement membranes of the arteries, while the homologous beta 1 chain is confined to the subendothelial basement membranes. The beta 2 chain was found in the basement membranes of ovarian carcinomas......Overlapping cDNA clones that encode the full-length human laminin beta 2 chain, formerly called the S chain, were isolated. The cDNA of 5680 nt contains a 5391-nt open reading frame encoding 1797 amino acids. At the amino terminus is a 32-amino-acid signal peptide that is followed by the mature...... beta 2 chain polypeptide of 1765 amino acids with a calculated molecular mass of 192,389 Da. The human beta 2 chain is predicted to have all of the seven structural domains typical of the beta chains of laminin, including the short cysteine-rich alpha region. The amino acid sequence of human beta 2...

  12. Analysis by rotavirus gene 6 reverse transcriptase-polymerase chain reaction assay of rotavirus-positive gastroenteritis cases observed during the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST)

    Science.gov (United States)

    Matson, David O; Vesikari, Timo; Dennehy, Penelope; Dallas, Michael D; Goveia, Michelle G; Itzler, Robbin F; Ciarlet, Max

    2014-01-01

    During the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST), the period between the administration of dose 1 through 13 days after the administration of dose 3, there were more wild-type rotavirus gastroenteritis (RVGE) cases among vaccine recipients compared with placebo recipients using the protocol-specified microbiological plaque assay in the clinical-efficacy cohort, a subset of subjects where vaccine efficacy against RVGE of any severity was assessed. In this study, a rotavirus genome segment 6-based reverse transcriptase–polymerase chain reaction assay was applied post hoc to clarify the accuracy of type categorization of all these RVGE cases in vaccine recipients during the vaccination phase of REST. The assay characterized 147 (90%) of 163 re-assayed RVGE cases or rotavirus-associated health care contacts as type-determinable: either wild-type or vaccine-type rotavirus strains. In the clinical-efficacy cohort (N = 5673), 19 (18.8%) of 101 samples from RVGE cases contained wild-type rotavirus, 70 (69.3%) vaccine virus, and 12 (11.9%) were indeterminable. In the large-scale cohort (N = 68,038), 10 (34.5%) of 29 samples from RVGE-related health care contacts contained wild-type rotavirus strains, 15 (51.7%) vaccine-type rotavirus strains, and 4 (13.8%) were indeterminable. Of the 33 samples from RVGE cases in placebo recipients, all were confirmed to contain wild-type rotaviruses. Altogether, this post-hoc re-evaluation showed that the majority (75%) of type-determinable RVGE cases or health care contacts that occurred during the vaccination phase of REST in vaccine recipients were associated with vaccine-type rotavirus strains rather than wild-type rotavirus strains. PMID:25424931

  13. Analysis by rotavirus gene 6 reverse transcriptase-polymerase chain reaction assay of rotavirus-positive gastroenteritis cases observed during the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST).

    Science.gov (United States)

    Matson, David O; Vesikari, Timo; Dennehy, Penelope; Dallas, Michael D; Goveia, Michelle G; Itzler, Robbin F; Ciarlet, Max

    2014-01-01

    During the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST), the period between the administration of dose 1 through 13 days after the administration of dose 3, there were more wild-type rotavirus gastroenteritis (RVGE) cases among vaccine recipients compared with placebo recipients using the protocol-specified microbiological plaque assay in the clinical-efficacy cohort, a subset of subjects where vaccine efficacy against RVGE of any severity was assessed. In this study, a rotavirus genome segment 6-based reverse transcriptase-polymerase chain reaction assay was applied post hoc to clarify the accuracy of type categorization of all these RVGE cases in vaccine recipients during the vaccination phase of REST. The assay characterized 147 (90%) of 163 re-assayed RVGE cases or rotavirus-associated health care contacts as type-determinable: either wild-type or vaccine-type rotavirus strains. In the clinical-efficacy cohort (N = 5673), 19 (18.8%) of 101 samples from RVGE cases contained wild-type rotavirus, 70 (69.3%) vaccine virus, and 12 (11.9%) were indeterminable. In the large-scale cohort (N = 68,038), 10 (34.5%) of 29 samples from RVGE-related health care contacts contained wild-type rotavirus strains, 15 (51.7%) vaccine-type rotavirus strains, and 4 (13.8%) were indeterminable. Of the 33 samples from RVGE cases in placebo recipients, all were confirmed to contain wild-type rotaviruses. Altogether, this post-hoc re-evaluation showed that the majority (75%) of type-determinable RVGE cases or health care contacts that occurred during the vaccination phase of REST in vaccine recipients were associated with vaccine-type rotavirus strains rather than wild-type rotavirus strains.

  14. A chain of microphones

    International Nuclear Information System (INIS)

    1994-07-01

    In order to discover a more accurate and selective measuring method for the identification of individual flow-noise pollution sources on wind turbines blades, measuring equipment based on a chain of microphones was developed. The principle underlying the design of this equipment is that signals from a number of microphones can be interpreted. Thus the microphones can register noise from sections of the rotary blade and unwished-for noise is eliminated. The gating technique ensures that noises from individual blades can be separated and that clarity is improved. In addition to this, noise can be determined close to the source. The chain consists of 8 microphones placed in a row at adjustable distances. Measurements are registered on tapes as are the trigger signals for the blade passage. The computer processes the measurement results and unnecessary noise is depressed. The listening angles can also be changed electronically so that the doppler effect can be corrected. Results confirmed that the equipment operated satisfactorily and could also be used in relation to noise pollution in power plants as it is especially effective in depressing excess, and cutting out outside, noise and registers accurately individual sources of noise helped by its ability to ''listen '' at varying angles to the source. (AB)

  15. Creative industries value chain: The value chain logic in supply chain relationships

    OpenAIRE

    Emilia Madudová

    2017-01-01

    The purpose of this paper is to provide a deeper look into value chain logic in supply chain relationships in a creative industries value chains. In recent years, value has been recognized as a key factor in better understanding of consumer behavior and gaining a competitive advantage. In a value chain, added value should be defined at every step of the chain. There should be defined activity which adds value as well as the activity that subtracts any value. The total value can be then calcul...

  16. Polyneutron Chain Reactions

    International Nuclear Information System (INIS)

    John C. Fisher

    2000-01-01

    Although helium atoms do not form molecules, a sufficiently large number will bind into a stable liquid droplet. A comparable situation is expected for neutrons, with a sufficiently large number binding into a stable droplet of neutron matter. Such polyneutron droplets can be viewed as isotopes of an element with nuclear charge Z=0, tentatively denoted neutrium, symbol Nt. Because of the relatively weak binding of neutrons compared with that of a mix of neutrons and protons, the minimum number of neutrons required for stability of a droplet is fairly large. Early estimates of ∼60 may be reduced to a dozen or so by the BCS pairing interaction. The Nt entries with N≥12 are new to the table of isotopes. Because all of them are beta-unstable, none is expected to persist as a free particle. Yet, some may occasionally be produced by means to be described below, and it is of interest to examine their decay chains and their interactions with charged nuclei to ascertain how their presence might be revealed. Although these reactions are interesting, they cannot be taken seriously without identifying a source for the initial Nt isotope that begins the chain. Here, we consider possible interactions between 16 O and A Nt. Although there is no strong interaction between them, we can expect a very weak residual attraction that can form a loosely bound 16 O A Nt nuclear molecule. This is not a compound nucleus in the usual sense because, considered as fluids, the 16 O and A Nt droplets are immiscible. For a droplet with fewer than about 60 neutrons, beta decay of A Nt is prevented by the buildup of Coulomb energy associated with transforming A Nt into A H in close proximity to 16 O. Thus, it is possible that 16 O A Nt molecules can persist indefinitely and that a few of them may be present in ordinary water as supermassive oxygen nuclei. Because the binding of these molecules is weak, the A Nt component can tunnel to an adjacent nucleus, and if the adjacent nucleus is 18 O, a

  17. One long chain among shorter chains : the Flory approach revisited

    OpenAIRE

    Raphaël , E.; Fredrickson , G.; Pincus , P.

    1992-01-01

    We consider the mean square end-to-end distance of a long chain immersed in a monodisperse, concentrated solution of shorter, chemically identical chains. In contrast with the earlier work of Flory, no simplifying assumption on the wave vector dependence of the effective potential between segments is made. In order to obtain a closed form expression for the dimension of the long chain, we first derive a general expression for the mean square end-to-end distance of a flexible chain with arbitr...

  18. Baby Skyrmion chains

    International Nuclear Information System (INIS)

    Foster, David

    2010-01-01

    The Baby Skyrmion model is a two-dimensional analogue of the full three-dimensional Skyrme model. It is not just useful for guiding investigations in the Skyrme model, it also has applications in condensed matter physics. Previous results on multi-charged Baby Skyrmion solutions have pointed to a modular structure, comprised of charge two rings and single charge one Skyrmions, which combine to form higher charged structures. In this paper we present alternative numerical solutions that correspond to new finite Baby Skyrmion chains, which have lower energy than those found previously, and are also good candidates for the global minimum energy solutions. We then proceed from the infinite plane, to Baby Skyrmions on a cylinder and then a torus, to obtain the solutions of periodic Baby Skyrmions, of which periodic segments will correspond to sections of large charge Baby Skyrmions in the plane

  19. Supply chain reaction

    International Nuclear Information System (INIS)

    Snieckus, Darius

    2000-01-01

    'Logic' (Leading Oil and Gas Industry Competitiveness) is a government-industry supply chain management initiative which aims to improve the competitiveness of the UK's North Sea business by 1 billion UK pounds by 2002 and its export performance by 50% inside 5 years. Much of the article is devoted to the background and views of Logic's chief executive Chris. Freeman. Freeman makes clear that 'unlike Crine, we are not a cost-reduction initiative: that may be one of the outcomes, but we are really focusing on the co-operation side of things'. Logic aims to change the culture of the UK offshore industry through example. Freeman believes that the creation of collaborative success will flag up industry and give credence to Logics objectives

  20. Polymerase chain reaction for the detection of Mycobacterium leprae

    NARCIS (Netherlands)

    Hartskeerl, R. A.; de Wit, M. Y.; Klatser, P. R.

    1989-01-01

    A polymerase chain reaction (PCR) using heat-stable Taq polymerase is described for the specific detection of Mycobacterium leprae, the causative agent of leprosy. A set of primers was selected on the basis of the nucleotide sequence of a gene encoding the 36 kDa antigen of M. leprae. With this set

  1. Polymorphism in TNP-1 gene of Murrah buffalo bulls | Panigrahi ...

    African Journals Online (AJOL)

    ... spermatids, have been reported to be important for histone displacement and chromatin ... (TNP-1) gene was analyzed using polymerase chain reaction-single strand ... Analysis of TNP-1 gene sequence of Murrah buffalo revealed 3 single ...

  2. Flux through a Markov chain

    International Nuclear Information System (INIS)

    Floriani, Elena; Lima, Ricardo; Ourrad, Ouerdia; Spinelli, Lionel

    2016-01-01

    Highlights: • The flux through a Markov chain of a conserved quantity (mass) is studied. • Mass is supplied by an external source and ends in the absorbing states of the chain. • Meaningful for modeling open systems whose dynamics has a Markov property. • The analytical expression of mass distribution is given for a constant source. • The expression of mass distribution is given for periodic or random sources. - Abstract: In this paper we study the flux through a finite Markov chain of a quantity, that we will call mass, which moves through the states of the chain according to the Markov transition probabilities. Mass is supplied by an external source and accumulates in the absorbing states of the chain. We believe that studying how this conserved quantity evolves through the transient (non-absorbing) states of the chain could be useful for the modelization of open systems whose dynamics has a Markov property.

  3. Advances in single chain technology.

    Science.gov (United States)

    Gonzalez-Burgos, Marina; Latorre-Sanchez, Alejandro; Pomposo, José A

    2015-10-07

    The recent ability to manipulate and visualize single atoms at atomic level has given rise to modern bottom-up nanotechnology. Similar exquisite degree of control at the individual polymeric chain level for producing functional soft nanoentities is expected to become a reality in the next few years through the full development of so-called "single chain technology". Ultra-small unimolecular soft nano-objects endowed with useful, autonomous and smart functions are the expected, long-term valuable output of single chain technology. This review covers the recent advances in single chain technology for the construction of soft nano-objects via chain compaction, with an emphasis in dynamic, letter-shaped and compositionally unsymmetrical single rings, complex multi-ring systems, single chain nanoparticles, tadpoles, dumbbells and hairpins, as well as the potential end-use applications of individual soft nano-objects endowed with useful functions in catalysis, sensing, drug delivery and other uses.

  4. A remark on Dickey's stabilizing chain

    International Nuclear Information System (INIS)

    Svinin, Andrei K.

    2011-01-01

    We observe that Dickey's stabilizing chain can be naturally included into two-dimensional chain of infinitely many copies of equations of KP hierarchy. -- Highlights: → In this study we consider Dickey's stabilizing chain. → We construct two-dimensional chain of dressing truncated operators. → We show that Dickey's stabilizing chain can be included into two-dimensional chain of KP hierarchies.

  5. Chain formation of metal atoms

    DEFF Research Database (Denmark)

    Bahn, Sune Rastad; Jacobsen, Karsten Wedel

    2001-01-01

    The possibility of formation of single-atomic chains by manipulation of nanocontacts is studied for a selection of metals (Ni, Pd, Pt, Cu, Ag, Au). Molecular dynamics simulations show that the tendency for chain formation is strongest for Au and Pt. Density functional theory calculations indicate...... that the metals which form chains exhibit pronounced many-atom interactions with strong bonding in low coordinated systems....

  6. Non-stationary Markov chains

    OpenAIRE

    Mallak, Saed

    1996-01-01

    Ankara : Department of Mathematics and Institute of Engineering and Sciences of Bilkent University, 1996. Thesis (Master's) -- Bilkent University, 1996. Includes bibliographical references leaves leaf 29 In thi.s work, we studierl the Ergodicilv of Non-Stationary .Markov chains. We gave several e.xainples with different cases. We proved that given a sec[uence of Markov chains such that the limit of this sec|uence is an Ergodic Markov chain, then the limit of the combination ...

  7. DOS cones along atomic chains

    Science.gov (United States)

    Kwapiński, Tomasz

    2017-03-01

    The electron transport properties of a linear atomic chain are studied theoretically within the tight-binding Hamiltonian and the Green’s function method. Variations of the local density of states (DOS) along the chain are investigated. They are crucial in scanning tunnelling experiments and give important insight into the electron transport mechanism and charge distribution inside chains. It is found that depending on the chain parity the local DOS at the Fermi level can form cone-like structures (DOS cones) along the chain. The general condition for the local DOS oscillations is obtained and the linear behaviour of the local density function is confirmed analytically. DOS cones are characterized by a linear decay towards the chain which is in contrast to the propagation properties of charge density waves, end states and Friedel oscillations in one-dimensional systems. We find that DOS cones can appear due to non-resonant electron transport, the spin-orbit scattering or for chains fabricated on a substrate with localized electrons. It is also shown that for imperfect chains (e.g. with a reduced coupling strength between two neighboring sites) a diamond-like structure of the local DOS along the chain appears.

  8. DOS cones along atomic chains

    International Nuclear Information System (INIS)

    Kwapiński, Tomasz

    2017-01-01

    The electron transport properties of a linear atomic chain are studied theoretically within the tight-binding Hamiltonian and the Green’s function method. Variations of the local density of states (DOS) along the chain are investigated. They are crucial in scanning tunnelling experiments and give important insight into the electron transport mechanism and charge distribution inside chains. It is found that depending on the chain parity the local DOS at the Fermi level can form cone-like structures (DOS cones) along the chain. The general condition for the local DOS oscillations is obtained and the linear behaviour of the local density function is confirmed analytically. DOS cones are characterized by a linear decay towards the chain which is in contrast to the propagation properties of charge density waves, end states and Friedel oscillations in one-dimensional systems. We find that DOS cones can appear due to non-resonant electron transport, the spin–orbit scattering or for chains fabricated on a substrate with localized electrons. It is also shown that for imperfect chains (e.g. with a reduced coupling strength between two neighboring sites) a diamond-like structure of the local DOS along the chain appears. (paper)

  9. Markov Chain Monte Carlo Methods

    Indian Academy of Sciences (India)

    Keywords. Markov chain; state space; stationary transition probability; stationary distribution; irreducibility; aperiodicity; stationarity; M-H algorithm; proposal distribution; acceptance probability; image processing; Gibbs sampler.

  10. Risikobasiertes Supply-Chain-Management

    DEFF Research Database (Denmark)

    Wieland, Andreas; Schinz, Philipp

    2015-01-01

    Das Management erlebt seit einigen Jahren dahingehend einen Paradigmenwechsel, dass nicht mehr Unternehmen als einzelne autonome Einheiten im Wettbewerb zueinander stehen, sondern die Supply Chains, in denen diese Unternehmen eingebunden sind. Ursachen hierfür sind beispielsweise die Auslagerung...... von Nicht-Kernprozessen auf vorgelagerte Stufen der Supply Chain oder auf Supply-Chain-Dienstleister sowie die Internationalisierung der Beschaffung. Dieser Paradigmenwechsel hat traditionelles Denken in den Schranken des Systems „Unternehmen“ abgelöst und zum Supply- Chain-Denken geführt, das vor......- und nachgelagerte Stufen des Systems „End-to-End-Supply-Chain“ einbezieht, etwa auch die Lieferanten von Lieferanten....

  11. Requirements of supply chain management in differentiating European pork chains

    NARCIS (Netherlands)

    Trienekens, J.H.; Wognum, P.M.

    2013-01-01

    This paper summarizes results obtained by research into pork chain management in the EU Integrated Project Q-Porkchains. Changing demands for intrinsic and extrinsic quality attributes of pork products impact the way supply chain management should be organized from the farmer down to the consumer.

  12. Chain Dynamics in Magnetorheological Suspensions

    Science.gov (United States)

    Gast, A. P.; Furst, E. M.

    1999-01-01

    Magnetorheological (MR) suspensions are composed of colloidal particles which acquire dipole moments when subjected to an external magnetic field. At sufficient field strengths and concentrations, the dipolar particles rapidly aggregate to form long chains. Subsequent lateral cross-linking of the dipolar chains is responsible for a rapid liquid-to-solid-like rheological transition. The unique, magnetically-activated rheological properties of MR suspensions make them ideal for interfacing mechanical systems to electronic controls. Additionally, the ability to experimentally probe colloidal suspensions interacting through tunable anisotropic potentials is of fundamental interest. Our current experimental work has focused on understanding the fluctuations of dipolar chains. It has been proposed by Halsey and Toor (HT) that the strong Landau-Peierls thermal fluctuations of dipolar chains could be responsible for long-range attractions between chains. Such interactions will govern the long-time relaxation of MR suspensions. We have synthesized monodisperse neutrally buoyant MR suspensions by density matching stabilized ferrofluid emulsion droplets with D2O. This allows us to probe the dynamics of the dipolar chains using light scattering without gravitational, interfacial, and polydispersity effects to resolve the short-wavelength dynamics of the dipolar chains. We used diffusing wave spectroscopy to measure these dynamics. The particle displacements at short times that show an independence to the field strength, but at long times exhibit a constrained, sub-diffusive motion that slows as the dipole strength is increased. The experiments are in good qualitative agreement with Brownian dynamics simulations of dipolar chains. Although there have been several important and detailed studies of the structure and interactions in MR suspensions, there has not been conclusive evidence that supports or contradicts the HT model prediction that long-range interactions exist between

  13. Requirements of supply chain management in differentiating European pork chains.

    Science.gov (United States)

    Trienekens, Jacques; Wognum, Nel

    2013-11-01

    This paper summarizes results obtained by research into pork chain management in the EU Integrated Project Q-Porkchains. Changing demands for intrinsic and extrinsic quality attributes of pork products impact the way supply chain management should be organized from the farmer down to the consumer. The paper shows the importance of Quality Management Systems for integrating supply chains and enhancing consumer confidence. The paper also presents innovations in information system integration for aligning information exchange in the supply chain and logistics concepts based on innovative measurement technologies at the slaughterhouse stage. In the final section research challenges towards sustainable pork supply chains satisfying current consumer demands are presented. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Association between genetic polymorphisms in the human interleukin-7 receptor alpha-chain and inhalation allergy

    DEFF Research Database (Denmark)

    Shamim, Z; Müller, K; Svejgaard, A

    2007-01-01

    Thymic stromal-derived lymphopoietin (TSLP) and interleukin-7 share a common receptor chain, IL-7Ralpha. IL-7 is involved in T-cell homeostasis, and TSLP induces production of pro-allergic cytokines. The gene encoding the IL-7Ralpha chain is polymorphic, and investigation of inhalation allergic p...

  15. SUPPLY CHAIN STRATEGIES IN THE CONTEXT OF AN E-COMMERCE CHAIN (E-CHAIN

    Directory of Open Access Journals (Sweden)

    João Gilberto Mendes do Reis

    2014-05-01

    Full Text Available This paper purpose to explore the relationships between supply chain strategies and product performance in retail e-commerce. In this case, we concern that in current, in order to bear up under competition, organizations have to manage their supply chains so that they meet the needs of their final customers. With this concept in mind, the research presented in this study focuses on establishing the right strategy for supply chains according to their product segment. Thus, after a Literature Review, the paper explain a methodology based in different authors studies. Finally the article focuses on a pratical case in e-commerce retail that describes its application in this field. The research shows that it is possible to use a methodology for classifying supply chains using chain strategies and product features. The use of the right strategy for supply chains will improve the competitive advantage of businesses. One limitation is that the methodology study focuses on only two e-commerce segment; future studies may go further in refining the proposed framework for other segments. The aim of this research is to offer businesses a model for evaluating supply chains, allowing them to improve the performance of their products and services by using the right strategy for supply chains. The classification proposal of this paper presents an original model for classification of supply chains based on different studies on the theme.

  16. The organization structure and regulatory elements of Chlamydomonas histone genes reveal features linking plant and animal genes.

    Science.gov (United States)

    Fabry, S; Müller, K; Lindauer, A; Park, P B; Cornelius, T; Schmitt, R

    1995-09-01

    The genome of the green alga Chlamydomonas reinhardtii contains approximately 15 gene clusters of the nucleosomal (or core) histone H2A, H2B, H3 and H4 genes and at least one histone H1 gene. Seven non-allelic histone gene loci were isolated from a genomic library, physically mapped, and the nucleotide sequences of three isotypes of each core histone gene species and one linked H1 gene determined. The core histone genes are organized in clusters of H2A-H2B and H3-H4 pairs, in which each gene pair shows outwardly divergent transcription from a short (< 300 bp) intercistronic region. These intercistronic regions contain typically conserved promoter elements, namely a TATA-box and the three motifs TGGCCAG-G(G/C)-CGAG, CGTTGACC and CGGTTG. Different from the genes of higher plants, but like those of animals and the related alga Volvox, the 3' untranslated regions contain no poly A signal, but a palindromic sequence (3' palindrome) essential for mRNA processing is present. One single H1 gene was found in close linkage to a H2A-H2B pair. The H1 upstream region contains the octameric promoter element GGTTGACC (also found upstream of the core histone genes) and two specific sequence motifs that are shared only with the Volvox H1 promoters. This suggests differential transcription of the H1 and the core histone genes. The H1 gene is interrupted by two introns. Unlike Volvox H3 genes, the three sequenced H3 isoforms are intron-free. Primer-directed PCR of genomic DNA demonstrated, however, that at least 8 of the about 15 H3 genes do contain one intron at a conserved position. In synchronized C. reinhardtii cells, H4 mRNA levels (representative of all core histone mRNAs) peak during cell division, suggesting strict replication-dependent gene control. The derived peptide sequences place C. reinhardtii core histones closer to plants than to animals, except that the H2A histones are more animal-like. The peptide sequence of histone H1 is closely related to the V. carteri VH1-II

  17. Negotiations in Supply Chain Management

    DEFF Research Database (Denmark)

    Zachariassen, Frederik

    2008-01-01

    negotiation literature. Subsequently, an empirical case study is presented, which explicitly aims at exploring the role of negotiations between members of a supply chain. Based on the findings, the paper concludes on specific, normative guidelines for negotiations for improved supply chain competitiveness...

  18. Monitoring the vaccine cold chain.

    OpenAIRE

    Cheriyan, E

    1993-01-01

    Maintaining the vaccine cold chain is an essential part of a successful immunisation programme. A continuous electronic temperature monitor helped to identify breaks in the cold chain in the community and the study led to the issue of proper guidelines and replacement of faulty equipment.

  19. Spatial Configuration of Macromolecular Chains

    Indian Academy of Sciences (India)

    where C reaches a constant value with increase in n for any series of finitely flexible chains. The smoothed density within the domain of a linear macromolecule having a molecular weight of 100,000 or greater (i.e., n > 1000) is low, only on the order of one percent or less of the space being occupied by chain segments.

  20. Spatial Configuration of Macromolecular Chains

    Indian Academy of Sciences (India)

    where C reaches a constant value with increase in n for any series of finitely flexible chains. ... entirely free of overlaps between any pair of segments is very small for a long chain. The attrition of .... Excerpts froin Flory's Nobel Banquet Speech.

  1. Academic Education Chain Operation Model

    NARCIS (Netherlands)

    Ruskov, Petko; Ruskov, Andrey

    2007-01-01

    This paper presents an approach for modelling the educational processes as a value added chain. It is an attempt to use a business approach to interpret and compile existing business and educational processes towards reference models and suggest an Academic Education Chain Operation Model. The model

  2. Integrable multiparametric quantum spin chains

    CERN Document Server

    Förster, A; Roditi, I; Foerster, Angela; Links, Jon; Roditi, Itzhak

    1998-01-01

    Using Reshetikhin's construction for multiparametric quantum algebras we obtain the associated multiparametric quantum spin chains. We show that under certain restrictions these models can be mapped to quantum spin chains with twisted boundary conditions. We illustrate how this general formalism applies to construct multiparametric versions of the supersymmetric t-J and U models.

  3. Realities of Supply Chain Collaboration

    NARCIS (Netherlands)

    Kampstra, R.P.; Ashayeri, J.; Gattorna, J.

    2006-01-01

    Successful supply chain collaboration (SCC) practices are rather exceptional, yet collaboration is believed to be the single most pressing need in supply chain management.In this paper we discuss the realities of SCC, present prerequisites for the collaboration process, indicate where the process

  4. Environmental Management in Product Chains

    DEFF Research Database (Denmark)

    Jørgensen, Michael Søgaard; Forman, Marianne

    2009-01-01

    between existing resources, norms and values and external pressures for environmental management (second section). A model for the types of corporate network relations that need to be mapped and understood in order to analyze and/or develop environmental management in a product chain (third section......The chapter aims at giving background to companies, consultants, governmental regulators, NGOs etc. for the analysis and planning of environmental management in specific product chains through: A framework for understanding environmental management in product chains as shaped by the interaction......). An overview of examples from our own research and from literature of the type and the role of environmental issues and initiatives in product chains (fourth section). A typology for characterizing corporate strategies as part of environmental management in product chains and characterizing those competencies...

  5. The Service-profit Chain

    DEFF Research Database (Denmark)

    Grønholdt, Lars; Martensen, Anne

    2016-01-01

    This paper examines the links between employee attitudes, customer loyalty and company profitability. From a conceptual point of view, this employee-customer-profit chain, also known as the service-profit chain, is well founded and generally accepted. But for many companies, it seems difficult...... to demonstrate such links, and several issues must be addressed to uncover the links. To investigate these links empirically, a hotel chain provided data matching employee and customer measures with measures of profitability. We have successfully employed a modeling approach, and the paper reports empirical...... evidence of the employee-customer-profit chain. As it is possible to estimate the links, we have demonstrated their effect on company profitability. The research findings provide a better understanding of the service-profit chain and may help practitioners in improving company financial performance....

  6. Replicator dynamics in value chains

    DEFF Research Database (Denmark)

    Cantner, Uwe; Savin, Ivan; Vannuccini, Simone

    2016-01-01

    The pure model of replicator dynamics though providing important insights in the evolution of markets has not found much of empirical support. This paper extends the model to the case of firms vertically integrated in value chains. We show that i) by taking value chains into account, the replicator...... dynamics may revert its effect. In these regressive developments of market selection, firms with low fitness expand because of being integrated with highly fit partners, and the other way around; ii) allowing partner's switching within a value chain illustrates that periods of instability in the early...... stage of industry life-cycle may be the result of an 'optimization' of partners within a value chain providing a novel and simple explanation to the evidence discussed by Mazzucato (1998); iii) there are distinct differences in the contribution to market selection between the layers of a value chain...

  7. Managing the Global Supply Chain

    DEFF Research Database (Denmark)

    Skjøtt-Larsen, Tage; Schary, Philip B.; Mikkola, Juliana Hsuan

    The world today faces global competition. The supply chain is vital part of the globalization process. Presenting a global view of the scope and complexity of supply chain management, this book reflects the rapid change that has taken place within the supply chain and its environment. This new...... edition has been fully updated with recent changes in concepts, technology and practice. Integration and collaboration are keywords in future competition. Firms must be agile and lean at the same time. The book gives an insightful overview of the conceptual foundations of the global supply chain, as...... well as current examples of best practice of managing supply chains in a global context....

  8. Thermodynamics of Dipolar Chain Systems

    International Nuclear Information System (INIS)

    Armstrong, J.R.; Zinner, N.T.; Fedorov, D.V.; Jensen, A.S.

    2013-01-01

    The thermodynamics of a quantum system of layers containing perpendicularly oriented dipolar molecules is studied within an oscillator approximation for both bosonic and fermionic species. The system is assumed to be built from chains with one molecule in each layer. We consider the effects of the intralayer repulsion and quantum statistical requirements in systems with more than one chain. Specifically, we consider the case of two chains and solve the problem analytically within the harmonic Hamiltonian approach which is accurate for large dipole moments. The case of three chains is calculated numerically. Our findings indicate that thermodynamic observables, such as the heat capacity, can be used to probe the signatures of the intralayer interaction between chains. This should be relevant for near future experiments on polar molecules with strong dipole moments. (author)

  9. Developing sustainable food supply chains.

    Science.gov (United States)

    Smith, B Gail

    2008-02-27

    This paper reviews the opportunities available for food businesses to encourage consumers to eat healthier and more nutritious diets, to invest in more sustainable manufacturing and distribution systems and to develop procurement systems based on more sustainable forms of agriculture. The important factors in developing more sustainable supply chains are identified as the type of supply chain involved and the individual business attitude to extending responsibility for product quality into social and environmental performance within their own supply chains. Interpersonal trust and working to standards are both important to build more sustainable local and many conserved food supply chains, but inadequate to transform mainstream agriculture and raw material supplies to the manufactured and commodity food markets. Cooperation among food manufacturers, retailers, NGOs, governmental and farmers' organizations is vital in order to raise standards for some supply chains and to enable farmers to adopt more sustainable agricultural practices.

  10. Managing the Global Supply Chain

    DEFF Research Database (Denmark)

    Hsuan, Juliana; Skjøtt-Larsen, Tage; Kinra, Aseem

    The world today faces global competition. The supply chain is vital part of the globalization process. Presenting a global view of the scope and complexity of supply chain management, this book reflects the rapid change that has taken place within the supply chain and its environment. This new...... well as current examples of best practice of managing supply chains in a global context....... edition has been fully updated with recent changes in concepts, technology and practice. Integration and collaboration are keywords in future competition. Firms must be agile and lean at the same time. The book gives an insightful overview of the conceptual foundations of the global supply chain, as...

  11. Twenty Years of European Union Support to Gene Therapy and Gene Transfer.

    Science.gov (United States)

    Gancberg, David

    2017-11-01

    For 20 years and throughout its research programmes, the European Union has supported the entire innovation chain for gene transfer and gene therapy. The fruits of this investment are ripening as gene therapy products are reaching the European market and as clinical trials are demonstrating the safety of this approach to treat previously untreatable diseases.

  12. Screening for Residual Disease in Pediatric Burkitt Lymphoma Using Consensus Primer Pools

    Directory of Open Access Journals (Sweden)

    Melissa Agsalda

    2009-01-01

    Full Text Available Assessing molecular persistent or minimal residual disease (PD/MRD in childhood Burkitt lymphoma (BL is challenging because access to original tumor is usually needed to design patient-specific primers (PSPs. Because BL is characterized by rearranged immunoglobulin heavy chain (IgVH genes, IgVH primer pools from IgVH1–IgVH7 regions were tested to detect PD/MRD, thus eliminating the need for original tumor. The focus of the current study was to assess the feasibility of using IgVH primer pools to detect disease in clinical specimens. Fourteen children diagnosed with B-NHL had follow-up repository specimens available to assess PD/MRD. Of the 14 patients, 12 were PD/MRD negative after 2 months of therapy and remained in remission at the end of therapy; 2/14 patients were PD/MRD positive at 2-3 months and later relapsed. PSP-based assays from these 14 patients showed 100% concordance with the current assay. This feasibility study warrants further investigation to assess PD/MRD using IgVH primer pools, which could have clinical significance as a real-time assessment tool to monitor pediatric BL and possibly other B-cell non-Hodgkin lymphoma therapy.

  13. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific...... Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively....

  14. Gene Therapy

    Science.gov (United States)

    Gene therapy Overview Gene therapy involves altering the genes inside your body's cells in an effort to treat or stop disease. Genes contain your ... that don't work properly can cause disease. Gene therapy replaces a faulty gene or adds a new ...

  15. Regeneration and general Markov chains

    Directory of Open Access Journals (Sweden)

    Vladimir V. Kalashnikov

    1994-01-01

    Full Text Available Ergodicity, continuity, finite approximations and rare visits of general Markov chains are investigated. The obtained results permit further quantitative analysis of characteristics, such as, rates of convergence, continuity (measured as a distance between perturbed and non-perturbed characteristics, deviations between Markov chains, accuracy of approximations and bounds on the distribution function of the first visit time to a chosen subset, etc. The underlying techniques use the embedding of the general Markov chain into a wide sense regenerative process with the help of splitting construction.

  16. Markov chains theory and applications

    CERN Document Server

    Sericola, Bruno

    2013-01-01

    Markov chains are a fundamental class of stochastic processes. They are widely used to solve problems in a large number of domains such as operational research, computer science, communication networks and manufacturing systems. The success of Markov chains is mainly due to their simplicity of use, the large number of available theoretical results and the quality of algorithms developed for the numerical evaluation of many metrics of interest.The author presents the theory of both discrete-time and continuous-time homogeneous Markov chains. He carefully examines the explosion phenomenon, the

  17. Markov Chains and Markov Processes

    OpenAIRE

    Ogunbayo, Segun

    2016-01-01

    Markov chain, which was named after Andrew Markov is a mathematical system that transfers a state to another state. Many real world systems contain uncertainty. This study helps us to understand the basic idea of a Markov chain and how is been useful in our daily lives. For some times there had been suspense on distinct predictions and future existences. Also in different games there had been different expectations or results involved. That is the reason why we need Markov chains to predict o...

  18. Quadratic Variation by Markov Chains

    DEFF Research Database (Denmark)

    Hansen, Peter Reinhard; Horel, Guillaume

    We introduce a novel estimator of the quadratic variation that is based on the the- ory of Markov chains. The estimator is motivated by some general results concerning filtering contaminated semimartingales. Specifically, we show that filtering can in prin- ciple remove the effects of market...... microstructure noise in a general framework where little is assumed about the noise. For the practical implementation, we adopt the dis- crete Markov chain model that is well suited for the analysis of financial high-frequency prices. The Markov chain framework facilitates simple expressions and elegant analyti...

  19. The Supply Chain Risk Management

    OpenAIRE

    Skitsko Volodymyr I.; Voynikov Mykola Yu.

    2018-01-01

    The article considers current approaches of risk-management in supply chains, the main steps of the risk management process are analyzed and detailed both for a separate enterprise – participant of supply chain, for the supply chain in general, and for the Beer game, based on the international risk management standards. The article provides a way to assess the risks of the «producer» in the Beer game according to the three strategies of its behavior, which presumably can correspond to differe...

  20. New sensors and processing chain

    CERN Document Server

    Yaakoubi, Nourdin

    2014-01-01

    A vital tool for researchers, engineers, and students, New Sensors and Processing Chain focuses on the processing chain to set up in order to extract relevant information on various systems. Highlighting the design of new microsensors and various applications, the authors present recent progress in instrumentation and microsystem design, providing insight to the modification of the sensor itself as well as its environment. Various applications illustrate the presentations, which show how a processing chain is organized from the data acquired by a specific sensor.

  1. Who Manages Our Supply Chains?

    DEFF Research Database (Denmark)

    Flöthmann, Christoph

    to manage the manifold tasks they face on a daily basis. Unfortunately, as globalization has simultaneously increased the complexity of supply chains and the demand for highly qualified personnel, companies are facing a significant undersupply of talent (Cottrill, 2010). Moreover, organizations appear......While managers in traditional management functions focus on developing strong expertise to become “specialists” in their own discipline, supply chain managers are a different species: They have to combine a cross-functional understanding of various business fields and multi-faceted competencies......-based dissertation is to address the research gap between human resource management (HRM) and supply chain management (SCM)....

  2. Development of anti-bovine IgA single chain variable fragment and its application in diagnosis of foot-and-mouth disease

    Science.gov (United States)

    Sridevi, N. V.; Shukra, A. M.; Neelakantam, B.; Anilkumar, J.; Madhanmohan, M.; Rajan, S.; Dev Chandran

    2014-01-01

    Recombinant antibody fragments like single chain variable fragments (scFvs) represent an attractive yet powerful alternative to immunoglobulins and hold great potential in the development of clinical diagnostic/therapeutic reagents. Structurally, scFvs are the smallest antibody fragments capable of retaining the antigen-binding capacity of whole antibodies and are composed of an immunoglobulin (Ig) variable light (VL) and variable heavy (VH) chain joined by a flexible polypeptide linker. In the present study, we constructed a scFv against bovine IgA from a hybridoma cell line IL-A71 that secretes a monoclonal antibody against bovine IgA using recombinant DNA technology. The scFv was expressed in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The binding activity and specificity of the scFv was established by its non-reactivity toward other classes of immunoglobulins as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Kinetic measurement of the scFv indicated that the recombinant antibody fragment had an affinity in picomolar range toward purified IgA. Furthermore, the scFv was used to develop a sensitive ELISA for the detection of foot and mouth disease virus (FMDV) carrier animals. PMID:24678404

  3. Insights into the post-transcriptional regulation of the mitochondrial electron transport chain.

    Science.gov (United States)

    Sirey, Tamara M; Ponting, Chris P

    2016-10-15

    The regulation of the mitochondrial electron transport chain is central to the control of cellular homeostasis. There are significant gaps in our understanding of how the expression of the mitochondrial and nuclear genome-encoded components of the electron transport chain are co-ordinated, and how the assembly of the protein complexes that constitute the electron transport chain are regulated. Furthermore, the role post-transcriptional gene regulation may play in modulating these processes needs to be clarified. This review summarizes the current knowledge regarding the post-transcriptional gene regulation of the electron transport chain and highlights how noncoding RNAs may contribute significantly both to complex electron transport chain regulatory networks and to mitochondrial dysfunction. © 2016 The Author(s).

  4. Supply Chain Shipment Pricing Data

    Data.gov (United States)

    US Agency for International Development — This data set provides supply chain health commodity shipment and pricing data. Specifically, the data set identifies Antiretroviral (ARV) and HIV lab shipments to...

  5. Designing the Reverse Supply Chain

    DEFF Research Database (Denmark)

    Gobbi, Chiara

    2011-01-01

    Purpose – The purpose of this paper is to explore the impact of the product residual value (PRV) and the loss of value over time of returned products in the reverse supply chain configuration. It also examines whether or not the distinction of Fisher's functional and innovative products holds...... that allows for recapturing most of the PRV. These notions have then been tested by analyzing two reverse supply chains with a case study research methodology. Findings – The findings show that low PRV is associated with second-class recovery options (recycling and energy recovery) and that high PRV...... is associated with first-class recovery options (reconditioning and remarketing). When the recovery option is recycling, time is not relevant, the primary objective is cost reduction (efficiency), the chain is centralized, and actors and phases of the reverse chain are determined by the specificity...

  6. The chain re-action

    CERN Multimedia

    2009-01-01

    On 18 March, beam commissioning started in the first ‘link’ of the accelerator chain – LINAC 2. This marks the start of what will be the longest period of beam operations in CERN’s history, with the accelerator complex remaining operational throughout the winter to supply the LHC. The Bulletin finds out what is being done to make sure the whole chain is ready for this historic run.

  7. Who Manages Our Supply Chains?

    OpenAIRE

    Flöthmann, Christoph H.

    2017-01-01

    While managers in traditional management functions focus on developing strong expertise to become “specialists” in their own discipline, supply chain managers are a different species: They have to combine a cross-functional understanding of various business fields and multi-faceted competencies to manage the manifold tasks they face on a daily basis. Unfortunately, as globalization has simultaneously increased the complexity of supply chains and the demand for highly qualified ...

  8. ARCHITECTURAL CONCEPTS FOR VALUE CHAINS

    OpenAIRE

    A.C. Janse van Rensburg

    2012-01-01

    ENGLISH ABSTRACT:The Porter Value Chain has been widely adopted by the business community as a mechanism to understand and comprehend complexity in business environments, with the ultimate goal of structuring the business to maximize its competitive advantage. Implementing the Value Chain is not easy: a number of organizations can testify to their failure to derive any benefits at all from this concept. To overcome this problem, an approach is presented to assist the organization in ...

  9. Osteoradionecrosis of the ossicular chain

    International Nuclear Information System (INIS)

    Kveton, J.F.; Sotelo-Avila, C.

    1986-01-01

    Osteoradionecrosis of the temporal bone is a well-known sequela of radiotherapy for neoplasms at the base of the skull. Presenting symptoms include otalgia, chronic aural drainage, and hearing loss, which can be conductive, sensorineural, or mixed. The conductive component has been hypothesized to be caused by radionecrosis of the ossicular chain. This case report documents the existence of osteoradionecrosis of the ossicular chain in a 10-year-old boy operated on for resection of extensive osteoradionecrosis of the temporal bone

  10. Health Care Provider Value Chain

    OpenAIRE

    Kawczynski , Lukasz; Taisch , Marco

    2009-01-01

    International audience; In every society there is a need for an efficient health care system. This paper aims to propose a value definition and a value chain model within the health care. In order to define value patients and experts were surveyed. The proposed definition offers a complex way of looking at the value within the health care sector. The proposal of the value chain model is anticipated with a value stream mapping activities and experts interviews. Proposed model offers consistent...

  11. Intermodal Supply Chain Risk Management

    OpenAIRE

    Maslarić, Marinko; Brnjac, Nikolina; Bago, Drago

    2016-01-01

    Efficient and secure global supply chains contribute to the Improvement of the competitiveness of the products traded on international markets by reducing their costs and delivery time while increasing the reliability and security. Global supply chains are unthinkable without transport integration, which is usually accomplished through the form of intermodal transport systems. Intermodal transport systems are much more complex than the unimodal ones due to the number of stakeholders, included...

  12. Symmetry chains and adaptation coefficients

    International Nuclear Information System (INIS)

    Fritzer, H.P.; Gruber, B.

    1985-01-01

    Given a symmetry chain of physical significance it becomes necessary to obtain states which transform properly with respect to the symmetries of the chain. In this article we describe a method which permits us to calculate symmetry-adapted quantum states with relative ease. The coefficients for the symmetry-adapted linear combinations are obtained, in numerical form, in terms of the original states of the system and can thus be represented in the form of numerical tables. In addition, one also obtains automatically the matrix elements for the operators of the symmetry groups which are involved, and thus for any physical operator which can be expressed either as an element of the algebra or of the enveloping algebra. The method is well suited for computers once the physically relevant symmetry chain, or chains, have been defined. While the method to be described is generally applicable to any physical system for which semisimple Lie algebras play a role we choose here a familiar example in order to illustrate the method and to illuminate its simplicity. We choose the nuclear shell model for the case of two nucleons with orbital angular momentum l = 1. While the states of the entire shell transform like the smallest spin representation of SO(25) we restrict our attention to its subgroup SU(6) x SU(2)/sub T/. We determine the symmetry chains which lead to total angular momentum SU(2)/sub J/ and obtain the symmetry-adapted states for these chains

  13. "Bullwhip Effect" in Supply Chains

    Directory of Open Access Journals (Sweden)

    Anđelko Ščukanec

    2007-09-01

    Full Text Available The system which connects all the elements between theproducer and the consumer is called the supply chain. This definitionof the supply chain shows its complexity, dynamics anduncertainty. Successful operation of supply chains requires thebest possible coordination of its elements, which obviously indicatesthe utmost importance of the information flow alongthem. The operation of supply chains on the whole has to bemore successful than the operation of their individual elements.It should be noted that such a complex process may give rise toa series of problems, unwanted events, as well as substantial reductionin profits and the level of service. One of such unwantedphenomena which may occur in the supply chain is the"bullwhip effect". This is a possible increase in the diversity oforders which occurs when we move along the elements of thesupply chain (from the customers' orders to the producers' orders.The work analyses the processes within the supply chainsand the occurrence of the bullwhip effect. The main causes ofthe bullwhip effect that we encounter daily in the supply chainshave been clearly defined. The paper also presents the mainnegative consequences of such a disturbance within the supplychains, and provides possible solutions for avoiding the bullwhipeffect.

  14. Reinforcement learning in supply chains.

    Science.gov (United States)

    Valluri, Annapurna; North, Michael J; Macal, Charles M

    2009-10-01

    Effective management of supply chains creates value and can strategically position companies. In practice, human beings have been found to be both surprisingly successful and disappointingly inept at managing supply chains. The related fields of cognitive psychology and artificial intelligence have postulated a variety of potential mechanisms to explain this behavior. One of the leading candidates is reinforcement learning. This paper applies agent-based modeling to investigate the comparative behavioral consequences of three simple reinforcement learning algorithms in a multi-stage supply chain. For the first time, our findings show that the specific algorithm that is employed can have dramatic effects on the results obtained. Reinforcement learning is found to be valuable in multi-stage supply chains with several learning agents, as independent agents can learn to coordinate their behavior. However, learning in multi-stage supply chains using these postulated approaches from cognitive psychology and artificial intelligence take extremely long time periods to achieve stability which raises questions about their ability to explain behavior in real supply chains. The fact that it takes thousands of periods for agents to learn in this simple multi-agent setting provides new evidence that real world decision makers are unlikely to be using strict reinforcement learning in practice.

  15. CHALLENGES OF SUPPLY CHAIN MANAGEMENT

    Directory of Open Access Journals (Sweden)

    Natalia GALANTON

    2017-11-01

    Full Text Available The present article highlights the basic characteristics of the supply chain, specific to changes in the business environment of companies at the beginning of the 21st century. Supply chain activities turn natural resources, raw materials and components into a finished product, which is then delivered to the final customer. The supply chain incorporates the production process alongside the raw material acquisition and distribution of finished products, encompassing all the existing process responsible for transforming the materials taken from the supplier to the finished products delivered to customers. Today, competitiveness and performance of an organization are dependent on belonging to an efficient and effective supply-delivery chain where synergies exist due to the establishment and implementation of common goals and strategies by partners. Competition does not take place between independent organizations or between small groups of organizations, but between supply and delivery chains, made up of interconnected networks of producers, distributors and logistics providers. Under these circumstances, supply chain managers must seek solutions to respond promptly to the most important challenges in the field.

  16. Sports Nutrition Food Industry Chain Development Research

    OpenAIRE

    Jie Yin

    2015-01-01

    Through the study of Henan sports nutrition food industry chain optimization, the study analyses development advantage and competitive advantage of Henan in sports nutrition food industry chain and existing problems and challenges in Henan sports nutrition food industry chain and at the same time introduces the theory of supply chain management to the development of sports nutrition food industry chain, clearly optimizes countermeasures of sports nutrition food industry chain. Pointing out sp...

  17. Risk Management in Biopharmaceutical Supply Chains

    OpenAIRE

    Ma, Yao

    2011-01-01

    Biopharmaceutical supply chains present considerable complexity issue for the formulation of optimal plans due to significant uncertainty in the supply chain. The primary goal of biopharmaceutical supply chain planning is to provide reliable supply to patients while coping with various supply chain risks. In chapter 1 first I discuss the key elements and basic characteristics of the biopharmaceutical supply chain . Then I present the major challenges in biopharmaceutical supply chain planning...

  18. Polymerase chain reaction methods (PCR in agrobiotechnology

    Directory of Open Access Journals (Sweden)

    Taški-Ajduković Ksenija

    2006-01-01

    Full Text Available The agricultural biotechnology applies polymerase chain reaction (PCR technology at numerous steps throughout product development. The major uses of PCR technology during product development include gene discovery and cloning, vector construction, transformant identification, screening and characterization as well as seed quality control. Commodity and food companies as well as testing laboratories rely on PCR technology to verify the presence or absence of genetically modification (GM in a product or to quantify the amount of GM material present in the product. This article describes the fundamental elements of PCR analysis and its application to the testing of grains and highlights some of areas to which attention must be paid in order to produce reliable test results. The article also discuses issues related to the analysis of different matrixes and the effect they may have on the accuracy of the PCR analytical results.

  19. Ig Light Chain Precedes Heavy Chain Gene Rearrangement during Development of B Cells in Swine

    Czech Academy of Sciences Publication Activity Database

    Šinkora, Marek; Šinkorová, Jana; Štěpánová, Kateřina

    2017-01-01

    Roč. 198, č. 4 (2017), s. 1543-1552 ISSN 0022-1767 R&D Projects: GA ČR GA15-02274S Institutional support: RVO:61388971 Keywords : ANTIBODY REPERTOIRE DEVELOPMENT * ILEAL PEYERS-PATCHES * DELTA T-LYMPHOCYTES Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 4.856, year: 2016

  20. Demand chain management - The evolution

    Directory of Open Access Journals (Sweden)

    D Ericsson

    2011-06-01

    Full Text Available The concepts of Supply Chain Management (SCM and Demand Chain Management (DCM are among the new and debated topics concerning logistics in the literature. The question considered in this paper is: “Are these concepts needed or will they just add to the confusion?” Lasting business concepts have always evolved in close interaction between business and academia. Different approaches start out in business and they are then, more or less si- multaneously, aligned, integrated, systemised and structured in academia. In this way a terminology (or language is provided that helps in further diffusion of the concepts. There is a lack of consensus on the definition of the concept of SCM. This may be one of the major reasons for the difficulty in advancing the science and measuring the results of implementation in business. Relationships in SCM span from rather loose coalitions to highly structured virtual network integrations. DCM is a highly organised chain in which the key is mutual interdependence and partnership. The purpose is to create a distinctive competence for the chain as a whole that helps to identify and satisfy customer needs and wishes. The classical research concerning vertical marketing systems is very helpful in systemising the rather unstructured discussions in current SCM research. The trend lies in increasing competition between channels rather than between companies, which in turn leads to the creation of channels with a high degree of partnership and mutual interdependence between members. These types of channels are known as organised vertical marketing systems in the classic marketing channel research. The behaviour in these types of channels, as well as the formal and informal structures, roles in the network, power and dependence relations, etc. are well covered topics in the literature. The concept of vertical marketing systems lies behind the definition of demand chains and demand chain management proposed in this paper. A

  1. Supply Chain Management Practices in Toy Supply Chains

    DEFF Research Database (Denmark)

    Arlbjørn, Jan Stentoft; Johansen, J.; Wong, C. Y.

    2005-01-01

    Purpose - Innovative products usually experience highly unpredictable and variable demand. This is especially valid for the volatile and seasonal toy industry, which produces high obsolete inventory, lost sales and markdown. In such a volatile industry, what supply chain management (SCM) practices...... (traditional mass-production or push-models). These low-responsive practices in the toy supply chain are not caused only by slow knowledge diffusion. SCM know-how is not yet capable of managing such levels of volatility and seasonality. Therefore, explanations of these theoretical gaps and what new theories...... are required for such extreme volatility and seasonality are proposed. Originality/value - It reveals actual SCM practices in a volatile and seasonal supply chain, such that theoretical and practical gaps are identified. Also, it proposes a model to match manufacturing SCM-practices with retailer SCM-practices....

  2. Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis

    Science.gov (United States)

    dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana

    2015-01-01

    Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis. PMID:26393928

  3. Gluon chains and multiquark hadrons

    International Nuclear Information System (INIS)

    Jadach, S.; Jezabek, M.

    1979-01-01

    A monopole approximation to the confining potential is proposed. In this approximation spatially separated groups of quarks carry a definite total colour charge. The potentials which lead to the formation of gluon chains are discussed. The generalization of a (3,3bar) chain notion, studied by Tiktopoulos, to the case of arbitrary colour charges is given. It is argued that these generalized chains may be unstable with respect to splitting into a system of weakly interacting chains of the (3,3bar) type. A unified picture of the high energy hadronic collisions, based on the gluon chain notion and the monopole approximation is proposed. In the meson-meson sector this picture is equivalent to the topological approach. For the other processes it is similar to the approach of Rossi and Veneziano. However, it is argued that the introduction of the junction line into the quark frame is superfluous. The results are expressed in the language of the coloured dual diagrams which provide a generalization of those of Harari and Rosner. (author)

  4. Evolution of SUMO Function and Chain Formation in Insects.

    Science.gov (United States)

    Ureña, Enric; Pirone, Lucia; Chafino, Silvia; Pérez, Coralia; Sutherland, James D; Lang, Valérie; Rodriguez, Manuel S; Lopitz-Otsoa, Fernando; Blanco, Francisco J; Barrio, Rosa; Martín, David

    2016-02-01

    SUMOylation, the covalent binding of Small Ubiquitin-like Modifier (SUMO) to target proteins, is a posttranslational modification that regulates critical cellular processes in eukaryotes. In insects, SUMOylation has been studied in holometabolous species, particularly in the dipteran Drosophila melanogaster, which contains a single SUMO gene (smt3). This has led to the assumption that insects contain a single SUMO gene. However, the analysis of insect genomes shows that basal insects contain two SUMO genes, orthologous to vertebrate SUMO1 and SUMO2/3. Our phylogenetical analysis reveals that the SUMO gene has been duplicated giving rise to SUMO1 and SUMO2/3 families early in Metazoan evolution, and that later in insect evolution the SUMO1 gene has been lost after the Hymenoptera divergence. To explore the consequences of this loss, we have examined the characteristics and different biological functions of the two SUMO genes (SUMO1 and SUMO3) in the hemimetabolous cockroach Blattella germanica and compared them with those of Drosophila Smt3. Here, we show that the metamorphic role of the SUMO genes is evolutionary conserved in insects, although there has been a regulatory switch from SUMO1 in basal insects to SUMO3 in more derived ones. We also show that, unlike vertebrates, insect SUMO3 proteins cannot form polySUMO chains due to the loss of critical lysine residues within the N-terminal part of the protein. Furthermore, the formation of polySUMO chains by expression of ectopic human SUMO3 has a deleterious effect in Drosophila. These findings contribute to the understanding of the functional consequences of the evolution of SUMO genes. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  5. Labour in Global Value Chains

    DEFF Research Database (Denmark)

    Lund-Thomsen, Peter; Nadvi, Khalid; Chan, Anita

    2012-01-01

    production contexts that influence work conditions. In doing so, it argues that current debates on the role of labour in global value chains have to go beyond a narrow focus on labour standards and corporate social responsibility compliance and engage with economic, technological and social upgrading......A critical challenge facing developing country producers is to meet international labour standards and codes of conduct in order to engage in global value chains. Evidence of gains for workers from compliance with such standards and codes remains limited and patchy. This article focuses...... on the global football industry, a sector dominated by leading global brands that manage dispersed global value chains. It assesses the working conditions for football stitchers engaged in different forms of work organization, factories, stitching centres and home-based settings in Pakistan, India and China...

  6. Environmental management in product chains

    DEFF Research Database (Denmark)

    Jørgensen, Michael Søgaard; Forman, Marianne; Hansen, Anne Grethe

    of environmental initiatives, a number of recommendations for governmental regulation, which can support the further diffusion of environmental management in product chains, are developed. Furthermore, the report describes a number of theoretical perspectives from sociology of technology, organisation theory......This report presents the analyses of the shaping, implementation and embedding of eight types of environmental initiatives in product chains. The analyses focus on • the role of the type of product and branch, of the size of the companies and of governmental regulation • the focus...... of the environmental concerns and the reductions in environmental impact • organisational changes which have been part of the embedding of the initiatives The analyses are based on 25 cases from national and international product chains involving one or more Danish companies. Based on the analyses of the eight types...

  7. Markov chains and mixing times

    CERN Document Server

    Levin, David A

    2017-01-01

    Markov Chains and Mixing Times is a magical book, managing to be both friendly and deep. It gently introduces probabilistic techniques so that an outsider can follow. At the same time, it is the first book covering the geometric theory of Markov chains and has much that will be new to experts. It is certainly THE book that I will use to teach from. I recommend it to all comers, an amazing achievement. -Persi Diaconis, Mary V. Sunseri Professor of Statistics and Mathematics, Stanford University Mixing times are an active research topic within many fields from statistical physics to the theory of algorithms, as well as having intrinsic interest within mathematical probability and exploiting discrete analogs of important geometry concepts. The first edition became an instant classic, being accessible to advanced undergraduates and yet bringing readers close to current research frontiers. This second edition adds chapters on monotone chains, the exclusion process and hitting time parameters. Having both exercises...

  8. Neuroimaging findings in Joubert syndrome with C5orf42 gene mutations: A milder form of molar tooth sign and vermian hypoplasia.

    Science.gov (United States)

    Enokizono, Mikako; Aida, Noriko; Niwa, Tetsu; Osaka, Hitoshi; Naruto, Takuya; Kurosawa, Kenji; Ohba, Chihiro; Suzuki, Toshifumi; Saitsu, Hirotomo; Goto, Tomohide; Matsumoto, Naomichi

    2017-05-15

    Little is known regarding neuroimaging-genotype correlations in Joubert syndrome (JBTS). To elucidate one of these correlations, we investigated the neuroimaging findings of JBTS patients with C5orf42 mutations. Neuroimaging findings in five JBTS patients with C5orf42 mutations were retrospectively assessed with regard to the infratentorial and supratentorial structures on T1-magnetization prepared rapid gradient echo (MPRAGE), T2-weighted images, and color-coded fractional anisotropy (FA) maps; the findings were compared to those in four JBTS patients with mutations in other genes (including three with AHI1 and one with TMEM67 mutations). In C5orf42-mutant patients, the infratentorial magnetic resonance (MR) images showed normal or minimally thickened and minimally elongated superior cerebellar peduncles (SCP), normal or minimally deepened interpeduncular fossa (IF), and mild vermian hypoplasia (VH). However, in other patients, all had severe abnormalities in the SCP and IF, and moderate to marked VH. Supratentorial abnormalities were found in one individual in other JBTS. In JBTS with all mutations, color-coded FA maps showed the absence of decussation of the SCP (DSCP). The morphological neuroimaging findings in C5orf42-mutant JBTS were distinctly mild and made diagnosis difficult. However, the absence of DSCP on color-coded FA maps may facilitate the diagnosis of JBTS. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Chain and network science: A research framework

    NARCIS (Netherlands)

    Omta, S.W.F.; Trienekens, J.H.; Beers, G.

    2001-01-01

    In this first article of the Journal on Chain and Network Science the base-line is set for a discussion on contents and scope of chain and network theory. Chain and network research is clustered into four main ‘streams’: Network theory, social capital theory, supply chain management and business

  10. Leading a supply chain turnaround.

    Science.gov (United States)

    Slone, Reuben E

    2004-10-01

    Just five years ago, salespeople at Whirlpool were in the habit of referring to their supply chain organization as the "sales disablers." Now the company excels at getting products to the right place at the right time--while managing to keep inventories low. How did that happen? In this first-person account, Reuben Slone, Whirlpool's vice president of Global Supply Chain, describes how he and his colleagues devised the right supply chain strategy, sold it internally, and implemented it. Slone insisted that the right focal point for the strategy was the satisfaction of consumers at the end of the supply chain. Most supply chain initiatives do the opposite: They start with the realities of a company's manufacturing base and proceed from there. Through a series of interviews with trade customers large and small, his team identified 27 different capabilities that drove industry perceptions of Whirlpool's performance. Knowing it was infeasible to aim for world-class performance across all of them, Slone weighed the costs of excelling at each and found the combination of initiatives that would provide overall competitive advantage. A highly disciplined project management office and broad training in project management were key to keeping work on budget and on benefit. Slone set an intense pace--three "releases" of new capabilities every month--that the group maintains to this day. Lest this seem like a technology story, however, Slone insists it is just as much a "talent renaissance." People are proud today to be part of Whirlpool's supply chain organization, and its new generation of talent will give the company a competitive advantage for years to come.

  11. Green supply chains: a new priority for supply chain managers

    CSIR Research Space (South Africa)

    Ittmann, H

    2011-01-01

    Full Text Available This article is not a comprehensive overview of green logistics. What it endeavours to do is to sensitise, in a very summarised way, those involved in logistics and supply chain management about the importance of green logistics and to highlight...

  12. Towards biotracing in food chains

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Wagner, Martin; Jordan, Kieran

    2011-01-01

    Biotracing is tracing (backward)/tracking (forward) biological contamination in the food/feed chain. Advances in detection technologies, improvements in molecular marker identification, clearer understanding of pathogenicity markers, improved modelling methodologies and, more importantly......, and 21 cross-disciplinary work packages that cover tracing and tracking of contamination in feed, meat and dairy chains, in addition to accidental and deliberate contamination of bottled water. The BIOTRACER Consortium consists of 46 partners, including Europe's largest food/feed industries, several SMEs...... and food retailers. The outcomes will ensure a more reliable and rapid response to a microbial contamination event....

  13. The first nuclear chain reaction

    International Nuclear Information System (INIS)

    Zinn, W.H.

    1989-01-01

    The author offers his recollections of the experimental efforts beginning in 1939 which culminated in the Chain Reaction in the squash court on December 2, 1942. Recalled are Columbia University experiments which did much to establish the feasibility of the chain in natural uranium and which stimulated the creation of the Manhattan District. The Columbia group moved to the University of Chicago, where, in early summer of 1942, construction and analysis of a number of subcritical reactors (piles) gave assurance with a high probability that only a reasonable amount of uranium and moderator would be required

  14. Thermodynamics of Dipolar Chain Systems

    DEFF Research Database (Denmark)

    R. Armstrong, J.; Zinner, Nikolaj Thomas; V. Fedorov, D.

    2012-01-01

    The thermodynamics of a quantum system of layers containing perpendicularly oriented dipolar molecules is studied within an oscillator approximation for both bosonic and fermionic species. The system is assumed to be built from chains with one molecule in each layer. We consider the effects...... numerically. Our findings indicate that thermodynamic observables, such as the heat capacity, can be used to probe the signatures of the intralayer interaction between chains. This should be relevant for near future experiments on polar molecules with strong dipole moments....

  15. Markov chains and mixing times

    CERN Document Server

    Levin, David A; Wilmer, Elizabeth L

    2009-01-01

    This book is an introduction to the modern approach to the theory of Markov chains. The main goal of this approach is to determine the rate of convergence of a Markov chain to the stationary distribution as a function of the size and geometry of the state space. The authors develop the key tools for estimating convergence times, including coupling, strong stationary times, and spectral methods. Whenever possible, probabilistic methods are emphasized. The book includes many examples and provides brief introductions to some central models of statistical mechanics. Also provided are accounts of r

  16. Loads applied to fixations for chain stretching

    Energy Technology Data Exchange (ETDEWEB)

    Ahrens, K; Brychta, P

    1985-06-01

    The chains of scraper chain conveyors must be pre-stretched during standstill in order to compensate the elongations occurring during operation. They require frequent retensiening in order to meet the varying operational requirements. During tensioning, the chains are fixed in a point in the top run by means of fixation elements. The authors present a method for calculating the retaining force needed in the fixations. There are three different initial conditions of the chain before trensioning: Tensionsfree chain, pretensioned chain (stressed chain), slack chain. In all three cases, it is important to find out whether or nor the tensioning drive reaches full speed. The method of calculation is illustrated by the example of a scraper chain conveyor; it enables the establishment of rules for tensioning without damaging the chain and is a good basis for the dimensioning of new types of fixation elements.

  17. Information flow in the pharmaceutical supply chain

    OpenAIRE

    Yousefi, Nazila; Alibabaei, Ahmad

    2015-01-01

    Managing the supply chain plays an important role in creating competitive advantages for companies. Adequate information flow in supply chain is one of the most important issues in SCM. Therefore, using certain Information Systems can have a significant role in managing and integrating data and information within the supply chain. Pharmaceutical supply chain is more complex than many other supply chains, in the sense that it can affect social and political perspectives. On the other hand, man...

  18. SUPPLY CHAIN MANAGEMENT – KEY FACTORS

    OpenAIRE

    Magdalena Daniela DINU

    2014-01-01

    This paper exposes Supply Chain Management by its key factors. Briefly, where the Supply Chain Management is treated as strategic part of a company then maintaining both control and influence throughout the entire supply chain are key factors and critical to success. On the other hand, finding the right partner to manage the non-strategic Supply Chains would be another key factor too. To define the most important key factors within Supply Chain Management means a deeply understanding of bot...

  19. Management Accounting and Supply Chain Strategy

    OpenAIRE

    Hald, Kim S.; Thrane, Sof

    2016-01-01

    Research positioned in the intersection between management accounting and supply chain management is increasing. However, the relationship between management accounting and supply chain strategies has been neglected in extant research. This research adds to literature on management accounting and supply chain management through exploring how supply chain strategy and management accounting is related, and how supply chain relationship structure modifies this relation. Building on a contingency...

  20. Supply Chain Management In Construction Revealed

    Directory of Open Access Journals (Sweden)

    Dr. Ghaith Al-Werikat

    2017-03-01

    Full Text Available The construction supply chain plays a major role in the construction market competition. Construction supply chain management assists enterprises by helping to improve competitiveness increase profits and have more control over the different factors and variables within the project. This paper discusses the construction supply chain characteristics challenges and problems supply chains encounter and the benefits of an integrated supply chain in the construction sector.