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Sample records for cadmium-resistant cho variants

  1. Heterologous Expression of Rat Testis GABAA Receptor β3t Splicing Variant in CHO Cells

    Institute of Scientific and Technical Information of China (English)

    Shi-feng LI; Yu-guang CHEN; Yuan-chang YAN; Yi-ping LI

    2004-01-01

    Objective To characterize a possible retention function of unique sequence in the 5'end of rat testis GABAA receptor β3t splicing variantMethods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method. Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined.Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively. The chimera product failed to be translocated into the cell surface when expressed in CHO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane.Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.

  2. Eliminating tyrosine sequence variants in CHO cell lines producing recombinant monoclonal antibodies.

    Science.gov (United States)

    Feeney, Lauren; Carvalhal, Veronica; Yu, X Christopher; Chan, Betty; Michels, David A; Wang, Yajun Jennifer; Shen, Amy; Ressl, Jan; Dusel, Brendon; Laird, Michael W

    2013-04-01

    Amino acid sequence variants are defined as unintended amino acid sequence changes that contribute to product variation with potential impact to product safety, immunogenicity, and efficacy. Therefore, it is important to understand the propensity for sequence variant (SV) formation during the production of recombinant proteins for therapeutic use. During the development of clinical therapeutic products, several monoclonal antibodies (mAbs) produced from Chinese Hamster Ovary (CHO) cells exhibited SVs at low levels (≤3%) in multiple locations throughout the mAbs. In these examples, the cell culture process depleted tyrosine, and the tyrosine residues in the recombinant mAbs were replaced with phenylalanine or histidine. In this work, it is demonstrated that tyrosine supplementation eliminated the tyrosine SVs, while early tyrosine starvation significantly increased the SV level in all mAbs tested. Additionally, it was determined that phenylalanine is the amino acid preferentially misincorporated in the absence of tyrosine over histidine, with no other amino acid misincorporated in the absence of tyrosine, phenylalanine, and histidine. The data support that the tyrosine SVs are due to mistranslation and not DNA mutation, most likely due to tRNA(Tyr) mischarging due to the structural similarities between tyrosine and phenylalanine.

  3. Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulating metallothionein gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Crawford, B.D.; Enger, M.D.; Griffith, B.B.; Griffith, J.K.; Hanners, J.L.; Longmire, J.L.; Munk, A.C.; Stallings, R.L.; Tesmer, J.G.; Walters, R.A.; Hildebrand, C.E.

    1985-02-01

    The authors describe here the derivation, characterization, and use of clonal cadmium-resistance (Cd/sup r) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylaminde gel electrophoresis, the authors showed that the stable Cd/sup r/ phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 M, coordinate amplifications of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which the authors have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cd/sup r/ variants expressing abundant MT and their corresponding Cd/sup s/ parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expressions of the isometallothioneins. 59 references, 8 figures.

  4. Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli

    OpenAIRE

    Achard-Joris, M; van Saparoea, HBV; Driessen, AJM; Bourdineaud, JP; Bourdineaud, Jean-Paul

    2005-01-01

    The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC mutant strain which proved to be hypersensitive to cadmium. Both the human and bacterial MDR genes conferred cadmium resistance to E. coli up to 0.4 mM concentration. Protection was abolished by 10...

  5. Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli

    NARCIS (Netherlands)

    Achard-Joris, M; van Saparoea, HBV; Driessen, AJM; Bourdineaud, JP; Bourdineaud, Jean-Paul

    2005-01-01

    The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC

  6. Detection,isolation,and identification of cadmium-resistant bacteria based on PCR-DGGE

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    This study focused on the screening of cadmium-resistant bacterial strains from Pb-Zn tailing.We investigated the diversity of microbial community inhabiting Dong-san-cha Pb-Zn tailing in Beijing,China,by polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene of bacterial strain,and found two dominant strains in the DGGE profile.Using special culture media,We isolated two strong cadmium-resistant bacterial strains.On the basis of morphological,physiological,and biochemical characteristics,BIOLOG,and 16S rDNA sequencing,the two strains were identified as Bacillus cereus and Enterobaeter cloacae.Minimal inhibitory concentrations (MICs) of heavy metals for the bacteria were determined.E.cloacae showed higher MIC values for heavy metals and a larger range of antibiotic resistance than B.cereus.

  7. Isolation, Identification, and Characterization of Cadmium Resistant Pseudomonas sp. M3 from Industrial Wastewater

    OpenAIRE

    Syed Zaghum Abbas; Mohd. Rafatullah; Norli Ismail; Japareng Lalung

    2014-01-01

    The present study deals with the isolation, identification, and characterization of the cadmium resistant bacteria from wastewater collected from industrial area of Penang, Malaysia. The isolate was selected based on high level of the cadmium and antibiotic resistances. On the basis of morphological, biochemical characteristics, 16S rDNA gene sequencing and phylogeny analysis revealed that the strain RZCd1 was authentically identified as Pseudomonas sp. M3. The industrial isolate showed more ...

  8. Plasmid-borne cadmium resistant determinants are associated with the susceptibility of Listeria monocytogenes to bacteriophage.

    Science.gov (United States)

    Zhang, Hui; Zhou, Yan; Bao, Hongduo; Zhang, Lili; Wang, Ran; Zhou, Xiaohui

    2015-03-01

    Listeria monocytogenes is an intracellular pathogen causing gastroenteritis, central nervous system infections and abortions. Chromosomal virulence determinants have been extensively investigated. However, the function of genes encoded by plasmids in L. monocytogenes has not been fully understood. In this study, we determined the prevalence and molecular profile of plasmids in food isolates of L. monocytogenes and examined the contribution of four plasmid-borne cadmium-resistant genes to the susceptibility of L. monocytogenes to bacteriophage infection. The results showed that plasmids were isolated from 55% (11/20) of the isolates and the plasmids exhibited 10 molecular types as determined by restriction enzyme digestion. Furthermore, 65% and 15% of the isolates were tolerant to cadmium and benzalkonium chloride (BC), respectively. All the BC-resistant isolates were resistant to cadmium. The prevalence of predicted cadmium resistance determinants (cadA1, cadA2, cadA3 and cadC) was determined and the results showed that cadA1 (35%) in isolates of serotypes 1/2a and 1/2b was much more prevalent than cadC (15%). As expected, both cadA and cadC mutants had reduced resistance to cadmium, while the resistance to BC was not significantly affected. Interestingly, both cadA and cadC mutants showed significantly higher susceptibility against L. monocytogenes phage LipG2-5 and FWLLm3 compared with the wide-type strain. Based on these results, we concluded that plasmids from L. monocytogenes encoded important functional determinants that are not only associated with cadmium resistance, but also phage susceptibility. PMID:25721472

  9. Biochemical characterization of N-methyl N' -nitro-N-nitrosoguanidine-induced cadmium resistant mutants of Aspergillus niger

    Indian Academy of Sciences (India)

    Samar Kumar Pal; Tapan Kumar Das

    2005-12-01

    Two cadmium resistant mutants (Cd1 and Cd2) of Aspergillus niger, among the six isolated by mutagenization with N-methyl N′-nitro-N-nitrosoguanidine (MNNG) at pH 6.4 were selected for the study. Analysis of lipid composition of the mutants and the wildtype indicated that total lipid as well as individual lipids of the cadmium resistant mutants were changed as compared with that of the wildtype. The increased activities of metal-lothionein and reduced activities of D-xylose isomerase and L-phenylalanine ammonia lyase in cell free extract of the cadmium resistant mutants suggested that mutants could allow high concentration of cadmium salt as compared with that of the wildtype. The respiratory activity and intracellular as well as extracellular Cd2+ concentration of the mutants reflected the high tolerance of the Cd mutants to cadmium ion.

  10. Isolation, Identification, and Characterization of Cadmium Resistant Pseudomonas sp. M3 from Industrial Wastewater

    Directory of Open Access Journals (Sweden)

    Syed Zaghum Abbas

    2014-01-01

    Full Text Available The present study deals with the isolation, identification, and characterization of the cadmium resistant bacteria from wastewater collected from industrial area of Penang, Malaysia. The isolate was selected based on high level of the cadmium and antibiotic resistances. On the basis of morphological, biochemical characteristics, 16S rDNA gene sequencing and phylogeny analysis revealed that the strain RZCd1 was authentically identified as Pseudomonas sp. M3. The industrial isolate showed more than 70% of the cadmium removal in log phase. The cadmium removal capacity of strain RZCd1 was affected by temperature and pH. At pH 7.0 and 35°C, strain RZCd1 showed maximum cadmium removal capacity. The minimal inhibitory concentration of strain RZCd1 against the cadmium was 550 µg/mL. The resistance against the cadmium was associated with resistance to multiple antibiotics: amoxicillin, penicillin, cephalexin, erythromycin, and streptomycin. The strain RZCd1 also gave thick bands of proteins in front of 25 kDa in cadmium stress condition after 3 h of incubation. So the identified cadmium resistant bacteria may be useful for the bioremediation of cadmium contaminated industrial wastewater.

  11. Galsang Cering Climbs Cho Oyu

    Institute of Scientific and Technical Information of China (English)

    XUEWENXIAN

    2004-01-01

    Gongbo is one of the three Chinese mountaineers who climbed Qomolangma, the highest peak of the world, from the northern side for the first time. Nowadays his son, Galsang Cering,last autumn successfully climbed Cho Oyu, which is the world's sixth highest peak at 8.201 meters.

  12. Cadmium resistance from Staphylococcus aureus plasmid pI258 cadA gene results from a cadmium-efflux ATPase.

    OpenAIRE

    Nucifora, G; Chu, L; Misra, T K; Silver, S

    1989-01-01

    Cadmium resistance specified by the cadA determinant of Staphylococcus aureus plasmid pI258 results from the functioning of a cadmium-efflux system. In the nucleotide sequence of the DNA fragment containing the cadA determinant, two open reading frames were identified. The larger one, corresponding to a predicted polypeptide of 727 amino acid residues, is necessary and sufficient for expression of cadmium resistance. Comparison of the CadA amino acid sequence with known protein sequences sugg...

  13. Improving expression of recombinant human IGF-1 using IGF-1R knockout CHO cell lines.

    Science.gov (United States)

    Romand, Sandrine; Jostock, Thomas; Fornaro, Mara; Schmidt, Joerg; Ritter, Anett; Wilms, Burkhard; Laux, Holger

    2016-05-01

    Chinese Hamster Ovary (CHO) cells are widely used for the large-scale production of recombinant biopharmaceuticals. However, attempts to express IGF-1 (a mutated human Insulin-like growth factor 1 Ea peptide (hIGF-1Ea mut)) in CHO cells resulted in poor cell growth and low productivity (0.1-0.2 g/L). Human IGF-1 variants negatively impacted CHO cell growth via the IGF-1 receptor (IGF-1R). Therefore knockout (KO) of the IGF-1R gene in two different CHO cell lines as well as knockdown (KD) of IGF-1R in one CHO cell line were performed. These cell line engineering approaches decreased significantly the hIGF-1 mediated cell growth inhibition and increased productivity of both KO CHO cell lines as well as of the KD CHO cell line. A productivity increase of 10-fold at pool level and sevenfold at clone level was achieved, resulting in a titer of 1.3 g/L. This data illustrate that cell line engineering approaches are powerful tools to improve the yields of recombinant proteins which are difficult to produce in CHO cells. Biotechnol. Bioeng. 2016;113: 1094-1101. © 2015 Wiley Periodicals, Inc. PMID:26523469

  14. The Role of Exopolymers in Protection of Ralstonia sp., a Cadmium-resistant Bacterium, from Cadmium Toxicity

    OpenAIRE

    Anchulee Watcharamusik; Benjaphorn Prapagdee

    2008-01-01

    Production of exopolymers is one of heavy metal resistance mechanisms in bacteria. Ralstonia sp. TAK1, a cadmium-resistant bacterium, was isolated from a high cadmium (Cd) contaminated soil at the zinc mine, Tak province, Thailand. The bacterium was cultivated in LB broth and its growth was monitored. The yields of exopolymers were measured by the phenol-sulfuric method at different growth phases. The levels of Cd resistance were quantitatively determined by survival cell assay. The highest a...

  15. Mechanism of cadmium resistance and adsorption of a yeast strain Rhodotorula sp. Y11

    Institute of Scientific and Technical Information of China (English)

    YUAN; Hongli; LI; Zhijian; WANG; Nengfei; HUANG; Huaizeng

    2005-01-01

    The mechanism of cadmium resistance of a yeast strain Rhodotorula sp. Y11 isolated from mine soil was investigated. We found that the yeast cells treated with different methods showed different cadmium-adsorption models. Grown in medium supplied with 100 mg/L of cadmium, 3.29% of the cell-absorbed cadmium was accounted in the cytoplasm. However, only 1% was taken into the cytoplasm and 99% was bound to the cell wall using the lyophilized biomass to adsorb cadmium in double distilled water. Treatments with alkali, ethanol-chloroform and proteinase showed different influences on the biosorption of whole cells and isolated cell walls. FT-IR analysis showed that acetyl of chitin was the active compound in the cells to absorb cadmium. The production of Metallothioneins, proteins related to the resistance to heavy metal in yeast, was evidently induced by cadmium, achieving 638.8 μg/g wet weight, which was about 85 folds higher than that in the uninduced biomass and was also much higher than that reported previously. The molecular weight of Metallothioneins was 6500 Da estimated by SDS-PAGE.

  16. Improvement of cadmium phytoremediation after soil inoculation with a cadmium-resistant Micrococcus sp.

    Science.gov (United States)

    Sangthong, Chirawee; Setkit, Kunchaya; Prapagdee, Benjaphorn

    2016-01-01

    Cadmium-resistant Micrococcus sp. TISTR2221, a plant growth-promoting bacterium, has stimulatory effects on the root lengths of Zea mays L. seedlings under toxic cadmium conditions compared to uninoculated seedlings. The performance of Micrococcus sp. TISTR2221 on promoting growth and cadmium accumulation in Z. mays L. was investigated in a pot experiment. The results indicated that Micrococcus sp. TISTR2221significantly promoted the root length, shoot length, and dry biomass of Z. mays L. transplanted in both uncontaminated and cadmium-contaminated soils. Micrococcus sp. TISTR2221 significantly increased cadmium accumulation in the roots and shoots of Z. mays L. compared to uninoculated plants. At the beginning of the planting period, cadmium accumulated mainly in the shoots. With a prolonged duration of cultivation, cadmium content increased in the roots. As expected, little cadmium was found in maize grains. Soil cadmium was significantly reduced with time, and the highest percentage of cadmium removal was found in the bacterial-inoculated Z. mays L. after transplantation for 6 weeks. We conclude that Micrococcus sp. TISTR2221 is a potent bioaugmenting agent, facilitating cadmium phytoextraction in Z. mays L.

  17. Cadmium resistance and uptake by bacterium, Salmonella enterica 43C, isolated from industrial effluent.

    Science.gov (United States)

    Khan, Zaman; Rehman, Abdul; Hussain, Syed Z; Nisar, Muhammad A; Zulfiqar, Soumble; Shakoori, Abdul R

    2016-12-01

    Cadmium resistant bacterium, isolated from industrial wastewater, was characterized as Salmonella enterica 43C on the basis of biochemical and 16S rRNA ribotyping. It is first ever reported S. enterica 43C bared extreme resistance against heavy metal consortia in order of Pb(2+)>Cd(2+)>As(3+)>Zn(2+)>Cr(6+)>Cu(2+)>Hg(2+). Cd(2+) stress altered growth pattern of the bacterium in time dependent manner. It could remove nearly 57 % Cd(2+) from the medium over a period of 8 days. Kinetic and thermodynamic studies based on various adsorption isotherm models (Langmuir and Freundlich) depicted the Cd(2+) biosorption as spontaneous, feasible and endothermic in nature. Interestingly, the bacterium followed pseudo first order kinetics, making it a good biosorbent for heavy metal ions. The S. enterica 43C Cd(2+) processivity was significantly influenced by temperature, pH, initial Cd(2+) concentration, biomass dosage and co-metal ions. FTIR analysis of the bacterium revealed the active participation of amide and carbonyl moieties in Cd(2+) adsorption confirmed by EDX analysis. Electron micrographs beckoned further surface adsorption and increased bacterial size due to intracellular Cd(2+) accumulation. An overwhelming increase in glutathione and other non-protein thiols levels played a significant role in thriving oxidative stress generated by metal cations. Presence of metallothionein clearly depicted the role of such proteins in bacterial metal resistance mechanism. The present study results clearly declare S. enterica 43C a suitable candidate for green chemistry to bioremediate environmental Cd(2+).

  18. Molecular mechanisms of plasmid-determined mercury and cadmium resistances in bacteria

    International Nuclear Information System (INIS)

    The structural basis for induction of the broad spectrum mercurial resistance operon of pDU1358 with inorganic mercury and with phenylmercury acetate was addressed by DNA sequencing analysis (that showed that a major difference occurred in the 3' 29 base pairs of the ital merR gene compared to the merR genes of Tn501 and R100) and by lac-fusion transcription experiments regulated by merR in trans. The lac-fusion results were compared with those from a narrow spectrum operon, and the pDU1358 merR deleted at the 3' end. A hybrid mer operon containing the merR gene from pDU1358 and lacking the merB gene was inducible by both phenylmercury and inorganic Hg2+, showing that organomercurial lyase is not needed for induction by organomercurials. A mutant form of pDU1358 merR missing the C-terminal 17 amino acids responded to inorganic Hg2+ but not to phenylmercury, indicating that the C-terminal region of the MerR protein of the pDU1358 mer operon is required for the recognition of phenylmercury acetate. The down regulation of the mer operon by the merD gene was also measured in trans with complementing mer operons of pDU1358 or R100 or merD- mutants. In the presence of the merD gene, beta-galactosidase activity was lowered by 2 to 4 fold. The merD gene gene product was visualized by autoradiography. The Cd2+ resistance determinant cadA of S. aureus was investigated. The nucleotide sequence of the DNA fragment containing the cadA determinant revealed two open reading frames the larger one of which is essential for expression of cadmium resistance

  19. Cadmium resistance and uptake by bacterium, Salmonella enterica 43C, isolated from industrial effluent.

    Science.gov (United States)

    Khan, Zaman; Rehman, Abdul; Hussain, Syed Z; Nisar, Muhammad A; Zulfiqar, Soumble; Shakoori, Abdul R

    2016-12-01

    Cadmium resistant bacterium, isolated from industrial wastewater, was characterized as Salmonella enterica 43C on the basis of biochemical and 16S rRNA ribotyping. It is first ever reported S. enterica 43C bared extreme resistance against heavy metal consortia in order of Pb(2+)>Cd(2+)>As(3+)>Zn(2+)>Cr(6+)>Cu(2+)>Hg(2+). Cd(2+) stress altered growth pattern of the bacterium in time dependent manner. It could remove nearly 57 % Cd(2+) from the medium over a period of 8 days. Kinetic and thermodynamic studies based on various adsorption isotherm models (Langmuir and Freundlich) depicted the Cd(2+) biosorption as spontaneous, feasible and endothermic in nature. Interestingly, the bacterium followed pseudo first order kinetics, making it a good biosorbent for heavy metal ions. The S. enterica 43C Cd(2+) processivity was significantly influenced by temperature, pH, initial Cd(2+) concentration, biomass dosage and co-metal ions. FTIR analysis of the bacterium revealed the active participation of amide and carbonyl moieties in Cd(2+) adsorption confirmed by EDX analysis. Electron micrographs beckoned further surface adsorption and increased bacterial size due to intracellular Cd(2+) accumulation. An overwhelming increase in glutathione and other non-protein thiols levels played a significant role in thriving oxidative stress generated by metal cations. Presence of metallothionein clearly depicted the role of such proteins in bacterial metal resistance mechanism. The present study results clearly declare S. enterica 43C a suitable candidate for green chemistry to bioremediate environmental Cd(2+). PMID:27491862

  20. Elucidation of the CHO Super-Ome (CHO-SO) by Proteoinformatics

    DEFF Research Database (Denmark)

    Kumar, Amit; Baycin-Hizal, Deniz; Wolozny, Daniel;

    2015-01-01

    Chinese hamster ovary (CHO) cells are the preferred host cell line for manufacturing a variety of complex biotherapeutic drugs including monoclonal antibodies. We performed a proteomics and bioinformatics analysis on the spent medium from adherent CHO cells. Supernatant from CHO-K1 culture was co...

  1. CHO Quasispecies—Implications for Manufacturing Processes

    Directory of Open Access Journals (Sweden)

    Florian M. Wurm

    2013-10-01

    Full Text Available Chinese hamster ovary (CHO cells are a source of multi-ton quantities of protein pharmaceuticals. They are, however, immortalized cells, characterized by a high degree of genetic and phenotypic diversity. As is known for any biological system, this diversity is enhanced by selective forces when laboratories (no sharing of gene pools grow cells under (diverse conditions that are practical and useful. CHO cells have been used in culture for more than 50 years, and various lines of cells are available and have been used in manufacturing. This article tries to represent, in a cursory way, the history of CHO cells, particularly the origin and subsequent fate of key cell lines. It is proposed that the name CHO represents many different cell types, based on their inherent genetic diversity and their dynamic rate of genetic change. The continuing remodeling of genomic structure in clonal or non-clonal cell populations, particularly due to the non-standardized culture conditions in hundreds of different labs renders CHO cells a typical case for “quasispecies”. This term was coined for families of related (genomic sequences exposed to high mutation rate environments where a large fraction of offspring is expected to carry one or more mutations. The implications of the quasispecies concept for CHO cells used in protein manufacturing processes are significant. CHO genomics/transcriptomics may provide only limited insights when done on one or two “old” and poorly characterized CHO strains. In contrast, screening of clonal cell lines, derived from a well-defined starting material, possibly within a given academic or industrial environment, may reveal a more narrow diversity of phenotypes with respect to physiological/metabolic activities and, thus, allow more precise and reliable predictions of the potential of a clone for high-yielding manufacturing processes.

  2. Chromosomal Locus for Cadmium Resistance in Pseudomonas putida Consisting of a Cadmium-Transporting ATPase and a MerR Family Response Regulator

    OpenAIRE

    Lee, Seon-Woo; Glickmann, Eric; Cooksey, Donald A.

    2001-01-01

    Pseudomonads from environmental sources vary widely in their sensitivity to cadmium, but the basis for this resistance is largely uncharactarized. A chromosomal fragment encoding cadmium resistance was cloned from Pseudomonas putida 06909, a rhizosphere bacterium, and sequence analysis revealed two divergently transcribed genes, cadA and cadR. CadA was similar to cadmium-transporting ATPases known mostly from gram-positive bacteria, and to ZntA, a lead-, zinc-, and cadmium-transporting ATPase...

  3. The Role of Exopolymers in Protection of Ralstonia sp., a Cadmium-resistant Bacterium, from Cadmium Toxicity

    Directory of Open Access Journals (Sweden)

    Anchulee Watcharamusik

    2008-07-01

    Full Text Available Production of exopolymers is one of heavy metal resistance mechanisms in bacteria. Ralstonia sp. TAK1, a cadmium-resistant bacterium, was isolated from a high cadmium (Cd contaminated soil at the zinc mine, Tak province, Thailand. The bacterium was cultivated in LB broth and its growth was monitored. The yields of exopolymers were measured by the phenol-sulfuric method at different growth phases. The levels of Cd resistance were quantitatively determined by survival cell assay. The highest amount of exopolymers (0.69 mg glucose equivalent/ mg dry weight was found at the stationary phase and sharply decreased at the late-stationary phase. In addition to high production of exopolymers at the stationary phase, Ralstonia sp. TAK1 was more resistant to Cd than that of exponential phase cells. These results suggested that the resistance to Cd toxicity in Ralstonia sp. TAK1 at the stationary phase is mediated by exopolymer production. Contradictorily, there was no correlation between Cd resistance level and exopolymer production of cells at exponential phase indicating that other mechanism(s is responsible for Cd resistance of exponential phase cells. In addition, 0.4 mM CdCl2 was able to induce the increasing of exopolymers at the mid-exponential phases compared to uninduced cells. Exopolymer production of Cd-induced cells was constant from the mid-stationary to late-stationary phase. However, the highest exopolymers was found in uninduced cells at the stationary phase.

  4. Proteomic differences in recombinant CHO cells producing two similar antibody fragments.

    Science.gov (United States)

    Sommeregger, Wolfgang; Mayrhofer, Patrick; Steinfellner, Willibald; Reinhart, David; Henry, Michael; Clynes, Martin; Meleady, Paula; Kunert, Renate

    2016-09-01

    Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. "Omics" studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label-free LC-MS proteomic analyses to investigate product-specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single-chain Fv-Fc homodimeric antibody fragments (scFv-Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase-mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label-free proteomic analysis. LC-MS-MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902-1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26913574

  5. Plasmid-borne cadmium resistance genes in Listeria monocytogenes are similar to cadA and cadC of Staphylococcus aureus and are induced by cadmium.

    OpenAIRE

    Lebrun, M; AUDURIER, A.; Cossart, P

    1994-01-01

    pLm74 is the smallest known plasmid in Listeria monocytogenes. It confers resistance to the toxic divalent cation cadmium. It contains a 3.1-kb EcoRI fragment which hybridizes with the cadAC genes of plasmid pI258 of Staphylococcus aureus. When introduced into cadmium-sensitive L. monocytogenes or Bacillus subtilis strains, this fragment conferred cadmium resistance. The DNA sequence of the 3.1-kb EcoRI fragment contains two open reading frames, cadA and cadC. The deduced amino acid sequences...

  6. Analysis of a new skip list variant

    OpenAIRE

    Louchard, Guy; Prodinger, Helmut

    2006-01-01

    International audience; For a skip list variant, introduced by Cho and Sahni, we analyse what is the analogue of horizontal plus vertical search cost in the original skip list model. While the average in Pugh's original version behaves like Q log_Q n, with Q = 1/q a parameter, it is here given by (Q+1) log_Q n.

  7. Methods for modeling chinese hamster ovary (cho) cell metabolism

    DEFF Research Database (Denmark)

    2015-01-01

    for identifying a CHO cell line having a desired genetic trait, as well as for generating a desired CHO cell line having a genetic basis for a desired phenotype. Additionally, described herein are methods for constructing and analyzing in silico models of biological networks for CHO cells.......Embodiments of the present invention generally relate to the computational analysis and characterization biological networks at the cellular level in Chinese Hamster Ovary (CHO) cells. Based on computational methods utilizing a hamster reference genome, the invention provides methods...

  8. Towards dynamic metabolic flux analysis in CHO cell cultures.

    Science.gov (United States)

    Ahn, Woo Suk; Antoniewicz, Maciek R

    2012-01-01

    Chinese hamster ovary (CHO) cells are the most widely used mammalian cell line for biopharmaceutical production, with a total global market approaching $100 billion per year. In the pharmaceutical industry CHO cells are grown in fed-batch culture, where cellular metabolism is characterized by high glucose and glutamine uptake rates combined with high rates of ammonium and lactate secretion. The metabolism of CHO cells changes dramatically during a fed-batch culture as the cells adapt to a changing environment and transition from exponential growth phase to stationary phase. Thus far, it has been challenging to study metabolic flux dynamics in CHO cell cultures using conventional metabolic flux analysis techniques that were developed for systems at metabolic steady state. In this paper we review progress on flux analysis in CHO cells and techniques for dynamic metabolic flux analysis. Application of these new tools may allow identification of intracellular metabolic bottlenecks at specific stages in CHO cell cultures and eventually lead to novel strategies for improving CHO cell metabolism and optimizing biopharmaceutical process performance. PMID:22102428

  9. The Products of the Thermal Decomposition of CH3CHO

    Energy Technology Data Exchange (ETDEWEB)

    Vasiliou, AnGayle; Piech, Krzysztof M.; Zhang, Xu; Nimlos, Mark R.; Ahmed, Musahid; Golan, Amir; Kostko, Oleg; Osborn, David L.; Daily, John W.; Stanton, John F.; Ellison, G. Barney

    2011-04-06

    We have used a heated 2 cm x 1 mm SiC microtubular (mu tubular) reactor to decompose acetaldehyde: CH3CHO + DELTA --> products. Thermal decomposition is followed at pressures of 75 - 150 Torr and at temperatures up to 1700 K, conditions that correspond to residence times of roughly 50 - 100 mu sec in the mu tubular reactor. The acetaldehyde decomposition products are identified by two independent techniques: VUV photoionization mass spectroscopy (PIMS) and infrared (IR) absorption spectroscopy after isolation in a cryogenic matrix. Besides CH3CHO, we have studied three isotopologues, CH3CDO, CD3CHO, and CD3CDO. We have identified the thermal decomposition products CH3(PIMS), CO (IR, PIMS), H (PIMS), H2 (PIMS), CH2CO (IR, PIMS), CH2=CHOH (IR, PIMS), H2O (IR, PIMS), and HC=CH (IR, PIMS). Plausible evidence has been found to support the idea that there are at least three different thermal decomposition pathways for CH3CHO: Radical decomposition: CH3CHO + DELTA --> CH3 + [HCO] --> CH3 + H + CO Elimination: CH3CHO + DELTA --> H2 + CH2=C=O. Isomerization/elimination: CH3CHO + DELTA --> [CH2=CH-OH] --> HC=CH + H2O. Both PIMS and IR spectroscopy show compelling evidence for the participation of vinylidene, CH2=C:, as an intermediate in the decomposition of vinyl alchohol: CH2=CH-OH + DELTA --> [CH2=C:] + H2O --> HC=CH + H2O.

  10. CRISPR/Cas9-mediated genome engineering of CHO cell factories: application and perspectives

    DEFF Research Database (Denmark)

    Lee, Jae Seong; Grav, Lise Marie; Lewis, Nathan E.;

    2015-01-01

    Chinese hamster ovary (CHO) cells are the most widely used production host for therapeutic proteins.With the recent emergence of CHO genome sequences, CHO cell line engineering has takenon a new aspect through targeted genome editing. The bacterial clustered regularly interspacedshort palindromic...... highlighting both future perspectivesand challenges. As one of the main drivers for the CHO systems biology era, genome engineeringwith CRISPR/Cas9 will pave the way for rational design of CHO cell factories....

  11. Comprehensive genome and epigenome characterization of CHO cells in response to evolutionary pressures and over time.

    Science.gov (United States)

    Feichtinger, Julia; Hernández, Inmaculada; Fischer, Christoph; Hanscho, Michael; Auer, Norbert; Hackl, Matthias; Jadhav, Vaibhav; Baumann, Martina; Krempl, Peter M; Schmidl, Christian; Farlik, Matthias; Schuster, Michael; Merkel, Angelika; Sommer, Andreas; Heath, Simon; Rico, Daniel; Bock, Christoph; Thallinger, Gerhard G; Borth, Nicole

    2016-10-01

    The most striking characteristic of CHO cells is their adaptability, which enables efficient production of proteins as well as growth under a variety of culture conditions, but also results in genomic and phenotypic instability. To investigate the relative contribution of genomic and epigenetic modifications towards phenotype evolution, comprehensive genome and epigenome data are presented for six related CHO cell lines, both in response to perturbations (different culture conditions and media as well as selection of a specific phenotype with increased transient productivity) and in steady state (prolonged time in culture under constant conditions). Clear transitions were observed in DNA-methylation patterns upon each perturbation, while few changes occurred over time under constant conditions. Only minor DNA-methylation changes were observed between exponential and stationary growth phase; however, throughout a batch culture the histone modification pattern underwent continuous adaptation. Variation in genome sequence between the six cell lines on the level of SNPs, InDels, and structural variants is high, both upon perturbation and under constant conditions over time. The here presented comprehensive resource may open the door to improved control and manipulation of gene expression during industrial bioprocesses based on epigenetic mechanisms. Biotechnol. Bioeng. 2016;113: 2241-2253. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:27072894

  12. Identification of a novel temperature sensitive promoter in cho cells

    Directory of Open Access Journals (Sweden)

    Hesse Friedemann

    2011-05-01

    Full Text Available Abstract Background The Chinese hamster ovary (CHO expression system is the leading production platform for manufacturing biopharmaceuticals for the treatment of numerous human diseases. Efforts to optimize the production process also include the genetic construct encoding the therapeutic gene. Here we report about the successful identification of an endogenous highly active gene promoter obtained from CHO cells which shows conditionally inducible gene expression at reduced temperature. Results Based on CHO microarray expression data abundantly transcribed genes were selected as potential promoter candidates. The S100a6 (calcyclin and its flanking regions were identified from a genomic CHO-K1 lambda-phage library. Computational analyses showed a predicted TSS, a TATA-box and several TFBSs within the 1.5 kb region upstream the ATG start signal. Various constructs were investigated for promoter activity at 37°C and 33°C in transient luciferase reporter gene assays. Most constructs showed expression levels even higher than the SV40 control and on average a more than two-fold increase at lower temperature. We identified the core promoter sequence (222 bp comprising two SP1 sites and could show a further increase in activity by duplication of this minimal sequence. Conclusions This novel CHO promoter permits conditionally high-level gene expression. Upon a shift to 33°C, a two to three-fold increase of basal productivity (already higher than SV40 promoter is achieved. This property is of particular advantage for a process with reduced expression during initial cell growth followed by the production phase at low temperature with a boost in expression. Additionally, production of toxic proteins becomes feasible, since cell metabolism and gene expression do not directly interfere. The CHO S100a6 promoter can be characterized as cold-shock responsive with the potential for improving process performance of mammalian expression systems.

  13. Cellulase variants

    Energy Technology Data Exchange (ETDEWEB)

    Blazej, Robert; Toriello, Nicholas; Emrich, Charles; Cohen, Richard N.; Koppel, Nitzan

    2015-07-14

    This invention provides novel variant cellulolytic enzymes having improved activity and/or stability. In certain embodiments the variant cellulotyic enzymes comprise a glycoside hydrolase with or comprising a substitution at one or more positions corresponding to one or more of residues F64, A226, and/or E246 in Thermobifida fusca Cel9A enzyme. In certain embodiments the glycoside hydrolase is a variant of a family 9 glycoside hydrolase. In certain embodiments the glycoside hydrolase is a variant of a theme B family 9 glycoside hydrolase.

  14. 耐镉细菌与土壤胶体作用对镉形态的影响%Effect of cadmium resistant bacteria and soil colloids on the forms of cadmium

    Institute of Scientific and Technical Information of China (English)

    王晶; 蒋代华; 刘立龙; 罗雅; 蒋雄英; 尹辉; 韩祖龑

    2011-01-01

    The present study was conducted to explore the effect of cadmium resistant bacteria on cadmium adsorption property of red soil, lateritic red soil and brown calcareous soil colloids, and transformation of Cd forms of bacteria-soil colloid complex, and to provide theoretical basis for bioremediation of heavy metal contaminated soils. [Method]A cadmium resistant bacteria was selected from heavy metal contaminated soil in a mining area, three colloids of red soil, lateritic red earth and brown calcareous soil were extracted to find out the effect of cadmium resistant bacteria on cadmium adsorption, and transformation of Cd forms of bacteria-soil colloid complex. [Result]Cd adsorption capacity of three soil colloids increased significantly after inoculating cadmium resistant bacteria, and the range of increase in inorganic and organic colloidal complex in the red soils was the highest, while the contents of soluble weak acid and residual Cd form of three soil colloids reduced after inoculation in contrast to contents of reducible and oxidizable Cd form. After inoculating cadmium resistant bacteria, the content of different Cd forms on natural lateritic red earth colloid and red soil colloid were as follows: residual form>reducible form>weak acid soluble form>oxidable form. However, the content of Cd forms on farming lateritic red earth colloid and brown calcareous soil colloid were: reducible form>residual form>weak acid soluble for> oxidizable form. [Conclusion]Cadmium resistant bacteria could promote transformation of Cd forms on bacteria soil colloid complex from unavailable forms to bioavailable forms, and improve the effectiveness of cadmium.%[目的]探讨耐镉细菌对红壤、赤红壤和棕色石灰土3类型土壤胶体镉吸附性能的影响及细菌—土壤胶体复合体上所吸附镉形态的变化,为重金属污染土壤的生物修复提供理论依据.[方法]从矿区镉污染土壤中筛选分离出—株耐镉细菌,同时提取红壤

  15. Complete Genome Sequence of Vibrio anguillarum Phage CHOED Successfully Used for Phage Therapy in Aquaculture

    OpenAIRE

    Romero, Jaime; Higuera, Gastón; Gajardo, Felipe; Castillo, Daniel; Middleboe, Mathias; García, Katherine; Ramírez, Carolina; Espejo, Romilio T.

    2014-01-01

    Vibrio anguillarum phage CHOED was isolated from Chilean mussels. It is a virulent phage showing effective inhibition of V. anguillarum. CHOED has potential in phage therapy, because it can protect fish from vibriosis in fish farms. Here, we announce the completely sequenced genome of V. anguillarum phage CHOED.

  16. Jeep variants

    CERN Document Server

    de Bondt, Michiel

    2010-01-01

    The jeep problem was first solved by O. Helmer and N.J. Fine. But not much later, C.G. Phipps formulated a more general solution. He formulated a so-called convoy or caravan variant of the jeep problem and reduced the original problem to it. The convoy idea of Phipps was refined in [3]. Here we will apply this refined idea to several variants of the jeep problem.

  17. 嗜镉茵对重金属Cd2+的吸附研究%Investigation on Cd2+ adsorption by cadmium-resistant bacteria

    Institute of Scientific and Technical Information of China (English)

    赵玉清; 朴永哲; 孙天竹; 杨洪泽; 海华; 李晋

    2011-01-01

    In order to solve the problem of cadmium pollution within the environment, the adsorptive conditions and mechanism of cadmium ion with cadmium-resistant strain cultivated by laboratories are conducted. The effects of adsorption time, mass concentration of cadmium ion, mass concentration of resistant strain on adsorption are discussed. Results indicate that adsorption can reach a balance when the adsorption time is 1.5 h; cadmium ion removal can reach up to 80 %. The lowest adsorbance of the strain is 10 mg/g and the highestis 50 mg/g. IR spectra of the strain shows that the hydroxyl and carbonyl groups of polysaccharide and protein exhibit red shifts in various degrees,the peak width of hydroxyl and carbonyl groups widens a little bit, which shows that the adsorption is mainly resulted from the function of the surface groups. AFM detections indicate that the mycelium shrinks a little after the adsorption, which illustrates that the adsorption mainly occurs on thesurface and can affect the stress adsorption in cells to a certain extent.%为了解决环境中镉污染问题,研究了实验室培养的嗜镉茵吸附镉的条件和机理,讨论了时间、镉离子的质量浓度和菌液的质量浓度对吸附效果的影响.试验结果表明:在一定试验条件下.吸附时间1.5 h,吸附趋于平衡,镉离子吸附率可达80%;嗜镉茵对镉的最低吸附量为10 mg/g,最高吸附量可达50 mg/g.红外表征表明:嗜镉茵表面多糖、蛋白质的羟基峰和羰基峰均有不同程度的紫移,且糖苷峰、羟基峰的峰宽略增,说明菌体吸附主要是表面基团的作用所致.原子力显微镜表征可见,吸附后的茵体略收缩.这进一步说明该吸附是以表面基团作用为主的过程,在一定程度上对茵细胞内部的胁迫吸附有影响.

  18. Metabolite profiling of CHO cells: Molecular reflections of bioprocessing effectiveness.

    Science.gov (United States)

    Sellick, Christopher A; Croxford, Alexandra S; Maqsood, Arfa R; Stephens, Gill M; Westerhoff, Hans V; Goodacre, Royston; Dickson, Alan J

    2015-09-01

    Whilst development of medium and feeds has provided major advances in recombinant protein production in CHO cells, the fundamental understanding is limited. We have applied metabolite profiling with established robust (GC-MS) analytics to define the molecular loci by which two yield-enhancing feeds improve recombinant antibody yields from a model GS-CHO cell line. With data across core metabolic pathways, that report on metabolism within several cellular compartments, these data identify key metabolites and events associated with increased cell survival and specific productivity of cells. Of particular importance, increased process efficiency was linked to the functional activity of the mitochondria, with the amount and time course of use/production of intermediates of the citric acid cycle, for uses such as lipid biosynthesis, precursor generation and energy production, providing direct indicators of cellular status with respect to productivity. The data provide clear association between specific cellular metabolic indicators and cell process efficiency, extending from prior indications of the relevance of lactate metabolic balance to other redox sinks (glycerol, sorbitol and threitol). The information, and its interpretation, identifies targets for engineering cell culture efficiency, either from genetic or environmental perspectives, and greater understanding of the significance of specific medium components towards overall CHO cell bioprocessing. PMID:26198903

  19. MAR characteristic motifs mediate episomal vector in CHO cells.

    Science.gov (United States)

    Lin, Yan; Li, Zhaoxi; Wang, Tianyun; Wang, Xiaoyin; Wang, Li; Dong, Weihua; Jing, Changqin; Yang, Xianjun

    2015-04-01

    An ideal gene therapy vector should enable persistent transgene expression without limitations in safety and reproducibility. Recent researches' insight into the ability of chromosomal matrix attachment regions (MARs) to mediate episomal maintenance of genetic elements allowed the development of a circular episomal vector. Although a MAR-mediated engineered vector has been developed, little is known on which motifs of MAR confer this function during interaction with the host genome. Here, we report an artificially synthesized DNA fragment containing only characteristic motif sequences that served as an alternative to human beta-interferon matrix attachment region sequence. The potential of the vector to mediate gene transfer in CHO cells was investigated. The short synthetic MAR motifs were found to mediate episomal vector at a low copy number for many generations without integration into the host genome. Higher transgene expression was maintained for at least 4 months. In addition, MAR was maintained episomally and conferred sustained EGFP expression even in nonselective CHO cells. All the results demonstrated that MAR characteristic sequence-based vector can function as stable episomes in CHO cells, supporting long-term and effective transgene expression.

  20. Choline uptake in Agrobacterium tumefaciens by the high-affinity ChoXWV transporter.

    Science.gov (United States)

    Aktas, Meriyem; Jost, Kathinka A; Fritz, Christiane; Narberhaus, Franz

    2011-10-01

    Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called "Venus flytrap mechanism" of substrate binding. PMID:21803998

  1. Stable Expression of Recombinant Factor VIII in CHO Cells Using Methotrexate-Driven Transgene Amplification.

    Science.gov (United States)

    Orlova, N A; Kovnir, S V; Vorobiev, I I; Yuriev, A S; Gabibov, A G; Vorobiev, A I

    2012-01-01

    Prophylaxis and treatment of inherited clotting disorder hemophilia A requires regular administration of factor VIII. Recombinant factor VIII, which is produced in CHO or BHK cells, is equivalent to the plasma-derived one and is prevalent in current clinical practice in developed countries. Development of a biosimilar recombinant FVIII requires the creation of a highly productive clonal cell line and generation of monoclonal antibodies suitable for affinity purification of the product. Methotrexate-driven transgene amplification of genetic cassettes that code full-length and truncated variants of FVIII under the control of the CMV promoter was studied. It was shown that the expression level of the truncated variant of FVIII is 6.5 times higher than that of the full-length molecule. The transgene amplification procedure was sufficient for a twofold increase of the expression level in the transfected cells pool and subsequent selection of the clonal line, stably producing truncated FVIII at the level of 0.52 IU/ml during cultivation in a chemically defined protein-free culture medium. Four generated mouse monoclonal antibodies toward the heavy chain of FVIII were found suitable for binding the truncated variant of FVIII directly from the conditioned medium and elution of the FVIII with a more than 85% yield and normal pro-coagulant activity. The producer cell line and monoclonal antibodies obtained are sufficient for the development of upstream and downstream processes of biosimilar FVIII production. Generation of more productive cell lines by the use of stronger, nonviral promoters and shorter cDNA of FVIII will be the subject of further studies. PMID:22708069

  2. HeI photoelectron spectroscopy of the isoproxy (CH 3) 2CHO radical

    Science.gov (United States)

    Zheng, Sun; Shijun, Zheng; Lingpeng, Meng; Dianxun, Wang

    2003-02-01

    A continuous (CH 3) 2CHO radical beam is generated by pyrolysis of (CH 3) 2CHONO at 145(±0.5) °C. The HeI photoelectron spectrum of (CH 3) 2CHO is recorded in situ. The IP of (CH 3) 2CHO is 9.70 eV and the spectrum of the X3A″ state of (CH 3) 2CHO + exhibits a vibrational progression of 1200±60 cm-1. The removal of an electron from the highest occupied molecular orbital 11a ', which corresponds to ionization process of ( CH3) 2CHO+( 1A')←( CH3) 2CHO( X2A') , leads to a very sharp peak at 10.21 eV. This study provides new experimental and theoretical ionization energies of several ionic states of (CH 3) 2CHO.

  3. Isolation and characterization of Chinese hamster ovary cell variants defective in adhesion to fibronectin-coated collagen

    OpenAIRE

    1980-01-01

    Variant clones of Chinese hamster ovary (CHO) cells were selected for reduced adhesion to serum-coated tissue culture plates. These clones also displayed reduced adhesion to substrata composed of collagen layers coated with bovine serum or with fibronectin (cold-insoluble globulin). Wild-type (WT) and adhesion variant (ADv) cells grew at comparable rates in suspension culture, but the adhesion variants could not be grown in monolayer culture because of their inability to attach to the substra...

  4. Synthesis of human prolactin in Chinese hamster ovary (CHO) cells

    International Nuclear Information System (INIS)

    Three different eukaryotic expression vectors, based on the same selectable gene marker (dhfr), have been used for dhf- CHO cells transfection to rapidly isolate stable cell lines capable of secreting high levels of recombinant human prolactin (rec-hPRL). Two vectors, one codifying a human prolactin (p658-hPRL) and the other a tag-prolactin (p658-tagPRL), contain the complete hepatitis B virus-X (HBV-X) gene coding for a viral transactivator and a sequence derived from the granulocyte-macrophage colony-stimulating factor (GM-CSF) that mediates selective dhfr mRNA degradation. These vectors have the advantage of rapidly obtaining stable cell lines without methotrexate amplification. The highest secretion obtained by these vectors was of approximately 10 μg hPRU106 cells/day. The other vector (pEDdc-hPRL) is based on a dicistronic expression system, containing an internal ribosome entry site isolated from the encephalomyocarditis (EMC) virus. This vector before amplification provided secretion levels at least 10 fold lower than that obtained with the other two vectors. However, after three steps of methotrexate amplification, it provided some clones able to secrete up to 30 μg hPRU106 cells/day. This is the first report describing the production and purification of rec-hPRL from CHO cells, obtaining secretion levels with both vectors higher than those reported so far for this hormone in other eukaryotic systems. CHO-derived rec-hPRL contained approximately 10 % of the glycosylated form, a value that is consistent with results reported for hPRL purified from the pituitary or from transformed murine C-127 cells. CHO-derived rec-hPRL was purified with good yield, obtaining also a good resolution between non-glycosylated and glycosylated prolactin. The latter, when its potency was determined via an in vitro bioassay, presented a 47 % lower bioactivity. A qualitative and quantitative analysis of these forms was also possible thanks to the setting up of a reversed

  5. Measurements of the absorption cross section of (13)CHO(13)CHO at visible wavelengths and application to DOAS retrievals.

    Science.gov (United States)

    Goss, Natasha R; Waxman, Eleanor M; Coburn, Sean C; Koenig, Theodore K; Thalman, Ryan; Dommen, Josef; Hannigan, James W; Tyndall, Geoffrey S; Volkamer, Rainer

    2015-05-14

    The trace gas glyoxal (CHOCHO) forms from the atmospheric oxidation of hydrocarbons and is a precursor to secondary organic aerosol. We have measured the absorption cross section of disubstituted (13)CHO(13)CHO ((13)C glyoxal) at moderately high (1 cm(-1)) optical resolution between 21 280 and 23 260 cm(-1) (430-470 nm). The isotopic shifts in the position of absorption features were found to be largest near 455 nm (Δν = 14 cm(-1); Δλ = 0.29 nm), whereas no significant shifts were observed near 440 nm (Δν DOAS) in a series of sensitivity tests using synthetic spectra, and laboratory measurements of mixtures containing (12)C and (13)C glyoxal, nitrogen dioxide, and other interfering absorbers. We find the changes in apparent spectral band shapes remain significant at the moderately high optical resolution typical of CE-DOAS (0.55 nm fwhm). CE-DOAS allows for the selective online detection of both isotopes with detection limits of ∼200 pptv (1 pptv = 10(-12) volume mixing ratio), and sensitivity toward total glyoxal of few pptv. The (13)C absorption cross section is available for download from the Supporting Information. PMID:25551419

  6. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

    DEFF Research Database (Denmark)

    Xu, Xun; Pan, Shengkai; Liu, Xin;

    2011-01-01

    Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of...

  7. Sequencing the CHO DXB11 genome reveals regional variations in genomic stability and haploidy

    DEFF Research Database (Denmark)

    Kaas, Christian Schrøder; Kristensen, Claus; Betenbaugh, Michael J.;

    2015-01-01

    Background: The DHFR negative CHO DXB11 cell line (also known as DUX-B11 and DUKX) was historically the first CHO cell line to be used for large scale production of heterologous proteins and is still used for production of a number of complex proteins.  Results: Here we present the genomic sequen...

  8. RNA-seq based expression analysis of the CHO cell protein secretion pathway

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kaas, Christian Schrøder; Kildegaard, Helene Faustrup;

    The Chinese hamster ovary (CHO) cell-line is the predominant mammalian industrial cell line being used to produce recombinant therapeutic proteins. Although CHO cells have been used for more than 25 years, the genome sequence was first published in 2011. So far there have been limited studies of ...

  9. The emerging CHO systems biology era: harnessing the ‘omics revolution for biotechnology

    DEFF Research Database (Denmark)

    Kildegaard, Helene Faustrup; Baycin-Hizal, Deniz; Lewis, Nathan;

    2013-01-01

    Chinese hamster ovary (CHO) cells are the primary factories for biopharmaceuticals because of their capacity to correctly fold and post-translationally modify recombinant proteins compatible with humans. New opportunities are arising to enhance these cell factories, especially since the CHO-K1 cell...... line was recently sequenced. Now, the CHO systems biology era is underway. Critical ‘omics data sets, including proteomics, transcriptomics, metabolomics, fluxomics, and glycomics, are emerging, allowing the elucidation of the molecular basis of CHO cell physiology. The incorporation of these data sets...... into mathematical models that describe CHO phenotypes will provide crucial biotechnology insights. As ‘omics technologies and computational systems biology mature, genome-scale approaches will lead to major innovations in cell line development and metabolic engineering, thereby improving protein production...

  10. Complete genome sequence of Vibrio anguillarum phage CHOED successfully used for phage therapy in aquaculture

    DEFF Research Database (Denmark)

    Romero, Jaime; Higuera, Gastón; Gajardo, Felipe;

    2014-01-01

    Vibrio anguillarum phage CHOED was isolated from Chilean mussels. It is a virulent phage showing effective inhibition of V. anguillarum. CHOED has potential in phage therapy, because it can protect fish from vibriosis in fish farms. Here, we announce the completely sequenced genome of V....... anguillarum phage CHOED....

  11. CRISPR/Cas9-mediated genome engineering of CHO cell factories: Application and perspectives.

    Science.gov (United States)

    Lee, Jae Seong; Grav, Lise Marie; Lewis, Nathan E; Faustrup Kildegaard, Helene

    2015-07-01

    Chinese hamster ovary (CHO) cells are the most widely used production host for therapeutic proteins. With the recent emergence of CHO genome sequences, CHO cell line engineering has taken on a new aspect through targeted genome editing. The bacterial clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system enables rapid, easy and efficient engineering of mammalian genomes. It has a wide range of applications from modification of individual genes to genome-wide screening or regulation of genes. Facile genome editing using CRISPR/Cas9 empowers researchers in the CHO community to elucidate the mechanistic basis behind high level production of proteins and product quality attributes of interest. In this review, we describe the basis of CRISPR/Cas9-mediated genome editing and its application for development of next generation CHO cell factories while highlighting both future perspectives and challenges. As one of the main drivers for the CHO systems biology era, genome engineering with CRISPR/Cas9 will pave the way for rational design of CHO cell factories.

  12. Cho Decomposition of One-Half Integer Monopoles Solutions

    Science.gov (United States)

    Teh, Rosy; Ng, Ban-Loong; Wong, Khai-Ming

    2013-11-01

    We performed the Cho decomposition of the SU(2) Yang-Mills-Higgs gauge potentials of the finite energy (1) one-half monopole solution and (2) the one and a half monopoles solution into Abelian and non-Abelian components. We found that the semi-infinite string singularity in the gauge potentials is a contribution from the Higgs field of the one-half monopole in both of the solutions. The non-Abelian components of the gauge potentials are able to remove the point singularity of the Abelian components of the 't Hooft-Polyakov monopole but not the string singularity of the one-half monopole which is topological in nature. Hence the total energy of a one monopole is infinite in the Maxwell electromagnetic theory but the total energy of a one-half monopole is finite. By analyzing the magnetic fields and the gauge covariant derivatives of the Higgs field, we are able to conclude that both the one-half integer monopoles solutions are indeed non-BPS even in the limit of vanishing Higgs self-coupling constant.

  13. Cho decomposition of electrically charged one-half monopole

    Science.gov (United States)

    Ng, Ban-Loong; Teh, Rosy; Wong, Khai-Ming

    2014-03-01

    Recently we have carried out some work on the Cho decomposition of the electrically neutral, finite energy one-half monopole solution of the SU(2) Yang-Mills-Higgs field theory. In this paper, we performed the decomposition of the electrically charged solution using the same numerical procedure. The gauge potential of the one-half dyon solution is decomposed into Abelian and non-Abelian components. The semi-infinite string singularity in the gauge potential is a contribution of the Higgs field and hence topological in nature. The string singularity cannot be cancelled by the non-Abelian components of the gauge potential. However, the string singularity is integrable and the energy of the solution is finite. By decomposing the magnetic fields and covariant derivatives of the Higgs field into three isospin space directions, we are able to provide conclusive evidence that the constructed one-half dyon is certainly a non-BPS solution even in the limit of vanishing Higgs self-coupling constant and electric charge. Furthermore, we found that the time component of gauge function is parallel to the Higgs field in isospace only at large distances, elsewhere they are non-parallel.

  14. Screening, Identification of Fungi with High Cadmium-Resistance and its Solubilization Capacity of Insoluble Phosphates%强抗镉真菌的分离鉴定及溶磷能力研究

    Institute of Scientific and Technical Information of China (English)

    冯宏; 李永涛; 张干; 罗春玲

    2013-01-01

    A fungus with high cadmium resistance was isolated from the soil polluted by e-waste primitive recycling activity by adding definite concentration of Cd (200 mg/L) as 3CdSO4 o 8H2O in the medium. The fungus belonged to Cochliobolus lunatus according to ITS rDNA sequences. This strain was able to grow well on PDA plate containing 2 000 mg/L Cd + , and its cadmium resistance might be ascribed to the production of alkaline materials during its growth. Apart from Cd 2+ , this stain could tolerate Pb + , Zn 2+ and Cu2 + efficiently as well. The resistance decreased in the order of Zn 2+ , Cd 2+ > Pb 2+ > Cu 2+ > Co2 + > Ni2 + , Cr2 + > Hg2+ , Ag+. This strain could grow in the liquid medium with three different inorganic phosphates, and the phosphate solubilization capacity was in the order of Ca3 (PO4 )2(104 mg/L) > A1PO4(86 mg/L) >FePO4 o 4H2O (17 mg/L).%通过在培养基中加入200 mg/L镉(3CdSO4·8H2O),从电子垃圾污染区土壤中筛选到1株强抗镉真菌,经ITS序列鉴定为月状旋孢腔菌Cochliobolus lunatus.该菌株可在镉质量浓度为2 000 mg/L的PDA平板上生长良好,抗性试验结果表明,菌株对镉的抗性可能与该菌在生长过程中产生碱性物质有关;菌株对镉、铅、锌和铜的最小抑菌浓度(MIC)均超过500 mg/L,菌体对9种重金属离子抗性的强弱顺序大致为:Zn2+、Cd2+ >Pb2+ >Cu2+ >Co2+>Ni2+ 、Cr2+> Hg2+、Ag+.该菌株对3种难溶性的磷酸盐具有溶磷作用,溶磷能力强弱顺序为:Ca3(PO4)2(104 mg/L)>AlPO4 (86 mg/L)> FePO4·4 H2O (17 mg/L).

  15. Benchmarking of commercially available CHO cell culture media for antibody production

    OpenAIRE

    Reinhart, David; Damjanovic, Lukas; Kaisermayer, Christian; Kunert, Renate

    2015-01-01

    In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but hi...

  16. CHO: A Benchmark Suite for OpenCL-based FPGA Accelerators

    OpenAIRE

    Ndu, Geoffrey; Lujan, Mikel; Navaridas, Javier

    2014-01-01

    Programming FPGAs with OpenCL-based high-level synthesis frameworks is gaining attention with a number of commercial and research frameworks announced. However, there are no benchmarks for evaluating these frameworks. To this end, we present CHO benchmark suite an extension of CHStone, a commonly used C-based high-level synthesis benchmark suite, for OpenCl. We characterise CHO at various levels and use it to investigate compiling non-trivial software to FPGAs.

  17. Interview with Dr. Seokhee CHO About Gifted Education and Its Future

    OpenAIRE

    Hüseyin MERTOL

    2014-01-01

    The purpose of this study, which is a major name in the education of gifted Dr. Cho 's about gifted education is to put forward their views. Dr. Seokhee Cho is a Professor at the School of Education, St. John’s University. She is currently conducting three research projects funded by US Department of Education: Project HOPE as a Principal Investigator, Project WIN and Project LEADER as a research director.

  18. Cultivos de células CHO-K1

    Directory of Open Access Journals (Sweden)

    M.C. Nóvoa-Valiñas

    2005-01-01

    Full Text Available El uso de determinados metales pesados y pesticidas es la estrategia más empleada para el control de plagas. Estas sustancias, una vez aplicadas a los cultivos, pueden pasan al medio ambiente, permaneciendo en él como xenobióticos que van a afectar, en mayor o menor medida, a los seres vivos. En el presente estudio se ha evaluado la toxicidad basal de un metal, cobre, y un pesticida organoclorado, lindano, así como mezclas de ambos a distintas concentraciones. Para llevar a cabo este trabajo se ha utilizado la línea celular CHO-K1 (células epiteliales de ovario de hamster, usándose como criterio de citotoxicidad la muerte celular, determinada mediante la técnica del rojo neutro. Las concentraciones iniciales fueron: 0,03; 0,06 y 0,9 mM de cobre y 0,01; 0,03 y 0,1 mM de lindano. Y en las mezclas, las concentraciones estuvieron comprendidas entre 0,01-0,9 de cobre y 0,001-0,1de lindano. Como resultados, la citotoxicidad del cobre y lindano fue dosis-dependiente. En las exposiciones a mezclas se observa que a concentraciones fijas de lindano, la viabilidad desciende al aumentar la concentración de cobre, mientras que, dentro de un cierto rango, a concentraciones fijas de cobre, la viabilidad celular se incrementa al aumentar la concentración de lindano

  19. Intraclonal protein expression heterogeneity in recombinant CHO cells.

    Directory of Open Access Journals (Sweden)

    Warren Pilbrough

    Full Text Available Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean, approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations. Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50

  20. Controllability analysis of protein glycosylation in CHO cells.

    Directory of Open Access Journals (Sweden)

    Melissa M St Amand

    Full Text Available To function as intended in vivo, a majority of biopharmaceuticals require specific glycan distributions. However, achieving a precise glycan distribution during manufacturing can be challenging because glycosylation is a non-template driven cellular process, with the potential for significant uncontrolled variability in glycan distributions. As important as the glycan distribution is to the end-use performance of biopharmaceuticals, to date, no strategy exists for controlling glycosylation on-line. However, before expending the significant amount of effort and expense required to develop and implement on-line control strategies to address the problem of glycosylation heterogeneity, it is imperative to assess first the extent to which the very complex process of glycosylation is controllable, thereby establishing what is theoretically achievable prior to any experimental attempts. In this work, we present a novel methodology for assessing the output controllability of glycosylation, a prototypical example of an extremely high-dimensional and very non-linear system. We first discuss a method for obtaining the process gain matrix for glycosylation that involves performing model simulations and data analysis systematically and judiciously according to a statistical design of experiments (DOE scheme and then employing Analysis of Variance (ANOVA to determine the elements of process gain matrix from the resulting simulation data. We then discuss how to use the resulting high-dimensional gain matrix to assess controllability. The utility of this method is demonstrated with a practical example where we assess the controllability of various classes of glycans and of specific glycoforms that are typically found in recombinant biologics produced with Chinese Hamster Ovary (CHO cells. In addition to providing useful insight into the extent to which on-line glycosylation control is achievable in actual manufacturing processes, the results also have important

  1. CYP3A4 overexpression enhances the cytotoxicity of the antitumor triazoloacridinone derivative C-1305 in CHO cells

    Institute of Scientific and Technical Information of China (English)

    Ewa AUGUSTIN; Barbara BOROWA-MAZGAJ; Agnieszka KIKULSKA; Milena KORDALEWSKA; Monika PAW(L)OWS KA

    2013-01-01

    Aim:To examine how the higher expression level of CYP3A4 isoenzyme influenced the cytotoxicity of the antitumor triazoloacridinone derivative C-1305 in Chinese hamster ovary (CHO) cells.Methods:Three CHO cell lines were examined:wild-type CHO cells; CHO-HR cells with overexpression of human cytochrome P450 reductase (CPR); and CHO-HR-3A4 cells with coexpression of human CYP3A4 and CPR.Cellular responses caused by C-1305 were monitored using DAPI staining,cell cycle analysis,phosphatydilserine externalization analysis and SA-β-galactosidase expression analysis.Cell viability was assessed with simultaneous FDA and PI staining.Results:Treatment with C-1305 for 72 h exhibited different levels of cytotoxicity in the 3 cell lines,and the values of IC80 in CHO,CHO-HR and CHO-HR-3A4 cells were 0.087+0.005,0.032+0.0001,and 0.064+0.0095 μmol/L,respectively.The cell cycle analysis revealed that both CHO and CHO-HR cells underwent transient G2/M arrest,whereas CHO-HR-3A4 cells did not accumulate in this phase.Prolonged exposure up to 120 h caused time-dependent increase in the sub-G1 fraction in all the 3 cell lines.Treatment with C-1305 caused cell death through apoptosis and necrosis.However,these processes were more pronounced in the transfected CHO cells than in the wild-type cells.The cells surviving after C-1305 exposure underwent senescence.Conclusion:CYP3A4 overexpression potently enhances the cellular responses (apoptosis,necrosis and senescence) caused by C-1305 in CHO cells.

  2. Quantitative mammalian cell mutagenesis and a preliminary study of the mutagenic potential of metallic compounds. [Cell system used was CHO/HGPRT

    Energy Technology Data Exchange (ETDEWEB)

    Hsie, A.W.; Johnson, N.P.; Couch, D.B.; San Sebastian, J.R.; O' Neill, J.P.; Forbes, N.L.

    1978-01-01

    We have defined a set of stringent conditions required to quantify specific gene mutation in a mammalian cell system, CHO/HGPRT. Greater than 98% of the 6-thioguanine (TG)-resistant variants were shown to be deficient in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in Chinese hamster ovary (CHO) cells. The sensitive and quantitative nature of this assay was utilized to study the structure-activity (mutagenicity) relationship of various classes of chemicals. Mutagenicity as determined in the CHO/HGPRT assay, appears to correlate well (76/83 (92%)) with the reported animal carcinogenicity of 108 chemicals studied. The system also appears to be suitable for studying the mutagenicity and cytotoxicity of metallic compounds. We found that cis-dichlorodiammine Pt(II) (cis-Pt(NH/sub 3/)/sub 2/Cl/sub 2/) (cis-DDP), one of the widely used inorganic antitumor agents, is cytotoxic and mutagenic. Mutagenicity of cis-DDP correlates with its binding to DNA. However, trans-DDP, (Pt(NH/sub 3/)/sub 4/)Cl/sub 2/, and K/sub 2/(PtCl/sub 4/) exhibit greatly reduced biological activities. Among 14 other metals studied, we found that carcinogenic metallic compounds, such as MnCl/sub 2/, NiCl/sub 2/, and BeSO/sub 4/ are mutagenic, while non-carcinogenic compounds such as MgCl/sub 2/ and H/sub 2/SeO/sub 3/ are not. Determination of metal mutagenicity is apparently complicated by the ionic composition of the medium. This may account in part for varying results in studies of the mutagenicity of other metallic compounds. Further refinement of the assay conditions, especially with respect to the ionic environment necessary for quantifying mutagenesis of each metallic agent, is in progress.

  3. Enhancement of Human Prolactin Synthesis by Sodium Butyrate Addition to Serum-Free CHO Cell Culture

    Directory of Open Access Journals (Sweden)

    Herbert Rodrigues Goulart

    2010-01-01

    Full Text Available Sodium butyrate (NaBu has been used as a productivity enhancer for the synthesis of recombinant proteins in Chinese hamster ovary (CHO cells. Thus, the influence of NaBu on the production of recombinant human prolactin (hPRL from CHO cells was investigated for the first time. CHO cell cultures were submitted to a treatment with different concentrations of NaBu (0.25 to 4 mM. Quantitative and qualitative analyses by reverse-phase high-performance liquid chromatography (RP-HPLC and Western blot or SDS-PAGE, carried out directly on CHO-conditioned medium, showed that the highest hPRL expression was obtained with 1 mM NaBu. In vitro biological assays based on noble rat lymphoma (Nb2 and mouse pro-B lymphoma (Ba/F3-LLP cells were carried out on purified hPRL. Its bioactivity in the presence of NaBu was not apparently different from that of the First International Reference Reagent of recombinant hPRL (WHO 97/714. Our results show that NaBu increased the synthesis of recombinant hPRL in CHO cells, apparently without compromising either its structure or function.

  4. 分泌吲哚乙酸的蒌蒿内生耐镉细菌的筛选与鉴定%Screening and Identification of Indole Acetic Acid-Producing Cadmium-Resistant Endophytic Bacteria fromArtemisia Selengensis

    Institute of Scientific and Technical Information of China (English)

    周小梅; 赵运林; 胥正钢; 董萌; 库文珍

    2014-01-01

    To isolate indole acetic acid (IAA)-producing cadmium-resistant endophytic bacteria fromArtemisia selengensis is to help build an effective combined remediation system of plant and microorganism. Using IAA-producing and cadmium-tolerance as the screening indexes, isolated IAA-producing cadmium-resistant endophytic bacteria from the root, stem and leafofArtemisia selengensis by the grinding method, and determinated the tolerance of lead, copper, antimony and the effect of the strains on growth ofAremisia selengensis. Strains were identified based on morphological, physiological and biochemical properties as well as 16S rDNA sequence analysis. The results showed that, two cadmium-resistant endophytic bacteria which both have strong abilities to secrete IAA, named as J2 and Y5, were isolated fromArtemisia selengensis. The cadmium tolerance mass concentration of strains J2 and Y5 were up to 90 mg·L-1. The IAA production of strains J2 and Y5 respectively were 23.108 mg·L-1, 15.192 mg·L-1. Strain J2 could significantly increase the plant height, the longest root length, the average root length, the fresh weight and the dry weight of Artemisia selengensis; strain Y5 could significantly improve the plant height, the fresh weight and the dry weight ofArtemisia selengensis. The lead tolerance mass concentration of strains J2 and Y5 were up to 1200 mg·L-1, while the copper tolerance mass concentration of strains J2 and Y5 respectively were 120 mg·L-1 and 160 mg·L-1, the antimony tolerancemass concentration of strains J2 and Y5 respectively were 50 mg·L-1 and 150 mg·L-1. The colony of strain J2 was yellow, suborbicular and sticky on LB tablet. The colony of strain Y5 was white, suborbicular and moist on LB tablet. Strains J2 and Y5 respectively obtained one about 1500 bp band by amplifying 16S rDNA sequences, and by comparing strains J2 and Y5 showed the closest similarity of 16S rDNA sequences to Pantoea agglomerans STY29(HQ220151) andPseudomonas fluorescens V7c10(KC

  5. Production of Highly Sialylated Recombinant Glycoproteins Using Ricinus communis Agglutinin-I-Resistant CHO Glycosylation Mutants.

    Science.gov (United States)

    Goh, John S Y; Chan, Kah Fai; Song, Zhiwei

    2015-01-01

    The degree of sialylation of therapeutic glycoproteins affects its circulatory half-life and efficacy because incompletely sialylated glycoproteins are cleared from circulation by asialoglycoprotein receptors present in the liver cells. Mammalian expression systems, often employed in the production of these glycoprotein drugs, produce heterogeneously sialylated products. Here, we describe how to produce highly sialylated glycoproteins using a Chinese hamster ovary (CHO) cell glycosylation mutant called CHO-gmt4 with human erythropoietin (EPO) as a model glycoprotein. The protocol describes how to isolate and characterize the CHO glycosylation mutants and how to assess the sialylation of the recombinant protein using isoelectric focusing (IEF). It further describes how to inactivate the dihydrofolate reductase (DHFR) gene in these cells using zinc finger nuclease (ZFN) technology to enable gene amplification and the generation of stable cell lines producing highly sialylated EPO.

  6. Gas phase UV and IR absorption spectra of CxF2x+1CHO (x=1-4)

    DEFF Research Database (Denmark)

    Hashikawa, Y; Kawasaki, M; Waterland, RL;

    2004-01-01

    The UV and IR spectra of CxF2x+1 CHO (x = 1-4) were investigated using computational and experimental techniques. CxF2x+1CHO (x = 1-4) have broad UV absorption features centered at 300-310 nm. The maximum absorption cross-section increases significantly and shifts slightly to the red with increas...

  7. Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

    DEFF Research Database (Denmark)

    Lee, Jae Seong; Beuchert Kallehauge, Thomas; Pedersen, Lasse Ebdrup;

    2015-01-01

    gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following...

  8. Mannose metabolism in recombinant CHO cells and its effect on IgG glycosylation.

    Science.gov (United States)

    Slade, Peter G; Caspary, R Guy; Nargund, Shilpa; Huang, Chung-Jr

    2016-07-01

    Understanding the causes of high-mannose (HM) glycosylation of recombinant IgG in CHO cells would facilitate the production of therapeutics. CHO cells grown with mannose as the major carbon source demonstrated a dramatic increase in total HM glycosylation in recombinant IgG, with no effect on cell growth, viability, or titer. Quantitative metabolomics and (13) C flux analysis were used to explore the mechanism for increased HM glycosylation and understand the metabolism of mannose in CHO cells. It was demonstrated that mannose was a good carbon source for CHO cell growth and IgG production, readily entering both glycolysis and the TCA Cycle. Previous mechanisms for increased HM glycosylation during antibody production have been attributed to changes in pH, osmolality, increased specific productivity, and nutrient limitation. The results from this study propose a novel mechanism where an increased carbon flux in the GDP-mannose synthetic pathway increased the intracellular concentration of mannose-containing metabolites. The abnormally high concentration of mannose and mannose-metabolites were shown to inhibit α-mannosidase activity and it was proposed that this inhibition in the ER and Golgi caused the production of IgG with increased high-mannose glycosylation. Biotechnol. Bioeng. 2016;113: 1468-1480. © 2016 Wiley Periodicals, Inc. PMID:26724786

  9. A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40: gene cloning and sequencing

    Digital Repository Service at National Institute of Oceanography (India)

    Khandeparker, R.; Verma, P.; Deobagkar, D.

    induced (140%) when pre-incubated with 0.5 M NaCl for 4 h. The xylanase gene, xyn40, from marine bacterium B. subtilis cho40 was cloned, and expressed in Escherichia coli. The xylanase gene was 645 bp long and had a 215 amino acid ORF protein with a...

  10. BEHAVIOR OF CHO CELLS ON MODIFIED POLYPROPYLENE BY LOW TEMPERATURE AMMONIA PLASMA

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hong; YU Yaoting; PAN Jilun; XU Yuanping; ZHU Hesun

    2001-01-01

    The surface of polypropylene (PP) membrane was modified by low temperature plasma with ammonia. The effect of exposure time was investigated by means of contact angle measurement. The results show that low temperature ammonia plcsma treatment can enhance its hydrophilicity. Chinese hamster ovary (CHO) cells attachment on the modified membrane was enhanced and the growth rate on the membrane was faster than unmodified one.

  11. On Certain Class of Analytic Functions Related to Cho-Kwon-Srivastava Operator

    Directory of Open Access Journals (Sweden)

    F. Ghanim

    2011-01-01

    Full Text Available Motivated by a multiplier transformation and some subclasses of meromorphic functions which were defined by means of the Hadamard product of the Cho-Kwon-Srivastava operator, we define here a similar transformation by means of the Ghanim and Darus operator. A class related to this transformation will be introduced and the properties will be discussed.

  12. Hemoglobin variants in Cyprus.

    Science.gov (United States)

    Kyrri, Andreani R; Felekis, Xenia; Kalogerou, Eleni; Wild, Barbara J; Kythreotis, Loukas; Phylactides, Marios; Kleanthous, Marina

    2009-01-01

    Cyprus, located at the eastern end of the Mediterranean region, has been a place of eastern and western civilizations, and the presence of various hemoglobin (Hb) variants can be considered a testimony to past colonizations of the island. In this study, we report the structural Hb variants identified in the Cypriot population (Greek Cypriots, Maronites, Armenians, and Latinos) during the thalassemia screening of 248,000 subjects carried out at the Thalassaemia Centre, Nicosia, Cyprus, over a period of 26 years. A sample population of 65,668 people was used to determine the frequency and localization of several of the variants identified in Cyprus. The localization of some of the variants in regions where the presence of foreign people was most prevalent provides important clues to the origin of the variants. Twelve structural variants have been identified by DNA sequencing, nine concerning the beta-globin gene and three concerning the alpha-globin gene. The most common beta-globin variants identified were Hb S (0.2%), Hb D-Punjab (0.02%), and Hb Lepore-Washington-Boston (Hb Lepore-WB) (0.03%); the most common alpha-globin variant was Hb Setif (0.1%). The presence of some of these variants is likely to be directly linked to the history of Cyprus, as archeological monuments have been found throughout the island which signify the presence for many years of the Greeks, Syrians, Persians, Arabs, Byzantines, Franks, Venetians, and Turks. PMID:19373583

  13. A proteomic study of cMyc improvement of CHO culture

    Directory of Open Access Journals (Sweden)

    Dunn Michael J

    2010-03-01

    Full Text Available Abstract Background The biopharmaceutical industry requires cell lines to have an optimal proliferation rate and a high integral viable cell number resulting in a maximum volumetric recombinant protein product titre. Nutrient feeding has been shown to boost cell number and productivity in fed-batch culture, but cell line engineering is another route one may take to increase these parameters in the bioreactor. The use of CHO-K1 cells with a c-myc plasmid allowing for over-expressing c-Myc (designated cMycCHO gives a higher integral viable cell number. In this study the differential protein expression in cMycCHO is investigated using two-dimensional gel electrophoresis (2-DE followed by image analysis to determine the extent of the effect c-Myc has on the cell and the proteins involved to give the new phenotype. Results Over 100 proteins that were differentially expressed in cMycCHO cells were detected with high statistical confidence, of which 41 were subsequently identified by tandem mass spectrometry (LC-MS/MS. Further analysis revealed proteins involved in a variety of pathways. Some examples of changes in protein expression include: an increase in nucleolin, involved in proliferation and known to aid in stabilising anti-apoptotic protein mRNA levels, the cytoskeleton and mitochondrial morphology (vimentin, protein biosysnthesis (eIF6 and energy metabolism (ATP synthetase, and a decreased regulation of all proteins, indentified, involved in matrix and cell to cell adhesion. Conclusion These results indicate several proteins involved in proliferation and adhesion that could be useful for future approaches to improve proliferation and decrease adhesion of CHO cell lines which are difficult to adapt to suspension culture.

  14. Cell-free protein expression based on extracts from CHO cells.

    Science.gov (United States)

    Brödel, Andreas K; Sonnabend, Andrei; Kubick, Stefan

    2014-01-01

    Protein expression systems are widely used in biotechnology and medicine for the efficient and economic production of therapeutic proteins. Today, cultivated Chinese hamster ovary (CHO) cells are the market dominating mammalian cell-line for the production of complex therapeutic proteins. Despite this outstanding potential of CHO cells, no high-yield cell-free system based on translationally active lysates from these cells has been reported so far. To date, CHO cell extracts have only been used as a foundational research tool for understanding mRNA translation (Lodish et al., 1974; McDowell et al., 1972). In the present study, we address this fact by establishing a novel cell-free protein expression system based on extracts from cultured CHO cells. Lysate preparation, adaptation of in vitro reaction conditions and the construction of particular expression vectors are considered for high-yield protein production. A specific in vitro expression vector, which includes an internal ribosome entry site (IRES) from the intergenic region (IGR) of the Cricket paralysis virus (CrPV), has been constructed in order to obtain optimal performance. The IGR IRES is supposed to bind directly to the eukaryotic 40S ribosomal subunit thereby bypassing the process of translation initiation, which is often a major bottleneck in cell-free systems. The combination of expression vector and optimized CHO cell extracts enables the production of approximately 50 µg/mL active firefly luciferase within 4 h. The batch-type cell-free coupled transcription-translation system has the potential to perform post-translational modifications, as shown by the glycosylation of erythropoietin. Accordingly, the system contains translocationally active endogenous microsomes, enabling the co-translational incorporation of membrane proteins into biological membranes. Hence, the presented in vitro translation system is a powerful tool for the fast and convenient optimization of expression constructs, the

  15. Synthesis of human prolactin in Chinese hamster ovary (CHO) cells; Sintese de prolactina humana em celulas de ovario de hamster chines (CHO)

    Energy Technology Data Exchange (ETDEWEB)

    Soares, Carlos Roberto Jorge

    2000-07-01

    Three different eukaryotic expression vectors, based on the same selectable gene marker (dhfr), have been used for dhf- CHO cells transfection to rapidly isolate stable cell lines capable of secreting high levels of recombinant human prolactin (rec-hPRL). Two vectors, one codifying a human prolactin (p658-hPRL) and the other a tag-prolactin (p658-tagPRL), contain the complete hepatitis B virus-X (HBV-X) gene coding for a viral transactivator and a sequence derived from the granulocyte-macrophage colony-stimulating factor (GM-CSF) that mediates selective dhfr mRNA degradation. These vectors have the advantage of rapidly obtaining stable cell lines without methotrexate amplification. The highest secretion obtained by these vectors was of approximately 10 {mu}g hPRU10{sup 6} cells/day. The other vector (pEDdc-hPRL) is based on a dicistronic expression system, containing an internal ribosome entry site isolated from the encephalomyocarditis (EMC) virus. This vector before amplification provided secretion levels at least 10 fold lower than that obtained with the other two vectors. However, after three steps of methotrexate amplification, it provided some clones able to secrete up to 30 {mu}g hPRU10{sup 6} cells/day. This is the first report describing the production and purification of rec-hPRL from CHO cells, obtaining secretion levels with both vectors higher than those reported so far for this hormone in other eukaryotic systems. CHO-derived rec-hPRL contained approximately 10 % of the glycosylated form, a value that is consistent with results reported for hPRL purified from the pituitary or from transformed murine C-127 cells. CHO-derived rec-hPRL was purified with good yield, obtaining also a good resolution between non-glycosylated and glycosylated prolactin. The latter, when its potency was determined via an in vitro bioassay, presented a 47 % lower bioactivity. A qualitative and quantitative analysis of these forms was also possible thanks to the setting up of a

  16. Quantitative analysis of the supernatant from host and transfected CHO cells using iTRAQ 8-plex technique.

    Science.gov (United States)

    Zhu, Guijie; Sun, Liangliang; Albanetti, Thomas; Linkous, Travis; Larkin, Christopher; Schoner, Ronald; McGivney, James B; Dovichi, Norman J

    2016-10-01

    We employed UPLC-MS/MS with iTRAQ 8-plex labeling to quantitatively analyze the supernatant produced by two Chinese hamster ovary (CHO) cell lines (CHO K1SV and CHO CAT-S). In each case, the supernatant from the host and three transfected clones were analyzed at days 5, 7, and 10 of culture. A total of eight iTRAQ 8-plex experiments were performed. For each cell line, the overlap of supernatant protein identifications between transfected clones is over 60%. Over 70% of the supernatant proteins in the CHO K1SV host cell line are present in the CHO CAT-S cell line. For the CHO K1SV cell line, the overlap in supernatant protein identifications between the host cell line and the transfected clones is >59%. For the CHO CAT-S cell line, the overlap between supernatant protein identifications for the transfected clone and host cell is >45%. These differences in the supernatant protein identifications between transfected clones in each cell line and between the two host cell lines are not significant. We used cluster analysis to characterize the change in supernatant protein expression as a function of cell culture time. Roughly 1.3 or clones at each time point. Greater than 65% of the common proteins in the CHO K1SV cell line supernatant and over 54% in the CHO CAT-S cell line supernatant show no significant expression difference between host and the three transfected clones. Data are available via ProteomeXchange with identifier PXD003462. Biotechnol. Bioeng. 2016;113: 2140-2148. © 2016 Wiley Periodicals, Inc. PMID:27070921

  17. Pyrolysis of the Simplest Carbohydrate, Glycolaldehyde (CHO-CH2OH), and Glyoxal in a Heated Microreactor.

    Science.gov (United States)

    Porterfield, Jessica P; Baraban, Joshua H; Troy, Tyler P; Ahmed, Musahid; McCarthy, Michael C; Morgan, Kathleen M; Daily, John W; Nguyen, Thanh Lam; Stanton, John F; Ellison, G Barney

    2016-04-14

    Both glycolaldehyde and glyoxal were pyrolyzed in a set of flash-pyrolysis microreactors. The pyrolysis products resulting from CHO-CH2OH and HCO-CHO were detected and identified by vacuum ultraviolet (VUV) photoionization mass spectrometry. Complementary product identification was provided by argon matrix infrared absorption spectroscopy. Pyrolysis pressures in the microreactor were about 100 Torr, and contact times with the microreactors were roughly 100 μs. At 1200 K, the products of glycolaldehyde pyrolysis are H atoms, CO, CH2═O, CH2═C═O, and HCO-CHO. Thermal decomposition of HCO-CHO was studied with pulsed 118.2 nm photoionization mass spectrometry and matrix infrared absorption. Under these conditions, glyoxal undergoes pyrolysis to H atoms and CO. Tunable VUV photoionization mass spectrometry provides a lower bound for the ionization energy (IE)(CHO-CH2OH) ≥ 9.95 ± 0.05 eV. The gas-phase heat of formation of glycolaldehyde was established by a sequence of calorimetric experiments. The experimental result is ΔfH298(CHO-CH2OH) = -75.8 ± 1.3 kcal mol(-1). Fully ab initio, coupled cluster calculations predict ΔfH0(CHO-CH2OH) of -73.1 ± 0.5 kcal mol(-1) and ΔfH298(CHO-CH2OH) of -76.1 ± 0.5 kcal mol(-1). The coupled-cluster singles doubles and noniterative triples correction calculations also lead to a revision of the geometry of CHO-CH2OH. We find that the O-H bond length differs substantially from earlier experimental estimates, due to unusual zero-point contributions to the moments of inertia. PMID:26979134

  18. Cytotoxicity of acrylamide and its epoxide glycidamide in CHO cells expressing human cytochrome P450 2E1

    Institute of Scientific and Technical Information of China (English)

    Shoulin Wang; Xiaoyang He; Xinru Wang; Junyan Hong

    2006-01-01

    Objective: To investigate whether CYP2E1 is responsible for the acrylamide metabolic activation in Flp-In CHO cell system. Methods: CYP2E1 cDNA was subcloned from the human liver full-length cDNA library and subsequently transfected into the Flp-In CHO cells to generate the stable transfectant of CYP2E1. The CYP2E1 mRNA expression was determined by RT-PCR. Acrylamide and its epoxide glycidamide induced cytotoxicity and cell cycle arrest in G2/M were conducted using MTS assay and flow cytometry, respectively. Results: In the CHO cell stably expressing CYP2E1 (CHO-2E1), a ~1.5 kbsize of band was detected from the mRNA in the cells while no corresponding band in the CHO-vector cells, which indicated that CYP2E1 was successfully transfected in the CHO cells. Compared with the CHO-vector cells, acrylamide showed a concentrationdependent loss of viability in the CHO-2E1 cells but no significant change of G2/M arrest was found. As expected, glycidamide induced similar profile of cytotoxicity in both of the cells, and G2/M arrest presented a concentration-dependent increased in the CHO-2E1 cells. Conclusion: The result suggested that CYP2E1 might be responsible for the acrylamide metabolism, and its metabolite glycidamide was a direct cytotoxic and genotoxic agent. It should be further considered whether acrylamide-induced toxicity is through its epoxide glycidamide in the presence of CYP2E1.

  19. Human cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells

    DEFF Research Database (Denmark)

    Merz, G S; Benedikz, Eirikur; Schwenk, V;

    1997-01-01

    in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half-life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely...... dimeric cystatin C and immunoreactive material, > or = 70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C indicating...... that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin...

  20. Stable Expression of Recombinant Factor VIII in CHO Cells Using Methotrexate-Driven Transgene Amplification

    OpenAIRE

    Orlova, N.; Kovnir, S.; Vorobiev, I.; Yuriev, A.; Gabibov, A.; Vorobiev, A.

    2012-01-01

    Prophylaxis and treatment of inherited clotting disorder hemophilia A requires regular administration of factor VIII. Recombinant factor VIII, which is produced in CHO or BHK cells, is equivalent to the plasma derived one and is prevalent in current clinical practice in developed countries. Development of a biosimilar recombinant FVIII requires the creation of a highly productive clonal cell line and generation of monoclonal antibodies suitable for affinity purification of the product. Methot...

  1. BEHAVIOR OF CHO CELLS ON MODIFIED POLYPROPYLENE BY LOW TEMPERATURE AMMONIA PLASMA

    Institute of Scientific and Technical Information of China (English)

    ZHANGHong; ZHUHesun; 等

    2001-01-01

    The surface of polypropylene(PP) membrane was modified by low temperature plasma with ammonia.The effect of exposure time was investigated by means of contact angle measurement.The results show that low temperature ammonia plasma treatment can enhance its hydrophilicity.Chinese hamster ovary(CHO)cells attachment on the modified membrane was enhanced and the growth rate on the membrane was faster than unmodified one.

  2. Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells.

    Directory of Open Access Journals (Sweden)

    Qi He

    Full Text Available The endocytosis of transferrin receptor (TfR has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

  3. Silver nanoparticle induced cytotoxicity, oxidative stress, and DNA damage in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Awasthi, Kumud Kant [University of Rajasthan, Department of Zoology (India); Awasthi, Anjali; Kumar, Narender; Roy, Partha [Indian Institute of Technology Roorkee, Department of Biotechnology (India); Awasthi, Kamlendra, E-mail: kamlendra.awasthi@gmail.com [Malaviya National Institute of Technology, Department of Physics (India); John, P. J., E-mail: placheriljohn@yahoo.com [University of Rajasthan, Department of Zoology (India)

    2013-09-15

    Silver nanoparticles (Ag NPs) are being used increasingly in wound dressings, catheters, and in various household products due to their antimicrobial activity. The present study reports the toxicity evaluation of synthesized and well characterized Ag NPs using Chinese hamster ovary (CHO) cells. The UV-Vis spectroscopy reveals the formation of silver nanoparticles by exhibiting the typical surface plasmon absorption maxima at 408-410 nm. Transmission electron microscopy (TEM) reveals that the average diameter of silver nanoparticles is about 5.0 {+-} 1.0 nm and that they have spherical shape. Cell visibility and cell viability percentage show dose-dependent cellular toxicity of Ag NPs. The half maximal inhibitory concentration (IC{sub 50}) for CHO cells is 68.0 {+-} 2.65 {mu}g/ml after 24 h Ag NPs exposure. Toxicity evaluations, including cellular morphology, mitochondrial function (MTT assay), reactive oxygen species (ROS), and DNA fragmentation assay (Ladder pattern) were assessed in unexposed CHO cells (control) and the cells exposed to Ag NPs concentrations of 15, 30, and 60 {mu}g/ml for 24 h. The findings may assist in the designing of Ag NPs for various applications and provide insights into their toxicity.

  4. Silver nanoparticle induced cytotoxicity, oxidative stress, and DNA damage in CHO cells

    International Nuclear Information System (INIS)

    Silver nanoparticles (Ag NPs) are being used increasingly in wound dressings, catheters, and in various household products due to their antimicrobial activity. The present study reports the toxicity evaluation of synthesized and well characterized Ag NPs using Chinese hamster ovary (CHO) cells. The UV–Vis spectroscopy reveals the formation of silver nanoparticles by exhibiting the typical surface plasmon absorption maxima at 408–410 nm. Transmission electron microscopy (TEM) reveals that the average diameter of silver nanoparticles is about 5.0 ± 1.0 nm and that they have spherical shape. Cell visibility and cell viability percentage show dose-dependent cellular toxicity of Ag NPs. The half maximal inhibitory concentration (IC50) for CHO cells is 68.0 ± 2.65 μg/ml after 24 h Ag NPs exposure. Toxicity evaluations, including cellular morphology, mitochondrial function (MTT assay), reactive oxygen species (ROS), and DNA fragmentation assay (Ladder pattern) were assessed in unexposed CHO cells (control) and the cells exposed to Ag NPs concentrations of 15, 30, and 60 μg/ml for 24 h. The findings may assist in the designing of Ag NPs for various applications and provide insights into their toxicity

  5. In what time scale proton transfer takes place in a live CHO cell?

    Science.gov (United States)

    Mojumdar, Supratik Sen; Chowdhury, Rajdeep; Mandal, Amit Kumar; Bhattacharyya, Kankan

    2013-06-01

    Excited state proton transfer (ESPT) of pyranine (8-hydroxypyrene-1,3,6-trisulfonate, HPTS) in a live Chinese hamster ovary (CHO) cell is studied by time resolved confocal microscopy. The cytoplasm region of the cell is stained by a photoacid, HPTS (HA). The time constant of initial proton transfer (τPT) in the cell is found to be ˜10 times longer than that in bulk water, while the time constants of recombination (τrec) and dissociation (τdiss) in the cell are ˜3 times and ˜2 times longer, respectively. The slower rate of proton transfer (˜10 times) inside the CHO cell compared to that in bulk water is ascribed to slower solvation dynamics, lower availability of free water molecules, and disruption of hydrogen-bond network inside the cell. Translational and rotational diffusion of HPTS inside a single CHO cell have been investigated by fluorescence correlation spectroscopy (FCS) and picosecond anisotropy measurement, respectively. Both the translational and rotational diffusion slow down inside the live cell. FCS studies indicate that HPTS remains tightly bound to a macromolecule inside the cell.

  6. Rapid amplification system for recombinant protein production in Chinese Hamster Ovary (CHO) Cells.

    Science.gov (United States)

    Metta, M K; Kunaparaju, R K; Tantravahi, S

    2016-01-01

    Recombinant therapeutic proteins have changed the face of modern medicine in the present trend and they continue to provide innovative therapies for deadly diseases. This study describes the development of a novel stable expression system for rapid amplification of genes in Chinese Hamster Ovary (CHO) cells. The expression system consists of a host CHO cell line and an expression vector (pUB-PyOri-D-C) which encodes for Polyomavirus (Py) Origin of Replication (PyOri) for amplification of integrated genes in the presence of Py Large T Antigen (PyLT) and Dihydrofolate Reductase (DHFR) selectable marker gene for selection in the presence of Methotrexate (MTX). Use of both PyOri/PyLT and DHFR can reduce the number of rounds of selection and amplification required for isolation of high producing clones. The efficiency of pUB-PyOri-D-C was compared with that of pUB-D-C plasmid using Green fluorescent protein (GFP) and Erythropoietin (EPO) as reporter proteins. Our results showed that pUB-PyOri-D-C-EPO can help development of high expressing clone in one round of selection/amplification as compared to multiple rounds of selection/amplification with pUB-D-C-EPO plasmid. CHO-DG44/EPO clone generated using pUB-PyOri-D-C-EPO gave a productivity of 119 mg/L in shake flask. PMID:26950459

  7. Precision control of recombinant gene transcription for CHO cell synthetic biology.

    Science.gov (United States)

    Brown, Adam J; James, David C

    2016-01-01

    The next generation of mammalian cell factories for biopharmaceutical production will be genetically engineered to possess both generic and product-specific manufacturing capabilities that may not exist naturally. Introduction of entirely new combinations of synthetic functions (e.g. novel metabolic or stress-response pathways), and retro-engineering of existing functional cell modules will drive disruptive change in cellular manufacturing performance. However, before we can apply the core concepts underpinning synthetic biology (design, build, test) to CHO cell engineering we must first develop practical and robust enabling technologies. Fundamentally, we will require the ability to precisely control the relative stoichiometry of numerous functional components we simultaneously introduce into the host cell factory. In this review we discuss how this can be achieved by design of engineered promoters that enable concerted control of recombinant gene transcription. We describe the specific mechanisms of transcriptional regulation that affect promoter function during bioproduction processes, and detail the highly-specific promoter design criteria that are required in the context of CHO cell engineering. The relative applicability of diverse promoter development strategies are discussed, including re-engineering of natural sequences, design of synthetic transcription factor-based systems, and construction of synthetic promoters. This review highlights the potential of promoter engineering to achieve precision transcriptional control for CHO cell synthetic biology. PMID:26721629

  8. Mucopolysaccharidosis: A New Variant?

    Science.gov (United States)

    Primrose, D. A.

    1972-01-01

    Described is a possibly new variant of mucopolysaccharidosis characterized by progressive mental and motor deficiency, bone abnormalities, a generalized skin lesion, and abnormal mucopolysaccharides in the urine as seen in a 20-year-old female. (DB)

  9. Some Applications of Differential Subordination of p-Valent Functions Associated with Cho-Kwon-Srivastava Operator

    Institute of Scientific and Technical Information of China (English)

    A. O. MOSTAFA; M.K. AOUF

    2009-01-01

    Making use of the principle of differential subordination, we investigate some inclusion relationships of certain subclasses of p-valent analytic functions which are defined by Cho-Kwon-Srivastava Operator.

  10. Amino acid consumption in naïve and recombinant CHO cell cultures: producers of a monoclonal antibody

    OpenAIRE

    Carrillo-Cocom, L. M.; Genel-Rey, T.; Araíz-Hernández, D.; López-Pacheco, F.; López-Meza, J.; Rocha-Pizaña, M. R.; Ramírez-Medrano, A.; Alvarez, M. M.

    2014-01-01

    Most commercial media for mammalian cell culture are designed to satisfy the amino acid requirements for cell growth, but not necessarily those for recombinant protein production. In this study, we analyze the amino acid consumption pattern in naïve and recombinant Chinese hamster ovary (CHO) cell cultures. The recombinant model we chose was a CHO-S cell line engineered to produce a monoclonal antibody. We report the cell concentration, product concentration, and amino acid concentration prof...

  11. One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment

    DEFF Research Database (Denmark)

    Grav, Lise Marie; Lee, Jae Seong; Thomsen, Signe Gerling;

    2015-01-01

    The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously....... Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further....

  12. Hemoglobin Variants in Mice

    Energy Technology Data Exchange (ETDEWEB)

    Popp, Raymond A.

    1965-04-22

    Variability among mammalian hemoglobins was observed many years ago (35). The chemical basis for differences among hemoglobins from different species of mammals has been studied by several investigators (5, 11, 18, 48). As well as interspecies differences, hemoglobin variants are frequently found within a species of mammals (2, 3, 7, 16) The inheritance of these intraspecies variants can be studied, and pedigrees indicate that the type of hemoglobin synthesized in an individual is genetically controlled (20). Several of the variant human hemoglobins are f'unctionally deficient (7, 16). Such hemoglobin anomalies are of basic interest to man because of the vital role of hemoglobin for transporting oxygen to all tissues of the body.

  13. Components of yeast (Sacchromyces cervisiae) extract as defined media additives that support the growth and productivity of CHO cells.

    Science.gov (United States)

    Spearman, Maureen; Chan, Sarah; Jung, Vince; Kowbel, Vanessa; Mendoza, Meg; Miranda, Vivian; Butler, Michael

    2016-09-10

    Yeast and plant hydrolysates are used as media supplements to support the growth and productivity of CHO cultures for biopharmaceutical production. Through fractionation of a yeast lysate and metabolic analysis of a fraction that had bioactivity equivalent to commercial yeast extract (YE), bioactive components were identified that promoted growth and productivity of two recombinant CHO cell lines (CHO-Luc and CHO-hFcEG2) equivalent to or greater than YE-supplemented media. Autolysis of the yeast lysate was not necessary for full activity, suggesting that the active components are present in untreated yeast cells. A bioactive fraction (3KF) of the yeast lysate was isolated from the permeate using a 3kDa molecular weight cut-off (MWCO) filter. Supplementation of this 3KF fraction into the base media supported growth of CHO-Luc cells over eight passages equivalent to YE-supplemented media. The 3KF fraction was fractionated further by a cation exchange spin column using a stepwise pH elution. Metabolomic analysis of a bioactive fraction isolated at high pH identified several arginine and lysine-containing peptides as well as two polyamines, spermine and spermidine, with 3.5× and 4.5× higher levels compared to a fraction showing no bioactivity. The addition of a mixture of polyamines and their precursors (putrescine, spermine, spermidine, ornithine and citrulline) as well as increasing the concentration of some of the components of the original base medium resulted in a chemically-defined (CD) formulation that produced an equivalent viable cell density (VCD) and productivity of the CHO-Luc cells as the YE-supplemented medium. The VCD of the CHO-hFcEG2 culture in the CD medium was 1.9× greater and with equivalent productivity to the YE-supplemented media.

  14. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells.

    Science.gov (United States)

    Chng, Jake; Wang, Tianhua; Nian, Rui; Lau, Ally; Hoi, Kong Meng; Ho, Steven C L; Gagnon, Peter; Bi, Xuezhi; Yang, Yuansheng

    2015-01-01

    Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing. PMID:25621616

  15. Citotoxicidad del fungicida mancozeb en cultivos de CHO-K1

    OpenAIRE

    A.E. Bayoumi; Ordóñez, C.; Y. Pérez Pertejo; R. Balaña Fouce; Ordóñez, D.

    2002-01-01

    Se ha determinado la citotoxicidad del fungicida ditiocarbámico mancozeb, en cultivos celulares de ovario de hámster (CHO-K1), usando los bioensayos estandarizados de incorporación de rojo neutro (RN) y del contenido total de proteínas (PT). Las dos técnicas mostraron ser comparables en la determinación del efecto citotóxico, mostrando valores de RN50 menores de 15 mg/ml después de 24 h de exposición al plaguicida. La citotoxicidad fue mayor cuanto mayor fue el tiempo ...

  16. Direct Dynamics Study on CH2O + CH·3 → CHO + CH4 Reaction

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    It is still a formidable challenge to study CH2O + CH·3 → CHO + CH4 reaction in the gas phase by traditional dynamics, because of the large number of freedom degrees for the system.In this paper, direct dynamics, in which trajectories were run directly on the DFT potential energy surface, have been applied to the reaction, which gave a direct look in the reaction processes.Two sets of trajectories at different initial orientations of reactants and temperature have been simulated. And the detailed reaction mechanisms have been described.

  17. C-terminal KDEL-modified cystatin C is retained in transfected CHO cells

    DEFF Research Database (Denmark)

    Johansen, Teit Eliot; Vogel, Charlotte Katrine; Schwartz, Thue W.

    1990-01-01

    The significance of a C-terminal tetrapeptide, Lys-Asp-Glu-Leu (KDEL), as a retention signal for the endoplasmatic reticulum was studied using cystatin C, a general thiol protease inhibitor, as the reporter protein. Clones of CHO cells were analyzed after stable transfection with eukaryotic...... expression vectors encoding either cystatin C, KDEL extended cystatin C, or cystatin C extended with a control sequence. It is concluded that cystatin C with the KDEL tetrapeptide as a C-terminal extension is retained intracellularly without apparent accumulation of the molecule....

  18. Enhancement of monoclonal antibody production in CHO cells by exposure to He–Ne laser radiation

    OpenAIRE

    Ghaleb, Rana; Naciri, Mariam; Al-Majmaie, Rasoul; Maki, Amel; Al-Rubeai, Mohamed

    2013-01-01

    This study tested the effectiveness of laser biostimulation in small-scale cultures in vitro. We investigated the response of recombinant CHO cells, which are used for the production of monoclonal antibody, to low level laser radiation. The cells were irradiated using a 632.8 nm He–Ne laser in a continuous wave mode at different energy doses. We incubated the irradiated cells in small batch cultures and assessed their proliferation and productivity at various time intervals. Compared to untre...

  19. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

    OpenAIRE

    Chng, Jake; Wang, Tianhua; Nian, Rui; Lau, Ally; Hoi, Kong Meng; Ho, Steven CL; Gagnon, Peter; Bi, Xuezhi; Yang, Yuansheng

    2015-01-01

    Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC ...

  20. Cross-cultural adaptation of the CHO-KLAT for boys with hemophilia in rural and urban china

    Directory of Open Access Journals (Sweden)

    Wu Runhui

    2012-09-01

    Full Text Available Abstract Background Quality of life (QoL is increasingly recognized as an important outcome measure in clinical trials. The Canadian Hemophilia Outcomes-Kids Life Assessment Tool (CHO-KLAT shows promise for use in China. Objective To adapt the CHO-KLAT version 2.0 for use in clinical trials in China. Methods Forward and back translations of the CHO-KLAT2.0 were completed in 2008. Between October 2009 and June 2010, a series of 3 focus groups were held with 20 boys and 31 parents in rural and urban China to elicit additional concepts, important to their QoL, for the Chinese CHO-KLAT2.0. All of the items identified by boys and parents were reviewed by a group of experts, resulting in a Chinese version of the CHO-KLAT2.0. This version underwent a detailed cognitive debriefing process between October 2010 and June 2011. Thirteen patient-parent pairs participated in this cognitive debriefing process until a stable and clearly understood Chinese version of the CHO-KLAT2.0 was obtained. Results The initial back translation of the Chinese CHO-KLAT2.0 was slightly discrepant from the original English version on 12 items. These were all successfully adjudicated. The focus groups identified 9 new items that formed an add-on Socio-Economic Context (SEC module for China. Linguistic improvements were made after the 2nd, 5th, 7th and 13th cognitive debriefings pairs and affected a total of 18 items. The result was a 35 item CHO-KLAT2.0 and a SEC module in Simplified Chinese, both of which have good content validity. Conclusion This detailed process proved to be extremely valuable in ensuring the items were accurately interpreted by Chinese boys with hemophilia ages ≤18 years. The need for the additional SEC module highlighted the different context that currently exists in China with regard to hemophilia care as compared to many Western countries, and will be important in tracking progress within both rural and urban China over time. Changes based on the

  1. Identifying the differences in mechanisms of mycophenolic acid controlling fucose content of glycoproteins expressed in different CHO cell lines.

    Science.gov (United States)

    Zhang, An; Tsang, Valerie Liu; Markely, Lam R; Kurt, Lutfiye; Huang, Yao-Ming; Prajapati, Shashi; Kshirsagar, Rashmi

    2016-11-01

    In the biopharmaceutical industry, glycosylation is a critical quality attribute that can modulate the efficacy of a therapeutic glycoprotein. Obtaining a consistent glycoform profile is desired because molecular function can be defined by its carbohydrate structures. Specifically, the fucose content of oligosaccharides in glycoproteins is one of the most important attributes that can significantly affect antibody-dependent cellular cytotoxicity (ADCC) activity. It is therefore important to understand the fucosylation pathway and be able to control fucosylation at the desired level to match predecessor materials in late stage and biosimilar programs. Several strategies were explored in this study and mycophenolic acid (MPA) was able to finely modulate the fucose content with the least undesired side effects. However, the response was significantly different between CHO cell lines of different lineages. Further experiments were then performed for a deeper understanding of the mechanism of fucosylation in different CHO cell lines. Results indicated that changes in the intracellular nucleotide involved in fucosylation pathway after MPA treatment are the main cause of the differences in fucosylation level response in different CHO cell lines. Differences in MPA metabolism in the various CHO cell lines directly resulted in different levels of afucosylation measured in antibodies produced by the CHO cell lines. Biotechnol. Bioeng. 2016;113: 2367-2376. © 2016 Wiley Periodicals, Inc.

  2. Potential bone-inducing activity in vitro of recombinant human bone morphogenetic protein-7 from a CHO expression system

    Institute of Scientific and Technical Information of China (English)

    LI Xiao-yan; SHI Wei-wei; WANG Hao; LI Bo-hua; YANG Yang; TAN Min; XUE Jing-ya; GUO Ya-jun

    2005-01-01

    Objective: To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary(CHO) cells, and to establish the in vitro biological activity assay of rhBMP-7.Methods: Human BMP-7 cDNA was subcloned into p114 mammalian expression vector and transfected to CHO cells by using the Lipofectamine2000 transfection method. CHO cell supernatants were harvested and analyzed to identify the molecule mass of secreted rhBMP-7 and examine its biological activity in vitro to stimulate the synthesis of alkaline phophatase(ALP), a characteristic of osteoblast phenotypes. Results:rhBMP-7 was produced stably in CHO cells, as a processed mature disulfide-linked homodimer, with an apparent molecular mass of 36 000. Examination of the rhBMP-7 biological activity showed that rhBMP-7 specifically stimulated the production of ALP(4-fold increase at 100 ng of rhBMP-7/ml). Conclusion: The rhBMP-7 from CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates rhBMP-7 has the potential bone regeneration activity.

  3. Tracking dipeptides at work-uptake and intracellular fate in CHO culture.

    Science.gov (United States)

    Sánchez-Kopper, Andres; Becker, Max; Pfizenmaier, Jennifer; Kessler, Christian; Karau, Andreas; Takors, Ralf

    2016-12-01

    Market demands for monoclonal antibodies (mAbs) are steadily increasing worldwide. As a result, production processes using Chinese hamster ovary cells (CHO) are in the focus of ongoing intensification studies for maximizing cell-specific and volumetric productivities. This includes the optimization of animal-derived component free (ADCF) cultivation media as part of good cell culture practice. Dipeptides are known to improve CHO culture performance. However, little or even conflicting assumptions exist about their putative import and functionality inside the cells. A set of well-known performance boosters and new dipeptide prospects was evaluated. The present study revealed that dipeptides are indeed imported in the cells, where they are decomposed to the amino acids building blocks. Subsequently, they are metabolized or, unexpectedly, secreted to the medium. Monoclonal antibody production boosting additives like L-alanine-L-glutamine (AQ) or glycyl-L-glutamine (GQ) can be assigned to fast or slow dipeptide uptake, respectively, thus pinpointing to the need to study dipeptide kinetics and to adjust their feeding individually for optimizing mAb production. PMID:27447702

  4. H-atom bombardment of CO2, HCOOH and CH3CHO containing ices

    CERN Document Server

    Bisschop, S E; Van Dishoeck, E F; Linnartz, H

    2007-01-01

    Context: Hydrogenation reactions are expected to be among the most important surface reactions on interstellar ices. However, solid state astrochemical laboratory data on reactions of H-atoms with common interstellar ice constituents are largely lacking. Aims: The goal of our laboratory work is to determine whether and how carbon dioxide (CO2), formic acid (HCOOH) and acetaldehyde (CH3CHO) react with H-atoms in the solid state at low temperatures and to derive reaction rates and production yields. Methods: Pure CO2, HCOOH and CH3CHO interstellar ice analogues are bombarded by H-atoms in an ultra-high vacuum experiment. The ices are monitored by reflection absorption infrared spectroscopy and the reaction products are detected in the gas phase through temperature programmed desorption to determine the destruction and formation yields as well as the corresponding reaction rates. Results: Within the sensitivity of our set-up we conclude that H-atom bombardment of pure CO2 and HCOOH ice does not result in detecta...

  5. Effect of 5-aminolevulinic acid on kinetics of protoporphyrin IX production in CHO cells.

    Directory of Open Access Journals (Sweden)

    W Warchoł

    2004-07-01

    Full Text Available 5-aminolevulinic acid (ALA is utilized in a photodynamic therapy as a compound capable of augmenting intracellular pool of protoporphyrin IX (PpIX, which exhibits properties of a photosensitizer. The studies were aimed at monitoring accumulation of endogenous protoporphyrin IX in CHO cells under effect of various concentrations of ALA in culture medium and following removal of the compound from the culture medium. Cell content of PpIX was determined following incubation of the cells for 72 h in a culture medium containing different concentration of ALA. Moreover, the cells were preincubated for 2 h in ALA at various concentrations and separated from the compound by medium change and their PpIX content was monitored following incubation. PpIX content was defined by a fluorescent technique under the confocal microscope. In the course of continuous incubation of cells with ALA, biphasic alterations were noted in cellular PpIX concentration. Removal of ALA from the incubation medium resulted at first in a decrease in PpIX content in cells, which was followed by an evidently augmented accumulation of the compound in the cells. The results suggested that in the case of CHO cells, exogenous ALA was not an exclusive source of PpIX synthesis and that alterations in enzyme activities were responsible for production of PpIX.

  6. Novel Stable Compounds in the C-H-O Ternary System at High Pressure.

    Science.gov (United States)

    Saleh, Gabriele; Oganov, Artem R

    2016-01-01

    The chemistry of the elements is heavily altered by high pressure, with stabilization of many new and often unexpected compounds, the emergence of which can profoundly change models of planetary interiors, where high pressure reigns. The C-H-O system is one of the most important planet-forming systems, but its high-pressure chemistry is not well known. Here, using state-of-the-art variable-composition evolutionary searches combined with quantum-mechanical calculations, we explore the C-H-O system at pressures up to 400 GPa. Besides uncovering new stable polymorphs of high-pressure elements and known molecules, we predicted the formation of new compounds. A 2CH4:3H2 inclusion compound forms at low pressure and remains stable up to 215 GPa. Carbonic acid (H2CO3), highly unstable at ambient conditions, was predicted to form exothermically at mild pressure (about 1 GPa). As pressure rises, it polymerizes and, above 314 GPa, reacts with water to form orthocarbonic acid (H4CO4). This unexpected high-pressure chemistry is rationalized by analyzing charge density and electron localization function distributions, and implications for general chemistry and planetary science are also discussed. PMID:27580525

  7. Effects of aldicarb and propoxur on cytotoxicity and lipid peroxidation in CHO-K1 cells.

    Science.gov (United States)

    Maran, E; Fernández-Franzón, M; Font, G; Ruiz, M J

    2010-06-01

    Cytotoxic effects of aldicarb, its sulfone and sulfoxide, and propoxur, lipid peroxidation and antioxidant parameters in Chinese Hamster Ovary (CHO-K1) cells were determined. D,L-buthionine-(S,R)-sulfoximine (BSO) was assayed to determine the role of GSH in the protection against carbamate cytotoxicity. Pre-treatment with 60 microM BSO, induced a significant decrease in the glutathione reductase (GR; 64-141%), the glutathione peroxidase (GPx; 10-30%) and the glutathione S-transferase (GST; 59-93%) activities, and its GSH levels (79-85%), while the oxidized glutathione (GSSG) levels significantly increased (64-78%) respect to experiment non-BSO-pretreated. Carbamates BSO pre-treated vs. non-BSO pre-treated showed a significant increase in malondialdehyde (MDA) production (from 13% to 52% vs. 25% to 93%). These data suggest that carbamates could injure CHO-K1 cells via oxidative stress by the increase of MDA production; moreover, BSO enhance the oxidative damage caused by carbamates. However, the glutathione system protects cells from carbamates damage.

  8. Novel Stable Compounds in the C-H-O Ternary System at High Pressure

    Science.gov (United States)

    Saleh, Gabriele; Oganov, Artem R.

    2016-09-01

    The chemistry of the elements is heavily altered by high pressure, with stabilization of many new and often unexpected compounds, the emergence of which can profoundly change models of planetary interiors, where high pressure reigns. The C-H-O system is one of the most important planet-forming systems, but its high-pressure chemistry is not well known. Here, using state-of-the-art variable-composition evolutionary searches combined with quantum-mechanical calculations, we explore the C-H-O system at pressures up to 400 GPa. Besides uncovering new stable polymorphs of high-pressure elements and known molecules, we predicted the formation of new compounds. A 2CH4:3H2 inclusion compound forms at low pressure and remains stable up to 215 GPa. Carbonic acid (H2CO3), highly unstable at ambient conditions, was predicted to form exothermically at mild pressure (about 1 GPa). As pressure rises, it polymerizes and, above 314 GPa, reacts with water to form orthocarbonic acid (H4CO4). This unexpected high-pressure chemistry is rationalized by analyzing charge density and electron localization function distributions, and implications for general chemistry and planetary science are also discussed.

  9. Reversal of acquired resistance to adriamycin in CHO cells by tamoxifen and 4-hydroxy tamoxifen: role of drug interaction with alpha 1 acid glycoprotein.

    OpenAIRE

    Chatterjee, M.; Harris, A. L.

    1990-01-01

    Tamoxifen and 4-OH tamoxifen were used to reverse multidrug resistance (MDR) in CHO cells with acquired resistance to adriamycin (CHO-Adrr). Because alpha 1 acid glycoprotein (AAG) can bind a range of calcium channel blockers that also reverse MDR and rises in malignancy, its interactions with tamoxifen and 4-OH tamoxifen were also studied. Tamoxifen decreased the IC50 of 10 microM adriamycin 4.8-fold in the parent CHO-K1 cell line and 16-fold in CHO-Adrr. Similarly 4-OH tamoxifen decreased t...

  10. Chemical inhibition of autophagy: Examining its potential to increase the specific productivity of recombinant CHO cell lines.

    Science.gov (United States)

    Baek, Eric; Kim, Che Lin; Kim, Mi Gyeom; Lee, Jae Seong; Lee, Gyun Min

    2016-09-01

    Chinese hamster ovary (CHO) cells activate and undergo apoptosis and autophagy for various environmental stresses. Unlike apoptosis, studies on increasing the production of therapeutic proteins in CHO cells by targeting the autophagy pathway are limited. In order to identify the effects of chemical autophagy inhibitors on the specific productivity (qp ), nine chemical inhibitors that had been reported to target three different phases of autophagy (metformin, dorsomorphin, resveratrol, and SP600125 against initiation and nucleation; 3-MA, wortmannin, and LY294002 against elongation, and chloroquine and bafilomycin A1 against autophagosome fusion) were used to treat three recombinant CHO (rCHO) cell lines: the Fc-fusion protein-producing DG44 (DG44-Fc) and DUKX-B11 (DUKX-Fc) and antibody-producing DG44 (DG44-Ab) cell lines. Among the nine chemical inhibitors tested, 3-MA, dorsomorphin, and SP600125 significantly increased the qp of DG44-Fc and DUKX-Fc. In contrast, for DG44-Ab, only 3-MA significantly increased the qp . The autophagy-inhibiting activity of the nine chemical inhibitors on the rCHO cell lines was evaluated through Western blot analysis and flow cytometry. Unexpectedly, some chemical inhibitors did not exhibit any apparent inhibition activity on autophagy. The chemical inhibitors that enhanced the qp , 3-MA, dorsomorphin, and SP600125, exhibited instead an increased autophagic flux. Taken all together, the chemical inhibition of autophagy was not effective in increasing the qp in rCHO cell lines and the positive effect of 3-MA, dorsomorphin, and SP600125 on the qp was not due to the inhibition of autophagy. Biotechnol. Bioeng. 2016;113: 1953-1961. © 2016 Wiley Periodicals, Inc.

  11. Chemical inhibition of autophagy: Examining its potential to increase the specific productivity of recombinant CHO cell lines.

    Science.gov (United States)

    Baek, Eric; Kim, Che Lin; Kim, Mi Gyeom; Lee, Jae Seong; Lee, Gyun Min

    2016-09-01

    Chinese hamster ovary (CHO) cells activate and undergo apoptosis and autophagy for various environmental stresses. Unlike apoptosis, studies on increasing the production of therapeutic proteins in CHO cells by targeting the autophagy pathway are limited. In order to identify the effects of chemical autophagy inhibitors on the specific productivity (qp ), nine chemical inhibitors that had been reported to target three different phases of autophagy (metformin, dorsomorphin, resveratrol, and SP600125 against initiation and nucleation; 3-MA, wortmannin, and LY294002 against elongation, and chloroquine and bafilomycin A1 against autophagosome fusion) were used to treat three recombinant CHO (rCHO) cell lines: the Fc-fusion protein-producing DG44 (DG44-Fc) and DUKX-B11 (DUKX-Fc) and antibody-producing DG44 (DG44-Ab) cell lines. Among the nine chemical inhibitors tested, 3-MA, dorsomorphin, and SP600125 significantly increased the qp of DG44-Fc and DUKX-Fc. In contrast, for DG44-Ab, only 3-MA significantly increased the qp . The autophagy-inhibiting activity of the nine chemical inhibitors on the rCHO cell lines was evaluated through Western blot analysis and flow cytometry. Unexpectedly, some chemical inhibitors did not exhibit any apparent inhibition activity on autophagy. The chemical inhibitors that enhanced the qp , 3-MA, dorsomorphin, and SP600125, exhibited instead an increased autophagic flux. Taken all together, the chemical inhibition of autophagy was not effective in increasing the qp in rCHO cell lines and the positive effect of 3-MA, dorsomorphin, and SP600125 on the qp was not due to the inhibition of autophagy. Biotechnol. Bioeng. 2016;113: 1953-1961. © 2016 Wiley Periodicals, Inc. PMID:26914152

  12. Mediation of buprenorphine analgesia by a combination of traditional and truncated mu opioid receptor splice variants.

    Science.gov (United States)

    Grinnell, Steven G; Ansonoff, Michael; Marrone, Gina F; Lu, Zhigang; Narayan, Ankita; Xu, Jin; Rossi, Grace; Majumdar, Susruta; Pan, Ying-Xian; Bassoni, Daniel L; Pintar, John; Pasternak, Gavril W

    2016-10-01

    Buprenorphine has long been classified as a mu analgesic, although its high affinity for other opioid receptor classes and the orphanin FQ/nociceptin ORL1 receptor may contribute to its other actions. The current studies confirmed a mu mechanism for buprenorphine analgesia, implicating several subsets of mu receptor splice variants. Buprenorphine analgesia depended on the expression of both exon 1-associated traditional full length 7 transmembrane (7TM) and exon 11-associated truncated 6 transmembrane (6TM) MOR-1 variants. In genetic models, disruption of delta, kappa1 or ORL1 receptors had no impact on buprenorphine analgesia, while loss of the traditional 7TM MOR-1 variants in an exon 1 knockout (KO) mouse markedly lowered buprenorphine analgesia. Loss of the truncated 6TM variants in an exon 11 KO mouse totally eliminated buprenorphine analgesia. In distinction to analgesia, the inhibition of gastrointestinal transit and stimulation of locomotor activity were independent of truncated 6TM variants. Restoring expression of a 6TM variant with a lentivirus rescued buprenorphine analgesia in an exon 11 KO mouse that still expressed the 7TM variants. Despite a potent and robust stimulation of (35) S-GTPγS binding in MOR-1 expressing CHO cells, buprenorphine failed to recruit β-arrestin-2 binding at doses as high as 10 µM. Buprenorphine was an antagonist in DOR-1 expressing cells and an inverse agonist in KOR-1 cells. Buprenorphine analgesia is complex and requires multiple mu receptor splice variant classes but other actions may involve alternative receptors. PMID:27223691

  13. Developement of serum-free media in CHO-DG44 cells using a central composite statistical design

    OpenAIRE

    Parampalli, Ananth; Eskridge, Kent; Smith, Leonard; Meagher, Michael M.; Mowry, Mark C.; Subramanian, Anuradha

    2007-01-01

    A serum free medium was developed for the production of recombinant antibody against Botulinum A (BoNTA) using dihydrofolate reductase deficient Chinese Hamster Ovary Cells (CHO-DG44) in suspension culture. An initial control basal medium was prepared, which was similar in composition to HAM’s F12: IMDM (1:1) supplemented with insulin, transeferrin, selenium and a lipid mixture. The vitamin concentration of the basal medium was twice that of HAM’s F12: IMDM (1:1). CHO-DG44 cells expressing S2...

  14. The GalNAc-type O-Glycoproteome of CHO Cells Characterized by the SimpleCell Strategy

    DEFF Research Database (Denmark)

    Zhang, Yang; Halim, Adnan; Narimatsu, Yoshiki;

    2014-01-01

    of glycan structures (glycostructures) on glycoproteins are well established, our knowledge of the capacity of CHO cells for attaching GalNAc-type O-glycans to proteins (glycosites) is minimal. This type of O-glycosylation is one of the most abundant forms of glycosylation, and it is differentially...... regulated in cells by expression of a subset of homologous polypeptide GalNAc-transferases. Here, we have genetically engineered CHO cells to produce homogeneous truncated O-glycans, so-called SimpleCells, which enabled lectin enrichment of O-glycoproteins and characterization of the O-glycoproteome. We...

  15. Comparison of the Production of Recombinant Protein in Suspension Culture of CHO Cells in Spinner Flask and Shake Flask System

    Directory of Open Access Journals (Sweden)

    S.N.Z Zainul Abidin

    2011-12-01

    Full Text Available Chinese hamster ovary (CHO cells have been most widely used as the production host for the commercial production of biopharmaceuticals product. They have been extensively studied and developed, and today provide a stable platform for producing monoclonal antibodies and recombinant proteins. This study was focusing on comparison of suspension culture system by using spinner flask and shake flask for the growth and production of recombinant protein in CHO cell line. The CHO cells were transfected with an expression of DNA plasmid containing lac Z gene which codes for β-galactosidase. The recombinant genes in these CHO cells and the β-galactosidase expressing cells were adapted to suspension culture. The agitation speed for both spinner and shake flask were adjusted accordingly. The experiments were carried out in duplicate and samples were taken for cell count, determination of glucose consumption, lactate production and protein level by using biochemical assay. The result showed that, the cell growth in spinner flask is more favorable then in shake flask. The cell concentration in spinner flask is 58% higher than in shake flask. On the other hand, specific activity of β-galactosidase is 25% higher in spinner flask compared to shake flask, at the same agitation speed.ABSTRAK: Sel ovari hamster China (Chinese hamster ovary (CHO digunakan secara meluas dalam hos pembiakan untuk tujuan komersil produk biofarmaseutikal. Ia telah dikaji dan dibangunkan secara ekstensif, dan kini ia menyediakan landasan yang stabil untuk penghasilan antibodi monoklon dan protein rekombinan. Kajian ini memfokuskan tentang penghasilan protein rekombinan menggunakan kultur ampaian sel CHO di dalam kelalang putar dan kelalang goncang. Sel CHO dimasukkan dengan plasmid DNA yang mengandungi gen lac Z yang juga memberikan kod untuk β-galaktosidase. Sel CHO β-galaktosidase-terungkap dimasukkan ke dalam kultur ampaian. Kelajuan agitasi untuk kedua-dua kelalang putar

  16. Benchmarking of commercially available CHO cell culture media for antibody production.

    Science.gov (United States)

    Reinhart, David; Damjanovic, Lukas; Kaisermayer, Christian; Kunert, Renate

    2015-06-01

    In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but high amino acid concentrations alone were not sufficient to ensure superior cell growth and high antibody production. However, some key amino acids that were limiting in most media could be identified. Unbalanced glucose and amino acids led to high cell-specific lactate and ammonium production rates. In some media, persistently high glucose concentrations probably induced the suppression of respiration and oxidative phosphorylation, known as Crabtree effect, which resulted in high cell-specific glycolysis rates along with a continuous and high lactate production. In additional experiments, two of the eight basal media were supplemented with feeds from two different manufacturers in six combinations, in order to understand the combined impact of media and feeds on cell metabolism in a CHO fed-batch process. Cell growth, nutrient consumption and metabolite production rates, antibody production, and IgG quality were evaluated in detail. Concentrated feed supplements boosted cell concentrations almost threefold and antibody titers up to sevenfold. Depending on the fed-batch strategy, fourfold higher peak cell concentrations and eightfold increased IgG titers (up to 5.8 g/L) were achieved. The glycolytic flux was remarkably similar among the fed-batches; however, substantially different specific lactate production rates were observed in the different media and feed combinations. Further analysis revealed that in addition to the feed additives, the basal medium can make a considerable

  17. Analysis of CHO cells metabolic redistribution in a glutamate-based defined medium in continuous culture.

    Science.gov (United States)

    Altamirano, C; Illanes, A; Casablancas, A; Gámez, X; Cairó, J J; Gòdia, C

    2001-01-01

    The effect of glutamine replacement by glutamate and the balance between glutamate and glucose metabolism on the redistribution of t-PA-producing recombinant CHO cells metabolism is studied in a series of glucose shift down and shift up experiments in continuous culture. These experiments reveal the existence of multiple steady states, and experimental data are used to perform metabolic flux analysis to gain a better insight into cellular metabolism and its redistribution. Regulation of glucose feed rate promotes a higher efficiency of glucose and nitrogen source utilization, with lower production of metabolic byproducts, but this reduces t-PA specific production rate. This reduction under glucose limitation can be attributed to the fact that the cells are forced to efficiently utilize the carbon and energy source for growth, impairing the production of dispensable metabolites. It is, therefore, the combination of growth rate and carbon and energy source availability that determines the level of t-PA production in continuous culture.

  18. Heating of low-density CHO-foam layers by means of soft X-rays

    Energy Technology Data Exchange (ETDEWEB)

    Rosmej, O.N., E-mail: o.rosmej@gsi.de [GSI Helmholtzzentrum fuer Schwerionenforschung, Planckstrasse 1, 164291 Darmstadt (Germany); Bagnoud, V.; Eisenbarth, U. [GSI Helmholtzzentrum fuer Schwerionenforschung, Planckstrasse 1, 164291 Darmstadt (Germany); Vatulin, V.; Zhidkov, N.; Suslov, N.; Kunin, A.; Pinegin, A. [All Russian Scientific Research Institute of Experimental Physics, RFNC-VNIIEF, Mira St. 37, Sarov (Russian Federation); Schaefer, D.; Nisius, Th.; Wilhein, Th. [RheinAhrCampus Remagen, Institute for X-optics, Suedallee 2, 53424 Remagen (Germany); Rienecker, T.; Wiechula, J.; Jacoby, J. [Goethe University, Frankfurt am Main (Germany); Zhao, Y. [Institute of Modern Physics, CAS, Nanchang Road 509, 730000 Lanzhou (China); Vergunova, G.; Borisenko, N. [Lebedev Physical Institute, Leninskii Prospekt, 65 Moscow (Russian Federation); Orlov, N. [Joint Institute for High Temperatures RAS, Institute for High Energy Density, Izhorskaya. 13, building 2, 125412 Moscow (Russian Federation)

    2011-10-11

    Interaction of soft X-ray thermal radiation with polymer foam layers has been studied experimentally. Indirectly heated CHO-foams were used to create a plasma target for applications in combined heavy ion beam-laser experiments that are aimed at investigation of the heavy ion energy loss in ionized matter. In this work, we report experimental results on heating of low Z foams by means of the Planckian radiation generated in gold hohlraums. The experimental goal was to study the hohlraum radiation field, duration of the soft X-ray pulse, the conversion efficiency of the laser energy into soft X-rays, measurements of the absorption properties of foam layers and parameters of the foam targets heated by the Plankian radiation.

  19. N-Glycosylation optimization of recombinant antibodies in CHO cell through process and metabolic engineering

    DEFF Research Database (Denmark)

    Fan, Yuzhou

    monoclonal antibody (mAb) towards desired patterns, and at the same time try to understand the underlying mechanisms of that from a systems biology perspective. Two different strategies were used and achieved great success in glyco-optimization: 1) optimize media and culture process; 2) Genetically optimize...... CHO cell factory. In the early part of the thesis, the first strategy was displayed by a number of successful case studies, in which process and media engineering approach was successfully used to direct N-glycosylation. Controlling the balance between glucose and amino acid metabolism, using...... galactose as feed additives, changing process parameters such as seeding density and cultivation duration are all demonstrated to be effective. The causal explanation of their impact on glycosylation can be various, including product, metabolism, proteome and physiology-associated mechanism. In the middle...

  20. Performance evaluation of CHO-K1 cell in culture medium supplemented with hemolymph

    Directory of Open Access Journals (Sweden)

    Tássia Raffoul

    2005-06-01

    Full Text Available The aim of this work was to evaluate the potential of hemolymph utilization as a culture medium supplement to cultivate the animal cell CHO-K1. For this purpose 1% v/v of hemolymph was added to DMEM medium containing 10% v/v of FBS and 1 or 4.5 g/L of glucose. The culture was grown in spinner flasks incubated in a 10% v/v CO2 environment, at 37ºC, with the Cytodex 1 microcarrier. Comparing the results obtained from the culture with hemolymph against those without hemolymph, a positive influence of the hemolymph was observed, as the experiment with hemolymph presented a 52% higher cell concentration and a higher productivity of up to 40%.Desenvolvimento de meios de cultura isentos de soro fetal bovino (SFB é uma das grandes prioridades de pesquisa em desenvolvimento de processos com célula animal. O objetivo do presente trabalho foi realizar uma análise do potencial de uso da hemolinfa como suplemento do meio utilizado no cultivo da célula animal ancorante CHO-K1. Para isso, foi adicionado 1% v/v de extrato de hemolinfa ao meio DMEM contendo 10% v/v de SFB e 1,0 ou 4,5 g/L de glicose. O cultivo foi realizado em frascos tipo spinner em um ambiente de 10% v/v de CO2, a 37ºC, utilizando o microcarregador Cytodex 1. Comparando os resultados obtidos no ensaio com hemolinfa com um sem hemolinfa pode-se notar uma influência positiva da hemolinfa no cultivo, já que o ensaio com hemolinfa apresentou uma concentração máxima de células 52% maior e uma produtividade máxima de até 40% maior.

  1. Fibrinogen interaction of CHO cells expressing chimeric αIIb/αvβ3 integrin

    Institute of Scientific and Technical Information of China (English)

    Juan-juan CHEN; Xiao-yu SU; Xiao-dong XI; Li-ping LIN; Jian DING; He LU

    2008-01-01

    Aim: The molecular mechanisms of the affinity regulation of αvβ3 integrin are important in tumor development, wound repairing, and angiogenesis. It has been established that the cytoplasmic domains of αvβ3 integrin play an important role in integrin-ligand affinity regulation. However, the relationship of structure-func-tion within these domains remains unclear. Methods: The extracellular and trans-membrane domain of αⅡb was fused to the αv integrin cytoplasmic domain, and the chimeric α subunit was coexpressed in Chinese hamster ovary (CHO) cells with the wild-type β3 subunit or with 3 mutant 133 sequences bearing truncations at the positions of T741, Y747, and F754, respectively. The CHO cells expressing these recombinant integrins were tested for soluble fibrinogen binding and the cell adhesion and spreading on immobilized fibrinogen. Results: All 4 types of integrins bound soluble fibrinogen in the absence of agonist stimulation, and only the cells expressing the chimeric α subunit with the wild-type β3 subunit, but not those with truncated β3, could adhere to and spread on immobilized fibrinogen. Conclusion: The substitution αⅡb at the cytoplasmic domain with the ctv cyto-plasmic sequence rendered the extracellular αⅡbβ3 a constitutively activated con-formation for ligands without the need of "inside-out" signals. Our results also indicated that the COOH-terminal sequence of β3 might play a key role in integrin αⅡb/αvβ3-mediated cell adhesion and spreading on immobilized fibrinogen. The cells expressing αⅡb/αvβ3 have enormous potential for facilitating drug screen-ing for antagonists either to αvβ3 intracellular interactions or to αⅡbβ3 receptor functions.

  2. Quantitative mammalian cell mutagenesis and mutagen screening: study with CHO cells

    International Nuclear Information System (INIS)

    The CHO/HGPRT system has been developed and defined for quantifying mutation induced by various physical and chemical agents at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells. In all direct-acting chemical mutagens studied, mutation induction increases linearly as a function of the concentration, with no apparent threshold. Some chemicals induce mutation at non-cytotoxic concentrations. The mutagenicity of ethyl methanesulfonate has been quantified as a function of exposure concentration x treatment time. The sensitive and quantitative nature of the system enables studies of the structure-activity (mutagenicity) relationships of various classes of chemicals, including alkylating agents, heterocyclic nitrogen mustards, and platinum compounds. When rat liver S9-mediated metabolic activation is present, procarcinogens such as benzo(a)pyrene, 2-acetylaminofluorene, and dimethylnitrosamine are mutagenic, whereas their noncarcinogenic structural analogues pyrene, fluorene, and dimethylamine are not. The system has been shown to be useful in determining the interactive effects between physical and chemical agents, and in screening for mutagenicity of fractionated organic mixtures and industrial chemicals in both liquid and gaseous state. For the system to be used successfully in routine screening, further studies should be directed toward the development of a metabolic activation system suitable for a broad spectrum of chemicals, a sensitive and reliable statistical method, and an experimental design to determine compounds with low mutagenicity. The system has been expanded for determination of mutagen-induced chromosome aberration, sister-chromatid exchange, and micronucleus formation in addition to gene mutation and cytotoxicity; it can also be used to study inhibition of DNA synthesis

  3. Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group 2 mutants.

    NARCIS (Netherlands)

    M. van Duin (Mark); J.H. Janssen; J. de Wit (Jan); J.H.J. Hoeijmakers (Jan); L.H. Thompson; D. Bootsma (Dirk); A. Westerveld (Andries)

    1988-01-01

    textabstractThe human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In ord

  4. CHO-S antibody titers >1 gram/liter using flow electroporation-mediated transient gene expression followed by rapid migration to high-yield stable cell lines.

    Science.gov (United States)

    Steger, Krista; Brady, James; Wang, Weili; Duskin, Meg; Donato, Karen; Peshwa, Madhusudan

    2015-04-01

    In recent years, researchers have turned to transient gene expression (TGE) as an alternative to CHO stable cell line generation for early-stage antibody development. Despite advances in transfection methods and culture optimization, the majority of CHO-based TGE systems produce insufficient antibody titers for extensive use within biotherapeutic development pipelines. Flow electroporation using the MaxCyte STX Scalable Transfection System is a highly efficient, scalable means of CHO-based TGE for gram-level production of antibodies without the need for specialized expression vectors or genetically engineered CHO cell lines. CHO cell flow electroporation is easily scaled from milligram to multigram quantities without protocol reoptimization while maintaining transfection performance and antibody productivity. In this article, data are presented that demonstrate the reproducibility, scalability, and antibody production capabilities of CHO-based TGE using the MaxCyte STX. Data show optimization of posttransfection parameters such as cell density, media composition, and feed strategy that result in secreted antibody titers >1 g/L and production of multiple grams of antibody within 2 weeks of a single CHO-S cell transfection. In addition, data are presented to demonstrate the application of scalable electroporation for the rapid generation of high-yield stable CHO cell lines to bridge the gap between early- and late-stage antibody development activities.

  5. CHO cells knocked out for TSC2 display an improved productivity of antibodies under fed batch conditions.

    Science.gov (United States)

    McVey, Duncan; Aronov, Michael; Rizzi, Giovanni; Cowan, Alexis; Scott, Charo; Megill, John; Russell, Reb; Tirosh, Boaz

    2016-09-01

    The kinase mTOR operates in two cellular complexes, mTORC1 and mTORC2. mTORC1 adjusts metabolic activity according to external growth conditions and nutrients availability. When conditions are prosperous, mTOR facilitates protein and lipid biosyntheses and inhibits autophagy, while under metabolic constraints, however, its attenuation induces a catabolic program, energy preservation and autophagy. CHO is a key cell line for manufacturing of biologics owing to its remarkable ability to grow to high densities and maintain protein production and secretion for extended times. While high mTOR activity has been associated with high productivity in CHO cells, its inhibition by rapamycin has also been documented to augment productivity via promotion of viability. Here using CRISPR/Cas9 editing we engineered CHO cells to enforce high mTORC1 activity by knocking-out TSC2, a major mTOR inhibitory protein, or PTEN, a phosphatase that attenuates the PI3K/AKT/mTOR pathway. Only TSC2-deleted cells exhibited a constitutive activation of mTORC1 under fed batch conditions. Cells grew larger in size, synthesized more proteins and displayed an over twofold elevation in their specific productivity. While peak viable cell density was compromised, overall titers increased to an extent dependent upon the parental clone. Our data underscore manipulation of TSC as a strategy to improve performance of CHO cell in bioreactors. Biotechnol. Bioeng. 2016;113: 1942-1952. © 2016 Wiley Periodicals, Inc. PMID:26888596

  6. Glycoprofiling effects of media additives on IgG produced by CHO cells in fed-batch bioreactors

    DEFF Research Database (Denmark)

    Kildegaard, Helene Faustrup; Fan, Yuzhou; Wagtberg Sen, Jette;

    2016-01-01

    . In this study, the effect on IgG N-glycosylation from feeding CHO cells with eight glycosylation precursors during cultivation was investigated. The study was conducted in fed-batch mode in bioreactors with biological replicates to obtain highly controlled and comparable conditions. We assessed charge...

  7. High-level recombinant protein production in CHO cells using an adenoviral vector and the cumate gene-switch.

    Science.gov (United States)

    Gaillet, Bruno; Gilbert, Rénald; Amziani, Rachid; Guilbault, Claire; Gadoury, Christine; Caron, Antoine W; Mullick, Alaka; Garnier, Alain; Massie, Bernard

    2007-01-01

    To facilitate and accelerate the production of eukaryotic proteins with correct post-translational modifications, we have developed a protein production system based on the transduction of Chinese hamster ovary (CHO) cells using adenovirus vectors (AdVs). We have engineered a CHO cell line (CHO-cTA) that stably expresses the transactivator (cTA) of our newly developed cumate gene-switch transcription system. This cell line is adapted to suspension culture and can grow in serum-free and protein-free medium. To increase the transduction level of AdVs, we have also generated a cell line (CHO-cTA-CAR) that expresses additional amounts of the coxackievirus and adenovirus receptor (CAR) on its surface. Recombinant protein production was tested using an AdV carrying the secreted alkaline phosphatase (SEAP) under the control of the CR5 promoter, which is strongly and specifically activated by binding to cTA. The SEAP expression was linked to the expression of the green fluorescent protein (GFP) through an internal ribosome entry site (IRES) to facilitate titration of the AdV. We monitored SEAP expression on a daily basis for 9 days after transduction of CHO-cTA and CHO-cTA-CAR using different quantities of AdVs at 37 and 30 degrees C. Incubation at the latter temperature increased the production of SEAP at least 10-fold, and the presence of CAR increased the transduction level of the AdV. Maximum SEAP production (63 mg/L) was achieved at 6-7 days post-infection at 30 degrees C by transducing CHO-cTA-CAR with 500 infectious particles/cell. Because numerous AdVs can now be generated within a few weeks and large-scale production of AdVs is now a routine procedure, this system could be used to produce rapidly milligram quantities of a battery of recombinant proteins as well as for large-scale protein production.

  8. Identification of potential molecular markers of ionizing radiation-induced mutations at the hprt locus in CHO cells

    International Nuclear Information System (INIS)

    Using multiplex polymerase chain reaction-based exon deletion analysis, we have analyzed mutations at the hprt locus from independent CHO cell mutants isolated from untreated, 60Co x-ray-, and 212Bi-exposed CHO-K1 cello and its radiation-sensitive derivative, xrs-5. In the 71 spontaneous CHO-K1 mutants analyzed, 78% showed no change in exon number or size, 20% showed loss of 1-8 exons (partial deletion), and 3% showed loss of all nine hprt exons (total deletion). Exposure of CHO-K1 cells to 6 Gy of γ rays (10% survival) produced 45% of the 20 mutants analyzed showing partial deletion, and 30% showing total deletion. Exposure to an equitoxic dose of a radiation from 212Bi, a 220Rn daughter, resulted in a spectrum similar to the γ-ray spectrum in that more than 75% of the 49 mutants analyzed were deletions. The α-radiation, however, tended to produce larger intragenic deletions that γ radiation. Of the 87 spontaneous xrs-5 mutants analyzed for deletions 44% showed partial deletion, and 14% showed total deletion. Exposure to α radiation (10% survival) resulted in a deletion spectrum similar to that seen in CHO-K1 cells. Of the 49 mutants analyzed, 43% showed no change in exon number or size, 16% showed partial deletion, and 41% showed total deletion. While the defect in xrs-5 has a profound effect on spontaneous mutation spectra, it does not appear to affect α-induced mutation spectra

  9. Identification and characterization of seven new exon 11-associated splice variants of the rat mu opioid receptor gene, OPRM1

    Directory of Open Access Journals (Sweden)

    Pasternak Gavril W

    2011-01-01

    Full Text Available Abstract Background The mouse mu opioid receptor (OPRM1 gene undergoes extensive alternative splicing at both the 3'- and 5'-ends of the gene. Previously, several C-terminal variants generated through 3' splicing have been identified in the rat OPRM1 gene. In both mice and humans 5' splicing generates a number of exon 11-containing variants. Studies in an exon 11 knockout mouse suggest the functional importance of these exon 11-associated variants in mediating the analgesic actions of a subset of mu opioids, including morphine-6β-glucuronide (M6G and heroin, but not others such as morphine and methadone. We now have examined 5' splicing in the rat. Results The current studies identified in the rat a homologous exon 11 and seven exon 11-associated variants, suggesting conservation of exon 11 and its associated variants among mouse, rat and human. RT-PCR revealed marked differences in the expression of these variants across several brain regions, implying region-specific mRNA processing of the exon 11-associated variants. Of the seven rat exon 11-associated variants, four encoded the identical protein as found in rMOR-1, two predicted 6 TM variants, and one, rMOR-1H2, generated a novel N-terminal variant in which a stretch of an additional 50 amino acids was present at the N-terminus of the previously established rMOR-1 sequence. When expressed in CHO cells, the presence of the additional 50 amino acids in rMOR-1H2 significantly altered agonist-induced G protein activation with little effect on opioid binding. Conclusion The identification of the rat exon 11 and its associated variants further demonstrated conservation of 5' splicing in OPRM1 genes among rodents and humans. The functional relevance of these exon 11 associated variants was suggested by the region-specific expression of their mRNAs and the influence of the N-terminal sequence on agonist-induced G protein coupling in the novel N-terminal variant, rMOR-1H2. The importance of the exon

  10. Variants of windmill nystagmus.

    Science.gov (United States)

    Choi, Kwang-Dong; Shin, Hae Kyung; Kim, Ji-Soo; Kim, Sung-Hee; Choi, Jae-Hwan; Kim, Hyo-Jung; Zee, David S

    2016-07-01

    Windmill nystagmus is characterized by a clock-like rotation of the beating direction of a jerk nystagmus suggesting separate horizontal and vertical oscillators, usually 90° out of phase. We report oculographic characteristics in three patients with variants of windmill nystagmus in whom the common denominator was profound visual loss due to retinal diseases. Two patients showed a clock-like pattern, while in the third, the nystagmus was largely diagonal (in phase or 180° out of phase) but also periodically changed direction by 180°. We hypothesize that windmill nystagmus is a unique manifestation of "eye movements of the blind." It emerges when the central structures, including the cerebellum, that normally keep eye movements calibrated and gaze steady can no longer perform their task, because they are deprived of the retinal image motion that signals a need for adaptive recalibration. PMID:27159990

  11. Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system.

    Science.gov (United States)

    Nakamura, Tsuyoshi; Omasa, Takeshi

    2015-09-01

    Therapeutic antibodies are commonly produced by high-expressing, clonal and recombinant Chinese hamster ovary (CHO) cell lines. Currently, CHO cells dominate as a commercial production host because of their ease of use, established regulatory track record, and safety profile. CHO-K1SV is a suspension, protein-free-adapted CHO-K1-derived cell line employing the glutamine synthetase (GS) gene expression system (GS-CHO expression system). The selection of high-producing mammalian cell lines is a crucial step in process development for the production of therapeutic antibodies. In general, cloning by the limiting dilution method is used to isolate high-producing monoclonal CHO cells. However, the limiting dilution method is time consuming and has a low probability of monoclonality. To minimize the duration and increase the probability of obtaining high-producing clones with high monoclonality, an automated single cell-based clone selector, the ClonePix FL system, is available. In this study, we applied the high-throughput ClonePix FL system for cell line development using CHO-K1SV cells and investigated efficient conditions for single cell-based clone selection. CHO-K1SV cell growth at the pre-picking stage was improved by optimizing the formulation of semi-solid medium. The efficiency of picking and cell growth at the post-picking stage was improved by optimization of the plating time without decreasing the diversity of clones. The conditions for selection, including the medium formulation, were the most important factors for the single cell-based clone selection system to construct a high-producing CHO cell line. PMID:25792187

  12. Histone variants and lipid metabolism

    NARCIS (Netherlands)

    Borghesan, Michela; Mazzoccoli, Gianluigi; Sheedfar, Fareeba; Oben, Jude; Pazienza, Valerio; Vinciguerra, Manlio

    2014-01-01

    Within nucleosomes, canonical histones package the genome, but they can be opportunely replaced with histone variants. The incorporation of histone variants into the nucleosome is a chief cellular strategy to regulate transcription and cellular metabolism. In pathological terms, cellular steatosis i

  13. Role of the Nfa1 protein in pathogenic Naegleria fowleri cocultured with CHO target cells.

    Science.gov (United States)

    Kang, Su-Yeon; Song, Kyoung-Ju; Jeong, Seok-Ryoul; Kim, Jong-Hyun; Park, Sun; Kim, Kyongmin; Kwon, Myung-Hee; Shin, Ho-Joon

    2005-07-01

    Naegleria fowleri, a free-living amoeba, exists as a virulent pathogen which causes fatal primary amoebic meningoencephalitis in experimental animals and humans. Using infected and immune mouse sera, we previously cloned an nfa1 gene from a cDNA library of N. fowleri by immunoscreening. The nfa1 gene (360 bp) produced a recombinant 13.1-kDa protein, and the Nfa1 protein showed pseudopodium-specific immunolocalization on a trophozoite of N. fowleri. In this study, the role of the Nfa1 protein as a cell contact mechanism of N. fowleri cocultured with target cells was observed by an immunofluorescence assay with an anti-Nfa1 polyclonal antibody. Using confocal microscopic findings, the Nfa1 protein was located on the pseudopodia of N. fowleri trophozoites. The Nfa1 protein in N. fowleri trophozoites cocultured with CHO target cells was also located on pseudopodia, as well as in a food cup formed as a phagocytic structure in close contact with target cells. The amount of nfa1 mRNA of N. fowleri was strongly increased 6 h after coculture.

  14. Induction of proline prototrophs in CHO-K1 cells by heavy ions

    International Nuclear Information System (INIS)

    Using an established mammalian cell line, Chinese hamster ovary cells (CHO-K1), we have observed the induction of prototrophs by various heavy ions. This cell line requires proline for normal growth in medium with low serum concentration. X-rays, three types of heavy particles (600 MeV/u iron, 670 MeV/u neon, and 320 MeV/u silicon ions), ethylmethane sulphonate and 5-azacytidine were used to induce revertants which were proline independent. Log-phase cells treated with 5-azacytidine showed a very high reversion frequency. The induction frequency per viable cell appears to be dose dependent for these four types of radiation, and the dose-response curves are approximately linear. Our results also indicate that the effectiveness of high-LET particles in inducing proline prototrophs is much greater than that of low-LET radiation. The RBE value for the induction of prototrophs was calculated for neon, silicon, and iron particles and found to be about 1.3, 1.7 and 4.5, respectively. At equal survival level, the reversion frequency for X-rays and EMS was about the same. (author)

  15. Effects of glutamine and asparagine on recombinant antibody production using CHO-GS cell lines.

    Science.gov (United States)

    Xu, Ping; Dai, Xiao-Ping; Graf, Erica; Martel, Richard; Russell, Reb

    2014-01-01

    A unique and nontraditional approach using glutamine and asparagine supplements for CHO-glutamine synthetase (GS) cell lines was studied. In our experiments, we found that a decrease in pH and an increase in cell death occurred in production phase of a GS cell line, leading to reduced antibody expression and lower antibody yields. The experimental results and the statistical analysis (ANOVA) indicated that additions of glutamine and asparagine in the basal and feed media were effective to buffer the cell culture pH, reduce lactate generation, maintain a higher cell viability profile, and improve antibody productivity. In bench-top bioreactors, glutamine and asparagine supplementation helped to prevent cell death, improve antibody yield, and reduce base usage. Glutamine is normally excluded from culture media for GS cell lines to prevent the bypass of selection pressure. In this study, however, the addition of glutamine did not affect cell population homogeneity, protein quality, or decrease antibody yield of two GS cell lines.

  16. In situ monitoring of intracellular glucose and glutamine in CHO cell culture.

    Directory of Open Access Journals (Sweden)

    Alireza Behjousiar

    Full Text Available The development of processes to produce biopharmaceuticals industrially is still largely empirical and relies on optimizing both medium formulation and cell line in a product-specific manner. Current small-scale (well plate-based process development methods cannot provide sufficient sample volume for analysis, to obtain information on nutrient utilization which can be problematic when processes are scaled to industrial fermenters. We envision a platform where essential metabolites can be monitored non-invasively and in real time in an ultra-low volume assay in order to provide additional information on cellular metabolism in high throughput screens. Towards this end, we have developed a model system of Chinese Hamster Ovary cells stably expressing protein-based biosensors for glucose and glutamine. Herein, we demonstrate that these can accurately reflect changing intracellular metabolite concentrations in vivo during batch and fed-batch culture of CHO cells. The ability to monitor intracellular depletion of essential nutrients in high throughput will allow rapid development of improved bioprocesses.

  17. Removal of endogenous retrovirus-like particles from CHO-cell derived products using Q sepharose fast flow chromatography.

    Science.gov (United States)

    Strauss, Daniel M; Lute, Scott; Brorson, Kurt; Blank, Gregory S; Chen, Qi; Yang, Bin

    2009-01-01

    Retrovirus-like particles (RVLPs) that are expressed during the production of monoclonal antibodies in Chinese hamster ovary (CHO) cell cultures must be removed during product recovery. Anion exchange chromatography (AEX) performed in product flow-through mode, a common component in the purification of monoclonal antibodies, has been shown to provide robust removal of a related retrovirus model, but it's ability to remove the actual RVLP impurities has not been directly investigated. We have determined the ability of a typical Q sepharose process to remove actual CHO RVLP impurities. Using small scale experiments with three model antibodies, we observe that this AEX process is capable of effectively removing both in-process and spiked RVLPs from different feedstocks containing different mAb products. In addition, we show that this AEX process also achieves a similarly high degree of RVLP removal during large scale manufacturing operations.

  18. Degradable Dextran Nanopolymer as a Carrier for Choline Kinase (ChoK siRNA Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Zhihang Chen

    2016-02-01

    Full Text Available Although small interfering RNA (siRNA therapy has proven to be a specific and effective treatment in cells, the delivery of siRNA is a challenge for the applications of siRNA therapy. We present a degradable dextran with amine groups as an siRNA nano-carrier. In our nano-carrier, the amine groups are conjugated to the dextran platform through the acetal bonds, which are acid sensitive. Therefore this siRNA carrier is stable in neutral and basic conditions, while the amine groups can be cleaved and released from dextran platform under weak acid conditions (such as in endosomes. The cleavage and release of amine groups can reduce the toxicity of cationic polymer and enhance the transfection efficiency. We successfully applied this nano-carrier to deliver choline kinase (ChoK siRNA for ChoK inhibition in cells.

  19. Accelerating Genome Editing in CHO Cells Using CRISPR Cas9 and CRISPy, a Web-Based Target Finding Tool

    DEFF Research Database (Denmark)

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Hansen, Henning Gram;

    2014-01-01

    of the CRISPR Cas9 technology in CHO cells by generating site-specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved...... mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user-friendly bioinformatics tool, named “CRISPy” for rapid identification of sgRNA target sequences in the CHO-K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27...

  20. Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

    Institute of Scientific and Technical Information of China (English)

    易继林; 田耕

    2003-01-01

    To clone the murine α-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine α-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. A fter transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

  1. The effect of different media composition and temperatures on the production of recombinant human growth hormone by CHO cells

    OpenAIRE

    M. Rezaei; Zarkesh-Esfahani, S. H.; Gharagozloo, M.

    2013-01-01

    Cell lines derived from mammalian are dominant systems for the production of recombinant therapeutic proteins because of their capacity for correct protein folding, assembly and post-translational modification. In the search of an efficient method for the production of a recombinant protein using animal cell culture, we investigated the effects of different treatment including fetal calf serum concentration, glycerol and culture temperature on a Chinese hamster ovary (CHO) cell line on the pr...

  2. Site-specific demethylation and normal chromatin structure of the human dihydrofolate reductase gene promoter after transfection into CHO cells.

    OpenAIRE

    Shimada, T.; Inokuchi, K; Nienhuis, A W

    1987-01-01

    The effect of in vitro methylation on the function and chromatin structure of the human dihydrofolate reductase (DHFR) promoter linked to the DHFR coding sequences (minigene) was studied after DNA-mediated gene transfer into DHFR- CHO cells. Methylation of HhaI sites reduced the transforming frequency to about 10% of control, whereas methylation of HpaII sites had a less significant effect. The integrated genes were demethylated at specific sites in the promoter sequence, namely, HpaII sites ...

  3. Using Gene Essentiality and Synthetic Lethality Information to Correct Yeast and CHO Cell Genome-Scale Models

    Science.gov (United States)

    Chowdhury, Ratul; Chowdhury, Anupam; Maranas, Costas D.

    2015-01-01

    Essentiality (ES) and Synthetic Lethality (SL) information identify combination of genes whose deletion inhibits cell growth. This information is important for both identifying drug targets for tumor and pathogenic bacteria suppression and for flagging and avoiding gene deletions that are non-viable in biotechnology. In this study, we performed a comprehensive ES and SL analysis of two important eukaryotic models (S. cerevisiae and CHO cells) using a bilevel optimization approach introduced earlier. Information gleaned from this study is used to propose specific model changes to remedy inconsistent with data model predictions. Even for the highly curated Yeast 7.11 model we identified 50 changes (metabolic and GPR) leading to the correct prediction of an additional 28% of essential genes and 36% of synthetic lethals along with a 53% reduction in the erroneous identification of essential genes. Due to the paucity of mutant growth phenotype data only 12 changes were made for the CHO 1.2 model leading to an additional correctly predicted 11 essential and eight non-essential genes. Overall, we find that CHO 1.2 was 76% less accurate than the Yeast 7.11 metabolic model in predicting essential genes. Based on this analysis, 14 (single and double deletion) maximally informative experiments are suggested to improve the CHO cell model by using information from a mouse metabolic model. This analysis demonstrates the importance of single and multiple knockout phenotypes in assessing and improving model reconstructions. The advent of techniques such as CRISPR opens the door for the global assessment of eukaryotic models. PMID:26426067

  4. Cytotoxic effects of zearalenone and its metabolites and antioxidant cell defense in CHO-K1 cells.

    Science.gov (United States)

    Tatay, Elena; Font, Guillermina; Ruiz, Maria-Jose

    2016-10-01

    Zearalenone (ZEA) and its metabolites (α-zearalenol; α-ZOL, β-zearalenol; β-ZOL) are secondary metabolites of Fusarium fungi that produce cell injury. The present study explores mycotoxin-induced cell damage and cellular protection mechanisms in CHO-K1 cells. Cytotoxicity has been determined by reactive oxygen species (ROS) production and DNA damage. ROS production was determined using the fluorescein assay and DNA strand breakage by comet assay. Intracellular protection systems were glutathione (GSH), glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD). The results demonstrated that all mycotoxins increased the ROS levels up to 5.3-fold the control levels in CHO-K1 cells. Zearalenone metabolites, but not ZEA, increased DNA damage 43% (α-ZOL) and 28% (β-ZOL) compared to control cells. The GSH levels decreased from 18% to 36%. The GPx and SOD activities respectively increased from 26% to 62% and from 23% to 69% in CHO-K1 cells, whereas CAT activity decreased from 14% to 52%. In addition, intracellular ROS production was induced by ZEA and its metabolites. The endogenous antioxidant system components GSH, GPx and SOD were activated against ZEA and its metabolites. These antioxidant system components thus could contribute to decrease cell injury by ZEA and its metabolites. PMID:27465603

  5. Recombinant human bone morphogenetic protein-7 expressed from CHO cells possessing the activity of bone-induced in vitro

    Institute of Scientific and Technical Information of China (English)

    LI Xiaoyan; WANG Hao; YANG Yang; TAN Min; XUE Jingya; NI Haidong; GUO Yajun

    2006-01-01

    Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was subcloned into pcDNA3.1 mammalian expression vector and transfected to CHO cells by using the lipofectin transfection method. BMP-7 expression cell culture supernatants were harvested and purified for target protein. To analyze the bioactivity of the secreted rhBMP-7, a novel in vitro assay was established by measuring its alkaline phosphatase (ALP) stimulating of osteoblast cell line, W-20-17. Results BMP-7 stably expressing cell clone was selected, which secreted mature disulfide-linked homodimer form of hBMP-7 and had an apparent molecular weight of 36kDa. rhBMP-7 with >95% purity was obtained using 3 step chromatography method. Bioactivity assay showed that the purified protein specifically stimulated W-20-17 cell producing ALP, with a 4-fold increase of ALP activity at 100ng/ml or more, and the EC50 of 15.6ng/ml. Conclusion Purified rhBMP-7 from this CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates potential bone regeneration activity.

  6. Caspase-3 and its inhibitor Ac-DEVD-CHO in rat lens epithelial cell apoptosis induced by hydrogen in vitro

    Institute of Scientific and Technical Information of China (English)

    姚克; 王凯军; 徐雯; 孙朝晖; 申屠形超; 邱培瑾

    2003-01-01

    Objective To investigate the role of caspase-3 and its inhibitor Ac-DEVD-CHO in rat lens epithelial cell apoptosis induced by hydrogen peroxide (H2O2) in vitro.Methods Rat lenses were incubated in modified Eagle' s medium containing 2 mmol/L H2O2 to induce apoptosis in vitro. Apoptosis in lens epithelial cells was assessed by transmission electron microscopy and annexin V-propidium iodide (PI) double staining flow cytometry after 12, 24 and 48 h of incubation. The activity of caspase-3 was analyzed by western blotting.Results Observations under transmission electron microscopy revealed that 2 mmol/L H2O2 could effectively induce lens epithelial cell apoptosis in vitro. Caspase-3 activity increased during cell apoptosis and the peak measurement occurred at 24 h after treatment with H2O2. Cell apoptosis was blocked by caspase-3 inhibitor Ac-DEVD-CHO.Conclusions The activation of caspase-3 plays an important role in executing apoptosis in H2O2-treated lens epithelial cells and in the formation of cataract. The caspase-3 inhibitor Ac-DEVD-CHO may effectively prevent lens epithelial cell apoptosis caused by oxidative injury.

  7. Creation of Tenecteplase-Producing CHO Cell Line Using Site-Specific Integrase from the Phage φC31

    Directory of Open Access Journals (Sweden)

    Khadijeh Karbalaie

    2010-01-01

    Full Text Available Objective: The aim of this study was to produce a stable CHO cell line expressing tenecteplase.Materials and Methods: In the first step, the tenecteplase coding sequence was clonedin a pDB2 vector containing attB recognition sites for the phage φC31 integrase. Then,using lipofection, the CHO cells were co-transfected with constructed recombinant plasmidencoding tenecteplase and attB recognition sites and the integrase coding sequencecontaining pCMV-Int plasmid. As the recombinant plasmid contained the neomycin resistancegene (neo, stable cells were then selected using G418 as an antibiotic. Stabletransformed cells were assessed using genomic PCR and RT-PCR. Finally, the functionalityof tenecteplase was evaluated on the cell culture media.Results: our results indicated that tenecteplase coding sequence was inserted into theCHO cell genome and was successfully expressed. Moreover, tenecteplase activity assessmentindicated the presence of our functional tenecteplase in the cell culture medium.Conclusion: Considering the data obtained from this study, φC31 integrase can be usedfor the production of a stable cell line and it be used to introduce ectopic genes into mammaliancells.

  8. Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs.

    Science.gov (United States)

    Li, Wenhua; Yang, Bin; Zhou, Dongmei; Xu, Jun; Ke, Zhi; Suen, Wen-Chen

    2016-07-01

    Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars. PMID:27214604

  9. Repeated integration of antibody genes into a pre-selected chromosomal locus of CHO cells using an accumulative site-specific gene integration system

    OpenAIRE

    Kawabe, Yoshinori; Makitsubo, Hirokatsu; Kameyama, Yujiro; Huang, Shuohao; Ito, Akira; Kamihira, Masamichi

    2011-01-01

    We previously reported an accumulative site-specific gene integration system using Cre recombinase and mutated loxP sites, where a recombinase-mediated cassette exchange (RMCE) reaction is repeatable. This gene integration system was applied for antibody production using recombinant Chinese hamster ovary (CHO) cells. We introduced an exchange cassette flanked by wild-type and mutated loxP sites into the chromosome of CHO cells for the establishment of recipient founder cells. Then, the donor ...

  10. [Expression of human IL-35-IgG4 (Fc) fusion protein in CHO/DG44 cells].

    Science.gov (United States)

    Tang, Jing; Gao, Wenda; Zhang, Qing; Zhang, Dawei; Chen, Yang; He, Bo; Liu, Quansheng

    2009-01-01

    We constructed the eukaryotic expression vector of human IL-35-IgG4 (Fc)-pOptiVEC-TOPO by gene recombination technique and expressed the fusion protein human IL-35-IgG4 (Fc) in CHO/DG44 cells. The two components of the newly discovered cytokine human IL-35, EBI3 and IL-12p35, were amplified by PCR from the cDNA library derived from the KG-I cells after LPS induction. The two PCR-amplified cDNA fragments of human IL-35 were linked by over-lapping PCR and then cloned into the IgG4 (Fc)-pOptiVEC-TOPO vector. The constructed plasmid with the recombinant cDNA IL-35-IgG4 (Fc) was verified by restriction enzyme digestion analysis, PCR and DNA sequencing. The verified plasmid with the recombinant cDNA was transfected into CHO/DG44 cells using Lipofectamine 2000. The success of the transfection was examined and confirmed by RT-PCR. After selection in alpha-MEM (-) medium, the IL-35-Ig G4 (Fc) positive CHO/DG44 clones were chosen and the media from these positive clones were collected to be used to purify the fusion protein. The positive CHO/DG44 clones were further cultured in increasing concentrations of MTX and the expression levels of the fusion protein IL-35-Ig G4 (Fc) were repetitively induced by MTX-induced gene amplification. The IL-35-IgG4 (Fc) fusion protein was purified from the media collected from the positive CHO/DG44 clones by protein G affinity chromatography and then identified by SDS-PAGE and Western blotting. The results showed that one protein band was found to match well with the predicted relative molecular mass of human IL-35-IgG4 (Fc) and this protein could specifically bind to anti-human IgG4 (Fc) monoclonal antibody. In conclusion, our study successfully established an IL-35-IgG4 (Fc) positive DG44 cell line which could stably express IL-35-IgG4 (Fc) fusion protein.

  11. Identification of active elementary flux modes in mitochondria using selectively permeabilized CHO cells.

    Science.gov (United States)

    Nicolae, Averina; Wahrheit, Judith; Nonnenmacher, Yannic; Weyler, Christian; Heinzle, Elmar

    2015-11-01

    Metabolic compartmentation is a key feature of mammalian cells. Mitochondria are the powerhouse of eukaryotic cells, responsible for respiration and the TCA cycle. We accessed the mitochondrial metabolism of the economically important Chinese hamster ovary (CHO) cells using selective permeabilization. We tested key substrates without and with addition of ADP. Based on quantified uptake and production rates, we could determine the contribution of different elementary flux modes to the metabolism of a substrate or substrate combination. ADP stimulated the uptake of most metabolites, directly by serving as substrate for the respiratory chain, thus removing the inhibitory effect of NADH, or as allosteric effector. Addition of ADP favored substrate metabolization to CO2 and did not enhance the production of other metabolites. The controlling effect of ADP was more pronounced when we supplied metabolites to the first part of the TCA cycle: pyruvate, citrate, α-ketoglutarate and glutamine. In the second part of the TCA cycle, the rates were primarily controlled by the concentrations of C4-dicarboxylates. Without ADP addition, the activity of the pyruvate carboxylase-malate dehydrogenase-malic enzyme cycle consumed the ATP produced by oxidative phosphorylation, preventing its accumulation and maintaining metabolic steady state conditions. Aspartate was taken up only in combination with pyruvate, whose uptake also increased, a fact explained by complex regulatory effects. Isocitrate dehydrogenase and α-ketoglutarate dehydrogenase were identified as the key regulators of the TCA cycle, confirming existent knowledge from other cells. We have shown that selectively permeabilized cells combined with elementary mode analysis allow in-depth studying of the mitochondrial metabolism and regulation.

  12. Genotoxic effects of environmental estrogen-like compounds in CHO-K1 cells.

    Science.gov (United States)

    Tayama, Sumiko; Nakagawa, Yoshio; Tayama, Kuniaki

    2008-01-01

    Some environmental estrogen-like compounds, such as bisphenol A (BPA), 4-nonylphenol (NP), 4-octylphenol (OP), propyl p-hydroxybenzoate (P-PHBA), and butyl p-hydroxybenzoate (B-PHBA), synthetic estrogen, diethylstilbestrol (DES), and natural estrogen, 17beta-estradiol (E2), were studied for their genotoxicity in CHO-K1 cells using sister-chromatid exchange (SCE), chromosome aberration (CA), and DNA strand break (comet) assays. Six of the chemicals, excluding E2, caused DNA migration in the comet assay and induced SCEs at one or more of the highest doses. Among the chemicals, OP produced an especially high incidence of SCEs. Structural CA was induced by five of the chemicals, excluding OP and NP, and BPA, E2, and DES also induced aneuploid cells. E2 and DES particularly increased the rate of polyploidy at high doses. The incidence of colchicine-mitosis-like (c-mitotic) figures suggesting spindle disrupting effects was also detected with five of the chemicals, excluding OP and NP, and six of the chemicals, excluding E2, caused endoreduplication (ERD), a form of nuclear polyploidization induced by block of cell cycle at G2 phase, at one or more high doses. Our present results suggest that OP and NP cause repairable DNA damage, including SCEs, and do not result in CA, while the damage caused by DES, BPA, P-PHBA, and B-PHBA results in the induction of CAs together with SCEs probably because of imperfect repair. We are unable to explain the observation that the DNA damage caused by E2 resulted in CA induction but not DNA migration or SCE induction, except for speculating that the DNA damage is different from that caused by DES and the estrogen-like chemicals. Our findings also suggest that E2, DES and BPA have aneuploidogenic properties, and that the former two of chemicals also are polyploidy-inducing agents. PMID:17913570

  13. Optimization of the medium perfusion rate in a packed-bed bioreactor charged with CHO cells.

    Science.gov (United States)

    Meuwly, F; von Stockar, U; Kadouri, A

    2004-09-01

    In the present study, the optimal medium perfusion rate to be used for the continuous culture of a recombinant CHO cell line in a packed-bed bioreactor made of Fibra-Cel((R)) disk carriers was determined. A first-generation process had originally been designed with a high perfusion rate, in order to rapidly produce material for pre-clinical and early clinical trials. It was originally operated with a perfusion of 2.6 vvd during production phase in order to supply the high cell density (2.5x10(7) cell ml(-1) of packed-bed) with sufficient fresh medium. In order to improve the economics of this process, a reduction of the medium perfusion rate by -25% and -50% was investigated at small-scale. The best option was then implemented at pilot scale in order to further produce material for clinical trials with an improved second-generation process. With a -25% reduction of the perfusion rate, the volumetric productivity was maintained compared to the first-generation process, but a -30% loss of productivity was obtained when the medium perfusion rate was further reduced to -50% of its original level. The protein quality under reduced perfusion rate conditions was analyzed for purity, N-glycan sialylation level, abundance of dimers or aggregates, and showed that the quality of the final drug substance was comparable to that obtained in reference conditions. Finally, a reduction of -25% medium perfusion was implemented at pilot scale in the second-generation process, which enabled to maintain the same productivity and the same quality of the molecule, while reducing costs of media, material and manpower of the production process. For industrial applications, it is recommended to test whether and how far the perfusion rate can be decreased during the production phase - provided that the product is not sensitive to residence time - with the benefits of reduced cost of goods and to simplify manufacturing operations. PMID:19003257

  14. Understanding and Controlling Sialylation in a CHO Fc-Fusion Process.

    Science.gov (United States)

    Lewis, Amanda M; Croughan, William D; Aranibar, Nelly; Lee, Alison G; Warrack, Bethanne; Abu-Absi, Nicholas R; Patel, Rutva; Drew, Barry; Borys, Michael C; Reily, Michael D; Li, Zheng Jian

    2016-01-01

    A Chinese hamster ovary (CHO) bioprocess, where the product is a sialylated Fc-fusion protein, was operated at pilot and manufacturing scale and significant variation of sialylation level was observed. In order to more tightly control glycosylation profiles, we sought to identify the cause of variability. Untargeted metabolomics and transcriptomics methods were applied to select samples from the large scale runs. Lower sialylation was correlated with elevated mannose levels, a shift in glucose metabolism, and increased oxidative stress response. Using a 5-L scale model operated with a reduced dissolved oxygen set point, we were able to reproduce the phenotypic profiles observed at manufacturing scale including lower sialylation, higher lactate and lower ammonia levels. Targeted transcriptomics and metabolomics confirmed that reduced oxygen levels resulted in increased mannose levels, a shift towards glycolysis, and increased oxidative stress response similar to the manufacturing scale. Finally, we propose a biological mechanism linking large scale operation and sialylation variation. Oxidative stress results from gas transfer limitations at large scale and the presence of oxygen dead-zones inducing upregulation of glycolysis and mannose biosynthesis, and downregulation of hexosamine biosynthesis and acetyl-CoA formation. The lower flux through the hexosamine pathway and reduced intracellular pools of acetyl-CoA led to reduced formation of N-acetylglucosamine and N-acetylneuraminic acid, both key building blocks of N-glycan structures. This study reports for the first time a link between oxidative stress and mammalian protein sialyation. In this study, process, analytical, metabolomic, and transcriptomic data at manufacturing, pilot, and laboratory scales were taken together to develop a systems level understanding of the process and identify oxygen limitation as the root cause of glycosylation variability. PMID:27310468

  15. Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation.

    Science.gov (United States)

    Yang, M; Butler, M

    2000-05-20

    The effect of ammonium chloride was determined on a culture of CHO cells transfected with the human erythropoietin (EPO) gene. Cell growth was inhibited above a culture concentration of 5 mM NH(4)Cl with an IC-50 determined to be 33 mM. The specific production of EPO increased with the addition of NH(4)Cl above 5 mM. At 10 mM NH(4)Cl, the final cell density after 4 days in culture was significantly lower but the final yield of EPO was significantly higher. This appeared to be due to continued protein production after cell growth had ceased. The metabolic effects of added NH(4)Cl included higher specific consumption rates of glucose and glutamine and an increased rate of production of alanine, glycine, and glutamate. The EPO analyzed from control cultures had a molecular weight range of 33-39 kDa and an isoelectric point range of 4.06-4.67. Seven distinct isoforms of the molecule were identified by two-dimensional electrophoresis. This molecular heterogeneity was ascribed to variable glycosylation. Complete enzymatic de-glycosylation resulted in a single molecular form with a molecular mass of 18 kDa. Addition of NH(4)Cl to the cultures caused a significant increase in the heterogeneity of the glycoforms as shown by an increased molecular weight and pI range. Enzymatic de-sialylation of the EPO from the ammonia-treated and control cultures resulted in identical electrophoretic patterns. This indicated that the effect of ammonia was in the reduction of terminal sialylation of the glycan structures which accounted for the increased pI. Selective removal of the N-glycan structures by PNGase F resulted in two bands identified as the O-glycan linked structure (19 kDa) and the completely de-glycosylated structure (18 kDa). The proportion of the O-linked glycan structure was reduced, and its pI increased in cultures to which ammonia was added. Thus, the glycosylation pattern altered by the presence of ammonia included a reduction in terminal sialylation of all the glycans

  16. Task-based detectability comparison of exponential transformation of free-response operating characteristic (EFROC) curve and channelized Hotelling observer (CHO)

    Science.gov (United States)

    Khobragade, P.; Fan, Jiahua; Rupcich, Franco; Crotty, Dominic J.; Gilat Schmidt, Taly

    2016-03-01

    This study quantitatively evaluated the performance of the exponential transformation of the free-response operating characteristic curve (EFROC) metric, with the Channelized Hotelling Observer (CHO) as a reference. The CHO has been used for image quality assessment of reconstruction algorithms and imaging systems and often it is applied to study the signal-location-known cases. The CHO also requires a large set of images to estimate the covariance matrix. In terms of clinical applications, this assumption and requirement may be unrealistic. The newly developed location-unknown EFROC detectability metric is estimated from the confidence scores reported by a model observer. Unlike the CHO, EFROC does not require a channelization step and is a non-parametric detectability metric. There are few quantitative studies available on application of the EFROC metric, most of which are based on simulation data. This study investigated the EFROC metric using experimental CT data. A phantom with four low contrast objects: 3mm (14 HU), 5mm (7HU), 7mm (5 HU) and 10 mm (3 HU) was scanned at dose levels ranging from 25 mAs to 270 mAs and reconstructed using filtered backprojection. The area under the curve values for CHO (AUC) and EFROC (AFE) were plotted with respect to different dose levels. The number of images required to estimate the non-parametric AFE metric was calculated for varying tasks and found to be less than the number of images required for parametric CHO estimation. The AFE metric was found to be more sensitive to changes in dose than the CHO metric. This increased sensitivity and the assumption of unknown signal location may be useful for investigating and optimizing CT imaging methods. Future work is required to validate the AFE metric against human observers.

  17. Protective effect of propolis on radiation-induced chromosomal damage on Chinese hamster ovary cells (CHO-K1)

    International Nuclear Information System (INIS)

    In the last years, particular interest has been given to investigations concerning natural, effective and nontoxic compounds with radioprotective capacity in concert with increasing utilization of different types of ionizing radiation for various applications. Among them, propolis, a resinous mixture of substances collected by honey bees (Apis mellifera) has been considered promising since it presents several advantageous characteristics, i.e., antiinflammatory, anticarcinogenic, antimicrobial and free radical scavenging action. It is, therefore, a direct antioxidant that protects cells and organisms from the adverse effects of ionizing radiation. These relevant biological activities are mainly mediated by the flavonoids, present at relatively high concentrations in the propolis. Considering that the chemical composition and, consequently, the biological activity of propolis is variable according to the environmental plant ecology, the present study was conducted in order to evaluate the radioprotective capacity of Brazilian propolis, collected in the State of Rio Grande do Sul, against genotoxic damages induced by 60Co γ-radiation in Chinese hamster ovary cells (CHO-K1). for this purpose, micronucleus induction was analyzed concerning irreparable damage, specifically related to DNA double-strand breaks, that are potentially carcinogenic. CHO-K1 cells were submitted to different concentrations of propolis (3 - 33 μg/ml), 1 h before irradiation, with 1 Gy of γ radiation (0.722 Gy/min). The data obtained showed a decreasing tendency in the quantity of radioinduced damage on cells previously treated with propolis. The radioprotective effect was more prominent at higher propolis concentration. The treatment with propolis alone did not induce genotoxic effects on CHO-K1 cells. Beside that, the treatment with propolis, associated or not with radiation, did not influence the kinetics of cellular proliferation. (author)

  18. Cytotoxicity Evaluation of Anatase and Rutile TiO2 Thin Films on CHO-K1 Cells in Vitro

    Directory of Open Access Journals (Sweden)

    Blanca Cervantes

    2016-07-01

    Full Text Available Cytotoxicity of titanium dioxide (TiO2 thin films on Chinese hamster ovary (CHO-K1 cells was evaluated after 24, 48 and 72 h of culture. The TiO2 thin films were deposited using direct current magnetron sputtering. These films were post-deposition annealed at different temperatures (300, 500 and 800 °C toward the anatase to rutile phase transformation. The root-mean-square (RMS surface roughness of TiO2 films went from 2.8 to 8.08 nm when the annealing temperature was increased from 300 to 800 °C. Field emission scanning electron microscopy (FESEM results showed that the TiO2 films’ thickness values fell within the nanometer range (290–310 nm. Based on the results of the tetrazolium dye and trypan blue assays, we found that TiO2 thin films showed no cytotoxicity after the aforementioned culture times at which cell viability was greater than 98%. Independently of the annealing temperature of the TiO2 thin films, the number of CHO-K1 cells on the control substrate and on all TiO2 thin films was greater after 48 or 72 h than it was after 24 h; the highest cell survival rate was observed in TiO2 films annealed at 800 °C. These results indicate that TiO2 thin films do not affect mitochondrial function and proliferation of CHO-K1 cells, and back up the use of TiO2 thin films in biomedical science.

  19. Effect of Caspase Inhibitor Ac-DEVD-CHO on Apoptosis of Vascular Smooth Muscle Cells Induced by Artesunate

    Directory of Open Access Journals (Sweden)

    Jingwen Zhang

    2014-05-01

    Full Text Available Numerous studies have shown that the proliferation and apoptosis of vascular smooth muscle cells play a key role in restenosis. Artesunate is a triterpenoid with a peroxide structure and its antimalarial, antitumor, and antiangiogenetic activities can inhibit the proliferation and apoptosis of multifarious cells. Apoptosis is caused by the activation of a series of intracellular proteolytic enzymes, among which caspase-dependent apoptosis was the earliest to be recognized. The purpose of this article is to study the effects of caspase-3 inhibitor Ac-DEVD-CHO on proliferation and apoptosis of vascular smooth muscle cells induced by Artesunate and to explore the mechanism of Artesunate-induced apoptosis of vascular smooth muscle cells. By using the method based on methyl thiazolyl tetrazolium to observe the effects of Artesunate on the growth and proliferation of vascular smooth muscle cells; observing the change in cell shape before and after Artesunate administration by transmission electron microscopy; detecting the changes in cell cycle and apoptosis rates before and after drug administration by flow cytometry; detecting the activity of caspase-3 in the caspase apoptosis pathway by the Western Blot method, we found that Artesunate inhibits the growth and proliferation of vascular smooth muscle cells in a dose- and time-dependent manner within the concentration range of 7.5–120 μg/mL, and the inhibition rate of Artesunate can be as high as 89.49 % at a concentration of 120 μg/mL after acting for 72 hours; vascular smooth muscle cells show a typical apoptosis peak due to the effects of higher concentration of Artesunate. Compared with the control group, the higher-concentration group shows major variability, Ac-DEVD-CHO, however, can significantly decrease this induction; it has been detected by Western Blot that Artesunate can induce caspase-3 activity dramatically in vascular smooth muscle cells, but this activation may be remarkably

  20. Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media.

    Science.gov (United States)

    Miki, Hideo; Takagi, Mutsumi

    2015-08-01

    The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.

  1. A kinetic-metabolic model based on cell energetic state: study of CHO cell behavior under Na-butyrate stimulation.

    Science.gov (United States)

    Ghorbaniaghdam, Atefeh; Henry, Olivier; Jolicoeur, Mario

    2013-04-01

    A kinetic-metabolic model approach describing and simulating Chinese hamster ovary (CHO) cell behavior is presented. The model includes glycolysis, pentose phosphate pathway, TCA cycle, respiratory chain, redox state and energetic metabolism. Growth kinetic is defined as a function of the major precursors for the synthesis of cell building blocks. Michaelis-Menten type kinetic is used for metabolic intermediates as well as for regulatory functions from energy shuttles (ATP/ADP) and cofactors (NAD/H and NADP/H). Model structure and parameters were first calibrated using results from bioreactor cultures of CHO cells expressing recombinant t-PA. It is shown that the model can simulate experimental data for all available experimental data, such as extracellular glucose, glutamine, lactate and ammonium concentration time profiles, as well as cell energetic state. A sensitivity analysis allowed identifying the most sensitive parameters. The model was then shown to be readily adaptable for studying the effect of sodium butyrate on CHO cells metabolism, where it was applied to the cases with sodium butyrate addition either at mid-exponential growth phase (48 h) or at the early plateau phase (74 h). In both cases, a global optimization routine was used for the simultaneous estimation of the most sensitive parameters, while the insensitive parameters were considered as constants. Finally, confidence intervals for the estimated parameters were calculated. Results presented here further substantiate our previous findings that butyrate treatment at mid-exponential phase may cause a shift in cellular metabolism toward a sustained and increased efficiency of glucose utilization channeled through the TCA cycle. PMID:22976819

  2. Monocarbaborane anion chemistry. [COOH], [CH2OH] and [CHO] units as functional groups on ten-vertex monocarbaborane anionic compounds.

    Science.gov (United States)

    Franken, Andreas; Carr, Michael J; Clegg, William; Kilner, Colin A; Kennedy, John D

    2004-11-01

    B(10)H(14) reacts with para-C(6)H(4)(CHO)(COOH) in aqueous KOH solution to give the [nido-6-CB(9)H(11)-6-(C(6)H(4)-para-COOH)](-) anion 1, which undergoes cage closure with iodine in alkaline solution to give the [closo-2-CB(9)H(9)-2-(C(6)H(4)-para-COOH)](-) anion 2. Upon heating, anion 2 rearranges to form the [closo-1-CB(9)H(9)-1-(C(6)H(4)-para-COOH)](-) anion 3. Similarly, B(10)H(14) with glyoxylic acid OHCCOOH in aqueous KOH gives the [arachno-6-CB(9)H(13)-6-(COOH)](-) anion 4, which undergoes cage closure with iodine in alkaline solution to give the [closo-2-CB(9)H(9)-2-(COOH)](-) anion 5. Upon heating, anion 5 rearranges to give the [closo-1-CB(9)H(9)-1-(COOH)](-) anion 6. Reduction of the [COOH] anions 3 and 6 with diisobutylaluminium hydride gives the [CH(2)OH] hydroxy anions [closo-1-CB(9)H(9)-1-(C(6)H(4)-para-CH(2)OH)](-) and [closo-1-CB(9)H(9)-1-(CH(2)OH)](-) 8 respectively. The [closo-1-CB(9)H(9)-1-(C(6)H(4)-para-CH(2)OH)](-) anion 7 can also be made via isomerisation of the [closo-2-CB(9)H(9)-2-(C(6)H(4)-para-CH(2)OH)](-) anion 9, in turn obtained from the [nido-6-CB(9)H(11)-6-(C(6)H(4)-para-CH(2)OH)](-) anion 10, which is obtained from the reaction of B(10)H(14) with terephthaldicarboxaldehyde, C(6)H(4)-para-(CHO)(2), in aqueous KOH solution. Oxidation of the hydroxy anions 7 and 8 with pyridinium dichromate gives the aldehydic [closo-1-CB(9)H(9)-1-(C(6)H(4)-para-CHO)](-) anion 11 and the aldehydic [closo-1-CB(9)H(9)-1-(CHO)](-) anion 12 respectively, characterised as their 2,4-dinitrophenylhydrazone derivatives, the [closo-1-CB(9)H(9)-1-C(6)H(4)-para-CH=N-NHC(6)H(3)(NO(2))(2)](-) anion 13 and the [closo-1-CB(9)H(9)-1-CH=N-NHC(6)H(3)(NO(2))(2)](-) anion respectively.

  3. Sensitivity to DNA-damaging agents and mutation induction by UV light in UV-sensitive CHO cells

    International Nuclear Information System (INIS)

    Three UV-sensitive (UVs) mutants isolated from a CHO cell line were analyzed for survival after exposure to H2O2, EMS, MMC, CCNU, X-rays and for mutation induction after UV-irradiation. The UVs mutants showed normal sensitivities to EMS and H2O2, whereas they were hypersensitive to the bifunctional alkylating agents MMC and CCNU and to hypoxic X-irradiation. Compared to parental cells, one of the UV-sensitive clones showed approximately 3- and 7-fold enhancement in the mutagenic response per unit UV dose for 6-thioguanine and ouabain resistance, respectively. (Auth.)

  4. DNA double-strand break induction in Ku80-deficient CHO cells following Boron Neutron Capture Reaction

    Directory of Open Access Journals (Sweden)

    Masunaga Shinichiro

    2011-09-01

    Full Text Available Abstract Background Boron neutron capture reaction (BNCR is based on irradiation of tumors after accumulation of boron compound. 10B captures neutrons and produces an alpha (4He particle and a recoiled lithium nucleus (7Li. These particles have the characteristics of high linear energy transfer (LET radiation and have marked biological effects. The purpose of this study is to verify that BNCR will increase cell killing and slow disappearance of repair protein-related foci to a greater extent in DNA repair-deficient cells than in wild-type cells. Methods Chinese hamster ovary (CHO-K1 cells and a DNA double-strand break (DSB repair deficient mutant derivative, xrs-5 (Ku80 deficient CHO mutant cells, were irradiated by thermal neutrons. The quantity of DNA-DSBs following BNCR was evaluated by measuring the phosphorylation of histone protein H2AX (gamma-H2AX and 53BP1 foci using immunofluorescence intensity. Results Two hours after neutron irradiation, the number of gamma-H2AX and 53BP1 foci in the CHO-K1 cells was decreased to 36.5-42.8% of the levels seen 30 min after irradiation. In contrast, two hours after irradiation, foci levels in the xrs-5 cells were 58.4-69.5% of those observed 30 min after irradiation. The number of gamma-H2AX foci in xrs-5 cells at 60-120 min after BNCT correlated with the cell killing effect of BNCR. However, in CHO-K1 cells, the RBE (relative biological effectiveness estimated by the number of foci following BNCR was increased depending on the repair time and was not always correlated with the RBE of cytotoxicity. Conclusion Mutant xrs-5 cells show extreme sensitivity to ionizing radiation, because xrs-5 cells lack functional Ku-protein. Our results suggest that the DNA-DSBs induced by BNCR were not well repaired in the Ku80 deficient cells. The RBE following BNCR of radio-sensitive mutant cells was not increased but was lower than that of radio-resistant cells. These results suggest that gamma-ray resistant cells have

  5. Stable expression of human thyrotropin (hTSH) in mammalian cells (CHO) expressing {alpha}2,6 sialyltransferase; Expressao estavel tireotrofina humana (r-hTSH) em celulas de mamifero (CHO) que expressam {alpha}2,6 sialiltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Damiani, Renata

    2009-07-01

    A CHO cell line, previously genetically modified by the introduction of rat {alpha}2,6-sialyltransferase cDNA, generated for the first time a human-like sialylated recombinant hTSH (hlsr-hTSH) more similar to the native hormone, with 61% of {alpha}2,3- and 39% of {alpha}2,6-linked sialic acid residues. The best clone, when submitted to gene amplification with up to 8 {mu}M methotrexate, presented a secretion level of {approx}2 {mu}g hTSH/10{sup 6} cells/day, useful for product purification and characterization. The relative molecular masses (M{sub r}) of the heterodimer and of the {alpha}- and {beta}-subunits of purified hlsr-hTSH, determined by MALDI-TOF mass spectrometry, and the relative hydrophobicities, determined by RP-HPLC, were not remarkably different from those presented by two r-hTSH preparations secreted by normal CHO cells. Some differences were observed, though, in N-glycan composition, with more tri- and much more tetra-sialylated structures in hlsr-hTSH. When analyzed via an in vivo bioassay based on hTSH-induced T{sub 4} release in mice, hlsr-hTSH was shown to be equipotent (p > 0.05) with the commercial preparation of r-hTSH (Thyrogen), and 1.5-fold more potent than native hTSH (p < 0.001). (author)

  6. Data-variant kernel analysis

    CERN Document Server

    Motai, Yuichi

    2015-01-01

    Describes and discusses the variants of kernel analysis methods for data types that have been intensely studied in recent years This book covers kernel analysis topics ranging from the fundamental theory of kernel functions to its applications. The book surveys the current status, popular trends, and developments in kernel analysis studies. The author discusses multiple kernel learning algorithms and how to choose the appropriate kernels during the learning phase. Data-Variant Kernel Analysis is a new pattern analysis framework for different types of data configurations. The chapters include

  7. Antioxidant and DNA Repair Stimulating Effect of Extracts from Transformed and Normal Roots of Rhaponticum carthamoides against Induced Oxidative Stress and DNA Damage in CHO Cells.

    Science.gov (United States)

    Skała, Ewa; Sitarek, Przemysław; Różalski, Marek; Krajewska, Urszula; Szemraj, Janusz; Wysokińska, Halina; Śliwiński, Tomasz

    2016-01-01

    Rhaponticum carthamoides has a long tradition of use in Siberian folk medicine. The roots and rhizomes of this species are used in various dietary supplements or nutraceutical preparations to increase energy level or eliminate physical weakness. This is the first report to reveal the protective and DNA repair stimulating abilities of R. carthamoides root extracts in Chinese hamster ovary (CHO) cells exposed to an oxidative agent. Both transformed root extract (TR extract) and extract of soil-grown plant roots (NR extract) may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, but CHO cells stimulated with extract from the transformed roots demonstrated significantly stronger properties than cells treated with the soil-grown plant root extract. These differences in biological activity may be attributed to the differences in the content of phenolic compounds in these root extracts. Preincubation of the CHO cells with TR and NR extracts showed an increase in gene expression and protein levels of catalase (CAT) and superoxide dismutase (SOD2). R. carthamoides may possess antioxidant properties that protect CHO cells against oxidative stress. PMID:27034736

  8. Antioxidant and DNA Repair Stimulating Effect of Extracts from Transformed and Normal Roots of Rhaponticum carthamoides against Induced Oxidative Stress and DNA Damage in CHO Cells

    Science.gov (United States)

    Skała, Ewa; Sitarek, Przemysław; Różalski, Marek; Krajewska, Urszula; Szemraj, Janusz; Wysokińska, Halina; Śliwiński, Tomasz

    2016-01-01

    Rhaponticum carthamoides has a long tradition of use in Siberian folk medicine. The roots and rhizomes of this species are used in various dietary supplements or nutraceutical preparations to increase energy level or eliminate physical weakness. This is the first report to reveal the protective and DNA repair stimulating abilities of R. carthamoides root extracts in Chinese hamster ovary (CHO) cells exposed to an oxidative agent. Both transformed root extract (TR extract) and extract of soil-grown plant roots (NR extract) may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, but CHO cells stimulated with extract from the transformed roots demonstrated significantly stronger properties than cells treated with the soil-grown plant root extract. These differences in biological activity may be attributed to the differences in the content of phenolic compounds in these root extracts. Preincubation of the CHO cells with TR and NR extracts showed an increase in gene expression and protein levels of catalase (CAT) and superoxide dismutase (SOD2). R. carthamoides may possess antioxidant properties that protect CHO cells against oxidative stress. PMID:27034736

  9. Antioxidant and DNA Repair Stimulating Effect of Extracts from Transformed and Normal Roots of Rhaponticum carthamoides against Induced Oxidative Stress and DNA Damage in CHO Cells

    Directory of Open Access Journals (Sweden)

    Ewa Skała

    2016-01-01

    Full Text Available Rhaponticum carthamoides has a long tradition of use in Siberian folk medicine. The roots and rhizomes of this species are used in various dietary supplements or nutraceutical preparations to increase energy level or eliminate physical weakness. This is the first report to reveal the protective and DNA repair stimulating abilities of R. carthamoides root extracts in Chinese hamster ovary (CHO cells exposed to an oxidative agent. Both transformed root extract (TR extract and extract of soil-grown plant roots (NR extract may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, but CHO cells stimulated with extract from the transformed roots demonstrated significantly stronger properties than cells treated with the soil-grown plant root extract. These differences in biological activity may be attributed to the differences in the content of phenolic compounds in these root extracts. Preincubation of the CHO cells with TR and NR extracts showed an increase in gene expression and protein levels of catalase (CAT and superoxide dismutase (SOD2. R. carthamoides may possess antioxidant properties that protect CHO cells against oxidative stress.

  10. Increased repair of {gamma}-induced DNA double-strand breaks at lower dose-rate in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Boucher, D.; Hindo, J.; Averbeck, D. [Centre Universitaire d' Orsay, Inst. Curie-Section de Recherche, Orsay CEDEX (France)]. E-mail: dietrich.averbeck@curie.u-psud.fr

    2004-02-01

    DNA double-strand breaks (DSBs) are highly cell damaging. We asked whether for a given dose a longer irradiation time would be advantageous for the repair of DSBs. Varying the {gamma}-irradiation dose and its delivery time (0.05 Gy/min low dose-rate (LDR) compared with 3.5 Gy/min high dose-rate), confluent Chinese hamster ovary cells (CHO-K1) and Ku80 mutant cells (xrs-6) deficient in nonhomologous end-joining (NHEJ) were irradiated in agarose plugs at room temperature using a cesium-137 {gamma}-ray source. We used pulsed-field gel electrophoresis (PFGE) to measure DSBs in terms of the fraction of activity released (FAR). At LDR, one third of DSBs were repaired in CHO-K1 but not in xrs-6 cells, indicating the involvement of NHEJ in the repair of {gamma}-induced DSBs at a prolonged irradiation incubation time. To improve DSB measurements, we introduced in our PFGE protocol an antioxidant at the cell lysis step, thus avoiding free-radical side reactions on DNA and spurious DSBs. Addition of the metal chelator deferoxamine (DFO) decreased more efficiently the basal DSB level than did reduced glutathione (GSH), showing that measuring DSBs in the absence of DFO reduces precision and underestimates the role of NHEJ in the dose-rate effect on DSB yield. (author)

  11. Fractionation of yeast extract by nanofiltration process to assess key compounds involved in CHO cell culture improvement.

    Science.gov (United States)

    Mosser, Mathilde; Kapel, Romain; Chevalot, Isabelle; Olmos, Eric; Marc, Ivan; Marc, Annie; Oriol, Eric

    2015-01-01

    Yeast extract (YE) is known to greatly enhance mammalian cell culture performances, but its undefined composition decreases process reliability. Accordingly, in the present study, the nature of YE compounds involved in the improvement of recombinant CHO cell growth and IgG production was investigated. First, the benefits of YE were verified, revealing that it increased maximal concentrations of viable cells and IgG up to 73 and 60%, respectively compared to a reference culture. Then, the analyses of YE composition highlighted the presence of molecules such as amino acids, vitamins, salts, nucleobase, and glucose that were contained in reference medium, while others including peptides, trehalose, polysaccharides, and nucleic acids were not. Consequently, YE was fractionated by a nanofiltration process to deeper evaluate its effects on CHO cell cultures. The YE molecules already contained in reference medium were mainly isolated in the permeate fraction together with trehalose and short peptides, while other molecules were concentrated in the retentate. Permeate, which was free of macromolecules, exhibited a similar positive effect than raw YE on maximal concentrations. Additional studies on cell energetic metabolism underlined that dipeptides and tripeptides in permeate were used as an efficient source of nitrogenous substrates.

  12. Women in church and society: Report of research done by a research team at the PU vir CHO

    Directory of Open Access Journals (Sweden)

    F.J. van Rensburg

    2002-08-01

    Full Text Available The research project “Women in Church and Society” was conducted under the auspices of one of the focus areas for research and postgraduate education at the Potchefstroomse Universiteit vir Christelike Hoër Onderwys: “Reformed Theology and the Development of the South African Society”. This focus area is based in the Faculty of Theology (PU vir CHO and is directed by Herrie van Rooy. Project 2 of this focus area is “The socio-historic context of the Bible and its implications for the development of South African Society” and is under the leadership of Fika J. van Rensburg. The first sub-project of Project 2 to be completed is “Women in Church and Society”. It commenced in 2000 and had its fourth and final workshop in September 2002. It was managed by a five-person executive committee and had the following categories of collaborators: 16 PU vir CHO researchers, 10 researchers from other South African universities, 6 international researchers, 19 masters’ and doctoral students, and 21 researchers with special expertise in relevant areas. In total 48 papers1 were read and discussed at the four workshops; and most of them have either been published or are in the process of being published as articles in accredited journals. This article is a report on the activities and outcome of the research project.

  13. Stable expression of human thyrotropin (hTSH) in mammalian cells (CHO) expressing α2,6 sialyltransferase

    International Nuclear Information System (INIS)

    A CHO cell line, previously genetically modified by the introduction of rat α2,6-sialyltransferase cDNA, generated for the first time a human-like sialylated recombinant hTSH (hlsr-hTSH) more similar to the native hormone, with 61% of α2,3- and 39% of α2,6-linked sialic acid residues. The best clone, when submitted to gene amplification with up to 8 μM methotrexate, presented a secretion level of ∼2 μg hTSH/106 cells/day, useful for product purification and characterization. The relative molecular masses (Mr) of the heterodimer and of the α- and β-subunits of purified hlsr-hTSH, determined by MALDI-TOF mass spectrometry, and the relative hydrophobicities, determined by RP-HPLC, were not remarkably different from those presented by two r-hTSH preparations secreted by normal CHO cells. Some differences were observed, though, in N-glycan composition, with more tri- and much more tetra-sialylated structures in hlsr-hTSH. When analyzed via an in vivo bioassay based on hTSH-induced T4 release in mice, hlsr-hTSH was shown to be equipotent (p > 0.05) with the commercial preparation of r-hTSH (Thyrogen), and 1.5-fold more potent than native hTSH (p < 0.001). (author)

  14. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

    Directory of Open Access Journals (Sweden)

    Ryan Haryadi

    Full Text Available Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig heavy chain (HC and kappa light chain (LC was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

  15. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

    Science.gov (United States)

    Haryadi, Ryan; Ho, Steven; Kok, Yee Jiun; Pu, Helen X; Zheng, Lu; Pereira, Natasha A; Li, Bin; Bi, Xuezhi; Goh, Lin-Tang; Yang, Yuansheng; Song, Zhiwei

    2015-01-01

    Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells. PMID:25706993

  16. Hypersensitivity to mutation and sister-chromatid-exchange induction in CHO cell mutants defective in incising DNA containing UV lesions

    International Nuclear Information System (INIS)

    Five UV-sensitive mutant strains of CHO cells representing different genetic complementation groups were analyzed for their ability to perform the incision step of nucleotide excision repair after UV exposure. The assay utilized inhibitors of DNA synthesis to accumulate the short-lived strand breaks resulting from repair incisions. After 6 J/m2, each of the mutants showed 2, the rate in AA8 was similar to that at 6 J/m2, but the rates in the mutants were significantly higher (approx. 20% of the rate of AA8). Thus by this incision assay the mutants were phenotypically indistinguishable. Each of the mutants were hypersensitive to mutation induction at both the hprt and aprt loci by a factor of 10, and in the one strain tested ouabain resistance was induced sevenfold more efficiently than in AA8 cells. Sister chromatid exchange was also induced with sevenfold increased efficiency in the two mutant strains examined. Thus, here CHO mutants resemble xeroderma pigmentosum cells in terms of their incision defects and their hypersensitivity to DNA damage by UV

  17. Effects of Iron-Oxide Nanoparticle Surface Chemistry on Uptake Kinetics and Cytotoxicity in CHO-K1 Cells.

    Science.gov (United States)

    Hanot, Camille C; Choi, Young Suk; Anani, Tareq B; Soundarrajan, Dharsan; David, Allan E

    2016-01-01

    Superparamagnetic iron-oxide nanoparticles (SPIONs) show great promise for multiple applications in biomedicine. While a number of studies have examined their safety profile, the toxicity of these particles on reproductive organs remains uncertain. The goal of this study was to evaluate the cytotoxicity of starch-coated, aminated, and PEGylated SPIONs on a cell line derived from Chinese Hamster ovaries (CHO-K1 cells). We evaluated the effect of particle diameter (50 and 100 nm) and polyethylene glycol (PEG) chain length (2k, 5k and 20k Da) on the cytotoxicity of SPIONs by investigating cell viability using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine B (SRB) assays. The kinetics and extent of SPION uptake by CHO-K1 cells was also studied, as well as the resulting generation of intracellular reactive oxygen species (ROS). Cell toxicity profiles of SPIONs correlated strongly with their cellular uptake kinetics, which was strongly dependent on surface properties of the particles. PEGylation caused a decrease in both uptake and cytotoxicity compared to aminated SPIONs. Interestingly, 2k Da PEG-modifed SPIONs displayed the lowest cellular uptake and cytotoxicity among all studied particles. These results emphasize the importance of surface coatings when engineering nanoparticles for biomedical applications. PMID:26729108

  18. Effects of Iron-Oxide Nanoparticle Surface Chemistry on Uptake Kinetics and Cytotoxicity in CHO-K1 Cells

    Directory of Open Access Journals (Sweden)

    Camille C. Hanot

    2015-12-01

    Full Text Available Superparamagnetic iron-oxide nanoparticles (SPIONs show great promise for multiple applications in biomedicine. While a number of studies have examined their safety profile, the toxicity of these particles on reproductive organs remains uncertain. The goal of this study was to evaluate the cytotoxicity of starch-coated, aminated, and PEGylated SPIONs on a cell line derived from Chinese Hamster ovaries (CHO-K1 cells. We evaluated the effect of particle diameter (50 and 100 nm and polyethylene glycol (PEG chain length (2k, 5k and 20k Da on the cytotoxicity of SPIONs by investigating cell viability using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT and sulforhodamine B (SRB assays. The kinetics and extent of SPION uptake by CHO-K1 cells was also studied, as well as the resulting generation of intracellular reactive oxygen species (ROS. Cell toxicity profiles of SPIONs correlated strongly with their cellular uptake kinetics, which was strongly dependent on surface properties of the particles. PEGylation caused a decrease in both uptake and cytotoxicity compared to aminated SPIONs. Interestingly, 2k Da PEG-modifed SPIONs displayed the lowest cellular uptake and cytotoxicity among all studied particles. These results emphasize the importance of surface coatings when engineering nanoparticles for biomedical applications.

  19. Variant Creutzfeldt-Jakob disease

    NARCIS (Netherlands)

    E.A. Croes (Esther); C.M. van Duijn (Cock)

    2003-01-01

    textabstractA variant form of Creutzfeldt-Jakob disease (vCJD) has had major impact in Europe during the last decade. In this article, we review the aetiology of vCJD and its relation with bovine spongiform encephalopathy. Further, treatment of the disease, the strategies focusing on prevention of t

  20. Variant (Swine Origin) Influenza Viruses in Humans

    Science.gov (United States)

    ... Past Newsletters Variant (Swine Origin) Influenza Viruses in Humans Language: English Español Recommend on Facebook Tweet ... Page Background Reporting Additional Information Key Facts about Human Infections with Variant Viruses (Swine Origin Influenza Viruses ...

  1. Swine Influenza/Variant Influenza Viruses

    Science.gov (United States)

    ... Documents (General) Workers Employed at Commercial Swine Farms Influenza Types Seasonal Avian Swine Variant Pandemic Other Get ... this? Submit Button Past Newsletters Information on Swine Influenza/Variant Influenza Viruses Language: English Español Recommend ...

  2. Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells.

    Science.gov (United States)

    Galbraith, Douglas J; Tait, Andrew S; Racher, Andrew J; Birch, John R; James, David C

    2006-01-01

    In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG4 monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 microg 10(6) cells(-1) mL(-1) at a PEI nitrogen:DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 degrees C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from approximately 13 to approximately 39

  3. Cadmium Resistance Screening in Nitrilotriacetate-Buffered Minimal Media

    OpenAIRE

    Angle, J. S.; Chaney, R. L.

    1989-01-01

    Media used to determine the MICs of heavy metals for bacteria are unreliable because organic components in the media bind or chelate most of the metal being studied. To define specific metal activity in media and to maintain metal activity at a constant level, HEPES-MES [N-2-hydroxyethylpiperazine-N′ -2-ethanesulfonic acid−2-(N-morpholine)ethanesulfonic acid] salts medium with arabinose medium was modified, and the modified medium was used to examine the MIC of cadmium for Rhizobium fredii US...

  4. Variants of lumbosacral elastic band.

    Directory of Open Access Journals (Sweden)

    Carlos Cesar Santín Alfaro

    2011-06-01

    Full Text Available It is made an intervention research, qualitative and quantitative of two variants of lumbosacral elastic bands used in Provincial Laboratory of Technical Orthopedics in Sancti Spiritus Province, taking into account the high demand for this device and that the laboratory do not often count with the raw material needed for the original lumbosacral belt made by denim cloth which is the conventional belt. The main goal of this research is to explain the technological process and to compare the cost of production of both elastic variants with lumbosacral belt made by cloth which are offer to patients who look for this service , giving them a rapid solution so that they can feel comfortable.

  5. [Mirizzi syndrome and its variants].

    Science.gov (United States)

    Meyer, G J; Runge, D; Gebhardt, J

    1990-04-01

    Between 1981 and 1987 5434 patients were studied by ERCP in Allgemeines Krankenhaus Hamburg-Barmbeck. 26 (i.e. 0.43%) suffered from Mirizze syndrome with the triad of cholelithiasis, cholecystitis and obstructive biliary disease. They were classified in four different types according to the variable localisation and origin of the biliary obstruction. 16 patients corresponded to the classical type (I and II) with compression, penetration, and obturation by the concrement, five patients matched borderline with infiltration (III) and five patients were classified as variants of this syndrome. A mild elevation of serum bilirubine and alkaline phosphatase indicated more likely the benign etiology of type I to III, however, a marked elevation of alkaline phosphatase in the variants suggested more likely a malignant underlying disease. The diagnosis was ascertained in all cases by ERC and sonography preoperatively and was verified by laparotomy (n = 18) and follow-up (n = 6).

  6. An unexpected caffeine-enhanced survival in x-ray-sensitive variant cells

    International Nuclear Information System (INIS)

    The sensitivity of normal Chinese hamster cell lines, V79 and CHO, mouse cell lines, L5178Y and L, and human HeLa cells to the killing effect of x-ray is enhanced with addition of caffeine following x-ray irradiation in a dose-dependent fashion. However, the survival rate of variant cell (V79-AL162/S-10) increased with addition of low concentration of caffeine (caffeine-enhanced survival phenomenon). Therefore, the effects of protein synthesis-inhibiting agents, such as cycloheximide and puromycin, on caffeine-enhanced survival phenomenon were examined. This phenomenon was completely abolished by the inhibitory agents, but not abolished by DNA synthesis-damaging agents, such as excess thymidine and aphidicolin. DNA-damaging physiochemical factors, such as neutrons, U.V., methyl methanesulfonate and mitomycin C, were examined in relation to variant cells' sensitivity and caffeine-enhanced survival phenomenon. V79-AL162/S-10 cells showed high sensitivity to the killing effect of mitomycin C, but their survival rate returned to the rate of normal V79-B310H cells with addition of caffeine. (Namekawa, K.)

  7. Microcystic Variant of Urothelial Carcinoma

    Directory of Open Access Journals (Sweden)

    Anthony Kodzo-Grey Venyo

    2013-01-01

    Full Text Available Background. Microcystic variant of urothelial carcinoma is one of the new variants of urothelial carcinoma that was added to the WHO classification in 2004. Aims. To review the literature on microcystic variant of urothelial carcinoma. Methods. Various internet search engines were used to identify reported cases of the tumour. Results. Microscopic features of the tumour include: (i Conspicuous intracellular and intercellular lumina/microcysts encompassed by malignant urothelial or squamous cells. (ii The lumina are usually empty; may contain granular eosinophilic debris, mucin, or necrotic cells. (iii The cysts may be variable in size; round, or oval, up to 2 mm; lined by urothelium which are either flattened cells or low columnar cells however, they do not contain colonic epithelium or goblet cells; are infiltrative; invade the muscularis propria; mimic cystitis cystica and cystitis glandularis; occasionally exhibit neuroendocrine differentiation. (iv Elongated and irregular branching spaces are usually seen. About 17 cases of the tumour have been reported with only 2 patients who have survived. The tumour tends to be of high-grade and high-stage. There is no consensus opinion on the best option of treatment of the tumour. Conclusions. It would prove difficult at the moment to be dogmatic regarding its prognosis but it is a highly aggressive tumour. New cases of the tumour should be reported in order to document its biological behaviour.

  8. Study on Aldol Condensation of HCHO and CH3CHO over MgO Catalysts Modified by Lanthanum and Cerium

    Institute of Scientific and Technical Information of China (English)

    王幸宜; 李春香; 杨菊群

    2004-01-01

    Aldol condensation of HCHO and CH3CHO over MgO, modified MgO and Al2O3 with rare earth oxides, was studied. The measurement of adsorption of pyrrole on catalysts by in-situ FT-IR and NH3 TPD indicated that the addition of elements La or Ce into MgO increased the acidity of the solid. In-situ FT-IR showed that the activation of-C=O in HCHO adsorbed on CeO-MgO and La2O3-MgO occurred. The measurement of catalytic activity implied that the modified catalysts can promote the formation of pentaerythritol, dipentaerthritol and tripentaerythritol.

  9. Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation

    DEFF Research Database (Denmark)

    Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian;

    2015-01-01

    and feeds were studied using two IgG-producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N-glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards......Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process....... Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case-by-case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found...

  10. Phylosenetic identification and microbial diversity in snow of the summit (8201 m) of Cho Oyu Mountain,Tibet

    Institute of Scientific and Technical Information of China (English)

    TONG XiaoMei; WANG Jian; CHEN Fang; YU Jun; HUA Sang; ASAN Ciren; LUOSANG JiangBai; WANG Wei; YU Liang; ZHENG XiaoGuang

    2008-01-01

    The bacterial diversity and abundance in snow of the summit (8201 m) of Cho Oyu mountain, Tibet,were analyzed by 16S rRNA gene sequencing followed by scanning electronic microscopy analysis. Most of bacteria were found to be of spherical or oval shape (>95%). Bacterial 16S rDNA sequences were classified into 5 genera (Caulobacter, Ralstonia, Cupriavidus, Pelomonas and Pseudornonas).Gammaproteobacteria were the most abundant (91.25%) among the library that consists of 594 clones. The sequences found in this study are highly similar to those previously retrieved from other cold en-vironments, such as ice core, sea ice, permafrost and snow. The results showed that the cold and barren environments strongly influence the survival of bacteria. The high similarity among sequences retrieved from snow sample and other places, such as ocean, soil and water, suggested that the bacte-ria in snow, soil and water environments have the same origin.

  11. Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

    LENUS (Irish Health Repository)

    Burleigh, Susan C

    2011-10-18

    Abstract Background The glycosylation of recombinant proteins can be altered by a range of parameters including cellular metabolism, metabolic flux and the efficiency of the glycosylation process. We present an experimental set-up that allows determination of these key processes associated with the control of N-linked glycosylation of recombinant proteins. Results Chinese hamster ovary cells (CHO) were cultivated in shake flasks at 0 mM glutamine and displayed a reduced growth rate, glucose metabolism and a slower decrease in pH, when compared to other glutamine-supplemented cultures. The N-linked glycosylation of recombinant human chorionic gonadotrophin (HCG) was also altered under these conditions; the sialylation, fucosylation and antennarity decreased, while the proportion of neutral structures increased. A continuous culture set-up was subsequently used to understand the control of HCG glycosylation in the presence of varied glutamine concentrations; when glycolytic flux was reduced in the absence of glutamine, the glycosylation changes that were observed in shake flask culture were similarly detected. The intracellular content of UDP-GlcNAc was also reduced, which correlated with a decrease in sialylation and antennarity of the N-linked glycans attached to HCG. Conclusions The use of metabolic flux analysis illustrated a case of steady state multiplicity, where use of the same operating conditions at each steady state resulted in altered flux through glycolysis and the TCA cycle. This study clearly demonstrated that the control of glycoprotein microheterogeneity may be examined by use of a continuous culture system, metabolic flux analysis and assay of intracellular nucleotides. This system advances our knowledge of the relationship between metabolic flux and the glycosylation of biotherapeutics in CHO cells and will be of benefit to the bioprocessing industry.

  12. Developement of serum-free media in CHO-DG44 cells using a central composite statistical design.

    Science.gov (United States)

    Parampalli, Ananth; Eskridge, Kent; Smith, Leonard; Meagher, Michael M; Mowry, Mark C; Subramanian, Anuradha

    2007-05-01

    A serum free medium was developed for the production of recombinant antibody against Botulinum A (BoNTA) using dihydrofolate reductase deficient Chinese Hamster Ovary Cells (CHO-DG44) in suspension culture. An initial control basal medium was prepared, which was similar in composition to HAM's F12: IMDM (1:1) supplemented with insulin, transeferrin, selenium and a lipid mixture. The vitamin concentration of the basal medium was twice that of HAM's F12: IMDM (1:1). CHO-DG44 cells expressing S25 antibody grew from 2 x 10(5) cells to maximum cell density of 1.04 x 10(6) cells/ml after 5 days in this control medium. A central composite design was used to identify optimal levels and interaction among five groups of medium components. These five groups were glutamine, Essential Amino Acids (EAA), Non Essential Amino Acids (NEAA), Insulin, Transferrin, Selenium (ITS), and lipids. Fifty experiments were carried out in four batches, with two controls in each batch. There was little effect of ITS and Lipid concentrations over the range studied, and glutamine concentration showed a strong interaction with EAA. The optimal concentrations of the variables studied were 2.5 mM Glutamine, 7.4 mM (2x) EAA, 1.4 mM (0.5x) NEAA, 1x ITS supplement, 0.7x Lipids supplement. The maximum viable cell density attained in the optimized medium was 1.4 x 10(6) cells/ml, a 35% improvement over the control culture, while the final antibody titer attained was 22 +/- 3.4 mug/mL, a 50% improvement.

  13. Lysophosphatidic acid activates peroxisome proliferator activated receptor-γ in CHO cells that over-express glycerol 3-phosphate acyltransferase-1.

    Directory of Open Access Journals (Sweden)

    Cliona M Stapleton

    Full Text Available Lysophosphatidic acid (LPA is an agonist for peroxisome proliferator activated receptor-γ (PPARγ. Although glycerol-3-phosphate acyltransferase-1 (GPAT1 esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression.

  14. Protective activity of a novel resveratrol analogue, HS-1793, against DNA damage in 137Cs-irradiated CHO-K1 cells

    International Nuclear Information System (INIS)

    Resveratrol has received considerable attention as a polyphenol with anti-oxidant, anti-carcinogenic, and anti-inflammatory effects. Radiation is an important component of therapy for a wide range of malignant conditions. However, it causes damage to normal cells and, hence, can result in adverse side effects. This study was conducted to examine whether HS-1793, a novel resveratrol analogue free from the restriction of metabolic instability and the high dose requirement of resveratrol, induces a protective effect against radiation-induced DNA damage. HS-1793 effectively scavenged free radicals and inhibited radiation-induced plasmid DNA strand breaks in an in vitro assay. HS-1793 significantly decreased reactive oxygen species and cellular DNA damage in 2 Gy-irradiated Chinese hamster ovary (CHO)-K1 cells. In addition, HS-1793 dose-dependently reduced the levels of phosphorylated H2AX in irradiated CHO-K1 cells. These results indicate that HS-1793 has chemical radioprotective activity. Glutathione levels and superoxide dismutase activity in irradiated CHO-K1 cells increased significantly following HS-1793 treatment. The enhanced biological anti-oxidant activity and chemical radioprotective activity of HS-1793 maintained survival of irradiated CHO-K1 cells in a clonogenic assay. Therefore, HS-1793 may be of value as a radioprotector to protect healthy tissue surrounding tumor cells during radiotherapy to obtain better tumor control with a higher dose. (author)

  15. COMPARISON OF THE TOXICITY OF ACRYLAMIDE, CYCLOPHOSPHAMIDE, CHLRODECONE, AND DIETHYLSTILBESTROL IN CHINESE HAMSTER OVARY (CHO) CELLS WITH THEIR TOXICITY IN VIVO

    Science.gov (United States)

    In order to compare in vitro toxicity with in vivo toxicity, four chemicals that have been tested in the in vivo/in vitro toxicological screen proposed by the Health Effects Research Laboratory, EPA were tested in a Chinese Hamster Ovary (CHO) cytotoxicity assay. Viability index,...

  16. CT skull base & calvarium normal variant pitfalls

    OpenAIRE

    Lockwood, P

    2013-01-01

    Intended learning outcomes - To recognise the varied neurological appearances of skull based normal variants with the brain. Highlighting the importance of differentiation of normal and variant anatomy from the pitfalls of misdiagnosing a pathological condition Content of Presentation -Pictorial review of 12 common examples of neuroradiological normal variant conditions of skull base and calvarium anatomical areas of the brain, including sutures, asymmetry of bones, benign growths, thicken...

  17. Integrated cell and process engineering for improved transient production of a "difficult-to-express" fusion protein by CHO cells.

    Science.gov (United States)

    Johari, Yusuf B; Estes, Scott D; Alves, Christina S; Sinacore, Marty S; James, David C

    2015-12-01

    Based on an optimized electroporation protocol, we designed a rapid, milliliter-scale diagnostic transient production assay to identify limitations in the ability of Chinese hamster ovary (CHO) cells to produce a model "difficult-to-express" homodimeric Fc-fusion protein, Sp35Fc, that exhibited very low volumetric titer and intracellular formation of disulfide-bonded oligomeric aggregates post-transfection. As expression of Sp35Fc induced an unfolded protein response in transfected host cells, we utilized the transient assay to compare, in parallel, multiple functionally diverse strategies to engineer intracellular processing of Sp35Fc in order to increase production and reduce aggregation as two discrete design objectives. Specifically, we compared the effect of (i) co-expression of ER-resident molecular chaperones (BiP, PDI, CypB) or active forms of UPR transactivators (ATF6c, XBP1s) at varying recombinant gene load, (ii) addition of small molecules known to act as chemical chaperones (PBA, DMSO, glycerol, betaine, TMAO) or modulate UPR signaling (PERK inhibitor GSK2606414) at varying concentration, (iii) a reduction in culture temperature to 32°C. Using this information, we designed a biphasic, Sp35Fc-specific transient manufacturing process mediated by lipofection that utilized CypB co-expression at an optimal Sp35Fc:CypB gene ratio of 5:1 to initially maximize transfected cell proliferation, followed by addition of a combination of PBA (0.5 mM) and glycerol (1% v/v) at the onset of stationary phase to maximize cell specific production and eliminate Sp35Fc aggregation. Using this optimal, engineered process transient Sp35Fc production was significantly increased sixfold over a 12 day production process with no evidence of disulfide-bonded aggregates. Finally, transient production in clonally derived sub-populations (derived from parental CHO host) screened for a heritably improved capability to produce Sp35Fc was also significantly improved by the optimized

  18. Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor.

    Science.gov (United States)

    Zhang, Ye; Stobbe, Per; Silvander, Christian Orrego; Chotteau, Véronique

    2015-11-10

    Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200 pF/cm (where 1 pF/cm is equivalent to 1 × 10(6)viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37 °C to 29 °C was studied during 13 days at cell densities above 100 pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31 °C was applied at cell density 100 pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period.

  19. Mitochondrial DNA variants in obesity.

    Directory of Open Access Journals (Sweden)

    Nadja Knoll

    Full Text Available Heritability estimates for body mass index (BMI variation are high. For mothers and their offspring higher BMI correlations have been described than for fathers. Variation(s in the exclusively maternally inherited mitochondrial DNA (mtDNA might contribute to this parental effect. Thirty-two to 40 mtDNA single nucleotide polymorphisms (SNPs were available from genome-wide association study SNP arrays (Affymetrix 6.0. For discovery, we analyzed association in a case-control (CC sample of 1,158 extremely obese children and adolescents and 435 lean adult controls. For independent confirmation, 7,014 population-based adults were analyzed as CC sample of n = 1,697 obese cases (BMI ≥ 30 kg/m2 and n = 2,373 normal weight and lean controls (BMI<25 kg/m2. SNPs were analyzed as single SNPs and haplogroups determined by HaploGrep. Fisher's two-sided exact test was used for association testing. Moreover, the D-loop was re-sequenced (Sanger in 192 extremely obese children and adolescents and 192 lean adult controls. Association testing of detected variants was performed using Fisher's two-sided exact test. For discovery, nominal association with obesity was found for the frequent allele G of m.8994G/A (rs28358887, p = 0.002 located in ATP6. Haplogroup W was nominally overrepresented in the controls (p = 0.039. These findings could not be confirmed independently. For two of the 252 identified D-loop variants nominal association was detected (m.16292C/T, p = 0.007, m.16189T/C, p = 0.048. Only eight controls carried the m.16292T allele, five of whom belonged to haplogroup W that was initially enriched among these controls. m.16189T/C might create an uninterrupted poly-C tract located near a regulatory element involved in replication of mtDNA. Though follow-up of some D-loop variants still is conceivable, our hypothesis of a contribution of variation in the exclusively maternally inherited mtDNA to the observed larger correlations for BMI between mothers and

  20. A control strategy to investigate the relationship between specific productivity and high-mannose glycoforms in CHO cells.

    Science.gov (United States)

    Zalai, Dénes; Hevér, Helga; Lovász, Krisztina; Molnár, Dóra; Wechselberger, Patrick; Hofer, Alexandra; Párta, László; Putics, Ákos; Herwig, Christoph

    2016-08-01

    The integration of physiological knowledge into process control strategies is a cornerstone for the improvement of biopharmaceutical cell culture technologies. The present contribution investigates the applicability of specific productivity as a physiological control parameter in a cell culture process producing a monoclonal antibody (mAb) in CHO cells. In order to characterize cell physiology, the on-line oxygen uptake rate (OUR) was monitored and the time-resolved specific productivity was calculated as physiological parameters. This characterization enabled to identify the tight link between the deprivation of tyrosine and the decrease in cell respiration and in specific productivity. Subsequently, this link was used to control specific productivity by applying different feeding profiles. The maintenance of specific productivity at various levels enabled to identify a correlation between the rate of product formation and the relative abundance of high-mannose glycoforms. An increase in high mannose content was assumed to be the result of high specific productivity. Furthermore, the high mannose content as a function of cultivation pH and specific productivity was investigated in a design of experiment approach. This study demonstrated how physiological parameters could be used to understand interactions between process parameters, physiological parameters, and product quality attributes. PMID:26910040

  1. Prevalence of Dementia and Mild Cognitive Impairment in the Rural Island Town of Ama-cho, Japan

    Directory of Open Access Journals (Sweden)

    Kenji Wada-Isoe

    2012-04-01

    Full Text Available Aims: In order to determine the prevalence of dementia and mild cognitive impairment (MCI, we conducted a population-based study in Japan. Methods: Participants included 924 subjects aged 65 years or older who resided in the town of Ama-cho. In phase 1 of the study, the Mini-Mental State Examination and Clinical Dementia Rating were administered for screening purposes. In phase 2 of the study, the subjects who screened positive were further examined by neurologists. Dementia and MCI were diagnosed by means of DSM-IV and International Working Group on MCI criteria, respectively. Results: By the prevalence date of June 1, 2010, 24 subjects had deceased or lived outside the town. In total, 723 of the remaining 900 subjects received a phase 1 test. In phase 2, 98 subjects were diagnosed with amnestic MCI, 113 subjects with non-amnestic MCI, and 82 subjects with dementia. Of the subjects who did not receive the phase 1 test, 66 subjects were diagnosed as having dementia according to data from their town medical card or the Long-term Care Insurance System. The crude prevalence of amnestic MCI, non-amnestic MCI, and dementia were 10.9, 12.6, and 16.4%, respectively. Conclusion: Consistent with the striking increase in the number of elderly individuals, we report higher prevalence of MCI and dementia in Japan than previously described.

  2. Effects of BrdUrd incorporation on radiation sensitivity recovery from PLD and repair of DNA damage in CHO cells

    International Nuclear Information System (INIS)

    CHO cells grown in the presence of various concentrations (0-10 μM) of BrdUrd were exposed to X-rays either in the logarithmic or in the plateau-phase of growth and survival was measured either immediately after irradiation or 6 hr later. An increase in radiosensitivity (immediate plating) was observed in cells growing in the presence of BrdUrd that was similar for exponentially growing and plateau-phase cells when related to the amount of incorporated BrdUrd. The rate of induction of DNA damage as assayed by hydroxylapatite chromatography was similar for cells grown in the presence or absence of BrdUrd. This resulted in an increase in the amount of unrejoined breaks measured in BrdUrd-containing cells 1 hr after irradiation and which was more pronounced at higher radiation doses. These results are compared to similar results obtained with untransformed C/sub 3/H 10T1/2 cells, and implications on the mechanism of radiosensitization by BrdUrd are discussed

  3. Modification of pGH cDNA using the first intron and adenovirus-mediated expression in CHO cells

    Institute of Scientific and Technical Information of China (English)

    李秀锦; 仲飞; 齐顺章

    2003-01-01

    Objective This study was conducted to investigate the function of the first intron of porcine growth hormone (pGH) gene in the gene expression.Methods PCR method was used to amplify the first intron from pig genomic DNA. The intron was then inserted into pGH cDNA to construct pGH cDNA-intron (pGH cDNA-in). The recombinant adenoviruses containing pGH cDNA and pGH cDNA-in genes under control of CMV promoter were generated by homologous recombination method in HEK 293 cells respectively. The effect of the first intron on gene expression was evaluated by comparing the expression levels of pGH cDNA-in and pGH cDNA mediated by adenovirus vectors in CHO cells.Results The expression level of pGH cDNA containing the first intron increased by 117%, which was significantly higher than that of pGH cDNA without the intron (P<0.001). Conclusion The first intron of pGH gene has the function to improve pGH gene expression.

  4. Radiation enhancement of the efficiency of DNA-mediated gene transfer in CHO UV-sensitive mutants

    International Nuclear Information System (INIS)

    We have employed the Chinese hamster ovary (CHO) UV-sensitive mutant cell lines, UV5 and UV20, to determine whether ionizing and ultraviolet irradiation enhance the efficiency of DNA-mediated gene transfer in cells deficient in excision repair. Confluent AA8 (wild type), UV5, and UV20 cells were transfected (via polybrene and dimethyl sulfoxide treatments) with the recombinant DNA plasmid, pSV2-gpt, trypsinized, irradiated with either X rays or ultraviolet in suspension, and then plated into flasks. After a 48-h expression time, cells were trypsinized, counted, and plated in XMAT media to select for pSV2-gpt transformation. We report that X-ray irradiation enhances gene transfer in wild-type AA8 and in both UV-sensitive cell lines. Ultraviolet irradiation enhances gene transfer in AA8 and UV20, but not in UV5. Since both UV20 and UV5 are deficient in excision repair, we suggest that ultraviolet-enhanced gene transfer may involve a postreplication repair mechanism deficient in UV5

  5. Comparison of three transposons for the generation of highly productive recombinant CHO cell pools and cell lines.

    Science.gov (United States)

    Balasubramanian, Sowmya; Rajendra, Yashas; Baldi, Lucia; Hacker, David L; Wurm, Florian M

    2016-06-01

    Several naturally occurring vertebrate transposable elements have been genetically modified to enable the transposition of recombinant genes in mammalian cells. We compared three transposons-piggyBac, Tol2, and Sleeping Beauty-for their ability to generate cell pools (polyclonal cultures of recombinant cells) and clonal cell lines for the large-scale production of recombinant proteins using Chinese hamster ovary cells (CHO-DG44) as the host. Transfection with each of the dual-vector transposon systems resulted in cell pools with volumetric yields of tumor necrosis factor receptor-Fc fusion protein (TNFR:Fc) that were about ninefold higher than those from cell pools generated by conventional plasmid transfection. On average, the cell pools had 10-12 integrated copies of the transgene per cell. In the absence of selection, the volumetric productivity of the cell pools decreased by 50% over a 2-month cultivation period and then remained constant. The average volumetric TNFR:Fc productivity of clonal cell lines recovered from cell pools was about 25 times higher than that of cell lines generated by conventional transfection. In 14-day fed-batch cultures, TNFR:Fc levels up to 900 mg/L were obtained from polyclonal cell pools and up to 1.5 g/L from clonal cell lines using any of the three transposons. Biotechnol. Bioeng. 2016;113: 1234-1243. © 2015 Wiley Periodicals, Inc. PMID:26616356

  6. Cho Abelian decomposition to the exact A-M-A solutions of the SU(2) Yang-Mills-Higgs theory

    Science.gov (United States)

    Wong, Khai-Ming; Teh, Rosy; Tie, Timothy

    2015-04-01

    We consider Cho Abelian decomposition to the exact A-M-A configurations in the SU(2) Yang-Mills-Higgs theory. The non-Abelian Yang-Mills gauge potential is decomposed into the restricted and the valence part. With the decomposition, the complete Abelian picture that draws to the various monopoles configurations can be seen clearly. The singularities for the two accompanying antimonopoles and the vortex ring are removed by the corresponding valence potential. However the singularity of the composite monopole at the origin is not removed, but strengthened. Hence the composite monopole is a different kind of monopole entity. Elsewhere, the plane singularity in the solution is not readily be removed by the valence potential. On the other hand, we also solve the decomposed equations to study the solutions that lead to the spatial infinity boundary conditions of the various numerical monopoles configurations. The decomposed equations are also solved in the near-origin region for exact solutions and their properties such as the magnetic field are plotted, which confirms the correspondence with their properties at the near infinity region.

  7. Dynamic distribution of Ser-10 phosphorylated histone H3 in cytoplasm of MCF-7 and CHO cells during mitosis

    Institute of Scientific and Technical Information of China (English)

    Deng Wen LI; Qin YANG; Jia Tong CHEN; Hao ZHOU; Ru Ming LIU; Xi Tai HUANG

    2005-01-01

    The dynamic distribution of phosphorylated Histone H3 on Ser10 (phospho-H3) in cells was investigated to determine its function during mitosis. Human breast adenocarcinoma cells MCF-7, and Chinese hamster cells CHO were analyzed by indirect immunofluorescence staining with an antibody against phospho-H3. We found that the phosphorylation begins at early prophase, and spreads throughout the chromosomes at late prophase. At metaphase, most of the phosphoH3 aggregates at the end of the condensed entity of chromosomes at equatorial plate. During anaphase and telophase,the fluorescent signal of phospho-H3 is detached from chromosomes into cytoplasm. At early anaphase, phospho-H3shows ladder bands between two sets of separated chromosome, and forms "sandwich-like structure" when the chromosomes condensed. With the cleavage progressing, the "ladders" of the histone contract into a bigger bright dot. Then the histone aggregates and some of compacted microtubules in the midbody region are composed into a "bar-like"complex to separate daughter cells. The daughter cells seal their plasma membrane along with the ends of the "bar",inside which locates microtubules and modified histones, to finish the cytokinesis and keep the "bar complex" out of the cells. The specific distribution and kinetics of phospho-H3 in cytoplasm suggest that the modified histones may take part in the formation of midbody and play a crucial role in cytokinesis.

  8. Beta-glucosidase I variants with improved properties

    Energy Technology Data Exchange (ETDEWEB)

    Bott, Richard R.; Kaper, Thijs; Kelemen, Bradley; Goedegebuur, Frits; Hommes, Ronaldus Wilhelmus; Kralj, Slavko; Kruithof, Paulien; Nikolaev, Igor; Van Der Kley, Wilhelmus Antonious Hendricus; Van Lieshout, Johannes Franciscus Thomas; Van Stigt Thans, Sander

    2016-09-20

    The present disclosure is generally directed to enzymes and in particular beta-glucosidase variants. Also described are nucleic acids encoding beta-glucosidase variants, compositions comprising beta-glucosidase variants, methods of using beta-glucosidase variants, and methods of identifying additional useful beta-glucosidase variants.

  9. Cytoskeleton, endoplasmic reticulum and nucleus alterations in CHO-K1 cell line after Crotalus durissus terrificus (South American rattlesnake venom treatment

    Directory of Open Access Journals (Sweden)

    B. P. Tamieti

    2007-01-01

    Full Text Available Snake venoms are toxic to a variety of cell types. However, the intracellular damages and the cell death fate induced by venom are unclear. In the present work, the action of the South American rattlesnake Crotalus durissus terrificus venom on CHO-K1 cell line was analyzed. The cells CHO-K1 were incubated with C. d. terrificus venom (10, 50 and 100g/ml for 1 and 24 hours, and structural alterations of actin filaments, endoplasmic reticulum and nucleus were assessed using specific fluorescent probes and agarose gel electrophoresis for DNA fragmentation. Significant structural changes were observed in all analyzed structures. DNA fragmentation was detected suggesting that, at the concentrations used, the venom induced apoptosis.

  10. Local binary patterns new variants and applications

    CERN Document Server

    Jain, Lakhmi; Nanni, Loris; Lumini, Alessandra

    2014-01-01

    This book introduces Local Binary Patterns (LBP), arguably one of the most powerful texture descriptors, and LBP variants. This volume provides the latest reviews of the literature and a presentation of some of the best LBP variants by researchers at the forefront of textual analysis research and research on LBP descriptors and variants. The value of LBP variants is illustrated with reported experiments using many databases representing a diversity of computer vision applications in medicine, biometrics, and other areas. There is also a chapter that provides an excellent theoretical foundation for texture analysis and LBP in particular. A special section focuses on LBP and LBP variants in the area of face recognition, including thermal face recognition. This book will be of value to anyone already in the field as well as to those interested in learning more about this powerful family of texture descriptors.

  11. Fundamental Characteristics of Industrial Variant Specification Systems

    DEFF Research Database (Denmark)

    Hansen, Benjamin Loer; Hvam, Lars

    2004-01-01

    This paper focuses on the operational task of creating customised variants of industrial specifications (e.g. drawings, routings and bill-of-materials). Rooted in a lack of existing literature on the subject the paper describes the nature of variant specification systems. It introduces some funda...... examples. In general the paper discusses an important focus area within mass customization and build-to-order production: the nature of industrial variant specification systems.......This paper focuses on the operational task of creating customised variants of industrial specifications (e.g. drawings, routings and bill-of-materials). Rooted in a lack of existing literature on the subject the paper describes the nature of variant specification systems. It introduces some...

  12. COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS

    OpenAIRE

    Vasila Packeer Mohamed; Yumi Z. H-Y. Hashim; A. Amid; M. Mel

    2014-01-01

    ABSTRACT: Various methods have been described to extract RNA from adherent mammalian cells. RNA isolation in conjunction with reverse transcription polymerase chain reaction (RT-PCR) is a valuable tool used to study gene expression profiling. This approach is now being used in mammalian cell bioprocessing to help understand and improve the system. The objective of this study was to compare and determine the most suitable RNA extraction method for CHO-K1 cells in a setting where a relatively l...

  13. Identification of an MRAP-Independent Melanocortin-2 Receptor: Functional Expression of the Cartilaginous Fish, Callorhinchus milii, Melanocortin-2 Receptor in CHO Cells

    OpenAIRE

    Reinick, Christina L.; Liang, Liang; Angleson, Joseph K.; Dores, Robert M.

    2012-01-01

    Phylogenetic analyses indicate that the genome of the cartilaginous fish, Callorhynchus milii (elephant shark), encodes a melanocortin-2 receptor (MC2R) ortholog. Expression of the elephant shark mc2r cDNA in Chinese hamster ovary (CHO) cells revealed that trafficking to the plasma membrane and functional activation of the receptor do not require coexpression with an exogenous melanocortin receptor-2 accessory protein (mrap) cDNA. Ligand selectivity studies indicated that elephant shark MC2R-...

  14. Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP

    Energy Technology Data Exchange (ETDEWEB)

    Sugita, Kazuya; Yamamura, Chiaki; Tabata, Ken-ichi [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); Fujita, Norihisa, E-mail: nori@ph.ritsumei.ac.jp [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); School of Pharmacy, Ristumeikan University, Kusatsu, Shiga 525-8577 (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. Black-Right-Pointing-Pointer These are due to increase in intracellular cAMP concentration. Black-Right-Pointing-Pointer Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. Black-Right-Pointing-Pointer The potencies of fatty acid moiety on LysoPS were oleoyl Greater-Than-Or-Slanted-Equal-To stearoyl > palmitoyl. Black-Right-Pointing-Pointer We propose that GPR174 is a lysophosphatidylserine receptor. -- Abstract: We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell-cell adhesion with localization of actin filaments to cell membrane, and revealed significant delay of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of G{alpha}{sub s} protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with G{alpha}{sub s}, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.

  15. Influence of the host (Cho) and of the cultivation strategy on glycan structures and molecular properties of human thyrotrophin; Influencia do hospedeiro (Cho) e da estrategia de cultivo nas estruturas glicidicas e propriedades moleculares da tireotrofina humana

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, Joao Ezequiel de

    2007-07-01

    A novel, fast and practical two-step purification strategy, consisting of a classical ion exchange and a reversed-phase high performance liquid chromatography (RP-HPLC), for rapidly obtaining CHO-derived hTSH, was set up providing r-hTSH with 70% yield and > 99% purity. A consistent increase of {approx} 60% in the secretion yields of r-hTSH-IPEN was observed by changing cell culture CO{sub 2} conditions from 5% CO{sub 2} to air environment (0.03% CO{sub 2}). The overall quality of the products obtained under both conditions was evaluated for what concerns N-glycan structure, charge isomers and biological activity in comparison with a well known recombinant biopharmaceutical (Thyrogen{sup R}) and with a pituitary reference preparation (p-hTSH) from National Hormone and Pituitary Program (NIDDK, USA). The N-glycans identified in the recombinant preparations were of the complex type, presenting bi-, tri- and tetra-antennary structures, sometimes fucosylated, 86-88% of the identified structures being sialylated at variable levels. The three most abundant structures were monosialylated glycans, representing {approx} 69% of all identified forms in the three preparations. The main difference was found in terms of antennarity, with 8-10% more bi-antennary structures obtained in the absence of CO{sub 2} and 7-9% more tri-antennary structures in its presence. In the case of p-hTSH, complex, high-mannose and hybrid N-glycan structures were identified, most of them containing sialic acid and/or sulphate terminal residues. The two most abundant structures were shown to contain one or two sulphate residues, one of which unexpectedly bound to galactose. The sialic acid-galactose linkage was also determined, having found that 68 3 {+-} 10% was in the {alpha} 2,6 and 32 {+-} 10% in the {alpha}2,3 conformation. No remarkable difference in charge isomers was observed between the three recombinant preparations, the isoelectric focusing profiles showing six distinct bands in the 5

  16. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids.

    Science.gov (United States)

    Sakuma, Tetsushi; Takenaga, Mitsumasa; Kawabe, Yoshinori; Nakamura, Takahiro; Kamihira, Masamichi; Yamamoto, Takashi

    2015-01-01

    Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins. PMID:26473830

  17. The Effect of Leonurus sibiricus Plant Extracts on Stimulating Repair and Protective Activity against Oxidative DNA Damage in CHO Cells and Content of Phenolic Compounds

    Directory of Open Access Journals (Sweden)

    Przemysław Sitarek

    2016-01-01

    Full Text Available Leonurus sibiricus L. has been used as a traditional and medicinal herb for many years in Asia and Europe. This species is known to have antibacterial, anti-inflammatory, and antioxidant activity and has demonstrated a reduction of intracellular reactive oxygen species. All tested extracts of L. sibiricus showed protective and DNA repair stimulating effects in Chinese hamster ovary (CHO cells exposed to H2O2. Preincubation of the CHO cells with 0.5 mg/mL of plant extracts showed increased expression level of antioxidant genes (SOD2, CAT, and GPx. LC-MS/MS and HPLC analyses revealed the presence of nine phenolic compounds in L. sibiricus plant extracts: catechin, verbascoside, two flavonoids (quercetin and rutin, and five phenolic acids (4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid. The roots and aerial parts of in vitro L. sibiricus plant extracts, which had the strongest antioxidant properties, may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, as well as protecting DNA via enhanced activation of the antioxidant genes (SOD2, CAT, and GPx regulating intracellular antioxidant capacity. The content of phenolic compounds in in vitro raised plants was greater than the levels found in plants propagated from seeds.

  18. [Stable and efficient expression of hepatitis B virus S antigen and preS1 epitope fusion protein (S/preS1) in CHO cells].

    Science.gov (United States)

    Yang, Zhenxi; Li, Shichong; Liu, Hong; Zhang, Miao; Ye, Lingling; Wu, Yanzhuo; Xu, Mingbo; Chen, Zhaolie

    2013-12-01

    Hepatitis B surface antigen (HBsAg) carrying preS sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. Here we report the success in achieving efficient and stable expression of hepatitis B virus S antigen and preS1 epitope fusion protein (S/preS1) in CHO cells. The HMRCHEF53u/Neo-S/preS1 expression vector carrying S/preS1 gene was constructed and transfected into CHO-S cells. A stable and high-expression CHO cell line, named 10G6, was selected by ELISA and limiting dilution analysis. Western blotting analysis showed S/preS1 expressed from 10G6 cells possessed both S and preS1 antigenicity. 10G6 cells displayed characters of favorable growth and stable S/preS1 expression in repeated batch cultures as evaluated by viable cell density, viability and S/preS1 concentration. And cultivation of 10G6 cells in fed-batch mode resulted in S/preS1 production at 17-20 mg/L with viable cell density at 7 x 10(6)-10 x 10(6) cells/mL. PMID:24660628

  19. Simultaneous hydrogenation and UV-photolysis experiments of NO in CO-rich interstellar ice analogues; linking HNCO, OCN-, NH2CHO, and NH2OH

    Science.gov (United States)

    Fedoseev, G.; Chuang, K.-J.; van Dishoeck, E. F.; Ioppolo, S.; Linnartz, H.

    2016-08-01

    The laboratory work presented here simulates the chemistry on icy dust grains as typical for the `CO freeze-out stage' in dark molecular clouds. It differs from previous studies in that solid-state hydrogenation and vacuum UV photoprocessing are applied simultaneously to co-depositing molecules. In parallel, the reactions at play are described for fully characterized laboratory conditions. The focus is on the formation of molecules containing both carbon and nitrogen atoms, starting with NO in CO-, H2CO-, and CH3OH-rich ices at 13 K. The experiments yield three important conclusions. (1) Without UV processing hydroxylamine (NH2OH) is formed, as reported previously. (2) With UV processing (energetic) NH2 is formed through photodissociation of NH2OH. This radical is key in the formation of species with an N-C bond. (3) The formation of three N-C bearing species, HNCO, OCN-, and NH2CHO, is observed. The experiments put a clear chemical link between these species; OCN- is found to be a direct derivative of HNCO and the latter is shown to have the same precursor as formamide (NH2CHO). Moreover, the addition of VUV competing channels decreases the amount of NO molecules converted into NH2OH by at least one order of magnitude. Consequently, this decrease in NH2OH formation yield directly influences the amount of NO molecules that can be converted into HNCO, OCN-, and NH2CHO.

  20. A Single Dynamic Metabolic Model Can Describe mAb Producing CHO Cell Batch and Fed-Batch Cultures on Different Culture Media.

    Science.gov (United States)

    Robitaille, Julien; Chen, Jingkui; Jolicoeur, Mario

    2015-01-01

    CHO cell culture high productivity relies on optimized culture medium management under fed-batch or perfused chemostat strategies enabling high cell densities. In this work, a dynamic metabolic model for CHO cells was further developed, calibrated and challenged using datasets obtained under four different culture conditions, including two batch and two fed-batch cultures comparing two different culture media. The recombinant CHO-DXB11 cell line producing the EG2-hFc monoclonal antibody was studied. Quantification of extracellular substrates and metabolites concentration, viable cell density, monoclonal antibody concentration and intracellular concentration of metabolite intermediates of glycolysis, pentose-phosphate and TCA cycle, as well as of energetic nucleotides, were obtained for model calibration. Results suggest that a single model structure with a single set of kinetic parameter values is efficient at simulating viable cell behavior in all cases under study, estimating the time course of measured and non-measured intracellular and extracellular metabolites. Model simulations also allowed performing dynamic metabolic flux analysis, showing that the culture media and the fed-batch strategies tested had little impact on flux distribution. This work thus paves the way to an in silico platform allowing to assess the performance of different culture media and fed-batch strategies.

  1. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

    Directory of Open Access Journals (Sweden)

    Tetsushi Sakuma

    2015-10-01

    Full Text Available Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc gene, in Chinese hamster ovary (CHO cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

  2. Comparative study of the cytotoxic and genotoxic effects of titanium oxide and aluminium oxide nanoparticles in Chinese hamster ovary (CHO-K1) cells

    International Nuclear Information System (INIS)

    The aim of this study was to analyze the cytotoxicity and genotoxicity of titanium oxide (TiO2) and aluminium oxide (Al2O3) nanoparticles (NPs) on Chinese hamster ovary (CHO-K1) cells using neutral red (NR), mitochondrial activity (by MTT assay), sister chromatid exchange (SCE), micronucleus (MN) formation, and cell cycle kinetics techniques. Results showed a dose-related cytotoxic effect evidenced after 24 h by changes in lysosomal and mitochondrial dehydrogenase activity. Interestingly, transmission electronic microscopy (TEM) showed the formation of perinuclear vesicles in CHO-K1 cells after treatment with both NPs during 24 h but no NP was detected in the nuclei. Genotoxic effects were shown by MN frequencies which significantly increased at 0.5 and 1 μg/mL TiO2 and 0.5-10 μg/mL Al2O3. SCE frequencies were higher for cells treated with 1-5 μg/mL TiO2. The absence of metaphases evidenced cytotoxicity for higher concentrations of TiO2. No SCE induction was achieved after treatment with 1-25 μg/mL Al2O3. In conclusion, findings showed cytotoxic and genotoxic effects of TiO2 and Al2O3 NPs on CHO-K1 cells. Possible causes of controversial reports are discussed further on.

  3. A bovine papillomavirus-1 based vector restores the function of the low-density lipoprotein receptor in the receptor-deficient CHO-ldlA7 cell line

    Directory of Open Access Journals (Sweden)

    Ustav Mart

    2002-04-01

    Full Text Available Abstract Background The rationale of using bovine papillomavirus-1 (BPV-1 derived vectors in gene therapy protocols lies in their episomal maintenance at intermediate to high copy number, and stable, high-level expression of the gene products. We constructed the BPV-1 based vector harbouring the human low-density lipoprotein receptor (LDLR gene cDNA and tested its ability to restore the function of the LDLR in the receptor-deficient cell line CHO-ldlA7. Results The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles. Conclusion Bovine papillomavirus type-1 (BPV-1-derived vectors could be suitable for gene therapy due to their episomal maintenance at intermediate to high copy number and stable, high-level expression of the gene products. The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation. In vivo experiments should reveal, whether 1–5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles.

  4. Different Variants of Fundamental Portfolio

    Directory of Open Access Journals (Sweden)

    Tarczyński Waldemar

    2014-06-01

    Full Text Available The paper proposes the fundamental portfolio of securities. This portfolio is an alternative for the classic Markowitz model, which combines fundamental analysis with portfolio analysis. The method’s main idea is based on the use of the TMAI1 synthetic measure and, in limiting conditions, the use of risk and the portfolio’s rate of return in the objective function. Different variants of fundamental portfolio have been considered under an empirical study. The effectiveness of the proposed solutions has been related to the classic portfolio constructed with the help of the Markowitz model and the WIG20 market index’s rate of return. All portfolios were constructed with data on rates of return for 2005. Their effectiveness in 2006- 2013 was then evaluated. The studied period comprises the end of the bull market, the 2007-2009 crisis, the 2010 bull market and the 2011 crisis. This allows for the evaluation of the solutions’ flexibility in various extreme situations. For the construction of the fundamental portfolio’s objective function and the TMAI, the study made use of financial and economic data on selected indicators retrieved from Notoria Serwis for 2005.

  5. Influence of the host (Cho) and of the cultivation strategy on glycan structures and molecular properties of human thyrotrophin

    International Nuclear Information System (INIS)

    A novel, fast and practical two-step purification strategy, consisting of a classical ion exchange and a reversed-phase high performance liquid chromatography (RP-HPLC), for rapidly obtaining CHO-derived hTSH, was set up providing r-hTSH with 70% yield and > 99% purity. A consistent increase of ∼ 60% in the secretion yields of r-hTSH-IPEN was observed by changing cell culture CO2 conditions from 5% CO2 to air environment (0.03% CO2). The overall quality of the products obtained under both conditions was evaluated for what concerns N-glycan structure, charge isomers and biological activity in comparison with a well known recombinant biopharmaceutical (ThyrogenR) and with a pituitary reference preparation (p-hTSH) from National Hormone and Pituitary Program (NIDDK, USA). The N-glycans identified in the recombinant preparations were of the complex type, presenting bi-, tri- and tetra-antennary structures, sometimes fucosylated, 86-88% of the identified structures being sialylated at variable levels. The three most abundant structures were monosialylated glycans, representing ∼ 69% of all identified forms in the three preparations. The main difference was found in terms of antennarity, with 8-10% more bi-antennary structures obtained in the absence of CO2 and 7-9% more tri-antennary structures in its presence. In the case of p-hTSH, complex, high-mannose and hybrid N-glycan structures were identified, most of them containing sialic acid and/or sulphate terminal residues. The two most abundant structures were shown to contain one or two sulphate residues, one of which unexpectedly bound to galactose. The sialic acid-galactose linkage was also determined, having found that 68 3 ± 10% was in the α 2,6 and 32 ± 10% in the α2,3 conformation. No remarkable difference in charge isomers was observed between the three recombinant preparations, the isoelectric focusing profiles showing six distinct bands in the 5.39 - 7.35 pl range. A considerably different distribution

  6. Detecting Malware Variants by Byte Frequency

    Directory of Open Access Journals (Sweden)

    Sheng Yu

    2011-04-01

    Full Text Available In order to make lots of new malwares fast and cheaply, attacker can simply modify the existing malwares based on their binary files to produce new ones, malware variants. Malware variants refer to all the new malwares manually or automatically produced from any existing malware. However, such simple approach to produce malwares can change signatures of the original malware so that the new malware variants can confuse and bypass most of popular signature-based anti-malware tools. In this paper we propose a novel byte frequency based detecting model (BFBDM to deal with the malware variants identification issue. The byte frequency of software refers to the frequency of the different unsigned bytes in the corresponding binary file. In order to implement BFBDM, two metrics, the distance and the similarity between the suspicious software and base sample, a known malware, are defined and calculated. According to the experimental results, we found out that if the distance is low and the similarity is high, the suspicious software is a variant of the selected malware with very high probability. The primary experimental results show that our model is efficient and effective for the identification of malware variants, especially for the manual variant.

  7. Genetic variants in adult liver diseases.

    Science.gov (United States)

    Dröge, C; Häussinger, D; Keitel, V

    2015-12-01

    In the last decades, understanding of genetic variants contributing to liver disease development has considerably improved through novel genotyping techniques. Genetic variants of single genes are known to be decisive for the development of monogenetic liver diseases of varying severity. Identification of genetic variants is an important part of the diagnostic process, e. g. the majority of patients with high iron [Fe] (HFE)-associated hemochromatosis carry the homozygous mutation p.C282Y. Detection of mutations in genes encoding hepatobiliary transport proteins like familial intrahepatic cholestasis 1 (FIC1), bile salt export pump (BSEP), or multidrug resistance protein 3 (MDR3) is the basis to differentiate various forms of intrahepatic cholestasis. Moreover, genetic variants in a variety of genes are known to act as disease modifiers and represent risk factors for disease progression and the development of cirrhosis or even hepatocellular carcinoma. Success of drug treatment or appearance of severe side effects can also be influenced by specific genetic variants. All these aspects underscore the increasing importance of genetic variants, which in the future may help to identify patients at risk for disease progression or help to guide treatment decisions. In the present overview, specific frequent genetic variants are summarized that play roles in monogenetic liver diseases, forms of intrahepatic cholestasis, gallstone development, fatty liver disease, drug-induced liver injury, and liver disease progression as well as hepatocellular carcinoma development. PMID:26666282

  8. Effects of lunar and mars dust simulants on HaCaT keratinocytes and CHO-K1 fibroblasts

    Science.gov (United States)

    Rehders, Maren; Grosshäuser, Bianka B.; Smarandache, Anita; Sadhukhan, Annapurna; Mirastschijski, Ursula; Kempf, Jürgen; Dünne, Matthias; Slenzka, Klaus; Brix, Klaudia

    2011-04-01

    Exposure to lunar dust during Apollo missions resulted in occasional reports of ocular, respiratory and dermal irritations which showed that lunar dust has a risk potential for human health. This is caused by its high reactivity as well as its small size, leading to a wide distribution also inside habitats. Hence, detailed information regarding effects of extraterrestrial lunar dusts on human health is required to best support future missions to moon, mars or other destinations. In this study, we used several methods to assess the specific effects of extraterrestrial dusts onto mammalian skin by exposing HaCaT keratinocytes and CHO-K1 fibroblasts to dusts simulating lunar or mars soils. These particular cell types were chosen because the skin protects the human body from potentially harmful substances and because a well orchestrated program ensures proper wound healing. Keratinocytes and fibroblasts were exposed to the dusts for different durations of time and their effects on morphology and viability of the cells were determined. Cytotoxicity was measured using the MTT assay and by monitoring culture impedance, while phalloidin staining of the actin cytoskeleton was performed to address structural integrity of the cells which was also investigated by propidium iodide intake. It was found that the effects of the two types of dust simulants on the different features of both cell lines varied to a considerable extent. Moreover, proliferation of HaCaT keratinocytes, as analyzed by Ki67 labeling, was suppressed in sub-confluent cultures exposed to lunar dust simulant. Furthermore, experimental evidence is provided for a delay in regeneration of keratinocyte monolayers from scratch-wounding when exposed to lunar dust simulant. The obtained results will facilitate further investigations of dust exposure during wound healing and will ease risk assessment studies e.g., for lunar lander approaches. The investigations will help to determine safety measures to be taken during

  9. Cross-scale predictive modeling of CHO cell culture growth and metabolites using Raman spectroscopy and multivariate analysis.

    Science.gov (United States)

    Berry, Brandon; Moretto, Justin; Matthews, Thomas; Smelko, John; Wiltberger, Kelly

    2015-01-01

    Multi-component, multi-scale Raman spectroscopy modeling results from a monoclonal antibody producing CHO cell culture process including data from two development scales (3 L, 200 L) and a clinical manufacturing scale environment (2,000 L) are presented. Multivariate analysis principles are a critical component to partial least squares (PLS) modeling but can quickly turn into an overly iterative process, thus a simplified protocol is proposed for addressing necessary steps including spectral preprocessing, spectral region selection, and outlier removal to create models exclusively from cell culture process data without the inclusion of spectral data from chemically defined nutrient solutions or targeted component spiking studies. An array of single-scale and combination-scale modeling iterations were generated to evaluate technology capabilities and model scalability. Analysis of prediction errors across models suggests that glucose, lactate, and osmolality are well modeled. Model strength was confirmed via predictive validation and by examining performance similarity across single-scale and combination-scale models. Additionally, accurate predictive models were attained in most cases for viable cell density and total cell density; however, these components exhibited some scale-dependencies that hindered model quality in cross-scale predictions where only development data was used in calibration. Glutamate and ammonium models were also able to achieve accurate predictions in most cases. However, there are differences in the absolute concentration ranges of these components across the datasets of individual bioreactor scales. Thus, glutamate and ammonium PLS models were forced to extrapolate in cases where models were derived from small scale data only but used in cross-scale applications predicting against manufacturing scale batches. PMID:25504860

  10. Investigation of superparamagnetic (Fe3O4) nanoparticles and magnetic field exposures on CHO-K1 cell line

    Science.gov (United States)

    Coker, Zachary; Estlack, Larry; Hussain, Saber; Choi, Tae-Youl; Ibey, Bennett L.

    2016-03-01

    Rapid development in nanomaterial synthesis and functionalization has led to advanced studies in actuation and manipulation of cellular functions for biomedical applications. Often these actuation techniques employ externally applied magnetic fields to manipulate magnetic nanomaterials inside cell bodies in order to drive or trigger desired effects. While cellular interactions with low-frequency magnetic fields and nanoparticles have been extensively studied, the fundamental mechanisms behind these interactions remain poorly understood. Additionally, modern investigations on these concurrent exposure conditions have been limited in scope, and difficult to reproduce. This study presents an easily reproducible method of investigating the biological impact of concurrent magnetic field and nanoparticle exposure conditions using an in-vitro CHO-K1 cell line model, with the purpose of establishing grounds for in-depth fundamental studies of the mechanisms driving cellular-level interactions. Cells were cultured under various nanoparticle and magnetic field exposure conditions from 0 to 500 μg/ml nanoparticle concentrations, and DC, 50 Hz, or 100 Hz magnetic fields with 2.0 mT flux density. Cells were then observed by confocal fluorescence microscopy, and subject to biological assays to determine the effects of concurrent extreme-low frequency magnetic field and nanoparticle exposures on cellnanoparticle interactions, such as particle uptake and cell viability by MTT assay. Current results indicate little to no variation in effect on cell cultures based on magnetic field parameters alone; however, it is clear that deleterious synergistic effects of concurrent exposure conditions exist based on a significant decrease in cell viability when exposed to high concentrations of nanoparticles and concurrent magnetic field.

  11. Cryptanalysis of SIMON Variants with Connections

    DEFF Research Database (Denmark)

    Alizadeh, Javad; Alkhzaimi, Hoda A.; Aref, Mohammad Reza;

    2014-01-01

    for a key-recovery attack and extend them further to attack other variants of SIMON. Moreover, we provide results of key recovery analysis using several impossible differential characteristics starting from 14 out of 32 rounds for SIMON32/64 to 22 out of 72 rounds for SIMON128/256. In some cases...... attacks extend to all variants of SIMON covering more rounds compared to any known results using linear cryptanalysis. We present a key recovery attack against SIMON128/256 which covers 35 out of 72 rounds with data complexity 2123. We have implemented our attacks for small scale variants of SIMON and our...

  12. Characterization of form variants of Xenorhabdus luminescens.

    OpenAIRE

    Gerritsen, L.J.M.; Raay, van, C.; Smits, P H

    1992-01-01

    From Xenorhabdus luminescens XE-87.3 four variants were isolated. One, which produced a red pigment and antibiotics, was luminescent, and could take up dye from culture media, was considered the primary form (XE-red). A pink-pigmented variant (XE-pink) differed from the primary form only in pigmentation and uptake of dye. Of the two other variants, one produced a yellow pigment and fewer antibiotics (XE-yellow), while the other did not produce a pigment or antibiotics (XE-white). Both were le...

  13. TCM Differential Treatment of Cough Variant Asthma

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhong-de; DENG Yi-qi; ZHANG Yu; HAN Yun; LIN Lin; CHAO En-xiang

    2010-01-01

    @@ Cough variant asthma (CVA), also called latent asthma or cough asthma, is a special type of asthma. With gradually deepened understanding of CVA in recent years, good curative effect has been achieved in TCM treatment of CVA.

  14. Development of industrial variant specification systems

    DEFF Research Database (Denmark)

    Hansen, Benjamin Loer

    acquisition and order fulfilment, i.e. the creation of drawings, bill-of-materials, routings, product descriptions, quote letters etc. The present thesis is rooted in the assumption that variant specification systems supporting the cross-functional processes of order acquisition and order fulfilment must...... be developed from a holistic and strategically anchored point of view. Another assumption is that this is a challenge for many industrial companies. Even though the literature presents many considerations on general issues covering new information technology, little work is found on the business perspectives...... to the four research tasks of the Ph.D. project: • Define and describe the variant specification system. • Create a procedure for the development of variant specification systems. • Create concepts, methods and tools to support the analysis and determination of the variant specification task. • Identify...

  15. Splicing variants of porcine synphilin-1

    DEFF Research Database (Denmark)

    Larsen, Knud Erik; Madsen, Lone Bruhn; Farajzadeh, Leila;

    2015-01-01

    %) and to mouse (84%) synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel......RNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa) synphilin-1 cDNA (SNCAIP) and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1) of 919 amino acids which shows a high similarity to human (90...... splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation....

  16. Bisalbuminemia. A new molecular variant, albumin Vancouver.

    Science.gov (United States)

    Frohlich, J; Kozier, J; Campbell, D J; Curnow, J V; Tárnoky, A L

    1978-11-01

    Of 18 members of a Fiji Indian family investigated, eight of the 12 males and two of the six females had an electrophoretically slow-type bisalbuminemia (alloalbuminemia). The albumin was characterized by the hiterto unique ratio of the two bands (Al A 35%: variant 65%), and by dye-binding studies and electrophoretic mobility in different media. The data suggest that this is a new variant, which we propose to call albumin Vancouver (Al Va).

  17. Conditionally replicating HIV and SIV variants.

    Science.gov (United States)

    Das, Atze T; Berkhout, Ben

    2016-05-01

    Conditionally replicating human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) variants that can be switched on and off at will are attractive tools for HIV and SIV research. We constructed HIV and SIV variants in which the natural transcription control mechanism was replaced by the doxycycline (dox)-inducible Tet-On gene expression mechanism. These HIV-rtTA and SIV-rtTA variants are fully replication-competent, but replication is critically dependent on dox administration. We here describe how the dox-dependent virus variants may improve the safety of live-attenuated virus vaccines and how they can be used to study the immune responses that correlate with vaccine-induced protection. Furthermore, we review how these variants were initially designed and subsequently optimized by spontaneous viral evolution. These efforts yielded efficiently replicating and tightly dox-controlled HIV-rtTA and SIV-rtTA variants that replicate in a variety of cell and tissue culture systems, and in human immune system (HIS) mice and macaques, respectively. These viruses can be used as a tool in HIV and SIV biology studies and in vaccine research. We review how HIV-rtTA and SIV-rtTA were used to study the role of the viral TAR and Tat elements in virus replication. PMID:25982510

  18. Discovery of rare variants for complex phenotypes.

    Science.gov (United States)

    Kosmicki, Jack A; Churchhouse, Claire L; Rivas, Manuel A; Neale, Benjamin M

    2016-06-01

    With the rise of sequencing technologies, it is now feasible to assess the role rare variants play in the genetic contribution to complex trait variation. While some of the earlier targeted sequencing studies successfully identified rare variants of large effect, unbiased gene discovery using exome sequencing has experienced limited success for complex traits. Nevertheless, rare variant association studies have demonstrated that rare variants do contribute to phenotypic variability, but sample sizes will likely have to be even larger than those of common variant association studies to be powered for the detection of genes and loci. Large-scale sequencing efforts of tens of thousands of individuals, such as the UK10K Project and aggregation efforts such as the Exome Aggregation Consortium, have made great strides in advancing our knowledge of the landscape of rare variation, but there remain many considerations when studying rare variation in the context of complex traits. We discuss these considerations in this review, presenting a broad range of topics at a high level as an introduction to rare variant analysis in complex traits including the issues of power, study design, sample ascertainment, de novo variation, and statistical testing approaches. Ultimately, as sequencing costs continue to decline, larger sequencing studies will yield clearer insights into the biological consequence of rare mutations and may reveal which genes play a role in the etiology of complex traits. PMID:27221085

  19. Histone variants: key players of chromatin.

    Science.gov (United States)

    Biterge, Burcu; Schneider, Robert

    2014-06-01

    Histones are fundamental structural components of chromatin. Eukaryotic DNA is wound around an octamer of the core histones H2A, H2B, H3, and H4. Binding of linker histone H1 promotes higher order chromatin organization. In addition to their structural role, histones impact chromatin function and dynamics by, e.g., post-translational histone modifications or the presence of specific histone variants. Histone variants exhibit differential expression timings (DNA replication-independent) and mRNA characteristics compared to canonical histones. Replacement of canonical histones with histone variants can affect nucleosome stability and help to create functionally distinct chromatin domains. In line with this, several histone variants have been implicated in the regulation of cellular processes such as DNA repair and transcriptional activity. In this review, we focus on recent progress in the study of core histone variants H2A.X, H2A.Z, macroH2A, H3.3, and CENP-A, as well as linker histone H1 variants, their functions and their links to development and disease.

  20. Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation.

    Science.gov (United States)

    Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian; Wagtberg Sen, Jette; Rasmussen, Søren Kofoed; Kontoravdi, Cleo; Weilguny, Dietmar; Andersen, Mikael Rørdam

    2015-03-01

    Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation-related product quality. In this work, different fed-batch processes with two chemically defined proprietary media and feeds were studied using two IgG-producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N-glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by-products such as NH4(+) and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case-by-case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP-GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (GnTI) and UDP-GlcNAc transporter activities, which may be attributed to high level of NH4+ in the cell culture. Furthermore, galactosylation of the mAb Fc glycans

  1. Conversion of a CHO cell culture process from perfusion to fed-batch technology without altering product quality.

    Science.gov (United States)

    Meuwly, F; Weber, U; Ziegler, T; Gervais, A; Mastrangeli, R; Crisci, C; Rossi, M; Bernard, A; von Stockar, U; Kadouri, A

    2006-05-01

    During the development of a new drug product, it is a common strategy to develop a first-generation process with the aim to rapidly produce material for pre-clinical and early stage clinical trials. At a later stage of the development, a second-generation process is then introduced with the aim to supply late-stage clinical trials as well as market needs. This work was aimed at comparing the performance of two different CHO cell culture processes (perfusion and fed-batch) used for the production of a therapeutically active recombinant glycoprotein at industrial pilot-scale. The first-generation process was based on the Fibra-Cel packed-bed perfusion technology. It appeared during the development of the candidate drug that high therapeutic doses were required (>100mg per dose), and that future market demand would exceed 100 kg per year. This exceeded by far the production capacity of the first-generation process, and triggered a change of technology from a packed-bed perfusion process with limited scale-up capabilities to a fed-batch process with scale-up potential to typical bioreactor sizes of 15m(3) or more. The productivity per bioreactor unit volume (in product m(-3)year(-1)) of the fed-batch process was about 70% of the level reached with the first-generation perfusion process. However, since the packed-bed perfusion system was limited in scale (0.6m(3) maximum) compared to the volumes reached in suspension cultures (15m(3)), the fed-batch was selected as second-generation process. In fact, the overall process performance (in product year(-1)) was about 18-fold higher for the fed-batch compared to the perfusion mode. Data from perfusion and fed-batch harvests samples indicated that comparable product quality (relative abundance of monomers dimers and aggregates; N-glycan sialylation level; isoforms distribution) was obtained in both processes. To further confirm this observation, purification to homogeneity of the harvest material from both processes, followed

  2. A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1.

    Science.gov (United States)

    Gonçalves, Cristine; Gross, Fabian; Guégan, Philippe; Cheradame, Hervé; Midou, Patrick

    2014-11-01

    Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 × 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 μg/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000™, and gene expression is higher than observed with FreeStyle™ and JetPEI®. Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications. PMID:25215936

  3. Comparative study of the cytotoxic and genotoxic effects of titanium oxide and aluminium oxide nanoparticles in Chinese hamster ovary (CHO-K1) cells

    Energy Technology Data Exchange (ETDEWEB)

    Di Virgilio, A.L. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA), Diag. 113 y 64, Correo 16, Suc. 4, La Plata (1900) (Argentina); Reigosa, M. [Instituto Multidisciplinario de Biologia Celular (IMBICE), Calle 526 y Camino Gral. Belgrano (entre 10 y 11), La Plata 1900 (Argentina); Arnal, P.M. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA), Diag. 113 y 64, Correo 16, Suc. 4, La Plata 1900 (Argentina); Fernandez Lorenzo de Mele, M., E-mail: mmele@inifta.unlp.edu.ar [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA), Diag. 113 y 64, Correo 16, Suc. 4, La Plata 1900 (Argentina)

    2010-05-15

    The aim of this study was to analyze the cytotoxicity and genotoxicity of titanium oxide (TiO{sub 2}) and aluminium oxide (Al{sub 2}O{sub 3}) nanoparticles (NPs) on Chinese hamster ovary (CHO-K1) cells using neutral red (NR), mitochondrial activity (by MTT assay), sister chromatid exchange (SCE), micronucleus (MN) formation, and cell cycle kinetics techniques. Results showed a dose-related cytotoxic effect evidenced after 24 h by changes in lysosomal and mitochondrial dehydrogenase activity. Interestingly, transmission electronic microscopy (TEM) showed the formation of perinuclear vesicles in CHO-K1 cells after treatment with both NPs during 24 h but no NP was detected in the nuclei. Genotoxic effects were shown by MN frequencies which significantly increased at 0.5 and 1 {mu}g/mL TiO{sub 2} and 0.5-10 {mu}g/mL Al{sub 2}O{sub 3}. SCE frequencies were higher for cells treated with 1-5 {mu}g/mL TiO{sub 2}. The absence of metaphases evidenced cytotoxicity for higher concentrations of TiO{sub 2}. No SCE induction was achieved after treatment with 1-25 {mu}g/mL Al{sub 2}O{sub 3}. In conclusion, findings showed cytotoxic and genotoxic effects of TiO{sub 2} and Al{sub 2}O{sub 3} NPs on CHO-K1 cells. Possible causes of controversial reports are discussed further on.

  4. miRNA profiling of high, low and non-producing CHO cells during biphasic fed-batch cultivation reveals process relevant targets for host cell engineering.

    Science.gov (United States)

    Stiefel, Fabian; Fischer, Simon; Sczyrba, Alexander; Otte, Kerstin; Hesse, Friedemann

    2016-05-10

    Fed-batch cultivation of recombinant Chinese hamster ovary (CHO) cell lines is one of the most widely used production modes for commercial manufacturing of recombinant protein therapeutics. Furthermore, fed-batch cultivations are often conducted as biphasic processes where the culture temperature is decreased to maximize volumetric product yields. However, it remains to be elucidated which intracellular regulatory elements actually control the observed pro-productive phenotypes. Recently, several studies have revealed microRNAs (miRNAs) to be important molecular switches of cell phenotypes. In this study, we analyzed miRNA profiles of two different recombinant CHO cell lines (high and low producer), and compared them to a non-producing CHO DG44 host cell line during fed-batch cultivation at 37°C versus a temperature shift to 30°C. Taking advantage of next-generation sequencing combined with cluster, correlation and differential expression analyses, we could identify 89 different miRNAs, which were differentially expressed in the different cell lines and cultivation phases. Functional validation experiments using 19 validated target miRNAs confirmed that these miRNAs indeed induced changes in process relevant phenotypes. Furthermore, computational miRNA target prediction combined with functional clustering identified putative target genes and cellular pathways, which might be regulated by these miRNAs. This study systematically identified novel target miRNAs during different phases and conditions of a biphasic fed-batch production process and functionally evaluated their potential for host cell engineering. PMID:27002234

  5. Macrophage receptor with collagenous structure (MARCO) is a dynamic adhesive molecule that enhances uptake of carbon nanotubes by CHO-K1 Cells

    International Nuclear Information System (INIS)

    The toxicity of carbon nanotubes (CNTs), a highly promising nanomaterial, is similar to that of asbestos because both types of particles have a fibrous shape and are biopersistent. Here, we investigated the characteristics of macrophage receptor with collagenous structure (MARCO), a membrane receptor expressed on macrophages that recognizes environmental or unopsonized particles, and we assessed whether and how MARCO was involved in cellular uptake of multi-walled CNTs (MWCNTs). MARCO-transfected Chinese hamster ovary (CHO-K1) cells took up polystyrene beads irrespective of the particle size (20 nm–1 μm). In the culture of MARCO-transfected CHO-K1 cells dendritic structures were observed on the bottom of culture dishes, and the edges of these dendritic structures were continually renewed as the cell body migrated along the dendritic structures. MWCNTs were first tethered to the dendritic structures and then taken up by the cell body. MWCNTs appeared to be taken up via membrane ruffling like macropinocytosis, rather than phagocytosis. The cytotoxic EC50 value of MWCNTs in MARCO-transfected CHO-K1 cells was calculated to be 6.1 μg/mL and transmission electron microscopic observation indicated that the toxicity of MWCNTs may be due to the incomplete inclusion of MWCNTs by the membrane structure. -- Highlights: ►Carbon nanotubes (CNTs) were tethered to MARCO in vitro. ►CNTs were taken up rapidly into the cell body via MARCO by membrane ruffling. ►The incomplete inclusion of CNTs by membranes caused cytotoxicity.

  6. Macrophage receptor with collagenous structure (MARCO) is a dynamic adhesive molecule that enhances uptake of carbon nanotubes by CHO-K1 Cells

    Energy Technology Data Exchange (ETDEWEB)

    Hirano, Seishiro, E-mail: seishiro@nies.go.jp [Environmental Nanotoxicology Project, RCER, National Institute for Environmental Studies (Japan); Fujitani, Yuji; Furuyama, Akiko [Environmental Nanotoxicology Project, RCER, National Institute for Environmental Studies (Japan); Kanno, Sanae [Department of Legal Medicine, St. Marianna School of Medicine (Japan)

    2012-02-15

    The toxicity of carbon nanotubes (CNTs), a highly promising nanomaterial, is similar to that of asbestos because both types of particles have a fibrous shape and are biopersistent. Here, we investigated the characteristics of macrophage receptor with collagenous structure (MARCO), a membrane receptor expressed on macrophages that recognizes environmental or unopsonized particles, and we assessed whether and how MARCO was involved in cellular uptake of multi-walled CNTs (MWCNTs). MARCO-transfected Chinese hamster ovary (CHO-K1) cells took up polystyrene beads irrespective of the particle size (20 nm–1 μm). In the culture of MARCO-transfected CHO-K1 cells dendritic structures were observed on the bottom of culture dishes, and the edges of these dendritic structures were continually renewed as the cell body migrated along the dendritic structures. MWCNTs were first tethered to the dendritic structures and then taken up by the cell body. MWCNTs appeared to be taken up via membrane ruffling like macropinocytosis, rather than phagocytosis. The cytotoxic EC{sub 50} value of MWCNTs in MARCO-transfected CHO-K1 cells was calculated to be 6.1 μg/mL and transmission electron microscopic observation indicated that the toxicity of MWCNTs may be due to the incomplete inclusion of MWCNTs by the membrane structure. -- Highlights: ►Carbon nanotubes (CNTs) were tethered to MARCO in vitro. ►CNTs were taken up rapidly into the cell body via MARCO by membrane ruffling. ►The incomplete inclusion of CNTs by membranes caused cytotoxicity.

  7. The Protective Effect of Rosemary Acid on DNA Damage of CHO Cells%迷迭香酸对CHO细胞DNA损伤的防护作用

    Institute of Scientific and Technical Information of China (English)

    夭建华; 高茜; 李雪梅; 黄海涛; 管莹; 米其利; 罗瑛

    2016-01-01

    采用高内涵分析技术测定DNA损伤/双键断裂的标志物γH2AX,来研究迷迭香酸对细胞DNA损伤的防护作用。即通过在体外对CHO细胞施予迷迭香酸,再诱导DNA损伤,对致损后的细胞进行γH2AX及Hoechst双荧光染色标记,最后通过分析γH2AX的荧光强度从而对细胞DNA损伤程度进行半定量检测。结果发现,施予迷迭香酸的细胞与对照相比较,DNA损伤程度降低,表明迷迭香酸对CHO细胞的DNA损伤具有防护作用。%To investigate the protective effect of rosemary acid on DNA damage,the DNA damage/double strand breaks biomarkerγH2AX was detected by using high content analysis technique. CHO cells were treated with rosemary acid, and then DNA damage was induced. Cells were double staining withγH2AX and Hoechst, andfinally the obtainedfluorescence intensity ofγH2AX was used to semi - quantitatively characterize the degree of DNA damage. The results showed that comparing with the control, CHO cells treated with rosemary acid had a lower degree of DNA damage, which indicated that the rosemary acid had protective effect on the DNA damage of CHO cells.

  8. Evidence for transketolase-like TKTL1 flux in CHO cells based on parallel labeling experiments and (13)C-metabolic flux analysis.

    Science.gov (United States)

    Ahn, Woo Suk; Crown, Scott B; Antoniewicz, Maciek R

    2016-09-01

    The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. It provides precursors for the biosynthesis of nucleotides and contributes to the production of reducing power in the form of NADPH. It has been hypothesized that mammalian cells may contain a hidden reaction in PPP catalyzed by transketolase-like protein 1 (TKTL1) that is closely related to the classical transketolase enzyme; however, until now there has been no direct experimental evidence for this reaction. In this work, we have applied state-of-the-art techniques in (13)C metabolic flux analysis ((13)C-MFA) based on parallel labeling experiments and integrated flux fitting to estimate the TKTL1 flux in CHO cells. We identified a set of three parallel labeling experiments with [1-(13)C]glucose+[4,5,6-(13)C]glucose, [2-(13)C]glucose+[4,5,6-(13)C]glucose, and [3-(13)C]glucose+[4,5,6-(13)C]glucose and developed a new method to measure (13)C-labeling of fructose 6-phosphate by GC-MS that allows intuitive interpretation of mass isotopomer distributions to determine key fluxes in the model, including glycolysis, oxidative PPP, non-oxidative PPP, and the TKTL1 flux. Using these tracers we detected a significant TKTL1 flux in CHO cells at the stationary phase. The flux results suggest that the main function of oxidative PPP in CHO cells at the stationary phase is to fuel the TKTL1 reaction. Overall, this study demonstrates for the first time that carbon atoms can be lost in the PPP, by means other than the oxidative PPP, and that this loss of carbon atoms is consistent with the hypothesized TKTL1 reaction in mammalian cells. PMID:27174718

  9. Effects of γ (60Co) and β (90Sr) radiations in Chinese hamster ovarian cells (CHO-K1): induction of micronuclei and cell death

    International Nuclear Information System (INIS)

    Among various types of ionising radiation, the beta emitter radionuclides are involved in many sectors of human activity, such as nuclear medicine, nuclear industries and biomedicine, with a consequent increased risk of accidental, occupational or therapeutic exposure. Despite their recognized importance, there is little information about the effect of beta particles at the cellular level when compared to other types of ionizing radiation. Thus, the objective of the present study was to evaluate the genotoxic and cytotoxic effects of 90Sr, a pure, highly energetic beta source, on CHO-K1 cells and to compare them with data obtained with 60Co. CHO-K1 cells irradiated with different doses (1.0, 2.5, 5.0, 7.5 Gy) of 60Co (0.34 Gy.min-1) and 90Sr (0.23 Gy.min-1) were processed for analysis of clonogenic death, induction of micronuclei (MN) and necrotic and apoptotic death. The survival curves obtained for both types of radiation were better fitted by the linear-quadratic model and were similar. However, the cytogenetic results showed that both the proportion of micronucleated cells and the magnitude of radioinduced lesions demonstrated by the analysis of MN distribution were significantly higher in cells irradiated with 60Co than in cells irradiated with 90Sr, whereas 90Sr was more damaging than 60Co in terms of cell death induction. Necrosis was the major type of death observed in CHO-K1 cells. The data obtained suggest that the low incidence of micronucleated cells after exposure to 90Sr may be a consequence of selective elimination of severely damaged cells from the population by the necrotic process at a higher rate than observed with 60Co exposure. The data obtained also demonstrated the need to use several parameters for a better estimate of cellular sensitivity to the action of genotoxic agents, which would be important in terms of radiobiology, oncology and therapeutics. (author)

  10. Endoplasmic reticulum-directed recombinant mRNA displays subcellular localization equal to endogenous mRNA during transient expression in CHO cells

    DEFF Research Database (Denmark)

    Beuchert Kallehauge, Thomas; Kol, Stefan; Andersen, Mikael Rørdam;

    2016-01-01

    When expressing pharmaceutical recombinant proteins in mammalian cells, the protein is commonly directed through the secretory pathway, in a signal peptide-dependent manner, to acquire specific post-translational modifications and to facilitate secretion into the culture medium. One key premise...... for this is the direction of the mRNA encoding the recombinant protein to the surface of the endoplasmic reticulum (ER) for subsequent protein translocation into the secretory pathway. To evaluate the efficiency of this process in Chinese hamster ovary (CHO) cells, the subcellular localization of recombinant mRNA encoding...

  11. MeIQx-induced DNA adduct formation and mutagenesis in DNA repair deficient CHO cells expressing human CYP1A1 and rapid or slow acetylator NAT2

    OpenAIRE

    Bendaly, Jean; Zhao, Shuang; Neale, Jason R.; Metry, Kristin J.; Doll, Mark A; States, J. Christopher; Pierce, William M.; Hein, David W.

    2007-01-01

    2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) is one of the most potent and abundant mutagens in the western diet. Bioactivation includes N-hydroxylation catalyzed by cytochrome P450s followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). Nucleotide excision repair-deficient chinese hamster ovary (CHO) cells were constructed by stable transfection of human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alle...

  12. Evaluation of the radio modifier effect of propolis on chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60-CO

    International Nuclear Information System (INIS)

    In the last decades, it has been given a great interest to investigations concerning natural, effective, nontoxic compounds with radioprotective potential together with the increasing utilization of different types of ionizing radiation for various applications. Among them propolis, a resinous compound produced by honeybees (Apis mellifera), has been considered quite promising, since it presents several advantageous biological characteristics, i. e., anti-inflammatory, antimicrobial, anticarcinogenic, antioxidant and also free radical scavenging action. The purpose of the present study was to evaluate the effect of Brazilian propolis, collected in the State of Rio Grande do Sul, on Chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60Co γ radiation. For this purpose, three interlinked parameters were analyzed: micronucleus induction, cell viability and clonogenic death. The choice of these parameters was justified by their biological significance, in addition to the fact that they are readily observable and measurable in irradiated cells. The cytogenetic data obtained showed a radioprotective effect of propolis (5-100 μg/ml) in the induction of DNA damage for both cell lines, irradiated with doses of 1 - 4 Gy. The cytotoxicity assay, however, showed a prominent antiproliferative effect of propolis (50 - 400μ/ml) in PC3 cells irradiated with 5 Gγ. The survival curves obtained were adequately fitted by a linear-quadratic model, where the α coefficient was higher in CHO-K1 cells. Concerning the clonogenic capacity, PC3 cells were more radiosensitive than CHO-K1 cells at the higher doses of the survival curve. Propolis at the concentrations of 30 - 100 μg/ml, did not influence the clonogenic potential of PC3 cells, since the survival curves, associated or not with propolis, were found similar, although the combined treatment in CHO-K1 cells exhibited a stimulating proliferative effect. The data obtained in vitro showed a

  13. Functional bioassays for immune monitoring of high-risk neuroblastoma patients treated with ch14.18/CHO anti-GD2 antibody.

    Directory of Open Access Journals (Sweden)

    Nikolai Siebert

    Full Text Available Effective treatment of high-risk neuroblastoma (NB remains a major challenge in pediatric oncology. Human/mouse chimeric monoclonal anti-GD2 antibody (mAb ch14.18 is emerging as a treatment option to improve outcome. After establishing a production process in Chinese hamster ovary (CHO cells, ch14.18/CHO was made available in Europe for clinical trials. Here, we describe validated functional bioassays for the purpose of immune monitoring of these trials and demonstrate GD2-specific immune effector functions of ch14.18/CHO in treated patients. Two calcein-based bioassays for complement-dependent- (CDC and antibody-dependent cellular cytotoxicity (ADCC were set up based on patient serum and immune cells tested against NB cells. For this purpose, we identified LA-N-1 NB cells as best suited within a panel of cell lines. Assay conditions were first established using serum and cells of healthy donors. We found an effector-to-target (E:T cell ratio of 20:1 for PBMC preparations as best suited for GD2-specific ADCC analysis. A simplified method of effector cell preparation by lysis of erythrocytes was evaluated revealing equivalent results at an E:T ratio of 40:1. Optimal results for CDC were found with a serum dilution at 1:8. For validation, both within-assay and inter-assay precision were determined and coefficients of variation (CV were below 20%. Sample quality following storage at room temperature (RT showed that sodium-heparin-anticoagulated blood and serum are stable for 48 h and 96 h, respectively. Application of these bioassays to blood samples of three selected high-risk NB patients treated with ch14.18/CHO (100 mg/m(2 revealed GD2-specific increases in CDC (4.5-9.4 fold and ADCC (4.6-6.0 fold on day 8 compared to baseline, indicating assay applicability for the monitoring of multicenter clinical trials requiring sample shipment at RT for central lab analysis.

  14. Nuclear receptor variants in liver disease.

    Science.gov (United States)

    Zimmer, Vincent; Liebe, Roman; Lammert, Frank

    2015-01-01

    This snapshot reviews the current state of knowledge on genetic variants of nuclear receptors (NRs) involved in regulating various aspects of liver metabolism. Interindividual differences in responses to diet and other 'in-' and environmental stressors can be caused by variants in components of the NR regulatory gene network. We recapitulate recent evidence for the application of NRs in genetic diagnosis of monogenic liver disease. Genetic analysis of multifactorial liver diseases, such as nonalcoholic fatty liver disease and diabetes mellitus, pinpoints key players in disease predisposition and progression. In particular, NR1H4 variants have been associated with intrahepatic cholestasis of pregnancy and gallstone disease. Other examples include studies of NR1I2 and NR1I3 polymorphisms in patients with drug-induced liver injury and NR5A2 variation in cholangiocarcinoma. Associations of NR gene variants have been identified in patients with dyslipidemia and other metabolic syndrome-associated traits by genome-wide studies. Evidence from these analyses confirms a role for NR variation in common diseases, linking regulatory networks to complex and variable phenotypes. These new insights into the impact of NR variants offer perspectives for their future use in diagnosis and treatment of common diseases. PMID:26045277

  15. Word Variant Identification in Old French

    Directory of Open Access Journals (Sweden)

    Peter Willett

    1997-01-01

    Full Text Available Increasing numbers of historical texts are available in machine-readable form, which retain the original spelling, which can be very different from the modern-day equivalents due to the natural evolution of a language, and because the concept of standardisation in spelling is comparatively modern. Among medieval vernacular writers, the same word could be spelled in different ways and the same author (or scribe might even use several alternative spellings in the same passage. Thus, we do not know,a priori, how many variant forms of a particular word there are in such texts, let alone what these variants might be. Searching on the modern equivalent, or even the commonest historical variant, of a particular word may thus fail to retrieve an appreciable number of occurrences unless the searcher already has an extensive knowledge of the language of the documents. Moreover, even specialist scholars may be unaware of some idiosyncratic variants. Here, we consider the use of computer methods to retrieve variant historical spellings.

  16. Hemoglobin variants: biochemical properties and clinical correlates.

    Science.gov (United States)

    Thom, Christopher S; Dickson, Claire F; Gell, David A; Weiss, Mitchell J

    2013-03-01

    Diseases affecting hemoglobin synthesis and function are extremely common worldwide. More than 1000 naturally occurring human hemoglobin variants with single amino acid substitutions throughout the molecule have been discovered, mainly through their clinical and/or laboratory manifestations. These variants alter hemoglobin structure and biochemical properties with physiological effects ranging from insignificant to severe. Studies of these mutations in patients and in the laboratory have produced a wealth of information on hemoglobin biochemistry and biology with significant implications for hematology practice. More generally, landmark studies of hemoglobin performed over the past 60 years have established important paradigms for the disciplines of structural biology, genetics, biochemistry, and medicine. Here we review the major classes of hemoglobin variants, emphasizing general concepts and illustrative examples.

  17. Genetics in psychiatry: common variant association studies

    Directory of Open Access Journals (Sweden)

    Buxbaum Joseph D

    2010-03-01

    Full Text Available Abstract Many psychiatric conditions and traits are associated with significant heritability. Genetic risk for psychiatric conditions encompass rare variants, identified due to major effect, as well as common variants, the latter analyzed by association analyses. We review guidelines for common variant association analyses, undertaking after assessing evidence of heritability. We highlight the importance of: suitably large sample sizes; an experimental design that controls for ancestry; careful data cleaning; correction for multiple testing; small P values for positive findings; assessment of effect size for positive findings; and, inclusion of an independent replication sample. We also note the importance of a critical discussion of any prior findings, biological follow-up where possible, and a means of accessing the raw data.

  18. Evaluación Genotóxica del D-003 en el ensayo de aberraciones cromosómicas en cultivo de células CHO

    Directory of Open Access Journals (Sweden)

    Rafael Gámez

    2010-01-01

    Full Text Available El D-003 es una mezcla de ácidos alifáticos primarios de alto peso molecular purificada de la cera de caña, con efectos antiosteoporóticos, hipolipemiantes y antioxidantes. El objetivo de este estudio fue investigar si el D-003 induce aberraciones cromosómicas en cultivo de células CHO. Diferentes concentraciones de D-003 (50, 500, 1000, 2500 y 5000¿g/ml se añadieron a los cultivos en presencia o ausencia de activación metabólica, incluyéndose controles positivos y negativos. Se examinaron 200 metafases por grupo, cuantificando el número de aberraciones cromosómicas por metafases y las frecuencias de células con aberraciones. El D-003 añadido a dosis de hasta 5000¿g/mL a los cultivos de células CHO no indujo aberraciones cromosómicas ni citotoxicidad en presencia o ausencia de activación metabólica respecto al control negativo, ni tampoco se apreciaron tendencias con las dosis. El índice mitótico no se modificó con el tratamiento, lo que indica que el D-003 no afectó la proliferación celular.

  19. Optimization protein productivity of human interleukin-2 through codon usage, gene copy number and intracellular tRNA concentration in CHO cells.

    Science.gov (United States)

    Ou, Kua-Chun; Wang, Chih-Yang; Liu, Kuan-Ting; Chen, Yi-Ling; Chen, Yi-Chen; Lai, Ming-Derg; Yen, Meng-Chi

    2014-11-14

    Transfer RNA (tRNA) abundance is one of the critical factors for the enhancement of protein productivity in prokaryotic and eukaryotic hosts. Gene copy number of tRNA and tRNA codon usage bias are generally used to match tRNA abundance of protein-expressing hosts and to optimize the codons of recombinant proteins. Because sufficient concentration of intracellular tRNA and optimized codons of recombinant proteins enhanced translation efficiency, we hypothesized that sufficient supplement of host's tRNA improved protein productivity in mammalian cells. First, the small tRNA sequencing results of CHO-K1 cells showed moderate positive correlation with gene copy number and codon usage bias. Modification of human interleukin-2 (IL-2) through codons with high gene copy number and high codon usage bias (IL-2 HH, modified on Leu, Thr, Glu) significantly increased protein productivity in CHO-K1 cells. In contrast, modification through codons with relatively high gene copy number and low codon usage bias (IL-2 HL, modified on Ala, Thr, Val), or relatively low gene copy number and low codon usage bias (IL-2 LH, modified on Ala, Thr, Val) did not increase IL-2 productivity significantly. Furthermore, supplement of the alanine tRNA or threonine tRNA increased IL-2 productivity of IL-2 HL. In summary, we revealed a potential strategy to enhance productivity of recombinant proteins, which may be applied in production of protein drug or design of DNA vaccine.

  20. Quantitative definition and monitoring of the host cell protein proteome using iTRAQ - a study of an industrial mAb producing CHO-S cell line.

    Science.gov (United States)

    Chiverton, Lesley M; Evans, Caroline; Pandhal, Jagroop; Landels, Andrew R; Rees, Byron J; Levison, Peter R; Wright, Phillip C; Smales, C Mark

    2016-08-01

    There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO-S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs. PMID:27214759

  1. DNA display selection of peptide ligands for a full-length human G protein-coupled receptor on CHO-K1 cells.

    Directory of Open Access Journals (Sweden)

    Nobuhide Doi

    Full Text Available The G protein-coupled receptors (GPCRs, which form the largest group of transmembrane proteins involved in signal transduction, are major targets of currently available drugs. Thus, the search for cognate and surrogate peptide ligands for GPCRs is of both basic and therapeutic interest. Here we describe the application of an in vitro DNA display technology to screening libraries of peptide ligands for full-length GPCRs expressed on whole cells. We used human angiotensin II (Ang II type-1 receptor (hAT1R as a model GPCR. Under improved selection conditions using hAT1R-expressing Chinese hamster ovary (CHO-K1 cells as bait, we confirmed that Ang II gene could be enriched more than 10,000-fold after four rounds of selection. Further, we successfully selected diverse Ang II-like peptides from randomized peptide libraries. The results provide more precise information on the sequence-function relationships of hAT1R ligands than can be obtained by conventional alanine-scanning mutagenesis. Completely in vitro DNA display can overcome the limitations of current display technologies and is expected to prove widely useful for screening diverse libraries of mutant peptide and protein ligands for receptors that can be expressed functionally on the surface of CHO-K1 cells.

  2. Communication: Photodissociation of CH3CHO at 308 nm: Observation of H-roaming, CH3-roaming, and transition state pathways together along the ground state surface

    Science.gov (United States)

    Li, Hou-Kuan; Tsai, Po-Yu; Hung, Kai-Chan; Kasai, Toshio; Lin, King-Chuen

    2015-01-01

    Following photodissociation of acetaldehyde (CH3CHO) at 308 nm, the CO(v = 1-4) fragment is acquired using time-resolved Fourier-transform infrared emission spectroscopy. The CO(v = 1) rotational distribution shows a bimodal feature; the low- and high-J components result from H-roaming around CH3CO core and CH3-roaming around CHO radical, respectively, in consistency with a recent assignment by Kable and co-workers (Lee et al., Chem. Sci. 5, 4633 (2014)). The H-roaming pathway disappears at the CO(v ≥ 2) states, because of insufficient available energy following bond-breaking of H + CH3CO. By analyzing the CH4 emission spectrum, we obtained a bimodal vibrational distribution; the low-energy component is ascribed to the transition state (TS) pathway, consistent with prediction by quasiclassical trajectory calculations, while the high-energy component results from H- and CH3-roamings. A branching fraction of H-roaming/CH3-roaming/TS contribution is evaluated to be (8% ± 3%)/(68% ± 10%)/(25% ± 5%), in which the TS pathway was observed for the first time. The three pathways proceed concomitantly along the electronic ground state surface.

  3. Quantitative definition and monitoring of the host cell protein proteome using iTRAQ - a study of an industrial mAb producing CHO-S cell line.

    Science.gov (United States)

    Chiverton, Lesley M; Evans, Caroline; Pandhal, Jagroop; Landels, Andrew R; Rees, Byron J; Levison, Peter R; Wright, Phillip C; Smales, C Mark

    2016-08-01

    There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO-S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs.

  4. Sustained productivity in recombinant Chinese Hamster Ovary (CHO cell lines: proteome analysis of the molecular basis for a process-related phenotype

    Directory of Open Access Journals (Sweden)

    Gammell Patrick

    2011-07-01

    Full Text Available Abstract Background The ability of mammalian cell lines to sustain cell specific productivity (Qp over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture. Results Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis and LC-MS/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3 that were differentially expressed in the same direction. Conclusion These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.

  5. Sustained productivity in recombinant Chinese Hamster Ovary (CHO) cell lines: proteome analysis of the molecular basis for a process-related phenotype

    LENUS (Irish Health Repository)

    Meleady, Paula

    2011-07-24

    Abstract Background The ability of mammalian cell lines to sustain cell specific productivity (Qp) over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO) cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture. Results Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis) and LC-MS\\/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3) that were differentially expressed in the same direction. Conclusion These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.

  6. Development of a recombinant CHO cell model for the investigation of CAR and DAF role during early steps of echovirus 6 infection.

    Science.gov (United States)

    Renois, Fanny; Hong, Saw-See; Le Naour, Richard; Gafa, Valérie; Talmud, Déborah; Andréoletti, Laurent; Lévêque, Nicolas

    2011-06-01

    The early steps of echovirus 6 (E6) infection remain poorly understood and the only described receptor for haemagglutinating E6 strains is the decay accelerating factor (DAF). There is, however, accumulating evidence suggesting that E6 interaction with DAF is necessary but not sufficient for infection. In this report, we investigated the role of the coxsackie-adenovirus-receptor (CAR) as a potential DAF co-receptor during E6 infection. Using stably transfected Chinese Hamster Ovary (CHO) cells expressing CAR and DAF receptors, we found that DAF expression allowed attachment of both haemagglutinating and non-haemagglutinating E6 strains but was not sufficient for promoting E6 cell entry. Interestingly, the co-expression of DAF and CAR rendered 0.1-0.2% of cells permissive to some E6 strains' infection. Although our results did not show a major role of the CAR/DAF cooperation for E6 infection, it nevertheless indicated the use of CAR in the cell entry step of some minor E6 quasispecies. Moreover, the present report validates the use of recombinant CHO cells as valuable cellular model for the further characterisation of E6 receptors. PMID:21420451

  7. Histone variants and melanoma: facts and hypotheses.

    Science.gov (United States)

    Konstantinov, Nikifor K; Ulff-Møller, Constance J; Dimitrov, Stefan

    2016-07-01

    Melanoma is the most aggressive form of skin cancer with rising incidence and morbidity. Despite advances in treatment, the 10-yr survival for patients with metastatic disease is less than 10%. During the past few years, ongoing research on different epigenomic aberrations in melanoma has catalyzed better understanding of its pathogenesis and identification of new therapeutics. In our review, we will focus on the role of histone variants, key epigenetic players in melanoma initiation and progression. Specifically, incorporation of histone variants enables additional layers of chromatin structure, and here, we will describe how alterations in this epigenetic behavior impact melanoma.

  8. XVCL: XML-based Variant Configuration Language

    DEFF Research Database (Denmark)

    Jarzabek, Stan; Basset, Paul; Zhang, Hongyu;

    2003-01-01

    XVCL (XML-based Variant Configuration Language) is a meta-programming technique and tool that provides effective reuse mechanisms. XVCL is an open source software developed at the National University of Singapore. Being a modern and versatile version of Bassett's frames, a technology that has...

  9. Cellobiohydrolase I gene and improved variants

    Science.gov (United States)

    Adney, William S.; Decker, Stephen R.; Mc Carter, Suzanne; Baker, John O.; Nieves, Raphael; Himmel, Michael E.; Vinzant, Todd B.

    2008-05-20

    The disclosure provides a method for preparing an active exoglucanase in a heterologous host of eukaryotic origin. The method includes mutagenesis to reduce glycosylation of the exoglucanase when expressed in a heterologous host. It is further disclosed a method to produce variant cellobiohydrolase that is stable at high temperature through mutagenesis.

  10. Splicing variants of porcine synphilin-1

    Directory of Open Access Journals (Sweden)

    Knud Larsen

    2015-09-01

    Full Text Available Parkinson's disease (PD, idiopathic and familial, is characterized by degradation of dopaminergic neurons and the presence of Lewy bodies (LB in the substantia nigra. LBs contain aggregated proteins of which α-synuclein is the major component. The protein synphilin-1 interacts and colocalizes with α-synuclein in LBs. The aim of this study was to isolate and characterize porcine synphilin-1 and isoforms hereof with the future perspective to use the pig as a model for Parkinson's disease. The porcine SNCAIP cDNA was cloned by reverse transcriptase PCR. The spatial expression of SNCAIP mRNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa synphilin-1 cDNA (SNCAIP and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1 of 919 amino acids which shows a high similarity to human (90% and to mouse (84% synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation.

  11. New genetic variants associated with prostate cancer

    Science.gov (United States)

    Researchers have newly identified 23 common genetic variants -- one-letter changes in DNA known as single-nucleotide polymorphisms or SNPs -- that are associated with risk of prostate cancer. These results come from an analysis of more than 10 million SNP

  12. Truncated variants of apolipoprotein B cause hypobetalipoproteinaemia

    Energy Technology Data Exchange (ETDEWEB)

    Collins, D.R.; Knott, T.J.; Pease, R.J.; Powell, L.M.; Wallis, S.C.; Robertson, S.; Pullinger, C.R.; Lloyd, K.; Miller, N.E.; Muller, D.; Scott, J. (MRC Clinical Research Centre, Harrow (England)); Humphries, S.E.; Talmud, P.J. (Charing Cross Sunley Research Centre, London (England)); Milne, R.W.; Marcel, Y.L. (Clinical Research Institute of Montreal, Quebec (Canada))

    1988-09-12

    Familial hypobetalipoproteinaemia is a rare autosomal dominant disorder in which levels of apo-B-containing plasma lipoproteins are approximately half-normal in heterozygotes and virtually absent in homozygotes. Here the authors describe mutations of the apo-B gene that cause two different truncated variants of apo-B in unrelated individuals with hypobetalipoproteinaemia. One variant is predicted to be 1,799 amino acids long and arises from deletion of a single nucleotide (G) from leucine codon 1,794. This protein was found at low levels in very low density and low density lipoprotein fractions in the blood. The second, shorter variant is caused by mutation of a CpG dinucleotide in arginine codon 1,306 converting it to a stop codon and predicting a protein of 1,305 residues. The differences in size and behavior of these two variants compared to apo-B100 or apo-B48 point to domains that may be important for the assembly, secretion or stability of apo-B-containing lipoproteins.

  13. Histone Variants in Development and Diseases

    Institute of Scientific and Technical Information of China (English)

    Ping Chen; Jicheng Zhao; Guohong Li

    2013-01-01

    Eukaryotic genomic DNA is highly packaged into chromatin by histones to fit inside the nucleus.Other than the bulk packaging role of canonical histones with an expression peak at S phase and replication-coupled deposition,different histone variants have evolved distinct regulatory mechanisms for their expression,deposition and functional implications.The diversity of histone variants results in structural plasticity of chromatin and highlights functionally distinct chromosomal domain,which plays critical roles in development from a fertilized egg into a complex organism,as well as in aging and diseases.However,the mechanisms of this fundamental process are poorly understood so far.It is of particular interest to investigate how the variants are incorporated into chromatin and mark specific chromatin states to regulate gene expression,and how they are involved in development and diseases.In this review,we focus on recent progress in studies of epigenetic regulation of three extensively investigated variants including H2A.Z,macroH2A and H3.3,and their functional implications in development and diseases.

  14. Report of a rare anatomic variant

    DEFF Research Database (Denmark)

    De Brucker, Y; Ilsen, B; Muylaert, C;

    2015-01-01

    We report the CT findings in a case of partial anomalous pulmonary venous return (PAPVR) from the left upper lobe in an adult. PAPVR is an anatomic variant in which one to three pulmonary veins drain into the right atrium or its tributaries, rather than into the left atrium. This results in a lef...

  15. Combined analyses of 20 common obesity susceptibility variants

    DEFF Research Database (Denmark)

    Sandholt, Camilla Helene; Sparsø, Thomas; Grarup, Niels;

    2010-01-01

    Genome-wide association studies and linkage studies have identified 20 validated genetic variants associated with obesity and/or related phenotypes. The variants are common, and they individually exhibit small-to-modest effect sizes.......Genome-wide association studies and linkage studies have identified 20 validated genetic variants associated with obesity and/or related phenotypes. The variants are common, and they individually exhibit small-to-modest effect sizes....

  16. Managing Process Variants in the Process Life Cycle

    OpenAIRE

    Hallerbach, A.; Bauer, Th.; Reichert, M.U.

    2007-01-01

    When designing process-aware information systems, often variants of the same process have to be specified. Each variant then constitutes an adjustment of a particular process to specific requirements building the process context. Current Business Process Management (BPM) tools do not adequately support the management of process variants. Usually, the variants have to be kept in separate process models. This leads to huge modeling and maintenance efforts. In particular, more fundamental proces...

  17. Variants in CUL4B are Associated with Cerebral Malformations

    NARCIS (Netherlands)

    Vulto-van Silfhout, Anneke T.; Nakagawa, Tadashi; Bahi-Buisson, Nadia; Haas, Stefan A.; Hu, Hao; Bienek, Melanie; Vissers, Lisenka E. L. M.; Gilissen, Christian; Tzschach, Andreas; Busche, Andreas; Muesebeck, Joerg; Rump, Patrick; Mathijssen, Inge B.; Avela, Kristiina; Somer, Mirja; Doagu, Fatma; Philips, Anju K.; Rauch, Anita; Baumer, Alessandra; Voesenek, Krysta; Poirier, Karine; Vigneron, Jacqueline; Amram, Daniel; Odent, Sylvie; Nawara, Magdalena; Obersztyn, Ewa; Lenart, Jacek; Charzewska, Agnieszka; Lebrun, Nicolas; Fischer, Ute; Nillesen, Willy M.; Yntema, Helger G.; Jarvela, Irma; Ropers, Hans-Hilger; de Vries, Bert B. A.; Brunner, Han G.; van Bokhoven, Hans; Raymond, F. Lucy; Willemsen, Michel A. A. P.; Chelly, Jamel; Xiong, Yue; Barkovich, A. James; Kalscheuer, Vera M.; Kleefstra, Tjitske; de Brouwer, Arjan P. M.

    2015-01-01

    Variants in cullin 4B (CUL4B) are a known cause of syndromic X-linked intellectual disability. Here, we describe an additional 25 patients from 11 families with variants in CUL4B. We identified nine different novel variants in these families and confirmed the pathogenicity of all nontruncating varia

  18. Variants in CUL4B are associated with cerebral malformations

    NARCIS (Netherlands)

    Vulto-van Silfhout, A.T.; Nakagawa, T.; Bahi-Buisson, N.; Haas, S.A.; Hu, H; Bienek, M.; Vissers, L.E.L.M.; Gilissen, C.F.H.A.; Tzschach, A.; Busche, A.; Musebeck, J.; Rump, P.; Mathijssen, I.B.; Avela, K.; Somer, M.; Doagu, F.; Philips, A.K.; Rauch, A.; Baumer, A.; Voesenek, K.E.J.; Poirier, K.; Vigneron, J.; Amram, D.; Odent, S.; Nawara, M.; Obersztyn, E.; Lenart, J.; Charzewska, A.; Lebrun, N.; Fischer, U.; Nillesen, W.M.; Yntema, H.G.; Jarvela, I.; Ropers, H.H.; Vries, B. de; Brunner, H.G.; Bokhoven, H. van; Raymond, F.L.; Willemsen, M.A.A.P.; Chelly, J.; Xiong, Y.; Barkovich, A.J.; Kalscheuer, V.M.; Kleefstra, T.; Brouwer, A.P.M. de

    2015-01-01

    Variants in cullin 4B (CUL4B) are a known cause of syndromic X-linked intellectual disability. Here, we describe an additional 25 patients from 11 families with variants in CUL4B. We identified nine different novel variants in these families and confirmed the pathogenicity of all nontruncating varia

  19. Variant of Rett syndrome and CDKL5 gene

    DEFF Research Database (Denmark)

    Pini, Giorgio; Bigoni, Stefania; Engerström, Ingegerd Witt;

    2012-01-01

    UNLABELLED: Rett syndrome (RTT) is a severe neurodevelopmental disorder affecting almost exclusively females. The Hanefeld variant, or early-onset seizure variant, has been associated with mutations in CDKL5 gene. AIMS: In recent years more than 60 patients with mutations in the CDKL5 gene have...... the general Rett population, suggesting a specific behavioral and cardiorespiratory phenotype of the RTT the Hanefeld variant....

  20. Genetics Home Reference: GM2-gangliosidosis, AB variant

    Science.gov (United States)

    ... of GM2-gangliosidosis, AB variant: Genetic Testing Registry: Tay-Sachs disease, variant AB These resources from MedlinePlus offer ... AB variant Activator Deficiency/GM2 Gangliosidosis Activator-deficient Tay-Sachs disease GM2 Activator Deficiency Disease GM2 gangliosidosis, type ...

  1. Evaluación del potencial bioactivo de fracciones obtenidas de Licania tomentosa en líneas celulares Jurkat y CHO

    OpenAIRE

    Estrada Ortiz, Natalia

    2012-01-01

    En este estudio se evaluó el potencial bioactivo de la planta Licania tomentosa sobre las líneas celulares Jurkat y CHO. Para las fracciones F8 y F10 se obtuvo un IC50 aproximado de 100 µg/mL por medio de MTT en las líneas celulares. Ambas fracciones mostraron citotóxico sobre las líneas celulares, evidenciado en la disminución de la viabilidad celular evaluada por medio del colorante azul de tripano y MTT, y la disminución de la capacidad de clonación. La fracción F8 mostró efecto genotóxic...

  2. Use of a transfected and amplified Drosophila heat shock promoter construction for inducible production of toxic mouse c-myc proteins in CHO cells

    International Nuclear Information System (INIS)

    After transfection and selection with methotrexate, CHO cell lines were established which contained up to 2000 copies of an expression vector for c-myc protein. The vector contained the Drosophila heat shock protein 70 (hsp70) promoter fused with the coding region of the mouse c-myc gene. Incubation of cells for up to 3 hours at 430C resulted in at least a 100-fold induction of recombinant c-myc mRNA. When cells were shifted back to 370C, within 1 to 4 hours, this RNA was translated into protein to yield about 250 μg per 109 cells. Cells died a few hours later, suggesting that high concentrations of intracellular c-myc are cytotoxic. 47 refs., 5 figs

  3. COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS

    Directory of Open Access Journals (Sweden)

    Vasila Packeer Mohamed

    2014-05-01

    Full Text Available ABSTRACT: Various methods have been described to extract RNA from adherent mammalian cells. RNA isolation in conjunction with reverse transcription polymerase chain reaction (RT-PCR is a valuable tool used to study gene expression profiling. This approach is now being used in mammalian cell bioprocessing to help understand and improve the system. The objective of this study was to compare and determine the most suitable RNA extraction method for CHO-K1 cells in a setting where a relatively large amount of samples was involved. Total RNA was extracted using Total RNA purification kit (without DNase treatment; Norgen, Canada and RNeasy mini kit (with DNase treatment; Qiagen, USA respectively. The extracted RNA was then reverse transcribed, and the cDNA was subjected to PCR-amplifying 18S. Yield from RNeasy kit was significantly higher (0.316 ± 0.033 µg/µl; p=0.004 than Total RNA purification kit (0.177 ± 0.0243 µg/µl. However, RNA purity for both methods was close to 2.0 and there was no significant difference between the methods. Total RNA purification kit is less expensive than RNeasy kit. Since there is no DNase treatment step in the former, extraction time for RNA is shorter. When the extracted RNA was subjected to RT-PCR, both methods were able to show detection of 18S at 219 bp.   Therefore, this study demonstrates that both protocols are suitable for RNA extraction for CHO-K1 cells. RNeasy mini kit (Qiagen is recommended if higher yields is the primary concern and Total RNA Purification kit (Norgen is recommended if time and cost are concerned. ABSTRAK: Pelbagai kaedah telah digunakan untuk mengekstrak RNA daripada sel mamalia lekat.  Pemencilan RNA dengan menggunakan reaksi rantai polimerase transkripsi berbalik (RT-PCR merupakan kaedah penting yang digunakan dalam mengkaji pernyataan gen berprofil.  Pendekatan ini kini digunakan dalam pemprosesan bio sel mamalia untuk memahami dan menambah baik sistem.  Tujuan kajian dijalankan

  4. Radiation induced enhancement of the efficiency of DNA mediated gene transfer in UV and x-ray sensitive CHO mutant cell lines

    International Nuclear Information System (INIS)

    The authors present results of experiments studying the enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer in mutant CHO cell lines. The established cell lines UV-5, UV-20 and EM9 are transfected with the recombinant DNA plasmid, pSV2-gpt irradiated with either UV or X-rays and plated in MAX selective media. MAX-resistant colonies are the result of the integration of pSV2-gpt and the expression of the gene coding for the bacterial xanthine-guanine phosphoribosyl transferase. Dose response curves for UV and X-rays are generated for the frequency of MAX-resistant colonies/survivor. From these experiments, the authors hope to delineate the role of DNA repair enzymes in the phenomenon at plasmid DNA integration after DNA mediated gene transfer

  5. Use of a transfected and amplified Drosophila heat shock promoter construction for inducible production of toxic mouse c-myc proteins in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Wurm, F.M.; Gwinn, K.A.; Papoulas, O.; Pallavicini, M.; Kingston, R.E.

    1987-07-24

    After transfection and selection with methotrexate, CHO cell lines were established which contained up to 2000 copies of an expression vector for c-myc protein. The vector contained the Drosophila heat shock protein 70 (hsp70) promoter fused with the coding region of the mouse c-myc gene. Incubation of cells for up to 3 hours at 43/sup 0/C resulted in at least a 100-fold induction of recombinant c-myc mRNA. When cells were shifted back to 37/sup 0/C, within 1 to 4 hours, this RNA was translated into protein to yield about 250 ..mu..g per 10/sup 9/ cells. Cells died a few hours later, suggesting that high concentrations of intracellular c-myc are cytotoxic. 47 refs., 5 figs.

  6. Landfill leachate sludge use as soil additive prior and after electrocoagulation treatment: A cytological assessment using CHO-k1 cells.

    Science.gov (United States)

    Morozesk, M; Bonomo, M M; Rocha, L D; Duarte, I D; Zanezi, E R L; Jesus, H C; Fernandes, M N; Matsumoto, S T

    2016-09-01

    Electrocoagulation has recently attracted attention as a potential technique for treating toxic effluents due to its versatility and environmental compatibility, generating a residue chemically suitable to be used as a soil additive. In the present study, landfill leachate sludge hazardous effects were investigated prior and after electrocoagulation process using in vitro assays with the mammalian cells CHO-k1. An integrated strategy for risk assessment was used to correctly estimate the possible adverse landfill leachate sludge effects on human health and ecosystem. Electrocoagulation process proved to be an effective treatment due to possibility to improve effluent adverse characteristics and produce sludge with potential to be used as soil additive. Despite low cytoxicity, the residue presented genotoxic and mutagenic effects, indicating a capacity to induce genetic damages, probably due to induction of polyploidization process in cells. The observed effects demand an improvement of waste management methods for reduce negative risks of landfill leachate sludge application.

  7. Landfill leachate sludge use as soil additive prior and after electrocoagulation treatment: A cytological assessment using CHO-k1 cells.

    Science.gov (United States)

    Morozesk, M; Bonomo, M M; Rocha, L D; Duarte, I D; Zanezi, E R L; Jesus, H C; Fernandes, M N; Matsumoto, S T

    2016-09-01

    Electrocoagulation has recently attracted attention as a potential technique for treating toxic effluents due to its versatility and environmental compatibility, generating a residue chemically suitable to be used as a soil additive. In the present study, landfill leachate sludge hazardous effects were investigated prior and after electrocoagulation process using in vitro assays with the mammalian cells CHO-k1. An integrated strategy for risk assessment was used to correctly estimate the possible adverse landfill leachate sludge effects on human health and ecosystem. Electrocoagulation process proved to be an effective treatment due to possibility to improve effluent adverse characteristics and produce sludge with potential to be used as soil additive. Despite low cytoxicity, the residue presented genotoxic and mutagenic effects, indicating a capacity to induce genetic damages, probably due to induction of polyploidization process in cells. The observed effects demand an improvement of waste management methods for reduce negative risks of landfill leachate sludge application. PMID:27243586

  8. Photodissociation of CH3CHO at 248 nm by time-resolved Fourier-transform infrared emission spectroscopy: Verification of roaming and triple fragmentation

    Science.gov (United States)

    Hung, Kai-Chan; Tsai, Po-Yu; Li, Hou-Kuan; Lin, King-Chuen

    2014-02-01

    By using time-resolved Fourier-transform infrared emission spectroscopy, the HCO fragment dissociated from acetaldehyde (CH3CHO) at 248 nm is found to partially decompose to H and CO. The fragment yields are enhanced by the Ar addition that facilitates the collision-induced internal conversion. The channels to CH2CO + H2 and CH3CO + H are not detected significantly. The rotational population distribution of CO, after removing the Ar collision effect, shows a bimodal feature comprising both low- and high-rotational (J) components, sharing a fraction of 19% and 81%, respectively, for the vibrational state v = 1. The low-J component is ascribed to both roaming pathway and triple fragmentation. They are determined to have a branching ratio of 0.06, respectively, relative to the whole v = 1 population. The CO roaming is accompanied by a highly vibrational population of CH4 that yields a vibrational bimodality.

  9. A three-step purification strategy for isolation of hamster TIG2 from CHO cells: characterization of two processed endogenous forms.

    Science.gov (United States)

    Busmann, Annette; Walden, Michael; Wendland, Martin; Kutzleb, Christian; Forssmann, Wolf-Georg; John, Harald

    2004-11-25

    We have recently isolated a bioactive, circulating protein of human tazarotene-induced gene-2 (TIG2) as the natural ligand of the orphan receptor ChemR23. Here we describe a simplified method for the isolation of hamster TIG2 protein from Chinese hamster ovary (CHO) cell supernatant. Using a heparin-affinity column followed by two reversed phase chromatography steps resulted in the isolation of pure biologically active material. Two processed bioactive forms of Chinese hamster TIG2 were identified by Edman sequencing and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) mass fingerprint analysis, representing the amino acid residues T20 to F156, and T20 to A155 of the 163 amino acid propeptide. Comparison with the predicted aa-sequence indicates a mutation or modification within the C-terminal end of the peptide. PMID:15522723

  10. Evaluation of cytogenetic effects of a naturally occurring non-ice-nucleation Pseudomonas fluorescens strain in Chinese hamster ovary (CHO) cells.

    Science.gov (United States)

    Caruso, P; Andreozzi, L; Motta, S; Mosesso, P

    1995-01-01

    One of the main methods for eliminating ice-nucleation-active (INA+) bacteria the micro-organisms responsible for frost injuries to plants at mild freezing temperatures, is the use, as competitors, of other naturally occurring non-nucleating strains (non-INA). In the present article we investigated the cytogenetic effects of a naturally occurring non-INA strain of Pseudomonas fluorescens (MS 1640 R3), evaluating the induction of chromosomal aberrations and sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells in the absence and presence of rat S9 metabolism. The results obtained did not show any increase in either chromosomal aberrations or SCEs, both in the absence and presence of rat S9 metabolism when used as i) intact bacteria cells, ii) sonicated bacteria (i.e., potential endotoxins), or iii) metabolic bacterial products (i.e., potential exotoxins) released in the growth medium. PMID:8584981

  11. Méthode impact-écho basse fréquence pour la détection de vide dans les gaines de précontrainte

    OpenAIRE

    ABRAHAM, O; Cote, P.

    2002-01-01

    Un des problèmes majeurs des ouvrages d'art est la détection de vide dans les gaines de précontrainte. Les méthodes couramment utilisées pour leur détection sont la radiographie X et la gammagraphie. Ces techniques requièrent malheureusement des zones de protection étendues contre les radiations. Depuis la fin des années 1980, la méthode impact-écho est aussi proposée. Deux indicateurs de présence de vide sont décrits dans la littérature : le changement de la fréquence de résonance liée à la ...

  12. Mutations induced by 1,3-butadiene metabolites, butadiene diolepoxide, and 1,2,3,4-diepoxybutane at the Hprt locus in CHO-K1 cells.

    Science.gov (United States)

    Lee, Dong-Hyun; Kim, Tae-Ho; Lee, Sun-Young; Kim, Hyun-Jo; Rhee, Seung Keun; Yoon, ByoungSu; Pfeifer, Gerd P; Lee, Chong-Soon

    2002-12-31

    Butadiene (BD) is an important industrial chemical that is classified as a probable human carcinogen. Butadiene diolepoxide (BDE) and 1,2,3,4-diepoxybutane (DEB) are metabolites of carcinogenic BD and contain the DNA-reactive one and two epoxides, respectively. In this study, the mutation frequencies and mutation spectra that are induced by BDE and DEB have been investigated at the hprt locus in CHO-K1 cells. The BDE- and DEB-treated CHO-K1 cells were allowed to grow for several days, then seeded in a medium that contained 6-thioguanine in order to select the hprt mutants. BDE exhibited the mutagenic activity at concentrations that were approximately 100-times higher than DEB. The mutation spectra for BDE and DEB were determined by a reverse transcription-polymerase chain reaction of hprt mRNA, which was followed by automatic DNA sequencing of the PCR products. The mutational spectrum for BDE was exon deletions (16/41), G x C --> A x T transitions (11/41), and A x T --> G x C transitions (5/41). The mutational spectrum for DEB was exon deletions (15/39), G x C --> A x T transitions (11/39), and A x T --> T x A transversions (5/39). The most common base substitution that was induced by both BDE and DEB was G x C --> A x T transitions. The sites of the single base substitutions that were induced by BDE and DEB were guanine and adenine, which was consistent with the DNA adduct profiles. The high frequencies of the exon deletions by each metabolite occurred in the regions of exons 2, 3, or 4. These data indicate that BDE and DEB are mutagenic carcinogens by forming DNA adducts at the site of adenine and guanine, and inducing large exon deletions and single base substitutions. PMID:12521305

  13. ARP2, a novel pro-apoptotic protein expressed in epithelial prostate cancer LNCaP cells and epithelial ovary CHO transformed cells.

    Directory of Open Access Journals (Sweden)

    Jaime Mas-Oliva

    Full Text Available Neoplastic epithelial cells generate the most aggressive types of cancers such as those located in the lung, breast, colon, prostate and ovary. During advanced stages of prostate cancer, epithelial cells are associated to the appearance of androgen-independent tumors, an apoptotic-resistant phenotype that ultimately overgrows and promotes metastatic events. We have previously identified and electrophysiologically characterized a novel Ca(2+-permeable channel activated during apoptosis in the androgen-independent prostate epithelial cancer cell line, LNCaP. In addition, we reported for the first time the cloning and characterization of this channel-like molecule named apoptosis regulated protein 2 (ARP2 associated to a lethal influx of Ca(2+ in Xenopus oocytes. In the present study, LNCaP cells and Chinese hamster ovary cells (CHO cell line transfected with arp2-cDNA are induced to undergo apoptosis showing an important impact on cell viability and activation of caspases 3 and 7 when compared to serum deprived grown cells and ionomycin treated cells. The subcellular localization of ARP2 in CHO cells undergoing apoptosis was studied using confocal microscopy. While apoptosis progresses, ARP2 initially localized in the peri-nuclear region of cells migrates with time towards the plasma membrane region. Based on the present results and those of our previous studies, the fact that ARP2 constitutes a novel cation channel is supported. Therefore, ARP2 becomes a valuable target to modulate the influx and concentration of calcium in the cytoplasm of epithelial cancer cells showing an apoptotic-resistant phenotype during the onset of an apoptotic event.

  14. The Saccharomyces Genome Database Variant Viewer.

    Science.gov (United States)

    Sheppard, Travis K; Hitz, Benjamin C; Engel, Stacia R; Song, Giltae; Balakrishnan, Rama; Binkley, Gail; Costanzo, Maria C; Dalusag, Kyla S; Demeter, Janos; Hellerstedt, Sage T; Karra, Kalpana; Nash, Robert S; Paskov, Kelley M; Skrzypek, Marek S; Weng, Shuai; Wong, Edith D; Cherry, J Michael

    2016-01-01

    The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org) is the authoritative community resource for the Saccharomyces cerevisiae reference genome sequence and its annotation. In recent years, we have moved toward increased representation of sequence variation and allelic differences within S. cerevisiae. The publication of numerous additional genomes has motivated the creation of new tools for their annotation and analysis. Here we present the Variant Viewer: a dynamic open-source web application for the visualization of genomic and proteomic differences. Multiple sequence alignments have been constructed across high quality genome sequences from 11 different S. cerevisiae strains and stored in the SGD. The alignments and summaries are encoded in JSON and used to create a two-tiered dynamic view of the budding yeast pan-genome, available at http://www.yeastgenome.org/variant-viewer. PMID:26578556

  15. Nuclear Receptor Variants in Liver Disease

    Directory of Open Access Journals (Sweden)

    Roman Müllenbach

    2012-01-01

    Full Text Available This review aims to provide a snapshot of the actual state of knowledge on genetic variants of nuclear receptors (NR involved in regulating important aspects of liver metabolism. It recapitulates recent evidence for the application of NR in genetic diagnosis of monogenic (“Mendelian” liver disease and their use in clinical diagnosis. Genetic analysis of multifactorial liver diseases such as viral hepatitis or fatty liver disease identifies key players in disease predisposition and progression. Evidence from these analyses points towards a role of NR polymorphisms in common diseases, linking regulatory networks to complex and variable phenotypes. The new insights into NR variants also offer perspectives and cautionary advice for their use as handles towards diagnosis and treatment.

  16. Wham: Identifying Structural Variants of Biological Consequence.

    Science.gov (United States)

    Kronenberg, Zev N; Osborne, Edward J; Cone, Kelsey R; Kennedy, Brett J; Domyan, Eric T; Shapiro, Michael D; Elde, Nels C; Yandell, Mark

    2015-12-01

    Existing methods for identifying structural variants (SVs) from short read datasets are inaccurate. This complicates disease-gene identification and efforts to understand the consequences of genetic variation. In response, we have created Wham (Whole-genome Alignment Metrics) to provide a single, integrated framework for both structural variant calling and association testing, thereby bypassing many of the difficulties that currently frustrate attempts to employ SVs in association testing. Here we describe Wham, benchmark it against three other widely used SV identification tools-Lumpy, Delly and SoftSearch-and demonstrate Wham's ability to identify and associate SVs with phenotypes using data from humans, domestic pigeons, and vaccinia virus. Wham and all associated software are covered under the MIT License and can be freely downloaded from github (https://github.com/zeeev/wham), with documentation on a wiki (http://zeeev.github.io/wham/). For community support please post questions to https://www.biostars.org/.

  17. Wham: Identifying Structural Variants of Biological Consequence.

    Directory of Open Access Journals (Sweden)

    Zev N Kronenberg

    2015-12-01

    Full Text Available Existing methods for identifying structural variants (SVs from short read datasets are inaccurate. This complicates disease-gene identification and efforts to understand the consequences of genetic variation. In response, we have created Wham (Whole-genome Alignment Metrics to provide a single, integrated framework for both structural variant calling and association testing, thereby bypassing many of the difficulties that currently frustrate attempts to employ SVs in association testing. Here we describe Wham, benchmark it against three other widely used SV identification tools-Lumpy, Delly and SoftSearch-and demonstrate Wham's ability to identify and associate SVs with phenotypes using data from humans, domestic pigeons, and vaccinia virus. Wham and all associated software are covered under the MIT License and can be freely downloaded from github (https://github.com/zeeev/wham, with documentation on a wiki (http://zeeev.github.io/wham/. For community support please post questions to https://www.biostars.org/.

  18. Evaluation of the radio modifier effect of propolis on chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60-CO; Avaliacao do efeito radiomodificador da propolis em celulas de ovario de hamster chines (CHO-K1) e em celulas tumorais de prostata (PC3), irradiadas com CO-60

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Geyza Spigoti

    2011-07-01

    In the last decades, it has been given a great interest to investigations concerning natural, effective, nontoxic compounds with radioprotective potential together with the increasing utilization of different types of ionizing radiation for various applications. Among them propolis, a resinous compound produced by honeybees (Apis mellifera), has been considered quite promising, since it presents several advantageous biological characteristics, i. e., anti-inflammatory, antimicrobial, anticarcinogenic, antioxidant and also free radical scavenging action. The purpose of the present study was to evaluate the effect of Brazilian propolis, collected in the State of Rio Grande do Sul, on Chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with {sup 60}Co {gamma} radiation. For this purpose, three interlinked parameters were analyzed: micronucleus induction, cell viability and clonogenic death. The choice of these parameters was justified by their biological significance, in addition to the fact that they are readily observable and measurable in irradiated cells. The cytogenetic data obtained showed a radioprotective effect of propolis (5-100 {mu}g/ml) in the induction of DNA damage for both cell lines, irradiated with doses of 1 - 4 Gy. The cytotoxicity assay, however, showed a prominent antiproliferative effect of propolis (50 - 400{mu}/ml) in PC3 cells irradiated with 5 G{gamma}. The survival curves obtained were adequately fitted by a linear-quadratic model, where the {alpha} coefficient was higher in CHO-K1 cells. Concerning the clonogenic capacity, PC3 cells were more radiosensitive than CHO-K1 cells at the higher doses of the survival curve. Propolis at the concentrations of 30 - 100 {mu}g/ml, did not influence the clonogenic potential of PC3 cells, since the survival curves, associated or not with propolis, were found similar, although the combined treatment in CHO-K1 cells exhibited a stimulating proliferative effect. The data

  19. Establishment of a Stable CHO-677 Cell Line Expressing Murine αvβ6 Integrin%稳定表达鼠源整联蛋白αvβ6的CHO-677细胞系的构建

    Institute of Scientific and Technical Information of China (English)

    朱志坚; 连凯琪; 杨帆; 张伟; 郑海学; 杨孝朴

    2015-01-01

    口蹄疫是由口蹄疫病毒(FMDV)引起的一种高度接触性传染病,主要侵害偶蹄动物.乳鼠常作为一种重要的实验动物模型用于FMDV的研究;整联蛋白αvβ6是FMDV的重要受体之一.为深入研究整联蛋白αvβ6在FMDV感染乳鼠中所发挥的作用,克隆了乳鼠整联蛋白αvβ6的两个亚基,并将其导入中国仓鼠卵巢细胞(Chinese hamster ovary,CHO-677)基因组中,构建了稳定表达乳鼠整联蛋白αV和β6亚基的细胞系CHO-677-mαvβ6,并分别选用两种不同血清型的野生型FMDV毒株Asia1/HN/CHA/06和O/BY/CHA/2010感染细胞系来分析细胞系对FMDV的易感性.首先通过PCR和间接免疫荧光试验证明了细胞系中整联蛋白αvβ6在基因水平成功导入,在蛋白水平成功表达.然后,通过实时荧光定量RT-PCR检测病毒RNA拷贝数,并结合TCID50试验测定了代表毒株在两个细胞上的生长曲线.结果表明,与亲本细胞CHO-677相比,细胞系CHO-677-mαvβ6对FMDV更易感,从αvβ6的功能性上进一步验证了细胞系被成功构建.

  20. Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population

    DEFF Research Database (Denmark)

    Berquist, Brian R; Singh, Dharmendra Kumar; Fan, Jinshui;

    2010-01-01

    XRCC1 operates as a scaffold protein in base excision repair, a pathway that copes with base and sugar damage in DNA. Studies using recombinant XRCC1 proteins revealed that: a C389Y substitution, responsible for the repair defects of the EM-C11 CHO cell line, caused protein instability; a V86R...... mutation abolished the interaction with POLbeta, but did not disrupt the interactions with PARP-1, LIG3alpha and PCNA; and an E98K substitution, identified in EM-C12, reduced protein integrity, marginally destabilized the POLbeta interaction, and slightly enhanced DNA binding. Two rare (P161L and Y576S......, but did reduce DNA-binding ability. When expressed in HeLa cells, the XRCC1 variants-excluding E98K, which was largely nucleolar, and C389Y, which exhibited reduced expression-exhibited normal nuclear distribution. Most of the protein variants, including the V86R POLbeta-interaction mutant, displayed...

  1. Cough variant asthma and atopic cough

    OpenAIRE

    Magni Chiara; Chellini Elisa; Zanasi Alessandro

    2010-01-01

    Abstract Chronic cough has been reported to be the fifth most common complaint seen by primary care physicians in the world, the third in Italy. Chronic cough in non-smoking, non-treated with ACE-inhibitor adults with normal chest radiogram could be a symptom of asthma and can be sub-classified into: cough-variant asthma, atopic cough, and eosinophilic bronchitis. This review discusses the differential diagnosis of these three disorders.

  2. Variant position of the medial plantar nerve

    OpenAIRE

    Astik RB; Dave UH; Gajendra KS

    2011-01-01

    Knowledge of variation of position of the medial plantar nerve is important for the forefoot surgeon for plantar reconstruction, local injection therapy and an excision of interdigital neuroma. During routine dissection of 50-year-old female cadaver, we found the medial plantar nerve and vessels variably located between plantar aponeurosis and the muscles of the first layer of the sole of the right foot. Due to this variant position, the medial plantar nerve and vessels lose their protection ...

  3. [Lynch syndrome, Muir Torre variant: 2 cases].

    Science.gov (United States)

    Castro-Mujica, María Del Carmen; Barletta-Carrillo, Claudia; Acosta-Aliaga, Marisa; Montenegro-Garreaud, Ximena

    2016-01-01

    Lynch syndrome (LS) is an autosomal-dominant inherited cancer predisposition syndrome caused by germline mutations in DNA mismatch repair genes (MLH1, MSH2, MSH6 or PMS2). Muir-Torre syndrome (MTS) is a phenotypic variant of LS that includes a predisposition to sebaceous glands tumors and keratoacanthomas. We report two patients with MTS, with more than one LS-related cancer, skin lesions, family history of cancer andmicrosatellite instability and immunohistochemistry analysis. PMID:27131946

  4. A Secure Variant of the Hill Cipher

    CERN Document Server

    Toorani, Mohsen; 10.1109/ISCC.2009.5202241

    2010-01-01

    The Hill cipher is a classical symmetric encryption algorithm that succumbs to the know-plaintext attack. Although its vulnerability to cryptanalysis has rendered it unusable in practice, it still serves an important pedagogical role in cryptology and linear algebra. In this paper, a variant of the Hill cipher is introduced that makes the Hill cipher secure while it retains the efficiency. The proposed scheme includes a ciphering core for which a cryptographic protocol is introduced.

  5. Orosomucoid-1 Expression in Ameloblastoma Variants.

    Science.gov (United States)

    García-Muñoz, Alejandro; Bologna-Molina, Ronell; A Rodríguez, Mario; Liceága-Reyes, Rodrigo; Farfán-Morales, Jose Eduardo; Aranda-Romo, Saray; Molina-Frechero, Nelly; González-González, Rogelio

    2016-01-01

    Odontogenic tumors constitute a group of heterogeneous lesions of benign and malignant neoplasms with variable aggressiveness. Ameloblastomas are a group of benign but locally invasive neoplasms that occur in the jaws and are derived from epithelial elements of the tooth-forming apparatus. We previously described orosomucoid-1 protein expression in odontogenic myxomas. However, whether orosomucoid-1 is expressed in other odontogenic tumors remains unknown. Since orosomucoid-1 belongs to a group of acute-phase proteins and has many functions in health and disease, we identified and analyzed orosomucoid-1 expression in ameloblastoma variants and ameloblastic carcinoma using western blot and immunohistochemical techniques. Thirty cases of ameloblastoma were analyzed for orsomucoid-1; five specimens were fresh for western blot study (four benign ameloblastomas and one ameloblastic carcinoma), and 25 cases of benign ameloblastoma for immunohistochemical assays. Orosomucoid-1 was widely expressed in each tumor variant analyzed in this study, and differential orosomucoid-1 expression was observed between benign and malignant tumor. Orosomucoid-1 may play an important role in the behavior of ameloblastomas and influence the biology and development of the variants of this tumor. PMID:27386438

  6. Association of genetic variants with diabetic nephropathy

    Institute of Scientific and Technical Information of China (English)

    Saliha; Rizvi; Syed; Tasleem; Raza; Farzana; Mahdi

    2014-01-01

    Diabetic nephropathy accounts for the most serious microvascular complication of diabetes mellitus. It is suggested that the prevalence of diabetic nephropathy will continue to increase in future posing a major challenge to the healthcare system resulting in increased morbidity and mortality. It occurs as a result of interaction between both genetic and environmental factors in individuals with both type 1 and type 2 diabetes. Genetic susceptibility has been proposed as an important factor for the development and progression of diabetic nephropathy, and various research efforts are being executed worldwide to identify the susceptibility gene for diabetic nephropathy. Numerous single nucleotide polymorphisms have been found in various genes giving rise to various gene variants which have been found to play a major role in genetic susceptibility to diabetic nephropathy. The risk of developing diabetic nephropathy is increased several times by inheriting risk alleles at susceptibility loci of various genes like ACE, IL, TNF-α, COL4A1, e NOS, SOD2, APOE, GLUT, etc. The identification of these genetic variants at a biomarker level could thus, allow the detection of those individuals at high risk for diabetic nephropathy which could thus help in the treatment, diagnosis and early prevention of the disease. The present review discusses about the various gene variants found till date to be associated with diabetic nephropathy.

  7. Primary Aldosteronism and ARMC5 Variants

    Science.gov (United States)

    Zilbermint, Mihail; Xekouki, Paraskevi; Faucz, Fabio R.; Berthon, Annabel; Gkourogianni, Alexandra; Schernthaner-Reiter, Marie Helene; Batsis, Maria; Sinaii, Ninet; Quezado, Martha M.; Merino, Maria; Hodes, Aaron; Abraham, Smita B.; Libé, Rossella; Assié, Guillaume; Espiard, Stéphanie; Drougat, Ludivine; Ragazzon, Bruno; Davis, Adam; Gebreab, Samson Y.; Neff, Ryan; Kebebew, Electron; Bertherat, Jérôme; Lodish, Maya B.

    2015-01-01

    Context: Primary aldosteronism is one of the leading causes of secondary hypertension, causing significant morbidity and mortality. A number of genetic defects have recently been identified in primary aldosteronism, whereas we identified mutations in ARMC5, a tumor-suppressor gene, in cortisol-producing primary macronodular adrenal hyperplasia. Objective: We investigated a cohort of 56 patients who were referred to the National Institutes of Health for evaluation of primary aldosteronism for ARMC5 defects. Methods: Patients underwent step-wise diagnosis, with measurement of serum aldosterone and plasma renin activity followed by imaging, saline suppression and/or oral salt loading tests, plus adrenal venous sampling. Cortisol secretion was also evaluated; unilateral or bilateral adrenalectomy was performed, if indicated. DNA, protein, and transfection studies in H295R cells were conducted by standard methods. Results: We identified 12 germline ARMC5 genetic alterations in 20 unrelated and two related individuals in our cohort (39.3%). ARMC5 sequence changes in 6 patients (10.7%) were predicted to be damaging by in silico analysis. All affected patients carrying a variant predicted to be damaging were African Americans (P = .0023). Conclusions: Germline ARMC5 variants may be associated with primary aldosteronism. Additional cohorts of patients with primary aldosteronism and metabolic syndrome, particularly African Americans, should be screened for ARMC5 sequence variants because these may underlie part of the known increased predisposition of African Americans to low renin hypertension. PMID:25822102

  8. Atmospheric chemistry of C2F5CHO: reaction with Cl atoms and OH radicals, IR spectrum of C2F5C(O)O2NO2

    DEFF Research Database (Denmark)

    Andersen, Mads Peter Sulbæk; Hurley, MD; Wallington, TJ;

    2003-01-01

    Smog chamber/FTIR techniques were used to measure k(Cl + C2F5CHO) = (1. 96 +/- 0.28) X 10(-12) and k(OH + C2F5CHO) = (5.26 +/- 0.80) x 10(-13) cm(3) molecule(-1) s(-1) in 700 Torr of N-2 or air at 296 +/- 2 K. The C1 initiated oxidation Of C2F5CHO in the presence of NO in air diluent gave COF2, CF3......ONO2, and C2F5C(O)O2NO2 in molar yields of 180 +/- 19 6.1 +/- 0.6 and 1.0 +/- 0.3 respectively. The IR spectrum for C2F5C(O)O2NO2 is reported. Results are discussed with respect to the potential for the atmospheric degradation of fluorinated aldehydes, CnF2n+1CHO, to contribute to the observed...... environmental burden of fluorinated carboxylic acids, CnF2n+1C(O)OH. (C) 2003 Elsevier B.V. All rights reserved....

  9. Inactivation of GDP-fucose transporter gene (Slc35c1) in CHO cells by ZFNs, TALENs and CRISPR-Cas9 for production of fucose-free antibodies.

    Science.gov (United States)

    Chan, Kah Fai; Shahreel, Wahyu; Wan, Corrine; Teo, Gavin; Hayati, Noor; Tay, Shi Jie; Tong, Wen Han; Yang, Yuansheng; Rudd, Pauline M; Zhang, Peiqing; Song, Zhiwei

    2016-03-01

    Removal of core fucose from N-glycans attached to human IgG1 significantly enhances its affinity for the receptor FcγRIII and thereby dramatically improves its antibody-dependent cellular cytotoxicity activity. While previous works have shown that inactivation of fucosyltransferase 8 results in mutants capable of producing fucose-free antibodies, we report here the use of genome editing techniques, namely ZFNs, TALENs and the CRISPR-Cas9, to inactivate the GDP-fucose transporter (SLC35C1) in Chinese hamster ovary (CHO) cells. A FACS approach coupled with a fucose-specific lectin was developed to rapidly isolate SLC35C1-deficient cells. Mass spectrometry analysis showed that both EPO-Fc produced in mutants arising from CHO-K1 and anti-Her2 antibody produced in mutants arising from a pre-existing antibody-producing CHO-HER line lacked core fucose. Lack of functional SLC35C1 in these cells does not affect cell growth or antibody productivity. Our data demonstrate that inactivating Slc35c1 gene represents an alternative approach to generate CHO cells for production of fucose-free antibodies.

  10. An analysis of the dispute process regarding high-level nuclear waste repository siting in Toyo-cho, Japan: Decisive factors in the dispute and roles of the governments and experts

    International Nuclear Information System (INIS)

    The siting policy of HLW repository in Japan was 'application-based' until 2007 and Toyo-cho is the only municipality which applied for the Literature Survey. In Toyo-cho, however, a serious antagonism among citizens occurred and the application was withdrawn after the mayor was replaced by election. Our detailed analysis of the process based on the methods of political science and psychology shows five decisive factors: 1) opposing activists both in the town and from outside successfully changed citizens' perceptions of HLW by rhetorical expressions, 2) the mayor lacks careful actions and effective policy adviser, 3) NUMO, an organization which runs HLW projects, didn't effectively coordinate Toyo-cho and stakeholders, 4) the municipal government and council exercised very limited influences on the dispute despite their political authority, and 5) the existence of grant adversely influenced the citizens since it causes criticism that Toyo-cho applies a repository for grant. We finally conclude that the substantial problems, caused by the five decisive factors, were the propagation of enthusiastic opposition and the lack of peaceful deliberation based on local governance. In order to avoid enthusiastic opposition and to realize responsible decision making, or negotiation, we suggest that A) active and prompt response of experts, especially political/administrative ones, to radical opposing activities, B) solution to the adverse influence of the grant by the government's agenda

  11. Pharmacokinetics and pharmacodynamics of ch14.18/CHO in relapsed/refractory high-risk neuroblastoma patients treated by long-term infusion in combination with IL-2.

    Science.gov (United States)

    Siebert, Nikolai; Eger, Christin; Seidel, Diana; Jüttner, Madlen; Zumpe, Maxi; Wegner, Danilo; Kietz, Silke; Ehlert, Karoline; Veal, Gareth J; Siegmund, Werner; Weiss, Michael; Loibner, Hans; Ladenstein, Ruth; Lode, Holger N

    2016-04-01

    Ch14.18 manufactured in Chinese hamster ovary (CHO) cells is currently being evaluated in clinical trials. Short-term infusion (STI) (8-20 h/day; 4-5 days) of 100 mg/m2 ch14.18/CHO (dinutiximab β) per cycle in combination with cytokines is standard treatment of neuroblastoma (NB) patients. As pain is a limiting factor, we investigated a novel delivery method by continuous long-term infusion (LTI) of 100 mg/m2 over 10 days. 53 NB patients were treated with 5-6 cycles of 6 × 106 IU/m2 subcutaneous interleukin-2 (d 1-5, 8-12), LTI of 100 mg/m2 ch14.18/CHO (d 8-18) and 160 mg/m2 oral 13-cis-retinoic acid (d 22-35). Human anti-chimeric antibody (HACA), antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity were determined. With LTI, we observed a maximum concentration of ch14.18/CHO (Cmax) of 12.56 ± 0.68 µg/ml and a terminal half-life time (t1/2 β) of 32.7 ± 16.2 d. The clearance values for LTI and STI of 0.54 ± 0.13 and 0.41 ± 0.29 L/d m2 and area under the serum concentration-time curve (AUC) values of 189.6 ± 41.4 and 284.8 ± 156.8 µg×d/ml, respectively, were not significantly different. Importantly, we detected ch14.18/CHO trough concentration of ≥ 1 µg/ml at time points preceding subsequent antibody infusions after cycle 1, allowing a persistent activation of antibody effector mechanisms over the entire treatment period of 6 months. HACA responses were observed in 10/53 (19%) patients, similar to STI (21%), indicating LTI had no effect on the immunogenicity of ch14.18/CHO. In conclusion, LTI of ch14.18/CHO induced effector mechanisms over the entire treatment period, and may therefore emerge as the preferred delivery method of anti-GD2 immunotherapy to NB patients. PMID:26785755

  12. Splicing analysis of 14 BRCA1 missense variants classifies nine variants as pathogenic

    DEFF Research Database (Denmark)

    Ahlborn, Lise B; Dandanell, Mette; Steffensen, Ane Y;

    2015-01-01

    needed to classify whether these uncertain variants are pathogenic or benign. In this study, we investigated 14 BRCA1 variants by in silico splicing analysis and mini-gene splicing assay. All 14 alterations were missense variants located within the BRCT domain of BRCA1 and had previously been examined...... by functional analysis at the protein level. Results from a validated mini-gene splicing assay indicated that nine BRCA1 variants resulted in splicing aberrations leading to truncated transcripts and thus can be considered pathogenic (c.4987A>T/p.Met1663Leu, c.4988T>A/p.Met1663Lys, c.5072C>T/p.Thr1691Ile, c...... to have no or an uncertain effect on the protein level, whereas one variant (c.5072C>T/p.Thr1691Ile) were shown to have a strong effect on the protein level as well. In conclusion, our study emphasizes that in silico splicing prediction and mini-gene splicing analysis are important for the classification...

  13. A new variant selection criterion for twin variants in titanium alloys. Pt. 2

    Energy Technology Data Exchange (ETDEWEB)

    Schuman, Christophe [Laboratoire d' Etude des Microstructures et de Mecanique des Materiaux, LEM3, CNRS 7239, Universite Paul Verlaine - Metz, Ile du Saulcy, Metz (France); Bao, Lei; Lecomte, Jean Sebastien; Zhang, Yudong; Raulot, Jean Marc; Philippe, Marie Jeanne; Esling, Claude [LEM3, CNRS 7239, Universite Paul Verlaine - Metz, Ile du Saulcy, Metz (France)

    2012-05-15

    A new selection criterion to explain the activation of the twinning variant is proposed. This criterion is based on the calculation of the deformation energy to create a primary twin. The calculation takes into account the effect of the grain size using a Hall-Petch type relation. This criterion allows to obtain a very good prediction for the twin family selection and twin variant selection. The calculations are compared with the experimental results obtained on T40 (ASTM grade 2) deformed by Channel Die compression. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  14. Joint Analysis of Multiple Traits in Rare Variant Association Studies.

    Science.gov (United States)

    Wang, Zhenchuan; Wang, Xuexia; Sha, Qiuying; Zhang, Shuanglin

    2016-05-01

    The joint analysis of multiple traits has recently become popular since it can increase statistical power to detect genetic variants and there is increasing evidence showing that pleiotropy is a widespread phenomenon in complex diseases. Currently, the majority of existing methods for the joint analysis of multiple traits test association between one common variant and multiple traits. However, the variant-by-variant methods for common variant association studies may not be optimal for rare variant association studies due to the allelic heterogeneity as well as the extreme rarity of individual variants. Current statistical methods for rare variant association studies are for one single trait only. In this paper, we propose an adaptive weighting reverse regression (AWRR) method to test association between multiple traits and rare variants in a genomic region. AWRR is robust to the directions of effects of causal variants and is also robust to the directions of association of traits. Using extensive simulation studies, we compare the performance of AWRR with canonical correlation analysis (CCA), Single-TOW, and the weighted sum reverse regression (WSRR). Our results show that, in all of the simulation scenarios, AWRR is consistently more powerful than CCA. In most scenarios, AWRR is more powerful than Single-TOW and WSRR. PMID:26990300

  15. Performance comparison of various time variant filters

    Energy Technology Data Exchange (ETDEWEB)

    Kuwata, M. [JEOL Engineering Co. Ltd., Akishima, Tokyo (Japan); Husimi, K.

    1996-07-01

    This paper describes the advantage of the trapezoidal filter used in semiconductor detector system comparing with the other time variant filters. The trapezoidal filter is the compose of a rectangular pre-filter and a gated integrator. We indicate that the best performance is obtained by the differential-integral summing type rectangular pre-filter. This filter is not only superior in performance, but also has the useful feature that the rising edge of the output waveform is linear. We introduce an example of this feature used in a high-energy experiment. (author)

  16. Research progress of behavioral variant frontotemporal dementia

    Directory of Open Access Journals (Sweden)

    Xiao-hua GU

    2015-07-01

    Full Text Available There is no epidemiological data of frontotemporal dementia (FTD in China. The application of updated diagnostic criteria, publishing of frontotemporal lobar degeneration (FTLD consensus in China, development of multimodal imaging and biomarkers promote the clinical understanding on behavioral variant frontotemporal dementia (bvFTD. There is still no drugs treating FTD approved by U.S. Food and Drug Administration (FDA. Multidisciplinary intervention may delay the progression of bvFTD. DOI: 10.3969/j.issn.1672-6731.2015.07.006

  17. Pitfalls and variants in pediatric chest imaging.

    Science.gov (United States)

    García Asensio, D; Fernández Martín, M

    2016-05-01

    Most pitfalls in the interpretation of pediatric chest imaging are closely related with the technique used and the characteristics of pediatric patients. To obtain a quality image that will enable the correct diagnosis, it is very important to use an appropriate technique. It is important to know how technical factors influence the image and to be aware of the possible artifacts that can result from poor patient cooperation. Moreover, radiologists need to be familiar with the normal anatomy in children, with the classic radiologic findings, and with the anatomic and developmental variants to avoid misinterpreting normal findings as pathological.

  18. Variant position of the medial plantar nerve

    Directory of Open Access Journals (Sweden)

    Astik RB

    2011-01-01

    Full Text Available Knowledge of variation of position of the medial plantar nerve is important for the forefoot surgeon for plantar reconstruction, local injection therapy and an excision of interdigital neuroma. During routine dissection of 50-year-old female cadaver, we found the medial plantar nerve and vessels variably located between plantar aponeurosis and the muscles of the first layer of the sole of the right foot. Due to this variant position, the medial plantar nerve and vessels lose their protection from the muscles of the first layer of the sole of the foot and became vulnerable for compression.

  19. Oral fibrolipoma: A rare histological variant

    Directory of Open Access Journals (Sweden)

    Treville Pereira

    2014-01-01

    Full Text Available Lipomas are benign soft tissue mesenchymal neoplasms. Fibrolipoma is a histological variant of lipoma that mostly affects the buccal mucosa and causes functional and cosmetic disabilities. The diagnosis and differentiation of fibrolipoma with clinically similar lesions such as fibroma and pleomorphic adenoma is very essential for a correct treatment plan and complete follow-up. This article presents a case of a 35-year-old female with a fibrolipoma on the lingual marginal gingiva of the mandibular left third molar.

  20. Genetic variants associated with lung function

    DEFF Research Database (Denmark)

    Thyagarajan, Bharat; Wojczynski, Mary; Minster, Ryan L;

    2014-01-01

    BACKGROUND: Reduced forced expiratory volume in 1 second (FEV1) and the ratio of FEV1 to forced vital capacity (FVC) are strong predictors of mortality and lung function is higher among individuals with exceptional longevity. However, genetic factors associated with lung function in individuals...... with exceptional longevity have not been identified. METHOD: We conducted a genome wide association study (GWAS) to identify novel genetic variants associated with lung function in the Long Life Family Study (LLFS) (n = 3,899). Replication was performed using data from the CHARGE/SpiroMeta consortia...

  1. Space-variant polarized Airy beam

    CERN Document Server

    Chen, Hao

    2015-01-01

    We experimentally generate an Airy beam with polarization structure while keeping its original amplitude and phase profile intact. This class of Airy beam preserves the acceleration properties. By monitoring their initial polarization structure we have provided insight concerning the self-healing mechanism of Airy beams. We investigate both theoretically and experimentally the self-healing polarization properties of the space-variant polarized Airy beams. Amplitude as well as the polarization structure tends to reform during propagation in spite of the severe truncation of the beam by finite apertures.

  2. Variants of P Systems with Toxic Objects

    OpenAIRE

    Alhazov, Artiom; Freund, Rudolf; Ivanov, Sergiu; Research Group on Natural Computing (Universidad de Sevilla) (Coordinador)

    2015-01-01

    Toxic objects have been introduced to avoid trap rules, especially in (purely) catalytic P systems. No toxic object is allowed to stay idle during a valid derivation in a P system with toxic objects. In this paper we consider special variants of toxic P systems where the set of toxic objects is prede ned { either by requiring all objects to be toxic or all catalysts to be toxic or all objects except the catalysts to be toxic. With all objects staying inside and being toxic, pur...

  3. MeIQx-induced DNA adduct formation and mutagenesis in DNA repair deficient CHO cells expressing human CYP1A1 and rapid or slow acetylator NAT2

    Science.gov (United States)

    Bendaly, Jean; Zhao, Shuang; Neale, Jason R.; Metry, Kristin J.; Doll, Mark A.; States, J. Christopher; Pierce, William M.; Hein, David W.

    2007-01-01

    2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) is one of the most potent and abundant mutagens in the western diet. Bioactivation includes N-hydroxylation catalyzed by cytochrome P450s followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). Nucleotide excision repair-deficient chinese hamster ovary (CHO) cells were constructed by stable transfection of human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. CYP1A1 and NAT2 catalytic activities were undetectable in untransfected CHO cell lines. CYP1A1 activity did not differ significantly (p > 0.05) among the CYP1A1-transfected cell lines. Cells transfected with NAT2*4 had significantly higher levels of sulfamethazine N-acetyltransferase (p = 0.0001) and N-hydroxy-MeIQx O-acetyltransferase (p = 0.0093) catalytic activity than cells transfected with NAT2*5B. Only cells transfected with both CYP1A1 and NAT2*4 showed concentration-dependent cytotoxicity and hypoxanthine phosphoribosyl transferase (hprt) mutagenesis following MeIQx treatment. dG-C8-MeIQx was the primary DNA adduct formed and levels were dose-dependent in each cell line and in the order: untransfected < transfected with CYP1A1 < transfected with CYP1A1 & NAT2*5B < transfected with CYP1A1 & NAT2*4. MeIQx DNA adduct levels were significantly higher (p < 0.001) in CYP1A1/NAT2*4 than CYP1A1/NAT2*5B cells at all concentrations of MeIQx tested. MeIQx-induced DNA adduct levels correlated very highly (r2 = 0.88) with MeIQx-induced mutants. These results strongly support extrahepatic activation of MeIQx by CYP1A1 and a robust effect of human NAT2 genetic polymorphism on MeIQx –induced DNA adducts and mutagenesis. The results provide laboratory-based support for epidemiological studies reporting higher frequency of heterocyclic amine-related cancers in rapid NAT2 acetylators. PMID:17627018

  4. In vitro genotoxic and cytotoxic effects of ivermectin and its formulation ivomec on Chinese hamster ovary (CHO{sub K1}) cells

    Energy Technology Data Exchange (ETDEWEB)

    Molinari, G.; Soloneski, S.; Reigosa, M.A. [Catedra de Citologia, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, La Plata (Argentina); Larramendy, M.L., E-mail: m_larramendy@hotmail.com [Catedra de Citologia, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, La Plata (Argentina)

    2009-06-15

    The effects of ivermectin (IVM) and its commercial formulation ivomec (IVM 1.0%) were studied on Chinese hamster ovary (CHO{sub K1}) cells by several genotoxicity [sister chromatid exchange (SCE) and single cell gel electrophoresis (SCGE)] and cytotoxicity [cell-cycle progression (CCP), mitotic index (MI), proliferative replication index (PRI), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and neutral red (NR)] bioassays within the 1.0-250 {mu}g/ml concentration-range. While IVM and ivomec did not modified SCE frequencies, they induced DNA-strand breaks revealed by SCGE. An enhancement of slightly damaged cells and a decrease in undamaged cells were observed in IVM-treated cultures with 5.0-50.0 {mu}g/ml. In ivomec-treated cells, while an increase in slightly damaged cells was induced with 5.0-50.0 {mu}g/ml, the damaged and undamaged cells increased and decreased only with 50.0 {mu}g/ml. Both compounds exerted a delay in CCP and a reduction in PRI when 25.0 {mu}g/ml was employed whereas cytotoxicity was observed at higher concentration than 50.0 {mu}g/ml. No MI alteration was observed with 1.0-10.0 and 1.0-5.0 {mu}g/ml of IVM and ivomec, respectively. A concentration-related trend to an increase in MI was achieved within 1.0-10.0 {mu}g/ml. An increase in the MI was induced in 10.0 {mu}g/ml ivomec-treated cultures. A marked reduction of about 89% and 62% in regard to controls was observed with 25.0 {mu}g/ml of IVM and ivomec, respectively. NR and MTT assays revealed a cell growth inhibition when 0.25-250.0 {mu}g/ml of both compounds was employed. The results highlighted that IVM and ivomec exert both genotoxicity and cytotoxicity in mammalian cells in vitro, at least in CHO{sub K1} cells.

  5. Using simple models to describe the kinetics of growth, glucose consumption, and monoclonal antibody formation in naive and infliximab producer CHO cells.

    Science.gov (United States)

    López-Meza, Julián; Araíz-Hernández, Diana; Carrillo-Cocom, Leydi Maribel; López-Pacheco, Felipe; Rocha-Pizaña, María Del Refugio; Alvarez, Mario Moisés

    2016-08-01

    Despite their practical and commercial relevance, there are few reports on the kinetics of growth and production of Chinese hamster ovary (CHO) cells-the most frequently used host for the industrial production of therapeutic proteins. We characterize the kinetics of cell growth, substrate consumption, and product formation in naive and monoclonal antibody (mAb) producing recombinant CHO cells. Culture experiments were performed in 125 mL shake flasks on commercial culture medium (CD Opti CHO™ Invitrogen, Carlsbad, CA, USA) diluted to different glucose concentrations (1.2-4.8 g/L). The time evolution of cell, glucose, lactic acid concentration and monoclonal antibody concentrations was monitored on a daily basis for mAb-producing cultures and their naive counterparts. The time series were differentiated to calculate the corresponding kinetic rates (rx = d[X]/dt; rs = d[S]/dt; rp = d[mAb]/dt). Results showed that these cell lines could be modeled by Monod-like kinetics if a threshold substrate concentration value of [S]t = 0.58 g/L (for recombinant cells) and [S]t = 0.96 g/L (for naïve cells), below which growth is not observed, was considered. A set of values for μmax, and Ks was determined for naive and recombinant cell cultures cultured at 33 and 37 °C. The yield coefficient (Yx/s) was observed to be a function of substrate concentration, with values in the range of 0.27-1.08 × 10(7) cell/mL and 0.72-2.79 × 10(6) cells/mL for naive and recombinant cultures, respectively. The kinetics of mAb production can be described by a Luedeking-Piret model (d[mAb]/dt = αd[X]/dt + β[X]) with values of α = 7.65 × 10(-7) µg/cell and β = 7.68 × 10(-8) µg/cell/h for cultures conducted in batch-agitated flasks and batch and instrumented bioreactors operated in batch and fed-batch mode. PMID:26091615

  6. A novel control scheme for inducing angiostatin-human IgG fusion protein production using recombinant CHO cells in a oscillating bioreactor.

    Science.gov (United States)

    Wang, Ing-Kae; Hsieh, Sing-Ying; Chang, King-Ming; Wang, Yu-Chi; Chu, Andy; Shaw, Shyh-Yu; Ou, Jung-Jung; Ho, Lewis

    2006-02-10

    In this study, a novel control scheme for inducing protein production using a recombinant CHO cell line in a BelloCell bioreactor was developed. This control scheme was applied in a simple regular semi-batch process. Production of angiostatin-human IgG fusion protein in a suspension recombinant CHO cell culture and a protein-free medium was used for this study. The bottom holding time (BH) was the sole operating variable to control the exposure time of the cells immobilized on the carriers to the air and allow the nutrient remained on the liquid film of the carriers to be consumed to a threshold level so that the cells can be arrested and promoted for protein production. In the cell cultures with various BH (1.5-90 min), final cell densities of 1.6-4.0 x 10(9) have been obtained in 20 days while total angiostatin-human IgG production of 228-388 mg have been harvested. In general, low BH will minimize the nutrient limitation and favor the cell growth, while high BH will restrict the nutrient and promote the production in this type of non-growth associated production systems. It was found that specific production rate was generally inversely proportional to the specific growth rate. In this case of study, BH of 30 and 60 min were found to be about 72% better than BH of 1.5 min and 35% better than BH of 9 and 90 min in term of the total angiostatin-human IgG production. In comparison to a conventional spinner flask study, a 3.8-fold increase of the total angiostatin-human IgG production was realized in a 35-day culture. This study illustrated that a simple method of using BH in a semi-batch process can effectively control the apparent nutrient concentration to the cells, and thus regulate the cell growth and protein production in a novel oscillating bioreactor. PMID:16162365

  7. Clinical relevance of hepatitis B virus variants

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    The hepatitis B virus (HBV) is a global public health problem with more than 240 million people chronicallyinfected worldwide, who are at risk for end-stage liverdisease and hepatocellular carcinoma. There are anestimated 600000 deaths annually from complications ofHBV-related liver disease. Antiviral therapy with nucleos/tide analogs (NA) targeting the HBV polymerase (P) caninhibit disease progression by long-term suppression ofHBV replication. However, treatment may fail with firstgeneration NA therapy due to the emergence of drugresistantmutants, as well as incomplete medicationadherence. The HBV replicates via an error-prone reversetranscriptase leading to quasispecies. Due to overlappingopen reading frames mutations within the HBV P cancause concomitant changes in the HBV surface gene (S )and vice versa. HBV quasispecies diversity is associatedwith response to antiviral therapy, disease severity andlong-term clinical outcomes. Specific mutants havebeen associated with antiviral drug resistance, immuneescape, liver fibrosis development and tumorgenesis.An understanding of HBV variants and their clinicalrelevance may be important for monitoring chronichepatitis B disease progression and treatment response.In this review, we will discuss HBV molecular virology,mechanism of variant development, and their potentialclinical impact.

  8. The power of multiplexed functional analysis of genetic variants.

    Science.gov (United States)

    Gasperini, Molly; Starita, Lea; Shendure, Jay

    2016-10-01

    New technologies have recently enabled saturation mutagenesis and functional analysis of nearly all possible variants of regulatory elements or proteins of interest in single experiments. Here we discuss the past, present, and future of such multiplexed (functional) assays for variant effects (MAVEs). MAVEs provide detailed insight into sequence-function relationships, and they may prove critical for the prospective clinical interpretation of genetic variants. PMID:27583640

  9. Effects of {gamma} ({sup 60}Co) and {beta} ({sup 90}Sr) radiations in Chinese hamster ovarian cells (CHO-K1): induction of micronuclei and cell death; Efeitos das radiacoes {gamma} ({sup 60}Co) e {beta} ({sup 90}Sr) em celulas de ovario de hamster chines (CHO-K1): inducao de micronucleos e morte celular

    Energy Technology Data Exchange (ETDEWEB)

    Murakami, Daniella

    2003-07-01

    Among various types of ionising radiation, the beta emitter radionuclides are involved in many sectors of human activity, such as nuclear medicine, nuclear industries and biomedicine, with a consequent increased risk of accidental, occupational or therapeutic exposure. Despite their recognized importance, there is little information about the effect of beta particles at the cellular level when compared to other types of ionizing radiation. Thus, the objective of the present study was to evaluate the genotoxic and cytotoxic effects of {sup 90}Sr, a pure, highly energetic beta source, on CHO-K1 cells and to compare them with data obtained with {sup 60}Co. CHO-K1 cells irradiated with different doses (1.0, 2.5, 5.0, 7.5 Gy) of {sup 60}Co (0.34 Gy.min{sup -1}) and {sup 90}Sr (0.23 Gy.min{sup -1}) were processed for analysis of clonogenic death, induction of micronuclei (MN) and necrotic and apoptotic death. The survival curves obtained for both types of radiation were better fitted by the linear-quadratic model and were similar. However, the cytogenetic results showed that both the proportion of micronucleated cells and the magnitude of radioinduced lesions demonstrated by the analysis of MN distribution were significantly higher in cells irradiated with {sup 60}Co than in cells irradiated with {sup 90}Sr, whereas {sup 90}Sr was more damaging than {sup 60}Co in terms of cell death induction. Necrosis was the major type of death observed in CHO-K1 cells. The data obtained suggest that the low incidence of micronucleated cells after exposure to {sup 90}Sr may be a consequence of selective elimination of severely damaged cells from the population by the necrotic process at a higher rate than observed with {sup 60}Co exposure. The data obtained also demonstrated the need to use several parameters for a better estimate of cellular sensitivity to the action of genotoxic agents, which would be important in terms of radiobiology, oncology and therapeutics. (author)

  10. Population structure analysis using rare and common functional variants

    Directory of Open Access Journals (Sweden)

    Ding Lili

    2011-11-01

    Full Text Available Abstract Next-generation sequencing technologies now make it possible to genotype and measure hundreds of thousands of rare genetic variations in individuals across the genome. Characterization of high-density genetic variation facilitates control of population genetic structure on a finer scale before large-scale genotyping in disease genetics studies. Population structure is a well-known, prevalent, and important factor in common variant genetic studies, but its relevance in rare variants is unclear. We perform an extensive population structure analysis using common and rare functional variants from the Genetic Analysis Workshop 17 mini-exome sequence. The analysis based on common functional variants required 388 principal components to account for 90% of the variation in population structure. However, an analysis based on rare variants required 532 significant principal components to account for similar levels of variation. Using rare variants, we detected fine-scale substructure beyond the population structure identified using common functional variants. Our results show that the level of population structure embedded in rare variant data is different from the level embedded in common variant data and that correcting for population structure is only as good as the level one wishes to correct.

  11. A unified phylogeny-based nomenclature for histone variants

    Directory of Open Access Journals (Sweden)

    Talbert Paul B

    2012-06-01

    Full Text Available Abstract Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.

  12. Behavioral variant frontotemporal dementia with dominant gait disturbances - case report.

    Directory of Open Access Journals (Sweden)

    Wojciech Guenter

    2016-04-01

    Presented case emphasises the significance of accurately gathered anamnesis with patient and his family. Behavioural variant frontotemporal dementia should be considered in cases of unexplained gait abnormalities.

  13. Assessment of cytotoxic and cytogenetic effects of a 1,2,5-thiadiazole derivative on CHO-K1 cells. Its application as corrosion inhibitor

    International Nuclear Information System (INIS)

    This work focuses on the possible use of phenanthro[9,10-c]-1,2,5-thiadiazole 1,1-dioxide (TDZ) as a harmless corrosion inhibitor. TDZ range-dose providing minimum adverse effects to the environment and human health, with satisfactory corrosion-inhibiting properties was evaluated. Cytotoxicity and genotoxicity of TDZ at 0.57-12.50 μM concentration range were tested by neutral red, chromosomal aberrations, mitotic index, and colony formation assays. Results showed a significant increase of chromatid-type aberrations for the highest concentration of TDZ assayed (12.50 μM). Additionally, a reduction in the proliferative rate for lower concentrations was detected by the MI assay. We concluded that TDZ should be used at concentrations lower than 1.16 μM. Corrosion assays performed showed good inhibition effect (ca. 50%) at low (0.65 μM) TDZ concentration. Consequently, our results indicated that TDZ induced a time- and dose-dependent genotoxic and cytotoxic response on CHO-K1 cells. Short assays should be complemented with long exposure tests to simulate chronic contact with TDZ since lower threshold levels may be found for shorter exposures and a wrong safety range could be determined.

  14. Changes in Al and Fe associated with amorphous soil minerals over one year after a wildfire at Pico Cho Marcial (Tenerife, Canary Islands, Spain

    Directory of Open Access Journals (Sweden)

    J. Notario

    2013-05-01

    Full Text Available Amorphous-linked Al, Fe and Si were determined both in burned and unburnt soil samples after a wildfire close to Pico Cho Marcial (Tenerife, Canary Islands, Spain that affected 7.1 ha of high mountain Teide broom scrub along four successive samplings held in September 2003 (three months after the wildfire, February 2004, June 2004 and October 2004. Soils in the area are Lithic Xerorthents, with a scarcely developed AC-type profile. The chemical elements under study were extracted using selective dissolutions (0.2M acid ammonium oxalate and 0.1N sodium pyrophosphate, and determined by Atomic Absorption Spectrophotometry. The average contents of total amorphous Al (oxalate-extractable and organo-metallic complexes-bound Al (pyrophosphate extractable were significantly higher in burned samples along the study. Also, the oxalate-extractable Al varied significantly along the different samplings, and so consequently did the Alp:Alox ratio. A progressive decrease in the Al:Si ratio in allophanes was also found throughout the study period. No differences were found for oxalate-extractable Fe, either between sample groups or samplings, which was also applicable to the (Alox+0.5Feox percentage.

  15. Assessment of cytotoxic and cytogenetic effects of a 1,2,5-thiadiazole derivative on CHO-K1 cells. Its application as corrosion inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Grillo, C.A. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA, CCT La Plata-CONICET), Facultad de Ciencias Exactas, Departamento de Quimica, Universidad Nacional de La Plata, Casilla de Correo 16, Sucursal 4, 1900 La Plata (Argentina); Mirifico, M.V. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA, CCT La Plata-CONICET), Facultad de Ciencias Exactas, Departamento de Quimica, Universidad Nacional de La Plata, Casilla de Correo 16, Sucursal 4, 1900 La Plata (Argentina); Facultad de Ingenieria, Areas Departamentales Ingenieria Quimica and Mecanica, Universidad Nacional de La Plata, Calle 47 y 1, 1900 La Plata (Argentina); Morales, M.L. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA, CCT La Plata-CONICET), Facultad de Ciencias Exactas, Departamento de Quimica, Universidad Nacional de La Plata, Casilla de Correo 16, Sucursal 4, 1900 La Plata (Argentina); Reigosa, M.A. [IMBICE (Instituto Multidisciplinario de Biologia Celular), CICPBA, CONICET, Calle 526 entre 10 y 11, 1900 La Plata (Argentina); Mele, M. Fernandez Lorenzo de, E-mail: mmele@inifta.unlp.edu.ar [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA, CCT La Plata-CONICET), Facultad de Ciencias Exactas, Departamento de Quimica, Universidad Nacional de La Plata, Casilla de Correo 16, Sucursal 4, 1900 La Plata (Argentina); Facultad de Ingenieria, Areas Departamentales Ingenieria Quimica and Mecanica, Universidad Nacional de La Plata, Calle 47 y 1, 1900 La Plata (Argentina)

    2009-10-30

    This work focuses on the possible use of phenanthro[9,10-c]-1,2,5-thiadiazole 1,1-dioxide (TDZ) as a harmless corrosion inhibitor. TDZ range-dose providing minimum adverse effects to the environment and human health, with satisfactory corrosion-inhibiting properties was evaluated. Cytotoxicity and genotoxicity of TDZ at 0.57-12.50 {mu}M concentration range were tested by neutral red, chromosomal aberrations, mitotic index, and colony formation assays. Results showed a significant increase of chromatid-type aberrations for the highest concentration of TDZ assayed (12.50 {mu}M). Additionally, a reduction in the proliferative rate for lower concentrations was detected by the MI assay. We concluded that TDZ should be used at concentrations lower than 1.16 {mu}M. Corrosion assays performed showed good inhibition effect (ca. 50%) at low (0.65 {mu}M) TDZ concentration. Consequently, our results indicated that TDZ induced a time- and dose-dependent genotoxic and cytotoxic response on CHO-K1 cells. Short assays should be complemented with long exposure tests to simulate chronic contact with TDZ since lower threshold levels may be found for shorter exposures and a wrong safety range could be determined.

  16. Galactose supplementation enhance sialylation of recombinant Fc-fusion protein in CHO cell: an insight into the role of galactosylation in sialylation.

    Science.gov (United States)

    Liu, Jintao; Wang, Jie; Fan, Li; Chen, Xinning; Hu, Dongdong; Deng, Xiancun; Poon, H Fai; Wang, Haibin; Liu, Xuping; Tan, Wen-Song

    2015-07-01

    Sialic acid levels of therapeutic glycoprotein play an important role in plasma half-life. An undesirable decrease of sialic acid content was observed when we increased Fc-fusion protein productivity fourfold in a GS-CHO cell line by bioprocess optimization. We investigated the potential mechanism for the sialic acid content reduction. We found that limited nucleotide sugar precursor and the extracellular sialidase were not responsible for the reduction of the sialic acid content after titer improvement. Oligosaccharide analysis revealed that the lack of protein galactosylation was the potential cause for the reduction of sialic acid content. Thus we validated this notion by evaluated galactose supplementation in 2 L bioreactors. Cell culture performance was not impacted by addition of up to 40 mM galactose except for the glucose consumption rate. Addition of 20 mM galactose to the bioreactor resulted in the increase of 44 % for total sialic acid content and 20.3 % for sialylated glycans. These data were further validated when the process was run on 200 L scaled bioreactor. These data together show that the galactosylation plays an apparent role in sialylation in our current system. PMID:25931375

  17. A multi-pronged investigation into the effect of glucose starvation and culture duration on fed-batch CHO cell culture

    DEFF Research Database (Denmark)

    Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian;

    2015-01-01

    In this study, omics-based analysis tools were used to explore the effect of glucose starvation and culture duration on monoclonal antibody (mAb) production in fed-batch CHO cell culture to gain better insight into how these parameters can be controlled to ensure optimal mAb productivity...... and quality. Titer and N-glycosylation of mAbs, as well as proteomic signature and metabolic status of the production cells in the culture were assessed. We found that the impact of glucose starvation on the titer and N-glycosylation of mAbs was dependent on the degree of starvation during early stationary...... phase of the fed-batch culture. Higher degree of glucose starvation reduced intracellular concentrations of UDP-GlcNAc and UDP-GalNAc, but increased the levels of UDP-Glc and UDP-Gal. Increased GlcNAc and Gal occupancy correlated well with increased degree of glucose starvation, which can be attributed...

  18. Quantitative modeling of viable cell density, cell size, intracellular conductivity, and membrane capacitance in batch and fed-batch CHO processes using dielectric spectroscopy.

    Science.gov (United States)

    Opel, Cary F; Li, Jincai; Amanullah, Ashraf

    2010-01-01

    Dielectric spectroscopy was used to analyze typical batch and fed-batch CHO cell culture processes. Three methods of analysis (linear modeling, Cole-Cole modeling, and partial least squares regression), were used to correlate the spectroscopic data with routine biomass measurements [viable packed cell volume, viable cell concentration (VCC), cell size, and oxygen uptake rate (OUR)]. All three models predicted offline biomass measurements accurately during the growth phase of the cultures. However, during the stationary and decline phases of the cultures, the models decreased in accuracy to varying degrees. Offline cell radius measurements were unsuccessfully used to correct for the deviations from the linear model, indicating that physiological changes affecting permittivity were occurring. The beta-dispersion was analyzed using the Cole-Cole distribution parameters Deltaepsilon (magnitude of the permittivity drop), f(c) (critical frequency), and alpha (Cole-Cole parameter). Furthermore, the dielectric parameters static internal conductivity (sigma(i)) and membrane capacitance per area (C(m)) were calculated for the cultures. Finally, the relationship between permittivity, OUR, and VCC was examined, demonstrating how the definition of viability is critical when analyzing biomass online. The results indicate that the common assumptions of constant size and dielectric properties used in dielectric analysis are not always valid during later phases of cell culture processes. The findings also demonstrate that dielectric spectroscopy, while not a substitute for VCC, is a complementary measurement of viable biomass, providing useful auxiliary information about the physiological state of a culture.

  19. Variation through the cell cycle in the dose-response of DNA neutral filter elution in X-irradiated synchronous CHO-cells

    International Nuclear Information System (INIS)

    Dose-response curves for DNA neutral (pH 9.6) filter elution were obtained with synchronized CHO cells exposed to X-rays at various phases of cell cycle. The dose response was similar in synchronized and plateau-phase G1 cells, as well as in cells arrested at the G1/S border using aphidicolin; it flattened as cells progressed into S phase and reached a minimum in the middle of this phase. An increase in DNA elution dose response, to values only slightly lower than those obtained with G1 cells, was observed as cells entered G2 phase. Significant alterations in the sedimentation properties of the DNA during S phase were also observed in Ehrlich ascites tumor cells using the neutral sucrose gradient centrifugation technique. A significant proportion of the DNA from S cells irradiated with 10 Gy sedimented at speeds (350S-700S) well above the maximum sedimentation speed expected for free sedimenting DNA molecules (ssub(max) = 350S), indicating the formation of a DNA complex. DNA from G1, G1/S, or G2 + M cells sedimented as expected for free sedimenting molecules. (author)

  20. Detecting Association of Rare Variants by Testing an Optimally Weighted Combination of Variants for Quantitative Traits in General Families

    OpenAIRE

    Fang, Shurong; Zhang, Shuanglin; Sha, Qiuying

    2013-01-01

    Although next-generation sequencing technology allows sequencing the whole genome of large groups of individuals, the development of powerful statistical methods for rare variant association studies is still underway. Even though many statistical methods have been developed for mapping rare variants, most of these methods are for unrelated individuals only, whereas family data have been shown to improve power to detect rare variants. The majority of the existing methods for unrelated individu...

  1. Detecting association of rare and common variants by testing an optimally weighted combination of variants with longitudinal data

    OpenAIRE

    Wang, Shuaicheng; Fang, Shurong; Sha, Qiuying; Zhang, Shuanglin

    2014-01-01

    Increasing evidence shows that complex diseases are caused by both common and rare variants. Recently, several statistical methods for detecting associations of rare variants have been developed, including the test for testing the effect of an optimally weighted combination of variants (TOW) developed by our group in 2012. These methodologies consider phenotype measurement at only one time point. Because many sequence data have been developed on population cohorts that contain phenotype measu...

  2. αIIbβ3 variants defined by next-generation sequencing: Predicting variants likely to cause Glanzmann thrombasthenia

    OpenAIRE

    Buitrago, Lorena; Rendon, Augusto; Liang, Yupu; Negri, Ana; Filizola, Marta; Ouwehand, Willem H.; Coller, Barry S.; Alessi, Marie-Christine; Ballmaier, Matthias; Bariana, Tadbir; Bellissimo, Daniel; Bertoli, Marta; Bray, Paul; Bury, Loredana; Carrell, Robin

    2015-01-01

    Next-generation sequencing is identifying millions of novel gene variants, presenting challenges to researchers and clinicians. Variations in the genes ITGA2B and ITGB3 affect integrin αIIbβ3, leading to the bleeding disorder Glanzmann thrombasthenia. We analyzed novel missense variants on ∼32,000 alleles of ITGA2B and ITGB3 and found missense variants affecting ∼10% of the amino acids in each protein in ∼1.3% of the population. Almost all variants are rare, indicating recent entry into the p...

  3. A look-ahead variant of TFQMR

    Energy Technology Data Exchange (ETDEWEB)

    Freund, R.W. [AT& T Bell Labs., Murray Hill, NJ (United States); Nachtigal, N.M. [Oak Ridge National Lab., TN (United States)

    1994-12-31

    Recently, Freund proposed a Krylov subspace iteration, the transpose-free quasi-minimal residual method (TFQMR), for solving general nonsingular non-Hermitian linear systems. The algorithm relies on a version of the squared Lanczos process to generate the basis vectors for the underlying Krylov subspace. It then constructs iterates defined by a quasi-minimization property, which leads to a smooth and nearly monotone convergence behavior. The authors investigate a variant of TFQMR that uses look-ahead to avoid some of the problems associated with breakdowns in the underlying squared Lanczos procedure. They also present some numerical examples that illustrate the properties of the new method, as compared to the original TFQMR algorithm.

  4. Genetic variants in periodontal health and disease

    Energy Technology Data Exchange (ETDEWEB)

    Dumitrescu, Alexandrina L. [Tromsoe Univ. (Norway). Inst. of Clinical Dentistry; Kobayashi, Junya [Kyoto Univ. (Japan). Dept. of Genome Repair Dynamics

    2010-07-01

    Periodontitis is a complex, multifactorial disease and its susceptibility is genetically determined. The present book systematically reviews the evidence of the association between the genetic variants and periodontitis progression and/or treatment outcomes. Genetic syndromes known to be associated with periodontal disease, the candidate gene polymorphisms investigated in relation to periodontitis, the heritability of chronic and aggressive periodontitis, as well as common guidelines for association studies are described. This growing understanding of the role of genetic variation in inflammation and periodontal chronic disease presents opportunities to identify healthy persons who are at increased risk of disease and to potentially modify the trajectory of disease to prolong healthy aging. The book represents a new concept in periodontology with its pronounced focus on understanding through knowledge rather than presenting the presently valid answers. Connections between genetics and periodontology are systematically reviewed and covered in detail. (orig.)

  5. Current conveyors variants, applications and hardware implementations

    CERN Document Server

    Senani, Raj; Singh, A K

    2015-01-01

    This book serves as a single-source reference to Current Conveyors and their use in modern Analog Circuit Design. The authors describe the various types of current conveyors discovered over the past 45 years, details of all currently available, off-the-shelf integrated circuit current conveyors, and implementations of current conveyors using other, off-the-shelf IC building blocks. Coverage includes prominent bipolar/CMOS/Bi-CMOS architectures of current conveyors, as well as all varieties of starting from third generation current conveyors to universal current conveyors, their implementations and applications. •Describes all commercially available off-the-shelf IC current conveyors, as well as hardware implementations of current conveyors using other off-the-shelf ICs; • Describes numerous variants of current conveyors evolved over the past forty five years; • Describes a number of Bipolar/CMOS/Bi-CMOS architectures of current conveyors, along with their characteristic features; • Includes a comprehe...

  6. Lipoleiomyoma: A rare variant of uterine leiomyoma

    Directory of Open Access Journals (Sweden)

    D Manimaran

    2014-01-01

    Full Text Available Uterine fatty tumors are rare variants of benign leiomyoma. Lipoleiomyoma, lipomyoma, fibromyolipoma are various synonyms for this lesion. They usually occur in the obese perimenopausal and postmenopausal females in the age group 50-70 years and 90% cases occur in patients older than 40 years. There were only few cases reported in the literature. These lesions are interesting due to the occasional diagnostic confusion with sarcomas and the curiosity regarding its histogenesis. We are presenting three cases of lipoleiomyoma whose age ranged from 40 to 50 years with clinical, radiologic and pathologic correlation. All three cases came with complaints of abnormal vaginal bleeding and found to have intramural heteroechoic nodule in the ultrasonogram.

  7. Some variants of SAT and their properties

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A new model for the well-known problem, the satisfiablility problem of boolean formula (SAT), is introduced. Based on this model, some variants of SAT and their properties are presented. Denote by NP the class of all languages which can be decided by a non-deterministic polynomial Turing machine and by P the class of all languages which can be decided by a deterministic polynomial-time Turing machine. This model also allows us to give another candidate for the natural problems in ((NP-NPC)-P), denoted as NPI, under the assumption P≠NP, where NPC represents NP-complete. It is proven that this candidate is not in NPC under P≠NP. While, it is indeed in NPI under some stronger but reasonable assumption, specifically, under the Exponential-Time Hypothesis (ETH). Thus we can partially solve this long standing important open problem.

  8. Possible new variant of Nijmegen breakage syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Der Kaloustian, V.M.; Booth, A.; Elliott, A.M. [Montreal Children`s Hospital and McGill Univ., Montreal (Canada)] [and others

    1996-10-02

    We report on a child with microcephaly, small facial and body size, and immune deficiency. The phenotype is consistent with Nijmegen breakage syndrome (NBS), with additional clinical manifestations and laboratory findings not reported heretofore. Most investigations, including the results of radiation-resistant DNA synthesis, concurred with the diagnosis of NBS. Cytogenetic analysis documented abnormalities in virtually all cells examined. Along with the high frequency of breaks and rearrangements of chromosomes 7 and 14, we found breakage and monosomies involving numerous other chromosomes. Because of some variation in the clinical presentation and some unusual cytogenetic findings, we suggest that our patient may represent a new variant of Nijmegen breakage syndrome. 34 refs., 3 figs., 7 tabs.

  9. Glycolipid binding preferences of Shiga toxin variants.

    Directory of Open Access Journals (Sweden)

    Sayali S Karve

    Full Text Available The major virulence factor of Shiga toxin producing E. coli, is Shiga toxin (Stx, an AB5 toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. The two major isoforms, Stx1 and Stx2, and Stx2 variants (Stx2a-h significantly differ in toxicity. The exact reason for this toxicity difference is unknown, however different receptor binding preferences are speculated to play a role. Previous studies used enzyme linked immunosorbent assay (ELISA to study binding of Stx1 and Stx2a toxoids to glycolipid receptors. Here, we studied binding of holotoxin and B-subunits of Stx1, Stx2a, Stx2b, Stx2c and Stx2d to glycolipid receptors globotriaosylceramide (Gb3 and globotetraosylceramide (Gb4 in the presence of cell membrane components such as phosphatidylcholine (PC, cholesterol (Ch and other neutral glycolipids. In the absence of PC and Ch, holotoxins of Stx2 variants bound to mixtures of Gb3 with other glycolipids but not to Gb3 or Gb4 alone. Binding of all Stx holotoxins significantly increased in the presence of PC and Ch. Previously, Stx2a has been shown to form a less stable B-pentamer compared to Stx1. However, its effect on glycolipid receptor binding is unknown. In this study, we showed that even in the absence of the A-subunit, the B-subunits of both Stx1 and Stx2a were able to bind to the glycolipids and the more stable B-pentamer formed by Stx1 bound better than the less stable pentamer of Stx2a. B-subunit mutant of Stx1 L41Q, which shows similar stability as Stx2a B-subunits, lacked glycolipid binding, suggesting that pentamerization is more critical for binding of Stx1 than Stx2a.

  10. Nuclear variants of bone morphogenetic proteins

    Directory of Open Access Journals (Sweden)

    Meinhart Christopher A

    2010-03-01

    Full Text Available Abstract Background Bone morphogenetic proteins (BMPs contribute to many different aspects of development including mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. They have previously been recognized only as secreted growth factors, but the present study detected Bmp2, Bmp4, and Gdf5/CDMP1 in the nuclei of cultured cells using immunocytochemistry and immunoblotting of nuclear extracts. Results In all three proteins, a bipartite nuclear localization signal (NLS was found to overlap the site at which the proproteins are cleaved to release the mature growth factors from the propeptides. Mutational analyses indicated that the nuclear variants of these three proteins are produced by initiating translation from downstream alternative start codons. The resulting proteins lack N-terminal signal peptides and are therefore translated in the cytoplasm rather than the endoplasmic reticulum, thus avoiding proteolytic processing in the secretory pathway. Instead, the uncleaved proteins (designated nBmp2, nBmp4, and nGdf5 containing the intact NLSs are translocated to the nucleus. Immunostaining of endogenous nBmp2 in cultured cells demonstrated that the amount of nBmp2 as well as its nuclear/cytoplasmic distribution differs between cells that are in M-phase versus other phases of the cell cycle. Conclusions The observation that nBmp2 localization varies throughout the cell cycle, as well as the conservation of a nuclear localization mechanism among three different BMP family members, suggests that these novel nuclear variants of BMP family proteins play an important functional role in the cell.

  11. Managing Process Variants in the Process Life Cycle

    NARCIS (Netherlands)

    Hallerbach, A.; Bauer, Th.; Reichert, M.U.

    2007-01-01

    When designing process-aware information systems, often variants of the same process have to be specified. Each variant then constitutes an adjustment of a particular process to specific requirements building the process context. Current Business Process Management (BPM) tools do not adequately supp

  12. Association analysis identifies ZNF750 regulatory variants in psoriasis

    Directory of Open Access Journals (Sweden)

    Birnbaum Ramon Y

    2011-12-01

    Full Text Available Abstract Background Mutations in the ZNF750 promoter and coding regions have been previously associated with Mendelian forms of psoriasis and psoriasiform dermatitis. ZNF750 encodes a putative zinc finger transcription factor that is highly expressed in keratinocytes and represents a candidate psoriasis gene. Methods We examined whether ZNF750 variants were associated with psoriasis in a large case-control population. We sequenced the promoter and exon regions of ZNF750 in 716 Caucasian psoriasis cases and 397 Caucasian controls. Results We identified a total of 47 variants, including 38 rare variants of which 35 were novel. Association testing identified two ZNF750 haplotypes associated with psoriasis (p ZNF750 promoter and 5' UTR variants displayed a 35-55% reduction of ZNF750 promoter activity, consistent with the promoter activity reduction seen in a Mendelian psoriasis family with a ZNF750 promoter variant. However, the rare promoter and 5' UTR variants identified in this study did not strictly segregate with the psoriasis phenotype within families. Conclusions Two haplotypes of ZNF750 and rare 5' regulatory variants of ZNF750 were found to be associated with psoriasis. These rare 5' regulatory variants, though not causal, might serve as a genetic modifier of psoriasis.

  13. Rare variants in PLXNA4 and Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Eva C Schulte

    Full Text Available Approximately 20% of individuals with Parkinson's disease (PD report a positive family history. Yet, a large portion of causal and disease-modifying variants is still unknown. We used exome sequencing in two affected individuals from a family with late-onset familial PD followed by frequency assessment in 975 PD cases and 1014 ethnically-matched controls and linkage analysis to identify potentially causal variants. Based on the predicted penetrance and the frequencies, a variant in PLXNA4 proved to be the best candidate and PLXNA4 was screened for additional variants in 862 PD cases and 940 controls, revealing an excess of rare non-synonymous coding variants in PLXNA4 in individuals with PD. Although we cannot conclude that the variant in PLXNA4 is indeed the causative variant, these findings are interesting in the light of a surfacing role of axonal guidance mechanisms in neurodegenerative disorders but, at the same time, highlight the difficulties encountered in the study of rare variants identified by next-generation sequencing in diseases with autosomal dominant or complex patterns of inheritance.

  14. Pathological assessment of mismatch repair gene variants in Lynch syndrome

    DEFF Research Database (Denmark)

    Rasmussen, Lene Juel; Heinen, Christopher D; Royer-Pokora, Brigitte;

    2012-01-01

    . Also, identifying family members that do not carry the variant is important so they can be released from the intensive surveillance. Determining which genetic variants are pathogenic and which are neutral is a major challenge in clinical genetics. The profound mechanistic knowledge on the genetics...

  15. Detecting rare variants in case-parents association studies.

    Directory of Open Access Journals (Sweden)

    Kuang-Fu Cheng

    Full Text Available Despite the success of genome-wide association studies (GWASs in detecting common variants (minor allele frequency ≥0.05 many suggested that rare variants also contribute to the genetic architecture of diseases. Recently, researchers demonstrated that rare variants can show a strong stratification which may not be corrected by using existing methods. In this paper, we focus on a case-parents study and consider methods for testing group-wise association between multiple rare (and common variants in a gene region and a disease. All tests depend on the numbers of transmitted mutant alleles from parents to their diseased children across variants and hence they are robust to the effect of population stratification. We use extensive simulation studies to compare the performance of four competing tests: the largest single-variant transmission disequilibrium test (TDT, multivariable test, combined TDT, and a likelihood ratio test based on a random-effects model. We find that the likelihood ratio test is most powerful in a wide range of settings and there is no negative impact to its power performance when common variants are also included in the analysis. If deleterious and protective variants are simultaneously analyzed, the likelihood ratio test was generally insensitive to the effect directionality, unless the effects are extremely inconsistent in one direction.

  16. Clinical and laboratory update on the DEL variant.

    Science.gov (United States)

    Nuchnoi, Pornlada; Thongbus, Jairak; Srisarin, Apapan; Kerdpin, Usanee; Prachayasittikul, Virapong

    2014-01-01

    Serological assays for the RhD blood group are based on detection of the RhD antigen on human red blood cells using a specific anti-D antibody. The weak expression of the RhD antigen in the DEL variant hinders the sensitivity of conventional serological assays. Evidence of anti-D immunization in patients with D-negativity who have received DEL-variant blood units has been reported in various populations. This observation has prompted the need for genetic epidemiological and clinical data on the DEL variant in the development of DEL molecular diagnostic testing. This review highlights the molecular features of the DEL variant, the clinical consequences of DEL-blood transfusion, and current approaches for detection of the DEL-variant for donor screening and transfusion. PMID:25316658

  17. Alzheimer's disease: rare variants with large effect sizes.

    Science.gov (United States)

    Del-Aguila, Jorge L; Koboldt, Daniel C; Black, Kathleen; Chasse, Rachel; Norton, Joanne; Wilson, Richard K; Cruchaga, Carlos

    2015-08-01

    Recent advances in sequencing technology and novel genotyping arrays (focused on low-frequency and coding variants) have made it possible to identify novel coding variants with large effect sizes and also novel genes (TREM2, PLD3, UNC5C, and AKAP9) associated with Alzheimer's disease (AD) risk. The major advantages of these studies over the classic genome-wide association studies (GWAS) include the identification of the functional variant and the gene-driven association. In addition to the large effect size, these studies make it possible to model these variants and genes using cell and animal systems. On the other hand, the underlying population-variability of these very low allele frequency variants poses a great challenge to replicating results. Studies that include very large datasets (>10,000 cases and controls) and combine sequencing and genotyping approaches will lead to the identification of novel genes for Alzheimer's disease. PMID:26311074

  18. Electrophoretic variants of blood proteins in Japanese, 7

    International Nuclear Information System (INIS)

    A total of 16,835 children, of whom 11,737 are unrelated, from Hiroshima and Nagasaki were examined for erythrocyte cytoplasmic glutamate-oxaloacetate transaminase (GOT1) by starch gel electrophoresis. A variant allele named GOT1*2HR1 which seems to be identical with GOT1*2 was encountered in polymorphic frequency. Five kinds of rare variants, 3NG1, 4NG1, 5NG1, 6HR1, and 7NG1 were encountered in a total of 109 children. Except for 7NG1 for which complete family study was unable, family studies confirmed the genetic nature of these rare variants, since for all instances in which both parents could be examined, one of the parents exhibited the same variant as that of their child. Thermostability profiles of these six variants were normal. The enzyme activities of five were decreased, while the value of one was normal compared to that of GOT1 1. (author)

  19. Genetic variants in Alzheimer disease - molecular and brain network approaches.

    Science.gov (United States)

    Gaiteri, Chris; Mostafavi, Sara; Honey, Christopher J; De Jager, Philip L; Bennett, David A

    2016-07-01

    Genetic studies in late-onset Alzheimer disease (LOAD) are aimed at identifying core disease mechanisms and providing potential biomarkers and drug candidates to improve clinical care of AD. However, owing to the complexity of LOAD, including pathological heterogeneity and disease polygenicity, extraction of actionable guidance from LOAD genetics has been challenging. Past attempts to summarize the effects of LOAD-associated genetic variants have used pathway analysis and collections of small-scale experiments to hypothesize functional convergence across several variants. In this Review, we discuss how the study of molecular, cellular and brain networks provides additional information on the effects of LOAD-associated genetic variants. We then discuss emerging combinations of these omic data sets into multiscale models, which provide a more comprehensive representation of the effects of LOAD-associated genetic variants at multiple biophysical scales. Furthermore, we highlight the clinical potential of mechanistically coupling genetic variants and disease phenotypes with multiscale brain models. PMID:27282653

  20. Bayesian detection of causal rare variants under posterior consistency.

    Directory of Open Access Journals (Sweden)

    Faming Liang

    Full Text Available Identification of causal rare variants that are associated with complex traits poses a central challenge on genome-wide association studies. However, most current research focuses only on testing the global association whether the rare variants in a given genomic region are collectively associated with the trait. Although some recent work, e.g., the Bayesian risk index method, have tried to address this problem, it is unclear whether the causal rare variants can be consistently identified by them in the small-n-large-P situation. We develop a new Bayesian method, the so-called Bayesian Rare Variant Detector (BRVD, to tackle this problem. The new method simultaneously addresses two issues: (i (Global association test Are there any of the variants associated with the disease, and (ii (Causal variant detection Which variants, if any, are driving the association. The BRVD ensures the causal rare variants to be consistently identified in the small-n-large-P situation by imposing some appropriate prior distributions on the model and model specific parameters. The numerical results indicate that the BRVD is more powerful for testing the global association than the existing methods, such as the combined multivariate and collapsing test, weighted sum statistic test, RARECOVER, sequence kernel association test, and Bayesian risk index, and also more powerful for identification of causal rare variants than the Bayesian risk index method. The BRVD has also been successfully applied to the Early-Onset Myocardial Infarction (EOMI Exome Sequence Data. It identified a few causal rare variants that have been verified in the literature.

  1. Bayesian detection of causal rare variants under posterior consistency.

    KAUST Repository

    Liang, Faming

    2013-07-26

    Identification of causal rare variants that are associated with complex traits poses a central challenge on genome-wide association studies. However, most current research focuses only on testing the global association whether the rare variants in a given genomic region are collectively associated with the trait. Although some recent work, e.g., the Bayesian risk index method, have tried to address this problem, it is unclear whether the causal rare variants can be consistently identified by them in the small-n-large-P situation. We develop a new Bayesian method, the so-called Bayesian Rare Variant Detector (BRVD), to tackle this problem. The new method simultaneously addresses two issues: (i) (Global association test) Are there any of the variants associated with the disease, and (ii) (Causal variant detection) Which variants, if any, are driving the association. The BRVD ensures the causal rare variants to be consistently identified in the small-n-large-P situation by imposing some appropriate prior distributions on the model and model specific parameters. The numerical results indicate that the BRVD is more powerful for testing the global association than the existing methods, such as the combined multivariate and collapsing test, weighted sum statistic test, RARECOVER, sequence kernel association test, and Bayesian risk index, and also more powerful for identification of causal rare variants than the Bayesian risk index method. The BRVD has also been successfully applied to the Early-Onset Myocardial Infarction (EOMI) Exome Sequence Data. It identified a few causal rare variants that have been verified in the literature.

  2. Regulated expression of the rat recombinant P2X(3) receptor in stably transfected CHO-K1 tTA cells.

    Science.gov (United States)

    Lachnit, W G; Oglesby, I B; Gever, J R; Gever, M; Huang, C; Li, X C; Jin, H; McGivern, J G; Ford, A P

    2000-07-01

    In this report, the regulatable expression by tetracycline of the rat recombinant P2X(3) receptor in stably transfected Chinese hamster ovary (CHO-K1) expressing the tetracycline-controlled transactivator (tTA) is described. cDNA encoding the rat P2X(3)-receptor was subcloned into pTRE (a tetracycline-repressible expression vector) which was used to transfect stably CHO-K1 tTA cells. Using whole cell patch clamp techniques, 100 microM ATP evoked inward currents of 2.9+/-1.6 nA in transfected cells grown in the absence of tetracycline (tet-). The P2X(3) receptor protein was detectable by immunoblot as early as 24 h and protein expression levels continued to increase as much as 192 h following activation of tTA by the removal of the antibiotic. Saturation binding isotherms using [35S]ATP gamma S yielded a pK(d) of 8.2+/-0.1 and a B(max) of 31.9+/-3.5 pmol/mg protein in tet- cell membranes and a pK(d) of 8.1+/-0.1 and a B(max) of 5.8+/-0.8 pmol/mg protein in tet+ cell membranes. The agonist ligands 2MeSATP and alpha beta MeATP displaced the binding of [35S]ATP gamma S in tet- cell membranes with very high affinity, yielding pIC(50) values of 9.4+/-0.2 and 7.5+/-0. 2, respectively. In tet+ cell membrane, displacement of [35S]ATP gamma S by 2MeSATP and alpha beta MeATP was of much lower affinity (pIC(50) values of 7.8 and 6.2, respectively). ATP, ADP and UTP showed similar displacement of [35S]ATP gamma S binding in tet- and tet+ cell membranes. In other experiments, cytosolic Ca(2+) was monitored using the fluorescent indicator, fluo-3. Increases in cytosolic Ca(2+) were elicited by 100 nM alpha beta MeATP in tet- cells while no increases in cytosolic Ca(2+) were detected below 100 microM alpha beta MeATP in either tet+ cells or untransfected cells. These calcium responses to alpha beta MeATP had a pEC(50) of 6.7 and were transient, returning to baseline within 120 s. Suramin produced concentration-dependent, parallel, dextral shifts of E/[A] curves to alpha beta Me

  3. An Optimized Method for Suspension Culture of CHO Cells to Produce Recombinant Human Erythropoietin (EPO)%悬浮培养CHO细胞生产重组人促红细胞生成素条件的优化

    Institute of Scientific and Technical Information of China (English)

    杨栋; 牛红军; 陆刚; 史嘉林; 孙浩明; 李晖

    2012-01-01

    Objective: To screen and domesticate the adherent cultured CHO cells to obtain high expression of cell suspension culture for production of recombinant human erythropoietin erythropoietin (rHuEPO). Methods: Using 96-well and 24-well plates culture method to screen and domesticate the highly expressing CHO cell strain. Acclimate the high expression cell strain and make it suitable for suspension culture. It's inoculated into the bioreactor in serum-free culture after amplified by the shake flask, and monitoring of glucose content, measuring rHuEPO expression of daily. Results: The suspension culture of CHO cell production of rHuEPO has short production period, higher expression than adherent culture. On the other hand, it is easy to operate and scale-up, but not easy to pollute. Furthermore, we established of the CHO cell strain for suspension culture,which provided a technical basis for industrial production of CHO cells the rHuEPO. Conclusion: After process optimization, the use of serum-free suspension culture production of erythropoietin average expression has high, short production period, low cost of production.than adherent culture.%目的:通过对贴壁培养CHO细胞筛选驯化,得到高表达的细胞后进行悬浮培养生产重组人促红细胞生成素(rHuEPO).方法:利用96孔板和24孔板对CHO细胞进行筛选,得到高表达细胞株后进行驯化,使其适合悬浮培养,经过摇瓶扩增后接种到生物反应器中无血清培养,每天监测葡萄糖含量,测rHuEPO表达量.结果:悬浮培养CHO细胞生产rHuEPO,生产周期短,表达量比贴壁培养高出很多,操作方便,减少污染,易于放大,并建立了适合悬浮培养的CHO细胞株,为工业化悬浮培养CHO细胞生产rHuEPO提供了技术基础.结论:经过工艺优化后利用无血清悬浮培养生产促红细胞生成素平均表达量较贴壁培养高,生产周期短,有利于降低生产成本.

  4. Yellow fluorescent protein-based assay to measure GABA(A channel activation and allosteric modulation in CHO-K1 cells.

    Directory of Open Access Journals (Sweden)

    Teres Johansson

    Full Text Available The γ-aminobutyric acid A (GABA(A ion channels are important drug targets for treatment of neurological and psychiatric disorders. Finding GABA(A channel subtype selective allosteric modulators could lead to new improved treatments. However, the progress in this area has been obstructed by the challenging task of developing functional assays to support screening efforts and the generation of cells expressing functional GABA(A ion channels with the desired subtype composition. To address these challenges, we developed a yellow fluorescent protein (YFP-based assay to be able to study allosteric modulation of the GABA(A ion channel using cryopreserved, transiently transfected, assay-ready cells. We show for the first time how the MaxCyte STX electroporation instrument can be used to generate CHO-K1 cells expressing functional GABA(A α2β3γ2 along with a halide sensing YFP-H148Q/I152L (YFP-GABA(A2 cells. As a basis for a cell-based assay capable of detecting allosteric modulators, experiments with antagonist, ion channel blocker and modulators were used to verify GABA(A subunit composition and functionality. We found that the I(- concentration used in the YFP assay affected both basal quench of YFP and potency of GABA. For the first time the assay was used to study modulation of GABA with 7 known modulators where statistical analysis showed that the assay can distinguish modulatory pEC50 differences of 0.15. In conclusion, the YFP assay proved to be a robust, reproducible and inexpensive assay. These data provide evidence that the assay is suitable for high throughput screening (HTS and could be used to discover novel modulators acting on GABA(A ion channels.

  5. Multifrequency permittivity measurements enable on-line monitoring of changes in intracellular conductivity due to nutrient limitations during batch cultivations of CHO cells.

    Science.gov (United States)

    Ansorge, Sven; Esteban, Geoffrey; Schmid, Georg

    2010-01-01

    Lab and pilot scale batch cultivations of a CHO K1/dhfr(-) host cell line were conducted to evaluate on-line multifrequency permittivity measurements as a process monitoring tool. The beta-dispersion parameters such as the characteristic frequency (f(C)) and the permittivity increment (Deltaepsilon(max)) were calculated on-line from the permittivity spectra. The dual-frequency permittivity signal correlated well with the off-line measured biovolume and the viable cell density. A significant drop in permittivity was monitored at the transition from exponential growth to a phase with reduced growth rate. Although not reflected in off-line biovolume measurements, this decrease coincided with a drop in OUR and was probably caused by the depletion of glutamine and a metabolic shift occurring at the same time. Sudden changes in cell density, cell size, viability, capacitance per membrane area (C(M)), and effects caused by medium conductivity (sigma(m)) could be excluded as reasons for the decrease in permittivity. After analysis of the process data, a drop in f(C) as a result of a fall in intracellular conductivity (sigma(i)) was identified as responsible for the observed changes in the dual-frequency permittivity signal. It is hypothesized that the beta-dispersion parameter f(C) is indicative of changes in nutrient availability that have an impact on intracellular conductivity sigma(i). On-line permittivity measurements consequently not only reflect the biovolume but also the physiological state of mammalian cell cultures. These findings should pave the way for a better understanding of the intracellular state of cells and render permittivity measurements an important tool in process development and control.

  6. Intracellular pH and 42.00 C heat response of CHO cells cultured at pH 6.6

    International Nuclear Information System (INIS)

    The authors previously reported that cells under chronic low pH (6.6) conditions have altered thermotolerance. They further characterized both the doubling time (t/sub d/) and the internal pH (pH/sub 1/) of CHO cells continuously cultured at pH 6.6 for times greater than one year. The following differences were noted: 1) A t/sub d/ of 16 hr compared to a t/sub d/ of 12 hr for cells at normal pH (7.3) and a t/sub d/ of 25 hr for the acute low pH cells (pH = 6.6; incubation time = 4 hr). 2) A pH/sub i/ 0.1-0.15 pH units > normal cells and 0.3 pH units > acute low pH cells. 3) Survival at 42.00C which differed from both normal and acute low pH cells. The chronic culture was still quite sensitive to 42.00C treatments during the first 5 hr, but developed tolerance at a higher level than cells under acute low pH conditions. The pH/sub i/ of the chronic culture responded to 42.00C heating in a manner similar to that for acute low pH cells. Whether this culture represents a normal response to long term low pH exposure, or was the response of a mutant population is at the present unknown

  7. Expression of the nfa1 gene cloned from pathogenic Naegleria fowleri in nonpathogenic N. gruberi enhances cytotoxicity against CHO target cells in vitro.

    Science.gov (United States)

    Jeong, Seok-Ryoul; Lee, Sang-Chul; Song, Kyoung-Ju; Park, Sun; Kim, Kyongmin; Kwon, Myung-Hee; Im, Kyung-Il; Shin, Ho-Joon

    2005-07-01

    The pathogenic amoeba Naegleria fowleri has a 360-bp nfa1 gene that encodes the Nfa1 protein (13.1 kDa), which is located in the pseudopodia of the amoeba, and an anti-Nfa1 antibody reduces N. fowleri-induced mammalian-cell cytotoxicity in vitro. In contrast, an anti-Nfa1 antibody cannot detect Nfa1 protein expression in the nonpathogenic amoeba Naegleria gruberi, which also possesses the nfa1 gene. In the present study, the nfa1 gene cloned from pathogenic N. fowleri was transfected into nonpathogenic N. gruberi to determine whether it was related to pathogenicity. The nfa1 gene was initially inserted into a eukaryotic transfection vector, pEGFP-C2, containing a cytomegalovirus promoter and the green fluorescent protein (GFP) gene, and was designed as pEGFP-C2/nfa1UTR (nfa1UTR contains 5' upstream regions, the nfa1 open reading frame, and 3' downstream regions). After transfection, the green fluorescence was observed in the cytoplasm of N. gruberi trophozoites. These transfectants were preserved for more than 9 months after selection. The transfected nfa1 gene was observed by PCR using nfa1- and vector-specific primers in the genomic DNA of N. gruberi transfected with the pEGFP-C2/nfa1UTR vector. In addition, the nfa1 and GFP genes were identified by reverse transcription-PCR in transgenic N. gruberi. The Nfa1 protein expressed in transgenic N. gruberi was identified as a 13.1-kDa band by Western blotting using an anti-Nfa1 antibody. Finally, N. gruberi transfected with the pEGFP-C2/nfa1UTR vector was found to have enhanced cytotoxicity against CHO cells compared with naïve N. gruberi.

  8. Comparison of internal ribosome entry site (IRES and Furin-2A (F2A for monoclonal antibody expression level and quality in CHO cells.

    Directory of Open Access Journals (Sweden)

    Steven C L Ho

    Full Text Available Four versions of tricistronic vectors expressing IgG1 light chain (LC, IgG1 heavy chain (HC, and dihydrofolate reductase (DHFR in one transcript were designed to compare internal ribosome entry site (IRES and furin-2A (F2A for their influence on monoclonal antibody (mAb expression level and quality in CHO DG44 cells. LC and HC genes are arranged as either the first or the second cistron. When using mAb quantification methods based on the detection antibodies against HC Fc region, F2A-mediated tricistronic vectors appeared to express mAb at higher levels than the IRES-mediated tricistronic vectors in both transient and stable transfections. Further analysis revealed that more than 40% of products detected in stably transfected pools generated using the two F2A-mediated tricistronic vectors were aggregates. LC and HC from the F2A stably transfected pools were not properly processed, giving rise to LC+F2A+HC or HC+F2A+LC fusion proteins, LC and HC polypeptides with F2A remnants, and incorrectly cleaved signal peptides. Both IRES-mediated tricistronic vectors express mAb with correct sizes and signal peptide cleavage. Arrangement of LC as the first cistron in the IRES-mediated tricistronic vectors exhibits increased mAb expression level, better growth, and minimized product aggregation, while arrangement of HC as first cistron results in low expression, slower growth, and high aggregation. The results obtained will be beneficial for designing vectors that enhance mAb expression level and quality in mammalian cells.

  9. Use of a Plackett-Burman statistical design to determine the effect of selected amino acids on monoclonal antibody production in CHO cells.

    Science.gov (United States)

    González-Leal, I J; Carrillo-Cocom, L M; Ramírez-Medrano, A; López-Pacheco, F; Bulnes-Abundis, D; Webb-Vargas, Y; Alvarez, M M

    2011-01-01

    Culture media design is central to the optimization of monoclonal antibody (mAb) production. Although general strategies do not currently exist for optimization of culture media, the combined use of statistical design and analysis of experiments and strategies based on simple material balances can facilitate culture media design. In this study, we evaluate the effect of selected amino acids on the growth rate and monoclonal antibody production of a Chinese hamster ovary DG-44 (CHO-DG44) cell line. These amino acids were selected based on their relative mass fraction in the specific mAb produced in this study, their consumption rate during bioreactor experiments, and also through a literature review. A Plackett-Burman statistical design was conducted to minimize the number of experiments needed to obtain statistically relevant information. The effect of this set of amino acids was evaluated during exponential cell culture (considering viable cell concentration and the specific growth rate as main output variables) and during the high cell-density stage (considering mAb final concentration and specific productivity as relevant output variables). For this particular cell line, leucine (Leu) and arginine (Arg) had the highest negative and positive effects on cell viability, respectively; Leu and threonine (Thr) had the highest negative effect on growth rate, and valine (Val) and Arg demonstrated the highest positive impact on mAb final concentration. Results suggest the pertinence of a two-stage strategy for amino acid supplementation, with a mixture optimized for cell growth and a different amino acid mixture for mAb production at high density.

  10. The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate

    Energy Technology Data Exchange (ETDEWEB)

    Bajinskis, Ainars, E-mail: ainars.bajinskis@gmt.su.se [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden); Olsson, Gunilla; Harms-Ringdahl, Mats [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden)

    2012-03-01

    The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO{sub 3}). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure.

  11. HIV-1 genetic variants in Kyrgyzstan

    Directory of Open Access Journals (Sweden)

    V Laga

    2012-11-01

    Full Text Available Objectives: During the last two decades, HIV-1 has been spreading rapidly in former Soviet Union republics including Kyrgyzstan. The current molecular monitoring of HIV-infection epidemic is carried out in Russia only with no or limited data from the other FSU countries. The aim of this work was to investigate the prevalence of HIV-1 genetic variants circulating in Kyrgyzstan. Methods: Blood collection from the HIV-infected patients was carried out by local specialists with the informed consent and the questionnaire was answered by each of the patients. The total number of samples was 100. The washed cell pellets were transferred to Moscow following with proviral DNA extraction, PCR amplification and gag, pol and env genes sequencing. The phylogenetic analysis of nucleotide sequences using neighbor-joining method was carried out by MEGA 3 program. The preliminary data were obtained in 22 samples isolated from PBMC of HIV-infected patients from Kyrgyzstan. Results: Among the samples studied 6 (27.3% samples belonged to a subtype CRF02_AG, 16 samples - to subtype A (A1. One of the samples belonging to CRF02_AG, probably, is a recombinant between CRF02_AG and A1. There was no major drug resistance mutations in the samples studied. The minor mutations were presented in small proportions: 1 in PR (L10I, 6 in RT (A62V - in 3 samples, V108G, E138A, Y181F, M184I, L210M - on one sample and 1 in IN (L74M. It was impossible to associate the distribution of mutations with HIV-1 genetic variant. The V3 loop (env gene in 17 samples was analyzed for tropism using geno2pheno program; all samples were found to be R5-viruses. Conclusion: The HIV-1 subtype A seems to dominate in Kyrgyzstan like in other FSU countries. The recombinant CRF02_AG epidemiologically linked to Uzbekistan is quite widespread. The rest of Kyrgyzstan collection is under investigation and the data will be refined soon.

  12. αIIbβ3 variants defined by next-generation sequencing: Predicting variants likely to cause Glanzmann thrombasthenia

    Science.gov (United States)

    Buitrago, Lorena; Rendon, Augusto; Liang, Yupu; Simeoni, Ilenia; Negri, Ana; Filizola, Marta; Ouwehand, Willem H.; Coller, Barry S.; Alessi, Marie-Christine; Ballmaier, Matthias; Bariana, Tadbir; Bellissimo, Daniel; Bertoli, Marta; Bray, Paul; Bury, Loredana; Carrell, Robin; Cattaneo, Marco; Collins, Peter; French, Deborah; Favier, Remi; Freson, Kathleen; Furie, Bruce; Germeshausen, Manuela; Ghevaert, Cedric; Gomez, Keith; Goodeve, Anne; Gresele, Paolo; Guerrero, Jose; Hampshire, Dan J.; Hadinnapola, Charaka; Heemskerk, Johan; Henskens, Yvonne; Hill, Marian; Hogg, Nancy; Johnsen, Jill; Kahr, Walter; Kerr, Ron; Kunishima, Shinji; Laffan, Michael; Natwani, Amit; Neerman-Arbez, Marguerite; Nurden, Paquita; Nurden, Alan; Ormiston, Mark; Othman, Maha; Ouwehand, Willem; Perry, David; Vilk, Shoshana Ravel; Reitsma, Pieter; Rondina, Matthew; Simeoni, Ilenia; Smethurst, Peter; Stephens, Jonathan; Stevenson, William; Szkotak, Artur; Turro, Ernest; Van Geet, Christel; Vries, Minka; Ward, June; Waye, John; Westbury, Sarah; Whiteheart, Sidney; Wilcox, David; Zhang, Bi

    2015-01-01

    Next-generation sequencing is transforming our understanding of human genetic variation but assessing the functional impact of novel variants presents challenges. We analyzed missense variants in the integrin αIIbβ3 receptor subunit genes ITGA2B and ITGB3 identified by whole-exome or -genome sequencing in the ThromboGenomics project, comprising ∼32,000 alleles from 16,108 individuals. We analyzed the results in comparison with 111 missense variants in these genes previously reported as being associated with Glanzmann thrombasthenia (GT), 20 associated with alloimmune thrombocytopenia, and 5 associated with aniso/macrothrombocytopenia. We identified 114 novel missense variants in ITGA2B (affecting ∼11% of the amino acids) and 68 novel missense variants in ITGB3 (affecting ∼9% of the amino acids). Of the variants, 96% had minor allele frequencies (MAF) < 0.1%, indicating their rarity. Based on sequence conservation, MAF, and location on a complete model of αIIbβ3, we selected three novel variants that affect amino acids previously associated with GT for expression in HEK293 cells. αIIb P176H and β3 C547G severely reduced αIIbβ3 expression, whereas αIIb P943A partially reduced αIIbβ3 expression and had no effect on fibrinogen binding. We used receiver operating characteristic curves of combined annotation-dependent depletion, Polyphen 2-HDIV, and sorting intolerant from tolerant to estimate the percentage of novel variants likely to be deleterious. At optimal cut-off values, which had 69–98% sensitivity in detecting GT mutations, between 27% and 71% of the novel αIIb or β3 missense variants were predicted to be deleterious. Our data have implications for understanding the evolutionary pressure on αIIbβ3 and highlight the challenges in predicting the clinical significance of novel missense variants. PMID:25827233

  13. CRY2 genetic variants associate with dysthymia.

    Directory of Open Access Journals (Sweden)

    Leena Kovanen

    Full Text Available People with mood disorders often have disruptions in their circadian rhythms. Recent molecular genetics has linked circadian clock genes to mood disorders. Our objective was to study two core circadian clock genes, CRY1 and CRY2 as well as TTC1 that interacts with CRY2, in relation to depressive and anxiety disorders. Of these three genes, 48 single-nucleotide polymorphisms (SNPs whose selection was based on the linkage disequilibrium and potential functionality were genotyped in 5910 individuals from a nationwide population-based sample. The diagnoses of major depressive disorder, dysthymia and anxiety disorders were assessed with a structured interview (M-CIDI. In addition, the participants filled in self-report questionnaires on depressive and anxiety symptoms. Logistic and linear regression models were used to analyze the associations of the SNPs with the phenotypes. Four CRY2 genetic variants (rs10838524, rs7121611, rs7945565, rs1401419 associated significantly with dysthymia (false discovery rate q<0.05. This finding together with earlier CRY2 associations with winter depression and with bipolar type 1 disorder supports the view that CRY2 gene has a role in mood disorders.

  14. Identification of copy number variants in horses

    KAUST Repository

    Doan, R.

    2012-03-01

    Copy number variants (CNVs) represent a substantial source of genetic variation in mammals. However, the occurrence of CNVs in horses and their subsequent impact on phenotypic variation is unknown. We performed a study to identify CNVs in 16 horses representing 15 distinct breeds (Equus caballus) and an individual gray donkey (Equus asinus) using a whole-exome tiling array and the array comparative genomic hybridization methodology. We identified 2368 CNVs ranging in size from 197 bp to 3.5 Mb. Merging identical CNVs from each animal yielded 775 CNV regions (CNVRs), involving 1707 protein- and RNA-coding genes. The number of CNVs per animal ranged from 55 to 347, with median and mean sizes of CNVs of 5.3 kb and 99.4 kb, respectively. Approximately 6% of the genes investigated were affected by a CNV. Biological process enrichment analysis indicated CNVs primarily affected genes involved in sensory perception, signal transduction, and metabolism. CNVs also were identified in genes regulating blood group antigens, coat color, fecundity, lactation, keratin formation, neuronal homeostasis, and height in other species. Collectively, these data are the first report of copy number variation in horses and suggest that CNVs are common in the horse genome and may modulate biological processes underlying different traits observed among horses and horse breeds.

  15. How important are rare variants in common disease?

    Science.gov (United States)

    Saint Pierre, Aude; Génin, Emmanuelle

    2014-09-01

    Genome-wide association studies have uncovered hundreds of common genetic variants involved in complex diseases. However, for most complex diseases, these common genetic variants only marginally contribute to disease susceptibility. It is now argued that rare variants located in different genes could in fact play a more important role in disease susceptibility than common variants. These rare genetic variants were not captured by genome-wide association studies using single nucleotide polymorphism-chips but with the advent of next-generation sequencing technologies, they have become detectable. It is now possible to study their contribution to common disease by resequencing samples of cases and controls or by using new genotyping exome arrays that cover rare alleles. In this review, we address the question of the contribution of rare variants in common disease by taking the examples of different diseases for which some resequencing studies have already been performed, and by summarizing the results of simulation studies conducted so far to investigate the genetic architecture of complex traits in human. So far, empirical data have not allowed the exclusion of many models except the most extreme ones involving only a small number of rare variants with large effects contributing to complex disease. To unravel the genetic architecture of complex disease, case-control data will not be sufficient, and alternative study designs need to be proposed together with methodological developments.

  16. The Clinical Significance of Unknown Sequence Variants in BRCA Genes

    Energy Technology Data Exchange (ETDEWEB)

    Calò, Valentina; Bruno, Loredana; Paglia, Laura La; Perez, Marco; Margarese, Naomi [Department of Surgery and Oncology, Regional Reference Center for the Biomolecular Characterization and Genetic Screening of Hereditary Tumors, University of Palermo, Via del Vespro 127, 90127 Palermo (Italy); Gaudio, Francesca Di [Department of Medical Biotechnologies and Legal Medicine, University of Palermo, Palermo (Italy); Russo, Antonio, E-mail: lab-oncobiologia@usa.net [Department of Surgery and Oncology, Regional Reference Center for the Biomolecular Characterization and Genetic Screening of Hereditary Tumors, University of Palermo, Via del Vespro 127, 90127 Palermo (Italy)

    2010-09-10

    Germline mutations in BRCA1/2 genes are responsible for a large proportion of hereditary breast and/or ovarian cancers. Many highly penetrant predisposition alleles have been identified and include frameshift or nonsense mutations that lead to the translation of a truncated protein. Other alleles contain missense mutations, which result in amino acid substitution and intronic variants with splicing effect. The discovery of variants of uncertain/unclassified significance (VUS) is a result that can complicate rather than improve the risk assessment process. VUSs are mainly missense mutations, but also include a number of intronic variants and in-frame deletions and insertions. Over 2,000 unique BRCA1 and BRCA2 missense variants have been identified, located throughout the whole gene (Breast Cancer Information Core Database (BIC database)). Up to 10–20% of the BRCA tests report the identification of a variant of uncertain significance. There are many methods to discriminate deleterious/high-risk from neutral/low-risk unclassified variants (i.e., analysis of the cosegregation in families of the VUS, measure of the influence of the VUSs on the wild-type protein activity, comparison of sequence conservation across multiple species), but only an integrated analysis of these methods can contribute to a real interpretation of the functional and clinical role of the discussed variants. The aim of our manuscript is to review the studies on BRCA VUS in order to clarify their clinical relevance.

  17. Private mitochondrial DNA variants in danish patients with hypertrophic cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Christian M Hagen

    Full Text Available Hypertrophic cardiomyopathy (HCM is a genetic cardiac disease primarily caused by mutations in genes coding for sarcomeric proteins. A molecular-genetic etiology can be established in ~60% of cases. Evolutionarily conserved mitochondrial DNA (mtDNA haplogroups are susceptibility factors for HCM. Several polymorphic mtDNA variants are associated with a variety of late-onset degenerative diseases and affect mitochondrial function. We examined the role of private, non-haplogroup associated, mitochondrial variants in the etiology of HCM. In 87 Danish HCM patients, full mtDNA sequencing revealed 446 variants. After elimination of 312 (69.9% non-coding and synonymous variants, a further 109 (24.4% with a global prevalence > 0.1%, three (0.7% haplogroup associated and 19 (2.0% variants with a low predicted in silico likelihood of pathogenicity, three variants: MT-TC: m.5772G>A, MT-TF: m.644A>G, and MT-CYB: m.15024G>A, p.C93Y remained. A detailed analysis of these variants indicated that none of them are likely to cause HCM. In conclusion, private mtDNA mutations are frequent, but they are rarely, if ever, associated with HCM.

  18. VCF-Miner: GUI-based application for mining variants and annotations stored in VCF files

    OpenAIRE

    Steven N Hart; Duffy, Patrick; Quest, Daniel J.; Hossain, Asif; Meiners, Mike A; Kocher, Jean-Pierre

    2015-01-01

    Next-generation sequencing platforms are widely used to discover variants associated with disease. The processing of sequencing data involves read alignment, variant calling, variant annotation and variant filtering. The standard file format to hold variant calls is the variant call format (VCF) file. According to the format specifications, any arbitrary annotation can be added to the VCF file for downstream processing. However, most downstream analysis programs disregard annotations already ...

  19. Human papillomavirus type-16 variants in Quechua aboriginals from Argentina.

    Science.gov (United States)

    Picconi, María Alejandra; Alonio, Lidia Virginia; Sichero, Laura; Mbayed, Viviana; Villa, Luisa Lina; Gronda, Jorge; Campos, Rodolfo; Teyssié, Angélica

    2003-04-01

    Cervical carcinoma is the leading cause of cancer death in Quechua indians from Jujuy (northwestern Argentina). To determine the prevalence of HPV-16 variants, 106 HPV-16 positive cervical samples were studied, including 33 low-grade squamous intraepithelial lesions (LSIL), 28 high-grade squamous intraepithelial lesions (HSIL), 9 invasive cervical cancer (ICC), and 36 samples from women with normal colposcopy and cytology. HPV genome variability was examined in the L1 and E6 genes by PCR-hybridization. In a subset of 20 samples, a LCR fragment was also analyzed by PCR-sequencing. Most variants belonged to the European branch with subtle differences that depended on the viral gene fragment studied. Only about 10% of the specimens had non-European variants, including eight Asian-American, two Asian, and one North-American-1. E6 gene analysis revealed that 43% of the samples were identical to HPV-16 prototype, while 57% corresponded to variants. Interestingly, the majority (87%) of normal smears had HPV-16 prototype, whereas variants were detected mainly in SIL and ICC. LCR sequencing yielded 80% of variants, including 69% of European, 19% Asian-American, and 12% Asian. We identified a new variant, the Argentine Quechua-51 (AQ-51), similar to B-14 plus two additional changes: G7842-->A and A7837-->C; phylogenetic inference allocated it in the Asian-American branch. The high proportion of European variants may reflect Spanish colonial influence on these native Inca descendants. The predominance of HPV-16 variants in pathologic samples when compared to normal controls could have implications for the natural history of cervical lesions.

  20. Variante de Dandy Walker: relato de caso = Dandy Walker variant: a case report

    Directory of Open Access Journals (Sweden)

    Khan, Richard Lester et al.

    2009-01-01

    Full Text Available Objetivos: relatar o caso de um paciente com variante de Dandy Walker, chamando atenção para a importância da suspeita, investigação e manejo das repercussões clínicas. Descrição do caso: é relatado o caso de um paciente do sexo masculino, com quadro clínico e radiológico típico da Variante de Dandy Walker. Durante o pré-natal, através de ecografia obstétrica com 23 semanas e 3 dias, apresentou alterações sugestivas de Síndrome de Dandy Walker. Ao nascimento apresentou exame físico com fenda palatina, criptorquidia à direita, hexodactilia em ambos os pés. Apresentava ainda ecocardiograma com forame oval patente e persistência do canal arterial. O diagnóstico foi estabelecido através da ressonância magnética realizada após o nascimento, que evidenciava hipoplasia do vermis cerebelar, alargamento da fossa posterior e leve dilatação ventricular. Conclusões: este artigo procura caracterizar a variante de Dandy Walker, que é uma malformação congênita do sistema nervoso central e é o tipo mais comum da Síndrome de Dandy Walker. Seu fenótipo é variável, devendo-se sempre pesquisar malformações tanto intra quanto extracranianas, visto que o risco de mortalidade pós-natal aumenta quando existe esta associação. O tratamento envolve equipe multidisciplinar e o prognóstico é reservado, variando conforme o fenótipo.

  1. Geometric Structures of Stable Time-Variant State Feedback Systems

    Institute of Scientific and Technical Information of China (English)

    ZHONG Feng-wei; SUN Hua-fei; ZHANG Zhen-ning

    2007-01-01

    A new technique for considering the stabilizing time-variant state feedback gains is proposed from the viewpoint of information geometry. First, parametrization of the set of all stabilizing time-variant state feedback gains is given. Moreover, a diffeomorphic structure between the set of stabilizing time-variant state feedback gains and the Cartesian product of positive definite matrix and skew symmetric matrix satisfying certain algebraic conditions is constructed. Furth ermore, an immersion and some results about the eigenvalue locations of stable state feedback systems are derived.

  2. Cloning, expression, purification and characterization of tryptophan hydroxylase variants

    DEFF Research Database (Denmark)

    Boesen, Jane

    such as depression and obsessive-compulsive disorder (OCD). Characterization of TPH and elucidation of the enzymes regulation and catalytic mechanism is therefore vital to our understanding of the serotonin balance. This study concerns variants of both human TPH isoform 1 (hTPH1) and human TPH isoform 2 (h PH2...... the variant. The GST-rhTPH1 variant could be purified using affinity chromatography followed by gel filtration with high purity and a yield of 40 mg/l culture. The purified GST-rhTPH1 exists as a dimer in solution due to dimerization of GST. The GST could be cleaved successfully from the fusion protein using...

  3. Multiple Sodium Channel Variants in the Mosquito Culex quinquefasciatus

    Directory of Open Access Journals (Sweden)

    Lin He, Ting Li, Lee Zhang, Nannan Liu

    2012-01-01

    Full Text Available Voltage-gated sodium channels are the target sites of both DDT and pyrethroid insecticides. The importance of alternative splicing as a key mechanism governing the structural and functional diversity of sodium channels and the resulting development of insecticide and acaricide resistance is widely recognized, as shown by the extensive research on characterizing alternative splicing and variants of sodium channels in medically and agriculturally important insect species. Here we present the first comparative study of multiple variants of the sodium channel transcripts in the mosquito Culex quinquefasciatus. The variants were classified into two categories, CxNa-L and CxNa-S based on their distinguishing sequence sizes of ~6.5 kb and ~4.0 kb, respectively, and generated via major extensive alternative splicing with minor small deletions/ insertions in susceptible S-Lab, low resistant HAmCqG0, and highly resistant HAmCqG8 Culex strains. Four alternative Cx-Na-L splice variants were identified, including three full length variants with three optional exons (2, 5, and 21i and one with in-frame-stop codons. Large, multi-exon-alternative splices were identified in the CxNa-S category. All CxNa-S splicing variants in the S-Lab and HAmCqG0 strains contained in-frame stop codons, suggesting that any resulting proteins would be truncated. The ~1000 to ~3000-fold lower expression of these splice variants with stop codons compared with the CxNa-L splicing variants may support the lower importance of these variants in S-Lab and HAmCqG0. Interestingly, two alternative splicing variants of CxNa-S in HAmCqG8 included entire ORFs but lacked exons 5 to18 and these two variants had much higher expression levels in HAmCqG8 than in S-Lab and HAmCqG0. These results provide a functional basis for further characterizing how alternative splicing of a voltage-gated sodium channel contributes to diversity in neuronal signaling in mosquitoes in response to pyrethroids, and

  4. Myostatin: genetic variants, therapy and gene doping

    Directory of Open Access Journals (Sweden)

    André Katayama Yamada

    2012-09-01

    Full Text Available Since its discovery, myostatin (MSTN has been at the forefront of muscle therapy research because intrinsic mutations or inhibition of this protein, by either pharmacological or genetic means, result in muscle hypertrophy and hyperplasia. In addition to muscle growth, MSTN inhibition potentially disturbs connective tissue, leads to strength modulation, facilitates myoblast transplantation, promotes tissue regeneration, induces adipose tissue thermogenesis and increases muscle oxidative phenotype. It is also known that current advances in gene therapy have an impact on sports because of the illicit use of such methods. However, the adverse effects of these methods, their impact on athletic performance in humans and the means of detecting gene doping are as yet unknown. The aim of the present review is to discuss biosynthesis, genetic variants, pharmacological/genetic manipulation, doping and athletic performance in relation to the MSTN pathway. As will be concluded from the manuscript, MSTN emerges as a promising molecule for combating muscle wasting diseases and for triggering wide-ranging discussion in view of its possible use in gene doping.Desde sua descoberta, a miostatina (MSTN entrou na linha de frente em pesquisas relacionadas às terapias musculares porque mutações intrínsecas ou inibição desta proteína tanto por abordagens farmacológicas como genéticas resultam em hipertrofia muscular e hiperplasia. Além do aumento da massa muscular, a inibição de MSTN potencialmente prejudica o tecido conectivo, modula a força muscular, facilita o transplante de mioblastos, promove regeneração tecidual, induz termogênese no tecido adiposo e aumenta a oxidação na musculatura esquelética. É também sabido que os atuais avanços em terapia gênica têm uma relação com o esporte devido ao uso ilícito de tal método. Os efeitos adversos de tal abordagem, seus efeitos no desempenho de atletas e métodos para detectar doping genético s

  5. A Unique Hairy Cell Leukemia Variant.

    Science.gov (United States)

    Jian, Charles; Hsia, Cyrus C

    2016-01-01

    A 65-year-old woman presented with easy bruising, left upper quadrant pain, decreased appetite, and weight loss. She had splenomegaly and lymphocytosis (lymphocyte count of 11.6 × 10(9)/l), with remarkably abnormal appearing morphology. Her hemoglobin and platelet counts were normal. Peripheral blood flow cytometry revealed a monoclonal B-cell population expressing CD11c, CD25, CD19, CD20, and CD103. An initial diagnosis of hairy cell leukemia (HCL) was made, and the patient was treated with a standard 5-day course of cladribine. However, her lymphocytosis improved transiently, with a relapse 4 months later. There was no improvement in her splenomegaly. An HCL variant (HCL-v) was considered based on her resistance to treatment with a purine nucleoside analog. A subsequent splenectomy improved symptoms. Two years after, the patient suffered a relapse and underwent 6 cycles of CHOP-R (cyclophosphamide, hydroxydaunomycin, oncovin, prednisone, and rituximab), achieving partial remission. While under observation, she progressed with lymphocytosis 6 months later and was treated with pentostatin. There was no significant improvement in her disease, and she died 8 weeks following treatment initiation. HCL-v is a clinically more aggressive mature B-cell lymphoma than HCL with worse splenomegaly, higher lymphocyte counts, and resistance to typical HCL therapy with purine nucleoside analogs. Early recognition of HCL-v in the history, physical examination, and investigations with morphology and flow cytometry is key to patient management. Further, as in our case of HCL-v, cell morphology can be distinctly atypical, with large nucleoli and extremely convoluted nuclei. The distinction between HCL and HCL-v is important as HCL-v patients require more aggressive therapy and closer follow-up. PMID:27462230

  6. Behavioral variant of frontotemporal dementia mimicking Huntington's disease

    DEFF Research Database (Denmark)

    Nielsen, T Rune; Bruhn, Peter; Nielsen, Jørgen E;

    2010-01-01

    Behavioral changes and cognitive decline are the core clinical manifestations in the behavioral variant of frontotemporal dementia (bv-FTD). The behavioral changes may include characteristic stereotypic movements. These movements, although without clear purpose, are not involuntary. Involuntary...

  7. Role of common and rare variants in SCN10A

    DEFF Research Database (Denmark)

    Behr, Elijah R.; Savio-Galimberti, Eleonora; Barc, Julien;

    2015-01-01

    , CASQ2, TKT, TBX3, and TBX5) in 156 Caucasian SCN5A mutation-negative BrS patients (80% male; mean age 48) with symptoms (64%) and/or a family history of sudden death (47%) or BrS (18%). Forty-nine variants were identified: 18 were rare (MAF ..., were predicted as pathogenic using multiple bioinformatics tools. Allele frequencies were compared with the Exome Sequencing and UK10K Projects. SKAT methods tested rare variation in SCN10A finding no statistically significant difference between cases and controls. Co-segregation analysis was possible.......8 were performed for SCN10A common variants V1073, A1073, and rare variants of interest: A200V and I671V. V1073, A200V and I671V, demonstrated significant reductions in peak INa compared with ancestral allele A1073 (rs6795970). CONCLUSION: Rare variants in the screened QRS-associated genes (including SCN...

  8. Neuropathology in classical and variant ataxia-telangiectasia.

    NARCIS (Netherlands)

    Verhagen, M.M.M.; Martin, J.J.; Deuren, M. van; Ceuterick-de Groote, C.; Weemaes, C.M.R.; Kremer, B.; Taylor, M.A.; Willemsen, M.A.A.P.; Lammens, M.M.Y.

    2012-01-01

    Ataxia-telangiectasia (A-T) is classically characterized by progressive neurodegeneration, oculocutaneous telangiectasia, immunodeficiency and elevated alpha-fetoprotein levels. Some patients, classified as variant A-T, exhibit a milder clinical course. In the latter patients extrapyramidal symptoms

  9. Radioimmunological activity of 22K variant of human growth hormone

    International Nuclear Information System (INIS)

    From a preparation of human growth hormone its integral variant (hGH-22K) was isolated by isoelectric focusing, having a pI of 5,20 and relative mobility (Rm) of 0,621 in the polyacrylamide gel electrophoresis. Several experiments for the characterization of the isolated variant were carried out. The immunological properties was tested by radioimmunoassay (RIE), in which the activity of the isolated variant and the activity of the total preparation were compared. The dose response-curves obtained by RIE were found to be considered parallels (p < 0,01). It was checked using the F test between the slope of the two curves. The parallelism shown the immunochemical identity of the two preparation and indicates that the separation process developed does not produce alterations in the immunological properties of the variant. (Author)

  10. Leapfrog variants of iterative methods for linear algebra equations

    Science.gov (United States)

    Saylor, Paul E.

    1988-01-01

    Two iterative methods are considered, Richardson's method and a general second order method. For both methods, a variant of the method is derived for which only even numbered iterates are computed. The variant is called a leapfrog method. Comparisons between the conventional form of the methods and the leapfrog form are made under the assumption that the number of unknowns is large. In the case of Richardson's method, it is possible to express the final iterate in terms of only the initial approximation, a variant of the iteration called the grand-leap method. In the case of the grand-leap variant, a set of parameters is required. An algorithm is presented to compute these parameters that is related to algorithms to compute the weights and abscissas for Gaussian quadrature. General algorithms to implement the leapfrog and grand-leap methods are presented. Algorithms for the important special case of the Chebyshev method are also given.

  11. Common Gene Variants Account for Most Genetic Risk for Autism

    Science.gov (United States)

    ... Releases News Release Sunday, July 20, 2014 Common gene variants account for most genetic risk for autism ... genetic risk for autism comes from versions of genes that are common in the population rather than ...

  12. Vitiligo, con énfasis en su variante inflamatoria Vitiligo, with emphasis in its inflammatory variant

    Directory of Open Access Journals (Sweden)

    C I Vera

    2009-03-01

    Full Text Available El vitiligo inflamatorio es un trastorno melanocitopénico adquirido, de baja frecuencia con características clínicas e histológicas propias. Ocurre en ambos sexos, a cualquier edad. Su fisiopatogenia parece involucrar mecanismos autoinmunes. En su evolución es frecuente la desaparición del componente inflamatorio que resulta en una mácula hipopigmentada clásica; a ello debe anticiparse también nuestra elección terapéutica. Se describe un paciente con vitiligo inflamatorio de resolución espontánea, junto a una revisión crítica de la bibliografía. Se discute si representa una variante de vitiligo vulgar o una entidad independiente.Inflammatory vitiligo is a low frequency melanocytopenic acquired entity with clinical and histological features of it 's own. It occurs in both sexes at any age. Fisiopathogenesis may involve autoimmune mechanisms. It's evolution most commonly ends up in a classic hypopigmented maculae, therefore our therapeutic choice has to be oriented to this outcome. A patient with inflammatory vitiligo is described along a critical review of the literature. It is discussed if it represents a variant of common vitiligo or an independent entity.

  13. The activity of superoxide-dismutase in animal cell culture CHO-K1 after treatment with fullerenol and mytomicine C

    Directory of Open Access Journals (Sweden)

    Bogdanović Višnja

    2009-01-01

    Full Text Available Eukaryotic cell survives in predominantly reduced conditions. Homeostasis of cellular redox system is an imperative of cell surviving and its normal metabolism. ROS are well recognized for playing a dual role as both deleterious and beneficial species, since they can be either harmful or beneficial to living systems. These species are mutagenic compounds known to lead to DNA damage, favor cell transformation, and contribute to the development of a variety of malignant diseases. All the effects of oxidants are influenced by the cellular antioxidant defenses. This multilayer system consists of low molecular weight components and several antioxidant enzymes. Superoxide dismutases (SODs are the only enzymes dismuting superoxide radicals. Mitomycin C, a cross-linking agent, demonstrated genotoxicity in all in vitro and in vivo test systems in mammalian cells and animals. Water-soluble fullerenes are well known as cytotoxic agents for many cell lines in vitro. At the other side, fullerenols are good free radical scavengers and antioxidants both in vitro and in vivo. This paper investigates the effects of fullerenol on survival and fullerenol/ /mytomicine (MMC treatment on superoxide-dismutase (SOD activity in CHO-K1 cells. Samples were treated 3 and 24 h with fullerenol (C60(OH24 at concentration range 0.01-0.5 mg/mL and survival was monitored with dye exclusion test (DET. The activity of total SOD was estimated in samples treated with chosen concentrations of fullerenol and MMC (0.5 and 0.1 mg/mL after 3 and 24 h of cell incubation. Increasing of C60(OH24 concentration leads to decreasing of percent of surviving cells 3 and 24 h after incubation. The activity of total SOD enhanced with higher concentration of fullerenol, while decreased in the highest concentration at both experimental points. In samples treated with MMC, as well as in samples treated with fullerenol (0.0625 mg/mL + MMC was noticed boost in total SOD activity in comparison with

  14. Novel micro-bioreactor high throughput technology for cell culture process development: Reproducibility and scalability assessment of fed-batch CHO cultures.

    Science.gov (United States)

    Amanullah, Ashraf; Otero, Jose Manuel; Mikola, Mark; Hsu, Amy; Zhang, Jinyou; Aunins, John; Schreyer, H Brett; Hope, James A; Russo, A Peter

    2010-05-01

    With increasing timeline pressures to get therapeutic and vaccine candidates into the clinic, resource intensive approaches such as the use of shake flasks and bench-top bioreactors may limit the design space for experimentation to yield highly productive processes. The need to conduct large numbers of experiments has resulted in the use of miniaturized high-throughput (HT) technology for process development. One such high-throughput system is the SimCell platform, a robotically driven, cell culture bioreactor system developed by BioProcessors Corp. This study describes the use of the SimCell micro-bioreactor technology for fed-batch cultivation of a GS-CHO transfectant expressing a model IgG4 monoclonal antibody. Cultivations were conducted in gas-permeable chambers based on a micro-fluidic design, with six micro-bioreactors (MBs) per micro-bioreactor array (MBA). Online, non-invasive measurement of total cell density, pH and dissolved oxygen (DO) was performed. One hundred fourteen parallel MBs (19 MBAs) were employed to examine process reproducibility and scalability at shake flask, 3- and 100-L bioreactor scales. The results of the study demonstrate that the SimCell platform operated under fed-batch conditions could support viable cell concentrations up to least 12 x 10(6) cells/mL. In addition, both intra-MB (MB to MB) as well as intra-MBA (MBA to MBA) culture performance was found to be highly reproducible. The intra-MB and -MBA variability was calculated for each measurement as the coefficient of variation defined as CV (%) = (standard deviation/mean) x 100. The % CV values for most intra-MB and intra-MBA measurements were generally under 10% and the intra-MBA values were slightly lower than those for intra-MB. Cell growth, process parameters, metabolic and protein titer profiles were also compared to those from shake flask, bench-top, and pilot scale bioreactor cultivations and found to be within +/-20% of the historical averages.

  15. Molecular basis for Duarte and Los Angeles variant galactosemia.

    OpenAIRE

    Langley, S D; Lai, K.; Dembure, P P; Hjelm, L. N.; Elsas, L. J.

    1997-01-01

    Human orythrocytes that are homozygous for the Duarte enzyme variant of galactosemia (D/D) have a characteristic isoform on isoelectric focusing and 50% reduction in galactose-1-phosphate uridyltransferase (GALT) enzyme activity. The Duarte biochemical phenotype has a molecular genotype of N314D/N314D. The characteristic Duarte isoform is also associated with a variant called the "Los Angeles (LA) phenotype," which has increased GALT enzyme activity. We evaluated GALT enzyme activity and scre...

  16. Variants at the 9p21 locus and melanoma risk

    International Nuclear Information System (INIS)

    The influence of variants at the 9p21 locus on melanoma risk has been reported through investigation of CDKN2A variants through candidate gene approach as well as by genome wide association studies (GWAS). In the present study we genotyped, 25 SNPs that tag 273 variants on chromosome 9p21 in 837 melanoma cases and 1154 controls from Spain. Ten SNPs were selected based on previous associations, reported in GWAS, with either melanocytic nevi or melanoma risk or both. The other 15 SNPs were selected to fine map the CDKN2A gene region. All the 10 variants selected from the GWAS showed statistically significant association with melanoma risk. Statistically significant association with melanoma risk was also observed for the carriers of the variant T-allele of rs3088440 (540 C>T) at the 3’ UTR of CDKN2A gene with an OR 1.52 (95% CI 1.14-2.04). Interaction analysis between risk associated polymorphisms and previously genotyped MC1R variants, in the present study, did not show any statistically significant association. Statistical significant association was observed for the interaction between phototypes and the rs10811629 (located in intron 5 of MTAP). The strongest association was observed between the homozygous carrier of the A–allele and phototype II with an OR of 15.93 (95% CI 5.34-47.54). Our data confirmed the association of different variants at chromosome 9p21 with melanoma risk and we also found an association of a variant with skin phototypes

  17. Active Object Recognition with a Space-Variant Retina

    OpenAIRE

    Christopher Kanan

    2013-01-01

    When independent component analysis (ICA) is applied to color natural images, the representation it learns has spatiochromatic properties similar to the responses of neurons in primary visual cortex. Existing models of ICA have only been applied to pixel patches. This does not take into account the space-variant nature of human vision. To address this, we use the space-variant log-polar transformation to acquire samples from color natural images, and then we apply ICA to the acquired samples....

  18. Population structure analysis using rare and common functional variants

    OpenAIRE

    Ding Lili; Baye Tesfaye M; He Hua; Kurowski Brad G; Zhang Xue; Martin Lisa J

    2011-01-01

    Abstract Next-generation sequencing technologies now make it possible to genotype and measure hundreds of thousands of rare genetic variations in individuals across the genome. Characterization of high-density genetic variation facilitates control of population genetic structure on a finer scale before large-scale genotyping in disease genetics studies. Population structure is a well-known, prevalent, and important factor in common variant genetic studies, but its relevance in rare variants i...

  19. Digital subtraction angiography of a persistent trigeminal artery variant.

    Science.gov (United States)

    Temizöz, Osman; Genchellac, Hakan; Unlü, Ercüment; Cağli, Bekir; Ozdemir, Hüseyin; Demir, M Kemal

    2010-09-01

    Persistent trigeminal artery variants are described as cerebellar arteries that directly originate from the precavernous segment of the internal carotid artery. This has been observed in 0.18% of cerebral catheter angiograms. On the other hand, a persistent trigeminal artery variant feeding both the anterior inferior cerebellar artery and the posterior inferior cerebellar artery territory is very rare. We present this uncommon anomalous artery along with digital subtraction angiography findings and discuss its clinical significance in light of the literature. PMID:19821254

  20. PIGMENTED BASAL CELL CARCINOMA: A RARE CLINICAL AND HISTOPATHOLOGICAL VARIANT

    OpenAIRE

    Chandralekha; Vijaya Bhaskar; Bhagyalakshmi; Sudhakar; Sumanlatha

    2015-01-01

    Basal cell carcinoma is a common malignant tumour of skin , commonly referred to as „rodent ulcer‟. It is common in the head and neck region. Exposure to ultraviolet radiation is an important risk factor. Pigmented basal cell carcinoma is a clinical and histological variant of basal cell carcinoma that exhibits inc reased pigmentation. It is a rare variant that can clinically mimic malignant melanoma. It is more common in males than females. Herein , we are...

  1. Long-term follow-up of HPV16-positive women: persistence of the same genetic variant and low prevalence of variant co-infections.

    Directory of Open Access Journals (Sweden)

    Daan T Geraets

    Full Text Available HPV16 variants correlate with geographic origin and ethnicity. The association between infection with a specific variant and the cervical disease risk remains unclear. We studied the prevalence, persistence and association with cervical intraepithelial neoplasia (CIN of different HPV16 variants, using cervical swabs and whole tissue sections (WTS of biopsies from 548 women in the placebo group of a HPV16/18 vaccine trial. In HPV16-positive samples, HPV16 variants were identified by a reverse hybridization assay (RHA. Laser-capture micro-dissection (LCM was performed for localized detection of HPV. HPV16 variants were determined in 47 women. Frequency of mixed HPV16 variant infections was lower (8.5% than for multiple HPV genotypes (39.1%. Among 35 women having consecutive HPV16 variant-positive swabs, 32 (91.4% had the same variant while in three (8.6% women a change in variant(s was observed. HPV16-positive WTS were obtained from 12 women having consecutive HPV16 variant-positive swabs. The same variant was present in WTS of 10 women, while two were negative. WTS of five women were histologically normal. A single HPV16 variant was detected in four women having CIN1-3, while additional HPV genotypes were found in three other women having CIN2 and CIN3. In the WTS of one woman with mixed genotypes, the HPV16 variant was assigned to a CIN2 lesion by LCM. HPV16 variant infections can be effectively studied in cervical swabs and tissue specimens by the HPV16 variant RHA. Multiple HPV16 variants in one woman are rare. The HPV16 genotype consistently detected in follow-up samples usually involves a persistent infection with the same variant.

  2. Five Rare β Globin Chain Hemoglobin Variants in India.

    Science.gov (United States)

    Colah, Roshan B; Nadkarni, Anita; Gorakshakar, Ajit; Sawant, Pratibha; Gorivale, Manju; Mehta, Pallavi; Sawant, Madhavi; Ghosh, Kanjaksha

    2016-06-01

    Thalassemias as well as structural hemoglobin (Hb) variants are common monogenic inherited disorders of Hb in India. In this paper we describe 5 rare β-chain Hb variants identified in the Indian population on the basis of high performance liquid chromatography (HPLC). Of these 3 were identified during antenatal screening of β-thalassemia while the other 2 cases were referred to us for a diagnostic work up. These 5 Hb variants were Hb British Columbia (β CD 101 GAG → AAG), Hb Saint Louis (β CD28 CTG → CAG), Hb G Coushatta (β CD 22 GAA → GCA), Hb Pyrgos (β CD 83 GGC → GAC) and Hb Agenogi (β CD 90 GAG → AAG). Hb Saint Louis and Hb G Coushatta eluted in the HbA2 window, Hb British Columbia and Hb Agenogi eluted in the Hb C window while Hb Pyrgos eluted in an unknown window on HPLC. They were all identified by DNA sequencing. The child having Hb St. Louis had hepatosplenomegaly and anemia while the individuals with the other 4 variants were asymptomatic. Rare Hb variants are diagnostic curiosities that may be encountered by laboratories. Correct identification requires the application of more than one technique to avoid misdiagnosing them as more common variants (e.g. St. Louis and G Coushatta as E or D Iran on HPLC. Some, like G Coushatta may interfere with HPLC-based HbA1c estimation). PMID:27408413

  3. Integrated analysis of germline and somatic variants in ovarian cancer.

    Science.gov (United States)

    Kanchi, Krishna L; Johnson, Kimberly J; Lu, Charles; McLellan, Michael D; Leiserson, Mark D M; Wendl, Michael C; Zhang, Qunyuan; Koboldt, Daniel C; Xie, Mingchao; Kandoth, Cyriac; McMichael, Joshua F; Wyczalkowski, Matthew A; Larson, David E; Schmidt, Heather K; Miller, Christopher A; Fulton, Robert S; Spellman, Paul T; Mardis, Elaine R; Druley, Todd E; Graubert, Timothy A; Goodfellow, Paul J; Raphael, Benjamin J; Wilson, Richard K; Ding, Li

    2014-01-01

    We report the first large-scale exome-wide analysis of the combined germline-somatic landscape in ovarian cancer. Here we analyse germline and somatic alterations in 429 ovarian carcinoma cases and 557 controls. We identify 3,635 high confidence, rare truncation and 22,953 missense variants with predicted functional impact. We find germline truncation variants and large deletions across Fanconi pathway genes in 20% of cases. Enrichment of rare truncations is shown in BRCA1, BRCA2 and PALB2. In addition, we observe germline truncation variants in genes not previously associated with ovarian cancer susceptibility (NF1, MAP3K4, CDKN2B and MLL3). Evidence for loss of heterozygosity was found in 100 and 76% of cases with germline BRCA1 and BRCA2 truncations, respectively. Germline-somatic interaction analysis combined with extensive bioinformatics annotation identifies 222 candidate functional germline truncation and missense variants, including two pathogenic BRCA1 and 1 TP53 deleterious variants. Finally, integrated analyses of germline and somatic variants identify significantly altered pathways, including the Fanconi, MAPK and MLL pathways. PMID:24448499

  4. Sonographic and cytopathologic correlation of papillary thyroid carcinoma variants.

    Science.gov (United States)

    Lee, Ji Hyun; Shin, Jung Hee; Lee, Hyun-Woo; Oh, Young Lyun; Hahn, Soo Yeon; Ko, Eun Young

    2015-01-01

    Papillary thyroid carcinoma (PTC) is the most common thyroid cancer and constitutes more than 70% of thyroid malignancies. Although TNM staging is the most widely used parameter for determination of therapeutic plans, recent studies have suggested that different histopathologic variants of PTC can also have different clinical courses and patient prognoses. Sonographic criteria for PTC are well established and include a taller-than-wide shape, an irregular margin, microcalcifications, and marked hypoechogenicity. The role of sonography has expanded to enable the characterization of PTC variants based on their sonographic features. Tall cell and diffuse sclerosing variants appear to have more aggressive clinical courses with unfavorable prognoses, whereas the more recently described cribriform-morular and Warthin-like variants have relatively indolent clinical courses. The prognoses of patients with follicular, solid, columnar cell, and oncocytic variants are still controversial and may be similar to the prognosis of conventional PTC. Understanding the sonographic characteristics of PTC variants with clinicopathologic correlation may be helpful for suggesting an appropriate treatment plan.

  5. Prebiotic Competition between Information Variants, With Low Error Catastrophe Risks

    Directory of Open Access Journals (Sweden)

    Radu Popa

    2015-07-01

    Full Text Available During competition for resources in primitive networks increased fitness of an information variant does not necessarily equate with successful elimination of its competitors. If variability is added fast to a system, speedy replacement of pre-existing and less-efficient forms of order is required as novel information variants arrive. Otherwise, the information capacity of the system fills up with information variants (an effect referred as “error catastrophe”. As the cost for managing the system’s exceeding complexity increases, the correlation between performance capabilities of information variants and their competitive success decreases, and evolution of such systems toward increased efficiency slows down. This impasse impedes the understanding of evolution in prebiotic networks. We used the simulation platform Biotic Abstract Dual Automata (BiADA to analyze how information variants compete in a resource-limited space. We analyzed the effect of energy-related features (differences in autocatalytic efficiency, energy cost of order, energy availability, transformation rates and stability of order on this competition. We discuss circumstances and controllers allowing primitive networks acquire novel information with minimal “error catastrophe” risks. We present a primitive mechanism for maximization of energy flux in dynamic networks. This work helps evaluate controllers of evolution in prebiotic networks and other systems where information variants compete.

  6. Variants affecting exon skipping contribute to complex traits.

    Directory of Open Access Journals (Sweden)

    Younghee Lee

    Full Text Available DNA variants that affect alternative splicing and the relative quantities of different gene transcripts have been shown to be risk alleles for some Mendelian diseases. However, for complex traits characterized by a low odds ratio for any single contributing variant, very few studies have investigated the contribution of splicing variants. The overarching goal of this study is to discover and characterize the role that variants affecting alternative splicing may play in the genetic etiology of complex traits, which include a significant number of the common human diseases. Specifically, we hypothesize that single nucleotide polymorphisms (SNPs in splicing regulatory elements can be characterized in silico to identify variants affecting splicing, and that these variants may contribute to the etiology of complex diseases as well as the inter-individual variability in the ratios of alternative transcripts. We leverage high-throughput expression profiling to 1 experimentally validate our in silico predictions of skipped exons and 2 characterize the molecular role of intronic genetic variations in alternative splicing events in the context of complex human traits and diseases. We propose that intronic SNPs play a role as genetic regulators within splicing regulatory elements and show that their associated exon skipping events can affect protein domains and structure. We find that SNPs we would predict to affect exon skipping are enriched among the set of SNPs reported to be associated with complex human traits.

  7. Generalization of Rare Variant Association Tests for Longitudinal Family Studies.

    Science.gov (United States)

    Chien, Li-Chu; Hsu, Fang-Chi; Bowden, Donald W; Chiu, Yen-Feng

    2016-02-01

    Given the functional relevance of many rare variants, their identification is frequently critical for dissecting disease etiology. Functional variants are likely to be aggregated in family studies enriched with affected members, and this aggregation increases the statistical power to detect rare variants associated with a trait of interest. Longitudinal family studies provide additional information for identifying genetic and environmental factors associated with disease over time. However, methods to analyze rare variants in longitudinal family data remain fairly limited. These methods should be capable of accounting for different sources of correlations and handling large amounts of sequencing data efficiently. To identify rare variants associated with a phenotype in longitudinal family studies, we extended pedigree-based burden (BT) and kernel (KS) association tests to genetic longitudinal studies. Generalized estimating equation (GEE) approaches were used to generalize the pedigree-based BT and KS to multiple correlated phenotypes under the generalized linear model framework, adjusting for fixed effects of confounding factors. These tests accounted for complex correlations between repeated measures of the same phenotype (serial correlations) and between individuals in the same family (familial correlations). We conducted comprehensive simulation studies to compare the proposed tests with mixed-effects models and marginal models, using GEEs under various configurations. When the proposed tests were applied to data from the Diabetes Heart Study, we found exome variants of POMGNT1 and JAK1 genes were associated with type 2 diabetes.

  8. Construction of anti-VEGFR-2 IgG1 like human antibody and its expression in CHO-k cells%抗VEGFR-2全人源IgG1抗体的构建及其在CHO-k细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    李致科; 何远; 张娟; 解伟; 曹婉璐; 王泽根; 王旻

    2013-01-01

    本文在实验室构建的单链抗体-Fc融合抗体[scFv(AK404R)-Fc]的基础上构建抗VEGFR-2全人源IgG1样全长抗体(Mab-04).利用重叠PCR,获得Mab-04的轻链和重链的核酸序列后分别克隆到真核表达载体pcDNA3.1,获得重组质粒.脂质体法将重组质粒转染至CHO-k细胞,经ProteinA柱纯化细胞培养上清液获得目的蛋白,利用Western blotting检测目的蛋白,ELISA检测Mab-04与抗原亲和力.测序表明重组质粒构建成功,Westem blotting检测显示目的蛋白成功表达(1μg.mL-1),ELISA检测阐明该抗体能与抗原结合并呈浓度依赖性(IC50为50 nmol.L-1),表明Mab-04成功表达并正确装配,为进一步大量制备该抗体及其活性研究打下基础.

  9. Sustained Uptake of a Hospital-Based Handwashing with Soap and Water Treatment Intervention (Cholera-Hospital-Based Intervention for 7 Days [CHoBI7]): A Randomized Controlled Trial.

    Science.gov (United States)

    George, Christine Marie; Jung, Danielle S; Saif-Ur-Rahman, K M; Monira, Shirajum; Sack, David A; Mahamud-ur Rashid; Mahmud, Md Toslim; Mustafiz, Munshi; Rahman, Zillur; Bhuyian, Sazzadul Islam; Winch, Peter J; Leontsini, Elli; Perin, Jamie; Begum, Farzana; Zohura, Fatema; Biswas, Shwapon; Parvin, Tahmina; Sack, R Bradley; Alam, Munirul

    2016-02-01

    Diarrhea is the second leading cause of death in children under 5 years of age globally. The time patients and caregivers spend at a health facility for severe diarrhea presents the opportunity to deliver water, sanitation, and hygiene (WASH) interventions. We recently developed Cholera-Hospital-Based Intervention for 7 days (CHoBI7), a 1-week hospital-based handwashing with soap and water treatment intervention, for household members of cholera patients. To investigate if this intervention could lead to sustained WASH practices, we conducted a follow-up evaluation of 196 intervention household members and 205 control household members enrolled in a randomized controlled trial of the CHoBI7 intervention 6 to 12 months post-intervention. Compared with the control arm, the intervention arm had four times higher odds of household members' handwashing with soap at a key time during 5-hour structured observation (odds ratio [OR]: 4.71, 95% confidence interval [CI]: 2.61, 8.49) (18% versus 50%) and a 41% reduction in households in the World Health Organization very high-risk category for stored drinking water (OR: 0.38, 95% CI: 0.15, 0.96) (58% versus 34%) 6 to 12 months post-intervention. Furthemore, 71% of observed handwashing with soap events in the intervention arm involved the preparation and use of soapy water, which was promoted during the intervention, compared to 9% of control households. These findings demonstrate that the hospital-based CHoBI7 intervention can lead to significant increases in handwashing with soap practices and improved stored drinking water quality 6 to 12 months post-intervention. PMID:26728766

  10. Sustained Uptake of a Hospital-Based Handwashing with Soap and Water Treatment Intervention (Cholera-Hospital-Based Intervention for 7 Days [CHoBI7]): A Randomized Controlled Trial.

    Science.gov (United States)

    George, Christine Marie; Jung, Danielle S; Saif-Ur-Rahman, K M; Monira, Shirajum; Sack, David A; Mahamud-ur Rashid; Mahmud, Md Toslim; Mustafiz, Munshi; Rahman, Zillur; Bhuyian, Sazzadul Islam; Winch, Peter J; Leontsini, Elli; Perin, Jamie; Begum, Farzana; Zohura, Fatema; Biswas, Shwapon; Parvin, Tahmina; Sack, R Bradley; Alam, Munirul

    2016-02-01

    Diarrhea is the second leading cause of death in children under 5 years of age globally. The time patients and caregivers spend at a health facility for severe diarrhea presents the opportunity to deliver water, sanitation, and hygiene (WASH) interventions. We recently developed Cholera-Hospital-Based Intervention for 7 days (CHoBI7), a 1-week hospital-based handwashing with soap and water treatment intervention, for household members of cholera patients. To investigate if this intervention could lead to sustained WASH practices, we conducted a follow-up evaluation of 196 intervention household members and 205 control household members enrolled in a randomized controlled trial of the CHoBI7 intervention 6 to 12 months post-intervention. Compared with the control arm, the intervention arm had four times higher odds of household members' handwashing with soap at a key time during 5-hour structured observation (odds ratio [OR]: 4.71, 95% confidence interval [CI]: 2.61, 8.49) (18% versus 50%) and a 41% reduction in households in the World Health Organization very high-risk category for stored drinking water (OR: 0.38, 95% CI: 0.15, 0.96) (58% versus 34%) 6 to 12 months post-intervention. Furthemore, 71% of observed handwashing with soap events in the intervention arm involved the preparation and use of soapy water, which was promoted during the intervention, compared to 9% of control households. These findings demonstrate that the hospital-based CHoBI7 intervention can lead to significant increases in handwashing with soap practices and improved stored drinking water quality 6 to 12 months post-intervention.

  11. In Vitro Evolution and Preliminary Characterization of a Cadmium-Resistant Population of Chlamydomonas reinhardtii

    OpenAIRE

    Nagel, Klaus; Voigt, Jürgen

    1989-01-01

    A cadmium-tolerant population of Chlamydomonas reinhardtii was derived from a Cd-sensitive cell wall-deficient strain by long-term selection in liquid culture. A comparison of Cd-sensitive and Cd-tolerant cells revealed that Cd tolerance was due to genetically determined alterations of metabolism rather than to increased efficiency of a detoxification system.

  12. Contribution of cell walls, nonprotein thiols, and organic acids to cadmium resistance in two cabbage varieties.

    Science.gov (United States)

    Sun, Jianyun; Cui, Jin; Luo, Chunling; Gao, Lu; Chen, Yahua; Shen, Zhenguo

    2013-02-01

    To study possible cadmium (Cd) resistance mechanisms in cabbage (Brassica oleracea L.), several parameters of metal uptake, distribution, and complexation were compared between two varieties Chunfeng [CF (Cd-tolerant)] and Lvfeng [LF (Cd-sensitive)]. Results showed that CF contained significantly lower Cd concentrations in leaves and higher Cd concentrations in roots than LF. Approximately 70 to 74 % and 66 to 68 % of Cd taken up by LF and CF, respectively, was transported to shoots. More Cd was bound to the cell walls of leaves, stems, and roots in CF than in LF. The higher capacity of CF to limit Cd uptake into shoots could be explained by immobilization of Cd in root cell walls. Compared with control groups, Cd treatment also significantly increased concentrations of nonprotein thiols, phytochelatins (PCs), and citric acid in the leaves and roots of the two varieties; the increases were more pronounced in CF than in LF. Taken together, the results suggest that the greater Cd resistance in CF than in LF may be attributable to the greater capacity of CF to limit Cd uptake into shoots and complex Cd in cell walls and metal binding ligands, such as PCs and citric acid. However, the contributions of PCs and citric acid to Cd detoxification might be smaller than those in cell walls.

  13. Molecular Characterization of Copper and Cadmium Resistance Determinants in the Biomining Thermoacidophilic Archaeon Sulfolobus metallicus

    Directory of Open Access Journals (Sweden)

    Alvaro Orell

    2013-01-01

    Full Text Available Sulfolobus metallicus is a thermoacidophilic crenarchaeon used in high-temperature bioleaching processes that is able to grow under stressing conditions such as high concentrations of heavy metals. Nevertheless, the genetic and biochemical mechanisms responsible for heavy metal resistance in S. metallicus remain uncharacterized. Proteomic analysis of S. metallicus cells exposed to 100 mM Cu revealed that 18 out of 30 upregulated proteins are related to the production and conversion of energy, amino acids biosynthesis, and stress responses. Ten of these last proteins were also up-regulated in S. metallicus treated in the presence of 1 mM Cd suggesting that at least in part, a common general response to these two heavy metals. The S. metallicus genome contained two complete cop gene clusters, each encoding a metallochaperone (CopM, a Cu-exporting ATPase (CopA, and a transcriptional regulator (CopT. Transcriptional expression analysis revealed that copM and copA from each cop gene cluster were cotranscribed and their transcript levels increased when S. metallicus was grown either in the presence of Cu or using chalcopyrite (CuFeS2 as oxidizable substrate. This study shows for the first time the presence of a duplicated version of the cop gene cluster in Archaea and characterizes some of the Cu and Cd resistance determinants in a thermophilic archaeon employed for industrial biomining.

  14. Increased cadmium and lead uptake of a cadmium hyperaccumulator tomato by cadmium-resistant bacteria.

    Science.gov (United States)

    He, Lin-Yan; Chen, Zhao-Jin; Ren, Gai-Di; Zhang, Yan-Feng; Qian, Meng; Sheng, Xia-Fang

    2009-07-01

    Two cadmium (Cd)-resistant strains Pseudomonas sp. RJ10 and Bacillus sp. RJ16 were investigated for their effects on the soil Cd and lead (Pb) solubilization and promotion of plant growth and Cd and Pb uptakes of a Cd-hyperaccumulator tomato. In the heavy metal-contaminated inoculated soil, the CaCl(2)-extractable Cd and Pb were increased by 58-104% and 67-93%, respectively, compared to the uninoculation control. The bacteria produced indole acetic acid, siderophore and 1-aminocyclopropane-1-carboxylate deaminase. Root elongation assay conducted on tomato under gnotobiotic conditions demonstrated increase in root elongation of inoculated tomato seedlings compared to the control plants. An increase in Cd and Pb contents of above-ground tissues varied from 92% to 113% and from 73% to 79% in inoculated plants growing in heavy metal-contaminated soil compared to the uninoculation control, respectively. These results show that the bacteria could be exploited for bacteria enhanced-phytoextraction of Cd- and Pb-polluted soils. PMID:19368973

  15. Alternative Technical Summary Report: Electrometallurgical Treatment Variant

    Energy Technology Data Exchange (ETDEWEB)

    Gray, L.W.

    1995-11-30

    Immobilization is the fixation of the surplus fissile materials in an acceptable matrix such as glass or ceramics to create an environmentally benign form for disposal in a repository. In addition to the traditional characteristics required of an immobilization form to achieve isolation of the fissile material from the biosphere over geologic times, the immobilization form for the Fissile Materials Disposition Program (FMDP) must also possess the property that it is inherently as unattractive and inaccessible as the fissile material from commercial spent fuel. This latter requirement is similar to the wording of the ''spent fuel standard'' invoked in the National Academy of Sciences (NAS) study on plutonium disposition. High-level wastes (HLW) or separated cesium ({sup 137}Cs), can be added with the fissile material into the waste form to create a radiation field that increases the proliferation resistance and decreases reuse by the host nation in the following ways: (1) Plutonium will be diluted with elements that must be removed by extensive chemical processing to return it to weapons-usable purity; (2) The immobilized plutonium canisters will contain approximately 2 tonnes (2000 kg; 2.2 tons) of mass, thereby forcing the use of heavy equipment to move the canisters; (3) A gamma radiation barrier will be added to the immobilized plutonium canisters; the present concept is to add a radiation barrier that is greater than 1 Gy (100 rad) per hour at 1 m (3 ft) 30 years after fabrication; (4) These canisters will then be sealed in casks and emplaced into drifts in a federal repository where they will be monitored for 100 years before the repository is sealed. This immobilization process is shown conceptually in Figure 1. In the electrometallurgical treatment (ET) variant, plutonium-rich residues are shipped to existing Argonne National Laboratory-West (ANL-W) facilities where the plutonium is converted to plutonium chloride, dissolved in a molten

  16. Control of carbohydrate processing: increased beta-1,6 branching in N-linked carbohydrates of Lec9 CHO mutants appears to arise from a defect in oligosaccharide-dolichol biosynthesis.

    OpenAIRE

    Rosenwald, A G; Stanley, P; Krag, S S

    1989-01-01

    A correlation between increased beta-1,6 branching of N-linked carbohydrates and the ability of a cell to metastasize or to form a tumor has been observed in several experimental models. Lec9 Chinese hamster ovary (CHO) mutants exhibit a drastic reduction in tumorigenicity in nude mice, and this phenotype directly correlates with their ability to attach an increased proportion of beta-1,6-branched carbohydrates to the G glycoprotein of vesicular stomatitis virus (J. Ripka, S. Shin, and P. Sta...

  17. Acantholytic Variant of Bowen's Disease with Micro-invasive Squamous Cell Carcinoma: A Case Report of a Unique Variant

    OpenAIRE

    Kanthilatha Pai; Shricharith Shetty; J Padmapriya; Sathish Pai; Lakshmi Rao

    2014-01-01

    Bowen′s disease is generally regarded as premalignant dermatoses. The disease affects both skin and the mucosa and has the potential to progress to invasive squamous cell carcinoma. There are descriptions of several histological variants of Bowen′s disease like psoriasiform, atrophic, pagetoid, etc. Acantholysis of anaplastic keratinocytes with bullae/cleft formation is described in premalignant condition like actinic keratosis and adenoid variant of squamous cell carcinoma, but there is lack...

  18. Two insular regions are differentially involved in behavioral variant FTD and nonfluent/agrammatic variant PPA.

    Science.gov (United States)

    Mandelli, Maria Luisa; Vitali, Paolo; Santos, Miguel; Henry, Maya; Gola, Kelly; Rosenberg, Lynne; Dronkers, Nina; Miller, Bruce; Seeley, William W; Gorno-Tempini, Maria Luisa

    2016-01-01

    The non-fluent/agrammatic variant of primary progressive aphasia (nfvPPA) and the behavioral variant frontotemporal dementia (bvFTD) are focal neurodegenerative disorders belonging to the FTD-spectrum clinical syndromes. NfvPPA is characterized by effortful speech and/or agrammatism and left frontal atrophy, while bvFTD is characterized by social-emotional dysfunction often accompanied by right-lateralized frontal damage. Despite their contrasting clinical presentations, both disorders show prominent left anterior insula atrophy. We investigated differential patterns of insular sub-region atrophy in nfvPPA and bvFTD. Based on knowledge of insular connectivity and physiology, we hypothesized that the left superior precentral region of the dorsal anterior insula (SPGI) would be more atrophic in nvfPPA due to its critical role in motor speech, whereas the ventral anterior region would be more atrophied in bvFTD reflecting its known role in social-emotional-autonomic functions. Early stage nfvPPA and bvFTD patients matched for disease severity, age, gender and education and healthy controls participated in the study. Detailed clinical history, neurological examination, neuropsychological screening evaluation, and high-resolution T1-weighted brain magnetic resonance imaging (MRI) were collected. Voxel-based morphometry (VBM) was applied to perform group comparisons across the whole brain and in bilateral insula region of interest (ROI). Correlation analyses between insular sub-region atrophy and relevant clinical features were performed. Whole brain group comparisons between nfvPPA and bvFTD showed the expected predominantly left or right anterior insular atrophy pattern. ROI analysis of bilateral insula showed that the left SPGI was significantly more atrophied in nfvPPA compared to bvFTD, while the bilateral ventral anterior and right dorsal anterior insula sub-regions were more atrophied in bvFTD than nfvPPA. Only left SPGI volume correlated with speech production

  19. Two insular regions are differentially involved in behavioral variant FTD and nonfluent/agrammatic variant PPA.

    Science.gov (United States)

    Mandelli, Maria Luisa; Vitali, Paolo; Santos, Miguel; Henry, Maya; Gola, Kelly; Rosenberg, Lynne; Dronkers, Nina; Miller, Bruce; Seeley, William W; Gorno-Tempini, Maria Luisa

    2016-01-01

    The non-fluent/agrammatic variant of primary progressive aphasia (nfvPPA) and the behavioral variant frontotemporal dementia (bvFTD) are focal neurodegenerative disorders belonging to the FTD-spectrum clinical syndromes. NfvPPA is characterized by effortful speech and/or agrammatism and left frontal atrophy, while bvFTD is characterized by social-emotional dysfunction often accompanied by right-lateralized frontal damage. Despite their contrasting clinical presentations, both disorders show prominent left anterior insula atrophy. We investigated differential patterns of insular sub-region atrophy in nfvPPA and bvFTD. Based on knowledge of insular connectivity and physiology, we hypothesized that the left superior precentral region of the dorsal anterior insula (SPGI) would be more atrophic in nvfPPA due to its critical role in motor speech, whereas the ventral anterior region would be more atrophied in bvFTD reflecting its known role in social-emotional-autonomic functions. Early stage nfvPPA and bvFTD patients matched for disease severity, age, gender and education and healthy controls participated in the study. Detailed clinical history, neurological examination, neuropsychological screening evaluation, and high-resolution T1-weighted brain magnetic resonance imaging (MRI) were collected. Voxel-based morphometry (VBM) was applied to perform group comparisons across the whole brain and in bilateral insula region of interest (ROI). Correlation analyses between insular sub-region atrophy and relevant clinical features were performed. Whole brain group comparisons between nfvPPA and bvFTD showed the expected predominantly left or right anterior insular atrophy pattern. ROI analysis of bilateral insula showed that the left SPGI was significantly more atrophied in nfvPPA compared to bvFTD, while the bilateral ventral anterior and right dorsal anterior insula sub-regions were more atrophied in bvFTD than nfvPPA. Only left SPGI volume correlated with speech production

  20. Biochemical characterization of the GM2 gangliosidosis B1 variant

    Directory of Open Access Journals (Sweden)

    J.C. Tutor

    2004-06-01

    Full Text Available The deficiency of the A isoenzyme of ß-hexosaminidase (Hex produced by different mutations of the gene that codes for the alpha subunit (Tay-Sachs disease has two variants with enzymological differences: the B variant consists of the absence of Hex A isoenzyme and the B1 variant produces an inactive Hex A isoenzyme for the hydrolysis of the GM2 ganglioside and synthetic substrates with negative charge. In contrast to the early childhood form of the B variant, the B1 variant appears at a later clinical stage (3 to 7 years of age with neurodegenerative symptoms leading to the death of the patient in the second decade of life. The most frequent mutation responsible for the GM2 gangliosidosis B1 variant is R178H, which has a widespread geographic and ethnic distribution. The highest incidence has been described in Portugal, which has been suggested as the point of origin of this mutation. Biochemical characterization of this lysosomal disease is carried out using negatively charged synthetic alpha subunit-specific sulfated substrates, since Hex A isoenzyme heat-inactivation assays are not applicable. However, the determination of the apparent activation energy of Hex using the neutral substrate 3,3'-dichlorophenolsulfonphthaleinyl N-acetyl-ß-D-glucosaminide, may offer a valid alternative. The presence of an alpha subunit in the alphaß heterodimer Hex A means that its activation energy (41.8 kJ/mol is significantly lower than that of the ßß homodimer Hex B (75.1 kJ/mol; however, as mutation inactivates the alpha subunit, the Hex A of the B1 variant presents an activation energy that is similar to that of the Hex B isoenzyme.

  1. HABP2 G534E Variant in Papillary Thyroid Carcinoma.

    Directory of Open Access Journals (Sweden)

    Jerneja Tomsic

    Full Text Available The main nonmedullary form of thyroid cancer is papillary thyroid carcinoma (PTC that accounts for 80-90% of all thyroid malignancies. Only 3-10% of PTC patients have a positive family history of PTC yet the familiality is one of the highest of all cancers as measured by case control studies. A handful of genes have been implicated accounting for a small fraction of this genetic predisposition. It was therefore of considerable interest that a mutation in the HABP2 gene was recently implicated in familial PTC. The present work was undertaken to examine the extent of HABP2 variant involvement in PTC. The HABP2 G534E variant (rs7080536 was genotyped in blood DNA from 179 PTC families (one affected individual per family, 1160 sporadic PTC cases and 1395 controls. RNA expression of HABP2 was tested by qPCR in RNA extracted from tumor and normal thyroid tissue from individuals that are homozygous wild-type or heterozygous for the variant. The variant was found to be present in 6.1% familial cases, 8.0% sporadic cases (2 individuals were homozygous for the variant and 8.7% controls. The variant did not segregate with PTC in one large and 6 smaller families in which it occurred. In keeping with data from the literature and databases the expression of HABP2 was highest in the liver, much lower in 3 other tested tissues (breast, kidney, brain but not found in thyroid. Given these results showing lack of any involvement we suggest that the putative role of variant HABP2 in PTC should be carefully scrutinized.

  2. Common genetic variants influence human subcortical brain structures.

    Science.gov (United States)

    Hibar, Derrek P; Stein, Jason L; Renteria, Miguel E; Arias-Vasquez, Alejandro; Desrivières, Sylvane; Jahanshad, Neda; Toro, Roberto; Wittfeld, Katharina; Abramovic, Lucija; Andersson, Micael; Aribisala, Benjamin S; Armstrong, Nicola J; Bernard, Manon; Bohlken, Marc M; Boks, Marco P; Bralten, Janita; Brown, Andrew A; Chakravarty, M Mallar; Chen, Qiang; Ching, Christopher R K; Cuellar-Partida, Gabriel; den Braber, Anouk; Giddaluru, Sudheer; Goldman, Aaron L; Grimm, Oliver; Guadalupe, Tulio; Hass, Johanna; Woldehawariat, Girma; Holmes, Avram J; Hoogman, Martine; Janowitz, Deborah; Jia, Tianye; Kim, Sungeun; Klein, Marieke; Kraemer, Bernd; Lee, Phil H; Olde Loohuis, Loes M; Luciano, Michelle; Macare, Christine; Mather, Karen A; Mattheisen, Manuel; Milaneschi, Yuri; Nho, Kwangsik; Papmeyer, Martina; Ramasamy, Adaikalavan; Risacher, Shannon L; Roiz-Santiañez, Roberto; Rose, Emma J; Salami, Alireza; Sämann, Philipp G; Schmaal, Lianne; Schork, Andrew J; Shin, Jean; Strike, Lachlan T; Teumer, Alexander; van Donkelaar, Marjolein M J; van Eijk, Kristel R; Walters, Raymond K; Westlye, Lars T; Whelan, Christopher D; Winkler, Anderson M; Zwiers, Marcel P; Alhusaini, Saud; Athanasiu, Lavinia; Ehrlich, Stefan; Hakobjan, Marina M H; Hartberg, Cecilie B; Haukvik, Unn K; Heister, Angelien J G A M; Hoehn, David; Kasperaviciute, Dalia; Liewald, David C M; Lopez, Lorna M; Makkinje, Remco R R; Matarin, Mar; Naber, Marlies A M; McKay, D Reese; Needham, Margaret; Nugent, Allison C; Pütz, Benno; Royle, Natalie A; Shen, Li; Sprooten, Emma; Trabzuni, Daniah; van der Marel, Saskia S L; van Hulzen, Kimm J E; Walton, Esther; Wolf, Christiane; Almasy, Laura; Ames, David; Arepalli, Sampath; Assareh, Amelia A; Bastin, Mark E; Brodaty, Henry; Bulayeva, Kazima B; Carless, Melanie A; Cichon, Sven; Corvin, Aiden; Curran, Joanne E; Czisch, Michael; de Zubicaray, Greig I; Dillman, Allissa; Duggirala, Ravi; Dyer, Thomas D; Erk, Susanne; Fedko, Iryna O; Ferrucci, Luigi; Foroud, Tatiana M; Fox, Peter T; Fukunaga, Masaki; Gibbs, J Raphael; Göring, Harald H H; Green, Robert C; Guelfi, Sebastian; Hansell, Narelle K; Hartman, Catharina A; Hegenscheid, Katrin; Heinz, Andreas; Hernandez, Dena G; Heslenfeld, Dirk J; Hoekstra, Pieter J; Holsboer, Florian; Homuth, Georg; Hottenga, Jouke-Jan; Ikeda, Masashi; Jack, Clifford R; Jenkinson, Mark; Johnson, Robert; Kanai, Ryota; Keil, Maria; Kent, Jack W; Kochunov, Peter; Kwok, John B; Lawrie, Stephen M; Liu, Xinmin; Longo, Dan L; McMahon, Katie L; Meisenzahl, Eva; Melle, Ingrid; Mohnke, Sebastian; Montgomery, Grant W; Mostert, Jeanette C; Mühleisen, Thomas W; Nalls, Michael A; Nichols, Thomas E; Nilsson, Lars G; Nöthen, Markus M; Ohi, Kazutaka; Olvera, Rene L; Perez-Iglesias, Rocio; Pike, G Bruce; Potkin, Steven G; Reinvang, Ivar; Reppermund, Simone; Rietschel, Marcella; Romanczuk-Seiferth, Nina; Rosen, Glenn D; Rujescu, Dan; Schnell, Knut; Schofield, Peter R; Smith, Colin; Steen, Vidar M; Sussmann, Jessika E; Thalamuthu, Anbupalam; Toga, Arthur W; Traynor, Bryan J; Troncoso, Juan; Turner, Jessica A; Valdés Hernández, Maria C; van 't Ent, Dennis; van der Brug, Marcel; van der Wee, Nic J A; van Tol, Marie-Jose; Veltman, Dick J; Wassink, Thomas H; Westman, Eric; Zielke, Ronald H; Zonderman, Alan B; Ashbrook, David G; Hager, Reinmar; Lu, Lu; McMahon, Francis J; Morris, Derek W; Williams, Robert W; Brunner, Han G; Buckner, Randy L; Buitelaar, Jan K; Cahn, Wiepke; Calhoun, Vince D; Cavalleri, Gianpiero L; Crespo-Facorro, Benedicto; Dale, Anders M; Davies, Gareth E; Delanty, Norman; Depondt, Chantal; Djurovic, Srdjan; Drevets, Wayne C; Espeseth, Thomas; Gollub, Randy L; Ho, Beng-Choon; Hoffmann, Wolfgang; Hosten, Norbert; Kahn, René S; Le Hellard, Stephanie; Meyer-Lindenberg, Andreas; Müller-Myhsok, Bertram; Nauck, Matthias; Nyberg, Lars; Pandolfo, Massimo; Penninx, Brenda W J H; Roffman, Joshua L; Sisodiya, Sanjay M; Smoller, Jordan W; van Bokhoven, Hans; van Haren, Neeltje E M; Völzke, Henry; Walter, Henrik; Weiner, Michael W; Wen, Wei; White, Tonya; Agartz, Ingrid; Andreassen, Ole A; Blangero, John; Boomsma, Dorret I; Brouwer, Rachel M; Cannon, Dara M; Cookson, Mark R; de Geus, Eco J C; Deary, Ian J; Donohoe, Gary; Fernández, Guillén; Fisher, Simon E; Francks, Clyde; Glahn, David C; Grabe, Hans J; Gruber, Oliver; Hardy, John; Hashimoto, Ryota; Hulshoff Pol, Hilleke E; Jönsson, Erik G; Kloszewska, Iwona; Lovestone, Simon; Mattay, Venkata S; Mecocci, Patrizia; McDonald, Colm; McIntosh, Andrew M; Ophoff, Roel A; Paus, Tomas; Pausova, Zdenka; Ryten, Mina; Sachdev, Perminder S; Saykin, Andrew J; Simmons, Andy; Singleton, Andrew; Soininen, Hilkka; Wardlaw, Joanna M; Weale, Michael E; Weinberger, Daniel R; Adams, Hieab H H; Launer, Lenore J; Seiler, Stephan; Schmidt, Reinhold; Chauhan, Ganesh; Satizabal, Claudia L; Becker, James T; Yanek, Lisa; van der Lee, Sven J; Ebling, Maritza; Fischl, Bruce; Longstreth, W T; Greve, Douglas; Schmidt, Helena; Nyquist, Paul; Vinke, Louis N; van Duijn, Cornelia M; Xue, Luting; Mazoyer, Bernard; Bis, Joshua C; Gudnason, Vilmundur; Seshadri, Sudha; Ikram, M Arfan; Martin, Nicholas G; Wright, Margaret J; Schumann, Gunter; Franke, Barbara; Thompson, Paul M; Medland, Sarah E

    2015-04-01

    The highly complex structure of the human brain is strongly shaped by genetic influences. Subcortical brain regions form circuits with cortical areas to coordinate movement, learning, memory and motivation, and altered circuits can lead to abnormal behaviour and disease. To investigate how common genetic variants affect the structure of these brain regions, here we conduct genome-wide association studies of the volumes of seven subcortical regions and the intracranial volume derived from magnetic resonance images of 30,717 individuals from 50 cohorts. We identify five novel genetic variants influencing the volumes of the putamen and caudate nucleus. We also find stronger evidence for three loci with previously established influences on hippocampal volume and intracranial volume. These variants show specific volumetric effects on brain structures rather than global effects across structures. The strongest effects were found for the putamen, where a novel intergenic locus with replicable influence on volume (rs945270; P = 1.08 × 10(-33); 0.52% variance explained) showed evidence of altering the expression of the KTN1 gene in both brain and blood tissue. Variants influencing putamen volume clustered near developmental genes that regulate apoptosis, axon guidance and vesicle transport. Identification of these genetic variants provides insight into the causes of variability in human brain development, and may help to determine mechanisms of neuropsychiatric dysfunction. PMID:25607358

  3. Breast cancer susceptibility variants alter risk in familial ovarian cancer.

    Science.gov (United States)

    Latif, A; McBurney, H J; Roberts, S A; Lalloo, F; Howell, A; Evans, D G; Newman, W G

    2010-12-01

    Recent candidate gene and genome wide association studies have revealed novel loci associated with an increased risk of breast cancer. We evaluated the effect of these breast cancer associated variants on ovarian cancer risk in individuals with familial ovarian cancer both with and without BRCA1 or BRCA2 mutations. A total of 158 unrelated white British women (54 BRCA1/2 mutation positive and 104 BRCA1/2 mutation negative) with familial ovarian cancer were genotyped for FGFR2, TNRC9/TOX3 and CASP8 variants. The p.Asp302His CASP8 variant was associated with reduced ovarian cancer risk in the familial BRCA1/2 mutation negative ovarian cancer cases (P = 0.016). The synonymous TNRC9/TOX3 (Ser51) variant was present at a significantly lower frequency than in patients with familial BRCA1/2 positive breast cancer (P = 0.0002). Our results indicate that variants in CASP8 and TNRC9/TOX3 alter the risk of disease in individuals affected with familial ovarian cancer.

  4. Identification and characterization of variant alleles at CODIS STR loci.

    Science.gov (United States)

    Allor, Catherine; Einum, David D; Scarpetta, Marco

    2005-09-01

    Short tandem repeat (STR) profiles from 32,671 individuals generated by the ABI Profiler Plus and Cofiler systems were screened for variant alleles not represented within manufacturer-provided allelic ladders. A total of 85 distinct variants were identified at 12 of the 13 CODIS loci, most of which involve a truncated tetranucleotide repeat unit. Twelve novel alleles, identified at D3S1358, FGA, D18S51, D5S818, D7S820 and TPOX, were confirmed by nucleotide sequence analysis and include both insertions and deletions involving the repeat units themselves as well as DNA flanking the repeat regions. Population genetic data were collected for all variants and frequencies range from 0.0003 (many single observations) to 0.0042 (D7S820 '10.3' in North American Hispanics). In total, the variant alleles identified in this study are carried by 1.6% of the estimated 1 million individuals tested annually in the U.S. for the purposes of parentage resolution. A paternity case involving a recombination event of paternal origin is presented and demonstrates how variant alleles can significantly strengthen the genetic evidence in troublesome cases. In such instances, increased costs and turnaround time associated with additional testing may be eliminated.

  5. Common genetic variants influence human subcortical brain structures

    Science.gov (United States)

    Hibar, Derrek P.; Stein, Jason L.; Renteria, Miguel E.; Arias-Vasquez, Alejandro; Desrivières, Sylvane; Jahanshad, Neda; Toro, Roberto; Wittfeld, Katharina; Abramovic, Lucija; Andersson, Micael; Aribisala, Benjamin S.; Armstrong, Nicola J.; Bernard, Manon; Bohlken, Marc M.; Boks, Marco P.; Bralten, Janita; Brown, Andrew A.; Chakravarty, M. Mallar; Chen, Qiang; Ching, Christopher R. K.; Cuellar-Partida, Gabriel; den Braber, Anouk; Giddaluru, Sudheer; Goldman, Aaron L.; Grimm, Oliver; Guadalupe, Tulio; Hass, Johanna; Woldehawariat, Girma; Holmes, Avram J.; Hoogman, Martine; Janowitz, Deborah; Jia, Tianye; Kim, Sungeun; Klein, Marieke; Kraemer, Bernd; Lee, Phil H.; Olde Loohuis, Loes M.; Luciano, Michelle; Macare, Christine; Mather, Karen A.; Mattheisen, Manuel; Milaneschi, Yuri; Nho, Kwangsik; Papmeyer, Martina; Ramasamy, Adaikalavan; Risacher, Shannon L.; Roiz-Santiañez, Roberto; Rose, Emma J.; Salami, Alireza; Sämann, Philipp G.; Schmaal, Lianne; Schork, Andrew J.; Shin, Jean; Strike, Lachlan T.; Teumer, Alexander; van Donkelaar, Marjolein M. J.; van Eijk, Kristel R.; Walters, Raymond K.; Westlye, Lars T.; Whelan, Christopher D.; Winkler, Anderson M.; Zwiers, Marcel P.; Alhusaini, Saud; Athanasiu, Lavinia; Ehrlich, Stefan; Hakobjan, Marina M. H.; Hartberg, Cecilie B.; Haukvik, Unn K.; Heister, Angelien J. G. A. M.; Hoehn, David; Kasperaviciute, Dalia; Liewald, David C. M.; Lopez, Lorna M.; Makkinje, Remco R. R.; Matarin, Mar; Naber, Marlies A. M.; McKay, D. Reese; Needham, Margaret; Nugent, Allison C.; Pütz, Benno; Royle, Natalie A.; Shen, Li; Sprooten, Emma; Trabzuni, Daniah; van der Marel, Saskia S. L.; van Hulzen, Kimm J. E.; Walton, Esther; Wolf, Christiane; Almasy, Laura; Ames, David; Arepalli, Sampath; Assareh, Amelia A.; Bastin, Mark E.; Brodaty, Henry; Bulayeva, Kazima B.; Carless, Melanie A.; Cichon, Sven; Corvin, Aiden; Curran, Joanne E.; Czisch, Michael; de Zubicaray, Greig I.; Dillman, Allissa; Duggirala, Ravi; Dyer, Thomas D.; Erk, Susanne; Fedko, Iryna O.; Ferrucci, Luigi; Foroud, Tatiana M.; Fox, Peter T.; Fukunaga, Masaki; Gibbs, J. Raphael; Göring, Harald H. H.; Green, Robert C.; Guelfi, Sebastian; Hansell, Narelle K.; Hartman, Catharina A.; Hegenscheid, Katrin; Heinz, Andreas; Hernandez, Dena G.; Heslenfeld, Dirk J.; Hoekstra, Pieter J.; Holsboer, Florian; Homuth, Georg; Hottenga, Jouke-Jan; Ikeda, Masashi; Jack, Clifford R.; Jenkinson, Mark; Johnson, Robert; Kanai, Ryota; Keil, Maria; Kent, Jack W.; Kochunov, Peter; Kwok, John B.; Lawrie, Stephen M.; Liu, Xinmin; Longo, Dan L.; McMahon, Katie L.; Meisenzahl, Eva; Melle, Ingrid; Mohnke, Sebastian; Montgomery, Grant W.; Mostert, Jeanette C.; Mühleisen, Thomas W.; Nalls, Michael A.; Nichols, Thomas E.; Nilsson, Lars G.; Nöthen, Markus M.; Ohi, Kazutaka; Olvera, Rene L.; Perez-Iglesias, Rocio; Pike, G. Bruce; Potkin, Steven G.; Reinvang, Ivar; Reppermund, Simone; Rietschel, Marcella; Romanczuk-Seiferth, Nina; Rosen, Glenn D.; Rujescu, Dan; Schnell, Knut; Schofield, Peter R.; Smith, Colin; Steen, Vidar M.; Sussmann, Jessika E.; Thalamuthu, Anbupalam; Toga, Arthur W.; Traynor, Bryan J.; Troncoso, Juan; Turner, Jessica A.; Valdés Hernández, Maria C.; van ’t Ent, Dennis; van der Brug, Marcel; van der Wee, Nic J. A.; van Tol, Marie-Jose; Veltman, Dick J.; Wassink, Thomas H.; Westman, Eric; Zielke, Ronald H.; Zonderman, Alan B.; Ashbrook, David G.; Hager, Reinmar; Lu, Lu; McMahon, Francis J.; Morris, Derek W.; Williams, Robert W.; Brunner, Han G.; Buckner, Randy L.; Buitelaar, Jan K.; Cahn, Wiepke; Calhoun, Vince D.; Cavalleri, Gianpiero L.; Crespo-Facorro, Benedicto; Dale, Anders M.; Davies, Gareth E.; Delanty, Norman; Depondt, Chantal; Djurovic, Srdjan; Drevets, Wayne C.; Espeseth, Thomas; Gollub, Randy L.; Ho, Beng-Choon; Hoffmann, Wolfgang; Hosten, Norbert; Kahn, René S.; Le Hellard, Stephanie; Meyer-Lindenberg, Andreas; Müller-Myhsok, Bertram; Nauck, Matthias; Nyberg, Lars; Pandolfo, Massimo; Penninx, Brenda W. J. H.; Roffman, Joshua L.; Sisodiya, Sanjay M.; Smoller, Jordan W.; van Bokhoven, Hans; van Haren, Neeltje E. M.; Völzke, Henry; Walter, Henrik; Weiner, Michael W.; Wen, Wei; White, Tonya; Agartz, Ingrid; Andreassen, Ole A.; Blangero, John; Boomsma, Dorret I.; Brouwer, Rachel M.; Cannon, Dara M.; Cookson, Mark R.; de Geus, Eco J. C.; Deary, Ian J.; Donohoe, Gary; Fernández, Guillén; Fisher, Simon E.; Francks, Clyde; Glahn, David C.; Grabe, Hans J.; Gruber, Oliver; Hardy, John; Hashimoto, Ryota; Hulshoff Pol, Hilleke E.; Jönsson, Erik G.; Kloszewska, Iwona; Lovestone, Simon; Mattay, Venkata S.

    2015-01-01

    The highly complex structure of the human brain is strongly shaped by genetic influences1. Subcortical brain regions form circuits with cortical areas to coordinate movement2, learning, memory3 and motivation4, and altered circuits can lead to abnormal behaviour and disease2. To investigate how common genetic variants affect the structure of these brain regions, here we conduct genome-wide association studies of the volumes of seven subcortical regions and the intracranial volume derived from magnetic resonance images of 30,717 individuals from 50 cohorts. We identify five novel genetic variants influencing the volumes of the putamen and caudate nucleus. We also find stronger evidence for three loci with previously established influences on hippocampal volume5 and intracranial volume6. These variants show specific volumetric effects on brain structures rather than global effects across structures. The strongest effects were found for the putamen, where a novel intergenic locus with replicable influence on volume (rs945270; P = 1.08 × 10−33; 0.52% variance explained) showed evidence of altering the expression of the KTN1 gene in both brain and blood tissue. Variants influencing putamen volume clustered near developmental genes that regulate apoptosis, axon guidance and vesicle transport. Identification of these genetic variants provides insight into the causes of variability inhuman brain development, and may help to determine mechanisms of neuropsychiatric dysfunction. PMID:25607358

  6. Persistent trigeminal artery variants detected by MR angiography

    Energy Technology Data Exchange (ETDEWEB)

    Uchino, A.; Kato, A.; Takase, Y.; Kudo, S. [Dept. of Radiology, Saga Medical School (Japan)

    2000-11-01

    Persistent trigeminal artery (PTA) variants are cerebellar arteries that originate directly from the precavernous portion of the internal carotid artery (ICA). The goal of our study was to determine the incidence and MR angiographic features of PTA variants. Between April 1996 and September 1999, 523 cranial MR angiographies were performed at our institution. Most of the patients examined had or were suspected of having cerebrovascular disease. We retrospectively reviewed these 523 MR angiograms. A 1.5-T scanner was used in all studies, and maximum intensity projection (MIP) images obtained using the three-dimensional time-of-flight (3D TOF) technique were displayed stereoscopically. Four PTA variants were detected on MR angiograms, at a rate of 0.76 %. At least three of the four PTA variants were anterior inferior cerebellar arteries (AICAs), small tortuous arteries arising from the precavernous portions of the ICAs and taking a posterior course. Although the clinical significance is not great, we found a relatively high incidence of PTA variants on MR angiograms. We stress that knowledge and recognition of these anomalous cerebellar arteries are useful and important in the interpretation of cranial MR angiograms. (orig.)

  7. HGVS Recommendations for the Description of Sequence Variants: 2016 Update.

    Science.gov (United States)

    den Dunnen, Johan T; Dalgleish, Raymond; Maglott, Donna R; Hart, Reece K; Greenblatt, Marc S; McGowan-Jordan, Jean; Roux, Anne-Francoise; Smith, Timothy; Antonarakis, Stylianos E; Taschner, Peter E M

    2016-06-01

    The consistent and unambiguous description of sequence variants is essential to report and exchange information on the analysis of a genome. In particular, DNA diagnostics critically depends on accurate and standardized description and sharing of the variants detected. The sequence variant nomenclature system proposed in 2000 by the Human Genome Variation Society has been widely adopted and has developed into an internationally accepted standard. The recommendations are currently commissioned through a Sequence Variant Description Working Group (SVD-WG) operating under the auspices of three international organizations: the Human Genome Variation Society (HGVS), the Human Variome Project (HVP), and the Human Genome Organization (HUGO). Requests for modifications and extensions go through the SVD-WG following a standard procedure including a community consultation step. Version numbers are assigned to the nomenclature system to allow users to specify the version used in their variant descriptions. Here, we present the current recommendations, HGVS version 15.11, and briefly summarize the changes that were made since the 2000 publication. Most focus has been on removing inconsistencies and tightening definitions allowing automatic data processing. An extensive version of the recommendations is available online, at http://www.HGVS.org/varnomen. PMID:26931183

  8. Common genetic variants influence human subcortical brain structures.

    Science.gov (United States)

    Hibar, Derrek P; Stein, Jason L; Renteria, Miguel E; Arias-Vasquez, Alejandro; Desrivières, Sylvane; Jahanshad, Neda; Toro, Roberto; Wittfeld, Katharina; Abramovic, Lucija; Andersson, Micael; Aribisala, Benjamin S; Armstrong, Nicola J; Bernard, Manon; Bohlken, Marc M; Boks, Marco P; Bralten, Janita; Brown, Andrew A; Chakravarty, M Mallar; Chen, Qiang; Ching, Christopher R K; Cuellar-Partida, Gabriel; den Braber, Anouk; Giddaluru, Sudheer; Goldman, Aaron L; Grimm, Oliver; Guadalupe, Tulio; Hass, Johanna; Woldehawariat, Girma; Holmes, Avram J; Hoogman, Martine; Janowitz, Deborah; Jia, Tianye; Kim, Sungeun; Klein, Marieke; Kraemer, Bernd; Lee, Phil H; Olde Loohuis, Loes M; Luciano, Michelle; Macare, Christine; Mather, Karen A; Mattheisen, Manuel; Milaneschi, Yuri; Nho, Kwangsik; Papmeyer, Martina; Ramasamy, Adaikalavan; Risacher, Shannon L; Roiz-Santiañez, Roberto; Rose, Emma J; Salami, Alireza; Sämann, Philipp G; Schmaal, Lianne; Schork, Andrew J; Shin, Jean; Strike, Lachlan T; Teumer, Alexander; van Donkelaar, Marjolein M J; van Eijk, Kristel R; Walters, Raymond K; Westlye, Lars T; Whelan, Christopher D; Winkler, Anderson M; Zwiers, Marcel P; Alhusaini, Saud; Athanasiu, Lavinia; Ehrlich, Stefan; Hakobjan, Marina M H; Hartberg, Cecilie B; Haukvik, Unn K; Heister, Angelien J G A M; Hoehn, David; Kasperaviciute, Dalia; Liewald, David C M; Lopez, Lorna M; Makkinje, Remco R R; Matarin, Mar; Naber, Marlies A M; McKay, D Reese; Needham, Margaret; Nugent, Allison C; Pütz, Benno; Royle, Natalie A; Shen, Li; Sprooten, Emma; Trabzuni, Daniah; van der Marel, Saskia S L; van Hulzen, Kimm J E; Walton, Esther; Wolf, Christiane; Almasy, Laura; Ames, David; Arepalli, Sampath; Assareh, Amelia A; Bastin, Mark E; Brodaty, Henry; Bulayeva, Kazima B; Carless, Melanie A; Cichon, Sven; Corvin, Aiden; Curran, Joanne E; Czisch, Michael; de Zubicaray, Greig I; Dillman, Allissa; Duggirala, Ravi; Dyer, Thomas D; Erk, Susanne; Fedko, Iryna O; Ferrucci, Luigi; Foroud, Tatiana M; Fox, Peter T; Fukunaga, Masaki; Gibbs, J Raphael; Göring, Harald H H; Green, Robert C; Guelfi, Sebastian; Hansell, Narelle K; Hartman, Catharina A; Hegenscheid, Katrin; Heinz, Andreas; Hernandez, Dena G; Heslenfeld, Dirk J; Hoekstra, Pieter J; Holsboer, Florian; Homuth, Georg; Hottenga, Jouke-Jan; Ikeda, Masashi; Jack, Clifford R; Jenkinson, Mark; Johnson, Robert; Kanai, Ryota; Keil, Maria; Kent, Jack W; Kochunov, Peter; Kwok, John B; Lawrie, Stephen M; Liu, Xinmin; Longo, Dan L; McMahon, Katie L; Meisenzahl, Eva; Melle, Ingrid; Mohnke, Sebastian; Montgomery, Grant W; Mostert, Jeanette C; Mühleisen, Thomas W; Nalls, Michael A; Nichols, Thomas E; Nilsson, Lars G; Nöthen, Markus M; Ohi, Kazutaka; Olvera, Rene L; Perez-Iglesias, Rocio; Pike, G Bruce; Potkin, Steven G; Reinvang, Ivar; Reppermund, Simone; Rietschel, Marcella; Romanczuk-Seiferth, Nina; Rosen, Glenn D; Rujescu, Dan; Schnell, Knut; Schofield, Peter R; Smith, Colin; Steen, Vidar M; Sussmann, Jessika E; Thalamuthu, Anbupalam; Toga, Arthur W; Traynor, Bryan J; Troncoso, Juan; Turner, Jessica A; Valdés Hernández, Maria C; van 't Ent, Dennis; van der Brug, Marcel; van der Wee, Nic J A; van Tol, Marie-Jose; Veltman, Dick J; Wassink, Thomas H; Westman, Eric; Zielke, Ronald H; Zonderman, Alan B; Ashbrook, David G; Hager, Reinmar; Lu, Lu; McMahon, Francis J; Morris, Derek W; Williams, Robert W; Brunner, Han G; Buckner, Randy L; Buitelaar, Jan K; Cahn, Wiepke; Calhoun, Vince D; Cavalleri, Gianpiero L; Crespo-Facorro, Benedicto; Dale, Anders M; Davies, Gareth E; Delanty, Norman; Depondt, Chantal; Djurovic, Srdjan; Drevets, Wayne C; Espeseth, Thomas; Gollub, Randy L; Ho, Beng-Choon; Hoffmann, Wolfgang; Hosten, Norbert; Kahn, René S; Le Hellard, Stephanie; Meyer-Lindenberg, Andreas; Müller-Myhsok, Bertram; Nauck, Matthias; Nyberg, Lars; Pandolfo, Massimo; Penninx, Brenda W J H; Roffman, Joshua L; Sisodiya, Sanjay M; Smoller, Jordan W; van Bokhoven, Hans; van Haren, Neeltje E M; Völzke, Henry; Walter, Henrik; Weiner, Michael W; Wen, Wei; White, Tonya; Agartz, Ingrid; Andreassen, Ole A; Blangero, John; Boomsma, Dorret I; Brouwer, Rachel M; Cannon, Dara M; Cookson, Mark R; de Geus, Eco J C; Deary, Ian J; Donohoe, Gary; Fernández, Guillén; Fisher, Simon E; Francks, Clyde; Glahn, David C; Grabe, Hans J; Gruber, Oliver; Hardy, John; Hashimoto, Ryota; Hulshoff Pol, Hilleke E; Jönsson, Erik G; Kloszewska, Iwona; Lovestone, Simon; Mattay, Venkata S; Mecocci, Patrizia; McDonald, Colm; McIntosh, Andrew M; Ophoff, Roel A; Paus, Tomas; Pausova, Zdenka; Ryten, Mina; Sachdev, Perminder S; Saykin, Andrew J; Simmons, Andy

    2015-04-01

    The highly complex structure of the human brain is strongly shaped by genetic influences. Subcortical brain regions form circuits with cortical areas to coordinate movement, learning, memory and motivation, and altered circuits can lead to abnormal behaviour and disease. To investigate how common genetic variants affect the structure of these brain regions, here we conduct genome-wide association studies of the volumes of seven subcortical regions and the intracranial volume derived from magnetic resonance images of 30,717 individuals from 50 cohorts. We identify five novel genetic variants influencing the volumes of the putamen and caudate nucleus. We also find stronger evidence for three loci with previously established influences on hippocampal volume and intracranial volume. These variants show specific volumetric effects on brain structures rather than global effects across structures. The strongest effects were found for the putamen, where a novel intergenic locus with replicable influence on volume (rs945270; P = 1.08 × 10(-33); 0.52% variance explained) showed evidence of altering the expression of the KTN1 gene in both brain and blood tissue. Variants influencing putamen volume clustered near developmental genes that regulate apoptosis, axon guidance and vesicle transport. Identification of these genetic variants provides insight into the causes of variability in human brain development, and may help to determine mechanisms of neuropsychiatric dysfunction.

  9. [Genetic variants associated to male infertility in Mexican patients].

    Science.gov (United States)

    Piña-Aguilar, Raúl Eduardo; Chima-Galán, María del Carmen; Yerena-de-vega, María de la Concepción A; Regalado-Hernández, Miguel Angel; Sánchez-Guerrero, Cecilia; García-Ortiz, Liliana; Santillán-Hernández, Yuritzi; Moreno-García, Jesús Daniel

    2013-05-01

    Recently Mexican Federation of Obstetrics and Gynecology Colleges (Federación Mexicana de Colegios de Obstetricia y Ginecologia, FEMECOG) published the Mexican guideline forthe management of male infertility, which suggests performing genetic laboratory tests as part of diagnosis and management of infertile patients and states that these should receive genetic counseling. This paper reviews the genetic approach proposed by Mexican guideline. A systematic review of medical literature was performed in Pubmed and Web of Knowledge from 1980 to 2012 in order to find reports of genetic variants associated to male infertility in Mexican patients. Also it is discussed the current knowledge of these variants, their clinical implications and finally the guidelines and recommendations for their molecular diagnosis. Most genetic variants in Mexican infertile patients are chromosome abnormalities. In relation to other variants there is only a report of Y chromosome microdeletions, repeated CAG in androgen receptor and more common mutations in CFTR, and other article reporting mutations in CFTR in patients with congenital absence of vas deferens. Little is known about the genetics of Mexican infertile patients apart from chromosome abnormalities. However, the contribution of genetics as etiology of male infertility is taking more relevance and currently the consensual management of infertile male should include the screening of genetic background. This review pretends to be a quick guide for clinicians who want to know about reports of genetic variants related to male infertility in Mexican population and how to approach their diagnosis.

  10. Comparison of protein patterns of xrs-5, a radiosensitive Chinese hamster ovary cell line, and CHO-K1, its radioresistant parent, using two-dimensional gel-electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Kramer, J.M. (Miami Univ., Oxford, OH (USA). Dept. of Zoology)

    1991-01-01

    X-ray sensitive strains of Chinese hamster ovary cell lines have been used to analyze radiation repair mechanisms. One cell line, xrs-5, has been shown to be very sensitive to ionizing radiation and radical forming chemical mutagens. This sensitivity is thought to be a result a mutation in the DNA double strand break (DSB) repair mechanism, and its characterization has been a goal of several repair mechanism studies. Using two-dimensional gel electrophoresis, we have detected a protein (MW approximately 55KD) in the DNA/Nuclear Matrix (nucleoid) cell fraction of CHO-Kl cells that is absent in the nucleoid fraction of xrs-5. This protein is present, however, in both CHO-Kl and xrs-5 whole cell protein maps. To determine whether the 55KD protein is responsible for the radiosensitive and defective DSB repair phenotype of xrs-5 cells, studies are now underway to analyze revertants of xrs-5 that are proficient in DSB repair. Furthermore, an effort to sequence the protein in question is planned. 23 refs., 2 figs.

  11. Comparison of protein patterns of xrs-5, a radiosensitive Chinese hamster ovary cell line, and CHO-K1, its radioresistant parent, using two-dimensional gel-electrophoresis

    International Nuclear Information System (INIS)

    X-ray sensitive strains of Chinese hamster ovary cell lines have been used to analyze radiation repair mechanisms. One cell line, xrs-5, has been shown to be very sensitive to ionizing radiation and radical forming chemical mutagens. This sensitivity is thought to be a result a mutation in the DNA double strand break (DSB) repair mechanism, and its characterization has been a goal of several repair mechanism studies. Using two-dimensional gel electrophoresis, we have detected a protein (MW approximately 55KD) in the DNA/Nuclear Matrix (nucleoid) cell fraction of CHO-Kl cells that is absent in the nucleoid fraction of xrs-5. This protein is present, however, in both CHO-Kl and xrs-5 whole cell protein maps. To determine whether the 55KD protein is responsible for the radiosensitive and defective DSB repair phenotype of xrs-5 cells, studies are now underway to analyze revertants of xrs-5 that are proficient in DSB repair. Furthermore, an effort to sequence the protein in question is planned. 23 refs., 2 figs

  12. Communication: Photodissociation of CH{sub 3}CHO at 308 nm: Observation of H-roaming, CH{sub 3}-roaming, and transition state pathways together along the ground state surface

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hou-Kuan; Tsai, Po-Yu; Hung, Kai-Chan; Kasai, Toshio; Lin, King-Chuen, E-mail: kclin@ntu.edu.tw [Department of Chemistry, National Taiwan University, Taipei 106, Taiwan and Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan (China)

    2015-01-28

    Following photodissociation of acetaldehyde (CH{sub 3}CHO) at 308 nm, the CO(v = 1–4) fragment is acquired using time-resolved Fourier-transform infrared emission spectroscopy. The CO(v = 1) rotational distribution shows a bimodal feature; the low- and high-J components result from H-roaming around CH{sub 3}CO core and CH{sub 3}-roaming around CHO radical, respectively, in consistency with a recent assignment by Kable and co-workers (Lee et al., Chem. Sci. 5, 4633 (2014)). The H-roaming pathway disappears at the CO(v ≥ 2) states, because of insufficient available energy following bond-breaking of H + CH{sub 3}CO. By analyzing the CH{sub 4} emission spectrum, we obtained a bimodal vibrational distribution; the low-energy component is ascribed to the transition state (TS) pathway, consistent with prediction by quasiclassical trajectory calculations, while the high-energy component results from H- and CH{sub 3}-roamings. A branching fraction of H-roaming/CH{sub 3}-roaming/TS contribution is evaluated to be (8% ± 3%)/(68% ± 10%)/(25% ± 5%), in which the TS pathway was observed for the first time. The three pathways proceed concomitantly along the electronic ground state surface.

  13. Variant of partially mapped crossover for the Travelling Salesman problems

    Directory of Open Access Journals (Sweden)

    Kusum Deep

    2012-01-01

    Full Text Available In this paper a variant of partially mapped crossover (VPMX is designed using cut point positions and is tested for its performance with the existing partially mapped crossover (PMX.In order to test the performance ,two mutation operators are used. These mutation operators are inverted displacement and inversion mutations. Partially mapped crossover (PMX with inversion and with inverted displacement and a variant of partially mapped crossover (VPMX with inversion and with inverted displacement are programmed in C++ and implemented on a set of ten benchmark problems taken from the Travelling salesman problem library (TSPLIB. The results indicate that the designed variant of PMX is superior by showing a better performance in eight instances in combination with the inverted displacement mutation. In two instances PMX has obtained a better result. One is PMX with inversion mutation and the other is PMX with inverted displacement mutation.

  14. Grinder Variant System Design and Implementation Based on Ontology

    Science.gov (United States)

    Yang, G. H.; Zhang, T. P.

    In order to improve the efficiency of product design and reuse in heterogeneous system of knowledge sharing, this paper introduced the concept of ontology into product variant design, and grinding machine design was as an example. A lot of experience and accumulated knowledge in product design was shared and reused. It is precisely to formulate ontology knowledge such as variant design features and parameter, and applied the software protégé4.3 to construct ontology model, as well as runed resoning on model data information. It developed a set of complete product intelligent system of variant design, which can effectively solve the problem of the repeated design and greatly shorten product development cycle.

  15. KD4v: Comprehensible Knowledge Discovery System for Missense Variant.

    Science.gov (United States)

    Luu, Tien-Dao; Rusu, Alin; Walter, Vincent; Linard, Benjamin; Poidevin, Laetitia; Ripp, Raymond; Moulinier, Luc; Muller, Jean; Raffelsberger, Wolfgang; Wicker, Nicolas; Lecompte, Odile; Thompson, Julie D; Poch, Olivier; Nguyen, Hoan

    2012-07-01

    A major challenge in the post-genomic era is a better understanding of how human genetic alterations involved in disease affect the gene products. The KD4v (Comprehensible Knowledge Discovery System for Missense Variant) server allows to characterize and predict the phenotypic effects (deleterious/neutral) of missense variants. The server provides a set of rules learned by Induction Logic Programming (ILP) on a set of missense variants described by conservation, physico-chemical, functional and 3D structure predicates. These rules are interpretable by non-expert humans and are used to accurately predict the deleterious/neutral status of an unknown mutation. The web server is available at http://decrypthon.igbmc.fr/kd4v.

  16. Arrhythmogenic KCNE gene variants: current knowledge and future challenges

    Directory of Open Access Journals (Sweden)

    Shawn M Crump

    2014-01-01

    Full Text Available There are twenty-five known inherited cardiac arrhythmia susceptibility genes, all of which encode either ion channel pore-forming subunits or proteins that regulate aspects of ion channel biology such as function, trafficking and localization. The human KCNE gene family comprises five potassium channel regulatory subunits, sequence variants in each of which are associated with cardiac arrhythmias. KCNE gene products exhibit promiscuous partnering and in some cases ubiquitous expression, hampering efforts to unequivocally correlate each gene to specific native potassium currents. Likewise, deducing the molecular etiology of cardiac arrhythmias in individuals harboring rare KCNE gene variants, or more common KCNE polymorphisms, can be challenging. In this review we provide an update on putative arrhythmia-causing KCNE gene variants, and discuss current thinking and future challenges in the study of molecular mechanisms of KCNE-associated cardiac rhythm disturbances.

  17. Analysis of Plasminogen Genetic Variants in Multiple Sclerosis Patients

    Science.gov (United States)

    Sadovnick, A. Dessa; Traboulsee, Anthony L.; Bernales, Cecily Q.; Ross, Jay P.; Forwell, Amanda L.; Yee, Irene M.; Guillot-Noel, Lena; Fontaine, Bertrand; Cournu-Rebeix, Isabelle; Alcina, Antonio; Fedetz, Maria; Izquierdo, Guillermo; Matesanz, Fuencisla; Hilven, Kelly; Dubois, Bénédicte; Goris, An; Astobiza, Ianire; Alloza, Iraide; Antigüedad, Alfredo; Vandenbroeck, Koen; Akkad, Denis A.; Aktas, Orhan; Blaschke, Paul; Buttmann, Mathias; Chan, Andrew; Epplen, Joerg T.; Gerdes, Lisa-Ann; Kroner, Antje; Kubisch, Christian; Kümpfel, Tania; Lohse, Peter; Rieckmann, Peter; Zettl, Uwe K.; Zipp, Frauke; Bertram, Lars; Lill, Christina M; Fernandez, Oscar; Urbaneja, Patricia; Leyva, Laura; Alvarez-Cermeño, Jose Carlos; Arroyo, Rafael; Garagorri, Aroa M.; García-Martínez, Angel; Villar, Luisa M.; Urcelay, Elena; Malhotra, Sunny; Montalban, Xavier; Comabella, Manuel; Berger, Thomas; Fazekas, Franz; Reindl, Markus; Schmied, Mascha C.; Zimprich, Alexander; Vilariño-Güell, Carles

    2016-01-01

    Multiple sclerosis (MS) is a prevalent neurological disease of complex etiology. Here, we describe the characterization of a multi-incident MS family that nominated a rare missense variant (p.G420D) in plasminogen (PLG) as a putative genetic risk factor for MS. Genotyping of PLG p.G420D (rs139071351) in 2160 MS patients, and 886 controls from Canada, identified 10 additional probands, two sporadic patients and one control with the variant. Segregation in families harboring the rs139071351 variant, identified p.G420D in 26 out of 30 family members diagnosed with MS, 14 unaffected parents, and 12 out of 30 family members not diagnosed with disease. Despite considerably reduced penetrance, linkage analysis supports cosegregation of PLG p.G420D and disease. Genotyping of PLG p.G420D in 14446 patients, and 8797 controls from Canada, France, Spain, Germany, Belgium, and Austria failed to identify significant association with disease (P = 0.117), despite an overall higher prevalence in patients (OR = 1.32; 95% CI = 0.93–1.87). To assess whether additional rare variants have an effect on MS risk, we sequenced PLG in 293 probands, and genotyped all rare variants in cases and controls. This analysis identified nine rare missense variants, and although three of them were exclusively observed in MS patients, segregation does not support pathogenicity. PLG is a plausible biological candidate for MS owing to its involvement in immune system response, blood-brain barrier permeability, and myelin degradation. Moreover, components of its activation cascade have been shown to present increased activity or expression in MS patients compared to controls; further studies are needed to clarify whether PLG is involved in MS susceptibility. PMID:27194806

  18. Analysis of Plasminogen Genetic Variants in Multiple Sclerosis Patients

    Directory of Open Access Journals (Sweden)

    A. Dessa Sadovnick

    2016-07-01

    Full Text Available Multiple sclerosis (MS is a prevalent neurological disease of complex etiology. Here, we describe the characterization of a multi-incident MS family that nominated a rare missense variant (p.G420D in plasminogen (PLG as a putative genetic risk factor for MS. Genotyping of PLG p.G420D (rs139071351 in 2160 MS patients, and 886 controls from Canada, identified 10 additional probands, two sporadic patients and one control with the variant. Segregation in families harboring the rs139071351 variant, identified p.G420D in 26 out of 30 family members diagnosed with MS, 14 unaffected parents, and 12 out of 30 family members not diagnosed with disease. Despite considerably reduced penetrance, linkage analysis supports cosegregation of PLG p.G420D and disease. Genotyping of PLG p.G420D in 14446 patients, and 8797 controls from Canada, France, Spain, Germany, Belgium, and Austria failed to identify significant association with disease (P = 0.117, despite an overall higher prevalence in patients (OR = 1.32; 95% CI = 0.93–1.87. To assess whether additional rare variants have an effect on MS risk, we sequenced PLG in 293 probands, and genotyped all rare variants in cases and controls. This analysis identified nine rare missense variants, and although three of them were exclusively observed in MS patients, segregation does not support pathogenicity. PLG is a plausible biological candidate for MS owing to its involvement in immune system response, blood-brain barrier permeability, and myelin degradation. Moreover, components of its activation cascade have been shown to present increased activity or expression in MS patients compared to controls; further studies are needed to clarify whether PLG is involved in MS susceptibility.

  19. CEACAM6 gene variants in inflammatory bowel disease.

    Directory of Open Access Journals (Sweden)

    Jürgen Glas

    Full Text Available BACKGROUND: The carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6 acts as a receptor for adherent-invasive E. coli (AIEC and its ileal expression is increased in patients with Crohn's disease (CD. Given its contribution to the pathogenesis of CD, we aimed to investigate the role of genetic variants in the CEACAM6 region in patients with inflammatory bowel diseases (IBD. METHODOLOGY: In this study, a total of 2,683 genomic DNA samples (including DNA from 858 CD patients, 475 patients with ulcerative colitis (UC, and 1,350 healthy, unrelated controls was analyzed for eight CEACAM6 SNPs (rs10415946, rs1805223 = p.Pro42Pro, rs4803507, rs4803508, rs11548735 = p.Gly239Val, rs7246116 = pHis260His, rs2701, rs10416839. In addition, a detailed haplotype analysis and genotype-phenotype analysis were performed. Overall, our genotype analysis did not reveal any significant association of the investigated CEACAM6 SNPs and haplotypes with CD or UC susceptibility, although certain CEACAM6 SNPs modulated CEACAM6 expression in intestinal epithelial cell lines. Despite its function as receptor of AIEC in ileal CD, we found no association of the CEACAM6 SNPs with ileal or ileocolonic CD. Moreover, there was no evidence of epistasis between the analyzed CEACAM6 variants and the main CD-associated NOD2, IL23R and ATG16L1 variants. CONCLUSIONS: This study represents the first detailed analysis of CEACAM6 variants in IBD patients. Despite its important role in bacterial attachment in ileal CD, we could not demonstrate a role for CEACAM6 variants in IBD susceptibility or regarding an ileal CD phenotype. Further functional studies are required to analyze if these gene variants modulate ileal bacterial attachment.

  20. Complexity on Acute Myeloid Leukemia mRNA Transcript Variant

    Directory of Open Access Journals (Sweden)

    Carlo Cattani

    2011-01-01

    Full Text Available This paper deals with the sequence analysis of acute myeloid leukemia mRNA. Six transcript variants of mlf1 mRNA, with more than 2000 bps, are analyzed by focusing on the autocorrelation of each distribution. Through the correlation matrix, some patches and similarities are singled out and commented, with respect to similar distributions. The comparison of Kolmogorov fractal dimension will be also given in order to classify the six variants. The existence of a fractal shape, patterns, and symmetries are discussed as well.

  1. GPI Mount Scopus--a variant of glucosephosphate isomerase deficiency.

    Science.gov (United States)

    Shalev, O; Shalev, R S; Forman, L; Beutler, E

    1993-10-01

    Glucosephosphate isomerase (GPI) deficiency is an unusual cause of hereditary nonspherocytic hemolytic anemia. The disease, inherited as an autosomal recessive disorder, is most often manifested by symptoms and signs of chronic hemolysis, ameliorated by splenectomy. We recently diagnosed GPI deficiency in a 23-year-old Ashkenazi Jewish man who displayed the typical clinical course of this disorder. The biophysical characteristics of the GPI variant are slow electrophoretic mobility, presence of only one of the two bands normally present, and extreme thermolability. To the best of our knowledge, this is the first report of GPI deficiency in a patient of Jewish descent, and we propose to designate this enzyme variant "GPI Mount Scopus".

  2. Integrated Analysis of Germline and Somatic Variants in Ovarian Cancer

    OpenAIRE

    Kanchi, Krishna L.; Johnson, Kimberly J.; Lu, Charles; McLellan, Michael D; Mark D M Leiserson; Wendl, Michael C; Zhang, Qunyuan; Koboldt, Daniel C.; Xie, Mingchao; Kandoth, Cyriac; McMichael, Joshua F.; Wyczalkowski, Matthew A.; Larson, David E.; Schmidt, Heather K.; Christopher A Miller

    2014-01-01

    We report the first large-scale exome-wide analysis of the combined germline-somatic landscape in ovarian cancer. Here we analyze germline and somatic alterations in 429 ovarian carcinoma cases and 557 controls. We identify 3,635 high confidence, rare truncation and 22,953 missense variants with predicted functional impact. We find germline truncation variants and large deletions across Fanconi pathway genes in 20% of cases. Enrichment of rare truncations is shown in BRCA1, BRCA2, and PALB2. ...

  3. Diffuse sclerosing variant of papillary thyroid carcinoma: case report

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung Chan; Kim, Dong Wook [Masan Samsung Hospital, Sungkyunkwan University, School of Medicine, Masan (Korea, Republic of)

    2006-07-15

    Diffuse sclerosing papillary carcinoma (DSPC) is a variant of papillary thyroid carcinoma (PTC), but it shows more aggressive clinical course and a poorer prognosis than the other types of PTC. Most PTCs show a focal nodular pattern in the thyroid on the imaging modalities, but DSPC reveals a diffuse infiltrating configuration in the thyroid without any focal nodular lesion. To our knowledge, there are scant radiological reports of diffuse sclerosing variant of papillary thyroid carcinoma. In this report, we present the case of a patient with DSPC who showed the characteristic findings on sonography and computed tomography.

  4. Cribriform-Morular Variant of Papillary Thyroid Carcinoma

    Directory of Open Access Journals (Sweden)

    Bahar AKKAYA

    2009-09-01

    Full Text Available Cribriform-morular variant of papillary thyroid carcinoma is a rare histological subtype of papillary thyroid carcinoma. This subtype is commonly reported in patients with familial adenomatous polyposis. However, cases not associated with polyposis have also been reported. The differential diagnosis of this entity from other aggressive thyroid neoplasms is important. A 29-year old man presented with a solitary mass in the left thyroid lobe underwent total thyroidectomy. Pathologic examination of the specimen revealed cribriform-morular variant of papillary thyroid carcinoma. After diagnosis, colonoscopy revealed a normal colon without polyposis. Herein, we report a case not associated with polyposis and discuss with the literature.

  5. Pharmacogenomic variants have larger effect sizes than genetic variants associated with other dichotomous complex traits.

    Science.gov (United States)

    Maranville, J C; Cox, N J

    2016-08-01

    It has been suggested that pharmacogenomic phenotypes are influenced by genetic variants with larger effect sizes than other phenotypes, such as complex disease risk. This is presumed to reflect the fact that relevant environmental factors (drug exposure) are appropriately measured and taken into account. To test this hypothesis, we performed a systematic comparison of effect sizes between pharmacogenomic and non-pharmacogenomic phenotypes across all genome-wide association studies (GWAS) reported in the NHGRI GWAS catalog. We found significantly larger effect sizes for studies focused on pharmacogenomic phenotypes, as compared with complex disease risk, morphological phenotypes and endophenotypes. We found no significant differences in effect sizes between pharmacogenomic studies focused on adverse events versus those focused on drug efficacy. Furthermore, we found that this pattern persists among sample size-matched studies, suggesting that this pattern does not reflect overestimation of effect sizes due to smaller sample sizes in pharmacogenomic studies.The Pharmacogenomics Journal advance online publication, 7 July 2015; doi:10.1038/tpj.2015.47. PMID:26149738

  6. Variant of a Klippel-Trenaunay syndrome - Case report; Variante eines Klippel-Trenaunay-Syndroms - ein Fallbericht

    Energy Technology Data Exchange (ETDEWEB)

    Irlbacher, K.; Behse, F.; Roericht, S.; Meyer, B.U. [Charite-Campus Virchow Klinikum, Humboldt-Univ. Berlin (Germany); Hoffmann, K.T. [Charite-Campus Virchow Klinikum, Humboldt-Univ. Berlin (Germany). Radiologische Klinik

    2000-07-01

    We report a patient with a variant of this syndrome presenting with extensive varicose veins and arteriovenous shunts within the left arm, bony hypotrophy of the left hand, mucocutaneous melanin spots in the face and thrombocytopenia. Imaging techniques play a major role in making a diagnosis in angiophakomatoses. (orig.) [German] Hier wird eine Patientin mit einer Variante eines KT vorgestellt, bei der klinisch und mit bildgebenden Verfahren folgende Befunde erhoben wurden: Segmentale varikoese Venektasien und arteriovenoese Kurzschluesse im linken Arm und Hypotrophie von Handknochen, Phlebolithen, Venenektasien in der linken Wangenschleimhaut, Pigmentnaevi in der Gesichts-, Lippen-, und Wangenschleimhaut und Thrombozytopenie. Bildgebende Verfahren helfen hier eine diagnostische Einstufung vorzunehmen. (orig.)

  7. Coinfection with human herpesvirus 6 variants A and B in lung tissue.

    OpenAIRE

    Cone, R W; Huang, M.L.; Hackman, R C; Corey, L

    1996-01-01

    Human herpesvirus 6 (HHV-6) variant B is frequently identified in peripheral blood, but identification of HHV-6 variant A is relatively rare. We devised a PCR-based method for sensitive, simultaneous detection of both HHV-6 variants. The method was applied to 34 lung tissue specimens that were previously shown to contain HHV-6 DNA. A total of 22 lung tissue samples showed coinfections with HHV-6 variants A and B, 2 had only HHV-6 variant A DNA, and 10 had only HHV-6 variant B DNA. The prevale...

  8. 炭疽受体CMG2-Fc融合蛋白在CHO细胞中的表达、纯化与鉴定%Expression,Purification and Identification of Anthrax Receptor CMG2-Fc Fusion Protein in CHO Cell

    Institute of Scientific and Technical Information of China (English)

    郗永义; 胥照平; 高丽华; 邵勇; 胡显文; 陈惠鹏

    2011-01-01

    目的:构建炭疽受体CMG2和人IgG1 Fc片段融合基因载体,转染CHO细胞并通过毒素中和试验检测CMG2-Fc拮抗炭疽毒素(PA+LF)的能力.方法:将含有CMG2胞外区1-217AA片度基因和人IgG1的Fc片段基因共同连接入pcDNA3.1载体转染CHO细胞并筛选高表达CMG2-Fc的CHO细胞系,通过小鼠RAW264.7巨噬细胞保护试验检测 CMG2-Fc拮抗炭疽毒素的能力.结果:获得了表达CMG2-Fc的细胞株,毒素中和实验显示该蛋白可以有效抑制炭疽毒素引起的细胞损伤.结论:CMG2-Fc能够保护小鼠巨噬细胞免受炭疽毒素攻击,提示其可以作为抗毒素治疗炭疽感染.%Objective: To construct fusion gene vector of anthrax receptor CMG2 and human IgG1 Fc fragent, transfect CHO cell and to testify the inhibit ability of CMG2-Fc on anthrax toxin (PA and LF) by toxin neutralization assay.Methods: An expression vector including CMG2 (1-225AA) gene and Fc fragment of human immunoglobulin G (IgG) were constructed, and CHO cell line with higher CMG2-Fc expression were got.The effect of CMG2-Fc on inhibiting anthrax toxin was detected by mouse macrophage protection assay.Result: The CHO cell line expressing CMG2-Fc protein were got, and toxin neutralization assay showed that CMG2-Fc could protect mouse RAW264.7 macrophage cell against anthrax toxins.Conclusion: CMG2-Fc can protect mouse macrophage against anthrax toxin challenge, which indicated that it maybe used as drugs against anthrax in future.

  9. A Variant of Young's Double Slit Experiment for Educational Purposes

    Science.gov (United States)

    Henault, Francois; Spang, Alain

    2011-01-01

    We describe a variant of the classical Young's double slit experiment that can be easily realized in any classroom, in order to evidence the wave nature of light. The proposed apparatus and its simplified theory are described and pictures of fringes, readily obtained using only cheap and off-the-shelf optical components, are reproduced. The…

  10. Analysis of Trivium by a Simulated Annealing variant

    DEFF Research Database (Denmark)

    Borghoff, Julia; Knudsen, Lars Ramkilde; Matusiewicz, Krystian

    2010-01-01

    . A characteristic of equation systems that may be efficiently solvable by the means of such algorithms is provided. As an example, we investigate equation systems induced by the problem of recovering the internal state of the stream cipher Trivium. We propose an improved variant of the simulated annealing method...

  11. Structure of chymotrypsin variant B from Atlantic cod, Gadus morhua

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Asgeirsson, B; Thórólfsson, M;

    1996-01-01

    The amino-acid sequence of chymotrypsin variant B isolated from the pyloric caeca of Atlantic cod has been elucidated. The characterization of the primary structure is based on N-terminal Edman degradation and mass spectrometry of the native protein and enzymatically derived peptides. Chymotrypsi...

  12. Neuropathology in classical and variant ataxia-telangiectasia

    NARCIS (Netherlands)

    Verhagen, Mijke M. M.; Martin, Jean-Jacques; van Deuren, Marcel; Groote, Chantal Ceuterick-de; Weemaes, Corry M. R.; Kremer, Berry H. P. H.; Taylor, Malcolm A. R.; Willemsen, Michel A. A. P.; Lammens, Martin

    2012-01-01

    Ataxia-telangiectasia (A-T) is classically characterized by progressive neurodegeneration, oculocutaneous telangiectasia, immunodeficiency and elevated a-fetoprotein levels. Some patients, classified as variant A-T, exhibit a milder clinical course. In the latter patients extrapyramidal symptoms, in

  13. Interaction between 5 genetic variants and allergy in glioma risk

    DEFF Research Database (Denmark)

    Schoemaker, Minouk J; Robertson, Lindsay; Wigertz, Annette;

    2010-01-01

    The etiology of glioma is barely known. Epidemiologic studies have provided evidence for an inverse relation between glioma risk and allergic disease. Genome-wide association data have identified common genetic variants at 5p15.33 (rs2736100, TERT), 8q24.21 (rs4295627, CCDC26), 9p21.3 (rs4977756...

  14. Histone variants of the insect Plodia interpunctella during metamorphosis.

    Science.gov (United States)

    Pataryas, T A; Sekeri-Pataryas, K T; Bonner, W M; Marinou, V A

    1984-01-01

    The pattern of histone variants from the meal moth Plodia interpunctella was compared to the mouse histone variant pattern. Plodia contains histones which comigrate on two dimensional gels with H3.2, H3.3, H4 and H2A.Z in mouse. Plodia H2A.1 and H2B.1 migrate somewhat differently from the respective mouse histones. Comparison of the iodinated tryptic peptides of H2A.1 and H2A.Z from mouse and Plodia showed that the H2A.Z proteins have two iodinated peptides that comigrate in the two species and three more that are different. The H2A.1 proteins in the two species have one iodinated peptide which comigrates and two more which migrate very close to each other. The histone variants from three developmental stages, larval, pupal and adult of Plodia interpunctella were also identified and compared. The same histone variant pattern is found through all stages of development. It is concluded that histone gene expression does not change during metamorphosis in Plodia .

  15. Status quo of annotation of human disease variants

    NARCIS (Netherlands)

    Venselaar, H.; Camilli, F.; Gholizadeh, S.; Snelleman, M.; Brunner, H.G.; Vriend, G.

    2013-01-01

    BACKGROUND: The ever on-going technical developments in Next Generation Sequencing have led to an increase in detected disease related mutations. Many bioinformatics approaches exist to analyse these variants, and of those the methods that use 3D structure information generally outperform those that

  16. The Role of Constitutional Copy Number Variants in Breast Cancer.

    Science.gov (United States)

    Walker, Logan C; Wiggins, George A R; Pearson, John F

    2015-01-01

    Constitutional copy number variants (CNVs) include inherited and de novo deviations from a diploid state at a defined genomic region. These variants contribute significantly to genetic variation and disease in humans, including breast cancer susceptibility. Identification of genetic risk factors for breast cancer in recent years has been dominated by the use of genome-wide technologies, such as single nucleotide polymorphism (SNP)-arrays, with a significant focus on single nucleotide variants. To date, these large datasets have been underutilised for generating genome-wide CNV profiles despite offering a massive resource for assessing the contribution of these structural variants to breast cancer risk. Technical challenges remain in determining the location and distribution of CNVs across the human genome due to the accuracy of computational prediction algorithms and resolution of the array data. Moreover, better methods are required for interpreting the functional effect of newly discovered CNVs. In this review, we explore current and future application of SNP array technology to assess rare and common CNVs in association with breast cancer risk in humans. PMID:27600231

  17. The Role of Constitutional Copy Number Variants in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Logan C. Walker

    2015-09-01

    Full Text Available Constitutional copy number variants (CNVs include inherited and de novo deviations from a diploid state at a defined genomic region. These variants contribute significantly to genetic variation and disease in humans, including breast cancer susceptibility. Identification of genetic risk factors for breast cancer in recent years has been dominated by the use of genome-wide technologies, such as single nucleotide polymorphism (SNP-arrays, with a significant focus on single nucleotide variants. To date, these large datasets have been underutilised for generating genome-wide CNV profiles despite offering a massive resource for assessing the contribution of these structural variants to breast cancer risk. Technical challenges remain in determining the location and distribution of CNVs across the human genome due to the accuracy of computational prediction algorithms and resolution of the array data. Moreover, better methods are required for interpreting the functional effect of newly discovered CNVs. In this review, we explore current and future application of SNP array technology to assess rare and common CNVs in association with breast cancer risk in humans.

  18. Inferring causative variants in microRNA target sites.

    Science.gov (United States)

    Thomas, Laurent F; Saito, Takaya; Sætrom, Pål

    2011-09-01

    MicroRNAs (miRNAs) regulate genes post transcription by pairing with messenger RNA (mRNA). Variants such as single nucleotide polymorphisms (SNPs) in miRNA regulatory regions might result in altered protein levels and disease. Genome-wide association studies (GWAS) aim at identifying genomic regions that contain variants associated with disease, but lack tools for finding causative variants. We present a computational tool that can help identifying SNPs associated with diseases, by focusing on SNPs affecting miRNA-regulation of genes. The tool predicts the effects of SNPs in miRNA target sites and uses linkage disequilibrium to map these miRNA-related variants to SNPs of interest in GWAS. We compared our predicted SNP effects in miRNA target sites with measured SNP effects from allelic imbalance sequencing. Our predictions fit measured effects better than effects based on differences in free energy or differences of TargetScan context scores. We also used our tool to analyse data from published breast cancer and Parkinson's disease GWAS and significant trait-associated SNPs from the NHGRI GWAS Catalog. A database of predicted SNP effects is available at http://www.bigr.medisin.ntnu.no/mirsnpscore/. The database is based on haplotype data from the CEU HapMap population and miRNAs from miRBase 16.0.

  19. Genetic variants regulating immune cell levels in health and disease.

    Science.gov (United States)

    Orrù, Valeria; Steri, Maristella; Sole, Gabriella; Sidore, Carlo; Virdis, Francesca; Dei, Mariano; Lai, Sandra; Zoledziewska, Magdalena; Busonero, Fabio; Mulas, Antonella; Floris, Matteo; Mentzen, Wieslawa I; Urru, Silvana A M; Olla, Stefania; Marongiu, Michele; Piras, Maria G; Lobina, Monia; Maschio, Andrea; Pitzalis, Maristella; Urru, Maria F; Marcelli, Marco; Cusano, Roberto; Deidda, Francesca; Serra, Valentina; Oppo, Manuela; Pilu, Rosella; Reinier, Frederic; Berutti, Riccardo; Pireddu, Luca; Zara, Ilenia; Porcu, Eleonora; Kwong, Alan; Brennan, Christine; Tarrier, Brendan; Lyons, Robert; Kang, Hyun M; Uzzau, Sergio; Atzeni, Rossano; Valentini, Maria; Firinu, Davide; Leoni, Lidia; Rotta, Gianluca; Naitza, Silvia; Angius, Andrea; Congia, Mauro; Whalen, Michael B; Jones, Chris M; Schlessinger, David; Abecasis, Gonçalo R; Fiorillo, Edoardo; Sanna, Serena; Cucca, Francesco

    2013-09-26

    The complex network of specialized cells and molecules in the immune system has evolved to defend against pathogens, but inadvertent immune system attacks on "self" result in autoimmune disease. Both genetic regulation of immune cell levels and their relationships with autoimmunity are largely undetermined. Here, we report genetic contributions to quantitative levels of 95 cell types encompassing 272 immune traits, in a cohort of 1,629 individuals from four clustered Sardinian villages. We first estimated trait heritability, showing that it can be substantial, accounting for up to 87% of the variance (mean 41%). Next, by assessing ∼8.2 million variants that we identified and confirmed in an extended set of 2,870 individuals, 23 independent variants at 13 loci associated with at least one trait. Notably, variants at three loci (HLA, IL2RA, and SH2B3/ATXN2) overlap with known autoimmune disease associations. These results connect specific cellular phenotypes to specific genetic variants, helping to explicate their involvement in disease. PMID:24074872

  20. Andes hantavirus variant in rodents, southern Amazon Basin, Peru.

    Science.gov (United States)

    Razuri, Hugo; Tokarz, Rafal; Ghersi, Bruno M; Salmon-Mulanovich, Gabriela; Guezala, M Claudia; Albujar, Christian; Mendoza, A Patricia; Tinoco, Yeny O; Cruz, Christopher; Silva, Maria; Vasquez, Alicia; Pacheco, Víctor; Ströher, Ute; Guerrero, Lisa Wiggleton; Cannon, Deborah; Nichol, Stuart T; Hirschberg, David L; Lipkin, W Ian; Bausch, Daniel G; Montgomery, Joel M

    2014-02-01

    We investigated hantaviruses in rodents in the southern Amazon Basin of Peru and identified an Andes virus variant from Neacomys spinosus mice. This finding extends the known range of this virus in South America and the range of recognized hantaviruses in Peru. Further studies of the epizoology of hantaviruses in this region are warranted.

  1. Two new splice variants in porcine PPARGC1A

    Directory of Open Access Journals (Sweden)

    Peelman Luc J

    2008-12-01

    Full Text Available Abstract Background Peroxisome proliferator-activated receptor γ coactivator 1α (PPARGC1A is a coactivator with a vital and central role in fat and energy metabolism. It is considered to be a candidate gene for meat quality in pigs and is involved in the development of obesity and diabetes in humans. How its many functions are regulated, is however still largely unclear. Therefore a transcription profile of PPARGC1A in 32 tissues and 4 embryonic developmental stages in the pig was constructed by screening its cDNA for possible splice variants with exon-spanning primers. Findings This led to the discovery of 2 new splice variants in the pig, which were subsequently also detected in human tissues. In these variants, exon 8 was either completely or partly (the last 66 bp were conserved spliced out, potentially coding for a much shorter protein of respectively 337 and 359 amino acids (aa, of which the first 291 aa would be the same compared to the complete protein (796 aa. Conclusion Considering the functional domains of the PPARGC1A protein, it is very likely these splice variants considerably affect the function of the protein and alternative splicing could be one of the mechanisms by which the diverse functions of PPARGC1A are regulated.

  2. The Main Social Variants in the English Language

    Institute of Scientific and Technical Information of China (English)

    权红梅

    2002-01-01

    To use a language properly, we of course have to know the grammatical structures of the language and their meanings ,but we also have to know what forms of language are appropriate for given situations,and for this purpose,this thesis briefly explains the social variants of English,such as (informal English), (written English)and (American English).

  3. The Main Social Variants in the English Language

    Institute of Scientific and Technical Information of China (English)

    QUANHong-mei

    2002-01-01

    To use a language properly, we of course have to know the grammatical structures of the language and their meanings,but we also have to know what forms of language are appropriate for given situations,and for this purpose,this thesis briefly explains the social variants of English,such asl (informal English), (written English)and (American English).

  4. New common variants affecting susceptibility to basal cell carcinoma.

    Science.gov (United States)

    Stacey, Simon N; Sulem, Patrick; Masson, Gisli; Gudjonsson, Sigurjon A; Thorleifsson, Gudmar; Jakobsdottir, Margret; Sigurdsson, Asgeir; Gudbjartsson, Daniel F; Sigurgeirsson, Bardur; Benediktsdottir, Kristrun R; Thorisdottir, Kristin; Ragnarsson, Rafn; Scherer, Dominique; Hemminki, Kari; Rudnai, Peter; Gurzau, Eugene; Koppova, Kvetoslava; Botella-Estrada, Rafael; Soriano, Virtudes; Juberias, Pablo; Saez, Berta; Gilaberte, Yolanda; Fuentelsaz, Victoria; Corredera, Cristina; Grasa, Matilde; Höiom, Veronica; Lindblom, Annika; Bonenkamp, Johannes J; van Rossum, Michelle M; Aben, Katja K H; de Vries, Esther; Santinami, Mario; Di Mauro, Maria G; Maurichi, Andrea; Wendt, Judith; Hochleitner, Pia; Pehamberger, Hubert; Gudmundsson, Julius; Magnusdottir, Droplaug N; Gretarsdottir, Solveig; Holm, Hilma; Steinthorsdottir, Valgerdur; Frigge, Michael L; Blondal, Thorarinn; Saemundsdottir, Jona; Bjarnason, Hjördis; Kristjansson, Kristleifur; Bjornsdottir, Gyda; Okamoto, Ichiro; Rivoltini, Licia; Rodolfo, Monica; Kiemeney, Lambertus A; Hansson, Johan; Nagore, Eduardo; Mayordomo, José I; Kumar, Rajiv; Karagas, Margaret R; Nelson, Heather H; Gulcher, Jeffrey R; Rafnar, Thorunn; Thorsteinsdottir, Unnur; Olafsson, Jon H; Kong, Augustine; Stefansson, Kari

    2009-08-01

    In a follow-up to our previously reported genome-wide association study of cutaneous basal cell carcinoma (BCC), we describe here several new susceptibility variants. SNP rs11170164, encoding a G138E substitution in the keratin 5 (KRT5) gene, affects risk of BCC (OR = 1.35, P = 2.1 x 10(-9)). A variant at 9p21 near CDKN2A and CDKN2B also confers susceptibility to BCC (rs2151280[C]; OR = 1.19, P = 6.9 x 10(-9)), as does rs157935[T] at 7q32 near the imprinted gene KLF14 (OR = 1.23, P = 5.7 x 10(-10)). The effect of rs157935[T] is dependent on the parental origin of the risk allele. None of these variants were found to be associated with melanoma or fair-pigmentation traits. A melanoma- and pigmentation-associated variant in the SLC45A2 gene, L374F, is associated with risk of both BCC and squamous cell carcinoma. Finally, we report conclusive evidence that rs401681[C] in the TERT-CLPTM1L locus confers susceptibility to BCC but protects against melanoma. PMID:19578363

  5. New common variants affecting susceptibility to basal cell carcinoma.

    NARCIS (Netherlands)

    Stacey, S.N.; Sulem, P.; Masson, G.; Gudjonsson, S.A.; Thorleifsson, G.; Jakobsdottir, M.; Sigurdsson, A.; Gudbjartsson, D.F.; Sigurgeirsson, B.; Benediktsdottir, K.R.; Thorisdottir, K.; Ragnarsson, R.; Scherer, D.; Hemminki, K.; Rudnai, P.; Gurzau, E.; Koppova, K.; Botella-Estrada, R.; Soriano, V.; Juberias, P.; Saez, B.; Gilaberte, Y.; Fuentelsaz, V.; Corredera, C.; Grasa, M.; Hoiom, V.; Lindblom, A.; Bonenkamp, J.J.; Rossum, M.M. van; Aben, K.K.H.; Vries, E. de; Santinami, M.; Mauro, M.G. Di; Maurichi, A.; Wendt, J.; Hochleitner, P.; Pehamberger, H.; Gudmundsson, J.; Magnusdottir, D.N.; Gretarsdottir, S.; Holm, H.; Steinthorsdottir, V.; Frigge, M.L.; Blondal, T.; Saemundsdottir, J.; Bjarnason, H.; Kristjansson, K.; Bjornsdottir, G.; Okamoto, I.; Rivoltini, L.; Rodolfo, M.; Kiemeney, L.A.L.M.; Hansson, J.; Nagore, E.; Mayordomo, J.I.; Kumar, R.; Karagas, M.R.; Nelson, H.H.; Gulcher, J.R.; Rafnar, T.; Thorsteinsdottir, U.; Olafsson, J.H.; Kong, A.; Stefansson, K.

    2009-01-01

    In a follow-up to our previously reported genome-wide association study of cutaneous basal cell carcinoma (BCC), we describe here several new susceptibility variants. SNP rs11170164, encoding a G138E substitution in the keratin 5 (KRT5) gene, affects risk of BCC (OR = 1.35, P = 2.1 x 10(-9)). A vari

  6. Clear cell variant of syringoma as a rare case

    Directory of Open Access Journals (Sweden)

    Özben Yalçın

    2014-12-01

    Full Text Available Syringoma is a benign skin tumor derived from eccrine glands characterized by yellowish-pink color and firm papular lesions of the skin especially on the lower eyelid. Typical histopathological features of syringoma are dilated cystic eccrine sweat gland ducts. In this paper, we report a case of clear cell variant syringoma with neck and trunk lesions.

  7. Retracing Atypical Development: A Preserved Speech Variant of Rett Syndrome

    Science.gov (United States)

    Marschik, Peter B.; Einspieler, Christa; Oberle, Andreas; Laccone, Franco; Prechtl, Heinz F. R.

    2009-01-01

    The subject of the present study is the development of a girl with the preserved speech variant of Rett disorder. Our data are based on detailed retrospective and prospective video analyses. Despite achieving developmental milestones, movement quality was already abnormal during the girl's first half year of life. In addition, early hand…

  8. Observational consequences of the standard model Higgs inflation variants

    International Nuclear Information System (INIS)

    We consider the possibility to observationally differentiate the Standard Model (SM) Higgs driven inflation with non-minimal coupling to gravity from other variants of SM Higgs inflation based on the scalar field theories with non-canonical kinetic term such as Galileon-like kinetic term and kinetic term with non-minimal derivative coupling to the Einstein tensor. In order to ensure consistent results, we study the SM Higgs inflation variants by using the same method, computing the full dynamics of the background and perturbations of the Higgs field during inflation at quantum level. Assuming that all the SM Higgs inflation variants are consistent theories, we use the MCMC technique to derive constraints on the inflationary parameters and the Higgs boson mass from their fit to WMAP7+SN+BAO data set. We conclude that a combination of the SM Higgs mass measurement by the LHC and accurate determination by the PLANCK satellite of the spectral index of curvature perturbations and tensor-to-scalar ratio will enable to distinguish among these models. We also show that the consistency relations of the SM Higgs inflation variants are distinct enough to differentiate among them

  9. Protein variants in human cells: enumeration by protein indexing

    International Nuclear Information System (INIS)

    In any attempt to construct a catalog of the proteins of a given species, the genetic heterogeneity of natural plant and animal populations makes it necessary to consider variants of each protein. Thus in compiling a Human Protein Index using high resolution two-dimensional electrophoresis as a separating technique, protein variants differing from the wild type by charge or by polypeptide length will be recognized as different and must be accounted for. Results of several recent investigations have argued for average heterozygosities of approximately 1% for human cellular proteins examined by two-dimensional electrophoresis, while the results of classical biochemical genetics lead to higher results. The ability to observe genetic variation in large numbers of proteins can be valuable in several contexts. More than 2000 human genetic diseases have been identified, the vast majority of which have not as yet been associated with a defect in a particular protein. The present study of 63 human fibroblast cell lines was initiated in order to determine the level of genetic variation at many loci and to see whether known genetic diseases were associated with any obvious variant proteins that might be expressed in fibroblasts in culture. The main result is a group of ten new putative variants available in permanent cell lines

  10. Effects of Variant Rates and Noise on Epidemic Spreading

    Institute of Scientific and Technical Information of China (English)

    LI Wei; GAO Zong-Mao; GU Jiao

    2011-01-01

    We introduce variant rates, for both infection and recovery and noise into the susceptible-infected-removed (SIR)model for epidemic spreading. The changing rates are taken mainly due to the changing profiles of an epidemic during its evolution. However, the noise parameter which is taken from a given distribution, i.e. Gaussian can describe the fluctuations of the infection and recovery rates. The numerical simulations show that the SIR model with variant rates and noise and can improve the fitting with real SARS data in the near-stationary stage.%@@ We introduce variant rates,for both infection and recovery and noise into the susceptible-infected-removed(SIR)modelfor epidemic spreading.The changing rates are taken mainly due to the changing profiles of an epidemic during its evolution.However,the noise parameter which is taken from a given distribution,i.e.Gaussian can describe the fluctuations of the infection and recovery rates.The numerical simulations show that the SIR model with variant rates and noise and can improve the fitting with real SARS data in the near-stationary stage.

  11. STUDY ON AN SIS EPIDEMIC MODEL WITH TIME VARIANT DELAY

    Institute of Scientific and Technical Information of China (English)

    YUAN Sanling; MA Zhien

    2002-01-01

    In this paper, we study an SIS epidemic model with a time variant delay.By means of Liapunov functional, some sufficient conditions of global stability to endemic equilibrium and disease free equilibrium have been obtained. The influence of time delay on the stability of equilibria is displayed.

  12. STUDY ON AN SIS EPIDEMIC MODEL WITH TIME VARIANT DELAY

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    In this paper,we study an SIS epidemic model with a time variant delay.By means of Liapunov functional,some sufficient conditions of global stability to endemic equilibrium and disease free equilibrium have been obtained.The influence of time delay on the stability of equilibria is displayed.

  13. A PYY Q62P variant linked to human obesity

    Energy Technology Data Exchange (ETDEWEB)

    Ahituv, Nadav; Kavaslar, Nihan; Schackwitz, Wendy; Ustaszewska,Anna; Collier, John Michael; Hebert, Sybil; Doelle, Heather; Dent,Robert; Pennacchio, Len A.; McPherson, Ruth

    2005-06-27

    Members of the pancreatic polypeptide family and the irreceptors have been implicated in the control of food intake in rodents and humans. To investigate whether nucleotide changes in these candidate genes result in abnormal weight in humans, we sequenced the coding exons and splice sites of seven family members (NPY, PYY, PPY, NPY1R, NPY2R, NPY4R, and NPY5R) in a large cohort of extremely obese (n=379) and lean (n=378) individuals. In total we found eleven rare non-synonymous variants, four of which exhibited familial segregation, NPY1R L53P and PPY P63L with leanness and NPY2R D42G and PYY Q62P with obesity. Functional analysis of the obese variants revealed NPY2R D42G to have reduced cell surface expression, while previous cell culture based studies indicated variant PYY Q62P to have altered receptor binding selectivity and we show that it fails to reduce food intake through mouse peptide injection experiments. These results support that rare non-synonymous variants within these genes can alter susceptibility to human body mass index extremes.

  14. Reliability and Validation Study of the Online Instinctual Variant Questionnaire

    Science.gov (United States)

    Andre, Sherry

    2014-01-01

    Leaders often manage both chaos and diversity. We can improve our leadership effectiveness by better understanding our motives and behaviors, and those of our followers. A potential tool for leadership development is the Instinctual Variant Questionnaire (IVQ). Based on Enneagram theory (pronounced "ANY-a-gram"), this online instrument…

  15. Interaction of human butyrylcholinesterase variants with bambuterol and terbutaline.

    Science.gov (United States)

    Kovarik, Zrinka; Simeon-Rudolf, Vera

    2004-04-01

    Bambuterol, a dimethylcarbamate, carbamoylates butyrylcholinesterase (BChE; EC 3.1.1.8). The carbamoylated enzyme is not very stable and the final product of the two-step hydrolysis is a bronchodilator drug, terbutaline (1-(3,5-dihydroxyphenyl)-2-t-butylamino-ethanol sulphate). Both bambuterol and terbutaline inhibit BChE, but their affinities differ in human serum BChE variants (U, A, F, K and S) due to their positive charge. Bambuterol inhibition rate constants for the homozygous usual (UU), Kalow (KK), fluoride-resistant (FF) or atypical (AA) variant ranged from 4.4 to 0.085min (-1)microM(-1). Terbutaline showed competitive reversible inhibition for all BChE variants. The dissociation constants for UU, FF and AA homozygotes were 0.18, 0.31 and 3.3 mM, respectively. The inhibition rate or dissociation constants for heterozygotes were distributed between the respective constants for the corresponding homozygotes. A 50-fold difference in inhibition between the UU and AA enzyme might affect terbutaline release in humans. The affinity of all studied BChE variants for terbutaline was low, which suggests that terbutaline originating from bambuterol hydrolysis should not affect the hydrolysis of bambuterol by BChE. PMID:15449725

  16. Molecular Mechanisms of Disease Pathogenesis Differ in Krabbe Disease Variants.

    Science.gov (United States)

    Spratley, Samantha J; Hill, Chris H; Viuff, Agnete H; Edgar, James R; Skjødt, Karsten; Deane, Janet E

    2016-08-01

    Krabbe disease is a severe, fatal neurodegenerative disorder caused by defects in the lysosomal enzyme galactocerebrosidase (GALC). The correct targeting of GALC to the lysosome is essential for the degradation of glycosphingolipids including the primary lipid component of myelin. Over 100 different mutations have been identified in GALC that cause Krabbe disease but the mechanisms by which they cause disease remain unclear. We have generated monoclonal antibodies against full-length human GALC and used these to monitor the trafficking and processing of GALC variants in cell-based assays and by immunofluorescence microscopy. Striking differences in the secretion, processing and endosomal targeting of GALC variants allows the classification of these into distinct categories. A subset of GALC variants are not secreted by cells, not proteolytically processed, and remain trapped in the ER; these are likely to cause disease due to protein misfolding and should be targeted for pharmacological chaperone therapies. Other GALC variants can be correctly secreted by cells and cause disease due to catalytic defects in the enzyme active site, inappropriate post-translational modification or a potential inability to bind essential cofactors. The classification of disease pathogenesis presented here provides a molecular framework for appropriate targeting of future Krabbe disease therapies. PMID:27126738

  17. Maternal inheritance and mitochondrial DNA variants in familial Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Pfeiffer Ronald F

    2010-04-01

    Full Text Available Abstract Background Mitochondrial function is impaired in Parkinson's disease (PD and may contribute to the pathogenesis of PD, but the causes of mitochondrial impairment in PD are unknown. Mitochondrial dysfunction is recapitulated in cell lines expressing mitochondrial DNA (mtDNA from PD patients, implicating mtDNA variants or mutations, though the role of mtDNA variants or mutations in PD risk remains unclear. We investigated the potential contribution of mtDNA variants or mutations to the risk of PD. Methods We examined the possibility of a maternal inheritance bias as well as the association between mitochondrial haplogroups and maternal inheritance and disease risk in a case-control study of 168 multiplex PD families in which the proband and one parent were diagnosed with PD. 2-tailed Fisher Exact Tests and McNemar's tests were used to compare allele frequencies, and a t-test to compare ages of onset. Results The frequency of affected mothers of the proband with PD (83/167, 49.4% was not significantly different from the frequency of affected females of the proband generation (115/259, 44.4% (Odds Ratio 1.22; 95%CI 0.83 - 1.81. After correcting for multiple tests, there were no significant differences in the frequencies of mitochondrial haplogroups or of the 10398G complex I gene polymorphism in PD patients compared to controls, and no significant associations with age of onset of PD. Mitochondrial haplogroup and 10398G polymorphism frequencies were similar in probands having an affected father as compared to probands having an affected mother. Conclusions These data fail to demonstrate a bias towards maternal inheritance in familial PD. Consistent with this, we find no association of common haplogroup-defining mtDNA variants or for the 10398G variant with the risk of PD. However, these data do not exclude a role for mtDNA variants in other populations, and it remains possible that other inherited mitochondrial DNA variants, or somatic m

  18. Estrogen withdrawal, increased breast cancer risk and the KRAS-variant

    Science.gov (United States)

    McVeigh, Terri P; Jung, Song-Yi; Kerin, Michael J; Salzman, David W; Nallur, Sunitha; Nemec, Antonio A; Dookwah, Michelle; Sadofsky, Jackie; Paranjape, Trupti; Kelly, Olivia; Chan, Elcie; Miller, Nicola; Sweeney, Karl J; Zelterman, Daniel; Sweasy, Joann; Pilarski, Robert; Telesca, Donatello; Slack, Frank J; Weidhaas, Joanne B

    2015-01-01

    The KRAS-variant is a biologically functional, microRNA binding site variant, which predicts increased cancer risk especially for women. Because external exposures, such as chemotherapy, differentially impact the effect of this mutation, we evaluated the association of estrogen exposures, breast cancer (BC) risk and tumor biology in women with the KRAS-variant. Women with BC (n = 1712), the subset with the KRAS-variant (n = 286) and KRAS-variant unaffected controls (n = 80) were evaluated, and hormonal exposures, KRAS-variant status, and pathology were compared. The impact of estrogen withdrawal on transformation of isogenic normal breast cell lines with or without the KRAS-variant was studied. Finally, the association and presentation characteristics of the KRAS-variant and multiple primary breast cancer (MPBC) were evaluated. KRAS-variant BC patients were more likely to have ovarian removal pre-BC diagnosis than non-variant BC patients (p = 0.033). In addition, KRAS-variant BC patients also appeared to have a lower estrogen state than KRAS-variant unaffected controls, with a lower BMI (P < 0.001). Finally, hormone replacement therapy (HRT) discontinuation in KRAS-variant patients was associated with a diagnosis of triple negative BC (P < 0.001). Biologically confirming our clinical findings, acute estrogen withdrawal led to oncogenic transformation in KRAS-variant positive isogenic cell lines. Finally, KRAS-variant BC patients had greater than an 11-fold increased risk of presenting with MPBC compared to non-variant patients (45.39% vs 6.78%, OR 11.44 [3.42–37.87], P < 0.001). Thus, estrogen withdrawal and a low estrogen state appear to increase BC risk and to predict aggressive tumor biology in women with the KRAS-variant, who are also significantly more likely to present with multiple primary breast cancer. PMID:25961464

  19. Delineation of concentration ranges and longitudinal changes of human plasma protein variants.

    Directory of Open Access Journals (Sweden)

    Olgica Trenchevska

    Full Text Available Human protein diversity arises as a result of alternative splicing, single nucleotide polymorphisms (SNPs and posttranslational modifications. Because of these processes, each protein can exists as multiple variants in vivo. Tailored strategies are needed to study these protein variants and understand their role in health and disease. In this work we utilized quantitative mass spectrometric immunoassays to determine the protein variants concentration of beta-2-microglobulin, cystatin C, retinol binding protein, and transthyretin, in a population of 500 healthy individuals. Additionally, we determined the longitudinal concentration changes for the protein variants from four individuals over a 6 month period. Along with the native forms of the four proteins, 13 posttranslationally modified variants and 7 SNP-derived variants were detected and their concentration determined. Correlations of the variants concentration with geographical origin, gender, and age of the individuals were also examined. This work represents an important step toward building a catalog of protein variants concentrations and examining their longitudinal changes.

  20. VPA: an R tool for analyzing sequencing variants with user-specified frequency pattern

    Directory of Open Access Journals (Sweden)

    Hu Qiang

    2012-01-01

    Full Text Available Abstract Background The massive amounts of genetic variant generated by the next generation sequencing systems demand the development of effective computational tools for variant prioritization. Findings VPA (Variant Pattern Analyzer is an R tool for prioritizing variants with specified frequency pattern from multiple study subjects in next-generation sequencing study. The tool starts from individual files of variant and sequence calls and extract variants with user-specified frequency pattern across the study subjects of interest. Several position level quality criteria can be incorporated into the variant extraction. It can be used in studies with matched pair design as well as studies with multiple groups of subjects. Conclusions VPA can be used as an automatic pipeline to prioritize variants for further functional exploration and hypothesis generation. The package is implemented in the R language and is freely available from http://vpa.r-forge.r-project.org.

  1. Dominance of variant A in Human Herpesvirus 6 viraemia after renal transplantation

    OpenAIRE

    Csoma Eszter; Mészáros Beáta (1985-) (molekuláris biológus, mikrobiológus); Gáll Tamás (1982-) (molekuláris biológus, mikrobiológus); Asztalos László; Kónya József (1964-) (szakorvos, klinikai mikrobiológus); Gergely Lajos (1965-) (belgyógyász, haematológus)

    2011-01-01

    Abstract Background Human herpesvirus 6 (HHV-6), mostly variant B reactivation in renal transplant patients has been published by other authors, but the pathogenetic role of HHV-6 variant A has not been clarified. Our aims were to examine the prevalence of HHV-6, to determine the variants, and to investigate the interaction between HHV-6 viraemia, human cytomegalovirus (HCMV) infection and clinical symptoms. Methods Variant-specific HHV-6 nested PCR and quantitative real-time PCR were used to...

  2. Variant-Specific Tropism of Human Herpesvirus 6 in Human Astrocytes

    OpenAIRE

    Donati, Donatella; Martinelli, Elena; Cassiani-Ingoni, Riccardo; Ahlqvist, Jenny; Hou, Jean; Major, Eugene O.; Jacobson, Steve

    2005-01-01

    Though first described as a lymphotropic virus, human herpesvirus 6 (HHV-6) is highly neuropathogenic. Two viral variants are known: HHV-6A and HHV-6B. Both variants can infect glial cells and have been differentially associated with central nervous system diseases, suggesting an HHV-6 variant-specific tropism for glial cell subtypes. We have performed infections with both viral variants in human progenitor-derived astrocytes (HPDA) and monitored infected cell cultures for cytopathic effect (...

  3. rVarBase: an updated database for regulatory features of human variants

    OpenAIRE

    Guo, Liyuan; Du, Yang; Qu, Susu; Wang, Jing

    2015-01-01

    We present here the rVarBase database (http://rv.psych.ac.cn), an updated version of the rSNPBase database, to provide reliable and detailed regulatory annotations for known and novel human variants. This update expands the database to include additional types of human variants, such as copy number variations (CNVs) and novel variants, and include additional types of regulatory features. Now rVarBase annotates variants in three dimensions: chromatin states of the surrounding regions, overlapp...

  4. Bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease: how safe is eating beef?

    Science.gov (United States)

    Roma, Andres A; Prayson, Richard A

    2005-03-01

    Cases of bovine spongiform encephalopathy (BSE, mad cow disease) have been found in North American cattle. Its human counterpart, called variant Creutzfeldt-Jakob disease (variant CJD), is rare but seems to be linked to eating diseased beef. Many questions remain about these diseases, such as why young people seem at greater risk of variant CJD. Also, are some people more genetically at risk for acquiring variant CJD than others? PMID:15825799

  5. Family- and population-based designs identify different rare causal variants

    OpenAIRE

    Zhang Xue; He Hua; Ding Lili; Baye Tesfaye M; Kurowski Brad G; Martin Lisa J

    2011-01-01

    Abstract Both family- and population-based samples are used to identify genetic variants associated with phenotypes. Each strategy has demonstrated advantages, but their ability to identify rare variants and genes containing rare variants is unclear. To compare these two study designs in the identification of rare causal variants, we applied various methods to the population- and family-based data simulated by the Genetic Analysis Workshop 17 with knowledge of the simulated model. Our results...

  6. Effect of population stratification analysis on false-positive rates for common and rare variants

    OpenAIRE

    He Hua; Zhang Xue; Ding Lili; Baye Tesfaye M; Kurowski Brad G; Martin Lisa J

    2011-01-01

    Abstract Principal components analysis (PCA) has been successfully used to correct for population stratification in genome-wide association studies of common variants. However, rare variants also have a role in common disease etiology. Whether PCA successfully controls population stratification for rare variants has not been addressed. Thus we evaluate the effect of population stratification analysis on false-positive rates for common and rare variants at the single-nucleotide polymorphism (S...

  7. Segregation of naturally occurring mitochondrial DNA variants in a mini-pig model

    Science.gov (United States)

    Within cells and tissues, the maternally inherited mitochondrial genome (mtDNA) is present in multimeric form and can harbour naturally occurring variants. Whilst high variant load can cause mitochondrial disease, naturally occurring mtDNA variants likely persist at low levels across generations of ...

  8. Choosing variants of pragmatic argumentation in anticipation of countermoves in health brochures

    NARCIS (Netherlands)

    L. van Poppel

    2011-01-01

    In this paper, I will determine the strategic function of the use of four variants of pragmatic argumentation in the context of advisory health brochures. I argue that each variant functions as a strategic manoeuvre that deals with potential countermoves: with variant I and II writers can address an

  9. A framework for the definition of variants of high-level Petri nets

    DEFF Research Database (Denmark)

    Kindler, Ekkart; Petrucci, Laure

    2009-01-01

    There exist many different variants of high-level Petri nets. Many differences between these variants, however, do not concern the features of the particular versions of Petri nets, but they concern the data types that can be defined and used in the different variants of high-level nets. One famous...

  10. Fast single-pass alignment and variant calling using sequencing data

    Science.gov (United States)

    Sequencing research requires efficient computation. Few programs use already known information about DNA variants when aligning sequence data to the reference map. New program findmap.f90 reads the previous variant list before aligning sequence, calling variant alleles, and summing the allele counts...

  11. A protein-truncating R179X variant in RNF186 confers protection against ulcerative colitis

    NARCIS (Netherlands)

    Rivas, Manuel A; Graham, Daniel; Sulem, Patrick; Stevens, Christine; Desch, A Nicole; Goyette, Philippe; Gudbjartsson, Daniel; Jonsdottir, Ingileif; Thorsteinsdottir, Unnur; Degenhardt, Frauke; Mucha, Sören; Kurki, Mitja I; Li, Dalin; D'Amato, Mauro; Annese, Vito; Vermeire, Severine; Weersma, Rinse K; Halfvarson, Jonas; Paavola-Sakki, Paulina; Lappalainen, Maarit; Lek, Monkol; Cummings, Beryl; Tukiainen, Taru; Haritunians, Talin; Halme, Leena; Koskinen, Lotta L E; Ananthakrishnan, Ashwin N; Luo, Yang; Heap, Graham A; Visschedijk, Marijn C; MacArthur, Daniel G; Neale, Benjamin M; Ahmad, Tariq; Anderson, Carl A; Brant, Steven R; Duerr, Richard H; Silverberg, Mark S; Cho, Judy H; Palotie, Aarno; Saavalainen, Päivi; Kontula, Kimmo; Färkkilä, Martti; McGovern, Dermot P B; Franke, Andre; Stefansson, Kari; Rioux, John D; Xavier, Ramnik J; Daly, Mark J; Barrett, J; de Lane, K; Edwards, C; Hart, A; Hawkey, C; Jostins, L; Kennedy, N; Lamb, C; Lee, J; Lees, C; Mansfield, J; Mathew, C; Mowatt, C; Newman, B; Nimmo, E; Parkes, M; Pollard, M; Prescott, N; Randall, J; Rice, D; Satsangi, J; Simmons, A; Tremelling, M; Uhlig, H; Wilson, D; Abraham, C; Achkar, J P; Bitton, A; Boucher, G; Croitoru, K; Fleshner, P; Glas, J; Kugathasan, S; Limbergen, J V; Milgrom, R; Proctor, D; Regueiro, M; Schumm, P L; Sharma, Y; Stempak, J M; Targan, S R; Wang, M H

    2016-01-01

    Protein-truncating variants protective against human disease provide in vivo validation of therapeutic targets. Here we used targeted sequencing to conduct a search for protein-truncating variants conferring protection against inflammatory bowel disease exploiting knowledge of common variants associ

  12. Schizophrenia genetic variants are not associated with intelligence

    DEFF Research Database (Denmark)

    Van Scheltinga, A.F.T.; Bakker, S.C.; Van Haren, N.E.M.;

    2013-01-01

    BACKGROUND: Schizophrenia is associated with lower pre-morbid intelligence (IQ) in addition to (pre-morbid) cognitive decline. Both schizophrenia and IQ are highly heritable traits. Therefore, we hypothesized that genetic variants associated with schizophrenia, including copy number variants (CNVs......) and a polygenic schizophrenia (risk) score (PSS), may influence intelligence. Method IQ was estimated with the Wechsler Adult Intelligence Scale (WAIS). CNVs were determined from single nucleotide polymorphism (SNP) data using the QuantiSNP and PennCNV algorithms. For the PSS, odds ratios for genome-wide SNP data...... were calculated in a sample collected by the Psychiatric Genome-Wide Association Study (GWAS) Consortium (8690 schizophrenia patients and 11 831 controls). These were used to calculate individual PSSs in our independent sample of 350 schizophrenia patients and 322 healthy controls. RESULTS: Although...

  13. On Bijective Variants of the Burrows-Wheeler Transform

    CERN Document Server

    Kufleitner, Manfred

    2009-01-01

    The sort transform (ST) is a modification of the Burrows-Wheeler transform (BWT). Both transformations map an arbitrary word of length n to a pair consisting of a word of length n and an index between 1 and n. The BWT sorts all rotation conjugates of the input word, whereas the ST of order k only uses the first k letters for sorting all such conjugates. If two conjugates start with the same prefix of length k, then the indices of the rotations are used for tie-breaking. Both transforms output the sequence of the last letters of the sorted list and the index of the input within the sorted list. In this paper, we discuss a bijective variant of the BWT (due to Scott), proving its correctness and relations to other results due to Gessel and Reutenauer (1993) and Crochemore, Desarmenien, and Perrin (2005). Further, we present a novel bijective variant of the ST.

  14. Multidetector CT angiography of renal vasculature: normal anatomy and variants

    Energy Technology Data Exchange (ETDEWEB)

    Tuerkvatan, Aysel; Oezdemir, Mustafa; Cumhur, Turhan; Oelcer, Tuelay [Tuerkiye Yueksek ihtisas Hospital, Department of Radiology, Sihhiye, Ankara (Turkey)

    2009-01-15

    Knowledge of the variations in renal vascular anatomy is important before laparoscopic donor or partial nephrectomy and vascular reconstruction for renal artery stenosis or abdominal aortic aneurysm. Recently, multidetector computed tomographic (MDCT) angiography has become a principal imaging investigation for assessment of the renal vasculature and has challenged the role of conventional angiography. It is an excellent imaging technique because it is a fast and non-invasive tool that provides highly accurate and detailed evaluation of normal renal vascular anatomy and variants. The number, size and course of the renal arteries and veins are easily identified by MDCT angiography. The purpose of this pictorial essay is to illustrate MDCT angiographic appearance of normal anatomy and common variants of the renal vasculature. (orig.)

  15. Dentinogenic Ghost Cell Tumor of the Peripheral Variant Mimicking Epulis

    Directory of Open Access Journals (Sweden)

    Uddipan Kumar

    2010-01-01

    Full Text Available Dentinogenic ghost cell tumor (DGCT is an uncommon locally invasive odontogenic tumor regarded by many as a variant of calcifying odontogenic cyst. The peripheral variant of this clinical rarity appears as a well-circumscribed mass mimicking a nonspecific gingival enlargement. Microscopic appearance of odontogenic epithelium admixed with focal areas of dentinoid formation and sheets of ghost cells giving the definitive diagnosis of dentinogenic ghost cell tumor imply that microscopic examination is compulsory for any gingival mass. Van Gieson histochemical stain further confirmed the nature of dentinoid-like material. A complete workup of a case of peripheral dentinogenic ghost cell tumor is presented in this paper and the current concept as well as the appraisal of literature is presented.

  16. Dandy-Walker variant associated with bipolar affective disorder

    Directory of Open Access Journals (Sweden)

    Anand Lingeswaran

    2009-01-01

    Full Text Available The Dandy-Walker malformation is a congenital brain malformation, typically involving the fourth ventricle and the cerebellum. To date, the Dandy-Walker syndrome has not been described in association with bipolar disorder type I mania, and therefore we briefly report the case of a Dandy-Walker variant associated with acute mania. A 10-year-old boy was brought by his mother to the outpatient clinic of the Department of Psychiatry of a tertiary care hospital, with symptoms of mania. The MRI brain of the patient showed a posterior fossa cystic lesion, a giant cisterna magna communicating with the fourth ventricle and mild hypoplasia of the cerebellar vermis, with the rest of the structures being normal and no signs of hydrocephalus. These findings showed that the patient had a Dandy-Walker variant. He responded partially to valproate and olanzepine, which controlled the acute manic symptoms in the ward.

  17. A Survey: variants of TCP in Ad-hoc networks

    Directory of Open Access Journals (Sweden)

    Komal Zaman

    2013-11-01

    Full Text Available MANET (Mobile Ad-hoc network forms a temporary network of wireless mobile nodes without any infrastructure where all nodes are allowed to move freely, configure themselves and interconnect with its neighbors to perform peer to peer communication and transmission. TCP (Transmission Control Protocol offers reliable, oriented connection and mechanism of end to end delivery. This article provides the review and comparison of existing variants of TCP for instance: The TCP Tahoe, The TCP Reno, The TCP New Reno, The Lite, The Sack, The TCP Vegas, Westwood and The TCP Fack. TCP’s performance depends on the type of its variants due to missing of congestion control or improper activation procedures such as Slow Start, Fast Retransmission, and Congestion Avoidance, Retransmission, Fast Recovery, Selective Acknowledgement mechanism and Congestion Control. This analysis is essential to be aware about a better TCP implementation for a specific scenario and then nominated a suitable one.

  18. Short-Coupled Variant of Torsade de Pointes

    Institute of Scientific and Technical Information of China (English)

    阮燕菲; 王琳

    2001-01-01

    Presented in this paper were 3 cases of a special kind of polymorphic ventricular tachycardia (VT). The clinical manifestation was recurrent syncope without organic heart disease. The electrocardiogram (ECG) was characterized by normal QT intervals with short-coupled variant of torsade de pointes. The efficacy of treatment with class Ⅰ,Ⅱ,Ⅲ antiarrhythmic drugs was not apparent but verapamil had an excellent therapeutic effect for it. This kind of VT had a high incidence of sudden death, so it was very important for physicians to identify and treat it promptly with long-term verapamil. The mechanism of short-coupled variant of torsade de pointes was unclear. It probably had some relationship with triggered activity or imbalance of autonomic nervous system.

  19. New common variants affecting susceptibility to basal cell carcinoma

    OpenAIRE

    Stacey, Simon N.; Sulem, Patrick; Masson, Gisli; Gudjonsson, Sigurjon A.; Thorleifsson, Gudmar; Jakobsdottir, Margret; Sigurdsson, Asgeir; Daniel F Gudbjartsson; Sigurgeirsson, Bardur; Benediktsdottir, Kristrun R.; Thorisdottir, Kristin; Ragnarsson, Rafn; Scherer, Dominique; Hemminki, Kari; Rudnai, Peter

    2009-01-01

    In a follow-up to our previously reported genome-wide association study of cutaneous basal cell carcinoma (BCC)1, we describe here several new susceptibility variants. SNP rs11170164, encoding a G138E substitution in the keratin 5 (KRT5) gene, affects risk of BCC (OR = 1.35, P = 2.1 × 10−9). A variant at 9p21 near CDKN2A and CDKN2B also confers susceptibility to BCC (rs2151280[C]; OR = 1.19, P = 6.9 × 10−9), as does rs157935[T] at 7q32 near the imprinted gene KLF14 (OR = 1.23, P = 5.7 × 10−10...

  20. A new hypercube variant: Fractal Cubic Network Graph

    Directory of Open Access Journals (Sweden)

    Ali Karci

    2015-03-01

    Full Text Available Hypercube is a popular and more attractive interconnection networks. The attractive properties of hypercube caused the derivation of more variants of hypercube. In this paper, we have proposed two variants of hypercube which was called as “Fractal Cubic Network Graphs”, and we have investigated the Hamiltonian-like properties of Fractal Cubic Network Graphs FCNGr(n. Firstly, Fractal Cubic Network Graphs FCNGr(n are defined by a fractal structure. Further, we show the construction and characteristics analyses of FCNGr(n where r=1 or r=2. Therefore, FCNGr(n is a Hamiltonian graph which is obtained by using Gray Code for r=2 and FCNG1(n is not a Hamiltonian Graph. Furthermore, we have obtained a recursive algorithm which is used to label the nodes of FCNG2(n. Finally, we get routing algorithms on FCNG2(n by utilizing routing algorithms on the hypercubes.