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clearance prediction of hiv protease inhibitors in man: role of hepatic uptake.the aim of this work was to explore the contribution of the organic anion transporting polypeptide-1b (oatp1b) drug transporters in the hepatic clearance (cl) of all marketed hiv protease inhibitors (pi) in humans. hiv pi uptake rates in oatp1b1/1b3-transfected chinese hamster ovary cells were converted to uptake cl values in human hepatocytes via a relative activity factor, which was determined by comparing uptake of known substrates between oatp1b1/3-transfected cells and human hepatocytes. me ...201626213315
elucidating the role of copper in cho cell energy metabolism using (13)c metabolic flux analysis.(13)c-metabolic flux analysis was used to understand copper deficiency-related restructuring of energy metabolism, which leads to excessive lactate production in recombinant protein-producing cho cells. stationary-phase labeling experiments with u-(13)c glucose were conducted on cho cells grown under high and limiting copper in 3 l fed-batch bioreactors. the resultant labeling patterns of soluble metabolites were measured by gc-ms and used to estimate metabolic fluxes in the central carbon metab ...201626097228
imac capture of recombinant protein from unclarified mammalian cell feed streams.fusion-tag affinity chromatography is a key technique in recombinant protein purification. current methods for protein recovery from mammalian cells are hampered by the need for feed stream clarification. we have developed a method for direct capture using immobilized metal affinity chromatography (imac) of hexahistidine (his6) tagged proteins from unclarified mammalian cell feed streams. the process employs radial flow chromatography with 300-500 μm diameter agarose resin beads that allow free ...201626174988
glycoprofiling effects of media additives on igg produced by cho cells in fed-batch bioreactors.therapeutic monoclonal antibodies (mabs) are mainly produced by heterologous expression in chinese hamster ovary (cho) cells. the glycosylation profile of the mabs has major impact on the efficacy and safety of the drug and is therefore an important parameter to control during production. 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 bio ...201626222761
coupling recombinase-mediated cassette exchange with somatic hypermutation for antibody affinity maturation in cho cells.heterologous expression of activation-induced cytidine deaminase (aid) can induce somatic hypermutation (shm) for genes of interest in various cells, and several research groups (including ours) have successfully improved antibody affinity in mammalian or chicken cells using aid-induced shm. these affinity maturation systems are time-consuming and inefficient. in this study, we developed an antibody affinity maturation platform in chinese hamster ovary (cho) cells by coupling recombinase-mediate ...201626235363
enhanced plasmid dna utilization in transiently transfected cho-dg44 cells in the presence of polar solvents.although the protein yields from transient gene expression (tge) with chinese hamster ovary (cho) cells have recently improved, the amount of plasmid dna (pdna) needed for transfection remains relatively high. we describe a strategy to reduce the pdna amount by transfecting cho-dg44 cells with 0.06 μg pdna/10(6) cells (10% of the optimal amount) in the presence of nonspecific (filler) dna and various polar solvents including dimethylsufoxide, dimethyl formamide, acetonitrile, dimethyl acetamide ...201626260195
the present state of the art in expression, production and characterization of monoclonal antibodies.monoclonal antibodies (mab's) have become one the most powerful therapeutic and diagnostic tools in modern medicine. some estimates target the worldwide market of mab's on the order of $125 billion in the next four years. recent advances in molecular biology, immunology, and development of robust production platforms will drive the development of more mab's suitable to treat an ever increasing number of disease states. this circumstance combined with the fact that many of the original antibody t ...201626299798
c-terminal region of gadd34 regulates eif2α dephosphorylation and cell proliferation in cho-k1 cells.gadd34 is a member of a growth arrest and dna damage (gadd)-inducible gene family. here, we established a novel chinese hamster ovary (cho)-k1-derived cell line, cho-k1-g34m, which carries a nonsense mutation (termed the q525x mutation) in the gadd34 gene. the q525x mutant protein lacks the c-terminal 66 amino acids required for gadd34 to bind to and activate protein phosphatase 1 (pp1). we investigated the effects of gadd34 with or without the q525x mutation on the phosphorylation status of pp1 ...201626318739
monitoring utilizations of amino acids and vitamins in culture media and chinese hamster ovary cells by liquid chromatography tandem mass spectrometry.monitoring amino acids and vitamins is important for understanding human health, food nutrition and the culture of mammalian cells used to produce therapeutic proteins in biotechnology. a method including ion pairing reversed-phase liquid chromatography with tandem mass spectrometry was developed and optimized to quantify 21 amino acids and 9 water-soluble vitamins in chinese hamster ovary (cho) cells and culture media. by optimizing the chromatographic separation, scan time, monitoring time win ...201626355770
combining mechanistic and data-driven approaches to gain process knowledge on the control of the metabolic shift to lactate uptake in a fed-batch cho process.a growing body of knowledge is available on the cellular regulation of overflow metabolism in mammalian hosts of recombinant protein production. however, to develop strategies to control the regulation of overflow metabolism in cell culture processes, the effect of process parameters on metabolism has to be well understood. in this study, we investigated the effect of ph and temperature shift timing on lactate metabolism in a fed-batch chinese hamster ovary (cho) process by using a design of exp ...201626439213
evaluation of in vivo cytogenetic toxicity of europium hydroxide nanorods (ehns) in male and female swiss albino mice.our group already demonstrated that europium hydroxide nanorods (ehns) show none or mild toxicity in c57bl/6 mice even at high dose and exhibited excellent pro-angiogenic activity towards in vitro and in vivo models. in the present study, we evaluated the in vivo cytogenetic toxicity of intraperitoneally administered ehns (12.5-250 mg/kg/b.w.) in male and female swiss albino mice by analyzing chromosomal aberrations (cas), mitotic index (mi), micronucleus (mn) from bone marrow and peripheral blo ...201626444710
the influence of bisphenol a on mammalian cell cultivation.bisphenol a (bpa) plays a substantial role in industry, as it is used for polycarbonate (pc) plastics and epoxy resins which are required for various plastic consumer products. however, bpa is known to be an endocrine disruptor, and its influence on humans, animals, and various cell lines was addressed in diverse studies. as the burden of bpa can be increased by using disposable plastic articles and single-use technologies for cultivation, it is essential to examine the consequences of bpa prese ...201626381666
arginine as an eluent overcomes the hindrance of monoclonal antibody quantification by dextran sulfate in protein a affinity chromatography.analytical chromatography using protein a affinity columns was employed for the fast and simple quantitative analysis of monoclonal antibodies (mab) from suspension cultures of recombinant chinese hamster ovary (rcho) cells. reliable results could not be obtained from analysis of rcho cell culture supernatants containing dextran sulfate using elution buffers such as phosphate, glycine, or mgcl2 . these problems increased as the number of analysis and the concentration of dextran sulfate in sampl ...201626363185
cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of glanzmann thrombasthenia.several patients have been reported to have variant dominant forms of glanzmann thrombasthenia, associated with macrothrombocytopenia and caused by gain-of-function mutations of itgb3 or itga2b leading to reduced surface expression and constitutive activation of integrin αiibβ3. the mechanisms leading to a bleeding phenotype of these patients have never been addressed. the aim of this study was to unravel the mechanism by which itgb3 mutations causing activation of αiibβ3 lead to platelet dysfun ...201626452979
actn1 rod domain mutation associated with congenital macrothrombocytopenia.mutations in actn1, the gene encoding the actin-crosslinking protein α-actinin-1, cause autosomal dominant macrothrombocytopenia. α-actinin-1 exists as antiparallel dimers, composed of an n-terminal actin-binding domain (abd), four spectrin-like repeats (slrs), which form the spacer rod, and a c-terminal calmodulin-like (cam) domain. all of the previously reported actn1 mutations associated with macrothrombocytopenia reside within the abd and the cam domain and not within the slr domain. in this ...201626453073
temperature-sensitive mir-483 is a conserved regulator of recombinant protein and viral vector production in mammalian cells.cell engineering and bioprocess optimizations such as low temperature cultivation represent powerful tools to improve cellular performance and product yields of mammalian production cells. besides monoclonal antibodies (mabs), novel biotherapeutic formats such as viral vectors will gain increasing importance. here, we demonstrate that similar to chinese hamster ovary (cho) cells, product yields of recombinant adeno-associated virus (raav) producing hela cells can be markedly increased by low tem ...201626461143
g-protein inhibition profile of the reported gq/11 inhibitor ubo-qic.ubo-qic (fr900359) is the only currently available gq/11 protein inhibitor. however, its characterization has not been published, and we thus set out to do this. gi, gs and gq protein-mediated responses were assessed utilizing endogenous or heterologously expressed receptors in chinese hamster ovary cells. ubo-qic, at 1 μm, was an effective inhibitor of the gq-mediated responses, but was inactive at gi- and gs-mediated responses. gq/11 and g16 responses were additionally compared in hel92.1.7 ce ...201626614908
expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in cho cells and its use for insulin analogue modification.the availability of catalytically active peptidylglycine α-amidating monooxygenase (pam) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. hypoglycemic activity is one of the most important features of insulin derivatives. insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. the aim of the study was ...201626614892
high-yield production of a human monoclonal igg by rhizosecretion in hydroponic tobacco cultures.rhizosecretion of recombinant pharmaceuticals from in vitro hydroponic transgenic plant cultures is a simple, low cost, reproducible and controllable production method. here, we demonstrate the application and adaptation of this manufacturing platform to a human antivitronectin igg1 monoclonal antibody (mab) called m12. the rationale for specific growth medium additives was established by phenotypic analysis of root structure and by lc-esi-ms/ms profiling of the total protein content profile of ...201626038982
the dependence of efficiency of transmembrane molecular transfer using electroporation on medium viscosity.in the present study, we aimed to evaluate the efficiency of drug and gene electrotransfer into cells in vitro depending on medium viscosity.201625761762
stirring rate regulates the proliferation and metabolism of microencapsulated recombinant cho cells.stirred tank bioreactors are the most widely used method for the large-scale culture of mammalian cells. however, the scale of stirred tank bioreactors is limited by insufficient oxygen/nutrient mixing and the accumulation of waste products in high cell density cultures. the most effective method to solve these problems is to increase the stirring rate; this usually leads to increased cell proliferation, but can decrease the utilization of nutrients for recombinant protein synthesis. to investig ...201625524589
clones of chinese hamster cells cultivated in vitro not permanently resistant to azaguanine. 20164843976
effects of isoleucine deficiency on nucleic acid and protein metabolism in cultured chinese hamster cells. continued ribonucleic acid and protein synthesis in the absence of deoxyribonucleic acid synthesis. 20165061874
studies on induced aberrations in diplochromosomes of chinese hamster cells. 20164796813
[anatomical studies on the urinary tract and genitalia of the chinese dwarf hamster (cricetulus griseus)]. 20165036384
islet and beta cell volumes in offspring of severely diabetic (ketotic) chinese hamsters. 20164914657
temporal variation of adenine ribonucleotides during the cell cycle of chinese hamster fibroblasts in culture. 20164855877
chromosomal aberrations induced by caffeine in endoreduplicated chinese hamster cells. 20164822595
chinese hamster ovary (cho) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression.n-glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (sa). recombinant glycoproteins produced in chinese hamster overy (cho) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (st6gal1) expression. the chinese hamster st6gal1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin g (igg)-producing cho cell lines. the stably transfected cell lines were enriched for st6gal1 overexpression using fitc-sambu ...201725641927
understanding the cholesterol metabolism-perturbing effects of docosahexaenoic acid by gas chromatography-mass spectrometry targeted metabonomic profiling.over the past few decades, docosahexaenoic acid (dha) has gained special attention for management of cholesterol-associated metabolic disorders and neurodegenerative diseases such as alzheimer's disease (ad) owing to its neuroprotective, anti-inflammatory and hypolipidemic properties. several epidemiological studies have reported the effect of dha in reducing the risk of developing ad by lowering cholesterol. hypercholesterolemia is a pro-amyloidogenic factor influencing the enzymatic processing ...201726428672
reciprocal translocation observed in end-of-production cells of a commercial cho-based process.during the validation of an additional working cell bank derived from a validated master cell bank to support the commercial production continuum of a recombinant protein, we observed an unexpected chromosomal location of the gene of interest in some end-of-production cells. this event-identified by fluorescence in situ hybridization and multicolour chromosome painting as a reciprocal translocation involving a chromosome region containing the gene of interest with its integral coding and flankin ...201726242789
restoration of mitochondrial gene expression using a cloned human gene in chinese hamster lung cell mutant.gal(-)32 is a chinese hamster lung cell nuclear mutant that is unable to grow in galactose due to a defect in mitochondrial protein synthesis. since the product of the gal(-)32 gene was unknown, it was imperative to use phenotypic complementation to clone a human gene that corrected the gal(-)32 mutation.201726052559
xgfr*, a novel affinity-matured bispecific antibody targeting igf-1r and egfr with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer.the epidermal growth factor receptor (egfr) and the insulin-like growth factor-1 receptor (igf-1r) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of igf-1r and egfr signaling in cancer therapy. we describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-igf-1r/egfr antibody xgfr*. xgfr* is based on the bispecific igg antibody xgfr, which enabled heterodimerization of an igf-1r binding scfab heavy chain w ...201726984378
increased in vivo effector function of human igg4 isotype antibodies through afucosylation.for some antibodies intended for use as human therapeutics, reduced effector function is desired to avoid toxicities that might be associated with depletion of target cells. since effector function(s), including antibody-dependent cell-mediated cytotoxicity (adcc), require the fc portion to be glycosylated, reduced adcc activity antibodies can be obtained through aglycosylation of the human igg1 isotype. an alternative is to switch to an igg4 isotype in which the glycosylated antibody is known t ...201727216702
polymorphisms in dna repair gene and susceptibility to glioma: a systematic review and meta-analysis based on 33 studies with 15 snps in 9 genes.at present, many publications have evaluated the correlation between the dna repair gene polymorphisms and glioma susceptibility. however, the results remain inconclusive. the aim of this research is to exhaustively assess the association of genetic polymorphisms in dna repair genes with glioma risk in human. meta-analysis method was conducted, and 33 studies with 15 snps in 9 genes were included (12553 glioma cases and 17178 controls). correlation strength was evaluated by odds ratio with a 95  ...201727055523
use of in vitro assays to assess the potential cytotoxic, genotoxic and antigenotoxic effects of vanillic and cinnamic acid.vanillic acid (va) found in vanilla and cinnamic acid (ca) the precursor of flavonoids and found in cinnamon oil, are natural plant phenolic acids which are secondary aromatic plant products suggested to possess many physiological and pharmacological functions. in vitro and in vivo experiments have shown that phenolic acids exhibit powerful effects on biological responses by scavenging free radicals and eliciting antioxidant capacity. in the present study, we investigated the antioxidant capacit ...201727309403
droplet size influences division of mammalian cell factories in droplet microfluidic cultivation.the potential of using droplet microfluidics for screening mammalian cell factories has been limited by the difficulty in achieving continuous cell division during cultivation in droplets. here, we report the influence of droplet size on mammalian cell division and viability during cultivation in droplets. chinese hamster ovary (cho) cells, the most widely used mammalian host cells for biopharmaceuticals production were encapsulated and cultivated in 33, 180 and 320 pl droplets for 3 days. perio ...201727535608
fish-based analysis of clonally derived cho cell populations reveals high probability for transgene integration in a terminal region of chromosome 1 (1q13).a basic goal in the development of recombinant proteins is the generation of cell lines that express the desired protein stably over many generations. here, we constructed engineered chinese hamster ovary cell lines (cho-s) with a pcho-hvr1 vector that carried an extracellular domain of a vegf receptor (vr) fusion gene. forty-five clones with high hvr1 expression were selected for karyotype analysis. using fluorescence in situ hybridization (fish) and g-banding, we found that pcho-hvr1 was integ ...201727684722
cell-free systems based on cho cell lysates: optimization strategies, synthesis of "difficult-to-express" proteins and future perspectives.nowadays, biotechnological processes play a pivotal role in target protein production. in this context, chinese hamster ovary (cho) cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called "difficult-to-express" proteins. this class of proteins comprises in particular several ...201727684475
a novel fusion protein, achr-fc, ameliorates myasthenia gravis by neutralizing antiacetylcholine receptor antibodies and suppressing acetylcholine receptor-reactive b cells.most patients with myasthenia gravis (mg) have elevated levels of autoantibodies against the nicotinic acetylcholine receptor (achr) at the neuromuscular junction, which leads to muscle weakness. we developed a fusion protein, achr-fc, as a novel therapeutic biomolecule for patients with mg and examined its efficacy. achr-fc was expressed by chinese hamster ovary cells and purified. we examined the neutralizing activity and cellular cytotoxicity of achr-fc using anti-achr antibody-producing hybr ...201727677608
reduction of ammonia and lactate through the coupling of glutamine synthetase selection and downregulation of lactate dehydrogenase-a in cho cells.chinese hamster ovary (cho) cell cultivation for production of therapeutic proteins is accompanied by production of metabolic wastes, mostly ammonia and lactate. to reduce ammonia production, the glutamine synthetase (gs) system was used to develop therapeutic monoclonal antibody (mab)-producing cho cells (sm-0.025). additionally, the lactate dehydrogenase-a (ldh-a) was downregulated with shrna to reduce lactate production in sm-0.025. the resulting mab-producing cell lines (#2, #46, and #52) pr ...201727704181
lengthening of high-yield production levels of monoclonal antibody-producing chinese hamster ovary cells by downregulation of breast cancer 1.the establishment process of high-producing chinese hamster ovary (cho) cells for therapeutic protein production is usually laborious and time consuming because of the low probability of obtaining stable, high-producing clones over a long term. thus, development of an efficient approach is required to establish stable, high-producing cells. this study presents a novel method that can efficiently establish sustainably high-producing cell lines by acceleration of transgene amplification and suppre ...201727742176
specific immune response to phospholipase b-like 2 protein, a host cell impurity in lebrikizumab clinical material.host cell proteins are manufacturing process-related impurities that may co-purify with the product despite extensive efforts to optimize the purification process. the risks associated with these impurities can vary and may be patient and/or therapeutic dependent. therefore, it is critical to monitor and control the levels of these impurities in products and their potential impact on safety and efficacy. lebrikizumab is a humanized immunoglobulin g4 monoclonal antibody (mab) that binds specifica ...201727739010
the effect of particle size on the genotoxicity of gold nanoparticles.despite the increasing biomedical applications of gold nanoparticles (aunps), their toxicological effects need to be thoroughly understood. in the present study, the genotoxic potential of commercially available aunps with varying size (5, 20, and 50 nm) were assessed using a battery of in vitro and in vivo genotoxicity assays. in the comet assay, 20 and 50 nm aunps did not induce obvious dna damage in hepg2 cells at the tested concentrations, whereas 5 nm nps induced a dose-dependent increment ...201727770565
mar-mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering.untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. the formation of plasmid concatemers and their genomic integration has been ascribed either to non-homologous end-joining (nhej) or homologous recombination (hr) dna repair pathways. however, a direct involvement of these pathways has remained unclear. here, we show that the silencing of many hr factors enhanced plasmid concatemer formation and stable expression of the gene of interest ...201727575535
detection of a phosphorylated glycine-serine linker in an igg-based fusion protein.molecular mass determination by electrospray ionization mass spectrometry of a recombinant igg-based fusion protein (mab1-f) produced in human embryonic kidney (hek) cells demonstrated the presence of a dominant +79 da product variant. using lc-ms tryptic peptide mapping analysis and collision-induced dissociation (cid) and electron-transfer/higher-energy collision dissociation fragmentations, the modification was localized to the c-terminal serine residue of a glycine-serine linker [(g4s)2] of ...201727661266
identification of an igg cdr sequence contributing to co-purification of the host cell protease cathepsin d.recombinant therapeutic monoclonal antibodies (mabs) must be purified from host cell proteins (hcps), dna, and other impurities present in chinese hamster ovary (cho) cell culture media. hcps can potentially result in adverse clinical responses in patients and, in specific cases, have caused degradation of the final mab product. as reported previously, residual traces of cathepsin d caused particle formation in the final product of mab-1. the current work was focused on identification of a prima ...201727798957
charge heterogeneity: basic antibody charge variants with increased binding to fc receptors.we identified active isoforms of the chimeric anti-gd2 antibody, ch14.18, a recombinant antibody produced in chinese hamster ovary cells, which is already used in clinical trials. (1,2,3) we separated the antibody by high resolution ion-exchange chromatography with linear ph gradient elution into acidic, main and basic charge variants on a preparative scale yielding enough material for an in-depth study of the sources and the effects of microheterogeneity. the binding affinity of the charge vari ...201727559765
'click'-xylosides as initiators of the biosynthesis of glycosaminoglycans: comparison of mono-xylosides with xylobiosides.different mono-xylosides and their corresponding xylobiosides obtained by a chemo-enzymatic approach featuring various substituents attached to a triazole ring were probed as priming agents for glycosaminoglycan (gag) biosynthesis in the xylosyltransferase-deficient pgsa-745 chinese hamster ovary cell line. xylosides containing a hydrophobic aglycone moiety were the most efficient priming agents. mono-xylosides induced higher gag biosynthesis in comparison with their corresponding xylobiosides. ...201727618481
fam60a plays a role for production stabilities of recombinant cho cell lines.recombinant cho (chinese hamster ovary) cell lines producing therapeutic proteins often lose their production capability during long-term cultivation. to ensure that cho production cell lines can be up-scaled to high-volume bioreactors, labor intensive stability studies of several months have to be performed to deselect clones that are losing productivity over time. the ability to predict whether clones will produce recombinant proteins at constant high levels, for example, through determination ...201727617904
label-free live cell imaging by confocal raman microscopy identifies cho host and producer cell lines.as a possible viable and non-invasive method to identify high producing cells, confocal raman microscopy was shown to be able to differentiate cho host cell lines and derivative production clones. cluster analysis of spectra and their derivatives was able to differentiate between different producer cell lines and a host, and also distinguished between an intracellular region of high lipid and protein content that in structure resembles the endoplasmic reticulum. this ability to identify the er m ...201727440252
fx knockout cho hosts can express desired ratios of fucosylated or afucosylated antibodies with high titers and comparable product quality.during antibody dependent cell cytotoxicity (adcc) the target cells are killed by monocytes and natural killer cells. adcc is enhanced when the antibody heavy chain's core n-linked glycan lacks the fucose molecule(s). several strategies have been utilized to generate fully afucosylated antibodies. a commonly used and efficient approach has been knocking out the fut8 gene of the chinese hamster ovary (cho) host cells, which results in expression of antibody molecules with fully afucosylated glyca ...201727666939
cationic lipids bearing succinic-based, acyclic and macrocyclic hydrophobic domains: synthetic studies and in vitro gene transfer.in this communication we describe the construction of four succinic-based cationic lipids, their formulation with plasmid dna (pdna), and an evaluation of their in vitro gene delivery into chinese hamster ovarian (cho-k1) cells. the cationic lipids employed in this work possess either a dimethylamine or trimethylamine headgroup, and a macrocyclic or an acyclic hydrophobic domain composed of, or derived from two 16-atom, succinic-based acyl chains. the synthesized lipids and a co-lipid of neutral ...201727662033
the use of transcription terminators to generate transgenic lines of chinese hamster ovary cells (cho) with stable and high level of reporter gene expression.mammalian cell lines are widely used to produce recombinant proteins. stable transgenic cell lines usually contain many insertions of the expression vector in one genomic region. transcription through transgene can be one of the reasons for target gene repression after prolonged cultivation of cell lines. in the present work, we used the known transcription terminators from the sv40 virus, as well as the human β- and γ-globin genes, to prevent transcription through transgene. the transcription t ...201726483962
biogenesis of silver nanoparticles using endophytic fungus pestalotiopsis microspora and evaluation of their antioxidant and anticancer activities.an endophytic fungal strain isolated from the leaves of gymnema sylvestre was identified as pestalotiopsis microspora vj1/vs1 based on nucleotide sequencing of internal transcribed spacer region (its 1-5.8s-its 2) of 18s rrna gene (ncbi accession number kx213894). in this study, an efficient and ecofriendly approach has been reported for the synthesis of silver nanoparticles (agnps) using aqueous culture filtrate of p. microspora. ultraviolet-visible analysis confirmed the synthesis of agnps by ...201727826190
network reconstruction of the mouse secretory pathway applied on cho cell transcriptome data.protein secretion is one of the most important processes in eukaryotes. it is based on a highly complex machinery involving numerous proteins in several cellular compartments. the elucidation of the cell biology of the secretory machinery is of great importance, as it drives protein expression for biopharmaceutical industry, a 140 billion usd global market. however, the complexity of secretory process is difficult to describe using a simple reductionist approach, and therefore a promising avenue ...201728298216
murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor viii.nowadays recombinant factor viii is produced in murine cells including in chinese hamster ovary (cho) and baby hamster kidney cells (bhk). previous studies, using the murine leukemia virus-derived retroviral vector pmfg-fviii-p140k, modified two recombinant human cell lines, hepg2 and hek293 to produce recombinant factor viii. in order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pmfg-fviii-p140k.201725031062
in vitro protective effects of botryosphaeran, a (1→3;1→6)-β-d-glucan, against mutagens in normal and tumor rodent cells.botryosphaeran (bot) is an exocellular β-d-glucan (carbohydrate biopolymer) of the (1→3;1→6)-linked type produced by botryosphaeria rhodina mamb-05. the cytotoxic, mutagenic, genotoxic, and protective effects of this substance were evaluated in chinese hamster lung fibroblasts (v79) and rat hepatocarcinoma cells (htc) by the micronucleus test (mn) and the comet assay. bot was not genotoxic in either cell line; it decreased the clastogenic effects of doxorubicin, h2o2, and benzo[a]pyrene. these r ...201728137365
genetic engineering of cho cells for viral resistance to minute virus of mice.contamination by the parvovirus minute virus of mice (mvm) remains a challenge in chinese hamster ovary (cho) biopharmaceutical production processes. although infrequent, infection of a bioreactor can be catastrophic for a manufacturer, can impact patient drug supply and safety, and can have regulatory implications. we evaluated engineering a cho parental cell line (chozn(®) gs(-/-) ) to create a new host cell line that is resistant to mvm infection by modifying the major receptors used by the v ...201727642072
identification of human junctional adhesion molecule 1 as a functional receptor for the hom-1 calicivirus on human cells.the hom-1 vesivirus was reported in 1998 following the inadvertent transmission of the animal calicivirus san miguel sea lion virus to a human host in a laboratory. we characterized the hom-1 strain and investigated the mechanism by which human cells could be infected. an expression library of 3,559 human plasma membrane proteins was screened for reactivity with hom-1 virus-like particles, and a single interacting protein, human junctional adhesion molecule 1 (hjam1), was identified. transient e ...201728196955
vesivirus 2117 capsids more closely resemble sapovirus and lagovirus particles than other known vesivirus structures.vesivirus 2117 is an adventitious agent that, in 2009, was identified as a contaminant of chinese hamster ovary cells propagated in bioreactors at a pharmaceutical manufacturing plant belonging to genzyme. the consequent interruption in supply of fabrazyme and cerezyme (drugs used to treat fabry and gaucher diseases, respectively) caused significant economic losses. vesivirus 2117 is a member of the caliciviridae, a family of small icosahedral viruses encoding a positive-sense rna genome. we hav ...201727902397
evaluation of the virus clearance capacity and robustness of the manufacturing process for the recombinant factor viii protein, turoctocog alfa.turoctocog alfa is a b-domain-truncated recombinant factor viii protein produced in a chinese hamster ovary (cho) cell line. the aim of this study was to evaluate the virus clearance capacity and robustness of the turoctocog alfa purification process. virus clearance evaluation studies were conducted utilising a scaled-down version of the manufacturing process. total virus clearance was evaluated using the ecotropic murine leukaemia virus (emulv) as a model for non-infectious retrovirus-like par ...201727620499
a scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay.hydroxylysine aldehyde-derived collagen cross-links (hlccs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. hlcc formation is initiated by lysyl hydroxylase 2 (lh2), an fe(ii) and α-ketoglutarate (αkg)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. development of lh2 antagonists for the treatment of these diseases will require a reliable source of recombinant lh2 protein and a non-radioactive ...201728216326
parameter estimation in large-scale systems biology models: a parallel and self-adaptive cooperative strategy.the development of large-scale kinetic models is one of the current key issues in computational systems biology and bioinformatics. here we consider the problem of parameter estimation in nonlinear dynamic models. global optimization methods can be used to solve this type of problems but the associated computational cost is very large. moreover, many of these methods need the tuning of a number of adjustable search parameters, requiring a number of initial exploratory runs and therefore further ...201728109249
cellular engineering for therapeutic protein production: product quality, host modification, and process improvement.recombinant proteins offer many therapeutic advantages unavailable in traditional small molecule drugs, but the need for cellular versus chemical synthesis complicates production. avenues for producing therapeutic biologics are continuously expanding, and developments in biochemistry, cell biology, and bioengineering fuel new discoveries that promise safer, more efficient, and cheaper drugs for consumers. numerous approaches to express recombinant proteins exist, but escherichia coli, saccharomy ...201727933725
genotoxicity evaluation of hwanglyeonhaedok-tang, an herbal formula.hwanglyeonhaedok-tang (huanglianjiedu-tang, orengedoku-to), a traditional herbal formula, is used for the treatment of inflammatory, gastrointestinal and cardiovascular diseases.201727916588
stable high-level expression of factor viii in chinese hamster ovary cells in improved elongation factor-1 alpha-based system.recombinant factor viii (fviii), used for haemophilia a therapy, is one of the most challenging among the therapeutic proteins produced in heterologous expression systems. deletion variant of fviii, in which the entire domain b is replaced by a short linker peptide, was approved for medical use. efficacy and safety of this fviii deletion variant are similar to full-length fviii preparations while the level of production in cho cells is substantially higher. typical levels of productivity for cho ...201728340620
matrix attachment region combinations increase transgene expression in transfected chinese hamster ovary cells.matrix attachment regions (mars) are cis-acting dna elements that can increase transgene expression levels in a cho cell expression system. to investigate the effects of mar combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (egfp) gene flanked by different combinations of human β-interferon and β-globin mar (imar and gmar, respectively), which was driven by the cytomegalovirus (cmv) or simian vir ...201728216629
in vitro antimicrobial and cytotoxic effects of anacardium occidentale and mangifera indica in oral care.oral health is an integral and important component of general health. infectious diseases such as caries, periodontal, and gingivitis indicate the onset of imbalance in homeostasis between oral micro biota and host. the present day medicaments used in oral health care have numerous side effects. the uses of herbal plants as an alternative have gained popularity due to side effects of antibiotics and emergence of multidrug resistant strains. anacardium occidentale (cashew) and mangifera indica (m ...201725709341
design and characterization of plasmids encoding antigenic peptides of aha1 from aeromonas hydrophila as prospective fish vaccines.the current investigation aimed at designing dna vaccines against aeromonas hydrophila infections. the dna vaccine candidates were designed to express two antigenic outer membrane protein (aha1) peptides and to be delivered by a nanoparticle-based delivery system. gene sequences of conserved regions of antigenic aha1 [aha1(211-381), aha1(211-381)opt, aha1(703-999) and aha1(703-999)opt] were cloned into pvax-gfp expression vector. the selected dna vaccine candidates were purified from e. coli dh5 ...201727899335
glycan region of gpi anchored-protein is required for cytocidal oligomerization of an anticancer parasporin-2, cry46aa1 protein, from bacillus thuringiensis strain a1547.parasporin-2 (ps2), alternatively named cry46aa1, an anticancer protein derived from bacillus thuringiensis strain a1547, causes specific cell damage via ps2 oligomerization in the cell membrane. although ps2 requires glycosylphosphatidylinositol (gpi)-anchored proteins for its cytocidal action, their precise role is unknown. here, we report that the glycan of gpi induces ps2 oligomerization, which causes cell death. cytotoxicity, cell-binding and oligomerization of the toxin were not observed i ...201727863961
whole-genome sequencing of invasion-resistant cells identifies laminin α2 as a host factor for bacterial invasion.to understand the role of glycosaminoglycans in bacterial cellular invasion, xylosyltransferase-deficient mutants of chinese hamster ovary (cho) cells were created using clustered regularly interspaced short palindromic repeat (crispr) and crispr-associated gene 9 (crispr-cas9) gene targeting. when these mutants were compared to the pgsa745 cell line, a cho xylosyltransferase mutant generated previously using chemical mutagenesis, an unexpected result was obtained. bacterial invasion of pgsa745 ...201728074024
novel citronellyl-based photoprobes designed to identify er proteins interacting with dolichyl phosphate in yeast and mammalian cells.dolichyl phosphate-linked mono- and oligosaccharides (dlo) are essential intermediates in protein n-glycosylation, c- and o-mannosylation and gpi anchor biosynthesis. while many membrane proteins in the endoplasmic reticulum (er) involved in the assembly of dlos are known, essential proteins believed to be required for the transbilayer movement (flip-flopping) and proteins potentially involved in the regulation of dlo synthesis remain to be identified.201727099830
characterization of membrane occupation and recognition nexus repeat containing 3, meiosis expressed gene 1 binding partner, in mouse male germ cells.mammalian spermatogenesis is a well-organized process of cell development and differentiation. meiosis expressed gene 1 (meig1) plays an essential role in the regulation of spermiogenesis. to explore potential mechanisms of meig1's action, a yeast two-hybrid screen was conducted, and several potential binding partners were identified; one of them was membrane occupation and recognition nexus repeat containing 3 (morn3). morn3 mrna is only abundant in mouse testis. in the testis, morn3 mrna is hi ...201725248657
molecular characterizations of natalisin and its roles in modulating mating in the oriental fruit fly, bactrocera dorsalis (hendel).initially, natalisin (ntl) was identified from three holometabolous insect species, drosophila melanogaster, tribolium castaneum and bombyx mori, and was documented to regulate reproductive behaviours in d. melanogaster and t. castaneum. in this study, we report the sequences of the ntl precursor and its receptor (ntlr) from an important agricultural pest, bactrocera dorsalis (hendel). ntlr is a typical g-protein coupled receptor and phylogenetic analysis showed that b. dorsalis ntlr was closely ...201727862548
enhancing the toolbox to study il-17a in cattle and sheep.the development of methods to detect cytokine expression by t cell subsets in ruminants is fundamental to strategic development of new livestock vaccines for prevention of infectious diseases. it has been possible to detect t cell expression of ifn-γ, il-4 and il-10 in ruminants for many years but methods to detect expression of il-17a are relatively limited. to address this gap in capability we have cloned bovine and ovine il-17a cdnas and expressed biologically-active recombinant proteins in c ...201728388924
optimizing conditions for calcium phosphate mediated transient transfection.calcium phosphate mediated transfection has been used for delivering dna into mammalian cells in excess of 30 years due to its most low cost for introducing recombinant dna into culture cells. however, multiple factors affecting the transfect efficiency are commonly recognized meanwhile for years, the low transfection efficiency of this approach on higher differentiated and non-tumor cells such as cho and c2c12 limits its application on research.201728386188
production and purification of recombinant glucocerebrosidase in transgenic rice cell suspension cultures.gaucher disease, which is caused by deficiency of glucocerebrosidase (gcd), is currently treated by enzyme replacement therapy. plant-based systems produce glycoproteins and can be combined with targeting strategies to generate proteins with terminal mannose structures for macrophage uptake. however, the gliding step for the purification is essential since the produced protein still exists inside cells. in the case of rice-amylase 1a (ramy1a) secretion signal peptide, gcd protein is secreted out ...201727817046
lysosomal alpha-mannosidase and alpha-mannosidosis.lysosomal alpha-mannosidase with acidic ph optimum is ubiquitous in human tissues where is expressed in two major forms, a and b that are the product of a single gene located on chromosome 19. mutations in the gene encoding for alpha-mannosidase cause alpha- mannosidosis, an autosomal recessive disease, resulting in the accumulation of unprocessed mannose containing oligosaccharide material. this rare disease has an estimated incidence of 1/500.0.00 live births and clinically is divided into thr ...201727814608
improved recombinant antibody production by cho cells using a production enhancer dna element with repeated transgene integration at a predetermined chromosomal site.chinese hamster ovary (cho) cells are one of the most useful host cell lines for the production of biopharmaceutical proteins. although a series of production processes have been refined to improve protein productivity and cost performance, establishing producer cells is still time-consuming and labor-intensive. recombinase-mediated site-specific gene integration into a predetermined chromosomal locus may enable predictable protein expression, reducing the laborious process of cell screening. we ...201727856232
differences in trpc3 and trpc6 channels assembly in mesenteric vascular smooth muscle cells in essential hypertension.canonical transient receptor potential (trpc)3 and trpc6 channels of vascular smooth muscle cells (vsmcs) mediate stretch- or agonist-induced cationic fluxes, contributing to membrane potential and vascular tone. native trpc3/c6 channels can form homo- or heterotetrameric complexes, which can hinder individual trpc channel properties. the possibility that the differences in their association pattern may change their contribution to vascular tone in hypertension is unexplored. functional characte ...201727861908
reduced cytotoxicity in pcb-exposed chinese hamster ovary (cho) cells pretreated with vitamin e.the aim of this study was to evaluate protective effects of vitamin e (50 -150 μm) in ovary cells upon cytotoxic effects induced by two structurally distinct pcb congeners - planar "dioxin-like" pcb 77 and non-planar di-ortho-substituted pcb 153 with an emphasis on identifying differences in the mechanism of vitamin e action depending on the structure of congeners. application of three bioassays confirmed that pcbs decrease ovarian cell proliferation with slightly profound effects of pcb 77. pcb ...201727865896
establishment of monoclonal antibodies against cell surface domains of asct2/slc1a5 and their inhibition of glutamine-dependent tumor cell growth.human alanine-serine-cysteine transporter 2 (asct2; slc1a5) is a major transporter of the amino acid glutamine that is known to be overexpressed in certain malignant tumors. in this study, we generated specific monoclonal antibodies (mabs) against asct2 by establishing an asct2-expressing chinese hamster ovary cell line that was used to immunize mice and rats. the mabs km4008, km4012, and km4018 against asct2 were isolated through a cell-based screen; these specifically bound to asct2-positive c ...201727865832
selection of chemically defined media for cho cell fed-batch culture processes.two cho cell clones derived from the same parental cho(bc®) cell line and producing the same monoclonal antibody (bc-g, a low producing clone; bc-p, a high producing clone) were tested in four basal media in all possible combinations with three feeds (=12 conditions) in fed-batch cultures. higher amino acid feeding did not always lead to higher mab production. the two clones showed differences in cell physiology, metabolism and optimal medium-feed combinations. during the phase transitions of al ...201727900626
segmented linear modeling of cho fed-batch culture and its application to large scale production.we describe a systematic approach to model cho metabolism during biopharmaceutical production across a wide range of cell culture conditions. to this end, we applied the metabolic steady state concept. we analyzed and modeled the production rates of metabolites as a function of the specific growth rate. first, the total number of metabolic steady state phases and the location of the breakpoints were determined by recursive partitioning. for this, the smoothed derivative of the metabolic rates wi ...201727869296
rational design of glycoengineered interferon-β analogs with improved aggregation state: experimental validation.recombinant human interferon-β (rhifn-β) used clinically has lower efficacy than expected due to protein instabilities such as aggregation. increasing molecular stability, glycoengineering has been used to improve clinical efficacy for a number of therapeutics; however, often labor-intensive trail-and-error approaches are used to identify additional glycosylation sites. in this study two rhifn-β analogs with one additional glycosylation site, l6t and s75n, identified by a rational in silico appr ...201727881683
flavonoids from heliotropium subulatum exudate and their evaluation for antioxidant, antineoplastic and cytotoxic activities ii.the flavonoids are the largest group of phenolic compounds isolated from a wide range of higher plants. these compounds work as antimicrobials, anti-insect agents and protect plants from other types of biotic and abiotic stresses. various researchers have suggested that flavonoids possessed antioxidant, antineoplastic and cytotoxic activities. the main objective of this study was to test dichloromethane fraction of resinous exudate of heliotropium subulatum for their antioxidant, antineoplastic ...201727905025
a framework to quantify karyotype variation associated with cho cell line instability at a single-cell level.chinese hamster ovary (cho) cells, the major mammalian host cells for biomanufacturing of therapeutic proteins, have been extensively investigated to enhance productivity and product quality. however, cell line instability resulting in unexpected changes in productivity or product quality continues to be a challenge. based on previous reports about causes and characteristics of production instability, we hypothesized that chromosomal rearrangements due to genomic instability are associated with ...201727922175
role of a tachykinin-related peptide and its receptor in modulating the olfactory sensitivity in the oriental fruit fly, bactrocera dorsalis (hendel).insect tachykinin-related peptide (trp), an ortholog of tachykinin in vertebrates, has been linked with regulation of diverse physiological processes, such as olfactory perception, locomotion, aggression, lipid metabolism and myotropic activity. in this study, we investigated the function of trp (bdtrp) and its receptor (bdtrpr) in an important agricultural pest, the oriental fruit fly bactrocera dorsalis. bdtrpr is a typical g-protein coupled-receptor (gpcr), and it could be activated by the pu ...201727923683
integrin αiibβ3-dependent erk signaling is regulated by src and rho kinases in both leu33 and pro33 polymorphic isoforms.platelet integrin αiibβ3 possesses a leu/pro polymorphism at residue 33 (leu33/hpa-1a or pro33/hpa-1b). the pro33 isoform has been suggested to exhibit prothrombotic features. αiibβ3-expressing cho (chinese hamster ovary) cells on immobilized fibrinogen show activation of the map kinase family member erk2, with an enhanced erk2 activity in pro33 cells compared to leu33 cells. in our present work, we examined how the leu/pro polymorphism modulates the erk2 activation stimulated by 2 differently t ...201727923225
improving the secretory capacity of chinese hamster ovary cells by ectopic expression of effector genes: lessons learned and future directions.chinese hamster ovary (cho) cells are the preferred cell factory for the production of therapeutic glycoproteins. although efforts primarily within bioprocess optimization have led to increased product titers of recombinant proteins (r-proteins) expressed in cho cells, post-transcriptional bottlenecks in the biosynthetic pathway of r-proteins remain to be solved. to this end, the ectopic expression of transgenes (effector genes) offers great engineering potential. however, studies on effector ge ...201727931938
integrated genome and protein editing swaps α-2,6 sialylation for α-2,3 sialic acid on recombinant antibodies from cho.immunoglobin g with α-2,6 sialylation has been reported to have an impact on antibody-dependent cellular cytotoxicity and anti-inflammatory efficacy. however, production of antibodies with α-2,6 sialylation from chinese hamster ovary cells is challenging due to the inaccessibility of sialyltransferases for the heavy chain n-glycan site and the presence of exclusively α-2,3 sialyltransferases. in this study, combining mutations on the fc regions to allow sialyltransferase accessibility with overe ...201727943633
knockout of a difficult-to-remove cho host cell protein, lipoprotein lipase, for improved polysorbate stability in monoclonal antibody formulations.while the majority of host cell protein (hcp) impurities are effectively removed in typical downstream purification processes, a small population of hcps are particularly challenging. previous studies have identified hcps that are challenging for a variety of reasons. lipoprotein lipase (lpl)-a chinese hamster ovary (cho) hcp that functions to hydrolyze esters in triglycerides-was one of ten hcps identified in previous studies as being susceptible to retention in downstream processing. lpl may d ...201727943242
preferential hydrolysis of truncated oxidized glycerophospholipids by lysosomal phospholipase a2.truncated oxidized glycerophospholipids (ox-pls) are bioactive lipids resulting from oxidative stress. the catabolic pathways for truncated ox-pls are not fully understood. lysosomal phospholipase a2 (lpla2) with phospholipase a and transacylase activities is a key enzyme in phospholipid homeostasis. the present study assessed whether lpla2 could hydrolyze truncated ox-pls. incubation of lpla2 with liposomes consisting of 1,2-o-octadecenyl-sn-glycero-3-phosphocholine (dodpc)/1,2-dioleoyl-sn-glyc ...201727993948
hiv protease inhibitors alter amyloid precursor protein processing via β-site amyloid precursor protein cleaving enzyme-1 translational up-regulation.mounting evidence implicates antiretroviral (arv) drugs as potential contributors to the persistence and evolution of clinical and pathological presentation of hiv-associated neurocognitive disorders in the post-arv era. based on their ability to induce endoplasmic reticulum (er) stress in various cell types, we hypothesized that arv-mediated er stress in the central nervous system resulted in chronic dysregulation of the unfolded protein response and altered amyloid precursor protein (app) proc ...201727993242
high-throughput lipidomic and transcriptomic analysis to compare sp2/0, cho, and hek-293 mammalian cell lines.a combined lipidomics and transcriptomics analysis was performed on mouse myeloma sp2/0, chinese hamster ovary (cho), and human embryonic kidney (hek) cells in order to compare widely used mammalian expression systems. initial thin layer chromatography (tlc) analysis indicated that phosphatidylethanolamine (pe) and phosphatidylcholine (pc) were the major lipid components in all cell lines with lower amounts of sphingomyelin (sm) in sp2/0 compared to cho and hek, which was subsequently confirmed ...201727991764
design, synthesis and biological activity of novel molecules designed to target parp and dna.in order to enhance the cytotoxic potential of poly(adp-ribose) polymerase (parp) inhibitors in brca1 or 2 deficient tumours, we designed a series of molecules containing a 1,2,3-triazene moiety tethered to a parp targeting scaffold. a cell-based selectivity assay involving a brca2-deficient chinese hamster cell line and its corresponding brca2 wild type transfectant, was used to predict the parp targeting potential of the latter agents. the results showed that adding a dna damaging function to ...201728003142
assessing the genotoxicity of two commonly occurring byproducts of water disinfection: chloral hydrate and bromal hydrate.water disinfection treatments result in the formation of disinfection byproducts (dbps) that have been linked to adverse human health outcomes including higher incidence of bladder and colorectal cancer. however, data about the genotoxicity of dbps is limited to only a small fraction of compounds. chloral hydrate (ch) and bromal hydrate (bh) are two trihaloacetaldehydes commonly detected in disinfected waters, but little is known about their genotoxicity, especially bh. we investigated the genot ...201728010927
tbx20 controls the expression of the kcnh2 gene and of herg channels.long qt syndrome (lqts) exhibits great phenotype variability among family members carrying the same mutation, which can be partially attributed to genetic factors. we functionally analyzed the kcnh2 (encoding for kv11.1 or herg channels) and tbx20 (encoding for the transcription factor tbx20) variants found by next-generation sequencing in two siblings with lqts in a spanish family of african ancestry. affected relatives harbor a heterozygous mutation in kcnh2 that encodes for p.t152hfsx180 kv11 ...201728049825
production of human mutant biologically active hepatocyte growth factor in chinese hamster ovary cells.hepatocyte growth factor (hgf) is a potent multifunctional cytokine that affects proliferation, migration, and morphogenesis of various cells. hgf is secreted as an inactive single-chain precursor protein and activated by the cleavage of serine proteases to form heterodimers. in our current study, the cleavage site of hgf was blocked by replaced arg 494 of glu (r494e) that resulted in the single-chain hgf (r494e) unable to be cleaved by serine proteases. we established chinese hamster ovary (cho ...201728045643
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