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biophysical studies on calcium and carbohydrate binding to carbohydrate recognition domain of gal/galnac lectin from entamoeba histolytica: insights into host cell adhesion.entamoeba histolytica, an enteric parasite expresses a gal/galnac-specific lectin that contributes to its virulence by establishing adhesion to host cell. in this study, carbohydrate recognition domain of hgl (ehcrd) was purified and biophysical studies were conducted to understand the thermodynamic basis of its binding to carbohydrate and ca(++) here, we show that carbohydrate recognition domain (crd) of the lectin binds to calcium through dpn motif. to decipher the role of calcium in carbohydr ...201627008865
rapid screening by cell-based fusion assay for identifying novel antivirals of glycoprotein b-mediated herpes simplex virus type 1 infection.herpes simplex virus type 1 (hsv-1) is a causative agent for a variety of diseases. although antiherpetic drugs such as acyclovir have been developed to inhibit virus replication through interaction with dna kinases, their continuous administration leads to an increase in the frequency of drug-resistant hsv-1, which is an important clinical issue that requires urgent solution. recently, we reported that the sialylated o-linked sugar t antigen (stn) and its attached peptide region (o-glycosylated ...201627803463
high-efficiency secretory expression of human neutrophil gelatinase-associated lipocalin from mammalian cell lines with human serum albumin signal peptide.human neutrophil gelatinase associated lipocalin (ngal) is a secretory glycoprotein initially isolated from neutrophils. it is thought to be involved in the incidence and development of immunological diseases and cancers. urinary and serum levels of ngal have been investigated as a new biomarker of acute kidney injury (aki), for an earlier and more accurate detection method than with creatinine level. however, expressing high-quality recombinant ngal is difficult both in escherichia coli and mam ...201626518367
liposome-mediated herpes simplex virus uptake is glycoprotein-d receptor-independent but requires heparan sulfate.cationic liposomes are widely used to facilitate introduction of genetic material into target cells during transfection. this study describes a non-receptor mediated herpes simplex virus type-1 (hsv-1) entry into the chinese hamster ovary (cho-k1) cells that naturally lack glycoprotein d (gd)-receptors using a commercially available cationic liposome: lipofectamine. presence of cell surface heparan sulfate (hs) increased the levels of viral entry indicating a potential role of hs in this mode of ...201627446014
generation and characterization of a bivalent hiv-1 subtype c gp120 protein boost for proof-of-concept hiv vaccine efficacy trials in southern africa.the viral envelope glycoprotein (env) is the major target for antibody (ab)-mediated vaccine development against the human immunodeficiency virus type 1 (hiv-1). although several recombinant env antigens have been evaluated in clinical trials, only the surface glycoprotein, gp120, (from hiv-1 subtype b, mn, and subtype crf_01ae, a244) used in the alvac prime-aidsvax gp120 boost rv144 phase iii hiv vaccine trial was shown to contribute to protective efficacy, although modest and short-lived. henc ...201627442017
high levels of histone h3 acetylation at the cmv promoter are predictive of stable expression in chinese hamster ovary cells.chinese hamster ovary cells (cho) are widely used in the production of glycosylated therapeutic proteins such as antibodies. during expansion and maintenance, cho cell lines are prone to production instability, which may be caused by promoter silencing, loss of transgene copies, or post-transcriptional effects. silencing of recombinant genes may be accompanied by dna methylation and histone modification. previously, we demonstrated that cytosine methylation of the human cytomegalovirus major imm ...201627073121
novel epstein-barr virus-like particles incorporating gh/gl-ebna1 or gb-lmp2 induce high neutralizing antibody titers and ebv-specific t-cell responses in immunized mice.previous epstein-barr virus (ebv) prophylactic vaccines based on the major surface glycoprotein gp350/220 as an immunogen have failed to block viral infection in humans, suggesting a need to target other viral envelope glycoproteins. in this study, we reasoned that incorporating gh/gl or gb, critical glycoproteins for viral fusion and entry, on the surface of a virus-like particle (vlp) would be more immunogenic than gp350/220 for generating effective neutralizing antibodies to prevent viral inf ...201627926486
rabbits immunized with epstein-barr virus gh/gl or gb recombinant proteins elicit higher serum virus neutralizing activity than gp350.epstein-barr virus (ebv) is the primary cause of infectious mononucleosis and has been strongly implicated in the etiology of multiple epithelial and lymphoid cancers, such as nasopharyngeal carcinoma, gastric carcinoma, hodgkin lymphoma, burkitt lymphoma, non-hodgkin lymphoma and post-transplant lymphoproliferative disorder. there is currently no licensed prophylactic vaccine for ebv. most efforts to develop prophylactic vaccines have focused on ebv gp350, which binds to cd21/cd35 to gain entry ...201627291087
xrcc1 rs25487 genetic variant and tp53 mutation at codon 249 predict clinical outcomes of hepatitis b virus-related hepatocellular carcinoma after hepatectomy: a cohort study for 10 years' follow up.to investigate the effects of rs25487 (the dna repair gene: x-ray repair complementing defective repair in chinese hamster cells 1 [xrcc1]) and codon 249 mutation (tp53 gene) on clinical outcomes of post-hepatectomy hepatitis b virus (hbv)-related hcc.201626519348
influenza virus hemagglutinin glycoproteins with different n-glycan patterns activate dendritic cells in vitro.influenza virus hemagglutinin (ha) n-glycans play important regulatory roles in the control of virus virulence, antigenicity, receptor-binding specificity, and viral escape from the immune response. considered essential for controlling innate and adaptive immune responses against influenza virus infections, dendritic cells (dcs) trigger proinflammatory and adaptive immune responses in hosts. in this study, we engineered chinese hamster ovary (cho) cell lines expressing recombinant ha from pandem ...201627099319
n-ethylmaleimide and the cyclic response to x-rays of synchronous chinese hamster cells. 20164736799
studies on induced aberrations in diplochromosomes of chinese hamster cells. 20164796813
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
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.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-gro ...201627034736
ultrastructural changes in a-cells exposed to diabetic hyperglycaemia. observations made on pancreas of chinese hamsters. 20164914660
the retinal vasculature of the chinese hamster: a preliminary study. 20164914659
intermittent dna synthesis and periodic expression of enzyme activity in the cell cycle of wi-38.synchronous cultures of wi-38 were obtained using an automated system for detachment and partitioning of mitotic cells which operates without the use of inhibitors, altered medium, or lowered temperatures. the generation time in synchronous wi-38 is 19.5 h and the duration of s phase when determined from the percentage of labeled metaphase cells or nuclei is 12 h. dna replication in wi-38 occurs in three temporally distinct and rapid bursts separated by intervals of greatly reduced synthesis wit ...20164795861
effect of circadian rhythm on the radiation response of the chinese hamster (cricetulus griseus). 20165030088
effect of bcl-x(l) overexpression on lactate metabolism in chinese hamster ovary cells producing antibody.previously, overexpression of anti-apoptotic proteins, such as e1b-19k and aven, was reported to alter lactate metabolism of cho cells in culture. to investigate the effect of bcl-xl , a well-known anti-apoptotic protein, on lactate metabolism of recombinant cho (rcho) cells, two antibody-producing rcho cell lines with regulated bcl-xl overexpression (cs13*-0.02-off-bcl-xl and cs13*-1.00-off-bcl-xl ) were established using the tet-off system. when cells were cultivated without bcl-xl overexpress ...201624039207
analytical characterization of a monoclonal antibody therapeutic reveals a three-light chain species that is efficiently removed using hydrophobic interaction chromatography.size exclusion high performance liquid chromatography analysis of a human monoclonal antibody (mab) showed the presence of a new species that eluted with a retention time between the dimeric and monomeric species of the antibody. extensive characterization of this species, referred to as "shoulder," indicated that it was a mab containing an extra light chain and had a molecular weight of approximately 175 kda. the extra light chain was found to be non-covalently associated with the fab portion o ...201623995619
bystander effect induced by uvc radiation in chinese hamster v79 cells.in past decades, researches on radiation-induced bystander effect mainly focused on ionizing radiation such as α-particle, β-particle, x-ray and γ-ray. but few researches have been conducted on the ability of ultraviolet (uv) radiation-induced bystander effect, and knowledge of uvc-induced bystander effect is far limited. here, we adopted medium transfer experiment to detect whether uvc could cause bystander effect in chinese hamster v79 cells. we determined the cell viability, apoptosis rate, c ...201624517728
a cho cell line engineered to express xbp1 and ero1-lα has increased levels of transient protein expression.transient gene expression (tge) systems currently provide rapid and scalable (up to 100 l) methods for generating multigram quantities of recombinant heterologous proteins. product titers of up to 1 g/l have been demonstrated in hek293 cells but reported yields from chinese hamster ovary (cho) cells are lower at ∼300 mg/l. we report on the establishment of an engineered chos cell line, which has been developed for tge. this cell line has been engineered to express both x-box binding protein (xbp ...201623335490
impact of aeration strategy on cho cell performance during antibody production.stirred tank bioreactors using suspension adapted mammalian cells are typically used for the production of complex therapeutic proteins. the hydrodynamic conditions experienced by cells within this environment have been shown to directly impact growth, productivity, and product quality and therefore an improved understanding of the cellular response is critical. here we investigate the sub-lethal effects of different aeration strategies on chinese hamster ovary cells during monoclonal antibody p ...201623074084
fed-batch cho cell t-pa production and feed glutamine replacement to reduce ammonia production.industrial therapeutic protein production has been greatly improved through fed-batch development. in this study, improvement to the productivity of a tissue-plasminogen activator (t-pa) expressing chinese hamster ovary (cho) cell line was investigated in shake flask culture through the optimization of the fed-batch feed and the reduction of ammonia generation by glutamine replacement. the t-pa titer was increased from 33 mg/l under batch conditions to 250 mg/l with daily feeding starting after ...201623125190
identifying bottlenecks in transient and stable production of recombinant monoclonal-antibody sequence variants in chinese hamster ovary cells.the increasing demand for antibody-based therapeutics has emphasized the need for technologies to improve recombinant antibody titers from mammalian cell lines. moreover, as antibody therapeutics address an increasing spectrum of indications, interest has increased in antibody engineering to improve affinity and biological activity. however, the cellular mechanisms that dictate expression and the relationships between antibody sequence and expression level remain poorly understood. fundamental u ...201622467228
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
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
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
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
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
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
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
inactivation of gdp-fucose transporter gene (slc35c1) in cho cells by zfns, talens and crispr-cas9 for production of fucose-free antibodies.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 chi ...201626471004
a rapid method for simultaneous evaluation of free light chain content and aggregate content in culture media of chinese hamster ovary cells expressing monoclonal antibodies for cell line screening.the goal of developing a monoclonal antibody (mab) production process is high productivity and high quality. because the productivity and quality of mabs depend on cell line properties, the selection of cell lines suitable for large-scale production is an important stage in process development for mab production. the light chain (lc) is important for antibody folding and assembly in the endoplasmic reticulum; cell lines that secrete a large amount of lcs in the medium secrete high-quality antibo ...201626467692
high zinc ion supplementation of more than 30 μm can increase monoclonal antibody production in recombinant chinese hamster ovary dg44 cell culture.effects of high znso4·7h2o supplementation on cell growth and monoclonal antibody (mab) production in chemically defined suspension cultures of recombinant chinese hamster ovary (rcho) dg44 cells were examined. the supplementation of znso4·7h2o up to 120 μm gradually increased specific mab production rate of rcho dg44 cells in the early growth phase (0-4 days of culture). the znso4·7h2o concentration for enhancing mab production without any cytotoxic effects on cell growth was 30-60 μm. in addit ...201626512008
host cell protein impurities in chromatographic polishing steps for monoclonal antibody purification.downstream purification of monoclonal antibodies (mabs) is normally performed using a platform process that is empirically tuned to optimize impurity removal for each new product. a more fundamental understanding of impurities and the product itself would provide insights into the rational design of efficient downstream processes. this work examines the chromatographic properties of chinese hamster ovary host cell protein (hcp) impurities in non-affinity chromatographic resins commonly used in p ...201626550778
manool, a salvia officinalis diterpene, induces selective cytotoxicity in cancer cells.manool, a diterpene isolated from salvia officinalis, was evaluated by the xtt colorimetric assay for cytotoxicity and selectivity against different cancer cell lines: b16f10 (murine melanoma), mcf-7 (human breast adenocarcinoma), hela (human cervical adenocarcinoma), hepg2 (human hepatocellular carcinoma), and mo59j, u343 and u251 (human glioblastoma). a normal cell line (v79, chinese hamster lung fibroblasts) was used to compare the selectivity of the test substance. manool exhibited higher cy ...201626547581
human cyp2e1-dependent mutagenicity of mono- and dichlorobiphenyls in chinese hamster (v79)-derived cells.polychlorinated biphenyls (pcbs) are a group of persistent organic pollutants with confirmed carcinogenicity to humans. metabolic activation of lower chlorinated pcbs to genotoxic metabolites may involve hydroxylation and further oxidation, and some hydroxylated metabolites may be sulfo-conjugated. however, the genotoxicity of individual pcb compounds is largely unknown. in this study, 15 mono- and dichlorobiphenyls were investigated for genotoxicity using the micronucleus and hprt mutagenicity ...201626547025
cloning and expression of codon-optimized recombinant darbepoetin alfa in leishmania tarentolae t7-tr.darbepoetin alfa is an engineered and hyperglycosylated analog of recombinant human erythropoietin (epo) which is used as a drug in treating anemia in patients with chronic kidney failure and cancer. this study desribes the secretory expression of a codon-optimized recombinant form of darbepoetin alfa in leishmania tarentolae t7-tr. synthetic codon-optimized gene was amplified by pcr and cloned into the plexsy-i-blecherry3 vector. the resultant expression vector, plexsydarbo, was purified, diges ...201626546410
the multidrug transporter mate1 sequesters ocs within an intracellular compartment that has no influence on oc secretion in renal proximal tubules.secretion of organic cations (ocs) across renal proximal tubules (rpts) involves basolateral oc transporter (oct)2-mediated uptake from the blood followed by apical multidrug and toxin extruder (mate)1/2-mediated efflux into the tubule filtrate. whereas oct2 supports electrogenic oc uniport, mate is an oc/h(+) exchanger. as assessed by epifluorescence microscopy, cultured chinese hamster ovary (cho) cells that stably expressed human mate1 accumulated the fluorescent oc n,n,n-trimethyl-2-[methyl( ...201626538438
a markov chain model for n-linked protein glycosylation--towards a low-parameter tool for model-driven glycoengineering.glycosylation is a critical quality attribute of most recombinant biotherapeutics. consequently, drug development requires careful control of glycoforms to meet bioactivity and biosafety requirements. however, glycoengineering can be extraordinarily difficult given the complex reaction networks underlying glycosylation and the vast number of different glycans that can be synthesized in a host cell. computational modeling offers an intriguing option to rationally guide glycoengineering, but the h ...201626537759
improving expression of recombinant human igf-1 using igf-1r knockout cho cell lines.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- ...201626523469
deletion of a telomeric region on chromosome 8 correlates with higher productivity and stability of cho cell lines.chinese hamster ovary (cho) cells are widely used for large scale production of recombinant biopharmaceuticals. although these cells have been extensively used, a demand to further increase the performance, for example, to facilitate the process of clone selection to isolate the highest producing cell lines that maintain stability of production over time is still existing. we compared gene expression profiles of high versus low producing cho clones to identify regulated genes which can be used a ...201626523402
genetic analysis of the clonal stability of chinese hamster ovary cells for recombinant protein production.chinese hamster ovary (cho) cells are frequently used for the production of recombinant proteins for therapeutical applications. however, the recombinant protein expression level of cho cells may reduce during long-term culture. the physiological changes related to the stability of expression were not well understood. in this study, we performed a series of genetic analysis on stable and unstable clonal derived populations. transcriptome analysis revealed that a large number of differentially ex ...201626563441
chemically defined media modifications to lower tryptophan oxidation of biopharmaceuticals.oxidation of biopharmaceuticals is a major product quality issue with potential impacts on activity and immunogenicity. at eli lilly and company, high tryptophan oxidation was observed for two biopharmaceuticals in development produced in chinese hamster ovary cells. a switch from historical hydrolysate-containing media to chemically defined media with a reformulated basal powder was thought to be responsible, so mitigation efforts focused on media modification. shake flask studies identified th ...201626560440
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
assays for dna double-strand break repair by microhomology-based end-joining repair mechanisms.dna double stranded breaks (dsbs) are one of the most deleterious types of dna lesions. the main pathways responsible for repairing these breaks in eukaryotic cells are homologous recombination (hr) and non-homologous end-joining (nhej). however, a third group of still poorly characterized dsb repair pathways, collectively termed microhomology-mediated end-joining (mmej), relies on short homologies for the end-joining process. here, we constructed gfp reporter assays to characterize and distingu ...201626657630
quick generation of raman spectroscopy based in-process glucose control to influence biopharmaceutical protein product quality during mammalian cell culture.mitigating risks to biotherapeutic protein production processes and products has driven the development of targeted process analytical technology (pat); however implementing pat during development without significantly increasing program timelines can be difficult. the development of a monoclonal antibody expressed in a chinese hamster ovary (cho) cell line via fed-batch processing presented an opportunity to demonstrate capabilities of altering percent glycated protein product. glycation is cau ...201626587969
slashing the timelines: opting to generate high-titer clonal lines faster via viability-based single cell sorting.chinese hamster ovary (cho) cell line development (cld) is a long and laborious process, which requires up to 5 - 6 months in order to generate and bank cho lines capable of stably expressing therapeutic molecules. additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and sin ...201626587808
hydroxycinnamic acid amides from scopolia tangutica inhibit the activity of m1 muscarinic acetylcholine receptor in vitro.scopolia tangutica maxim (s. tangutica) extracts have been traditionally used as antispasmodic, sedative, and analgesic agents in tibet and in the qinghai province of china. their active compositions are however poorly understood. we have recently isolated five new hydroxycinnamic acid (hca) amides along with two known hca amides, one cinnamic acid amide from these extracts. in this study, we evaluate their abilities to inhibit carbacol-induced activity of m1 muscarinic acetylcholine receptor al ...201626586621
ox2 orexin/hypocretin receptor signal transduction in recombinant chinese hamster ovary cells.there are two subtypes of orexin receptors, ox1 and ox2. signalling pathways have been mapped in much higher detail for ox1 receptors than ox2 receptors. almost all the detailed studies have been performed in chinese hamster ovary cells, and we thus chose the same cell background for the studies on human ox2 receptors to allow comparison to human ox1 receptors. adenylyl cyclase, phospholipase a2, c and d and diacylglycerol lipase activities were assessed by precursor radiolabelling and chromatog ...201626582739
an ultra scale-down approach identifies host cell protein differences across a panel of mab producing cho cell line variants.during the manufacture of biopharmaceutical products, the final product must lie within strict pre-set specifications, for example the host cell protein (hcp) content. a number of specific hcps have been identified in particular products and the interactions between product/hcps have also been recently investigated; however, a comparison of the hcp dynamics between related cell lines and their response to early downstream processing to aid process development and cell line selection has not been ...201626579700
development and characterization of a novel long-acting recombinant follicle stimulating hormone agonist by fusing fc to an fsh-β subunit.does a novel long-acting recombinant human fsh, kn015, a heterodimer composed of fshα and fshβ-fc/fc, offer a potential fsh alternative?201626621853
microfluidic assessment of mechanical cell damage by extensional stress.mammalian cells have been widely used in bioreactors to produce biological products such as pharmaceutical materials. the productivity of such bioreactors is vastly affected by flow-induced cell damage in complicated flow environments, such as agitation-driven turbulence and oxygen bubble bursting at the interface between the culturing medium and air. however, there is no systematic approach to diagnose the cell damage caused by the hydrodynamic stress. in this work, we propose a novel microflui ...201626621113
comparison of three transposons for the generation of highly productive recombinant cho cell pools and cell lines.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 s ...201626616356
identification and characterization of a -1 reading frameshift in the heavy chain constant region of an igg1 recombinant monoclonal antibody produced in cho cells.frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. we report here the identification and characterization of a novel -1 frameshift variant in a recombinant igg1 therapeutic monoclonal antibody (mab) produced in chinese hamster ovary cells during the cell line selection studies. the variant was initially observed as an atypical post-monomer fragment peak i ...201626652198
discovery and characterization of hydroxylysine in recombinant monoclonal antibodies.tryptic peptide mapping analysis of a chinese hamster ovary (cho)-expressed, recombinant igg1 monoclonal antibody revealed a previously unreported +16 da modification. through a combination of ms(n) experiments, and preparation and analysis of known synthetic peptides, the possibility of a sequence variant (ala to ser) was ruled out and the presence of hydroxylysine was confirmed. post-translational hydroxylation of lysine was found in a consensus sequence (xkg) known to be the site of modificat ...201626651858
ubiquitous chromatin opening elements (ucoes) effect on transgene position and expression stability in cho cells following methotrexate (mtx) amplification.the requirement for complex therapeutic proteins has resulted in mammalian cells, especially cho cells, being the dominant host for recombinant protein manufacturing. in creating recombinant cho cell lines, the expression vectors integrate into various parts of the genome leading to variable levels of expression and stability of protein production. this makes mammalian cell line development a long and laborious process. therefore, with the intention to accelerate process development of recombina ...201626632501
enhanced sialylation of a human chimeric igg1 variant produced in human and rodent cell lines.glycosylation of the igg-fc is essential for optimal binding and activation of fcγ receptors and the c1q component of complement. however, it has been reported that the effector functions are down-regulated when the fc glycans terminate in sialic acid residues and that sialylated igg mediates anti-inflammatory effects of intravenous immunoglobulin (ivig). although recombinant igg is hypo-sialylated, fc sialylation is shown to be markedly increased when a mouse/human chimeric igg3 phe243ala (f243 ...201626627984
a method for specifically targeting two independent genomic integration sites for co-expression of genes in cho cells.stable mammalian production cell lines in suspension culture enable the reproducible expression of target genes in any desired scale using bioreactor technology. targeted integration methods have been developed to cut down timelines for the generation of stable producer cell lines. the powerful flp recombinase mediated cassette exchange (rmce) technique allows fast integration of target genes in preselected and optimized high expression loci in so called master cell lines. up to now, these cells ...201626658354
valeric acid induces cell cycle arrest at g1 phase in cho cell cultures and improves recombinant antibody productivity.to find a more effective chemical reagent for improved monoclonal antibody (mab) production, eight chemical reagents (curcumin, quercein, dl-sulforaphane, thymidine, valeric acid, phenyl butyrate, valproic acid, and lithium chloride) known to induce cell cycle arrest were examined individually as chemical additives to recombinant cho (rcho) cell cultures producing mab. among these chemical additives, valeric acid showed the best production performance. valeric acid decreased specific growth rate ...201626663903
overexpression of yy1 increases the protein production in mammalian cells.the production of therapeutic antibodies using mammalian cells remains a high-priority in the biopharmaceutical manufacturing industry. bioengineers have targeted different cellular processes, including transcription, translation, secretion and post-translational modifications, to overcome the metabolic bottlenecks limiting production capacity and create high-producing mammalian cell lines. the polycomb group (pcg) proteins belong to a family of chromatin regulators with important roles in multi ...201626686315
dysbindin-1 modifies signaling and cellular localization of recombinant, human d₃ and d₂ receptors.dystrobrevin binding protein-1 (dysbindin-1), a candidate gene for schizophrenia, modulates cognition, synaptic plasticity and frontocortical circuitry and interacts with glutamatergic and dopaminergic transmission. loss of dysbindin-1 modifies cellular trafficking of dopamine (da) d2 receptors to increase cell surface expression, but its influence upon signaling has never been characterized. further, the effects of dysbindin-1 upon closely related d3 receptors remain unexplored. hence, we exami ...201626685100
glycosylphosphatidylinositol anchor modification machinery deficiency is responsible for the formation of pro-prion protein (prp) in bxpc-3 protein and increases cancer cell motility.the normal cellular prion protein (prp) is a glycosylphosphatidylinositol (gpi)-anchored cell surface glycoprotein. however, in pancreatic ductal adenocarcinoma cell lines, such as bxpc-3, prp exists as a pro-prp retaining its glycosylphosphatidylinositol (gpi) peptide signaling sequence. here, we report the identification of another pancreatic ductal adenocarcinoma cell line, aspc-1, which expresses a mature gpi-anchored prp. comparison of the 24 genes involved in the gpi anchor modification pa ...201626683373
importance of interaction between integrin and actin cytoskeleton in suspension adaptation of cho cells.the biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. this environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as chinese hamster ovary (cho) cells. use of cho cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. the underlying molecu ...201626679704
cd147 overexpression promotes tumorigenicity in chinese hamster ovary cells.cd147 overexpresses in many epithelium-originated tumors and plays an important role in tumor migration and invasion. most studies aim at the role of cd147 in tumor progression using tumor cell models. however, the influence of abnormal overexpression of cd147 on neoplastic transformation of normal cells is unknown. here, the role of cd147 in malignant phenotype transformation in cho cells was investigated. three cho cell lines that stably overexpressed cd147 (cho-cd147), egfp-cd147 (cho-egfp-cd ...201626676266
hypertension enhances aβ-induced neurovascular dysfunction, promotes β-secretase activity, and leads to amyloidogenic processing of app.hypertension (htn) doubles the risk of alzheimer’s disease (ad), but the mechanisms remain unclear. amyloid-β (aβ), a key pathogenic factor in ad, induces cerebrovascular dysfunction. we hypothesized that htn acts in concert with aβ to amplify its deleterious cerebrovascular effects and to increase aβ production. infusion of angiotensin ii (angii; intravenously) elevated blood pressure and attenuated the cerebral blood flow (cbf) response to whisker stimulation or the endothelium-dependent vasod ...201625920959
effect of electromagnetic field on cyclic adenosine monophosphate (camp) in a human mu-opioid receptor cell model.during the cell communication process, endogenous and exogenous signaling affect normal as well as pathological developmental conditions. exogenous influences such as extra-low-frequency electromagnetic field (emf) have been shown to effect pain and inflammation by modulating g-protein receptors, down-regulating cyclooxygenase-2 activity, and affecting the calcium/calmodulin/nitric oxide pathway. investigators have reported changes in opioid receptors and second messengers, such as cyclic adenos ...201626151161
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
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
preclinical safety evaluation of low molecular weight heparin-deoxycholate conjugates as an oral anticoagulant.the preclinical safety of a newly developed oral anticoagulant, the low molecular weight heparin-deoxycholate conjugate (oh09208), was evaluated by a comprehensive evaluating program in compliance with standard guidelines. the single dose oral toxicity study in rats receiving 2000 and 5000 mg kg(-1) of oh09208 did not reveal any mortality, unusual body weight changes or necropsy findings. the results of the 4-week oral toxicity study with a 4-week recovery program in rats receiving oh09208 in do ...201625900269
in vitro evaluation of low-intensity light radiation on murine melanoma (b16f10) cells.changes in the energy state of biomolecules induced by electromagnetic radiation lead to changes in biological functions of irradiated biomolecules. using the rrm approach, it was computationally predicted that far-infrared light irradiation in the range of 3500-6000 nm affects biological activity of proto-oncogene proteins. this in vitro study evaluates quantitatively and qualitatively the effects of selected far-infrared exposures in the computationally determined wavelengths on mouse melanoma ...201626002595
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
using simple models to describe the kinetics of growth, glucose consumption, and monoclonal antibody formation in naive and infliximab producer cho cells.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™ in ...201626091615
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
detection of adventitious agents using next-generation sequencing.next-generation sequencing has been evaluated at genzyme as a means of identifying bioreactor contaminants due to its capability for detection of known and novel microbial species. in this approach, data obtained from next-generation sequencing is used to interrogate databases containing genomic sequences and identities of potential adventitious agents. we describe here the use of this approach to help identify the causative agent of a bioreactor contamination. we also present the results of spi ...201625475640
meeting report-workshop on spike characterizations and virus removal by filtration: trends and new developments.this workshop was held on june 3, 2013, in berlin, germany, in conjunction with the pda virus & tse safety forum 2013. a total of nine speakers presented on key considerations of virus filtration, including a historical overview and emerging trends in evaluating parvovirus filters. several talks addressed understanding the mechanism of virus capture and breakthrough by filters, as well as addressing this risk by carefully controlling transmembrane pressure. improvements to validation studies wer ...201625188344
cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics.protein glycosylation is an important post-translational modification toward the structure and function of recombinant therapeutics. the addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. it is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. in this work, we ...201625132658
combination of dextran sulfate and recombinant trypsin on aggregation of chinese hamster ovary cells.in laboratory scale therapeutical protein production, cell clumps form typically in shake flasks, which hinders cell growth and decreases protein yield. to minimize clumps during the culture of chinese hamster ovary cells, we employed the combination of two reagents, dextran sulfate (ds) and recombinant trypsin (r-trypsin). our results showed that both ds and r-trypsin could diminish cell aggregation when adding them respectively, but clumps were still noticed obviously. in order to further miti ...201625087075
comparison of three different cell viability assays for evaluation of vanadyl sulphate cytotoxicity in a chinese hamster ovary k1 cell line.previously, evaluation of sodium metavanadate (navo3) cytotoxicity after 24 h exposure of chinese hamster ovary k1 (cho-k1) cells revealed different sensitivity of the in vitro assays used starting from the neutral red (nr, 3-amino-7-dimethylamino-2-methylphenazine hydrochloride) test (detecting lysosomal and possibly the golgi apparatus damage) as the most sensitive followed by the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2h-tetrazolium-5-carboxyanilide inner salt (xtt) and resazurin (7-hydroxy ...201625107459
induction of cad gene amplification by restriction endonucleases in v79,b7 chinese hamster cells.the restriction endonucleases pvuii, bamhi and ecori were tested for their ability to induce gene amplification in v79,b7 chinese hamster cells. the results indicate that treatment with these enzymes efficiently increases the frequency of clones resistant to n-phosphonacetyl-l-aspartate, indicating induction of cad gene amplification.20162563307
the pathobiology of ozone-induced damage.ozone remains one of the three most important air pollutants worldwide, yet little direct documented evidence of its genotoxicity exists. the interest in the pathology of ozone exposure and the molecular events that underlie its course stems from dna damage caused by oxygen stress including hydroxyl radicals, superoxide, singlet oxygen, and hydrogen peroxide. although the tissue damage associated with ozone inhalation occurs at both the conducting airway and the alveolus, the cellular and mechan ...20162185706
embryotoxicity and mutagenicity of mycotoxins.embryotoxicity: aflatoxin b1 (afb1), g1 (afg1), and patulin (pa) were investigated in nmri mice for embryotoxic and teratogenic activity. these three mycotoxins were injected intraperitoneally or given orally on day 12 and 13 of pregnancy. afb1 (15, 45, and 90 mg/kg ip or 45 mg/kg po) produced moderate retardation in fetal development and a dose-related increase of cleft palates, wavy ribs, and diaphragm changes. the effects after injection of afg1 (45 to 90 mg/kg ip) were reduction of fetal wei ...20162121956
[antimutagenic activity of dextran gammaphos derivatives].in experiments with v-79 chinese hamster cell culture the influence of dextran gammaphos derivatives on the mutagenic effects of gamma-radiation was studied by the number of cells with micronuclei and fragmented nuclei. products of interaction between gammaphos and dialdehyde dextran were shown to have a higher antimutagenic activity than gammaphos.20161719580
[the antimutagenic activity of compounds of modified polyglucin].the effect of modified polygluquin compounds on gamma-irradiated nuclei of v-79 chinese hamster cells has been studied. antimutagenic properties of the compounds are determined by aldehyde groups and oxidized chains.20161703176
[the lethal and mutagenic actions of radiations with different lets on mammalian cells].inactivation and induction of mutations in the hgprt locus in chinese hamster cells after irradiation with accelerated heavy ions in the let range of 20 to 367 kev/micron were studied. in both cases, inactivation and induction of mutations, the let dependence of rbe is described by a curve with a local maximum in the range of 80 to 100 kev/micron. the maximum rbe value for the mutagenic action is almost twice as high as that for inactivation. however, the rbe coefficients of the mutation inducti ...20169181964
inhibition of methotrexate-induced chromosomal damage by vanillin and chlorophyllin in v79 cells.methotrexate (mtx), a chemotherapeutic agent used to treat cancer, produces cytogenetic damage and has a cytostatic effect in a variety of test systems. several antigenotoxic agents have been studied in various in vitro and in vivo systems. however, data are limited regarding their ability to modulate mtx-induced genotoxicity. in the present study, vanillin (va) and chlorophyllin (chl) were used as antigenotoxic agents to study their ability to minimize the dna damage caused by mtx. exponentiall ...20169485540
[the mutagenic action of gamma radiation on chinese hamster cells. the cytogenetic characteristics of the mutants at the hprt locus].the peculiarities of mutation induction and cytogenetic characteristics of spontaneous and radiation-induced hprt mutant clones have been studied. the linear-quadratic dependence of the mutation induction on radiation dose has been found. high heterogeneity of cytogenetic parameters (aneuploidy and chromosome aberration frequency) has been shown in the mutants.20169026291
selection of n-(phosphonacetyl)-l-aspartate resistant chinese hamster mutants in the presence of the uridine uptake inhibitor dipyridamole.in mammalian cells selected in culture for resistance to pala the cad gene is amplified and these cells are a widely used model system to study gene amplification. selection of resistant mutants is routinely performed in medium supplemented with dialyzed serum, because the cytotoxic effect of pala is reversed by uridine, which is contained in serum. we have shown that in chinese hamster cells dipyridamole reduced uridine uptake to less than 5% with limited effect on cell survival. moreover, in m ...20167733632
hyperthermic sensitization by the radical initiator 2,2'-azobis (2-amidinopropane) dihydrochloride (aaph). i. in vitro studies.aaph (2,2'-azobis-(2-amidinopropane dihydrochloride)) is a water-soluble, heat-labile azo compound which undergoes thermal decomposition to produce carbon-centred free radicals. these carbon-centred radicals might be directly cytotoxic or may react with oxygen to produce potentially cytotoxic alkoxyl and peroxyl radicals. the rate of free radical production as a result of aaph thermal decomposition increases with increasing temperature. we have evaluated the efficacy of aaph as a heat sensitizer ...20168064185
[effect of chronic neutron irradiation on fibroblast cell culture in chinese hamster].the effect of monoenergy (30 gev) neutron radiation on chinese hamster fibroblasts has been studied by micronuclear test. it has been shown that in the range of doses up to 60 cgy rbe is equal to unity.20168293117
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