Publications

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the long intron 1 of growth hormone gene from reeves' turtle (chinemys reevesii) correlates with negatively regulated gh expression in four cell lines.turtles grow slowly and have a long lifespan. ultrastructural studies of the pituitary gland in reeves' turtle (chinemys reevesii) have revealed that the species possesses a higher nucleoplasmic ratio and fewer secretory granules in growth hormone (gh) cells than other animal species in summer and winter. c. reevesii gh gene was cloned and species-specific similarities and differences were investigated. the full gh gene sequence in c. reevesii contains 8517 base pairs (bp), comprising five exons ...201627077853
one-step conjugation method for site-specific antibody-drug conjugates through reactive cysteine-engineered antibodies.engineered cysteine residues are particularly convenient for site-specific conjugation of antibody-drug conjugates (adc), because no cell engineering and additives are required. usually, unpaired cysteine residues form mixed disulfides during fermentation in chinese hamster ovarian (cho) cells; therefore, additional reduction and oxidization steps are required prior to conjugation. in this study, we prepared light chain (lc)-q124c variants in igg and examined the conjugation efficiency. intrigui ...201627074832
comprehensive genome and epigenome characterization of cho cells in response to evolutionary pressures and over time.the most striking characteristic of cho cells is their adaptability, which enables efficient production of proteins as well as growth under a variety of culture conditions, but also results in genomic and phenotypic instability. to investigate the relative contribution of genomic and epigenetic modifications towards phenotype evolution, comprehensive genome and epigenome data are presented for six related cho cell lines, both in response to perturbations (different culture conditions and media a ...201627072894
quantitative analysis of the supernatant from host and transfected cho cells using itraq 8-plex technique.we employed uplc-ms/ms with itraq 8-plex labeling to quantitatively analyze the supernatant produced by two chinese hamster ovary (cho) cell lines (cho k1sv and cho cat-s). in each case, the supernatant from the host and three transfected clones were analyzed at days 5, 7, and 10 of culture. a total of eight itraq 8-plex experiments were performed. for each cell line, the overlap of supernatant protein identifications between transfected clones is over 60%. over 70% of the supernatant proteins i ...201627070921
isotetrandrine reduces astrocyte cytotoxicity in neuromyelitis optica by blocking the binding of nmo-igg to aquaporin 4.neuromyelitis optica (nmo) is a severe neurological demyelinating autoimmune disease that affects the optic nerves and spinal cord with no cure and no fda-approved therapy. research over the last decade revealed that the binding of nmo-igg to the water channel protein astrocyte aquaporin 4 (aqp4) might be the primary cause of nmo pathogenesis. the purpose of this study was to identify potential blockers of nmo-igg and aqp4 binding.201627064690
characterization of recombinant human diamine oxidase (rhdao) produced in chinese hamster ovary (cho) cells.human diamine oxidase (hdao) efficiently degrades polyamines and histamine. reduced enzyme activities might cause complications during pregnancy and be involved in histamine intolerance. so far hdao has been characterized after isolation from either native sources or the heterologous production in insect cells. accessibility to human enzyme is limited and insect cells produce non-human glycosylation patterns that may alter its biochemical properties. we present the heterologous expression of hda ...201627063138
poly-pathway model, a novel approach to simulate multiple metabolic states by reaction network-based model - application to amino acid depletion in cho cell culture.mammalian cell lines are characterized by a complex and flexible metabolism. a single model that could describe the variations in metabolic behavior triggered by variations in the culture conditions would be a precious tool in bioprocess development. in this paper, we introduce an approach to generate a poly-pathway model and use it to simulate diverse metabolic states triggered in response to removal, reduction or doubling of amino acids in the culture medium of an antibody-producing cho cell l ...201627060554
polysorbates 20 and 80 degradation by group xv lysosomal phospholipase a2 isomer x1 in monoclonal antibody formulations.decreases in polysorbate (ps80) content were observed while evaluating the long-term storage stability of chinese hamster ovary-derived, purified monoclonal antibodies. it was determined that polysorbate had been enzymatically degraded; therefore, studies were performed to identify and characterize the protein(s) responsible. polysorbate degrading activity was enriched from chinese hamster ovary media leading to the identification of group xv lysosomal phospholipase a2 isomer x1 (lpla2) by shotg ...201627056628
predominant expression of hybrid n-glycans has distinct cellular roles relative to complex and oligomannose n-glycans.glycosylation modulates growth, maintenance, and stress signaling processes. consequently, altered n-glycosylation is associated with reduced fitness and disease. therefore, expanding our understanding of n-glycans in altering biological processes is of utmost interest. herein, clustered regularly interspaced short palindromic repeats/caspase9 (crispr/cas9) technology was employed to engineer a glycosylation mutant chinese hamster ovary (cho) cell line, k16, which expresses predominantly hybrid ...201627304954
graphene quantum dots from graphite by liquid exfoliation showing excitation-independent emission, fluorescence upconversion and delayed fluorescence.facile synthesis of 2-10 nm-sized graphene quantum dots (gqds) from graphite powder by organic solvent-assisted liquid exfoliation using a sonochemical method is reported in this study. synthesized gqds are well dispersed in organic solvents like ethyl acetoacetate (eaa), dimethyl formamide (dmf) and also in water. maldi-tof mass spectrometry reveals its selective mass fragmentation. detailed characterizations by various techniques like x-ray diffraction (xrd), fourier transform infrared spectro ...201627302411
targeted delivery of epirubicin to cancer cells by polyvalent aptamer system in vitro and in vivo.the clinical use of epirubicin (epi), as an anthracycline drug, is limited because of its cardiotoxicity. here, an epirubicin (epi)-modified polyvalent aptamer system (mpas) conjugate was developed for the treatment of both murine colon carcinoma cells (c26) and breast cancer cells (mcf-7).201627283831
corrigendum to "characterization of recombinant human diamine oxidase (rhdao) produced in chinese hamster ovary (cho) cells" [j. biotechnol. 227 (2016) 120-130]. 201627268045
[effect of intron orientation on the expression of transgene imposed by mar expression vector in stably recombinant cho cells].to determine the effect of intron orientation on the transgene expression level imposed by matrix attachment region (mar) expression vector.201627263292
effect of ionic liquids on zebrafish (danio rerio) viability, behavior, and histology; correlation between toxicity and ionic liquid aggregation.the effect of 11 common amidinium, imidazolium, and phosphonium based ionic liquids (ils) on zebrafish (danio rerio) and chinese hamster ovary cells (cho) was investigated with specific emphasis on the effect of anion and cation chain length and aggregation of phosphonium based ils. viability and behavioral alteration in the locomotor activity and place preference, after il treatment of 5 days postfertilization larvae, was recorded. behavior and histological damage evaluation was performed for a ...201627253865
application of unfolded protein response for production of biologics.improvements in the productivity of established mammalian cell lines used for biopharmaceutical production are desirable to increase product yields. chinese hamster ovary (cho) cells are the workhorse used for more than 60% of industrial therapeutic antibody production. glycoprotein secretion by cho cells requires intracellular processes including transcription, translation, glycosylation, and secretion. within these intracellular steps, post-translational processes are rate limiting for product ...201627252063
the cyclic amp assay using human cannabinoid cb2 receptor-transfected cells.the cyclic amp assay is a functional assay that is commonly used to determine the pharmacological behavior (agonists, antagonists, inverse agonists) of g-protein-coupled receptor (gpcr) ligands. here, we describe the cyclic amp assay that is carried out with commercially available non-radioligand ready-to-use kits and chinese hamster ovarian (cho) cells stably transfected with the human cannabinoid cb2 receptor.201627245894
the displacement binding assay using human cannabinoid cb2 receptor-transfected cells.displacement binding assays are nonfunctional assays mostly used with the aim of determining whether a certain compound (plant-derived or synthetic) is able to bind to a specific receptor with high affinity. here, we describe the displacement binding assay that is carried out with a radioligand and cho (chinese hamster ovarian) cells stably transfected with the human cannabinoid cb2 receptor.201627245891
contribution of antibody hydrodynamic size to vitreal clearance revealed through rabbit studies using a species-matched fab.we have developed a tool fab fragment of a rabbit monoclonal antibody that is useful for early evaluation in rabbit models of technologies for long acting delivery (lad) of proteins to the eye. using this fab we show that vitreal clearance can be slowed through increased hydrodynamic size. fab (g10rabfab) and fab' (g10rabfab') fragments of a rabbit monoclonal antibody (g10rabigg) were expressed in chinese hamster ovary (cho) cells and purified using antigen-based affinity chromatography. g10rabf ...201627244474
g-protein-dependency of orexin/hypocretin receptor signalling in recombinant chinese hamster ovary cells.multiple signalling pathways for orexin receptors have been discovered, and most thoroughly mapped in chinese hamster ovary k1 (cho-k1) cells. it is also known that orexin receptors can couple to the g-protein families gi, gs and gq. however, the connection between the g-proteins and the downstream signals is only vaguely established, and we now set out to resolve this for human orexin receptors expressed in cho-k1 cells. adenylyl cyclase (ac), phospholipase a2, c and d, and diacylglycerol lipas ...201627237973
isolation and characterization of the corticotropin-releasing factor-related diuretic hormone receptor in rhodnius prolixus.rhodnius prolixus, the vector of human chagas disease, is a hemipteran insect that undergoes rapid post-feeding diuresis following ingestion of a blood meal that can be up to 10 times its initial body weight. corticotropin-releasing factor-related diuretic hormone (rhopr-crf/dh) and serotonin are neurohormones that are synergistic in increasing rates of fluid secretion by malpighian tubules during this rapid post-feeding diuresis. a rhopr-crf/dh receptor transcript has now been isolated and char ...201627237375
novel glyceryl glucoside is a low toxic alternative for cryopreservation agent.glyceryl glucoside (gg, α-d-glucosyglycerol) is a natural glycerol derivative found in alcoholic drinks. recently gg has been used as an alternative for glycerol in cosmetic products. however, the safety of using gg is still unclear. currently, dimethyl sulfoxide (dmso) and glycerol are wildly used in cryopreservation. despite gg being a derivative of glycerol, the ability of gg in cryopreservation is still unknown. by using a system of chinese hamster ovary cells (cho), a549 cells and ag1522 ce ...201627235553
expression and characterization of a potent long-acting glp-1 receptor agonist, glp-1-igg2σ-fc.human glp-1 (glucagon-like peptide-1) can produce a remarkable improvement in glycemic control in patients with type 2 diabetes. however, its clinical benefits are limited by its short half-life, which is less than 2 min because of its small size and rapid enzymatic inactivation by dipeptidyl peptidase iv. we engineered glp-1-igg2σ-fc, a 68-kda fusion protein linking a variant human glp-1 (a8g/g26e/r36g) to a human igg2σ constant heavy-chain. a stably transfected chinese hamster ovary cell line ...201627232339
moderate and strong static magnetic fields directly affect egfr kinase domain orientation to inhibit cancer cell proliferation.static magnetic fields (smfs) can affect cell proliferation in a cell-type and intensity-dependent way but the mechanism remains unclear. at the same time, although the diamagnetic anisotropy of proteins has been proposed decades ago, the behavior of isolated proteins in magnetic fields has not been directly observed. here we show that smfs can affect isolated proteins at the single molecular level in an intensity-dependent manner. we found that epidermal growth factor receptor (egfr), a protein ...201627223425
physiological modulators of kv3.1 channels adjust firing patterns of auditory brain stem neurons.many rapidly firing neurons, including those in the medial nucleus of the trapezoid body (mntb) in the auditory brain stem, express "high threshold" voltage-gated kv3.1 potassium channels that activate only at positive potentials and are required for stimuli to generate rapid trains of actions potentials. we now describe the actions of two imidazolidinedione derivatives, aut1 and aut2, which modulate kv3.1 channels. using chinese hamster ovary cells stably expressing rat kv3.1 channels, we found ...201627052580
characterization of alanine to valine sequence variants in the fc region of nivolumab biosimilar produced in chinese hamster ovary cells.nivolumab is a therapeutic fully human igg4 antibody to programmed death 1 (pd-1). in this study, a nivolumab biosimilar, which was produced in our laboratory, was analyzed and characterized. sequence variants that contain undesired amino acid sequences may cause concern during biosimilar bioprocess development. we found that low levels of sequence variants were detected in the heavy chain of the nivolumab biosimilar by ultra performance liquid chromatography (uplc) and tandem mass spectrometry. ...201627050807
biphasic cultivation strategy to avoid epo-fc aggregation and optimize protein expression.in biphasic cultivations, the culture conditions are initially kept at an optimum for rapid cell growth and biomass accumulation. in the second phase, the culture is shifted to conditions ensuring maximum specific protein production and the protein quality required. the influence of specific culture parameters is cell line dependent and their impact on product quality needs to be investigated. in this study, a biphasic cultivation strategy for a chinese hamster ovary (cho) cell line expressing a ...201627050504
measurement of rapid amiloride-dependent ph changes at the cell surface using a proton-sensitive field-effect transistor.we present a novel method for the rapid measurement of ph fluxes at close proximity to the surface of the plasma membrane in mammalian cells using an ion-sensitive field-effect transistor (isfet). in conjuction with an efficient continuous superfusion system, the isfet sensor was capable of recording rapid changes in ph at the cells' surface induced by intervals of ammonia loading and unloading, even when using highly buffered solutions. furthermore, the system was able to isolate physiologicall ...201627043644
cell growth on 3d microstructured surfaces.chinese hamster ovary (cho) cell cultures were grown on surfaces lithographed with periodic 3d hexagonal microcavity array morphology. the range of microcavity size (inscribed circle diameter) was from 12 μm to 560 μm. cho cells were grown also on flat surfaces. the characterization was performed with respect to cell growth density (number of nuclei per unit area) by fluorescence optical microscopy and evaluated by correlation function analysis. we found that optimum microcavity radius was 80 μm ...201627040266
helium ions for radiotherapy? physical and biological verifications of a novel treatment modality.modern facilities for actively scanned ion beam radiotherapy allow in principle the use of helium beams, which could present specific advantages, especially for pediatric tumors. in order to assess the potential use of these beams for radiotherapy, i.e., to create realistic treatment plans, the authors set up a dedicated (4)he beam model, providing base data for their treatment planning system trip98, and they have reported that in this work together with its physical and biological validations.201627036594
characterization of melanocortin receptors from stingray dasyatis akajei, a cartilaginous fish.melanocortin (mc) systems are composed of mc peptides such as adrenocorticotropic hormone (acth), several molecular forms of melanocyte-stimulating hormones (mshs) and mc receptors (mcrs). here we demonstrated that the cartilaginous fish, dasyatis akajei (stingray) expresses five subtypes of mcr genes-mc1r to mc5r-as in the case of teleost and tetrapod species. this is the first evidence showing the presence of the full repertoire of melanocortin receptors in a single of cartilaginous fish. expr ...201627021018
oxytocin neuron activation prevents hypertension that occurs with chronic intermittent hypoxia/hypercapnia in rats.hypertension is a common outcome associated with obstructive sleep apnea (osa), a prevalent yet poorly treated cardiovascular disease. recent studies showed oxytocin (oxt), released from hypothalamic paraventricular nucleus (pvn) neurons, activates cardiac vagal neurons in the dorsal motor nucleus of the vagus (dmnx) and may blunt cardiovascular responses to stress. this study tests whether the release of oxt from pvn fibers in the dmnx is diminished with chronic intermittent hypoxia-hypercapnia ...201627016581
putting the spotlight back on plant suspension cultures.plant cell suspension cultures have several advantages that make them suitable for the production of recombinant proteins. they can be cultivated under aseptic conditions using classical fermentation technology, they are easy to scale-up for manufacturing, and the regulatory requirements are similar to those established for well-characterized production systems based on microbial and mammalian cells. it is therefore no surprise that taliglucerase alfa (elelyso®)-the first licensed recombinant ph ...201627014320
glycan-deficient prp stimulates vegfr2 signaling via glycosaminoglycan.whether the two n-linked glycans are important in prion, prp, biology is unresolved. in chinese hamster ovary (cho) cells, the two glycans are clearly not important in the cell surface expression of transfected human prp. compared to fully-glycosylated prp, glycan-deficient prp preferentially partitions to lipid raft. in cho cells glycan-deficient prp also interacts with glycosaminoglycan (gag) and vascular endothelial growth factor receptor 2 (vegfr2), resulting in vegfr2 activation and enhance ...201627006333
genetic mutation analysis at early stages of cell line development using next generation sequencing.a central goal for most biopharmaceutical companies is to reduce the development timeline to reach clinical proof of concept. this objective requires the development of tools that ensure the quality of biotherapeutic material destined for the clinic. recent advances in high throughput protein analytics provide confidence in our ability to assess productivity and product quality attributes at early stages of cell line development. however, one quality attribute has, until recently, been absent fr ...201627004436
mirna profiling of high, low and non-producing cho cells during biphasic fed-batch cultivation reveals process relevant targets for host cell engineering.fed-batch cultivation of recombinant chinese hamster ovary (cho) cell lines is one of the most widely used production modes for commercial manufacturing of recombinant protein therapeutics. furthermore, fed-batch cultivations are often conducted as biphasic processes where the culture temperature is decreased to maximize volumetric product yields. however, it remains to be elucidated which intracellular regulatory elements actually control the observed pro-productive phenotypes. recently, severa ...201627002234
biocompatibility and degradation of gold-covered magneto-elastic biosensors exposed to cell culture.magneto-elastic materials (me) have important advantages when applied as biosensors due to the possibility of wireless monitoring. commercial metglas 2826mb3™ (fenimob) is widely used, however sensor stabilization is an important factor for biosensor performance. this study compared the effects of biocompatibility and degradation of the metglas 2826mb3™ alloy, covered or not with a gold layer, when in contact with cell culture medium. strips of amorphous metglas 2826mb3™ were cut and coated with ...201626998872
experimental evaluation of proposed small-molecule inhibitors of water channel aquaporin-1.the aquaporin-1 (aqp1) water channel is a potentially important drug target, as aqp1 inhibition is predicted to have therapeutic action in edema, tumor growth, glaucoma, and other conditions. here, we measured the aqp1 inhibition efficacy of 12 putative small-molecule aqp1 inhibitors reported in six recent studies, and one aqp1 activator. osmotic water permeability was measured by stopped-flow light scattering in human and rat erythrocytes that natively express aqp1, in hemoglobin-free membrane ...201626993802
a signature of 12 micrornas is robustly associated with growth rate in a variety of cho cell lines.as chinese hamster ovary (cho) cells are the cell line of choice for the production of human-like recombinant proteins, there is interest in genetic optimization of host cell lines to overcome certain limitations in their growth rate and protein secretion. at the same time, a detailed understanding of these processes could be used to advantage by identification of marker transcripts that characterize states of performance. in this context, micrornas (mirnas) that exhibit a robust correlation to ...201626993211
characterization of endogenous sodium channels in the nd7-23 neuroblastoma cell line: implications for use as a heterologous ion channel expression system suitable for automated patch clamp screening.the rodent neuroblastoma cell line, nd7-23, is used to express voltage-dependent sodium (nav) and other neuronal ion channels resistant to heterologous expression in chinese hamster ovary (cho) or human embryonic kidney (hek) cells. their advantage is that they provide endogenous factors and signaling pathways to promote ion channel peptide folding, expression, and function at the cell surface and are also amenable to automated patch clamping. however, nd7-23 cells exhibit endogenous tetrodotoxi ...201626991361
effect of temperature downshift on the transcriptomic responses of chinese hamster ovary cells using recombinant human tissue plasminogen activator production culture.recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. hence, improvement of bioprocess productivity is greatly needed. a temperature downshift (tds) from 37°c to 28-34°c is an effective strategy to expand the productive life period of cells and increase their productivity (qp). here, tds in chinese hamster ovary (cho) cell cultures, initially grown at 37°c and switched to 30°c during the exponential growth phase, resulted in a 1. ...201626991106
carboxypeptidase d is the only enzyme responsible for antibody c-terminal lysine cleavage in chinese hamster ovary (cho) cells.heterogeneity of c-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. identifying the responsible carboxypeptidase(s) for c-terminal lysine cleavage in cho cells would provide valuable insights for antibody production cell culture processes development and optimization. in this study, five carboxypeptidases, cpd, cpm, cpn, cpb, and cpe, were studied for message rna ...201626989081
double targeting and aptamer-assisted controlled release delivery of epirubicin to cancer cells by aptamers-based dendrimer in vitro and in vivo.clinical use of epirubicin (epi) in the treatment of cancer has been limited, due to its cardiotoxicity. targeted delivery of chemotherapeutic agents could increase their efficacy and reduce their off-target effects. high drug loading and excellent stability of dna dendrimers make these dna nanostructures unique candidates for biological applications. in this study a modified and promoted dendrimer using three kinds of aptamers (muc1, as1411 and atp aptamers) was designed for targeted delivery o ...201626987703
granular activated carbon treatment may result in higher predicted genotoxicity in the presence of bromide.certain unregulated disinfection byproducts (dbps) are more of a health concern than regulated dbps. brominated species are typically more cytotoxic and genotoxic than their chlorinated analogs. the impact of granular activated carbon (gac) on controlling the formation of regulated and selected unregulated dbps following chlorine disinfection was evaluated. the predicted cyto- and genotoxicity of dbps was calculated using published potencies based on the comet assay for chinese hamster ovary cel ...201627467860
tumor targeting with novel 6-substituted pyrrolo [2,3-d] pyrimidine antifolates with heteroatom bridge substitutions via cellular uptake by folate receptor α and the proton-coupled folate transporter and inhibition of de novo purine nucleotide biosynthesis.targeted antifolates with heteroatom replacements of the carbon vicinal to the phenyl ring in 1 by n (4), o (8), or s (9), or with n-substituted formyl (5), acetyl (6), or trifluoroacetyl (7) moieties, were synthesized and tested for selective cellular uptake by folate receptor (fr) α and β or the proton-coupled folate transporter. results show increased in vitro antiproliferative activity toward engineered chinese hamster ovary cells expressing frs by 4-9 over the ch2 analogue 1. compounds 4-9 ...201627458733
high expression of xrcc6 promotes human osteosarcoma cell proliferation through the β-catenin/wnt signaling pathway and is associated with poor prognosis.increasing evidences show that xrcc6 (x-ray repair complementing defective repair in chinese hamster cells 6) was upregulated and involved in tumor growth in several tumor types. however, the correlation of xrcc6 and human osteosarcoma (os) is still unknown. this study was conducted with the aim to reveal the expression and biological function of xrcc6 in os and elucidate the potential mechanism. the mrna expression level of xrcc6 was measured in osteosarcoma cells and os samples by quantitative ...201627455247
tracking dipeptides at work-uptake and intracellular fate in cho culture.market demands for monoclonal antibodies (mabs) are steadily increasing worldwide. as a result, production processes using chinese hamster ovary cells (cho) are in the focus of ongoing intensification studies for maximizing cell-specific and volumetric productivities. this includes the optimization of animal-derived component free (adcf) cultivation media as part of good cell culture practice. dipeptides are known to improve cho culture performance. however, little or even conflicting assumption ...201627447702
unexpected potency differences between b-cell-activating factor (baff) antagonist antibodies against various forms of baff: trimer, 60-mer, and membrane-bound.therapeutic agents antagonizing b-cell-activating factor/b-lymphocyte stimulator (baff/blys) are currently in clinical development for autoimmune diseases; belimumab is the first food and drug administration-approved drug in more than 50 years for the treatment of lupus. as a member of the tumor necrosis factor superfamily, baff promotes b-cell survival and homeostasis and is overexpressed in patients with systemic lupus erythematosus and other autoimmune diseases. baff exists in three recognize ...201627440419
parallelized cytoindentation using convex micropatterned surfaces.here we present a high-throughput, parallelized cytoindentor for local compression of live cells. the cytoindentor uses convex lens-induced confinement (clic) to indent micrometer-sized areas in single cells and/or populations of cells with submicron precision. this is accomplished using micropatterned poly(dimethylsiloxane) (pdms) films that are adhered to a convex lens to create arrays of extrusions referred to here as "posts." these posts caused local deformation of subcellular regions withou ...201627528072
[micronucleus in vitro induced by inhalable particulate matters in moxa smoke].to investigate whether inhalable particulate matters can cause the damage of chromosome or mitotic apparatus to produce micronucleus, and to evaluate genetic toxicology of moxa smoke on chromosome.201627509611
electrophysiology-based assays to detect subtype-selective modulation of human nicotinic acetylcholine receptors.the family smoking prevention and tobacco control act of 2009 (public law 111-31) gave the us food and drug administration (fda) the responsibility for regulating tobacco products. nicotine is the primary addictive component of tobacco and its effects can be modulated by additional ingredients in manufactured products. nicotine acts by mimicking the neurotransmitter acetylcholine on neuronal nicotinic acetylcholine receptors (nachrs), which function as ion channels in cholinergic modulation of n ...201627505073
a simultaneous assessment metric for mab quantity and glycan quality.as a critical quality attribute, glycosylation represents an important consideration when analyzing the success of a glycoprotein production process. though critical, glycosylation is not the only measure of culture success; other factors, including culture size, maintenance, and productivity, are also critical. a new metric was developed to address both product quality, as measured through glycosylation, and product quantity, as measured through product concentration. a monoclonal antibody chin ...201627502107
an omics approach to rational feed: enhancing growth in cho cultures with nmr metabolomics and 2d-dige proteomics.expression of recombinant proteins exerts stress on cell culture systems, affecting the expression of endogenous proteins, and contributing to the depletion of nutrients and accumulation of waste metabolites. in this work, 2d-dige proteomics was employed to analyze differential expression of proteins following stable transfection of a chinese hamster ovary (cho) cell line to constitutively express a heavy-chain monoclonal antibody. thirty-four proteins of significant differential expression were ...201627496566
elucidating the effects of ph shift on igg1 monoclonal antibody acidic charge variant levels in chinese hamster ovary cell cultures.charge variants, especially acidic charge variants, in recombinant monoclonal antibodies are critical quality attributes, which can affect antibodies' properties in vitro and in vivo. meanwhile, charge variants are cumulative effects of various post-translational modifications and chemical degradations on antibody. in this work, to investigate the effect of lowering culture ph in the stationary phase on acidic charge variant contents in fed-batch cultures and its mechanism, cell culture experime ...201627484582
evaluating the efficiency of phic31 integrase-mediated monoclonal antibody expression in cho cells.traditional methods to generate cho cell lines rely on random integration(s) of the gene of interest and result in unpredictable and unstable protein expression. in comparison, site-specific recombination methods increase the recombinant protein expression by inserting transgene at a locus with specific expression features. phic31 serine integrase, catalyze unidirectional integration that occurs at higher frequency in comparison with the reversible integration carried out by recombinases such as ...201627604579
acylation of lysine residues in human plasma high density lipoprotein increases stability and plasma clearance in vivo.although human plasma high density lipoproteins (hdl) concentrations negatively correlate with atherosclerotic cardiovascular disease, underlying mechanisms are unknown. thus, there is continued interest in hdl structure and functionality. numerous plasma factors disrupt hdl structure while inducing the release of lipid free apolipoprotein (apo) ai. given that hdl is an unstable particle residing in a kinetic trap, we tested whether hdl could be stabilized by acylation with acetyl and hexanoyl a ...201627594697
mirorna-127-3p targets xrcc3 to enhance the chemosensitivity of esophageal cancer cells to a novel phenanthroline-dione derivative.micrornas are small non-coding rnas with 18-22 nucleotides in length and have been proposed to function in various biological processes by targeting genes for post-transcriptional degradation via their 3' untranslated region. moreover, they have been suggested to improve the chemosensitivity in a panel of tumors. however, the biological functions of microrna-127-3p in esophageal carcinogenesis are still enigmatic. thus, in the study, we firstly analyzed the roles of microrna-127-3p in regulating ...201627590853
temperature-dependent changes in neuronal dynamics in a patient with an scn1a mutation and hyperthermia induced seizures.dravet syndrome is the prototype of scn1a-mutation associated epilepsies. it is characterised by prolonged seizures, typically provoked by fever. we describe the evaluation of an scn1a mutation in a child with early-onset temperature-sensitive seizures. the patient carries a heterozygous missense variant (c3818c > t; pala1273val) in the nav1.1 brain sodium channel. we compared the functional effects of the variant vs. wild type nav1.1 using patch clamp recordings from channels expressed in chine ...201627582020
tetrahydrofolate increases suspension growth of dihydrofolate reductase-deficient chinese hamster ovary dg44 cells in chemically defined media.adaptation of dihydrofolate reductase (dhfr)-deficient chinese hamster ovary (cho) dg44 cells to chemically defined suspension culture conditions is a time-consuming and labor-intensive process because nonadapted dhfr-deficient cho dg44 cells normally show poor growth in chemically defined medium (cdm). we examined the effects of folate derivatives, ribonucleotides, and nucleobases on the growth of suspension-adapted dhfr-deficient cho dg44 cells in cdm. among the tested additives, tetrahydrofol ...201627578320
progestin withdrawal at parturition in the mare.mammalian pregnancies need progestogenic support and birth requires progestin withdrawal. the absence of progesterone in pregnant mares, and the progestogenic bioactivity of 5α-dihydroprogesterone (dhp), led us to reexamine progestin withdrawal at foaling. systemic pregnane concentrations (dhp, allopregnanolone, pregnenolone, 5α-pregnane-3β, 20α-diol (3β,20αdhp), 20α-hydroxy-5α-dihydroprogesterone (20αdhp)) and progesterone) were monitored in mares for 10days before foaling (n=7) by liquid chrom ...201627568209
c-terminal of human histamine h1 receptors regulates their agonist-induced clathrin-mediated internalization and g-protein signaling.it has been suggested that the agonist-induced internalization of g-protein-coupled receptors from the cell surface into intracellular compartments regulates cellular responsiveness. we previously reported that gq/11 -protein-coupled human histamine h1 receptors internalized via clathrin-dependent mechanisms upon stimulation with histamine. however, the molecular determinants of h1 receptors responsible for agonist-induced internalization remain unclear. in this study, we evaluated the roles of ...201627566099
efficient expression of functional human coagulation factor ix in stably-transfected drosophila melanogaster s2 cells; comparison with the mammalian cho system.to compare stably-transfected drosophila melanogaster s2 and mammalian chinese hamster ovary (cho) cells for the expression and secretion efficiency of biologically-active human coagulation factor ix (hfix).201627565667
multi-omic profiling of epo-producing chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production. 201627563854
development of genetically modified chinese hamster ovary host cells for the enhancement of recombinant tissue plasminogen activator expression.chinese hamster ovary (cho) cells are the most commonly used host system for the expression of high quality recombinant proteins. however, the development of stable, high-yielding cho cell lines is a major bottleneck in the industrial manufacturing of therapeutic proteins. therefore, different strategies such as the generation of more efficient expression vectors and establishment of genetically engineered host cells have been employed to increase the efficiency of cell line development. in orde ...201627547109
identification of regulatory motifs in the cho genome for stable monoclonal antibody production.chinese hamster ovary (cho) cell lines are widely used for therapeutic protein production. when a transgene is integrated into the genome of a cho cell, the expression level is highly dependent on the site of integration because of positional effects such as gene silencing. to overcome negative positional effects and establish stable cho cell lines with high productivity, several regulatory dna elements are used in vector construction. previously, we established the cho dr1000l-4n cell line, a s ...201627544513
isothiouronium modification empowers pyrimidine-substituted curcumin analogs potent cytotoxicity and golgi localization.most of protein post-translational modifications occur in the golgi and many human diseases are associated with abnormal golgi function or improper post translational modifications of proteins in the golgi. in this study, we designed and synthesized 4 × 6 series of novel isothiouronium-modified (e,e)-4,6-bis(styryl)-pyrimidine analogs and found that they localized at the golgi as visualized by the intrinsic fluorescence of the analogs. the isothiouronium-modified analogs had potent cytotoxicity ...201627543879
detection of msiglec-e, in solution and expressed on the surface of chinese hamster ovary cells, using sialic acid functionalised gold nanoparticles.sialic acids are widespread in biology, fulfilling a wide range of functions. their cognate lectin receptors - siglecs - are equally diverse and widely distributed, with different siglecs found within distinct populations of cells in the haemopoietic, immune and nervous systems. a convenient way to assay ligand recognition of soluble siglecs would be useful, as would methods for the concomitant assessment of siglec distribution on cell surfaces. here we report the use of gold nanoparticles funct ...201627537280
systems glycobiology: integrating glycogenomics, glycoproteomics, glycomics, and other 'omics data sets to characterize cellular glycosylation processes.the number of proteins encoded in the human genome has been estimated at between 20,000 and 25,000, despite estimates that the entire proteome contains more than a million proteins. one reason for this difference is due to many post-translational modifications of protein that contribute to proteome complexity. among these, glycosylation is of particular relevance because it serves to modify a large number of cellular proteins. glycogenomics, glycoproteomics, glycomics, and glycoinformatics are h ...201627423401
application of the protein maker as a platform purification system for therapeutic antibody research and development.within the research and development environment, higher throughput, parallelized protein purification is required for numerous activities, from small scale purification of monoclonal antibodies (mabs) and antibody fragments for in vitro and in vivo assays to process development and optimization for manufacturing. here, we describe specific applications and associated workflows of the protein maker liquid handling system utilized in both of these contexts. to meet the requirements for various in ...201627418955
lack of influence of substrate on ligand interaction with the human multidrug and toxin extruder, mate1.multidrug and toxin extruder (mate) 1 plays a central role in mediating renal secretion of organic cations, a structurally diverse collection of compounds that includes ∼40% of prescribed drugs. because inhibition of transport activity of other multidrug transporters, including the organic cation transporter (oct) 2, is influenced by the structure of the transported substrate, the present study screened over 400 drugs as inhibitors of the mate1-mediated transport of four structurally distinct or ...201627418674
electrochemical potential gradient as a quantitative in vitro test platform for cellular oxidative stress.oxidative stress in a biological system is often defined as a redox imbalance within cells or groups of cells within an organism. reductive-oxidative (redox) imbalances in cellular systems have been implicated in several diseases, such as cancer. to better understand the redox environment within cellular systems, it is important to be able to characterize the relationship between the intensity of the oxidative environment, characterized by redox potential, and the biomolecular consequences of ox ...201627409641
quantification of the mutagenic potency and repair of glycidol-induced dna lesions.glycidol (gly) is an electrophilic low-molecular weight epoxide that is classified by iarc as probably carcinogenic to humans. humans might be exposed to gly from food, e.g. refined vegetable oils, where gly has been found as a food process contaminant. it is therefore important to investigate and quantify the genotoxicity of gly as a primary step towards cancer risk assessment of the human exposure. here, quantification of the mutagenic potency expressed per dose (auc: area under the concentrat ...201627402481
characterization of neuropeptide b (npb), neuropeptide w (npw), and their receptors in chickens: evidence for npw being a novel inhibitor of pituitary gh and prolactin secretion.the 2 structurally and functionally related peptides, neuropeptide b (npb) and neuropeptide w (npw), together with their receptor(s) (npbwr1/npbwr2) constitute the npb/npw system, which acts mainly on the central nervous system to regulate many physiological processes in mammals. however, little is known about this npb/npw system in nonmammalian vertebrates. in this study, the functionality and expression of this npb/npw system and its actions on the pituitary were investigated in chickens. the ...201627399877
over-expressed human trek-1 inhibits cho cell proliferation via inhibiting pka and p38 mapk pathways and subsequently inducing g1 arrest.recent studies have shown that the two-pore-domain potassium channel trek-1 is involved in the proliferation of neural stem cells, astrocytes and human osteoblasts. in this study, we investigated how trek-1 affected the proliferation of chinese hamster ovary (cho) cells in vitro.201627397543
exploring the capabilities of fluorometric online monitoring on chinese hamster ovary cell cultivations producing a monoclonal antibody.online monitoring of chinese hamster ovary fed-batch cell cultures via two-dimensional fluorescence spectroscopy (2dfs) was evaluated in this work. particular attention was directed toward different process strategies regarding the use of nutrient-rich feed media and temperature shifts. these intentionally performed process manipulations broadened the variances in the obtained fluorescence spectra and this was suspected to hamper the generation of reliable soft sensors. principal component analy ...201627390298
exposure to sirna-galnac conjugates in systems of the standard test battery for genotoxicity.registration of pharmaceuticals requires an assessment of their genotoxic potential using in vitro and in vivo tests outlined in the international conference on harmonisation (ich) guidance s2(r1). we have evaluated numerous sirna-n-acetylgalactosamine (galnac) conjugates containing phosphorothioate linkages and various combinations of 2'-fluoro and 2'-o-methyl ribose modifications of multiple nucleotides in the ich battery of assays, all of which have uniformly yielded negative results. to veri ...201627387336
heparin: past, present, and future.heparin, the most widely used anticoagulant drug in the world today, remains an animal-derived product with the attendant risks of adulteration and contamination. a contamination crisis in 2007-2008 increased the impetus to provide non-animal-derived sources of heparin, produced under cgmp conditions. in addition, recent studies suggest that heparin may have significant antineoplastic activity, separate and distinct from its anticoagulant activity, while other studies indicate a role for heparin ...201627384570
pingyangmycin and bleomycin share the same cytotoxicity pathway.pingyangmycin is an anticancer drug known as bleomycin a5 (a5), discovered in the pingyang county of zhejiang province of china. bleomycin (blm) is a mixture of mainly two compounds (a2 and b2), which is on the world health organization's list of essential medicines. both blm and a5 are hydrophilic molecules that depend on transporters or endocytosis receptors to get inside of cells. once inside, the anticancer activities rely on their abilities to produce dna breaks, thus leading to cell death. ...201627376254
augmenting chinese hamster genome assembly by identifying regions of high confidence.chinese hamster ovary (cho) cell lines are the dominant industrial workhorses for therapeutic recombinant protein production. the availability of genome sequence of chinese hamster and cho cells will spur further genome and rna sequencing of producing cell lines. however, the mammalian genomes assembled using shot-gun sequencing data still contain regions of uncertain quality due to assembly errors. identifying high confidence regions in the assembled genome will facilitate its use for cell engi ...201627374913
percutaneous treatment of herniated lumbar discs with ozone: investigation of the mechanisms of action.to elucidate the mechanism of action of intradiscal oxygen-ozone therapy for herniated intervertebral disc therapy.201627363296
[domestication of suspension cho cells and its application in the expression of anti-psma antibody].to domesticate adherent chinese hamster ovary (cho) cells into suspension cho cells (cho-s) cultured in serum-free medium,and evaluate the application of the cho-s cells in antibody expression.201627358992
the infectious bac genomic dna expression library: a high capacity vector system for functional genomics.gene dosage plays a critical role in a range of cellular phenotypes, yet most cellular expression systems use heterologous cdna-based vectors which express proteins well above physiological levels. in contrast, genomic dna expression vectors generate physiologically-relevant levels of gene expression by carrying the whole genomic dna locus of a gene including its regulatory elements. here we describe the first genomic dna expression library generated using the high-capacity herpes simplex virus- ...201627353647
subchronic and genetic safety evaluation of a calcium fructoborate in rats.a branded calcium fructoborate product, a nature-identical calcium salt of bis (fructose) ester of boric acid found in plants and a natural source of boron in the human diet and sold under the trade name fruitex-b(®) brand calcium fructoborate ("frxb"), was evaluated in a 90-day dietary toxicity study and two genotoxicity studies. in the 90-day study, four groups of 10 male and 10 female crl:sd cd(®) igs rats were fed diets with frxb admixtures of 0.56, 1.12, and 1.68% dietary concentration, pro ...201627350145
effects of heterocyclic-based head group modifications on the structure-activity relationship of tocopherol-based lipids for non-viral gene delivery.gene therapy, a promising strategy for the delivery of therapeutic nucleic acids, is greatly dependent on the development of efficient vectors. in this study, we designed and synthesized several tocopherol-based lipids varying in the head group region. here, we present the structure-activity relationship of stable aqueous suspensions of lipids that were synthetically prepared and formulated with 1,2-dioleoyl phosphatidyl ethanolamine (dope) as the co-lipid. the physicochemical properties such as ...201627348545
a theoretical estimate for nucleotide sugar demand towards chinese hamster ovary cellular glycosylation.glycosylation greatly influences the safety and efficacy of many of the highest-selling recombinant therapeutic proteins (rtps). in order to define optimal cell culture feeding strategies that control rtp glycosylation, it is necessary to know how nucleotide sugars (nss) are consumed towards host cell and rtp glycosylation. here, we present a theoretical framework that integrates the reported glycoproteome of cho cells, the number of n-linked and o-galnac glycosylation sites on individual host c ...201627345611
genotoxicity biomarkers for airborne particulate matter (pm2.5) in an area under petrochemical influence.the effects of fine inhalable particles (pm2.5) were evaluated in an area under the influence of a petrochemical industry, investigating the sensitivity of different genotoxicity biomarkers. organic extracts were obtained from pm2.5 samples at two sites, positioned in the first and second preferential wind direction in the area. the extracts were evaluated with salmonella/microsome assay, microsuspension method, strains ta98, yg1021 and yg1024. the mammalian metabolization fraction (s9) was used ...201627343868
understanding and controlling sialylation in a cho fc-fusion process.a chinese hamster ovary (cho) bioprocess, where the product is a sialylated fc-fusion protein, was operated at pilot and manufacturing scale and significant variation of sialylation level was observed. in order to more tightly control glycosylation profiles, we sought to identify the cause of variability. untargeted metabolomics and transcriptomics methods were applied to select samples from the large scale runs. lower sialylation was correlated with elevated mannose levels, a shift in glucose m ...201627310468
non-proton ligand-sensing domain of acid-sensing ion channel 3 is required for itch sensation.itch, the unpleasant sensation that evokes a desire to scratch, accompanies numerous skin and nervous system disorders. however, the molecular mechanisms of itch are unclear. acid-sensing ion channel 3 (asic3) is a sensor of acidic and primary inflammatory pain. the whole-cell patch clamp technique was used to determine the effect of chloroquine (cq) on asics currents in primary sensory neurons or the chinese hamster ovary cells transfected with rat asic1a or asic3. site-directed mutagenesis of ...201627770439
akt activation by evodiae fructus extract protects ovary against 4-vinylcyclohexene diepoxide-induced ovotoxicity.evodiae fructus (ef) is the dried, unripe fruit of evodia rutaecarpa benth., and one of the main components of traditional herbal prescriptions issued for the treatment of sterility caused by irregular menstruation in korea. however, scientific evidence regarding the efficacy and action mechanism of ef is lacking.201627769945
a comparison of orbitally-shaken and stirred-tank bioreactors: ph modulation and bioreactor type affect cho cell growth and protein glycosylation.orbitally shaken bioreactors (osrs) support the suspension cultivation of animal cells at volumetric scales up to 200 l and are a potential alternative to stirred-tank bioreactors (strs) due to their rapid and homogeneous mixing and high oxygen transfer rate. in this study, a chinese hamster ovary cell line producing a recombinant antibody was cultivated in a 5 l osr and a 3 l str, both operated with or without ph control. effects of bioreactor type and ph control on cell growth and metabolism a ...201627453130
multigene expression in stable cho cell pools generated with the piggybac transposon system.heterogenous populations of recombinant cells (cell pools) stably expressing 1-4 transgenes were generated from chinese hamster overy (cho) cells with the piggybac (pb) transposon system. the cell pools produced different combinations of three model proteins-enhanced green fluorescent protein (egfp), secreted alkaline phosphatase (seap), and a monoclonal igg1 antibody. each transgene was present on a separate pb donor plasmid with either the same or a different selection gene. in both cases, we ...201627302570
generation of stable chinese hamster ovary pools yielding antibody titers of up to 7.6 g/l using the piggybac transposon system.chinese hamster ovary (cho) cells remain the default production host for many biopharmaceutical drugs, particularly monoclonal antibodies (mab). production of gram and kilogram quantities of protein typically requires the generation of stable cho clones. unfortunately, this process takes several months, significantly slowing down the drug discovery and development process. therefore, improved technologies are needed to accelerate biopharmaceutical drug discovery and final drug substance manufact ...201627254818
elucidation of molecular mechanism(s) of cognition enhancing activity of bacomind(®): a standardized extract of bacopa monnieri.bacopa monnieri (l.) wettst., commonly known as brahmi, is renowned in indian traditional system for its potent memory enhancing activity, which has been validated by various scientific studies.201627761079
mtorc1 signalling and eif4e/4e-bp1 translation initiation factor stoichiometry influence recombinant protein productivity from gs-chok1 cells.many protein-based biotherapeutics are produced in cultured chinese hamster ovary (cho) cell lines. recent reports have demonstrated that translation of recombinant mrnas and global control of the translation machinery via mammalian target of rapamycin (mtor) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mtor complex 1 (mtorc1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relatio ...201627760840
evaluating the use of a cpg free promoter for long-term recombinant protein expression stability in chinese hamster ovary cells.methylated cpg dinucleotides in promoters are associated with the loss of gene expression in recombinant chinese hamster ovary (cho) cells during large-scale commercial manufacturing. we evaluated a promoter devoid of cpg dinucleotides, cpgfree, in parallel with a similar cpg containing promoter, cpgrich, for their ability to maintain the expression of recombinant enhanced green fluorescent protein (egfp) after 8 weeks of culturing.201627756290
antinociceptive effects of the 6-o-sulfate ester of morphine in normal and diabetic rats: comparative role of mu- and delta-opioid receptors.this study determined the antinociceptive effects of morphine and morphine-6-o-sulfate (m6s) in both normal and diabetic rats, and evaluated the comparative role of mu-opioid receptors (mu-ors) and delta-opioid receptors (delta-ors) in the antinociceptive action of these opioids. in vitro characterization of mu-or and delta-or-mediated signaling by m6s and morphine in stably transfected chinese hamster ovary (cho-k1) cells showed that m6s exhibited a 6-fold higher affinity for delta-ors and modu ...201627637375
inhibition of the ubiquitin ligase activity improves the production of biologically active fusion protein hsa-hgf in chinese hamster ovary cells.hepatocyte growth factor (hgf) is a potent multi-functional protein that stimulates proliferation, survival, motility, scattering and differentiation during growth and development, and has been considered to be a potential therapeutic agent for the treatment of a number of intractable diseases. the aim of this study was to enhance the expression of recombinant fusion protein hsa-hgf (r494e) in cho cells by inhibiting the intracellular ubiquitin ligase activity. the high stable expression sub-clo ...201627753513
molecular and functional characterization of toll-like receptor 21 in large yellow croaker (larimichthys crocea).toll-like receptor 21 (tlr21) is a non-mammalian tlr that functions similar to mammalian tlr9 in recognizing cpg dna. in the present study, we identified a tlr21 homologue, lyctlr21, from large yellow croaker (larimichthys crocea). the complete coding sequence of lyctlr21 is 2946 nucleotides long, encoding a protein of 981 amino acids. the deduced lyctlr21 protein has typical tlr domain architecture, including a signal peptide, 13 leucine-rich repeats (lrrs) in the extracellular region, a transm ...201627751918
chronic opioid treatment augments caveolin-1 scaffolding: relevance to stimulatory μ-opioid receptor adenylyl cyclase signaling.caveolin-1 is the predominant structural protein of caveolae, a subset of (lipid) membrane rafts that compartmentalize cell signaling. caveolin-1 binds most to g protein-coupled receptors and their signaling partners, thereby enhancing interactions among signaling cascade components and the relative activation of specific g protein-coupled pathways. this study reveals that chronic opioid exposure of μ-opioid receptor (mor) expressing chinese hamster ovary cells (mor-cho) and chronic in vivo morp ...201627726130
adaptation of cho cells in serum-free conditions for erythropoietin production: application of evop technique for process optimization.mammalian cell cultures are the preferred expression systems for the production of biopharmaceuticals requiring posttranslational processing. usually, cell cultures are cultivated in medium supplemented with serum, which supports cell proliferation, viability, and productivity. however, due to scientific and regulatory concerns, serum-free conditions are required in recombinant protein production. cell lines that are intended for commercial recombinant protein production have to adapt to serum- ...201626661088
understanding central carbon metabolism of rapidly proliferating mammalian cells based on analysis of key enzymatic activities in gs-cho cell lines.the central carbon metabolism (glycolysis, the pentose phosphate pathway [ppp], and the tricarboxylic acid [tca] cycle) plays an essential role in the supply of biosynthetic precursors and energy. how the central carbon metabolism changes with the varying growth rates in the in vitro cultivation of rapidly proliferating mammalian cells, such as cancer cells and continuous cell lines for recombinant protein production, remains elusive. based on relationships between the growth rate and the activi ...201626108557
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