Publications

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low glucose concentrations within typical industrial operating conditions have minimal effect on the transcriptome of recombinant cho cells.typically, mammalian cell culture medium contains high glucose concentrations that are analogous to diabetic levels in humans, suggesting that mammalian cells are cultivated in excessive glucose. using rna-seq, this study characterized the chinese hamster ovary (cho) cell transcriptome under two glucose concentrations to assess the genetic effects associated with metabolic pathways, in addition to other global responses. the initial extracellular glucose concentrations used represented high (30 ...201728371311
investigation of toxicity and mutagenicity of cold atmospheric argon plasma.cold atmospheric argon plasma is recognized as a new contact free approach for the decrease of bacterial load on chronic wounds in patients. so far very limited data are available on its toxicity and mutagenicity on eukaryotic cells. thus, the toxic/mutagenic potential of cold atmospheric argon plasma using the microplaster β(®) , which has been used efficiently in humans treating chronic and acute wounds, was investigated using the xtt assay in keratinocytes and fibroblasts and the hgprt (hypox ...201728370324
n-terminal or signal peptide sequence engineering prevents truncation of human monoclonal antibody light chains.monoclonal antibodies (mabs) contain short n-terminal signal peptides on each individual polypeptide that comprises the mature antibody, targeting them for export from the cell in which they are produced. the signal peptide is cleaved from each heavy chain (hc) and light chain (lc) polypeptide after translocation to the er and prior to secretion. this process is generally highly efficient, producing a high proportion of correctly cleaved hc and lc polypeptides. however, mis-cleavage of the signa ...201728369727
segmentation and quantitative analysis of apoptosis of chinese hamster ovary cells from fluorescence microscopy images.accurate and fast quantitative analysis of living cells from fluorescence microscopy images is useful for evaluating experimental outcomes and cell culture protocols. an algorithm is developed in this work to automatically segment and distinguish apoptotic cells from normal cells. the algorithm involves three steps consisting of two segmentation steps and a classification step. the segmentation steps are: (i) a coarse segmentation, combining a range filter with a marching square method, is used ...201728367787
the hybrid search: a mass spectral library search method for discovery of modifications in proteomics.we present a mass spectral library-based method to identify tandem mass spectra of peptides that contain unanticipated modifications and amino acid variants. we describe this as a "hybrid" method because it combines matching both ion m/z and mass losses. the mass loss is the difference between the mass of an ion peak and the mass of its precursor. this difference, termed deltamass, is used to shift the product ions in the library spectrum that contain the modification, thereby allowing library p ...201728367633
label-free protein quantification of sodium butyrate treated cho cells by esi-uhr-tof-ms.effects of butyrate on cho producer cells are contradictory, promoting productivity and at the same time repressing proliferation. though in previous omics studies the background of butyrate impact on producer cells has been investigated, the knowledge about the mechanism is still very limited. as previous proteomic results on this field are mainly based on 2de-gels, we conducted a label-free ms quantification, based on fast high resolution esi-ms and a straight forward software solution, to gai ...201728363874
production of recombinant human acid α-glucosidase with high-mannose glycans in gnt1 rice for the treatment of pompe disease.lysosomal storage diseases are a group of inherited metabolic disorders. patients are treated with enzyme replacement therapy (ert), in which the replacement enzymes are required to carry terminal mannose or mannose 6-phosphate residues to allow efficient uptake into target cells and tissues. n-acetylglucosaminyltransferase-i (gnti) mediates n-glycosylation in the cis cisternae of the golgi apparatus by adding n-acetylglucosamine to the exposed terminal mannose residue of core n-glycan structure ...201728363873
development of a cho-based cell-free platform for synthesis of active monoclonal antibodies.chinese hamster ovary (cho) cells are routinely optimized to stably express monoclonal antibodies (mabs) at high titers. at the early stages of lead isolation and optimization, hundreds of sequences for the target protein of interest are screened. typically, cell-based transient expression technology platforms are used for expression screening, but these can be time- and resource-intensive. here, we have developed a cell-free protein synthesis (cfps) platform utilizing a commercially available c ...201728350472
antioxidant and cytoprotective effects of morin against hydrogen peroxide-induced oxidative stress are associated with the induction of nrf-2‑mediated ho-1 expression in v79-4 chinese hamster lung fibroblasts.natural phytochemicals of plant origin, including flavonoids, have been found to be potent antioxidants providing beneficial effects against oxidative stress-related diseases. the present study was carried out to investigate the antioxidant properties of morin, a flavonoid originally isolated from the flowering plants of the moraceae family. superoxide dismutase (sod)‑like activity and 2,2'‑azino‑bis‑(3‑ethylbenzothiazoline‑6‑sulfonic acid) (abts•+) radical scavenging activity were determined. w ...201728350063
a high density cho-s transient transfection system: comparison of expicho and expi293.chinese hamster ovary (cho) cells are the principal mammalian host used for stable cell line generation and biotherapeutic protein production. until recently, production of milligrams to grams of protein in cho transient systems was challenging. as such, human embryonic kidney (hek293) cells are the most common mammalian cell type used for transient transfection. the post-translational modifications (ptms) of a protein are dictated in part by the cell line used for expression, and changes in ptm ...201728342833
studies to examine potential tolerability differences between the 5-ht2c receptor selective agonists lorcaserin and cp-809101.lorcaserin (lor) is a selective 5-ht2c receptor agonist that has been fda approved as a treatment for obesity. the most frequently reported side-effects of lor include nausea and headache, which can be dose limiting. we have previously reported that in the rat, while lor produced unconditioned signs characteristic of nausea/malaise, the highly selective 5-ht2c agonist cp-809101 (cp) produced fewer equivalent signs. because this may indicate a subclass of 5-ht2c agonists having better tolerabilit ...201728338324
an aromatic amino acid within intracellular loop 2 of the prostaglandin ep2 receptor is a prerequisite for selective association and activation of gαs.we previously demonstrated that the aromatic moiety of tyr(143) within the intracellular loop 2 (icl2) region of the prostaglandin ep2 receptor plays a crucial role in gs coupling. here we investigated whether the icl2 of the ep2 receptor directly binds to gαs and whether an aromatic moiety affects this interaction. in chinese hamster ovary cells, mutations of tyr(143) reduced the ability of the ep2 receptor to interact with g proteins as demonstrated by gtpγs sensitivity, as well as the ability ...201728336329
antitumor activity of chlpmab-2, a human-mouse chimeric cancer-specific antihuman podoplanin antibody, via antibody-dependent cellular cytotoxicity.human podoplanin (hpdpn), a platelet aggregation-inducing transmembrane glycoprotein, is expressed in different types of tumors, and it binds to c-type lectin-like receptor 2 (clec-2). the overexpression of hpdpn is involved in invasion and metastasis. anti-hpdpn monoclonal antibodies (mabs) such as nz-1 have shown antitumor and antimetastatic activities by binding to the platelet aggregation-stimulating (plag) domain of hpdpn. recently, we developed a novel mouse anti-hpdpn mab, lpmab-2, using ...201728332312
transcriptomic changes in cho cells after adaptation to suspension growth in protein-free medium analysed by a species-specific microarray.chinese hamster ovary (cho) cells are the preferred cell line for production of biopharmaceuticals. these cells are capable to grow without serum supplementation, but drastic changes in their phenotype occur during adaptation to protein-free growth, which typically include the change to a suspension phenotype with reduced growth rate. a possible approach to understand this transformation, with the intention to counteract the reduction in growth by targeted supplementation of protein-free media, ...201728302587
evaluation of two public genome references for chinese hamster ovary cells in the context of rna-seq based gene expression analysis.rna-seq is a powerful transcriptomics tool for mammalian cell culture process development. successful rna-seq data analysis requires a high quality reference for read mapping and gene expression quantification. currently, there are two public genome references for chinese hamster ovary (cho) cells, the predominant mammalian cell line in the biopharmaceutical industry. in this study, we compared these two references by analyzing 60 rna-seq samples from a variety of cho cell culture conditions. am ...201728295162
copper(i)-phosphine polypyridyl complexes: synthesis, characterization, dna/hsa binding study, and antiproliferative activity.a series of copper(i)-phosphine polypyridyl complexes have been investigated as potential antitumor agents. the complexes [cu(pph3)2dpq]no3 (2), [cu(pph3)2dppz]no3 (3), [cu(pph3)2dppa]no3 (4), and [cu(pph3)2dppme]no3 (5) were synthesized by the reaction of [cu(pph3)2no3] with the respective planar ligand under mild conditions. these copper complexes were fully characterized by elemental analysis, molar conductivity, fab-ms, and nmr, uv-vis, and ir spectroscopies. interactions between these coppe ...201728290674
dielectrophoresis study of temporal change in internal conductivity of single cho cells after electroporation by pulsed electric fields.applying sufficiently strong pulsed electric fields to a cell can permeabilize the membrane and subsequently affect its dielectric properties. in this study, we employ a microfluidic dielectrophoresis cytometry technique to simultaneously electroporate and measure the time-dependent dielectric response of single chinese hamster ovary cells. using experimental measurements along with numerical simulations, we present quantitative results for the changes in the cytoplasm conductivity of single cel ...201728289483
multiple expression cassette exchange via tp901-1, r4, and bxb1 integrase systems on a mouse artificial chromosome.the site-specific excision of a target dna sequence for genetic knockout or lineage tracing is a powerful tool for investigating biological systems. currently, site-specific recombinases (ssrs), such as cre or flp recombination target cassettes, have been successfully excised or inverted by a single ssr to regulate transgene expression. however, the use of a single ssr might restrict the complex control of gene expression. this study investigated the potential for expanding the multiple regulati ...201728286726
high glucose enhances camp level and extracellular signal-regulated kinase phosphorylation in chinese hamster ovary cell: usage of br-camp in foreign protein β-galactosidase expression.glucose is a carbon source for chinese hamster ovary (cho) cell growth, while low growth rate is considered to enhance the production of recombinant proteins. the present study reveals that glucose concentrations higher than 1 g/l reduce the growth rate and substantially increase in camp (∼300%) at a high glucose concentration (10 g/l). high glucose also enhances the phosphorylation of extracellular signal-regulated kinase (erk) and p27(kip) by western blot analysis. to determine whether the pho ...201728286121
seroprevalence for 2117-like vesiviruses in italian household dogs.in 2003, a novel calicivirus, the vesivirus (vev) strain 2117, was identified incidentally as a contaminant in chinese hamster ovary (cho) cell cultures by a german pharmaceutical company. similar contaminations have been documented in three additional episodes, in bio-reactors used for production of recombinant drugs. more, recently 2117-like vevs have also been identified at high prevalence in the stools from asymptomatic kennel dogs and only sporadically in diarrhoeic and healthy household do ...201728284600
cytotoxicities and genotoxicities of cements based on calcium silicate and of dental formocresol.increasing interest is being paid to the toxicities of dental materials. the purpose of this study was to determine the cytotoxicities and genotoxicities of endodontic compounds to chinese hamster ovary (cho-k1) reproductive cells. cultured cho-k1 cells were treated with dental formocresol, two types of calcium hydroxide paste, and two types of mineral trioxide aggregate cement for 24h. a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed on each culture, and the m ...201728283090
efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in chinese hamster ovary cells.insulin-like growth factor-1 (igf-1) plays a crucial role in cell development, differentiation, and metabolism, and has been a potential therapeutic agent for many diseases. chinese hamster ovary (cho) cells are widely used for production of recombinant therapeutic proteins, but the expression level of igf-1 in cho cells is very low (1,500 µg/l) and the half-life of igf-1 in blood circulation is only 4.5 min according to previous studies. therefore, igf-1 was fused to long-circulating serum prot ...201728281882
binding and biologic characterization of recombinant human serum albumin-etgfbr2 fusion protein expressed in cho cells.transforming growth factor-β1 (tgf-β1) signaling is involved in cell metabolism, growth, differentiation, carcinoma invasion and fibrosis development, which suggests tgf-β1 can be treated as a therapeutic target extensively. because tgf-β1 receptor type α(tgfbr2) is the directed and essential mediator for tgf-β1 signals, the extracellular domain of tgfbr2 (etgfbr2), binding partner for tgf-β1, has been produced in a series of expression systems to inhibit tgf-β1 signaling. however, etgfbr2 is un ...201728281868
proteomic analysis of host cell protein dynamics in the culture supernatants of antibody-producing cho cells.chinese hamster ovary (cho) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mabs). host cell proteins (hcps), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant cho (rcho) cells, potentially impairing product quality. in an effort to maintain good mab quality during the cultures, hcps accumulated extracellularly in batch and fed-batch cultures of a mab-producing rcho cell line ...201728281648
enzyme replacement therapy for farber disease: proof-of-concept studies in cells and mice.a series of studies were carried out in farber disease (omim #228000) cells and mice to evaluate the feasibility of enzyme replacement therapy (ert) for this disorder. media from chinese hamster ovary (cho) cells overexpressing human recombinant acid ceramidase (rhac) was used to treat fibroblasts from a farber disease patient, leading to significantly reduced ceramide. we also found that chondrocytes from farber disease mice had a markedly abnormal chondrogenic phenotype, and this was corrected ...201728275553
antioxidant effect of thiazolidine molecules in cell culture media improves stability and performance.the ability of cell culture media components to generate reactive species as well as their sensitivity to oxidative degradation, affects the overall stability of media and the behavior of cells cultured in vitro. this study investigates the influence of thiazolidine molecules, formed from the condensation between cysteine and alpha-ketoacids, on the stability of these complex mixtures and on the performance of cell culture processes aiming to produce therapeutically relevant monoclonal antibodie ...201728268250
optimizing cell-free protein expression in cho: assessing small molecule mass transfer effects in various reactor configurations.cell-free protein synthesis (cfps) is an ideal platform for rapid and convenient protein production. however, bioreactor design remains a critical consideration in optimizing protein expression. using turbo green fluorescent protein (tgfp) as a model, we tracked small molecule components in a chinese hamster ovary (cho) cfps system to optimize protein production. here, three bioreactors in continuous-exchange cell-free (cecf) format were characterized. a gfp optical sensor was built to monitor t ...201728266026
understanding of decreased sialylation of fc-fusion protein in hyperosmotic recombinant chinese hamster ovary cell culture: n-glycosylation gene expression and n-linked glycan antennary profile.to understand the effects of hyperosmolality on protein glycosylation, recombinant chinese hamster ovary (rcho) cells producing the fc-fusion protein were cultivated in hyperosmolar medium resulting from adding nacl (415 mosm/kg). the hyperosmotic culture showed increased specific fc-fusion protein productivity (qfc ) but a decreased proportion of acidic isoforms and sialic acid content of the fc-fusion protein. the intracellular and extracellular sialidase activities in the hyperosmotic culture ...201728266015
mirna engineering of cho cells facilitates production of difficult-to-express proteins and increases success in cell line development.in recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (dte) therapeutic proteins has increased considerably. dte proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mabs), multi-specific affinity scaffolds (e.g. bispecific antibodies), cytokines or fusion proteins. hence, the availability of robust and versatile chinese hamster ovary (cho) host cell factories is ...201728262952
a role of corazonin receptor in larval-pupal transition and pupariation in the oriental fruit fly bactrocera dorsalis (hendel) (diptera: tephritidae).corazonin (crz) is a neuropeptide hormone, but also a neuropeptide modulator that is internally released within the cns, and it has a widespread distribution in insects with diverse physiological functions. here, we identified and cloned the cdnas of bactrocera dorsalis that encode crz and its receptor crzr. mature bdcrz has 11 residues with a unique ser(11) substitution (instead of the typical asn) and a his in the evolutionary variable position 7. the bdcrzr cdna encodes a putative protein of ...201728261106
screening for small molecule inhibitors of statin-induced app c-terminal toxic fragment production.alzheimer's disease (ad) is characterized by neuronal and synaptic loss. one process that could contribute to this loss is the intracellular caspase cleavage of the amyloid precursor protein (app) resulting in release of the toxic c-terminal 31-amino acid peptide app-c31 along with the production of appδc31, full-length app minus the c-terminal 31 amino acids. we previously found that a mutation in app that prevents this caspase cleavage ameliorated synaptic loss and cognitive impairment in a mu ...201728261092
gold nanoparticles and electroporation impose both separate and synergistic radiosensitizing effects in ht-29 tumor cells: an in vitro study.radiation therapy (rt) is the gold standard treatment for more than half of known tumors. despite recent improvements in rt efficiency, the side effects of ionizing radiation (ir) in normal tissues are a dose-limiting factor that restricts higher doses in tumor treatment. one approach to enhance the efficiency of rt is the application of radiosensitizers to selectively increase the dose at the tumor site. gold nanoparticles (gnps) and electroporation (ep) have shown good potential as radiosensit ...201728260889
a systematic approach to time-series metabolite profiling and rna-seq analysis of chinese hamster ovary cell culture.chinese hamster ovary (cho) cells are the primary host used for biopharmaceutical protein production. the engineering of cho cells to produce higher amounts of biopharmaceuticals has been highly dependent on empirical approaches, but recent high-throughput "omics" methods are changing the situation in a rational manner. omics data analyses using gene expression or metabolite profiling make it possible to identify key genes and metabolites in antibody production. systematic omics approaches using ...201728252038
the enhancement of antibody concentration and achievement of high cell density cho cell cultivation by adding nucleoside.recently, with the dramatic increase in demand for therapeutic antibodies, chinese hamster ovary (cho) cell culture systems have made significant progress in recombinant antibody production. over the past two decades, recombinant antibody productivity has been improved by more than 100-fold. medium optimization has been identified as an important key approach for increasing product concentrations. in this study, we evaluated the effects of deoxyuridine addition to fed-batch cultures of antibody- ...201728251404
effects of lysosomal biotherapeutic recombinant protein expression on cell stress and protease and general host cell protein release in chinese hamster ovary cells.recombinant human acid alpha glucosidase (gaa) is the therapeutic enzyme used for the treatment of pompe disease, a rare genetic disorder characterized by gaa deficiency in the cell lysosomes (raben et al., curr mol med. 2002; 2:145-166). the manufacturing process for gaa can be challenging, in part due to protease degradation. the overall goal of this study was to understand the effects of gaa overexpression on cell lysosomal phenotype and host cell protein (hcp) release, and any resultant cons ...201728249362
antibody purification from cho cell supernatant using new multimodal membranes.this contribution describes strategies to purify monoclonal antibodies from chinese hamster ovary (cho) cell culture supernatant using newly designed multimodal membranes (mmms). the mmms were used for the capture step purification of human igg1 following a size-exclusion desalting column to remove chaotropic salts that interfere with igg binding. the mmm column attained higher dynamic binding capacity than a protein a resin column at an equivalent residence time of 1 min. the two-step mmm chrom ...201728248007
an automated micro-total immunoassay system for measuring cancer-associated α2,3-linked sialyl n-glycan-carrying prostate-specific antigen may improve the accuracy of prostate cancer diagnosis.the low specificity of the prostate-specific antigen (psa) for early detection of prostate cancer (pca) is a major issue worldwide. the aim of this study to examine whether the serum pca-associated α2,3-linked sialyl n-glycan-carrying psa (s2,3psa) ratio measured by automated micro-total immunoassay systems (μtas system) can be applied as a diagnostic marker of pca. the μtas system can utilize affinity-based separation involving noncovalent interaction between the immunocomplex of s2,3psa and ma ...201728241428
antiglycopeptide mouse monoclonal antibody lpmab-21 exerts antitumor activity against human podoplanin through antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity.the interaction between podoplanin (pdpn) and c-type lectin-like receptor 2 (clec-2) is involved in tumor malignancy. we have established many monoclonal antibodies (mabs) against human podoplanin using the cancer-specific mab (casmab) technology. lpmab-21, one of the mouse antipodoplanin mabs, is of the igg2a subclass, and its minimum epitope was determined to be thr76-arg79 of the human podoplanin. importantly, sialic acid is linked to thr76; therefore, lpmab-21 is an antiglycopeptide mab (gpm ...201728234556
what can mathematical modelling say about cho metabolism and protein glycosylation?chinese hamster ovary cells have been in the spotlight for process optimization in recent years, due to being the major, long established cell factory for the production of recombinant proteins. a deep, quantitative understanding of cho metabolism and mechanisms involved in protein glycosylation has proven to be attainable through the development of high throughput technologies. here we review the most notable accomplishments in the field of modelling cho metabolism and protein glycosylation.201728228925
evaluating the expression profile and stability of different ucoe containing vector combinations in mab-producing cho cells.as the demand for monoclonal antibodies (mab) increases, more efficient expression methods are required for their manufacturing process. transcriptional gene silencing is a common phenomenon in recombinant cell lines which leads to expression reduction and instability. there are reports on improved antibody expression in ubiquitous chromatin opening element (ucoe) containing both heavy and light chain gene constructs. here we investigate the impact of having these elements as part of the light c ...201728228095
how important is thermodynamics for identifying elementary flux modes?we present a method for computing thermodynamically feasible elementary flux modes (tefms) using equilibrium constants without need of internal metabolite concentrations. the method is compared with the method based on a binary distinction between reversible and irreversible reactions. when all reactions are reversible, adding the constraints based on equilibrium constants reduces the number of elementary flux modes (efms) by a factor of two. declaring in advance some reactions as irreversible, ...201728222104
interaction of approved drugs with synaptic vesicle protein 2a.levetiracetam (lev) and its recently approved derivative brivaracetam are anti-epileptic drugs with a unique mechanism of action. the synaptic vesicle protein 2a (sv2a) was previously identified as their main target. in the current study, we tested a collection of 500 approved drugs for interaction with the human sv2a protein expressed in chinese hamster ovary cells. competition binding studies were performed using cell lysates with high sv2a expression and [(3) h]brivaracetam as a radioligand. ...201728220535
glycoengineering of esterase activity through metabolic flux-based modulation of sialic acid.this report describes the metabolic glycoengineering (mge) of intracellular esterase activity in human colon cancer (ls174t) and chinese hamster ovary (cho) cells. in silico analysis of the carboxylesterases ces1 and ces2 suggested that these enzymes are modified with sialylated n-glycans, which are proposed to stabilize the active multimeric forms of these enzymes. this premise was supported by treating cells with butanolylated mannac to increase sialylation, which in turn increased esterase ac ...201728218815
unveiling gene trait relationship by cross-platform meta-analysis on chinese hamster ovary cell transcriptome.in the past few years, transcriptome analysis has been increasingly employed to better understand the physiology of chinese hamster ovary (cho) cells at a global level. as more transcriptome data accumulated, meta-analysis on data sets collected from various sources can potentially provide better insights on common properties of those cells. here, we performed meta-analysis on transcriptome data of different cho cell lines obtained using nimblegen or affymetrix microarray platforms. hierarchical ...201728218403
manipulating cholesterol status within cells.cellular cholesterol levels are intricately controlled to maintain homeostasis. here, we describe ways in which cellular cholesterol status can be manipulated for the study of cholesterol homeostasis, including sterol starvation (by culturing cells in lipoprotein-deficient serum and pretreating/treating with the cholesterol-lowering drug, statin) and sterol enrichment (using cholesterol complexed to cyclodextrin, and low-density lipoprotein). we also describe how to prepare lipoprotein-deficient ...201728205165
antioxidant and cytoprotective effects of morin against hydrogen peroxide-induced oxidative stress are associated with the induction of nrf-2‑mediated ho-1 expression in v79-4 chinese hamster lung fibroblasts.natural phytochemicals of plant origin, including flavonoids, have been found to be potent antioxidants providing beneficial effects against oxidative stress-related diseases. the present study was carried out to investigate the antioxidant properties of morin, a flavonoid originally isolated from the flowering plants of the moraceae family. superoxide dismutase (sod)‑like activity and 2,2'‑azino‑bis‑(3‑ethylbenzothiazoline‑6‑sulfonic acid) (abts•+) radical scavenging activity were determined. w ...201728204816
distinct c-mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian dpy19l1 and dpy19l3.thrombospondin type 1 repeats (tsrs) occur in diverse proteins involved in adhesion and signaling. the two extracellular tsrs of the netrin receptor unc5a contain wxxwxxwxxc motifs that can be c-mannosylated on all tryptophans. a single c-mannosyltransferase (dumpy-19, dpy-19), modifying the first two tryptophans, occurs in caenorhabditis elegans, but four putative enzymes (dpy-19-like 1-4, dpy19l1-4) exist in mammals. single and triple crispr-cas9 knockouts of the three homologs that are expres ...201728202721
multiple blood-brain barrier transport mechanisms limit bumetanide accumulation, and therapeutic potential, in the mammalian brain.there is accumulating evidence that bumetanide, which has been used over decades as a potent loop diuretic, also exerts effects on brain disorders, including autism, neonatal seizures, and epilepsy, which are not related to its effects on the kidney but rather mediated by inhibition of the neuronal na-k-cl cotransporter isoform nkcc1. however, following systemic administration, brain levels of bumetanide are typically below those needed to inhibit nkcc1, which critically limits its clinical use ...201728192112
magnetic sorting of membrane associated igg for phenotype-based selection of stable antibody producing cells.to establish a simple and widely accessible technique for rapidly selecting high producing chinese hamster ovary (cho) cells engineered to express a monoclonal antibody (mab), we have exploited the transient display of recombinant protein on their cell surface. in combination with magnetic bead-based methods, we demonstrate the ability to select for cells of high productivity in the absence of any metabolic-based selection method. this technique is sufficient to obtain genetically stable enginee ...201728189705
fatty-acyl chain profiles of cellular phosphoinositides.phosphoinositides are rapidly turning-over phospholipids that play key roles in intracellular signaling and modulation of membrane effectors. through technical refinements we have improved sensitivity in the analysis of the phosphoinositide pi, pip, and pip2 pools from living cells using mass spectrometry. this has permitted further resolution in phosphoinositide lipidomics from cell cultures and small samples of tissue. the technique includes butanol extraction, derivatization of the lipids, po ...201728189644
ultra-deep next generation mitochondrial genome sequencing reveals widespread heteroplasmy in chinese hamster ovary cells.recent sequencing of the chinese hamster ovary (cho) cell and chinese hamster genomes has dramatically advanced our ability to understand the biology of these mammalian cell factories. in this study, we focus on the powerhouse of the cho cell, the mitochondrion. utilizing a high-resolution next generation sequencing approach we sequenced the chinese hamster mitochondrial genome for the first time and surveyed the mutational landscape of cho cell mitochondrial dna (mtdna). depths of coverage rang ...201728188893
bioreactor scale up and protein product quality characterization of piggybac transposon derived cho pools.chinese hamster ovary (cho) cells remain the most popular host for the production of biopharmaceutical drugs, particularly monoclonal antibodies (mabs), bispecific antibodies, and fc-fusion proteins. creating and characterizing the stable cho clonally-derived cell lines (cdcls) needed to manufacture these therapeutic proteins is a lengthy and laborious process. therefore, cho pools have increasingly been used to rapidly produce protein to support and enable preclinical drug development. we recen ...201728188692
evaluation of heavy chain c-terminal deletions on productivity and product quality of monoclonal antibodies in chinese hamster ovary (cho) cells.monoclonal antibodies (mabs) have been well established as potent therapeutic agents and are used to treat many different diseases. during cell culture production, antibody charge variants can be generated by cleavage of heavy chain (hc) c-terminal lysine and proline amidation. differences in levels of charge variants during manufacturing process changes make it challenging to demonstrate process comparability. in order to reduce heterogeneity and achieve consistent product quality, we generated ...201728188688
proteomics profiling of chimeric-truncated tissue plasminogen activator producing- chinese hamster ovary cells cultivated in a chemically defined medium supplemented with protein hydrolysatesculture media enrichment through the addition of protein hydrolysates is beneficial for achieving higher protein expression.201728187683
site-specific monitoring of n-glycosylation profiles of a ctla4-fc-fusion protein from the secretory pathway to the extracellular environment.glycosylation often plays a key role in the safety and efficacy of therapeutic proteins to patients, thus underlying the need for consistent control of this important post-translational modification during biologics production. in this study, we profiled the site-specific evolution of n-glycans on a ctla4-fc-fusion protein, from the intracellular secretory pathway to the conditioned medium (cm) in fed-batch cell culture. for this, we developed an approach that combined sub-cellular fractionation ...201728186328
fabrication of electrospun hpgl scaffolds via glycidyl methacrylate cross-linker: morphology, mechanical and biological properties.electrospinning is a suitable method to produce scaffolds composed of nanoscale to microscale fibers, which are comparable to the extracellular matrix (ecm). hyperbranched polyglycerol (hpgl) is a highly biocompatible polyether polyol potentially useful for the design of fibrous scaffolds mimicking the ecm architecture. however, scaffolds developed from hpgl have poor mechanical properties and morphological stability in the aqueous environments required for tissue engineering applications. this ...201728183666
method comparison for analyzing wound healing rates.wound healing scratch assay is a frequently used method to characterize cell migration, which is an important biological process in the course of development, tissue repair, and immune response for example. the measurement of wound healing rate, however, varies among different studies. here we summarized these measurements into three types: i) direct rate average; ii) regression rate average; and iii) average distance regression rate. using chinese hamster ovary (cho) cells as a model, we compar ...201728177756
the vdac2-bak axis regulates peroxisomal membrane permeability.peroxisomal biogenesis disorders (pbds) are fatal genetic diseases consisting of 14 complementation groups (cgs). we previously isolated a peroxisome-deficient chinese hamster ovary cell mutant, zp114, which belongs to none of these cgs. using a functional screening strategy, vdac2 was identified as rescuing the peroxisomal deficiency of zp114 where vdac2 expression was not detected. interestingly, knockdown of bak or overexpression of the bak inhibitors bcl-xl and mcl-1 restored peroxisomal bio ...201728174205
small-molecule-biased formyl peptide receptor agonist compound 17b protects against myocardial ischaemia-reperfusion injury in mice.effective treatment for managing myocardial infarction (mi) remains an urgent, unmet clinical need. formyl peptide receptors (fpr) regulate inflammation, a major contributing mechanism to cardiac injury following mi. here we demonstrate that fpr1/fpr2-biased agonism may represent a novel therapeutic strategy for the treatment of mi. the small-molecule fpr1/fpr2 agonist, compound 17b (cmpd17b), exhibits a distinct signalling fingerprint to the conventional fpr1/fpr2 agonist, compound-43 (cmpd43). ...201728169296
high-resolution imaging and quantification of plasma membrane cholesterol by nanosims.cholesterol is a crucial lipid within the plasma membrane of mammalian cells. recent biochemical studies showed that one pool of cholesterol in the plasma membrane is "accessible" to binding by a modified version of the cytolysin perfringolysin o (pfo*), whereas another pool is sequestered by sphingomyelin and cannot be bound by pfo* unless the sphingomyelin is destroyed with sphingomyelinase (smase). thus far, it has been unclear whether pfo* and related cholesterol-binding proteins bind unifor ...201728167768
atf6β-based fine-tuning of the unfolded protein response enhances therapeutic antibody productivity of chinese hamster ovary cells.the dynamics of protein folding and secretion are key issues in improving the productivity and robustness of chinese hamster ovary (cho) producer cells. high recombinant protein secretion in cho producer clones triggers the activation of the unfolded protein response (upr), an intracellular response to the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (er). we previously reported that the human microrna (mirna) mir-1287 enhances productivity in igg expressing cho c ...201728165157
the fibrous form of intracellular inclusion bodies in recombinant variant fibrinogen-producing cells is specific to the hepatic fibrinogen storage disease-inducible variant fibrinogen.fibrinogen storage disease (fsd) is a rare disorder that is characterized by the accumulation of fibrinogen in hepatocytes and induces liver injury. six mutations in the γc domain (γg284r, γt314p, γd316n, the deletion of γg346-q350, γg366s, and γr375w) have been identified for fsd. our group previously established γ375w fibrinogen-producing chinese hamster ovary (cho) cells and observed aberrant large granular and fibrous forms of intracellular inclusion bodies. the aim of this study was to inve ...201728161763
a vector based on the chicken hypersensitive site 4 insulator element replicates episomally in mammalian cell.gene therapy in mammalian cells requires vectors exhibiting long-term stability and high expression. episomal gene expression vectors offer a safe and attractive alternative to those that integrate into the host cell genome. in the present study, we developed a new episomal vector based on the insulator, chicken hypersensitive site 4 (chs4). the chs4 element was artificially synthesized, cloned into the pegfp-c1 vector, and used to transfect chinese hamster ovary (cho) and human chang liver cell ...201728155604
dedicated high dose rate (192)ir brachytherapy radiation fields for in vitro cell exposures at variable source-target cell distances: killing of mammalian cells depends on temporal dose rate fluctuation.afterloading brachytherapy is conducted by the stepwise movement of a radioactive source through surgically implanted applicator tubes where at predefined dwell positions calculated dwell times optimize spatial dose delivery with respect to a planned dose level. the temporal exposure pattern exhibits drastic fluctuations in dose rate at a given coordinate and within a single treatment session because of the discontinuous and repeated source movement into the target volume. this could potentially ...201728145285
engineering the expression of an anti-interleukin-13 antibody through rational design and mutagenesis.high levels of protein expression are key to the successful development and manufacture of a therapeutic antibody. here, we describe two related antibodies, ab001 and ab008, where ab001 shows a markedly lower level of expression relative to ab008 when stably expressed in chinese hamster ovary cells. we use single-gene expression vectors and structural analysis to show that the reduced titer is associated with the vl cdr2 of ab001. we adopted two approaches to improve the expression of ab001. fir ...201728130326
cell-controlled hybrid perfusion fed-batch cho cell process provides significant productivity improvement over conventional fed-batch cultures.a simple method originally designed to control lactate accumulation in fed-batch cultures of chinese hamster ovary (cho) cells has been modified and extended to allow cells in culture to control their own rate of perfusion to precisely deliver nutritional requirements. the method allows for very fast expansion of cells to high density while using a minimal volume of concentrated perfusion medium. when the short-duration cell-controlled perfusion is performed in the production bioreactor and is i ...201728128436
taming hyperactive hdnase i: stable inducible expression of a hyperactive salt- and actin-resistant variant of human deoxyribonuclease i in cho cells.while the most common causes of clonal instability are dna copy number loss and silencing, toxicity of the expressed protein(s) may also induce clonal instability. human dnase i (hdnase i) is used therapeutically for the treatment of cystic fibrosis (cf) and may have potential benefit for use in systemic lupus erythematosus (sle). hdnase i is an endonuclease that catalyzes degradation of extracellular dna and is inhibited by both salt and g-actin. engineered versions of hdnase i, bearing multipl ...201728127892
application of (13)c flux analysis to identify high-productivity cho metabolic phenotypes.industrial bioprocesses place high demands on the energy metabolism of host cells to meet biosynthetic requirements for maximal protein expression. identifying metabolic phenotypes that promote high expression is therefore a major goal of the biotech industry. we conducted a series of (13)c flux analysis studies to examine the metabolic response to igg expression during early stationary phase of cho cell cultures grown in 3l fed-batch bioreactors. we examined eight clones expressing four differe ...201728122259
a novel approach to characterize host cell proteins associated with therapeutic monoclonal antibodies.recombinant monoclonal antibody (mab) products are widely produced in the pharmaceutical industry using chinese hamster ovary (cho) cells. host cell proteins (hcps) are one of many process-related impurities generated during the production of mab products. the multi-analyte hcp enzyme linked immunosorbent assay (elisa) is the industry standard accepted assay to measure clearance of hcps from recombinant protein therapeutics. while similar platform processes are used for expression and purificati ...201728112396
expression and characterization of a codon-optimized blood coagulation factor viii.essentials recombinant factor viii (fviii) is known to be expressed at a low level in cell culture. to increase expression, we used codon-optimization of a b-domain deleted fviii (bdd-fviii). this resulted in 7-fold increase of the expression level in cell culture. the biochemical properties of codon-optimized bdd-fviii were similar to the wild-type protein.201728109042
from the cover: an investigation of the genotoxicity and interference of gold nanoparticles in commonly used in vitro mutagenicity and genotoxicity assays.the suitability of 4 in vitro assays, commonly used for mutagenicity and genotoxicity assessment, was investigated in relation to treatment with 14 nm citrate-stabilized gold nanoparticles (aunps). specifically, the ames test was conducted without metabolic activation, where no mutagenic effects were observed. high resolution transmission electron microscopy and cytoviva dark-field image analysis showed that aunps did not enter the bacterial cells, thus confirming the unreliability of the ames t ...201728108664
a highly efficient modified human serum albumin signal peptide to secrete proteins in cells derived from different mammalian species.signal peptides (sps) are key elements in the production of recombinant proteins; however, little information is available concerning different sp in mammalian cells other than cho. in order to study the efficiency of different sps to direct the traffic along the secretory pathway of the green fluorescence protein (gfp) and a scfv-fc fusion protein; cho-k1, hek293 and ns0 cell lines were transfected in a transient and stable way. sp of human azurocidin (az), modified human albumin (msa), modifie ...201728089882
secretory pathway optimization of cho producer cells by co-engineering of the mitosrna-1978 target genes cers2 and tbc1d20.chinese hamster ovary (cho) cells are the most commonly used host for the production of biopharmaceuticals. although transcription and translation engineering strategies have been employed to generate high-producer cell clones, the secretory pathway still remains a bottleneck in cellular productivity. in this study we show that ectopic expression of a human mitochondrial genome-encoded small rna (mitosrna-1978) in an igg expressing cho cell line strongly improved specific productivity by functio ...201728088541
adverse and beneficial effects in chinese hamster lung fibroblast cells following radiofrequency exposure.in this study, the effect of radiofrequency (rf) exposure to 1950 mhz, universal mobile telecommunication system signal, was investigated in chinese hamster lung fibroblast cell line (v79). genotoxic and cytotoxic effects of 20-h exposure at specific absorption rate (sar) values from 0.15 w/kg to 1.25 w/kg were measured by means of cytokinesis-block micronucleus (mn) assay. exposure was carried out blinded under strictly controlled conditions of dosimetry and temperature. the effect of rf exposu ...201728072461
development, qualification, validation and application of the neutral red uptake assay in chinese hamster ovary (cho) cells using a vitrocell® vc10® smoke exposure system.cytotoxicity assessment of combustible tobacco products by neutral red uptake (nru) has historically used total particulate matter (tpm) or solvent captured gas vapor phase (gvp), rather than fresh whole smoke. here, the development, validation and application of the nru assay in chinese hamster ovary (cho) cells, following exposure to fresh whole smoke generated with the vitrocell® vc10® system is described. whole smoke exposure is particularly important as both particulate and vapor phases of ...201728062357
tbx20 controls the expression of the kcnh2 gene and of herg channels.long qt syndrome (lqts) exhibits great phenotype variability among family members carrying the same mutation, which can be partially attributed to genetic factors. we functionally analyzed the kcnh2 (encoding for kv11.1 or herg channels) and tbx20 (encoding for the transcription factor tbx20) variants found by next-generation sequencing in two siblings with lqts in a spanish family of african ancestry. affected relatives harbor a heterozygous mutation in kcnh2 that encodes for p.t152hfsx180 kv11 ...201728049825
production of human mutant biologically active hepatocyte growth factor in chinese hamster ovary cells.hepatocyte growth factor (hgf) is a potent multifunctional cytokine that affects proliferation, migration, and morphogenesis of various cells. hgf is secreted as an inactive single-chain precursor protein and activated by the cleavage of serine proteases to form heterodimers. in our current study, the cleavage site of hgf was blocked by replaced arg 494 of glu (r494e) that resulted in the single-chain hgf (r494e) unable to be cleaved by serine proteases. we established chinese hamster ovary (cho ...201728045643
asp73-dependent and -independent regulation of the affinity of ligands for human histamine h1 receptors by na().the affinity of ligands for g-protein-coupled receptors (gpcrs) is allosterically regulated by na(+) via a highly conserved aspartate residue (asp(2.50)) in the second transmembrane domain of gpcrs. in the present study, we examined the na(+)-mediated regulation of the affinity of ligands for gq/11-protein-coupled human histamine h1 receptors in chinese hamster ovary cells. the affinities of 3 agonists and 20 antihistamines were evaluated by their displacement curves against the binding of [(3)h ...201728040476
assessing the genotoxicity of two commonly occurring byproducts of water disinfection: chloral hydrate and bromal hydrate.water disinfection treatments result in the formation of disinfection byproducts (dbps) that have been linked to adverse human health outcomes including higher incidence of bladder and colorectal cancer. however, data about the genotoxicity of dbps is limited to only a small fraction of compounds. chloral hydrate (ch) and bromal hydrate (bh) are two trihaloacetaldehydes commonly detected in disinfected waters, but little is known about their genotoxicity, especially bh. we investigated the genot ...201728010927
design, synthesis and biological activity of novel molecules designed to target parp and dna.in order to enhance the cytotoxic potential of poly(adp-ribose) polymerase (parp) inhibitors in brca1 or 2 deficient tumours, we designed a series of molecules containing a 1,2,3-triazene moiety tethered to a parp targeting scaffold. a cell-based selectivity assay involving a brca2-deficient chinese hamster cell line and its corresponding brca2 wild type transfectant, was used to predict the parp targeting potential of the latter agents. the results showed that adding a dna damaging function to ...201728003142
preferential hydrolysis of truncated oxidized glycerophospholipids by lysosomal phospholipase a2.truncated oxidized glycerophospholipids (ox-pls) are bioactive lipids resulting from oxidative stress. the catabolic pathways for truncated ox-pls are not fully understood. lysosomal phospholipase a2 (lpla2) with phospholipase a and transacylase activities is a key enzyme in phospholipid homeostasis. the present study assessed whether lpla2 could hydrolyze truncated ox-pls. incubation of lpla2 with liposomes consisting of 1,2-o-octadecenyl-sn-glycero-3-phosphocholine (dodpc)/1,2-dioleoyl-sn-glyc ...201727993948
hiv protease inhibitors alter amyloid precursor protein processing via β-site amyloid precursor protein cleaving enzyme-1 translational up-regulation.mounting evidence implicates antiretroviral (arv) drugs as potential contributors to the persistence and evolution of clinical and pathological presentation of hiv-associated neurocognitive disorders in the post-arv era. based on their ability to induce endoplasmic reticulum (er) stress in various cell types, we hypothesized that arv-mediated er stress in the central nervous system resulted in chronic dysregulation of the unfolded protein response and altered amyloid precursor protein (app) proc ...201727993242
high-throughput lipidomic and transcriptomic analysis to compare sp2/0, cho, and hek-293 mammalian cell lines.a combined lipidomics and transcriptomics analysis was performed on mouse myeloma sp2/0, chinese hamster ovary (cho), and human embryonic kidney (hek) cells in order to compare widely used mammalian expression systems. initial thin layer chromatography (tlc) analysis indicated that phosphatidylethanolamine (pe) and phosphatidylcholine (pc) were the major lipid components in all cell lines with lower amounts of sphingomyelin (sm) in sp2/0 compared to cho and hek, which was subsequently confirmed ...201727991764
integrated genome and protein editing swaps α-2,6 sialylation for α-2,3 sialic acid on recombinant antibodies from cho.immunoglobin g with α-2,6 sialylation has been reported to have an impact on antibody-dependent cellular cytotoxicity and anti-inflammatory efficacy. however, production of antibodies with α-2,6 sialylation from chinese hamster ovary cells is challenging due to the inaccessibility of sialyltransferases for the heavy chain n-glycan site and the presence of exclusively α-2,3 sialyltransferases. in this study, combining mutations on the fc regions to allow sialyltransferase accessibility with overe ...201727943633
knockout of a difficult-to-remove cho host cell protein, lipoprotein lipase, for improved polysorbate stability in monoclonal antibody formulations.while the majority of host cell protein (hcp) impurities are effectively removed in typical downstream purification processes, a small population of hcps are particularly challenging. previous studies have identified hcps that are challenging for a variety of reasons. lipoprotein lipase (lpl)-a chinese hamster ovary (cho) hcp that functions to hydrolyze esters in triglycerides-was one of ten hcps identified in previous studies as being susceptible to retention in downstream processing. lpl may d ...201727943242
role of a tachykinin-related peptide and its receptor in modulating the olfactory sensitivity in the oriental fruit fly, bactrocera dorsalis (hendel).insect tachykinin-related peptide (trp), an ortholog of tachykinin in vertebrates, has been linked with regulation of diverse physiological processes, such as olfactory perception, locomotion, aggression, lipid metabolism and myotropic activity. in this study, we investigated the function of trp (bdtrp) and its receptor (bdtrpr) in an important agricultural pest, the oriental fruit fly bactrocera dorsalis. bdtrpr is a typical g-protein coupled-receptor (gpcr), and it could be activated by the pu ...201727923683
integrin αiibβ3-dependent erk signaling is regulated by src and rho kinases in both leu33 and pro33 polymorphic isoforms.platelet integrin αiibβ3 possesses a leu/pro polymorphism at residue 33 (leu33/hpa-1a or pro33/hpa-1b). the pro33 isoform has been suggested to exhibit prothrombotic features. αiibβ3-expressing cho (chinese hamster ovary) cells on immobilized fibrinogen show activation of the map kinase family member erk2, with an enhanced erk2 activity in pro33 cells compared to leu33 cells. in our present work, we examined how the leu/pro polymorphism modulates the erk2 activation stimulated by 2 differently t ...201727923225
improving the secretory capacity of chinese hamster ovary cells by ectopic expression of effector genes: lessons learned and future directions.chinese hamster ovary (cho) cells are the preferred cell factory for the production of therapeutic glycoproteins. although efforts primarily within bioprocess optimization have led to increased product titers of recombinant proteins (r-proteins) expressed in cho cells, post-transcriptional bottlenecks in the biosynthetic pathway of r-proteins remain to be solved. to this end, the ectopic expression of transgenes (effector genes) offers great engineering potential. however, studies on effector ge ...201727931938
a framework to quantify karyotype variation associated with cho cell line instability at a single-cell level.chinese hamster ovary (cho) cells, the major mammalian host cells for biomanufacturing of therapeutic proteins, have been extensively investigated to enhance productivity and product quality. however, cell line instability resulting in unexpected changes in productivity or product quality continues to be a challenge. based on previous reports about causes and characteristics of production instability, we hypothesized that chromosomal rearrangements due to genomic instability are associated with ...201727922175
selection of chemically defined media for cho cell fed-batch culture processes.two cho cell clones derived from the same parental cho(bc®) cell line and producing the same monoclonal antibody (bc-g, a low producing clone; bc-p, a high producing clone) were tested in four basal media in all possible combinations with three feeds (=12 conditions) in fed-batch cultures. higher amino acid feeding did not always lead to higher mab production. the two clones showed differences in cell physiology, metabolism and optimal medium-feed combinations. during the phase transitions of al ...201727900626
rational design of glycoengineered interferon-β analogs with improved aggregation state: experimental validation.recombinant human interferon-β (rhifn-β) used clinically has lower efficacy than expected due to protein instabilities such as aggregation. increasing molecular stability, glycoengineering has been used to improve clinical efficacy for a number of therapeutics; however, often labor-intensive trail-and-error approaches are used to identify additional glycosylation sites. in this study two rhifn-β analogs with one additional glycosylation site, l6t and s75n, identified by a rational in silico appr ...201727881683
flavonoids from heliotropium subulatum exudate and their evaluation for antioxidant, antineoplastic and cytotoxic activities ii.the flavonoids are the largest group of phenolic compounds isolated from a wide range of higher plants. these compounds work as antimicrobials, anti-insect agents and protect plants from other types of biotic and abiotic stresses. various researchers have suggested that flavonoids possessed antioxidant, antineoplastic and cytotoxic activities. the main objective of this study was to test dichloromethane fraction of resinous exudate of heliotropium subulatum for their antioxidant, antineoplastic ...201727905025
segmented linear modeling of cho fed-batch culture and its application to large scale production.we describe a systematic approach to model cho metabolism during biopharmaceutical production across a wide range of cell culture conditions. to this end, we applied the metabolic steady state concept. we analyzed and modeled the production rates of metabolites as a function of the specific growth rate. first, the total number of metabolic steady state phases and the location of the breakpoints were determined by recursive partitioning. for this, the smoothed derivative of the metabolic rates wi ...201727869296
reduced cytotoxicity in pcb-exposed chinese hamster ovary (cho) cells pretreated with vitamin e.the aim of this study was to evaluate protective effects of vitamin e (50 -150 μm) in ovary cells upon cytotoxic effects induced by two structurally distinct pcb congeners - planar "dioxin-like" pcb 77 and non-planar di-ortho-substituted pcb 153 with an emphasis on identifying differences in the mechanism of vitamin e action depending on the structure of congeners. application of three bioassays confirmed that pcbs decrease ovarian cell proliferation with slightly profound effects of pcb 77. pcb ...201727865896
establishment of monoclonal antibodies against cell surface domains of asct2/slc1a5 and their inhibition of glutamine-dependent tumor cell growth.human alanine-serine-cysteine transporter 2 (asct2; slc1a5) is a major transporter of the amino acid glutamine that is known to be overexpressed in certain malignant tumors. in this study, we generated specific monoclonal antibodies (mabs) against asct2 by establishing an asct2-expressing chinese hamster ovary cell line that was used to immunize mice and rats. the mabs km4008, km4012, and km4018 against asct2 were isolated through a cell-based screen; these specifically bound to asct2-positive c ...201727865832
differences in trpc3 and trpc6 channels assembly in mesenteric vascular smooth muscle cells in essential hypertension.canonical transient receptor potential (trpc)3 and trpc6 channels of vascular smooth muscle cells (vsmcs) mediate stretch- or agonist-induced cationic fluxes, contributing to membrane potential and vascular tone. native trpc3/c6 channels can form homo- or heterotetrameric complexes, which can hinder individual trpc channel properties. the possibility that the differences in their association pattern may change their contribution to vascular tone in hypertension is unexplored. functional characte ...201727861908
improved recombinant antibody production by cho cells using a production enhancer dna element with repeated transgene integration at a predetermined chromosomal site.chinese hamster ovary (cho) cells are one of the most useful host cell lines for the production of biopharmaceutical proteins. although a series of production processes have been refined to improve protein productivity and cost performance, establishing producer cells is still time-consuming and labor-intensive. recombinase-mediated site-specific gene integration into a predetermined chromosomal locus may enable predictable protein expression, reducing the laborious process of cell screening. we ...201727856232
production and purification of recombinant glucocerebrosidase in transgenic rice cell suspension cultures.gaucher disease, which is caused by deficiency of glucocerebrosidase (gcd), is currently treated by enzyme replacement therapy. plant-based systems produce glycoproteins and can be combined with targeting strategies to generate proteins with terminal mannose structures for macrophage uptake. however, the gliding step for the purification is essential since the produced protein still exists inside cells. in the case of rice-amylase 1a (ramy1a) secretion signal peptide, gcd protein is secreted out ...201727817046
lysosomal alpha-mannosidase and alpha-mannosidosis.lysosomal alpha-mannosidase with acidic ph optimum is ubiquitous in human tissues where is expressed in two major forms, a and b that are the product of a single gene located on chromosome 19. mutations in the gene encoding for alpha-mannosidase cause alpha- mannosidosis, an autosomal recessive disease, resulting in the accumulation of unprocessed mannose containing oligosaccharide material. this rare disease has an estimated incidence of 1/500.0.00 live births and clinically is divided into thr ...201727814608
detection of a phosphorylated glycine-serine linker in an igg-based fusion protein.molecular mass determination by electrospray ionization mass spectrometry of a recombinant igg-based fusion protein (mab1-f) produced in human embryonic kidney (hek) cells demonstrated the presence of a dominant +79 da product variant. using lc-ms tryptic peptide mapping analysis and collision-induced dissociation (cid) and electron-transfer/higher-energy collision dissociation fragmentations, the modification was localized to the c-terminal serine residue of a glycine-serine linker [(g4s)2] of ...201727661266
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