135,952 research outputs found
CHO microRNA engineering is growing up : recent successes and future challenges
No full textmicroRNAs with their ability to regulate complex pathways that control cellular behavior and phenotype have been proposed as potential targets for cell engineering in the context of optimization of biopharmaceutical production cell lines, specifically of Chinese Hamster Ovary cells. However, until recently, research was limited by a lack of genomic sequence information on this industrially important cell line. With the publication of the genomic sequence and other relevant data sets for CHO cells since 2011, the doors have been opened for an improved understanding of CHO cell physiology and for the development of the necessary tools for novel engineering strategies. In the present review we discuss both knowledge on the regulatory mechanisms of microRNAs obtained from other biological models and proof of concepts already performed on CHO cells, thus providing an outlook of potential applications of microRNA engineering in production cell lines
TG, T-cho, H-cho, and cortisol levels in the serum.
No full text<p>CMS group, chronic mild stress group; C group, control group; TG, triglyceride; T-cho, total cholesterol; H-cho, HDL-cholesterol; SD, standard deviation.</p><p>TG, T-cho, H-cho, and cortisol levels in the serum.</p
Unimodality of Betti numbers for Hamiltonian circle actions with index-increasing moment Maps
No full textThe unimodality conjecture posed by Tolman in [L. Jeffrey, T. Holm, Y. Karshon, E. Lerman and E. Meinrenken, Moment maps in various geometries, http://www.birs.ca/workshops/2005/05w5072/report05w5072.pdf] states that if (M,ω) is a 2n-dimensional smooth compact symplectic manifold equipped with a Hamiltonian circle action with only isolated fixed points, then the sequence of Betti numbers {b0(M),b2(M),...,b2n(M)} is unimodal, i.e. bi(M) ≤ bi+2(M) for every i < n. Recently, the author and Kim [Y. Cho and M. Kim, Unimodality of the Betti numbers for Hamiltonian circle action with isolated fixed points, Math. Res. Lett. 21(4) (2014) 691-696] proved that the unimodality holds in eight-dimensional case by using equivariant cohomology theory. In this paper, we generalize the idea in [Y. Cho and M. Kim, Unimodality of the Betti numbers for Hamiltonian circle action with isolated fixed points, Math. Res. Lett. 21(4) (2014) 691-696] to an arbitrary dimensional case. We prove the conjecture in arbitrary dimension under the assumption that the moment map H : M → R is index-increasing, which means that ind(p) < ind(q) implies H(p) < H(q) for every pair of critical points p and q of H, where ind(p) is the Morse index of p with respect to H. © World Scientific Publishing Company1111sciescopu
Cell Apoptosis Induced by a Synthetic Carbazole Compound Lcy-2-Cho Is Mediated through Activation of Caspase and Mitochondrial Pathways
No full textThe mechanisms involved in the apoptotic effect of LCY-2-CHO [9-(2- chlorobenzyl)-9H-carbazole-3-carbaldehyde], a synthetic carbazole derivative identified as an anti- inflammatory compound, were studied. Cell cycle analysis by propidium iodide staining in human THP-1 monocytic leukemia cells showed the ability of LCY-2-CHO to increase cell population in sub-G1 stage with time- and concentration- dependent manners. LCY-2- CHO-mediated cell death was also demonstrated by DNA laddering and was not related to the release of lactate dehydrogenase. Apoptosis in THP-1 cells induced by LCY-2-CHO was accompanied by the Bid cleavage, collapse of mitochondrial transmembrane potential, the release of cytochrome c and the activation of caspase-3. The apoptotic effect of LCY-2-CHO was diminished by the presence of zVEID-fmk (caspase-6 inhibitor), zIETD-fmk ( caspase-8 inhibitor), and zVAD-fmk (non-selective caspase inhibitor), but was not altered by several antioxidants, and cathepsin inhibitor. The Bid cleavage and loss of mitochondrial transmembrane potential, but not the cytochrome c release, were reversed by zIETD-fmk. Comparing the cell selectivity of LCY-2-CHO, we found T-cell acute lymphoblastic CEM leukemia cells were sensitive to 1 μM LCY -2-CHO, acute myeloid leukemia HL-60 cells underwent apoptosis at 10 μM, while adherent cancer cells, such as PC 3, HT29 and MCF-7, were resistant to 30 μM LCY-2-CHO within 24-h incubation. Taken together in the present study, we demonstrated LCY-2-CHO might be apoptotic for malignant hematopoietic cells but not anchorage-dependent cells. This action is mediated by an intrinsic caspase-dependent apoptotic event involving mitochondria
8-Chloro-cAMP inhibits transforming growth factor alpha transformation of mammary epithelial cells by restoration of the normal mRNA patterns for cAMP-dependent protein kinase regulatory subunit isoforms which show disruption upon transformation
No full textDifferential regulation of the regulatory subunits of cAMP-dependent protein kinase isozymes correlates with the growth inhibitory effect of site-selective 8-Cl-cAMP demonstrated in cancer cell lines (Ally, S., Tortora, G., Clair, T., Grieco, D., Merlo, G., Katsaros, D., Ogreid, D., Døskeland, S.O., Jahnsen, T., and Cho-Chung, Y.S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6319-6322). Such selective modulation of protein kinase isozyme regulatory subunits was also found in the 8-Cl-cAMP-induced inhibition of both transformation and transforming growth factor alpha (TGF alpha) production in Ki-ras-transformed rat kidney fibroblasts (Tortora, G., Ciardiello, F., Ally, S., Clair, T., Salomon, D. S., and Cho-Chung, Y. S. (1989) FEBS Lett. 242, 363-367). In this work, we have demonstrated that 8-Cl-cAMP antagonizes the TGF alpha effect in TGF alpha-transformed mouse mammary epithelial cells (NOG-8TFC17) at the level of gene expression for cAMP receptor protein isoforms, RI and RII (the regulatory subunits of protein kinase isozymes). Northern blot analysis demonstrated that in the transformed NOG-8TFC17 cells, compared with the nontransformed counterpart NOG-8 cells, the mRNA levels for the RI alpha cAMP receptor protein markedly increased, whereas the mRNA levels for the RII alpha and RII beta cAMP receptor proteins decreased. 8-Cl-cAMP, which induced growth inhibition and phenotypic reversion in NOG-8TFC17 cells, caused an inverse change in the mRNA patterns of the cAMP receptor proteins; RI alpha cAMP receptor mRNA sharply decreased to levels comparable with that of the nontransformed NOG-8 cells, whereas RII beta mRNA increased to a level even greater than that in the NOG-8 cells. In addition, one mRNA species of RII alpha increased, whereas the other RII alpha mRNA species decreased during the treatment. The mRNA level for the catalytic subunit of protein kinase, however, did not change during 8-Cl-cAMP treatment. In addition, 8-Cl-cAMP brought about a reduction in both TGF alpha mRNA and protein levels. These coordinated changes in the expression of the cAMP receptor proteins and TGF alpha were not observed during cis-hydroxyprolineor TGF beta-induced growth inhibition of the NOG-8TFC17 cells. Thus, the antagonistic effect of 8-Cl-cAMP toward TGF alpha-induced transformation involves modulation of the expression of a specific set of cellular genes
Molecular analysis and engineering of CHO production cell lines
No full textChinese hamster ovary (CHO) cell lines are considered as the “gold standard” host for production of therapeutic proteins such as monoclonal antibodies (mAbs). Given the increasing importance of antibody products and the growing number of complex artificial modalities that are oftentimes difficult to produce, highly optimized production hosts and processes are necessary to meet the growing demand. Although intensive cell line characterization has been conducted to increase the understanding of production hosts, the growing diversity of biopharmaceutical products requires product specific and knowledge-based process improvements. However, in order to identify further targets for cell line engineering, deep insight into cellular processes is required. To address this issue, the current dissertation focused on molecular analysis and engineering of CHO production cell lines. Especially cell line characterization, where omics studies focusing on certain subsets, so-called sub-omics, enable high resolution and deeper insight into cellular properties is of special interest. Since this approach represents an attractive tool for future targeted cell line engineering, the current dissertation highlighted the advances and the potential of sub-omics in CHO cells in a systematic and comprehensive overview. Subsequently, the recently published subset of CHO surface proteins was utilized for CHO cell line characterization. Here, pathway enrichment analysis as well as supplementary messenger ribonucleic acid (mRNA) expression data provided insight into enriched growth and survival related pathways as well as expression or absence of related surface receptors and their associated ligands. Hence, a valuable knowledge-base for future engineering or optimization approaches in CHO cells was established, emphasizing the relevance of sub-omics analyses for cell line characterization. However, as cellular production bottlenecks still represent a critical obstacle for efficient bioprocessing, a focused molecular analysis of cellular production bottlenecks was furthermore addressed. Here, it could be demonstrated that analysis of relevant steps of protein expression in a CHO production cell line resulted in identification of the root cause for production limitations of difficult-to-express (DTE) half-life extended (HLE) bispecific T-cell Engager (BiTE®) molecules, which underlined the application of focused exploration of cellular bottlenecks for targeted and product specific optimization strategies. Finally, aiming on providing an application of targeted cell line engineering based on previous cell line characterization, genome editing in CHO cells was conducted. For that purpose, the recently published CHO surfaceome provided valuable insight into surface protein composition, which enabled the identification of suitable targets for large-scale gene excision ultimately leading to a first step towards the generation of a genome-reduced CHO production host chassis enabling e.g. a cleaner production host with improved cellular energetic economy. In sum, this dissertation demonstrated the relevance of tailored cell characterization strategies for different scopes as well as their applicability
High productivity of t-PA in CHO cells using hypoxia response element
No full textThe dissolved oxygen level of any cell Culture environment has a critical effect on cellular metabolism. Specifically, hypoxia condition decreases cell viability and recombinant protein productivity. In this work, to develop CHO cells producing recombinant protein with high productivity, mammalian expression vectors containing a human tissue-type plasminogen activator (t-PA) gene with hypoxia response element (HR-E) were constructed and stably transfected into CHO cells. CHO/2HRE-t-PA cells produced 2-folds higher 2 recombinant t-PA production than CHO/t-PA cells in a Ba2+-alginate immobilized culture, and 16.8-folds in a repeated batch culture. In a non-aerated batch Culture of suspension-adapted cells, t-PA productivity of CHO/2HRE/t-PA cells was 4.2-folds higher than that of CHO/t-PA cells. Our results indicate that HRE is a useful tool for the enhancement of protein productivity in mammalian cell cultures
Non feasible EFMs of the CHO model at T = 0.
No full textNon feasible EFMs of the CHO model at T = 0.</p
Letter, [Author unclear] to Paulina T. Merritt
No full textHandwritten letter to Paulina Merritt from an unknown author, October 1, 1876.
1H NMR Spectroscopy Profiling of Metabolic Reprogramming of Chinese Hamster Ovary Cells upon a Temperature Shift during Culture
No full textWe report an NMR based approach to determine the metabolic reprogramming of Chinese hamster ovary cells upon
a temperature shift during culture by investigating the extracellular cell culture media and intracellular metabolome of
CHOK1 and CHO-S cells during culture and in response to cold-shock and subsequent recovery from hypothermic
culturing. A total of 24 components were identified for CHOK1 and 29 components identified for CHO-S cell systems
including the observation that CHO-S media contains 5.6 times the level of glucose of CHOK1 media at time zero.
We confirm that an NMR metabolic approach provides quantitative analysis of components such as glucose and
alanine with both cell lines responding in a similar manner and comparable to previously reported data. However,
analysis of lactate confirms a differentiation between CHOK1 and CHO-S and that reprogramming of metabolism in
response to temperature was cell line specific. The significance of our results is presented using principal component
analysis (PCA) that confirms changes in metabolite profile in response to temperature and recovery. Ultimately, our
approach demonstrates the capability of NMR providing real-time analysis to detect reprogramming of metabolism
upon cellular perception of cold-shock/sub-physiological temperatures. This has the potential to allow manipulation of
metabolites in culture supernatant to improve growth or productivity
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