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Find video protocols related to scientific articles indexed in Pubmed.
Gene expression measurements normalized to cell number reveal large scale differences due to cell size changes, transcriptional amplification and transcriptional repression in CHO cells.
J. Biotechnol.
PUBLISHED: 07-09-2014
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Conventional approaches to differential gene expression comparisons assume equal cellular RNA content among experimental conditions. We demonstrate that this assumption should not be universally applied because total RNA yield from a set number of cells varies among experimental treatments of the same Chinese Hamster Ovary (CHO) cell line and among different CHO cell lines expressing recombinant proteins. Conventional normalization strategies mask these differences in cellular RNA content and, consequently, skew biological interpretation of differential expression results. On the contrary, normalization to synthetic spike-in RNA standards added proportional to cell numbers reveals these differences and allows detection of global transcriptional amplification/repression. We apply this normalization method to assess differential gene expression in cell lines of different sizes, as well as cells treated with a cell cycle inhibitor (CCI), an mTOR inhibitor (mTORI), or subjected to high osmolarity conditions. CCI treatment of CHO cells results in a cellular volume increase and global transcriptional amplification, while mTORI treatment causes global transcriptional repression without affecting cellular volume. Similarly to CCI treatment, high osmolarity increases cell size, total RNA content and antibody expression. Furthermore, we show the importance of spike-in normalization for studies involving multiple CHO cell lines and advocate normalization to spike-in controls prior to correlating gene expression to specific productivity (qP). Overall, our data support the need for cell number specific spike-in controls for all gene expression studies where cellular RNA content differs among experimental conditions.
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Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.
Biotechnol. Bioeng.
PUBLISHED: 04-29-2014
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The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110?pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures. Biotechnol. Bioeng. 2014;9999: 1-15. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.
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Connecting Small Molecules with Similar Assay Performance Profiles Leads to New Biological Hypotheses.
J Biomol Screen
PUBLISHED: 01-24-2014
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High-throughput screening allows rapid identification of new candidate compounds for biological probe or drug development. Here, we describe a principled method to generate "assay performance profiles" for individual compounds that can serve as a basis for similarity searches and cluster analyses. Our method overcomes three challenges associated with generating robust assay performance profiles: (1) we transform data, allowing us to build profiles from assays having diverse dynamic ranges and variability; (2) we apply appropriate mathematical principles to handle missing data; and (3) we mitigate the fact that loss-of-signal assay measurements may not distinguish between multiple mechanisms that can lead to certain phenotypes (e.g., cell death). Our method connected compounds with similar mechanisms of action, enabling prediction of new targets and mechanisms both for known bioactives and for compounds emerging from new screens. Furthermore, we used Bayesian modeling of promiscuous compounds to distinguish between broadly bioactive and narrowly bioactive compound communities. Several examples illustrate the utility of our method to support mechanism-of-action studies in probe development and target identification projects.
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Quantitative-proteomic comparison of alpha and Beta cells to uncover novel targets for lineage reprogramming.
PLoS ONE
PUBLISHED: 01-01-2014
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Type-1 diabetes (T1D) is an autoimmune disease in which insulin-secreting pancreatic beta cells are destroyed by the immune system. An emerging strategy to regenerate beta-cell mass is through transdifferentiation of pancreatic alpha cells to beta cells. We previously reported two small molecules, BRD7389 and GW8510, that induce insulin expression in a mouse alpha cell line and provide a glimpse into potential intermediate cell states in beta-cell reprogramming from alpha cells. These small-molecule studies suggested that inhibition of kinases in particular may induce the expression of several beta-cell markers in alpha cells. To identify potential lineage reprogramming protein targets, we compared the transcriptome, proteome, and phosphoproteome of alpha cells, beta cells, and compound-treated alpha cells. Our phosphoproteomic analysis indicated that two kinases, BRSK1 and CAMKK2, exhibit decreased phosphorylation in beta cells compared to alpha cells, and in compound-treated alpha cells compared to DMSO-treated alpha cells. Knock-down of these kinases in alpha cells resulted in expression of key beta-cell markers. These results provide evidence that perturbation of the kinome may be important for lineage reprogramming of alpha cells to beta cells.
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Cellular responses to individual amino-acid depletion in antibody-expressing and parental CHO cell lines.
Biotechnol. Bioeng.
PUBLISHED: 07-22-2013
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Depletion of two nonessential amino acids, asparagine (Asn) and glutamine (Gln), occurred during a fed-batch production process with a CHO cell line expressing a recombinant antibody. This depletion coincided with growth suppression and the onset of the stationary phase. Experimental withdrawal of Asn led to cell cycle arrest of cell line A in G0/G1 phase. On a mechanistic level, withdrawal of either Asn or Gln stimulated the amino-acid response (AAR) pathway, indicating that depletion of nonessential amino acids can induce AAR in this cell line. Compared to withdrawal of an essential amino acid, leucine (Leu), withdrawal of either Asn or Gln induced fewer changes in downstream effectors of mammalian target of rapamycin (mTOR) signaling involved in regulation of global protein synthesis. Global transcriptional analysis followed by pathway analysis revealed that the cultures experienced a down-regulation of cell-cycle progression, DNA replication and nucleotide biosynthesis in an E2F-dependent manner, as well as a down-regulation of lipid metabolism in a SREBP1/2-dependent manner as a result of individual amino-acid withdrawal. Timing and magnitude of observed phenotypic and transcriptional responses to amino-acid withdrawal differed between essential (Leu) and nonessential (Asn and Gln) amino acids examined. Observed responses were similar in parental (CS9 and CHOK-1) and two other antibody-producing CHO cell lines, but the magnitude of the transcriptional response was both cell-line and amino-acid dependent. Overall, these results suggest that depletion of nonessential amino acids in cell culture plays a role in the onset of the stationary phase of production process and offer mechanistic insights into the observed growth attenuation phenotype. Biotechnol. Bioeng. 2013;9999: 1-15. © 2013 Wiley Periodicals, Inc.
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A human islet cell culture system for high-throughput screening.
J Biomol Screen
PUBLISHED: 12-07-2011
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A small-molecule inducer of beta-cell proliferation in human islets represents a potential regeneration strategy for treating type 1 diabetes. However, the lack of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted an islet cell culture system to high-throughput screening to identify such small molecules. We prepared microtiter plates containing extracellular matrix from a human bladder carcinoma cell line. Dissociated human islets were seeded onto these plates, cultured for up to 7 days, and assessed for proliferation by simultaneous Ki67 and C-peptide immunofluorescence. Importantly, this environment preserved beta-cell physiological function, as measured by glucose-stimulated insulin secretion. Adenoviral overexpression of cdk-6 and cyclin D(1), known inducers of human beta cell proliferation, was used as a positive control in our assay. This induction was inhibited by cotreatment with rapamycin, an immunosuppressant often used in islet transplantation. We then performed a pilot screen of 1280 compounds, observing some phenotypic effects on cells. This high-throughput human islet cell culture method can be used to assess various aspects of beta-cell biology on a relatively large number of compounds.
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Small-molecule inducers of insulin expression in pancreatic alpha-cells.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-09-2010
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High-content screening for small-molecule inducers of insulin expression identified the compound BRD7389, which caused alpha-cells to adopt several morphological and gene expression features of a beta-cell state. Assay-performance profile analysis suggests kinase inhibition as a mechanism of action, and we show that biochemical and cellular inhibition of the RSK kinase family by BRD7389 is likely related to its ability induce a beta-cell-like state. BRD7389 also increases the endocrine cell content and function of donor human pancreatic islets in culture.
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Comparative analysis of mutant tyrosine kinase chemical rescue.
Biochemistry
PUBLISHED: 03-06-2009
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Protein tyrosine kinases are critical cell signaling enzymes. These enzymes have a highly conserved Arg residue in their catalytic loop which is present two residues or four residues downstream from an absolutely conserved Asp catalytic base. Prior studies on protein tyrosine kinases Csk and Src revealed the potential for chemical rescue of catalytically deficient mutant kinases (Arg to Ala mutations) by small diamino compounds, particularly imidazole; however, the potency and efficiency of rescue was greater for Src. This current study further examines the structural and kinetic basis of rescue for mutant Src as compared to mutant Abl tyrosine kinase. An X-ray crystal structure of R388A Src revealed the surprising finding that a histidine residue of the N-terminus of a symmetry-related kinase inserts into the active site of the adjacent Src and mimics the hydrogen-bonding pattern seen in wild-type protein tyrosine kinases. Abl R367A shows potent and efficient rescue more comparable to Src, even though its catalytic loop is more like that of Csk. Various enzyme redesigns of the active sites indicate that the degree and specificity of rescue are somewhat flexible, but the overall properties of the enzymes and rescue agents play an overarching role. The newly discovered rescue agent 2-aminoimidazole is about as efficient as imidazole in rescuing R/A Src and Abl. Rate vs pH studies with these imidazole analogues suggest that the protonated imidazolium is the preferred form for chemical rescue, consistent with structural models. The efficient rescue seen with mutant Abl points to the potential of this approach to be used effectively to analyze Abl phosphorylation pathways in cells.
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Chromatin-targeting small molecules cause class-specific transcriptional changes in pancreatic endocrine cells.
Proc. Natl. Acad. Sci. U.S.A.
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Under the instruction of cell-fate-determining, DNA-binding transcription factors, chromatin-modifying enzymes mediate and maintain cell states throughout development in multicellular organisms. Currently, small molecules modulating the activity of several classes of chromatin-modifying enzymes are available, including clinically approved histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors. We describe the genome-wide expression changes induced by 29 compounds targeting HDACs, DNMTs, histone lysine methyltransferases (HKMTs), and protein arginine methyltransferases (PRMTs) in pancreatic ?- and ?-cell lines. HDAC inhibitors regulate several hundred transcripts irrespective of the cell type, with distinct clusters of dissimilar activity for hydroxamic acids and orthoamino anilides. In contrast, compounds targeting histone methyltransferases modulate the expression of restricted gene sets in distinct cell types. For example, we find that G9a/GLP methyltransferase inhibitors selectively up-regulate the cholesterol biosynthetic pathway in pancreatic but not liver cells. These data suggest that, despite their conservation across the entire genome and in different cell types, chromatin pathways can be targeted to modulate the expression of selected transcripts.
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GW8510 increases insulin expression in pancreatic alpha cells through activation of p53 transcriptional activity.
PLoS ONE
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Expression of insulin in terminally differentiated non-beta cell types in the pancreas could be important to treating type-1 diabetes. Previous findings led us to hypothesize involvement of kinase inhibition in induction of insulin expression in pancreatic alpha cells.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.