Abstract 426: A Novel Method to Study Viral Cardiomyopathy

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Millie Shah ◽  
Cheryl Borgman ◽  
Kevin Janes
Keyword(s):  
Cell Lines ◽  
High Throughput ◽  
Viral Rna ◽  
Extracellular Matrix Protein ◽  
Cytokine Signaling ◽  
Rna Expression ◽  
Live Virus ◽  
Signaling Protein ◽  
Human Cardiomyocytes ◽  
Cvb3 Infection

Latent coxsackievirus B3 (CVB3) cardiac infections are implicated in the development of DCM with persistent enteroviral RNA found in up to 66% (30/45) of DCM patient biopsies who are at a 6-fold higher risk of fatality. Current treatments aim at delaying heart failure but none address the viral etiology of the disease. Viral protein presence is thought to disrupt normal cellular signaling leading to tissue dysfunction. The downstream effects of viral perturbations are complex and wide-ranging; especially in proinflammatory contexts seen clinically. Thus, comprehensively understanding the molecular mechanisms of CVB3-mediated disease is key in developing treatments for viral DCM patients. In this study we built a novel in vitro model of chronic CVB3 infection to facilitate high-throughput, systems-level studies of viral cardiomyopathy. Current methods of studying CVB3 rely on animals or animal derived cardiac cells, making large-scale intracellular signaling studies difficult, time intensive, and expensive. To facilitate high-throughput studies we used immortalized human cardiomyocytes to engineer single-cell derived cell lines that express a maturation deficient CVB3 genome. CVB3 RNA expression was validated in each line by two independent methods: gene-specific nested qRT-PCR and single molecule RNA fluorescence in situ hydridization (smFISH). Plaque-assay verified that viral RNA expression did not result in live virus release. Microarray analysis shows that CVB3 expressing cell lines have altered immune cytokine, extracellular matrix protein, and stress-signaling protein expression. Further, differences between CVB3 expressing lines suggest differential responses to viral RNA expression which may identify a set of beneficial adaptations. Future studies will include high-throughput signaling protein activity assays we have developed specifically for phosphatase and kinase activity quantification with the aim of linking immune cytokine signaling dysregulation to pathogenic gene expression. These studies will deepen our knowledge of CVB3-mediated DCM and identify proteins whose targeted modulation could offer new treatment strategies to patients whose current options are either palliative care or heart transplantation.

High Throughput Sequencing of Acute Lymphoblastic Leukemia Small RNAs.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3650-3650
Author(s):  
Michael C Wei ◽  
Elizabeth O Osborn ◽  
Michael Cleary
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
High Throughput ◽  
Small Rna ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Rna Expression ◽  
Leukemia Cell Lines

Abstract Abstract 3650 MicroRNAs are small, non-coding RNAs that regulate gene expression and play key roles in cancer by modulating oncogene and tumor suppressor pathways. We are investigating the clinical and prognostic roles of miRNA expression in pediatric leukemias using high-throughput sequencing as a profiling tool. To establish the methodology, we have utilized high-throughput sequencing to quantify small RNA expression from eight acute lymphoblastic leukemia cell lines and one MLL-rearranged infant ALL patient sample. We generated sequencing libraries from these cells, conducted high-throughput sequencing using the Illumina platform, and established a custom bioinformatics pipeline for data analysis. Over 50 million individual sequence reads were analyzed. These sequences were mapped against a database of human miRNAs, and the frequency of miRNA expression among samples was enumerated. Expression of hematopoietic-specific miR-142 and miR-181 cluster miRs was found in these leukemia samples, while the liver-specific miR-122 was not expressed. miR-196b, previously reported to be over-expressed in MLL-rearranged leukemias, was expressed in 3/3 MLL-rearranged leukemia cell lines and 1/5 non-MLL cell lines. Expression of individual miRNAs was validated by quantitative PCR. Additional analysis of MLL-associated miRNAs and novel small RNAs will be presented. Our results demonstrate the feasibility and potential of high-throughput sequencing to profile the expression of small RNAs from leukemia cells, and we plan to apply these methods to additional primary patient samples to examine prognostic and clinical correlations with small RNA expression patterns. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

2019 ◽  
Vol 25 (1) ◽  
pp. 9-20 ◽  
Author(s):  
Olivia W. Lee ◽  
Shelley Austin ◽  
Madison Gamma ◽  
Dorian M. Cheff ◽  
Tobie D. Lee ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Large Scale ◽  
Early Stage ◽  
Cancer Cell Line ◽  
Hek 293 ◽  
Cytotoxic Compounds

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes, and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed the cell-based cytotoxicity of nearly 10,000 compounds in the National Institutes of Health, National Center for Advancing Translational Sciences annotated libraries and more than 100,000 compounds in a diversity library against four normal cell lines (HEK 293, NIH 3T3, CRL-7250, and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity, and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitute a valuable resource for the scientific community and provide insight into the extent of cytotoxic compounds in screening libraries, allowing for the identification and avoidance of compounds with cytotoxicity during high-throughput screening campaigns.

scholarly journals Epithelium-Specific Ets-Like Transcription Factor 1, ESE-1, Regulates ICAM-1 Expression in Cultured Lung Epithelial Cell Lines

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Zhiqi Yu ◽  
Jun Xu ◽  
Jinbao Liu ◽  
Jing Wu ◽  
Chan Mi Lee ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Airway Inflammation ◽  
Cell Lines ◽  
Leukemia Cell ◽  
A549 Cells ◽  
Leukemia Cell Line ◽  
Intercellular Adhesion Molecule ◽  
Cytokine Signaling ◽  
Target Cells ◽  
Transcription Factor 1

Cystic fibrosis (CF) patients suffer from chronic airway inflammation with excessive neutrophil infiltration. Migration of neutrophils to the lung requires chemokine and cytokine signaling as well as cell adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), which plays an important role in mediating adhesive interactions between effector and target cells in the immune system. In this study, we investigated the relationship betweenICAM-1and epithelium-specific ETS-like transcription factor 1 (ESE-1) and found thatICAM-1expression is upregulated in cell lines of CF (IB3-1) as well as non-CF (BEAS-2B and A549) epithelial origin in response to inflammatory cytokine stimulation. SinceESE-1is highly expressed in A549 cells without stimulation, we examined the effect ofESE-1knockdown onICAM-1expression in these cells. We found thatICAM-1expression was downregulated whenESE-1was knocked down in A549 cells. We also tested the effect ofESE-1knockdown on cell-cell interactions and demonstrate that the knocking downESE-1in A549 cells reduce their interactions with HL-60 cells (human promyelocytic leukemia cell line). These results suggest thatESE-1may play a role in regulating airway inflammation by regulatingICAM-1expression.

scholarly journals A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

10.3791/3010 ◽  
2011 ◽  
Author(s):  
Shuangping Shi ◽  
Russ G.G. Condon ◽  
Liang Deng ◽  
Jason Saunders ◽  
Finn Hung ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Throughput ◽  
Protein Therapeutics ◽  
Manufacturing Cell ◽  
Automated Platform

scholarly journals Quantitative measurement of infectious virus in SARS-CoV-2 Alpha, Delta and Epsilon variants reveals higher infectivity (viral titer:RNA ratio) in clinical samples containing the Delta and Epsilon variants.

2021 ◽  
Author(s):  
Hannah W Despres ◽  
Margaret G Mills ◽  
David J Shirley ◽  
Madaline M Schmidt ◽  
Meei-Li Huang ◽  
...  
Keyword(s):  
Quantitative Measurement ◽  
Infectious Virus ◽  
Viral Rna ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Genome Copy ◽  
Live Virus ◽  
High Degree ◽  
The Relationship ◽  
Rna Levels

ABSTRACT Background Novel SARS-CoV-2 Variants of Concern (VoC) pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between viral RNA and infectious virus for individual variants is unknown. Methods We measured infectious viral titer (using a micro-focus forming assay) as well as total and subgenomic viral RNA levels (using RT-PCR) in a set of 165 clinical samples containing SARS-CoV-2 Alpha, Delta and Epsilon variants that were processed within two days of collection from the patient. Results We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite the variability we observed for individual samples the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (6 and 4 times as much, p=0.0002 and 0.009 respectively) or subgenomic E RNA (11 and 7 times as much, p<0.0001 and 0.006 respectively). Conclusion In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may also be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity of the Delta variant may further explain increased spread and suggests a need for increased measures to prevent viral transmission.

scholarly journals High-Throughput 3D Tumor Spheroid Array Platform for Evaluating Sensitivity of Proton-Drug Combinations

2022 ◽  
Vol 23 (2) ◽  
pp. 587
Author(s):  
Dong Woo Lee ◽  
Jung Eun Kim ◽  
Ga-Haeng Lee ◽  
Arang Son ◽  
Hee Chul Park ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Throughput ◽  
High Throughput Screening ◽  
Quantitative Data ◽  
Survival Rates ◽  
Drug Combinations ◽  
Targeted Drugs ◽  
Tumor Spheroid ◽  
Array Platform ◽  
Hnscc Cell

Proton beam therapy (PBT) is a critical treatment modality for head and neck squamous cell carcinoma (HNSCC). However, not much is known about drug combinations that may improve the efficacy of PBT. This study aimed to test the feasibility of a three-dimensional (3D) tumor-spheroid-based high-throughput screening platform that could assess cellular sensitivity against PBT. Spheroids of two HNSCC cell lines—Fadu and Cal27—cultured with a mixture of Matrigel were arrayed on a 384-pillar/well plate, followed by exposure to graded doses of protons or targeted drugs including olaparib at various concentrations. Calcein staining of HNSCC spheroids revealed a dose-dependent decrease in cell viability for proton irradiation or multiple targeted drugs, and provided quantitative data that discriminated the sensitivity between the two HNSCC cell lines. The combined effect of protons and olaparib was assessed by calculating the combination index from the survival rates of 4 × 4 matrices, showing that Cal27 spheroids had greater synergy with olaparib than Fadu spheroids. In contrast, adavosertib did not synergize with protons in both spheroids. Taken together, we demonstrated that the 3D pillar/well array platform was a useful tool that provided rapid, quantitative data for evaluating sensitivity to PBT and drug combinations. Our results further supported that administration of the combination of PBT and olaparib may be an effective treatment strategy for HNSCC patients.

scholarly journals Complex Tumor Spheroids, a Tissue-Mimicking Tumor Model, for Drug Discovery and Precision Medicine

2021 ◽  
pp. 247255522110383
Author(s):  
Gurmeet Kaur ◽  
David M. Evans ◽  
Beverly A. Teicher ◽  
Nathan P. Coussens
Keyword(s):  
Drug Discovery ◽  
Precision Medicine ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cell Types ◽  
Response To Therapy ◽  
Cancer Drug ◽  
Malignant Cells ◽  
Pancreatic Cell

Malignant tumors are complex tissues composed of malignant cells, vascular cells, structural mesenchymal cells including pericytes and carcinoma-associated fibroblasts, infiltrating immune cells, and others, collectively called the tumor stroma. The number of stromal cells in a tumor is often much greater than the number of malignant cells. The physical associations among all these cell types are critical to tumor growth, survival, and response to therapy. Most cell-based screens for cancer drug discovery and precision medicine validation use malignant cells in isolation as monolayers, embedded in a matrix, or as spheroids in suspension. Medium- and high-throughput screening with multiple cell lines requires a scalable, reproducible, robust cell-based assay. Complex spheroids include malignant cells and two normal cell types, human umbilical vein endothelial cells and highly plastic mesenchymal stem cells, which rapidly adapt to the malignant cell microenvironment. The patient-derived pancreatic adenocarcinoma cell line, K24384-001-R, was used to explore complex spheroid structure and response to anticancer agents in a 96-well format. We describe the development of the complex spheroid assay as well as the growth and structure of complex spheroids over time. Subsequently, we demonstrate successful assay miniaturization to a 384-well format and robust performance in a high-throughput screen. Implementation of the complex spheroid assay was further demonstrated with 10 well-established pancreatic cell lines. By incorporating both human stromal and tumor components, complex spheroids might provide an improved model for tumor response in vivo.

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