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1.
J Pharmacol Toxicol Methods ; 99: 106612, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31319140

RESUMO

INTRODUCTION: Voltage-sensitive optical (VSO) sensors offer a minimally invasive method to study the time course of repolarization of the cardiac action potential (AP). This Comprehensive in vitro Proarrhythmia Assay (CiPA) cross-platform study investigates protocol design and measurement variability of VSO sensors for preclinical cardiac electrophysiology assays. METHODS: Three commercial and one academic laboratory completed a limited study of the effects of 8 blinded compounds on the electrophysiology of 2 commercial lines of human induced pluripotent stem-cell derived cardiomyocytes (hSC-CMs). Acquisition technologies included CMOS camera and photometry; fluorescent voltage sensors included di-4-ANEPPS, FluoVolt and genetically encoded QuasAr2. The experimental protocol was standardized with respect to cell lines, plating and maintenance media, blinded compounds, and action potential parameters measured. Serum-free media was used to study the action of drugs, but the exact composition and the protocols for cell preparation and drug additions varied among sites. RESULTS: Baseline AP waveforms differed across platforms and between cell types. Despite these differences, the relative responses to four selective ion channel blockers (E-4031, nifedipine, mexiletine, and JNJ 303 blocking IKr, ICaL, INa, and IKs, respectively) were similar across all platforms and cell lines although the absolute changes differed. Similarly, four mixed ion channel blockers (flecainide, moxifloxacin, quinidine, and ranolazine) had comparable effects in all platforms. Differences in repolarisation time course and response to drugs could be attributed to cell type and experimental method differences such as composition of the assay media, stimulated versus spontaneous activity, and single versus cumulative compound addition. DISCUSSION: In conclusion, VSOs represent a powerful and appropriate method to assess the electrophysiological effects of drugs on iPSC-CMs for the evaluation of proarrhythmic risk. Protocol considerations and recommendations are provided toward standardizing conditions to reduce variability of baseline AP waveform characteristics and drug responses.

2.
Int Rev Cell Mol Biol ; 306: 275-332, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24016528

RESUMO

It is of interest to quantify the size, shape, and metabolic subtype of skeletal muscle fibers in many areas of biomedical research. To do so, skeletal muscle samples are sectioned transversely to the length of the muscle and labeled for extracellular or membrane proteins to delineate the fiber boundaries and additionally for biomarkers related to function or metabolism. The samples are digitally photographed and the fibers "outlined" for quantification of fiber cross-sectional area (CSA) using pointing devices interfaced to a computer, which is tedious, prone to error, and can be nonobjective. Here, we review methods for characterizing skeletal muscle fibers and describe new automated techniques, which rapidly quantify CSA and biomarkers. We discuss the applications of these methods to the characterization of mitochondrial dysfunctions, which underlie a variety of human afflictions, and we present a novel approach, utilizing images from the online Human Protein Atlas to predict relationships between fiber-specific protein expression, function, and metabolism.


Assuntos
Automação/métodos , Tamanho Celular , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Animais , Humanos
3.
Assay Drug Dev Technol ; 9(3): 262-80, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21186937

RESUMO

Lipolysis in adipocytes is associated with phosphorylation of hormone sensitive lipase (HSL) and translocation of HSL to lipid droplets. In this study, adipocytes were cultured in a high-throughput format (96-well dishes), exposed to lipolytic agents, and then fixed and labeled for nuclei, lipid droplets, and HSL (or HSL phosphorylated on serine 660 [pHSLser660]). The cells were imaged via automated digital fluorescence microscopy, and high-content analysis (HCA) methods were used to quantify HSL phosphorylation and the degree to which HSL (or pHSLser660) colocalizes with the lipid droplets. HSL:lipid droplet colocalization was quantified through use of Pearson's correlation, Mander's M1 Colocalization, and the Tanimoto coefficient. For murine 3T3L1 adipocytes, isoproterenol, Lys-γ3-melanocyte stimulating hormone, and forskolin elicited the appearance and colocalization of pHSLser660, whereas atrial natriuretic peptide (ANP) did not. For human subcutaneous adipocytes, isoproterenol, forskolin, and ANP activated HSL phosphorylation/colocalization, but Lys-γ3-melanocyte stimulating hormone had little or no effect. Since ANP activates guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase, HSL serine 660 is likely a substrate for cGMP-dependent protein kinase in human adipocytes. For both adipocyte model systems, adipocytes with the greatest lipid content displayed the greatest lipolytic responses. The results for pHSLser660 were consistent with release of glycerol by the cells, a well-established assay of lipolysis, and the HCA methods yielded Z' values >0.50. The results illustrate several key differences between human and murine adipocytes and demonstrate advantages of utilizing HCA techniques to study lipolysis in cultured adipocytes.


Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Hormônios/metabolismo , Lipase/metabolismo , Lipólise/efeitos dos fármacos , Lipólise/fisiologia , Microscopia/métodos , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Animais , Células Cultivadas , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/química , Camundongos , Reconhecimento Automatizado de Padrão/métodos , Fosforilação/efeitos dos fármacos , Processamento de Sinais Assistido por Computador , Pele/citologia , Fenômenos Fisiológicos da Pele/efeitos dos fármacos
4.
J Biomol Screen ; 15(7): 798-805, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20639500

RESUMO

Hepatic lipid droplets (LDs) are associated with metabolic syndrome, type 2 diabetes, hepatitis C, and both alcoholic and nonalcoholic fatty liver disease. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the level of translation. Approximately 1000 different miRNA species are encoded within the human genome, and many are differentially expressed by healthy and diseased liver. However, few studies have investigated the role of miRNAs in regulating LD expression. Accordingly, a high-content assay (HCA) was performed in which human hepatocytes (Huh-7 cells) were transiently transfected with 327 unique human miRNAs; the cells were then fixed, labeled for nuclei and lipid droplets, and imaged with an automated digital microscopy workstation. LD expression was analyzed on a cell-by-cell basis, using automated image analysis. Eleven miRNAs were identified that altered LDs. MiR-181d was the most efficacious inhibitor, decreasing LDs by about 60%. miRNA-181d was also confirmed to reduce cellular triglycerides and cholesterol ester via biochemical assays. Furthermore, a series of proteins was identified via miRNA target analysis, and siRNAs directed against many of these proteins also modified LDs. Thus, HCA-based screening identified novel miRNA and protein regulators of LDs and cholesterol metabolism that may be relevant to hepatic diseases arising from obesity and alcohol abuse.


Assuntos
Hepatócitos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Metabolismo dos Lipídeos , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Colesterol/metabolismo , Biblioteca Gênica , Humanos , Espaço Intracelular/metabolismo , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Triglicerídeos/metabolismo
5.
Assay Drug Dev Technol ; 7(5): 440-60, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19895345

RESUMO

Intracellular lipid droplets are associated with a myriad of afflictions including obesity, fatty liver disease, coronary artery disease, and infectious diseases (eg, HCV and tuberculosis). To develop high-content analysis (HCA) techniques to analyze lipid droplets and associated proteins, primary human preadipocytes were plated in 96-well dishes in the presence of rosiglitazone (rosi), a PPAR-(c) agonist that promotes adipogenesis. The cells were then labeled for nuclei, lipid droplets, and proteins such as perilipin, protein kinase C (PKC), and hormone-sensitive lipase (HSL). The cells were imaged via automated digital microscopy and algorithms were developed to quantify lipid droplet (Lipid Droplet algorithm) and protein expression and colocalization (Colocalization algorithm). The algorithms, which were incorporated into Vala Science Inc's CyteSeer((R)) image cytometry program, quantified the rosi-induced increases in lipid droplet number, size, and intensity, and the expression of perilipin with exceptional consistency (Z' values of 0.54-0.71). Regarding colocalization with lipid droplets, Pearson's correlation coefficients of 0.38 (highly colocalized), 0.16 (moderate), and -0.0010 (random) were found for perilipin, PKC, and HSL, respectively. For hepatocytes (AML12, HuH-7, and primary cells), the algorithms also quantified the stimulatory and inhibitory effect of oleic acid and triacsin C on lipid droplets (Z's > 0.50) and ADFP expression/colocalization. Oleic acid-induced lipid droplets in HeLa cells and macrophages (THP-1) were also well quantified. The results suggest that HCA techniques can be utilized to quantify lipid droplets and associated proteins in many cell models relevant to a variety of diseases.


Assuntos
Lipídeos/química , Lipoproteínas/química , Obesidade/patologia , Proteínas/química , Adipócitos/fisiologia , Algoritmos , Linhagem Celular , Interpretação Estatística de Dados , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Citometria de Fluxo , Hepatócitos/fisiologia , Humanos , Processamento de Imagem Assistida por Computador , Microscopia , Modelos Biológicos , Terminologia como Assunto , Triglicerídeos/análise
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