RESUMEN
Programmable liquid handling devices for cell culture systems have dramatically enhanced scalability and reproducibility. We previously reported a protocol to produce cell aggregates demonstrating growth plate-like structures containing hypertrophic chondrocytes from human induced pluripotent stem cells (hiPSCs). To apply this protocol to large-scale drug screening for growth plate-related diseases, we adapted it to the automated cell culture system (ACCS) consisting of programmable liquid handling devices connected to CO2 incubators, a refrigerator, and labware feeders, designed for up to 4 batches with several cell culture plates culturing for several months. We developed a new program preparing culture media with growth factors at final concentration immediately before dispensing them to each well and precisely positioning the tip for the medium change without damaging cell aggregates. Using these programs on the ACCS, we successfully cultured cell aggregates for 56 days, only needing to replenish the labware, medium, and growth factors twice a week. The size of cell aggregates in each well increased over time, with low well-to-well variability. Cell aggregates on day 56 showed histochemical, immunohistochemical, and gene expression properties of growth plate-like structures containing hypertrophic chondrocytes, indicating proper quality as materials for basic research and drug discovery of growth plate related diseases. The established program will be a suitable reference for making programs of experiments requiring long term and complex culture procedures using ACCS.
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Células Madre Pluripotentes Inducidas , Humanos , Reproducibilidad de los Resultados , Placa de Crecimiento , Técnicas de Cultivo de Célula/métodos , Células CultivadasRESUMEN
Due to their physiological relevance, cell-based assays using human-induced pluripotent stem cell (iPSC)-derived cells are a promising in vitro pharmacological evaluation system for drug candidates. However, cell-based assays involve complex processes such as long-term culture, real-time and continuous observation of living cells, and detection of many cellular events. Automating multi-sample processing through these assays will enhance reproducibility by limiting human error and reduce researchers' valuable time spent conducting these experiments. Furthermore, this integration enables continuous tracking of morphological changes, which is not possible with the use of stand-alone devices. This report describes a new laboratory automation system called the Screening Station, which uses novel automation control and scheduling software called Green Button Go to integrate various devices. To integrate the above-mentioned processes, we established three workflows in Green Button Go: 1) For long-term cell culture, culture plates and medium containers are transported from the automatic CO2 incubator and cool incubator, respectively, and the cell culture medium in the microplates is exchanged daily using the Biomek i7 workstation; 2) For time-lapse live-cell imaging, culture plates are automatically transferred between the CQ1 confocal quantitative image cytometer and the SCALE48W automatic CO2 incubator; 3) For immunofluorescence imaging assays, in addition to the above-mentioned devices, the 405LS microplate washer allows for formalin-fixation and immunostaining of cells. By scheduling various combinations of the three workflows, we successfully automated the culture and medium exchange processes for iPSCs derived from patients with facioscapulohumeral muscular dystrophy, confirmation of their differentiation status by live-cell imaging, and confirmation of the presence of differentiation markers by immunostaining. In addition, deep learning analysis enabled us to quantify the degree of iPSC differentiation from live-cell imaging data. Further, the results of the fully automated experiments could be accessed via the intranet, enabling experiments and analysis to be conducted remotely once the necessary reagents and labware were prepared. We expect that the ability to perform clinically and physiologically relevant cell-based assays from remote locations using the Screening Station will facilitate global research collaboration and accelerate the discovery of new drug candidates.
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To deal with the broad spectrum of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that threaten human health, it is essential to not only drugs develop that target viral proteins but also consider drugs that target host proteins/cellular processes to protect them from being hijacked for viral infection and replication. To this end, it has been reported that autophagy is deeply involved in coronavirus infection. In this study, we used airway organoids to screen a chemical library of autophagic modulators to identify compounds that could potentially be used to fight against infections by a broad range of coronaviruses. Among the 80 autophagy-related compounds tested, cycloheximide and thapsigargin reduced SARS-CoV-2 infection efficiency in a dose-dependent manner. Cycloheximide treatment reduced the infection efficiency of not only six SARS-CoV-2 variants but also human coronavirus (HCoV)-229E and HCoV-OC43. Cycloheximide treatment also reversed viral infection-induced innate immune responses. However, even low-dose (1 µM) cycloheximide treatment altered the expression profile of ribosomal RNAs; thus, side effects such as inhibition of protein synthesis in host cells must be considered. These results suggest that cycloheximide has broad-spectrum anti-coronavirus activity in vitro and warrants further investigation.
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COVID-19 , Coronavirus Humano 229E , Humanos , SARS-CoV-2 , Cicloheximida/farmacología , AutofagiaRESUMEN
Induced pluripotent stem cells (iPSCs) are attractive for use in early drug discovery because they can differentiate into any cell type. Maintenance cultures and differentiation processes for iPSCs, however, require a high level of technical expertise. To overcome this problem, technological developments such as enhanced automation are necessary to replace manual operation. In addition, a robot system with the flexibility and expandability to carry out maintenance culture and each of the required differentiation processes would also be important. In this study, we established a platform to enable the multiple processes required for iPSC experiments using the Maholo LabDroid, which is a humanoid robotic system with excellent reproducibility and flexibility. The accuracy and robustness of Maholo LabDroid enabled us to cultivate undifferentiated iPSCs for 63 days while maintaining their ability to differentiate into the three embryonic germ layers. Maholo LabDroid maintained and harvested iPSCs in six-well plates, then seeded them into 96-well plates, induced differentiation, and implemented immunocytochemistry. As a result, Maholo LabDroid was confirmed to be able to perform the processes required for myogenic differentiation of iPSCs isolated from a patient with muscular disease and achieved a high differentiation rate with a coefficient of variation (CV) <10% in the first trial. Furthermore, the expandability and flexibility of Maholo LabDroid allowed us to experiment with multiple cell lines simultaneously.
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Células Madre Pluripotentes Inducidas , Diferenciación Celular , Línea Celular , Descubrimiento de Drogas , Humanos , Reproducibilidad de los ResultadosRESUMEN
Dysferlinopathy is a progressive muscle disorder that includes limb-girdle muscular dystrophy type 2B and Miyoshi myopathy (MM). It is caused by mutations in the dysferlin (DYSF) gene, whose function is to reseal the muscular membrane. Treatment with proteasome inhibitor MG-132 has been shown to increase misfolded dysferlin in fibroblasts, allowing them to recover their membrane resealing function. Here, we developed a screening system based on myocytes from MM patient-derived induced pluripotent stem cells. According to the screening, nocodazole was found to effectively increase the level of dysferlin in cells, which, in turn, enhanced membrane resealing following injury by laser irradiation. Moreover, the increase was due to microtubule disorganization and involved autophagy rather than the proteasome degradation pathway. These findings suggest that increasing the amount of misfolded dysferlin using small molecules could represent an effective future clinical treatment for dysferlinopathy. Stem Cells Translational Medicine 2019;8:1017-1029.
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Evaluación Preclínica de Medicamentos/métodos , Células Madre Pluripotentes Inducidas/trasplante , Células Musculares/metabolismo , Distrofia Muscular de Cinturas/tratamiento farmacológico , Adulto , Femenino , Humanos , Persona de Mediana Edad , FenotipoRESUMEN
Since impaired mitochondrial ATP production in cardiomyocytes is thought to lead to heart failure, a drug that protects mitochondria and improves ATP production under disease conditions would be an attractive treatment option. In this study, we identified small-molecule drugs, including the anti-parasitic agent, ivermectin, that maintain mitochondrial ATP levels under hypoxia in cardiomyocytes. Mechanistically, transcriptomic analysis and gene silencing experiments revealed that ivermectin increased mitochondrial ATP production by inducing Cox6a2, a subunit of the mitochondrial respiratory chain. Furthermore, ivermectin inhibited the hypertrophic response of human induced pluripotent stem cell-derived cardiomyocytes. Pharmacological inhibition of importin ß, one of the targets of ivermectin, exhibited protection against mitochondrial ATP decline and cardiomyocyte hypertrophy. These findings indicate that maintaining mitochondrial ATP under hypoxia may prevent hypertrophy and improve cardiac function, providing therapeutic options for mitochondrial dysfunction.
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Adenosina Trifosfato/metabolismo , Cardiotónicos/farmacología , Hipoxia de la Célula/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Miocitos Cardíacos/citología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Células Cultivadas , Complejo IV de Transporte de Electrones/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Ivermectina/farmacología , Ratones , Mitocondrias/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , beta Carioferinas/metabolismoRESUMEN
Inositol hexakisphosphate kinase 3 (IP6K3) generates inositol pyrophosphates, which regulate diverse cellular functions. However, little is known about its own physiological role. Here, we show the roles of IP6K3 in metabolic regulation. We detected high levels of both mouse and human IP6K3 mRNA in myotubes and muscle tissues. In human myotubes, IP6K3 was upregulated by dexamethasone treatment, which is known to inhibit glucose metabolism. Furthermore, Ip6k3 expression was elevated under diabetic, fasting, and disuse conditions in mouse skeletal muscles. Ip6k3(-/-) mice demonstrated lower blood glucose, reduced circulating insulin, deceased fat mass, lower body weight, increased plasma lactate, enhanced glucose tolerance, lower glucose during an insulin tolerance test, and reduced muscle Pdk4 expression under normal diet conditions. Notably, Ip6k3 deletion extended animal lifespan with concomitant reduced phosphorylation of S6 ribosomal protein in the heart. In contrast, Ip6k3(-/-) mice showed unchanged skeletal muscle mass and no resistance to the effects of high fat diet. The current observations suggest novel roles of IP6K3 in cellular regulation, which impact metabolic control and lifespan.
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Metabolismo Energético/fisiología , Longevidad/fisiología , Fosfotransferasas (Aceptor del Grupo Fosfato)/fisiología , Animales , Composición Corporal , Peso Corporal , Línea Celular , Dexametasona/farmacología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Dieta Alta en Grasa/efectos adversos , Metabolismo Energético/genética , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Islotes Pancreáticos/enzimología , Longevidad/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Fibras Musculares Esqueléticas/enzimología , Músculo Esquelético/enzimología , Miocardio/metabolismo , Especificidad de Órganos , Fosforilación , Fosfotransferasas (Aceptor del Grupo Fosfato)/deficiencia , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Procesamiento Proteico-Postraduccional , Ratas , Proteína S6 Ribosómica/metabolismoRESUMEN
Acyl-CoA: cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes cholesterol esterification. ACAT inhibitors are expected to be potent therapeutic agents for the treatment of atherosclerosis. A series of potent ACAT inhibitors based on an (4-phenylcoumarin)acetanilide scaffold was identified. Evaluation of the structure-activity relationships of a substituent on this scaffold, with an emphasis on improving the pharmacokinetic profile led to the discovery of 2-[7-chloro-4-(3-chlorophenyl)-6-methyl-2-oxo-2H-chromen-3-yl]-N-[4-chloro-2-(trifluoromethyl)phenyl]acetamide (23), which exhibited potent ACAT inhibitory activity (IC50=12 nM) and good pharmacokinetic profile in mice. Compound 23 also showed regressive effects on atherosclerotic plaques in apolipoprotein (apo)E knock out (KO) mice at a dose of 0.3 mg/kg per os (p.o.).
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Acetamidas/síntesis química , Acetamidas/farmacología , Acetamidas/farmacocinética , Acilcoenzima A/antagonistas & inhibidores , Anticolesterolemiantes/farmacología , Aterosclerosis/metabolismo , Benzopiranos/síntesis química , Benzopiranos/farmacología , Benzopiranos/farmacocinética , Inhibidores Enzimáticos/farmacología , Acetamidas/química , Acetanilidas/química , Administración Oral , Animales , Anticolesterolemiantes/síntesis química , Anticolesterolemiantes/química , Anticolesterolemiantes/farmacocinética , Apolipoproteínas/metabolismo , Benzopiranos/química , Colesterol/metabolismo , Cumarinas/química , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Terapia Molecular Dirigida , Relación Estructura-ActividadRESUMEN
BACKGROUND: Apelin is an endogenous ligand for the G-protein-coupled 7-transmembrane receptor, APJ. The administration of apelin-13, a truncated 13-amino acid apelin peptide, in diet-induced obese mice is reported to result in a decrease in adiposity due to the increase of energy expenditure with an increase in the expression of uncoupling proteins. METHODS: We systematically compared the phenotype of human apelin-transgenic (apelin-Tg) mice fed standard or high-fat diets (HFD) with that of non-Tg control mice to clarify the effect of apelin on obesity. The beneficial effects of apelin were evaluated by multiple assay methods including indirect calorimetrical measurements, gene expression analysis, and immunohistochemical staining. RESULTS: Apelin-Tg mice inhibited HFD-induced obesity without altering food intake and exhibited increased oxygen consumption and body temperature compared to non-Tg controls. Interestingly, the mRNA expressions of angiopoietin-1 (Ang1), a key molecule for vascular maturation, and its receptor, endothelium-specific receptor tyrosine kinase 2 (Tie2), were significantly upregulated in the skeletal muscle of HFD-fed apelin-Tg mice, and the areas of anti-CD31 antibody-positive endothelial cells also increased. Furthermore, both the aerobic type-I muscle fibre ratio and the DNA copy number of mitochondrial NADH dehydrogenase subunit 1 increased 2.0- and 1.4-fold in skeletal muscle, respectively. CONCLUSIONS: These findings suggest that apelin stimulates energy expenditure via increase vascular mass and mitochondrial biogenesis in skeletal muscle. GENERAL SIGNIFICANCE: Apelin is a prerequisite factor for anti-obesity by stimulating energy expenditure via regulating homeostatic energy balance.
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Grasas de la Dieta/efectos adversos , Péptidos y Proteínas de Señalización Intercelular/genética , Mitocondrias Musculares/genética , Obesidad/fisiopatología , Tejido Adiposo/crecimiento & desarrollo , Angiopoyetina 1/biosíntesis , Animales , Apelina , Temperatura Corporal/fisiología , Humanos , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/fisiología , Obesidad/metabolismo , Consumo de Oxígeno , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptor TIE-2RESUMEN
The angiotensin II (AII) antagonistic action of azilsartan (AZL) [2-ethoxy-1-{[2'-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methyl}-1H-benzimidazole-7-carboxylic acid] was investigated in radioligand binding and function studies. AZL inhibited the specific binding of ¹²5I-Sar¹-Ile8-AII to human angiotensin type 1 receptors with an IC50 of 2.6 nM. The inhibitory effect of AZL persisted after washout of the free compound (IC(50) value of 7.4 nM). Olmesartan, telmisartan, valsartan, and irbesartan also inhibited the specific binding with IC50 values of 6.7, 5.1, 44.9, and 15.8 nM, respectively. However, their inhibitory effects were markedly attenuated with washout (IC50 values of 242.5, 191.6, >10,000, and >10,000 nM). AZL also inhibited the accumulation of AII-induced inositol 1-phosphate (IP1) in the cell-based assay with an IC50 value of 9.2 nmol; this effect was resistant to washout (IC50 value of 81.3 nM). Olmesartan and valsartan inhibited IP1 accumulation with IC50 values of 12.2 and 59.8 nM, respectively. The activities of these compounds were markedly reduced after washout (IC50 value of 908.5 and 22,664.4 nM). AZL was defined as an inverse agonist in an experiment by using a constitutively active mutant of human angiotensin type 1 receptors. In isolated rabbit aortic strips, AZL reduced the maximal contractile response to AII with a pD'2 value of 9.9. The inhibitory effects of AZL on contractile responses induced by AII persisted after the strips were washed; these inhibitory effects were more potent than those of olmesartan. These results suggest that AZL is a highly potent and slowly dissociating AII receptor blocker. Its tight receptor binding might be expected to produce potent and long-lasting antihypertensive effects in preclinical and clinical settings.
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Bloqueadores del Receptor Tipo 1 de Angiotensina II/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Bencimidazoles/metabolismo , Bencimidazoles/farmacología , Oxadiazoles/metabolismo , Oxadiazoles/farmacología , Receptor de Angiotensina Tipo 1/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/química , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Bencimidazoles/química , Células COS , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Oxadiazoles/química , Unión Proteica/fisiología , Conejos , Receptor de Angiotensina Tipo 1/fisiologíaRESUMEN
Human LCAT-like lysophospholipase (LLPL), or lysophospholipase 3, was first identified in vitro, in foam cells derived from THP-1 cells. We demonstrated that LLPL was present in foam cells in the severe atherosclerotic lesions that develop in apolipoprotein E-null (apoE(-/-)) mice. This indicated that LLPL might affect lipid metabolisms in foam cells and, therefore, atherogenesis. Accordingly, we created LLPL-knockout mice by gene targeting and crossed them with apoE(-/-) mice. We showed that the absence of LLPL increased lesion formation markedly in apoE(-/-) mice but had little effect on the plasma-lipid profile. In addition, LLPL-deficient peritoneal macrophages were more sensitive to apoptosis induced by exposure to oxidized low-density lipoprotein. LLPL might provide a link between apoptosis in macrophages and atherogenesis. Our data demonstrate that LLPL activity is anti-atherogenic and indicate that the regulation of this enzyme might be a novel drug target for the treatment of atherosclerosis.