RESUMO
3D cell cultures enable the in vitro study of dynamic biological processes such as the cell cycle, but their use in high-throughput screens remains impractical with conventional fluorescent microscopy. Here, we present a screening workflow for the automated evaluation of mitotic phenotypes in 3D cell cultures by light-sheet microscopy. After sample preparation by a liquid handling robot, cell spheroids are imaged for 24â h in toto with a dual-view inverted selective plane illumination microscope (diSPIM) with a much improved signal-to-noise ratio, higher imaging speed, isotropic resolution and reduced light exposure compared to a spinning disc confocal microscope. A dedicated high-content image processing pipeline implements convolutional neural network-based phenotype classification. We illustrate the potential of our approach using siRNA knockdown and epigenetic modification of 28 mitotic target genes for assessing their phenotypic role in mitosis. By rendering light-sheet microscopy operational for high-throughput screening applications, this workflow enables target gene characterization or drug candidate evaluation in tissue-like 3D cell culture models.
Assuntos
Processamento de Imagem Assistida por Computador , Esferoides Celulares , Microscopia de Fluorescência , Mitose , FenótipoRESUMO
Delivery of large and functionally active biomolecules across cell membranes presents a challenge in cell biological experimentation. For this purpose, we developed a novel solid-phase reverse transfection method that is suitable for the intracellular delivery of proteins into mammalian cells with preservation of their function. We show results for diverse application areas of the method, ranging from antibody-mediated inhibition of protein function to CRISPR/Cas9-based gene editing in living cells. Our method enables prefabrication of "ready to transfect" substrates carrying diverse proteins. This allows their easy distribution and standardization of biological assays across different laboratories.
Assuntos
Anticorpos/farmacologia , Sistemas CRISPR-Cas , Edição de Genes/métodos , Transfecção/métodos , Células HEK293 , Células HeLa , HumanosRESUMO
Carnosinase 1 (CN1) is encoded by the Cndp1 gene and degrades carnosine and anserine, two natural histidine-containing dipeptides. In vitro and in vivo studies suggest carnosine- and anserine-mediated protection against long-term sequelae of reactive metabolites accumulating, e.g., in diabetes mellitus. We have characterized the metabolic impact of CN1 in 11- and 55-week-old Cndp1-knockout (Cndp1-KO) mice and litter-matched wildtypes (WT). In Cndp1-KO mice, renal carnosine and anserine concentrations were gender-specifically increased 2- to 9-fold, respectively in the kidney and both most abundant in the renal cortex, but remained unchanged in all other organs and in serum. Renal oxidized/reduced glutathione concentrations, renal morphology and function were unaltered. In Cndp1-KO mice at week 11, renal asparagine, serine and glutamine levels and at week 55, renal arginine concentration were reduced. Renal heat-shock-protein 70 (Hspa1a/b) mRNA declined with age in WT but not in Cndp1-KO mice, transcription factor heat-shock-factor 1 was higher in 55-week-old KO mice. Fasting blood glucose concentrations decreased with age in WT mice, but were unchanged in Cndp1-KO mice. Blood glucose response to intraperitoneal insulin was gender- but not genotype-dependent, the response to intraperitoneal glucose injection was similar in all groups. A global Cndp1-KO selectively, age- and gender-specifically, increases renal carnosine and anserine concentrations, alters renal amino acid- and HSP70 profile and modifies systemic glucose homeostasis. Increase of the natural occurring carnosine and anserine levels in the kidney by modulation of CN1 represents a promising therapeutic approach to mitigate or prevent chronic kidney diseases such as diabetic nephropathy.
Assuntos
Anserina/metabolismo , Carnosina/metabolismo , Dipeptidases/metabolismo , RNA Mensageiro/metabolismo , Aminoácidos/metabolismo , Animais , Glicemia/metabolismo , Nefropatias Diabéticas/metabolismo , Feminino , Glucose/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Insulina/metabolismo , Rim , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
PURPOSE: Understanding of the mechanisms of vascular smooth muscle cells (VSMCs) phenotypic regulation is critically important to identify novel candidates for future therapeutic intervention. While HTS approaches have recently been used to identify novel regulators in many cell lines, such as cancer cells and hematopoietic stem cells, no studies have so far systematically investigated the effect of gene inactivation on VSMCs with respect to cell survival and growth response. METHODS AND RESULTS: 257 out of 2000 genes tested resulted in an inhibition of cell proliferation in HaoSMCs. After pathway analysis, 38 significant genes were selected for further study. 23 genes were confirmed to inhibit proliferation, and 13 genes found to induce apoptosis in the synthetic phenotype. 11 genes led to an aberrant nuclear phenotype indicating a central role in cell mitosis. 4 genes affected the cell migration in synthetic HaoSMCs. Using computational biological network analysis, 11 genes were identified to have an indirect or direct interaction with the Osteopontin pathway. For 10 of those genes, levels of proteins downstream of the Osteopontin pathway were found to be down-regulated, using RNAi methodology. CONCLUSIONS: A phenotypic high-throughput siRNA screen could be applied to identify genes relevant for the cell biology of HaoSMCs. Novel genes were identified which play a role in proliferation, apoptosis, mitosis and migration of HaoSMCs. These may represent potential drug candidates in the future.
Assuntos
Aorta/citologia , Miócitos de Músculo Liso/metabolismo , Osteopontina/metabolismo , Apoptose , Movimento Celular , Proliferação de Células , Células Cultivadas , Humanos , Osteopontina/genética , Fenótipo , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de SinaisRESUMO
OBJECTIVE: Prolonged catabolic states in type 2 diabetes (T2D), exacerbated by excess substrate flux and hyperglycemia, can challenge metabolic flexibility and antioxidative capacity. We investigated cellular responses to glucose load after prolonged fasting in T2D. RESEARCH DESIGN AND METHODS: Glucose-tolerant individuals (CON, n = 10), T2D individuals with (T2D+, n = 10) and without diabetes complications (T2D-, n = 10) underwent oral glucose tolerance test before and after a 5-day fasting-mimicking diet. Peripheral blood mononuclear cells' (PBMC) resistance to ex vivo dicarbonyl methylglyoxal (MG) exposure after glucose load was assessed. Markers of dicarbonyl detoxification, oxidative stress, and mitochondrial biogenesis were analyzed by quantitative PCR, with mitochondrial complex protein expression assessed by western blotting. RESULTS: T2D+ exhibited decreased PBMC resistance against MG, while T2D- resistance remained unchanged, and CON improved postglucose load and fasting (-19.0% vs.-1.7% vs. 12.6%; all P = 0.017). T2D+ showed increased expression in dicarbonyl detoxification (mRNA glyoxalase-1, all P = 0.039), oxidative stress (mRNA glutathione-disulfide-reductase, all P = 0.006), and mitochondrial complex V protein (all P = 0.004) compared with T2D- and CON postglucose load and fasting. Citrate synthase activity remained unchanged, indicating no change in mitochondrial number. Mitochondrial biogenesis increased in T2D- compared with CON postglucose load and fasting (mRNA HspA9, P = 0.032). T2D-, compared with CON, exhibited increased oxidative stress postfasting, but not postglucose load, with increased mRNA expression in antioxidant defenses (mRNA forkhead box O4, P = 0.036, and glutathione-peroxidase-2, P = 0.034), and compared with T2D+ (glutathione-peroxidase-2, P = 0.04). CONCLUSIONS: These findings suggest increased susceptibility to glucose-induced oxidative stress in individuals with diabetes complications after prolonged fasting and might help in diet interventions for diabetes management.
RESUMO
Carnosine and anserine supplementation markedLy reduce diabetic nephropathy in rodents. The mode of nephroprotective action of both dipeptides in diabetes, via local protection or improved systemic glucose homeostasis, is uncertain. Global carnosinase-1 knockout mice (Cndp1-KO) and wild-type littermates (WT) on a normal diet (ND) and high fat diet (HFD) (n = 10/group), with streptozocin (STZ)-induced type-1 diabetes (n = 21-23/group), were studied for 32 weeks. Independent of diet, Cndp1-KO mice had 2- to 10-fold higher kidney anserine and carnosine concentrations than WT mice, but otherwise a similar kidney metabolome; heart, liver, muscle and serum anserine and carnosine concentrations were not different. Diabetic Cndp1-KO mice did not differ from diabetic WT mice in energy intake, body weight gain, blood glucose, HbA1c, insulin and glucose tolerance with both diets, whereas the diabetes-related increase in kidney advanced glycation end-product and 4-hydroxynonenal concentrations was prevented in the KO mice. Tubular protein accumulation was lower in diabetic ND and HFD Cndp1-KO mice, interstitial inflammation and fibrosis were lower in diabetic HFD Cndp1-KO mice compared to diabetic WT mice. Fatalities occurred later in diabetic ND Cndp1-KO mice versus WT littermates. Independent of systemic glucose homeostasis, increased kidney anserine and carnosine concentrations reduce local glycation and oxidative stress in type-1 diabetic mice, and mitigate interstitial nephropathy in type-1 diabetic mice on HFD.
RESUMO
Anserine and carnosine have nephroprotective actions; hydrogen sulfide (H2S) protects from ischemic tissue damage, and the underlying mechanisms are debated. In view of their common interaction with HSP70, we studied possible interactions of both dipeptides with H2S. H2S formation was measured in human proximal tubular epithelial cells (HK-2); three endothelial cell lines (HUVEC, HUAEC, MCEC); and in renal murine tissue of wild-type (WT), carnosinase-1 knockout (Cndp1-KO) and Hsp70-KO mice. Diabetes was induced by streptozocin. Incubation with carnosine increased H2S synthesis capacity in tubular cells, as well as with anserine in all three endothelial cell lines. H2S dose-dependently reduced anserine/carnosine degradation rate by serum and recombinant carnosinase-1 (CN1). Endothelial Hsp70-KO reduced H2S formation and abolished the stimulation by anserine and could be restored by Hsp70 transfection. In female Hsp70-KO mice, kidney H2S formation was halved. In Cndp1-KO mice, kidney anserine concentrations were several-fold and sex-specifically increased. Kidney H2S formation capacity was increased 2-3-fold in female mice and correlated with anserine and carnosine concentrations. In diabetic Cndp1-KO mice, renal anserine and carnosine concentrations as well as H2S formation capacity were markedly reduced compared to non-diabetic Cndp1-KO littermates. Anserine and carnosine induce H2S formation in a cell-type and Hsp70-specific manner within a positive feedback loop with CN1.
RESUMO
Due to high associated costs and considerable time investments of cell-based screening, there is a strong demand for new technologies that enable preclinical development and tests of diverse biologicals in a cost-saving and time-efficient manner. For those reasons we developed the high-density cell array (HD-CA) platform, which miniaturizes cell-based screening in the form of preprinted and ready-to-run screening arrays. With the HD-CA technology, up to 24,576 samples can be tested in a single experiment, thereby saving costs and time for microscopy-based screening by 75%. Experiments on the scale of the entire human genome can be addressed in a real parallel manner, with screening campaigns becoming more comfortable and devoid of robotics infrastructure on the user side. The high degree of miniaturization enables working with expensive reagents and rare and difficult-to-obtain cell lines. We have also optimized an automated imaging procedure for HD-CA and demonstrate the applicability of HD-CA to CRISPR-Cas9- and RNAi-mediated phenotypic assessment of the gene function.