RESUMEN
BACKGROUND: In tissues and organisms, the coordination of neighboring cells is essential to maintain their properties and functions. Therefore, knowing which cells are adjacent is crucial to understand biological processes that involve physical interactions among them, e.g. cell migration and proliferation. In addition, some signaling pathways, such as Notch or extrinsic apoptosis, are highly dependent on cell-cell communication. While this is straightforward to obtain from membrane images, nuclei labelling is much more ubiquitous for technical reasons. However, there are no automatic and robust methods to find neighboring cells based only on nuclear markers. RESULTS: In this work, we describe Nfinder, a method to assess the cell's local neighborhood from images with nuclei labeling. To achieve this goal, we approximate the cell-cell interaction graph by the Delaunay triangulation of nuclei centroids. Then, links are filtered by automatic thresholding in cell-cell distance (pairwise interaction) and the maximum angle that a pair of cells subtends with shared neighbors (non-pairwise interaction). We systematically characterized the detection performance by applying Nfinder to publicly available datasets from Drosophila melanogaster, Tribolium castaneum, Arabidopsis thaliana and C. elegans. In each case, the result of the algorithm was compared to a cell neighbor graph generated by manually annotating the original dataset. On average, our method detected 95% of true neighbors, with only 6% of false discoveries. Remarkably, our findings indicate that taking into account non-pairwise interactions might increase the Positive Predictive Value up to + 11.5%. CONCLUSION: Nfinder is the first robust and automatic method for estimating neighboring cells in 2D and 3D based only on nuclear markers and without any free parameters. Using this tool, we found that taking non-pairwise interactions into account improves the detection performance significantly. We believe that using our method might improve the effectiveness of other workflows to study cell-cell interactions from microscopy images. Finally, we also provide a reference implementation in Python and an easy-to-use napari plugin.
Asunto(s)
Arabidopsis , Drosophila melanogaster , Animales , Caenorhabditis elegans , Microscopía/métodos , Núcleo Celular/metabolismo , Algoritmos , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Background: Matrix metalloproteinase 12 (MMP12), a member of MMPs, can take lots of roles including extracellular matrix component degradation, viral infection, inflammation, tissue remodeling and tumorigenesis. To explore the transcriptional regulation of MMP12 gene, a sensitive luciferase reporter HEK293 cell line for endogenous MMP12 promoter was generated by CRISPR/Cas9 technology. Results: The HEK293-MMP12-T2A-luciferase-KI cell line was successfully established by CRISPR/Cas9 technology. The sequencing results indicated that one allele of the genome was proven to have a site-directed insertion of luciferase gene and another allele of the genome was confirmed to have additional 48 bp insertion in this cell line. The cell line was further demonstrated to be a sensitive reporter of the endogenous MMP12 promoter by applying transcription factors STAT3, AP-1 and SP-1 to the cell line. The reporter cell line was then screened with bioactive small molecule library, and a small molecule Tanshinone I was found to significantly inhibit the transcriptional activity of MMP12 gene in HEK293-MMP12-T2A-luciferase-KI cell line by luciferase activity assay, which was further confirmed to inhibit the expression of MMP12 mRNA in wild-type HEK293 cells. Conclusions: This novel luciferase knock-in reporter system will be helpful for investigating the transcriptional regulation of MMP12 gene and screening the drugs targeting MMP12 gene.
Asunto(s)
Humanos , Metaloproteinasa 12 de la Matriz/genética , Sistemas CRISPR-Cas , Luciferasas/genética , Transcripción Genética , Comunicación Celular , Línea Celular , Regiones Promotoras Genéticas/genética , Técnicas de Cultivo de Célula , Matriz Extracelular , Técnicas de Sustitución del Gen , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente EspaciadasRESUMEN
Intercellular junctions play a role in regulating islet cytoarchitecture, insulin biosynthesis and secretion. In this study, we investigated the animal metabolic state as well as islet histology and cellular distribution/expression of CAMs and F-actin in the endocrine pancreas of C57BL/6/JUnib mice fed a high-fat diet (HFd) for a prolonged time period (8 months). Mice fed a HFd became obese and type 2 diabetic, displaying significant peripheral insulin resistance, hyperglycemia and moderate hyperinsulinemia. Isolated islets of HFd-fed mice displayed a significant impairment of glucose-induced insulin secretion associated with a diminished frequency of intracellular calcium oscillations compared with control islets. No marked change in islet morphology and cytoarchitecture was observed; however, HFd-fed mice showed higher beta cell relative area in comparison with controls. As shown by immunohistochemistry, ZO-1, E-, N-cadherins, α- and ß-catenins were expressed at the intercellular contact site of endocrine cells, while VE-cadherin, as well as ZO-1, was found at islet vascular compartment. Redistribution of N-, E-cadherins and α-catenin (from the contact region to the cytoplasm in endocrine cells) associated with increased submembranous F-actin cell level as well as increased VE-cadherin islet immunolabeling was observed in diabetic mice. Increased gene expression of VE-cadherin and ZO-1, but no change for the other proteins, was observed in islets of diabetic mice. Only in the case of VE-cadherin, a significant increase in islet content of this CAM was detected by immunoblotting in diabetic mice. In conclusion, CAMs are expressed by endocrine and endothelial cells of pancreatic islets. The distribution/expression of N-, E- and VE-cadherins as well as α-catenin and F-actin is significantly altered in islet cells of obese and diabetic mice.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Diabetes Mellitus Experimental/metabolismo , Dieta Alta en Grasa , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Animales , Cadherinas/análisis , Cadherinas/metabolismo , Cateninas/análisis , Cateninas/metabolismo , Moléculas de Adhesión Celular/análisis , Diabetes Mellitus Experimental/patología , Secreción de Insulina , Islotes Pancreáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína de la Zonula Occludens-1/análisis , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Crotoxin (CTX) is the main neurotoxic component of Crotalus durissus terrificus snake venom. It inhibits tumour growth and modulates the function of macrophages, which are essential cells in the tumour microenvironment. The present study investigated the effect of CTX on the secretory activity of monocultured macrophages and macrophages co-cultivated with LLC-WRC 256 cells. The effect of the macrophage secretory activities on tumour cell proliferation was also evaluated. Macrophages pre-treated with CTX (0.3 µg/mL) for 2 h were co-cultivated with LLC-WRC 256 cells, and the secretory activity of the macrophages was determined after 12, 24 and 48 h. The co-cultivation of CTX-treated macrophages with the tumour cells caused a 20% reduction in tumour cell proliferation. The production of both H2O2 and NO was increased by 41% and 29% after 24 or 48 h of co-cultivation, respectively, compared to the values for the co-cultures of macrophages of control. The level of secreted IL-1ß increased by 3.7- and 3.2-fold after 12 h and 24 h of co-cultivation, respectively. Moreover, an increased level of LXA4 (25%) was observed after 24 h of co-cultivation, and a 2.3- and 2.1-fold increased level of 15-epi-LXA4 was observed after 24 h and 48 h, respectively. Boc-2, a selective antagonist of formyl peptide receptors, blocked both the stimulatory effect of CTX on the macrophage secretory activity and the inhibitory effect of these cells on tumour cell proliferation. Taken together, these results indicate that CTX enhanced the secretory activity of macrophages, which may contribute to the antitumour activity of these cells, and that activation of formyl peptide receptors appears to play a major role in this effect.