Quantification of gap junctional intercellular communication based on digital image analysis

2009 ◽  
Vol 297 (2) ◽  
pp. R243-R247 ◽  
Author(s):  
Johannes P. Hofgaard ◽  
Sarah Mollerup ◽  
Niels-Henrik Holstein-Rathlou ◽  
Morten Schak Nielsen
Keyword(s):  
Image Analysis ◽  
Digital Image ◽  
Intercellular Communication ◽  
Connexin 43 ◽  
Digital Image Analysis ◽  
Fluorescent Dyes ◽  
Cell Counting ◽  
C6 Glioma Cells ◽  
Gap Junctional Intercellular Communication ◽  
Metabolic Coupling

Intercellular communication via gap junction channels can be quantified by several methods based on diffusion of fluorescent dyes or metabolites. Given the variation in intercellular coupling of cells, even under untreated control conditions, it is of essence to quantify the coupling between numerous cells to obtain reliable estimates of metabolic coupling. Quantification is often based on manual counting of fluorescent cells, which is time consuming and may include some degree of subjectivity. In this report, we introduce a technique based on digital image analysis, and the software for the analysis is presented together with a detailed protocol in the online supplemental material ( http://bmi.ku.dk/matlab_program/ ). Fluorescent dye was introduced in connexin 43-expressing C6 glioma cells by in situ electroporation, and fluorescence intensity was measured in the electroporated cells and in cells receiving dye by intercellular diffusion. The analysis performed is semiautomatic, and comparison with traditional cell counting shows that this method reliably determines the effect of uncoupling by several interventions. This new method of analysis yields a rapid and objective quantification process with a high degree of reproducibility.

scholarly journals Cancer-Associated Fibroblasts Accelerate Malignant Progression of Non-Small Cell Lung Cancer via Connexin 43-Formed Unidirectional Gap Junctional Intercellular Communication

2018 ◽  
Vol 51 (1) ◽  
pp. 315-336 ◽  
Author(s):  
Min Luo ◽  
Yanmei Luo ◽  
Naiquan Mao ◽  
Guolin Huang ◽  
Cuifang Teng ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Intercellular Communication ◽  
Connexin 43 ◽  
Small Cell ◽  
Migration And Invasion ◽  
Cancer Associated Fibroblasts ◽  
Gap Junctional Intercellular Communication ◽  
Small Cell Lung ◽  
Metabolic Coupling ◽  
Gap Junctional

Background/Aims: Gap junctions, which are assembled by connexins, can directly connect the cytoplasm of adjacent cells and enable gap junctional intercellular communication (GJIC) as well as metabolic coupling between neighboring cells. Here, we investigated the role of connexin 43 (Cx43) and its derived GJIC in the interplay between non-small cell lung cancer (NSCLC) cells and cancer-associated fibroblasts (CAFs). Methods: CAFs and NSCLC cells were co-cultured with direct contact and separated using flow cytometry. Glucose uptake, lactate production, and the expression and activity of PKM-2 and LDH-A in sorted CAFs were measured by a colorimetric assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). Meanwhile, E-cadherin and N-cadherin expression and the migration and invasion of sorted NSCLC cells were detected by western blotting, wound width, and Transwell assays. Pyruvate, acetyl-CoA, and citric acid levels, ATP levels, and LDH-B and α-KG activity in sorted NSCLC cells were determined by a colorimetric or fluorometric assay and ELISA, respectively. Functional GJIC between cells and the subcellular location of connexins were detected by a “Parachute” assay and immunofluorescence. Levels of α-SMA, Cx43, and LDH-B in tissue from patients with NSCLC were determined by immunohistochemistry. Results: Cx43 accumulated in the plasma membrane, which favored the assembly of asymmetric unidirectional GJIC from CAFs to NSCLC cells. CAFs underwent increased aerobic glycolysis and promoted the epithelial-mesenchymal transition, migration, and invasion of NSCLC cells. In contrast, NSCLC cells experienced enhanced oxidative phosphorylation upon CAF stimulation, with an increase in ATP generation and thereby activation of the PI3K/Akt and MAPK/ERK pathways. Metabolic coupling between CAFs and NSCLC cells was under the strict control of Cx43-formed unidirectional GJIC. Patients with high tri-expression of α-SMA, Cx43, and LDH-B had the shortest overall survival and relapse-free survival compared with those with individual overexpression or high bi-expression. Conclusion: Cx43-formed unidirectional GJIC plays a critical role in mediating close metabolic cooperation between CAFs and NSCLC cells to support the malignant progression of NSCLC.

Digital Image Analysis of the Larynx

2000 ◽  
Vol 10 (2) ◽  
pp. 7-9
Author(s):  
Yaser Natour ◽  
Christine Sapienza ◽  
Mark Schmalz ◽  
Savita Collins
Keyword(s):  
Image Analysis ◽  
Digital Image ◽  
Digital Image Analysis

scholarly journals Digital Image Analysis of BAP-1 Accurately Predicts Uveal Melanoma Metastasis

2019 ◽  
Vol 8 (3) ◽  
pp. 11 ◽  
Author(s):  
Gustav Stålhammar ◽  
Thonnie Rose O. See ◽  
Stephen Phillips ◽  
Stefan Seregard ◽  
Hans E. Grossniklaus
Keyword(s):  
Image Analysis ◽  
Uveal Melanoma ◽  
Digital Image ◽  
Digital Image Analysis ◽  
Melanoma Metastasis

Quantitative evaluation of elderly skin based on digital image analysis

2008 ◽  
Vol 14 (2) ◽  
pp. 192-200 ◽  
Author(s):  
Hiromasa Tanaka ◽  
Gojiro Nakagami ◽  
Hiromi Sanada ◽  
Yunita Sari ◽  
Hiroshi Kobayashi ◽  
...  
Keyword(s):  
Image Analysis ◽  
Quantitative Evaluation ◽  
Digital Image ◽  
Digital Image Analysis

scholarly journals In vitro validation of Digital Image Analysis Sequence (DIAS) for the assessment of the marginal fit of cement-retained implant-supported experimental crowns

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Aristeidis A. Villias ◽  
Stefanos G. Kourtis ◽  
Hercules C. Karkazis ◽  
Gregory L. Polyzois
Keyword(s):  
Image Analysis ◽  
Digital Image ◽  
Confidence Level ◽  
Digital Image Analysis ◽  
Statistical Tests ◽  
Interobserver Reliability ◽  
Intraclass Correlation ◽  
Marginal Fit ◽  
Smallest Detectable Change ◽  
The Mean

Abstract Background The replica technique with its modifications (negative replica) has been used for the assessment of marginal fit (MF). However, identification of the boundaries between prosthesis, cement, and abutment is challenging. The recently developed Digital Image Analysis Sequence (DIAS) addresses this limitation. Although DIAS is applicable, its reliability has not yet been proven. The purpose of this study was to verify the DIAS as an acceptable method for the quantitative assessment of MF at cemented crowns, by conducting statistical tests of agreement between different examiners. Methods One hundred fifty-one implant-supported experimental crowns were cemented. Equal negative replicas were produced from the assemblies. Each replica was sectioned in six parts, which were photographed under an optical microscope. From the 906 standardized digital photomicrographs (0.65 μm/pixel), 130 were randomly selected for analysis. DIAS included tracing the profile of the crown and the abutment and marking the margin definition points before cementation. Next, the traced and marked outlines were superimposed on each digital image, highlighting the components’ boundaries and enabling MF measurements. One researcher ran the analysis twice and three others once, independently. Five groups of 130 measurements were formed. Intra- and interobserver reliability was evaluated with intraclass correlation coefficient (ICC). Agreement was estimated with the standard error of measurement (SEM), the smallest detectable change at the 95% confidence level (SDC95%), and the Bland and Altman method of limits of agreement (LoA). Results Measured MF ranged between 22.83 and 286.58 pixels. Both the intra- and interobserver reliability were excellent, ICC = 1 at 95% confidence level. The intra- and interobserver SEM and SDC95% were less than 1 and 3 pixels, respectively. The Bland–Altman analysis presented graphically high level of agreement between the mean measurement of the first observer and each of the three other observers’ measurements. Differences between observers were normally distributed. In all three cases, the mean difference was less than 1 pixel and within ± 3 pixels LoA laid at least 95% of differences. T tests of the differences did not reveal any fixed bias (P > .05, not significant). Conclusion The DIAS is an objective and reliable method able to detect and quantify MF at ranges observed in clinical practice.

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