Detachment Assay Research Articles

Host infectivity of Pyricularia pathogen of finger millet (Eleusine coracana (L.) Gaertn.) on Poaceae weeds

Blast disease of finger millet is a serious disease all over the world and causes significant yield loss. Pyricularia populations consist of various pathotypes with different host ranges within the Poaceae family and express host specificities mediated by Avr and R genes. So, identification of the host range of Pyricularia on weeds within the poaceae family will give an idea for further research on understanding of genes responsible for host specificity reaction of Pyricularia among various host ranges. Pyricularia isolated from finger millet was tested on different weed host plants to study the infectivity of Pyricularia on weed species. The Pyricularia pathogen isolated from the infected leaf of finger millet plants having spindle-shaped lesions. The Poaceae weeds viz., Chloris barbeta, Cynodon dactylon, Cyperus rotundus, Dactylacterium aegyptium, and Echinochloa colonum are observed regularly in the field of finger millet. Among the weed species, spindle shaped lesion was observed in C. barbeta, C. dactylon, C. rotundus and D. aegyptium in leaf detachment assay with Pyricularia at Centre of Excellence in Millets, Athiyandal, Tiruvannamalai, India. Whereas E. colonum did not express any lesion. The incidence of Pyricularia in finger millet was studied under various weed infestation levels under field conditions during 2022-23 and 2023- 24 growing seasons. It was found that the experimental plot of finger millet with all Poaceae weeds recorded the highest incidence of leaf blast and the largest number of Pyricularia colonies in both years. This was followed by the plot with finger millet + D. aegyptium alone and finger millet + C. rotundus alone. The leaf blast symptoms were noticed in finger millet after one week of sowing in the case of the crop with all weed species during 2022-23 and 2023-24. A significant correlation was obtained between the incidence of the leaf blast and colonies of Pyricularia from 28 days after sowing till the maturity of the crop in both years. Hence, the incidence, as well as colonies of Pyricularia, were higher in plots with finger millet + all weeds, followed by plots with finger millet + D. aegyptium alone and plots with finger millet + Cyp. rotundus alone.

Characterization of antibiofilm compound from marine sponge Stylissa carteri.

The fouling phenomenon grabbed global attention and caused huge economic losses specifically in marine-related industries. Sessile behavior exposed the sponge to the risk of fouling. However, their bodies remained free from foulers, which were attributed to the chemical defense system. The objectives of this study were to determine the antibiofilm activity of the marine sponge, Stylissa carteri, and to characterize the isolated compound involved. The antibiofilm activity of S. carteri methanolic crude extract (MCE) and fractions was tested against biofilm-producing bacteria, Pseudomonas aeruginosa, using two different modes of crystal violet biofilm assays: preventive and detachment. Besides that, the disc-diffusion test was conducted to screen the antibacterial activity against gram-positive and gram-negative bacteria while a cytotoxicity assay was conducted on the HepG2 cell line. Bioassay-guided fractionation was carried out using vacuum liquid chromatography (VLC) and solid phase extraction using a C18 Sep-Pak Cartridge. The crystal compound was isolated and characterized through thin-layer chromatography (TLC), Fourier transform infrared (FTIR) spectroscopy, liquid chromatography-mass spectrometry (LCMS), and nuclear magnetic resonance (NMR) spectroscopy. The S. carteri MCE showed a promising result with a half-maximal inhibitory concentration (IC50) of 20.22μg/mL in the preventive assay, while no IC50 was determined in the detachment assay since all inhibitions < 50%. The S. carteri MCE exhibited broad-spectrum antibacterial activity and displayed a non-cytotoxic effect. Fraction 4 from MCE of S. carteri (IC50 = 2.40μg/mL) reduced the biofilm in the preventive assay at all concentrations and exhibited no antibacterial activity indicating the independence of antibiofilm from antibacterial properties. Based on the data obtained, an alkaloid named debromohymenialdisine (DBH) was identified from Fraction 4 of S. carteri MCE. In conclusion, S. carteri was able to reduce the establishment of the biofilm formed by P. aeruginosa and could serve as a prominent source of natural antifouling agents.

Improvement of protocol for detachment assays in B16F10 cells

B16F10 (B16) murine melanoma cells have the characteristic of strong adhesion to culture surfaces. The use of trypsin solutions in high doses to release this type of cell is not suitable. When B16F10 cells are released with high concentrations of trypsin they do not form a precipitate after centrifugation and consequently the cells will be lost when discarding the supernatant. To solve this problem, we established a solution with phosphate buffered saline (PBS) and ethylenediaminetetraacetic acid (EDTA) at low trypsin concentrations. Finally, we verified the efficiency of this solution on the quantity of cells recovered after centrifugation.

Achieving Complete Human Gingival Fibroblast Collagen Coverage on Implant Abutments through Vacuum Ultraviolet (VUV) Photofunctionalization.

The formation of a biological seal between implant abutments and the surrounding soft tissue is a preventive strategy against peri-implantitis. The aim of this study is to test the hypothesis that surfaces of prosthetic implant abutments treated with vacuum ultraviolet (VUV) light enhance the growth and function of human gingival fibroblasts. Implant abutments were treated with 172 nm VUV light for one minute. Untreated abutments were subjected as controls. Their surface properties were characterized using SEM, contact angle measurements, and chemical composition analysis. Human gingival fibroblasts were cultured on both untreated and VUV-treated abutments to evaluate cell attachment, proliferation, distribution, and collagen production. Cell detachment assays were also performed under various mechanical and chemical stimuli. After VUV treatment, implant abutments demonstrated a notable transition from hydrophobic to hydrophilic wettability. Surface element analysis revealed a considerable reduction in surface carbon and increases in oxygen and titanium elements on the VUV-treated surfaces. On day 1 of culture, 3.9 times more fibroblasts attached on VUV-treated abutments than on untreated control abutments. Fibroblastic proliferation increased 1.9-3.1 times on VUV-treated abutments, along with a significant improvement in the distribution of populating cells. Collagen production on VUV-treated abutments increased by 1.5-1.7 times. While untreated abutment surfaces showed voids and limited spread of collagen deposition, dense and full coverage of collagen was observed on VUV-treated abutments, with a great contrast in the challenging axial surface zone. Cell retention against mechanical and chemical detaching stimuli was increased 11.3 and 4.3 times, respectively, by VUV treatment. Treatment of implant abutments with VUV light for one minute resulted in a reduction of surface carbon and a transformation of the surface from hydrophobic to hydrophilic. This led to enhanced attachment, proliferation, and retention of human gingival fibroblasts, along with nearly complete collagen coverage on implant abutments. These in vitro results indicate the promising potential of utilizing VUV photofunctionalized implant abutments to enhance soft tissue reaction and sealing mechanisms.

Azithromycin effect on multidrug resistant Acinetobacter baumannii biofilm production and composition.

To evaluate the effect of azithromycin (AZM) on biofilm formation and composition in multidrug resistant (MDR) Acinetobacter baumannii. Ninety-six A. baumannii isolates were studied. Antimicrobial susceptibility and sub-minimum inhibitory concentration (sub-MIC) were determined by the broth microdilution method. Carbapenemase genes were detected by polymerase chain reaction and clonal diversity by pulsed-field gel electrophoresis (PFGE). Biofilm formation without AZM and AZM sub-MIC were determined by crystal violet staining. AZM-free biofilm composition and AZM sub-MIC were determined by detachment assays. The selected A. baumannii were MDR; 93.8% were carbapenem-resistant and 24 were OXA-24-positive. PFGE showed predominance of clones A (53%), B (34.7%) and C (12.5%). Biofilm production at AZM sub-MICs decreased in 53.1%, increased in 34.7% and showed no differences in 12.5% of isolates, in comparison with biofilm production without AZM. AZM sub-MIC can reduce biofilm production in A. baumannii MDR isolates with decreased protein and DNA in the biofilm. Our results may be useful in synergy studies for new therapeutic alternatives.

Effect of 3α-dihydroprogesterone and 5α-dihydroprogesterone on DCIS cells and possible impact for postmenopausal women

Objective Progesterone metabolites 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αP) have opposite effects on proliferation, apoptosis and metastasis in the breast. Evidence regarding their influence on ductal carcinoma in situ (DCIS) lesions is lacking. Methods MCF10DCIS.com cells were cultured in a 3D culture system and treated with 5αP or 3αP. After 5 and 12 days of treatment, polymerase chain reaction (PCR) of proliferation, invasion/metastasis, anti-apoptotic or other markers was performed. Cells treated with the tumor-promoting 5αP were observed under the light and confocal microscopes to reveal possible morphological changes that could indicate a transition from an in situ to an invasive phenotype. As a control, the morphology of the MDA-MB-231 invasive cell line was examined. The invasive potential after exposure to 5αP was also assessed using a detachment assay. Results The PCR analysis of the chosen markers showed no statistically significant difference between naive cells and cells treated with 5αP or 3αP. DCIS spheroids retained their in situ morphology after treatment with 5αP. The detachment assay showed no increased potential for invasion after exposure to 5αP. Progesterone metabolites 5αP and 3αP do not facilitate or prohibit tumor promotion/invasion in MCF10DCIS.com cells, respectively. Conclusion As oral micronized progesterone has been proved effective for hot flushes in postmenopausal women, first in vitro data propose that progesterone-only therapy could possibly be considered for women after DCIS suffering from hot flushes.

Analysis of biofilm production and expression of adhesion structures of circulating Clostridioides difficile strains from Mexico

IntroductionClostridioides difficile biofilms are believed to protect the pathogen from antibiotics, in addition to potentially contributing to recurrent infections. MethodologyBiofilm production of 102 C. difficile isolates was determined using the crystal violet staining technique, and detachment assays were performed. The expression levels of cwp84 and slpA genes were evaluated by real-time PCR on selected isolates. ResultsMore than 70% of isolates (75/102) were strong biofilm producers, and the highest detachment of biofilm was achieved with the proteinase K treatment (>90%). The overall mean expression of cwp84 was higher in RT027 than in RT001 (p=0.003); among strong biofilm-producing strains, the slpA expression was lower in RT027 than in RT001 (p<0.000). ConclusionsProteins seem to have an important role in the biofilm's initial adherence and maturation. slpA and cwp84 are differentially expressed by C. difficile ribotype and biofilm production level.

Rapid methicillin resistance detection and subspecies discrimination in Staphylococcus hominis clinical isolates by MALDI-TOF MS

PurposeStaphylococcus hominis is a coagulase-negative opportunistic pathogen responsible for implanted medical device infections. Rapid identification and virulence factors detection are crucial for appropriate antimicrobial therapy. We aimed to search protein biomarker peaks for rapid classification of antibiotic resistance and subspecies of S. hominis using MALDI-TOF MS. MethodsS. hominis clinical isolates (n = 148) were screened for subspecies differentiation by novobiocin resistance. Biofilm composition and formation were determined by detachment assay and crystal violet staining, respectively. Antibiotic susceptibility was performed by the broth microdilution method. The search for potential biomarkers peaks was enabled by ClinProTools 3.0, flexAnalysis 3.4, and Biotools 3.2 for statistical analysis, peak visualization, and protein/peptide alignment, respectively. ResultsOf 148 isolates, 12.16% were classified as S. hominis subsp. novobiosepticus, 77.77% were biofilm producers, and ˃ 50% were multidrug-resistant. Two potential biomarker peaks, 8975 m/z and 9035 m/z were detected for the discrimination of methicillin resistance with a sensitivity of 96.72%. The following peaks were detected for subspecies differentiation: 2582 m/z, 2823 m/z, and 2619 m/z with 88.89–98.28% of sensitivity. ConclusionsWe found potential biomarker peaks to predict methicillin resistance and discriminate S. hominis subspecies during routine MALDI-TOF MS identification in a clinical setting to enable better antibiotic treatment.

Determination of Inhibitory Effects of Allium sativum Extract on Biofilm Production by Clinical Staphylococcus aureus Isolates

Objectives: To determine anti-biofilm effect of fresh garlic extract (FGE) on Staphylococcus aureus biofilm production and the relationship between methicillin resistance and biofilm production intensity. Methods: Clinical S. aureus isolates were identified methicillin resistant S. aureus (MRSA) by cefoxitin disc diffusion method. The anti-biofilm effects of FGE on S. aureus biofilm biomass determination was done using crystal violet assay. Results: Among 71 S. aureus isolates, MRSA were 37 (52.1%). Among biofilm producers, comparison of biofilm biomass (absorbance at 492 nm) showed no significant differences in biofilm formation ability between MRSA and MSSA (p=0.325). Use of 10% FGE decreased biofilm production in MRSA and MSSA by 40.4% (p&lt;0.001) and 48.1% (p&lt;0.001) respectively. Detachment assay using sodium dodecyl sulfate showed that control group biofilm biomass was decreased by 41.2%, while test group was decreased by 61.7% (p&lt;0.001). Conclusion: Garlic extracts has potency as an anti-biofilm agent and could be developed and used to manage different S. aureus biofilm related infections.

High-Throughput Characterization of Cell Adhesion Strength Using Long-Channel Constriction-Based Microfluidics.

The adhesion strength of a cancer cell is a valuable biophysical marker of its metastatic potential, tightly associated with various metastatic processes; for example, cancer cells escape from a primary tumor, and circulating tumor cells (CTCs) are anchored to the vessel wall. Although constriction-based microfluidics can realize the high-throughput characterization of single-cell deformability, due to the influence of cell size heterogeneity, accurately evaluating the adhesion strength of a cancer cell at high throughputs in constriction remains difficult. In this paper, we first proposed an approach for the assessment of adhesion strength of BGC-823 and SGC-7901 cell lines at high throughputs based on a friction coefficient using the constant velocity stage of cell transit in a long-channel constriction. Cell size was proven to be independent of adhesion strength by cell detachment assay; however, it has large effects on cell transit velocity in constriction. Therefore, the linear elasticity of a completely deformed cell in constriction is simplified as a compressed spring model, effectively reducing the influence of cell size heterogeneity. Theoretically, our proposed model can well offset the influence of cell size by cell transit velocity, while our experimental results indicate that the friction coefficient has a good linear relationship with the logarithm of the adhesion strength too. Therefore, our proposed approach can realize accurate characterization of cell adhesion strength at high throughputs using long-channel constriction-based microfluidics. Hence, this work might enrich the functions of constriction-based microfluidics and bring new insights into the characterization of mechanical phenotypes.

Biotransformation and removal of arsenic oxyanions by Alishewanella agri PMS5 in biofilm and planktonic states

Arsenic oxyanions are toxic chemicals that impose a high risk to humans and other living organisms in the environment. The present study investigated indigenous heterotrophic bacteria in the tailings dam effluent (TDE) of a gold mining factory. Thirty-seven arsenic resistant bacteria were cultured on Reasoner's 2A agar supplemented with arsenic salts through filtration. One strain encoded as PMS5 with the highest resistance to 140-mM sodium arsenite and 600-mM sodium arsenate in tryptic soy broth was selected for further investigations. According to phenotypic examinations and 16S rDNA sequence analysis, PMS5 belonged to the genus Alishewanella and was sensitive to most of the examined antibiotics. The biosorption and bioaccumulation abilities of arsenic salts were observed in this isolate based on Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX) and biosorption and bioaccumulation data. PMS5 was also found to cause the volatilization and biotransformation of arsenic oxyanions through their oxidation and reduction. Moreover, the contribution of PMS5 to arsenic (3+, 5+) bioprocessing under oligotrophic conditions was confirmed in fixed-bed reactors fed with the TDE of the gold factory (R1) and synthetic water containing As5+ (R2). According to biofilm assays such as biofilm staining, cell count, detachment assay and SEM, the arsenic significantly reduced the biofilm density of the examined reactors compared to that of the control (R3). Arsenate reduction and arsenite oxidation under bioreactor conditions were respectively obtained as 75.5–94.7% and 8%. Furthermore, negligible arsenic volatilization (1.2 ppb) was detected.

Evaluating cell viability heterogeneity based on information fusion of multiple adhesion strengths.

Cell viability evaluation is significantly meaningful for cellular assays. Some cells with weak viability are easily killed in the detection of anticancer drugs, while others with strong viability survive and proliferate, ultimately leading to the treatment failure or the inaccuracy of biological assays. Accurately evaluating cell viability heterogeneity still remains difficult. This article proposed a multiphysical property information fusion method for evaluating cell viability heterogeneity based on polynomial regression in a single-channel integrated microfluidic chip. In this method, adhesion strengths τN , that are defined as the magnitude of shear stress needed to detach (100 - N) % of cell population, were extracted as the independent variables of polynomial regression model by calculating the nonlinear fitting of the impedance-response curves for shear stress (cell detachment assay). Besides, by calculating the nonlinear fitting of the drug dose-response curves for cancer cells (IC50 assay), the half-maximal inhibitory concentration (IC50 ) was extracted as the dependent variables of polynomial regression model. The results show that the mean relative error of our fusion method averagely reduces by 6.04% and 62.79% compared with the multiple linear regression method and the cell counting method. Moreover, a simplified theoretical model used to describe the quantitative relationship between cell viability and its adhesion strengths was built to provide a theoretical basis for our fusion method.

Analysis of biofilm production and expression of adhesion structures of circulating Clostridioides difficile strains from Mexico

IntroductionClostridioides difficile biofilms are believed to protect the pathogen from antibiotics, in addition to potentially contributing to recurrent infections. MethodologyBiofilm production of 102 C. difficile isolates was determined using the crystal violet staining technique, and detachment assays were performed. The expression levels of cwp84 and slpA genes were evaluated by real-time PCR on selected isolates. ResultsMore than 70% of isolates (75/102) were strong biofilm producers, and the highest detachment of biofilm was achieved with the proteinase K treatment (>90%). The overall mean expression of cwp84 was higher in RT027 than in RT001 (p=0.003); among strong biofilm-producing strains, the slpA expression was lower in RT027 than in RT001 (p<0.000). ConclusionsProteins seem to have an important role in the biofilm's initial adherence and maturation. slpA and cwp84 are differentially expressed by C. difficile ribotype and biofilm production level.

Influence of poly(N-isopropylacrylamide) (PIPAAm) graft density on properties of PIPAAm grafted poly(dimethylsiloxane) surfaces and their stability

A previous report shows that poly(N-isopropylacrylamide) (PIPAAm) gel grafted onto poly(dimethylsiloxane) (PDMS) (PI-PDMS) surfaces with large PIPAAm graft density (Lar-PI-PDMS), is prepared by using electron beam irradiation, demonstrating that applied mechanical stretching affects properties of the Lar-PI-PDMS surface. However, the influence of PIPAAm graft density on the properties of PI-PDMS surfaces and their stability are not understood. To provide insight into these points, the properties of PI-PDMS surfaces with low PIPAAm graft density (Low-PI-PDMS) surfaces with stretched (stretch ratio = 20%) and unstretched states were examined as stretchable temperature-responsive cell culture surface using contact angle measurement and cell attachment/detachment assays, compared to those with Lar-PI-PDMS, as previously reported. Long-term contact angle measurements (61 days) for unstretched Low-PI-PDMS and Lar-PI-PDMS surfaces indicated that the cross-linked structure of the grafted PIPAAm gel suppressed hydrophobic recovery of the basal PDMS surface. The cell attachment assay revealed that the stretched Low-PI-PDMS surface was less cell adhesive than that of the unstretched Low-PI-PDMS surface despite of a larger amount of adsorbed fibronectin (FN). The lower cell adhesiveness was possibly explained by denaturation of adsorbed FN, which was induced by the strong hydrophobic property of the stretched Low-PI-PDMS surface. The cell detachment assay revealed that dual stimuli, low temperature treatment and mechanical shrinking stress applied to the stretched Low-PI-PDMS surface promoted cell detachment compared to a single stimulus, low temperature treatment or mechanical shrinking stress. These results suggested that the PIPAAm gelgrafted PDMS surface was chemically stable and did not suffer from hydrophobic recovery. External mechanical stretching stress not only strongly dehydrated grafted PIPAAm chains, but also denatured the adsorbed FN when the grafted PIPAAm layer was extremely thin, as in Low-PI-PDMS surfaces. Thus, PI-PDMS may be utilized as a stretchable temperature-responsive cell culture surface without significant hydrophobic recovery.

An Evaluation of the Antibacterial Properties of Tormentic Acid Congener and Extracts From Callistemon viminalis on Selected ESKAPE Pathogens and Effects on Biofilm Formation.

ESKAPE pathogens, namely, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, are responsible for a majority of all healthcare-acquired infections (HAI). The bacteria cause nosocomial infections in immunocompromised patients. Extracts from Callistemon viminalis have been shown to have antibacterial, antifungal, and anti-inflammatory activities. Tormentic acid congener, a pentacyclic triterpene saponin, was isolated from C. viminalis leaves. This study aimed to investigate the antibacterial effects of tormentic acid congener and leaf extracts on biofilm formation by A. baumannii, S. aureus, S. pyogenes, and P. aeruginosa. The antibacterial effects were determined by the microbroth dilution method, and ciprofloxacin was used as the standard antibacterial drug. Biofilm formation and detachment assays were performed using crystal violet staining. Production of extracellular polymeric DNA and polysaccharides from biofilms was also determined. Tormentic acid congener showed time-dependent antibacterial activity against P. aeruginosa with a MIC of 100 µg/ml and caused significant protein leakage. Antibacterial activity was found when tormentic acid congener was tested against both S. aureus and P. aeruginosa. The MICs were found to be 25 µg/ml and 12.5 µg/ml for P. aeruginosa and S. aureus cells, respectively. S. pyogenes was found to be susceptible to tormentic acid congener and the hydroethanolic extract with an MIC of 100 µg/ml and 25 µg/ml, respectively. A. baumannii was found not to be susceptible to the compound or the extracts. The compound and the extracts caused a significant decrease in the biofilm extracellular polysaccharide content of S. pyogenes. The extracts and tormentic acid congener caused detachment of biofilms and decreased the release of extracellular DNA and capsular polysaccharides from biofilms of P. aeruginosa and S. aureus. Tormentic acid congener and extracts, thus, have significant antibacterial and antibiofilm activities on these selected ESKAPE bacteria and can act as source lead compounds for the development of antibacterial triterpenoids.

Effects of baohuoside-I on epithelial-mesenchymal transition and metastasis in nasopharyngeal carcinoma.

To investigate the effect of baohuoside-I against nasopharyngeal carcinoma (NPC) and its underlying mechanism, baohuoside-I was employed to treat NPC cell lines CNE1 and CNE2 in vitro, followed by attachment and detachment assays to evalute the epithelial-mesenchymal transition (EMT) phenotype markers. Baohuoside-I was also administered to experimental mice to assess its effect on xenograft tumor growth and NPC cell metastasis. A microRNA (miRNA, miR) microarray was performed to screen for miRNA altered by baohuoside-I in NPC cells. Bioinformatic tools and luciferase activity assay was conducted to identify the downstream molecules mediating the anti-tumor property of baohuoside-I. Baohuoside-I inhibited EMT and metastasis and upregulated miR-370-3p in NPC cells, which was shown to directly recognize and inhibit expression of Hedgehog pathway component Smoothened (SMO). Baohuoside-I suppresses metastasis as well as EMT of NPC cells through targeting the Hedgehog pathway component SMO, and may serve as a potent anti-tumor agent in the clinical management of NPC.

An oriented hydroxyapatite film with arrayed plate-like particles enhance chondrogenic differentiation of ATDC5 cells.

Osteochondral defects of articular cartilage cannot regenerate spontaneously. For its surgical treatment, advancements in cartilage tissue engineering have particularly focused on subchondral bone lesions that tend to delay healing. Therefore, it is important to understand interactions between subchondral bone and chondrocytes. This study aimed to investigate the behavior of chondrogenic ATDC5 cells on oriented hydroxyapatite (HAp) films that mimic bone surfaces. HAp nanoparticles prepared herein were needle like and plate like. HAp films were formed through self-organization of the nanoparticles and had 2D structures regularly arrayed with the particles. Both films prominently comprised a-plane orientation surfaces but differed in the degree of hydrophilicity because of the patterns of particle self-assembly. ATDC5 cells cultured on the HAp film with plate-like particles could adhered in a shorter period but could not spread. The adhesive force of cells was weaker with the hydrophilic surface than with other surfaces, as determined using a trypsin-based cell detachment assay. In addition, ATDC5 cells displayed enhanced proliferation and chondrogenic differentiation. Our results suggest that the oriented HAp film formed using plate-like particles provided chondrogenic cells with a desired scaffold as that of subchondral bone to increase cell proliferation and differentiation.

An Evaluation Approach of Cell Viability Based on Cell Detachment Assay in a Single-Channel Integrated Microfluidic Chip.

Due to the heterogeneity of cancer cell populations, the traditional evaluation approach of cell viability based on the cell counting assay is quite inaccurate for the dose-response test of anticancer drugs, cell toxicology assays, and other biochemical stimulations. In this paper, an evaluation approach of cell viability based on the cell detachment assay in a single-channel integrated microfluidic chip is proposed to improve the accuracy of cell viability assessment. The electrodes are coated by fibronectin for specific cell adhesion, and it is biologically significant to study the cell detachment assay in vitro. The maximum number of cells that can be detected by this sensor is about 105 cells (overgrowing), while the minimum is about 100 cells. This method is calibrated with the half-maximal inhibitory concentration assay, and the results show that the cell viability calculated by adhesion strength is more accurate than that evaluated using the cell counting assay. Meanwhile, the shear rate is transformed into shear stress for the comparability among the results in other papers. The most sensitive frequency is also determined as 1 kHz according to normalized impedance. Besides, the impedance of cell adhesion affected by different shear stresses is monitored to study the optimized plan for long-term culture of cells in the integrated microfluidic chip prepared for the cell detachment assay. Adhesion strength τ25, which is the magnitude of shear stress needed to detach 75% of cell population, is introduced to describe the cell adhesion forces. It is calculated and normalized based on the cell detachment assay to evaluate cell viability. The relative errors of the cell detachment method compared with those of the cell counting method decrease by 0.637 (0% FBS), 0.586 (0.5% FBS), and 0.342 (2% FBS).

TRPC6 Binds to and Activates Calpain, Independent of Its Channel Activity, and Regulates Podocyte Cytoskeleton, Cell Adhesion, and Motility.

Mutations in the transient receptor potential channel 6 (TRPC6) gene are associated with an inherited form of FSGS. Despite widespread expression, patients with TRPC6 mutations do not present with any other pathologic phenotype, suggesting that this protein has a unique yet unidentified role within the target cell for FSGS, the kidney podocyte. We generated a stable TRPC6 knockout podocyte cell line from TRPC6 knockout mice. These cells were engineered to express wild-type TRPC6, a dominant negative TRPC6 mutation, or either of two disease-causing mutations of TRPC6, G109S or K874*. We extensively characterized these cells using motility, detachment, and calpain activity assays; immunofluorescence; confocal or total internal reflection fluorescence microscopy; and western blotting. Compared with wild-type cells, TRPC6-/- podocytes are less motile and more adhesive, with an altered actin cytoskeleton. We found that TRPC6 binds to ERK1/2 and the actin regulatory proteins, caldesmon (a calmodulin- and actin-binding protein) and calpain 1 and 2 (calcium-dependent cysteine proteases that control the podocyte cytoskeleton, cell adhesion, and motility via cleavage of paxillin, focal adhesion kinase, and talin). Knockdown or expression of the truncated K874* mutation (but not expression of the gain-of-function G019S mutation or dominant negative mutant of TRPC6) results in the mislocalization of calpain 1 and 2 and significant downregulation of calpain activity; this leads to altered podocyte cytoskeleton, motility, and adhesion-characteristics of TRPC6-/- podocytes. Our data demonstrate that independent of TRPC6 channel activity, the physical interaction between TRPC6 and calpain in the podocyte is important for cell motility and detachment and demonstrates a scaffolding role of the TRPC6 protein in disease.

Abstract 1878: MicroRNA-200c (miRNA-200c) increases adhesive interactions between E-selectin ligands expressed by breast cancer cells and E-selectin under physiological blood flow conditions

Abstract The epithelial-to-mesenchymal transition (EMT), and its reverse process, the mesenchymal-to-epithelial transition (MET), are associated with metastatic tumor progression in breast cancer (BC). E-selectin, a cell adhesion molecule expressed by cytokine-activated vascular endothelium, is also involved in metastasis via binding to counter-receptors (E-selectin ligands) on the surface of BC cells. Consequently, efforts have been made to investigate a link between the EMT/MET and the adhesive interactions between E-selectin and their ligands. Our lab previously demonstrated that BC cells with an epithelial phenotype exhibit more adhesive interactions between E-selectin/ligands under blood flow conditions than cells with a mesenchymal phenotype. Therefore, molecular factors in the blood that activate the EMT or the MET in circulating tumor cells (CTCs) have been of particular interest. These factors may regulate the potential for CTCs to traffic to bone marrow endothelium, a frequent site of BC metastasis that constitutively expresses E-selectin. To advance knowledge in this area, we hypothesized that miRNA-200c, associated with poor prognosis in metastatic BC patients, induces the MET in MDA-MB-231 BC cells and promotes adhesion between E-selectin/ligands. Microscopy images show that MDA-MB-231 cells transfected with miRNA-200c displayed a cell morphology similar to that of epithelial cells, whereas cells transfected with a negative control miRNA (NC miRNA) retained their mesenchymal phenotype. Furthermore, cells transfected with miRNA-200c exhibited greater mRNA expression of E-cadherin and lower expression of Vimentin compared to cells transfected with a NC miRNA by qRT-PCR. The expression of Zeb1/2, both targets for miRNA-200c, were down-regulated in cells transfected with miRNA-200c compared to cells transfected with a NC miRNA. Both the microscopy images and mRNA expression analysis indicate induction of MET in miRNA-200c transfected cells. Shear flow adhesion assays and shear flow detachment assays were performed at bone marrow shear stresses to examine adhesive interactions between E-selectin/ligands. Using these assays, MDA-MB-231 cells transfected with miRNA-200c demonstrated slower rolling velocities, greater firm adhesion, lower detachment, and equivalent tethering activity when compared to cells transfected with a NC miRNA. Moreover, analysis of the mRNA expression of α1,3- and α1,4- fucosyltransferases (FT) revealed increased expression of FT3 and FT6 in cells transfected with miRNA-200c compared to cells transfected with a NC miRNA. Altogether, these results suggest that miRNA-200c increases fluid-shear resistant adhesion and that factors present in the blood may impact the metastatic potential of cancer cells in circulation by regulating cellular phenotypes. Citation Format: Christian A. Showalter, Monica M. Burdick. MicroRNA-200c (miRNA-200c) increases adhesive interactions between E-selectin ligands expressed by breast cancer cells and E-selectin under physiological blood flow conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1878.