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The objective of the present study was to compare recent methods for characterizing cell modifications. We studied the effect of extracellular reduced glutathione (GSH) on an olfactory neuroblast cell line (13s24). Three methods were used to monitor, in label-free, noninvasive real-time experiments, cell surface occupancy by measuring impedance (xCELLigence), cell behavior (HoloMonitor cytometry), cell ultrastructure by measuring refractive index (3D Nanolive microscopy). Reduced glutathione dose-dependently increased cell volume and motility and decreased cell adhesion. Cell sorting analyses revealed that after short-term exposure (6 h), GSH reduced F-actin polymerization and extracellular glycoproteins leading to adhesion strength loss. Results support the hypothesis that excreted GSH could modulate disulfide bound-dependent integrin conformations involved in neurogenesis and/or neuronal plasticity. This is the first evidence of a causal link between GSH and changes in cell volume and motility required for cell division, migration, and/or differentiation. Results show the importance of real-time analysis methods, without labelling, in the study of cell responses under culture conditions. The present findings highlight important criteria in the choice of methods, beyond the parameters studied, such as cell preparation time, plate filling time, number of cells studied, friendly use of the devices, and the complexity of data processing. [ABSTRACT FROM AUTHOR]

We investigate role of ATP sensitive potassium (KATP) channel in cytotoxic effect of cypermethrin on rat aortic smooth muscle cells. Cytotoxicity analysis was performed at 0, 0.1, 0.5, 10, 50, and 100 µM concentrations of cypermethrin and the cell index (CI) was calculated. KATP currents were recorded using patch clamp technique for 50 and 100 µM concentrations and channel conductivity was determined by obtaining current-voltage characteristics. No cytotoxic effect was observed in the first 72 hours. At the 96th hour, only at 100 µM concentration, the CI value decreased significantly compared to control group and at 120 and 144th hours, it was observed that the CI value decreased significantly at all concentrations. Currents and conductivities were significantly decreased at 50 and 100 µM concentrations. Results gave clues that cypermethrin causes a cytotoxic effect on vascular smooth muscles and that KATP channels may have a role in the emergence of this effect. [ABSTRACT FROM AUTHOR]

Pollen, in addition to allergens, comprise low molecular weight components (LMC) smaller than 3 kDa. Emerging evidence indicates the relevance of LMC in allergic immune responses. However, the interaction of birch pollen (BP)-derived LMC and epithelial cells has not been extensively studied. We investigated epithelial barrier modifications induced by exposure to BP LMC, using the human bronchial epithelial cell line 16HBE14o-. Epithelial cell monolayers were apically exposed to the major BP allergen Bet v 1, aqueous BP extract or BP-derived LMC. Barrier integrity after the treatments was monitored by measuring transepithelial electrical resistance at regular intervals and by using the xCELLigence Real-Time Cell Analysis system. The polarized release of cytokines 24 h following treatment was measured using a multiplex immunoassay. Epithelial barrier integrity was significantly enhanced upon exposure to BP LMC. Moreover, BP LMC induced the repair of papain-mediated epithelial barrier damage. The apical release of CCL5 and TNF-α was significantly reduced after exposure to BP LMC, while the basolateral release of IL-6 significantly increased. In conclusion, the results of our study demonstrate that BP-derived LMC modify the physical and immunological properties of bronchial epithelial cells and thus regulate airway epithelial barrier responses. [ABSTRACT FROM AUTHOR]

The objective of the present study was to compare recent methods for characterizing cell modifications. We studied the effect of extracellular reduced glutathione (GSH) on an olfactory neuroblast cell line (13s24). Three methods were used to monitor, in label-free, noninvasive real-time experiments, cell surface occupancy by measuring impedance (xCELLigence), cell behavior (HoloMonitor cytometry), cell ultrastructure by measuring refractive index (3D Nanolive microscopy). Reduced glutathione dose-dependently increased cell volume and motility and decreased cell adhesion. Cell sorting analyses revealed that after short-term exposure (6 h), GSH reduced F-actin polymerization and extracellular glycoproteins leading to adhesion strength loss. Results support the hypothesis that excreted GSH could modulate disulfide bound-dependent integrin conformations involved in neurogenesis and/or neuronal plasticity. This is the first evidence of a causal link between GSH and changes in cell volume and motility required for cell division, migration, and/or differentiation. Results show the importance of real-time analysis methods, without labelling, in the study of cell responses under culture conditions. The present findings highlight important criteria in the choice of methods, beyond the parameters studied, such as cell preparation time, plate filling time, number of cells studied, friendly use of the devices, and the complexity of data processing.

Asteraceae family plants have potential anticancer activity. In this study, the cytotoxic effects of C. iconica were investigated using different tumor cell lines: Non-small lung cancer (A549) and human endometrium carcinoma cell line (RL95-2). The cytotoxicity of the extracts was determined with MTT assay and the xCELLigence Real-Time Cell Analyser (RTCA). As a result, the ethyl acetate sub-extract and methanol extract of C. iconica were found cytotoxic to RL95-2 cell line with 271 and 51 µg/mL IC50 values, respectively. According to RTCA results, the IC50 values of ethyl acetate subextract were 44.82, 60.89, and 55.22 µg/mL and methanol extract 265.52, 250.48, and 246.65 µg/mL, for 24, 48, and 72-h exposure, respectively. RL95-2 cells are more sensitive to ethyl acetate sub-extract than methanol extract. This study is the known first study to show the cytotoxic effects of C. iconica extracts on both A549 and RL95-2 cell lines. [ABSTRACT FROM AUTHOR]

The prostaglandin transporter (PGT, SLCO2A1) mediates transport of prostanoids (a.o. prostaglandin E2 (PGE2)) into cells and thereby promotes their degradation. Overexpression of PGT leads to low extracellular PGE2 levels and has been linked to impaired wound healing of diabetic foot ulcers. Inhibition of PGT could thus be beneficial, however, no PGT inhibitors are currently on the market and drug discovery efforts are hampered by lack of high-through screening assays for this transporter. Here we report on a label-free impedance-based assay for PGT that measures transport activity through receptor activation (TRACT) utilizing prostaglandin E2 receptor subtype EP3 and EP4 that are activated by PGE2. We found that induction of PGT expression on HEK293-JumpIn-SLCO2A1 cells that also express EP3 and EP4 leads to an over 10-fold reduction in agonistic potency of PGE2. PGE2 potency could be recovered upon inhibition of PGT-mediated PGE2 uptake with PGT inhibitors olmesartan and T26A, the potency of which could be established as well. Moreover, the TRACT assay enabled the assessment of transport function of PGT natural variants. Lastly, HUVEC cells endogenously expressing prostanoid receptors and PGT were exploited to study wound healing properties of PGE2 and T26A in real-time using a novel impedance-based scratch-induced wound healing assay. These novel impedance-based assays will advance PGT drug discovery efforts and pave the way for the development of PGT-based therapies. [ABSTRACT FROM AUTHOR]

Equid herpesvirus 4 (EHV-4) is a common respiratory pathogen in horses. It sporadically induces abortion or neonatal death. Although its contribution in neurological disorders is not clearly demonstrated, there is a strong suspicion of its involvement. Despite preventive treatments using vaccines against EHV-1/EHV-4, the resurgence of alpha-EHV infection still constitutes an important threat to the horse industry. Yet very few studies have been conducted on the search for antiviral molecules against EHV-4. A screening of 42 antiviral compounds was performed in vitro on equine fibroblast cells infected with the EHV-4 405/76 reference strain (VR2230). The formation of cytopathic effects was monitored by real-time cell analysis (RTCA), and the viral load was quantified by quantitative PCR. Aciclovir, the most widely used antiviral against alpha-herpesviruses in vivo, does not appear to be effective against EHV-4 in vitro. Potential antiviral activities were confirmed for eight molecules (idoxuridine, vidarabine, pritelivir, cidofovir, valganciclovir, ganciclovir, aphidicolin, and decitabine). Decitabine demonstrates the highest efficacy against EHV-4 in vitro. Transcriptomic analysis revealed the up-regulation of various genes implicated in interferon (IFN) response, suggesting that decitabine triggers the immune antiviral pathway. [ABSTRACT FROM AUTHOR]

Lung cancer is a type of cancer that is mostly diagnosed at an advanced stage and has a short survival time despite standard chemotherapy and targeted therapies. Terbutaline and Orciprenaline are bronchodilator agents that are potent and selective β2 receptor agonists. The purpose of this study was to investigate to evaluate the effects of Terbutaline and Orciprenaline on A549 human lung carcinoma cell line and Beas-2b human bronchial epithelial cell line. Cells were treated with 1, 10, 100, 200, 400 μM concentrations of Terbutaline and Orciprenaline. 3- (4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay and xCELLigence real-time cell analyzer were used to determine their effects on cell viability. The cell index was monitored continuously by visualizing the impedance of the E-plate wells. Because of the roles of Glycogen Synthase Kinase 3β (GSK3β) in a diverse range of cellular processes like metabolism, cell proliferation, differentiation and survival and its key position at several signaling pathways, GSK3β inhibition by Terbutaline and Orciprenaline was also investigated. The results showed that Terbutaline and Orciprenaline inhibits GSK-3β. The overall results led to the conclusion that Terbutaline and especially Orciprenaline may have potential therapeutic effects in lung carcinoma. [ABSTRACT FROM AUTHOR]

The prostaglandin transporter (PGT, SLCO2A1) mediates transport of prostanoids (a.o. prostaglandin E2 (PGE2)) into cells and thereby promotes their degradation. Overexpression of PGT leads to low extracellular PGE2 levels and has been linked to impaired wound healing of diabetic foot ulcers. Inhibition of PGT could thus be beneficial, however, no PGT inhibitors are currently on the market and drug discovery efforts are hampered by lack of high-through screening assays for this transporter. Here we report on a label-free impedance-based assay for PGT that measures transport activity through receptor activation (TRACT) utilizing prostaglandin E2 receptor subtype EP3 and EP4 that are activated by PGE2. We found that induction of PGT expression on HEK293-JumpIn-SLCO2A1 cells that also express EP3 and EP4 leads to an over 10-fold reduction in agonistic potency of PGE2. PGE2 potency could be recovered upon inhibition of PGT-mediated PGE2 uptake with PGT inhibitors olmesartan and T26A, the potency of which could be established as well. Moreover, the TRACT assay enabled the assessment of transport function of PGT natural variants. Lastly, HUVEC cells endogenously expressing prostanoid receptors and PGT were exploited to study wound healing properties of PGE2 and T26A in real-time using a novel impedance-based scratch-induced wound healing assay. These novel impedance-based assays will advance PGT drug discovery efforts and pave the way for the development of PGT-based therapies.

• Tagetes minuta is a valuable medicinal plant with various uses, such as spice herb, traditional medicine, and a natural herbicide. • The study identified and quantified the secondary metabolites of T. minuta using colorimetric assays and liquid chromatography-mass spectrometry (LCMS). • The major classes of secondary metabolites identified in T. minuta were Phenolic acids, Flavonoids, Fatty acids, and Coumarins. • Ethanol extracts of T. minuta demonstrated higher antioxidant content, activity, and cytotoxicity against HepG2 and Vero cell lines, highlighting their pharmacological potential. The complexity of secondary metabolites in different medicinal plants, including Tagetes minuta , defines the general properties and uses of the plant. Tagetes minuta is reportedly a valuable medicinal plant that has long been used as a spice herb, medicine, a natural herbicide for weeds, and pest control in agriculture. In this study, the secondary metabolite profiles of the water and aqueous ethanol extracts of T. minuta were profiled through identification and quantification using colorimetric assays and a Waters Synapt G2 Quadrupole time-of-flight (QTOF) mass spectrometer (MS) connected to a Waters Acquity ultra-performance liquid chromatograph (UPLC). The antioxidant capacity of the extracts was evaluated with 2,2-Azino-di-3-ethylbenzthiazoline sulfonate and ferric reducing antioxidant power, as well as their toxicity on HepG2 (cancerous) and Vero (non-cancerous) cell lines. The major classes of secondary metabolites identified and quantified were phenolic acids (benzoic acids and hydroxycinnamic acids), flavonoids (chalcones, flavonols, flavanols, and flavones), fatty acids, coumarins and furanocoumarins. The bioavailability of these secondary metabolites is influenced by the polarity of the solvent of extraction. Extracts from aqueous ethanol showed higher secondary metabolites, corresponding to antioxidant content and activity as well as cytotoxicity in HepG2 and Vero cells, compared to the water extract. This study presents a comprehensive phytochemical knowledge of secondary metabolites, primarily phenolic compounds, identified in T. minuta, which can pave the way for future studies on the pharmacological importance and standardisation of the medicinal uses of the plant. [ABSTRACT FROM AUTHOR]

Purpose of the study: development of in vitro laboratory models to evaluate quality parameters and specific efficacy of dendritic cell vaccine (DCV).Material and Methods. Biological samples of malignant tumor patients treated with autologous dendritic cell vaccine (DC) were included into the study. Immature DCs (n=46) and mature DCs (n=56) were used to induce proliferation of antigen-specific T lymphocytes (n=227). Autologous tumor cells from skin melanoma (n=10) or sarcoma (n=8) patients in the xCELLigence® assay system were used to study the in vitro antitumor cytotoxic activity of generated CTLs (n=18). The secretion of cytokines and cytolytic proteins was studied by multiplex analysis. The subpopulation composition of effector T-lymphocytes was determined by flow cytometry.Results. We revealed that mature DCs (CD83+CD1a-) had a high expression of antigen presenting molecules (HLA-DR) and those providing migration of DCs into lymph nodes (CCR7) as well as costimulatory molecules CD80 and CD86 as compared to immature DCs (CD83-CD1a+). Induction of mature DCs was found to stimulate an increase in the relative content of proliferating T cells compared with stimulation of immature DCs (p

Candida species cause life-threatening infections with high morbidity and mortality rates and their resistance to conventional therapy is closely linked to biofilm formation. Thus, the development of new approaches to study Candida biofilms and the identification of novel therapeutic strategies could yield improved clinical outcomes. In the current study, we have set up an impedance-based in vitro system to study Candida spp. biofilms in real-time and to evaluate their sensitivity to two conventional antifungal groups used in clinical practice - azoles and echinocandins. Both fluconazole and voriconazole were unable to inhibit biofilm formation in most strains tested, while echinocandins showed biofilm inhibitory capacity at relatively low concentrations (starting from 0.625 mg/L). However, assays performed on 24 h Candida albicans and C. glabrata biofilms revealed that micafungin and caspofungin failed to eradicate mature biofilms at all tested concentrations, evidencing that once formed, Candida spp. biofilms are extremely difficult to eliminate using currently available antifungals. We then evaluated the antifungal and anti-biofilm effect of andrographolide, a natural compound isolated from the plant Andrographis paniculata with known antibiofilm activity on Gram-positive and Gram-negative bacteria. Optical density measures, impedance evaluation, CFU counts, and electron microscopy data showed that andrographolide strongly inhibits planktonic Candida spp. growth and halts Candida spp. biofilm formation in a dose-dependent manner in all tested strains. Moreover, andrographolide was capable of eliminating mature biofilms and viable cell numbers by up to 99.9% in the C. albicans and C. glabrata strains tested, suggesting its potential as a new approach to treat multi-resistant Candida spp. biofilm-related infections.

IntroductionPeriodontitis is a biofilm-mediated disease that is usually treated by non-surgical biofilm elimination with or without antibiotics. Antibiotic treatment in periodontal patients is typically selected empirically or using qPCR or DNA hybridization methods. These approaches are directed towards establishing the levels of different periodontal pathogens in periodontal pockets to infer the antibiotic treatment. However, current methods are costly and do not consider the antibiotic susceptibility of the whole subgingival biofilm.MethodsIn the current manuscript, we have developed a method to culture subgingival samples ex vivo in a fast, label-free impedance-based system where biofilm growth is monitored in real-time under exposure to different antibiotics, producing results in 4 hours. To test its efficacy, we performed a double-blind, randomized clinical trial where patients were treated with an antibiotic either selected by the hybridization method (n=32) or by the one with the best effect in the ex vivo growth system (n=32).ResultsAntibiotic selection was different in over 80% of the cases. Clinical parameters such as periodontal pocket depth, attachment level, and bleeding upon probing improved in both groups. However, dental plaque was significantly reduced only in the group where antibiotics were selected according to the ex vivo growth. In addition, 16S rRNA sequencing showed a larger reduction in periodontal pathogens and a larger increase in health-associated bacteria in the ex vivo growth group.DiscussionThe results of clinical and microbiological parameters, together with the reduced cost and low analysis time, support the use of the impedance system for improved individualized antibiotic selection.

Copyright of Journal of Faculty of Pharmacy of Ankara University / Ankara Üniversitesi Eczacilik Fakültesi Dergisi is the property of Ankara University and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas is the property of Universidad de Santiago de Chile and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Attention-deficit hyperactivity disorder (ADHD) is a neurodevelopmental polygenic disorder that affects more than 5% of children and adolescents around the world. Genetic and environmental factors play important roles in ADHD etiology, which leads to a wide range of clinical outcomes and biological phenotypes across the population. Brain maturation delays of a 4-year lag are commonly found in patients, when compared to controls of the same age. Possible differences in cellular growth rates might reflect the clinical observations in ADHD patients. However, the cellular mechanisms are still not elucidated. To test this hypothesis, we analysed the proliferation of induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) derived from male children and adolescents diagnosed with ADHD and with genetic predisposition to it (assessed using polygenic risk scores), as well as their respective matched controls. In the current pilot study, it was noticeable that NSCs from the ADHD group proliferate less than controls, while no differences were seen at the iPSC developmental stage. Our results from two distinct proliferation methods indicate that the functional and structural delays found in patients might be associated with these in vitro phenotypic differences, but start at a distinct neurodevelopmental stage. These findings are the first ones in the field of disease modelling of ADHD and might be crucial to better understand the pathophysiology of this disorder. [ABSTRACT FROM AUTHOR]

Objectives: Increasing the effectiveness and reliability of Akt inhibition in lung cancer treatment may pave the way for a more favorable use of this method in the future. Therefore, we aimed to evaluate the possible role of RhoB in the regulation of Akt inhibition in NSCLC., Material and Methods: The study was conducted using the NSCLC cell line A549. Small interfering RNA (siRNA)-mediated RhoB knockdown was performed and combined with perifosine (Akt inhibitor) treatment. Cell proliferation was detected by xCELLigence® RTCA. RhoB, pAkt, and Bcl2l11 expressions were analyzed by ELISA. Apoptosis rates were determined by flow cytometry., Results: We knocked down RhoB in A549 cells using siRNA. According to the results of the 72-hour experiment, RhoB upregulation was observed to reduce the antiproliferative ac-tivity of the Akt inhibitor. The pAkt level was significantly lower in the group where the Akt inhibitor was applied to RhoB-silenced cells via siRNA compared to other treatment groups. The percentage of apoptosis in the group where the Akt inhibitor was applied to RhoB-silenced cells was found to be significantly higher than in the RhoB-suppressed group., Conclusion: Both proliferation and apoptosis tests determined that the RhoB molecule is a negative regulator of the anti-tumor activity of Akt inhibition in non-small cell lung cancer. This study suggests that RhoB expression may play a critical role in the regulation of Akt inhibition in NSCLC, potentially opening new avenues for treatment strategies., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The aim of this study is to reveal the potentially protective role of ethanolic extract of agrimony (Agrimonia eupatoria L.) against the cytotoxic effect of bisphenol A (BPA) in vitro, using an intestinal porcine epithelial cell line (IPEC-1). The cells were exposed to different concentrations of BPA: 12.5, 25, 50, 100, and 200 µg.ml–1 alone and in combination with agrimony extract (250 µg.ml–1). The proliferative cell response was monitored for 72 h by a xCELLigence system or real-time cell analyser (RTCA), recorded as the cell index (CI) and expressed as a proliferative activity (% PA) compared to the control cells without treatment. The metabolic activity was measured by a MTS colorimetric test, performed after 48 h of treatment with the tested substances. The cytotoxic effect on cells exposed to BPA alone, in comparison to the control cells without treatment, was observed in both assays (P < 0.0001). It was confirmed that BPA reduces both the metabolic activity and the proliferation of cells. After the cell treatment with agrimony, the metabolic activity had increased to reach over the control (101.52 %), while reducing the proliferation of the cells. The protective role of agrimony against cytotoxicity caused by BPA was observed after cell treatment with agrimony in combination with lower concentrations of BPA (12.5; 25 and 50 µg.ml–1). The slight improvement in the adherence was observed in cells treated with these combinations, in comparison to the cells treated with BPA alone. On the other hand, the metabolic activity was slightly improved in cells treated with a combination of agrimony and BPA at higher concentrations (50 a 100 µg.ml–1). This supported our assumption that agrimony can protect a model organism against cytotoxicity caused by BPA.

Objective:The frequency of epileptic seizures is high in patients with brain tumors and its treatment is important. It is atypical mitotic proliferation that is effective in the proliferation of malignant tumor cells. It is known that antiepileptics have direct or indirect effects on mitotic proliferation. In our study, we aimed to maximize the use of both the antiepileptic effect and the cytoreductive effect by suppressing tumor growth while choosing the drug to stop the seizure.Methods:In our study, anti-tumoral activities of antiepileptic agents containing gabapentin, pregabalin, valproic acid, levetiracetam, zonisamide, phenytoin, carbamazepine in in vitro glioblastoma (c6) and neuroblastoma cell cultures (NA/An1) were evaluated with a real-time cell analysis system. Statistically, the difference between groups was investigated by analysis of variance, followed by post hoc Tukey’s test Statistical Package for the Social Sciences software 16.0 (IBM Inc, Chicago, IL, USA).Results:In our study, it was observed that all drugs except gabapentin had antimitotic effects in glioblastoma cell cultures, among which phenytoin, levetiracetam, and valproic acid had dose-dependent antimitotic effects, while carbamazepine had reduced antimitotic effects at concentrations above 25 μg/mL. In in vitro neuroblastoma cell cultures, it was found that only valproic acid and zonisamide had antimitotic effects, and other drugs studied did not have antimitotic effects.Conclusions:In our study, it was observed that the preference of antiepileptics with a high antimitotic effect on tumoral cells was important when arranging seizure treatment in patients with brain tumors.

Multi-walled carbon nanotubes (MWNTs) were investigated before and after carboxylic acid functionalization. Here, the comparative analysis of MWNTs with different bulk densities reveals similar Raman and FTIR spectra before and after acid functionalization except for minor differences. However thermal analyses exhibited some basic differences for both MWNTs before and after acid treatment. We investigated the cytotoxicity of two MWNTs on HT-29 and HEK293-T cells through three different methods: MTT assay, DAPI staining, and xCELLigence real-time cell analyzing method. It was observed that high bulk density affects the cytotoxicity for both cell lines and in all methods. Because the acid treatment lowered the bulk density, after acid treatment, the MWNTs with the higher bulk density (C150P) elicited similar cytotoxicity compared to the lower one (C70P). [ABSTRACT FROM AUTHOR]

We have previously shown that human melanoma cells rapidly decrease human brain endothelial barrier strength. Our findings showed a fast mechanism of melanoma mediated barrier disruption, which was localised to the paracellular junctions of the brain endothelial cells. Melanoma cells are known to release molecules which cleave the surrounding matrix and allow traversal within and out of their metastatic niche. Enzymatic families, such as matrix metalloproteinases (MMPs) and proteases are heavily implicated in this process and their complex nature in vivo makes them an intriguing family to assess in melanoma metastasis. Herein, we assessed the expression of MMPs and other proteases in melanoma conditioned media. Our results showed evidence of a high expression of MMP-2, but not MMP-1, -3 or -9. Other proteases including Cathepsins D and B were also detected. Recombinant MMP-2 was added to the apical face of brain endothelial cells (hCMVECs), to measure the change in barrier integrity using biosensor technology. Surprisingly, this showed no decrease in barrier strength. The addition of potent MMP inhibitors (batimastat, marimastat, ONO4817) and other protease inhibitors (such as aprotinin, Pefabloc SC and bestatin) to the brain endothelial cells, in the presence of various melanoma lines, showed no reduction in the melanoma mediated barrier disruption. The inhibitors batimastat, Pefabloc SC, antipain and bestatin alone decreased the barrier strength. These results suggest that although some MMPs and proteases are released by melanoma cells, there is no direct evidence that they are substantially involved in the initial melanoma-mediated disruption of the brain endothelium. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Health Sciences / Sağlık Bilimleri Dergisi is the property of Erciyes Universitesi Saglik Bilimleri Dergisi and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Biofilm formation and the appearance of persister cells with low metabolic rates are key factors affecting conventional treatment failure and antibiotic resistance. Using impedance-based measurements, crystal violet staining and traditional culture we have studied the biofilm growth dynamics of 13 Pseudomonas aeruginosa strains under the effect of seven conventional antibiotics. Real-time growth quantifications revealed that the exposure of established P. aeruginosa biofilms to certain concentrations of ciprofloxacin, ceftazidime and tobramycin induced the emergence of persister cells, that showed different morphology and pigmentation, as well increased antibiotic resistance. Whole-genome sequencing of wildtype and persister cells identified several SNPs, a genomic inversion and a genomic duplication in one of the strains. However, these mutations were not uniquely associated with persisters, suggesting that the persistent phenotype may be related to metabolic and transcriptional changes. Given that mannitol has been proposed to activate bacterial metabolism, the synergistic combination of mannitol and ciprofloxacin was evaluated on clinical 48 h P. aeruginosa biofilms. When administered at doses ≥320 mg/L, mannitol was capable of preventing persister cell formation by efficiently activating dormant bacteria and making them susceptible to the antibiotic. These results were confirmed using viable colony counting. As the tested ciprofloxacin-mannitol combination appeared to fully eradicate mature biofilms, we conclude that impedance-based biofilm diagnostics, which permits antibiotic susceptibility testing and the identification of persister cells, is of great potential for the clinical practice and could aid in establishing treatment breakpoints for emerging biofilm-related infections.

ABSTRACT Objectives: The purpose of this study was to explore the cytotoxic effects of five different universal bonding agents on mouse fibroblast cell lines (L929). Materials and Methods: Five different widely used universal adhesive systems were chosen that have different contents, pH levels, and polymerization methods. A real-time cell analyzer (RT-CES, xCELLigence; Roche Applied Science, Germany, and ACEA Biosciences, USA) was used for cytotoxic evaluation of light-cured polymerized G-Premio Bond (GC Europe, Belgium), Prime&Bond Universal (Dentsply Sirona, USA), Universal Bond Quick (Kuraray, USA), Single Bond Universal (3M ESPE, USA) and self-cured polymerized Tokuyama Universal Bond (Tokuyama, USA) experimental groups. L929 were cultured in Dulbecco’s modified Eagle’s medium and supplemented with 10% fetal bovine serum and 1 % antibiotics. The assay was performed E-plate-16 and monitored every 15 min for 72 h. Statistical analysis was performed using ANOVA and Tukey’s posthoc tests. Results: All tested universal adhesive systems showed a statistically significant difference in cytotoxicity values in different time periods (p

Introduction: The vast diverse products and applications of engineered nanoparticle bio-conjugates (ENPBCs) are increasing, and thus flooding the-markets. However, the data to support risk estimates of ENPBC are limited. While it is important to assess the potential benefits, acceptability and uptake, it is equally important to understand where ENPBCs safety is and how to expand and affirm consumer security concerns. Methods: Online articles were extracted from 2013 to 2016 that pragmatically used xCELLigence real-time cell analysis (RTCA) technology to describe the in-vitro toxicity of ENPBCs. The xCELLigence is a +noninvasive in vitro toxicity monitoring process that mimics exact continuous cellular bio-responses in real-time settings. On the other hand, articles were also extracted from 2008 to 2016 describing the in vivo animal models toxicity of ENPBCs with regards to safety outcomes. Results: Out of 32 of the 121 (26.4%) articles identified from the literature, 23 (71.9%) met the in-vitro xCELLigence and 9(28.1%) complied with the in vivo animal model toxicity inclusion criteria. Of the 23 articles, 4 of them (17.4%) had no size estimation of ENPBCs. The xCELLigence technology provided information on cell interactions, viability, and proliferation process. Eighty-three (19/23) of the in vitro xCELLigence technology studies described ENPBCs as nontoxic or partially nontoxic materials. The in vivo animal model provided further toxicity information where 1(1/9) of the in vivo animal model studies indicated potential animal toxicity while the remaining results recommended ENPPCs as potential candidates for drug therapy though with limited information on toxicity. Conclusion: The results showed that the bioimpacts of ENPBCs either at the in vitro or at in vivo animal model levels are still limited due to insufficient information and data. To keep pace with ENPBCs biomedical products and applications, in vitro, in vivo assays, clinical trials and long-term impacts are needed to validate their usability and uptake. Besides, more real-time ENPBCs-cell impact analyses using xCELLigence are needed to provide significant data and information for further in vivo testing.

Abstract In this current study, antiproliferative effect of Hec1/Nek2 Mitotic Pathway inhibitor INH1 was investigated in adenocarcinomic human alveolar basal epithelial cells A549 and human cervix carcinoma HeLa cell lines in vitro. To this end cell index values by xCELLigence Real‐Time Cell Analysis DP instrument, mitotic index, BrdU proliferation assay and apoptotic index analysis were used. The results of the current study showed that INH1 had cytostatic and cytoskeletal effects on A549 and cytostatic effect on HeLa cells. The IC50 concentration was determined as 56 µM with the xCelligence device for both cell lines. IC50 concentration was used for all other parameters. While this concentration decreased the mitotic index BrdU proliferation values, it increased the apoptotic index values of both of cell lines. There were significant differences between the control and the experimental groups (p

Sporopollenin-mediated control drug delivery has been studied extensively owing to its desirable physicochemical and biological properties. Herein, sporopollenin was successfully extracted from C. libani and P. nigra pollens followed by loading of a commonly known anticancer drug Oxaliplatin. Drug loading and physicochemical features were confirmed by using light microscopy, FT-IR, SEM and TGA. For the first-time, real-time cell analyzer system xCELLigence was employed to record the Oxaliplatin loaded sporopollenin-mediated cell death (CaCo-2 and Vero cells) in real time. Both the release assays confirmed the slow release of oxaliplatin from sporopollenin for around 40–45 h. The expression of MYC and FOXO-3 genes has been significantly increased in CaCo2 cell and decreased non-cancerous Vero cell confirming the fact that sporopollenin-mediated control release of oxaliplatin is promoting apoptosis cell death preventing the spread of negative effects on nearby healthy cells. All the results suggested that C. libani and P. nigra can be suitable candidates for the slow delivery of drugs. [ABSTRACT FROM AUTHOR]

Abstract Background Biofilm is a fundamental bacterial survival mode which proceeds through three main generalized phases: adhesion, maturation, and dispersion. Lactobacilli spp. (LB) are critical components of gut and reproductive health and are widely used probiotics. Evaluation of time-dependent mechanisms of biofilm formation is important for understanding of host-microbial interaction and development of therapeutic interventions. Time-dependent LB biofilm growth was studied in two systems: large biofilm output in continuous flow system (microfermenter (M), Institute Pasteur, France) and electrical impedance-based real time label-free cell analyzer (C) (xCELLigence, ACEA Bioscience Inc., San Diego, CA). L. plantarum biofilm growth in M system was video-recorded, followed by analyses using IMARIS software (Bitplane, Oxford Instrument Company, Concord, MA, USA). Additionally, whole genome expression and analyses of attached (A) and dispersed (D) biofilm phases at 24 and 48 h were performed. Results The dynamic of biofilm growth of L. plantarum was similar in both systems except for D phases. Comparison of the transcriptome of A and D phases revealed, that 121 transcripts differ between two phases at 24 h. and 35 transcripts – at 48 h. of M growth. The main pathways, down-regulated in A compared to D phases after 24 h. were transcriptional regulation, purine nucleotide biosynthesis, and L-aspartate biosynthesis, and the upregulated pathways were fatty acid and phospholipid metabolism as well as ABC transporters and purine nucleotide biosynthesis. Four LB species differed in the duration and amplitude of attachment phases, while growth phases were similar. Conclusion LB spp. biofilm growth and propagation area dynamic, time-dependent processes with species-specific and time specific characteristics. The dynamic of LB biofilm growth agrees with published pathophysiological data and points out that real time evaluation is an important tool in understanding growth of microbial communities.

Abstract Background Preeclampsia is a common obstetric syndrome affecting women in their first pregnancy and characterized by hypertension and proteinuria, which appears after 20 weeks of gestation. It is characterized by high blood pressure and occasional damage to another organ system most often the liver and kidneys. Currently, the etiology and pathogenesis of this syndrome are not fully understood. Since mesenchymal stem cells/stromal cells (MSCs) are intimately associated with endothelial cells that line vessel walls in the decidua they may play some role in the pathogenesis of this syndrome. In this study, we have partly, unveiled the mechanism of preeclampsia pathogenesis at the stem cells level. Methods We have isolated and characterized MSCs from decidua basalis of preeclampsia placenta (PE-DBMSCs) and showed their decreased functionality in terms of proliferation, migration, adhesion and clone formation potential as compared to MSCs isolated from decidua region of normal placentae (DBMSCs). The cells were preconditioned with H2O2 and the functional characteristics were evaluated. Differentially expressed genes were analyzed using mass spectrometry. Immunoblotting confirmed the expression of these proteins. Results Pre-conditioning with H2O2 restored the functional outcome of PE-DBMSCs. Mass spectrometry (MS) analysis of differentially expressed proteins revealed HMOX1 as one of the major candidates missing in PE-DBMSCs. HMOX1 inhibition by tin protoporphyrin (SnPP) in normal DBMSCs resulted in a reduction in proliferation, migration, adhesion, and clone formation processes as compared to the untreated controls. mRNA and protein analyses of PE-DBMSCs preconditioned with H2O2 at lower doses showed upregulation of HMOX1 expression. Conclusions We hereby show for the first time that loss of function of stem cells/stromal cells isolated from the patients with preeclampsia may contribute towards the disease exacerbation. Our results suggest that HMOX1 may be partially responsible for the loss of functionality in PE-DBMSCs and contribute significantly towards the pathophysiology of preeclampsia. However, further investigation is required to decipher its exact role in the development and onset of the disorder.

Extensive use of triclosan (2,4,4′‐trichloro‐2′‐hydroxydiphenyl ether) as an antimicrobial agent in household and personal care products has resulted in global exposure of the human population. Its presence in human tissues, including milk, and its oestrogen‐disrupting properties raise concerns for an involvement in breast cancer. Because metastatic tumour spread is the main cause of breast cancer mortality, we have investigated the effects of triclosan on cell migration and invasion using three human breast epithelial cell lines and using concentrations comparable with those in human tissues. Long‐term exposure to 10−7 M of triclosan resulted in increased migration and invasion as measured by xCELLigence technology for all three cell lines, for the immortalized but nontransformed MCF‐10F breast epithelial cells (after 28 weeks), the oestrogen‐responsive MCF‐7 breast cancer cells (after 17 weeks) and the oestrogen‐unresponsive MDA‐MB‐231 breast cancer cells (after 20 weeks). The effects were therefore not limited to cancerous cells or to oestrogen‐responsive cells. This was paralleled in the MCF‐10F and MCF‐7 (but not MDA‐MB‐231) cells by a reduction in levels of E‐cadherin mRNA as measured by reverse transcription–polymerase chain reaction (RT‐PCR) and of E‐cadherin protein as measured by western immunoblotting, suggesting a mechanism involving epithelial‐to‐mesenchymal transition. This adds triclosan to the increasing list of ingredients of personal care products that can not only enter human breast tissue and increase cell proliferation but also influence cell motility. If mixtures of components in household and personal care products contribute to increasing cell migration and invasion, then reduction in exposure could offer a strategy for reducing breast cancer spread. Long‐term (>15 weeks) exposure to 10−7 M of triclosan resulted in increased migration and invasion of three human breast epithelial cell lines as measured using xCELLigence technology. Effects were not limited to cancerous cells or to oestrogen‐responsive cells. In two cell lines, reduced levels of E‐cadherin (mRNA and protein) were observed, suggesting a mechanism involving epithelial‐to‐mesenchymal transition. Triclosan is therefore another ingredient of personal care products that is entering the human breast and increasing cell motility, which is a prerequisite for metastasis. [ABSTRACT FROM AUTHOR]

We have previously shown that human melanoma cells rapidly decrease human brain endothelial barrier strength. Our findings showed a fast mechanism of melanoma mediated barrier disruption, which was localised to the paracellular junctions of the brain endothelial cells. Melanoma cells are known to release molecules which cleave the surrounding matrix and allow traversal within and out of their metastatic niche. Enzymatic families, such as matrix metalloproteinases (MMPs) and proteases are heavily implicated in this process and their complex nature in vivo makes them an intriguing family to assess in melanoma metastasis. Herein, we assessed the expression of MMPs and other proteases in melanoma conditioned media. Our results showed evidence of a high expression of MMP-2, but not MMP-1, -3 or -9. Other proteases including Cathepsins D and B were also detected. Recombinant MMP-2 was added to the apical face of brain endothelial cells (hCMVECs), to measure the change in barrier integrity using biosensor technology. Surprisingly, this showed no decrease in barrier strength. The addition of potent MMP inhibitors (batimastat, marimastat, ONO4817) and other protease inhibitors (such as aprotinin, Pefabloc SC and bestatin) to the brain endothelial cells, in the presence of various melanoma lines, showed no reduction in the melanoma mediated barrier disruption. The inhibitors batimastat, Pefabloc SC, antipain and bestatin alone decreased the barrier strength. These results suggest that although some MMPs and proteases are released by melanoma cells, there is no direct evidence that they are substantially involved in the initial melanoma-mediated disruption of the brain endothelium.

Endometrial cancer is the most common gynecologic malignancy and is associated with increased morbidity each year, including young people. However, its mechanisms of proliferation and progression are not fully elucidated. It is well known that abnormal glycosylation is involved in oncogenesis, and fucosylation is one of the most important types of glycosylation. In particular, fucosyltransferase 8 (FUT8) is the only FUT responsible for α1, 6-linked fucosylation (core fucosylation), and it is involved in various physiological as well as pathophysiological processes, including cancer biology. Therefore, we aimed to identify the expression of FUT8 in endometrial endometrioid carcinoma and investigate the effect of the partial silencing of the FUT8 gene on the cell proliferation of Ishikawa cells, an epithelial-like endometrial cancer cell line. Quantitative real-time PCR analysis showed that FUT8 gene expression was significantly elevated in the endometrial endometrioid carcinoma, compared to the normal endometrium. The immunostaining of FUT8 and Ulex europaeus Agglutinin 1 (UEA-1), a kind of lectin family specifically binding to fucose, was detected endometrial endometrioid carcinoma. The proliferation assay showed FUT8 partial knockdown by transfection of siRNA significantly suppressed the proliferation of Ishikawa cells, concomitant with the upregulation in the gene expressions associated with the interesting pathways associated with de-ubiquitination, aspirin trigger, mesenchymal-epithelial transition (MET) et al. It was suggested that the core fucosylation brought about by FUT8 might be involved in the proliferation of endometrial endometrioid carcinoma cells.

Colon cancer is one of the causes of cancer-related mortality. Therefore, more efficient therapy strategies are required. There is an increasing interest in natural products due to the potential cytotoxic activity in various cancer cell lines. Osthole and imperatorin are major active coumarins found in a variety of plants. The present study aimed to assess the cytotoxic effects of two natural coumarins, osthole, and imperatorin, administered separately and combined with 5-fluorouracil (5-FU) in human colon carcinoma cells and to identify the action of a mechanism. Real-time cell analysis with the xCELLigence System was used to screen for appropriate concentrations of osthole, imperatorin, 5-FU, and API-1 with cytotoxic or cytostatic potential and to monitor cell proliferation and viability in CoLo 205 cells continuously. Furthermore, the effects of the compounds on p38MAPK-α levels and the expression of Akt mRNA were evaluated. As a result, osthole showed considerable antiproliferative activity in CoLo 205 cells and increased the efficacy of 5-FU by accelerating the cytotoxic effect. Osthole was found to be as effective as protein kinase inhibitor API-1. Osthole, osthole+5-FU, and imperatorin treatments were significantly able to increase the ratio of phospho-p38MAPK-α/ p38MAPK-α. It was demonstrated that osthole, 5-FU, and API-I+5-FU were significantly suppressed the Akt mRNA expression as positive control API-1. The findings indicate that osthole may be a promising anti-cancer agent in the treatment of colon cancer and may increase the efficacy of 5-FU and, therefore, may reduce common side effects of traditional chemotherapy in colon cancer with 5-FU. [ABSTRACT FROM AUTHOR]

It has been found that monasnicotinic acid (MNA) isolated from the fungus Aspergillus cavernicola VKM F-906 reduces the proliferation and migration of human prostate cancer cells LnCaP and inhibits the AKT–mTORC1 and FAK–Src signaling pathways. However, MNA also increases the level of HSP60 and the phosphorylation of c-Jun, factors that stimulate the vital activity of cancer cells. MNA-induced cellular responses appear only after 48 h, which indicates the involvement of either the products of MNA metabolism or tautomers arising during the MNA protonation due to the growth of cell cultures and utilization of the compound by human cells. These results suggest that MNA is a promising compound for the production of various derivatives that would inhibit oncogenic signaling pathways without stimulating the factors providing the survival of tumor cells. [ABSTRACT FROM AUTHOR]

We compared proliferative activity and hypoxic tolerance in a co-culture of neurons and astrocytes in vitro after preliminary exposure to normobaric hypoxia and/or permissive hypercapnia in vivo. Preliminary hypoxic exposure increased the cell index throughout the 72-h period of observation, the effect of hypercapnia was observed on days 1 and 3 of the experiment, and the effect of hypercapnic hypoxia was noted only on day 1. Preliminary hypoxic exposure has a protective effect on nerve cells under conditions of chemical hypoxia. This suggests that hypercapnia and hypoxia activate proliferative activity of nerve cells, which can be viewed as a mechanism of their neuroprotective effectiveness. [ABSTRACT FROM AUTHOR]

The emergence of novel venom extraction techniques over the last half-century has greatly facilitated advances in the field of cnidarian research. A new recovery protocol utilizing ethanol as the primary stimulant in nematocyst discharge was recently published, however in vitro examination of the venom on organic models was not performed. This present study reports an original comparison of the chemically-induced discharge technique in vitro with a commonly used saltwater extraction method. Size-exclusion chromatography revealed distinct differences in venom profiles between the two methods: the saltwater recovery method FPLC profile and SDS-PAGE gel were similar to previously published results, whereas the ethanol-induced method was not. SDS-PAGE gel revealed distinct 40–55 kDa bands of previously identified cardiotoxic proteins recovered from the saltwater method, whereas the ethanol-induced method yielded degraded venom protein bands. A concentration-response curve generated through xCELLigence Real-Time Cell Analysis (RTCA) revealed a dramatic decrease in human cardiomyocyte activity when venom recovered via saltwater discharge was applied to these cells. With the exception of one sample, all ethanol-induced recovered venom failed to prompt a concentration-dependent decrease in cell survival when applied to human cardiomyocytes, resulting in a significant difference in IC50 concentrations between the compared venom samples. The data presented here facilitates an improved understanding of the parameters and analyses that are essential when developing and utilizing novel techniques for future cnidarian venom extraction research and supports the conclusion that recovery of venom from the tentacles of the box jellyfish Chironex fleckeri by ethanol is not an effective, efficient, or comprehensive extraction method compared to the published method of saltwater degradation of tentacles and bead mill extraction. • Venom extracted via ethanol had a different venom profile than bead mill extraction. • Venom extracted via ethanol lacked cardiotoxicity and repeatability. • Venom yield was lower in ethanol extracted venom. • Venom profile was less complex in ethanol extracted venom. [ABSTRACT FROM AUTHOR]

Introduction: The vast diverse products and applications of engineered nanoparticle bioconjugates (ENPBCs) are increasing, and thus flooding the-markets. However, the data to support risk estimates of ENPBC are limited. While it is important to assess the potential benefits, acceptability and uptake, it is equally important to understand where ENPBCs safety is and how to expand and affirm consumer security concerns. Methods: Online articles were extracted from 2013 to 2016 that pragmatically used xCELLigence realtime cell analysis (RTCA) technology to describe the in-vitro toxicity of ENPBCs. The xCELLigence is a +noninvasive in vitro toxicity monitoring process that mimics exact continuous cellular bioresponses in real-time settings. On the other hand, articles were also extracted from 2008 to 2016 describing the in vivo animal models toxicity of ENPBCs with regards to safety outcomes. Results: Out of 32 of the 121 (26.4%) articles identified from the literature, 23 (71.9%) met the in-vitro xCELLigence and 9(28.1%) complied with the in vivo animal model toxicity inclusion criteria. Of the 23 articles, 4 of them (17.4%) had no size estimation of ENPBCs. The xCELLigence technology provided information on cell interactions, viability, and proliferation process. Eighty-three (19/23) of the in vitro xCELLigence technology studies described ENPBCs as nontoxic or partially nontoxic materials. The in vivo animal model provided further toxicity information where 1(1/9) of the in vivo animal model studies indicated potential animal toxicity while the remaining results recommended ENPPCs as potential candidates for drug therapy though with limited information on toxicity. Conclusion: The results showed that the bioimpacts of ENPBCs either at the in vitro or at in vivo animal model levels are still limited due to insufficient information and data. To keep pace with ENPBCs biomedical products and applications, in vitro, in vivo assays, clinical trials and long-term impacts are needed to validate their usability and uptake. Besides, more real-time ENPBCs-cell impact analyses using xCELLigence are needed to provide significant data and information for further in vivo testing. [ABSTRACT FROM AUTHOR]

Microorganisms grown in biofilms are more resistant to antimicrobial treatment and immune system attacks compared to their planktonic forms. In fact, infections caused by biofilm-forming Staphylococcus aureus and Staphylococcus epidermidis are a large threat for public health, including patients with medical devices. The aim of the current manuscript was to test the effect of dalbavancin, a recently developed lipoglycopeptide antibiotic, alone or in combination with compounds contributing to bacterial cell disaggregation, on staphylococcal biofilm formation and elimination. We used real-time impedance measurements in microtiter plates to study biofilm growth dynamics of S. aureus and S. epidermidis strains, in the absence or presence of dalbavancin, linezolid, vancomycin, cloxacillin, and rifampicin. Further experiments were undertaken to check whether biofilm-detaching compounds such as N -acetylcysteine (NAC) and ficin could enhance dalbavancin efficiency. Real-time dose–response experiments showed that dalbavancin is a highly effective antimicrobial, preventing staphylococcal biofilm formation at low concentrations. Minimum biofilm inhibitory concentrations were up to 22 higher compared to standard E -test values. Dalbavancin was the only antimicrobial that could halt new biofilm formation on established biofilms compared to the other four antibiotics. The addition of NAC decreased dalbavancin efficacy while the combination of dalbavancin with ficin was more efficient than antibiotic alone in preventing growth once the biofilm was established. Results were confirmed by classical biofilm quantification methods such as crystal violet (CV) staining and viable colony counting. Thus, our data support the use of dalbavancin as a promising antimicrobial to treat biofilm-related infections. Our data also highlight that synergistic and antagonistic effects between antibiotics and biofilm-detaching compounds should be carefully tested in order to achieve an efficient treatment that could prevent both biofilm formation and disruption. [ABSTRACT FROM AUTHOR]

Cytotoxicity of fluorescent carbon fluoroxide (CFO) nanoparticles (NPs) was studied in a label-free manner on several cancer and non-cancer cell lines. A direct cytotoxic effect of the CFO NPs was clearly observed by a suppression of cell proliferation. The real-time measurement of cell activities allowed to quantify the impact of the uptaken NPs on cell proliferation and after washout of the NPs from the cell culture medium. The results show more toxic effects of the CFO NPs on cancer than on non-cancer cell lines. The notion of NPs biocompatibility must be related to a maximum concentration value of the NPs acceptable for a given cell type. Furthermore, the cytotoxicity effects of NPs should be studied not only during their direct exposure to cells but also after their washout from the culture medium.

Abstract Background Local injections of anesthetics, NSAIDs, and corticosteroids for tendinopathies are empirically used. They are believed to have some cytotoxicity toward tenocytes. The maximal efficacy dosages of local injections should be determined. A commercial 2D microfluidic xCELLigence system had been developed to detect real-time cellular proliferation and their responses to different stimuli and had been used in several biomedical applications. The purpose of this study is to determine if human tenocytes can successfully proliferate inside xCELLigence system and the result has high correlation with conventional cell culture methods in the same condition. Methods First passage of human tenocytes was seeded in xCELLigence and conventional 24-well plates. Ketorolac tromethamine, bupivacaine, methylprednisolone, and betamethasone with different concentrations (100, 50, and 10% diluted of clinical usage) were exposed in both systems. Gene expression of type I collagen, type III collagen, tenascin-C, decorin, and scleraxis were compared between two systems. Results Human tenocytes could proliferate both in xCELLigence and conventional cell culture systems. Cytotoxicity of each drug revealed dose-dependency when exposed to tenocytes in both systems. Significance was found between groups. All the four drugs had comparable cytotoxicity in their 100% concentration. When 50% concentration was used, betamethasone had a relatively decreased cytotoxicity among them in xCELLigence but not in conventional culture. When 10% concentration was used, betamethasone had the least cytotoxicity. Strong and positive correlation was found between cell index of xCELLigence and result of WST-1 assay (Pearson’s correlation [r] = 0.914). Positive correlation of gene expression between tenocytes in xCELLigence and conventional culture was also observed. Type I collagen: [r] = 0.823; type III collagen: [r] = 0.899; tenascin-C: [r] = 0.917; decorin: [r] = 0.874; and scleraxis: [r] = 0.965. Conclusions Human tenocytes could proliferate inside xCELLigence system. These responses varied when tenocytes were exposed to different concentrations of ketorolac tromethamine, bupivacaine, methylprednisolone, and betamethasone. The result of cell proliferation and gene expression of tenocytes in both xCELLigence and conventional culture system is strongly correlated. Clinical relevance xCELLigence culture system may replace conventional cell culture, which made real-time tenocyte proliferation monitoring possible.

Objective: to evaluate the effect of NSAIDs eye drops with different preservatives on the viability of epithelial cells of human eyes in vitro. Materials and methods. The object of the study was four of the drug Broxinac®, Acular LS®, Diclo-F ®, Indocollyre®. As test-systems were used permanent cell lines of transformed conjunctival (Chang Conjunctiva, Clone 1-5c-4) and corneal (HCEC). The cytotoxicity of NSAID drops were assessed by the morphology and functional cells activity by the methods of phase contrast microscopy (PCM), MTT test and cell analysis in real time using the xCELLigence system. Results. It was found with MTT-test that cell lines HCEC are more sensitive to the cytotoxic action of the studied drugs than cell lines of Chang Conjunctiva, Clone 1-5C-4. The metabolic activity of conjunctiva cells in the presence of drugs, Acular LS® and Broxinac® was three times lower than in control; in the presence of drugs Indocollyre® and Diclo-F® — 20 times lower vs. the control. The metabolic activity of the cornea cells e in the presence of drugs, Acular LS® and Broxinac® was four times lower than in control; in the presence of drugs Indocollyre® and Diclo-F® viable cells were not identified. Drugs Indocollyre® and Diclo-F® has had a very high toxic effect on both cell types. The results of cell analysis xCELLIgence for both types of cells are consistent with the results of MTT test and in vivo observation of cells. Conclusion. Based on these data, there is the following gradation of NSAID eye drop’s cytotoxic potential: (reduce toxicity): Indocollyre® = Diclo-F® > Acular LS® = Broxinac®.

Background: Biofilm is a fundamental bacterial survival mode which proceeds through three main generalized phases: adhesion, maturation, and dispersion. Lactobacilli spp. (LB) are critical components of gut and reproductive health and are widely used probiotics. Evaluation of time-dependent mechanisms of biofilm formation is important for understanding of host-microbial interaction and development of therapeutic interventions. Time-dependent LB biofilm growth was studied in two systems: large biofilm output in continuous flow system (microfermenter (M), Institute Pasteur, France) and electrical impedance-based real time label-free cell analyzer (C) (xCELLigence, ACEA Bioscience Inc., San Diego, CA). L. plantarum biofilm growth in M system was video-recorded, followed by analyses using IMARIS software (Bitplane, Oxford Instrument Company, Concord, MA, USA). Additionally, whole genome expression and analyses of attached (A) and dispersed (D) biofilm phases at 24 and 48 h were performed. Results: The dynamic of biofilm growth of L. plantarum was similar in both systems except for D phases. Comparison of the transcriptome of A and D phases revealed, that 121 transcripts differ between two phases at 24 h. and 35 transcripts – at 48 h. of M growth. The main pathways, down-regulated in A compared to D phases after 24 h. were transcriptional regulation, purine nucleotide biosynthesis, and L-aspartate biosynthesis, and the upregulated pathways were fatty acid and phospholipid metabolism as well as ABC transporters and purine nucleotide biosynthesis. Four LB species differed in the duration and amplitude of attachment phases, while growth phases were similar. Conclusion: LB spp. biofilm growth and propagation area dynamic, time-dependent processes with species-specific and time specific characteristics. The dynamic of LB biofilm growth agrees with published pathophysiological data and points out that real time evaluation is an important tool in understanding growth of microbial communities. [ABSTRACT FROM AUTHOR]