Crystal Violet Test Research Articles

Xanthone-rich extract from Gentiana dinarica transformed roots and its active component norswertianin induce autophagy and ROS-dependent differentiation of human glioblastoma cell line

BackgroundGlioblastoma multiforme (GMB) is the most malignant of all brain tumors with poor prognosis. Anticancer potential of xanthones, bioactive compounds found in Gentiana dinarica, is well-documented. Transformation of G. dinarica roots with Agrobacterium rhizogenes provides higher xanthones accumulation, which enables better exploitation of these anticancer compounds. Hypothesis/PurposeThe aim of this study was to investigate antiglioma effect of three different G. dinarica extracts: E1—derived from untransformed roots, E2—derived from roots transformed using A. rhizogenes strain A4M70GUS, and E3—derived from roots transformed using A. rhizogenes strain 15834/PI. Further, mechanisms involved in anticancer potential of the most potent extract were examined in detail, and its active component was determined. MethodsThe cell viability was assessed using MTT and crystal violet test. Cell cycle analysis, the expression of differentiation markers, the levels of autophagy, and oxidative stress were analyzed by flow cytometry. Autophagy and related signaling pathways were assessed by immunoblotting. ResultsE3, in contrast to E1 and E2, strongly reduced growth of U251 human glioblastoma cells, triggered cell cycle arrest in G2/M phase, changed cellular morphology, and increased expression of markers of differentiated astrocytes (glial fibrillary acidic protein) and neurons (β-tubulin). E3 stimulated autophagy, as demonstrated by enhanced intracellular acidification, increased microtubule-associated light chain 3B (LC3-I) conversion to autophagosome associated LC3-II, and decreased level of selective autophagy target p62. Induction of autophagy was associated with Akt-dependent inhibition of main autophagy suppressor mammalian target of rapamycin (mTOR). Both genetic and pharmacological inhibition of autophagy suppressed the expression of differentiation markers, but had no effect on cell cycle arrest in E3-treated cells. E3 stimulated oxidative stress, and antioxidants vitamin E and N-acetyl cysteine inhibited autophagy and differentiation of E3-treated U251 cells. The most prevalent compound of E3, xanthone aglycone norswertianin, also arrested glioblastoma cell proliferation in G2/M phase and induced glioblastoma cell differentiation through induction of autophagy and oxidative stress. ConclusionThese results indicate that E3 and its main active component norswertianin may serve as a potential candidate for differentiation therapy of glioblastoma.

Chemical composition, anti-biofilm activity and potential cytotoxic effect on cancer cells of Rosmarinus officinalis L. essential oil from Tunisia

BackgroundRosmarinus officinalis L. from Tunisia, popularly known as rosemary, is of a considerable importance for its medicinal uses and aromatic value. The aim of this study was to examine the chemical composition of Rosmarinus officinalis essential oil (ROEO) and to evaluate its antibiofilm activity on biofilm-forming bacterium and its anticancer activity on cancer cell lines.MethodsThe chemical composition of Rosmarinus officinalis essential oil (ROEO) was analyzed by GC-MS and its antibacterial activity was evaluated by micro-dilution method. The antibofilm activity of ROEO was evaluated using the crystal violet test and the cytotoxicity activity was determined by the MTT assay.ResultsIn this research, thirty-six compounds were identified in ROEO using GC-MS analyses. The main components were 1,8-cineole (23.56%), camphene (12.78%), camphor (12.55%) and β-pinene (12.3%). The antibacterial activity of ROEO was evaluated by micro-dilution method. The oil exhibited inhibition and bactericidal effect against two strains: Staphylococcus aureus ATCC 9144 and Staphylococcus epidermidis S61. It was found that the minimum inhibitory concentration (MIC) obtained for S. aureus and S. epidermidis ranged from 1.25 to 2.5 and from 0.312 to 0.625 μl ml−1, respectively and the minimum bactericidal concentration (MBC) were in the order of 5 and 2.5 μl ml−1, respectively. Furthermore, this oil showed a S. epidermidis biofilm inhibition more than 57% at a concentration of 25 μl ml−1. The eradication of 67% of the established biofilm was observed at a concentration of 50 μl ml−1 of ROEO, whereas the dose of 25 μl ml−1 removed only 38% of preformed biofilm. ROEO strongly inhibited the proliferation of Hela and MCF-7 cells with IC50 values of 0.011 and 0.253 μl ml−1, respectively.ConclusionOur results demonstrate that ROEO could have a potential role in the treatment of diseases related to infection by microorganisms or proliferation of cancer cells.

Phytochemical and Bioactive Potential of in vivo and in vitro Grown Plants of Centaurea ragusina L. - Detection of DNA/RNA Active Compounds in Plant Extracts via Thermal Denaturation and Circular Dichroism.

The phytochemical composition and biological activity of non-volatile components of Centaurea ragusina L. has not been studied previously. Our aim was to evaluate the phytochemical and bioactive potential (including interactions with polynucleotides) of C. ragusina L. depending on the origin of plant material (in vivo - leaves from natural habitats, ex vitro - leaves from plants acclimated from culture media, in vitro - leaves and calli from plants grown in culture media) and polarity of solvents used in extract preparation (80 and 96% ethanol and water combinations or single solvents). The polyphenol composition was determined by spectrophotometric and HPLC analysis. Biological activity of extracts was evaluated by following methods: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods for antioxidative activity, 2,3,5-triphenyl tetrazolium chloride (TTC) microdilution method for antibacterial activity, crystal-violet test for cytotoxic activity and thermal denaturation (TD) and circular dichroism (CD) for DNA/RNA interactions. Conditions for the most efficient polyphenol extraction were determined: the 80% ethanol/water solvent system was the most suitable for callus and leaf ex vitro samples and 80 or 96% ethanol for leaf in vivo samples. Significantly higher levels of chlorogenic acid and naringenin were detected in callus tissue than in vivo plant. Ethanolic extracts exhibited the significant antibacterial activity against Staphylococcus aureus ATCC 25923. DNA/RNA active compounds in plant extracts were detected by TD and CD methods. Callus tissue and ex vitro leaves represent a valuable source of polyphenols as in vivo leaves. TD and CD can be applied for detection of DNA/RNA active compounds in extracts from natural resources. Copyright © 2017 John Wiley & Sons, Ltd.

The Cytoprotective Effect of Crocin on Oxaliplatin Treated VERO cell line In Vitro Study

AbstractBackgroundCancer is a disease characterized by a shifted in the control mechanism that governs cell survival, proliferation and differentiation.There are many types of treatment to the cancer like surgery, radiation, chemotherapy, immunotherapy and others. Each of these above treatments has its indication effect and has Sid effect and / or adverse effect. The chemotherapy is one type of this treatment to the cancer that have the indication effect, side effect and adverse effect. There are many type of chemotherapeutic agent that used for this type of cancer one of them it is platinum compound (cisplatin, oxaliplatin and others).There are two types of limitation in the use of these agents one of them is the side effect which is neurotoxicity, Ototoxicity, nephrotoxicity, and etc. and the second one is the resistance of cancer cells to these agents.Aim of the studyTaste the cytoprotection effect of Crocin on oxaliplatin treated VERO cell lineMaterial and methods:-By using the tissue culture techniques which is a newly used techniques to check the cytotoxicity of many compound and the expected mechanism of action these techniques made a good jump in the research in many fieldIn this study we use VERO cell line and after exposure of this cell line firstly to different concentrations of oxaliplatin ( 1, 0.5, 0.25 and 0.125 mg/ml) for 48hr, secondly to different concentrations of Crocin (0.5, 0.25 and 0.125 mg/ ml ) for 48hr. And thirdly to mix concentrations of oxaliplatin and Crocin in 48hr for the VERO cell line and measure the cytotoxicity of these exposures by crystal violet test (C.V. test) to show and compare the effect of these agents specially the Crocin.The oxaliplatin has cytotoxic effect, its inhibitory concentration 50% (IC50) in 48hr is (0.031mg/ml) which is the maximum cytotoxic effect in vitro. While the Crocin don’t have cytotoxic effect at this tested concentration and for this cell line. After mixed different concentration of Crocin with different concentration of oxaliplatin the cytotoxicity of oxaliplatin reduce significantly.Conclusion:-From our study we can conclude the following:-The Crocin has cytoprotective effect. The Crocin has no anti tumor and anti proliferative activity within these tasted concentration and this cell line

Virulence factors of fluconazole-susceptible and fluconazole-resistant Candida albicans after antimicrobial photodynamic therapy

This study evaluated the effects of antimicrobial photodynamic therapy (aPDT) mediated by Photodithazine® (PDZ) and LED light on the virulence factors of fluconazole-susceptible (CaS) and fluconazole-resistant (CaR) Candida albicans. Standardized suspensions of strains were prepared (107), and after 48h of biofilm formation, these strains were incubated with PDZ (100mg/L) for 20min and exposed to LED light (660nm, 37.5J/cm2). Additional samples were treated with PDZ or light only, and the control consisted of biofilms that received no treatment. After aPDT, the cells were recovered and the virulence factors were evaluated. To analyze the capacity of adhesion, cells were recovered after aPDT and submitted to the adhesion process in the bottom of a 96-well plate. After this, metabolic activity tests (XTT assay) and cell viability (colony forming units per milliliter, CFU/mL) were applied. To evaluate the biofilm-forming ability after aPDT, the cells recovered were submitted to biofilm formation procedures, and the biofilm formed was evaluated by XTT, CFU/mL, and total biomass (crystal violet) tests. Lastly, the capacity for synthesizing protease and phospholipase enzymes after aPDT was evaluated by fluorimetric tests. Data were analyzed by two- or three-way ANOVA tests (p ≤ 0.05). It was verified that aPDT reduced the viability of both strains, fluconazole-susceptible and fluconazole-resistant C. albicans. It was also observed that the CaR strain had lower susceptibility to the aPDT when compared with the CaS strain. However, regarding the virulence factors evaluated, it was demonstrated that aPDT did not alter the adherence and biofilm formation ability and enzymatic production.

Microwave synthesis of functionally graded tricalcium phosphate for osteoconduction

Porosity is an important parameter of biomaterials used to replace bone. Crystalline tricalcium phosphate (TCP) is a common ceramic used to substitute bone in small quantities and coat medical devices. Using microwave combustion synthesis (MCS) to provide volumetric heating of the reactant materials, along with varied urea to calcium phosphate concentration ratios, a porosity structure is synthesized. The microwave power provided 145W and the reactants are exposed for 5min. Results from in-situ IR thermal analysis show heating in two phases: initially (i.e., first 30s) thermal energy is localized along the surface of the sample followed by (i.e., remaining 5min) volumetric heating and MCS. The product material is confirmed to be tricalcium phosphate via FTIR analysis. Scanning electron microscopy (SEM) images show increased porosity with increased urea concentration. By controlling the concentration gradient in the reactant sample, the product material produced a tailored porosity gradient. Moreover, the MCS tricalcium phosphate is demonstrated to be biocompatible and does not affect cell proliferation by crystal violet tests. This study provides a new perspective on volumetric combustion synthesis by using weak microwave energy to facilitate the formation of a tailored TCP structure.

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The percentage of cancer-related deaths attributable to diet and tobacco globally is reported to be as high as 60-70%. A large number of dietary compounds have gained research interest and tested to determine their chemopreventive / antiproliferative effects on in-vivo/in-vitro models. The present study effect of flavonoid fisetin on proliferation of human lung carcinoma cells A549 and NCI-H460 was investigated. The effect of fisetin on cell growth was investigated by MTT, Crystal violet, XTT, LDH release, and colony forming assays. Cell proliferation was assessed by BrdU incorporation test. Further, cell morphology and cytopathology was analyzed by crystal violet test and hematoxylin and eosin staining. Results of study revealed that fisetin treatment exerted a dose dependent decrease in the growth of A549 and NCI-H460 cells with IC50 values of 190µM and 210µM respectively. It also inhibited cell proliferation and colony formation in both A549 and NCI-H460 cells. Cytopathology studies exhibited features such as nuclear fragmentation, cytoplasmic vacuolation, karyorrhexis, chromatin condensation, which were suggestive of apoptotic cell death. Results of the study indicate growth inhibitory, anti-proliferative and pro-apoptotic effects of fisetin on lung carcinoma cells.

Attenuation of 7-ketocholesterol-induced overproduction of reactive oxygen species, apoptosis, and autophagy by dimethyl fumarate on 158 N murine oligodendrocytes

Mitochondrial dysfunctions and oxidative stress are involved in several non demyelinating or demyelinating neurodegenerative diseases. Some of them, including multiple sclerosis (MS), are associated with lipid peroxidation processes leading to increased levels of 7-ketocholesterol (7KC). So, the eventual protective effect of dimethylfumarate (DMF), which is used for the treatment of MS, was evaluated on 7KC-treated oligodendrocytes, which are myelin synthesizing cells. To this end, murine oligodendrocytes 158N were exposed to 7KC (25, 50μM) for 24h without or with DMF (1, 25, 50μM). The biological activities of DMF associated or not with 7KC were evaluated by phase contrast microscopy, crystal violet and MTT tests. The impact on transmembrane mitochondrial potential (ΔYm), O2− and H2O2 production, apoptosis and autophagy was measured by microscopical and flow cytometric methods by staining with DiOC6(3), dihydroethidine and dihydrorhodamine 123, Hoechst 33342, and by Western blotting with the use of specific antibodies raised against uncleaved and cleaved caspase-3 and PARP, and LC3-I/II. DMF attenuates the different effects of 7KC, namely: cell growth inhibition and/or loss of cell adhesion, decrease of ΔΨm, O2− and H2O2 overproduction, PARP and caspase-3 cleavage, nuclear condensation and fragmentation, and activation of LC3-I into LC3-II. The ability of DMF to attenuate 7KC-induced reactive oxygen species overproduction, apoptosis, and autophagy on oligodendrocytes reinforces the interest for this molecule for the treatment of MS or other demyelinating diseases.

Evidence of hormesis on human neuronal SK-N-BE cells treated with sodium arsenate: impact at the mitochondrial level.

Exposure of human neuronal SK-N-BE cells to sodium arsenate (AsV 0.1-400μM; 48h) induced a biphasic toxic effect evoking hormesis. Indeed, at low concentrations, AsV stimulates cell proliferation visualized by phase contrast microscopy, whereas at high concentrations, an induction of cell death associated with a loss of cell adhesion was observed. These side effects were confirmed with crystal violet test, cell cycle analysis, evaluation of the percentage of Ki67 positive cells, and staining with propidium iodide. The impact of AsV on mitochondrial functions, which was determined by the MTT assay, the measurement of mitochondrial transmembrane potential with DiOC6(3), and the rate of mitochondrial ATP, also support an hormesis process. In addition, in the presence of high concentrations of AsV, a significant decrease of the protein expression of OXPHOS complexes of the respiratory chain was observed by western blot supporting that AsV-induced cell death is associated with mitochondrial alterations. Therefore, there are some evidences of hormesis on AsV-treated SK-N-BE cells, and at high concentrations, the mitochondria are a target of toxicity induced by AsV.

Pb and Hg heavy metal tolerance of single- and mixedspecies biofilm (Rhodotorula mucilaginosa and Escherichia coli)

The aim of this study was to examine heavy metal tolerance (lead (Pb2+) and mercury (Hg2+)) of single- and mixed-species biofilms, formed by yeast Rhodotorula mucilaginosa and bacteria Escherichia coli LM1. Single- and mixed-species biofilms were quantified by crystal violet test and the absorbance was measured using microplate reader (OD570). The minimal inhibitory concentration (MIC) and the minimal lethal concentration (MLC) were determined and the results were confirmed by fluorescence microscopy. The significant difference in lead tolerance was observed between the mixed- and the single-species biofilms. The MIC of lead (Pb2+) for the examined biofilms (E. coli LM1, R. mucilaginosa and R. mucilaginosa / E. coli) was recorded at concentrations of 4000 μg/ml, 4000 μg/ml and 16000 μg/ml, respectively. The MIC of mercury (Hg2+) for the biofilms was noticed at concentrations of 31.25 μg/ml, 250 μg/ml and 250 μg/ml, respectively. Standard antibiotics (amphotericin B and tetracycline) were used as positive control. Results obtained for single-species biofilms were compared in between and with the results obtained for mixed-species biofilm. The tolerance of the mixed- species biofilm was higher in comparison to the singlespecies biofilms and the results were confirmed by a fluoresecence microscope. The obtained results suggest that the R. mucilaginosa / E. coli biofilm may have a potential to be used in bioremediation of wastewaters contaminated with lead and mercury.

Prospective study on the diagnostics and susceptibility testing of infections associated with osseointegrated percutaneous implants

Event Abstract Back to Event Prospective study on the diagnostics and susceptibility testing of infections associated with osseointegrated percutaneous implants Magdalena Zaborowska1, 2, Rickard Brånemark2, 3, 4, Joakim Strömberg3, Peter Thomsen1, 2 and Margarita Trobos1, 2 1 Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Department of Biomaterials, Sweden 2 University of Gothenburg, BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Sweden 3 Center for Advanced Reconstruction of Extremities, Sahlgrenska University Hospital, Department of Orthopaedics, Sweden 4 Department of Orthopaedics, University of California, International Center for Osseointegration Research Education and Surgery (iCORES), United States Introduction: Biomaterial-associated infections (BAI) are among the main reasons for implant failure, often leading to long-term antimicrobial treatment and implant removal. BAI are mainly caused by biofilm producing bacteria with increased antimicrobial resistance. Proper and rapid diagnosis is important for successful treatment. Sonication is an alternative method for the diagnosis of prosthetic infections with higher sensitivity than standard tissue cultures[1]. The aims of the study were to: i) identify and quantify bacteria causing osteomyelitis associated with osseointegrated percutaneous amputation prostheses by sonication of explanted implants; ii) characterise the isolated strains by their biofilm production abilities and antimicrobial susceptibility. In parallel, the diagnostic laboratory at the hospital performed standard culturing techniques of bone tissue and blood marrow samples. Materials and Methods: A bone-anchored part (fixture) of a prosthesis was extracted due to suspicion of infection according to clinical evaluation. The retrieved fixture was placed in sterile saline, sonicated at 47 KHz for 5 min and vortexed for 1 min. The implant was removed, the remaining solution was centrifuged, pellet was re-suspended in saline and quantitative cultures were made on general purpose agar (blood agar for aerobic and brucella agar for anaerobic cultures) and selective agar plates (staphylococci agar, enterococci agar, streptococci agar, chromogenic orientation agar and pseudomonas agar). Crystal violet (CV) staining and Congo Red (CR) agar plate test was performed to evaluate the biofilm production. Identification of the staphylococcal strains was performed using API Staph. The Calgary Biofilm Device and a custom-made antimicrobial susceptibility plate were used to determine minimum biofilm eradication concentrations (MBEC) and minimum inhibitory concentrations (MIC) to 10 antimicrobial agents. Results and Discussion: We report the first results of an ongoing prospective study where the method has been tested real-time. In the first patient, Enterococcus faecalis (5x104 CFU/implant) and Escherichia coli (102 CFU/implant) were detected after sonication. These strains were both biofilm-producing strains according to CV and CR tests. Biofilms showed increased tolerance to the antimicrobials compared with the standard planktonic MIC values (Table 1). Escherichia coli was the only species detected by standard tissue cultures; in contrast, by the use of sonication, Enterococcus faecalis was further identified indicating the possibility of a polymicrobial infection with potential implications for the choice of the optimal treatment strategy for the patient. Conclusions: An increased detection of the potential causative agents by sonication of retrieved implants may improve the current diagnostics of BAI. In addition, determination of MBEC for clinically relevant antimicrobial agents has the potential to optimize the treatment choices for patients. The BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, the Västra Götaland Region, the Swedish Research Council (K2015-52X-09495-28-4), an LUA/ALF Research Grant (ALFGBG-138721), the IngaBritt and Arne Lundberg Foundation, the Wilhelm and Martina Lundgren Foundation, the Handlanden Hjalmar Svensson Foundation, Doctor Felix Neubergh Foundation and the Area of Advance Materials of Chalmers and GU Biomaterials within the Strategic Research Area initiative launched by the Swedish Government

Upregulation of cugbp2 increases response of pancreatic cancer cells to chemotherapy.

Altered expression and/or function of ribosomal RNA (rRNA)-binding proteins CUGBP2/CELF2 might influence post-transcriptional regulation of the HO-1- and COX-2-mediated cytoprotective pathways and represents an important therapeutic target. The aim of this study was to assess the effects of CUGBP2-mediated post-transcriptional regulation of COX-2 and HO-1 in pancreatic cancer cells in regard of response to gemcitabine (GEM) treatment. Expression of CUGBP2, COX-2, and HO-1 was evaluated using qRT-PCR and Western blot methods. Cell viability after treatment with GEM and/or curcumin and siCUGBP2 was evaluated using MTT and crystal violet tests. RNA immunoprecipitation analysis was used to confirm COX-2 and HO-1 post-transcriptional regulation by CUGBP2 protein. CUGBP2 expression at the messenger RNA (mRNA) level was 2.2-fold lower (p = 0.007), but HO-1 and COX-2 expression was increased 6.9- (p = 0.023) and 2.3- (p = 0.046) fold in pancreatic cancer tissues. The median survival of patients with low CUGBP2 expression from the lowest tercile was 13.8 months. The median survival of patients in terciles of middle and high CUGBP2 expression levels was 21.9 month (p = 0.123). Induction of CUGBP2 expression by curcumin resulted in the downregulation of HO-1 and COX-2 and strongly sensitized tumor cells to GEM treatment. However, CUGBP2 silencing upregulated HO-1 and COX-2 protein expression and had a high effect on cells viability. Decreased activity of CUGBP2 could be associated with high chemoresistance and early dissemination of pancreatic cancer through the HO-1- and COX-2-mediated cytoprotective and carcinogenesis pathways. Curcumin significantly increased the effectiveness of GEM treatment in vitro via the CUGBP2-mediated post-transcriptional regulation pathway.

Heavy metals produce toxicity, oxidative stress and apoptosis in the marine teleost fish SAF-1 cell line

The use of cell lines to test the toxicity of aquatic pollutants is a valuable alternative to fish bioassays. In this study, fibroblast SAF-1 cells from the marine gilthead seabream (Sparus aurata L.) were exposed for 24 h to the heavy metals Cd, Hg, MeHg (Methylmercury), As or Pb and the resulting cytotoxicity was assessed. Neutral red (NR), MTT-tetrazolio (MTT), crystal violet (CV) and lactate dehydrogenase (LDH) viability tests showed that SAF-1 cells exposed to the above heavy metals produced a dose-dependent reduction in the number of viable cells. Methylmercury showed the highest toxicity (EC50 = 0.01 mM) followed by As, Cd, Hg and Pb. NR was the most sensitive method followed by MTT, CV and LDH. SAF-1 cells incubated with each of the heavy metals also exhibited an increase in the production of reactive oxygen species and apoptosis cell death. Moreover, the corresponding gene expression profiles pointed to the induction of the metallothionein protective system, cellular and oxidative stress and apoptosis after heavy metal exposure for 24 h. This report describes and compares tools for evaluating the potential effects of marine contamination using the SAF-1 cell line.

Biofilm Forming Ability Of Salmonella Enteritidis In Vitro

Abstract Salmonella enterica serotype Enteritidis is an important alimentary pathogen that recently gained special attention due to the ability of a large number of strains to form biofilms. Qualitative testing of biofilm forming ability was performed by observing the morphotype of the colonies on Congo Red agar and by conducting the pellicle test, while quantitative testing was carried out by Cristal violet assay on microtiter plates. A total of 14 isolates of S. Enteritidis were tested for biofilm forming ability, while Salmonella Enteritidis ATTC 13076 was used as the reference strain. Based on the morphotype of colonies cultivated on Congo Red agar at 25°C incubation temperature, among tested isolates three morphotypes were detected – red, dry and rough (rdar), brown, dry and rough (bdar) and smooth and white (saw). Half of the tested isolates demonstrated rdar morphotype. All isolates that showed a specific morphotype at this incubation temperature also formed the corresponding type of pellicle at the air-liquid interface. Additionally, comparing OD (optical density) values obtained by crystal violet test between groups of isolates that represent one of the three detected morphotypes (rdar, bdar and saw), statistically significant differences were detected. Based on OD values obtained by crystal violet test at both applied incubation temperatures, isolates were classified into three categories, regarding their ability to form biofilms: strong, moderate and weak biofilm producers. By comparing the amounts of the biofilms formed after 48h at 25°C and 37°C, statistically significant differences were noted (P<0.05). In this research we presented micrographs and a reconstruction of three-dimensional projections of biofilm developing phases of rdar morphotype isolates, which were obtained using confocal laser scanning microscopy.

Protective effect of 14-3-3 antibodies on stressed neuroretinal cells via the mitochondrial apoptosis pathway.

BackgroundPrevious studies demonstrate changes of autoantibody concentrations against retinal and optic nerve head antigens in the serum of glaucoma patients in comparison to healthy persons. These antibodies belong to the natural autoimmunity. Previous studies showed up regulated, but also significantly down-regulated autoantibody levels. These antibodies have the ability to influence protein profiles of neuroretinal cells and possibly hold neuroprotective potential, as we have been able to demonstrate before. Aim of this study was to analyse the serum and antibody effect of glaucoma patients on neuroretinal cells in more detail and also determine the impact of antibodies found down-regulated in glaucoma patients on the pathogenesis of the neurodegenerative disease glaucoma.MethodsNeuroretinal cells (RGC-5) were incubated with serum either from glaucoma patients or healthy controls for 24 h. Mass spectrometric analysis was performed after cell lysis. Furthermore the neuroretinal cells were preincubated with different and concentrations of 14-3-3 antibodies (0.005, 0.1, 0.5, 1, 5 and 10 μg/ml) and then stressed with H2O2, staurosporine or glutamate. Viability tests were performed with crystal violet and ROS tests with DCFH-DA. Antibody location in the cell after antibody incubation was performed with immunoccytochemical methods. Additionally mass spectrometric analysis was performed with the cells after antibody incubation.ResultsProtein expression analysis with Maldi-Orbitrap MS showed changes in the expression level of regulatory proteins in cells incubated with glaucoma serum, e.g. an up-regulation of 14-3-3 and a down-regulation of Calmodulin. After preincubation of the cells with anti-14-3-3 antibody and stressing the cells, we detected an increase in viability of up to 22 % and a decrease in reactive oxygen species (ROS) of up to 31 %. Proteomic 1 analysis involvement of the mitochondrial apoptosis pathway in this protective effect and immunohistochemical analysis showed an antibody uptake in the cells.ConclusionWe found significant effects of serum antibodies on proteins of neuroretinal cells especially of the mitochondrial apoptosis pathway. Furthermore we detected a protective potential of antibodies down-regulated in glaucoma patients. The changed autoantibodies belong to the natural autoimmunity. We conclude that changes in the natural autoimmunity of patients with glaucoma can negatively impact regulatory functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12886-015-0044-9) contains supplementary material, which is available to authorized users.

An expeditious synthesis of spinasterol and schottenol, two phytosterols present in argan oil and in cactus pear seed oil, and evaluation of their biological activities on cells of the central nervous system

Spinasterol and schottenol, two phytosterols present in argan oil and in cactus pear seed oil, were synthesized from commercially available stigmasterol by a four steps reactions. In addition, the effects of these phytosterols on cell growth and mitochondrial activity were evaluated on 158N murine oligodendrocytes, C6 rat glioma cells, and SK-N-BE human neuronal cells with the crystal violet test and the MTT test, respectively. The effects of spinasterol and schottenol were compared with 7-ketocholesterol (7KC) and ferulic acid, which is also present in argan and cactus pear seed oil. Whatever the cells considered, dose dependent cytotoxic effects of 7KC were observed whereas no or slight effects of ferulic acid were found. With spinasterol and schottenol, no or slight effects on cell growth were detected. With spinasterol, reduced mitochondrial activities (30–50%) were found on 158N and C6 cells; no effect was found on SK-N-BE. With schottenol, reduced mitochondrial activity were revealed on 158N (50%) and C6 (10–20%) cells; no effect was found on SK-N-BE. Altogether, these data suggest that spinasterol and schottenol can modulate mitochondrial activity and might therefore influence cell metabolism.

Evaluation of the photobiomodulation in L929 cell culture.

Low-level laser therapy has become an important tool for bio-modulation process. It can induce stimulatory or inhibitory effects according to cell behavior at specific irradiation. Our objective was to determine L929 cell line response to irradiation at λ 685 and 830 nm, concentrations of 5 and 10% fetal bovine serum and different energy densities of 0.1, 0.5, 1, 2, 3, 5, 7, 10, 20, and 30 J/cm(2). Thus, cells were plated at 1 × 10(5) cells/mL and irradiated with semiconductor laser As-Ga-Al. Twenty-four hours after irradiation, cells were subjected to MTT, neutral red, crystal violet tests, and cell staining was performed using the kit Alexa Fluor 488 Annexin V-FITC and propidium iodide. Our results showed that low-level laser therapy stimulates effect when the energy density is 5 to 3030 J/cm(2) and inhibits effects on energy density 0.1 to 3 J/cm(2). This inhibitory effect was evidenced by the absence of dead cells labeled, decreased cell density, and by the absorption of neutral red in intact cells. The study also demonstrated that fetal bovine serum, at different concentrations, did not affect response of the cells after irradiation.

Temperature and pH affect the production of bacterial biofilm

The effect of different cultivation temperatures (30 and 37 degrees C) and pH of the media (5.5, 7.5, 8.5) on the biofilm production was compared in Pseudomonas aeruginosa, Klebsiella pneumoniae, and Vibrio cholerae non-O1 and O1 using the crystal-violet test for estimation of quantitative production of the biofilm. Decrease (46.4-98.4 %) in the biofilm production was observed at 37 degrees C in 8 of the tested strains (P. aeruginosa three strains, K pneumoniae two, V. cholerae non-O1 two, and V. cholerae O1 one strain) compared with the production at 30 degrees C. On the other hand, five strains (P. aeruginosa 1, K. pneumoniae 3, V. cholerae non-O1 1) exhibited under these conditions a higher biofilm production (103-143 %). However, this difference was not significant (p = 0.196). Increased pH lead to a higher biofilm production using all media tested. In P. aeruginosa the biofilm production at pH 8.5 was 139-244 %, at pH 7.5 136-164 % in comparison with pH 5.5. Similarly, in K. pneumoniae the biofilm production increased to 151-319 % at pH 8.5 while with the drop of pH to 7.5 the biofilm production was 113-177 % compared with pH 5.5. In V. cholerae non-O1 and O1 the biofilm production reached 204-329 % at pH 8.5, and 123-316 % at pH 7.5 (compared with the production at pH 5.5). An increase in biofilm production represented an average of 169 % (p = 0.001) at pH change from 5.5 to 7.5, with the rise of pH from 5.5 to 8.5 caused an average difference of 229 % (p = 0.001).

Bradykinin protects against brain microvascular endothelial cell death induced by pathophysiological stimuli

The morphological and functional integrity of the microcirculation is compromised in many cardiovascular diseases such as hypertension, diabetes, stroke, and sepsis. Angiotensin converting enzyme inhibitors (ACEi), which are known to favor bradykinin (BK) bioactivity by reducing its metabolism, may have a positive impact on preventing the microvascular structural rarefaction that occurs in these diseases. Our study was designed to test the hypothesis that BK, via B2 receptors (B2R), protects the viability of the microvascular endothelium exposed to the necrotic and apoptotic cell death inducers H(2)O(2) and LPS independently of hemodynamics. Expression (RT-PCR and radioligand binding) and functional (calcium mobilization with fura-2AM, and p42/p44MAPK and Akt phosphorylation assays) experiments revealed the presence of functional B2R in pig cerebral microvascular endothelial cells (pCMVEC). In vitro results showed that the cytocidal effects of H(2)O(2) and LPS on pCMVEC were significantly decreased by a BK pretreatment (MTT and crystal violet tests, annexin-V staining/FACS analysis), which was countered by the B2R antagonist HOE 140. BK treatment coincided with enhanced expression of the cytoprotective proteins COX-2, Bcl-2, and (Cu/Zn)SOD. Ex vivo assays on rat brain explants showed that BK impeded (by approximately 40%) H(2)O(2)-induced microvascular degeneration (lectin-FITC staining). The present study proposes a novel role for BK in microvascular endothelial protection, which may be pertinent to the complex mechanism of action of ACEi explaining their long-term beneficial effects in maintaining vascular integrity.

Expression of Fucosyltransferases Contributes to Melanoma Invasive Phenotype

During carcinogenesis aberrant N-glycosylation may lead to the development of subpopulations of tumor cells with altered adhesion properties and increased invasive potential. Biosynthesis of glycans and oligosaccharides is tissue-specific and developmentally regulated by number of glycosyltransferases of which fucosyl-, sialyl- and N-acetylglucosaminyltransferases often participate in synthesis of tumor type glycans. We analyzed the expression of selected glycosyltransferases (real-time PCR): fucosyltransferases FUT-1 and FUT-4, sialyltransferase SIAT4C and beta 1,6-N-acetylglucosaminyltransferase V (MGAT-5), in human melanoma cell lines: WM35 from primary tumor site and WM239, WM9, A375 from metastatic sites. In parallel their proliferation (crystal violet test) and adhesion to fibronectin and collagen IV (BD Biocoat assay) was assessed. Examined cell lines showed expression of all studied glycosyltransferases. The level of expression of fucosyltransferases was significantly higher in melanoma cell lines from metastatic site than from primary cell line: mRNA expression of FUT-1 was 100 times higher in A375 melanoma cell line from metastatic site (A375, solid tumor) than in WM35 primary cell line. The expression of FUT-4 in cell lines from metastatic sites: WM9 (lymph node) and WM239 (skin) was respectively 80 and 37 times higher than in WM 35 primary cell line. In all melanoma cell lines very low expression of MGAT-5 and high expression of SIAT4C was observed. Melanoma cells bound both to fibronectin and to collagen IV. LTA (Lotus tetragonolobus agglutinin), the lectin that specifically recognizes fucose residue of glycans and 20mM L-fucose by itself significantly reduced adhesion of all studied cell lines, both primary and metastatic, to fibronectin (20-50 %) and to collagen IV (20-50 %). In addition LTA reduced the proliferation (20-30 %) of metastatic cell lines (A375, WM9, WM239) and did not affect the growth of primary cell line (WM35). The results suggest that higher expression of fucosyltransferases (FUT-1, FUT-4) might be an important step in the formation of surface structures that facilitate metastasis of melanoma.