24-well Microplates Research Articles

A novel microplate-based spectrophotometric method for the quantitative assessment of freshwater bacterial coaggregation kinetics

Coaggregation, the specific recognition and adhesion of genetically distinct bacteria, is proposed to contribute to the development of freshwater biofilms. This work aimed to develop a microplate-based system to measure and model the kinetics of freshwater bacterial coaggregation. Blastomonas natatoria 2.1 and Micrococcus luteus 2.13 were evaluated for coaggregation ability using 24-well microplates containing novel dome shaped wells (DSWs) and standard flat-bottom wells. Results were compared to a tube-based visual aggregation assay. The DSWs facilitated the reproducible detection of coaggregation via spectrophotometry and the estimation of coaggregation kinetics using a linked mathematical model. Quantitative analysis using DSWs was more sensitive than the visual tube aggregation assay and subject to substantially less variation than flat-bottom wells. Collectively these results demonstrate the utility of the DSW-based method and improve upon the current toolkit for studying freshwater bacterial coaggregation.

The effect of ethyl acetate fraction of Marsilea crenata presl. leaves in increasing osterix expression in hFOB 1.19 cells

Marsilea crenata Presl. leaves contain phytoestrogens that is suspected to play a role in increasing bone formation. Bone formation activity is defined by the expression of Osterix, a transcription factor that plays a key role in bone development. The aim of this study is to show that the ethyl acetate fraction of Marsilea crenata Presl. leaves can increase bone formation process in hFOB 1.19 cells in a TNF-α dependent manner, by measurement of Osterix. The hFOB 1.19 cells were grown in 24-well microplates and treated with 10 ng/ml TNF-α for 24 hours. The ethyl acetate fraction of Marsilea crenata Presl. leaves were also added in concentrations of 62.5, 125, and 250 ppm. A positive control was employed, at a concentration of 2.5 g/L of genistein. The expression of osterix was examined using an immunocytochemistry technique with CLSM to assess bone forming activity. The results show that ethyl acetate fraction Marsilea crenata Presl. leaves can boost osterix expression in hFOB 1.19 cells, with optimal concentration was 250 ppm at p<0.005. The ethyl acetate fraction of Marsilea crenata Presl. leaves can increase Osterix in osteoblast hFOB 1.19 cell, indicating improved bone forming activity.

Plant extract incorporated into glass ionomer cement as a photosensitizing agent for antimicrobial photodynamic therapy on Streptococcus mutans

BackgroundA plant extract (EB) incorporated into glass ionomer cement (GIG) could be a potential photosensitizer for Antimicrobial PDT (aPDT) against caries-microorganisms, replacing methylene blue (MB), due to the presence of chlorophyll. GIC + EB + aPDT could be an therapeutic alternative to dentin decontamination and sealing, allowing reduction of operative time. ObjectiveEvaluate Dioscorea altissima (EB) incorporated into GIC as a photosensitizer for aPDT against Streptococcus mutans. MethodsGroups (n = 24; ntotal = 192): G1-GIC; G2-GIC + LASER; G3-GIC/EB; G4-GIC/EB + LASER; G5-GIC+MB; G6-GIC + aPDT; G7-GIC/EB + MB; and G8 – GIC/EB+aPDT. In aPDT groups, MB was the photosensitizer. In LASER groups, MB was not used. The irradiation protocol was 660 nm/100 mW/5 J/150 J/cm²/50 s, with a 5-min pre-irradiation time for the MB groups. Antibacterial assays were carried out in 24-well microplates. The wells were completed with one milliliter of a S. mutans in BHI at 1.3 × 108 CFU/mL suspension. After incubation, PDT or laser was performed. After MTT bacteria viability test, the data were submitted to the Kolgomorov-Smirnoff normality test, followed by one-way ANOVA and Tukey's posterior test, α < 0.05. ResultsGroup G6 showed significant inhibition (p < 0.001), followed by groups G4, G5, G7, and G8, which did not show significant differences among them (p > 0.05). Groups G2 and G3 also showed similar results (p > 0.05) and were the least active compared to the others. ConclusionsEB potentiated the antimicrobial action of GIC against S. mutans and laser irradiation over GIC/EB presented better antimicrobials results. The results indicate that EB could be a potential photosensitizer for aPDT.

IN VITRO ANTI-NEUROINFLAMMATORY EFFECT OF GENISTEIN (4',5,7-TRIHYDROXYISOFLAVONE) ON MICROGLIA HMC3 CELL LINE, AND IN SILICO EVALUATION OF ITS INTERACTION WITH ESTROGEN RECEPTOR-β

Objective: This study was aimed to evaluate the role of genistein or 4',5,7-trihydroxyisoflavone as a phytoestrogen in the treatment of estrogen deficiency-induced neuroinflammatory. The specific objectives of this study were to determine the anti-neuroinflammatory effect of genistein through measurement of MHC II and Arg1 expressions on microglia HMC3 cell line, as well as to prove that the effect occurs in an ER-dependent manner, through the measurement of free-ERβ expression.&#x0D; Methods: The cells were cultured in 24-well microplates, induced with 10 ng IFN-γ, and incubated for 24 h to activate the cell to M1 phenotype which has pro-inflammatory characteristics. Genistein with a concentration of 50 μM was added to the cells. The expression of MHC II, Arg1, and free-ERβ as markers was tested through an immunocytochemistry method and measured using the CLSM instrument. In silico approach was also conducted to determine the interaction between genistein and ERβ, compared to 17β-estradiol. Genistein structure was prepared with Avogadro 1.0.1, and molecular docking was done using PyRx 0.8 software. Biovia Discovery Studio Visualizer 2016 was used to visualize the structure of genistein against 3OLS protein. The physicochemical characteristics of genistein were analyzed using the SwissADME web tool.&#x0D; Results: Genistein can decrease MHC II expression and increase Arg1 expression in microglia HMC3 cells compared to negative controls (p&lt;0.005), with expression value of 472.577±26.701 AU and 114.299±6.578 AU. But, genistein cannot decrease the free-ERβ expression in cells (p&lt;0.005). The results of in silico analysis showed that genistein is an ERβ agonist.&#x0D; Conclusion: Genistein shows anti-neuroinflammatory effects by decreasing the MHC II expression and increasing Arg1 expression in the microglia HMC3 cell line. However, this effect does not occur through the binding of genistein to ERβ, but it is likely to occur through the binding of genistein with other types of ER.

Role of biofilms in the survival of Legionella pneumophila to sodium chloride treatment.

Background and Objectives:Legionnaires’ disease continues to be a public health concern. Colonized water distribution systems are often implicated in Legionella transmission, despite the use of various disinfection strategies, the bacterium is capable to persist and survive in water systems. The aim of this study was to investigate the persistence of Legionella pneumophila to sodium chloride over time at different temperatures and analysing the role of biofilms in the survival of this bacteria.Materials and Methods:L. pneumophila serogroup 1 and L. pneumophila serogroup 2–15 were used to study the effect of sodium chloride on planktonic and sessile cells. The tested concentrations were: 0.5%, 1%, 2%, 3%, 4%, 6% and 8% (W/V) NaCl. Biofilms were grown on 24-well microplates.Results:At 20°C, L. pneumophila planktonic cells were able to survive in sodium chloride concentrations up to 2%. However, at 37°C, a sodium chloride concentration over 1.5%, reduced systematically the numbers of bacterial cells. Biofilms were grown for 20 days in the absence and presence of sodium chloride. The results show that bacterial strains were able to survive and regrow after the sodium chloride shock (2–3%). Moreover, it seems that this effect is less expressed with the age of the biofilm; old biofilms were more persistent than the young ones.Conclusion:Results from this study demonstrate that the sodium chloride disinfection strategy was effective on Legionella pneumophila planktonic cells but not on biofilms, which demonstrate the role of biofilms in the persistence and recolonization of L. pneumophila in water distribution systems.

In vitro and in silico analysis of phytochemical compounds of 96% ethanol extract of semanggi (Marsilea crenata Presl.) leaves as a bone formation agent.

Osteoporosis is the result of an imbalance in the rate of bone resorption and bone formation due to a decrease in estrogen. Phytoestrogens are plant compounds with structures and functions similar to estrogen. Phytoestrogens that bind to estrogen receptors in bone cells are able to modulate bone formation. Semanggi (Marsilea crenata Presl.) is a plant that contains phytoestrogens. The purpose of this study was to observe the expression of osteocalcin and predict the content of extract phytoestrogens through a computer simulation study to study the bone formation activity of the 96% ethanol extract of M. crenata leaves on hFOB 1.19cells. hFOB 1.19cells were cultured in 24-well microplates, and 96% ethanol extract of M. crenata Presl. leaves was added at 62.5, 125 and 250ppm. The expression of osteocalcin was analyzed using CLSM immunocytochemistry. Using PyRx 0.8 software and 1ERE protein for molecular docking, the compound was analyzed by computer. The 96% ethanol extract of M. crenata Presl. leaves can increase the expression of osteocalcin, the optimal dose is 125ppm, and p<0.05 is 881.658 AU. In silico study was obtained six compounds that showed similar activity 17β-estradiol as ER-β agonists. The 96% ethanol extract of M. crenata Presl. leaves contain six compounds that are thought to be phytoestrogens and ER-β agonists, and play a role in increasing bone formation activity and have the potential to be used as an oral drug.

A Validated "Pool and Split" Approach to Screening and Optimization of Copper-Catalyzed C-N Cross-Coupling Reactions.

A general method for the quick identification of effective catalytic systems for copper-catalyzed C-N cross-couplings is described. This is based on evaluating mixtures of copper sources, ancillary ligands, and bases in different solvents followed by two deconvolution procedures, which aim at identifying the most proper reagent combination in only three distinct steps. Despite being a high-throughput approach in nature, the proposed method utilizes only frugal technological platforms such as 24-well microplates while offering a screening efficiency-the number of executed experiments vs the total number of possible experiments-higher than 95%. To facilitate visualization and mining of the high-throughput experimentation (HTE) data, Visual Basic scripts have been developed, which allow streamlining the extraction of raw HPLC data into TIBCO Spotfire for the graphical display in the form of pie charts. The unique capabilities of this "pool and split" approach have been demonstrated by applying it to literature known cross-coupling reactions. In every case, the described experimental setup was validated by retrieving the original literature conditions in addition to exposing several additional solutions with a minimum number of parallel experiments. Finally, examples are provided for the successful application of this HTE screening workflow to internal projects.

A microplate assay for extracellular hydrolase detection

This article presents a new qualitative method to detect enzyme activity replacing the conventional Agar-Petri dishes. This new method is a simple rapid and low-cost technique that uses 24-well microplates. The detection of hydrolases producing microorganisms in bioprospecting studies by qualitative methods is time consuming, costly and requires a large quantity of strains or enzymatic extracts. Tests with different substrate concentrations (0 to 20 g/L) in agar solution for the enzymatic hydrolysis analysis were performed to determine the best substrate concentrations in 24-well microplates. Other quantitative and analytical methods, such as enzymatic assays and thin layer chromatography, were performed to validate this new method and to compare the relationship between enzymatic activity and substrate degradation. Statistically relevant results were observed for amylase, endoglucanase and polygalacturonase enzymes, even when there was a low substrate concentration in agar, where the halo diameter was high. The results also indicated that the concentrations for efficient enzyme index measurements were 4 g/L carboxymethylcellulose for endoglucanase detection and 8 g/L for amylase and polygalacturonase assays. The results were presented according to the traditional methods for detection of enzymatic activity. This new method can be used as a general test for the detection of important industrial hydrolases. It is a faster and less costly alternative for screening microbial enzyme producing microorganisms and is useful for studying the production of microbial enzymes under different growing conditions.

Drug Release from Polymer Thin Films and Gel Pellets: Insights from Programmed Microplate Electroanalysis.

Robotic electroanalysis in 24-well microplates was used to determine Paracetamol (PCT) release from thin films of chitosan and two pH-sensitive synthetic polymers as well as blends of the polymers with each other and with agarose. Square-wave voltammograms were recorded automatically in a potential window of 0.35 V-0.85 V vs. Ag/AgCl/0.1 M KCl and their evaluation revealed time-dependent PCT release into acidic and basic media. Comparison of the release profiles showed that pure chitosan layers released PCT quickly in a single-phase process while liberation from synthetic polymer thin films was slower with a sigmoidal shape at pH 1.2 and pH 8.0 with a maximum release of PCT after approximately 150 and 140 min, respectively. The release profile from thicker agarose films was between those of the thin films. Agarose blended with chitosan or synthetic polymers formed films with biphasic release behavior. Chitosan linearized the initial section of the release profile in chitosan/polymer blends. The automated procedure for release testing offers the advantage of low-cost, labor-effective and error-free data acquisition. The procedure has been validated as a useful microplate assay option for release profile testing.

Antineuroinflammation activity of n-butanol fraction of Marsilea crenata Presl. in microglia HMC3 cell line.

Background Neuroinflammation is one of the main causes of neurodegenerative events. Phytoestrogen is a group compounds that have an estrogen-like structure or function. Phytoestrogen has a high potential to overcome neuroinflammation caused by estrogen deficiency in postmenopausal women. Marsilea crenata Presl. is a plant known to contain phytoestrogens. This research aimed to analyze the activity of an n-butanol fraction of M. crenata leaves in inhibiting the classical pathway activation of microglia HMC3 cell line to M1 polarity, which has proinflammatory characteristics. Methods Microglia HMC3 cell line was cultured in Eagle's minimum essential medium and induced with IFN-γ for 24 h to activate the cell to M1 polarity in 24-well microplates. The n-butanol fraction was added with various doses of 62.5, 125, and 250 ppm and genistein 50 μM as a positive control. The expression of major histocompatibility complex II (MHC II) as a marker was tested using a confocal laser scanning microscope. Results The result of MHC II measurement shows a significant difference in the MHC II expression in the microglia HMC3 cell line between the negative control and all treatment groups at p<0.05, indicating a non-monotonic dose-response profile. Conclusions The best dosage to inhibit MHC II expression was 250 ppm with the value of 200.983 AU. It is then concluded that n-butanol fraction of M. crenata leaves has antineuroinflammation activity due to its phytoestrogens.

Effect of betaine and selenium on the growth and photosynthetic pigment production in Dunaliella salina as biostimulants.

The aim of our study was to establish the effect of selenium and betaine on the growth of D. salina, accumulation of photosynthetic pigments and antioxidant activity of the hydrophobic fraction. This approach was an attempt to demonstrate 'microalgae biostimulant' effects, similar to 'plant biostimulant' effects, i.e. increased tolerance to abiotic stress and enhanced accumulation of bioactive compounds. A high-throughput assay was done in 24-well microplates, at 15% NaCl and different concentrations of sodium selenite (0, 0.5, 2 and 8 µM) or betaine (0, 5, 50 and 500 µM). Both selenium and betaine induced a slight delay in algae growth during the actively growing stage but the final density reached similar values to the control. Betaine significantly enhanced (50%-100%) carotenoids and chlorophyll a accumulation, in a concentration depending manner. Antioxidant activity increased almost 3-fold in extracts of algae treated with 50 µM betaine. Selenium had a much more discrete effect than betaine on pigments biosynthesis. The antioxidant activity of the extracts increased 2-fold in the presence of Se compared to the control. Our work proves that it is possible to enhance production and activity of bioactive compounds from microalgae by using ingredients, which already proved to act as plant biostimulants.

Comparison and Optimization of Different Protein Nitrogen Quantitation and Residual Protein Characterization Methods in Dietary Fiber Preparations.

Proteins are plant cell wall components but they are not included in the definition of dietary fiber. Therefore, dietary fiber preparations have to be corrected for their residual protein contents. This is commonly done by calculating the residual protein concentrations from the nitrogen contents after Kjeldahl digestion. Here, three different methods to determine nitrogen in Kjeldahl digests were compared: conventional titration with hydrochloric acid after steam distillation, a colorimetric assay (24-well microplates and cuvettes), and the determination by using an ammonia electrode. All assays gave similar results but detection using the ammonia electrode was found to be the most time-efficient approach. Also, an amino-acid profiling method, which is not based on commercial kits and which is suitable for routine analysis of dietary fiber preparations, was established. For this purpose, an HPLC-FLD method following amino acid derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was optimized for fiber samples. Although all commonly used dietary fiber preparation methods involve the application of proteases the amino acid profiles of fiber samples from different sources were shown to be quite diverse. Considering the amino acid composition of the residual protein in various dietary fiber preparations, residual protein is probably not only based on structural proteins.

Design and validation of an open-source modular Microplate Photoirradiation System for high-throughput photobiology experiments.

Research in photobiology is currently limited by a lack of devices capable of delivering precise and tunable irradiation to cells in a high-throughput format. This limits researchers to using expensive commercially available or custom-built light sources which make it difficult to replicate, standardize, optimize, and scale experiments. Here we present an open-source Microplate Photoirradiation System (MPS) developed to enable high-throughput light experiments in standard 96 and 24-well microplates for a variety of applications in photobiology research. This open-source system features 96 independently controlled LEDs (4 LEDs per well in 24-well), Wi-Fi connected control and programmable graphical user interface (GUI) for control and programming, automated calibration GUI, and modular control and LED boards for maximum flexibility. A web-based GUI generates light program files containing irradiation parameters for groups of LEDs. These parameters are then uploaded wirelessly, stored and used on the MPS to run photoirradiation experiments inside any incubator. A rapid and semi-quantitative porphyrin metabolism assay was also developed to validate the system in wild-type fibroblasts. Protoporphyrin IX (PpIX) fluorescence accumulation was induced by incubation with 5-aminolevulinic acid (ALA), a photosensitization method leveraged clinically to destroy malignant cell types in a process termed photodynamic therapy (PDT), and cells were irradiated with 405nm light with varying irradiance, duration and pulsation parameters. Immediately after light treatment with the MPS, subsequent photobleaching was measured in live, adherent cells in both 96-well and a 24-well microplates using a microplate reader. Results demonstrate the utility and reliability of the Microplate Photoirradiation System to irradiate cells with precise irradiance and timing parameters in order to measure PpIx photobleaching kinetics in live adherent cells and perform comparable experiments with both 24 and 96 well microplate formats. The high-throughput capability of the MPS enabled measurement of enough irradiance conditions in a single microplate to fit PpIX fluorescence to a bioexponential decay model of photobleaching, as well as reveal a dependency of photobleaching on duty-cycle—but not frequency—in a pulsed irradiance regimen.

The Effects of Nitric Oxide Synthase Inhibitors on Mitochondrial Respiration in Isolated Mouse Brain Mitochondria

ObjectiveNitric oxide synthase (NOS) plays an important role in intracellular and intercellular signaling pathways. Experimental studies in mitochondria from various tissues, including the brain, have shown the possibility of the presence of a novel mitochondrial variant of NOS (mtNOS) located within the mitochondrial matrix. We hypothesized that administration of NOS inhibitors significantly affects the mitochondrial respiration in isolated brain mitochondria.MethodsMitochondria were isolated from the mice aged 8–12 weeks old and plated in 24‐well microplates for oxygen consumption rate (OCR) readings in the Seahorse XFe24 Analyzer. Mitochondria were incubated with inhibitors of nNOS (ARL‐17477 and N‐ω‐propyl‐L‐arginine), eNOS (N5‐(1‐iminoethyl)‐L‐ornithine), or a non‐specific NOS inhibitor (L‐NG‐Nitroarginine methyl ester) for 30 minutes prior to the mitochondrial stress test in the XFe24 Analyzer and compared to an untreated control group.ResultsPharmacological inhibition of nNOS in isolated brain mitochondria reduced basal respiration and ATP production. Treatment with nNOS‐specific and non‐specific NOS inhibitors showed reduced basal OCR compared to control mitochondria. Control OCR values of 90.76±1.89 pmol/min were significantly higher than treatment with L‐NAME (74.17±2.12 pmol/min), NPA (77.92±2.83 pmol/min) and ARL (76.60±2.85 pmol/min). eNOS‐specific inhibitor treatment showed no significant change in mitochondrial respiration. ATP production as a bioenergetics parameter represents the portion of basal oxygen consumption that was used to drive ATP synthesis. Isolated mitochondria treated with non‐specific inhibitor L‐NAME and nNOS‐specific ARL‐17477 showed a significant decrease in ATP production. Isolated mitochondria treated with NPA show a slight trend towards decreased ATP production, but the statistical analysis did not show significance. Treatment with NIO demonstrated no significant change in ATP production.ConclusionsPharmacological inhibition of NOS isoforms affects brain mitochondrial respiration. Given the significant effects demonstrated by both nNOS‐specific inhibitors and L‐NAME, it may be postulated that mtNOS may be a new isoform of NOS sharing specific properties of the traditional three isoforms.Support or Funding InformationAmerican Heart Association (PVG: 14SDG20490359 and VNS: 16PRE31450006); National Institute of Health: National Institute of Neurological Disorders and Stroke and National Institute of General Medical Sciences (PVK: R01NS094834); and National Institute of Diabetes and Digestive and Kidney Diseases (RS: DK107694).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Effects of Quinoline and Non-Quinoline Inhibitors on the Kinetics of Lipid-Mediated β-Hematin Crystallization.

The throughput of a biomimetic lipid-mediated assay used to investigate the effects of inhibitors on the kinetics of β-hematin formation has been optimized through the use of 24-well microplates. The rate constant for β-hematin formation mediated by monopalmitoyl-rac-glycerol was reduced from 0.17 ± 0.04 min-1 previously measured in Falcon tubes to 0.019 ± 0.002 min-1 in the optimized assay. While this necessitated longer incubation times, transferring aliquots from multiple 24-well plates to a single 96-well plate for final absorbance measurements actually improved the overall turnaround time per inhibitor. This assay has been applied to investigate the effects of four clinically relevant antimalarial drugs (chloroquine, amodiaquine, quinidine, and quinine) as well as several short-chain 4-aminoquinoline derivatives and non-quinoline (benzamide) compounds on the kinetics of β-hematin formation. The adsorption strength of these inhibitors to crystalline β-hematin (Kads) was quantified using a theoretical kinetic model that is based on the Avrami equation and the Langmuir isotherm. Statistically significant linear correlations between lipid-mediated β-hematin inhibitory activity and Kads values for quinoline (r2 = 0.76, P-value = 0.0046) and non-quinoline compounds (r2 = 0.99, P-stat = 0.0006), as well as between parasite inhibitory activity (D10) and Kads values for quinoline antimalarial drugs and short-chain chloroquine derivatives (r2 = 0.64, P-value = 0.0098), provide a strong indication that drug action involves adsorption to the surface of β-hematin crystals. Independent support in this regard is provided by experiments that spectrophotometrically monitor the direct adsorption of antimalarial drugs to preformed β-hematin.

Automated Quantitative Enzyme Biosensing in 24-Well Microplates.

We report a novel system for glucose estimation in model and real samples, utilizing enzyme-modified pencil leads (PL) as effective electrochemical biosensors for robotic substrate quantification in 24-well microplates. Electrochemically formed carboxyl groups on the surface of the graphite were cross-linked to amino groups in the enzyme so as to attach glucose oxidase to the PL surface. Automated amperometric sensing of glucose solutions in microtiter-plate wells used computer-controlled stepper motors to move the biosensor/counter/reference electrode assemblies sequentially between the samples. This setup achieved stable analyte response and, in calibration trials, a linear response range and detection limit of 0.1-8 mM and 0.05 ± 0.01 mM, respectively. The biosensor microplate assay offered accurate "hands-off" evaluation of 4 or 20 samples per plate run, in the standard addition or calibration curve mode, respectively. Mode-independent glucose assays in standard solutions and human serum samples worked reproducibly with close to 100% recovery. The choice of cheap and practical PL enzyme biosensors and simple nonmicrofluidic measurement automation offers a convenient, labor- and cost-efficient form of quantitative biosensing, with a reduced risk of operator errors. The robotic approach is best suited to repetitive measurements of sample series, with academic research and clinical, environmental, pharmaceutical, or biotechnological analysis being potential areas for future exploitations of the methodology.

Antiviral Activity of Hatay Propolis Against Replication of Herpes Simplex Virus Type 1 and Type 2.

BackgroundPropolis is a bee product widely used in folk medicine and possessing many pharmacological properties. In this study we aimed to investigate: i) the antiviral activities of Hatay propolis samples against HSV-1 and HSV-2 in HEp-2 cell line, and ii) the presence of the synergistic effects of propolis with acyclovir against these viruses.Material/MethodsAll experiments were carried out in HEp-2 cell cultures. Proliferation assays were performed in 24-well flat bottom microplates. We inoculated 1×105 cells per ml and RPMI 1640 medium with 10% fetal calf serum into each well. Studies to determine cytotoxic effect were performed. To investigate the presence of antiviral activity of propolis samples, different concentrations of propolis (3200, 1600, 800, 400, 200, 100, 75, 50, and 25 μg/mL) were added into the culture medium. The amplifications of HSV-1 and HSV-2 DNA were performed by real-time PCR method. Acyclovir (Sigma, USA) was chosen as a positive control. Cell morphology was evaluated by scanning electron microscopy (SEM).ResultsThe replication of HSV-1 and HSV-2 was significantly suppressed in the presence of 25, 50, and 100 μg/mL of Hatay propolis. We found that propolis began to inhibit HSV-1 replication after 24 h of incubation and propolis activity against HSV-2 was found to start at 48 h following incubation. The activity of propolis against both HSV-1 and HSV-2 was confirmed by a significant decrease in the number of viral copies.ConclusionsWe determined that Hatay propolis samples have important antiviral effects compared with acyclovir. In particular, the synergy produced by antiviral activity of propolis and acyclovir combined had a stronger effect against HSV-1 and HSV-2 than acyclovir alone.

Effect of two TiO2 nanoparticles on the growth of unicellular green algae using the OECD 201 test guideline: influence of the exposure system

ABSTRACTThis work aimed at assessing the influence of different exposure systems to perform the commonly used OECD 201 freshwater algal growth inhibition test in the context of nanoparticles hazard assessment. Two distinct TiO2 nanoparticles were considered and three different exposure systems were investigated: Erlenmeyers flasks and 24-well microplates (both using an orbital shake system), and an alternative system using cylindrical vials and magnetic stirring. All three systems are in accordance with the OECD 201 test guideline recommendations. We concluded that the exposure systems applied to achieve the test can substantially affect the ecotoxicological results and the subsequent calculated ECx. The selected systems influenced both the interaction between algal cells and TiO2 nanoparticles as well as the growth inhibition recorded. Disparities in ecotoxicity relative to the TiO2 nanoparticles tested were also observed and are finally discussed.

A miniaturized solid contact test with Arthrobacter globiformis for the assessment of the environmental impact of silver nanoparticles

Silver nanoparticles (AgNPs) are widely applied for their antibacterial activity. Their increasing use in consumer products implies that they will find their way into the environment via wastewater-treatment plants. The aim of the present study was to compare the ecotoxicological impact of 2 differently designed AgNPs using the solid contact test for the bacterial strain Arthrobacter globiformis. In addition, a miniaturized version of this test system was established, which requires only small-sized samples because AgNPs are produced in small quantities during the design level. The results demonstrate that the solid contact test can be performed in 24-well microplates and that the miniaturized test system fulfills the validity criterion. Soils spiked with AgNPs showed a concentration-dependent reduction of Arthrobacter dehydrogenase activity for both AgNPs and Ag ions (Ag(+)). The toxic effect of the investigated AgNPs on the bacterial viability differed by 1 order of magnitude and can be related to the release of dissolved Ag(+). The release of dissolved Ag(+) can be attributed to particle size and surface area or to the fact that AgNPs are in either metallic or oxide form. Environ

Influence of PVM/MA copolymer on bacterial adherence to resin surfaces

Purpose: The aim of this pilot study was to investigate the effects of methylvinyl ether and maleic acid (PVM/MA) copolymer containing self-etch (SE) adhesive bonding agent in preventing colonization of the composite material surfaces by Streptococcus gordonii. Methods and materials: Twelve polished cylindrical composite resin discs (8mm in diameter and 2mm in thickness) were divided into two test groups: 1. SE adhesive resin (Clearfil SE Bond, Kuraray) application to the top and bottom surfaces of the discs; 2. PVM/MA containing SE adhesive resin application. For PVM/MA containing adhesive resin, 3mg PVM/MA powder was mixed in 2 drops of bonding agent of SE Bond adhesive material. The specimens were placed in 24-well microplates and sterilized by UV irradiation for 30min before incubation with bacteria. Bacterial adherence to composite surfaces was assessed by determining colony-forming units (CFU) and viewing by scanning electron microscopy (SEM). The specimens were immersed in clear and filtered whole saliva which had been collected from a single donor. After washing with phosphate-buffered saline (PBS), the specimens were placed in individual wells containing 1× 108 cells/ml of Streptococcus gordonii in PBS. Immersed specimens were incubated at 37 ◦C for 24h and washed again with PBS to remove unattached bacteria. Half of the specimens were then placed into tubes containing 1mL of PBS. The attached bacterial cells were dispersed by sonication. Serial dilutions were made and inoculated ontoBrainHeart InfusionBrothplates to determine CFU. For SEM analysis, the other half of the specimens were immersed in Karnovsky’s fixative for 24h, dehydrated, coated with gold-palladium. ANOVA and post hoc Tukey’s test were used to assess statistical differences. Results: Bacterial adherence (log CFU/mL) to PVM/MA containing SE adhesive resin-covered composite surfaces was significantly less (9.20E+02) than adherence (7.92E+03) to regular SE adhesive resin covered surfaces (p=0.05). SEM also confirmed these results showing reduced bacterial attachment to PVM/MA containing adhesive resin applied surfaces. Conclusion: The PVM/MA copolymer significantly reduces bacterial adherence to resin materials.