Colorimetric Microplate Assay Research Articles

Toxicity Evaluation of Silver Nanoparticle in the Kidneys of Wistar Rats

This study aimed to evaluate the nephrotoxic effects of silver nanoparticles (AgNPs) in Wistar rats using biochemical, oxidative stress and histopathological changes. Three groups of six rats were orally administered AgNPs once a day for 28 days with doses of 100, 500, 1000 mg/kg bodyweight. A control group was administered with deionized water. Blood and kidneys were collected 24 h after the last treatment following standard protocols. The activities of creatinine and blood urea nitrogen against AgNP-induced toxicity was determined in the serum by colorimetric microplate assay. Various activity levels of oxidative stress including, Catalase (CAT), Superoxide dismutase (SOD), Glutathione peroxidase (GPx) and Lipid hydroperoxides (LPO) were evaluated in the kidney tissue. Scanning (SEM) and transmission electron microscopy (TEM) was used to determine the histopathological evaluation of the kidneys. A significant increase in the levels of serum creatinine, blood urea nitrogen, CAT and LPO, were noted in AgNPs exposed rats compared to that in control rats. In contrast, decreased activities of SOD and GPx in a dose-dependent manner was observed in AgNPs exposed rats relative to control rats. SEM and TEM study showed significant morphological alterations in kidneys of AgNPs exposed rats in accordance with the biochemical markers. The results of the study demonstrate that AgNPs might be nephrotoxic, and its toxicity is mediated through oxidative stress mechanism.

Development of a high-throughput screening method for exopolysaccharide-producing Streptococcus thermophilus based on Congo red

As a thickening or stabilizing agent, exopolysaccharide (EPS) can significantly enhance food texture. However, the conventional analytical and screening methods of EPS have been unable to meet the current needs of industrial production, owing to low efficiency and heavy time-consumption. A simple and rapid qualitative/quantitative method is needed to accelerate the selection of high EPS-producing strains. Here, a high-throughput screening (HTS) program for EPS production was established in S. thermophilus by combining Congo red agar method (CRA, primary screening) and microplate colorimetric assay (MPC, secondary screening). The correlation coefficient (R2) between CRA/MPC and the phenol-sulfuric acid assay (a classical EPS quantification method) were 0.779 and 0.862, respectively, suggesting the feasibility of this HTS method. A mutant library (>300 colonies) of S. thermophilus was constructed using ribosome engineering strategy, and EPS-producing mutants with the titer from 50 mg/L to 200 mg/L were rapidly obtained by our developed HTS method, confirming that the HTS method is simple and efficient for the isolation of EPS-producing S. thermophilus. Taken together, this HTS method was developed for rapidly screening of EPS-producing S. thermophilus, which could be a valuable tool not only for the rapid detection of EPS production capabilities, but also for the screening of strain libraries from genetic engineering with desirable characteristics.

The Effects of a High-Fat/Cholesterol Diet on the Intestine of Rats Were Attenuated by Sparassis latifolia Polysaccharides.

Sparassis latifolia polysaccharides can regulate lipids and cholesterol in serum and liver. However, little is known about the regulation mechanism of the polysaccharides on cholesterol metabolism and especially the causal relationship with gut microbiota regulation. This study will provide a theoretical basis for the cholesterol-lowering mechanism of S. latifolia polysaccharides and further development of functional foods. In this study, we investigated how the regulation mechanism of Sparassis latifolia polysaccharides affects intestinal cholesterol metabolism in high-fat and high-cholesterol diet-fed rats. Briefly, enzymatic colorimetric microplate assay was used to determine the concentration of faecal bile acid. Gas chromatography-mass spectrometry was used to detect the content of cholesterol and alcohol in faeces. Haematoxylin and eosin staining method was applied to observe the changes in the structure of the small intestine tissue. The related gene expressions in jejunum and ileum were detected by real-time fluorescent quantitative polymerase chain reaction. The related protein expressions in jejunum were studied by using Western blot. High-throughput sequencing was used to detect the intestinal flora changes of the caecal contents. Gas chromatography-mass spectrometry was applied to detect the concentration of short-chain fatty acids in the caecal content. The results showed that Sparassis latifolia polysaccharides could improve the intestinal morphological structure and physiological indices in rats fed high-fat and high-cholesterol diet. Moreover, it could improve intestinal cholesterol metabolism disorder induced by high-fat and high-cholesterol diets via the reduction of the expression of HMGCR, NPC1L1, ACAT2, MTP, ASBT and IBABP mRNA or protein, increasing ABCG8 mRNA expression. In addition, it could also increase the relative abundance of Bacteroides, Butyricicoccus, Parabacteroides, Parasutteerella and Alloprevotella and the short-chain fatty acid concentration, to comprehensively regulate the intestinal cholesterol metabolism. The metabolomics analysis found that Sparassis latifolia polysaccharides could affect lipid, carbohydrate and other related metabolites. Some biomarkers associated with cholesterol metabolism correlated significantly with the abundance of specific intestinal microbiota. These findings indicate that Sparassis latifolia polysaccharides could attenuate intestinal cholesterol metabolism disorder, correlating with modulating gut microbiota and improving host metabolism. They provide theoretical support for the development of Sparassis latifolia as a new food resource.

Rolling circle amplification based colorimetric determination of Staphylococcus aureus.

A colorimetric microplate assay for determination of Staphylococcus aureus DNAis described. Linear padlock probes were designed to recognize target sequences. After DNA binding, the linear padlock probes were circularized by ligation and then hybridize with biotin-labeled capture probes. Biotin-labeled capture probes act as primers to initiate the RCA. The biotin-labeled RCA products hybridize with digoxin-labeled signal probes fixed on streptavidin-functionalized wells of a 96-well plate. To enhance sensitivity, an AuNP-anti-digoxigenin-POx-HRP conjugate was added to the wells and then bound to digoxin-labeled signalling probes. The oxidation of tetramethylbenzidine (TMB) by H2O2 produces a color change from colorless to blue via HRP catalysis. After the reaction was terminated, absorbance is measured at 450nm. For target sequences of Staphylococcus aureus, the detection limit is 1.2 pM. For genomic DNA, the detection limit is 7.4 pg.μL-1. The potential application of the method was verified by analyzing spiked food samples. Graphical abstractSchematic representation of rolling circle amplification and functionalized AuNP-based colorimetric determination ofStaphylococcus aureus. The method uses streptavidin-functionalized 96-well plates and RCA as a molecular tool and AuNP-anti-digoxigenin-POx-HRP as signal transduction markers to increasesensitivity.

A fluorometric hybridization assay for detecting and genotyping high-risk human papillomavirus 16 and 18 in archival tissues of cervical specimens.

Early diagnosis and genotyping of high-risk human papillomavirus (HR-HPV) in cervical tissue specimens is significant for cervical cancer prevention. A sensitive microplate fluorometric hybridization assay (MFHA) was designed for the detection of HPV DNA 16 and 18 in cervical tissue. Following optimization and validation of the method, 60 formalin-fixed and paraffin-embedded cervical samples representing different cervical intraepithelial neoplasia grades of HPV-associated lesions were tested to determine the sensitivity and specificity of the assay. Using consensus GP5+/6+ biotin-labeled primers to amplify a conserved region within the L1 gene, the amplicons were added to the microplate wells coated with specific probes for the hybridization of HPV 16 and 18 individually. Final detection was performed with streptavidin-AlexaFluor488 conjugated. The results were then compared with type-specific nested polymerase chain reaction (PCR) and colorimetric microplate assay. While the agreement between the results obtained by the type-specific nested PCR and fluorometric assay for the detection of both HR-HPV types was 100%, this agreement for the detection of HPV type 16 and 18 using microplate colorimetric assay was 94.2% and 85% respectively. Overall, the results of the fluorometric and colorimetric assays are promising for detecting both HR-HPV types in a large number of cervical tissue samples with the higher MFHA assay sensitivity.

Effects of macular xanthophyll supplementation on brain-derived neurotrophic factor, pro-inflammatory cytokines, and cognitive performance

PurposeOxidative and inflammatory processes play a major role in stress-induced neural atrophy. There is a wide body of literature linking oxidative and inflammatory stress with reductions in neurotrophic factors, stress resilience, and cognitive function. Based on their antioxidant and anti-inflammatory capacity, we investigated the effect of the dietary carotenoids lutein and zeaxanthin, along with the zeaxanthin isomer meso-zeaxanthin (collectively the “macular xanthophylls” [MXans]) on systemic brain-derived neurotrophic factor (BDNF) and anti-oxidant capacity (AOC), and the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β. To investigate higher-order effects, we assessed cognitive performance. Methods59 young (18–25 yrs.), healthy subjects participated in a 6-month, double-blind, placebo-controlled trial to evaluate the effects of MXan supplementation on the aforementioned serum parameters and cognitive performance. Subjects were randomly assigned to one of three groups: placebo, 13 mg, or 27 mg/day total MXans; all measures were taken at baseline and 6 months. Blood was obtained via fasting blood draw, and MXan concentration in the retina (termed macular pigment optical density [MPOD]) was measured via customized heterochromatic flicker photometry. Serum BDNF and cytokines were assessed via ELISA. Serum antioxidant capacity (AOC) and serum MXan concentrations were quantified via colorimetric microplate assay, and high-performance liquid chromatography, respectively. Cognitive performance was measured via a computer-based assessment tool (CNS Vital Signs). ResultsBDNF, MPOD, serum MXans, and AOC all increased significantly versus placebo in both treatment groups over the 6-month study period (p < .05 for all). IL-1β decreased significantly versus placebo in both treatment groups (p = .0036 and p = .006, respectively). For cognitive measures, scores for composite memory, verbal memory, sustained attention, psychomotor speed, and processing speed all improved significantly in treatment groups (p < .05 for all) and remained unchanged in the placebo group. Several measures were found to be significantly associated in terms of relational changes over the course of the study. Notably, change in BDNF was related to change in IL-1β (r = −0.47; p < .001) and MPOD (r = 0.44; p = .0086). Additionally, changes in serum MXans were strongly related to AOC (r = 0.79 & 0.61 for lutein and zeaxanthin isomers respectively; p < .001). For cognitive scores, change in BDNF was correlated to change in composite memory (r = 0.32; p = .014) and verbal memory (r = 0.35; p = .007), whereas change in MPOD was correlated with change in both psychomotor speed (r = 0.38; p = .003), and processing speed (r = 0.35; p = .007). Change in serum lutein was found to be significantly correlated to change in verbal memory (r = 0.41; p < .001), composite memory (r = 0.31; p = .009), and sustained attention (r = 0.28; p = .036). Change in serum zeaxanthin isomers was significantly correlated with change in verbal memory (r = 0.33; p = .017). Lastly, change in AOC was significantly associated with verbal memory (r = 0.34; p = .021), composite memory (r = 0.29; p = .03), and sustained attention (r = 0.35; p = .016). No significant relational changes in any cognitive parameter were found for the placebo group. ConclusionsSix months of daily supplementation with at least 13 mg of MXans significantly reduces serum IL-1β, significantly increases serum MXans, BDNF, MPOD, and AOC, and improves several parameters of cognitive performance. Findings suggest that increased systemic antioxidant/anti-inflammatory capacity (and not necessarily deposition of the carotenoids in neural tissues), may explain many of the effects determined in this study. The significant relationship between change in BDNF and IL-1β over the course of the study suggests that regular consumption of MXans interrupts the inflammatory cascade that can lead to reduction of BDNF. Changes in MPOD and BDNF appear to account for enhancement in cognitive parameters that involve speed of processing and complex processing, respectively.ISRCTN Clinical Trial Registration: ISRCTN16156382.

Development of ultrafast colorimetric microplate assay method for ubiquinone utilizing the redox cycle of the quinone

A simple, rapid, and sensitive microplate assay method for determination of ubiquinone in cosmetic products has been developed and validated. This method is based on measuring reactive oxygen species (ROS) generated from the redox cycle of quinone using redox colorimetric reagent, 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT). The reaction with ROS converts INT to a formazan dye having a strong absorbance at 510 nm that is increased with increasing concentration of ubiquinone. On the basis of this reaction, we developed a microplate assay method that allows high-throughput measurement of ubiquinone. This method uses microplate reader for the measurement of absorbance and it can determine many samples at one time. Under the optimized conditions, the proposed method was linear in the range of 0.02–4 μM and the limit of detection (blank +3SD) was 14.8 nM. We applied the proposed method to determine ubiquinone contents in cosmetic products, and the measurement values were in good agreement with the indicated value. The proposed colorimetric method should be useful as a high-throughput analysis for quality management of ubiquinone containing products.

Acquired Enamel Pellicle Engineered Peptides: Effects on Hydroxyapatite Crystal Growth

The aim of this study was to test the hypothesis that duplication/hybridization of functional domains of naturally occurring pellicle peptides amplified the inhibitory effect of hydroxyapatite crystal growth, which is related to enamel remineralization and dental calculus formation. Histatin 3, statherin, their functional domains (RR14 and DR9), and engineered peptides (DR9-DR9 and DR9-RR14) were tested at seven different concentrations to evaluate the effect on hydroxyapatite crystal growth inhibition. A microplate colorimetric assay was used to quantify hydroxyapatite crystal growth. The half-maximal inhibitory concentration (IC50) was determined for each group. ANOVA and Student-Newman-Keuls pairwise comparisons were used to compare the groups. DR9-DR9 increased the inhibitory effect of hydroxyapatite crystal growth compared to single DR9 (p < 0.05), indicating that functional domain multiplication represented a strong protein evolution pathway. Interestingly, the hybrid peptide DR9-RR14 had an intermediate inhibitory effect compared to DR9 and DR9-DR9. This study used an engineered peptide approach to investigate a potential evolution protein pathway related to duplication/hybridization of acquired enamel pellicle’s natural peptide constituents, contributing to the development of synthetic peptides for therapeutic use against dental caries and periodontal disease.

A micro-plate colorimetric assay for rapid determination of trace zinc in animal feed, pet food and drinking water by ion masking and statistical partitioning correction

A new micro-plate colorimetric assay was developed for rapid determination of zinc in animal feed, pet food and drinking water. Zinc ion was extracted from sample by trichloroacetic acid and then reacted with 2-(5-Bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS) to form a Zn-PAPS complex to be detected by a micro-plate reader at 552 nm. An ion masking formula including salicylaldoxime, deferoxamine and sodium citrate were screened and applied to exclude the interference from other heavy metals and a partitioning correction approach was proposed to eliminate the matrix effect derived from feed sample. The entire procedure can be completed within 40 min and the detection range was 0.038–8.0 μg mL−1 zinc in buffer solution. Moreover, the analysis in real samples revealed the consistency of results by this assay and those by atomic absorption spectrometry analysis. These features highlighted the possibility for this proposed assay to be used for rapid determination of zinc in complex samples.

Further characterization and independent validation of a DNA aptamer-quantum dot-based magnetic sandwich assay for Campylobacter

Previously reported DNA aptamers developed against surface proteins extracted from Campylobacter jejuni were further characterized by aptamer-based Western blotting and shown to bind epitopes on proteins weighing ~16 and 60kD from reduced C. jejuni and Campylobacter coli lysates. Proteins of these approximate weights have also been identified in traditional antibody-based Western blots of Campylobacter spp. Specificity of the capture and reporter aptamers from the previous report was further validated by aptamer-based ELISA-like (ELASA) colorimetric microplate assay. Finally, the limit of detection of the previously reported plastic-adherent aptamer-magnetic bead and aptamer-quantum dot sandwich assay (PASA) was validated by an independent food safety testing laboratory to lie between 5 and 10 C. jejuni cells per milliliter in phosphate buffered saline and repeatedly frozen and thawed chicken rinsate. Such ultrasensitive and rapid (30min) aptamer-based assays could provide alternative or additional screening tools to enhance food safety testing for Campylobacter and other foodborne pathogens.

Investigation of Mammal Arginase Inhibitory Properties of Natural Ubiquitous Polyphenols by Using an Optimized Colorimetric Microplate Assay.

Polyphenols are plant secondary metabolites which possess many positive effects on human health. Although these beneficial effects could be mediated through an increase in nitric oxide synthase activity, little is known regarding the inhibitory effect of polyphenols on mammal arginase, an enzyme which competes with nitric oxide synthase for their common substrate, L-arginine. The aim of the present study was to determine the potential of a series of polyphenols as mammalian arginase inhibitors and to identify some structure-activity relationships. For this purpose, we first developed a simple and cost-effective in vitro colorimetric microplate method using commercially-available mammal bovine liver arginase (b-ARG 1). Among the ten tested polyphenolic compounds [chlorogenic acid, piceatannol, resveratrol, (-)-epicatechin, taxifolin, quercetin, fisetin, caffeic acid, quinic acid, and kaempferol], cholorogenic acid and piceatannol exhibited the highest inhibitory activities (IC50 = 10.6 and 12.1 µM, respectively) but were however less active as (S)-(2-Boronoethyl)-L-cysteine (IC50 = 3.3 µM), used as reference compound. Enzyme kinetic studies showed that both chlorogenic acid and piceatannol are competitive arginase inhibitors. Structural data identified the importance of the caffeoyl (3,4-dihydroxycinnamoyl)-part and of the catechol function in the inhibitory activity of the tested compounds. These results identified chlorogenic acid and piceatannol as two potential core structures for the design of new arginase inhibitors.

Therapeutic effects of a combined antibiotic-enzyme treatment on subclinical mastitis in lactating dairy cows

The objective of this study was to evaluate a combined antibiotic-enzyme therapy for Staphylococcus aureus mastitis and biofilm formation. A total of 141 cases of S. aureus chronic mastitis from three farms were divided into two groups: the control group (n = 54) were treated with Nafpenzal® ointment; the enzyme + antibiotic group (n = 87) were treated with Nafpenzal® plus an enzymatic ointment (MastiVeyxym®). Quantitative determination of biofilm formation was determined using a colorimetric microplate assay and the detection of ica genes by PCR. Enzyme + antibiotic therapy did not significantly improve cure rates compared to control (48.3 vs 38.9%). The cure rate for infections caused by biofilm-positive strains was 42.6 and 46.6% for control and enzyme + antibiotic groups, respectively (P > 0.05). Comparison of cure rates between farms showed a relationship with somatic cell count (SCC), parity and oxacillin resistance. 79.4% of the isolates produced biofilm and antibacterial resistance rates for oxacillin, penicillin and streptomycin were 25.5, 71.6 and 95.7%, respectively. These results indicate that while the increase in mastitis cure rate using enzymatic therapy was not significant, this treatment could be useful in some situations.

A Sensitive Microplate Assay for Lipase Activity Measurement Using Olive Oil Emulsion Substrate: Modification of the Copper Soap Colorimetric Method.

The present work involves a sensitive high-throughput microtiter plate based colorimetric assay for estimating lipase activity using cupric acetate pyridine reagent (CAPR). In the first approach, three factors two levels factorial design methodology was used to evaluate the interactive effect of different parameters on the sensitivity of the assay method. The optimization study revealed that the optimum CAPR concentration was 7.5% w/v, the optimum solvent was heptane and the optimum CAPR pH was 6. In the second approach, the optimized colorimetric microplate assay was used to measure lipase activity based on enzymatic hydrolysis of olive oil emulsion substrate at 37°C and 150 rpm. The emulsion substrates were formulated by using olive oil, triton X-100 (10% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 1:1:1 in the case of Candida sp. lipase. While in the case of immobilized lipozyme RMIM, The emulsion substrates were formulated by using olive oil, triton X-100 (1% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 2:1:1. Absorbance was measured at 655 nm. The stability of this assay (in terms of colored heptane phase absorbance readings) retained more than 92.5% after 24 h at 4°C compared to the absorbance readings measured at zero time. In comparison with other lipase assay methods, beside the developed sensitivity, the reproducibility and the lower limit of detection (LOD) of the proposed method, it permits analyzing of 96 samples at one time in a 96-well microplate. Furthermore, it consumes small quantities of chemicals and unit operations.

Bordonein-L, a new L-amino acid oxidase from Crotalus durissus terrificus snake venom: isolation, preliminary characterization and enzyme stability.

BackgroundCrotalus durissus terrificus venom (CdtV) is one of the most studied snake venoms in Brazil. Despite presenting several well known proteins, its L-amino acid oxidase (LAAO) has not been studied previously. This study aimed to isolate, characterize and evaluate the enzyme stability of bordonein-L, an LAAO from CdtV.MethodsThe enzyme was isolated through cation exchange, gel filtration and affinity chromatography, followed by a reversed-phase fast protein liquid chromatography to confirm its purity. Subsequently, its N-terminal amino acid sequence was determined by Edman degradation. The enzyme activity and stability were evaluated by a microplate colorimetric assay and the molecular mass was estimated by SDS-PAGE using periodic acid-Schiff staining and determined by mass spectrometry.ResultsThe first 39 N-terminal amino acid residues exhibited high identity with other snake venom L-amino acid oxidases. Bordonein-L is a homodimer glycoprotein of approximately 101 kDa evaluated by gel filtration. Its monomer presents around 53 kDa estimated by SDS-PAGE and 58,702 Da determined by MALDI-TOF mass spectrometry. The enzyme exhibited maximum activity at pH 7.0 and lost about 50 % of its activity after five days of storage at 4 °C. Bordonein-L’s activity was higher than the control when stored in 2.8 % mannitol or 8.5 % sucrose.ConclusionsThis research is pioneering in its isolation, characterization and enzyme stability evaluation of an LAAO from CdtV, denominated bordonein-L. These results are important because they increase the knowledge about stabilization of LAAOs, aiming to increase their shelf life. Since the maintenance of enzymatic activity after long periods of storage is essential to enable their biotechnological use as well as their functional studies.

Hydroxyapatite Growth Inhibition Effect of Pellicle Statherin Peptides

In our recent studies, we have shown that in vivo–acquired enamel pellicle is a sophisticated biological structure containing a significant portion of naturally occurring salivary peptides. From a functional aspect, the identification of peptides in the acquired enamel pellicle is of interest because many salivary proteins exhibit functional domains that maintain the activities of the native protein. Among the in vivo–acquired enamel pellicle peptides that have been newly identified, 5 peptides are derived from statherin. Here, we assessed the ability of these statherin pellicle peptides to inhibit hydroxyapatite crystal growth. In addition, atomistic molecular dynamics (MD) simulations were performed to better understand the underlying physical mechanisms of hydroxyapatite growth inhibition. A microplate colorimetric assay was used to quantify hydroxyapatite growth. Statherin protein, 5 statherin-derived peptides, and a peptide lacking phosphate at residues 2 and 3 were analyzed. Statherin peptide phosphorylated on residues 2 and 3 indicated a significant inhibitory effect when compared with the 5 other peptides (P < 0.05). MD simulations showed a strong affinity and fast adsorption to hydroxyapatite for phosphopeptides, whereas unphosphorylated peptides interacted weakly with the hydroxyapatite. Our data suggest that the presence of a covalently linked phosphate group (at residues 2 and 3) in statherin peptides modulates the effect of hydroxyapatite growth inhibition. This study provides a mechanism to account for the composition and function of acquired enamel pellicle statherin peptides that will contribute as a base for the development of biologically stable and functional synthetic peptides for therapeutic use against dental caries and/or periodontal disease.

Arginase inhibitory activity of several natural polyphenols using a novel in vitro test on purified bovine arginase

Interest in arginase, metalloenzyme hydrolyzing L-arginine into L-ornithine and urea, was stimulated by the finding that arginases might compete with nitric oxide synthases for their common substrate, L-arginine and therefore regulate NO availability. Thus, emerged the concept that selective arginase inhibitors might be a valuable strategy for the treatment of various diseases associated to decreased NO [1,2]. Therefore, research of new candidates targeting on arginase has been attracting scientists but the methods used, in terms of models and of biochemical conditions for the arginase assay are currently too different from one study to another to allow valid comparisons. In this work, we developed a novel in vitro microplate colorimetric assay for evaluation of arginase inhibitory effect using commercially available purified liver bovine arginase 1. This assay adapted from [3], is based on the reaction of urea with α-isonitrosopropiophenone and allows using low quantities (0.5 U) of arginase. The evaluation of S-(2-boronoethyl)-L-cystein, a known reversible competitive arginase inhibitor, validated our method (IC50= 12,0µM) that is thus applicable for routine screening analyses. This assay was used to evaluate several natural polyphenols (IC50): rutoside (458.2µM), epicatechin (94µM), taxifolin (79.25µM), piceatannol (37.7µM), chlorogenic acid (37.4µM) and its two moieties caffeic (571.4µM) and quinic (5500µM) acids. These results suggested that piceatannol and chlorogenic acid are suitable as lead compounds to develop new natural or semi-synthetic arginase inhibitors and that caffeoyl part is crucial for the activity.

The Kampo medicine Rokumigan possesses antibiofilm, anti-inflammatory, and wound healing properties.

Periodontal diseases, which are inflammatory diseases of bacterial origin affecting the tooth-supporting tissues, are characterized by inflammation and destruction of gingival connective tissue and alveolar bone, and may lead to tooth loss. The aim of the study was to investigate Rokumigan, a Kampo Japanese traditional medicine made of six different plants, for its capacity to prevent biofilm formation by Fusobacterium nucleatum, to inhibit interleukin-6 (IL-6) and interleukin-8 (IL-8) secretion by mucosal cells, and to promote wound healing in a fibroblast model. Using a microplate colorimetric assay, Rokumigan prevented biofilm formation by F. nucleatum, while it had no effect on bacterial growth. Rokumigan inhibited IL-6 secretion in both epithelial cells and fibroblasts stimulated with lipopolysaccharide. However, it caused no significant inhibition of IL-8 secretion by both cell types. Rokumigan significantly increased proliferation and migration of gingival fibroblasts in a wound healing assay. In conclusion, the Kampo formulation Rokumigan, through suppression of biofilm formation by F. nucleatum, inhibition of IL-6 secretion by gingival epithelial cells and fibroblasts, and promotion of wound healing in a fibroblast model, may have potential application for periodontal diseases.

Combining Ultrasound-Assisted Extraction and a Microliter Colorimetric Assay for the Streamlined Determination of Urea in Animal Feedstuff

This paper proposes a novel analytical strategy for the routine determination of urea in animal feedstuffs, combining an ultrasound-assisted miniaturized extraction protocol with a microplate colorimetric assay based on the reaction between 4-dimethylaminobenzaldehyde and urea. In order to accelerate the extraction, we introduced an ultrasound-assisted miniaturized protocol and compared it with both classic and miniaturized alternatives. The potential interference of amino acids was bypassed by shifting the detection wavelength from 435 to 450 nm. Urea could be quantified in the range 0.05-1.00% (w/w) with high precision (RSD < 5%). The results were in agreement with a commercial enzymatic method, demonstrating the accuracy and selectivity of the assay. The miniaturization led to a 50 times downscale when compared to the official method, resulting in a reduction of at least 90% in chemical consumption per determination, contributing to a more "green" and sustainable analytical methodology.

A specific and rapid colorimetric method to monitor the activity of methionine sulfoxide reductase A

Considerable evidence indicates that methionine sulfoxide (MetO) reductase A (MsrA) plays an important role in cytoprotection against oxidative stress and serves as a potential drug target. To screen for MsrA regulators, a rapid and specific assay to monitor MsrA activity is required. Most of current assays for MsrA activity are based on the reduction of radioactive substrates such as [3H]-N-acetyl-MetO or fluorescent derivatives such as dimethylaminoazo-benzenesulfonyl-MetO. However, these assays require extraction procedures and special instruments. Here, we developed a specific colorimetric microplate assay for testing MsrA activity quickly, which was based on the fact that MsrA can catalyze the reduction of methyl sulfoxides and simultaneously oxidize dithiothreitol (DTT), whose color can be produced by reacting with Ellman's reagent (dithio-bis-nitrobenzoic acid, DTNB). The corresponding absorbance change at 412nm was recorded with a microplate reader as the reaction proceeded. This method to monitor MsrA activity is easy to handle. Our findings may serve as a rapid method for the characterization of recombinant enzyme and for the screening of enzyme inhibitors, pharmacological activators, gene expression regulators and novel substrates.

Synthesis of hydrophobic N-acylated isonicotinic acid hydrazide derivatives as potential enoyl–acyl carrier protein reductase (InhA) inhibitors

Enoyl–acyl carrier protein reductase (InhA) is an important enzyme for the biosynthesis of mycolic acids which are central constituents of the mycobacterium cell wall. In the present study, we synthesized, characterized hydrophobic N-acylated isonicotinic acid hydrazide derivatives (2a–2m) as potential enoyl–acyl carrier protein reductase (InhA) inhibitors and tested them against Mycobacterium tuberculosis H37Rv and two human clinical isolates by means of colorimetric tetrazolium micro-plate assay. Compounds 2c–2m displayed good-to-excellent inhibitory activity against all tested strains as compared with isoniazid. In vivo acute toxicity studies were performed by Up-and-Down procedure. The results showed that all the tested compounds were non-toxic and well tolerated considering their LD50 values ranging from 3,898 to 5,000 mg/kg body weight.