Inhibition Curves Research Articles

Inhibitory effect of flavonoids from Scutellariae Radix on human cytochrome P450 1A

This study investigated the inhibitory effect of eight natural flavonoids in Chinese herb Scutellariae Radix on huamn cytochrome P450 1 A(CYP1 A), a key cancer chemo-preventive target. In this study, phenacetin was used as a probe substrate for CYP1 A, while human liver microsomes and recombinant human CYP1 A enzymes were used as enzyme sources. Liquid chromatography-tandem mass spectrometry was used to monitor the formation rates of acetaminophen, the O-deethylated metabolite of phenacetin. The dose-dependent inhibition curves were depicted based on the changes of the formation rates of acetaminophen, while the IC_(50) were determined. Inhibition kinetic analyses and docking simulations were used to investigate the inhibition modes and mechanism of wogonin(the most potent CYP1 A inhibitor in this herb), while the inhibition constants(K_i) of wogonin against both CYP1 A1 and CYP1 A2 were determined. Among all tested flavonoids, wogonin, 7-methoxyflavanone and oroxylin A displayed a strong inhibitory effect on CYP1 A(IC_(50)<1 μmol·L~(-1)), baicalein exhibited a moderate inhibitory effect on CYP1 A(IC_(50) between 1-10 μmol·L~(-1)), and baicalin, scutellarein and wogonoside displayed a very weak inhibitory effect on CYP1 A(IC_(50) between 10-25 μmol·L~(-1)), but scutellarin displayed a negligible inhibitory effect on CYP1 A(IC_(50)>100 μmol·L~(-1)). Further investigations demonstrated that wogonin had a weak inhibitory effect on other human CYP enzymes, suggesting that it could be used as a lead compound for the development of specific inhibitors of CYP1 A. Furthermore, the inhibition kinetic analyses clearly demonstrated that wogonin could strongly inhibit phenacetin O-deethylation in both CYP1 A1 and CYP1 A2 in a competitive manner, with K_i values at 0.118 and 0.262 μmol·L~(-1), respectively. Molecular docking demonstrated that wogonin could strongly interact with CYP1 A1 and CYP1 A2 via hydrophobic and π-π interactions, as well as Ser120 and Ser116 in CYP1 A1 via hydrogen-bonding. In conclusion, this study found that some flavonoids in Scutellariae Radix displayed a strong inhibitory effect on CYP1 A, while wogonin is the most potent CYP1 A inhibitor with a relatively high selectivity towards CYP1 A over other human CYPs.

Change of Autophagic Activity of U266 Cells after Bortezomib -Resistance and Its Mechanisms

To explore the changes of autophagic activity after resistance of U266 cells to bortezomib (Bor) and its mechanisms. The proliferation inhibition rate, 50% inhibitory concentration (IC50), drug-resistance coefficient, drug-resistance reversed multiple by 3-methyladenine (3-MA) in U266 and U266/Bor cells treated with Bor were detected and calculated by using MTT method, then the proliferation inhibition curve was drawed. The Western blot was used to detect the expression of LC3-I, IC3-II, p-mTOR, Beclin-1, ATG5 and ATG7 proteins. The Bor showed the its proliferation inhibition effect on U266 cells and U266/Bor cells, IC50 of Bor on U266 and U266/Bor cells on 24 hours were 35.7 nmol/L and 526.5 nmol/L respectively; the drug-resistance coefficient was 14.7; the drug-resistance reversed multiple by 3-MA was 2.7. The expression of LC3-II, Beclin11, ATG5 and ATG7 in U266/Bor cells was higher than that in U266 cells; after the treatment with Bor for 24 h, the expression levels of LC3-II, Beclin-1, ATG5 and ATG7 in U266 cells all decreased, as compared with levels before treatment; while the expression levels of LC3-II, Beclin-1, ATG5 and ATG7 in U266/Bor cells were higher than those before treatment. There were no significant difference of p-mTOR expression among U266, U266+Bor, U266/Bor, U266/Bor+Bor cells. The increase of autophagy closely relates with resistance of U266 cells to bortezomib, moreover with up-regulation of Beclin-1, ATG5 and ATG7 expression.

Oil sands process-affected water impairs the olfactory system of rainbow trout (Oncorhynchus mykiss)

Oil sands process-affected water (OSPW), a byproduct of the extraction of bitumen in the surface mining of oil sands, is currently stored in massive on-site tailings ponds. Determining the potential effects of OSPW on aquatic ecosystems is of main concern to oil sands companies and legislators concerned about the reclamation of mining sites. In the present study, the interaction of OSPW with the chemosensory system of rainbow trout was studied. Using an electro-olfactography (EOG) technique, a 24 h inhibition curve was established and concentrations that inhibit the olfactory system by 20% and 80% (IC20 and IC80) were estimated at 3% and 22% OSPW, respectively. To study the interaction of exposure time and concentration along with the mechanism of the toxic effects, rainbow trout were exposed to 3% and 22% OSPW for 2, 24, and 96 h. An EOG investigation of olfactory sensitivity demonstrated a positive interaction between exposure time and concentration of OSPW concentration, because an increase in either or both elevated the inhibitory effect. To investigate whether or not structural damage of the olfactory epithelium could account for the observed inhibitory effects of OSPW on fish olfaction, the ultrastructure of the olfactory epithelium of exposed fish was investigated using scanning electron microscopy (SEM) and light microscopy (LM). The SEM micrographs showed no changes in the structure of the olfactory epithelium. The light micrographs revealed an increase in the number of mucous cells in 22% OSPW. The results of the present study demonstrated that exposure to OSPW impairs the olfactory system of rainbow trout and its effects increase gradually with increasing exposure time. The present study demonstrated that structural epithelial damage did not contribute to the inhibitory effects of OSPW on the olfactory system.

Baicalin Weakens Staphylococcus aureus Pathogenicity by Targeting Sortase B.

Staphylococcus aureus (S. aureus) is a human and other animal pathogen that contributes to the primary etiology of nosocomial pneumonia, a disease with high mortality rates and costs. Treatment of multidrug-resistant S. aureus infection is extremely challenging, and new therapeutic strategies beyond antibiotic treatment are needed. Anti-virulence agents that specifically target the molecular determinants of virulence may be a novel method for treating drug-resistant nosocomial infections. Sortase B (SrtB) is a crucial virulence factor in S. aureus and plays an important role during infection. In this study, we find that baicalin suppresses the activity of SrtB. Minimum inhibitory concentration and growth curve assays confirmed that baicalin has no anti-S. aureus properties. We performed live/dead, lactate dehydrogenase (LDH), adherence, and enzyme-linked immunosorbent assays to confirm that baicalin reduced human alveolar epithelial A549 cell injury caused by S. aureus, reduced the adherence of S. aureus to A549 cells, and significantly attenuated the inflammatory response of mouse macrophage J774 cells to S. aureus. Additionally, we were able to elucidate the binding mechanics and identify the interacting sites of baicalin and SrtB via a molecular dynamics simulation, site-directed mutagenesis, and fluorescence spectroscopy quenching. Finally, we confirmed that baicalin directly binds to the active center of SrtB, and the residues Asn92 and Tyr128 perform an important function in the interaction of SrtB and baicalin. Taken together, these data indicate that baicalin is a promising candidate to combat S. aureus infections.

Development and validation of a mouse-based primary screening method for testing relative allergenicity of proteins from different wheat genotypes

BackgroundWheat allergy is a major food allergy that has reached significant levels of global public health concern. Potential variation in allergenicity among different wheat genotypes is not well studied at present largely due to the unavailability of validated methods. Here, we developed and validated a novel mouse-based primary screening method for this purpose. MethodsGroups of Balb/c mice weaned on-to a plant protein-free diet were sensitized with salt-soluble protein (SSP) extracted from AABB genotype of wheat (durum, Carpio variety). After confirming clinical sensitization for anaphylaxis, mice were boosted 7 times over a 6-month period. Using a pooled-plasma mini bank, a wheat-specific IgE-inhibition (II)-ELISA was optimized. Then the relative allergenicity of SSPs from tetraploid (AABB), hexaploid (AABBDD) and diploid (DD) wheat genotypes were determined. The IC50/IC75 values were estimated using IgE inhibition curves. ResultsThe optimized II-ELISA with an inhibition time of 2.5 h had a co-efficient of variation of <2%. Primary screening for relative allergenicity demonstrated that IgE binding to AABB-SSP was significantly abolished by the other two wheat genotypes. Compared to AABB, the relative allergenicity of SSPs of AABBDD and DD were significantly lower (p < .01). Furthermore, IgE inhibition curves showed significant differences in IC50 and IC75 values among the three wheat genotypes. ConclusionWe report a novel mouse-based primary screening method of testing relative allergenicity of wheat proteins from three different wheat genotypes for the first time. This method is expected to have broad applications in wheat allergy research.

Patterns of Phenol in a Continuous Toxicity Monitoring System Using Bioluminescent Bacteria

The purpose of this study is to evaluate effect of phenol toxicity of Vibrio fischeri using MicroTox Ⓡ CTM. A continuous flow system for biomonitoring tool has been used to assess the toxicity of phenol. The bioluminescence inhibition of different dillutions of phenol (1.5, 2.0, 2.5, 3.0 mg/L) with deionized water for reference was evaluated from 3.5 to 97.4% and the EC20 and EC50 from inhibition curve were estimated in 1.7 and 2.2 mg/L, respectively. Meanwhile, the bioluminescence inhibition of mixtures of phenol (2.0, 3.0, 5.0, 7.0 mg/L) with effluent wastewater for reference was evaluated from 5.4 to 99.2% and the EC20 and EC50 from inhibition curve were estimated in 2.9 and 4.4 mg/L, respectively. Using MicroTox Ⓡ CTM provides a simple, rapid and sensitive method for accessing the toxic risks posed by phenol to aquatic ecosystems and has practical applications for municipal and industrial wastewater management and screening of contaminants in fresh water streams and lakes. Key words: Bioassay, Bioluminescent Bacteria, Vibrio fischeri, Biosensor, Phenol

Interaction of environmental steroids with organic anion transporting polypeptide (Oatp1d1) in zebrafish (Danio rerio).

Steroid hormones in the aquatic environment may pose a risk to fish health due to their ubiquitous presence and high biological activity. At present, the uptake process and toxicokinetics of steroids in fish are poorly known, in particular the role of cell membrane transporters. We investigated the interaction of 17 endogenous and environmental steroids with the zebrafish organic anion transporting peptide (Oatp1d1) uptake transporter, which is prominently expressed in liver and kidneys. We selected steroids of different classes including androstenedione (A4), progesterone (P4), and its metabolites, as well as glucocorticoids and spironolactone, and analyzed their interaction with Oatp1d1 by competitive inhibition of the uptake of the fluorescent substrate Lucifer Yellow. The half-maximal inhibition (IC50) values derived from sigmoid inhibition curves were lowest for P4, and the order of increasing IC50 values was as follows: 17α-hydroxyprogesterone > clobetasol propionate > spironolactone > 21α-hydroxyprogesterone > fludrocortisone acetate and additional glucocorticoids. The interaction activity showed a positive correlation with the lipophilicity of the steroids. Our data show that different classes of steroids interact with Oatp1d1 with different activity (either by uptake or inhibition, or both). This is of importance, because in consequence, steroids may interfere with the transport of endogenous substrates, and thus physiological processes. Moreover, steroids may alter cellular trafficking of environmental contaminants by competitive inhibition of this transporter. Environ Toxicol Chem 2018;37:2670-2676. © 2018 SETAC.

How to extend range linearity in enzyme inhibition-based biosensing assays

Bioassays based on enzyme inhibition are analytical tools widely employed for inhibitor analysis. Beside the conventional analytical techniques such as chromatography and mass spectrometry, these bioassays are cost-effective, easy to use, and suitable for in situ measurement but they are often characterised by a quite narrow linear range.Herein, we report a novel graphical method based on integrated Michaelis-Menten equation, valid for all types of reversible inhibition, which provides an extended linear range. The suitability of this innovative approach was demonstrated in the case of fluoride quantification using a colorimetric bioassay based on acetylcholinesterase inhibition. The “half time reaction”, estimated by the progress curve of cholinesterase inhibition, was plotted versus the fluoride inhibitor concentration, observing an extended linear range up to 5 mM, instead of 0.6 mM using initial rate measurements. The applicability of this new concept was further demonstrated in the case of catalase enzyme inhibited by cyanide.Furthermore, it was demonstrated that fixed substrate conversion at level of 10–50% allows determination of inhibitor concentration in a wide linear range with high precision and in short time of analysis. This novel theoretical and practical approach allows for the extension of the linear range without any further experiments, with several advantages including low reagent consumption, reduced waste generation and time of measurement.

Novel strategy for obtaining uniformly dispersed silver nanoparticles on soluble cotton wound dressing through carboxymethylation and in-situ reduction: antimicrobial activity and histological assessment in animal model

A simple but cost-effective method for preparing a novel partially carboxymethylated cotton gauze (PCG) with silver nanoparticles (AgNPs; i.e., PCG/Ag) was developed through carboxymethylation modification and reduction of Ag nitrate using polyvinylpyrrolidone (PVP) as a coating and complexing agent. The Fourier transform infrared spectrum of PCG/Ag revealed an absorption band at 1610 cm−1 and no chemical conversion in the synthesis of AgNPs. The synthesis of spherical AgNPs with a diameter of 5–20 nm was confirmed by ultraviolet–visible spectroscopy and showed plasmon resonance peaks at 425 nm; X-ray diffraction analysis revealed peaks at 2θ = 38.3° and 44.2° and X-ray photoelectron spectroscopy showed two peaks at 374.2–376.9 and 368.2–371.0 eV. AgNPs attached to partly carboxymethylated cotton gauze were optimized by a one-factor-at-a-time approach; the optimized conditions for an antimicrobial effect with no cytotoxicity was 30 min of soaking in 0.4% (w/v) Ag nitrate and reduction in 0.5% (w/v) ascorbic acid in the presence of defined amounts of PVP with stirring. PCG/Ag-4 showed excellent water absorption exceeding 2000% of the initial value. The inhibition zone test and bacterial growth inhibition curves suggested that PCG/Ag had antimicrobial activity against Escherichia coli and Staphylococcus aureus. The cell viability and cell attachment assays confirmed that the PCG/Ag was not cytotoxic and promoted wound healing in an animal model. These results indicate that PCG/Ag is a safe and effective material for wound dressing. This novel in-situ reduction method for obtaining uniformly dispersed nanoparticles on a biomaterial for wound dressing can be applied on a large scale without sacrificing low cost and efficiency.

Characterization of Respiration in Paraburkholderia tropica Ppe8T under Static Culture Conditions

Objective: To investigate the respiratory chain of Paraburkholderia tropica under limited oxygen conditions would allow us to better understand their functions in plant tissue. Methods: We first optimized the medium for P. tropica biomass production in static culture. Oxidase and Oxidoreductase activities were determined in absence and presence de inhibitors. Spectroscopic analysis of cytochromes was obtained at room and liquid nitrogen temperature. Findings: Isolated membranes showed respiratory oxidase activities with succinate, glucose, NADH and ascorbate-N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD). Succinate and glucose respiration were inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide, antimycin A and myxothiazol, suggesting the presence of a bc1 complex.KCN inhibition curves in the presence of ascorbate-TMPD, succinate or glucose were biphasic, with KCN-sensitive and -resistant oxidases. Spectral analysis with physiological or artificial substrates revealed the presence of only cytochromes b and c, with remarkable cytochrome reduction by ascorbate alone. The CO difference and photodissociation spectra of glucosereduced membranes revealed one component with the spectral features of a cytochrome o´-like CO complex. Air-oxidation spectra of the cytochromes with KCN showed that cytochrome c is an obligate participant in the KCN-sensitive pathway and revealed the presence of a cyanide-resistant oxidase cytochrome bo. Novelty: The characterization of the electron transport system of P. tropica allowed us to propose the following components: 1. Dehydrogenases for succinate, glucose and NADH, 2. A ubiquinone, 3. A bc complex and 4. Two oxidases, cbb-type cytochrome c oxidase and bo-type quinol oxidase. Keywords: bc1 complex, cbb oxidase, Cytochromes, Paraburkholderia tropica, Quinol Oxidase, Respiration

Abstract 1525: SY-1365, a selective CDK7 inhibitor, exhibits potent antitumor activity against ovarian cancer models in vitro and in vivo

Abstract Introduction: CDK7, a key regulator of transcription and cell cycle progression, has been implicated in the pathogenesis of ovarian carcinoma. SY-1365, a potent and selective inhibitor of CDK7, was developed to exploit tumor dependencies driven by CDK7 and is in phase 1 clinical development. Here we report, for the first time, on the activity of SY-1365 in preclinical models of ovarian carcinoma and on potential biomarkers of SY-1365 response. Methods: SY-1365 growth rate inhibition (GR) curves were determined in 314 solid tumor cell lines, including 22 ovarian carcinoma lines. Cell lines were clustered into cytotoxic and cytostatic response groups and assessed for RNA expression to identify markers associated with SY-1365 sensitivity. In vivo, SY-1365 activity was evaluated at the maximum tolerated dose in 17 independent ovarian PDX models derived from heavily pretreated patients; a signal-finding design was used in which tumor growth in SY-1365 treated mice (n=2-3 per PDX model) was compared to vehicle-treated mice or historical growth in untreated mice. Results: SY-1365 induced cytotoxicity (GR &amp;lt;0) in 61% (190/314) of all tumor lines at &amp;lt;50nM. In ovarian carcinoma lines, 36% (8/22) were cytotoxic at &amp;lt;50nM, with deep responses (GRmax &amp;lt; -0.5) seen in 23% (5/22). Lower expression of BCL2L1, which encodes the mitochondrial apoptosis antagonist BCLXL, was the most predictive marker of sensitivity across all lines (accuracy = 70%; FDR=0.006). Reasoning that low BCL2L1 may be a marker of dependence on other mitochondrial apoptosis antagonists (MCL1 and/or BCL2) for survival, we evaluated expression of BCL2, BCLXL, and MCL1 protein at baseline and post-SY-1365 treatment in ovarian carcinoma lines. As with BCL2L1 RNA, BCLXL expression was lower at baseline in cytotoxic- versus cytostatic-responsive lines. In addition, SY-1365 induced MCL1 downregulation in cytotoxic, but not in cytostatic lines. In vivo, SY-1365 induced responses in 52% (9/17) of ovarian carcinoma PDX models. Of the 9 responders, 2 exhibited 40-60% tumor growth inhibition (TGI), 7 exhibited ≥ 100% TGI. As in cell lines, low BCLXL, in combination with low BCL2, was associated with SY-1365 response in PDXs. In addition, 7/9 responsive models had alterations in the RB signaling pathway including loss of RB1 expression (n=3), high CCNE1 expression (n=3), or CDKN2A loss (n=1). Conclusions: SY-1365 induces cytotoxicity in ovarian carcinoma cell lines and induces robust responses in heavily pretreated (including PARP-inhibitor and platinum-resistant) ovarian PDX models. Alterations in expression of mitochondrial apoptosis antagonists and RB pathway regulators are associated with SY-1365 response and support exploration of these as predictive biomarkers of SY-1365 clinical activity in ovarian carcinoma. SY-1365 is currently being assessed in a phase 1 trial in adult patients with advanced solid tumors (NCT03134638). Citation Format: Panagiotis A. Konstantinopoulos, Graeme Hodgson, Nisha Rajagopal, Liv Johannessen, Joyce F. Liu, Paul T. Kirschmeier, Shan Zhou, Cam Anh Tran, David Orlando, Christian Fritz, Emmanuelle di Tomaso, Ursula A. Matulonis. SY-1365, a selective CDK7 inhibitor, exhibits potent antitumor activity against ovarian cancer models in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1525.

Lethal and sublethal responses in the fish, Odontesthes bonariensis, exposed to chlorpyrifos alone or under mixtures with endosulfán and lambda-cyhalothrin.

Need for ecotoxicological information on local species has been recently highlighted as a priority issue in Latin America. In addition, little information has been found on concentration distances between lethal and sublethal effects, and the effect of mixtures at these two levels of analysis. Chlorpyrifos (CPF) is an organophosphate insecticide broadly used in soybean crops which has dramatically expanded in Latin America and other regions of the world. The aim of the present study was to evaluate lethal and sublethal effects of CPF, singly or in mixtures, on the inland "Pejerrey" (Odontesthes bonariensis) under laboratory conditions. Bioassays were performed using 15-30 d post hatch Pejerrey larvae. Six toxicity tests were run for estimating the average inter-assay dose-response curve of CPF and other six for assessing the effects of mixtures of CPF with endosulfan (EN) or lambda-cyhalothrin (LC), at three toxic units (TU) proportions (25:75, 50:50, 75:25). In addition, four assays were performed to describe the average inter-assay dose-response inhibition curve of acetylcholinesterase (AchE) for CPF alone and two for assessing the mixtures. The estimated 96 h-LC50 for CPF was 2.26 ± 1.11 µg/L and the incipiency value was 0.048 ± 0.012 µg/L, placing this Neotropical species among the 13% of worldwide fish more sensitive to CPF. In addition, the 96 h-LC50 for EN and LC were 0.30 ± 0.012 µg/L and 0.043 ± 0.031 µg/L, respectively. Therefore, relative toxicity of the three soybean insecticides for O. bonariensis was LC > EN > CPF. Effects of mixtures with EN and LC were variable, but in general fitted to both, independent action (IA) and concentration addition (CA) models. Slight antagonism was found when CPF TU proportions were above 50%. Therefore, from the regulatory point of view, the use of both mixture models, CA or IA, would be precautionary. Differential sensitivity to CPF was found for AchE inhibition at the head (96 h-IC50 = 0.065 ± 0.058 µg/L) and the body (96 h-IC50 = 0.48 ± 0.17 µg/L). In addition, whereas no significant effects induced by mixtures was observed in body AchE activity, antagonism was induced in head AchE inhibition in presence of both, EN and LC in the mixture. The lethal to sublethal ratio was close to 25.2 and 3.4 when comparing the CPF-LC50 and IC50s for head and body AchE activity, respectively. However, considerable overlapping was observed between concentration-response curves, indicating that the use of AchE as biomarker for environmental monitoring would be limited.

CD8+ T cell-mediated inhibition of HIV-1.

Abstract CD8+ T cells contribute to the control of viral infections, including HIV-1. Previous studies have reported that CD8+-mediated virus inhibition is predictive of subsequent HIV plasma viremia and CD4+T cell decline in HIV-infected, antiretroviral naïve individuals; higher virus inhibition is associated with a better clinical outcomes. Ex vivo CD8+ T cell-mediated inhibition of HIV replication is measured after co-culture of CD8+ T cells with HIV-1 super-infected, autologous CD4+ T cells, at different effector to target ratios. We adapted and standardized the CD8+ T cell virus inhibition assay (CD8-VIA) for use as an endpoint assay in therapeutic testing of HIV-1 T cell vaccines. CD8+ T cells from HIV infected, durably (&amp;gt;2 years) antiretroviral suppressed participants (n=11) were co-cultured with JR-CSF infected autologous CD4+ T cells at E:T ratios ranging from 1/2:1 to 1/160:1, generating CD8+ T cell virus inhibition curves for each participant. IC50 values varied by 100-fold across participants suggesting that CD8+ T cells harbor a broad range of virus inhibitory capacity even when viremia in participants is durably suppressed. Ongoing studies are comparing IC50 values to other measurements (magnitude, specificity, avidity, proliferation and production of cytokines and lytic molecules) of HIV-specific T cell immunity to understand the critical determinants of CD8+ T cell inhibitory capacity.

Pathogen Box screening for hit identification against Mycobacterium abscessus.

Mycobacterium abscessus is a rapidly growing life-threatening mycobacterium with multiple drug-resistance mechanisms. However, there is no official regimen for M. abscessus therapy. In this study, we screened the Pathogen Box, which contains 400 drug-like molecules active against neglected diseases, to identify active molecules targeting Mycobacterium abscessus using resazurin live/dead assays. In this screening assay, the Z-factor was 0.7, as an indicator of the statistical confidence of the assay. A cut-off of 80% growth inhibition in the screening resulted in the identification of four different compounds at a single concentration (20 μM). Dose-response curves identified three different hit candidates, i.e., MMV688508, MMV688844, and MMV688845, which generated good inhibitory curves. All hit candidates were expected to have different molecular targets. Among them, MMV688844 showed the best minimum inhibitory concentration value for not only wild-type M. abscessus but also for nine different R and S morphotype clinical isolates. Thus, we found that MMV688844, identified from the Pathogen Box screen, may be a promising candidate in the M. abscessus drug discovery pipeline.

Influence of naringenin on the biofilm formation of Streptococcus mutans

ObjectivesTo evaluate the effect of naringenin on the biofilm formation of Streptococcus mutans (S. mutans), and to investigate its mechanisms of action and biological toxicity. MethodsMinimum inhibitory concentrations, growth curves, and biofilm inhibition rates of naringenin were determined to assess its antimicrobial effect on S. mutans. The morphology of S. mutans and the structure of biofilm were observed by FESEM and CLSM. Bacterial aggregation, bacterial surface hydrophobicity, and real-time PCR for gtfB, gtfC, comD, comE, and luxS mRNA expression were assessed to preliminarily investigate the mechanisms of action. MTT test using human dental pulp cells (HDPCs) was also performed to investigate cytotoxicity. ResultsThe S.mutans growth curves, FESEM, CLSM showed that both 100 and 200 μg/mL of naringenin obviously inhibited S. mutans growth and biofilm formation, increased S. mutans surface hydrophobicity, reduced bacterial aggregation, and downregulated the mRNA expression of gtfB, gtfC, comD, comE, and luxS. However, naringenin at 200 μg/mL slightly decreased the growths of HDPCs compared with 100 μg/mL. ConclusionNaringenin at 100 and 200 μg/mL suppressed the second (bacterial adhesion) and third stages (biofilm maturation) of S. mutans biofilm formation. Clinical significanceNaringenin is promising for dental clinic promotion to prevent the biofilm formation of S. mutans, serving as a safe anti-caries agent at an appropriate concentration.

Azoles additively inhibit cytochrome P450 1 (EROD) and 19 (aromatase) in rainbow trout (Oncorhynchus mykiss)

Antifungal azoles are widely used in medicine, agriculture, and material protection and several antifungal azoles have been found in environmental samples. Although these compounds were designed to inhibit fungal enzymes such as lanosterol-14-demethylase (cytochrome P450 (CYP) 51), it is well established that the inhibitory actions of azoles are not specific for fungal CYP isozymes.We refined a gill filament assay to determine the inhibition of CYP1, measured as reduced 7-ethoxyresorufin-O-deethylase (EROD) activity, in rainbow trout (Oncorhynchus mykiss) gill tissue ex vivo. The advantage of this method is that both induction and inhibition of EROD are performed ex vivo. Among thirteen azoles studied, the five that caused the strongest inhibition of gill EROD activity at a concentration of 5 μM were selected for concentration–response assessment. These compounds (bifonazole, clotrimazole, imazalil, miconazole, and prochloraz) showed IC50 values ranging from 0.1 to 1.5 μM. CYP19 (aromatase) inhibition was measured using microsomes from rainbow trout brains. Concentration-response curves for CYP19 inhibition were determined for letrozole, bifonazole, clotrimazole, imazalil, miconazole and prochloraz, which gave IC50 values ranging from 0.02 to 3.3 μM. It was further found that mixtures of the five most potent azoles reduced both CYP1 and 19 catalytic activity in an additive fashion (IC50 = 0.7 μM and 0.6 μM, in the respective assay). Bifonazole (IC50 = 0.1 μM) is not previously known to inhibit CYP1 activity.The additive inhibition of CYP1 and CYP19 catalytic activity is an important finding of the present study. We conclude that this additive action of azoles could mediate adverse impacts on CYP regulated physiological functions in environmentally exposed fish.

Effect of pH and Mexican Oregano (Lippia berlandieri Schauer) Essential Oil Added to Carboxymethyl Cellulose and Starch Edible Films on Listeria monocytogenes and Staphylococcus aureus

The aim of this work was to evaluate the effect of pH and Mexican oregano essential oil (MOEO) added to carboxymethyl cellulose (CMC) and starch (S) edible films on Listeria monocytogenes and Staphylococcus aureus. CMC and S edible films were formulated with different concentrations (0%, 0.25%, 0.50%, 0.75%, and 1%) of MOEO at different pH (5, 6, or 7). Antimicrobial assay was performed. Inhibition curves were fitted to the Fermi model. Significant differences (p&lt;0.05) were found in tc (time to reduce 50% of microbial population) and a (slope of the curve around tc), being lower at acidic pH. For L. monocytogenes, CMC films exhibited a higher antimicrobial effectiveness (0.50% of MOEO) compared to S films which need a higher concentration of MOEO (0.75%). S. aureus was inhibited with CMC films at 0.50% MOEO and pH 5 and 6. Microbial modeling has allowed estimating key intrinsic factors as pH and MOEO concentration with the synergistic effect against two important food-borne pathogens.

Antifungal Activity of Osthole on Microsporum canis through Interfering with Biosynthesis of Fungal Cell Wall

Osthole extracted from Cnidii Fructus, a herb used in traditional Chinese medicine, was reported to possess multiple activities such as antibacterial, antiinflammatory, antitumor and insecticidal. However, its antifungal activity against Microsproum canis has not been investigated yet. The present study aimed to investigate the antifungal effect of osthole against Microsproum canis. The minimum inhibitory concentration and a growth inhibitory curve of osthole on Microsporum canis were according to the program of M38-A2 of Clinical and Laboratory Standards Institute. HPLC analysis, aniline blue test and spectrophotometry were used to determine contents of ergosterol, 1,3-β-D-glucan and chitin from Microsporum canis, respectively. The results indicated that minimum inhibitory concentration of osthole against Microsporum canis was 1.95 µg/ml. HPLC analysis demonstrated that there was no significant change in the content of ergosterol between the osthole and control group. Compared with the control group, the content of 1,3-β-D-glucan and chitin in the cell wall of Microsporum canis was decreased. Taken together, this research showed that osthole could notably inhibit the growth of Microsporum canis and the antifungal activity was related to inhibiting the biosynthesis of 1,3-β-D-glucan and chitin in the cell wall of Microsporum canis, making osthole a potential future antifungal agent.

Assay for Heavy Metals Using an Inhibitive Assay Based on the Acetylcholinesterase from Puntius schwanenfeldii

Acetylcholinesterase (AChE) is frequently used as an inhibitive assay for insecticides. Relatively little is known about AChE inhibition properties on heavy metals. This present study assesses the potential of the AChE from the brain of Puntius schwanenfeldii, which forms as a waste from the aquaculture industry, as an inhibitive assay for heavy metals. The results of the study revealed that Ag+, Cu2+, and Hg2+ completely inhibited AChE activity during initial screening. Furthermore, when tested at various concentrations, the heavy metals demonstrated exponential decay type inhibition curves. The calculated IC50 for the heavy metals Ag+, Cu2+, and Hg2+ were observed to be 0.2498, 0.2427 and 0.2255 mg/L, respectively. The present assay for copper was comparable in sensitivity to assay methods such as immobilized urease, 15-min MicrotoxTM,48-h Daphnia magna, 96-h Rainbow trout, papain and bromelain assays while the present assay for mercury was significantly more sensitive than immobilized urease, equivalent in sensitivity to the rest of the assays, but less sensitive than the papain and bromelain assays. The present assay for silver was more sensitive to all of the assays with the exception of the rainbow trout assay. In conclusion, the findings in this study indicate that the assay, which can be carried out in less than 30 minutes at ambient temperature can be a useful assay for monitoring both insecticides and heavy metals pollution.

Difference in the PK of ceftiofur in the presence and absence of nimesulide and implications for dose determination through PK/PD integration

The study was planned to determine the pharmacokinetic (PK) and pharmacodynamic (PD) interactions between ceftiofur crystalline free acid (CCFA) and nimesulide (NS). The CCFA was administered to six adult non-lactating goats alone and in combination with NS in a two period cross-over study so that each goat receives each treatment. CCFA (6.6 mg/kg) and NS (2 mg/kg) were injected SC and IM, respectively. Blood samples were collected from jugular vein before and at 0, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 30, 36, 48, 72, 96, 120, 144, 168, 192, 240, 288 and 336 h post CCFA + NS administration. The MIC, MBC, MPC and ex-vivo antibacterial activity of ceftiofur against P. multocida were determined in presence of NS. PK-PD data were integrated and modelled to determine AUC24h/MIC values for three levels of effect on the bacteria to predict ceftiofur doses. The MIC, MBC, MPC and exvivo growth inhibition curves indicated time-dependent bactericidal activity of ceftiofur against P. multocida. The mean peak concentration (Cmax), absorption (K01HL) and terminal half-lives (K10HL) of ceftiofur were 2.89 μg/mL, 2.31 h and 54.8 h, respectively. The predicted AUC0-24h/MIC ratios required to produce bacteriostatic, bactericidal and eradication activity for P. multocida were higher (15.4, 70.1, 114.9 h) with CCFA alone compared to CCFA + NS treatment (12.8 h, 57.6 h, 92.1 h). The study supports using CCFA at 6.6 mg/kg in combination with NS to treat respiratory infectious diseases in goats caused by P. multocida since it may decrease the selection for resistant mutants.