Development of a chemically defined medium for Paenibacillus polymyxa by parallel online monitoring of the respiration activity in microtiter plates.

BACKGROUND: One critical parameter in microbial cultivations is the composition of the cultivation medium. Nowadays, the application of chemically defined media increases, due to a more defined and reproducible fermentation performance than in complex media. In order, to improve cost-effectiveness of fermentation processes using chemically defined media, the media should not contain nutrients in large excess. Additionally, to obtain high product yields, the nutrient concentrations should not be limiting. Therefore, efficient medium optimization techniques are required which adapt medium compositions to the specific nutrient requirements of microorganisms. RESULTS: Since most Paenibacillus cultivation protocols so far described in literature are based on complex ingredients, in this study, a chemically defined medium for an industrially relevant Paenibacillus polymyxa strain was developed. A recently reported method, which combines a systematic experimental procedure in combination with online monitoring of the respiration activity, was applied and extended to identify growth limitations for Paenibacillus polymyxa. All cultivations were performed in microtiter plates. By systematically increasing the concentrations of different nutrient groups, nicotinic acid was identified as a growth-limiting component. Additionally, an insufficient buffer capacity was observed. After optimizing the growth in the chemically defined medium, the medium components were systematically reduced to contain only nutrients relevant for growth. Vitamins were reduced to nicotinic acid and biotin, and amino acids to methionine, histidine, proline, arginine, and glutamate. Nucleobases/-sides could be completely left out of the medium. Finally, the cultivation in the reduced medium was reproduced in a laboratory fermenter. CONCLUSION: In this study, a reliable and time-efficient high-throughput methodology was extended to investigate limitations in chemically defined media. The interpretation of online measured respiration activities agreed well with the growth performance of samples measured in parallel via offline analyses. Furthermore, the cultivation in microtiter plates was validated in a laboratory fermenter. The results underline the benefits of online monitoring of the respiration activity already in the early stages of process development, to avoid limitations of medium components, oxygen limitation and pH inhibition during the scale-up.

Improvement of multicatalytic properties of nitrilase from Paraburkholderia graminis for efficient biosynthesis of 2-chloronicotinic acid.

Nitrilases are promising biocatalysts to produce high-value-added carboxylic acids through hydrolysis of nitriles. However, since the enzymes always show low activity and sometimes with poor reaction specificity toward 2-chloronicotinonitrile (2-CN), very few robust nitrilases have been reported for efficient production of 2-chloronicotinic acid (2-CA) from 2-CN. Herein, a nitrilase from Paraburkholderia graminis (PgNIT) was engineered to improve its catalytic properties. We identified the beneficial residues via computational analysis and constructed the mutant library. The positive mutants were obtained and the activity of the "best" mutant F164G/I130L/N167Y/A55S/Q260C/T133I/R199Q toward 2-CN was increased from 0.14 × 10-3  to 4.22 U/mg. Its reaction specificity was improved with elimination of hydration activity. Molecular docking and molecular dynamics simulation revealed that the conformational flexibility, the nucleophilic attack distance, as well as the interaction forces between the enzyme and substrate were the main reason alternating the catalytic properties of PgNIT. With the best mutant as biocatalyst, 150 g/L 2-CN was completely converted, resulting in 2-CA accumulated to 169.7 g/L. When the substrate concentration was increased to 200 g/L, 203.1 g/L 2-CA was obtained with yield of 85.7%. The results laid the foundation for industrial production of 2-CA with the nitrilase-catalyzed route.

Crystal structures of the M1 and M4 muscarinic acetylcholine receptors.

Muscarinic M1-M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.

Mechanism of 6-Hydroxynicotinate 3-Monooxygenase, a Flavin-Dependent Decarboxylative Hydroxylase Involved in Bacterial Nicotinic Acid Degradation.

6-Hydroxynicotinate 3-monooxygenase (NicC) is a Group A FAD-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP) with concomitant oxidation of NADH in nicotinic acid degradation by aerobic bacteria. Two mechanisms for the decarboxylative hydroxylation half-reaction have been proposed [Hicks, K., et al. (2016) Biochemistry 55, 3432-3446]. Results with Bordetella bronchiseptica RB50 NicC here show that a homocyclic analogue of 6-HNA, 4-hydroxybenzoic acid (4-HBA), is decarboxylated and hydroxylated by NicC with a 420-fold lower catalytic efficiency than is 6-HNA. The 13( V/ K), measured with wild-type NicC by isotope ratio mass spectrometry following the natural abundance of 13C in the CO2 product, is inverse for both 6-HNA (0.9989 ± 0.0002) and 4-HBA (0.9942 ± 0.0004) and becomes negligible (0.9999 ± 0.0004) for 5-chloro-6-HNA, an analogue that is 10-fold more catalytically efficient than 6-HNA. Covalently bound 6-HNA complexes of NicC are not observed by mass spectrometry. Comparative steady-state kinetic and Kd6HNA analyses of active site NicC variants (C202A, H211A, H302A, H47E, Y215F, and Y225F) identify Tyr215 and His47 as critical determinants both of 6-HNA binding ( KdY215F/ KdWT > 240; KdH47E/ KdWT > 350) and in coupling rates of 2,5-DHP and NAD+ product formation ([2,5-DHP]/[NAD+] = 1.00 (WT), 0.005 (Y215F), and 0.07 (H47E)]. Results of these functional analyses are in accord with an electrophilic aromatic substitution reaction mechanism in which His47-Tyr215 may serve as the general base to catalyze substrate hydroxylation and refine the structural model for substrate binding by NicC.

The Bordetella bronchiseptica nic locus encodes a nicotinic acid degradation pathway and the 6-hydroxynicotinate-responsive regulator BpsR.

The classical Bordetella species use amino acids as carbon sources and can catabolize organic acids and tricarboxylic acid cycle intermediates. They are also auxotrophic for nicotinamide adenine dinucleotide (NAD) pathway precursors such as nicotinic acid. Bordetellae have a putative nicotinate catabolism gene locus highly similar to that characterized in Pseudomonas putida KT2440. This study determined the distribution of the nic genes among Bordetella species and analyzed the regulation of this nicotinic acid degradation system. Transcription of the Bordetella bronchiseptica nicC gene was repressed by the NicR ortholog, BpsR, previously shown to regulate extracellular polysaccharide synthesis genes. nicC expression was derepressed by nicotinic acid or by the first product of the degradation pathway, 6-hydroxynicotinic acid, which was shown to be the inducer. Results using mutants with either a hyperactivated pathway or an inactivated pathway showed a marked effect on growth on nicotinic acid that indicated this degradation pathway influences NAD biosynthesis. Pathway dysregulation also affected Bordetella BvgAS-mediated virulence gene regulation, demonstrating that fluctuation of intracellular nicotinic acid pools impacts Bvg phase transition responses.

Stability indicating liquid chromatographic method for simultaneous quantification of betamethasone valerate and tazarotene in in vitro and ex vivo studies of complex nanoformulation.

Low-potency corticosteroid betamethasone valerate and vitamin-A tazarotene are used in combination for effective treatment of psoriasis. There is no robust high-performance liquid chromatography analytical technique available for simultaneous estimation of betamethasone valerate and tazarotene in conventional and nanocarriers based formulations. A simple, accurate, robust isocratic high-performance liquid chromatography method was developed for simultaneous estimation of betamethasone valerate and tazarotene in topical pharmaceutical formulations. The developed method was validated as per the regulatory guidelines. The validated method was linear over the concentration range of 150-6000 ng/mL (r2  > 0.999) at 239 nm wavelength. Limits of detection and quantification of two analytes were 50 and 150 ng/mL, respectively. The %relative standard deviation for intraday and interday precision was less than 2%. The method was also evaluated in the presence of forced degradation conditions. The developed method was successfully applied for in vitro and ex vivo drug release studies of in-house designed nanoformulations.

Metabolism and excretion of (S)-6-(3-cyclopentyl-2-(4-trifluoromethyl)-1H-imidazol-1-yl)propanamido)nicotinic acid (PF-04991532), a hepatoselective glucokinase activator, in humans: confirmation of the MIST potential noted in first-in-Human metabolite scouting studies.

1. The absorption, metabolism, and excretion of a single oral 450-mg dose of [14C]-(S)-6-(3-cyclopentyl-2-(4-trifluoromethyl)-1H-imidazol-1-yl)propanamido)nicotinic acid (PF-04991532), a hepatoselective glucokinase activator, was investigated in humans. Mass balance was achieved with ∼94.6% of the administered dose recovered in urine and feces. The total administered radioactivity excreted in feces and urine was 70.6% and 24.1%, respectively. Unchanged PF-04991532 collectively accounted for ∼47.2% of the dose excreted in feces and urine, suggestive of moderate metabolic elimination in humans. 2. The biotransformation pathways involved acyl glucuronidation (M1), amide bond hydrolysis (M3), and CYP3A4-mediated oxidative metabolism on the cyclopentyl ring in PF-04991532 yielding monohydroxylated isomers (M2a-d). Unchanged PF-04991532 was the major circulating component (64.4% of total radioactivity) whereas M2a-d collectively represented 28.9% of the total plasma radioactivity. 3. Metabolites M2a-d were not detected systemically in rats and dogs, the preclinical species for the toxicological evaluation of PF-04991532. In contrast, cynomologus monkeys dosed orally with unlabeled PF-04991532 revealed M2a-d in circulation, whose UV abundance was comparable to the profile in humans. This observation suggested that monkeys could potentially serve as a non-rodent alternative for studying the toxicity of PF-04991532 and its metabolites M2a-d. 4. The present results are in excellent agreement with our previously generated metabolite scouting data, which provided preliminary evidence for the disproportionate metabolism of PF-04991532 in humans.

Tissue-specific and maturity-dependent distribution of pyridine alkaloids in Areca triandra.

Areca nuts (seeds of Areca catechu L.) are a traditional and popular masticatory in India, Bangladesh, Malaysia, certain parts of China, and some other countries. Four related pyridine alkaloids (arecoline, arecaidine, guvacoline, and guvacine) are considered being the main functional ingredients in areca nut. Until now, A. catechu is the only known species producing these alkaloids in the Arecaceae family. In the present study, we investigated alkaloid contents in 12 Arecaceae species and found that only Areca triandra Roxb. contained these pyridine alkaloids. We further analyzed in more detail tissue-specific and development-related distribution of these alkaloids in leaves, male and female flowers and fruits in different stages of maturity in A. triandra by ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry. Results revealed that the alkaloids were most abundant in young leaves, the pericarp of ripe fruits and the endosperm of unripe fruits in developmental stage 2. Abundance of the 4 different alkaloids in A. triandra fruits varied during maturation. Pericarps of ripe fruits had the highest arecaidine concentration (4.45 mg g-1) and the lowest guvacoline concentration (0.0175 mg g-1), whereas the endosperm of unripe fruits of developmental stage 2 contained the highest guvacoline concentration (3.39 mg g-1) and the lowest guvacine concentration (0.245 mg g-1). We conclude that A. triandra is useful in future as a further valuable source of Areca alkaloids.

Development of a simple method for simultaneous determination of tazarotene and betamethasone dipropionate and their metabolites using LC-MS method and its application to dermatopharmacokinetic study.

In our study, a method for the determination for tazarotene and betamethasone dipropionate in human tissue-engineered skin was established. Tazarotene gel, betamethasone dipropionate cream or a combination cream was administered to the skin. Then the skin was taken off at 0.25, 0.75, 1.75, 3, 5, 8, 12, 24, 36, 48 h time points after the residual drug was removed. The concentrations of tazarotene, betamethasone dipropionate and their major metabolites in skin were determined by LC-MS. Tazarotene and tazarotenic acid were detected in the concentration range of 2-200 µg/mL with an LLOQ of 2 µg/mL. Betamethasone dipropionate was detected in the concentration range 0.5-300 µg/mL with an LLOQ of 0.5 µg/mL, and betamethasone was detected at 2-200 µg/mL with an LLOQ of 2 µg/mL. The intra- and inter-day precisions of the four analytes in the skin homogenate were all <15% (RSD, %). The results showed that tazarotene could be metabolized to tazarotenic acid and betamethasone dipropionate could be metabolized to betamethasone in tissue-engineered skin. The results also revealed that this method was suitable for the simultaneous determination of tazarotene, betamethasone dipropionate and their metabolites in tissue-engineered skin.

Comparison of the Photodegradation of Imazethapyr in Aqueous Solution, on Epicuticular Waxes, and on Intact Corn (Zea Mays) and Soybean (Glycine Max) Leaves.

A direct, controlled comparison of the photodegradation of imazethapyr has been made between imazethapyr in aqueous solutions, imazethapyr on the surface of epicuticular waxes of corn and soybean plants, and imazethapyr on the surface of intact corn and soybean plant leaves. In some experiments, the imazethapyr solutions were allowed to evaporate partially or fully after application to better model environmental conditions. The photodegradation of imazethapyr was fastest in aqueous solutions (k = 0.16 ± 0.02 h-1) and slowest on the surface of corn and soybean plants (kcorn = 0.00048 ± 0.001 h-1 and ksoy = 0.00054 ± 0.003 h-1). Experiments allowing evaporation during irradiation have intermediate rate constants (e.g., kcorn = 0.082 ± 0.005 h-1). Finally, identification of photoproducts was also examined on epicuticular waxes of corn and soybean plants for the first time.

Hyperthermophilic Archaeon Thermococcus kodakarensis Utilizes a Four-Step Pathway for NAD+ Salvage through Nicotinamide Deamination.

Many organisms possess pathways that regenerate NAD+ from its degradation products, and two pathways are known to salvage NAD+ from nicotinamide (Nm). One is a four-step pathway that proceeds through deamination of Nm to nicotinic acid (Na) by Nm deamidase and phosphoribosylation to nicotinic acid mononucleotide (NaMN), followed by adenylylation and amidation. Another is a two-step pathway that does not involve deamination and directly proceeds with the phosphoribosylation of Nm to nicotinamide mononucleotide (NMN), followed by adenylylation. Judging from genome sequence data, the hyperthermophilic archaeon Thermococcus kodakarensis is supposed to utilize the four-step pathway, but the fact that the adenylyltransferase encoded by TK0067 recognizes both NMN and NaMN also raises the possibility of a two-step salvage mechanism. Here, we examined the substrate specificity of the recombinant TK1676 protein, annotated as nicotinic acid phosphoribosyltransferase. The TK1676 protein displayed significant activity toward Na and phosphoribosyl pyrophosphate (PRPP) and only trace activity with Nm and PRPP. We further performed genetic analyses on TK0218 (quinolinic acid phosphoribosyltransferase) and TK1650 (Nm deamidase), involved in de novo biosynthesis and four-step salvage of NAD+, respectively. The ΔTK0218 mutant cells displayed growth defects in a minimal synthetic medium, but growth was fully restored with the addition of Na or Nm. The ΔTK0218 ΔTK1650 mutant cells did not display growth in the minimal medium, and growth was restored with the addition of Na but not Nm. The enzymatic and genetic analyses strongly suggest that NAD+ salvage in T. kodakarensis requires deamination of Nm and proceeds through the four-step pathway.IMPORTANCE Hyperthermophiles must constantly deal with increased degradation rates of their biomolecules due to their high growth temperatures. Here, we identified the pathway that regenerates NAD+ from nicotinamide (Nm) in the hyperthermophilic archaeon Thermococcus kodakarensis The organism utilizes a four-step pathway that initially hydrolyzes the amide bond of Nm to generate nicotinic acid (Na), followed by phosphoribosylation, adenylylation, and amidation. Although the two-step pathway, consisting of only phosphoribosylation of Nm and adenylylation, seems to be more efficient, Nm mononucleotide in the two-step pathway is much more thermolabile than Na mononucleotide, the corresponding intermediate in the four-step pathway. Although NAD+ itself is thermolabile, this may represent an example of a metabolism that has evolved to avoid the use of thermolabile intermediates.

Identification and characterization of a new three-component nicotinic acid hydroxylase NahAB1 B2 from Pusillimonas sp. strain T2.

Nicotinic acid (NA) is ubiquitous in nature and its microbial degradation mechanisms are diverse. In this study, Pusillimonas sp. strain T2 was found to be capable of utilizing NA as sole carbon and nitrogen sources. This strain could completely degrade 300 mg l-1 NA within 3·5 h at 30°C and pH 7·0 and one of the degradation intermediate of NA was identified as 6-hydroxynicotinic acid (6HNA). The draft genome sequences of strain T2 were determined to have a total length of 3·3 M bp and 3054 proteins were predicted. The encoding genes of three-component NA hydroxylase (NahAB1 B2 ) genes were identified. The nahAB1 B2 genes were heterologously expressed in the non-NA-degrading Shinella sp. strain HZN7. The recombinant HZN7-pBBR-nahAB1 B2 converted NA into equimolar 6HNA, while the recombinants HZN7-pBBR-nahAB1 (lacking component B2 ) and HZN7-pBBR-nahAB2 (lacking component B1 ) could not convert NA. Cell-free extracts of HZN7-pBBR-nahAB1 B2 exhibited NA hydroxylase activity. After addition of an artificial electron acceptor (such as phenazine methosulphate, PMS), the NA hydroxylase activity was significantly increased. The Km and Vmax values for NA were 65·94 µmol l-1 and 260·80 ± 5·69 mU mg-1 , respectively, using PMS as an electron acceptor. This study provides a novel insight into the NA degradation by bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Nicotinic acid (NA) serves as a model system for the degradation of N-heterocyclic aromatic compounds and the microbial degradation mechanisms are diverse. This is the first time that a three-component hydroxylase has been identified. This study provides a novel insight into the NA degradation by bacteria.

1H NMR Metabolic Profiling of Earthworm (Eisenia fetida) Coelomic Fluid, Coelomocytes, and Tissue: Identification of a New Metabolite-Malylglutamate.

Earthworm metabolism is recognized as a useful tool for monitoring environmental insults and measuring ecotoxicity, yet extensive earthworm metabolic profiling using 1H nuclear magnetic resonance (NMR) spectroscopy has been limited in scope. This study aims to expand the embedded metabolic material in earthworm coelomic fluid, coelomocytes, and tissue to aid systems toxicology research. Fifty-nine metabolites within Eisenia fetida were identified, with 47 detected in coelomic fluid, 41 in coelomocytes, and 54 in whole-worm samples and tissue extracts. The newly detected but known metabolites 2-aminobutyrate, nicotinurate, Nδ,Nδ,Nδ-trimethylornithine, and trigonelline are reported along with a novel compound, malylglutamate, elucidated using 2D NMR and high-resolution MS/MS. We postulate that malylglutamate acts as a glutamate/malate store, chelator, and anionic osmolyte and helps to provide electrolyte balance.

Integrated Analytical and Statistical Two-Dimensional Spectroscopy Strategy for Metabolite Identification: Application to Dietary Biomarkers.

A major purpose of exploratory metabolic profiling is for the identification of molecular species that are statistically associated with specific biological or medical outcomes; unfortunately, the structure elucidation process of unknowns is often a major bottleneck in this process. We present here new holistic strategies that combine different statistical spectroscopic and analytical techniques to improve and simplify the process of metabolite identification. We exemplify these strategies using study data collected as part of a dietary intervention to improve health and which elicits a relatively subtle suite of changes from complex molecular profiles. We identify three new dietary biomarkers related to the consumption of peas (N-methyl nicotinic acid), apples (rhamnitol), and onions (N-acetyl-S-(1Z)-propenyl-cysteine-sulfoxide) that can be used to enhance dietary assessment and assess adherence to diet. As part of the strategy, we introduce a new probabilistic statistical spectroscopy tool, RED-STORM (Resolution EnhanceD SubseT Optimization by Reference Matching), that uses 2D J-resolved 1H NMR spectra for enhanced information recovery using the Bayesian paradigm to extract a subset of spectra with similar spectral signatures to a reference. RED-STORM provided new information for subsequent experiments (e.g., 2D-NMR spectroscopy, solid-phase extraction, liquid chromatography prefaced mass spectrometry) used to ultimately identify an unknown compound. In summary, we illustrate the benefit of acquiring J-resolved experiments alongside conventional 1D 1H NMR as part of routine metabolic profiling in large data sets and show that application of complementary statistical and analytical techniques for the identification of unknown metabolites can be used to save valuable time and resources.

Characterization and Genome Analysis of a Nicotine and Nicotinic Acid-Degrading Strain Pseudomonas putida JQ581 Isolated from Marine.

The presence of nicotine and nicotinic acid (NA) in the marine environment has caused great harm to human health and the natural environment. Therefore, there is an urgent need to use efficient and economical methods to remove such pollutants from the environment. In this study, a nicotine and NA-degrading bacterium-strain JQ581-was isolated from sediment from the East China Sea and identified as a member of Pseudomonas putida based on morphology, physio-biochemical characteristics, and 16S rDNA gene analysis. The relationship between growth and nicotine/NA degradation suggested that strain JQ581 was a good candidate for applications in the bioaugmentation treatment of nicotine/NA contamination. The degradation intermediates of nicotine are pseudooxynicotine (PN) and 3-succinoyl-pyridine (SP) based on UV, high performance liquid chromatography, and liquid chromatography-mass spectrometry analyses. However, 6-hydroxy-3-succinoyl-pyridine (HSP) was not detected. NA degradation intermediates were identified as 6-hydroxynicotinic acid (6HNA). The whole genome of strain JQ581 was sequenced and analyzed. Genome sequence analysis revealed that strain JQ581 contained the gene clusters for nicotine and NA degradation. This is the first report where a marine-derived Pseudomonas strain had the ability to degrade nicotine and NA simultaneously.

Biodegradation of imidacloprid in liquid media by an isolated wastewater fungus Aspergillus terreus YESM3.

In the present study, a new fungal strain capable of imidacloprid degradation was isolated from agricultural wastewater drain. The fungal strain of YESM3 was identified as Aspergillus terreus based on ITS1-5.8S rDNA-ITS2 gene sequence by PCR amplification of a 500 bp sequence. Screening of A. terreus YESM3 to the insecticide imidacloprid tolerance was achieved by growing fungus in Czapek Dox agar for 6 days at 28°C. High values (1.13 and 0.94 cm cm-1) of tolerance index (TI) were recorded at 25 and 50 mg L-1 of imidacloprid, respectively in the presence and absence of sucrose. However, at 400 mg L-1 the fungus did not grow. Effects of the imidacloprid concentration, pH, and inoculum size on the biodegradation percentage were tested using Box-Behnken statistical design and the biodegradation was monitored by HPLC analysis at different time intervals. Box-Behnken results indicated that optimal conditions for biodegradation were at pH 4 and two fungal discs (10 mm diameter) in the presence of 61.2 mg L-1 of imidacloprid. A. terreus YESM3 strain was capable of degrading 85% of imidacloprid 25 mg L-1 in Czapek Dox broth medium at pH 4 and 28°C for 6 days under static conditions. In addition, after 20 days of inoculation, biodegradation recorded 96.23% of 25 mg L-1 imidacloprid. Degradation kinetics showed that the imidacloprid followed the first order kinetics with half-life (t50) of 1.532 day. Intermediate product identified as 6-chloronicotinic acid (6CNA) as one of the major metabolites during degradation of imidacloprid by using HPLC. Thus, A. terreus YESM3 showed a potential to reduce pollution by pesticides and toxicity in the effected environment. However, further studies should be conducted to understand the biodegradation mechanism of this pesticide in liquid media.

Imazethapyr persistence in sandy loam detected using white mustard bioassay.

Field experiments were conducted during two years at Srem region to investigate the influence of meteorological conditions, time and rate of application on soil persistence of imazethapyr in sandy loam type of soil. Imazethapyr was applied PRE- and POST-EM and in both cases in three application rates: 80, 120 and 160 g a.i./ha. Soil samples were collected from the day of herbicide application in predetermined intervals up to one year after application and residual concentrations were determined with a white mustard root bioassay. Imazetapyr persistence was significantly influenced by meteorological conditions with average half-life being 6 days longer in season with lower precipitation level. Time of application induced slower imazethapyr dissipation resulting in higher average t1/2 (seven and nine days in first and second year of examination, respectively). Application rates had no consistent effect on imazethapyr persistence. Imazethapyr residue level one year after application caused no visible injuries on white mustard shoots, while root growth reduction ranged from 4.6 to 27.7%. Obtained residue levels were further compared with known data on crop sensitivity in order to assess possibility of crop injuries one year after imazethapyr application.

Identification of Tazarotenic Acid as the First Xenobiotic Substrate of Human Retinoic Acid Hydroxylase CYP26A1 and CYP26B1.

Cytochrome P450 (CYP) 26A1 and 26B1 are heme-containing enzymes responsible for metabolizing all-trans retinoic acid (at-RA). No crystal structures have been solved, and therefore homology models that provide structural information are extremely valuable for the development of inhibitors of cytochrome P450 family 26 (CYP26). The objectives of this study were to use homology models of CYP26A1 and CYP26B1 to characterize substrate binding characteristics, to compare structural aspects of their active sites, and to support the role of CYP26 in the metabolism of xenobiotics. Each model was verified by dockingat-RA in the active site and comparing the results to known metabolic profiles ofat-RA. The models were then used to predict the metabolic sites of tazarotenic acid with results verified by in vitro metabolite identification experiments. The CYP26A1 and CYP26B1 homology models predicted that the benzothiopyranyl moiety of tazarotenic acid would be oriented toward the heme of each enzyme and suggested that tazarotenic acid would be a substrate of CYP26A1 and CYP26B1. Metabolite identification experiments indicated that CYP26A1 and CYP26B1 oxidatively metabolized tazarotenic acid on the predicted moiety, with in vitro rates of metabolite formation by CYP26A1 and CYP26B1 being the highest across a panel of enzymes. Molecular analysis of the active sites estimated the active-site volumes of CYP26A1 and CYP26B1 to be 918 Å(3)and 977 Å(3), respectively. Overall, the homology models presented herein describe the enzyme characteristics leading to the metabolism of tazarotenic acid by CYP26A1 and CYP26B1 and support a potential role for the CYP26 enzymes in the metabolism of xenobiotics.

Optimizing the dermal accumulation of a tazarotene microemulsion using skin deposition modeling.

CONTEXT: It is well known that microemulsions are mainly utilized for their transdermal rather than their dermal drug delivery potential due to their low viscosity, and the presence of penetration enhancing surfactants and co-surfactants. OBJECTIVE: Applying quality by design (QbD) principles, a tazarotene microemulsion formulation for local skin delivery was optimized by creating a control space. MATERIALS AND METHODS: Critical formulation factors (CFF) were oil, surfactant/co-surfactant (SAA/CoS), and water percentages. Critical quality attributes (CQA) were globular size, microemulsion viscosity, tazarotene skin deposition, permeation, and local accumulation efficiency index. RESULTS AND DISCUSSION: Increasing oil percentage increased globular size, while the opposite occurred regarding SAA/CoS, (p = 0.001). Microemulsion viscosity was reduced by increasing oil and water percentages (p < 0.05), due to the inherent high viscosity of the utilized SAA/CoS. Drug deposition in the skin was reduced by increasing SAA/CoS due to the increased hydrophilicity and viscosity of the system, but increased by increasing water due to hydration effect (p = 0.009). Models with very good fit were generated, predicting the effect of CFF on globular size, microemulsion viscosity, and drug deposition. A combination of 40% oil and 45% SAA/CoS showed the maximum drug deposition of 75.1%. Clinical skin irritation study showed that the aforementioned formula was safe for topical use. CONCLUSION: This article suggests that applying QbD tools such as experimental design is an efficient tool for drug product design.

Occurrence and Profile Characteristics of the Pesticide Imidacloprid, Preservative Parabens, and Their Metabolites in Human Urine from Rural and Urban China.

Knowledge of human exposure to imidacloprid, the most extensively used insecticide, and para-hydroxybenzoic acid esters (parabens), the most extensively used preservative, is insufficient. In this study, 295 urine samples collected from subjects in rural and urban areas in China were analyzed for imidacloprid and four parabens (namely, methyl paraben, ethyl paraben, propyl paraben, and butyl paraben) as well as their major metabolites (namely, 6-chloronicotinic acid (6-ClNA) and para-hydroxybenzoic acid (p-HB)). Imidacloprid was detected in 100% of the urine samples from rural Chinese subjects and 95% of the urine samples from urban Chinese subjects. Concentrations of urinary imidacloprid detected in rural Chinese subjects (geometric mean (GM) = 0.18 ng/mL) were slightly higher than those detected in urban Chinese subjects (GM = 0.15 ng/mL) when the effect of pesticide spraying was excluded. However, concentrations of urinary imidacloprid detected in rural adults increased significantly in the subsequent days of pesticide spraying (GM = 0.62 ng/mL), which could return to the normal levels within 3 days. In contrast, concentrations of urinary parabens detected in rural Chinese subjects (GM = 6.90 ng/mL) were lower than that in urban Chinese subjects (GM = 30.5 ng/mL). In addition, the metabolism characteristics of imidacloprid to 6-ClNA and parabens to p-HB were discussed preliminarily.