A cytochrome P450 system initiates 4-nitroanisole degradation in Rhodococcus sp. strain JS3073.

Nitroanisoles are used widely as synthetic intermediates and explosives. Although bacteria have been reported to degrade 4-nitroanisole (4NA) under aerobic conditions, the key enzymes and the catalytic mechanism have remained elusive. Rhodococcus sp. strain JS3073 was isolated for its ability to grow on 4NA as the sole carbon and energy source. In this study, whole cell biotransformation experiments indicated that 4NA degradation is initiated by O-demethylation to form 4-nitrophenol (PNP), which undergoes subsequent degradation by a previously established pathway involving formation of 1,2,4-benzenetriol and release of nitrite. Based on comparative transcriptomics and heterologous expression, a novel three-component cytochrome P450 system encoded by pnaABC initiates the O-demethylation of 4NA to yield formaldehyde and PNP. The pnaABC genes encode a phthalate dioxygenase type reductase (PnaA), a cytochrome P450 monooxygenase (PnaB), and an EthD family protein (PnaC) with putative function similar to ferredoxins. This unusual P450 system also has a broad substrate specificity for nitroanisole derivatives. Sequence analysis of PnaAB revealed high identity with multiple self-sufficient P450s of the CYP116B subfamily. The findings revealed the molecular basis of the catabolic pathway for 4NA initiated by an unusual O-demethylase PnaABC and extends the understanding of the diversity among P450s and their electron transport chains.

Biodegradation of insensitive munition (IM) formulations: IMX-101 and IMX-104 using aerobic granule technology.

A laboratory-scale aerobic granular sludge (AGS) sequencing batch bioreactor (SBR) was initiated in this study for the biodegradation of hazardous insensitive munition (IM) formulation constituents; 2,4-dinitroanisole (DNAN), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 1-nitroguanidine (NQ), and 3-nitro-1,2,4-triazol-5-one (NTO). Efficient (bio)transformation of the influent DNAN and NTO was achieved throughout reactor operation with removal efficiencies greater than 95%. An average removal efficiency of 38.4 ± 17.5% was recorded for RDX. NQ was only slightly removed (3.96 ± 4.15%) until alkalinity was provided in the influent media, which subsequently increased the NQ removal efficiency up to an average of 65.8 ± 24.4%. Batch experiments demonstrated a competitive advantage for aerobic granular biofilms over flocculated biomass for the (bio)transformation DNAN, RDX, NTO, and NQ, as aerobic granules were capable of reductively (bio)transforming each IM compound under bulk aerobic conditions while flocculated biomass could not, thus demonstrating the contribution of inner oxygen-free zones within aerobic granules. A variety of catalytic enzymes were identified in the extracellular polymeric matrix of the AGS biomass. 16 S rDNA amplicon sequencing found Proteobacteria (27.2-81.2%) to be the most abundant phyla, with many genera associated with nutrient removal as well as genera previously described in relation to the biodegradation of explosives or related compounds.

New derivatives from dehydrodieugenol B and its methyl ether displayed high anti-Trypanosoma cruzi activity and cause depolarization of the plasma membrane and collapse the mitochondrial membrane potential.

In the present work, dehydrodieugenol B (1) and its methyl ether (2), isolated from Nectandra leucantha twigs, were used as starting material for the preparation of two new derivatives (1a and 2a) containing an additional methoxycarbonyl unit on allyl side chains. Compounds 1a and 2a demonstrated activity against trypomastigotes (EC50 values of 13.5 and 23.0 µM, respectively) and against intracellular amastigotes (EC50 values of 10.2 and 6.1 µM, respectively). Additionally, compound 2a demonstrated no mammalian cytotoxicity up to 200 µM whereas compound 1a exhibited a CC50 value of 139.8 µM. The mechanism of action studies of compounds 1a and 2a demonstrated a significant depolarization of the plasma membrane potential in trypomastigotes, followed by a mitochondrial membrane potential collapse. Neither calcium level nor reactive oxygen species alterations were observed after a short-time incubation. Considering the potential of compound 2a against T. cruzi and its simple preparation from the natural product 2, isolated from N. leucantha, this compound could be considered a new hit for future drug design studies in Chagas disease.

Designing bacterial consortia for the complete biodegradation of insensitive munitions compounds in waste streams.

Insensitive munitions compounds (IMCs), such as 2,4-dinitroanisole (DNAN) and 3-nitro-1,2,4-triazol-5-one (NTO), are replacing conventional explosives in munitions formulations. Manufacture and use of IMCs generate waste streams in manufacturing plants and load/assemble/pack facilities. There is a lack of practical experience in executing biodegradation strategies to treat IMCs waste streams. This study establishes a proof-of-concept that bacterial consortia can be designed to mineralize IMCs and co-occurring nitroaromatics in waste streams. First, DNAN, 4-nitroanisole (4-NA), and 4-chloronitrobenzene (4-CNB) in a synthetic DNAN-manufacturing waste stream were biodegraded using an aerobic fluidized-bed reactor (FBR) inoculated with Nocardioides sp. JS 1661 (DNAN degrader), Rhodococcus sp. JS 3073 (4-NA degrader), and Comamonadaceae sp. LW1 (4-CNB degrader). No biodegradation was detected when the FBR was operated under anoxic conditions. Second, DNAN and NTO were biodegraded in a synthetic load/assemble/pack waste stream during a sequential treatment comprising: (i) aerobic DNAN biodegradation in the FBR; (ii) anaerobic NTO biotransformation to 3-amino-1,2,4-triazol-5-one (ATO) by an NTO-respiring enrichment; and (iii) aerobic ATO mineralization by an ATO-oxidizing enrichment. Complete biodegradation relied on switching redox conditions. The results provide the basis for designing consortia to treat mixtures of IMCs and related waste products by incorporating microbes with the required catabolic capabilities.

Modeling the hydroxylation of estragole via human liver cytochrome P450.

Natural compounds derived from plants are generally regarded safe and devoid of adverse effects. However, there are individual ingredients that possess toxic, genotoxic, and carcinogenic activities. These compounds when exposed at specific level become hazardous to health. Estragole (1-allyl-4-methoxybenzene) is a common component of spice plants. Its toxicity gets activated with the hydroxylation at benzylic carbon (C1') position by P450 enzymes present in the human liver. The present study grounds to explore the reaction mechanism of conversion of estragole to hydroxylated metabolite using computational methodology. Density functional theory (DFT)-based calculations were employed to explore the cytochrome P450-catalyzed mechanism at C1 position aliphatic hydroxylation of estragole. Overall reaction energy profile, electronic configuration, and 3D structure of all intermediates, transition states, and product complexes formed during the reaction along with their free energies were tried to be investigated.

Bioactivation of estragole and anethole leads to common adducts in DNA and hemoglobin.

Estragole and anethole are secondary metabolites occurring in a variety of commonly used herbs like fennel, basil, and anise. Estragole is genotoxic and carcinogenic in rodents, which depends on the formation of 1'-sulfoxyestragole after hydroxylation and subsequent sulfoconjugation catalyzed by CYP and SULT, respectively. It was hypothesized recently that anethole may be bioactivated via the same metabolic pathways. Incubating estragole with hepatic S9-fractions from rats and humans, specific adducts with hemoglobin (N-(isoestragole-3-yl)-valine, IES-Val) and DNA (isoestragole-2'-deoxyguanosine and isoestragole-2'-deoxyadenosine) were formed. An isotope-dilution technique was developed for the quantification of IES-Val after cleavage with fluorescein isothiocyanate (FITC) according to a modified Edman degradation. The same adducts, albeit at lower levels, were also detected in reactions with anethole, indicating the formation of 3'-hydroxyanethole and the reactive 3'-sulfoxyanethole. Finally, we conducted a pilot investigation in which IES-Val levels in human blood were determined during and after the consumption of an estragole- and anethole-rich fennel tea for four weeks. A significant increase of IES-Val levels was observed during the consumption phase and followed by a continuous decrease during the washout period. IES-Val may be used to monitor the internal exposure to the common reactive genotoxic metabolites of estragole and anethole, 1'-sulfoxyestragole and 3'-sulfoxyanethole, respectively.

Impact of physiologically relevant temperatures on dermal absorption of active substances - an ex-vivo study in human skin.

Skin temperature plays a certain role in the dermal absorption of substances, but the extent and mechanisms of skin temperatures-induced modulation in ranges caused by physiological thermoregulation or environmental conditions are largely unknown. The influence of dermal temperature on the absorption of the model lipophilic compound (anisole) and the model hydrophilic compounds (1,4-dioxane, ethanol) through human skin was investigated at three dermal temperatures (25, 32 and 39 °C) in an ex-vivo diffusion cell model. The substances were applied to the skin and transdermal penetration was monitored. All substances showed temperature dependent variations in their penetration behavior (3 h: 25-39 °C: 202-275% increase in cumulative, transdermally penetrated amounts). The relative differences in absorption in relation to temperature were greatest within 45 min after exposure (25-39 °C: 347-653% rise in cumulated penetration), although absolute amounts absorbed were small (45 min vs. 3 h: 4.5-14.5%). Regardless of blood circulation, skin temperature significantly influences the amount and kinetics of dermal absorption. Substance-dependent, temperature-related changes of the lipid layer order or the porous pathway may facilitate penetration. The early-stage modulation of transdermal penetration indicates transappendageal absorption, which may be relevant for short-term exposures. For both, toxicological evaluation and perfusion cell studies, it is important to consider the thermal influence on absorption or to perform the latter at a standardized temperature (32±1 °C).

Evidence from stable-isotope labeling that catechol is an intermediate in salicylic acid catabolism in the flowers of Silene latifolia (white campion).

MAIN CONCLUSION: A stable isotope-assisted mass spectrometry-based platform was utilized to demonstrate that the plant hormone, salicylic acid, is catabolized to catechol, a widespread secondary plant compound. The phytohormone salicylic acid (SA) plays a central role in the overall plant defense program, as well as various other aspects of plant growth and development. Although the biosynthetic steps toward SA are well documented, how SA is catabolized in plants remains poorly understood. Accordingly, in this study a series of stable isotope feeding experiments were performed with Silene latifolia (white campion) to explore possible routes of SA breakdown. S. latifolia flowers that were fed a solution of [2H6]-salicylic acid emitted the volatile and potent pollinator attractant, 1,2-dimethoxybenzene (veratrole), which contained the benzene ring-bound deuterium atoms. Extracts from these S. latifolia flowers revealed labeled catechol as a possible intermediate. After feeding flowers with [2H6]-catechol, the stable isotope was recovered in veratrole as well as its precursor, guaiacol. Addition of a trapping pool of guaiacol in combination with [2H6]-salicylic acid resulted in the accumulation of the label into catechol. Finally, we provide evidence for catechol O-methyltransferase enzyme activity in a population of S. latifolia that synthesizes veratrole from guaiacol. This activity was absent in non-veratrole emitting flowers. Taken together, these results imply the conversion of salicylic acid to veratrole in the following reaction sequence: salicylic acid > catechol > guaiacol > veratrole. This catabolic pathway for SA may also be embedded in other lineages of the plant kingdom, particularly those species which are known to accumulate catechol.

DNA double strand break repair as cellular response to genotoxic asarone isomers considering phase I metabolism.

The phenylpropenes α-asarone and ß-asarone are widely spread in the marsh plant Acorus calamus. Both isomers are classified as carcinogenic in rodents. However, the respective genotoxic mechanisms are not elucidated so far. The present study gives deeper insights into the genotoxic effects of asarone isomers as well as their known oxidative phase I metabolites, (E)-3'-oxoasarone and asarone epoxide. We show that asarone metabolites highly increase DNA strand breaks after 1 h of incubation, markedly metabolic activation contributes to their carcinogenic mode of action. All test compounds act as aneugens and potently enhance the amounts of micronuclei in binuclear cells. However, a prolonged incubation time of 24 h results in a decrease of DNA damage. This work suggests that asarone metabolites also induce DNA double strand breaks , why we put a strong focus on homologous recombination and non-homologous end joining. The obtained results herein indicate that asarone epoxide-induced DNA strand breaks are repaired via a homologous repair pathway.

ß­asarone modulates Beclin­1, LC3 and p62 expression to attenuate Aß40 and Aß42 levels in APP/PS1 transgenic mice with Alzheimer's disease.

Alzheimer's disease (AD) is a common neurodegenerative disease in the elderly population. Autophagy is a well­known regulator of neurodegenerative diseases and ß­asarone has been discovered to have certain neuropharmacological effects. Thus, the present study aimed to analyze the potential effects of ß­asarone in AD and its possible mechanism of action in relation to autophagy. The present study investigated the effects of ß­asarone on the number of senile plaques and amyloid ß(Aß)40, Aß42, amyloid precursor protein (APP) and Beclin­1 mRNA levels in the hippocampus of APP/presenilin­1 (PS1) transgenic mice. The possible mechanism of ß­asarone on autophagy­related proteins, including Beclin­1, light chain (LC)3A, LC3B and p62 levels, and the number of autophagosomes was also investigated. Mice were divided into a normal control group, a model group, a ß­asarone­treated group, a 3­MA­treated group and a rapamycin­treated group. Treatments were continuously administered to all mice for 30 days by intragastric administration. The mice, including those in the normal and model control groups, were given equal volumes of saline. It was demonstrated that ß­asarone treatment reduced the number of senile plaques and autophagosomes, and decreased Aß40, Aß42, APP and Beclin­1 expression in the hippocampus of model mice compared with untreated model mice. ß­asarone also inhibited LC3A/B expression levels, but increased p62 expression. It was deduced that the neuroprotective effects of ß­asarone in APP/PS1 transgenic mice resulted from its inhibition of autophagy. In conclusion, the data suggested that ß­asarone should be explored further as a potential therapeutic agent in AD.

Unravelling the antifungal and anti-aflatoxin B1 mechanism of chitosan nanocomposite incorporated with Foeniculum vulgare essential oil.

The study reports the antifungal and aflatoxin B1 inhibitory efficacy of chemically characterized chitosan-based nanoencapsulated Foeniculum vulgare Mill. essential oil (Ne-FvEO), and its effect on sensory and nutritional properties of Sorghum bicolor. The Ne-FvEO was characterized through SEM, FTIR and XRD. The Ne-FvEO demonstrated superior antifungal (0.4 µl/ml) and aflatoxin B1 inhibitory (0.3 µl/ml) performance than the free FvEO. The biochemical studies reveal the significant alteration in ergosterol content, ions leakage, mitochondrial membrane potential, C-sources utilization and antioxidant defense system in A. flavus exposed to Ne-FvEO. The 3D modeling of the Nor-1 gene product was done using the I-TASSER server and validated by the Ramachandran plot. The in-situ result reveals that the Ne-FvEO significantly preserved the nutritional and sensory characteristics of S. bicolor seeds. Therefore, Ne-FvEO could be used as a green preservative agent to enhance the shelf-life of the food commodities.

Metabolism of carcinogenic alpha-asarone by human cytochrome P450 enzymes.

Major metabolites of alpha-asarone in liver microsomes are epoxide-derived side-chain diols. The intermediately formed epoxides are mutagenic and form DNA adducts and thus are likely responsible for the (hepato) carcinogenic effect of alpha-asarone observed in male mice. We here investigated the role of eight human cytochrome P450 enzymes (CYP1A1, 1A2, 2A6, 2B6, 2C19, 2D6, 2E1, and 3A4) in the metabolism of alpha-asarone using Supersomes™. The epoxidation of the side-chain of alpha-asarone was mainly catalyzed by CYP3A4 and to a lesser extent by 2B6 and 1A1 whereas the hydroxylation of the side-chain leading to (E)-3'-hydroxyasarone was catalyzed by all investigated CYPs excluding CYP2A6. O-demethylation was catalyzed by CYP1A1, 2A6, 2B6, and 2C19. Applying relative activity factors (RAF) to the observed formation rates revealed that CYP3A4, at least at lower substrate concentrations, is nearly solely responsible for the formation of the mutagenic side-chain epoxides of alpha-asarone. Comparison of the RAF-corrected formation rates of all metabolites with those found in incubation with human liver microsomes revealed that the side-chain hydroxylation and epoxidation can be explained in good approximation by the tested hepatic CYPs, whereas other CYPs or enzymes may contribute to the O-demethylation of alpha-asarone. Therefore, the capacity for metabolic activation of alpha-asarone has to be expected to be widely present among the general population.

Mechanism of H2S Formation from the Metabolism of Anetholedithiolethione and Anetholedithiolone by Rat Liver Microsomes.

The drug anetholedithiolethione (ADT) and its analogs have been extensively used as H2S donors. However, the mechanism of H2S formation from ADT under biologic conditions remains almost completely unknown. This article shows that only small amounts of H2S are formed during incubation of ADT and of its metabolite anetholedithiolone (ADO) with rat liver cytosol or with rat liver microsomes (RLM) in the absence of NADPH, indicating that H2S formation under these conditions is of hydrolytic origin only to a minor extent. By contrast, much greater amounts of H2S are formed upon incubation of ADT and ADO with RLM in the presence of NADPH and dioxygen, with a concomitant formation of H2S and para-methoxy-acetophenone (pMA). Moreover, H2S and pMA formation under those conditions are greatly inhibited in the presence of N-benzyl-imidazole indicating the involvement of cytochrome P450-dependent monooxygenases. Mechanistic studies show the intermediate formation of the ADT-derived 1,2-dithiolium cation and of the ADO sulfoxide during microsomal metabolism of ADT and ADO, respectively. This article proposes the first detailed mechanisms for the formation of H2S from microsomal metabolism of ADT and ADO in agreement with those data and with previously published data on the metabolism of compounds involving a C=S bond. Finally, this article shows for the first time that ADO is a better H2S donor than ADT under those conditions. SIGNIFICANCE STATEMENT: Incubation of anetholedithiolethione (ADT) or its metabolite anetholedithiolone (ADO) in the presence of rat liver microsomes, NADPH, and O2 leads to H2S. This article shows for the first time that this H2S formation involves several steps catalyzed by microsomal monooxygenases and that ADO is a better H2S donor than ADT. We propose the first detailed mechanisms for the formation of H2S from the microsomal metabolism of ADT and ADO.

Formation of odorant haloanisoles and variation of microorganisms during microbial O-methylation in annular reactors equipped with different coupon materials.

Taste and odor (T & O) issues in drinking water have become serious problems which cannot be ignored by customers. Several studies have confirmed that microbes in water can biotransform halophenols (HPs) to haloanisoles (HAs) with earthy and musty flavors via microbial O-methylation. In this paper, the formation of 2-chloroanisole (2-CA), 2,4-dichloroanisole (2,4-DCA), 2,4,6-trichloroanisole (2,4,6-TCA), 2,3,6-trichloroanisole (2,3,6-TCA) and 2,4,6-tribromoanisole (2,4,6-TBA), and the microbial variation during the microbial O-methylation were investigated in annular reactors (ARs) with three coupon materials. For precursors, 42.5% of 2-CP and 68.9% of 2,4-DCP decayed during the reaction. Among the five HAs, the formation rate constant followed an order of 2,4,6-TCA > 2-CA > 2,4,6-TBA > 2,4-DCA ~ 2,3,6-TCA, while [HA]max followed a totally opposite one. The simulated flow velocity had no significant effect (p > 0.05) on HA formation. Ductile iron (DI) AR could produce more HAs than stainless steel (SS) and polyvinyl chloride (PVC) ARs. The final HA molar concentration followed an order of 2,3,6-TCA > 2,4-DCA > 2,4,6-TBA ~ 2,4,6-TCA > 2-CA, which might be explained by multiple factors including HP's dissociation degree, halogen atom's steric hindrance and specificity of HP O-methyltransferases. During the reaction, the microbial biomass dramatically increased 6.8-9.0 times in bulk water but dropped significantly on coupon biofilms. The effect of HPs significantly changed the bacterial communities on coupon in terms of composition and diversity, and declined the relative abundance of HA-producing bacteria, while fungi and their HA-producing genus showed better resistance ability towards HPs. By using Pearson correlation analysis, a significant correlation (p = 0.0003) was found between [HA]max and initial coupon biofilm biomass. Finally, a linear relationship was established between initial total biomass and HA formation potential.

Asarone and metformin delays experimentally induced hepatocellular carcinoma in diabetic milieu.

AIMS: The evidence suggests that the hyperglycemia and hyperinsulinemia of diabetes mellitus (DM) are risk factors for the development of hepatocellular carcinoma (HCC). The aim of the present study was to examine the effect of streptozotocin (STZ)-induced DM on promoting diethylnitrosamine (DEN) induced HCC in male wistar rats. Further, we investigated the administration of (α)-and (ß)-asarone and metformin HCl on experimentally induced diabetic-hepatocellular carcinoma. MATERIALS AND METHODS: Diabetes was induced by single dose of STZ (55 mg/2 ml/kg b.w. i.p.) and HCC by single dose of DEN (200 mg/ml/kg b.w. i.p.). Another group received the STZ followed by DEN two weeks later to mimic diabetic-HCC. The combined dose of (α)-and (ß)-asarone (50 µg/1.5 ml/kg b.w. p.o. in the ratio of 1:1) and metformin HCl (250 mg/1.5 ml/kg b.w. p.o.) treatment was compared with the STZ + DEN group. The blood and liver samples were collected at the end of 12 and 18-weeks to study biochemical and histopathological changes in liver. KEY FINDINGS: The STZ induced diabetes promoted the tumor progression due to administration of DEN. The treatment of asarones and metformin significantly reduced the levels of glucose, glycosylated hemoglobin, liver dysfunction markers and tumor biomarkers along with an increase in level of insulin when compared to diabetic-HCC group. Histopathological examination indicated that asarones and metformin attenuate the inflammation, fibrosis, cirrhosis and development of spontaneous HCC. SIGNIFICANCE: The STZ can be used to promote the DEN induced HCC. Treatment with (α)-and (ß)-asarone attenuates the effect of STZ + DEN induced HCC akin to metformin.

Anti-inflammatory treatment with ß-asarone improves impairments in social interaction and cognition in MK-801 treated mice.

The aim of this study investigates whether ß-asarone can improve cognition deficits in dizocilpine (MK-801) treated mice. Six-week-old male C57BL/6 mice were divided into four groups: control group (CON), MK-801-treated group (MK-801), MK-801 plus ß-asarone group (MK-801+ß-asa) and ß-asarone group (ß-asa). Behavioral tests, including sociability test, open field test (OPT) and Morris water-maze (MWM), were performed. Extracellular field excitatory postsynaptic potentials were recorded in the hippocampal dentate gyrus (DG) region. Western blot was employed to measure the expression of cognitive function-associated proteins and pro-inflammatory cytokines in the hippocampus. Immunofluorescence was performed to assess the microglial activation in the hippocampus DG region. The data show that social interactions and spatial learning and memory were impaired by MK-801. However, ß-asarone significantly mitigated the impairments. Furthermore, it was found that MK-801 aggravated the hyperactivity and anxiety-like behavior, but ß-asarone alleviated them. Moreover, ß-asarone alleviated the impairments of hippocampal synaptic plasticity and enhanced the expression of hippocampal synaptophysin (SYP) and postsynaptic density protein 95 (PSD95) in MK-801-treated mice. In addition, it suppressed the expression of interleukin-6 (IL-6), interleukin-1ß (IL-1ß), inducible nitric oxide synthase (i-Nos) and cyclo-oxygenase-2 (COX-2) expression in MK-801-treated mice. The results suggest that ß-asarone improved the impairment of cognition and synaptic plasticity possibly through modulating the excess release of pro-inflammatory cytokines and microglia activation in MK-801-treated mice.

APOBEC3G is a restriction factor of EV71 and mediator of IMB-Z antiviral activity.

Enterovirus 71 (EV71), a single-stranded positive-sense RNA virus, is the causative agent of hand, foot, and mouth disease (HFMD), for which no effective antiviral therapy is currently available. Apolipoprotein B messenger RNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) is a cytidine deaminase that inhibits the replication of several viruses, such as human immunodeficiency virus-1, hepatitis B virus and hepatitis C virus. In our efforts toward understanding the antiviral spectrum and mechanism of A3G, we found that ectopic expression of A3G inhibited EV71 replication, whereas knockdown of endogenous A3G expression promoted EV71 replication. Moreover, inhibition of EV71 replication by IMB-Z, a N-phenylbenzamide derivative, is associated with increased levels of intracellular A3G, but reducing the level of A3G by RNA interference diminished the antiviral activity of IMB-Z. Mechanistically, we obtained evidence suggesting that the cytidine deaminase activity is not required for A3G inhibition of EV71 replication. Instead, we demonstrated that A3G can interact with viral 3D RNA-dependent RNA polymerase (RdRp) and viral RNA and be packaged into progeny virions to reduce its infectivity. Taken together, our results indicate that A3G is a cellular restriction factor of EV71 and mediator of the antiviral activity of IMB-Z. Pharmacological induction and/or stabilization of A3G is a potential therapeutic approach to treat diseases caused by EV71 infection, such as HFMD.

Structure of Hierridin C, Synthesis of Hierridins B and C, and Evidence for Prevalent Alkylresorcinol Biosynthesis in Picocyanobacteria.

Small, single-celled planktonic cyanobacteria are ubiquitous in the world's oceans yet tend not to be perceived as secondary metabolite-rich organisms. Here we report the isolation and structure elucidation of hierridin C, a minor metabolite obtained from the cultured picocyanobacterium Cyanobium sp. LEGE 06113. We describe a simple, straightforward synthetic route to the scarcely produced hierridins that relies on a key regioselective halogenation step. In addition, we show that these compounds originate from a type III PKS pathway and that similar biosynthetic gene clusters are found in a variety of bacterial genomes, most notably those of the globally distributed picocyanobacteria genera Prochlorococcus, Cyanobium and Synechococcus.

Biosynthesis of methyleugenol and methylisoeugenol in Daucus carota leaves: Characterization of eugenol/isoeugenol synthase and O-Methyltransferase.

Carrot (Daucus carota subsp. sativus) is a widely cultivated root vegetable of high economic importance. The aroma of carrot roots and aboveground organs is mainly defined by terpenes. We found that leaves of orange carrot cultivar also produce considerable amounts of the phenylpropenes methyleugenol and methylisoeugenol. Notably, methyleugenol is most abundant in young leaves, while methylisoeugenol is the dominant phenylpropene in mature leaf tissue. The goal of the present study was to shed light on the biochemistry and molecular biology of these compounds' biosynthesis and accumulation. Using the available genomic and transcriptomic data, we isolated a cDNA encoding eugenol/isoeugenol synthase (DcE(I)GS1), an NADPH-dependent enzyme that converts coniferyl acetate to eugenol. This enzyme exhibits dual product specificity and yields propenylphenol isoeugenol alongside allylphenol eugenol. Furthermore, we identified a cDNA encoding S-adenosyl-L-methionine:eugenol/isoeugenol O-methyltransferase 1 (DcE(I)OMT1) that produces methyleugenol and methylisoeugenol via methylation of the para-OH-group of their respective precursors. Both DcE(I)GS1 and DcE(I)OMT1 were expressed in seeds, roots, young and mature leaves, and the DcE(I)OMT1 transcript levels were the highest in leaves. The DcE(I)GS1 protein is 67% identical to anise t-anol/isoeugenol synthase and displays an apparent Km of 247 µM for coniferyl acetate. The catalytic efficiency of DcEOMT1 with eugenol is more than five-fold higher than that with isoeugenol, with Km values of 40 µM for eugenol, and of 115 µM for isoeugenol. This work expands the current knowledge of the enzymes involved in phenylpropene biosynthesis and would enable studies into structural elements defining the regioselectivity of phenylpropene synthases.

Doping control analysis of four JWH-250 metabolites in equine urine by liquid chromatography-tandem mass spectrometry.

JWH-250 is a synthetic cannabinoid. Its use is prohibited in equine sport according to the Association of Racing Commissioners International (ARCI) and the Fédération Équestre Internationale (FEI). A doping control method to confirm the presence of four JWH-250 metabolites (JWH-250 4-OH-pentyl, JWH-250 5-OH-pentyl, JWH-250 5-OH-indole, and JWH-250 N-pentanoic acid) in equine urine was developed and validated. Urine samples were treated with acetonitrile and evaporated to concentrate the analytes prior to the analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The chromatographic separation was carried out using a Phenomenex Lux® 3 µm AMP column (150 x 3.0 mm). A triple quadrupole mass spectrometer was used for detection of the analytes in positive mode electrospray ionization using multiple reaction monitoring (MRM). The limits of detection, quantification, and confirmation for these metabolites were 25, 50, and 50 pg/mL, respectively. The linear dynamic range of quantification was 50-10000 pg/mL. Enzymatic hydrolysis indicated that JWH-250 4-OH-pentyl, JWH-250 5-OH-pentyl, and JWH-250 5-OH indole are highly conjugated whereas JWH-250 N-pentanoic acid is not conjugated. Relative retention time and product ion intensity ratios were employed as the criteria to confirm the presence of these metabolites in equine urine. The method was successfully applied to post-race urine samples collected from horses suspected of being exposed to JWH-250. All four JWH-250 metabolites were confirmed in these samples, demonstrating the method applicability for equine doping control analysis.