Julius Ben-Ari

Uptake of carbamazepine by cucumber plants - A case study related to irrigation with reclaimed wastewater

Reclaimed wastewater is an important source of irrigation in semiarid and arid zones. Here we report data on carbamazepine (CBZ) uptake by cucumber plants in hydroponic culture and greenhouse experiments using different soil types irrigated with fresh water or reclaimed wastewater. Data obtained from the hydroponic culture experiments suggest that CBZ is mainly translocated by water mass flow, and thus it is concentrated and accumulated to the largest extent in the mature/older leaves. Carbamazepine concentration in cucumber fruits and leaves was negatively correlated with soil organic matter content. The concentrations of CBZ in the roots and stems were relatively low, and most CBZ in the plant (76-84% of total uptake) was detected in the leaves. A greenhouse experiment using fresh water and reclaimed wastewater spiked, or not, with CBZ at 1 μg L(-1) (typical concentration in effluents) revealed that CBZ can be taken up and bioaccumulated from its background concentration in reclaimed wastewater. Bioaccumulation factor (calculated as the ratio of CBZ concentration in the plant to that in the soil solution) for the fruits (0.8-1) was significantly lower than the value calculated for the leaves (17-20). This study emphasizes the potential uptake of active pharmaceutical compounds by crops in organic-matter-poor soils irrigated with reclaimed wastewater and highlights the potential risks associated with this agricultural practice.

Generation of the potent anti-malarial drug artemisinin in tobacco

volume 29 number 12 DeCember 2011 nature biotechnology To the Editor: The emergence of multidrug-resistant strains of Plasmodium spp., the etiological agent of malaria, constitutes a major threat to controlling the disease1,2. Artemisinin, a natural compound from Artemisia annua (sweet wormwood) plants, is highly effective against drug-resistant malaria. Even so, lowcost artemisinin-based drugs are lacking because of the high cost of obtaining natural or chemically synthesized artemisinin1,2. Martin et al.3 were the first to report the generation of an artemisinin precursor in a microbial system. They engineered Escherichia coli with a synthetic mevalonate pathway from Saccharomyces cerevisiae. Expression of amorphadiene synthase (ADS) from A. annua in this strain allowed production of amorpha4,11-diene, the sesquiterpene olefin precursor to artemisinin. However, despite extensive effort invested in the past decade in metabolic engineering of artemisinin and its precursors in both microbial and heterologous plant systems2–6, production of artemisinin itself has never been achieved. Here we report the metabolic engineering of tobacco to produce artemisinin, generating transgenic plants that express five plantand yeast-derived genes involved in the mevalonate and artemisinin pathways, all expressed from a single vector. Our experiments demonstrate that artemisinin can be fully biosynthesized in a heterologous (that is, other than A. annua) plant system, such as tobacco. Although the artemisinin levels we have generated in transgenic tobacco are currently lower than those in A. annua, our experimental platform should lead to the design of new routes for the drug’s commercial production in heterologous plant systems. The World Health Organisation (WHO; Geneva) promotes the use of artemisinin as a first-line treatment for malaria, and it is heavily involved in facilitating the development of artemisinin-based anti-malaria drugs1. Artemisinin is biosynthesized from terpenoid backbones generated by the mevalonate and methyl-erythritol phosphate (MEP) pathways7–9 (Fig. 1a). Although detailed knowledge of the artemisinin-biosynthesis pathway is still lacking, it initiates with the cyclization of farnesyl diphosphate by ADS to form amorpha-4,11-diene, which is then oxidized by the cytochrome P450 CYP71AV1, reduced by artemisinic aldehyde reductase (DBR2) and possibly reoxidized by aldehyde dehydrogenase to yield dihydroartemisinic acid—the presumed precursor of artemisinin in plants6,7,10. Normally, dihydroartemisinic acid accumulates in A. annua and slowly converts to artemisinin, a process that can be stimulated after harvest by drying in the sun. The transformation of dihydroartemisinic acid to artemisinin in A. annua has been proposed to be nonenzymatic, requiring only the presence of light and molecular oxygen6,7; a singlet oxygen formed as a consequence of exposure to UV/visible light may react with dihydroartemisinic acid to form a ketoenol, which can then react with ground-state oxygen to form a second hydroperoxide that spontaneously forms artemisinin. Despite the great interest in enhancing yields of artemisinin in its host A. annua, classic breeding and genetic engineering strategies have met with only limited success6,11. Moreover, recent attempts to produce the artemisinin precursors artemisinic and dihydroartemisinic acids in heterologous plants4,5 did not lead to their accumulation due to internal glycosylation and insufficient oxidation toward the acids. Harnessing E. coli and S. cerevisiae has allowed the production of high titers of amorpha-4,11diene and artemisinic acid2,3, but not of the active artemisinin drug itself. To reconstruct the artemisinin-producing pathway in Nicotiana tabacum, we first generated a mega-vector carrying cytochrome P450 reductase (CPR) from A. annua to prevent the accumulation of inactive oxidized P450, as well as ADS, CYP71AV1 and DBR2 (Fig. 1b and Supplementary Methods). The mega-vector also contained a truncated and deregulated 3-hydroxy-3-methylglutarylcoenzyme A reductase (tHMG) from yeast to increase the supply of precursor from the mevalonate pathway for artemisinin production. To ensure genetic stability and Generation of the potent anti-malarial drug artemisinin in tobacco

Transformation of the recalcitrant pharmaceutical compound carbamazepine by Pleurotus ostreatus: role of cytochrome P450 monooxygenase and manganese peroxidase.

Carbamazepine (CBZ) is an environmentally recalcitrant compound highly stable in soil and during wastewater treatment. In this study, we examined the mechanisms by which the white-rot fungus Pleurotus ostreatus metabolizes CBZ in liquid culture using a physiological approach. P. ostreatus PC9 was grown in media known to support different levels of a multiplicity of enzyme systems such as cytochrome P450 (CYP450) and manganese peroxidase (MnP). When both CYP450 and MnP systems were active, 99% of the added CBZ was eliminated from the solution and transformed to 10,11-epoxycarbamazepine. High removal of CBZ was also obtained when either MnP or CYP450 was active. When both CYP450 and MnP were inactivated, only 10 to 30% of the added CBZ was removed. In this latter system, removal of CBZ might be partially attributed to the activity of versatile peroxidase. P. ostreatus was able to eliminate CBZ in liquid culture even when CBZ was added at an environmentally relevant concentration (1 μg L(-1)). On the basis of our study, we suggest that two families of enzymes are involved in the oxidation of CBZ in liquid culture: MnP in a Mn(2+)-dependent or independent manner and CYP450. Our study also highlights the potential of using P. ostreatus for bioremediation systems.

Quorum‐sensing quenching by rhizobacterial volatiles

We show that volatile organic compounds (VOCs) produced by rhizospheric strains Pseudomonas fluorescens B-4117 and Serratia plymuthica IC1270 may act as inhibitors of the cell-cell communication quorum-sensing (QS) network mediated by N-acyl homoserine lactone (AHL) signal molecules produced by various bacteria, including strains of Agrobacterium, Chromobacterium, Pectobacterium and Pseudomonas. This quorum-quenching effect was observed when AHL-producing bacteria were treated with VOCs emitted by strains B-4117 and IC1270 or with dimethyl disulfide (DMDS), the major volatile produced by strain IC1270. LC-MS/MS analysis revealed that treatment of strains Pseudomonas chlororaphis 449, Pseudomonas aeruginosa PAO1 or Ps. fluorescens 2-79 with VOCs emitted by strain IC1270 or DMDS drastically decreases the amount of AHLs produced by these bacteria. Volatile organic compounds produced by Ps. chlororaphis 449 were able to suppress its own QS-induction activity, suggesting a negative interaction between VOCs and AHL molecules in the same strain. Quantitative RT-PCR analysis showed that treatment of Ps. chlororaphis 449 with VOCs emitted by cells of IC1270, B-4117 or 449 itself, or with DMDS, leads to significant suppression of transcription of AHL synthase genes phzI and csaI. Thus, along with AHLs, bacterial volatiles might be considered another type of signal molecule involved in microbial communication in the rhizosphere.

Amelioration of diabesity-induced colorectal ontogenesis by omega-3 fatty acids in mice

Postnatal intestinal ontogenesis in an animal model of diabesity may recapitulate morphological and transduction features of diabesity-induced intestinal dysplasia and its amelioration by endogenous (n-3) polyunsaturated fatty acids (PUFA). Proliferation, differentiation, and transduction aspects of intestinal ontogenesis have been studied here in obese, insulin-resistant db/db mice, in fat-1 transgene coding for desaturation of (n-6) PUFA into (n-3) PUFA, in db/db crossed with fat-1 mice, and in control mice. Diabesity resulted in increased colonic proliferation and dedifferentiation of epithelial colonocytes and goblet cells, with increased colonic β-catenin and hepatocyte nuclear factor (HNF)-4α transcriptional activities accompanied by enrichment in HNF-4α–bound (n-6) PUFA. In contrast, in fat-1 mice, colonic proliferation was restrained, accompanied by differentiation of crypt stem cells into epithelial colonocytes and goblet cells and by decrease in colonic β-catenin and HNF-4α transcriptional activities, with concomitant enrichment in HNF-4α-bound (n-3) PUFA at the expense of (n-6) PUFA. Colonic proliferation and differentiation, the profile of β-catenin and HNF-4α-responsive genes, and the composition of HNF-4α-bound PUFA of db/db mice reverted to wild-type by introducing the fat-1 gene into the db/db context. Suppression of intestinal HNF-4α activity by (n-3) PUFA may ameliorate diabesity-induced intestinal ontogenesis and offer an effective preventive modality for colorectal cancer.

Mn2+-deficiency reveals a key role for the Pleurotus ostreatus versatile peroxidase (VP4) in oxidation of aromatic compounds

The manganese peroxidase gene family (mnps) is a part of the ligninolytic system of Pleurotus ostreatus. This gene family is comprised of nine members, mnp1–9, encoding short manganese peroxidases (short-MnPs) or versatile peroxidases (VPs). We show that unlike in Mn2+-amended glucose–peptone (GP) medium, where redundancy among mnps was reported, in Mn2+-deficient GP medium mnp4 [encoding versatile peroxidase isoenzyme 4 (VP4)] has a key and nonredundant function. The abundance of mnps transcripts at time points corresponding to the tropophase (active growth), early idiophase, and idiophase indicates that mnp4 is the predominantly expressed mnp gene and that its relative predominance is dependent on the age of the culture. In this medium, azo dye, Orange II (OII) decolorization occurs only during the idiophase and a Δmnp4 strain showed a drastic reduction in this decolorization. Three degradation metabolites were identified by liquid chromatography-mass spectroscopy (LC-MS), indicating both asymmetric and symmetric enzymatic cleavage of the azo-bond. In addition, the culture filtrate of Δmnp4 showed negligible values of oxidation capability of four typical VP substrates: Mn2+, 2,6-dimethoxyphenol, phenol red, and Reactive Black 5 (RB5), compared to the wild-type strain PC9. We concluded that under Mn2+-deficient GP culture, VP4 (encoded by mnp4) is the main active ligninolytic enzyme able to oxidize Mn2+ as well as high and low redox potential aromatic substrate, including dyes. Furthermore, other VPs/MnPs do not compensate for the lack of VP4 activity.

Carryover Effects of Acute DEHP Exposure on Ovarian Function and Oocyte Developmental Competence in Lactating Cows

We examined acute exposure of Holstein cows to di(2-ethylhexyl) phthalate (DEHP) and its carryover effects on ovarian function and oocyte developmental competence. Synchronized cows were tube-fed with water or 100 mg/kg DEHP per day for 3 days. Blood, urine and milk samples were collected before, during and after DEHP exposure to examine its clearance pattern. Ovarian follicular dynamics was monitored through an entire estrous cycle by ultrasonographic scanning. Follicular fluids were aspirated from the preovulatory follicles on days 0 and 29 of the experiment and analyzed for phthalate metabolites and estradiol concentration. The aspirated follicular fluid was used as maturation medium for in-vitro embryo production. Findings revealed that DEHP impairs the pattern of follicular development, with a prominent effect on dominant follicles. The diameter and growth rate of the first- and second-wave dominant follicles were lower (P < 0.05) in the DEHP-treated group. Estradiol concentration in the follicular fluid was lower in the DEHP-treated group than in controls, and associated with a higher number of follicular pathologies (follicle diameter >25 mm). The pattern of growth and regression of the corpus luteum differed between groups, with a lower volume in the DEHP-treated group (P < 0.05). The follicular fluid aspirated from the DEHP-treated group, but not the controls, contained 23 nM mono(2-ethylhexyl) phthalate. Culturing of cumulus oocyte complexes in the follicular fluid aspirated from DEHP-treated cows reduced the proportion of oocytes progressing to the MII stage, and the proportions of 2- to 4-cell-stage embryos (P < 0.04) and 7-day blastocysts (P < 0.06). The results describe the risk associated with acute exposure to DEHP and its deleterious carryover effects on ovarian function, nuclear maturation and oocyte developmental competence.

Effect of CMV infection and high temperatures on the enzymes involved in raffinose family oligosaccharide biosynthesis in melon plants.

Ultrastructural and molecular studies have provided experimental evidence for the classification of cucurbits as symplastic loaders, mainly translocating the raffinose family oligosaccharides (RFOs) raffinose and stachyose. Earlier studies established that cucumber mosaic virus (CMV) infection causes a significant increase in the sucrose-to-RFO ratio in the phloem sap of melon plants. The alteration in phloem sap sugar composition was associated with upregulation of CmSUT1 transcript within the vascular bundles. The current research aimed to explore the effect of CMV infection on the enzymes involved in symplastic phloem loading and RFO biosynthesis. Viral infection did not affect the activity of either raffinose or stachyose synthases in source leaves, but caused upregulation of the respective transcripts. Interestingly, activity of galactinol synthase was higher in CMV-infected leaves, associated with upregulation of CmGAS2. A significant increase in CmGAS2 expression in source leaves of melon plants exposed to high temperatures indicated that this response is common for both biotic and abiotic stresses. However, the effect of CMV or heat stress on phloem sap sugar composition is not due to alteration in RFO biosynthesis.

Mechanochemically enhanced degradation of pyrene and phenanthrene loaded on magnetite.

The enhancement of the degradation of polycyclic aromatic hydrocarbons (PAHs), exemplified by pyrene and phenanthrene, using mild grinding in the presence of common minerals was investigated. Magnetite, birnessite, and Na- and Cu-montmorillonite samples were loaded with pyrene or phenanthrene and ground manually or in a ball mill for short periods of time. The ground samples were analyzed for PAHs and for their metabolites, using high-performance liquid chromatography and liquid chromatography-mass spectrometry. No degradation of pyrene occurred when it was in contact with Na-montmorillonite or birnessite. Sorption of pyrene on Cu-montmorillonite enhanced its degradation, but grinding of the loaded clay actually inhibited pyrenes degradation. Phenanthrene hardly degraded on Cu-montmorillonite. Grinding magnetite loaded with either PAH resulted in a significant degradation of both (∼50% after grinding for 5 min), while in the nonground samples, negligible degradation was detected. The extent of degradation increased with the duration of grinding. The degradation of either PAH loaded on magnetite yielded oxidized products. In soil samples contaminated with PAHs and mixed with magnetite, a similar grinding-induced degradation pattern was observed, but with a lower rate. A liquid phase was required to initiate degradation in the soil. The liquid phase apparently served as the medium through which the pollutants reached the surface of the degradation-enhancing mineral.