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Plant secondary metabolites play a major role in plant adaptation to stress. Species belonging to Solanaceae family and Datura genus produce tropane alkaloids: D. stramonium, D. tatula and D. innoxia. These species are cultivated for their hyoscyamine (HS) content, whence the interest of this study to induce transformed roots of these species with strain A4 of Agrobacterium rhizogenes. Hairy roots (HRs) of Datura were established at high frequency by infecting vitroplants. All HRs (343 independent lines) were next employed to study the production of HS and growth. A screening of HRs alkaloid content by GC/MS is performed; it reveals, for the first time, the production of 13 alkaloids by the selected root lines. The selection of high productive line offers an interesting option to enhance the productivity. As HS is the dominant compound, the lines of Datura species were selected for their characteristics for biomass and HS production. The elicitors salicylic acid (SA) and acetyl salicylic acid (ASA) were also used to increase HS production. The results showed that the optimal concentration of the two elicitors (AS and ASA) was 0.1 mM. The highest HS content (17.94 +/- 0.14 mg g(-1) D. W.) obtained in HRs of D. tatula treated with 0.1 mM of acetyl salicylic acid.

In this research, tropane alkaloids in Radix physochlainae were extracted by tropine-type ionic liquid (IL) aqueous solutions under ultrasound assistance, and N-propyltropine hexafluorophosphate ([C3Tr][PF6]) was found to be the most ideal IL in this extraction mode after comprehensive screening. When 0.03 mol/L [C3Tr][PF6] aqueous solution was chosen as the extraction solvent, the solid-liquid ratio of raw material powders and ionic liquid aqueous solution was 1:20 (g/mL), ultrasonic power was 90 W and extraction time was 30 min, the extraction efficiency of tropane alkaloids has reached 121.3%. Compared with common heating extraction, it can further shorten the extraction time, improve extraction efficiency and decrease IL consumption. Furthermore, extraction mechanism together with potential toxicity of IL have been explored and discussed.

The rise in cyanobacterial blooms due to eutrophication and climate change has increased cyanotoxin presence in water. Most current water treatment plants do not effectively remove these toxins, posing a potential risk to public health. This study introduces a water treatment approach using nanostructured beads containing magnetic nanoparticles (MNPs) for easy removal from liquid suspension, coated with different adsorbent materials to eliminate cyanotoxins. Thirteen particle types were produced using activated carbon, CMK-3 mesoporous carbon, graphene, chitosan, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidised cellulose nanofibers (TOCNF), esterified pectin, and calcined lignin as an adsorbent component. The particles’ effectiveness for detoxification of microcystin-LR (MC-LR), cylindrospermopsin (CYN), and anatoxin-A (ATX-A) was assessed in an aqueous solution. Two particle compositions presented the best adsorption characteristics for the most common cyanotoxins. In the conditions tested, mesoporous carbon nanostructured particles, P1-CMK3, provide good removal of MC-LR and Merck-activated carbon nanostructured particles, P9-MAC, can remove ATX-A and CYN with high and fair efficacy, respectively. Additionally, in vitro toxicity of water treated with each particle type was evaluated in cultured cell lines, revealing no alteration of viability in human renal, neuronal, hepatic, and intestinal cells. Although further research is needed to fully characterise this new water treatment approach, it appears to be a safe, practical, and effective method for eliminating cyanotoxins from water.

Hairy root cultures of Hyoscyamus niger were cultivated in shake-flasks, a bubble-column bioreactor and a hybrid bubble-column/spray bioreactor and evaluated for alkaloid production. The latter gave the highest anisodamine content (0.67 mg/g dry wt) whereas scopolamine, hyoscyamine and cuscohygrine concentrations were highest in the bubble-column reactor (5.3, 1.6 and 26.5 mg/g dry wt, respectively). Both bioreactors gave similar productivities of scopolamine (1 and 0.98 mg/l day) and cuscohygrine (5 and 5.4 mg/l day), but anisodamine productivity was 3.5-fold higher in the hybrid bioreactor (HB) (0.02 and 0.07 mg/l day, respectively). Elicitation with methyl jasmonate increased scopolamine productivity by 146 % in roots grown in the HB whereas their permeabilization with DMSO caused 4-, 5-, 25- and 28-fold increase in scopolamine, hyoscyamine, anisodamine and cuscohygrine concentrations in the growth medium. In situ extraction with Amberlite XAD-2 doubled scopolamine productivity in the hybrid reactor after 50 days.

An open-tubular capillary electrochromatography column was prepared by chemically immobilized β-cyclodextrin modified gold nanoparticles onto new surface with the prederivatization of (3-mercaptopropyl)-trimethoxysilane. The synthesized nanoparticles and the prepared column were characterized by transmission electron microscopy, scanning electron microscopy, infrared spectroscopy and ultraviolet visible spectroscopy. When the column was employed as the chiral stationary phase, no enantioselectivity was observed for ten model basic drugs. So β-cyclodextrin was added to the background electrolyte as chiral additive to expect a possible synergistic effect occurring and resulting in a better separation. Fortunately, significant improvement in enantioselectivity was obtained for ten pairs of drug enantiomers. Then, the effects of β-cyclodextrin concentration and background electrolyte pH on the chiral separation were investigated. With the developed separation mode, all the enantiomers (except for venlafaxine) were baseline separated in resolutions of 4.49, 1.68, 1.88, 1.57, 2.52, 2.33, 3.24, 1.63 and 3.90 for zopiclone, chlorphenamine maleate, brompheniramine maleate, dioxopromethazine hydrochloride, carvedilol, homatropine hydrobromide, homatropine methylbromide, venlafaxine, sibutramine hydrochloride and terbutaline sulfate, respectively. Further, the possible separation mechanism involved was discussed.

SINCE the 19th century, fatal poisonings of domestic livestock, pets and wild animals have been attributed to cyanobacterial toxins through exposure to aquatic media (eg, lakes and reservoirs).

Benthic algae fuel summer food webs in many sunlit rivers, and are hotspots for primary and secondary production and biogeochemical cycling. Concerningly, riverine benthic algal assemblages can become dominated by toxic cyanobacteria, threatening water quality and public health. In the Eel River in Northern California, over a dozen dog deaths have been attributed to cyanotoxin poisonings since 2000. During the summers of 2013-2015, we documented spatial and temporal patterns of cyanotoxin concentrations in the watershed, showing widespread distribution of anatoxin-a in benthic cyanobacterial mats. Solid phase adsorption toxin tracking (SPATT) samplers were deployed weekly to record dissolved microcystin and anatoxin-a levels at 10 sites throughout the watershed, and 187 Anabaena-dominated or Phormidium-dominated cyanobacterial mat samples were collected from 27 locations to measure intracellular anatoxin-a (ATX) and microcystins (MCY). Anatoxin-a levels were higher than microcystin for both SPATT (mean MCY = 0.8 and ATX = 4.8 ng g resin-1 day-1) and cyanobacterial mat samples (mean MCY = 0.074 and ATX = 1.89 μg g-1 DW). Of the benthic mats sampled, 58.9% had detectable anatoxin-a (max = 70.93 μg g-1 DW), while 37.6% had detectable microcystins (max = 2.29 μg g-1 DW). SPATT cyanotoxin levels peaked in mid-summer in warm mainstem reaches of the watershed. This is one of the first documentations of widespread anatoxin-a occurrence in benthic cyanobacterial mats in a North American watershed.

Blooms of planktonic cyanobacteria have long been of concern in lakes, but more recently, harmful impacts of riverine benthic cyanobacterial mats been recognized. As yet, we know little about how various benthic cyanobacteria are distributed in river networks, or how environmental conditions or other associated microbes in their consortia affect their biosynthetic capacities. We performed metagenomic sequencing for 22 Oscillatoriales-dominated (Cyanobacteria) microbial mats collected across the Eel River network in Northern California and investigated factors associated with anatoxin-a producing cyanobacteria. All microbial communities were dominated by one or two cyanobacterial species, so the key mat metabolisms involve oxygenic photosynthesis and carbon oxidation. Only a few metabolisms fueled the growth of the mat communities, with little evidence for anaerobic metabolic pathways. We genomically defined four cyanobacterial species, all which shared <96% average nucleotide identity with reference Oscillatoriales genomes and are potentially novel species in the genus Microcoleus . One of the Microcoleus species contained the anatoxin-a biosynthesis genes, and we describe the first anatoxin-a gene cluster from the Microcoleus clade within Oscillatoriales. Occurrence of these four Microcoleus species in the watershed was correlated with total dissolved nitrogen and phosphorus concentrations, and the species that contains the anatoxin-a gene cluster was found in sites with higher nitrogen concentrations. Microbial assemblages in mat samples with the anatoxin-a gene cluster consistently had a lower abundance of Burkholderiales (Betaproteobacteria) species than did mats without the anatoxin-producing genes. The associations of water nutrient concentrations and certain co-occurring microbes with anatoxin-a producing Microcoleus motivate further exploration for their roles as potential controls on the distributions of toxigenic benthic cyanobacteria in river networks.

Anatoxin-a (ATX-a) is a neurotoxin produced by some species of cyanobacteria. Due to its water solubility and stability in natural water, it could pose health risks to human, animals, and plants. Conventional water treatment techniques are not only insufficient for the removal of ATX-a, but they also result in cell lysis and toxin release. The elimination of this toxin through biodegradation may be a promising strategy. This study examines for the first time the biodegradation of ATX-a to a non-toxic metabolite (Epoxy-ATX-a) by a strain of Bacillus that has a history of dealing with toxic cyanobacteria in a eutrophic lake. The Bacillus strain AMRI-03 thrived without lag phase in a lake water containing ATX-a. The strain displayed fast degradation of ATX-a, depending on initial toxin concentration. At the highest initial concentrations (50 & 100 µg L− 1), total ATX-a degradation took place in 4 days, but it took 6 & 7 days at lower concentrations (20, 10, and 1 µg L− 1, respectively). The ATX-a biodegradation rate was also influenced by the initial toxin concentration, reaching its maximum value (12.5 µg L− 1 day− 1) at the highest initial toxin concentrations (50 & 100 µg L− 1). Temperature and pH also had an impact on the rate of ATX-a biodegradation, with the highest rates occurring at 25 and 30 ºC and pH 7 and 8. This nontoxic bacterial strain could be immobilized within a biofilm on sand filters and/or sludge for the degradation and removal of ATX-a and other cyanotoxins during water treatment processes, following the establishment of mesocosm experiments to assess the potential effects of this bacterium on water quality.

Withania somnifera is one of the most reputed medicinal plants of Indian systems of medicine synthesizing diverse types of secondary metabolites such as withanolides, alkaloids, withanamides etc. Present study comprises cloning and E. coli over-expression of a tropinone reductase gene (WsTR-I) from W. somnifera, and elucidation of biochemical characteristics and physiological role of tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The recombinant enzyme was demonstrated to catalyze NADPH-dependent tropinone to tropine conversion step in tropane metabolism, through TLC, GC and GC-MS-MS analyses of the reaction product. The functionally active homodimeric ~60 kDa enzyme catalyzed the reaction in reversible manner at optimum pH 6.7. Catalytic kinetics of the enzyme favoured its forward reaction (tropine formation). Comparative 3-D models of landscape of the enzyme active site contours and tropinone binding site were also developed. Tissue-wide and ontogenic stage-wise assessment of WsTR-I transcript levels revealed constitutive expression of the gene with relatively lower abundance in berries and young leaves. The tissue profiles of WsTR-I expression matched those of tropine levels. The data suggest that, in W. somnifera, aerial tissues as well possess tropane alkaloid biosynthetic competence. In vivo feeding of U-[(14)C]-sucrose to orphan shoot (twigs) and [(14)C]-chasing revealed substantial radiolabel incorporation in tropinone and tropine, confirming the de novo synthesizing ability of the aerial tissues. This inherent independent ability heralds a conceptual novelty in the backdrop of classical view that these tissues acquire the alkaloids through transportation from roots rather than synthesis. The TR-I gene expression was found to be up-regulated on exposure to signal molecules (methyl jasmonate and salicylic acid) and on mechanical injury. The enzyme's catalytic and structural properties as well as gene expression profiles are discussed with respect to their physiological overtones.