Dietary Exposure to Particles of Polytetrafluoroethylene (PTFE) and Polymethylmethacrylate (PMMA) Induces Different Responses in Periwinkles Littorina brevicula.

The marine and ocean water pollution with different-sized plastic waste poses a real threat to the lives of the next generations. Plastic, including microplastics, is found in all types of water bodies and in the organisms that live in them. However, given the chemical diversity of plastic particles, data on their toxicity are currently incomplete. Moreover, it is clear that different organisms, depending on their habitat and feeding habits, are at different risks from plastic particles. Therefore, we performed a series of experiments on feeding the gastropod scraping mollusk Littorina brevicula with two types of polymeric particles-polymethylmethacrylate (PMMA) and polytetrafluoroethylene (PTFE)-using a special feeding design. In the PMMA-exposed group, changes in gastrointestinal biochemical parameters such as increases in malondialdehyde (MDA) and protein carbonyls (PC) were detected, indicating the initiation of oxidative stress. Similarly, a comet assay showed an almost twofold increase in DNA damage in digestive gland cells compared to the control group. In mollusks fed with PTFE-containing food, no similar changes were recorded.

Bio-evaluation of Untapped Alkaloids from Vinca minor Enriched by Methyl-jasmonate-induced Stress: an Integrated Approach.

The low amount of metabolites isolated from natural products is one of the challenges preventing their biological evaluation. The modulation of biosynthetic pathways by stimulating stress-induced responses in plants was proven to be a valuable tool for diversification of already known natural products. Recently, we reported the dramatic effect of methyl jasmonate (MeJA) on Vinca minor alkaloids distribution. In this study, three compounds identified as 9-methoxyvincamine, minovincinine, and minovincine are successfully isolated in good yield and subjected to several bioassays based on a network pharmacology study. The extracts and isolated compounds show weak to moderate antimicrobial and cytotoxic activities. Also, they are found to significantly promote wound healing in scratch assay, and transforming growth factor-ß (TGF-ß) modulation is suggested to be the potential pathway based on bioinformatic analysis. Hence, Western blotting is used to assess the expression of several markers related to this pathway and wound healing. The extracts and isolated compounds are able to increase the expression of Smad3 and Phosphatidylinositol-3-kinase (PI3K), while downregulating the levels of cyclin D1 and the mammalian target of rapamycin (mTOR) except for minovincine, which increases the mTOR expression, inferring that it might act through a different mechanism. Molecular docking is used to give insights on the ability of isolated compounds to bind with different active sites in mTOR. Collectively, the integrated phytochemical, in silico, and molecular biology approach reveal that V. minor and its metabolite could be repurposed for the management of dermatological disorders where these markers are dysregulated, which opens the gate to develop new therapeutics in the future.

Structural convergence for tubulin binding of CPAP and vinca domain microtubule inhibitors.

Microtubule dynamics is regulated by various cellular proteins and perturbed by small-molecule compounds. To what extent the mechanism of the former resembles that of the latter is an open question. We report here structures of tubulin bound to the PN2-3 domain of CPAP, a protein controlling the length of the centrioles. We show that an α-helix of the PN2-3 N-terminal region binds and caps the longitudinal surface of the tubulin ß subunit. Moreover, a PN2-3 N-terminal stretch lies in a ß-tubulin site also targeted by fungal and bacterial peptide-like inhibitors of the vinca domain, sharing a very similar binding mode with these compounds. Therefore, our results identify several characteristic features of cellular partners that bind to this site and highlight a structural convergence of CPAP with small-molecule inhibitors of microtubule assembly.

Identifying Genes Involved in alkaloid Biosynthesis in Vinca minor Through Transcriptomics and Gene Co-Expression Analysis.

The lesser periwinkle Vinca minor accumulates numerous monoterpene indole alkaloids (MIAs) including the vasodilator vincamine. While the biosynthetic pathway of MIAs has been largely elucidated in other Apocynaceae such as Catharanthus roseus, the counterpart in V. minor remains mostly unknown, especially for reactions leading to MIAs specific to this plant. As a consequence, we generated a comprehensive V. minor transcriptome elaborated from eight distinct samples including roots, old and young leaves exposed to low or high light exposure conditions. This optimized resource exhibits an improved completeness compared to already published ones. Through homology-based searches using C. roseus genes as bait, we predicted candidate genes for all common steps of the MIA pathway as illustrated by the cloning of a tabersonine/vincadifformine 16-O-methyltransferase (Vm16OMT) isoform. The functional validation of this enzyme revealed its capacity of methylating 16-hydroxylated derivatives of tabersonine, vincadifformine and lochnericine with a Km 0.94 ± 0.06 µM for 16-hydroxytabersonine. Furthermore, by combining expression of fusions with yellow fluorescent proteins and interaction assays, we established that Vm16OMT is located in the cytosol and forms homodimers. Finally, a gene co-expression network was performed to identify candidate genes of the missing V. minor biosynthetic steps to guide MIA pathway elucidation.

Green Synthesis of Ag-MnO2 Nanoparticles using Chelidonium majus and Vinca minor Extracts and Their In Vitro Cytotoxicity.

Medicinal plants are often used as reducing agents to prepare metal nanoparticles through green-synthesis due to natural compounds and their potential as chemotherapeutic drugs. Thus, three types of eco-friendly Ag-MnO2 nanoparticles (Ag-MnO2NPs) were synthesized using C. majus (CmNPs), V. minor (VmNPs), and a 1:1 mixture of the two extracts (MNPs). These NPs were characterized using S/TEM, EDX, XRD, and FTIR methods, and their biological activity was assessed in vitro on normal keratinocytes (HaCaT) and skin melanoma cells (A375). All synthesized NPs had manganese oxide in the middle, and silver oxide and plant extract on the exterior. The NPs had different forms (polygonal, oval, and spherical), uniformly distributed, with crystalline structures and different sizes (9.3 nm for MNPs; 10 nm for VmNPs, and 32.4 nm for CmNPs). The best results were obtained with VmNPs, which reduced the viability of A375 cells up 38.8% and had a moderate cytotoxic effect on HaCaT (46.4%) at concentrations above 500 µg/mL. At the same concentrations, CmNPs had a rather proliferative effect, whereas MNPs negatively affected both cell lines. For the first time, this paper proved the synergistic action of the combined C. majus and V. minor extracts to form small and uniformly distributed Ag-MnO2NPs with high potential for selective treatments.

Tyrosinase inhibitory effects of Vinca major and its secondary metabolites: Enzyme kinetics and in silico inhibition model of the metabolites validated by pharmacophore modelling.

In the present study, we aimed to identify the tyrosinase enzyme inhibitory potential of Vinca major L. extract and its secondary metabolites. The extract possessed remarkable tyrosinase enzyme inhibitory effect with IC50 value of 20.39 ±â€¯0.44 µg/mL compared to the positive control, kojic acid (IC50 8.56 ±â€¯0.17 µg/mL). Compounds 1 and 5 were the most potent isolates with IC50 values of 32.41 ±â€¯0.99 and 31.34 ±â€¯0.75 µM, they were more potent than kojic acid (IC50: 60.25 ±â€¯0.54 µM). Compound 2 also exhibited remarkable tyrosinase inhibition with an IC50 value of 64.51 ±â€¯1.29 µM. An enzyme kinetics analysis revealed that 1 was a mixed-type, 2 and 5 were noncompetitive inhibitors. Using molecular docking, we predicted binding affinity and interactions of the compounds, which were in good alignment with a pharmacophore hypothesis generated out of a number of known tyrosinase inhibitors. The modelling studies underlined crucial interactions with the copper ions and residues around them such as Asn260, His263, and Met280.

Gastropod pedal mucus retains microplastics and promotes the uptake of particles by marine periwinkles.

The rapid dissemination of microplastics in many habitats of the oceans has raised concerns about the consequences for marine biota and ecosystems. Many adverse effects of microplastics on marine invertebrates are consequences of ingestion. Accordingly, the identification of mechanisms that facilitate the uptake of microplastics is essential for the evaluation of possible implications for marine organisms and food webs. Gastropods produce mucus for locomotion. Gastropod pedal mucus naturally retains formerly suspended micro-organisms, such as bacteria, microalgae, and seaweed spores. The retained organisms are consumed by gastropods that forage on pedal mucus. Here, we investigated the potential of gastropod pedal mucus to retain suspended microplastic particles and make them available for ingestion by periwinkles that forage on the contaminated mucus. In laboratory experiments, mucus of the periwinkles Littorina littorea and Littorina obtusata efficiently retained microplastics. Retention of microplastics varied between mucus from conspecifics of different size but not between mucus from either species. The density of microplastics in mucus trails increased concomitantly with the experimental particle concentration but was independent of incubation time. Aging of mucus and, particularly, desiccation affected the retention of microplastics. Periwinkles ingested microplastics when foraging on the contaminated mucus. Our results reveal a functional link between biogenic accumulation of microplastics and their trophic transfer by marine benthic herbivores into marine food webs.

Stress-induced modification of indole alkaloids:Phytomodificines as a new category of specialized metabolites.

This study focuses on the elucidation of the stress-induced reverse changes of major indole alkaloids in Vinca minor, primarily on the postulated conversion of vincamine and vincadifformine to yield 9-methoxyvincamine, minovincine, and minovincinine, respectively. By applying the P450 enzyme inhibitors, naproxen and resveratrol, it was shown that the oxidative reaction involved in the postulated conversion of vincamine and vincadifformine is catalyzed by cytochrome P450 enzymes. In combination with the identification of 9-hydroxyvincamine as a postulated intermediate, this result confirms that the observed stress-induced reverse changes in the alkaloid pattern are caused by modifications of the alkaloids which regularly accumulate in the healthy Vinca minor plants. Up to now, just two main types of defense compounds are distinguished: phytoalexins, which are synthesized de novo from primary metabolites and phytoanticipins, which are constitutively present in plants - either intrinsically active or are activated after cell death by hydrolysis or oxidation of the precursors. In contrast, the results presented in this paper demonstrate that indole alkaloids, representing typical phytoanticipins, are just slightly modified in response to a stress-related elicitation. Accordingly, these modified alkaloids neither represent classical phytoalexins (being synthesized de novo), nor can they be classified as phytoanticipins, since modification does not occur postmortem. Consequently, we propose a new category for these modified alkaloids that we call phytomodificines.

Metabolomics Characterization of Two Apocynaceae Plants, Catharanthus roseus and Vinca minor, Using GC-MS and LC-MS Methods in Combination.

Catharanthus roseus (C. roseus) and Vinca minor (V. minor) are two common important medical plants belonging to the family Apocynaceae. In this study, we used non-targeted GC-MS and targeted LC-MS metabolomics to dissect the metabolic profile of two plants with comparable phenotypic and metabolic differences. A total of 58 significantly different metabolites were present in different quantities according to PCA and PLS-DA score plots of the GC-MS analysis. The 58 identified compounds comprised 16 sugars, eight amino acids, nine alcohols and 18 organic acids. We subjected these metabolites into KEGG pathway enrichment analysis and highlighted 27 metabolic pathways, concentrated on the TCA cycle, glycometabolism, oligosaccharides, and polyol and lipid transporter (RFOS). Among the primary metabolites, trehalose, raffinose, digalacturonic acid and gallic acid were revealed to be the most significant marker compounds between the two plants, presumably contributing to species-specific phenotypic and metabolic discrepancy. The profiling of nine typical alkaloids in both plants using LC-MS method highlighted higher levels of crucial terpenoid indole alkaloid (TIA) intermediates of loganin, serpentine, and tabersonine in V. minor than in C. roseus. The possible underlying process of the metabolic flux from primary metabolism pathways to TIA synthesis was discussed and proposed. Generally speaking, this work provides a full-scale comparison of primary and secondary metabolites between two medical plants and a metabolic explanation of their TIA accumulation and phenotype differences.

The ATP binding cassette transporter, VmTPT2/VmABCG1, is involved in export of the monoterpenoid indole alkaloid, vincamine in Vinca minor leaves.

Vinca minor is a herbaceous plant from the Apocynaceae family known to produce over 50 monoterpene indole alkaloids (MIAs). These include several biologically active MIAs that have a range of pharmaceutical activities. The present study shows that the MIAs, vincamine, akuammicine, minovincinine, lochnericine and vincadifformine tend to be secreted on V. minor leaf surfaces. A secretion mechanism of MIAs, previously described for Catharanthus roseus, appears to be mediated by a member (CrTPT2) of the pleiotropic drug resistance ABC transporter subfamily. The molecular cloning of an MIA transporter (VmTPT2/VmABCG1) that is predominantly expressed in V. minor leaves was functionally characterized in yeast and established it as an MIA efflux transporter. The similar function of VmTPT2/VmABCG1 to CrTPT2 increases the likelihood that this MIA transporter family may have co-evolved within members of Apocynaceae family to secrete selected MIAs and to regulate leaf MIA surface chemistry.

Envisaging the Regulation of Alkaloid Biosynthesis and Associated Growth Kinetics in Hairy Roots of Vinca minor Through the Function of Artificial Neural Network.

Artificial neural network based modeling is a generic approach to understand and correlate different complex parameters of biological systems for improving the desired output. In addition, some new inferences can also be predicted in a shorter time with less cost and labor. As terpenoid indole alkaloid pathway in Vinca minor is very less investigated or elucidated, a strategy of elicitation with hydroxylase and acetyltransferase along with incorporation of various precursors from primary shikimate and secoiridoid pools via simultaneous employment of cyclooxygenase inhibitor was performed in the hairy roots of V. minor. This led to the increment in biomass accumulation, total alkaloid concentration, and vincamine production in selected treatments. The resultant experimental values were correlated with algorithm approaches of artificial neural network that assisted in finding the yield of vincamine, alkaloids, and growth kinetics using number of elicits. The inputs were the hydroxylase/acetyltransferase elicitors and cyclooxygenase inhibitor along with various precursors from shikimate and secoiridoid pools and the outputs were growth index (GI), alkaloids, and vincamine. The approach incorporates two MATLAB codes; GRNN and FFBPNN. Growth kinetic studies revealed that shikimate and tryptophan supplementation triggers biomass accumulation (GI = 440.2 to 540.5); while maximum alkaloid (3.7 % dry wt.) and vincamine production (0.017 ± 0.001 % dry wt.) was obtained on supplementation of secologanin along with tryptophan, naproxen, hydrogen peroxide, and acetic anhydride. The study shows that experimental and predicted values strongly correlate each other. The correlation coefficient for growth index (GI), alkaloids, and vincamine was found to be 0.9997, 0.9980, 0.9511 in GRNN and 0.9725, 0.9444, 0.9422 in FFBPNN, respectively. GRNN provided greater similarity between the target and predicted dataset in comparison to FFBPNN. The findings can provide future insights to calculate growth index, alkaloids, and vincamine in combination to different elicits.

Over-expression of Catharanthus roseus tryptophan decarboxylase and strictosidine synthase in rol gene integrated transgenic cell suspensions of Vinca minor.

Tryptophan decarboxylase (TDC) and strictosidine synthase (STR) genes from Catharanthus roseus have been successfully over-expressed in the rol gene integrated cell suspensions of V. minor. Thirty seconds SAAT (sonication-assisted Agrobacterium transformation) treatment of plant cell suspension with LBA1119 having construct () generated three stable TDC + STR over-expressing cell lines--PVG1, PVG2, and PVG3. The transgenes were confirmed by ß-glucuronidase GUS histochemical assay and PCR amplification of rol genes/GUS gene. All the three cell suspension lines were found to be slow growing. In comparison to the control cell suspensions (GI = 241.0 ± 5.8), PVG3 cell line registered a growth index (GI) of 208.0 ± 10.0 followed by PVG1 (GI = 140.0 ± 14.2) and PVG2 (GI = 85.0 ± 9.6). The PVG3 cell line was also up-scaled in the 5-l stirred tank bioreactor with GI of 745.6 ± 35.3 under optimized parameters. Only PVG3 line registered a twofold increase in total alkaloid content (2.1 ± 0.1% dry wt.) and showed vincamine presence (0.003 ± 0.001% dry wt.) which was further enhanced at the bioreactor level (2.7 ± 0.3 and 0.005 ± 0.001% dry wt., respectively). Real-time (RT) qPCR analysis of PVG3 showed more than sevenfold to eightfold increase in TDC and STR expression [relative quantity value (RQ) = 7.6 ± 0.8 (TDC); RQ = 8.5 ± 0.9 (STR)].

Regulation of vincamine biosynthesis and associated growth promoting effects through abiotic elicitation, cyclooxygenase inhibition, and precursor feeding of bioreactor grown Vinca minor hairy roots.

Hydroxylase/acetyltransferase elicitors and cyclooxygenase inhibitor along with various precursors from primary shikimate and secoiridoid pools have been fortified to vincamine less hairy root clone of Vinca minor to determine the regulatory factors associated with vincamine biosynthesis. Growth kinetic studies revealed that acetyltransferase elicitor acetic anhydride and terpenoid precursor loganin significantly reduce the growth either supplemented alone or in combination (GI = 140.6 ± 18.5 to 246.7 ± 24.3), while shikimate and tryptophan trigger biomass accumulation (GI = 440.2 ± 31.5 to 540.5 ± 40.3). Loganin also downregulates total alkaloid biosynthesis. Maximum flux towards vincamine production (0.017 ± 0.001 % dry wt.) was obtained when 20-day-old hairy roots were fortified with secologanin (10 mg/l) along with tryptophan (100 mg/l), naproxen (8.4 mg/l), hydrogen peroxide (20 µg/l), and acetic anhydride (32.4 mg/l). This was supported by RT PCR (qPCR) analysis where 2- and 3-fold increase in tryptophan decarboxylase (TDC; RQ = 2.0 ± 0.09) and strictosidine synthase (STR; RQ = 3.3 ± 0.36) activity, respectively, was recorded. The analysis of variance (ANOVA) for growth kinetics, total alkaloid content, and gene expression studies favored highly significant data (P < 0.05-0.01). Above treated hairy roots were also up-scaled in a 5-l stirred-tank bioreactor where a 40-day cycle yielded 8-fold increase in fresh root mass.

Effect of metal accumulation on metallothionein level and condition of the periwinkle Littorina littorea along the Scheldt estuary (the Netherlands).

Metal (i.e. Ag, As, Ca, Cd, Co, Cu, Mn, Pb and Zn) and metallothionein (MT) concentrations in the soft tissue of Littorina littorea were measured along the heavily polluted Western Scheldt (WS) and relatively clean Eastern Scheldt (ES) estuary. Along the WS metal and MT levels in periwinkles reflected the known downstream decreasing pollution gradient. Surprisingly in ES animals As, Mn and Zn concentrations decreased from east to west reflecting past pollution. Compared to the WS metal concentrations of ES periwinkles were significantly lower and both estuaries were maximally discriminated from each other based on their Cd soft tissue concentration using a canonical discriminant analysis. Furthermore, no overall difference was found in MT levels among animals from both estuaries. Using previously obtained condition data (i.e. dry/wet weight ratio and lipid content) the relation between soft tissue metal concentration (i.e. Cd, Cu and Zn) and fitness indicators (i.e. MT and condition data) was examined using a canonical correlation analysis. Periwinkles with a high metal load (i.e. Cd and Zn) also had high MT levels but were in a relatively poor condition.

A weight-of-evidence approach to assessing the ecological impact of organotin pollution in Dutch marine and brackish waters; combining risk prognosis and field monitoring using common periwinkles (Littorina littorea).

In the present study an integrated ecological risk assessment based on multiple lines of evidence (LOEs) was evaluated in order to better assess the risk from TBT in Dutch harbours and open coastal waters. On the basis of spatial distributions of measured tributyltin (TBT) concentrations in sediments and suspended matter, predictions of the intersex index (ISI) in Littorina littorea and the ecological risk expressed as the Potentially Affected Fraction (PAF) of species were made. The results were compared to actual ISI measurements and presence of L. littorea in the field. The PAF calculated on the basis of TBT levels for open coastal waters ranged from 4.2% to 15.3%; for harbours it ranged from 3.5% to 26.9%. Significant intersex levels were observed only in waters where the risk was calculated above 10% PAF. This study suggests that the absence of L. littorea from some harbours with high ecological risk values can be explained by high TBT concentrations. A call is made for the use of integrated approaches like weight-of-evidence (WOE) to help practitioners improve ecological risk assessment.

Regulation of Vinca alkaloid-induced apoptosis by NF-kappaB/IkappaB pathway in human tumor cells.

Antimicrotubule Vinca alkaloids, such as vinblastine and vincristine, interfere with the dynamics of microtubules and have shown significant cell killing activity in a variety of tumor cells through induction of apoptosis. The mechanism by which Vinca alkaloids induce apoptosis is not entirely clear. In this study, we found that glucocorticoids inhibit Vinca alkaloid-induced apoptosis without affecting G(2)-M arrest in human breast cancer BCap37 cells and human epidermoid tumor KB cells, suggesting that Vinca alkaloid-induced apoptosis may occur via a pathway independent of cell cycle arrest. Further analyses indicated that Vinca alkaloids cause significant degradation of IkappaBalpha, which in turn results in nuclear factor-kappaB (NF-kappaB) activation. Transfection of antisense IkappaBalpha in BCap37 cells sensitizes Vinca alkaloid-induced apoptosis. Moreover, in vitro kinase assays show that the activity of IkappaB kinase (IKK) was activated by Vinca alkaloids and was not affected by glucocorticoids. Stable transfection of dominant-negative deletional mutant IkappaBalpha, which is insensitive to IKK-mediated phosphorylation and degradation, resulted in the inhibition of Vinca alkaloid-induced NF-kappaB activation and reduced sensitivity of tumor cells to Vinca alkaloid-induced apoptosis. These findings suggest that the NF-kappaB/IkappaB signaling pathway may contribute to the mediation of Vinca alkaloid-induced apoptosis in human tumor cells.