Establishment of the Daucus carota SMC-1 Cell Suspension Line for Poliovirus Vaccine Development.

The development of virus-free, oral vaccines against poliovirus capable of inducing mucosal protective immunity is needed to safely combat this pathogen. In the present study, a carrot cell line expressing the poliovirus VP2 antigen was established at the level of callus and cell suspensions, exploring the effects of culture media (MS and B5), supplementation with urea, phytoregulators (2,4-D : KIN), and light conditions (continuous light, photoperiod, and total darkness). The best callus growth was obtained on B5 medium supplemented with 2 mg/L of 2,4-D + 2 mg/L kinetin and 0.0136 g/L of urea and in continuous light conditions. Suspension cultures of the SMC-1 line in 250 mL Erlenmeyer flasks had a maximum growth of 16.07 ± 0.03 g/L DW on day 12 with a growth rate of µ=0.3/d and a doubling time of 2.3 days. In a 2 L airlift bioreactor, the biomass yield achieved was 25.6 ± 0.05 g/L DW at day 10 with a growth rate of µ= 0.58/d and doubling time of 1.38 d. Cell growth was 1.5 times higher in bioreactors than in shake flasks, highlighting that both systems resulted in the accumulation of VP2 throughout the time in culture. The maximum VP2 yield in flasks was 387.8 µg/g DW at day 21, while in the reactor it was 550.2 µg/g DW at day 18. In conclusion, bioreactor-based production of the VP2 protein by the SMC-1 suspension cell line offers a higher productivity when compared to flask cultures, offering a key perspective to produce low-cost vaccines against poliomyelitis.

Statistical Experimental Designs for cLTB-Syn Vaccine Production Using Daucus carota Cell Suspension Cultures.

The carrot-made LTB-Syn antigen (cLTB-Syn) is a vaccine candidate against synucleinopathies based on carrot cells expressing the target antigen LTB and syn epitopes. Therefore, the development of an efficient production process is required with media culture optimization to increase the production yields as the main goal. In this study, the effect of two nitrogen sources (urea and glutamate) on callus cultures producing cLTB-Syn was studied, observing that the addition of 17 mM urea to MS medium favored the biomass yield. To optimize the MS media composition, the influence of seven medium components on biomass and cLTB-Syn production was first evaluated by a Plackett-Burman design (PBD). Then, three factors were further analyzed using a central composite design (CCD) and response surface methodology (RSM). The results showed a 1.2-fold improvement in biomass, and a 4.5-fold improvement in cLTB-Syn production was achieved at the shake-flask scale. At the bioreactor scale, there was a 1.5-fold increase in biomass and a 2.8-fold increase in cLTB-Syn yield compared with the standard MS medium. Moreover, the cLTB-Syn vaccine induced humoral responses in BALB/c mice subjected to either oral or subcutaneous immunization. Therefore, cLTB-Syn is a promising vaccine candidate that will aid in developing immunotherapeutic strategies to combat PD and other neurodegenerative diseases without the need for cold storage, making it a financially viable option for massive immunization.

In Vitro and In Vivo Cysticidal Effects of Carica Papaya Cell Suspensions.

Parasitic diseases are a major public health problem worldwide. Plant-derived products appear to be ideal candidates from a biotechnological perspective, being sustainable and environmentally friendly. The antiparasitic properties of Carica papaya have been attributed to some of its components, including papain and other compounds that are concentrated in the latex and seeds. This study demonstrated in vitro a high and insignificantly different cysticidal activity of soluble extract that was obtained after the disruption of nontransformed wild-type (WT) cells as well as transformed papaya calluses (PC-9, PC-12, and PC-23) and papaya cell suspensions (CS-9, CS-12, and CS-23). In vivo, cell suspensions of CS-WT and CS-23 that had been previously lyophilized were tested with respect to their cysticidal effects, compared with those of three commercial antiparasitic drugs. CS-WT and CS-23 together reduced the number of cysticerci, the number of buds, and the percentage of calcified cysticerci in a similar extent to albendazole and niclosamide, whereas ivermectin was less effective. Mice were then orally immunized with CS-23 that expressed the anti-cysticercal KETc7 antigen (10 µg/mouse), CS-WT (10 mg/mouse), or both together to evaluate their preventive properties. CS-23 and CS-WT significantly reduced the expected parasite and increased the percentage of calcified cysticerci as well as recovery, being more effective when employed together. The results reported in this study support the feasibility of the development of an anti-cysticercosis vaccine from cells of C. papaya in in vitro cultures, as they are a source of an anthelmintic, natural, and reproducible product.

Establishment of the Carrot-Made LTB-Syn Antigen Cell Line in Shake Flask and Airlift Bioreactor Cultures.

Carrot (Daucus carota) cells have been used to effectively manufacture recombinant biopharmaceuticals such as cytokines, vaccines, and antibodies. We generated the carrot cell line Z4, genetically modified to produce the LTB-Syn antigen, which is a fusion protein proposed for immunotherapy against synucleinopathies. In this work, the Z4 cell suspension line was cultivated to produce the LTB-Syn protein in a 250 mL shake flask and 2 L airlift bioreactor cultures grown for 45 and 30 days, respectively. Maximum biomass was obtained on day 15 in both the airlift bioreactor (35.00 ± 0.04 g/L DW) and shake flasks (17.00 ± 0.04 g/L DW). In the bioreactor, the highest LTB-Syn protein yield (1.52 ± 0.03 µg/g FW) was obtained on day 15; while the same occurred on day 18 for shake flasks (0.92 ± 0.02 µg/g FW). LTB-Syn protein levels were analyzed by GM1-ELISA and western blot. PCR analysis confirmed the presence of the transgene in the Z4 line. The obtained data demonstrate that the carrot Z4 cell suspension line grown in airlift bioreactors shows promise for a scale-up cultivation producing an oral LTB-Syn antigen.

Identification of a Huperzine A-producing endophytic fungus from Phlegmariurus taxifolius.

Highly prized huperzine A (Hup A), a natural alkaloid formerly isolated from the Chinese medicinal plant Huperzia serrata, has been widely used for the treatment of Alzheimer disease, inspiring us to search for endophytic fungi that produce this compound. In this study, we obtained the C17 fungus isolate from the Mexican club moss Phlegmariurus taxifolius, which produced a yield of 3.2 µg/g Hup A in mycelial dry weight, when cultured in potato dextrose broth medium. The C17 isolate was identified as belonging to the genus Fusarium with reference to the colony´s morphological characteristics and the presence of macroconidia and microconidia structures; and this was confirmed by DNA-barcoding analysis, by amplifying and sequencing the ribosomal internal transcribed spacer (rITS).

Anticholinesterase activity and identification of huperzine A in three Mexican lycopods: Huperzia cuernavacensis, Huperzia dichotoma and Huperzia linifolia (Lycopodiaceae).

Huperzine A (Hup A), the alkaloid produced by the Chinese medicinal plant Huperzia serrata, has been documented to be a promising agent for the treatment of Alzheimer's disease due to its potent acetylcholinesterase inhibitory (AChEI) activity. The search for anticholinesterase natural products, as well as for alternative sources of Hup A in Mexican lycopods, prompted us to investigate these plants. The action of methanolic and alkaloidal extracts of three Huperzia species (H. cuernavacensis, H. dichotoma, and H. linifolia) was evaluated using an in vitro anticholinesterase activity assay. Also, chromatographic and spectroscopic analyses were employed to detect the presence of Hup A. Methanolic and alkaloidal extracts of H. cuernavacensis showed IC50 =5.32±0.8µg/mL and 0.74±0.05µg/mL; H. dichotoma displayed AChEI with IC50 values =14.11±2.1µg/mL and 0.64±0.09µg/mL; and H. linifolia presented IC50 =158.37±8.7µg/mL and 4.2±1.24µg/mL, respectively, compared to the control Hup A (IC50= 0.16±0.03µg/mL). Hup A was identified in the extracts of H. dichotoma, but it was not detected in the extracts of H. cuernavacensis and H. linifolia by 1H NMR techniques. This study reveals H. dichotoma as a new source of Hup A, and presents H. linifolia and H. cuernavacensis as potential candidates to obtain other anticholinesterase compounds useful in the Alzheimer's disease treatment.

Preclinical evaluation of anti-Helicobacter spp. activity of Hippocratea celastroides Kunth and its acute and sub-acute toxicity.

BACKGROUND: Hippocratea celastroides Kunth, commonly known as "cancerina", is used in Mexican Traditional Medicine for the treatment of gastric and intestinal infections, systemic and skin inflammation, injuries and gastritis. The aim of this research was to assess the anti-Helicobacter pylori activities of hydro-ethanolic root-bark extracts from Hippocratea celastroides Kunth in naturally infected dogs, after testing their acute and subacute toxicities in mice. METHODS: To determine in vivo acute toxicity, a hydro-ethanolic extract was obtained and administered orally in female and male Balb-C mice, at doses ranging from 2000 to 5000 mg/kg. For the subacute study, a hydro-ethanolic extract was given to male and female Balb-C mice at doses ranging from 200 to 2000 mg/kg body weight. The animals were observed daily over a period of 42 days for signs of toxicity. In the pre-clinical anti-Helicobacter spp. assay, 60 dogs were included. Eighteen and 19 dogs for the experimental and control groups respectively, concluded the study. The experimental treatment consisted of H. celastroides hydro-ethanolic extract and the control treatment of amoxicillin-clarithromycin-omeprazole. RESULTS: Oral LD50 (lethal dose 50) values for hydro-ethanolic extract were indeterminable at the highest tested doses. Under the subacute administration, neither mortality nor any sign of toxicity were observed when the hydro-ethanolic extract was administered. There were no significant alterations in biochemical parameters. The prevalence of Helicobacter spp. infection in dogs was 97.1 % for the experimental group and 100 % for the control group. Effectiveness was of 33.3 and 55 % in the experimental and control group respectively. The oral administration of H. celastroides was well-tolerated and safe. CONCLUSION: The root-bark of H. celastroides produced no signs of toxicity, and manifested pharmacological activity that indicated the possibility of an alternative treatment for H. pylori infection. Effectiveness is still low so it is necessary to continue research.

Cytotoxicity, Post-Treatment Recovery, and Selectivity Analysis of Naturally Occurring Podophyllotoxins from Bursera fagaroides var. fagaroides on Breast Cancer Cell Lines.

Despite prevention and treatment options, breast cancer (BC) has become one of the most important issues in the present day. Therefore, the need for more specific and efficient compounds remains paramount. We evaluated four previously isolated aryltetralin lignans: 5'-demethoxy-ß-peltatin-A-methylether (1), acetylpodophyllotoxin (2), 5'-demethoxydeoxypodophyllotoxin (3), and 7',8'-dehydroacetylpodophyllotoxin (4) for cytotoxicity, clonogenicity, and selectivity against three BC cell lines: MCF-7, MDA-MB-231, and BT-549, as well as the non-tumorigenic mammary epithelial cell line MCF-10A. Cytotoxicity was evaluated after 72 h of treatment, and clonogenicity was determined at 72 h post-treatment; experiments were performed using the sulforhodamine B staining assay. Selective-index (SI) was calculated by comparing pure compound IC50 values in MCF-10A cell line against the IC50 of the same compound in cancer cell lines. Structural similarities among lignans and controls (podophyllotoxin and etoposide) were analyzed using the Tanimoto coefficient (Tc). Lignans were cytotoxic against all tested cell lines (0.011-7.22 µM) and clonogenicity testing showed a dose-dependent cytocidality for all lignans (≥0.08 µg/mL); compounds 2 and 3 were more potent (14.1 and 7.6 respectively) than etoposide in BT-549 cell line, while compound 2 displayed selectivity (SI = 28.17) in BT-549 cell line. Tc values of lignans suggested a greater similarity with podophyllotoxin structure.

Radical scavenging, antioxidant, and cytotoxic activities of the methanolic extracts from different organs of Ternstroemia pringlei.

Ternstroemia pringlei (Rose) Standl. (Theaceae) is widely used in Mexican traditional medicine to treat a diverse array of illnesses including rheumatoid pains, and is listed as one of the most consumed medicinal plants in the country. We selected T. pringlei given the strong relationship between oxidative stress and arthritis pathology, and investigated antioxidant potential of leaf, petal, fruit and seed methanolic extracts. Our method included assessing the in vitro free radical scavenger activity using the 2,2´-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) test, as well as the in vivo antioxidant action in the H2O2 protection model with Saccharomyces cerevisiae. Leaves and fruits afforded the most active extract in the ABTS assay, with antiradical activity of IC50=33.91 and 38.09µg/mL, respectively; while fruit extracts at 250µg/mL proved the most protective action against H2O2 oxidative stress. All extracts were non-cytotoxic against HF-6 (colon), PC-3 (prostate), MCF-7 (breast), SiHa (cervical) cancer cell lines and also toward the HFS-30 fibroblast normal skin cell line (IC50&>20µg/mL). Leaf methanolic extracts afforded ternstroside B, a known phenylethanoid glycoside with a strong free radical scavenging action. The presence of this kind of metabolites opens new research perspectives for the plant.

Phytochemical Profiles and Cytotoxic Activity of Bursera fagaroides (Kunth) Engl. Leaves and Its Callus Culture.

Bursera fagaroides, popularly used in México, possesses bioactive lignans. These compounds are low in the bark, and its extraction endangers the life of the trees. The aim of the present investigation was to search for alternative sources of cytotoxic compounds in B. fagaroides prepared as leaves and in vitro callus cultures. The friable callus of B. fagaroides was established using a combination of plant growth regulators: 4 mgL-1 of 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mgL-1 Naphthaleneacetic Acid (NAA) and 1 mgL-1 Zeatin. The maximum cell growth was at day 28 with a specific growth rate of µ = 0.059 days-1 and duplication time td = 11.8 days. HPLC quantification of the dichloromethane callus biomass extract showed that Scopoletin, with a concentration of 10.7 µg g-1 dry weight, was the main compound inducible as a phytoalexin by the addition of high concentrations of 2,4-D, as well as by the absence of nutrients in the culture medium. In this same extract, the compounds γ-sitosterol and stigmasterol were also identified by GC-MS analysis. Open column chromatography was used to separate and identify yatein, acetyl podophyllotoxin and 7',8'-dehydropodophyllotoxin in the leaves of the wild plant. Cytotoxic activity on four cancer cell lines was tested, with PC-3 prostate carcinoma (IC50 of 12.6 ± 4.6 µgmL-1) being the most sensitive to the wild-type plant extract and HeLa cervical carcinoma (IC50 of 72 ± 5 µgmL-1) being the most sensitive to the callus culture extract.

Antioxidant Active Phytochemicals in Ternstroemia lineata Explained by Aquaporin Mechanisms.

The antioxidant action of terngymnoside C (1) and hydroxytyrosol-1-glucoside (2), isolated for the first time from the flower buds of Ternstroemia lineata, as well as katsumadin (3), obtained from the seedless fruits, was evaluated using ABTS•+ and H2O2-Saccharomyces cerevisiae models. In silico docking analysis of 1, 2, and 3 determined their affinity forces to the aquaporin monomers of the modeled S. cerevisiae protein 3 (AQP3) and human protein 7 (AQP7) channels that regulate the H2O2 cell transport. The ABTS•+ antiradical capacity of these compounds showed IC50 values of 22.00 µM (1), 47.64 µM (2), and 73.93 µM (3). The S. cerevisiae antioxidant assay showed that at 25 µM (1) and 50 µM (2 and 3), the cells were protected from H2O2-oxidative stress. These compounds, together with quercetin and vitamin C, were explored through the modeled S. cerevisiae AQP3 and human AQP7 by molecular docking analysis. To explain these results, an antioxidant mechanism for the isolated compounds was proposed through blocking H2O2 passage mediated by aquaporin transport. On the other hand, 1, 2, and 3 were not cytotoxic in a panel of three cancer cell lines.

1H-NMR Metabolomic Study of the Mushroom Pleurotus djamor for the Identification of Nematocidal Compounds.

Due to the increasing populations of anthelmintic-resistant gastrointestinal nematodes and as a consequence of the adverse effects of synthetic drugs, this study focuses on the search for secondary metabolites with nematocidal activity from the edible mushroom Pleurotus djamor using The proton nuclear magnetic resonance (1H-NMR) metabolomics. The highest activity was shown by the ethyl acetate fractions of mycelium (EC50 290.8 µg/mL) and basidiomes (EC50 282.7 µg/mL). Principal component analysis (PCA) and hierarchical data analysis (HCA) of the 1H-NMR metabolic profiles data showed that the ethanolic extracts, the ethyl acetate, butanol, and water fractions from mycelium have different metabolic profiles than those from basidiomes, while low polarity (hexane) fractions from both stages of fungal development show similar profiles. Orthogonal partial least squares discriminant analysis (OPLS-DA) allowed the identification of signals in the 1H-NMR metabolic profile associated with nematocidal activity. The signals yielded via OPLS-DA and bidimensional NMR analysis allowed the identification of uracil as a component in the ethyl acetate fraction from basidiomes, with an EC50 of 237.7 µg/mL. The results obtained showed that chemometric analyses of the 1H-NMR metabolic profiles represent a viable strategy for the identification of bioactive compounds from samples with complex chemical profiles.

Production and characterization of a chimeric protein targeting synuclein epitopes for immunotherapy against synucleinopathies.

The aggregation and spread of alpha-synuclein (αSyn) is associated with several pathogenic pathways that lead to neurodegeneration and, ultimately, to synucleinopathies development. Hence, the establishment of a safe and effective disease-modifying therapy that limits or prevents the spread of toxic αSyn aggregation could lead to positive clinical outcomes. A rational vaccine design can be focused on the selection of specific epitopes able to induce the immune response desired, for example, antibodies able to mediate the clearance of αSyn aggregates without the induction of inflammatory responses. To develop a rapid system for the evaluation of a vaccine candidate against synucleinopathies, rLTB-Syn (an antigen based on three B cell epitopes from αSyn and the B subunit of the heat-labile Escherichia coli enterotoxin [LTB] as adjuvant/carrier) was produced using recombinant E. coli (Rosetta DE3) as the expression host. The bacterial version of rLTB-Syn was produced as soluble protein at yields up to 1.72 mg/g biomass. A method for the purification of rLTB-Syn (~18 kDa) was developed based on ion exchange chromatography, reaching purity >93% with a final concentration of 82.6 µg/mL. Furthermore, the purified soluble rLTB-Syn retained GM1 binding activity, suggesting proper folding and pentameric structure. The results from this study establish a fast and effective method to obtain rLTB-Syn, making it useful in the design of novel vaccine formulations targeting synucleinopathies.

(1) H and (13) C NMR characterization of new cycloartane triterpenes from Mangifera indica.

From the stem bark of Mangifera indica, seven cycloartane-type secondary metabolites were isolated. Compound 1 has been isolated for the first time from M. indica, whereas compounds 2 (2a and 2b, as an epimeric mixture), 3, and 4 are new triterpenoid-type cycloartanes. Unambiguous (13) C and (1) H NMR assignments for these compounds and the known compounds mangiferonic acid (compound 5), isomangiferolic acid (compound 6), ambolic acid (compound 7), and friedelin (compound 8) are reported; the latter because full NMR data for these compounds are not available in the literature.

Cytotoxic podophyllotoxin type-lignans from the steam bark of Bursera fagaroides var. fagaroides.

The hydroalcoholic extract of the steam bark of B. fagaroides var. fagaroides displayed potent cytotoxic activity against four cancer cell lines, namely KB (ED50 = 9.6 × 10(-2) µg/mL), PC-3 (ED50 = 2.5 × 10(-1) µg/mL), MCF-7 (ED50 = 6.6 µg/mL), and HF-6 (ED50 = 7.1 × 10(-3) µg/mL). This extract also showed anti-tumour activity when assayed on mice inoculated with L5178Y lymphoma cells. Bioactivity-directed isolation of this extract, afforded seven podophyllotoxin-type lignans identified as podophyllotoxin (1), ß-peltatin-A-methylether (2), 5'-desmethoxy-ß-peltatin-A-methylether (3), desmethoxy-yatein (4), desoxypodophyllotoxin (5), burseranin (6), and acetyl podophyllotoxin (7) by 1D and 2DNMR and FAB-MS analyses, and comparison with reported values. All the isolated compounds showed potent cytotoxic activity in the cell lines tested, especially compound 3, which exhibited greater activity than camptothecin and podophyllotoxin against PC-3 (ED50= 1.0 × 10(-5) µg/mL), and KB (ED50 = 1.0 × 10(-5) µg/mL). This is the first report of the isolation of podophyllotoxin and its acetate in a Bursera species.

Identification of Putative Candidate Genes from Galphimia spp. Encoding Enzymes of the Galphimines Triterpenoids Synthesis Pathway with Anxiolytic and Sedative Effects.

Galphimia spp. is popularly used in Mexican traditional medicine. Some populations of Galphimia exert anxiolytic and sedative effects due to the presence of the modified triterpenoids galphimines. However, the galphimine synthesis pathway has not yet been elucidated. Hence, in this study, a comparative transcriptome analysis between two contrasting populations of Galphimia spp., a galphimine-producer, and a non-galphimine-producer, is performed using RNA-Seq in the Illumina Next Seq 550 platform to identify putative candidates genes that encode enzymes of this metabolic pathway. Transcriptome functional annotation was performed using the Blast2GO in levels of gene ontology. For differential expression analysis, edgeR, pheatmap, and Genie3 library were used. To validate transcriptome data, qPCR was conducted. In producer and non-producer plants of both populations of Galphimia spp., most of the transcripts were grouped in the Molecular Function level of gene ontology. A total of 680 differentially expressed transcripts between producer and non-producer plants were detected. In galphimine-producer plants, a larger number of highly expressed transcripts related to acyclic and polycyclic terpene synthesis were identified. As putative candidate genes involved in the galphimine synthesis pathway, P450 family members and enzymes with kinase activity were identified.

Updated biotechnological approaches developed for 2,7'-cyclolignan production1.

2,7'-Cyclolignans constitute a group of compounds regularly employed in clinical contexts due to their antiviral and anticancer properties as expressed in a number of already available designed drugs. Possibilities for other therapeutic developments are indicated. All commercial preparations actually originate from a single compound, podophyllotoxin, currently extracted from a few limited-in-abundance species of Podophyllum. This supply problem has stimulated considerable interest in the development of alternative strategies offering greater sustainable availability of the compound at affordable costs. Approaches such as plant breeding and micropropagation of the high-producing Podophyllum species have been explored with the aim of establishing production of these compounds. Since 20 years of exploration of the total chemical synthesis of podophyllotoxin has not proved economically viable, extensive research has now been undertaken to find methods for stimulating the accumulation of 2,7'-cyclolignans using tissue cultures. Both undifferentiated and differentiated cell cultures, mainly from Linum, Podophyllum, Juniperus, Callitris, Anthriscus and Forsynthia genera, have been reported. Although considerable progress has been made concerning the concentration and productivity of certain strains of Linum album, no economically viable production system has been established. Exploration and development of biosynthetical pathways leading to 2,7'-cyclolignans may open paths for future metabolic engineering, such as the bioconversion of deoxypodophyllotoxin to epipodophyllotoxin employing a human hepatic enzyme heterologously expressed in Escherichia coli.

Transformed cell suspension culture of Galphimia glauca producing sedative nor-friedelanes.

The Mexican species GALPHIMIA GLAUCA (Cav.) Kuntze (Malphigiaceae) synthesises a family of sedative and anxiolytic nor-secofriedelanes, designated as galphimines. These active principles accumulate at low concentration in the aerial parts of plants from wild populations. Transformed calluses and cell suspension cultures of this species were established in order to induce a greater production of nor-friedelanes. The cell suspension line GgBa was selected and grown over a period of two years of continuous subculturing in MS nutrient medium in the absence of growth regulators. PCR and Southern blot analyses were employed in order to confirm that the ROL A gene had been integrated into the plant genome. Batch cultures of the GgBa cell line were grown over a 32-day period and first-order growth kinetics was observed, reaching a specific growth rate (micro) of 0.13 d (-1). The production of glaucacetalin A ( 10), a triterpenoid related to the known galphimines, was quantified in the nutrient medium by HPLC. The transformed cell suspension culture GgBa also synthesised a novel nor-friedelane, given the name glaucacetalin D ( 13). High-resolution spectroscopic and spectrometric techniques were employed to elucidate the structure of 13. This triterpene has never been observed in wild plant tissues or in other IN VITRO cultures. Maslinic acid ( 14) was identified in cell biomasses. The triterpene production of the cell line GgBa was as follows: glaucacetalin A, 2.7 mg/L; glaucacetalin D, 2.9 mg/L and maslinic acid, 2.4 mg/g dry weight. The sedative activity of compounds 10 and 13 was demonstrated in ICR mice by using the sodium pentobarbital-induced hypnosis model. No cytotoxicity of 10 and 13 was exhibited against KB, MCF-7 and HF6 human cancer cell lines.

Characterization of Fungal Endophytes Isolated from the Metal Hyperaccumulator Plant Vachellia farnesiana Growing in Mine Tailings.

Heavy metal pollution has become an environmental and health problem worldwide. With the aim of finding novel strategies for metal bioremediation, endophytic fungi from the heavy metal hyperaccumulator plant Vachellia farnesiana were isolated and characterized. The plants were growing in mine tailings, rich in Zn, Pb, and Cu. Morphological and phylogenetic analyses indicated that the fungal strains belonged to Neocosmospora and Aspergillus genera. The Neocosmospora isolate belongs to the Fusarium solani species complex (FSSC) that groups phytopathogen species. However, in this case the plants from which it was isolated did not show any signs of disease. Both fungal strains were able to remove significant amounts of heavy metals from liquid cultures, either in a mixture of the three metals or each metal in a single culture. In response to lead exposure, the Neocosmospora sp. strain secreted specific novel phenolic compounds other than anthraquinones or naphtoquinones, which have been described in similar situations. The Aspergillus sp. dropped the pH in the medium. High-performance liquid chromatography determinations indicated that this strain secreted mainly glutamic acid in response to lead, a novel mechanism, which has not been reported elsewhere. Malic and succinic acids were also produced in response to lead exposure. Possibly, glutamic and succinic acids (synthesized in the Krebs cycle) can be used to cope with metal toxicity due to the plant providing photosynthates to the fungus. These fungi showed the potential to be used for bioremediation or restoration of metal-polluted environments.