Investigation of Linum flavum (L.) Hairy Root Cultures for the Production of Anticancer Aryltetralin Lignans
Linum flavum hairy root lines were established from hypocotyl pieces using Agrobacterium rhizogenes strains LBA 9402 and ATCC 15834. Both strains were effective for transformation but induction of hairy root phenotype was more stable with strain ATCC 15834. Whereas similar accumulation patterns were observed in podophyllotoxin-related compounds (6-methoxy-podophyllotoxin, podophyllotoxin and deoxypodophyllotoxin), significant quantitative variations were noted between root lines. The influence of culture medium and various treatments (hormone, elicitation and precursor feeding) were evaluated. The highest accumulation was obtained in Gamborg B5 medium. Treatment with methyl jasmonate, and feeding using ferulic acid increased the accumulation of aryltetralin lignans. These results point to the use of hairy root culture lines of Linum flavum as potential sources for these valuable metabolites as an alternative, or as a complement to Podophyllum collected from wild stands.
Biogenic zinc oxide nanoparticles-enhanced biosynthesis of lignans and neolignans in cell suspension cultures of Linum usitatissimum L
Zinc oxide nanoparticles (NPs) have emerged as a novel elicitor for enhanced biosynthesis of secondary metabolites in in vitro plant cell cultures. The current study was aimed to explore elicitation abilities of ZnO-NPs for enhanced accumulation of lignans and neolignans in cell cultures of Linum usitatissimum. We optimized concentration of zinc oxide NPs before carrying out a full-fledged experiment. Subsequently, an optimum dose of 100 mg/l was introduced into the culture medium on day 0, days 0 and 15, and finally days 0 and 25. We observed that repeated elicitation stimulated various parameters and physiological responses in Linum usitatissimum cell cultures than one-time elicitation. Repeated elicitation of cell cultures on day 0 and 15 resulted in highest fresh weight (412.16 g/l) and lignans production (secoisolariciresinol diglucoside 284.12 mg/l: lariciresinol diglucoside 86.97 mg/l). Contrarily, repeated elicitation on day 0 and 25 resulted in highest DW (13.53 g/l), total phenolic production (537.44 mg/l), total flavonoid production (123.83 mg/l) and neolignans production (dehydrodiconiferyl alcohol glucoside 493.28 mg/l: guaiacylglycerol-β-coniferyl alcohol ether glucoside 307.69 mg/l). Enhancement in plant growth and secondary metabolites accumulation was several fold higher than controls. Furthermore, a linear relationship existed between total phenolic and flavonoid contents which in turn was correlated with higher antioxidant activities.
Single Laboratory Validation of a Quantitative Core Shell-Based LC Separation for the Evaluation of Silymarin Variability and Associated Antioxidant Activity of Pakistani Ecotypes of Milk Thistle (Silybum Marianum L.)
Fruits of Silybum marianum (L.) Gaernt are the main source of taxifolin derived flavonolignans. Together, these molecules constitute a mixture called silymarin with many useful applications for cosmetic and pharmaceutic industries. Here, a validated method for the separation of the silymarin constituents has been developed to ensure precision and accuracy in their quantification. Each compound was separated with a high reproducibility. Precision and repeatability of the quantification method were validated according to the AOAC recommendations. The method was then applied to study the natural variability of wild accessions of S. marianum. Analysis of the variation in the fruits composition of these 12 accessions from Pakistan evidenced a huge natural diversity. Correlation analysis suggested a synergistic action of the different flavonolignans to reach the maximal antioxidant activity, as determined by cupric ion reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP) assays. Principal component analysis (PCA) separated the 12 accessions into three distinct groups that were differing from their silymarin contents, whereas hierarchical clustering analysis (HCA) evidenced strong variations in their silymarin composition, leading to the identification of new silybin-rich chemotypes. These results proved that the present method allows for an efficient separation and quantification of the main flavonolignans with potent antioxidant activities.
Melatonin-stimulated biosynthesis of anti-microbial ZnONPs by enhancing bio-reductive prospective in callus cultures of Catharanthus roseus var. Alba
Melatonin as plant growth regulator induces differential effects on metabolites that are responsible for reduction, capping and stabilization of zinc oxide nanoparticles. Phytochemical analysis of callus cultures was performed and results were compared with callus cultures supplemented with other plant growth regulators (α-napthalene acetic acid, 2,4-dichlorophenoxy acetic acid and thidiazuron). Highest total phenolic and flavonoid content [42.23 mg of gallic acid equivalent (GAE) g−1 DW and 36.4 mg of (quercetin equivalent) g−1 DW, respectively] were recorded at melatonin (1.0 µM) + NAA (13.5 µM). ZnONPs were synthesized from NAA (13.5 µM) and melatonin (1.0 µM) + NAA (13.5 µM)-induced calli extracts separately and characterized via X-ray diffraction, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). FTIR analysis confirmed the presence of phenolics and flavonoids that were mainly found responsible for reduction and capping of ZnONPs. SEM analysis showed triangular shaped ZnONPs synthesized from melatonin + NAA callus extract and these NPs were more dispersed as compared to the spherical-agglomerates of ZnONPs synthesized from NAA-mediated callus extract. Melatonin + NAA callus extract-mediated ZnONPs (having smaller size) were more potent against multiple drug resistant bacterial strains, e.g. Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa by producing zone of inhibitions 17 ± 0.76 mm,10 ± 0.57 mm and 13 ± 0.54 mm, respectively.
Enhanced production of lignans and neolignans in chitosan-treated flax (Linum usitatissimum L.) cell cultures
Linum usitatissimum is a source of pharmacologically active lignans and neolignans. An effective protocol has been established for the enhanced biosynthesis of lignans and neolignans in cell cultures of Linum usitatissimum by using chitosan addition. Gene expression analysis of monolignols (PAL, CCR and CAD), lignans (DIR, PLR and UGT) and neolignans (PCBER) biosynthetic genes by RT-qPCR as well as monolignol biosynthetic PAL, CCR and CAD enzyme activities evidenced a stimulation following chitosan treatment. Validated reverse phase high-performance liquid chromatography coupled to diode array detection was used to quantify secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG), dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol-β-coniferyl alcohol ether glucoside (GGCG) showed that chitosan treated cell cultures had better accumulation of these metabolites. Maximum enhancements of 7.3-fold (28 mg/g DW) occurred for LDG, 3.5-fold (58.85 mg/g DW) in DCG and while the least enhancement of 2-fold (18.42 mg/g DW) for SDG was observed in 10 mg/l chitosan treated cell cultures than to controls. Furthermore, same concentration of chitosan also resulted in 1.3-fold increase in antioxidant activity. Compared to the lignans and neolignans accumulations observed in wild type and RNAi-PLR transgenic flaxseeds, chitosan-treated cell cultures appeared to be a very effective production system for these compounds.
Malaria is one of the most important parasitic infection in the world. Cerebral and pulmonary complications may occur after infection and are often lethal. Immune response plays an important role in controlling malaria infection; however, excessive inflammatory response can lead to severe disease. The present work aims to decipher the cellular and molecular events associated with brain and pulmonary pathology in response to blood stage Plasmodium berghei ANKA (PbA) infection. PbA infection in C57BL/6 wild-type (WT) mice induces experimental cerebral malaria (ECM), associated with strong pro-inflammatory response, brain damage, as well as paralysis, coma early death (around day 7 p.i.). Interestingly, IFNγ receptor deficient mice (IFNγR1-/-, C57BL/6 background) are resistant to ECM and died at a later time-point, due to the hyperparasitaemia and severe anemia. Here, we addressed the impact of IFNγR1 deficiency in the development of pulmonary damage during PbA infection. At day 7 post-infection, the broncho-alveolar lavage (BAL) allowed the quantitative analysis of total cells and proteins in the broncho-alveolar space of the animals. In addition, histological analysis and Western blot were performed to compare the cerebral and pulmonary compartments. As compared to PbA-infected WT mice, the histological sections confirmed a less intense accumulation of leukocytes as well as an absence of hemorrhages in the brains of IFNγR1-/- mice. In addition, the quantification of pro-apoptotic proteins (Granzyme B and cleaved caspase-3) in olfactory bulbs showed lower levels in IFNγR1-/- mice. While IFNγR1 deficient mice were fully resistant to brain pathology, those mice were partially protected for pulmonary damage, as observed by the levels of Granzyme B and cleaved caspase-3 in the lung parenchyma, leukocyte number in the broncho-alveolar space and pulmonary edema.
Thermal Shift Assay for Characterizing the Stability of RNA Helicases and Their Interaction with Ligands
Thermofluor or thermal shift assay is an easily implementable, high-throughput method for assessing the thermostability of proteins and the influence of various ligands on that stability. It is particularly useful for the assaying of ligands that may stabilize oligomeric helicases, which rely on both substrates (oligonucleotides) and nucleotide cofactors (ATP analogues) for their stability in a functional state. In this chapter, we describe the rationale and present a basic protocol for the use of this technique. Multi-ligand screening is also discussed via a worked example of the stabilization of a hexameric RNA helicase, a target protein for structural studies in our laboratories.
Cryo-EM structure of transcription termination factor Rho from Mycobacterium tuberculosis reveals bicyclomycin resistance mechanism
The bacterial Rho factor is a ring-shaped motor triggering genome-wide transcription termination and R-loop dissociation. Rho is essential in many species, including in Mycobacterium tuberculosis where rho gene inactivation leads to rapid death. Yet, the M. tuberculosis Rho [MtbRho] factor displays poor NTPase and helicase activities, and resistance to the natural Rho inhibitor bicyclomycin [BCM] that remain unexplained. To address these issues, we solved the cryo-EM structure of MtbRho at 3.3 Å resolution. The MtbRho hexamer is poised into a pre-catalytic, open-ring state wherein specific contacts stabilize ATP in intersubunit ATPase pockets, thereby explaining the cofactor preference of MtbRho. We reveal a leucine-to-methionine substitution that creates a steric bulk in BCM binding cavities near the positions of ATP γ-phosphates, and confers resistance to BCM at the expense of motor efficiency. Our work contributes to explain the unusual features of MtbRho and provides a framework for future antibiotic development.
This paper aims to reflect on the perspectives of a critical return to certain aspects of the Postelian heritage, while in the recent decades the figure of this heterodox Renaissance thinker has been downgraded from fascinating to merely secondary. Indeed, his equation between intercultural communication and universal concordia remains generally valid to the present day, even for those who do not share his Adamitic and cabbalistic conceptions of language. On the other hand, his concept of congregator mundi appears as a valuable starting point for the discussion on the role and prerogatives of the intellectual as a mediator between human societies and the transcendent sphere. One may compare it with the recent thought of Giorgio Agamben, re-collocating the intellectual and the cultural critic in the line of the monotheistic prophets.
Les résultats que nous présentons ici ont été obtenus dans le cadre d’un projet de recherche actuellement en cours, portant sur la monnaie ducale normande au Xe siècle. Ce projet a été rendu possible grâce à une bourse de recherche accordée par Le Studium, qui a pour mission de faire venir des chercheurs étrangers (in casu JCM) dans la Région Centre-Val de Loire pendant un an pour mener un projet de recherche en collaboration avec des chercheurs de la région (in casu Marc et GS de l’IRAMAT-CEB, CNRS/Université d’Orléans, UMR 5060). Prenant comme point de départ l’étude incontournable de Françoise Dumas portant sur les 8584 monnaies conservées du trésor de Fécamp, enfoui vers 980/9853 , notre projet aboutira à une monographie sur la monnaie normande du Xe siècle. Il nous a cependant semblé que l’aspect que nous présentons ici a une valeur exemplaire qui justifie sa publication en avant-première. Un de nos objectifs est l’étude de la composition élémentaire des pièces, spécialité de l’IRAMAT-CEB. Nous avons pour l’heure analysé 134 monnaies normandes par LA-ICP-MS (spectrométrie de masse couplée à un plasma inductif avec prélèvement par ablation laser), une méthode d’analyse quasi-non destructive qui permet de doser les constituants majeurs des alliages monétaires (dans ce cas l’argent et le cuivre) ainsi qu’un grand nombre d’éléments mineurs et traces.