Enhanced Biosynthesis of Withanolides by Elicitation and Precursor Feeding in Cell Suspension Culture of Withania somnifera (L.) Dunal in Shake-Flask Culture and Bioreactor

The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg), withanolide B (4826.05 mg), withaferin A (3732.81 mg), withanone (6538.65 mg), 12 deoxy withanstramonolide (3176.63 mg), withanoside IV (2623.21 mg) and withanoside V (2861.18 mg)] were achieved in the combined treatment of chitosan (100 mg/l) and squalene (6 mM) along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28^th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold) as well as bioreactor (1.66-fold) when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period.     

A novel acid-producing fungus isolated from bauxite residue: the potential to reduce the alkalinity

Abstract

Globally, bauxite residue is creating extensive environmental problems. Nevertheless, there are numerous functional microorganisms that exist in this extreme environment, and fungus has an essential role in the pedogenesis of bauxite residue. This study describes a microbial method to reduce the alkalinity of bauxite residue through the actions of a functional fungus to produce acidic substances. Bauxite residue samples were screened for fungal activity, and fungal tolerance was assessed using saline and alkaline media of varying concentrations, using aniline blue-PDA medium to evaluate acid production. A tolerant fungus with the ability to produce acidic substances was selected and named EEEL01 (Environmental Ecological Engineering Laboratory No. 01). Further morphological and molecular characterization identified this fungus as Penicillium oxalicum. Factors including pH, NaCl concentration, carbon and nitrogen sources were used to test the efficiency of acid production by the fungus. Based on optimal growth conditions, bauxite residue inoculated with EEEL01 reduced pH from 10.26 to 6.48 over 11?days. These results suggest that EEEL01 could effectively grow and release organic acids under extreme alkaline and saline conditions, which provide the potential for remediation.     

Fisetin modulates mitochondrial enzymes and apoptotic signals in benzo(a)pyrene-induced lung cancer

The present study was aimed to delineate in vivo mechanisms of orally administered fisetin with special reference to mitochondrial dysfunction in lung tissues employing benzo(a)pyrene (B(a)P) as the model lung carcinogen. The recent revival of interest in the study of mitochondria has been stimulated by the evidence that genetic and/or metabolic alterations in this organelle lead to a variety of human diseases including cancer. These alterations could be either causative or contributing factors. Hence, the activities of mitochondrial-specific enzymes of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and tumor marker, carcinogenic embryonic antigen were analyzed in control and experimental groups of mice. The induction of apoptotic and anti-apoptotic proteins such as Bcl-2/Bax, cytochrome c, caspase-9 and caspase-3 was confirmed by the immunohistochemistry and Western blot analyses. Furthermore, transmission electron microscopy study of lung sections of B(a)P-induced mice showed the presence of phaemorphic cells with dense granules and increased mitochondria. All the aberrations were alleviated when the mice were treated with fisetin (25 mg/kg body weight). The results proved fisetin to be a very successful drug in combating the mitochondrial dysfunction in an experimental model of lung carcinogenesis induced by B(a)P.     

Exploration of protein–protein interaction effects on α-2-macroglobulin in an inhibition of serine protease through gene expression and molecular simulations studies

In Prophenoloxidase (ProPO) cascade, two targets namely serine protease and α-2-macroglobulin are key regulators involved in the defense system of crustaceans. In biological systems, routine role of cell systems requires the understanding in protein–protein interactions through experimental and theoretical concepts, which might yield useful insights into the cellular responses. Response of cells to regulating the immune system is governed by the interactions-involved biomolecular simulations. Unfortunately, studies on the inhibitors (SP and α-2M) that negatively regulate the proPO system or melanization in penaeid shrimp are not yet available. In order to understand how these interactions change the proPO mechanism in Indian white shrimp Fenneropenaeus indicus was determined. In F. indicus, innate immune system is in a sensitive balance of intricate interactions; elucidating these interactions by the integration of in silico and in vitro has great potential. We have determined the expression of both the SP and α-2M enzymes in regulatory mechanism, which are analyzed through qRT-PCR, protein–protein docking, and simulation studies. From this work, we propose a novel approach for studying an organism at the systems level by integrating genome-wide computational analysis and the gene expression data.     

Sonication,Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal

In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants.     

Molecular docking analysis of phytocompounds from Andrographis paniculata binding with proteins in the notch-signaling pathway

It is of interest to document the molecular docking analysis of phytocompounds from Andrographis paniculata binding with protein NOTCH1 in the Notch-signaling pathway in the context of cancer. Hence, we document the binding features of neoandrographolide, 14-deoxyandrographolide, androgapholide and andrograpanin with proteins in the notch-signaling pathway for further consideration.     

Karyomorphological effects of two new oil formulations on Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

The cotton bollworm Helicoverpa armigera is a serious pest of many economically important crops. Since this pest has become resistant to the conventional synthetic insecticides, newer compounds and formulations are being developed against this insect pest. Many natural compounds isolated from the plants were tested against this pest. Among them Hyptis suaveolens and Melochia corchorifolia showed insecticidal properties against H. arnigera. Based on bioefficacy studies, caryophyllene and β-sitosterol were isolated from H. suaveolens and M. corchorifolia respectively. The isolated natural compounds were further developed as formulations in various combinations with neem (Azadirachta indica) and karanj (Pongamia pinnata) oils. The present study indicated that the formulations influenced the karyomorphology of H. armigera.