Correlation of CYP2R1 gene promoter methylation with circulating vitamin D levels among healthy adults.

Background & objectives: Despite being a tropical country, vitamin D deficiency is highly prevalent in India with studies indicating 40-99 per cent prevalence. Apart from calcium and phosphate metabolism, vitamin D is involved in cell cycle regulation, cardiovascular, hepatoprotection. The metabolism of vitamin D is regulated by vitamin D tool genes (CYP2R1/CYP27B1/CYP24A1/VDR). The promoter regions of some of these genes have CpG islands, making them prone to methylation induced gene silencing, which may cause a reduction in circulating vitamin D levels. Epigenetic basis of vitamin D deficiency is yet to be studied in India, and hence, this pilot study was aimed to analyze whether methylation levels of CYP2R1 gene were correlated with the levels of 25(OH)D in healthy, adult individuals in Indian population. Methods: In this cross-sectional study, healthy adults of 18-45 yr of age with no history of malabsorption, thyroidectomy, chronic illness or therapeutic vitamin D supplementation were recruited. DNA methylation analysis was carried out by methylation specific quantitative PCR. Serum calcium, phosphate and vitamin D levels were also quantified. Statistical analysis was done by R 4.0.5 software. Results: A total of 61 apparently healthy adults were analyzed. The serum vitamin D levels did not correlate with CYP2R1 methylation levels in our study population. Significant positive correlation was observed between age and serum vitamin D levels. Significant association of gender was found with CYP2R1 methylation levels. Interpretation & conclusions: This study found no significant correlation between levels of CYP2R1 methylation and circulating 25(OH)D deficiency. Further studies on the Indian population having a larger sample size including entire vitamin D tool genes, among different ethnic groups may be conducted to elucidate molecular etiology of circulating 25(OH)D deficiency. The high prevalence of normal serum calcium and phosphate levels among vitamin D deficient subjects in this study coupled with the strikingly high prevalence of the deficiency at the national level, may suggest the need to revise the cut-off criteria for vitamin D deficiency in the Indian population.

Phytoremediation potential of Solanum viarum Dunal and functional aspects of their capitate glandular trichomes in lead, cadmium, and zinc detoxification.

In the present scenario, remediation of heavy metals (HMs) contaminated soil has become an important work to be done for the well-being of human and their environment. Phytoremediation can be regarded as an excellent method in environmental technologies. The present contemporary research explores the Solanum viarum Dunal function as a potential accumulator of hazardous HMs viz. lead (Pb), cadmium (Cd), zinc (Zn), and their combination (CHM). On toxic concentrations of Pb, Cd, Zn, and their synergistic exposure, seeds had better germination percentage and their 90d old aerial tissues accumulated Pb, Cd, and Zn concentrations ranging from 44.53, 84.06, and 147.29 mg kg-1 DW, respectively. Pattern of accumulation in roots was as Zn 70.08 > Pb 48.55 > Cd 42.21 mg kg-1DW. Under HMs treatment, positive modulation in physiological performances, antioxidant activities suggested an enhanced tolerance along with higher membrane stability due to increased levels of lignin, proline, and sugar. Phenotypic variations were recorded in prickles and roots of 120 d old HM stressed plants, which are directly correlated with better acclimation. Interestingly, trichomes of the plant also showed HM accumulation. Later, SEM-EDX microanalysis suggested involvement of S. viarum capitate glandular trichomes as excretory organs for Cd and Zn. Thus, the present study provides an understanding of the mechanism that makes S. viarum to function as potent accumulator and provides information to generate plants to be used for phytoremediation.

Single-cell sequencing: A cutting edge tool in molecular medical research.

The rapid development of advanced high throughput technologies and introduction of high resolution "omics" data through analysis of biological molecules has revamped medical research. Single-cell sequencing in recent years, is in fact revolutionising the field by providing a deeper, spatio-temporal analyses of individual cells within tissues and their relevance to disease. Like conventional sequencing, the single-cell approach deciphers the sequence of nucleotides in a given Deoxyribose Nucleic Acid (DNA), Ribose Nucleic Acid (RNA), Micro Ribose Nucleic Acid (miRNA), epigenetically modified DNA or chromatin DNA; however, the unit of analyses is changed to single cells rather than the entire tissue. Further, a large number of single cells analysed from a single tissue generate a unique holistic perception capturing all kinds of perturbations across different cells in the tissue that increases the precision of data. Inherently, execution of the technique generates a large amount of data, which is required to be processed in a specific manner followed by customised bioinformatic analysis to produce meaningful results. The most crucial role of single-cell sequencing technique is in elucidating the inter-cell genetic, epigenetic, transcriptomic and proteomic heterogeneity in health and disease. The current review presents a brief overview of this cutting-edge technology and its applications in medical research.

Growth and alkaloid production along with expression profiles of biosynthetic pathway genes in two contrasting morphotypes of prickly and prickleless Solanum viarum Dunal.

Growth and production kinetics of three important glycoalkaloids viz. α-solanine, solanidine, and solasodine in two contrasting prickly and prickleless plants of Solanum viarum Dunal were evaluated under in vitro conditions. The prickleless plants showed improved accumulation of total glycoalkaloid content [7.11 and 6.85 mg g-1 dry weight (DW)] and growth (GI = 11.08 and 19.26) after 45 and 50 days of culture cycle, respectively. For higher biomass (91.18 g l-1) as well as glycoalkaloid (52.56 mg l-1) recovery, the prickleless plants served as highly profitable platform. All the three studied glycoalkaloids were identified and quantified by mass spectrometry and HPLC. All the three studied glycoalkaloids accumulated in age-dependent manner. The presence of two constituents, i.e., solasodine and solanidine mainly contributed for higher accumulation of total glycoalkaloid content in the prickleless plants. However, the synthesis of α-solanine was highly age specific and could be detected after 4 to 5 weeks of culture cycle in both prickle containing as well as prickleless plants of S. viarum. The higher accumulation of glycoalkaloids in prickleless plants was also supported with the expression analysis of six key pathway enzymes viz. mevalonate kinase (MVK), 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR), farnesyl diphosphate synthase (FPS), UDP-galactose/solanidine galactosyltransferase (SGT1), UDP-glucose/solanidine glucosyltransferase (SGT2), and cytochrome P450 monooxygenase (CYP). The results indicated that the plants harvested after 45 and 50 days of culture cycle accumulated maximum bioactive in-demand glycoalkaloids in the prickly and prickleless plants of S. viarum Dunal, respectively.

Elicitation Enhanced the Yield of Glycyrrhizin and Antioxidant Activities in Hairy Root Cultures of Glycyrrhiza glabra L.

Glycyrrhiza glabra L. has become an endangered medicinal plant due to the unabated extraction of glycyrrhizin. Glycyrrhizin is a triterpenoid saponin that is a root centric secondary metabolite having numerous pharmacological properties, such as anti-inflammatory, immunomodulatory, antiallergic, antiulcer, and is found to be effective even against HIV. Harvesting of the roots for high value glycyrrhizin destroys the whole plant causing existential threat to the plant itself and consequent damage to biodiversity. The present study establishes that hairy root cultures of G. glabra, using an optimized elicitor, can dramatically enhance focused production of glycyrrhizin at a much faster pace year-round without causing destruction of the plant. Hairy root cultures of G. glabra were developed using the Agrobacterium rhizogenes A4 strain. The glycyrrhizin content was enhanced using different biotic and abiotic elicitors, for example, PEG (polyethylene glycol), CdCl2, cellulase, and mannan at different concentrations and durations. PEG at 1% concentration enhanced the yield of glycyrrhizin up to 5.4-fold after 24 h of exposure, whereas 200 µg mL-1 cellulase enhanced glycyrrhizin yield to 8.6-fold after 7 days of treatment. Mannan at 10 mg L-1 concentration enhanced the production of glycyrrhizin up to 7.8-fold after 10 days of stress. Among different antioxidant enzymes, SOD activity was significantly enhanced under drought, cellulase and mannan stress. This identification of elicitors can result in abundant supply of valuable glycyrrhizin to meet broad spectrum demand through commercial production without endangering G. glabra L.

Transcriptome analysis provides insight into prickle development and its link to defense and secondary metabolism in Solanum viarum Dunal.

Prickles are epidermal outgrowth found on the aerial surface of several terrestrial plants. Microscopic studies on prickles of S. viarum Dunal indicated a crucial role of glandular trichomes (GTs) in their development. A spontaneously obtained prickleless mutant showed normal epidermal GTs, but its downstream developmental process to prickle was perturbed. Thus, prickleless mutant offers an ideal opportunity to unveil molecular regulators working downstream to GTs in the prickle formation. Differential transcriptome analysis of epidermis of prickly and prickleless mutant revealed that expression of several defense regulators like ethylene, salicylic acid, PR-proteins, etc. were significantly down-regulated in prickleless mutant, provide an important link between defense and prickle development. It was also noteworthy that the expression of few essential development related TFs like MADS-box, R2R3-MYB, REM, DRL1, were also down-regulated in the stem, petioles, and leaves of prickleless mutant indicating their potential role in prickle development. Interestingly, the gene expression of terpenoid, steroid, flavonoid, glucosinolate, and lignin biosynthesis pathways were up-regulated in prickleless mutant. The biochemical and qRT-PCR analysis also confirmed metabolite elevation. These results indicated that the loss of prickle was compensated by elevated secondary metabolism in the prickleless mutant which played important role in the biotic and abiotic stress management.

Withania somnifera: Advances and Implementation of Molecular and Tissue Culture Techniques to Enhance Its Application.

Withania somnifera, commonly known as Ashwagandha an important medicinal plant largely used in Ayurvedic and indigenous medicine for over 3,000 years. Being a medicinal plant, dried powder, crude extract as well as purified metabolies of the plant has shown promising therapeutic properties. Withanolides are the principal metabolites, responsible for the medicinal properties of the plant. Availability and amount of particular withanolides differ with tissue type and chemotype and its importance leads to identification characterization of several genes/ enzymes related to withanolide biosynthetic pathway. The modulation in withanolides can be achieved by controlling the environmental conditions like, different tissue culture techniques, altered media compositions, use of elicitors, etc. Among all the in vitro techniques, hairy root culture proved its importance at industrial scale, which also gets benefits due to more accumulation (amount and number) of withanolides in roots tissues of W. somnifera. Use of media compostion and elicitors further enhances the amount of withanolides in hairy roots. Another important modern day technique used for accumulation of desired secondary metabolites is modulating the gene expression by altering environmental conditions (use of different media composition, elicitors, etc.) or through genetic enginnering. Knowing the significance of the gene and the key enzymatic step of the pathway, modulation in withanolide contents can be achieved upto required amount in therapeutic industry. To accomplish maximum productivity through genetic enginnering different means of Withania transformation methods have been developed to obtain maximum transformation efficiency. These standardized transformation procedues have been used to overexpress/silence desired gene in W. somnifera to understand the outcome and succeed with enhanced metabolic production for the ultimate benefit of human race.

Sterol glycosyltransferases required for adaptation of Withania somnifera at high temperature.

Heat is a major environmental stress factor that confines growth, productivity, and metabolism of plants. Plants respond to such unfavorable conditions through changes in their physiological, biochemical and developmental processes. Withania somnifera, an important medicinal plant, grows in hot and dry conditions, however, molecular mechanisms related to such adaptive properties are not known. Here, we elucidated that members of the sterol glycosyltransferases (SGT) gene family play important roles in the survival of W. somnifera under adverse conditions through maintaining the integrity of the membrane. SGTs are enzymes involved in sterol modifications and participate in metabolic flexibility during stress. Silencing of WsSGT members, for instance WsSGTL1, WsSGTL2 and WsSGTL4, was inimical for important physiological parameters, such as electron transport rate, photochemical quantum yield, acceptor side limitation, non-photochemical quenching (NPQ), Fv/Fm and net photosynthetic rate, whereas stomatal conductance, transpiration rate and dark respiration rates (Rds) were increased. Decreased NPQ and increased Rds helped to generate significant amount of ROS in the Wsamisgt lines. After heat stress, H2 O2 , lipid peroxidation and nitric oxide production increased in the Wsamisgt lines due to high ROS generation. The expression of HSPs in Wsamisgt lines might be involved in regulation of physiological processes during stress. We have also observed increased proline accumulation which might be involved in restricting water loss in the Wsamisgt lines. Taken together, our observations revealed that SGTL enzyme activity is required to maintain the internal damages of the cell against high temperature by maintaining the sterol vs sterol glycosides ratio in the membranes of W. somnifera.

Comparative interactions of withanolides and sterols with two members of sterol glycosyltransferases from Withania somnifera.

BACKGROUND: Sterol glycosyltransferases (SGTs) are ubiquitous but one of the most diverse group of enzymes of glycosyltransferases family. Members of this family modulate physical and chemical properties of secondary plant products important for various physiological processes. The role of SGTs has been demonstrated in the biosynthesis of pharmaceutically important molecules of medicinal plants like Withania somnifera. RESULTS: Analysis suggested conserved behaviour and high similarity in active sites of WsSGTs with other plant GTs. Substrate specificity of WsSGTs were analysed through docking performance of WsSGTs with different substrates (sterols and withanolides). Best docking results of WsSGTL1 in the form of stable enzyme-substrate complex having lowest binding energies were obtained with brassicasterol, transandrosteron and WsSGTL4 with solasodine, stigmasterol and 24-methylene cholesterol. CONCLUSION: This study reveals topological characters and conserved nature of two SGTs from W. somnifera (WsSGTs) i.e. WsSGTL1 and WsSGTL4. However, besides being ubiquitous in nature and with broad substrate specificity, difference between WsSGTL1 and WsSGTL4 is briefly described by difference in stability (binding energy) of enzyme-substrate complexes through comparative docking.

WsSGTL1 gene from Withania somnifera, modulates glycosylation profile, antioxidant system and confers biotic and salt stress tolerance in transgenic tobacco.

Glycosylation of sterols, catalysed by sterol glycosyltransferases (SGTs), improves the sterol solubility, chemical stability and compartmentalization, and helps plants to adapt to environmental changes. The SGTs in medicinal plants are of particular interest for their role in the biosynthesis of pharmacologically active substances. WsSGTL1, a SGT isolated from Withania somnifera, was expressed and functionally characterized in transgenic tobacco plants. Transgenic WsSGTL1-Nt lines showed an adaptive mechanism through demonstrating late germination, stunted growth, yellowish-green leaves and enhanced antioxidant system. The reduced chlorophyll content and chlorophyll fluorescence with decreased photosynthetic parameters were observed in WsSGTL1-Nt plants. These changes could be due to the enhanced glycosylation by WsSGTL1, as no modulation in chlorophyll biogenesis-related genes was observed in transgenic lines as compared to wildtype (WT) plants. Enhanced accumulation of main sterols like, campesterol, stigmasterol and sitosterol in glycosylated form was observed in WsSGTL1-Nt plants. Apart from these, other secondary metabolites related to plant's antioxidant system along with activities of antioxidant enzymes (SOD, CAT; two to fourfold) were enhanced in WsSGTL1-Nt as compared to WT. WsSGTL1-Nt plants showed significant resistance towards Spodoptera litura (biotic stress) with up to 27 % reduced larval weight as well as salt stress (abiotic stress) with improved survival capacity of leaf discs. The present study demonstrates that higher glycosylation of sterols and enhanced antioxidant system caused by expression of WsSGTL1 gene confers specific functions in plants to adapt under different environmental challenges.

Agrobacterium tumefaciens-mediated transformation of Withania somnifera (L.) Dunal: an important medicinal plant.

This report describes Agrobacterium tumefaciens-mediated transformation of Withania somnifera--an important Indian medicinal plant. A. tumefaciens strain LBA4404, containing the binary vector pIG121Hm was used for transformation, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter. The leaf segments from two-and-a-half-month-old green house-grown seedlings were more efficient in transformation, as compared to those from the in vitro-grown shoots. Second expanded leaf from the shoot tip gave the highest transient transformation efficiency. Selection of transgenic shoots was done in the presence of 50 mg l(-1) kanamycin. Polymerase chain reaction analysis of T(0) transgenic plants showed the presence of gusA and nptII genes. The expression of these transgenes in T(1) progeny was confirmed by RT-PCR. The integration of gusA gene was confirmed by Southern blot analysis. The transformation efficiency was found to be 1.67%.