Fokker-Planck modeling of the stochastic dynamics of a Rijke tube.

We derive and numerically validate a low-order oscillator model to capture the stochastic dynamics of a prototypical thermoacoustic system (a Rijke tube) undergoing a subcritical Hopf bifurcation in the presence of additive noise. We find that on the fixed-point branch before the bifurcation, the system is dominated by the first duct mode, and the Fokker-Planck solution for the first Galerkin mode can adequately predict the stochastic dynamics of the overall system. We also find that this analytical framework predicts well the dominant mode on the limit-cycle branch, but underperforms in the hysteretic bistable zone where the role of nonlinearities is more pronounced. Besides offering new insights into stochastic thermoacoustic behavior, this study shows that an analytical framework based on the Fokker-Planck equation can facilitate the early detection of thermoacoustic instabilities in a Rijke-tube model subjected to noise.

Immunoglobulin G4 disease-related retroperitoneal fibrosis: A series of five cases.

Immunoglobulin G4 (IgG4)-related disease has the potential to impact any part of the body, including the walls of large- and medium-sized blood vessels and the ureters. While histopathologic examination is currently the standard method for identifying organ involvement and diagnosing IgG4-related disease (IgG4-RD), obtaining biopsy or surgical samples from vessel or ureteral walls is challenging. Given that patients may display only mild symptoms, non-invasive imaging plays a vital role in both diagnosing and managing IgG4-related diseases. Multidetector CT scans are valuable in establishing the primary diagnosis, identifying anatomical landmarks and assessing their relationships. Involvement of the genitourinary organs, such as the ureter, bladder, urethra, and male and female reproductive organs in IgG4-RD, is infrequent when compared to kidney involvement. The imaging findings may include the presence of a localised mass within or surrounding the affected organ or a generalised enlargement of the organ. This report includes cross-sectional images of five cases of IgG4-RD involving large- and medium-sized blood vessels (the aorta and superior mesenteric artery) and the ureters. Contribution: This case series provides insight into the various imaging appearances of IgG4-related retroperitoneal organ involvement and helps differentiate it radiologically from retroperitoneal fibrosis.

Effect of correlation time of combustion noise on early warning indicators of thermoacoustic instability.

In this paper, we analyze the effects of finite correlation time (noise color) of combustion noise on noise-induced coherence and early warning indicators (EWIs) via numerical and experimental studies. We consider the Rijke tube as a prototypical combustion system and model combustion noise as an additive Ornstein-Uhlenbeck process while varying noise intensity and correlation time. We numerically investigate corresponding effects on coherence resonance and multi-fractal properties of pressure fluctuations. Subsequently, we experimentally validate results and elucidate the influence of noise color and intensity on trends in coherence resonance and multi-fractal measures that can be expected in a practical scenario using an electroacoustic simulator. We find that the coherence factor, which quantifies the relative contribution of coherent oscillations in a noisy signal, increases as the system approaches the thermoacoustic instability-irrespective of the correlation time. It works at most levels of combustion noise (except for too low and too high noise levels). The Hurst exponent reduces as the system approaches thermoacoustic instability only when the correlation time is small. These results have implications on the prediction and monitoring of thermoacoustic instability in practical combustors.

Model-free forecasting of partially observable spatiotemporally chaotic systems.

Reservoir computing is a powerful tool for forecasting turbulence because its simple architecture has the computational efficiency to handle high-dimensional systems. Its implementation, however, often requires full state-vector measurements and knowledge of the system nonlinearities. We use nonlinear projector functions to expand the system measurements to a high dimensional space and then feed them to a reservoir to obtain forecasts. We demonstrate the application of such reservoir computing networks on spatiotemporally chaotic systems, which model several features of turbulence. We show that using radial basis functions as nonlinear projectors enables complex system nonlinearities to be captured robustly even with only partial observations and without knowing the governing equations. Finally, we show that when measurements are sparse or incomplete and noisy, such that even the governing equations become inaccurate, our networks can still produce reasonably accurate forecasts, thus paving the way towards model-free forecasting of practical turbulent systems.

Application of Vesical Imaging-Reporting and Data System in Evaluation of Urinary Bladder Cancer Using Multiparametric Magnetic Resonance Imaging: A Hospital-Based Cross-Sectional Study.

Background Multiparametric magnetic resonance imaging (mp-MRI) of urinary bladder (UB) is a novel imaging to predict detrusor muscle invasion in Bladder cancer (BC). The Vesical Imaging-Reporting and Data System (VI-RADS) was introduced in 2018 to standardize the reporting of BC with mp-MRI and to diagnose muscle invasion. This study was performed to evaluate the role of mp-MRI using VI-RADS to predict muscle invasive BC. Methods This prospective study was carried from June 2020 to May 2021 in a tertiary care institute. Thirty-six patients with untreated BC underwent mp-MRI followed by transuretheral resection of the tumor (TURBT). Mp-MRI findings were evaluated by two radiologists and BC was categorized according to VI-RADS scoring system. Resected tumors along with separate biopsy from the base were reported by two pathologists. Histopathological findings were compared with VI-RADS score and the performance of VI-RADS for determining detrusor muscle invasion was analyzed. Results VI-RADS scores of 4 and 5 were assigned to 9 (25%) and 15 (41.7%) cases, respectively, while 4 (13.3%) cases had VI-RADS score 3 on mp-MRI. VI-RADS 1 and 2 lesions were observed in six (16.7%) and two (5.5%) cases, respectively. On histopathology, 23 cases (63.9%) had muscle-invasive cancer and 13 cases (36.1%) had non-muscle-invasive cancer. The sensitivity and diagnostic accuracy of mp-MRI in predicting muscle invasive BC was 95.6 and 80.6%, respectively. Conclusion Mp-MRI has high sensitivity and diagnostic accuracy in predicting muscle invasive BC and should be advocated for evaluation of BC prior to surgery.

Evaluation of clinically significant prostate cancer using biparametric magnetic resonance imaging: An evolving concept.

Background: Multiparametric magnetic resonance imaging (mp-MRI) of prostate involves a combination of T2-weighted imaging, diffusion-weighted imaging, and dynamic contrast-enhanced (DCE) scans. However, controversy exists in the literature regarding the true value of DCE in the detection of clinically significant (CS) prostate cancer (PCa). Aim: The aim of this study is to compare the role of biparametric MRI (bp-MRI) and mp-MRI in the detection of CS PCa. Materials and Methods: Thirty-six patients with raised serum prostate-specific antigen levels were included. Bp-MRI was performed in all patients, whereas mp-MRI was performed in 30 cases only. The findings were characterized on the basis of prostate imaging reporting and data system (PI-RADS) v2 grading. PI-RADS v2 score of 3 or more was considered CS PCa. All patients underwent transrectal ultrasound-guided biopsy. Gleason score >6 was considered CS. Statistical analysis was done using the SPSS software and results interpreted. Results: CS PCa was observed in 31 cases on histopathology. On bp-MRI, CS PCa was seen in 31 patients. Five cases of PI-RADS v2 score 3 were seen on bp-MRI and 3 of them were upgraded to PI-RADS 4 on DCE images. One case of PI-RADS 3 had low Gleason score on biopsy, whereas 1 case of PI-RADS 2 had CS PCa on biopsy. No significant difference was observed between bp-MRI and mp-MRI in the detection of CS PCa. Conclusions: Both bp-MRI and mp-MRI have high sensitivity, specificity, and diagnostic accuracy and were nearly identical in the detection of CS PCa with no significant advantage of DCE images.

Quenching and amplification of thermoacoustic oscillations in two nonidentical Rijke tubes interacting via time-delay and dissipative coupling.

We numerically explore the quenching and amplification of self-excited thermoacoustic oscillations in two nonidentical Rijke tubes interacting via time-delay and dissipative coupling. On applying either type of coupling separately, we find that the presence of nonidentical heater powers can shrink the regions of amplitude death in both oscillators, while producing new regions of amplitude amplification in the weaker oscillator. We find that the magnitude of amplitude amplification grows with the heater power mismatch and with the total power input. These effects are also present when both types of coupling are applied simultaneously. This study highlights the critical role that nonidentical thermal loads can play in determining the amplitude response of coupled thermoacoustic systems, facilitating the design of control strategies for coupled oscillatorlike devices such as gas turbines.

Comparative evaluation of severity of COVID-19 pneumonia on computed tomography of the chest in vaccinated and non-vaccinated individuals: an observational study.

Purpose: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel virus causing an infectious disease, coronavirus disease 2019 (COVID-19). Computed tomography (CT) of the chest plays a significant role in the diagnosis and prognosis of COVID-19 using computed tomography severity scoring (CT-SS). Numerous vaccines are being made available in the world to lessen the effect of the COVID-19 pandemic. The purpose of the current study is to compare the severity of COVID-19 pneumonia using CT-SS in COVID-19-positive vaccinated (Covishield/Oxford-AstraZeneca) and non-vaccinated individuals and to compare the final outcome wherever possible. Material and methods: This observational study was carried out from March 2021 to April 2021. Forty vaccinated and 40 non-vaccinated RT-PCR-positive COVID-19 patients who underwent CT chest during the 4-12th day of illness formed the material of the study. Semi-quantitative scoring was used, and CT-SS was calculated based on the extent of lobar involvement in all the patients. CT-SS was then compared between the vaccinated and non-vaccinated groups and the results analysed. Results: CT scans were performed in 80 patients (40 patients each in the vaccinated and non-vaccinated groups). The majority of patients in the vaccinated group had mild (42.5%) and moderate (37.5%) CT-SS while the majority of patients in the non-vaccinated group had moderate (52.5%) and severe (27.5%) CT-SS score on chest CT. Also, no mortality was observed in the vaccinated group, with 2 deaths in the non-vaccinated group. Conclusions: Covishield vaccine administration reduces the severity of COVID-19 pneumonia as compared to the nonvaccinated group, with a marked reduction in mortality.

Azadirachta indica MicroRNAs: Genome-Wide Identification, Target Transcript Prediction, and Expression Analyses.

MicroRNAs are short, endogenous, non-coding RNAs, liable for essential regulatory function. Numerous miRNAs have been identified and studied in plants with known genomic or small RNA resources. Despite the availability of genomic and transcriptomic resources, the miRNAs have not been reported in the medicinal tree Azadirachta indica (Neem) till date. Here for the first time, we report extensive identification of miRNAs and their possible targets in A. indica which might help to unravel their therapeutic potential. A comprehensive search of miRNAs in the A. indica genome by C-mii tool was performed. Overall, 123 miRNAs classified into 63 families and their stem-loop hairpin structures were predicted. The size of the A. indica (ain)-miRNAs ranged between 19 and 23 nt in length, and their corresponding ain-miRNA precursor sequence MFEI value averaged as -1.147 kcal/mol. The targets of ain-miRNAs were predicted in A. indica as well as Arabidopsis thaliana plant. The gene ontology (GO) annotation revealed the involvement of ain-miRNA targets in developmental processes, transport, stress, and metabolic processes including secondary metabolism. Stem-loop qRT-PCR was carried out for 25 randomly selected ain-miRNAs and differential expression patterns were observed in different A. indica tissues. Expression of miRNAs and its targets shows negative correlation in a dependent manner.

Input-output system identification of a thermoacoustic oscillator near a Hopf bifurcation using only fixed-point data.

We present a framework for performing input-output system identification near a Hopf bifurcation using data from only the fixed-point branch, prior to the Hopf point itself. The framework models the system with a van der Pol-type equation perturbed by additive noise, and identifies the system parameters via the corresponding Fokker-Planck equation. We demonstrate the framework on a prototypical thermoacoustic oscillator (a flame-driven Rijke tube) undergoing a supercritical Hopf bifurcation. We find that the framework can accurately predict the properties of the Hopf bifurcation and the limit cycle beyond it. This study constitutes an experimental demonstration of system identification on a reacting flow using only prebifurcation data, opening up pathways to the development of early warning indicators for nonlinear dynamical systems near a Hopf bifurcation.

Enhanced expression of ginsenoside biosynthetic genes and in vitro ginsenoside production in elicited Panax sikkimensis (Ban) cell suspensions.

Dual metabolite, i.e., ginsenoside and anthocyanin, co-accumulating cell suspensions of Panax sikkimensis were subjected to elicitation with culture filtrates of Serratia marcescens (SD 21), Bacillus subtilis (FL11), Trichoderma atroviridae (TA), and T. harzianum (TH) at 1.25% and 2.5% v/v for 1- and 3-week duration. The fungal-derived elicitors (TA and TH) did not significantly affect biomass accumulation; however, bacterial elicitors (SD 21 and FL11), especially SD 21, led to comparable loss in biomass growth. In terms of ginsenoside content, differential responses were observed. A maximum of 3.2-fold increase (222.2 mg/L) in total ginsenoside content was observed with the use of 2.5% v/v TH culture filtrate for 1 week. Similar ginsenoside accumulation was observed with the use of 1-week treatment with 2.5% v/v SD 21 culture filtrate (189.3 mg/L) with a 10-fold increase in intracellular Rg2 biosynthesis (31 mg/L). Real-time PCR analysis of key ginsenoside biosynthesis genes, i.e., FPS, SQS, DDS, PPDS, and PPTS, revealed prominent upregulation of particularly PPTS expression (20-23-fold), accounting for the observed enhancement in protopanaxatriol ginsenosides. However, none of the elicitors led to successful enhancement in in vitro anthocyanin accumulation as compared to control values.

Comparative transcriptome analysis reveals candidate genes for the biosynthesis of natural insecticide in Tanacetum cinerariifolium.

BACKGROUND: Pyrethrins are monoterpenoids and consist of either a chrysanthemic acid or pyrethric acid with a rethrolone moiety. Natural pyrethrins are safe and eco-friendly while possessing strong insecticidal properties. Despite such advantages of commercial value coming with the eco-friendly tag, most enzymes/genes involved in the pyrethrin biosynthesis pathway remain unidentified and uncharacterized. Since the flowers of Tanacetum cinerariifolium are rich in major pyrethrins, next generation transcriptome sequencing was undertaken to compare the flowers and the leaves of the plant de novo to identify differentially expressed transcripts and ascertain which among them might be involved in and responsible for the differential accumulation of pyrethrins in T. cinerariifolium flowers. RESULTS: In this first tissue specific transcriptome analysis of the non-model plant T. cinerariifolium, a total of 23,200,000 and 28,500,110 high quality Illumina next generation sequence reads, with a length of 101 bp, were generated for the flower and leaf tissue respectively. After functional enrichment analysis and GO based annotation using public protein databases such as UniRef, PFAM, SMART, KEGG and NR, 4443 and 8901 unigenes were identified in the flower and leaf tissue respectively. These could be assigned to 13344 KEGG pathways and the pyrethrin biosynthesis contextualized. The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway was involved in the biosynthesis of acid moiety of pyrethrin and this pathway predominated in the flowers as compared to the leaves. However, enzymes related to oxylipin biosynthesis were found predominantly in the leaf tissue, which suggested that major steps of pyrethrin biosynthesis occurred in the flowers. CONCLUSIONS: Transcriptome comparison between the flower and leaf tissue of T. cinerariifolium provided an elaborate list of tissue specific transcripts that was useful in elucidating the differences in the expression of the biosynthetic pathways leading to differential presence of pyrethrin in the flowers. The information generated on genes, pathways and markers related to pyrethrin biosynthesis in this study will be helpful in enhancing the production of these useful compounds for value added breeding programs. Related proteome comparison to overlay our transcriptome comparison can generate more relevant information to better understand flower specific accumulation of secondary metabolites in general and pyrethrin accumulation in particular.

Transcriptome-wide identification of Rauvolfia serpentina microRNAs and prediction of their potential targets.

MicroRNAs (miRNAs) are small non-coding RNAs of ∼ 19-24 nucleotides (nt) in length and considered as potent regulators of gene expression at transcriptional and post-transcriptional levels. Here we report the identification and characterization of 15 conserved miRNAs belonging to 13 families from Rauvolfia serpentina through in silico analysis of available nucleotide dataset. The identified mature R. serpentina miRNAs (rse-miRNAs) ranged between 20 and 22nt in length, and the average minimal folding free energy index (MFEI) value of rse-miRNA precursor sequences was found to be -0.815 kcal/mol. Using the identified rse-miRNAs as query, their potential targets were predicted in R. serpentina and other plant species. Gene Ontology (GO) annotation showed that predicted targets of rse-miRNAs include transcription factors as well as genes involved in diverse biological processes such as primary and secondary metabolism, stress response, disease resistance, growth, and development. Few rse-miRNAs were predicted to target genes of pharmaceutically important secondary metabolic pathways such as alkaloids and anthocyanin biosynthesis. Phylogenetic analysis showed the evolutionary relationship of rse-miRNAs and their precursor sequences to homologous pre-miRNA sequences from other plant species. The findings under present study besides giving first hand information about R. serpentina miRNAs and their targets, also contributes towards the better understanding of miRNA-mediated gene regulatory processes in plants.

Promoters of AaGL2 and AaMIXTA-Like1 genes of Artemisia annua direct reporter gene expression in glandular and non-glandular trichomes.

Herein, we report cloning and analysis of promoters of GLABRA2 (AaGL2) homolog and a MIXTA-Like (AaMIXTA-Like1) gene from Artemisia annua. The upstream regulatory regions of AaGL2 and AaMIXTA-Like1 showed the presence of several crucial cis-acting elements. Arabidopsis and A. annua seedlings were transiently transfected with the promoter-GUS constructs using a robust agro-infiltration method. Both AaGL2 and AaMIXTA-Like1 promoters showed GUS expression preferentially in Arabidopsis single-celled trichomes and glandular as well as T-shaped trichomes of A. annua. Transgenic Arabidopsis harboring constructs in which AaGL2 or AaMIXTA-Like1 promoters would control GFP expression, showed fluorescence emanating specifically from trichome cells. Our study provides a fast and efficient method to study trichome-specific expression, and 2 promoters that have potential for targeted metabolic engineering in plants.

Unravelling the genome of Holy basil: an "incomparable" "elixir of life" of traditional Indian medicine.

BACKGROUND: Ocimum sanctum L. (O. tenuiflorum) family-Lamiaceae is an important component of Indian tradition of medicine as well as culture around the world, and hence is known as "Holy basil" in India. This plant is mentioned in the ancient texts of Ayurveda as an "elixir of life" (life saving) herb and worshipped for over 3000 years due to its healing properties. Although used in various ailments, validation of molecules for differential activities is yet to be fully analyzed, as about 80 % of the patents on this plant are on extracts or the plant parts, and mainly focussed on essential oil components. With a view to understand the full metabolic potential of this plant whole nuclear and chloroplast genomes were sequenced for the first time combining the sequence data from 4 libraries and three NGS platforms. RESULTS: The saturated draft assembly of the genome was about 386 Mb, along with the plastid genome of 142,245 bp, turning out to be the smallest in Lamiaceae. In addition to SSR markers, 136 proteins were identified as homologous to five important plant genomes. Pathway analysis indicated an abundance of phenylpropanoids in O. sanctum. Phylogenetic analysis for chloroplast proteome placed Salvia miltiorrhiza as the nearest neighbor. Comparison of the chemical compounds and genes availability in O. sanctum and S. miltiorrhiza indicated the potential for the discovery of new active molecules. CONCLUSION: The genome sequence and annotation of O. sanctum provides new insights into the function of genes and the medicinal nature of the metabolites synthesized in this plant. This information is highly beneficial for mining biosynthetic pathways for important metabolites in related species.

Identification of conserved and novel microRNAs in Catharanthus roseus by deep sequencing and computational prediction of their potential targets.

MicroRNAs are small endogenous non-coding RNAs of ~19-24 nucleotides and perform regulatory roles in many plant processes. To identify miRNAs involved in regulatory networks controlling diverse biological processes including secondary metabolism in Catharanthus roseus, an important medicinal plant, we employed deep sequencing of small RNA from leaf tissue. A total of 88 potential miRNAs comprising of 81 conserved miRNAs belonging to 35 families and seven novel miRNAs were identified. Precursors for 16 conserved and seven novel cro-miRNAs were identified, and their stem-loop hairpin structures were predicted. Selected cro-miRNAs were analyzed by stem-loop qRT-PCR and differential expression patterns were observed in different vegetative tissues of C. roseus. Targets were predicted for conserved and novel cro-miRNAs, which were found to be involved in diverse biological role(s) including secondary metabolism. Our study enriches available resources and information regarding miRNAs and their potential targets for better understanding of miRNA-mediated gene regulation in plants.

De novo sequencing and comparative analysis of holy and sweet basil transcriptomes.

BACKGROUND: Ocimum L. of family Lamiaceae is a well known genus for its ethnobotanical, medicinal and aromatic properties, which are attributed to innumerable phenylpropanoid and terpenoid compounds produced by the plant. To enrich genomic resources for understanding various pathways, de novo transcriptome sequencing of two important species, O. sanctum and O. basilicum, was carried out by Illumina paired-end sequencing. RESULTS: The sequence assembly resulted in 69117 and 130043 transcripts with an average length of 1646 ± 1210.1 bp and 1363 ± 1139.3 bp for O. sanctum and O. basilicum, respectively. Out of the total transcripts, 59648 (86.30%) and 105470 (81.10%) from O. sanctum and O. basilicum, and respectively were annotated by uniprot blastx against Arabidopsis, rice and lamiaceae. KEGG analysis identified 501 and 952 transcripts from O. sanctum and O. basilicum, respectively, related to secondary metabolism with higher percentage of transcripts for biosynthesis of terpenoids in O. sanctum and phenylpropanoids in O. basilicum. Higher digital gene expression in O. basilicum was validated through qPCR and correlated to higher essential oil content and chromosome number (O. sanctum, 2n = 16; and O. basilicum, 2n = 48). Several CYP450 (26) and TF (40) families were identified having probable roles in primary and secondary metabolism. Also SSR and SNP markers were identified in the transcriptomes of both species with many SSRs linked to phenylpropanoid and terpenoid pathway genes. CONCLUSION: This is the first report of a comparative transcriptome analysis of Ocimum species and can be utilized to characterize genes related to secondary metabolism, their regulation, and breeding special chemotypes with unique essential oil composition in Ocimum.

Comparative transcripts profiling of fruit mesocarp and endocarp relevant to secondary metabolism by suppression subtractive hybridization in Azadirachta indica (neem).

Azadirachta indica (neem) is a medicinally important plant that is valued for its bioactive secondary metabolites. Higher levels of the bioactive phytochemicals are accumulated in fruits than in other tissues. In the present study, a total of 387 and 512 ESTs, respectively, from endocarp and mesocarp of neem fruits were isolated and analyzed. Out of them 318 ESTs (82.17%) clones from endocarp and 418 ESTs (81.64%) from mesocarp encoded putative proteins that could be classified into three major gene ontology categories: biological process, molecular function and cellular component. From the analyses of contigs, 73 unigenes from the forward subtracted library and 35 unigenes from the reverse subtracted library were obtained. The ESTs from mesocarp encoded cytochrome P450 enzymes, which indicated hydroxylation to be a major metabolic event and that biogeneration of hydroxylated neem fruit phytochemicals was differentially regulated with developmental stage-specificity of synthesis. Through this study, we present the first report of any gene expression data in neem tissues. Neem hydroxy-methyl glutaryl-coenzyme A reductase (NHMGR) gene was used as expressing control vis-a-vis subtracted tissues. NHMGR was present in fruit, endocarp and mesocarp tissues, but absent in subtractive libraries, revealing that it was successfully eliminated during subtraction. Eight genes of interest from subtracted libraries were profiled for their expression in fruit, mesocarp and endocarp. Expression profiles validated the quality of the libraries and functional diversity of the tissues. The subtractive cDNA library and EST database described in this study represent a valuable transcript sequence resource for future research aimed at improving the economically important medicinal plant.

Differentially expressed genes during contrasting growth stages of Artemisia annua for artemisinin content.

Artemisia annua is the source of antimalarial phytomolecule, artemisinin. It is mainly produced and stored in the glandular secretory trichomes present in the leaves of the plant. Since, the artemisinin biosynthesis steps are yet to be worked out, in this investigation a microarray chip was strategized for the first time to shortlist the differentially expressing genes at a stage of plant producing highest artemisinin compared to the stage with no artemisinin. As the target of this study was to analyze differential gene expression associated with contrasting artemisinin content in planta and a genotype having zero/negligible artemisinin content was unavailable, it was decided to compare different stages of the same genotype with contrasting artemisinin content (seedling--negligible artemisinin, mature leaf--high artemisinin). The SCAR-marked artemisinin-rich (~1.2%) Indian variety 'CIM-Arogya' was used in the present study to determine optimal plant stage and leaf ontogenic level for artemisinin content. A representative EST dataset from leaf trichome at the stage of maximal artemisinin biosynthesis was established. The high utility small scale custom microarray chip of A. annua containing all the significant artemisinin biosynthesis-related genes, the established EST dataset, gene sequences isolated in-house and strategically selected candidates from the A. annua Unigene database (NCBI) was employed to compare the gene expression profiles of two stages. The expression data was validated through semiquantitative and quantitative RT-PCR followed by putative annotations through bioinformatics-based approaches. Many candidates having probable role in artemisinin metabolism were identified and described with scope for further functional characterization.

Activated charcoal-mediated RNA extraction method for Azadirachta indica and plants highly rich in polyphenolics, polysaccharides and other complex secondary compounds.

BACKGROUND: High quality RNA is a primary requisite for numerous molecular biological applications but is difficult to isolate from several plants rich in polysaccharides, polyphenolics and other secondary metabolites. These compounds either bind with nucleic acids or often co-precipitate at the final step and many times cannot be removed by conventional methods and kits. Addition of vinyl-pyrollidone polymers in extraction buffer efficiently removes polyphenolics to some extent, but, it failed in case of Azadirachta indica and several other medicinal and aromatic plants. FINDINGS: Here we report the use of adsorption property of activated charcoal (0.03%-0.1%) in RNA isolation procedures to remove complex secondary metabolites and polyphenolics to yield good quality RNA from Azadirachta indica. We tested and validated our modified RNA isolation method across 21 different plants including Andrographis paniculata, Aloe vera, Rosa damascena, Pelargonium graveolens, Phyllanthus amarus etc. from 13 other different families, many of which are considered as tough system for isolating RNA. The A260/280 ratio of the extracted RNA ranged between 1.8-2.0 and distinct 28S and 18S ribosomal RNA bands were observed in denaturing agarose gel electrophoresis. Analysis using Agilent 2100 Bioanalyzer revealed intact total RNA yield with very good RNA Integrity Number. CONCLUSIONS: The RNA isolated by our modified method was found to be of high quality and amenable for sensitive downstream molecular applications like subtractive library construction and RT-PCR. This modified RNA isolation procedure would aid and accelerate the biotechnological studies in complex medicinal and aromatic plants which are extremely rich in secondary metabolic compounds.