Nucleic acid extraction from complex biofluid using toothpick-actuated over-the-counter medical-grade cotton.

Nucleic acid amplification technique (NAAT)-assisted detection is the primary intervention for pathogen molecular diagnostics. However, NAATs such as quantitative real-time polymerase chain reaction (qPCR) require prior purification or extraction of target nucleic acid from the sample of interest since the latter often contains polymerase inhibitors. Similarly, genetic disease screening is also reliant on the successful extraction of pure patient genomic DNA from the clinical sample. However, such extraction techniques traditionally utilize spin-column techniques that in turn require centralized high-speed centrifuges. This hinders any potential deployment of qPCR- or PCR-like NAAT methods in resource-constrained settings. The development of instrument-free nucleic acid extraction methods, especially those utilizing readily available materials would be of great interest and benefit to NAAT-mediated molecular diagnosis workflows in resource-constrained settings. In this report, we screened medical-grade cotton, a readily available over-the-counter biomaterial to extract genomic DNA (gDNA) spiked in 30 %, 45 %, and 60 % serum or cell lysate. The extraction was carried out in a completely instrument-free manner using cotton and a sterilized toothpick and was completed in 30 min (with using chaotropic salt) or 10 min (without using chaotropic salt). The quality of the extracted DNA was then probed using PCR followed by agarose gel analysis for preliminary validation of the study. The qPCR experiments then quantitatively established the extraction efficiency (0.3-27 %, depending on serum composition). Besides, percent similarity score obtained from the Sanger sequencing experiments probed the feasibility of extracted DNA towards polymerase amplification with fluorescent nucleotide incorporation. Overall, our method demonstrated that DNA extraction could be performed utilizing toothpick-mounted cotton both with or without using a chaotropic salt, albeit with a difference in the quality of the extracted DNA.

Capturing acyltransferase(s) transforming final step in the biosynthesis of a major Iridoid Glycoside, (Picroside-II) in a Himalayan Medicinal Herb, Picrorhiza kurroa.

BACKGROUND: Picrorhiza kurroa has been reported as an age-old ayurvedic hepato-protection to treat hepatic disorders due to the presence of iridoids such as picroside-II (P-II), picroside-I, and kutkoside. The acylation of catalpol and vanilloyl coenzyme A by acyltransferases (ATs) is critical step in P-II biosynthesis. Since accumulation of P-II occurs only in roots, rhizomes and stolons in comparison to leaves uprooting of this critically endangered herb has been the only source of this compound. Recently, we reported that P-II acylation likely happen in roots, while stolons serve as the vital P-II storage compartment. Therefore, developing an alternate engineered platform for P-II biosynthesis require identification of P-II specific AT/s. METHODS AND RESULTS: In that direction, egg-NOG function annotated 815 ATs from de novo RNA sequencing of tissue culture based 'shoots-only' system and nursery grown shoots, roots, and stolons varying in P-II content, were cross-compared in silico to arrive at ATs sequences unique and/or common to stolons and roots. Verification for organ and accession-wise upregulation in gene expression of these ATs by qRT-PCR has shortlisted six putative 'P-II-forming' ATs. Further, six-frame translation, ab initio protein structure modelling and protein-ligand molecular docking of these ATs signified one MBOAT domain containing AT with preferential binding to the vanillic acid CoA thiol ester as well as with P-II, implying that this could be potential AT decorating final structure of P-II. CONCLUSIONS: Organ-wise comparative transcriptome mining coupled with reverse transcription real time qRT-PCR and protein-ligand docking led to the identification of an acyltransferases, contributing to the final structure of P-II.

Transcriptome profiling reveal key hub genes in co-expression networks involved in Iridoid glycosides biosynthetic machinery in Picrorhiza kurroa.

Picrorhiza kurroa is a medicinal herb rich in hepatoprotective iridoid glycosides, picroside-I (P-I) and picroside-II (P-II). The biosynthetic machinery of picrosides is poorly understood, therefore, 'no-direction' gene co-expression networks were used to extract linked/closed and separated interactions in terpenoid glycosides-specific sub-networks. Transcriptomes generated from different organs, varying for P-I and P-II contents such as shoots grown at 15 and 25 °C and nursery-grown shoots, stolons, and roots resulted in 47,726, 44,958, 40,117, 66,979, and 55,578 annotated transcripts, respectively. Occurrence of 2810 ± 136 nodes and 15,626 ± 696 edges in these networks indicated intense, co-expressed, closed loop interactions. Either deregulation/inhibition of abscisic acid (ABA) biosynthesis/signaling or constitutive degradation of ABA resulted in organ-specific accumulation of P-I and P-II. Biosynthesis, condensation and glucosylation of isoprene units may occur in shoots, roots or stolons; but addition of phenylpropanoid moiety and further modification/s of the iridoid backbone occurs mainly inside vacuoles in roots.

Genomics and relative expression analysis identifies key genes associated with high female to male flower ratio in Jatropha curcas L.

Jatropha curcas, has been projected as a major source of biodiesel due to high seed oil content (42 %). A major roadblock for commercialization of Jatropha-based biodiesel is low seed yield per inflorescence, which is affected by low female to male flower ratio (1:25-30). Molecular dissection of female flower development by analyzing genes involved in phase transitions and floral organ development is, therefore, crucial for increasing seed yield. Expression analysis of 42 genes implicated in floral organ development and sex determination was done at six floral developmental stages of a J. curcas genotype (IC561235) with inherently higher female to male flower ratio (1:8-10). Relative expression analysis of these genes was done on low ratio genotype. Genes TFL1, SUP, AP1, CRY2, CUC2, CKX1, TAA1 and PIN1 were associated with reproductive phase transition. Further, genes CUC2, TAA1, CKX1 and PIN1 were associated with female flowering while SUP and CRY2 in female flower transition. Relative expression of these genes with respect to low female flower ratio genotype showed up to ~7 folds increase in transcript abundance of SUP, TAA1, CRY2 and CKX1 genes in intermediate buds but not a significant increase (~1.25 folds) in female flowers, thereby suggesting that these genes possibly play a significant role in increased transition towards female flowering by promoting abortion of male flower primordia. The outcome of study has implications in feedstock improvement of J. curcas through functional validation and eventual utilization of key genes associated with female flowering.

Discerning picroside-I biosynthesis via molecular dissection of in vitro shoot regeneration in Picrorhiza kurroa.

KEY MESSAGE: Expression analysis of primary and secondary metabolic pathways genes vis-à-vis shoot regeneration revealed developmental regulation of picroside-I biosynthesis in Picrorhiza kurroa. Picroside-I (P-I) is an important iridoid glycoside used in several herbal formulations for treatment of various disorders. P-I is synthesized in shoots of Picrorhiza kurroa and Picrorhiza scrophulariiflora. Current study reports on understanding P-I biosynthesis in different morphogenetic stages, viz. plant segment (PS), callus initiation (CI), callus mass (CM), shoot primordia (SP), multiple shoots (MS) and fully developed (FD) stages of P. kurroa. Expression analysis of genes involved in primary and secondary metabolism revealed that genes encoding HMGR, PMK, DXPS, ISPE, GS, G10H, DAHPS and PAL enzymes of MVA, MEP, iridoid and shikimate/phenylpropanoid pathways showed significant modulation of expression in SP, MS and FD stages in congruence with P-I content compared to CM stage. While HK, PK, ICDH, MDH and G6PDH showed high expression in MS and FD stages of P. kurroa, RBA, HisK and CytO showed high expression with progress in regeneration of shoots. Quantitative expression analysis of secondary metabolism genes at two temperatures revealed that 7 genes HMGR, PMK, DXPS, GS, G10H, DAHPS and PAL showed high transcript abundance (32-87-folds) in FD stage derived from leaf and root segments at 15 °C compared to 25 °C in P. kurroa. Further screening of these genes at species level showed high expression pattern in P. kurroa (6-19-folds) vis-à-vis P. scrophulariiflora that was in corroboration with P-I content. Therefore, current study revealed developmental regulation of P-I biosynthesis in P. kurroa which would be useful in designing a suitable genetic intervention study by targeting these genes for enhancing P-I production.

Retraction: Characterization of cellulolytic activities of newly isolated Thelephora sowerbyi from North-Western Himalayas on different lignocellulosic substrates.

Characterization of cellulolytic activities of newly isolated Thelephora sowerbyi from North-Western Himalayas on different lignocellulosic substrate J. Basic Microbiol. 2015, 55, 1-11 - DOI: 10.1002/jobm.201500107 The above article from the Journal of Basic Microbiology, published online on 08 June 2015 in Wiley Online Library as Early View (http://onlinelibrary.wiley.com/doi/10.1002/jobm.201500107/pdf), has been retracted by agreement between the authors, the Editor-in-Chief and Wiley-VCH GmbH & Co. KGaA. The retraction has been agreed because the microorganism studied in the described experiments has been identified as the fungus Cotylidia pannosa (Gene Accession No. KT008117) instead of Thelephora sowerbyi. The culture has been identified on the basis of the sequence of the amplified ITS region of the microorganism which was submitted by the authors to the NCBI database.

Next-generation sequencing (NGS) transcriptomes reveal association of multiple genes and pathways contributing to secondary metabolites accumulation in tuberous roots of Aconitum heterophyllum Wall.

MAIN CONCLUSION: The transcriptomes of Aconitum heterophyllum were assembled and characterized for the first time to decipher molecular components contributing to biosynthesis and accumulation of metabolites in tuberous roots. Aconitum heterophyllum Wall., popularly known as Atis, is a high-value medicinal herb of North-Western Himalayas. No information exists as of today on genetic factors contributing to the biosynthesis of secondary metabolites accumulating in tuberous roots, thereby, limiting genetic interventions towards genetic improvement of A. heterophyllum. Illumina paired-end sequencing followed by de novo assembly yielded 75,548 transcripts for root transcriptome and 39,100 transcripts for shoot transcriptome with minimum length of 200 bp. Biological role analysis of root versus shoot transcriptomes assigned 27,596 and 16,604 root transcripts; 12,340 and 9398 shoot transcripts into gene ontology and clusters of orthologous group, respectively. KEGG pathway mapping assigned 37 and 31 transcripts onto starch-sucrose metabolism while 329 and 341 KEGG orthologies associated with transcripts were found to be involved in biosynthesis of various secondary metabolites for root and shoot transcriptomes, respectively. In silico expression profiling of the mevalonate/2-C-methyl-D-erythritol 4-phosphate (non-mevalonate) pathway genes for aconites biosynthesis revealed 4 genes HMGR (3-hydroxy-3-methylglutaryl-CoA reductase), MVK (mevalonate kinase), MVDD (mevalonate diphosphate decarboxylase) and HDS (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase) with higher expression in root transcriptome compared to shoot transcriptome suggesting their key role in biosynthesis of aconite alkaloids. Five genes, GMPase (geranyl diphosphate mannose pyrophosphorylase), SHAGGY, RBX1 (RING-box protein 1), SRF receptor kinases and ß-amylase, implicated in tuberous root formation in other plant species showed higher levels of expression in tuberous roots compared to shoots. A total of 15,487 transcription factors belonging to bHLH, MYB, bZIP families and 399 ABC transporters which regulate biosynthesis and accumulation of bioactive compounds were identified in root and shoot transcriptomes. The expression of 5 ABC transporters involved in tuberous root development was validated by quantitative PCR analysis. Network connectivity diagrams were drawn for starch-sucrose metabolism and isoquinoline alkaloid biosynthesis associated with tuberous root growth and secondary metabolism, respectively, in root transcriptome of A. heterophyllum. The current endeavor will be of practical importance in planning a suitable genetic intervention strategy for the improvement of A. heterophyllum.

Mining NGS transcriptomes for miRNAs and dissecting their role in regulating growth, development, and secondary metabolites production in different organs of a medicinal herb, Picrorhiza kurroa.

MAIN CONCLUSION: This study is the first endeavor on mining of miRNAs and analyzing their involvement in development and secondary metabolism of an endangered medicinal herb Picrorhiza kurroa (P. kurroa ). miRNAs are ubiquitous non-coding RNA species that target complementary sequences of mRNA and result in either translational repression or target degradation in eukaryotes. The role of miRNAs has not been investigated in P. kurroa which is a medicinal herb of industrial value due to the presence of secondary metabolites, picroside-I and picroside-II. Computational identification of miRNAs was done in 6 transcriptomes of P. kurroa generated from root, shoot, and stolon organs varying for growth, development, and culture conditions. All available plant miRNA entries were retrieved from miRBase and used as backend datasets to computationally identify conserved miRNAs in transcriptome data sets. Total 18 conserved miRNAs were detected in P. kurroa followed by target prediction and functional annotation which suggested their possible role in controlling various biological processes. Validation of miRNA and expression analysis by qRT-PCR and 5' RACE revealed that miRNA-4995 has a regulatory role in terpenoid biosynthesis ultimately affecting the production of picroside-I. miR-5532 and miR-5368 had negligible expression in field-grown samples as compared to in vitro-cultured samples suggesting their role in regulating P. kurroa growth in culture conditions. The study has thus identified novel functions for existing miRNAs which can be further validated for their potential regulatory role.

Mining whole genomes and transcriptomes of Jatropha (Jatropha curcas) and Castor bean (Ricinus communis) for NBS-LRR genes and defense response associated transcription factors.

Jatropha (Jatropha curcas L.) and Castor bean (Ricinus communis) are oilseed crops of family Euphorbiaceae with the potential of producing high quality biodiesel and having industrial value. Both the bioenergy plants are becoming susceptible to various biotic stresses directly affecting the oil quality and content. No report exists as of today on analysis of Nucleotide Binding Site-Leucine Rich Repeat (NBS-LRR) gene repertoire and defense response transcription factors in both the plant species. In silico analysis of whole genomes and transcriptomes identified 47 new NBS-LRR genes in both the species and 122 and 318 defense response related transcription factors in Jatropha and Castor bean, respectively. The identified NBS-LRR genes and defense response transcription factors were mapped onto the respective genomes. Common and unique NBS-LRR genes and defense related transcription factors were identified in both the plant species. All NBS-LRR genes in both the species were characterized into Toll/interleukin-1 receptor NBS-LRRs (TNLs) and coiled-coil NBS-LRRs (CNLs), position on contigs, gene clusters and motifs and domains distribution. Transcript abundance or expression values were measured for all NBS-LRR genes and defense response transcription factors, suggesting their functional role. The current study provides a repertoire of NBS-LRR genes and transcription factors which can be used in not only dissecting the molecular basis of disease resistance phenotype but also in developing disease resistant genotypes in Jatropha and Castor bean through transgenic or molecular breeding approaches.

Expression pattern of fifteen genes of non-mevalonate (MEP) and mevalonate (MVA) pathways in different tissues of endangered medicinal herb Picrorhiza kurroa with respect to picrosides content.

Picrorhiza kurroa, has become an endangered medicinal herb due to excessive utilization, therefore it necessitates the understanding of biology and molecular basis of major chemical constituents i.e. Picroside-I (P-I) and Picroside-II (P-II). Estimation of P-I and P-II in different tissues of P. kurroa showed that shoots contain only P-I whereas P-II is present only in roots. Differential conditions with varying concentrations of P-I (0-27 µg/mg) and P-II (0-4 µg/mg) were selected. Four genes of MEP pathway; DXPS, ISPD, ISPE, MECPS and one gene of MVA pathway PMK showed elevated levels of transcripts in shoots (57-166 folds) and stolons (5-15 folds) with P-I contents 0-27 µg/mg and 2.9-19.7 µg/mg, respectively. Further HDS and DXPR genes of MEP pathway showed higher expression ~9-12 folds in roots having P-II (0-4 µg/mg). The expression of ISPH and ISPE was also high ~5 folds in roots accumulating P-II. GDPS was the only gene with high transcript level in roots (9 folds) and shoots (20 folds). Differential biosynthesis and accumulation of picrosides would assist in regulating quality of plant material for herbal drug formulations.

A proposed biosynthetic pathway of picrosides linked through the detection of biochemical intermediates in the endangered medicinal herb Picrorhiza kurroa.

INTRODUCTION: Picrorhiza kurroa Royle ex Benth is an important medicinal herb used in the preparation of several herbal drug formulations due to the presence of picroside-I (P-I) and picroside-II (P-II) along with other iridoid-glucosides derivatives. OBJECTIVE: The endangered status of P. kurroa coupled with lack of information on biosynthesis of P-I and P-II necessitate deciphering the biosynthetic pathway for picrosides. METHODS: LC with electrospray ionisation (ESI) and quadrupole time of flight combined with MS/MS was used to detect intermediates and assemble the picrosides biosynthetic pathway in P. kurroa. RESULTS: The presence of catalpol and aucubin, the major backbone structures of picrosides, along with intermediate metabolites boschnaloside, bartsioside and mussaenosidic acid, was confirmed in ESI negative mode with pseudomolecular ion peaks, that is, m/z 361, m/z 343, m/z 345, m/z 329 and m/z 375 ions and their fragmentation patterns. CONCLUSION: The picrosides biosynthetic pathway is expected to provide a reliable platform towards understanding the molecular components (genes/enzymes) of P-I and P-II biosynthesis in P. kurroa for their eventual utilisation in various applications.

ABC transporters mined through comparative transcriptomics associate with organ-specific accumulation of picrosides in a medicinal herb, Picrorhiza kurroa.

Picrorhiza kurroa Royle ex Benth is a valuable medicinal herb of North-Western Himalayas due to presence of two major bioactive compounds, picroside-I and picroside-II used in the preparation of several hepatoprotective herbal drugs. These compounds accumulate in stolons/rhizomes; however, biosynthesized in different organs, viz., picroside-I in shoots and picroside-II in roots. As of today, no information exists on what transporters are transporting these metabolites from shoots and roots to the final storage organ, stolon, which ultimately transforms into rhizome. The ATP-binding cassette (ABC) transporters are reported to transport majority of secondary metabolites, including terpenoids in plants, therefore, we mined P. kurroa transcriptomes to identify and shortlist potential candidates. A total of 99 ABC transporter-encoding transcripts were identified in 3 differential transcriptomes, PKSS (shoots), PKSTS (stolons), and PKSR (roots) of P. kurroa, based on in silico comparative analysis and transcript abundance. 15 of these transcripts were further validated for their association using qRT-PCR in shoots, roots and stolon tissues in P. kurroa accessions varying for picroside-I and picroside-II contents. Organ-specific expression analysis revealed that PkABCA1, PkABCG1, and PkABCB5 had comparatively elevated expression in shoots; PkABCB2 and PkABCC2 in roots; PkABCB3 and PkABCC1 in stolon tissues of P. kurroa. Co-expression network analysis using ABC genes as hubs further unravelled important interactions with additional components of biosynthetic machinery. Our study has provided leads, first to our knowledge as of today, on putative ABC transporters possibly involved in long distance and local transport of picrosides in P. kurroa organs, thus opening avenues for designing a suitable genetic intervention strategy.

Differential transcript profiling through cDNA-AFLP showed complexity of rutin biosynthesis and accumulation in seeds of a nutraceutical food crop (Fagopyrum spp.).

BACKGROUND: Buckwheat, consisting of two cultivated species Fagopyrum tataricum and F. esculentum, is the richest source of flavonoid rutin. Vegetative tissues of both the Fagopyrum species contain almost similar amount of rutin; however, rutin content in seed of F. tataricum are ~50 folds of that in seed of F. esculentum. In order to understand the molecular basis of high rutin content in F. tataricum, differential transcript profiling through cDNA-AFLP has been utilized to decipher what genetic factors in addition to flavonoid structural genes contribute to high rutin content of F. tataricum compared to F. esculentum. RESULTS: Differential transcript profiling through cDNA-AFLP in seed maturing stages (inflorescence to seed maturation) with 32 primer combinations generated total of 509 transcript fragments (TDFs). 167 TDFs were then eluted, cloned and sequenced from F. tataricum and F. esculentum. Categorization of TDFs on the basis of their presence/absence (qualitative variation) or differences in the amount of expression (quantitative variation) between both the Fagopyrum species showed that majority of variants are quantitative (64%). The TDFs represented genes controlling different biological processes such as basic and secondary metabolism (33%), regulation (18%), signal transduction (14%), transportation (13%), cellular organization (10%), and photosynthesis & energy (4%). Most of the TDFs except belonging to cellular metabolism showed relatively higher transcript abundance in F. tataricum over F. esculentum. Quantitative RT-PCR analysis of nine TDFs representing genes involved in regulation, metabolism, signaling and transport of secondary metabolites showed that all the tested nine TDFs (Ubiquitin protein ligase, ABC transporter, sugar transporter) except MYB 118 showed significantly higher expression in early seed formation stage (S7) of F. tataricum compared to F. esculentum. qRT-PCR results were found to be consistent with the cDNA-AFLP results. CONCLUSIONS: The present study concludes that in addition to structural genes, other classes of genes such as regulators, modifiers and transporters are also important in biosynthesis and accumulation of flavonoid content in plants. cDNA-AFLP technology was successfully utilized to capture genes that are contributing to differences in rutin content in seed maturing stages of Fagopyrum species. Increased transcript abundance of TDFs during transition from flowers to seed maturation suggests their involvement not only in the higher rutin content of F. tataricum over F. esculentum but also in nutritional superiority of the former.

In silico identification and comparative genomics of candidate genes involved in biosynthesis and accumulation of seed oil in plants.

Genes involved in fatty acids biosynthesis, modification and oil body formation are expected to be conserved in structure and function in different plant species. However, significant differences in the composition of fatty acids and total oil contents in seeds have been observed in different plant species. Comparative genomics was performed on 261 genes involved in fatty acids biosynthesis, TAG synthesis, and oil bodies formation in Arabidopsis, Brassica rapa, castor bean and soybean. In silico expression analysis revealed that stearoyl desaturase, FatB, FAD2, oleosin and DGAT are highly abundant in seeds, thereby considered as ideal candidates for mining of favorable alleles in natural population. Gene structure analysis for major genes, ACCase, FatA, FatB, FAD2, FAD3 and DGAT, which are known to play crucial role in oil synthesis revealed that there are uncommon variations (SNPs and INDELs) which lead to varying content and composition of fatty acids in seed oil. The predicted variations can provide good targets for seed oil QTL identification, understanding the molecular mechanism of seed oil accumulation, and genetic modification to enhance seed oil yield in plants.

Repertoire of SSRs in the Castor Bean Genome and Their Utilization in Genetic Diversity Analysis in Jatropha curcas.

Castor bean and Jatropha contain seed oil of industrial importance, share taxonomical and biochemical similarities, which can be explored for identifying SSRs in the whole genome sequence of castor bean and utilized in Jatropha curcas. Whole genome analysis of castor bean identified 5,80,986 SSRs with a frequency of 1 per 680 bp. Genomic distribution of SSRs revealed that 27% were present in the non-genic region whereas 73% were also present in the putative genic regions with 26% in 5'UTRs, 25% in introns, 16% in 3'UTRs and 6% in the exons. Dinucleotide repeats were more frequent in introns, 5'UTRs and 3'UTRs whereas trinucleotide repeats were predominant in the exons. The transferability of randomly selected 302 SSRs, from castor bean to 49 J. curcas genotypes and 8 Jatropha species other than J. curcas, showed that 211 (∼70%) amplified on Jatropha out of which 7.58% showed polymorphisms in J. curcas genotypes and 12.32% in Jatropha species. The higher rate of transferability of SSR markers from castor bean to Jatropha coupled with a good level of PIC (polymorphic information content) value (0.2 in J. curcas genotypes and 0.6 in Jatropha species) suggested that SSRs would be useful in germplasm analysis, linkage mapping, diversity studies and phylogenetic relationships, and so forth, in J. curcas as well as other Jatropha species.

Bioconversion of herbal industry waste into vermicompost using an epigeic earthworm Eudrilus eugeniae.

The aim of the present study was to investigate the potential of bioconversion of industrial herbal waste to vermicompost using Eudrilus eugeniae. Vermibeds were made using a mixture of herbal waste and cowdung (1 : 1) in comparison with the use of cowdung alone as substrate, resulting in vermicomposts 1 and 2, respectively. Different parameters were studied and it was observed that the nutrient profile of vermicompost 1 strongly influenced the growth of pea (Pisum sativum) and marigold plant (Tagetus erectus). The dry and fresh weight of shoots and roots, number of flowers, total yield in terms of fruit showed significant increase with vermicompost 1. Furthermore, vermicompost 1 (herbal waste and cow dung as substrate) resulted in a significant reduction in TOC by 58% in comparison with vermicompost 2 (cowdung as substrate). The C : N ratio was less than 20 in vermicompost 1 as well as in vermicompost 2, which indicated an advanced degree of stabilization and mineralization. The ability of earthworms to survive, grow and breed in the vermibed fed with the herbal waste indicates the sustainability and efficiency of a heterogeneous kind of organic waste. The results of the study suggested that bulk industrial herbal waste can be utilized as a substrate for vermicomposting and this can be proposed as an alternative for waste disposal in a clean green manner, promoting the concept of organic farming.

Publisher Correction: A novel miRNA analysis framework to analyze differential biological networks.

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Comparative transcriptomics reveals a reduction in carbon capture and flux between source and sink in cytokinin-treated inflorescences of Jatropha curcas L.

The low seed yield of Jatropha curcas has been a stumbling block in realizing its full potential as an ideal bioenergy crop. Low female to male flower ratio is considered as a major limiting factor responsible for low seed yield in Jatropha. An exogenous cytokinin application was performed on floral meristems to increase the seed yield. This resulted in an increase of total flowers count with a higher female to male flower ratio. However, the seed biomass did not increase in the same proportion. The possible reason for this was hypothesized to be the lack of increased photosynthesis efficiency at source tissues which could fulfil the increased demand of photosynthates and primary metabolites in maturing seeds. After cytokinin application, possible molecular mechanisms underlying carbon capture and flux affected between the source and sink in developing flowers, fruits and seeds were investigated. Comparative transcriptome analysis was performed on inflorescence meristems (treated with cytokinin) and control (untreated inflorescence meristems) at time intervals of 15 and 30 days, respectively. KEGG-based functional annotation identified various metabolic pathways associated with carbon capture and flux. Pathways such as photosynthesis, carbon fixation, carbohydrate metabolism and nitrogen metabolism were upregulated after 15 days of cytokinin treatment; however, those were downregulated after 30 days. Five genes FBP, SBP, GS, GDH and AGPase showed significant increase in transcript abundance after 15 days of treatment but showed a significant decrease after 30 days. These genes, after functional validation, can be suitable targets in designing a suitable genetic intervention strategy to increase overall seed yield in Jatropha.

A novel miRNA analysis framework to analyze differential biological networks.

For understanding complex biological systems, a systems biology approach, involving both the top-down and bottom-up analyses, is often required. Numerous system components and their connections are best characterised as networks, which are primarily represented as graphs, with several nodes connected at multiple edges. Inefficient network visualisation is a common problem related to transcriptomic and genomic datasets. In this article, we demonstrate an miRNA analysis framework with the help of Jatropha curcas healthy and disease transcriptome datasets, functioning as a pipeline derived from the graph theory universe, and discuss how the network theory, along with gene ontology (GO) analysis, can be used to infer biological properties and other important features of a network. Network profiling, combined with GO, correlation, and co-expression analyses, can aid in efficiently understanding the biological significance of pathways, networks, as well as a studied system. The proposed framework may help experimental and computational biologists to analyse their own data and infer meaningful biological information.