Comprehensive analysis of alternative splicing in Digitalis purpurea by strand-specific RNA-Seq.
ABSTRACT: Digitalis purpurea (D. purpurea) is one of the most important medicinal plants and is well known in the treatment of heart failure because of the cardiac glycosides that are its main active compounds. However, in the absence of strand specific sequencing information, the post-transcriptional mechanism of gene regulation in D. purpurea thus far remains unknown. In this study, a strand-specific RNA-Seq library was constructed and sequenced using Illumina HiSeq platforms to characterize the transcriptome of D. purpurea with a focus on alternative splicing (AS) events and the effect of AS on protein domains. De novo RNA-Seq assembly resulted in 48,475 genes. Based on the assembled transcripts, we reported a list of 3,265 AS genes, including 5,408 AS events in D. purpurea. Interestingly, both glycosyltransferases and monooxygenase, which were involved in the biosynthesis of cardiac glycosides, are regulated by AS. A total of 2,422 AS events occurred in coding regions, and 959 AS events were located in the regions of 882 unique protein domains, which could affect protein function. This D. purpurea transcriptome study substantially increased the expressed sequence resource and presented a better understanding of post-transcriptional regulation to further facilitate the medicinal applications of D. purpurea for human health.
Project description:BACKGROUND: Digitalis purpurea is an important ornamental and medicinal plant. There is considerable interest in exploring its transcriptome. RESULTS: Through high-throughput 454 sequencing and subsequent assembly, we obtained 23532 genes, of which 15626 encode conserved proteins. We determined 140 unigenes to be candidates involved in cardiac glycoside biosynthesis. It could be grouped into 30 families, of which 29 were identified for the first time in D. purpurea. We identified 2660 mRNA-like npcRNA (mlncRNA) candidates, an emerging class of regulators, using a computational mlncRNA identification pipeline and 13 microRNA-producing unigenes based on sequence conservation and hairpin structure-forming capability. Twenty five protein-coding unigenes were predicted to be targets of these microRNAs. Among the mlncRNA candidates, only 320 could be grouped into 140 families with at least two members in a family. The majority of D. purpurea mlncRNAs were species-specific and many of them showed tissue-specific expression and responded to cold and dehydration stresses. We identified 417 protein-coding genes with regions significantly homologous or complementary to 375 mlncRNAs. It includes five genes involved in secondary metabolism. A positive correlation was found in gene expression between protein-coding genes and the homologous mlncRNAs in response to cold and dehydration stresses, while the correlation was negative when protein-coding genes and mlncRNAs were complementary to each other. CONCLUSIONS: Through comprehensive transcriptome analysis, we not only identified 29 novel gene families potentially involved in the biosynthesis of cardiac glycosides but also characterized a large number of mlncRNAs. Our results suggest the importance of mlncRNAs in secondary metabolism and stress response in D. purpurea.
Project description:Cardiac glycosides, steroid derivatives extracted from the foxglove plants, have been used for the treatment of heart failure since the 18th century. A method based on liquid chromatography coupled with high-resolution tandem mass spectrometry (LC/MS2) has been developed to characterize and quantify cardiac glycosides in fresh-leaf extracts of the foxglove (Digitalis sp.) plants . In this report, the fragmentation spectra of additional authentic standards of cardiac glycoside (digitoxigenin, digoxigenin, ?-acetyldigoxin) and cardenolides identified in the leaves of Digitalis lanata (D. lanata) and Digitalis purpurea (D. purpurea) were provided with high resolution. The exact mass of signature peaks for the aglycones and the sugar units of cardenolides were measured. This dataset is valuable to researchers interested in characterizing cardenolides in plants, or quantifying cardenolides in drug tablets, or studying cardenolide toxicities in animals. The fragmentation patterns of authentic cardenolide standards provided in these data can be used to validate relevant cardenolides in various biological samples and to infer chemical structures of unknown cardiac glycosides.
Project description:There are four isoforms of the alpha subunit (alpha1-4) and three isoforms of the beta subunit (beta1-3) of Na,K-ATPase, with distinct tissue-specific distribution and physiological functions. alpha2 is thought to play a key role in cardiac and smooth muscle contraction and be an important target of cardiac glycosides. An alpha2-selective cardiac glycoside could provide important insights into physiological and pharmacological properties of alpha2. The isoform selectivity of a large number of cardiac glycosides has been assessed utilizing alpha1beta1, alpha2beta1, and alpha3beta1 isoforms of human Na,K-ATPase expressed in Pichia pastoris and the purified detergent-soluble isoform proteins. Binding affinities of the digitalis glycosides, digoxin, beta-methyl digoxin, and digitoxin show moderate but highly significant selectivity (up to 4-fold) for alpha2/alpha3 over alpha1 (K(D) alpha1 > alpha2 = alpha3). By contrast, ouabain shows moderate selectivity ( approximately 2.5-fold) for alpha1 over alpha2 (K(D) alpha1 <or= alpha3 < alpha2). Binding affinities for the three isoforms of digoxigenin, digitoxigenin, and all other aglycones tested are indistinguishable (K(D) alpha1 = alpha3 = alpha2), showing that the sugar determines isoform selectivity. Selectivity patterns for inhibition of Na,K-ATPase activity of the purified isoform proteins are consistent with binding selectivities, modified somewhat by different affinities of K(+) ions for antagonizing cardiac glycoside binding on the three isoforms. The mechanistic insight on the role of the sugars is strongly supported by a recent structure of Na,K-ATPase with bound ouabain, which implies that aglycones of cardiac glycosides cannot discriminate between isoforms. In conclusion, several digitalis glycosides, but not ouabain, are moderately alpha2-selective. This supports a major role of alpha2 in cardiac contraction and cardiotonic effects of digitalis glycosides.
Project description:The botanical genus <i>Digitalis</i> is equal parts colorful, toxic, and medicinal, and its bioactive compounds have a long history of therapeutic use. However, with an extremely narrow therapeutic range, even trace amounts of <i>Digitalis</i> can cause adverse effects. Using chemical methods, the United States Food and Drug Administration traced a 1997 case of <i>Digitalis</i> toxicity to a shipment of <i>Plantago</i> (a common ingredient in dietary supplements marketed to improve digestion) contaminated with <i>Digitalis lanata</i>. With increased accessibility to next generation sequencing technology, here we ask whether this case could have been cracked rapidly using shallow genome sequencing strategies (e.g., genome skims). Using a modified implementation of the Site Identification from Short Read Sequences (SISRS) bioinformatics pipeline with whole-genome sequence data, we generated over 2 M genus-level single nucleotide polymorphisms in addition to species-informative single nucleotide polymorphisms. We simulated dietary supplement contamination by spiking low quantities (0-10%) of <i>Digitalis</i> whole-genome sequence data into a background of commonly used ingredients in products marketed for "digestive cleansing" and reliably detected Digitalis at the genus level while also discriminating between <i>Digitalis</i> species. This work serves as a roadmap for the development of novel DNA-based assays to quickly and reliably detect the presence of toxic species such as <i>Digitalis</i> in food products or dietary supplements using genomic methods and highlights the power of harnessing the entire genome to identify botanical species.
Project description:In this report, we have investigated the influence of different light qualities on Digitalis purpurea under a controlled environment. For this purpose, red (R), blue (B), fluorescent lamp (FL, control), along with combined red and blue (R:B) LEDs were used. Interestingly, the plant growth parameters such as number of leaf, longest root, width of leaf, width of stomata, width of trichome, leaf area, leaf or root fresh weight (FW), weight (DW) as well as length of trichome were maximum under R:B (8:2), and significantly larger than control plants. The stomatal conductance or anthocyanin was maximum under B LED than those under FL, however the photosynthesis rate was greater under FL. RuBisCO activity was maximum under R:B (1:1) LEDs while the quantity of the UV absorbing substances was highest under R LED than under FL. The maximum amount of cardenolides were obtained from leaf tissue under R:B (2:8) LED than those under FL. The R:B LEDs light was suitable for Digitalis plant growth, development, micro- and macro-elements, as well as cardenolides accumulation in the plant factory system. The adaptation of the growth strategy developed in this study would be useful for the production of optimized secondary metabolites in Digitalis spp.
Project description:Digitalis drugs are selective inhibitors of the plasma membrane Na+/K+-ATPase. There are many studies on molecular mechanisms of digitalis interaction with purified pig kidney enzyme, with the tacit assumption that it is a good model of human kidney enzyme. However, previous studies on crude or recombinant human kidney enzymes are limited, and have not resulted in consistent findings on their digitalis sensitivities. Hence, we prepared comparably purified enzymes from human and pig kidneys and determined inhibitory constants of digoxin, ouabain, ouabagenin, bufalin, and marinobufagenin (MBG) on enzyme activity under optimal turnover conditions. We found that each compound had the same potency against the two enzymes, indicating that (i) the pig enzyme is an appropriate model of the human enzyme, and (ii) prior discrepant findings on human kidney enzymes were either due to structural differences between the natural and recombinant enzymes or because potencies were determined using binding constants of digitalis for enzymes under nonphysiological conditions. In conjunction with previous findings, our newly determined inhibitory constants of digitalis compounds for human kidney enzymes indicate that (i) of the compounds that have long been advocated to be endogenous hormones, only bufalin and MBG may act as such at kidney tubules, and (ii) beneficial effects of digoxin, the only digitalis with extensive clinical use, does not involve its inhibitory effect on renal tubular Na+/K+-ATPase.
Project description:<h4>Background</h4>Gastrointestinal cancers are characterized by a male predominance, suggesting a role of sex hormones. We hypothesized that digitalis medication, due to its estrogenic properties, decreases the risk of male-predominated gastrointestinal cancers.<h4>Results</h4>Long -term digitalis use (?2 years) was followed by decreased risk for several gastrointestinal cancers, but associations were statistically significant only for liver cancer (hazard ratio [HR]=0.40, 95% confidence interval (CI) 0.16-0.98). Short-term (<1 year) use was associated with an increased risk of esophageal squamous cell carcinoma (HR=1.79, 95% CI 1.01-3.17), colorectal cancer (HR=1.72, 95% CI 1.57-1.89), gallbladder cancer (HR=1.93, 95% CI 1.04-3.59), and pancreatic cancer (HR=1.33, 95% CI 1.00-1.76), but no such increase was found among long-term users.<h4>Methods</h4>We performed a nationwide population-based cohort study in Sweden. Participants included 156,385 individuals using digitalis and a reference group of 551,933 users of organic nitrates between 2005 and 2013, who were identified in the Swedish Prescribed Drug Register. New diagnoses of gastrointestinal cancers were identified from the Swedish Cancer Register. Hazard ratios of gastrointestinal cancers in digitalis users compared to users of organic nitrates were calculated from Cox proportional hazards regression with adjustment for sex, age, municipality of residence and comorbidity.<h4>Conclusions</h4>This study suggests a decreased risk of male-predominated gastrointestinal cancers, particularly of liver cancer, in long-term users of digitalis. Short-term use may be associated with an increased risk of esophageal squamous cell carcinoma, colorectal cancer, gallbladder cancer, and pancreatic cancer.The use of digitalis as preventive or therapeutic agents remains to be fully evaluated.