De novo transcriptome assembly of Conium maculatum L. to identify candidate genes for coniine biosynthesis

Poison hemlock (Conium maculatum L.) is a notorious weed containing the potent alkaloid coniine. Only some of the enzymes in the coniine biosynthesis have so far been characterized. Here, we utilize the next-generation RNA sequencing approach to report the first-ever transcriptome sequencing of five organs of poison hemlock: developing fruit, flower, root, leaf, and stem. Using a de novo assembly approach, we derived a transcriptome assembly containing 123,240 transcripts. The assembly is deemed high quality, representing over 88% of the near-universal ortholog genes of the Eudicots clade. Nearly 80% of the transcripts were functionally annotated using a combination of three approaches. The current study focuses on describing the coniine pathway by identifying in silico transcript candidates for polyketide reductase, l-alanine:5-keto-octanal aminotransferase, gamma-coniceine reductase, and S-adenosyl-l-methionine:coniine methyltransferase. In vitro testing will be needed to confirm the assigned functions of the selected candidates.Peer reviewe

Contrasting Dihydronaphthoquinone Patterns in Closely Related Drosera (Sundew) Species Enable Taxonomic Distinction and Identification

Dihydronaphthoquinones are described as constituents of sundews (Drosera), Venus flytraps (Dionaea), and dewy pines (Drosophyllum) for the first time. As in the corresponding naphthoquinones, these reduced derivatives may occur in two regio-isomeric series distinguished by the relative position of a methyl group (at position 2 or 7 in the naphthalene skeleton), depending on the taxon. Species producing plumbagin (2-methyljuglone, 1) do commonly contain the corresponding dihydroplumbagin (5), while species containing ramentaceone (7-methyljuglone, 2) also contain dihydroramentaceone (7-methyl-β-dihydrojuglone, 6). So far, only few species containing plumbagin (1) and dihydroplumbagin (5) additionally form dihydroramentaceone (6) but not ramentaceone (2). Thus, subtle but constant differences in the chemism of closely related and morphologically similar species reliably define and distinguish taxa within D. sect. Arachnopus, which is taken to exemplify their chemotaxonomic utility. The joint presence of quinones and hydroquinones allows observations and predictions on the chemical structures and the reactions of these intriguing natural products

Variability in the production of tannins and other polyphenols in cell cultures of 12 Nordic plant species

MAIN CONCLUSION: The polyphenol profiles of 18 cell cultures from 12 plant species were screened. The detected polyphenol fingerprints were diverse and differed from polyphenol profiles typically found in corresponding plant species. Cell cultures originating from 12 different plant species growing or grown in the Nordic countries were screened for their ability to synthesize polyphenols to assess their suitability for future studies and applications. The focus was on plant families Rosaceae and Ericaceae. On average, the Rosaceae cultures were the most efficient to produce hydrolysable tannins and the Ericaceae cultures were the most efficient to produce proanthocyanidins. This is in line with the general trend of polyphenols found in Rosaceae and Ericaceae leaves and fruits, even though several individual cell cultures differed from natural plants in their polyphenolic composition. Overall, several of the studied cell cultures exhibited capability in producing a large variety of polyphenols, including tannins with a high molecular weight, thus also showing promise for further studies concerning, for example, the accumulation of specific polyphenols or biosynthesis of polyphenols in the cell cultures.</p

Optimization of Invasion-Specific Effects of Betulin Derivatives on Prostate Cancer Cells through Lead Development

The anti-invasive and anti-proliferative effects of betulins and abietane derivatives was systematically tested using an organotypic model system of advanced, castration-resistant prostate cancers. A preliminary screen of the initial set of 93 compounds was performed in two-dimensional (2D) growth conditions using non-transformed prostate epithelial cells (EP156T), an androgen-sensitive prostate cancer cell line (LNCaP), and the castration-resistant, highly invasive cell line PC-3. The 25 most promising compounds were all betulin derivatives. These were selected for a focused secondary screen in three-dimensional (3D) growth conditions, with the goal to identify the most effective and specific anti-invasive compounds. Additional sensitivity and cytotoxicity tests were then performed using an extended cell line panel. The effects of these compounds on cell cycle progression, mitosis, proliferation and unspecific cytotoxicity, versus their ability to specifically interfere with cell motility and tumor cell invasion was addressed. To identify potential mechanisms of action and likely compound targets, multiplex profiling of compound effects on a panel of 43 human protein kinases was performed. These target de-convolution studies, combined with the phenotypic analyses of multicellular organoids in 3D models, revealed specific inhibition of AKT signaling linked to effects on the organization of the actin cytoskeleton as the most likely driver of altered cell morphology and motility.</p

The c4h, tat, hppr and hppd Genes Prompted Engineering of Rosmarinic Acid Biosynthetic Pathway in Salvia miltiorrhiza Hairy Root Cultures

Rational engineering to produce biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Here we capitalized on our previously described gene-to-metabolite network in order to engineer rosmarinic acid (RA) biosynthesis pathway for the production of beneficial RA and lithospermic acid B (LAB) in Salvia miltiorrhiza hairy root cultures. Results showed their production was greatly elevated by (1) overexpression of single gene, including cinnamic acid 4-hydroxylase (c4h), tyrosine aminotransferase (tat), and 4-hydroxyphenylpyruvate reductase (hppr), (2) overexpression of both tat and hppr, and (3) suppression of 4-hydroxyphenylpyruvate dioxygenase (hppd). Co-expression of tat/hppr produced the most abundant RA (906 mg/liter) and LAB (992 mg/liter), which were 4.3 and 3.2-fold more than in their wild-type (wt) counterparts respectively. And the value of RA concentration was also higher than that reported before, that produced by means of nutrient medium optimization or elicitor treatment. It is the first report of boosting RA and LAB biosynthesis through genetic manipulation, providing an effective approach for their large-scale commercial production by using hairy root culture systems as bioreactors

Pyrosequencing of the Camptotheca acuminata transcriptome reveals putative genes involved in camptothecin biosynthesis and transport

Background: Camptotheca acuminata is a Nyssaceae plant, often called the "happy tree", which is indigenous in Southern China. C. acuminata produces the terpenoid indole alkaloid, camptothecin (CPT), which exhibits clinical effects in various cancer treatments. Despite its importance, little is known about the transcriptome of C. acuminata and the mechanism of CPT biosynthesis, as only few nucleotide sequences are included in the GenBank database.Results: From a constructed cDNA library of young C. acuminata leaves, a total of 30,358 unigenes, with an average length of 403 bp, were obtained after assembly of 74,858 high quality reads using GS De Novo assembler software. Through functional annotation, a total of 21,213 unigenes were annotated at least once against the NCBI nucleotide (Nt), non-redundant protein (Nr), Uniprot/SwissProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Arabidopsis thaliana proteome (TAIR) databases. Further analysis identified 521 ESTs representing 20 enzyme genes that are involved in the backbone of the CPT biosynthetic pathway in the library. Three putative genes in the upstream pathway, including genes for geraniol-10-hydroxylase (CaPG10H), secologanin synthase (CaPSCS), and strictosidine synthase (CaPSTR) were cloned and analyzed. The expression level of the three genes was also detected using qRT-PCR in C. acuminata. With respect to the branch pathway of CPT synthesis, six cytochrome P450s transcripts were selected as candidate transcripts by detection of transcript expression in different tissues using qRT-PCR. In addition, one glucosidase gene was identified that might participate in CPT biosynthesis. For CPT transport, three of 21 transcripts for multidrug resistance protein (MDR) transporters were also screened from the dataset by their annotation result and gene expression analysis.Conclusion: This study produced a large amount of transcriptome data from C. acuminata by 454 pyrosequencing. According to EST annotation, catalytic features prediction, and expression analysis, novel putative transcripts involved in CPT biosynthesis and transport were discovered in C. acuminata. This study will facilitate further identification of key enzymes and transporter genes in C. acuminata

Metabolic Profiles and cDNA-AFLP Analysis of Salvia miltiorrhiza and Salvia castanea Diel f. tomentosa Stib

Plants of the genus Salvia produce various types of phenolic compounds and tanshinones which are effective for treatment of coronary heart disease. Salvia miltiorrhiza and S. castanea Diels f. tomentosa Stib are two important members of the genus. In this study, metabolic profiles and cDNA-AFLP analysis of four samples were employed to identify novel genes potentially involved in phenolic compounds and tanshinones biosynthesis, including the red roots from the two species and two tanshinone-free roots from S. miltiorrhiza. The results showed that the red roots of S. castanea Diels f. tomentosa Stib produced high contents of rosmarinic acid (21.77 mg/g) and tanshinone IIA (12.60 mg/g), but low content of salvianolic acid B (1.45 mg/g). The red roots of S. miltiorrhiza produced high content of salvianolic acid B (18.69 mg/g), while tanshinones accumulation in this sample was much less than that in S. castanea Diels f. tomentosa Stib. Tanshinones were not detected in the two tanshinone-free samples, which produced high contents of phenolic compounds. A cDNA-AFLP analysis with 128 primer pairs revealed that 2300 transcript derived fragments (TDFs) were differentially expressed among the four samples. About 323 TDFs were sequenced, of which 78 TDFs were annotated with known functions through BLASTX searching the Genbank database and 14 annotated TDFs were assigned into secondary metabolic pathways through searching the KEGGPATHWAY database. The quantitative real-time PCR analysis indicated that the expression of 9 TDFs was positively correlated with accumulation of phenolic compounds and tanshinones. These TDFs additionally showed coordinated transcriptional response with 6 previously-identified genes involved in biosynthesis of tanshinones and phenolic compounds in S. miltiorrhiza hairy roots treated with yeast extract. The sequence data in the present work not only provided us candidate genes involved in phenolic compounds and tanshinones biosynthesis but also gave us further insight into secondary metabolism in Salvia

Family physicians' perspectives on practice guidelines related to cancer control

BACKGROUND: Family physicians (FPs) play an important role in cancer control. While FPs' attitudes towards, and use of guidelines in general have been explored, no study has looked at the needs of FPs with respect to guidelines for the continuum of cancer control. The objective of this study was to understand which guideline topics FPs consider important. METHODS: Five group interviews were conducted by telephone with FPs from across Ontario, Canada. Transcripts were analyzed inductively. Content analysis identified emergent themes. Themes are illustrated by representative quotes taken from the transcripts. RESULTS: The main areas where FPs felt guidelines were needed most included screening – a traditional area of responsibility for FPs – and treatment and follow-up – areas where they felt they lacked the knowledge to best support patients. Confusion over best practice when faced with conflicting guidelines varied according to disease site. FPs defined good guideline formats; the most often cited forms of presentation were tear-off sheets to use interactively with patients, or a binder. Computer-based dissemination was acknowledged as the best way of widely distributing material that needs frequent updates. However, until computer use is a common aspect of practice, mail was considered the most viable method of dissemination. Guidelines designed for use by patients were supported by FPs. CONCLUSIONS: Preferred guideline topics, format, dissemination methods and role of patient guidelines identified by FPs in this study reflect the nature of their practice situations. Guideline developers and those supporting use of evidence-based guidelines (e.g., Canadian Strategy for Cancer Control) have a responsibility to ensure that FPs are provided with the resources they identify as important, and to provide them in a format that will best support their use

Evaluation of metabolomics profiles of grain from maize hybrids derived from near-isogenic GM positive and negative segregant inbreds demonstrates that observed differences cannot be attributed unequivocally to the GM trait

Introduction: Past studies on plant metabolomes have highlighted the influence of growing environments and varietal differences in variation of levels of metabolites yet there remains continued interest in evaluating the effect of genetic modification (GM). Objectives: Here we test the hypothesis that metabolomics differences in grain from maize hybrids derived from a series of GM (NK603, herbicide tolerance) inbreds and corresponding negative segregants can arise from residual genetic variation associated with backcrossing and that the effect of insertion of the GM trait is negligible. Methods: Four NK603-positive and negative segregant inbred males were crossed with two different females (testers). The resultant hybrids, as well as conventional comparator hybrids, were then grown at three replicated field sites in Illinois, Minnesota, and Nebraska during the 2013 season. Metabolomics data acquisition using gas chromatography–time of flight-mass spectrometry (GC–TOF-MS) allowed the measurement of 367 unique metabolite features in harvested grain, of which 153 were identified with small molecule standards. Multivariate analyses of these data included multi-block principal component analysis and ANOVA-simultaneous component analysis. Univariate analyses of all 153 identified metabolites was conducted based on significance testing (α = 0.05), effect size evaluation (assessing magnitudes of differences), and variance component analysis. Results: Results demonstrated that the largest effects on metabolomic variation were associated with different growing locations and the female tester. They further demonstrated that differences observed between GM and non-GM comparators, even in stringent tests utilizing near-isogenic positive and negative segregants, can simply reflect minor genomic differences associated with conventional back-crossing practices. Conclusion: The effect of GM on metabolomics variation was determined to be negligible and supports that there is no scientific rationale for prioritizing GM as a source of variation.</p