The EPSR Co-ordinator would like to thank everyone who has registered by returning their EPSR Website Access T&Cs. If this is still on your to do list, the form is available in the EPSR section in the members only area of the website.
Also available is a filterable Excel spreadsheet containing the species, tissue and finding of each EPSR case for ease of identifying lesions of interest and we are now able to share the original case descriptions and discussions if any more detail is required on any of the cases in the EPSR archive.
I hope you agree that this is an exciting offering from the BSTP that will benefit all our members as well as enhancing future Webinars, Continuing Education Symposia and Annual Scientific Meetings, where we can also make use of this facility to share scanned slides. New cases for future use are always very welcome so if you have any interesting or unusual lesions or cases please contact the EPSR Co-ordinator via the BSTP Secretariat.


You've lived in your beloved home for 30 years. It's filled with stuff that you've collected over time—the huge sofa, the dining room table with room enough for the whole family.
Now you're thinking of moving into a two-bedroom apartment in a retirement community, and just the thought of what you're going to do with all that stuff makes you nervous.
Ingleside at King Farm is pleased to present a Health and Wellness Fair from 10 a.m. to 1 p.m. Wednesday, March 11, in the Multipurpose Room.
To help you learn about the wellness resources that are available within the community and in the King Farm area, we've invited a number of vendors who provide wellness services to conduct demonstrations and provide you with healthy living tips.


Air France-KLM CEO Ben Smith temporarily Becomes CEO of Air France. Ben Smith, the new CEO of Air France-KLM, may also become the temporary CEO of subsidiary Air France.
According to reports, the appointment will take place on Thursday.
The current CEO of Air France, Franck Turner, will then have to make way for Smith.
Also have been rumours for some weeks about a new administrative structure at Air France.
will also be directly responsible for French society.
Air France is currently engaged in a wage conflict with the employees.
The previous CEO of Air France-KLM left the company after a majority of the staff voted opposite a proposal from the then board on remuneration.
In the run-up to that offer from Air France, airline employees have frequently resigned.
It has cost Air France-KLM estimated hundreds of millions.
therefore, one of the more significant challenges for Smith.
The Canadian was selected in mid-August as the new CEO of Air France-KLM.
Smith before worked at Air Canada, where he was also responsible for negotiations with the staff on the collective labour agreement.


Winner of the 2017 MPI RISE Award for Educational Programming, CMP University is the dynamic way to prepare for the CMP examination. CMP U covers all domains and topics contained in the CMP International Standards (CMP-IS) within a university extension course format. With the generosity of our venue sponsor, SPUR, conveniently located in San Francisco within a few blocks of public transportation including BART, CMP U will be held on select Saturday mornings preceding the CMP examination periods offered in August 2019 and November 2019. This series of regular infusions of knowledge will better prepare you throughout the months leading up to the exam and help you stay on track with your studying.
If you have questions about CMP University, please read through the CMP U FAQ page. If you still have questions, please contact the CMP/CMM Committee Chairs, Allyson Gee, CMM, CMP or Noelle Boesenberg, CMP, CIS, CTE.
For more information about the CMP designation, exam dates, application deadlines and fees, please visit the Events Industry Council website.
Important Note about Registration: It is the attendee's responsibility to ensure that the email address used to register for CMP U is the same email address on your EIC account. After the final class, MPINCC will upload the CMP U attendee list to the EIC's website, which uses an email address matching system. You will then receive an email from the EIC prompting you to login to your EIC account and claim your hours for CMP U.


On Monday, indie folk rockers Mumford & Sons announced dates and locations for their upcoming "An Arrow Through the Heartland" U.S. tour, which kicks off in April.
The British band's tour will support of Wilder Mind, Mumford & Sons' third LP released in May, and subsequently climbed to the top of Billboard U.K. album charts.
"Touring the United States of America in many ways fulfills the childhood dream of what touring might be like," the band's keyboardist, pianist and backup vocalist Ben Lovett wrote in a release. "Rolling from one great city to the next, each with its own personality and uniqueness whilst remaining familiar enough at the same time. Waking up every morning with a conversation amongst us about who wants to visit 'that' bookshop or 'the' ultimate music shop or whatever the various cities had to offer.
"And on this note, we're very excited to announce some new tour dates for 2016... Hope you can make it!"
For a full list of tour dates click here. Tickets can be purchased via the band's web page, and presale begins 10 a.m. local time Wednesday. Ticket shoppers are required to sign up for Mumford & Sons' newsletter in order to access presale tickets. General sale for tour dates will begin at 10 a.m. local time Friday.


Epolene® C-16P by Westlake Chemical is a branched low density polyethylene modified with maleic anhydride. Exhibits excellent thermal stability, low color and medium molecular weight. The maleic anhydride modification allows improved adhesion, greater filler tolerance, broader capabilities, and improved aging properties. Shows compatibility with many polymers, resins, and natural- & synthetic waxes. Epolene® C-16P is used as a base polymer for solvent-based adhesives & hot-melt adhesives, and applications which require compatibility with polyamide.


Only offered as a bundle with an embroidery machine purchase.
Wilcom is the best and most complete digitizing software in the world and now you can purchase your license at a reduced price only when you purchase a CAMFive embroidery machine because we are Wilcom's integrator and authorized distributor for OEM versions. We offer 3 different Wilcom versions DecoStudio Lite, DecoStudio Lite PLUS and EmbroideryStudio Digitizing E3, so you can choose the one that would better fit your needs.


text:
Tesla Sees Highest Vehicle Production Month in Q2 2022, Revenue Climbs 42% YoY
Despite seeing its highest vehicle production month in history, Tesla also saw its automotive gross margin fall for the first time in a long while.
Shares of EV giant Tesla Inc (NASDAQ: TSLA) jumped in extended trading as the company beat analysts' expectations in Q2 2022 despite the market conditions. The company released its quarterly financial result and shareholders' letter after the market closed on the 20th of July. Analysts' prediction on Tesla for Q2 2022 was $16.521 billion in revenue. Also, the Wall Street consensus for earnings per share was $1.81. These predictions were fueled by the fact that Tesla had to shut down its Gigafactory Shanghai.
Tesla in Q2 2022
Due to the forceful closure, the EV maker recorded around 250,000 deliveries instead of the expected total of over 300,000. However, Tesla has remained afloat, exceeding revenue and earnings expectations for 2022 Q2. Wedbush analysts Dan Ives noted that Tesla seems to be approaching delivering 1.4 million units this year.
Tesla posted revenue of $16.934 billion for the 2022 Q2, representing a 42% increase YoY. Also, adjusted earnings per share were $2.27, higher than the expected $1.81, according to Refinitiv. In reaction to the 2022 Q2 financial report, Tesla recorded a 3% gain. At press time, however, the company is up 1.45% in after-hours trading to $753.30.
In a statement, the automaker acknowledged that it experienced challenges during the last quarter. However, it came out strong.
"We continued to make significant progress across the business during the second quarter of 2022. Though we faced certain challenges, including limited production and shutdowns in Shanghai for the majority of the quarter, we achieved an operating margin among the highest in the industry of 14.6%, positive free cash flow of $621M and ended the quarter with the highest vehicle production month in our history."
Despite seeing its highest vehicle production month in history, Tesla also saw its automotive gross margin fall for the first time in a long while. The automotive gross margin was 27.9% in the quarterly financial report. Meanwhile, it came in at 32.9% in the previous quarter and 18.4% in 2021 Q2. The automaker struggled with automotive gross margin due to inflation and increased competition for battery cells and other supplies required for EV production.
Tesla Coverts 75% Bitcoin Holdings to Cash
Furthermore, Tesla revealed that it sold 75% of its Bitcoin holdings in Q2 of 2022. As a result of the crypto conversion to fiat currency, the company added $936 million in cash to its balance sheet. CEO Elon Musk explained that the EV giant sold BTC because it was not sure when China would ease its lockdowns. He added that it was important for the company to "maximize" its "cash position."
Despite the inflation, lockdowns, and factory closure, TSLA has managed to add 4.83% over the past month. The company's stock is also up 4.41% in the last five days, amassing more than 13% in the last twelve months.
Crypto Lender Celsius Network Finally Files for Chapter 11 Bankruptcy
Robinhood Adds New Features for Shiba Inu, Bitcoin, and Others
Red Hot US Inflation Brings Strong Volatility to Bitcoin and Crypto
Fundamentals of Finhaven Token
Skybridge Capital Halts Withdrawals from Funds with Exposure to Crypto
Crypto Fund Manager Fintonia Group Obtains Provisional Virtual Asset Licence in Dubai | Coinspeaker
10 Million New Users Join KuCoin, Up 219% from 2021 | Coinspeaker

redpajama_set_name: RedPajamaCommonCrawl


text:
Identification of candidate genes related to calanolide biosynthesis by transcriptome sequencing of Calophyllum brasiliense (Calophyllaceae)
Hilda-Beatriz Gómez-Robledo1,2,
Francisco Cruz-Sosa3,
Antonio Bernabé-Antonio4,
Antonio Guerrero-Analco5,
José Luis Olivares-Romero5,
Alexandro Alonso-Sánchez5,
Emanuel Villafán5 &
Enrique Ibarra-Laclette ORCID: orcid.org/0000-0003-1441-19385
Calophyllum brasiliense is highlighted as an important resource of calanolides, which are dipyranocoumarins that inhibit the reverse transcriptase of human immunodeficiency virus type 1 (HIV-1 RT). Despite having great medicinal importance, enzymes involved in calanolide, biosynthesis and the pathway itself, are still largely unknown. Additionally, no genomic resources exist for this plant species.
In this work, we first analyzed the transcriptome of C. brasiliense leaves, stem, and roots using a RNA-seq strategy, which provided a dataset for functional gene mining. According to the structures of the calanolides, putative biosynthetic pathways were proposed. Finally, candidate unigenes in the transcriptome dataset, potentially involved in umbelliferone and calanolide (angular pyranocoumarin) biosynthetic pathways, were screened using mainly homology-based BLAST and phylogenetic analyses.
The unigene dataset that was generated in this study provides an important resource for further molecular studies of C. brasiliense, especially for functional analysis of candidate genes involved in the biosynthetic pathways of linear and angular pyranocoumarins.
Calophyllum sp. (Calophyllaceae) is a large group of tropical trees with more than 180–200 species [1]. Currently, some species of this genus have aroused great interest in the scientific community due to their promising phytochemical aspects. In Mexico, the most widely distributed species among the eight found in America is Calophyllum brasiliense Cambes [2], which grows in tropical rain forests from Brazil to northwest of Mexico [3]. This species contains a large number and variety of secondary metabolites including flavonoids, triterpenes, coumarins, chromones, and xanthones [4], some of which exhibit interesting anti-leishmanial, anti-bacterial, anti-cancer, anti-parasitic, and anti-viral properties [4, 5]. Two chemotypes have been classified according to their geographical origin. Chemotype 1 (CTP 1), which grows in Sierra de Santa Marta, State of Veracruz, Mexico, produces mammea type coumarins with high in vitro cytotoxic activity against human tumor cells and antibacterial properties against Staphyloccoccus aureus, S. epidermidis and Bacillus subtilis [6]. Meanwhile, chemotype 2 (CTP 2) grows in San Andres Tuxtla, State of Veracruz, Mexico, and produces calanolides, a series of tetracyclic dipyranocoumarins that exhibit an inhibitory effect against the reverse transcriptase of the human immunodeficiency virus type 1 (HIV-1 RT) [2, 7]. There are three different calanolides (A, B and C) that have been found in C. brasiliense and exhibit a significant inhibition on replication of the HIV-1 virus. Interestingly, these bioactive compounds show no toxicity to MT2 human lymphocytes [7]. Additional studies have shown that a high dose of B and C calanolides causes an increased number of spleen megakaryocytes and no alteration of hepatocytes [8]. Calanolide A, which possesses the highest inhibition of viral replication, has been synthesized and has been reported to have similar actions to the natural product [9, 10]. This molecule is in fact in clinical development as a novel therapeutic agent against HIV-1 infection [11, 12]. In plants, calanolides can be detected mainly in leaves (from CTP2), even if they come from seedlings of C. brasiliense that were germinated from seed and grown in a greenhouse [13]. Calanolides can be also detected in plant callus [13], cell suspension cultures, and leaves from 12-month-old plants that were in vitro regenerated from the young, nodal-stems of C. brasiliense plants [14].
The metabolic pathways in the biosynthesis of calanolides involve multiple and complex series of enzymatic reactions in which L-phenylalanine and trans-cinnamic acids can be considered as primary precursors. It is important to emphasize that some intermediates such as 7-hydroxycoumarin (umbelliferone) have interesting properties such as anti-fungal [15, 16] and insecticidal [17] activities, and they have also been shown to have strong inhibitory activity on proliferation of human bladder carcinoma E-J cell lines ([18] cited in [15]).
Despite the great pharmacological importance of C. brasiliense, the genomic basis of the synthesis and function of metabolic compounds such as calanolides remains poorly understood. Here, we present the first report of a complete transcriptome analysis of C.brasiliense. The main goal of this study was to characterize the transcriptome of C.brasiliense (CT2) for future gene identification and functional genomics studies of this species. We carried out de novo transcriptome sequencing and assembly of RNA libraries derived from terminal leaves, stems, and roots that come from in vitro regenerated C. brasiliense seedlings. We provide annotation to public databases and categorize the transcripts into biological functions and pathways. In addition, calanolide biosynthetic pathways are suggested, and based on the homologies of some genes, we propose some of them to be promising candidates for future analyses of the calanolide biosynthetic pathway.
Results and discussions
Sequencing and assembly
A total of 16,842,368 paired-end reads (2x150) were generated (5,276,841 for leaves, 5,000,558 for stem and 6,240,602 for roots). Prior to the assembly process, the paired reads were trimmed and/or merged together using the SeqPrep pipeline (see methods for more details). A de novo assembly was generated using Oases [19], a Bruijn graph-based assembler designed as an extension of Velvet [20] mainly used to assemble short-read sequences derived from transcriptomics data. Velvet/Oases produced a total of 61,620 contigs ranged from 0.1 to 10 kb, with an average length of 547.28 bp (Additional file 1). The GC contents of the contig set was approximately 44.7 %, which is similar to the GC content of the coding regions from other species within the Malpighiales order (reviewed in [21]). The N50 of these contigs was also estimated and resulted in a moderately high value of 867 bp. A fairly large number (40,727) of assembled contigs (40,727, which represents a 66.01 % of the total), were between 200 bp and 500 bp in length, indicating the presence of assembled fragments. For practical purposes, in the presented work, all contigs from the dataset are referred to as unigenes. The BLASTx algorithm [22] was used to annotate the unigenes based on the traditional top-BLAST-hit annotation method. As references, a collection of protein databases including the Arabidopsis thaliana (Arabidopsis) and plant RefSeq databases were used for this purpose. A significant value (e-value) of 10−5 was applied as threshold in the BLASTx similarity searches (Additional file 2: Table S1).
Based on the Arabidopsis top hits, Gene Ontology (GO) annotations for the C. brasiliense unigenes were obtained. WEGO software [23] was used to perform GO functional classification into the three major categories (biological process, molecular function, and cellular components). Among the unigenes with Arabidopsis hits, 42,090 (68.30 %) were assigned to different gene ontology categories with a total of 367,994 functional terms, of which 103,865 are unique. Biological processes comprised the majority of the functional terms (178,629; 48.54 %), followed by cellular components (95,428; 25.93 %) and molecular functions (93,937; 25.52 %) (Additional file 3: Figure S1; see also Additional file 4: Table S2). Top-ranked categories of GO biological processes were the sub-categories corresponding to cellular (27,090 unigenes) and metabolic (24,653 unigenes) processes. Interestingly, response to stimulus (14,101 unigenes) and biological regulation (12,646 unigenes) were also prominently represented among GO biological processes categories. In addition to functional annotation based on GOs, C. brasiliense unigenes were classified based on metabolic pathways available and described in Kyoto Encyclopedia of Genes and Genomes (KEGG). KEGG Automatic Annotation Server (KAAS; [24]) was used to assign to C.brasiliense unigenes the KEGG Orthology (KO) codes and enzyme commission (EC) numbers. KO codes were assigned to 4,881 unigenes, of which 1,733 could be associated to specific EC numbers related to 226 different metabolic pathways (Additional file 2: Table S1).
Gene expression profiles of C. brasiliense organs
The gene expression profiles of roots, stems, and leaves were analyzed by mapping paired reads to the transcriptome assembly. An expression profile matrix containing the contigs (rows) and the number of mapped reads in each transcriptome sample (columns) was created (Additional file 4: Table S3). To make read counts comparable among samples, a normalization of reads per kilobase per million (RPKM) was performed. In order to distinguish expressed genes in at least one of the organs sampled from background, a threshold of RPKM ≥ 3 was used [25]. According to the stringency levels, 26,299 unigenes (59.26 % of the total) were expressed in all three tissues sampled (ubiquitous genes). The remaining unigenes were expressed in a single tissue (organ specifics) or they were distributed among different tissue combinations. The likelihood ratio statistic (R-statistic) [26] was calculated and used to identify those genes with significant expression level differences among sampled organs. Higher R-values indicate a greater probability of differential expression, whereas R-values near zero represent constitutive expression. In this context, it was considered as preferentially expressed genes the unigenes with R-values ≥ 8 (true positive rate of ~98 %) [26]. A total of 3,173 (7.15 %) C. brasiliense unigenes were identified as preferentially expressed genes, 1,274 in leaves, 965 in stems, and 934 in roots, some of which (247, 143 and 231, respectively) could be considered as organ-specific. (Fig. 1 and Additional file 4: Table S4).
a Hierarchical clustering of C. brasiliense preferentially expressed unigenes based on RPKM values. The log2 RPKM values are presented with varying colors. The darker brown represents higher expression values, and the gray in the heat map lowest values. b RT-qPCR-based expression of eight-selected C. brasiliense preferentially expressed unigenes based on expression levels across organs (leaves, stem, and roots). Expression levels are given as 40-ΔCT, where ΔCT is the difference in threshold cycle number of the respective gene and the reference UN18770 (homolog to AT4G05050 which codify to UBQ11 on Arabidopsis); the number 40 was chosen because the PCR run stops after 40 cycles and a constant value was required as calibrator (see as example [76]). The results are shown as the averages ± SE of three biological replicate c RNA-Seq expression levels measured as reads per kb per million of reads (RPKM)
In order to gain insight into the organ-function connection, the top 10 organ-specific unigenes were surveyed. In leaves, three C. brasiliense unigenes (UN36044, UN28345 and UN34582), which are homologous to Arabidopsis members of the glycine-aspartic acid-serine-leucine motif lipase/ hydrolase (GDSL lipase) family, were highly expressed as well as some homologs (UN09544 and UN13106) of 3-ketoacyl-CoA synthase proteins (KSC). Consistently, members of the GDSL lipase gene family, such as AT5G33370 and AT3G04290, are co-regulated with genes involved in cutin biosynthesis as CER6/KCS6/CUT1 (AT1G68530), which has a dominant role in the elongation of very-long-chain fatty acids for cuticular wax synthesis [27–29]. The lipoxygenase/peroxygenase pathway is also involved in biosynthesis of cutin monomers [30]; this could explain the expression profile of the UN00603, which was identified as a leaf-specific unigene and annotated as homologous to chloroplast lipoxygenase LOX2 (AT3G45140), an enzyme required for wound-induced jasmonic acid accumulation in Arabidopsis [31]. The presence of all of these genes in the leaf transcriptome was expected considering that epicuticular waxes are produced either exclusively during leaf development and expansion, or during the entire lifetime of the leaf [18].
Regarding stems, a homolog of fasciclin-like arabinogalactan protein FLA12 (AT5G60490; unigene UN35075) exhibited one of the highest expression levels as a stem-specific gene, which is consistent with previous reports showing that the expression of some members of the FLA gene family are correlated with the onset of secondary-wall cellulose synthesis in Arabidopsis stems, and with wood formation in the stems and branches of trees. This data suggests that unigene UN35075 may play a biological role in C. brasiliense stem development [32]. Additionally, genes encoding enzymes related to monolignol biosynthesis, such as phenylalanine ammonia-lyase (PAL1; UN01310), caffeoyl-CoA 3-O-methyltransferase (CCoAOMT; UN21637 and UN12250), and 4-coumarate: CoA ligase (4CL; UN01988), were identified as preferentially expressed in stems, although transcripts could be detected in all three organs sampled. Lignin, which plays a crucial role in conducting tissue in plant stems, is synthesized from the oxidative coupling of monolignols [33]. In addition, 16 unigenes homologous to Arabidopsis IRX proteins were also classified as specifically or preferentially expressed in stems; this was expected considering that the irregular xylem (irx) mutant is characterized by a reduction in cellulose in stem tissue [34]. Finally, two C. barsiliense unigenes (UN02173 and UN03226), which were homologous to members of the family of high affinity phosphorous transporters (PHT1), were identified as highly expressed only in roots, as well as the UN03833 unigene, a homolog of ARSK (AT2G26290), a poorly characterized gene encoding a root-specific kinase [35].
In order to validate the expression profiles obtained by normalized read counts, RT-qPCR was performed using nine chosen genes. All genes evaluated showed RT-qPCR expression profiles in complete agreement with the profiles derived from read counts analyses (Fig. 1).
Functional annotation of preferentially expressed genes
An enrichment analysis of GO terms was performed in order to identify, among the biological process categories, those subcategories that were over-represented in at least one of the organs analyzed (Additional file 4: Table S5). To compare the abundance of some preferentially expressed genes with specific biological processes, a modification of the same method applied to the preferentially expressed genes was used (the percentage of unigenes annotated with a given GO term in the organ-preferentially expressed genes was compared with the percentage of genes annotated with the same GO term in the complete set of the C. brasiliense transcriptome; Additional file 4: Table S6). Amoung the GO terms assigned to the sub-set of 3,173 of C. brasiliense preferentially expressed unigenes within biological process category, some over-represented biological processes were photosynthesis, alkaloid, and terpenoid metabolic-related (Additional file 3: Figure S2; see also Additional file 4: Table S7). This is consistent with the notion that the carbon skeletons of all secondary plant products are derived from carbohydrates synthesized by photosynthesis and that the synthesis of some of them depends on primary metabolites (Fig. 2). It should be noted that some biological processes showed no significant differences when the number of unigenes grouped for each process in each organ were compared. This was the case for the phenylpropanoid biosynthesis pathway (GO: 0009699), which showed an average of 39 genes that were grouped within this category in each organ sampled (Additional file 4: Table S6).
Principal biosynthetic pathways leading to synthesis of secondary metabolites (modified from [77]). Metabolic pathways shown in cyan represent the over-represented biological processes identified in leaves preferentially expressed genes
The biosynthesis of umbelliferone, a key precursor in the calanolides formation
Coumarins are synthesized in plants via the shikimate pathway, in which phenylalanine is an end product that also gives rise to the aromatic amino acids tyrosine and tryptophan and other small molecules such as flavonoids and hydroxycinnamic acid conjugates [36]. Successive para- and ortho- hydroxylation of trans-cinnamate (conjugate base of trans-cinnamic acid) leads to the formation of coumarin via 2-coumarate, or via 4-coumarate, to the formation of hydroxycoumarins such as umbelliferone (7-hydroxycoumarin). Other hydroxycoumarins lacking oxygenation at C-7 also share the trans-cinnamic acid as its precursor. According to EC numbers assigned to C. brasiliense unigenes, with only one exception (glutamate-prephenate aminotransferase; EC: 2.6.1.79), homologs from all enzymes required for the formation of L-phenylalanine via the shikimate pathway were identified (Additional file 3: Figure S3).
The key enzymes involved in the umbelliferone biosynthetic pathway (via 4-coumarate) are: a) phenylalanine ammonia lyase [PAL; EC:4.3.1.24], b) cinnamate 4 hydroxylase [C4H; EC:1.14.13.11], c) 4-coumarate: CoA ligase [4CL; EC:6.2.1.12], and d) 4-coumaroyl 2′-hydroxylase [EC:1.14.11.-] (Fig. 3). These enzymes involved in umbelliferone biosynthesis have been functionally characterized in Ruta graveolens species [39].
Biosynthetic pathway from umbelliferone starting with L-phenylalanine. The figure is an image taken and modified from the MetaCyc (http://metacyc.org/) search for umbelliferone biosynthesis
Proteins encoded by PAL gene family members can catalyze the conversion of L-phenylalanine to ammonia and trans-cinnamic acid, and are found in angiosperms and gymnosperms and also in some mosses, algae, fungi, and bacteria [37]. Based on the annotations extracted from KEGG, unigene UN01310 was identified as a homolog of PAL (K10775, EC:4.3.1.24). The WISE2 program [38] (http://www.ebi.ac.uk/Tools/psa/genewise/) and the Arabidopsis PAL1 protein (AT2G37040) were used to identify coding sequences in their correct open reading frames (this program uses a protein sequence as a template to predict a related protein sequence in the analyzed DNA sequence), then using the SeaView program [39], the protein-coding nucleotide sequences were aligned based on their corresponding amino acid translations to calculate the percent of identity at nucleotide and amino acid levels (Additional file 5). The identity between C. brasiliense/Arabidopsis PAL genes was estimated to be 69.05 % at the nucleotide level (CDS) and 79.9 % at the amino acid level. The distinctive aromatic amino acid lyase conserved domain (PF00221) was identified by motif/domain search against the Pfam database [40] (http://pfam.xfam.org/). Through a BLAST search based on the PF00221 domain, a total of eighteen C. brasiliense unigenes were identified as homologous to PAL genes, all of them annotated as homologs of some of the four existing Arabidopsis PAL genes ([41] PAL1; AT2G37040), PAL2; AT3G53260, PAL3; AT5G04230 and PAL4; AT3G10340) during the top-blast-hit annotation process (Additional file 2: Table S1). A phylogenetic tree from C. brasiliense PAL proteins was generated. Only the unigenes for which the proteins derived from their corresponding coding sequences represent at least 65 % of the length of their homologs were included (UN01310, UN03680 and UN02730). The PAL family members from Arabidopsis were also included in phylogenetic analysis. The multiple sequence alignments (Additional file 6) show that C. brasiliense proteins have high identity (ranged from 49.2 to 80.0 %) with PAL family proteins from Arabidopsis, and a three-dimensional structure analysis indicated that these proteins also contain a highly similar putative structure to other PAL plant proteins previously reported (Fig. 4). The high similarities of protein sequences among the C. brasiliense and Arabidopsis homologs and 3D models of the PAL family suggest that they may play similar functional roles.
a Maximum likelihood phylogenetic tree depicting the relationship between the different C. brasiliense (blue) and Arabidopsis (green) PAL family members. Protein sequences were aligned with MUSCLE [69], and phylogenetic analyses were performed in maximum likelihood framework with SeaView v4.0 software [39]. b Primary protein structures in which the aromatic amino acid lyase conserved domain (PF00221) are represented by gray boxes. c Matrix of the percentage identities between the aligned protein sequences of PAL family members from C. brasiliense and Arabidopsis. d Structural model superposition of PAL proteins from C. brasiliense [UN01310 (pink), UN02730 (yellow) and UN03680 (cyan)]. The available structure of PAL protein from Petroselinum crispum in the Protein Database (PDB entry 1w27.1) was used as a template. The homology modeling was carried out using the SWISS-MODEL program and SWISS-PDB viewer v4.1.0 ([73, 75]; http://swissmodel.expasy.org/) was used to superpose the C. brasiliense PAL proteins
Similar approaches to those described above were used to identify the remaining C. brasiliense genes involved in umbelliferone biosynthesis. A total of three unigenes were identified as homologous to the only trans-cinnamate 4-hydroxylase (AT2G30490; C4H) in the Arabidopsis genome (Additional file 3: Figure S4 and Additional file 7). C4H is a plant-specific cytochrome P450 (PF00067) that catalyzes the second step of the multibranched phenylpropanoid pathway [42]. Regarding 4-coumarate CoA ligase [4CL; EC:6.2.1.12], a total of six unigenes were detected as homologous to these proteins; however, only in three of them (UN01603, UN01988 and UN01725), were complete open reading frames identified (Additional file 8). The motif/domain searches revealed that both the AMP-binding C-terminal domain (PF13193) and common AMP-binding central domain (PF00501) are present in the translated sequences corresponding to these C. brasiliense unigenes (Additional file 3: Figure S5). Commonly, most angiosperms encode to a small family of 4CL (e.g., seven members in case of Arabidopsis). These enzymes are involved in the last step of the general phenylpropanoid pathway, and in addition to using 4-coumarate as substrate, they also convert p-coumaric acid, ferulic acid, caffeic acid and 5-OH-ferulic acid with different catalytic efficiency [43, 44].
Finally, the bi-directional best hit (BBH) method was used to identify a homolog of 4-coumaroyl 2′-hydroxylase [EC:1.14.11.-] from Ruta graveolens. The 4-coumaroyl 2′-hydroxylase isolated/characterized from R. graveolens (Accession JF799117.1) is the only enzyme that has been specifically assigned to coumarin synthesis, and to a lesser extent this enzyme also accepts 4-coumaroyl-CoA to produce umbelliferone [45]. The C. brasiliense unigene UN02124, in which the cytochrome P450 conserved domain PF00067 is present, showed 87.2 % identity with the published protein from R. graveolens (Additional file 3: Figure S6 and Additional file 9). The final step of the in vivo pathway for the synthesis of umbelliferone involves a trans-cis isomerization followed by a subsequent lactonization of the 2, 4-dihydroxy-cinnamoyl-CoA that closes the side chain, and this reaction occurs spontaneously (Fig. 3).
Considering that we were able to identify homologs to all genes involved in the umbelliferone biosynthetic pathway via 4-coummarate, and due the absence of several transcripts that potentially encode for enzymes such as cinnamate 2-hydroxylase (EC: 1.14.13.14), 2-coumarate O-β-glucosyltransferase (EC: 2.4.1.114), 2-coumarate β-D-glucoside isomerase (EC: 5.2.1.-) and coumarinic acid glucoside β-glucosidase (EC:3.2.1.21), which were first characterized in Melilotus alba [46, 47] were they are all involved in coumarin biosynthetic pathway via 2-coumarate, we suggest that in C. brasiliense, umbelliferone is synthesized via the 4-coumarate pathway. This was expected, because in contrast with mammals, only in a few plant species (e.g. Catharanthus roseus and Conium maculatum) has it been suggested that enzymes capable of carrying out the hydroxylation of coumarin in C-7 to produce umbelliferone exist (reviewed in [48]).
Analysis of putative candidate genes involved in the calanolide (angular pyranocoumarins) biosynthetic pathway
The calanolides are pyranocoumarins, one class of coumarin derivates. The biosynthesis of pyranocoumarins is still poorly understood, but it has been reported that remarkable structural similarities between furanocoumarins and pyranocoumarins exist. Both classes of coumarins (furano- and pyrano- coumarins) can be grouped into linear and angular types. The determination of the type, either linear or angular, is based on the prenylation position of the common precursor (umbelliferone). In the case of the linear type, the pyran (or furan) ring is attached at C-6 and C-7, while in the angular type, the substitution is carried out at C-7 and C-8 [49]. Both types of pyranocoumarins are derived from the same precursors that give rise to linear and angular furanocoumarins (demethylsuberonosin and osthenol, respectively) (Fig. 5).
Biosynthetic pathways of both, linear and angular, furano- a and pyrano-coumarins b. Abbreviations of identified/characterized enzymes are shown in red: PS, umbelliferone prenyltransferase; MS, marmesin synthase; PS, psoralen synthase; CS, columbianetin synthase; AS, angelicin synthase; BMT, bergaptol O-methyltransferase. Details of the psoralen synthase-catalyzed reaction are in brackets. This figure was modified from [56]
Previous studies conducted in Pimpinella magna and Pastinaca sativa plants could be considered the first experimental evidence to prove that linear and angular furanocuoumarins are derived from umbelliferone, prenylated at either the C-6 (leading to the formation of demethylsuberonosin; DMS) or C-8 position (osthenol), respectively [50]. In later years, additional investigations revealed that the cyclization of demethylsuberosin leads to (+)-marmesin formation, taking place through an enzymatic reaction that occurs in the presence of NADPH and molecular oxygen [51]. These 'mamersin synthases' have been identified as cytochrome P450 monooxygenases in Petroselinum crispum and Ammi maju plant species (reviewed in [52]). The range of reactions catalyzed by P450s include the epoxidation of olefins by insertion of an 'oxen' [53], and the reactive product of this reaction often inactivates the enzyme by alkylation of the prosthetic heme group [54]. However, no intermediate was released from mamersin synthase reaction, and it is likely that the 7-hydroxyl group of demethylsuberosin delocalizes the double bond electrons and favors the instantaneous cyclization to the dihydrofuranocoumarin (Fig. 5a). Model mechanisms have been proposed for the reactions mediated by catalytic action of the P450 enzymes, and one of these mechanisms consists of primary interaction of the catalytic P450 oxo-derivative, formed by heterolytic cleavage of the oxygen-oxygen bond in the ferric-hydroperoxy species, with aliphatic double bonds [52, 54]. This mechanism of reaction is compatible with such a cyclization of demethylsuberosin to (+)-marmesin, avoiding the formation of an intermediate epoxide (Fig. 5a). The formation of (−)-columbianetin from osthenol is catalyzed in an analogous fashion and the subsequent activities of both enzymes, angelicin and psoralen synthases, have been supported experimentally [55, 56]. It appears feasible that the synthesis of linear and angular pyranocoumarins such xanthyletin [57], graveolone [58], or seselin [59] may be produced concomitantly with furanocoumarins in a very similar way as from demethylsuberosin or osthenol, respectively (Fig. 5b).
Here, we propose two closely related biosynthetic pathways for calanolide biosynthesis (Fig. 6). These metabolic pathways imply some steps in the biosynthesis upon the basis of the presence of some related-compounds previously identified in other plant species. Starting from umbelliferone (1), a highly abundant metabolite identified in several plant species belonging to the Rutaceae and Compositae families [60], it is likely that the C-8 prenylated derivative (osthenol), which has been previously considered as a precursor at angular pyranocoumarins biosynthesis, could be one of the first intermediates. However, considering that some umbelliferone-related metabolites such as esculetin (2), daphnetin (3), 6,7,8-trimethoxycoumarin (4) and 5,7,8-trimethoxycoumarin (5), have been previously identified in several different plant species (reviewed in [49, 61]), it may be suggested that the formation of 5,7-dihydroxycoumarin (6) could be an initial step before the c-prenylation of umbelliferone at C-6, which leads to osthenol. Additional steps in calanolide biosinthesis include the epoxidation of 5,7-dihydroxy-6-(3-methyl-2-butenyl) coumarin (7) to (8), followed by the cyclization of (8) to (9), which after a dehydration step leads to (10). (10) could be a precursor of alloxanthoxyletin (11) [61], or in a similar way, a second c-prenylation at C-8 (12) may lead to (13) after their corresponding epoxidation-cyclization. Successive steps from (13) may involve the C-3 alkylation and the mono-hydroxylation of the pyran ring to produce (14), and then by Wagner-Meerwein rearrangement (1,2-methyl shifts), followed by the subsequent hydroxylation, the calanolides A, B and C could be produced (Fig. 6). Details from Wagner-Meerwein rearrangements that might lead to a calanolide formation (perhaps favoring the biosynthesis of one over the others) are shown in Additional file 3, Figure S7. The second suggested pathway could occur in a very similar way, starting with the c-prenylation of umbelliferone at C-8, which would lead to seselin production [59] and followed by a second c-prenylation at position C-4 (Fig. 6). Apparently, unlike the biosynthesis of furanocoumarins, in which the formation of an intermediary epoxide is avoided, the pyran ring formation in the pyranocoumarin biosynthetic pathway depends precisely on that intermediate.
Proposed biosynthetic pathway for the calonolides a, b and c in C. brasiliense
Considering the remarkable structural similarities between furanocoumarins and pyranocoumarins (linear and angular), and the fact that both classes of compounds are derived from the same precursors (DMS and osthenol), it is possible to hypothesize that the enzymes involved in these biosynthetic pathways might share common ancestry. This hypothesis is consistent with the observation that furanocoumarins and pyranocuoumarins do not usually coexist in the same species. Even when many unknowns about biosynthesis and the function of furanocoumarins remain unresolved, it is clear that our knowledge about closely related compounds, such as pyranocoumarins, appears to be even less.
Concerning furanocoumarins, it has been reported that they are produced by a wide variety of plants in response to pathogen or herbivore attack. They are activated by ultraviolet light and can be highly toxic to certain vertebrate and invertebrate herbivores due to their integration into DNA, which contributes to rapid cell death [49, 62]. It has been suggested that linear and angular furanocoumarins are the results of a co-evolutionary process between plant and insects. Plant-insect interaction studies reveal that linear furanocoumarins are more toxic than angular ones; however, angular structures apparently produce a synergistic effect when they are combined with linear ones [62]. When mixed, linear and angular furanocoumarins result in a combination that is more difficult for insects to detoxify [62]. Apparently, during evolution, angular furanocoumarins appeared later than linear ones, a hypothesis that finds support based on the observation that angular furanocoumarins are always found concomitantly with linear structures, while linear types can be found alone (reviewed in [49, 63]).
During recent years, many enzymes involved in furanocoumarin biosynthesis have been described at the molecular level, including three P450 monooxygenases (psoralen-, angelicin-, and (+)-marmesin- synthases [55, 56]), bergaptol O-methyltransferase [64], as well as the key enzyme, a prenyltransferase [63]) involved in the critical step leading to precursors synthesis of linear and angular furanocoumarins (DMS and osthenol respectively; see Figs. 5 and 6). Additional P450-dependent enzymatic steps have remained unresolved, but the participation of this class of enzymes has been suggested in many steps of furanocoumarin biosynthesis. (Figs. 5 and 6, see [49] for an extended revision). Furanocoumarins are produced by a wide variety of plants in response to pathogen or herbivore attack.
The biosynthesis of pyranocoumarins has not yet been investigated. However, considering that psoralen synthase shows 70 % identity with angelisin synthase, and that its participation in linear and angular furanocoumarins biosynthesis has been previously demonstrated [55, 56], and based on the assumption that enzymes involved in pyranocoumarin biosynthesis might share a common ancestor of unknown functionality (perhaps as a result of gene duplications and subsequent molecular evolution), we used these and other enzymes involved in furanocoumarin biosynthesis as sequence references to identify homologs in the C. brasiliense unigenes set.
First, the prenyltransferase (PT) identified in parsley (Petroselinum crispum) [63], the angelicin synthase (CYP71AJ4) from Pastinaca sativa, the psoralen synthase (CYP71AJ1) identified from Ammi majus and their orthologs (CYP71AJ2 and CYP71AJ3 from Apium graveolens and Pastinaca sativa, respectively) [55, 56], were used as references in tBLASTn similarity searches (e-value 10−6) against the C. brasiliense unigene dataset. Additionally, the poorly characterized CYP82H1 isolated from Ammi majus, which increases its expression levels in plant-fungi interactions and accompanying furanocoumarin biosynthesis, was also included [65]. Sequences of unigenes that upon translation show at least 20 % identity over a resides window that represents a complete coding sequence (CDS), or at least 70 % of the homologous protein, were retained for future analysis.
A total of six PT-like sequences were identified. The subsequent comparison against PFAM databases confirmed the presence of the UbiA prenyltransferase domain (PF01040). The unigene UN01964 (with 35 % shared identity) was resolved in the same clade as the parsley prenyltransferase (Additional file 3: Figure S8 and Additional file 10). Considering these results, we propose that UN01964 should be considered a leading candidate to encode a putative prenyl transferase involved in biosynthesis of the precursors that lead to synthesis of linear and angular pyranocoumarins.
Regarding the subsequent steps in pyranocoumarin biosynthesis, a total of 34 unigenes were identified as homologous to the previously characterized P450 monooxygeneases involved in the furanocoumarin biosynthetic pathway (stringency levels: coverage ≥ 70 %, identity ≥ 20 %). Three major clades were recognized on the phylogenetic tree, and one of these included only three C. brasiliense unigenes. The second clade brought together all CYP71AJ proteins that were included in phylogenetic analyses and a total of fourteen C. brasiliense unigenes. Finally, the third clade included the CYP82H1 P450 monooxygenase and the remaining C. brasiliense CYP-like sequences that were identified (Additional file 3: Figure S9, Additional file 11). The CYP71-related clade comprises two sister clades named as classes I and II, respectively. The translated Class I unigenes showed a percent identity that ranged from 31 to 46 % with respect to CYP71AJ proteins, while class II members were on average ~10 % less similar (Additional file 4: Table S8). Low percent similarities at the protein level were expected considering that the presence of furanocoumarins has not been reported in C. brasiliense, which is instead capable of producing pyranocoumarins (linear and angular, including the calonolide compounds) in young seedlings (mainly at leaves) and in callus cultures [13]. According to the expression profiles of these genes, 44 % were identified as preferentially expressed in some of the organs sampled. UN02363, UN03063, UN04124, and UN02841 were selected as preferentially expressed in leaves. With only one exception (UN02841), these genes were classified as CYP71-related; two of them (UN02363 and UN03063) grouped in class II and the other one into class I (UN04124). In addition, UN04124 possesses 35.6 and 35.8 % identity with the psoralen and angelicin synthases of Pastinaca sativa. Altogether, this data suggests that UN04124 can be considered a prime candidate for involvement in angular and/or linear pyranocoumarin biosynthesis.
The unigene dataset generated in this study provides an important resource for further molecular studies of C. brasiliense, especially for characterizing candidate genes in the biosynthetic pathways of linear and angular pyranocoumarins. Using appropriate approaches, a series of candidate genes were identified. Consecutive analyses were conducted to determine their corresponding expression patterns and phylogenetic relationships. The candidate genes identified in C. brasiliense transcriptome that were suggested to possibly be involved in the biosynthesis of calanolides (angular coumarins), could be cloned and characterized in further studies. Additional bioinformatic analyses could be conducted in order to reduce the number of candidate proteins that may catalyze specific reactions at particular steps. We suggest that at least for P450 monooxygenase enzymes, docking and molecular dynamics analyses could be performed in order to reduce the number of candidate genes involved in the calanolide biosynthetic pathway.
Plan material
Previously, Bernabé et al., [13], have shown that callus cultures or young plants of C. brasiliense grown in a greenhouse are capable of producing calanolides. In order to guarantee the production of calanolides, in vitro seedlings were regenerated from the young nodal-stem of the same plants (chemotype 2) used previously by Bernabé et al. These plants were germinated from seeds collected in San Andrés Tuxtla, State of Veracruz. The plants were grown in a green house during 5–6 months approximately. Young nodal-stem were collected from the plants and used as the source of explants. Leaves, stems, and roots were collected from in vitro regenerated 12-month-old plants. According to the manufacturer's instructions, total RNAs were isolated with TRIzol® Reagent (Life technologies). RNAs isolated from the three organs sampled, were re-purified with the RNeasy kit (Qiagen) and treated with RNase-free DNase I (Invitrogen) in order to remove the DNA residues. The quality and purity of RNAs were assessed with OD260/230 ratio by using the NanoDrop 2000 (Thermo Fisher). RNA integrity was evaluated by RNA integrity number (RIN) using an Agilent 2100 Bioanalyzer (Agilent Technologies). Only RNAs with RIN values ≥ 8.5 were used from library generation.
cDNA library preparation and sequencing (RNA-seq)
cDNA preparation, library construction, and sequencing were performed at the Genomic Services laboratory, LANGEBIO-CINVESTAV, Mexico by using the MiSeq™ platform according to the manufacturer's instructions (Illumina, San Diego, CA). Briefly, poly (A) RNA was isolated from 20 μg of total RNA using Sera-mag Magnetic Oligo (dT) Beads (Illumina). To avoid priming bias when synthesizing cDNA, the purified mRNA was first fragmented into small pieces. Then the double-stranded cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen, Camarillo, CA) with random hexamer (N6) primers (Illumina). The synthesized cDNA was subjected to end-repair and phosphorylation using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. These repaired cDNA fragments were 3′ adenylated using Klenow Exo- (3′ to 5′ exo minus, Illumina). Illumina paired-end adapters were ligated to the ends of these 3′-adenylated cDNA fragments, using specific barcodes for each sample (roots, stem, and leaves). Fifteen rounds of PCR amplification were performed to enrich the purified cDNA template using PCR Primer PE 1.0 and PE 2.0 (Illumina) with Phusion DNA Polymerase. The cDNA library was constructed with a fragment length range of 278 bp (±0.5 SD). Finally, after validating on an Agilent Technologies 2100 Bioanalyzer using the Agilent DNA 1000 chip kit, cDNA libraries were sequenced on a paired-end (2x150) flow cell using Illumina MiSeq sequencer. Files containing sequence reads and quality scores were deposited in the Short Read Archive of the National Center for Biotechnology Information (NCBI) [Accession number SRP079249].
Data filtering and de novo assembly
Forward and reverse read pairs (generated by Illumina-MiSeq) were merged to form a single "longer-reads" using the SeqPrep pipeline (https://github.com/jstjohn/SeqPrep), with default parameters (a quality score cutoff of phred 33, a minimum merged read length of 15 bp and no mismatches in the overlapping region). Paired-end reads that did not overlap were trimmed with a sliding window approach (window size 10 bases, shift 1 base). The FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html) was used to this purpose. Reads were discarded if they were smaller than 30 bases after trimming, orphan reads were also removed in order to keep pairs only. Velvet assembler using the Oases module [19] was used for sequence assembly. A unigenes set from C. brasiliense was generated considering only resulting contigs with a minimum size of 100 bp.
Annotation of C. brasiliense unigenes
To annotate unigenes obtained by de novo assembly, we performed sequence similarity searches using the BLASTx algorithm (e-value 10−5) on Arabidopsis thaliana (TAIR v11), and other plant proteins (NCBI; ftp://ftp.ncbi.nlm.nih.gov/refseq/release/plant/) databases. Top protein matches from Arabidopsis or additional plant proteins were assigned to each of the C. brasiliense unigenes. The gene ontology (GO) functional classes and pathways for each C. brasiliense unigene were assigned based on Arabidopsis GO SLIM and pathway annotation (ftp://ftp.arabidopsis.org/home/tair/Ontologies/). The data were statistically analyzed using the WEGO software [23] which is a useful tool for plotting GO annotation results. Additionally, the unigenes were also analyzed using the KEGG Automatic Annotation Server (KAAS; http://www.genome.jp/tools/kaas/) to provide annotations of KEGG Orthology (KO) codes. The bi-directional best hit (BBH) method was used. Enzyme Commission (EC) numbers were also assigned based on the annotations extracted from Kyoto Encyclopedia of Genes and Genomes (KEGG) and were cross-checked with orthologous gene annotation projections from Plant Metabolic Network (PMN; http://www.plantcyc.org/), and if available, corresponding EC-formatted MetaCyc [66] cross-references were added.
Expression profile analysis of C. brasiliense transcriptome
After assembling of the C. brasiliense transcriptome, every RNA-seq library was separately aligned to the generated transcriptome assembly using Bowtie [67]. Counting of alignments was done using RSEM [68]. Differential expression statistical analysis was done using the FPKM (fragments per Kilo bases of contigs for per million mapped reads) values and statistical method described by Sketel [26]. Briefly, all clusters were submitted to a log likelihood ratio statistics which tends asymptotically to a χ2 distribution as described by Stekel. It is based on a single statistical test to describe the extent to which a gene is differentially expressed between libraries. This method permits in any number of libraries to identify differential expressed genes.
RT-qPCR
In order to identify the coding sequence in their correct open reading frame, eight of the unigenes of C. brasiliense identified as differentially expressed genes were aligned versus their Arabidopsis homologues. Using the SeaView program, protein-coding nucleotide sequences were aligned based on their corresponding amino acid translations (Additional file 12). Gene-specific primer pairs (Additional file 4: Table S9), were designed using the Primer3 v.0.4.0 web tool (http://bioinfo.ut.ee/primer3-0.4.0/primer3/) and then used in RT-qPCR assays.
A total of 10 ug of total RNA was reverse transcribed using SuperScript® III Reverse Transcriptase (Life Technologies) according to the manufacturer's instructions. qPCR of selected genes was carried out through SYBR green chemistry (Applied Biosystem) on a real time thermal cycler (AB7500, Applied Biosystem). UBQ11 (UN18770) was used as an internal control. The thermal cycling program was set to 95 °C for 5 min, 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min. All reactions were run in duplicates of three biological replicates.
Phylogenetic analyses
A maximum likelihood framework was used in phylogenetic analyses, which were performed with SeaView v2.4 software [39]. The alignments and phylogenetic analysis were drived by SeaView using Muscle [69] and PhyML [70] programs, respectively. Topology, branch lengths and, equilibrium frequencies, were optimized and the PhyML option was used under LG (Le and Gascuel) model [71]. The starting tree was determined using BioNJ, and both nearest-neighbor interchange (NNI) and subtree pruning and regrafting (SPR) algorithms for tree searching were used. Branch robustness was analyzed by approximate likelihood-ratio test (aLRT) [72].
Proteins modeling
The C. brasiliense's 3D protein structures were modeled by the rigid body grouping method, using the Swiss-Model workspace (http://swissmodel.expasy.org/) [73, 74]. This server is used to align the target sequences and template structure available in the Protein Data Bank (PDB). Once the template has been selected the 3D structure of the target sequences can be modeled. Templates used for modeling were 1w27.1 for phenylalanine ammonia lyases (PAL) and 3a9u.1 for 4-coumarate: CoA ligases (4CL). Each model generated was checked for various parameters that include Z, GMQE (Global Model Quality Estimation) and QMEAN (Qualitative Model Energy ANalysis) scores to assess the accuracy of the model. The modeled C. brasiliense proteins were superimposed onto their corresponding homologues structures using the SWISS-PDB viewer v4.1.0 program [75].
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Bourgaud F, Hehn A, Larbat R, Doerper S, Gontier E, Kellner S, Matern U. Biosynthesis of coumarins in plants: a major pathway still to be unravelled for cytochrome P450 enzymes. Phytochem Rev. 2006;5(2):293–308.
Harborne JB. The natural coumarins: occurrence, chemistry and biochemistry (Book). Plant Cell Environ. 1982;5(6):435–6.
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Larbat R, Kellner S, Specker S, Hehn A, Gontier E, Hans J, Bourgaud F, Matern U. Molecular cloning and functional characterization of psoralen synthase, the first committed monooxygenase of furanocoumarin biosynthesis. J Biol Chem. 2007;282(1):542–54.
Larbat R, Hehn A, Hans J, Schneider S, Jugdé H, Schneider B, Matern U, Bourgaud F. Isolation and functional characterization of CYP71AJ4 encoding for the first P450 monooxygenase of angular furanocoumarin biosynthesis. J Biol Chem. 2009;284(8):4776–85.
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Karamat F, Olry A, Munakata R, Koeduka T, Sugiyama A, Paris C, Hehn A, Bourgaud F, Yazaki K. A coumarin-specific prenyltransferase catalyzes the crucial biosynthetic reaction for furanocoumarin formation in parsley. Plant J. 2014;77(4):627–38.
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We would like to thank Victor A. Albert for his positive and relevant comments which improved the quality of this manuscript. We also thank Victor's support for language editing. Finally, we wish to thank Aldo Segura-Cabrera, Ana-Luisa Kiel-Martínez and Ofelia Ferrera-Rodríguez for fruitful discussions and valuable suggestions.
This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT), grant 223323 (EIL).
All supporting data are included within the article or in the additional files.
HBG-R wrote the paper with significant contributions by EI-L, HBG-R, FC-S, AB-A, AG-A, JLO-R, AA-S, EV and EI-L collected data. HBG-R, AG-A, JLO-R, AA-S, EV and EI-L analyzed data. EI-L conceived of and led the study. All authors read and approved the final manuscript.
Facultad de Ciencias de la Salud, Universidad Anáhuac, 52786, Estado de México, México
Hilda-Beatriz Gómez-Robledo
Laboratorio de Bioquímica y Biología Molecular, Escuela Médico Militar, Universidad del Ejército y Fuerza Aérea, 11200, Ciudad de México, México
Departamento de Biotecnología, Universidad Autónoma Metropolitana Unidad Iztapalapa (UAM-Iztapalapa), 09340, Ciudad de México, México
Francisco Cruz-Sosa
Departamento de Madera, Celulosa y Papel, Centro Universitario de Ciencias Exactas e Ingenierías, Universidad de Guadalajara, 45100, Zapopan, Jalisco, México
Antonio Bernabé-Antonio
Red de Estudios Moleculares Avanzados, Instituto de Ecología A.C, 91070, Xalapa, Veracruz, México
Antonio Guerrero-Analco, José Luis Olivares-Romero, Alexandro Alonso-Sánchez, Emanuel Villafán & Enrique Ibarra-Laclette
Antonio Guerrero-Analco
José Luis Olivares-Romero
Alexandro Alonso-Sánchez
Emanuel Villafán
Enrique Ibarra-Laclette
Correspondence to Enrique Ibarra-Laclette.
C. brasiliense unigenes set. (TXT 68107 kb)
Additional file 2: Table S1.
Annotation of C. brasiliense unigenes. (XLSX 20582 kb)
Additional file 3: Figures S1-S9.
Figure S1: Gene ontology classification of C. brasiliense unigenes. Figure S2: Biological processes assigned to differentially expressed genes. Figure S3: Re-constructed C. brasiliense metabolic network from glucose to trans-cinnamate. Figure S4: Phylogenetic relationships, primary protein structures and identities percent of trans-cinnamate 4-hydroxylases proteins. Figure S5: Structural and phylogenetic analysis of 4-coumarate: CoA ligases (4CL) from C. brasiliense. Figure S6: Alignment of the 4-coumaroyl 2′-hydroxylases from C. brasiliense. Figure S7: Schematic representation of the Wagner-Meerwein rearrangement. Figure S8: C. brasiliense prenyltransferases. Figure S9: Phylogenetic tree of selected C. brasiliense cytochrome P450. (PPTX 1219 kb)
Additional file 4: Tables S2-S9.
Table S2: Functional classification of C. brasiliense unigenes set based on Arabidopsis GO annotation. Table S3: Expression profile matrix of C. brasiliense unigenes. Table S4: Preferentially expressed genes in the organs sampled (leaves, stem and roots) of C. brasiliense. Table S5: Functional classification of preferentially expressed genes. Table S6: Number of preferentially expressed genes associated to specific GO terms. Table S7: Over-represented biological processes in specific organs. Table S8: Matrix of the percentage identities between the aligned cytochrome P450. Table S9: Primers used in RT-qPCR assays. (XLSX 16223 kb)
Coding sequences alignment between PAL-like C. brasiliense unigenes and Arabidopsis PAL1 protein. (TXT 8 kb)
Coding sequences alignment between C. brasiliense and Arabidopsis PAL proteins. (TXT 16 kb)
Coding sequences alignment between C4H-like C. brasiliense and Arabidopsis C4H protein. (TXT 6 kb)
Coding sequences alignment between C. brasiliense and Arabidopsis 4CL proteins. (TXT 18 kb)
Coding sequences alignment between unigene UN02124 of C. brasiliense and Ruta graveolens CYP98A22 protein. (TXT 3 kb)
Additional file 10:
Coding sequences alignment between PT-like unigenes from C. brasiliense and Petroselinum crispum PT protein. (TXT 9 kb)
Coding sequences alignment between CYP-like unigenes from C. brasiliense and CYP proteins from different plant species involved in furanocoumarins biosynthesis. (TXT 74 kb)
Coding sequences alignment between those unigenes from C. brasiliense and their corresponding Arabidopsis homologs used in RT-qPCR assays. (TXT 29 kb)
Gómez-Robledo, HB., Cruz-Sosa, F., Bernabé-Antonio, A. et al. Identification of candidate genes related to calanolide biosynthesis by transcriptome sequencing of Calophyllum brasiliense (Calophyllaceae). BMC Plant Biol 16, 177 (2016). https://doi.org/10.1186/s12870-016-0862-9
Phenylalanine Ammonia Lyase
Enzyme Commission
KEGG Orthology


In this file photo from 2008, Keith Loneker Sr., a former University of Kansas football standout, works as a substitute teacher at Free State High School.
Former Kansas and NFL offensive lineman Keith Loneker Sr. died Thursday morning after a battle with cancer.
Loneker, whose son Keith Loneker Jr., is a current linebacker for the KU football team, has been actively involved both with his alma mater and Lawrence's youth athletics scene throughout most of his adult life and was beloved by athletes and coaches at both Lawrence High and Free State.
In addition to his broad and successful football career, Loneker starred in a handful of Hollywood films, including Superbad, which developed a wild cult following, Lakeview Terrace, with Samuel L. Jackson, and Leatherheads, a football movie starring George Clooney.
He also was actively involved in the independent film scene and dabbled with music.
Loneker, who turned 46 on Wednesday and was a substitute teacher in the Lawrence School District, fought his battle with cancer since at least last December and did so with the kind of positive attitude and spirit he was known to have throughout his life.
After starring at KU from 1989-92 — a stint that include all-Big 8 honors during his final season in crimson and blue and a trip to the 1992 Aloha Bowl, Loneker joined the Los Angeles/St. Louis Rams and played in 19 games, including five starts, with the franchise from 1993-95.
Rest In Peace to my hero, warrior and idol. My father has passed this morning and put up a hell of a fight. Appreciate the love.
I remember watching Superbad awhile back and seeing him and thinking "this guy looks really familiar." Well there it is, he was a KU football player. I also saw him a few times at the Ranch when it was still up and running. Very sad news though.
What a huge, huge loss. A first-class guy in every way.
Sorry for your loss. He seemed like a great guy.
I remember him from Out of Sight with George Clooney and Jennifer Lopez (as well as from KU football of course). My condolences to the Loneker family.
I've known Lonny since his playing days when he would come into The Jayhawk Cafe w Chris Maumalanga all the time. Extremely nice guy and a true Jayhawk. He and I had a chance to talk some over the last 6-8 months on FB. I knew he was sick but didn't know how bad it really was. Super talented on the field and off - Rest easy my friend!
So sad to hear this. Condolences to all family and friends. I watched him play, loved his attitude and truly appreciated his commitment to Jayhawk football. Rest easy big fella'.
So young, so sad. My thoughts are with the Loneker family.
Sad. My heart goes out to Keith and family.


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Tsygankin DV in a small country (part 1)
By Редакция | Video | 24 July, 2015
A trip to the village Mokshaley (Republic of Mordovia)
Dmitry Vasilyevich Tsygankin – a bright representative of generation, for youth who last war. He came to Konigsberg, was awarded the Order of the Patriotic War II degree, the medal «For Courage».
DV Tsygankin born October 22, 1925 in with. Mokshaley Mokshaleyskoy parish Saransk County Penza province (now – Chamzinsky District of the Republic of Mordovia).
Candidate of Philology (1958), Doctor of Philology (1978). Associate Professor (1961), Professor (1979). Double «Laureate of the Republic of Mordovia State Prize in Science and Technology» (2000, 2005), Laureate of the Prize of the Republic of Mordovia (2005).
#Cygankin ,#Mokshalej ,#Mordovia


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Creative Arts & Design, Valley of the Arts - Creative minds seek Greater Palm Springs for work and play
Valley of the Arts
Creative minds seek Greater Palm Springs for work and play
Joan McKenna September 23, 2013 Vision, Watch & Listen - Vision 0 Comments
World-class art exhibitions, national touring and locally produced theater, classical to contemporary music concerts, couture fashion shows, architecture and design tours, film festivals, and culinary experiences with top chefs weave a culture-rich tapestry for the creative set to enjoy as a spectator or pursue as a career.
Celebrating its 75th year, Palm Springs Art Museum houses a wide-ranging permanent collection of contemporary and Modern art, studio glass, and Native American artifacts, as well as four classrooms, an artists' center, 85-seat lecture hall, and the 433-seat Annenberg Theater in its main venue in downtown Palm Springs. Its new 8,400-square-foot location in Palm Desert, The Galen, mounts changing exhibitions, and includes the Faye Sarkowsky Sculpture Garden and a community education wing.
Art aficionados can stroll among more than 25 galleries on El Paseo in Palm Desert. Palm Springs boasts a vibrant art scene as well, peaking each February with the Palm Springs Fine Art Fair at Palm Springs Convention Center.
La Quinta Arts Festival, named by Art Fair SourceBook as the top U.S. arts festival, unfolds in March, while La Quinta Arts Foundation promotes one-day Art Under the Umbrella shows, with live entertainment, throughout the season.
Other offerings and venues include Sunnylands Center and Gardens, which hosts rotating exhibitions; Indio's Southwest Arts Festival in January; Indian Wells Arts Festival and Desert Mountains Art Fair (Palm Desert) in April; and Rancho Mirage Art Affaire in November; as well as works from emerging artists at the 18,000-square-foot Coachella Valley Arts Center in downtown Indio.
Palm Springs Modernism Week, held every February, draws architecture and design enthusiasts from around the globe. The celebration of the midcentury aesthetic includes more than 100 events, such as exhibitions, tours, films, lectures, receptions, and parties. The fall kickoff — a "mini Modernism Week" — incorporates additional architecture tours and events. Many organizations get in the game: the museum's Architecture and Design Council, Palm Springs Modernism Committee, Palm Springs Preservation Foundation, and others.
The popular event has breathed life into Uptown Palm Springs, which has fashioned itself as a design district, and inspired a community of architects, interior designers, event planners, and Realtors who specialize in perpetuating the modern vibe.
Palm Springs Art Museum has begun transforming the midcentury modern, E. Stewart Williams-designed Santa Fe Federal Savings & Loan building into the Architecture and Design Center, Edwards Harris Pavilion in downtown Palm Springs.
The museum's Annenberg Theater opens the season big (Tommy Tune leads the 2013-2014 schedule) and continues with intimate cabaret shows, live theater, and much more. At McCallum Theatre in Palm Desert, audiences enjoy fully staged Broadway musicals, such as Hello, Dolly! and Man of La Mancha, and world-class dance, opera, jazz, comedy, magic, and even acrobatic shows.
Palm Springs Opera Guild, OperaArts, and Desert Symphony fill the bill with classical music programming, while local theater productions pack the schedule all season at Indio Performing Arts Center, Indian Wells Theater, Palm Canyon Theatre, Black Box Theater, Pollock Theatre at College of the Desert, and the new Helene Galen Theatre for the Performing Arts in Rancho Mirage.
The annual Coachella Valley Music and Arts Festival, known simply as "Coachella," has expanded to two weekends each spring at Eldorado Polo Club in Indio, attracting major music headliners from around the globe and generating more than $250 million in local economic impact. The annual Stagecoach festival, also held at Eldorado, features country music's top artists.
Casinos also bring in high-profile music and comedy acts.
Palm Springs International Film Festival, possibly the area's most prestigious entertainment event, kicks off the film awards season in January, drawing A-list stars to the red carpet at Palm Springs Convention Center. In June, the festival's popular Palm Springs International ShortFest offers forums, screening, and festivities for film buffs. Other film events include the American Documentary Film Festival, the Native American FilmFest, Arthur Lyons Film Noir Festival, Cinema Diverse: The Palm Springs Gay and Lesbian Film Festival, and the new AMFM Fest, featuring films, music, and art.
In addition to programming, the area offers film producers an ideal shooting location. Several cities and organizations provide location incentives and support.
FASHION, FOOD, AND WINE
Fashion enthusiasts converge on Palm Desert every March for Fashion Week El Paseo, showcasing the newest collections by emerging and established West Coast designers, such as Beverly Hills couturier Gilbert Chagoury, Palm Springs' homegrown Michael Costello, and many others. Highlights include runway shows, the Le Chien benefit show for the local humane society, trunk shows, and parties galore.
On the heels of Fashion Week, foodies and wine lovers enjoy the three-day Food+Wine Festival Palm Desert, a celebration of culinary finery with celebrity and Jame Beard award-winning chefs, grand tastings, and demonstrations.
• Total annual salaries in creative sector: $372 million
• Total self-employed receipts: $308+ million
• 1,000 creative businesses in the Greater Palm Springs area
• 9,000 creative employees (does not include freelance and contract talent)
• More than 10,000 independent contractors and creative artists
Source: 2012 Creative California Desert Economic Study
WORKFORCE EXCELLENCE
Greater Palm Springs has a creative workforce of more than 19,000 people working in film, advertising, marketing, publishing, video, television, architecture, performing and visual arts, music, public relations, and the culinary arts. These fields contribute more than $1 billion locally in economic activity, according to the 2012 Creative California Desert Economic Study.
ArtsOasis, an initiative of the Coachella Valley Economic Partnership, is building the creative segment by supporting school programs and attracting high-paying jobs.
ArtsOasis works with Palm Springs Women in Film and Television International and CVEP's Workforce Excellence and Arts, Media and Entertainment Industry Councils to prepare students for creative careers. "Sixty percent of high school students live in families below the poverty level in this community," says Robert Stearns, president of ArtsOasis. "Many don't know they could have a job as a gaffer on a movie set. Here, they can learn about careers, graduate with a high school degree, and work in an exciting theater or film production capacity."
Others building the film segment include Independent Filmmakers of the Inland Empire, Palm Springs Film Office, Palm Springs Film Alliance, and Inland Empire Film Commission.
ArtsOasis is developing a web-based skills marketplace for the region, starting with media, television, and video production. The estimated economic impact from this sector is $25 million through 2018. Additional wages for the burgeoning workforce could hit $2.5 million through 2018.
All three local public school districts offer media arts programs, including the Digital Arts Technology Academy at Cathedral City High School and the Helene Galen Theatre for the Performing Arts at Rancho Mirage High School, which also plans a culinary arts career pathway.
College of the Desert offers several certificate and transfer programs, including visual arts, music, dance, digital design and production, theater, and culinary arts. Its new 13,948-square-foot visual arts facility in Palm Desert will open in early 2014. Meanwhile, the University of California, Riverside Palm Desert Graduate Center offers a masters of fine arts in creative writing and writing for the performing arts.
Meanwhile, creative types in the culinary realm gravitate to ShareKitchen, a Cathedral City-based nonprofit business incubator helping to develop, package, and market product and create food industry jobs. Visit www.sharekitchen.org
REALIZING A VISION
Meet Levi Vincent of pstalent.com, a virtual production and animation company that operates inside the Sony PlayStation Home network, with license agreements to develop entertainment platforms. Vincent is one of the first five graduates of the Coachella Valley Innovation Hub Accelerator Campus.
Meet Carole Sumner Krechman, CEO of CV Studios Entertainment Network, a digital multi-platform company that creates content for television, websites, Facebook, Twitter, and mobile apps.
Palm Desert International Dance Festival at McCallum Theatre
Valley of the Arts - Creative minds seek Greater Palm Springs for work and play
GREGORY BATARDON/MCCALUM THEATRE
Stagecoach country music festival
GREATER PALM SPRINGS CVB
Sunnylands Center and Gardens
MARK DAVIDSON/THE ANNENBERG FOUNDATION TRUST AT SUNNYLANDS
GARY BIND,MAN/FASHION WEEK EL PASEO
Arts and Culture in the Coachella Valley
Taking the Valley's Temperature
Tourism in the Coachella Valley
Health and Medicine in the Coachella Valley


8:30 a.m. – 4:00 p.m., closed between 12:30 p.m. – 1:30 p.m.
Open for arraignments on weekends and holidays.
Take I-787 south to the Madison Avenue exit. Make a left hand turn onto South Pearl Street. Go south on South Pearl street until the intersection of Morton Avenue, make a right on Morton Avenue. The court is located on One Morton Avenue - the Public Safety Building. Parking is available on the street.


Nigerians groan under what has become the toughest economic downturn in decades. The Buhari-led administration keeps promising palliatives and requesting understanding from the citizens but things having gotten out of hand. Bags of rice that used to go for under 10 thousand Naira have shot up to as much as 20 thousand Naira in certain parts of the country. One minister warns that prices could rise to as high as 40 thousand Naira by December this year! And to think that this is the main food staple of the large African country of 180 million people. One report conservatively puts rice importation at 365 billion Naira a year, not including local production.
The chant for change from the masses has become a dirge of death. Enough of the talk and semantics. President Buhari's government needs to work fast. What good is a corruption fight that leaves the innocent populace dead from starvation?


Here as your request! Simple but healthy food. It can take you only 10 minutes or less to do this meal. Try it and let me know. I'm looking forward to see your stir fried.
Tip: look for very fresh vegetable & fry in very hot oil/pan!
If you like it spicy, you can add fresh ground chili for 1-2 chili with garlic.
Next Post News: New menu selection is coming next week!


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Announcing Our 2021 Season
The Princeton Festival's
17th Annual Season:
After some intense planning and careful discussion, we are excited to announce our 17th season, which will include both digital and in-person performances!
Our events are scheduled to take place June 3 – 20 on the beautiful grounds of the historic Morven Museum in Princeton, NJ. Musicians and singers will perform from the safety of the gorgeous new Stockton Education Center (pictured above), while patrons will be seated in socially distanced pods on the lawn.
There will be opportunities to picnic and engage with your fellow audience members at a distance, and most importantly, four of the performances will be live-streamed in high-definition to watch at home.
Tickets will go on sale in March 2021.
The Princeton Festival's 2021 season is grounded at once in the reality of an evolving public health crisis, along with the unique opportunities that situation provides for us to offer great performances to a worldwide audience of arts lovers.
Jazz (live)
The Princeton Festival is bringing a major jazz musician to perform on the beautiful grounds at Morven Museum. More details will be released soon, but our patrons will enjoy an evening of exquisite jazz in socially distanced pods that adhere to local COVID-19 guidelines.
Chamber Music (virtual)
Each season, The Princeton Festival presents the Concordia Chamber Players, a world-class ensemble under the artistic direction of cellist Michelle Djokic. Concordia brings together instrumentalists that perform at arts festivals such as Marlboro and Mostly Mozart. This year, we are pleased to present a specially commissioned video of the Concordia Chamber Players featuring chamber music favorites and some delightfully unusual works.
15th Annual Piano Competition (virtual)
Our 2021 Piano Competition has just opened to applications and, for the first time this year, it is open to pianists of all ages, thanks to a new Open Class category for artists ages 26 and older. The 2021 competition will be adjudicated by three outstanding educators and the finalists will be presented in a virtual format in June.
Baroque Chamber Series (live and virtual)
The enduring popularity of The Princeton Festival's Baroque series is a testament to its artistic and programming integrity. The 2021 Baroque series will feature two chamber concerts at the Morven Museum and Garden's Stockton Education Center. Our patrons will enjoy seeing and hearing the performances either in socially distanced pods or via livestream on the festival's website.
Favorite Opera Selections (live and virtual)
Opera is a foundational part of The Princeton Festival's programming. Our 2021 season includes two unique concerts of arias, duets, and trios from popular operas by Mozart, Verdi, Richard Strauss, and more. We will present both concerts at the Morven Museum and Garden's Stockton Education Center. Our patrons will enjoy the concerts either in socially distanced pods or via livestream on the festival's website.
Interdisciplinary Arts Event (virtual)
The Princeton Festival has commissioned an original interdisciplinary art video from Chicago-based Kosmologia, an artists' collective that develops works intersecting music with other arts and new technologies. Kosmologia's presentations may include singers, instrumentalists, composers, video and sound designers, digital graphic artists and dancers to explore vivid experiences of our community and our time.


THIS IS A LARGE ESTATE LOT FROM 1960'S. THE GROUP HAS INSTRUCTIONS, DECALS, PARTS STILL ON TREES, LOOSE PARTS AND ARE STILL IN EXCELLENT SHAPE.
YOU COULD USE THIS GREAT LOT FOR YOUR COLLECTION. The item "AMT 3 in 1 1960's BIG ESTATE GROUP Model Parts Lot Wheels" is in sale since Tuesday, March 21, 2017.
This item is in the category "Toys & Hobbies\Models & Kits\Automotive\Vintage". The seller is "dlaumbach" and is located in Burnsville, Minnesota. This item can be shipped worldwide.


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Diode Silicon Lattice. How Stuff Works - 2005 Most materials fall into two categories: conductors and insulators. These materials conduct…
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The advent of the semiconductor has revolutionised our lives, since it is the basis of all integrated circuits and microprocessors…
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The PN Junction Diode The effect described in the previous tutorial is achieved without any external voltage being applied to the actual…
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The idea about this page is that anybody who came into contact with the Series 32 family can tell his or her experience here. Udo…
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Hidden inside nearly every modern electronic is a technology - called power electronics - that is quietly making our world run. Yet…
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WASHINGTON—Nov. 2, 2015—The Semiconductor Industry Association (SIA), representing U.S. leadership in semiconductor manufacturing…
Rothschild Semiconductor
Along with the Rockefellers and Bushs, the Rothschilds are the most well-known Illuminati family. Few people know that there really…
Physics of Semiconductors
The Physics of Semiconductors contains ample material for a comprehensive upper-level undergraduate or beginning graduate course, guiding…
Definition of "semiconductor diode
The most common type of diode in use today is a semiconductor diode that is equipped with p-n junction for connection between two…
Applications of Semiconductor diodes
C. F. Bohman, The laser and microsoldering, society of Manufacturing Engineers, Tech. paper No. AD74-810 Mich. 48128 (1974) p. 19…
Diodo Semiconductor
Comprende cómo funciona un diodo. Un diodo está compuesto de un semiconductor tipo N unido a un semiconductor tipo P. El semiconductor…
Interesting fact
Did you know that there are some games that can be used by scientists to spawn the research on human reactions? The World of Craft with a bug that allowed players to infect other players and to kill each low-level palyer is the one. Also, the bug reflected the real disease models exiting. On gamerbolt, you will find everything you need to know about the world of video games.
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