Elucidating the Mesocarp Drupe Transcriptome of Açai (Euterpe oleracea Mart.): An Amazonian Tree Palm Producer of Bioactive Compounds.

Euterpe oleracea palm, endemic to the Amazon region, is well known for açai, a fruit violet beverage with nutritional and medicinal properties. During E. oleracea fruit ripening, anthocyanin accumulation is not related to sugar production, contrarily to grape and blueberry. Ripened fruits have a high content of anthocyanins, isoprenoids, fibers, and proteins, and are poor in sugars. E. oleracea is proposed as a new genetic model for metabolism partitioning in the fruit. Approximately 255 million single-end-oriented reads were generated on an Ion Proton NGS platform combining fruit cDNA libraries at four ripening stages. The de novo transcriptome assembly was tested using six assemblers and 46 different combinations of parameters, a pre-processing and a post-processing step. The multiple k-mer approach with TransABySS as an assembler and Evidential Gene as a post-processer have shown the best results, with an N50 of 959 bp, a read coverage mean of 70x, a BUSCO complete sequence recovery of 36% and an RBMT of 61%. The fruit transcriptome dataset included 22,486 transcripts representing 18 Mbp, of which a proportion of 87% had significant homology with other plant sequences. Approximately 904 new EST-SSRs were described, and were common and transferable to Phoenix dactylifera and Elaeis guineensis, two other palm trees. The global GO classification of transcripts showed similar categories to that in P. dactylifera and E. guineensis fruit transcriptomes. For an accurate annotation and functional description of metabolism genes, a bioinformatic pipeline was developed to precisely identify orthologs, such as one-to-one orthologs between species, and to infer multigenic family evolution. The phylogenetic inference confirmed an occurrence of duplication events in the Arecaceae lineage and the presence of orphan genes in E. oleracea. Anthocyanin and tocopherol pathways were annotated entirely. Interestingly, the anthocyanin pathway showed a high number of paralogs, similar to in grape, whereas the tocopherol pathway exhibited a low and conserved gene number and the prediction of several splicing forms. The release of this exhaustively annotated molecular dataset of E. oleracea constitutes a valuable tool for further studies in metabolism partitioning and opens new great perspectives to study fruit physiology with açai as a model.

Deep evolutionary origin of limb and fin regeneration.

Salamanders and lungfishes are the only sarcopterygians (lobe-finned vertebrates) capable of paired appendage regeneration, regardless of the amputation level. Among actinopterygians (ray-finned fishes), regeneration after amputation at the fin endoskeleton has only been demonstrated in polypterid fishes (Cladistia). Whether this ability evolved independently in sarcopterygians and actinopterygians or has a common origin remains unknown. Here we combine fin regeneration assays and comparative RNA-sequencing (RNA-seq) analysis of Polypterus and axolotl blastemas to provide support for a common origin of paired appendage regeneration in Osteichthyes (bony vertebrates). We show that, in addition to polypterids, regeneration after fin endoskeleton amputation occurs in extant representatives of 2 other nonteleost actinopterygians: the American paddlefish (Chondrostei) and the spotted gar (Holostei). Furthermore, we assessed regeneration in 4 teleost species and show that, with the exception of the blue gourami (Anabantidae), 3 species were capable of regenerating fins after endoskeleton amputation: the white convict and the oscar (Cichlidae), and the goldfish (Cyprinidae). Our comparative RNA-seq analysis of regenerating blastemas of axolotl and Polypterus reveals the activation of common genetic pathways and expression profiles, consistent with a shared genetic program of appendage regeneration. Comparison of RNA-seq data from early Polypterus blastema to single-cell RNA-seq data from axolotl limb bud and limb regeneration stages shows that Polypterus and axolotl share a regeneration-specific genetic program. Collectively, our findings support a deep evolutionary origin of paired appendage regeneration in Osteichthyes and provide an evolutionary framework for studies on the genetic basis of appendage regeneration.

Salamander-like tail regeneration in the West African lungfish.

Salamanders, frog tadpoles and diverse lizards have the remarkable ability to regenerate tails. Palaeontological data suggest that this capacity is plesiomorphic, yet when the developmental and genetic architecture of tail regeneration arose is poorly understood. Here, we show morphological and molecular hallmarks of tetrapod tail regeneration in the West African lungfish Protopterus annectens, a living representative of the sister group of tetrapods. As in salamanders, lungfish tail regeneration occurs via the formation of a proliferative blastema and restores original structures, including muscle, skeleton and spinal cord. In contrast with lizards and similar to salamanders and frogs, lungfish regenerate spinal cord neurons and reconstitute dorsoventral patterning of the tail. Similar to salamander and frog tadpoles, Shh is required for lungfish tail regeneration. Through RNA-seq analysis of uninjured and regenerating tail blastema, we show that the genetic programme deployed during lungfish tail regeneration maintains extensive overlap with that of tetrapods, with the upregulation of genes and signalling pathways previously implicated in amphibian and lizard tail regeneration. Furthermore, the lungfish tail blastema showed marked upregulation of genes encoding post-transcriptional RNA processing components and transposon-derived genes. Our results show that the developmental processes and genetic programme of tetrapod tail regeneration were present at least near the base of the sarcopterygian clade and establish the lungfish as a valuable research system for regenerative biology.

First genomic microsatellite markers developed for Platonia insignis (Clusiaceae), a Brazilian fruit tree.

Platonia insignis is a fruit tree native of Brazil with allogamous and asexual reproduction. The production of fruits is mainly obtained by exploitation of natural populations and the impact of genetic structuring on plant production may be evaluated. For this purpose, codominant and multiallelic markers such as microsatellite are the most suitable, but they need to be developed for this species. Thus, the aim of this work was to develop and validate microsatellite markers for P. insignis. We used Roche 454 GS FLX sequencing platform of a single P. insignis genotype and 1702 microsatellite sequences were identified. Based on some pre-requisites, we could develop 50 primer pairs to be tested. Twenty-two primer pairs successfully amplified fragments and they were tested in 31 genotypes of P. insignis that belong to a germplasm bank and were sampled in the northeast of Pará State, Brazil. Thirteen primers were polymorphic and the number of alleles per loci varied from 5 (PI18 and PI27) to 2 (PI08, PI25, PI31, PI33 and PI 37). Expected heterozygosity (HE) varied from 0.74 (PI27) to 0.12 (PI31) and observed heterozygosity (HO) varied from 1.00 (PI25) to 0.00 (PI08, PI31, PI33 and PI37). Principal coordinates could separate the genotypes of P. insignis in clusters and we can conclude that the primers can estimate the genetic diversity of P. insignis populations.

Inhibition of Phytosterol Biosynthesis by Azasterols.

Inhibitors of enzymes in essential cellular pathways are potent probes to decipher intricate physiological functions of biomolecules. The analysis of Arabidopsis thaliana sterol profiles upon treatment with a series of azasterols reveals a specific in vivo inhibition of SMT2, a plant sterol-C-methyltransferase acting as a branch point between the campesterol and sitosterol biosynthetic segments in the pathway. Side chain azasteroids that modify sitosterol homeostasis help to refine its particular function in plant development.

Secure and Sustainable Sourcing of Plant Tissues for the Exhaustive Exploration of Their Chemodiversity.

The main challenge of plant chemical diversity exploration is how to develop tools to study exhaustively plant tissues. Their sustainable sourcing is a limitation as bioguided strategies and dereplication need quite large amounts of plant material. We examine if alternative solutions could overcome these difficulties by obtaining a secure, sustainable, and scalable source of tissues able to biosynthesize an array of metabolites. As this approach would be as independent of the botanical origin as possible, we chose eight plant species from different families. We applied a four steps culture establishment procedure, monitoring targeted compounds through mass spectrometry-based analytical methods. We also characterized the capacities of leaf explants in culture to produce diverse secondary metabolites. In vitro cultures were successfully established for six species with leaf explants still producing a diversity of compounds after the culture establishment procedure. Furthermore, explants from leaves of axenic plantlets were also analyzed. The detection of marker compounds was confirmed after six days in culture for all tested species. Our results show that the first stage of this approach aiming at easing exploration of plant chemodiversity was completed, and leaf tissues could offer an interesting alternative providing a constant source of natural compounds.

Metabolism and Biological Activities of 4-Methyl-Sterols.

4,4-Dimethylsterols and 4-methylsterols are sterol biosynthetic intermediates (C4-SBIs) acting as precursors of cholesterol, ergosterol, and phytosterols. Their accumulation caused by genetic lesions or biochemical inhibition causes severe cellular and developmental phenotypes in all organisms. Functional evidence supports their role as meiosis activators or as signaling molecules in mammals or plants. Oxygenated C4-SBIs like 4-carboxysterols act in major biological processes like auxin signaling in plants and immune system development in mammals. It is the purpose of this article to point out important milestones and significant advances in the understanding of the biogenesis and biological activities of C4-SBIs.

The non-visual opsins: eighteen in the ancestor of vertebrates, astonishing increase in ray-finned fish, and loss in amniotes.

Non-visual opsins were discovered in the early 1990s. These genes play roles in circadian rhythm in mammals, seasonal reproduction in birds, light avoidance in amphibian larvae, and neural development in fish. However, the interpretation of such studies and the success of future work are compromised by the fact that non-visual opsin repertoires have not been properly characterized in any of these lineages. Here, we show that non-visual opsins from tetrapods and ray-finned fish are distributed among 18 monophyletic subfamilies. An amphibian sequence occurs in every subfamily, whereas mammalian orthologs occur in only seven. Species in the major ray-finned fish lineages, Holostei, Osteoglossomorpha, Otomorpha, Protacanthopterygii, and Neoteleostei, have large numbers of non-visual opsins (22-32 genes) as a result of gene duplication events including, but not limited to, the teleost genome duplication (TGD). In contrast to visual opsins, where lineage-specific duplication is common, the ray-finned fish non-visual opsin repertoire appears to have stabilized shortly after the TGD event and consequently even distantly related species have repertoires of similar size and composition. Most non-visual opsins have been named without the benefit of a phylogenetic perspective and, accordingly, major revisions are proposed.

Modes-of-action of antifungal compounds: Stressors and (target-site-specific) toxins, toxicants, or toxin-stressors.

Fungi and antifungal compounds are relevant to the United Nation's Sustainable Development Goals. However, the modes-of-action of antifungals-whether they are naturally occurring substances or anthropogenic fungicides-are often unknown or are misallocated in terms of their mechanistic category. Here, we consider the most effective approaches to identifying whether antifungal substances are cellular stressors, toxins/toxicants (that are target-site-specific), or have a hybrid mode-of-action as toxin-stressors (that induce cellular stress yet are target-site-specific). This newly described 'toxin-stressor' category includes some photosensitisers that target the cell membrane and, once activated by light or ultraviolet radiation, cause oxidative damage. We provide a glossary of terms and a diagrammatic representation of diverse types of stressors, toxic substances, and toxin-stressors, a classification that is pertinent to inhibitory substances not only for fungi but for all types of cellular life. A decision-tree approach can also be used to help differentiate toxic substances from cellular stressors (Curr Opin Biotechnol 2015 33: 228-259). For compounds that target specific sites in the cell, we evaluate the relative merits of using metabolite analyses, chemical genetics, chemoproteomics, transcriptomics, and the target-based drug-discovery approach (based on that used in pharmaceutical research), focusing on both ascomycete models and the less-studied basidiomycete fungi. Chemical genetic methods to elucidate modes-of-action currently have limited application for fungi where molecular tools are not yet available; we discuss ways to circumvent this bottleneck. We also discuss ecologically commonplace scenarios in which multiple substances act to limit the functionality of the fungal cell and a number of as-yet-unresolved questions about the modes-of-action of antifungal compounds pertaining to the Sustainable Development Goals.

High-throughput sequencing of black pepper root transcriptome.

BACKGROUND: Black pepper (Piper nigrum L.) is one of the most popular spices in the world. It is used in cooking and the preservation of food and even has medicinal properties. Losses in production from disease are a major limitation in the culture of this crop. The major diseases are root rot and foot rot, which are results of root infection by Fusarium solani and Phytophtora capsici, respectively. Understanding the molecular interaction between the pathogens and the host's root region is important for obtaining resistant cultivars by biotechnological breeding. Genetic and molecular data for this species, though, are limited. In this paper, RNA-Seq technology has been employed, for the first time, to describe the root transcriptome of black pepper. RESULTS: The root transcriptome of black pepper was sequenced by the NGS SOLiD platform and assembled using the multiple-k method. Blast2Go and orthoMCL methods were used to annotate 10338 unigenes. The 4472 predicted proteins showed about 52% homology with the Arabidopsis proteome. Two root proteomes identified 615 proteins, which seem to define the plant's root pattern. Simple-sequence repeats were identified that may be useful in studies of genetic diversity and may have applications in biotechnology and ecology. CONCLUSIONS: This dataset of 10338 unigenes is crucially important for the biotechnological breeding of black pepper and the ecogenomics of the Magnoliids, a major group of basal angiosperms.

Phytosterol Profiles, Genomes and Enzymes - An Overview.

The remarkable diversity of sterol biosynthetic capacities described in living organisms is enriched at a fast pace by a growing number of sequenced genomes. Whereas analytical chemistry has produced a wealth of sterol profiles of species in diverse taxonomic groups including seed and non-seed plants, algae, phytoplanktonic species and other unicellular eukaryotes, functional assays and validation of candidate genes unveils new enzymes and new pathways besides canonical biosynthetic schemes. An overview of the current landscape of sterol pathways in the tree of life is tentatively assembled in a series of sterolotypes that encompass major groups and provides also peculiar features of sterol profiles in bacteria, fungi, plants, and algae.

Distinct triterpene synthases in the laticifers of Euphorbia lathyris.

Euphorbia lathyris was proposed about fifty years ago as a potential agroenergetic crop. The tremendous amounts of triterpenes present in its latex has driven investigations for transforming this particular biological fluid into an industrial hydrocarbon source. The huge accumulation of terpenes in the latex of many plant species represent a challenging question regarding cellular homeostasis. In fact, the enzymes, the mechanisms and the controllers that tune the amount of products accumulated in specialized compartments (to fulfill ecological roles) or deposited at important sites (as essential factors) are not known. Here, we have isolated oxidosqualene cyclases highly expressed in the latex of Euphorbia lathyris. This triterpene biosynthetic machinery is made of distinct paralogous enzymes responsible for the massive accumulation of steroidal and non-steroidal tetracyclic triterpenes. More than eighty years after the isolation of butyrospermol from shea butter (Heilbronn IM, Moffet GL, and Spring FS J. Chem. Soc. 1934, 1583), a butyrospermol synthase is characterized in this work using yeast and in folia heterologous expression assays.

Tetrapod limb and sarcopterygian fin regeneration share a core genetic programme.

Salamanders are the only living tetrapods capable of fully regenerating limbs. The discovery of salamander lineage-specific genes (LSGs) expressed during limb regeneration suggests that this capacity is a salamander novelty. Conversely, recent paleontological evidence supports a deeper evolutionary origin, before the occurrence of salamanders in the fossil record. Here we show that lungfishes, the sister group of tetrapods, regenerate their fins through morphological steps equivalent to those seen in salamanders. Lungfish de novo transcriptome assembly and differential gene expression analysis reveal notable parallels between lungfish and salamander appendage regeneration, including strong downregulation of muscle proteins and upregulation of oncogenes, developmental genes and lungfish LSGs. MARCKS-like protein (MLP), recently discovered as a regeneration-initiating molecule in salamander, is likewise upregulated during early stages of lungfish fin regeneration. Taken together, our results lend strong support for the hypothesis that tetrapods inherited a bona fide limb regeneration programme concomitant with the fin-to-limb transition.

Enzymological properties of sterol-C4-methyl-oxidase of yeast sterol biosynthesis.

Despite genes of the sterol methyl-oxidase component (SMO) of the sterol-C4-demethylation multienzymatic complex have been identified in a variety of organisms and the key role played by SMO in yeast sterol biosynthesis, the enzymological properties of yeast SMO have not been investigated. An enzymatic assay for measuring specifically sterol 4alpha-methyl-oxidase activity in Saccharomyces cerevisiae has been developed for the first time by using [14C]-4,4-dimethyl-zymosterol as substrate. It allowed enzymatically formed C4 mono- and di-demethylated products to be characterized as well as two novel C4-hydroxymethyl-zymosterol derivatives to be identified as immediate oxidative metabolites by the yeast 4,4-dimethyl-zymosterol 4alpha-methyl-oxidase (ScSMO). The properties of microsomal ScSMO have been established with respect to cofactor requirements and kinetics and the substrate selectivity examined with a number of 4,4-dimethyl- and 4alpha-methyl-sterols. Remarkably, ScSMO showed very low activity with 24-methylene-24-dihydrocycloartenol, the natural substrate of maize 4,4-dimethyl-sterol-C4-methyl-oxidase. Conversely, maize sterol-C4-methyl-oxidases showed extremely reduced activity with the natural substrate of ScSMO. The previously described antifungal agent, 6-amino-2-n-pentylbenzothiazole was shown to directly inhibit the microsomal ScSMO activity in vitro. The yeast system was more than 500 times more sensitive to this derivative than the maize systems. These distinct substrate specificities and inhibitor sensitivities between yeast and plant sterol-4alpha-methyl-oxidases probably reflect diversity in the structure of their active sites in relation to the distinct sterol biosynthetic pathways.

Plant sterol biosynthesis: identification of two distinct families of sterol 4alpha-methyl oxidases.

In plants, the conversion of cycloartenol into functional phytosterols requires the removal of the two methyl groups at C-4 by an enzymic complex including a sterol 4alpha-methyl oxidase (SMO). We report the cloning of candidate genes for SMOs in Arabidopsis thaliana, belonging to two distinct families termed SMO1 and SMO2 and containing three and two isoforms respectively. SMO1 and SMO2 shared low sequence identity with each other and were orthologous to the ERG25 gene from Saccharomyces cerevisiae which encodes the SMO. The plant SMO amino acid sequences possess all the three histidine-rich motifs (HX3H, HX2HH and HX2HH), characteristic of the small family of membrane-bound non-haem iron oxygenases that are involved in lipid oxidation. To elucidate the precise functions of SMO1 and SMO2 gene families, we have reduced their expression by using a VIGS (virus-induced gene silencing) approach in Nicotiana benthamiana. SMO1 and SMO2 cDNA fragments were inserted into a viral vector and N. benthamiana inoculated with the viral transcripts. After silencing with SMO1, a substantial accumulation of 4,4-dimethyl-9beta,19-cyclopropylsterols (i.e. 24-methylenecycloartanol) was obtained, whereas qualitative and quantitative levels of 4alpha-methylsterols were not affected. In the case of silencing with SMO2, a large accumulation of 4alpha-methyl-Delta7-sterols (i.e. 24-ethylidenelophenol and 24-ethyllophenol) was found, with no change in the levels of 4,4-dimethylsterols. These clear and distinct biochemical phenotypes demonstrate that, in contrast with animals and fungi, in photosynthetic eukaryotes, these two novel families of cDNAs are coding two distinct types of C-4-methylsterol oxidases controlling the level of 4,4-dimethylsterol and 4alpha-methylsterol precursors respectively.

High-Throughput Sequencing of miRNAs Reveals a Tissue Signature in Gastric Cancer and Suggests Novel Potential Biomarkers.

Gastric cancer has a high incidence and mortality rate worldwide; however, the use of biomarkers for its clinical diagnosis remains limited. The microRNAs (miRNAs) are biomarkers with the potential to identify the risk and prognosis as well as therapeutic targets. We performed the ultradeep miRnomes sequencing of gastric adenocarcinoma and gastric antrum without tumor samples. We observed that a small set of those samples were responsible for approximately 80% of the total miRNAs expression, which might represent a miRNA tissue signature. Additionally, we identified seven miRNAs exhibiting significant differences, and, of these, hsa-miR-135b and hsa-miR-29c were able to discriminate antrum without tumor from gastric cancer regardless of the histological type. These findings were validated by quantitative real-time polymerase chain reaction. The results revealed that hsa-miR-135b and hsa-miR-29c are potential gastric adenocarcinoma occurrence biomarkers with the ability to identify individuals at a higher risk of developing this cancer, and could even be used as therapeutic targets to allow individualized clinical management.

MiRNA expression profile for the human gastric antrum region using ultra-deep sequencing.

BACKGROUND: MicroRNAs are small non-coding nucleotide sequences that regulate gene expression. These structures are fundamental to several biological processes, including cell proliferation, development, differentiation and apoptosis. Identifying the expression profile of microRNAs in healthy human gastric antrum mucosa may help elucidate the miRNA regulatory mechanisms of the human stomach. METHODOLOGY/PRINCIPAL FINDINGS: A small RNA library of stomach antrum tissue was sequenced using high-throughput SOLiD sequencing technology. The total read count for the gastric mucosa antrum region was greater than 618,000. After filtering and aligning using with MirBase, 148 mature miRNAs were identified in the gastric antrum tissue, totaling 3,181 quality reads; 63.5% (2,021) of the reads were concentrated in the eight most highly expressed miRNAs (hsa-mir-145, hsa-mir-29a, hsa-mir-29c, hsa-mir-21, hsa-mir-451a, hsa-mir-192, hsa-mir-191 and hsa-mir-148a). RT-PCR validated the expression profiles of seven of these highly expressed miRNAs and confirmed the sequencing results obtained using the SOLiD platform. CONCLUSIONS/SIGNIFICANCE: In comparison with other tissues, the antrum's expression profile was unique with respect to the most highly expressed miRNAs, suggesting that this expression profile is specific to stomach antrum tissue. The current study provides a starting point for a more comprehensive understanding of the role of miRNAs in the regulation of the molecular processes of the human stomach.

High-throughput sequencing of a South American Amerindian.

The emergence of next-generation sequencing technologies allowed access to the vast amounts of information that are contained in the human genome. This information has contributed to the understanding of individual and population-based variability and improved the understanding of the evolutionary history of different human groups. However, the genome of a representative of the Amerindian populations had not been previously sequenced. Thus, the genome of an individual from a South American tribe was completely sequenced to further the understanding of the genetic variability of Amerindians. A total of 36.8 giga base pairs (Gbp) were sequenced and aligned with the human genome. These Gbp corresponded to 95.92% of the human genome with an estimated miscall rate of 0.0035 per sequenced bp. The data obtained from the alignment were used for SNP (single-nucleotide) and INDEL (insertion-deletion) calling, which resulted in the identification of 502,017 polymorphisms, of which 32,275 were potentially new high-confidence SNPs and 33,795 new INDELs, specific of South Native American populations. The authenticity of the sample as a member of the South Native American populations was confirmed through the analysis of the uniparental (maternal and paternal) lineages. The autosomal comparison distinguished the investigated sample from others continental populations and revealed a close relation to the Eastern Asian populations and Aboriginal Australian. Although, the findings did not discard the classical model of America settlement; it brought new insides to the understanding of the human population history. The present study indicates a remarkable genetic variability in human populations that must still be identified and contributes to the understanding of the genetic variability of South Native American populations and of the human populations history.