Unravelling Chemical Composition of Agave Spines: News from Agave fourcroydes Lem.

Spines are key plant modifications developed to deal against herbivores; however, its physical structure and chemical composition have been little explored in plant species. Here, we took advantage of high-throughput chromatography to characterize chemical composition of Agave fourcroydes Lem. spines, a species traditionally used for fiber extraction. Analyses of structural carbohydrate showed that spines have lower cellulose content than leaf fibers (52 and 72%, respectively) but contain more than 2-fold the hemicellulose and 1.5-fold pectin. Xylose and galacturonic acid were enriched in spines compared to fibers. The total lignin content in spines was 1.5-fold higher than those found in fibers, with elevated levels of syringyl (S) and guaiacyl (G) subunits but similar S/G ratios within tissues. Metabolomic profiling based on accurate mass spectrometry revealed the presence of phenolic compounds including quercetin, kaempferol, (+)-catechin, and (-)-epicatechin in A. fourcroydes spines, which were also detected in situ in spines tissues and could be implicated in the color of these plants' structures. Abundance of (+)-catechins could also explain proanthocyanidins found in spines. Agave spines may become a plant model to obtain more insights about cellulose and lignin interactions and condensed tannin deposition, which is valuable knowledge for the bioenergy industry and development of naturally dyed fibers, respectively

Entamoeba histolytica Up-Regulates MicroRNA-643 to Promote Apoptosis by Targeting XIAP in Human Epithelial Colon Cells

MicroRNAs (miRNAs) are small non-coding RNAs that function as negative regulators of gene expression. Recent evidences suggested that host cells miRNAs are involved in the progression of infectious diseases, but its role in amoebiasis remains largely unknown. Here, we reported an unexplored role for miRNAs of human epithelial colon cells during the apoptosis induced by Entamoeba histolytica. We demonstrated for the first time that SW-480 colon cells change their miRNAs profile in response to parasite exposure. Our data showed that virulent E. histolytica trophozoites induced apoptosis of SW-480 colon cells after 45 min interaction, which was associated to caspases-3 and -9 activation. Comprehensive profiling of 667 miRNAs using Taqman Low-Density Arrays showed that 6 and 15 miRNAs were significantly (FC > 1.5; p < 0.05) modulated in SW-480 cells after 45 and 75 min interaction with parasites, respectively. Remarkably, no significant regulation of the 6-miRNAs signature (miR-526b-5p, miR-150, miR-643, miR-615-5p, miR-525, and miR-409-3p) was found when SW-480 cells were exposed to non-virulent Entamoeba dispar. Moreover, we confirmed that miR-150, miR-643, miR-615-5p, and miR-525 exhibited similar regulation in SW-480 and Caco2 colon cells after 45 min interaction with trophozoites. Exhaustive bioinformatic analysis of the six-miRNAs signature revealed intricate miRNAs-mRNAs co-regulation networks in which the anti-apoptotic XIAP, API5, BCL2, and AKT1 genes were the major targets of the set of six-miRNAs. Of these, we focused in the study of functional relationships between miR-643, upregulated at 45 min interaction, and its predicted target X-linked inhibitor of apoptosis protein (XIAP). Interestingly, interplay of amoeba with SW-480 cells resulted in downregulation of XIAP consistent with apoptosis activation. More importantly, loss of function studies using antagomiRs showed that forced inhibition of miR-643 leads to restoration of XIAP levels and suppression of both apoptosis and caspases-3 and -9 activation. Congruently, mechanistic studies using luciferase reporter assays confirmed that miR-643 exerts a postranscripcional negative regulation of XIAP by targeting its 3′-UTR indicating that it's a downstream effector. In summary, we provide novel lines of evidence suggesting that early-branched eukaryote E. histolytica may promote apoptosis of human colon cells by modulating, in part, the host microRNome which highlight an unexpected role for miRNA-643/XIAP axis in the host cellular response to parasites infection

Life and Death of mRNA Molecules in Entamoeba histolytica

In eukaryotic cells, the life cycle of mRNA molecules is modulated in response to environmental signals and cell-cell communication in order to support cellular homeostasis. Capping, splicing and polyadenylation in the nucleus lead to the formation of transcripts that are suitable for translation in cytoplasm, until mRNA decay occurs in P-bodies. Although pre-mRNA processing and degradation mechanisms have usually been studied separately, they occur simultaneously and in a coordinated manner through protein-protein interactions, maintaining the integrity of gene expression. In the past few years, the availability of the genome sequence of Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, coupled to the development of the so-called “omics” technologies provided new opportunities for the study of mRNA processing and turnover in this pathogen. Here, we review the current knowledge about the molecular basis for splicing, 3′ end formation and mRNA degradation in amoeba, which suggest the conservation of events related to mRNA life throughout evolution. We also present the functional characterization of some key proteins and describe some interactions that indicate the relevance of cooperative regulatory events for gene expression in this human parasite

Phage Therapy for Crops: Concepts, Experimental and Bioinformatics Approaches to Direct Its Application

Phage therapy consists of applying bacteriophages, whose natural function is to kill specific bacteria. Bacteriophages are safe, evolve together with their host, and are environmentally friendly. At present, the indiscriminate use of antibiotics and salt minerals (Zn2+ or Cu2+) has caused the emergence of resistant strains that infect crops, causing difficulties and loss of food production. Phage therapy is an alternative that has shown positive results and can improve the treatments available for agriculture. However, the success of phage therapy depends on finding effective bacteriophages. This review focused on describing the potential, up to now, of applying phage therapy as an alternative treatment against bacterial diseases, with sustainable improvement in food production. We described the current isolation techniques, characterization, detection, and selection of lytic phages, highlighting the importance of complementary studies using genome analysis of the phage and its host. Finally, among these studies, we concentrated on the most relevant bacteriophages used for biocontrol of Pseudomonas spp., Xanthomonas spp., Pectobacterium spp., Ralstonia spp., Burkholderia spp., Dickeya spp., Clavibacter michiganensis, and Agrobacterium tumefaciens as agents that cause damage to crops, and affect food production around the world

Pollen Types Reveal Floral Diversity in Natural Honeys from Campeche, Mexico

The Yucatan Peninsula, located in southern Mexico, is a central honey-producing region with extraordinary biodiversity of melliferous plants. Approximately 900 plant species have been described as being a source of nectar and pollen for bees and other pollinators. They provide ecosystem services that help to keep plant biodiversity high and mitigate the effects of climate change. This study aimed to reveal the diversity of the pollen content in honey through a melissopalynological analysis of 22 honey samples collected in February–August 2021 from the north-central area of Campeche, Mexico. The extraction of pollen from the honey was carried out using standard methods for melissopalynological analysis. The honeys were classified by botanical origin to determine their floral sources and a diverse spectrum of 19 pollen types from 13 families was identified. Only eight were predominant: Milleria quinqueflora, Gymnopodium floribundum, Terminalia buceras, Amaranthus spinosus, Zea mays, Talisia floresii, Guazuma ulmifolia, and Croton icche. Our research shows the high quality of the honey analyzed and highlights the diversity and critical role of local melliferous flora and crops in beekeeping development in southern Mexico. The results in this study are useful for confirming the botanical origins of honey, generating information for designing nature conservation and agroecosystem management strategies, and increasing the knowledge of beekeepers in Campeche, Mexico

Single-Step Protocol for Isolating the Recombinant Extracellular Domain of the Luteinizing Hormone Receptor from the Ovis aries Testis

The luteinizing hormone receptor (LHR) is a glycoprotein member of the G protein-coupled receptors superfamily. It participates in corpus luteum formation and ovulation in females and acts in testosterone synthesis and spermatogenesis in males. In this study, we extracted RNA from sheep testicles and synthetized the cDNA to amplify the gene lhr-bed. This gene consists of 762 bp and encodes 273 amino acids of the extracellular domain of LHR. The lhr-bed was cloned into pJET1.2/blunt, then subcloned into pCOLD II, and finally, transformed in E. coli BL21 (DE3) cells. Because the induced rLHR-Bed protein was found in the insoluble fraction, we followed a modified purification protocol involving induction at 25 °C, subjection to denaturing conditions, and on-column refolding to increase solubility. We confirmed rLHR-Bed expression by means of Western blot and mass spectrometry analysis. It is currently known that the structure stem-loop 5′UTR on pCOLD II vector is stable at 15 °C. We predicted and obtained RNAfold stability at 25 °C. We successfully obtained the recombinant LHR extracellular domain, with protein yields of 0.2 mg/L, and purity levels of approximately 90%, by means of a single chromatographic purification step. The method described here may be used to obtain large quantities of rLHR-Bed in the future

Unravelling Chemical Composition of Agave Spines: News from Agave fourcroydes Lem.

International audienceSpines are key plant modifications developed to deal against herbivores; however, its physical structure and chemical composition have been little explored in plant species. Here, we took advantage of high-throughput chromatography to characterize chemical composition of Agave fourcroydes Lem. spines, a species traditionally used for fiber extraction. Analyses of structural carbohydrate showed that spines have lower cellulose content than leaf fibers (52 and 72%, respectively) but contain more than 2-fold the hemicellulose and 1.5-fold pectin. Xylose and galacturonic acid were enriched in spines compared to fibers. The total lignin content in spines was 1.5-fold higher than those found in fibers, with elevated levels of syringyl (S) and guaiacyl (G) subunits but similar S/G ratios within tissues. Metabolomic profiling based on accurate mass spectrometry revealed the presence of phenolic compounds including quercetin, kaempferol, (+)-catechin, and (−)-epicatechin in A. fourcroydes spines, which were also detected in situ in spines tissues and could be implicated in the color of these plants’ structures. Abundance of (+)-catechins could also explain proanthocyanidins found in spines. Agave spines may become a plant model to obtain more insights about cellulose and lignin interactions and condensed tannin deposition, which is valuable knowledge for the bioenergy industry and development of naturally dyed fibers, respectively

Transcriptome Mining Provides Insights into Cell Wall Metabolism and Fiber Lignification in Agave tequilana Weber.

Resilience of growing in arid and semiarid regions and a high capacity of accumulating sugar-rich biomass with low lignin percentages have placed Agave species as an emerging bioenergy crop. Although transcriptome sequencing of fiber-producing agave species has been explored, molecular bases that control wall cell biogenesis and metabolism in agave species are still poorly understood. Here, through RNAseq data mining, we reconstructed the cellulose biosynthesis pathway and the phenylpropanoid route producing lignin monomers in A. tequilana, and evaluated their expression patterns in silico and experimentally. Most of the orthologs retrieved showed differential expression levels when they were analyzed in different tissues with contrasting cellulose and lignin accumulation. Phylogenetic and structural motif analyses of putative CESA and CAD proteins allowed to identify those potentially involved with secondary cell wall formation. RT-qPCR assays revealed enhanced expression levels of AtqCAD5 and AtqCESA7 in parenchyma cells associated with extraxylary fibers, suggesting a mechanism of formation of sclerenchyma fibers in Agave similar to that reported for xylem cells in model eudicots. Overall, our results provide a framework for understanding molecular bases underlying cell wall biogenesis in Agave species studying mechanisms involving in leaf fiber development in monocots

mRNA Decay Proteins Are Targeted to poly(A)⁺ RNA and dsRNA-Containing Cytoplasmic Foci That Resemble P-Bodies in <em>Entamoeba histolytica</em>

<div><p>In higher eukaryotes, mRNA degradation and RNA-based gene silencing occur in cytoplasmic foci referred to as processing bodies (P-bodies). In protozoan parasites, the presence of P-bodies and their putative role in mRNA decay have yet to be comprehensively addressed. Identification of P-bodies might provide information on how mRNA degradation machineries evolved in lower eukaryotes. Here, we used immunofluorescence and confocal microscopy assays to investigate the cellular localization of mRNA degradation proteins in the human intestinal parasite <em>Entamoeba histolytica</em> and found evidence of the existence of P-bodies. Two mRNA decay factors, namely the <em>Eh</em>XRN2 exoribonuclease and the <em>Eh</em>DCP2 decapping enzyme, were localized in cytoplasmic foci in a pattern resembling P-body organization. Given that amoebic foci appear to be smaller and less rounded than those described in higher eukaryotes, we have named them “P-body-like structures”. These foci contain additional mRNA degradation factors, including the <em>Eh</em>CAF1 deadenylase and the <em>Eh</em>AGO2-2 protein involved in RNA interference. Biochemical analysis revealed that <em>Eh</em>CAF1 co-immunoprecipitated with <em>Eh</em>XRN2 but not with <em>Eh</em>DCP2 or <em>Eh</em>AGO2-2, thus linking deadenylation to 5′-to-3′ mRNA decay. The number of <em>Eh</em>CAF1-containing foci significantly decreased after inhibition of transcription and translation with actinomycin D and cycloheximide, respectively. Furthermore, results of RNA-FISH assays showed that (i) <em>Eh</em>CAF1 colocalized with poly(A)⁺ RNA and (ii) during silencing of the <em>Ehpc4</em> gene by RNA interference, <em>Eh</em>AGO2-2 colocalized with small interfering RNAs in cytoplasmic foci. Our observation of decapping, deadenylation and RNA interference proteins within P-body-like foci suggests that these structures have been conserved after originating in the early evolution of eukaryotic lineages. To the best of our knowledge, this is the first study to report on the localization of mRNA decay proteins within P-body-like structures in <em>E. histolytica</em>. Our findings should open up opportunities for deciphering the mechanisms of mRNA degradation and RNA-based gene silencing in this deep-branching eukaryote.</p> </div

mRNA expression profiles of genes for <i>E. histolytica</i> mRNA degradation proteins.

<p>Quantitative real-time PCR assays were performed to analyze the relative expression of representative mRNA degradation genes after heat shock, UV-C induced DNA damage and sodium nitroprusside treatments. For each triplicate experiment, the mean of the relative concentrations obtained for the tested mRNA were divided by the mean of the corresponding values obtained for endogenous ribosomal <i>L31</i> amplification. Each PCR experiment was carried out three times and three independent biological samples were analyzed. The relative expression (fold change) of mRNA degradation genes in the different treatments was calculated by the 2(−ΔΔCt method) using as reference the Ct data for the untretated condition. Bars indicate the mean ± SD.</p