Identification of an Amylomaltase from the Halophilic Archaeon Haloquadratum walsbyi by Functional Metagenomics: Structural and Functional Insights

Amylomaltases are prokaryotic 4-α-glucanotransferases of the GH77 family. Thanks to the ability to modify starch, they constitute a group of enzymes of great interest for biotechnological applications. In this work we report the identification, by means of a functional metagenomics screening of the crystallization waters of the saltern of Margherita di Savoia (Italy), of an amylomaltase gene from the halophilic archaeon Haloquadratum walsbyi, and its expression in Escherichia coli cells. Sequence analysis indicated that the gene has specific insertions yet unknown in homologous genes in prokaryotes, and present only in amylomaltase genes identified in the genomes of other H. walsbyi strains. The gene is not part of any operon involved in the metabolism of maltooligosaccharides or glycogen, as it has been found in bacteria, making it impossible currently to assign a precise role to the encoded enzyme. Sequence analysis of the H. walsbyi amylomaltase and 3D modelling showed a common structure with homologous enzymes characterized in mesophilic and thermophilic bacteria. The recombinant H. walsbyi enzyme showed starch transglycosylation activity over a wide range of NaCl concentrations, with maltotriose as the best acceptor substrate compared to other maltooligosaccharides. This is the first study of an amylomaltase from a halophilic microorganism.

CsAGA1 and CsAGA2 Mediate RFO Hydrolysis in Partially Distinct Manner in Cucumber Fruits

A Raffinose family oligosaccharides (RFOs) is one of the major translocated sugars in the vascular bundle of cucumber, but little RFOs can be detected in fruits. Alpha-galactosidases (α-Gals) catalyze the first catabolism step of RFOs. Six α-Gal genes exist in a cucumber genome, but their spatial functions in fruits remain unclear. Here, we found that RFOs were highly accumulated in vascular tissues. In phloem sap, the stachyose and raffinose content was gradually decreased, whereas the content of sucrose, glucose and fructose was increased from pedicel to fruit top. Three alkaline forms instead of acid forms of α-Gals were preferentially expressed in fruit vascular tissues and alkaline forms have stronger RFO-hydrolysing activity than acid forms. By inducible gene silencing of three alkaline forms of α-Gals, stachyose was highly accumulated in RNAi-CsAGA2 plants, while raffinose and stachyose were highly accumulated in RNAi-CsAGA1 plants. The content of sucrose, glucose and fructose was decreased in both RNAi-CsAGA1 and RNAi-CsAGA2 plants after β-estradiol treatment. In addition, the fresh- and dry-weight of fruits were significantly decreased in RNAi-CsAGA1 and RNAi-CsAGA2 plants. In cucurbitaceous plants, the non-sweet motif within the promoter of ClAGA2 is widely distributed in the promoter of its homologous genes. Taken together, we found RFOs hydrolysis occurred in the vascular tissues of fruits. CsAGA1 and CsAGA2 played key but partly distinct roles in the hydrolysis of RFOs.

Comprehensive analysis of miRNA–mRNA regulatory network and potential drugs in chronic chagasic cardiomyopathy across human and mouse

Background Chronic chagasic cardiomyopathy (CCC) is the leading cause of heart failure in Latin America and often causes severe inflammation and fibrosis in the heart. Studies on myocardial function and its molecular mechanisms in patients with Chronic chagasic cardiomyopathy are very limited. In order to understand the development and progression of Chronic chagasic cardiomyopathy and find targets for its diagnosis and treatment, the field needs to better understand the exact molecular mechanisms involved in these processes. Methods The mRNA microarray datasets GSE84796 (human) and GSE24088 (mouse) were obtained from the Gene Expression Omnibus (GEO) database. Homologous genes between the two species were identified using the online database mining tool Biomart, followed by differential expression analysis, gene enrichment analysis and protein–protein interaction (PPI) network construction. Cytohubba plug-in of Cytoscape software was used to identify Hub gene, and miRNet was used to construct the corresponding miRNA–mRNA regulatory network. miRNA-related databases: miRDB, Targetscan and miRWalk were used to further evaluate miRNAs in the miRNA–mRNA network. Furthermore, Comparative Toxicogenomics Database (CTD) and L1000 Platform were used to identify hub gene-related drugs. Results A total of 86 homologous genes were significantly differentially expressed in the two datasets, including 73 genes with high expression and 13 genes with low expression. These differentially expressed genes were mainly enriched in the terms of innate immune response, signal transduction, protein binding, Natural killer cell mediated cytotoxicity, Tuberculosis, Chemokine signaling pathway, Chagas disease and PI3K−Akt signaling pathway. The top 10 hub genes LAPTM5, LCP1, HCLS1, CORO1A, CD48, TYROBP, RAC2, ARHGDIB, FERMT3 and NCF4 were identified from the PPI network. A total of 122 miRNAs were identified to target these hub genes and 30 of them regulated two or more hub genes at the same time. miRDB, Targetscan and miRWalk were further analyzed and screened out hsa-miR-34c-5p, hsa-miR-34a-5p and hsa-miR-16-5p as miRNAs regulating these hub genes. Finally, Progesterone, Flutamide, Nimesulide, Methotrexate and Temozolomide were identified to target these hub genes and might be targeted therapies for Chronic chagasic cardiomyopathy. Conclusions In this study, the potential genes associated with Chronic chagasic cardiomyopathy are identified and a miRNA–mRNA regulatory network is constructed. This study explores the molecular mechanisms of Chronic chagasic cardiomyopathy and provides important clues for finding new therapeutic targets.

Developing functional markers for vitamin E biosynthesis in oil palm

Vitamin E is essential for human health and plays positive roles in anti-oxidation. Previously, we detected large variation in vitamin E content among 161 oil palm accessions. In this study, twenty oil palm accessions with distinct variation in vitamin E contents (171.30 to 1 258.50 ppm) were selected for genetic variation analysis and developing functional markers associated with vitamin E contents. Thirty-seven homologous genes in oil palm belonging to vitamin E biosynthesis pathway were identified via BLASTP analysis, the lengths of which ranged from 426 to 25 717 bp (average 7 089 bp). Multiplex PCR sequencing for the 37 genes found 1 703 SNPs and 85 indels among the 20 oil palm accessions, with 226 SNPs locating in the coding regions. Clustering analysis for these polymorphic loci showed that the 20 oil palm accessions could be divided into five groups. Among these groups, group I included eight oil palm accessions whose vitamin E content (mean value: 893.50 ppm) was far higher than other groups (mean value 256.29 to 532.94 ppm). Correlation analysis between the markers and vitamin E traits showed that 134 SNP and 7 indel markers were significantly (p < 0.05) related with total vitamin E content. Among these functional markers, the indel EgTMT-1-24 was highly correlated with variation in vitamin E content, especially tocotrienol content. Our study identified a number of candidate function associated markers and provided clues for further research into molecular breeding for high vitamin E content oil palm.

Gcorn fungi: A Web Tool for Detecting Biases between Gene Evolution and Speciation in Fungi

(1) Background: Fungi contain several millions of species, and the diversification of fungal genes has been achieved by speciation, gene duplication, and horizontal gene transfer. Although several databases provide information on orthologous and paralogous events, these databases show no information on biases between gene mutation and speciation. Here, we designed the Gcorn fungi database to better understand such biases. (2) Methods: Amino acid sequences of fungal genes in 249 species, which contain 2,345,743 sequences, were used for this database. Homologous genes were grouped at various thresholds of the homology index, which was based on the percentages of gene mutations. By grouping genes that showed highly similar homology indices to each other, we showed functional and evolutionary traits in the phylogenetic tree depicted for the gene of interest. (3) Results: Gcorn fungi provides well-summarized information on the evolution of a gene lineage and on the biases between gene evolution and speciation, which are quantitatively identified by the Robinson–Foulds metric. The database helps users visualize these traits using various depictions. (4) Conclusions: Gcorn fungi is an open access database that provides a variety of information with which to understand gene function and evolution.

Gene recruitments and dismissals in argonaut octopus genome provide insights to pelagic lifestyle adaptation and shell-like eggcase reacquisition

The paper nautilus, Argonauta argo, also known as the greater argonaut, is a species of octopods distinctly characterized by its pelagic lifestyle and by the presence of a spiral-shaped shell-like eggcase in females. The eggcase functions by protecting the eggs laid inside it, and by building and keeping air intakes for buoyancy. To reveal the genomic background of the species′ adaptation to pelagic lifestyle and the acquisition of its shell-like eggcase, we sequenced the draft genome sequence of the species. The genome size was 1.1 Gb, which is the smallest among the cephalopods known to date, with the top 215 scaffolds (average length 5,064,479 bp) covering 81% (1.09 Gb) of the total assembly. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified nearly intact HOX, Parahox, Wnt clusters and some gene clusters probably related to the pelagic lifestyle, such as reflectin, tyrosinase, and opsin. For example, opsin might have undergone an extensive duplication in order to adapt to the pelagic lifestyle, as opposed to other octopuses, which are mostly the benthic. Our gene models also discovered several genes homologous to those related to calcified shell formation in Conchiferan Mollusks, such as Pif-like, SOD, and TRX. Interestingly, comparative genomics analysis revealed that the homologous genes for such genes were also found in the genome of the octopus, which does not have a shell, as well as the basal cephalopods Nautilus. Therefore, the draft genome sequence of A. argo we presented here had not only helped us to gain further insights into the genetic background of the dynamic recruitment and dismissal of genes for the formation of an important, converging extended phenotypic structure such as the shell and the shell-like eggcase, but also the evolution of lifestyles in Cephalopods and the octopods, from benthic to pelagic.

AIP1, Encoding the Small Subunit of Acetolactate Synthase, Is Partially Responsible for Resistance to Hypoxic Stress in Arabidopsis thaliana

Flooding is a significant stress to land plants, depriving them of essential oxygen. Plants have evolved diverse strategies with variable success to survive flooding. Similar strategies have been described in organisms from other kingdoms. Several fungal species can successfully survive a low-oxygen environment by increasing their branched-chain amino acid (BCAA) contents. BCAAs may act as alternative electron acceptors in the respiratory chain under an oxygen-limited environment. The key and first enzyme for BCAA biosynthesis is acetolactate synthase (ALS). We identified two homologous genes encoding the small subunit of ALS in Arabidopsis (Arabidopsis thaliana). We determined that ALS INTERACTING PROTEIN1 (AIP1), which encodes the small subunit of ALS, is strongly expressed in all organs and highly expressed under submergence and low-oxygen stresses. We also showed that the overexpression of AIP1 confers tolerance to low-oxygen stress. These results indicate that ALS may play an essential role under prolonged flooding or oxygen deficiency in Arabidopsis.

A Recombinant Multivalent Vaccine (rCpa1) Induces Protection for C57BL/6 and HLA Transgenic Mice Against Pulmonary Infection with Both Species of Coccidioides

Coccidioidomycosis is caused by Coccidioides posadasii (Cp) and Coccidioides immitis (Ci) that have 4-5% differences in their genomic sequences. There is an urgent need to develop a human vaccine against both species. A previously created recombinant antigen (rCpa1) that contains multiple peptides derived from Cp isolate C735 is protective against the autologous isolate. The focus of this study is to evaluate cross-protective efficacy and immune correlates by the rCpa1-based vaccine against both species of Coccidioides. DNA sequence analyses of the homologous genes for the rCpa1 antigen were conducted for 39 and 17 clinical isolates of Cp and Ci, respectively. Protective efficacy and vaccine-induced immunity were evaluated for both C57BL/6 and human HLA-DR4 transgenic mice against 5 highly virulent isolates of Cp and Ci. There are a total of 7 amino acid substitutions in the rCpa1 antigen between Cp and Ci. Both C57BL/6 and HLA-DR4 mice that were vaccinated with a rCpa1 vaccine resulted in a significant reduction of fungal burden and increased numbers of IFN-γ- and IL-17-producing CD4+ T cells in the first 2 weeks post-challenge. These data support that rCpa1 has cross-protection activity against Cp and Ci pulmonary infection through activation of early Th1 and Th17 responses.

De novo SNP calling reveals the candidate genes regulating days to flowering through interspecies GWAS of Amaranthus genus

Amaranths serve as pseudo cereals and also as traditional leafy vegetables worldwide. In addition to high vigor and richness in nutrients, drought and salinity tolerance of amaranth makes it a promising vegetable to acclimatize to the effects of global climate change. The World Vegetable Center genebank conserves about 1,000 amaranth accessions and various agronomic properties of these accessions were recorded during seed regeneration over decades. In this study, we verified the taxonomic annotation of the germplasm based on a 15K SNP set. Besides, in the assumption that the yield components of grain amaranths are different from those of leaf amaranths, we observed that grain amaranths presented larger inflorescences and earlier flowering than leaf amaranths. Dual-purpose amaranth showed larger leaves than leaf amaranths and later flowering than grain amaranths, which seemed reasonable because farmers can harvest more leaves during the prolonged vegetable stage, which also provides recovery time to enrich grain production. Considering frequent interspecies hybridization among grain amaranth complex, we proposed an interspecies GWAS for days to flowering, identifying a AGL20/SOC1 homolog. Meanwhile, another GWAS using only A. tricolor accessions revealed six candidate genes homologous to lba1, bri1, sgs1 and fca. These homologous genes were involved in the regulation of flowering time in Arabidopsis. This study revealed the usefulness of genotypes for species demarcation in the genus Amaranthus and the potential of interspecies GWAS to detect QTLs across different species, opening up the possibility of targeted introduction of specific genetic variants into different Amaranthus species.