Identification of differentially accumulated proteins associated with embryogenic and non-embryogenic calli in saffron (Crocus sativus L.)

<p>Abstract</p> <p>Background</p> <p>Somatic embryogenesis (SE) is a complex biological process that occurs under inductive conditions and causes fully differentiated cells to be reprogrammed to an embryo like state. In order to get a better insight about molecular basis of the SE in <it>Crocus sativus </it>L. and to characterize differentially accumulated proteins during the process, a proteomic study based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry has been carried out.</p> <p>Results</p> <p>We have compared proteome profiles of non-embryogenic and embryogenic calli with native corm explants. Total soluble proteins were phenol-extracted and loaded on 18 cm IPG strips for the first dimension and 11.5% sodium dodecyl sulfate-polyacrylamide gels for the second dimension. Fifty spots with more than 1.5-fold change in abundance were subjected to mass spectrometry analysis for further characterization. Among them 36 proteins could be identified, which are classified into defense and stress response, protein synthesis and processing, carbohydrate and energy metabolism, secondary metabolism, and nitrogen metabolism.</p> <p>Conclusion</p> <p>Our results showed that diverse cellular and molecular processes were affected during somatic to embryogenic transition. Differential proteomic analysis suggests a key role for ascorbate metabolism during early stage of SE, and points to the possible role of ascorbate-glutathione cycle in establishing somatic embryos.</p

Western Blot Analysis of Leishmania infantum Antigens in Se-ra of Patients with Visceral Leishmaniasis

Background: Visceral leishmaniasis (VL) is endemic in the northwest and south of Iran. Untreated cases of VL could cause death. The aim of the present study was to evaluate the diagnostic performance of western blotting to detect a specific immunodominant proteins pattern for Leishmania infantum infection using human sera infected with VL. Methods: We studied a panel of 122 cryopreserved human serum samples from the leishmaniasis Research Laboratory, Tehran University of Medical Sciences, Tehran, Iran from 2010 to 2017. Serum samples were collected from visceral (Group I, n: 43) and cutaneous leishmaniasis (CL) (Group II, n: 8) patients, healthy individuals from endemic (Group III, n: 13) and non-endemic (Group IV, n: 16) areas for VL, and patients with other infectious diseases (Group V, n: 42). Total antigens were prepared from the Iranian strain of L. infantum promastigote form. Results: In western blotting method, 34 protein bands of 14 to 163 kDa were recognized using the sera of VL pa­tients. The polypeptide fractions with the highest frequency including 29, 51, and 62 kDa fractions were detected using 81.4%, 79%, and 81.4% of the sera, respectively. These bands were not detected using the sera of the negative control. Moreover, 19-23, 27, 31-35, 143-163, and 109 kDa fractions were detected specifically using the sera of the patients with VL. Conclusion: This technique could be a primary step for further exploration of VL immunodominant antigens for cloning (or any technique) further investigations for future planning

A metagenomic analysis of the camel rumen’s microbiome identifies the major microbes responsible for lignocellulose degradation and fermentation

Abstract Background The diverse microbiome present in the rumen of ruminant animals facilitates the digestion of plant-based fiber. In this study, a shotgun metagenomic analysis of the microbes adhering to plant fiber in the camel rumen was undertaken to identify the key species contributing to lignocellulose degradation and short chain volatile fatty acids (VFA) fermentation. Results The density of genes in the metagenome encoding glycoside hydrolases was estimated to be 25 per Mbp of assembled DNA, which is significantly greater than what has been reported in other sourced metagenomes, including cow rumen. There was also a substantial representation of sequences encoding scaffoldins, dockerins and cohesins, indicating the potential for cellulosome-mediated lignocellulose degradation. Binning of the assembled metagenome has enabled the definition of 65 high-quality genome bins which showed high diversity for lignocellulose degrading enzymes. Species associated to Bacteroidetes showed a high proportion of genes for debranching and oligosaccharide degrading enzymes, while those belonging to Firmicutes and Fibrobacteres were rich in cellulases and hemicellulases and thus these lineages were probably the key for ensuring the degradation of lignocellulose. The presence of many “polysaccharide utilization loci” (PULs) in Bacteroidetes genomes indicates their broad substrate specificity and high potential carbohydrate degradation ability. An analysis of VFA biosynthesis pathways showed that genes required for the synthesis of acetate were present in a range of species, except for Elusimicrobiota and Euryarchaeota. The production of propionate, exclusively via the succinate pathway, was carried out by species belonging to the phyla Bacteroidetes, Firmicutes, Spirochaetes and Fibrobacteres. Butyrate was generated via the butyrylCoA: acetate CoA-transferase pathway by Bacteroidetes and Lentisphaerae species, but generally via the butyrate kinase pathway by Firmicutes species. Conclusion The analysis confirmed the camel rumen’s microbiome as a dense and yet largely untapped source of enzymes with the potential to be used in a range of biotechnological processes including biofuel, fine chemicals and food processing industries

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Additional file 2: Table S1. Details of the number of predicted glycoside hydrolases (GHs) detected in the assembled metagenomes (contigs ≥ 1000 bp). The abundances of GHs were compared between variously sourced metagenomes including the bovine and the moose rumen, elephant faces, and the biogas reactors. Statistical significant differences in CAZyme profiles between the camel rumen’s metagenome and the other metagenomes were assessed using Fisher’s exact test. P values were corrected using FDR method. ns = non-significant, * = FDR-corrected p value < 0.05, ** = FDR-corrected p value < 0.01, *** = FDR-corrected p value < 0.001. Table S2. Counts of carbohydrate binding modules (CBMs) containing proteins predicted in the assembled metagenomes (contigs ≥ 1000 bp). Statistical significant differences in CAZyme profiles between the camel rumen’s metagenome and the other metagenomes were assessed using Fisher’s exact test. P values were corrected using FDR method. ns = non-significant, * = FDR-corrected p value < 0.05, ** = FDR-corrected p value < 0.01, *** = FDR-corrected p value < 0.001. Table S3. The predicted carbohydrate esterases (CEs) characterized in the assembled metagenomes (contigs ≥ 1000 bp). Statistical significant differences in CAZyme profiles between the camel rumen’s metagenome and the other metagenomes were assessed using Fisher’s exact test. P values were corrected using FDR method. ns = non-significant, * = FDR-corrected p value < 0.05, ** = FDR-corrected p value < 0.01, *** = FDR-corrected p value < 0.001. Table S4. Table shows the auxiliary activity domain containing proteins (AAs) identified in the assembled metagenomes (contigs ≥ 1000 bp). Statistical significant differences in CAZyme profiles between the camel rumen’s metagenome and the other metagenomes were assessed using Fisher’s exact test. P values were corrected using FDR method. ns = non-significant, * = FDR-corrected p value < 0.05, ** = FDR-corrected p value < 0.01, *** = FDR-corrected p value < 0.001. Table S5. Table presents the distribution of dockerin, cohesion, and surface layer homology (SLH) domain containing proteins predicted in the assembled metagenomes (contigs ≥ 1000 bp). Statistical significant differences in CAZyme profiles between the camel rumen’s metagenome and the other metagenomes were assessed using Fisher’s exact test. P values were corrected using FDR method. ns = non-significant, * = FDR-corrected p value < 0.05, ** = FDR-corrected p value < 0.01, *** = FDR-corrected p value < 0.001. Table S6. The numbers of predicted polysaccharide lyases (PLs) detected in the assembled metagenomes (contigs ≥ 1000 bp). Statistical significant differences in CAZyme profiles between the camel rumen’s metagenome and the other metagenomes were assessed using Fisher’s exact test. P values were corrected using FDR method. ns = non-significant, * = FDR-corrected p value < 0.05, ** = FDR-corrected p value < 0.01, *** = FDR-corrected p value < 0.001

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Additional file 1: Figure S1. The taxonomic distribution of GHs (A), PLs (B), CBMs (C), AAs (D), and CEs (E) predicted in the camel rumen’s metagenome. Figure S2. Additional examples of PULs identified in the Bacteroidetes bins reconstituted from the camel rumen’s metagenome

Enhanced crystalline cellulose degradation by a novel metagenome-derived cellulase enzyme

Abstract Metagenomics has revolutionized access to genomic information of microorganisms inhabiting the gut of herbivorous animals, circumventing the need for their isolation and cultivation. Exploring these microorganisms for novel hydrolytic enzymes becomes unattainable without utilizing metagenome sequencing. In this study, we harnessed a suite of bioinformatic analyses to discover a novel cellulase-degrading enzyme from the camel rumen metagenome. Among the protein-coding sequences containing cellulase-encoding domains, we identified and subsequently cloned and purified a promising candidate cellulase enzyme, Celcm05-2, to a state of homogeneity. The enzyme belonged to GH5 subfamily 4 and exhibited robust enzymatic activity under acidic pH conditions. It maintained hydrolytic activity under various environmental conditions, including the presence of metal ions, non-ionic surfactant Triton X-100, organic solvents, and varying temperatures. With an optimal temperature of 40 °C, Celcm05-2 showcased remarkable efficiency when deployed on crystalline cellulose (> 3.6 IU/mL), specifically Avicel, thereby positioning it as an attractive candidate for a myriad of biotechnological applications spanning biofuel production, paper and pulp processing, and textile manufacturing. Efficient biodegradation of waste paper pulp residues and the evidence of biopolishing suggested that Celcm05-2 can be used in the bioprocessing of cellulosic craft fabrics in the textile industry. Our findings suggest that the camel rumen microbiome can be mined for novel cellulase enzymes that can find potential applications across diverse biotechnological processes