Identification of the grass carp interleukin-23 receptor and its proinflammatory role in intestinal inflammation.

The interleukin 23 receptor (IL-23R) is associated with a variety of inflammatory diseases in humans and other mammals. However, whether IL-23R is involved in inflammatory diseases in teleost fish is less understood. Thus, to investigate the potential involvement of IL-23R in fish inflammatory diseases, the full-length cDNA of IL-23R from grass carp Ctenopharyngodon idella was cloned and used to generate a recombinant protein (rgcIL-23R) containing the extracellular domain of IL-23R, against which a polyclonal antibody (rgcIL-23R pAb) was then developed. qPCR analysis revealed that IL-23R mRNA was significantly upregulated in most grass carp tissues in response to infection with Gram-negative Aeromonas hydrophila. Treatment with rgcIL-23R significantly induced IL-17A/F1 expression in C. idella kidney (CIK) cells. By contrast, knockdown of IL-23R caused significant decreases in IL-23R, STAT3, and IL-17N expression in CIK cells after lipopolysaccharide (LPS) stimulation. Similarly, rgcIL-23R pAb treatment effectively inhibited the LPS-induced increase in the expression of IL-23 subunit genes and those of the IL-23/IL-17 pathway in CIK cells. Furthermore, intestinal symptoms identical to those caused by A. hydrophila were induced by anal intubation with rgcIL-23R, but suppressed by rgcIL-23R pAb. Therefore, these results suggest that IL-23R has a crucial role in the regulation of intestinal inflammation and, thus, is a promising target for controlling inflammatory diseases in farmed fish.

Engineering Escherichia coli for l-homoserine production.

l-homoserine, a nonprotein amino acid, is used to synthesize many active substances in the industry. Here, to develop a robust l-homoserine-producing strain, Escherichia coli W3110 was used as a chassis to be engineered. Based on a previous construct with blocked competing routes for l-homoserine synthesis, five genes were overexpressed by promoter replacement strategy to increase the l-homoserine production, including enhancement of precursors for l-homoserine synthesis (ppc, thrA, and asd), reinforcement of the NADPH supply (pntAB) and efflux transporters (rhtA) to improve the l-homoserine production. However, the plasmid losing was to blame for the wildly fluctuating fermentation performance of engineered strains, ranging between 2.1 and 6.2 g/L. Then, a hok/sok toxin/antitoxin system was introduced into the free plasmid expression cassette to maintain the genetic stability of the episomal plasmid; consequently, the plasmid-losing rate sharply decreased, resulting in the engineered strain SHL17, which exhibited excellent stability in l-homoserine production, with 6.3 g/L in shake flasks and 44.4 g/L in a 5-L fermenter without antibiotic addition. This work verified the effective use of the hok/sok toxin/antitoxin system combined with promoter engineering to improve the genetic stability of E. coli episomal plasmids without antibiotics.

Structure-guided engineering of a flavin-containing monooxygenase for the efficient production of indirubin.

Indirubin is a bisindole compound for the treatment of chronic myelocytic leukemia. Here, we presented a structure-guided method to improve the activity of a flavin-containing monooxygenase (bFMO) for the efficient production of indirubin in Escherichia coli. A flexible loop interlocked with the active pocket through a helix and the substrate tunnel rather than the active pocket in bFMO were identified to be two reconfigurable structures to improve its activity, resulting in K223R and N291T mutants with enhanced catalytic activity by 2.5- and 2.0-fold, respectively. A combined modification at the two regions (K223R/D317S) achieved a 6.6-fold improvement in catalytic efficiency (kcat/Km) due to enhancing π-π stacking interactions stabilization. Finally, an engineered E. coli strain was constructed by metabolic engineering, which could produce 860.7 mg/L (18 mg/L/h) indirubin, the highest yield ever reported. This work provides new insight into the redesign of FMOs to boost their activities and an efficient approach to produce indirubin.

Interleukin-12 receptor ß2 from grass carp: Molecular characterization and its involvement in Aeromonas hydrophila-induced intestinal inflammation.

Interleukin-12 receptor ß2 (IL-12Rß2) is a signaling subunit of heterodimeric receptors for IL-12 and IL-35. It plays important regulatory functions in the development of Th1 cells and in the expression of inflammatory cytokines in mammals and other higher vertebrates. However, little is known about IL-12Rß2 in teleost fish. In this work, we have cloned and characterized IL-12Rß2 from grass carp (Ctenopharyngodon idella). The full-length cDNA of grass carp IL-12Rß2 is 2875 bp, which encodes a mature protein with 741 amino acids. This mature protein contains three fibronectin type III domains, a transmembrane helix, and CXW and WSXWS-like motifs that are characteristic of the type I cytokine receptor family. Phylogenetic analysis revealed that cyprinid fish IL-12Rß2 formed a single branch, clearly separated from those of other vertebrates. We expressed and purified a recombinant grass carp IL-12Rß2 protein containing major antigenic regions, which was used to raise a polyclonal antibody. The specificity of the antibody was assessed by Western blotting analysis of whole cell lysates from Escherichia coli cells expressing the recombinant IL-12Rß2, grass carp intestinal intraepithelial lymphocytes, and cultured C. idella kidney cells. To explore the potential regulatory role of IL-12Rß2 in inflammation, we generated an intestinal inflammation model by anal intubation of fish with Aeromonas hydrophila. Immunohistochemical staining of the inflamed intestines revealed that IL-12Rß2 expression is consistent with inflammatory cell recruitment during intestinal inflammation. Real-time quantitative PCR revealed that IL-12Rß2 is widely expressed in normal tissues and is up-regulated in most tissues after infecting with A. hydrophila. We found that IL-12Rß2, IL-12p35, and interferon-γ were expressed in similar patterns in the intestines during inflammation. Taken together, our results suggest that IL-12Rß2 is involved in the regulation of intestinal inflammation.

A transcriptome analysis focusing on inflammation-related genes of grass carp intestines following infection with Aeromonas hydrophila.

Inflammation is a protective response that is implicated in bacterial enteritis and other fish diseases. The inflammatory mechanisms behind Aeromonas hydrophila infections in fish remain poorly understood. In this study, we performed a de novo grass carp transcriptome assembly using Illumina's Solexa sequencing technique. On this basis we carried out a comparative analysis of intestinal transcriptomes from A. hydrophila-challenged and physiological saline solution (PSS/mock) -challenged fish, and 315 genes were up-regulated and 234 were down-regulated in the intestines infected with A. hydrophila. The GO enrichment analysis indicated that the differentially expressed genes were enriched to 12, 4, and 8 GO terms in biological process, molecular function, and cellular component, respectively. A KEGG analysis showed that 549 DEGs were involved in 165 pathways. Moreover, 15 DEGs were selected for quantitative real-time PCR analysis to validate the RNA-seq data. The results confirmed the consistency of the expression levels between RNA-seq and qPCR data. In addition, a time-course analysis of the mRNA expression of 12 inflammatory genes further demonstrated that the intestinal inflammatory responses to A. hydrophila infection simultaneously modulated gene expression variations. The present study provides intestine-specific transcriptome data, allowing us to unravel the mechanisms of intestinal inflammation triggered by bacterial pathogens.

Characterization of interleukin-1ß as a proinflammatory cytokine in grass carp (Ctenopharyngodon idella).

Interleukin-1ß (IL-1ß) is a well-characterized cytokine that plays key roles in cellular responses to infection, inflammation, and immunological challenges in mammals. In this study, we identified and analyzed a grass carp (Ctenopharyngodon idella) ortholog of IL-1ß (gcIL-1ß), examined its expression patterns in various tissues in both healthy and lipopolysaccharide (LPS)-stimulated specimens, and evaluated its proinflammatory activities. The gcIL-1ß gene consists of seven exons and six introns. The full-length cDNA sequence contains an open reading frame of 813 nucleotides. The deduced amino acid sequence exhibits a characteristic IL-1 signature but lacks the typical IL-1ß converting enzyme cleavage site that is conserved in mammals. In the phylogenetic tree, IL-1ßs from grass carp and other members of the Cyprinidae family clustered into a single group. Expression pattern analysis revealed that gcIL-1ß is constitutively expressed in all 11 tissues examined, and LPS stimulation leads to significant up-regulation in muscle, liver, intestine, skin, trunk kidney, head kidney, and gill. Recombinant grass carp IL-1ß (rgcIL-1ß) was generated prokaryotically as a fusion protein of Trx-rgcIL-1ß. An anti-rgcIL-1ß polyclonal antibody (rgcIL-1ß pAb) was raised in mice against the purified Trx-rgcIL-1ß. Western blot analysis confirmed that rgcIL-1ß pAb reacted specifically with gcIL-1ß in C. idella kidney (CIK) cells. Quantitative real-time PCR data indicated that intestinal mRNA expression levels of endogenous IL-1ß, IL-1R2, and TNF-α were significantly up-regulated following Trx-rgcIL-1ß exposure. The inhibitory activities of rgcIL-1ß pAb against the inflammatory response were confirmed in a model of Aeromonas hydrophila-induced intestinal inflammation. Our immunohistochemical study revealed that the degree and intensity of inflammatory cell infiltration are fully consistent with the observed mRNA expression patterns of these key inflammatory genes. Taken together, these data suggest that gcIL-1ß plays a critical role in the proinflammatory response in the grass carp intestine.

Molecular identification of one event of Ds excision and re-insertion at two loci in rice genome.

The maize Ac/Ds transposable elements are members of the hAT transposon superfamily, and have stable transpositional activity in transgenic rice plants. Ac/Ds transposable elements are considered to transpose via a conservative non-replicative "cut and paste" model, though their transposition mechanism is not completely understood. Previous studies have shown that Ds preferentially transposes to genetically linked sites after being excised from its original site in the presence of Ac-transposase. In this study, genomic sequences flanking Ds insertions from a Ds-tagged rice mutant and its rever- tant were determined by TAIL-PCR. The Ds insertion site, the excision footprint and the re-insertion sites in the mutant were identified using bioinformatics tool. The results showed that Ds element excised from its original insertion site on chromosome 3 by leaving an 8 bp footprint (CATCATGA), which resulted in exon changes in tagged gene. After the excision, Ds element was re-inserted into the coding sequences of two genes on chromosome 2 and chromosome 6, which encode a nicotianamine aminotransferase and a senescence-associated protein, respectively. The transposition behavior of Ds element in this study could not be fully explained by the "cut and paste" mechanism, while it is likely to transpose in a "cut and copy and paste" way.

[Amplification of Ds flanking sequences from rice genomic DNA of hybrids of Ac x Ds lines and the analysis of Ds insertions].

Ac and Ds insertions among the genomic DNAs of hybrids of Ac x Ds lines were screened by PCR. The genomic DNAs, which were proved to harbour both Ac and Ds, were used as templates in TAIL-PCR to clone the Ds flanking sequences. The cloned specific fragments were sequenced, and the sequenced Ds flanking sequences were used as query sequences to perform on-line sequence comparing analysis against GenBank by employing BLAST program of NCBI. The information about the chromosome location of Ds-inserted genes, or genes immediately downstream of the inserted sites, and their functional innotations were achieved. Based on the analysis from the cloned 93 Ds-flanking sequences, it was found that 21 hybrid plants had Ds insertions in genic regions, whereas the remaining 72 samples's intergenic regions were inserted by Ds element. Moreover, among the 72 regions, 12 were inserted immediately upstream (within 3 kb) of specific genes. Also, the strategies to improve the performance in cloning the Ds flanking sequences and in screening the Ac/Ds lines were emphasized.

Rapid, large-scale generation of Ds transposant lines and analysis of the Ds insertion sites in rice.

Rapid, large-scale generation of a Ds transposant population was achieved using a regeneration procedure involving tissue culture of seed-derived calli carrying Ac and inactive Ds elements. In the F(2) progeny from genetic crosses between the same Ds and Ac starter lines, most of the crosses produced an independent germinal transposition frequency of 10-20%. Also, many Ds elements underwent immobilization even though Ac was expressed. By comparison, in a callus-derived regenerated population, over 70% of plants carried independent Ds insertions, indicating transposition early in callus formation. In the remaining population, the majority of plants carried only Ac. Most of the new Ds insertions were stably transmitted to a subsequent generation. An exceptionally high proportion of independent transposants in the regenerated population means that selection markers for transposed Ds and continual monitoring of Ac/Ds activities may not necessarily be required. By analyzing 1297 Ds-flanking DNA sequences, a genetic map of 1072 Ds insertion sites was developed. The map showed that Ds elements were transposed onto all of the rice chromosomes, with preference not only near donor sites (36%) but also on certain physically unlinked arms. Populations from both genetic crossing and tissue culture showed the same distribution patterns of Ds insertion sites. The information of these mapped Ds insertion sites was deposited in GenBank. Among them, 55% of Ds elements were on predicted open-reading frame (ORF) regions. Thus, we propose an optimal strategy for the rapid generation of a large population of Ds transposants in rice.

[cDNA cloning and sequence analysis of leginsulin gene in broad bean (Vicia faba)].

In order to elucidate the relationship between the structural features of leginsulin gene in legume plants and their phylogenetic significance, we have cloned the cDNA sequence of leginsulin gene from radicles of broad bean (Vicia faba) via RT-PCR techniques according to the leginsulin gene sequence we previously obtained from soybean (Glycine max). The cloned cDNA encoded for a precursor protein consisting of the signal peptide, mature leginsulin and an additional 45 amino acids of another polypeptide. A sequence search for homology comparison revealed the cloned leginsulin cDNA fragment shares 62.5% and 58.7% similarity to soybean and pea, respectively. The results also shown that leginsulin cDNA from broad bean presents 44.2% and 43.6% amino acid sequence homology with soybean and pea (Pisum sativum), respectively, and that there exists highly conserved cysteine sites among the leginsulin cDNAs, which may play a crucial role in maintaining the three-dimensional structure and the physiological functions of leginsulin.