Transcriptome analysis revealed changes of multiple genes involved in muscle hardness in grass carp (Ctenopharyngodon idellus) fed with faba bean meal.

An 8-week feeding trial and transcriptome analysis were conducted to investigate the potential mechanism of muscle-hardening caused by faba bean in grass carp (Ctenopharyngodon idellus). Ordinary grass carp (fed with practical diet) and crisp grass carp (fed with faba bean meal) groups were designed. Lower water holding capacity and higher some texture parameters were observed in the muscle of crisp grass carp compared with another group. 19.62 GB clean reads were generated, and total 1354 genes exhibiting differentially expression were identified (FDR < 0.05). Genes function enrichment revealed up-regulated genes in crisp grass carp mainly in response to myofibroblast proliferation, while down-regulated genes in response to immune regulation. Consistent with this, the tight junction pathway and the NF-κB signaling pathway were likewise significantly enriched. In summary, this study identified several candidate genes and putative signaling pathways deserving further investigation to the mechanism of muscle-hardening in fish fed with faba bean.

Identification of duplicated Cited3 genes and their responses to hypoxic stress in blunt snout bream (Megalobrama amblycephala).

The CITED3 protein is a non-DNA-binding transcriptional co-regulator involved in the regulation of various transcriptional responses against hypoxia stress. Here, we characterized two paralogs Cited3 genes (Cited3a and Cited3b) from blunt snout bream (Megalobrama amblycephala), which is a hypoxia-sensitive species. Both genes have an open reading frame of 756 and 723 bp; encoded a protein of 251 amino acid and 240 amino acid, respectively; and they shared a sequence identity of 67%. In adult fish, both Cited3a and Cited3b mRNAs were highly expressed in kidney tissues. In contrast, they were detected in the skin, muscle, and gonad at extraordinarily low levels. During embryogenesis, both Cited3a and Cited3b mRNAs were maternally deposited in eggs and fluctuated from the zygote to the 44-hpf (hours post-fertilization) larvae. Whole-mount in situ hybridization demonstrated that both Cited3a and Cited3b mRNAs were transcribed in the brain, gut, and tailbud at 12 hpf, and at the brain and gut at 24 hpf, and at the brain at 36 hpf embryos. Hypoxic treatment led to upregulated expression of the Cited3 genes during embryogenesis. Under hypoxia, both Cited3a and Cited3b genes in the kidney and brain and Cited3a genes in the liver were significantly upregulated. These results suggest that hypoxia was associated with increases in mRNA levels for both Cited3a (kidney, brain, liver) and Cited3b (kidney and liver).

Insertional mutagenesis in ChordinA induced by endogenous ΔTgf2 transposon leads to bifurcation of axial skeletal systems in grass goldfish.

The grass goldfish appeared early in the evolutionary history of goldfish, and shows heritable stability in the development of the caudal fin. The twin-tail phenotype is extremely rare, however, some twin-tail individuals were produced in the process of breeding for ornamental value. From mutations in the twin-tail goldfish genome, we identified two kinds of Tgf2 transposons. One type was completely sequenced Tgf2 and the other type was ΔTgf2, which had 858 bp missing. We speculate that the bifurcation of the axial skeletal system in goldfish may be caused by an endogenous ΔTgf2 insertion mutation in Chordin A, as ΔTgf2 has no transposition activity and blocks the expression of Chordin A. The twin-tail showed doubled caudal fin and accumulation of red blood cells in the tail. In addition, in situ hybridization revealed that ventral embryonic tissue markers (eve1, sizzled, and bmp4) were more widely and strongly expressed in the twin-tail than in the wild-type embryos during the gastrula stage, and bmp4 showed bifurcated expression patterns in the posterior region of the twin-tail embryos. These results provide new insights into the artificial breeding of genetically stable twin-tail grass goldfish families.

Divergence of Genes Encoding CITED1 and CITED2 in Blunt Snout Bream (Megalobrama amblycephala) and Their Transcriptional Responses to Hypoxia.

The proteins CITED belong to a family of non-DNA-binding transcriptional co-regulators involved in the regulation of various transcriptional responses. Previous studies suggest that members of CITED family may function in response to hypoxia in mammals. however, the molecular and functional information on CITED genes in aquaculture fish is unclear. Here, we characterized and examined the transcriptional patterns of CITED1 and CITED2 genes in the hypoxia-sensitive blunt snout bream (Megalobrama amblycephala). Blunt snout bream CITED1 and CITED2 genes shared a relatively low sequence identity of 45%. CITED1 and CITED2 mRNAs were widely transcribed in adult tissues. During embryogenesis, CITED1 mRNA was significantly transcribed at 4, 24, 28, 40, and 44 hpf, whereas CITED2 mRNA levels fluctuated from the zygote to 44 hpf larval stage. Whole-mount in situ hybridization demonstrated that CITED1 and CITED2 mRNAs were detected in the brain at 12 hpf, brain and gut at 24 hpf, and brain at 36 hpf. In addition, low expression of CITED1 mRNA was detected in the tailbud at 24 hpf. The results of acute hypoxia experiment showed that CITED1 and CITED2 mRNAs were markedly upregulated in the kidney and downregulated in the liver, brain, gill, and heart under hypoxia. Embryos in hypoxic conditions at different developmental stages showed a significant increase in mRNA levels of CITED1 and CITED2. These results provide a new insight into the divergence of CITED1 and CITED2 genes and their transcriptional responses to hypoxia.

[Characterization of the size variants of a recombinant humanized monoclonal antibody (rhumAb1)].

The composition and potency of the high temperature (40 ℃) stress induced size variants of a recombinant humanized monoclonal antibody(rhumAb1) were characterized by means of SEC-HPLC, non- reduced CE-SDS, liquid chromatography coupled with mass spectrometry (LC-MS) and antibody dependent cell-mediated cytotoxicity (ADCC) assay. The molecular masses of the four size variants (SEC-1-SEC-4) separated by SEC-HPLC and seven size variants(NR-1-NR-7) detected by non-reduced CE-SDS were all characterized by LC-MS. The major low molecular weight variants were generated due to the hinge region fragmentation of heavy chain. The hinge region cleavage was found mainly in the Ser221-Cys-Asp-Lys-Thr- His-Thr-Cys228 sequence, in which C222-D223 and H226-T227 were the major cleavage sites. The size variants of rhumAb1, namely dimer and fragments, have significantly reduced ADCC activity in comparison with the intact rhumAb1 drug product. This study provided insights into the stability profiling for rhumAb1 drug product. The study protocols presented here may be applicable to the analytical characterization of other monoclonal antibody-based therapeutic products.