A Comprehensive Analysis of Codon Usage Patterns in Blunt Snout Bream (Megalobrama amblycephala) Based on RNA-Seq Data

Blunt snout bream (Megalobrama amblycephala) is an important fish species for its delicacy and high economic value in China. Codon usage analysis could be helpful to understand its codon biology, mRNA translation and vertebrate evolution. Based on RNA-Seq data for M. amblycephala, high-frequency codons (CUG, AGA, GUG, CAG and GAG), as well as low-frequency ones (NUA and NCG codons) were identified. A total of 724 high-frequency codon pairs were observed. Meanwhile, 14 preferred and 199 avoided neighboring codon pairs were also identified, but bias was almost not shown with one or more intervening codons inserted between the same pairs. Codon usage bias in the regions close to start and stop codons indicated apparent heterogeneity, which even occurs in the flanking nucleotide sequence. Codon usage bias (RSCU and SCUO) was related to GC₃ (GC content of 3rd nucleotide in codon) bias. Six GO (Gene ontology) categories and the number of methylation targets were influenced by GC₃. Codon usage patterns comparison among 23 vertebrates showed species specificities by using GC contents, codon usage and codon context analysis. This work provided new insights into fish biology and new information for breeding projects.     

A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli

Adiponectin is one of the most bioactive substances secreted by adipose tissue and is involved in the protection against metabolic syndrome, artherosclerosis and type II diabetes. Research into the use of adiponectin as a promising drug for metabolic syndromes requires production of this hormone in high quantities considering its molecular isoforms. The objective of this study is to produce recombinant human adiponectin by Pichia pastoris (P-ADP) as a cheap and convenient eukaryotic expression system for potential application in pharmaceutical therapy. For comparison, adiponectin was also expressed using the Escherichia coli (E-ADP) expression system. Adiponectin was constructed by overlap-extension PCR, and cloned in standard cloning vector and hosts. Recombinant expression vectors were cloned in the P. pastoris and E. coli host strains, respectively. SDS-PAGE and western blotting were used to detect and analyse expressed recombinant protein in both systems. Adiponectin was purified by affinity chromatography and quantified using the Bradford Assay. The results of this study indicated that P-ADP quantity (0.111 mg/mL) was higher than that of E-ADP (0.04 mg/mL) and both were produced in soluble form. However, P-ADP was able to form high molecular weights of adiponectin molecules, whilst E-ADP was not able to form isoforms higher than trimer. In addition, P-ADP was more active in lowering blood glucose compared with E-ADP. The two types of proteins were equally efficient and significantly decreased blood triglyceride and increased high density lipoprotein. We conclude that P. pastoris is able to produce high quantity of bioactive adiponectin for potential use in treatment of metabolic syndromes.     

Ectopic Expression of CsCTR1, a Cucumber CTR-Like Gene,Attenuates Constitutive Ethylene Signaling in an Arabidopsis ctr1-1 Mutant and Expression Pattern Analysis of CsCTR1 in Cucumber (Cucumis sativus)

The gaseous plant hormone ethylene regulates many aspects of plant growth, development and responses to the environment. Constitutive triple response 1 (CTR1) is a central regulator involved in the ethylene signal transduction pathway. To obtain a better understanding of this particular pathway in cucumber, the cDNA-encoding CTR1 (designated CsCTR1) was isolated from cucumber. A sequence alignment and phylogenetic analyses revealed that CsCTR1 has a high degree of homology with other plant CTR1 proteins. The ectopic expression of CsCTR1 in the Arabidopsis ctr1-1 mutant attenuates constitutive ethylene signaling of this mutant, suggesting that CsCTR1 indeed performs its function as negative regulator of the ethylene signaling pathway. CsCTR1 is constitutively expressed in all of the examined cucumber organs, including roots, stems, leaves, shoot apices, mature male and female flowers, as well as young fruits. CsCTR1 expression gradually declined during male flower development and increased during female flower development. Additionally, our results indicate that CsCTR1 can be induced in the roots, leaves and shoot apices by external ethylene. In conclusion, this study provides a basis for further studies on the role of CTR1 in the biological processes of cucumber and on the molecular mechanism of the cucumber ethylene signaling pathway.     

Functional Expression of an Orchid Fragrance Gene in Lactococcus lactis

Vanda Mimi Palmer (VMP), an orchid hybrid of Vanda tesselata and Vanda Tan Chay Yan is a highly scented tropical orchid which blooms all year round. Previous studies revealed that VMP produces a variety of isoprenoid volatiles during daylight. Isoprenoids are well known to contribute significantly to the scent of most fragrant plants. They are a large group of secondary metabolites which may possess valuable characteristics such as flavor, fragrance and toxicity and are produced via two pathways, the mevalonate (MVA) pathway or/and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. In this study, a sesquiterpene synthase gene denoted VMPSTS, previously isolated from a floral cDNA library of VMP was cloned and expressed in Lactococcus lactis to characterize the functionality of the protein. L. lactis, a food grade bacterium which utilizes the mevalonate pathway for isoprenoid production was found to be a suitable host for the characterization of plant terpene synthases. Through recombinant expression of VMPSTS, it was revealed that VMPSTS produced multiple sesquiterpenes and germacrene D dominates its profile.     

Functional Expression of Thyroid-Stimulating Hormone Receptor on Nano-Sized Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1

The measurement of autoantibodies to thyroid-stimulating hormone receptor (TSHR) is important for the diagnosis of autoimmune thyroid disease such as Graves’ disease (GD). Although TSHR from porcine thyroid membrane is commonly used for the measurement of TSHR autoantibodies (TRAb), recombinant human TSHR (hTSHR) remains ideal in terms of stable supply and species identity. Here we set out to express recombinant hTSHR on the lipid-bilayer surface of magnetic nanoparticles from a magnetotactic bacterium, Magnetospirillum magneticum AMB-1. Using a tetracycline-inducible expression system, we successfully overexpressed functional hTSHR on bacterial magnetic particles (BacMPs) in AMB-1 via an anchor protein specific for BacMPs. The overexpressed hTSHR was membrane integrated and possessed both ligand and autoantibody binding activity. Our data suggest that hTSHR-displayed BacMPs have potential as novel tools for ligand-receptor interaction analysis or for TRAb immunoassay in GD patients.     

Modulation of P1 and EGF Expression by Baicalin

Mycoplasma pneumoniae (M. pneumoniae) is increasingly recognized as a major cause of acute respiratory tract infections. Today, macrolides are used in the primary treatment of M. pneumoniae infection. However, with the increasing prevalence of strains resistant to macrolides, as well as reports of toxicity and adverse side effects, it is necessary to develop an alternative therapeutic agent. A compound recipe—Qinbaiqingfei pellets (Qinbai)—have already been approved in China as the first effective traditional Chinese medicine to be used against M. pneumoniae. Herein, we characterize the mechanism by which Qinbai interacts with M. pneumoniae and lung epithelial cells. The fact that Baicalin is the key component of Qingbai leads us to believe its study is important to elucidating the mechanism of the action of Qinbai. In this study, we describe the complex impact of Baicalin on the adhesin protein P1 of M. pneumoniae and on the expression of epidermal growth factor (EGF) in BALB/c mice and A549 cells infected with M. pneumonia. We draw the conclusion that Baicalin not only cured M. pneumoniae infection by inhibiting P1 expression, but also enhanced the repair of lung epithelial cells by upregulating EGF. Finally, we demonstrate that Baicalin plays a role in Qinbai treatment.     

BTG1 Expression Correlates with the Pathogenesis and Progression of Ovarian Carcinomas

BTG (B-cell translocation gene) can inhibit cell proliferation, metastasis, and angiogenesis and regulate cell cycle progression and differentiation in a variety of cell types. We aimed to clarify the role of BTG1 in ovarian carcinogenesis and progression. A BTG1-expressing plasmid was transfected into ovarian carcinoma cells and their phenotypes and related proteins were examined. BTG1 mRNA expression was detected in ovarian normal tissue (n = 17), ovarian benign tumors (n = 12), and ovarian carcinoma (n = 64) using real-time RT-PCR. Ectopic BTG1 expression resulted in lower growth rate, high cisplatin sensitivity, G₁ arrest, apoptosis, and decreased migration and invasion. Phosphoinositide 3-kinase, protein kinase B, Bcl-xL, survivin, vascular endothelial growth factor, and matrix metalloproteinase-2 mRNA and protein expression was reduced in transfectants as compared to control cells. There was higher expression of BTG1 mRNA in normal tissue than in carcinoma tissue (p = 0.001) and in benign tumors than in carcinoma tissue (p = 0.027). BTG1 mRNA expression in International Federation of Gynecology and Obstetrics (FIGO) stage I/II ovarian carcinomas was higher than that in FIGO stage III/IV ovarian carcinomas (p = 0.038). Altered BTG1 expression might play a role in the pathogenesis and progression of ovarian carcinoma by modulating proliferation, migration, invasion, the cell cycle, and apoptosis.     

Zebrafish Cyclin-Dependent Protein Kinase-Like 1 (zcdkl1): Identification and Functional Characterization

The cyclin-dependent protein kinase family regulates a wide range of cellular functions such as cell cycle progression, differentiation, and apoptosis. In this study, we identified a zebrafish cyclin-dependent protein kinase-like 1 protein called zebrafish cdkl1 (zcdkl1), which shared a high degree of homology and conserved synteny with mammalian orthologs. zcdkl1 exhibited abilities for phosphorylation of myelin basic protein and histone H1. RT-PCR analysis revealed that zcdkl1 was expressed starting from fertilization and continuing thereafter. In adult tissues, zcdkl1 was predominantly detected in brain, ovary, and testis, and was expressed at low levels in other tissues. At 50% epiboly stage, zcdkl1 was widely expressed. At 12 to 48 h post-fertilization, zcdkl1 was predominantly expressed in the hypochord, the medial and lateral floor plate, and the pronephric duct. Interference of zcdkl1 expression resulted in abnormalities, such as brain and eye malformation, pericardial edema, and body axis curvature. Disruption of zcdkl1 reduced neurogenin-1 in the brain and sonic hedgehog expression in the floor plate region. These deformities were apparently rescued by co-injection of zcdkl1 mRNA. Findings of this study indicate that zcdkl1 plays an essential role in zebrafish development.     

Genetic Variations of TAP1 Gene Exon 3 Affects Gene Expression and Escherichia coli F18 Resistance in Piglets

Firstly, our research group identified Sutai pigs’ phenotypes that exhibited extreme resistance and susceptibility to the Escherichia coli F18 respectively, and then eight ETEC (Enterotoxigenic Escherichia coli) F18-resistant piglets and eight ETEC F18-sensitive piglets were selected. Then, the TAP1 (Transporter associated with antigen processing) mRNA relative expression levels were analyzed in 11 tissues of the resistant and susceptible phenotypes. Simultaneously, we detected the genetic variations in exon 3 of the TAP1 gene and evaluated the TAP1 mRNA expression levels among the different genotype pigs to study the effects of the genetic variation on gene expression, and the E. coli F18 resistance. The results revealed higher expression levels in the resistant genotypes than that in the susceptible genotypes in 11 tissues, with significant differences in the spleen, lymph node, lung, thymus, duodenum and jejunum. Furthermore, a G729A mutation was identified in the TAP1 gene exon 3, and this mutation deviates from Hardy-Weinberg equilibrium (p < 0.01). The TAP1 mRNA levels in GG genotype were significantly higher than that in the other two genotypes, with significant differences in the liver, lung, kidney, thymus, lymph node, duodenum and jejunum tissues. We speculated that high expression of the TAP1 gene might confer resistance against the E. coli F18, the G729A mutation had a significant effect on the mRNA expression, and individuals with the GG genotype possessed a stronger ability to resist the E. coli F18 infection.     

Exercise Pretreatment Promotes Mitochondrial Dynamic Protein OPA1 Expression after Cerebral Ischemia in Rats

Exercise training is a neuroprotective strategy in cerebral ischemic injury, but the underlying mechanisms are not yet clear. In the present study, we investigated the effects of treadmill exercise pretreatment on the expression of mitochondrial dynamic proteins. We examined the expression of OPA1/DLP1/MFF/Mfn1/Mfn2, which regulatesmitochondrial fusion and fission, and cytochrome C oxidase subunits (COX subunits), which regulatemitochondrial functions, after middle cerebral artery occlusion (MCAO) in rats. T2-weighted magnetic resonance imaging (MRI) was evaluated as indices of brain edema after ischemia as well. Treadmill training pretreatment increased the expression levels of OPA1 and COXII/III/IV and alleviated brain edema, indicating that exercise pretreatment provided neuroprotection in cerebral ischemic injury via the regulation of mitochondrial dynamics and functions.