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Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a predominant DNA sensor inducing the activation of the innate immune responses that produce proinflammatory cytokines and type I interferons, which has been well-investigated in mammals. However, chicken cGAS (chcGAS), which participates in avian innate immunity, has not been well-investigated. Here, we cloned the complete open reading frame sequence of chcGAS. Multiple sequence alignment and phylogenetic analysis revealed that chcGAS was homologous to mammalian cGAS. The chcGAS mRNA was highly expressed in the bone marrow and ileum. The subcellular localization of chcGAS was mainly in the cytoplasm, and partial co-localization was observed in the endoplasmic reticulum. Through overexpression and RNA interference, we demonstrated that chcGAS responded to exogenous dsDNA, HS-DNA, and poly(dA:dT), and to self dsDNA from the DNA damage response, thereby triggering the activation of STING/TBK1/IRF7-mediated innate immunity in both chicken embryonic fibroblasts and chicken liver cancer cells. Furthermore, downregulation of chcGAS enhanced the infection of fowl adenovirus serotype 4 in LMH cells. Our results demonstrated that chcGAS was an important cytosolic DNA sensor activating innate immune responses and may shed light on a strategy for preventing infectious diseases in the poultry industry.

Colon adenocarcinoma (COAD) is one of the most common malignant tumors with high morbidity and mortality rates worldwide. Due to the poor clinical outcomes, it is indispensable to investigate novel biomarkers for the diagnosis and prognosis of COAD. The aim of this study is to explore key genes as potential biomarkers for the diagnosis and prognosis of COAD for clinical utility. Gene expression profiles (GSE44076 and GSE44861) and gene methylation profile (GSE29490) were analyzed to identify the aberrantly methylated-differentially expressed genes by R language and Perl software. Function enrichments were performed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Moreover, hub genes were identified through protein-protein interaction (PPI) network. Besides, key genes were found by the module analysis and The Cancer Genome Atlas (TCGA) survival analysis. Finally, TCGA data and quantitative real-time polymerase chain reaction (RT-qPCR) was used to validate the key genes involved in COAD. Our study found two hypomethylation-high-expression genes (CXCL3 and CXCL8) in COAD tissues compared with the adjacent normal tissues. These results were also confirmed by RT-qPCR with 25 pairs of COAD and adjacent normal tissues. Meanwhile, low expression of the two genes was associated with poor survival in patients with COAD. CXCL3 and CXCL8 may serve as key genes in the diagnosis and prognosis for COAD.

In aquatic ecosystems, mercuric compounds are considered to be highly toxic to a great variety of aquatic biota ranging from bacteria to fishes. In the present study, the effects of different concentrations (0, 2.0, 6.0, 10.0, 14.0 and 18.0 μg·L − 1 ) of Hg 2 + on the life table demography of Brachionus calyciflorus were investigated at three densities (1.0 × 10 6 , 2.0 × 10 6 and 4.0 × 10 6 cells·mL − 1 ) of Scenedesmus obliquus . The results showed that compared to the controls, Hg 2 + at 14.0 and 18.0 μg·L − 1 significantly decreased both the average lifespan and the life expectancy at hatching, Hg 2 + at 2.0–18.0 μg·L − 1 shortened the generation time, and Hg 2 + at 18.0 μg·L − 1 decreased the net reproductive rate of B. calyciflorus cultured at 1.0 × 10 6 cells·mL − 1 of S. obliquus . Increase in algal food level reduced the negative effects of high concentrations of Hg 2 + on survival, reproduction and population growth of the rotifers. Hg 2 + at 6.0 μg·L − 1 , and 6.0 and 18.0 μg·L − 1 increased the intrinsic rate of population increase of the rotifers cultured at 1.0 × 10 6 and 2.0 × 10 6 cells·mL − 1 of S. obliquus , respectively. But Hg 2 + at 18.0 μg·L − 1 decreased the intrinsic rate of population increase of the rotifers cultured at 4.0 × 10 6 cells·mL − 1 of S. obliquus . Significantly nominal concentration–response relationships existed between Hg 2 + concentration and each of all the five life history variables of B. calyciflorus cultured at 1.0 × 10 6 cells·mL − 1 of S. obliquus , the generation time of the rotifers cultured at 2.0 × 10 6 cells·mL − 1 of S. obliquus , as well as the intrinsic rate of population increase of the rotifers cultured at 4.0 × 10 6 cells·mL − 1 of S. obliquus . Among all the life history variables, generation time was the most sensitive to mercury pollution. Since algal levels chosen in the present study are much higher than those available in natural waters, except in eutrophic water bodies, the effects of mercury on survival, reproduction and population growth of the rotifers in natural waters may be more severe than those extrapolated from the present study.

The strawberry vein banding virus (SVBV) open reading frame (ORF) VI encodes a P6 protein known as the RNA silencing suppressor. This protein is known to form inclusion like granules of various sizes and accumulate in both the nuclei and the cytoplasm of SVBV-infected plant cells. In this study, we have determined that the P6 protein is the only trans-activator (TAV) encoded by SVBV, and can efficiently trans-activate the translation of downstream gfp mRNA in a bicistron derived from the SVBV. Furthermore, the P6 protein can trans-activate the expression of different bicistrons expressed by different caulimovirus promoters. The P6 protein encoded by SVBV from an infectious clone can also trans-activate the expression of bicistron. Through protein-protein interaction assays, we determined that the P6 protein could interact with the cell translation initiation factor FveIF3g of Fragaria vesca and co-localize with it in the nuclei of Nicotiana benthamiana cells. This interaction reduced the formation of P6 granules in cells and its trans-activation activity on translation.

Spatial distribution characteristics of available phosphorus (AP) and total phosphorus (TP) in wetland soils withPhragmites australis,Suaeda salsaandTamarix chinnensiswere investigated and their influencing factors was also indentified using principal correspondence analysis for ordination in the Yellow River Delta of China. Our results showed that TP and AP contents in both sites withPhragmites australisandSuaeda salsa, and the horizontal distributions of AP content in three sites exhibited similar distribution characteristics to that of AP:TP ratios. Additionally, soil properties such as soil moisture, salinity, total nitrogen, total carbon and pH values were the important influencing factors of higher spatial variability of AP and TP.

PEI and chitosan are considered to be promising non-viral gene delivery vectors. To improve the transfection efficiency of chitosan, we linked chitosan with polyethylenimine (PEI, Mw = 1.8 kDa) by 1,1′-carbonyldiimidazole to form a complex. The composition, particle size, as well as the zeta potential of this chitosan-linked-PEI (CP) complex were measured. And the DNA binding ability, cytotoxicity, and gene transfection efficiency of CP complex were also investigated in cancer cells. In HepG2, A549 and HeLa cells, CP complex exhibited lower cytotoxicity as compared with PEI25KDa (Mw = 25 kDa), a positive control proved to be an efficient gene transfection polymer. Likewise, it showed good transfection efficiency in these cancer cell lines. Specifically, the long-term transfection efficiency of CP was higher than PEI25KDa as demonstrated by the in vitro cancer cell model. The confocal laser scanning microscopy data showed the time for CP to enter the nucleus was 4 h, which was longer than that of PEI25KDa but shorter than that of chitosan. Furthermore, CP complexes were used as a gene carrier to deliver the CCL22 gene into H22 cells. When these gene-altered cells were inoculated in mice, the tumor growth rate was significantly decreased, indicating the CP copolymer was a promising vector for the therapeutic gene delivery.

In this study, the cytotoxicity and transfection efficiency of using chitosan/DNA complex combined with poly(ethylenimine) (PEI) were investigated. The combination of PEI with the chitosan/DNA complex markedly enhanced the gene expression of HeLa cells to 1000-fold of that induced by chitosan alone. PEI's cytotoxicity was considerably decreased upon combination with the chitosan/DNA complex. Furthermore, the PEI/chitosan/DNA could maintain the gene expression efficiency in the presence of serum.

For designing a complex vector that has the advantages of both polyethylenimine (PEI) and chitosan for gene delivery, a PEI/chitosan/DNA complex was constructed at various N/P ratios (the ratios of moles of the amine groups of cationic polymers to those of the phosphate ones of DNA) and both the cytotoxicity and the transfection efficiency of the vector were evaluated. The results demonstrated that the chitosan/DNA binding degree was depended on the N/P ratio. The mean size of the complex vector was between 100 nm and 150 nm. Compared with PEI/DNA, the complex vector (PEI/chitosan/DNA with chitosan/DNA N/P=4, PEI/DNA N/P=10) appeared to have low cytotoxicity, which maintained the cell survival rate at greater than 80%, and showed higher transfection efficiency of nearly 1000 fold compared with that using chitosan/DNA alone. Furthermore, the expression efficiency of the complex vector carrying enhanced green fluorescent protein was not inhibited in the presence of serum in both HeLa cells and A549 cells. The PEI/chitosan complex may be a promising gene carrier that has high transfection efficiency as well as low cytotoxicity.

Qing-Qing Zhao,1,2 Yu-Lan Hu,1 Yang Zhou,3 Ni Li,1 Min Han,1 Gu-Ping Tang,4 Feng Qiu,2 Yasuhiko Tabata,5 Jian-Qing Gao,11Institute of Pharmaceutics, Zhejiang University, Hangzhou, China; 2Department of Pharmacy, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China; 3Institute of Biochemistry, Iowa State University, Ames, IA, USA; 4Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou, China; 5Institute for Frontier Medical Sciences, Kyoto University, Kyoto, JapanBackground: The success of gene transfection is largely dependent on the development of a vehicle or vector that can efficiently deliver a gene to cells with minimal toxicity.Methods: A liver cancer-targeted specific peptide (FQHPSF sequence) was successfully synthesized and linked with chitosan-linked polyethylenimine (CP) to form a new targeted gene delivery vector called CPT (CP/peptide). The structure of CPT was confirmed by 1H nuclear magnetic resonance spectroscopy and ultraviolet spectrophotometry. The particle size of CPT/DNA complexes was measured using laser diffraction spectrometry and the cytotoxicity of the copolymer was evaluated by methylthiazol tetrazolium method. The transfection efficiency evaluation of the CP copolymer was performed using luciferase activity assay. Cellular internalization of the CP/DNA complex was observed under confocal laser scanning microscopy. The targeting specificity of the polymer coupled to peptide was measured by competitive inhibition transfection study. The liver targeting specificity of the CPT copolymer in vivo was demonstrated by combining the copolymer with a therapeutic gene, interleukin-12, and assessed by its abilities in suppressing the growth of ascites tumor in mouse model.Results: The results showed that the liver cancer-targeted specific peptide was successfully synthesized and linked with CP to form a new targeted gene delivery vector called CPT. The composition of CPT was confirmed and the vector showed low cytotoxicity and strong targeting specificity to liver tumors in vitro. The in vivo study results showed that interleukin-12 delivered by the new gene vector CPT/DNA significantly enhanced the antitumor effect on ascites tumor-bearing imprinting control region mice as compared with polyethylenimine (25 kDa), CP, and other controls, which further demonstrate the targeting specificity of the new synthesized polymer.Conclusion: The synthesized CPT copolymer was proven to be an effective liver cancer-targeted vector for therapeutic gene delivery, which could be a potential candidate for targeted cancer gene therapy.Keywords: targeting, peptide, polyethylenimine, chitosan, antitumor