Isolation and expression of grass carp toll-like receptor 5a (CiTLR5a) and 5b (CiTLR5b) gene involved in the response to flagellin stimulation and grass carp reovirus infection

Toll-like receptor 5 (TLR5), a member of Toll-like receptors (TLRs) family and is responsible for the bacterial flagellin recognition in vertebrates, play an important role in innate immunity. In the study, two TLR5 genes of grass carp (Ctenopharyngodon idellus), named CiTLR5a and CiTLR5b, were cloned and analyzed. Both CiTLR5a and CiTLR5b are typical TLR proteins, including LRR motif, transmembrane region and TIR domain. The full-length cDNA of CiTLR5a is 3054 bp long, with a 2646 bp open reading frame (ORF), 78 bp 5' untranslated regions (UTR), and 330 bp 3' UTR. The full-length cDNA of CiTLR5b is 3326 bp, with a 2627 bp ORF, 95 bp 5' UTR, and 594 bp 3' UTR. Phylogenetic analysis showed that CiTLR5a and CiTLR5b were closed to the TLR5 of cirrhinus mrigala, cyprinus_carpio, and danio redo. Subcellular localization indicated that CiTLR5a and CiTLR5b shared similar localization pattern and may locate in the plasma membrane of transfected cells. Real-time quantitative PCR revealed CiTLR5a and CiTLR5b were constitutively expressed in all examined tissues, whereas the highest expressed tissue differed. Following exposure to flagellin and GCRV, CiTLR5a and CiTLR5b were up-regulated significantly. Moreover, the downstream genes of TLR5 signal pathway such as MyD88, NF-kappa B, IRF7, IL-1 beta, and TNF-alpha also up-regulated significantly, whereas the I kappa B gene was down-regulated, suggesting that CiTLR5a and CiTLR5b involved in response to flagellin stimulation and GCRV infection. The results obtained in the study would provide a new insight for further understand the function of TLR5 in teleost fish. (C) 2015 Elsevier Ltd. All rights reserved

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

RNA-seq profiles from grass carp tissues after reovirus (GCRV) infection based on singular and modular enrichment analyses

Hemorrhagic disease of the grass carp, Ctenopharyngodon idella, is a fatal disease in fingerlings and yearlings caused by a reovirus, GCRV. RNA-seq data from four diseased grass carp tissues (gill, intestine, liver and spleen) were obtained at 2 h before and six times after (2 h, 24 h, 48 h, 72 h, 96 h and 120 h) GCRV challenge. A total of 7.25 +/- 0.18 million (M) clean reads and 3.53 +/- 0.37 M unique reads were obtained per RNA-seq analysis. Compared with controls, there were 9060 unique differentially expressed genes (DEGs) in the four tissues at the six time points post-GCRV challenge. Hierarchical clustering analysis of the DEGs showed that the data from the six time points fell into three branches: 2 h, 24 h/48 h, and 72 h/96 h/120 h. Singular (SEA) and modular enrichment analyses of DEGs per RNA-seq dataset were performed based on gene ontology. The results showed that immune responses occurred in all four tissues, indicating that GCRV probably does not target any tissue specifically. Moreover, during the course of disease, disturbances were observed in lipid and carbohydrate metabolism in each of the organs. SEA of DEGs based on the Kyoto Encyclopedia of Genes and Genomes database was also performed, and this indicated that the complement system and cellular immunity played an important role during the course of hemorrhagic disease. The qPCR of pooled samples of duplicate challenge experiment were used to confirm our RNA-seq approach. (C) 2014 Elsevier Ltd. All rights reserved

El Diario de Pontevedra : periódico liberal: Ano XVIII Número 5130 - 1901 xuño 27

Background: Grass carp is an important farmed fish in China that is affected by serious disease, especially hemorrhagic disease caused by grass carp reovirus (GCRV). The mechanism underlying the hemorrhagic symptoms in infected fish remains to be elucidated. Although GCRV can be divided into three distinct subtypes, differences in the pathogenesis and host immune responses to the different subtypes are still unclear. The aim of this study was to provide a comprehensive insight into the grass carp response to different GCRV subtypes and to elucidate the mechanism underlying the hemorrhagic symptoms

Additional file 3: of Differences in responses of grass carp to different types of grass carp reovirus (GCRV) and the mechanism of hemorrhage revealed by transcriptome sequencing

Detailed information of the GO terms of the differentially expressed genes in the two groups. (XLSX 1291 kb

REGγ Mediated Regulation of p21Waf/Cip1, p16INK4a and p14ARF/p19ARF in Vivo

peer reviewedp21Waf/Cip1, p16INK4a and p14ARF (p19ARF in mice) have been demonstrated to be degraded by REGγ-proteasome pathway in an ATP- and ubiquitin-independent manner in vitro. However, the in vivo roles of REGγ mediated-degradation of p21Waf/Cip1, p16INK4a and p14ARF remain unclear. In this study, we showed enhanced expression of p21Waf/Cip1, p16INK4a and p19ARF in multiple tissues from REG–/– mice compared to REG+/+ mice. Furthermore, we examined the expression of p21Waf/Cip1, p16INK4a and p14ARF in different cancer tissues and observed that the REGγ protein levels were highly expressed in different human cancers while the level of p21Waf/Cip1, p16INK4a and p14ARF appears to be inversely corre- lated. These results demonstrate that REGγ may exert its function in physiological and pathological conditions through degradation of p21Waf/Cip1, p16INK4a and p14ARF in vivo

Global gene expression patterns of grass carp following compensatory growth

Background: Compensatory growth is accelerated compared with normal growth and occurs when growth-limiting conditions are overcome. Most animals, especially fish, are capable of compensatory growth, but the mechanisms remain unclear. Further investigation of the mechanism of compensatory growth in fish is needed to improve feeding efficiency, reduce cost, and explore growth-related genes

The REGγ-proteasome forms a regulatory circuit with IκBɛ and NFκB in experimental colitis.

Increasing incidence of inflammatory bowel disorders demands a better understanding of the molecular mechanisms underlying its multifactorial aetiology. Here we demonstrate that mice deficient for REGγ, a proteasome activator, show significantly attenuated intestinal inflammation and colitis-associated cancer in dextran sodium sulfate model. Bone marrow transplantation experiments suggest that REGγ's function in non-haematopoietic cells primarily contributes to the phenotype. Elevated expression of REGγ exacerbates local inflammation and promotes a reciprocal regulatory loop with NFκB involving ubiquitin-independent degradation of IκBɛ. Additional deletion of IκBɛ restored colitis phenotypes and inflammatory gene expression in REGγ-deficient mice. In sum, this study identifies REGγ-mediated control of IκBɛ as a molecular mechanism that contributes to NFκB activation and promotes bowel inflammation and associated tumour formation in response to chronic injury

The REG gamma-proteasome forms a regulatory circuit with I kappa B epsilon and NF kappa B in experimental colitis

Increasing incidence of inflammatory bowel disorders demands a better understanding of the molecular mechanisms underlying its multifactorial aetiology. Here we demonstrate that mice deficient for REG gamma, a proteasome activator, show significantly attenuated intestinal inflammation and colitis-associated cancer in dextran sodium sulfate model. Bone marrow transplantation experiments suggest that REG gamma's function in non-haematopoietic cells primarily contributes to the phenotype. Elevated expression of REG gamma exacerbates local inflammation and promotes a reciprocal regulatory loop with NF kappa B involving ubiquitin-independent degradation of I kappa B epsilon. Additional deletion of I kappa B epsilon restored colitis phenotypes and inflammatory gene expression in REG gamma-deficient mice. In sum, this study identifies REG gamma-mediated control of I kappa B epsilon as a molecular mechanism that contributes to NF kappa B activation and promotes bowel inflammation and associated tumour formation in response to chronic injury