Protection against Atlantic halibut nodavirus in turbot is induced by recombinant capsid protein vaccination but not following DNA vaccination

Fish nodaviruses (betanodaviruses) are small, non-enveloped icosahedral single-stranded positive-sense RNA viruses that can cause viral encephalopathy and retinopathy (VER) in a number of cultured marine teleost species, including Atlantic halibut (Hippoglossus hippoglossus). A recombinant protein vaccine and a DNA vaccine were produced, based on the same capsid-encoding region of the Atlantic halibut nodavirus (AHNV) genome, and tested for protection in juvenile turbot (Scophthalmus maximus). Vaccine efficacy was demonstrated in the fish vaccinated with recombinant capsid protein but not in the DNA-vaccinated fish, despite the fact that in vivo expression of the DNA vaccine-encoded antigen was confirmed by RNA in situ hybridisation and immunohistochemistry. Combined DNA and recombinant vaccine administration did not improve the effect of the latter. Surprisingly, fish vaccinated with 50 microg recombinant protein demonstrated a threefold lower survival rate than the two groups that received 10 microg recombinant protein. Neither the recombinant protein vaccine nor the DNA vaccine induced anti-viral antibodies 9 weeks after immunisation, while antibodies reactive with the recombinant protein were detectable mainly in fish vaccinated with 50 microg recombinant protein. The study also demonstrates evidence of viral replication inside the myocytes of intramuscularly challenged fish.     

An intron splicing enhancer containing a G-rich repeat facilitates inclusion of a vertebrate micro-exon.

The average length of a vertebrate axon is approximately 130 nt. Decreasing the size of an internal axon to less than 51 nt induces axon skipping, implying a minimal size for exons. A few constitutively included internal exons, however, are extremely small. To investigate if such micro-exons require special mechanisms for their inclusion, we studied the sequences necessary for inclusion of a 6-nt axon from chicken cardiac troponin T (cTNT). In vivo, the cTNT micro-exon was not included in mRNA unless accompanied by a 134-nt sequence located next to the micro-exon in the downstream intron. Increasing the length of the micro-exon alleviated the requirement for the intron element, indicating that the lack of inclusion of the micro-exon in the absence of a facilitating sequence was due to its small size, rather than suboptimal splice sites. The intron element contained six copies of a G-rich 7-nt sequence. Multimers of the repeat supported exon inclusion, indicating that the repeat sequence is an important part of the intron element. The entire intron element activated inclusion of a heterologous 7-nt exon, suggesting that the intron element is a general enhancer for the splicing of micro-exons. In vitro, the intron element and the repeated sequence facilitated splicing of a heterologous exon. Because of the ability of the cTNT intron element to facilitate the splicing of heterologous exons, we have termed the element an intron splicing enhancer (ISE). Interestingly, the ISE demonstrated position independence in that it facilitated inclusion of the heterologous micro-exon when placed either upstream or downstream of the micro-exon. In vitro, the ISE or copies of the ISE G-rich repeat stimulated splicing of an adjacent intron. The ISE thus becomes one of only a few characterized ISEs containing a G-rich repeat and the first to work both upstream and downstream of a target axon.     

Energy landscapes shape microbial communities in hydrothermal systems on the Arctic Mid-Ocean Ridge

Methods developed in geochemical modelling combined with recent advances in molecular microbial ecology provide new opportunities to explore how microbial communities are shaped by their chemical surroundings. Here, we present a framework for analyses of how chemical energy availability shape chemotrophic microbial communities in hydrothermal systems through an investigation of two geochemically different basalt-hosted hydrothermal systems on the Arctic Mid-Ocean Ridge: the Soria Moria Vent field (SMVF) and the Loki''s Castle Vent Field (LCVF). Chemical energy landscapes were evaluated through modelling of the Gibbs energy from selected redox reactions under different mixing ratios between seawater and hydrothermal fluids. Our models indicate that the sediment-influenced LCVF has a much higher potential for both anaerobic and aerobic methane oxidation, as well as aerobic ammonium and hydrogen oxidation, than the SMVF. The modelled energy landscapes were used to develop microbial community composition models, which were compared with community compositions in environmental samples inside or on the exterior of hydrothermal chimneys, as assessed by pyrosequencing of partial 16S rRNA genes. We show that modelled microbial communities based solely on thermodynamic considerations can have a high predictive power and provide a framework for analyses of the link between energy availability and microbial community composition.     

Validation of an in vitro RNA processing system for CT/CGRP precursor mRNA.

The pre-mRNA encoding calcitonin (CT) and calcitonin gene-related peptide (CGRP) is differentially processed in a tissue-specific fashion to include or exclude the calcitonin-specific exon 4. A minigene containing a viral first exon and exons 4, 5, and 6 from the human CT/CGRP gene was correctly processed in transfected HeLa or F9 teratocarcinoma cells to produce mRNA that included or excluded exon 4, respectively. This processing decision could be reproduced in vitro using nuclear extracts from these two cell lines and an RNA precursor from a similar minigene. Supplementation of extract from HeLa cells with extract from F9 cells resulted in the F9 splicing pattern in which exon 4 was excluded. This model system may be useful for the purification of splicing factors important in the regulation of this splice choice.     

饲料中植物油替代鱼油对大西洋鲑肝细胞油酸跨膜吸收的影响简

以[1-14C]油酸(oleic acid;18:1n-9,OA)为指示剂,研究了不同饲料油源饲喂下大西洋鲑肝细胞膜脂肪酸组成受到改变时该细胞对OA吸收的状况,以探讨植物油(Vegetable oil,VO)替代鱼油(Fish oil,FO)对大西洋鲑肝细胞脂肪酸跨膜吸收的影响,为大西洋鲑饲料中植物油替代鱼油的可行性提供理论依据。试验先以鱼油和大豆油为油源配制两种全价配合饲料,分别饲喂大西洋鲑幼鲑5个月,使其产生不同的脂肪酸组成。在饲养结束后,分离并培养试验鱼肝细胞,将细胞与[1-14C]OA及37.5μmol/L OA(1/30,mol/mol,0.3μCi/瓶)共孵育2h,收集并测定细胞内OA放射活性,再计算细胞内OA吸收量[nmol/(h·million cells)]。同时,试验采取RT-PCR方法测定了细胞脂肪酸运送蛋白(Fatty acid transport protein,FATP)、脂肪酸移位蛋白(Fatty acid translocase,FAT/CD36)的基因表达量。结果表明,FO和VO组肝细胞对OA吸收分别为(0.924±0.258)及(0.888±0.179)nmol/(h·million cells),两组间无显著差异(P0.05)。RT-PCR的检测结果表明,FAT/CD36和FATP基因表达量在FO与VO两组间均无显著差异(P0.05)。结果表明,从植物油替代鱼油不影响大西洋鲑肝细胞对长链脂肪酸的跨膜吸收方面来看,大西洋鲑饲料中以植物油替代鱼油具有可行性。     

Elevated serum MMP-9/TIMP-1 ratio in patients with homozygous familial hypercholesterolemia: Effects of LDL-apheresis

ObjectiveMatrix degradation within an atherosclerotic plaque is an important pathogenic factor in atherosclerosis, and is largely modulated by the balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors (i.e., tissue inhibitor of MMPs [TIMPs]). Familial hypercholesterolemia (FH) is a rare inherited disorder associated with premature coronary heart disease. The aim of the present study was to examine MMP-9 and TIMP-1 on plasma and cellular mRNA levels in homozygous FH patients (n = 7) compared with age- and sex-matched heterozygous FH patients (n = 6), and with healthy subjects (n = 7), and to test whether once-weekly LDL-apheresis (three consecutive sessions) of homozygous FH patients show short-term effects on these variables.ResultsThe main findings were that (i) Compared to healthy control subjects, homozygous FH patients have significantly higher serum levels of MMP-9 and lower levels of TIMP-1, and consequently significantly higher MMP-9/TIMP-1 ratio, potentially reflecting higher MMP-9 activity. (ii) Peripheral blood mononuclear cells (PBMC) isolated from FH homozygotes have significantly higher mRNA levels of MMP-9 compared to cells from heterozygotes. (iii) TNFα-stimulated PBMC from FH homozygotes released borderline-significantly more MMP-9 than cells from heterozygotes and healthy controls. (iv) LDL-apheresis (one day before treatment versus fifteen days later, on the day after the weekly treatment) had no significant short-term effect on any of the MMP-9 and TIMP-1 variables measured in serum and cells.ConclusionsThe data may suggest that homozygous FH patients have an enhanced matrix degrading potential as compared with heterozygous FH patients and healthy controls, potentially contributing to the increased cardiovascular risk observed in these patients.     

Phage P22 tail protein: gene and amino acid sequence

The tail structure of the Salmonella phage P22 mediates both adsorption of the phage to its host and enzymatic hydrolysis of the bacterial O-antigen. The tail is an oligomeric structure, which is assembled from a single polypeptide species. We report here the amino- and carboxyl-terminal sequences of the P22 tail protein and the nucleotide sequence of its gene (gene 9). These data specify the complete amino acid sequence of the tail protein. The tail protein is a slightly acidic protein containing 666 amino acids. Comparison of the gene and protein sequences indicates that mature tail protein arises by cleavage of the initiator N-formyl-methionine from the nascent chain.