Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

Aims: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo-induced antigen technology (IVIAT). Methods and Results: The convalescent-phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo-induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion-mutated, and the growth and 50% lethal dose (LD50) of these mutants were evaluated. Conclusions: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. Significance and Impact of the Study: To the best of our knowledge, this is the first report describing in vivo-expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.

Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

Aims: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo-induced antigen technology (IVIAT). Methods and Results: The convalescent-phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo-induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion-mutated, and the growth and 50% lethal dose (LD50) of these mutants were evaluated. Conclusions: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. Significance and Impact of the Study: To the best of our knowledge, this is the first report describing in vivo-expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.

Construction and characterization of a live, attenuated esrB mutant of Edwardsiella tarda and its potential as a vaccine against the haemorrhagic septicaemia in turbot, Scophthamus maximus (L.)

The esrB gene of Edwardsiella tarda, which encodes a regulator protein of the type III secretion system, was mutated by the unmarked deletion method and reintroduced by allelic exchange into the chromosome of E. tarda LSE40 by means of the suicide vector pRE 112. The LSE40 esrB mutant was highly attenuated when inoculated intraperitoneally into turbot Scophthamus maximus L., showing a 50% lethal dose of 10(8.1) cfu/fish. The esrB mutants were not recoverable from the internal organs at 14 days post-inoculation. Vaccination with a single dose of 10(5)-10(7) cfu/fish of the esrB mutant elicited significant protection against the wildtype strain of E. tarda LSE40 (relative percentage survival > 50%). The protection correlated well with the antibody titres in the serum of vaccinated fish. (c) 2006 Elsevier Ltd. All rights reserved.

Intraperitoneal injection of extracted microcystins results in hypovolemia and hypotension in crucian carp (Carassius auratus)

Circulatory responses of crucian carp injected intraperitoneally with extracted micro-cystins (MCs) were studied at sublethal and lethal doses (150 and 600 mu g MC kg(-1) body mass, respectively). Mean arterial blood pressure (MAP), heart rate, hematocrit (Hct), red blood cell (RBC) counts, and circulating blood volume (BV) were assayed at 0, 1, 3, 12, 24, and 48 h post-toxin administration. MAP decreased significantly in a dose-dependent manner over time. Within the 48-h test period, the lethal dose as well as the sublethal dose resulted in a steady decline of MAP without recovery. Heart rate significantly increased within 24 h post-injection as blood pressure significantly dropped, then showed a terminal decline to the control level. The dose-dependent decreases in BV and Hct were directly related to the drop in MAP. Intraperitoneal injection of a lethal dose of MCs led to hepatic and gill hemorrhage. Consequently, crucian carp given MCs suffered from hypovolemic hypotensive shock. (C) 2009 Elsevier Ltd. All rights reserved.

Investigation on intake, accumulation and toxicity of microcystins to silver carp

Daily intake and accumulation of microcystins (MCYSTs, MCs) in silver carp (Hypophthalmichthys molitrix) were investigated under lab conditions by feeding the fish exclusively with fresh toxic Microcystis bloom at a density of 6 x 10(9) algal cells L-1. The medial lethal dose (LD50) of microcystin-LR to silver carp was estimated to be 270 mu g kg(-1) body-weight, underlining its strong resistance to toxic Microcystis bloom. It can survive after being ingested with high doses of microcystins (about 10 mg kg(-1)) during the 28-days feeding experiment. Enzyme-linked immuno-sorbent assay results show that microcystin concentrations in muscle and liver are 1.57 +/- 0.31 mu g kg(-1) and 4.28 +/- 1.64 mg kg(-1) fresh weight. The former is much lower than the World Health Organization limit recommended for human consumption. These results suggest that silver carps can be widely used in cyanobacterial bloom control, and consumption of fish muscles is safe for human beings.

Isolation and analysis of the vaccine potential of an attenuated Edwardsiella tarda strain

Edwardsiella tarda is an important aquaculture pathogen that can infect a wide range of marine and freshwater fish worldwide. In this study, a modified E. tarda strain, TX5RM, was selected by multiple passages of the pathogenic E. tarda strain TX5 on growth medium containing the antibiotic rifampicin. Compared to the wild type strain, the rifampicin-resistant mutant TX5RM (i) shows drastically increased median lethal dose and reduced capacity to disseminate in and colonize fish tissues and blood; (ii) exhibits slower growth rates when cultured in rich medium or under conditions of iron depletion; and (iii) differs in the production profile of whole-cell proteins. The immunoprotective potential of TX5RM was examined in a Japanese flounder (Paralichthys olivaceus) model as a vaccine delivered via intraperitoneal injection, oral feeding, bath immersion, and oral feeding plus immersion. All the vaccination trials, except those of injection, were performed with a booster at 3-week after the first vaccination. The results showed that TX5RM administered via all four approaches produced significant protection, with the highest protection levels observed with TX5RM administered via oral feeding plus immersion, which were, in terms of relative percent of survival (RPS), 80.6% and 69.4% at 5- and 8-week post-vaccination, respectively. Comparable levels of specific serum antibody production were induced by TX5RM-vaccinated via different routes. Microbiological analyses showed that TX5RM was recovered from the gut, liver, and spleen of the fish at 1-10 days post-oral vaccination and from the spleen, liver, kidney, and blood of the fish at 1-14 days post-immersion vaccination. Taken together, these results indicate that TX5RM is an attenuated E. tarda strain with good vaccine potential and that a combination of oral and immersion vaccinations may be a good choice for the administration of live attenuated vaccines. (C) 2010 Elsevier Ltd. All rights reserved.

Tissue distribution and depuration of the extracted hepatotoxic cyanotoxin microcystins in crucian carp (Carassius carassius) intraperitoneally injected at a sublethal dose

An acute toxicity experiment was conducted by intraperitoneal injection with a sublethal dose of extracted microcystins (MCs), 50 mu g MC-LR (where L = leucine and R = arginine) equivalent/kg body weight (BW), to examine tissue distribution and depuration of MCs in crucian carp (Carassius carassius). Liver to body weight ratio increased at 3, 12, 24, and 48 h postinjection compared with that at 0 h (p < 0.05). MC concentrations in various tissues and aquaria water were analyzed at 1, 3, 12, 24, 48, and 168 h postinjection using liquid chromatography coupled with mass spectrometry (LC-MS). The highest concentration of MCs (MC-RR + MC-LR) was found in blood, 2 -270 ng/g dry weight (DW), followed by heart (3 -100 ng/g DW) and kidney (13 -88 ng/g DW). MC levels were relatively low in liver, gonad, intestine, spleen, and brain. MC contents in gills, gallbladder, and muscle were below the limit of detection. Significant negative correlation was present between MC-RR concentration in blood and that in kidney, confirming that blood was important in the transportation of MC-RR to kidney for excretion. Rapid accumulation and slow degradation of MCs were observed in gonad, liver, intestine, spleen, and brain. Only 0.07% of injected MCs were detected in liver. The recovery of MCs in liver of crucian carp seemed to be dose dependent.

Isolation of a lethal rhabdovirus from the cultured Chinese sucker Myxocyprinus asiaticus

A rhabdovirus was found to be associated with a lethal hemorrhagic disease in the cultured Chinese sucker Myxocyprinus asiaticus Bleeker. The rhabdovirus was amplified and isolated from the infected GCO, (grass carp ovary) cells. In ultrathin sections of liver cells from the diseased fish, the virus particles exhibited the characteristic bacilliform morphology, and budded through vesicle membranes of the infected cells. The isolated rhabdovirus particles were found to have a bacilliform morphology with 2 rounded ends rather than a typical flat base. The virus particles were measured and ranged in size from 150 to 200 nm in length and 50 to 60 nm in diameter. Most other characteristics, including their size, extensive virus infectivity to fish cell Lines, strong cytopathogenic effects, stability at high temperatures, vesicle formation in infected cells, structure protein electrophoretic patterns and the presence of an RNA genome, very closely resembled those of other fish rhabdoviruses. At present it is not known if this is a novel virus species or if it is an isolate of a known fish rhabdovirus. Until a confirmed identification can be made, we will temporarily refer to this virus as Chinese sucker rhabdovirus (CSRV).

Pre-irradiation with low-dose C-12(6+) beam significantly enhances the efficacy of AdCMV-p53 gene therapy in human non-small lung cancer

The combination of ionizing radiation and gene therapy has been investigated. However, there are very few reports about the combination of heavy-ion irradiation and gene therapy. To determine if the pre-exposure to low-dose heavy ion beam enhances the suppression of AdCMV-p53 on non-small lung cancer (NSLC), the cells pre-irradiated or non-irradiated were infected with 20, 40 MOI of AdCMV-p53. Survival fraction and the relative biology effect (RBE) were determined by clonogenic assay. The results showed that the proportions of p53 positive cells in C-12(6+) beam induced AdCMV-p53 infected cells were more than 90%, which were significantly more than those in gamma-ray induced AdCMV-p53 infected cells. The pre-exposure to low-dose 12C6+ beam significantly prevented the G(0)/G(1) arrest and activated G(2)/M checkpoints. The pre-exposure to C-12(6+) beam significantly improved cell to apoptosis. RBEs for the C-12(6+)+ AdCMV-p53 infection groups were 30%-60%,20% -130% and 30%-70% more than those for the C-12(6+)_irradiated only, AdCMV-p53 infected only, and gamma-irradiation induced AdCMVp53 infected groups, respectively. The data suggested that the pre-exposure to low-dose C-12(6+) beam significantly promotes exogenous p53 expression in NSLC, and the suppression of AdCMV-p53 gene therapy on NSLC.

The hyper-radiosensitivity effect of human hepatoma SMMC-7721 cells exposed to low dose gamma-rays and C-12 ions

Hypersensitive response of mammalian cells in cell killing to X- and gamma-rays has been reported at doses below 1 Gy. The purpose of this study was to examine the low dose sensitivity of human hepatoma SMMC-7721 cells irradiated with Co-60 gamma-rays and 50 MeV/u C-12 ions. Experiments using gamma-rays and charged particle irradiation were performed, particularly in the low dose range from 0 to 2 Gy. The survival effect of SMMC-7721 cells was measured by means of standard clonogenic assay in conjunction with a cell sorter. The result indicates SMMC-7721 cells showed hyper-radiosensitive response at low doses and increased radio-resistance at larger single doses for the carbon ions (LET = 45.2 keV/mu m) and the gamma-rays. However, the HRS/IRR effect caused by high-LET irradiation is different from that by low-LET radiation. This might possibly be due to the difference in the mode of energy deposition by particle beam and low-LET irradiation.

High-efficiency transfer and expression of AdCMV-p53 in human hepatocellular carcinoma cells induced by low-dose carbon-ion radiation

Objective To investigate whether the irradiation with C-beam could enhance adenovirus-mediated transfer and expression of p53 in human hepatocellular carcinoma. Materials and methods HepG2 cells were exposed to C-beam or gamma-ray and then infected with replicationdeficient adenovirus recombinant vectors containing human wild-type p53 or green fluorescent protein, respectively. The transfer efficiency and expression level of the exogenous gene were detected by flow cytometric analysis. Cell survival fraction was detected by clonogenic assay. Results The transfer frequency in C-beam or gamma-irradiated groups increased by 50-83% and 5.7-38.0% compared with the control, respectively (P < 0.05). Compared with C-beam alone, p53 alone, and gamma-ray with p53, the percentages of p53 positive cells for 1 Gy C-beam with p53 increased by 56.0-72.0%, 63.5-82.0%, and 31.3-72.5% on first and third day after the treatments, respectively (P < 0.05). The survival fractions for the 2Gy C-bearn and AdCMV-p53 infection groups decreased to similar to 2%. Conclusion C-beam irradiation could significantly promote AdCMV-green fluorescent protein transfer and expression of p53.

应用力学的50年变迁——兼评美国《应用力学杂志》创刊50周年特刊

<正> 1983年是美国机械工程协会主办的《应用力学杂志》(Journal of Applied Mechanics)创刊50周年。50年前,该刊编辑部登载了Timoshenko等人合写的一篇题为“应用力学的进展”的文章作为发刊词,该文仅涉及当时一般力学与经典连续介质力学若干领域的一些研究状况。50年后的今天,该刊编辑部继承了以往的传统,邀请37位专家(包括力学界的前辈与

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1955年，钱学森冲破重重阻挠回到祖国，并与钱伟长等一道创建了中国科学院力学研究所。2005年12月6日，我所召开“庆祝中国科学院力学研究所建所50周年暨钱学森同国50周年大会”，中国科学院副院长曹健林、两弹一星功勋奖章获得者陈能宽院士、钱学森的学术秘书涂季同志、钱学森的儿子钱永刚、女儿钱永真、国防科工委、总装备部、中国工程物理研究院、中国海洋石油总公司、国家自然科学基金委、兄弟院所和高校的有关领导和专家学者以及力学所职工共计300余人出席了大会，路甬祥院长、白春礼常务副院长、原国务委员、中国科学院原党组书记张劲夫同志为力学所建所50年和钱老回国50年题词。

中国力学学会50年

<正>序1955年8月,通过外交途径,钱学森将离美回国。在他向导师冯?卡门教授告别时,呈上的是两本新著:《物理力学》和《工程控制论》,此时此刻,两人心中无比激动。同年10月2日,钱学森乘坐克里夫兰号邮轮到达九龙,随即过罗湖,踏上了阔别20年的故土。次年10月,郭永怀也回到北

Growth of alpha-Al2O3:C crystal with highly sensitive optically stimulated luminescence

In this work. an alpha-Al2O3:C crystal was directly grown by the temperature gradient technique (TGT) using Al2O3 and graphite powders as the raw materials. The optical, optically stimulated luminescence (OSL) properties and dosimetric characteristics of as-grown crystal were investigated. As-grown alpha-Al2O3:C crystal shows strong absorption band at 205, 230 and 256 nm. Three-dimensional thermoluminescence (TL) emission spectrum of the crystal shows a single emission peak at similar to 415 nm. The OSL decay curve can be fitted to two exponentials, the faster component and the slower component. The OSL response of the crystal shows a linear-sublinear-saturation characteristic. As-grown alpha-Al2O3:C crystal shows excellent linearity in the dose range from 5 x 10(-6) to 50 Gy. For doses higher than the saturation dose (100 Gy). the OSL sensitivity decreases as the dose increases. Crown Copyright (C) 2008 Published by Elsevier B.V. All rights reserved.