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Several cases of humans infected with the H9N2 avian influenza virus (AIV) have been described since 1999; however, the infectivity and pathogenicity of H9N2 in humans is not well defined. A non-human primate model in rhesus macaques was developed to study H9N2 virus infections as a means of better understanding the pathogenesis and virulence of this virus, in addition to testing antiviral drugs. Rhesus macaques inoculated with H9N2 AIV presented with biphasic fever and viral pneumonia. H9N2 was recovered from nasal washes and pharyngeal samples up to days 7-9 postinfection, followed by an increase in HI (hemagglutination inhibition) antibody titers. Tissue tropism and immunohistochemistry indicated that H9N2 AIV replicated in the upper respiratory tract (turbinate, trachea, and bronchus) and in all lobes of the lung. Our data suggest that rhesus macaques are a suitable animal model to study H9N2 influenza virus infections, particularly in the context of viral evolution and pathogenicity.

Proline-rich proteins contribute to cell wall structure of specific cell types and are involved in plant growth and development. In this study, a fiber-specific gene, GhPRP5, encoding a proline-rich protein was functionally characterized in cotton. GhPRP5 promoter directed GUS expression only in trichomes of both transgenic Arabidopsis and tobacco plants. The transgenic Arabidopsis plants with overexpressing GhPRP5 displayed reduced cell growth, resulting in smaller cell size and consequently plant dwarfs, in comparison with wild type plants. In contrast, knock-down of GhPRP5 expression by RNA interference in cotton enhanced fiber development. The fiber length of transgenic cotton plants was longer than that of wild type. In addition, some genes involved in fiber elongation and wall biosynthesis of cotton were up-regulated or down-regulated in the transgenic cotton plants owing to suppression of GhPRP5. Collectively, these data suggested that GhPRP5 protein as a negative regulator participates in modulating fiber development of cotton.

Replication and transmission of avian influenza virus (AIV) in domestic dogs and cats may pose a risk to humans. The susceptibility of cats and dogs to H9N2 influenza virus was evaluated by intranasally or orally inoculating animals with an H9N2 influenza virus. Cats had recoverable virus in respiratory tissues and the olfactory bulb three days post-inoculation and shed H9N2 virus into nasal washes and pharyngeal swabs from day 2 through day 10 post-inoculation. Virus was recovered from respiratory tissues of dogs three days post-inoculation, but was not detected in nasal washes or pharyngeal swabs. While no virus shedding or replication was detected in cats or dogs following consumption of H9N2-infected chicks, one of two cats and one of two dogs seroconverted. Two of three naïve contact cats seroconverted following co-housing with cats that were intranasally inoculated with H9N2 virus, whereas none of the three naïve contact dogs seroconverted. Our results demonstrate that H9N2 AIV can infect domestic cats and dogs via the upper respiratory tract and indicate that cats are more susceptible than dogs to H9N2 AIV. These findings suggest that domestic dogs and cats may serve as host species contributing to the adaptation of H9N2 viruses in mammals., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objective: To sequence the complete genome of CVS-11 strain and establish a reverse genetic system of CVS-11 to further study its pathogenic mechanism, virulence genes and antigenic sites., Methods: We amplified12 fragments covering the complete genome of the CVS-11 strain by RT-PCR, and then cloned to pEASY-Blunt vector for sequencing the complete genome of CVS-11. We analyzed single restriction enzyme sites of the full length cDNA of the CVS-11 strain by DNAMAN and designed 4 pairs of specific primers. We amplified the full-length cDNA of CVS-11 by RT-PCR. We obtained four fragments and cloned into pcDNA3. 1. We named the full-length cDNA plasmid pcDNA3. 1-CVS-11. We also cloned helper plasmids pcDNA3.1-N, P, L and G expressing N, P, L and G protein of CVS-11 strain, respectively. We co-transfected NA cells with the full-length plasmid and four helper plasmids. We identified the supernatant of the transfected and then passaged NA cells by immunofluorescence staining and RT-PCR and found the recombinant virus rCVS-11 rescued successfully., Results: Sequencing results showed that the complete genome of CVS-11 was composed of 11 927 nucleotides. The complete genome of CVS-11 encoded 5 structure proteins and gene array was the same as other reported rabies viruses. We successfully constructed a reverse genetic system of CVS-11, namely the full length plasmid pcDNA3. 1-CVS-11 and 4 help plasmids pcDNA3. 1-N, P, L, G and rescued the rCVS-11 from a full-length infectious cDNA clone., Conclusion: The reverse genetic system of the CVS-11 strain laid the foundation for future studies on rabies virus.

Arabinogalactan proteins (AGPs) are involved in many aspects of plant development. In this study, biochemical and genetic approaches demonstrated that AGPs are abundant in developing fibers and may be involved in fiber initiation and elongation. To further investigate the role of AGPs during fiber development, a fasciclin-like arabinogalactan protein gene (GhFLA1) was identified in cotton (Gossypium hirsutum). Overexpression of GhFLA1 in cotton promoted fiber elongation, leading to an increase in fiber length. In contrast, suppression of GhFLA1 expression in cotton slowed down fiber initiation and elongation. As a result, the mature fibers of the transgenic plants were significantly shorter than those of the wild type. In addition, expression levels of GhFLAs and the genes related to primary cell wall biosynthesis were remarkably enhanced in the GhFLA1 overexpression transgenic fibers, whereas the transcripts of these genes were dramatically reduced in the fibers of GhFLA1 RNA interference plants. An immunostaining assay indicated that both AGP composition and primary cell wall composition were changed in the transgenic fibers. The levels of glucose, arabinose, and galactose were also altered in the primary cell wall of the transgenic fibers compared with those of the wild type. Together, our results suggested that GhFLA1 may function in fiber initiation and elongation by affecting AGP composition and the integrity of the primary cell wall matrix.

The asymmetric unit of the title compound, C6H9N3, contains three crystallographically independent mol-ecules of similar geometry. All of the mol-ecules are almost planar, with r.m.s. deviations of 0.003, 0.016 and 0.005 Å. In the crystal, the mol-ecules are linked by N-H⋯N hydrogen bonds into zigzag ribbons parallel to the c axis, generating rings of R2(2)(8) graph-set motif.

To develop a specific, rapid, and convenient immunochromatography assay (ICA) to detect the rabies antibody in clinical sample from immuned dogs by rabies vaccine. Colloidal gold particles labeled with purified rabies virus (CVS11) were used as the detector reagent. The staphylococcal protein A (SPA) and pured rabbit anti-rabies virus IgG were blotted on the test and control regions of nitrocellulose membrane. Then the strip was assembled with sample pad, absorbing pad, and dorsal shield. The assay samples (261 dog's serum) were collected from Wildlife Rabies Disease Diagnostic Laboratories of Ministry of Agriculture in China, Institute of Military Veterinary, Academy of Military Medical Sciences and other six provinces, including rabies virus positive and negative serum. The performance of the strip was compared to fluorescent antibody virus neutralization test. The neutralizing antibody titer could be detected above 0.5 IU. The strip did not change of performance when stored at room temperature for 12 months. It may offer reference of neutralizing antibody titer level after dogs immuned rabies vaccine and determin whether the dogs need to be immuned again.

Copper is vitally required for plants at low concentrations but extremely toxic for plants at elevated concentrations. Plants have evolved a series of mechanisms to prevent the consequences of the excess or deficit of copper. These mechanisms require copper-interacting proteins involved in copper trafficking. Blue copper-binding proteins (BCPs) are a class of copper proteins containing one blue copper-binding domain binding a single type I copper. To investigate the role of BCPs in plant development and in response to stresses, we isolated nine cDNAs encoding the putative blue copper-binding proteins (GhBCPs) from cotton (Gossypium hirsutum). Meanwhile, four corresponding genes (including GhBCP1-GhBCP4), which contain a single intron inserted in their conserved position, were isolated from cotton genome. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that the nine GhBCP genes are differentially expressed in cotton tissues. Among them, GhBCP1 and GhBCP4 were predominantly expressed in fibers, while the transcripts of GhBCP2 and GhBCP3 were accumulated at relatively high levels in fibers. These four genes were strongly expressed in early fiber elongation, but dramatically declined with further fiber development. In addition, these GhBCP genes were upregulated in fibers by Cu(2+) , Zn(2+) , high-salinity and drought stresses, but downregulated in fibers by Al(3+) treatment. Overexpression of GhBCP1 and GhBCP4 in yeast (Schizosaccharomyces pombe) significantly increased the cell growth rate under Cu(2+) , Zn(2+) and high-salinity stresses. These results suggested that these GhBCPs may participate in the regulation of fiber development and in response to high-salinity and heavy metal stresses in cotton., (Copyright © Physiologia Plantarum 2010.)

An immunochromatographic test strip (ICTS) for detecting antibodies to rabies virus was developed, using colloidal gold particles labeled with rabies virus glycoprotein as the tracer. The assay was evaluated using sera from dogs immunized with various commercial rabies vaccines, or from dogs in the clinics and sera from dogs immunized with vaccines against pathogens other than rabies virus, and negative sera from a wide variety of animal sources, including dogs, mice, and cats which had never been vaccinated. The ICTS was found to be highly specific for antibodies against rabies virus, with a detection limit of 0.5IU/ml as measured by the fluorescent antibody virus neutralization (FAVN) test. Compared with the FAVN test, the specificity and sensitivity of ICTS were 98.2% and 90.4%, respectively. There was an excellent agreement between results obtained by the ICTS and FAVN tests (kappa=0.888). Strips stored at 4°C in a plastic bag with a desiccant retained their specificity and sensitivity for at least 15 months, and strips stored at ambient temperature remained stable for 12 months. The immunochromatographic test strip may therefore be useful for clinical laboratories lacking specialized equipment and for diagnosis in the field for rapid detection of rabies virus-specific antibodies., (Copyright © 2010 Elsevier B.V. All rights reserved.)

To investigate whether the high expression levels of actin-depolymerizing factor genes are related to pollen development, three GhADF genes (cDNAs) were isolated and characterized in cotton. Among them, GhADF6 and GhADF8 were preferentially expressed in petals, whereas GhADF7 displayed the highest level of expression in anthers, revealing its anther specificity. The GhADF7 transcripts in anthers reached its peak value at flowering, suggesting that its expression is developmentally-regulated in anthers. The GhADF7 gene including the promoter region was isolated from the cotton genome. To demonstrate the specificity of the GhADF7 promoter, the 5'-flanking region, including the promoter and 5'-untranslated region, was fused with the GUS gene. Histochemical assays demonstrated that the GhADF7:GUS gene was specifically expressed in pollen grains. When pollen grains germinated, very strong GUS staining was detected in the elongating pollen tube. Furthermore, overexpression of GhADF7 gene in Arabidopsis thaliana reduced the viable pollen grains and, consequently, transgenic plants were partially male-sterile. Overexpression of GhADF7 in fission yeast (Schizosaccharomyces pombe) altered the balance of actin depolymerization and polymerization, leading to the defective cytokinesis and multinucleate formation in the cells. Given all the above results together, it is proposed that the GhADF7 gene may play an important role in pollen development and germination.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death in China. We aimed to first present the expression of endocan in HCC tissue and its correlation with the clinicopathological features and overall survival of patients with HCC after curative hepatectomy. Immunohistochemical detection of endocan, CD34, and vascular endothelial growth factor (VEGF) were performed on samples from 100 patients with HCC. Endocan protein was expressed in endothelium of HCC tissue in all specimens, but was not expressed in endothelium of pericarcinomatous liver tissue and normal liver tissue. Microvessel density (MVD) denoted by endocan (endocan-MVD) in HCC was correlated with microscopic venous invasion and VEGF expression (P < 0.05). Survival analysis showed that overall survival of patients was inversely associated with endocan-MVD (P < 0.01). Multivariate analysis showed that endocan-MVD was an independent prognostic marker for overall survival of HCC (P < 0.01). In conclusion, endocan-MVD was a significant factor to predict the prognosis of HCC patients after curative hepatectomy.

Fasciclin-like arabinogalactan proteins (FLAs), a subclass of arabinogalactan proteins (AGPs), are usually involved in cell development in plants. To investigate the expression profiling as well as the role of FLA genes in fiber development, 19 GhFLA genes (cDNAs) were isolated from cotton (Gossypium hirsutum). Among them, 15 are predicted to be glycosylphosphatidylinositol anchored to the plasma membranes. The isolated cotton FLAs could be divided into four groups. Real-time quantitative reverse transcriptase polymerase chain reaction results indicated that the GhFLA genes are differentially expressed in cotton tissues. Three genes (GhFLA1/2/4) were specifically or predominantly expressed in 10 days post-anthesis fibers, and the transcripts of the other four genes (GhFLA6/14/15/18) were accumulated at relatively high levels in cotton fibers. Furthermore, expressions of the GhFLA genes are regulated in fiber development and in response to phytohormones and NaCl. The identification of cotton FLAs will facilitate the study of their roles in cotton fiber development and cell wall biogenesis.

Safe and effective vaccination is important for rabies prevention. Here, genetically engineered rabies vaccine CAV2-deltaE3-Rgp was developed and characterized. The recombinant genome pPoly2-CAV2-deltaE3-Rgp carrying the rabies glycoprotein (Rgp) cDNA was generated by a series of strictly gene cloning steps and infectious recombinant virus CAV2-deltaE3-Rgp was obtained by transfecting the recombinant genome into a canine kidney cell line, MDCK. To efficiently construct cloned recombinant canine adenovirus type 2 genome pPoly2-CAV2-deltaE3-Rgp bearing exogenous Rgp gene, The Rgp gene was first subcloned from the clone vector pMD18-T into the eukaryon expression vector pVAX1. The Rgp expression cassette was then subcloned into the shuttle vector pVAXdeltaE3 and subsequently into the canine adenovirus type 2 backbone vector pPoly2-CAV2. To indirectly confirm pPoly2-CAV2-deltaE3-Rgp, conventional restriction endonuclease digestion was performed. CAV2-deltaE3-Rgp can generate typical CPE of CAV-2. CAV2-deltaE3-Rgp was tested by restriction endonuclease digestion, PCR, DNA sequencing. As a result, The Rgp expression cassette was successfully integrated into the target region of the CAV2 genome. It is confirmed by RT-PCR, Western blot that CAV2-deltaE3-Rgp can express Rgp antigen in MDCK cell. This recombinant virus, CAV2-deltaE3-Rgp, was intramuscularly injected into dogs. All vaccinated dogs produced effective antibodies against CAV and RV after three inoculations. This recombinant virus would be prospective in immunizing dogs against CAV and RV.

The microtubule cytoskeleton may play an important role in the polarized growth of fibre cells that are single-cell trichomes on the surface of cotton ovules. To investigate whether the high expression levels of alpha-tubulin genes are correlated with fibre elongation, nine GhTUA genes (cDNAs) encoding alpha-tubulins with 449-451 amino acid residues were isolated and characterized in cotton. The GhTUA genes share high sequence homology at the nucleotide level (62-93% identity) in the coding region and at the amino acid level (89-99% identity), and can be classified into two subgroups. Real-time quantitative RT-PCR analysis revealed that seven out of the nine GhTUA genes are predominantly expressed in developing fibres. Among them, GhTUA9 displays the highest level of expression, revealing its fibre specificity. The GhTUA9 transcripts in fibres reached its peak value between 5-10 DPA, and dramatically declined to undetectable levels as the ovule matured further, suggesting that its expression is developmentally-regulated in fibres. The GhTUA9 gene including the promoter region was isolated from the cotton genome. To demonstrate the specificity of the GhTUA9 promoter, the 5'-flanking region, including the promoter and 5'-untranslated region, was fused with the GUS gene. Histochemical assays demonstrated that the GhTUA9:GUS gene was specifically expressed in elongating fibres. Overexpression of GhTUA9 in fission yeast (Schizosaccharomyces pombe) promoted atypical longitudinal growth of the host cells by 1.4-1.7-fold, indicating that the GhTUA9 gene is involved in cell elongation. Given all the above results, it is proposed that the GhTUA9 gene may play an important role in fibre elongation.

Plant aquaporins, belonging to the MIP superfamily, are a series of transmembrane proteins that facilitate water transport through cell membranes. In this study, a cDNA clone encoding the PIP1-like protein was isolated from cotton (Gossypium hirsutum) cDNA libraries, and designated as GhAQP1 (Fig.1). We also isolated the GhAQP1 gene from cotton genome by PCR. The gene is 2,096 bp in length, including an open reading frame (ORF) and 5'-/3'-untranslated regions (UTR). It contains two introns in its ORF. The first intron is inserted between codons 209 and 210 in the fifth transmembrane helix, and another is located between codons 256 and 257 in the sixth transmembrane helix of GhAQP1, respectively (Figs.2 and 3). Northern blot analysis showed that GhAQP1 gene is expressed specifically in 6-15 DPA ovule, and reaches a peak in 9 DPA ovule (Figs.4 and 5), suggesting that its expression is ovule-specific and developmentally regulated in cotton.

H5N1 highly pathogenic avian influenza virus was highly pathogenic and sometimes even fatal for tigers and cats. To develop a new type of vaccine for Felidae influenza prevention, recombinant replication-competent canine adenovirus Type 2 expressing hemagglutinin gene of H5N1 subtype tiger influenza virus was constructed. A/tiger/Harbin/01/2003 (HSN1) HA gene was cloned into PVAX1. The HA expression cassette which included CMV and HA and PolyA was ligated into the E3 deletion region of pVAXdeltaE. The recombinant plasmid was named pdeltaEHA. The pdelta EHA and the pPoly2-CAV2 were digested with Nru I /Sal I, respectively. The purified Nru I/Sal I DNA fragment containing the HA expression cassette was cloned into pPoly2-CAV2 to generate the recombinant plasmid pCAV-2/HA. The recombinant genome was released from pCAV-2/HA, and was transfected into MDCK cells by Lipofectamine. The recombinant virus named CAV2/HA was gained. Anti-H5N1 influenza virus HI antibody (1:8 - 1:16) was detected in the cat immunized with CAV-2/HA.

Objective: To analyze the risk factors for metastasis and recurrence of hepatocellular carcinoma (HCC) postoperatively., Methods: Data of 270 cases of postoperative HCC were analyzed by SPSS software retrospectively., Results: Out of the 270 cases, 162 got follow-up study and 136 showed metastasis and recurrence. Lots of risk factors induced the recurrence of HCC, such as AFP, tumor form, venous blood invasion, HBV infection, resection dimension and perioperative transfusion. There were different risk factors at different stages., Conclusion: The early recurrence of HCC may be mediated by macro- or micro-vessel blood invasion and metastasis, the late recurrence by multicentric carcinogenesis or introhepatic cacinoma de novo.

Aim: To identify the role of alpha-fetoprotein (AFP) mRNA expression in peripheral blood one week after surgery as a predictor for recurrence of hepatocellular carcinoma (HCC)., Methods: Published studies fulfilling the selection criteria were identified by searching several databases online. After a methodology assessment using a quality scale designed by European Lung Cancer Working Party, data in each research were aggregated by means of meta-analysis., Results: Altogether 368 cases were included in the 9 selected studies, which fulfilled the selection criteria. The quality scores ranged from 35% to 84% with a median score of 55%. The 'design' subscore had the lowest median value (38%). By aggregating the data, a high chi2 value (77.576) was presented. The fail-safe number was 136 and 64 for P = 0.05 and 0.01, respectively., Conclusion: AFP mRNA expression in peripheral blood 1 wk after surgery correlated with the recurrence of HCC and was a good predictor for tumor recurrence.

Aim: To comprehend the risk factors of recurrence of hepatocellular carcinoma (HCC) and its relationship with the infection patterns of hepatitis B virus (HBV)., Methods: All materials of 270 cases of postoperative HCC were statistically analyzed by SPSS software. Recurrence and metastasis were classified into early (< or =2 years) and late phase (>2 years). Risk factors for recurrence and metastasis after surgery in each group were analyzed., Results: Out of 270 cases of HCC, 162 cases were followed up in which recurrence and metastasis occurred in 136 cases. There were a lot of risk factors related to recurrence and metastasis of HCC; risk factors contributing to early phase recurrence were serum AFP level, vascular invasion, incisal margin and operative transfusion, gross tumor classification and number of intrahepatic node to late phase recurrence. The HBV infective rate of recurrent HCC was 94.1%, in which "HBsAg, HBeAb, HbcAb" positive pattern reached 45.6%. The proportion of HBV infection in solitary large hepatocellular carcinoma (SLHCC) evidently decreased compared to nodular hepatocellular carcinoma (NHCC) (P<0.05)., Conclusion: The early and late recurrence and metastasis after hepatectomy of HCC were associated with different risk factors. The early recurrence may be mediated by vascular invasion and remnant lesion, the late recurrence by tumor's clinical pathology propert, as multicentric carcinogenesis or intrahepatic carcinoma de novo. HBV replication takes a great role in this process. From this study, we found that SLHCC has more satisfactory neoplasm biological behavior than NHCC.

Objective: Rho, a ras homologous gene, encodes a group of GTP-binding proteins. Our previous study suggested that one member of the Rho gene family, RhoC, was related to the progression of human hepatocellular carcinoma (HCC). This study is to elucidate correlation of Rho overexpression with invasion and metastasis of HCC., Methods: The expression level of RhoC mRNA and protein in 25 cases of HCC and adjacent non-cancerous liver tissue was examined by RT-PCR and Western blot. Mutation of RhoC gene was examined by PCR-SSCP., Results: The expression of RhoC mRNA and protein was found in all HCC and adjacent non-cancerous liver tissue. The expression level of RhoC mRNA and protein was significantly higher in tumor tissue than in adjacent non-cancerous liver tissue (1.8 +/- 1.1 vs 1.0 +/- 0.7; 33 992 +/- 10 384 vs 17 342 +/- 9998, P < 0.01). The degree of RhoC overexpression was even more marked in metastatic lesions than in primary tumors (P < 0.01). Overexpression of the rhoC gene was significantly correlated with such clinic-pathological findings as cell differentiation, portal vein invasion, number of primary tumor nodules and metastatic lesions (P < 0.05). Mutation of RhoC gene was found in none of the HCC specimens examined., Conclusion: Overexpression of RhoC gene may play an important role in carcinogenesis and progression of HCC.

Aim: To investigate the role of tri-iodothyronine supplement in protecting gut barrier in septic rats., Methods: Twenty-two rats were randomized into three groups: sham group (n=6), sepsis group (n=8), and sepsis plus tri-iodothyronine (T(3)) group (n=8). Septic rat model was established through cecal ligation and puncture (CLP). After 5 h, sham and sepsis groups received saline, and the remaining group received T(3) intraperitoneally. Twenty-one hrs After CLP, intestinal permeability and serum free T(3) and T(4) were measured with fluorescence spectrophotometer and by radioimmunoassay, respectively. Intestinal ultrastructure and histologic morphology were observed under transmission electron microscopy (TEM) and light microscopy, respectively., Results: After 21 h, septic symptoms and signs in sepsis plus T(3) group were milder than those in sepsis group. Serum FT(3) or FT(4) concentration in sepsis group was lower than that in sham group (1.59+/-0.20, 3.41+/-2.14 pmol/L vs 3.44+/-1.40, 9.53+/-3.39 pmol/L, P<0.05), and FT(3) concentration in sepsis plus T(3) group (3.40+/-1.65 pmol/L, P<0.05) was corrected. Portal concentration of fluorescein isothiocyanate-dextran (FITC-D) in sepsis group (2.51+/-0.56 mg/L) was higher than that in sham group (1.22+/-0.21 mg/L) (P<0.01), and in sepsis plus T(3) group (1.68+/-0.38 mg/L) it was decreased significantly(P<0.01). TEM and light microscopy showed that T(3) supplement preserved well ultrastructure and morphology of intestinal mucosa in septic rats., Conclusion: Tri-iodothyronine supplement protects gut barrier in septic rats.