A highly conserved amino acid in VP1 regulates maturation of enterovirus 71.

Enterovirus 71 (EV71) is the major causative agent of hand, foot and mouth disease (HFMD) in children, causing severe clinical outcomes and even death. Here, we report an important role of the highly conserved alanine residue at position 107 in the capsid protein VP1 (VP1A107) in the efficient replication of EV71. Substitutional mutations of VP1A107 significantly diminish viral growth kinetics without significant effect on viral entry, expression of viral genes and viral production. The results of mechanistic studies reveal that VP1A107 regulates the efficient cleavage of the VP0 precursor during EV71 assembly, which is required, in the next round of infection, for the transformation of the mature virion (160S) into an intermediate or A-particle (135S), a key step of virus uncoating. Furthermore, the results of molecular dynamic simulations and hydrogen-bond networks analysis of VP1A107 suggest that flexibility of the VP1 BC loop or the region surrounding the VP1107 residue directly correlates with viral infectivity. It is possible that sufficient flexibility of the region surrounding the VP1107 residue favors VP0 conformational change that is required for the efficient cleavage of VP0 as well as subsequent viral uncoating and viral replication. Taken together, our data reveal the structural role of the highly conserved VP1A107 in regulating EV71 maturation. Characterization of this novel determinant of EV71 virulence would promote the study on pathogenesis of Enteroviruses.

[Determination of degree of polymerization of natural cellulose pulp using near-infrared diffuse reflectance spectroscopy].

A new method of near-infrared (NIR) diffuse reflectance spectroscopy is proposed to rapidly determine the degree of polymerization (DP) of natural cellulose (cotton and wood) pulp produced by a new clean pulping process. One hundred and ninety five samples were collected and their DP data were determined by standard method GB/T 9107-1999. The spectroscopy measurement method of the samples was studied and their near-infrared diffuse reflectance spectra were collected. The quantitative DP calibration models of one mixed cotton & wood and two separate cotton and wood pulps were established by partial least squares (PLS). The optimum models were developed using the spectra pretreated by derivative, autoscaling and mean-centering, and their performance is as follows: correlation coefficient of 0.980, 0.993 and 0.886, and RMSEP of 147, 143 and 53, respectively. The accuracy of NIR method was also studied. The results show that the accuracy of the two separate models of cotton and wood is better than that of the mixed model, and the precision of the two separate models is better than that of GB/T9107-1999. The identification model of cotton and wood was also established using principal component analysis (PCA). The result shows that the spectra of cotton and wood pulp have obvious difference, and the model can identify successfully the two kinds of pulp. The result indicates that the new NIR method is feasible to realize the on-line analysis of polymerization degree of natural cellulose pulp with its advantage of rapidness and easy operation.

Construction and characterization of an infectious cDNA clone of enterovirus type 71 subgenotype C4.

Enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease and induces fatal neurological complications. In recent years, this virus has become a major threat to public health in the Asia-Pacific region, while no effective antiviral therapies and vaccines are currently available. In this study, we constructed and characterized for the first time an infectious full-length EV71 cDNA clone derived from the SHZH98 strain, which was the first subgenotype C4 strain isolated in China. Our data demonstrate that the rescued EV71 viruses exhibited growth kinetics in vitro and morphologies similar to those of the BrCr-TR strain and reached a maximum titer of 10(7.5) TCID50/ml. Although the rescued viruses were able to infect suckling mice, no typical symptoms of EV71 infection were observed for up to 18 days post-inoculation. Taken together our research provides an important tool to study the epidemic strains of EV71 in the Asia-Pacific region and promote the development of vaccines.

[Determination of alpha-cellulose content of natural cellulose pulp in a new clean pulping process using near infrared diffuse reflectance spectroscopy].

A new near infrared diffuse reflectance spectroscopy method is proposed to rapidly detect alpha-cellulose content of natural cellulose (plant fiber: cotton, wood) pulp in a new clean pulping process. One hundred forty two samples were collected and their alpha-cellulose content data were determined by standard method GB/T 9107-1999. The samples were homogenized by grinding pretreatment to improve spectroscopy measurement accuracy. Effective classification models were built by SIMCA, with the total correct identification. Using partial least squares (PLS) quantitative calibration, alpha-cellulose of the whole and separate cotton and wood pulp was established, with the correlation coefficients of 0.954, 0.911, 0.839, SEP, 0.024, 0.012 and 0.016, respectively. The repeatability results obtained by the new method are in agreement with the results from GB/T 9107-1999. The new method is feasible for determining alpha-cellulose content of natural cellulose (plant fiber: cotton, wood) in clean pulping process.

Discrepancy of lignin dissolution from eucalyptus during formic acid fractionation.

Understanding the structure and properties of lignin has important practical significance for its further applications. In this case, eucalyptus was fractionated with 88% formic acid at 101 °C for different durations, and the removal efficiency as well as the chemical structure of lignin at various stages were comparatively analyzed. The obtained data indicated that with increasing reaction time, lignin was continuously removed and the process could be divided into three stages. The lignin dissolution rate was fast first and then slow, and the molecular weight of the dissolved lignin increased with time. The lignin structure was condensed and the molecular weight increased with prolonged of reaction time. Structural analysis indicated that the ß-O-4' structure was largely destroyed, the G-type lignin dissolved early, and the degradation of the S-type lignin became more intensive with increasing reaction time. This is of great help for reaction control as well as the further processing of lignin byproducts.

Patterning of 293T fibroblasts on a mica surface.

Controllable cell growth on the defined areas of surfaces is important for potential applications in biosensor fabrication and tissue engineering. In this study, controllable cell growth was achieved by culturing 293 T fibroblast cells on a mica surface which had been patterned with collagen strips by a microcontact printing (microCP) technique. The collagen area was designed to support cell adhesion and the native mica surface was designed to repel cell adhesion. Consequently, the resulting cell patterns should follow the micro-patterns of the collagen. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) measurement, atomic-force microscope (AFM) observation, and force-curve measurement were used to monitor property changes before and after the collagen adsorption process. Further data showed that the patterned cells were of good viability and able to perform a gene-transfection experiment in vitro. This technique should be of potential applications in the fields of biosensor fabrication and tissue engineering.

Preparation and Imaging Investigation of Dual-targeted C3F8-filled PLGA Nanobubbles as a Novel Ultrasound Contrast Agent for Breast Cancer.

Molecularly-targeted contrast enhanced ultrasound (US) imaging is a promising imaging strategy with large potential for improving diagnostic accuracy of conventional US imaging in breast cancer detection. Therefore, we constructed a novel dual-targeted nanosized US contrast agent (UCA) directed at both vascular endothelial growth factor receptor 2 (VEGFR2) and human epidermal growth factor receptor 2 (HER2) based on perfluoropropane (C3F8)-filled poly(lactic-co-glycolic acid) (PLGA) (NBs) for breast cancer detection. In vitro, single- or dual-targeted PLGA NBs showed high target specificities and better effects of target enhancement in VEGFR2 or HER2-positive cells. In vivo, US imaging signal in the murine breast cancer model was significantly higher (P < 0.01) for dual-targeted NBs than single-targeted and non-targeted NBs. Small animal fluorescence imaging further confirmed the special affinity of the dual-targeted nanosized contrast agent to both VEGFR2 and HER2. Immunofluorescence and immunohistochemistry staining confirmed the expressions of VEGFR2 and HER2 on tumor neovasculature and tumor cells of breast cancer. In conclusions, the feasibility of using dual-targeted PLGA NBs to enhance ultrasonic images is demonstrated in vitro and in vivo. This may be a promising approach to target biomarkers of breast cancer for two site-specific US molecular imaging.

Au-poly(lactic-co-glycolic) acid complex nanoparticles as ultrasound contrast agents: Preparation, characterization and in vitro study.

The present study aimed to prepare and characterize an Au-poly(lactic-co-glycolic) acid (PLGA) complex, surfaced with gold nanoparticles (GNPs), and to assess its use as an ultrasound contrast agent for in vitro imaging. PLGA nanoparticles (NPs) were prepared by a modified double­emulsion solvent evaporation method. GNPs were prepared via reduction with sodium citrate. Au­PLGA complexes were created through the binding of polyethylene glycosylated GNPs to the surface of PLGA NPs. Various physical characteristics of PLGA NPs, GNPs and Au­PLGA complexes, including morphology, size, dispersion, ζ potential and stability were observed and measured. The study of the in vitro imaging capability of the Au­PLGA complex was conducted by setting multi­parameter imaging conditions. The average sizes of PLGA NPs, GNPs and the Au­PLGA complex were 144.5±30.28, 21.42±1.56 and 359.4±67.94 nm, respectively. Transmission electron microscopy (TEM) revealed that PLGA NPs and GNPs were spherically shaped, but GNPs had smooth surface while PLGA NPs had a capsular morphology with a wrinkled surface and large voids. The maximum absorption peak of 20 nm GNPs was 508 nm while that of the Au­PLGA complex was 515 nm on an ultraviolet­visible absorption spectrum curve. The results of TEM and the redshift spectrum demonstrated that GNPs successfully bound to the surface of the PLGA NPs. The complex demonstrated good stability and its size changed little over time. In vitro imaging experiments revealed that the Au­PLGA complex had a good imaging ability. The present study demonstrated that the Au­PLGA complex is a promising novel ultrasound contrast agent.

Study on the concentration effects in size exclusion chromatography VII. A quantitative verification for the model theory of concentration and molecular mass dependences of hydrodynamic volumes for polydisperse polymers.

A model theory of concentration effects for polydisperse polymers was proposed in 1988. It is successful in relating the concentration of the injected solution to the effective hydrodynamic volumes of peak, the retention volumes of peak and the polydispersity index (Dc = (Vhcw)/(Vhcn) of hydro-dynamic volume distribution for polydisperse polymers at a given concentration. The dependence of the concentration of injected polymer solution on the effective hydrodynamic volumes, the retention volumes of peak and the polydispersity index of hydrodynamic volume distribution for narrow disperse and polydisperse polystyrene, poly(dodecyl methacrylate), poly(tridecyl methacrylate) and poly(methyl methacrylate) in tetrahydrofuran solvent were studied. The proposed theory was verified by these experimental data. Results show that the proposed theory can predict the concentration effects in GPC for polydisperse polymers quantitatively and can provide a theoretical foundation for the two methods of calibrating the universal calibration curves with polydisperse polymers and of determining the second virial coefficients (A2) of polymers. It is found that the determined values of A2 for narrow disperse and polydisperse polymers by the proposed method are in agreement with those obtained by the LALLS method, and the two universal calibration curves with narrow disperse and polydisperse polymers are in excellent agreement.

[Construction of cDNA infectious clones of EV71 highly-pathogenic and cell-culture-adapted strains].

The highly-pathogenic EV71 strain is the primary cause of mortality in hand-foot-and-mouth disease (HFMD) associated with neurological symptoms, for which no clinically effective drugs or vaccines exist. This study aimed to construct infectious cDNA clones of the EV71 highly-pathogenic strain and the cell-culture adapted strain using a reverse genetics approach. The genomic RNAs of EV71 parent strains were used as the templates for RT-PCR amplification, and then the PCR products that overlapped the full-length genome were connected into the pBR322 vector to produce infectious clones of pEV71 (HP) and pEV71 (CCA), respectively. The results showed that the HP strain propagated much more quickly than the CCA strain. The rescued viruses derived from the infectious clones not only maintained their consistency with their parent strains in terms of genomic sequences, but also retained their respective biological phenotypes. This research will contribute to our understanding of EV71 pathogenesis and the development of novel vaccines against HFMD.

[A randomized single-blind controlled clinical trial of tacrolimus mouth rinse on erosive oral lichen planus].

PURPOSE: To evaluate the efficacy and safety of tacrolimus mouth rinse on the treatment of erosive and ulcerative oral lichen planus (OLP). METHODS: A randomized single-blind open trial of tacrolimus mouth rinse with dexamethasone as control was designed. The VAS and REU scoring system was utilized to compare the signs and symptoms. The scores and therapeutic effects were analyzed with SPSS 17.0 software package. RESULTS: There was no significant difference in effective rate between the treatment group and control group (X(2)=0.295,0.413, P>0.01) at 4-week and 12-week after treatment. There was significant difference in REU scores between the 2 groups (P<0.01) 4 weeks after treatment. CONCLUSIONS: Tacrolimus mouth rinse effects quickly and is worthy of application in the treatment of erosive and ulcerative OLP.

[Matrix metalloproteinase 8 and 9 regulations of polymorphonuclear leukocytes stimulated by Porphyromonas gingivalis with different fimA genotypes].

OBJECTIVE: To investigate the pathogenicity of matrix metalloproteinase 8, 9 (MMP-8, MMP-9) regulations of polymorphonuclear leukocytes (PMNs) by challenge of Porphyromonas gingivalis (P. gingivalis) with different fimA genotypes. METHODS: The studies mainly adopt the isopycnic sedimentation separation to separate the PMNs from human peripheral blood. P. gingivalis ATCC 33277 (type I), WCSP 115 (type II), WCSP 1.5 (type III), W83 (type IV), WCSP 559 (type IV) were assessed for their inductions of MMP-8, MMP-9 expression in PMNs. MMP-8, MMP-9 protein levels in culture supernatant were determined by ELISA at different time intervals (5 min, 30 min, 1 h, 2 h) following continuous co-culture of bacteria with PMNs. RESULTS: MMP-8 and MMP-9 protein levels produced by PMNs co-culture with the I fimA-IV fimA P. gingivalis were significantly stronger than unsimulated group. The velocity and quantity of MMP-8 produced by PMNs co-culture with the II fimA P. gingivalis and IV fimA P. gingivalis were more than III fimA, IVfimA P. gingivalis. The MMP-9 protein levels produced by PMNs co-culture with the I fimA, II fimA, IV fimA P. gingivalis was significantly stronger than III fimA and IV fimA P. gingivalis. CONCLUSION: II fimA and IV fimA P. gingivalis have stronger pathogenicity relatively, which indicate that fimA genotype is associated with pathogenesis of P. gingivalis.

[Clinical analysis of midfacial fractures with orbital floor fractures].

OBJECTIVE: To study the characters of the diagnosis and treatment for midfacial fractures with orbital floor fractures. METHODS: 136 cases of midfacial fractures with orbital floor fractures were diagnosed and treated. All patients underwent CT scans and accepted surgical intervention. Orbital floor fracture was restituted in 49 cases. Orbital floor defects were reconstructed with autogenous bone, titanium mesh or porous polyethylene implant (Medpor) in 21 cases. RESULTS: 136 cases recovered well, their facial appearance and function were improved significantly. There were no severe complications happened postoperatively in all cases. 2 cases had postoperative wounds infection and 1 case had temporary ablepsia, but healed completely. CONCLUSION: CT is the best method for diagnosing orbital floor fractures. The objective of treatment is restoration of normal orbital floor and orbital capacity, the reduction of orbital contents prolapsed and reconstruction of orbital floor fractures with repair materials.