Objective INF2 is a member of the formins family.Abnormal expression and regulation of INF2 have been associated with the progression of various tumors,but the expression and role of INF2 in hepatocellular carcinoma(H...Objective INF2 is a member of the formins family.Abnormal expression and regulation of INF2 have been associated with the progression of various tumors,but the expression and role of INF2 in hepatocellular carcinoma(HCC)remain unclear.HCC is a highly lethal malignant tumor.Given the limitations of traditional treatments,this study explored the expression level,clinical value and potential mechanism of INF2 in HCC in order to seek new therapeutic targets.Methods In this study,we used public databases to analyze the expression of INF2 in pan-cancer and HCC,as well as the impact of INF2 expression levels on HCC prognosis.Quantitative real time polymerase chain reaction(RT-qPCR),Western blot,and immunohistochemistry were used to detect the expression level of INF2 in liver cancer cells and human HCC tissues.The correlation between INF2 expression and clinical pathological features was analyzed using public databases and clinical data of human HCC samples.Subsequently,the effects of INF2 expression on the biological function and Drp1 phosphorylation of liver cancer cells were elucidated through in vitro and in vivo experiments.Finally,the predictive value and potential mechanism of INF2 in HCC were further analyzed through database and immunohistochemical experiments.Results INF2 is aberrantly high expression in HCC samples and the high expression of INF2 is correlated with overall survival,liver cirrhosis and pathological differentiation of HCC patients.The expression level of INF2 has certain diagnostic value in predicting the prognosis and pathological differentiation of HCC.In vivo and in vitro HCC models,upregulated expression of INF2 triggers the proliferation and migration of the HCC cell,while knockdown of INF2 could counteract this effect.INF2 in liver cancer cells may affect mitochondrial division by inducing Drp1 phosphorylation and mediate immune escape by up-regulating PD-L1 expression,thus promoting tumor progression.Conclusion INF2 is highly expressed in HCC and is associated with poor prognosis.High expression of INF2 may promote HCC progression by inducing Drp1 phosphorylation and up-regulation of PD-L1 expression,and targeting INF2 may be beneficial for HCC patients with high expression of INF2.展开更多
The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose⁃catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monit...The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose⁃catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monitoring xylitol concentration.In this study,the gene encoding the thermophilic fungus Talaromyces emersonii XDH(TeXDH)was heterologously expressed in Escherichia coli BL21(DE3)at 16℃in the soluble form.Recombinant TeXDH with high purity was purified by using a Ni⁃NTA affinity column.Size⁃exclusion chromatography and SDS⁃PAGE analysis demonstrated that the puri⁃fied recombinant TeXDH exists as a native trimer with a molecular mass of approximately 116 kD,and is composed of three identical subunits,each with a molecular weight of around 39 kD.The TeXDH strictly preferred NAD^(+)as a coenzyme to NADP^(+).The optimal temperature and pH of the TeXDH were 40℃and 10.0,respectively.After EDTA treatment,the enzyme activity of TeXDH decreased to 43.26%of the initial enzyme activity,while the divalent metal ions Mg^(2+)or Ca^(2+)could recover the enzyme activity of TeXDH,reaching 103.32%and 110.69%of the initial enzyme activity,respectively,making them the optimal divalent metal ion cofactors for TeXDH enzyme.However,the divalent metal ions of Mn^(2+),Ni^(2+),Cu^(2+),Zn^(2+),Co^(2+),and Cd^(2+)significantly inhibited the activity of TeXDH.ICP⁃MS and molecular doc⁃king studies revealed that 1 mol/L of TeXDH bound 2 mol/L Zn^(2+)ions and 1 mol/L Mg^(2+)ion.Further⁃more,TeXDH exhibited a high specificity for xylitol,laying the foundation for the development of future xylitol biosensors.展开更多
Starch-nanoparticles were synthesized in water-in-oil microemusion at room temperature, and the starch-nanoparticles were coated with poly-L-lysine. The surface of the starch-nanoparticles was combined with fluorescen...Starch-nanoparticles were synthesized in water-in-oil microemusion at room temperature, and the starch-nanoparticles were coated with poly-L-lysine. The surface of the starch-nanoparticles was combined with fluorescence material Ru(bpy)32+·6H2O, and then the particles were characterized via transmission electron microscope. The fluorescence nanoparticles were conjugated with plasmid DNA to form complexes, and then treated with ultrasound and DNase I. pEGAD plasmid DNA-nanoparticle complexes were co-cultured with plant suspension cells of Dioscrea Zigiberensis G H Wright, and treated with ultrasound. The results show that the diameter of the fluorescence starch-nanoparticles is 50-100 nm. DNA-nanoparticle complexes can protect DNA from ultrasound damage as well as from DNase I cleavage. Mediated by ultrasound, pEGAD plasmid DNA-nanoparticle complexes can pierce into the cell wall, cell membrane and nucleus membrane of plant suspension cells. The green fluorescence protein(GFP) gene at a high frequency exceeds 5%. This nano-biomaterial can efficiently solve the problem that exterior genes cannot traverse the plant cell wall easily.展开更多
The transient response of an unlimited cylindrical cavity buried in the infinite elastic soil subjected to an anti-plane impact load along the cavern axis direction was studied.Using Laplace transform combining with c...The transient response of an unlimited cylindrical cavity buried in the infinite elastic soil subjected to an anti-plane impact load along the cavern axis direction was studied.Using Laplace transform combining with contour integral of the Laplace inverse transform specifically,the general analytical expressions of the soil displacement and stress are obtained in the time domain,respectively.And the numerical solutions of the problem computed by analytical expressions are presented.In the time domain,the dynamic responses of the infinite elastic soil are analyzed,and the calculation results are compared with those from numerical inversion proposed by Durbin and the static results.One observes good agreement between analytical and numerical inversion results,lending the further support to the method presented.Finally,some valuable shear wave propagation laws are gained: the displacement of the soil remains zero before the wave arrival,and after the shear wave arrival,the stress and the displacement at this point increase abruptly,then reduce and tend to the static value gradually at last.The wave attenuates along the radial,therefore the farther the wave is from the source,the smaller the stress and the displacement are,and the stress and the displacement are just functions of the radial distance from the axis.展开更多
To provide a seepage-stress coupling constitutive model that can directly describe the seepage-stress coupling relationship, a series of one-dimensional seepage-stress coupling tests on two kinds of soft rock (argilla...To provide a seepage-stress coupling constitutive model that can directly describe the seepage-stress coupling relationship, a series of one-dimensional seepage-stress coupling tests on two kinds of soft rock (argillaceous siltstone and brown mudstone) were performed by using an MTS-815.02 tri-axial rock mechanics test system, with which the stress-strain curves according to the seepage variation were obtained. Based on the experimental results and by employing Hooke's law, the formulation of the coefficient of strain-dependent permeability was presented and introduced to establish a coupling model. In addition, the mathematical expression and the incremental formulation for coupling model were advanced, in which five parameters that can be respectively determined by using the experimental results were included. The calculated results show that the proposed coupling model is capable of simulating the stress-strain relationship with considering the seepage-stress coupling in the nonlinear elastic stage of two kinds of soft rock.展开更多
In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A,...In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.展开更多
Seepage and stress redistribution are the main factors affecting the stability of surrounding rock in high-pressure hydraulic tunnels.In this work,the effects of the seepage field were firstly simplified as a seepage ...Seepage and stress redistribution are the main factors affecting the stability of surrounding rock in high-pressure hydraulic tunnels.In this work,the effects of the seepage field were firstly simplified as a seepage factor acting on the stress field,and the equilibrium equation of high pressure inner water exosmosis was established based on physical theory.Then,the plane strain theory was used to solve the problem of elasticity,and the analytic expression of surrounding rock stress was obtained.On the basis of criterion of Norway,the influences of seepage,pore water pressure and buried depth on the characteristics of the stress distribution of surrounding rocks were studied.The analyses show that the first water-filling plays a decisive role in the stability of the surrounding rock; the influence of seepage on the stress field around the tunnel is the greatest,and the change of the seepage factor is approximately consistent with the logarithm divergence.With the effects of the rock pore water pressure,the circumferential stress shows the exchange between large and small,but the radial stress does not.Increasing the buried depth can enhance the arching effect of the surrounding rock,thus improving the stability.展开更多
An analytical approach was presented for estimating the factor of safety(FS) for slope failure, with consideration of the impact of a confined aquifer. An upward-moving wetting front from the confined water was assume...An analytical approach was presented for estimating the factor of safety(FS) for slope failure, with consideration of the impact of a confined aquifer. An upward-moving wetting front from the confined water was assumed and the pore water pressure distribution was then estimated and used to obtain the analytical expression of FS. Then, the validation of the theoretical analysis was applied based on an actual case in Hong Kong. It is shown that the presence of a confined aquifer leads to a lower FS value, and the impact rate of hydrostatic pressure on FS increases as the confined water pressure increases, approaching to a maximum value determined by the ratio of water density to saturated soil density. It is also presented that the contribution of hydrostatic pressure and hydrodynamic pressure to the slope stability vary with the confined aquifer pressure.展开更多
Gp96, a member of HSP90 family, is a versatile molecular chaperone with various newly-discovered functions, for example to serve as a low affinity, high capacity calcium binding protein, a natural adjuvant for therape...Gp96, a member of HSP90 family, is a versatile molecular chaperone with various newly-discovered functions, for example to serve as a low affinity, high capacity calcium binding protein, a natural adjuvant for therapeutic cancer vaccines, a tumor rejection antigen, an immune regulator to pathological cell death. Its multi-functional and structural characteristics make it also an interesting target to develop antibody-based therapeutics. However, its low immunogenicity to mice, because of its high-sequence similarity among different species, is an obstacle to obtain valuable monoclonal antibodies (MAbs). This is a common problem for any low immunogenic proteins, whose sequences share close identity between mice and other species. Here, a new strategy of priming was employed by swine endogenous full-length gp96 and then boosting by E. coli-system heterologously expressed gp96 N-terminal fragment (N-355) to generate MAbs. Twelve different highly-specific MAbs against swine/human endogenous gp96 were successfully obtained. The binding activities of these MAbs were confirmed by enzyme-linked immunosorbent assay (ELISA), Western blot (WB), immunofluorescence and flow cytometry analysis. This provides some important reagents for further research and potential therapeutics. The methods employed can be used for MAb production of any sequence-highly-conserved proteins between mice and swine/human (or any other species).展开更多
Xylanase 1 (Xyn1) is one of the two major representatives of the xylanase system of T. reesei; the mechanisms governing its expression were analysed throughout this study. All factors and regulatory motifs responsible...Xylanase 1 (Xyn1) is one of the two major representatives of the xylanase system of T. reesei; the mechanisms governing its expression were analysed throughout this study. All factors and regulatory motifs responsible for transcriptional regulation and the model of their interplay in induction and repression will be presented. Using in vivo foot printing analysis of xylan-induced and glucose repressed mycelia, we detected three adjacent nucleotide sequences contacted by DNA-binding proteins. Protection within the inverted repeat of the Cre1 (SYGGRG) consensus sequence on the non coding strand under repressing conditions is in perfect agreement with the previously reported Cre1 dependent glucose repression of xyn1. Constitutive protein binding could be observed to a CCAAT-box and an inverted repeat of a 5′ GGCTAA 3′ sequence. EMSA with crude extracts from induced and repressed mycelia revealed that the latter motifs are sufficient for formation of the basal transcriptional complex under all conditions. The inverted repeat of GGCTAA closely resembles the consensus sequences of the cellulase and xylanase regulators Ace1, Ace2 and, Xyr1 (encoded by xyr1, cloned and characterised in this study) EMSA with heterologously expressed components of each factor and of the T. reesei Hap2/3/5 protein complex revealed that the basal transcriptional complex is formed by Xyr1 and the Hap2/3/5. Additionally to the Cre1 mediated carbon catabolite repression a yet unknown mechanism antagonizing induction of xyn1 expression could be elucidated. Latter occurs through competition of the repressor Ace1 and Xyr1 for the GGCTAA motif. In vivo proof for the relevance of identified motifs could be given through analysis of T. reesei transformants containing correspondingly mutated versions of the xyn1 promoter fused to the A. niger goxA gene. The results indicated that the basal as well as the induction level of xyn1 gene transcription is dependent on an interaction of Xyr1 with the GGCTAA motif while formation of the CCAAT-Hap2/3/5 complex slightly reduces induction. It can be concluded that mutations impairing protein binding in vitro lead to a loss of distinct regulatory functions in xyn1 gene expression in vivo. A respective model of gene regulation will be presented.展开更多
Mycoparasitic species of Trichoderma are commercially applied as biological control agents against various fungal pathogens. The mycoparasitic interaction is host specific and includes recognition, attack and subseque...Mycoparasitic species of Trichoderma are commercially applied as biological control agents against various fungal pathogens. The mycoparasitic interaction is host specific and includes recognition, attack and subsequent penetration and killing of the host. Investigations on the underlying events revealed that Trichoderma responds to multiple signals from the host (e.g. lectins or other ligands such as low molecular weight components released from the host’s cell wall) and host attack is accompanied by morphological changes and the secretion of hydrolytic enzymes and antibiotics. Degradation of the cell wall of the host fungus is-besides glucanases and proteases-mainly achieved by chitinases. In vivo studies showed that the ech42 gene (encoding endochitinase 42) is expressed before physical contact of Trichoderma with its host, probably representing one of the earliest events in mycoparasitism, whereas Nag1 (N-acetylglucosaminidase) plays a key role in the general induction of the chitinolytic enzyme system of T. atroviride . Investigations on the responsible signal transduction pathways of T. atroviride led to the isolation of several genes encoding key components of the cAMP and MAP kinase signaling pathways, as alpha and β subunits of heterotrimeric G proteins, the regulatory subunit of cAMP-dependent protein kinase, adenylate cyclase, and three MAP kinases. Analysis of knockout mutants, generated by Agrobacterium-mediated transformation, revealed that at least two alpha-subunits of heterotrimeric G proteins are participating in mycoparasitism-related signal transduction. The Tga1 G alpha subunit was shown to be involved in mycoparasitism-related processes such as chitinase expression and overproduction of toxic secondary metabolites, whereas Tga3 was found to be completely avirulent showing defects in chitinase formation and host recognition.展开更多
A model of vibrating device coupling two pendulums (VDP) which is highly nonlinear was put forward to conduct vibration analysis. Based on energy analysis, dynamic equations with cubic nonlinearities were established ...A model of vibrating device coupling two pendulums (VDP) which is highly nonlinear was put forward to conduct vibration analysis. Based on energy analysis, dynamic equations with cubic nonlinearities were established using Lagrange's equation. In order to obtain approximate solution, multiple time scales method, one of perturbation technique, was applied. Cases of non-resonant and 1:1:2:2 internal resonant were discussed. In the non-resonant case, the validity of multiple time scales method is confirmed, comparing numerical results derived from fourth order Runge-Kutta method with analytical results derived from first order approximate expression. In the 1:1:2:2 internal resonant case, modal amplitudes of Aa1 and Ab2 increase, respectively, from 0.38 to 0.63 and from 0.19 to 0.32, while the corresponding frequencies have an increase of almost 1.6 times with changes of initial conditions, indicating the existence of typical nonlinear phenomenon. In addition, the chaotic motion is found under this condition.展开更多
The sparse recovery algorithms formulate synthetic aperture radar （SAR） imaging problem in terms of sparse representation （SR） of a small number of strong scatters＇ positions among a much large number of potentia...The sparse recovery algorithms formulate synthetic aperture radar （SAR） imaging problem in terms of sparse representation （SR） of a small number of strong scatters＇ positions among a much large number of potential scatters＇ positions, and provide an effective approach to improve the SAR image resolution. Based on the attributed scatter center model, several experiments were performed with different practical considerations to evaluate the performance of five representative SR techniques, namely, sparse Bayesian learning （SBL）, fast Bayesian matching pursuit （FBMP）, smoothed 10 norm method （SL0）, sparse reconstruction by separable approximation （SpaRSA）, fast iterative shrinkage-thresholding algorithm （FISTA）, and the parameter settings in five SR algorithms were discussed. In different situations, the performances of these algorithms were also discussed. Through the comparison of MSE and failure rate in each algorithm simulation, FBMP and SpaRSA are found suitable for dealing with problems in the SAR imaging based on attributed scattering center model. Although the SBL is time-consuming, it always get better performance when related to failure rate and high SNR.展开更多
文摘Objective INF2 is a member of the formins family.Abnormal expression and regulation of INF2 have been associated with the progression of various tumors,but the expression and role of INF2 in hepatocellular carcinoma(HCC)remain unclear.HCC is a highly lethal malignant tumor.Given the limitations of traditional treatments,this study explored the expression level,clinical value and potential mechanism of INF2 in HCC in order to seek new therapeutic targets.Methods In this study,we used public databases to analyze the expression of INF2 in pan-cancer and HCC,as well as the impact of INF2 expression levels on HCC prognosis.Quantitative real time polymerase chain reaction(RT-qPCR),Western blot,and immunohistochemistry were used to detect the expression level of INF2 in liver cancer cells and human HCC tissues.The correlation between INF2 expression and clinical pathological features was analyzed using public databases and clinical data of human HCC samples.Subsequently,the effects of INF2 expression on the biological function and Drp1 phosphorylation of liver cancer cells were elucidated through in vitro and in vivo experiments.Finally,the predictive value and potential mechanism of INF2 in HCC were further analyzed through database and immunohistochemical experiments.Results INF2 is aberrantly high expression in HCC samples and the high expression of INF2 is correlated with overall survival,liver cirrhosis and pathological differentiation of HCC patients.The expression level of INF2 has certain diagnostic value in predicting the prognosis and pathological differentiation of HCC.In vivo and in vitro HCC models,upregulated expression of INF2 triggers the proliferation and migration of the HCC cell,while knockdown of INF2 could counteract this effect.INF2 in liver cancer cells may affect mitochondrial division by inducing Drp1 phosphorylation and mediate immune escape by up-regulating PD-L1 expression,thus promoting tumor progression.Conclusion INF2 is highly expressed in HCC and is associated with poor prognosis.High expression of INF2 may promote HCC progression by inducing Drp1 phosphorylation and up-regulation of PD-L1 expression,and targeting INF2 may be beneficial for HCC patients with high expression of INF2.
基金湖南省教育厅基金优秀青年项目(No.22B0482)湖南科技大学博士启动基金(No.E51992 and E51993)资助。
文摘The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose⁃catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monitoring xylitol concentration.In this study,the gene encoding the thermophilic fungus Talaromyces emersonii XDH(TeXDH)was heterologously expressed in Escherichia coli BL21(DE3)at 16℃in the soluble form.Recombinant TeXDH with high purity was purified by using a Ni⁃NTA affinity column.Size⁃exclusion chromatography and SDS⁃PAGE analysis demonstrated that the puri⁃fied recombinant TeXDH exists as a native trimer with a molecular mass of approximately 116 kD,and is composed of three identical subunits,each with a molecular weight of around 39 kD.The TeXDH strictly preferred NAD^(+)as a coenzyme to NADP^(+).The optimal temperature and pH of the TeXDH were 40℃and 10.0,respectively.After EDTA treatment,the enzyme activity of TeXDH decreased to 43.26%of the initial enzyme activity,while the divalent metal ions Mg^(2+)or Ca^(2+)could recover the enzyme activity of TeXDH,reaching 103.32%and 110.69%of the initial enzyme activity,respectively,making them the optimal divalent metal ion cofactors for TeXDH enzyme.However,the divalent metal ions of Mn^(2+),Ni^(2+),Cu^(2+),Zn^(2+),Co^(2+),and Cd^(2+)significantly inhibited the activity of TeXDH.ICP⁃MS and molecular doc⁃king studies revealed that 1 mol/L of TeXDH bound 2 mol/L Zn^(2+)ions and 1 mol/L Mg^(2+)ion.Further⁃more,TeXDH exhibited a high specificity for xylitol,laying the foundation for the development of future xylitol biosensors.
基金Project(200501) supported the "985" Program of China
文摘Starch-nanoparticles were synthesized in water-in-oil microemusion at room temperature, and the starch-nanoparticles were coated with poly-L-lysine. The surface of the starch-nanoparticles was combined with fluorescence material Ru(bpy)32+·6H2O, and then the particles were characterized via transmission electron microscope. The fluorescence nanoparticles were conjugated with plasmid DNA to form complexes, and then treated with ultrasound and DNase I. pEGAD plasmid DNA-nanoparticle complexes were co-cultured with plant suspension cells of Dioscrea Zigiberensis G H Wright, and treated with ultrasound. The results show that the diameter of the fluorescence starch-nanoparticles is 50-100 nm. DNA-nanoparticle complexes can protect DNA from ultrasound damage as well as from DNase I cleavage. Mediated by ultrasound, pEGAD plasmid DNA-nanoparticle complexes can pierce into the cell wall, cell membrane and nucleus membrane of plant suspension cells. The green fluorescence protein(GFP) gene at a high frequency exceeds 5%. This nano-biomaterial can efficiently solve the problem that exterior genes cannot traverse the plant cell wall easily.
文摘The transient response of an unlimited cylindrical cavity buried in the infinite elastic soil subjected to an anti-plane impact load along the cavern axis direction was studied.Using Laplace transform combining with contour integral of the Laplace inverse transform specifically,the general analytical expressions of the soil displacement and stress are obtained in the time domain,respectively.And the numerical solutions of the problem computed by analytical expressions are presented.In the time domain,the dynamic responses of the infinite elastic soil are analyzed,and the calculation results are compared with those from numerical inversion proposed by Durbin and the static results.One observes good agreement between analytical and numerical inversion results,lending the further support to the method presented.Finally,some valuable shear wave propagation laws are gained: the displacement of the soil remains zero before the wave arrival,and after the shear wave arrival,the stress and the displacement at this point increase abruptly,then reduce and tend to the static value gradually at last.The wave attenuates along the radial,therefore the farther the wave is from the source,the smaller the stress and the displacement are,and the stress and the displacement are just functions of the radial distance from the axis.
基金Projects(50378069, 50639090) supported by the National Natural Science Foundation of ChinaProject(50639090) supported by the Joint Fund of Yalong River Hydropower Development, China
文摘To provide a seepage-stress coupling constitutive model that can directly describe the seepage-stress coupling relationship, a series of one-dimensional seepage-stress coupling tests on two kinds of soft rock (argillaceous siltstone and brown mudstone) were performed by using an MTS-815.02 tri-axial rock mechanics test system, with which the stress-strain curves according to the seepage variation were obtained. Based on the experimental results and by employing Hooke's law, the formulation of the coefficient of strain-dependent permeability was presented and introduced to establish a coupling model. In addition, the mathematical expression and the incremental formulation for coupling model were advanced, in which five parameters that can be respectively determined by using the experimental results were included. The calculated results show that the proposed coupling model is capable of simulating the stress-strain relationship with considering the seepage-stress coupling in the nonlinear elastic stage of two kinds of soft rock.
基金Project(13JJ9002)supported by Hunan Provincial Natural Science Foundation of ChinaProject(2012XK4081)supported by the Key Science Technology Plan Project of Hunan Provincial Science&Technology Department,ChinaProject(CX2012B124)supported by the Graduate Degree Thesis Innovation Program of Hunan Province,China
文摘In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.
基金Projects(51374112/E0409,51109084/E090701) supported by the National Natural Science Foundation of ChinaProject(ZQN-PY112) supported by the Promotion Program for Young and Middle-aged Teacher in Science and Technology Research of Huaqiao University,China+1 种基金Project(SKLGP2013K014) supported by the Opening Fund of State Key Laboratory of Geohazard Prevention and Geoenvironment Protection(Chengdu University of Technology),ChinaProject(SKLGDUEK1304) supported by the Open Research Fund of State Key Laboratory for Geomechanics and Deep Underground Engineering,China University of Mining and Technology,China
文摘Seepage and stress redistribution are the main factors affecting the stability of surrounding rock in high-pressure hydraulic tunnels.In this work,the effects of the seepage field were firstly simplified as a seepage factor acting on the stress field,and the equilibrium equation of high pressure inner water exosmosis was established based on physical theory.Then,the plane strain theory was used to solve the problem of elasticity,and the analytic expression of surrounding rock stress was obtained.On the basis of criterion of Norway,the influences of seepage,pore water pressure and buried depth on the characteristics of the stress distribution of surrounding rocks were studied.The analyses show that the first water-filling plays a decisive role in the stability of the surrounding rock; the influence of seepage on the stress field around the tunnel is the greatest,and the change of the seepage factor is approximately consistent with the logarithm divergence.With the effects of the rock pore water pressure,the circumferential stress shows the exchange between large and small,but the radial stress does not.Increasing the buried depth can enhance the arching effect of the surrounding rock,thus improving the stability.
基金Project(R5110012)supported by Special Foundation for Distinguished Young Scholars of Zhejiang Province,ChinaProject(2009C33117)supported by The General Program of Natural Science Foundation of Zhejiang Province,China
文摘An analytical approach was presented for estimating the factor of safety(FS) for slope failure, with consideration of the impact of a confined aquifer. An upward-moving wetting front from the confined water was assumed and the pore water pressure distribution was then estimated and used to obtain the analytical expression of FS. Then, the validation of the theoretical analysis was applied based on an actual case in Hong Kong. It is shown that the presence of a confined aquifer leads to a lower FS value, and the impact rate of hydrostatic pressure on FS increases as the confined water pressure increases, approaching to a maximum value determined by the ratio of water density to saturated soil density. It is also presented that the contribution of hydrostatic pressure and hydrodynamic pressure to the slope stability vary with the confined aquifer pressure.
基金Project(31030030) supported by the National Natural Science Foundation of China
文摘Gp96, a member of HSP90 family, is a versatile molecular chaperone with various newly-discovered functions, for example to serve as a low affinity, high capacity calcium binding protein, a natural adjuvant for therapeutic cancer vaccines, a tumor rejection antigen, an immune regulator to pathological cell death. Its multi-functional and structural characteristics make it also an interesting target to develop antibody-based therapeutics. However, its low immunogenicity to mice, because of its high-sequence similarity among different species, is an obstacle to obtain valuable monoclonal antibodies (MAbs). This is a common problem for any low immunogenic proteins, whose sequences share close identity between mice and other species. Here, a new strategy of priming was employed by swine endogenous full-length gp96 and then boosting by E. coli-system heterologously expressed gp96 N-terminal fragment (N-355) to generate MAbs. Twelve different highly-specific MAbs against swine/human endogenous gp96 were successfully obtained. The binding activities of these MAbs were confirmed by enzyme-linked immunosorbent assay (ELISA), Western blot (WB), immunofluorescence and flow cytometry analysis. This provides some important reagents for further research and potential therapeutics. The methods employed can be used for MAb production of any sequence-highly-conserved proteins between mice and swine/human (or any other species).
文摘Xylanase 1 (Xyn1) is one of the two major representatives of the xylanase system of T. reesei; the mechanisms governing its expression were analysed throughout this study. All factors and regulatory motifs responsible for transcriptional regulation and the model of their interplay in induction and repression will be presented. Using in vivo foot printing analysis of xylan-induced and glucose repressed mycelia, we detected three adjacent nucleotide sequences contacted by DNA-binding proteins. Protection within the inverted repeat of the Cre1 (SYGGRG) consensus sequence on the non coding strand under repressing conditions is in perfect agreement with the previously reported Cre1 dependent glucose repression of xyn1. Constitutive protein binding could be observed to a CCAAT-box and an inverted repeat of a 5′ GGCTAA 3′ sequence. EMSA with crude extracts from induced and repressed mycelia revealed that the latter motifs are sufficient for formation of the basal transcriptional complex under all conditions. The inverted repeat of GGCTAA closely resembles the consensus sequences of the cellulase and xylanase regulators Ace1, Ace2 and, Xyr1 (encoded by xyr1, cloned and characterised in this study) EMSA with heterologously expressed components of each factor and of the T. reesei Hap2/3/5 protein complex revealed that the basal transcriptional complex is formed by Xyr1 and the Hap2/3/5. Additionally to the Cre1 mediated carbon catabolite repression a yet unknown mechanism antagonizing induction of xyn1 expression could be elucidated. Latter occurs through competition of the repressor Ace1 and Xyr1 for the GGCTAA motif. In vivo proof for the relevance of identified motifs could be given through analysis of T. reesei transformants containing correspondingly mutated versions of the xyn1 promoter fused to the A. niger goxA gene. The results indicated that the basal as well as the induction level of xyn1 gene transcription is dependent on an interaction of Xyr1 with the GGCTAA motif while formation of the CCAAT-Hap2/3/5 complex slightly reduces induction. It can be concluded that mutations impairing protein binding in vitro lead to a loss of distinct regulatory functions in xyn1 gene expression in vivo. A respective model of gene regulation will be presented.
文摘Mycoparasitic species of Trichoderma are commercially applied as biological control agents against various fungal pathogens. The mycoparasitic interaction is host specific and includes recognition, attack and subsequent penetration and killing of the host. Investigations on the underlying events revealed that Trichoderma responds to multiple signals from the host (e.g. lectins or other ligands such as low molecular weight components released from the host’s cell wall) and host attack is accompanied by morphological changes and the secretion of hydrolytic enzymes and antibiotics. Degradation of the cell wall of the host fungus is-besides glucanases and proteases-mainly achieved by chitinases. In vivo studies showed that the ech42 gene (encoding endochitinase 42) is expressed before physical contact of Trichoderma with its host, probably representing one of the earliest events in mycoparasitism, whereas Nag1 (N-acetylglucosaminidase) plays a key role in the general induction of the chitinolytic enzyme system of T. atroviride . Investigations on the responsible signal transduction pathways of T. atroviride led to the isolation of several genes encoding key components of the cAMP and MAP kinase signaling pathways, as alpha and β subunits of heterotrimeric G proteins, the regulatory subunit of cAMP-dependent protein kinase, adenylate cyclase, and three MAP kinases. Analysis of knockout mutants, generated by Agrobacterium-mediated transformation, revealed that at least two alpha-subunits of heterotrimeric G proteins are participating in mycoparasitism-related signal transduction. The Tga1 G alpha subunit was shown to be involved in mycoparasitism-related processes such as chitinase expression and overproduction of toxic secondary metabolites, whereas Tga3 was found to be completely avirulent showing defects in chitinase formation and host recognition.
基金Projects(50574091, 50774084) supported by the National Natural Science Foundation of ChinaProject supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions+1 种基金Project(CXLX12_0949) supported by Research and Innovation Project for College Graduates of Jiangsu Province, ChinaProject(2013DXS03) supported by the Fundamental Research Funds for the Central Universities, China
文摘A model of vibrating device coupling two pendulums (VDP) which is highly nonlinear was put forward to conduct vibration analysis. Based on energy analysis, dynamic equations with cubic nonlinearities were established using Lagrange's equation. In order to obtain approximate solution, multiple time scales method, one of perturbation technique, was applied. Cases of non-resonant and 1:1:2:2 internal resonant were discussed. In the non-resonant case, the validity of multiple time scales method is confirmed, comparing numerical results derived from fourth order Runge-Kutta method with analytical results derived from first order approximate expression. In the 1:1:2:2 internal resonant case, modal amplitudes of Aa1 and Ab2 increase, respectively, from 0.38 to 0.63 and from 0.19 to 0.32, while the corresponding frequencies have an increase of almost 1.6 times with changes of initial conditions, indicating the existence of typical nonlinear phenomenon. In addition, the chaotic motion is found under this condition.
基金Project(61171133)supported by the National Natural Science Foundation of ChinaProject(11JJ1010)supported by the Natural Science Fund for Distinguished Young Scholars of Hunan Province,ChinaProject(61101182)supported by National Natural Science Foundation for Young Scientists of China
文摘The sparse recovery algorithms formulate synthetic aperture radar （SAR） imaging problem in terms of sparse representation （SR） of a small number of strong scatters＇ positions among a much large number of potential scatters＇ positions, and provide an effective approach to improve the SAR image resolution. Based on the attributed scatter center model, several experiments were performed with different practical considerations to evaluate the performance of five representative SR techniques, namely, sparse Bayesian learning （SBL）, fast Bayesian matching pursuit （FBMP）, smoothed 10 norm method （SL0）, sparse reconstruction by separable approximation （SpaRSA）, fast iterative shrinkage-thresholding algorithm （FISTA）, and the parameter settings in five SR algorithms were discussed. In different situations, the performances of these algorithms were also discussed. Through the comparison of MSE and failure rate in each algorithm simulation, FBMP and SpaRSA are found suitable for dealing with problems in the SAR imaging based on attributed scattering center model. Although the SBL is time-consuming, it always get better performance when related to failure rate and high SNR.
