一种新型广谱的原核型分泌表达载体的建立及人表皮生长因子的表达

通过Cyanobase藻类学数据库查询,从集胞藻6803转译后加工过程中的某些蛋白的分泌信号序列得到了可以用于分泌型表达的信号序列,进一步通过SignalP 3.0 Server分析软件预验证,从集胞藻6803得到4种不同蛋白的信号序列A、B、C、D,将这些信号序列插入pET-His-EGF表达载体hegf基因上游,得到了带有分泌型信号的pS-X系列分泌型表达载体,人表皮生长因子(hEGF)在其中的pS-A载体中实现了分泌型表达,hEGF分泌到周质空间的相对表达量约为1%,在其它3个载体中未见表达.获得的pS-A分泌型表达载体是一种新型分泌型表达载体,它利用蓝藻信号序列作为分泌表达的信号肽,实现了hEGF多肽在大肠杆菌中分泌表达,通过融合白亚细胞定位法处理得到的hEGF多肽在胞质、周质空间和培养基中表达的比例为85:33:25(density intensity/mm2),利用渗透休克处理方法得到的比例为50:37:25.由于A信号序列本身是集胞藻6803膜蛋白(ID:s110172/NCBI-GI:1001433)的分泌信号肽,说明pS-A载体是一种广谱的原核型分泌表达载体.这一结果为实现hEGF在螺旋藻中的分泌型表达奠定了基础.     

Fundamental analysis of recombinant human epidermal growth factor in solution with biophysical methods

Correlation of thermodynamic and secondary structural stability of proteins at various buffer pHs was investigated using differential scanning calorimetry (DSC), dynamic light scattering (DLS) and attenuated total reflection Fourier-transform infrared spectroscopy (ATR FT-IR). Recombinant human epithelial growth factor (rhEGF) was selected as a model protein at various pHs and in different buffers, including phosphate, histidine, citrate, HEPES and Tris. Particle size and zeta potential of rhEGF at each selected pH of buffer were observed by DLS. Four factors were used to characterize the biophysical stability of rhEGF in solution: temperature at maximum heat flux (T_m), intermolecular β-sheet contents, zeta size and zeta potential. It was possible to predict the apparent isoelectric point (pI) of rhEGF as 4.43 by plotting pH against zeta potential. When the pH of the rhEGF solution increased or decreased from pI, the absolute zeta potential increased indicating a reduced possibility of protein aggregation, since T_m increased and β-sheet contents decreased. The contents of induced intermolecular β-sheet in Tris and HEPES buffers were the lowest. Thermodynamic stability of rhEGF markedly increased when pH is higher than 6.2 in histidine buffer where T_m of first transition was all above 70?°C. Moreover, rhEGF in Tris buffer was more thermodynamically stable than in HEPES with higher zeta potential. Tris buffer at pH 7.2 was concluded to be the most favorable.     

Epidermal growth factor-ferritin h-chain protein nanoparticles for tumor active targeting

Human ferritin H-chain protein (FTH1)-based nanoparticles possess a precisely assembled nanometer-scale structure and high safety. However, their applications for imaging and drug delivery towards cancer cells remain limited due to a lack of target specificity. Epidermal growth factor receptor (EGFR) is overexpressed in many malignant tissues including breast cancer, and has been used as a therapeutic target for cancer treatment. Herein, a genetic method is shown to generate EGF-FTH1 chimeric proteins. EGF-FTH1 nanoparticles with EGF on the surface are then produced. The data demonstrate that EGF-FTH1 nanoparticles, with a small size (11.8 ± 1.8 nm), narrow size distribution, and high biosafety, can specifically bind to and then be taken up by breast cancer MCF-7 cells and MDA-MB-231 cells, but not normal breast epithelial MCF-10A cells. In contrast, binding and absorption of nontargeted ferritin-based nanoparticles to breast cancer cells are negligible. In vivo studies show that EGF-FTH1 nanoparticles are accumulated in breast tumors in a mouse xenograft model. Interestingly, the concentration of EGF-FTH1 nanoparticles in the tumor site is significantly reduced when mice are pretreated with an excess of free EGF. These results imply that EGF-EGFR interaction plays an important role in regulating the tumor retention of EGF-FTH1 nanoparticles.     

Immobilization of Photo‐Immunoconjugates on Nanoparticles Leads to Enhanced Light‐Activated Biological Effects

The past three decades have witnessed notable advances in establishing photosensitizer–antibody photo‐immunoconjugates for photo‐immunotherapy and imaging of tumors. Photo‐immunotherapy minimizes damage to surrounding healthy tissue when using a cancer‐selective photo‐immunoconjugate, but requires a threshold intracellular photosensitizer concentration to be effective. Delivery of immunoconjugates to the target cells is often hindered by I) the low photosensitizer‐to‐antibody ratio of photo‐immunoconjugates and II) the limited amount of target molecule presented on the cell surface. Here, a nanoengineering approach is introduced to overcome these obstacles and improve the effectiveness of photo‐immunotherapy and imaging. Click chemistry coupling of benzoporphyrin derivative (BPD)–Cetuximab photo‐immunoconjugates onto FKR560 dye‐containing poly(lactic‐co‐glycolic acid) nanoparticles markedly enhances intracellular photo‐immunoconjugate accumulation and potentiates light‐activated photo‐immunotoxicity in ovarian cancer and glioblastoma. It is further demonstrated that co‐delivery and light activation of BPD and FKR560 allow longitudinal fluorescence tracking of photoimmunoconjugate and nanoparticle in cells. Using xenograft mouse models of epithelial ovarian cancer, intravenous injection of photo‐immunoconjugated nanoparticles doubles intratumoral accumulation of photo‐immunoconjugates, resulting in an enhanced photoimmunotherapy‐mediated tumor volume reduction, compared to “standard” immunoconjugates. This generalizable “carrier effect” phenomenon is attributed to the successful incorporation of photo‐immunoconjugates onto a nanoplatform, which modulates immunoconjugate delivery and improves treatment outcomes.     

A Gold Nanocage/Cluster Hybrid Structure for Whole‐Body Multispectral Optoacoustic Tomography Imaging,EGFR Inhibitor Delivery,and Photothermal Therapy

Gold nanocages (AuNCs) and gold nanoclusters (AuClusters) are two classes of advantageous nanostructures with special optical properties, and many other attractive properties. Integrating them into one nanosystem may achieve greater and smarter performance. Herein, a hybrid gold nanostructure for fluorescent and optoacoustic tomography imaging, controlled release of drugs, and photothermal therapy (PTT) is demonstrated. For this nanodrug (EA–AB), an epidermal growth factor receptor (EGFR) inhibitor erlotinib (EB) is loaded into AuNCs, which are then capped and functionalized by biocompatible AuCluster@BSA (BSA = bovine serum albumin) conjugates via electrostatic interaction. Upon cell internalization, the lysosomal proteases and low pH cause the release of EB from EA–AB, and also induce fluorescence restoration of the AuCluster for imaging. Irradiation with near‐infrared light further promotes the drug release and affords a PTT effect as well. The AuNC‐based nanodrug is optoacoustically active, and its biodistribution and metabolic process have been successfully monitored by whole‐body and 3D multispectral optoacoustic tomography imaging. Owing to the combined actions of PTT and EGFR pathway blockage, EA–AB exhibits marked tumor inhibition efficacy in vivo.     

Rapid and accurate ranking of binding affinities of epidermal growth factor receptor sequences with selected lung cancer drugs

The epidermal growth factor receptor (EGFR) is a major target for drugs in treating lung carcinoma. Mutations in the tyrosine kinase domain of EGFR commonly arise in human cancers, which can cause drug sensitivity or resistance by influencing the relative strengths of drug and ATP-binding. In this study, we investigate the binding affinities of two tyrosine kinase inhibitors—AEE788 and Gefitinib—to EGFR using molecular dynamics simulation. The interactions between these inhibitors and the EGFR kinase domain are analysed using multiple short (ensemble) simulations and the molecular mechanics/Poisson–Boltzmann solvent area (MM/PBSA) method. Here, we show that ensemble simulations correctly rank the binding affinities for these systems: we report the successful ranking of each drug binding to a variety of EGFR sequences and of the two drugs binding to a given sequence, using petascale computing resources, within a few days.     

纯化肝细胞生长因子对肝癌细胞的增殖刺激作用

利用ＤＥＡＥ纤维素层析、ＦＰＬＣ和ＨＰＬＣ分离，从人胎肝中获得了纯化的特异刺激肝来源细胞ＤＮＡ合成的肝细胞生长因子（ＨＧＦ）。进一步研究表明，ＨＧＦ明显刺激人肝癌细胞的增殖，其刺激活性具有量效关系，并诱导肝癌细胞从Ｇ０／Ｇ１期进入Ｓ期。本研究为探讨ＨＧＦ作用机理建立了模型。     

MFI型分子筛膜的制备及气体渗透性能研究

采用原位水热合成和无模板剂二次生长合成的方法在α-Al2O3基膜上合成了MFI型分子筛膜,并用XRD,SEM和气体渗透实验等方法进行表征,结果表明合成在α-Al2O3基膜的物质为MFI型分子筛.对于水热合成的分子筛膜,氢/异丁烷的理想分离系数在298 K和473 K时分别为97和52;对于二次生长合成的分子筛膜,氢/异丁烷的理想分离系数在298 K和473 K时分别为497和370,远大于它们Knudsen扩散5.34的比值.表明气体是通过MFI型分子筛的孔道透过.水热合成分子筛膜的正/异丁烷理想分离系数在298 K和473 K时分别为22和13;二次生长分子筛膜的正/异丁烷理想分离系数在298 K和473 K时分别为77和70,气体分离数据表明,两种分子筛膜对气体分离是由分子筛分占主导,同时分子筛膜没有裂缺.     

Nanofiber‐Reinforced Silver Nanowires Network as a Robust,Ultrathin, and Conformable Epidermal Electrode for Ambulatory Monitoring of Physiological Signals

Extremely soft and thin electrodes with high skin conformability have potential applications in wearable devices for personal healthcare. Here, a submicrometer thick, highly robust, and conformable nanonetwork epidermal electrode (NEE) is reported. Electrospinning of polyamide nanofibers and electrospraying of silver nanowires are simultaneously performed to form a homogeneously convoluted network in a nonwoven way. For a 125 nm thick NEE, a low sheet resistance of ≈4 Ω sq^?1 with an optical transmittance of ≈82% is achieved. Due to the nanofiber‐based scaffold that undertakes most of the stress during deformation, the electric resistance of the NEE shows very little variation; less than 1.2% after 50 000 bending cycles. The NEE can form a fully conformal contact to human skin without additional adhesives, and the NEE shows a contact impedance that is over 50% lower than what is found in commercial gel electrodes. Due to conformal contact even under deformation, the NEE proves to be a stable, robust, and comfortable approach for measuring electrocardiogram signals, especially when a subject is in motion. These features make the NEE promising for use in the ambulatory measurement of physiological signals for healthcare applications.     

An experimental investigation on fatigue crack growth of AL6XN stainless steel

The crack growth behavior of AL6XN stainless steel was experimentally investigated using round compact tension (CT) specimens. The influences of the R-ratio (the ratio of the minimum load over the maximum applied load in a cycle), the tensile and compressive overloads, and the loading sequence on crack growth were studied in detail. The results from the constant-amplitude experiments show a sensitivity of the crack growth rate to the R-ratio. The application of a tensile overload has a profound effect on crack growth, resulting in a significant retardation in the crack propagation rate. A compressive overload (underload) leads to a short-lived acceleration in crack growth. Results from the two-step high-low loading reveal a period of crack growth retardation at the beginning of the lower amplitude step, an effect similar to that of a single overload. A crack driving force parameter together with a modified Wheeler model is found to correlate the crack growth experiments well.     

Polarization of hippocampal neurons with competitive surface stimuli: contact guidance cues are preferred over chemical ligands

Neuronal behaviour is profoundly influenced by extracellular stimuli in many developmental and regeneration processes. Understanding neuron responses and integration of environmental signals could impact the design of successful therapies for neurodegenerative diseases and nerve injuries. Here, we have investigated the influence of localized extracellular cues on polarization (i.e. axon formation) of hippocampal neurons. Electron-beam lithography, microfabrication techniques and protein immobilization were used to create a unique system that provided simultaneous and independent chemical and physical cues to individual neurons. In particular, we analysed competitive responses between simultaneous stimulation with chemical ligands, including immobilized nerve growth factor and laminin, and contact guidance cues mediated by surface topography (i.e. microchannels). Contact guidance cues were preferred 70% of the time over chemical ligands by neurons extending axons, which suggests a stronger stimulation mechanism triggered by topography. This investigation contributes to the understanding of neuronal behaviour on artificial substrates, which is applicable to the creation of artificial environments for neural engineering applications.     

The influence of cross-sectional thickness on fatigue crack growth

For thin structures, fatigue crack growth rates may vary with the structure's thickness for a given stress intensity factor range. This effect is mainly due to the change in the nature of the plastic deformation when the plastic zone size becomes comparable with, or greater than, the cross-sectional thickness. Variations in the constraint affect both the crack tip plastic blunting behaviour as well as the fatigue crack closure level. Approximate expressions are constructed for the constraint factor based on asymptotic values and numerical results, which are shown to correlate well with finite element results. It is demonstrated that the present results not only permit predictions of the specimen thickness effects on fatigue crack propagation under spectrum loading, but also eliminate the need to determine the constraint factor by curve-fitting crack growth data.     

主客体胶束水凝胶的制备及对神经生长因子的包载

通过可逆加成-断裂链转移聚合(RAFT)反应制备了一种亲水性两嵌段聚合物——聚乙二醇/聚N-异丙基丙烯酰胺(PEG-PNIPAM),利用α-环糊精(α-CD)与其中PEG链段的选择性包合形成了一种独特的主客体胶束,这种胶束由于具备中空结构,可对亲水性药物起到很好的负载作用。其对神经生长因子的包载率为经典普朗尼克F127胶束的2倍。进一步利用主客体胶束为大分子交联剂制备丙烯酰胺水凝胶,可赋予水凝胶优异的力学性能,拉伸伸长率为化学交联对照组5倍,并且强度高于对照组2倍以上。循环拉伸测试表明,该水凝胶具有良好的应力耗散机制。     

Mixed-mode fatigue crack growth under biaxial loading

Studies on crack growth in a panel with an inclined crack subjected to biaxial tensile fatigue loading are presented. The strain energy density factor approach is used to characterize the fatigue crack growth. The crack growth trajectory as a function of the initial crack angle and the biaxiality ratio is also predicted. The analysis is applied to 7075-T6 aluminium alloy to predict the dependence of crack growth rate on the crack angle. The effect of crack angle on the cyclic life of the component and on the cyclic life ratio is presented and discussed.     

A NEW METHOD FOR THE MEASUREMENT OF MODE II FATIGUE THRESHOLD STRESS INTENSITY FACTOR RANGE ΔKτth

Abstract— In order to evaluate the threshold value Δ K _τth for mode II fatigue crack growth, a new measurement method of mode II fatigue crack growth has been developed. This method uses a conventional closed-loop tension—compression fatigue testing machine without additional loading attachments. Mode II fatigue tests for structural steel and rail steel have been carried out. This method has proved successful and has reproduced mode II fatigue fracture surfaces similar to those found in the spalling of industrial steel-making rolls. The crack length during testing was measured by an AC potential method. The relationships between d a /d N and Δ K _τ and AK _τth for several materials have been obtained.     

Experimental Study of Crack Growth in a Bimetal Under Fatigue and Fatigue-Creep Conditions

Fatigue and fatigue-creep crack growth rate in a bimetal of continuous caster rolls for different loading frequencies and waveforms is investigated with the use of stress, deformation and energy criteria of fracture mechanics.     

MFI型分子筛膜的制备及气体渗透特性

采用原位水热合成和无模板剂二次生长合成的方法,在α-Al2O3基膜上合成了MFI型分子筛膜,用XRD,SEM和气体渗透实验等方法进行表征,表明合成在α-Al2O3基膜的物质为MFI型分子筛.原位合成的分子筛膜,氢/异丁烷的理想分离系数在298K和473K时分别为97和52;二次生长合成的分子筛膜,氢/异丁烷的理想分离系数在298K和473K时分别为497和370,远大于它们Knudsen扩散5.34的比值,表明气体是通过MFI型分子筛的孔道透过.SF6分子动力学直径大于分子筛膜孔径,H2/SF6分离因子远大于Knudsen扩散值,几乎不透过分子筛膜.原位合成分子筛膜的正/异丁烷理想分离系数在298K和473K时分别为24和17;二次生长分子筛膜的正/异丁烷理想分离系数在298K和473K时分别为77和74.气体渗透分离实验结果表明,两种分子筛膜对气体分离是由分子筛分占主导,分子筛膜完整没有缺陷.     

The Effect of Micro Damage on Time-Dependent Crack Growth in a Composite Solid Propellant

The damage characteristics near the crack tip and crack growth behaviorin a centrally cracked sheet specimen of a solid propellant wereinvestigated. The specimen was subjected to a constant crosshead speed of0.508 mm/min at room temperature. The effects of local damage andfracture process near the crack tip on crack growth behavior wereinvestigated and results are discussed.     

Control of cell proliferation by a porous chitosan scaffold with multiple releasing capabilities

Abstract

The aim of this study was to develop a porous chitosan scaffold with long-acting drug release as an artificial dressing to promote skin wound healing. The dressing was fabricated by pre-freezing at different temperatures (?20 and ?80 °C) for different periods of time, followed by freeze-drying to form porous chitosan scaffolds with different pore sizes. The chitosan scaffolds were then used to investigate the effect of the controlled release of fibroblast growth factor-basic (bFGF) and transforming growth factor-β1 (TGFβ1) on mouse fibroblast cells (L929) and bovine carotid endothelial cells (BEC). The biocompatibility of the prepared chitosan scaffold was confirmed with WST-1 proliferation and viability assay, which demonstrated that the material is suitable for cell growth. The results of this study show that the pore sizes of the porous scaffolds prepared by freeze-drying can change depending on the pre-freezing temperature and time via the formation of ice crystals. In this study, the scaffolds with the largest pore size were found to be 153 ± 32 μm and scaffolds with the smallest pores to be 34 ± 9 μm. Through cell culture analysis, it was found that the concentration that increased proliferation of L929 cells for bFGF was 0.005 to 0.1 ng/mL, and the concentration for TGFβ1 was 0.005 to 1 ng/mL. The cell culture of the chitosan scaffold and growth factors shows that 3.75 ng of bFGF in scaffolds with pore sizes of 153 ± 32 μm can promote L929 cell proliferation, while 400 pg of TGFβ1 in scaffolds with pore size of 34 ± 9 μm can enhance the proliferation of L929 cells, but also inhibit BEC proliferation. It is proposed that the prepared chitosan scaffolds can form a multi-drug (bFGF and TGFβ1) release dressing that has the ability to control wound healing via regulating the proliferation of different cell types.