A method for detecting hydrophobic patches on protein surfaces

A method for the detection of hydrophobic patches on the surfaces of protein tertiary structures is presented. It delineates explicit contiguous pieces of surface of arbitrary size and shape that consist solely of carbon and sulphur atoms using a dot representation of the solvent-accessible surface. The technique is also useful in detecting surface segments with other characteristics, such as polar patches. Its potential as a tool in the study of protein-protein interactions and substrate recognition is demonstrated by applying the method to myoglobin, Leu/Ile/Val-binding protein, lipase, lysozyme, azurin, triose phosphate isomerase, carbonic anhydrase, and phosphoglycerate kinase. Only the largest patches, having sizes exceeding random expectation, are deemed meaningful. In addition to well-known hydrophobic patches on these proteins, a number of other patches are found, and their significance is discussed. The method is simple, fast, and robust. The program text is obtainable by anonymous ftp. © 1996 Wiley-Liss, Inc.     

Influence of tool rotation on 3D surface topography at the nanometric scale

The influence of the tool rotation on the 3D surface topography produced by the nano-cutting process is investigated using molecular dynamics simulations. The least square mean method is utilized to model the evaluation criteria for the surface roughness parameters. The effects of the tool rotation on the cutting force and the chip formation at the nano-metric scale are also evaluated. It is found that the chip formation produced with tool rotation is dominated by the ploughing and the shearing forces. With increase of the adopted rotation velocity, the cutting force is sharply increased and the smaller elastic recovery of the machined surface is observed. The 3D surface roughness parameters at the nano-metric scale are significantly influenced by the tool rotation velocity and the feed speed, and the surface quality can be improved by decreasing the tool rotation velocity and the feed speed.     

A biomimetic platform for studying root-environment interaction

Aims

Microstructure plays an important role in biological systems. Microstructural features are critical in the interaction between two biological organisms, for example, a microorganism and the surface of a plant. However, isolating the structural effect of the interaction from all other parameters is challenging when working directly with the natural system. Replicating microstructure of leaves was recently shown to be a powerful research tool for studying leaf-environment interaction. However, no such tool exists for roots. Roots present a special challenge because of their delicacy (specifically of root hairs) and their 3D structure. We aim at developing such a tool for roots.

Methods

Biomimetics use synthetic systems to mimic the structure of biological systems, enabling the isolation of structural function. Here we present a method which adapts tools from leaf microstructure replication to roots. We introduce new polymers for this replication.

Results

We find that Polyurethane methacrylate (PUMA) with fast UV curing gives a reliable replication of the tomato root surface microstructure. We show that our system is compatible with the pathogenic soilborne bacterium Ralstonia solanacearum.

Conclusions

This newly developed tool may be used to study the effect of microstructure, isolated from all other effects, on the interaction of roots with their environment.

     

Surface Modeling of Workpiece and Tool Trajectory Planning for Spray Painting Robot

Automated tool trajectory planning for spray-painting robots is still a challenging problem, especially for a large free-form surface. A grid approximation of a free-form surface is adopted in CAD modeling in this paper. A free-form surface model is approximated by a set of flat patches. We describe here an efficient and flexible tool trajectory optimization scheme using T-Bézier curves calculated in a new way from trigonometrical bases. The distance between the spray gun and the free-form surface along the normal vector is varied. Automotive body parts, which are large free-form surfaces, are used to test the scheme. The experimental results show that the trajectory planning algorithm achieves satisfactory performance. This algorithm can also be extended to other applications.     

Determining functionally important amino acid residues of the E1 protein of Venezuelan equine encephalitis virus

A new method for predicting interacting residues in protein complexes, InterProSurf, was applied to the E1 envelope protein of Venezuelan equine encephalitis (VEEV). Monomeric and trimeric models of VEEV-E1 were constructed with our MPACK program, using the crystal structure of the E1 protein of Semliki forest virus as a template. An alignment of the E1 sequences from representative alphavirus sequences was used to determine physical chemical property motifs (likely functional areas) with our PCPMer program. Information on residue variability, propensity to be in protein interfaces, and surface exposure on the model was combined to predict surface clusters likely to interact with other viral or cellular proteins. Mutagenesis of these clusters indicated that the predictions accurately detected areas crucial for virus infection. In addition to the fusion peptide area in domain 2, at least two other surface areas play an important role in virus infection. We propose that these may be sites of interaction between the E1–E1 and E1–E2 subdomains of the envelope proteins that are required to assemble the functional unit. The InterProSurf method is, thus, an important new tool for predicting viral protein interactions. These results can aid in the design of new vaccines against alphaviruses and other viruses.     

Monitoring surface chemical changes in the bacterial cell wall: multivariate analysis of cryo-x-ray photoelectron spectroscopy data

Gram-negative bacteria can alter the composition of the lipopolysaccharide (LPS) layer of the outer membrane as a response to different growth conditions and external stimuli. These alterations can, for example, promote attachment to surfaces and biofilm formation. The changes occur in the outermost layer of the cell and may consequently influence interactions between bacterial cells and surrounding host tissue, as well as other surfaces. Microscopic analyses, fractionation of bacterial cells, or other traditional microbiological assays have previously been used to study these alterations. These methods can, however, be time consuming and do not always give detailed chemical information about the bacterial cell surface. We here present an analytical method that provides chemical information on the outermost portion of bacterial cells with respect to protein, peptidoglycan, lipid, and polysaccharide content. The method involves cryo-x-ray photoelectron spectroscopy analyses of the outermost portion (within ～10 nm of the surface) of intact bacterial cells followed by a multivariate curve resolution analysis of carbon spectra. It can be used as a tool for characterizing and monitoring variations in the chemical composition of bacterial cell walls or of isolated outer membrane vesicles, variations that result from e.g. mutations or external stimuli. The method enabled us to predict accurately the alterations in polysaccharide content and surface chemistries of a set of well characterized Escherichia coli LPS mutants. The described approach may moreover be applied to monitor surface chemical composition of other biological samples.     

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.     

Analysis of single nucleotide polymorphisms with solid phase invasive cleavage reactions

Using microparticles as the capture surface and fluorescence resonance energy transfer as the detection technology, we have demonstrated the feasibility of performing the invasive cleavage reaction on a solid phase. An effective tool for many genomic applications, the solution phase invasive cleavage assay is a signal amplification method capable of distinguishing nucleic acids that differ by only a single base mutation. The method positions two overlapping oligonucleotides, the probe and upstream oligonucleotides, on the target nucleic acid to create a complex recognized and cleaved by a structure-specific 5′-nuclease. For microarray and other multiplex applications, however, the method must be adapted to a solid phase platform. Effective cleavage of the probe oligonucleotide occurred when either of the two required overlapping oligonucleotides was configured as the particle-bound reagent and also when both oligonucleotides were attached to the solid phase. Positioning probe oligonucleotides away from the particle surface via long tethers improved both the signal and the reaction rates. The particle-based invasive cleavage reaction was capable of distinguishing the ApoE Cys158 and Arg158 alleles at target concentrations as low as 100 amol/assay (0.5 pM).     

Direct monitoring of molecular recognition processes using fluorescence enhancement at colloid-coated microplates

Direct monitoring of recognition processes at the molecular level is a valuable tool for studying reaction kinetics to assess affinity constants (e.g. drugs to receptors) and for designing rapid single step immunoassays. Methods currently used to gain information about binding processes predominantly depend on surface plasmon resonance. These systems use excitation with coherent light in attenuated total reflection geometry to obtain discrimination between surface-bound and free molecules in solution. Therefore labeling of the compounds is not necessary, but due to the complexity of the measuring setup the method is rather costly. In this contribution we present a simple method for performing kinetic single step biorecognition assays with fluorophore labeled compounds using the fluorescence enhancement properties of surface bound silver colloids. Silver colloids are bound to standard microplates via silanization of the plastic surface. Fluorophores close to this colloid coated surface show a significant gain in fluorescence compared to fluorophores farther away in the bulk solution. Therefore discrimination between surface bound and free fluorophores is possible and the binding of, for example, fluorophore labeled antibodies to antigens immobilized on the colloid surface results in increasing fluorescence intensity. Utilization of standard microplates makes this method fully compatible with conventional microplate processing and reading devices. Neither excitation with coherent laser light nor ATR geometry is required, the measurement is performed in a standard fluorescence microplate reader in front face geometry with a xenon flash lamp as excitation source. Methods for the preparation of colloid-coated microplates and fluorescence-enhanced biorecognition assays are presented. Additionally the dependence of the system performance on the structure and properties of the metal colloid coated surface is described. A two-component biorecognition model system shows a detection limit in the subnanomolar range. The ease of colloid-surface preparation and the high sensitivity makes fluorescence enhancement at colloid-coated microplates a valuable tool for studying reaction kinetics and performing rapid single-step immunoassays.     

Cellulosome-based, Clostridium-derived multi-functional enzyme complexes for advanced biotechnology tool development: Advances and applications

The cellulosome is one of nature's most elegant and elaborate nanomachines and a key biological and biotechnological macromolecule that can be used as a multi-functional protein complex tool. Each protein module in the cellulosome system is potentially useful in an advanced biotechnology application. The high-affinity interactions between the cohesin and dockerin domains can be used in protein-based biosensors to improve both sensitivity and selectivity. The scaffolding protein includes a carbohydrate-binding module (CBM) that attaches strongly to cellulose substrates and facilitates the purification of proteins fused with the dockerin module through a one-step CBM purification method. Although the surface layer homology (SLH) domain of CbpA is not present in other strains, replacement of the cell surface anchoring domain allows a foreign protein to be displayed on the surface of other strains. The development of a hydrolysis enzyme complex is a useful strategy for consolidated bioprocessing (CBP), enabling microorganisms with biomass hydrolysis activity. Thus, the development of various configurations of multi-functional protein complexes for use as tools in whole-cell biocatalyst systems has drawn considerable attention as an attractive strategy for bioprocess applications. This review provides a detailed summary of the current achievements in Clostridium-derived multi-functional complex development and the impact of these complexes in various areas of biotechnology.     

Quantification analysis of yeast-displayed lipase

We describe a method for quantification of displayed lipase on yeast cell surface. The strategy uses an organophosphonate ester to irreversibly inhibit the active lipase and release a detectable fluorescent group. The amount of displayed lipase can be represented as “g/g cell” or “molecules/cell”. The results obtained correlated well with those obtained by existing methods. Therefore, this method is credible and will provide a powerful tool to promote research of lipase yeast surface display.     

Pre-absorbed immunoproteomics: a novel method for the detection of Streptococcus suis surface proteins

Streptococcus suis serotype 2 (SS2) is a zoonotic pathogen that can cause infections in pigs and humans. Bacterial surface proteins are often investigated as potential vaccine candidates and biomarkers of virulence. In this study, a novel method for identifying bacterial surface proteins is presented, which combines immunoproteomic and immunoserologic techniques. Critical to the success of this new method is an improved procedure for generating two-dimensional electrophoresis gel profiles of S. suis proteins. The S. suis surface proteins identified in this study include muramidase-released protein precursor (MRP) and an ABC transporter protein, while MRP is thought to be one of the main virulence factors in SS2 located on the bacterial surface. Herein, we demonstrate that the ABC transporter protein can bind to HEp-2 cells, which strongly suggests that this protein is located on the bacterial cell surface and may be involved in pathogenesis. An immunofluorescence assay confirmed that the ABC transporter is localized to the bacterial outer surface. This new method may prove to be a useful tool for identifying surface proteins, and aid in the development of new vaccine subunits and disease diagnostics.     

Altering the orientation of proteins on self-assembled monolayers: a computational study

A combined computational docking-molecular dynamics study has been performed on a system consisting of cytochrome c protein and alkanethiol self-assembled monolayers of various geometries. The results suggest that the orientation of the protein on the surface may be controlled or altered by means of designing specific structural motifs on the surface. The proposed computational approach may be used as a fast and reliable tool to complement other theoretical and experimental techniques of exploring other protein-surface interfaces.     

Versatile tool for the manipulation of electrophoresis chips

This communication describes a versatile tool allowing standard operations (i.e. washing, pre-conditioning, separation, inner surface modification of the chip channel) with capillary electrophoresis chips. Through currently designed for a chip of maximal dimensions 30 x 60 mm, other formats of the chip require only a minimum adjustment of the equipment, namely setting of the chip sliding rails and adequate arrangement of the exchangeable heads. The application of the tool is demonstrated by the separation of the standard set of inorganic cations.     

表面肌电信号的肌肉疲劳判别研究进展

肌肉在周期的收缩或静态的拉伸过程中,会渐渐进入疲劳状态,肌肉疲劳特性的研究在康复医学、运动医学领域具有重要作用。表面肌电信号是从肌肉表面通过电极记录下来的反映神经肌肉系统活动的一维时间序列非平稳生物电信号,是评价局部肌肉疲劳的有效工具。本研究从时域和频域、时频域线性方法下的测量指标和非线性方法下的指标来综述表面肌电信号的疲劳研究进展,同时比较各种方法的优缺点,并对使用表面肌电信号来判别疲劳研究做了进一步的展望。     

Evaluation of surface hydrophobicity of immobilized protein with a surface plasmon resonance sensor

Immobilization is widely used to isolate agglutinative and associative proteins with large hydrophobic surfaces. Surface hydrophobicities of immobilized proteins were quantified by measuring the adsorption amounts of Triton X-100 as a hydrophobic probe with a biosensor that utilizes the phenomena of surface plasmon resonance (SPR). We measured SPR signal changes derived from adsorption of Triton X-100 to five kinds proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller equation, partly modified with a term for correcting an influence of the net charge of immobilized protein. SPR signal changes obtained by this method correlated with the values of surface hydrophobicities obtained by conventional assay using a hydrophobic probe. Thus this measuring method using an SPR sensor and Triton X-100 is expected to be a tool for quantifying surface hydrophobicities of immobilized proteins.     

Quick Construction of Femoral Model Using Surface Feature Parameterization

To facilitate the modifying of femoral surface model, by dividing the femoral mesh into surface feature units bearing medical significance based on surface feature technology, a new approach of constructing femoral models using surface feature technology is proposed. Firstly, considering of femoral anatomy, the femoral triangle mesh model generated from the averaged point-clouds is divided into several specific regions, which are called feature regions; Secondly, feature parameters are defined and the constraints among them are set up, and feature surfaces are created by skinning the contours; Finally, the adjacent feature surfaces are connected by transition surfaces, and the parametric CAD surface model of femur is constructed. Experimental results show that, with the proposed method, the surface feature model can be intuitively constructed and edited with high-level parameters. Therefore, the proposed method provides a basic tool for the design of implants and the digital restoration of incomplete femurs.     

响应面法优化獐牙菜苦苷生物转化条件

采用响应面法对獐牙菜苦苷的生物转化条件进行优化,首先应用Plackett-Burman方法筛选出对转化具有显著性影响的因素,再用中心响应面法的Response Optimizer工具对最优转化条件进行了分析和预测,最后对预测结果进行了验证。结果表明,獐牙菜新碱的最优化转化条件(mg/mL)为MgSO4.7H2O 5.14、MnSO4.4H2O 3.42、葡萄糖9.95、蛋白胨9.24、pH5.80。在此条件下獐牙菜新碱的最大转化产率为17.64%;红百金花内酯的最优转化条件(mg/mL)为MgSO4.7H2O 4.09、MnSO4.4H2O6.07、葡萄糖7.37、酵母膏7.37,pH5.73。在此条件下红百金花内酯的最大转化产率为8.81%。     

Path planning for minimally-invasive knee surgery using a hybrid optimization procedure

The aim of this study was to develop a procedure for medical tool path planning in minimally-invasive knee surgery. The collision-free paths for the tool were obtained using the control locations method with a hybrid optimization strategy. The tool and knee elements were described with surface meshes. The knee model allowed for bones displacement and variable incision size and location. The proposed procedure was proven to be effective in path planning for minimally-invasive surgery. It can serve as a valuable aid in surgery planning and may also be used in systems for autonomous or semi-autonomous knee surgery.     

Single-base mutational analysis of cancer and genetic diseases using membrane bound modified oligonucleotides.

A convenient format for the detection of PCR amplified sequences is the hybridization of the PCR products to oligonucleotide probes which are immobilized on a solid phase. We describe a new method for site-specific attachment of such probe oligonucleotides to nylon membranes. The method is based on the formation of an amide bond between carboxyl groups present on the membranes and amino-linkers situated on the 5' end of the oligonucleotides. The covalent attachment is via a carbodiimide mediated condensation. The single, 5' end attachment of the oligonucleotides to the membrane surface leaves the probe free to interact with complementary sequences, thus increasing the hybridization efficiency relative to methods where heat or ultraviolet light is used for non-specific fixation. Using biotinylated PCR products in hybridization reactions along with a non-radioactive chemiluminescent detection system, high efficiency hybridization is obtained as well as a very good signal to noise ratio. The method has been applied successfully to the detection of RAS point mutations, cystic fibrosis deletion and point mutations and others. The sensitivity, simplicity and reproducibility of this method make it an ideal tool for the diagnosis of infectious and genetic diseases, as well as analysis of mutations in neoplasias, HLA typing and other areas.