利用MALDI-TOF MS鉴定芽苗菜中解鸟氨酸拉乌尔菌和肺炎克雷伯菌的研究

应用基质解析电离飞行时间质谱(MALDI-TOF MS)方法对芽苗菜中检出率较高的2种微生物进行鉴定,结果为解鸟氨酸拉乌尔菌(33.3%)和肺炎克雷伯菌(43.5%)。应用SARAMIS Premium软件对这2种微生物的蛋白质图谱进行分析,获得解鸟氨酸拉乌尔菌特征标识峰16组,肺炎克雷伯菌特征标识峰10组;对分离微生物进行全标识峰和加权特征峰相关性分析,结果表明特征峰的加权会影响微生物鉴定结果间的相关关系,增强微生物的辨识度;对两种微生物进行多方面比较,发现两种微生物生化特性比较接近,但16Sr DNA分辨率较高,质谱相关分析虽有相交但可辨识。实验结果表明MALDI-TOF MS具有检测芽苗菜中微生物的能力并在其特征分析及溯源研究上具有一定的优势,MALDI-TOF MS为开放平台,对农产品微生物蛋白质特征图谱的收集与优化可提高其鉴定准确性。     

Identification of cellular pathways affected by Sortin2, a synthetic compound that affects protein targeting to the vacuole in Saccharomyces cerevisiae

Background

Sortin2 is a low mass compound that interferes with vacuolar delivery of proteins in plants and yeast. The Sortin2 phenotype was tested in Arabidopsis thaliana and found to be reversible upon drug removal, demonstrating the ability of chemical genomics to induce reversible phenotypes that would be difficult to achieve using conventional genetics [1]. However, standard genetic methods can be used to identify drug target pathways in a high-throughput manner.

Results

In this study, we analyzed structure-function relationships of Sortin2 using structural analogues. The results show the key roles of sulphite substitution and a benzoic acid group. A Sortin 2 hypersensitivity screen for the induced secretion of a vacuolar cargo protein was done utilizing a yeast haploid deletion library. Using bioinformatics approaches, we highlighted functional information about the cellular pathways affected by drug treatment which included protein sorting and other endomembrane system-related processes.

Conclusion

Chemical, genomic and genetics approaches were used to understand the mode of action of Sortin2, a bioactive chemical that affects the delivery of a vacuolar protein. Critical features of Sortin2 structure necessary for bioactivity suggest a binding pocket that may recognize two ends of Sortin2. The genome-wide screen shows that Sortin2 treatment in yeast affects primarily components within the endomembrane system. This approach allowed us to assign putative functions in protein sorting for fifteen genes of previously unknown function.     

The effect of thiolation on the mechanical and protein adsorption properties of polyurethanes

Segmented polyurethanes are important polymers for a number of industrial and technological applications. The purpose of this work was to synthesize polybutadiene-based polyurethanes and subsequently graft carboxylate and sulfonate side chains via thiol-ene reaction. Spectroscopic investigations showed that grafting yielded good conversion for the vinyl unsaturation of the polybutadiene soft segment. DSC and tensile testing revealed that grafted polyurethanes had a better segmental compatibility and superior mechanical properties than the control polyurethane without grafting. The carboxylic side chains of the soft segment were responsible for the observed improved mechanical properties. Initial protein adsorption tests on these polymers were found to be higher than the control surface. The polyurethanes of the current study could be used for biomedical applications where protein attachment to the surface is needed for specific cell adhesion and tissue repair.     

Identification and characterization of human polyserase-3, a novel protein with tandem serine-protease domains in the same polypeptide chain

Background  

We have previously described the identification and characterization of polyserase-1 and polyserase-2, two human serine proteases containing three different catalytic domains within the same polypeptide chain. Polyserase-1 shows a complex organization and it is synthesized as a membrane-bound protein which can generate three independent serine protease domains as a consequence of post-translational processing events. The two first domains are enzymatically active. By contrast, polyserase-2 is an extracellular glycosylated protein whose three protease domains remain embedded in the same chain, and only the first domain possesses catalytic activity.     

Dimensional considerations on the mechanical properties of 3D printed polymer parts

Additive manufacturing offers a useful and accessible tool for prototyping and manufacturing small volume functional parts. Polylactic acid (PLA) and thermoplastic polyurethane (TPU) are amongst the most commonly used materials. Characterising 3D printed PLA and TPU is potentially important for both designing and finite element modelling of functional parts. This work explores the mechanical properties of additively manufactured PLA/TPU specimens with consideration to design parameters including size, and infill percentage. PLA/TPU specimens are 3D-printed in selected ISO standard geometries with 20%, 60%, 100% infill percentage. Tensile and compression test results suggest that traditional ISO testing standards might be insufficient in characterising 3D printed materials for finite element modelling or application purposes. Infill percentage in combination to design size, may significantly affect the mechanical performance of 3D printed parts. Dimensional variation may cause inhomogeneity in mechanical properties between large and small cross section areas of the same part. The effect was reduced in small cross section parts where reducing the nominal infill had less effect on the resulting specimens. The results suggest that for 3D printed functional parts with significant dimensional differences between sections, the material properties are not necessarily homogeneous. This consideration may be significant for designers using 3D printing for applications, which include mechanical loading.     

Water-induced reconstruction that affects mobile ions on the surface of calcite

Time-sequenced contact-force micrographs show that the (104) calcite cleavage surface reconstructs in humid air through pit formation and film growth. After 8 h at 80% relative humidity (RH), 50% to 80% of the surface is covered by islands that are flat-topped and 1-nm high. The lateral growth rates of individual islands are 4.2+/-0.4 nm min-1 in the 41 direction and 1.8+/-0.4 nm min-1 in the 48 direction, resulting in islands having distinct major and minor axes. On some samples, a contiguous, 1.5-nm-high film rapidly grows between the islands and the pits. The areal expansion rate of the film is 500 times faster than that of the islands. Gaps between the contiguous film and the islands expand and contract, which suggests that mass is exchanged between them and that both are loosely bound. Complementing the topographic images, polarization heights are simultaneously measured by polarization-force microscopy. The polarization heights of the islands and the contiguous film are -6 to -10 nm and -4 to -5 nm, respectively, compared to their respective topographic heights of +1.0 and +1.5 nm. Under our experimental conditions, the polarization heights are a surrogate for the local dielectric constant of the sample epsilon and arise from a convolution of the mobility and the density of surface ions. The polarization heights imply that epsilonsubstrate>epsilonfilm>epsilonisland. Changes in topographic and polarization heights at 20% and 50% RH suggest that the structures of the islands are in dynamic equilibrium with the adsorbed water. Our evidence suggests that the islands contain loosely bound water and may therefore be a hydrated calcium carbonate phase stabilized by the calcite surface.     

In Vitro Investigation of the Impact of Bacterial–Fungal Interaction on Carbapenem-Resistant Klebsiella pneumoniae

Fungal–bacterial co-culturing is a potential technique for the production of secondary metabolites with antibacterial activity. Twenty-nine fungal species were screened in a co-culture with carbapenem-resistant Klebsiella pneumoniae at different temperatures. A temperature of 37 ° showed inhibition of bacterial growth. Antimicrobial susceptibility testing for K. pneumoniae was conducted to compare antibiotic resistance patterns before and after the co-culture. Genotypic comparison of the K. pneumonia was performed using next generation sequencing (NGS). It was shown that two out of five K. pneumoniae, with sequence type ST 101 isolates, lost bla-_OXA48, bla-_CTX-M-14, tir, strA and strB genes after the co-culture with Scopulariopsis brevicaulis fungus. The other three isolates (ST 383 and 147) were inhibited in the co-culture but did not show any changes in resistance. The total ethyl acetate extract of the fungal–bacterial co-culture was tested against K. pneumoniae using a disc diffusion method. The concentration of the crude extract was 0.97 mg/µL which resulted in total inhibition of the bacteria. Using chromatographic techniques, the purified compounds were identified as 11-octadecenoic acid, 2,4-Di-tert-butylphenol, 2,3-Butanediol and 9-octadecenamide. These were tested against K. pneumoniae using the well diffusion method at a concentration of 85 µg/µL which resulted in total inhibition of bacteria. The co-culture results indicated that bacteria under chemical stress showed variable responses and induced fungal secondary metabolites with antibacterial activities.     

Exploring the electronic and mechanical properties of protein using conducting atomic force microscopy

In interfacing man-made electronic components with specifically folded biomacromolecules, the perturbative effects of junction structure on any signal generated should be considered. We report herein on the electron-transfer characteristics of the blue copper metalloprotein, azurin, as characterized at a refined level by conducting atomic force microscopy (C-AFM). Specifically, the modulation of current-voltage (I-V) behavior with compressional force has been examined. In the absence of assignable resonant electron tunneling within the confined bias region, from -1 to 1 V, the I-V behavior was analyzed with a modified Simmons formula. To interpret the variation of tunneling barrier height and barrier length obtained by fitting with the modified Simmons formula, an atom packing density model associated with protein mechanical deformation was proposed and simulated by molecular dynamics. The barrier heights determined at the minimum forces necessary for stable electrical contact correlate reasonably well with those estimated from bulk biophysical (electroanalytical and photochemical) experiments previously reported. At higher forces, the tunnel barrier decreases to fall within the range observed with saturated organic systems. Molecular dynamics simulations revealed changes in secondary structure and atomic density of the protein with respect to compression. At low compression, where transport measurements are made, secondary structure is retained, and atomic packing density is observed to increase linearly with force. These predictions, and those made at higher compression, are consistent with both experimentally observed modulations of tunneling barrier height with applied force and the applicability of the atom packing density model of electron tunneling in proteins to molecular-level analyses.     

Molecular recognition properties of FN3 monobodies that bind the Src SH3 domain

We have constructed a phage-displayed library based on the human fibronectin tenth type III domain (FN3) scaffold by randomizing residues in its FG and BC loops. Screening against the SH3 domain of human c-Src yielded six different clones. Five of these contained proline-rich sequences in their FG loop that resembled class I (i.e., +xxPxxP) peptide ligands for the Src SH3 domain. The sixth clone lacked the proline-rich sequence and showed particularly high binding specificity to the Src SH3 domain among various SH3 domains tested. Competitive binding, loop replacement, and NMR perturbation experiments were conducted to analyze the recognition properties of selected binders. The strongest binder was able to pull down full-length c-Src from murine fibroblast cell extracts, further demonstrating the potential of this scaffold for use as an antibody mimetic.     

Aging effect on the mechanical properties of hybrid gels

Sol-gel derived unsupported films and thin rods have been obtained from co-hydrolysis of triethoxysilane and methyldiethoxysilane. The materials are flexible, dense and transparent. Films and rods have been aged for different periods of time in air at room temperature. The elastic modulus has been measured by means of tensile or flexural tests. Measurements showed an increase of elastic modulus with aging time and showed different values for films and rods. The observed evolution of mechanical properties has been related to a corresponding structural modification as highlighted mainly by MAS-NMR studies. Analyses pointed out the crucial role of condensation processes and showed that the stiffness increase arises from the formation of relatively few bonds which link and constrain pre-existing mobile network regions.     

Structural properties of an artificial protein that regulates the nucleation of inorganic and organic crystals

Technological advances have facilitated the generation of artificial proteins that possess the capabilities of recognizing and binding to inorganic solids and/or controlling nucleation processes that form inorganic solids. However, very little is known regarding the structure of these interesting polypeptides and how their structure contributes to functionality. To address this deficiency, we report structural investigations of an artificial protein, p288, that self-assembles and controls the nucleation of simple salts and organic compounds into dendrite-like crystals. Under aqueous conditions at low pH and in the presence of high salt, p288 is conformationally labile and exists primarily as a random coil conformer in equilibrium with other undefined secondary structures, including polyproline type II and beta turn. We note that p288 can fold into either a partial beta strand (at neutral pH) or a predominantly alpha helical (in the presence of TFE) conformation. Solid-state 13C-15N NMR experiments also reveal that p288 in the lyophilized, hydrated state possesses some degree of nonrandom coil structure. These results indicate that p288 is conformationally labile but can undergo conformational transitions to a more stable structure when water solvent loss/displacement occurs and protein concentrations increase. We believe that conformational instability and the ability to adopt different structures as a function of different environmental conditions represent important molecular features that impact p288 supramolecular assembly and crystal nucleation processes.     

The effect of 3D printing on the morphological and mechanical properties of polycaprolactone filament and scaffold

Three‐dimensional (3D) printing becomes an attractive technique to fabricate tissue engineering scaffolds through its high control on fabrication and repeatability using the printing parameters. This technique can be combined by the finite element method (FEM), and tissue‐specific scaffolds with desirable morphological and mechanical properties can be designed and manufactured. In this study, the influential 3D printing parameters on the morphological and mechanical properties of polycaprolactone (PCL) filament and scaffold were studied experimentally and numerically. First, the effects of printing parameters and process on the properties of extruded PCL filament were investigated. Then, using FEM, the effects of filament specifications on the overall characteristics of the scaffold were evaluated. Results showed that both the printing process in terms of resting time and remaining time and the printing parameters like pressure, printing speed, and printing path length have influenced the filament properties. In addition, both the filament diameter and elastic modulus had significant effects on the properties of scaffold especially, a 20% increase in the filament diameter caused the scaffold compressive elastic modulus to rise by around 72%. It is concluded that the printing parameters and process must be tuned very well in fabricating scaffolds with the desired morphology and mechanical property.     

Synthesis of a derivative of a pentasaccharide repeating unit of the O-antigenic polysaccharide of the bacterium Klebsiella pneumoniae O3 as a benzoylated 2-methoxycarbonylethyl thioglycoside

Block synthesis of a fully benzoylated derivative of the pentasaccharide α-d-Manp-(1→3)-α-d-Manp-(1→2)-α-d-Manp-(1→2)-α-d-Manp-(1→2)-α-d-Manp-SCH₂CH₂CO₂Me, the glycoside of the repeating unit of the O-antigenic polysaccharide of the bacterium Klebsiella pneumoniae O3, was performed.     

Thermodynamics of binding of 2-methoxy-3-isopropylpyrazine and 2-methoxy-3-isobutylpyrazine to the major urinary protein

In the present study we examine the thermodynamics of binding of two related pyrazine-derived ligands to the major urinary protein, MUP-I, using a combination of isothermal titration calorimetry (ITC), X-ray crystallography, and NMR backbone (15)N and methyl side-chain (2)H relaxation measurements. Global thermodynamics data derived from ITC indicate that binding is driven by favorable enthalpic contributions, rather than the classical entropy-driven hydrophobic effect. Unfavorable entropic contributions from the protein backbone and side-chain residues in the vicinity of the binding pocket are partially offset by favorable entropic contributions at adjacent positions, suggesting a "conformational relay" mechanism whereby increased rigidity of residues on ligand binding are accompanied by increased conformational freedom of side chains in adjacent positions. The principal driving force governing ligand affinity and specificity can be attributed to solvent-driven enthalpic effects from desolvation of the protein binding pocket.     

Effect of water on the mechanical properties of collagen films

The effect on water on mechanical properties of collagen films has been studied. The S-shaped sorption isotherm is separated into an adsorption curve C₁ and a solution curve C₂. From the C₂ curve, a value of 0.8 is calculated for the Flory-Huggins interaction parameter χ₁. The dynamic shear modulus G′, loss modulus G″, and loss tangent tan δ determined as functions of water content indicate two dispersions at low and at high water content. The region of water content from about 0.05 to 0.1 g/g, G′ decreases suddenly, G″ has a peak, and tan δ increases, corresponds to the region where the C₂ component of sorption becomes detectable. Another dispersion occurs at water contents above 0.2 g/g. A composite curve can be obtained by shifting stress-relaxation curves obtained at different humidities along the log time axis. When only the C₂ component of sorbed water is taken into account, the shift factor a_c is explained by a relation of Fujita and Kishimotos' based on free-volume theory. Shift factor for the relaxation curves of wool fibers, except for an initial part at times of less than 1 sec, are described by the same equation. The parameter β in the equation has the same value of 0.16 for both collagen and wool.     

Study of the influence of plasticizers on the thermal and mechanical properties of poly(3-hydroxybutyrate) compounds

The influence of four types of plasticizers, dioctyl phthalate (DOP), dioctyl adipate (DOA), triacetyl glycerol (TAG) and polyadipate (PA), in the thermal and mechanical properties of Poly(3-hydroxybutyrate) (PHB), a highly crystalline biodegradable polyester, was evaluated in this work. The plasticizers were introduced alone or mixtures of them, using concentrations that varied from 5 to 30% wt. Their influence in some important polymer parameters as Tg, Tm and degree of crystallinity, and on its mechanical behavior, elongation and tensile strain were investigated. The best results were obtained for the sample with 30% TAG and that one using a binary mixture of plasticizers PA 20% and TAG 10%.     

Theoretical exploration on the vibrational and mechanical properties of M3C2/M3C2T2 MXenes

MXenes have attracted intensive attention in chemistry and material science for their special structures and properties. In order to understand the basic physical properties of the M₃C₂/M₃C₂T₂ (MSc, Ti, V, Cr, Zr, Nb, Mo, Hf, Ta, W; TF, O, OH) MXenes, first-principles calculations are carried out to investigate the structural, vibrational, and mechanical properties in this work. Both the metal atoms and surface groups can significantly influence the configurations or mechanical behaviors of the MXenes. The dehydrogenation tendency is calculated to evaluate the possible forms of the M₃C₂(OH)₂ toward M₃C₂O₂. The work functions of MXenes functionalized by different groups are compared, and the lower work functions for the  OH functionalized ones, which can be as low as 1.358 eV for the Sc₃C₂(OH)₂, suggest potential good performance in electron emission. In addition, the stability, mechanical properties, and the Raman and infrared (IR) activity modes of the MXenes are reported. Generally, functionalized MXenes would present smaller lattice parameters, lower free energies, and stronger mechanical strength compared to their counterparts. The data obtained may provide important theoretical ground for the investigations of the applications of MXenes.     

Characterization of the mechanical properties of cross-linked serum albumin microcapsules: effect of size and protein concentration

A microfluidic technique is used to characterize the mechanical behavior of capsules that are produced in a two-step process: first, an emulsification step to form droplets, followed by a cross-linking step to encapsulate the droplets within a thin membrane composed of cross-linked proteins. The objective is to study the influence of the capsule size and protein concentration on the membrane mechanical properties. The microcapsules are fabricated by cross-linking of human serum albumin (HSA) with concentrations from 15 to 35 % (w/v). A wide range of capsule radii (～40–450 μm) is obtained by varying the stirring speed in the emulsification step. For each stirring speed, a low threshold value in protein concentration is found, below which no coherent capsules could be produced. The smaller the stirring speed, the lower the concentration can be. Increasing the concentration from the threshold value and considering capsules of a given size, we show that the surface shear modulus of the membrane increases with the concentration following a sigmoidal curve. The increase in mechanical resistance reveals a higher degree of cross-linking in the membrane. Varying the stirring speed, we find that the surface shear modulus strongly increases with the capsule radius: its increase is two orders of magnitude larger than the increase in size for the capsules under consideration. It demonstrates that the cross-linking reaction is a function of the emulsion size distribution and that capsules produced in batch through emulsification processes inherently have a distribution in mechanical resistance.