Mechanism of Targeted Regulation of Ovalbumin Epitopes by Pulsed Electric Field-Assisted Alcalase Treatment.

Egg is one of the eight major food allergens, and ovalbumin (OVA) is the most abundant allergenic protein in eggs. In this study, the effects of pulsed electric field (PEF)-assisted Alcalase hydrolysis on the spatial conformation and potential allergenicity of OVA were studied, and the mechanism of its inhibiting allergic reactions effect was revealed. PEF-assisted Alcalase hydrolysis increased the degree of hydrolysis, surface hydrophobicity, and free sulfhydryl group content. Moreover, the reduction in the α-helix content, fluorescence intensity, and disulfide bond content suggested that PEF promoted the OVA hydrolysis by Alcalase. Additionally, enzyme-linked immunosorbent assay data indicated that PEF-assisted Alcalase hydrolysis hindered OVA binding to immunoglobulins E and G1. Finally, based on bioinformatics combined with mass spectrometry, PEF-assisted Alcalase reduced OVA-induced allergic reactions by destroying epitopes in OVA. Overall, PEF technology further destroyed the epitopes of allergens by targeting the binding sites of substrates and enzymes to improve the affinity of enzymes and substrates, reducing allergic reactions.

Pulsed Electric Field-Assisted Alcalase Treatment Reduces the Allergenicity and Eliminates the Antigenic Epitopes of Ovomucoid.

Physically assisted chemical modifications can effectively reduce the allergenicity of ovomucoid (OVM). However, only a few studies have used pulsed electric field (PEF)-assisted alcalase hydrolysis to reduce the allergenicity of OVM. Herein, we investigated the effect of PEF-assisted alcalase treatment on the spatial conformation, allergenicity, and antigenic epitopes of OVM based on multispectroscopic analyses, bioinformatics, and mass spectrometry. The results showed that PEF-assisted alcalase treatment promoted the hydrolysis of OVM; moreover, the α-helix content and surface hydrophobicity of OVM significantly decreased, which disordered its spatial conformation and weakened its intermolecular interactions. Additionally, enzyme-linked immunosorbent assay (ELISA) results showed that the PEF-assisted alcalase treatment significantly reduced the binding levels of IgE and IgG1, which were 47.66 and 36.41%, respectively. Finally, eight epitopes of OVM were obtained by immunoinformatic tools. Nano-high performance liquid chromatography coupled to tandem mass spectrometry (nano-HPLC MS/MS) results showed that the hydrolysate of OVM and alcalase (HOVM) had nine more peptide-containing epitopes than the hydrolysate of PEF-treated OVM and PEF-treated alcalase (HOVM-PP'), indicating that PEF could promote the elimination of linear epitopes in OVM, thereby reducing OVM allergenicity.

Mechanism of trypsin activation by pulsed electric field treatment revealed based on chemical experiments and molecular dynamics simulations.

A pulsed electric field (PEF) treatment exhibits different effects on trypsin; however, the mechanism of enzyme activation remains unclear. Herein, chemical experiments combined with molecular dynamics simulations revealed the mechanism of trypsin activation by PEF treatment at the molecular level. The results indicated that compared with the values at 0 kV/cm, the enzyme activity, Vmax, and Kcat at 20 kV/cm increased by 9.30%, 4.74%, and 4.30%, respectively, and Km decreased by 11.14%, indicating an improved interaction between the enzyme and substrate. The simulation results revealed that PEF treatment increased the number of molecular hydrogen bonds and the solvent-accessible surface area, while decreasing the rotation radius and random coil content by 5.00% and 3.37%, respectively. Molecular docking indicated that PEF treatment altered the active center and increased the affinity between the enzyme and substrate. The simulation results were consistent with those of the spectroscopic experiments conducted on trypsin after PEF treatment.

Evaluation of the structure-activity relationship between allergenicity and spatial conformation of ovalbumin treated by pulsed electric field.

The aim of the present study was to investigate the effect of pulsed electric field (PEF) treatment on ovalbumin (OVA) induced allergens and reveal potential allergy regulatory mechanisms. At 10 kV/cm, OVA-induced allergic symptoms were significantly reduced, and the capacity of OVA to bind with specific IgG1 and IgE was reduced by 10.32% and 3.61%, respectively. Furthermore, the degranulation of RBL-2H3 cells and allergen activity were also reduced by 4.63% and 22.15%, respectively. Interestingly, the α-helix content was reduced by 5.81% and the fluorescence intensity was increased by 6.90% with PEF treatment. At 10 kV/cm, water contact angle and surface hydrophobicity increased by 8.40% and 0.18%, respectively, indicating that PEF treatment increased the exposure of hydrophobic amino acid residues. PEF treatment alters the hydrogen bonding and hydrophobic interactions in the protein, which masks the binding sites of sensitized epitopes, and consequently reduces allergies.

Internal cavity amplification of shell-like ferritin regulated with the change of the secondary and tertiary structure induced by PEF technology.

In this study, the effect of pulsed electric field (PEF) on apparent morphology and molecular structure of shell-like ferritin obtained from horse spleen was determined by circular dichroic (CD), fluorescence spectroscopy, Raman spectroscopy, cold field emission scanning electron microscopy (CF-SEM) and transmission electron microscopy (TEM), and verified by molecule dynamics (MD) simulation. After PEF treatment, the α-helix content of the samples reached a minimum value at 10 kV/cm, which indicated that the ferritin structure has been partially unfolded. However, the α-helix content peaked again after resting for 2 h at 25 ± 1 °C. This indicated that the PEF-treated ferritin tended to restore its original spherical morphology probably owing to the reversible assembly characteristic of ferritin. In addition, microstructure analysis revealed that ferritin particles aggregated after PEF treatment. Therefore, PEF treatment could induce the "exposure" of hydrophobic amino acids and conversion of disulfide bond configuration, and consequently, regulate the internal cavity stability of ferritin. The research will be beneficial to expand the application of PEF treatment in the modification of protein structure, and provide a theoretical basis for the application of ferritin as a carrier of bioactive molecules in food.

In-situ analysis of the water distribution and protein structure of dough during ultrasonic-assisted freezing based on miniature Raman spectroscopy.

The effect of ultrasonic-assisted freezing (UAF) on the water distribution of dough and molecular structure of gluten was in-situ monitored by low field nuclear magnetic resonance (LF-NMR) and micro-miniature Raman spectroscopy in this research. The results showed that UAF treatment increased the bound water content between 5 min and 30 min, and weakened the signal intensity of hydrogen protons due to the ultrasound enhanced heat and mass transfer during the freezing process. In-situ Raman spectra analysis indicated that UAF delayed the transition from embedded to exposure of tyrosine and tryptophan residues during the freezing process. Meanwhile, UAF reduced the number of hydrogen bonds, gauche-gauche-gauche (g-g-g) conformation breakage, the degree of α-helix to random coil conversion and damage to the gluten network during the freezing process. UAF treatment reduced the water mobility and breakage of non-covalent bonds, which prevented a dramatic shift in the protein advanced conformation during the freezing process, thereby improving the quality of frozen dough.

Effect of ultrasound-assisted freezing on the textural characteristics of dough and the structural characterization of wheat gluten.

To better understand the effects of ultrasonic treatment in the whole freezing process (UWF) and the maximum ice crystal formation zone (UMF) on the quality of frozen dough, the textural properties of dough and the structure of gluten were investigated. The results showed that the UWF and UMF treatments improved the textural properties of frozen dough and obtain the best effect at the 60 W/L power densities. Ultrasound-assisted freezing reduced the destructive effect of disulfide bonds on dough, and led to a state of dynamic equilibrium of hydrophobic groups. UWF treatment at 80 W/L and UMF treatment at 40 W/L had positive effects prevented the secondary structure from destruction by freezing. The network of gluten treated by ultrasound-assisted freezing was more uniform and smaller than that of traditional freezing samples, which was similar to the network structure of fresh protein. According to Pearson's correlation analysis, there was a high correlation between SH, α-helix content and springiness. There was a significant positive correlation between ß-turn and G', G″, and there was a significant negative correlation between ß-turn and hardness. These results suggest that ultrasound-assisted freezing improved the process quality of dough though reducing the damage to gluten structure caused by freezing.

Controllable Fabrication of Au-Coated AFM Probes via a Wet-Chemistry Procedure.

Tip-enhanced Raman spectroscopy (TERS), which offers a spatial resolution far beyond the limitations of the optical diffraction and detection sensitivity down to a single molecular level, has become one of the powerful techniques applied in current nanoscience and technology. However, the excellent performance of a TERS system is very much dependent on the quality of metallized probes used in TERS characterization. Thus, how to prepare higher-quality probes plays a vital role in the development and application of TERS technique. In this work, one simple wet-chemistry procedure was designed to fabricate atomic force microscopy-based TERS (AFM-TERS) probes. Through the controlled growth of a gold film on a commercial silicon AFM probe, TERS probes with different apex diameters were prepared successfully. A series of TERS results indicated that the probes with the apex size of 50~60 nm had the maximum TERS enhancement, and the Raman enhancement factor was in the range of 106 to 107. Compared with those prepared by other fabrication methods, our TERS probes fabricated by this wet-chemistry method have the virtues of good stability, high reproducibility, and strong enhancement effect.

In Situ Monitoring of the Effect of Ultrasound on the Sulfhydryl Groups and Disulfide Bonds of Wheat Gluten.

Ultrasound treatment can improve enzymolysis efficiency by changing the amounts of sulfhydryl groups (SH) and disulfide bonds (SS) in protein. This paper proposes an in-situ and real-time monitoring method for SH and SS during ultrasound application processes using a miniature near-infrared (NIR) optical fiber spectrometer and a chemometrics model to determine the endpoint of ultrasonic treatment. The results show that SH and SS contents fluctuated greatly with the extension of ultrasonic time. The optimal spectral intervals for SH content were 869⁻947, 1207⁻1284, 1458⁻1536 and 2205⁻2274 nm, the optimal spectral intervals of SS content were 933⁻992, 1388⁻1446, 2091⁻2148 and 2217⁻2274 nm. According to the optimal spectral intervals, the synergy interval partial least squares (Si-PLS) and error back propagation neural network (BP-ANN) for SH, SS contents were established. The BP-ANN model was better than the Si-PLS model. The correlation coefficient of the prediction set (Rp) and the root mean square error of prediction (RMSEP) for the BP-ANN model of SH were 0.9113 and 0.38 µmol/g, respectively, the Rp² and residual prediction deviation of SH were 0.8305 and 2.91, respectively. For the BP-ANN model of SS, the Rp and the RMSEP were 0.7523 and 6.56 µmol/g, respectively. The Rp² and residual prediction deviation (RPD) of SS were 0.8305 and 2.91, respectively. However, the Rp² and RPD of SS was 0.5660 and 1.64, respectively. This work demonstrated that the miniature NIR combined with BP-ANN algorithms has high potential for in-situ monitoring of SH during the ultrasonic treatment process, while the spectral prediction model of SS needs to be further developed.

Investigating the Co-Adsorption Behavior of Nucleic-Acid Base (Thymine and Cytosine) and Melamine at Liquid/Solid Interface.

The co-adsorption behavior of nucleic-acid base (thymine; cytosine) and melamine was investigated by scanning tunneling microscopy (STM) technique at liquid/solid (1-octanol/graphite) interface. STM characterization results indicate that phase separation happened after dropping the mixed solution of thymine-melamine onto highly oriented pyrolytic graphite (HOPG) surface, while the hetero-component cluster-like structure was observed when cytosine-melamine binary assembly system is used. From the viewpoints of non-covalent interactions calculated by using density functional theory (DFT) method, the formation mechanisms of these assembled structures were explored in detail. This work will supply a methodology to design the supramolecular assembled structures and the hetero-component materials composed by biological and chemical compound.

Exploring the complex mechanical properties of xanthan scaffolds by AFM-based force spectroscopy.

The polysaccharide xanthan has been extensively studied owing to its potential application in tissue engineering. In this paper, xanthan scaffold structures were investigated by atomic force microscope (AFM) in liquid, and the mechanical properties of the complex xanthan structures were investigated by using AFM-based force spectroscopy (FS). In this work, three types of structures in the xanthan scaffold were identified based on three types of FS stretching events. The fact that the complex force responses are the combinations of different types of stretching events suggests complicated intermolecular interactions among xanthan fibrils. The results provide crucial information to understand the structures and mechanical properties of the xanthan scaffold.

Ab initio investigation of the first hydration shell of protonated glycine.

The first hydration shell of the protonated glycine is built up using Monte Carlo multiple minimum conformational search analysis with the MMFFs force field. The potential energy surfaces of the protonated glycine and its hydration complexes with up to eight water molecules have been scanned and the energy-minimized structures are predicted using the ab initio calculations. First, three favorable structures of protonated glycine were determined, and the micro-hydration processes showed that water can significantly stabilize the unstable conformers, and then their first hydration shells were established. Finally, we found that seven water molecules are required to fully hydrate the first hydration shell for the most stable conformer of protonated glycine. In order to analyse the hydration process, the dominant hydration sites located around the ammonium and carboxyl groups are studied carefully and systemically. The results indicate that, water molecules hydrate the protonated glycine in an alternative dynamic hydration process which is driven by the competition between different hydration sites. The first three water molecules are strongly attached by the ammonium group, while only the fourth water molecule is attached by the carboxyl group in the ultimate first hydration shell of the protonated glycine. In addition, the first hydration shell model has predicted most identical structures and a reasonable accord in hydration energy and vibrational frequencies of the most stable conformer with the conductor-like polarizable continuum model.

Nanomechanics of phospholipid LB film studied layer by layer with AFM.

BACKGROUND: Phospholipid, a main component of cell membrane, has been explored as a model system of the cell membrane and temporary scaffold materials in recent studies. The mechanical properties of phospholipid layers are essentially interesting since it is involved in several biological processes. RESULTS: Here, the nanomechanical properties such as indentation force, adhesion force and DMT (Derjaguin-Müller-Toporov) modulus of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) Langmuir-Blodgett (LB) films were analyzed layer by layer with Atomic Force Microscope (AFM) under deionized water condition. CONCLUSIONS: The penetration distances in the indentation force curves are equal to the thicknesses of phospholipid films, and the yield forces of DSPC LB films in deionized water are smaller than that of similar lipid films in buffered solutions due to the influence of ions. Moreover, the DMT modulus of upper layer DSPC LB film is different from that of monolayer DSPC LB film due to the influence of their different substrates. Our results suggest that environment such as surrounding ions and substrate will strongly influence the measured nano-mechanical properties of the lipid bilayer, especially that of the down layer. Graphical AbstractA process about the exploration of nanomechanics of DSPC LB film.

Collagen coated tantalum substrate for cell proliferation.

The extracellular matrix (ECM) plays a key role in cell culture in various physiological and pathological processes in the field of tissue engineering. Recently, the type I collagen ECM has been widely utilized in vitro model systems for the attachment of many different cell lines since it has multi-functions in human tissues. For example it accounts for 6% of the weight of strong, tendinous muscles. In this paper, we reported a new material by coating tantalum (Ta), one highly biocompatible metal, with type I collagen fibrils. The morphology of the new material was studied by high resolution atomic force microscope. It was shown that the adhesion force between type I collagen fibrils network and Ta was strong enough to overcome surface defects. A possible way to explain the phenomenon is that the longitudinal periodicity of collagen fibrils matches the grain size of the Ta domains, which results in increase of the physical adsorption contact area, thereby inducing the dramatic adhesion enhancement between collagen fibrils and Ta. The obtained material was then employed as a template for cell proliferation. Although the surface of this template is more hydrophobic by comparison with the bare Ta surface, the cells on this material were successfully incubated, indicating that the collagen coated Ta might be used as the buffer layer for proliferating cells in hydrophobic biomaterials.

Building layer-by-layer 3D supramolecular nanostructures at the terephthalic acid/stearic acid interface.

By using the layer-by-layer deposition method, we build three dimensional (3D) supramolecular nanostructures by stacking small molecular species on top of the first buffer layer, which can be utilized to fabricate novel 3D supramolecular functional nanostructures.

Building the first hydration shell of deprotonated glycine by the MCMM and ab initio methods.

The first hydration shell of the deprotonated glycine is built up by the discrete hydration model. The potential energy surfaces (PESs) of the deprotonated glycine and its hydration complexes with different number of water molecules have been scanned by the Monte Carlo multiple minimum (MCMM) conformational search analysis with the MMFFs force field. Then the energy-minimized structures are predicted using the high-level ab initio calculations/MP2/6-311++G(d,p). The results of the structural parameters and the infrared spectra indicate that the first-shell water molecules around the anion of deprotonated glycine play a more important role in determining the hydration process of deprotonated glycine. The competition between the hydrate site I and the hydrate site II represents a dynamic process of hydrated complexes. The vibrational properties of C═O and N-H are determined to characterize the structure of deprotonated glycine in solution by the discrete hydration model and the conductor-like polarizable continuum model (CPCM) in the gas phase, respectively.

Two-dimensional scaffold layer formations on a solid surface through xanthan polysaccharide: temperature effect.

The influences of temperature on xanthan biopolymer assemblies on a two-dimensional surface have been thoroughly studied. High resolution atomic force microscope images show that the xanthan nanofibrils can be used to build up well-dispersed 2D scaffold layer after 1 day annealing at 35 degrees C. By increasing annealing temperatures (60 degrees C, 90 degrees C) of xanthan solutions, the well-dispersed layers can be produced rapidly (6h, 0.5h) with micro-sized pore structures. The xanthan scaffold with pore structures potentially allows accommodating micro-sized cells for tissue engineering.

Influence of tunable external stimuli on the self-assembly of guanosine supramolecular nanostructures studied by atomic force microscope.

The self-assembly of guanosine (G) molecules on solid surfaces is investigated by tapping-mode atomic force microscopy (AFM) upon controlling and introducing external factors (stimuli) to the G stock solution such as incubation time, presence/absence of metal cations, and mechanical shaking. Surprisingly, at different stages of incubation time at room temperature and in the absence of any metal cations in the G stock solution, which are known to be one of the governing factors in forming G-nanostructures, two assembly pathways resulting into two distinct supramolecular nanostructures were revealed. Astonishingly, by introducing a mechanical shaking of the tube containing the G stock solution, one-dimensional (1D) wires of G molecules are observed by AFM, and very interestingly, novel "branched" supramolecular nanostructures are formed. We have also observed that the later branched G nanostructures can grow further into a two-dimensional (2D) thin film by increasing the incubation time of the G stock solution at room temperature after it is exposed to the external mechanical stimuli. The self-assembled nanostructures of G molecules are changed significantly by tuning the assembly conditions, which show that it is indeed possible to grow complex 2D nanostructures from simple nucleoside molecules.

A separation of the refractive index and topography in photon-scanning tunneling microscopy: simulations and experiments.

In order to separate the purely optical and topographic information from images in constant-gap mode simultaneously, we proposed the atomic force/photon-scanning tunneling microscopy (AF/PSTM). In this paper, we focus on the principle of separation of the refractive index image from the images of photon-scanning tunneling microscopy. We prove the formula of refractive index imaging by using a three-dimensional finite-difference time-domain method. The formula indicates that the refractive index of a sample is approximately proportional to photon tunneling information (DeltaI/I )2. From the viewpoint of practical use, we simulated the refractive index images for the realistic experiments. We present line scans along two orthogonal directions and the transmitted intensity as a function of the tip position under the constant-gap mode. The experimental results are presented and are in good agreement with the numerical results.