Efficient preparation of high-purity and intact mesenchymal stem cell-derived extracellular vesicles.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown great promise for regeneration and immunomodulation. However, efficient and scalable methods for their preparation are still lacking. In this study, we present the adoption of a label-free technique known as "EXODUS" to isolate and purify MSC-EVs from the conditioned medium. Our findings indicate that EXODUS can rapidly isolate EVs from 10 mL of conditioned medium with a 5-fold higher yield compared to conventional approaches, including ultracentrifugation (UC) and polyethylene glycol precipitation (PEG) methods. Additionally, pre-storing the conditioned medium at 4°C for 1 week resulted in a ~2-fold higher yield of MSC-EVs compared to the freshly prepared medium. However, storing the purified EV particles at 4°C for 1 month led to a 2-fold reduction in particle concentration. Furthermore, we found that MSC-EVs isolated using EXODUS exhibit higher expression levels of EV markers such as Alix, Flotillin1, CD81, and TSG101 in comparison to PEG and UC methods. We also discovered that MSC-EVs isolated using EXODUS are enriched in response to cytokine, collagen-containing extracellular matrix, and calcium ion binding compared to PEG method and enriched in extracellular structure organization, extracellular matrix, and extracellular matrix structure constituents compared to UC. Finally, we demonstrated that MSC-EVs isolated using EXODUS exhibit greater potential in animal organ development, tissue development, and anatomical structure morphogenesis compared to the UC. These findings suggest that EXODUS is a suitable method for the large-scale preparation of high-quality MSC-EVs for various clinical applications.

Enhancing viability of Lactobacillus plantarum encapsulated by alginate-gelatin hydrogel beads during gastrointestinal digestion, storage and in the mimic beverage systems.

To improve the viability of Lactobacillus plantarum (P) during digestion and storage, the probiotics were encapsulated by alginate (ALG) and alginate-gelatin (ALG-GE) hydrogels beads. ALG-P-GE showed much better physicochemical properties than ALG-P. The scanning electron microscopy (SEM) results validated the incorporation of bacterial cells into the beads. ALG-P-GE exhibited good encapsulation efficiency of 97.7 %, and the storage and thermal stability of probiotic were increased by 15 % and 8 %, respectively, when comparing with ALG-P. ALG-P-GE beads could protect the probiotics from inactivation in simulated gastric fluid and then release it in simulated intestinal fluid. The protective mechanism of ALG-GE for probiotics was further studied by fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and found that ALG and GE can form gel network through hydrogen bonding and electrostatic interactions. In the mimic beverage systems, ALG-P-GE beads could protect the encapsulated probiotics and increase its viability. The storage, thermal, and digestion stability of encapsulated probiotic were significantly increased and showed high viability in the mimic beverage systems. ALG-P-GE beads have great potential for the protection and delivery of probiotics in food systems.

Preparation of alginate-whey protein isolate and alginate-pectin-whey protein isolate composites for protection and delivery of Lactobacillus plantarum.

Probiotics are sensitive to external conditions, resulting in low survival rates after being ingested or during food production, transportation and storage. In order to improve the survival rate of Lactobacillus plantarum (LP) during gastrointestinal digestion, storage, and freeze-drying, alginate-whey protein isolate (ALG-WPI) and alginate-pectin-whey protein isolate (ALG-PEC-WPI) composites were employed to encapsulate LP. The encapsulation efficiency of ALG-WPI-LP and ALG-PEC-WPI-LP beads both reached more than 99 %. Scanning electron microscopy (SEM) indicated that dense and rough aggregates were formed on the surface of both composites, and attached LP cells could be observed inside the beads. The ALG-WPI and ALG-PEC-WPI composites can protect the viability of LP in simulated gastric fluid (SGF) and release the probiotics in simulated intestinal fluid (SIF). The storage stability of LP at 4 °C was improved by about 15 % in comparison with bare LP and the survival rates of LP in ALG-WPI-LP and ALG-PEC-WPI-LP powders after freeze-drying were increased by 65.37 % and 72.06 %, respectively. The formation mechanism of ALG-WPI and ALG-PEC-WPI composites was further explored by fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and thermogravimetric analysis (TGA). The ALG-WPI and ALG-PEC-WPI composites have great potential to protect and deliver probiotics in food systems.

Preparation of zein-lecithin-EGCG complex nanoparticles stabilized peppermint oil emulsions: Physicochemical properties, stability and intelligent sensory analysis.

Peppermint oil emulsions were prepared by using zein-lecithin-EGCG (Z-L/E) complex nanoparticles as emulsifiers. The preparation conditions of emulsions were optimized via measuring the particle size, surface tension and stability of emulsions, and peppermint oil of 3% (particle size = 375 nm, polydispersity index (PDI) = 0.45), the zein:lecithin ratio of 4:1 (w/w) (particle size = 396 nm), and the zein:EGCG ratio of 10:1 (w/w) (surface tension = 47.32 N/m) was the optimal condition. The rapid stability analysis showed that the instability mechanism of emulsions was ascribed to creaming and stratification, and the stability mechanism of emulsions was explored, indicating that the complex nanoparticles adsorbed on the surface of oil droplets to give Pickering emulsions. Electronic tongue experiments showed that the Z-E/L4:1 stabilized emulsion was distinguished from the other three samples due to its good stability. The electronic nose experiment could distinguish the emulsions with different droplet sizes.

The fabrication of novel zein and resveratrol covalent conjugates: Enhanced thermal stability, emulsifying and antioxidant properties.

Novel zein and resveratrol conjugates were fabricated by alkaline and free radical grafting reactions. The grafting efficiency and total phenolic content of alkaline treated conjugates were slightly higher than those of free radical grafting. Compared to native and alkaline treated zein, the sulfhydryl contents of conjugates were obviously decreased, confirming that nucleophilic addition of resveratrol to sulfhydryl group of zein formed stable CS covalent bonds. The conformation changes of zein modified by resveratrol were revealed by fourier transform infrared spectroscopy and fluorescence spectroscopy. Moreover, covalent modification changed isoelectric point of zein from 6.5 to 5.4 (alkaline) or 5.6 (free radical grafting), and broadening the pH application range of zein. It was worth mentioning that the conjugates showed much higher thermal stability, antioxidant activity, and emulsify activity than those of native zein. This study provides an effective way for the design of novel delivery systems to encapsulate bioactive substances.

Characterization and stability of peppermint oil emulsions using polyglycerol esters of fatty acids and milk proteins as emulsifiers.

Three peppermint oil emulsions using polyglycerol esters of fatty acids-casein (PGFE-CN), polyglycerol esters of fatty acids-sodium caseinate (PGFE-NaCN), and polyglycerol esters of fatty acids-whey protein isolate (PGFE-WPI) as emulsifiers were fabricated, and the droplet size, zeta potential, viscosity, and stability of emulsions were determined. The experimental results showed that the emulsion containing PGFE-CN has relatively smaller droplet size of 231.77 ± 0.49 nm. No significant changes were observed on the average particle size, polydispersity index and zeta potential during 4-week of storage, indicating that the emulsions kept stable against pH, salt ion, freeze-thaw, and storage. Fourier transform infrared spectrometer (FTIR) results showed that the electrostatic interaction occurs between CN and PGFE in the emulsion. The confocal laser scanning microscope (CLSM) was used to observe the microstructure of the emulsion, proving that droplets were evenly distributed throughout the aqueous phase by PGFE-CN emulsifier. The protein-stabilized emulsions can be used as potential carriers for the delivery of the lipophilic nutrients such as peppermint oil. PRACTICAL APPLICATION: PGFE-CN emulsifier can be directly added to the beverage systems containing oil or protein, such as coconut milk, peanut milk, and walnut milk. It can enhance the stability of beverage, prevent the precipitation, stratification, and oil floating, improve the homogeneity of the system and therefore extend the shelf life.

Fabrication and characterization of oil-in-water pickering emulsions stabilized by ZEIN-HTCC nanoparticles as a composite layer.

In this work, the ZEIN-HTCC nanoparticles formed by zein and N-(2-hydroxy)propyl-3-trimethylammonium chitosan chloride (HTCC) were used as stabilizers to prepare oil-in-water (O/W) Pickering emulsions. The preparation conditions including shearing time, volume fraction of corn oil, mass ratio of ZEIN:HTCC and total concentration of ZEIN-HTCC of emulsions were optimized by measuring the droplet size, zeta potential, PDI and surface tension of emulsions. The ZEIN-HTCC emulsions are stable at the pH range of 4-9 and in the low salt ion concentrations up to 0.2 mol L-1, and can keep stable up to 21 d during low temperature storage. Fourier transform infrared spectroscopy (FTIR), the confocal laser scanning microscope (CLSM) and scanning electron microscopy (SEM) were used to analyze the interaction between emulsion components, revealing that zein and HTCC form a composite layer by flocculation to adsorb on the surface of oil droplets, thus preventing emulsion droplets from aggregation. This novel, long-term stable, surfactant-free, and edible zein-based Pickering emulsion could be used as potential carriers for lipophilic nutrients delivery.

Fabrication of Zein-Lecithin-EGCG complex nanoparticles: Characterization, controlled release in simulated gastrointestinal digestion.

The Zein-Lecithin-Epigallocatechin gallate (EGCG) complex nanoparticles were fabricated by anti-solvent coprecipitation method. The Zein-Lecithin (Z-L) nanocomplexes exhibited great encapsulation efficiency of 68.5% for EGCG, and the encapsulated EGCG still had good antioxidative capacity. The cumulative release of EGCG in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were 19% and 92%, respectively, and the release was closest to Fick release in SGF and First release in SIF. Fluorescence spectroscopy (FL), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) experiments revealed that the EGCG was successfully encapsulated by Z-L nanocomplexes through electrostatic, hydrophobic and hydrogen bonding interactions. The Zein-Lecithin-EGCG complex nanoparticles exhibited excellent stability and great sustained-release performance, which will be the alternative for potential application in the food industry.

The regulation of sodium alginate on the stability of ovalbumin-pectin complexes for VD3 encapsulation and in vitro simulated gastrointestinal digestion study.

The ovalbumin (OVA)-pectin (PEC)-sodium alginate (SA)-Vitamin D3 (VD3) complex nanoparticles were fabricated by antisolvent precipitation method, and the excellent encapsulation efficiency and loading capacity of VD3 were obtained by 96.6% and 2.8%, respectively. Compared with ternary OVA-PEC-VD3 complexes, the addition of SA with strong negative charge effectively regulated the OVA-PEC complexes and significantly improved the stability of OVA-PEC-SA-VD3 complex nanoparticles with preferable size as small as 126 nm. The storage stability was also investigated after low temperature storage for 31 d, and the particle size of quaternary complexes was increased only 40 nm. In vitro digestion results elucidated that the complex nanoparticles had good stability in the simulated gastric fluid, and almost completely released in the simulated intestinal fluid confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) experiments and scanning electron microscope (SEM) images. The release kinetics study clarified that it was close to Fick release. Fluorescence and Fourier transform infrared spectroscopy (FTIR) experiments showed that quaternary complex nanoparticles were mainly combined by electrostatic, hydrophobic and hydrogen bonding interactions. The novel quaternary protein-polysaccharide complexes have excellent stability and great sustained-release performance for VD3, which may be helpful for the digestion and absorption of vitamin by human body, thus have potential applications in the food and drug industry.

Complexation of ß-lactoglobulin with gum arabic: Effect of heat treatment and enhanced encapsulation efficiency.

Heat treatment is widely used in food industry. Proteins and polysaccharides as important natural polymers in food, under heat treatment, the interactions between them could mediate the conformation and functional properties of proteins. Thermally induced ß-lactoglobulin-gum arabic complexes (ß-Lg-GA) were fabricated, and the effect of heat treatment on physicochemical properties of the complexes was systematically investigated. The average particle size of ß-Lg-GA complexes decreased with temperature increased, at 85°C, a smaller size of 273 nm was obtained. A saturated adsorption of GA was found when mass ratio of ß-Lg/GA was <1:2. At pH = 4.2-7.0, electrostatic attraction between ß-Lg and GA was low and a fairly constant turbidity was observed, the formed composite particles had good stability to the pH value. Through UV, fluorescence, and FTIR spectroscopy, it was found that formation of the nanoparticles relied on thermal denaturation and aggregation of protein, the electrostatic, hydrophobic, and hydrogen bonding interactions between ß-Lg and GA were also important. Scanning electron microscope further indicated ß-Lg and GA had good compatibility, and the complexes had a spherical core-shell structure at molecular level. In addition, these prepared natural nanoparticles by heat treatment show significantly higher encapsulation efficiency for (-)-epigallocatechin-3-gallate (EGCG) than that of unheated, thus could be used as a promising carrier for biologically active substances.

Detection of Tear Components Using Matrix-Assisted Laser Desorption Ionization/Time-of-Flight Mass Spectrometry for Rapid Dry Eye Diagnosis.

The tear is a biological fluid that has the diagnostic potential for ocular diseases. Extracellular vesicles (EVs), widly detected in various biofluids including tears, are nanoparticles released by living cells and considered as promising detection sources for noninvasive liquid biopsy. Understanding the roles of tears and tear-EVs in ocular diseases such as dry eye can facilitate the studies of clinical diagnosis, which usually entails detecting such liquid objects with a rapid and effective method. In this study, we used a mass spectrometry-based strategy to analyze peptidome/proteome profiles of tears and EVs for rapid dry eye diagnosis. Nanosized EVs were isolated from tears of both healthy control (HC) individuals and dry eye syndrome (DES) patients, and the tear compositions were further analyzed by tracking their fingerprints with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. The fingerprints of tear-EVs could be observed in a dose-dependent manner and tears, allowing for comparison of the discriminant peaks between tears and EVs. By analyzing these peaks, the fingerprints of both tear and tear-EVs were showed to have the capability of distinguishing patients with DES from HC donors and providing an efficient way for screening potential DES biomarkers. The proposed tear and EV fingerprinting approach is expected to be a potential tool in the rapid diagnosis of ocular diseases and in-depth research on pathogenesis. Data are available via ProteomeXchange with identifier PXD020217.

Spectroscopic investigation and comprehensive analysis of the polychrome clay sculpture of Hua Yan Temple of the Liao Dynasty.

This article shows the investigation results of the polychrome clay sculptures in Hua Yan Temple of the Liao Dynasty in Datong, China. The mineral pigments, adhesive and painting techniques used in these cultural relics were systematically analyzed in this project. Optical microscope (OM), Scanning electron microscope coupled with an energy-dispersive X-ray spectroscopy (SEM-EDS), micro-Raman, Fourier transform infrared spectrometer (FT-IR) and Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) analyses were selected as scientific micro-destructive analytical methods. The results show that the pigments in the polychrome clay sculptures include cinnabar, lead red, malachite, atacamite, azurite, orpiment and gold. Meanwhile, the presence of nitrogen-containing substances and glue-marker characteristic pyrolysis products clearly indicates that the adhesive used in most of the polychrome clay sculptures was glue-protein. Additionally, the combination of heat-bodied tung oil and glue-protein was also found in the golden areas.

Inhibition of Autism-Related Crm1 Disrupts Mitosis and Induces Apoptosis of the Cortical Neural Progenitors.

De novo microdeletion of chromosome 2p15-16.1 presents clinically recognizable phenotypes that include mental retardation, autism, and microcephaly. Chromosomal maintenance 1 (CRM1) is a gene commonly missing in patients with 2p15-16.1 microdeletion and one of two genes found in the smallest deletion case. In this study, we investigate the role and mechanism of Crm1 in the developing mouse brain by inhibiting the protein or knocking down the gene in vivo. Inhibition of Crm1 reduces the proliferation and increases p53-dependent apoptosis of the cortical neural progenitors, thereby impeding the growth of embryonic cerebral cortex. Live imaging of mitosis in ex vivo embryonic brain slices reveals that inhibition of CRM1 arrests the cortical progenitors at metaphase. The arrested cells eventually slip into a pseudo-G1 phase without chromosome segregation. The mitotic slippage cells are marked by persistent expression of the spindle assembly checkpoint (SAC), repressing of which rescues the cells from apoptosis. Our study reveals that activating the SAC and inducing the mitotic slippage may lead to apoptosis of the cortical neural progenitors. The resulting cell death may well contribute to microcephaly associated with microdeletion of chromosome 2p15-16.1 involving CRM1.

Comparisons of the restoring and reinforcement effects of carboxymethyl chitosan-silk fibroin (Bombyx Mori/Antheraea Yamamai/Tussah) on aged historic silk.

This work presents the results of the reinforcement effects of regenerated silk fibroin solutions (SF) of Bombyx-Mori, Antheraea-Yamamai and Tussah on aged historic silk. Furthermore, Carboxymethyl-chitosan (CMC) was utilized as reinforcement and antibacterial filler to further improving the mechanical properties and antibacterial effects. To clarify the rationale behind this process, comprehensive characterization was applied, and a speculative explanation was provided. The results showed that Bombyx-mori and Tussah have better restoring effects than Antheraea-yamamai. CMC has good compatibility to the SF, and the addition of CMC has significantly contributed to the improvement the mechanical properties and thermal stability of the restored silk, which is due to the formation of chemical bonding, strong hydrogen bonding and the construction of polymer network structure. The enhancement of crystallinity and reduction of ß-turns structure indicate that the micro-defects in the crystallization zone of the aged silk has been restored, and the ordered arrangement in the long-range ordered structure has been improved within a certain range. It was found that the CMC acted as antifungal agents when introduced on the aged historic silk, reducing the growth of Aspergillus niger, Aspergillus flavus and Paecilomyces variotii to a certain extent, which were commonly found in storage areas of libraries.

Proliferation and differentiation of direct co­culture of bone marrow mesenchymal stem cells and pigmented cells from the ciliary margin.

Damage of retinal ganglion cells (RGCs) is the major consequence of glaucoma and regeneration of RGCs is extremely difficult once the damage has occurred. Retinal stem cells (RSCs) are considered an ideal choice for RGC regeneration. Pigmented cells from the ciliary margin (PCMs) have great retinal differentiation potential and may be an ideal RSC candidate. However, the ciliary margin is too small, so the number of cells that can be obtained is limited. Bone marrow­derived mesenchymal stem cells (BMMSCs) are another type of stem cell that have been previously investigated for RGC regeneration. BMMSCs expand sufficiently, whereas the retinal differentiation of BMMSCs is insufficient. The aim of the present study was to investigate whether the co­culture of PCMs and BMMSCs may combine the advantages of both cell types to establish a novel and effective stem cell source for RGC regeneration. Primary rat PCMs and BMMSCs were isolated and co­cultured. Cell growth was observed by an inverted microscope and proliferation was monitored by an MTT assay. Cell cycle analysis was performed by using a flow cytometer, while the expression of the photoreceptor­specific homeobox gene (cone­rod homeobox, Crx) was determined by reverse transcription­quantitative polymerase chain reaction and western blot analysis. In addition, retinal differentiation was confirmed by immunofluorescence staining of major markers of retinal differentiation, including rhodopsin, visual system homeobox 2 and heparin sulfate. The co­cultured cells expanded successfully, in a similar way to BMMSCs. In addition, the expression of Crx and retinal markers were significantly upregulated following BMMSC and PCM co­culture. The results of the present study demonstrated that the co­culture of BMMSCs and PCMs may be used as a source of RSCs.

ß-Furan-Fused bis(Difluoroboron)-1,2-bis((1H-pyrrol-2-yl)methylene)hydrazine Fluorescent Dyes in the Visible Deep-Red Region.

Novel ß-furan-fused bis(difluoroboron)-1,2-bis((1H-pyrrol-2-yl)methylene)hydrazine (BOPHY) fluorescent dyes (F-BOPHY1-3) were prepared through an efficient process, and their structures were confirmed by (1)H NMR spectroscopy, (13)C NMR spectroscopy, MALDI-TOF HRMS, and element analysis. Their optical properties were then characterized by UV-vis absorption and photoluminescence (PL) spectroscopy. The UV-vis absorption and PL spectra of the dyes shifted to longer wavelengths relative to those of BOPHY because of the fusion of their furan rings, which extended π-conjugation of the molecules. All of the dyes exhibited large extinction coefficients (109700-12300 M(-1) cm(-1)), deep-red fluorescence emission (646-667 nm), moderate fluorescence quantum yields (0.30-0.45), as well as high chemical stability and photostability in solution. These advantageous properties show that these compounds are important to the design of efficient long-wavelength fluorescent dyes and are suitable for various applications in biotechnology and materials science.