Improving the solubility and interfacial absorption of hempseed protein via a novel high pressure homogenization-assisted pH-shift strategy.

A pH shift treatment aided by high pressure homogenization (HPH) with various pressures (0-120 MPa) was employed to structurally modify hempseed protein isolate (HPI). Compared with individual pH shift or HPH treatment, HPH-assisted pH shift improved the structural flexibility of HPI, as revealed by the increased random coil in protein secondary structure. With the incorporation of HPH into pH shift, the intrinsic fluorescence intensity was remarkably attenuated and redshifted, whereas the surface hydrophobicity was pronouncedly boosted, indicating the extensive unfolding of protein structure. Moreover, the cotreated HPI exhibited a smaller and more homogenous particle size, notably at a higher pressure. Consequently, the solubility was drastically raised by the cooperated treatments, to the maximum value (62.8 %) at 120 MPa. These physicochemical changes in the cotreated HPI facilitated a consolidated interfacial activity. Moreover, the cooperated treatment, especially highly pressured (120 MPa), facilitated the penetration and rearrangement of proteins at the oil-water interface.

Characterization of CM-Dil-labeled Muse cells in culture and in skin wounds in rats.

To investigate the characteristics of multilineage-differentiating stress-enduring (Muse) cells labeled with chloromethyl dialkylcarbocyanine (CM-Dil) in culture and in skin wounds of rats. Normal human dermal fibroblasts (NHDFs) were obtained from foreskins and were confirmed by immunocytochemistry with vimentin. Muse cells were derived from NHDFs using long-term trypsinization (LTT), were confirmed using immunocytochemistry with antibodies against stage specific embryonic antigen-3 (SSEA-3) and CD105 and were expanded in suspension cultures. The Muse cells were labeled with CM-Dil and were further evaluated with respect to their biological properties using CCK-8 assays and scratch tests. One hundred µl CM-Dil-labeled Muse cells at a concentration of 5 × 103/µl were injected subcutaneously at the edges of skin wounds in adult male SD rats. At weeks 1, 3 and 5 after the injection, the distribution of CM-Dil-labeled Muse cells in skin tissues was observed using immunofluorescence microscopy. Muse cells were double-positive for CD105 and SSEA-3. ALP staining of the M-clusters were positive and they displayed orange-red fluorescence after labelling with CM-Dil, which had no adverse effects on their viability, migration or differentiation capacity. One week after the subcutaneous injection of CM-Dil-labeled Muse cells, many cells with orange-red fluorescence were observed at the edges of the skin injuries; those fluorescent spots gradually decreased over time, and only a few Muse cells with fluorescence could be detected by week 5. CM-Dil can be used to label Muse cells without affecting their proliferation, migration or differentiation, and can be used for short-term tracking of Muse cells for the treatment of skin wounds in a rat model.

Modulating in vitro digestion of whey protein cold-set emulsion gels via gel properties modification with gallic acid and EGCG.

Gallic acid (GA) and epigallocatechin gallate (EGCG), cooperated at varied ratios (1:0, 3:1, 1:1, 1:3, and 0:1), were employed to modify gel properties of calcium induced-whey protein emulsion gel. The effects of GA/EGCG on emulsion morphology, as well as gel properties and in vitro digestive behavior of the emulsion gels were investigated. Compared with emulsions without phenolics, GA/EGCG induced slightly smaller particle size and stronger electrostatic repulsion between emulsion droplets. Moreover, GA/EGCG, notably at a ratio of 3:1, promoted electrostatic and hydrophobic interactions between protein molecules and the formation of a compact and filamentous gel microstructure, resulting in a remarkable increment in the gel strength (up to 106 %). Furthermore, in vitro oral digestion, dynamic gastric digestion (using an artificial gastric digestive system, AGDS), and intestinal digestion of the emulsion gels were simulated. Particle size and protein hydrolysis results revealed that GA/EGCG was prone to weaken the physical disintegration of gels, reduce protein hydrolysis, and enhance the stability of emulsified oil droplets during dynamic gastric digestion. As a consequence, delayed release of oil droplets was observed in the gels and more free fatty acids were released in the intestinal digestion, particularly in the gel with GA/EGCG (3:1). These findings would provide novel strategies for application of phenolic compounds in developing protein gel-based delivery systems.

Characteristics of multilineage-differentiating stress-enduring cell clusters in different culture conditions.

OBJECTIVE: To observe the morphological characteristics of clusters of Muse cells from normal human dermal fibroblasts (NHDFs) under different culture conditions. METHODS: Muse cells were sorted by magnetic activated cell sorting (MACS) from NHDFs, and were evaluated by flow cytometry. Muse cells were cultured in suspension and in adherent conditions to obtain Muse cell clusters (M-clusters), which were further characterized by alkaline phosphatase (AP) staining, immunofluorescence (IF) staining and transmission electron microscopy (TEM). The M-clusters were further cultured on Lando artificial dermal regeneration matrix (LADRM) for analysis by scanning electron microscopy (SEM) and IF staining of frozen sections. RESULTS: The proportion of SSEA3 and CD105 double-positive cells obtained by MACS was 87.4%. The sorted cells rapidly formed M-clusters after suspension culture, and showed internal characteristics of stem cells under TEM. After adherent culture, M-clusters stained positively for AP, SSEA-3 and OCT-4. Each M-cluster on the surface of the LADRM displayed an outer membrane of amorphous materials under SEM. Frozen sections and fluorescence staining of LADRM loaded with M-clusters showed an uneven fluorescence intensity of SSEA-3 within the clusters. CONCLUSIONS: Muse cells sorted by MACS from NHDFs could generate M-clusters, which included cells of different stemness and are wrapped in membrane-like structures.

Single-cell RNA sequencing combined with whole exome sequencing reveals the landscape of the immune pathogenic response to chronic mucocutaneous candidiasis with STAT1 GOF mutation.

Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent or persistent infections with Candida of the skin, nails, and mucous membranes (e.g., mouth, esophagus, and vagina). Compared with that of other infectious diseases, the immune pathogenic mechanism of CMC is still poorly understood. We identified a signal transducer and activator of transcription 1 gain-of-function (c.Y289C) mutation in a CMC patient. Single-cell transcriptional profiling on peripheral blood mononuclear cells from this patient revealed decreases in immature B cells and monocytes. Further analysis revealed several differentially expressed genes related to immune regulation, including RGS1, TNFAIP3, S100A8/A9, and CTSS. In our review of the literature on signal transducer and activator of transcription 1 gain-of-function (c.Y289C) mutations, we identified seven cases in total. The median age of onset for CMC (n=4, data lacking for three cases) was 10.5 years (range: birth to 11 years), with an average onset age of 8 years. There were no reports linking tumors to the c.Y289C mutation, and the incidence of pre-existing clinical disease in patients with the c.Y289C mutation was similar to previous data.

Preparation of Gum Arabic-Maltose-Pea Protein Isolate Complexes for 1-Octacosanol Microcapsule: Improved Storage Stability, Sustained Release in the Gastrointestinal Tract, and Its Effect on the Lipid Metabolism of High-Fat-Diet-Induced Obesity Mice.

1-Octacosanol (Octa) is a natural compound with several beneficial properties. However, its poor water solubility and metabolism in the digestive tract reduce its efficacy. The Octa-GA-Malt-PPI microcapsule was prepared as follows: gum Arabic (GA):maltose (Malt):pea protein isolate (PPI) = 2:1:2; core:shell = 1:7.5; emulsification temperature 70 °C; pH 9.0. An in vitro simulated gastrointestinal tract was used to analyze the digestion behavior. C57BL/6 mice were selected to establish an obesity model induced by a high-fat diet (HFD) to evaluate the effect of Octa monomer and the microcapsule. The diffusivity in water and storage stability of Octa improved after encapsulation. The microcapsule was ascribed to electrostatic interactions, hydrogen bonding, and hydrophobic interactions. The sustained release of Octa from the microcapsule was observed in a simulated gastrointestinal tract. Compared with Octa monomer, the microcapsule was more effective in alleviating the symptoms of weight gain, hypertension, and hyperlipidemia induced by HFD in mice. In conclusion, the construction of microcapsule structure can improve the dispersibility and stability of Octa in water, achieve sustained release of Octa in the gastrointestinal tract, and improve its efficiency in alleviating the effects of HFD on the body.

Clinical retrospective analysis of long-pulsed 1064-nm Nd:YAG laser in the treatment of onychomycosis and its effect on the ultrastructure of fungus pathogen.

The objective of this study was to analyze retrospectively the clinical efficacy and fungal clearance of long-pulsed 1064-nm Nd:YAG laser for treating onychomycosis and explore the inhibitory effects of laser on the fungus pathogen-induced onychomycosis in vitro. We performed a systematic retrospective analysis of clinical patients (162 effected nails) of onychomycosis treatment applying laser with or without topical ketoconazole ointment and followed up 3 months after treatment. Trichophyton rubrum- and Trichophyton mentagrophytes-induced onychomycosis was irradiated with laser superimposed for different cumulative energy, respectively; then, the areas of fungus colonies and growth curve in different days were showed, and changes in ultrastructures were observed under SEM and TEM. The clinical effective rate and fungal clearance rate in the combined group were higher than those in the laser group; however, there was no significant difference between the two groups. In vitro, the areas of T. rubrum colonies were significantly reduced at days 1, 3, and 5 after irradiation with cumulative laser energy ≥ 6400 J/cm2. When irradiated with cumulative laser energy ≥ 25600 J/cm2, significant difference in the areas of T. mentagrophytes colonies was found at day 5. And ultrastructure of the two strains before and after laser irradiation was damaged at different degrees. This study confirmed that long-pulsed 1064-nm Nd:YAG laser is effective for treating onychomycosis, and the laser irradiation can inhibit the colony growth of T. rubrum and T. mentagrophytes and change their cellular ultrastructures. The mechanism of laser treatment of onychomycosis may be related to direct damage of fungus pathogen.

Identification and validation of differentially expressed proteins in serum of CSU patients with different duration of wheals using an iTRAQ labeling, 2D-LC-MS/MS.

Chronic spontaneous urticaria (CSU) is one of the most common types of chronic urticaria (CU), with symptoms that recur easily, migrate and are refractory. It is unclear whether association between the differentiation of protein expression levels in the serum of CSU patients and the different duration of wheals exists. In the present study the samples were divided according to the duration of the wheals into group A (wheal duration <2 h) and group B (wheal duration 12-24 h). Differentially expressed proteins in sera of CSU patients with different durations of wheals were identified and validated with isobaric tags for relative and absolute quantitation (iTRAQ) in combination with two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS). Three hundred and seventy CSU serum-related proteins were initially identified. Among these proteins, ~30 had significant differences between the groups. According to the classification of biological functions and upregulated/downregulated values, serum amyloid A (SAA), CFL1, TPM4 and monocyte differentiation antigen (CD14) were chosen and validated by enzyme-linked immunosorbent assay (ELISA). The expression levels of CD14 in sera were not significantly different among the groups. SAA, CFL1 and TPM4 were associated with the wheal duration in CSU patients and therefore could be considered as new potential inflammatory biomarkers associated with CSU.

Binding of Gallic Acid and Epigallocatechin Gallate to Heat-Unfolded Whey Proteins at Neutral pH Alters Radical Scavenging Activity of in Vitro Protein Digests.

Preheated (80 °C for 9 min) whey protein isolate (HWPI) was reacted with 20, 120, and 240 µmol/g (protein basis) gallic acid (GA) or epigallocatechin gallate (EGCG) at neutral pH and 25 °C. Isothermal titration calorimetry and fluorometry showed a similar trend that GA binding to HWPI was moderate but weaker than EGCG binding. However, the shift of maximal fluorescence emission wavelength in opposite directions in response to GA (blue) and EGCG (red) suggests discrepant binding patterns. Electrophoresis results showed that EGCG induced formation of HWPI complexes while GA only had a marginal effect. Both free and phenolic-bound HWPI exhibited mild antiradical activity. However, when subjected to in vitro digestion, synergistic radical-scavenging activity was produced between the phenolics and peptides with the highest synergism being observed on 120 µmol/g phenolics.

Interaction of Whey Proteins with Phenolic Derivatives Under Neutral and Acidic pH Conditions.

Integration of gallic acid (GA) and its derivative epigallocatechin gallate (EGCG; 20, 120, and 240 µmol/g, protein basis) into whey protein isolate (WPI) at room temperature and pH 3.0 and pH 7.0 was investigated. At pH 7.0, both phenolics caused significant structural changes and EGCG induced greater digestibility of WPI. Total sulfhydryl in WPI decreased from 28.6 to 7.6 µmol/g and surface hydrophobicity declined by nearly 50% with 240 µmol/g EGCG at pH 7.0. Similar but less appreciable changes were produced by GA and at pH 3.0. Isothermal titration and fluorescence quenching tests showed moderately weak binding of WPI with GA but strong binding with EGCG at pH 7.0. Both phenolics at high concentrations lowered the thermal transition temperature of ß-lactoglobulin by 0.5 °C to 1.4 °C and promoted its digestion. The phenolics also displayed a remarkable synergism with peptides in the WPI digest exerting radical scavenging activity.

A live, attenuated pseudorabies virus strain JS-2012 deleted for gE/gI protects against both classical and emerging strains.

Emerging pseudorabies virus (PRV) variant have led to pseudorabies outbreaks in Chinese pig farms. The commercially available PRV vaccine provides poor protection against the PRV variant. In this study, a gE/gI deleted PRV strain JS-2012-△gE/gI was generated from a PRV variant strain using homologous DNA recombination. Compared to the parental strain JS-2012, JS-2012-△gE/gI grew slowly and showed small plaque morphology on Vero cells. The safety and immunological efficacy of JS-2012-△gE/gI was evaluated as a vaccine candidate. JS-2012-△gE/gI was avirulent to suckling piglets, but was able to provide full protection for young piglets against challenge with both the classical virulent PRV and the emerging PRV variant. After sows were vaccinated with the gE/gI-deleted strain, their suckling offspring were resistant to an otherwise lethal challenge with the classical and the variant PRVs. Piglets inoculated with JS-2012-△gE/gI did not develop PRV-specific gE-ELISA antibodies. Thus, JS-2012-△gE/gI appears to be a promising marker vaccine candidate to control PRV variant circulating in pig farms in China.

The characterization of soybean oil body integral oleosin isoforms and the effects of alkaline pH on them.

Oil body, an organelle in seed cell (naturally pre-emulsified oil), has great potentials to be used in food, cosmetics, pharmaceutical and other applications requiring stable oil-in-water emulsions. Researchers have tried to extract oil body by alkaline buffers, which are beneficial for removing contaminated proteins. But it is not clear whether alkaline buffers could remove oil body integral proteins (mainly oleosins), which could keep oil body integrity and stability. In this study, seven oleosin isoforms were identified for soybean oil body (three isoforms, 24 kDa; three isoforms, 18 kDa; one isoform, 16kDa). Oleosins were not glycoproteins and 24 kDa oleosin isoforms possessed less thiol groups than 18 kDa ones. It was found that alkaline pH not only removed contaminated proteins but also oleosins, and more and more oleosins were removed with increasing alkaline pH.

Asthma outcomes in children and adolescents with multiple morbidities: Findings from the National Health Interview Survey.

BACKGROUND: More Americans are managing multiple chronic conditions (MCCs), and trends are particularly alarming in youth. OBJECTIVE: The purpose of this study was to examine the prevalence and distribution of 9 chronic conditions in children and adolescents with and without asthma, and adverse asthma outcomes associated with having MCCs. METHODS: Cross-sectional interview data from the National Health Interview Survey were analyzed (N = 66,790) between 2007 and 2012 in youth 0 to 17 years of age. Bivariate analysis methods and multivariate generalized linear regression were used to examine associations. RESULTS: Five percent of children with asthma had 1 or more coexisting health conditions. The prevalence of 1 or more comorbidities was greater among those with asthma than those without (5.07% [95% CI: 4.5-5.6] vs. 2.73% [95% CI: 2.6-2.9]). Those with asthma were twice as likely to have co-occurring hypertension (prevalence ratio [PR] = 2.2 [95% CI: 1.5-3.2]) and arthritis (PR = 2.7 [95% CI: 1.8-4.0]) compared with those without asthma. Every additional chronic condition with asthma was associated with a greater likelihood of an asthma attack (PR = 1.1 [95% CI: 1.0-1.2]), all-cause emergency department visits (PR = 1.3 [95% CI: 1.1-1.5]), and missed school days (PR = 2.3 [95% CI: 1.7-3.2]). CONCLUSIONS: Children and adolescents with asthma in the US who suffer from MCCs have increased asthma symptoms, missed school days, and all-cause emergency department visits. Further research on optimal management strategies for this group is needed.

Macronutrients and micronutrients of soybean oil bodies extracted at different pH.

UNLABELLED: In this study, the macronutrients and micronutrients of pH 6.8, 8.0, 9.5, and 11.0 extracted soybean oil bodies (OBs) were examined, revealing that soybean OBs might be used as a natural carrier for bioactive components (unsaturated fatty acids, phospholipid, tocopherol, and phytosterol). pH 6.8 extracted OBs (dry basis) contained 85.88% neutral lipid, 8.18% protein, and 5.85% polar lipid (mainly phospholipid) by gravimetric analysis. The percentage of neutral lipid was increased, while those of protein and polar lipid were decreased with increasing pH. Tocopherol (about 75 mg/100 g neutral lipid of OBs) was not affected, while phytosterol was decreased (136 to 110 mg/100 g neutral lipid of OBs) with increasing pH. The detectable total monosaccaride (galactosamine, glucosamine, and glucose) content of extracted OBs was low and also decreased (35.80 to 6.13 mg/100 g neutral lipid of OBs) with increasing pH. The protein of extracted OBs had higher percentage of essential amino acids than soybean protein isolate with tryptophan and methionine as limited amino acids. The fatty acid composition of extracted OBs was rich in linoleic acid (about 59%), oleic acid (about 20%), and α-linolenic acid (about 7%). PRACTICAL APPLICATION: Oil bodies (OBs) from soybean and other plant seeds are greatly examined owing to their potential utilizations in food ingredients. The determination of its macronutrients and micronutrients would be very meaningful for its efficient utilization in the future.

Oleosins (24 and 18 kDa) are hydrolyzed not only in extracted soybean oil bodies but also in soybean germination.

After oil bodies (OBs) were extracted from ungerminated soybean by pH 6.8 extraction, it was found that 24 and 18 kDa oleosins were hydrolyzed in the extracted OBs, which contained many OB extrinsic proteins (i.e., lipoxygenase, ß-conglycinin, γ-conglycinin, ß-amylase, glycinin, Gly m Bd 30K (Bd 30K), and P34 probable thiol protease (P34)) as well as OB intrinsic proteins. In this study, some properties (specificity, optimal pH and temperature) of the proteases of 24 and 18 kDa oleosins and the oleosin hydrolysis in soybean germination were examined, and the high relationship between Bd 30K/P34 and the proteases was also discussed. The results showed (1) the proteases were OB extrinsic proteins, which had high specificity to hydrolyze 24 and 18 kDa oleosins, and cleaved the specific peptide bonds to form limited hydrolyzed products; (2) 24 and 18 kDa oleosins were not hydrolyzed in the absence of Bd 30K and P34 (or some Tricine-SDS-PAGE undetectable proteins); (3) the protease of 24 kDa oleosin had strong resistance to alkaline pH while that of 18 kDa oleosin had weak resistance to alkaline pH, and Bd 30K and P34, resolved into two spots on two-dimensional electrophoresis gel, also showed the same trend; (4) 16 kDa oleosin as well as 24 and 18 kDa oleosins were hydrolyzed in soybean germination, and Bd 30K and P34 were always contained in the extracted OBs from germinated soybean even when all oleosins were hydrolyzed; (5) the optimal temperature and pH of the proteases were respectively determined as in the ranges of 35-50 °C and pH 6.0-6.5, while 60 °C or pH 11.0 could denature them.

The integral and extrinsic bioactive proteins in the aqueous extracted soybean oil bodies.

Soybean oil bodies (OBs), naturally pre-emulsified soybean oil, have been examined by many researchers owing to their great potential utilizations in food, cosmetics, pharmaceutical, and other applications requiring stable oil-in-water emulsions. This study was the first time to confirm that lectin, Gly m Bd 28K (Bd 28K, one soybean allergenic protein), Kunitz trypsin inhibitor (KTI), and Bowman-Birk inhibitor (BBI) were not contained in the extracted soybean OBs even by neutral pH aqueous extraction. It was clarified that the well-known Gly m Bd 30K (Bd 30K), another soybean allergenic protein, was strongly bound to soybean OBs through a disulfide bond with 24 kDa oleosin. One steroleosin isoform (41 kDa) and two caleosin isoforms (27 kDa, 29 kDa), the integral bioactive proteins, were confirmed for the first time in soybean OBs, and a considerable amount of calcium, necessary for the biological activities of caleosin, was strongly bound to OBs. Unexpectedly, it was found that 24 kDa and 18 kDa oleosins could be hydrolyzed by an unknown soybean endoprotease in the extracted soybean OBs, which might give some hints for improving the enzyme-assisted aqueous extraction processing of soybean free oil.