DNA methylation variations of DNA damage response correlate survival and local immune status in melanomas.

AIM: We aimed to explore the impact of DNA methylation alterations on the DNA damage response (DDR) in melanoma prognosis and immunity. MATERIAL & METHODS: Different melanoma cohorts with molecular and clinical data were included. RESULTS: Hierarchical clustering utilizing different combinations of DDR-relevant CpGs yielded distinct melanoma subtypes, which were characteristic of different prognoses, transcriptional function profiles of DDR, and immunity and immunotherapy responses but were associated with similar tumor mutation burdens. We then constructed and validated a clinically applicable 4-CpG risk-score signature for predicting survival and immunotherapy response. CONCLUSION: Our study describes the close interrelationship among DNA methylation, DDR machinery, local tumor immune status, melanoma prognosis, and immunotherapy response.

Dynamic high-pressure microfluidization assisted with galactooligosaccharide-modified whey protein isolate: Investigating its effect on relieving intestinal barrier damage.

The study aimed to investigating the mechanisms of relieved intestinal barrier damage by dynamic high-pressure microfluidization assisted with galactooligosaccharide- glycated whey protein isolate. The modifications changed the multi-structure, and the modified whey protein isolate could promote the proliferation of IEC-6 cells and contributed to the restoration of LPS-induced occludin damage in IEC-6 cells. Also, it could repair cyclophosphamide-induced ileal villus rupture and crypt destruction in BALB/c mice, significantly altered the abundance of dominant bacteria, which were associated with propionic acid, butyric acid, isovaleric acid, and valeric acid. Ileum transcriptomics revealed that the modified whey protein isolate significantly regulate of the levels of Cstad, Cyp11a1, and Hs6st2 genes, relating to the increase of propionic acid, isovaleric acid, and valeric acid. In conclusion, galactooligosaccharide- modified whey protein isolate could regulate the level of Cstad, Cyp11a1 and Hs6st2 genes by altering the gut microbial structure and the level of SCFAs, thereby repairing the intestinal barrier.

Galacto-oligosaccharides modified whey protein isolate ameliorates cyclophosphamide-induced immunosuppression.

The effect of whey protein isolate (WPI)- galacto-oligosaccharides (GOS)/fructo-oligosaccharides (FOS) conjugates on RAW264.7 cells, and further the effect of WPI-GOS conjugates on CTX-induced immunosuppressed mice were investigated. Compared to WPI-FOS conjugates, WPI-GOS conjugates exhibited deeper glycation extent, more pronounced structural unfolding and helix-destabilizing, and obviously improved functional indicators of RAW264.7 macrophages. In addition, WPI-GOS conjugates also repaired immune organ and intestinal barrier and increased IL-1ß and IFN-γ levels in immunosuppressed mice. The alteration of gut microbiota induced by WPI-GOS conjugates changed the serum metabolites, causing the activation of NFκB pathway, which strengthens the immune system. The activation of NFκB pathway maybe associated with the mTOR signal pathway and ABC transporters. However, the precise mechanisms by which NFκB pathway interacts with mTOR signal pathway and ABC transporters to modulate the immune response need for further research.

Allergenicity assessment of ß-lactoglobulin ferulic acid-glucose conjugates.

We prepared the ß-lactoglobulin (BLG)-ferulic acid (FA)-glucose (Glu) conjugates by alkaline method and Maillard reaction to assess the allergenicity. FA and Glu can form a ternary covalent conjugate with BLG, as evidenced by the shortening of SEC retention time, upward migration of SDS-PAGE protein bands, considerable decrease in free amino and sulfhydryl content, and changes in multistructure. BLG-Glu-FA conjugates weakly bound to immunoglobulin E in allergic sera was weak, reduced interleukin 4 and tumor necrosis factor α levels in RBL-2H3 cells and histamin and interleukin 6 secretion levels in KU812 cells, and inhibited the nuclear factor-κB signaling pathway. In vivo experiments showed that the conjugates regulated T-cell homeostasis in mouse splenic and mesenteric lymphocytes and attenuated splenic and duodenal immune injury. Therefore, the conjugates of BLG with FA combined with Glu altered the epitope structure and exhibited low allergenicity.

Gut Microbiome-Serum Metabolism Revealed the Allergenicity of Ferulic Acid Combined with Glucose-Modified ß-Lactoglobulin.

A novel strategy combining ferulic acid and glucose was proposed to reduce ß-lactoglobulin (BLG) allergenicity and investigate whether the reduction in allergenicity was associated with gut microbiome and serum metabolism. As a result, the multistructure of BLG changed, and the modified BLG decreased significantly the contents of IgE, IgG, IgG1, and mMCP-1 in serum, improved the diversity and structural composition of gut microbiota, and increased the content of short-chain fatty acids (SCFAs) in allergic mice. Meanwhile, allergic mice induced by BLG affected arachidonic acid, tryptophan, and other metabolic pathways in serum, the modified BLG inhibited the production of metabolites in arachidonic acid metabolism pathway and significantly increased tryptophan metabolites, and this contribution helps in reducing BLG allergenicity. Overall, reduced allergenicity of BLG after ferulic acid was combined with glucose modification by regulating gut microbiota, the metabolic pathways of arachidonic acid and tryptophan. The results may offer new thoughts alleviating the allergy risk of allergenic proteins.

Simulated in vitro digestion of α-lactalbumin modified by phosphorylation: Detection of digestive products and allergenicity.

The effects of phosphorylation on the allergenicity of bovine α-lactalbumin (BLA) and digestive products were studied in vitro digestion. Two components with different molecular weight and conformation were obtained from natural and phosphorylated BLA. In vivo and in vitro assessment of allergenicity showed that phosphorylation prior to digestion significantly decreased the IgE/IgG binding capacity and allergic response in KU812 cells, and reduced the levels of IgG, IgE, IL-4 and histamine, with an increase in IFN-γ levels in mouse serum, depending on the changes in BLA structures, producing numerous small peptides. There were four phosphorylated sites (S22, T29, S47 and S70) in the high molecular weight components of phosphorylated BLA after digestion. These phosphorylated sites could mask the linear epitopes of digestive products, resulting in reduced allergic activity. Phosphorylation prior to digestion of dairy products can reduce the risk of anaphylaxis in patients with milk allergy to some extent.

Bovine ß-Lactoglobulin Covalent Modification by Flavonoids: Effect on the Allergenicity and Human Intestinal Microbiota.

The present study aims to investigate the structure of covalent conjugates of bovine ß-lactoglobulin (BLG) and flavonoids (luteolin, myricetin, and hyperoside), and their effect on the allergenicity and human intestinal microbiota. Covalent modification of amino acids in BLG by flavonoids was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and o-phthaldialdehyde assay. The secondary and conformational structures of BLG were changed by the covalent modification, which were determined by the circular dichroism, Fourier transform infrared spectroscopy, fluorescence spectroscopy, and UV spectroscopy. The enzyme-linked immunosorbent assay (ELISA) and cell experiments indicated that BLG covalent conjugates could reduce IgE/IgG binding capacities and suppress the allergy reactivity of RBL-2H3 cells, suggesting that the covalent modification modulated the balance of T cells. Meanwhile, covalent modification of BLG with these flavonoids can alter the diversity of human intestinal microbiota and the community abundance at phylum, family, and genus levels. The results revealed that covalent modification of BLG with flavonoids alters human intestinal microbiota, might result in the reduction of allergenicity, which could provide information for confirming the relationship between food allergy and the intestinal microbial ecosystem.

Improved antitumor activity and IgE/IgG-binding ability of α-Lactalbumin/ß-lactoglobulin induced by ultrasonication prior to binding with oleic acid.

Bovine α-lactalbumin (α-La)/ß-lactoglobulin (ß-Lg) was pretreated through ultrasonic treatment and subsequently binding with oleic acid (OA) by heat treatment. And, the antitumor activity, IgE/IgG-binding ability, and structural modifications were investigated. After α-La/ß-Lg were treated by ultrasonic prior to binding with OA, the treated α-La/ß-Lg showed high antitumor activity and IgE/IgG-binding ability, and significantly affected the structural modifications, which reflected by the reduction in α-helix content, the increase of molecular weight, intrinsic fluorescence intensity, and surface hydrophobicity. Molecular docking studies indicated that OA bound to α-La/ß-Lg by hydrogen bonds and hydrophobic interaction. Therefore, ultrasonic prior to binding with OA could improve antitumor activity and IgE/IgG-binding ability of α-La/ß-Lg as a result of structural modifications. And, ultrasonic prior to binding with fatty acid processing of milk products alone may increase the antitumor activity, this change may enhance the risk of an allergenic reaction in milk allergy patients to some extent. PRACTICAL APPLICATIONS: Fatty acids, natural ligands associated with the bovine milk proteins, and milk protein-fatty acid complex has a variety of functional applications in the food industry. This study revealed that antitumor activity, IgE/IgG-binding ability, and structural modifications of α-La/ß-Lg induced by ultrasonic prior to binding with oleic acid. It will be beneficial to understand the mechanism of the functional changes of protein. Ultrasonic prior to binding with oleic acid will be more likely to develop a practical technology to improve the functional characteristics of milk protein and design the optimal nutritional performance of milk food.

The mechanism of the reduction in allergenic reactivity of bovine α-lactalbumin induced by glycation, phosphorylation and acetylation.

Bovine α-lactalbumin (α-Lac) allergy is a common health problem. This study assesses the allergenic reactivity and the structural properties of α-Lac after protein modification (glycation, phosphorylation and acetylation) by ELISA, cells experiment and high-resolution mass spectrometry. Three modified methods significantly reduced the IgE/IgG-binding capacity, and the release of histamine and interleukin-6, and changed the conformational structure of α-Lac. α-Lac was glycated at K13, K16, K94, K98, and K108, phosphorylated at Y18, S22, Y103, and S112, and acetylated at K13, T33, S34, T38, S47, K62, S69, S70, K108, and K114, respectively, leading to masking the linear epitopes of α-Lac. Therefore, the decrease of allergenic reactivity of α-Lac induced by glycation, phosphorylation and acetylation depends upon not only the shielding effect of their modified sites, but also the change of conformational structure. This study confirmed that protein modification was a promising method for decreasing the allergenic reactivity of allergic proteins.

[Inhibitory effect of kaempferol against binding of platelet activating factor to its receptor].

OBJECTIVE: To observe the platelet activating factor (PAF) antagonistic effect of kaempferol. METHOD: The specific binding of [3H] PAF to rabbit platelet receptor was investigatedwith radio ligand binding assay (RLBA). Platelet adhesion induced by PAF was measured with spectrophotometry. The elevation of inner free calcium concentration in rabbit polymorphonuclear leukocytes (PMNs) induced by PAF was determined with Fura-2 fluorescent technique. RESULT: The 1, 2 or 4 nmol x L(-1) [3H]PAF specific binding to rabbit platelet receptor was inhibited by Kae dosage dependently and the IC50 were 30.8, 74.6 and 92.0 micro mol x L(-1), respectively. The PAF induced reactions of rabbit platelet adhesion and PMNs inner free calcium concentration elevation were inhibited by Kae in a dose-dependent manner. The IC50 of Kae to inhibit platelet adhesion was 65 micromol x L(-1). CONCLUSION: Kae is effective in inhibiting the action of PAF and it is a new PAF receptor antagonist.

Preparation of glucose-sensitive microcapsules with a porous membrane and functional gates.

A glucose-sensitive microcapsule with a porous membrane and with linear-grafted polyacrylic acid (PAAC) chains and covalently bound glucose oxidase (GOD) enzymes in the membrane pores acting as functional gates was successfully prepared. Polyamide microcapsules with a porous membrane were prepared by interfacial polymerization, PAAC chains were grafted into the pores of the microcapsule membrane by plasma-graft pore-filling polymerization, and GOD enzymes were immobilized onto the PAAC-grafted microcapsules by a carbodiimide method. The release rates of model drug solutes from the fabricated microcapsules were significantly sensitive to the existence of glucose in the environmental solution. In solution, the release rate of either sodium chloride or VB(12) molecules from the microcapsules was low but increased dramatically in the presence of 0.2mol/L glucose. The prepared PAAC-grafted and GOD-immobilized microcapsules showed a reversible glucose-sensitive release characteristic. The proposed microcapsules provide a new mode for injection-type self-regulated drug delivery systems having the capability of adapting the release rate of drugs such as insulin in response to changes in glucose concentration, which is highly attractive for diabetes therapy.

Preparation of submicrometer-sized monodispersed thermoresponsive core-shell hydrogel microspheres.

We have successfully prepared monodispersed thermoresponsive core-shell hydrogel microspheres with a mean diameter of 200-400 nm with poly(N-isopropylacrylamide-co-styrene) [P(NIPAM-co-St)] cores and poly(N-isopropylacrylamide) (PNIPAM) shells. The submicrometer-sized monodispersed P(NIPAM-co-St) core seeds were prepared by using a surfactant-free emulsion polymerization method, and the PNIPAM shell layers were fabricated onto the core seeds by using a seed polymerization method. The particle size, morphology and monodispersity, and thermoresponsive characteristics of the prepared microspheres were experimentally studied. In the preparation of P(NIPAM-co-St) seeds, with increasing the initiator dosage, the mean diameters and the dispersal coefficients were almost at the same levels at first; however, when the initiator dosage increased further to a critical amount, the mean diameters decreased drastically and the monodispersity became worse significantly. With increasing the stirring rate, the particle diameter decreased, and when the stirring rate was larger than 600 rpm, the monodispersity became worse obviously. With increasing the phase ratio, the mean diameter became larger simply, and the monodispersity became worse first and then became better again. With increasing the reaction time, the particle sizes nearly did not change, while the monodispersity gradually became better slightly. For the core-shell microspheres, with increasing the NIPAM dosage in the preparation of the PNIPAM shell layers, the mean diameters became larger simply, the monodispersity became better, and the thermoresponsive swelling ratio of the hydrodynamic diameters increased.

Study of SPG membrane emulsification processes for the preparation of monodisperse core-shell microcapsules.

Experimental investigations on the Shirasu-porous-glass (SPG)-membrane emulsification processes for preparing monodisperse core-shell microcapsules with porous membranes were carried out systematically. The results showed that, to get monodisperse oil-in-water (O/W) emulsions by SPG membrane emulsification, it was more important to choose an anionic surfactant than to consider hydrophile-lipophile balance (HLB) matching. Increasing the viscosity of either the disperse phase or the continuous phase or decreasing the solubility of the disperse phase in the continuous phase could improve both the monodispersity and the stability of emulsions. With increasing monomer concentration inside the disperse phase, the monodispersity of emulsions became slightly worse and the mean diameter of emulsions gradually became smaller. Monodisperse monomer-containing emulsions were obtained when the SPG membrane pore size was larger than 1.0 micro m, and from these emulsions satisfactory monodisperse core-shell microcapsules with a porous membrane were prepared. On the other hand, when the SPG membrane pore size was smaller than 1.0 mciro m, no monodisperse emulsions were obtained because of the formation and chokage of solid monomer crystals in the pores or at the end of the pores of the SPG membrane. This was due to the remarkable solvation and diffusion of the solvent in water. With increasing the emulsification time the average emulsion diameter generally decreased, and the monodispersity of the emulsions gradually became worse.

[Antagonistic effect of myricetin on platelet activing factor].

AIM: To study the antagonistic effect of myricetin on platelet activing factor (PAF). METHODS: The specific binding of [3H] PAF to rabbit platelet receptor was investigated using radio ligand binding assay (RLBA). Platelet adhesion induced by PAF was measured with spectrophotometry. The elevation of inner free calcium concentration in rabbit polymorphonuclear leukocytes (PMNs) induced by PAF was assayed by Fura-2 fluorescent technique. RESULTS: The specific binding inhibition potency of Myr was found to be concentration-dependent. The IC50 of Myr in [3H] PAF 1, 2 and 4 nmol.L-1 were 34.8, 85.7 and 118.6 mumol.L-1, respectively. The PAF induced reactions of rabbit platelet adhesion and PMNs inner free calcium concentration increase were inhibited by Myr in a dose-dependent manner. The IC50 of Myr to inhibit platelet adhesion was 13.1 mumol.L-1. CONCLUSION: The specific receptor binding of PAF can be antagonized by myricetin.

[Experimental study on inhibitory effect of rutin against platelet activation induced by platelet activating factor in rabbits].

OBJECTIVE: To explore the inhibitory effect and mechanism of rutin against platelet activating factor (PAF) induced platelet aggregation, 5-HT release and intra-platelet free calcium concentration. METHODS: The rate of washed rabbit platelet (WRP) aggregation was measured by turbidimetry and O-phthaldialdehyde (OPT) fluoro-spectrophotometry (FSPM) was used to determine 5-HT content. The intraplatelet free calcium concentration was measured with Fura-2/AM FSPM assay. RESULTS: Rutin in vitro was concentration-dependently inhibiting PAF (9.55 x 10(-9) mol/L) induced WRP aggregation, the IC50 of 5-HT release was 0.73, 1.13 mmol/L respectively and the intraplatelet free calcium concentration elevation evoked by PAF (4.78 x 10(-10) mol/L) were inhibited by 68.3, 136, 274, 545 mumol/L of rutin dose-dependently. CONCLUSION: Rutin could inhibit PAF induced platelet aggregation, 5-HT release and the increase of intraplatelet free calcium.