Study on the interaction mechanism of whey protein isolate with phosphatidylcholine: By multispectral methods and molecular docking.

The elucidation of the interaction mechanism between phospholipids and milk proteins within emulsions is pivotal for comprehending the properties of infant formula fat globules. In this study, multispectral methods and molecular docking were employed to explore the relationship between phosphatidylcholine (PC) and whey protein isolate (WPI). Observations indicate that the binding constant, alongside thermodynamic parameters, diminishes as temperature ascends, hinting at a predominantly static quenching mechanism. Predominantly, van der Waals forces and hydrogen bonds constitute the core interactions between WPI and PC. This assertion is further substantiated by Fourier transform infrared spectroscopy, which verifies PC's influence on WPI's secondary structure. A detailed assessment of thermodynamic parameters coupled with molecular docking reveals that PC predominantly adheres to specific sites within α-lactalbumin, ß-lactoglobulin, and bovine serum albumin, propelled by a synergy of hydrophobic interactions, hydrogen bonding, and van der Waals forces, with binding energies noted at -5.59, -6.71, and -7.85 kcal/mol, respectively. An increment in PC concentration is observed to amplify the emulsification properties of WPI whilst concurrently diminishing the zeta potential. This study establishes a theoretical foundation for applying the PC-WPI interaction mechanism in food.

High-intensity ultrasound improved the physicochemical and gelling properties of Litopenaeus vannamei myofibrillar protein.

The effects of high-intensity ultrasound on the physicochemical and gelling properties of Litopenaeus vannamei (L. vannamei) myofibrillar protein (MP) were investigated. MP solutions were subjected to ultrasound treatment (power 100 W, 300 W, and 500 W). It was found that the carbonyl and free amino contents of MP increased significantly with increasing ultrasound power, accompanied by enhanced emulsification properties. The increase of free radical and carbonyl content indicated that ultrasound induced the oxidation of MP. With the increase of ultrasound power, it was found that the total sulfhydryl content of the shrimp MP decreased, but the surface hydrophobicity increased significantly, which might be closely related to the conformational changes of MP. Meanwhile, a significant increase of ß-sheet but a decrease of α-helix in the secondary structure of MP was observed with increasing ultrasound power, indicating that ultrasound treatment induced the stretching and flexibility of MP molecules. SDS-PAGE showed that L. vannamei MP consisted of myosin heavy chain, actin, myosin light chain, paramyosin and tropomyosin. Ultrasound treatment could lead to some degree of oxidative aggregation of MP. The results of rheological properties indicated that ultrasound treatment enhanced the viscoelasticity of MP and further improved the gel strength of MP gel. This study can provide a theoretical basis for the functional modification of shrimp MP and the processing of its surimi products.

[Platelet HPA Typing of Platelet Donors in Zhangjiakou Area].

OBJECTIVE: To analyze the polymorphism of the HPA1-5,15 system of the donors in Zhangjiakou area. METHODS: DNA was extracted from the blood samples of the donors, PCR- SSP method was used to divide HPA1-6, 15 genotype. The gene frequency and genotype frequency were calculated, compared with the difference and regiahal specificity of the populations in our country and foregiens was compared other populations. RESULTS: The gene expression in the HPA-1, HPA-2 and HPA-4 systems were all homozygous aa, and the donors who expressed homozygous bb was not exessed. Among them, one heterozygous ab expression was found in both HPA-1 and HPA-4 systems (1%), and 14 cases of heterozygous ab expression were found in HPA-2 system (14%). The gene expression in the HPA-5 system was mainly homozygous aa (98%), and a very few expressed homozygous bb (2%) was found. The degree of heterozygosity of gene expression in the HPA-3 and HPA-15 systems was relatively high. The proprotion of the expression of aa, ab and bb in the HPA-3 system was respectively 46%, 40% and 14%, the proprotion of the expression of aa, ab and bb in the HPA-15 system was respectively 21%, 64% and 15%. CONCLUSION: The gene frequency of platelet-specific antigen HPA1-5,15 system in zhangjiakou region shows local characteristics. The heterozygosity degree of gene expression in the HPA-3 and HPA-15 systems are both high, suggesting that they are more likely to result in alloimmunization and ineffective platelet transfusion, which should be pays attention to.

Evaluation of the properties of whey protein films with modifications.

Whey protein concentrate (WPC) has been widely studied as a biodegradable bio-based packaging material in the food industry. In this study, different whey protein films were obtained through physical, chemical, enzymatic, and composite modifications. The molecular structure, micro-morphology, mechanical properties, barrier properties, and other characteristics of the films were evaluated. The results illustrated that the thickness of WPC with composite modification increased and the transmittance decreased, but the mechanical properties and barrier properties were more prominent. The WPC film prepared by physical modification combined with transglutaminase has the best film-forming effect, the tensile strength (TS) was 5.45 MPa, the elongation at break (EAB) was 25.19%, the WVP was 5.53 g·mm/m2 ·hr·kPa, and the Oxygen permeability (OP) was 1.83 meq/K, and its microstructure was and uniform. In addition, based on the the results of SDS-PAGE and Fourier transform infrared spectroscopy (FTIR), the intermolecular and intramolecular interactions of various modification methods on WPC were studied, thus contributing to analyze the properties of the film. This study provides theoretical basis and technical support for the industrial production of protein-based films.

Correlation between histone H3K4 trimethylation and DNA methylation and evaluation of the metabolomic features in acute rejection after kidney transplantation.

OBJECTIVE: To investigate the difference between trimethylation of monocyte histone H3K4 and DNA methylation in acute rejection (AR) after renal transplantation in rats and reveal the epigenetic mechanism of the AR rats based on metabolomics. METHOD: Peripheral blood mononuclear cells (PBMCs) were isolated, and CD4 +CD25 + Treg cells were sorted by flow cytometry. The Foxp3 mRNA and protein levels of CD4 +CD25 + Treg cells were detected by real-time RT-PCR and Western blotting, respectively. High-throughput screening was applied to evaluate the H3K4 methylation of monocytes using chromatin immunoprecipitation with DNA microarray (ChIP-chip) and verified by ChIP with real-time PCR (ChIP-qPCR). Methylated DNA immunoprecipitation sequencing was combined with real-time PCR (MeDIP-qPCR) to detect the DNA methylation level of positive genes (ABCC4, Mef2d, Tbx1 and Eif6). Real-time quantitative PCR (qRT-PCR) and Western blotting were used to detect the mRNA and protein levels of positive genes. The difference in lipid metabolism in the blood of (non) acute rejection rats was analysed by HPLC/MS. RESULTS: AR rats showed an apparent increase in BUN and Cr levels, as well as IL-2, IL-10 and IFN-γ. Compared with non-AR rats, the expression of CD4 +CD25 + Treg cells and Foxp3 mRNA and protein were significantly lower in AR rats. AR rats also showed an increase in H3K4 trimethylation of CD4 +CD25 + Treg and decrease in DNA methylation. There were significant differences in the DNA methylation level of four positive genes between AR and non-AR rats (P<0.05), in addition to differences in the expression levels of mRNA and protein. Pathological examination of the transplanted kidney indicated that AR rats had more severe pathological injury of the kidney than the non-AR rats. There were significant increase in the contents of several phosphatidylcholines, lysophosphatidylcholine, free fatty acids and carnitine in AR rats which detected by HPLC/MS. CONCLUSION: H3K4 trimethylation increased in PBMCs in AR rats, while DNA methylation decreased, indicating the presence of epigenetic differences between AR and non-AR rats. Metabolomic studies indicated a significant increase in AR rats in the contents of several metabolites, such as phosphatidylcholines, lysophosphatidylcholine, free fatty acids and carnitine, suggesting an increasein phospholipase activity and leading to an energy metabolism imbalance with intensification of ß-oxidation. DNA methylation may be associated with an increase in phosphatidylcholines, lysophosphatidylcholine and free fatty acids in AR rats, which may further affect energy metabolism by enhancing the tricarboxylic acid cycle in AR rats.

Association between Nod-like receptor protein 3 inflammasome and gouty nephropathy.

Crystalized deposits of monosodium urate activate the Nod-like receptor protein 3 (NLRP3) inflammasome, resulting in kidney damage. The present study investigated whether the NLRP3 inflammasome is associated with the progression of hyperuricaemia and gouty nephropathy. Adult male patients were recruited at the Affiliated Baoan Hospital of Shenzhen and divided into three groups of 15 patients each: The control group, the hyperuricaemia group and the gouty nephropathy group. General characteristics and organ function indicators were also measured for each patient. NLRP3, apoptosis-associated speck like protein (ASC) and caspase-1 mRNA and protein expressions in peripheral blood mononuclear cells were detected. The expression of certain downstream inflammatory factors, including interleukin (IL)-1ß and IL-18 were also assessed in plasma. The results demonstrated that the concentration of uric acid and creatinine were increased in the hyperuricaemia and gouty nephropathy groups compared with the control group. NLRP3, ASC and caspase-1 mRNA and protein expression, and IL-1ß and IL-18 expression were increased in the hyperuricaemia and gouty nephropathy groups compared with the control group. In addition, ASC and caspase-1 mRNA and protein expression, and IL-1ß expression were higher in the gouty nephropathy group compared with the hyperuricaemia group. In conclusion, the present results supported the hypothesis that the NLRP3 inflammasome signalling pathway is associated with gouty nephropathy leading to initiation of the inflammatory response and causing renal damage.

NLRP3 inflammasome and lipid metabolism analysis based on UPLC-Q-TOF-MS in gouty nephropathy.

To determine the differences in plasma metabolism between healthy patients and patients with hyperuricaemia and gouty nephropathy, the present study identified differentially expressed metabolites associated with gouty nephropathy. Furthermore, the NLRP3 inflammasome signalling pathway in gouty nephropathy was explored, and the mechanism of hyperuricaemia­induced renal damage. Adult male patients examined between July 2016 and June 2017 were selected as the patient cohort for the present study from the Affiliated Bao'an Hospital of Shenzhen, Southern Medical University (Shenzhen, China). These patients were divided into three groups of 30 patients each: Control, hyperuricaemia and gouty nephropathy groups. The expression levels of NLRP3, ASC and caspase­1 mRNA and protein were detected in peripheral blood mononuclear cells, and the plasma levels of IL­1ß and IL­18. Ultra­performance liquid chromatography coupled with quadrupole time­of­flight mass spectrometry was used to determine differential levels of metabolites between patients from different groups, in order to identify potential biomarkers. The expression of the NLRP3 inflammasome in peripheral blood mononuclear cells, and the levels of IL­1ß and IL­18 in the plasma were increased in the gouty nephropathy group compared with the control and hyperuricaemia groups. In addition, 46 metabolites were identified as potential plasma metabolic biomarkers that were able to distinguish gouty nephropathy from hyperuricaemia. The majority of these metabolites were involved in lipid metabolism, in particular the activity of phospholipase Α2 and ß­oxidation. These data indicated that lipid metabolism and the NLRP3 inflammasome serve a pivotal role in gouty nephropathy. In addition, the results suggested that lipids may mediate the progression of gouty nephropathy through the activity of phospholipase A2, ß­oxidation and activation of the NLRP3 inflammasome.

Monocytes, endoplasmic reticulum stress and metabolomics in dogs with multiple organ dysfunction syndrome treated by continuous venovenous hemodiafiltration.

OBJECTIVES: We tried to investigate the mechanism of continuous venovenous hemodiafiltration (CVVHDF) treatment in monocytes function, endoplasmic reticulum (ER) stress signaling pathways, metabolomics and histopathological changes of MODS dogs, and aimed to enhance the understanding of pathogenesis and provide novel avenues to potential therapies. METHODS: 12 male Beagle dogs were used to develop the stable models of MODS by using hemorrhagic shock plus resuscitation and endotoxemia, and assigned randomly to CVVHDF group (n=6) and MODS group (n=6). The dogs in CVVHDF group were given the typical CVVHDF treatment for 24h after the completion of endotoxin intravenous infusion, while those in MODS group were offered the i.v heparin instead only. Serum sample were collected at five time points, i.e. before anesthesia, 0h, 6h, 12h and 24h after the endotoxin injection (T1~T5, respectively), and meanwhile, the changes of mRNA, protein and human umbilical vein endothelial cells (HUVECs) apoptosis rates in JNK, CHOP and Caspase-12 were observed before and after interfered by RNA interference technology. RESULTS: The levels of DLA-DR, IL-1ß and IL-4 were higher than those in MODS group after the CVVHDF treatment, and the early and late apoptosis rates showed downward trend compared with MODS group. In vitro and prior to RNA interference (RNAi), the levels of mRNA and protein expression and HUVECs apoptosis rates of JNK, CHOP and Caspase-12 in CVVHDF group were significantly lower compared to T1 and MODS group respectively. However, the levels of mRNA and protein expression and HUVECs apoptosis rates were significantly lower than those before interfered by RNAi in both two groups. The serum levels of LPCs, ornithine, proline, methionine, etc. were down-regulated while carnitines, FFAs, PC, etc. were increased significantly in MODS (T4), and the serum levels of methionine, proline, arginine and lysine were increased while carnitine, LPCs, PCs, SMs and orthophosporic acid were decreased after 12 hours CVVHDF treatment (T4). CONCLUSION: CVVHDF treatment could reduce the apoptosis of the cells by enhancing the antigen presentation, improving the anti-inflammatory and proinflammatory imbalance and even correcting the metabolic disorder of amino acids and phospholipids.

[Cloning and bioinformatics analysis of rhoptry protein 11 of Toxoplasma gondii].

Total RNA was extracted from tachyzoites of RH strain of Toxoplasma gondii. The open reading frame of ROP11 gene was amplified by using a pair of specific primers designed according to the coding sequence of ROP11 gene (Accession No. DQ077905). The RT-PCR product was digested by restriction enzyme EcoR I and Not I, and then ligated into a pGEX-6P-2 vector. The recombinant plasmid was transferred into E. coli XL-Blue. The positive clones was selected by colony PCR, and confirmed by the double restriction enzyme digestion and sequencing. The RT-PCR product was 1,548 bp. The recombinant plasmid was confirmed by colony PCR and double restriction enzyme digestion. Sequencing results showed that the obtained ROP11 gene was 1 548 bp (Accession No. KC456639). There was a high sequence consistency (99%) between the obtained ROP11 gene sequence and the Toxoplasma ROP11 gene from GenBank. Bioinformatics analysis showed that the ROP11 protein (Mr 57,020) consisted of the signal peptide (amino acids 1-26), 12 conservative domains, a serine/threonine protein kinase catalytic domain (amino acids 170-511), and two potential N-glycosylation sites.