Effects of High-Intensity Ultrasound Pretreatment on Structure, Properties, and Enzymolysis of Walnut Protein Isolate.

The purpose of this paper was to investigate the effect of high-intensity ultrasonication (HIU) pretreatment before enzymolysis on structural conformations of walnut protein isolate (WPI) and antioxidant activity of its hydrolysates. Aqueous WPI suspensions were subjected to ultrasonic processing at different power levels (600-2000 W) and times (5-30 min), and then changes in the particle size, zeta (ζ) potential, and structure of WPI were investigated, and antioxidant activity of its hydrolysates was determined. The particle size of the particles of aqueous WPI suspensions was decreased after ultrasound, indicating that sonication destroyed protein aggregates. The ζ-potential values of a protein solution significantly changed after sonication, demonstrating that the original dense structure of the protein was destroyed. Fourier transform infrared spectroscopy indicated a change in the secondary structure of WPI after sonication, with a decrease in ß-turn and an increase in α-helix, ß-sheet, and random coil content. Two absorption peaks of WPI were generated, and the fluorescence emission intensity of the proteins decreased after ultrasonic treatment, indicating that the changes in protein tertiary structure occurred. Moreover, the degree of hydrolysis and the antioxidant activity of the WPI hydrolysates increased after sonication. These results suggest that HIU pretreatment is a potential tool for improving the functional properties of walnut proteins.

Serum myeloid-related protein 8/14 is a potential predictor of diabetic kidney disease.

BACKGROUND: Myeloid-related protein 8/14 (MRP8/14) is secreted by macrophages and formed by MRP8 and MRP14, which is closely related to vascular inflammation. Chronic vascular inflammation plays a significant role in the development and progression of diabetic kidney disease (DKD). This study aims to investigate the relationship between MRP8/14 and DKD. METHODS: A total of 80 individuals with type 2 diabetes were divided into four groups, according to the baseline urinary albumin/creatinine ratio (ACR) levels Serum concentrations of MRP8/14 were measured by ELISA. The clinical variables were obtained through physical examination, illness history, or laboratory evidence. RESULTS: As DKD worsened, the level of serum MRP8/14 increased gradually, and MRP8/14 has a significantly positive correlation with ACR (r = 0.349, P = 0.002), body mass index (BMI) (r = 0.288, P = 0.009), serum creatinine (Cre) (r = 0.392, P < 0.001), blood urine nitrogen (BUN) (r = 0.333, P = 0.003), systolic blood pressure (SBP) (r = 0.301, P = 0.007), and a negative correlation with the estimated glomerular filtration rate (eGFR) (r = -0.478, P < 0.001). Logistic regression analysis showed that age, Cre, eGFR, ACR, and MRP8/14 were associated with the progression of DKD (P < 0.05). CONCLUSIONS: The serum MRP8/14 is correlated significantly with the progression of DKD, suggesting that MRP8/14 may be an independent predictor of the progression of DKD.

The Effect of MicroRNA-331-3p on Preadipocytes Proliferation and Differentiation and Fatty Acid Accumulation in Laiwu Pigs.

OBJECTIVE: The proliferation and differentiation of preadipocytes are regulated by microRNAs (miRNAs), hormones, and other factors. This study aimed to investigate the effects of miR-331-3p on the proliferation and differentiation of preadipocytes in addition to fatty acid metabolism. METHODS: Preadipocytes were transfected with miR-331-3p mimics, miR-NC, or miR-331-3p inhibitor to explore its effect on cell proliferation and fatty acid accumulation. Furthermore, preadipocytes were transfected with pre-miR-331-3p, pcDNA3.1(+), or miR-331-3p inhibitor to explore its effect on differentiation. RESULTS: It was observed that miR-331-3p could inhibit preadipocytes proliferation. Furthermore, miR-331-3p was highly expressed during cellular differentiation and appeared to promote the process. In addition, dual fluorescein analysis showed that dihydrolipoamide S-succinyltransferase (DLST) is a target gene of miR-331-3p, and overexpression of miR-331-3p could regulate the metabolism of fatty acids in the citrate pyruvate cycle by targeting DLST expression. CONCLUSION: In summary, these findings indicated that miR-331-3p exerts contrasting effects on the processes of fat deposition.

[Construction of miR-331-3p overexpression vector and its effect on cell proliferation].

To investigate the effect of miR-331-3p on the proliferation of porcine renal epithelial cells (PK15) and its mechanism, the pcDNA 3.1(+) overexpression vector of miRNA-331-3p (pcDNA 3.1(+)-miR-331-3p) was constructed. PK15 cells were divided into four groups, including experimental group, experimental control group, inhibitor group and inhibitor control group. Experimental group and experimental control group were transfected with pcDNA 3.1(+)-miR-331-3p and pcDNA 3.1(+), respectively. Inhibitor group and inhibitor control group were transfected with miR-331-3p inhibitor and miR-331-3p negative control (miR-331-3p NC), respectively. Above all, CCK-8 reagent was used to plot the cell proliferation curve and Propidium (PI) staining was used to detect the proportion of cell stages. Secondly, its expression change were detected by quantitative real-time PCR that included the growth inhibitory protein family member 5 (ING5), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 3 (CDK3), cyclin-dependent kinase 4 (CDK4), Cyclin B and cyclin-dependent kinase inhibitor 1A (CDKN1A). The results showed that the expression of miRNA-331-3p was significantly increased in the experimental group. The cell proliferation curve showed that the number of cells in experimental group was significantly higher than that in experimental control group or inhibitor control group at 48 h and 72 h (P<0.05). Simultaneously, Inhibitor group was significantly lower than experimental control group or inhibitor control group in the number of cells at 48 h and 72 h (P<0.05), but there was no significant difference between the experimental group and the control group. Compared with the experimental control group, the proportion of cells of experimental group in G0/G1 phase decreased, the proportion of S phase and G2/M phase increased, and the inhibitor control group showed the opposite trend. Simultaneously, the expression levels of CDK2, CDK3, CDK4 and Cyclin B genes in the experimental group were significantly increased, while ING5 and CDKN1A genes inhibiting proliferation showed a significant downward trend. These results demonstrate that the miR-331-3p overexpression vector was successfully constructed, and miR-331-3p has the ability to promote the proliferation of PK15 cells. The study lays a solid foundation for further research for its role in pig growth and development.

Study on quantitative expression of PPARγ and ADRP in muscle and its association with intramuscular fat deposition of pig.

BACKGROUND: Intramuscular fat (intramuscular fat, IMF) is one of the important traits of pork quality. How to reasonably improve the intramuscular fat content is the most focus researchers. Some possible regulation of intramuscular fat deposition of candidate genes to cause the attention of people. The objective of this study was to elucidate the relationship between peroxisome proliferator-activated receptor γ (PPARγ) and adipose differentiation-related protein (ADRP) mRNA expression and intramuscular fat (IMF) deposition in the muscle tissue of three breeds of pig: Laiwu (LW), Lulai Black (LL), and Large White (LY). RESULTS: qPCR analysis of the PPARγ and ADRP genes in the three breeds of pig revealed PPARγ and ADRP mRNA expression profiles of LW > LL > LY and LL > LW > LY, respectively. PPARγ mRNA expression was significantly and positively correlated with IMF deposition (p < 0.05). There were significant correlations between PPARγ and ADRP mRNA expression levels (p < 0.01). CONCLUSIONS: These results suggest correlations between PPARγ and ADRP in fat deposition and regulation in pigs, PPARγ gene may be a main effector of IMF content and play an important role during adipocyte differentiation in pigs, thereby providing new information to further elucidate molecular mechanisms associated with intramuscular fat deposition in Laiwu pigs and provides new data for further molecular studies of mechanisms underlying intramuscular fat deposition in human obesity. The continued elucidation of specific genetic mechanisms between PPARγ and ADRP warrants further studies.

[Cloning, expression and functional analysis of CuZnSOD gene in swine.].

In order to understand the structure and function of CuZnSOD gene, reveal the effect of the anti-oxidant in swine, and find the molecule marker correlated with meat traits, the cDNA of CuZnSOD gene was cloned and sequenced from muscle of Laiwu black swine by RACE (rapid amplification of cDNA end) techniques. The structure and function of CuZnSOD were analyzed by bioinformatics, and the gene expression profile in different tissues was examined by real-time PCR. The results showed that the full sequence of CuZnSOD cDNA is 658 bp (GU944822), containing 76 bp sequence of 5' UTR and 120 bp sequence of 3' UTR, and coding region (CDS, 462 bp) encodes 153 amino acids. The isoelectric point (pI) of the protein is 6.03, and the molecular weight is 15.9 kDa. There were one O-glycosylation site at the third amino acid and one N-glycosylation site at the eighty-fourth amino acid. The percentage of alpha helix was 1.31%. The alignment similarities of the CDS sequence of swine CuZnSOD with those of cattle, human, rat, and mouse were 87.74%, 87.66%, 83.44%, and 83.23%, and the similarities of amino acid sequence were 90.26%, 94.12%, 92.21%, and 91.50%, respectively. CuZnSOD possesses the typical metal binding ligands (GFHVHQFGDNT). The phylogenic tree based on CuZnSOD protein sequence detected the closest relationship between swine and cattle. CuZnSOD mRNA is a broad-spectrum expression gene, which was detected in brain, heart, spleen, liver, kidney, lung, large intestine, small intestine, spinal cord, muscle, backfat, and stomach. In particular, high expression levels of CuZnSOD mRNA were detected in kidney, small intestine and lung, but low expressions were observed in heart and muscle tissues.

[CDS cloning and relationship between intramuscular fat content and mRNA expression of PID1 gene in pig].

To explore the relationship between the expression of PID1 gene and fat deposition, we cloned CDS of PID1 from porcine fat and muscle tissues by RT-PCR using degenerate primers, and investigated expression of this gene in various tissues (i.e., liver, backfat, and muscle tissues) of different breeds (i.e., Yorkshire, Laiwu, and Lulai Black) by real-time fluorescence quantitative PCR. The results showed that 654 bp CDS of porcine PID1 was obtained by sequencing and was 93.88%, 66.94% and 88.07% identical to those of the human, rat, and Bos taurus, respectively. The expression of PID1 mRNA in various tissues and breeds, on the whole, tended to be liver > fat > muscle and Laiwu > Lulai Black > Yorkshire, respectively. For different breeds, PID1 mRNA abundance in liver had significant difference (P < 0.05), but had no significant differences in fat and muscle tissues between Laiwu and Lulai Black (P > 0.05). For the three groups of Laiwu pigs with high (LWH), intermediate (LWI), and low IMF content (LWL), PID1 mRNA level was higher in liver tissue of LWH than that of LWL significantly (P < 0.05), and was higher in muscle tissue of LWH than that of LWI and LWL significantly (P < 0.05). PID1 mRNA abundance was not correlated with IMF in these three tissues of Laiwu breed, but it was positively correlated with IMF in the tissues of these three breeds (P < 0.05). These results implied that the expression of PID1 may be related to fat deposition.