Effect of Lactoferrin on the Expression Profiles of Long Non-coding RNA during Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells.

Lactoferrin (LF) has demonstrated stimulation of osteogenic differentiation of mesenchymal stem cells (MSCs). Long non-coding RNAs (lncRNAs) participate in regulating the osteogenic differentiation processes. However, the impact of LF on lncRNA expression in MSC osteogenic differentiation is poorly understood. Our aim was to investigate the effects of LF on lncRNAs expression profiles, during osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs), by RNA sequencing. A total number of 1331 putative lncRNAs were identified in rBMSCs during osteogenic differentiation in the study. LF influenced the expression of 120 lncRNAs (differentially expressed lncRNAs [DELs], Fold change > 1.5 or < -1.5; p < 0.05) in rBMSCs on day 14 of osteogenic differentiation, consisted of 60 upregulated and 60 down-regulated. Furthermore, the potential functions of DELs were of prediction by searching their target cis- and trans-regulated protein-coding genes. The bioinformatic analysis of DELs target gene revealed that LF led to the disfunction of transforming growth factor beta stimulus (TGF-ß) and positive regulation of I-κappa B kinase/NF-κappa B signaling pathway, which may relate to osteogenic differentiation of rBMSCs. Our work is the first profiling of lncRNA in osteogenic differentiation of rBMSCs induced by LF, and provides valuable insights into the potential mechanisms for LF promoting osteogenic activity.

[Effect of pH on circular dichroism and Raman spectroscopy of secondary structure of beta-casein from Chinese human milk].

To obtain a structural basis for the beta-casein in Chinese human milk, structural transitions of the beta-casein in response to variation of pH were investigated using Raman and circular dichroism (CD) spectroscopy. Both methods indicated that the secondary structures of beta-casein in the solution were induced by the pH. Secondary structural analysis of beta-casein by CD spectroscopy yielded 0.5%-2% alpha-helical, 16%-18% beta-sheet, 30%-34% beta-turn and 49%-51% random coil contents. Another result was that as pH increases, these structures change. Several distinct transitions were observed by circular dichroism in alpha-helix at pH 8 and pH 10. Raman spectrum also showed random coil as the major secondary structure in native beta-casein, for the characteristic band of the beta-casein amide I was at 1662 cm(-1): Calculations from I850/I830 suggested that the tyrosine residues of beta-casein tended to "exposure". CD and Raman spectra both showed that at neutral and alkaline pH the beta-casein existed predominantly in random coil conformation, and the proportion of alpha-helix was higher at pH 8 than under other pH conditions. Over the range of pH studied, the sheet and turn areas remained relatively constant, and in the condition of pH 8, the content of alpha-helical was higher than in the other pH conditions.

[Comparison of the function and conformation of human ß-casein and bovine ß-casein by spectroscopic study].

ß-Casein was the main component of human milk casein, but the content of ß-casein in the bovine milk was less. The difference in ß-casein content of the two samples was one of the reasons why human milk is more digestible than bovine milk. Studying the differences of structure and function in human and bovine milk ß-casein can help us develop a new human milk simulated infant formula which will be more suitable for the infant gut. The UV spectrophotometer was used to study the solubility, sulfhydryl and emulsification of human milk ß-casein and bovine milk ß-casein, Fluorescence spectroscopy and the infrared spectroscopy were used to study the structural characteristics of human milk ß-casein and bovine milk ß-casein. The two samples shared a similar isoelectric point (pH 4.0~5.0), the solubility of human milk ß-casein (10.83%) was lower than which in bovine milk ß-casein (11.83%) near the pI, while it was higher when it deviated the pI. The emulsion ability (110~140 m2 · g(-1)) of human milk ß-casein was higher than that in bovine milk ß-casein (70~130 m2 · g(-1)) and surface sulfhydryl group (SH) of two kinds of milk protein were similar [(18.47±0.08) µmol · g(-1) and (18.67±0.17) µmol · g(-1)]. The total sulfhydryl group [(47.46±0.23) µmol · g(-1)] in bovine milk ß-casein was more than that in human milk ß-casein [(26.17±0.12) µmol · g(-1)]. Functional groups in two samples were similar and they both contained beta sheet, human milk ß-casein had less H-bond and internal hydrophobic than bovine milk ß-casein. The results showed that the two samples had similar functional groups, while human milk ß-casein had much less secondary structure such as α-helix and ß-sheet, a looser tertiary structure and a better interfacial activity.

Investigation of the formation mechanism and ß-carotene encapsulation stability of emulsion gels based on egg yolk granules and sodium alginate.

The formation mechanism of heat-induced egg yolk granules (EYGs)/sodium alginate (SA) emulsion gel was studied under pH 6.2 and 7.5. Particle size, water holding capacity, LF NMR, and protein solubility revealed that pH 7.5 increased the surface charge of EYGs and enhanced non-covalent interaction with SA, and hydrogen bonding dominated of the gel formation process. Microscopy and rheological analysis indicated that samples with 0.75% SA had the smallest particle size and highest G', with chain-like oil droplets. Excess SA (1%) led to depletion flocculation due to SA structural rearrangements around oil droplets caused by the increase in negatively charged, causing uneven network structure. The in vitro release property and storage stability of ß-carotene loaded in the EYGs/SA emulsion gel showed that SA increased storage stability and decreased bioaccessibility of ß-carotene with delayed digestion rate. These results provide a theoretical basis for the nutrient delivery system in gel foods.

UHPLC-Q-Exactive Orbitrap mass spectrometry reveals the lipidomics of bovine milk and yogurt.

Compared with milk intake, yogurt intake appears to cause a lower risk of cardiovascular disease (CVD) and type 2 diabetes (T2D). The molecular components responsible for the phenomenon are elusive. We hypothesized that the fermentation would change the lipid profile and fatty acid composition of milk. Untargeted analysis of lipids in milk and yogurt was performed using ultra-high-performance liquid chromatography (UHPLC) coupled with Q-Exactive Orbitrap mass spectrometry and gas chromatography (GC) with a flame ionization detector (FID). The results showed that yogurt had increased C4:0-C10:0 fatty acid, rumenic acid (cis-9 and trans-11-18:2), vaccenic acid (trans-11-18:1), linoleic acid (LA), and polyunsaturated fatty acid (PUFA) contents, and decreased triglyceride (TG), C16:0 and C18:0 fatty acids, and saturated fatty acid (SFA) contents compared with milk. These results advance the understanding of the difference between yogurt and milk regarding reduced risk of CVD and T2D.

A dipeptidyl peptidase IV inhibitory peptide relieves palmitic acid-induced endoplasmic reticulum stress in HepG2 cells independent of inhibiting dipeptidyl peptidase IV activity.

The peptide VLATSGPG (VLA) is known to inhibit dipeptidyl peptidase IV (DPP-IV), although its mechanism in relieving endoplasmic reticulum (ER) stress is unclear. In this study, we found that treating HepG2 cells with 1.0 mM VLA reduced DPP-IV activity by 73.7 ± 4.8% without changing the DPP-IV mRNA expression level. In addition, 1.0 and 0.5 mM VLA alleviated palmitic acid (PA)-induced cell death and intracellular calcium imbalances. The levels of apoptosis-related proteins (caspase-3, caspase-9, and CHOP) were reduced by VLA treatment, which was presumed to be related to ER stress. Further studies confirmed that VLA alleviated PA-induced morphological damage to the ER and reduced the levels of the ER stress marker proteins (BIP, p-PERK, and p-IRE1α). VLA alleviated PA-induced ER stress in HepG2 cells independent of DPP-IV enzymatic activity inhibition. These findings have implications for developing novel treatment approaches for liver diseases caused by ER stress.

Characterization of DPP-IV Inhibitory Peptides Using an In Vitro Cell Culture Model of the Intestine.

Here, we characterize the activities of two depeptidyl peptidase-IV (DPP-IV) inhibitory peptides, VLATSGPG and LDKVFER, using the Caco-2 monolayer model for the intestine. VLATSGPG and LDKVFR inhibited the DPP-IV in the cells via a mixed-type inhibition mode, with in situ IC50 values of 207.3 and 148.5 µM, respectively. Furthermore, VLATSGPG and LDKVFR were transported intact across the cells, with Papp values of 2.41 ± 0.16 and 4.23 ± 0.29 × 10-7 cm/s, respectively. Fragmented peptides were identified in the basolateral side of the membrane. Two of these, GPG and VLA, exhibited high inhibitory activities of 83.6 ± 3.3 and 58.5 ± 2.5%, respectively, at 100 µM concentration. Although 3 mM VLATSGPG and LDKVFR were transported across the epithelium in a concentration-dependent manner, their transport did not damage the tight junction proteins, ZO-1 and occludin. This study demonstrates that the two peptides potentially regulate DPP-IV activity in the intestine.

Exopolysaccharide Produced by Lactobacillus casei Promotes the Differentiation of CD4+ T Cells into Th17 Cells in BALB/c Mouse Peyer's Patches in Vivo and in Vitro.

The immunomodulatory activity of a few Lactobacillus exopolysaccharides (EPS) has been reported. However, whether Lactobacillus EPS can promote the differentiation of CD4 T lymphocytes (CD4+T) cells into T-helper 17 cells (Th17 cells) in the Peyer's Patches (PPs) of mice has not been addressed. In this study, we found the molecular weight (Mw) of the purified EPS from L. casei ranged from 2.7 × 106 Da to 1.7 × 107 Da, and the average Mw was approximately 8.4 × 106 Da. In healthy BALB/c mice, EPS elevated the numbers of Th17 cells and levels of Th17-related cytokines. In vitro, EPS induced BMDCs to stimulate the differentiation of CD4+T cells of PPs into Th17 cells and the related cytokine secretions. Results suggest that L. casei EPS can effectively induce and promote the differentiation of CD4+T cells of PPs into Th17 cells in healthy mice and has the potential ability to improve intestinal mucosa immunity.

Urinary Metabolic Profiling via LC-MS/MS Reveals Impact of Bovine Lactoferrin on Bone Formation in Growing SD Rats.

Lactoferrin (LF) exerts a promoting bone health function. The effects of LF on bone formation at the metabolic level have been less explored. Urinary metabolic profiling of growing Sprague-Dawley (SD) rats LF-supplemented (1000 mg/kg bw) for four weeks were explored by Liquid chromatography-tandem mass spectrometry (LC-MS/MS). The serum markers of bone formation and bone resorption, the bone mass, and the osteogenesis markers of femur were measured by an enzyme-linked immunosorbent assay, micro-computerized tomography, and immunohistochemistry, respectively. Compared with the control, LF supplementation improved bone formation (p < 0.05), reduced bone resorption (p < 0.05), enhanced femoral bone mineral density and microarchitecture (p < 0.05), and upregulated osteocalcin, osterix, and Runx-2 expression (p < 0.05) of femur. LF upregulated 69 urinary metabolites. KEGG and pathway enrichment analyses of those urinary metabolites, and the Person's correlation analyses among those urinary metabolites and bone status revealed that LF impacted on bone formation via regulatory comprehensive pathways including taurine and hypotaurine metabolism, arginine and proline metabolism, cyanoamino acid metabolism, nitrogen metabolism, nicotinate and nicotinamide metabolism, and fatty acid biosynthesis. The present study indicated the metabolomics is a useful and practical tool to elucidate the mechanisms by which LF augments bone mass formation in growing animals.

[Separation and identification of beta-casein from Chinese human milk by ion exchange chromatography-matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry].

The selective precipitation of whole casein from skimmed milk was achieved by the addition of calcium salt under acidic pH. The effects of pH, centrifugal force and final concentration of CaCl2 on the separation of casein were studied by measuring the purity of final products using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that casein with the highest purity could be obtained with the pH of 4.3, the centrifugal force of 10 400 g and the final concentration of CaCl2 of 60 mmol/L. The casein was processed with DEAE anion exchange chromatography and three peaks were obtained. Then the third peak (peak III) was identified with Western-Blot method and matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). The identification of Western-Blot showed that peak III can combine with the specificity of human milk beta-casein antibody, and it is proved to be human milk beta-casein. The fingerprints of peak III were nalyzed by Mascot searching, and the sequence coverage was 50%, further supporting it is human milk beta-casein. In conclusion, an effective method to obtain human milk beta-casein from milk samples through DEAE anion exchange chromatography was established, and it is suitable for the proteomics research requirements of the beta-casein from human milk.