Yak milk-derived exosomes alleviate lipopolysaccharide-induced intestinal inflammation by inhibiting PI3K/AKT/C3 pathway activation.

Intestinal epithelial cells (IEC) are important parts of the mucosal barrier, whose function can be impaired upon various injury factors such as lipopolysaccharide. Although food-derived exosomes are preventable against intestinal barrier injuries, there have been few studies on the effect of yak milk-derived exosomes and the underlying mechanism that remains poorly understood. This study aimed to characterize the effect of exosomal proteins derived from yak and cow milk on the barrier function of IEC-6 treated with lipopolysaccharide and the relevant mechanism involved. Proteomics study revealed 392 differentially expressed proteins, with 58 higher expressed and 334 lower expressed in yak milk-derived exosomes than those in cow exosomes. Additionally, the top 20 proteins with a relatively consistent higher expression in yak milk exosomes than cow milk exosomes were identified. Protein CD46 was found to be a regulator for alleviating inflammatory injury of IEC-6. In vitro assay of the role of yak milk exosomes on survival of IEC-6 in inflammation by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay confirmed the effectiveness of yak milk exosomes to increase IEC-6 survival up to 18% for 12 h compared with cow milk exosomes (up to 12%), indicating a therapeutic effect of yak milk exosomes in the prevention of intestinal inflammation. Furthermore, yak and cow milk exosomes were shown to activate the PI3K/AKT/C3 signaling pathway, thus promoting IEC-6 survival. Our findings demonstrated an important relationship between yak and cow milk exosomes and intestinal inflammation, facilitating further understanding of the mechanisms of inflammation-driven epithelial homeostasis. Interestingly, compared with cow milk exosomes, yak milk exosomes activated the PI3K/AKT/C3 signaling pathway more to lower the incidence and severity of intestine inflammation, which might represent a potential innovative therapeutic option for intestinal inflammation.

Yak milk-derived exosomal microRNAs regulate intestinal epithelial cells on proliferation in hypoxic environment.

Intestinal epithelial cells (IEC) act as an important intestinal barrier whose function can be impaired upon induction by hypoxia. Although intestinal barrier injuries are preventable by milk-derived exosomal microRNAs (miRNAs), the underlying mechanism remains poorly understood. This study aimed to characterize the effect of yak and cow milk-derived exosomal miRNA on the barrier function of IEC-6 under hypoxic conditions, and explore the mechanism of yak milk exosomal miRNA to relieve the hypoxia stress. First, by Illumina HiSeq 2500 (Illumina Inc., San Diego, CA) sequencing, the miRNA expression was systematically screened, and differential expression of 130 miRNAs was identified with 51 being upregulated and 79 downregulated in yak and cow milk-derived exosomes. Furthermore, the top 20 miRNAs that had a relatively consistent high expression in yak milk exosome were identified, and bta-miR-34a was found to be an effective regulator for alleviating hypoxic injury of IEC-6. In vitro assay of the role of bta-miR-34a on survival of IEC-6 in hypoxia by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) confirmed its effectiveness to significantly increase IEC-6 survival up to 13% for 12 h, and up to 9.5% for 24 h. Investigation on the regulatory relationship between bta-miRNA-34a and the hypoxia-inducible factor/apoptosis signaling pathway provided insights into the possible mechanisms by which bta-miR-34a activated the hypoxia-inducible factor and apoptosis signaling pathway, thus promoting IEC-6 survival. The results of this study suggest an important relationship between miRNA expression and intestine barrier integrity, which facilitated further understanding of the physiological function of yak and cow milk exosomal miRNAs, as well as mechanisms of hypoxia-driven epithelial homeostasis.

Yak-milk-derived exosomes promote proliferation of intestinal epithelial cells in an hypoxic environment.

Intestinal epithelial cells (IEC) are an important part of the intestinal barrier. Barrier function was disrupted under hypoxia, but milk-derived exosomes can regulate the intestinal barrier function. However, the mechanisms underlying the association between yak milk exosomes and hypoxia in IEC remain poorly understood. In this follow-up study, we proposed an effective optimization method for purifying yak-milk-derived exosomes. The Western blot analyses indicated that the expression of the proteins of the endosomal sorting complexes required for transport (TSG101), proteins of the tetraspanin family (CD63), and heat shock protein 70 (Hsp-70) proteins from yak-milk-derived exosomes were significantly higher than those in cow-milk-derived exosomes. Flow cytometry analysis showed that yak milk had 3.7 times the number of exosomes compared with cow milk. Moreover, we explored whether yak milk exosomes could facilitate intestinal cell survival under hypoxic conditions in vitro. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide results showed that yak-milk-derived exosomes significantly increased survival of IEC-6 cells with rates of up to 29% for cells incubated in hypoxic conditions for 12 h, compared with those of cow-milk-derived exosomes posttreatment (rates of up to 22% for cells incubated in hypoxic conditions for 12 h). Confocal microscopy revealed that the IEC-6 cells uptake more yak-milk-derived exosomes than cow milk in hypoxic conditions. Furthermore, the Western blot analyses indicated that yak-milk-derived exosomes significantly promote oxygen-sensitive prolyl hydroxylase (PHD)-1 expression and decrease the expression of hypoxia-inducible factor-α and its downstream target vascular endothelial growth factor (VEGF) in the IEC-6 cells. Further, yak-milk-derived exosomes significantly inhibited p53 levels. In conclusion, our findings demonstrate that yak-milk-derived exosomes more effectively activate the hypoxia-inducible factor signaling pathway, thus promoting IEC-6 cell survival, which may result in higher hypoxia tolerance than cow-milk-derived exosomes.

d-Glucose and amino acid deficiency inhibits casein synthesis through JAK2/STAT5 and AMPK/mTOR signaling pathways in mammary epithelial cells of dairy cows.

Amino acids and energy deficiency lead to lower milk protein content in dairy cows. However, the known mechanisms involved in this process do not adequately explain the variability of milk protein concentration in the mammary gland. We hypothesized that a deficiency in d-glucose (d-Glc) or AA would inhibit casein synthesis by regulating signaling pathways in mammary epithelial cells. Cow mammary epithelial cells (CMEC) were subjected to combinations of 1 of 3 concentrations of d-Glc (0, 2.50, or 17.5 mM) and 1 of 3 concentrations of AA (0, 1.03, or 7.20 mM). The effect of each mixture on cell cycle stage was assessed by flow cytometry. The expression levels of ß-casein and κ-casein (encoded by CSN2 and CSN3) were measured by quantitative real-time PCR and Western blotting. Phosphorylation of Janus kinase 2 (Jak2), signal transducer and activator of transcription 5a (Stat5a), AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), and eukaryotic factor 4E-binding protein 1 (4EBP1) were analyzed by Western blotting. The percentages of cells in the DNA postsynthetic (G2) and DNA synthesis (S) phases would decrease, with the level of d-Glc or AA declining individually, but no interaction was observed between the d-Glc and AA effects. The CSN2 and CSN3 mRNA and protein were downregulated when d-Glc or AA decreased individually from 17.5 to 2.50 mM or from 7.20 to 1.03 mM, but d-Glc deficiency had a greater effect according to the regression analysis. The phosphorylation ratio of Jak2 (Tyr1007/1008), Stat5a (Tyr694), mTOR (Ser2448), S6K1 (Thr389), and 4EBP1 (Thr37) was downregulated with the level of d-Glc or AA decline, whereas the phosphorylation ratio of AMPK (Thr183/172) was upregulated. And the change of d-Glc level had a more marked effect than AA in regulating the activity of these signaling protein above according to the regression analysis. Thus, d-Glc or AA deficiency likely reduced casein transcription via inhibition of the Jak2/Stat5 pathway, and reduced translation via suppression of the mTOR pathway by activation of AMPK, but d-Glc deficiency had a more marked effect. These indicated that deficiency of AA, and especially Glc, suppressed proliferation of CMEC and casein gene and protein expression, associated with inhibition of JAK2/STAT5 and AMPK/mTOR signaling pathways.

Combination of histidine, lysine, methionine, and leucine promotes ß-casein synthesis via the mechanistic target of rapamycin signaling pathway in bovine mammary epithelial cells.

The ratio of different AA in the diets of cows is vital to improve milk protein yield. ß-Casein is one of the important milk proteins with high nutritive value. However, the suitable ratio of essential amino acids (EAA) for the expression of ß-casein in the immortalized bovine mammary epithelial cell line is not fully characterized. This study employed response surface methodology to determine the optimal ratio of His, Lys, Met, and Leu on ß-casein expression level in vitro and clarified the effect of the 4 EAA on ß-casein via the mechanistic target of rapamycin (mTOR) signaling pathway. A central composite design containing 5 axial points per EAA and 28 combinations of the 4 EAA was used in our study. The results of response surface methodology and the changes of the mTOR-related signaling proteins were determined by western blot. The results showed that ß-casein level was significantly affected by all 4 EAA (R2 = 0.71). The optimum conditions for ß-casein expression are found to be 5.47 mM of His, 7.48 mM of Lys, 1.17 mM of Met, and 8.21 mM of Leu (His:Lys:Met:Leu = 5:6:1:7) in the designed scope of concentration. The interaction of Leu and Met significantly affected ß-casein expression (P < 0.01). The phosphorylation of mTOR (Ser2481), regulatory associated protein of target of rapamycin (Ser792), ribosomal protein S6 kinase 1 (Thr389), ribosomal protein S6 (Ser235/236), and eukaryotic elongation factor 2 (Thr56) was increased with the supplementation of either single EAA or an optimal combination of EAA. However, the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (Thr37) was decreased with the addition of Lys, Met, or Leu alone. Furthermore, the phosphorylation (P) of eIF2α (Ser51) was decreased when Met was supplemented alone. Under the optimal mixture of 4 EAA, the phosphorylation of mechanistic target of rapamycin complex 1 signaling proteins was significantly greater than the single EAA supplementations and the expression of ß-casein was 98% as high as the positive control (i.e., medium with all AA). A similar trend was found with P-ribosomal protein S6 kinase 1 and P-ribosomal protein S6. In conclusion, the extracellular concentrations of His, Lys, Met, and Leu at a ratio of 5:6:1:7 maximized ß-casein expression in the immortalized bovine mammary epithelial cell line may occur via activation of the mechanistic target of rapamycin complex 1 signaling pathway.

Opportunistic posttransplantation virus infections in renal transplant recipients.

BACKGROUND: Opportunistic virus infection is one of the most common complications in renal transplant (RT) recipients. Cytomegalovirus (CMV) and BK virus (BKV) are important pathogens and each of these infections affects the other. In contrast, there is only limited information on JC virus (JCV) infection and its relation to CMV infection in RT recipients. This prospective study investigated the rates of JCV and CMV infections and their risk factors and correlations. METHODS: We studied 52 RT recipients. JCV and CMV were detected using nested qualitative polymerase chain reaction assays of urine. The clinical characteristics of JCV and CMV infection were compared and risk factors analyzed with the use of binary logistic regression. RESULTS: JCV and CMV were detected in 40.4% and 34.6% of the RT recipients, respectively. Cyclosporine (CsA) was a risk factor for both JCV and CMV infection (odds ratio [OR] 7.187; P=.002; OR 4.182; P=.021); CMV infection was a risk factor for JCV infection (OR 3.900; P=.039). CONCLUSIONS: JCV and CMV infections are common in RT recipients. CsA is a risk factor for both JCV and CMV infection. JCV infection is related to CMV infection.

Monitoring of human cytomegalovirus glycoprotein B genotypes using real-time quantitative PCR in immunocompromised Chinese patients.

Based on sequence variation in the N-terminus of glycoprotein B (gB), human cytomegalovirus (HCMV) can be classified into four gBn genotypes, and these genotypes are associated with different clinical outcomes. The distribution of gBn genotypes and the level of gBn DNA load were examined in immunocompromised Chinese patients using real-time quantitative PCR. In addition, the PCR and pp65 antigenemia results were compared. In 1480 specimens, 81.4% were antigen-positive, 12.6% were PCR-positive. The gB genotype distribution was as follows among PCR-positive samples: gBn1, 63.1%; gBn2, 13.4%; gBn3, 8.6%; gBn4, not detected; mixed genotypes, 14.9% (gBn1 and gBn3, 14.4%; gBn2 and gBn3, 0.5%). The gBn3 and gBn1 genotypes had the highest and lowest copy numbers, respectively (p<0.05). The quantity of gBn DNA found in PCR-positive, pp65-negative samples was significantly lower than that found in PCR-positive, pp65-positive samples (p<0.05). The PCR and antigenemia results did not differ among bone marrow transplant patients, solid organ transplant patients, and immunocompromised patients without transplantation (p>0.05). HCMV gBn genotyping using real-time quantitative PCR was established successfully, and the distribution of gBn genotypes in immunocompromised Chinese patients was investigated. This method may help to understand better the relationship between gBn genotype and clinical outcome and aid in clinical detection.

Monitoring of human cytomegalovirus infection in bone marrow and liver transplant recipients by antigenaemia assay and enzyme-linked immunosorbent assay.

Human cytomegalovirus (HCMV) infection is a common complication in transplant recipients. Sensitive, specific and timely diagnostic tests for the detection of HCMV infection remain essential for successful therapy. The results of three tests to detect HCMV in bone marrow and liver transplant recipients were compared: a pp65 antigenaemia assay, an immediate-early (IE) antigenaemia assay and an anti-HCMV immunoglobulin M (IgM) antibody enzyme-linked immunosorbent assay (ELISA). Of 1344 samples, 911 (67.8%) and 917 (68.2%) samples were positive for pp65 and IE, respectively. The coincidence level was 85.1%. There was no statistical difference after transplantation to the first positive detection of HCMV (mean first checkout time) between the pp65 and IE antigenaemia assays. Moreover, the levels of HCMV detected by the pp65 and IE antigenaemia assays were significantly correlated. The HCMV-positivity rate as detected by the anti-HCMV IgM ELISA was 11.1%, which was significantly different from the IE and pp65 antigenaemia assays. We suggest that the IE antigenaemia assay could replace the pp65 antigenaemia assay for monitoring active HCMV infection and early detection of HCMV infection.