The BLV-miRNAs pathway of BLV inhibits the expression of Lactoferrin, Lactoperoxidase, Alpha-lactalbumin and Beta-lactoglobulin proteins.

Bovine leukemia virus (BLV) is a widespread virus that decreases milk production and quality in dairy cows. As crucial components of BLV, BLV-encoded microRNAs (BLV-miRNAs) affect BLV replication and may impact the synthesis of Lactoferrin (LTF), Lactoperoxidase (LPO), Alpha-lactalbumin (alpha-LA), and Beta-lactoglobulin (beta-LG). In this study, we investigated the targeting relationship between BLV-miRNAs and LTF, LPO, alpha-LA, and beta-LG in cow's milk. Additionally, we investigated the possible mechanisms by which BLV reduces milk quality. The results showed that cow's milk had significantly lower levels of LTF, LPO, and alpha-LA proteins in BLV-positive cows than in BLV-negative cows. BLV-△miRNAs (miRNA-deleted BLV) enhanced the reduction of LPO, alpha-LA, and beta-LG protein levels caused by BLV infection. Multiple BLV-miRNAs have binding sites with LTF and LPO mRNA; however, only BLV-miR-B1-5 P has a targeting relationship with LPO mRNA. The results revealed that BLV-miR-B1-5 P inhibits LPO protein expression by targeting LPO mRNA. However, BLV does not directly regulate the expression of LTF, alpha-LA, or beta-LG proteins through BLV-miRNAs.

The effect of bta-miR-1296 on the proliferation and extracellular matrix synthesis of bovine mammary fibroblasts.

Mammary fibrosis in dairy cows is a chronic condition caused by mastitis, and can lead to serious culling of dairy cows resulting in huge economic losses in the dairy industry. MicroRNAs (miRNAs) exert an important role in regulating mammary gland health in dairy cows. This study investigated whether exosomal miRNAs in mammary epithelial cells can regulate the proliferation of bovine mammary fibroblasts (BMFBs) in mastitis. Liposome transfection technology was used to construct a cellular model of the overexpression and inhibition of miRNAs. The STarMir software, dual luciferase reporter gene test, real-time quantitative PCR (qRT-PCR), a Cell Counting Kit-8 (CCK-8), and a Western Blot and plate clone formation test were used to investigate the mechanism by which bta-miR-1296 regulates the proliferation of BMFBs. Target gene prediction results revealed that glutamate-ammonia ligase was a direct target gene by which bta-miR-1296 regulates cell proliferation. It was found that bta-miR-1296 significantly inhibited the proliferation of BMFBs. After BMFBs were transfected with a bta-miR-1296 mimic, mRNA expression in the extracellular matrix (ECM), α-smooth muscle actin (α-SMA), collagen type I alpha 1 chain (COL1α1) and collagen type III alpha 1 chain (COL3α1), and various cell growth factors (basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-ß1 (TGF-ß1)) were down-regulated, and the expressions of α-SMA, COL1α1, COL3α1, phospho-extracellular regulated protein kinases, phospho-protein kinase B, TGF-ß1, and phospho-Smad family member3 proteins were inhibited. In conclusion, bta-miR-1296 can inhibit the proliferation of BMFBs and the synthesis of ECM in BMFBs, thus affecting the occurrence and development of mammary fibrosis in dairy cows and laying the foundation for further studies to clarify the regulatory mechanism of mammary fibrosis.

Enhancement of recombinant human interleukin-22 production by fusing with human serum albumin and supplementing N-acetylcysteine in Pichia Pastoris.

Interleukin-22 (IL-22) plays an important role in the treatment of organ failure, which can induce anti-apoptotic and proliferative signaling pathways; Nevertheless, the practical utilization of IL-22 is hindered by the restricted efficacy of its production. Pichia pastoris presents a viable platform for both industrial and pharmaceutical applications. In this study, we successfully generated a fusion protein consisting of truncated human serum albumin and human IL-22 (HSA-hIL-22) using P. pastoris, and examined the impact of antioxidants on HSA-hIL-22 production. We have achieved the production of HSA-hIL-22 in the culture medium at a yield of approximately 2.25 mg/ml. Moreover, 0-40 mM ascorbic acid supplementation did not significantly affect HSA-hIL-22 production or the growth rate of the recombinant strain. However, 80 mM ascorbic acid treatment had a detrimental effect on the expression of HSA-hIL-22. In addition, 5-10 mM N-acetyl-l-cysteine (NAC) resulted in an increase of HSA-hIL-22 production, accompanied by a reduction in the growth rate of the recombinant strain. Conversely, 20-80 mM NAC supplementation inhibited the growth of the recombinant strains and reduced intact HSA-hIL-22 production. However, neither NAC nor ascorbic acid exhibited any effect on superoxide dismutase (SOD) and malondialdehyde (MDA) levels, except that NAC increased GSH content. Furthermore, our findings indicate that recombinant HSA-hIL-22, which demonstrated the ability to stimulate the proliferation of HepG2 cells, possesses bioactivity. In addition, NAC did not affect HSA-hIL-22 bioactivity. In conclusion, our study demonstrates that NAC supplementation can enhance the secretion of functional HSA-hIL-22 proteins produced in P. pastoris without compromising their activity.

Effect of N-acetyl-l-cysteine on Cell Phenotype and Autophagy in Pichia pastoris Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein.

Pichia pastoris is widely used for the production of recombinant proteins, but the low secretion efficiency hinders its wide application in biopharmaceuticals. Our previous study had shown that N-acetyl-l-cysteine (NAC) promotes human serum albumin and porcine follicle-stimulating hormone fusion protein (HSA-pFSHß) secretion by increasing intracellular GSH levels, but the downstream impact mechanism is not clear. In this study, we investigated the roles of autophagy as well as cell phenotype in NAC promoting HSA-pFSHß secretion. Our results showed that NAC slowed down the cell growth rate, and its effects were unaffected by Congo Red and Calcofluor White. Moreover, NAC affected cell wall composition by increasing chitin content and decreasing ß-1,3-glucan content. In addition, the expressions of vesicular pathway and autophagy-related genes were significantly decreased after NAC treatment. Further studies revealed that autophagy, especially the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy and pexophagy, was significantly increased with time, and NAC has a promoting effect on autophagy, especially at 48 h and 72 h of NAC treatment. However, the disruption of mitophagy receptor Atg32, but not pexophagy receptor Atg30, inhibited HSA-pFSHß production, and neither of them inhibited the NAC-promoted effect of HSA-pFSHß. In conclusion, vesicular transport, autophagy and cell wall are all involved in the NAC-promoted HSA-pFSHß secretion and that disruption of the autophagy receptor alone does not inhibit the effect of NAC.

Enhancement of HSA-pFSHß production by disrupting YPS1 and supplementing N-acetyl-L-cysteine in Pichia pastoris.

Introduction: Pichia pastoris is widely used for the production of recombinant proteins, but the low production efficiency hinders its wide application in biopharmaceuticals. Moreover, many biopharmaceutical-like proteins are accompanied by degradation during secretory expression in P. pastoris. Objective: In this study, we used human serum albumin and porcine follicle-stimulating hormone ß (HSA-pFSHß) fusion protein as a model protein to investigate whether YPS1 and YPT7 gene disruption and N-acetyl-L-cysteine (NAC) supplementation have synergistic effects to inhibit the degradation of recombinant proteins. Results and discussion: Our results showed that YPS1 gene disruption reduced the degradation of intact HSA-pFSHß and increased the yield of intact protein in the culture medium and cells without affecting the integrity of the cell wall. Moreover, the beneficial effects of YPS1 gene disruption were associated with the upregulation of the MAPK signaling pathway and maintenance of redox homeostasis. YPS1 gene disruption and NAC supplementation had synergistic effects on HSA-pFSHß production. In addition, disruption of vacuolar morphology by YPT7 gene disruption or NH4Cl treatment affected the production of recombinant HSA-pFSHß protein. Furthermore, YPT7 gene disruption inhibited the processing of signal peptide in high-level produced HSA-pFSHß strain. In conclusion, our results demonstrated that YPS1 disruption could reduce the degradation of intact HSA-pFSHß proteins, and synergistically increase the yield of intact HSA-pFSHß with NAC supplementation. This study provided a valuable reference for reducing recombinant protein degradation and therefore improving the yield of recombinant proteins in P. pastoris.

LPS-induced SOCS3 antagonizes the JAK2-STAT5 pathway and inhibits ß-casein synthesis in bovine mammary epithelial cells.

Bovine mammary epithelial cells (BMECs) are essential for lactation in the dairy cow mammary gland, and are often used as a cellular model to study changes in inflammatory responses and lactation functions with exogenous stimuli. Prolactin (PRL) promotes milk protein synthesis by continuously activating the Janus kinase 2 and signal transducer and activator of transcription 5 (JAK2-STAT5) pathway. Lipopolysaccharides (LPS) activates inflammatory responses in cells and inhibits casein synthesis, but the exact mechanism is still unclear. Suppressor of cytokine signaling-3 (SOCS3) is a negative regulator of the JAK-STATs signaling pathway, and regulates a variety of inflammatory responses by inhibiting STAT3. Previous studies also suggested that SOCS3 plays a role in the development and involution of bovine mammary glands. The purpose of this study was to investigate whether LPS activated SOCS3, and whether SOCS3 resisted the regulation of casein synthesis by PRL in a JAK2-STAT5-dependent manner. We treated in vitro BMECs with 125 ng/mL PRL, 10 µg/mL LPS, SOCS3 siRNA (silencing), a SOCS3-GFP adenovirus overexpression vector, or combinations, to determine ß-casein expression. We demonstrated that PRL up-regulated phospho-JAK2, phsopho-STAT5 and ß-casein expression, whereas LPS caused the opposite effects, and activated SOCS3. SOCS3 overexpression interrupted the JAK2-STAT5 pathway in BMECs. With SOCS3 was silenced, LPS could not activate the JAK2-STAT5 pathway, and no inhibition of ß-casein expression was observed. In conclusion, we showed that LPS activated SOCS3 in BMECs, antagonized the JAK2-STAT5 pathway via SOCS3 regulation, and ultimately reduced ß-casein expression in these cells.