Proteomic analysis of milk fat globule membranes from small-sized milk fat globules and their function in promoting lipid droplet fusion in bovine mammary epithelial cells.

In mammary epithelial cells, milk fat is synthesized as lipid droplets and secreted in the form of globules. Milk fat globules (MFGs) are covered by a lipid-protein membrane known as the milk fat globule membrane (MFGM). We randomly divided 12 Holstein cows into control and conjugated linoleic acid (CLA) groups. The control group was fed a basal diet, while the CLA group was fed the basal diet + CLA (15 g per kg DM) for 10 days. Cow performance, milk composition, and MFG size were measured daily. On day 10, we extracted MFGM proteins (n = 3) and identified them via quantitative proteomic analysis. We investigated the effects of the MFGM proteins from control and CLA-treated milk on the lipid droplet formation in MAC-T cells. Compared with the control group, the CLA group had reduced milk fat content (3.39 g/100 mL vs. 2.45 g/100 mL) and MFG size parameters (D[4,3] of 3.85 µm vs. 3.37 µm; D[3,2] of 3.24 µm vs. 2.83 µm). The specific surface area (SSA) increased in the CLA group. A total of 361 differentially expressed proteins were identified in the CLA group by iTRAQ quantitative proteomic analysis. Among these proteins, 100 were upregulated and 251 were downregulated (p < 0.05). In MAC-T cells, CLA-MFGM proteins increased the diameter of the lipid droplets to 1.32 µm. CLA-MFGM proteins decreased the proportion of the small lipid droplets (15.33% vs. 47.78%) and increased the proportion of the large lipid droplets (25.04% vs. 11.65%). CLA-MFGM proteins promoted lipid droplet fusion. Therefore, MFGM proteins play an important role in the regulation of the lipid droplet size.

Alphaherpesvirus upregulates NDRG1 expression to facilitate the nuclear import of viral UL31 and UL34 proteins.

Proteins UL31 and UL34 encoded by alphaherpesvirus are critical for viral primary envelopment and nuclear egress. We report here that pseudorabies virus (PRV), a useful model for research on herpesvirus pathogenesis, uses N-myc downstream regulated 1 (NDRG1) to assist the nuclear import of UL31 and UL34. PRV promoted NDRG1 expression through DNA damage-induced P53 activation, which was beneficial to viral proliferation. PRV induced the nuclear translocation of NDRG1, and its deficiency resulted in the cytosolic retention of UL31 and UL34. Therefore, NDRG1 assisted the nuclear import of UL31 and UL34. Furthermore, in the absence of the nuclear localization signal (NLS), UL31 could still translocate to the nucleus, and NDRG1 lacked an NLS, thus suggesting the existence of other mediators for the nuclear import of UL31 and UL34. We demonstrated that heat shock cognate protein 70 (HSC70) was the key factor in this process. UL31 and UL34 interacted with the N-terminal domain of NDRG1 and the C-terminal domain of NDRG1 bound to HSC70. Replenishment of HSC70ΔNLS in HSC70-knockdown cells, or interference in importin α expression, abolished the nuclear translocation of UL31, UL34, and NDRG1. These results indicated that NDRG1 employs HSC70 to facilitate viral proliferation in the nuclear import of PRV UL31 and UL34.

Prevalence and Characteristics of Canine Parvovirus Type 2 in Henan Province, China.

To investigate the epidemic profile and genetic diversity of canine parvovirus type 2 (CPV-2), a total of 111 clinical samples collected from dogs suspected of CPV-2 infection in 10 cities of Henan province of China during 2020 to 2021 were screened by PCR. The results showed a CPV-2-positive rate of 88.29% (98/111). Nearly full-length genomes of 98 CPV-2 strains were sequenced and analyzed. CPV-2c strains (91.84%, 90/98) were significantly higher than that of new CPV-2a strains (8.16%, 8/98) in Henan province without detecting other CPV genotypes, indicating that CPV-2c has become the dominant genotype in Henan province. A phylogenetic analysis of NS1 and VP2 amino acids grouped the strains in this study with Asian strains, which clustered into an identical branch. Based on the CPV-2 VP2 sequences in this study and available in the NCBI database, the adaptation analyses showed that 17 positive selection sites and 10 parallel evolution sites were identified in the VP2 protein of CPV-2, of which three sites (sites 5, 370, and 426) were both under positive selection pressure and parallel evolution. Interestingly, two amino acid mutations (A5G and Q370R) were also observed in the VP2 proteins of 82 CPV-2c strains in this study, which differed from the earlier CPV-2c strain (GU380303) in China. In addition, a unique mutation (I447M) was observed in the VP2 protein of five CPV-2c strains, which was first reported in China. This study provides powerful insight to further our understanding of the epidemic status and evolution of CPV-2 in China. IMPORTANCE CPV-2 was the original virus strain identified in dogs, which cause an acute and lethal disease in dogs. Subsequently, the original CPV-2 was replaced throughout the world by novel antigenic variants (e.g., CPV-2a, CPV-2b, new CPV-2a, new CPV-2b, and CPV-2c). Currently, the epidemiological characteristics of CPV-2 in Henan province of China is still unclear. In our study, a total of 98 nearly full-length genomes of CPV-2 strains were obtained to explore prevalence and genetic evolution of CPV-2 in Henan Province. Moreover, the epidemiological and genetic evolution of CPV-2 in China since its discovery was also investigated. The results of this study will provide valuable information regarding the evolution of CPV-2 strains in China.

Acute transcriptomic changes in murine RAW 264.7 cells following pseudorabies virus infection.

Next-generation sequencing enables the evaluation of gene expression changes resulting from virus-host interactions at the RNA level. Pseudorabies virus (PRV) causes substantial economic loss in the swine industry. Recent research has revealed that PRV can be transmitted to and infect humans as well. To identify physiopathological and pathological responses post-PRV infection, we characterized transcriptomic changes in the murine RAW 264.7 cell line over the course of 36 h. In total, 156, 153, and 190 differentially expressed genes were identified at 2 h, 12 h, and 36 h, respectively. Seven differentially expressed genes (Trim27, Ccdc117, Mrps12, Ccl4, Cerkl, Ubald1, and Hmga1-rs1) were present across all treatment groups. Our findings expand our knowledge of gene regulation and immune response following PRV infection. These differentially expressed genes can subsequently improve our understanding of PRV pathogenesis.

Inhibition of USP14 influences alphaherpesvirus proliferation by degrading viral VP16 protein via ER stress-triggered selective autophagy.

Alphaherpesvirus infection results in severe health consequences in a wide range of hosts. USPs are the largest subfamily of deubiquitinating enzymes that play critical roles in immunity and other cellular functions. To investigate the role of USPs in alphaherpesvirus replication, we assessed 13 USP inhibitors for PRV replication. Our data showed that all the tested compounds inhibited PRV replication, with the USP14 inhibitor b-AP15 exhibiting the most dramatic effect. Ablation of USP14 also influenced PRV replication, whereas replenishment of USP14 in USP14 null cells restored viral replication. Although inhibition of USP14 induced the K63-linked ubiquitination of PRV VP16 protein, its degradation was not dependent on the proteasome. USP14 directly bound to ubiquitin chains on VP16 through its UBL domain during the early stage of viral infection. Moreover, USP14 inactivation stimulated EIF2AK3/PERK- and ERN1/IRE1-mediated signaling pathways, which were responsible for VP16 degradation through SQSTM1/p62-mediated selective macroautophagy/autophagy. Ectopic expression of non-ubiquitinated VP16 fully rescued PRV replication. Challenge of mice with b-AP15 activated ER stress and autophagy and inhibited PRV infection in vivo. Our results suggested that USP14 was a potential therapeutic target to treat alphaherpesvirus-induced infectious diseases.Abbreviations ATF4: activating transcription factor 4; ATF6: activating transcription factor 6; ATG5: autophagy related 5; ATG12: autophagy related 12; CCK-8: cell counting kit-8; Co-IP: co-immunoprecipitation; CRISPR: clustered regulatory interspaced short palindromic repeat; Cas9: CRISPR associated system 9; DDIT3/CHOP: DNA-damage inducible transcript 3; DNAJB9/ERdj4: DnaJ heat shock protein family (Hsp40) member B9; DUBs: deubiquitinases; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EP0: ubiquitin E3 ligase ICP0; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum (ER) to nucleus signaling 1; FOXO1: forkhead box O1; FRET: Förster resonance energy transfer; HSPA5/BiP: heat shock protein 5; HSV: herpes simplex virus; IE180: transcriptional regulator ICP4; MAP1LC3/LC3: microtube-associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; PPP1R15A/GADD34: protein phosphatase 1, regulatory subunit 15A; PRV: pseudorabies virus; PRV gB: PRV glycoprotein B; PRV gE: PRV glycoprotein E; qRT-PCR: quantitative real-time polymerase chain reaction; sgRNA: single guide RNA; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TCID50: tissue culture infective dose; UB: ubiquitin; UBA: ubiquitin-associated domain; UBL: ubiquitin-like domain; UL9: DNA replication origin-binding helicase; UPR: unfolded protein response; USPs: ubiquitin-specific proteases; VHS: virion host shutoff; VP16: viral protein 16; XBP1: X-box binding protein 1; XBP1s: small XBP1; XBP1(t): XBP1-total.

Inhibition of PI3K/Akt/mTOR pathway by ammonium chloride induced apoptosis and autophagy in MAC-T cell.

Ammonia is a harmful gas with a pungent odor, participates in the regulation of a variety of apoptosis and autophagy, which in turn affects the growth and differentiation of cells. To test the regulation of NH3 on the apoptosis and autophagy of mammary epithelial cells, we selected NH4Cl as NH3 donor in vitro model. MTT and CCK-8 assay kits were employed to detect cell activity. Real-time quantitative PCR and western blot methods were used to detect the abundance of inflammatory molecules, apoptosis markers, and autophagy genes. We selected TUNEL kit and the Annexin-FITC/PI method to detect apoptosis. TEM analysis was used to detect autophagic vesicles, and MDC stain evaluated the formation of autophagosome. The results indicated that NH4Cl reduced cell viability in a concentration-dependent manner and promoted cell inflammatory response, apoptosis, and autophagy. NH4Cl stimulation notable increased the autophagosomes number. Interestingly, we also detected that the addition of LY294002 and Rapamycin inhibited the PI3K/Akt pathway and the mTOR pathway, respectively, resulting in changes in both apoptosis and autophagy. Therefore, we draw a conclusion that NH3 may regulate the apoptosis and autophagic response of bovine mammary epithelial cells through the PI3K/Akt/mTOR signaling pathway. Further investigations on ammonia's function in other physiological respects, will be critical to provide theoretical help for the improvement of production performance. It will be also helpful for controlling the harmful gas ammonia concentration in the livestock house to protect the health of dairy cows.

Effect of melatonin on visceral fat deposition, lipid metabolism and hepatic lipo-metabolic gene expression in male rats.

Melatonin (MT) influences lipid metabolism in animals; however, the mechanistic effect of melatonin on liver fat and abdominal adipose deposition requires further clarity. In order to study the effects of melatonin on lipid metabolism, and hepatic fat and abdominal adipose deposition in animals, twenty Sprague-Dawley (SD) rats of 6 weeks of age with similar bodyweight were randomly divided into two groups: control (CTL) and MT-treated (10 mg/kg/day). During a 60-day experiment, food intake and bodyweight were measured daily and weekly respectively. At the end of treatment, blood samples were collected to collect plasma to quantify hormones and metabolic indicators of lipid metabolism. In addition, organ and abdominal adipose depots including liver, and omental, perirenal, and epididymal fat were weighed. Liver tissue was sampled for sectioning, long-chain fatty acid (LCFA) quantification, and gene chip and Real-time quantitative PCR (qPCR) analyses. The results showed that liver weight and index (ratio of liver weight to body weight) in MT group reduced by 20.69% and 9.63% respectively; omentum weight and index reduced by 59.88% and 54.93% respectively, and epididymal fat weight reduced by 45.34% (p = 0.049), relative to CTL. Plasma lipid indices, triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and total cholesterol (TC) with MT treatment decreased significantly compared with the control. Fat and 8 LCFA content in liver in MT group also decreased. Gene chip and qPCR demonstrated that there were 289 genes up-regulated and 293 genes down-regulated by MT. Further analysis found that the mRNA expression of lipolysis-related genes increased, while the mRNA expression of lipogenesis-related enzymes decreased (p < 0.05) with MT. This study concluded that melatonin greatly affected fat deposition, and hepatic LCFA supply and the expression of genes associated with lipogenesis and lipolysis.

Viperin inhibits classical swine fever virus replication by interacting with viral nonstructural 5A protein.

Classical swine fever virus (CSFV) is a single-stranded RNA flavivirus that can cause serious diseases in porcine species, including symptoms of infarction, systemic hemorrhage, high fever, or depression. Viperin is an important interferon-inducible antiviral gene that has been shown to inhibit CSFV, but the exact mechanisms by which it is able to do so remain poorly characterized. In the present study, we determined that CSFV infection led to viperin upregulation in PK-15 cells (porcine kidney cell). When viperin was overexpressed in these cells, this markedly attenuated CSFV replication, with clear reductions in viral copy number after 12 to 48 hours postinfection. Immunofluorescence microscopy revealed that the viral NS5A protein colocalized with viperin in infected cells, and this was confirmed via confocal laser scanning microscopy using labeled versions of these proteins, and by co-immunoprecipitation which confirmed that NS5A directly interacts with viperin. When NS5A was overexpressed, this inhibited the replication of CSFV, and we determined that the radical SAM domain and N-terminal domain of viperin was critical for its ability to bind to NS5A, with the latter being most important for this interaction. Together, our in vitro results highlight a potential mechanism whereby viperin is able to inhibit CSFV replication. These results have the potential to assist future efforts to prevent or treat systemic CSFV-induced disease, and may also offer more general insights into the antiviral role of viperin in innate immunity.

H2S mediates apoptosis in response to inflammation through PI3K/Akt/NFκB signaling pathway.

OBJECTIVES: Hydrogen sulfide (H2S) is involved in regulating cell apoptosis and proliferation. However, The effects and mechanism of H2S on the apoptosis of mammary epithelial cells that suffer from an inflammatory response remain unknown. RESULTS: An inflammatory cell model was used to explore whether exogenous H2S regulates lipopolysaccharides (LPS)-induced cell proliferation and apoptosis. We found that H2S affected cell viability, the inflammatory response and apoptosis in LPS-treated cells in a concentration-dependent manner. Moreover, exogenous H2S rescued LPS-induced cystathionine γ-lyase (CSE) inhibition and cystathionine ß-synthase (CBS) synthesis. Interestingly, in cells undergoing inflammation-induced apoptosis, H2S activated the PI3K/Akt and NFκB signal pathways both tested concentrations. Akt appeared to be a key crosstalk molecule that played a "bridge" role. CONCLUSIONS: H2S regulates LPS-induced inflammation and apoptosis by activating the PI3K/Akt/NFκB signaling pathway. Hence, NaHS may be clinically useful for preventing or treating mastitis.

S100 calcium-binding protein A12 as a diagnostic index for subclinical mastitis in cows.

Diagnosis of subclinical mastitis is very important in management of the dairy industry and improvement of dairy cow productivity. S100A12, that is found in related tissues of mammals, is considered as an index for diagnosing inflammatory reaction. To evaluate whether S100A12 is involved in subclinical mastitis, milk somatic cell mRNA from 276 dairy cows was used to detect the transcriptional level of S100A12 by real-time quantitative polymerase chain reaction. A predictive analysis for mastitis was performed, and the correlation between S100A12 and other subclinical mastitis indicators was also assessed. The transcriptional levels of S100A12 in the milk of cows with mastitis were significantly higher than those in the milk of healthy cows (p < 0.05). The correlation analysis showed that S100A12 was positively associated with the somatic cell count and the sodium and chloride concentrations of milk. In contrast, a negative correlation was found between S100A12 and the potassium concentration and pH of milk. However, no significant correlation was detected between S100A12 and the other parameters, such as protein, lactose, ash, fat, density, Ca2+ and SNF. These results suggested that the S100A12 level in milk may serve as a diagnostic tool for subclinical mastitis in cows without obvious clinical signs.

GABA regulates the proliferation and apoptosis of MAC-T cells through the LPS-induced TLR4 signaling pathway.

Subacute ruminal acidosis (SARA) can cause rapid lipopolysaccharide (LPS) elevation and milk yield decline in lactating ruminants. LPS has been shown to promote apoptosis and reduce the proliferation of mammary epithelial cells. Previous studies have shown that γ- amino butyric acid (GABA) can enhance production performance, regulating ß-cell apoptosis and proliferation. Whether GABA can regulate apoptosis and proliferation induced by LPS in mammary epithelial cells is unknown. In this paper, we detected the role of GABA on proliferation and apoptosis as well as inflammation induced by LPS in bovine mammary epithelial cells (MAC-T cell line). In addition, we explored the role mechanism of GABA in LPS-induced MAC-T cells response through detecting the NFκB signaling pathway key molecules. The results suggested that GABA reduced the effects of cell apoptosis induced by LPS. Furthermore, GABA inhibited the expression of inflammatory cytokines activated by LPS. More importantly, blocking GABA receptors with its antagonist, GABA could not reduce the expression of inflammatory and pro-apoptotic factors activated by LPS. Notably, GABA significantly decreased the TLR4, NFκB p65, and MyD88 mRNA expression levels that were elevated by LPS. Our data indicated that GABA can improve cell viability and decrease apoptosis induced by LPS, while exerting an anti-inflammatory effect through the NFκB signaling pathway.

Characterization and anti-inflammation role of swine IFITM3 gene.

IFITM3 is involved in cell adhesion, apoptosis, immune, and antivirus activity. Furthermore, IFITM3 gene has been considered as a preferential marker for inflammatory diseases, and positive correlation to pathological grades. Therefore, we assumed that IFITM3 was regulated by different signal pathways. To better understand IFITM3 function in inflammatory response, we cloned swine IFITM3 gene, and detected IFITM3 distribution in tissues, as well as characterized this gene. Results indicated that the length of swine IFITM3 gene was 438 bp, encoding 145 amino acids. IFITM3 gene expression abundance was higher in spleen and lungs. Moreover, we next constructed the eukaryotic expression vector PBIFM3 and transfected into PK15 cells, finally obtained swine IFITM3 gene stable expression cell line. Meanwhile, we explored the effects of LPS on swine IFITM3 expression. Results showed that LPS increased IFITM3 mRNA abundance and exhibited time-dependent effect for LPS treatment. To further demonstrate the mechanism that IFITM3 regulated type I IFNs production, we also detected the important molecules expression of TLR4 signaling pathway. In transfected and non-transfected IFITM3 PK15 cells, LPS exacerbated the relative expression of TLR4-NFκB signaling molecules. However, the IFITM3 overexpression suppressed the inflammatory development of PK15 cells. In conclusion, these data indicated that the overexpression of swine IFITM3 could decrease the inflammatory response through TLR4 signaling pathway, and participate in type I interferon production. These findings may lead to an improved understanding of the biological function of IFITM3 in inflammation.

Swine IRF3/IRF7 attenuates inflammatory responses through TLR4 signaling pathway.

To explore the role of IRF3/IRF7 during inflammatory responses, we investigated the effects of swine IRF3/IRF7 on TLR4 signaling pathway and inflammatory factors expression in porcine kidney epithelial PK15 cell lines. We successfully constructed eukaryotic vectors PB-IRF3 and PB-IRF7, transfected these vectors into PK15 cells and observed GFP under a fluorescence microscope. In addition, RT-PCR was also used to detect transfection efficiency. We found that IRF3/IRF7 was efficiently overexpressed in PK15 cells. Moreover, we evaluated the effects of IRF3/IRF7 on the TLR4 signaling pathway and inflammatory factors by RT-PCR. Transfected cells were treated with lipopolysaccharide (LPS) alone, or in combination with a TBK1 inhibitor (LiCl). We revealed that IRF3/IRF7 enhanced IFNα production, and decreased IL-6 mRNA expression. Blocking the TBK1 pathway, inhibited the changes in IFNα, but not IL-6 mRNA. This illustrated that IRF3/IRF7 enhanced IFNα production through TLR4/TBK1 signaling pathway and played an anti-inflammatory role, while IRF3/IRF7 decreased IL-6 expression independent of the TBK1 pathway. Trends in MyD88, TRAF6, TBK1 and NFκB mRNA variation were similar in all treatments. LPS increased MyD88, TRAF6, TBK1 and NFκB mRNA abundance in PBR3/PBR7 and PBv cells, while LiCl blocked the LPS-mediated effects. The levels of these four factors in PBR3/PBR7 cells were higher than those in PBv. These results demonstrated that IRF3/IRF7 regulated the inflammatory response through the TLR4 signaling pathway. Overexpression of swine IRF3/IRF7 in PK15 cells induced type I interferons production, and attenuated inflammatory responses through TLR4 signaling pathway.

NF-κB is involved in the LPS-mediated proliferation and apoptosis of MAC-T epithelial cells as part of the subacute ruminal acidosis response in cows.

OBJECTIVES: To determine the effect of NF-κB on cell proliferation and apoptosis, we investigate the expression of inflammation and apoptosis-related factors in the bovine mammary epithelial cell line, MAC-T. RESULTS: MAC-T cells were cultured in vitro and MTT and LDH assays used to determine the effects of lipopolysaccharide (LPS) on proliferation and cytotoxicity respectively. RT-PCR and western blotting were used to evaluate the effect of LPS and NF-κB inhibition [pyrrolidine dithiocarbamate (PDTC) treatment] on the expression of inflammation and apoptosis-related factors. LPS significantly inhibited MAC-T cell proliferation in a dose- and time-dependent manner. Furthermore, LPS promoted apoptosis while the NF-кB inhibitor PDTC attenuated this effect. After LPS treatment, the NF-кB signaling pathway was activated, and the expression of inflammation and apoptosis-related factors increased. When PDTC blocked NF-кB signaling, the expression of inflammation and apoptosis-related factors were decreased in MAC-T cells. CONCLUSIONS: LPS activates the TLR4/NF-κB signaling pathway, inhibits proliferation and promotes apoptosis in MAC-T cells. NF-кB inhibition attenuates MAC-T cell apoptosis and TLR4/NF-κB signaling pathway. NF-кB inhibitor alleviating MAC-T cell apoptosis is presumably modulated by NF-кB.