Effects of choline deficiency and supplementation on lipid droplet accumulation in bovine primary liver cells in vitro.

Rumen-protected choline (RPC) supplementation in the periparturient period has in some instances prevented and alleviated fatty liver disease in dairy cows. Mechanistically, however, it is unclear how choline prevents the accumulation of lipid droplets (LD) in liver cells. In this study, primary liver cells isolated from liver tissue obtained via puncture biopsy from 3 nonpregnant mid-lactation multiparous Holstein cows (∼160 d postpartum) were used. Analyses of LD via oil red O staining, protein abundance via Western blotting, and phospholipid content and composition measured by thin-layer chromatography and HPLC/mass spectrometry were performed in liver cells cultured in choline-deficient medium containing 150 µmol/L linoleic acid for 24 h. In a subsequent experiment, lipophagy was assessed in liver cells cultured with 30, 60, or 90 µmol/L choline-chloride. All data were analyzed statistically using SPSS 20.0 via t-tests or one-way ANOVA. Compared with liver cells cultured in Dulbecco's Modified Eagle Medium alone, choline deficiency increased the average diameter of LD (1.59 vs. 2.10 µm), decreased the proportion of small LD (<2 µm) from 75.3% to 56.6%, and increased the proportion of large LD (>4 µm) from 5.6% to 15.0%. In addition, the speed of LD fusion was enhanced by the absence of choline. Among phospholipid species, the phosphatidylcholine (PC) content of liver cells decreased by 34.5%. Seventeen species of PC (PC [18:2_22:6], PC [15:0_16:1], PC [14:0_20:4], and so on) and 6 species of lysophosphatidylcholine (LPC; LPC [15:0/0:0]), PC (22:2/0:0), LPC (20:2/0:0), and so on] were decreased, while PC (14:1_16:1) and LPC (0:0/20:1) were increased. Choline deficiency increased the triglyceride (TAG) content (0.57 vs. 0.39 µmol/mg) in liver cells and increased the protein abundance of sterol regulatory element binding protein 1, sterol regulatory element binding protein cleavage activation protein, and fatty acid synthase by 23.5%, 17%, and 36.1%, respectively. Upon re-supplementation with choline, the phenotype of LD (TAG content, size, proportion, and phospholipid profile) was reversed, and the ratio of autophagy marker LC3II/LC3I protein was significantly upregulated in a dose-dependent manner. Overall, at least in vitro in mid-lactation cows, these data demonstrated that PC synthesis is necessary for normal LD formation, and both rely on choline availability. According to the limitation of the source of liver cells used, further work should be conducted to ascertain that these effects are applicable to liver cells from postpartum cows, the physiological stage where the use of RPC has been implemented for the prevention and treatment of fatty liver.

Milk fat globule membrane proteins are involved in controlling the size of milk fat globules during conjugated linoleic acid-induced milk fat depression.

Milk fat globule membrane (MFGM) proteins surround the triacylglycerol core comprising milk fat globules (MFG). We previously detected a decrease in the size of fat globules during conjugated linoleic acid (CLA)-induced milk fat depression (MFD), and other studies have reported that some MFGM proteins play a central role in regulating mammary cellular lipid droplet size. However, little is known about the relationship between MFD, MFG size, and MFGM proteins in bovine milk. The aim of this study was to investigate the profile of MFGM proteins during MFD induced by CLA. Sixteen mid-lactating Holstein cows (145 ± 24 d in milk) with similar body condition and parity were divided into control and CLA groups over a 10-d period. Cows were fed a basal diet (control, n = 8) or control plus 15 g/kg of dry matter (DM) CLA (n = 8) to induce MFD. Cow performance, milk composition, and MFG size were measured daily. On d 10, MFGM proteins were extracted and identified by quantitative proteomic analysis, and western blotting was used to verify a subset of the identified MFGM proteins. Compared with controls, supplemental CLA did not affect milk production, DM intake, or milk protein and lactose contents. However, CLA reduced milk fat content (3.73 g/100 mL vs. 2.47 g/100 mL) and the size parameters volume-related diameter D[4,3] (3.72 µm vs. 3.35 µm) and surface area-related diameter D[3,2] (3.13 µm vs. 2.80 µm), but increased specific surface area of MFG (1,905 m2/kg vs. 2,188 m2/kg). In total, 177 differentially expressed proteins were detected in milk from cows with CLA-induced MFD, 60 of which were upregulated and 117 downregulated. Correlation analysis showed that MFG size was negatively correlated with various proteins, including XDH and FABP3, and positively correlated with MFG-E8, RAB19, and APOA1. The results provide evidence for an important role of MFGM proteins in regulating MFG diameter, and they facilitate a mechanistic understanding of diet-induced MFD.

Construction of a rAAV-SaCas9 system expressing eGFP and its application to improve muscle mass.

An ideal rAAV gene editing system not only effectively edits genes at specific site, but also prevents the spread of the virus from occurring off-target or carcinogenic risks. This is important for gene editing research at specific site in vivo. We report a single rAAV containing SaCas9 and guide RNAs under the control of subtle EF1a and tRNA promoters. The capacity of rAAV was compressed, and the editing efficiency was similar to that of the classical Cas9 system in vitro and in vivo. And we inserted the sequence of the green fluorescent protein eGFP into rAAV. The number of cells infected with the rAAV and the region in which the rAAV spreads were known by the fluorescent expression of eGFP in cells. In addition, we demonstrated that myostatin gene in the thigh muscles of C57BL/10 mice was knocked out by the rAAV9-SaCas9 system to make muscle mass increased obviously. The protein eGFP into rAAV has significant implications for our indirect analysis of the editing efficiency of SaCas9 in the genome of the target tissue and reduces the harm caused by off-target editing and prevents other tissue mutations. The rAAV system has substantial potential in improving muscle mass and preventing muscle atrophy.

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.

Lipidomic profiling analysis of the phospholipid molecules in SCAP-induced lipid droplet formation in bovine mammary epithelial cells.

The accumulation of lipid droplets (LDs) in the cytoplasm plays an important role in energy balance, membrane synthesis and cell signal transduction. The aim of this study was to investigate the profile of phospholipids after SCAP-induced LD formation in bovine mammary epithelial cells (BMECs). A shRNA-SCAP vector and a SCAP/SREBP vector were used to knock down and overexpress the SCAP gene in BMECs prior to evaluating the effects on LDs using Western blotting, real-time PCR, LD staining and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The average LD diameter was determined following oil red O staining. The overexpression of SCAP increased the abundance of SCD, ACACA and FASN genes and nuclear SREBP1a. In contrast, knocking down SCAP decreased the abundance of the nuclear SREBP1a protein and downregulated the abundance of target genes. Lipid droplet staining revealed that knocking down SCAP reduced LD formation and average LD diameter. In contrast, overexpression of SCAP increased the formation and size of the LDs. The results from an analysis of cellular lipids revealed that phospholipids are the predominant species in the profile of cell lipids. phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are important for determining the size of LDs. The LD formation induced by SCAP gene overexpression and knockdown underscored the role of phospholipids involved in lipid droplet formation and fusion.

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.

Synthesis and biological evaluation of curcumin derivatives modified with α-amino boronic acid as proteasome inhibitors.

Curcumin is a well-known pharmacophore and some of its derivatives are shown to target 20S proteasome recently. In this report, we designed and synthesized two series of curcumin derivatives modified with different α-amino boronic acids as potent proteasome inhibitors. The synthesized compounds were evaluated for their cytotoxic activities against HCT116 cells, and the results showed that all of them exhibited excellent cell growth inhibitory activity comparing with curcumin, with the IC50 values varying from 0.17 µM to 1.63 µM. Compound II-2F with free boronic acid was assayed for its proteasome inhibitory activity and the results indicated that II-2F exhibited more potent inhibitory activity against ChT-L with high subunit selectivity than any other reported curcumin derivatives.

Effect of the Transition Points Mismatch on Quanta Image Sensors.

Mathematical models and imaging models that show the relationship between the transition points mismatch of analog-to-digital converters (ADCs) and the bit error rate (BER) in single-bit and multi-bit quanta image sensors (QISs) are established. The mathematical models suggest that when the root-mean-square (r.m.s.) of the read noise in jots is 0.15e-, the standard deviation of the transition points should be less than 0.15e- to ensure that the BER is lower than 1% in the single-bit QIS, and 0.21e- to ensure that the BER is lower than 5% in the multi-bit QIS. Based on the mathematical models, the imaging models prove that the fixed-pattern noise (FPN) increases with a stronger transition point mismatch. The imaging models also compare the imaging quality in the case of different spatial oversampling factors and bit depths. The grayscale similarity index (GSI) is 3.31 LSB and 1.74 LSB when the spatial oversampling factors are 256 and 4096, respectively, in the single-bit QIS. The GSI is 1.93 LSB and 1.13 LSB when the bit depth is 3 and 4, respectively, in the multi-bit QIS. It indicates that a higher bit depth and a larger spatial oversampling factor could reduce the effect of the transition points mismatch of1-bit or n-bit ADCs.

Synthesis and biological activity of peptide proline-boronic acids as proteasome inhibitors.

On the basis of the application of proline-boronic acid as pharmacophore in the kinase inhibitors and our previous research results, using proline-boronic acid as warhead, two series of peptide proline-boronic acids, dipeptide proline-boronic acids (I) and tripeptide proline-boronic acids (II), were designed and synthesized. All the synthesized compounds were first evaluated for their biological activity against MGC803 cell, and then, the best compound II-7 was selected to test its anti-tumor spectrum on six human tumor cell lines and proteasome inhibition against three subunits. The results indicated that series II have much better biological activities than series I. The compound II-7 exhibited not only excellent biological activities with IC50 values of nM level in both cell and proteasome models, but also much better subunit selectivity. Thus, proline-boronic acid as warhead is reasonable in the design of proteasome inhibitors.

The Ruminal Microbiome Alterations Associated with Diet-Induced Milk Fat Depression and Milk Fat Globule Size Reduction in Dairy Goats.

The aim of this study was to evaluate the effect of conjugated linoleic acid (CLA) on milk fat globule (MFG) size and the ruminal microbiome of goats. Twenty-four mid-lactation Saanen dairy goats weighing 49 ± 4.5 kg (168 ± 27 d in milk, 1.2 ± 0.1 kg milk/d, 2-3 years old) were randomly divided into four groups-a control (CON) group, which was fed a basal diet, and three CLA supplementation groups, in which 30 g CLA (low-dose group, L-CLA), 60 g CLA (medium-dose group, M-CLA), or 90 g CLA (high-dose group, H-CLA) was added to the basal diet daily. The experiment lasted for 21 days, during which time goat milk was collected for composition and MFG size analysis. On day 21 of feeding, ruminal fluid was collected from the CON and H-CLA groups for analysis of the changes in microorganismal abundance. The results showed that CLA supplementation did not affect milk production, milk protein, or lactose content in the dairy goats (p > 0.05), but significantly reduced the milk fat content (p < 0.01) compared with the CON group. The CLA supplementation significantly decreased the D[3,2] and D[4,3] of the MFGs in a dose-dependent manner (p < 0.01). Moreover, dietary CLA inclusion increased the proportion of small-sized MFGs and decreased that of large-sized ones. The results of 16S rRNA gene sequencing showed that CLA-induced milk fat depression in dairy goats was accompanied by significant changes in the relative abundance of ruminal bacterial populations, most of which belonged to the Firmicutes and Bacteroidetes phyla. The relative abundance of Rikenellaceae_RC9_gut_group and Prevolellaceae_UCG-003 in Bacteroidetes and UCG-002, Succiniclasticum, and norank_f__norank_o__Clostridia_vadinBB60_group in Firmicutes was significantly higher in the CON group than in the H-CLA group. In contrast, the relative abundance of norank_f__UCG-011, norank_f_Eubacterium_coprostanoligenes_group, unclassified_f__Lachnospiraceae, and UCG-001 in Firmicutes and norank_f__Muribaculaceae in Bacteroidetes was significantly higher in the H-CLA group than in the CON group. Correlation analysis showed that the milk fat content was negatively correlated with the relative abundance of some bacteria, including members of Firmicutes and Bacteroidetes. Similarly, MFG size (D[3,2] and D[4,3]) was negatively correlated with several members of Firmicutes and Bacteroidetes, including Lachnospiraceae, norank_f__UCG-011, UCG-001, norank_f__Eubacterium_coprostanoligenes_group (Firmicutes), and norank_f__Muribaculaceae (Bacteroidetes), while positively correlated with the relative abundance of some members of Firmicutes and Bacteroidetes, including Mycoplasma, Succiniclasticum, norank_f__norank_o__Clostridia_vadinBB60_group, UCG-002 (Firmicutes), and Rikenellaceae_RC9_gut_group (Bacteroidetes). Overall, our data indicated that CLA treatment affected milk fat content and MFG size in dairy goats, and these effects were correlated with the relative abundance of ruminal bacterial populations. These results provide the first evidence to explain the mechanism underlying diet-induced MFG from the perspective of the ruminal microbiome in dairy goats.

Is milk fat globule size correlated with milk fat content in Ruminants?

In milk, fat exists in the form of milk fat globules (MFGs). The average size (average fat globules of different particle sizes) is the most common parameter when describing MFG size. There are different views on whether there is a correlation between MFG size and milk fat content. Is the MFG size correlated with milk fat content in ruminants? To address this question, we conducted two experiments. In experiment Ⅰ, dairy cows (n = 40) and dairy goats (n = 30) were each divided into a normal group and a low-fat group according to the milk fat content. In experiment Ⅱ, dairy cows (n = 16) and dairy goats (n = 12) were each divided into a normal group and a conjugated linoleic acid (CLA)-induced low-fat group. The normal groups were fed a basal diet, and the CLA-induced low-fat groups were fed the basal diet + 300 g/d CLA (cows) or the basal diet + 90 g/d CLA (goats). In both experiments, we determined the correlation between MFG size and milk composition and MFG distribution. The results showed that in the normal and low-fat groups of cows and goats, MFG size was not correlated with milk fat, protein, or lactose content or fat-to-protein ratio. Additionally, there was no difference in the distribution of large, medium, and small MFGs (P > 0.05). However, in the CLA-induced low-fat groups, we found a correlation between MFG size and milk fat content and fat-to-protein ratio (R2 > 0.3). Moreover, there was a significant change in the size distribution of MFGs. Therefore, in natural milk, MFG size was not correlated with milk fat content. Following CLA supplementation, MFG size was correlated with milk fat content. Our findings revealed that CLA and not milk fat affects MFG distribution and size.

Role of optical coherence tomography in pipeline embolization device for the treatment of vertebral-basilar artery dissecting aneurysms.

BACKGROUND: Vertebral-basilar artery dissecting aneurysms (VADAs) are an uncommon phenomenon in all fields of cerebrovascular disease. The flow diverter (FD) can be used as an endoluminal reconstruction device that promotes neointima formation at the aneurysmal neck and preserves the parent artery. To date, imaging examinations such as CT angiography, MR angiography, and DSA are the main methods used to evaluate the vasculature of patients. However, none of these imaging methods can reveal the situation of neointima formation, which is of great importance in evaluating occlusion of VADAs, especially those treated with a FD. METHODS: Three patients were included in the study from August 2018 to January 2019. All patients underwent preprocedural, postprocedural, and follow-up evaluations with high resolution MRI, DSA, and optical coherence tomography (OCT), as well as the formation of intima on the surface of the scaffold at the 6 month follow-up. RESULTS: Preprocedural, postoperative, and follow-up high resolution MRI, DSA, and OCT of all three cases successfully evaluated occlusion of the VADAs and occurrence of in stent stenosis from different views of intravascular angiography and neointima formation. CONCLUSIONS: OCT was feasible and useful to further evaluate VADAs treated with FD from a near pathological perspective, which may contribute toward guiding the duration of antiplatelet medication and early intervention of in stent stenosis.

Sirtuin 5 alleviates apoptosis and autophagy stimulated by ammonium chloride in bovine mammary epithelial cells.

Ammonia (NH3) is an irritating and harmful gas that affects cell apoptosis and autophagy. Sirtuin 5 (SIRT5) has multiple enzymatic activities and regulates NH3-induced autophagy in tumor cells. In order to determine whether SIRT5 regulates NH3-induced bovine mammary epithelial cell apoptosis and autophagy, cells with SIRT5 overexpression or knockdown were generated and in addition, bovine mammary epithelial cells were treated with SIRT5 inhibitors. The results showed that SIRT5 overexpression reduced the content of NH3 and glutamate in cells by inhibiting glutaminase activity in glutamine metabolism, and reduced the ratio of ADP/ATP. The results in the SIRT5 knockdown and inhibitor groups were comparable, including increased content of NH3 and glutamate in cells by activating glutaminase activity, and an elevated ratio of ADP/ATP. It was further confirmed that SIRT5 inhibited the apoptosis and autophagy of bovine mammary epithelial cells through reverse transcription-quantitative PCR, western blot, flow cytometry with Annexin V FITC/PI staining and transmission electron microscopy. In addition, it was also found that the addition of LY294002 or Rapamycin inhibited the PI3K/Akt or mTOR kinase signal, decreasing the apoptosis and autophagy activities of bovine mammary epithelial cells induced by SIRT5-inhibited NH3. In summary, the PI3K/Akt/mTOR signal involved in NH3-induced cell autophagy and apoptosis relies on the regulation of SIRT5. This study provides a new theory for the use of NH3 to regulate bovine mammary epithelial cell apoptosis and autophagy, and provides guidance for improving the health and production performance of dairy cows.

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells.

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety of metabolic diseases, such as obesity, fatty liver disease, and diabetes. In hepatic cells, the sizes and numbers of LDs are signs of fatty liver disease. Moreover, the oxidative stress reaction, cell autophagy, and apoptosis are often accompanied by changes in the sizes and numbers of LDs. As a result, the dimensions and quantity of LDs are the basis of the current research regarding the mechanism of LD biogenesis. Here, in fatty acid-induced bovine hepatic cells, we describe how to use oil red O to stain LDs and to investigate the sizes and numbers of LDs. The size distribution of LDs is statistically analyzed. The process of small LDs fusing into large LDs is also observed by a live cell imaging system. The current work provides a way to directly observe the size change trend of LDs under different physiological conditions.

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.

Conjugated linoleic acid supplementation caused reduction of perilipin1 and aberrant lipolysis in epididymal adipose tissue.

Perilipin1, a coat protein of lipid droplet, plays a key role in adipocyte lipolysis and fat formation of adipose tissues. However, it is not clear how the expression of perilipin1 is affected in the decreased white adipose tissues (WAT) of mice treated with dietary supplement of conjugated linoleic acids (CLA). Here we obtained lipodystrophic mice by dietary administration of CLA which exhibited reduced epididymal (EPI) WAT, aberrant adipocytes and decreased expression of leptin in this tissue. We found both transcription and translation of perilipin1 was suppressed significantly in EPI WAT of CLA-treated mice compared to that of control mice. The gene expression of negative regulator tumor necrosis factor α (TNFα) and the positive regulator Peroxisome Proliferator-Activated Receptor-γ (PPARγ) of perilipin1 was up-regulated and down-regulated, respectively. In cultured 3T3-L1 cells the promoter activity of perilipin1 was dramatically inhibited in the presence of CLA. Using ex vivo experiment we found that the basal lipolysis was elevated but the hormone-stimulated lipolysis blunted in adipose explants of CLA-treated mice compared to that of control mice, suggesting that the reduction of perilipin1 in white adipose tissues may at least in part contribute to CLA-mediated alternation of lipolysis of WAT.

[mRNA expression of lipogenic genes in mouse mammary gland in different lactation stages].

To investigate the expression pattern of lipogenic genes in mammary gland of mouse in different lactation stages, the relative mRNA abundance and expression of 20 genes involved in milk fat synthesis and secretion of lactating mammary tissues were assessed using real-time PCR. Results revealed the significant upregulation of mRNA expression for both high abundant genes (abundance > 5%), such as LPL, ACACA, SCD, XDH, BTN, and ADFP and moderate abundant genes (5% > abundance > 1%), such as CD36, FASN, AGPAT6, and DGAT in lactation stages compared to pregnancy (P < 0.05). The mRNA expression of lipogenic enzyme genes for ACACA, SCD, FASN, AGPAT6, and DGAT exhibited lower expression lever in early (6 d) and late (18 d) lactation stages but higher expression level at middle stage (12 d), demonstrating a low-high-low pattern. Besides, the mRNA levels of some gene regulators were also measured. The mRNA expression of SREBF gene increased gradually along with the lactation, which showed a 10-fold elevation at middle stage (12 d). The expression pattern of SREBF gene was the same as lipogenic enzyme genes, suggesting that SREBF may play an important role in the regulation of lipogenic enzyme genes in the lactating mammary gland.

The anti-apoptotic and anti-autophagic effects of EPO through PI3K/Akt/mTOR signaling pathway in MAC-T cells.

Lipopolysaccharide (LPS) is an important inflammatory and infected factor of bacterial mastitis, which treated bovine mammary epithelial cells (MAC-T) in our previous studies, as mastitis cells model in vitro. Erythropoietin (EPO) is a well-known hematopoietic hormone with antioxidative, anti-apoptotic, and anti-inflammatory roles. We hypothesized that EPO might regulate the apoptosis and autophagy to attenuate the inflammation of mastitis. Western blot, RT-PCR, transmission electron microscope analysis and Annexin V-FITC/PI were used to evaluate the regulation of EPO on apoptosis and autophagy in inflammatory MAC-T cells. These results demonstrated that EPO promoted the proliferation of MAC-T cells. Meanwhile, EPO had a better anti-inflammatory effect in MAC-T cells with LPS treatment. Certainly, EPO also showed anti-apoptotic and anti-autophagic effects. Interestingly, we found that the beneficial effect of EPO on inflammatory MAC-T cells depended on the PI3K/Akt/mTOR signaling pathway, which was involved in the regulation of apoptosis and autophagy. Generally, this study provides an insight for EPO to inhibit apoptosis and autophagy of inflammatory MAC-T cells via PI3K/Akt/mTOR signaling pathway.

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.

Nano-selenium Supplementation Increases Selenoprotein (Sel) Gene Expression Profiles and Milk Selenium Concentration in Lactating Dairy Cows.

Supplementation with selenium is common for dairy cows, but the importance of selenium source is not clear. This study aimed to compare nano-selenium (Nano-Se) and sodium selenite supplements for dairy cows on lactation performance, milk Se levels and selenoprotein (Sel) gene expression. Twelve multiparous Holstein cows were randomly divided into two groups: a control group fed a basal diet plus 0.30 mg Se/kg of DM as sodium selenite or Nano-Se for 30 days. Dry matter intake, milk yield and composition were not affected by dietary Se source (P > 0.05); however, the milk total Se levels and milk glutathione peroxidase (GSH-Px) activities were higher with Nano-Se supplementation than sodium selenite (P < 0.05). At the end of the experiment, Nano-Se supplementation significantly increased plasma Se levels and GSH-Px activity, compared with the sodium selenite supplement. The mRNA expression levels of glutathione peroxidase 1, 2 and 4; thioredoxin reductase 2 and 3; and selenoproteins W, T, K and F were markedly upregulated (P < 0.05) in the mammary gland of the Nano-Se group. Thus, the source of selenium plays an important role in the antioxidant status and in particular the Sel gene expression in the mammary glands of dairy cows, both being stimulated by nano sources.