Prenatal diagnosis of fetal intracranial medulloepithelioma: a case report.

Intracranial medulloepithelioma is a very rare and highly malignant tumor that is typically diagnosed in childhood and has an inferior prognosis. In the current report, we described a case of fetal intracranial medulloepithelioma that was detected during the third trimester by prenatal ultrasonography, which displayed homogenous echogenicity with well-circumscribed margins and abundant blood flow. On magnetic resonance imaging, it was hyperintense on both T1- and T2-weighted magnetic resonance imaging. The fetal intracranial tumor was progressive, with rapid expansion within 3 weeks. The report aimed to provide knowledge on the clinical characteristics of fetal intracranial medulloepithelioma in prenatal diagnosis, particularly the radiological features.

Metabolite Comparison between Serum and Follicular Fluid of Dairy Cows with Inactive Ovaries Postpartum.

Inactive ovaries (IO) accounts for 50% of ovarian disease in postpartum dairy cows, which seriously affects their reproductive efficiency. To investigate the metabolic changes in the serum and follicular fluid of dairy cows with IO during lactation, six estrus (E) cows and six IO cows at 50 to 55 days in milk were selected based on B ultrasonic detection and clinical manifestations. The differential metabolites in serum and follicular fluid between the E cows and IO cows were identified by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry, combined with multidimensional statistical methods. The results showed that dairy cows with IO were in a subclinical ketosis status where beta-hydroxybutyrate (BHB) exceeded 1.20 mmol/L, 14 differential metabolites in the serum of IO cows included 10 increased metabolites and 4 decreased metabolites, and 14 differential metabolites in the follicular fluid of IO cows included 8 increased metabolites and 6 decreased metabolites. These differential metabolites mainly involved nine metabolic pathways. The common enrichment pathway of different metabolites in serum and follicular fluid were glycerophospholipid metabolism and pentose and glucuronate interconversions. In conclusion, there were significant differences in the differential metabolites and enrichment pathways between serum and follicular fluid of IO cows, implying that there were complex changes in blood metabolism and local follicular metabolism of IO cows, whose interactions need further investigation.

Plasma Proteomics Characteristics of Subclinical Vitamin E Deficiency of Dairy Cows During Early Lactation.

Vitamin E (VE) is an essential fat-soluble nutrient for dairy cows. Vitamin E deficiency leads to immune suppression and oxidative stress and increases the susceptibility of cows to reproductive disorders in the early post-partum period. However, studies on plasma proteomics of VE deficiency have not been reported so far. Therefore, the purpose of this study was to understand the changes of blood protein profile in cows with subclinical VE deficiency in the early post-partum period. In this study, plasma protein levels of 14 healthy cows (>4 µg/ml α-tocopherol) and 13 subclinical VE-deficient cows (2-3 µg/ml α-tocopherol) were analyzed by tandem mass tag (TMT). The results showed that there were 26 differentially expressed proteins (DEPs) in the plasma of cows with subclinical VE deficiency compared with healthy controls. Twenty-one kinds of proteins were downregulated, and five kinds were upregulated, among which eight proteins in protein-protein interactions (PPI) network had direct interaction. These proteins are mainly involved in the MAPK signaling pathway, pantothenic acid and coenzyme A (CoA) biosynthesis, PPAR signaling pathway, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. The top four DEPs in PPI (APOC3, APOC4, SAA4, PHLD) and one important protein (VNN1) by literature review were further verified by ELISA and Western blot. The expression levels of APOC3, VNN1, and SAA4 were significantly lower than those of healthy controls by ELISA. VNN1 was significantly lower than those of healthy controls by Western blot. VNN1 is closely related to dairy cow subclinical VE deficiency and can be a potential biomarker. It lays a foundation for further research on the lack of pathological mechanism and antioxidative stress of VE.

Inhibitory Effect of Bovine Adipose-Derived Mesenchymal Stem Cells on Lipopolysaccharide Induced Inflammation of Endometrial Epithelial Cells in Dairy Cows.

Endometritis is a disease that affects reproductive health in dairy cows and causes serious economic damage to the dairy industry world-wide. Although in recent years, the application of mesenchymal stem cell (MSC) therapy for the treatment of inflammatory diseases has attracted much attention, there are few reports of the use of MSCs in dairy cows. In the present study, our objective was to explore the inhibitory effects of bovine adipose-derived mesenchymal stem cells (bAD-MSCs) on lipopolysaccharide (LPS) induced inflammation in bovine endometrial epithelial cells (bEECs) along with the potential underlying molecular mechanisms. We characterized isolated bAD-MSCs using cell surface marker staining and adipogenic/osteogenic differentiation, and analyzed them using immunofluorescence, flow cytometry (surface marker staining), and adipogenic and osteogenic differentiation. Furthermore, to understand the anti-inflammatory effects of bAD-MSCs on LPS induced bEEC inflammation, we used a bAD-MSC/bEEC co-culture system. The results showed that bAD-MSC treatments could significantly decrease LPS induced bEEC apoptosis and pro-inflammatory cytokine expression levels, such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). Furthermore, our results showed that bAD-MSC treatments could also significantly downregulate LPS induced p38, IkB-a, and JAK1 phosphorylation and Bax protein expression levels, which are closely related to inflammatory progress and cellular apoptosis in bEECs. Our findings demonstrate that bAD-MSCs play an inhibitory role in LPS induced bEEC inflammation and provide new insights for the clinical therapy of endometritis in dairy cows.

GC/MS and LC/MS Based Serum Metabolomic Analysis of Dairy Cows With Ovarian Inactivity.

Metabolic disorders may lead to the inactive ovaries of dairy cows during early lactation. However, the detailed metabolic profile of dairy cows with inactive ovaries around 55 days postpartum has not been clearly elucidated. The objective of this study was to investigate the metabolic difference in cows with inactive ovaries and estrus from the perspective of serum metabolites. According to clinical manifestations, B-ultrasound scan, rectal examination, 15 cows were assigned to the estrus group (E; follicular diameter 15-20 mm) and 15 to the inactive ovary group (IO; follicular diameter <8 mm and increased <2 mm within 5 days over two examinations). The blood was collected from the tail vein of the cow to separate serum 55-60 days postpartum, and then milked and fasted in the morning. Serum samples were analyzed using gas chromatography time-of-flight mass spectrometry technology (GC-TOF-MS) and ultra-high-pressure liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF-MS). Differences in serum metabolites were identified using multivariate statistical analysis and univariate analysis. Thirty differentially abundant metabolites were identified between the two groups. In cows with inactive ovaries compared with cows in estrus, 20 serum metabolites were significantly higher (beta-cryptoxanthin (p = 0.0012), 9-cis-retinal (p = 0.0030), oxamic acid (p = 0.0321), etc.) while 10 metabolites were significantly lower (monostearin (p = 0.0001), 3-hydroxypropionic acid (p = 0.0005), D-talose (p = 0.0018), etc.). Pathway analysis indicated that the serum differential metabolites of multiparous cows in estrus obtained by the two metabolomics techniques were mainly involved in ß-alanine metabolism and steroid biosynthesis metabolism, while other involved metabolic pathways were related to metabolism of glyoxylate; dicarboxylate metabolism; fructose, mannose, glutathione, glycerolipid, glycine, serine, threonine, propanoate, retinol, and pyrimidine metabolism. This indicates that the abnormalities in glucose metabolism, lipid metabolism, amino acid metabolism, and glutathione metabolism of postpartum dairy cows obstructed follicular development.

Knockdown of phosphatase and tensin homolog (PTEN) inhibits fatty acid oxidation and reduces very low density lipoprotein assembly and secretion in calf hepatocytes.

Dairy cows with fatty liver exhibit hepatic lipid accumulation and disturbances in fatty acid oxidation and lipid transport. Phosphatase and tensin homolog (PTEN), a lipid phosphatase, regulates intrahepatic fatty acid oxidation and lipid transport in mice. Whether PTEN play a role in fatty acid oxidation and very low density lipoprotein (VLDL) assembly in calf hepatocytes are unknown. Hepatocytes isolated from 3 healthy female Holstein calves (1 d old, 30-40 kg) were infected with empty adenovirus with green fluorescent protein for 48 h (Ad-GFP group) or infected with PTEN knockdown adenovirus for 48 h (Ad-shPTEN group), or cultured in RPMI-1640 without Ad-shPTEN or Ad-GFP (control group). Compared with the Ad-GFP group, PTEN knockdown decreased mRNA and protein abundance and the activity of fatty acid oxidation-related molecules, including acyl-coA synthetase long-chain 1, carnitine palmitoyltransferase 1, carnitine palmitoyltransferase 2, and 3-hydroxy acyl-coA dehydrogenase. Furthermore, PTEN knockdown decreased mRNA and protein abundance of VLDL assembly-related molecules, including apolipoprotein B100, apolipoprotein E, microsomal triglyceride transfer protein, and low density lipoprotein receptor. Importantly, PTEN knockdown promoted triglyceride accumulation in hepatocytes and reduced the VLDL content in culture medium. A subsequent study was conducted on the following 4 groups: cells infected with Ad-GFP for 48 h and then treated with 2% BSA for another 24 h (Ad-GFP + BSA); cells infected with Ad-GFP for 48 h and then treated with 1.2 mM free fatty acids (FFA) and 2% BSA for another 24 h (Ad-GFP + 1.2 mM FFA); cells infected with Ad-shPTEN for 48 h and then treated with 2% BSA for another 24 h (Ad-shPTEN + BSA); cells infected with Ad-shPTEN for 48 h and then treated with 1.2 mM FFA and 2% BSA for another 24 h (Ad-shPTEN + 1.2 mM FFA). Compared with Ad-GFP + BSA, the abundances of PTEN and of fatty acid oxidation- and VLDL assembly-related proteins were lower in the Ad-GFP + 1.2 mM FFA group. Importantly, PTEN knockdown heightened the increase in triglyceride accumulation of hepatocytes and the decrease in VLDL content in culture medium induced by FFA. Overall, these in vitro data indicate that FFA inhibits PTEN expression, leading to triglyceride accumulation and the inhibition of VLDL assembly in calf hepatocytes. These findings suggest that PTEN may be a potential therapeutic target for FFA-induced hepatic steatosis in dairy cows.

Metabolic alterations in dairy cows with subclinical ketosis after treatment with carboxymethyl chitosan-loaded, reduced glutathione nanoparticles.

BACKGROUND: Subclinical ketosis (SCK) causes economic losses in the dairy industry because it reduces the milk production and reproductive performance of cows. HYPOTHESIS/OBJECTIVES: To evaluate whether carboxymethyl chitosan-loaded reduced glutathione (CMC-rGSH) nanoparticles can alleviate the incidence or degree of SCK in a herd. ANIMALS: Holstein dairy cows 21 days postpartum (n = 15). METHODS: The trial uses a prospective study. Five cows with serum ß-hydroxybutyric acid (BHBA) ≥1.20 mmol/L and aspartate aminotransferase (AST) <100 IU/L were assigned to group T1, 5 cows with BHBA ≥1.20 mmol/L and AST >100 IU/L to group T2, and 5 cows with BHBA <1.00 mmol/L and AST <100 IU/L to group C. Carboxymethyl chitosan-loaded reduced glutathione (0.012 mg/kg body weight per cow) was administered to cows in T1 and T2 once daily via jugular vein for 6 days after diagnosis. Serum from all groups were collected 1 day before administration, then on days 1, 3, 5, 7, 10, and 15 after administration to determine the changes in biochemical index and 1 H-NMR. RESULTS: The difference in liver function or energy metabolism indices in T1, T2, and C disappeared at day 7 and day 10 after the administration (P > .05). Valine, lactate, alanine, lysine, creatinine, glucose, tyrosine, phenylalanine, formate, and oxalacetic acid levels, and decrease in isoleucine, leucine, proline, acetate, trimethylamine N-oxide, glycine, and BHBA levels were greater (P < .05) at day 7 than day 0 for cows in T2. CONCLUSIONS AND CLINICAL IMPORTANCE: Carboxymethyl chitosan-loaded reduced glutathione treatment might alleviate SCK by enhancing gluconeogenesis and reducing ketogenesis in amino acids.

Blood urea nitrogen may serve as a predictive indicator of retained placenta in dairy cows.

Retained placentae (RP) results in significant economic losses to dairy farmers. In Experiment 1, to screen biochemical indicators of RP, 21 cows with RP and 21 cows with no retained placenta (NRP) were selected as a control group, and blood was collected at -7 d, 0 h (parturition) and 12 h. Serum biochemical indicators were ascertained. Results indicate serum concentrations of phosphorus (P) and blood urea nitrogen (BUN) in cows of the RP group were markedly greater than in cows of the NRP group at -7 d (P < 0.01). In Experiment 2, to evaluate predictive indicators for RP, 34 cows with RP and 34 cows with NRP were selected, and there was blood sampling at -15 d, -10 d, -7 d, -4 d, and -1 d. Serum P, BUN, and total protein (TP) were evaluated. Associations of values among the three indicators and occurrence of RP were analyzed using binary logistic regression. Results indicate there was a negative correlation between only the values for BUN and RP (P = 0.016). In Experiment 3, to test hypothesis that relatively greater concentrations of BUN effects immune function in placental tissues, four cows were selected, placentae were collected at 0 and 12 h, and hematoxylin-eosin (HE) staining was performed. Results indicated that the extent of inflammatory cell infiltration and vascular proliferation were less at the 12 than 0-hour timepoint. Taken together, BUN at -7 d may serve as a predictive indicator of RP in cows.

Comparison of Metabolic Alterations in Serum and Milk Whey Between Inactive Ovaries and Estrus Dairy Cows.

Inactive ovaries (IOs) affect the estrus cycle and timed artificial insemination (TAI) efficiency in dairy cows during early lactation. The objective of the experiment was to determine metabolic changes in the serum and milk whey of dairy cows with IO and estrus. Twenty-eight healthy postpartum Holstein cows in similar age, milk production, and body condition were selected at 30 days postpartum for tracking to 70 days postpartum, and estrus performance was recorded through Afi Farm® software. The ovarian status and follicular diameter of dairy cows were examined by an experienced breeder through B-ultrasound and rectal examination. Fourteen normal estrus cows were allocated to control group A and 14 cows with IO to group B, all at 30-70 days postpartum. The serum and milk whey in the two groups of cows at 70 days postpartum were used for non-targeted nuclear magnetic resonance (1H-NMR) analysis to measure the different metabolites of cows with IO. In group B compared with group A at 70 days postpartum, there was an increase in the milk whey of six different metabolites including succinate, creatine phosphate, glycine, myo-inositol, glycolate, and orotate and a decrease in the milk whey of seven metabolites, including alanine, creatinine, o-phosphorylcholine, lactose, taurine, galactose, and glucose-1-phosphate. There was an increase in the serum of group B cows of four differential metabolites, including 3-hydroxybutyrate, acetate, glutamine, and glycine and a decrease in the serum of nine differential metabolites, including alanine, succinate, citrate, creatinine, o-phosphocholine, glucose, myo-inositol, tyrosine, and histidine compared with group A. Group B cows with IO had decreased glucose metabolism and impaired tricarboxylic acid cycle, increased lipid mobilization, and abnormal amino acid metabolism. The study provides a potential prevention strategy for IO in dairy cows in future.

SREBP-1c gene silencing can decrease lipid deposits in bovine hepatocytes cultured in vitro.

BACKGROUND: Fatty liver is a major metabolic disorder that occurs during early lactation in high-producing dairy cows. Sterol regulatory element-binding protein-1c (SREBP-1c) is an important transcription factor that regulates lipid synthesis by regulating the expression of lipid metabolism genes. METHODS: In this study, we reduced the expression of SREBP-1c by adenovirus-mediated SREBP-1c with a low expression vector (AD-GFP-SREBP-1c) to study the effects of SREBP-1c on lipid deposits in bovine hepatocytes. The expression levels and enzyme activities of SERBP-1c and its target genes were determined by real-time PCR, western blot, and ELISA. RESULTS: These results showed that Ad-GFP-SREBP-1c could inhibit SREBP-1c expression. The expression of the lipid synthesis enzyme acetyl-CoA carboxylase (ACC) was down-regulated. The expression levels of the lipid oxidation enzymes long-chain fatty acyl-COA synthetase (ACSL-1), carnitine palmitoyltransferase І (CPT-І), carnitine palmitoyltransferase II (CPT- II), and ß-hydroxyacyl-CoA-DH (HADH) were significantly elevated. Furthermore, the expression levels of factors involved in the assembly and transport of very low-density lipoproteins (VLDLs), such as apolipoprotein B100 (ApoB), apolipoprotein E (ApoE), and microsomal triglyceride transfer protein (MTTP) were decreased comparison with the negative control and the blank control groups, but the low-density lipoprotein receptor (LDLR) was elevated. The concentrations of TG (triglyceride) and VLDL were also reduced. CONCLUSION: These data suggest that low SREBP-1c expression can decrease lipid synthesis, increase lipid oxidation, and decrease the TG and VLDL content in bovine hepatocytes.

SREBP-1c overexpression induces triglycerides accumulation through increasing lipid synthesis and decreasing lipid oxidation and VLDL assembly in bovine hepatocytes.

The natural incidence of fatty liver in ruminants is significantly higher than in monogastric animals. Fatty liver is associated with sterol regulatory element-binding protein 1c (SREBP-1c). The aim of this study was to investigate the regulatory network effects of SREBP-1c on the lipid metabolic genes involved in fatty acid uptake, activation, oxidation, synthesis, and very low-density lipoprotein (VLDL) assembly in bovine hepatocytes. In vitro, bovine hepatocytes were transfected with an adenovirus-mediated SREBP-1c overexpression vector. SREBP-1c overexpression significantly up-regulated the expression and activity of the fatty acid uptake, activation, and synthesis enzymes: liver fatty acid binding protein, fatty acid translocase, acyl-CoA synthetase long-chain 1, acetyl-CoA carboxylase 1, and fatty acid synthase, increasing triglyceride (TG) synthesis and accumulation. SREBP-1c overexpression down-regulated the expression and activity of the lipid oxidation enzymes: carnitine palmitoyltransferase 1 and carnitine palmitoyltransferase 2. Furthermore, the apolipoprotein B100 expression and microsomal triglyceride transfer protein activity were significantly decreased. SREBP-1c overexpression reduced lipid oxidation and VLDL synthesis, thereby decreasing TG disposal and export. Therefore, large amounts of TG accumulated in the bovine hepatocytes. Taken together, these results indicate that SREBP-1c overexpression increases lipid synthesis and decreases lipid oxidation and VLDL export, thereby inducing TG accumulation in bovine hepatocytes.

Non-esterified fatty acids activate the AMP-activated protein kinase signaling pathway to regulate lipid metabolism in bovine hepatocytes.

Non-esterified fatty acids (NEFAs) act as signaling molecules involved in regulating genes expression to modulate lipid metabolism. However, the regulation mechanism of NEFAs on lipid metabolism in dairy cows is unclear. The AMP-activated protein kinase (AMPK) signaling pathway plays a key role in regulating hepatic lipid metabolism. In vitro, bovine hepatocytes were cultured and treated with different concentrations of NEFAs and AMPKα inhibitors (BML-275). NEFAs increased AMPKα phosphorylation through up-regulating the protein levels of liver kinase B1. Activated AMPKα increased the expression and transcriptional activity of peroxisome proliferator-activated receptor α (PPARα). NEFAs also directly activate the PPARα independent of AMPKα. Activated PPARα increased the lipolytic genes expression to increase lipid oxidation. Furthermore, activated AMPKα inhibited the expression and transcriptional activity of the sterol regulatory element-binding protein 1c and carbohydrate responsive element-binding protein, which reduced the expression of lipogenic genes, thereby decreasing lipid synthesis. Activated AMPKα phosphorylated and inhibited acetyl-CoA carboxylase and increased carnitine palmitoyltransferase-1 activity, which increased lipid oxidation. Consequently, the triglyceride content in the NEFAs-treated hepatocytes was significantly decreased. These results indicate that NEFAs activate the AMPKα signaling pathway to increase lipid oxidation and decrease lipid synthesis in hepatocytes, which in turn, generates more ATP to relieve the negative energy balance in transition dairy cows.

Increase of fatty acid oxidation and VLDL assembly and secretion overexpression of PTEN in cultured hepatocytes of newborn calf.

BACKGROUND: Phosphatase and tensin homolog (PTEN) is a potent tumor suppressor gene that also plays a vital role in regulating fatty acid metabolism. Here we attempted to elucidate the role of PTEN in the regulation of fatty acid oxidation and the assembly and secretion of very low density lipoprotein (VLDL) in dairy cow liver. METHODS: We transfected primary culture calf hepatocytes with adenovirus-mediated PTEN overexpression vector (AD-GFP-PTEN).PTEN-overexpressing hepatocytes and control hepatocytes were obtained. RESULTS: Compared with controls, overexpression of PTEN significantly up-regulated CPT I, ACSL, HADH expression (p<0.05), which are all involved in fatty acid oxidation. At the same time, the expression of ApoB100 (p<0.01), ApoE (p<0.05) and MTP (p<0.01) increased. Therefore, the assembly and secretion of VLDL was enhanced (p<0.05). The expression of LDLR was slightly up-regulated, but there was no significant difference (p>0.05). To demonstrate that fatty acid metabolism was changed, we measured the concentrations of TG and VLDL. The concentration of TG was significantly decreased in hepatocytes (p<0.01), while the concentration of VLDL was significantly increased in the medium (P<0.05). CONCLUSIONS: Overexpressing PTEN enhanced fatty acid oxidation and assembly and secretion of VLDL. PTEN gene therapy could have therapeutic potential for fatty liver diseases of dairy cattle.

Effect of NEFA and glucose levels on CPT-I mRNA expression and translation in cultured bovine hepatocytes.

The objective of this study was to determine the effects of NEFA and glucose on carnitine palmitoyltransferase-I (CPT-I) mRNA expression in cultured bovine hepatocytes using real-time reverse transcription polymerase chain reaction and ELISA methods. The results indicated that CPT-I transcription increased gradually, but that CPT-I translation was not significantly changed, with glucose concentrations ranging from 0 to 3.0 mmol/L (P<0.01). Furthermore CPT-I transcription and translation were enhanced significantly when the NEFA concentrations increased from 0 to 1.2 mmol/L and decreased significantly when the NEFA concentrations increased from 1.2 to 4.8 mmol/L (P<0.01). A high concentration NEFA was found to reduce fatty acid oxidation, potentially explaining the development from NEB to ketosis in dairy cows.