trans-10,cis-12 conjugated linoleic acid alters lipid metabolism of goat mammary epithelial cells by regulation of de novo synthesis and the AMPK signaling pathway.

The trans-10,cis-12 isomer of conjugated linoleic acid (t10c12-CLA) is a biohydrogenation intermediate in the rumen and has been shown to cause milk fat depression in dairy goats. However, few studies have focused on the in vitro molecular mechanisms involved in the response of the goat mammary gland to t10c12-CLA. In the present study, RNA sequencing technology was used to investigate the effects of t10c12-CLA on goat mammary epithelial cells. From the data, 25,153 annotated transcripts were obtained, and differentially expressed genes were selected based on a false discovery rate <0.05. Candidate genes and potent cellular signaling pathways were identified through Gene Ontology (GO) and pathway analysis. Next, real-time quantitative PCR and Western blot analyses were used to verify the results of the RNA sequencing data. The results indicated that t10c12-CLA inhibits fatty acid synthesis through downregulation of genes involved in de novo fatty acid synthesis, and this process is likely correlated with the activation of the AMP-activated protein kinase signaling pathways.

Overexpression of SREBP1 (sterol regulatory element binding protein 1) promotes de novo fatty acid synthesis and triacylglycerol accumulation in goat mammary epithelial cells.

Sterol regulatory element binding protein 1 (SREBP1; gene name SREBF1) is known to be the master regulator of lipid homeostasis in mammals, including milk fat synthesis. The major role of SREBP1 in controlling milk fat synthesis has been demonstrated in bovine mammary epithelial cells. Except for a demonstrated role in controlling the expression of FASN, a regulatory role of SREBP1 on milk fat synthesis is very likely, but has not yet been demonstrated in goat mammary epithelial cells (GMEC). To explore the regulatory function of SREBP1 on de novo fatty acids and triacylglycerol synthesis in GMEC, we overexpressed the mature form of SREBP1 (active NH2-terminal fragment) in GMEC using a recombinant adenovirus vector (Ad-nSREBP1), with Ad-GFP (recombinant adenovirus of green fluorescent protein) as control, and infected the GMEC for 48 h. In infected cells, we assessed the expression of 20 genes related to milk fat synthesis using real time-quantitative PCR, the protein abundance of SREBP1 and FASN by Western blot, the production of triacylglycerol, and the fatty acid profile. Expression of SREBF1 was modest in mammary compared with the other tissues in dairy goats but its expression increased approximately 30-fold from pregnancy to lactation. The overexpression of the mature form of SREBP1 was confirmed by >200-fold higher expression of SREBF1 in Ad-nSREBP1 compared with Ad-GFP. We observed no changes in amount of the precursor form of SREBP1 protein but a >10-fold increase of the mature form of SREBP1 protein with Ad-nSREBP1. Compared with Ad-GFP cells (control), Ad-nSREBP1 cells had a significant increase in expression of genes related to long-chain fatty acid activation (ACSL1), transport (FABP3), desaturation (SCD1), de novo synthesis of fatty acids (ACSS2, ACLY, IDH1, ACACA, FASN, and ELOVL6), and transcriptional factors (NR1H3 and PPARG). We observed a >10-fold increase in expression of INSIG1 but SCAP was downregulated by Ad-nSREBP1. Among genes related to milk fat synthesis and lipid droplet formation, only LPIN1 and DGAT1 were upregulated by Ad-nSREBP1. Compared with the Ad-GFP, the cellular triacylglycerol content was higher and the percentage of C16:0 and C18:1 increased, whereas that of C16:1, C18:0, and C18:2 decreased in Ad-nSREBP1 cells. Overall, the data provide strong support for a central role of SREBP1 in the regulation of milk fat synthesis in goat mammary cells.

Sterol regulatory element binding protein-1 (SREBP-1)c promoter: Characterization and transcriptional regulation by mature SREBP-1 and liver X receptor α in goat mammary epithelial cells.

Sterol regulatory element binding protein-1 (SREBP-1) is a key transcription factor that regulates lipogenesis in rodent liver. Two isoforms (SREBP-1a and SREBP-1c) of SREBP-1 are transcribed by an alternative promoter on the same gene (SREBF1), and the isoforms differ only in their first exon. Although the regulatory effects of SREBP-1 on lipid and milk fat synthesis have received much attention in ruminants, SREBP-1c promoter and its regulatory mechanisms have not been characterized in the goat. In the present study, we cloned and sequenced a 2,012-bp fragment of the SREBP-1c 5'-flanking region from goat genomic DNA. A luciferase reporter assay revealed that SREBP-1c is transcriptionally activated by the liver X receptor α (LXRα) agonist T0901317, and is decreased by SREBP-1 small interfering (si)RNA. A 5' deletion analysis revealed a core promoter region located -395 to +1 bp upstream of the transcriptional start site (TSS). Site-directed mutagenesis of LXRα binding elements (LXRE1 and LXRE2) and sterol regulatory elements (SRE1 and SRE2) revealed that the full effects of T 4506585 require the presence of both LXRE and SRE. We also characterized a new SRE (SRE1) and demonstrated a direct role of SREBP-1 (auto-loop regulation) in maintaining its basal transcription activity. Results suggest that goat SREBP-1c gene is transcriptionally regulated by mature SREBP-1 (auto-loop circuit regulation) and LXRα in goat mammary epithelial cells.

Adipocyte differentiation-related protein promotes lipid accumulation in goat mammary epithelial cells.

Milk fat originates from the secretion of cytosolic lipid droplets (CLD) synthesized within mammary epithelial cells. Adipocyte differentiation-related protein (ADRP; gene symbol PLIN2) is a CLD-binding protein that is crucial for synthesis of mature CLD. Our hypothesis was that ADRP regulates CLD production and metabolism in goat mammary epithelial cells (GMEC) and thus plays a role in determining milk fat content. To understand the role of ADRP in ruminant milk fat metabolism, ADRP (PLIN2) was overexpressed or knocked down in GMEC using an adenovirus system. Immunocytochemical staining revealed that ADRP localized to the surface of CLD. Supplementation with oleic acid (OA) enhanced its colocalization with CLD surface and enhanced lipid accumulation. Overexpression of ADRP increased lipid accumulation and the concentration of triacylglycerol in GMEC. In contrast, morphological examination revealed that knockdown of ADRP decreased lipid accumulation even when OA was supplemented. This response was confirmed by the reduction in mass of cellular TG when ADRP was knocked down. The fact that knockdown of ADRP did not completely eliminate lipid accumulation at a morphological level in GMEC without OA suggests that some other compensatory factors may also aid in the process of CLD formation. The ADRP reversed the decrease of CLD accumulation induced by adipose triglyceride lipase. This is highly suggestive of ADRP promoting triacylglycerol stability within CLD by preventing access to adipose triglyceride lipase. Collectively, these data provide direct in vitro evidence that ADRP plays a key role in CLD formation and stability in GMEC.

Association between nasal colonization of Staphylococcus aureus and surgical site infections in spinal surgery patients: a systematic review and meta-analysis.

OBJECTIVE: The study aimed at examining the relationship between nasal colonization of Staphylococcus aureus (SA) or methicillin-resistant Staphylococcus aureus (MRSA) and the risk of SSI after spinal surgeries MATERIALS AND METHODS: PubMed, CENTRAL, Scopus, Web of Science, and Embase databases up to 24th September 2022 for articles on nasal colonization of SA/MRSA and spine surgeries. RESULTS: Ten studies were included. Meta-analysis revealed that the incidence of SSI was not significantly different between SA-positive and SA-negative patients (RR: 0.75, 95% CI: 0.47, 1.18 I2=2% p=0.21). It was noted that when no decolonization was done, there was no statistically significant difference in the risk of SSI between MRSA positive and MRSA negative patients, but a tendency of higher SSI in MRSA carriers (RR: 2.40, 95% CI: 0.91, 6.32, I2=37% p=0.08). However, in the subgroup analysis with decolonization, the risk of SSI was significantly higher in the MRSA-positive group (RR: 2.99, 95% CI: 1.27, 7.03, I2=24% p=0.01). Specifically, the risk of MRSA-SSI was significantly higher in MRSA carriers with (RR: 6.05, 95% CI: 1.14, 31.99, I2=43% p=0.03) and without decolonization (RR: 7.54, 95% CI: 1.43, 39.85, I2=38% p=0.02). CONCLUSIONS: Evidence from observational studies indicates that only MRSA nasal colonization increases the risk of SSIs in spinal surgery patients. Nasal decolonization was unable to reduce the risk of overall or MRSA-specific SSIs in MRSA carriers. Evidence was biased due to the extremely small number of MRSA-positive patients in the studies and the lack of adjustment of confounding factors.

MicroRNA-150 alleviates acute myocardial infarction through regulating cardiac fibroblasts in ventricular remodeling.

OBJECTIVE: The aim of this study was to investigate the effect of microRNA-150 on the regulation of myocardial fibrosis and ventricular remodeling in rats with acute myocardial infarction (AMI). MATERIALS AND METHODS: The AMI rats model was established by the ligation of the left anterior descending coronary artery (LAD) in vivo. After AMI procedures, the rats were injected with microRNA-150 lentivirus overexpression or negative control, respectively. Cardiac function of rats was evaluated by echocardiography. Hematoxylin and eosin (HE) staining and Masson trichrome were performed to evaluate myocardial fibrosis in each rat. Meanwhile, cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method. The expression levels of microRNA-150, col1α1, col1α2, col3 and α smooth muscle actin (α-SMA) in the border zone of rat infarct myocardium were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. RESULTS: MicroRNA-150 expression in the border zone of infarct myocardium decreased significantly at day 28 after AMI (p<0.05). Overexpressing microRNA-150 significantly improved cardiac function, decreased collagen volume fraction (CVF) and attenuated cardiomyocyte apoptosis in rats. Furthermore, the expression levels of col1ɑ1, col1ɑ2, col3 and α-SMA in the border zone of infarct myocardium were remarkably down-regulated in rats overexpressing microRNA-150 compared with those of controls (p<0.001). CONCLUSIONS: MicroRNA-150 expression in the border zone of rat infarct myocardium decreased at day 28 after AMI. In addition, the upregulation of microRNA-150 in myocardial tissue could inhibit myocardial fibrosis and improve ventricular remodeling at post-AMI.

Derivation of a simple clinical model to categorize the probability of acute myocardial infarction in patients with atrial fibrillation.

OBJECTIVE: Atrial fibrillation (AF) is independently associated with a higher risk of acute myocardial infarction (AMI). The occurrence of AMI in AF patients may lead to dismal prognosis. Risk assessment is a fundamental component of prevention for AMI. PATIENTS AND METHODS: 2419 consecutive patients with nonvalvular AF were enrolled in this retrospective study. A logistic regression analysis was performed on clinical variables to create a simple clinical prediction rule. The following nine variables and assigned scores (in brackets) were included in the prediction rule: age ≥65 years (1.0), heart failure (1.0), hypertension (1.0), diabetes mellitus (1.0), hyperlipidemia (0.5), history of stroke/TIA (0.5), vascular disease (1.0), current smoking (0.5), and resting heart rate >90 beats/min (1.0). Patients were considered to have a low probability if the score was ≤2.5, moderate if the score was 3.0 to 4.0, and high if the score was ≥4.5. The AMI unlikely was assigned to patients with scores <3.5 and AMI likely if the score was ≥3.5. To evaluate the score, we included an external validation cohort of 1810 nonvalvular AF patients from the Cardiology Center, Shandong Provincial Hospital affiliated to Shandong University, Jinan, China. RESULTS: The score showed a good ability in discriminating AF patients experiencing AMI both in the internal derivation cohort, with a c-index of 0.80 [95% Confidence Interval (CI) 0.77-0.83, p<0.001] and in the external validation cohort (c-index 0.73, 95% CI 0.69-0.77, p<0.001). Our scoring system offered significantly better predictive performance than the CHA2DS2-VASc score (c-index 0.80 vs 0.71, p<0.001). CONCLUSIONS: Our scoring system is a simple and accurate way of predicting the risk of AMI in AF patients. Therefore, more accurate targeting of preventive therapy will be allowed.

Leucine regulates α-amylase and trypsin synthesis in dairy calf pancreatic tissue in vitro via the mammalian target of rapamycin signalling pathway.

Starch digestion in the small intestines of the dairy cow is low, to a large extent, due to a shortage of syntheses of α-amylase. One strategy to improve the situation is to enhance the synthesis of α-amylase. The mammalian target of rapamycin (mTOR) signalling pathway, which acts as a central regulator of protein synthesis, can be activated by leucine. Our objectives were to investigate the effects of leucine on the mTOR signalling pathway and to define the associations between these signalling activities and the synthesis of pancreatic enzymes using an in vitro model of cultured Holstein dairy calf pancreatic tissue. The pancreatic tissue was incubated in culture medium containing l-leucine for 3 h, and samples were collected hourly, with the control being included but not containing l-leucine. The leucine supplementation increased α-amylase and trypsin activities and the messenger RNA expression of their coding genes (P <0.05), and it enhanced the mTOR synthesis and the phosphorylation of mTOR, ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1 (P <0.05). In addition, rapamycin inhibited the mTOR signal pathway factors during leucine treatment. In sum, the leucine regulates α-amylase and trypsin synthesis in dairy calves through the regulation of the mTOR signal pathways.