FOXO1 regulates bovine skeletal muscle cells differentiation by targeting MYH3.

The growth and development of bovine skeletal muscle and beef yield is closely intertwined. Our previous research found that forkhead box O1 (FOXO1) plays an important role in the regulation of beef muscle formation, but its specific mechanism is still unknown. In this study, we aimed to clarify the regulatory mechanism of FOXO1 in proliferation and differentiation of bovine skeletal muscle cells (BSMCs). The results showed that interfering with FOXO1 can promote proliferation and the cell G1/S phase of BSMCs by up-regulating the expression of PCNA, CDK1, CDK2, CCNA2, CCNB1, CCND1 and CCNE2. Besides, interfering with FOXO1 inhibited the apoptosis of BSMCs by up-regulating the expression of anti-apoptosis gene BCL2, while simultaneously down-regulating the expression of the pro-apoptosis genes BAD and BAX. Inversely, interfering with FOXO1 can promote the differentiation of BSMCs by up-regulating the expression of myogenic differentiation marker genes MYOD, MYOG, MYF5, MYF6 and MYHC. Furthermore, RNA-seq combined with western bolt, immunofluorescence and chromatin immunoprecipitation analysis showed that FOXO1 could regulate BSMCs differentiation process by influencing PI3K-Akt, Relaxin and TGF-beta signaling pathways, and target MYH3 for transcriptional inhibition. In conclusion, this study provides a basis for studying the role and molecular mechanism of FOXO1 in BSMCs.

A case report of cardia carcinoma: Underwent Heller myotomy for Achalasia.

BACKGROUND: One of the risk factors for esophageal adenocarcinoma is achalasia, an esophageal motility disorder that is typically treated surgically through laparotomy or laparoscopic surgery. The risk factors of gastric cardia cancer are also similar to esophageal adenocarcinoma due to the anatomical location of the gastric cardia close to the esophagus. There is currently no clinical evidence that achalching has a correlation with gastric cardia cancer. CASE SUMMARY: We report the case of an 85-year-old female patient was admitted to our department with dysphagia for 6 months. She underwent a dissecting Heller myotomy for pancreatic achalasia in 2006, with occasional postoperative symptoms of reflux and heartburn. Outpatient upper gastrointestinal imaging was suggestive of cardia cancer, and gastroscopic pathological findings were suggestive of moderately-lowly-differentiated adenocarcinoma. The patient was admitted to the operating room on August 30, 2022 to undergo radical pancreatic cancer surgery plus abdominal adhesion release, and postoperative review of the upper gastrointestinal imaging showed a patent anastomosis with no spillage, filling of the residual stomach, and duodenal visualization. CONCLUSION: Postoperative patients with achalasia often have symptoms of reflux, which may be one of the factors for the development of pancreatic cancer in this patient, thus requiring clinicians to pay more attention to the use of antireflux procedures in the surgical treatment of pancreatic achalasia. And the choice of which modality to perform surgery in patients with previous surgical history is also one of the points to be discussed.

RNA-seq reveals the role of miR-223 in alleviating inflammation of bovine mammary epithelial cells.

Bovine mammary epithelial cells (bMECs) are involved in the early defense against the invasion of intramammary pathogens and are essential for the health of bovine mammary gland. MicroRNA (MiRNA) is a key factor that regulates cell state and physiological function. In the present study, the transcriptome profiles of miR-223 inhibitor transfection group (miR-223_Inhibitor) and negative control inhibitor transfection group (NC_Inhibitor) within bMECs were detected via the RNA sequencing (RNA-seq) platform. Based on these experiments, the differentially expressed mRNAs (DE-mRNAs) of the miR-223_Inhibitor transfection group were screened, and the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional analyses of DE-mRNAs were performed. The results revealed that compared with the NC_Inhibitor, 224 differentially expressed genes (DEGs) were identified in the miR-223_Inhibitor, including 184 upregulated and 40 downregulated genes. The functional annotation of the above DEGs indicated that some of these genes are involved in the immune response generated by extracellular substance stimulation, regulation of the activity of cytokines and chemokines, and the immune signaling pathways of NF-κB and TNF. Meanwhile, miR-223_inhibitor upregulated the immune key genes IRF1 and NFκBIA, cytokines IL-6 and IL-24, as well as chemokines CXCL3, CXCL5, and CCR6, triggering a signaling cascade response that exacerbated inflammation in bMECs. These results suggested that miR-223 plays an important role in inhibiting the inflammatory response and maintaining the stability of bMECs, and is a potential target for treating mastitis in dairy cows.

NAD+ Biosynthesis Impairment and Acute Kidney Injury after Major Vascular Surgery.

Acute kidney injury (AKI) is a serious complication after vascular surgery. Reduced synthesis of nicotinamide adenine dinucleotide (NAD+) from tryptophan is associated with an increased risk of AKI in critically ill patients, patients hospitalized with COVID-19, and cardiac surgery patients, and is marked by elevated urinary quinolinate and quinolinate to tryptophan ratios. We measured quinolinate concentrations in vascular surgery patients to determine if impaired NAD+ synthesis was associated with AKI in this patient population. Eight preoperative and eight postoperative vascular surgery patients who developed AKI were selected from a parent study to participate in this single-center case-control study. They were matched with controls who did not develop AKI based on age, sex, BMI, eGFR, hypertension, and diabetes. Urinary quinolinate and tryptophan concentrations were measured at anesthetic induction and on postoperative day one. Two-sided Mann-Whitney U tests were used to compare quinolinate and quinolinate to tryptophan ratios. Multivariate linear regression modeling was used to estimate the relationship between quinolinate and serum creatinine. There was no difference in preoperative or postoperative urine quinolinate concentrations or the preoperative quinolinate to tryptophan ratio between patients that did and did not develop AKI (p = 0.07, 0.50, and 0.32, respectively). However, postoperative quinolinate to tryptophan ratios were higher in AKI patients (p = 0.04). Further, after adjustment for AKI risk factors, higher preoperative quinolinate concentrations and higher postoperative quinolinate to tryptophan ratios were associated with greater postoperative creatinine increases (p = 0.04 and 0.04, respectively). These data suggest that impaired NAD+ synthesis may contribute to AKI development in vascular surgery patients.

miR-302b promotes bovine preadipocyte differentiation and inhibits proliferation by targeting CDK2.

MicroRNAs have been recently reported to act as key regulators of adipogenesis, a multifactorial complex process. One miRNA, miR-302b, is an important regulator of cell proliferation and differentiation and controls cancer development, but we speculate that miR-302b may also regulate bovine adipogenesis. Herein we have evaluated the role of this miRNA in bovine adipocyte differentiation using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), Oil Red O staining, a dual-luciferase reporter. CDK2 was identified as the target gene of miR-302b, and miR-302b agomir promoted mRNA and protein expression levels of adipocyte-specific genes. In addition, a CCK-8 kit was used to show that miR-302b agomir, but not the negative control, inhibits preadipocyte proliferation. In conclusion, miR-302b promotes bovine preadipocyte differentiation and inhibits proliferation by targeting CDK2.

Bta-miR-199a-3p Inhibits LPS-Induced Inflammation in Bovine Mammary Epithelial Cells via the PI3K/AKT/NF-κB Signaling Pathway.

Mastitis is characterized by inflammatory damage to mammary gland tissue, which could decline milk production and quality and significantly affect the economic benefits of ranching. MicroRNAs (miRNAs), such as miR-199a-3p, are novel therapeutic targets in inflammation, and their regulation is an effective strategy for inflammation control. Despite its importance in humans and animals, the molecular mechanism of bovine miR-199a-3p (bta-miR-199a-3p) in dairy cow mastitis and bovine mammary epithelial cell (bMEC) inflammation is unclear. In our study, a bovine mammary epithelial cell line (MAC-T) induced by lipopolysaccharide (LPS) was used as an inflammatory cell model to investigate the molecular mechanism of bta-miR-199a-3p in the MAC-T inflammatory response. bta-miR-199a-3p was up-regulated in the LPS-induced MAC-T cells, while CD2-associated protein (CD2AP) was revealed as its target gene in a double luciferase reporter gene experiment. In addition, the overexpression of bta-miR-199a-3p negatively regulated the expression of CD2AP and the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/nuclear factor kappa-B (NF-κB) signaling pathway. These subsequently inhibited the secretion of related inflammatory factors (TNF-α, IL-1ß, and IL-6) and the expression of apoptotic genes (CASP3 and CASP9), thereby alleviating the LPS-challenged inflammatory response in the MAC-T cells. Silencing of bta-miR-199a-3p, however, reversed the above effects. Thus, bta-miR-199a-3p inhibits LPS-induced inflammation in bMECs by directly targeting CD2AP and regulating the PI3K/AKT/NF-κB signaling pathway. This study reveals the potential regulatory mechanism of bta-miR-199a-3p in bMEC inflammatory immune response and may serve as a useful target for the treatment of mastitis.

Roles of MEF2A and MyoG in the transcriptional regulation of bovine LATS2 gene.

As an important downstream effector gene in the hippo signaling pathway, large tumor suppressor gene 2 (LATS2) is involved in cell proliferation and differentiation, organ size and tissue regeneration, and plays an important role in regulating the growth and development of animal muscles. The purpose of this study is to explore the temporal expression of bovine LATS2 gene, and determine the key transcription factors for regulating bovine LATS2 gene. The result showed that bovine LATS2 gene was highly expressed in liver and longissimus dorsi, and was up-regulated in infancy muscle. In addition, it was highly expressed on the 2th day during the differentiation stage of myoblast. The upstream 1.7 Kb sequence of the 5 'translation region of bovine LATS2 gene was cloned, and 7 different deletion fragments were amplified by the upstream primers. These fragments were constructed into double luciferase reporter vectors and transfected into myoblasts and myotubes cells, respectively to detect the core promoter regions. In addition, the key transcription factors of the core promoter sequence of the bovine LATS2 gene were analyzed and predicted by online software. Combining with site-directed mutations, siRNA interference and chromatin immunoprecipitation technology, it was identified that MEF2A and MyoG combined in core promoter region (-248/-56) to regulate the transcription activity of bovine LATS2 gene. The results have laid a theoretical foundation for exploring the molecular regulation mechanism of LATS2 gene in the process of muscle growth.

Tissue Expression Analysis, Cloning, and Characterization of the 5'-Regulatory Region of the Bovine LATS1 Gene.

As a member of the large tumor suppressor (LATS) gene family, LATS1 plays an important role in regulating muscle growth and development. In this study, we determined the distinct exhibit patterns of tissue expression of bovine LATS1. Further, we determined the functional proximal minimal promoter of bovine LATS1 and identified the key transcription factors in the core promoter region to elucidate its molecular regulation mechanism. The results showed that bovine LATS1 was highly expressed in the longissimus thoracis and upregulation in infancy muscle. An electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay in combination with site-directed mutation and small interfering RNA (siRNA) interference demonstrated that myogenic differentiation 1 (Myod1) and myocyte enhancer factor 2A (MEF2A) binding in the core promoter region (-298/-123 bp) play important roles in the transcriptional regulation of the bovine LATS1 promoter. Taken together, these interactions provide insight into the regulatory mechanisms of LATS1 transcription in mediating skeletal muscle growth in cattle.

RNA-Seq Reveals the Role of miR-29c in Regulating Inflammation and Oxidative Stress of Bovine Mammary Epithelial Cells.

Healthy mammary gland is essential for milk performance in dairy cows. MicroRNAs (miRNAs) are the key molecules to regulate the steady state of mammary gland in dairy cows. This study investigated the potential role of miR-29c in bovine mammary epithelial cells (bMECs). RNA sequencing (RNA-seq) was used to measure the transcriptome profile of bovine mammary epithelial cells line (MAC-T) transfected with miR-29c inhibitor or negative control (NC) inhibitor, and then differentially expressed genes (DEGs) were screened. The results showed that a total of 42 up-regulated and 27 down-regulated genes were found in the miR-29c inhibitor group compared with the NC inhibitor group. The functional enrichment of the above DEGs indicates that miR-29c is a potential regulator of oxidative stress and inflammatory response in bMECs through multiple genes, such as forkhead box O1 (FOXO1), tumor necrosis factor-alpha (TNF-α), and major histocompatibility complex, class II, DQ alpha 5 (BoLA-DQA5) in the various biological process and signaling pathways of stress-activated mitogen-activated protein kinase (MAPK) cascade, Epstein-Barr virus infection, inflammatory bowel disease, etc. The results imply that miR-29c plays an important role in a steady state of bMECs or cow mammary gland and may be a potential therapeutic target for mastitis in dairy cows.

Differential expression of circRNAs related to lipopolysaccharide-induced inflammation in bovine mammary epithelial cells.

Circular RNAs (circRNAs) are widely involved in inflammatory responses, but their specific regulatory roles in cow mastitis remain controversial. In this study, RNA-seq was used to generate a circRNA expression profile, which identified 71 differentially expressed circRNAs (DEcircRNAs) in lipopolysaccharide (LPS)-stimulated MAC-T bovine mammary epithelial cells (bMECs) at different stages of inflammation. Functional analyses revealed that these DEcircRNAs may be involved in cellular proliferation, apoptosis, migration, and the inflammatory responses through regulation of numerous related signaling pathways. In addition, these data suggest that 2 novel circRNAs, named novel_circ_0004830 and novel_circ_0003097, may act as the key competing endogenous RNAs (ceRNAs) in the regulation of bovine mastitis through binding to inflammation-related microRNAs (miRNAs). These results provide a new angle for the study of the molecular regulatory mechanisms in dairy cow mastitis.

MiR-125b regulates inflammation in bovine mammary epithelial cells by targeting the NKIRAS2 gene.

Mastitis is a complex inflammatory disease caused by pathogenic infection of mammary tissue in dairy cows. The molecular mechanism behind its occurrence, development, and regulation consists of a multi-gene network including microRNA (miRNA). Until now, there is no report on the role of miR-125b in regulating mastitis in dairy cows. This study found that miR-125b expression is significantly decreased in lipopolysaccharide (LPS)-induced MAC-T bovine mammary epithelial cells. Also, its expression is negatively correlated with the expression of NF-κB inhibitor interacting Ras-like 2 (NKIRAS2) gene. MiR-125b target genes were identified using a double luciferase reporter gene assay, which showed that miR-125b can bind to the 3' untranslated region (3' UTR) of the NKIRAS2, but not the 3'UTR of the TNF-α induced protein 3 (TNFAIP3). In addition, miR-125b overexpression and silencing were used to investigate the role of miR-125b on inflammation in LPS-induced MAC-T. The results demonstrate that a reduction in miR-125b expression in LPS-induced MAC-T cells increases NKIRAS2 expression, which then reduces NF-κB activity, leading to low expression of the inflammatory factors IL-6 and TNF-α. Ultimately, this reduces the inflammatory response in MAC-T cells. These results indicate that miR-125b is a pro-inflammatory regulator and that its silencing can alleviate bovine mastitis. These findings lay a foundation for elucidating the molecular regulation mechanism of cow mastitis.

miR-223: An Effective Regulator of Immune Cell Differentiation and Inflammation.

MicroRNAs (miRNAs) play a critical role in regulating various biological processes, such as cell differentiation and immune modulation by binding to their target genes. miR-223 is a miRNA with important functions and has been widely investigated in recent years. Under certain physiological conditions, miR-223 is regulated by different transcription factors, including sirtuin1 (Sirt1), PU.1 and Mef2c, and its biological functions are mediated through changes in its cellular or tissue expression. This review paper summarizes miR-223 biosynthesis and its regulatory role in the differentiation of granulocytes, dendritic cells (DCs) and lymphocytes, macrophage polarization, and endothelial and epithelial inflammation. In addition, it describes the molecular mechanisms of miR-223 in regulating lung inflammation, rheumatoid arthritis, enteritis, neuroinflammation and mastitis to provide insights into the existing molecular regulatory networks and therapies for inflammatory diseases in humans and animals.

KLF15 promotes transcription of KLF3 gene in bovine adipocytes.

The Krüppel-like factors (KLF) family plays an important role in adipogenesis, which is subject to internal hierarchical regulation. KLF3 is a member of KLF family, mainly responsible for adipocyte differentiation and fat deposition. However, the transcriptional regulation of bovine KLF3 gene and its relationship with KLF15 gene remains unclear during bovine adipogenesis. Here, we report that the expression pattern of KLF3 and KLF15 genes during bovine adipogenesis, when KLF15 gene was overexpressed through adenoviral vector (Ad-KLF15) in bovine adipocytes the expression level of KLF3 gene was increased, similarly when KLF15 was down regulated through siRNA the expression level of KLF3 was also reduced. To explore the transcriptional regulation of bovine KLF3 gene and its relationship with KLF15, serial deletion constructs of the 5'flanking region of bovine KLF3gene revealed through dual-luciferase reporter assay that the core promoter is located in -264 to -76 regions. The most proximal GGGG element in the promoter of the bovine KLF3 gene (located in -264 to -76 regions) is required for promotion by KLF15. Electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays further confirmed that KLF15 gene binds to the KLF3 gene core promoter region in bovine adipocytes. These findings conclude that KLF15 promotes the transcriptional activity of KLF3 in bovine adipocytes. This mechanism to provides a new direction for further study of adipogenesis by internal regulation of members within KLF family.

Overexpression of Mycothiol Disulfide Reductase Enhances Corynebacterium glutamicum Robustness by Modulating Cellular Redox Homeostasis and Antioxidant Proteins under Oxidative Stress.

Mycothiol (MSH) is the dominant low-molecular-weight thiol (LMWT) unique to high-(G+C)-content Gram-positive Actinobacteria, such as Corynebacterium glutamicum, and is oxidised into its disulfide form mycothiol disulfide (MSSM) under oxidative conditions. Mycothiol disulfide reductase (Mtr), an NADPH-dependent enzyme, reduces MSSM to MSH, thus maintaining intracellular redox homeostasis. In this study, a recombinant plasmid was constructed to overexpress Mtr in C. glutamicum using the expression vector pXMJ19-His6. Mtr-overexpressing C. glutamicum cells showed increased tolerance to ROS induced by oxidants, bactericidal antibiotics, alkylating agents, and heavy metals. The physiological roles of Mtr in resistance to oxidative stresses were corroborated by decreased ROS levels, reduced carbonylation damage, decreased loss of reduced protein thiols, and a massive increase in the levels of reversible protein thiols in Mtr-overexpressing cells exposed to stressful conditions. Moreover, overexpression of Mtr caused a marked increase in the ratio of reduced to oxidised mycothiol (MSH:MSSM), and significantly enhanced the activities of a variety of antioxidant enzymes, including mycothiol peroxidase (MPx), mycoredoxin 1 (Mrx1), thioredoxin 1 (Trx1), and methionine sulfoxide reductase A (MsrA). Taken together, these results indicate that the Mtr protein functions in C. glutamicum by protecting cells against oxidative stress.