Effect of coating with combined chitosan and gallic acid on shelf-life stability of Jeju black cattle beef.

OBJECTIVE: Beef of Jeju black cattle (JBC) is considered as a healthy meat type due to its significantly higher unsaturated fatty acids (UFA). Lipid (e.g., UFA) is highly susceptible to oxidizing agents, which results in the quality deterioration and economic value loss of meat products. Therefore, development and application of novel preservative techniques is necessary to improve the shelf-life stability of high-UFA beef. The objective of this study was to assess the applicability of chitosan-based coatings in preservation of JBC beef. METHODS: Different coating solutions: 2% chitosan alone, and 2% chitosan containing 0.1% or 0.3% gallic acid were prepared to investigate their applicability in preservation of fresh beef during storage. Jeju black cattle beef (2-cm thick steaks) were non-coated (control) or coated with the above coating solutions, placed on trays, over-wrapped with plastic film and stored at 4°C. The microbiological indices, color, total volatile basic nitrogen (TVBN) and lipid oxidation of the beef were investigated after 1, 10, and 21 days of storage. RESULTS: Coating with 2% chitosan alone reduced the spoilage bacteria count, TVBN and thiobarbituric acid reactive substances levels in the beef compared with control during storage (p<0.05). Noticeably, coating with 2% chitosan containing 0.1% or 0.3% gallic acid was more effective on retardation of spoilage bacteria growth, lipid oxidation and discoloration in the beef compared to the chitosan coating alone over the storage period (21 days) (p<0.05). CONCLUSION: Taken together, the combined chitosan and gallic acid coating could be used as a bio-preservative technique in the meat industry.

Rabbit Meat Extract Induces Browning in 3T3-L1 Adipocytes via the AMP-Activated Protein Kinase Pathway.

The browning of white adipocytes may be an innovative approach to address obesity. This study investigated the effects of rabbit meat extract on 3T3-L1 adipocytes, with a specific emphasis on inducing browning. The browning effects of rabbit meat extract were evaluated by analyzing genes specifically expressed in 3T3-L1 adipocytes using quantitative PCR and immunoblotting. Rabbit meat extract increased the expression of brown adipocyte-specific markers, UCP1 and PGC1α, and mitochondrial biogenesis factors, TFAM and NRF1, without affecting cell viability in fully differentiated 3T3-L1 adipocytes. Moreover, adipocyte differentiation and the triglyceride content were decreased; hormone-sensitive lipase activity was promoted. Rabbit meat extract activated the AMPK pathway in the differentiated 3T3-L1 cells. However, in adipocytes treated with rabbit meat extract, the expression of genes related to browning was reduced by the AMP-activated protein kinase (AMPK) inhibitor, dorsomorphin dihydrochloride. To the best of our knowledge, this is the first study to demonstrate that rabbit meat extract induces the browning of white adipocytes via the activation of the AMPK pathway, thereby demonstrating its therapeutic potential in preventing obesity.

A Comparative Study on the Carcass and Meat Chemical Composition, and Lipid-Metabolism-Related Gene Expression in Korean Hanwoo and Brindle Chikso Cattle.

The objective of this study was to elucidate the effect of cattle breed on carcass and meat chemical composition, fatty acid profiles, and lipid-metabolism-related genes. For this study, same-age Hanwoo and Chikso steers (n = 6 per breed) reared under identical conditions were used. Immediately after slaughter, muscle tissues were collected for analysis of mRNA expression. At 24 h post-mortem, the carcasses were assessed for carcass traits (marbling score, meat yield, etc.), and meat quality and fatty acid profiles in the longissimus lumborum (LL) and semimembranosus (SM) muscles. The results showed that no differences in the slaughter weight, dressing rate, back-fat thickness, trimmed fat, and total meat yield occurred between the two breeds (p > 0.05). However, Hanwoo cattle had a higher marbling score, intramuscular fat (IMF) content, and expression level of lipid-metabolism-related genes such as lipoprotein lipase, peroxisome proliferator-activated receptor gamma, and fatty acid binding protein 4, compared with Chikso (p < 0.05). Contrastingly, Chikso had a higher total unsaturated fatty acid content and expression level of stearoyl CoA desaturase 1 (p < 0.05). It may be said that the difference in the expression levels of lipid-metabolism-related genes could be the molecular factors underlying IMF deposition and fatty acid profile differences in the beef from the two breeds.

A Comparative Study on the Meat Quality, Taste and Aroma Related Compounds between Korean Hanwoo and Chikso Cattle.

The aim of this study was to compare the meat quality and taste-and-aroma-related components of beef between breeds. For this purpose, Hanwoo and Chikso steers (n = 7 per breed) raised under identical conditions until 30 months old were used. After 24 h of slaughter, longissimus lumborum (LL) and semimembranosus (SM) muscles were collected and analyzed for technological quality, free amino acids, metabolites, and volatile compounds. The Chikso meat showed lower values for shear force and color traits (lightness, redness, and yellowness) compared to Hanwoo (p < 0.05). The Chikso presented a higher amount of sweetness-related free amino acids (alanine, proline, and threonine) in the LL muscle, while Hanwoo had a higher amount of methionine and glutamine associated with umami taste (p < 0.05). A total of 36 metabolites were identified and quantified in the meat samples; out of them, 7 compounds were affected by breed (p < 0.05). Regarding aroma compounds, a significantly higher amount of fat-derived aldehydes associated with fatty and sweet notes was found in Hanwoo, whereas a higher amount of pyrazines associated with roasty notes was found in Chikso (p < 0.05). Thus, under identical feeding conditions, breed showed a significant effect on the quality and taste-and-aroma-related components that may influence the eating quality of beef between the two breeds studied.

Milk Exosome-Derived MicroRNA-2478 Suppresses Melanogenesis through the Akt-GSK3ß Pathway.

Exosomes participate in intercellular communication by transferring molecules from donor to recipient cells. Exosomes are found in various body fluids, including blood, urine, cerebrospinal fluid and milk. Milk exosomes contain many endogenous microRNA molecules. MicroRNAs are small noncoding RNAs and have important roles in biological processes. The specific biological functions of milk exosomes are not well understood. In this study, we investigated the effects of milk exosomes on melanin production in melanoma cells and melanocytes. We found that milk exosomes decreased melanin contents, tyrosinase activity and the expression of melanogenesis-related genes in melanoma cells and melanocytes. Bovine-specific miR-2478 in exosomes inhibited melanin production. We found that Rap1a is a direct target gene of miR-2478 in melanoma cells and melanocytes. MiR-2478 overexpression decreased Rap1a expression, which led to downregulated melanin production and expression of melanogenesis-related genes. Inhibition of Rap1a expression decreased melanogenesis through the Akt-GSK3ß signal pathway. These results support the role of miR-2478 derived from milk exosomes as a regulator of melanogenesis through direct targeting of Rap1a. These results show that milk exosomes could be useful cosmeceutical ingredients to improve whitening.

Sinapic Acid Promotes Browning of 3T3-L1 Adipocytes via p38 MAPK/CREB Pathway.

Sinapic acid is a plant-derived phenolic compound, which acts as an antioxidant, anticancer, and anti-inflammatory agent. Although sinapic acid is valuable in a variety of therapeutic applications, its role in the improvement of obesity-related metabolic disease is relatively unexplored. Brown-like adipocytes (beige adipocytes) are characterized by a high concentration of mitochondria and high expression of uncoupling protein 1 (UCP1), which has specific functions in energy expenditure and thermogenesis. This study assessed the browning effects of sinapic acid in 3T3-L1 adipocytes. We investigated the expression of beige marker genes in 3T3-L1 adipocytes treated with sinapic acid. Sinapic acid increased the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and UCP1. Sinapic acid also promoted mitochondrial biogenesis by dose-dependently upregulating the oxygen consumption rate and enhancing the expression of representative subunits of oxidative phosphorylation complexes. In addition, treatment with p38 mitogen-activated protein kinase (MAPK) inhibitor and cAMP response element binding (CREB) inhibitor decreased the expressions of genes associated with thermogenesis, mitochondrial biogenesis, and oxidative phosphorylation. In summary, sinapic acid initiates browning 3T3-L1 adipocytes via the p38 MAPK/CREB signaling pathway. Thus, sinapic acid may have potential therapeutic implication in obesity.

Expression and Secretion of an Atrial Natriuretic Peptide in Beige-Like 3T3-L1 Adipocytes.

The browning of white adipose tissue (beige adipocytes) stimulates energy expenditure. Omega-3 fatty acids have been shown to induce thermogenic action in adipocytes via G-protein coupled receptor 120 (GPR120). Atrial natriuretic peptide (ANP) is a peptide hormone that plays the role of maintaining normal blood pressure in kidneys to inhibit Na+ reuptake. Recently, ANP was found to induce adipocyte browning by binding to NPR1, an ANP receptor. However, the expression of ANP in adipocytes has not yet been studied. Therefore, in this study, we investigate the expression of ANP in beige-like adipocytes induced by docosahexaenoic acids (DHA), T3, or a PPAR agonist, rosiglitazone. First, we found that brown adipocyte-specific genes were upregulated in beige-like adipocytes. DHA promoted ANP expression in beige-like cells, whereas DHA-induced ANP expression was abolished by GPR120 knockout. ANP secretion of beige-like adipocytes was increased via PKC/ERK1/2 signaling in the GPR120 pathway. Furthermore, ANP secreted from beige-like adipocytes acted on HEK-293 cells, the recipient cells, leading to increased cGMP activity. After the NPR1 knockdown of HEK-293 cells, cGMP activity was not changed. Taken together, our findings indicate that beige-like adipocytes induce ANP secretion, which may contribute to improving obesity-associated metabolic disease.

Identification of endogenous microRNA references in porcine serum for quantitative real-time PCR normalization.

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that regulate the expression of genes, and they affect important biological and physiological states. Circulating miRNAs in blood are useful markers of metabolism and economic traits. Expression levels of circulating miRNAs have been estimated using quantitative real-time PCR (qPCR). Proper normalization is critical for accurate miRNA expression analysis. However, there is no study which systematically presented endogenous reference genes for evaluating circulating miRNA expression in pigs. In this study, ten porcine miRNAs (let-7a, miR-16, miR-17, miR-23a, miR-26a, miR-93, miR-103, miR-107, miR-127 and miR-191), based on the literature, were chosen as candidate reference miRNAs in serum. We evaluated the expression stability value of these miRNAs in Berkshire, Duroc, Landrace and Yorkshire pigs using geNorm and NormFinder. We determined the optimal combination of reference miRNAs for qPCR experiments: miR-127 and miR-17 in Berkshire pigs; miR-127 and miR-93 in Duroc and Landrace pigs; miR-127 and miR-16 in Yorkshire pigs. miR-127 was the best reference gene in pigs, regardless of the breed. Our study is crucial for the discovery of novel biomarkers in pigs. The reference miRNAs presented in this study could be used as appropriate reference genes for the measurement of circulating miRNA levels in studies of physiological blood metabolites.

MicroRNA-196b enhances the radiosensitivity of SNU-638 gastric cancer cells by targeting RAD23B.

Gastric cancer is characterized by resistance to ionizing radiation. The development of resistance to radiotherapy in gastric cancer patients is one of the obstacles to effective radiotherapy. MicroRNAs are small well-conserved non-coding RNA species that regulate post-transcriptional activation. Our study aimed to investigate the role of miR-196b in radiation-induced gastric cancer. In the present study, we found that miR-196b expression was significantly reduced following radiation. The ectopic miR-196b expression sensitized SNU-638 gastric cancer cells and increased γ-H2AX foci upon radiation treatment. Bioinformatics analysis suggested that the DNA repair protein RAD23B was a putative target gene of miR-196b. Overexpression of miR-196b suppressed RAD23B expression in SNU-638 cells. Reporter assays further showed that miR-196b inhibited RAD23B 3'-UTR luciferase activity. Knockdown of RAD23B by small interfering RNA transfection closely mimicked the outcomes of miR-196b transfection, leading to impaired DNA damage repair in gastric cancer cells. Our results show that miR-196b improved radiosensitivity of SNU-638 cells by targeting RAD23B. Our data indicate that miR-196b is a potential target to enhance the effect of radiation treatment on gastric cancer cells. These findings will provide evidence for a new therapeutic target in radiotherapy.

PPARγ-mediated G-protein coupled receptor 120 signaling pathway promotes transcriptional activation of miR-143 in adipocytes.

MicroRNAs (miRNAs), the small noncoding RNAs, regulate various biological processes such as adipogenesis. MicroRNA-143 (miR-143) promotes adipocyte differentiation, and is correlated with obesity in mice fed a high-fat diet. However, the transcriptional regulation of miR-143 is largely unknown. In this study, we identified that miR-143 is a target of peroxisome proliferator-activated receptor γ (PPARγ), a key transcription factor in adipogenesis. Four putative peroxisome proliferator response elements (PPREs) were identified in the miR-143 promoter region. Using chromatin immune-precipitation, we observed that PPARγ was bound with two PPRE regions of the miR-143 promoter in 3T3-L1 adipocytes. A luciferase reporter assay showed that the PPRE1 region (-1330/-1309) of the miR-143 promoter played an important role in PPARγ transcriptional activation. In addition, we determined that G-protein coupled receptor 120 (GPR 120), which functions as an omega 3 fatty acid receptor, affected miR-143 expression in adipocytes. GPR120 silencing in adipocytes inhibited the expression of PPARγ and miR-143, whereas GPR120 overexpression led to increased expressions of PPARγ and miR-143. Silencing of PPARγ inhibited the induction of miR-143 by GPR-120. These results suggested that a PPARγ-mediated GPR120 signaling pathway promotes transcriptional activation of miR-143 in adipocytes.

Identification of reference genes for relative quantification of circulating microRNAs in bovine serum.

Circulating microRNAs in body fluids have been implicated as promising biomarkers for physiopathology disorders. Currently, the expression levels of circulating microRNAs are estimated by reverse transcription quantitative real-time polymerase chain reaction. Use of appropriate reference microRNAs for normalization is critical for accurate microRNA expression analysis. However, no study has systematically investigated reference genes for evaluating circulating microRNA expression in cattle. In this study, we describe the identification and characterization of appropriate reference microRNAs for use in the normalization of circulating microRNA levels in bovine serum. We evaluated the expression stability of ten candidate reference genes in bovine serum by using reverse transcription quantitative real-time polymerase chain reaction. Data were analyzed using geNorm, NormFinder, and BestKeeper statistical algorithms. The results consistently showed that a combination of miR-93 and miR-127 provided the most stably expressed reference. The suitability of these microRNAs was validated, and even when compared among different genders or breeds, the combination of miR-93 and miR-127 was ranked as the most stable microRNA reference. Therefore, we conclude that this combination is the optimal endogenous reference for reverse transcription quantitative real-time polymerase chain reaction-based detection of microRNAs in bovine serum. The data presented in this study are crucial to successful biomarker discovery and validation for the diagnosis of physiopathological conditions in cattle.