IRE1α-JNK axis activation contributes to intracellular lipid accumulation in calf hepatocytes.

During the perinatal period, dairy cows undergo negative energy balance (NEB), resulting in elevated circulating levels of nonesterified fatty acids (NEFA). Although increased blood NEFA concentrations are a physiological adaptation of early lactation, excessive NEFA in dairy cows is a major cause of fatty liver. Aberrant lipid metabolism leads to hepatic lipid accumulation and subsequently the development of fatty liver. Both IRE1α and JNK have been validated for their association with hepatic lipid accumulation, including their regulatory functions in calf hepatocyte insulin resistance, oxidative stress and apoptosis. Meanwhile, both IRE1α and JNK are involved in lipid metabolism in non-ruminants. Therefore, the aim of this study was to investigate how IRE1α and JNK regulate lipid metabolism in bovine hepatocytes. An experiment was conducted on randomly selected 10 healthy cows (hepatic TG content <1%) and 10 cows with fatty liver (hepatic TG content >5%). Liver tissue and blood samples were collected from experimental cows. Serum concentrations of NEFA and ß-hydroxybutyrate (BHB) were greater, while serum concentrations of glucose and milk production were lower in cows with fatty liver. The Western blot results revealed that dairy cows with fatty liver had higher phosphorylation levels of c-Jun N-terminal kinase (JNK), c-Jun and inositol-requiring enzyme 1α (IRE1α) in the liver tissue. Three in vitro experiments were conducted using primary calf hepatocytes isolated from 5 healthy calves (body weight: 30-40 kg; 1d old). First, hepatocytes were treated with NEFA (1.2 mM) for 0.5, 1, 2, 3, 5, 7, 9, or 12 h, which showed that the phosphorylated levels of JNK, c-Jun and IRE1α increased in both linear and quadratic effects. In the second experiment, hepatocytes were treated with high concentrations of NEFA (1.2 mM) for 12 h with or without SP600125, a canonical inhibitor of JNK. Western blot results showed that SP600125 treatment could decrease the expression of lipogenesis-associated proteins (PPARγ and SREBP-1c) and increase the expression of fatty acid oxidation (FAO)-associated proteins (CPT1A and PPARα) in NEFA-treated hepatocytes. The perturbed expression of lipogenesis-associated genes (FASN, ACACA and CD36), and FAO-associated gene ACOX1 were also recovered by JNK inhibition, indicating that JNK reduced excessive NEFA-induced lipogenesis and FAO dysregulation in calf hepatocytes. Third, shRNA targeting IRE1α (sh-IRE1α) was transfected into calf hepatocytes to silence IRE1α, and KIRA6 was used to inhibit the kinase activity of IRE1α. The blockage of IRE1α could at least partially suppressed NEFA-induced JNK activation. Moreover, the blockage of IRE1α downregulated the expression of lipogenesis genes and upregulated the expression of FAO genes in NEFA-treated hepatocytes. In conclusion, these findings indicate that targeting the IRE1α-JNK axis can reduce NEFA-induced lipid accumulation in bovine hepatocytes by modulating lipogenesis and fatty acid oxidation. This may offer a prospective therapeutic target for fatty liver in dairy cows.

Impaired autophagy aggravates oxidative stress in mammary gland of dairy cows with clinical ketosis.

When ketosis occurs, supraphysiological levels of free fatty acids (FFA) can cause oxidative injury to the mammary gland and autophagy can regulate the cellular oxidative status. The aim of this study was to investigate the autophagy status of mammary tissue and its associations with oxidative stress in healthy and clinically ketotic dairy cows. Mammary tissue and blood samples were collected from healthy cows [n = 15, ß-hydroxybutyrate (BHB) <0.6 mM] and clinically ketotic cows (n = 15, BHB >3.0 mM) at 3 to 15 (average = 7) days in milk. For in vitro study, bovine mammary epithelial cells (BMEC) isolated from healthy cows were treated with 0, 0.3, 0.6, or 1.2 mM FFA for 24 h. Furthermore, BMEC were pretreated with 100 nM rapamycin, an autophagy activator, for 4 h or 50 mM 3-methyladenine (3-MA), an autophagy inhibitor, for 1 h, followed by treatment with or without FFA (1.2 mM) for another 24 h. Oxidation indicators and autophagy-related protein abundance were measured. Compared with healthy cows, serum concentrations of FFA, BHB, and malondialdehyde were greater in clinically ketotic cows, but milk production (kg/d), milk protein (kg/d), activities of superoxide dismutase, catalase, and glutathione peroxidase were lower. Abundances of mRNA and protein of autophagy-related gene 5 (ATG5) and 7 (ATG7) were lower, but sequestosome-1 (SQSTM1, also called p62) greater in mammary tissue of clinically ketotic cows. The mRNA abundance of microtubule-associated protein 1 light chain 3 (MAP1LC3, also called LC3) and protein abundance of LC3-II were lower in mammary tissue of clinically ketotic cows. In vitro, exogenous FFA increased the content of malondialdehyde and reactive oxygen species, but decreased the activities of superoxide dismutase, catalase, and plasma glutathione peroxidase. Compared with the 0 mM FFA group, abundance of ATG5, ATG7, LC3-II was greater, but p62 was lower in the 0.6 mM FFA-treated cells. Similarly, abundance of ATG5, ATG7, and LC3-II was lower, but p62 greater in the 1.2 mM FFA-treated cells relative to 0 mM FFA group. Culture with rapamycin alleviated oxidative stress induced by 1.2 mM FFA, whereas 3-MA aggravated it. Overall, results indicated that a low concentration (0.6 mM) of FFA can induce oxidative stress and activate autophagy in BMEC. At higher concentrations of FFA (1.2 mM), autophagy is impaired and oxidative stress is aggravated. Autophagy is a mechanism for BMEC to counteract FFA-induced stress. As such, it could serve as a potential target for further development of novel strategies against oxidative stress.

Increased adipose tissue lipolysis in dairy cows with fatty liver is associated with enhanced autophagy activity.

Lipolysis is increased in adipose tissue of cows with fatty liver during the transition period. Autophagy, a major cellular degradation process, plays a critical role in adipose tissue homeostasis. The objective of this study was to explore the relationship between lipolysis and autophagy in adipose tissue of cows with fatty liver. Using a nested case-control design, we compared blood and adipose tissue samples from 10 control cows [parity: median = 3, range = 2-4; days in milk: median = 8 d, range = 5-10 d; hepatic triacylglycerol content: median = 0.55% liver wt, range = 0.48-0.61% liver wt] and 10 lactation stage-matched cows with fatty liver (parity: median = 3, range = 2-4; days in milk: median = 9 d, range = 5-11 d; hepatic triacylglycerol content: median = 6.28% liver wt, range = 2.86-7.75% liver wt). Data were analyzed using paired t-tests. Serum concentrations of free fatty acids and ß-hydroxybutyrate were greater and glucose concentration was lower in cows with fatty liver, which we determined by using commercially-available kits. Furthermore, western blotting showed that increased protein abundance of ATGL (adipose triglyceride lipase), ATG5 (autophagy-related gene 5), and ATG7; ratio of phosphorylated (p)-HSL (hormone-sensitive lipase) to HSL and MAP1LC3 (microtubule-associated protein 1 light chain 3, also called LC3-II) to LC3-I along with decreased abundance of PLIN1 (perilipin 1), SQSTM1 (sequestosome-1, also called p62), and the ratio of p-mTOR (phosphorylated mechanistic target of rapamycin) to mTOR in cows with fatty liver. Quantitative reverse-transcription PCR revealed an increase in abundance of MAP1LC3 mRNA and a decrease in SQSTM1 mRNA in cows with fatty liver. These findings were replicated using an adipocyte model. Primary cultures of calf adipocytes isolated from the adipose tissue of the peritoneal omentum and mesentery were treated with 10 mM 3-methyladenine (3-MA), 5 nM rapamycin, 1 µM isoproterenol (ISO), and 1 µM ISO + 10 mM 3-MA. Comparisons among groups were analyzed using one-way ANOVA. Compared with the control, the 1 µM ISO treatment upregulated the abundance of ATGL, the ratio of p-HSL to HSL and LC3-II to LC3-I, and the glycerol content, whereas it downregulated the abundance of PLIN1 and p62 in calf adipocytes. Compared with the 1 µM ISO treatment group, 1 µM ISO + 10 mM 3-MA downregulated the abundance of ATGL, the ratio of p-HSL to HSL and LC3-II to LC3-I, and the glycerol content, whereas it upregulated the abundance of PLIN1 and p62. Compared with the control, the 5 nM rapamycin treatment upregulated the abundance of ATGL, the ratio of p-HSL to HSL and LC3-II to LC3-I, and the glycerol content, whereas it downregulated the abundance of PLIN1 and p62 in calf adipocytes. Overall, these data indicated that increased lipolysis in adipose tissue of cows with fatty liver was associated with enhanced autophagy. However, the specific molecular mechanisms that link lipolysis and autophagy need to be further investigated.

Mitochondrial membrane protein mitofusin 2 as a potential therapeutic target for treating free fatty acid-induced hepatic inflammation in dairy cows during early lactation.

Inflammation is critical in the progression from benign hepatic lipidosis to pathological hepatic steatosis. The objective of this study was to examine the potential role of the outer mitochondrial membrane protein mitofusin 2 (MFN2) in the etiology of hepatic steatosis in dairy cows during early lactation. Using a nested case-control design, we compared blood and liver samples from 10 healthy cows and 10 age-matched cows with moderate fatty liver. Cows with moderate fatty liver had high liver triacylglycerols, elevated plasma concentrations of free fatty acids (FFA) and ß-hydroxybutyrate, and low concentrations of glucose. Cows with moderate fatty liver had overactivated inflammatory pathways in the liver, as indicated by increased abundance of phosphorylated nuclear factor κB (NF-κB) p65, NLR family pyrin domain containing 3 (NLRP3) and caspase-1 inflammasome protein, and elevated plasma concentrations and hepatic mRNA abundance of their molecular targets IL-1ß, IL-6, and tumor necrosis factor α (TNF-α). In the cell culture model, we were able to replicate our findings in cows with moderate fatty liver: 1.2 mM exogenous FFA decreased the abundance of MFN2 and upregulated phosphorylation levels of the inhibitor of NF-κB (IκB) α and NF-κB p65, the IκB kinase ß activity, and the abundance of NLRP3, caspase-1, IL-1ß, IL-6, and TNF-α. Whereas MFN2 knockdown potentiated the FFA-induced activation of these inflammatory pathways, overexpression of MFN2 attenuated the detrimental effect of excess exogenous FFA by improving mitochondrial function and decreasing the release of reactive oxygen species, suggesting that MFN2 may be a potential therapeutic target for FFA-induced hepatic inflammation in dairy cows during early lactation.

A scientometric review of the research on the impacts of climate change on water quality during 1998-2018.

Research on the impacts of climate change on water quality helps to better formulate water quality strategies under the challenge of an uncertain future, which is critical for human survival and development. As a result, in recent years, there has been growing attention given to research in the field, and the attention has led to an increasing number of publications, which is why a systematic literature review on this topic has been proposed in the current paper. This study reviewed 2998 related articles extracted from the Science Citation Index-Expanded (SCI-E) database from 1998 to 2018 to analyse and visualize historical trend evolution, current research hotspots, and promising ideas for future research by combining a traditional literature review, bibliometric analysis, and scientific knowledge mapping. The results revealed that the impacts of climate change on water quality mainly included the aggravation of eutrophication, changes in the flow, hydrological and thermal conditions, and the destruction of ecosystems and biodiversity. Further exploration of the influence mechanism of climate change on cyanobacteria is an emerging research topic. Additionally, the water quality conditions of shallow lakes and drinking water are promising future research objects. In the context of climate change, the general rules of water quality management and the scientific planning of land use are of great significance and need to be further studied. This study provides a practical and valuable reference for researchers to help with the selection of future research topics, which may contribute to further development in this field.

Increased autophagy mediates the adaptive mechanism of the mammary gland in dairy cows with hyperketonemia.

Hyperketonemia is a metabolic disease in dairy cows, associated with negative nutrition balance (NNB) induced by low dry matter intake (DMI) and increased nutrient requirements. Hyperketonemia could induce metabolic stress, which might indirectly affect mammary tissue. Autophagy is a highly conserved physiological process that results in the turnover of intracellular material, and is involved in maintaining cellular homeostasis under the challenge of metabolic stress induced by NNB. The aim of this study was to investigate the autophagy status and autophagy-related pathways AMP-activated kinase α (AMPKα) and mechanistic target of rapamycin (mTOR) in the mammary glands of dairy cows with hyperketonemia. Cows with hyperketonemia [CWH, n = 10, blood ß-hydroxybutyrate (BHB) concentration 1.2 to 3.0 mmol/L] and cows without hyperketonemia (CWOH, n = 10, BHB < 1.2 mmol/L) from 3 to 12 DIM were randomly selected from the herd. The mammary tissue and blood samples were collected from these cows between 0630 and 0800 h, before feeding, at 3 to 12 d in milk. Serum concentrations of glucose, BHB, and fatty acids were determined using an autoanalyzer with commercial kits between 0630 and 0800 h, before feeding. Concentrations of fatty acids, BHB (median and interquartile range: CWH, 2.44 and 1.3, 2.82 mM; CWOH, 0.49 and 0.41, 0.57 mM), and milk fat were greater in CWH. The DMI, glucose concentration, milk production, and milk protein levels were lower in CWH. The mRNA abundance of autophagosome formation-related gene, beclin 1 (BECN1), phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), autophagy-related gene (ATG) 5, ATG7, ATG12, microtubule-associated protein 1 light chain 3 (MAP1LC3, also called LC3) and sequestosome-1 (SQSTM1, also called p62) were greater in the mammary glands of CWH. The protein abundance of LC3-II and phosphorylation level of Unc-51-like kinase 1 (ULK1) were greater in CWH, but the total ubiquitinated proteins and protein abundance of p62 were lower. Transmission electron microscopy showed an increased number of autophagosomes in the mammary glands of CWH. Furthermore, the phosphorylation of AMPKα was greater, but the phosphorylation of mTOR was lower in the mammary glands of CWH. These results indicate that activity of mTOR pathways and autophagy activity, and upregulation of AMPKα, may be response mechanisms to mitigate metabolic stress induced by hyperketonemia in the mammary glands of dairy cows.

Enhanced lake-eutrophication model combined with a fish sub-model using a microcosm experiment.

Eutrophication models are effective tools for assessing aquatic environments. The lake ecosystem consists of at least three trophic levels: phytoplankton, zooplankton, and fish. However, only a few studies have included fish sub-models in existing eutrophication models. In addition, no specific value or range is available for certain parameters of the fish sub-model. In the present study, a lake microcosm experimental system was established to determine the range of fish sub-model parameters. A three-trophic-level eutrophication model was established by combining the fish sub-model and eutrophication model. The Bayesian Markov Chain Monte Carlo and genetic algorithm method was used to calibrate the parameters of the eutrophication model. The results show that the maximum relative errors were due to phosphate (5.31%), the minimum relative error was due to nitrate (1.94%), and the relative error of dissolved oxygen, ammonia N, zooplankton, and chlorophyll ranged from 3 to 4%. Compared with the two-trophic-level eutrophication model, the relative errors of ammonia nitrogen (4.17%), phosphate (- 5.31%), and nitrate (1.94%) in the three-trophic-level eutrophication model were lower than those in the two-trophic-level eutrophication model, indicating that the three-trophic-level eutrophication model can obtain highly accurate simulation results and provide a better understanding of eutrophication models for future use.

Perilipin 5 promotes hepatic steatosis in dairy cows through increasing lipid synthesis and decreasing very low density lipoprotein assembly.

Fatty liver is a common metabolic disorder in dairy cows during the transition period. Perilipin 5 (PLIN5), a lipid droplet coat protein, plays important roles in the development of hepatic steatosis in mice and humans. Whether PLIN5 plays a role in the development of fatty liver in dairy cows is unknown. An in vivo study consisting of 10 healthy and 10 cows with fatty liver was performed to harvest liver tissue and blood samples. In addition, hepatocytes isolated from calves were infected with PLIN5 overexpression adenovirus for 48 h; treated with 0, 0.6, 1.2, or 2.4 mM nonesterified fatty acids (NEFA) for 24 h; or infected with PLIN5 silencing adenovirus for 48 h and then treated with 1.2 mM NEFA for 24 h. Serum concentrations of NEFA and ß-hydroxybutyrate were greater in cows with fatty liver. Milk production and plasma glucose concentrations were lower in cows with fatty liver. The results revealed that PLIN5 is highly expressed in steatotic liver and localized to lipid droplets. The abundance of fatty acid and triacylglycerol (TAG) synthesis-related proteins including sterol regulatory element binding protein-1c, fatty acid synthase, acetyl-coA carboxylase 1, diacylglycerol acyltransferase 1, and diacylglycerol acyltransferase 2 was greater in the liver of cows with fatty liver. In contrast, the abundance of microsomal triglyceride transfer protein (MTP), apolipoprotein B100, and apolipoprotein E was lower in the liver of cows with fatty liver. Consequently, cows with fatty liver exhibited severe hepatic TAG accumulation and lower blood concentration of very low density lipoprotein apolipoprotein B (VLDL-ApoB). Overexpression of PLIN5 and exogenous NEFA in cultured hepatocytes increased the abundance of sterol regulatory element binding protein-1, fatty acid synthase, acetyl-coA carboxylase 1, diacylglycerol acyltransferase 1, and diacylglycerol acyltransferase 2 but decreased the abundance of microsomal triglyceride transfer protein, apolipoprotein B100, and apolipoprotein E, which promoted TAG synthesis and inhibited VLDL-ApoB assembly, inducing lipid accumulation. Importantly, knockdown of PLIN5 attenuated the upregulation of TAG synthesis and downregulation of VLDL-ApoB assembly induced by NEFA. Overall, these data suggest that NEFA activate PLIN5, leading to TAG accumulation and inhibition of VLDL assembly. As such, these mechanisms explain in part the development of hepatic steatosis in dairy cows.

Surface Activity and Aggregation Behavior of Siloxane-Based Ionic Liquids in Aqueous Solution.

Six novel siloxane-based surface-active ionic liquids (SAILs)--siloxane ammonium carboxylate [Si(n)N(2)-CA(1), (n = 3, 4)]--were designed and synthesized. Their melting points, surface activities, and self-aggregation behavior in aqueous solution were studied. The results showed that because of the bulky hydrophobic siloxane chains at the end of the tail, all six siloxane-based SAILs are room-temperature ionic liquids (RT-SAILs). The introduction of the siloxane group can reduce the melting point of ionic liquids to below room temperature and can promote the micellization and aggregation behavior more efficiently. These siloxane-based SAILs can greatly reduce the surface tension of water, as shown by the critical aggregation concentration (γCAC) values of 20 mN·m(-1); all six siloxane RT-SAILs can form a vesicle spontaneously in aqueous solution, indicating potential uses as model systems for biomembranes and vehicles for drug delivery.

Molecular Features of Triple Negative Breast Cancer: Microarray Evidence and Further Integrated Analysis.

PURPOSE: Breast cancer is a heterogeneous disease usually including four molecular subtypes such as luminal A, luminal B, HER2-enriched, and triple-negative breast cancer (TNBC). TNBC is more aggressive than other breast cancer subtypes. Despite major advances in ER-positive or HER2-amplified breast cancer, there is no targeted agent currently available for TNBC, so it is urgent to identify new potential therapeutic targets for TNBC. METHODS: We first used microarray analysis to compare gene expression profiling between TNBC and non-TNBC. Furthermore an integrated analysis was conducted based on our own and published data, leading to more robust, reproducible and accurate predictions. Additionally, we performed qRT-PCR in breast cancer cell lines to verify the findings in integrated analysis. RESULTS: After searching Gene Expression Omnibus database (GEO), two microarray studies were obtained according to the inclusion criteria. The integrated analysis was conducted, including 30 samples of TNBC and 77 samples of non-TNBC. 556 genes were found to be consistently differentially expressed (344 up-regulated genes and 212 down-regulated genes in TNBC). Functional annotation for these differentially expressed genes (DEGs) showed that the most significantly enriched Gene Ontology (GO) term for molecular functions was protein binding (GO: 0005515, P = 6.09E-21), while that for biological processes was signal transduction (GO: 0007165, P = 9.46E-08), and that for cellular component was cytoplasm (GO: 0005737, P = 2.09E-21). The most significant pathway was Pathways in cancer (P = 6.54E-05) based on Kyoto Encyclopedia of Genes and Genomes (KEGG). DUSP1 (Degree = 21), MYEOV2 (Degree = 15) and UQCRQ (Degree = 14) were identified as the significant hub proteins in the protein-protein interaction (PPI) network. Five genes were selected to perform qRT-PCR in seven breast cancer cell lines, and qRT-PCR results showed that the expression pattern of selected genes in TNBC lines and non-TNBC lines was nearly consistent with that in the integrated analysis. CONCLUSION: This study may help to understand the pathogenesis of different breast cancer subtypes, contributing to the successful identification of therapeutic targets for TNBC.

siRNA-Mediated Suppression of Synuclein γ Inhibits MDA-MB-231 Cell Migration and Proliferation by Downregulating the Phosphorylation of AKT and ERK.

PURPOSE: Synuclein-γ (SNCG), which was initially identified as breast cancer specific gene 1, is highly expressed in advanced breast cancers, but not in normal or benign breast tissue. This study aimed to evaluate the effects of SNCG siRNA-treatment on breast cancer cells and elucidate the associated mechanisms. METHODS: Vectors containing SNCG and negative control (NC) siRNAs were transfected into MDA-MB-231 cells; mRNA levels were determined by real-time polymerase chain reaction. Cell proliferation was evaluated using the MTT assay, cell migration was assessed by the Transwell assay, apoptosis and cell cycle analyses were conducted with the flow cytometer, and Western blot analysis was performed to determine the relative levels of AKT, ERK, p-AKT, and p-ERK expression. RESULTS: SNCG mRNA levels were significantly reduced in MDA-MB-231 cells transfected with SNCG siRNA. Our results indicate that in SNCG siRNA-treated cells, cell migration and proliferation decreased significantly, apoptosis was induced, and the cell cycle was arrested. Western blot analysis indicated that the protein levels of p-AKT and p-ERK were much lower in the SNCG siRNA-treated groups, than in the control and NC groups. CONCLUSION: SNCG siRNA could decrease the migration and proliferation of breast cancer cells by downregulating the phosphorylation of AKT and ERK.