Expression of hepatic genes involved in bile acid metabolism in dairy cows with fatty liver.

Bile acids are cholesterol-derived molecules that are primarily produced in the liver. In nonruminants with fatty liver, overproduction of bile acids is associated with liver injury. During the transition period, fatty liver is a metabolic disorder that can affect up to 50% of high-producing dairy cows. The purpose of this study was to provide a comprehensive evaluation on hepatic bile acid metabolism in dairy cows with fatty liver by assessing expression changes of genes involved in bile acid synthesis, export and uptake. The serum activities of aspartate aminotransferase, alanine aminotransferase and glutamate dehydrogenase and concentration of total bile acids were all greater, whereas serum concentration of total cholesterol was lower in cows with fatty liver than in healthy cows. Content of total bile acids was higher but total cholesterol was slightly lower in liver tissues from fatty liver cows than from healthy cows. The hepatic mRNA abundance of cholesterol 7a-hydroxylase (CYP7A1), hydroxy-delta-5-steroid dehydrogenase, 3 ß- and steroid delta-isomerase 7 (HSD3B7) and sterol 12α-hydroxylase (CYP8B1), enzymes involved in the classic pathway of bile acid synthesis, was higher in fatty liver cows than in healthy cows. Compared with healthy cows, the hepatic mRNA abundance of alternative bile acid synthesis pathway-related genes sterol 27-hydroxylase (CYP27A1) and oxysterol 7α-hydroxylase (CYP7B1) did not differ in cows with fatty liver. The protein and mRNA abundance of bile acid transporter bile salt efflux pump (BSEP) were lower in the liver of dairy cow with fatty liver. Compared with healthy cows, the hepatic mRNA abundance of bile acid transporters solute carrier family 51 subunit α (SLC51A), ATP binding cassette subfamily C member 1 (ABCC1) and 3 (ABCC3) was greater in cows with fatty liver, whereas the solute carrier family 51 subunit ß (SLC51B) did not differ. The expression of genes involved in bile acid uptake, including solute carrier family 10 member 1 (NTCP), solute carrier organic anion transporter family member 1A2 (SLCO1A2) and 2B1 (SLCO2B1) was upregulated in dairy cows with fatty liver. Furthermore, the hepatic protein and mRNA abundance of bile acid metabolism regulators farnesoid X receptor (FXR) and small heterodimer partner (SHP) were lower in cows with fatty liver than in healthy cows. Overall, these data suggest that inhibition of FXR signaling pathway may lead to the increased bile acid synthesis and uptake and decreased secretion of bile acids from hepatocytes to the bile, which elevates hepatic bile acids content in dairy cows with fatty liver. As the hepatotoxicity of bile acids has been demonstrated on nonruminant hepatocytes, it is likely that the liver injury is induced by increased hepatic bile acids content in dairy cows with fatty liver.

Nuciferine protects bovine hepatocytes against free fatty acid-induced oxidative damage by activating the transcription factor EB/peroxisome proliferator-activated receptor γ coactivator 1 alpha pathway.

Excessive free fatty acid (FFA) oxidation and related metabolism are the major cause of oxidative stress and liver injury in dairy cows during the early postpartum period. In nonruminants, activation of transcription factor EB (TFEB) can improve cell damage and reduce the overproduction of mitochondrial reactive oxygen species. As a downstream target of TFEB, peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α, gene name PPARGC1A) is a critical regulator of oxidative metabolism. Nuciferine (Nuc), a major bioactive compound isolated from the lotus leaf, has been reported to possess hepatoprotective activity. Therefore, the objective of this study was to investigate whether Nuc could protect bovine hepatocytes from FFA-induced lipotoxicity and the underlying mechanisms. A mixture of FFA was diluted in RPMI-1640 basic medium containing 2% low fatty acid bovine serum albumin to treat hepatocytes. Bovine hepatocytes were isolated from newborn calves and treated with various concentrations of FFA mixture (0, 0.3, 0.6, or 1.2 mM) or Nuc (0, 25, 50, or 100 µM), as well as co-treated with 1.2 mM FFA and different concentrations of Nuc. For the experiments of gene silencing, bovine hepatocytes were transfected with small interfering RNA targeted against TFEB or PPARGC1A for 36 h followed by treatment with 1.2 mM FFA for 12 h in presence or absence of 100 µΜ Nuc. The results revealed that FFA treatment decreased protein abundance of nuclear TFEB, cytosolic TFEB, total (t)-TFEB, lysosome-associated membrane protein 1 (LAMP1) and PGC-1α and mRNA abundance of LAMP1, but increased phosphorylated (p)-TFEB. In addition, FFA treatment increased the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2) and decreased the activities of catalase (CAT) and glutathione peroxidase (GSH-Px) in bovine hepatocytes. Moreover, FFA administration enhanced the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactose dehydrogenase (LDH) in the medium of FFA-treated hepatocytes, but reduced the content of urea. In FFA-treated bovine hepatocytes, Nuc administration increased TFEB nuclear localization and the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, decreased the contents of MDA and H2O2 and the protein abundance of p-TFEB, and enhanced the activities of CAT and GSH-Px in a dose-dependent manner. Consistently, Nuc administration reduced the activities of ALT, AST, and LDH and increased the content of urea in the medium of FFA-treated hepatocytes. Importantly, knockdown of TFEB reduced the protein abundance of p-TFEB, t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, and impeded the beneficial effects of Nuc on FFA-induced oxidative damage in bovine hepatocytes. In addition, PPARGC1A silencing did not alter Nuc-induced nuclear translocation of TFEB, increase of the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, or decrease of the protein abundance of p-TFEB, whereas it partially reduced the beneficial effects of Nuc on FFA-caused oxidative injury. Taken together, Nuc exerts protective effects against FFA-induced oxidative damage in bovine hepatocytes through activation of the TFEB/PGC-1α signaling pathway.

Evaluation of hepatic AMPK, mTORC1, and autophagy-lysosomal pathway in cows with mild or moderate fatty liver.

The aim of the present study was to investigate the activity of AMPK and mTORC1 as well as TFEB transcriptional activity and autophagy-lysosomal function in the liver of dairy cows with mild fatty liver (FL) and cows with moderate FL. Liver and blood samples were collected from healthy dairy cows (n = 10; hepatic triglyceride content <1% wet weight) and cows with mild FL (n = 10; 1% ≤ hepatic triglyceride content < 5% wet weight) or moderate FL (n = 10; 5% ≤ hepatic triglyceride content < 10% wet weight) that had a similar number of lactations (median = 3, range = 2-4) and days in milk (median = 6 d, range = 3-9). Blood parameters were determined using a Hitachi 3130 autoanalyzer with commercially available kits. Protein and mRNA abundances were determined using western blotting and quantitative real-time PCR, respectively. Activities of calcineurin and ß-N-acetylglucosaminidase were measured with commercial assay kits. Data were analyzed using one-way ANOVA with subsequent Bonferroni correction. Blood concentrations of glucose were lower in moderate FL cows (3.03 ± 0.21 mM) than in healthy (3.71 ± 0.14 mM) and mild FL cows (3.76 ± 0.14 mM). Blood concentrations of ß-hydroxybutyrate (BHB, 1.37 ± 0.15 mM in mild FL, 1.88 ± 0.17 mM in moderate FL) and free fatty acids (FFA, 0.69 ± 0.05 mM in mild FL, 0.96 ± 0.09 mM in moderate FL) were greater in FL cows than in healthy cows (BHB, 0.76 ± 0.12 mM; FFA, 0.42 ± 0.04 mM). Compared with healthy cows, phosphorylation of AMPK was greater and phosphorylation of its downstream target acetyl-CoA carboxylase 1 was lower in cows with mild and moderate FL. Phosphorylation of mTOR was lower in cows with mild FL compared with healthy cows. In cows with moderate FL, phosphorylation of mTOR and its downstream effectors was greater than in healthy cows and cows with mild FL. The mRNA abundance of TFEB was downregulated in cows with moderate FL compared with healthy cows and mild FL cows. In mild FL cows, the mRNA and protein abundances of TFEB were greater than in healthy cows. Compared with healthy cows, the mRNA abundances of autophagy markers sequestosome-1 and microtubule-associated protein 1 light chain 3-II, and the protein and mRNA abundances of lysosome-associated membrane protein 1 and cathepsin D were increased in mild FL cows but decreased in moderate FL cows. Compared with healthy cows, the mRNA abundance of mucolipin 1 and activities of ß-N-acetylglucosaminidase and calcineurin were higher in cows with mild FL but lower in cows with moderate FL. These data demonstrate that hepatic AMPK signaling pathway, TFEB transcriptional activity, and autophagy-lysosomal function are increased in dairy cows with mild FL; the hepatic mTORC1 signaling pathway is inhibited in mild FL cows but activated in moderate FL cows; and activities of AMPK and TFEB as well as autophagy-lysosomal function are impaired in moderate FL cows.

Thioredoxin-2 suppresses hydrogen peroxide-activated nuclear factor kappa B signaling via alleviating oxidative stress in bovine adipocytes.

During the periparturient period, both oxidative stress, and inflammation of adipose tissue are considered high risk factors for metabolic disorder of dairy cows. Oxidative stress can activate transcription factor nuclear factor kappa B (NF-κB), which lead to the upregulation of genes involved in inflammatory pathways. Thioredoxin-2 (TXN2) is a mitochondrial protein that regulates cellular redox by suppressing mitochondrial reactive oxygen species (ROS) generation in nonruminant, whereas the function of TXN2 in bovine adipocytes was unclear. Thus, the objective of this study was to evaluate how or by which mechanisms TXN2 regulates oxidative stress and NF-κB signaling pathway in bovine adipocytes. Bovine pre-adipocytes isolated from 5 healthy Holstein cows were differentiated and used for (1) treatment with different concentrations of hydrogen peroxide (H2O2; 0, 25, 50, 100, 200, or 400 µM) for 2 h; (2) transfection with or without TXN2 small interfering RNA (si-TXN2) for 48 h and then treated with or without 200 µM H2O2 for 2 h; (3) transfection with scrambled negative control siRNA (si-control) or si-TXN2 for 48 h, and then treatment with or without 10 mM N-acetylcysteine (NAC) for 2 h; (4) transfection with or without TXN2-overexpressing plasmid for 48 h and then treatment with or without 200 µM H2O2 for 2 h. High concentrations of H2O2 (200 and 400 µM) decreased protein and mRNA abundance of TXN2, reduced total antioxidant capacity (T-AOC) and ATP content in adipocytes. Moreover, 200 and 400 µM H2O2 reduced protein abundance of inhibitor of kappa B α (IκBα), increased phosphorylation of NF-κB and upregulated mRNA abundance of tumor necrosis factor-α (TNFA) and interleukin-1B (IL-1B), suggesting that H2O2-induced oxidative stress and activated NF-κB signaling pathway. Silencing of TXN2 increased intracellular ROS content, phosphorylation of NF-κB and mRNA abundance of TNFA and IL-1B, decreased ATP content and protein abundance of IκBα in bovine adipocytes. Knockdown of TXN2 aggravated H2O2-induced oxidative stress and inflammation. In addition, treatment with antioxidant NAC ameliorated oxidative stress and inhibited NF-κB signaling pathway in adipocytes transfected with si-TXN2. In bovine adipocytes treated with H2O2, overexpression of TXN2 reduced the content of ROS and elevated the content of ATP and T-AOC. Overexpression of TXN2 alleviated H2O2-induced inflammatory response in adipocytes, as demonstrated by decreased expression of phosphorylated NF-κB, TNFA, IL-1B, as well as increased expression of IκBα. Furthermore, the protein and mRNA abundance of TXN2 was lower in adipose tissue of dairy cows with clinical ketosis. Overall, our studies contribute to the understanding of the role of TXN2 in adipocyte oxidative stress and inflammatory response.

RNA Interference against ATP as a Gene Therapy Approach for Prostate Cancer.

Chemotherapeutic agents targeting energy metabolism have not achieved satisfactory results in different types of tumors. Herein, we developed an RNA interference (RNAi) method against adenosine triphosphate (ATP) by constructing an interfering plasmid-expressing ATP-binding RNA aptamer, which notably inhibited the growth of prostate cancer cells through diminishing the availability of cytoplasmic ATP and impairing the homeostasis of energy metabolism, and both glycolysis and oxidative phosphorylation were suppressed after RNAi treatment. Further identifying the mechanism underlying the effects of ATP aptamer, we surprisingly found that it markedly reduced the activity of membrane ionic channels and membrane potential which led to the dysfunction of mitochondria, such as the decrease of mitochondrial number, reduction in the respiration rate, and decline of mitochondrial membrane potential and ATP production. Meanwhile, the shortage of ATP impeded the formation of lamellipodia that are essential for the movement of cells, consequently resulting in a significant reduction of cell migration. Both the downregulation of the phosphorylation of AMP-activated protein kinase (AMPK) and endoplasmic reticulum kinase (ERK) and diminishing of lamellipodium formation led to cell apoptosis as well as the inhibition of angiogenesis and invasion. In conclusion, as the first RNAi modality targeting the blocking of ATP consumption, the present method can disturb the respiratory chain and ATP pool, which provides a novel regime for tumor therapies..

Rapid and visual detection of dichloroacetonitrile in water.

Dichloroacetonitrile (DCAN) is a common biotoxic disinfection by-product (DBP) of chlorine. The current methods used for detecting DCAN are tedious and heavily instrument-dependent, and are not suitable for on-site detection. In the present study, we developed a colorimetric assay for rapid detection of DCAN. DCAN in water acted as a complexing agent that formed a complex with cuprous species. The cuprous species was then extracted by chloroform and visualized using dithizone. The visual detection limit for DCAN was 20 ng mL-1, while fluorescence quantification could detect DCAN at a concentration as low as 8.75 ng mL-1. Moreover, haloacetonitriles (HANs) derived from chlorine disinfection and structurally similar to DCAN, including TCAN, BCAN, and DBAN, could also be detected using this method. Other DBPs at concentrations as high as 200 ng mL-1 did not affect the detection process. The low cost and instrument-independence characteristic of the present method enables its routine determination of the concentration of DCAN in water.

Tumor necrosis factor-α reduces adiponectin production by decreasing transcriptional activity of peroxisome proliferator-activated receptor-γ in calf adipocytes.

Adiponectin (encoded by ADIPOQ) is an adipokine that orchestrates energy homeostasis by modulating glucose and fatty acid metabolism in peripheral tissues. During the periparturient period, dairy cows often develop adipose tissue inflammation and decreased plasma adiponectin levels. Proinflammatory cytokine tumor necrosis factor-α (TNF-α) plays a pivotal role in regulating the endocrine functions of adipocytes, but whether it affects adiponectin production in calf adipocytes remains obscure. Thus, the present study aimed to determine whether TNF-α could affect adiponectin production in calf adipocytes and to identify the underlying mechanism. Adipocytes isolated from Holstein calves were differentiated and used for (1) BODIPY493/503 staining; (2) treatment with 0.1 ng/mL TNF-α for different times (0, 8, 16, 24, or 48 h); (3) transfection with peroxisome proliferator-activated receptor-γ (PPARG) small interfering RNA for 48 h followed by treatment with or without 0.1 ng/mL TNF-α for 24 h; and (4) overexpression of PPARG for 48 h followed by treatment with or without 0.1 ng/mL TNF-α for 24 h. After differentiation, obvious lipid droplets and secretion of adiponectin were observed in adipocytes. Treatment with TNF-α did not alter mRNA abundance of ADIPOQ but reduced the total and high molecular weight (HMW) adiponectin content in the supernatant of adipocytes. Quantification of mRNA abundance of endoplasmic reticulum (ER)/Golgi resident chaperones involved in adiponectin assembly revealed that ER protein 44 (ERP44), ER oxidoreductase 1α (ERO1A), and disulfide bond-forming oxidoreductase A-like protein (GSTK1) were downregulated in TNF-α-treated adipocytes, while 78-kDa glucose-regulated protein and Golgi-localizing γ-adaptin ear homology domain ARF binding protein-1 were unaltered. Moreover, TNF-α diminished nuclear translocation of PPARγ and downregulated mRNA abundance of PPARG and its downstream target gene fatty acid synthase, suggesting that TNF-α suppressed the transcriptional activity of PPARγ. In the absence of TNF-α, overexpression of PPARG enhanced the total and HMW adiponectin content in supernatant and upregulated the mRNA abundance of ADIPOQ, ERP44, ERO1A, and GSTK1 in adipocytes. However, knockdown of PPARG reduced the total and HMW adiponectin content in supernatant and downregulated the mRNA abundance of ADIPOQ, ERP44, ERO1A, and GSTK1 in adipocytes. In the presence of TNF-α, overexpression of PPARG decreased, while knockdown of PPARG further exacerbated TNF-α-induced reductions in total and HMW adiponectin secretion and gene expression of ERP44, ERO1A, and GSTK1. Overall, TNF-α reduces adiponectin assembly in the calf adipocyte, which may be partly mediated by attenuation of PPARγ transcriptional activity. Thus, locally elevated levels of TNF-α in adipose tissue may be one reason for the decrease in circulating adiponectin in periparturient dairy cows.

A Novel Analytical Model for the IEEE 802.11p/bd Medium Access Control, with Consideration of the Capture Effect in the Internet of Vehicles.

The traditional vehicular ad hoc network (VANET), which is evolving into the internet of vehicles (IoV), has drawn great attention for its enormous potential in road safety improvement, traffic management, infotainment service support, and even autonomous driving. IEEE 802.11p, as the vital standard for wireless access in vehicular environments, has been released for more than one decade and its evolution, IEEE 802.11bd, has also been released for a few months. Since the analytical models for the IEEE 802.11p/bd medium access control (MAC) play important roles in terms of performance evaluation and MAC protocol optimization, a lot of analytical models have been proposed. However, the existing analytical models are still not accurate as a result of ignoring some important factors of the MAC itself and real communication scenarios. Motivated by this, a novel analytical model is proposed, based on a novel two-dimensional (2-D) Markov chain model. In contrast to the existing studies, all the important factors are considered in this proposed model, such as the backoff freezing mechanism, retry limit, post-backoff states, differentiated packet arrival probabilities for empty buffer queue, and queue model of packets in the buffer. In addition, the influence of the capture effect under a Nakagami-m fading channel has also been considered. Then, the expressions of successful transmission, collided transmission, normalized unsaturated throughput, and average packet delay are all meticulously derived, respectively. At last, the accuracy of the proposed analytical model is verified via the simulation results, which show that it is more accurate than the existing analytical models.

Comparative Transcriptome Analysis Reveals a Potential Regulatory Network for Ogura Cytoplasmic Male Sterility in Cabbage (Brassica oleracea L.).

Ogura cytoplasmic male sterility (CMS) lines are widely used breeding materials in cruciferous crops and play important roles in heterosis utilization; however, the sterility mechanism remains unclear. To investigate the microspore development process and gene expression changes after the introduction of orf138 and Rfo, cytological observation and transcriptome analysis were performed using a maintainer line, an Ogura CMS line, and a restorer line. Semithin sections of microspores at different developmental stages showed that the degradation of tapetal cells began at the tetrad stage in the Ogura CMS line, while it occurred at the bicellular microspore stage to the tricellular microspore stage in the maintainer and restorer lines. Therefore, early degradation of tapetal cells may be the cause of pollen abortion. Transcriptome analysis results showed that a total of 1287 DEGs had consistent expression trends in the maintainer line and restorer line, but were significantly up- or down-regulated in the Ogura CMS line, indicating that they may be closely related to pollen abortion. Functional annotation showed that the 1287 core DEGs included a large number of genes related to pollen development, oxidative phosphorylation, carbohydrate, lipid, and protein metabolism. In addition, further verification elucidated that down-regulated expression of genes related to energy metabolism led to decreased ATP content and excessive ROS accumulation in the anthers of Ogura CMS. Based on these results, we propose a transcriptome-mediated induction and regulatory network for cabbage Ogura CMS. Our research provides new insights into the mechanism of pollen abortion and fertility restoration in Ogura CMS.

Hospital information system-based pharmacovigilance for elderly patients: practices and results.

OBJECTIVE: To evaluate the effect of a pharmacovigilance system on potentially inappropriate medication prescriptions for elderly patients. METHODS: The retrospective study was conducted at Shaanxi Provincial People's Hospital, China, after approval from the ethics review committee, and comprised data from May 2020 to April 2021, and comprised prescriptions related to elderly patients aged at least 65 years. Number of medication risk assessment entries, number of intervened medical orders on outpatients and inpatients number of medical order prompts, and number of physician communication with prescription-checking pharmacists were noted. Potential drug interaction rate was compared between pre- implementation from May to October 2020 and post-implementation from November 2020 to April 2021. Besides, the usage of sedatives and hypnotics and potentially inappropriate medication was noted for the period from January to June 2021 to evaluate the sustained effect of pharmacovigilance system. Data was analysed using SPSS 19. Results: A total of 118 drugs were involved in the 3911 entries of outpatient prescription warnings, of which 19 drugs accounted for 3156 (80%). Besides, a total of 113 drugs were involved in the 3999 entries of inpatient prescription warnings, of which 19 drugs accounted for 3199 (80%) The overall prevalence of potentially inappropriate medication related to sedatives and hypnotics decreased post-intervention as warning percentage was 16.1% in January and 6.7% in June among outpatients. On inpatients, the warning percentage was 30.6% in January and 6.1% in June. CONCLUSIONS: The pharmacovigilance system could reduce potentially inappropriate medication and provide deeper technical support for the safety of medical behaviour and individualised treatment of patients.

Low abundance of mitophagy markers is associated with reactive oxygen species overproduction in cows with fatty liver and causes reactive oxygen species overproduction and lipid accumulation in calf hepatocytes.

Mitochondria are the main site of fatty acid oxidation and reactive oxygen species (ROS) formation. Damaged or dysfunctional mitochondria induce oxidative stress and increase the risk of lipid accumulation. During the process of mitophagy, PTEN induced kinase 1 (PINK1) accumulates on damaged mitochondria and recruits cytoplasmic Parkin to mitochondria. As an autophagy receptor protein, sequestosome-1 (p62) binds Parkin-ubiquitinated outer mitochondrial membrane proteins and microtubule-associated protein 1 light chain 3 (LC3) to facilitate degradation of damaged mitochondria. In nonruminants, clearance of dysfunctional mitochondria through the PINK1/Parkin-mediated mitophagy pathway contributes to reducing ROS production and maintaining metabolic homeostasis. Whether PINK1/Parkin-mediated mitophagy plays a similar role in dairy cow liver is not well known. Thus, the objective of this study was to investigate mitophagy status in dairy cows with fatty liver and its role in free fatty acid (FFA)-induced oxidative stress and lipid accumulation. Liver and blood samples were collected from healthy dairy cows (n = 10) and cows with fatty liver (n = 10) that had a similar number of lactations (median = 3, range = 2 to 4) and days in milk (median = 6 d, range = 3 to 9 d). Calf hepatocytes were isolated from 5 healthy newborn female Holstein calves (1 d of age, 30-40 kg). Hepatocytes were transfected with small interfering RNA targeted against PRKN for 48 h or transfected with PRKN overexpression plasmid for 36 h, followed by treatment with FFA (0.3 or 1.2 mM) for 12 h. Mitochondria were isolated from fresh liver tissue or calf hepatocytes. Serum concentrations of ß-hydroxybutyrate were higher in dairy cows with fatty liver. Hepatic malondialdehyde (MDA) and hydrogen peroxide (H2O2) were greater in cows with fatty liver. The lower protein abundance of PINK1, Parkin, p62, and LC3-II in hepatic mitochondrial fraction of dairy cows with fatty liver indicated the mitophagy was impaired. In hepatocytes, knockdown of PRKN decreased protein abundance of p62 and LC3-II in the mitochondrial fraction, and increased contents of triacylglycerol (TG), MDA, and H2O2. In addition, protein abundances of PINK1, Parkin, p62, and LC3-II were lower in the mitochondrial fraction from hepatocytes treated with 1.2 mM FFA than the hepatocytes treated with 0.3 mM FFA, whereas the content of TG, MDA, and H2O2 increased. In 1.2 mM FFA-treated hepatocytes, PRKN overexpression increased protein abundance of p62 and LC3-II in the mitochondrial fraction and decreased contents of TG, MDA, and H2O2. Together, our data demonstrate that low abundance of mitophagy markers is associated with ROS overproduction in dairy cows with fatty liver and impaired mitophagy induced by a high concentration of FFA promotes ROS production and lipid accumulation in female calf hepatocytes.

Activated autophagy-lysosomal pathway in dairy cows with hyperketonemia is associated with lipolysis of adipose tissues.

Activated autophagy-lysosomal pathway (ALP) can degrade virtually all kinds of cellular components, including intracellular lipid droplets, especially during catabolic conditions. Sustained lipolysis and increased plasma fatty acids concentrations are characteristic of dairy cows with hyperketonemia. However, the status of ALP in adipose tissue during this physiological condition is not well known. The present study aimed to ascertain whether lipolysis is associated with activation of ALP in adipose tissues of dairy cows with hyperketonemia and in calf adipocytes. In vivo, blood and subcutaneous adipose tissue (SAT) biopsies were collected from nonhyperketonemic (nonHYK) cows [blood ß-hydroxybutyrate (BHB) concentration <1.2 mM, n = 10] and hyperketonemic (HYK) cows (blood BHB concentration 1.2-3.0 mM, n = 10) with similar days in milk (range: 3-9) and parity (range: 2-4). In vitro, calf adipocytes isolated from 5 healthy Holstein calves (1 d old, female, 30-40 kg) were differentiated and used for (1) treatment with lipolysis inducer isoproterenol (ISO, 10 µM, 3 h) or mammalian target of rapamycin inhibitor Torin1 (250 nM, 3 h), and (2) pretreatment with or without the ALP inhibitor leupeptin (10 µg/mL, 4 h) followed by ISO (10 µM, 3 h) treatment. Compared with nonHYK cows, serum concentration of free fatty acids was greater and serum glucose concentration, DMI, and milk yield were lower in HYK cows. In SAT of HYK cows, ratio of phosphorylated hormone-sensitive lipase to hormone-sensitive lipase, and protein abundance of adipose triacylglycerol lipase were greater, but protein abundance of perilipin 1 (PLIN1) and cell death-inducing DNA fragmentation factor-α-like effector c (CIDEC) was lower. In addition, mRNA abundance of autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and microtubule-associated protein 1 light chain 3 beta (MAP1LC3B), protein abundance of lysosome-associated membrane protein 1, and cathepsin D, and activity of ß-N-acetylglucosaminidase were greater, whereas protein abundance of sequestosome-1 (p62) was lower in SAT of HYK cows. In calf adipocytes, treatment with ISO or Torin1 decreased protein abundance of PLIN1, and CIDEC, and triacylglycerol content in calf adipocytes, but increased glycerol content in the supernatant of calf adipocytes. Moreover, the mRNA abundance of ATG5, ATG7, and MAP1LC3B was upregulated, the protein abundance of lysosome-associated membrane protein 1, cathepsin D, and activity of ß-N-acetylglucosaminidase were increased, whereas the protein abundance of p62 was decreased in calf adipocytes treated with ISO or Torin1 compared with control group. Compared with treatment with ISO alone, the protein abundance of p62, PLIN1, and CIDEC, and triacylglycerol content in calf adipocytes were higher, but the glycerol content in the supernatant of calf adipocytes was lower in ISO and leupeptin co-treated group. Overall, these data indicated that activated ALP is associated with increased lipolysis in adipose tissues of dairy cows with hyperketonemia and in calf adipocytes.

Targeting IRE1α and PERK in the endoplasmic reticulum stress pathway attenuates fatty acid-induced insulin resistance in bovine hepatocytes.

Endoplasmic reticulum (ER) stress can be induced by various stimuli and triggers the unfolded protein response to activate intracellular signaling pathways that are mediated by 3 ER-resident sensors: inositol requiring protein-1α (IRE1α), PKR-like ER kinase (PERK), and activating transcription factor-6 (ATF6). In nonruminants, ER stress plays a critical role in hepatic insulin resistance. However, whether ER stress plays a role in nonesterified fatty acid (NEFA)-induced hepatic insulin resistance in dairy cows is still unknown. Experiments were conducted using primary bovine hepatocytes isolated from 5 healthy calves (body weight: 30-40 kg; 1 d old). First, hepatocytes were treated with NEFA (1.2 mM) for 0.5, 1, 2, 3, 5, 7, 9, or 12 h. Treatment with NEFA elevated abundance of phosphorylated IRE1α and PERK, and cleavage of ATF6, along with the ER stress-associated genes XBP1, ATF4, and DNAJC3, resulting in both linear and quadratic effects. Furthermore, ER Tracker red staining and transmission electron microscopy results indicated that ER was dilated and degranulated in response to NEFA treatment, suggesting that ER stress was induced by NEFA treatment in bovine hepatocytes. Second, to assess the effect of ER stress on NEFA-induced insulin resistance, hepatocytes were treated with different concentrations of NEFA (0, 0.6, 1.2, or 2.4 mM) for 5 h with or without tauroursodeoxycholic acid (TUDCA, a canonical inhibitor of ER stress). Here, NEFA induced insulin resistance by increasing the abundance of insulin receptor substrate-1 (IRS1) phosphorylation at the inhibitory residue Ser 307 (S307) and decreasing the abundance of phosphorylated protein kinase B (AKT) and glycogen synthase kinase-3ß (GSK3ß) in a dose-dependent manner. This was accompanied by upregulation of an abundance of gluconeogenic genes [phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6-Pase)]. These detrimental effects of NEFA on insulin signaling could be reversed with TUDCA treatment, indicating a mechanistic link between ER stress and NEFA-induced insulin resistance. In a third experiment, pGPU6/GFP/Neo vectors containing short hairpin RNA targeting IRE1α were used to silence IRE1α transcription, and GSK2656157 (PERK phosphorylation inhibitor) and 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF; an inhibitor of ATF6) were used to block PERK and ATF6 branches, respectively. Notably, the silencing of the IRE1α branch improved NEFA-induced insulin resistance by decreasing phosphorylation of IRS1 (S307) and increasing phosphorylation of AKT and GSK3ß, and reducing PEPCK and G6-Pase mRNA abundance, which was likely dependent on IRE1α kinase activity. Similarly, blockage of the PERK branch increased phosphorylation of AKT and GSK3ß, and reduced PEPCK and G6-Pase mRNA abundance, but had no effect on phosphorylation of IRS1 (S307). However, results showed that inhibition of the ATF6 branch had no effects on phosphorylation of IRS1, AKT, and GSK3ß, and instead found increasing PEPCK and G6-Pase mRNA abundance. Taken together, data in the present study found that impeding IRE1α and PERK signaling might aid in relieving hepatic insulin resistance. However, the more detailed mechanisms of how IRE1α and PERK signaling contribute to hepatic insulin resistance in dairy cows remain to be determined.

Overactivation of hepatic mechanistic target of rapamycin kinase complex 1 (mTORC1) is associated with low transcriptional activity of transcription factor EB and lysosomal dysfunction in dairy cows with clinical ketosis.

Ketosis occurs most frequently in the peripartal period and is associated with liver injury and steatosis. Lysosomes serve as the terminal degradative station and contribute to liver homeostasis through their role in the digestion of dysfunctional organelles and lipid droplets. Transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function. Thus, the objective of the present study was to investigate the status of lysosomal function and TFEB transcriptional activity and potential changes in abundance of upstream effectors of TFEB identified in nonruminants, including mechanistic target of rapamycin kinase complex 1 (mTORC1), protein kinase B (Akt), glycogen synthase kinase ß (GSK3ß), and extracellular signal-regulated kinase1/2 (ERK1/2), and to explore which factor induces the above changes. Liver and blood samples were collected from healthy cows (n = 10) and ketotic cows (n = 10) that had a similar number of lactations (median = 3, range = 2-4) and days in milk (median = 6 d, range = 3-9 d). Calf hepatocytes were isolated from Holstein calves and treated with 10 ng/mL growth hormone (GH), 3.0 mM ß-hydroxybutyrate (BHB), 1.5 ng/mL interleukin-18 (IL-18), 0.15 ng/mL tumor necrosis factor-α (TNF-α), or 1.2 mM free fatty acid (FFA) for 12 h. Serum levels of FFA and activities of alanine aminotransferase and aspartate aminotransferase were greater in ketotic cows, whereas glucose was lower. Additionally, ketotic dairy cows exhibited higher serum concentrations of GH, IL-18, and TNF-α, and lower serum concentration of insulin. The lower protein abundance of lysosome-associated membrane protein 1 (LAMP1) and mRNA abundance of LAMP1 indicated that hepatic lysosomal mass was lower in ketotic cows. Furthermore, lower protein abundance of cathepsin D (CTSD) and mRNA abundance of CTSD and V0 domain of the vacuolar ATPase along with lower activity of ß-N-acetylglucosaminidase indicated impairment in hepatic lysosomal function due to ketosis. The lower nuclear abundance, total protein, and mRNA abundance of TFEB and peroxisome proliferator-activated receptor γ coactivator 1 α along with greater phosphorylated (p)-TFEB in the liver of ketotic cows indicated an impairment of hepatic TFEB transcriptional activity. The protein abundances of phosphorylated mTOR (p-mTOR) and its downstream effectors ribosomal protein S6 kinase B (RPS6KB) and eukaryotic factor 4E-binding protein 1 (EIF4EBP1) were greater, whereas p-Akt, p-GSK3ß, and p-ERK1/2 were lower in the liver of ketotic cows. Importantly, elevated phosphorylation of mTOR, RPS6KB, and EIF4EBP1 was observed in calf hepatocytes treated with GH, BHB, IL-18, TNF-α, and FFA. Moreover, BHB, TNF-α, and FFA, not GH and IL-18, reduced TFEB transcriptional activity and impaired lysosomal function in calf hepatocytes. Taken together, these data suggest that BHB, TNF-α, and FFA overactivate the hepatic mTORC1 signaling pathway during ketosis and further impaired TFEB transcriptional activity and lysosomal function, which may contribute to liver injury and steatosis.

Tumor necrosis factor-α promotes lipolysis and reduces insulin sensitivity by activating nuclear factor kappa B and c-Jun N-terminal kinase in primary bovine adipocytes.

Sustained lipolysis and insulin resistance increase the risk of metabolic dysfunction in dairy cows during the transition period. Proinflammatory cytokines are key regulators of adipose tissue metabolism in nonruminants, but biological functions of these molecules in ruminants are not well known. Thus, the objective of this study was to investigate whether tumor necrosis factor-α (TNF-α) could affect insulin sensitivity and lipolysis in bovine adipocytes as well as the underlying mechanisms. Bovine adipocytes (obtained from the omental and mesenteric adipose depots) isolated from 5 Holstein female calves (1 d old) with similar body weight (median: 36.9 kg, range: 35.5-41.2 kg) were differentiated and used for (1) treatment with different concentrations of TNF-α (0, 0.1, 1, or 10 ng/mL) for 12 h; (2) pretreatment with 10 µM lipolytic agonist isoproterenol (ISO) for 3 h, followed by treatment with or without 10 ng/mL TNF-α for 12 h; and (3) pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 (20 µM for 2 h) and nuclear factor kappa B (NF-κB) inhibitor BAY 11-7082 (10 µM for 1 h) followed by treatment with or without 10 ng/mL TNF-α for 12 h. The TNF-α increased glycerol content in supernatant, decreased triglyceride content and insulin-stimulated phosphorylation of protein kinase B suggesting activation of lipolysis and impairment of insulin sensitivity. The TNF-α reduced cell viability, upregulated mRNA abundance of Caspase 3 (CASP3), an apoptosis marker, and increased activity of Caspase 3. In addition, increased phosphorylation of NF-κB and JNK, upregulation of mRNA abundance of interleukin-6 (IL-6), TNFA, and suppressor of cytokine signaling 3 (SOCS3) suggested that TNF-α activated NF-κB and JNK signaling pathways. Furthermore, ISO plus TNF-α-activated NF-κB and JNK signaling pathway to a greater extent than TNF-α alone. Combining TNF-α and ISO aggravated TNF-α-induced apoptosis, insulin insensitivity and lipolysis. In the absence of TNF-α, inhibition of NF-κB and JNK did not alter glycerol content in supernatant, triglyceride content or insulin-stimulated phosphorylation of protein kinase B. In the presence of TNF-α, inhibition of NF-κB and JNK alleviated TNF-α-induced apoptosis, insulin insensitivity and lipolysis. Overall, TNF-α impairs insulin sensitivity and induces lipolysis and apoptosis in bovine adipocytes, which may be partly mediated by activation of NF-κB and JNK. Thus, the data suggested that NF-κB and JNK are potential therapeutic targets for alleviating lipolysis dysregulation and insulin resistance in adipocytes.

Design of Lidar Data Acquisition and Control System in High Repetition Rate and Photon-Counting Mode: Providing Testing for Space-Borne Lidar.

For ground-based lidars in atmospheric observation, their data acquisition unit and control unit usually work independently. They usually require the cooperation of large-volume, high-power-consumption Industrial Personal Computer (IPC). However, the space-borne lidar has high requirements on the stability and integration of the acquisition control system. In this paper, a new data acquisition and lidar control system (DALCS) was developed based on System-on-Chip Field-Programmable Gate Array (SoC FPGA) technology. It can be used in lidar systems with high repetition rate and photon-counting mode and has functions such as data storage, laser control, automatic collimation, wireless communication, and fault self-test. DALCS has two working modes: in online mode, the echo data collected by DALCS are transmitted to the computer for display in real-time and then stored with the current time as the file name; in offline mode, the data are stored in local non-volatile memory, which can be read remotely and can work autonomously when there is no IPC. The test results showed that in the frequency range of 0-70 M, the counting linearity of DALCS reached 0.9999, and the maximum relative error between the DALCS card and the standard signal source was 0.211%. The comparison results showed that the correlation coefficient between DALCS and MCS-PCI was as high as 0.99768. The DALCS was placed in a self-developed lidar sensor system for continuous observation, and the system worked stably under different weather conditions. The range-squared-corrected signal profiles obtained from the observations reflect the spatial and temporal distribution characteristics of aerosols and clouds well. This provides scheme verification and experimental support for the development of space-borne lidar data acquisition and control system.

Mechanism and Utilization of Ogura Cytoplasmic Male Sterility in Cruciferae Crops.

Hybrid production using lines with cytoplasmic male sterility (CMS) has become an important way to utilize heterosis in vegetables. Ogura CMS, with the advantages of complete pollen abortion, ease of transfer and a progeny sterility rate reaching 100%, is widely used in cruciferous crop breeding. The mapping, cloning, mechanism and application of Ogura CMS and fertility restorer genes in Brassica napus, Brassica rapa, Brassica oleracea and other cruciferous crops are reviewed herein, and the existing problems and future research directions in the application of Ogura CMS are discussed.

Global DNA Methylation and mRNA-miRNA Variations Activated by Heat Shock Boost Early Microspore Embryogenesis in Cabbage (Brassica oleracea).

Microspore culture, a type of haploid breeding, is extensively used in the cultivation of cruciferous crops such as cabbage. Heat shock (HS) treatment is essential to improve the embryo rate during the culture process; however, its molecular role in boosting early microspore embryogenesis (ME) remains unknown. Here we combined DNA methylation levels, miRNAs, and transcriptome profiles in isolated microspores of cabbage '01-88' under HS (32 °C for 24 h) and normal temperature (25 °C for 24 h) to investigate the regulatory roles of DNA methylation and miRNA in early ME. Global methylation levels were significantly different in the two pre-treatments, and 508 differentially methylated regions (DMRs) were identified; 59.92% of DMRs were correlated with transcripts, and 39.43% of miRNA locus were associated with methylation levels. Significantly, the association analysis revealed that 31 differentially expressed genes (DEGs) were targeted by methylation and miRNA and were mainly involved in the reactive oxygen species (ROS) response and abscisic acid (ABA) signaling, indicating that HS induced DNA methylation, and miRNA might affect ME by influencing ROS and ABA. This study revealed that DNA methylation and miRNA interfered with ME by modulating key genes and pathways, which could broaden our understanding of the molecular regulation of ME induced by HS pre-treatment.

Transcriptome Analysis Reveals Key Genes and Pathways Associated with the Petal Color Formation in Cabbage (Brassica oleracea L. var. capitata).

Petal color is an important agronomic trait in cabbage (Brassica oleracea L. var. capitata). Although the key gene BoCCD4 has been functionally characterized, the underlying molecular regulatory mechanism of petal color formation in cabbage is still unclear. In this study, we applied the transcriptome analysis of yellow petals from the cabbage inbred line YL-1 and white petals from the Chinese kale inbred line A192-1 and the BoCCD4-overexpressing transgenic line YF-2 (YL-1 background), which revealed 1928 DEGs common to both the A192-1 vs. YL-1 and the YL-1 vs. YF-2 comparison groups. One key enzyme-encoding gene, BoAAO3, and two key TF-encoding genes, Bo2g151880 (WRKY) and Bo3g024180 (SBP), related to carotenoid biosynthesis were significantly up-regulated in both the A192-1 and YF-2 petals, which was consistent with the expression pattern of BoCCD4. We speculate that these key genes may interact with BoCCD4 to jointly regulate carotenoid biosynthesis in cabbage petals. This study provides new insights into the molecular regulatory mechanism underlying petal color formation in cabbage.

Genetic mapping and candidate gene identification of BoGL5, a gene essential for cuticular wax biosynthesis in broccoli.

BACKGROUND: The aerial organs of most terrestrial plants are covered by cuticular waxes, which impart plants a glaucous appearance and play important roles in protecting against various biotic and abiotic stresses. Despite many glossy green (wax-defective) mutants being well characterized in model plants, little is known about the genetic basis of glossy green mutant in broccoli. RESULTS: B156 is a spontaneous broccoli mutant showing a glossy green phenotype. Detection by scanning electron microscopy (SEM) and chromatography-mass spectrometry (GC-MS) revealed that B156 is a cuticular wax-defective mutant, lacking waxes mostly longer than C28. Inheritance analysis revealed that this trait was controlled by a single recessive gene, BoGL5. Whole-genome InDel markers were developed, and a segregating F2 population was constructed to map BoGL5. Ultimately, BoGL5 was mapped to a 94.1 kb interval on C01. The BoCER2 gene, which is homologous to the Arabidopsis CER2 gene, was identified as a candidate of BoGL5 from the target interval. Sequence analyses revealed that Bocer2 in B156 harbored a G-to-T SNP mutation at the 485th nucleotide of the CDS, resulting in a W-to-L transition at the 162nd amino acid, a conserved site adjacent to an HXXXD motif of the deduced protein sequence. Expression analysis revealed that BoCER2 was significantly down-regulated in the leaves, stems, and siliques of B156 mutant than that of B3. Last, ectopic expression of BoCER2 in A. thaliana could, whereas Bocer2 could not, rescue the phenotype of cer2 mutant. CONCLUSIONS: Overall, this study mapped the locus determining glossy phenotype of B156 and proved BoCER2 is functional gene involved in cuticular wax biosynthesis which would promotes the utilization of BoCER2 to enhance plant resistance to biotic and abiotic stresses, and breeding of B. oleracea cultivars with glossy traits.