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ABSTRACT: During the perinatal period, dairy cows undergo negative energy balance, 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 inositol-requiring enzyme 1α (IRE1α) and c-Jun N-terminal kinase (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 nonruminants. 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 triglyceride [TG] content 5%). Liver tissue and blood samples were collected from experimental cows. Serum concentrations of NEFA and β-hydroxybutyrate (BHB) were greater, whereas 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 JNK, c-Jun, and 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; 1 d 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, short hairpin RNA 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 FAO. This may offer a prospective therapeutic target for fatty liver in dairy cows.

ABSTRACT: 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

Objective The analysis of pore structure and movable fluid distribution characteristics is the key element of reservoir research and is also the focus and hotspot of current research. It is of great significance for exploring tight sandstone oil and gas and improving oil and gas recovery. Methods The Chang-7 Member tight sandstone reservoir in the Qingcheng area of Ordos Basin was selected as the research object in this paper. Combined with fractal theory, through physical property tests, casting thin section, scanning electron microscopy (SEM), high-pressure mercury injection, and nuclear magnetic resonance (NMR) experiments, the pore throat structure, heterogeneity, and movable fluid distribution characteristics of tight sandstone reservoirs were analysed, and the influence of the pore throat structure and heterogeneity on movable fluid occurrence was discussed. Results The results show that the reservoir space of the Chang-7 Member reservoir in the study area is mainly contributed by micro-nanopores, which have poor pore connectivity. The pore throat radius is mainly 0.050-0.500 μm. The heterogeneity of the pore throat structure is strong, and the fractal dimension distribution is between 2.65 and 2.90. The fluid mobility is poor, and the movable fluid saturation is distributed between 16.68 % and 51.74 %, the movable fluid is mostly distributed in medium and small pores. The Chang-7 Member reservoirs in the study area can be divided into three types. From the type Ⅰ reservoir to the type Ⅲ reservoir, the development of residual intergranular pores and intergranular dissolution pores decreases, and the pore connectivity becomes poorer, and the pore throat size decreases, and the content of larger pore throats decreases, and the heterogeneity becomes stronger, and the fluid mobility is worse, and the movable fluid content in the medium and large pores tends to decrease, and movable fluid tends to occur in the small pores. Conclusion This study provides a theoretical basis for tight sandstone oil and gas exploration and improving oil and gas recovery.

Abstract PARP inhibitors and HDAC inhibitors have been approved for the clinical treatment of malignancies, but acquired resistance of or limited effects on solid tumors with a single agent remain as challenges. Bioinformatics analyses and a combination of experiments had demonstrated the synergistic effects of PARP and HDAC inhibitors in triple-negative breast cancer. A series of novel dual PARP and HDAC inhibitors were rationally designed and synthesized, and these molecules exhibited high enzyme inhibition activity with excellent antitumor effects in vitro and in vivo. Mechanistically, dual PARP and HDAC inhibitors induced BRCAness to restore synthetic lethality and promoted cytosolic DNA accumulation, which further activates the cGAS–STING pathway and produces proinflammatory chemokines through type I IFN-mediated JAK–STAT pathway. Moreover, these inhibitors promoted neoantigen generation, upregulated antigen presentation genes and PD-L1, and enhanced antitumor immunity when combined with immune checkpoint blockade therapy. These results indicated that novel dual PARP and HDAC inhibitors have antitumor immunomodulatory functions in triple-negative breast cancer. Novel dual PARP and HDAC inhibitors induce BRCAness to restore synthetic lethality, activating tumoral IFN signaling via the cGAS–STING pathway and inducing cytokine production, promoting neoantigen generation and presentation to enhance the immune response.

ABSTRACT: 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.

ABSTRACT: When ketosis occurs, supraphysiological concentrations of nonesterified fatty acids (NEFA) display lipotoxicity and are closely related to the occurrence of hepatic lipid accumulation, oxidative stress, and inflammation, resulting in hepatic damage and exacerbating the progression of ketosis. However, the mechanism of these lipotoxic effects caused by high concentrations of NEFA in ketosis is still unclear. Cluster antigen 36 (CD36), a fatty acid transporter, plays a vital role in the development of hepatic pathological injury in nonruminants. Thus, the aim of this study was to investigate whether CD36 plays a role in NEFA-induced hepatic lipotoxicity in dairy cows with clinical ketosis. Liver tissue and blood samples were collected from healthy (n = 10) and clinically ketotic (n = 10) cows at 3 to 15 d in milk. In addition, hepatocytes isolated from healthy calves were treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 12 h; or infected with CD36 expressing adenovirus or CD36 silencing small interfering RNA for 48 h and then treated with 1.2 mM NEFA for 12 h. Compared with healthy cows, clinically ketotic cows had greater concentrations of serum NEFA and β-hydroxybutyrate and activities of aspartate aminotransferase and alanine aminotransferase but lower serum glucose. In addition, dairy cows with clinical ketosis displayed excessive hepatic lipid accumulation. More importantly, these alterations were accompanied by an increased abundance of hepatic CD36. In the cell culture model, exogenous NEFA (0, 0.6, 1.2, or 2.4 mM) treatment could dose-dependently increase the abundance of CD36. Meanwhile, NEFA (1.2 mM) increased the content of triacylglycerol, reactive oxygen species and malondialdehyde, and decreased the activities of glutathione peroxidase and superoxide dismutase. Moreover, NEFA upregulated phosphorylation levels of nuclear factor κB (NF-κB) and the inhibitor of NF-κB (IκB) α, along with the upregulation of protein abundance of NLR family pyrin domain containing 3 (NLRP3) and caspase-1, and mRNA abundance of IL1B, IL6, and tumor necrosis factor α (TNFA). These alterations induced by NEFA in bovine hepatocytes were associated with increased lipid accumulation, oxidative stress and inflammation, which could be further aggravated by CD36 overexpression. Conversely, silencing CD36 attenuated these NEFA-induced detriments. Overall, these data suggest that CD36 may be a potential therapeutic target for NEFA-induced hepatic lipid accumulation, oxidative stress, and inflammation in dairy cows.

ABSTRACT: Dairy cows have high incidence of ketosis during perinatal. According to our previous studies, elevated ketone bodies (mainly β-hydroxybutyrate, BHB) in the peripheral blood are believed to contribute to the impairment of neutrophils mobility and directionality thereby contributing to the immunosuppression and further infectious disease secondary to ketosis. However, the specific effect of BHB on the directionality of bovine neutrophils needs further study and the underlying molecular mechanisms are still unclear. According to the concentration of serum BHB, 40 multiparous cows (within 3 wk postpartum) were selected and divided into the control (n = 20, BHB 3.0 mM) group. Blood samples were collected for baseline serum characteristics analysis and neutrophil mobility and directionality detection. Platelet activation factor was used as a chemoattractant in cell migration experiments. Our ex-vivo data showed ketotic cows, compared with control cows, were in a negative energy balance state, and their neutrophils had shorter migration distance, lower migration speed, and impaired migration directionality. Neutrophils from control cows were incubated with 3.0 mM BHB for 6 h in vitro. Similarly, BHB stimulation resulted in impaired mobility and directionality of bovine neutrophils. We further specifically studied the underlying molecular mechanism of BHB regulating neutrophil migration directionality in the present study. Cell division control protein 42 homolog (Cdc42) and Ras-related C3 botulinum toxin substrate 1 (Rac1), 2 key markers in the regulation of migration directionality, were found increased after BHB treatment in their total and activated protein levels while decreasing in their transcription level, suggesting that an imbalance of the protein degradation system may be involved. Interestingly, transmission electron microscopy data revealed a decrease in autophagosome number in neutrophils from ketotic cows. Western blotting data showed the accumulation of sequestosome-1 (p62) protein and a decrease in microtubule-associated protein 1 light chain 3-II (LC3-II) protein abundance after BHB treatment, further confirming that the autophagy flux was inhibited in neutrophils from ketotic cows. Additionally, rapamycin (RAPA), a specific autophagy activator, was used with or without BHB treatment in vitro. Accordingly, the BHB-induced impairment of migration directionality but not mobility was relieved by RAPA. Furthermore, as verified by in vivo experiments, compared with the control cows, the protein abundance of total and activated Cdc42 and Rac1 increased and their mRNA abundance decreased in neutrophils from ketotic cows. Overall, the present study revealed that pathological concentration of BHB impairs neutrophil migration directionality through inhibiting the autophagy-mediated degradation of Cdc42 and Rac1. These findings help explain the immunosuppression caused by ketosis.

ABSTRACT: Ketosis is often accompanied by a reduction in milk production in dairy cows, but the molecular mechanism has not been fully elucidated. Ketotic cows possess systemic oxidative stress (OS), which may implicate apoptosis in mammary glands. Sirtuin 3 (SIRT3) is a vital regulator of cellular redox homeostasis and is under the control of AMP-activated protein kinase (AMPK) signaling in nonruminants. Thus, we aimed to investigate (1) the AMPK-SIRT3 and apoptosis status of mammary glands from ketotic cows, (2) the effect of SIRT3 on OS-induced apoptosis in bovine mammary epithelial cells (BMEC), and (3) the role of AMPK signaling on SIRT3-mediated effects on apoptosis. Mammary gland samples were reused from a previous study, which contained healthy and ketotic cows (both n = 15). BMEC were incubated with 0, 0.3, 0.6, or 0.9 mM H2O2 for 6 h with/without a 30 min incubation of an antioxidant MitoQ (1 μM). Then BMEC were incubated with SIRT3 overexpression adenovirus (Ad-SIRT3) for 6 h followed by a 6 h incubation with 0.6 mM H2O2. Finally, BMEC were treated with the AMPK inhibitor Compound C (Cd C,10 μM) for 30 min before the H2O2 challenge, or cells were initially treated with the AMPK agonist MK8722 (10 μM) for 30 min followed by a 30-h culture with/without si-SIRT3 and eventually the H2O2 exposure. Ketotic cows displayed higher levels of Bax, Caspase-3 and Bax/Bcl-2 but lower levels of Bcl-2 in mammary glands. H2O2 incubation displayed similar results, exhibiting a dose-dependent manner between the H2O2 concentration and the apoptosis degree. Mito Q pretreatment reduced cellular reactive oxygen species and rescued cells from apoptosis. Ketotic cows had a lower mammary protein abundance of SIRT3. Similarly, H2O2 incubation downregulated both mRNA and protein levels of SIRT3 in a dose- and time-dependent manner. Ad-SIRT3 infection lowered levels of cellular reactive oxygen species, Bax, Caspase-3 and Bax/Bcl-2 but increased levels of Bcl-2. TUNEL assays confirmed that Ad-SIRT3 infection mitigated H2O2-induced apoptosis. Both ketotic cows and H2O2-induced BMEC had lower levels of p-AMPK and p-AMPK/AMPK. Additionally, Cd C pretreatment decreased SIRT3 and Bcl-2 expression but increased levels of Bax and Caspase-3. Contrary to the inhibitor, MK8722 had opposite effects and reduced the percentage of apoptotic cells. However, these effects of MK8722 were reversed upon SIRT3 silencing. In conclusion, in vivo data confirmed that ketosis is associated with greater apoptosis and restricted AMPK-SIRT3 signaling in mammary glands; in vitro data indicated that SIRT3 mitigates OS-induced apoptosis via AMPK signaling. As such, there may be potential benefits for targeting the AMPK-SIRT3 axis to help counteract the negative effects of mammary glands during ketosis.

Reversible regulation of N6-methyladenosine (m6A) methylation of eukaryotic RNA via methyltransferases is an important epigenetic event affecting RNA metabolism. As such, m6A methylation plays crucial roles in regulating animal growth, development, reproduction, and disease progression. Herein, we review the latest research advancements in m6A methylation modifications and discuss regulatory aspects in the context of growth, development, and reproductive traits of livestock. New insights are highlighted and perspectives for the study of m6A methylation modifications in shaping economically important traits are discussed.

ABSTRACT: During the transition period in dairy cows, high circulating concentrations of nonesterified fatty acids (NEFA) increase hepatic lipid deposits and are considered a major pathological factor for liver damage. We investigated whether AdipoRon, a synthetic small-molecule agonist of adiponectin receptors 1 and 2 shown to prevent liver lipid accumulation in nonruminants, could alleviate NEFA-induced lipid accumulation and mitochondrial dysfunction. Bovine hepatocytes were isolated from 5 healthy Holstein female newborn calves (1 d of age, 30–40 kg, fasting), and independently isolated hepatocytes from at least 3 different calves were used for each subsequent experiment. The composition and concentration of NEFA used in this study were selected according to hematological criteria of dairy cows with fatty liver or ketosis. First, hepatocytes were cultured with various concentrations of NEFA (0, 0.6, 1.2, or 2.4 mM) for 12 h. In a second experiment, hepatocytes were treated with AdipoRon at different concentrations (0, 5, 25, or 50 μM for 12 h) and times (25 μM for 0, 6, 12, or 24 h) with or without NEFA (1.2 mM) treatment. In the last experiment, hepatocytes were treated with AdipoRon (25 μM), NEFA (1.2 mM), or both for 12 h after treatment with or without the autophagy inhibitor chloroquine. Hepatocytes treated with NEFA had increased protein abundance of sterol regulatory element-binding protein 1c (SREBP-1c) and mRNA abundance of acetyl-CoA carboxylase 1 (ACACA), and decreased protein abundance of peroxisome proliferator-activated receptor α (PPARA), proliferator-activated receptor gamma coactivator-1 α (PGC-1α), mitofusin 2 (MFN2), cytochrome c oxidase subunit IV (COX IV), and mRNA abundance of carnitine palmitoyltransferase 1A (CPT1A), along with lower ATP concentrations. AdipoRon treatment reversed these effects, suggesting this compound had a positive effect on lipid metabolism and mitochondrial dysfunction during the NEFA challenge. In addition, upregulated expression of microtubule-associated protein 1 light chain 3-II (LC3-II, encoded by MAP1LC3) and downregulated expression of sequestosome-1 (SQSTM1, also called p62) indicated that AdipoRon enhanced autophagic activity in hepatocytes. The fact that chloroquine impeded the beneficial effects of AdipoRon on lipid accumulation and mitochondrial dysfunction suggested a direct role for autophagy during NEFA challenge. Our results suggest that autophagy is an important cellular mechanism to prevent NEFA-induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes, which is consistent with other studies. Overall, AdipoRon may represent a promising therapeutic agent to maintain hepatic lipid homeostasis and mitochondrial function in dairy cows during the transition period.

ABSTRACT: Fatty liver is a major metabolic disorder of high-producing dairy cows during the transition period. In nonruminants, it is well established that insulin-induced gene 1 (INSIG1) plays a crucial role in regulating hepatic lipogenesis by controlling the anchoring of sterol regulatory element-binding protein 1 (SREBP-1) on the endoplasmic reticulum along with SREBP cleavage-activating protein (SCAP). Whether the INSIG1-SCAP-SREBP-1c transport axis is affected in cows experiencing fatty liver is unknown. Thus, the aim of this study was to investigate the potential role of INSIG1-SCAP-SREBP-1c axis in the progression of fatty liver in dairy cows. For in vivo experiments, 24 dairy cows at the start of their fourth lactation (median; range 3–5) and 8 d in milk (median; range 4–12 d) were selected into a healthy group [n = 12; triglyceride (TG) content 10%) according to their hepatic TG content. Blood samples were collected for detecting serum concentrations of free fatty acids, β-hydroxybutyrate, and glucose. Compared with healthy cows, cows with severe fatty liver had higher serum concentrations of β-hydroxybutyrate and free fatty acids and lower concentration of glucose. Liver biopsies were used to detect the status of INSIG1-SCAP-SREBP-1c axis, and the mRNA expression of SREBP-1c-target lipogenic genes acetyl-CoA carboxylase α (ACACA), fatty acid synthase (FASN), and diacylglycerol acyltransferase 1 (DGAT1). Cows with severe fatty liver had lower protein expression of INSIG1 in the hepatocyte endoplasmic reticulum fraction, greater protein expression of SCAP and precursor SREBP-1c in the hepatocyte Golgi fraction, and greater protein expression of mature SREBP-1c in the hepatocyte nuclear fraction. In addition, the mRNA expression of SREBP-1c-target lipogenic genes ACACA, FASN, and DGAT1 was greater in the liver of dairy cows with severe fatty liver. In vitro experiments were conducted on hepatocytes isolated from 5 healthy 1-d-old female Holstein calves, and hepatocytes from each calf were run independently. First, hepatocytes were treated with 0, 200, or 400 μM palmitic acid (PA) for 12 h. Exogenous PA treatment decreased INSIG1 protein abundance, enhanced the endoplasmic reticulum to Golgi export of SCAP–precursor SREBP-1c complex and the nuclear translocation of mature SREBP-1c, all of which was associated with increased transcriptional activation of lipogenic genes and TG synthesis. Second, hepatocytes were transfected with INSIG1-overexpressing adenovirus for 48 h and treated with 400 μM PA 12 h before the end of transfection. Overexpressing INSIG1 inhibited PA-induced SREBP-1c processing, upregulation of lipogenic genes, and TG synthesis in hepatocytes. Overall, the present in vivo and in vitro results indicated that the low abundance of INSIG1 contributed to SREBP-1c processing and hepatic steatosis in dairy cows. Thus, the INSIG1-SCAP-SREBP-1c axis may be a novel target for treatment of fatty liver in dairy cows.

ABSTRACT: 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.

Acetaminophen (APAP) overdose is a major cause of liver injury. Neural precursor cell expressed developmentally downregulated 4–1 (NEDD4-1) is an E3 ubiquitin ligase that has been implicated in the pathogenesis of numerous liver diseases; however, its role in APAP-induced liver injury (AILI) is unclear. Thus, this study aimed to investigate the role of NEDD4-1 in the pathogenesis of AILI. We found that NEDD4-1 was dramatically downregulated in response to APAP treatment in mouse livers and isolated mouse hepatocytes. Hepatocyte-specific NEDD4-1 knockout exacerbated APAP-induced mitochondrial damage and the resultant hepatocyte necrosis and liver injury, while hepatocyte-specific NEDD4-1 overexpression mitigated these pathological events both in vivo and in vitro. Additionally, hepatocyte NEDD4-1 deficiency led to marked accumulation of voltage-dependent anion channel 1 (VDAC1) and increased VDAC1 oligomerization. Furthermore, VDAC1 knockdown alleviated AILI and weakened the exacerbation of AILI caused by hepatocyte NEDD4-1 deficiency. Mechanistically, NEDD4-1 was found to interact with the PPTY motif of VDAC1 through its WW domain and regulate K48-linked ubiquitination and degradation of VDAC1. Our present study indicates that NEDD4-1 is a suppressor of AILI and functions by regulating the degradation of VDAC1.

ABSTRACT: Fatty liver (i.e., hepatic lipidosis) is a prevalent metabolic disorder in dairy cows during the transition period, characterized by excess hepatic accumulation of triglyceride (TG), tissue dysfunction, and cell death. Detailed pathological changes, particularly hepatic fibrosis, during fatty liver remain to be determined. Liver fibrosis occurs as a consequence of liver damage, resulting from the excessive accumulation of extracellular matrix, which distorts the architecture of the normal liver, compromising its normal synthetic and metabolic functions. Thus, we aimed to investigate liver fibrosis status and its potential causal factors including oxidative stress, hepatocyte apoptosis, and production of inflammatory cytokines in the liver of cows with fatty liver. Forty-five dairy cows (parity, 3–5) were selected, and liver biopsy and blood were collected on the second week postpartum (days in milk, 10–14 d). On the basis of the degree of lipid accumulation in liver, selected cows were categorized into normal (n = 25; TG

The unconventional tight oil and gas resources in the Xinzhao East belt of the Hangjinqi Prospect area in the Ordos Basin of China are abundant. However, the reservoir’s internal storage space is complex, and the microscopic pore throat structural features are not well recognized, which has led to some trouble in the deployment of oil and gas exploration. To reveal the microscopic characteristics of the dense sandstone gas layer in the first member of the Lower Stone Box Formation of the D-well Zone in the Xinzhao East belt of the Hangjinqi Prospect area, a three-dimensional space digital core was built, and the stored set spatial data were extracted, based on rock sheet and coring data and X-CT scanning technology. Quartz grain size was segmented and analyzed based on an adaptive approach. The microscopic characteristics of the gas layer in the studied section and the factors influencing its development were studied, combining the use of a field emission scanning electron microscope, helium porosimeter, and gas permeability meter. We found that in the studied section, the porosity is relatively high, the pore throat size is large, and the pore permeability correlation is good. The reservoir space, which consists of intergranular pores, intragranular pores, and microcracks at the grain edges in the study area, is characterized by a complex distribution pattern. Within the gas layer, isolated pores are connected by microcracks to form a network of reservoir spaces, which increases the pore throat size, enhances the connectivity of the pore throat, and makes the microscopic characteristics of the reservoir space better. The first member of the Lower Stone Box Formation could be an advantageous reservoir. Hole–throat connectivity is poor because of the gas layer having underdeveloped primary pores, the blockage of pores by unstable minerals (kaolinite, etc.), and poorly connected pore throats based on insoluble mud cementation. The high content of quartz brittle minerals and the development of natural microcracks within the gas formation are favorable conditions for fracking development. The quartz grain size within the gas layer is positively correlated with the pore throat size, which suggests that the quartz grain size somewhat influences the microscopic characteristics of the reservoir space. This comprehensive study shows that the methodology of the study is more advantageous than traditional methods in the fine and three-dimensional spatial characterization of the microstructure of dense sandstone reservoirs. The research results of this paper have certain guiding significance for further reservoir evaluation and advantageous reservoir prediction in the Hangjinqi Prospect area in the Ordos Basin. We also provide the basis for the subsequent efficient development of the gas reservoir.

Abstract Androgen receptor (AR) is strong association with breast cancer (BRCA). We aimed to investigate the effect of the androgen receptor signaling pathway-related long non-coding RNAs (ARSP-related lncRNAs) on the process of subtype classification and the tumor microenvironment (TME) of breast cancer (BRCA). Our study screen ARSP-related lncRNAs for the construction of a risk model. The single-sample gene set enrichment analysis (ssGSEA) method was used to detect the differences between the immune responses generated by the patients belonging to the low- and high-risk groups. The relationship between the ARSP-related lncRNAs and TME was explored following the process of cluster analysis. The univariate Cox analysis and the Lasso regression analysis method was used to screen nine of these lncRNAs to develop a risk model. It was observed that risk score could function as an independent prognostic factor, affecting the prognoses of patients suffering from BRCA. The validity of the model was assessed by analyzing the generated calibration curves and a nomogram. Additionally, the effect of the risk score on the extent of immune cell infiltration realized in TME was explored. M2 macrophages correlated positively, whereas NK cells, CD4+ T cells, and naive B cells correlated negatively with the risk score. Results obtained using the cluster analysis indicated that immune scores correlated with clustered subtypes. Finally, the risk score and cluster subtypes were analyzed to study the sensitivity of the patients toward different drugs to identify the appropriate therapeutic agents. The prognoses of patients suffering from BRCA can be accurately predicted by ARSP-related lncRNAs.

With the rapid development of modern technology and economy, mineral resources such as oil and natural gas have attracted more and more attention. Oil and gas accumulation is a key link in China’s national economy and major national safety production. The prediction of favorable hydrocarbon accumulation assemblages has always been a matter of great concern to people. However, the current oil and gas exploration methods have undergone tremendous changes and the prospecting environment has become increasingly complex, and the exploration methods have gradually developed in a vectorized direction. The exploration focus has also shifted to complex deep deposits, overburdens and concealed deposits. With the increasing abundance of mineral resources, the challenges faced are also increasing, so it is particularly important to develop new exploration technologies and new technical means. With the continuous development of big data, artificial intelligence, machine learning, swarm intelligence and other technologies have good application prospects in various aspects. In this paper, machine learning technology was used for oil and gas accumulation combination classification and favorable accumulation combination prediction. The prediction model was established by using machine learning method, and the prediction data of the model was compared with the conventional prediction method, so that the prediction effect of the model was verified. The test results showed that the average relative errors of oil wells No. 1, No. 2, No. 3, and No. 4 in the proposed model were 23.33%, 24.93%, 26.38%, and 22.60%, respectively; the average relative errors of No. 1, No. 2, No. 3 and No. 4 in the conventional prediction model were 26.19%, 32.37%, 36.16, and 27.87%, respectively. Therefore, it can be concluded that the prediction effect of the prediction model using the machine learning method was significantly better than that of the traditional prediction model.

ABSTRACT: Peripartum dairy cows experience negative energy balance, characterized by high concentrations of blood free fatty acids (FFA) and immune dysfunction. Palmitic acid (PA), the most abundant saturated fatty acid in cow blood, is not only an energy precursor, but causes cellular dysfunction when in excess. Neutrophil extracellular traps (NET) are one of the arsenals of weapons neutrophils use to fight invading pathogens. However, given the marked increase in circulating PA during the peripartum period, it remains to be determined what effect (if any) PA has on NET release. Thus, the objective of this study was to evaluate the effect of PA on NET release and the underlying mechanism in vitro. Phorbol-12-myristate-13-acetate (PMA; 100 ng/mL, 3 h) was used to induce the release of NET in vitro. We isolated neutrophils from the peripheral blood of 5 healthy postpartum dairy cows with similar parity (median = 3, range = 2–4), milk yield (median = 27.84 kg/d per cow, range = 25.79–31.43 kg/d per cow), days in milk (median = 7 d, range = 4–10 d), and serum FFA 3.5 mM. Inhibition of double-stranded DNA (dsDNA) level, a marker of NET release, in response to PA was used to determine an optimal incubation time and concentration for in vitro experiments. Cells were maintained in RPMI-1640 basic medium without phenol red, treated with 600 μM PA for different times (4, 5, 6, and 7 h) in the presence or absence of PMA. There was a decrease for dsDNA level in the supernatant due to increased duration of PA treatment, with a peak response at 6 h. Thus, 6 h was selected as the challenge time. Then, cells were treated with different concentrations of PA (100, 200, 400, and 600 μM) for 6 h in the presence or absence of PMA. There was a decrease for dsDNA level in the supernatant due to increased dose of PA, with a peak response at 400 μM. Finally, 400 μM PA for 6 h was selected as the treatment for subsequent experiments. Protein abundance of citrullinated histone in the presence or absence of PMA was markedly lower in response to incubation with PA. Morphological observations by laser confocal microscopy and scanning electron microscopy showed that the ratio of NET-releasing cells decreased in response to incubation with PA. Autophagy is a potential key intermediate process in the regulation of NET by PA. To investigate the effect of PA on autophagy, we used chloroquine to block lysosomal degradation. Exogenous PA led to accumulation of sequestosome-1 and microtubule-associated protein 1 light chain 3-II, and no further accumulation in the presence of chloroquine, all of which suggested an impairment of autophagic flux. To verify the role of autophagy in NET, we used rapamycin to promote autophagic flux; 100 nM rapamycin attenuated the suppressive effect of PA on NET release indicated by greater dsDNA levels, accumulation of citrullinated histone, and ratio of NET-releasing neutrophils. Overall, these data demonstrate PA inhibits NET release by suppressing autophagic flux, which provides information for understanding the immune dysfunction in postpartum cows.

ABSTRACT: 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.

ABSTRACT: 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 β-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.

ABSTRACT: Subacute ruminal acidosis (SARA), a common digestive disease in dairy cows, is accompanied by systemic inflammation and high concentrations of histamine in blood. Histamine-induced neutrophil adhesion may play an important role in the systemic inflammation experienced by cows during SARA. Autophagy, an intracellular degradation system, regulates recycling of membrane-associated integrin and may be involved in histamine-induced adhesion of bovine neutrophils. In the present study, 20 multiparous mid-lactation cows (average body weight 486 ± 24 kg) fitted with ruminal fistula were assigned to a control group (n = 10) or a SARA group (n = 10). We induced SARA by feeding different combinations of wheat-barley pellets and chopped alfalfa hay for 8 wk; SARA was defined as a ruminal pH

ABSTRACT: 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 β-hydroxybutyrate (BHB) concentration

ABSTRACT: 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-3β (GSK3β) 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 GSK3β, 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 GSK3β, 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 GSK3β, 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.

ABSTRACT: 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, β-hydroxybutyrate (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.

Abstract Understanding the association of pyroptosis with tumor progression, prognosis and effect on immunotherapeutic response in breast cancer (BC) is limited. This study analysed forty pyroptosis-related genes to construct the pyroptosis score. Association of the pyroptosis score with the overall survival, clinical features, tumor mutation load, immune cell infiltration, and treatment sensitivity of patients with BC was analysed. Out of 983 BC samples, 304 (30.93%) had genetic alterations with the highest TP53 frequency. We identified three separate subtypes associated with pyroptosis action. These subtypes correlate with the clinicopathological characteristics, TME immune cell infiltration, and disease prognosis. Based on the expression levels of the pyroptosis genes, we divided the pyroptosis score into a high group and a low group. The immune-activated pyroptosis subtype had a higher score with a better prognosis. We also observed that the pyroptosis score correlates with the tumor mutation burden. The pyroptosis score and disease prognosis were directly proportional. A higher pyroptosis score indicated a better prognosis. Results suggest that the pyroptosis-related gene prognosis model is closely related to the immune cell infiltration of BC. The three pyroptosis subtypes associated with BC assist in accurately identifying the tumor subtype, the prognosis of immunotherapy drugs and the patient’s therapeutic response.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis and insulin resistance and there are currently no approved drugs for its treatment. Hyperactivation of mTOR complex 1 (mTORC1) and subsequent impairment of the transcription factor EB (TFEB)-mediated autophagy–lysosomal pathway (ALP) are implicated in the development of NAFLD. Accordingly, agents that augment hepatic TFEB transcriptional activity may have therapeutic potential against NAFLD. The objective of this study was to investigate the effects of nuciferine, a major active component from lotus leaf, on NAFLD and its underlying mechanism of action. Here we show that nuciferine activated ALP and alleviated steatosis, insulin resistance in the livers of NAFLD mice and palmitic acid-challenged hepatocytes in a TFEB-dependent manner. Mechanistic investigation revealed that nuciferine interacts with the Ragulator subunit hepatitis B X-interacting protein and impairs the interaction of the Ragulator complex with Rag GTPases, thereby suppressing lysosomal localization and activity of mTORC1, which activates TFEB-mediated ALP and further ameliorates hepatic steatosis and insulin resistance. Our present results indicate that nuciferine may be a potential agent for treating NAFLD and that regulation of the mTORC1–TFEB–ALP axis could represent a novel pharmacological strategy to combat NAFLD.

ABSTRACT: Reduced feed intake during the transition period renders cows unable to meet their energy needs for maintenance and lactation, leading to a state of negative energy balance. Severe negative energy balance initiates fat mobilization and increases circulating levels of free fatty acids (FFA), which could induce hepatic mitochondrial dysfunction, oxidative stress, and apoptosis. Enhancing the hepatic supply of propionate (major gluconeogenic substrate) is a feasible preventive and therapeutic strategy to alleviate hepatic metabolic disorders during the transition period. Whether propionate supply affects pathways beyond gluconeogenesis during high FFA loads is not well known. Thus, the objective of this study was to investigate whether propionate supply could protect calf hepatocytes from FFA-induced mitochondrial dysfunction, oxidative stress, and apoptosis. Hepatocytes were isolated from 5 healthy calves (1 d old, female, 30–40 kg, fasting) and treated with various concentrations of propionate (0, 1, 2, and 4 mM propionate for 12 h) or for different times (2 mM propionate for 0, 3, 6, 12 and 24 h). Furthermore, hepatocytes were treated with propionate (2 mM), fatty acids (1.2 mM), or both for 12 h with or without 50 nM PGC-1α (peroxisome proliferator-activated receptor-gamma coactivator-1 alpha) small interfering RNA. Compared with the control group, protein abundance of PGC-1α was greater with 2 and 4 mM propionate treatment groups. Furthermore, protein abundance of TFAM (mitochondrial function marker mitochondrial transcription factor A) and VDAC1 (voltage-dependent anion channel 1) was greater with 1, 2, and 4 mM propionate, and COX4 (cyclooxygenase 4) was greater with 2 and 4 mM propionate groups. In addition, propionate supply led to an increase in protein abundance of PGC-1α, TFAM, VDAC1, and COX4 over time. Flow cytometry revealed that propionate treatment increased the number of mitochondria in hepatocytes compared with control group, but inhibition of PGC-1α abolished these beneficial effects. The lower protein abundance of PGC-1α, TFAM, COX4, and VDAC1 and activities of superoxide dismutase and glutathione peroxidase, along with greater production of reactive oxygen species, malondialdehyde, and apoptosis rate in response to treatment with high concentrations of FFA suggested an impairment of mitochondrial function and induction of oxidative stress and apoptosis. In contrast, propionate treatment hastened these negative effects. Knockdown of PGC-1α by small interfering RNA impeded the beneficial role of propionate on FFA-induced mitochondrial dysfunction, oxidative stress, and apoptosis. Overall, results demonstrated that propionate supply alleviates mitochondrial dysfunction, oxidative stress, and apoptosis in FFA-treated calf hepatocytes by upregulating PGC-1α. Together, the data suggest that PGC-1α may be a promising target for preventing or improving hepatic function during periods such as the transition into lactation where the FFA load on the liver increases.

ABSTRACT: 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 β (GSK3β), 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 β-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 β-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-GSK3β, 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.

ABSTRACT: Ketosis in dairy cows often occurs in the peripartal period and is accompanied by immune dysfunction. High concentrations of β-hydroxybutyrate (BHB) in peripheral blood during ketosis inhibits the release of neutrophil extracellular traps (NET) and contributes to immune dysfunction. However, the mechanisms whereby BHB affects NET release remains unclear. In this study, 5 healthy peripartal dairy cows (within 3 wk postpartum) with serum BHB concentrations 3.5 mM were used as blood donors. Blood samples were collected before feeding, and the isolated polymorphonuclear neutrophils were incubated with 3 mM BHB for different times. Inhibition of Cit-H3 (citrullinated histone 3) protein abundance, a marker of NET activation, in response to BHB was used to determine an optimal incubation time for in vitro experiments. Four hours was selected as the optimal duration of BHB treatment. Phorbol-12-myristate-13-acetate (PMA) was used to induce the release of NET in vitro. The BHB treatment with or without PMA treatment decreased protein abundance of Cit-H3 and PAD4 (arginine deiminase 4) and increased neutrophil elastase. Immunofluorescence and scanning electron microscope analyses revealed that BHB treatment inhibited PMA-induced NET release. The BHB treatment also decreased double strain DNA content in the supernatant, further confirming the inhibitory effect of BHB on NET release. Furthermore, BHB treatment decreased the level of intracellular reactive oxygen species (ROS), phosphorylation level of p47, and protein abundance of Rac2, suggesting that BHB-induced NET inhibition may have been caused by decreased NADPH oxidase-derived ROS. The phosphorylation level of phosphoinositide 3-kinase (PI3K), an important upstream regulator of NADPH oxidase, was attenuated by BHB treatment. To confirm the involvement of PI3K signaling pathway in BHB-induced NET inhibition, 740Y-P, a potent activator of PI3K signaling pathway, was used. Data indicated that 740Y-P relieved the inhibitory effects of BHB on ROS production and NADPH oxidase activation. Importantly, as revealed by immunofluorescence and scanning electron microscopy analyses, 740Y-P also dampened the inhibitory effect of BHB on NET release and the protein abundance of Cit-H3 and PAD4. Overall, the present study revealed that high concentration of BHB impairs NET release through inhibiting PI3K-mediated NADPH oxidase ROS production. These findings help partly explain the immune dysfunction in cows experiencing negative energy balance or ketosis in early lactation.

ABSTRACT: Ketosis in dairy cows, a common metabolic disorder during the peripartal period, is accompanied by systemic inflammation and high concentrations of blood β-hydroxybutyrate (BHB). Neutrophil apoptosis plays a key role in maintaining the balance of inflammation and functional capacity of circulating neutrophils in ketotic cows. The kinases ERK1/2 and AKT, as well as their downstream Bcl-2 family-mediated mitochondrial signaling, are important apoptosis-regulating pathways in neutrophils. The objective of our study was to investigate the effects of BHB on neutrophil apoptosis and the underlying regulatory mechanisms during ketosis. Neutrophils were isolated from 5 multiparous cows (within 3 wk postpartum) with serum BHB concentrations 3.5 mM. In a series of experiments, neutrophils were treated with increasing concentrations of BHB (0, 0.6, 2, and 3 mM for 10 h) and time (0, 2, 4, 6, 8, and 10 h with 2 mM). Subsequently, a 2 mM BHB dose was used to challenge neutrophils for 8 h. Apoptosis rate of neutrophils and protein abundance of cleaved caspase 3 were lower after BHB treatment. Treatment with BHB decreased protein and mRNA abundance of the pro-apoptotic genes Bax (BAX) and Bad (BAD), whereas it increased mitochondrial membrane potential (MMP) and protein and mRNA of the anti-apoptotic genes Bcl-xL (BCL2L1) and Mcl-1 (MCL1). This indicated that a mitochondrial pathway was involved in the inhibition of neutrophil apoptosis via BHB. In addition, both SCH772984 (an inhibitor of the ERK1/2 signaling pathway) and MK-2206 (an inhibitor of the AKT signaling pathway) alleviated the BHB-induced anti-apoptotic function of the Bcl-2 family and the inhibition of MMP. Overall, our data demonstrated that high concentrations of BHB inhibit apoptosis in bovine neutrophils by activating the ERK1/2 and AKT signaling pathways. These findings provide a theoretical basis for the understanding of systemic inflammation in ketotic cows.

IntroductionSubclinical ketosis (SCK) in dairy cows, a common metabolic disorder during the perinatal period, is accompanied by systemic inflammation and a high concentration of blood β-hydroxybutyrate (BHB). BHB induced adhesion of neutrophils may play a crucial role in the development of systemic inflammation in SCK cows. Autophagy, an intracellular degradation system, regulates the recycling of membrane adhesion molecules and may be involved in BHB regulating adhesion and pro-inflammatory activation of bovine neutrophils. Thus, the objective of this study was to determine the relationship between BHB, autophagy, and neutrophil adhesion. Results and discussionHere, elevated abundance of serum amyloid A, haptoglobin, C-reactive protein, interleukin-1β, interleukin-6, and tumor necrosis factor-α were found in SCK cows, and all these pro-inflammatory factors had a strong positive correlation with serum BHB. After BHB treatment, the number of adherent neutrophils and the adhesion associated protein abundance of both total and membrane CD11a, CD11b, and CD18 was greater, confirming that BHB promoted the adhesion of bovine neutrophils. However, the mRNA abundance of ITGAL (CD11a), ITGAM (CD11b), and ITGB2 (CD18) did not show a significant difference, suggesting that the degradation of adhesion molecules may be impaired. Transmission electron microscopy revealed a decreased number of autophagosomes and a decrease in mRNA abundance of SQSTM1 (p62) and MAP1LC3B (LC3) after BHB treatment. In parallel, protein abundance of p62 increased while the ratio of protein LC3 II to LC3 I decreased after BHB treatment, indicating that BHB inhibits autophagy of bovine neutrophils. To confirm the regulatory role of autophagy in BHB promoting neutrophil adhesion, we used an autophagy activator rapamycin (RAPA). Data showed that RAPA relieved the inhibitory effect on autophagy and the promotive effect on cell adhesion induced by BHB. Importantly, BHB inhibited the colocalization of LC3 and CD11b, which was relieved by RAPA, further confirming the regulatory role of autophagy in the recycling of the above adhesion molecules. Furthermore, BHB treatment increased the mRNA abundance and the release of pro-inflammatory factors IL-1B, IL-6, and TNF of bovine neutrophils, and these effects were attenuated by RAPA. Overall, the present study revealed that BHB promotes the adhesion of bovine neutrophils by inhibiting autophagy.

ABSTRACT: Subclinical ketosis (SCK) in dairy cows, a common metabolic disorder during the peripartal period, is accompanied by systemic inflammation. Excessive release of azurophil granule (AG) contents during degranulation of polymorphonuclear neutrophils (PMN) could contribute to systemic inflammation in SCK cows. Although the increase in blood free fatty acids (FFA) in SCK cows may promote AG degranulation from PMN, the underlying mechanisms are unclear. Thirty multiparous cows (within 3 wk postpartum) with similar lactation numbers (median = 3, range = 2–4) and days in milk (median = 6, range = 3–15) were classified based on serum β-hydroxybutyrate (BHB) level as control (n = 15, BHB < 0.6 mM) or SCK (n = 15, 1.2 mM < BHB < 3.0 mM). Cows with SCK had greater levels of serum haptoglobin, serum amyloid A, IL-1β, IL-6, IL-8 and tumor necrosis factor-α. These proinflammatory factors had strong positive correlations with myeloperoxidase (MPO), a marker protein of PMN AG, whose content was greater in the serum of SCK cows. Both the number of AG and the protein abundance of MPO were lower in PMN isolated from SCK cows. Additionally, we found a greater ratio of blood CH138A+/CD63high cells and greater mean fluorescence intensity of CD63 on the PMN membrane, further confirming the greater degree of AG degranulation in cows with SCK. In vitro FFA dose response (0, 0.3, 0.6, 1.2, and 2.4 mM for 4 h) and time course (0, 0.5, 1, 2, and 4 h with 0.6 mM) experiments were performed on PMN isolated from control cows. The increase in MPO content in extracellular supernatant resulting from those experiments led to the selection of 0.6 mM FFA for 1 h duration as conditions for subsequent studies. After FFA treatment, release of intracellular MPO was increased along with increased levels of CD63 mean fluorescence intensity on the PMN membrane, confirming that FFA promoted degranulation of AG. In addition, FFA treatment increased reactive oxygen species (ROS) production by PMN, an effect that was attenuated by incubation with diphenyleneiodonium chloride (DPI), a NADPH oxidase-derived ROS inhibitor. The mitochondrial-derived ROS inhibitor carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) did not affect ROS in response to FFA treatment. Treatment with FFA increased p47 phosphorylation and mRNA abundance of NCF1, NCF2, and CYBB in PMN. Furthermore, DPI, but not FCCP, dampened the degranulation of PMN AG induced by FFA in vitro. These data suggested that the degranulation of AG in PMN induced by FFA was mediated by NADPH oxidase-derived ROS. As verified ex vivo, PMN from SCK cows had greater levels of ROS, phosphorylation of p47, and mRNA abundance of NCF1, NCF2, and CYBB. Overall, the present study revealed that high blood concentrations of FFA in SCK cows induce the production of NADPH oxidase-derived ROS, thereby promoting degranulation of AG in PMN. The stimulatory effect of FFA on the release of AG content during degranulation, especially MPO, provides a new insight into the systemic inflammation experienced by peripartal cows with SCK.

ABSTRACT: 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 β-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.

ABSTRACT: Ketosis in dairy cows often occurs in the peripartal period and is accompanied by immune dysfunction. High concentrations of β-hydroxybutyrate (BHB) in peripheral blood during ketosis are closely related to the impairment of polymorphonuclear neutrophil (PMN) chemotaxis and contribute to immune dysfunction. The specific effect of BHB on PMN chemotaxis in dairy cows and the underlying molecular mechanisms are unclear. Here, 30 multiparous cows (within 3 wk postpartum) classified based on serum BHB as control (n = 15, BHB 3.0 mM) were used. Blood samples were collected before feeding, and the isolated PMN were treated with platelet-activating factor for 0.5 h to activate their migration. Scanning electron microscopy revealed a longer tail in PMN of ketotic cows. In addition, the phosphorylation and transcription levels of myosin light chain 2 (MLC2) increased in PMN of ketotic cows. Polymorphonuclear neutrophils from control dairy cows were incubated with 3.0 mM BHB for different times in vitro, and 6 h was selected as the proper duration of BHB stimulation according to its inhibition effect on PMN migration using an under-agarose PMN chemotaxis model. Similarly, BHB stimulation in vitro resulted in inhibition of migration distance and deviation of migration direction of PMN, as well as a longer tail in morphology in the scanning electron microscope data, suggesting that BHB-induced PMN migration inhibition may be mediated by impairing the trailing edge contraction. To confirm this hypothesis, sotrastaurin (Sotra)—a specific inhibitor of protein kinase C (PKC), which is the core regulator of cell contraction—was used with or without BHB treatment in vitro. Sotra was pretreated 0.5 h before BHB treatment. Accordingly, BHB treatment increased the phosphorylation level of PKC and MLC2, the protein abundance of RhoA and rho-kinase 1 (ROCK1), and the mRNA abundance of PRKCA, MYL2, RHOA, and ROCK1 in PMN. In contrast, these effects of BHB on PMN were dampened by Sotra. As demonstrated by immunofluorescence experiments in vitro, the BHB-induced inhibition of trailing edge contraction of PMN was relieved by Sotra. In addition, Sotra also dampened the effects of BHB on PMN migration in vitro. Furthermore, as verified by in vivo experiments, compared with the control cows, both abundance and activation of PKC signaling were enhanced in PMN of ketotic cows. Overall, the present study revealed that high concentrations of blood BHB impaired PMN migration distance through inhibition of the trailing edge contraction, mediated by enhancing the activation of PKC-MLC2 signaling. These findings help explain the dysfunctional immune state in ketotic cows and provide information on the pathogenesis of infectious diseases secondary to ketosis.

This article describes a closed-loop detection MEMS accelerometer for acceleration measurement. This paper analyzes the working principle of MEMS accelerometers in detail and explains the relationship between the accelerometer zero bias, scale factor and voltage reference. Therefore, a combined compensation method is designed via reference voltage source compensation and terminal temperature compensation of the accelerometer, which comprehensively improves the performance over a wide temperature range of the accelerometer. The experiment results show that the initial range is reduced from 3679 ppm to 221 ppm with reference voltage source compensation, zero-bias stability of the accelerometer over temperature is increased by 14.3% on average and the scale factor stability over temperature is increased by 88.2% on average. After combined compensation, one accelerometer zero-bias stability over temperature was reduced to 40 μg and the scale factor stability over temperature was reduced to 16 ppm, the average value of the zero-bias stability over temperature was reduced from 1764 μg to 36 μg, the average value of the scale factor stability over temperature was reduced from 2270 ppm to 25 ppm, the average stability of the zero bias was increased by 97.96% and the average stability of the scale factor was increased by 98.90%.

The research evaluated the effects of Aflatoxin B1 on growth performance, antioxidant status, immune response, and pro-inflammatory cytokine mRNA expression in ISA chicks. In total, 240 7-day-old ISA chicks were randomly assigned to four treatment groups. The control group comprised chicks fed a basal diet. The aflatoxin (AFB1)-treatment groups (T1, T2, and T3) comprised chicks fed the basal diet supplemented with AFB1 at concentrations of 5, 8, and 10 μg/kg, respectively. The growth performance, antioxidant status, immune responses, and pro-inflammatory cytokine mRNA expression in all groups were measured. In the T1 treatment group (receiving the lowest AFB1 dose), a reduction in the Newcastle disease virus antibody (NDV-Ab) titer, and increases in interleukin 2 (IL-2), IL-6, and interferon γ (IFN-γ) mRNA levels were observed on days 21 and 42 (P < 0.05). Treatment with the higher AFB1 doses (groups T2 and T3) reduced the chicks' growth performance on days 21 and 42, measured as reductions in body weight (BW) and average daily gain (ADG) compared with the control group. In the T2 and T3 groups, the total antioxidant capacity (T-AOC), glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities, serum immunoglobulin A (IgA) and IgG levels, and IL-2, IL-6, and IFN-γ levels were also lower than in the control group. On days 21 and 42, these two groups also showed increased malondialdehyde (MDA) content, higher feed to gain ratio (F/G), and higher IL-2, IL-6, and IFN-γ mRNA levels than the control group (P < 0.05). The T2 and T3 groups also showed reduced T-AOC, NDV-Ab titer, IL-2 content, and GPx-1 mRNA levels on days 21 and 42 (P < 0.05), increased IL-6 and IFN-γ mRNA levels on day 21, and increased F/G and MDA content on day 42 (P < 0.05) compared with group (T1). Increased MDA content and IL-6 mRNA levels in the liver and ileum were observed in group T3 compared with group T2 on day 21, and lower IgM and IL-6 levels were observed on days 21 and 42 (P < 0.05). In conclusion, our data showed that AFB1 exposure resulted in dose-dependent oxidative and inflammatory damage, immunosuppression, and a decline in the growth performance of chicks.

This paper presents a novel method for the performance of an all-silicon accelerometer by adjusting the ratio of the Si-SiO2 bonding area, and the Au-Si bonding area in the anchor zone, with the aim of eliminating stress in the anchor region. The study includes the development of an accelerometer model and simulation analysis which demonstrates the stress maps of the accelerometer under different anchor–area ratios, which have a strong impact on the performance of the accelerometer. In practical applications, the deformation of the comb structure fixed by the anchor zone is influenced by the stress in the anchor region, causing a distorted nonlinear response signal. The simulation results demonstrate that when the area ratio of the Si-SiO2 anchor zone to the Au-Si anchor zone decreases to 0.5, the stress in the anchor zone decreases significantly. Experimental results reveal that the full-temperature stability of zero-bias is optimized from 133 μg to 46 μg when the anchor–zone ratio of the accelerometer decreases from 0.8 to 0.5. At the same time, the full-temperature stability of the scale factor is optimized from 87 ppm to 32 ppm. Furthermore, zero-bias full-temperature stability and scale factor full-temperature stability are improved by 34.6% and 36.8%, respectively.

The role of lipocalin 2 (LCN2) in pulmonary hypertension (PH) in pediatric patients with congenital heart disease (CHD) remains unclear. We sought to investigate whether LCN2 could be a potential biomarker for PH in pediatric patients who underwent surgery for CHD. From December 2018 to February 2020, patients undergoing surgical repair for congenital defects with and without PH were identified. Healthy children without CHD and PH served as controls. A mean pulmonary artery pressure (mPAP) >20 mmHg was used as the definition of PH. Blood samples and echocardiograms were obtained in all patients and right heart catheterization was performed in 79 patients. Multivariable logistic regression analysis was used to determine potential predictors for PH. Among 102 patients, the median age was 10 [Interquartile range (IQR) 7.0–13] months, and 37.5% were female. Compared to non-PH patients and controls, PH patients showed elevated levels of LCN2 (P < 0.001). In addition, LCN2 levels positively correlated with the invasive haemodynamic indices of PH. In univariate regression, LCN2 (odds ratio = 2.69 [1.06–5.31], P < 0.001), N-Terminal pro Brain Natriuretic Peptide (NT-proBNP) (OR = 1.91 [1.21–7.56], P = 0.03) and high-sensitive troponin T (hsTnT) (OR = 1.36 [1.01–3.57], P = 0.01) were associated with PH; however, only LCN2 (OR = 1.68 [1.04–4.52], P = 0.03) was significantly associated with PH on multivariate analysis. In conclusion, children with PH had increased LCN2 expression. LCN2 levels positively correlated with invasive indices of PH. These results indicate LCN2 could be a useful biomarker for prediction of PH in pediatric CHD cases.

Abstract Ivosidenib is a once daily (q.d.), orally available, potent mutant isocitrate dehydrogenase 1 (mIDH1) inhibitor approved for treatment of patients with relapsed or refractory (R/R) acute myeloid leukemia (AML) and intensive chemotherapy ineligible AML with a susceptible IDH1 mutation. Population pharmacokinetics (PKs; N = 253), exposure‐response (efficacy [n = 201] and safety [n = 253]), and concentration‐corrected electrocardiogram QT interval (QTc; n = 171) analyses were performed using phase I data (100 mg twice daily and 300–1200 mg q.d.). Ivosidenib disposition was well‐described by a two‐compartment PK model with first‐order absorption and elimination. Between‐subject variability was moderate for PK parameters. Intrinsic factors did not affect ivosidenib PKs. Moderate/strong CYP3A4 inhibitors increased the area under the plasma ivosidenib concentration‐time curve at steady state (AUCss) by 60%. Efficacy responders and nonresponders had similar ivosidenib exposures. Based on AUCss, there was no apparent relationship between ivosidenib exposure and efficacy or adverse events. The plasma ivosidenib concentration‐QT analysis showed a mean change in QTc using Fridericia’s method (ΔQTcF) of 17.2 msec at the approved 500 mg q.d. dose. Because of the direct association between ivosidenib exposure and QTcF, patients should have their electrocardiograms and electrolytes monitored, and comedications that increase ivosidenib exposure or prolong the QT interval should be avoided. These model‐based analyses quantitatively provide a framework to describe the relationship among ivosidenib dose, exposure, and clinical end points. With precautions for QTc prolongation, the exposure‐response analyses support the 500 mg q.d. dose in patients with AML with a susceptible IDH1 mutation.

The regulation of resistance to IDH inhibitors in acute myeloid leukaemia is not completely understood. Here the authors reveal with integrative multi-omics analyses that stemness features are major drivers of primary resistance, while high-risk mutations drive acquired resistance.

Abstract Background Left displaced abomasum (LDA) occurs at high frequency in the early postpartum period and can affect production performance of dairy cows. Clinical diagnosis of LDA is usually done by abdominal auscultation and percussion. The purpose of this study was to explore the potential applicability of blood biomarkers for early warning and diagnosis of LDA in dairy cows. Results Twenty early postpartum healthy cows and thirty early postpartum LDA cows of similar parity were used. A receiver operating characteristic curve (ROC) method was used to analyze the sensitivity of hematological biomarkers to LDA including energy balance metabolic biomarkers, liver/kidney function biomarkers, and minerals. A cut-off point was defined for each of the selected hematological biomarkers deemed sensitive markers of LDA. Compared with healthy cows, body condition score (BCS), dry matter intake (DMI) and milk production were lower in LDA cows. Among energy metabolism markers, serum non-esterified fatty acid (NEFA), β-hydroxybutyric acid (BHBA), insulin (INS), and revised quantitative insulin sensitivity check index (RQUICKI) levels were lower while serum glucagon (GC) was greater in LDA cows. Among the liver/kidney function biomarkers, activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), lactate dehydrogenase (LDH), the ratio of AST/ALT and levels of total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), albumin (ALB), blood urea nitrogen (BUN), creatinine, and total protein (TP) were greater in LDA cows. Among minerals analyzed, serum Cl, Ca, and K were lower in LDA cows. After ROC analysis, it was determined that serum Ca, INS, RQUICKI, ALT, GGT, and creatinine are potential indicators for early warning and diagnosis of LDA for early postpartum dairy cows. Conclusions Dairy cows with LDA were under severe negative energy balance (NEB), had signs of liver damage and potentially lower insulin sensitivity. A combination of multi-hematological biomarkers including Ca, INS, RQUICKI, ALT, GGT and creatinine has the potential to help identify cows at risk of LDA in the early postpartum period.

Excessive lipid mobilization for adipose tissue caused by severe negative energy balance is the pathological basis for subclinical ketosis (SCK) in dairy cows. In non-ruminants, transcription factor EB (TFEB) was reported to play a role in the regulation of lipid catabolism, but its role in the control of lipolysis in the bovine is unknown. The present study aimed to determine whether the enhanced TFEB transcriptional activity contributes to lipolysis of adipose tissue in SCK cows, and to explore the possibility of establishing a therapeutic strategy by using TFEB as a target to control lipolysis. Thirty cows with similar lactation number (median = 3, range = 2–4) and days in milk (median = 6 d, range = 3–9) were selected into a healthy control (n = 15) and SCK (n = 15) group, and used for subcutaneous adipose tissue biopsies and blood sampling. Adipocytes from healthy Holstein calves were used as a model for in vitro studies involving treatment with 10 μM isoproterenol (ISO) for 0, 1, 2 and 3 h, 250 nM of the TFEB activator Torin1 for 3 h, or used for transfection with TFEB small interfering RNA for 48 h followed by treatment with 10 μM ISO for 3 h. Compared with healthy cows, adipose tissue in SCK cows showed increased lipolysis accompanied by enhanced TFEB transcriptional activity. In vitro, ISO and Torin1 treatment increased lipolysis and enhanced TFEB transcriptional activity in calf adipocytes. However, knockdown of TFEB attenuated ISO-induced lipolysis in adipocytes. Overall, these findings indicated that enhanced transcriptional activity of TFEB may contribute to lipolysis of adipose tissue in dairy cows with SCK. The regulation of TFEB activity may be an effective therapeutic strategy for controlling overt lipolysis in ketotic cows.

Transition dairy cows are often in a state of negative energy balance because of decreased dry matter intake and increased energy requirements, initiating lipid mobilization and leading to high serum β-hydroxybutyrate (BHBA) and non-esterified fatty acid (NEFAs) levels, which can induce ketosis and fatty liver in dairy cows. Inflammation and insulin resistance are also common diseases in the perinatal period of dairy cows. What is the relationship between negative energy balance, insulin resistance and inflammation in dairy cows? To study the role of non-esterified fatty acids in the nuclear factor kappa beta (NF-κB) inflammatory and insulin signaling pathways through Toll-like receptor 4 (TLR4), we cultured primary calf hepatocytes and added different concentrations of NEFAs to assess the mRNA and protein levels of inflammatory and insulin signaling pathways. Our experiments indicated that NEFAs could activate the NF-κB inflammatory signaling pathway and influence insulin resistance through TLR4. However, an inhibitor of TLR4 alleviated the inhibitory effects of NEFAs on the insulin pathway. In conclusion, all of these results indicate that high-dose NEFAs (2.4 mM) can activate the TLR4/NF-κB inflammatory signaling pathway and reduce the sensitivity of the insulin pathway through the TLR4/PI3K/AKT metabolic axis.

β2 integrins are critical for neutrophil firm adhesion, trans-endothelial migration, and the recruitment to the inflamed tissue. Autophagy is implicated in cell migration and tumor metastasis through facilitating the turnover of β1 integrins; however, whether autophagy is able to control neutrophil migration by promoting the degradation of β2 integrins is unexplored. Here, we show that high blood levels of palmitic acid (PA) strongly triggered neutrophil autophagy activation, leading to adhesion deficiency in dairy cows with fatty liver. The three neutrophil granule subtypes, namely, azurophil granules (AGs), specific granules (SGs), and gelatinase granules (GGs), were engulfed by the autophagosomes for degradation, resulting in an increased vacuolation in fatty liver dairy cow neutrophils. Importantly, the adhesion-associated molecules CD11b and CD18 distributed on AGs, SGs, and GGs were degraded with the three granule subtypes by autophagy. Moreover, FGA, Hsc70, and TRIM21 mediated the degradation of cytosolic oxidized–ubiquitinated CD11b and CD18. Collectively, our results demonstrate that high blood PA triggers neutrophil autophagy-dependent vacuolation and granule-dependent adhesion deficiency, decreasing neutrophil mobility, and impairing the innate immune system of dairy cow with fatty liver. This theory extends the category of autophagy in maintaining granule homeostasis and provides a novel strategy to improve the immune of dairy cows with metabolic disease.

Abstract Background Dairy cows with metabolic disorder in peripartal period display high inflammatory levels. Adipose tissue is a major endocrine organ, which is closely related to systemic inflammation. Retinoic acid (RA), an active metabolite of vitamin A, has shown potential therapeutic immunomodulatory properties. The objective of the study was to examine the effect of all-trans-RA (ATRA), the biologically most active metabolite of vitamin A, on lipopolysaccharide (LPS)-induced bovine adipocytes inflammatory responses and elucidate the underlying mechanisms. Primary cultured bovine adipocytes were treated with ATRA in the presence or absence of LPS. The treated cells were examined for the inflammatory responses and the activity of transforming growth factor beta 1 (TGFβ1) /Smad3 signaling pathway. Results LPS treatment significantly decreased the expression levels of TGFβ1/Smad3 components and increased the content of pro-inflammatory cytokines. Treatment with ATRA could over-activate TGFβ1/Smad3 signaling pathway in bovine adipocytes and reversed the over-production of pro-inflammatory cytokines and inhibition of anti-inflammatory cytokines induced by LPS. Importantly, inhibition of TGFβ1/Smad3 signaling diminished the effects of ATRA on suppressing the proinflammatory responses induced by LPS. Furthermore, activation of TGFβ1/Smad3 signaling further extended the effects of ATRA on suppressing the proinflammatory responses on LPS stimulation. Conclusion In conclusion, ATRA stimulates TGFβ1/Smad3 signaling pathway and further suppresses bovine adipocytes inflammatory responses induced by LPS.

Rumen fluid of cows with subacute ruminal acidosis (SARA) has high concentrations of short chain fatty acids (SCFA). However, the mechanism of SCFA accumulation is unknown. The solute-linked carrier 5a8 (SLC5A8) plays a key role in the transportation and absorption of SCFA in the intestinal epithelium. The objective of the current study was to investigate (1) SLC5A8 gene and protein expression in various parts of the bovine gastrointestinal tract, (2) the effect of SCFA on SLC5A8 expression in rumen epithelial cells, and (3) SLC5A8 gene and protein expression in SARA and healthy cows. A total of 10 dairy cows, 84 ± 26 days in milk and in their second to fourth parity were allocated to control (n = 5) and SARA groups (n = 5). Three cows from the control group and three calves (1-day-old, female, 45–50 kg, healthy, fasting) were chosen to collect a total of 10 sections of digestive tract, from rumen to rectum, and then bovine ruminal epithelial cells were isolated from the three calves. Gene and protein expression of SLC5A8 was detected in all tested regions of the gastrointestinal tract in calves and adult cows by Western blot and quantitative real-time PCR and were both highest in the rumen. Gene and protein expression of SLC5A8 was more than 50% lower in the rumen epithelium of SARA vs. control cows and was partly restored after therapy of SARA cows. Compared with SCFA concentrations typical for control cows (60 mM acetate, 30 mM propionate, and 20 mM butyrate), gene and protein expression of SLC5A8 in rumen epithelium was lower at elevated SCFA concentrations typical for SARA cows (90 mM acetate, 40 mM propionate, and 30 mM butyrate), specifically for elevated concentrations of propionate or butyrate in contrast to elevated concentrations of acetate increased gene and protein expression of SLC5A8 in rumen epithelium. In conclusion, the elevated concentrations of propionate and butyrate inhibit ruminal absorption of SCFA via downregulation of SLC5A8 in SARA cows; the expression of SLC5A8 plays an important role in the etiology of SARA.

Elevated plasma non-esterified fatty acid (NEFA) levels and hepatocytes damage are characteristics of ketosis in dairy cows. Oxidative stress is associated with the pathogenesis of NEFA-induced liver damage. However, the exact mechanism by which oxidative stress mediates NEFA-induced hepatocytes apoptosis and liver injury remains poorly understood. The results of the present study demonstrated that NEFA contribute to reactive oxygen species (ROS) generation, resulting in an imbalance between oxidative and antioxidant species, transcriptional activation of p53, transcriptional inhibition of nuclear factor E2-related factor 2 (Nrf2), loss of mitochondria membrane potential (MMP) and release of apoptosis-inducing factor (AIF) and cytochrome c (cyt c) into the cytosol, leading to hepatocytes apoptosis. Besides, NEFA triggered apoptosis in dairy cow hepatocytes via the regulation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), Bcl-2-associated X protein (Bax), B-cell lymphoma gene 2 (Bcl-2), caspase 9 and poly (ADP-ribose) polymerase (PARP). Pretreatment with the inhibitor SP600125 or PD98059 or the antioxidant N-acetylcysteine (NAC) revealed that NEFA-ROS-JNK/ERK-mediated mitochondrial signaling pathway plays a crucial role in NEFA-induced hepatocytes apoptosis. Moreover, the results suggested that the transcription factors p53 and Nrf2 function downstream of this NEFA-ROS-JNK/ERK pathway and are involved in NEFA-induced hepatocytes apoptosis. In conclusion, these findings indicate that the NEFA-ROS-JNK/ERK-mediated mitochondrial pathway plays an important role in NEFA-induced dairy cow hepatocytes apoptosis and strongly suggests that the inhibitors SP600125 and PD98059 and the antioxidant NAC may be developed as therapeutics to prevent hyperlipidemia-induced apoptotic damage in ketotic dairy cows.

Abstract Background During peripartum period, dairy cows are highly susceptible to energy metabolism disorders such as fatty liver and ketosis. Angiopoietin-like protein 4 (ANGPTL4) and fibroblast growth factor 21 (FGF21), known as hepatokines, play important roles in lipid metabolism. The purposes of our study were to evaluate variations of serum ANGPTL4 and FGF21 concentrations in periparturient dairy cows and changes in these serum analyte concentrations of energy-related metabolic disorders in early lactation dairy cows. This study was divided into two experiments. Experiment I: Blood parameters were measured in healthy periparturient Holstein cows from 4 wk antepartum to 4 wk postpartum (n = 219). In this experiment, weekly blood samples were obtained from 4 wk before the expected calving date through 4 wk after calving. Experiment II: Blood parameters were measured in healthy cows (n = 30) and cows with clinical ketosis (n = 29) and fatty liver (n = 25) within the first 4 wk of lactation. In the present study, all blood samples were collected from the coccygeal vein in the early morning before feeding. Results Serum ANGPTL4 and FGF21 concentrations peaked at parturition, and declined rapidly over the following 2 wk Serum ANGPTL4 and FGF21 concentrations were positively correlated with serum non-esterified fatty acids (NEFA) concentration (r = 0.856, P = 003; r = 0.848, P = 0.004, respectively). Cows with clinical ketosis and fatty liver had significantly higher serum ANGPTL4 and FGF21 concentrations than healthy cows (P