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Monocrotaline (MCT), a major pyrrolizidine alkaloid, is well-known for its high liver toxicity. Dysregulation of autophagy induced apoptosis can lead to various liver diseases, including those induced by chemical compounds. Therefore, we aim to explore whether autophagy might serve as a potential strategy for addressing liver apoptosis caused by MCT. In primary rat hepatocytes (PRHs), MCT significantly increased the number of autophagosomes and the expression levels of LC3II, Becline-1, and Atg5, while it decreased the expression of p62 in a concentration-dependent manner at doses of 100, 200, 300, and 400 μM. Western blot assays revealed MCT inhibited the phosphorylation levels of the PI3K/AKT/mTOR pathway. To elucidate the role of autophagy in mediating MCT-induced apoptosis, we further pretreated PRHs with the autophagy agonist Rapamycin and the inhibitors Bafilomycin A1 and Chloroquine, respectively, and assessed the apoptosis of PRHs induced by MCT. The results displayed that Rapamycin increased the apoptosis rate and the expression of cleaved caspase-3, whereas Bafilomycin A1 and Chloroquine reduced the apoptosis and the expression of cleaved caspase-3 in PRHs. This study confirms that autophagy enhances PRHs apoptosis induced by MCT. In summary, this study demonstrates that MCT-induced autophagy via inhibition of the PI3K/AKT/mTOR pathway can lead to apoptosis in PRHs.

Abstract Background Strontium (Sr) has similar physicochemical properties as calcium (Ca) and is often used to evaluate the absorption of this mineral. Because the major route of Ca absorption in the bovine occurs in the rumen, it is essential to understand whether Sr impacts the ruminal epithelial cells and to what extent. Results In the present study, RNA sequencing and assembled transcriptome assembly were used to identify transcription factors (TFs), screening and bioinformatics analysis in bovine ruminal epithelial cells treated with Sr. A total of 1405 TFs were identified and classified into 64 families based on an alignment of conserved domains. A total of 174 differently expressed TFs (DE-TFs) were increased and 52 DE-TFs were decreased; the biological process-epithelial cell differentiation was inhibited according to the GSEA-GO analysis of TFs; The GO analysis of DE-TFs was enriched in the DNA binding. Protein-protein interaction network (PPI) found 12 hubs, including SMAD4, SMAD2, SMAD3, SP1, GATA2, NR3C1, PPARG, FOXO1, MEF2A, NCOA2, LEF1, and ETS1, which verified genes expression levels by real-time PCR. Conclusions In this study, SMAD2, PPARG, LEF1, ETS1, GATA2, MEF2A, and NCOA2 are potential candidates that could be targeted by Sr to mediate cell proliferation and differentiation, as well as lipid metabolism. Hence, these results enhance the comprehension of Sr in the regulation of transcription factors and provide new insight into the study of Sr biological function in ruminant animals.

Locoweed is a poisonous plant widely present in grasslands around the world. Swainsonine (SW), an indole alkaloid that, is the main toxic component of the locoweed. To understand the mechanism of SW-induced toxicity and to delineate the metabolic profile of locoweed poisoning we performed the LC–MS/MS untargeted metabolomic study to analyze metabolites in SW-treated renal tubular epithelial cells (0.8 mg/mL, 12 h) and in order to identify the SW-induced metabolomic changes. The analysis identified 2,563 metabolites in positive ion mode and 1,990 metabolites in negative ion mode. Our results showed that the metabolites were mainly benzenoids, lipids and lipid-like molecules, nucleosides, nucleotides, and analogs, organic acids, and derivatives. The differential metabolites were primarily enriched in pathways involving bile secretion, primary bile acid biosynthesis, riboflavin metabolism, ferroptosis, drug metabolism-cytochrome P450, and primidine metabolism. We have screened out substances such as swainsonine, 3alpha,7alpha-Dihydroxy-5beta-cholestanate, 2-Hydroxyiminostilbene, and glycochenodeoxycholate, which may have the potential to serve as biomarkers for swainsonine poisoning. This study provides insights into the types of metabolomic alteration in renal tubular epithelial cells induced by swainsonine.

Abstract Dairy goats experience metabolic stress during the peripartal period, and their ability to navigate this stage of lactation is related to the occurrence and development of metabolic diseases. Unlike dairy cows, there is a lack of comprehensive analysis of changes in the plasma profiles of peripartal dairy goats, particularly using high-throughput techniques. A subset of 9 clinically-healthy dairy goats were used from a cohort of 96 primiparous Guanzhong dairy goats (BCS, 2.75 ± 0.15). Blood samples were collected at seven time points around parturition (d 21, 14, 7 before parturition, the day of kidding, and d 7, 14, 21 postpartum), were analyzed using untargeted metabolomics and targeted lipidomics. The orthogonal partial least squares discriminant analysis model revealed a total of 31 differential metabolites including p-cresol sulfate, pyruvic acid, cholic acid, and oxoglutaric acid. The pathway enrichment analysis identified phenylalanine metabolism, aminoacyl-tRNA biosynthesis, and citrate cycle as the top three significantly-altered pathways. The Limma package identified a total of 123 differentially expressed lipids. Phosphatidylserine (PS), free fatty acids (FFA), and acylcarnitines (ACs) were significantly increased on the day of kidding, while diacylglycerols (DAG) and triacylglycerols (TAG) decreased. Ceramides (Cer) and lyso-phosphatidylinositols (LPI) were significantly increased during postpartum period, while PS, FFA, and ACs decreased postpartum and gradually returned to antepartum levels. Individual species of FFA and phosphatidylcholines (PC) were segregated based on the differences in the saturation and length of the carbon chain. Overall, this work generated the largest repository of the plasma lipidome and metabolome in dairy goats across the peripartal period, which contributed to our understanding of the multifaceted adaptations of transition dairy goats.

Abstract Background Artemisia is important medicinal plants in China and are widely used in medicine, agriculture, and food. Pharmacologically active components of the plants remain to be investigated. Methods This study sought to identify and compare the chemical constituents of three species of Artemisia in Tibet using a widely-targeted metabolomics approach and their antibacterial and antioxidant capacities were determined. Result A total of 1109 metabolites within 10 categories were detected from the three species of Artemisia, including lipids, amino acids, nucleotides, flavonoids, terpenes, coumarins, organic acids, and phenolic acids. 732 different metabolites have been identified between Artemisia sieversiana and Artemisia annua, 751 different metabolites were identified between Artemisia wellbyi and A. sieversiana, and 768 differential metabolites were differentially detected from A. wellbyi and A. annua. Differentially identified compounds included flavonoids, phenolic acids, artemisinins and coumarin. A. annua contained the highest relative content of artemisinin among three Artemisia. The antimicrobial experiments showed that the three Artemisia species had strong antibiotic activities against Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Proteus mirabilis and Pseudomonas aeruginosa. The biochemical analysis showed that the three species of Artemisia have strong antioxidant capacity. Conclusions This is the first reported attempt to comparatively determine the types of the metabolites of the three widely distributed Artemisia species in Tibet. The information should help medicinal research and facilitate comprehensive development and utilization of Artemisia species in Tibet.

The research on the significant toxic weed Oxytropis glabra, which adversely affects the grazing industry and the ecological integrity of natural grasslands in the arid and semi-arid regions of northern China, aims to delineate its potential distribution amidst changing climate conditions. This analysis involves both current conditions (1970–2000) and future projections (2050s and 2070s) under four climate scenarios using an R-optimized MaxEnt model. The results indicate that the distribution of O. glabra was primarily influenced by the temperature of the coldest quarter (bio11, ranging from −12.04 to −0.07 °C), precipitation of the coldest quarter (bio19, 0 to 15.17 mm), and precipitation of the warmest quarter (bio18, 0 to 269.50 mm). Currently, the weed predominantly occupies parts of Xinjiang, Inner Mongolia, Gansu, Qinghai, Ningxia, and Tibet. Projections indicate that, across four future climate scenarios, the area of suitable habitats for O. glabra is expected to expand and shift toward higher latitudes and elevations. The research provides valuable information and a theoretical foundation for the management of O. glabra, alongside advancing grassland ecological research and grazing practices.

Influenza A virus (IAV) nonstructural protein 1 (NS1) is a multifunctional virulence factor that interacts with several host factors such as phosphatidylinositol-3-kinase (PI3K). NS1 binds specifically to the p85β regulatory subunit of PI3K and subsequently activates PI3K signaling. Here, Kim et al. show that functionally near-neutral, strain-specific NS1 mutations lead to variations in binding kinetics to p85β exhibit long-range epistatic interactions. Applying NMR they provide evidence that the structural dynamics of the NS1 hydrophobic core have evolved over time and contributed to epistasis.

Abstract Background The Artemisia species are widely distributed around the world, and have found important usage in traditional medicinal practice. This study was designed to investigate the metabolites of Tibetan Artemisia species and understand the metabolic pathways. Methods The metabolites from three Artemisia species in Tibet, were analyzed using LC–MS/MS. The differential metabolites were classified and analyzed by principal component analysis (PCA), partial least squares analysis and hierarchical clustering. KEGG Pathway enrichment analysis was used to identify the key metabolic pathways involved in the differential metabolites of three Artemisia species. Result The metabolites of three Artemisia species were analyzed. Under the positive ion mode in LC–MS/MS, 262 distinct metabolites were differentially detected from Artemisia sieversiana and Artemisia annua, 312 differential metabolites were detected from Artemisia wellbyi and Artemisia sieversiana, 306 differential metabolites were screened from Artemisia wellbyi and Artemisia annua. With the negative ion mode, 106 differential metabolites were identified from Artemisia sieversiana and Artemisia annua, 131 differential metabolites were identified from Artemisia wellbyi and Artemisia sieversiana,133 differential metabolites were differentially detected from Artemisia wellbyi and Artemisia annua. The selected differential metabolites were mainly organic acids and their derivatives, ketones, phenols, alcohols and coumarins. Among these natural compounds, artemisinin, has the highest relative content in Artemisia annua. Conclusions This is the first reported attempt to comparatively determine the types of the metabolites of the three widely distributed Artemisia species in Tibet. The information should help medicinal research and facilitate comprehensive development and utilization of Artemisia species in Tibet.

BackgroundsPrevious studies identified the extent of lymph node dissection for esophagogastric junction (EGJ) carcinoma based on the metastatic incidence. The study aimed to determine the optimal extent and priority of lymphadenectomy based on the therapeutic efficacy from each station.MethodsThe studies on the lymph node metastasis (LNM) and therapeutic efficacy index (EI) for EGJ carcinomas were identified until April 2022. The obligatory stations with the LNM rates over 5% and therapeutic EI exceeding 2% should be routinely resected for D2 dissection, whereas the optional stations with EI between 0.5% and 2% should be resected for D3 dissection in selective cases.ResultsThe survey yielded 16 eligible articles including 6,350 patients with EGJ carcinoma. The metastatic rates exceeded 5% at no. 1, 2, 3, 7, 9, 11p, and 110 stations and were less than 5% in abdominal no. 4sa~6, 8a, 10, 11d, 12a, and 16a2/b1 and mediastinal no. 105~112 stations. Consequently, obligatory stations with EI over 2% were largely determined by the epicenter location and located at the upper perigastric, lower mediastinal, and suprapancreatic zones, corresponding to those with rates of LNM over 5%. Consistent with the LNM rates less than 5%, the optional stations with EI between 0.5% and 2% were largely dependent on the degree of tumor extension toward the lower perigastric, splenic hilar (grecurvature), para-aortic (less curvature of the cardia), and middle or upper mediastinal zones.ConclusionsThe obligatory stations can be resected as an “envelope-like” wrap by transhiatal proximal gastrectomy with lower esophagectomy, whereas the optional stations for dissection are indicated by the tumor extension. The extended gastrectomy is required for the lower perigastric in the stomach-predominant tumor with gastric involvement exceeding 5.0 cm, para-aortic dissection in the less curvature-predominant tumor and splenic hilar dissection in the grecurvature-predominant tumor whereas transthoracic subtotal esophagectomy is required for complete mediastinal dissection and adequate negative margin in the esophagus-predominant tumor with esophageal invasion exceeding 3.0 cm.

Astragalus variabilis is a locoweed of northwest China that can seriously impede livestock development. However, it also plays various ecological roles, such as wind protection and sand fixation. Here, we used an optimized MaxEnt model to predict the distribution of suitable habitat of A. variabilis under current (1970–2000) conditions and future (2021–2080) climate change scenarios based on recent occurrence records. The most important environmental variables (suitability ranges in parentheses) affecting the distribution of A. variabilis were average maximum temperature of February (–2.12–5.34°C), followed by total precipitation of June (2.06–37.33 mm), and topsoil organic carbon (0.36–0.69%). The habitat suitability of A. variabilis was significantly correlated with the frequency of livestock poisoning (p < 0.05). Under current climate conditions, the suitable environment of A. variabilis was distributed in central and western Inner Mongolia, Ningxia, central and northwestern Gansu, central and northwestern Qinghai, and the four basins around the Tianshan Mountains in Xinjiang. Under future climate conditions, the suitable habitat of A. variabilis shifted to higher latitudes and altitudes. No previous studies have used niche models to predict the suitable environment of this species nor analyzed the relationship between the habitat suitability of poisonous plants and the frequency of animal poisoning. Our findings provide new insights that will aid the prevention of livestock animal poisoning and the control of poisonous plants, promote the development of the livestock husbandry industry, and provide basic information that will facilitate the maintenance of the ecological balance of grassland ecosystems.

Abstract Background A better comprehension of the redox status during the periparturient period may facilitate the development of management and nutritional solutions to prevent subclinical hyperketonemia (SCHK) and subclinical hypocalcemia (SCHC) in dairy goats. We aimed to evaluate the variation in the redox status of dairy goats with SCHK and SCHC during their periparturient periods. Guanzhong dairy goats (n = 30) were assigned to SCHK (n = 10), SCHC (n = 10), and healthy (HEAL, n = 10) groups based on their blood β-hydroxybutyrate (BHBA) and calcium (Ca) concentrations. Blood were withdrawn from goats every week from 3 weeks before the expected parturition date to 3 weeks post-kidding. On the same day, the body condition scores (BCS) were evaluated, and the milk yield was recorded for each goat. The metabolic profile parameters and the indicators of oxidative status were determined by using the standard biochemical techniques. Results In comparison with the HEAL goats, SCHK and SCHC goats presented with a more dramatic decline of BCS post-kidding and a significant decrease in the milk yield at 2- and 3-weeks postpartum, ignoring the obvious increase at 1-week postpartum. The levels of non-esterified fatty acids (NEFA) peaked at parturition, exhibiting significantly higher levels from 1-week prepartum to the parturition day in the SCHK and SCHC groups. The malondialdehyde (MDA) concentration was increased in the SCHK goats from 1-week antepartum until 3-weeks postpartum, with its concentration being significantly higher in the SCHC goats at parturition. The hydrogen peroxide (H2O2) concentration was significantly lower in the SCHK and SCHC goats from 2-weeks antepartum to 1-week post-kidding. The total antioxidant capacity (T-AOC) and the superoxide dismutase (SOD) level were decreased at 1-week antepartum in the SCHK and SCHC goats, respectively. The glutathione peroxidase (GSH-Px) level was increased in the SCHK and SCHC goats during the early lactation period. Conclusions The SCHK and SCHC goats exerted more efforts to maintain their redox homeostasis and to ensure the production performance than the HEAL goats during their periparturient period, probably owing to more intense fat mobilization and lipid peroxidation in the former.

Endotoxins are toxic substances that widely exist in the environment and can enter the intestine with food and other substances. Intestinal epithelial cells are protected by a mucus layer that contains MUC2 as its main structural component. However, a detailed understanding of the mechanisms involved in the function of the mucus barrier in endotoxin penetration is lacking. Here, we established the most suitable proportion of Caco-2/HT-29 co-culture cells as a powerful tool to evaluate the intestinal mucus layer. Our findings significantly advance current knowledge as focal adhesion and ECM-receptor interaction were identified as the two most significantly implicated pathways in MUC2 small interfering RNA (siRNA)-transfected Caco-2/HT-29 co-culture cells after 24 h of LPS stimulation. When the mucus layer was not intact, LPS was found to damage the tight junctions of Caco-2/HT29 co-cultured cells. Furthermore, LPS was demonstrated to inhibit the integrin-mediated focal adhesion structure and damage the matrix network structure of the extracellular and actin microfilament skeletons. Ultimately, LPS inhibited the interactive communication between the extracellular matrix and the cytoskeleton for 24 h in the siMUC2 group compared with the LPS(+) and LPS(-) groups. Overall, we recognized the potential of MUC2 as a tool for barrier function in several intestinal bacterial diseases.

Baicalin is one of the most abundant flavonoids found in the dried roots of Scutellaria baicalensis Georgi (SBG) belonging to the genus Scutellaria. While baicalin is demonstrated to have anti-inflammatory, antiviral, antitumor, antibacterial, anticonvulsant, antioxidant, hepatoprotective, and neuroprotective effects, its low hydrophilicity and lipophilicity limit the bioavailability and pharmacological functions. Therefore, an in-depth study of baicalin’s bioavailability and pharmacokinetics contributes to laying the theoretical foundation for applied research in disease treatment. In this view, the physicochemical properties and anti-inflammatory activity of baicalin are summarized in terms of bioavailability, drug interaction, and inflammatory conditions.

Strontium (Sr) belongs to the same group in the periodic table as calcium (Ca). Sr level can serve as an index of rumen Ca absorption capacity; however, the effects of Sr on Ca2+ metabolism are unclear. This study aims to investigate the effect of Sr on Ca2+ metabolism in bovine rumen epithelial cells. The bovine rumen epithelial cells were isolated from the rumen of newborn Holstein male calves (n = 3, 1 day old, 38.0 ± 2.8 kg, fasting). The half maximal inhibitory concentration (IC50) of Sr-treated bovine rumen epithelial cells and cell cycle were used to establish the Sr treatment model. Transcriptomics, proteomics, and network pharmacology were conducted to investigate the core targets of Sr-mediated regulation of Ca2+ metabolism in bovine rumen epithelial cells. The data of transcriptomics and proteomics were analyzed using bioinformatic analysis (Gene Ontology and Kyoto Encyclopedia of genes/protein). Quantitative data were analyzed using one-way ANOVA in GraphPad Prism 8.4.3 and the Shapiro–Wilk test was used for the normality test. Results presented that the IC50 of Sr treatment bovine rumen epithelial cells for 24 h was 43.21 mmol/L, and Sr increased intracellular Ca2+ levels. Multi-omics results demonstrated the differential expression of 770 mRNAs and 2436 proteins after Sr treatment; network pharmacology and reverse transcriptase polymerase chain reaction (RT-PCR) revealed Adenosylhomocysteine hydrolase-like protein 2 (AHCYL2), Semaphoring 3A (SEMA3A), Parathyroid hormone-related protein (PTHLH), Transforming growth factor β2 (TGF-β2), and Cholesterol side-chain cleavage enzyme (CYP11A1) as potential targets for Sr-mediated Ca2+ metabolism regulation. Together these results will improve the current comprehension of the regulatory effect of Sr on Ca2+ metabolism and pave a theoretical basis for Sr application in bovine hypocalcemia.

Aconitine is the principal toxic ingredient of Aconitum, which can cause systemic poisoning involving multiple organs and systems after animal ingestion. The purpose of this study was to investigate the effects of aconitine on hematological indices and histological changes in mice. One hundred twenty mice were divided into a control group (normal saline), low-dose group (0.14 μmol/L), middle-dose group (0.28 μmol/L) and high-dose group (0.56 μmol/L), which were continuously lavaged for 30 days. The blood of 10 mice were collected randomly and analyzed by group at the 10th, 20th, and 30th days, and some tissues were collected and stained with hematoxylin-eosin to observe histological changes at the 30th day. Compared with the control group, the organ coefficient (%) of liver, spleen, lungs, and brain of the high-dose group were significantly increased (p < 0.05 or p < 0.01). WBC and Gran initially decreased and then increased in each poisoning group, with significant differences in the high-dose group (p < 0.05 or p < 0.01). RBC, HGB, HCT, and PLT decreased continuously in all groups except the low-dose group at the 20th and 30th days (p < 0.05 or p < 0.01). Moreover, BUN, ALT and AST increased in each poisoning group, in comparison with the control group, with significant differences except for the low-dose group (p < 0.05 or p < 0.01). CRE initially increased and then decreased, the TP and ALB decreased, with significant differences observed in the high-dose and middle-dose groups (p < 0.05). All the mice in the poison-treated groups showed varying degrees of histopathological changes such as degeneration and necrosis of tissues, especially heart and cerebellum. Our data suggest that different doses of aconitine have remarkable effects on hematological and histopathological changes in mice, in a significant time and dose-effect relationship.

Swainsonine (SW), an indolizidine alkaloid extracted from locoweeds, was shown toxic effects in multiple studies, but the underlying action mechanism remains unclear. SW is known to cause autophagy and apoptosis, but there has been no report on paraptosis mediated cell death. Here, we showed that SW induced rat primary renal tubular epithelial cells (RTECs) death accompanied by vacuolation in vitro. The fluorescence with the endoplasmic reticulum (ER)-Tracker Red and transmission electron microscopy (TEM) results indicated that the vacuoles were of ER origin, typical of paraptosis. The level of ER stress markers, such as polyubiquitinated proteins, Bip, CHOP and cytoplasmic concentration of Ca2+ have drastically increased. Interestingly, autophagy inhibitor could not interrupt but enhanced the induction of cytoplasmic vacuolization. Furthermore, MAPK pathways were activated by SW and inhibitors of ERK and JNK pathways could prevent the formation of cytoplasmic vacuolization. In this study, we confirmed that SW induced cell paraptosis through ER stress and MAPK signaling pathway, thus further laying a theoretical foundation for the study of SW toxicity mechanism.

Abstract Background The indolizidine alkaloid-swainsonine is produced by an endophytic fungus Alternaria oxytropis, which was isolated from locoweeds. Swainsonine has many biological activities such as anti-tumorigenic, anti-viral and bacteriostatic. However, the full complement of metabolites produced by Alternaria oxytropis is not known. This study is a chemical analysis of Alternaria oxytropis metabolites, which not only unravels the potential compounds from the fermentation broth but also in which solvent are they extracted, facilitating industrial application. Results Alternaria oxytropis isolated from Oxytropis gansuensis was cultured in Czapek’s medium for 30d to collect the fermentation broth. The fermentation broth is treated with methanol and then evaporated to dryness to obtain a concentrate of the fermentation broth. The concentrate is added with water for the subsequent fractional extraction with petroleum ether, chloroform, ethyl acetate and n-butanol. Different fractions of the extract were eluted by wet packing and dry loading. The obtained eluate was combined by TLC to detect the same fraction, and then characterized by GC-MS and LC-MS. The results of GC-MS showed that 105 different compounds existed in the petroleum ether, chloroform, and ethyl acetate phases of Alternaria oxytropis fermentation broth. Moreover, the results of LC-MS indicated that the fermentation broth of Alternaria oxytropis contained five alkaloids, 2-hydroxy-indolizidine, retronecine, lentiginosine, swainsonine and swainsonine N-oxide. Conclusions In addition to swainsonine and swainsonine N-oxide, 2-hydroxy-indolizidine, retronecine and lentiginosine were identified as the secondary metabolites of Alternaria oxytropis. Other compounds were also detected including 5,6-dihydroergosterol, eburicol, lanosterol, and L-phenylalanyl-L-proline lactam, which have potential applications as drugs.

Abstract Background TRAF3 is known as a central mediator of type I interferon (IFN) induction by various pattern recognition receptors, but the in vivo function of TRAF3 in host defense against viral infection is poorly defined due to the lack of a viable mouse model. Results Here we show that mice carrying conditional deletion of TRAF3 in myeloid cells or dendritic cells do not have a significant defect in host defense against vesicular stomatitis virus (VSV) infection. However, whole-body inducible deletion of TRAF3 renders mice more sensitive to VSV infection. Consistently, TRAF3 was essential for type I IFN induction in mouse embryonic fibroblasts (MEFs) but not in macrophages. In dendritic cells, TRAF3 was required for type I IFN induction by TLR ligands but not by viruses. We further show that the IFN-regulating function is not unique to TRAF3, since TRAF2 is an essential mediator of type I IFN induction in several cell types, including macrophages, DCs, and MEFs. Conclusions These findings suggest that both TRAF2 and TRAF3 play a crucial role in type I IFN induction, but their functions are cell type- and stimulus-specific.

Monocrotaline (MCT), a pyrrolizidine alkaloid, is the major toxin in Crotalaria, which causes cell apoptosis in humans and animals. It has been reported that the liver is a vulnerable target of MCT. However, the exact molecular mechanism of the interaction between endoplasmic reticulum (ER) stress and liver injury induced by MCT is still unclear. In this study, the cytotoxicity of MCT on primary rat hepatocytes was analyzed by a CCK-8 assay and Annexin V-FITC/PI assay. Protein expression was detected by western blotting and immunofluorescence staining. As a result, MCT significantly decreased the cell viability and mediated the apoptosis of primary rat hepatocytes. Meanwhile, MCT could also induce ER stress in hepatocytes, indicated by the expression of ER stress-related proteins, including GRP78, p-IRE1α, ATF6, p-eIF2α, ATF4, and CHOP. Pretreatment with 4-PBA, an inhibitor of ER stress, or knockdown of CHOP by siRNA could partly enhance cell viability and relieve the apoptosis. Our findings indicate that ER stress is involved in the hepatotoxicity induced by MCT, and CHOP plays an important role in this process.

During the periparturient period, dairy cows suffer drastic metabolic stress because of plasma increased non-esterified fatty acids (NEFAs) that stem from a negative energy balance. Fibroblast growth factor 21 (FGF21) is a hepatokine that activates the AMP-activated protein kinase (AMPK) signaling pathway to maintain intracellular energy balance and tissue integrity via the promotion of catabolism and the inhibition of anabolic regulation. FGF21 treatment caused a 50% reduction in triglyceride (TG) content in liver in dairy cows. However, it is not clear whether FGF21 regulates lipid metabolism in bovine liver. The purpose of this study was to evaluate the influence of FGF21 on lipid metabolism via AMPK signaling in bovine hepatocytes. The hepatocytes isolated from calves were treated with different concentrations of FGF21 or co-treated with AMPK inhibitor (BML-275). Herein, the study showed that FGF21 significantly reduced TG content in a dose–response manner and promoted very-low-density lipoprotein (VLDL) secretion via an up-regulation of the proteins (ApoB 100, ApoE and MTTP) involved in VLDL secretion. Otherwise, the genes associated with lipid transport (LDLR and CD36) and lipid oxidation (PPARGC1A, ACOX1 and CPT1A), were up-regulated following FGF21 treatment. Moreover, FGF21 treatment inhibited lipogenesis via SREBF1, ACACA, FASN and ACLY inhibition. After being co-treated with the AMPK inhibitor, FGF21-induced changes were reversed in some genes. In conclusion, these results indicate that FGF21 adaptively regulates energy metabolism for a negative impact on lipogenesis, strengthens lipid oxidation, and inhibited lipid transportation via AMPK signaling in bovine hepatocytes. The present data suggest the possibility that FGF21 has potential value in alleviating perinatal metabolic diseases in dairy cows, and specific research in vivo should be studied in more detail.

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

The present study was conducted regarding four acute-phase proteins (APPs) including C-reactive protein (CRP), ceruloplasmin (CP), serum amyloid A (SAA), and haptoglobin (HP) in dairy goats during the periparturient period. The aim of this study was to detect the changes in APPs in plasma during the periparturient period of healthy dairy goats. Guanzhong dairy goats with no other symptoms (n = 15) were selected on the basis of their blood calcium (Ca) and β-hydroxybutyrate (BHBA) concentration. The plasma was collected once a week for ±3 weeks delivery. The concentrations of the four APPs mentioned above were determined using goat-specific ELISA kits. The results showed the CRP level in plasma decreased from 3 weeks to 1 week antepartum and increased later until 1 week postpartum and then decreased to a similar level with antepartum between 1 and 3 weeks postpartum. The content of CP showed a decline in 3 weeks before parturition and an upward trend between 1 week antepartum and 3 weeks postpartum. The SAA concentration decreased from 3 weeks antepartum to 2 weeks postpartum and rebounded later. The level of HP decreased during 3 weeks before parturition and increased until 1 week postpartum, then reached a stable value. Clear variation range and rules of APPs contribute to perinatal health monitoring of dairy goats.

During the perinatal period, the abnormally high plasma non-esterified fatty acids (NEFA) concentration caused by the negative energy balance (NEB) can impose a significant metabolic stress on the liver of dairy cows. Endoplasmic reticulum (ER) stress is an important adaptive response that can serve to maintain cell homeostasis in the event of stress. The protein kinase R-like endoplasmic reticulum kinase (PERK) pathway is the most rapidly activated cascade when ER stress occurs in cells and has an important impact on the regulation of hepatic lipid metabolism and autophagy modulation. However, it is unknown whether NEFA can affect autophagy through modulating the PERK pathway, under NEB conditions. In this study, we provide evidence that NEFA treatment markedly increased lipid accumulation, the phosphorylation level of PERK and eukaryotic initiation factor 2α (eIF2α), and the expression of glucose-regulated protein 78 (Grp78), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). More importantly, NEFA treatment can cause a substantial increase in the protein levels of autophagy-related gene 7 (ATG7), Beclin-1 (BECN1), sequestosome-1 (p62), and microtubule-associated protein 1 light chain 3 (LC3)-II, and in the number of autophagosomes in primary bovine hepatocytes. The addition of GSK2656157 (PERK phosphorylation inhibitor) can significantly inhibit the effect of NEFA on autophagy and can further increase lipid accumulation. Overall, our results indicate that NEFA could promote autophagy via the PERK pathway in bovine hepatocytes. These findings provide novel evidence about the potential role of the PERK signaling pathway in maintaining bovine hepatocyte homeostasis.

Background: Dairy goats are highly susceptible to subclinical hyperketonemia (SCHK) during the transition period. This study aimed to compare the variation in metabolic parameters and surrogate indexes of insulin resistance (sIR) between goats with SCHK and clinically healthy (HEAL) goats during the transition period. Methods: Twenty Guanzhong dairy goats were assorted to HEAL (n = 10) and SCHK (n = 10) groups according to the blood β-hydroxybutyrate (BHBA) concentrations. The blood samples were taken from the jugular vein of each goat at −3, −2, −1, 0 (partum), +1, +2, and +3 weeks relative to kidding to analyses GLU and INS. The sIR was calculated from blood metabolic parameters. Results: Compared with the HEAL goats, the insulin concentrations were significantly higher in SCHK goats during the first three weeks postpartum. The QUICKI, revised QUICKI (RQUICKI), and RQUICKIBHBA were significantly lower in goats with SCHK at 1 week postpartum, while the homeostasis model assessment-IR (HOMA-IR) was significantly higher. Conclusion: Goats with SCHK made more efforts through elevated insulin levels at early lactation than HEAL goats, thereby maintaining the normal glucose concentrations.

Zika virus infection can cause human birth defects and Guillain-Barré syndrome. Here the authors present the structures of the full-length nonstructural protein 5 and its RNA-dependent RNA polymerase domain of Zika virus, which are targets for inhibitors of virus replication.

Background: The optimal extent of gastrectomy and lymphadenectomy for esophagogastric junction (EGJ) cancer is controversial. Our study aimed to compare the long-term survival of transhiatal proximal gastrectomy with extended periproximal lymphadenectomy (THPG with EPL) and transhiatal total gastrectomy with complete perigastric lymphadenectomy (THTG with CPL) for patients with the stomach-predominant EGJ cancer.Methods: Between January 2004, and August 2015, 306 patients with Siewert II tumors were divided into the THTG group (n = 148) and the THPG group (n = 158). Their long-term survival was compared according to Nishi's classification. The Kaplan–Meier method and Cox proportional hazards models were used for survival analysis.Results: There were no significant differences between the two groups in the distribution of age, gender, tumor size or Nishi's type (P > 0.05). However, a significant difference was observed in terms of pathological tumor stage (P < 0.05). The 5-year overall survival rates were 62.0% in the THPG group and 59.5% in the THTG group. The hazard ratio for death was 0.455 (95% CI, 0.337 to 0.613; log-rank P < 0.001). Type GE/E = G showed a worse prognosis compared with Type G (P < 0.05). Subgroup analysis stratified by Nishi's classification, Stage IA-IIB and IIIA, and tumor size ≤ 30 mm indicated significant survival advantages for the THPG group (P < 0.05). However, this analysis failed to show a survival benefit in Stage IIIB (P > 0.05).Conclusions: Nishi's classification is an effective method to clarify the subdivision of Siewert II tumors with a diameter ≤ 40 mm above or below the EGJ. THPG with EPL is an optimal procedure for the patients with the stomach-predominant EGJ tumors ≤30 mm in diameter and in Stage IA-IIIA. For more advanced and larger EGJ tumors, further studies are required to confirm the necessity of THTG with CPL.

The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]). uPAR is organized as a dynamic modular protein structure composed of three homologous Ly6/uPAR domains (LU).This internally flexible protein structure of uPAR enables an allosteric regulation of the interactions with its two principal ligands: the serine protease urokinase-type plasminogen activator (uPA) and the provisional matrix protein vitronectin (Vn) (Mertens et al., 2012; Gårdsvoll et al., 2011; Madsen et al., 2007 [2–4]). The data presented here relates to the non-covalent trapping of one of these biologically relevant uPAR-conformations by a novel class of monoclonal antibodies (Zhao et al., 2015 [5]) and to the general mapping of the topographic epitope landscape on uPAR. The methods required to achieve these data include: (1) recombinant expression and purification of a uPAR-hybrid protein trapped in the desired conformation [patent; WO 2013/020898 A12013]; (2) developing monoclonal antibodies with unique specificities using this protein as antigen; (3) mapping the functional epitope on uPAR for these mAbs by surface plasmon resonance with a complete library of purified single-site uPAR mutants (Zhao et al., 2015; Gårdsvoll et al., 2006 [5,6]); and finally (4) solving the three-dimensional structures for one of these mAbs by X-ray crystallography alone and in complex with uPAR [deposited in the PDB database as 4QTH and 4QTI, respectively].

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.

Oral squamous cell carcinoma (OSCC) is a common tumor in the world. Despite the rapid development of medical care, OSCC is also accompanied by high incidence and mortality every year. Therefore, it is still necessary to continuously develop new methods or find new targets to treat OSCC. Previous research showed that scavenger receptor class A member 5 (SCARA5) was one of the potential biomarkers of OSCC, and its expression is significantly low in OSCC. This study aimed to explore the role and related molecular mechanisms of SCARA5 in OSCC. In this study, we found that the SCARA5 expression was lower in CAL-27 and SCC-9 cells than that in human normal oral epithelial keratinocytes. SCARA5 overexpression significantly inhibited cell proliferation and induced apoptosis of CAL-27 and SCC-9 cells. In addition, SCARA5 repressed OSCC cell epithelial-mesenchymal transformation (EMT), evidenced by increased E-cadherin expression and reduced N-cadherin expression. Finally, we found that SCARA5 could suppress STAT3, PI3K, and AKT phosphorylation. Therefore, SCARA5 was related to STAT3 and PI3K/AKT signaling pathways in OSCC. In conclusion, SCARA5 inhibited the proliferation and EMT and induced the apoptosis of OSCC cells through the inhibition of STAT3 and PI3K/AKT signaling pathways, thereby exerting a tumor suppressor effect. [ABSTRACT FROM AUTHOR]

The innate immune system is the first line of defense against bacterial and viral infections. The recognition of pathogen-associated molecular patterns by the RIG-I-like receptors, TLRs, and cGAS leads to the induction of IFN-I by activating the transcription factor IRF-3. Although the mechanism of IRF-3 activation has been extensively studied, the structural basis of IRF-3 activation upon phosphorylation is not fully understood. In this study, we determined the crystal structures of phosphorylated human and mouse IRF-3 bound to CREB-binding protein (CBP), which reveal that phosphorylated IRF-3 forms a dimer via pSer386 (pSer379 in mouse IRF-3) and a downstream pLxIS motif. Size-exclusion chromatography and cell-based studies show that mutations of key residues interacting with pSer386 severely impair IRF-3 activation and IFN-β induction. By contrast, phosphorylation of Ser396 within the pLxIS motif of human IRF-3 only plays a moderate role in IRF-3 activation. The mouse IRF-3/CBP complex structure reveals that the mechanism of mouse IRF-3 activation is similar but distinct from human IRF-3. These structural and functional studies reveal the detailed mechanism of IRF-3 activation upon phosphorylation. [ABSTRACT FROM AUTHOR]

The 1918 influenza A virus (IAV) caused themost severe flu pandemic in recorded human history. Nonstructural protein 1 (NS1) is an important virulence factor of the 1918 IAV. NS1 antagonizes host defense mechanisms through interactions with multiple host factors. One pathway by which NS1 increases virulence is through the activation of phosphoinositide 3-kinase (PI3K) by binding to its p85β subunit. Here we present the mechanism underlying the molecular recognition of the p85β subunit by 1918 NS1. Using X-ray crystallography, we determine the structure of 1918 NS1 complexed with p85β of human PI3K. We find that the 1918 NS1 effector domain (1918 NS1ED) undergoes a conformational change to bind p85β. Using NMR relaxation dispersion and molecular dynamics simulation, we identify that free 1918 NS1ED exists in a dynamic equilibrium between p85β-binding-competent and -incompetent conformations in the submillisecond timescale. Moreover, we discover that NS1ED proteins of 1918 (H1N1) and Udorn (H3N2) strains exhibit drastically different conformational dynamics and binding kinetics to p85β. These results provide evidence of strain-dependent conformational dynamics of NS1. Using kinetic modeling based on the experimental data, we demonstrate that 1918 NS1ED can result in the faster hijacking of p85β compared to Ud NS1ED, although the former has a lower affinity to p85β than the latter. Our results suggest that the difference in binding kinetics may impact the competition with cellular antiviral responses for the activation of PI3K. We anticipate that our findings will increase the understanding of the straindependent behaviors of influenza NS1 proteins. [ABSTRACT FROM AUTHOR]

Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)-like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses. [ABSTRACT FROM AUTHOR]

Background: Livestock that consume locoweed exhibit multiple neurological symptoms, including dispirited behavior, staggered gait, trembling, ataxia, impaired reproductive function and cellular vacuolar degeneration of multiple tissues due to toxicity from plant-derived alkaloids such as swainsonine. Results: Swainsonine was administered to F0 and F1 mice by intraperitoneal injection before, during and after pregnancy at the following doses: 0.525 mg/kg BW(I), 0.2625 mg/kg BW(II), 0.175 mg/kg BW(III) and 0 mg/kg BW(IV). Hemosiderin deposits were observed the lamina propria of endometrium in uterus and the red pulp of spleen. Ovary corpus lutea counts in F0 mice were higher in swainsonine-treated mice compared to control mice. Indirect bilirubin content and reticulocyte numbers were increased in swainsonine-treated F0 and F1 generation mice compared to control group (P < 0.05). Lactate dehydrogenase, alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase content in F0-I and F0-II mice were significantly increased compared with F0-IV group mice (P < 0.05). Red blood cells, hemoglobin and mean corpuscular hemoglobin levels were significantly decreased in F0 and F1 mice compared with the control group (P < 0.05). Conclusions: Swainsonine exerts effects on estrus period and reproductive ability, and offspring of dams dosed with swainsonine were affected in-utero or from nursing. Damage to liver, uterus and spleen, as well as hematological changes, are observable before neurological symptoms present. [ABSTRACT FROM AUTHOR]

Matriptase, a type II trans-membrane serine protease of the S1 trypsin-like family, is expressed on the surface of nearly all normal human epithelium and found in biological fluid-like human milk. Matriptase overexpression has been implicated in tumor progression in certain epithelium-derived cancer cells. Matriptase is tightly regulated by its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). It has been demonstrated that the Kunitz domain I (KD1) but not Kunitz domain II (KD2) of HAI-1 is responsible for the inhibitory activity of HAI-1 against matriptase. To investigate the molecular basis of inhibition of matriptase by HAI-1, we solved several crystal structures of matriptase serine protease domain in complex with the fragments of HAI-1. Based on these structures, we found that the binding of KD1 was different from previously predicted binding mode. The P3 arginine residue occupies the S3 specificity pocket of matriptase, but not the S4 pocket as in the cases of hepatocyte growth factor activator·HAI-1 KD1 and matriptase·sunflower trypsin inhibitor-1 complexes. The long 60-loop of matriptase makes direct contact with HAI-1 but remains flexible even in the complexes, and its apex does not bind with KD1 tightly. The interactions between this unique 60-loop and KD1 may provide an opportunity to increase the specificity and inhibitory activity of KD1 for matriptase. Furthermore, comparison between KD1 and a homology model of HAI-1 KD2 rationalizes the structural basis of why KD1 but not KD2 is responsible for the inhibitory activity of HAI-1 against matriptase. [ABSTRACT FROM AUTHOR]

Hepatocyte growth factor activator inhibitor 1 (HAI-1) is a membrane-bound multidomain protein essential to the integrity of the basement membrane during placental development and is also important in maintaining postnatal homeostasis in many tissues. HAI-1 is a Kunitz-type serine protease inhibitor, and soluble fragments of HAI-1 with variable lengths have been identified in vivo. The full-length extracellular portion of HAI-1 (sHAI-1) shows weaker inhibitory activity toward target proteases than the smaller fragments, suggesting auto-inhibition of HAI-1. However, this possible regulatory mechanism has not yet been evaluated. Here, we solved the crystal structure of sHAI-1 and determined the solution structure by small-angle X-ray scattering. These structural analyses revealed that, despite the presence of long linkers, sHAI-1 exists in a compact conformation in which sHAI-1 active sites in Kunitz domain 1 are sterically blocked by neighboring structural elements. We also found that in the presence of target proteases, sHAI-1 adopts an extended conformation that disables the auto-inhibition effect. Our results also reveal the roles of non-inhibitory domains of this multidomain protein and explain the low activity of the full-length protein. The structural insights gained here improve our understanding of the regulation of HAI-1 inhibitory activities and point to new approaches for better controlling these activities. [ABSTRACT FROM AUTHOR]

Abstract: Locoweeds are perennial herbaceous plants included in Astragalus spp. and Oxytropis spp. that contain the toxic indolizidine alkaloid swainsonine. The livestock that consume locoweed feeding can suffer from a type of toxicity called “locoism.” There are aliphatic nitro compounds, selenium, selenium compounds and alkaloids in locoweed. The toxic component in locoweeds has been identified as swainsonine, an indolizidine alkaloid. Swainsonine inhibits lysosomal α-mannosidase and mannosidase II, resulting in altered oligosaccharide degradation and incomplete glycoprotein processing. As a result, livestock that consume locoweeds exhibit several symptoms, including dispirited behavior, staggering gait, chromatopsia, trembling, ataxia, and cellular vacuolar degeneration of most tissues by pathological observation. Locoism results in significant annual economic losses. Recently, locoweed populations have increased domestically in China and abroad, resulting in an increase in the incidence of poisoning. Therefore, in this paper, we review the current research on locoweed, including on species variation, pathogenesis, damage and poisoning prevention measures. [Copyright &y& Elsevier]