Effect of strontium on transcription factors identified by transcriptome analyses of bovine ruminal epithelial cells.

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.

Strontium Attenuates LPS-Induced Inflammation via TLR4/MyD88/NF-κB Pathway in Bovine Ruminal Epithelial Cells.

Subacute ruminal acidosis (SARA) is a common nutritional metabolic disease in ruminants that causes significant economic losses to dairy farming. Strontium (Sr) is known to be involved in bone metabolism and exhibits potent anti-inflammatory effects. To evaluate the effect of Sr on inflammation in bovine ruminal epithelial cells, a model of LPS-induced inflammation was established in this study, and the cell viability of bovine ruminal epithelial cells was measured using CCK-8. The production of pro-inflammatory cytokines was measured by ELISA and real-time PCR, respectively. The related proteins of the TLR4/MyD88/NF-κB pathway were assayed through Western blotting, and the fluorescence of p-p65 and p-IκB were assayed by immunofluorescence. Molecular docking of Sr and TLR4/MyD88/NF-κB pathway-related proteins was performed using MIB2 ( http://bioinfo.cmu.edu.tw/MIB2/ ). Results showed that after treatment for 24 h, the cell viability was decreased at the high concentration of Sr (≥ 10 mmol/L). Sr significantly decreased the production of TNF-α, IL-1ß, and IL-6, downregulated the related proteins expression of the TLR4/MyD88/NF-κB pathway, and reduced the fluorescence levels of p-p65 and p-IκB. The NF-κB pathway inhibitor PDTC and molecular docking further revealed that Sr reduced LPS-induced pro-inflammatory cytokines production via the TLR4/MyD88/NF-κB pathway. These results suggest that Sr reduces LPS-induced pro-inflammatory cytokines production via the TLR4/MyD88/NF-κB pathway, thereby exerting an anti-inflammatory effect in bovine ruminal epithelial cells, providing a basis for Sr in the treatment of bovine rumen acidosis disease.

Effects of perinatal stress on the metabolites and lipids in plasma of dairy goats.

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.

Effects of Chromium Propionate and Calcium Propionate on Lactation Performance and Rumen Microbiota in Postpartum Heat-Stressed Holstein Dairy Cows.

Chromium propionate (Cr-Pro) and calcium propionate (Ca-Pro) are widely applied in dairy production, especially in the alleviation of heat stress (HS). HS can reduce the abundance of rumen microbiota and the lactation performance of dairy cows. The present work mainly focused on evaluating the effects of Cr-Pro and Ca-Pro on the performance, ruminal bacterial community, and stress of postpartum HS dairy cows as well as identifying the differences in their mechanisms. Fifteen multiparous postpartum Holstein cows with equivalent weights (694 ± 28 kg) and milk yields (41.2 ± 1.21 kg/day) were randomly divided into three groups: control (CON), Cr-Pro (CRPR), and Ca-Pro (CAPR). The control cows received the basal total mixed ration (TMR) diet, while the CRPR group received TMR with 3.13 g/day of Cr-Pro, and the CAPR group received TMR with 200 g/day of Ca-Pro. The rumen microbial 16S rRNA was sequenced using the Illumina NovaSeq platform along with the measurement of ruminal volatile fatty acids (VFAs) and milking performance. Cr-Pro and Ca-Pro improved lactation performance, increased the rumen VFA concentration, and altered the rumen microbiota of the HS dairy cows. Cr-Pro significantly improved the milk yield (p < 0.01). The richness and diversity of the microbial species significantly increased after feeding on Ca-Pro (p < 0.05). Gene function prediction revealed increased metabolic pathways and biological-synthesis-related function in the groups supplemented with Cr-Pro and Ca-Pro. Our results indicate that the application of Cr-Pro or Ca-Pro can provide relief for heat stress in dairy cows through different mechanisms, and a combination of both is recommended for optimal results in production.

Sodium Propionate Relieves LPS-Induced Inflammation by Suppressing the NF-ĸB and MAPK Signaling Pathways in Rumen Epithelial Cells of Holstein Cows.

Subacute ruminal acidosis (SARA) is a prevalent disease in intensive dairy farming, and the rumen environment of diseased cows acidifies, leading to the rupture of gram-negative bacteria to release lipopolysaccharide (LPS). LPS can cause rumentitis and other complications, such as liver abscess, mastitis and laminitis. Propionate, commonly used in the dairy industry as a feed additive, has anti-inflammatory effects, but its mechanism is unclear. This study aims to investigate whether sodium propionate (SP) reduces LPS-induced inflammation in rumen epithelial cells (RECs) and the underlying mechanism. RECs were stimulated with different time (0, 1, 3, 6, 9, 18 h) and different concentrations of LPS (0, 1, 5, 10 µg/mL) to establish an inflammation model. Then, RECs were treated with SP (15, 25, 35 mM) or 10 µM PDTC in advance and stimulated by LPS for the assessment. The results showed that LPS (6h and 10 µg/mL) could stimulate the phosphorylation of NF-κB p65, IκB, JNK, ERK and p38 MAPK through TLR4, and increase the release of TNF-α, IL-1ß and IL-6. SP (35 mM) can reduce the expression of cytokines by effectively inhibiting the NF-κB and MAPK inflammatory pathways. This study confirmed that SP inhibited LPS-induced inflammatory responses through NF-κB and MAPK in RECs, providing potential therapeutic targets and drugs for the prevention and treatment of SARA.

Swainsonine inhibits autophagic degradation and causes cytotoxicity by reducing CTSD O-GlcNAcylation.

Swainsonine (SW) is the primary toxin in locoweed, a poisonous plant. SW can cause animal poisoning, affect the quality and safety of meat products and threaten human health, but the mechanism of its toxicity is little defined. Here, we identified 159 differentially expressed proteins, many of which are involved in autophagy and glycosylation modification processes, using proteomics sequencing analysis. O-linked-N-acetylglucosamylation (O-GlcNAcylation) is a glycosylation modification widely involved in various biological processes. Our results show that SW toxicity is related to O-GlcNAcylation. In addition, increased O-GlcNAcylation with the O-GlcNAcase (OGA) inhibitor TMG promoted autophagy, while decreased O-GlcNAcylation with the O-GlcNAc transferase (OGT) inhibitor OSMI inhibited autophagy. Further analysis by Immunoprecipitation (IP) showed that SW could change the O-GlcNAcylation of Cathepsin D (CTSD), reducing the expression of mature CTSD (m-CTSD). In summary, these findings suggest that SW inhibits the O-GlcNAcylation of CTSD, affecting its maturation and leading to the impairment of lysosome function. Consequently, it inhibits autophagy degradation, and causes cytotoxicity, providing a new theoretical basis for SW toxicological mechanism.

A Network Pharmacology and Multi-Omics Combination Approach to Reveal the Effect of Strontium on Ca2+ Metabolism in Bovine Rumen Epithelial Cells.

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 ß2 (TGF-ß2), 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.

Untargeted metabolomics and lipidomics to assess plasma metabolite changes in dairy goats with subclinical hyperketonemia.

Subclinical hyperketonemia (SCHK) is the major metabolic disease observed during the transition period in dairy goats, and is characterized by high plasma levels of nonesterified fatty acids (NEFA) and ß-hydroxybutyrate (BHB). However, no prior study has comprehensively assessed metabolomic profiles of dairy goats with SCHK. Plasma samples were collected within 1 h after kidding from SCHK goats (BHB concentration >0.8 mM, n = 7) and clinically healthy goats (BHB concentration <0.8 mM, n = 7) with similar body condition score (2.75 ± 0.15, mean ± standard error of the mean) and parity (primiparous). A combination of targeted and untargeted mass spectrometric approaches was employed for analyzing the various changes in the plasma lipidome and metabolome. Statistical analyses were performed using the GraphPad Prism 8.0, SIMCA-P software (version 14.1), and R packages (version 4.1.3). Plasma aminotransferase, nonesterified fatty acids, and BHB concentrations were greater in the SCHK group, but plasma glucose concentrations were lower. A total of 156 metabolites and 466 lipids were identified. The analysis of untargeted metabolomics data by principal component analysis and orthogonal partial least squares discriminant analysis revealed a separation between SCHK and clinically healthy goats. According to the screening criteria (unpaired t-test, P < 0.05), 30 differentially altered metabolites and 115 differentially altered lipids were detected. Pathway enrichment analysis identified citrate cycle, alanine, aspartate and glutamate metabolism, glyoxylate and dicarboxylate metabolism, and phenylalanine metabolism as significantly altered pathways. A greater concentration of plasma isocitric acid and cis-aconitic acid levels was observed in SCHK goats. In addition, AA such as lysine and isoleucine were greater, whereas alanine and phenylacetylglycine were lower in SCHK dairy goats. Dairy goats with SCHK also exhibited greater oleic acid, acylcarnitine, and phosphatidylcholine and lower choline and sphingomyelins. Acylcarnitines, oleic acid, and tridecanoic acid displayed positive correlations with several lipid species. Alanine, hippuric acid, and histidinyl-phenylalanine were negatively correlated with several lipids. Overall, altered metabolites in SCHK dairy goats indicated a more severe degree of negative energy balance. Data also indicated an imbalance in the tricarboxylic acid (TCA) cycle, lipid metabolism, and AA metabolism. The findings provide a more comprehensive understanding of the pathogenesis of SCHK in dairy goats.

Strontium Regulates the Proliferation and Differentiation of Isolated Primary Bovine Chondrocytes via the TGFß/SMAD Pathway.

The present study evaluated the effects of strontium (Sr) on proliferation and differentiation of chondrocytes isolated from dairy cows, and whether Sr exerts its effects via transforming growth factor ß (TGFß) signaling. The chondrocytes were isolated from patellar cartilage from newborn Holstein bull calves (n = 3, 1 day old, 38.0 ± 2.8 kg, fasting) within 15 min after euthanasia, and treated with different concentrations of Sr (0, 0.1, 1, and 10 µg/ml, as SrCl2·6H2O). After pretreatment with or without activin receptor-like kinase 5 (ALK5) inhibitor (10 µM SB-505124) for 4 h, chondrocytes were incubated with Sr for another 4 h. Overall effects of Sr were evaluated relative to NaCl as the control. In contrast, the 1 µg/ml Sr-treated group served as the control to determine effects of preincubating with SB-505124. Western blot and qRT-PCR were used for measuring expression of proliferation-, differentiation-, and TGFß1-responsive factors. Data were analyzed using one-way ANOVA in GraphPad Prism 7.0. Incubation with all doses of Sr increased TGFß1/ALK5-induced SMAD3 phosphorylation, and at 10 µg/ml it inhibited ALK1-induced SMAD1/5/9 phosphorylation. Expression of mRNA and protein of the proliferation-responsive factors type Ⅱ Collagen α1 (COL2A1) and aggrecan (ACAN) was induced by Sr at 1 µg/ml. In contrast, Sr at 10 µg/ml inhibited the expression of differentiation-responsive factors type Ⅹ Collagen α1 (COL10A1) and secreted phosphoprotein 1 (SPP1), and at 1 µg/ml it had the same effect on alkaline phosphatase (ALPL) mRNA and protein levels. Cells were stained with PI/RNase Staining buffer to assess cell cycle activity using flow-cytometry. Incubation with Sr at 1 and 10 µg/ml induced an increase in the number of cells in the S-phase, leading to an increase in the proliferation index. Incubation with SB-505124 inhibited phosphorylation of SMAD3. Abundance of ACAN and COL2A1 mRNA and protein was lower when cells were pre-incubated with SB-505124. Overall, data indicated that Sr promotes proliferation and inhibits differentiation of primary chondrocytes by directing TGFß1 signaling towards SMAD3 phosphorylation rather than SMAD1/5/9 phosphorylation. Whether these effects occur in vivo remains to be determined and could impact future application of Sr as an experimental tool in livestock.

FGF21 Reduces Lipid Accumulation in Bovine Hepatocytes by Enhancing Lipid Oxidation and Reducing Lipogenesis via AMPK Signaling.

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.

NEFA Promotes Autophagosome Formation through Modulating PERK Signaling Pathway in Bovine Hepatocytes.

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.

Changes in Acute-Phase Proteins in Plasma during the Periparturient Period of Dairy Goats.

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 ß-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.

Elucidation of the mechanism of NEFA-induced PERK-eIF2α signaling pathway regulation of lipid metabolism in bovine hepatocytes.

During the periparturient transition period, negative energy balance (NEB) characterized by high concentrations of non-esterified fatty acids (NEFA) may cause fatty liver and ketosis in dairy cows. Previous studies have shown that the protein kinase R-like endoplasmic reticulum kinase (PERK) branch of the endoplasmic reticulum stress (ERS) response plays an important role in lipid metabolism in hepatocytes. This study, therefore, investigated the role of the PERK-branch in NEFA-induced fatty liver. Different concentrations of NEFA or GSK2656157 (a novel catalytic inhibitor of PERK) were used to treat hepatocytes isolated from calves. The NEFA treatment significantly increased the triacylglycerol (TG) content, the phosphorylation level of PERK and eukaryotic initiation factor 2α (eIF2α), and the abundance of glucose-regulated protein 78 (Grp78), C/EBP homologous protein (CHOP), sterol regulatory element-binding protein 1c (SREBP-1c), fatty acid synthase (FASN), peroxisome proliferator-activated receptor-α (PPARα), carnitine palmitoyltransferase 1A (CPT1A), apolipoprotein B (APOB), and the low-density lipoprotein receptor (LDLR). Compared with the 1.2 mM NEFA group, inhibition of PERK activity further increased the TG content in hepatocytes, the very-low-density lipoprotein (VLDL) content in the supernatant and the protein abundance of APOB while reducing the expression and nuclear levels of SREBP-1c and PPARα, as well as the expression of CPT1A and CPT2. In conclusion, the results showed that the NEFA-induced PERK-eIF2α signaling pathway promotes lipid synthesis, lipid oxidation, but inhibits the assembly and secretion of VLDL. Therefore, during the transition period, the activation of the PERK-eIF2α signaling pathway in the liver of dairy cows could defeat the acid-induced lipotoxicity and provide energy to alleviate NEB.

Based on G-Series Mouse TH17 Array Study the Effect of Fluoride on C2C12 Cells Cytokines Expression.

C2C12 cells were cultured on medium containing fluoride (0, 1, and 2.5 mmol/L) for 48 h to investigate the effect of excessive fluoride on T helper 17 (Th17)-related cytokine expression profile in skeletal muscle cells, and the culture supernatant was collected and subjected for the detection of 18 cytokines via Th17 array. Results showed that compared with the control group, no differential expression proteins (DEPs) were found in the 1 mmol/L fluoride group; however, eight DEPs were upregulated in the 2.5 mmol/L fluoride group, including macrophage inflammatory protein-3α (MIP-3α), interleukin-21 (IL-21), IL-13, IL-17F, IL-28A, transforming growth factor type beta 1 (TGF-ß1), IL-23, and IL-17A. In addition, five DEPs (MIP-3α, IL-13, IL-21, TGF-ß1, and IL-17F) were upregulated in the 2.5 mmol/L fluoride group compared with the 1 mmol/L fluoride group. Gene ontology analysis revealed that the positive regulation of cytokine production, cytokine activity, receptor ligand activity, and cytokine receptor binding accounted for high percent of DEPs present. Kyoto Encyclopedia of Genes and Genomes analysis showed that these DEPs primarily involved 12 pathways enriched in the cytokine-cytokine receptor interaction and IL-17 signaling pathway after 2.5 mmol/L fluoride treatment. The results indicated that fluoride might induce cytotoxicity by disturbing Th17-related cytokine expression.

Technological characterization of gold jewellery from the Sogdian tomb of Shi Jun (d. 579 CE) in Xi'an, Shaanxi Province.

Northern dynasties (386-581 CE) of China witnessed extensive cultural contacts with the outside world. Several gold objects of this period indicate multiple culture influences. However, very few of them were testified by metallurgic analysis. The gold jewellery including a finger ring and an earring with exotic features were uncovered from the joint elite Sogdian tomb of Shi Jun and his wife of the Northern Zhou dynasty (557-581 CE) in Xi'an. The current study applied multiple non-destructive analyses to investigate the decorative techniques and materials of the two objects. The results showed that both ornaments were made of refined gold. Autogenous welding and brazing were employed for joining the granules of the earring, indicating different technical choices. More interestingly, niello made of silver sulfide was identified as an innovative technology to decorate the finger ring, presenting the earliest evidence of niello inlay in ancient China. It is noteworthy that powders of silver and sulfur were applied separately, deferring from the traditional method of silver sulfide being synthesised prior to being used. These findings help us gain insights into understanding the technical features of early Medieval gold jewellery, as well as the goldsmith's methods and intentions.

Key Role of Pro-inflammatory Cytokines in the Toxic Effect of Fluoride on Hepa1-6 Cells.

The role of pro-inflammatory cytokines in the toxicity of fluoride to tumor cells was investigated by culturing Hepa1-6 cells in medium containing gradient concentrations of fluoride (0, 0.5, 1, 1.5, 2, 3, 4, and 5 mmol/L). The viability of Hepa1-6 cells was detected via MTT assay. Interleukin (IL)-2, IL-6, tumor necrosis factor (TNF)-α, and IL-1ß levels in the supernatant were determined via an enzyme-linked immunosorbent assay, and the protein expression levels of these enzymes in Hepa1-6 cells were evaluated by immunofluorescence staining. Results showed that the viability of Hepa1-6 cells remarkably decreases after fluoride exposure, especially at concentration of 3, 4, and 5 mmol/L fluoride. Levels of IL-2, TNF-α, and IL-1ß in the supernatant markedly decreased when cells were exposed to fluoride at concentrations of 1 mmol/L or higher. However, levels of TNF-α and IL-1ß substantially increased and IL-2 showed no remarkable change when the fluoride concentration was 0.5 mmol/L. The content of IL-6 remarkably increased with increasing fluoride concentrations up to 2 mmol/L, and then markedly decreased at 3, 4, and 5 mmol/L fluoride; the decreasing trend of IL-6 content under high fluoride exposure is consistent with the decrease in Hepa1-6 cell viability observed at the same concentration. The protein expression levels of IL-2, IL-6, TNF-α, and IL-1ß were in accordance with their contents in the supernatant. In summary, our study demonstrated that fluoride inhibits Hepa1-6 cell growth and results in disorders in the expression and secretion pro-inflammatory cytokines.

Evaluation of the protective immunity of Riemerella anatipestifer OmpA.

Riemerella anatipestifer is responsible for an economically important disease of commercially raised ducks. No or only few cross-protection was observed between different serotypes of R. anatipestifer strains, and so far no protective antigen in this bacterium has been identified. OmpA is a predominant immunogenic protein of R. anatipestifer, and within the 1467 bp ompA ORF (ompA1467), there is another 1164 bp ORF (ompA1164) with the same C-terminal. In this study, our results showed that the full sequence of ompA1467 from some R. anatipestifer strains with different serotypes shared the same amino acid sequence. Animal experiments showed that the soluble recombinant protein rOmpA1164, but not rOmpA1467, could provide partial protective immunity against challenge. Moreover, there was no significant difference in protective immunity between ducklings immunized with Th4△ompA bacterin and those immunized with Th4 bacterin. In addition, OmpA1467 was the main existing form of OmpA in R. anatipestifer cells by gel electrophoresis and western blot analyses. The results suggested that OmpA1467 was not a protective antigen of R. anatipestifer, and antibodies against proteins other than OmpA play a critical role in the process of anti-R. anatipestifer infection.

Mitochondrial respiratory chain dysfunction mediated by ROS is a primary point of fluoride-induced damage in Hepa1-6 cells.

To evaluate the mechanism of fluoride (F) mitochondrial toxicity, we cultured Hepa1-6 cells with different F concentrations (0, 1 and 2 mmoL/L) and determined cell pathological morphology, mitochondrial respiratory chain damage and cell cycle change. Results showed that the activities and mRNA expression levels of antioxidant enzymes considerably decreased, whereas the contents of reactive oxygen species (ROS), malondialdehyde (MDA) and nitric oxide (NO) markedly increased. Breakage of mitochondrial cristae and substantial vacuolated mitochondria were observed by transmission electron microscopy. These results indicate the F-induced oxidative damage in Hepa1-6 cells. The enzyme activities of mitochondrial complexes I, II, III and IV were disordered in Hepa1-6 cells treated by excessive F, thereby indicating a remarkable down-regulation. Further research showed that complex subunits also demonstrated the development of disorder, in which the protein expressions levels of NDUFV2 and SDHA were substantially down-regulated, whereas those of CYC1 and COX Ⅳ were markedly up-regulated. Reductions in ATP and mitochondrial membrane potential were detected with the dysfunction of the mitochondrial respiratory chain. The G2/M phase arrest of the cell cycle in Hepa1-6 cells was measured via flow cytometry, and the up-regulated protein expressions of Cyt c, caspase 9, caspase 3 and substantial apoptotic cells were determined. In summary, this study demonstrated that ROS-mediated mitochondrial respiratory chain dysfunction causes F-induced Hepa1-6 cell damage.

Carbocisteine Improves Histone Deacetylase 2 Deacetylation Activity via Regulating Sumoylation of Histone Deacetylase 2 in Human Tracheobronchial Epithelial Cells.

Histone deacetylase (HDAC) 2 plays a vital role in modifying histones to mediate inflammatory responses, while HDAC2 itself is commonly regulated by post-translational modifications. Small ubiquitin-related modifier (SUMO), as an important PTM factor, is involved in the regulation of multiple protein functions. Our previous studies have shown that carbocisteine (S-CMC) reversed cigarette smoke extract (CSE)-induced down-regulation of HDAC2 expression/activity in a thiol/GSH-dependent manner and enhanced sensitivity of steroid therapy. However, the mechanism by which S-CMC regulates HDAC2 is worth further exploring. Our study aimed to investigate the relationships between HDAC2 sumoylation and its deacetylase activity under oxidative stress and the molecular mechanism of S-CMC to regulate HDAC2 activity that mediates inflammatory responses in human bronchial epithelial cells. We found that modification of HDAC2 by SUMO1 and SUMO2/3 occurred in 16HBE cells under physiological conditions, and CSE induced SUMO1 modification of HDAC2 in a dose and time-dependent manner. K462 and K51 of HDAC2 were the two major modification sites of SUMO1, and the K51 site mediated deacetylation activity and function of HDAC2 on histone H4 that regulates IL-8 secretion. S-CMC inhibited CSE-induced SUMO1 modification of HDAC2 in the presence of thiol/GSH, increased HDAC activity, and decreased IL-8 expression. Our study may provide novel mechanistic explanation of S-CMC to ameliorate steroid sensitivity treatment in chronic obstructive pulmonary disease.

Direct SUMOylation of M1 muscarinic acetylcholine receptor increases its ligand-binding affinity and signal transduction.

SUMOylation is a significant post-translational modification (PTM) by the small ubiquitin-related modifier (SUMO). Increasing evidence shows SUMOylation regulates GPCR signaling; however, very few GPCRs have been shown to be SUMOylation targets to date. In this study, we identified M1 muscarinic acetylcholine receptor (M1 mAChR), a member of the GPCRs, as a new SUMO substrate. When the mAChR was activated by the agonist carbachol, the colocalization of the M1 mAChR and SUMO-1 protein markedly decreased in immunoprecipitation and immunofluorescence assays. SUMOylation of the M1 mAChR played an important role in increasing the ligand-binding affinity to M1 mAChR, signaling efficiencies, and receptor endocytosis. Through the site-directed mutagenesis approach, K327 was identified as the SUMOylation site of the M1 mAChR. Mutation of the consensus SUMOylation site of the M1 mAChR reduces not only the colocalization of SUMO-1, but also the ligand-binding affinity and signal transduction. The function of M1 mAChR was regulated by SUMOylation through the stabilization of active-state conformation revealed by molecular dynamics simulations. Our results provide evidence that M1 SUMOylation is an important PTM involved in regulation of the affinity for agonists and for activation of signaling pathways.-Xu, J., Tan, P., Li, H., Cui, Y., Qiu, Y., Wang, H., Zhang, X., Li, J., Zhu, L., Zhou, W., Chen, H. Direct SUMOylation of M1 muscarinic acetylcholine receptor increases its ligand-binding affinity and signal transduction.