Development and application of an indirect ELISA for the serological detection of bovine viral diarrhea virus infection based on the protein E2 antigen.

BACKGROUND: Bovine viral diarrhea virus (BVDV) causes continuous economic losses to the livestock industry. Monitoring antibodies with enzyme-linked immunosorbent assay (ELISA) is a valuable tool to ensure the purification of BVDV in cattle. However, currently available ELISA kits based on the whole BVDV virion are both costly and time-consuming. The E2 protein has good immunogenicity, induces the secretion of neutralizing antibodies and is an essential immunogen for serological detection. METHODS AND RESULTS: We developed a novel recombinant E2 protein-based indirect ELISA (rE2-iELISA) and conducted a serological survey for BVDV antibodies in 2021-2022 in Beijing, China. The results showed that E2 protein was successfully expressed with high immunogenicity and the optimal rE2-iELISA displayed high sensitivity, reproducibility and specificity. Clinical testing of 566 serum specimens indicated that 318 BVDV positive samples and 194 BVDV negative samples were tested by rE2-iELISA and the IDEXX BVDV ELISA-Ab kit, with a positive coincidence rate of 93.3%, a negative coincidence rate of 86.3%, and an overall coincidence rate of 90.5%. CONCLUSION: This study established an rE2-iELISA method, which is a highly sensitive, specific and robust ELISA-test validated to detect anti-BVDV antibodies. These findings indicate that the newly developed rE2-iELISA method has the potential to be used as a rapid, reliable and cost-effective screening tool for BVDV infection and provides technical support for the evaluation of vaccine efficacy in cattle herds in the future.

A Novel Small Molecular Inhibitor of DNMT1 Enhances the Antitumor Effect of Radiofrequency Ablation in Lung Squamous Cell Carcinoma Cells.

Radiofrequency ablation (RFA) is a relatively new and effective therapeutic strategy for treating lung squamous cell carcinomas (LSCCs). However, RFA is rarely used in the clinic for LSCC which still suffers from a lack of effective comprehensive treatment strategies. In the present work, we investigate iDNMT, a novel small molecular inhibitor of DNMT1 with a unique structure. In clinical LSCC specimens, endogenous DNMT1 was positively associated with methylation rates of miR-27-3p's promoter. Moreover, endogenous DNMT1 was negatively correlated with miR-27-3p expression which targets PSEN-1, the catalytic subunit of γ-secretase, which mediates the cleavage and activation of the Notch pathway. We found that DNMT1 increased activation of the Notch pathway in clinical LSCC samples while downregulating miR-27-3p expression and hypermethylation of miR-27-3p's promoter. In addition of inhibiting activation of the Notch pathway by repressing methylation of the miR-27-3p promoter, treatment of LSCC cells with iDNMT1 also enhanced the sensitivity of LSCC tumor tissues to RFA treatment. These data suggest that iDNMT-induced inhibition of DNMT-1 enhances miR-27-3p expression in LSCC to inhibit activation of the Notch pathway. Furthermore, the combination of iDNMT and RFA may be a promising therapeutic strategy for LSCC.

Berberine activates the ß-catenin/TCF4 signaling pathway by down-regulating miR-106b to promote GLP-1 production by intestinal L cells.

Berberine facilitates the production of glucagon-like peptide-1 (GLP-1) by intestinal L cells. Here, we aimed to reveal the mechanism of berberine facilitating the production of GLP-1 by intestinal L cells. In this study, we confirmed that the 100 mg/kg berberine daily through diet decreased the miR-106b expression and elevated the expressions of ß-catenin and T-cell factor 4 (TCF4) in colon tissues of high-fat diet mice; berberine decreased the concentrations of triglycerides, total cholesterol and the ratio of low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in mouse serum samples; berberine decreased the blood glucose in the mouse tail vein blood and promoted GLP-1 production by intestinal L cells in mouse serum samples and elevated the GLP-1 expression in mouse colon tissues. Meanwhile, the mechanism analysis demonstrated that a dose of 100 µM berberine down-regulated the miR-106b expression by elevating the methylation levels of miR-106b in STC-1 cells and miR-106b bound to TCF4 in 293T cells. Moreover, the 100 mg/kg berberine daily through diet activated the ß-catenin/TCF4 signaling pathway by decreasing miR-106b, thereby facilitating GLP-1 production in intestinal L cells through the in vivo assays. Conclusively, our experimental data illustrated that berberine decreased miR-106b expression by increasing its methylation levels and then activated the ß-catenin/TCF4 signaling pathway, thereby facilitating GLP-1 production by intestinal L cells.

Metabolic profiling of nanosilver toxicity in the gills of common carp.

Studies have shown silver nanoparticles (AgNPs) exposure can result in a series of toxic effects in fish gills. However, it is still unclear how AgNPs affect metabolite expression and their related molecular metabolic pathways in fish gills. In this study, we employed untargeted metabolomics to study the effects of AgNPs and silver supernatant ions on fish gill metabolites. The results showed that AgNPs can induce significant changes in 96 differentially expressed metabolites, which mainly affect amino acid metabolism and energy metabolism in fish gills. Among these metabolites, AgNPs specifically induce significant changes in 72 differentially expressed metabolites, including L-histidine, L-isoleucine, L-phenylalanine, and citric acid. These metabolites were significantly enriched in the pathways of aminoacyl-tRNA biosynthesis, ABC transporters, and the citrate cycle. In contrast, Ag+ supernatant exposure can specifically induce significant changes in 14 differentially expressed metabolites that mainly interfere with sphingolipid metabolism in fish gills. These specifically regulated fish gill metabolites include sphinganine, sphingosine, and phytosphingosine, which were significantly enriched in the sphingolipid metabolism pathway. Our results clearly reveal the effects and potential toxicity mechanisms of AgNPs on fish gill metabolites. Furthermore, our study further determined the unique functions of released silver ions in AgNPs toxicity in fish gills.

MicroRNA-194: a novel regulator of glucagon-like peptide-1 synthesis in intestinal L cells.

In the status of obesity, the glucagon-like peptide-1 (GLP-1) level usually declines and results in metabolic syndrome. This study aimed to investigate the intracellular mechanism of GLP-1 synthesis in L cells from the perspective of microRNA (miRNA). In the present study, we found that GLP-1 level was down-regulated in the plasma and ileum tissues of obese mice, while the ileac miR-194 expression was up-regulated. In vitro experiments indicated that miR-194 overexpression down-regulated GLP-1 level, mRNA levels of proglucagon gene (gcg) and prohormone convertase 1/3 gene (pcsk1), and the nuclear protein level of beta-catenin (ß-catenin). Further investigation confirmed that ß-catenin could promote gcg transcription through binding to transcription factor 7-like 2 (TCF7L2). miR-194 suppressed gcg mRNA level via negatively regulating TCF7L2 expression. What's more, forkhead box a1 (Foxa1) could bind to the promoter of pcsk1 and enhanced its transcription. miR-194 suppressed pcsk1 transcription through targeting Foxa1. Besides, the interference of miR-194 reduced palmitate (PA)-induced cell apoptosis and the anti-apoptosis effect of miR-194 inhibitor was abolished by TCF7L2 knockdown. Finally, in HFD-induced obese mice, the silence of miR-194 significantly elevated GLP-1 level and improved the metabolic symptoms caused by GLP-1 deficiency. To sum up, our study found that miR-194 suppressed GLP-1 synthesis in L cells via inhibiting TCF7L2-mediated gcg transcription and Foxa1-mediated pcsk1 transcription. Meanwhile, miR-194 took part in the PA-induced apoptosis of L cells.

Ecological risk assessment of heavy metals in fish from the Dianchi Lake, China using the integrated biomarker response approach.

This study used the integrated biomarker response (IBR) index approach to assess the ecological risks of heavy metals in different regions of Dianchi Lake, combined with active monitoring and passive monitoring. The contents of five heavy metals (Cu, As, Cd, Hg, and Pb) and six biomarkers (acetylcholinesterase, sodium-potassium ATPase, metallothionein, superoxide dismutase, glutathione peroxidase, and malondialdehyde) in the muscles of crucians (Carassius auratus) were measured to calculate the IBR value. The results indicate that the contents of heavy metal in the fish under active monitoring and passive monitoring were rather low and did not exceed the National Food Safety Standards of China. The IBR value of day 14 of active monitoring correlated with the heavy metal Cd content in the fish, suggesting a potential risk of Cd pollution in the aquatic environment of Dianchi Lake. The IBR values obtained for different regions of the lake on day 14 can be arranged in the following order: West S3 (9.24) > East S1 (3.97) > South S2 (2.39) > North S4 (0.36). These results suggest a potential risk of heavy metal contamination in the western part of Dianchi Lake.

Effect of silver nanoparticles on gill membranes of common carp: Modification of fatty acid profile, lipid peroxidation and membrane fluidity.

Although the toxicity of silver nanoparticles (AgNPs) in aquatic organisms has been extensively investigated, the mechanism by which AgNPs damage membranes remains unclear. This study investigated the toxic effects of a series of sub-lethal concentrations of AgNPs on the membranes of freshwater carp (Cyprinus carpio) gills, based on changes in membrane fatty acid (FA) profile, membrane fluidity, membrane lipid peroxidation, and histopathology. Most of the FAs in fish gill membrane was not significantly affected by exposure to multiple AgNPs concentrations, only few significant changes occurred in some specific FAs species at a high concentration of AgNPs exposure. In particular, high concentrations of AgNPs significantly decreased the proportions of two important long-chain n-3 series polyunsaturated FAs (C20: 5n3, and C22: 6n3), resulting in a decreased ratio of n-3 polyunsaturated FAs to n-6 polyunsaturated FAs (Σn-3UFA/Σn-6UFA). The AgNPs also caused a dose-dependent decrease in fish gill membrane fluidity, increased the level of lipid peroxidation, and inhibited Na+/K+-ATPase enzyme activity. Further histopathological examination revealed that exposure to AgNPs can cause toxic responses in the lamellae, including the thinning of the basement membrane, malformation, and inflammation. Together, the results suggest that the mechanism of AgNPs membrane toxicity involves the oxidization of long-chain omega-3 unsaturated FAs to saturated FAs via lipid peroxidation, resulting in, decreased membrane fluidity and ultimately the destruction of the normal physiological function of the fish gill membrane. The findings contribute significantly to our understanding of nanoparticle-induced membrane toxicity and potential risks in aquatic environments.

The apoptosis and GLP-1 hyposecretion induced by LPS via RIP/ROS/mTOR pathway in GLUTag cells.

Lipopolysaccharide (LPS) as a component of the outer structure of cell wall of gram-negative bacteria, could induce apoptosis in the intestinal endocrine cell line STC-1. However, the signaling cascades involved in this process have not been elucidated. Hence, we investigated the mechanism of cell apoptosis and hyposecretion of glucagon-like peptide 1 (GLP-1) induced by LPS in the GLUTag enteroendocrine cell line. LPS decreased the cell viability of GLUTag cells, up-regulated the TNF-α level, induced the apoptosis and down-regulated the mRNA and protein levels of GLP-1. In addition, TNF-α promoted LPS-induced apoptosis of GLUTag cells through mediating the formation of the RIP1/RIP3 necrosome. RIP1 and RIP3 knockdown increased cell viability, the mRNA and protein levels of GLP-1 and the mTOR signaling pathway-related proteins (p-mTOR and p-S6), and decreased the relative caspase 3/7 activity, cell apoptosis and ROS production. Further studies showed that ROS inhibited the mTOR signaling pathway. Moreover, the antioxidant N-acetyl-l-cysteine increased cell viability, GLP-1 expressions and the mTOR signaling pathway-related proteins, and inhibited the ROS production. However, the mTOR specific inhibitor (Rapa) reversed all these above effects. Taken together, our result revealed that LPS induced the apoptosis of GLUTag cells and GLP-1 hyposecretion through the RIP/ROS/mTOR pathway.

High glucose contributes to the proliferation and migration of non-small cell lung cancer cells via GAS5-TRIB3 axis.

Despite the growing number of studies exhibited an association of diabetes mellitus (DM) and lung cancer progression, the concrete mechanism of DM aggravating lung cancer has not been elucidated. This study was to investigate whether and how high glucose (HG) contribute to the proliferation and migration of non-small cell lung cancer (NSCLC) cells in vitro. In the present study, we confirmed that HG promoted the proliferation and migration of NSCLC cells, and also induced an anti-apoptosis effect on NSCLC cells. Moreover, HG inhibited the expression of GAS5 in NSCLC cells but elevated the protein level of TRIB3. GAS5 overexpression promoted the degradation of TRIB3 protein by ubiquitination and inhibited the HG induced-proliferation, anti-apoptosis and migration of NSCLC cells. Importantly, TRIB3 overexpression reversed the effects of GAS5 on the HG-treated NSCLC cells. Taken together, down-regulated GAS5 by HG significantly enhanced the proliferation, anti-apoptosis and migration in NSCLC cells through TRIB3, thus promoting the carcinogenesis of NSCLC.

Intensive epidermal adsorption and specific venous deposition of carboxyl quantum dots in zebrafish early-life stages.

To properly assess the environmental risk of quantum dots (QDs), it is necessary to determine their fate in living organisms, including adsorption, distribution and bioaccumulation under representative environmental or physiological conditions. We comprehensively investigated the fate of QDs with carboxyl terminal functional groups (carboxyl-QDs) in zebrafish (Danio rerio) embryo and larvae subjected to either waterborne exposure or cardiovascular system microinjection. On waterborne exposure, carboxyl-QDs exhibited an intensive adsorption and accumulation in the chorion of embryos, and their predominate target organs were the gill and intestinal tract in larvae. On microinjection, carboxyl-QDs were rapidly delivered into the cardiovascular system and specifically deposited in veins and the capillary network system of zebrafish larvae, but not in the arterial system. Taken together, we found that the exact tissue condition including epidermal structures, mucus secretion and vascular microstructures strongly affected the adsorption, uptake and distribution of carboxyl-QDs in zebrafish. This work highlights the intensive tissue epidermal adsorption and accumulation of carboxyl-QDs and their specific vein and capillary deposition in the cardiovascular system in zebrafish early-life stages.

Nanotoxicity of silver nanoparticles to red blood cells: size dependent adsorption, uptake, and hemolytic activity.

Silver nanoparticles (AgNPs) are increasingly being used as antimicrobial agents and drug carriers in biomedical fields. However, toxicological information on their effects on red blood cells (RBCs) and the mechanisms involved remain sparse. In this article, we examined the size dependent nanotoxicity of AgNPs using three different characteristic sizes of 15 nm (AgNPs15), 50 nm (AgNPs50), and 100 nm (AgNPs100) against fish RBCs. Optical microscopy and transmission electron microscopy observations showed that AgNPs exhibited a size effect on their adsorption and uptake by RBCs. The middle sized AgNPs50, compared with the smaller or bigger ones, showed the highest level of adsorption and uptake by the RBCs, suggesting an optimal size of ∼50 nm for passive uptake by RBCs. The toxic effects determined based on the hemolysis, membrane injury, lipid peroxidation, and antioxidant enzyme production were fairly size and dose dependent. In particular, the smallest sized AgNPs15 displayed a greater ability to induce hemolysis and membrane damage than AgNPs50 and AgNPs100. Such cytotoxicity induced by AgNPs should be attributed to the direct interaction of the nanoparticle with the RBCs, resulting in the production of oxidative stress, membrane injury, and subsequently hemolysis. Overall, the results suggest that particle size is a critical factor influencing the interaction between AgNPs and the RBCs.

[Effect of transfection of microRNA-146a on expression of tumor necrosis factor-a in alveolar macrophages].

OBJECTIVE: To observe the effect of transfected microRNA-146a (miR-146a) on expression of tumor necrosis factor-alpha ( TNF-alpha ) in alveolar macrophages, and to analyze its regulatory mechanism in the inflammatory response of alveolar macrophages. METHODS: Cy.11113 labeled with 25, 50, 100 nmol/L of Pre-miR", respectively, were transfected into rat alveolar macrophages NR8383 cultured in vitro. The highest transfection efficiency was selected to he the experimental concentration. NR8383 cells were divided into two groups: transfected group was tranfected with 50 nmol/L Pre-miR1111 miR-146a precursor, and control group with SO nmol/L Cym3 labeled Pre-miR1" as the negative control. The mRNA expression of miR-146a of cells was detected hy real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Then cells were stimulated with lipopolysaccharide (LPS, 1 mg/L) for 6 hours. The production of TNF-alpha protein in the supernatant of cells was assayed by enzyme-linked immunosorhent assay (ELISA) , and the expression of TNF-alpha mRNA of cells was detected hy RT-qPCR. RESULTS: Transfection rate was highest in the 50 nmol/L Cy11113 labeled Pre-miR"' cells, and it reached 80%. Compared with control group (set at 1), the expression of miR-146a increased by (24.55 ±6.14) folds in transfected alveolar marrophages (P<0.01). After the cells were stimulated with LPS, the production of TNF-alpha protein (ng/L) in the supernatant of cell was decreased from 616.6 ± 42.3 to 211.5 ± 30.4 (P<0.01), and the expression of TNF-alpha mRNA was decreased by (47 ± 6) % (P<0.05) in transfected alveolar macrophages when compared with that of the control. CONCLUSIONS: Transfection alveolar macrophages with miR-146a precursors could down-regulate the expression of TNF-alpha. It is therefore suggests that up-regulation of miR-146a can inhibit inflammatory responses as induced hy LPS, in alveolar macrophages.

[Effect of Shenfu injection on expression of lipopolysaccharide --induced microRNA-146a in alveolar macrophages].

OBJECTIVE: To study the effects of Shenfu injection (SF) on the expression of lipopolysaccharide(LPS)-induced microRNA-146a (miR-146a) in rat alveolar macrophages (AMs), and to extrapolate its potential anti-inflammatory mechanisms. METHODS: In vitro cultured rat AMs (NR8383 cells) were randomly divided into control group, LPS stimulation group, and SF stimulation group. The LPS stimulation group was challenged with a final concentration of 1 mg/L LPS, and to the control group an equal volume of phosphate buffer solution (PBS) was added instead. For SF treated group, SF in different concentrations (1 ml/L or 10 ml/L) was used during incubation of AMs for half an hour, and then LPS was added (1 mg/L final concentration). After 6 hours, the cells and were collected. MiRNA-146a expression [reverse transcription-polymerase chain reaction (RT-PCR)] in cells and tumor necrosis factor-α (TNF-α ) content [enzyme-linked immunosorbent assay (ELISA)] in culture supernatant were determined for each group. RESULTS: Both the expression of miR-146a and TNF-α content in LPS stimulation group were significantly elevated compared with control group [miR-146a (expression folds): 5.92 + 1.57 vs. 1.04 +0.38; TNF-α (ng/L): 636.93 _ 30.21 vs. 20.46 + 2.81; both P<0.05]. Compared with LPS stimulation group, the expression of miR-146a was significantly upregulated in cells in both 1 ml/L and 10 ml/L SF stimulation groups, but TNF- α content was significantly reduced in the supernatant [miR-146a (expression folds): 7.02 + 0.91, 8.11 ± 1.07 vs. 5.92 -1.57; TNF-α (ng/L): 447.24 +21.29, 357.83 +19.73 vs. 636.93 +30.21, all P<0.05] in a dose-dependent manner (both P<0.05). CONCLUSION: SF could up-regulate miR-146a expression in AMs in a dose-dependent manner, and it was speculated that miR-146a might be involved in the anti-inflammatory processes with SF treatment.

[Expression of microRNA-146a in lipopolysaccharide challenged alveolar macrophages in vitro].

OBJECTIVE: To investigate the expression of microRNA-146a (miRNA-146a) in NR8383 alveolar macrophages treated with lipopolysaccharides (LPS). METHODS: NR8383 alveolar macrophages were divided into two groups: LPS treated group and phosphate buffer saline (PBS) control group, and they cultured for 6 hours. The production of tumor necrosis factor-α (TNF-α) in the supernatant of cells was determined with enzyme-linked immunosorbent assay (ELISA), and the expression of miRNA-146a of cells was detected by real-time polymerase chain reaction (PCR). RESULTS: Compared with PBS control group, the TNF-α content (ng/L) in LPS treated group was significantly increased (650.26±40.53 vs. 6.23±1.76, P<0.01), and miRNA-146a in LPS treated group increased by about (5.33±0.81) folds (P<0.01). CONCLUSION: The expression of miRNA-146a was increased in LPS treated NR8383 cells, and miRNA-146a may be involved in the modulation inflammatory response of the NR8383 alveolar macrophage.