A fluorescent probe for ultrarapid H2O2 detection during reagent-stimulated oxidative stress in cells and zebrafish.

Hydrogen peroxide(H2O2), as a reliable signaling biomolecule for oxidative stress, its accurate detection during agent-stimulated oxidative stress plays a vital role in pathological and physiological mechanism exploration for disease theranostics. It's necessary to develop an efficient method for their detection. In view of the advantages of fluorescent probes, we rationally constructed a novel fluorescent probe Compound 2 based on 4-(Bromomethyl)benzeneboronic acid pinacol ester_Herein, a small molecule fluorescent probe was fabricated using isoflore nitrile as fluorescent group, phenylboronic acid pinacol ester as the response group, to detect H2O2. The probe Compound 2 has a strong fluorescence intensity at 575 nm, indicating that the structure of the probe molecule is reasonably designed, and the Stokes shift is up to 172 nm. While the detection time is as low as 30 s and the LOD of the probe for H2O2 is as low as 3.7 µmol/L,the quantum yield is Φ = 40.31 %. It has been successfully used for imaging detection of H2O2 in HepG2 cells and zebrafish for its low toxicity. It can be found that this small molecule fluorescent probe can identify H2O2 in tumor cells significantly and efficiently, which would realize the early diagnosis of tumor.

Design, Synthesis of Hydrogen Peroxide Response AIE Fluorescence Probes Based on Imidazo [1,2-a] Pyridine.

Hydrogen peroxide (H2O2), a significant member of reactive oxygen species, plays a crucial role in oxidative stress and cell signaling. Abnormal levels of H2O2 in the body can induce damage or even impair body function, leading to the development of certain diseases. Therefore, real-time monitoring of H2O2 in living cells is very important. In this work, the aggregation-induced emission fluorescence probe 2-(2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzyl) oxy) phenyl) imidazo [1,2-a] pyridine (B2) was designed and synthesized, which enables the long-term tracing of H2O2 in living cells. The addition of H2O2 to probe B2 results in a dramatic fluorescence enhancement around 500 nm. Notably, B2 can visualize both exogenous and endogenous H2O2 in living cells. The synthesis method for B2 is simple, has a high yield, and utilizes readily available materials. It exhibits advantages such as low toxicity, photostability, and good biocompatibility. Consequently, the developed fluorescent probe in this study has great potential as a reliable tool for determining H2O2 in living cells.

Click Triazole as a Linker for Pretargeting Strategies: Synthesis, Docking Investigations, Fluorescence Diagnosis, and Antibacterial Action Studies.

In this study, three compounds A1, A2, and A3 and fluorescent probes T1, T2, T3, and T4 were designed and synthesized. 1H NMR, 13C NMR, and MS characterization and elemental analysis were used to confirm A1-A3 and T1-T4. A1-A3 and T1-T4 formed diagnostic molecules by "click" reactions. A1-A3 and T1-T4 did not significantly increase cell death at concentrations of 80 µmol/L. Preliminary screening of the compounds for antibacterial activity revealed that A2 has better antibacterial activity against Agrobacterium tumefaciens. The synthesized compounds and fluorescent probes can be targeted and combined in the physiological condition to form diagnostic molecules for fluorescence detection of Agrobacterium tumefaciens. The binding sites of A1-A3 were deduced theoretically using the AutoDock Vina software docking tool. Further study of the mechanism of the antibacterial action of these compounds is likely to identify new agents against resistant bacterial strains.

Anti-Invasion and Antimetastatic Effects of Porcine Recombinant NK-lysin on SMMC-7721 Human Hepatocellular Carcinoma Cells.

The high invasion and metastasizing abilities of hepatocellular carcinoma (HCC) are the primary reasons for the high mortality rate of patients. Therefore, identification of agents to inhibit invasion and metastasis is very important for treatment of HCC. We analyzed the anti-invasion and antimetastatic effects of porcine recombinant NK-lysin, which was designed and expressed in vitro by our research group, on SMMC-7721 hepatocellular carcinoma cells via wound-healing assays, adhesion assays, invasion assays, real-time polymerase chain reaction (PCR), and Western blot analysis. MTT assay results indicated that NK-lysin inhibited the growth of SMMC-7721 cells in a dose- and time-dependent manner. NK-lysin reduced the ability of cell migration, adhesion, and invasion. Based on gene and protein expression analysis, NK-lysin decreased ß-catenin and MMP-2 expression. These results suggested that NK-lysin has anti-invasion and antimetastatic effects on hepatocellular carcinoma cells in vitro by reducing the level of the ß-catenin and MMP-2.

Induction of apoptosis by an oleanolic acid derivative in SMMC-7721 human hepatocellular carcinoma cells is associated with mitochondrial dysfunction.

The aim of the present study was to investigate the effects of an oleanolic acid derivative, a novel antitumor drug, on the growth of SMMC-7721 human hepatocellular carcinoma cells and the underlying mechanism. An MTT assay was performed to determine the cytotoxicity of the oleanolic acid derivative. Cell membrane integrity was assessed using fluorescence microscopy to assess the uptake of annexin V-FITC/propidium iodide (PI). Western blotting was used to detect the apoptosis-associated proteins B cell lymphoma-2 (Bcl-2), Bax, caspase-9 and caspase-3. A spectrophotometer was used to analyze the intracellular adenosine triphosphate (ATP) expression level. The loss of mitochondrial membrane potential was detected by performing the JC-1 assay. ELISA was used to evaluate the content of cytochrome c (Cyt-C). The oleanolic acid derivative reduced the cell viability of SMMC-7721 cells in a dose- and time-dependent manner. The half maximal inhibitory concentration values of the oleanolic acid derivative in SMMC-7721 cells at 24, 48 and 72 h were 26.80, 11.85, and 6.66 µM, respectively. The antiapoptotic-protein Bcl-2 was downregulated, and the proapoptotic protein Bax was upregulated following treatment with the oleanolic acid derivative for 48 h. The oleanolic acid derivative induced the cleavage of caspase-9 and caspase-3 as well as promoted annexin V-FITC/PI uptake in SMMC-7721 cells. Furthermore, treatment of SMMC-7721 cells with the oleanolic acid derivative induced a reduction of the intracellular ATP expression level, loss of ΔΨm and Cyt-C release from the mitochondria. The oleanolic acid derivative induced apoptosis in SMMC-7721 human cells. Mitochondrial dysfunction was involved in the anticancer effects of this derivative on SMMC-7721 human cells.

Suppression of the Wnt signaling pathway may contribute to the inhibition of proliferation of human hepatocellular carcinoma SMMC-7721 cells by esculetin.

The aims of the present study were to investigate the effects of esculetin on the proliferation of human hepatocellular carcinoma SMMC-7721 cells and to determine the underlying mechanism behind this activity. An MTT assay was used to assess cell proliferation, reverse transcription-quantitative polymerase chain reaction was used to determine the relative mRNA expression levels of ß-catenin, c-Myc and cyclin D1, and western blot analysis was utilized to determine the levels of the associated proteins. Compared with the dimethyl sulfoxide control, esculetin reduced the cell viability of SMMC-7721 and HL-7702 cells in a dose- and time-dependent manner. Treatment of SMMC-7721 cells with esculetin resulted in downregulation of the mRNA and protein levels of ß-catenin, c-Myc and cyclin D1. Esculetin increased the phosphorylation of ß-catenin at Ser33/Ser37/Thr41 and inhibited the proliferation of human hepatoma SMMC-7721 cells by suppressing the Wnt signaling pathway. The results of the present study suggest that esculetin inhibited the Wnt/ß-catenin signaling pathway in SMMC-7721 cells and may have potential as an effective anti-cancer drug, acting to inhibit the Wnt/ß-catenin signaling pathway.

Complement receptor activity of recombinant porcine CR1-like protein expressed in a eukaryotic system.

Primate complement receptor type 1 (CR1) protein, a single-chain transmembrane glycoprotein, plays an important role in immune adherence and clearing complement-opsonized immune complexes. Here, the mRNA of the porcine primate-like complement receptor (CR1-like) gene was analyzed, and two domain sequences with potential functions were cloned into the pwPICZalpha vector for expression in Pichia pastoris. The recombinant proteins were purified with both Protein Pure Ni-NTA resin and strong anion exchange resin. The activities of the purified recombinant proteins were evaluated by SDS-PAGE, western blotting, and complement receptor assays. The results indicated that two domains of the CR1-like protein, CCP36 and CCP811 with molecular weights of 29.8 kDa and 30 kDa, respectively, were successfully expressed in P. pastoris. These two recombinant proteins possess some of the functions of the primate CR1 protein. Using these two proteins coupled with an antibody blocking technique, we also showed that CR1-like is expressed on natural porcine erythrocytes.

Expression and purification of the recombinant porcine NK-lysin in Pichia pastoris and observation of anticancer activity in vitro.

Natural killer (NK)-lysin, a broad-spectrum antimicrobial peptide, has antitumor and antibactericidal activities against both gram-positive and gram-negative bacteria. In this study the recombinant porcine NK-lysin was expressed and purified in the Pichia pastoris system, and then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to assess its anticancer activity in vitro. The results showed that the recombinant porcine NK-lysin possesses potent antitumor activity against the human hepatocellular carcinoma cell line SMMC-7721 in a time- and dose-dependent manner, but has negligible hemolysis activity against human erythrocytes. Scanning electronic microscopy was used to directly observe the ultrastructure of SMMC-7721 cells treated with NK-lysin; untreated cells showed lamellipodia and filopodia scattered with the cell surface, with good cell-cell contacts among neighboring cells. In contrast, treated tumor cells exhibited marked alterations in cell morphology, and cell-cell contacts disappeared among neighboring cells. Compared with the untreated tumor cells, the tumor cells treated with NK-lysin for 12 and 24 hr were suppressed for the expression of fascin 1. Thus, the recombinant porcine NK-lysin potentially could be developed as a therapeutic agent for inhibiting tumor growth.

Oleanolic acid derivatives inhibit the Wnt/ß-catenin signaling pathway by promoting the phosphorylation of ß-catenin in human SMMC-7721 cells.

Oleanolic acid, isolated from privet, has shown antitumor effects in several cancers. However, the underlying molecular mechanism associated with these effects is largely unknown. In this study, we explored the effect of oleanolic acid derivatives on the Wnt/ß-catenin signaling pathway in human hepatocellular carcinoma SMMC-7721 cells. The mRNA and protein levels of related genes were determined by real-time quantitative PCR and Western blot, respectively. Treatment of SMMC-7721 cells with oleanolic acid derivatives led to the downregulation of the mRNA and protein levels of ß-catenin, c-myc, and cyclin D1. Treatment with oleanolic acid derivatives decreased the levels of ß-catenin in both the cytoplasm and the nucleus. Moreover, oleanolic acid derivatives promoted the phosphorylation of ß-catenin (Ser33/37/Thr41) in the cytoplasm. Our results suggest that oleanolic acid derivatives inhibit the Wnt/ß-catenin signaling pathway by stimulating the phosphorylation of ß-catenin (Ser33/37/Thr41) in human SMMC-7721 cells.

The immune adherence receptor CR1-like existed on porcine erythrocytes membrane.

In the present study, we obtain a mouse anti-porcine complement receptor type 1 (CR1)-like monoclonal antibody (McAb) and use this McAb to verify the existence of CR1-like protein on porcine erythrocytes. Our results confirm that CR1-like protein is localized on the surface of porcine erythrocytes. Mouse immunoglobulin G inhibited the binding of serum-opsonized green fluorescent protein-expressing Escherichia coli to porcine erythrocytes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicates that CR1-like McAb reacts with biochemically-purified porcine erythrocyte membrane fractions, with a clear band at 135 kDa to 140 kDa. We postulate that the 135 kDa to 140 kDa membrane protein is the equivalent of the porcine erythrocyte CR1-like protein.

Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors.

Hepatocyte growth factor (HGF)/c-Met signaling has been considered as a key pathway in both melanocyte development and melanogenesis. To understand better the expression patterns and tissue distribution characterization of HGF and its receptor c-Met in skin of white versus brown alpaca (Vicugna pacos), we detected the tissue distribution of HGF and c-Met using immunohistochemistry and analyzed the expression patterns by using Western blot and quantitative real time PCR (qPCR). Immunohistochemistry analysis demonstrated that HGF staining robustly increased in the dermal papilla and mesenchymal cells of white alpaca skin compared with that of brown. However, c-Met staining showed strongly positive result, particularly inhair matrix and root sheath in brown alpaca skin. Western blot and qPCR results suggested that HGF and c-Met were expressed at significantly high levels in white and brown alpaca skins, respectively, and protein and transcripts possessed the same expression pattern in white and brown alpaca skins. The results suggested that HGF/c-Met signaling functions in alpaca coat color formation offer essential theoretical basis for further exploration of the role of HGF/c-Met signaling in pigment formation.

Expression and purification of soluble porcine cystatin 11 in Pichia pastoris.

Cystatin 11 (CST11) belongs to the cystatin type 2 family of cysteine protease inhibitors and exhibits antimicrobial activity in vitro. In this study, we describe the expression and purification of recombinant porcine CST11 in the Pichia pastoris system. We then assess its antimicrobial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis by liquid growth inhibition assay. Kinetic studies indicate that the recombinant porcine CST11 has high potency against E. coli and S. aureus. Scanning electronic microscope analysis showed that CST11 might be targeting the bacterial membrane and, thus, could potentially be developed as a therapeutic agent for inhibiting microbe infection without the risk of antibiotic resistance.

Screening compounds of Chinese medicinal herbs anti-Marek's disease virus.

CONTEXT: Marek's disease (MD) seriously threatens the world poultry industry and has resulted in great economic losses. Chinese medicinal herbs are a rich source for lead compounds and drug candidates for antiviral treatments. OBJECTIVE: To investigate the anti-MDV activity and mechanism of 20 compounds extracted from Chinese medicinal herbs. MATERIALS AND METHODS: Antiviral assay, time of addition experiments, and virucidal assay were performed on chicken embryo fibroblast cells. The 50% cytotoxic concentration and 50% effective concentration were determined and, accordingly, selectivity index and inhibition ratio were calculated. RESULTS: Antiviral assay showed dipotassium glycyrrhizinate (DG) and sodium tanshinone IIA sulfonate (STS) exhibited significantly inhibitory activity against MDV in a dose-dependent manner. EC50 of DG and STS were 893.5 ± 36.99 µg/mL and 54.82 ± 2.99 µg/mL, and selective index (SI) were >3.36 and >9.12, respectively. Time of addition experiment and virucidal assay demonstrated DG inhibited viral replication in the full replication cycle and inactivated MDV particles in non-time-dependent manner, but STS interfered with the early stage of MDV replication and inactivated MDV particles in a time-dependent manner. Moreover, both DG and STS promoted apoptosis of cells infected by MDV. DISCUSSION AND CONCLUSION: DG and STS have great potential for developing new anti-MDV drugs for clinic application.

Sodium tanshinone IIA sulfonate inhibits the meq, ul49 and VP22 expression of Marek's disease virus.

BACKGROUND: Our previous studies have demonstrated that sodium tanshinone IIA sulfonate (STS), a natural compound derived from Salviae Miltiorrhizae Radix et Rhizoma (Danshen), could effectively inhibit Marek's disease virus (MDV) infection both in vitro and in vivo, but the underlying mechanisms remain unclear. The main objective of the study was to explore the effect of STS on the meq, ul49 and VP22 expression of MDV in vitro. METHODS: Quantitative real-time PCR (qRT-PCR) was used to analyse the effect of STS on meq and ul49 expression at both the DNA and messenger RNA (mRNA) level, and the effect of STS on VP22 was assessed by immunofluorescence assay and western blotting. RESULTS: The DNA and mRNA copy numbers of meq and ul49 significantly decreased in the groups treated with STS compared with MDV control (P<0.05), which indicated that STS could inhibit the expression of meq and ul49 at both the DNA and mRNA level. Moreover, the expression of VP22 encoded by ul49 was also significantly inhibited (P<0.05). CONCLUSIONS: STS possessed anti-MDV activity in chicken embryo fibroblasts. Its antiviral mechanisms may be ascribed to inactivating MDV directly, disturbing meq and ul49 replication and inhibiting the expression of VP22 encoded by ul49. These results suggested that STS is a promising natural compound to be further developed as an antiviral agent against MDV infection.

In vitro antiviral activity and underlying molecular mechanisms of dipotassium glycyrrhetate against porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) has caused large economic losses in the swine industry. Currently, there is no effective way to prevent PRRSV infection. In this study, we investigated the inhibitory effect of dipotassium glycyrrhetate (DG), a derivative of glycyrrhetinic acid, on PRRSV infection ability. METHODS: The cytotoxicity of DG was measured by MTT assay, and the effects of DG on PRRSV N gene/protein were investigated using real-time PCR, western blot and immunofluorescence assay. In addition, the effect of DG on cell apoptosis was analysed by fluorescence staining. RESULTS: Our results indicated that DG could effectively inhibit virus replication and N gene expression in MARC-145 cells infected with PRRSV. When the infected cells received DG, the numbers of apoptotic cells were decreased, and the cleaved caspase-3 contents were decreased dramatically. CONCLUSIONS: Our study demonstrates that DG could effectively inhibit the PRRS virus via multiple pathways including inhibition of virus replication and N gene expression and reduction of apoptotic cells. DG can serve as a potential chemical for PRRSV prevention and control.

In vitro screening for compounds derived from traditional chinese medicines with antiviral activities against porcine reproductive and respiratory syndrome virus.

Seventeen compounds derived from traditional Chinese medicines (TCMs) were tested for their antiviral activity against porcine reproductive and respiratory syndrome virus (PRRSV) in vitro. Visualization with the cytopathologic effect (CPE) assay and the 3-(4, 5-dimethyithiazol- 2-yl)-2,5-diphenyltetrazolium bromide test were used to determine the 50% cytotoxic concentration (CC50) and 50% effective concentration (EC50) in cultured Marc-145 cells. Among the tested compounds, chlorogenic acid and scutellarin showed potential anti-PRRSV activity. The EC50 values were 270.8 ± 14.6 µg/ml and 28.21 ± 26.0 µg/ml and the selectivity indexes were >5.54 and 35.5, respectively. The time-of-addition and virucidal assay indicated that the anti-PRRSV activity of the two compounds could be due to their inhibiting the early stage of virus replication and/or inactivating the virus directly. The inhibition of the virus attachment was not observed in the adsorption inhibition assay. The inhibition ratios of chlorogenic acid and scutellarin were, respectively, 90.8% and 61.1% at the maximum non-cytotoxic concentrations. The results have provided a basis for further exploration of their antiviral properties and mechanisms in vivo. We believe that the chlorogenic acid and scutellarin have a great potential to be developed as new anti-PRRSV drugs for clinical application.

Antiviral effects of the constituents derived from Chinese herb medicines on infectious bursal disease virus.

CONTEXT: The prevalence of infectious bursal disease has brought about enormous financial losses to the world poultry industry. Chinese herb medicines can provide valuable materials for discovery and development of new drugs. OBJECTIVE: To screen constituents derived from Chinese herb medicines for their antiviral activity against infectious bursal disease virus (IBDV) in vitro. MATERIALS AND METHODS: Twenty constituents derived from Chinese herb medicines and B87 strain of IBDV were used. The 50% cytotoxic concentration (CC50) and 50% effective concentration (EC50) were determined by visualization of cytopathologic effect (CPE) and 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) test on chicken embryo fibroblast. Selectivity index (SI) and inhibition ratio (%I) were calculated from the data obtained from the MTT test. RESULTS: Antiviral assays showed dipotassium glycyrrhizinate and ligustrazine hydrochloride among the 20 constituents tested exhibited significant inhibitory activity against IBDV in a dose-dependent manner. EC50 of dipotassium glycyrrhizinate and ligustrazine hydrochloride were 663.2 ± 268.4 and 92.52 ± 21.13 µg/mL, and SI were >4.52 and >21.62, respectively. The time-of-addition and virucidal assay indicated that anti-IBDV activity of the two constituents could be due to their inhibiting virus replication and/or inactivating virus directly. The inhibition of virus attachment was not observed in the adsorption inhibition assay. Dipotassium glycyrrhizinate and ligustrazine hydrochloride exhibited more than 70% and 80% inhibition of IBDV, respectively, at the maximum safe concentration. DISCUSSION AND CONCLUSION: We believe that dipotassium glycyrrhizinate and ligustrazine hydrochloride can be used to develop a new anti-IBDV compound, and it is worth applying the constituents in clinical practice.

The cytological observation of immune adherence of porcine erythrocyte.

The immune adherence (IA) between the porcine erythrocytes and the opsonized Escherichia coli carried green fluorescent protein gene (GFP-E.coli) were detected by the fluorescence microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) with an attempt to verify the existence of IA between the porcine erythrocytes and complemented-opsonized microbes. Under fluorescence microscopy, GFP-E.coli opsonized by fresh rabbit serum complement adhered to the erythrocytes and could not be detached by PBS washing, and no IA was observed between the erythrocytes and nonopsonized GFP-E.coli after co-incubation. SEM and TEM also revealed the existence of IA between the serum complement-opsonized GFP-E.coli membrane and the erythrocyte membrane. The partial complement receptor type 1 (CR1)-like gene from porcine was generated by RT-PCR and rapid amplification of cDNA 3' end (3' RACE) (157bp and 578bp), both of which have high similarity with published mammal's CR1 gene. The sequences were spliced based on homology comparison and submitted to GenBank (GenBank Accession No. JX033989). These results indicated that the porcine erythrocytes were able to bind to the opsonized microorganisms. Furthermore, the sequencing results confirmed that the CR1-like gene exists in porcine.

Identification and characterization of microRNAs in white and brown alpaca skin.

UNLABELLED: AB BACKGROUND: MicroRNAs (miRNAs) are small, non-coding 21-25 nt RNA molecules that play an important role in regulating gene expression. Little is known about the expression profiles and functions of miRNAs in skin and their role in pigmentation. Alpacas have more than 22 natural coat colors, more than any other fiber producing species. To better understand the role of miRNAs in control of coat color we performed a comprehensive analysis of miRNA expression profiles in skin of white versus brown alpacas. RESULTS: Two small RNA libraries from white alpaca (WA) and brown alpaca (BA) skin were sequenced with the aid of Illumina sequencing technology. 272 and 267 conserved miRNAs were obtained from the WA and BA skin libraries, respectively. Of these conserved miRNAs, 35 and 13 were more abundant in WA and BA skin, respectively. The targets of these miRNAs were predicted and grouped based on Gene Ontology and KEGG pathway analysis. Many predicted target genes for these miRNAs are involved in the melanogenesis pathway controlling pigmentation. In addition to the conserved miRNAs, we also obtained 22 potentially novel miRNAs from the WA and BA skin libraries. CONCLUSION: This study represents the first comprehensive survey of miRNAs expressed in skin of animals of different coat colors by deep sequencing analysis. We discovered a collection of miRNAs that are differentially expressed in WA and BA skin. The results suggest important potential functions of miRNAs in coat color regulation.