Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method

BACKGROUND: Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. Traditional detection methods including polymerase chain reaction (PCR)-based assays and enzyme-linked immunosorbent assays (ELISA) are expensive and time-consuming. In our assay, a protein chip assay using Nano-gold Immunological Amplification and Silver Staining (NIASS) method was applied to detect HBV and HCV antibodies rapidly and simultaneously. METHODS: Chemically modified glass slides were used as solid supports (named chip), on which several antigens, including HBsAg, HBeAg, HBcAg and HCVAg (a mixture of NS3, NS5 and core antigens) were immobilized respectively. Colloidal nano-gold labelled staphylococcal protein A (SPA) was used as an indicator and immunogold silver staining enhancement technique was applied to amplify the detection signals, producing black image on array spots, which were visible with naked eyes. To determine the detection limit of the protein chip assay, a set of model arrays in which human IgG was spotted were structured and the model arrays were incubated with different concentrations of anti-IgG. A total of 305 serum samples previously characterized with commercial ELISA were divided into 4 groups and tested in this assay. RESULTS: We prepared mono-dispersed, spherical nano-gold particles with an average diameter of 15 ± 2 nm. Colloidal nano-gold-SPA particles observed by TEM were well-distributed, maintaining uniform and stable. The optimum silver enhancement time ranged from 8 to 12 minutes. In our assay, the protein chips could detect serum antibodies against HBsAg, HBeAg, HBcAg and HCVAg with the absence of the cross reaction. In the model arrays, the anti-IgG as low as 3 ng/ml could be detected. The data for comparing the protein chip assay with ELISA indicated that no distinct difference (P > 0.05) existed between the results determined by our assay and ELISA respectively. CONCLUSION: Results showed that our assay can be applied with serology for the detection of HBV and HCV antibodies rapidly and simultaneously in clinical detection

Advances in understanding cartilage remodeling

Cartilage remodeling is currently among the most popular topics in osteoarthritis research. Remodeling includes removal of the existing cartilage and replacement by neo-cartilage. As a loss of balance between removal and replacement of articular cartilage develops (particularly, the rate of removal surpasses the rate of replacement), joints will begin to degrade. In the last few years, significant progress in molecular understanding of the cartilage remodeling process has been made. In this brief review, we focus on the discussion of some current “controversial” observations in articular cartilage degeneration: (1) the biological effect of transforming growth factor-beta 1 on developing and mature articular cartilages, (2) the question of whether aggrecanase 1 (ADAMTS4) and aggrecanase 2 (ADAMTS5) are key enzymes in articular cartilage destruction, and (3) chondrocytes versus chondron in the development of osteoarthritis. It is hoped that continued discussion and investigation will follow to better clarify these topics. Clarification will be critical for those in search of novel therapeutic targets for the treatment of osteoarthritis

Roles of TGF-beta 1 signaling in the development of osteoarthritis

Osteoarthritis (OA) is a degenerative joint disorder characterized by the destruction of articular cartilage, subchondral bone and other joint tissues. Although multiple growth factors and cytokines have been shown to be involved in articular cartilage degeneration and subchondral bone destruction, which eventually leads to OA, the molecular mechanisms underlying the pathogenesis of OA are largely unknown. The canonical transforming growth factor beta 1 (TGFβ1) signaling functions as one of the key factors in cartilage and bone formation, remodeling, and maintenance. However, the effects of TGF-β1 signaling on the development of OA are unclear. Numerous studies provide evidence that TGF-β1 is required for the formation of articular cartilage at early stages of joint development. In contrast, other investigations indicate that TGF-β1 may, in fact, be a factor in joint destruction. Therefore, we, in this review article, discuss the “conflicting” roles of TGF-β1 signaling in the development of OA

Primary structure of the long and short splice variants of mouse collagen XII and their tissue‐specific expression during embryonic development

Type XII collagen, a member of the FACIT group of extracellular matrix proteins, consists of molecules that are trimers of α1(XII) chains. The three chains in each molecule form a cross‐shaped structure with a central globule from which a triple‐helical tail and three finger‐like regions (containing von Willebrand factor A‐like domains and fibronectin type III repeats) extend. cDNA cloning/sequencing of chicken α1(XII) collagen and protein studies with mouse, bovine, and human material suggest that the α1(XII) collagen gene gives rise to two molecular variants, differing in the length of the finger‐like regions, by alternative splicing of the primary transcript. To provide a basis for studies of the function of the two variants in an organism that can be genetically manipulated, we have isolated and sequenced mouse cDNAs encoding both splice variants. The sequence provides the first complete nucleotide and amino acid sequence of mammalian type XII collagen. From these cDNAs we have generated digoxigenin‐labeled RNA probes for in situ hybridization of developing mouse embryos to find out whether the splicing mechanism responsible for generation of the two forms is developmentally regulated. The results, combined with Northern blot and RT‐PCR analysis of RNA from embryos at various developmental stages, demonstrate that the long form of collagen XII, XIIA, is the predominant form at early stages (ED7 and 11); at later stages of development (ED15 and 17) the short form, XIIB, becomes the major form. As the short form becomes the major product, the long splice variant continues to be expressed in several tissues, even after birth. An exception is dermis, which is positive for the long form up to embryonic day 15, but negative at day 18, when only the short form RNA can be detected. © 1995 wiley‐Liss, Inc. Copyright © 1995 Wiley‐Liss, Inc

Induction of high temperature requirement A1, a serine protease, by TGF-beta1 in articular chondrocytes of mouse models of OA

The goal of this study is to determine whether transforming growth factor beta 1 (Tgf-ß1) induces the high temperature requirement A1 (HtrA1) in articular chondrocytes of two mouse models of osteoarthritis (OA). Early onset articular cartilage degeneration in the mouse models was characterized by histology. Expression profiles of Tgf-ß1, p-Smad1, pSmad2/3 and HtrA1 in knee joints of the mouse models were examined by immunofluorescene. By in vitro and ex vivo experiments, human primary chondrocytes and articular cartilages from femoral condyles of mice were treated with recombinant human TGF-ß1 and an ALK5 chemical inhibitor, SB431542. The level of HTRA1 mRNA in human and mouse articular chondrocytes was examined by real-time PCR. Chondrocyte clusters were present in the articular cartilage of knee joints in the mouse models. Increased expressions of Tgf-ß1, pSmad2/3 and HtrA1 were detected in the articular chondrocyte of knee joints in the mouse models. The increased expressions of p-Smad2/3 and HtrA1 were colocalized in the articular chondrocyte of the knee joints. The expression of p-Smad1 was hardly detectable in the mouse models and their corresponding wild-type littermates. The level of HTRA1 mRNA was increased in human and mouse articular chondrocytes treated with TGF-ß1, compared with that in chondrocytes without the treatment of TGF-ß1. The TGF-ß1-induced expression of HTRA1 in human and mouse articular chondrocytes was inhibited by SB431542. These results suggest that the Tgf-ß1 canonical signaling was activated to induce HtrA1 in the articular chondrocytes of the mouse models of OA

High lability of global niche and range in the Giant African Snail (Lissachatina fulica): Small niche expansions resulting in large range shifts

As a major source of global change, biological invasions have received increasing attention in past decades. Niche and range dynamics of alien invasive species provide essential evidence for understanding mechanisms underlying biological invasions. However, to our knowledge few relevant studies on mollusks have been reported. Here, from the perspective of climatic suitability, we investigated niche and range shifts of the Giant African Snail (Lissachatina fulica), one of the world’s most invasive mollusks. We observed that, compared with its native counterpart, this invasive snail could survive in environments with wider temperature ranges. The invasive snail showed a wider niche breadth and larger potential range than its native counterpart, and occupied different niche and range positions, indicating its high lability in terms of niche and range. This invasive Giant African Snail did not conform to the niche and range conservatism hypotheses, probably due to the influence of human introductions and niche releasing. Given that large shifts in range size can result from small niche expansions, making niche shifts a more important indicator for biological invasion assessments than range shifts. While most invasive species show low lability in niche space, highly labile niche was observed in the Giant African Snail, indicating that strategies to prevent biological invasions by this invasive mollusk should be prioritized. Our study, to certain extent, could further our understanding on the niche shifts and range dynamics between the invasive mollusks and their native counterparts

Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method

Abstract Background Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. Traditional detection methods including polymerase chain reaction (PCR)-based assays and enzyme-linked immunosorbent assays (ELISA) are expensive and time-consuming. In our assay, a protein chip assay using Nano-gold Immunological Amplification and Silver Staining (NIASS) method was applied to detect HBV and HCV antibodies rapidly and simultaneously. Methods Chemically modified glass slides were used as solid supports (named chip), on which several antigens, including HBsAg, HBeAg, HBcAg and HCVAg (a mixture of NS3, NS5 and core antigens) were immobilized respectively. Colloidal nano-gold labelled staphylococcal protein A (SPA) was used as an indicator and immunogold silver staining enhancement technique was applied to amplify the detection signals, producing black image on array spots, which were visible with naked eyes. To determine the detection limit of the protein chip assay, a set of model arrays in which human IgG was spotted were structured and the model arrays were incubated with different concentrations of anti-IgG. A total of 305 serum samples previously characterized with commercial ELISA were divided into 4 groups and tested in this assay. Results We prepared mono-dispersed, spherical nano-gold particles with an average diameter of 15 ± 2 nm. Colloidal nano-gold-SPA particles observed by TEM were well-distributed, maintaining uniform and stable. The optimum silver enhancement time ranged from 8 to 12 minutes. In our assay, the protein chips could detect serum antibodies against HBsAg, HBeAg, HBcAg and HCVAg with the absence of the cross reaction. In the model arrays, the anti-IgG as low as 3 ng/ml could be detected. The data for comparing the protein chip assay with ELISA indicated that no distinct difference (P > 0.05) existed between the results determined by our assay and ELISA respectively. Conclusion Results showed that our assay can be applied with serology for the detection of HBV and HCV antibodies rapidly and simultaneously in clinical detection.</p

SAP Binds to CD22 and Regulates B Cell Inhibitory Signaling and Calcium Flux

The signaling lymphocyte activation molecule (SLAM)-associated protein (SAP or SH2D1A) is an important regulator of immune function which, when mutated or deleted, causes the X-linked lymphoproliferative syndrome (XLP). Because B cell lymphoma is a major phenotype of XLP, it is important to understand the function of SAP in B cells. Here we report that SAP is expressed endogenously in mouse splenic B cells, is inducibly expressed in the human BJAB cells, and co-localizes and interacts with CD22. We also show that SAP binding to the inhibitory immunoreceptor CD22 regulates calcium mobilization in B cells. Moreover, forced expression of SAP leads to constitutive CD22 tyrosine phosphorylation and decreased Ca(2+) response in B cells. Biochemical analysis reveals that, in response to IgM cross-linking, the phosphorylation of Syk, Blnk, or PLCgamma2 and their interactions with one another were either diminished or completely abolished in SAP-expressing cells compared to cells that lack SAP. Collectively our work identifies a novel role for SAP in B cells and extends its function to inhibitory immunoreceptor signaling and calcium mobilization

Expression, purification and antimicrobial activity analysis of recombination peptide subtilosin A in Escherichia coli using SUMO fusion technology

138-144Subtilosin A is an antimicrobial peptide isolated from Bacillus subtilis, and it possess broad-spectrum antibacterial activity. As subtilosin A prevents biofilm formation by inhibiting bacterial quorum sensing processes against Gram-positive, Gram-negative, and Gram-variable bacteria, it is urgent to obtain high-quality subtilosin A production in an economical and effective way. Escherichia coli is undoubtedly a most preferred host system for production of heterologous recombinant proteins. However, subtilosin A has the inhibitory effects against E. coli and it is easily to be degraded. To produce subtilosin A in E. coli, the application of small ubiquitin-related modifier (SUMO) fusion system with the purpose of stability and prevention of antimicrobial activity is the best practices in this regard. In this study, subtilosin A gene with codon optimization was cloned into the Stu I/Hind III sites of pSUMO vector after SUMO tag and transformed to E. coli BL21 (DE3). The SUMO-subtilosin A fusion protein was induced at 15ºC for 16 hours with 0.5 mM isopropyl thio-β-D- galactoside (IPTG) induction. The fusion protein with a molecular weight of approximately 20 kDa was confirmed by SDS-PAGE. The subtilosin A was then released by SUMO protease cleavage at the junction site and isolated and purified by affinity and cation exchange chromatography. The recombinant subtilosin A demonstrated a good antibacterial activity against Pseudomonas aeruginosa (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria), with minimum inhibitory concentrations (MICs) of 100 mg/L and 100 mg/L, respectively. These results showed an efficient method for synthesis functional subtilosin A

Protective effects of the pericellular matrix of chondrocyte on articular cartilage against the development of osteoarthritis

Understanding the pathogenesis of osteoarthritis (OA) provides invaluable information in the search of therapeutic targets for the development of disease-modifying OA drugs. Emerging results from investigations demonstrate that the pericellular matrix of chondrocytes plays important roles in protecting articular cartilages from being degraded. Thus, maintaining the structural integrity of the pericellular matrix may be an effective approach to prevent the development of osteoarthritic joints. In this review article, we discuss the consequences of lacking one or more components of the pericellular matrix, and biological effects of the destruction of the pericellular matrix in the development of OA. We believe that more attention should be directed towards the pericellular matrix for the identification of novel biomarkers and therapeutic targets for the prevention and treatment of OA