A novel C-type lectin from Crassostrea gigas involved in the innate defense against Vibrio alginolyticus.

C-type lectins (CTLs) are important immune molecules that participate in invertebrate defense response. In the present work, a novel structural CTL (CgLec-4E) was identified from Crassostrea gigas, which encodes 237 amino acids (aa) with an extra long chain of aa and in the C-type CRD domain with EPA, QPG and WHD mutated motifs respectively. rCgLec-4E could agglutinate and inhibit the growth of Vibrio alginolyticus, except Chlorella, which might be relevant to three mutated motifs. CgLec-4E was mainly expressed in digestive gland, and its expression level was significantly up-regulated post V. alginolyticus challenge, indicating that the high expression of CgLec-4E could provide necessary mucosal immune protections and might involve in food particle recognition for C. gigas. Moreover, the subcellular locations indicated that CgLec-4E might play different roles in the immune response. Taken together, our results enrich our understanding of the structures and function of CTLs in invertebrates.

A simple QD-FRET bioprobe for sensitive and specific detection of hepatitis B virus DNA.

We report here a simple quantum dot-FRET (QD-FRET) bioprobe based on fluorescence resonance energy transfer (FRET) for the sensitive and specific detection of hepatitis B virus DNA (HBV DNA). The proposed one-pot HBV DNA detection method is very simple, rapid and convenient due to the elimination of the washing and separation steps. In this study, the water-soluble CdSe/ZnS QDs were prepared by replacing the trioctylphosphine oxide on the surface of QDs with mercaptoacetic acid (MAA). Subsequently, DNA was attached to QDs surface to form the functional QD-DNA bioconjugates by simple surface ligand exchange. After adding 6-carboxy-X-rhodamine (ROX)-modified HBV DNA (ROX-DNA) into the QD-DNA bioconjugates solution, DNA hybridization between QD-DNA bioconjugates and ROX-DNA was formed. The resulting hybridization brought the ROX fluorophore, the acceptor, and the QDs, the donor, into proximity, leading to energy transfer from QDs to ROX. When ROX-DNA was displaced by the unlabeled HBV DNA, the efficiency of FRET was dramatically decreased. Based on the changes of both fluorescence intensities of QDs and ROX, HBV DNA could be detected with high sensitivity and specificity. Under the optimized conditions, the linear range of HBV DNA determination was 2.5 - 30 nmol L(-1), with a correlation coefficient (R) of 0.9929 and a limit of detection (3σ black) of 1.5 nmol L(-1). The relative standard deviation (R.S.D.) for 12 nmol L(-1) HBV DNA was 0.9% (n = 5). There was no interference to non-complementary DNA. Time-resolved fluorescence spectra and fluorescence images were performed to verify the validity of this method and the results were satisfying.

Systematic investigation of interactions between papain and MPA-capped CdTe quantum dots.

Fluorescent quantum dots (QDs) have been widely applied in biological and biomedical areas, but relatively little is known about the interaction of QDs with some natural enzymes. Herein, the interactions between 3-mercaptopropionic acid-capped CdTe QDs (MPA-QDs) and papain were systematically investigated by UV-Vis absorption spectra, fluorescence spectra and circular dichroism (CD) spectra under the physiological conditions. The fluorescence spectra results indicated that MPA-QDs quenched the fluorescence intensity of papain. The modified Stern-Volmer quenching constant K a at different temperatures and the corresponding thermodynamic parameters ΔH, ΔG and ΔS were also calculated. The binding of MPA-QDs and papain is a result of the formation of QDs-papain complex and the electrostatic interactions play a major role in stabilizing the complex. The CD technique was further used to analyze the conformational changes of papain induced by MPA-QDs and the results indicated that the biological activity of papain was affected by MPA-QDs dramatically.

A novel toll-like receptor from Crassostrea gigas is involved in innate immune response to Vibrio alginolyticus.

Based on previous reports，toll-like receptors (TLRs) are recognition molecules common in various aquatic animals and play a vital role in innate immunity. In this study, a novel TLR CgToll-3 with leucine-rich repeats (LRRs) and a TIR (Toll-interleukin 1-resistance) domain was cloned in Crassostrea gigas. CgToll-3 with sixteen potential extracellular N-linked glycosylation sites and shares the closest phylogenic relationship with molluscan TLRs. Alignment of LRRs and TIR domains indicated that CgToll-3 was highly conserved compared to other LRRs of mollusks which could respond against Vibrio or other bacterial molecules, and contained three conserved functionally important motifs (Box 1, Box 2, and Box 3). The Hex Molecular Docking result showed that CgToll-3 could interact with CgMyd88 via the TIR domain. Subcellular Co-localization and BiFC Assay confirmed this interaction, and they could induce NF-κB activation. CgToll-3 was moderately expressed in the digestive gland, and its expression level was significantly up-regulated after Vibrio alginolyticus challenge. Taken together, CgToll-3 might be involved in the innate immune response to V. alginolyticus for C. gigas through a MyD88-dependent TLR mediated signaling pathway.

Systematical investigation of in vitro interaction of InP/ZnS quantum dots with human serum albumin by multispectroscopic approach.

Cadmium-free quantum dots (QDs) have attracted great attention in biological and biomedical applications due to their less content of toxic metals, but their potential toxicity investigations on molecular biology level are rarely involved. Since few studies have addressed whether InP/ZnS QDs could bind and alter the structure and function of human serum albumin (HSA), in vitro interaction between InP/ZnS QDs and HSA was systematically characterized by multispectroscopic approaches. InP/ZnS QDs could quench the intrinsic fluorescence of HSA via static mode. The binding site of InP/ZnS QDs was mainly located at subdomain IIA of HSA. Some thermodynamic parameters suggested that InP/ZnS QDs interacted with HSA mainly through electrostatic interactions. As further revealed by three-dimensional spectrometry, FT-IR spectrometry and circular dichroism technique, InP/ZnS QDs caused more global and local conformational change of HSA than CdSe/ZnS QDs, which illustrated the stronger binding interaction and higher potential toxicity of InP/ZnS QDs on biological function of HSA. Our results offer insights into the in vitro binding mechanism of InP/ZnS QDs with HSA and provide important information for possible toxicity risk of these cadmium-free QDs to human health.

Study on the molecular interaction of graphene quantum dots with human serum albumin: combined spectroscopic and electrochemical approaches.

Graphene quantum dots (GQDs) have attracted great attention in biological and biomedical applications due to their super properties, but their potential toxicity investigations are rarely involved. Since few studies have addressed whether GQDs could bind and alter the structure and function of human serum albumin (HSA), the molecular interaction between GQDs and HSA was systematically characterized by the combination of multispectroscopic and electrochemical approaches. GQDs could quench the intrinsic fluorescence of HSA via static mode. The competitive binding fluorescence assay revealed that the binding site of GQDs was site I of HSA. Some thermodynamic parameters suggested that GQDs interacted with HSA mainly through van der Waals interactions and hydrogen bonding interactions, and protonation might also participate in the process. As further revealed by FT-IR spectroscopy and circular dichroism technique, GQDs could cause the global and local conformational change of HSA, which illustrated the potential toxicity of GQDs that resulted in the structural damage of HSA. Electrochemical techniques demonstrated the complex formation between GQDs and HSA. Our results offered insights into the binding mechanism of GQDs with HSA and provided important information for possible toxicity risk of GQDs to human health.

Molecular interaction investigation between three CdTe:Zn(2+) quantum dots and human serum albumin: A comparative study.

Water-soluble Zn-doped CdTe quantum dots (CdTe:Zn(2+) QDs) have attracted great attention in biological and biomedical applications. In particular, for any potential in vivo application, the interaction of CdTe:Zn(2+) QDs with human serum albumin (HSA) is of greatest importance. As a step toward the elucidation of the fate of CdTe:Zn(2+) QDs introduced to organism, the molecular interactions between CdTe:Zn(2+) QDs with three different sizes and HSA were systematically investigated by spectroscopic techniques. Three CdTe:Zn(2+) QDs with maximum emission of 514 nm (green QDs, GQDs), 578 nm (yellow QDs, YQDs), and 640 nm (red QDs, RQDs) were tested. The binding of CdTe:Zn(2+) QDs with HSA was a result of the formation of HSA-QDs complex and electrostatic interactions played major roles in stabilizing the complex. The Stern-Volmer quenching constant, associative binding constant, and corresponding thermodynamic parameters were calculated. The site-specific probe competitive experiments revealed that the binding location of CdTe:Zn(2+) QDs with HSA was around site I. The microenvironmental and conformational changes of HSA induced by CdTe:Zn(2+) QDs were analyzed. These results suggested that the conformational change of HSA was dramatically at secondary structure level and the biological activity of HSA was weakened in the present of CdTe:Zn(2+) QDs with bigger size.

A sensitive quantum dots-based "OFF-ON" fluorescent sensor for ruthenium anticancer drugs and ctDNA.

In this contribution, a simple and sensitive fluorescent sensor for the determination of both the three ruthenium anticancer drugs (1 to 3) and calf thymus DNA (ctDNA) was established based on the CdTe quantum dots (QDs) fluorescence "OFF-ON" mode. Under the experimental conditions, the fluorescence of CdTe QDs can be effectively quenched by ruthenium anticancer drugs because of the surface binding of these drugs on CdTe QDs and the subsequent photoinduced electron transfer (PET) process from CdTe QDs to ruthenium anticancer drugs, which render the system into fluorescence "OFF" status. The system can then be "ON" after the addition of ctDNA which brought the restoration of CdTe QDs fluorescence intensity, since ruthenium anticancer drugs broke away from the surface of CdTe QDs and inserted into double helix structure of ctDNA. The fluorescence quenching effect of the CdTe QDs-ruthenium anticancer drugs systems was mainly concentration dependent, which could be used to detect three ruthenium anticancer drugs. The limits of detection were 5.5 × 10(-8) M for ruthenium anticancer drug 1, 7.0 × 10(-8) M for ruthenium anticancer drug 2, and 7.9× 10(-8) M for ruthenium anticancer drug 3, respectively. The relative restored fluorescence intensity was directly proportional to the concentration of ctDNA in the range of 1.0 × 10(-8) M ∼ 3.0 × 10(-7) M, with a correlation coefficient (R) of 0.9983 and a limit of detection of 1.1 × 10(-9) M. The relative standard deviation (RSD) for 1.5 × 10(-7) M ctDNA was 1.5% (n = 5). There was almost no interference to some common chemical compounds, nucleotides, amino acids, and proteins. The proposed method was applied to the determination of ctDNA in three synthetic samples with satisfactory results. The possible reaction mechanism of CdTe QDs fluorescence "OFF-ON" was further investigated. This simple and sensitive approach possessed some potential applications in the investigation of interaction between drug molecules and DNA.