A novel role of acellular hemoglobin in hemolytic thrombosis.

BACKGROUND: Hemolytic thrombosis has been associated with acellular hemoglobin released from damaged red blood cells during hemolysis. However, the precise molecular mechanism underlying acellular hemoglobin-induced thrombosis remains arguable. In this study, we examined the interaction between hemoglobin and the A1 domain of von Willebrand factor (VWF), which is a critical mediator of platelet activation. METHODS: Previous studies have suggested that the interaction between hemoglobin and the A1 domain of VWF enhances VWF's hemostatic activity. We employed a multidisciplinary investigation to re-examine this interaction, and identified significant differences in binding affinity between the active and inactive forms of A1. RESULTS: We found that hemoglobin binds more strongly to the active A1 than the inactive form. Using hydrogen­deuterium exchange mass spectrometry, we identified the specific residues involved in this interaction, which are located on the α1-ß2 and ß3-α2 loops that are typically covered by the "autoinhibitory module" in the inactive A1. This observation provides a structural explanation for the differential binding affinity between the active and inactive forms of A1. We demonstrated that the binding of hemoglobin to A1 blocks the interaction between GPIbα and VWF, and inhibits VWF-mediated thrombosis in vivo. Furthermore, we found that administration of hemoglobin led to similar levels of thrombocytopenia and microthrombosis in both wildtype and VWF-deficient mice, indicating that the mechanism underlying acellular hemoglobin-induced thrombosis is VWF-independent. CONCLUSIONS: These findings challenge the previous theory that hemoglobin-induced thrombosis occurs solely through binding with VWF, and provide evidence supporting a novel role for hemoglobin in hemolytic thrombosis.

Sema7A protects against high-fat diet-induced obesity and hepatic steatosis by regulating adipo/lipogenesis.

OBJECTIVE: Obesity and related diseases are becoming a growing risk for public health around the world due to the westernized lifestyle. Sema7A, an axonal guidance molecule, has been known to play a role in neurite growth, bone formation, and immune regulation. Whether Sema7A participates in obesity and metabolic diseases is unknown. As several SNPs in SEMA7A and its receptors were found to correlate with BMI and metabolic parameters in the human population, we investigated the potential role of Sema7A in obesity and hepatic steatosis. METHODS: GWAS and GEPIA database was used to analyze SNPs in SEMA7A and the correlation of Sema7A expression with lipid metabolism related genes. Sema7A-/- mice and recombinant Sema7A (rSema7A) were used to study the role of Sema7A in HFD-induced obesity and hepatic steatosis. Adipose tissue-derived mesenchymal stem cells (ADSCs) were used to examine the role of Sema7A in adipogenesis, lipogenesis and downstream signaling. RESULTS: Deletion of Sema7A aggravated HFD-induced obesity. Sema7A deletion enhanced adipogenesis in both subcutaneous and visceral ADSCs, while the addition of rSema7A inhibited adipogenesis of ADSCs and lipogenesis of differentiated mature adipocytes. Sema7A inhibits adipo/lipogenesis potentially through its receptor integrin ß1 and downstream FAK signaling. Importantly, administration of rSema7A had protective effects against diet-induced obesity in mice. In addition, deletion of Sema7A led to increased hepatic steatosis and insulin resistance in mice. CONCLUSIONS: Our findings reveal a novel inhibitory role of Sema7A in obesity and hepatic steatosis, providing a potential new therapeutic target for obesity and metabolic diseases.

Tissue dynamics of von Willebrand factor characterized by a novel fluorescent protein-von Willebrand factor chimera.

BACKGROUND: Tissue dynamics of von Willebrand factor (VWF) that are vital to its biological function have not been fully characterized. OBJECTIVE: To develop a new fluorescent protein--VWF chimera (FP-VWF) that has similar hematologic function to wild-type VWF and use it to monitor the tissue dynamics of VWF distribution. METHODS: Genotyping, platelet counting, tail bleeding time assay, agarose gels, western blot, platelet aggregation, proteolytic analysis, and ELISA were applied in characterizing the function of FP-VWF; fluorescence spectrometer and confocal fluorescence microscope were used to monitor the plasma and tissue distribution of FP-VWF. RESULTS: The transgenic mice that carry the FP-VWF retain hematologic activity of VWF with plasma levels of FP-VWF reduced by 50% and there are reduced high molecular weight FP-VWF multimers compared to the wild-type mice. The GPIb-binding and ADAMTS-13 (A Disintegrin and Metalloprotease with ThrombSpondin type 1 motif, member 13) proteolytic efficiency of FP-VWF are similar to wild-type VWF. The tissue distribution of FP-VWF was probed directly through its intrinsic fluorescence at normal or stimulated status, which indicated that the medicine-stimulated endogenous FP-VWF seems primarily released from the aorta and cleared in the spleen. Similar results were observed in non-fluorescent mice through a standard immunofluorescence approach. The fluorescence signals of FP-VWF were also similar to the standard dye-based approach in detecting the FeCl3 -induced blood clotting in vivo. CONCLUSIONS: Together, these results suggest that this novel FP-VWF chimera is valuable in probing the tissue dynamics of VWF in quite a few biological and pharmaceutical applications.

Imaging sensor array coupled with dual-signal amplification strategy for ultrasensitive chemiluminescence immunoassay of multiple mycotoxins.

A novel chemiluminescence (CL) immunosensor array in combination with a dual-signal amplification strategy was developed for rapid and ultrasensitive detection of multiple mycotoxins in herbal medicine. The multi-component immunosensor array was constructed by immobilizing different bovine serum albumin combined mycotoxins on the corresponding sites of the aldehyde-modified glass slide. After competitive immunoreactions, CL triggered by signal tags captured on all the sensing sites can be collected by a charge-coupled device simultaneously for joint detection of several mycotoxins. By assembling a high ratio of horseradish peroxidase (HRP) and IgG on the surface of gold nanoparticles (AuNPs), a biofunctionalized complex named HRP@AuNP-IgG was prepared and served as the primary signal tag to amplify the CL signals. Then, by introducing tyramine signal amplification (TSA) technique, a new round of HRP could deposit around the HRP@AuNP-IgG, which brought the secondary CL amplification. As a proof of concept, the CL imaging sensor array was applied to the trace detection of citrinin, aflatoxin B1 and ochratoxins A in red yeast rice samples. Under optimal conditions, it exhibited wide linear ranges over 4 orders of magnitude and much lower limits of detection than previous works. Owing to the excellent sensitivity (50-57-fold signal amplification and detection limits down to sub-pM level), acceptable throughput (20 tests h-1), small amounts of reagents (3.5 µL for each test), simple sample pretreatment (no necessary of separation for 3 mycotoxins), high selectivity, acceptable stability and accuracy, the proposed sensing platform showed broad prospects in the joint monitoring of low-abundant mycotoxins and safety evaluation of herbal medicines.

Porous TiO2 Film Immobilized with Gold Nanoparticles for Dual-Polarity SALDI MS Detection and Imaging.

Surface-assisted laser desorption/ionization (SALDI) mass spectrometry (MS) has become an attractive complementary approach to matrix-assisted laser desorption/ionization (MALDI) MS. SALDI MS has great potential for the detection of small molecules because of the absence of applied matrix. In this work, a functionalized porous TiO2 film immobilized with gold nanoparticles (AuNPs-FPTDF) was prepared to enhance SALDI MS performance. The porous TiO2 films were prepared by the facile sol-gel method and chemically functionalized for dense loading of AuNPs. The prepared AuNPs-FPTDF showed superior performance in the detection and imaging of small molecules in dual-polarity modes, with high detection sensitivity in the low pmol range, good repeatability, and low background noise compared to common organic MALDI matrixes. Its usage efficiently enhanced SALDI MS detection of various small molecules, such as amino acids and neurotransmitters, fatty acids, saccharides, alkaloids, and flavonoids, as compared with α-cyano-4-hydroxycinnamic acid, 9-aminoacridine, and the three precursor substrates of AuNPs-FPTDF. In addition, the blood glucose level in rats was successfully determined from a linearity concentration range of 0.5-9 mM, as well as other biomarkers in rat serum with SALDI MS. More importantly, the spatial distribution of metabolites from the intact flowers of the medicinal plant Catharanthus roseus was explored by using the AuNPs-FPTDF as an imprint SALDI MS substrate in dual-polarity modes. These results demonstrate wide applications and superior performances of the AuNPs-FPTDF as a multifunctional SALDI surface with enhanced detection sensitivity and imaging capabilities.

Visualization of Latent Fingermarks by Enhanced Chemiluminescence Immunoassay and Pattern Recognition.

Herein we report the combination of enzyme-linked immunoassay and pattern recognition analysis for extracting both chemical and spatial information from latent fingermarks (LFMs). The development approach basically involves two steps, namely, specific recognition of protein and polypeptide secretions present in the ridge residues of LFMs by horseradish peroxidase (HRP)-labeled antibodies and the HRP-catalyzed chemiluminescent (CL) reaction between luminol and H2O2. The emitted light can spatially resolve the ridges, generating a bright image against the dark object surface for visualization of an LFM. Meanwhile, thanks to the molecular specificity of the immunoassay step, the emission also provides us additional information on the existence of specific substances in LFMs. The developed LFMs are further processed by a set of digital image processing procedures. Quantitative analysis based on minutia features shows that even poorly developed fingermarks can be matched successfully. This work offers the promise of facilitating cross-disciplinary studies between data-processing approaches and fingermark development techniques, such as the extraction of more information from LFM evidence, as well as the establishment of evaluation criteria for an enhancement technique.

Vascular Semaphorin 7A Upregulation by Disturbed Flow Promotes Atherosclerosis Through Endothelial ß1 Integrin.

OBJECTIVE: Accumulating evidence suggests a role of semaphorins in vascular homeostasis. Here, we investigate the role of Sema7A (semaphorin 7A) in atherosclerosis and its underlying mechanism. APPROACH AND RESULTS: Using genetically engineered Sema7A-/-ApoE-/- mice, we showed that deletion of Sema7A attenuates atherosclerotic plaque formation primarily in the aorta of ApoE-/- mice on a high-fat diet. A higher level of Sema7A in the atheroprone lesser curvature suggests a correlation of Sema7A with disturbed flow. This notion is supported by elevated Sema7A expression in human umbilical venous endothelial cells either subjected to oscillatory shear stress or treated with the PKA (protein kinase A)/CREB (cAMP response element-binding protein) inhibitor H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl hydrate). Further studies using the partial carotid artery ligation model showed that disturbed flow in the left carotid artery of Sema7A+/+ApoE-/- mice promoted the expression of endothelial Sema7A and cell adhesion molecules, leukocyte adhesion, and plaque formation, whereas such changes were attenuated in Sema7A-/-ApoE-/- mice. Further studies showed that blockage of ß1 integrin, a known Sema7A receptor, or inhibition of FAK (focal adhesion kinase), MEK1/2 (mitogen-activated protein kinase kinase 1/2), or NF-κB (nuclear factor-κB) significantly reduced the expression of cell adhesion molecules and THP-1 (human acute monocytic leukemia cell line) monocyte adhesion in Sema7A-overexpressing human umbilical venous endothelial cells. Studies using chimeric mice suggest that vascular, most likely endothelial, Sema7A plays a major role in atherogenesis. CONCLUSIONS: Our findings indicate a significant role of Sema7A in atherosclerosis by mediating endothelial dysfunction in a ß1 integrin-dependent manner.

A simple microdroplet chip consisting of silica nanochannel-assisted electrode and paper cover for highly sensitive electrochemiluminescent detection of drugs in human serum.

This paper describes a simple and miniaturized microdroplet chip (µchip) that is constructed with a silica nanochannel (SNC)-assisted electrode array and a hydrophobic paper cover (SNC&P-µchip). Vertically aligned SNCs with uniform pore size of 2-3 nm in diameter and negatively charged surface can significantly accelerate the mass transport of the positively charged tris(2,2'-bipyridyl) ruthenium (II), resulting in a remarkably enhanced electrochemiluminescence (ECL) signal. The SNC-assisted electrode array was coupled to a low cost paper cover to achieve simultaneous detection of six samples in 1 min. The feasibility and universality of the SNC&P-µchip was evaluated by detecting a series of alkaloidal drugs both in buffers and in human serum. The performance of the SNC&P-µchip was fully validated with respect to linearity (0.9999 > R > 0.9939), sensitivity (limits of detection from 1.799 nM to 11.43 nM), and accuracy (recovery rate between 94.38% and 109.12%). The facile and economic SNC&P-µchip shows promising potential for rapid drug detection in complex biofluids.

Electrochemiluminescence bipolar electrode array for the multiplexed detection of glucose, lactate and choline based on a versatile enzymatic approach.

A simple, efficient and versatile biosensing platform capable of the multiplexed detection for glucose, lactate and choline was developed by the integration of bipolar electrochemistry and electrochemiluminescence (ECL) imaging. The sensing bipolar electrodes (BPEs) were simply modified via a one-step method adaptable to different enzymes. The biorecognition event happening between the substrate and the corresponding enzyme could be directly reported by the ECL emitted on the same pole from luminol and in situ generated H2O2. Under optimized conditions, the BPEs array was successfully applied for the determination of glucose, lactate and choline in the ranges of 0.01-1mM, 0.01-1mM and 0.02-5mM, with the LOD of 7.57µM, 8.25µM and 43.19µM, respectively. Owing to the improved stability of in situ generated H2O2, a whole series of analytes testing could be completed in the same BPE biochip. Subsequently, an array chip consisting of nine BPEs enabled the concomitant detection of glucose, lactate and choline, demonstrating the capability for multifunctional detection of biomolecules. This versatile analytical system could be easily extended to sensitive screening in a miniaturized device and point of care testing.

Two orders-of-magnitude enhancement in the electrochemiluminescence of Ru(bpy)3²âº by vertically ordered silica mesochannels.

In this paper, we report significantly enhanced electrochemiluminescence (ECL) of tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)3²âº) at indium tin oxide (ITO) electrodes modified with a thin film consisting of vertically aligned silica mesochannels (SMCs). The perpendicular SMCs allow for the effective mass transport of Ru(bpy)3²âº and its co-reactants from the solution to the underlying ITO electrode surface. Moreover, due to the ultrasmall diameter (namely 2-3 nm) and the negatively charged surface, SMCs exerted a strong electrostatic attraction to the positively charged Ru(bpy)3²âº to accelerate its mass transport, resulting in a remarkably increased ECL signal. As a model system, when using tri-n-propylamine (TPrA) as the co-reactant, the intensity of ECL generated at the SMCs-modified ITO electrode was stronger than that at a bare ITO electrode by more than two orders of magnitude (i.e., ∼107-fold increase). In this case, the amount of Ru(bpy)3²âº required for ECL-based analysis can be considerably reduced. A very low concentration of Ru(bpy)3²âº, namely 9 µM, was demonstrated to be enough for achieving the sensitive detection of TPrA, nicotine and atropine. Both photomultiplier (PMT) and charge coupled device (CCD) were used to measure the luminescence from the ECL reaction. The obtained detection limits were 0.17 nM for TPrA on the basis of the ECL intensity measurement, 72 nM for nicotine and 38 nM for atropine based on the gray value analysis of captured ECL images.

Immunological multimetal deposition for rapid visualization of sweat fingerprints.

A simple method termed immunological multimetal deposition (iMMD) was developed for rapid visualization of sweat fingerprints with bare eyes, by combining the conventional MMD with the immunoassay technique. In this approach, antibody-conjugated gold nanoparticles (AuNPs) were used to specifically interact with the corresponding antigens in the fingerprint residue. The AuNPs serve as the nucleation sites for autometallographic deposition of silver particles from the silver staining solution, generating a dark ridge pattern for visual detection. Using fingerprints inked with human immunoglobulin G (hIgG), we obtained the optimal formulation of iMMD, which was then successfully applied to visualize sweat fingerprints through the detection of two secreted polypeptides, epidermal growth factor and lysozyme. In comparison with the conventional MMD, iMMD is faster and can provide additional information than just identification. Moreover, iMMD is facile and does not need expensive instruments.

A novel biosensor array with a wheel-like pattern for glucose, lactate and choline based on electrochemiluminescence imaging.

Electrochemiluminescence (ECL) imaging provides a superior approach to achieve array detection because of its ability for ultrasensitive multiplex analysis. In this paper, we reported a novel ECL imaging biosensor array modified with an enzyme/carbon nanotubes/chitosan composite film for the determination of glucose, choline and lactate. The biosensor array was constructed by integrating a patterned indium tin oxide (ITO) glass plate with six perforated poly(dimethylsiloxane) (PDMS) covers. ECL is generated by the electrochemical reaction between luminol and hydrogen peroxide that is produced by the enzyme catalysed oxidation of different substrates with molecular oxygen, and ECL images were captured by a charge-coupled device (CCD) camera. The separated electrochemical micro-cells enabled simultaneous assay of six samples at different concentrations. From the established calibration curves, the detection limits were 14 µM for glucose, 40 µM for lactate and 97 µM for choline, respectively. Moreover, multicomponent assays and cross reactivity were also studied, both of which were satisfied for the analysis. This biosensing platform based on ECL imaging shows many distinct advantages, including miniaturization, low cost, and multi-functionalization. We believe that this novel ECL imaging biosensor platform will have potential applications in clinical diagnostics, medicine and food inspection.

Electrochemiluminescence imaging of latent fingermarks through the immunodetection of secretions in human perspiration.

We present the combination of electrochemiluminescence imaging with enzyme immunoassay for the highly sensitive detection of protein/polypeptide residues in latent fingermarks. This technique provides an effective method for fingermark detection that enables both identification of an individual and recognition of the secretions in the human perspiration.

Image-contrast technology based on the electrochemiluminescence of porous silicon and its application in fingerprint visualization.

The electrochemiluminescence (ECL) of porous silicon (pSi) has attracted great interest for its potential application in display technology and chemical sensors. In this study, we found that pSi with a different surface chemistry displayed an apparently different dynamic ECL process. An image-contrast technology was established on the basis of the intrinsic mechanism of the ECL dynamic process. As a proof of principle, the visualization of latent fingerprints (LFPs) and in situ detection of TNT in fingerprints was demonstrated by using the ECL-based image-contrast technology.

Integrating bipolar electrochemistry and electrochemiluminescence imaging with microdroplets for chemical analysis.

Here we develop a microdroplet sensor based on bipolar electrochemistry and electrochemiluminescence (ECL) imaging. The sensor was constructed with a closed bipolar cell on a hybrid poly(dimethylsioxane) (PDMS)-indium tin oxide (ITO) glass microchip. The ITO microband functions as the bipolar electrode and its two poles are placed in two spatially separate micro-reservoirs predrilled on the PDMS cover. After loading microliter-sized liquid droplets of tris(2,2'-bipyridyl) ruthenium (II)/2-(dibutylamino) ethanol (Ru(bpy)3(2+)/DBAE) and the analyte to the micro-reservoirs, an appropriate external voltage imposed on the driving electrodes could induce the oxidation of Ru(bpy)3(2+)/DBAE and simultaneous reduction of the analyte at the anodic and cathodic poles, respectively. ECL images generated by Ru(bpy)3(2+)/DBAE oxidation at the anodic pole and the electrical current flowing through the bipolar electrode can be recorded for quantitative analyte detection. Several types of quinones were selected as model analytes to demonstrate the sensor performance. Furthermore, the cathodic pole of bipolar electrode can be modified with (3-aminopropyl)triethoxysilane-gold nanoparticles-horseradish peroxidase composites for hydrogen peroxide detection. This microdroplet sensor with a closed bipolar cell can avoid the interference and cross-contamination between analyte solutions and ECL reporting reagents. It is also well adapted for chemical analysis in the incompatible system, e.g., detection of organic compounds insoluble in water by aqueous ECL generation. Moreover, this microdroplet sensor has advantages of simple structure, high sensitivity, fast response and wide dynamic response, providing great promise for chemical and biological analysis.

Non-destructive enhancement of latent fingerprints on stainless steel surfaces by electrochemiluminescence.

Visualization and detection of latent fingerprints (LFPs) on metal surfaces are of highly practical importance, e.g., in identifying gun cartridges. We report herein the visualization of LFPs on stainless steel surfaces by electrochemiluminescence (ECL). Since organic residues, such as fatty acids, in the fingerprint deposit make the underlying surface electrochemically inert or less active, an ECL reaction occurs only on the metal portions untouched by the fingertip, hence generating a negative image of the fingerprint. The popular ECL reaction solution, consisting of ruthenium(ii) tris(2,2'-bipyridyl) and tri-n-propylamine, was used for this imaging purpose. Factors, including the applied potential and the concentration of ECL luminophore, as well as the stability of ECL negative images, were investigated to achieve a satisfactory visualization enhancement. This imaging approach is simple, rapid, non-invasive, and no pre-treatment either on the background or on the fingerprint itself is needed. It constitutes a powerful tool for visualizing LFPs on metal surfaces. This method was also demonstrated to be suitable for enhancing LFPs collected from various surfaces.

Hydrothermal synthesis of GSH-TGA co-capped CdTe quantum dots and their application in labeling colorectal cancer cells.

We have successfully synthesized GSH and TGA co-capped CdTe quantum dots (QDs) with good biological compatibility and high fluorescence intensity. The effects of different reaction time, temperature, pH value, ligand concentration and the molar ratio of GSH/TGA were carefully investigated to optimize the synthesis condition. The optical properties of as-prepared CdTe QDs were studied by UV-visible absorption spectrum and fluorescence spectrum, meanwhile their structure and morphology were characterized using transmission electron microscope (TEM), Fourier transform infrared spectra (FT-IR) and X-ray powder diffraction (XRD). Compared with the CdTe QDs that are single-capped with either GSH or TGA, the GSH-TGA co-capped CdTe QDs demonstrated significantly improved fluorescence intensity and optical stability. In addition, GSH-TGA co-capped CdTe QDs were conjugated to amonoclonal antibody ND-1. The GSH-TGA co-capped CdTe QDs-antibody probe was successfully used to label colorectal cancer cells, CCL187, in vitro.