The ability to quantitate and visualize microRNAs (miRNAs) in situ in single cells would greatly facilitate the elucidation of miRNA-mediated regulatory circuits and their disease associations. A toehold-initiated strand-displacement process was used to initiate rolling circle amplification of specific miRNAs, an approach that achieves both stringent recognition and in situ amplification of the target miRNA. This assay, termed toehold-initiated rolling circle amplification (TIRCA), can be utilized to identify miRNAs at physiological temperature with high specificity and to visualize individual miRNAs in situ in single cells within 3 h. TIRCA is a competitive candidate technique for in situ miRNA imaging and may help us to understand the role of miRNAs in cellular processes and human diseases in more detail.
Photocontrolled organocatalyzed living radical polymerization was conducted over a wide range of irradiation wavelengths (350-750 nm). The polymerization was induced and controlled at the desired wavelengths by exploiting suitable organic catalysts. This system was finely responsive to the irradiation wavelength; the polymerization was instantly switched on and off, and the polymerization rate was sensitively modulated by altering the irradiation wavelength. The polymer molecular weight and its distribution (M(w)/M(n) = 1.1-1.4) were well controlled for methacrylate monomers up to fairly high conversions in many cases. The monomer scope encompassed various functional methacrylates, and their block copolymers were obtained. The feasibility of such a wide range of wavelengths and the fine response to the wavelength are unprecedented features. As a unique application of the wavelength-responsive nature of this system, we demonstrated "one-pot" selective regulation of living radical polymerization and another type of polymerization (ring opening polymerization), where the regulation was achieved by simply altering the irradiation wavelength. Facile operation and applicability to a wide range of polymer designs are advantages of this polymerization.
In February 2016, the World Health Organization declared a Public Health Emergency of International Concern on Zika Virus (ZIKV), because of its association with severe fetal anomalies of congenitally infected humans. This has led to urgent efforts by academic, federal, and industry research groups to improve our understanding of the pathogenesis of ZIKV and to develop detection methods, therapeutic strategies, and vaccines. Although we still do not have the entire picture of the pathogenesis of ZIKV, extensive research has been conducted on related pathogenic flaviviruses (i.e., dengue virus, West Nile virus, and yellow fever virus). Binding to glycosaminoglycans (GAGs) through its envelope protein is the first step in successful host cell invasion of dengue virus. In this study, we examined ZIKV envelope protein (ZIKV E) binding to GAGs in a real time interaction study using surface plasmon resonance (SPR) to explore the role of GAGs in host cell entry of ZIKV into placenta and brain. ZIKV E strongly binds (K = 443 nM) pharmaceutical heparin (HP), a highly sulfated GAG, and binds with lower avidity to less sulfated GAGs, suggesting that the ZIKV E-GAG interaction may be electrostatically driven. Using SPR competition assays with various chain length HP oligosaccharides (from 4 to 18 saccharide units in length), we observed that ZIKV E preferentially binds to longer HP oligosaccharides (with 8-18 saccharides). Next, we examined GAGs prepared from human placentas to determine if they bound ZIKV E, possibly mediating placental cell invasion of ZIKV. Compositional analysis of these GAGs as well as SPR binding studies showed that both chondroitin sulfate and heparan sulfate GAGs, present on the placenta, showed low-micromolar interactions with ZIKV E. Both porcine brain CS and HS also showed micromolar binding with ZIKV E. Moreover, heparan sulfate with a higher TriS content, the dominant repeating unit of HP, shows a high affinity for ZIKV E. These results suggest that GAGs may be utilized as attachment factors for host cell entry of Zika virus as they do in other pathogenic flaviviruses. They may also assist us in advancing our understanding of the pathogenesis of ZIKV and guide us in designing therapeutics to combat ZIKV with more insight.
The endothelial glycocalyx is a heparan sulfate (HS)-rich endovascular structure critical to endothelial function. Accordingly, endothelial glycocalyx degradation during sepsis contributes to tissue edema and organ injury. We determined the endogenous mechanisms governing pulmonary endothelial glycocalyx reconstitution, and if these reparative mechanisms are impaired during sepsis. We performed intravital microscopy of wild-type and transgenic mice to determine the rapidity of pulmonary endothelial glycocalyx reconstitution after nonseptic (heparinase-III mediated) or septic (cecal ligation and puncture mediated) endothelial glycocalyx degradation. We used mass spectrometry, surface plasmon resonance, and in vitro studies of human and mouse samples to determine the structure of HS fragments released during glycocalyx degradation and their impact on fibroblast growth factor receptor (FGFR) 1 signaling, a mediator of endothelial repair. Homeostatic pulmonary endothelial glycocalyx reconstitution occurred rapidly after nonseptic degradation and was associated with induction of the HS biosynthetic enzyme, exostosin (EXT)-1. In contrast, sepsis was characterized by loss of pulmonary EXT1 expression and delayed glycocalyx reconstitution. Rapid glycocalyx recovery after nonseptic degradation was dependent upon induction of FGFR1 expression and was augmented by FGF-promoting effects of circulating HS fragments released during glycocalyx degradation. Although sepsis-released HS fragments maintained this ability to activate FGFR1, sepsis was associated with the downstream absence of reparative pulmonary endothelial FGFR1 induction. Sepsis may cause vascular injury not only via glycocalyx degradation, but also by impairing FGFR1/EXT1-mediated glycocalyx reconstitution.
Conspectus Glycosaminoglycans (GAGs) are a family of structurally complex heteropolysaccharides composed of alternating hexosamine and uronic acid or galatose residue that include hyaluronan, chondroitin sulfate and dermatan sulfate, heparin and heparan sulfate, and keratan sulfate. GAGs display a range of critical biological functions, including regulating cell–cell interactions and cell proliferation, inhibiting enzymes, and activating growth factor receptors during various metabolic processes. Indeed, heparin is a widely used GAG-based anticoagulant drug. Unfortunately, naturally derived GAGs are highly heterogeneous, limiting studies of their structure–activity relationships and even resulting in safety concerns. For example, the heparin contamination crisis in 2007 reportedly killed more than a hundred people in the United States. Unfortunately, the chemical synthesis of GAGs, or their oligosaccharides, based on repetitive steps of protection, activation, coupling, and deprotection, is incredibly challenging. Recent advances in chemoenzymatic synthesis integrate the flexibility of chemical derivatization with enzyme-catalyzed reactions, mimicking the biosynthetic pathway of GAGs, and represent a promising strategy to solve many of these synthetic challenges. In this critical Account, we examine the recent progress made, in our laboratory and by others, in the chemoenzymatic synthesis of GAGs, focusing on heparan sulfate and heparin, a class of GAGs with profound physiological and pharmacological importance. A major challenge for the penetration of the heparin market by homogeneous heparin products is their cost-effective large-scale synthesis. In the past decade, we and our collaborators have systematically explored the key factors that impact this process, including better enzyme expression, improved biocatalysts using protein engineering and immobilization, low cost production of enzyme cofactors, optimization of the order of enzymatic transformations, as well as development of efficient technologies, such as using ultraviolet absorbing or fluorous tags, to detect and purify synthetic intermediates. These improvements have successfully resulted in multigram-scale synthesis of low-molecular-weight heparins (LMWHs), with some showing excellent anticoagulant activity and even resulting in more effective protamine reversal than commercial, animal-sourced LMWH drugs. Sophisticated structural analysis is another challenge for marketing heparins, since impurities and contaminants can be present that are difficult to distinguish from heparin drug products. The availability of the diverse library of structurally defined heparin oligosaccharides has facilitated the systematic analytical studies undertaken by our group, resulting in important information for characterizing diverse heparin products, safeguarding their quality. Recently, a series of chemically modified nucleotide sugars have been investigated in our laboratory and have been accepted by synthases to obtain novel GAGs and GAG oligosaccharides. These incl...
Heparan sulfate (HS) is a complex linear polysaccharide that modulates a wide range of biological functions. Elucidating the structure-function relationship of HS has been challenging. Here we report the generation of a HS mutant mouse lung endothelial cell library by systematic deletion of HS genes expressing in the cell. We applied this library to answer several fundamental questions about HS biology including: 1) determining that strictly defined fine structure of HS, not its overall sulfation degree, is more important for FGF2-FGFR1 signaling; 2) defining the epitope features of commonly used anti-HS phage display antibodies; and 3) delineating the fine inter-regulation networks of HS modification and chain length by HS genes in mammalian cells and at a cell type specific level. Our mutant cell library will enable robust and systematic interrogation of the roles and related structures of HS in a cellular context.
Platinum group metal‐based (PGM) catalysts are widely applied in many electrochemical systems such as fuel cells or metal–air batteries because of their excellent catalytic performance. But the high raw material cost of PGM catalysts has become a significant issue. In recent years, huge efforts have been made to reduce the material cost of electrochemical systems by developing non‐PGM catalysts, and as one of the promising non‐PGM catalysts, nitrogen‐doped graphene (N‐G) has emerged. In this research, nanoscale high‐energy wet ball milling methodology was investigated as an effective synthesis method for N‐G catalysts by using graphene oxide and melamine as raw materials. The main purpose is to study reaction mechanism of the synthesis process and the physical, chemical, and electrochemical properties of N‐G catalysts generated by this mechanochemical approach. The elemental composition, chemical bonding composition, and electron transfer number of the synthesized products were characterized. The results show that the electron transfer number of the N‐G catalyst with 23.2 at% nitrogen doping content, synthesized by the high‐energy wet ball milling method, has attained a value of 3.87, which is close to the number (3.95) of Pt/C catalysts, and the grinding time was found to be a significant factor in the properties of N‐G catalysts in the experiments. The results also show that the high‐energy wet ball milling developed in this research is a promising method to synthesize high‐performance N‐G catalysts with a simple and easy controllable approach. Copyright © 2016 John Wiley & Sons, Ltd.
The risk of extensive exposure of the human epidermis to solar ultraviolet radiation is significantly increased nowadays. It not only induces skin aging and solar erythema but also increases the possibility of skin cancer. Therefore, a simply prepared, highly sensitive, and optically readable device for monitoring the solar ultraviolet radiation is highly desired for the skin health management. Because of the photoinitiated polymerization triggered by graphene-carbon nitride (g-C 3 N 4 ) under ultraviolet radiation, g-C 3 N 4 is homogeneously distributed in the hybrid hydrogels containing N-isopropylacrymide (NIPAM), poly(ethylene glycol) methyl ether methacrylate (OEGMA 300 ), and sodium alginate (SA). By further immersing the hybrid hydrogels into calcium chloride solution, hybrid alginate-Ca 2+ /P(NIPAM-co-OEGMA 300 )/g-C 3 N 4 interpenetrating polymeric network (IPN) hydrogels are obtained. Due to the homogeneous distribution of g-C 3 N 4 and the existence of thermoresponsive polymers, the hybrid IPN hydrogels present good adsorption capability and high degradation efficiency for methylene blue (MB) especially at high temperature under ultraviolet radiation. Based on this unique property, the bracelet monitoring skin health is prepared by simply immersing the hybrid IPN hydrogels into the MB solution and then wrapping it with PET foil. Because the immersion time for the top, middle, and bottom parts of the hybrid IPN hydrogels is gradually increased, their colors vary from light to dark blue. A longer time is required for the discoloration of the darker part under solar ultraviolet radiation. Thus, the bracelet can be used to conveniently monitor the dose of solar ultraviolet radiation by simply checking the discoloration in the bracelet under sunshine. Due to the facile preparation and low cost of the bracelet, it is a promising candidate for wearable devices for skin health management.
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