Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells

Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture.     

Surface segregation of silicon impurities in organic materials

The present revolution in novel organic materials is driven by the synthesis of new materials exhibiting specific functional properties. Traces of silicon compounds are often present in these materials and, although the bulk concentrations of these impurities may be low, segregation can seriously modify the surface composition. Surfaces and interfaces play an important role in many applications, and the intrinsic properties of the materials are thus often obscured by the presence of segregated impurities. By studying silicon impurity segregation in poly‐dialkoxy phenylenevinylene (PPV), polycarbonate and dendrimer macromolecules, we demonstrate how low‐energy ion scattering may be used to determine the surface impurity fraction and to observe which groups at the surface are shielded by the segregated species. We demonstrate that the performance of PPV‐ based light‐emitting diodes is significantly reduced for submonolayer coverages of siloxanes. We find that the kinetics of the segregation process depend strongly on the materials and the sample preparation conditions. We find that the presence of solvents is needed to enable segregation at room temperature. Heating does enable siloxane impurity segregation in polycarbonate in the solid phase, whereas for polydimethylsiloxane in PPV films we find that segregation in the solid phase does not occur up to 200 °C. The siloxane molecules are found to segregate to preferential sites at the surface, shielding the polar groups. Finally, we demonstrate that purification of the surface is often possible through simple procedures that provide an easy way to study the intrinsic properties of the materials. Copyright © 2004 John Wiley & Sons, Ltd.     

Recent developments and applications of screen-printed electrodes in environmental assays—A review

Screen-printed electrodes (SPEs), which are used as economical electrochemical substrates, have gone through significant improvements over the past few decades with respect to both their format and their printing materials. Because of their advantageous material properties, such as disposability, simplicity, and rapid responses, SPEs have been successfully utilised for the rapid in situ analysis of environmental pollutants. This critical review describes the basic fabrication principles, the configuration designs of SPEs and the hybrid analytical techniques based on SPEs. We mainly overview the electrochemical applications of SPEs in environmental analysis over the past 3 years, including the determination of organic compounds, heavy metals and gas pollutants.     

Correlative microscopy: a powerful tool for exploring neurological cells and tissues

Imaging tools for exploring the neurological samples have seen a rapid transformation over the last decade. Approaches that allow clear and specific delineation of targeted tissues, individual neurons, and their cell–cell connections as well as subcellular constituents have been especially valuable. Considering the significant complexity and extent to which the nervous system interacts with every organ system in the body, one non-trivial challenge has been how to identify and target specific structures and pathologies by microscopy. To this end, correlative methods enable one to view the same exact structure of interest utilizing the capabilities of typically separate, but powerful, microscopy platforms. As such, correlative microscopy is well-positioned to address the three critical problems of identification, scale, and resolution inherent to neurological systems. Furthermore, the application of multiple imaging platforms to the study of singular biological events enables more detailed investigations of structure–function relationships to be conducted, greatly facilitating our understanding of relevant phenomenon. This comprehensive review provides an overview of methods for correlative microscopy, including histochemistry, transgenic markers, immunocytochemistry, photo-oxidation as well as various probes and tracers. An emphasis is placed on correlative light and electron microscopic strategies used to facilitate relocation of neurological structures. Correlative microscopy is an invaluable tool for neurological research, and we fully anticipate developments in automation of the process, and the increasing availability of genomic and transgenic tools will facilitate the adoption of correlative microscopy as the method of choice for many imaging experiments.     

Sensing colorimetric approaches based on gold and silver nanoparticles aggregation: Chemical creativity behind the assay. A review

Localized surface plasmon resonance (LSPR) is one of the most remarkable features of gold nanoparticles (Au NPs) and silver nanoparticles (Ag NPs). Due to these inherent optical properties, colloidal solutions of Au and Ag NPs have high extinction coefficients and different colour in the visible region of the spectrum when they are well-spaced in comparison with when they are aggregated. Therefore, a well-designed chemical interaction between the analyte and NPs surroundings leads to a change of colour (red to blue for Au NPs and yellow to brown for Ag NPs from well-spaced to aggregated ones, respectively) allowing the visual detection of the target analyte.     

Tracing nanoparticles and photosensitizing molecules at transmission electron microscopy by diaminobenzidine photo-oxidation

During the last three decades, diaminobenzidine photo-oxidation has been applied in a variety of studies to correlate light and electron microscopy. Actually, when a fluorophore is excited by light, it can induce the oxidation of diaminobenzidine into an electron-dense osmiophilic product, which precipitates in close proximity to the fluorophore, thereby allowing its ultrastructural detection. This method has very recently been developed for two innovative applications: tracking the fate of fluorescently labeled nanoparticles in single cells, and detecting the subcellular location of photo-active molecules suitable for photodynamic therapy. These studies established that the cytochemical procedures exploiting diaminobenzidine photo-oxidation represent a reliable tool for detecting, inside the cells, with high sensitivity fluorescing molecules. These procedures are trustworthy even if the fluorescing molecules are present in very low amounts, either inside membrane-bounded organelles, or at the surface of the plasma membrane, or free in the cytosol. In particular, diaminobenzidine photo-oxidation allowed elucidating the mechanisms responsible for nanoparticles internalization in neuronal cells and for their escape from lysosomal degradation. As for the photo-active molecules, their subcellular distribution at the ultrastructural level provided direct evidence for the lethal multiorganelle photo-damage occurring after cell photo-sensitization. In addition, DAB photo-oxidized samples are suitable for the ultrastructural detection of organelle-specific molecules by post-embedding gold immunolabeling.     

3,3′-二氨基联苯胺柱前衍生高效液相色谱法测定酒中双乙酰

以3,3′-二氨基联苯胺(DAB)为衍生化试剂,建立了柱前衍生高效液相色谱测定酒中双乙酰含量的分析方法。双乙酰与衍生化试剂DAB在室温条件下反应10 min进行柱前衍生,并采用Shim-pack VP-ODS色谱柱(250mm×4.6 mm,4.6μm)对衍生化产物进行分离分析,以水-甲醇为流动相进行梯度洗脱,流速为0.7 mL/min,并采用配有二极管阵列检测器(DAD)的高效液相色谱仪测定,检测波长为254 nm。该法在双乙酰浓度为0.20~180μmol/L的范围内呈现良好的线性关系,相关系数(R2)为0.999,检出限(S/N=3)为0.09μmol/L,定量限(S/N=10)为0.20μmol/L,日内精密度(RSD)为1.28%(n=6)。实际酒样品的加标回收率为92.0%~103.6%,RSD为0.69%~3.45%(n=3)。该法简便快捷,结果准确,稳定性好,可以用于白酒及红酒中双乙酰含量的测定。     

A versatile approach to pyrrolidine azasugars and homoazasugars based on a highly diastereoselective reductive benzyloxymethylation of protected tartarimide

A highly diastereoselective synthesis of enantio-enriched all trans-3,4-dibenzyloxyl-5-benzyloxymethyl-2-pyrrolidinone 13a was developed based on SmI₂-mediated benzyloxymethylation of O,O′-dibenzyltartarimide. The versatility of 13a and its antipode as the key building blocks for the asymmetric synthesis of pyrrolidine azasugars and homoazasugars has been demonstrated by elaborating them into naturally occurring DAB 1 (1), LAB 1 (2), N-hydroxyethyl-DAB 1 (4), 6-deoxy-DMDP 7, and 5-epi-radicamine B 36 as well as the reductive ring-opening product 35.     

Monitoring tissue inflammation and responses to drug treatments in early stages of mice bone fracture using 50 MHz ultrasound

Bone fracture induces moderate inflammatory responses that are regulated by cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LO) for initiating tissue repair and bone formation. Only a handful of non-invasive techniques focus on monitoring acute inflammation of injured bone currently exists. In the current study, we monitored in vivo inflammation levels during the initial 2 weeks of the inflammatory stage after mouse bone fracture utilizing 50 MHz ultrasound. The acquired ultrasonic images were correlated well with histological examinations. After the bone fracture in the tibia, dynamic changes in the soft tissue at the medial-posterior compartment near the fracture site were monitored by ultrasound on the days of 0, 2, 4, 7, and 14. The corresponding echogenicity increased on the 2nd, 4th, and 7th day, and subsequently declined to basal levels after the 14th day. An increase of cell death was identified by the positive staining of deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay and was consistent with ultrasound measurements. The increases of both COX-2 and Leukotriene B4 receptor 1 (BLT₁, 5-LO-relative receptor), which are regulators for tissue inflammation, in the immunohistochemistry staining revealed their involvement in bone fracture injury. Monitoring the inflammatory response to various non-steroidal anti-inflammatory drugs (NSAIDs) treatments was investigated by treating injured mice with a daily oral intake of aspirin (Asp), indomethacin (IND), and a selective COX-2 inhibitor (SC-236). The Asp treatment significantly reduced fracture-increased echogenicity (hyperechogenicity, p < 0.05) in ultrasound images as well as inhibited cell death, and expression of COX-2 and BLT₁. In contrast, treatment with IND or SC-236 did not reduce the hyperechogenicity, as confirmed by cell death (TUNEL) and expression levels of COX-2 or BLT₁. Taken together, the current study reports the feasibility of a non-invasive ultrasound method capable of monitoring post-fracture tissue inflammation that positively correlates with histological findings. Results of this study also suggest that this approach may be further applied to elucidate the underlying mechanisms of inflammatory processes and to develop therapeutic strategies for facilitating fracture healing.