Synthesis and evaluation of fluorogenic 2-amino-1,8-naphthyridine derivatives for the detection of bacteria

Several novel fluorogenic N-aminoacylnaphthyridine substrates were synthesized in good yield and tested for their ability to detect pathogenic bacteria in agar-based cell culture. Simple 2-N-(β-alanyl)amino-5,7-dialkylnaphthyridine substrates were selectively hydrolysed by β-alanylaminopeptidase expressing bacteria, but were subject to diffusion in the agar medium. Diffusion was reduced in the 2-N-(β-alanyl)amino-7-alkylnaphthyridine substrates with longer alkyl chains, but inhibition of growth was increased. 2-N-(β-Alanyl)amino-7-octylnaphthyridine inhibited the growth of all species tested, except for strains resistant to colistin/polymyxin, providing a rationale for the development of substrates for the selective detection of drug resistant species in clinical samples.     

The effect of surface hydrophilicity on the behavior of embryonic cortical neurons

The aim of this study was to investigate the interaction of mouse embryonic cortical neurons on P(L)LA and PLGA substrates, which were partially hydrolysed using potassium hydroxide (KOH). The chemical and topographical properties of the surfaces were characterized, and it was discovered that there was a decrease in the hydrophilicity for the P(L)LA with increasing concentration of KOH. This was due to chemical modifications to the surfaces of the substrates. Alternatively for the PLGA substrate, only the 0.1 M KOH treated sample had a significantly different hydrophilicity highlighting that surface erosion resulted at higher concentrations. The morphology of the neurons grown on the two substrates were compared to poly(D)lysine (positive control). The neurons formed colonies on all of the substrates, but were dramatically reduced in size in the case of the 0.1 M KOH treated substrates. This finding was attributed to the increases in cell spreading and the size of the cells, as they were larger, more elongated and bipolar like those on the positive control. However, there was a significant decrease in the total number of live cells per unit area. Therefore, on these materials when there was increased cellular spreading there was significantly higher cell death. Furthermore, unlike the 0, 0.2, and 0.4 M KOH treated substrates, there was an absence of large bundles of axons that extended between colonies on the 0.1 M sample, instead exhibiting short axons that grew in free space.     

The enzymatic hydrolysis of pretreated agricultural residues and the fermentation of the liberated sugars to ethanol

Agricultural residues were pretreated by steam explosion and the cellulosic component of these substrates were converted to ethanol using a combined enzymatic hydrolysis and fermentation (CHF) process. The enzymatic hydrolysis was carried out using culture filtrates ofTrichoderma harzianum E58 while the liberated sugars were fermented to ethanol byS. cerevisiae. Initially, pretreatment conditions were optimized to ensure that the substrates were readily hydrolyzed and fermented. The agricultural residues were steamed for various times between 30 and 120 s at approximately 240‡C prior to rapid decompression (explosion) in a small masonite-type gun. The various substrates were selectively extracted by water and alkali to see whether the enzymatic hydrolysis and fermentability of the substrates were enhanced. A comparison between the overall conversion of wheat and barley straw was made since these are the two most readily available agricultural residues in Canada. Steam explosion did not affect the hexosan content of the residues, although the pentosan content of the substrates decreased with increasing duration of steaming. The hexosan (cellulose) content of wheat straw was 50.7% of the total substrate while a slightly higher 52.9% cellulose content was detected in the barley straw. Wheat straw was more efficiently hydrolyzed after it had been steamed for 90 s while optimum hydrolysis of the barley straw was detected after 60 s. Steam exploded wheat and barley straw that was subsequently extracted with water was readily hydrolyzed to their component sugars.S. cerevisiae could almost quantitatively convert these sugars to ethanol. This indicated that water washing not only enhanced the enzymatic hydrolysis of the steam exploded substrates, it also removed inhibitory material that restricted the growth of S.cerevisiae. Maximum hydrolysis (78.5%) and ethanol yields (10 mg/mL) were obtained when wheat straw was steamed for 90 s. Slightly lower hydrolysis (76.0%) and ethanol yields (9.5 mg/mL) were obtained with barley straw that had been steamed for 120 s.     

Synthesis and evaluation of halogenated nitrophenoxazinones as nitroreductase substrates for the detection of pathogenic bacteria

The synthesis and microbiological evaluation of 7-, 8- and 9-nitro-1,2,4-trihalogenophenoxazin-3-one substrates with potential in the detection of nitroreductase-expressing pathogenic microorganisms are described. The 7- and 9-nitrotrihalogenophenoxazinone substrates were reduced by most Gram-negative microorganisms and were inhibitory to the growth of certain Gram-positive bacteria; however, the majority of Gram-positive strains that were not inhibited by these agents, along with the two yeast strains evaluated, did not reduce the substrates. These observations suggest there are differences in the active site structures and substrate requirements of the nitroreductase enzymes from different strains; such differences may be exploited in the future for differentiation between pathogenic microorganisms. The absence of reduction of the 8-nitrotrihalogenophenoxazinone substrates is rationalized according to their electronic properties and correlates well with previous findings.     

Increasing the enantioselectivity of cyclopentanone monooxygenase (CPMO): profile of new CPMO mutants

A series of cyclohexanones substituted at the 4-position with a selection of hydrophobic and hydrophilic groups were used as substrates in the evaluation of six new cyclopentanone monooxygenase (CPMO) mutants. These mutants were obtained through evolutionary modifications in two specific regions of the CPMO’s putative active site. Several mutant enzymes with improved enantioselectivity were identified. Analysis of the results, in terms of a diamond model, illustrates how a family of cyclohexanone substrates may be used to explore putative active sites of Baeyer–Villiger monooxygenases (BVMOs) and to design productive mutations for specific substrates.     

Substrate activity screening: a fragment-based method for the rapid identification of nonpeptidic protease inhibitors

A new fragment-based method for the rapid development of novel and distinct classes of nonpeptidic protease inhibitors, Substrate Activity Screening (SAS), is described. This method consists of three steps: (1) a library of N-acyl aminocoumarins with diverse, low molecular weight N-acyl groups is screened to identify protease substrates using a simple fluorescence-based assay, (2) the identified N-acyl aminocoumarin substrates are optimized by rapid analogue synthesis and evaluation, and (3) the optimized substrates are converted to inhibitors by direct replacement of the aminocoumarin with known mechanism-based pharmacophores. The SAS method was successfully applied to the cysteine protease cathepsin S, which is implicated in autoimmune diseases. Multiple distinct classes of nonpeptidic substrates were identified upon screening an N-acyl aminocoumarin library. Two of the nonpeptidic substrate classes were optimized to substrates with >8000-fold improvements in cleavage efficiency for each class. Select nonpeptidic substrates were then directly converted to low molecular weight, novel aldehyde inhibitors with nanomolar affinity to cathepsin S. This study demonstrates the unique characteristics and merits of this first substrate-based method for the rapid identification and optimization of weak fragments and provides the framework for the development of completely nonpeptidic inhibitors to many different proteases.     

Comparison of hydrothermal,hydrotropic and organosolv pretreatment for improving the enzymatic digestibility of bamboo

Pretreatment is the crucial step to disrupt the recalcitrant structure of lignocellulosic biomass for improving the enzymatic hydrolysis efficiency. Typically, hydrothermal, organosolv and hydrotropic pretreatments are environmentally benign and effective methods. In this work, effects of hydrothermal, organosolv and hydrotropic pretreatments on improving enzymatic hydrolysis of bamboo were comprehensively compared. Hydrotropic pretreatment was more effective in removal lignin and xylose from bamboo fiber cell wall. However, the surface coverage by lignin and extractives were dramatically displaced during organosolv pretreatment as investigation by X-ray photoelectron spectroscopy. After pretreatments, the crystallinity of cellulose in pretreated substrates has a significant reduction, and pores were exposed on fiber surface. The residual content of acetyl and phenolic groups in hydrotropic pretreated substrates is lower than organosolv pretreated substrates. In order to deeply assess the delignification of pretreatments, the isolated lignins obtaining from pretreatments process were characterized by Fourier transform infrared spectroscopy also. It was revealed that hydrotropic lignin contained more phenolic hydroxyl group and syringyl units than organosolv lignin. Compared to hydrothermal and organosolv pretreatment, cellulase adsorption capacity of pretreated substrates was notably improved by hydrotropic pretreatment, which indicating the better enzyme accessibility of cellulose. Eventually, the maximum glucose yield was obtained from hydrotropic pretreated substrates.     

Enzymatics synthesis and use of new fluorigenic substrates for a novel approach to the study of bacterialpeptidoglycan degradation

Two complex new fluorigenic substrates were synthesized enzymatically. Their use is demonstrated in the study of specific hydrolases concerned in the degradation of bacterial peptidoglycans. The substrates operate according to the “sequential fluorigenic enzyme assay” principle. Results are presented to illustrate the fluorigenic method for the estimation of lysozyme activities. The limitations of simple fluorigenic substrates in studies on the degradation of inter-sugar linkages in complex natural substances are demonstrated.     

Elasticity of cardiac cells on the polymer substrates with different stiffness: an atomic force microscopy study

Influences of substrate stiffness on mechanical properties of cardiac myocytes and fibroblasts were investigated by cell elasticity measurement with atomic force microscopy. The cells were cultured on collagen-coated polyacrylamide substrates with gradient rigidity. While cardiac myocytes showed no evident change in cell elasticity on different substrates, cardiac fibroblasts displayed the non-monotonic dependence on substrate stiffness with a maximum elastic modulus. Moreover, the elasticity change of cardiac fibroblasts with substrates stiffness was found to be regulated by actin filaments. Study of the effect of substrate stiffness on cell elasticity for different cardiac cells provides new information for the better understanding of cardiac physiology and pathology.     

Potentiality of reflectometry for the study of the adsorption on dielectric and metal substrates: application to the adsorption of polyvinylimidazole on silica and gold

Optical fixed-angle reflectometry was examined as a technique to study the adsorption of organics (polymers, surfactants) on metallic substrates. Calculations were made to evaluate the sensitivity of the method with regard to the refractive index and the thickness of the metal and the incidence angle. Theoretical results show that Ti, Cr, V, Zr, and gold have an appropriate sensitivity for detecting the adsorption accurately. Parallel experiments using gold and silica substrates and polyvinylimidazole (PVI) as an organic material were carried out. Adsorption measurements as a function of polymer concentration, pH, and ionic strength were in accordance with expected trends.     

Photoluminescence in the characterization and early detection of biomimetic bone-like apatite formation on the surface of alkaline-treated titanium implant: state of the art

Photoluminescence (PL) property is particularly important in the characterization of materials that contain significant proportions of noncrystalline components, multiple phases, or low concentrations of mineral phases. In this research, the ability of biomimetic bone-like apatite deposition on the surface of titanium alloy (Ti6Al4V) substrates in simulated body fluid (SBF) right after alkaline-treatment and subsequent heat-treatment was studied by the inherent luminescence properties of apatite. For this purpose, the metallic substrates were treated in 5 M NaOH solution at 60 °C. Subsequently, the substrates were heat-treated at 600 °C for 1 h for consolidation of the sodium titanate hydrogel layer. Then, they were soaked in SBF for different periods of time. Finally, the possibility to use of PL monitoring as an effective method and early detection tool is discussed. According to the obtained results, it was concluded that the PL emission peak did not have any significant shift to the shorter or higher wavelengths, and the PL intensity increased as the exposure time increased. This research proved that the observed inherent PL of the newly formed apatite coatings might be of specific interest for histological probing and bone remodelling monitoring.     

The effects of substrate orientation on the mechanism of a phosphotriesterase

While the underlying chemistry of enzyme-catalyzed reactions may be almost identical, the actual turnover rates of different substrates can vary significantly. This is seen in the turnover rates for the catalyzed hydrolysis of organophosphates by the bacterial phosphotriesterase OpdA. We investigate the variation in turnover rates by examining the hydrolysis of three classes of substrates: phosphotriesters, phosphothionates, and phosphorothiolates. Theoretical calculations were used to analyze the reactivity of these substrates and the energy barriers to their hydrolysis. This information was then compared to information derived from enzyme kinetics and crystallographic studies, providing new insights into the mechanism of this enzyme. We demonstrate that the enzyme catalyzes the hydrolysis of organophosphates through steric constraint of the reactants, and that the equilibrium between productively and unproductively bound substrates makes a significant contribution to the turnover rate of highly reactive substrates. These results highlight the importance of correct orientation of reactants within the active sites of enzymes to enable efficient catalysis.     

Peptide microarrays for the determination of protease substrate specificity

A method is described for the preparation of substrate microarrays that allow for the rapid determination of protease substrate specificity. Peptidyl coumarin substrates, synthesized on solid support using standard techniques, are printed onto glass slides using DNA microarraying equipment. The linkage from the peptide to the slide is formed through a chemoselective reaction, resulting in an array of uniformly displayed fluorogenic substrates. The arrays can be treated with proteases to yield substrate specificity profiles. Standard instrumentation for visualization of microarrays can be used to obtain comparisons of the specificity constants for all of the prepared substrates. The utility of these arrays is demonstrated by the selective cleavage of preferred substrates with trypsin, thrombin, and granzyme B, and by assessing the extended substrate specificity of thrombin using a microarray of 361 different peptidyl coumarin substrates.     

Electronic control of chiral quaternary center creation in the intramolecular asymmetric heck reaction

The Boehringer-Ingelheim phosphinoimidazoline (BIPI) ligands were applied to the formation of chiral quaternary centers in the asymmetric Heck reaction. Several different substrates were examined in detail, using more than 70 members of this new ligand class. Hammett relationships were determined through systematic variation of the ligand electronics. All substrates showed essentially the same Hammett behavior, where enantioselectivity increased as the ligands were made more electron-deficient. Ligand optimization has led to catalysts which give the highest enantioselectivities reported to date for these difficult systems.     

The influence of collecting substrates on the single-particle characterization of real atmospheric aerosols

This work investigated the influence of three different collecting substrate materials, Ag and Al foils and grids for transmission electron microscopy (TEM grid), on the morphological and chemical compositional analysis of individual particles collected at an underground shopping area in Seoul, Korea. The feasibility of using each substrate in a quantitative single-particle analysis was evaluated by comparing particle morphologies, X-ray spectra, and elemental quantification results obtained for the three substrates. The morphologies and the quality of X-ray spectra for crystalline mineral particles were very similar among the three substrates. However, water-soluble, CNO-rich aerosols showed different morphologies among the three substrates, mainly due to the differences in the hygroscopic properties of the substrates. The quality of the X-ray spectra of the CNO-rich particles was optimal when collected on the TEM grid. To reliably assess the characteristic X-rays of the CNO-rich particles collected on the Ag and Al foils, appropriate data analysis had to be applied. Especially, the X-ray spectra of the CNO-rich particles collected on Al foil required a new background subtraction procedure. The overall relative abundances of the chemical species, obtained from the three collecting substrates, were in good agreement with each other and single-particle characterization of the real aerosol sample was feasible on the different substrates. However, the TEM grid substrate was the most appropriate for single-particle analysis of the water-soluble CNO-rich particles as: (i) it retains the original morphology and size of the particles, (ii) it allows high contrast in the backscattered electron image (BSEI) mode, and (iii) it provides a high peak-to-background ratio (P/B) with small and correctable interferences in the X-ray spectra.     

Formation of Anatase Nanocrystals-Precipitated Silica Coatings on Plastic Substrates by the Sol-Gel Process with Hot Water Treatment

Anatase nanocrystals-precipitated silica coatings were formed on plastic substrates such as poly(ethylene terephtalate) (PET), acrylic resin (AC) and polycarbonate (PC) from silica-titania gel coatings with and without addition of poly(ethylene glycol) (PEG) by hot water treatment. The maximum thickness of the coatings which can be formed without cracking or peeling-off was 100 to 200 nm for PET and PC substrates, whereas it was less than 50 nm for AC substrates. After a hot water treatment at 90°C for 120 min, the size of the anatase nanocrystals increased to be 30 and 50 nm for the coatings obtained with and without PEG, respectively. Anatase nanocrystals were formed throughout the whole of the coatings obtained with PEG and were formed only on the surface of the coatings obtained without PEG. Both coatings obtained with and without PEG were highly transparent. The plastic substrates with coatings obtained without PEG showed good weathering resistance owing to the protective effects of the residual silica under-layer. The coatings obtained with PEG showed higher photocatalytic activities than those obtained without PEG due to smaller size and higher dispersion of anatase nanocrystals in the coatings.     

Effect of oxygen plasma treatment on the surface properties of tin-doped indium oxide substrates for polymer LEDs

The effect of oxygen plasma treatment on the surface properties of tin-doped indium oxide (ITO) substrates and the changes in surface properties of treated ITO substrates with ageing time were investigated by X-ray photoelectron spectroscopy (XPS), contact angle and surface free energy measurements. Experimental results show that oxygen plasma treatment increases the oxygen concentration, decreases the carbon concentration, and enhances the surface free energy and polarity, and thereby improves the surface properties of ITO substrates. However, the improved ITO surface properties tended to decay and the surface free energy decreased, with ageing time. In addition, the ageing effect of treated ITO substrates on the performance of polymer light-emitting diodes (LEDs) was studied with respect to the driving voltage, electroluminescent luminance and efficiency. We observe that the ITO substrates aged for various times result in significant differences in optical and electrical characteristics which become worse as the ageing time increases. The optical and electrical performance of polymer LEDs is closely related to the surface properties of ITO substrate and the interface characteristics of ITO/polymer.     

Disposable microfluidic substrates: transitioning from the research laboratory into the clinic

As more microfluidic applications emerge for clinical diagnostics, the choice of substrate and production method must be considered for eventual regulatory approval. In this review, we survey recent developments in disposable microfluidic substrates and their fabrication methods. We note regulatory approval for disposable microfluidic substrates will be more forthcoming if the substrates are developed with the United States Pharmacopeia's biocompatibility compliance guidelines in mind. We also review the recent trend in microfluidic devices constructed from a hybrid of substrates that takes advantage of each material's attributes.     

Palladium complexes of phosphinamine ligands in the intramolecular asymmetric Heck reaction

The synthesis of two novel cyclisation substrates for the asymmetric intramolecular Heck reaction is reported. Their cyclisation, in addition to a known substrate for cis-decalin formation, were tested with palladium complexes of BINAP and heterobidentate oxazoline-containing ligands. In general BINAP provides a more active catalyst system for the range of substrates tested although excellent enantioselectivities of up to 85% were obtained with the P,N ligands studied. A trend was noted whereby the t-leucine-derived oxazoline ligands were more reactive and enantioselective than the valine-derived analogues. Similarly, the diphenylphosphinoferrocenyloxazoline ligands were more reactive and selective than the corresponding diphenylphosphinophenyloxazoline ligands.