Development of a monoclonal antibody-based immunoassay for specific quantification of bovine milk alkaline phosphatase

The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0.02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.     

Texture and surface morphology effects on tetragonal phase stabilization in ZrO2 films deposited by metal-organic chemical vapor deposition

Tetragonal zirconia coatings (ZrO2) without doping any trivalent impurities have been deposited by metal-organic chemical vapor deposition (MOCVD) on (100) Si single crystals, using Zr(thd)4 precursor. The surface and cross-section morphologies were observed with Field-Emission-Gun Scanning Electron Microscopy (FEG-SEM). The crystalline structures were characterized by grazing incident X-ray diffraction (GIXRD). Crystallographic textures of these films were studied for both {011}t and {110}t planes by pole figure recording by X-ray diffraction under a 4-circle goniometer. The internal stresses were measured with the use of sin2 psi method. In order to study the relationship of microstructures and tetragonal phase stabilization in ZrO2 films, annealing experiments were taken at different temperature. The results show that the critical crystallite size for tetragonal to monoclinic phase transformation is different for samples with different initial microstructures. Besides the critical crystallite size and the residual stress, the texture and crystallite morphology of the ZrO2 films are responsible for the stabilization of the metastable tetragonal phase.     

Sensitive detection of Bacillus anthracis spores by immunocapture and liquid chromatography-tandem mass spectrometry

Bacillus anthracis is one of the most dangerous agents of the bioterrorism threat. We present here a sensitive immuno-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) approach to spore detection in complex environmental samples. It is based on the combined specificity and sensitivity of two techniques: immunocapture and targeted mass spectrometry. The immunocapture step, realized directly on the intact spores, is essential for their selective isolation and concentration from complex environmental samples. After parallel trypsin and Glu-C digestions, proteotypic peptides corresponding to small acid-soluble spore protein-B (SASP-B) are specifically monitored in the multiple reaction monitoring (MRM) mass spectrometry mode. Peptide ratio is carefully monitored and provides an additional level of specificity, which is shown to be highly useful for distinguishing closely related samples and avoiding false-positive/negative results. Sensitivity at the level of the infectious dose is demonstrated, with limits of detection of 7 × 10(3) spores/mL of milk or 10 mg of soil. This mass spectrometry approach is thus complementary to polymerase chain reaction (PCR) techniques.     

Analysis of a polydisperse polyrotaxane based on poly(ethylene oxide) and α-cyclodextrins using nanoelectrospray and LTQ-Orbitrap

Polyrotaxanes (PR) are among the most studied interlocked molecules in the field of supramolecular chemistry. Cyclodextrin based polyrotaxanes (CD based PRs) are well-known to be difficult to analyze by mass spectrometry (MS). Nanoelectrospray (nanoESI) employed during mass spectrometry (MS) and tandem mass spectrometry (MS/MS) experiments turns out to be particularly useful to analyze these noncovalent assemblies. While ESI/nanoESI based spectra usually contain multicharged species which greatly complicate the interpretation, particularly for such complex mixtures analysis, the hyphenation with a high resolution analyzer such as Orbitrap could overcome this limitation. This Article reports efforts to achieve a detailed structural deciphering by nanoESI-MS and nanoESI-MS/MS of CD based PRs constituted of αCDs, unmodified or surrounded by 1 or 2 sulfation(s), which were threaded along polydisperse poly(ethylene oxide) α,ω-dipyrenyl chains. The described method is more sensitive and less sample consuming than a typical NMR experiment and in good agreement with size-exclusion chromatography (SEC) results. Moreover, as compared to MALDI-TOF MS analysis, all populations were presumably elucidated without discrimination effect. Therefore, this MS development allowed us to estimate the PR sample content with 16 to 35 ethylene oxide units, 1 to 5 αCDs threaded, and 0 to 10 sulfo groups grafted on the overall CDs. Finally, the method afforded the possibility to unambiguously attribute supramolecular architectures from 2276.0278 to 7767.8342 g·mol(-1) corresponding to poly[2]- to poly[6]rotaxanes.     

Desorption electrospray ionization then MALDI mass spectrometry imaging of lipid and protein distributions in single tissue sections

Imaging mass spectrometry (MS) is a powerful technique for mapping the spatial distributions of a wide range of chemical compounds simultaneously from a tissue section. Co-localization of the distribution of individual molecular species, including particular lipids and proteins, and correlation with the morphological features of a single tissue section are highly desirable for comprehensive tissue analysis and disease diagnosis. We now report on the use, in turn, of desorption electrospray ionization (DESI), matrix assisted laser desorption ionization (MALDI), and then optical microscopy to image lipid and protein distributions in a single tissue section. This is possible through the use of histologically compatible DESI solvent systems, which allow for sequential analyses of the same section by DESI then MALDI. Hematoxylin and eosin (H&E) staining was performed on the same section after removal of the MALDI matrix. This workflow allowed chemical information to be unambiguously matched to histological features in mouse brain tissue sections. The lipid sulfatide (24:1), detected at m/z 888.8 by DESI imaging, was colocalized with the protein MBP isoform 8, detected at m/z 14117 by MALDI imaging, in regions corresponding to the corpus callosum substructure of the mouse brain, as confirmed in the H&E images. Correlation of lipid and protein distributions with histopathological features was also achieved for human brain cancer samples. Higher tumor cell density was observed in regions demonstrating higher relative abundances of oleic acid, detected by DESI imaging at m/z 281.4, and the protein calcyclin, detected by MALDI at m/z 10085, for a human glioma sample. Since correlation between molecular signatures and disease state can be achieved, we expect that this methodology will significantly enhance the value of MS imaging in molecular pathology for diagnosis.     

Melt processing of poly(L‐lactic acid) in the presence of organomodified anionic or cationic clays

Poly(L ‐lactic acid) (PLA) films are in use for various types of food packaging; however, a wider range of applications would be possible if the barrier properties of these films could be improved. To make such improvements, combinations of PLA with two nanofillers, laurate‐intercalated Mg‐Al layered double hydroxide (LDH‐C₁₂) and a cationic organomodified montmorillonite (MMT) clay (Cloisite® 30B), were investigated. The dispersion of these fillers in PLA by melt processing was explored using two methods, either by mixing the nanofillers with PLA granulate immediately before extrusion or by preparation and subsequent dilution of PLA‐nanofiller masterbatches. After melt processing of these materials, PLA molecular weight, thermal stability, film transparency, morphology, and permeability characteristics were determined. Direct addition of LDH‐C₁₂ drastically reduced the PLA molecular weight. Although this reduction in molecular weight was still very significant, it was less when a PLA/LDH‐C₁₂ masterbatch was processed. In contrast, there was no significant reduction in PLA molecular weight when processing with Cloisite® 30B. However, film transparency was compromised when either LDH or MMT nanofillers were used. Evidence from DSC analyses showed a significant increase in heat of fusion when LDH‐C₁₂ was dispersed in PLA compared with Cloisite® 30B, likely indicating a difference in nucleating properties. Complementary optical purity analyses suggested that racemization as a result of processing could influence the PLA crystallinity as determined by DSC in certain cases. A reduction in thermal stability when incorporating LDH‐C₁₂ could be a direct result of PLA molecular weight reduction. XRD and TEM analyses showed that both Cloisite® 30B‐ and LDH‐C₁₂‐based PLA composites yielded exfoliated and intercalated morphologies, but nanofiller agglomeration was also seen when LDH‐C₁₂ was used. PLA/Cloisite® 30B nanocomposite films exhibited significant enhancement in oxygen and water vapor barrier properties, but no such improvement was found in PLA/LDH‐C₁₂ nanocomposite films. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

C5-DNA methyltransferase inhibitors: from screening to effects on zebrafish embryo development

DNA methylation is involved in the regulation of gene expression and plays an important role in normal developmental processes and diseases, such as cancer. DNA methyltransferases are the enzymes responsible for DNA methylation on the position 5 of cytidine in a CpG context. In order to identify and characterize novel inhibitors of these enzymes, we developed a fluorescence-based throughput screening by using a short DNA duplex immobilized on 96-well plates. We have screened 114 flavones and flavanones for the inhibition of the murine catalytic Dnmt3a/3L complex and found 36 hits with IC(50) values in the lower micromolar and high nanomolar ranges. The assay, together with inhibition tests on two other methyltransferases, structure-activity relationships and docking studies, gave insights on the mechanism of inhibition. Finally, two derivatives effected zebrafish embryo development, and induced a global demethylation of the genome, at doses lower than the control drug, 5-azacytidine.     

Numerical study of the coupling between reaction and mass transfer for liquid‐liquid slug flow in square microchannels

While the benefits of miniaturization on processes have been widely demonstrated, its impact on microfluidics and local mechanisms such as mass transfer is still little understood. The coupling between reaction and mass transfer in microchannels is simulated for liquid‐liquid slug flow. First, the extrapolation to confined flow of the classical model used to calculate interfacial mass fluxes in reactive infinite media was studied. This model consists in estimating transferred fluxes between two phases as a function of the enhancement factor E. Its expression depends on the model used to represent interfacial mass transfer. In infinite media, Lewis and Whitman's stagnant film theory is generally preferred for its simplicity and its reliability. In the case of confined slug flow, the limitation of such a model to predict interfacial fluxes is highlighted. Second, the case of liquid‐liquid competitive consecutive reactions in microchannels is considered. The unfavorable impact of the length between droplets on selectivity is emphasized. This is a direct consequence of mass‐transport mechanisms in microchannels. © 2010 American Institute of Chemical Engineers AIChE J, 2011     

Turbulent liquid–liquid dispersion in SMV static mixer at high dispersed phase concentration

The aim of this paper is to investigate the influence of physico-chemical parameters on liquid–liquid dispersion at high dispersed phase concentration in Sulzer SMV™ mixer. Four different oil-in-water systems involving two different surfactants are used in order to evaluate the effect of interfacial tension, densities and viscosities ratio on mean droplets size diameters. Moreover the influence of the dispersed phase concentration on the pressure drop as well as on the droplet size distribution is investigated. Two different droplets size distribution analysis techniques are used in order to compare the resulting Sauter mean diameters. The comparison between residence time in the mixer and surfactants adsorption kinetics leads to take into account the evolution of the interfacial tension between both phases at short times. Finally experimental results are correlated as a function of dimensionless Reynolds and Weber numbers.     

Application of the guanidine-acylguanidine bioisosteric approach to argininamide-type NPY Y₂ receptor antagonists

Strongly basic groups such as guanidine moieties are crucial structural elements, but they compromise the drug‐likeness of numerous biologically active compounds, including ligands of G‐protein‐coupled receptors (GPCRs). As part of a project focused on the search for guanidine bioisosteres, argininamide‐type neuropeptide Y (NPY) Y₂ receptor (Y₂R) antagonists related to BIIE0246 were synthesized. Starting from ornithine derivatives, N^G‐acylated argininamides were obtained by guanidinylation with tailor‐made mono‐Boc‐protected N‐acyl‐S‐methylisothioureas. The compounds were investigated for Y₂R antagonism (calcium assays), Y₂R affinity, and NPY receptor subtype selectivity (flow cytometric binding assays). Most of the N^G‐substituted (S)‐argininamides showed Y₂R antagonistic activities and binding affinities similar to those of the parent compound, whereas N^G‐acylated or ‐carbamoylated analogues with a terminal amine were superior (Y₂R: K_i and K_B values in the low nanomolar range). This demonstrates that the basicity of the compounds, although 4–5 orders of magnitude lower than that of guanidines, is sufficient to form key interactions with acidic amino acids of the Y₂R. The acylguanidines bind with high affinity and selectivity to Y₂R over the Y₁, Y₄, and Y₅ receptors. As derivatization of the amino group is tolerated, these compounds can be considered building blocks for the preparation of versatile fluorescent and radiolabeled pharmacological tools for in vitro studies of the Y₂R. The results support the concept of bioisosteric guanidine–acylguanidine exchange as a broadly applicable approach to retain pharmacological activity despite decreased basicity.