Automated Morphological and Morphometric Analysis of Mass Spectrometry Imaging Data: Application to Biomarker Discovery

Mass spectrometry imaging datasets are mostly analyzed in terms of average intensity in regions of interest. However, biological tissues have different morphologies with several sizes, shapes, and structures. The important biological information, contained in this highly heterogeneous cellular organization, could be hidden by analyzing the average intensities. Finding an analytical process of morphology would help to find such information, describe tissue model, and support identification of biomarkers. This study describes an informatics approach for the extraction and identification of mass spectrometry image features and its application to sample analysis and modeling. For the proof of concept, two different tissue types (healthy kidney and CT-26 xenograft tumor tissues) were imaged and analyzed. A mouse kidney model and tumor model were generated using morphometric – number of objects and total surface – information. The morphometric information was used to identify m/z that have a heterogeneous distribution. It seems to be a worthwhile pursuit as clonal heterogeneity in a tumor is of clinical relevance. This study provides a new approach to find biomarker or support tissue classification with more information.

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Quantification of plasma phospholipids by ultra performance liquid chromatography tandem mass spectrometry

We describe a fast and robust ultra performance liquid chromatography tandem mass spectrometry method for the quantification of phospholipid (PL) species in EDTA-plasma samples. We quantified total phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylcholine (LPC), and sphingomyelin (SM) and several species within these classes using one or two external calibrators and one internal standard for each class. Inter-assay coefficients of variation were <10% for the most abundant species and <20% for all quantified PC, LPC, and SM species and the three most abundant PE species. Coefficients of linear regression were R ² > 0.98. Mean recoveries were between 83% and 123%. The limits of detection were 0.37 μmol/L for PC, 4.02 μmol/L for LPC, 3.75 μmol/L for PE, and 0.86 μmol/L for SM. Quantification was linear over the physiological ranges for PE, LPC, and SM and up to 500 μmol/L for PC. The concentrations of PLs in the plasma of healthy donors yielded results that were comparable with those of previous works.     

The Effects of Prolonged Storage on ARPE-19 Cells Stored at Three Different Storage Temperatures

This study aimed to investigate how prolonged storage of adult retinal pigment epithelial (ARPE-19) cell sheets affects cell metabolism, morphology, viability, and phenotype. ARPE-19 cell sheets were stored at three temperatures (4 °C, 16 °C, and 37 °C) for three weeks. Metabolic status and morphology of the cells were monitored by sampling medium and examining cells by phase-contrast microscopy, respectively, throughout the storage period. Cell viability was analyzed by flow cytometry, and phenotype was determined by epifluorescence microscopy after the storage. Lactate production and glucose consumption increased heavily, while pH dropped considerably, through storage at 37 °C compared to 4 °C and 16 °C. During storage, morphology started to deteriorate first at 4 °C, then at 37 °C, and was maintained the longest at 16 °C. Viability of the cells after three weeks of storage was best preserved at 16 °C, while cells stored at 4 °C and 37 °C had reduced viability. Dedifferentiation indicated by reduced expression of retinal pigment epithelium-specific protein 65 (RPE65), zonula occludens protein 1 (ZO-1), and occludin after three weeks of storage was noticed in all experimental groups compared to control. We conclude that storage temperature affects the metabolic status of ARPE-19 cells and that 16 °C reduces metabolic activity while protecting viability and morphology.     

Roles of diffusion and activation barrier on the appearance of Marcus inversion behavior: a study of a photoinduced electron-transfer reaction in aqueous triblock copolymer (p123) micellar solutions

Photoinduced electron transfer (ET) reactions between amines and a series of coumarins have been investigated using fluorescence-quenching measurements in aqueous P123 triblock copolymer micellar solutions. Fluorescence spectral characteristics and fluorescence anisotropy measurements indicated a nearly similar microenvironment for all of the coumarins used in P123 micelles. Substantial quenching of coumarin fluorescence in the presence of amines has been observed. The quenching rates (k(q)(TR)) are largely reduced in the P123 micelle as compared to those in other micelles (sodium dodecyl sulfate (SDS), Triton-X 100 (TX-100), cetyl trimethyl ammonium bromide (CTAB), and dodecyl trimethyl ammonium bromide (DTAB)), which is probably due to larger coumarin-amine separations in the micellar phase. The k(q)(TR) values, when plotted against free energy changes (DeltaG degrees), follow a Marcus predicted bell-shaped correlation. The estimated activation energy for the ET reactions follow an inverse bell-shaped correlation with DeltaG degrees. Present results indicate that the appearance of Marcus inversion is primarily related to the changes in the activation barrier, as predicted from the Marcus ET theory. As the k(q)(TR) values are higher than the estimated bimolecular diffusional rate constant, the role of reactant diffusion on the quenching kinetics in the P123 micelle is negligible. The appearance of Marcus inversion at unexpectedly lower exergonicity has been rationalized on the basis of slow solvent relaxation and by the application of the two-dimensional ET (2DET) theory. Critical analysis of the present results establishes that the inversion in the ET rates at high exergonicity is not due to the alteration in the diffusion parameters of the reactants, as has been suggested in some recent reports. Instead, it is evident that the inversion in quenching rates at high exergonicity is due to the alteration in the activation barrier for the ET reactions.     

Influence of electrolytes on the microenvironment of F127 triblock copolymer micelles: a solvation and rotational dynamics study of coumarin dyes

Dynamic Stokes' shift and fluorescence anisotropy measurements of coumarin 153 (C153) and coumarin 151 (C151) as fluorescence probes have been carried out to understand the influence of electrolytes (NaCl and LiCl) on the hydration behavior of aqueous (ethylene oxide)100-(propylene oxide)70-(ethylene oxide)100 (EO100-PO70-EO100, F127) block copolymer micelles. A small blue shift in the fluorescence spectra of C153 has been observed in presence of electrolytes due to the dehydration of the oxyethylene chains in the PEO-PPO region, although fluorescence spectra of C151 remain unaltered. The close vicinity of bulk water for C151 probably negates the effect of dehydration in the PEO region. Fluorescence anisotropy measurements indicate a gradual increase in microviscosity with electrolyte concentrations. The partial collapse of copolymer blocks in the presence of electrolytes has been suggested as a reason for the increase in microviscosity along with the strong hydration of ions in the corona region. The interplay between the ion hydration and the mechanically trapped water content, and specific interaction of ions, such as complexation of Li+ ions with the copolymer block, is found to control solvation dynamics in the corona region. In addition to that, it has been established that Na+ ions reside deep into the corona region whereas Li+ ions prefer to reside closer to the surface. Owing to its higher lyotropicity, LiCl influences the corona hydration to a greater extent than NaCl and sets in micelle-micelle interaction above the 2 M LiCl concentration, as reflected in the saturation of solvation time constants. The formation of larger clusters of F127 micelles above 2 M LiCl has been confirmed by dynamic light scattering measurements; however, such cluster formation is not evident with NaCl.     

Structurally original oxathioethers macrocycles containing an exocyclic double-bond: synthesis,characterization, reactivity,and coordination

New oxathioethers macrocycles have been synthesized and characterized. Each macrocycle consists in structurally defined ether and thioether moieties and an exocyclic double-bond (2a–c) or a hydroxymethyl group (3a–c). Macrocycles (2a–c) have been synthesized by reaction of dianions of thioethers diols (1a–c) with 3-chloro-2-chloromethylprop-1-ene. Their hydroboration/oxidation led to corresponding primary alcohols (3a–c). Structures of compounds (2b) and (3a) have been determined by X-ray diffraction. The reactivity of the hydroxyl group allowed the preparation of oxathioethers macrocycles bearing a polyether chain or a benzyl group (4a,b) and the synthesis of new bicyclic sandwich-type compounds (5a,b). The ability of these functionalized macrocycles to coordinate to palladium has been investigated.     

Enhanced visible light emission from Co doped ZnS nanoparticles

ZnS nanoparticles with Co²⁺ doping have been prepared at room temperature through a soft chemical route, namely the chemical co-precipitation method. The nanostructures of the prepared nanoparticles have been analyzed using X-ray diffraction (XRD), scanning electron microscope (SEM), transmission electron microscope (TEM), selected-area electron diffraction (SAED), and UV-vis spectrophotometer. The sizes of as prepared nanoparticles are found to be in 1–4 nm range. Room-temperature photoluminescence (PL) spectrum of the undoped sample exhibits emission in the blue region with multiple peaks under UV excitation. On the other hand, in the Co²⁺ doped ZnS samples enhanced visible light emissions with emission intensities of ~35 times larger than that of the undoped sample are observed under the same UV excitation wavelength of 280 nm.     

Radioprotective activity of metalladithioacetals derived from N‐substituted naphthylethylimidazoline

A number of organosilicon and organogermanium derivatives of N‐substituted 2‐[1‐(1‐naphthyl)ethyl]‐2‐imidazoline have been reported and the toxicity of these compounds has been determined in mice. In this paper we report the evaluation of the radioprotective activity of new sila‐ and germa‐dithioacetals derived from N‐substituted 2‐[1‐(1‐naphthyl)ethyl]‐2‐imidazoline. Copyright © 2003 John Wiley & Sons, Ltd.     

Identification of Non-Macrocyclic Small Molecule Inhibitors against the NS3/4A Serine Protease of Hepatitis C Virus through in Silico Screening

Drug discovery and design for inhibition of the Hepatitis C Virus (HCV) NS3/4A serine protease is a major challenge. The broad, shallow, and generally featureless nature of the active site makes it a difficult target for "hit" selection especially using standard docking programs. There are several macrocyclic NS3/4A protease inhibitors that have been approved or are in clinical trials to treat chronic HCV (alone or as combination therapy), but most of the current therapies for HCV infection have untoward side effects, indicating a continuing medical need for the discovery of novel therapeutics with improved efficacy. In this study, we designed and implemented a two-tiered and progressive docking regime that successfully identified five non-macrocyclic small molecules that show inhibitory activity in the low micromolar range. Of these, four compounds show varying inhibition against HCV subgenotypes 1b, 1a, 2a, and 4d. The top inhibitor (3) has an IC(50) value of 15 μM against both subgenotypes 1b and 2a of the NS3/4A protease enzyme. Another inhibitor, 1, inhibits all four subgenotypes with moderate activity, showing highest activity for genotype 2a (24 μM). The five inhibitors presented in this study could be valuable candidates for future hit to lead optimization. Additionally, enzyme-inhibitor interaction models presented herein provide key information regarding structural differences between the active sites of the NS3/4A protease of the HCV subgenotype 1a and 1b that might explain the variable inhibitory activity between subgenotypes of the small molecule inhibitors identified here.     

Specification and estimation of multiple output technologies: A primal approach

This paper addresses specification and estimation of multiple-outputs and multiple-inputs production technology in the presence of technical inefficiency. The primary focus is on the primal formulations. Several competing specifications such as production function, input (output) distance function, input requirement function are considered. We show that all these specifications come from the same transformation function and are algebraically identical. We also show that: (i) unless the transformation function is separable (i.e., outputs are separable from inputs), the input (output) ratios in the input (output) distance function can not be treated as exogenous (uncorrelated with technical inefficiency) resulting inconsistent estimates of the input (output) distance function parameters. (ii) Even if input (output) ratios are exogenous, estimation of the input (output) distance function will result in inconsistent parameter estimates if outputs (inputs) are endogenous. We address endogeneity and instrumental variable issues in details in the context of flexible (translog) functional forms. Estimation of several specifications using both single and system approaches are discussed using Norwegian dairy farming data.