Luca Quaroni

A molecular insight into the nature of crystallographic mismatches in self-assembled fibrillar networks under non-isothermal crystallization conditions

The lengths of the 12-hydroxystearic acid (12HSA) fibers are influenced by crystallographic mismatches resulting from the incorporation of 12HSA monomers into the crystal lattice in an imperfect manner. On a molecular level, this can be differentiated using synchrotron Fourier transform infrared (FTIR) spectroscopy by monitoring the change in area of the 1700 cm−1 and 3200 cm−1 peaks, corresponding, respectively, to the dimerization of the carboxylic acid groups and hydroxyl non-covalent interactions, during crystallization. The crystallographic mismatch is attributed to a plateau in the apparent rate constant for the dimerization of the carboxylic acid head groups while the hydroxyl interactions linearly increase as a function of cooling rate (ϕ). The rate constant for hydroxyl interactions linearly increases as a function of cooling rate while a plateau is observed for the rate of dimerization at cooling rates between 5 and 7 °C min−1. At cooling rates greater than 5 to 7 °C min−1 12HSA monomers do not effectively dimerize before being incorporated into the crystal lattice causing crystal imperfections impeding linear epitaxial crystal growth and produces branched fibers. At slow cooling rates (i.e., less than 5 to 7 °C min−1), long fibers are produced with a fractal dimension between 0.95 and 1.05 and for rapid cooling rates (i.e., greater than 5 to 7 °C min−1) short branched fibers are produced with a fractal dimension between 1.15 and 1.32.

Characterization of Barrett esophagus and esophageal adenocarcinoma by Fourier-transform infrared microscopy.

The objective of this exploratory study was to evaluate the feasibility of using Fourier-Transform Infrared (FTIR) spectromicroscopy to characterize formalin-fixed, paraffin-embedded human esophageal tissues. Matched histologically normal esophageal squamous epithelium (NS), premalignant Barrett esophagus (BE), and primary esophageal adenocarcinoma (EADC) tissues, each defined according to strict clinicopathologic criteria, were obtained from patients who underwent esophageal resection. Using confocal IR microscopy, measurements in the mid-IR spectral region were carried out in transflection configuration, scanning regions of interest in 15 microm steps. A multidimensional dataset reporting the spectroscopic properties at each sampled point were analyzed by performing a hierarchical cluster analysis on the second derivative of spectral traces. Normal esophageal epithelia were characterized by a few well defined regions, mostly of large size (tens of contiguous pixels), which correlated with tissue histology, specifically the basal cell layer. BE tissues had characteristic regions localized to gland crypts, ranging in size from one pixel to a few tens of pixels, which displayed IR spectra with defined absorption features characteristic of glycoproteins. The incorporation of synchrotron light to improve the resolution of individual cells in BE tissues has demonstrated that these glycoproteins are associated with goblet cells, the characteristic cell type defining BE. Whereas the highly fragmented regions identified in EADC likely reflect tumor heterogeneity, FTIR mapping would appear to be a potentially useful technique to identify premalignant BE tissues. The technical feasibility of using FTIR to characterize formalin-fixed, paraffin-embedded human esophageal tissues demonstrates the potential of this technique to study archival human BE tissue specimens via automated screening techniques.

Detection of lipid phase coexistence and lipid interactions in sphingomyelin/cholesterol membranes by ATR-FTIR spectroscopy.

The phase behavior of binary mixtures of egg sphingomyelin and cholesterol has been inspected by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy in the amide I band region of the spectrum. Because cholesterol does not have any major absorption bands in this region, effects seen in the spectra of mixtures of sphingomyelin and cholesterol can be attributed to the change in the lipid phase and to the interaction with cholesterol. It is shown that the temperature dependence of the overall bandwidth of the amide I band displays a phase-specific behavior. In addition, it is observed that the amide I band for a sample exhibiting phase coexistence can be described by a linear combination of the spectra of the individual lipid phases. Description of changes in the amide I band shape and by that the study of possible hydrogen bonding interactions of sphingomyelin with cholesterol was assisted by the use of curve fitting. It turns out that the presence of hydrogen bonding between hydroxyl group of cholesterol and carbonyl group of sphingomyelin is obscured by the complexity of different possible hydrogen bonding and coupling between the N-H (N-D) and the CO group vibrations.

Measurement of Molecular Orientation in a Subcellular Compartment by Synchrotron Infrared Spectromicroscopy

Subcellular dichroism at the diffraction limit: Synchrotron infrared spectromicroscopy provides a snapshot of the axial molecular architecture in the outer segment of an intact retinal rod cell (see picture), which reinforces the role of this technique as a tool for in vivo studies of biomolecular structure–function relationships.

Shining light on Barrett's esophagus

. However, over the past 30 years, a marked change in the epidemiology of esophageal malignancy has been reported in many Western populations. While the frequency of esophageal squamous cell car-cinoma has remained relatively stable, inci-dence rates for primary esophageal adeno-carcinoma (EADC) and adenocarcinomas involving the esophagogastric junction (cardia) have increased steadily, exceeding that of any other human solid tumor

Measurement of ethanol formation in single living cells of Chlamydomonas reinhardtii using synchrotron Fourier Transform Infrared spectromicroscopy

We demonstrate the capability of Fourier‐Transform Infra‐Red (FITR) spectroscopy to detect metabolite formation by the unicellular algae Chlamydomonas reinhardtii in solution. We show that using a synchrotron source in the microscopy configuration provides a sufficient s/n ratio to detect small molecular species accumulating at a single cell, allowing an increased sensitivity relative to measurements of bulk cultures. The formation of small molecular species, including ethanol and at least one carbonyl containing compound, can be detected with a time resolution of the order of one minute.

Two‐Dimensional Correlation Spectroscopy Analysis for the Recovery of Weak Bands from Time‐Resolved Infrared Spectra of Single Cells

We show that Generalized Two‐Dimensional Correlation Spectroscopy analysis can be used to extract weak spectral changes, of amplitude comparable or below noise levels, in time‐resolved spectra with relatively low signal‐to‐noise levels. We propose to use the technique as a tool for the analysis the Fourier Transform Infrared spectra of dynamic events in cellular samples measured at diffraction limited spatial resolution, where the presence of strong and complex background absorption and low signal‐to‐noise levels severely limit the possibility of extracting detailed spectroscopic information.

Abstract 3363: Identification and characterization of stem-like cells in human esophageal epithelial and adenocarcinoma cells

The aim of this study was to identify and characterize stem-like cells in human normal esophageal epithelial and adenocarcinoma cell lines. The immortalized esophageal epithelial cell line, Het-1A, and the primary esophageal adenocarcinoma (EADC) cell line, OE33, were first studied by flow cytometry and immunofluorescence using a panel of antibodies to putative stem cell biomarkers which included CD34, CD44, CD44/CD24, CD117/integrin α6, Musashi I, EpiCam, and Oct 4. Fluorescence activated cell sorting (FACS) was then used to isolate 15% of cells with the highest CD44 staining (CD44 high ) or reactive oxygen species (ROS) (ROS high ) and 15% of cells with the lowest (including negative) CD44 staining (CD44 low ) or ROS (ROS low ). Each cell population was further characterized using sphere-forming assays, soft agar clonogenic assays, flow cytometry for ROS, and synchrotron mid-infrared (IR) spectromicroscopy. CD44 positivity was found in 90% of Het-1A and 75% of OE33 cells overall. For both Het-1A and OE33 cell lines, spheroid formation (mean±SD/5000 cells) was significantly higher in cells expressing high levels of CD44 (Het-1A:138.33±2.08 vs. 97.67±1.53, p high (Het-1A: 15.33±4.51 vs. 8.00±1.00, p=0.051; OE33: 92.67±11.02 vs. 18.00±1.73, p low (Het-1A: 45.00±1.00 vs. 31.67±3.79, p high cells were found to express consistently lower levels of ROS compared with OE33 CD44 low cells (p We conclude that the higher rates of spheroid formation and clonogenicity of Het-1A and OE33 cells expressing high levels of CD44 (CD44 high ) and low levels of ROS (ROS low ) reflect an enrichment of stem-like cell populations in esophageal cell lines. However, the significant differences in ROS levels between CD44 high OE33 cells and CD44 high Het-1A cells suggests unique and biologically distinct pathways in stem-like cells derived from normal and malignant esophageal epithelia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3363.