Biological study of the effect of water soluble [N-(2-hydroxybenzyl)-L-aspartato] gallium complexes on breast carcinoma and fibrosarcoma cells.

Two water soluble gallium complexes described as [Ga(III)LCl], where L is the deprotonated form of N-2-hydroxybenzyl aspartic acid derivatives, were synthesized and characterized by (1)H NMR, (13)C NMR, FT-IR, mass spectrometry, and elemental analysis. The 2-(5-chloro-2-hydroxybenzylamino)succinic acid derivative (GS2) has been found to be a promising anticancer drug candidate. This compound was found to be more cytotoxic against human breast carcinoma MDA-MB231 and fibrosarcoma HT-1080 cell lines than the unsubstituted derivative and GaCl3. GS2 was able to induce apoptosis through downregulation of AKT phosphorylation, G2M arrest in cell cycle, and caspase 3/7 pathway. This gallium complex was found to induce an increase in mitochondrial ROS level in HT-1080 cells but not in MDA-MB231 cells. This suggests that the mechanism of action of GS2 would not be mediated by the drug-induced oxidative stress but probably by directly and indirectly inhibiting the AKT cell-signaling pathway.

Development of a memetic clustering algorithm for optimal spectral histology: application to FTIR images of normal human colon.

The coupling between Fourier-transform infrared (FTIR) imaging and unsupervised classification is effective in revealing the different structures of human tissues based on their specific biomolecular IR signatures; thus the spectral histology of the studied samples is achieved. However, the most widely applied clustering methods in spectral histology are local search algorithms, which converge to a local optimum, depending on initialization. Multiple runs of the techniques estimate multiple different solutions. Here, we propose a memetic algorithm, based on a genetic algorithm and a k-means clustering refinement, to perform optimal clustering. In addition, this approach was applied to the acquired FTIR images of normal human colon tissues originating from five patients. The results show the efficiency of the proposed memetic algorithm to achieve the optimal spectral histology of these samples, contrary to k-means.

Probing in vitro ribose induced DNA-glycation using Raman microspectroscopy.

To identify and characterize glycation, induced modifications of DNA are crucial toward understanding their functional significance due to their significant role in the long term control of aging and age-related diseases. In this study, we present the ability of Raman microspectroscopy as a novel analytical technique for a rapid and reliable identification of glycated DNA in a reagent-free manner. We have demonstrated that this technique has potential to provide very small conformational modifications. The combination of principal component analysis (PCA) and two-dimensional (2D) correlation spectroscopy has assisted us to explore in vitro DNA-glycation and provide more insights into the dynamics of the DNA-glycation process in an easier fashion. PCA analysis of Raman spectra shows a clear discrimination between native and glycated DNA samples. On the other hand, 2D correlation Raman analysis provides sequential order of the mechanism of the DNA-glycation process, and most likely, it occurs in the following sequence: Structural modifications of individual nucleobases (G > A > C) → DNA backbone modifications → partial transition of DNA conformations (A to B form). Our observations clearly suggest that the structure of DNA is altered, i.e., a partial transition of DNA backbone conformation from A to B form when glycated, but does not induce any final transition in DNA double helix conformation, and eventually, DNA presents in an intermediate A-B form, more toward the B form.

A microscopic and macroscopic study of aging collagen on its molecular structure, mechanical properties, and cellular response.

During aging, collagen structure changes, detrimentally affecting tissues' biophysical and biomechanical properties due to an accumulation of advanced glycation end-products (AGEs). In this investigation, we conducted a parallel study of microscopic and macroscopic properties of different-aged collagens from newborn to 2-yr-old rats, to examine the effect of aging on fibrillogenesis, mechanical and contractile properties of reconstituted hydrogels from these collagens seeded with or without fibroblasts. In addition to fibrillogenesis of collagen under the conventional conditions, some fibrillogenesis was conducted alongside a 12-T magnetic field, and gelation rate and AGE content were measured. A nondestructive indentation technique and optical coherence tomography were used to determine the elastic modulus and dimensional changes, respectively. It was revealed that in comparison to younger specimens, older collagens exhibited higher viscosity, faster gelation rates, and a higher AGE-specific fluorescence. Exceptionally, only young collagens formed highly aligned fibrils under magnetic fields. The youngest collagen demonstrated a higher elastic modulus and contraction in comparison to the older collagen. We conclude that aging changes collagen monomer structure, which considerably affects the fibrillogenesis process, the architecture of the resulting collagen fibers and the global network, and the macroscopic properties of the formed constructs.

Development of a hierarchical double application of crisp cluster validity indices: a proof-of-concept study for automated FTIR spectral histology.

Fourier-transform infrared (FTIR) spectral imaging is currently used as a non-destructive and label-free method for analyzing biological specimens. However, to highlight the different tissue regions, unsupervised clustering methods are commonly used leading to a subjective choice of the number of clusters. Here, we develop a hierarchical double application of 9 selected crisp cluster validity indices (CCVIs) using K-Means clustering. This approach when tested first on an artificial dataset showed that the indices Pakhira-Bandyopadhyay-Maulik (PBM) and Sym-Index (SI) perfectly estimated the expected 9 sub-clusters. Then, the concept was applied to a real dataset consisting of FTIR spectral images of normal human colon tissue samples originating from 5 patients. PBM and SI were revealed to be the most efficient indices that correctly identified the different colon histological components including crypts, lamina propria, muscularis mucosae, submucosa, and lymphoid aggregates. In conclusion, these results strongly suggest that the hierarchical double CCVI application is a promising method for automated and informative spectral histology.

Probing non-enzymatic glycation of type I collagen: a novel approach using Raman and infrared biophotonic methods.

BACKGROUND: Non-enzymatic glycation is the main post-translational modification of long-life proteins observed during aging and physiopathological processes such as diabetes and atherosclerosis. Type I collagen, the major component in matrices and tissues, represents a key target of this spontaneous reaction which leads to changes in collagen biomechanical properties and by this way to tissue damages. METHODS: The current study was performed on in vitro glycated type I collagens using vibrational microspectroscopies, FT-IR and Raman, to highlight spectral features related to glycation effect. RESULTS AND CONCLUSIONS: We report a conservation of the triple-helical structure of type I collagen and noticeable variations in the exposure of proline upon glycation. Our data also show that the carbohydrate band can be a good spectroscopic marker of the glycation level, correlating well with the fluorescent AGEs formation with sugar addition. GENERAL SIGNIFICANCE: These non-invasive and label-free methods can shed new light on the spectral features of glycated collagens and represent an effective tool to study changes in the extracellular matrix observed in vivo during aging or on the advent of a pathological situation.

Microfluidity mapping using fluorescence correlation spectroscopy: a new way to investigate plasma membrane microorganization of living cells.

Diffusion time distribution analysis has been employed to highlight the microfluidity fingerprint of plasma membrane of living cells. Diffusion time measurements were obtained through fluorescence correlation spectroscopy performed at the single cell level, over various eukaryotic cell lines (MCF7, LR73, KB3.1, MESSA and MDCKII). The nonsymmetric profile of the diffusion time distributions established experimentally, is discussed according to Monte Carlo simulations, which reproduce the diffusion of the fluorescent probe in heterogeneous membrane.

Automation of an algorithm based on fuzzy clustering for analyzing tumoral heterogeneity in human skin carcinoma tissue sections.

This study aims to develop a new FT-IR spectral imaging of tumoral tissue permitting a better characterization of tumor heterogeneity and tumor/surrounding tissue interface. Infrared (IR) data were acquired on 13 biopsies of paraffin-embedded human skin carcinomas. Our approach relies on an innovative fuzzy C-means (FCM)-based clustering algorithm, allowing the automatic and simultaneous estimation of the optimal FCM parameters (number of clusters K and fuzziness index m). FCM seems more suitable than classical 'hard' clusterings, as it permits the assignment of each IR spectrum to every cluster with a specific membership value. This characteristic allows differentiating the nuances in the assignment of pixels, particularly those corresponding to tumoral tissue and those located at the tumor/peritumoral tissue interface. FCM images permit to highlight a marked heterogeneity within the tumor and characterize the interconnection between tissular structures. For the infiltrative tumors, a progressive gradient in the membership values of the pixels of the invasive front was also revealed.

Raman imaging of single living cells: probing effects of non-cytotoxic doses of an anti-cancer drug.

Identifying cell response to a chemotherapy drug treatment, in particular at the single cell level, is an important issue in patient management. This study aims at evaluating the effect of gemcitabine on single living cells using micro-Raman imaging. We used as a model the non-small lung cancer cell line, Calu-1, exposed to cytostatic doses (1 nM to 1 µM for 24 h and 48 h) of gemcitabine, an antitumor drug currently used in the treatment of lung cancer. Following drug treatment as a function of doses and incubation times, the Raman maps of single living cells were acquired. Cell biomolecules (DNA, RNA, and proteins) were chemically extracted and their spectral signatures used to evaluate their respective distribution in the cellular spectral information of control and treated cells. The quantification of these distributions reveals a significant effect of 100 nM gemcitabine at 48 h incubation (concomitant decrease of nucleic acids and increase of proteins). PCA analyses performed both on nuclear and extracted biomolecules spectra show a time-dependent effect of the drug. These promising results reveal that effects of subtoxic doses can be monitored at the single cell level highlighting the importance of such studies for clinical applications.

IR spectral imaging of secreted mucus: a promising new tool for the histopathological recognition of human colonic adenocarcinomas.

AIMS: During colonic carcinogenesis, mucin-type glycoproteins are known to undergo quantitative and qualitative alterations. The aim of this study was to determine the value of infrared (IR) spectral histology for the histopathological recognition of colonic adenocarcinomas based on mucin-associated IR spectral markers. METHODS AND RESULTS: Paraffin-embedded tissue sections of normal human colon and adenocarcinomas were analysed directly by IR-microspectroscopy (IR-MSP), without prior chemical dewaxing. IR-MSP imaging combined with multivariate analysis permitted the construction of IR colour-coded images of the tissue sections providing spatially resolved biochemical information. This allowed localization of mucin-rich areas and provided label-free spectral-based staining of secreted mucus related to the biochemical heterogeneity of its mucin content. IR images of secreted mucus display the same spectral clusters in both normal and adenocarcinomatous colonic tissues, but with significant differences in surface percentages. Such differences allow a distinction between these two tissue types. Spectral variations associated with changes of mucin secondary structure were the most accurate mucus spectral marker for discriminating between normal colon and adenocarcinomas in the sample set. CONCLUSIONS: IR-MSP imaging provides a new type of histology, independent of visual morphology, presenting tremendous possibilities for discovery and clinical monitoring of cancer markers.