Classification of Gemcitabine resistant Cholangiocarcinoma cell lines using synchrotron FTIR microspectroscopy

Cholangiocarcinoma (CCA), a cancer of bile duct epithelium, is a major health problem in Thailand especially in the northeast. Overall treatment outcomes have not shown much improvement because the disease is usually detected at an advanced stage and often shows chemotherapeutic resistance. High‐throughput Fourier Transform Infrared (FTIR) microspectroscopy can be used for cell classification and has the potential to diagnose cancer and possibly predict chemo‐response. This study was aimed to differentiate gemcitabine‐sensitive and gemcitabine‐resistant induction in two CCA cell lines (KKU‐M139 and KKU‐M214) and xenograft tissues using synchrotron‐FTIR microspectroscopy. Partial Least Squares Discriminant Analysis (PLS‐DA) could discriminate between chemo‐sensitive and chemo‐resistant cells in the FTIR fingerprint spectral region (1800–1000 cm^–1) with more than 90% sensitivity and specificity. The chemo‐resistant and chemo‐sensitive phenotypes were different in protein (amide I, amide II), lipids (carbonyl group and CH₃ deformation) and phosphodiester from nucleic acids. Additionally, spectra from xenograft tissues showed similar results to the cell line study with marked differences between chemo‐resistant and chemo‐sensitive CCA tissues, and PLS‐DA could discriminate the chemotherapeutic response with 98% sensitivity and specificity. This is the first study to demonstrate the use of FTIR microspectroscopy to assess chemo‐response both in vitro and in vivo.

     

Discriminating nevus and melanoma on paraffin-embedded skin biopsies using FTIR microspectroscopy

FTIR microspectroscopy, in combination with cluster analysis, has been used to characterise skin tissues, in order to discriminate cancerous from non-cancerous ones. The main objective of this in vitro study was to demonstrate the applicability of infrared spectral imaging to separate, on paraffinised biopsies, pigmented nevi (benign skin lesions) from melanomas (malignant skin lesions). Infrared spectra were collected from paraffin-embedded samples of nevi and melanomas, without deparaffinisation. Despite the important contribution of the paraffin in these spectra, it was possible to find meaningful and discriminating spectral regions. Spectral imaging was first performed to localize different skin layers (dermis and epidermis). Spectra extracted from the images were subjected to hierarchical classification algorithm, which allowed the discrimination of melanomas from the nevi, using selected spectral windows that correspond to vibrations of DNA and melanin content. The diversity of skin lesions and direct accessibility to the skin make this organ an interesting field of investigation using this technique.     

Studies on acute human infections using FTIR microspectroscopy and cluster analysis

A novel methodology for the diagnosis of acute infections using FTIR microspectroscopy (FTIR-MSP) data on blood components and cluster analysis is presented. Blood samples were collected from 11 patients suffering from various infections and 16 age-matched healthy human controls. Blood components such as white blood cells, red blood cells, and plasma were isolated using standard procedures and FTIR-MSP of these components was utilized. A cluster analysis of the FTIR spectra was performed. The spectra obtained from the three blood components of patients were different from those of controls. The FTIR spectra of white blood cells from patients suffering infections were significantly different from the controls. Cluster analyses of averaged FTIR-MSP spectra of white blood cells provided 100% classification between patients and healthy controls.     

Novel methodology for the follow-up of acute lymphoblastic leukemia using FTIR microspectroscopy

In this report, we present a novel spectroscopic method of follow-up during chemotherapy treatment for B- and T-cell childhood leukemia patients. We isolated peripheral lymphocytes from blood drawn from patients before and after the chemotherapy and collected Microscopic FTIR (FTIR-MC) spectra of the isolated lymphocytes. Our results showed that nucleic acids content decreased in both types of patients. Changes in phospholipids and proteins level could be observed. The overall effects of drugs administered to the patients can be understood at the molecular level using FTIR-MC and these results are expected to stimulate wider applications of spectroscopy in leukemia research.     

Synchrotron FTIR microspectroscopy of single natural silk fibers

Synchrotron FTIR (S-FTIR) microspectroscopy was used to monitor the silk protein conformation in a range of single natural silk fibers (domestic and wild silkworm and spider dragline silk). With the selection of suitable aperture size, we obtained high-resolution S-FTIR spectra capable of semiquantitative analysis of protein secondary structures. For the first time, we have determined from S-FTIR the β-sheet content in a range of natural single silk fibers, 28 ± 4, 23 ± 2, and 17 ± 4% in Bombyx mori, Antheraea pernyi, and Nephila edulis silks, respectively. The trend of β-sheet content in different silk fibers from the current study accords quite well with published data determined by XRD, Raman, and (13)C NMR. Our results indicate that the S-FTIR microspectroscopy method has considerable potential for the study of single natural silk fibers.     

FTIR microspectroscopy study of composition fluctuations in extruded amylopectin-gelatin blends

The spatial variation in the composition of nonexpanded biopolymer blends prepared by extrusion of mixtures of gelatin with either native or pregelatinized waxy maize starch was studied using a 30-microm aperture FTIR microspectroscopy technique. The ratio of the areas of the "saccharide" bands (953-1180 cm(-1)) and the amide I and II bands (1483-1750 cm(-1)) was used to monitor the relative distributions of the two components of the blend. Two calibration methods were used to obtain amylopectin concentration values from the ratios of the IR bands. The results suggested a high degree of heterogeneity in these blends, despite the thorough mixing expected by twin-screw extrusion processing. The concentration fluctuations were greater for the blends produced by extruding gelatin and native waxy maize starch mixtures. This was in agreement with the reduced degree of conversion of the starch granules when extruded in the presence of gelatin. The FTIR 2-dimensional maps obtained suggested that in the blends produced from either native or pregelatinized starch at all concentrations studied (25/75, 50/50, and 75/25 amylopectin/gelatin) the gelatin constituted the continuous phase. The effect of the spatial resolution on the FTIR microspectroscopy results was considered and the proposed interpretation was verified by the use of polarized light microscopy and FTIR microspectroscopy acquired at higher spatial resolution (10 microm).     

FTIR microspectroscopy of malignant fibroblasts transformed by mouse sarcoma virus

Fourier transform infrared microspectroscopy (FTIR-MSP), which is based on the characteristic molecular vibrational spectra of cells, was used to investigate spectral differences between normal primary rabbit bone marrow (BM) cells and bone marrow cells transformed (BMT) by murine sarcoma virus (MuSV). Primary cells, rather than cell lines, were used for this research because primary cells are similar to normal tissue cells in most of their characteristics. Our results showed dramatic changes in absorbance between the control cells and MuSV124-transformed cells. Various biological markers, such as the phosphate level and the RNA/DNA obtained, based on the analysis of the FTIR-MSP spectra, also displayed significant differences between the control and transformed cells. Preliminary results suggested that the cluster analysis performed on the FTIR-MSP spectra yielded 100% accuracy in classifying both types of cells.     

Compositional mapping of the human dystrophin-encoding gene.

The genomes of warm-blooded vertebrates are mosaics of long DNA segments (> 300 kb, on the average), the isochores, homogeneous in GC levels, which belong to a small number of compositional families. In the present work, the human dystrophin-encoding gene, spanning more than 2.3 Mb in Giemsa band Xp21 (on the short arm of the X chromosome), was analyzed in its isochore organization by hybridizing cDNA probes, corresponding to eight contiguous segments of the coding sequence, on compositional fractions from human DNA. Five DNA regions of uniform (+/- 0.5%) GC content, separated by compositional discontinuities of about 2% GC, were found, so providing the first high-resolution compositional map obtained for a human genome locus and the first direct estimate of isochore size (360 kb to more than 770 kb, in the locus under consideration). One of the isochores contains 71% and another one 21% of deletion breakpoints found in patients suffering from Duchenne's and Becker's muscular dystrophies.     

Peptide mapping of proteins from acrylamide gels

A procedure is described by which proteins may be eluted from detergent gels, made radioactive by reaction with [¹²⁵I]sodium iodide, and analyzed by peptide mapping. The method permits a rapid and reliable means of identification of less than 10 μg of protein from a stained band.     

Direct evidence of lipid translocation between adipocytes and prostate cancer cells with imaging FTIR microspectroscopy

Various epidemiological studies show a positive correlation between high intake of dietary FAs and metastatic prostate cancer (CaP). Moreover, CaP metastasizes to the bone marrow, which harbors a rich source of lipids stored within adipocytes. Here, we use Fourier transform infrared (FTIR) microspectroscopy to study adipocyte biochemistry and to demonstrate that PC-3 cells uptake isotopically labeled FA [deuterated palmitic acid (D(31)-PA)] from an adipocyte. Using this vibrational spectroscopic technique, we detected subcellular locations in a single adipocyte enriched with D(31)-PA using the upsilon(as+s)(C-D)(2+3) (D(31)-PA): upsilon(as+s)(C-H)(2+3) (lipid hydrocarbon) signal. In addition, larger adipocytes were found to consist of a higher percentage of D(31)-PA of the total lipid found within the adipocyte. Following background subtraction, the upsilon(as)(C-D)(2+3) signal illuminated starved PC-3 cells cocultured with D(31)-PA-loaded adipocytes, indicating translocation of the labeled FA. This study demonstrates lipid-specific translocation between adipocytes and tumor cells and the use of FTIR microspectroscopy to characterize various biomolecular features of a single adipocyte without the requirement for cell isolation and lipid extraction.     

Amphetamine effects on brain protein structure and oxidative stress as revealed by FTIR microspectroscopy

Amphetamines are psychostimulants abused by man, that eventually leads to drug dependence. Amphetamine administration to rodents has been shown to provoke significant neurotoxicity involving dopaminergic nerve terminal degeneration. However, little information related to the effect of amphetamines on reactive oxygen species (ROS) production and neurotoxicity in brain is currently available. Herein we report the biochemical alterations of lipids and proteins in brain sections from amphetamine-treated rodents using infrared microspectroscopy, immunohistochemistry, and immunoblotting. The spectroscopic changes reveal for the first time the formation of beta-sheet-rich proteins in the cortex, but no significant protein alterations are visible in hippocampus region where hydroperoxide concentration is found to be lower relative to cortex. These result suggest that ROS generated by amphetamine-mediated oxidative stress induce formation beta-sheet-rich proteins which can be of amyloid beta-like character.     

Dense-core and diffuse Abeta plaques in TgCRND8 mice studied with synchrotron FTIR microspectroscopy

Plaques composed of the Abeta peptide are the main pathological feature of Alzheimer's disease. Dense-core plaques are fibrillar deposits of Abeta, showing all the classical properties of amyloid including beta-sheet secondary structure, while diffuse plaques are amorphous deposits. We studied both plaque types, using synchrotron infrared (IR) microspectroscopy, a technique that allows the chemical composition and average protein secondary structure to be investigated in situ. We examined plaques in hippocampal, cortical and caudal tissue from 5- to 21-month-old TgCRND8 mice, a transgenic model expressing doubly mutant amyloid precursor protein, and displaying impaired hippocampal function and robust pathology from an early age. Spectral analysis confirmed that the congophilic plaque cores were composed of protein in a beta-sheet conformation. The amide I maximum of plaque cores was at 1623 cm(-1), and unlike for in vitro Abeta fibrils, the high-frequency (1680-1690 cm(-1)) component attributed to antiparallel beta-sheet was not observed. A significant elevation in phospholipids was found around dense-core plaques in TgCRND8 mice ranging in age from 5 to 21 months. In contrast, diffuse plaques were not associated with IR detectable changes in protein secondary structure or relative concentrations of any other tissue components.     

Deposition and organisation of cell wall polymers during maturation of poplar tension wood by FTIR microspectroscopy

To advance our understanding of the formation of tension wood, we investigated the macromolecular arrangement in cell walls by Fourier transform infrared microspectroscopy (FTIR) during maturation of tension wood in poplar (Populus tremula x P. alba, clone INRA 717-1B4). The relation between changes in composition and the deposition of the G-layer in tension wood was analysed. Polarised FTIR measurements indicated that in tension wood, already before G-layer formation, a more ordered structure of carbohydrates at an angle more parallel to the fibre axis exists. This was clearly different from the behaviour of opposite wood. With the formation of the S₂ layer in opposite wood and the G-layer in tension wood, the orientation signals from the amorphous carbohydrates like hemicelluloses and pectins were different between opposite wood and tension wood. For tension wood, the orientation for these bands remains the same all along the cell wall maturation process, probably reflecting a continued deposition of xyloglucan or xylan, with an orientation different to that in the S₂ wall throughout the whole process. In tension wood, the lignin was more highly oriented in the S₂ layer than in opposite wood.     

Modified method for peptide mapping of collagen chains using cyanogen bromide-cleavage of protein within polyacrylamide gels

A highly efficient method for cyanogen bromide (CNBr)-mapping of collagen peptides is described. This method was developed based on polypeptide cleavage by CNBr within gel slices according to Barsh et al. [1981) Collagen Relat. Res. 1, 543-548). The proposed method has the following advantages: (i) Analysis of both radiolabeled and unlabeled collagens is possible; (ii) CNBr-cleavage of polypeptides is performed in gel pieces which contain individual bands; (iii) The peptide losses are minimized, offering a more complete analysis of collagens including the low molecular weight CNBr-peptides.     

Synchrotron-based FTIR microspectroscopy of chili resistance induced by Bacillus subtilis strain D604 against anthracnose disease

The aim of this study was to determine the resistance mechanisms of chili induced by the Bacillus subtilis strain D604 using synchrotron FTIR microspectroscopy (SR-FTIR). In this study, the strain D604 reduced anthracnose disease severity in chili plants by approximately 31.10%. The SR-FTIR spectral changes from the epidermis and mesophyll leaf tissue revealed higher integral areas for the C=O ester from lipids, lignin, or pectin (1770–1700?cm^?1) as well as polysaccharides (1200–900?cm^?1) in the treated samples of D606 and distilled water and then challenge inoculation with chili anthracnose pathogen, Colletotrichum acutatum. The secondary structure of the Amide I protein failed to convert from alpha helices (centered at 1650?cm^?1) to beta sheets (centered at 1600?cm^?1) in the mesophyll of samples not treated with D604. This study suggested that the strain D604 induced resistance against anthracnose pathogen in chili by inducing cellular changes related to defense compounds involved in plant defense mechanism.     

Multi-environment QTL mapping in blackcurrant (Ribes nigrum L.) using mixed models

The first genetic linkage map of blackcurrant, published by Brennan et al. (Euphytica 161:19–34, 2008), identified regions where quantitative trait loci (QTLs) for some important traits were located. The analysis was complicated by the fact that the mapping population was found to contain two subgroups, with segregation ratios consistent with these being crossed and selfed offspring. The QTL analysis was based on the trait mean over 3 years and focused on the crossed offspring. Here we proposed a mixed model multi-environment approach for this population. The 3 years are considered as three separate environments, the data from both the selfed and crossed offspring are combined and different residual terms are explored to model the correlation between the years. This permits tests for interactions between the QTLs, the year and the type of offspring (selfed or crossed). This is applied to re-analyse two important traits, anthocyanin concentration and budbreak. Several additional QTLs were identified, some affecting the trait in both the selfed and crossed offspring, others in just one.     

In situ monitoring of cell death using Raman microspectroscopy

We investigated the use of Raman microspectroscopy to monitor the molecular changes in human lung carcinoma epithelial cells (A549) when cell death was induced by a toxic chemical. We treated A549 cells with 100 microM Triton X-100 and carried out Raman microspectroscopy measurements in parallel with cell viability and DNA integrity assays at time points of 0, 24, 48, and 72 hours. We found that the important biochemical changes taking place during cell death, such as the degradation of proteins, DNA breakdown, and the formation of lipid vesicles, can be detected with Raman microspectroscopy. A decrease in the intensity of the O-P-O stretching Raman peak corresponding to the DNA molecule phosphate-sugar backbone at 788 cm(-1) indicated DNA disintegration, an observation which was confirmed by DNA integrity analysis. We also found a decrease in the intensity of the Raman peaks corresponding to proteins (1005 cm(-1), 1342 cm(-1)) and an increase in the concentration of lipids (1660 cm(-1), 1303 cm(-1)). These changes are the effects of the complex molecular mechanisms during the induction of cell death, such as protein cleavage due to the activation of caspases, followed by DNA fragmentation.     

Ultra-structural organisation of cell wall polymers in normal and tension wood of aspen revealed by polarisation FTIR microspectroscopy

Polarisation Fourier transform infra-red (FTIR) microspectroscopy was used to characterize the organisation and orientation of wood polymers in normal wood and tension wood from hybrid aspen (Populus tremula × Populus tremuloides). It is shown that both xylan and lignin in normal wood are highly oriented in the fibre wall. Their orientation is parallel with the cellulose microfibrils and hence in the direction of the fibre axis. In tension wood a similar orientation of lignin was found. However, in tension wood absorption peaks normally assigned to xylan exhibited a 90° change in the orientation dependence of the vibrations as compared with normal wood. The molecular origin of these vibrations are not known, but they are abundant enough to mask the orientation dependence of the xylan signal from the S₂ layer in tension wood and could possibly come from other pentose sugars present in, or associated with, the gelatinous layer of tension wood fibres.