A Fourier transform infrared microspectroscopic imaging investigation into an animal model exhibiting glioblastoma multiforme

Glioblastoma multiforme (GBM) is a highly malignant human brain tumour for which no cure is available at present. Numerous clinical studies as well as animal experiments are under way with the goal being to understand tumour biology and develop potential therapeutic approaches. C6 cell glioma in the adult rat is a frequently used and well accepted animal model for the malignant human glial tumour. By combining standard analytical methods such as histology and immunohistochemistry with Fourier Transform Infrared (FTIR) microspectroscopic imaging and multivariate statistical approaches, we are developing a novel approach to tumour diagnosis which allows us to obtain information about the structure and composition of tumour tissues that could not be obtained easily with either method alone. We have used a "Stingray" FTIR imaging spectrometer to analyse and compare the compositions of coronal brain tissue sections of a tumour-bearing animal and those from a healthy animal. We have found that the tumour tissue has a characteristic chemical signature, which distinguishes it from tumour-free brain tissue. The physical-chemical differences, determined by image and spectral comparison are consistent with changes in total protein absorbance, phosphodiester absorbance and physical dispersive artefacts. The results indicate that FTIR imaging analysis could become a valuable analytic method in brain tumour research and possibly in the diagnosis of human brain tumours.     

畜禽粪便堆肥的理化腐熟指标及其红外光谱

选择新鲜的牛粪、猪粪和鸡粪为堆肥原料,通过室内模拟直接堆肥试验,研究堆肥过程理化参数变化、种子发芽指数以及腐熟物红外光谱特征.结果表明: 堆肥过程温度变化明显,牛粪出现一次堆肥高温阶段,鸡粪和猪粪各两次,堆肥温度超过50 ℃以上时间均大于10 d,温度可以最直观地表征堆肥的腐熟程度.堆肥过程中pH值变化幅度较大,其中猪粪的pH值从6.63上升至7.74,鸡粪从7.73上升至8.66,牛粪则先从7.86上升至8.36再下降至7.52;有机碳含量逐渐降低,牛粪、猪粪和鸡粪分别下降23.3%、28.2%和31.7%;堆肥过程中,牛粪、猪粪和鸡粪的铵态氮含量分别下降87.8%、73.6%和79.7%,硝态氮含量分别增加至堆肥前的56.81、6.49和4.85倍,铵态氮/硝态氮均下降至2以下.温度、pH值、有机碳含量和铵态氮/硝态氮能较好地反映3种粪便高温堆肥的腐熟程度,且与种子发芽率相关性较高(P≤0.05).红外光谱分析表明,在堆肥过程中,牛粪和猪粪的脂肪族、多糖类物质减少,芳香结构化增强,腐殖化程度增加;鸡粪则相反.在建立理化指标体系的同时,应兼顾物质结构的稳定性.     

Evaluation of the biological stability of waste during landfill stabilization by thermogravimetric analysis and Fourier transform infrared spectroscopy

This study seeks to assess the biological stability of landfilled municipal solid waste (MSW) based on the changes in organic matter, as revealed by thermogravimetric analysis and Fourier transform infrared (FTIR) spectroscopy. Derivate thermogravimetry profiles (DTG) showed a reduction in peak intensity at 200-350 °C (DTG2), while an increase in peak intensity and a shift towards higher temperature at 400-600 °C (DTG3). The decrease in the peak intensity of the aliphatic methylene at 2920 and 2850 cm(-1), and the increase of aromatic substances and polysaccharide at 1640 cm(-1) in the FTIR spectra also confirm the changes. Well-fitted correlations of the peak intensity ratio (2920/1640) and peak area ratio (DTG2/DTG3) to C/N ratio were also established, confirming that the 2920/1640 and the DTG2/DTG3 ratios can be considered as reliable parameters for tracking the biological stability of MSW during landfill stabilization.     

Chemical imaging of biological tissue with synchrotron infrared light

Fourier transform infrared micro-spectroscopy (FTIRM) and imaging (FTIRI) have become valuable techniques for examining the chemical makeup of biological materials by probing their vibrational motions on a microscopic scale. Synchrotron infrared (S-IR) light is an ideal source for FTIRM and FTIRI due to the combination of its high brightness (i.e., flux density), also called brilliance, and broadband nature. Through a 10-μm pinhole, the brightness of a synchrotron source is 100-1000 times higher than a conventional thermal (globar) source. Accordingly, the improvement in spatial resolution and in spectral quality to the diffraction limit has led to a plethora of applications that is just being realized. In this review, we describe the development of synchrotron-based FTIRM, illustrate its advantages in many applications to biological systems, and propose some potential future directions for the technique.     

Phase behaviour and crystallinity of plant cuticular waxes studied by Fourier transform infrared spectroscopy

The phase behaviour of cuticular waxes from leaves of Hedera helix L. and Juglans regia L. was studied by Fourier transform infrared spectroscopy. For this purpose reconstituted waxes, isolated cuticular membranes, dewaxed polymer matrix membranes and whole leaves were studied in the horizontal attenuated total reflection and transmission modes. Melting curves of cuticular waxes were derived from temperature-dependent changes in the absorption maximum of the symmetric stretching mode of CH₂ groups (ν_s, at approx. 2856–2848 cm⁻¹). With increasing temperature absorption band doublets due to CH₂ scissoring (δ_sciss) and rocking (δ_rock) movements (at approx. 1473–1471 and 730–720 cm⁻¹, respectively) indicative of an orthorhombic arrangement of alkyl chains merged into a single peak. The area ratio of the peaks at approx. 720 and 730 cm⁻¹ was used as a measure for aliphatic crystallinity of plant cuticular waxes at a given temperature. The investigations of reconstituted cuticular waxes and those still embedded in isolated cuticles or in situ on the leaf produced comparable results. The findings are discussed in terms of the properties of the cuticular transport barrier. Received: 21 March 1997 / Accepted: 25 April 1997     

Molecular response of Anabaena flos-aquae to differing concentrations of phosphorus: A combined Fourier transform infrared and X-ray microanalytical study

The response of Anabaena flos‐aquae to varying levels of P availability was determined using the combined techniques of Fourier transform infrared (FTIR) microspectroscopy and energy‐dispersive X‐ray microanalysis (EDXRMA). Batch cultures of Anabaena were grown at initial P concentrations of 50 μg (low‐P culture), 500 μg (intermediate‐P) and 5000 μg (high‐P) (PO₄‐P L^?1) and were monitored for total biovolume, chlorophyll a, media nutrients (PO₄‐P and NO₃‐N), cellular P quota (EDXRMA) and carbon allocation (FTIR spectroscopy). The high‐P culture showed a sixfold increase in total biovolume over the sampling period. Phosphorus in the media remained at more than ～4000 μg L^?1 and intracellular P concentration (P quota) as determined by EDXRMA showed no decline, remaining at more than 0.20% dry weight (DW). The intermediate‐P culture showed a similar increase in total biovolume, but P concentrations in the media fell to ～20 μg L^?1, and this was reflected in a decline in the cellular P quota from 0.24% to 0.06% DW. Although the high‐ and intermediate‐P treatments differed markedly in both internal and external P, analysis of the FTIR spectra from the two treatments, using both hierarchical cluster analysis (HCA) and principal component analysis (PCA), indicated no difference in carbon allocation. Cells from the low‐P treatment showed strong evidence for P deficiency. P concentrations in the media were undetectable (<5 μg L^?1), total biovolume was much reduced, and P quotas were low, falling from 0.08% to 0.01% DW. HCA and PCA clearly separated FTIR spectra from low‐P cells from those of intermediate‐ and high‐P cultures, with low‐P cells having increased absorbance in the region of the spectra associated with carbohydrate. Both FTIR spectroscopy and EDXRMA have the resolution to study the elemental and macromolecular composition of single species within mixed phytoplankton populations and the combined use of these techniques has considerable potential for the study of cyanobacterial responses to environmental stress.     

Pressure dependence of human fibrinogen correlated to the conformational alpha-helix to beta-sheet transition: an Fourier transform infrared study microspectroscopic study

We used Fourier transform infrared (FTIR) microspectroscopy to investigate pressure-induced conformational changes in secondary structure of fibrinogen (FBG). Solid state FBG was compressed on a KBr pellet (1KBr method) or between two KBr pellets (2KBr method). The peak positions of the original and second-derivative ir spectra of compressed FBG samples prepared by the 1KBr method were similar to FBG sample without pressure. When FBG was prepared by the 2KBr method and pressure was increased up to 400 kg/cm(2), peaks at 1625 (intermolecular beta-sheet) and 1611 (beta-sheet aggregates structure and/or the side-chain absorption of the tyrosine residues) cm(-1) were enhanced. The peaks near 1661 (beta-sheet) and 1652 (alpha-helix) cm(-1) also exhibited a marked change with pressure. A linear correlation was found between the peak intensity ratio of 1611/1652 cm(-1) (r = 0.9879) or 1625/1652 cm(-1) (r = 0.9752) and applied pressure. The curve-fitted compositional changes in secondary structure of FBG also indicate that the composition of the alpha-helix structure (1657-1659 cm(-1)) was gradually reduced with the increase in compression pressure, but the composition of the beta-sheet structure (1681, 1629, and 1609 cm(-1)) gradually increased. This indicates that pressure-induced conformational changes in FBG include not only transformations from alpha-helix to beta-sheet structure, but also unfolding and denaturation of FBG and the formation of aggregates.     

Fourier transform infrared/vibrational circular dichroism spectroscopy as an informative tool for the investigation of large supramolecular complexes of biological macromolecules

A combination of ultraviolet (UV) and infrared (IR) absorption and circular dichroism (CD) spectroscopy was applied to investigate the structure and formation of large supramolecular DNA-protein complexes. This combination of techniques was used to overcome limitations of UV-CD (electronic, or ECD) spectroscopy due to considerable light scattering in such solutions. Based on the analysis of FTIR and UV-CD spectra, the interaction of DNA with nonhistone chromatin protein HMGB1 and linker histone H1 was studied. The data obtained showed that under the conditions of the experiment (15 mM NaCl, protein/DNA ratio r < 1 w/w) the proteins did not reveal any AT or GC specificity in binding to DNA. In the presence of both proteins, mainly interactions in the DNA minor groove were observed, which were attributed to HMGB1 binding. Histone H1 facilitated binding of HMGB1 to DNA by interacting with the negatively charged groups of the sugar-phosphate backbone and binding of aspartic and glutamic amino acid residues of HMGB1. Acting together, HMGB1 and H1 stimulated the assemblage of supramolecular DNA-protein structures. The structural organization of the ternary complexes depended not only on the properties of the protein-DNA interactions but also on the interactions between HMGB1 and H1 molecules.     

Evaluation of photosynthetic activities in thylakoid membranes by means of Fourier transform infrared spectroscopy

Light-induced Fourier transformed infrared (FTIR) difference spectroscopy is a powerful method to study the structures and reactions of redox cofactors involved in the photosynthetic electron transport chain. So far, most of the FTIR studies of the reactions of oxygenic photosynthesis have been performed using isolated photosystem I (PSI) and photosystem II (PSII) preparations, which, however, could be modified during isolation procedures. In this study, we developed a methodology to evaluate the photosynthetic activities of thylakoids using FTIR spectroscopy. FTIR difference spectra upon successive flashes using thylakoids from spinach exhibited signals typical of the S-state cycle at the Mn₄CaO₅ cluster and Q_B reactions in PSII with period-four and -two oscillations, respectively. Similar measurement in the presence of an artificial quinone as an exogenous electron acceptor showed features specific to the S-state cycle. Simulations of the oscillation patterns provided the quantum efficiencies of the S-state cycle and electron transfer in PSII. Moreover, FTIR measurement under continuous illumination on thylakoids in the presence of DCMU showed signals due to Q_A reduction and P700 oxidation simultaneously. From the relative amplitudes of marker bands of Q_A^? and P700⁺, the molar ratio of photoactive PSII and PSI centers in thylakoids was estimated. FTIR analyses of the photo-reactions in thylakoids, which are more intact than isolated photosystems, will be useful in investigations of the photosynthetic mechanism especially by genetic modification of photosystem proteins.     

Use of infrared microspectroscopy to determine leaf biochemical composition of cassava in response to Bacillus subtilis CaSUT007

The objective of this study was to investigate the growth stimulating properties of Bacillus subtilis CaSUT007 applied to cassava plants using fourier transform infrared (FTIR) microspectroscopy to monitor the production of cellular components involved in plant growth and development. Cassava stakes treated with CaSUT007 or sterile distilled water were germinated in soil. After incubation for 2 months, CaSUT007 treated plants had higher growth rate and greater biomass than the control. FTIR analysis revealed that the leaves of cassava plants treated with CaSUT007 display FTIR spectra changes in the epidermis and mesophyll tissue. These changes associated with proteins, lipids, and pectins, which are related to changes in plant cell growth and development. FTIR microspectroscopy can be used as a new tool to examine the biochemical changes within the plant tissue. This technique allows us to reveal structural chemical makeup and features of different tissue types.     

An adaptive modeling approach using spiking-augmentation method to improve chemometric model performance in bioprocess monitoring

Intensified and continuous processes require fast and robust methods and technologies to monitor product titer for faster analytical turnaround time, process monitoring, and process control. The current titer measurements are mostly offline chromatography-based methods which may take hours or even days to get the results back from the analytical labs. Thus, offline methods will not meet the requirement of real time titer measurements for continuous production and capture processes. FTIR and chemometric based multivariate modeling are promising tools for real time titer monitoring in clarified bulk (CB) harvests and perfusate lines. However, empirical models are known to be vulnerable to unseen variability, specifically a FTIR chemometric titer model trained on a given biological molecule and process conditions often fails to provide accurate predictions of titer in another molecule under different process conditions. In this study, we developed an adaptive modeling strategy: the model was initially built using a calibration set of available perfusate and CB samples and then updated by augmenting spiking samples of the new molecules to the calibration set to make the model robust against perfusate or CB harvest of the new molecule. This strategy substantially improved the model performance and significantly reduced the modeling effort for new molecules.     

A Fourier transform infrared spectroscopy study of cell membrane domain modifications induced by docosahexaenoic acid

Background

Detergent resistant membranes (DRMs) are a useful model system for the in vitro characterization of cell membrane domains. Indeed, DRMs provide a simple model to study the mechanisms underlying several key cell processes based on the interplay between specific cell membrane domains on one hand, and specific proteins and/or lipids on the other. Considering therefore their biological relevance, the development of methods capable to provide information on the composition and structure of membrane domains and to detect their modifications is highly desirable. In particular, Fourier transform infrared (FTIR) spectroscopy is a vibrational tool widely used for the study not only of isolated and purified biomolecules but also of complex biological systems, including intact cells and tissues. One of the main advantages of this non-invasive approach is that it allows obtaining a molecular fingerprint of the sample under investigation in a rapid and label-free way.

Methods

Here we present an FTIR characterization of DRM fractions purified from the human breast cancer cells MCF-7, before and after treatment with the omega 3 fatty acid docosahexaenoic acid (DHA), which was found to promote membrane microdomain reorganization.

Results and Conclusions

We will show that FTIR spectroscopy coupled with multivariate analysis enables to monitor changes in the composition of DRMs, induced in particular by the incorporation of DHA in cell membrane phospholipids.

General significance

This study paves the way for a new label-free characterization of specific membrane domains within intact cells, which could provide complementary information to the fluorescence approaches presently used.     

The interaction between N-n-undecyl-N'-(sodium p-aminobenzenesulfonate) thiourea and serum albumin studied using various spectroscopies and the molecular modeling method

The preparation and characteristics of N-n-undecyl-N'-(sodium-p-aminobenzenesulfonate) thiourea (UPT), a new water-soluble reagent with a saturated fatty hydrocarbon group, were described. The interactions of UPT with bovine serum albumin (BSA) and human serum albumin (HSA) were studied using fluorescence spectroscopy in combination with ultraviolet (UV) absorption spectroscopy, circular dichroism (CD) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and the molecular modeling method. UPT exhibited a strong ability to quench the intrinsic fluorescence of both BSA and HSA through a static quenching procedure. The binding constants of UPT and BSA or HSA were determined at different temperatures based on the relevant fluorescence data. The binding sites were obtained and the acting force was suggested to be mainly hydrophobic interaction, which was consistent with the result of the molecular modeling study, and there were also a number of hydrogen bonds between UPT and HSA. The results of determination of the proteins in bovine serum or human serum by this method were very close to those obtained by using Coomassie Brilliant Blue G-250 colorimetry. A practical method was proposed for the determination of UPT in bovine serum or human serum samples with satisfactory results.