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Macromolecular changes in Chlorella sp. FleB1 and Scenedesmus acutus YaA6 exposed to Pb were characterised by Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. Spectra were collected in the mid-infrared spectral region and a chemometric approach was used to analyse the spectra. Principal Component Analysis showed that under Pb treatment, carbohydrates and lipids increased while proteins and phosphorylated molecules decreased in both the isolates. These conclusions were corroborated by conventional measurements of photosynthesis and neutral lipids. In the two strains, Pb inhibited photosynthesis, which directly affects the synthesis of polysaccharides and other macromolecules. Neutral lipids as measured by Nile Red were increased in lead-treated samples. Modelling of spectral data against Pb exposure levels allowed Pb-exposed samples to be readily discriminated from unexposed controls and the level of Pb exposure could be predicted with good accuracy in independent validation testing. This study demonstrates the utility of the spectroscopic approach as a rapid, inexpensive, non-destructive method to understand changes in cellular composition induced by Pb and allowing detection and quantification of Pb levels in metal-contaminated cells in a "snapshot"., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The superior characteristics of high photon flux and diffraction-limited spatial resolution achieved by synchrotron-FTIR microspectroscopy allowed molecular characterization of individual live thraustochytrids. Principal component analysis revealed distinct separation of the single live cell spectra into their corresponding strains, comprised of new Australasian thraustochytrids (AMCQS5-5 and S7) and standard cultures (AH-2 and S31). Unsupervised hierarchical cluster analysis (UHCA) indicated close similarities between S7 and AH-7 strains, with AMCQS5-5 being distinctly different. UHCA correlation conformed well to the fatty acid profiles, indicating the type of fatty acids as a critical factor in chemotaxonomic discrimination of these thraustochytrids and also revealing the distinctively high polyunsaturated fatty acid content as key identity of AMCQS5-5. Partial least squares discriminant analysis using cross-validation approach between two replicate datasets was demonstrated to be a powerful classification method leading to models of high robustness and 100% predictive accuracy for strain identification. The results emphasized the exceptional S-FTIR capability to perform real-time in vivo measurement of single live cells directly within their original medium, providing unique information on cell variability among the population of each isolate and evidence of spontaneous lipid peroxidation that could lead to deeper understanding of lipid production and oxidation in thraustochytrids for single-cell oil development., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Rapid and sensitive methods for identifying stem cell differentiation state are required for facilitating future stem cell therapies. We aimed to evaluate the capability of focal plane array-Fourier transform infrared (FPA-FTIR) microspectroscopy for characterising the differentiation of chondrocytes from human mesenchymal stem cells (hMSCs). Successful induction was validated by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis for collagen and aggrecan expression as chondrocyte markers in parallel with the spectroscopy. Spectra derived from chondrocyte-induced cells revealed strong IR absorbance bands attributed to collagen near 1338 and 1234 cm(-1) and proteoglycan at 1245 and 1175-960 cm(-1) compared to the non-induced cells. In addition, spectra from control and induced cells are segregated into separate clusters in partial least squares discriminant analysis score plots at the very early stages of induction and discrimination of an independent set of validation spectra with 100% accuracy. The predominant bands responsible for this discrimination were associated with collagen and aggrecan protein concordant with those obtained from RT-PCR and Western blot techniques. Our findings support the capability of FPA-FTIR microspectroscopy as a label-free tool for stem cell characterization allowing rapid and sensitive detection of macromolecular changes during chondrogenic differentiation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In the last two decades the field of infrared spectroscopy has seen enormous advances in both instrumentation and the development of bioinformatic methods for spectral analysis, allowing the examination of a large variety of healthy and diseased samples, including biological fluids, isolated cells, whole tissues, and tissue sections. The non-destructive nature of the technique, together with the ability to directly probe biochemical changes without the addition of stains or contrast agents, enables a range of complementary analyses. This review focuses on the application of Fourier transform infrared (FTIR) microspectroscopy to analyse central nervous system tissues, with the aim of understanding the biochemical and structural changes associated with neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, transmissible spongiform encephalopathies, multiple sclerosis, as well as brain tumours. Modern biospectroscopic methods that combine FTIR microspectroscopy with bioinformatic analysis constitute a powerful new methodology that can discriminate pathology from normal healthy tissue in a rapid, unbiased fashion, with high sensitivity and specificity. Notably, the ability to detect protein secondary structural changes associated with Alzheimer's plaques, neurons in Parkinson's disease, and in some spectra from meningioma, as well as in the animal models of Alzheimer's disease, transmissible spongiform encephalopathies, and multiple sclerosis, illustrates the power of this technology. The capacity to offer insight into the biochemical and structural changes underpinning aetio-pathogenesis of diseases in tissues provides both a platform to investigate early pathologies occurring in a variety of experimentally induced and naturally occurring central nervous system diseases, and the potential to evaluate new therapeutic approaches., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The ability to detect DNA conformation in eukaryotic cells is of paramount importance in understanding how some cells retain functionality in response to environmental stress. It is anticipated that the B to A transition might play a role in resistance to DNA damage such as heat, desiccation and toxic damage. To this end, conformational detail about the molecular structure of DNA has been derived primarily from in vitro experiments on extracted or synthetic DNA. Here, we report that a B- to A-like DNA conformational change can occur in the nuclei of intact cells in response to dehydration. This transition is reversible upon rehydration in air-dried cells. By systematically monitoring the dehydration and rehydration of single and double-stranded DNA, RNA, extracted nuclei and three types of eukaryotic cells including chicken erythrocytes, mammalian lymphocytes and cancerous rodent fibroblasts using Fourier transform infrared (FTIR) spectroscopy, we unequivocally assign the important DNA conformation marker bands within these cells. We also demonstrate that by applying FTIR spectroscopy to hydrated samples, the DNA bands become sharper and more intense. This is anticipated to provide a methodology enabling differentiation of cancerous from non-cancerous cells based on the increased DNA content inherent to dysplastic and neoplastic tissue.

Multiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS). Despite progress in understanding immunogenetic aspects of this disease, the mechanisms involved in lesion formation are unknown. To gain new insights into the neuropathology of MS, we used an innovative integration of Fourier transform infrared (FT-IR) microspectroscopy, bioinformatics, and a synchrotron light source to analyze macromolecular changes in the CNS during the course and prevention of experimental autoimmune encephalomyelitis (EAE), an animal model for MS. We report that subtle chemical and structural changes not observed by conventional histology were detected before the onset of clinical signs of EAE. Moreover, trained artificial neural networks (ANNs) could discriminate, with excellent sensitivity and specificity, pathology from surrounding tissues and the early stage of the disease progression. Notably, we show that this novel measurement platform can detect characteristic differences in biochemical composition of lesion pathology in animals partially protected against EAE by vaccination with Nogo-A, an inhibitor of neural outgrowth, demonstrating the potential for automated screening and evaluation of new therapeutic agents.

A species of marine diatom, Toxarium undulatum, has emerged as a problematic biofouler of contemporary environmentally benign marine coatings. Previous analyses by atomic force microscopy (AFM) showed the cell-substratum adhesive of this alga contained macromolecules with a modular protein backbone assembled into nanofibers in which the domains of the macromolecules folded and unfolded in a co-ordinated manner. In the present study, we investigated further the composition and properties of the adhesive. A combination of energy dispersive X-ray analysis (EDXA) and Fourier transform infrared (FTIR) spectroscopy showed that the adhesive contained mainly protein, carbohydrate, sulfate, calcium, and magnesium. AFM demonstrated that EDTA treatment of native T. undulatum adhesive resulted in rapid disruption of the adhesive nanofiber (ANF) structure but ANFs were restored by subsequent treatment (within 1 h) with solutions containing divalent cations. Prolonged exposure to EDTA (≥18 h) led to cell detachment. The soluble EDTA extract was separated from the cells, dialyzed, concentrated, and analyzed further. The extract had a protein-to-carbohydrate-to-sulfate weight ratio of 1.0 : 0.2 : 0.9 and contained a single, high-molecular-mass (>220 kDa) band by SDS-PAGE which was visualized by Stains-All® but not by Coomassie blue, indicating that it was a highly anionic macromolecule. The most abundant amino acids in the extract were glycine (22 mol%), aspartic acid/aspartamine (14 mol%), and histidine (11 mol%). The adhesive contained 11 neutral sugars dominated by mannose (50 mol%) and xylose (29 mol%). On the basis of these data, we propose that the ANFs of T. undulatum are composed of sulfated high-molecular-mass glycoproteins cross-linked by calcium and magnesium ions. The cross-linking enables domains of adjacent protein backbones to unfold and re-fold in register.

Cultures of the marine diatoms Phaeodactylum tricornutum and Chaetoceros muelleri were grown in f/2 medium supplied with either nitrate (N-Nt), ammonium (N-Am) or urea (N-Ur) as the nitrogen (N) source at the same final N concentration (0.88 mM). Exponential growth phase cultures of the two diatoms were exposed to four different light regimes for 2 days: (UVAR) PAR (60 micromol quanta m-2 s-1) plus 8.22 W m-2 (unweighted) UVAR; (high UVBR) PAR (60 micromol quanta m-2 s-1) plus 1.04 W m-2 (unweighted) UVBR plus 13.73 W m-2 (unweighted) UVAR; (low UVBR) PAR (60 micromol quanta m-2 s-1) plus 0.19 W m-2 (unweighted) UVBR plus 2.76 W m-2 (unweighted) UVAR and (PAR) PAR (60 micromol quanta m-2 s-1) alone (control). No significant effects of N source on the growth rates of the two diatoms were detected. The maximum effective quantum yield of PSII, PhiPSIIe-max, and the initial slope of the light curve, alpha, of P. tricornutum and C. muelleri were all inhibited, whereas Ik was somewhat increased, as a consequence of 2 days of exposure to all the UVR treatments. Multiple factor ANOVA revealed that all the major fatty acids, both in P. tricornutum and C. muelleri, were influenced more strongly by N source than by UVR. The composition of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) in P. tricornutum and C. muelleri exhibited almost the same pattern of variation with N source and UVR. The maximum value of SFA was found in the N-Am treatment, that of MUFA in the N-Nt treatment and for PUFA in the N-Ur treatment irrespective of the UV radiation. On the other hand, the impact of UVR resulted in an increase of PUFA and a reduction of SFA both in P. tricornutum and C. muelleri under all N sources.

We report the coupling of a portable Raman spectrometer to an acoustic levitation device to enable environmental monitoring and the potential taxonomic identification of microalgae. Spectra of living cells were recorded at 785 nm using a fiber-optic probe coupled to a portable Raman spectrometer. The spectra exhibit an excellent signal-to-noise ratio and clearly show bands from chlorophyll a and beta-carotene. Spectra of levitated photobleached microalgae clearly show a reduction in chlorophyll a concentration relative to beta-carotene after 10 min of exposure to a quartz halogen lamp. Spectra recorded from levitated nitrogen-limited cells also show a significant reduction in bands associated with chlorophyll a, as compared to nitrogen-replete cells. To investigate the diagnostic capability of the technique, four species of microalgae were analyzed. Good quality spectra of all four species were obtained showing varying ratios of beta-carotene to chlorophyll. The combination of an acoustic levitation device and a portable Raman spectrometer shows potential as a taxonomic and environmental monitoring tool with direct application to field studies in remote environments.