ATR-FTIR spectroscopy reveals genomic loci regulating the tissue response in high fat diet fed BXD recombinant inbred mouse strains.

BACKGROUND: Obesity-associated organ-specific pathological states can be ensued from the dysregulation of the functions of the adipose tissues, liver and muscle. However, the influence of genetic differences underlying gross-compositional differences in these tissues is largely unknown. In the present study, the analytical method of ATR-FTIR spectroscopy has been combined with a genetic approach to identify genetic differences responsible for phenotypic alterations in adipose, liver and muscle tissues. RESULTS: Mice from 29 BXD recombinant inbred mouse strains were put on high fat diet and gross-compositional changes in adipose, liver and muscle tissues were measured by ATR-FTIR spectroscopy. The analysis of genotype-phenotype correlations revealed significant quantitative trait loci (QTL) on chromosome 12 for the content of fat and collagen, collagen integrity, and the lipid to protein ratio in adipose tissue and on chromosome 17 for lipid to protein ratio in liver. Using gene expression and sequence information, we suggest Rsad2 (viperin) and Colec11 (collectin-11) on chromosome 12 as potential quantitative trait candidate genes. Rsad2 may act as a modulator of lipid droplet contents and lipid biosynthesis; Colec11 might play a role in apoptopic cell clearance and maintenance of adipose tissue. An increased level of Rsad2 transcripts in adipose tissue of DBA/2J compared to C57BL/6J mice suggests a cis-acting genetic variant leading to differential gene activation. CONCLUSION: The results demonstrate that the analytical method of ATR-FTIR spectroscopy effectively contributed to decompose the macromolecular composition of tissues that accumulate fat and to link this information with genetic determinants. The candidate genes in the QTL regions may contribute to obesity-related diseases in humans, in particular if the results can be verified in a bigger BXD cohort.

Genetic diversity of Burkholderia contaminans isolates from cystic fibrosis patients in Argentina.

A total of 120 Burkholderia cepacia complex isolates collected during 2004-2010 from 66 patients in two cystic fibrosis reference centers in Argentina were analyzed. Burkholderia contaminans was the species most frequently recovered (57.6%), followed by Burkholderia cenocepacia (15%), a species distribution not reported so far. The recA-PCR-based techniques applied to the B. contaminans isolates revealed that 85% of the population carried the recA-ST-71 allele. Our results showed the utility of BOX-PCR genotyping in analyzing B. contaminans diversity. This approach allowed us to address clonal transmission during an outbreak and the genetic changes occurring in infecting bacteria over the course of chronic infection.

Direct optical detection of viral nucleoprotein binding to an anti-influenza aptamer.

We have demonstrated label-free optical detection of viral nucleoprotein binding to a polyvalent anti-influenza aptamer by monitoring the surface-enhanced Raman (SERS) spectra of the aptamer-nucleoprotein complex. The SERS spectra demonstrated that selective binding of the aptamer-nucleoprotein complex could be differentiated from that of the aptamer alone based solely on the direct spectral signature for the aptamer-nucleoprotein complex. Multivariate statistical methods, including principal components analysis, hierarchical clustering, and partial least squares, were used to confirm statistically significant differences between the spectra of the aptamer-nucleoprotein complex and the spectra of the unbound aptamer. Two separate negative controls were used to evaluate the specificity of binding of the viral nucleoproteins to this aptamer. In both cases, no spectral changes were observed that showed protein binding to the control surfaces, indicating a high degree of specificity for the binding of influenza viral nucleoproteins only to the influenza-specific aptamer. Statistical analysis of the spectra supports this interpretation. AFM images demonstrate morphological changes consistent with formation of the influenza aptamer-nucleoprotein complex. These results provide the first evidence for the use of aptamer-modified SERS substrates as diagnostic tools for influenza virus detection in a complex biological matrix.

Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element.

BACKGROUND: The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. RESULTS: By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C-terminally encoded glutamine- and proline-rich peptides, QP1 and QP2, spanning 23 and 19 amino acids, respectively. CONCLUSIONS: Expression of reconstituted sequences of original HERV elements is an important tool for studying fundamental aspects of the biology of these ancient viruses. The analysis of HERV-K(HML-2) Gag processing and the nature of the mature Gag proteins presented here will facilitate further studies of the discrete functions of these proteins and of their potential impact on the human host.

Observation of content and heterogeneity of poly-ß-hydroxybutyric acid (PHB) in Legionella bozemanii by vibrational spectroscopy.

Information on how cells respond to their environment, interact with each other, or undergo complex processes such as cellular differentiation or gene expression has been obtained mostly by interference from population-level data. Individual microorganisms, even those on supposedly "clonal" populations, may differ widely from each other in terms of their genetic composition, physiology, biochemistry, or behaviours. This genetic and phenotypic heterogeneity has important practical consequences for a number of relevant interests, including antibiotic or biocide resistance, the productivity and stability of industrial fermentations, the efficacy of food preservatives, and the potential of pathogens to cause disease. Here we introduce vibrational spectroscopy to characterize Legionella bozemanii with respect to its content of poly-hydroxybutyric acid (PHB) and its distribution on both the population level and the single cell level.

Evaluation of tip-enhanced Raman spectroscopy for characterizing different virus strains.

Tip-enhanced Raman spectroscopy (TERS) is a highly sensitive spectroscopic technique which combines the advantages of optical spectroscopy with the requirements needed for the characterization of biological nano-structures. In this study, TERS was used to investigate the applicability of this spectroscopic technique for the detection of different virus strains like avipoxvirus and adeno-associated virus. TERS spectra obtained from different particles of the same virus strain show variations in relative peak intensities and positions of most spectral features observed. These spectral variations were higher for the larger avipoxvirus particles (∅≈350 nm) than for the smaller adeno-associated virus particles (∅≈26 nm).

HLA-B27 heavy chains distinguished by a micropolymorphism exhibit differential flexibility.

OBJECTIVE: Although the products of the HLA subtypes B*2705 and B*2709 differ only in residue 116 (Asp versus His) within their peptide-binding grooves, they are differentially associated with inflammatory rheumatic diseases such as ankylosing spondylitis (AS): B*2705 occurs in AS patients, whereas B*2709 is only rarely encountered. The reasons for this distinct association are still unclear but could include subtype-specific conformational and dynamic properties of these antigens. The present study was undertaken to investigate structural and dynamic differences between B*2705 and B*2709 and their possible relationship to subtype-specific disease association. METHODS: The membrane-distal segments of the B*2705 and B*2709 heavy chains were expressed in vitro and reconstituted together with beta(2)-microglobulin and a peptide. HLA-B27 complexes loaded with 2 self peptides (TIS [RRLPIFSRL] and pVIPR [RRKWRRWHL]) and a sequence-related viral peptide (pLMP2 [RRRWRRLTV]) were studied by isotope-edited infrared spectroscopy to detect differences in their structure and flexibility at physiologic temperature. RESULTS: Our analyses revealed the existence of subtype-specific conformational differences between the 2 HLA-B27 heavy chains at physiologic temperature, which are undetectable using x-ray crystallography. Irrespective of the bound peptide, the heavy chain of the B*2705 complex exhibited higher conformational flexibility than the B*2709 heavy chain. CONCLUSION: The present study demonstrates the existence of previously undetected systematic conformational and dynamic differences between the heavy chains of the 2 HLA-B27 subtypes. Since effector cell recognition of cells expressing HLA antigens is dependent on the dynamic properties of the interacting cell surface molecules, this HLA-B27 subtype-specific heavy chain flexibility could have a role in the distinct association of HLA-B27 subtypes with spondylarthritides.

Characterization of Yersinia using MALDI-TOF mass spectrometry and chemometrics.

Yersinia are Gram-negative, rod-shaped facultative anaerobes, and some of them, Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis, are pathogenic in humans. Rapid and accurate identification of Yersinia strains is essential for appropriate therapeutic management and timely intervention for infection control. In the past decade matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) in combination with computer-aided pattern recognition has evolved as a rapid, objective, and reliable technique for microbial identification. In this comprehensive study a total of 146 strains of all currently known Yersinia species complemented by 35 strains of other relevant genera of the Enterobacteriaceae family were investigated by MALDI-TOF MS and chemometrics. Bacterial sample preparation included microbial inactivation according to a recently developed mass spectrometry compatible inactivation protocol. The mass spectral profiles were evaluated by supervised feature selection methods to identify family-, genus-, and species-specific biomarker proteins and--for classification purposes--by pattern recognition techniques. Unsupervised hierarchical cluster analysis revealed a high degree of correlation between bacterial taxonomy and subproteome-based MALDI-TOF MS classification. Furthermore, classification analysis by supervised artificial neural networks allowed identification of strains of Y. pestis with an accuracy of 100%. In-depth analysis of proteomic data demonstrated the existence of Yersinia-specific biomarkers at m/z 4350 and 6046. In addition, we could also identify species-specific biomarkers of Y. enterocolitica at m/z 7262, 9238, and 9608. For Y. pseudotuberculosis a combination of biomarkers at m/z 6474, 7274, and 9268 turned out to be specific, while a peak combination at m/z 3065, 6637, and 9659 was characteristic for strains of Y. pestis. Bioinformatic approaches and tandem mass spectrometry were employed to reveal the molecular identity of biomarker ions. In this way, the Y. pestis-specific biomarker at m/z 3065 could be identified as a fragment of the plasmid-encoded plasminogen activator, one of the major virulence factors in plague infections.

Multiplex detection of microbial and plant toxins by immunoaffinity enrichment and matrix-assisted laser desorption/ionization mass spectrometry.

Plant and microbial toxins such as ricin, staphylococcal enterotoxin B (SEB), and the botulinum neurotoxins (BoNT) are considered as potential biological warfare agents. Specific screening methods are, therefore, required that enable unambiguous and sensitive identification of these biohazards, particularly for the occurrence of the toxins in complex sample matrixes. The present study describes a combination of a multiplex-immunoaffinity purification approach, followed by matrix-assisted laser desorption/ionization (MALDI)-based detection for the simultaneous identification of ricin, SEB, BoNT/A, and BoNT/B. The method comprises an affinity enrichment step, using specific monoclonal antibodies for each of the four toxins which have been selected from a pool of antibodies. The selected antibodies allow for specific and simultaneous capture of ricin, SEB, BoNT/A, BoNT/B, and the corresponding BoNT complexes. These were subsequently identified by MALDI time-of-flight (TOF) mass spectrometry (MS), following tryptic digest. The sensitivity of the technique was approximately 500 fmol for each of the toxins. These toxins were detectable within 8 h, even when present in complex matrixes such as milk or juice. Furthermore, the MALDI-based multiplex assay allowed for the discrimination of closely related BoNT sero- and subtypes, including a real case of food-borne botulism in Germany.

Changes in protein structure and distribution observed at pre-clinical stages of scrapie pathogenesis.

Scrapie is a neurodegenerative disorder that involves the misfolding, aggregation and accumulation of the prion protein (PrP). The normal cellular PrP (PrP(C)) is rich in alpha-helical secondary structure, whereas the disease-associated pathogenic form of the protein (PrP(Sc)) has an anomalously high beta-sheet content. In this study, protein structural changes were examined in situ in the dorsal root ganglia from perorally 263K scrapie-infected and mock-infected hamsters using synchrotron Fourier Transform InfraRed Microspectroscopy (FTIRM) at four time points over the course of the disease (pre-clinical, 100 and 130 days post-infection (dpi); first clinical signs ( approximately 145 dpi); and terminal ( approximately 170 dpi)). Results showed clear changes in the total protein content, structure, and distribution as the disease progressed. At pre-clinical time points, the scrapie-infected animals exhibited a significant increase in protein expression, but the beta-sheet protein content was significantly lower than controls. Based on these findings, we suggest that the pre-clinical stages of scrapie are characterized by an overexpression of proteins low in beta-sheet content. As the disease progressed, the beta-sheet content increased significantly. Immunostaining with a PrP-specific antibody, 3F4, confirmed that this increase was partly - but not solely - due to the formation of PrP(Sc) in the tissue and indicated that other proteins high in beta-sheet were produced, either by overexpression or misfolding. Elevated beta-sheet was observed near the cell membrane at pre-clinical time points and also in the cytoplasm of infected neurons at later stages of infection. At the terminal stage of the disease, the protein expression declined significantly, likely due to degeneration and death of neurons. These dramatic changes in protein content and structure, especially at pre-clinical time points, emphasize the possibility for identifying other proteins involved in early pathogenesis, which are important for a further understanding of the disease.

Identification of Bacillus anthracis by using matrix-assisted laser desorption ionization-time of flight mass spectrometry and artificial neural networks.

This report demonstrates the applicability of a combination of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and chemometrics for rapid and reliable identification of vegetative cells of the causative agent of anthrax, Bacillus anthracis. Bacillus cultures were prepared under standardized conditions and inactivated according to a recently developed MS-compatible inactivation protocol for highly pathogenic microorganisms. MALDI-TOF MS was then employed to collect spectra from the microbial samples and to build up a database of bacterial reference spectra. This database comprised mass peak profiles of 374 strains from Bacillus and related genera, among them 102 strains of B. anthracis and 121 strains of B. cereus. The information contained in the database was investigated by means of visual inspection of gel view representations, univariate t tests for biomarker identification, unsupervised hierarchical clustering, and artificial neural networks (ANNs). Analysis of gel views and independent t tests suggested B. anthracis- and B. cereus group-specific signals. For example, mass spectra of B. anthracis exhibited discriminating biomarkers at 4,606, 5,413, and 6,679 Da. A systematic search in proteomic databases allowed tentative assignment of some of the biomarkers to ribosomal protein or small acid-soluble proteins. Multivariate pattern analysis by unsupervised hierarchical cluster analysis further revealed a subproteome-based taxonomy of the genus Bacillus. Superior classification accuracy was achieved when supervised ANNs were employed. For the identification of B. anthracis, independent validation of optimized ANN models yielded a diagnostic sensitivity of 100% and a specificity of 100%.

Phenotypic heterogeneity within microbial populations at the single-cell level investigated by confocal Raman microspectroscopy.

Single cells in genetically homogeneous microbial cultures exhibit marked phenotypic heterogeneity that is considered to bolster the fitness of the whole population. Heterogeneity on the single-cell level is typically masked in conventional studies of microbial populations, which rely on data averaged across thousands or millions of cells in a sample. Here we introduce confocal Raman microspectroscopy as a method for investigating and illustrating the spatial heterogeneity of microbial cell populations. By the use of three different test organisms as model systems, we show pronounced cellular heterogeneity even in colonies cultivated under laboratory conditions.

Correction of axial chromatic aberrations in confocal Raman microspectroscopic measurements of a single microbial spore.

Herein we describe a strategy for correcting the longitudinal or axial component of chromatic aberration in confocal Raman microspectroscopy. The method is based on measuring a vertical series of confocal Raman sections of samples by a high numerical aperture Raman microscope. Using the known characteristics of the wavelength-dependent focal shift of the optical system, the Raman intensities can be corrected to allow the rearrangement of Raman data from different focal planes. In the present study the computational correction routine was applied to an experimental data set of 4-dimensional (xyz spatial and the spectral dimension) confocal Raman spectra collected from single spores of Bacillus cereus. After correcting the axial component of the chromatic aberration, univariate and multivariate spectral parameters were obtained and used in the following for 3D segmentation and volume rendering on the basis of the structural and compositional information contained in the Raman spectra of the spore. Using univariate Raman intensities from defined functional group frequencies or k-means cluster membership values as a multivariate parameter for volume rendering, we demonstrate a high degree of correlation between confocal Raman microspectroscopy and the spores' morphology. In this paper we will also present cluster mean spectra which will be discussed in light of the presence of proteins and Ca-DPA, a calcium chelate of dipicolinic acid in the spore.

Resonance Raman microscopy in combination with partial dark-field microscopy lights up a new path in malaria diagnostics.

Our goal is to produce a rapid and accurate diagnostic tool for malaria using resonance Raman spectroscopy to detect small inclusions of haemozoin in Plasmodium falciparum infected red blood cells. In pursuit of this aim we serendipitously discovered a partial dark-field effect generated by our experimental setup, which helps identify in thick blood films potential parasites that are normally difficult to see with conventional bright-field microscopy. The haemozoin deposits 'light up' and these can be selectively targeted with the Raman microscope to confirm the presence or absence of haemozoin by the strong 1569 cm(-1) band, which is a marker for haemozoin. With newly developed imaging Raman microscopes incorporating ultra-sensitive rapid readout CCDs it is possible to obtain spectra with a good signal-to-noise ratio in 1 second. Moreover, images from a smear of potentially infected cells can be recorded and analysed with multivariate methods. The reconstructed images show what appear to be sub-micron-inclusions of haemozoin in some cells indicating that the technique has potential to identify low pigmented forms of the parasite including early trophozoite-stage infected cells. Further work is required to unambiguously confirm the presence of such forms through systematic staining but the results are indeed promising and may lead to the development of a new Raman-based malaria diagnostic.

Rapid identification of Burkholderia cepacia complex species including strains of the novel Taxon K, recovered from cystic fibrosis patients by intact cell MALDI-ToF mass spectrometry.

Two approaches based on intact cell matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (IC-MALDI-ToF MS) have been evaluated in order to discriminate and identify nine former Burkholderia cepacia complex (Bcc) species, Burkholderia contaminans belonging to the novel Taxon K, Burkholderia gladioli, and the most relevant non-fermentative (NF) Gram-negative rods recovered from cystic fibrosis (CF) sputum cultures. In total, 146 clinical isolates and 26 reference strains were analysed. IC mass spectra were obtained with high reproducibility applying a recently developed inactivation protocol which is based on the extraction of microbial proteins by trifluoroacetic acid (TFA). In a first approach, spectral analysis was carried out by means of a gel-view representation of mass spectra, which turned out to be useful to recognize specific identifying biomarker proteins (SIBPs). A series of prominent mass peaks, mainly assigned to constitutively expressed proteins, were selected as SIBPs for identifications at the genus and species level. Two distinctive mass peaks present in B. contaminans spectra (7501 and 7900 Da) were proposed as SIBPs for the identification of this novel species. A second approach of spectral analysis based on data reduction, feature selection and subsequent hierarchical cluster analysis was used to obtain an objective discrimination of all species analysed. Both complementary modalities of analyzing complex IC-MALDI-ToF MS data open the path towards a rapid, accurate and objective means of routine clinical microbiology diagnosis of pathogens from sputum samples of CF patients.

Early stages of misfolding and association of beta2-microglobulin: insights from infrared spectroscopy and dynamic light scattering.

Conformational changes associated with the assembly of recombinant beta 2-microglobulin in vitro under acidic conditions were investigated using infrared spectroscopy and static and dynamic light scattering. In parallel, the morphology of the different aggregated species obtained under defined conditions was characterized by electron microscopy. The initial salt-induced aggregate form of beta 2-microglobulin, composed of small oligomers (dimers to tetramers), revealed the presence of beta-strands organized in an intramolecular-like fashion. Further particle growth was accompanied by the formation of intermolecular beta-sheet structure and led to short curved forms. An increase in temperature by only 25 degrees C was able to disaggregate these assemblies, followed by the formation of longer filamentous structures. In contrast, a rise in temperature up to 100 degrees C was associated with a reorganization of the short curved forms at the level of secondary structure and the state of assembly, leading to a species with a characteristic infrared spectrum different from those of all the other aggregates observed before, suggesting a unique overall structure. The infrared spectral features of this species were nearly identical to those of beta 2-microglobulin assemblies formed at low ionic strength with agitation, indicating the presence of fibrils, which was confirmed by electron microscopy. The observed spectroscopic changes suggest that the heat-triggered conversion of the short curved assemblies into fibrils involves a reorganization of the beta-strands from an antiparallel arrangement to a parallel arrangement, with the latter being characteristic of amyloid fibrils of beta 2-microglobulin.

Fourier transform infrared spectroscopy for rapid identification of nonfermenting gram-negative bacteria isolated from sputum samples from cystic fibrosis patients.

The accurate and rapid identification of bacteria isolated from the respiratory tract of patients with cystic fibrosis (CF) is critical in epidemiological studies, during intrahospital outbreaks, for patient treatment, and for determination of therapeutic options. While the most common organisms isolated from sputum samples are Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, in recent decades an increasing fraction of CF patients has been colonized by other nonfermenting (NF) gram-negative rods, such as Burkholderia cepacia complex (BCC) bacteria, Stenotrophomonas maltophilia, Ralstonia pickettii, Acinetobacter spp., and Achromobacter spp. In the present study, we developed a novel strategy for the rapid identification of NF rods based on Fourier transform infrared spectroscopy (FTIR) in combination with artificial neural networks (ANNs). A total of 15 reference strains and 169 clinical isolates of NF gram-negative bacteria recovered from sputum samples from 150 CF patients were used in this study. The clinical isolates were identified according to the guidelines for clinical microbiology practices for respiratory tract specimens from CF patients; and particularly, BCC bacteria were further identified by recA-based PCR followed by restriction fragment length polymorphism analysis with HaeIII, and their identities were confirmed by recA species-specific PCR. In addition, some strains belonging to genera different from BCC were identified by 16S rRNA gene sequencing. A standardized experimental protocol was established, and an FTIR spectral database containing more than 2,000 infrared spectra was created. The ANN identification system consisted of two hierarchical levels. The top-level network allowed the identification of P. aeruginosa, S. maltophilia, Achromobacter xylosoxidans, Acinetobacter spp., R. pickettii, and BCC bacteria with an identification success rate of 98.1%. The second-level network was developed to differentiate the four most clinically relevant species of BCC, B. cepacia, B. multivorans, B. cenocepacia, and B. stabilis (genomovars I to IV, respectively), with a correct identification rate of 93.8%. Our results demonstrate the high degree of reliability and strong potential of ANN-based FTIR spectrum analysis for the rapid identification of NF rods suitable for use in routine clinical microbiology laboratories.

Spatial resolution in infrared microspectroscopic imaging of tissues.

Spatial resolution is one of the most critical measurement parameters in infrared microspectroscopy. Due to the distinct levels of morphologic heterogeneity in cells and tissues the spatial resolution in a given IR imaging setup strongly affects the character of the infrared spectral patterns obtained from the biomedical samples. This is particularly important when spectral data bases of reference microspectra from defined tissue structures are collected. In this paper we have also pointed out that the concept of spatial resolution in IR imaging is inseparable from the contrast. Based on infrared microspectroscopic transmittance data acquired from an USAF 1951 resolution target we have demonstrated how the spatial resolution can be determined experimentally and some numbers for the spatial resolution of popular IR imaging systems are provided. Finally, we have presented a new computational procedure which is suitable to improve the spatial resolution in IR imaging. A theoretical model of 3D-Fourier self-deconvolution (FSD) is given and advantages or pitfalls of this method are discussed. Based on synchrotron IR microspectroscopic data we have furthermore demonstrated that the technique of 3D-FSD can be successfully applied to increase the spatial resolution in a real IR imaging setup.

Structural differences between TSEs strains investigated by FT-IR spectroscopy.

Strain diversity in transmissible spongiform encephalopathies (TSEs) has been suggested to be "enciphered" in the structure of the misfolded prion protein isoform PrP(Sc). We have recently demonstrated the strain typing potential of the FT-IR spectroscopy technique, analyzing four different TSE agents adapted to Syrian hamsters [A. Thomzig, S. Spassov, M. Friedrich, D. Naumann and M. Beekes, Discriminating scrapie and BSE isolates by infrared spectroscopy of pathological prion protein J. Biol. Chem. 279 (2004) 33847-33854.] [1]. In the present paper, we have extended the FT-IR study, exploring the secondary structure, temperature stability, and hydrogen-deuterium exchange characteristics of PrP27-30, from the TSE agents 263K, ME7-H, 22A-H, and BSE-H. The strain differentiation capacity of the FT-IR approach was objectively proven for the first time by multivariate cluster analysis. The second derivative FT-IR spectra obtained from dried protein films or samples hydrated in H(2)O or D(2)O consistently exhibited strain-specific infrared characteristics in the secondary structure sensitive amide I region, complemented by strain dependent spectral traits in the amide II and amide A absorption regions, and the different H/D-exchange behaviour of the various PrP27-30 samples. FT-IR spectra of PrP27-30 samples from 263K, ME7-H and 22A-H exposed to increasing temperature (up to 90 degrees C) showed that a strain-specific response to heat treatment is associated with strain specific thermostability of distinct secondary structure elements, providing additional means for TSEs strain discrimination.

FTIR-microspectroscopy of prion-infected nervous tissue.

The family of transmissible spongiform encephalopathies (TSE), also termed prion diseases, is a group of fatal, neurodegenerative diseases characterized by the accumulation of a misfolded protein, the disease-associated prion protein PrPSc. This glycoprotein differs in secondary structure from its normal, cellular isoform PrPC, which is physiologically expressed mostly by neurons. Scrapie is a prion disease first described in the 18th century in sheep and goats, and has been established as a model in rodents to study the pathogenesis and pathology of prion diseases. Assuming a multitude of molecular parameters change in the tissue in the course of the disease, FTIR microspectroscopy has been proposed as a valuable new method to study and identify prion-affected tissues due to its ability to detect a variety of changes in molecular structure and composition simultaneously. This paper reviews and discusses results from previous FTIR microspectroscopic studies on nervous tissue of scrapie-infected hamsters in the context of histological and molecular alterations known from conventional pathogenesis studies. In particular, data from studies reporting on disease-specific changes of protein structure characteristics, and also results of a recent study on hamster dorsal root ganglia (DRG) are discussed. These data include an illustration on how the application of a brilliant IR synchrotron light source enables the in situ investigation of localized changes in protein structure and composition in nervous cells or tissue due to PrPSc deposition, and a demonstration on how the IR spectral information can be correlated with results of complementary studies using immunohistochemistry and x-ray fluorescence techniques. Using IR microspectroscopy, some neurons exhibited a high accumulation of disease-associated prion protein evidenced by an increased amount of beta-sheet at narrow regions in or around the infected nervous cells. However, not all neurons from terminally diseased hamsters showed PrPSc deposition. Generally, the average spectral differences between all control and diseased DRG spectra are small but consistent as demonstrated by independent experiments. Along with studies on the purified misfolded prion protein, these data suggest that synchrotron FTIR microspectroscopy is capable of detecting the misfolded prion protein in situ without the necessity of immunostaining or purification procedures.