Structural effects of charge destabilization and amino acid substitutions in amyloid fragments of CsgA.

The most studied functional amyloid is the CsgA, major curli subunit protein, which is produced by numerous strains of Enterobacteriaceae. Although CsgA sequences are highly conserved, they exhibit species diversity, which reflects the specific evolutionary and functional adaptability of the major curli subunit. Herein, we performed bioinformatics analyses to uncover the differences in the amyloidogenic properties of the R4 fragments in Escherichia coli and Salmonella enterica and proposed four mutants for more detailed studies: M1, M2, M3, and M4. The mutated sequences were characterized by various experimental techniques, such as circular dichroism, ATR-FTIR, FT-Raman, thioflavin T, transmission electron microscopy and confocal microscopy. Additionally, molecular dynamics simulations were performed to determine the role of buffer ions in the aggregation process. Our results demonstrated that the aggregation kinetics, fibril morphology, and overall structure of the peptide were significantly affected by the positions of charged amino acids within the repeat sequences of CsgA. Notably, substituting glycine with lysine resulted in the formation of distinctive spherically packed globular aggregates. The differences in morphology observed are attributed to the influence of phosphate ions, which disrupt the local electrostatic interaction network of the polypeptide chains. This study provides knowledge on the preferential formation of amyloid fibrils based on charge states within the polypeptide chain.

The application of the hierarchical approach for the construction of foldameric peptide self-assembled nanostructures.

In this paper, we show that a hierarchical approach for the construction of nanofibrils based on α,ß-peptide foldamers is a rational method for the design of novel self-assembled nanomaterials based on peptides. Incorporation of a trans-(1S,2S)-2-aminocyclopentanecarboxylic acid residue into the outer positions of the model coiled-coil peptide led to the formation of helical foldamers, which was determined by circular dichroism (CD) and vibrational spectroscopy. The oligomerization state of the obtained peptides in water was established by analytical ultracentrifugation (AUC). The thioflavin T assay and Congo red methods showed that the obtained α,ß-peptides possess a strong tendency to aggregate, leading to the formation of self-assembled nanostructures, which were assessed by microscopic techniques. The location of the ß-amino acid in the heptad repeat of the coiled-coil structure proved to have an influence on the secondary structure of the obtained peptides and on the morphology of the self-assembled nanostructures.

AmyloGraph: a comprehensive database of amyloid-amyloid interactions.

Information about the impact of interactions between amyloid proteins on their fibrillization propensity is scattered among many experimental articles and presented in unstructured form. We manually curated information located in almost 200 publications (selected out of 562 initially considered), obtaining details of 883 experimentally studied interactions between 46 amyloid proteins or peptides. We also proposed a novel standardized terminology for the description of amyloid-amyloid interactions, which is included in our database, covering all currently known types of such a cross-talk, including inhibition of fibrillization, cross-seeding and other phenomena. The new approach allows for more specific studies on amyloids and their interactions, by providing very well-defined data. AmyloGraph, an online database presenting information on amyloid-amyloid interactions, is available at (http://AmyloGraph.com/). Its functionalities are also accessible as the R package (https://github.com/KotulskaLab/AmyloGraph). AmyloGraph is the only publicly available repository for experimentally determined amyloid-amyloid interactions.

Exploring a diverse world of effector domains and amyloid signaling motifs in fungal NLR proteins.

NLR proteins are intracellular receptors constituting a conserved component of the innate immune system of cellular organisms. In fungi, NLRs are characterized by high diversity of architectures and presence of amyloid signaling. Here, we explore the diverse world of effector and signaling domains of fungal NLRs using state-of-the-art bioinformatic methods including MMseqs2 for fast clustering, probabilistic context-free grammars for sequence analysis, and AlphaFold2 deep neural networks for structure prediction. In addition to substantially improving the overall annotation, especially in basidiomycetes, the study identifies novel domains and reveals the structural similarity of MLKL-related HeLo- and Goodbye-like domains forming the most abundant superfamily of fungal NLR effectors. Moreover, compared to previous studies, we found several times more amyloid motif instances, including novel families, and validated aggregating and prion-forming properties of the most abundant of them in vitro and in vivo. Also, through an extensive in silico search, the NLR-associated amyloid signaling was identified in basidiomycetes. The emerging picture highlights similarities and differences in the NLR architectures and amyloid signaling in ascomycetes, basidiomycetes and other branches of life.

Indirectly assessing changes in corneal properties with OCT speckle after crosslinking in porcine eyes.

The aim of this study was to assess the effect of the standard crosslinking (CXL) procedure on corneal properties and subsequent changes in collagen bonds formation using optical coherence tomography (OCT) corneal speckle statistics and vibrational spectroscopy. Porcine eyes with intact corneal epithelium were randomly selected to one of the four study groups: (1) untreated eyes moistened with phosphate-buffered saline (PBS); (2) eyes after the epithelial debridement and riboflavin application; (3) eyes after CXL procedure according to the Dresden protocol; and (4) eyes after corneal epithelial debridement, regularly moistened with PBS. Before and after this selection, each eyeball was subjected to the constant intraocular pressure of 20 mmHg. Then, ocular biometry was performed and the central cornea was imaged using spectral-domain OCT. Following this, a nonparametric approach to speckle modeling (the Contrast Ratio (CR)) was utilized within the region of interest for each B-scan covering the central corneal stroma. To verify whether the CXL performed ex-vivo results in formation of new bonds in the cornea, Fourier Transform Infrared Attenuated Total Reflectance (ATR-FTIR) spectra of dried corneas, dissected from examined eyeballs, were collected and analyzed. Corneal epithelium removal alone or with the riboflavin application leads to a statistically significant decrease in the CR median value (the Wilcoxon signed-rank test, p < 0.05). However, the most pronounced change in CR median value, which decreases with the increased number of scatterers, was shown after the complete CXL procedure including riboflavin soaking and UVA irradiation (the Wilcoxon signed-rank test, p = 0.004). Analysis of ATR-FTIR spectra revealed influence of UVA irradiation on collagen matrix. The study has shown the increased dehydration accompanied by almost no alteration of collagen native triple-helical structure. Significant changes have been observed for bands related to collagen crosslinks. Specifically, the predominant changes occurred in the sugar region from 1150 to 975 cm-1, as well as in the absorbance of carbonyl groups. Furthermore, the ratio of two Amide I components at approximately 1660 cm-1 and 1675 cm-1 decreased after UVA irradiation. Together, these results provided the evidence for the creation of new corneal crosslinks. In conclusion, this study clearly indicates that the UVA exposure causes the substantial difference in optical scattering occurring in corneal stroma as a result of the induced biochemical changes at the molecular level in this tissue assessed with ATR-FTIR. The proposed speckle-based methodology brings a new insight into the development of OCT technology useful in an indirect assessment of some collagen changes.

Challenges in Experimental Methods.

Experimental studies of amyloids encounter many challenges. There are many methods available for studying proteins, which can be applied to amyloids: from basic staining techniques, allowing visualization of fibers, to complex methods, e.g., AFM-IR used to their detailed biochemical and structural characterization in nanoscale. Which method is appropriate depends on the goal of an experiment: verification of aggregational properties of a peptide, distinguishing oligomers from mature fibers, or kinetic studies. Insolubility, rapid aggregation, and the need of using a high-purity peptide may be a limiting factor in studies involving amyloids. Moreover, the results obtained by various experimental methods often differ significantly, which may lead to misclassification of amyloid peptides. Due to ambiguity of experimental results, laborious and time-consuming analysis, bioinformatical methods become more widely used for amyloids.

Variability of Amyloid Propensity in Imperfect Repeats of CsgA Protein of Salmonella enterica and Escherichia coli.

CsgA is an aggregating protein from bacterial biofilms, representing a class of functional amyloids. Its amyloid propensity is defined by five fragments (R1-R5) of the sequence, representing non-perfect repeats. Gate-keeper amino acid residues, specific to each fragment, define the fragment's propensity for self-aggregation and aggregating characteristics of the whole protein. We study the self-aggregation and secondary structures of the repeat fragments of Salmonella enterica and Escherichia coli and comparatively analyze their potential effects on these proteins in a bacterial biofilm. Using bioinformatics predictors, ATR-FTIR and FT-Raman spectroscopy techniques, circular dichroism, and transmission electron microscopy, we confirmed self-aggregation of R1, R3, R5 fragments, as previously reported for Escherichia coli, however, with different temporal characteristics for each species. We also observed aggregation propensities of R4 fragment of Salmonella enterica that is different than that of Escherichia coli. Our studies showed that amyloid structures of CsgA repeats are more easily formed and more durable in Salmonella enterica than those in Escherichia coli.

Bacteria Single-Cell and Photosensitizer Interaction Revealed by Quantitative Phase Imaging.

Quantifying changes in bacteria cells in the presence of antibacterial treatment is one of the main challenges facing contemporary medicine; it is a challenge that is relevant for tackling issues pertaining to bacterial biofilm formation that substantially decreases susceptibility to biocidal agents. Three-dimensional label-free imaging and quantitative analysis of bacteria-photosensitizer interactions, crucial for antimicrobial photodynamic therapy, is still limited due to the use of conventional imaging techniques. We present a new method for investigating the alterations in living cells and quantitatively analyzing the process of bacteria photodynamic inactivation. Digital holographic tomography (DHT) was used for in situ examination of the response of Escherichia coli and Staphylococcus aureus to the accumulation of the photosensitizers immobilized in the copolymer revealed by the changes in the 3D refractive index distributions of single cells. Obtained results were confirmed by confocal microscopy and statistical analysis. We demonstrated that DHT enables real-time characterization of the subcellular structures, the biophysical processes, and the induced local changes of the intracellular density in a label-free manner and at sub-micrometer spatial resolution.

Searching for universal model of amyloid signaling motifs using probabilistic context-free grammars.

BACKGROUND: Amyloid signaling motifs are a class of protein motifs which share basic structural and functional features despite the lack of clear sequence homology. They are hard to detect in large sequence databases either with the alignment-based profile methods (due to short length and diversity) or with generic amyloid- and prion-finding tools (due to insufficient discriminative power). We propose to address the challenge with a machine learning grammatical model capable of generalizing over diverse collections of unaligned yet related motifs. RESULTS: First, we introduce and test improvements to our probabilistic context-free grammar framework for protein sequences that allow for inferring more sophisticated models achieving high sensitivity at low false positive rates. Then, we infer universal grammars for a collection of recently identified bacterial amyloid signaling motifs and demonstrate that the method is capable of generalizing by successfully searching for related motifs in fungi. The results are compared to available alternative methods. Finally, we conduct spectroscopy and staining analyses of selected peptides to verify their structural and functional relationship. CONCLUSIONS: While the profile HMMs remain the method of choice for modeling homologous sets of sequences, PCFGs seem more suitable for building meta-family descriptors and extrapolating beyond the seed sample.

Bioinformatics methods for identification of amyloidogenic peptides show robustness to misannotated training data.

Several disorders are related to amyloid aggregation of proteins, for example Alzheimer's or Parkinson's diseases. Amyloid proteins form fibrils of aggregated beta structures. This is preceded by formation of oligomers-the most cytotoxic species. Determining amyloidogenicity is tedious and costly. The most reliable identification of amyloids is obtained with high resolution microscopies, such as electron microscopy or atomic force microscopy (AFM). More frequently, less expensive and faster methods are used, especially infrared (IR) spectroscopy or Thioflavin T staining. Different experimental methods are not always concurrent, especially when amyloid peptides do not readily form fibrils but oligomers. This may lead to peptide misclassification and mislabeling. Several bioinformatics methods have been proposed for in-silico identification of amyloids, many of them based on machine learning. The effectiveness of these methods heavily depends on accurate annotation of the reference training data obtained from in-vitro experiments. We study how robust are bioinformatics methods to weak supervision, encountering imperfect training data. AmyloGram and three other amyloid predictors were applied. The results proved that a certain degree of misannotation in the reference data can be eliminated by the bioinformatics tools, even if they belonged to their training set. The computational results are supported by new experiments with IR and AFM methods.

Hierarchical approach for the rational construction of helix-containing nanofibrils using α,ß-peptides.

The rational design of novel self-assembled nanomaterials based on peptides remains a great challenge in modern chemistry. A hierarchical approach for the construction of nanofibrils based on α,ß-peptide foldamers is proposed. The incorporation of a helix-promoting trans-(1S,2S)-2-aminocyclopentanecarboxylic acid residue in the outer positions of the model coiled-coil peptide led to its increased conformational stability, which was established consistently by the results of CD, NMR and FT-IR spectroscopy. The designed oligomerization state in the solution of the studied peptides was confirmed using analytical ultracentrifugation. Moreover, the cyclopentane side chain allowed additional interactions between coiled-coil-like structures to direct the self-assembly process towards the formation of well-defined nanofibrils, as observed using AFM and TEM techniques.

Antibody CD133 Biofunctionalization of Ammonium Acryloyldimethyltaurate and Vinylpyrrolidone Co-Polymer-Based Coating of the Vascular Implants.

Current vascular stents, such as drug eluting stents (DES), have some serious drawbacks, like in stent restenosis and thrombosis. Therefore, other solutions are sought to overcome these post-implantations complications. These include the strategy of biofunctionalization of the stent surface with antibodies that facilitate adhesion of endothelial cells (ECs) or endothelial progenitor cells (EPCs). Rapid re-endothelialization of the surface minimizes the risk of possible complications. In this study, we proposed ammonium acryloyldimethyltaurate/vinylpyrrolidone co-polymer-based surface (AVC), which was mercaptosilanized in order to expose free thiol groups. The presence of free thiol groups allowed for the covalent attachment of CD133 antibodies by disulfide bridges formation between mercaptosilanized surface and cysteine of the protein molecule thiol groups. Various examinations were performed in order to validate the procedure, including attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and Fourier transform Raman spectroscopy (FT-Raman), atomic force microscopy (AFM) and scanning electron microscopy (SEM). By means of ATR-FTIR spectroscopy presence of the CD133 antibody within coating was confirmed. In vitro studies proved good biocompatibility for blood cells without induction of hemolytic response. Thus, proposed biofunctionalized CD133 antibody AVC surface has shown sufficient stability for adapting as cardiovascular implant coating and biocompatibility. According to conducted in vitro studies, the modified surface can be further tested for applications in various biological systems.

Mechanical and structural properties of different types of human aortic atherosclerotic plaques.

Atherosclerotic plaques are characterized by structural heterogeneity affecting aortic behaviour under mechanical loading. There is evidence of direct connections between the structural plaque arrangement and the risk of plaque rupture. As a consequence of aortic plaque rupture, plaque components are transferred by the bloodstream to smaller vessels, resulting in acute cardiovascular events with a poor prognosis, such as heart attacks or strokes. Hence, evaluation of the composition, structure, and biochemical profile of atherosclerotic plaques seems to be of great importance to assess the properties of a mechanically induced failure, indicating the strength and rupture vulnerability of plaque. The main goal of the research was to determine experimentally under uniaxial loading the mechanical properties of different types of the human abdominal aorta and human aortic atherosclerotic plaques identified based on vibrational spectra (ATR-FTIR and FT-Raman spectroscopy) analysis and validated by histological staining. The potential of spectroscopic techniques as a useful histopathological tool was demonstrated. Three types of atherosclerotic plaques - predominantly calcified (APC), lipid (APL), and fibrotic (APF) - were distinguished and confirmed by histopathological examinations. Compared to the normal aorta, fibrotic plaques were stiffer (median of EH for circumferential and axial directions, respectively: 8.15 MPa and 6.56 MPa) and stronger (median of σM for APLc = 1.57 MPa and APLa = 1.64 MPa), lipidic plaques were the weakest (median of σM for APLc = 0.76 MPa and APLa = 0.51 MPa), and calcified plaques were the stiffest (median of EH for circumferential and axial directions, respectively: 13.23 MPa and 6.67 MPa). Therefore, plaques detected as predominantly lipid and calcified are most prone to rupture; however, the failure process reflected by the simplification of the stress-stretch characteristics seems to vary depending on the plaque composition.

Applicability of FTIR-ATR Method to Measure Carbonyls in Blood Plasma after Physical and Mental Stress.

INTRODUCTION: Oxidative stress is a state of imbalance between the production of reactive oxygen species and antioxidant defenses. It results in the oxidation of all cellular elements and, to a large extent, proteins, causing inter alia the formation of carbonyl groups in their structures. The study focused on assessment of changes in the plasma protein-bound carbonyls in police horses after combat training and after rest and the applicability of infrared spectroscopy with a Fourier transform, utilizing the attenuated total reflectance (FTIR-ATR) in detecting plasma protein oxidation. METHODS: We evaluated the influence of both the different concentrations of hydrogen peroxide and combat training on protein carbonylation in horse blood plasma. The oxidation of plasma proteins was assessed using a spectrophotometric method based on the carbonyl groups derivatization with 2,4-dinitrophenylhydrazine (DNPH). The measured values were correlated with the carbonyl groups concentrations determined by means of the FTIR-ATR method. RESULTS: The linear correlation between the DNPH and FTIR-ATR methods was shown. The concentration of plasma protein-bound carbonyls significantly deceased in police horses after one-day rest when compared to the values measured directly after the combat training (a drop by 23%, p<0.05 and 29%, p<0.01 measured by DNPH and FTIR-ATR methods, respectively). These results were consistent with the proteins phosphorylation analysis. CONCLUSION: The FTIR-ATR method may be applied to measure the level of plasma proteins peroxidation.

Changes of albumin secondary structure after palmitic acid binding. FT-IR spectroscopic study.

PURPOSE: Albumin is an universal transport protein. Plasma pool of free fatty acids arising from triglyceride hydrolysis, critical in energy metabolism and etiology of metabolic disorders is transported by albumin. According to various studies albumin has from seven to nine binding sites with diverse affinity to long chain fatty acids. X-ray diffraction crystallography measurements have provided data only for pure human serum albumin or albumin with fully saturated binding sites. These results have shown that amount of -helices is higher after fatty acids binding. Molecular mechanics simulations suggest that binding of fatty acids in two high-affinity sites leads to major conformational changes in albumin structure. The aim of this research was to investigate albumin secondary structure upon gradually increasing fatty acids to protein mole ratio. METHODS: Fourier transform infrared spectroscopy was applied to study changes of bovine serum albumin (as an analogue of human serum albumin) -helical structures after binding palmitic acid in a range of 0-20 palmitic acid: albumin molar ratios representing pure protein, partial, full saturation and excess binding sites capacity. RESULTS: Amount of -helices was increasing along with the amount of palmitic acid: bovine serum albumin molar ratio and reached maximum value around 2 mol/mol. CONCLUSIONS: Our studies confirmed molecular mechanics simulations and crystallographic studies. Palmitic acid binding in two high-affinity sites leads to major structural changes, filling another sites only slightly influenced bovine serum albumin secondary structure. The systematic study of fatty acids and albumin interactions, using an experimental model mimicking metabolic disorders, may results in new tools for personalized nanopharmacotherapy.

Effects of Near Infrared Radiation on DNA. DLS and ATR-FTIR Study.

We presume that the primary effect of Near Infrared (NIR) radiation on aqueous solutions of biological molecules concerns modification of hydrogen bonded structures mainly the global and the hydration shell water molecules. Since water has a significant influence on the DNA structure, we expect that the thermal stability of DNA could be modified by NIR radiation. The herring sperm DNA was exposed to NIR radiation (700-1100nm) for 5, 10, and 20min periods. The temperature dependent infrared measurements were done for the thin films formed on the diamond ATR crystal from evaporated DNA solutions exposed and unexposed to NIR radiation. For the NIR-treated samples (at room temperature) the B form was better conserved than in the control sample independently of the irradiation period. Above 50°C a considerable increase in the A form was only observed for 10min NIR exposed samples. The hydrodynamic radius, (Rh), studied by the dynamic light scattering, showed drastic decrease with the increasing irradiation time. Principal components analysis (PCA) allowed to detect the spectral features correlated with the NIR effect and thermal stability of the DNA films. Obtained results strongly support the idea that the photoionization of water by NIR radiation in presence of DNA molecules is the main factor influencing on its physicochemical properties.

Comparison of FTIR-ATR and Raman spectroscopy in determination of VLDL triglycerides in blood serum with PLS regression.

Hypertriglyceridemia, related with triglyceride (TG) in plasma above 1.7mmol/L is one of the cardiovascular risk factors. Very low density lipoproteins (VLDL) are the main TG carriers. Despite being time consuming, demanding well-qualified staff and expensive instrumentation, ultracentrifugation technique still remains the gold standard for the VLDL isolation. Therefore faster and simpler method of VLDL-TG determination is needed. Vibrational spectroscopy, including FT-IR and Raman, is widely used technique in lipid and protein research. The aim of this study was assessment of Raman and FT-IR spectroscopy in determination of VLDL-TG directly in serum with the isolation step omitted. TG concentration in serum and in ultracentrifugated VLDL fractions from 32 patients were measured with reference colorimetric method. FT-IR and Raman spectra of VLDL and serum samples were acquired. Partial least square (PLS) regression was used for calibration and leave-one-out cross validation. Our results confirmed possibility of reagent-free determination of VLDL-TG directly in serum with both Raman and FT-IR spectroscopy. Quantitative VLDL testing by FT-IR and/or Raman spectroscopy applied directly to maternal serum seems to be promising screening test to identify women with increased risk of adverse pregnancy outcomes and patient friendly method of choice based on ease of performance, accuracy and efficiency.

Anti-atherosclerotic effects of pravastatin in brachiocephalic artery in comparison with en face aorta and aortic roots in ApoE/LDLR-/- mice.

BACKGROUND: Cholesterol-dependent and independent mechanisms were proposed to explain anti-atherosclerotic action of statins in humans. However, their effects in murine models of atherosclerosis have not been consistently demonstrated. Here, we studied the effects of pravastatin on atherosclerosis in ApoE/LDLR-/- mice fed a control and atherogenic diet. METHODS: ApoE/LDLR-/- mice were fed a control (CHOW) or an atherogenic (Low Carbohydrate High Protein, LCHP) diet. Two doses of pravastatin (40mg/kg and 100mg/kg) were used. The anti-atherosclerotic effects of pravastatin in en face aorta, cross-sections of aortic roots and brachiocephalic artery (BCA) were analysed. The lipid profile was determined. Fourier Transform Infrared Spectroscopy followed by Fuzzy C-Means (FCM) clustering was used for the quantitative assessment of plaque composition. RESULTS: Treatment with pravastatin (100mg/kg) decreased total and LDL cholesterol only in the LCHP group, but displayed a pronounced anti-atherosclerotic effect in BCA and abdominal aorta. The anti-atherosclerotic effect of pravastatin (100mg/kg) in BCA was associated with significant alterations of the chemical plaque composition, including a fall in cholesterol and cholesterol esters contents independently on total cholesterol and LDL concentration in plasma. CONCLUSIONS: Pravastatin at high (100mg/kg), but not low dose displayed a pronounced anti-atherosclerotic effect in ApoE/LDLR-/- mice fed a CHOW or LCHP diet that was remarkable in BCA, visible in en face aorta, whereas it was not observed in aortic roots, suggesting that previous inconsistencies might have been due to the various sites of atherosclerotic plaque analysis.

Antiatherosclerotic Effects of 1-Methylnicotinamide in Apolipoprotein E/Low-Density Lipoprotein Receptor-Deficient Mice: A Comparison with Nicotinic Acid.

1-Methylnicotinamide (MNA), the major endogenous metabolite of nicotinic acid (NicA), may partially contribute to the vasoprotective properties of NicA. Here we compared the antiatherosclerotic effects of MNA and NicA in apolipoprotein E (ApoE)/low-density lipoprotein receptor (LDLR)-deficient mice. ApoE/LDLR(-/-) mice were treated with MNA or NicA (100 mg/kg). Plaque size, macrophages, and cholesterol content in the brachiocephalic artery, endothelial function in the aorta, systemic inflammation, platelet activation, as well as the concentration of MNA and its metabolites in plasma and urine were measured. MNA and NicA reduced atherosclerotic plaque area, plaque inflammation, and cholesterol content in the brachiocephalic artery. The antiatherosclerotic actions of MNA and NicA were associated with improved endothelial function, as evidenced by a higher concentration of 6-keto-prostaglandin F1 α and nitrite/nitrate in the aortic ring effluent, inhibition of platelets (blunted thromboxane B2 generation), and inhibition of systemic inflammation (lower plasma concentration of serum amyloid P, haptoglobin). NicA treatment resulted in an approximately 2-fold higher concentration of MNA and its metabolites in urine and a 4-fold higher nicotinamide/MNA ratio in plasma, compared with MNA treatment. In summary; MNA displays pronounced antiatherosclerotic action in ApoE/LDLR(-/-) mice, an effect associated with an improvement in prostacyclin- and nitric oxide-dependent endothelial function, inhibition of platelet activation, inhibition of inflammatory burden in plaques, and diminished systemic inflammation. Despite substantially higher MNA availability after NicA treatment, compared with an equivalent dose of MNA, the antiatherosclerotic effect of NicA was not stronger. We suggest that detrimental effects of NicA or its metabolites other than MNA may limit beneficial effects of NicA-derived MNA.

Vascular diseases investigated ex vivo by using Raman, FT-IR and complementary methods.

This work shows the application of vibrational spectroscopy supported by other complementary techniques in analysis of tissues altered by vascular diseases, in particular atherosclerosis. The analysis of atherosclerotic plaque components, as well as label-free imaging of vessels and identification of biochemical markers of endothelial dysfunction are reported. Additionally, the potential of vibrational spectroscopy imaging in following the disease progression (including calcification) and pathological changes in heart valves is described. The presented research shows the effectiveness of techniques used in the biochemical studies of altered tissues and summarizes their capabilities in research on vascular diseases. The scope of the paper is to collect previously published work connected with the application of Raman spectroscopy, FT-IR spectroscopy and complementary methods for the investigation of vascular diseases ex vivo and presenting it in a comprehensive overview.