Search

Acute lymphoblastic leukaemia (ALL) is a complex disease in pediatric oncology, necessitating accurate diagnostic strategies for effective treatment planning. The ability to differentiate between B-cell ALL (B-ALL) and T-cell ALL (T-ALL) is crucial for targeted interventions. However, current diagnostic methods are time-consuming and require rapid, dependable tests. This study explores the potential of label-free Raman imaging coupled with chemometrics for rapid blast phenotyping of B-ALL and T-ALL. Our findings demonstrate the efficacy of Raman spectroscopy in sensitively and specifically screening and classifying ALL, as well as its rapidity and reliability. The obtained molecular information allows for label-free and precise leukaemia diagnosis at the single-cell level, surpassing the capabilities of traditional diagnostic techniques. Raman spectra of cancer cells reveal distinctive molecular signatures, specifically heightened protein and nucleic acid content, revealing molecular signatures unique to leukemic phenotypes. Based on that, they could be distinguished from each other and their normal B and T lymphocyte counterparts. This research underscores the analytical power of Raman spectroscopy, positioning it as a valuable tool for identifying and classifying pediatric ALL subtypes. The potential translational applications in clinical practice offer a promising avenue for an expedited and accurate leukaemia diagnosis, paving the way for more targeted and personalised therapeutic approaches. [ABSTRACT FROM AUTHOR]

Introduction: The present study concerns a connection of the Q192RPON1 polymorphism with atherosclerosis requiring percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) in the Polish population. Methods: A total of 282 individuals who underwent coronary angiography took part in this study. The polymorphism was determined with the PCRRFLP method. Results: The odds ratio for atherosclerosis in carriers of the 192RR genotype was 2.50 (p = 0.002). The median HDL-C concentration was significantly lower in the study group than the control group (p = 0.02). Conclusions: The presence of the 192RR genotype and 192R allele are indicative of at least a two-fold increased risk of atherosclerosis requiring PCI or CABG in the Polish population. [ABSTRACT FROM AUTHOR]

Carotenoids tend to form supramolecular aggregates via non‐covalent interactions where the chirality of individual molecules is amplified to the macroscopic level. We show that this can also be achieved for non‐chiral carotenoid monomers interacting with polysaccharides. The chirality induction in canthaxanthin (CAX), caused by heparin (HP) and hyaluronic acid (HA), was monitored by chiroptical spectroscopy. Electronic circular dichroism (ECD) and Raman optical activity (ROA) spectra indicated the presence of multiple carotenoid formations, such as H‐ and J‐type aggregates. This is consistent with molecular dynamics (MD) and density functional theory (DFT) simulations of the supramolecular structures and their spectroscopic response. [ABSTRACT FROM AUTHOR]

Carotenoids tend to form supramolecular aggregates via non‐covalent interactions where the chirality of individual molecules is amplified to the macroscopic level. We show that this can also be achieved for non‐chiral carotenoid monomers interacting with polysaccharides. The chirality induction in canthaxanthin (CAX), caused by heparin (HP) and hyaluronic acid (HA), was monitored by chiroptical spectroscopy. Electronic circular dichroism (ECD) and Raman optical activity (ROA) spectra indicated the presence of multiple carotenoid formations, such as H‐ and J‐type aggregates. This is consistent with molecular dynamics (MD) and density functional theory (DFT) simulations of the supramolecular structures and their spectroscopic response. [ABSTRACT FROM AUTHOR]

Although the potential of Raman Optical Activity (ROA) spectroscopy in the field of organic and inorganic materials research has been well demonstrated, its practical application is still limited. This work shows historical background, current state of the art and challenges of ROA spectroscopy facing scientists today. Expectations and development prospects for the coming years are also discussed. Efforts have already been made to improve but also to properly interpret the ROA signal when the ROA technique is combined with other spectroscopies (i.e., ROA/Electronic Circular Dichroism and ROA/Circularly Polarized Luminescence). The paper shows how to intensify the ROA effect to shorten the spectrum accumulation time, control possible artefacts and support the analysis of ROA spectra with actual and new theoretical approaches. However, the challenge is to increase the interest in this technique among the academic community, develop reliable protocols and adapt them for practical use. National and international research networks can significantly contribute to the growth of methodologies and standardization in areas in which the ROA has so far been little used. This paper presents a perspective vision for the future progress of ROA alongside other chiroptical methods. [ABSTRACT FROM AUTHOR]

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with chromosome translocations like KMT2A gene rearrangement (KMT2A-r) and BCR-ABL1 fusion gene have been recognized as crucial drivers in both BCP-ALL leukemogenesis and treatment management. Standard diagnostic protocols for proliferative diseases of the hematopoietic system, like KMT2A-r-ALL, are genetically based and strongly molecularly oriented. Therefore, an efficient diagnostic procedure requires not only experienced and multidisciplinary laboratory staff but also considerable instrumentation and material costs. In recent years, a Raman spectroscopy method has been increasingly used to detect subtle chemical changes in individual cells resulting from stress or disease. Therefore, the objective of this study was to identify Raman signatures for the molecular subtypes and to develop a classification method based on the unique spectroscopic profile of in vitro models that represent specific aberrations aimed at KMT2A-r (RS4;11, and SEM) and the BCR-ABL1 fusion gene (SUP-B15, BV-173, and SD-1). Data analysis was based on chemometric methods, i.e. principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and support vector machine (SVM). The PCA-based multivariate model was used for pattern recognition of each investigated group of cells while PLS-DA and SVM were used to build models for the discrimination of spectra from the studied BCP-ALL molecular subtypes. The results showed that the studied molecular subtypes of ALL have characteristic spectroscopic profiles reflecting their peculiar biochemical state. The content of lipids (1600 cm−1), nucleic acids (789 cm−1), and haemoproteins (754, 1130, and 1315 cm−1), which are crucial in cell metabolism, was indicated as the main source of differentiation between subtypes. Identification of spectroscopic markers of cells with BCR-ABL1 or KMT2A-r may be useful in pharmacological studies to monitor the effectiveness of chemotherapy and further to understand differences in molecular responses between leukemia primary cells and cell lines. [ABSTRACT FROM AUTHOR]

Astaxanthin (AXT) is a lipophilic antioxidant and anti-inflammatory natural pigment whose cellular uptake and bioavailability could be improved via liposomal encapsulation. Endothelial cells (EC) line the lumen of all blood vessels and are tasked with multiple roles toward maintaining cardiovascular homeostasis. Endothelial dysfunction is linked to the development of many diseases and is closely interconnected with oxidative stress and vascular inflammation. The uptake of free and liposomal AXT into EC was investigated using Raman and fluorescence microscopies. AXT was either encapsulated in neutral or cationic liposomes. Enhanced uptake and anti-inflammatory effects of liposomal AXT were observed. The anti-inflammatory effects of liposomal AXT were especially prominent in reducing EC lipid unsaturation, lowering numbers of lipid droplets (LDs), and decreasing intercellular adhesion molecule 1 (ICAM-1) overexpression, which is considered a well-known marker for endothelial inflammation. These findings highlight the benefits of AXT liposomal encapsulation on EC and the applicability of Raman imaging to investigate such effects. [ABSTRACT FROM AUTHOR]

Endothelial cells (EC) in vivo buffer and regulate the transfer of plasma fatty acid (FA) to the underlying tissues. We hypothesize that inflammation could alter the functionality of the EC, i.e., their capacity and uptake of different FA. The aim of this work is to verify the functionality of inflamed cells by analyzing their ability to uptake and accumulate exogenous saturated FA. Control and inflammatory human microvascular endothelial cells stimulated in vitro with two deuterium-labeled saturated FA (D-FA), i.e., palmitic (D31-PA) and myristic (D27-MA) acids. Cells were measured both by spontaneous and stimulated Raman imaging to extract detailed information about uptaken FA, whereas coherent anti-Stokes Raman scattering and fluorescence imaging showed the global content of FA in cells. Additionally, we employed atomic force microscopy to obtain a morphological image of the cells. The results indicate that the uptake of D-FA in inflamed cells is dependent on their concentration and type. Cells accumulated D-FA when treated with a low concentration, and the effect was more pronounced for D27-MA, in normal cells, but even more so, in inflamed cells. In the case of D31-PA, a slightly increased uptake was observed for inflamed cells when administered at higher concentration. The results provide a better understanding of the EC inflammation and indicate the impact of the pathological state of the EC on their capacity to buffer fat. All the microscopic methods used showed complementarity in the analysis of FA uptake by EC, but each method recognized this process from a different perspective. [ABSTRACT FROM AUTHOR]

Experimental vibrational Raman and Raman optical activity (ROA) spectra for diacetyl L‐tartaric acid (DAT), two of its esters, namely, monomethyl and lauryl esters (T1OH and T12OH), and corresponding sodium salts (DATNa, T1ONa, and T12ONa), are measured. T12OH and T12ONa represent the first chiral surfactants investigated using ROA spectroscopy. The quantum chemical (QC) predictions using B3LYP functional and 6‐311++G(2d,2p) basis set are used to interpret the ROA spectra for DAT, DATNa, T1OH, and T1ONa. It is found that the use of implicit solvation, as represented in polarizable continuum model (PCM), for predicting the experimental ROA spectra in aqueous solutions is inadequate for DAT and T1OH. However, the same PCM predicts the experimental ROA spectra satisfactorily for the DATNa and T1ONa. This favorable observation for the latter is attributed to the absence of intra‐ and inter‐molecular hydrogen bonding interactions for sodium salts in aqueous solutions. The overwhelming number of conformations resulting from 12‐carbon alkyl chain, in T12OH and T12ONa, makes it impractical to undertake QC predictions for them. Nevertheless, it is found that the predictions made for shorter alkyl chain analogs, namely, T1OH and T1ONa, may be used to explain the experimental ROA spectra of T12OH and T12ONa. The current work highlights the importance of converting carboxylic acids to corresponding sodium salts and of QC predictions for shorter achiral alkyl chain analogs to interpret the ROA spectra of chiral surfactants that contain long achiral alkyl chains. [ABSTRACT FROM AUTHOR]

In this study, we found that a recently discovered ECD-Raman effect dominated over the natural Raman optical activity in a series of atropisomeric naphthalenediimides, and we investigated the kind of information about the molecular structure that could be obtained from the spectra. The ECD-Raman effect is polarised Raman scattering modulated by electronic circular dichroism. We showed that the spectra significantly depended on the substitution of the solute and/or the change of the solvent. Moreover, the spectra could be well-predicted by the theory, thus providing an interesting tool to monitor the chirality of the binaphthyl compounds. [ABSTRACT FROM AUTHOR]

Intermolecular interactions sensitive to chirality occur in many biological events. We report a complex formation between a versatile vanadium-based probe and a chiral co-ligand monitored via the combination of electronic circular dichroism (ECD) and Raman scattering. This "ECD-Raman" effect was discovered relatively recently and can be measured using a Raman optical activity (ROA) spectrometer. Simulated spectra based on experimental ECD and degree of circularity (DOC) values agree with the observed ones. Sensitive recognition of the chiral enantiopure co-ligand is thus enabled by a combination of resonance of the excitation light with the diastereoisomeric complex, co-ligand complexation, circular dichroism, and polarized Raman scattering from the achiral solvent. Relatively dilute solutions could be detected (10−4 mol dm−3), about 1000× less than is necessary for conventional ROA detection of the pure co-ligand and comparable to concentrations needed for conventional ECD spectroscopy. The results thus show that differential ECD-Raman measurements can be conveniently used to monitor molecular interactions and molecular spectroscopic properties. [ABSTRACT FROM AUTHOR]

Resonance Raman optical activity (RROA) possesses all aspects of a sensitive tool for molecular detection, but its measurement remains challenging. We demonstrate that reliable recording of RROA of chiral colorful compounds is possible, but only after considering the effect of the electronic circular dichroism (ECD) on the ROA spectra induced by the dissolved chiral compound. We show RROA for a number of model vitamin B12 derivatives that are chemically similar but exhibit distinctively different spectroscopic behavior. The ECD/ROA effect is proportional to the concentration and dependent on the optical pathlength of the light propagating through the sample. It can severely alter relative band intensities and signs in the natural RROA spectra. The spectra analyses are supported by computational modeling based on density functional theory. Neglecting the ECD effect during ROA measurement can lead to misinterpretation of the recorded spectra and erroneous conclusions about the molecular structure. [ABSTRACT FROM AUTHOR]

Resonance Raman optical activity (RROA) possesses all aspects of a sensitive tool for molecular detection, but its measurement remains challenging. We demonstrate that reliable recording of RROA of chiral colorful compounds is possible, but only after considering the effect of the electronic circular dichroism (ECD) on the ROA spectra induced by the dissolved chiral compound. We show RROA for a number of model vitamin B12 derivatives that are chemically similar but exhibit distinctively different spectroscopic behavior. The ECD/ROA effect is proportional to the concentration and dependent on the optical pathlength of the light propagating through the sample. It can severely alter relative band intensities and signs in the natural RROA spectra. The spectra analyses are supported by computational modeling based on density functional theory. Neglecting the ECD effect during ROA measurement can lead to misinterpretation of the recorded spectra and erroneous conclusions about the molecular structure. [ABSTRACT FROM AUTHOR]

Small molecules are frequently used as dyes, labels and markers to visualize and probe biophysical processes within cells. However, very little is generally known about the light-driven excited-state reactivity of such systems when placed in cells. Here an experimental approach to study ps time-resolved excited state dynamics of a benchmark molecular marker, astaxanthin, in live human cells is introduced. [ABSTRACT FROM AUTHOR]

Here we report a new Raman probe for cellular studies on lipids detection and distribution. It is (3S, 3'S)-astaxanthin (AXT), a natural xanthophyll of hydrophobic properties and high solubility in lipids. It contains a chromophore group, a long polyene chain of eleven conjugated C=C bonds including two in the terminal rings, absorbing light in the visible range that coincides with the excitation of lasers commonly used in Raman spectroscopy for studying of biological samples. Depending on the laser, resonance (excitation in the visible range) or pre-resonance (the near infrared range) Raman spectrum of astaxanthin is dominated by bands at ca. 1008, 1158, and 1520 cm−1 that now can be also a marker of lipids distribution in the cells. We showed that AXT accumulates in lipidic structures of endothelial cells in time-dependent manner that provides possibility to visualize e.g. endoplasmic reticulum, as well as nuclear envelope. As a non-toxic reporter, it has a potential in the future studies on e.g. nucleus membranes damage in live cells in a very short measuring time. [ABSTRACT FROM AUTHOR]

The importance of the electric‐dipole—electric‐quadrupole polarizability contribution to the vibrational Raman optical activity (VROA) and dimensionless circular intensity difference spectra of (1S)‐camphor is examined. The spectra are simulated with and without this tensor contribution using density functional theory calculations, and similarity is evaluated for each against the experimental spectra. Careful examination of the comparison between experimental and calculated spectra reveals multiple vibrational bands, in the ~1,130–950 cm−1 region, originating from −C–H bending vibrations that are dominated by the electric‐dipole—electric‐quadrupole polarizability contributions. The similarity overlap analysis also reveals that the similarity overlap between experimental and predicted spectra in the measured spectral range increases, by up to 12%, when electric‐dipole—electric‐quadrupole polarizability contributions are included. The negative VROA band at 1,125 cm−1 in the experimental spectrum of (1S)‐(‐)‐camphor can only be reproduced in the predicted spectra when electric‐dipole—electric‐quadrupole polarizability contribution is included. Investigations on additional molecules indicated that (a) two experimental VROA bands of (1S,4R)‐(+)‐fenchone at ~1,740 and 220 cm−1 originate from dominating electric‐dipole—electric‐quadrupole polarizability contribution and (b) the symmetric and antisymmetric ring deformation modes of dimethyloxirane have dominating electric‐dipole—electric‐quadrupole polarizability contribution. These observations establish the importance of electric‐dipole—electric‐quadrupole polarizability contribution to VROA for the first time. [ABSTRACT FROM AUTHOR]

A new type of aggregate, formed in human red blood cells (RBCs) in response to glutaraldehyde treatment, was discovered and analyzed with the classical and advanced biomolecular imaging techniques. Advanced Heinz body-like aggregates (AHBA) formed in a single human RBC are characterized by a higher level of hemoglobin (Hb) degradation compared to typical Heinz bodies, which consist of hemichromes. The complete destruction of the porphyrin structure of Hb and the aggregation of the degraded proteins in the presence of Fe3+ ions are observed. The presence of such aggregated, highly degraded proteins inside RBCs, without cell membrane destruction, has been never reported before. For the first time the spatial differentiation of two kinds of protein mixtures inside a single RBC, with different phenylalanine (Phe) conformations, is visualized. The non-resonant Raman spectra of altered RBCs with AHBA are characterized by the presence of a strong band located at 1037 cm−1, which confirms that glutaraldehyde interacts strongly with Phe. The shape-shifting of RBCs from a biconcave disk to a spherical structure and sinking of AHBA to the bottom of the cell are observed. Results reveal that the presence of AHBA should be considered when fixing RBCs and indicate the analytical potential of Raman spectroscopy, atomic force microscopy and scanning near-field optical microscopy in AHBA detection and analysis. [ABSTRACT FROM AUTHOR]

This paper describes how tunicamycin (Tu), the most widely used pharmacological agent for inducing endoplasmic reticulum (ER) stress, interacts with endothelial cells. Our results show that tunicamycin enters the cells and accumulates within the ER area. ER stress takes place when improperly folded or damaged proteins begin to accumulate; however, spectroscopic markers of these changes have not been identified as yet. In this work, Raman spectroscopy and scanning electron microscopy imaging of individual endothelial cells treated with Tu were performed. The changes in the biochemical composition of endothelial cells induced by Tu attributed to ER stress were studied in detail. A main feature of the Tu impact on the cells was a decrease of the phospholipid content in the area of ER, and the most abundant lipid with phosphorus groups found there, was identified as sphingomyelin. [ABSTRACT FROM AUTHOR]

Summary: Eosinophils are acidophilic granulocytes that develop in the bone marrow. Although their population contributes only to approximately 1–6% of all leucocytes present in the human blood, they possess a wide range of specific functions. They play a key role in inflammation‐regulating processes, when their numbers can increased to above 5 × 109/l of peripheral blood. Their characteristic feature is the presence of granules containing eosinophil peroxidase (EPO), the release of which can trigger a cascade of events promoting oxidative stress, apoptosis or necrosis, leading finally to cell death. Raman spectroscopy is a powerful technique to detect EPO, which comprises a chromophore protoporphyrin IX. Another cell structure associated with inflammation processes are lipid bodies (lipid‐rich organelles), also well recognized and imaged using high resolution confocal Raman spectroscopy. In this work, eosinophils isolated from the blood of a human donor were analysed versus their model, EoL‐1 human eosinophilic leukaemia cell line, by Raman spectroscopic imaging. We showed that EPO was present only in primary cells and not found in the cell line. Eosinophils were activated using phorbol 12‐myristate 13‐acetate, which resulted in lipid bodies formation. An effect of cells stimulation was studied and compared for eosinophils and EoL‐1. [ABSTRACT FROM AUTHOR]

Carotenoid microcrystals, extracted from cells of carrot roots and consisting of 95 % of achiral β‐carotene, exhibit a very intense chiroptical (ECD and ROA) signal. The preferential chirality of crystalline aggregates that consist mostly of achiral building blocks is a newly observed phenomenon in nature, and may be related to asymmetric information transfer from the chiral seeds (small amount of α‐carotene or lutein) present in carrot cells. To confirm this hypothesis, we synthesized several model aggregates from various achiral and chiral carotenoids. Because of the sergeant‐and‐soldier behavior, a small number of chiral sergeants (α‐carotene or astaxanthin) force the achiral soldier molecules (β‐ or 11,11′‐[D2]‐β‐carotene) to jointly form supramolecular assemblies of induced chirality. The chiral amplification observed in these model systems confirmed that chiral microcrystals appearing in nature might consist predominantly of achiral building blocks and their supramolecular chirality might result from the co‐crystallization of chiral and achiral analogues. [ABSTRACT FROM AUTHOR]

Carotenoid microcrystals, extracted from cells of carrot roots and consisting of 95 % of achiral β‐carotene, exhibit a very intense chiroptical (ECD and ROA) signal. The preferential chirality of crystalline aggregates that consist mostly of achiral building blocks is a newly observed phenomenon in nature, and may be related to asymmetric information transfer from the chiral seeds (small amount of α‐carotene or lutein) present in carrot cells. To confirm this hypothesis, we synthesized several model aggregates from various achiral and chiral carotenoids. Because of the sergeant‐and‐soldier behavior, a small number of chiral sergeants (α‐carotene or astaxanthin) force the achiral soldier molecules (β‐ or 11,11′‐[D2]‐β‐carotene) to jointly form supramolecular assemblies of induced chirality. The chiral amplification observed in these model systems confirmed that chiral microcrystals appearing in nature might consist predominantly of achiral building blocks and their supramolecular chirality might result from the co‐crystallization of chiral and achiral analogues. [ABSTRACT FROM AUTHOR]

Gaining knowledge on the biochemical profile of primary endothelial cells on a subcellular level can contribute to better understanding of cardiovascular disease. In this work, primary cardiac microvascular endothelial cells (CMECs) isolated from the mouse heart and murine H5V endothelial cell line were characterized with the use of a Raman imaging technique. Primary CMECs displayed a distinct Raman-based biochemical phenotype as compared with other cells isolated from the heart and were characterized by a low lipid content. In contrast to the murine H5V endothelial cell line, CMECs did not display lipid droplets (LDs) in the cytoplasm, while the former have many low-unsaturated LDs. In conclusion, Raman imaging is a fast and efficient tool to analyse single coronary endothelial cells in a non-invasive manner that can prove useful to characterize biochemical changes in a single isolated primary endothelial cell from a diseased heart. [ABSTRACT FROM AUTHOR]

The work presents the complementary approach to characterize the formation of various Hb species inside isolated human RBCs exposed to NO, with a focus on the formed Hb–NO adducts. This work presents a complementary approach based on Resonance Raman Spectroscopy (RRS) supported by Blood Gas Analysis, Electron Paramagnetic Resonance Spectroscopy, UV–Vis Absorption Spectroscopy and Mössbauer Spectroscopy to characterize the formation of various Hb species, with a focus on the Hb–NO adducts formed inside isolated human RBCs exposed to NO, under the experimental conditions of low and high levels of oxygen Hb saturation. In the present work, we induced Hb–NO adducts using PAPA–NONOate, a NO-donor with known chemistry and kinetics of NO release, and confirmed the formation of Hb–NO adducts in RBCs incubated with Human Aortic Endothelial Cells (HAECs) stimulated to produce NO. Our results provide a new insight into the formation of Hb–NO adducts after the exposure of RBCs with high oxyHb content to exogenous NO with special attention to the formation of LSHbIIINO in addition to LSHbIINO and metHb (HS/LSHbIIIH2O). We also point out that reliable characterization of Hb–NO adducts requires complementary techniques. Among them, RRS, as a label-free and non-destructive tool, appears to be an important discrimination technique in the studies of Hb–NO adducts inside intact RBCs. [ABSTRACT FROM AUTHOR]

Growing interest in the role of endothelium under physiological and pathological conditions has led to an increasing demand for its representative in vitro models especially suitable for drug tests and medical diagnostics. There are several endothelial cell lines commercially available whose biochemistry, and hence response to various stimuli, can be different. Recently, two vibrational techniques, Raman and Fourier-transform infrared microscopy, have been found to be potent tools for studying the biochemical composition of a single cell in an easy, rapid and label-free way. However, depending on the applied technique, the results may exhibit some divergence due to different selection rules as well as distinct experimental conditions. This paper presents the methodology of examination and characterization of three popular human endothelial cell lines: HAoEC (primary cells), HMEC-1 and EA.hy926 (immortalized cells). Based on high lateral resolution Raman imaging together with standard and high magnification Fourier-transform infrared measurements, the differences in spectral information and the distribution of biomolecules are presented and discussed. [ABSTRACT FROM AUTHOR]

Biomolecular aggregation is omnipresent in nature and important for metabolic processes or in medical treatment; however, the phenomenon is rather difficult to predict or understand on the basis of computational models. Recently, we found that electronic circular dichroism (ECD) spectroscopy and closely related resonance Raman optical activity (RROA) are extremely sensitive to the aggregation mechanism and structure of the astaxanthin dye. In the present study, molecular dynamics (MD) and quantum chemical (QC) computations (ZIndo/S, TDDFT) are used to link the aggregate structure with ECD spectral shapes. Realistic absorption and ECD intensities were obtained and the simulations reproduced many trends observed experimentally, such as the prevalent sign pattern and dependence of the aggregate structure on the solvent type. The computationally cheaper ZIndo/S method provided results very similar to those obtained by TDDFT. In the future, the accuracy of the combined MD/QC methodology of spectra interpretation should be improved to provide more detailed information on astaxanthin aggregates and similar macromolecular systems. [ABSTRACT FROM AUTHOR]

Purpose: We aimed at assessing the predisposition of A-kinase anchoring protein 10 (AKAP10) polymorphism toward acquired repolarization disorders in high-risk vascular surgery patients. Patients and methods: One hundred adult patients (age =44–85 years), scheduled for an elective high-risk "open" vascular surgery procedure, were recruited. The electrocardiogram Holter monitor was used to assess repolarization stability from the beginning of the operation up to 24 hours afterward. The AKAP10 gene rs203462 polymorphism and cardiac complications were analyzed. Results: Repolarization disturbances defined as QT interval duration corrected for heart rate (QTc) interval prolongation >500 ms and QTc interval dispersion >65 ms were recorded in 46 patients. A model of multivariate logistic regression showed that only the presence of allele G of the AKAP10 polymorphism was an independent risk factor for repolarization disturbances in the perioperative period (odds ratio =14.35; 95% CI =4.65–44.23; p<0.0001). Conclusion: When the acquired QTc interval prolongation or QTc dispersion is associated with AKAP10 polymorphism, it may remain clinically silent. [ABSTRACT FROM AUTHOR]

In this work, confocal Raman imaging was used to study the formation of lipid droplets (LDs) in vitro in a single endothelial cell upon incubation with polyunsaturated fatty acids (10 or 25 μM) including arachidonic acid (AA) and its deuterated analog (AA-d8), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Based on the Raman spectra obtained from a single endothelial cell, it was possible to investigate biochemical changes induced by addition of polyunsaturated fatty acids. In particular, the content of lipids in the formed LDs and the unsaturation degree were identified by Raman spectroscopy by marker bands at 1660 cm−1 due to the C=C stretching and at ∼3015 cm−1 due to the stretching mode of =C–H associated with C=C double bonds (except for a deuterated form where these bands are shifted respectively). To establish if the exogenous fatty acid was taken up by the cell and stored in LDs, a deuterium labelled polyunsaturated fatty acid was used. AA-d8 shows characteristic bands at around 2200–2300 cm−1 assigned to the =C–D stretching modes. We established the uptake of AA and the accumulation of EPA into newly formed LDs in the endothelial cells. In contrast, no accumulation of DHA in LDs was observed even though LDs were formed upon DHA incubation. Furthermore, using AFM we demonstrated that the presence of LDs in the endothelium affected endothelial stiffness which could have pathophysiological significance. In summary, the results suggest that the formation of LDs in the endothelium involves exogenous and endogenous polyunsaturated fatty acids, and their relative contribution to the LD formation seems distinct for AA, EPA and DHA. [ABSTRACT FROM AUTHOR]

Angiotensin‐converting enzyme inhibitors (ACE‐I) display vasoprotective activity and represent the cornerstone in the treatment of cardiovascular diseases. In this study, we tested whether Fourier transform infrared (FTIR)‐based analysis of blood plasma is sensitive to detect vasoprotective effects of treatment with perindopril including reversal of endothelial dysfunction in diabetes. For this purpose, plasma samples were collected from untreated db/db mice, db/db mice treated with 2 or 10 mg/kg perindopril and db+ mice. The effect of perindopril on endothelial function was examined in ex vivo aortic rings; 10 mg/kg but not 2 mg/kg of perindopril reversed endothelial dysfunction. In plasma of db/db mice, the balance between conformations of plasma proteins was noted, and treatment with perindopril at a high dose but not at a low dose reversed this effect. This was revealed by amide II/amide I ratio attributed to increased β‐sheet formation. Spectral markers at 3010, 1520/1238 cm−1, representative for unsaturation degree of lipids and phosphorylation of tyrosine, respectively, were also affected by perindopril treatment. In conclusion, although metabolic abnormalities associated with type 2 diabetes mellitus such as hypertriglyceridemia and hyperglycemia strongly affected spectral FTIR profile of diabetic plasma, we identified FTIR features that seem to be associated with the vasoprotective activity of ACE‐I. [ABSTRACT FROM AUTHOR]

Non-Alcoholic Fatty Liver Disease (NAFLD) is the most prevalent liver disorder worldwide, involving pathogenic mechanisms of liver sinusoidal endothelial cells (LSECs), hepatocytes and other liver cells. Here, we used a novel approach of label-free Raman confocal imaging to study primary LSECs and hepatocytes freshly isolated from the livers of mice with NAFLD induced by a high fat diet (HFD), in comparison to healthy controls. Our aim was to characterize changes in the biochemical composition in LSECs and hepatocytes that occur in a single cell at the subcellular level. LSECs from NAFLD livers displayed a significant increase in the intensity of marker bands of nuclear DNA that was not associated with changes in LSEC nucleus size. A number of changes in the cytoplasm of hepatocytes were identified. However, the most prominent change in hepatocytes was a substantial increase in the degree of unsaturation of LBs’ (lipid bodies) lipids in NAFLD, suggesting an increase in the de novo lipogenesis of unsaturated lipids. The confocal Raman imaging of single live cells isolated from the liver provided a unique tool to better understand disease-induced cell-specific changes in the biochemical phenotype of primary liver cells. [ABSTRACT FROM AUTHOR]

The scanning near-field optical microscopy (SNOM) shows a potential to study details of biological samples, since it provides the optical images of objects with nanometric spatial resolution (50-200 nm) and the topographic information at the same time. The goal of this work is to demonstrate the capabilities of SNOM in transmission configuration to study human endothelial cells and their morphological changes, sometimes very subtle, upon inflammation. Various sample preparations were tested for SNOM measurements and promising results are collected to show: 1) the influence of α tumor necrosis factor (TNF-α) on EA.hy 926 cells (measurements of the fixed cells); 2) high resolution images of various endothelial cell lines, i.e. EA.hy 926 and HLMVEC (investigations of the fixed cells in buffer environment); 3) imaging of live endothelial cells in physiological buffers. The study demonstrate complementarity of the SNOM measurements performed in air and in liquid environments, on fixed as well as on living cells. Furthermore, it is proved that the SNOM is a very useful method for analysis of cellular morphology and topography. Changes in the cell shape and nucleus size, which are the symptoms of inflammatory reaction, were noticed in TNF-α activated EA.hy 926 cells. The cellular structures of submicron size were observed in high resolution optical images of cells from EA.hy 926 and HLMVEC lines. [ABSTRACT FROM AUTHOR]

Experimental vibrational Raman, vibrational Raman optical activity (VROA), and dimensionless circular intensity difference (CID) spectra are analyzed with quantum chemical (QC) predictions of the corresponding spectra using the B3LYP functional and the aug-cc-pVDZ basis set, for five bicyclic terpenes as test cases. The experimentally measured vibrational Raman and VROA spectra have arbitrary y-axis intensities, because of their dependence on instrumental parameters. As a result, the absolute magnitudes of intensities in experimental and calculated vibrational Raman or vibrational ROA spectra are not normally analyzed. However, the experimental dimensionless CID spectrum is independent of the instrumental parameters and can be evaluated quantitatively with corresponding QC predicted CID spectrum. The similarity overlaps between experimental and quantum chemical predictions of vibrational Raman, VROA, and dimensionless CID spectra are evaluated. The quantum chemical predictions of dimensionless CID spectral magnitudes at B3LYP/aug-ccpVDZ level match well with the corresponding experimental magnitudes. The individual anisotropic contributions to VROA and CID spectra are also presented. The anisotropic contribution involving electric dipole-electric quadrupole polarizability derivative tensor is relatively smaller than that involving electric dipole-magnetic dipole polarizability derivative tensor. A new format, which contains all of the information needed for the spectral analysis, has been advanced for presenting the vibrational Raman, VROA, and CID spectra and their similarity analysis with corresponding QC-predicted spectra. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Raman spectroscopy is a label free, versatile, simple and fast method that is increasingly used to detect pathological changes in the cells and tissues that could be useful in medical diagnostics. In this work, we tested the hypothesis that Raman spectroscopy may serve to detect endothelial dysfunction in murine models of lifestyle diseases associated with endothelial dysfunction. For that purpose, we analysed spectra from ex vivo vessels taken from mice with diabetes, hypertension and cancer metastasis. We extracted 50-70 random, single spectra, recorded in 0.2 s, from endothelium of mice with diseases and respective control animals and subjected them to hierarchical cluster analysis. Independently on the sample preparation protocol, very good discrimination was obtained for three-tested murine models, i.e. diabetes, hypertension and cancer metastasis. Obtained sensitivity and specificity parameters were between 93% and 96% (with the exception of sensitivity in the diabetes model equalled to 88%). Our results show that single, random spectra of endothelium, recorded in less than a second, contains enough information on biochemical content of the endothelium to detect endothelial dysfunction. Furthermore, we demonstrated that biochemical profile of the endothelial dysfunction in diabetes, hypertension or cancer metastasis differs with a very high specificity and sensitivity. This conclusion can be a good starting point for the development of in vivo fast diagnostic methodology of endothelium in the future. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Mouse model of nitric oxide deficiency, induced by prolonged treatment with NG-nitro-L-arginine methyl ester (L-NAME) was used for infrared spectroscopy (FTIR) analysis of plasma. L-NAME leads to increased peripheral resistance and systemic hypertension. Classification of spectral response was by principal component analysis (PCA) and linear discriminant analysis (LDA). PCA allowed to separate each animal group showing that FTIR spectra are sensitive to development of NO-deficiency on contrary to blood pressure values indicating hypertension. Globally, the most pronounced spectral alternations were observed in the second and third week of L-NAME treatment indicating that infrared signature of blood plasma can serve as indicator of early and late stages of the disease. The PLS-DA method provided >95% classification accuracy. Spectral features characteristic for L-NAME treatment were mainly associated with an elevated level of proteins accompanied by a decrease of a tyrosine content and changes in lipids/phospholipid concentration. In our work we discuss these changes for which statistically significant differences ( p < 0.05 - 0.005) were observed between spectra collected for each time-point of the L-NAME treatment versus control subjects. We demonstrated for the first time that NO-deficiency and hypertension resulted in changes in biochemical profile of plasma that was detected by FTIR spectroscopy. [ABSTRACT FROM AUTHOR]

It was recently reported in the murine model of metastatic breast cancer (4T1) that tumor progression and development of metastasis is associated with systemic endothelial dysfunction characterized by impaired nitric oxide (NO) production. Using Raman 3D confocal imaging with the analysis of the individual layers of the vascular wall combined with AFM endothelial surface imaging, we demonstrated that metastasis-induced systemic endothelial dysfunction resulted in distinct chemical changes in the endothelium of the aorta. These changes, manifested as a significant increase in the protein content (18 %) and a slight decrease in the lipid content (4 %), were limited to the endothelium and did not occur in the deeper layers of the vascular wall. The altered lipid to protein ratio in the endothelium, although more pronounced in the fixed vascular wall, was also observed in the freshly isolated unfixed vascular wall samples in the aqueous environment (12 and 7 % change of protein and lipid content, respectively). Our results support the finding that the metastasis induces systemic endothelial dysfunction that may contribute to cancer progression. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

In this work the formation of lipid droplets (LDs) in human endothelial cells culture in response to the uptake of polyunsaturated fatty acids (PUFAs) was studied. Additionally, an effect of 1-methylnicotinamide (MNA) on the process of LDs formation was investigated. LDs have been previously described structurally and to some degree biochemically, however neither the precise function of LDs nor the factors responsible for LD induction have been clarified. Lipid droplets, sometimes referred in the literature as lipid bodies are organelles known to regulate neutrophil, eosinophil, or tumor cell functions but their presence and function in the endothelium is largely unexplored. 3D linear Raman spectroscopy was used to study LDs formation in vitro in a single endothelial cell. The method provides information about distribution and size of LDs as well as their composition. The incubation of endothelial cells with various PUFAs resulted in formation of LDs. As a complementary method for LDs identification a fluorescence microscopy was applied. Fluorescence measurements confirmed the Raman results suggesting endothelial cells uptake of PUFAs and subsequent LDs formation in the cytoplasm of the endothelium. Furthermore, MNA seem to potentiate intracellular uptake of PUFAs to the endothelium that may bear physiological and pharmacological significance. [ABSTRACT FROM AUTHOR]

High spatially resolved Raman microscopy was applied to study the early apoptosis in endothelial cells and chemical and structural changes induced by this process. Application of cluster analysis enabled separation of signals due to various subcellular organelles and compartments such as the nuclei, nucleoli, endoplasmic reticulum or cytoplasm and analysis of alterations locally at the subcellular level. Different stimuli, i.e. Fas ligand, a tumor necrosis factor, and cycloheximide, an inhibitor of eukaryotic protein biosynthesis, were applied to induce apoptotic mechanisms. Due to different mechanisms of action, the changes observed in subcellular structures were different for FasL and cycloheximide. Although in both cases a statistically significant decrease of the protein level was observed in all studied cellular structures, the increase of the nucleic acids content locally in apoptotic nuclei was considerably more pronounced upon FasL-induced apoptosis compared to the cycloheximide one. Additionally, apoptosis invokes also a decrease of the proteins with the α-helix protein structure selectively for FasL in the cytoplasm and endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Cinchona alkaloids are well-known antimalarial compounds also used in asymmetric synthesis in organic chemistry. In this work, vibrational spectra of quinine, quinidine, cinchonine, and cinchonidine were acquired and interpreted on the basis of theoretical calculations. Normal Raman spectra of the alkaloids in solution exhibit similar patterns and cannot be used for differentiation between the derivatives (e.g. quinine and cinchonidine) and corresponding pseudoenantiomers (e.g. quinine and quinidine). Thus, Raman Optical Activity (ROA) method was applied to show distinct differences related to the configuration of chiral atoms. ROA allowed unequivocal identification of the pseudoenantiomers based on the sign of the characteristic bands from a single measurement. The experiments were supported by the theoretical approach including conformational study followed by wavenumber calculations and Potential Energy Distribution (PED) analysis. For quinine, vibrational spectroscopy was additionally used to show its structural changes in aqueous solutions at various pH and its distribution in a pharmaceutical product. Spatial distribution of quinine in a drug was observed by the FT-Raman mapping technique. [ABSTRACT FROM AUTHOR]

Non-Alcoholic Fatty Liver Disease (NAFLD) is a common liver disorder, characterized by an excessive lipids deposition within the hepatic tissue. Due to the lack of clear-cut symptoms and optimal diagnostic method, the actual prevalence of NAFLD and its pathogenesis remains unclear, especially in the early stages of progression. In the presented work confocal Raman microspectroscopy was used to investigate alterations in the chemical composition of the NAFLD-affected liver. We have investigated two NAFLD models, representative for macrovesicular and microvesicular steatosis, induced by High Fat Diet (60 kcal %) and Low Carbohydrate High Protein Diet (LCHP), respectively. In both models we confirmed the development of NAFLD, manifested by the presence of lipid droplets (LDs), but of different sizes. Model of macrovesicular steatosis was characterized by large LDs, whereas in the microvesicular steatosis model small droplets were found. In both models, however, we observed a significant decrease in the degree of unsaturation of lipids, in comparison to the control. In addition, for both models, the impact of medical treatment with selected drugs (perindopril and nicotinic acid, respectively) was tested, indicating a significant influence of medicine not only on the occurrence and size of the droplets, but also on their composition. Inboth cases the drug treatment resulted in an increase of the degree of unsaturation of lipids forming droplets. Confocal Raman microspectroscopy was proven to be a powerful tool providing detailed insight into selected areas of hepatic tissue, following the NAFLD pathogenesis and diagnostic potential of the applied drugs. [ABSTRACT FROM AUTHOR]

Although the potential of vibrational spectroscopy for biomedical applications has been well demonstrated, translation into clinical practice has been relatively slow. This Editorial assesses the challenges facing the field and the potential way forward. While many technological challenges have been addressed to date, considerable effort is still required to gain acceptance of the techniques among the medical community, standardise protocols, extend to a clinically relevant scale, and ultimately assess the health economics underlying clinical deployment. National and international research networks can contribute much to technology development and standardisation. Ultimately, large-scale funding is required to engage in clinical trials and instrument development. [ABSTRACT FROM AUTHOR]

The intracellular pH plays an important role in various cellular processes. In this work, we describe a method for monitoring of the intracellular pH in endothelial cells by using surface enhanced Raman spectroscopy (SERS) and 4-mercaptobenzoic acid (MBA) anchored to gold nanoparticles as pH-sensitive probes. Using the Raman microimaging technique, we analysed changes in intracellular pH induced by buffers with acid or alkaline pH, as well as in endothelial inflammation induced by tumour necrosis factor-α (TNFα). The targeted nanosensor enabled spatial pH measurements revealing distinct changes of the intracellular pH in endosomal compartments of the endothelium. Altogether, SERS-based analysis of intracellular pH proves to be a promising technique for a better understanding of intracellular pH regulation in various subcellular compartments. [ABSTRACT FROM AUTHOR]

FTIR spectroscopy is a widely used technique that provides insights into disease processes at the molecular level. Due to its numerous advantages it is becoming an increasingly powerful tool for the study of biological materials and has the potential to become an excellent diagnostic method, especially considering the low cost of transflection substrates. However, questions about the usefulness of the transflection measurement mode due to the complicated nature of physical processes occurring during the measurement and in particular the Electric Field Standing Wave (EFSW) effect have been raised. In this paper we present a comparison of the two most common FT-IR measurement modes: transmission and transfection using healthy and pathologically altered tissue (histiocytic sarcoma). We found that the major differences between normal and cancerous tissue were associated with changes DNA and carbohydrate content. In particular we identified a band at 964 cm−1 assigned to a nucleic acid phosphodiester backbone mode, which appeared more pronounced in cancerous tissue irrespective of the substrate. We applied Principal Component Analysis, Unsupervised Hierarchical Cluster Analysis and k-means clustering to transmission and transflection substrates and found that both measurement modes were equally capable of discrimination normal form cancerous tissue. Moreover, the differences between spectra from cancerous and normal tissue were significantly more important than the ones arising from the measurement modes. [ABSTRACT FROM AUTHOR]

The main goal of this study was to find specific plasma spectral markers associated with pulmonary arterial hypertension (PAH) induced by monocrotaline injection in rats. FTIR was used to monitor biochemical changes in plasma caused by PAH as compared with the systemic hypertension induced by partial ligation on the left artery and with the control group. Both pathologies, systemic and pulmonary hypertension, induced a unique response in the biochemical content of plasma, mainly related to the composition and secondary structure of plasma proteins. For PAH, β-pleated sheet components of plasma proteins were identified whereas the protein composition in systemic hypertension was dominated by unordered structures. In addition, a higher concentration of tyrosine-rich proteins was found in plasma in PAH than in systemic hypertension. The differences between both pathologies were identified also in terms of lipid composition/metabolism as well as in the content of RNA and glucose, suggesting that lipid peroxidation appears upon pulmonary hypertension development. In summary, this work demonstrates that FTIR spectroscopy supported by principal component analysis (PCA) has the potential to become a fast and non-destructive method for biochemical characterization of plasma that consequently could have a diagnostic significance in pulmonary hypertension. [ABSTRACT FROM AUTHOR]

Raman microimaging was applied to study the biochemical composition in the aortic valves obtained from patients with calcific aortic stenosis. This progressive disease affects an increasing number of elderly patients with hyperlipidemia and hypercholesterolemia. Lipid accumulation in the tissue is associated with pathogenesis and progression of cardiac valve calcification. This is in line with our finding that lipid deposits, predominantly composed of cholesterol and its esters, are frequently co-localized with calcium salt deposits, even at an early stage of their development. Overall changes in the biochemical composition of the tissue upon pathology progression are less obvious. Globally, although the cholesterol level rises, the relative lipid-to-protein content decreases. The results broaden the knowledge of biochemical alterations in dysfunctional human aortic valves and may be helpful in designing lipid lowering therapies. [ABSTRACT FROM AUTHOR]

Fourier transform infrared (FTIR) microspectroscopy is assessed in terms of two techniques (i.e., transmission and transflection) as a method for rapid measurements of blood plasma. Apart from the expected effect of the electric field standing wave (EFSW), we also noticed that second-derivative IR spectra recorded in transflection mode exhibited a significant shift in the amide I band (up to 1667 cm−1) in comparison to the one recorded in transmission (1658 cm−1). This has not been reported thus far in studies of the EFSW distortion of IR spectra of biological material. The thinner the sample deposited on the low-e microscope slide, the lower the position of the amide I band found in FTIR spectra, suggesting various plasma compositions after stratification or certain changes in secondary protein conformations due to chemical and/or physical effects. There are potentially several phenomena that can occur at the surface of both IR substrates affecting the protein profile, including changes in optical properties (refractive index), variation in water content in the sample, and segregation of plasma components. All three hypotheses are discussed here, with the help of atomic force microscopy (AFM). [ABSTRACT FROM AUTHOR]

Raman microimaging of live Rhodotorula mucilaginosa cells, cultured under aerobic and anaerobic conditions, showed striking differences in the composition and distribution of cell components. The analysis of these differences and recovery of oxidative phosphorylation upon environmental changes enabled the interrelation of carotenoids, hemoproteins and the unknown species considered as the “Raman signature of life”. [ABSTRACT FROM AUTHOR]

Fluorescence and surface-enhanced Raman scattering (SERS) spectroscopy were employed to investigate the cellular uptake of rhodamine 6G (R6G) alone and of R6G loaded with gold nanoparticles (AuNPs) by endothelial cells. R6G plays the role of a Raman reporter in SERS but also displays strong fluorescence. The presence of bare R6G molecules and R6G-AuNPs in the cytoplasm of the cells is detected via the 2D fluorescence of the dye after a 0.5 h of the incubation with R6G and R6G-AuNPs, and then the concentration of the dye increases within 4 h of exposure. The examination of the cellular uptake of the R6G and R6G-AuNPs species at different temperatures suggests that the internalization of the R6G-AuNPs into endothelial cells occurs mainly via endocytosis. 3D fluorescence imaging of R6G inside cells reveals inhomogeneous distribution of the dye in the cytoplasm. The SERS signal of the Raman reporter inside the cell disappears after 2 h of incubation with R6G-AuNPs and then amino acid residues, purines and pyrimidines become SERS-active via their interactions with the gold. The results highlight the significance of using multiple techniques to cover a spectrum of issues in the application of SERS nanosensors for probing an intracellular environment under comparable and standardized conditions. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Raman confocal microscopy with 488 nm excitation wavelength supported with atomic force microscopy (AFM), scanning near-field optical microscopy (SNOM) and UV-Vis spectrometry was used to investigate air-dried erythrocytes (red blood cells, RBCs) in whole human blood smears. The central internal part of the cell was dominated by the laser-induced O2 dissociated oxyHb form as evidenced by the Fe2+ marker band appearing at 1356 cm−1. The existence of a thin outer layer of hemoglobin in the periphery of RBCs was assigned to hemichrome. Evidence for hemichrome includes the oxidation state marker band appearing at 1376 cm−1, the absence of FeO2 band at 570 cm−1 and a UV-Vis spectrum consistent with hemichrome. This is the first time that distributions of Fe2+/Fe3+ hemes inside the single RBC have been reported. The outer layer formation of hemichrome was additionally studied when RBCs were in contact with leucocytes and carotenoids of blood plasma. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]