DOX-DNA Interactions on the Nanoscale: In Situ Studies Using Tip-Enhanced Raman Scattering.

Chemotherapeutic anthracyclines, like doxorubicin (DOX), are drugs endowed with cytostatic activity and are widely used in antitumor therapy. Their molecular mechanism of action involves the formation of a stable anthracycline-DNA complex, which prevents cell division and results in cell death. It is known that elevated DOX concentrations induce DNA chain loops and overlaps. Here, for the first time, tip-enhanced Raman scattering was used to identify and localize intercalated DOX in isolated double-stranded calf thymus DNA, and the correlated near-field spectroscopic and morphologic experiments locate the DOX molecules in the DNA and provide further information regarding specific DOX-nucleobase interactions. Thus, the study provides a tool specifically for identifying intercalation markers and generally analyzing drug-DNA interactions. The structure of such complexes down to the molecular level provides mechanistic information about cytotoxicity and the development of potential anticancer drugs.

Predictors of outcomes of patients ≥ 80 years old admitted to intensive care units in Poland - a post-hoc analysis of the VIP2 prospective observational study.

INTRODUCTION: Elderly patients pose a significant challenge to intensive care unit (ICU) clinicians. In this study we attempted to characterise the population of patients over 80 years old admitted to ICUs in Poland and identify associations between clinical features and short-term outcomes. MATERIAL AND METHODS: The study is a post-hoc analysis of the Polish cohort of the VIP2 European prospective observational study enrolling patients > 80 years old admitted to ICUs over a 6-month period. Data including clinical features, clinical frailty scale (CFS), geriatric scales, interventions within the ICU, and outcomes (30-day and ICU mortality and length of stay) were gathered. Univariate analyses comparing frail (CFS > 4) to non-frail patients and survivors to non-survivors were performed. Multivariable models with CFS, activities of daily living score (ADL), and the cognitive decline questionnaire IQCODE as predictors and ICU or 30-day mortality as outcomes were formed. RESULTS: A total of 371 patients from 27 ICUs were enrolled. Frail patients had significantly higher ICU (58% vs. 44.45%, P = 0.03) and 30-day (65.61% vs. 54.14%, P = 0.01) mortality compared to non-frail counterparts. The survivors had significantly lower SOFA score, CFS, ADL, and IQCODE than non-survivors. In multivariable analysis CFS (OR 1.15, 95% CI: 1.00-1.34) and SOFA score (OR 1.29, 95% CI: 1.19-1.41) were identified as significant predictors for ICU mortality; however, CFS was not a predictor for 30-day mortality ( P = 0.07). No statistical significance was found for ADL, IQCODE, polypharmacy, or comorbidities. CONCLUSIONS: We found a positive correlation between CFS and ICU mortality, which might point to the value of assessing the score for every patient admitted to the ICU. The older Polish ICU patients were characterised by higher mortality compared to the other European countries.

Why Does One Measure Resonance Raman Optical Activity? A Unique Case of Measurements under Strong Resonance versus Far-from-Resonance Conditions.

Raman optical activity (ROA) spectroscopy exhibits significant potential in the study of (bio)molecules as it encodes information on their molecular structure, chirality, and conformations. Furthermore, the method reveals details on excited electronic states when applied under resonance conditions. Here, we present a combined study of the far from resonance (FFR)-ROA and resonance ROA (RROA) of a single relatively small molecular system. Notably, this study is the first to employ the density functional theory (DFT) analysis of both FFR-ROA and RROA spectra. This is illustrated for cobalamin derivatives using near-infrared and visible light excitation. Although the commonly observed monosignate RROA spectra lose additional information visible in bisignate nonresonance ROA spectra, the RROA technique acts as a complement to nonresonance ROA spectroscopy. In particular, the combination of these methods integrated with DFT calculations can reveal a complete spectral picture of the structural and conformational differences between tested compounds.

Alterations in lipid metabolism accompanied by changes in protein and carotenoid content as spectroscopic markers of human T cell activation.

This work aims to understand better the mechanism of cellular processes accompanying the activation of human T cells and to develop a novel, fast, label-free approach to identify molecular biomarkers for this process. The standard methodology for confirming the activation state of T cells is based on flow cytometry and using antibodies recognizing activation markers. The method provide high specificity detection but may be susceptible to background staining or non-specific secondary antibody reactions. Here, we evaluated the potential of Raman-based molecular imaging in distinguishing non-activated and activated human T cells. Confocal Raman microscopy was performed on T cells followed by chemometrics to obtain comprehensive molecular information, while Stimulated Raman Scattering imaging was used to quickly provide high-resolution images of selected cellular components of activated and non-activated cells. For the first time, carotenoids, lipids, and proteins were shown to be important biomarkers of T-cell activation. We found that T-cell activation was accompanied by lipid accumulation and loss of carotenoid content. Our findings on the biochemical, morphological, and structural changes associated with activated mature T cells provide insights into the molecular changes that occur during therapeutic manipulation of the immune response. The methodology for identifying activated T cells is based on a novel imaging method and supervised and unsupervised chemometrics. It unambiguously identifies specific and unique molecular changes without the need for staining, fixation, or any other sample preparation.

Induced Chirality in Canthaxanthin Aggregates Reveals Multiple Levels of Supramolecular Organization.

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.

The role of cardiolipin and cytochrome c in mitochondrial metabolism of cancer cells determined by Raman imaging: in vitro study on the brain glioblastoma U-87 MG cell line.

In this paper, we present Raman imaging as a non-invasive approach for studying changes in mitochondrial metabolism caused by cardiolipin-cytochrome c interactions. We investigated the effect of mitochondrial dysregulation on cardiolipin (CL) and cytochrome c (Cyt c) interactions for a brain cancer cell line (U-87 MG). Mitochondrial metabolism was monitored by checking the intensities of the Raman bands at 750 cm-1, 1126 cm-1, 1310 cm-1, 1337 cm-1, 1444 cm-1 and 1584 cm-1. The presented results indicate that under pathological conditions, the content and redox status of Cyt c in mitochondria can be used as a Raman marker to characterize changes in cellular metabolism. This work provides evidence that cardiolipin-cytochrome c interactions are crucial for mitochondrial energy homeostasis by controlling the redox status of Cyt c in the electron transport chain, switching from disabling Cyt c reduction and enabling peroxidase activity. This paper provides experimental support for the hypothesis of how cardiolipin-cytochrome c interactions regulate electron transfer in the respiratory chain, apoptosis and mROS production in mitochondria.

Raman spectroscopy can recognize the KMT2A rearrangement as a distinct subtype of leukemia.

Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are the two most common hematologic malignancies, challenging to treat and associated with high recurrence and mortality rates. This work aims to identify specific Raman biomarkers of ALL cells with the KMT2A gene rearrangement (KMT2A-r), representing a highly aggressive subtype of childhood leukemia with a poor prognosis. The proposed approach combines the sensitivity and specificity of Raman spectroscopy with machine learning and allows us to distinguish not only myelo- and lymphoblasts but also discriminate B-cell precursor (BCP) ALL with KMT2A-r from other blasts of BCP-ALL. We have found that KMT2A-r ALL cells fixed with 0.5% glutaraldehyde exhibit a unique spectroscopic profile that enables us to identify this subtype from other leukemias and normal cells. Therefore, a rapid and label-free method was developed to identify ALL blasts with KMT2A-r based on the ratio of the two Raman bands assigned to phenylalanine - 1040 and 1008 cm-1. This is the first time that a particular group of leukemic cells has been identified in a label-free way. The identified biomarker can be used as a screening method in diagnostic laboratories or non-reference medical centers.

New Highly Sensitive and Specific Raman Probe for Live Cell Imaging of Mitochondrial Function.

For Raman hyperspectral detection and imaging in live cells, it is very desirable to create novel probes with strong and unique Raman vibrations in the biological silent region (1800-2800 cm-1). The use of molecular probes in Raman imaging is a relatively new technique in subcellular research; however, it is developing very rapidly. Compared with the label-free method, it allows for a more sensitive and selective visualization of organelles within a single cell. Biological systems are incredibly complex and heterogeneous. Directly visualizing biological structures and activities at the cellular and subcellular levels remains by far one of the most intuitive and powerful ways to study biological problems. Each organelle plays a specific and essential role in cellular processes, but importantly for cells to survive, mitochondrial function must be reliable. Motivated by earlier attempts and successes of biorthogonal chemical imaging, we develop a tool supporting Raman imaging of cells to track biochemical changes associated with mitochondrial function at the cellular level in an in vitro model. In this work, we present a newly synthesized highly sensitive RAR-BR Raman probe for the selective imaging of mitochondria in live endothelial cells.

Automatic subtyping of Diffuse Large B-cell Lymphomas (DLBCL): Raman-based genetic and metabolic classification.

Diffuse large B-cell lymphoma (DLBCL), the most common non-Hodgkin's lymphoma in adults, is a genetically and metabolically heterogeneous group of aggressive malignancies. The complexity of their molecular composition and the variability in clinical presentation make clinical diagnosis and treatment selection a serious challenge. The challenge is therefore to quickly and correctly classify DLBCL cells. In this work, we show that Raman imaging is a tool with high diagnostic potential, providing unique information about the biochemical components of tumor cells and their metabolism. We present models of classification of lymphoma cells based on their Raman spectra. The models automatically and efficiently identify DLBCL cells and assign them to a given cell-of-origin (COO) subtype (activated B cell-like (ABC) or germinal center B cell-like (GCB)) or, respectively, to a comprehensive cluster classification (CCC) subtype (OxPhos/non-OxPhos). In addition, we describe each lymphoma subtype by its unique spectral profile, linking it to biochemical, genetic, or metabolic features.

Raman microscopy reveals how cell inflammation activates glucose and lipid metabolism.

Metabolism of endothelial cells (ECs) depends on the availability of the energy substrates. Since the endothelium is the first line of defence against inflammation in the cardiovascular system and its dysfunction can lead to the development of cardiovascular diseases, it is important to understand how glucose metabolism changes during inflammation. In this work, glucose uptake was studied in human microvascular endothelial cells (HMEC-1) in high glucose (HG), and additionally in an inflammatory state, using Raman imaging. HG state was induced by incubation of ECs with a deuterated glucose analogue, while the EC inflammation was caused by TNF-α pre-treatment. Spontaneous and stimulated Raman scattering spectroscopy provided comprehensive information on biochemical changes, including lipids and the extent of unsaturation induced by excess glucose in ECs., induced by excess glucose in ECs. In this work, we indicated spectroscopic markers of metabolic changes in ECs as a strong increase in the ratio of the intensity of lipids / (proteins + lipids) bands and an increase in the level of lipid unsaturation and mitochondrial changes. Inflamed ECs treated with HG, revealed enhanced glucose uptake, and intensified lipid production i.a. of unsaturated lipids. Additionally, increased cytochrome c signal in the mitochondrial region indicated higher mitochondrial activity and biogenesis. Raman spectroscopy is a powerful method for determining the metabolic markers of ED which will better inform understanding of disease onset, development, and treatment.

Raman classification of selected subtypes of acute lymphoblastic leukemia (ALL).

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.

Uptake and anti-inflammatory effects of liposomal astaxanthin on endothelial cells tracked by Raman and fluorescence imaging.

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.

Investigation of the Immunogenic Properties of Ovalbumin Modified by Urban Airborne Particulate Matter.

Exposure to air particulate matter (PM) is linked to the blood oxidative stress and systemic inflammation. The aim of this study was to elucidate whether oxidative PM modification of ovalbumin (OVA), the major antioxidant serum protein, may alter its antigenicity and/or immunogenicity. Ovalbumin was exposed via dialysis to the standard urban PM (SRM 1648a) or to PM with removed organic content (encoded as LAP). Both structural changes and biological properties of PM-modified OVA were measured. T lymphocytes and dendritic cells (the major antigen-presenting cells) isolated from C57BL/6 and OT-II (323-339 epitope) OVA-specific T cell receptor (TCR)-transgenic mice were used to test the effect of PM on OVA immunogenicity. The immunogenicity of both SRM 1648a and LAP-modified OVA was significantly higher than that of control OVA, as measured by the epitope-specific T cell proliferation and interferon γ production by the stimulated cells. This effect was associated with mild oxidative changes in the carrier molecule outside the structure of the OVA epitope and with increased resistance to proteolysis of PM-modified OVA. Interestingly, dendritic cells showed enhanced capacity for the uptake of proteins when the cells were cultured with PM-modified OVA. Our results suggest that the enhanced immunogenicity of PM-modified OVA is not associated with altered antigenicity or antigen presentation. However, it may result from slower degradation and longer persistence of modified antigens in dendritic cells. Whether this phenomenon is associated with enhanced risk prevalence of autoimmune diseases observed in the areas with high urban PM pollution needs to be explained.

Modified glucose as a sensor to track the metabolism of individual living endothelial cells - Observation of the 1602 cm-1 band called "Raman spectroscopic signature of life".

A relatively new approach to subcellular research is Raman microscopy with the application of sensors called Raman probes. This paper describes the use of the sensitive and specific Raman probe, 3-O-propargyl-d-glucose (3-OPG), to track metabolic changes in endothelial cells (ECs). ECs play a significant role in a healthy and dysfunctional state, the latter is correlated with a range of lifestyle diseases, particularly with cardiovascular disorders. The metabolism and glucose uptake may reflect the physiopathological conditions and cell activity correlated with energy utilization. To study metabolic changes at the subcellular level the glucose analogue, 3-OPG was used, which shows a characteristic and intense Raman band at 2124 cm-1.3-OPG was applied as a sensor to track both, its accumulation in live and fixed ECs and then metabolism in normal and inflamed ECs, by employing two spectroscopic techniques, i.e. spontaneous and stimulated Raman scattering microscopies. The results indicate that 3-OPG is a sensitive sensor to follow glucose metabolism, manifested by the Raman band of 1602 cm-1. The 1602 cm-1 band has been called the "Raman spectroscopic signature of life" in the cell literature, and here we demonstrate that it is attributed to glucose metabolites. Additionally, we have shown that glucose metabolism and its uptake are slowed down in the cellular inflammation. We showed that Raman spectroscopy can be classified as metabolomics, and its uniqueness lies in the fact that it allows the analysis of the processes of a single living cell. Gaining further knowledge on metabolic changes in the endothelium, especially in pathological conditions, may help in identifying markers of cellular dysfunction, and more broadly in cell phenotyping, better understanding of the mechanism of disease development and searching for new treatments.

Changes in the mitochondrial membrane potential in endothelial cells can be detected by Raman microscopy.

The role of mitochondria goes beyond their capacity to create molecular fuel and includes e.g. the production of reactive oxygen species and the regulation of cell death. In endothelial cells, mitochondria have a significant impact on cellular function under both healthy and pathological conditions. Endothelial dysfunction contributes to the development of various lifestyle diseases and the key players in their pathogenesis are among others vascular inflammation and oxidative stress. The latter is very closely related to mitochondrial dysfunction; however, it is not straightforward. First, because mitochondria are small cellular structures, and second, it requires a sensitive method to follow the subtle biochemical changes. For this purpose, Raman microscopy (RM) was used here, which is considered a high-resolution method and can be applied in situ, usually as a non-labeled technique. In this work, we show that RM can not only locate mitochondria in the cell but also track their functional changes. Moreover, we test if labeling cells with Raman probes (Rp) can improve the specificity and sensitivity of RM (compared to conventional labeled techniques such as fluorescence, and the non-labeled Raman technique). MitoBADY Rp was used to detect changes in mitochondrial membrane potential as an indicator of mitochondrial activity, e.g. hyperpolarization or distortion of the proton gradient in the intermembrane space (depolarization). Thus, we show and compare RM, in the form of a label and non-labeled, to such a subtle cellular analysis.

What is the ability of inflamed endothelium to uptake exogenous saturated fatty acids? A proof-of-concept study using spontaneous Raman, SRS and CARS microscopy.

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.

Raman-based spectrophenotyping of the most important cells of the immune system.

INTRODUCTION: Human peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of cells that includes T and B lymphocytes. The total number of lymphocytes and their percentage in the blood can be a marker for the diagnosis of several human diseases. Currently, cytometric methods are widely used to distinguish subtypes of leukocytes and quantify their number. These techniques use cell immunophenotyping, which is limited by the number of fluorochrome-labeled antibodies that can be applied simultaneously. OBJECTIVE: B and T lymphocytes were isolated from peripheral blood obtained from healthy human donors. METHODS: The immunomagnetic negative selection was used for the enrichment of B and T cells fractions, and their purity was assessed by flow cytometry. Isolated cells were fixed with 0.5% glutaraldehyde and measured using confocal Raman imaging. K-means cluster analysis, principal component analysis and partial least squares discriminant methods were applied for the identification of spectroscopic markers to distinguish B and T cells. HPLC was the reference method for identifying carotene in T cells. RESULTS: Reliable discrimination between T and B lymphocytes based on their spectral profile has been demonstrated using label-free Raman imaging and chemometric analysis. The presence of carotene in T lymphocytes (in addition to the previously reported in plasma) was confirmed and for the first time unequivocally identified as ß-carotene. In addition, the molecular features of the lymphocytes nuclei were found to support the discriminant analysis. It has been shown that although the presence of carotenoids in T cells depends on individual donor variability, the reliable differentiation between lymphocytes is possible based on Raman spectra collected from individual cells. CONCLUSIONS: This proves the potential of Raman spectroscopy in clinical diagnostics to automatically differentiate between cells that are an important component of our immune system.

EdU sensing: The Raman way of following endothelial cell proliferation in vitro and ex vivo.

Endothelial cells line the lumen of all vessels in the body and maintain vascular homeostasis. In particular, endothelial cell regeneration in response to insult sustain functional endothelial layer. EdU (5-ethynyl-2'-deoxyuridine) is an alkyne-tagged proliferation probe that incorporates into newly synthesized DNA and is used for fluorescence imaging of cell proliferation with the use of "click chemistry" reaction with a fluorescent azide. Here, we utilized EdU as a click-free Raman probe for tracking endothelial cell proliferation. Raman imaging of EdU was performed in live endothelial cells, showing an advantage over fluorescence imaging of EdU, as this technique did not require sample fixation and permeabilization. To validate Raman-based imaging of EdU to study endothelial cell proliferation, we showed that when endothelial cells were treated with cycloheximide or doxorubicin to impair the proliferation of endothelial cells, the Raman-based signal of EdU was diminished. Furthermore, endothelial cells proliferation detected using EdU-labelled Raman imaging was compared with fluorescence imaging. Finally, the method of Raman-based EdU imaging was used in the isolated murine aorta ex vivo. Altogether, our results show that Raman-based imaging of EdU provides a novel alternative for fluorescence-based assay to assess endothelial proliferation and regeneration.