Label-free tracking of cytochrome C oxidation state in live cells by resonance Raman imaging.

Free interconversion of cytochrome C (CytC) between native ferrous (Cyt-FeII) and oxidized ferric (CytC-FeIII) states is necessary to maintain the respiratory function of mitochondria. Disturbances in CytC-FeIII to total CytC ratio may indicate mitochondrial dysfunction and apoptosis. Thus, tracking CytC oxidation state delivers important information about cellular physiology. In this work, we propose a novel methodology based on resonance Raman (rR) imaging optimized uniquely to track and qualitatively analyze the transition of Cyt-FeII to CytC-FeIII within live cells without affecting their morphology. None of the commonly used excitation lines allows such clear-cut differentiation, contrary to the 405 nm applied in this work. The presented methodology provides a novel pathway in the label-free detection of ferrous and ferric heme proteins.

Surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS) in label-free characterization of erythrocyte membranes and extracellular vesicles at the nano-scale and molecular level.

The manuscript presents the potential of surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS) for label-free characterization of extracellular microvesicles (EVs) and their isolated membranes derived from red blood cells (RBCs) at the nanoscale and at the single-molecule level, providing detection of a few individual amino acids, protein and lipid membrane compartments. The study shows future directions for research, such as investigating the use of the mentioned techniques for the detection and diagnosis of diseases. We demonstrate that SERS and TERS are powerful techniques for identifying the biochemical composition of EVs and their membranes, allowing the detection of small molecules, lipids, and proteins. Furthermore, extracellular vesicles released from red blood cells (REVs) can be broadly classified into exosomes, microvesicles, and apoptotic bodies, based on their size and biogenesis pathways. Our study specifically focuses on microvesicles that range from 100 to 1000 nanometres in diameter, as presented in AFM images. Using SERS and TERS spectra obtained for REVs and their membranes, we were able to characterize the chemical and structural properties of microvesicle membranes with high sensitivity and specificity. This information may help better distinguish and categorize different types of EVs, leading to a better understanding of their functions and potential biomedical applications.

Secondary structure alterations of RBC assessed by FTIR-ATR in correlation to 2,3-DPG levels in ApoE/LDLR-/- Mice.

In the present study, we characterized the secondary structure alterations of intact red blood cells (RBCs) cytosol with special attention to the sex-related alterations in 8- and 24-week-old female and male ApoE/LDLR-/- mice, compared to age-matched female and male C57BL/6J control animals. Results were obtained with previously established methodology based on Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR). Additionally, we evaluated 2,3-DPG levels in the RBCs and showed its potential link to the hemoglobin (Hb) secondary structure alterations. Considering Hb structure alterations probed by FTIR-ATR, the ratio of turns to α-helices in 8-week-old ApoE/LDLR-/- mice suggested more pronounced secondary structure alterations within the RBCs than in the age-matched control. Sex-related differences were observed solely in 24-week-old male ApoE/LDLR-/- mice, which showed statistically significant increase in the secondary structure alterations compared to 24-week-old female ApoE/LDLR-/- mice. Similar to the secondary structure alterations, no sex-related differences were observed in the levels of 2,3-DPG in RBCs, except for 24-week-old male ApoE/LDLR-/- mice, which showed significantly higher levels compared to the age-matched female ApoE/LDLR-/- mice. Considering the age-related alterations, we observed significant increases in the intracellular 2,3-DPG of RBCs with animals' age in all studied groups, except for female ApoE/LDLR-/- mice, where a significant difference was not reported. This suggests the clear correlation between secondary structure of Hb alterations and 2,3-DPG levels for male and female murine RBC and proves a higher resistance of older female RBCs to the secondary structure changes with progression of atherosclerosis. Moreover, it may be concluded that higher 2,3-DPG levels in RBCs occurred in response to the secondary structure alterations of Hb in ApoE/LDLR-/- mice.

Label-free testing strategy to evaluate packed red blood cell quality before transfusion to leukemia patients.

Patients worldwide require therapeutic transfusions of packed red blood cells (pRBCs), which is applied to the high-risk patients who need periodic transfusions due to leukemia, lymphoma, myeloma and other blood diseases or disorders. Contrary to the general hospital population where the transfusions are carried out mainly for healthy trauma patients, in case of high-risk patients the proper quality of pRBCs is crucial. This leads to an increased demand for efficient technology providing information on the pRBCs alterations deteriorating their quality. Here we present the design of an innovative, label-free, noninvasive, rapid Raman spectroscopy-based method for pRBCs quality evaluation, starting with the description of sample measurement and data analysis, through correlation of spectroscopic results with reference techniques' outcomes, and finishing with methodology verification and its application in clinical conditions. We have shown that Raman spectra collected from the pRBCs supernatant mixture with a proper chemometric analysis conducted for a minimum one ratio of integral intensities of the chosen Raman marker bands within the spectrum allow evaluation of the pRBC quality in a rapid, noninvasive, and free-label manner, without unsealing the pRBCs bag. Subsequently, spectroscopic data were compared with predefined reference values, either from pRBCs expiration or those defining the pRBCs quality, allowing to assess their utility for transfusion to patients with acute myeloid leukemia (AML) and lymphoblastic leukemia (ALL).

Sex-Specific Differences of Adenosine Triphosphate Levels in Red Blood Cells Isolated From ApoE/LDLR Double-Deficient Mice.

In this study for the first time, we investigated the correlation between sex-specific differences in adenosine triphosphate (ATP) levels in red blood cells (RBCs) and their mechanical, biochemical, and morphological alterations during the progression of atherosclerosis in ApoE/LDLR double-deficient (ApoE/LDLR-/-) mice. Our results indicate that both sex and age affect alterations in RBCs of both ApoE/LDLR-/- and C57BL/6J mice. When compared with male RBCs, female RBCs were characterized by lower basal ATP and mean corpuscular hemoglobin concentration (MCHC), higher hemoglobin concentration (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), deformability, and phosphatidylserine (PS) exposure levels, regardless of age in both, ApoE/LDLR-/- and C57BL/6J mice. ApoE/LDLR-/- mice compared with age-matched controls showed lower basal ATP levels regardless of age and sex. Intracellular ATP level of RBCs was decreased solely in senescent female C57BL/6J mice, while it was elevated in males. Basal extracellular ATP levels were 400 times lower than corresponding intracellular level. In conclusion, basal ATP levels, RBC morphology, deformability, PS exposure levels alterations are sex-dependent in mice. Changes in basal ATP levels were correlated with PS exposure and trends of changes in MCV. Trends of changes of the most RBC parameters were similar in both sexes of ApoE/LDLR-/- mice compared with age-matched controls; however, their kinetics and levels vary greatly between different stages of disease progression.

Improvement in clinical symptoms in patients with the first episode of psychosis is associated with a decrease in systemic nitric oxide availability. A pilot study. / Zwiazek poprawy objawów klinicznych u pacjentów z pierwszym epizodem psychozy i jej zwiazek ze spadkiem ogólnoustrojowej dostepnosci tlenku azotu. Badanie pilotazowe.

OBJECTIVES: The aim of the study was to assess the relationship between the improvement of the clinical condition of patients with the first episode of psychosis (FEP) and changes in - nitric oxide (NO) plasma concentration based on the level of its metabolites NO2- and NO3, as well as changes in lipid profile and biomarkers of systemic inflammation. METHODS: The study was carried out in agroup of 25 young patients with FEP (aged 14-35). Blood samples were collected in the 1st and 12th week after admission to the hospital to assess NO metabolites, lipid profile and inflammatory biomarkers. Demographic and clinical data were also analysed. RESULTS: In the study group, three months after admission to the hospital, an improvement in the clinical symptoms was observed, as evidenced by a decrease in the Positive and Negative Syndrome Scale (PANSS) scores. This improvement was associated with a decrease in the plasma nitrite concentration, a deterioration of the lipid profile and the activation of systemic inflammation. Interestingly, in the 1st week after the hospital admission, a longer duration of untreated psychosis (DUP) was associated with a lower NO2- plasma concentration, and a higher intensity of positive symptoms (PANSS Positive Symptoms Scale) was associated with higher CRP plasma level. CONCLUSIONS: Our results suggest that adverse metabolic response, systemic inflammation and a fall in systemic NO bioavailability represent early systemic manifestations of FEP that are not controlled by short-term anti-psychotic treatment and may pose cardiovascular risk.

Spectroscopic Signature of Red Blood Cells in a D-Galactose-Induced Accelerated Aging Model.

This work presents a semi-quantitative spectroscopic approach, including FTIR-ATR and Raman spectroscopies, for the biochemical analysis of red blood cells (RBCs) supported by the biochemical, morphological and rheological reference techniques. This multi-modal approach provided the description of the RBC alterations at the molecular level in a model of accelerated aging induced by administration of D-galactose (D-gal), in comparison to natural aging. Such an approach allowed to conclude that most age-related biochemical RBC membrane changes (a decrease in lipid unsaturation and the level of phospholipids, or an increase in acyl chain shortening) as well as alterations in the morphological parameters and RBC deformability are well reflected in the D-gal model of accelerated aging. Similarly, as in natural aging, a decrease in LDL level in blood plasma and no changes in the fraction of glucose, creatinine, total cholesterol, HDL, iron, or triglycerides were observed during the course of accelerated aging. Contrary to natural aging, the D-gal model led to an increase in cholesterol esters and the fraction of total esterified lipids in RBC membranes, and evoked significant changes in the secondary structure of the membrane proteins. Moreover, a significant decrease in the phosphorous level of blood plasma was specific for the D-gal model. On the other hand, natural aging induced stronger changes in the secondary structures of the proteins of the RBCs' interior. This work proves that research on the aging mechanism, especially in circulation-related diseases, should employ the D-gal model with caution. Nonetheless, the D-gal model enables to imitate age-related rheological alterations in RBCs, although they are partially derived from different changes observed in the RBC membrane at the molecular level.

An Insight into the Stages of Ion Leakage during Red Blood Cell Storage.

Packed red blood cells (pRBCs), the most commonly transfused blood product, are exposed to environmental disruptions during storage in blood banks. In this study, temporal sequence of changes in the ion exchange in pRBCs was analyzed. Standard techniques commonly used in electrolyte measurements were implemented. The relationship between ion exchange and red blood cells (RBCs) morphology was assessed with use of atomic force microscopy with reference to morphological parameters. Variations observed in the Na+, K+, Cl-, H+, HCO3-, and lactate ions concentration show a complete picture of singly-charged ion changes in pRBCs during storage. Correlation between the rate of ion changes and blood group type, regarding the limitations of our research, suggested, that group 0 is the most sensitive to the time-dependent ionic changes. Additionally, the impact of irreversible changes in ion exchange on the RBCs membrane was observed in nanoscale. Results demonstrate that the level of ion leakage that leads to destructive alterations in biochemical and morphological properties of pRBCs depend on the storage timepoint.

Bardoxolone Methyl Displays Detrimental Effects on Endothelial Bioenergetics, Suppresses Endothelial ET-1 Release, and Increases Endothelial Permeability in Human Microvascular Endothelium.

Nrf2 is a master regulator of antioxidant cellular defence, and agents activating the Nrf2 pathway have been tested in various diseases. However, unexpected side effects of cardiovascular nature reported for bardoxolone methyl in patients with type 2 diabetes mellitus and stage 4 chronic kidney disease (the BEACON trial) still have not been fully explained. Here, we aimed to characterize the effects of bardoxolone methyl compared with other Nrf2 activators-dimethyl fumarate and L-sulforaphane-on human microvascular endothelium. Endothelial toxicity, bioenergetics, mitochondrial membrane potential, endothelin-1 (ET-1) release, endothelial permeability, Nrf2 expression, and ROS production were assessed in human microvascular endothelial cells (HMEC-1) incubated for 3 and 24 hours with 100 nM-5 µM of either bardoxolone methyl, dimethyl fumarate, or L-sulforaphane. Three-hour incubation with bardoxolone methyl (100 nM-5 µM), although not toxic to endothelial cells, significantly affected endothelial bioenergetics by decreasing mitochondrial membrane potential (concentrations ≥ 3 µM), decreasing spare respiratory capacity (concentrations ≥ 1 µM), and increasing proton leak (concentrations ≥ 500 nM), while dimethyl fumarate and L-sulforaphane did not exert such actions. Bardoxolone methyl at concentrations ≥ 3 µM also decreased cellular viability and induced necrosis and apoptosis in the endothelium upon 24-hour incubation. In turn, endothelin-1 decreased permeability in endothelial cells in picomolar range, while bardoxolone methyl decreased ET-1 release and increased endothelial permeability even after short-term (3 hours) incubation. In conclusion, despite that all three Nrf2 activators exerted some beneficial effects on the endothelium, as evidenced by a decrease in ROS production, bardoxolone methyl, the most potent Nrf2 activator among the tested compounds, displayed a distinct endothelial profile of activity comprising detrimental effects on mitochondria and cellular viability and suppression of endothelial ET-1 release possibly interfering with ET-1-dependent local regulation of endothelial permeability.

Irreversible alterations in the hemoglobin structure affect oxygen binding in human packed red blood cells.

The ability of hemoglobin (Hb) to transport respiratory gases is directly linked to its quaternary structure properties and reversible changes between T (tense) and R (relax) state. In this study we demonstrated that packed red blood cells (pRBCs) storage resulted in a gradual increase in the irreversible changes in the secondary and quaternary structures of Hb, with subsequent impairment of the T↔R transition. Such alteration was associated with the presence of irreversibly settled in the relaxed form, quaternary structure of Hb, which we termed R'. On the secondary structure level, disordered protein organization involved formation of ß-sheets and a decrease in α-helices related to the aggregation process stabilized by strong intermolecular hydrogen bonding. Compensatory changes in RBCs metabolism launched to preserve reductive microenvironment were disclosed as an activation of nicotinamide adenine dinucleotide phosphate (NADPH) production and increased reduced to oxidized glutathione (GSH/GSSG) ratio. For the first time we showed the relationship between secondary structure changes and the occurrence of newly discovered R', which through an artificial increase in oxyhemoglobin level altered Hb ability to bind and release oxygen.

Temporal sequence of the human RBCs' vesiculation observed in nano-scale with application of AFM and complementary techniques.

Based on the multimodal characterization of human red blood cells (RBCs), the link between the storage-related sequence of the nanoscale changes in RBC membranes in the relation to their biochemical profile as well as mechanical and functional properties was presented. On the background of the accumulation of RBCs waste products, programmed cell death and impaired rheological properties, progressive alterations in the RBC membranes including changes in their height and diameter as well as the in situ characterization of RBC-derived microparticles (RMPs) on the RBCs surface were presented. The advantage of atomic force microscopy (AFM) in RMPs visualization, even at the very early stage of vesiculation, was shown based on the results revealed by other reference techniques. The nanoscale characterization of RMPs was correlated with a decrease in cholesterol and triglycerides levels in the RBC membranes, proving the link between the lipids leakage from RBCs and the process of vesiculation.

Multimodal detection and analysis of a new type of advanced Heinz body-like aggregate (AHBA) and cytoskeleton deformation in human RBCs.

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.