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Heme released from damaged and senescent red blood cells (RBCs) may contribute to oxidant-mediated cell injury. One of the recently investigated physiological processes, essential in preventing the inflammatory impact of labile heme, is its uptake from the bloodstream by endothelial cells (ECs). In this study, we investigated heme uptake by ECs starting from the model studies on the in vitro cellular level, through the endothelium layer on the ex vivo murine aortic tissues. As the cellular model, Human Aortic Endothelial Cells (HAECs) were chosen, and the concentration of labile heme was adjusted so to avoid the excessive toxic effect of the labile heme. We utilized label-free Raman imaging with two different excitation wavelengths to capture the uptake process in situ and characterize the oxidation state of the iron ion in the intercalated heme. The phenomenon of heme uptake was demonstrated in both, the healthy control C57Bl/6J and FVB animals, as well as in mice with developed atherosclerosis (ApoE/LDLR-/- mice). In the presented work, we presented for the first time Raman-based evidence on the heme uptake process by endothelial cells in both, in vitro and ex vivo systems.
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Células Endoteliales , Hemo , Espectrometría Raman , Animales , Hemo/metabolismo , Espectrometría Raman/métodos , Células Endoteliales/metabolismo , Ratones , Humanos , Ratones Endogámicos C57BL , Aterosclerosis/metabolismo , Aterosclerosis/patologíaRESUMEN
Amyloid fibrils form remarkable, multi-layered chiral supramolecular architectures. The proximity of interacting oscillators in the chiral fibril supramolecules is responsible for the unusual sensitivity of vibrational circular dichroism (VCD) for fibril formation. Surprisingly, up to now, such characteristics have not been shown for ROA, although it displays the same vibrational markers of fibrils as VCD, including the amide I band. Here, we report an exceptionally large enhancement of the ROA signal detected for mature amyloid fibrils and their prefibrillar states. Remarkably, the same ROA signal has been obtained for fibrils of homologous lysozymes and the dissimilar protein, insulin, indicating a possible common enhanced ROA spectrum, analogous to that for VCD for all amyloid fibrils investigated to date. The ROA signal is observed at earlier stages of fibril formation than VCD and provides access to a considerably broader range of vibrations. Further studies are necessary to verify the applicability of ROA for the analysis of amyloid fibrils.
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Amidas , Amiloide , Rotación Óptica , Dicroismo Circular , CitoesqueletoRESUMEN
Amyloid fibrils are fascinating and complex structures with the multilayered chiral organization. Using the multimodal methodology, including VCD, ECD, cryo-EM, and TEM, we characterized in detail different levels of organization (secondary structure/protofilament/mesoscopic structure) of amyloid fibrils prepared from proteins highly homologous in the structure (hen egg white and human lysozymes). Our results demonstrate that small changes in the native protein structure or preparation conditions translate into significant differences in the handedness and architecture of the formed fibrils at various levels of their complexity. In particular, fibrils of hen egg white and human lysozymes obtained inâ vitro at the same preparation conditions, possess different secondary structure, protofilament twist and ultrastructure. Yet, formed fibrils adopted a relatively similar mesoscopic structure, as observed in high-resolution 3D cryo-EM, scarcely used up to now for fibrils obtained inâ vitro in denaturing condition. Our results add to other puzzling experiments implicating the indeterministic nature of fibril formation.
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Amiloide , Muramidasa , Humanos , Muramidasa/química , Amiloide/química , Dicroismo Circular , Estructura Secundaria de ProteínaRESUMEN
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).
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Transfusión de Eritrocitos , Leucemia , Humanos , Transfusión de Eritrocitos/efectos adversos , Transfusión Sanguínea , Eritrocitos , Leucemia/diagnóstico , Leucemia/terapia , Leucemia/etiologíaRESUMEN
The aggregation-sensitive chiroptical (ECD and RROA) output, provided by enantiopure (3S,3'S)-astaxanthin, was used to investigate and control the assembling processes of the carotenoid in Pluronic F-127 nanoparticles. The process of carotenoid J-aggregation inside nanocarriers is interfered with by the formation of kinetically stabilized H1 self-assemblies outside the micelles. Nanocarriers with encapsulated stable J-aggregates provide controlled release of carotenoid molecules to primary murine adipocytes.
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Carotenoides , Xantófilas , Animales , Carotenoides/farmacología , Ratones , Xantófilas/farmacologíaRESUMEN
The core size of iron oxide nanoparticles (IONPs) is a crucial factor defining not only their magnetic properties but also toxicological profile and biocompatibility. On the other hand, particular IONPs may induce different biological response depending on the dose, exposure time, but mainly depending on the examined system. New light on this problem may be shed by the information concerning biomolecular anomalies appearing in various cell lines in response to the action of IONPs with different core diameters and this was accomplished in the present study. Using Raman microscopy we studied the abnormalities in the accumulation of proteins, lipids and organic matter within the nucleus, cytoplasm and cellular membrane of macrophages, HEK293T and U87MG cell line occurring as a result of 24-hour long exposure to PEG-coated magnetite IONPs. The examined nanoparticles had 5, 10 and 30 nm cores and were administered in doses 5 and 25 µg Fe/ml. The obtained results showed significant anomalies in biochemical composition of macrophages and the U87MG cells, but not the HEK293T cells, occurring as a result of exposure to all of the examined nanoparticles. However, IONPs with 10 nm core diminished the accumulation of biomolecules in cells only when they were administered at a larger dose. The Raman spectra recorded for the macrophages subjected to 30 nm IONPs and for the U87MG cells exposed to 5 and 10 nm showed the presence of additional bands in the wavenumber range 1700-2400 cm-1, probably resulting from the appearance of Fe adducts within cells. Our results indicate, moreover, that smaller IONPs may be effectively internalized into the U87MG cells, which points at their diagnostic/therapeutic potential in the case of glioblastoma multiforme.
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Nanopartículas de Magnetita , Nanopartículas , Compuestos Férricos/toxicidad , Óxido Ferrosoférrico , Células HEK293 , Humanos , Macrófagos , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/toxicidad , Nanopartículas/químicaRESUMEN
AIMS: Endothelial dysfunction (ED) and red blood cell distribution width (RDW) are both prognostic factors in heart failure (HF), but the relationship between them is not clear. In this study, we used a unique mouse model of chronic HF driven by cardiomyocyte-specific overexpression of activated Gαq protein (Tgαq*44 mice) to characterize the relationship between the development of peripheral ED and the occurrence of structural nanomechanical and biochemical changes in red blood cells (RBCs). METHODS AND RESULTS: Systemic ED was detected in vivo in 8-month-old Tgαq*44 mice, as evidenced by impaired acetylcholine-induced vasodilation in the aorta and increased endothelial permeability in the brachiocephalic artery. ED in the aorta was associated with impaired nitric oxide (NO) production in the aorta and diminished systemic NO bioavailability. ED in the aorta was also characterized by increased superoxide and eicosanoid production. In 4- to 6-month-old Tgαq*44 mice, RBC size and membrane composition displayed alterations that did not result in significant changes in their nanomechanical and functional properties. However, 8-month-old Tgαq*44 mice presented greatly accentuated structural and size changes and increased RBC stiffness. In 12-month-old Tgαq*44 mice, the erythropathy was featured by severely altered RBC shape and elasticity, increased RDW, impaired RBC deformability, and increased oxidative stress (gluthatione (GSH)/glutathione disulfide (GSSG) ratio). Moreover, RBCs taken from 12-month-old Tgαq*44 mice, but not from 12-month-old FVB mice, coincubated with aortic rings from FVB mice, induced impaired endothelium-dependent vasodilation and this effect was partially reversed by an arginase inhibitor [2(S)-amino-6-boronohexanoic acid]. CONCLUSION: In the Tgαq*44 murine model of HF, systemic ED accelerates erythropathy and, conversely, erythropathy may contribute to ED. These results suggest that erythropathy may be regarded as a marker and a mediator of systemic ED in HF. RBC arginase and possibly other RBC-mediated mechanisms may represent novel therapeutic targets for systemic ED in HF.
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Insuficiencia Cardíaca , Enfermedades Vasculares , Acetilcolina/metabolismo , Animales , Arginasa/metabolismo , Enfermedad Crónica , Modelos Animales de Enfermedad , Eicosanoides/metabolismo , Endotelio Vascular/metabolismo , Eritrocitos/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Disulfuro de Glutatión/metabolismo , Ratones , Ratones Transgénicos , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , VasodilataciónRESUMEN
Iron phosphates are a wide group of compounds that possess versatile applications. Their properties are strongly dependent on the role and position of iron in their structure. Iron, because of its chemical character, is able to easily change its redox state and accommodate different chemical surroundings. Thus, iron-phosphate crystallography is relatively complex. In addition, the compounds possess intriguing magnetic and electric properties. In this paper, we present crystal structure properties of a newly developed iron-phosphate compound that was obtained by devitrification from iron-phosphate glass of pyrophosphate stoichiometry. Based on X-ray diffraction (XRD) studies, the new compound (Fe7P11O38) was shown to adopt the hexagonal space group P63 (No. 173) in which iron is present as Fe3+ in two inequivalent octahedral and one tetrahedral positions. The results were confirmed by Raman and Mössbauer spectroscopies, and appropriate band positions, as well as hyperfine interaction parameters, are assigned and discussed. The magnetic and electric properties of the compound were predicted by ab initio simulations. It was observed that iron magnetic moments are coupled antiferromagnetically and that the total magnetic moment of the unit cell has an integer value of 2 µB. Electronic band structure calculations showed that the material has half-metallic properties.
RESUMEN
Iron-phosphate glasses, due to their properties, have many potential applications. One of the most promising seems to be nuclear waste immobilization. Radioactive 90Sr isotope is the main short-lived product of fission and, due to its high solubility, it can enter groundwater and pose a threat to the environment. On the other hand, Sr is an important element in hard tissue metabolic processes, and phosphate glasses containing Sr are considered bioactive. This study investigated the effect of SrO addition on a glass structure of nominal 30Fe2O3-70P2O5 chemical composition using classical molecular dynamics simulations. To describe the interaction between Sr-O ion pairs, new interatomic potential parameters of the Buckingham-type were developed and tested for crystalline compounds. The short-range structure of the simulated glasses is presented and is in agreement with previous experimental and theoretical studies. The simulations showed that an increase in SrO content in the glass led to phosphate network depolymerization. Analysis demonstrated that the non-network oxygen did not take part in the phosphate network depolymerization. Furthermore, strontium aggregation in the glass structure was observed to lead to the non-homogeneity of the glass network. It was demonstrated that Sr ions prefer to locate near to Fe(II), which may induce crystallization of strontium phosphates with divalent iron.
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The present study aimed to investigate the influence of the gradual substitution of Fe2O3 by Al2O3 on the thermal properties of polyphosphate glasses. The conducted considerations based on differential scanning calorimetry (DSC) and heating microscopy thermal analysis provided much essential information about the correlation between glass chemical composition and its characteristic parameters, such as transformation temperature, specific heat, crystallization temperature, crystallization enthalpy, the activation energy of crystal growth, melting temperature, and Angell glass thermal stability. The obtained estimation of viscosity changes as a function of temperature could be very helpful for researchers to correctly plan the vitrification process and thus radioactive waste immobilization. A precise analysis of DSC curves and X-ray diffraction patterns revealed the possibility of crystallization process design in order to create materials with different levels of crystallinity and phase composition. The drawn conclusions allow choosing the glass with the optimal concentration of Al2O3 and Fe2O3, which ensures the relatively low melting temperature, viscosity, and glass crystallization ability, with application potential in nuclear waste immobilization.
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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.
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Conservación de la Sangre/métodos , Eritrocitos/metabolismo , Intercambio Iónico , Manejo de Especímenes/métodos , Carbonatos/metabolismo , Membrana Eritrocítica , Humanos , Iones/metabolismo , Ácido Láctico/metabolismo , Microscopía de Fuerza Atómica , Potasio/metabolismo , Sodio/metabolismoRESUMEN
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.
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Envejecimiento Prematuro/inducido químicamente , Envejecimiento/sangre , Modelos Animales de Enfermedad , Membrana Eritrocítica/química , Galactosa/toxicidad , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría Raman , Envejecimiento Prematuro/sangre , Animales , Citosol/química , Envejecimiento Eritrocítico/efectos de los fármacos , Deformación Eritrocítica/efectos de los fármacos , Índices de Eritrocitos/efectos de los fármacos , Membrana Eritrocítica/efectos de los fármacos , Radicales Libres/toxicidad , Galactosa/farmacología , Hemorreología/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Fósforo/sangre , Proyectos de InvestigaciónRESUMEN
The effects of Sm3+ content on the optical properties and bioactivity of 13-93 bioactive glass were presented. Sm3+ doped glass fibers drawn from bioactive glass were analyzed in simulated body fluid (SBF) for the determination of ion release. Optical analysis of the Sm3+ ions in bioactive glass fibers was used for degradation monitoring. While the fibers were immersed in SBF solution, changes in their luminescence spectra under 405 nm laser excitation were measured continuously for 48 h. The morphology of the fibers after the immersion process was determined by SEM/EDS. It was shown that the proposed approach to the analysis of changes in Sm3+ ion luminescence is a sensitive method for the monitoring of degradation processes and the formation of hydroxycarbonate-apatite (HCA) layers on glass fiber surfaces. SEM/EDS measurements showed a significant deterioration on the surface of the fibers and the formation of HCA on 13-93_02Sm bioactive glass. The optical analysis of the time constant indicated that bioactive glass fibers doped with 2 %mol Sm3+ degrade at a rate almost five times slower than 13-93_02Sm.
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In this work we applied a multimodal approach to define the age- and atherosclerosis-related biochemical and functional alterations in red blood cells (RBCs) in ApoE/LDLR-/- mice. Our results revealed that age-related changes in RBCs, such as decreases in RBC deformability and mean height, were more pronounced in ApoE/LDLR-/- mice than in age-matched control mice (C57BL/6J). The decreases in phospholipid content and level of lipid unsaturation were accompanied by an increase in cholesterol esters and esterified lipids in RBC membranes in aged C57BL/6J mice. The age-related decrease in the phospholipid content was more pronounced in ApoE/LDLR-/- mice. In contrast, the increase in the total lipid content in RBC membranes occurred only in ApoE/LDLR-/- mice with advanced atherosclerosis. The age-related alterations also included a decrease in the ratio of turns to α-helices in the secondary structure of hemoglobin (Hb) inside intact RBCs. On the other hand, an increase in the ratio of unordered conformations to α-helices of Hb was observed only in ApoE/LDLR-/- mice and occurred already at the age of 5-weeks. This was related to hypercholesterolemia and resulted in an increased oxygen-carrying capacity. In conclusion, progressive mechanical and functional alterations of RBCs in aged ApoE/LDLR-/- mice were more pronounced than in age-matched C57BL/6J mice. Although, several biochemical changes in RBCs in aged ApoE/LDLR-/- mice recapitulated age-dependent changes observed in control mice, some biochemical features of RBC membranes attributed to hypercholesterolemia were distinct and could contribute to the accelerated deterioration of RBC function in ApoE/LDLR-/- mice.
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Apolipoproteínas E/deficiencia , Aterosclerosis/metabolismo , Eritrocitos/metabolismo , Receptores de LDL/deficiencia , Factores de Edad , Animales , Apolipoproteínas E/metabolismo , Aterosclerosis/patología , Eritrocitos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de LDL/metabolismoRESUMEN
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 TâR 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.