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Molecular, morphological, and physiological heterogeneity is the inherent property of cells which governs differences in their response to external influence. Tumor cell metabolic heterogeneity is of a special interest due to its clinical relevance to tumor progression and therapeutic outcomes. Rapid, sensitive, and noninvasive assessment of metabolic heterogeneity of cells is a great demand for biomedical sciences. Fluorescence lifetime imaging (FLIM), which is an all-optical technique, is an emerging tool for sensing and quantifying cellular metabolism by measuring fluorescence decay parameters of endogenous fluorophores, such as NAD(P)H. To achieve accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this paper, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity is demonstrated. This has been achieved using an approach to data analysis based on the nonparametric analysis, which revealed a much better sensitivity to the presence of metabolically distinct subpopulations compared to more traditional approaches of FLIM measurements and analysis. The approach was further validated for imaging cultured cancer cells treated with chemotherapy. These results pave the way for accurate detection and quantification of cellular metabolic heterogeneity using FLIM, which will be valuable for assessing therapeutic vulnerabilities and predicting clinical outcomes.
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Neoplasias/metabolismo , Imagem Óptica/métodos , Progressão da Doença , Humanos , Neoplasias/patologiaRESUMO
OBJECTIVES: The aim of this work is to assess the performance of multimodal spectroscopic approach combined with single core optical fiber for detection of bladder cancer during surgery in vivo. METHODS: Multimodal approach combines diffuse reflectance spectroscopy (DRS), fluorescence spectroscopy in the visible (405 nm excitation) and near-infrared (NIR) (690 nm excitation) ranges, and high-wavenumber Raman spectroscopy. All four spectroscopic methods were combined in a single setup. For 21 patients with suspected bladder cancer or during control cystoscopy optical spectra of bladder cancer, healthy bladder wall tissue and/or scars were measured. Classification of cancerous and healthy bladder tissue was performed using machine learning methods. RESULTS: Statistically significant differences in relative total haemoglobin content, oxygenation, scattering, and visible fluorescence intensity were found between tumor and normal tissues. The combination of DRS and visible fluorescence spectroscopy allowed detecting cancerous tissue with sensitivity and specificity of 78% and 91%, respectively. The addition of features extracted from NIR fluorescence and Raman spectra did not improve the quality of classification. CONCLUSIONS: This study demonstrates that multimodal spectroscopic approach allows increasing sensitivity and specificity of bladder cancer detection in vivo. The developed approach does not require special probes and can be used with single-core optical fibers applied for laser surgery.
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Espectrometria de Fluorescência , Análise Espectral Raman , Neoplasias da Bexiga Urinária , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/patologia , Humanos , Análise Espectral Raman/métodos , Feminino , Espectrometria de Fluorescência/métodos , Masculino , Idoso , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Cistoscopia , Idoso de 80 Anos ou mais , Espectroscopia de Luz Próxima ao Infravermelho/métodosRESUMO
A reaction of acyl chlorides derived from 1,10-phenanthroline-2,9-dicarboxylic acids with piperazine allows the preparation of the corresponding 24-membered macrocycles in good yield. The structural and spectral properties of these new macrocyclic ligands were thoroughly investigated, revealing promising coordination properties towards f-elements (Am, Eu). It was shown that the prepared ligands can be used for selective extraction of Am(III) from alkaline-carbonate media in presence of Eu(III) with an SFAm/Eu up to 40. Their extraction efficiency is higher than calixarene-type extraction of the Am(III) and Eu(III) pair. Composition of macrocycle-metal complex with Eu(III) was investigated by luminescence and UV-vis spectroscopy. The possibility of such ligands to form complexes of L:Eu = 1:2 stoichiometry is revealed.
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Complexos de Coordenação , Diamida , Modelos Moleculares , Ligantes , Complexos de Coordenação/químicaRESUMO
The freezing-induced loading (FIL) method is a promising technique for encapsulation of bioactive substances as well as for preparation of nanocomposite materials. A critically important aspect for this method is the remote control of the freezing process. The knowledge of the moment of freezing process ending can allow us to increase the quality of loading and reduce the process duration, thus making this approach more controllable. Herein, we present a photonic technique based on Raman spectroscopy as one of the optimal solutions for remote control of FIL. As a result of our study, the setup for obtaining Raman spectra during the process of liquid vehicle crystallization in suspensions has been developed, which allowed us to analyze the sorption of nanoparticles onto micro- and submicron particles by the FIL method in situ. The main focus of the present work is the in situ Raman spectroscopy monitoring of the crystallization process, including technologically important parameters such as the ice/water interface velocity in water colloids/suspensions and the moment of the final adsorption of the nanoparticles on the microparticles. In contrast to other approaches, Raman spectroscopy allows to directly observe the hydrogen bond formation during crystallization. Additionally, a schematic and a detailed description of the setup are presented here. Thus, the developed technique has a good perspective for scaling up the FIL approach and increasing the area of application of this technology.
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Humification is a ubiquitous natural process of biomass degradation that creates multicomponent systems of nonliving organic matter, including dissolved organic matter (DOM) and humic substances (HS) in water environments, soils, and organic rocks. Despite significant differences in molecular composition, the optical properties of DOM and HS are remarkably similar, and the reason for this remains largely unknown. Here, we employed fluorescence spectroscopy with (sub)picosecond resolution to elucidate the role of electronic interactions within DOM and HS. We revealed an ultrafast decay component with a characteristic decay lifetime of 0.5-1.5 ps and spectral diffusion originating from excitation energy transfer (EET) in the system. The rate of EET was positively correlated to the fraction of aromatic species and tightness of aromatic species packing. Diminishing the number of EET donor-acceptor pairs by reduction with NaBH4 (decrease of the acceptor number), decrease of pH (decrease of the electron-donating ability), or decrease of the average particle size by filtration (less donor-acceptor pairs within a particle) resulted in a lower impact of the ultrafast component on fluorescence decay. Our results uncover the role of electronic coupling among fluorophores in the formation of DOM and HS optical properties and provide a framework for studying photophysical processes in heterogeneous systems of natural fluorophores.
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Substâncias Húmicas , Solo , Biomassa , Transferência de Energia , Substâncias Húmicas/análise , Espectrometria de FluorescênciaRESUMO
The self-assembly of peptides is a key direction for fabrication of advanced materials. Novel approaches for fine tuning of macroscopic and microscopic properties of peptide self-assemblies are of a high demand for constructing biomaterials with desired properties. In this work, while studying the kinetics of the Fmoc-Diphenylalanine (Fmoc-FF) dipeptide self-assembly using the Thioflavinâ T (ThT) dye, we observed that the presence of ThT strongly modifies structural and mechanical properties of the Fmoc-FF hydrogel. Notably, the presence of ThT resulted in a tenfold increase of the gelation time and in the formation of short and dense fibers in the hydrogel. As a result of these morphological alteration higher thermal stability, and most important, tenfold increase of the hydrogel rigidity was achieved. Hence, ThT not only slowed the kinetics of the Fmoc-FF hydrogel formation, but also strongly enhanced its mechanical properties. In this study, we provide a detailed description of the ThT effect on the hydrogel properties and suggest the mechanisms for this phenomenon, paving the way for the novel approach to the control of the peptide hydrogels' micro- and macroscale properties.
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Thioflavin T (ThT) assay is extensively used for studying fibrillation kinetics in vitro. However, the differences in the time course of ThT fluorescence intensity and lifetime and other physical parameters of the system, such as particle size distribution, raise questions about the correct interpretation of the aggregation kinetics. In this work, we focused on the investigation of the mechanisms, which underlay the difference in sensitivity of ThT fluorescence intensity and lifetime to the formation of protein aggregates during fibrillation by the example of insulin and during binding to globular proteins. The assessment of aggregate sizes and heterogeneity was performed using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Using the sub-nanosecond resolution measurements, it was shown that the ThT lifetime is sensitive to the appearance of as much as a few percent of ThT bound to the high-affinity sites that occur simultaneously with an abrupt increase of the average particle size, particles concentration, and size heterogeneity. The discrepancy between ThT fluorescence intensity and a lifetime can be explained as the consequence of a ThT molecule fraction with ultrafast decay and weak fluorescence. These ThT molecules can only be detected using time-resolved fluorescence measurements in the sub-picosecond time domain. The presence of a bound ThT subpopulation with similar photophysical properties was also demonstrated for globular proteins that were attributed to non-specifically bound ThT molecules with a non-rigid microenvironment.
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Amiloide/química , Benzotiazóis/química , Corantes Fluorescentes/química , Difusão Dinâmica da Luz , Humanos , Nanopartículas , Tamanho da PartículaRESUMO
Endogenous autofluorescence of biological tissues is an important source of information for biomedical diagnostics. Despite the molecular complexity of biological tissues, the list of commonly known fluorophores is strictly limited. Still, the question of molecular sources of the red and near-infrared excited autofluorescence remains open. In this work we demonstrated that the oxidation products of organic components (lipids, proteins, amino acids, etc.) can serve as the molecular source of such red and near-infrared excited autofluorescence. Using model solutions and cell systems (human keratinocytes) under oxidative stress induced by UV irradiation we demonstrated that oxidation products can contribute significantly to the autofluorescence signal of biological systems in the entire visible range of the spectrum, even at the emission and excitation wavelengths higher than 650 nm. The obtained results suggest the principal possibility to explain the red fluorescence excitation in a large class of biosystems-aggregates of proteins and peptides, cells and tissues-by the impact of oxidation products, since oxidation products are inevitably presented in the tissue. The observed fluorescence signal with broad excitation originated from oxidation products may also lead to the alteration of metabolic imaging results and has to be taken into account.
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Fluorescência , Imagem Molecular , Imagem Óptica , Oxirredução , Biomarcadores , Citometria de Fluxo , Humanos , Queratinócitos/metabolismo , Microscopia Confocal , Imagem Molecular/métodos , Imagem Óptica/métodos , Processos Fotoquímicos , Espectrometria de Fluorescência , Raios UltravioletaRESUMO
Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCPO) into the red-shifted, active state (OCPR) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCPR is converted to OCPO and dissociates from PBs; however, the mode and site of OCPR/FRP interactions remain elusive. Recently, we have introduced the purple OCPW288A mutant as a competent model for the signaling state OCPR (Sluchanko et al., Biochim Biophys Acta 1858:1-11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCPW288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCPO, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCPO, a substantial compaction of the OCPW288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCPW288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.
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Apoproteínas/metabolismo , Proteínas de Bactérias/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Apoproteínas/química , Proteínas de Bactérias/química , Varredura Diferencial de Calorimetria , Cromatografia em Gel , Cisteína/metabolismo , Difusão , Hidrodinâmica , Espectrometria de Massas , Reprodutibilidade dos Testes , Espectrometria de Fluorescência , Coloração e Rotulagem , Compostos de Sulfidrila/metabolismoRESUMO
In Fig. 1a in the original article, the amino acid side chains were incorrectly labeled in the structure representation of the orange carotenoid protein (OCP). The corrected figure is printed in this erratum.
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Pathogenesis of numerous diseases is associated with the formation of amyloid fibrils. Extrinsic fluorescent dyes, including Thioflavin T (ThT), are used to follow the fibrillation kinetics. It has recently been reported that the so-called deep-blue autofluorescence (dbAF) is changing during the aggregation process. However, the origin of dbAF and the reasons for its change remain debatable. Here, the kinetics of fibril formation in model proteins were comprehensively analyzed using fluorescence lifetime and intensity of ThT, intrinsic fluorescence of proteinaceous fluorophores, and dbAF. For all systems, intensity enhancement of the dbAF band with similar spectral parameters (â¼350â¯nm excitation; â¼450â¯nm emission) was observed. Although the time course of ThT lifetime (indicative of protofibrils formation) coincided with that of tyrosine residues in insulin, and the kinetic changes in the ThT fluorescence intensity (reflecting formation of mature fibrils) coincided with changes in ThT absorption spectrum, the dbAF band started to increase from the beginning of the incubation process without a lag-phase. Our mass-spectrometry data and model experiments suggested that dbAF could be at least partially related to oxidation of amino acids. This study scrutinizes the dbAF features in the context of the existing hypotheses about the origin of this spectral band.
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Amiloide/química , Insulina/química , Muramidase/química , Agregados Proteicos , Aminoácidos/química , Animais , Benzotiazóis/química , Fluorescência , Corantes Fluorescentes/química , Humanos , Cinética , Oxirredução , Espectrometria de FluorescênciaRESUMO
The formation of apoptosis-inducing amyloidal structures by metabolites has significantly extended the "amyloid hypothesis" to include non-proteinaceous, single metabolite building blocks. However, detection of metabolite assemblies is restricted compared to their larger protein-based counterparts owing to the hindrance of external labelling and limited immunohistochemical detection tools. Herein, we present the detection of the formation, dynamics, and cellular distribution of metabolite amyloid-like structures and provide mechanistic insights into the generation of supramolecular chromophores. Moreover, the intrinsic fluorescence properties allow the detection of metabolite assemblies in living cells without the use of external dyes. Altogether, this intrinsic fluorescence of metabolite assemblies further verifies their amyloidal nature, while providing an important tool for further investigation of their pathological role in inborn error of metabolism disorders.
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Amiloide/química , Amiloide/metabolismo , Linhagem Celular Tumoral , Fluorescência , Células HEK293 , Humanos , Microscopia ConfocalRESUMO
Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatus under excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stable orange to the signaling red state. This is thought to involve a 12-Å translocation of the carotenoid cofactor and separation of the N- and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion and associated photoprotection remains elusive. Here, we labeled the OCP of Synechocystis with tetramethylrhodamine-maleimide (TMR) and obtained a photoactive OCP-TMR complex, the fluorescence of which was highly sensitive to the protein state, showing unprecedented contrast between the orange and red states and reflecting changes in protein conformation and the distances from TMR to the carotenoid throughout the photocycle. The OCP-TMR complex was sensitive to the light intensity, temperature, and viscosity of the solvent. Based on the observed Förster resonance energy transfer, we determined that upon photoconversion, the distance between TMR (donor) bound to a cysteine in the C-terminal domain and the carotenoid (acceptor) increased by 18 Å, with simultaneous translocation of the carotenoid into the N-terminal domain. Time-resolved fluorescence anisotropy revealed a significant decrease of the OCP rotation rate in the red state, indicating that the light-triggered conversion of the protein is accompanied by an increase of its hydrodynamic radius. Thus, our results support the idea of significant structural rearrangements of OCP, providing, to our knowledge, new insights into the structural rearrangements of OCP throughout the photocycle and a completely novel approach to the study of its photocycle and non-photochemical quenching. We suggest that this approach can be generally applied to other photoactive proteins.
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Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Corantes Fluorescentes/química , Fotoperíodo , Modelos Moleculares , Conformação Proteica , Rodaminas/química , Solventes/química , Espectrometria de Fluorescência , Temperatura , ViscosidadeRESUMO
In Cyanobacteria, the Orange Carotenoid Protein (OCP) and Fluorescence Recovery Protein (FRP) are central to the photoprotective mechanism consisting in regulated quenching of phycobilisome (PBs) fluorescence. Due to a transient and flexible nature of the light-activated red quenching form, OCPR, which is obtained from the stable dark-adapted orange form, OCPO, by photoconversion, the detailed mechanism of photoprotection remains unclear. Here we demonstrate that our recently described W288A mutant of the Synechocystis OCP (hereinafter called OCPW288A) is a fully functional analogue of the OCPR form which is capable of constitutive PBs fluorescence quenching in vitro with no need of photoactivation. This PBs quenching effect is abolished in the presence of FRP, which interacts with OCPW288A with micromolar affinity and an apparent stoichiometry of 1:1, unexpectedly, implying dissociation of the FRP dimers. This establishes OCPW288A as a robust model system providing novel insights into the interplay between OCP and FRP to regulate photoprotection in cyanobacteria.
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Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ficobilissomas/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Carotenoides/metabolismo , Fluorescência , Luz , Modelos Moleculares , Mutação/genética , Multimerização Proteica/fisiologiaRESUMO
Non-photochemical quenching (NPQ) is a mechanism responsible for high light tolerance in photosynthetic organisms. In cyanobacteria, NPQ is realized by the interplay between light-harvesting complexes, phycobilisomes (PBs), a light sensor and effector of NPQ, the photoactive orange carotenoid protein (OCP), and the fluorescence recovery protein (FRP). Here, we introduced a biophysical model, which takes into account the whole spectrum of interactions between PBs, OCP, and FRP and describes the experimental PBs fluorescence kinetics, unraveling interaction rate constants between the components involved and their relative concentrations in the cell. We took benefit from the possibility to reconstruct the photoprotection mechanism and its parts in vitro, where most of the parameters could be varied, to develop the model and then applied it to describe the NPQ kinetics in the Synechocystis sp. PCC 6803 mutant lacking photosystems. Our analyses revealed that while an excess of the OCP over PBs is required to obtain substantial PBs fluorescence quenching in vitro, in vivo the OCP/PBs ratio is less than unity, due to higher local concentration of PBs, which was estimated as ~10-5 M, compared to in vitro experiments. The analysis of PBs fluorescence recovery on the basis of the generalized model of enzymatic catalysis resulted in determination of the FRP concentration in vivo close to 10% of the OCP concentration. Finally, the possible role of the FRP oligomeric state alteration in the kinetics of PBs fluorescence was shown. This paper provides the most comprehensive model of the OCP-induced PBs fluorescence quenching to date and the results are important for better understanding of the regulatory molecular mechanisms underlying NPQ in cyanobacteria.
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Fenômenos Biofísicos , Cianobactérias/metabolismo , Cianobactérias/efeitos da radiação , Luz , Processos Fotoquímicos , Proteínas de Bactérias/metabolismo , Fenômenos Biofísicos/efeitos da radiação , Carotenoides/metabolismo , Fluorescência , Cinética , Processos Fotoquímicos/efeitos da radiação , Synechocystis/metabolismo , Synechocystis/efeitos da radiaçãoRESUMO
Orange carotenoid protein (OCP) is the photoactive protein that is responsible for high light tolerance in cyanobacteria. We studied the kinetics of the OCP photocycle by monitoring changes in its absorption spectrum, intrinsic fluorescence, and fluorescence of the Nile red dye bound to OCP. It was demonstrated that all of these three methods provide the same kinetic parameters of the photocycle, namely, the kinetics of OCP relaxation in darkness was biexponential with a ratio of two components equal to 2:1 independently of temperature. Whereas the changes of the absorption spectrum of OCP characterize the geometry and environment of its chromophore, the intrinsic fluorescence of OCP reveals changes in its tertiary structure, and the fluorescence properties of Nile red indicate the exposure of hydrophobic surface areas of OCP to the solvent following the photocycle. The results of molecular-dynamics studies indicated the presence of two metastable conformations of 3'-hydroxyechinenone, which is consistent with characteristic changes in the Raman spectra. We conclude that rotation of the ß-ionylidene ring in the C-terminal domain of OCP could be one of the first conformational rearrangements that occur during photoactivation. The obtained results suggest that the photoactivated form of OCP represents a molten globule-like state that is characterized by increased mobility of tertiary structure elements and solvent accessibility.
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Proteínas de Bactérias/química , Proteínas Luminescentes/química , Simulação de Dinâmica Molecular , Absorção de Radiação , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Cianobactérias/química , Corantes Fluorescentes/farmacologia , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
Tyrosine fluorescence in native proteins is known to be effectively quenched, whereas its emission increases upon proteins' unfolding. This suggests that tyrosine fluorescence could be exploited for probing structural rearrangements of proteins in addition to the extensively used tryptophan emission. We studied the possibility of using tyrosine fluorescence as an indicator of surfactant-induced conformational changes in albumins. It was shown that fluorescence of tyrosine residues, which are uniformly distributed all over the protein molecules, allows the detection of subtle structural rearrangements of proteins upon surfactant binding, which do not influence the properties of a single tryptophan residue buried in the inner hydrophobic region of human serum albumin. Tyrosine fluorescence properties, including its fluorescence lifetime, revealed the multistage character of surfactant binding to albumin, consistent with the data provided by other methods. The obtained results demonstrate the possibility of probing conformational changes in proteins using tyrosine photophysical parameters as indicators.
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Fluorescência , Albumina Sérica/química , Tensoativos/química , Tirosina/química , Animais , Bovinos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Micelas , Conformação Proteica , Desdobramento de Proteína , Análise Espectral , Triptofano/químicaRESUMO
This study investigates the relationship between body hydration levels and skin hydration using spatially resolved diffuse reflectance spectroscopy. The research involved monitoring skin dehydration and rehydration under various conditions, including thermal and physical loads on healthy volunteers, and diuretic therapy in patients with edema syndrome. Findings indicate a correlation between body mass reduction and skin hydration: a 1% loss in body mass corresponds to a 10% decrease in skin hydration. During thermal stress, water absorption at 970 nm decreased monotonically without recovery. Physical activity resulted in approximately 10% changes in skin water content within 20 min, followed by rehydration. Patients with edema syndrome exhibited the most substantial decrease in water absorption amplitude, at nearly 30%, during diuretic treatment. These results support optical spectroscopy as a non-invasive tool for assessing body hydration, with implications for developing portable hydration monitoring devices for clinical and sports applications.
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Heterogeneity of tumor metabolism is an important, but still poorly understood aspect of tumor biology. Present work is focused on the visualization and quantification of cellular metabolic heterogeneity of colorectal cancer using fluorescence lifetime imaging (FLIM) of redox cofactor NAD(P)H. FLIM-microscopy of NAD(P)H was performed in vitro in four cancer cell lines (HT29, HCT116, CaCo2 and CT26), in vivo in the four types of colorectal tumors in mice and ex vivo in patients' tumor samples. The dispersion and bimodality of the decay parameters were evaluated to quantify the intercellular metabolic heterogeneity. Our results demonstrate that patients' colorectal tumors have significantly higher heterogeneity of energy metabolism compared with cultured cells and tumor xenografts, which was displayed as a wider and frequently bimodal distribution of a contribution of a free (glycolytic) fraction of NAD(P)H within a sample. Among patients' tumors, the dispersion was larger in the high-grade and early stage ones, without, however, any association with bimodality. These results indicate that cell-level metabolic heterogeneity assessed from NAD(P)H FLIM has a potential to become a clinical prognostic factor.
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Neoplasias Colorretais , NADP , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Animais , Camundongos , NADP/metabolismo , Linhagem Celular Tumoral , Imagem Óptica/métodos , Metabolismo EnergéticoRESUMO
Autofluorescence of blood plasma has been broadly considered as a prospective disease screening method. However, the assessment of such intrinsic fluorescence is mostly phenomenological, and its origin is still not fully understood, complicating its use in the clinical practice. Here we present the detailed evaluation of analytical capabilities, variability, and formation of blood plasma protein fluorescence based on the open dataset of excitation-emission matrices measured for â¼300 patients with suspected colorectal cancer, and our supporting model experiments. Using high-resolution size-exclusion chromatography coupled with comprehensive spectral analysis, we demonstrate, for the first time, the dominant role of HSA in the formation of blood plasma fluorescence in the visible spectral range (excitation wavelength >350 nm), presumably caused by its oxidative modifications. Furthermore, the diagnostic value of the tryptophan emission, as well as of the tyrosine fluorescence and visible fluorescence of proteins is shown by building a tree-based classification model that uses a small subset of physically interpretable fluorescence features for distinguishing between the control group and cancer patients with >80% accuracy. The obtained results extend current understanding and approaches used for the analysis of blood plasma fluorescence and pave the way for novel autofluorescence-based disease screening methods.