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Cytochrome c (CytC) is a single-electron carrier between complex bc1 and cytochrome c-oxidase (CcO) in the electron transport chain (ETC). It is also known as a good radical scavenger but its participation in electron flow through the ETC makes it impossible to use CytC as a radical sensor. To solve this problem, a series of mutants were constructed with substitutions of Lys residues in the universal binding site (UBS) which interact electrostatically with negatively charged Asp and Glu residues at the binding sites of CytC partners, bc1 complex and CcO. The aim of this study was to select a mutant that had lost its function as an electron carrier in the ETC, retaining the structure and ability to quench radicals. It was shown that a mutant CytC with substitutions of five (8Mut) and four (5Mut) Lys residues in the UBS was almost inactive toward CcO. However, all mutant proteins kept their antioxidant activity sufficiently with respect to the superoxide radical. Mutations shifted the dipole moment of the CytC molecule due to seriously changed electrostatics on the surface of the protein. In addition, a decrease in the redox potential of the protein as revealed by the redox titrations of 8Mut was detected. Nevertheless, the CD spectrum and dynamic light scattering suggested no significant changes in the secondary structure or aggregation of the molecules of CytC 8Mut. Thus, a variant 8Mut with multiple mutations in the UBS which lost its ability to electron transfer and saved most of its physico-chemical properties can be effectively used as a detector of superoxide generation both in mitochondria and in other systems.

Here, we present a direct comparison of different dyes and assays for the determination of protein concentrations. We compared the classical Bradford assay with two modern assays based on the fluorogenic dyes QuDye and ProteOrange and showed that the Bradford reagent achieved excellent results in the determination of protein concentrations as compared with more modern rivals. We also showed that standard approaches for determining the limit of detection (LoD) and limit of quantification (LoQ) may not work correctly with the tested dyes. We proposed a new approach that extends the standard algorithm for LoD and LoQ determination. This approach works well with both classical colorimetric and fluorogenic dyes, as well as with nontrivial fluorescent probes.

Compounds sensitive to reactive oxygen species are widely used in the study of processes in living cells and in the development of therapeutic agents for photodynamic therapy. In the present work, we have synthesized a dyad in which the BODIPY dye is chemically bound to 9,10-diphenylanthracene (DPA). Here, DPA acts as a specific sensor of singlet oxygen and BODIPY as a reference dye. We studied the photophysical properties of the BODIPY-DPA dyad and showed that energy transfer occurs between the chromophores. As a result, the compound has excitation maxima in the absorption region of both DPA and BODIPY, but the fluorescence emission occurs mainly from BODIPY. In the presence of singlet oxygen, the excitation maximum of DPA decreases, while the intensity of the excitation maximum of BODIPY remains almost unchanged. This allows the BODIPY-DPA dyad to be used as a ratiometric sensor of singlet oxygen.

Carotenoids are potent antioxidants with a wide range of biomedical applications. However, their delivery into human cells is challenging and relatively inefficient. While the use of natural water-soluble carotenoproteins capable to reversibly bind carotenoids and transfer them into membranes is promising, the quantitative estimation of the delivery remains unclear. In the present work, we studied echinenone (ECN) delivery by cyanobacterial carotenoprotein AnaCTDH (C-terminal domain homolog of the Orange Carotenoid Protein from Anabaena), into liposome membranes labelled with BODIPY fluorescent probe. We observed that addition of AnaCTDH-ECN to liposomes led to the significant changes in the fast-kinetic component of the fluorescence decay curve, pointing on the dipole-dipole interactions between the probe and ECN within the membrane. It may serve as an indirect evidence of ECN delivery into membrane. To study the delivery in detail, we carried out molecular dynamics modeling of the localization of ECN within the lipid bilayer and calculate its orientation factor. Next, we exploited FRET to assess concentration of ECN delivered by AnaCTDH. Finally, we used time-resolved fluorescence anisotropy to assess changes in microviscosity of liposomal membranes. Incorporation of liposomes with β-carotene increased membrane microviscosity while the effect of astaxanthin and its mono- and diester forms was less pronounced. At temperatures below 30 °C addition of AnaCTDH-ECN increased membrane microviscosity in a concentration-dependent manner, supporting the protein-mediated carotenoid delivery mechanism. Combining all data, we propose FRET-based analysis and assessment of membrane microviscosity as potent approaches to characterize the efficiency of carotenoids delivery into membranes.

In nonpolar solvents, hydrophobic organic fluorophores often show bright fluorescence, whereas in polar media, they usually suffer from aggregation-caused quenching (ACQ). Here, we harnessed this solvatochromic behavior of a 1,3,5,7-tetramethyl-BODIPY derivative for cell staining and applied it to live-cell imaging and flow cytometry. As opposed to commercially available dyes, this BODIPY derivative showed excellent contrast immediately after staining and did not require any wash-off.

The determination of pH in live cells and tissues is of high importance in physiology and cell biology. In this report, we outline the process of the creation of SypHerExtra, a genetically encoded fluorescent sensor that is capable of measuring extracellular media pH in a mildly alkaline range. SypHerExtra is a protein created by fusing the previously described pH sensor SypHer3s with the neurexin transmembrane domain that targets its expression to the cytoplasmic membrane. We showed that with excitation at 445 nm, the fluorescence lifetime of both SypHer3s and SypHerExtra strongly depend on pH. Using FLIM microscopy in live eukaryotic cells, we demonstrated that SypHerExtra can be successfully used to determine extracellular pH, while SypHer3s can be applied to measure intracellular pH. Thus, these two sensors are suitable for quantitative measurements using the FLIM method, to determine intracellular and extracellular pH in a range from pH 7.5 to 9.5 in different biological systems.

As a leading cause of death and morbidity, heart failure (HF) is responsible for a large portion of healthcare and disability costs worldwide. Current approaches to define specific HF subpopulations may fail to account for the diversity of etiologies, comorbidities, and factors driving disease progression, and therefore have limited value for clinical decision making and development of novel therapies. Here we present a novel and data-driven approach to understand and characterize the real-world manifestation of HF by clustering disease and symptom-related clinical concepts (complaints) captured from unstructured electronic health record clinical notes. We used natural language processing to construct vectorized representations of patient complaints followed by clustering to group HF patients by similarity of complaint vectors. We then identified complaints that were significantly enriched within each cluster using statistical testing. Breaking the HF population into groups of similar patients revealed a clinically interpretable hierarchy of subgroups characterized by similar HF manifestation. Importantly, our methodology revealed well-known etiologies, risk factors, and comorbid conditions of HF (including ischemic heart disease, aortic valve disease, atrial fibrillation, congenital heart disease, various cardiomyopathies, obesity, hypertension, diabetes, and chronic kidney disease) and yielded additional insights into the details of each HF subgroup's clinical manifestation of HF. Our approach is entirely hypothesis free and can therefore be readily applied for discovery of novel insights in alternative diseases or patient populations.