Calcium Indicators with Fluorescence Lifetime-Based Signal Readout: A Structure-Function Study.

The calcium cation is a crucial signaling molecule involved in numerous cellular pathways. Beyond its role as a messenger or modulator in intracellular cascades, calcium's function in excitable cells, including nerve impulse transmission, is remarkable. The central role of calcium in nervous activity has driven the rapid development of fluorescent techniques for monitoring this cation in living cells.

Crystal Structure of Bright Fluorescent Protein BrUSLEE with Subnanosecond Fluorescence Lifetime; Electric and Dynamic Properties.

The rapid development of new microscopy techniques for cell biology has exposed the need for genetically encoded fluorescent tags with special properties. Fluorescent biomarkers of the same color and spectral range and different fluorescent lifetimes (FLs) became useful for fluorescent lifetime image microscopy (FLIM). One such tag, the green fluorescent protein BrUSLEE (Bright Ultimately Short Lifetime Enhanced Emitter), having an extremely short subnanosecond component of fluorescence lifetime (FL~0.

[Prospects of Genetically Encoded Flim Indicators for the Quantitative Assessment of Intracellular Parameters].

A significant number of genetically encoded indicators based on fluorescent proteins that allow detecting changes in various parameters: membrane potential shift, pH, concentrations of hydrogen peroxide, lactate, pyruvate, NAD+/NADH, ATP, calcium cations, etc. have been created. Some of them (for example, indicators of calcium cations and hydrogen peroxide) are successfully used by numerous groups of researchers in experiments in vivo.

A Single Fluorescent Protein-Based Indicator with a Time-Resolved Fluorescence Readout for Precise pH Measurements in the Alkaline Range.

The real-time monitoring of the intracellular pH in live cells with high precision represents an important methodological challenge. Although genetically encoded fluorescent indicators can be considered as a probe of choice for such measurements, they are hindered mostly by the inability to determine an absolute pH value and/or a narrow dynamic range of the signal, making them inefficient for recording the small pH changes that typically occur within cellular organelles. Here, we study the pH sensitivity of a green-fluorescence-protein (GFP)-based emitter (EGFP-Y145L/S205V) with the alkaline-shifted chromophore's pKa and demonstrate that, in the pH range of 7.

Amino acid residue at the 165th position tunes EYFP chromophore maturation. A structure-based design.

For the whole GFP family, a few cases, when a single mutation in the chromophore environment strongly inhibits maturation, were described. Here we study EYFP-F165G - a variant of the enhanced yellow fluorescent protein - obtained by a single F165G replacement, and demonstrated multiple fluorescent states represented by the minor emission peaks in blue and yellow ranges (~470 and ~530 nm), and the major peak at ~330 nm. The latter has been assigned to tryptophan fluorescence, quenched due to excitation energy transfer to the mature chromophore in the parental EYFP protein.

Deciphering the Role of Positions 145 and 165 in Fluorescence Lifetime Shortening in the EGFP Variants.

The bright ultimately short lifetime enhanced emitter (BrUSLEE) green fluorescent protein, which differs from the enhanced green fluorescent protein (EGFP) in three mutations, exhibits an extremely short fluorescence lifetime at a relatively high brightness. An important contribution to shortening the BrUSLEE fluorescence lifetime compared to EGFP is provided by the T65G substitution of chromophore-forming residue and the Y145M mutation touching the chromophore environment. Although the influence of the T65G mutation was studied previously, the role of the 145th position in determining the GFPs physicochemical characteristics remains unclear.

[Algorithm for stratification of women for differentiated therapy of menopausal syndrome in combination with perimenopausal melatonin deficiency syndrome.].

Climacteric syndrome (CS) is considered to be a frequent manifestation of pathological menopause. Menopause associated not only with deficiency of sex steroids, decrease of melatonin secretion is observed. Perimenopausal melatonin deficiency syndrome (SPDM) is the complex of symptoms, which is often formed amid decrease of melatonin synthesis and clinically characterized by the prevalence of complaints of sleep disorders (problems), bodily pain, depression, anxiety/fears and somatic symptoms.

[Perimenopausal melatonin deficiency syndrome in system of personalized management of quality of life of women with climacteric syndrome.].

The aim of our study is to search diagnostic tools for early detection of prenosological melatonin deficiency in postmenopausal women and women in menopausal transition with climacteric syndrome for establishment effective personalized prevention and treatment programs. In this study 221 women were enrolled. They were divided into four groups: the 1st group - 39 women in menopausal transition with climacteric syndrome, the 2nd group - 104 menopausal women with climacteric syndrome, the 3rd group - 41 women with physiological menopause, the 4th group - 37 healthy women in reproductive-age.

Switching of cardiac troponin I between nuclear and cytoplasmic localization during muscle differentiation.

The nuclear accumulation of proteins may depend on the presence of short targeting sequences, which are known as nuclear localization signals (NLSs). Here, we found that NLSs are predicted in some cytosolic proteins and examined the hypothesis that these NLSs may be functional under certain conditions. As a model, human cardiac troponin I (hcTnI) was used.

Influence of the First Chromophore-Forming Residue on Photobleaching and Oxidative Photoconversion of EGFP and EYFP.

Enhanced green fluorescent protein (EGFP)-one of the most widely applied genetically encoded fluorescent probes-carries the threonine-tyrosine-glycine (TYG) chromophore. EGFP efficiently undergoes green-to-red oxidative photoconversion ("redding") with electron acceptors. Enhanced yellow fluorescent protein (EYFP), a close EGFP homologue (five amino acid substitutions), has a glycine-tyrosine-glycine (GYG) chromophore and is much less susceptible to redding, requiring halide ions in addition to the oxidants.

Bright GFP with subnanosecond fluorescence lifetime.

Fluorescence lifetime imaging microscopy (FLIM) measures fluorescence decay rate at every pixel of an image. FLIM can separate probes of the same color but different fluorescence lifetimes (FL), thus it is a promising approach for multiparameter imaging. However, available GFP-like fluorescent proteins (FP) possess a narrow range of FLs (commonly, 2.

[PREDICTORS OF MODERATE COGNITIVE DISORDERS IN PATIENTS WITH CORONARY HEART DISEASE AND TYPE 2 DIABETES MELLITUS].

Aim: To study predictors of moderate cognitive disorders (MCD) in patients with coronary heart disease (CHD) and type 2 diabetes mellitus (DM2).

Materials And Methods: The study included 54 men with CPD andDM2 (mean age 56.8 ± 4.

Turning On and Off Photoinduced Electron Transfer in Fluorescent Proteins by π-Stacking, Halide Binding, and Tyr145 Mutations.

Photoinduced electron transfer in fluorescent proteins from the GFP family can be regarded either as an asset facilitating new applications or as a nuisance leading to the loss of optical output. Photooxidation commonly results in green-to-red photoconversion called oxidative redding. We discovered that yellow FPs do not undergo redding; however, the redding is restored upon halide binding.

[THE COGNITIVE STATUS OF PATIENTS WITH TYPE 2 DIABETES MELLITUS AFTER CORONARY BYPASS SURGERY].

The aim of the study was to evaluate the neuropsychological status of patients with type 2 diabetes mellitus (DM2) before and I year after coronary bypass surgery performed under conditions of artificial circulation. It included 114 patients (54 with and 60 without DM2). Prior to surgery, the patients with DM2 had positive characteristics of neurodynamics and attention.

[Effect of Statins on Development of Early Cognitive Dysfunction After Coronary Artery Bypass Grafting].

We studied the effect of rosuvastatin on the development of early postoperative cognitive dysfunction (POCD) in patients after coronary artery bypass grafting (CABG). One hundred nine men aged 45-70 year was divided into two groups. Group 1 comprised 69 patients (mean age 56.

[Association of cognitive impairments with carbohydrate and lipid metabolic parameters in type 2 diabetes mellitus patients undergoing coronary bypass surgery].

Unlabelled: AIM. To comparatively analyze neuropsychological parameters in patients with coronary artery disease (CAD) depending on the presence of type 2 diabetes mellitus (DM) and to evaluate their relationship to carbohydrate and lipid metabolic parameters.

Subjects And Methods: Fifty-two male patients with type 2 DM (mean age 58.

[A role of carotid stenoses in the structure of early postoperative cognitive dysfunction in patients underwent coronary artery bypass grafting].

Objective: To analyze the structure and severity of early postoperative cognitive dysfunction as well the accompanying spectral electroencephalographic (EEG) changes in patients underwent coronary artery bypass grafting (CABG) depending on the presence or absence of small and moderate internal carotid stenosis.

Material And Methods: Fifty seven patients, aged from 45 to 70 years, were stratified into two groups: without stenosis (n=35, mean age 54.3±6.

Does preoperative mild cognitive impairment predict postoperative cognitive dysfunction after on-pump coronary bypass surgery?

Background: Mild cognitive impairment (MCI) may contribute to the development of postoperative cognitive dysfunction (POCD) after coronary artery bypass grafting (CABG).

Objective: The aim of this study was to investigate the incidence of early and long-term POCD after CABG in coronary heart disease patients with and without preoperative MCI.

Methods: The study enrolled two groups of males with coronary heart disease: 51 without MCI (mean age 56.

Influence of cell growth conditions and medium composition on EGFP photostability in live cells.

Photostability is a key characteristic of fluorescent proteins. It was recently demonstrated that green fluorescent protein (GFP) photobleaching in live cells can be suppressed by changes in medium composition. Here we show that Ham's F12 medium provides very high enhanced GFP (EGFP) photostability during fluorescence microscopy of live cells.

[Neuropsychological status of patients with low and moderate carotid artery stenoses after the coronary artery bypass surgery].

The purpose of the study was a comparative evaluation of a neurological and neuropsychological status of patients with and without carotid artery stenoses less than 50% after coronary artery bypass grafting. The study involved 65 patients divided into two groups: the first group included 35 patients (mean age 56.2±5.

Severe atherosclerosis and hypoalphalipoproteinemia in the staggerer mouse, a mutant of the nuclear receptor RORalpha.

Background: Hypoalphalipoproteinemia is the most common lipoprotein abnormality in patients with coronary artery disease, yet its causes are unknown.

Methods And Results: We show that the homozygous staggerer (sg/sg) mutant mouse, which carries a deletion within the nuclear receptor RORalpha gene, develops severe atherosclerosis when maintained on an atherogenic diet. In addition, sg/sg mice display a profound hypoalphalipoproteinemia, which is associated with decreased plasma levels of the major HDL proteins, apolipoprotein (apo) A-I and apoA-II.

C3a and C5a anaphylatoxins bind to heparin-based sorbent in low density lipoprotein apheresis: in vitro and in vivo investigations.

Plasmasorption on a heparin-based sorbent was performed in vitro. It demonstrated affinity of the C3a and C5a anaphylatoxins for the sorbent: C3a was removed almost completely (97%), and the C5a concentration decreased on average by 55%. The plasma level of C3a and C5a complement components was also monitored during the procedure of clinical extracorporeal low density lipoprotein (LDL) apheresis on the sorbent in patients with familial hypercholesterolemia.

Apheresis of low density lipoproteins using a heparin-based sorbent with low antithrombin III binding capacity.

A sorbent based on heparin fraction with low affinity for antithrombin III is proposed for low density lipoproteins apheresis in hypercholesterolemia. Heparin was fractionated on antithrombin III-Sepharose; fractions with high and low affinity for antithrombin III were immobilized on CNBr-activated Sepharose 4B. Both sorbents appeared to have an LDL-binding capacity essentially similar to that of the sorbent based on unfractionated heparin.

[Use of avidin for speedy clearance of biotinylated immunoglobulins from the blood flow].

The phenomenon of a hundredfold more rapid blood clearance of biotinylated immunoglobulins after post-injection of an equiponderate dose of avidin is described. The concentration of 125I-labeled biotinylated IgG in rat circulation slowly decreased to 20% of the initial level in 24 hours. Avidin injection at any moment of this period induced a 90-95% reduction of blood radioactivity in 15 minutes.