Blood Plasma Proteins Research Articles

Evaluation of the suitability of ionic liquid-based liquid-liquid microextractions for blood protein removal

The analysis of biological samples, such as whole blood, comes with several sample preparation challenges. Biological matrices often contain a variety of endogenous components that can interfere with the determination of xenobiotics. Especially blood plasma proteins (e.g. serum albumin) are known to interfere with electrospray ionization and result in analyte ion suppression. Sample preparation techniques should guarantee adequate removal of these biomolecules. The current study aims to determine to which extent proteins are removed from whole blood samples, using ionic liquid-based dispersive liquid-liquid microextraction (IL-DLLME). A qualitative comparison of the protein presence in extracts of IL-DLLME, solid-phase extraction (SPE) and protein precipitation (PP) was performed, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, UV/VIS spectrophotometry was used to determine the protein content of a whole blood sample and IL-DLLME, SPE and PP extracts of the same sample. Finally, a quantitative comparison of matrix effects of benzodiazepines present in both whole blood and water samples. SDS-PAGE results showed that IL-DLLME extracts still contained proteins (i.e. albumin, hemoglobin); however, band intensities were comparable to SPE extracts. Spectrophotometric tests showed a total protein content of approximately 2 mg/mL in the final extracts. PP showed the highest protein extraction rate (19 mg/mL). Quantitative ME results showed no significant differences (α = 0.05) between blood and water IL-DLLME extracts. Overall, this is the first study to conclude that IL-DLLME is able to sufficiently remove blood proteins from whole blood samples, in order to avoid significant ion suppression.

Prognosis of cardiovascular events after implantation of a cardioverterdefibrillator in patients with chronic heart failure: the value of increasing concentration of endothelin-1 and soluble forms of ST2 protein in blood plasma

Aim.The purpose of this study was to examine the association of increasing concentrations of endothelin-1 and soluble forms of ST2 protein (sST2) in the blood plasma of patients with chronic heart failure (CHF) who underwent implantation of a cardioverter-defibrillator, with the development of cardiovascular events (death from cardiovascular diseases, decompensated CHF, acute coronary syndrome or acute ischemic stroke) to determine whether these biomarkers can be used as predictors of an unfavorable course of the disease.Material and methods. A 6-month cohort study included 40 patients with CHF who received an ICD 4-12 weeks before their inclusion in the study. At the beginning of the study the concentrations of endothelin-1 and sST2 in plasma by ELISA were analyzed, along with conventional diagnostic techniques typical of a specialized cardiology clinic.Results.Phenotype with a favorable course of the disease was identified in 21 patients (group 1), and with a unfavorable course was seen in 19 (group 2). The concentration of endothelin-1 and sST2 in the blood plasma of the 2nd group patients was higher than in patients of the 1st group. The link of cardiovascular events with high levels of soluble ST2 protein and endothelin-1 in blood plasma has been identified. Variation of the decision rule threshold on the ROC-curve has allowed sST2 concentration of 34.93 ng/ml to be determined as a cutoff point. Accuracy of two-class classification (determination of the posterior probability of cardiovascular events) after identifying such cut-off point was characterized by 93% sensitivity and 72% specificity (area under ROC-curve – 0.87). If the value of the plasma concentrations of sST2 more 34.93 ng/ml the relative risk of cardiovascular events was 4.4 (95% CI 1.7–11.1). The use of endothelin-1 surplus in plasma (cut-off point – 0.34 fmol/ml) in predicting cardiovascular events was lower: the sensitivity – 88%, specificity – 68% (area under ROC-curve – 0.74). When the concentration of endothelin-1 in blood plasma was more than 0.34 fmol/ml the relative risk of cardiovascular events was 2.7 (95% CI 1.2–6.3).Conclusion.The increase in the sST2 and endothelin-1 concentration in blood plasma may be considered as a predictor of cardiovascular events in CHF patients after implantation of cardioverter-defibrillator.

Screening of lung cancer biomarker-proteins with a multiplex electrochemical sensor system based on aptamers

The aimof this work is the development and demonstration of the method of simultaneous detection of several biomarkers of lung cancer in the blood plasma of patients using a multiplex electrochemical testing system based on DNA aptamers. DNA aptamers are a new class of synthetic affinity probes obtained by in vitro or in vivo selection procedure by the systematic evolution of ligands by exponential enrichment (SELEX).Materials and methods.A set of aptamers obtained previously by selection for postoperative lung cancer tissue was used to create a multiplex electrochemical biochip. Identification of aptamer target proteins was performed using a modified affinity enrichment method (AptaBID). Molecular targets for the used set of aptamers to lung cancer were defined as vimentin, defensin, a light chain of myosin, tubulin alpha 1-B, neutrophil elastase and A1 elongation factor 1.Measurements of the presence of these biomarker proteins in blood plasma were carried out using electrochemical detection. The difference between peak heights before and after plasma deposition on the electrodes modified by aptamers was considered as a response of the system to the presence of protein onco-markers in blood plasma. Blood plasma of healthy volunteers was used as control.Results. Research showed that in the blood plasma of all the patients with lung cancer the content of biomarker proteins that bind to aptamers on electrode surfaces was increased. The increased content of these proteins in the blood plasma of patients suggests the presence of invasiveness and metastasis of tumors and their chemo-resistance.

Dynamics of oxidative modification of blood plasma proteins in patients with acute epididymitis under the influence of treatment

У нашому дослідженні була виконана спроба з'ясування діагностичної та прогностичної значимості рівня окисної модифікації білків (ОМБ) у плазмі крові хворих на гострий епідидиміт (ГЕ) протягом лікування. У всіх обстежених пацієнтів до лікування виявили підвищення рівня окисної модифікації білків. У період надходження до стаціонару у всіх хворих на ГЕ рівні ОМБ виявились високими, що свідчить про високий окисний стрес при даному захворюванні. Протягом лікування ці показники поступово знижувалися до норми. Отримані дані можуть бути використані як для моніторингу лікування, так і одужання хворих.

Modeling of a single nanoparticle interaction with the human blood plasma proteins.

When nanoparticles are introduced into a physiological environment, proteins and lipids immediately cover their surface, forming a protein "corona". It is well recognized that the corona structure influences the biological response of the body. Two deterministic models for corona formation of the human blood serum proteins around a single nanoparticle are presented and studied numerically in this paper. One of them is based on a coupled system of PDEs and involves diffusion of proteins toward the nanoparticle surface. The other one is described by ODEs and is a limit version of the first model as the protein diffusivity tends to infinity. The protein diffusivity influence on the temporal corona structure is studied in detail. Results are presented using figures and discussion.

Non-aggregated zwitterionic Zinc(II) phthalocyanine complexes in water with high singlet oxygen quantum yield

Syntheses of non-peripheral (4) and peripheral (5) 2-(4-methyl-1,3-thiazol-5-yl)ethanol substituted zinc (II) phthalocyanines were achieved in this study. These zinc(II) phthalocyanines were converted to their quaternized ionic (4a and 5a) and zwitterionic (4b and 5b) derivatives. The structures of these novel compounds were characterized by elemental analysis and different spectroscopic methods such as FT-IR, UV-Vis, NMR and mass. General trends were examined for aggregation behaviours, photochemical and photophysical properties of the non-ionic zinc (II) phthalocyanines (4 and 5) in DMSO and their water soluble cationic (4a and 5a) and zwitterionic (4b and 5b) derivatives in both of DMSO and phosphate solution (PBS). The effect of different position and quaternization or zwitterionization of the binding groups on the photophysical and photochemical properties were also announced. Water soluble zwitterionic compound 4b exhibited very high singlet oxygen generation which makes it ideal photosensitizer in treatment of cancer by photodynamic therapy method. The fluorescence of the tetra-substituted zinc (II) phthalocyanines (4 and 5) are effectively quenched by addition of 1,4-benzoquinone (BQ). Furthermore, the water soluble phthalocyanine derivatives (4a, 5a, 4b and 5b) can bind to bovine serum albumin (BSA) which is a blood plasma protein.

Age-related features of activity of free radical oxidation in erythrocyte membranes and blood plasma in postinfarction cardiosclerosis in rats

Aim. To determine the presence of lipid and protein peroxidation products in erythrocyte membranes and blood plasma in rats of different ages in an intact state and with developed postinfarction cardiosclerosis.
 Methods. In rats aged 4, 12, 24 months postinfarction cardiosclerosis was modeled by coronary occlusion. In blood plasma and erythrocyte membranes, products reacting with thiobarbituric acid and carbonyl derivatives of protein amino acid residues were determined.
 Results. In erythrocyte membranes of intact animals aged 12 months, a decrease of the content of thiobarbituric acid-reacting products was revealed in comparison with 4 month-old animals, and in 24 months-old animals the index significantly exceeded the values of young subjects. In the blood plasma of intact animals aged 12 and 24 months, an increased content of thiobarbituric acid-reacting products with respect to rats aged 4 months was revealed. In postinfarction cardiosclerosis, an increase in the accumulation of thiobarbituric acid-reacting products was observed in erythrocyte membranes of 4 and 24 months-old animals. In blood plasma, an increase of this parameter was noted only in the group of 4 months-old animals with postinfarction cardiosclerosis. In erythrocyte membranes of intact rats of all age groups, no differences in the level of carbonyl derivatives of protein amino acid residues were observed, and in blood plasma it significantly increased with age. In 4 and 12 months-old animals with postinfarction cardiosclerosis, significant increase of carbonyl derivatives of protein amino acid residues was mostly pronounced in erythrocyte membranes. In 24 months-old animals with postinfarction cardiosclerosis, this parameter in the membranes and blood plasma was stable.
 Conclusion. The intensity of processes of cell membrane and blood lipid peroxidation has pronounced age-dependent features that persist in postinfarction cardiosclerosis; proteins of erythrocyte membranes of intact animals are subject to peroxide modification to a lesser degree than blood plasma proteins; in postinfarction cardiosclerosis formation in 4 and 12 months-old animals, carbonyl derivatives of protein amino acid residues are more intensively accumulated in erythrocyte membranes compared to blood plasma.

Nontoxic silver nanocluster-induced folding, fibrillation, and aggregation of blood plasma proteins

In recent years, concerns have been raised considering the potential risks of nanocluster (NC) for the environment and human health. Since the blood circulation system is probably the first entry route of NC into the human body, adsorption of blood proteins on NC may change cellular responses, including cellular uptake efficiency, bio distribution patterns, and nanotoxicity profiles, besides other biological effects. Therefore, the interaction of NCs with proteins and the cellular implications can be therapeutically of great importance. Adsorption of human blood proteins on NCs has been methodically investigated. In the present study, the first analysis of fibrillation was conducted between MBI-AgNCs and human serum (a complex biofluid). AgNCs were prepared by coating with 2-mercaptobenzimidazole. Then, interactions with human blood proteins, such as immunoglobulin, albumin, and insulin were investigated using various experimental approaches. Upon protein association, the fluorescence of proteins significantly decreased, accompanied by a blue shift in the AgNCs-human serum albumin (HSA) system and a red shift in the AgNCs-insulin/γ-globulin. Concomitantly, circular dichroism spectroscopy and atomic force microscopy were employed to investigate the effects of protein binding to NCs. We found that AgNCs induced γ-globulin aggregation. HSA at the AgNC surface was partially unfolded and could promote protein self-assembly into amyloid fibrils, while the surface morphology remained unchanged after insulin incubation. The atomic force microscopy (AFM) data and the ThT and CR analysis of the proteins, as well as circular dichroism (CD) and fluorescence findings, support the use of AgNCs as an indicator for monitoring the progress of HSA fibrillogenesis. Additionally, cytotoxicity assays were used to ensure the biocompatibility of nanoparticles within the applicable limits.

Role of Glutathione Redox System on the T-2 Toxin Tolerance of Pheasant (Phasianus colchicus)

The purpose of the present study was to evaluate the effects of different dietary concentrations of T-2 toxin on blood plasma protein content, lipid peroxidation and glutathione redox system of pheasant (Phasianus colchicus). A total of 320 one-day-old female pheasants were randomly assigned to four treatment groups fed with a diet contaminated with different concentrations of T-2 toxin (control, 4 mg/kg, 8 mg/kg and 16 mg/kg). Birds were sacrificed at early (12, 24 and 72 hr) and late (1, 2 and 3 weeks) stages of the experiment to demonstrate the effect of T-2 toxin on lipid peroxidation and glutathione redox status in different tissues. Feed refusal and impaired growth were observed with dose dependent manner. Lipid-peroxidation was not induced in the liver, while the glutathione redox system was activated partly in the liver, but primarily in the blood plasma. Glutathione peroxidase activity has changed parallel with reduced glutathione concentration in all tissues. Based on our results, pheasants seem to have higher tolerance to T-2 toxin than other avian species, and glutathione redox system might contribute in some extent to this higher tolerance, in particular against free-radical mediated oxidative damage of tissues, such as liver.

PO-395 Cortisol triggers immune escape of human acute myeloid leukaemia cells by inducing expression of G protein-coupled receptor latrophilin 1 which facilitates exocytosis of immune suppressor galectin-9

IntroductionAcute myeloid leukaemia (AML) is a blood and bone marrow cancer originating from self-renewing malignant immature myeloid cells. AML cells are capable of escaping host immune surveillance. One of the mechanisms underlying this phenomenon is the ability of AML cells to secrete galectin-9, a tandem protein, which induces variety of intracellular and cell-to-cell signalling events leading to inactivation of NK cells as well as killing of cytotoxic T cells. Exocytosis of galectin-9 is mediated by the G protein-coupled receptor latrophilin 1 (LPHN1). However, molecular mechanisms underlying its expression and functional control remain unknown, and thus became the main goal of our work.Material and methodsWe used THP-1 and primary human AML cells, primary leukocytes obtained from healthy donors. Western blot analysis, ELISA and quantitative Real-Time PCR served as main instrumental in experimental set-ups.Results and discussionsWe discovered that cortisol induces LPHN1 expression on transcriptional level thus also upregulating its translation in AML cells, but not in primary healthy human leukocytes. We found that secreted form of fibronectin and leucine-rich transmembrane protein 3 (FLRT3, an endogenous LPHN1 ligand) is present in blood plasma of healthy donors and AML patients in equally high amounts. Therefore, LPHN1 expressed by human AML cells is not lacking its ligand and thus triggers galectin-9 secretion on permanent basis in order to provide continuous protection against host immune surveillance.ConclusionOur results demonstrate a fundamentally novel survival mechanism employed by human AML cells. They use a crucial human hormone (cortisol) to express receptor (LPHN1), which uses blood plasma protein (FLRT3) to escape immune surveillance. Thus AML cells employ normal physiological systems of the human body to support their survival and attenuate anti-cancer activity of cytotoxic lymphoid cells.

Surface Nanostructuring of Parylene-C Coatings for Blood Contacting Implants.

This paper investigates the effects on the blood compatibility of surface nanostructuring of Parylene-C coating. The proposed technique, based on the consecutive use of O2 and SF6 plasma, alters the surface roughness and enhances the intrinsic hydrophobicity of Parylene-C. The degree of hydrophobicity of the prepared surface can be precisely controlled by opportunely adjusting the plasma exposure times. Static contact angle measurements, performed on treated Parylene-C, showed a maximum contact angle of 158°. The nanostructured Parylene-C retained its hydrophobicity up to 45 days, when stored in a dry environment. Storing the samples in a body-mimicking solution caused the contact angle to progressively decrease. However, at the end of the measurement, the plasma treated surfaces still exhibited a higher hydrophobicity than the untreated counterparts. The proposed treatment improved the performance of the polymer as a water diffusion barrier in a body simulating environment. Modifying the nanotopography of the polymer influences the adsorption of different blood plasma proteins. The adsorption of albumin—a platelet adhesion inhibitor—and of fibrinogen—a platelet adhesion promoter—was studied by fluorescence microscopy. The adsorption capacity increased monotonically with increasing hydrophobicity for both studied proteins. The effect on albumin adsorption was considerably higher than on fibrinogen. Study of the proteins simultaneous adsorption showed that the albumin to fibrinogen adsorbed ratio increases with substrate hydrophobicity, suggesting lower thrombogenicity of the nanostructured surfaces. Animal experiments proved that the treated surfaces did not trigger any blood clot or thrombus formation when directly exposed to the arterial blood flow. The findings above, together with the exceptional mechanical and insulation properties of Parylene-C, support its use for packaging implants chronically exposed to the blood flow.

On the way to precision formulation additives: 2D-screening to select solubilizers with tailored host and release capabilities

A 2-dimensional high-throughput screening method is presented to select peptide sequences from large peptide libraries for precision formulation additives, having a high capacity to specifically host a drug of interest and provide tailored drug release properties. The identified sequences are conjugated with poly(ethylene glycol) (PEG) to obtain peptide-PEG conjugates that proved to be valuable as solubilizers for small organic molecule drugs to overcome limitations of poor water-solubility and low bio-availability. The 2D-screening method selects peptide sequences on both (i) high loading capacities and (ii) preferred drug-release capabilities as demonstrated on an experimental Tau-protein aggregation inhibitor/Tau- deaggregator with potentials for an anti-Alzheimer disease drug (BB17). To enable 2D-screening, a one-bead one-compound (OBOC) peptide library was immobilized on a glass slide, allocating individual beads to permanent positions. While the first screening step involved incubation of the supported OBOC library with BB17 to identify beads with high drug binding capacities by fluorescence scanner readouts, the second step reveals release properties of the high capacity binders by incubation with blood plasma protein model solutions. Efficiently peptides with high BB17 capacities and either keeper or medium or fast releaser properties can be identified by direct sequence readouts from the glass slide supported resin beads via matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Four peptides are synthesized as peptide-PEG solubilizers representing strong, medium, weak releasers and non-binders. Loading capacities reached up to 1:3.4 (mol drug per mol carrier) and release kinetics (fast/medium/slow) are in agreement with the selection process as investigated by fluorescence anisotropy and fluorescence correlation spectroscopy. The ability of BB17/conjugate complexes to inhibit the aggregation of Tau4RDΔK (four repeat Tau ((M)Q244-E372 with deletion of K280), 129 residues) in N2a cells is studied by a Tau-pelleting assay showing the modulation of cellular Tau aggregation. Promising effects such as the reduction of 55% of total Tau load are observed for the strong releaser additive. Studies of in vitro Thioflavin S Tau-aggregation assays show half-maximal inhibitory activities (IC50 values) of BB17/conjugates in the low micro-molar range.

Streptococcus pyogenes Infection and the Human Proteome with a Special Focus on the Immunoglobulin G-cleaving Enzyme IdeS

Infectious diseases are characterized by a complex interplay between host and pathogen, but how these interactions impact the host proteome is unclear. Here we applied a combined mass spectrometry-based proteomics strategy to investigate how the human proteome is transiently modified by the pathogen Streptococcus pyogenes, with a particular focus on bacterial cleavage of IgG in vivo. In invasive diseases, S. pyogenes evokes a massive host response in blood, whereas superficial diseases are characterized by a local leakage of several blood plasma proteins at the site of infection including IgG. S. pyogenes produces IdeS, a protease cleaving IgG in the lower hinge region and we find highly effective IdeS-cleavage of IgG in samples from local IgG poor microenvironments. The results show that IdeS contributes to the adaptation of S. pyogenes to its normal ecological niches. Additionally, the work identifies novel clinical opportunities for in vivo pathogen detection.

Scrotal circumference: A predictor of testosterone concentration and certain attributes of seminal vesicles influencing buffalo male fertility.

Aim:The aim of this study was to evaluate the relationship of scrotal circumference (SC) with plasma testosterone, seminal vesicles (SVs) weight, and its secretion as measurable indicators of fertility and also to sequence and establish phylogenetic relatedness of certain SV protein genes with other species as such integrated approach is lacking.Materials and Methods:Altogether, 59 apparently healthy male buffaloes sacrificed at slaughterhouse were selected (irrespective of breed) for measuring SC and collecting blood and paired SVs. The SC was measured at greater curvature using soft thread. In the present study, blood plasma testosterone, cholesterol, protein, and glucose in addition to SV fructose, citric acid and proteins in SV fluid were also estimated. The SV tissue was fixed in RNAlater for RNA extraction. Male buffaloes were categorized as per total SV weight into Group I (<5.0 g), Group II (5.0-7.84 g), and Group III (>8.0 g) and dentitions-I (≤18 months), II (18-24 months), and III (≥24 months) to assess the effect of weight and dentition age on SC, SV weight, and its certain secretions. Data were analyzed using linear model procedure including Tukey HSD test and Pearson’s correlation coefficient. Variance inflation and condition index were also used to assess multicollinearity.Results:Gross and histomorphological evaluation of SVs did not show any abnormality. Macronutrients (plasma protein, glucose, and cholesterol) showed non-significant (p>0.05) variation between groups. The SC and SV weight varied significantly (p<0.05) with a significant positive relationship with plasma testosterone, SV protein, fructose, and citric acid. In addition, testosterone concentration also showed increasing trend from Groups I to III but increased significantly (p<0.05) from Group II to III with positive and significant correlations with SV protein, fructose, and citric acid similar to SV weight and SC. Binders of sperm protein (BSP1, 3, and 5) genes (full length) were sequenced and established an evolutionary relationship which is lacking in buffalo.Conclusion:The present findings established a significant positive correlation of SC with that of other fertility parameters related to SVs weight and its secretions: Fructose, citric acid, and protein (inclusive of BSPs sequenced full length), and testosterone. Therefore, the present integrated approach along with certain semen quality attributes reflecting epididymis function could be used as a predictive fertility marker for grading and selection of breeding bulls and their progenies to develop outstanding bull mother farm.

Spectroscopic analysis, docking and molecular dynamics simulation of the interaction of cinnamaldehyde with human serum albumin

Human serum albumin (HSA) is one of the blood plasma proteins, which plays an important role in the transportation of various natural and pharmaceutical compounds in the body. Cinnamaldehyde is the main component of cinnamon extract which has several medicinal properties. In this study, the conformational changes of HSA in the presence of cinnamaldehyde were investigated by UV–Vis, fluorescence and FT-IR spectroscopic methods and the binding parameters of cinnamaldehyde to HSA were determined. The binding site and driving forces of this interaction were calculated using molecular docking softwares. On the other hand, molecular dynamics simulations were also performed with the GROMACS program package. Using the results of the intrinsic fluorescence spectroscopy data, binding constant of 1.14 × 104 M−1 and number of binding sites 0.883 was obtained. FT-IR measurements have shown that the secondary structure of the protein have been changed by the interaction of cinnamaldehyde with HSA. The free energy of binding (ΔG°) and binding constant (Ka) obtained from the intrinsic fluorescence results were − 5.54 kcal mol−1 and 1.14 × 104 M−1 respectively, which is in good agreement with molecular docking results. On the other hand, the molecular docking calculations showed that cinnamaldehyde binding site is located in the IA subdomain of HSA. Spectroscopy, molecular docking and MD simulations methods showed that the cinnamaldehyde is bound to the HSA protein and hydrogen bonding and hydrophobic forces play the most important role in this interaction. Excellent agreement was found between the experimental and theoretical results.

γ-Pyrone compounds: flavonoids and maltol glucoside derivatives from Herniaria glabra L. collected in the Ternopil region of the Ukraine

The phytochemical investigation of the whole plant extracts of Herniaria glabra L. (Caryophyllaceae) led to the identification and isolation of four known flavonoids, one known and three undescribed maltol derivatives, and benzyl β-gentiobioside. The structures were established by extensive 1D and 2D NMR spectroscopic analyses, as well as HRESIMS data. For the first time in Herniaria genus, as well as in Caryophylaceae family the presence of apiorutin {quercetin 3-O-[(D-apio-β-d-furanosyl-(1 → 2)-O-[-α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside]} and licoagroside B {maltol 3-O-[6-O-(3-hydroxy-3-methylglutaroyl)]-β-d-glucopyranoside} were revealed. Additionally, antioxidant actions of apiorutin, rutin, narcissin (isorhamentin 3-O-β-d-rutinoside) and licoagroside B were assessed in human blood plasma, exposed to the peroxynitrite-induced oxidative stress in vitro. The isolates partly reduced oxidative (oxidation of thiol groups) and nitrative (tyrosine nitration) damage to blood plasma proteins, decreased plasma lipid peroxidation as well as enhanced the non-enzymatic antioxidant capacity of blood plasma. No cytotoxicity of the examined substances towards peripheral blood mononuclear cells was found.

The Influence of Fatty Acids on Metoprolol - Human Serum Albumin Interaction in Low Affinity Binding Sites: A Multifactorial NMR Approach.

Metoprolol (MTP) is a cardio-selective β1-blocker used in hypertension, angina pectoris and chronic heart failure therapies. Serum albumin is the most frequently occurring protein in blood plasma. The binding of ligands to human serum albumin (HSA) has an important effect on pharmacokinetics and final clinical effects. The objectives of this study included a detailed analysis of metoprolol - serum albumin interactions in low affinity binding sites, on the surface or within the hydrophobic subdomain of a macromolecule, as well as an analysis of the competition between MTP and fatty acids in binding with protein. The analysis of the drug-albumin interaction was based on the observed chemical shifts in combination with correlation Times (T1 -1 = τ) [1/s], 2D NOESY 1H NMR spectra and association constants Ka [M-1]. For the determination of chemical shifts σ [ppm], relaxation times T1 [s] and for the NOESY experiment, the final concentrations of MTP and albumins (in the presence (HSA) and absence of fatty acids (dHSA)) were 5 x 10-3 M and 2 x 10-5 M - 4.55 x 10-4 M, respectively. In order to calculate the association constants, the final concentrations of MTP and both HSA and dHSA were 2.75 x 10-3 M - 6.25 x 10-2 M and 2.5 x 10-4 M, respectively. For the analysis, the MTP proton resonances of aliphatic H17, aromatic (H2/H6 and H3/H5) and the methoxy group H14 were chosen. Changes in the values of the 1H NMR chemical shift [ppm] are evidence of interaction between MTP, fatted (HSA) and defatted (dHSA) human serum albumin. With an increase of albumin concentration, changes in the chemical shift values were observed for the aromatic protons H2/H6 (Δσ = 0.013 ppm and 0.018 ppm) and H3/H5 (Δσ = 0.015 ppm and 0.019 ppm), the aliphatic proton H17 (Δσ = 0.018 ppm and 0.022 ppm) and the aliphatic protons of the methoxy group H14 (Δσ = 0.019 ppm and 0.022 ppm) for dHSA and HSA, respectively. Greater changes in chemical shifts in the presence of fatty acids (FA) were observed. Changes in the correlation times of MTP aromatic H2/H6 (Δτc = 0.224 1/s and 0.189 1/s) and H3/H5 (Δτc = 0.269 1/s and 0.210 1/s), aliphatic from the methoxy group H14 (Δτc = 0.472 1/s and 0.271 1/s) and aliphatic H17 protons (Δτc = 0.178 1/s and 0.137 1/s) for dHSA and HSA systems, respectively. It confirms the interaction between the drug and albumin are evidence for the dynamics of the process. In the presence of FA the relaxation time of all analyzed MTP proton resonance signals significantly increases (due to the decrease of correlation time). This phenomenon is due to the increase of electron density in the MTP protons' surroundings. Association constants for the MTP-dHSA complex in the low affinity site range between 0.29 x 102 M-1 and 0.47 x 102 M-1. The presence of FA results in a two to three-fold increase of the Ka values of protons from aromatic (H2/H6 and H3/H5), aliphatic H17 and methoxy (H14) groups. In 2D NOESY spectra proton magnetization transfer was observed between cysteine (Cys-34) and aromatic H3/H5 and H2/H6 protons. Cross-peaks were also observed between cysteine and aliphatic protons from the methoxy group. The selective changes in σ [ppm] and τc [1/s] values indicated the unequal participation of chemical groups of MTP in the interaction with HSA and dHSA. The data obtained suggest that the presence of fatty acids increases the accessibility of low affinity sites of serum albumin to MTP, which results in the higher affinity of albumin towards the drug. The results showed that the main binding site of MTP and fatty acid is probably a low affinity site in subdomain IB, where Cys-34 can be located.

Staphylococcus aureus clumping factor A is a force-sensitive molecular switch that activates bacterial adhesion

Clumping factor A (ClfA), a cell-wall-anchored protein from Staphylococcus aureus, is a virulence factor in various infections and facilitates the colonization of protein-coated biomaterials. ClfA promotes bacterial adhesion to the blood plasma protein fibrinogen (Fg) via molecular forces that have not been studied so far. A unique, yet poorly understood, feature of ClfA is its ability to favor adhesion to Fg at high shear stress. Unraveling the strength and dynamics of the ClfA-Fg interaction would help us better understand how S. aureus colonizes implanted devices and withstands physiological shear stress. By means of single-molecule experiments, we show that ClfA behaves as a force-sensitive molecular switch that potentiates staphylococcal adhesion under mechanical stress. The bond between ClfA and immobilized Fg is weak (∼0.1 nN) at low tensile force, but is dramatically enhanced (∼1.5 nN) by mechanical tension, as observed with catch bonds. Strong bonds, but not weak ones, are inhibited by a peptide mimicking the C-terminal segment of the Fg γ-chain. These results point to a model whereby ClfA interacts with Fg via two distinct binding sites, the adhesive function of which is regulated by mechanical tension. This force-activated mechanism is of biological significance because it explains at the molecular level the ability of ClfA to promote bacterial attachment under high physiological shear stress.

Intravital molecular tagging velocimetry of cerebral blood flow using Evans Blue.

The effects of light-driven enhancement of Evans Blue dye complexes with blood plasma proteins were observed for the first time, both in vitro and in vivo. The possible background of the effect concerns the photochemical cis-trans isomerization of the azo dye molecules. The effect was induced in the solution with a red laser with a wavelength of 638 nm, which corresponds to the peak of the dye absorption. The lifetime of the enhanced fluorescence is approximately 1 second and enables its use as an optically tagged molecular flow tracer for blood flow velocity measurements. Utilizing the effect, we performed for the first time the intravital molecular tagging velocimetry of the blood velocity in blood vessels in a living animal. The results of the measurements of the blood flow velocities in the cerebral veins of a group of healthy mice are presented.

Oxidation-induced modifications of the catalytic subunits of plasma fibrin-stabilizing factor at the different stages of its activation identified by mass spectrometry

Plasma fibrin-stabilizing factor (pFXIII) is a heterotetrameric proenzyme composed of two catalytic A subunits (FXIII-A2) and two inhibitory/carrier B subunits (FXIII-B2). The main function of the protein is the formation of cross-links between the polypeptide chains of the fibrin clot. The conversion of pFXIII into the enzymatic form FXIII-A2* is a multistage process. Like many other blood plasma proteins, pFXIII is an oxidant-susceptible target. The influence of distinct sites susceptible to oxidation-mediated modifications on the changes in the structural-functional characteristics of the protein remains fully unexplored. For the first time, a set of the oxidation sites within FXIII-A2 under ozone-induced oxidation of pFXIII at different stages of its activation have been identified by mass spectrometry, and the extent as well as the chemical nature of these modifications have been explored. It was shown that the set of amino acid residues susceptible to oxidative attack and the degree of oxidation of these residues in FXIII-A2 of non-activated pFXIII, pFXIII activated by Ca2+ and fully activated pFXIII treated with thrombin and Ca2+ significantly differ. The obtained data enable one to postulate that in the process of the proenzyme conversion into FXIII-A2*, new earlier-unexposed amino acid residues become available for the oxidizer while some of the initially surface-exhibited residues are buried within the protein globule.