Parallel imaging of coagulation pathway proteases activated protein C,thrombin, and factor Xa in human plasma

Activated protein C (APC), thrombin, and factor (f) Xa are vitamin K-dependent serine proteases that are key factors in blood coagulation. Moreover, they play important roles in inflammation, apoptosis, fibrosis, angiogenesis, and viral infections. Abnormal activity of these coagulation factors has been related to multiple conditions, such as bleeding and thrombosis, Alzheimer''s disease, sepsis, multiple sclerosis, and COVID-19. The individual activities of APC, thrombin, and fXa in coagulation and in various diseases are difficult to establish since these proteases are related and have similar substrate preferences. Therefore, the development of selective chemical tools that enable imaging and discrimination between coagulation factors in biological samples may provide better insight into their roles in various conditions and potentially aid in the establishment of novel diagnostic tests. In our study, we used a large collection of unnatural amino acids, and this enabled us to extensively explore the binding pockets of the enzymes'' active sites. Based on the specificity profiles obtained, we designed highly selective substrates, inhibitors, and fluorescent activity-based probes (ABPs) that were used for fast, direct, and simultaneous detection of APC, thrombin, and fXa in human plasma.

Using a collection of natural and unnatural amino acids, we synthesized a set of fluorescent activity-based probes for the fast, direct, and simultaneous detection of coagulation factors in human plasma.     

Effect of Grinding Process Parameters and Storage Time on Extraction of Antioxidants from Ginger and Nutmeg

The aim of this study was to optimize the grinding process parameters (mesh size of grinder sieve (X₁), the peripheral velocity of the grinding wheels (X₂)), and the storage time (X₃) of ground ginger rhizome and nutmeg to obtain ethanol and ethanol-water extracts with improved antioxidant properties. The optimal conditions were estimated using response surface methodology (RSM) based on a three-variable Box–Behnken design (BBD) in order to maximize the antioxidant capacity (AC) determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) methods, and the total phenolic content (TPC) was determined by the Folin–Ciocalteu (F–C) method in spice extracts. Additionally, the phenolic acid profiles in extracts from optimized conditions were analyzed using ultra-performance liquid chromatography (UPLC). It was found that the optimal preparation conditions for antioxidant extraction were dependent on the spice source and solvent type. The best antioxidant properties in nutmeg extracts were achieved for X₁ = 1.0 mm, X₂ = 40–41 Hz and X₃ = 7 days, whereas the optimized parameters for ginger extracts were more varied (1.0–2.0 mm, 43–50 Hz and 1–9 days, respectively). The ginger extracts contained 1.5–1.8 times more phenolic acids, and vanillic, ferulic, gallic, and p-OH-benzoic acids were dominant. In contrast, the nutmeg extracts were rich in protocatechuic, vanillic, and ferulic acids.     

Application of Dipotassium Glycoxides–Activated 18-Crown-6 for the Synthesis of Poly(propylene oxide) with Increased Molar Mass

Mono- and dipotassium salts of dipropylene glycol were applied for the polymerization of propylene oxide in mild conditions, i.e., tetrahydrofuran solution at ambient temperature. The structure of polymers was investigated by use of ¹³C NMR and MALDI-TOF techniques. The structure depends strongly on the kind of initiator and additives that are used such as coronand 18-crown-6 and dipropylene glycol. The lowest unsaturation, represented by allyloxy starting groups, has the polymer obtained by use of monopotassium salt without the ligand. The highest unsaturation degree is for the polymer synthesized in the presence of dipotassium salt–activated 18-crown-6. This polymer, obtained at high initial monomer concentration and low initial concentration of initiator, consists of two fractions, i.e., a low molar mass fraction (M_n = 9400) containing mainly macromolecules with alkoxide starting and end groups and a much higher molar mass fraction (M_n = 29500 g/mol) containing macromolecules with allyloxy starting groups and alkoxide or hydroxyl end groups. Addition of free glycol to this system decreases the molar mass of polymers. Similar results were obtained by use of dipotassium salts of other glycols. The mechanisms of the studied processes are discussed.     

Electron attachment to chlorouracil: a comparison between 6-ClU and 5-ClU

Low energy electron impact to the isomers 6-chlorouracil (6-ClU) and 5-chlorouracil (5-ClU) yields a variety of negative ion fragments with surprisingly high cross sections. These ions are dominantly formed via sharply structured resonance features at energies below the threshold for electronic excitation and result from dissociative electron attachment (DEA). The most dominant DEA channel is formation of (M-HCl)-, i.e., ejection of a neutral HCl molecule with the negative charge remaining on the ring. The reaction cross section is 9 x 10(-18) m2 and 5 x 10(-18) m2 for 6-Cl and 5-ClU, respectively, and thus about two orders of magnitude higher than the geometrical cross section of the molecule. Further reactions also operative via low energy resonances (<2.5 eV) are Cl- abstraction, dehydrogenation [formation of (M-H)-, M=ClU], and DEA processes associated with a ring opening. Most of the ion yield curves exhibit remarkably sharp structures which have not been observed before in DEA to a polyatomic system. Although some possibilities on their origin are discussed, their interpretation remains a challenge for theory and further experiments. While electron attachment to both 6-ClU and 5-ClU generates fragments of the same stoichiometric composition, their ion yields and also their relative intensities show some very pronounced differences which can be explained by the different structure but also the different energetic situation in the two isomers.     

Study of the temperature-dependent near-infrared spectra of water by two-dimensional correlation spectroscopy and principal components analysis

Near-infrared spectroscopy (NIR) is an important analytical tool in monitoring properties of systems for that water is a major constituent. For such objects of analysis a quality of information extracted from the NIR spectra depends essentially on used methods of analysis of a massive absorbance of water. Correctly chosen method should be able to identified rational number of overlapped components hidden under the broad band of water. The resolved components have to be justified on grounds of the structure of water and by relation to the properties a hydrogen-bonded network of water molecules. The interest in the correlation is imposed by a fact that hydrogen bonds of water around nonpolar group are significantly strengthened and weakened around polar groups. Intensity variations classified in this context could be valuable source of information on varying properties of the solute molecules embedded in water environment. Therefore, there is a big interest in methods that have a power for detailed analysis of the intensity changes in the broad NIR spectra. Two-dimensional correlation spectroscopy (2DCOS) and principal component analysis (PCA) are our proposition. In the analysis of the temperature-dependent NIR spectra of water by means of the two methods we have focused on the interpretation of the 2DCOS results through the concept of linear and nonlinear relationships. Moreover, a cascaded curve fitting procedure has been employed. Presented approach should be very instructive of how to interpret the features of the 2D results that frequently is not a straightforward task.     

The influence of the mobile phase pH and the stationary phase type on the selectivity tuning in high performance liquid chromatography nucleosides separation

Analysis of the modified nucleosides is particularly important in the medical area because of a possibility of cancerogenic processes studies. The aim of this work was to study the selectivity tuning of modified nucleosides through the investigations of interactions analyte (modified nucleoside) <==> stationary phase <==> mobile phase. A series of homemade stationary phases with different surface properties has been utilized. All of them contain various interaction sites such as: cholesterol (SG-CHOL); n-acylamide (SG-CHOL, SG-AP); aminopropyl (SG-CHOL, SG-AP, SG-NH2, SG-MIX); cyanopropyl, phenyl, octyl (SG-MIX), octadecyl (SG-MIX, SG-C18) and silanols localized on the silica gel surface of all packings. The attempt to predict the main interactions responsible for the retention between nucleosides and stationary phase ligands was done on the basis of the elemental analysis, and proportional part of an individual ligand bonded to silica surface results. In order to study the influence of different packing types on the analyzed nucleosides retention, the relationship between pH of the mobile phase buffer and the selectivity of a stationary phase was investigated.     

Measuring the Electrode Kinetics of Vitamin B2 at a Constant Time Window of a Square Wave Voltammetric Experiment

The electrode kinetics of vitamin B2 was measured at a constant scan rate of a square‐wave (SW) voltammetric experiment by recently introduced method based on alteration of the height (amplitude) of the SW potential pulses. The electrode reaction mechanism was analyzed in the light of the simplified model considering two‐electron one step surface electrode mechanism, as well as to more appropriate EE surface reaction pathway consisting of two successive one‐electron quasireversible reactions. Theoretical analysis indicated that the evolution of the voltammetric response under variation of the SW amplitude enables estimation of the rate determining step of the EE mechanism.     

Substituent effects on the electrophilic activity of nitroarenes in reactions with carbanions

The effect on electrophilic activity of substituents located para, ortho, and meta to the nitro group of nitrobenzenes was determined by using vicarious nucleophilic substitution of hydrogen (VNS) with the carbanion of chloromethyl phenyl sulfone (1) as the model process. Values for the relative activities of substituted nitroarenes are given relative to nitrobenzene, which was taken as the standard. This process was chosen as a model reaction because it meets key criteria, such as the wide range of substituents that can be present on the nitrobenzene ring, a low sensitivity to steric hindrance, and in particular the possibility of ensuring conditions in which the overall relative rates of reaction in competitive experiments are equal to the relative rates of nucleophilic addition. The values of relative rates of addition, which were taken to be a measure of electrophilic activity, were determined by competitive experiments in which pairs of nitroarenes competed for the VNS reaction with carbanion of 1. A comprehensive set of data for effects of substituents on the electrophilic activity of nitroarenes is presented for the first time.     

Cationic agents for DNA compaction

Fluorescence microscopy was used to investigate the conformational changes of individual T4 DNA molecules induced by different compacting agents, namely the cationic surfactants, cetyltrimethylammonium bromide (CTAB) and chloride (CTAC), iron(III), lysozyme, and protamine sulfate. A protocol for establishing size estimates is suggested to obtain reproducible results. Observations show that in the presence of lysozyme and protamine sulfate, DNA molecules exhibit a conformational change from an elongated coil structure to compact globules, usually interpreted as a first-order transition. The maximum degree of compaction that is attained when iron(III) or CTAB (CTAC) are used as compacting agents is considerably smaller, and intermediate structures (less elongated coils) are visible even for high concentrations of these agents. Dynamic light scattering experiments were carried out, for some of the systems, to assess the reliability of size estimates from fluorescence microscopy.     

Bond- and site-selective loss of H- from pyrimidine bases

Electron attachment to gas phase thymine and uracil leads to H- loss within a broad and structured feature in the energy range between about 5 and 12 eV consisting of 4 overlapping resonances. By using thymine and uracil methylated at the N1 and N3 positions, respectively, and taking into account recent results from partly deuterated thymine, we find that by tuning the electron energy, H- loss turns out to be not only bond selective, i.e., (C-H) versus (N-H) bonds, but also site selective (N1 versus N3 site). Such a bond and site selectivity by energy has not been observed before in dissociative electron attachment. Implications for the mechanism of strand breaks observed in plasmid DNA are considered.