Search Results (22)

This study introduces an innovative microfluidic-based approach for extracting drugs from oral fluids using self-assembled tripeptide hydrogels as sorbents. Peptide microfiber derived from the heterochiral tripeptide ^DLeu-^LPhe-^LPhe was formed in situ within the 14 mm-long microchannel of a two-inlet microfluidic device. The methodology enables the laminar flow-driven mixing of buffer solutions, inducing hydrogel formation at their interface. The resulting fiber exhibited a well-defined morphology and β-sheet structure, confirmed by Raman spectroscopy and Thioflavin T fluorescence. The peptide fibers co-assembled successfully with 5-fluorouracil (5-FU) and naproxen (39.8 ± 1.4 nmol of 5-FU and 27.4 ± 6.6 nmol of naproxen per 112 nmol of peptide used to prepare the fiber), resulting in a molar ratio drug/peptide ratio of approximately 1:3 and 1:4, respectively, demonstrating versatility in drug entrapment. The use of the gel fiber as a sorbent phase was first assessed in buffer, and subsequently, the optimized method was applied to saliva. Adsorption studies under stopped-flow conditions showed a significant drug adsorption capability from buffered solutions by the pre-formed hydrogel (32.8 ± 0.9% of 5-FU and 36.4 ± 3.3% of naproxen per fiber preformed with 112 nmol of peptide), demonstrating their suitability as sorbent material. The extension of the methodology to simulated saliva samples allowed extraction of 36% of 5-FU by the fiber, as determined by ¹⁹F NMR spectroscopy on microcoils, which enabled us to work with the small volume of fluid extracted from the microfluidic device and provided clean spectra and quantitative results. These findings highlight the potential of this tripeptide hydrogel as a sorbent material for therapeutic drug monitoring and toxicological analysis via a simple, non-invasive and rapid approach for drug detection in oral fluids. Full article

The selective inhibition of key enzymes, such as carbonic anhydrases (CAs IX and XII), which are overexpressed in cancer tissues, has emerged as a promising strategy in cancer research. However, a multitarget approach is often preferred to achieve enhanced therapeutic outcomes. In this study, aryl sulfonamides were conjugated with a thiosemicarbazone moiety to enable dual functionality: the inhibition of CAs and the chelation of metal cations. Several structural factors were systematically modified, including the position of the sulfonamido group, the length of the linker, the nature of the aromatic residue, and the type of substituents. Tumor-associated CAs IX and XII inhibition was evaluated using the stopped-flow CO₂ hydrase assay, and the inhibition constants (K_i) were determined. The most promising compounds were further analyzed through molecular docking simulations. Metal chelation capabilities were evaluated using UV–Vis spectroscopy, while antiproliferative activities were measured using the sulforhodamine B (SBR) assay. Additionally, holotomographic 3D microscopy was employed to investigate the mechanisms of cell death. Sulfonamido-derived Schiff bases were synthesized through a three-step procedure that did not require column chromatography purification: (1) isothiocyanation of amino-sulfonamides, (2) nucleophilic addition of hydrazine, and (3) acid-promoted condensation with different aldehydes (benzaldehydes or pyridine-2-carboxaldehyde). The synthesized compounds exhibited inhibition of CAs in the low nanomolar to submicromolar range, with selectivity largely influenced by structural features. Notably, the m-sulfonamide derivative 5b, bearing a pyridin-2-yl residue, demonstrated potent and selective inhibition of CA IX (K_i = 4.9 nM) and XII (K_i = 5.6 nM). Additionally, it efficiently chelated Fe²⁺, Fe³⁺, and Cu²⁺ and showed promising antiproliferative activity (GI₅₀ 4.5–10 µM). Mechanistic studies revealed that apoptosis was involved in its mode of action. Therefore, the synergistic integration of sulfonamides and thiosemicarbazones represents an effective strategy for the development of multimodal anticancer agents. Full article

Ascorbate peroxidases (APXs) are key components of the ascorbate–glytathione cycle, which plays an important role in removing excess reactive oxygen species (ROS) in plants. Herein, MaAPX1 was verified as being involved in the ripening and senescence of banana fruit, exhibiting responsiveness to the accumulation of ROS and the oxidation of proteins. Site-directed mutation was applied to explore the mechanism of MaAPX1 activity changes. We found that the 32-site cysteine (Cys, C) served as a potential S-nitrosylation site. The mutant MaAPX1^C32S activity was decreased significantly when Cys32 was mutated to serine (Ser, S). Intriguingly, the neighboring conserved 36-site methionine (Met, M), which is adjacent to Cys32, displayed an enzyme activity that was approximately five times higher than that of the wild-type MaAPX1 when mutated to lysine (Lys, K). Utilizing LC-MS/MS spectroscopy coupled with stopped-flow analysis showed that the enhanced MaAPX1^M36K activity might be due to the increased S-nitrosylation level of Cys32 and the promotion of intermediate (compound I, the first intermediate product of the reaction of APX with H₂O₂) production. Molecular docking simulations showed that the S-N bond between Cys32 and Lys36 in MaAPX1^M36K might have a function in protecting the thiol of Cys32 from oxidation. MaAPX1^M36K, a promising mutant, possesses immense potential for improving the antioxidant capabilities of APX in the realm of bioengineering technology research. Full article

2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO^•), a persistent nitronyl nitroxide radical, has been used for the detection and trapping of nitric oxide, as a redox mediator for batteries, for the activity estimation of antioxidants, and so on. However, there is no report on the reactivity of PTIO^• in the presence of redox-inactive metal ions. In this study, it is demonstrated that the addition of scandium triflate, Sc(OTf)₃ (OTf = OSO₂CF₃), to an acetonitrile (MeCN) solution of PTIO^• resulted in an electron-transfer disproportionation to generate the corresponding cation (PTIO⁺) and anion (PTIO⁻), the latter of which is suggested to be stabilized by Sc³⁺ to form [(PTIO)Sc]²⁺. The decay of the absorption band at 361 nm due to PTIO^•, monitored using a stopped-flow technique, obeyed second-order kinetics. The second-order rate constant for the disproportionation, thus determined, increased with increasing the Sc(OTf)₃ concentration to reach a constant value. A drastic change in the cyclic voltammogram recorded for PTIO^• in deaerated MeCN containing 0.10 M Bu₄NClO₄ was also observed upon addition of Sc(OTf)₃, suggesting that the large positive shift of the one-electron reduction potential of PTIO^• (equivalent to the one-electron oxidation potential of PTIO⁻) in the presence of Sc(OTf)₃ may result in the disproportionation. When H₂O was added to the PTIO^•–Sc(OTf)₃ system in deaerated MeCN, PTIO^• was completely regenerated. It is suggested that the complex formation of Sc³⁺ with H₂O may weaken the interaction between PTIO⁻ and Sc³⁺, leading to electron-transfer comproportionation to regenerate PTIO^•. The reversible disproportionation of PTIO^• was also confirmed by electron paramagnetic resonance (EPR) spectroscopy. Full article

Non-heme dioxygenases of the AlkB family hold a unique position among enzymes that repair alkyl lesions in nucleic acids. These enzymes activate the Fe(II) ion and molecular oxygen through the coupled decarboxylation of the 2-oxoglutarate co-substrate to subsequently oxidize the substrate. ALKBH3 is a human homolog of E. coli AlkB, which displays a specific activity toward N1-methyladenine and N3-methylcytosine bases in single-stranded DNA. Due to the lack of a DNA-bound structure of ALKBH3, the basis of its substrate specificity and structure–function relationships requires further exploration. Here we have combined biochemical and biophysical approaches with site-directed mutational analysis to elucidate the role of key amino acids in maintaining the secondary structure and catalytic activity of ALKBH3. Using stopped-flow fluorescence spectroscopy we have shown that conformational dynamics play a crucial role in the catalytic repair process catalyzed by ALKBH3. A transient kinetic mechanism, which comprises the steps of the specific substrate binding, eversion, and anchoring within the DNA-binding cleft, has been described quantitatively by rate and equilibrium constants. Through CD spectroscopy, we demonstrated that replacing side chains of Tyr143, Leu177, and His191 with alanine results in significant alterations in the secondary structure content of ALKBH3 and decreases the stability of mutant proteins. The bulky side chain of Tyr143 is critical for binding the methylated base and stabilizing its flipped-out conformation, while its hydroxyl group is likely involved in facilitating the product release. The removal of the Leu177 and His191 side chains substantially affects the secondary structure content and conformational flexibility, leading to the complete inactivation of the protein. The mutants lacking enzymatic activity exhibit a marked decrease in antiparallel β-strands, offset by an increase in the helical component. Full article

This study is a further development of our “Proposal of a new double-nozzle technique for in-gas-jet laser resonance ionization spectroscopy” paper published in the journal Atoms earlier this year. Here, we propose equipping the double-nozzle technique with the RF-only funnel and RF-buncher placed in a gas-jet chamber at a 70 mm distance downstream of the double-nozzle exit. It allows for highly effective extraction into vacuum heavy ion beams, produced in two-steps laser resonance ionization in the argon supersonic jet. We explored the operation of this new full version of the double-nozzle technique through detailed gas dynamic and Monte Carlo trajectory simulations, with the results presented and discussed. In particular, our calculations showed that more than 80% of all nobelium-254 neutral atoms, extracted by argon flow from the gas-stopping cell, can then be extracted into vacuum in a form of pulsed ion beam having low transverse and longitudinal emittance. Full article

Human Fe(II)/α-ketoglutarate-dependent dioxygenase ABH2 plays a crucial role in the direct reversal repair of nonbulky alkyl lesions in DNA nucleobases, e.g., N¹-methyladenine (m¹A), N³-methylcytosine (m³C), and some etheno derivatives. Moreover, ABH2 is capable of a less efficient oxidation of an epigenetic DNA mark called 5-methylcytosine (m⁵C), which typically is a specific target of DNA dioxygenases from the TET family. In this study, to elucidate the mechanism of the substrate specificity of ABH2, we investigated the role of several active-site amino acid residues. Functional mapping of the lesion-binding pocket was performed through the analysis of the functions of Tyr122, Ile168, and Asp173 in the damaged base recognition mechanism. Interactions of wild-type ABH2, or its mutants Y122A, I168A, or D173A, with damaged DNA containing the methylated base m¹A or m³C or the epigenetic marker m⁵C were analyzed by molecular dynamics simulations and kinetic assays. Comparative analysis of the enzymes revealed an effect of the substitutions on DNA binding and on catalytic activity. Obtained data clearly demonstrate the effect of the tested amino acid residues on the catalytic activity of the enzymes rather than the DNA-binding ability. Taken together, these data shed light on the molecular and kinetic consequences of the substitution of active-site residues for the mechanism of the substrate recognition. Full article

A complex heterogeneous intracellular environment seems to affect enzymatic catalysis by changing the mobility of biomolecules, their stability, and their conformational states, as well as by facilitating or hindering continuously occurring interactions. The evaluation and description of the influence of the cytoplasmic matrix components on enzymatic activity are problems that remain unsolved. In this work, we aimed to determine the mechanisms of action of two-component media with cosolvents of various molecular sizes on the complex multi-stage bioluminescent reaction catalyzed by bacterial luciferase. Kinetic and structural effects of ethylene glycol, glycerol, sorbitol, glucose, sucrose, dextran, and polyethylene glycol on bacterial luciferase were studied using stopped-flow and fluorescence spectroscopy techniques and molecular dynamics simulations. We have found that diffusion limitations in the presence of cosolvents promote the stabilization of flavin substrate and peroxyflavin intermediate of the reaction, but do not provide any advantages in bioluminescence quantum yield, because substrate binding is slowed down as well. The catalytic constant of bacterial luciferase has been found to be viscosity-independent and correlated with parameters of water–cosolvent interactions (Norrish constant, van der Waals interaction energies). Crowding agents, in contrast to low-molecular-weight cosolvents, had little effect on peroxyflavin intermediate decay and enzyme catalytic constant. We attributed specific kinetic effects to the preferential interaction of the cosolvents with enzyme surface and their penetration into the active site. Full article

Coproheme decarboxylases (ChdCs) are terminal enzymes of the coproporphyrin-dependent heme biosynthetic pathway. In this reaction, two propionate groups are cleaved from the redox-active iron-containing substrate, coproheme, to form vinyl groups of the heme b product. The two decarboxylation reactions proceed sequentially, and a redox-active three-propionate porphyrin, called monovinyl, monopropionate deuteroheme (MMD), is transiently formed as an intermediate. While the reaction mechanism for the first part of the redox reaction, which is initiated by hydrogen peroxide, has been elucidated in some detail, the second part of this reaction, starting from MMD, has not been studied. Here, we report the optimization of enzymatic MMD production by ChdC and purification by reversed-phase chromatography. With the obtained MMD, we were able to study the second part of heme b formation by actinobacterial ChdC from Corynebacterium diphtheriae, starting with Compound I formation upon the addition of hydrogen peroxide. The results indicate that the second part of the decarboxylation reaction is analogous to the first part, although somewhat slower, which is explained by differences in the active site architecture and its H-bonding network. The results are discussed in terms of known kinetic and structural data and help to fill some mechanistic gaps in the overall reaction catalyzed by ChdCs. Full article

Elucidation of physicochemical mechanisms of enzymatic processes is one of the main tasks of modern biology. High efficiency and selectivity of enzymatic catalysis are mostly ensured by conformational dynamics of enzymes and substrates. Here, we applied a stopped-flow kinetic analysis based on fluorescent spectroscopy to investigate mechanisms of conformational transformations during the removal of alkylated bases from DNA by ALKBH2, a human homolog of Escherichia coli AlkB dioxygenase. This enzyme protects genomic DNA against various alkyl lesions through a sophisticated catalytic mechanism supported by a cofactor (Fe(II)), a cosubstrate (2-oxoglutarate), and O₂. We present here a comparative study of conformational dynamics in complexes of the ALKBH2 protein with double-stranded DNA substrates containing N1-methyladenine, N3-methylcytosine, or 1,N6-ethenoadenine. By means of fluorescent labels of different types, simultaneous detection of conformational transitions in the protein globule and DNA substrate molecule was performed. Fitting of the kinetic curves by a nonlinear-regression method yielded a molecular mechanism and rate constants of its individual steps. The results shed light on overall conformational dynamics of ALKBH2 and damaged DNA during the catalytic cycle. Full article

NADPH-cytochrome P450 reductase (CPR) from Sorghum bicolor (SbCPR) serves as an electron donor for cytochrome P450 essential for monolignol and lignin production in this biofuel crop. The CPR enzymes undergo an ample conformational transition between the closed and open states in their functioning. This transition is triggered by electron transfer between the FAD and FMN and provides access of the partner protein to the electron-donating FMN domain. To characterize the electron transfer mechanisms in the monolignol biosynthetic pathway better, we explore the conformational transitions in SbCPR with rapid scanning stop-flow and pressure-perturbation spectroscopy. We used FRET between a pair of donor and acceptor probes incorporated into the FAD and FMN domains of SbCPR, respectively, to characterize the equilibrium between the open and closed states and explore its modulation in connection with the redox state of the enzyme. We demonstrate that, although the closed conformation always predominates in the conformational landscape, the population of open state increases by order of magnitude upon the formation of the disemiquinone state. Our results are consistent with several open conformation sub-states differing in the volume change (ΔV⁰) of the opening transition. While the ΔV⁰ characteristic of the oxidized enzyme is as large as −88 mL/mol, the interaction of the enzyme with the nucleotide cofactor and the formation of the double-semiquinone state of CPR decrease this value to −34 and −18 mL/mol, respectively. This observation suggests that the interdomain electron transfer in CPR increases protein hydration, while promoting more open conformation. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions. Full article

Lactoperoxidase (LPO) together with its (pseudo)halogenation cycle substrates, H₂O₂ and thiocyanate ions oxidized to hypothiocyanite ions, form one of the main systems involved in antimicrobial defense within the oral cavity. In bacterial diseases such as dental caries, lactoperoxidase is oxidized to a form known as Compound II, which is characterized by its inability to oxidize SCN^–, resulting in a decreased generation of antimicrobial products. Reynoutria sp. rizome extracts, due to their high polyphenol content, have been tested as a source of compounds able to regenerate the antimicrobial activity of lactoperoxidase through converting the Compound II to the native LPO state. In the presented study, acetone extracts of R. japonica, R. sachalinensis, and R. x bohemica, together with their five fractions and four selected polyphenols dominating in the studied in extracts, were tested toward lactoperoxidase reactivating potential. For this purpose, IC50, EC50, and activation percentage were determined by Ellman’s method. Furthermore, the rate constants for the conversion of Compound I–Compound II and Compound II–native-LPO in the presence of extracts, extracts fractions, and selected polyphenols were determined. Finally, the ability to enhance the antimicrobial properties of the lactoperoxidase system was tested against Streptococcus mutans. We proved that Reynoutria sp. rhizome is the source of lactoperoxidase peroxidation cycle substrates, which can act as activators and inhibitors of the antimicrobial properties of that system. The presented study shows that the reactivation of lactoperoxidase could become a potential therapeutic target in prevention and treatment support in some infectious oral diseases. Full article

Understanding the origin of reactive species following ionization in aqueous systems is an important aspect of radiation–matter interactions as the initial reactive species lead to production of radicals and subsequent long-term radiation damage. Tunable ultrafast X-ray free-electron pulses provide a new window to probe events occurring on the sub-picosecond timescale, supplementing other methodologies, such as pulse radiolysis, scavenger studies, and stop flow that capture longer timescale chemical phenomena. We review initial work capturing the fastest chemical processes in liquid water radiolysis using optical pump/X-ray probe spectroscopy in the water window and discuss how ultrafast X-ray pump/X-ray probe spectroscopies can examine ionization-induced processes more generally and with better time resolution. Ultimately, these methods will be applied to understanding radiation effects in complex aqueous solutions present in high-level nuclear waste. Full article

The country or mine of origin is an important economic and societal issue inherent in the diamond industry. Consumers increasingly want to know the provenance of their diamonds to ensure their purchase does not support inhumane working conditions. Governments around the world reduce the flow of conflict diamonds via paper certificates through the Kimberley Process, a United Nations mandate. However, certificates can be subject to fraud and do not provide a failsafe solution to stopping the flow of illicit diamonds. A solution tied to the diamonds themselves that can withstand the cutting and manufacturing process is required. Here, we show that multivariate analysis of LIBS (laser-induced breakdown spectroscopy) diamond spectra predicts the mine of origin at greater than 95% accuracy, distinguishes between natural and synthetic stones, and distinguishes between synthetic stones manufactured in different laboratories by different methods. Two types of spectral features, elemental emission peaks and emission clusters from C-N and C-C molecules, are significant in the analysis, indicating that the provenance signal is contained in the carbon structure itself rather than in inclusions. Full article

Nodulin 26-like intrinsic proteins (NIPs) of the plant aquaporin family majorly facilitate the transport of physiologically relevant solutes. The present study intended to investigate how substrate selectivity in grapevine NIPs is influenced by the aromatic/arginine (ar/R) selectivity filter within the pore and the possible underlying mechanisms. A mutational approach was used to interchange the ar/R residues between grapevine NIPs (VvTnNIP1;1 with VvTnNIP6;1, and VvTnNIP2;1 with VvTnNIP5;1). Their functional characterization by stopped-flow spectroscopy in Saccharomyces cerevisiae revealed that mutations in residues of H2/H5 helices in VvTnNIP1;1 and VvTnNIP6;1 caused a general decline in membrane glycerol permeability but did not impart the expected substrate conductivity in the mutants. This result suggests that ar/R filter substitution could alter the NIP channel activity, but it was not sufficient to interchange their substrate preferences. Further, homology modeling analyses evidenced that variations in the pore radius combined with the differences in the channel’s physicochemical properties (hydrophilicity/hydrophobicity) may drive substrate selectivity. Furthermore, yeast growth assays showed that H5 residue substitution alleviated the sensitivity of VvTnNIP2;1 and VvTnNIP5;1 to As, B, and Se, implying importance of H5 sequence for substrate selection. These results contribute to the knowledge of the overall determinants of substrate selectivity in NIPs. Full article