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Abstract The main protease (Mpro) of SARS-CoV-2 is critical for viral function and a key drug target. Mpro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized Mpro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. Mpro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein’s crystallization behavior is linked to its redox state. Oxidized Mpro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of Mpro and the crystallization conditions necessary to study this structurally.

NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

Serial crystallography (SX) has become an established technique for protein structure determination, especially when dealing with small or radiation-sensitive crystals and investigating fast or irreversible protein dynamics. The advent of newly developed multi-megapixel X-ray area detectors, capable of capturing over 1000 images per second, has brought about substantial benefits. However, this advancement also entails a notable increase in the volume of collected data. Today, up to 2 PB of data per experiment could be easily obtained under efficient operating conditions. The combined costs associated with storing data from multiple experiments provide a compelling incentive to develop strategies that effectively reduce the amount of data stored on disk while maintaining the quality of scientific outcomes. Lossless data-compression methods are designed to preserve the information content of the data but often struggle to achieve a high compression ratio when applied to experimental data that contain noise. Conversely, lossy compression methods offer the potential to greatly reduce the data volume. Nonetheless, it is vital to thoroughly assess the impact of data quality and scientific outcomes when employing lossy compression, as it inherently involves discarding information. The evaluation of lossy compression effects on data requires proper data quality metrics. In our research, we assess various approaches for both lossless and lossy compression techniques applied to SX data, and equally importantly, we describe metrics suitable for evaluating SX data quality.

The study is aimed to identify urgent problems in the field of communication between young men and women and other significant participants in unregulated interaction. Using standardised test methods, questionnaires were adapted for this study and were collected the primary material that reflects the essential characteristics of unregulated communication between high school students in Russia. The sample of the study was 378 people. The main results were the data indicating a serious gap between high school students' need to communicate and the actual satisfaction of this need in the interaction with other significant participants. The main meaning of young people's need for trustful reference unregulated communication is not so much the communicative component as the emotional-affective content, which consists of acceptance, support, emotional exchange and understanding on the part of other people. Currently, there is a gap between the need for unregulated confidential communication of young men and women with significant adults and the real system of such communication. The results were put into the development of recommendations that allow psychologists to build an optimal interaction with family systems and the pedagogical community to optimise communication between high school students. As a prospect for further research, the tasks of developing parent-child relationships are identified.

Background Management of bone defects with autologous bone grafting has always been the "gold standard" but it is not always possible to use it for a number of reasons. Preprocessed materials of biological and non-biological origin were developed as an alternative. A new branch of these materials is tissue-engineered constructs that fully imitate autologous bone in required volume. Aim is to study in vivo the possibility of using deproteinized human cancellous bone tissue as a matrix for creating tissue-engineered constructs. Methods The study was carried out on 24 NZW line rabbits, since this line has a fully characterized stromal-vascular fraction formula (SVF). The study design included 3 groups. Fiкst group (control) had surgical modeling of bone defects in the diaphysis of the contralateral femur without reconstruction; Group 2 had bone defect reconstruction using fragments of a deproteinized cancellous bone graft; group 3 underwent bone defect reconstruction using fragments of deproteinized cancellous bone matrix along with the autologous adipose tissue SVF (obtained according to ACP SVF technology). Animals were sacrificed with ether anesthesia at 2, 4 and 6 weeks after the operation and subsequent histological study followed. Result During all periods of the study, the newly formed bone tissue volume density in the 3rd group (reconstruction with deproteinized human cancellous bone + stromal-vascular fraction) was 1.78 times higher (p < 0.001) than in the first group (bone defect without reconstruction), 1.21 times higher (p < 0.001) than in the 2nd group (reconstruction with deproteinized cancellous bone alone). The dynamics of changes in the mature bone tissue volume density was similar to those of the newly formed bone tissue. Discussion The comparative analysis of reparative processes using a tissue engeneered construst based on deproteinized cancellous human bone with adipose tissue stromal vascular fraction revealed that the use of these bone substitute materials contributes not only to the early activation of reparative regeneration of the main structural elements of bone tissue at the site of bone defect, but also their timely differentiation. Conclusion The use of deproteinized cancellous bone matrix combined with stromal-vascular fraction to create a tissue-engineered construct could unleash several regeneration mechanisms and accelerate the process of bone defect site repair, compared with 1st and 2nd group of study.

Background. Pigment concentration is a species trait, and is an integral indicator of adaptive response to changes in environmental factors and the growing conditions. The intensive development of modern cities imposes certain requirements on the selection of plants for landscaping, one of which is a high level of adaptive responses. Purpose. The purpose of this study was to determine the content of photosynthetic pigments in the leaves of the genus Acer species in urban conditions of the city of Maуkop, the Adygea Republic. Materials and methods. We used the spectrophotometric method to determine the content of photosynthetic pigments in the leaves of the genus Acer L. species: A. platanoides, A. pseudoplatanus, A. saccharinum, A. negundo, A. campestre in an urban environment of the city of Maуkop and the zone of conditional control (Botanical garden). We carried out statistical processing of experimental data by using the MS Excel 2010 package. Results. We have revealed that in the conditions of an urbanized environment the total content of chlorophylls is 1.1-2.2 times higher in comparison with that of the zone of control. Taking into account the level of variability of the total content of green pigments in the city, we arranged the studied species in the following order: A. saccharinum A. campestre ˂ A. pseudoplatanus ˂ A. platanoides ˂ A. negundo. The ratio of the amount of chlorophylls to carotenoids reacted very sensitively to the impact of urban environmental factors. A. campestre and A. saccharinum differed from A. negundo, A. pseudoplatanus, and A. platanoides for this indicator. Conclusion. The study made allow us to state that in the conditions of an urbanized environment, the studied species of the genus Acer undergo restructuring in the structure of pigment complexes. An increase in the synthesis of chlorophylls and carotenoids raises their adaptive capabilities to the negative impact of urban environmental factors.

The paper presents results of the desktop analysis on the current status of the problem of the personal potential in terms of psychology. It provides the author's understanding of the term as the formation consistent and structured in its psychological aspect. This is the analysis of the characteristics that the career choice has and the difficulties that the graduates face in this process. We believe that substantial resources for the independent and responsible career choice are inherent in the personal potential. According to the research methods, the structure of the personal potential includes intra and interpsychological characteristics. Internal factors relate to the non-acceptance of the choice situation, the lack of skills that are necessary for subjective construction of its process. They also come from hesitation, lack of confidence, poor self-concept, internal conflicts, low community commitment, etc. In most cases, the powerful influence of relevant adults is an external obstacle. The data obtained justify a need in psychological and education counseling to develop the personal potential of graduates. In this regard, one of the main tasks is to provide graduates with competent and comprehensive support to overcome difficulties in the process of making the career choice and personal potential development.

Abstract For decades, researchers have elucidated essential enzymatic functions on the atomic length scale by tracing atomic positions in real-time. Our work builds on possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals. Here, we report in atomic detail (between 2.2 and 2.7 Å resolution) by room-temperature, time-resolved crystallography with millisecond time-resolution (with timepoints between 3 ms and 700 ms) how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating, cooperativity, induced fit, and conformational selection all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme noncovalently before reacting to a trans-enamine. This was made possible in part by the application of singular value decomposition to the MISC data using a program that remains functional even if unit cell parameters change up to 3 Å during the reaction.

Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme-ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.

Background. At present, for a number of reasons the complete bone defect replacement with autogenous bone is not always possible. Bone substitute materials are used as an alternative to autogenous bone tissue and can be of either biological or non-biological origin. One of the ways of development of reconstructive technologies is the use of tissue-engineered constructs that fully imitate autogenous bone tissue in the required volume. Aim of study to define in vivo the possibility of using deproteinized human cancellous bone tissue as a matrix for creating tissue-engineered constructs. Methods. An in vivo study was carried out on NZW rabbits. To create a construct, we used the fragments of deproteinized cancellous bone tissue of the human femoral head and stromal vascular fraction of rabbit adipose tissue as a matrix. Bone defect modeling with its subsequent replacement was performed to evaluate the efficacy of reparative osteogenesis during bone defects reconstruction. Study groups were defined: group 1 (control) surgical modeling of a bone defect of the femur without its reconstruction; group 2 surgical modeling of a bone defect of the femur with its reconstruction using fragments of deproteinized cancellous bone matrix; group 3 surgical modeling of a bone defect of the femur with its reconstruction using fragments of deproteinized cancellous bone matrix in combination with stromal vascular fraction of adipose tissue (according to ACP SVF technology). Results. Comparative analysis of reparative processes in case of applying tissue-engineered constructs based on deproteinized human cancellous bone matrix in combination with adipose tissue-derived stromal vascular fraction on in vivo experimental model revealed that the use of these bone substitute materials contributes not only to an early activation of reparative regeneration of main structural elements of the bone tissue in the area of the bone defect replacement, but also to its well-timed differentiation. This determines the restoration of structural and functional viability of the bone tissue at the damage site without developing discernible reactive inflammation. Moreover, the effect of the selected tissue-engineered construct with the combined influence of several factors (ACP SVF) in its composition turned out to be more effective in stimulating bone tissue repair and differentiation. Conclusion. Combination of SVF and deproteinized bone matrix for creating tissue-engineered constructs enables to engage several regeneration mechanisms and accelerate the process of bone defect replacement in comparison with isolated deproteinized bone matrix without bone defect reconstruction.

Metabolic syndrome is a complex of abnormalities involving impaired glucose and lipid metabolism, which needs effective pharmacotherapy. One way to reduce lipid and glucose levels associated with this pathology is the simultaneous activation of nuclear PPAR-alpha and gamma. For this purpose, we synthesized a number of potential agonists based on the pharmacophore fragment of glitazars with the inclusion of mono- or diterpenic moiety in the molecular structure. The study of their pharmacological activity in mice with obesity and type 2 diabetes mellitus (C57Bl/6Ay) revealed one substance that was capable of reducing the triglyceride levels in the liver and adipose tissue of mice by enhancing their catabolism and expressing a hypoglycemic effect connected with the sensitization of mice tissue to insulin. It has also been shown to have no toxic effects on the liver.

In paediatrics, it is a common practice to use pharmaceuticals outside the approved Summary of Product Characteristics (i.e. off-label). According to the literature, up to 45% of inpatient paediatric prescriptions and 10–20% of outpatient ones are for off-label uses. It is essential to analyse such uses, as it helps medical practitioners act reasonably and professionally.The aim of the study was to explore the possibility of using spontaneous reports to assess the risks of the off-label use of cardiac medicinal products in children in the Irkutsk region.Materials and methods. The authors analysed 25 reporting forms on adverse drug reactions (ADRs) in paediatric cardiac patients from the database of the Regional Centre for Drug Safety Monitoring of the Irkutsk Region and regional data from the Automated Information System (AIS) of the Federal Service for Surveillance in Healthcare of the Russian Federation (Roszdravnadzor) submitted in 2009–2020. The inclusion criterion for reporting forms was a causal relationship between the off-label use of a medicinal product and the ADR that was scored as “possible” or higher on the Naranjo probability scale.Results. According to the reporting forms, the off-label use of cardiac medicinal products in children was associated with ADRs, such as angiooedema, cutaneous symptoms, and bronchospasm. The majority of ADRs (84%) were considered severe. The medicinal products were prescribed for heart failure, arterial hypertension, and cardiac arrhythmias. The majority of ADR reports (75%) were submitted by inpatient medical organisations.Conclusions. The analysis of spontaneous ADR reporting databases is a simple and informative method for studying the safety of medicinal products. It is necessary to raise awareness of ADRs associated with off-label prescribing amongst paediatric cardiologists in order to reduce the incidence of ADRs. The following contributions will improve the quality of medical care: all parties involved with pharmaceuticals should adhere to good pharmacovigilance practices, medical practitioners should be actively involved in treatment safety monitoring, and the professional training curricula of paediatric cardiologists and paediatricians should include specific units on the practical functioning of pharmacovigilance.

Nimesulide (NIM) is a nonsteroid anti-inflammatory drug which acts as a selective cyclooxygenase 2 inhibitor and is widely used for acute pain treatment. In medical practice, a large amount of data has been collected describing the effect of NIM on the body, while a hepatotoxic side effect of the drug has been found. The exact mechanisms of such NIM-induced hepatotoxicity largely remain unknown but likely involve the intermediate reaction of its metabolism. Reduction of the hepatotoxic side effect of NIM is an actual problem for pharmacology. The aim of the present research was to evaluate the hepatotoxicity of the mechanochemically obtained composition of NIM with glycyrrhizic acid disodium salt (Na2GA) compared to pure NIM and a physical mixture of NIM with Na2GA. Material and methods. CD-1 mice were orally administered for 14 days: 1 group – mechanochemical composition NIM/Na2GA (1:10, m/m) at a dose of 1650 mg/kg; 2 group – physical mixture of NIM with Na2GA (1:10, m/m) at a dose of 1650 mg/kg; 3 group – pure NIM at a dose of 600 mg/kg (which pharmacokinetically corresponds to 1650 mg/kg of NIM/Na2GA); 4 group – vehicle (distilled water). The liver damage was assessed using histological studies and enzymatic activity of the alanine aminotransferase and aspartate aminotransferase in blood serum. Results. Histological analysis did not detect any changes in the liver of NIM/Na2GA-treated animals in comparison with a water-treated group. On the opposite, NIM given alone or as a physical mixture with Na2GA induced severe hepatotoxicity in experimental mice. Biochemical analysis of the blood serum revealed that mechanochemical NIM/Na2GA composition significantly reduced activity of the alanine aminotransferase (about 1.5 times) and aspartate aminotransferase (1.3 times) as compared with the pure NIM. Conclusions. The results obtained indicate a high potential for the practical application of the NIM/Na2GA mechanochemical composition.

Highly resistant to reduction nitroxides open new opportunities for structural studies of biological macromolecules in their native environment inside living cells and for functional imaging of pH and thiols, enzymatic activity and redox status in living animals. 3,4-Disubstituted nitroxides of 2,2,5,5-tetraethylpyrrolidine and pyrroline series with a functional group for binding to biomolecules and a polar moiety for higher solubility in water and for more rigid attachment via additional coordination to polar sites were designed and synthesized. The EPR spectra, lipophilicities, kinetics of the reduction in ascorbate-containing systems and the decay rates in liver homogenates were measured. The EPR spectra of all 3,4-disubstituted pyrrolidine nitroxides showed additional large splitting on methylene hydrogens of the ethyl groups, while the spectra of similar pyrroline nitroxides were represented with a simple triplet with narrow lines and hyperfine structure of the nitrogen manifolds resolved in oxygen-free conditions. Both pyrrolidine and pyrroline nitroxides demonstrated low rates of reduction with ascorbate, pyrrolidines being a bit more stable than similar pyrrolines. The decay of positively charged nitroxides in the rat liver homogenate was faster than that of neutral and negatively charged radicals, with lipophilicity, rate of reduction with ascorbate and the ring type playing minor role. The EPR spectra of N,N-dimethyl-3,4-bis-(aminomethyl)-2,2,5,5-tetraethylpyrrolidine-1-oxyl showed dependence on pH with pKa = 3, ΔaN = 0.055 mT and ΔaH = 0.075 mT.

The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. Here, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. The results connect the previously explored femtosecond PYP dynamics to timescales accessible at synchrotrons. This opens the door to a wide range of time-resolved studies at the EuXFEL.

A novel free fatty acid receptor 1 (FFAR1) agonist has been synthesized and evaluated in vitro. The synthesis of the title compound was performed from commercially available 4-hydroxybenzaldehyde, 2-thiophenecarboxaldehyde, and (+)-camphor. The compound was shown to have an affinity to FFAR1.

The aerosol inhalation delivery of composite particles consisting of Ag nanoparticles enveloped by polyvinylpyrrolidone was investigated in experiments with mice. An ultrasonic nebulizing system was created for the generation of aerosols with a mean diameter and mass concentration of 700 ± 50 nm and 65 ± 5 mg/m3, respectively. The mass fraction of Ag in the composite particles was α = 0.061. The aerosol delivery was performed in a whole-body chamber with an exposition time of 20 min. Pharmacokinetic measurements were taken and the silver concentrations in the blood and lungs of the mice were measured as a function of time after exposition by means of electrothermal (graphite furnace) atomic absorption spectrometry. The inhalation dose and other pharmacokinetic parameters were determined. The antibacterial effect of aerosolized silver was assessed for mice infected with Klebsiella pneumoniae 82 and Staphylococcus aureus ATCC 25953. The survival rate of the infected mice after the aerosol exposure demonstrated the high antibacterial efficiency of Ag nanoparticles after inhalation delivery.

One of the little-studied ways that climate warming or temperature increases in aquaculture could affect aquatic animals is through accelerated aging. This study is dedicated to understanding the principles of molecular and cellular aging in the target tissues of juvenile whitefishes (Yenisei hump-snout whitefish and its hybrid) under the influence of acute heat stress (up to 26 °C), and the effects of thermal preconditioning as pre-adaptation. Non-adapted stressed hump-snout whitefish showed a higher induction threshold for functionally active mitochondria in the blood and a decrease in telomerase activity in the liver after heat shock exposure as a long-term compensatory response to prevent telomere shortening. However, we observed heat-induced telomere shortening in non-adapted hybrids, which can be explained by a decrease in mitochondrial membrane stability and a gradual increase in energy demand, leading to a decrease in protective telomerase activity. The pre-adapted groups of hump-snout whitefish and hybrids showed a long-term or delayed response of telomerase activity to heat shock, which served as a therapeutic mechanism against telomere shortening. We concluded that the telomerase and telomere responses to thermal stress demonstrate plasticity of tolerance limits and greater stability in hump-snout whitefish compared with hybrids.

Berberine is a phytogenic isoquinoline alkaloid which demonstrates several pharmacological effects, including a hypoglycemic effect. Its medical use is limited by its very low bioavailability. Synthesizing new berberine derivatives might help in overcoming this problem. In this work, we report on the synthesis and biological evaluation of a novel berberine 9-O-derivative. At an oral dose of 25 mg/kg, the compound demonstrated hypoglycemic activity in an oral glucose tolerance test performed using C57BL/6 mice.

Derivatives of dihydropyridines are promising agents for research, as they have high biological activity. Their low toxicity and polytargeted properties make it possible to create new derivatives and identify new biological activity. As part of this work, we have obtained six new fluorinated derivatives of 4-phenyl-1,4-dihydropyridines. These compounds were synthesized by a modified Hantzsch reaction, the interaction of polyfluorinated sulfur-containing benzaldehydes with ethyl acetoacetate and ethyl 3-aminobut-2-enoate. Studies on the isolated atrium have shown that the possible mechanism of action of the dihydropyridine derivatives lies in the blockade of beta-adrenergic receptors. At the same time, it is not a blocker of potassium and calcium channels. The fluorinated 4-phenyl-1,4-dihydropyridine derivatives, with SCF2H (decrease pressure) and SO2CH3 (increase pressure) groups showed pronounced activity in the study of these derivatives on the blood pressure of rats.