Design of stable circular permutants of the GroEL chaperone apical domain.

Enhancing protein stability holds paramount significance in biotechnology, therapeutics, and the food industry. Circular permutations offer a distinctive avenue for manipulating protein stability while keeping intra-protein interactions intact. Amidst the creation of circular permutants, determining the optimal placement of the new N- and C-termini stands as a pivotal, albeit largely unexplored, endeavor. In this study, we employed PONDR-FIT's predictions of disorder propensity to guide the design of circular permutants for the GroEL apical domain (residues 191-345). Our underlying hypothesis posited that a higher predicted disorder value would correspond to reduced stability in the circular permutants, owing to the increased likelihood of fluctuations in the novel N- and C-termini. To substantiate this hypothesis, we engineered six circular permutants, positioning glycines within the loops as locations for the new N- and C-termini. We demonstrated the validity of our hypothesis along the set of the designed circular permutants, as supported by measurements of melting temperatures by circular dichroism and differential scanning microcalorimetry. Consequently, we propose a novel computational methodology that rationalizes the design of circular permutants with projected stability. Video Abstract.

The effect of oral administration of zeolite on the energy metabolism and reproductive health of Romanian spotted breed in advanced gestation and post partum period.

The dairy cow experiences the most significant impact from negative energy balance during this period, which adversely affects reproductive health. Consequently, most pathologies affect dairy cows during this time frame. Thus, with the primary objective of reducing the incidence of these pathologies on dairy farms, we questioned whether supplemental zeolite administration in cattle feed would affect metabolism and reproductive health. Therefore, we proposed introducing an antepartum and postpartum supplementation of 400 g of zeolite in the basal diet. The control group received only the basal diet without zeolite supplementation. Monitoring the results stemmed from the consideration that reproductive health can only be present based on an unaltered energy metabolism. Hence, we deemed it necessary to analyze several metabolic markers in light of the expected outcomes concerning reproductive health. Cows treated with zeolite exhibited a calving to first service interval 12.78 days earlier than those in the control group. Moreover, the average number of services per conception used for future gestation was 0.44 lower in the zeolite-treated group compared to the control group (p<0.05). Additionally, the treatment group showed a lower presence of pathogens in the uterus and displayed a more favorable average uterine score. Observations following the completion of the research point towards an improvement in the health of transition dairy cows, opening a new path for dairy farms in terms of preventing postpartum pathologies. Indeed, the benefits from this study primarily impact the animals rather than directly influencing milk production. Therefore, further research is necessary in this regard.

A New Method for Tungsten Oxide Nanopowder Deposition on Carbon-Fiber-Reinforced Polymer Composites for X-ray Attenuation.

A new method for the synthesis and deposition of tungsten oxide nanopowders directly on the surface of a carbon-fiber-reinforced polymer composite (CFRP) is presented. The CFRP was chosen because this material has very good thermal and mechanical properties and chemical resistance. Also, CFRPs have low melting points and are transparent under ionized radiation. The synthesis is based on the direct interaction between high-power-density microwaves and metallic wires to generate a high-temperature plasma in an oxygen-containing atmosphere, which afterward condenses as metallic oxide nanoparticles on the CFRP. During microwave discharge, the value of the electronic temperature of the plasma, estimated from Boltzmann plots, reached up to 4 eV, and tungsten oxide crystals with a size between 5 nm and 100 nm were obtained. Transmission electron microscopy (TEM) analysis of the tungsten oxide nanoparticles showed they were single crystals without any extended defects. Scanning electron microscopy (SEM) analysis showed that the surface of the CFRP sample does not degrade during microwave plasma deposition. The X-ray attenuation of CFRP samples covered with tungsten oxide nanopowder layers of 2 µm and 21 µm thickness was measured. The X-ray attenuation analysis indicated that the thin film with 2 µm thickness attenuated 10% of the photon flux with 20 to 29 KeV of energy, while the sample with 21 µm thickness attenuated 60% of the photon flux.

Correction: Finkelstein et al. How Can Ice Emerge at 0 °C? Biomolecules 2022, 12, 981.

We regret to state that our article "How Can Ice Emerge at 0 °C?" [...].

Utilizing Extraepitopic Amino Acid Substitutions to Define Changes in the Accessibility of Conformational Epitopes of the Bacillus cereus HlyII C-Terminal Domain.

Hemolysin II (HlyII)-one of the pathogenic factors of Bacillus cereus, a pore-forming ß-barrel toxin-possesses a C-terminal extension of 94 amino acid residues, designated as the C-terminal domain of HlyII (HlyIICTD), which plays an important role in the functioning of the toxin. Our previous work described a monoclonal antibody (HlyIIC-20), capable of strain-specific inhibition of hemolysis caused by HlyII, and demonstrated the dependence of the efficiency of hemolysis on the presence of proline at position 324 in HlyII outside the conformational antigenic determinant. In this work, we studied 16 mutant forms of HlyIICTD. Each of the mutations, obtained via multiple site-directed mutagenesis leading to the replacement of amino acid residues lying on the surface of the 3D structure of HlyIICTD, led to a decrease in the interaction of HlyIIC-20 with the mutant form of the protein. Changes in epitope structure confirm the high conformational mobility of HlyIICTD required for the functioning of HlyII. Comparison of the effect of the introduced mutations on the effectiveness of interactions between HlyIICTD and HlyIIC-20 and a control antibody recognizing a non-overlapping epitope enabled the identification of the amino acid residues N339 and K340, included in the conformational antigenic determinant recognized by HlyIIC-20.

Development and application of an RT‒PCR assay for the identification of the delta and omicron variants of SARS-COV-2.

The emergence of mutations in the coronavirus genome provides opportunities for occurrence new strains with higher transmissibility, severity and duration of the disease poses. In 2020, a new variant of the coronavirus SARS-COV-2 - Delta was identified in India. This genetic variant has spread rapidly and became dominant in many countries, including Russia. In November 2021, a new outbreak of COVID-19 occurred in Africa driven by a variant SARS-COV-2 named later Omicron. Both variants had increased transmissibility compared to previously encountered variants and quickly, replacing its around the world. To promptly monitor the epidemiological situation in the country, to assess the spread of dominant genetic variants of the virus and to take appropriate measures, we have developed an RT‒PCR reagent kit for the identification of Delta and Omicron by detecting a corresponding combination of major mutations. The minimum set of mutations was chosen which allows to differentiate Delta and Omicron variants, in order to increase the analysis productivity and reduce costs. Primers and LNA-modified probes were selected to detect mutations in the S gene, typical for the Delta and Omicron. Similar approach can be implemented for the rapid development of assays for differentiating important SARS-COV-2 variants or for other viruses genotyping for epidemiological surveillance or for diagnostic use in order to assist in making clinical decisions. It was demonstrated that the results of VOC Delta and Omicron detection and their typical mutations were concordant with genotyping based on WGS results for all 847 samples of SARS-CoV-2 RNA. The kit has high analytical sensitivity (1х103 copies/mL of SARS-CoV-2 RNA) for each of the detected genetic variants and possesses 100% analytic specificity for microorganism panel testing. The diagnostic sensitivity (95% confidence interval) obtained during pivotal trials was 91.1-100% for Omicron and 91.3-100% for Delta, while the diagnostic specificity with a 95% confidence interval was 92.2-100%. The use of a set of reagents in combination with sequencing of SARS-CoV-2 RNA as part of epidemiological monitoring made it possible to quickly track the dynamics of changes in Delta and Omicron prevalence in the Moscow region in the period from December 2021 to July 2022.

DETERMINATION OF SOMATIC, NEUROLOGICAL AND PSYCHO-EMOTIONAL MANIFESTATIONS OF THE ACUTE AND POSTCOVID PERIOD IN PATIENTS WITH A MILD COURSE OF COVID-19 IN WARTIME.

OBJECTIVE: The aim: To analyze the typical symptom complex at the stage of COVID-19 acute phase in the systemic relationship with somatic, psychosomatic, and neurological manifestations. PATIENTS AND METHODS: Materials and methods: The collection of primary material was performed by clinical-anamnestic method, laboratory, and sociological examination of patients treated out patiently. Summarizing of the results was performed according to the analysis of 100 completed cases of COVID-19 in patients aged 35-45 years (50 men and 50 women) who had no concomitant chronic pathology, and patients did not receive any vaccine dose before the disease (acute COVID-19) and during the next follow-up period (6 months). RESULTS: Results: The data of the analysis allowed us to make a grounded conclusion about the syndromic heterogeneity of COVID 19 in a standardized patients group with a mild course. CONCLUSION: Conclusions: the highest number of symptoms in the postcovid period by frequency, polymorphism, and life quality impact was found in the group of patients with subjectively tolerate acute COVID-19 most easily. Patients whose acute episode meets the mild criteria have pronounced neurological and psychoemotional manifestations during the postcovid period.

FUNCTIONAL CHARACTERISTICS OF THE CARDIOVASCULAR SYSTEM OF PATIENTS WITH ISCHEMIC HEART DISEASE WITH OBESITY.

OBJECTIVE: The aim: To determine the features of the functional characteristics of the cardiovascular system of patients with ischemic heart disease with obesity. PATIENTS AND METHODS: Materials and methods: Examined 130 persons (mostly military personnel and persons who were in the zone of active hostilities): 65 patients (the main group, 62,67±8,93 years) with coronary heart disease and obesity and 45 people of the control group (virtually healthy people, randomized by age and sex, 58,76±14,6 years). RESULTS: Results: Coronary heart disease and obesity compared to healthy individuals probably the exceed all values of the functional state of the cardiovascular system: systolic blood pressure (152.72±14.61 and 119.03±7.94 mmHg; p<0.001); diastolic blood pressure (90.74±7.36 and 80.36±6.74 mmHg; p<0.001); end-diastolic volume (103.17±40.84 and 52.48±8.58 mm3; р<0.001); end-systolic volume (47.98±29.92 and 31.47±8.42 mm3; р=0.001); end-diastolic size (4.74±0.81 and 4.12 ± 0.27 cm; р<0.001); end-systolic size (3.34±0.76 and 3.17±0.59 cm; р=0.014). CONCLUSION: Conclusions: The identified functional disorders of the heart in the comorbid course of coronary heart disease and obesity can be used for early diagnosis of cardiovascular complications in such patients and for the development of adequate therapeutic schemes.

An Occlusive Form of Acute Gangrenous Appendicitis With Periappendicular Abscess in an Elderly Patient: A Case Report and Literature Review.

Acute appendicitis represents one of the common causes of admission to the emergency department. In rare cases, patients with appendicitis can suffer complications such as intestinal obstruction. These particular cases of occlusive appendicitis with a periappendicular abscess usually occur in elderly patients and can develop in an aggressive form, nonetheless with a favorable evolution. We present a case of an 80-year-old male patient, reporting symptoms similar to an occlusive digestive pathology: abdominal pain, intestinal transit disorders, and fecal vomiting. A computerized tomography scan suggested a mechanical bowel obstruction. The patient had an exploratory laparotomy indication to find the cause of the obstruction. The peritoneal cavity inspection revealed an occlusive form of acute gangrenous appendicitis with a periappendicular abscess. An appendectomy was performed. In conclusion, as surgeons, we must always take into consideration that acute appendicitis can represent a cause of intestinal obstruction, especially in elderly patients.

Lane nucleation in complex active flows.

Laning is a paradigmatic example of spontaneous organization in active two-component flows that has been observed in diverse contexts, including pedestrian traffic, driven colloids, complex plasmas, and molecular transport. We introduce a kinetic theory that elucidates the physical origins of laning and quantifies the propensity for lane nucleation in a given physical system. Our theory is valid in the low-density regime, and it makes different predictions about situations in which lanes may form that are not parallel with the direction of flow. We report on experiments with human crowds that verify two notable consequences of this phenomenon: tilting lanes under broken chiral symmetry and lane nucleation along elliptic, parabolic, and hyperbolic curves in the presence of sources or sinks.

Physical literacy in Europe: The current state of implementation in research, practice, and policy.

Background/objective: The holistic concept of physical literacy (PL) embraces different person-centered qualities (physical, cognitive, affective/psychological) necessary to lead physically active lifestyles. PL has recently gained increasing attention globally and Europe is no exception. However, scientific endeavors summarizing the current state of PL in Europe are lacking. Therefore, the goal of this study was to comprehensively assess and compare the implementation of PL in research, policy, and practice across the continent. Methods: We assembled a panel of experts representing 25 European countries. Employing a complementary mixed-methods design, the experts first prepared reviews about the current state of PL in their countries (categories: research, practice/policy). The reviews underwent comparative document analysis, ensuring a transnational four-eyes principle. For re-validation purposes, the representatives completed a quantitative survey with questions reflecting the inductive themes from the document analysis. Results: The document analysis resulted in ten disjunct themes (related to "concept", "research", "practice/policy", "future/prospect") and yielded a heterogenous PL situation in Europe. The implementation state was strongly linked to conceptual discussions (e.g., existence of competing approaches), linguistic issues (e.g., translations), and country-specific traditions. Despite growing scholarly attention, PL hesitantly permeates practice and policy in most countries. Nevertheless, the experts largely anticipate increasing popularity of PL for the future. Conclusion: Despite the heterogeneous situation across Europe, the analysis has uncovered similarities among the countries, such as the presence of established yet not identical concepts. Research should intensify academic activities (conceptual-linguistic elaborations, empirical work) before PL may gain further access into practical and political spheres in the long term.

Physics of Ice Nucleation and Antinucleation: Action of Ice-Binding Proteins.

Ice-binding proteins are crucial for the adaptation of various organisms to low temperatures. Some of these, called antifreeze proteins, are usually thought to inhibit growth and/or recrystallization of ice crystals. However, prior to these events, ice must somehow appear in the organism, either coming from outside or forming inside it through the nucleation process. Unlike most other works, our paper is focused on ice nucleation and not on the behavior of the already-nucleated ice, its growth, etc. The nucleation kinetics is studied both theoretically and experimentally. In the theoretical section, special attention is paid to surfaces that bind ice stronger than water and thus can be "ice nucleators", potent or relatively weak; but without them, ice cannot be nucleated in any way in calm water at temperatures above -30 °C. For experimental studies, we used: (i) the ice-binding protein mIBP83, which is a previously constructed mutant of a spruce budworm Choristoneura fumiferana antifreeze protein, and (ii) a hyperactive ice-binding antifreeze protein, RmAFP1, from a longhorn beetle Rhagium mordax. We have shown that RmAFP1 (but not mIBP83) definitely decreased the ice nucleation temperature of water in test tubes (where ice originates at much higher temperatures than in bulk water and thus the process is affected by some ice-nucleating surfaces) and, most importantly, that both of the studied ice-binding proteins significantly decreased the ice nucleation temperature that had been significantly raised in the presence of potent ice nucleators (CuO powder and ice-nucleating bacteria Pseudomonas syringae). Additional experiments on human cells have shown that mIBP83 is concentrated in some cell regions of the cooled cells. Thus, the ice-binding protein interacts not only with ice, but also with other sites that act or potentially may act as ice nucleators. Such ice-preventing interaction may be the crucial biological task of ice-binding proteins.

Synthesis and Photophysical Properties of Novel Meta-Conjugated Organic Molecules with 1,3,5-Benzene Branching Units.

The synthesis and photophysical investigation of three novel meta-conjugated molecules based on 3,1,2-benzothiadiazole and thiophene-2,5-diyl derivatives linked through 1,3,5-benzene branching units are described. Each of them is a symmetrical molecule with two branching units, four identical lateral thiophene-containing fragments, and one central benzothiadiazole-containing fragment. To study the effect of the chemical structure on their photophysical properties, the molecules with different linearly conjugated lateral and central fragments due to incorporation of additional thiophene rings were synthesized and compared. It was shown that absorption spectra of the meta-conjugated molecules can be represented as a sum of absorption bands of model compounds for their peripheral and central fragments containing a common benzene ring being branched at the 1,3,5-benzene unit in the meta-conjugated molecules. Therefore, they cannot be considered simply as isolated π-conjugated systems of their peripheral and central fragments. Instead, DFT calculations showed that several transitions between the orbitals located in different regions of the meta-conjugated molecule are responsible for the formation of their absorption spectra, and they strongly depend on the degree of their overlapping. Theoretical absorption spectra reconstructed from the DFT data demonstrated a good agreement with the experimental results: the transitions with larger oscillator strength correspond to the bands with higher molar extinction coefficients and vice versa. It was shown that luminescence spectral maxima of the meta-conjugated molecules monotonically shift to the lower energy from 489 to 540 and 613 nm with increasing the number of thiophene rings in the peripheral and central fragments, respectively. However, luminescence quantum yield of the meta-conjugated molecules critically depends on the length of linearly conjugated fragments in its structure decreasing from 24% to 1.3% with increasing the number of thiophene rings in the lateral fragments but increasing to 90% in the molecule with more thiophene rings in both types of the fragments. The results obtained are well correlated to the ratio of radiative and nonradiative deactivation rate constants of the meta-conjugated molecules that indicates a high rate of internal conversion between the excited states corresponding to different fragments of the molecule. The CV measurements allowed estimating the HOMO, LUMO, and bandgap values of the target and model compounds, which confirm the presence of meta-conjugation within the molecules investigated. Thus, connection of linearly conjugated fragments through meta-positions (meta-conjugation) of a benzene ring leads to an intermediate option between fully conjugated and nonconjugated molecules due to partial delocalization of electron density through the 1,3,5-substituted benzene branching center.

Relationship between Changes in the Protein Folding Pathway and the Process of Amyloid Formation: The Case of Bovine Carbonic Anhydrase II.

Many proteins form amyloid fibrils only under conditions when the probability of transition from a native (structured, densely packed) to an intermediate (labile, destabilized) state is increased. It implies the assumption that some structural intermediates are more convenient for amyloid formation than the others. Hence, if a mutation affects the protein folding pathway, one should expect that this mutation could affect the rate of amyloid formation as well. In the current work, we have compared the effects of amino acid substitutions of bovine carbonic anhydrase II on its unfolding pathway and on its ability to form amyloids at acidic pH and an elevated temperature. Wild-type protein and four mutant forms (L78A, L139A, I208A, and M239A) were studied. We analyzed the change of the protein unfolding pathway by the time-resolved fluorescence technique and the process of amyloid formation by thioflavin T fluorescence assay and electron microscopy. It was revealed that I208A substitution accelerates amyloid formation and affects the structure of the late (molten globule-like)-intermediate state of carbonic anhydrase, whereas the other mutations slow down the growth of amyloids and have either no effect on the unfolding pathway (L78A, L139A) or alter the conformational states arising at the early unfolding stage (M239A).

Nanoscale Control of Structure and Composition for Nanocrystalline Fe Thin Films Grown by Oblique Angle RF Sputtering.

The use of Fe films as multi-element targets in space radiation experiments with high-intensity ultrashort laser pulses requires a surface structure that can enhance the laser energy absorption on target, as well as a low concentration and uniform distribution of light element contaminants within the films. In this paper, (110) preferred orientation nanocrystalline Fe thin films with controlled morphology and composition were grown on (100)-oriented Si substrates by oblique angle RF magnetron sputtering, at room temperature. The evolution of films key-parameters, crucial for space-like radiation experiments with organic material, such as nanostructure, morphology, topography, and elemental composition with varying RF source power, deposition pressure, and target to substrate distance is thoroughly discussed. A selection of complementary techniques was used in order to better understand this interdependence, namely X-ray Diffraction, Atomic Force Microscopy, Scanning and Transmission Electron Microscopy, Energy Dispersive X-ray Spectroscopy and Non-Rutherford Backscattering Spectroscopy. The films featured a nanocrystalline, tilted nanocolumn structure, with crystallite size in the (110)-growth direction in the 15-25 nm range, average island size in the 20-50 nm range, and the degree of polycrystallinity determined mainly by the shortest target-to-substrate distance (10 cm) and highest deposition pressure (10-2 mbar Ar). Oxygen concentration (as impurity) into the bulk of the films as low as 1 at. %, with uniform depth distribution, was achieved for the lowest deposition pressures of (1-3) × 10-3 mbar Ar, combined with highest used values for the RF source power of 125-150 W. The results show that the growth process of the Fe thin film is strongly dependent mainly on the deposition pressure, with the film morphology influenced by nucleation and growth kinetics. Due to better control of film topography and uniform distribution of oxygen, such films can be successfully used as free-standing targets for high repetition rate experiments with high power lasers to produce Fe ion beams with a broad energy spectrum.

PHYSICAL DEVELOPMENT OF STUDENTS AS AN INDICATOR OF THE PHYSICAL EDUCATION SYSTEM FUNCTIONING IN THE EDUCATIONAL INSTITUTION.

OBJECTIVE: The aim: To investigate the impact of organizational and methodological conditions of physical education system in higher educational institution on the students' physical development. PATIENTS AND METHODS: Materials and methods: The research involved 120 students of two higher educational institutions (30 men and 30 women each). Determination of physical development of students was carried out in the dynamics of learning from the first to the fourth instructional years in terms of 100 m run, 3000 m (boys) and 2000 m (girls) run, pull-ups on the crossbar (boys), push-ups (girls), 4 x 9 m shuttle run, standing long jump, lifting torso to the sitting position, torso leaning forward from the sitting position. RESULTS: Results: The research determined organizational and methodological conditions of physical education system in two educational institutions, which differ in the duration of "Physical Education" academic subject depending on the instructional year, the number of academic hours provided for the subject mastery, approaches to the basic and elective components of the curriculum, organization of fitness and health recreation as well as sports events during extracurricular activities. CONCLUSION: Conclusions: It was established that the level of students' physical development is better in the educational institutions where the physical education system functions more efficiently (compulsory physical education training sessions are held in the amount of 4 hours a week, more students are involved in educational and extracurricular physical education training sessions, where students' training motivation is higher).

How Can Ice Emerge at 0 °C?

The classical nucleation theory shows that bulk water freezing does not occur at temperatures above ≈ -30 °C, and that at higher temperatures ice nucleation requires the presence of some ice-binding surfaces. The temperature and rate of ice nucleation depend on the size and level of complementarity between the atomic structure of these surfaces and various H-bond-rich/depleted crystal planes. In our experiments, the ice nucleation temperature was within a range from -8 °C to -15 °C for buffer and water in plastic test tubes. Upon the addition of ice-initiating substances (i.e., conventional AgI or CuO investigated here), ice appeared in a range from -3 °C to -7 °C, and in the presence of the ice-nucleating bacterium Pseudomonas syringae from -1 °C to -2 °C. The addition of an antifreeze protein inhibited the action of the tested ice-initiating agents.

The influence of Pseudomonas syringae on water freezing and ice melting.

Pseudomonas syringae is a widely spread plant pathogen known to have ice-nucleating proteins that serve as crystallization sites promoting ice growth at near-zero temperatures. Three temperatures that characterize water freezing and ice melting are (i) the freezing point of water, (ii) the temperature of coexistence of ice and water, and (iii) the melting point of ice. Here we show the influence of different concentrations of P. syringae on these three parameters. P. syringae appears to affect both the freezing point of water and the temperature of the coexistence of ice and water. Additionally, we propose a research technique for studying the freezing/melting process that is simple and requires no complex equipment.

Design and Analysis of a Mutant form of the Ice-Binding Protein from Choristoneura fumiferana.

Ice-binding proteins are expressed in the cells of some cold adapted organisms, helping them to survive at extremely low temperatures. One of the problems in studying such proteins is the difficulty of their isolation and purification. For example, eight cysteine residues in the cfAF (antifreeze protein from the eastern spruce budworm Choristoneura fumiferana) form intermolecular bridges during the overexpression of this protein. This impedes the process of the protein purification dramatically. To overcome this issue, in this work, we designed a mutant form of the ice-binding protein cfAFP, which is much easier to isolate that the wild-type protein. The mutant form named mIBP83 did not lose the ability to bind to ice surface. Besides, observation of the processes of freezing and melting of ice in the presence of mIBP83 showed that this protein affects the process of ice melting, increasing its melting temperature, and does not decrease the water freezing temperature.

PRACTICAL ASPECTS AND RESULTS OF COGNITIVE THERAPY IN THE EARLY RECOVERY PERIOD OF ISCHEMIC STROKE.

OBJECTIVE: The aim: To determine the impact of cognitive training on the degree of cognitive functions recovery and quality of life in the early recovery period of ischemic stroke. PATIENTS AND METHODS: Materials and methods: 108 patients with cerebral infarction were examined outpatiently, follow-up from 1 to 3 months from the onset of the disease. Basic assessment methods: screening index of cognitive disorders according to the Montreal Cognitive Assessment Scale (MoCA), SF-36 questionnaire. RESULTS: Results and Conclusions: Comprehensive rehabilitation measures for the early recovery period of ischemic stroke achieve improvement of the cognitive sphere: a significant increase in the average score on the Montreal scale of cognitive functions assessment (MoCA scale) in both observation groups.