A key GPCR phosphorylation motif discovered in arrestin2⋠CCR5 phosphopeptide complexes.

The two non-visual arrestins, arrestin2 and arrestin3, bind hundreds of GPCRs with different phosphorylation patterns, leading to distinct functional outcomes. Structural information on these interactions is available only for very few GPCRs. Here, we have characterized the interactions between the phosphorylated human CC chemokine receptor 5 (CCR5) and arrestin2. We identified several new CCR5 phosphorylation sites necessary for stable arrestin2 complex formation. Structures of arrestin2 in the apo form and complexes with CCR5 C-terminal phosphopeptides, together with NMR, biochemical, and functional assays, revealed three phosphoresidues in a pXpp motif that are essential for arrestin2 binding and activation. The identified motif appears responsible for robust arrestin2 recruitment in many other GPCRs. An analysis of receptor sequences and available structural and functional information provides hints on the molecular basis of arrestin2/arrestin3 isoform specificity. Our findings demonstrate how multi-site phosphorylation controls GPCR⋅arrestin interactions and provide a framework to probe the intricate details of arrestin signaling.

The dynamic mechanism of 4E-BP1 recognition and phosphorylation by mTORC1.

The activation of cap-dependent translation in eukaryotes requires multisite, hierarchical phosphorylation of 4E-BP by the 1 MDa kinase mammalian target of rapamycin complex 1 (mTORC1). To resolve the mechanism of this hierarchical phosphorylation at the atomic level, we monitored by NMR spectroscopy the interaction of intrinsically disordered 4E binding protein isoform 1 (4E-BP1) with the mTORC1 subunit regulatory-associated protein of mTOR (Raptor). The N-terminal RAIP motif and the C-terminal TOR signaling (TOS) motif of 4E-BP1 bind separate sites in Raptor, resulting in avidity-based tethering of 4E-BP1. This tethering orients the flexible central region of 4E-BP1 toward the mTORC1 kinase site for phosphorylation. The structural constraints imposed by the two tethering interactions, combined with phosphorylation-induced conformational switching of 4E-BP1, explain the hierarchy of 4E-BP1 phosphorylation by mTORC1. Furthermore, we demonstrate that mTORC1 recognizes both free and eIF4E-bound 4E-BP1, allowing rapid phosphorylation of the entire 4E-BP1 pool and efficient activation of translation. Finally, our findings provide a mechanistic explanation for the differential rapamycin sensitivity of the 4E-BP1 phosphorylation sites.

The antibiotic darobactin mimics a ß-strand to inhibit outer membrane insertase.

Antibiotics that target Gram-negative bacteria in new ways are needed to resolve the antimicrobial resistance crisis1-3. Gram-negative bacteria are protected by an additional outer membrane, rendering proteins on the cell surface attractive drug targets4,5. The natural compound darobactin targets the bacterial insertase BamA6-the central unit of the essential BAM complex, which facilitates the folding and insertion of outer membrane proteins7-13. BamA lacks a typical catalytic centre, and it is not obvious how a small molecule such as darobactin might inhibit its function. Here we resolve the mode of action of darobactin at the atomic level using a combination of cryo-electron microscopy, X-ray crystallography, native mass spectrometry, in vivo experiments and molecular dynamics simulations. Two cyclizations pre-organize the darobactin peptide in a rigid ß-strand conformation. This creates a mimic of the recognition signal of native substrates with a superior ability to bind to the lateral gate of BamA. Upon binding, darobactin replaces a lipid molecule from the lateral gate to use the membrane environment as an extended binding pocket. Because the interaction between darobactin and BamA is largely mediated by backbone contacts, it is particularly robust against potential resistance mutations. Our results identify the lateral gate as a functional hotspot in BamA and will allow the rational design of antibiotics that target this bacterial Achilles heel.

Intraoperative cone-beam computed tomography for catheter placement verification in pediatric hydrocephalus: technical note.

Ventriculoperitoneal (VP) shunt placement, essential for managing hydrocephalus, often risks catheter malpositioning, especially in patients with small ventricles. We present a novel technique combining neuronavigation with intraoperative cone-beam computed tomography using the BrainLab system and Loop-X mobile imaging unit. This approach enables real-time verification of catheter placement by integrating preoperative MRI data with intraoperative CT imaging. In a 12-year-old boy with therapy-refractory idiopathic intracranial hypertension, neuronavigation was guided by the BrainLab Skull Fix and Cushing canula, ensuring precise catheter insertion into the right frontal horn. Post-placement, Loop-X facilitated immediate verification of the catheter's trajectory and positioning, corroborated by postoperative MRI. This technique demonstrated high precision and minimized radiation exposure, emphasizing its utility in reducing revision rates due to suboptimal catheter placement.

Effects of a dual-task activity on gait parameters of people with and without intellectual disabilities.

BACKGROUND: The main objective of this study was to evaluate gait parameters in people with intellectual disability (ID) and without intellectual disability (WID) in two different walking conditions [single task vs. dual task (DT)]. A secondary aim was to evaluate the dual-task cost (DTC) that the DT causes in each group. METHODS: A total of 119 participants joined in this study: 56 ID (30 men) and 63 WID (30 men). The OptoGait system was used to assess gait. In addition, Witty photocells were added to assess gait under the DT condition. RESULTS: Single support time was lower for participants with ID (P < 0.01), while double support time was higher (P < 0.05). All coefficients of variation for gait parameters were higher in participants with ID. Additionally, changes in gait were observed in both groups during the DT condition compared with the single-task condition. These changes were larger for participants with ID in step length, double support time and gait speed (P < 0.001), resulting in a higher DTC in these variables in the ID group (P < 0.01). CONCLUSIONS: Both groups reduced gait performance in the DT condition. However, greater gait variability occurred in the ID group. In addition, DTC was higher for the ID group in all variables analysed. Therefore, people with ID show worse gait performance during a DT than people WID.

Risk Versus Reward: Hospitals Incentivized More Than Surgeons to Care for Riskier Arthroplasty Patients.

BACKGROUND: The purpose of this study was to assess the relationship between risk and reimbursement for both surgeons and hospitals among Medicare patients undergoing primary total joint arthroplasty (TJA). METHODS: The "2021 Medicare Physician and Other Provider" and "2021 Medicare Inpatient Hospitals" files were used. Patient comorbidity profiles were collected, including the mean patient hierarchal condition category (HCC) risk score. Surgeon data included all primary TJA procedures (inpatient and outpatient) billed to Medicare in 2021, while hospital data included all such inpatient episodes. Surgeon and hospital reimbursements were collected. All episodes were split into a "sicker cohort" with an HCC risk score of 1.5 or more and a "healthier cohort" with HCC risk scores less than 1.5. Variables were compared across cohorts. RESULTS: In 2021, 386,355 primary total hip and knee arthroplasty procedures were billed to Medicare and were included. The mean surgeon reimbursement among the sicker cohort was $1,021.91, which was less than for the healthier cohort of $1,060.13 (P < .001). Meanwhile, for the hospital analysis, 112,012 Medicare TJA patients were admitted as inpatients and included. The mean reimbursement to hospitals was significantly greater for the sicker cohort at $13,950.66, compared to the healthier cohort of $8,430.46. For both analyses, the sicker patient cohorts had a significantly higher rate of all comorbidities assessed (P < .001). CONCLUSIONS: This study demonstrates that mean surgeon reimbursement was lower for primary TJA among sicker patients in comparison to their healthier counterparts, while hospital reimbursement was higher for sicker patients. This represents a discrepancy in the incentivization of care for complex patients, as hospitals receive increased remuneration for taking on extra risk, while surgeons get paid less on average for performing TJA on sicker patients. Such data should inform future policy to assure continued access to arthroplasty care among complex patients.

Bacterial Chaperone Domain Insertions Convert Human FKBP12 into an Excellent Protein-Folding Catalyst-A Structural and Functional Analysis.

Many folding enzymes use separate domains for the binding of substrate proteins and for the catalysis of slow folding reactions such as prolyl isomerization. FKBP12 is a small prolyl isomerase without a chaperone domain. Its folding activity is low, but it could be increased by inserting the chaperone domain from the homolog SlyD of E. coli near the prolyl isomerase active site. We inserted two other chaperone domains into human FKBP12: the chaperone domain of SlpA from E. coli, and the chaperone domain of SlyD from Thermococcus sp. Both stabilized FKBP12 and greatly increased its folding activity. The insertion of these chaperone domains had no influence on the FKBP12 and the chaperone domain structure, as revealed by two crystal structures of the chimeric proteins. The relative domain orientations differ in the two crystal structures, presumably representing snapshots of a more open and a more closed conformation. Together with crystal structures from SlyD-like proteins, they suggest a path for how substrate proteins might be transferred from the chaperone domain to the prolyl isomerase domain.

Comparative study of oral microbiota in the experimental long-term opioid exposure, after its withdrawal and the use of complex drug correction.

OBJECTIVE: Aim: To study changes of dental biofilm microbiota composition during experimental opioid exposure, after its withdrawal and when using of complex drug correction.. PATIENTS AND METHODS: Materials and Methods: Microbiological studies (48 rats) included microscopic and bacteriological methods, as well as determination of antibiotic susceptibility of microbial isolates. Ceftriaxone and pentoxifylline were used to correction the changes. RESULTS: Results: The action of opioid for 10 weeks caused considerable changes in the microbiocenosis, which was illustrated by a significant increasing of the opportunistic pathogens quantitative indicators and the emergence of pathogenic microbiota. Changes in the microbiocenosis at 6 weeks of opioid exposure and after its withdrawal for 4 weeks were expressed in the appearance of pathogenic microbiota and the absence of significant differences in quantitative indicators of saprophytic and opportunistic microflora compared to similar indicators in animals with 10 weeks opioid exposure. This indicated a slow progression of dysbiotic changes and the inflammatory process in the oral cavity of rats. CONCLUSION: Conclusions: After 10 weeks of experiment with opioid administration for 6 weeks and the use of ceftriaxone and pentoxifylline on the background of 4-week opioid withdrawal, a significant reduction of quantitative indicators of opportunistic bacteria and elimination of pathogenic species of microorganisms was determined. The use of complex drug correction on the background of 10 weeks of opioid exposure led to a significant reduction in the quantitative indicators of opportunistic pathogens and contributed to the elimination of most pathogenic species of microbiota under the action of ceftriaxone.

Strengthening an Intramolecular Non-Classical Hydrogen Bond to Get in Shape for Binding.

In this research article, we report on the strengthening of a non-classical hydrogen bond (C-H⋅⋅⋅O) by introducing electron withdrawing groups at the carbon atom. The approach is demonstrated on the example of derivatives of the physiological E-selectin ligand sialyl Lewisx (1, sLex). Its affinity is mainly due to a beneficial entropy term, which is predominantly caused by the pre-organization of sLex in its binding conformation. We have shown, that among the elements responsible for the pre-organization, the stabilization by a non-classical hydrogen bond between the H-C5 of l-fucose and the ring oxygen O5 of the neighboring d-galactose moiety is essential and yields 7.4 kJ mol-1. This effect could be further strengthened by replacing l-fucose by 6,6,6-trifluoro-l-fucose leading to an improved non-classical H-bond of 14.9 kJ mol-1, i.e., an improved pre-organization in the bioactive conformation. For a series of glycomimetics of sLex (1), this outcome could be confirmed by high field NMR-shifts of the H-C5Fuc, by X-ray diffraction analysis of glycomimetics co-crystallized with E-selectin as well as by isothermal titration calorimetry. Furthermore, the electron-withdrawing character of the CF3-group beneficially influences the pharmacokinetic properties of sLex mimetics. Thus, acid-stability, a prerequisite for gastrointestinal stability, could be substantially improved.

Summarizing the effects of different exercise types in chronic neck pain - a systematic review and meta-analysis of systematic reviews.

BACKGROUND: To date, no consensus exists as to whether one exercise type is more effective than another in chronic neck pain. This systematic review and meta-analysis of systematic reviews aimed to summarize the literature on the effect of various exercise types used in chronic neck pain and to assess the certainty of the evidence. METHODS: We searched the databases Ovid MEDLINE, Embase, Cochrane Library, SportDiscus, and Web of Science (Core Collection) for systematic reviews and meta-analyses on adults between 18 and 70 years with chronic neck pain lasting ≥ 12 weeks which investigated the effects of exercises on pain and disability. The included reviews were grouped into motor control exercise (MCE), Pilates exercises, resistance training, traditional Chinese exercise (TCE), and yoga. Study quality was assessed with AMSTAR-2 and the level of certainty for the effects of the exercise through GRADE. A narrative analysis of the results was performed and in addition, meta-analyses when feasible. RESULTS: Our database search resulted in 1,794 systematic reviews. We included 25 systematic reviews and meta-analyses including 17,321 participants (overlap not accounted for). The quality of the included reviews ranged from critically low to low (n = 13) to moderate to high (n = 12). We found low to high certainty of evidence that MCE, Pilates exercises, resistance training, TCE, and yoga have short-term positive effects on pain and that all exercise types except resistance training, show positive effects on disability compared to non-exercise controls. We found low to moderate certainty of evidence for conflicting results on pain and disability when the exercise types were compared to other exercise interventions in the short-term as well as in intermediate/long-term apart for yoga, as no long-term results were available. CONCLUSION: Overall, our findings show low to high certainty of evidence for positive effects on pain and disability of the various exercise types used in chronic neck pain compared to non-exercise interventions, at least in the short-term. Based on our results, no optimal exercise intervention for patients with chronic neck pain can be recommended, since no large differences between the exercise types were shown here. Because the quality of the included systematic reviews varied greatly, future systematic reviews need to increase their methodological quality. TRIAL REGISTRATION: Prospero CRD42022336014.

Protective Properties of Spheroidal Taxifolin Form in Streptozotocin-Induced Diabetic Rats.

One of the key factors in the pathogenesis of diabetes and its complications is oxidative stress. To inhibit this process, antioxidants may be helpful. Herein, we focused on the protective properties of taxifolin spheroidal form (TS) in the streptozotocin rat model of diabetes mellitus. After 4 weeks of treatment with TS, the fasting blood glucose level of the diabetic animals decreased by 12% compared with the level right after the injection of streptozotocin. While the feed intake in the untreated diabetic rats increased by 5.3% compared with the healthy group, the TS-treated group showed a pronounced 15.3% decrease. Therapeutic administration of TS has a protective effect on the pancreas and the liver against the cytotoxic action of streptozotocin. The plasma antioxidant capacity of all diabetic groups appeared to be approximately 15% lower than in healthy rats with no significant difference between the TS-treated and untreated diabetic animals. Apparently, this can be attributed to taxifolin and plasma proteins binding. These data demonstrate the potential of TS in antidiabetic therapy.

Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases.

Polyketide synthases (PKSs) are biosynthetic factories that produce natural products with important biological and pharmacological activities. Their exceptional product diversity is encoded in a modular architecture. Modular PKSs (modPKSs) catalyse reactions colinear to the order of modules in an assembly line, whereas iterative PKSs (iPKSs) use a single module iteratively as exemplified by fungal iPKSs (fiPKSs). However, in some cases non-colinear iterative action is also observed for modPKSs modules and is controlled by the assembly line environment. PKSs feature a structural and functional separation into a condensing and a modifying region as observed for fatty acid synthases. Despite the outstanding relevance of PKSs, the detailed organization of PKSs with complete fully reducing modifying regions remains elusive. Here we report a hybrid crystal structure of Mycobacterium smegmatis mycocerosic acid synthase based on structures of its condensing and modifying regions. Mycocerosic acid synthase is a fully reducing iPKS, closely related to modPKSs, and the prototype of mycobacterial mycocerosic acid synthase-like PKSs. It is involved in the biosynthesis of C20-C28 branched-chain fatty acids, which are important virulence factors of mycobacteria. Our structural data reveal a dimeric linker-based organization of the modifying region and visualize dynamics and conformational coupling in PKSs. On the basis of comparative small-angle X-ray scattering, the observed modifying region architecture may be common also in modPKSs. The linker-based organization provides a rationale for the characteristic variability of PKS modules as a main contributor to product diversity. The comprehensive architectural model enables functional dissection and re-engineering of PKSs.

Summarizing the effects of different exercise types in chronic low back pain - a systematic review of systematic reviews.

BACKGROUND: In chronic LBP (CLBP), guideline-endorsed treatment is to stay active, return to normal activity, and to exercise. Several reviews on various exercise types used in CLBP have been published. We aimed to identify systematic reviews of common exercise types used in CLBP, to appraise their quality, and to summarize and compare their effect on pain and disability. METHODS: We searched the databases OVID MEDLINE, EMBASE, COCHRANE LIBRARY, and WEB OF SCIENCE (Core collection) for systematic reviews and meta-analyses on adults between 18 and 70 years of age suffering from chronic or recurrent LBP for a period of at least 12 weeks, which investigated the effects of exercises on pain and disability. All searches were conducted without language restriction. The search was performed up until 2022-01-26. The included reviews were grouped into nine exercise types: aerobic training, aquatic exercises, motor control exercises (MCE), resistance training, Pilates, sling exercises, traditional Chinese exercises (TCE), walking, and yoga. The study quality was assessed with AMSTAR-2. For each exercise type, a narrative analysis was performed, and the level of evidence for the effects of exercise was assessed through GRADE. RESULTS: Our database search resulted in 3,475 systematic reviews. Out of the 253 full texts that were screened, we included 45 systematic reviews and meta-analyses. The quality of the included reviews ranged from high to critically low. Due to large heterogeneity, no meta-analyses were performed. We found low-to-moderate evidence of mainly short-term and small beneficial effects on pain and disability for MCE, Pilates, resistance training, TCE, and yoga compared to no or minimal intervention. CONCLUSIONS: Our findings show that the effect of various exercise types used in CLBP on pain and disability varies with no major difference between exercise types. Many of the included systematic reviews were of low-to-moderate quality and based on randomized controlled trials with high risk of bias. The conflicting results seen, undermine the certainty of the results leading to very-low-to-moderate quality of evidence for our results. Future systematic reviews should be of higher quality to minimize waste of resources. TRIAL REGISTRATION: PROSPERO: Reg no 190409 Registration date 01AUG 2020.

Solubility Enhancement of Dihydroquercetin via "Green" Phase Modification.

Dihydroquercetin (DHQ) is a promising antioxidant for medical applications. The poor water solubility of this flavanonol at ambient conditions inhibits its implementation in clinical practice as an injectable dosage form. Thus, increasing water solubility is a critical step toward solving this problem. Herein we attempted to deal with this problem via DHQ phase modification while at the same time adhering to the principles of green chemistry as much as possible. Lyophilization is an appropriate method to achieve phase modification in an environment-friendly way. This method was employed to generate new phase modifications of DHQ that were then characterized. Mixtures of water with ethanol or acetonitrile were used as solvents for the preparation of the lyophilizates, DHQE, and DHQA, respectively. The results of dissolution testing of the obtained DHQE and DHQA demonstrated that the lyophilization increased water solubility at least 30-fold times. These new DHQ modifications were studied by scanning electron microscopy, mass-spectrometry, nuclear magnetic resonance spectroscopy, infrared spectroscopy, X-ray powder diffraction, and thermal analysis. Their solid-state phases were confirmed to differ from the initial DHQ substance without any changes in the molecular structure. Both DHQE and DHQA showed as high antioxidant activity as the initial DHQ. These data demonstrate the potential of DHQE and DHQA as active pharmaceutical ingredients for injectable dosage forms.

OCCUPATIONAL SAFETY AND HYGIENE OF HEALTHCARE PROFESSIONALS IN THE CONTEXT OF HOSPITAL ENVIRONMENT SAFETY.

OBJECTIVE: The aim: This study was conducted to carry out a comprehensive assessment of the occupational safety and health of medical workers. PATIENTS AND METHODS: Materials and methods: The study involved a questionnaire survey and full-scale physiological and hygienic research using bibliosemantic, questionnaire, hygienic, and statistical methods. RESULTS: Results: In the course of research it was recorded that the work of surgeons (according to the criteria of the current "Hygienic classification of work") is characterized by high intensity (class 3.2), high probability of exposure to infectious agents (class 3.3, and 4), harmful effects of physical factors (class 3.1 ), which according to the criteria of the general hygienic assessment of working conditions according to the degree of harmfulness and danger allows being assigned to class 3.3 ("harmful") and 4 ("dangerous"), respectively. Such working conditions provoke an increase in chronic morbidity (industrial and temporary disability), lead to the development of occupational diseases, and can pose a threat to the lives of workers. The need to focus attention on assessing the safety of hospitals in emergencies not only on issues of occupational safety and health but also on the stability and reliability of hospitals in such extreme conditions. CONCLUSION: Conclusions: The work process of doctors is characterized by the impact on their body of a complex of unfavorable production factors of psycho-physiological, biological, physical, and chemical nature, among which the leaders are high labor intensity due to intellectual and emotional load, irrational mode of work and rest, and biological factors.

Sweet Drugs for Bad Bugs: A Glycomimetic Strategy against the DC-SIGN-Mediated Dissemination of SARS-CoV-2.

The C-type lectin receptor DC-SIGN is a pattern recognition receptor expressed on macrophages and dendritic cells. It has been identified as a promiscuous entry receptor for many pathogens, including epidemic and pandemic viruses such as SARS-CoV-2, Ebola virus, and HIV-1. In the context of the recent SARS-CoV-2 pandemic, DC-SIGN-mediated virus dissemination and stimulation of innate immune responses has been implicated as a potential factor in the development of severe COVID-19. Inhibition of virus binding to DC-SIGN, thus, represents an attractive host-directed strategy to attenuate overshooting innate immune responses and prevent the progression of the disease. In this study, we report on the discovery of a new class of potent glycomimetic DC-SIGN antagonists from a focused library of triazole-based mannose analogues. Structure-based optimization of an initial screening hit yielded a glycomimetic ligand with a more than 100-fold improved binding affinity compared to methyl α-d-mannopyranoside. Analysis of binding thermodynamics revealed an enthalpy-driven improvement of binding affinity that was enabled by hydrophobic interactions with a loop region adjacent to the binding site and displacement of a conserved water molecule. The identified ligand was employed for the synthesis of multivalent glycopolymers that were able to inhibit SARS-CoV-2 spike glycoprotein binding to DC-SIGN-expressing cells, as well as DC-SIGN-mediated trans-infection of ACE2+ cells by SARS-CoV-2 spike protein-expressing viruses, in nanomolar concentrations. The identified glycomimetic ligands reported here open promising perspectives for the development of highly potent and fully selective DC-SIGN-targeted therapeutics for a broad spectrum of viral infections.

Concurrent and discriminant validity of ActiGraph waist and wrist cut-points to measure sedentary behaviour, activity level, and posture in office work.

BACKGROUND: Sedentary Behaviour (SB) gets an increasing attention from ergonomics and public health due to its associated detrimental health effects. A large number of studies record SB with ActiGraph counts-per-minute cut-points, but we still lack valid information about what the cut-points tell us about office work. This study therefore analysed the concurrent and discriminant validity of commonly used cut-points to measure SB, activity level, and posture. METHODS: Thirty office workers completed four office tasks at three workplaces (conventional chair, activity-promoting chair, and standing desk) while wearing two ActiGraphs (waist and wrist). Indirect calorimetry and prescribed posture served as reference criteria. Generalized Estimation Equations analysed workplace and task effects on the activity level and counts-per-minute, and kappa statistics and ROC curves analysed the cut-point validity. RESULTS: The activity-promoting chair (p < 0.001, ES ≥ 0.66) but not the standing desk (p = 1.0) increased the activity level, and both these workplaces increased the waist (p ≤ 0.003, ES ≥ 0.63) but not the wrist counts-per-minute (p = 0.74) compared to the conventional chair. The concurrent and discriminant validity was higher for activity level (kappa: 0.52-0.56 and 0.38-0.45, respectively) than for SB and posture (kappa ≤0.35 and ≤ 0.19, respectively). Furthermore, the discriminant validity for activity level was higher for task effects (kappa: 0.42-0.48) than for workplace effects (0.13-0.24). CONCLUSIONS: ActiGraph counts-per-minute for waist and wrist placement were - independently of the chosen cut-point - a measure for activity level and not for SB or posture, and the cut-points performed better to detect task effects than workplace effects. Waist cut-points were most valid to measure the activity level in conventional seated office work, but they showed severe limitations for sit-stand desks. None of the placements was valid to detect the increased activity on the activity-promoting chair. Caution should therefore be paid when analysing the effect of workplace interventions on activity level with ActiGraph waist and wrist cut-points.

A folding nucleus and minimal ATP binding domain of Hsp70 identified by single-molecule force spectroscopy.

The folding pathways of large proteins are complex, with many of them requiring the aid of chaperones and others folding spontaneously. Along the folding pathways, partially folded intermediates are frequently populated; their role in the driving of the folding process is unclear. The structures of these intermediates are generally not amenable to high-resolution structural techniques because of their transient nature. Here we employed single-molecule force measurements to scrutinize the hierarchy of intermediate structures along the folding pathway of the nucleotide binding domain (NBD) of Escherichia coli Hsp70 DnaK. DnaK-NBD is a member of the sugar kinase superfamily that includes Hsp70s and the cytoskeletal protein actin. Using optical tweezers, a stable nucleotide-binding competent en route folding intermediate comprising lobe II residues (183-383) was identified as a critical checkpoint for productive folding. We obtained a structural snapshot of this folding intermediate that shows native-like conformation. To assess the fundamental role of folded lobe II for efficient folding, we turned our attention to yeast mitochondrial NBD, which does not fold without a dedicated chaperone. After replacing the yeast lobe II residues with stable E. coli lobe II, the obtained chimeric protein showed native-like ATPase activity and robust folding into the native state, even in the absence of chaperone. In summary, lobe II is a stable nucleotide-binding competent folding nucleus that is the key to time-efficient folding and possibly resembles a common ancestor domain. Our findings provide a conceptual framework for the folding pathways of other members of this protein superfamily.

Calciprotein Particles Link Disturbed Mineral Homeostasis with Cardiovascular Disease by Causing Endothelial Dysfunction and Vascular Inflammation.

An association between high serum calcium/phosphate and cardiovascular events or death is well-established. However, a mechanistic explanation of this correlation is lacking. Here, we examined the role of calciprotein particles (CPPs), nanoscale bodies forming in the human blood upon its supersaturation with calcium and phosphate, in cardiovascular disease. The serum of patients with coronary artery disease or cerebrovascular disease displayed an increased propensity to form CPPs in combination with elevated ionised calcium as well as reduced albumin levels, altogether indicative of reduced Ca2+-binding capacity. Intravenous administration of CPPs to normolipidemic and normotensive Wistar rats provoked intimal hyperplasia and adventitial/perivascular inflammation in both balloon-injured and intact aortas in the absence of other cardiovascular risk factors. Upon the addition to primary human arterial endothelial cells, CPPs induced lysosome-dependent cell death, promoted the release of pro-inflammatory cytokines, stimulated leukocyte adhesion, and triggered endothelial-to-mesenchymal transition. We concluded that CPPs, which are formed in the blood as a result of altered mineral homeostasis, cause endothelial dysfunction and vascular inflammation, thereby contributing to the development of cardiovascular disease.

PSYCHOLOGICAL AND PEDAGOGICAL DETERMINANTS OF THE STUDENTS' HEALTHY LIFESTYLE FORMATION BY MEANS OF HEALTH AND FITNESS ACTIVITIES.

OBJECTIVE: The aim: Is to investigate the psychological and pedagogical determinants of the students' healthy lifestyle formation by means of health and fitness activities. PATIENTS AND METHODS: Materials and methods: The study involved 882 students between the ages of 17 and 23. An analysis of the students' health dynamics during the period of study determined their motivational and value-based attitude to a healthy lifestyle, the level of health competence and the desire of students to implement it in future professional activities, studied the attitude of students to their own health. RESULTS: Results: It was found that during two years of study, the health of students had significantly deteriorated. The number of sick students increased to 34.8 % among males and to 31.9 % among females. It was found that only 12.3 % of male students and 10.6 % of female ones had a high level of motivation for a healthy lifestyle. CONCLUSION: Conclusions: It was determined that students were aware of the importance of a high level of health to ensure the effectiveness of vital activity (more than 50 % of students realized the value of health in the system of life values), but the vast majority did nothing to maintain it. Most students did not have the knowledge, skills, and abilities to use health technologies during studying and leisure.