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To study the problem of rough landings and aircraft runway excursions, a 22-item questionnaire, related to the subject under consideration, was drawn up for pilots. The questionnaire mainly focused on aircraft veering off. The range of issues covered different aspects of the problem, including: pilots' perception and a comparative evaluation of various risk factors; personal experience of landings with prerequisites for a runway excursion; decision-making for the go-around procedure; attitude to the airline policy regarding the landing quality assessment; reliability of information reported to the crew about weather and runway conditions; quality of simulator training for flights in terms of crosswind and slippery runways; modern aircraft systems to prevent a runway excursion, etc. More than 50 civil aviation pilots of different age, experience, including aircraft captains and co-pilots were interviewed. The survey was conducted remotely and anonymously. The processing of the questionnaires included a detailed analysis of the answers to each question. If a question was not answered or if there was some reasonable doubt as to its correctness, the questionnaire was excluded from consideration. The results of questionnaire processing for the entire group and for individual categories (young/experienced, captains/co-pilots, turboprop/turbojet aircraft) were presented and analyzed in this paper.

ABSTRACTThe World Health Organization’s goal to combat tuberculosis (TB) is hindered by the emergence of anti-microbial resistance, therefore necessitating the exploration of new drug targets. Multidrug regimens are indispensable in TB therapy as they provide synergetic bactericidal effects, shorten treatment duration, and reduce the risk of resistance development. The research within our European RespiriTB consortium explores Mycobacterium tuberculosis energy metabolism to identify new drug candidates that synergize with bedaquiline, with the aim of discovering more efficient combination drug regimens. In this study, we describe the development and validation of a luminescence-coupled, target-based assay for the identification of novel compounds inhibiting Mycobacterium tuberculosis mycothione reductase (MtrMtb), an enzyme with a role in the protection against oxidative stress. Recombinant MtrMtb was employed for the development of a highly sensitive, robust high-throughput screening (HTS) assay by coupling enzyme activity to a bioluminescent readout. Its application in a semi-automated setting resulted in the screening of a diverse library of ~130,000 compounds, from which 19 hits were retained after an assessment of their potency, selectivity, and specificity. The selected hits formed two clusters and four fragment molecules, which were further evaluated in whole-cell and intracellular infection assays. The established HTS discovery pipeline offers an opportunity to deliver novel MtrMtb inhibitors and lays the foundation for future efforts in developing robust biochemical assays for the identification and triaging of inhibitors from high-throughput library screens.IMPORTANCEThe growing anti-microbial resistance poses a global public health threat, impeding progress toward eradicating tuberculosis. Despite decades of active research, there is still a dire need for the discovery of drugs with novel modes of action and exploration of combination drug regimens. Within the European RespiriTB consortium, we explore Mycobacterium tuberculosis energy metabolism to identify new drug candidates that synergize with bedaquiline, with the aim of discovering more efficient combination drug regimens. In this study, we present the development of a high-throughput screening pipeline that led to the identification of M. tuberculosis mycothione reductase inhibitors.

Abstract Intermediate filaments (IFs) are essential constituents of the metazoan cytoskeleton. A vast family of cytoplasmic IF proteins are capable of self-assembly from soluble tetrameric species into typical 10–12 nm wide filaments. The primary structure of these proteins includes the signature central ‘rod’ domain of ~ 300 residues which forms a dimeric α-helical coiled coil composed of three segments (coil1A, coil1B and coil2) interconnected by non-helical, flexible linkers (L1 and L12). The rod is flanked by flexible terminal head and tail domains. At present, the molecular architecture of mature IFs is only poorly known, limiting our capacity to rationalize the effect of numerous disease-related mutations found in IF proteins. Here we addressed the molecular structure of soluble vimentin tetramers which are formed by two antiparallel, staggered dimers with coil1B domains aligned (A11 tetramers). By examining a series of progressive truncations, we show that the presence of the coil1A domain is essential for the tetramer formation. In addition, we employed a novel chemical cross-linking pipeline including isotope labelling to identify intra- and interdimeric cross-links within the tetramer. We conclude that the tetramer is synergistically stabilized by the interactions of the aligned coil1B domains, the interactions between coil1A and the N-terminal portion of coil2, and the electrostatic attraction between the oppositely charged head and rod domains. Our cross-linking data indicate that, starting with a straight A11 tetramer, flexibility of linkers L1 and L12 enables ‘backfolding’ of both the coil1A and coil2 domains onto the tetrameric core formed by the coil1B domains. Through additional small-angle X-ray scattering experiments we show that the elongated A11 tetramers dominate in low ionic strength solutions, while there is also a significant structural flexibility especially in the terminal domains.

We theoretically study the generation of attosecond XUV pulses via high-order frequency mixing (HFM) of two intense generating fields, and compare this process with the more common high-order harmonic generation (HHG) process. We calculate the macroscopic XUV signal by numerically integrating the 1D propagation equation coupled with the 3D time-dependent Schrödinger equation. We analytically find the length scales which limit the quadratic growth of the HFM macroscopic signal with propagation length. Compared to HHG these length scales are much longer for a group of HFM components, with orders defined by the frequencies of the generating fields. This results in a higher HFM macroscopic signal despite the microscopic response being lower than for HHG. In our numerical simulations in a gas target, the intensity of the HFM signal is several times higher than that for HHG, while for a plasma target the HFM generation efficiency is up to three orders of magnitude higher than for HHG. The HFM with relatively long generating pulses provides very narrow XUV lines ( $\delta \omega / \omega = 4.6 \times 10^{-4}$ ) with well-defined frequencies, thus allowing for a simple extension of optical frequency standards to the XUV range. Finally, we show that the group of HFM components effectively generated due to macroscopic effects provides a train of attosecond pulses such that the carrier-envelope phase of an individual attosecond pulse can be easily controlled by tuning the phase of one of the generating fields.

Crystal structures for all enzyme states of leucyl-tRNA synthetase in Neisseria gonorrhoeae reveal multi-domain conformational changes that correlate with a local peptide-plane flip in the active site to compartmentalize catalytic steps.

We investigate the production of an isolated attosecond pulse (IAP) via the phase-matching gating of high-harmonic generation with intense laser pulses. Our study is based on the integration of the propagation equation for the fundamental and generated fields with nonlinear polarisation found via the numerical solution of the time-dependent Schrödinger equation. We study the XUV energy as a function of the propagation distance (or the medium density) and find that the onset of the IAP production corresponds to the change from linear to quadratic dependence of this energy on the propagation distance (or density). Finally, we show that the upper limit of the fundamental pulse duration for which IAP generation is feasible is defined by the temporal spreading of the fundamental pulse during the propagation. This nonlinear spreading is defined by the difference in the group velocities for the neutral and photoionised medium.

Attosecond pulses created by high-order harmonic generation in gases often exhibit strong chromatic aberrations, arising from the broad bandwidth and wavelength-dependent nonlinear light–matter interaction. When the driving laser intensity varies spatially, as for Gaussian driving beams, the apparent source position of the harmonics differs significantly from one order to the next, thus affecting the achievable intensity and duration of the attosecond pulses when they are focused on a target. We show that these chromatic aberrations can be reduced by spatially shaping the fundamental beam to generate high-order harmonics with a driver having a flat-top profile inside the gas medium. By measuring both the intensity profile and wavefront for each harmonic in a plane, we access the extreme ultra-violet (XUV) beam properties and investigate these properties near focus. We observe that controlling chromatic aberrations by flat-top spatial shaping strongly reduces the variation of the XUV spectrum on the beam axis during propagation and, in return, the longitudinal sensitivity of both the temporal profiles and the temporal shifts of the focused attosecond pulses.

Mycothiol (MSH), the major cellular thiol in Mycobacterium tuberculosis (Mtb), plays an essential role in the resistance of Mtb to various antibiotics and oxidative stresses. MshC catalyzes the ATP-dependent ligation of 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol (GlcN-Ins) with l-cysteine (l-Cys) to form l-Cys-GlcN-Ins, the penultimate step in MSH biosynthesis. The inhibition of MshC is lethal to Mtb. In the present study, five new cysteinyl-sulfonamides were synthesized, and their binding affinity with MshC was evaluated using a thermal shift assay. Two of them bind the target with EC50 values of 219 and 231 µM. Crystal structures of full-length MshC in complex with these two compounds showed that they were bound in the catalytic site of MshC, inducing dramatic conformational changes of the catalytic site compared to the apo form. In particular, the observed closure of the KMSKS loop was not detected in the published cysteinyl-sulfamoyl adenosine-bound structure, the latter likely due to trypsin treatment. Despite the confirmed binding to MshC, the compounds did not suppress Mtb culture growth, which might be explained by the lack of adequate cellular uptake. Taken together, these novel cysteinyl-sulfonamide MshC inhibitors and newly reported full-length apo and ligand-bound MshC structures provide a promising starting point for the further development of novel anti-tubercular drugs targeting MshC.

Abstract We measured the temperature dependence of the probability of small heating and total losses of UCNs on the PFPE Fomblin Y surface with various molecular mass $$M_{{\bar{W}}}$$ MW¯ (2800, 3300, 6500 amu) in the temperature range of 100–300 K. The probability of small heating sharply decreases with increasing $$M_{{\bar{W}}}$$ MW¯ and decreasing temperature. The probability of total loss weakly decreases with decreasing temperature and takes the minimum value at $$M_{{\bar{W}}} =3300 \, \hbox {amu}$$ MW¯=3300amu . As this oil provides a homogeneous surface with minimal probabilities of small heating and total losses of UCNs, it is the preferred candidate for experiments on measuring the neutron lifetime.

Introduction. This article describes the procedure for personnel management in a pharmacy organization in terms of the current requirements of Good Pharmacy Practice (GAP) and Quality Management System (QMS). A key element of the NSP and QMS is staff, so the HR management system should be based on the principles and foundations inherent in these concepts and be adequate to the current quality system in the pharmacy organization.Text. Quality management involves the use of systematic and process approaches. In doing so, pharmacies need to take a number of steps to improve the quality of the workforce, its activities and involvement in the process of formation of NPS and QMS. In addition, the professional development of staff is an essential condition and key to the successful functioning of the quality system, as well as the efficiency, performance and well-being of the organization as a whole. To implement the requirements of the quality management system, specialists of the personnel department, managers of all levels, as well as ordinary employees should be involved. Achieving a high level of quality management system at the enterprise is possible only in the presence of highly qualified personnel with certain competences. According to the requirements of Good Pharmacy Practice, which came into effect on March 1, 2017, the Pharmacy should implement the Personnel Adaptation Programme. Adaptation is a complex and time-consuming process of adjusting new employees to the content, working conditions and social environment of the organization, the gradual inclusion of new employees in the professional activities and team, and the pharmaceutical industry has its own characteristics and specifics. In the article the variant of the program of adaptation for the young employee including various aspects and forms of its carrying out which can render the essential help to heads of the drugstore organizations and experts of service of the personnel at training of new employees is offered. In addition to the adaptation program, the pharmacy organization should develop and implement a training system that corresponds to the specifics of the organization's production processes. The training needs of staff are determined by the training specialist, who annually and on a quarterly basis forms a schedule for external training of staff. The training schedule takes into account the plans for the development and updating of documents of the organization, which relate to the working activities of the employee. Training according to the documents is carried out by the head of the structural subdivision, the results of knowledge control should be recorded by him in the internal training cards of the employee. In contrast to the traditional approach to the organization of personnel training in the pharmacy organization, it is now rational to introduce standard and individual training matrices. Conclusion. The authors express their vision of strategy, planning, professional growth management, training of new knowledge and skills of personnel in the quality management system.

The technology of augmented and mixed reality (AR/MR) is useful in various areas of modern surgery. We considered the use of augmented and mixed reality technologies as a method of preoperative planning and intraoperative navigation in abdominal cancer patients. Practical use of AM/MR raises a range questions, which demand suitable solutions. The difficulties and obstacles we encountered in the practical use of AR/MR are presented, along with the ways we chose to overcome them. The most demonstrative case is covered in detail. The three-dimensional anatomical model obtained from the CT scan needed to be rigidly attached to the patient’s body, and therefore an invasive approach was developed, using an orthopedic pin fixed to the pelvic bones. The pin is used both similarly to an X-ray contrast marker and as a marker for augmented reality. This solution made it possible, not only to visualize the anatomical structures of the patient and the border zone of the tumor, but also to change the position of the patient during the operation. In addition, a noninvasive (skin-based) marking method was developed that allows the application of mixed and augmented reality during operation. Both techniques were used (8 clinical cases) for preoperative planning and intraoperative navigation, which allowed surgeons to verify the radicality of the operation, to have visual control of all anatomical structures near the zone of interest, and to reduce the time of surgical intervention, thereby reducing the complication rate and improving the rehabilitation period.

Given the role of intermediate filaments (IFs) in normal cell physiology and scores of IF-linked diseases, the importance of understanding their molecular structure is beyond doubt. Research into the IF structure was initiated more than 30 years ago, and some important advances have been made. Using crystallography and other methods, the central coiled-coil domain of the elementary dimer and also the structural basis of the soluble tetramer formation have been studied to atomic precision. However, the molecular interactions driving later stages of the filament assembly are still not fully understood. For cytoplasmic IFs, much of the currently available insight is due to chemical cross-linking experiments that date back to the 1990s. This technique has since been radically improved, and several groups have utilized it recently to obtain data on lamin filament assembly. Here, we will summarize these findings and reflect on the remaining open questions and challenges of IF structure. We argue that, in addition to X-ray crystallography, chemical cross-linking and cryoelectron microscopy are the techniques that should enable major new advances in the field in the near future.

The paper considers the possibilities, prospects, and drawbacks of the mixed reality (MR) technology application using mixed reality smartglasses Microsoft HoloLens 2. The main challenge was to find and develop an approach that would allow surgeons to conduct operations using mixed reality on a large scale, reducing the preparation time required for the procedure and without having to create custom solutions for each patient. Research was conducted in three clinical cases: two median neck and one branchial cyst excisions. In each case, we applied a unique approach of hologram positioning in space based on mixed reality markers. As a result, we listed a series of positive and negative aspects related to MR surgery, along with proposed solutions for using MR in surgery on a daily basis.

Human cytosolic prolyl-tRNA synthetase (HcProRS) catalyses the formation of the prolyl-tRNAPro, playing an important role in protein synthesis. Inhibition of HcProRS activity has been shown to have potential benefits in the treatment of fibrosis, autoimmune diseases and cancer. Recently, potent pyrazinamide-based inhibitors were identified by a high-throughput screening (HTS) method, but no further elaboration was reported. The pyrazinamide core is a bioactive fragment found in numerous clinically validated drugs and has been subjected to various modifications. Therefore, we applied a virtual screening protocol to our in-house library of pyrazinamide-containing small molecules, searching for potential novel HcProRS inhibitors. We identified a series of 3-benzylaminopyrazine-2-carboxamide derivatives as positive hits. Five of them were confirmed by a thermal shift assay (TSA) with the best compounds 3b and 3c showing EC50 values of 3.77 and 7.34 µM, respectively, in the presence of 1 mM of proline (Pro) and 3.45 µM enzyme concentration. Co-crystal structures of HcProRS in complex with these compounds and Pro confirmed the initial docking studies and show how the Pro facilitates binding of the ligands that compete with ATP substrate. Modelling 3b into other human class II aminoacyl-tRNA synthetases (aaRSs) indicated that the subtle differences in the ATP binding site of these enzymes likely contribute to its potential selective binding of HcProRS. Taken together, this study successfully identified novel HcProRS binders from our anti-tuberculosis in-house compound library, displaying opportunities for repurposing old drug candidates for new applications such as therapeutics in HcProRS-related diseases.

High-energy photons in XUV range and attosecond pulses are generated from infrared laser pulses through high harmonic generation in gases and solids. Here, the authors demonstrate the microscopic origin of resonant harmonic generation involving the autoionizing states of Sn in plasma plumes.

Plasminogen activator inhibitor-1 (PAI-1), a key regulator of the fibrinolytic system, is the main physiological inhibitor of plasminogen activators. By interacting with matrix components, including vitronectin (Vn), PAI-1 plays a regulatory role in tissue remodeling, cell migration, and intracellular signaling. Emerging evidence points to a role for PAI-1 in various pathological conditions, including cardiovascular diseases, cancer, and fibrosis. Targeting PAI-1 is therefore a promising therapeutic strategy in PAI-1-related pathologies. A class of small molecule inhibitors including TM5441 and TM5484, designed to bind the cleft in the central β-sheet A of PAI-1, showed to be potent PAI-1 inhibitors in vivo. However, their binding site has not yet been confirmed. Here, we report two X-ray crystallographic structures of PAI-1 in complex with TM5484. The structures revealed a binding site at the flexible joint region, which is distinct from the presumed binding site. Based on the structural analysis and biochemical data we propose a mechanism for the observed dose-dependent two-step mechanism of PAI-1 inhibition. By binding to the flexible joint region in PAI-1, TM5484 might restrict the structural flexibility of this region, thereby inducing a substrate form of PAI-1 followed by a conversion to an inert form.

Aminoacyl-tRNA synthetases (aaRSs) have become viable targets for the development of antimicrobial agents due to their crucial role in protein translation. A series of six amino acids were coupled to the purine-like 7-amino-5-hydroxymethylbenzimidazole nucleoside analogue following an optimized synthetic pathway. These compounds were designed as aaRS inhibitors and can be considered as 1,3-dideazaadenine analogues carrying a 2-hydroxymethyl substituent. Despite our intentions to obtain N1-glycosylated 4-aminobenzimidazole congeners, resembling the natural purine nucleosides glycosylated at the N9-position, we obtained the N3-glycosylated benzimidazole derivatives as the major products, resembling the respective purine N7-glycosylated nucleosides. A series of X-ray crystal structures of class I and II aaRSs in complex with newly synthesized compounds revealed interesting interactions of these “base-flipped” analogues with their targets. While the exocyclic amine of the flipped base mimics the reciprocal interaction of the N3-purine atom of aminoacyl-sulfamoyl adenosine (aaSA) congeners, the hydroxymethyl substituent of the flipped base apparently loses part of the standard interactions of the adenine N1 and the N6-amine as seen with aaSA analogues. Upon the evaluation of the inhibitory potency of the newly obtained analogues, nanomolar inhibitory activities were noted for the leucine and isoleucine analogues targeting class I aaRS enzymes, while rather weak inhibitory activity against the corresponding class II aaRSs was observed. This class bias could be further explained by detailed structural analysis.

Plasminogen activator inhibitor-1 (PAI-1) is the main physiological inhibitor of tissue-type (tPA) and urokinase-type (uPA) plasminogen activators (PAs). Apart from being critically involved in fibrinolysis and wound healing, emerging evidence indicates that PAI-1 plays an important role in many diseases, including cardiovascular disease, tissue fibrosis, and cancer. Targeting PAI-1 is therefore a promising therapeutic strategy in PAI-1 related pathologies. Despite ongoing efforts no PAI-1 inhibitors were approved to date for therapeutic use in humans. A better understanding of the molecular mechanisms of PAI-1 inhibition is therefore necessary to guide the rational design of PAI-1 modulators. Here, we present a 1.9 Å crystal structure of PAI-1 in complex with an inhibitory nanobody VHH-s-a93 (Nb93). Structural analysis in combination with biochemical characterization reveals that Nb93 directly interferes with PAI-1/PA complex formation and stabilizes the active conformation of the PAI-1 molecule.

The molecular architecture and assembly mechanism of intermediate filaments have been enigmatic for decades. Among those, lamin filaments are of particular interest due to their universal role in cell nucleus and numerous disease-related mutations. Filament assembly is driven by specific interactions of the elementary dimers, which consist of the central coiled-coil rod domain flanked by non-helical head and tail domains. We aimed to investigate the longitudinal ‘head-to-tail’ interaction of lamin dimers (the so-called ACN interaction), which is crucial for filament assembly. To this end, we prepared a series of recombinant fragments of human lamin A centred around the N- and C-termini of the rod. The fragments were stabilized by fusions to heterologous capping motifs which provide for a correct formation of parallel, in-register coiled-coil dimers. As a result, we established crystal structures of two N-terminal fragments one of which highlights the propensity of the coiled-coil to open up, and one C-terminal rod fragment. Additional studies highlighted the capacity of such N- and C-terminal fragments to form specific complexes in solution, which were further characterized using chemical cross-linking. These data yielded a molecular model of the ACN complex which features a 6.5 nm overlap of the rod ends.

Ubiquitously expressed human small heat shock proteins (sHsps) HspB1, HspB5, HspB6 and HspB8 contain a conserved motif (S/G)RLFD in their N-terminal domain. For each of them, we prepared mutants with a replacement of the conserved R by A (R/A mutants) and a complete deletion of the pentapeptide (Δ mutants) and analyzed their heterooligomerization with other wild-type (WT) human sHsps. We found that WT HspB1 and HspB5 formed heterooligomers with HspB6 only upon heating. In contrast, both HspB1 mutants interacted with WT HspB6 even at low temperature. HspB1/HspB6 heterooligomers revealed a broad size distribution with equimolar ratio suggestive of heterodimers as building blocks, while HspB5/HspB6 heterooligomers had an approximate 2:1 ratio. In contrast, R/A or Δ mutants of HspB6, when mixed with either HspB1 or HspB5, resulted in heterooligomers with a highly variable molar ratio and a decreased HspB6 incorporation. No heterooligomerization of HspB8 or its mutants with either HspB1 or HspB5 could be detected. Finally, R/A or Δ mutations had no effect on heterooligomerization of HspB1 and HspB5 as analyzed by ion exchange chromatography. We conclude that the conserved N-terminal motif plays an important role in heterooligomer formation, as especially pronounced in HspB6 lacking the C-terminal IXI motif.

The efficient generation of the coherent XUV light via frequency conversion of intense laser drivers is a problem of both fundamental and technological importance. Increasing the intensity of the generated high harmonics by raising the intensity of the driving field works only up to a point: at high intensities, rapid ionisation of the medium limits the conversion efficiency. Considering the combined effect of the phase-matching and of the blue shift of the driving field during its propagation in a rapidly ionising medium, we show that the latter can be the dominant limiting mechanism. We introduce a new spatial scale, the blue-shift length, which sets the upper bound for the quadratic intensity growth of the generated harmonics. Moreover, we show that this seemingly fundamental restriction can be overcome by using an additional generating weak mid-IR field. For specific combinations of frequencies of the generating fields, the corresponding high-order frequency-mixing process does not suffer from the blue shift of the drivers and phase mismatch, and thus its efficiency grows quadratically with propagation distance. Our results thus open a new route for highly efficient generation of coherent XUV light.

The flavivirus family contains several important human pathogens, such as Zika virus (ZIKV), dengue, West Nile, and Yellow Fever viruses, that collectively lead to a large, global disease burden. Currently, there are no approved medicines that can target these viruses. The sudden outbreak of ZIKV infections in 2015⁻2016 posed a serious threat to global public health. While the epidemic has receded, persistent reservoirs of ZIKV infection can cause reemergence. Here, we have used X-ray crystallography-based screening to discover two novel sites on ZIKV NS3 helicase that can bind drug-like fragments. Both sites are structurally conserved in other flaviviruses, and mechanistically significant. The binding poses of four fragments, two for each of the binding sites, were characterized at atomic precision. Site A is a surface pocket on the NS3 helicase that is vital to its interaction with NS5 polymerase and formation of the flaviviral replication complex. Site B corresponds to a flexible, yet highly conserved, allosteric site at the intersection of the three NS3 helicase domains. Saturation transfer difference nuclear magnetic resonance (NMR) experiments were additionally used to evaluate the binding strength of the fragments, revealing dissociation constants (KD) in the lower mM range. We conclude that the NS3 helicase of flaviviruses is a viable drug target. The data obtained open opportunities towards structure-based design of first-in-class anti-ZIKV compounds, as well as pan-flaviviral therapeutics.

Although the N-terminal domain of vertebrate small heat shock proteins (sHsp) is poorly conserved, it contains a core motif preserved in many members of the sHsp family. The role of this RLFDQxFG motif remains elusive. We analyzed the specific role of the first arginine residue of this conserved octet sequence in five human sHsps (HspB1, HspB4, HspB5, HspB6, and HspB8). Substitution of this arginine with an alanine induced changes in thermal stability and/or intrinsic fluorescence of the related HspB1 and HspB8, but yielded only modest changes in the same biophysical properties of HspB4, HspB5, and HspB6 which together belong to another clade of vertebrate sHsps. Removal of the positively charged Arg side chain resulted in destabilization of the large oligomers of HspB1 and formation of smaller size oligomers of HspB5. The mutation induced only minor changes in the structure of HspB4 and HspB6. In contrast, the mutation in HspB8 was accompanied by shifting the equilibrium from dimers towards the formation of larger oligomers. We conclude that the RLFDQxFG motif plays distinct roles in the structure of several sHsp orthologs. This role correlates with the evolutionary relationship of the respective sHsps, but ultimately, it reflects the sequence context of this motif.

We suggest a new method of attosecond pulse train measurement based on two-photon Raman-type resonant interaction of the XUV with an atom (Attosecond pulse reconstruction using Raman-type Transitions, ART). We show analytically and numerically that the attosecond pulse auto-correlation function and chirp can be measured using this process. Its resonant nature allows obtaining reasonable signal even for relatively low XUV intensity. The interpretation of the results is remarkably simple for the XUV generated by bi-circular field. The experimental realization of the method can be based either on measuring the excitation of the gas or on four-wave mixing fully optical scheme.

Physico-chemical properties of the mutations G34R, P39L and E41K in the N-terminal domain of human heat shock protein B1 (HspB1), which have been associated with hereditary motor neuron neuropathy, were analyzed. Heat-induced aggregation of all mutants started at lower temperatures than for the wild type protein. All mutations decreased susceptibility of the N- and C-terminal parts of HspB1 to chymotrypsinolysis. All mutants formed stable homooligomers with a slightly larger apparent molecular weight compared to the wild type protein. All mutations analyzed decreased or completely prevented phosphorylation-induced dissociation of HspB1 oligomers. When mixed with HspB6 and heated, all mutants yielded heterooligomers with apparent molecular weights close to ~400 kDa. Finally, the three HspB1 mutants possessed lower chaperone-like activity towards model substrates (lysozyme, malate dehydrogenase and insulin) compared to the wild type protein, conversely the environmental probe bis-ANS yielded higher fluorescence with the mutants than with the wild type protein. Thus, in vitro the analyzed N-terminal mutations increase stability of large HspB1 homooligomers, prevent their phosphorylation-dependent dissociation, modulate their interaction with HspB6 and decrease their chaperoning capacity, preventing normal functioning of HspB1.

We propose a new method for production of ultracold neutrons (UCNs) in superfluid helium. The principal idea consists in installing a helium UCN source into an external beam of thermal or cold neutrons and in surrounding this source with a solid methane moderator/reflector cooled down to ~4 K. The moderator plays the role of an external source of cold neutrons needed to produce UCNs. The flux of accumulated neutrons could exceed the flux of incident neutrons due to their numerous reflections from methane; also the source size could be significantly larger than the incident beam diameter. We provide preliminary calculations of cooling of neutrons. These calculations show that such a source being installed at an intense source of thermal or cold neutrons like the ILL or PIK reactor or the ESS spallation source could provide the UCN density 105 cm−3, the production rate 107 UCN/s−1. Main advantages of such an UCN source include its low radiative and thermal load, relatively low cost, and convenient accessibility for any maintenance. We have carried out an experiment on cooling of thermal neutrons in a methane cavity. The data confirm the results of our calculations of the spectrum and flux of neutrons in the methane cavity.

HSPB6 is a member of the human small heat shock protein (sHSP) family, a conserved group of molecular chaperones that bind partially unfolded proteins and prevent them from aggregating. In vertebrate sHSPs the poorly structured N-terminal domain has been implicated in both chaperone activity and the formation of higher-order oligomers. These two functionally important properties are likely intertwined at the sequence level, complicating attempts to delineate the regions that define them. Differing from the prototypical α-crystallins human HSPB6 has been shown to only form dimers in solution making it more amendable to explore the determinants of chaperoning activity alone. Using a systematic and iterative deletion strategy, we have extensively investigated the role of the N-terminal domain on the chaperone activity of this sHSP. As determined by size-exclusion chromatography and small-angle X-ray scattering, most mutants had a dimeric structure closely resembling that of wild-type HSPB6. The chaperone-like activity was tested using three different substrates, whereby no single truncation, except for complete removal of the N-terminal domain, showed full loss of activity, pointing to the presence of multiple sites for binding unfolding proteins. Intriguingly, we found that the stretch encompassing residues 31 to 35, which is nearly fully conserved across vertebrate sHSPs, acts as a negative regulator of activity, as its deletion greatly enhanced chaperoning capability. Further single point mutational analysis revealed an interplay between the highly conserved residues Q31 and F33 in fine-tuning its function.

The anti-apoptotic Bcl-2 protein is the founding member and namesake of the Bcl-2-protein family. It has recently been demonstrated that Bcl-2, apart from its anti-apoptotic role at mitochondrial membranes, can also directly interact with the inositol 1,4,5-trisphosphate receptor (IP3R), the primary Ca(2+)-release channel in the endoplasmic reticulum (ER). Bcl-2 can thereby reduce pro-apoptotic IP3R-mediated Ca(2+) release from the ER. Moreover, the Bcl-2 homology domain 4 (Bcl-2-BH4) has been identified as essential and sufficient for this IP3R-mediated anti-apoptotic activity. In the present study, we investigated whether the reported inhibitory effect of a Bcl-2-BH4 peptide on the IP 3R1 was related to the distinctive α-helical conformation of the BH4 domain peptide. We therefore designed a peptide with two glycine "hinges" replacing residues I14 and V15, of the wild-type Bcl-2-BH4 domain (Bcl-2-BH4-IV/GG). By comparing the structural and functional properties of the Bcl-2-BH4-IV/GG peptide with its native counterpart, we found that the variant contained reduced α-helicity, neither bound nor inhibited the IP 3R1 channel, and in turn lost its anti-apoptotic effect. Similar results were obtained with other substitutions in Bcl-2-BH4 that destabilized the α-helix with concomitant loss of IP3R inhibition. These results provide new insights for the further development of Bcl-2-BH4-derived peptides as specific inhibitors of the IP3R with significant pharmacological implications.