Alternative Computational Protocols for Supercharging Protein Surfaces for Reversible Unfolding and Retention of Stability

Bryan S. Der, Ron Jacak, Brian Kuhlman, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of AmericaChristien Kluwe, Aleksandr E. Miklos, Andrew D. Ellington , Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas, United States of AmericaChristien Kluwe, Aleksandr E. Miklos, George Georgiou, Andrew D. Ellington, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, United States of AmericaAleksandr E. Miklos, Andrew D. Ellington , Applied Research Laboratories, University of Texas at Austin, Austin, Texas, United States of AmericaSergey Lyskov, Jeffrey J. Gray, Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States of AmericaBrian Kuhlman, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of AmericaReengineering protein surfaces to exhibit high net charge, referred to as “supercharging”, can improve reversibility of unfolding by preventing aggregation of partially unfolded states. Incorporation of charged side chains should be optimized while considering structural and energetic consequences, as numerous mutations and accumulation of like-charges can also destabilize the native state. A previously demonstrated approach deterministically mutates flexible polar residues (amino acids DERKNQ) with the fewest average neighboring atoms per side chain atom (AvNAPSA). Our approach uses Rosetta-based energy calculations to choose the surface mutations. Both protocols are available for use through the ROSIE web server. The automated Rosetta and AvNAPSA approaches for supercharging choose dissimilar mutations, raising an interesting division in surface charging strategy. Rosetta-supercharged variants of GFP (RscG) ranging from −11 to −61 and +7 to +58 were experimentally tested, and for comparison, we re-tested the previously developed AvNAPSA-supercharged variants of GFP (AscG) with +36 and −30 net charge. Mid-charge variants demonstrated ~3-fold improvement in refolding with retention of stability. However, as we pushed to higher net charges, expression and soluble yield decreased, indicating that net charge or mutational load may be limiting factors. Interestingly, the two different approaches resulted in GFP variants with similar refolding properties. Our results show that there are multiple sets of residues that can be mutated to successfully supercharge a protein, and combining alternative supercharge protocols with experimental testing can be an effective approach for charge-based improvement to refolding.This work was supported by the Defense Advanced Research Projects Agency (HR-0011-10-1-0052 to A.E.) and the Welch Foundation (F-1654 to A.E.), the National Institutes of Health grants GM073960 (B.K.) and R01-GM073151 (J.G. and S.L.), the Rosetta Commons (S.L.), the National Science Foundation graduate research fellowship (2009070950 to B.D.), the UNC Royster Society Pogue fellowship (B.D.), and National Institutes of Health grant T32GM008570 for the UNC Program in Molecular and Cellular Biophysics. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Center for Systems and Synthetic BiologyCellular and Molecular BiologyApplied Research LaboratoriesEmail: [email protected]

HRV Biofeedback in Neck Pain Effects of Heart Rate Variability Biofeedback in Subjects with Stress-related Chronic Neck Pain: a Pilot study

Abstract Recent studies focusing on autonomic nervous system (ANS) dysfunctions, together with theoretical pathophysiological models of musculoskeletal disorders, indicate the involvement of ANS regulation in development and maintenance of chronic muscle pain. Research has demonstrated the effectiveness of heart rate variability (HRV) biofeedback (BF) in increasing HRV and reducing the symptoms of different disorders characterized by ANS aberration. The study investigated the effects of resonance frequency HRV BF on autonomic regulation and perceived health, pain, stress and disability in 24 subjects with stress-related chronic neckshoulder pain. Twelve subjects participated in 10 weekly sessions of resonant HRV BF and were compared to a control group. Subjective reports and HRV measures during relaxation and in response to a standardized stress protocol were assessed for both groups pre-and postintervention. Group X time interactions revealed a significantly stronger increase over time in perceived health (SF-36) for the treatment group, including vitality, bodily pain and social functioning. Interactions were also seen for HRV during relaxation and reactivity to stress. The present pilot study indicates improvement in perceived health over a 10 week intervention with HRV-biofeedback in subjects with chronic neck-pain. Increased resting HRV as well as enhanced reactivity to hand grip and cold pressor tests might reflect beneficial effects on ANS regulation, and suggest that this intervention protocol is suitable for a larger controlled trial

Evolution of oxygen-ion and proton conductivity in Ca-Doped Ln2Zr2O7 (Ln = Sm, Gd), located near pyrochlore fluorite phase boundary

Sm2-xCaxZr2O7-x/2 (x = 0, 0.05, 0.1) and Gd2-xCaxZr2O7-x/2 (x = 0.05, 0.1) mixed oxides in a pyrochlore-fluorite morphotropic phase region were prepared via the mechanical activation of oxide mixtures, followed by annealing at 1600 ?C. The structure of the solid solutions was studied by X-ray diffraction and refined by the Rietveld method, water content was determined by thermogravimetry (TG), their bulk and grain-boundary conductivity was determined by impedance spectroscopy in dry and wet air (100-900 ?C), and their total conductivity was measured as a function of oxygen partial pressure in the temperature range: 700-950 ?C. The Sm2-xCaxZr2O7-x/2 (x = 0.05, 0.1) pyrochlore solid solutions, lying near the morphotropic phase boundary, have proton conductivity contribution both in the grain bulk and on grain boundaries below 600 ?C, and pure oxygen-ion conductivity above 700 ?C. The 500 ?C proton conductivity contribution of Sm2-xCaxZr2O7-x/2 (x = 0.05, 0.1) is ~ 1 ? 10-4 S/cm. The fluorite-like Gd2-xCaxZr2O7-x/2 (x = 0.1) solid solution has oxygen-ion bulk conductivity in entire temperature range studied, whereas proton transport contributes to its grain-boundary conductivity below 700 ?C. As a result, of the morphotropic phase transition from pyrochlore Sm2-xCaxZr2O7-x/2 (x = 0.05, 0.1) to fluorite-like Gd2-xCaxZr2O7-x/2 (x = 0.05, 0.1), the bulk proton conductivity disappears and oxygen-ion conductivity decreases. The loss of bulk proton conductivity of Gd2-xCaxZr2O7-x/2 (x = 0.05, 0.1) can be associated with the fluorite structure formation. It is important to note that the degree of Ca substitution in such solid solutions (Ln2-xCax)Zr2O7-? (Ln = Sm, Gd) is low, x < 0.1. In both series, grain-boundary conductivity usually exceeds bulk conductivity. The high grain-boundary proton conductivity of Ln2-xCaxZr2O7-x/2 (Ln = Sm, Gd; x = 0.1) is attributable to the formation of an intergranular CaZrO3-based cubic perovskite phase doped with Sm or Gd in Zr sublattice. ? 2019 by the authors.371C-9F16-EBDE | Eduarda GomesN/

Патентование лекарственных средств и государственная регистрация лекарственных препаратов: подводные камни

During the lifecycle of a medicine - from its development to consumption - the developer has to go through several stages, such as obtaining the exclusive right for a medicine, obtaining permission to produce the medicine, obtaining permission to distribute and use the medicine, i.e. the developer has to patent and register the invention and obtain a marketing authorization for it. In order to accomplish these procedures that fall within the remit of the Federal Service for Intellectual Property and the Ministry of Health of Russia, developers have to submit documents described in relevant legislative acts. The article describes the patenting procedure used by the Federal Service for Intellectual Property, including examination as to form and substantive examination. It also addresses some problems associated with patenting medicines and their state authorization. A conclusion is drawn on the importance of searching for unexpired patents and their analysis in order to prevent situations in which developers of medicines infringe the rights of others.На пути лекарственного средства от его создания до потребителя разработчику предстоит преодолеть такие стадии, как приобретение исключительного права на лекарственное сродство и получение разрешения на его производство, реализацию, применение, т.е. необходимо запатентовать и зарегистрировать разработку, получить регистрационное удостоверение. Для осуществления этих процедур, проводимых Роспатентом и Минздравом России, требуются разные документы, установленные соответствующими законодательными актами. Описана процедура патентования в Роспатенте, включающая в себя формальную экспертизу и экспертизу по существу. Рассмотрены проблемы, связанные с патентованием лекарственных средств и их государственной регистрацией. Сделаны выводы об актуальности поиска действующих патентов и их анализа на предмет нарушения чьих-либо прав

Association of Tinnitus and Electromagnetic Hypersensitivity: Hints for a Shared Pathophysiology?

BACKGROUND: Tinnitus is a frequent condition with high morbidity and impairment in quality of life. The pathophysiology is still incompletely understood. Electromagnetic fields are discussed to be involved in the multi-factorial pathogenesis of tinnitus, but data proofing this relationship are very limited. Potential health hazards of electromagnetic fields (EMF) have been under discussion for long. Especially, individuals claiming themselves to be electromagnetic hypersensitive suffer from a variety of unspecific symptoms, which they attribute to EMF-exposure. The aim of the study was to elucidate the relationship between EMF-exposure, electromagnetic hypersensitivity and tinnitus using a case-control design. METHODOLOGY: Tinnitus occurrence and tinnitus severity were assessed by questionnaires in 89 electromagnetic hypersensitive patients and 107 controls matched for age-, gender, living surroundings and workplace. Using a logistic regression approach, potential risk factors for the development of tinnitus were evaluated. FINDINGS: Tinnitus was significantly more frequent in the electromagnetic hypersensitive group (50.72% vs. 17.5%) whereas tinnitus duration and severity did not differ between groups. Electromagnetic hypersensitivity and tinnitus were independent risk factors for sleep disturbances. However, measures of individual EMF-exposure like e.g. cell phone use did not show any association with tinnitus. CONCLUSIONS: Our data indicate that tinnitus is associated with subjective electromagnetic hypersensitivity. An individual vulnerability probably due to an over activated cortical distress network seems to be responsible for, both, electromagnetic hypersensitivity and tinnitus. Hence, therapeutic efforts should focus on treatment strategies (e.g. cognitive behavioral therapy) aiming at normalizing this dysfunctional distress network

Proton Conductivity of La2 (Hf2−x Lax )O7−x/2 “Stuffed” Pyrochlores

The design of new oxygen-and proton-conducting materials is of paramount importance for their possible utilization in solid oxide fuel cells. In the present work, La2 (Hf2–x Lax )O7–x/2 (x = 0, 0.1) ceramics were prepared using ball milling of oxide mixtures (La2 O3 and HfO2 ) followed by high-temperature annealing at 1600◦ C for 10 h in air. La2 Hf2 O7 ceramics exhibit an ordered pyrochlore-type structure, whereas La2 (Hf1.9 La0.1)O6.95 has a defect pyrochlore structure type with oxygen vacancies at the 48f positions. The oxygen ion and proton conductivity of La2 (Hf1.9 La0.1 )O6.95 “stuffed” pyrochlore ceramics was investigated by electrochemical impedance spectroscopy (two-probe AC) and four-probe DC measurements in a dry and a wet atmosphere (air and nitrogen). The use of two distinct conductivity measurement techniques ensured, for the first time, the collection of reliable data on the proton conductivity of the La2 (Hf1.9 La0.1)O6.95 “stuffed” hafnate pyrochlore. La2 Hf2 O7 was found to be a dielectric in the range 400–900◦ C, whereas the La2 (Hf1.9 La0.1 )O6.95 “stuffed” pyrochlore had both oxygen ion and proton conductivities in this temperature range. The proton conductivity level was found to be equal to ~8 × 10−5 S/cm at 700◦ C. Clearly, the proton conductivity of the La2 (Hf1.9 La0.1 )O6.95 “stuffed” hafnate pyrochlore is mainly due to the hydration of oxygen vacancies at 48f positions. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

Mutation D816V Alters the Internal Structure and Dynamics of c-KIT Receptor Cytoplasmic Region: Implications for Dimerization and Activation Mechanisms

The type III receptor tyrosine kinase (RTK) KIT plays a crucial role in the transmission of cellular signals through phosphorylation events that are associated with a switching of the protein conformation between inactive and active states. D816V KIT mutation is associated with various pathologies including mastocytosis and cancers. D816V-mutated KIT is constitutively active, and resistant to treatment with the anti-cancer drug Imatinib. To elucidate the activating molecular mechanism of this mutation, we applied a multi-approach procedure combining molecular dynamics (MD) simulations, normal modes analysis (NMA) and binding site prediction. Multiple 50-ns MD simulations of wild-type KIT and its mutant D816V were recorded using the inactive auto-inhibited structure of the protein, characteristic of type III RTKs. Computed free energy differences enabled us to quantify the impact of D816V on protein stability in the inactive state. We evidenced a local structural alteration of the activation loop (A-loop) upon mutation, and a long-range structural re-organization of the juxta-membrane region (JMR) followed by a weakening of the interaction network with the kinase domain. A thorough normal mode analysis of several MD conformations led to a plausible molecular rationale to propose that JMR is able to depart its auto-inhibitory position more easily in the mutant than in wild-type KIT and is thus able to promote kinase mutant dimerization without the need for extra-cellular ligand binding. Pocket detection at the surface of NMA-displaced conformations finally revealed that detachment of JMR from the kinase domain in the mutant was sufficient to open an access to the catalytic and substrate binding sites