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This study aimed to better characterize a recently purified stable extracellular alkaline peptidase produced by Penicillium aurantiogriseum (URM 4622) through fluorescence spectroscopy, far-UV circular dichroism, kinetic and thermodynamic models to understand its' structure-activity and denaturation. Fluorescence data showed that changing pH leads to tryptophan residues exposure to more hydrophilic environments at optimum activity pH 9.0 and 10.0. When thermally treated, it displayed less unfolding at these pH values, along with 4-fold less photoproducts formation than at neutral pH. Different pH CD spectra showed more β-sheet (21.5-43.0%) than α-helix (1-6.2%). At pH9.0, more than 2-fold higher α-helix content than any other pH. The melting temperature (Tm) was observed between 50 and 60 °C at all pH studied, with lower Tm at pH 9.0-11.0 (54.9-50.3 °C). The protease displayed two phase transition, with two energies of denaturation, and a 4-fold higher thermal stability (ΔH°m) than reports for other microorganism's proteases. An irreversible folding transition occurs between 50 and 60 °C. It displayed energies of denaturation suggesting higher thermal stability than reported for other microorganism's proteases. These results help elucidating the applicability of this new stable protease.

Talk presented at the14th Conference on the Intersections of Particle and Nuclear Physics (CIPANP 2022), Lake Buena Vista, Florida. September 3 2022.&nbsp

The aim of this thesis was to increase our knowledge of the glassy state and the glass transition phenomenon and to evaluate the effect of water on the glassy state. To accomplish this, investigations were focused on the amorphous sucrose-water (paper I, II, III), trehalose-water (paper I), maltodextrin-water (paper I), and lysozyme-sucrose-water (paper IV) systems. We studied temperature-induced and isothermal glass transition (I, II, III, IV), as well as the impact of water on the activation energy of the relaxation process (II). It has been shown that water undergoes glass transition with disaccharides, but in polysaccharides water dynamics is uncoupled from the polymer matrix. This results in differences in the water diffusion coefficient: water moves several orders of magnitude faster in the polymers than in disaccharides (I). Water reduces the activation energy of the relaxation process in the sucrose-water system (II). Attenuated water diffusion at sub-zero temperatures leads to a delay in water crystallization/melting in the sucrose-water system, which does not happen in a polysaccharide - water system (III). The Tg of the lysozyme-sucrose system increases with increasing lysozyme concentration, i.e., the DCp of the mixtures does not follow the prediction based on the properties of the pure components. Consequently, lysozyme does not modulate the glass transition of the sucrose matrix and the increase of the Tg of the mixtures is a result of the confinement of amorphous sucrose in the space between lysozyme molecules. The amorphous structure and unfolding of lysozyme in the presence of sucrose was investigated by DSC and SAXS. These data revealed an increase of the protein-protein distance upon addition of sucrose and upon heating, as a result of lysozyme unfolding (IV).

As the ownership of electric vehicles keeps rising, an increasing power burden is inflicted upon the existing grid infrastructure, which will soon be unable to supply enough power from the medium voltage transmission lines. Replacing this infrastructure will come at a high cost and years of construction. Consequently, there has been a need for creative and adaptable solutions that are capable of supporting the existing grid infrastructure as well as meeting the increasing peak power demand. Grid supporting solutions include static wireless charging, dynamic wireless charging, and direct charging systems that are capable of processing high power from relatively high input voltages. The following thesis takes part in the development of a 560 kW, 4.16 kV medium-voltage AC-DC converter for high-power wireless charging of electric drayage trucks. This solution allows the increased power demands to be accessed directly from the medium voltage grid and bypasses the existing grid infrastructure. The scope of this thesis extends beyond the development of a single AC-DC converter by addressing complex control challenges and seeking a modularized control structure between high-power modules, which are used to form the full converter.

Human γD-crystallin (HGDC) is an abundant lens protein residing in the nucleus of the human lens. Aggregation of this and other structural proteins within the lens leads to the development of cataract. Much has been explored on the stability and aggregation of HGDC and where detailed investigation at the atomic resolution was needed, the X-ray structure was used as an initial starting conformer for molecular modeling. In this study, we implemented NMR-solution HGDC structures as starting conformers for molecular dynamics simulations to provide the missing pieces of the puzzle on the very early stages of HGDC unfolding leading up to the domain swap theories proposed by past studies. The high-resolution details of the conformational dynamics also revealed additional insights to possible early intervention for cataractogenesis.

In bacteria, Lon is a large hexameric ATP-dependent protease that targets misfolded and also folded substrates, some of which are involved in cell division and survival of cellular stress. The N-terminal domain of Lon facilitates substrate recognition, but how the domains confer such activity has remained unclear. Here, we report the full-length structure of Lon protease from Thermus thermophilus at 3.9 Å resolution in a substrate-engaged state. The six N-terminal domains are arranged in three pairs, stabilized by coiled-coil segments and forming an additional channel for substrate sensing and entry into the AAA+ ring. Sequence conservation analysis and proteolysis assays confirm that this architecture is required for the degradation of both folded and unfolded substrates in bacteria.

En este trabajo se analiza hasta qué punto el orden de administración de las opciones de respuesta afecta a la falta de respuesta y a la elección de respuestas 'no definidas'. Un segundo objetivo es considerar si la estrategia de 'descomponer' una pregunta en varias disminuye el número preguntas sin respuesta, así como de respuestas 'no definidas'. Un experimento con encuesta telefónica muestra que presentar las preguntas del cuestionario comenzando por las opciones desfavorables en preguntas descompuestas aumenta las respuestas 'no sabe', además de ampliar el tiempo de respuesta. Por el contrario, utilizar preguntas con opciones de respuesta que comienzan por las desfavorables reduce el rechazo a responder la pregunta. Las respuestas indefinidas se reducen cuando se emplea una pregunta que comienza por preguntas favorables. The impact of questionnaire format on responses has been long studied in survey research. This study seeks to contribute to this area of study by analyzing the extent to which the strategy of branching decreases the number of nonresponses and 'undefined' responses. A second objective is to consider whether nonresponse and the choice of 'undefined' responses are related to the order of administration of the response options. An experiment using a telephone survey showed that presenting a questionnaire starting with the unfavorable options and branched questions increased the 'don't know' answers and extended response time by 60 seconds. Using a reverse order with a single question reduces the number of item-nonresponse. Undefined responses are dropped when using a question that begins with favorable options.

I present an extension of https://arxiv.org/abs/2009.02913, which contained a method for solving unfolding problems without explicitly inverting the response matrix. In this extended work, the problem of setting an uncertainty in the point estimate is solved by learning a full Bayesian posterior using Markov chain Monte Carlo sampling and, where needed, Approximate Bayesian Computation., Presented at the 10th International Conference on New Frontiers in Physics (ICNFP 2021)

The SARS-CoV-2 spike is the primary target of virus-neutralizing antibodies and critical to the development of effective vaccines against COVID-19. Here, we demonstrate that the prefusion-stabilized two-proline "S2P" spike -widely employed for laboratory work and clinical studies- unfolds when stored at 4 °C, physiological pH, as observed by electron microscopy (EM) and differential scanning calorimetry, but that its trimeric, native-like conformation can be reacquired by low pH treatment. When stored for approximately one week, this unfolding does not significantly alter antigenic characteristics; however, longer storage diminishes antibody binding, and month-old spike elicits virtually no neutralization in mice despite inducing high ELISA-binding titers. Cryo-EM structures reveal the folded fraction of spike to decrease with aging, though its structure remains largely similar, although with varying mobility of the receptor-binding domain. Thus, the SARS-CoV-2 spike is susceptible to unfolding which affects immunogenicity, highlighting the need to monitor its integrity.

Talk given at theML4Jets 2021 conference (online)

Battery electric vehicles (BEVs) and plugin hybrid electric vehicles (PHEVs) are more efficient than internal combustion-based vehicles. Adaption of EVs will help reduce the carbon emissions produced by the transportation sector. The charging infrastructure has to grow at a rapid pace to encourage EV adaption. Installing higher capacity fast chargers will help alleviate the range anxiety of battery electric vehicle customers. More public charging stations are required for the full adaption of EVs. Utility power is distributed as ‘alternating current.’ A battery requires ‘direct current’ (DC) source to charge it. Hence a power converter that converts AC source to DC source is required to charge an electric vehicle battery. Public transportation is another sector that is adapting electric vehicles at a fast pace. These vehicles require more power to operate and hence have huge battery packs. These vehicles require ultra-high-power charger to keep the charging time reasonable. A 60 Hz stepdown transformer is required at the facility to use the power. The cost and time to install this heavy transformer will inhibit the setting up a charging station. Power converters than can connect to medium voltage directly will eliminate the need for the step-down transformer saving space and cost. Commercially available state-of-the-art fast charging converters are adapted from general purpose commercial and industrial application rectifiers. The efficiencies of these converters tend to be lower (around 94%) due to the two-stage power conversion architecture. All the power that flows from the AC utility grid to charge the battery will be processed and filtered through two power conversion stages. Due to the anticipated increase in demand, there is a renewed interest in developing power converter topologies specific to battery charging applications. The objective here is to develop cheaper and compact power converters for battery charging. This dissertation proposes an innovative quasi-single stage power converter topologies for battery charging applications and direct medium voltage connected converters. The proposed topology fundamentally can achieve higher efficiency and power density than the conventional two-stage based converters. Only one stage requires filtering and incurs power conversion losses. Control burden is usually higher for single stage topologies. Innovative control approaches are presented to simplify the control complexity.

Compared with more rigid objects, clothing items are inherently difficult for robots to recognize and manipulate. We propose a method for detecting how cloth is folded, to facilitate choosing a manipulative action that corresponds to a garment’s shape and position. The proposed method involves classifying the edges and corners of a garment by distinguishing between edges formed by folds and the hem or ragged edge of the cloth. Identifying the type of edges in a corner helps to determinate how the object is folded. This bottom-up approach, together with an active perception system, allows us to select strategies for robotic manipulation. We corroborate the method using a two-armed robot to manipulate towels of different shapes, textures, and sizes.

Talk given at theDIS-2021 is the 28th in the series of annual workshops on Deep-Inelastic Scattering (DIS) and Related Subjects, Santiago de Compostela, Spain.

In this paper we compare the notion of stochastic two-scale convergence in the mean (by Bourgeat, Mikeli\'c and Wright), the notion of stochastic unfolding (recently introduced by the authors), and the quenched notion of stochastic two-scale convergence (by Zhikov and Pyatnitskii). In particular, we introduce stochastic two-scale Young measures as a tool to compare mean and quenched limits. Moreover, we discuss two examples, which can be naturally analyzed via stochastic unfolding, but which cannot be treated via quenched stochastic two-scale convergence., Comment: 30 pages. arXiv admin note: substantial text overlap with arXiv:1805.09546

Significance The cerebellum has long been recognized as a partner of the cerebral cortex, and both have expanded greatly in human evolution. The thin cerebellar cortex is even more tightly folded than the cerebral cortex. By scanning a human cerebellum specimen at ultra-high magnetic fields, we were able to computationally reconstruct its surface down to the level of the smallest folds, revealing that the cerebellar cortex has almost 80% of the surface area of the cerebral cortex. By performing the same procedure on a monkey brain, we found that the surface area of the human cerebellum has expanded even more than that of the human cerebral cortex, suggesting a role in characteristically human behaviors, such as toolmaking and language., The surface of the human cerebellar cortex is much more tightly folded than the cerebral cortex. It was computationally reconstructed for the first time to the level of all individual folia from multicontrast high-resolution postmortem MRI scans. Its total shrinkage-corrected surface area (1,590 cm2) was larger than expected or previously reported, equal to 78% of the total surface area of the human neocortex. The unfolded and flattened surface comprised a narrow strip 10 cm wide but almost 1 m long. By applying the same methods to the neocortex and cerebellum of the macaque monkey, we found that its cerebellum was relatively much smaller, approximately 33% of the total surface area of its neocortex. This suggests a prominent role for the cerebellum in the evolution of distinctively human behaviors and cognition.

The temperature-dependent secondary structure of two monoclonal IgG antibodies, anti-IGF1R and anti-TSLP, were examined by transmission mode Fourier Transform Infrared (FTIR) spectroscopy. Anti-IGF1R and anti-TSLP are IgG monoclonal antibodies (mAbs) directed against human Insulin-like Growth Factor 1 Receptor for anti-tumor activity and Thymic Stromal Lymphopoietin cytokine for anti-asthma activity, respectively. Differential scanning calorimetry (DSC) clearly indicates both antibodies in their base formulations have a lower temperature protein conformational change near 70 °C (Tm1) and a higher temperature protein conformational change near 85 °C (Tm2). Thermal scanning dynamic light scatting (TS-DLS) indicates a significant particle size increase for both antibodies near Tm2 suggesting a high level of protein aggregation. The nature of these protein conformational changes associated with increasing the formulation temperature and decreasing sucrose concentration were identified by transmission mode FTIR and second derivative FTIR spectroscopy of temperature controlled aqueous solutions of both monoclonal antibodies. The transition from intra-molecular β sheets to inter-molecular β sheets was clearly captured for both monoclonal antibodies using FTIR spectroscopy. Finally, FTIR Spectroscopy was able to show the impact of a common excipient such as sucrose on the stability of each monoclonal antibody, further demonstrating the usefulness of FTIR spectroscopy for studying protein aggregation and formulation effects.

Pharmacogenomics is a powerful tool to prevent adverse reactions caused by different response of individuals to drug administration. Single nucleotide polymorphisms (SNPs) represent up to 90% of genetic variations among individuals. Drug metabolizing enzymes are highly polymorphic therefore the kinetic parameters of their catalytic reactions can be significantly influenced. This work reports on the unfolding process of a phase I drug metabolizing enzyme, human flavin-containing monooxygenase 3 (hFMO3) and its single nucleotide polymorphic variants (SNPs) V257M, E158K and E308G. Differential scanning calorimetry (DSC) indicates that the thermal denaturation of the enzyme is irreversible. The melting temperature (Tm) for the (Wild Type) WT and its polymorphic variants is found to be in a range from 46 °C to 50 °C. Also the activation energies of unfolding (Ea) show no significant differences among all proteins investigated (290–328 KJ/mol), except for the E308G variant that showed a significantly higher Ea of 412 KJ/mol. The presence of the bound NADP+ cofactor is found to stabilize all the variants by shifting the main Tm by 4–5 °C for all the proteins, exception made for E308G where no changes are observed. Isothermal titration calorimetry (ITC) was used to characterize the interaction of the protein with NADP+ in terms of dissociation constant (Kd), enthalpy (ΔH) and entropy (ΔS). Kd values of 1.6 and 0.7 μM, ΔH of −13.9 Kcal/mol and −16.8 Kcal/mol, ΔS of −20.5 cal/mol/deg, and −28.5 cal/mol/deg were found for V257M and E158K respectively. E308G was found to be unable to bind the NADP+ cofactor, a result that is in line with the Tm results. Circular dichroism also confirmed an overall lower stability of E308G, while NADP+ was found to give a strong positive shift of the Tm stabilizing the structure of E158K (46.2 to 50.6 °C). Previous data highlighted significant differences in terms of activity among the SNPs of hFMO3. In this work a minor impact of the SNPs was found on the stability of the enzyme in the ligand free form, except for E308G, whereas the binding of NADP+ reveals major differences among WT and polymorphic variants that are all measurable in terms of heat capacity, enthalpy and secondary structure content. These data provide the first direct evidence of ligand stabilization effects on hFMO3 that can explain the differences observed in catalytic efficiencies and serve as the starting point for the development of inhibitors of this enzyme.

The scientific study of consciousness is currently undergoing a critical transition in the form of a rapidly evolving scientific debate regarding whether or not currently proposed theories can be assessed for their scientific validity. At the forefront of this debate is Integrated Information Theory (IIT), widely regarded as the preeminent theory of consciousness because of its quantification of consciousness in terms a scalar mathematical measure called $\Phi$ that is, in principle, measurable. Epistemological issues in the form of the "unfolding argument" have provided a refutation of IIT by demonstrating how it permits functionally identical systems to have differences in their predicted consciousness. The implication is that IIT and any other proposed theory based on a system's causal structure may already be falsified even in the absence of experimental refutation. However, so far the arguments surrounding the issue of falsification of theories of consciousness are too abstract to readily determine the scope of their validity. Here, we make these abstract arguments concrete by providing a simple example of functionally equivalent machines realizable with table-top electronics that take the form of isomorphic digital circuits with and without feedback. This allows us to explicitly demonstrate the different levels of abstraction at which a theory of consciousness can be assessed. Within this computational hierarchy, we show how IIT is simultaneously falsified at the finite-state automaton (FSA) level and unfalsifiable at the combinatorial state automaton (CSA) level. We use this example to illustrate a more general set of criteria for theories of consciousness: to avoid being unfalsifiable or already falsified scientific theories of consciousness must be invariant with respect to changes that leave the inference procedure fixed at a given level in a computational hierarchy., Comment: 10 pages, 8 figures

In this paper, the induced sequence of folding and unfolding on the fundamental group will be obtained from a sequence of folding and unfolding on a manifold. The limit of folding and unfolding on the fundamental group is deduced. The sequences of the commutative diagram of fundamental groups will be achieved from the sequences of the commutative diagram of manifolds. The connection between a manifold and a fundamental group is assigned.

Friedreich's ataxia is a disease caused by a decrease in the levels of expression or loss of functionality of the mitochondrial protein frataxin (FXN). The development of an active and stable recombinant variant of FXN is important for protein replacement therapy. Although valuable data about the mature form FXN81‐210 has been collected, not enough information is available about the conformation of the frataxin precursor (FXN1‐210). We investigated the conformation, stability and function of a recombinant precursor variant (His6‐TAT‐FXN1‐210), which includes a TAT peptide in the N‐terminal region to assist with transport across cell membranes. His6‐TAT‐FXN1‐210 was expressed in Escherichia coli and conditions were found for purifying folded protein free of aggregation, oxidation or degradation, even after freezing and thawing. The protein was found to be stable and monomeric, with the N‐terminal stretch (residues 1–89) mostly unstructured and the C‐terminal domain properly folded. The experimental data suggest a complex picture for the folding process of full‐length frataxin in vitro: the presence of the N‐terminal region increased the tendency of FXN to aggregate at high temperatures but this could be avoided by the addition of low concentrations of GdmCl. The purified precursor was translocated through cell membranes. In addition, immune response against His6‐TAT‐FXN1‐210 was measured, suggesting that the C‐terminal fragment was not immunogenic at the assayed protein concentrations. Finally, the recognition of recombinant FXN by cellular proteins was studied to evaluate its functionality. In this regard, cysteine desulfurase NFS1/ISD11/ISCU was activated in vitro by His6‐TAT‐FXN1‐210. Moreover, the results showed that His6‐TAT‐FXN1‐210 can be ubiquitinated in vitro by the recently identified frataxin E3 ligase RNF126, in a similar way as the FXN1‐210, suggesting that the His6‐TAT extension does not interfere with the ubiquitination machinery. Fil: Castro, Ignacio Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Ferrari, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Herrera, Maria Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Noguera, Martín Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Maso, Lorenzo. Universita Di Padova. Dipartimento Di Biología; Italia Fil: Benini, Monica. Fratagene Therapeutics Srl; Italia. University Of Rome Tor Vergata; Italia Fil: Rufini, Alessandra. University Of Rome Tor Vergata; Italia. Fratagene Therapeutics Srl; Italia Fil: Testi, Roberto. University Of Rome Tor Vergata; Italia. Fratagene Therapeutics Srl; Italia Fil: Costantini, Paola. Universita Di Padova. Dipartimento Di Biología; Italia Fil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

This article focuses on computing minimal test suites for multithreaded programs. Based on previous work on test case generation for multithreaded programs using unfoldings, this article shows how this unfolding can be used to generate minimal test suites covering all local states of the program. Generating such minimal test suites is shown to be NP-complete in the size of the unfolding. We propose an SMT encoding for this problem and two methods based on heuristics which only approximate the solution, but scale better in practice. Finally, we apply our methods to compute the minimal test suites for several benchmarks.

Proteolytic susceptibility during endolysosomal degradation is decisive for allergic sensitization. In the major birch pollen allergen Bet v 1 most protease cleavage sites are located within its secondary structure elements, which are inherently inaccessible to proteases. The allergen thus must unfold locally, exposing the cleavage sites to become susceptible to proteolysis. Hence, allergen cleavage rates are presumed to be linked to their fold stability, i.e., unfolding probability. Yet, these locally unfolded structures have neither been captured in experiment nor simulation due to limitations in resolution and sampling time, respectively. Here, we perform classic and enhanced molecular dynamics (MD) simulations to quantify fold dynamics on extended timescales of Bet v 1a and two variants with higher and lower cleavage rates. Already at the nanosecond-timescale we observe a significantly higher flexibility for the destabilized variant compared to Bet v 1a and the proteolytically stabilized mutant. Estimating the thermodynamics and kinetics of local unfolding around an initial cleavage site, we find that the Bet v 1 variant with the highest cleavage rate also shows the highest probability for local unfolding. For the stabilized mutant on the other hand we only find minimal unfolding probability. These results strengthen the link between the conformational dynamics of allergen proteins and their stability during endolysosomal degradation. The presented approach further allows atomistic insights in the conformational ensemble of allergen proteins and provides probability estimates below experimental detection limits.

Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C12E8). Thus SDS converts β-lactoglobulin, which has mainly β-sheet secondary structure, into a state rich in α-helicality, while addition of C12E8 leads to refolding and recovery of the original β-sheet structure. Here we extend these studies to the large β-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C12E8 were investigated at pH 4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3–4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C12E8 recovers almost all the secondary structure, part of the tertiary structure, about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C12E8 was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state.

Agradecimentos: We congratulate our colleagues in the CERN accelerator departments for the excellent performance of the LHC and thank the technical and administrative staffs at CERN and at other CMS institutes for their contributions to the success of the CMS effort. In addition, we gratefully acknowledge the computing centers and personnel of the Worldwide LHC Computing Grid for delivering so effectively the computing infrastructure essential to our analyses. Finally, we acknowledge the enduring support for the construction and operation of the LHC and the CMS detector provided by the following funding agencies: BMWFW and FWF (Austria); FNRS and FWO (Belgium); CNPq, CAPES, FAPERJ, and FAPESP (Brazil); MES (Bulgaria); CERN; CAS, MoST, and NSFC (China); COLCIENCIAS (Colombia); MSES and CSF (Croatia); RPF (Cyprus); SENESCYT (Ecuador); MoER, ERC IUT, and ERDF (Estonia); Academy of Finland, MEC, and HIP (Finland); CEA and CNRS/IN2P3 (France); BMBF, DFG, and HGF (Germany); GSRT (Greece); OTKA and NIH (Hungary); DAE and DST (India); IPM (Iran); SFI (Ireland); INFN (Italy); MSIP and NRF (Republic of Korea); LAS (Lithuania); MOE and UM (Malaysia); BUAP, CINVESTAV, CONACYT, LNS, SEP, and UASLP-FAI (Mexico); MBIE (New Zealand); PAEC (Pakistan); MSHE and NSC (Poland); FCT (Portugal); JINR (Dubna); MON, ROSATOM, RAS, RFBR and RAEP (Russia); MESTD (Serbia); SEIDI, CPAN, PCTI and FEDER (Spain); Swiss Funding Agencies (Switzerland); MST (Taipei); ThEP-Center, IPST, STAR, and NSTDA (Thailand); TUBITAK and TAEK (Turkey); NASU and SFFR (Ukraine); STFC (United Kingdom); DOE and NSF (USA). Individuals have received support from the Marie -Curie program and the European Research Council and Horizon 2020 Grant, contract No. 675440 (European Union); the Leventis Foundation; the A.P. Sloan Foundation; the Alexander von Humboldt Foundation; the Belgian Federal Science Policy Office; the Fonds pour la Formation a la Recherche dans l'Industrie et dans l'Agriculture (FRIA-Belgium); the Agentschap voor Innovatie door Wetenschap en Technologie (IWT-Belgium); the Ministry of Education, Youth and Sports (MEYS) of the Czech Republic; the Council of Science and Industrial Research, India; the HOMING PLUS program of the Foundation for Polish Science, cofinanced from European Union, Regional Development Fund, the Mobility Plus program of the Ministry of Science and Higher Education, the National Science Center (Poland), contracts Harmonia 2014/14/M/ST2/00428, Opus 2014/13/B/ST2/02543, 2014/15/B/ST2/03998, and 2015/19/B/ST2/02861, Sonata-bis 2012/07/E/ST2/01406; the National Priorities Research Program by Qatar National Research Fund; the Programa Severo Ochoa del Principado de Asturias; the Thalis and Aristeia programs cofinanced by EU-ESF and the Greek NSRF; the Rachadapisek Sompot Fund for Postdoctoral Fellowship, Chulalongkorn University and the Chulalongkorn Academic into Its 2nd Century Project Advancement Project (Thailand); the Welch Foundation, contract C-1845; and the Weston Havens Foundation (USA) Abstract: Event-by-event fluctuations in the elliptic-flow coefficient v(2) are studied in PbPb collisions at root S-NN = 5.02 TeV using the CMS detector at the CERN LHC. Elliptic-flow probability distributions p(v(2)) for charged particles with transverse momentum 0.3 < p(T) < 3.0 GeV/c and pseudorapidity vertical bar eta vertical bar < 1.0 are determined for different collision centrality classes. The moments of the p(v(2)) distributions are used to calculate the v(2) coefficients based on cumulant orders 2, 4, 6, and 8. A rank ordering of the higher-order cumulant results and nonzero standardized skewness values obtained for the p(v(2)) distributions indicate non-Gaussian initial-state fluctuations. Bessel-Gaussian and elliptic power fits to the flow distributions are studied to characterize the initial-state spatial anisotropy CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES FUNDAÇÃO CARLOS CHAGAS FILHO DE AMPARO À PESQUISA DO ESTADO DO RIO DE JANEIRO - FAPERJ FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP Aberto

The CMS Collaboration: et al., Event-by-event fluctuations in the elliptic-flow coefficient v are studied in PbPb collisions at s=5.02 TeV using the CMS detector at the CERN LHC. Elliptic-flow probability distributions p(v) for charged particles with transverse momentum 0.3, Individuals have received support from the Marie-Curie program and the European Research Council and Horizon 2020 Grant, contract No. 675440 (European Union); the Programa Severo Ochoa del Principado de Asturias.

Metrics of model goodness-of-fit, model comparison, and model parameter estimation are the main categories of statistical problems in science. Bayesian and frequentist methods that address these questions often rely on a likelihood function, which is the key ingredient in order to assess the plausibility of model parameters given observed data. In some complex systems or experimental setups, predicting the outcome of a model cannot be done analytically, and Monte Carlo techniques are used. In this paper, we present a new analytic likelihood that takes into account Monte Carlo uncertainties, appropriate for use in the large and small sample size limits. Our formulation performs better than semi-analytic methods, prevents strong claims on biased statements, and provides improved coverage properties compared to available methods., Comment: 18 pages, 7 figures, 2 tables, code can be found at https://github.com/austinschneider/MCLLH

Event-by-event fluctuations in the elliptic-flow coefficient v(2) are studied in PbPb collisions at root S-NN = 5.02 TeV using the CMS detector at the CERN LHC. Elliptic-flow probability distributions p(v(2)) for charged particles with transverse momentum 0.3 < p(T) < 3.0 GeV/c and pseudorapidity vertical bar eta vertical bar < 1.0 are determined for different collision centrality classes. The moments of the p(v(2)) distributions are used to calculate the v(2) coefficients based on cumulant orders 2, 4, 6, and 8. A rank ordering of the higher-order cumulant results and nonzero standardized skewness values obtained for the p(v(2)) distributions indicate non-Gaussian initial-state fluctuations. Bessel-Gaussian and elliptic power fits to the flow distributions are studied to characterize the initial-state spatial anisotropy. (C) 2018 The Author(s). Published by Elsevier B.V.

Event-by-event fluctuations in the elliptic-flow coefficient v2 are studied in PbPb collisions at √sNN=5.02 TeV using the CMS detector at the CERN LHC. Elliptic-flow probability distributions p(v2) for charged particles with transverse momentum 0.3

Ricin is an A-B ribosome inactivating protein (RIP) toxin composed of an A-chain subunit (RTA) that contains a catalytic N-glycosidase and a B-chain (RTB) lectin domain that binds cell surface glycans. Ricin exploits retrograde transport to enter into the Golgi and the endoplasmic reticulum, and then dislocates into the cytoplasm where it can reach its substrate, the rRNA. A subset of isolated antibodies (Abs) raised against the RTA subunit protect against ricin intoxication, and RTA-based vaccine immunogens have been shown to provide long-lasting protective immunity against the holotoxin. Anti-RTA Abs are unlikely to cross a membrane and reach the cytoplasm to inhibit the enzymatic activity of the A-chain. Moreover, there is not a strict correlation between the apparent binding affinity (Ka) of anti-RTA Abs and their ability to successfully neutralize ricin toxicity. Some anti-RTA antibodies are toxin-neutralizing, whereas others are not. We hypothesize that neutralizing anti-RTA Abs may interfere selectively with conformational change(s) or partial unfolding required for toxin internalization. To test this hypothesis, we measured the melting temperatures (Tm) of neutralizing single-domain Ab (sdAb)-antigen (Ag) complexes relative to the Tm of the free antigen (Tm-shift = Tmcomplex – TmAg), and observed increases in the Tmcomplex of 9–20 degrees. In contrast, non-neutralizing sdAb-Ag complexes shifted the TmComplex by only 6–7 degrees. A strong linear correlation (r2 = 0.992) was observed between the magnitude of the Tm-shift and the viability of living cells treated with the sdAb and ricin holotoxin. The Tm-shift of the sdAb-Ag complex provided a quantitative biophysical parameter that could be used to predict and rank-order the toxin-neutralizing activities of Abs. We determined the first structure of an sdAb-RTA1-33/44-198 complex, and examined other sdAb-RTA complexes. We found that neutralizing sdAb bound to regions involved in the early stages of unfolding. These Abs likely interfere with steps preceding or following endocytosis that require conformational changes. This method may have utility for the characterization or rapid screening of other Ab that act to prevent conformational changes or unfolding as part of their mechanism of action.

The amount of heat-denatured whey protein is typically determined by pH 4.6 precipitation. Using this method, a significant amount of nondenatured protein was reported even after long heating times. Apparently, a fraction of the unfolded protein refolds into the "native" state rather than form aggregates. This fact is known and has been explained using kinetic models. How the conditions affect the refolding and aggregation is, however, not fully understood. Therefore, this study investigates the unfolding, refolding, and aggregation process of β-lactoglobulin using circular dichroism and size-exclusion chromatography to characterize different folding variants and to quantify their content. The proteins remaining in solution at pH 4.6 were confirmed to be native-like. The nonaggregated fraction contains proteins with a native-like and two types of non-native-like conformations. The nonaggregated fraction increased with decreasing temperature (60-90 °C) and concentration (1-50 g/L) and increasing electrostatic repulsion (pH 7-8; 0-50 mM). The native-like fraction in the nonaggregated fraction was independent of pH, ionic strength, and concentration but increased with decreasing temperature.

Regularization extensions to the Fully Bayesian Unfolding are implemented and studied with an algorithm of combined sampling to find, in a reasonable computational time, an optimal value of the regularization strength parameter in order to obtain an unfolded result of a desired property, like smoothness. Three regularization conditions using the curvature, entropy and derivatives are applied, as a model example, to several simulated spectra of top-pair quark pairs that are produced in high energy pp collisions. The existence of a minimum of a &chi, 2 between the unfolded and particle-level spectra is discussed, with recommendations on the checks and validity of the usage of the regularization feature in Fully Bayesian Unfolding (FBU).

α-Lactalbumin (α-LA) is a small (Mr 14,200), acidic (pI 4–5), Ca2+-binding protein. α-LA is a regulatory component of lactose synthase enzyme system functioning in the lactating mammary gland. The protein possesses a single strong Ca2+-binding site, which can also bind Mg2+, Mn2+, Na+, K+, and some other metal cations. It contains several distinct Zn2+-binding sites. Physical properties of α-LA strongly depend on the occupation of its metal binding sites by metal ions. In the absence of bound metal ions, α-LA is in the molten globule-like state. The binding of metal ions, and especially of Ca2+, increases stability of α-LA against the action of heat, various denaturing agents and proteases, while the binding of Zn2+ to the Ca2+-loaded protein decreases its stability and causes its aggregation. At pH 2, the protein is in the classical molten globule state. α-LA can associate with membranes at neutral or slightly acidic pH at physiological temperatures. Depending on external conditions, α-LA can form amyloid fibrils, amorphous aggregates, nanoparticles, and nanotubes. Some of these aggregated states of α-LA can be used in practical applications such as drug delivery to tissues and organs. α-LA and some of its fragments possess bactericidal and antiviral activities. Complexes of partially unfolded α-LA with oleic acid are cytotoxic to various tumor and bacterial cells. α-LA in the cytotoxic complexes plays a role of a delivery carrier of cytotoxic fatty acid molecules into tumor and bacterial cells across the cell membrane. Perhaps in the future the complexes of α-LA with oleic acid will be used for development of new anti-cancer drugs.

In this paper, we report the measurement relative to the production of forward neutrons in proton-proton collisions at $ \sqrt{s}=13 $ TeV obtained using the LHCf Arm2 detector at the Large Hadron Collider. The results for the inclusive differential production cross section are presented as a function of energy in three different pseudorapidity regions: η > 10.76, 8.99 < η < 9.22 and 8.81 < η < 8.99. The analysis was performed using a data set acquired in June 2015 that corresponds to an integrated luminosity of 0.194 nb$^{−1}$. The measurements were compared with the predictions of several hadronic interaction models used to simulate air showers generated by Ultra High Energy Cosmic Rays. None of these generators showed good agreement with the data for all pseudorapidity intervals. For η > 10.76, no model is able to reproduce the observed peak structure at around 5 TeV and all models underestimate the total production cross section: among them, QGSJET II-04 shows the smallest deficit with respect to data for the whole energy range. For 8.99 < η < 9.22 and 8.81 < η < 8.99, the models having the best overall agreement with data are SIBYLL 2.3 and EPOS-LHC, respectively: in particular, in both regions SIBYLL 2.3 is able to reproduce the observed peak structure at around 1.5–2.5 TeV. In this paper, we report the measurement relative to the production of forward neutrons in proton-proton collisions at $\mathrm{\sqrt{s} = 13~TeV}$ obtained using the LHCf Arm2 detector at the Large Hadron Collider. The results for the inclusive differential production cross section are presented as a function of energy in three different pseudorapidity regions: $\eta > 10.76$, $8.99 < \eta < 9.22$ and $8.81 < \eta < 8.99$. The analysis was performed using a data set acquired in June 2015 that corresponds to an integrated luminosity of $\mathrm{0.194~nb^{-1}}$. The measurements were compared with the predictions of several hadronic interaction models used to simulate air showers generated by Ultra High Energy Cosmic Rays. None of these generators showed good agreement with the data for all pseudorapidity intervals. For $\eta > 10.76$, no model is able to reproduce the observed peak structure at around $\mathrm{5~TeV}$ and all models underestimate the total production cross section: among them, QGSJET II-04 shows the smallest deficit with respect to data for the whole energy range. For $8.99 < \eta < 9.22$ and $8.81 < \eta < 8.99$, the models having the best overall agreement with data are SIBYLL 2.3 and EPOS-LHC, respectively: in particular, in both regions SIBYLL 2.3 is able to reproduce the observed peak structure at around $\mathrm{1.5-2.5~TeV}$.

Bacterial conjugation, a mechanism of horizontal gene transfer, is the major means by which antibiotic resistance spreads among bacteria (1, 2). Conjugative plasmids are transferred from one bacterium to another through a type IV secretion system (T4SS) in the form of single-stranded DNA covalently attached to a protein called relaxase. The relaxase is fully functional both in a donor cell (prior to conjugation) and recipient cell (after conjugation). Here, we demonstrate that the protein substrate has to unfold for efficient translocation through the conjugative T4SS. Furthermore, we present various relaxase modifications that preserve the function of the relaxase but block substrate translocation. This study brings us a step closer to deciphering the complete mechanism of T4SS substrate translocation, which is vital for the development of new therapies against multidrug-resistant pathogenic bacteria. IMPORTANCE Conjugation is the principal means by which antibiotic resistance genes spread from one bacterium to another (1, 2). During conjugation, a covalent complex of single-stranded DNA and a protein termed relaxase is transported by a type IV secretion system. To date, it is not known whether the relaxase requires unfolding prior to transport. In this report, we use functional assays to monitor the transport of wild-type relaxase and variants containing unfolding-resistant domains and show that these domains reduce conjugation and protein transport dramatically. Mutations that lower the free energy of unfolding in these domains do not block translocation and can even promote it. We thus conclude that the unfolding of the protein substrate is required during transport.

In this work, we present an in silico study of the dynamic behavior of a carbon nanotorus during the localized breaking of the C-C bonds in its atomic network. It is shown that the unfolding of a carbon nanotorus is accompanied by the appearance of deformation waves running along the atomic network of the carbon structure. We estimate energy stability of a carbon nanotorus under deformations using the original method for calculating the local stresses of an atomic network. A new physical phenomenon of the molecular current fl ow in the most deformed sites of the torus atomic network is discovered.

The unfolding (or deconvolution) technique is used in the development of digital pulse processing systems applied to particle detection. This technique is applied to digital signals obtained by digitization of analog signals that represent the combined response of the particle detectors and the associated signal conditioning electronics. This work describes a technique to determine if the signal is unfoldable. For unfoldable signals the characteristics of the unfolding system (unfolder) are presented. Finally, examples of the method applied to real experimental setup are discussed., Ministerio de Economía y Competitividad

Event structures are a well-accepted model of concurrency. In a seminal paper by Nielsen, Plotkin and Winskel, they are used to establish a bridge between the theory of domains and the approach to concurrency proposed by Petri. A basic role is played by an unfolding construction that maps (safe) Petri nets into a subclass of event structures, called prime event structures, where each event has a uniquely determined set of causes. Prime event structures, in turn, can be identified with their domain of configurations. At a categorical level, this is nicely formalised by Winskel as a chain of coreflections. Contrary to prime event structures, general event structures allow for the presence of disjunctive causes, i.e., events can be enabled by distinct minimal sets of events. In this paper, we extend the connection between Petri nets and event structures in order to include disjunctive causes. In particular, we show that, at the level of nets, disjunctive causes are well accounted for by persistent places. These are places where tokens, once generated, can be used several times without being consumed and where multiple tokens are interpreted collectively, i.e., their histories are inessential. Generalising the work on ordinary nets, Petri nets with persistence are related to a new subclass of general event structures, called locally connected, by means of a chain of coreflections relying on an unfolding construction., Logical Methods in Computer Science ; Volume 14, Issue 3 ; 1860-5974

Chitinase 60 from the psychrophilic bacterium Moritella marina (MmChi60) is a four-domain protein whose structure revealed flexible hinge regions between the domains, yielding conformations in solution that range from fully extended to compact. The catalytic domain is a shallow-grooved TIM-barrel. Heat-induced denaturation experiments of the wild-type and mutants resulting from the deletions of the two-Ig-like domains and the chitin binding domain reveal calorimetric profiles that are consistent with non-collaborative thermal unfolding of the individual domains, a property that must be associated to the “hinge-regions”. The calorimetric measurements of the (β/α)8 catalytic domain reveal that the thermal unfolding is a slow-relaxation transition exhibiting a stable, partially structured intermediate state. Circular dichroism provides evidence that the intermediate exhibits features of a molten globule i.e., loss of tertiary structure while maintaining the secondary structural elements of the native. GdnHCl-induced denaturation studies of the TIM-barrel demonstrate an extraordinarily high resistance to the denaturant. Slow-relaxation kinetics characterize the unfolding with equilibration times exceeding six days, a property that is for the first time observed for a psychrophilic TIM barrel. On the other hand, the thermodynamic stability is ΔG=6.75±1.3 kcal/mol, considerably lower than for structural-insertions-containing barrels. The mutant E153Q used for the crystallographic studies of MmChi60 complexes with NAG ligands has a much lower stability than the wild-type., Highlights • We use heat-induced and chemical denaturation to study MmChi60. • The impact of “hinge” regions upon the DSC calorimetric profiles is explored. • CD is used to characterize the thermal unfolding intermediate of the catalytic domain. • The thermodynamic stability of the TIM-barrel is measured via chemical denaturation. • High-resistance to denaturants is evidenced for the psychrophilic (β/α)8 domain.

The concept of non-interference has been introduced to characterise the absence of un- desired information flows in a computing system. Although it is often explained referring to an informal notion of causality - the activity involving the part of the system with higher level of confi- dentiality should not cause any observable effect at lower levels - it is almost invariably formalised in terms of interleaving semantics. Here we focus on Petri nets and on the BNDC (Bisimilarity-based Non-Deducibility on Composition) property, a formalisation of non-interference widely studied in the literature. We show that BNDC admits natural characterisations based on the unfolding seman- tics - a classical true concurrent semantics for Petri nets - in terms of causalities and conflicts between high and low level activities. This leads to algorithms for checking BNDC on various classes of Petri nets, based on the construction of suitable complete prefixes of the unfolding. We also developed a prototype tool UBIC (Unfolding-Based Interference Checker), working on safe Petri nets, which provides promising results in terms of efficiency.

Double differential neutron yields from a thick aluminum target irradiated by 9 MeV deuterons were measured at the Kyushu University Tandem accelerator Laboratory (KUTL). An NE213 liquid organic scintillator (50.4 mm thick and 50.4 mm in diameter) was used as a neutron detector. Neutron yields from the target were measured at nine angles between 0° and 140°. The neutron energy spectra were obtained by means of an unfolding method using FORIST code with the response function of the NE213 scintillator calculated by SCINFUL-QMD code. The experimental result was compared with other measured data at 5 and 40 MeV and the calculation based on intra-nuclear cascade of Liege (INCL) model in PHITS code.

Urea interacts with the Na,K-ATPase, leading to reversible as well as irreversible inhibition of the hydrolytic activity. The enzyme purified from shark rectal glands is more sensitive to urea than Na,K-ATPase purified from pig kidney. An immediate and reversible inhibition under steady-state conditions of hydrolytic activity at 37°C is demonstrated for the three reactions studied: the overall Na,K-ATPase activity, the Na-ATPase activity observed in the absence of K(+) as well as the K(+)-dependent phosphatase reaction (K-pNPPase) seen in the absence of Na(+). Half-maximal inhibition is seen with about 1M urea for shark enzyme and about 2M urea for pig enzyme. In the presence of substrates there is also an irreversible inhibition in addition to the reversible process, and we show that ATP protects against the irreversible inhibition for both the Na,K-ATPase and Na-ATPase reaction, whereas the substrate paranitrophenylphosphate leads to a slight increase in the rate of irreversible inhibition of the K-pNPPase. The rate of the irreversible inactivation in the absence of substrates is much more rapid for shark enzyme than for pig enzyme. The larger number of potentially urea-sensitive hydrogen bonds in shark enzyme compared to pig enzyme suggests that interference with the extensive hydrogen bonding network might account for the higher urea sensitivity of shark enzyme. The reversible inactivation is interpreted in terms of domain interactions and domain accessibilities using as templates the available crystal structures of Na,K-ATPase. It is suggested that a few interdomain hydrogen bonds are those mainly affected by urea during reversible inactivation.

Internal bremsstrahlung (IB) accompanying the beta(-) decay of Tl-204 was measured using a 5.08 x 5.08 cm(2) NaI(Tl) detector employing a magnetic deflection method in the range of 10-760 keV. A novel approach, the Singular Value Decomposition (SVD), is applied to unfold the raw detector spectrum of Tl-204. Unfolded IB spectrum is compared with the KUB theory. The measured spectrum is found to show fairly good agreement with the KUB theory in the energy range of 100-600 keV. The distribution beyond the 600 keV takes a positive deviation from the theory. (C) 2015 Published by Elsevier Ltd.

Neither the influence of high shear rates nor the impact of cavitation on protein aggregation is fully understood. The effect of cavitation bubble collapse‐derived hydroxyl radicals on the aggregation behavior of human serum albumin (HSA) was investigated. Radicals were generated by pumping through a micro‐orifice, ultra‐sonication, or chemically by Fenton's reaction. The amount of radicals produced by the two mechanical methods (0.12 and 11.25 nmol/(L min)) was not enough to change the protein integrity. In contrast, Fenton's reaction resulted in 382 nmol/(L min) of radicals, inducing protein aggregation. However, the micro‐orifice promoted the formation of soluble dimeric HSA aggregates. A validated computational fluid dynamic model of the orifice revealed a maximum and average shear rate on the order of 108 s−1 and 1.2 × 106 s−1, respectively. Although these values are among the highest ever reported in the literature, dimer formation did not occur when we used the same flow rate but suppressed cavitation. Therefore, aggregation is most likely caused by the increased surface area due to cavitation‐mediated bubble growth, not by hydroxyl radical release or shear stress as often reported.

AAA+ unfoldases are thought to unfold substrate through the central pore of their hexameric structures, but how this process occurs is not known. VAT, the Thermoplasma acidophilum homologue of eukaryotic CDC48/p97, works in conjunction with the proteasome to degrade misfolded or damaged proteins. We show that in the presence of ATP, VAT with its regulatory N-terminal domains removed unfolds other VAT complexes as substrate. We captured images of this transient process by electron cryomicroscopy (cryo-EM) to reveal the structure of the substrate-bound intermediate. Substrate binding breaks the six-fold symmetry of the complex, allowing five of the six VAT subunits to constrict into a tight helix that grips an ~80 Å stretch of unfolded protein. The structure suggests a processive hand-over-hand unfolding mechanism, where each VAT subunit releases the substrate in turn before re-engaging further along the target protein, thereby unfolding it. DOI: http://dx.doi.org/10.7554/eLife.25754.001, eLife digest Proteins perform many important jobs inside cells. Each protein is made of a chain of building blocks called amino acids that fold together to form precise shapes. Old, damaged or poorly constructed proteins tend to be harmful to cells so it is important that they are recycled. Ring-shaped enzymes called AAA+ unfoldases help to recycle these proteins by unfolding their amino acid chains. However, since these enzymes only interact with their target proteins for a short time it has been difficult to find out what happens when the target protein binds to the enzyme. AAA+ unfoldases use chemical energy provided by a molecule called ATP to drive protein unfolding. Ripstein et al. used a technique called electron cryomicroscopy to study a AAA+ unfoldase from a microbe called Thermoplasma acidophilum. The enzymes were supplied with a molecule called ATPγS, which is similar to ATP but releases its energy more slowly. Ripstein et al. used this slowed down reaction, as well as new computer algorithms to analyse the microscopy images, to examine the structure of the target bound to the enzyme. The experiments show that proteins move through the centre of the enzyme’s ring as they unfold. At any time the enzyme is attached to the amino acid chain of the target protein in several places, which allows it to pull the protein through the ring in a “hand-over-hand” motion. Energy from each ATP molecule drives the enzyme to release one contact with the amino acid chain and form a new contact slightly further along the chain. It remains unclear how AAA+ unfoldases identify and bind to the proteins they unfold and how they work alongside other recycling proteins inside cells. AAA+ unfoldases have an essential role in the biology of cells, and contribute to cancer and several other human diseases. Therefore, understanding how these proteins work may aid the development of new drugs to treat these diseases. DOI: http://dx.doi.org/10.7554/eLife.25754.002

© 2017 Elsevier Inc. All-atom explicit solvent molecular dynamics was used to study the process of unfolding of hen egg white lysozyme in water and mixtures of water with dimethyl sulfoxide at different compositions. We have determined the kinetic parameters of unfolding at a constant temperature 450 K. For each run, the time of disruption of the tertiary structure of lysozyme t u was defined as the moment when a certain structural criterion computed from the trajectory reaches its critical value. A good agreement is observed between the results obtained using several different criteria. The secondary structure according to DSSP calculations is found to be partially unfolded to the moment of disruption of tertiary structure, but some of its elements keep for a long time after that. The values of t u averaged over ten 30 ns-long trajectories for each solvent composition are shown to decrease very rapidly with addition of dimethyl sulfoxide, and rather small amounts of dimethyl sulfoxide are found to change the pathway of unfolding. In pure water, despite the loss of tertiary contacts and disruption of secondary structure elements, the protein preserves its compact globular state at least over 130 ns of simulation, while even at 5 mol percents of dimethyl sulfoxide it loses its compactness within 30 ns. The proposed methodology is a generally applicable tool to quantify the rate of protein unfolding in simulation studies.

© 2017 Elsevier Inc. All-atom explicit solvent molecular dynamics was used to study the process of unfolding of hen egg white lysozyme in water and mixtures of water with dimethyl sulfoxide at different compositions. We have determined the kinetic parameters of unfolding at a constant temperature 450 K. For each run, the time of disruption of the tertiary structure of lysozyme t u was defined as the moment when a certain structural criterion computed from the trajectory reaches its critical value. A good agreement is observed between the results obtained using several different criteria. The secondary structure according to DSSP calculations is found to be partially unfolded to the moment of disruption of tertiary structure, but some of its elements keep for a long time after that. The values of t u averaged over ten 30 ns-long trajectories for each solvent composition are shown to decrease very rapidly with addition of dimethyl sulfoxide, and rather small amounts of dimethyl sulfoxide are found to change the pathway of unfolding. In pure water, despite the loss of tertiary contacts and disruption of secondary structure elements, the protein preserves its compact globular state at least over 130 ns of simulation, while even at 5 mol percents of dimethyl sulfoxide it loses its compactness within 30 ns. The proposed methodology is a generally applicable tool to quantify the rate of protein unfolding in simulation studies.

Agri Ibrahim Cecen University;IC Foudation 2nd International Conference on Advances in Natural and Applied Sciences, ICANAS 2017 -- 18 April 2017 through 21 April 2017 -- -- 127507 Energy distribution of ?-rays emitted from standard sources, passing through various thicknesses of Al medium, were obtained by using 5.08cm x 5.08cm NaI(Tl) detector. The full energy peak and the total efficiency, photopeak/total (P/T) ratios and energy resolution of NaI(Tl) detector were measured using standard ?-ray sources. Detector response functions (DRFs) were obtained in every energy value of ?-ray rays by means of P/T ratios and energy resolutions. ?-rays incoming to the slice-shape geometry medium, can take all the energy values between 0 and the maximum energy. The energy range is divided into n lower energy region. DRFs are obtained for the energy values correspond to the midpoint of each energy range. In this way, the response matrix is developed. A bin to bin unfolding method is applied to the ?-ray spectra and the results are compared with the spectra obtained by the Monte Carlo method. © 2017 Author(s).