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The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a K i value of 2.9 μM against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV. IMPORTANCE Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti , continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors provide novel scaffolds that enable exploiting the prime side of the protease active site, with the aim of achieving better specificity and lower hydrophilicity than those of current scaffolds in the design of antiflaviviral inhibitors.

Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1′ and S2′ subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1′ subsite highly influences other subsites: the extension of the hydrophobic P1′ moiety results in 1) reduced van der Waals contacts in the P2′ subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor.

HIV-1 protease inhibitors (PIs) are competitive active-site inhibitors that mimic the transition state of the enzyme’s substrate, and are the most potent antiretroviral drugs against HIV infection. HIV-1 protease processes the viral polyproteins at specific cleavage sites and allows infectious mature virions and hence spread of the virus. Unfortunately rapid viral evolution combined with selective pressure of therapy causes selection of many drug-resistant variants that are no longer efficiently inhibited by the PIs. HIV-1 protease can tolerate extensive mutations, with close to half of the 99-residues making up each of the chains in the homodimeric protease and residues at substrate cleavage sites mutating to escape PI pressure. Structural and biophysical studies of many drug-resistant HIV-1 protease variants revealed insights into how mutations at and outside of the protease active site are able to confer PI resistance while still allowing recognition and processing of substrates, and why substrate mutations coevolve with primary protease mutations. We summarize the main molecular mechanisms underlying PI resistance due to primary, secondary, and substrate coevolved mutations and how this knowledge may guide the design of robust inhibitors to avoid resistance.

Clay soils are very useful as liners in geotechnical structures such as landfill sites, dams, water channels, etc . Swelling is a common problem in clay liners, however. To better understand swelling properties, in the present study clay nanocomposites were produced by means of the sol gel method, using a hydrophobic clay, polymers (locust bean gum, latex, glycerine, vinyl acrylic copolymer), and rubber powder. The study focused on the swelling and dynamic properties (secant shear modulus and damping ratio) of the clay nanocomposites researched experimentally in laboratory conditions. The dynamic tests were conducted on samples compacted using two different compaction energy levels. The test results were compared with those of natural clay and hydrophobic organo-clay. The test results revealed that the damping ratios and secant shear modulus of clay nanocomposites without rubber (CNC) and with rubber (CNCr) that were compacted with both the E1 and E2 energy levels were increased and decreased, respectively. In addition, with increasing percentage of vinyl acrylic in nanoclay composites, the secant shear modulus values were decreased and damping ratio values were increased. Consequently, the test results found that the swelling and dynamic properties of clay nanocomposites can be optimized in order to attenuate the negative effects of dynamic loads on clay liners.

Under the selective pressure of therapy, HIV-1 protease mutants resistant to inhibitors evolve to confer drug resistance. Such mutations can impact both the dynamics and structures of the bound and unbound forms of the enzyme. Flap+ is a multidrug-resistant variant of HIV-1 protease with a combination of primary and secondary resistance mutations (L10I, G48V, I54V, V82A) and a strikingly altered thermodynamic profile for darunavir (DRV) binding relative to the wild-type protease. We elucidated the impact of these mutations on protein dynamics in the DRV-bound state using molecular dynamics simulations and NMR relaxation experiments. Both methods concur in that the conformational ensemble and dynamics of protease are impacted by the drug resistance mutations in Flap+ variant. Surprisingly this change in ensemble dynamics is different from that observed in the unliganded form of the same variant (Cai, Y. et al. J. Chem. Theory Comput.2012, 8, 3452–3462). Our comparative analysis of both inhibitor-free and bound states presents a comprehensive picture of the altered dynamics in drug-resistant mutant HIV-1 protease and underlies the importance of incorporating dynamic analysis of the whole system, including the unliganded state, into revealing drug resistance mechanisms.

Clay soils have various areas of use in geotechnical engineering.The common use of clayey soils are for landfills, dams,nuclear plants, etc. However, when the clay soils interactwith water, the geotechnical properties such as the swelling,the shear strength, and the compressibility properties ofclay change gradually. Therefore, in this research, using ahydrophobic organo-clay, polymers (locust bean gum, latex,glycerine, and vinyl acrylic copolymer), and rubber powder,clay-nanocomposites were developed to solve the problemsdue to the clay-water interaction encountered in the clay liners.This study focuses on the geotechnical properties of theclay-nanocomposites researched experimentally in laboratoryconditions. The test results found that, the specific gravities,consistency limits, compaction parameters, and unconfinedcompressive strengths of clay-nanocomposites change significantly,when compared to those of natural clay and hydrophobicorgano-clay.

The rapid evolution of HIV under selective drug pressure has led to multidrug resistant (MDR) strains that evade standard therapies. We designed highly potent HIV-1 protease inhibitors (PIs) using the substrate envelope model, which confines inhibitors within the consensus volume of natural substrates, providing inhibitors less susceptible to resistance because a mutation affecting such inhibitors will simultaneously affect viral substrate processing. The designed PIs share a common chemical scaffold but utilize various moieties that optimally fill the substrate envelope, as confirmed by crystal structures. The designed PIs retain robust binding to MDR protease variants and display exceptional antiviral potencies against different clades of HIV as well as a panel of 12 drug-resistant viral strains. The substrate envelope model proves to be a powerful strategy to develop potent and robust inhibitors that avoid drug resistance., National Institute of General Medical Sciences (U.S.) (Grant P01-GM66524), National Institute of General Medical Sciences (U.S.) (Grant AI41404), National Institute of General Medical Sciences (U.S.) (Grant AI43198), National Institute of General Medical Sciences (U.S.) (Grant GM065418), National Institute of General Medical Sciences (U.S.) (Grant GM082209), United States. American Recovery and Reinvestment Act of 2009 (Supplement P01GM066524-08S1)

Drug resistance occurs through a series of subtle changes that maintain substrate recognition but no longer permit inhibitor binding. In HIV-1 protease, mutations at I50 are associated with such subtle changes that confer differential resistance to specific inhibitors. Residue I50 is located at the protease flap tips, closing the active site upon ligand binding. Under selective drug pressure, I50V/L substitutions emerge in patients, compromising drug susceptibility and leading to treatment failure. The I50V substitution is often associated with amprenavir (APV) and darunavir (DRV) resistance, while the I50L substitution is observed in patients failing atazanavir (ATV) therapy. To explain how APV, DRV, and ATV susceptibility are influenced by mutations at residue 50 in HIV-1 protease, structural and binding thermodynamics studies were carried out on I50V/L-substituted protease variants in the compensatory mutation A71V background. Reduced affinity to both I50V/A71V and I50L/A71V double mutants is largely due to decreased binding entropy, which is compensated for by enhanced enthalpy for ATV binding to I50V variants and APV binding to I50L variants, leading to hypersusceptibility in these two cases. Analysis of the crystal structures showed that the substitutions at residue 50 affect how APV, DRV, and ATV bind the protease with altered van der Waals interactions and that the selection of I50V versus I50L is greatly influenced by the chemical moieties at the P1 position for APV/DRV and the P2 position for ATV. Thus, the varied inhibitor susceptibilities of I50V/L protease variants are largely a direct consequence of the interdependent changes in protease inhibitor interactions.

Understanding the interdependence of multiple mutations in conferring drug resistance is crucial to the development of novel and robust inhibitors. As HIV-1 protease continues to adapt and evade inhibitors while still maintaining the ability to specifically recognize and efficiently cleave its substrates, the problem of drug resistance has become more complicated. Under the selective pressure of therapy, correlated mutations accumulate throughout the enzyme to compromise inhibitor binding, but characterizing their energetic interdependency is not straightforward. A particular drug resistant variant (L10I/G48V/I54V/V82A) displays extreme entropy-enthalpy compensation relative to wild-type enzyme but a similar variant (L10I/G48V/I54A/V82A) does not. Individual mutations of sites in the flaps (residues 48 and 54) of the enzyme reveal that the thermodynamic effects are not additive. Rather, the thermodynamic profile of the variants is interdependent on the cooperative effects exerted by a particular combination of mutations simultaneously present.

Hepatitis C virus (HCV), affecting an estimated 150 million people worldwide, is the leading cause of viral hepatitis, cirrhosis and hepatocellular carcinoma. HCV is genetically diverse with six genotypes (GTs) and multiple subtypes of different global distribution and prevalence. Recent development of direct-acting antivirals against HCV including NS3/4A protease inhibitors (PIs) has greatly improved treatment outcomes for GT-1. However, all current PIs exhibit significantly lower potency against GT-3. Lack of structural data on GT-3 protease has limited our ability to understand PI failure in GT-3. In this study the molecular basis for reduced potency of current inhibitors against GT-3 NS3/4A protease is elucidated with structure determination, molecular dynamics simulations and inhibition assays. A chimeric GT-1a3a NS3/4A protease amenable to crystallization was engineered to recapitulate decreased sensitivity of GT-3 protease to PIs. High-resolution crystal structures of this GT-1a3a bound to 3 PIs, asunaprevir, danoprevir and vaniprevir, had only subtle differences relative to GT-1 despite orders of magnitude loss in affinity. In contrast, hydrogen-bonding interactions within and with the protease active site and dynamic fluctuations of the PIs were drastically altered. The correlation between loss of intermolecular dynamics and inhibitor potency suggests a mechanism where polymorphisms between genotypes (or selected mutations) in the drug target confer resistance through altering the intermolecular dynamics of the protein-inhibitor complex.

The murine monoclonal antibody LA-2 recognizes a clinically protective epitope on outer surface protein (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease in North America. Human antibody equivalence to LA-2 is the best serologic correlate of protective antibody responses following OspA vaccination. Understanding the structural and functional basis of the LA-2 protective epitope is important for developing OspA-based vaccines and discovering prophylactic antibodies against Lyme disease. Here, we present a detailed structure-based analysis of the LA-2/OspA interaction interface and identification of residues mediating antibody recognition. Mutations were introduced into both OspA and LA-2 on the basis of computational predictions on the crystal structure of the complex and experimentally tested for in vitro binding and borreliacidal activity. We find that Y32 and H49 on the LA-2 light chain, N52 on the LA-2 heavy chain and residues A208, N228 and N251 on OspA were the key constituents of OspA/LA-2 interface. These results reveal specific residues that may be exploited to modulate recognition of the protective epitope of OspA and have implications for developing prophylactic passive antibodies.

Recent advances in direct-acting antivirals against Hepatitis C Virus (HCV) have led to the development of potent inhibitors, including MK-5172, that target the viral NS3/4A protease with relatively low susceptibility to resistance. MK-5172 has a P2–P4 macrocycle and a unique binding mode among current protease inhibitors where the P2 quinoxaline packs against the catalytic residues H57 and D81. However, the effect of macrocyclization on this binding mode is not clear, as is the relation between macrocyclization, thermodynamic stabilization, and susceptibility to the resistance mutation A156T. We have determined high-resolution crystal structures of linear and P1–P3 macrocyclic analogs of MK-5172 bound to WT and A156T protease and compared these structures, their molecular dynamics, and experimental binding thermodynamics to the parent compound. We find that the “unique” binding mode of MK-5172 is conserved even when the P2–P4 macrocycle is removed or replaced with a P1–P3 macrocycle. While beneficial to decreasing the entropic penalty associated with binding, the constraint exerted by the P2–P4 macrocycle prevents efficient rearrangement to accommodate the A156T mutation, a deficit alleviated in the linear and P1–P3 analogs. Design of macrocyclic inhibitors against NS3/4A needs to achieve the best balance between exerting optimal conformational constraint for enhancing potency, fitting within the substrate envelope and allowing adaptability to be robust against resistance mutations.

HIV-1 protease is a major drug target against AIDS as it permits viral maturation by processing the gag and pol polyproteins of the virus. The cleavage sites in these polyproteins do not have obvious sequence homology or a binding motif and the specificity of the protease is not easily determined. We used various threading approaches, together with the crystal structures of substrate complexes which served as template structures, to study the substrate specificity of HIV-1 protease with the aim of obtaining a better differentiation between binding and nonbinding sequences. The predictions from threading improved when distance-dependent interaction energy functions were used instead of contact matrices. To rank the peptides and properly account for the peptide's conformation in the total energy, the results from using short-range potentials on multiple template structures were averaged. Finally, a dynamic threading approach is introduced which is potentially useful for cases when there is only one template structure available. The conformational energy of the peptide—especially the term accounting for the side chains—was found to be important in differentiating between binding and nonbinding sequences. Hence, the substrate specificity, and thus the ability of the virus to mature, is affected by the compatibility of the substrate peptide to fit within the limited conformational space of the active site groove.

The dynamics of HIV-1 protease, both in unliganded and substrate-bound forms have been analyzed by using an analytical method, Gaussian network model (GNM). The method is applied to different conformations accessible to the protein backbone in the native state, observed in crystal structures and snapshots from fully atomistic molecular dynamics (MD) simulation trajectories. The modes of motion obtained from GNM on different conformations of HIV-1 protease are conserved throughout the MD simulations. The flaps and 40's loop of the unliganded HIV-1 protease structure are identified as the most mobile regions. However, in the liganded structure these flaps lose mobility, and terminal regions of the monomers become more flexible. Analysis of the fast modes shows that residues important for stability are in the same regions of all the structures examined. Among these, Gly86 appears to be a key residue for stability. The contribution of residues in the active site region and flaps to the stability is more pronounced in the substrate-bound form than in the unliganded form. The convergence of modes in GNM to similar regions of HIV-1 protease, regardless of the conformation of the protein, supports the robustness of GNM as a potentially useful and predictive tool.

The backbone dynamics of barnase has been studied by a recently developed off-lattice Monte Carlo (MC)/Metropolis simulation technique, where a low-resolution model (virtual-bond model) is used together with knowledge-based potentials, with the main emphasis on its cooperative motions at different time windows. The conformations generated around the native state are analysed by time-dependent auto- and cross-conformational correlation functions of the virtual bonds. There exists a correlation between the long time auto-correlated behaviour of the bond rotations and the potential stability of the respective regions. The analysis at different time windows reveals that there are cooperative motions between the bond rotations, which are only near neighbours and basically local motions at all time windows. However, as the time window widens, a progressive increase in the number of correlated pairs, which are separated far along the sequence and are not necessarily close in space, is observed. The structural distribution of these motions shows that the cooperative interactions are not bi-directional and that different residues have a different role within the network of interactions. Thus, the conditions yielding global motion coherence can be accounted for by the existence of anisotropic cooperative long-range interactions among the units in cooperation with the short-range interactions.

Drug resistance mutations in response to HIV-1 protease inhibitors are selected not only in the drug target but elsewhere in the viral genome, especially at the protease cleavage sites in the precursor protein Gag. To understand the molecular basis of this protease–substrate coevolution, we solved the crystal structures of drug resistant I50V/A71V HIV-1 protease with p1-p6 substrates bearing coevolved mutations. Analyses of the protease–substrate interactions reveal that compensatory coevolved mutations in the substrate do not restore interactions lost due to protease mutations, but instead establish other interactions that are not restricted to the site of mutation. Mutation of a substrate residue has distal effects on other residues’ interactions as well, including through the induction of a conformational change in the protease. Additionally, molecular dynamics simulations suggest that restoration of active site dynamics is an additional constraint in the selection of coevolved mutations. Hence, protease–substrate coevolution permits mutational, structural, and dynamic changes via molecular mechanisms that involve distal effects contributing to drug resistance.

An off-lattice dynamic Monte Carlo (MC) method is used to investigate the conformational dynamics of chymotrypsin inhibitor 2 (CI2) and subtilisin in both free and complex forms over two time windows, referring to short and long time scales. The conformational dynamics of backbone bonds analysed from several independent trajectories reveal that: Both the inhibitor and the enzyme are restricted in their bond rotations, excluding a few bonds, upon binding; the effect being greatest for the loop regions, and for the inhibitor. A cooperativity in the near-neighbor bond rotations are observed on both time scales, whereas the cooperative rotations of the bonds far along the sequence appear only in the long time window, and the latter time window is where most of the interactions between the inhibitor and the enzyme are observed. Upon binding, the cooperatively rotating parts of the inhibitor and the enzyme are readjusted compared to their free forms, and new correlations appear. The binding loop, although it is the closest contact region, is not the only part of the inhibitor involved in the interactions with the enzyme. Loops 3 and 8 and the helices F and G in bound enzyme and the binding loop of the inhibitor contribute at the most to the collective motions of whole structure on the slow time scale and are apparently important for enzyme-inhibitor interactions and function. The results in general provide evidence for the contribution of the loops with cooperative motions to the extensive communication network of the complex.

The murine monoclonal antibody LA-2 recognizes a clinically protective epitope on outer surface protein (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease in North America. Human antibody equivalence to LA-2 is the best serologic correlate of protective antibody responses following OspA vaccination. Understanding the structural and functional basis of the LA-2 protective epitope is important for developing OspA-based vaccines and discovering prophylactic antibodies against Lyme disease. Here, we present a detailed structure-based analysis of the LA-2/OspA interaction interface and identification of residues mediating antibody recognition. Mutations were introduced into both OspA and LA-2 on the basis of computational predictions on the crystal structure of the complex and experimentally tested for in vitro binding and borreliacidal activity. We find that Y32 and H49 on the LA-2 light chain, N52 on the LA-2 heavy chain and residues A208, N228 and N251 on OspA were the key constituents of OspA/LA-2 interface. These results reveal specific residues that may be exploited to modulate recognition of the protective epitope of OspA and have implications for developing prophylactic passive antibodies.

The engineering characteristics of clay are dependant on its mineral and chemical structure. When the clay soils interacted with water, their volume and shear strength and compressibility properties change gradually. Large amount of decrease in terms of the shear strength of clay soils due to swelling is observed. In order to solve these problems, the stabilization of clay soils using chemical additives is a prevalent subject of research. In view of this, previous research studies have employed surface active surfactants to modify the fundamental properties of soft clays. In this study, surfactant clay complexes (organoclays) were developed using a raw clay modification with four surfactants and their geotechnical properties were determined in laboratory conditions. Two of the surfactants are cationic (cetyl trimethyl ammonium chloride, denoted as CTAC and quaternised ethoxylated fatty amine, denoted as QEFA) and the others are anionic (linear alkylbenzene sulphonic acid, denoted as LABSA and sodium lauryl ether sulphate, denoted as SLES). The experimental results indicated that the geotechnical properties of organoclays show significant change when compared to those of natural clay. The specific gravities, unconfined compression strengths and maximum dry densities of all organoclays were decreased. Optimum moisture content and swelling pressure values were decreased in cationic surfactant modified clays. However, the optimum moisture content and swelling pressure values of anionic surfactant clays are increased. A direct shear test was performed to determine the shear strength of organoclays. The test results also showed that the internal friction angles of organoclays were increased. Additionally, the change in the hydraulic conductivity was found to be insignificant.

The development of HIV-1 protease inhibitors has been the historic paradigm of rational structure-based drug design, where structural and thermodynamic analyses have assisted in the discovery of novel inhibitors. While the total enthalpy and entropy change upon binding determine the affinity, often the thermodynamics are considered in terms of inhibitor properties only. In the current study, profound changes are observed in the binding thermodynamics of a drug-resistant variant compared to wild-type HIV-1 protease, irrespective of the inhibitor bound. This variant (Flap+) has a combination of flap and active site mutations and exhibits extremely large entropy-enthalpy compensation compared to wild-type protease, 5-15 kcal/mol, while losing only 1-3 kcal/mol in total binding free energy for any of six FDA-approved inhibitors. Although entropy-enthalpy compensation has been previously observed for a variety of systems, never have changes of this magnitude been reported. The co-crystal structures of Flap+ protease with four of the inhibitors were determined and compared with complexes of both the wild-type protease and another drug-resistant variant that does not exhibit this energetic compensation. Structural changes conserved across the Flap+ complexes, which are more pronounced for the flaps covering the active site, likely contribute to the thermodynamic compensation. The finding that drug-resistant mutations can profoundly modulate the relative thermodynamic properties of a therapeutic target independent of the inhibitor presents a new challenge for rational drug design.

In the rapidly evolving disease of HIV drug resistance readily emerges, nullifying the effectiveness of therapy. Drug resistance has been extensively studied in HIV-1 protease where resistance occurs when the balance between enzyme inhibition and substrate recognition and turn-over is perturbed to favor catalytic activity. Mutations which confer drug resistance can impact the dynamics and structure of both the bound and unbound forms of the enzyme. Flap+ is a multi-drug-resistant variant of HIV-1 protease with a combination of mutations at the edge of the active site, within the active site, and in the flaps (L10I, G48V, I54V, V82A). The impact of these mutations on the dynamics in the unliganded form in comparison with the wild-type protease was elucidated with Molecular Dynamic simulations and NMR relaxation experiments. The comparative analyses from both methods concur in showing that the enzyme's dynamics are impacted by the drug resistance mutations in Flap+ protease. These alterations in the enzyme dynamics, particularly within the flaps, likely modulate the balance between substrate turn-over and drug binding, thereby conferring drug resistance.

The aim of this study is to investigate the effects of epilepsy, valproic acid and oxcarbazepine on nitric oxide levels, lipid peroxidation and xanthine oxidase levels in newly diagnosed epileptic children and healthy controls. A total of 49 patients with newly diagnosed idiopathic epilepsy and 15 healthy children were enrolled in this study. Of these 49 patients, 16 children were treated with valproate and 16 treated with oxcarbazepine. Nitric oxide, malondialdehyde and xanthine oxidase levels prior to antiepileptic drug therapy were measured in the serum. Blood samples were drawn before antiepileptic drug therapy and after 3 and 6 months of the antiepileptic drug treatment. Nitric oxide levels were statistically higher in the newly diagnosed epileptic patients. In oxcarbazepine group, the nitric oxide and malondialdehyde levels were found to be decreased. No statistically significant differences were noted in nitric oxide, malondialdehyde and xanthine oxidase levels in valproic acid treated group. Oxcarbazepine which is a frequently used new antiepileptic drug in childhood epilepsy may modify nitric oxide levels and lipid peroxidation. These results suggest that decreased lipid peroxidation would play a role in the mechanism of antiepileptic effects by oxcarbazepine treatment. (C) 2010 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

Hsp70 chaperones are involved in the prevention of misfolding, and possibly the folding, of newly synthesized proteins. The members of this chaperone family are capable of interacting with polypeptide chains both co- and posttranslationally, but it is currently not clear how different structural domains of the chaperone affect binding specificity. We explored the interactions between the bacterial Hsp70, DnaK, and the sequence of a model all-R-helical globin (apoMb) by cellulose-bound peptide scanning. The binding specificity of the full-length chaperone was compared with that of its minimal substrate-binding domain, DnaK-‚. Six specific chaperone binding sites evenly distributed along the apoMb sequence were identified. Binding site locations are identical for the full-length chaperone and its substrate- binding domain, but relative affinities differ. The binding specificity of DnaK- ‚ is only slightly decreased relative to that of full-length DnaK. DnaK's binding motif is known to comprise hydrophobic regions flanked by positively charged residues. We found that the simple fractional mean buried area correlates well with Hsp70's binding site locations along the apoMb sequence. In order to further characterize the properties of the minimal binding host, the stability of DnaK-‚ upon chemical denaturation by urea and protons was investigated. Urea unfolding titrations yielded an apparent folding ¢G° of 3.1 ( 0.9 kcal mol -1 and an m value of 1.7 ( 0.4 kcal mol -1 M -1 . ‡ Joint first authors. §

A coarse-grained dynamic Monte Carlo (MC) simulation method is used to investigate the conformational dynamics of chymotrypsin inhibitor 2 (CI2). Each residue is represented therein by two interaction sites, one at the α-carbon and the other on the amino acid side-chain. The energy and geometry parameters extracted from databank structures are used. The calculated rms fluctuations of α-carbon atoms are in good agreement with crystallographic temperature factors. The two regions of the protein that pack against each other to form the main hydrophobic core exhibit negatively correlated fluctuations. The conformational dynamics could efficiently be probed by the time-delayed orientational and conformational correlation functions of the virtual bonds: the active site loop, excluding the active site bond, the turn region, and the N-terminal of the α-helix are relatively more mobile regions of the structure. A correlation is observed between the hydrogen/deuterium (H/D) exchange behavior and the long-time orientational and conformational autocorrelation function values for CI2. A cooperativity in the rotations of the bonds near in sequence is observed at all time windows, whereas the cooperative rotations of the bonds far along the sequence appear at long time windows; these correlations contribute to the stability of the secondary structures and the tertiary structure, respectively. Proteins 1999;37:454–464. ©1999 Wiley-Liss, Inc.

WOS: 000455572800006, This study was to determine the severity of the tandoor burns in a city in Eastern Anatolia and to attract attention for the prevention of tandoor burns. Subjects and Methods: A retrospective evaluation was made of the registration data of burns hospitalized. A record was made of patient variables including burn type, age, gender, socioeconomic status, and amputation site and level. Statistical Analysis Used: Descriptive statistics were used to summarize the data, and results were reported as number and percentage. Categorical variables were evaluated with the Chi-square test. P

WOS: 000341933600118, PubMed ID: 25102393, Partial-thickness wound models of rat skin have some difficulties in creating the wounds in equal size and depth. Moreover, making a split-thickness wound on the rat skin seems not to be simple and rapid. A new alternative method was presented here to overcome these obstacles, by using a waterjet device to create a split-thickness wound on rat skin. Twenty-four male Wistar rats were randomly divided into 3 groups. An area of 4 x 4 cm in diameter was marked on the center of the dorsal skin. Waterjet hydrosurgery system was used to create a wound on the dorsal rat skin, by removing the outer layers of the skin. In group 1, rat skin was wounded with setting 1 to create a superficial skin wound. In group 2, it was injured with setting 5 to make a deeper wound, and in group 3, skin wound was performed with setting 10 making the deepest wound in the experiment. After the wounds were created on the rat skin, a full-thickness skin biopsy was taken from the middle of the cranial margin of the wound, including both the wound surface and the healthy skin in a specimen. Healing time of the wounds of animals was recorded in the experiment groups. Then, the results were compared statistically between the groups. In the histologic assessment, both the thickness of the remnant of the epidermis in the wound surface and the thickness of the healthy epidermis were measured under light microscope. Thickness of the epidermis remaining after wounding was statistically compared among the groups and with the healthy epidermis. The mean thickness of the remaining epidermis was determined for each group. It was higher in the superficial wounds than in the deep wounds, because of the removal of the skin from its outer surface through the deep layers of the skin with waterjet device. The most superficial wound in the experiment was observed in group 1, which was statistically different from the wounds of group 3, whereas there was no difference between the wounds of groups 1 and 2. Compared with the wounds of groups 1 and 2, the wounds in group 3 were significantly deeper than the wounds of other groups, which was statistically significant. In all groups, mean thickness of epidermis in the wound surface showed statistically significant difference from that in the healthy skin. When compared with the healing times of the wounds in the groups, a statistically significant difference was found between them. Creation of a split-skin wound, by using the waterjet system, provides a wound in reproducible size and depth, also in a standardized and rapid manner. Moreover, it makes precise and controlled wound creation in the rat skin.

Over the last 5 years, cytosine base editors (CBEs) have emerged as a promising therapeutic tool for specific editing of single nucleotide variants and disrupting specific genes associated with disease. Despite this promise, the currently available CBE's have the significant liabilities of off-target and bystander editing activities, in part due to the mechanism by which they are delivered, causing limitations in their potential applications. In this study we engineeredhighly stabilized Cas-embedded CBEs (sCE_CBEs) that integrate several recent advances, andthat are highly expressible and soluble for direct delivery into cells as ribonucleoprotein (RNP) complexes. Our resulting sCE_CBE RNP complexes efficiently and specifically target TC dinucleotides with minimal off-target or bystander mutations. Additional uracil glycosylase inhibitor (UGI) protein in trans further increased C-to-T editing efficiency and target purity in a dose-dependent manner, minimizing indel formation to untreated levels. A single electroporation was sufficient to effectively edit the therapeutically relevant locus for sickle cell disease in hematopoietic stem and progenitor cells (HSPC) in a dose dependent manner without cellular toxicity. Significantly, these sCE_CBE RNPs permitted for the transplantation of edited HSPCs confirming highly efficient editing in engrafting hematopoietic stem cells in mice. The success of the designed sCBE editors, with improved solubility and enhanced on-target editing, demonstrates promising agents for cytosine base editing at other disease-related sites in HSPCs and other cell types.