bluuebunny/crossref_metadata_2025 huggingface dataset:
General energy decay estimates of Timoshenko systems with frictional versus viscoelastic damping
Authors: Guesmia Aissa , Messaoudi Salim A.
Published: February 2009

In this paper we consider the following Timoshenko system:

with Dirichlet boundary conditions and initial data where a, b, g and h are specific functions and ρ1, ρ2, k1, k2 and L are given positive constants. We establish a general stability estimate using the multiplier method and some properties of convex functions. Without imposing any growth condition on h at the origin, we show that the energy of the system is bounded above by a quantity, depending on g and h, which tends to zero as time goes to infinity. Our estimate allows us to consider a large class of functions h with general growth at the origin. We use some examples (known in the case of wave equations and Maxwell system) to show how to derive from our general estimate the polynomial, exponential or logarithmic decay. The results of this paper improve and generalize some existing results in the literature and generate some interesting open problems. Copyright © 2009 John Wiley & Sons, Ltd.
A bonded joint finite element for a symmetric double lap joint subjected to mechanical and thermal loads
Authors: Gustafson Peter A. , Waas Anthony M.
Published: February 2009

A bonded joint finite element (FE) for a symmetric double lap joint is developed that is capable of predicting field quantities in the lap region. The element is a hybrid method and incorporates features of classical analytical and numerical methods. The element stiffness and load vector formulations have unique, load dependent, non‐linear shape functions based on an analytical solution. The adaptive shape functions are formulated in terms of the dimensionless mechanical load fraction \documentclass{article}\footskip=0pc\pagestyle{empty}\begin{document}$(\bar{\bar{\phi}}_P)$\end{document} and total load \documentclass{article}\footskip=0pc\pagestyle{empty}\begin{document}$(\bar{\bar{\phi}}_{\rm {tot}})$\end{document} and are capable of predicting the thermal and mechanical load response. The bonded joint element has been implemented as a user element in the Abaqus® commercial FE code. A comparison of the stress predictions for the bonded joint element and a conventional 2D FE model is presented and are found to be in good agreement. Therefore, the element provides a computationally efficient and mesh‐independent stress prediction. The single element reproduces the analytical solution with minimal analyst input and can be easily incorporated into early design and sizing studies. Copyright © 2009 John Wiley & Sons, Ltd.
An enriched element‐failure method (REFM) for delamination analysis of composite structures
Authors: Sun X. S. , Tan V. B. C. , Liu G. , Tay T. E.
Published: February 2009

This paper develops an enriched element‐failure method for delamination analysis of composite structures. This method combines discontinuous enrichments in the extended finite element method and element‐failure concepts in the element‐failure method within the finite element framework. An improved discontinuous enrichment function is presented to effectively model the kinked discontinuities; and, based on fracture mechanics, a general near‐tip enrichment function is also derived from the asymptotic displacement fields to represent the discontinuity and local stress intensification around the crack‐tip. The delamination is treated as a crack problem that is represented by the discontinuous enrichment functions and then the enrichments are transformed to external nodal forces applied to nodes around the crack. The crack and its propagation are modeled by the ‘failed elements’ that are applied to the external nodal forces. Delamination and crack kinking problems can be solved simultaneously without remeshing the model or re‐assembling the stiffness matrix with this method. Examples are used to demonstrate the application of the proposed method to delamination analysis. The validity of the proposed method is verified and the simulation results show that both interlaminar delamination and crack kinking (intralaminar crack) occur in the cross‐ply laminated plate, which is observed in the experiment. Copyright © 2009 John Wiley & Sons, Ltd.
Pubic symphyseal face eburnation: an Egyptian sport story?
Authors: Judd M. A.
Published: February 2009

Strenuous physical activity leaves scars on bone that attest to the demands of occupation, sport, aggression and recreation. During the assessment of 74 C‐Group Nubians from Hierakonpolis (Egypt) dated to the Egyptian Middle Kingdom–Second Intermediate Period (2080–1700 BC), robust muscle insertions along the ilia and ischia were observed among some adults. In addition, a disproportionate degeneration of the pubic symphyseal faces when compared to other age‐related features was also noted. In the case of one male (Burial 32), the pubic symphyseal faces were completely flattened and polished so that they resembled the eburnation that is pathognomic of osteoarthritis. Differential diagnoses are discussed and osteitis pubis, an increasingly diagnosed injury among modern athletes who participate in intense activity that involves running, kicking, twisting or leaping, is proposed as the most likely etiology. The exaggerated muscle insertions and pubic symphyseal wear, epitomised by the individual interred in Burial 32, are unique features that may be linked to the unexplained presence of this Nubian group deep in Egyptian territory during a period of political instability. Artefactual, artistic and documentary evidence records how the Egyptian pharaohs and elites conscripted Nubian athletes to the royal courts for staged contests and entertainment, part of a propaganda program engineered to reinforce among the general populace the dogma of Egyptian supremacy over the enemy. This Nubian community, serviced by Cemetery HK27C, may have functioned as a source for individuals skilled in athletics or other activities that required exceptional physical dexterity. The extraordinary modification of these pubic symphyseal faces underscores the importance of recognising paleopathological conditions that may further confound current macroscopic methods used to ascertain the chronological age of an individual. Copyright © 2009 John Wiley & Sons, Ltd.
Preparation and characterization of polyurethan‐block‐poly(trifluoro‐ propylmethyl)siloxane elastomers
Authors: Shi Zhendong , Wang Xinling
Published: February 2009

A series of polyurethane‐block‐poly(trifluoropropylmethyl)siloxane (PUFS) elastomers were prepared via a two‐step process from toluenediisocyanate (TDI), α ω‐bis(3‐aminopropyldiethoxylsilane) poly(trifluoropropylmethyl)siloxane (APFS), and poly(tetramethylene oxide) (PTMO). The PUFS films were formed through moisture curing and characterized by DSC, DMTA, TGA, mechanical testing, and water contact angle. It was found that the extent of microphase separation of the PUFS system would increase with the increase in APFS content, and result in the decrease in the tensile strength and the thermal stability. On the other hand, the crosslink density of the PUFS system would apparently increase with the increase in the TDI content, which reduced the microphase separation and improved the tensile properties and the thermal stability of the PUFS elastomers. Copyright © 2009 John Wiley & Sons, Ltd.
Morphology of phase‐separated thermotropic layers based on UV cured acrylate resins
Authors: Resch Katharina , Wallner Gernot M.
Published: February 2009

In this paper, relationships between the scattering domain parameters (size and shape) and the light‐shielding properties of thermotropic systems with fixed domains (TSFD) are established. Specific focus is given to the effect of additive type on the formation of scattering domain size. Various functional layers are prepared by a variation of thermotropic additives. Scattering domains are investigated applying high resolution Atomic Force Microscopy (AFM) in a phase imaging mode. Thermotropic layers formulated with additive types exhibiting a short chain length display roughly spherical scattering particles with dimensions between 0.5 and 3 µm and a moderate reduction in hemispheric solar transmittance along with a significant increase in diffuse solar transmittance above the switching threshold. Additive types with long‐chain molecules develop anisotropic scattering domains resembling distorted disks with a diameter up to 50 µm and a thickness between 100 and 400 nm. Disk‐like scattering features yield enhanced light‐shielding properties. Copyright © 2009 John Wiley & Sons, Ltd.
Irradiation‐induced grafting of acrylonitrile onto activated carbon fiber
Authors: Zhang Sujuan , Zhang Qikun , Chen Shuixia , Yuan Qingyu , Li Peiyuan
Published: February 2009

Viscose‐based activated carbon fiber (VACF) was modified with acrylonitrile (AN) by γ‐irradiation‐induced grafting polymerization. Effects of the grafting conditions, such as concentrations of AN and divinylbenzene (DVB), pH value, and solvent on the grafting process were studied. The physicochemical properties of the fibers were characterized. The results show that AN can be effectively grafted onto the surface of VACF with the addition of DVB. The grafting yield is higher than 12% according to thermogravimetric (TG) analysis. The study shows that DVB can improve the grafting degree of AN in the form of grafting chains or agglomerate materials. After grafting modification, VACF shows a small decrease in the specific surface area. Copyright © 2009 John Wiley & Sons, Ltd.
Development of the vertebrate central nervous system: formation of the neural tube
Authors: Greene Nicholas D. E. , Copp Andrew J.
Published: February 2009

The developmental process of neurulation involves a series of coordinated morphological events, which result in conversion of the flat neural plate into the neural tube, the primordium of the entire central nervous system (CNS). Failure of neurulation results in neural tube defects (NTDs), severe abnormalities of the CNS, which are among the commonest of congenital malformations in humans. In order to gain insight into the embryological basis of NTDs, such as spina bifida and anencephaly, it is necessary to understand the morphogenetic processes and molecular mechanisms underlying neural tube closure. The mouse is the most extensively studied mammalian experimental model for studies of neurulation, while considerable insight into underlying developmental mechanisms has also arisen from studies in other model systems, particularly birds and amphibians. We describe the process of neural tube formation, discuss the cellular mechanisms involved and highlight recent findings that provide links between molecular signaling pathways and morphogenetic tissue movements. Copyright © 2009 John Wiley & Sons, Ltd.
Rhabdomyolysis reports show interaction between simvastatin and CYP3A4 inhibitors
Authors: Rowan Christopher , Brinker Allen D. , Nourjah Parivash , Chang Jennie , Mosholder Andrew , Barrett Jeffrey S. , Avigan Mark
Published: February 2009

Purpose

To assess spontaneous reports of rhabdomyolysis associated with simvastatin (SV) and pravastatin (PV) for evidence of CYP3A4 interaction. Clinical trial results advocate cholesterol lowering in high‐risk patients including diabetics and the elderly. Given the association between advancing age, metabolic, and cardiovascular disease, many patients are treated with concomitant medications upon statin initiation. Although statins are generally safe, minor and severe adverse reactions arise, especially when given to patients taking concomitant medications that inhibit the statin clearance and lead to increased statin plasma concentration.

Methods

We conducted a comparative case series of rhabdomyolysis reports associated with SV and PV. Domestic spontaneous reports were obtained from the FDA's Adverse Event Reporting System (AERS). Drug utilization data were obtained from IMS HEALTH and the National Ambulatory Medical Care Survey (NAMCS). Adverse event reporting rates (AER) and ratios (AERR) of rhabdomyolysis associated with SV and PV—with and without stratification by CYP3A4 inhibitor concomitancy were determined.

Results

Stratification by CYP3A4 inhibitor concomitancy did not change the rhabdomyolysis AER for PV with or without a CYP3A4 inhibitor (2.4 cases and 3.1 cases per 10 million Rx, respectively). However, stratification of SV reports with or without a concomitant CYP3A4 inhibitor resulted in a rhabdomyolysis AER (38.4 and 6.0 cases per 10 million Rx, respectively). The corresponding AERR with or without a CYP3A4 inhibitor were 0.77 for PV and 6.43 for SV.

Conclusions

Spontaneous adverse event reports provide evidence of increased risk for rhabdomyolysis based on interaction between SV and selected CYP3A4 inhibitors. Copyright © 2009 John Wiley & Sons, Ltd.
Plant oil‐based biofoam composites with balanced performance
Authors: Wu Su Ping , Qiu Jun Feng , Rong Min Zhi , Zhang Ming Qiu , Zhang Li Yang
Published: February 2009

BACKGROUND: Biofoam composites were prepared using short sisal fibers as reinforcement and acrylated epoxidized soybean oil as matrix, aiming at replacing traditional unsaturated polyester foams in structural applications. The compressive properties of the composites were examined as a function of fiber loading, fiber length and foam density.

RESULTS: The foam composite with 10 phr (parts per hundred of base resin by weight) sisal fiber possessed properties similar to those of commercial unsaturated polyester foams. A study of the failure mechanism revealed that debonding between fiber and matrix was a key issue responsible for catastrophic damage of the composites. According to this finding, surface pre‐treatment of the sisal using an alkali or silane coupling agent was carried out. This brought about positive effects on interfacial interaction and compressive strength of the composites, as desired. Also, soil burial tests proved that the foam composites could be biodegraded, and the incorporated sisal fibers accelerated the biodegradation of the composites.

CONCLUSION: This work shows the feasibility of making rigid biofoams from natural resources, which could be potential candidates for structural foams. Copyright © 2009 Society of Chemical Industry
Characterisation of Plasmodium invasive organelles; an ookinete microneme proteome
Authors: Lal Kalpana , Prieto Judith Helena , Bromley Elizabeth , Sanderson Sanya J. , Yates John R. , Wastling Jonathan M. , Tomley Fiona M. , Sinden Robert E.
Published: February 2009

Secretion of microneme proteins is essential to Plasmodium invasion but the molecular composition of these secretory organelles remains poorly defined. Here, we describe the first Plasmodium microneme proteome. Purification of micronemes by subcellular fractionation from cultured ookinetes was confirmed by enrichment of known micronemal proteins and electron microscopy. Quantitation of electron micrographs showed >14‐fold microneme enrichment compared to the intact ookinete, such that micronemes comprised 85% of the identifiable organelles in the fraction. Gel LC‐MS/MS of the most abundant protein constituents of the fraction identified three known micronemal proteins chitinase, CTRP, SOAP, together with protein disulphide isomerase (PDI) and HSP70. Highly sensitive MudPIT shotgun proteomics described a total of 345 proteins in the fraction. M1 aminopeptidase and PDI, the former a recognised target of drug development, were both shown to have a micronemal location by IFA. We further identified numerous proteins with established vesicle trafficking and signaling functions consistent with micronemes being part of a regulated secretory pathway. Previously uncharacterised proteins comprise the largest functional group of the microneme proteome and will include secreted proteins important to invasion.
A proteomic evaluation of Pyrenophora tritici‐repentis, causal agent of tan spot of wheat, reveals major differences between virulent and avirulent isolates
Authors: Cao Tiesen , Kim Yong Min , Kav Nat N. V. , Strelkov Stephen E.
Published: February 2009

Pyrenophora tritici‐repentis causes tan spot, an important foliar disease of wheat. The fungus produces multiple host‐specific toxins, including Ptr ToxB, a chlorosis‐inducing protein encoded by the ToxB gene. A homolog of ToxB is also found in avirulent isolates of the fungus. In order to improve understanding of the role of this homolog and evaluate the general pathogenic ability of P. tritici‐repentis, we compared the proteomes of avirulent race 4 and virulent race 5 isolates of the pathogen. Western blotting analysis revealed the presence of Ptr ToxB in spore germination and culture fluids of race 5 but not race 4. A comprehensive proteome‐level comparison by 2‐DE indicated 133 differentially abundant proteins in the secretome (29 proteins) and mycelium (104 proteins) of races 4 and 5, of which 63 were identified by MS/MS. A number of the proteins found to be up‐regulated in race 5 have been implicated in microbial virulence in other pathosystems, and included the secreted enzymes α‐mannosidase and exo‐β‐1,3‐glucanase, heat‐shock and BiP proteins, and various metabolic enzymes. These proteome‐level differences suggest a reduced general pathogenic ability in race 4 of P. tritici‐repentis, irrespective of toxin production. Such differences may reflect an adaptation to a saprophytic habit.
Actin, gelsolin and filamin‐A are dynamic actors in the cytoskeleton remodelling contributing to the blood brain barrier phenotype
Authors: Pottiez Gwënaël , Sevin Emmanuel , Cecchelli Roméo , Karamanos Yannis , Flahaut Christophe
Published: February 2009

The brain vascular endothelium operates as a dynamic regulatory interface to maintain the cell environment of the nervous system. In the vicinity of astrocytes, brain endothelial cells develop characteristic features conferring a strong cellular impermeability which limits the penetration of various compounds. The aim of our study was to determine by differential proteomic analysis the changes occurring in bovine brain capillary endothelial cells (BBCEC) differentiated in co‐culture with astrocytes compared with endothelial cells cultured alone. In order to obtain reproducible and meaningful protein profiles of in vitro blood–brain barrier models, three sample preparation procedures were carried out to provide the first 2‐D comparative proteomic study of BBCEC. Our study highlights advantages and drawbacks of each procedure. The cellular proteins prepared from mechanical scraping of collagen‐seeded BBCEC were strongly contaminated by serum proteins. Enzymatic dissociation of BBCEC by trypsin or collagenase solved this problem. A comparative 2‐DE profile study of collagenase‐harvested BBCEC revealed that cytoskeleton‐related proteins (actin, gelsolin and filamin‐A) show the most significant quantitative changes in the Triton soluble protein fraction from BBCEC that exhibit characteristics closest to the in vivo situation.
Mining the Leishmania genome for novel antigens and vaccine candidates
Authors: Herrera‐Najera Carla , Piña‐Aguilar Raúl , Xacur‐Garcia Fiona , Ramirez‐Sierra Maria Jesus , Dumonteil Eric
Published: February 2009

Leishmaniasis is a neglected disease with an estimated 12 million infected people. The recent completion of the sequencing of the Leishmania major genome has opened opportunities for the identification of targets for vaccine development. We present here the first attempt at identifying novel vaccine candidates by whole genome analysis. We predicted CD8+ T cell epitopes from the L. major proteome and validated in vivo in mice the immunogenicity of some of the best predicted epitopes. Consensus epitope predictions from 8272 annotated protein sequences with 5–8 different algorithms allowed the identification of 78 class I CD8+ epitopes. BALB/c mice were immunized with 26 synthetic peptides corresponding to the most likely epitopes. Fourteen (54%) resulted immunogenic, with eight being strong inducers of T cell IFNγ production. None of the proteins from which the epitopes are derived are differentially expressed, only two may be surface proteins, eight have putative enzymatic, and metabolic activities. These epitopes and proteins represent new antigen candidates for further studies. While pathogen genomes have not yet delivered their full promise in terms of human health applications, our study opens the way for extensive genome mining for antigen identification and vaccine development against Leishmania and other pathogens.
Internal and external factors influencing the Diels–Alder reaction
Authors: Kiselev Vladimir D. , Konovalov Alexander I.
Published: February 2009

The internal and external key factors influencing the reaction rates were considered for the Diels–Alder reaction (DAR) with normal electron demand. A simple dependence lgk2 = −28.81 +316.3/(IPD−EA)−69.9 R1–4/(IPD−EA)−0.054ΔHr‐n for the reaction rate as a function of the ionization potential of π‐donor dienes, electron affinity of π–acceptor dienophiles, heat of reaction and diene structure (R1–4) was obtained. This relation allows one to explain the origin of the conventional and ‘anomalous’ relations between activity and selectivity, and the variety of relationships between the kinetic activity and thermodynamic stability. It helps one to choose the conditions for the directed synthesis of desired products, to explain the acceleration effects in the presence of Lewis acids and to predict the rates of the DAR between the different reagents with CC bonds not explored earlier. Copyright © 2009 John Wiley & Sons, Ltd.
Synthesis and characterization of a new ethynyl‐linked alternating anthracene/fluorene copolymer for organic thin film transistor
Authors: Ju Jin Uk , Chung Dae Sung , Kim Seul Ong , Jung Sung Ouk , Park Chan Eon , Kim Yun‐Hi , Kwon Soon‐Ki
Published: February 2009

A new p‐type conjugated copolymer, poly(9,10‐diethynylanthracene‐alt‐9,9‐didodecylfluorene) (PDADF), which is composed of ethynyl‐linked alternating anthracene/fluorene, is synthesized via a palladium(II)‐catalyzed Sonogashira coupling reaction with 9,10‐diethynylanthracene and 2,7‐diiodo‐9,9‐didodecyl‐fluorene. The obtained polymer is confirmed by FTIR, 1H‐NMR, 13C‐NMR and elemental analysis. The PDADF had very good solubility in organic solvents such as chloroform and had a weight average molecular weight of 29,300 with a polydispersity index of 1.29. The PL maximum of the polymer was found at 533 and 568 nm for a solution and 608 nm for film, respectively. The highest occupied molecular orbital (HOMO) energy of the polymer is −5.62 eV as measured via cyclic voltammetry (CV). A solution‐processed thin film transistor device showed a carrier mobility value of 6.0 × 10−4 cm2/Vs with a threshold voltage of −17 V and a capacitance (Ci) of 10 nF/cm2. The out‐of‐plane and in‐plane GIXD pattern of spin‐coated polymer on SiO2 dielectric surfaces showed an amorphous halo near 2θ = 20°. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1609–1616, 2009
Anionic alternating copolymerization of a bifunctional six‐membered lactone and glycidyl phenyl ether: Selective synthesis of a linear polyester having lactone moiety
Authors: Uenishi Kazuya , Sudo Atsushi , Endo Takeshi
Published: February 2009

An imidazole‐initiated copolymerization of an aromatic bislactone, 10‐methyl‐2H,8H‐benzo[1,2‐b:5,4‐b′]bipyran‐2,8‐dione (1), and glycidyl phenyl ether (GPE) was investigated. In spite of the bifunctional nature of 1 that would potentially permit formation of networked and thus insoluble polymers upon its copolymerization, only one of the two lactone moieties of 1 exclusively underwent the copolymerization to give a linear polyester. Spectroscopic analysis of the polyester and its reductive scission into the corresponding fragment revealed that the polyester was formed by a 1:1 alternating copolymerization of GPE and the lactone moiety of 1. The other lactone in 1 that did not participate in the copolymerization was quantitatively incorporated into the side chain of the polyester as a reactive site, of which ring‐opening reactions by amine and alcohol as nucleophilic reagents allowed chemoselective polymer reactions. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1661–1672, 2009
Azine bridge formation during the electrografting of acrylonitrile
Authors: Easter Penelope A. , Taylor D. Martin
Published: February 2009

We report the electrografting of polyacrylonitrile (PAN) films onto evaporated gold, silver, and copper substrates, and onto platinum foil. The FTIR spectra of the films are virtually identical to library spectra of PAN and show no evidence of the cyclization observed by some authors. Raman spectra of the same films, however, display a broad absorption band centered at ∼1520 cm−1 and a second band at 1096 cm−1, which are neither present in spin‐coated films of commercially obtained PAN nor are they present in the FTIR spectra. These previously unreported Raman bands are dependent on a number of factors in the electrografting process; the working electrode metal, the monomer concentration, the solvent used, and the electrochemical protocol used for cleaning the working electrode. The relative intensity of the bands was found to be strongest in films grown on platinum and weakest in those grown on gold, and stronger in thin films (<50 nm) than in thick films (>100 nm). We suggest that these new spectral features are associated with azine‐linked polymer chains in a ladder‐like structure close to the electrode surface as a result of the predominantly isotactic brush‐like conformation of the polymer during the early stages of electrografting. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1685–1695, 2009
RAFT polymerization of activated 4‐vinylbenzoates
Authors: Nilles Katja , Theato Patrick
Published: February 2009

The reversible addition fragmentation chain transfer (RAFT) polymerization of five active ester monomers based on 4‐vinylbenzoic acid had been investigated. Pentafluorophenyl 4‐vinylbenzoate could be polymerized under RAFT conditions yielding polymers with very good control over molecular weight and narrow molecular weight distributions. Following the synthesis of diblock copolymers consisting of polystyrene, polypentafluorostyrene, poly(4‐octylstyrene), or poly(4‐acetoxystyrene) as an inert block and poly(pentafluorophenyl 4‐vinylbenzoate) as a reactive block was successfully performed. The diblock copolymer poly(pentafluoro styrene)‐block‐poly(pentafluorophenyl 4‐vinylbenzoate) had been analyzed by 19F NMR spectroscopy in solution, demonstrating the synthetic potential of pentafluorophenyl 4‐vinylbenzoate as an extremely valuable monomer for the synthesis of highly functionalized polymeric architectures. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1696–1705, 2009
Levels of pollutants in indoor air and respiratory health in preschool children: A systematic review
Authors: Fuentes‐Leonarte Virginia , Tenías José M. , Ballester Ferran
Published: February 2009

The aim of this paper is thus to identify, evluate, and summarize in a systematic fashion all the epidemiological studies that have analyzed the association between exposure to specific indoor air pollutants and respiratory disease among children under the age of five. A search was carried out in the main biomedical bibliographica sources in December 2006 and updated in February 2008. The study period covered 12 years (1996–2007). All the selected papers were carefully read. We focused on studies that analyzed at least one indicator of respiratory health and which included one or more indoor air pollutants in relation to the respiratory health of children under the age of 5. Studies that analyzed passive smoking as the sole source of indoor air pollution were not included. Fourteen studies were considered to be relevant. The most analyzed pollutant was nitrogen dioxide, followed by volative organic compounds, airborne particulates and other pollutants; phthalates and CO2. The literature reviewed within our criteria seems to indicate that several indoor pollutants, even at the moderate levels found in the developed countries, could be harmful to the respiratory health of very young children. Future research should focus on conducting more studies, preferably making use of cohorts, with adequate techniques for measuring indooor pollution levels. Pediatr Pulmonol. 2009; 44:231–243. © 2009 Wiley‐Liss, Inc.
The effect of obesity on pulmonary lung function of school aged children in Greece
Authors: Spathopoulos Diogenis , Paraskakis Emmanouil , Trypsianis Grigorios , Tsalkidis Aggelos , Arvanitidou Vasiliki , Emporiadou Maria , Bouros Demosthenes , Chatzimichael Athanasios
Published: February 2009

Obesity impacts on many issues of pulmonary medicine, where it is debated if obesity is linked to asthma, atopy or altered lung function tests. Our study aimed to investigate primarily the effect of obesity on the lung function tests and secondary the possible link of obesity with atopy and asthma in a large cohort of children in Greece.

Body mass index (BMI) and data from a questionnaire for lung health, atopy, nutritional habits and family history were obtained from 2,715 children aged 6–11 years. Six hundred fifty‐seven children with BMI>85th percentile (357 overweight, 300 obese) and a group of 196 normal weight children underwent spirometry. The % expected FVC, FEV1, FEF25–75, and FEV1/FVC were significantly reduced in overweight or obese children compared to children with normal weight (P = 0.007, P < 0.001, P < 0.001, and P < 0.001, respectively). Reported atopy was significantly higher in overweight or obese children compared to normal weight children (P = 0.008). High BMI remained a strong independent risk factor for asthma (OR = 2.17, 95% CI = 1.22–3.87, P = 0.009) and for atopy (OR = 2.06, 95% CI = 1.32–3.22, P = 0.002). The effect of increased BMI on asthma was significant in girls, but not in boys (OR = 2.73, 95% CI = 1.09–6.85, P = 0.032; OR = 1.74, 95% CI = 0.83–3.73, P = 0.137, respectively).

In conclusion we have shown that high BMI remains an important determinant of reduced spirometric parameters, a risk factor for atopy in both genders and for asthma in girls. Pediatr Pulmonol. 2009; 44:273–280. © 2009 Wiley‐Liss, Inc.
Crystal structure of domain‐swapped STE20 OSR1 kinase domain
Authors: Lee Seung‐Jae , Cobb Melanie H. , Goldsmith Elizabeth J.
Published: February 2009

OSR1 (oxidative stress‐responsive‐1) and SPAK (Ste20/Sps1‐related proline/alanine‐rich kinase) belong to the GCK‐VI subfamily of Ste20 group kinases. OSR1 and SPAK are key regulators of NKCCs (Na+/K+/2Cl− cotransporters) and activated by WNK family members (with‐no‐lysine kinase), mutations of which are known to cause Gordon syndrome, an autosomal dominant form of inherited hypertension. The crystal structure of OSR1 kinase domain has been solved at 2.25 Å. OSR1 forms a domain‐swapped dimer in an inactive conformation, in which P+1 loop and αEF helix are swapped between dimer‐related monomers. Structural alignment with nonswapped Ste20 TAO2 kinase indicates that the integrity of chemical interactions in the kinase domain is well preserved in the domain‐swapped interfaces. The OSR1 kinase domain has now been added to a growing list of domain‐swapped protein kinases recently reported, suggesting that the domain‐swapping event provides an additional layer of complexity in regulating protein kinase activity.
A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding
Authors: Moparthi Satish Babu , Hammarström Per , Carlsson Uno
Published: February 2009

The protein folding process is often in vitro rate‐limited by slow cis‐trans proline isomerization steps. Importantly, the rate of this process in vivo is accelerated by prolyl isomerases (PPIases). The archetypal PPIase is the human cyclophilin 18 (Cyp18 or CypA), and Arg 55 has been demonstrated to play a crucial role when studying short peptide substrates in the catalytic action of Cyp18 by stabilizing the transition state of isomerization. However, in this study we show that a R55A mutant of Cyp18 is as efficient as the wild type to accelerate the refolding reaction of human carbonic anhydrase II (HCA II). Thus, it is evident that the active‐site located Arg 55 is not required for catalysis of the rate‐limiting prolyl cis‐trans isomerization steps during the folding of a protein substrate as HCA II. Nevertheless, catalysis of cis‐trans proline isomerization in HCA II occurs in the active‐site of Cyp18, since binding of the inhibitor cyclosporin A abolishes rate acceleration of the refolding reaction. Obviously, the catalytic mechanisms of Cyp18 can differ when acting upon a simple model peptide, four residues long, with easily accessible Pro residues compared with a large protein molecule undergoing folding with partly or completely buried Pro residues. In the latter case, the isomerization kinetics are significantly slower and simpler mechanistic factors such as desolvation and/or strain might operate during folding‐assisted catalysis, since binding to the hydrophobic active site is still a prerequisite for catalysis.
Investigating protein unfolding kinetics by pulse proteolysis
Authors: Na Yu‐Ran , Park Chiwook
Published: February 2009

Investigation of protein unfolding kinetics of proteins in crude samples may provide many exciting opportunities to study protein energetics under unconventional conditions. As an effort to develop a method with this capability, we employed “pulse proteolysis” to investigate protein unfolding kinetics. Pulse proteolysis has been shown to be an effective and facile method to determine global stability of proteins by exploiting the difference in proteolytic susceptibilities between folded and unfolded proteins. Electrophoretic separation after proteolysis allows monitoring protein unfolding without protein purification. We employed pulse proteolysis to determine unfolding kinetics of E. coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H). The unfolding kinetic constants determined by pulse proteolysis are in good agreement with those determined by circular dichroism. We then determined an unfolding kinetic constant of overexpressed MBP in a cell lysate. An accurate unfolding kinetic constant was successfully determined with the unpurified MBP. Also, we investigated the effect of ligand binding on unfolding kinetics of MBP using pulse proteolysis. On the basis of a kinetic model for unfolding of MBP•maltose complex, we have determined the dissociation equilibrium constant (Kd) of the complex from unfolding kinetic constants, which is also in good agreement with known Kd values of the complex. These results clearly demonstrate the feasibility and the accuracy of pulse proteolysis as a quantitative probe to investigate protein unfolding kinetics.
Design of a heterotetrameric coiled coil
Authors: Root Benjamin C. , Pellegrino Laurel D. , Crawford Emily D. , Kokona Bashkim , Fairman Robert
Published: February 2009

We have successfully designed a simple peptide sequence that forms highly stable coiled‐coil heterotetramers. Our model system is based on the GCN4‐pLI parallel coiled‐coil tetramer, first described by Kim and coworkers (Harbury et al., Science 1993;262:1401–1407). We introduced glutamates at all of the e and c heptad positions of one sequence (ecE) and lysines at the same positions in a second sequence (ecK). Based on a modeling study, these sidechains are close enough in space to form structure‐stabilizing salt bridges. We show that ecE and ecK are highly unstable by themselves but form very stable parallel helical tetramers when mixed, as judged by circular dichroism, analytical ultracentrifugation, and disulfide crosslinking studies. The origin of the difference in stabilities between the homomeric structures and the heteromeric structures comes from a combination of the relief of electrostatic repulsions with concomitant formation of electrostatic attractive interactions based on pH and NaCl screening experiments. We quantify the stability of the heterotetrameric coiled coil from a thermodynamic analysis and compare the finding to other similar coiled‐coil systems.
Engineering antibody fragments to fold in the absence of disulfide bonds
Authors: Seo Min Jeong , Jeong Ki Jun , Leysath Clinton E. , Ellington Andrew D. , Iverson Brent L. , Georgiou George
Published: February 2009

Disulfide bonds play a critical role in the stabilization of the immunoglobulin β‐sandwich sandwich. Under reducing conditions, such as those that prevail in the cytoplasm, disulfide bonds do not normally form and as a result most antibodies expressed in that compartment (intrabodies) accumulate in a misfolded and inactive state. We have developed a simple method for the quantitative isolation of antibody fragments that retain full activity under reducing conditions from large mutant libraries. In E. coli, inactivation of the cysteine oxidoreductase DsbA abolishes protein oxidation in the periplasm, which leads to the accumulation of scFvs and other disulfide‐containing proteins in a reduced form. Libraries of mutant scFvs were tethered onto the inner membrane of dsbA cells and mutants that could bind fluorescently labeled antigen in the reducing periplasm were screened by Anchored Periplasmic Expression (APEx; Harvey et al., Proc Natl Acad Sci USA 2004;101:9193–9198.). Using this approach, we isolated scFv antibody variants that are fully active when expressed in the cytoplasm or when the four Cys residues that normally form disulfides are substituted by Ser residues.
Discovery of amyloid‐beta aggregation inhibitors using an engineered assay for intracellular protein folding and solubility
Authors: Lee Li Ling , Ha HyungHo , Chang Young‐Tae , DeLisa Matthew P.
Published: February 2009

Genetic and biochemical studies suggest that Alzheimer's disease (AD) is caused by a series of events initiated by the production and subsequent aggregation of the Alzheimer's amyloid β peptide (Aβ), the so‐called amyloid cascade hypothesis. Thus, a logical approach to treating AD is the development of small molecule inhibitors that either block the proteases that generate Aβ from its precursor (β‐ and γ‐secretases) or interrupt and/or reverse Aβ aggregation. To identify potent inhibitors of Aβ aggregation, we have developed a high‐throughput screen based on an earlier selection that effectively paired the folding quality control feature of the Escherichia coli Tat protein export system with aggregation of the 42‐residue AD pathogenesis effecter Aβ42. Specifically, a tripartite fusion between the Tat‐dependent export signal ssTorA, the Aβ42 peptide and the β‐lactamase (Bla) reporter enzyme was found to be export incompetent due to aggregation of the Aβ42 moiety. Here, we reasoned that small, cell‐permeable molecules that inhibited Aβ42 aggregation would render the ssTorA‐Aβ42‐Bla chimera competent for Tat export to the periplasm where Bla is active against β‐lactam antibiotics such as ampicillin. Using a fluorescence‐based version of our assay, we screened a library of triazine derivatives and isolated four nontoxic, cell‐permeable compounds that promoted efficient Tat‐dependent export of ssTorA‐Aβ42‐Bla. Each of these was subsequently shown to be a bona fide inhibitor of Aβ42 aggregation using a standard thioflavin T fibrillization assay, thereby highlighting the utility of our bacterial assay as a useful screen for antiaggregation factors under physiological conditions.
Residual structure within the disordered C‐terminal segment of p21Waf1/Cip1/Sdi1 and its implications for molecular recognition
Authors: Yoon Mi‐Kyung , Venkatachalam Veena , Huang Austin , Choi Byong‐Seok , Stultz Collin M. , Chou James J.
Published: February 2009

Probably the most unusual class of proteins in nature is the intrinsically unstructured proteins (IUPs), because they are not structured yet play essential roles in protein‐protein signaling. Many IUPs can bind different proteins, and in many cases, adopt different bound conformations. The p21 protein is a small IUP (164 residues) that is ubiquitous in cellular signaling, for example, cell cycle control, apoptosis, transcription, differentiation, and so forth; it binds to approximately 25 targets. How does this small, unstructured protein recognize each of these targets with high affinity? Here, we characterize residual structural elements of the C‐terminal segment of p21 encompassing residues 145–164 using a combination of NMR measurements and molecular dynamics simulations. The N‐terminal half of the peptide has a significant helical propensity which is recognized by calmodulin while the C‐terminal half of the peptide prefers extended conformations that facilitate binding to the proliferating cell nuclear antigen (PCNA). Our results suggest that the final bound conformations of p21 (145–164) pre‐exist in the free peptide even without its binding partners. While the conformational flexibility of the p21 peptide is essential for adapting to diverse binding environments, the intrinsic structural preferences of the free peptide enable promiscuous yet high affinity binding to a diverse array of molecular targets.
An approach to characterizing single‐subunit mutations in multimeric prepores and pores of anthrax protective antigen
Authors: Janowiak Blythe E. , Finkelstein Alan , Collier R. John
Published: February 2009

Heptameric pores formed by the protective antigen (PA) moiety of anthrax toxin translocate the intracellular effector moieties of the toxin across the endosomal membrane to the cytosol of mammalian cells. We devised a protocol to characterize the effects of individual mutations in a single subunit of heptameric PA prepores (pore precursors) or pores. We prepared monomeric PA containing a test mutation plus an innocuous Cys‐replacement mutation at a second residue (Lys563, located on the external surface of the prepore). The introduced Cys was biotinylated, and the protein was allowed to cooligomerize with a 20‐fold excess of wild‐type PA. Finally, biotinylated prepores were freed from wild‐type prepores by avidin affinity chromatography. For the proof of principle, we examined single‐subunit mutations of Asp425 and Phe427, two residues where Ala replacements have been shown to cause strong inhibitory effects. The single‐subunit D425A mutation inhibited pore formation by >104 and abrogated activity of PA almost completely in our standard cytotoxicity assay. The single‐subunit F427A mutation caused ∼100‐fold inhibition in the cytotoxicity assay, and this effect was shown to result from a combination of strong inhibition of translocation and smaller effects on pore formation and ligand affinity. Our results show definitively that replacing a single residue in one subunit of the heptameric PA prepore can inhibit the transport activity of the oligomer almost completely—and by different mechanisms, depending on the specific residue mutated.
Nonenzymatic biotinylation of histone H2A
Authors: Healy Shannon , Heightman Tom D. , Hohmann Laura , Schriemer David , Gravel Roy A.
Published: February 2009

Holocarboxylase synthetase (HCS, eukaryotic enzyme) and BirA (prokaryotic) are biotin protein ligases that catalyze the ATP‐dependent attachment of biotin to apocarboxylases via the reactive intermediate, bio‐5′‐AMP. In this study, we examined the in vitro mechanism of biotin attachment to histone H2A in the presence of HCS and BirA. The experiment derives from our observations that HCS is found in the nucleus of cells in addition to the cytoplasm, and it has the ability to attach biotin to histones in vitro (Narang et al., Hum Mol Genet 2004; 13:15–23). Using recombinant HCS or BirA, the rate of biotin attachment was considerably slower with histone H2A than with the biotin binding domain of an apocarboxylase. However, on incubation of recombinant H2A with chemically synthesized bio‐5′‐AMP, H2A was observed to be rapidly labeled with biotin in the absence of enzyme. Nonenzymatic biotinylation of a truncated apocarboxylase (BCCP87) has been previously reported (Streaker and Beckett, Protein Sci 2006; 15:1928–1935), though at a much slower rate than we observe for H2A. The specific attachment sites of nonenzymatically biotinylated recombinant H2A at different time points were identified using mass spectrometry, and were found to consist of a similar pattern of biotin attachment as seen in the presence of HCS, with preference for lysines in the highly basic N‐terminal region of the histone. None of the lysine sites within H2A resembles the biotin attachment consensus sequence seen in carboxylases, suggesting a novel mechanism for histone biotinylation.
StoneHinge: Hinge prediction by network analysis of individual protein structures
Authors: Keating Kevin S. , Flores Samuel C. , Gerstein Mark B. , Kuhn Leslie A.
Published: February 2009

Hinge motions are important for molecular recognition, and knowledge of their location can guide the sampling of protein conformations for docking. Predicting domains and intervening hinges is also important for identifying structurally self‐determinate units and anticipating the influence of mutations on protein flexibility and stability. Here we present StoneHinge, a novel approach for predicting hinges between domains using input from two complementary analyses of noncovalent bond networks: StoneHingeP, which identifies domain‐hinge‐domain signatures in ProFlex constraint counting results, and StoneHingeD, which does the same for DomDecomp Gaussian network analyses. Predictions for the two methods are compared to hinges defined in the literature and by visual inspection of interpolated motions between conformations in a series of proteins. For StoneHingeP, all the predicted hinges agree with hinge sites reported in the literature or observed visually, although some predictions include extra residues. Furthermore, no hinges are predicted in six hinge‐free proteins. On the other hand, StoneHingeD tends to overpredict the number of hinges, while accurately pinpointing hinge locations. By determining the consensus of their results, StoneHinge improves the specificity, predicting 11 of 13 hinges found both visually and in the literature for nine different open protein structures, and making no false‐positive predictions. By comparison, a popular hinge detection method that requires knowledge of both the open and closed conformations finds 10 of the 13 known hinges, while predicting four additional, false hinges.
Selection and structural analysis of de novo proteins from an α3β3 genetic library
Authors: Jumawid Mariejoy Therese , Takahashi Tsuyoshi , Yamazaki Toshimasa , Ashigai Hiroshi , Mihara Hisakazu
Published: February 2009

The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding α3β3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP‐based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8‐anilino‐1‐naphthalenesulfonate (ANS) binding assay, and analytical size‐exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both α‐helix and β‐sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten‐globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three α‐helices and three β‐strands as intended by its design. Thus, it would appear that artificially generated α3β3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.