Fluorescent probe studies of biomembranes and model systems. The design and use of anionic site reporters

The fluorescence properties of a homologous series of fluorescent alkylamines are described. The binding of the probes to crythrocyte membranes increases with the length of the alkyl chain. The probes are shown to interact more strongly with membranes than with protein and lipid model systems. The binding of the probes to the membrane is sensitive to the cation concentration of the medium.

Estimation of design basis earthquake using region-specific M-max, for the NPP site at Kalpakkam, Tamil Nadu, India

The objective of the paper is to estimate Safe Shutdown Earthquake (SSE) and Operating/Design Basis Earthquake (OBE/DBE) for the Nuclear Power Plant (NPP) site located at Kalpakkam, Tamil Nadu, India. The NPP is located at 12.558 degrees N, 80.175 degrees E and a 500 km circular area around NPP site is considered as `seismic study area' based on past regional earthquake damage distribution. The geology, seismicity and seismotectonics of the study area are studied and the seismotectonic map is prepared showing the seismic sources and the past earthquakes. Earthquake data gathered from many literatures are homogenized and declustered to form a complete earthquake catalogue for the seismic study area. The conventional maximum magnitude of each source is estimated considering the maximum observed magnitude (M-max(obs)) and/or the addition of 0.3 to 0.5 to M-max(obs). In this study maximum earthquake magnitude has been estimated by establishing a region's rupture character based on source length and associated M-max(obs). A final source-specific M-max is selected from the three M-max values by following the logical criteria. To estimate hazard at the NPP site, ten Ground-Motion Prediction Equations (GMPEs) valid for the study area are considered. These GMPEs are ranked based on Log-Likelihood (LLH) values. Top five GMPEs are considered to estimate the peak ground acceleration (PGA) for the site. Maximum PGA is obtained from three faults and named as vulnerable sources to decide the magnitudes of OBE and SSE. The average and normalized site specific response spectrum is prepared considering three vulnerable sources and further used to establish site-specific design spectrum at NPP site.

Development of design charts for stress intensity factors at tips of multi-site cracks in unstiffened curved panels

Multi-site damage need to be addressed and evaluated in order to assess the integrity of aging aircraft structures. One of the problems recognized in the recent times is the effect of interaction between two or more cracks in the close neighborhood in such structures. The present paper deals with such a problem and presents numerical estimates of stress intensity factors at a crack tip in an un-stiffened curved panel with a secondary crack in the vicinity of a primary crack. The results are presented in the form of design charts. These results should be useful in evaluation in the damage tolerance evaluation of aircraft structures with multi-site damage. (C) 2014 Elsevier Ltd. All rights reserved.

SUPPORTING ENVIRONMENTALLY-BENIGN DESIGN ELUCIDATING ENVIRONMENTAL IMPACT PROPAGATION IN CONCEPTUAL DESIGN PHASE BY SAPPHIRE MODEL OF CAUSALITY

Conceptual Design Phase is the most critical for design decisions and their impact on the Environment. It is also a phase of many `unknowns' making it flexible and allowing exploration of many solutions. Thus, it is a challenge to determine the most Environmentally-benign Solution or Concept to be translated in to a `good' product. The SAPPhIRE Model captures the various levels of abstractions present in Conceptual Design by Outcomes and defines a Solution-variant as a set of verifiable and quantifiable Outcomes. The Causality explains the propagation of Environmental Impact across Outcomes at varying levels of abstraction, suggesting that the Environmental Impact of an Outcome at a certain level can be represented as a collation of Environmental Impact information of all the Outcomes at each of its subsequent lower levels of abstraction. Thus a ball-park impact value can be associated with the higher-levels of abstraction, thereby supporting design decisions taken earlier on in Conceptual Design directing towards Environmentally-benign Design.

Site classification and estimation of surface level seismic hazard using geophysical data and probabilistic approach

This paper presents the site classification of Bangalore Mahanagar Palike (BMP) area using geophysical data and the evaluation of spectral acceleration at ground level using probabilistic approach. Site classification has been carried out using experimental data from the shallow geophysical method of Multichannel Analysis of Surface wave (MASW). One-dimensional (1-D) MASW survey has been carried out at 58 locations and respective velocity profiles are obtained. The average shear wave velocity for 30 m depth (Vs(30)) has been calculated and is used for the site classification of the BMP area as per NEHRP (National Earthquake Hazards Reduction Program). Based on the Vs(30) values major part of the BMP area can be classified as ``site class D'', and ``site class C'. A smaller portion of the study area, in and around Lalbagh Park, is classified as ``site class B''. Further, probabilistic seismic hazard analysis has been carried out to map the seismic hazard in terms spectral acceleration (S-a) at rock and the ground level considering the site classes and six seismogenic sources identified. The mean annual rate of exceedance and cumulative probability hazard curve for S. have been generated. The quantified hazard values in terms of spectral acceleration for short period and long period are mapped for rock, site class C and D with 10% probability of exceedance in 50 years on a grid size of 0.5 km. In addition to this, the Uniform Hazard Response Spectrum (UHRS) at surface level has been developed for the 5% damping and 10% probability of exceedance in 50 years for rock, site class C and D These spectral acceleration and uniform hazard spectrums can be used to assess the design force for important structures and also to develop the design spectrum.

Design of laterally loaded piles in clays based on cone penetration test data: a reliability-based approach

The behaviour of laterally loaded piles is considerably influenced by the uncertainties in soil properties. Hence probabilistic models for assessment of allowable lateral load are necessary. Cone penetration test (CPT) data are often used to determine soil strength parameters, whereby the allowable lateral load of the pile is computed. In the present study, the maximum lateral displacement and moment of the pile are obtained based on the coefficient of subgrade reaction approach, considering the nonlinear soil behaviour in undrained clay. The coefficient of subgrade reaction is related to the undrained shear strength of soil, which can be obtained from CPT data. The soil medium is modelled as a one-dimensional random field along the depth, and it is described by the standard deviation and scale of fluctuation of the undrained shear strength of soil. Inherent soil variability, measurement uncertainty and transformation uncertainty are taken into consideration. The statistics of maximum lateral deflection and moment are obtained using the first-order, second-moment technique. Hasofer-Lind reliability indices for component and system failure criteria, based on the allowable lateral displacement and moment capacity of the pile section, are evaluated. The geotechnical database from the Konaseema site in India is used as a case example. It is shown that the reliability-based design approach for pile foundations, considering the spatial variability of soil, permits a rational choice of allowable lateral loads.

Computer-Aided Drug Design of Pyranopyrazoles and Related Compounds for Checkpoint Kinase-1

Checkpoint-1 kinase plays an important role in the G(2)M cell cycle control, therefore its inhibition by small molecules is of great therapeutic interest in oncology. In this paper, we have reported the virtual screening of an in-house library of 2499 pyranopyrazole derivatives against the ATP-binding site of Chk1 kinase using Glide 5.0 program, which resulted in six hits. All these ligands were docked into the site forming most crucial interactions with Cys87, Glu91 and Leu15 residues. From the observed results these ligands are suggested to be potent inhibitors of Chk1 kinase with sufficient scope for further elaboration.

Agents Based Algorithms for Design Parameter Estimation in Contaminant Transport Inverse Problems

A considerable amount of work has been dedicated on the development of analytical solutions for flow of chemical contaminants through soils. Most of the analytical solutions for complex transport problems are closed-form series solutions. The convergence of these solutions depends on the eigen values obtained from a corresponding transcendental equation. Thus, the difficulty in obtaining exact solutions from analytical models encourages the use of numerical solutions for the parameter estimation even though, the later models are computationally expensive. In this paper a combination of two swarm intelligence based algorithms are used for accurate estimation of design transport parameters from the closed-form analytical solutions. Estimation of eigen values from a transcendental equation is treated as a multimodal discontinuous function optimization problem. The eigen values are estimated using an algorithm derived based on glowworm swarm strategy. Parameter estimation of the inverse problem is handled using standard PSO algorithm. Integration of these two algorithms enables an accurate estimation of design parameters using closed-form analytical solutions. The present solver is applied to a real world inverse problem in environmental engineering. The inverse model based on swarm intelligence techniques is validated and the accuracy in parameter estimation is shown. The proposed solver quickly estimates the design parameters with a great precision.

Design of mechanism-based inhibitors of transthyretin amyloidosis:Studies with biphenyl ethers and new structural templates

Transthyretin (TTR), a tetrameric thyroxine (T4) carrier protein, is associated with a variety of amyloid diseases. In this study, we explore the potential of biphenyl ethers (BPE), which are shown to interact with a high affinity to its T4 binding site thereby preventing its aggregation and fibrillogenesis. They prevent fibrillogenesis by stabilizing the tetrameric ground state of transthyretin. Additionally, we identify two new structural templates (2-(5-mercapto-[1,3,4]oxadiazol-2-yl)-phenol and 2,3,6-trichloro-N-(4H-[1,2,4]triazol-3-yl) represented as compounds 11 and 12, respectively, throughout the manuscript) exhibiting the ability to arrest TTR amyloidosis. The dissociation constants for the binding of BPEs and compound 11 and 12 to TTR correlate with their efficacies of inhibiting amyloidosis. They also have the ability to inhibit the elongation of intermediate fibrils as well as show nearly complete (> 90%) disruption of the preformed fibrils. The present study thus establishes biphenyl ethers and compounds 11 and 12 as very potent inhibitors of TTR fibrillization and inducible cytotoxicity.

Three-dimensional structure of heat shock protein 90 from Plasmodium falciparum: molecular modelling approach to rational drug design against malaria

We have recently implicated heat shock protein 90 from Plasmodium falciparum (PfHsp90) as a potential drug target against malaria. Using inhibitors specific to the nucleotide binding domain of Hsp90, we have shown potent growth inhibitory effects on development of malarial parasite in human erythrocytes. To gain better understanding of the vital role played by PfHsp90 in parasite growth, we have modeled its three dimensional structure using recently described full length structure of yeast Hsp90. Sequence similarity found between PfHsp90 and yeast Hsp90 allowed us to model the core structure with high confidence. The superimposition of the predicted structure with that of the template yeast Hsp90 structure reveals an RMSD of 3.31 angstrom. The N-terminal and middle domains showed the least RMSD (1.76 angstrom) while the more divergent C-terminus showed a greater RMSD (2.84 angstrom) with respect to the template. The structure shows overall conservation of domains involved in nucleotide binding, ATPase activity, co-chaperone binding as well as inter-subunit interactions. Important co-chaperones known to modulate Hsp90 function in other eukaryotes are conserved in malarial parasite as well. An acidic stretch of amino acids found in the linker region, which is uniquely extended in PfHsp90 could not be modeled in this structure suggesting a flexible conformation. Our results provide a basis to compare the overall structure and functional pathways dependent on PfHsp90 in malarial parasite. Further analysis of differences found between human and parasite Hsp90 may make it possible to design inhibitors targeted specifically against malaria.

Disulfide conformation and design at helix N-termini

To understand structural and thermodynamic features of disulfides within an alpha-helix, a non-redundant dataset comprising of 5025 polypeptide chains containing 2311 disulfides was examined. Thirty-five examples were found of intrahelical disulfides involving a CXXC motif between the N-Cap and third helical positions. GLY and PRO were the most common amino acids at positions 1 and 2, respectively. The N-Cap residue for disulfide bonded CXXC motifs had average values of (-112 +/- 25.2 degrees, 106 +/- 25.4 degrees). To further explore conformational requirements for intrahelical disulfides, CYS pairs were introduced at positions N-Cap-3; 1,4; 7,10 in two helices of an Escherichia coli thioredoxin mutant lacking its active site disulfide (nSS Trx). In both helices, disulfides formed spontaneously during purification only at positions N-Cap-3. Mutant stabilities were characterized by chemical denaturation studies (in both oxidized and reduced states) and differential scanning calorimetry (oxidized state only). All oxidized as well as reduced mutants were destabilized relative to nSS Trx. All mutants were redox active, but showed decreased activity relative to wild-type thioredoxin. Such engineered disulfides can be used to probe helix start sites in proteins of unknown structure and to introduce redox activity into proteins. Conversely, a protein with CYS residues at positions N-Cap and 3 of an alpha-helix is likely to have redox activity.

Triosephosphate isomerase from Plasmodium falciparum:. the crystal structure provides insights into antimalarial drug design

Background: Malaria caused by the parasite Plasmodium falciparum is a major public health concern. The parasite lacks a functional tricarboxylic acid cycle, making glycolysis its sole energy source. Although parasite enzymes have been considered as potential antimalarial drug targets, little is known about their structural biology. Here we report the crystal structure of triosephosphate isomerase (TIM) from P. falciparum at 2.2 Angstrom resolution. Results: The crystal structure of P. falciparum TIM (PfTIM), expressed in Escherichia coli, was determined by the molecular replacement method using the structure of trypanosomal TIM as the starting model. Comparison of the PfTIM structure with other TIM structures, particularly human TIM, revealed several differences, In most TIMs the residue at position 183 is a glutamate but in PtTIM it is a leucine, This leucine residue is completely exposed and together with the surrounding positively charged patch, may be responsible for binding TIM to the erythrocyte membrane. Another interesting feature is the occurrence of a cysteine residue at the dimer interface of PfTIM (Cys13), in contrast to human TIM where this residue is a methionine. Finally, residue 96 of human TIM (Ser96), which occurs near the active site, has been replaced by phenylalanine in PfTIM.

Design of mechanism-based inhibitors of transthyretin amyloidosis: Studies with biphenyl ethers and new structural templates

Transthyretin (TTR), a tetrameric thyroxine (T4) carrier protein, is associated with a variety of amyloid diseases. In this study, we explore the potential of biphenyl ethers (BPE), which are shown to interact with a high affinity to its T4 binding site thereby preventing its aggregation and fibrillogenesis. They prevent fibrillogenesis by stabilizing the tetrameric ground state of transthyretin. Additionally, we identify two new structural templates (2-(5-mercapto-[1,3,4]oxadiazol-2-yl)-phenol and 2,3,6-trichloro-N-(4H-[1,2,4]triazol-3-yl) represented as compounds 11 and 12, respectively, throughout the manuscript) exhibiting the ability to arrest TTR amyloidosis. The dissociation constants for the binding of BPEs and compound 11 and 12 to TTR correlate with their efficacies of inhibiting amyloidosis. They also have the ability to inhibit the elongation of intermediate fibrils as well as show nearly complete (> 90%) disruption of the preformed fibrils. The present study thus establishes biphenyl ethers and compounds 11 and 12 as very potent inhibitors of TTR fibrillization and inducible cytotoxicity.

Design of a Non-glycosylated Outer Domain-derived HIV-1 gp120 Immunogen That Binds to CD4 and Induces Neutralizing Antibodies

The outer domain (OD) of the HIV-1 envelope glycoprotein gp120 is an important target for vaccine design as it contains a number of conserved epitopes, including a large fraction of the CD4 binding site.Attempts to design OD-based immunogens in the past have met with little success. We report the design and characterization of an Escherichia coli-expressed OD-based immunogen (ODEC), based on the sequence of the HxBc2 strain. The ODEC-designed immunogen lacks the variable loops V1V2 and V3 and incorporates 11 designed mutations at the interface of the inner and the outer domains of gp120. Biophysical studies showed that ODEC is folded and protease-resistant, whereas ODEC lacking the designed mutations is highly aggregation-prone. In contrast to previously characterized OD constructs, ODEC bound CD4 and the broadly neutralizing antibody b12 but not the non-neutralizing antibodies b6 and F105. Upon immunization in rabbits, ODEC was highly immunogenic,and the sera showed measurable neutralization for four subtype B and one subtype C virus including two b12-resistant viruses. In contrast,sera from rabbits immunized with gp120 did not neutralize any of the viruses. ODEC is the first example of a gp120 fragment-based immunogen that yields significant neutralizing antibodies.