Generalized eigenvalue decomposition of the field autocorrelation in correlation diffusion of photons in turbid media

We propose a novel numerical method based on a generalized eigenvalue decomposition for solving the diffusion equation governing the correlation diffusion of photons in turbid media. Medical imaging modalities such as diffuse correlation tomography and ultrasound-modulated optical tomography have the (elliptic) diffusion equation parameterized by a time variable as the forward model. Hitherto, for the computation of the correlation function, the diffusion equation is solved repeatedly over the time parameter. We show that the use of a certain time-independent generalized eigenfunction basis results in the decoupling of the spatial and time dependence of the correlation function, thus allowing greater computational efficiency in arriving at the forward solution. Besides presenting the mathematical analysis of the generalized eigenvalue problem on the basis of spectral theory, we put forth the numerical results that compare the proposed numerical method with the standard technique for solving the diffusion equation.

Synthesis and application of weight function for edge cracked semicircular disk (ECSD)

We analyze the utility of edge cracked semicircular disk (ECSD) for rapid assessment of fracture toughness using compressive loading. Continuing our earlier work on ECSD, a theoretical examination here leads to a novel way for synthesizing weight functions using two distinct form factors. The efficacy of ECSD mode-I weight function synthesized using displacement and form factor methods is demonstrated by comparing with finite element results. Theory of elasticity in conjunction with finite element method is utilized to analyze crack opening potency of ECSD under eccentric compression to explore newer configurations of ECSD for fracture testing.

MPK-09, a small molecule inspired from bioactive styryllactone restores the wild-type function of mutant p53

In the search for more efficacious and less toxic cancer drugs, the tumor suppressor p53 protein has long been a desirable therapeutic target. In the recent past, few independent studies have demonstrated that the antitumor activity of wild-type p53 can be restored in cancer cells harboring mutant form of p53 using small molecule activators. In this study, we describe a novel small molecule MPK-09, which is selective and highly potent against allele specific p53 mutations mainly, R175H, R249S, R273H, R273C, and E285K. Except E285K, all other mutations tested are among the six ``hot spot'' p53 mutations reported in majority of human cancer. Furthermore, our study conclusively demonstrates that the apoptotic activity of the small molecule MPK-09 against cancer cells harboring R273C and E285K mutations is due to restoration of the wild-type conformation to the corresponding mutant form of p53.

N-Terminal disordered domain of saccharomyces cerevisiae Hop1 protein Is dispensable for DNA binding, bridging, and synapsis of double-stranded DNA molecules but is ecessary for spore formation

The cytological architecture of the synaptonemal complex (SC), a meiosis-specific proteinaceous structure, is evolutionarily conserved among eukaryotes. However, little is known about the biochemical properties of SC components or the mechanisms underlying their roles in meiotic chromosome synapsis and recombination. Functional analysis of Saccharomyces cerevisiae Hop1, a key structural component of SC, has begun to reveal important insights into its function in interhomolog recombination. Previously, we showed that Hop1 is a structure-specific DNA-binding protein, exhibits higher binding affinity for the Holliday junction, and induces structural distortion at the core of the junction. Furthermore, Hop1 promotes DNA condensation and intra- and intermolecular synapsis between duplex DNA molecules. Here, we show that Hop1 possesses a modular domain organization, consisting of an intrinsically disordered N-terminal domain and a protease-resistant C-terminal domain (Hop1CTD). Furthermore, we found that Hop1CTD exhibits strong homotypic as well as heterotypic protein protein interactions, and its biochemical activities were similar to those of the full-length Hop1 protein. However, Hop1CTD failed to complement the meiotic recombination defects of the Delta hop1 strain, indicating that both N- and C-terminal domains of Hop1 are essential for meiosis and spore formation. Altogether, our findings reveal novel insights into the structure-function relationships of Hop1 and help to further our understanding of its role in meiotic chromosome synapsis and recombination.

Mechanistic Insights into the Neutralization of Cytotoxic Abrin by the Monoclonal Antibody D6F10

Abrin, an A/B toxin obtained from the Abrus precatorius plant is extremely toxic and a potential bio-warfare agent. Till date there is no antidote or vaccine available against this toxin. The only known neutralizing monoclonal antibody against abrin, namely D6F10, has been shown to rescue the toxicity of abrin in cells as well as in mice. The present study focuses on mapping the epitopic region to understand the mechanism of neutralization of abrin by the antibody D6F10. Truncation and mutational analysis of abrin A chain revealed that the amino acids 74-123 of abrin A chain contain the core epitope and the residues Thr112, Gly114 and Arg118 are crucial for binding of the antibody. In silico analysis of the position of the mapped epitope indicated that it is present close to the active site cleft of abrin A chain. Thus, binding of the antibody near the active site blocks the enzymatic activity of abrin A chain, thereby rescuing inhibition of protein synthesis by the toxin in vitro. At 1: 10 molar concentration of abrin: antibody, the antibody D6F10 rescued cells from abrin-mediated inhibition of protein synthesis but did not prevent cell attachment of abrin. Further, internalization of the antibody bound to abrin was observed in cells by confocal microscopy. This is a novel finding which suggests that the antibody might function intracellularly and possibly explains the rescue of abrin's toxicity by the antibody in whole cells and animals. To our knowledge, this study is the first report on a neutralizing epitope for abrin and provides mechanistic insights into the poorly understood mode of action of anti-A chain antibodies against several toxins including ricin.

Layerwise decomposition of water dynamics in reverse micelles: A simulation study of two-dimensional infrared spectrum

We present computer simulation study of two-dimensional infrared spectroscopy (2D-IR) of water confined in reverse micelles (RMs) of various sizes. The present study is motivated by the need to understand the altered dynamics of confined water by performing layerwise decomposition of water, with an aim to quantify the relative contributions of different layers water molecules to the calculated 2D-IR spectrum. The 0-1 transition spectra clearly show substantial elongation, due to in-homogeneous broadening and incomplete spectral diffusion, along the diagonal in the surface water layer of different sized RMs. Fitting of the frequency fluctuation correlation functions reveal that the motion of the surface water molecules is sub-diffusive and indicate the constrained nature of their dynamics. This is further supported by two peak nature of the angular analogue of van Hove correlation function. With increasing system size, the water molecules become more diffusive in nature and spectral diffusion almost completes in the central layer of the larger size RMs. Comparisons between experiments and simulations establish the correspondence between the spectral decomposition available in experiments with the spatial decomposition available in simulations. Simulations also allow a quantitative exploration of the relative role of water, sodium ions, and sulfonate head groups in vibrational dephasing. Interestingly, the negative cross correlation between force on oxygen and hydrogen of O-H bond in bulk water significantly decreases in the surface layer of each RM. This negative cross correlation gradually increases in the central water pool with increasing RMs size and this is found to be partly responsible for the faster relaxation rate of water in the central pool. (C) 2013 AIP Publishing LLC.

Structural Insights into Saccharomyces cerevisiae Msh4-Msh5 Complex Function Using Homology Modeling

The Msh4-Msh5 protein complex in eukaryotes is involved in stabilizing Holliday junctions and its progenitors to facilitate crossing over during Meiosis I. These functions of the Msh4-Msh5 complex are essential for proper chromosomal segregation during the first meiotic division. The Msh4/5 proteins are homologous to the bacterial mismatch repair protein MutS and other MutS homologs (Msh2, Msh3, Msh6). Saccharomyces cerevisiae msh4/5 point mutants were identified recently that show two fold reduction in crossing over, compared to wild-type without affecting chromosome segregation. Three distinct classes of msh4/5 point mutations could be sorted based on their meiotic phenotypes. These include msh4/5 mutations that have a) crossover and viability defects similar to msh4/5 null mutants; b) intermediate defects in crossing over and viability and c) defects only in crossing over. The absence of a crystal structure for the Msh4-Msh5 complex has hindered an understanding of the structural aspects of Msh4-Msh5 function as well as molecular explanation for the meiotic defects observed in msh4/5 mutations. To address this problem, we generated a structural model of the S. cerevisiae Msh4-Msh5 complex using homology modeling. Further, structural analysis tailored with evolutionary information is used to predict sites with potentially critical roles in Msh4-Msh5 complex formation, DNA binding and to explain asymmetry within the Msh4-Msh5 complex. We also provide a structural rationale for the meiotic defects observed in the msh4/5 point mutations. The mutations are likely to affect stability of the Msh4/5 proteins and/or interactions with DNA. The Msh4-Msh5 model will facilitate the design and interpretation of new mutational data as well as structural studies of this important complex involved in meiotic chromosome segregation.

Expression and function of cyclooxygenase-2 is necessary for hamster blastocyst hatching

Blastocyst hatching is critical for successful implantation leading to pregnancy. Its failure causes infertility. The phenomenon of blastocyst hatching in humans is poorly understood and the available information on this stems from studies of rodents such as mice and hamsters. We and others showed that hamster blastocyst hatching is characterized by firstly blastocyst deflation followed by a dissolution of the zona pellucida (zona) and accompanied by trophectodermal projections (TEPs). We also showed that embryo-derived cathepsins (Cat) proteases, specifically Cat-L, -B and -P act as zonalysins and are responsible for hatching. In this study, we show the expression and function of one of the potential regulators of embryogenesis, cyclooxygenase (COX)-2 during blastocyst development and hatching. The expression of COX-2 mRNA and protein was observed in 8-cell through hatched blastocyst stages and it was also localized to blastocysts TEPs. Specific COX-2 inhibitors, NS-398 and CAY-10404, inhibited blastocyst hatching; percentages achieved were only 28.4 5.3 and 32.3 5.4, respectively, compared with 90 with untreated embryos. Interestingly, inhibitor-treated blastocysts failed to deflate, normally observed during hatching. Supplementation of prostaglandins (PGs)-E-2 or -I-2 to cultured embryos reversed the inhibitors effect on hatching and also the deflation behavior. Importantly, the levels of mRNA and protein of Cat-L, -B and -P showed a significant reduction in the inhibitor-treated embryos compared with untreated embryos, although its mechanism remains to be examined. These data provide the first evidence that COX-2 is critical for blastocyst hatching in the golden hamster.

Functional specificity among yeast mitochondrial Hsp70s paralogs and orthologs

The evolutionary diversity of the HSP70 gene family at the genetic level has generated complex structural variations leading to altered functional specificity and mode of regulation in different cellular compartments. By utilizing Saccharomyces cerevisiae as a model system for better understanding the global functional cooperativity between Hsp70 paralogs, we have dissected the differences in functional properties at the biochemical level between mitochondrial heat shock protein 70 (mtHsp70) Ssc1 and an uncharacterized Ssc3 paralog. Based on the evolutionary origin of Ssc3 and a high degree of sequence homology with Ssc1, it has been proposed that both have a close functional overlap in the mitochondrial matrix. Surprisingly, our results demonstrate that there is no functional cross-talk between Ssc1 and Ssc3 paralogs. The lack of in vivo functional overlap is due to altered conformation and significant lower stability associated with Ssc3. The substrate-binding domain of Ssc3 showed poor affinity toward mitochondrial client proteins and Tim44 due to the open conformation in ADP-bound state. In addition to that, the nucleotide-binding domain of Ssc3 showed an altered regulation by the Mge1 co-chaperone due to a high degree of conformational plasticity, which strongly promotes aggregation. Besides, Ssc3 possesses a dysfunctional inter-domain interface thus rendering it unable to perform functions similar to generic Hsp70s. Moreover, we have identified the critical amino acid sequence of Ssc1 and Ssc3 that can “make or break” mtHsp70 chaperone function. Together, our analysis provides the first evidence to show that the nucleotide-binding domain of mtHsp70s plays a critical role in determining the functional specificity among paralogs and orthologs across kingdoms.

mulPBA: an efficient multiple protein structure alignment method based on a structural alphabet

The increasing number of available protein structures requires efficient tools for multiple structure comparison. Indeed, multiple structural alignments are essential for the analysis of function, evolution and architecture of protein structures. For this purpose, we proposed a new web server called multiple Protein Block Alignment (mulPBA). This server implements a method based on a structural alphabet to describe the backbone conformation of a protein chain in terms of dihedral angles. This sequence-like' representation enables the use of powerful sequence alignment methods for primary structure comparison, followed by an iterative refinement of the structural superposition. This approach yields alignments superior to most of the rigid-body alignment methods and highly comparable with the flexible structure comparison approaches. We implement this method in a web server designed to do multiple structure superimpositions from a set of structures given by the user. Outputs are given as both sequence alignment and superposed 3D structures visualized directly by static images generated by PyMol or through a Jmol applet allowing dynamic interaction. Multiple global quality measures are given. Relatedness between structures is indicated by a distance dendogram. Superimposed structures in PDB format can be also downloaded, and the results are quickly obtained. mulPBA server can be accessed at www.dsimb.inserm.fr/dsimb_tools/mulpba/.

Common-Mode and Differential-Mode Active Damping for PWM Rectifiers

Modern pulse-width-modulated (PWM) rectifiers use LC L filters that can be applied in both the common mode and differential mode to obtain high-performance filtering. Interaction between the passive L and C components in the filter leads to resonance oscillations. These oscillations need to be damped either by the passive damping or active damping. The passive damping increases power loss and can reduce the effectiveness of the filter. Methods of active damping, using control strategy, are lossless while maintaining the effectiveness of the filters. In this paper, an active damping strategy is proposed to damp the oscillations in both line-to-line and line-to-ground. An approach based on pole placement by the state feedback is used to actively damp both the differential-and common-mode filter oscillations. Analytical expressions for the state-feedback controller gains are derived for both continuous and discrete-time model of the filter. Tradeoff in selection of the active damping gain on the lower order power converter harmonics is analyzed using a weighted admittance function. Experimental results on a 10-kVA laboratory prototype PWM rectifier are presented. The results validate the effectiveness of the active damping method, and the tradeoff in the settings of the damping gain.

Chaperoning mitochondrial permeability transition: regulation of transition pore complex by a J-protein, DnaJC15

Mitochondria have a central role in the intrinsic pathway of apoptosis and involve activation of several transmembrane channels leading to release of death factors. Reduced expression of a mitochondrial J-protein DnaJC15 was associated with the development of chemoresistance in ovarian cancer cells. DnaJC15 was found to be a part of mitochondrial protein-transport machinery, though its connection with cell death mechanisms is still unclear. In the present study, we have provided evidence towards a novel function of DnaJC15 in regulation of mitochondrial permeability transition pore (MPTP) complex in normal and cancer cells. Overexpression of DnaJC15 resulted in MPTP opening and induction of apoptosis, whereas reduced amount of protein suppressed MPTP activation, upon cisplatin treatment. DnaJC15 was found to exert its proapoptotic function through the essential component of MPTP, cyclophilin D (CypD). Our results reveal a specific role of DnaJC15 in recruitment and coupling of CypD with mitochondrial permeability transition. In summary, our analysis provides first-time insights on the functional connection between mitochondrial inner membrane protein translocation machinery-associated J-protein DnaJC15 and regulation of cell death pathways.

A Novel C-Terminal Homologue of Aha1 Co-Chaperone Binds to Heat Shock Protein 90 and Stimulates Its ATPase Activity in Entamoeba histolytica

Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with K-d values of 365.2 and 10.77 mu M, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12 x 10(-4) min(-1) mu M-1. Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire. (C) 2014 Elsevier Ltd. All rights reserved.

Unraveling the Intricate Organization of Mammalian Mitochondrial Presequence Translocases: Existence of Multiple Translocases for Maintenance of Mitochondrial Function

Mitochondria are indispensable organelles implicated in multiple aspects of cellular processes, including tumorigenesis. Heat shock proteins play a critical regulatory role in accurately delivering the nucleus-encoded proteins through membrane-bound presequence translocase (Tim23 complex) machinery. Although altered expression of mammalian presequence translocase components had been previously associated with malignant phenotypes, the overall organization of Tim23 complexes is still unsolved. In this report, we show the existence of three distinct Tim23 complexes, namely, B1, B2, and A, involved in the maintenance of normal mitochondrial function. Our data highlight the importance of Magmas as a regulator of translocase function and in dynamically recruiting the J-proteins DnaJC19 and DnaJC15 to individual translocases. The basic housekeeping function involves translocases B1 and B2 composed of Tim17b isoforms along with DnaJC19, whereas translocase A is nonessential and has a central role in oncogenesis. Translocase B, having a normal import rate, is essential for constitutive mitochondrial functions such as maintenance of electron transport chain complex activity, organellar morphology, iron-sulfur cluster protein biogenesis, and mitochondrial DNA. In contrast, translocase A, though dispensable for housekeeping functions with a comparatively lower import rate, plays a specific role in translocating oncoproteins lacking presequence, leading to reprogrammed mitochondrial functions and hence establishing a possible link between the TIM23 complex and tumorigenicity.

Discrete time second order sliding mode observer for uncertain linear multi-output system

This paper presents a second order sliding mode observer (SOSMO) design for discrete time uncertain linear multi-output system. The design procedure is effective for both matched and unmatched bounded uncertainties and/or disturbances. A second order sliding function and corresponding sliding manifold for discrete time system are defined similar to the lines of continuous time counterpart. A boundary layer concept is employed to avoid switching across the defined sliding manifold and the sliding trajectory is confined to a boundary layer once it converges to it. The condition for existence of convergent quasi-sliding mode (QSM) is derived. The observer estimation errors satisfying given stability conditions converge to an ultimate finite bound (within the specified boundary layer) with thickness O(T-2) where T is the sampling period. A relation between sliding mode gain and boundary layer is established for the existence of second order discrete sliding motion. The design strategy is very simple to apply and is demonstrated for three examples with different class of disturbances (matched and unmatched) to show the effectiveness of the design. Simulation results to show the robustness with respect to the measurement noise are given for SOSMO and the performance is compared with pseudo-linear Kalman filter (PLKF). (C) 2013 Published by Elsevier Ltd. on behalf of The Franklin Institute