D2N: Distance to the native

Root-mean-square-deviation (RMSD), of computationally-derived protein structures from experimentally determined structures, is a critical index to assessing protein-structure-prediction-algorithms (PSPAs). The development of PSPAs to obtain 0 Å RMSD from native structures is considered central to computational biology. However, till date it has been quite challenging to measure how far a predicted protein structure is from its native — in the absence of a known experimental/native structure. In this work, we report the development of a metric “D2N” (distance to the native) — that predicts the “RMSD” of any structure without actually knowing the native structure. By combining physico-chemical properties and known universalities in spatial organization of soluble proteins to develop D2N, we demonstrate the ability to predict the distance of a proposed structure to within ± 1.5 ? error with a remarkable average accuracy of 93.6% for structures below 5 ? from the native. We believe that this work opens up a completely new avenue towards assigning reliable structures to whole proteomes even in the absence of experimentally determined native structures. The D2N tool is freely available at http://www.scfbio-iitd.res.in/software/d2n.jsp.     

Synthesis,Characterization and In Vitro Study of Biocompatible Cinnamaldehyde Functionalized Magnetite Nanoparticles (CPGF Nps) For Hyperthermia and Drug Delivery Applications in Breast Cancer

Cinnamaldehyde, the bioactive component of the spice cinnamon, and its derivatives have been shown to possess anti-cancer activity against various cancer cell lines. However, its hydrophobic nature invites attention for efficient drug delivery systems that would enhance the bioavailability of cinnamaldehyde without affecting its bioactivity. Here, we report the synthesis of stable aqueous suspension of cinnamaldehyde tagged Fe₃O₄ nanoparticles capped with glycine and pluronic polymer (CPGF NPs) for their potential application in drug delivery and hyperthermia in breast cancer. The monodispersed superparamagnetic NPs had an average particulate size of ∼20 nm. TGA data revealed the drug payload of ∼18%. Compared to the free cinnamaldehyde, CPGF NPs reduced the viability of breast cancer cell lines, MCF7 and MDAMB231, at lower doses of cinnamaldehyde suggesting its increased bioavailability and in turn its therapeutic efficacy in the cells. Interestingly, the NPs were non-toxic to the non-cancerous HEK293 and MCF10A cell lines compared to the free cinnamaldehyde. The novelty of CPGF nanoparticulate system was that it could induce cytotoxicity in both ER/PR positive/Her2 negative (MCF7) and ER/PR negative/Her2 negative (MDAMB231) breast cancer cells, the latter being insensitive to most of the chemotherapeutic drugs. The NPs decreased the growth of the breast cancer cells in a dose-dependent manner and altered their migration through reduction in MMP-2 expression. CPGF NPs also decreased the expression of VEGF, an important oncomarker of tumor angiogenesis. They induced apoptosis in breast cancer cells through loss of mitochondrial membrane potential and activation of caspase-3. Interestingly, upon exposure to the radiofrequency waves, the NPs heated up to 41.6°C within 1 min, suggesting their promise as a magnetic hyperthermia agent. All these findings indicate that CPGF NPs prove to be potential nano-chemotherapeutic agents in breast cancer.     

Novel model to analyze the effect of a large compressive follower pre-load on range of motions in a lumbar spine

A 3-D finite element model (FEM) of the lumbar spine (L1-S1) was used to determine the effect of a large compressive follower pre-load on range of motions (ROM) in all three planes. The follower load modeled in the FEM produced minimal vertebral rotations in all the three planes. The model was validated by comparing the disc compression at all levels in the lumbar spine with the corresponding results obtained by compressing 10 cadevaric lumbar spines (L1-S1) using the follower load technique described by Patwardhan et al. [1999. A follower load increases the load-carrying capacity of the lumbar spine in compression. Spine 24(10), 1003-1009]. Further validation of the model was performed by comparing the lateral bending and torsion response without pre-load and the flexion-extension response without pre-load and with an 800 N follower pre-load with those obtained using cadaver lumbar spines. Following validation, the FEM was subjected to bending moments in all three planes with and without compressive follower pre-loads of up to 1200 N. Disc compression values and the flexion-extension range of motion under 800 N follower pre-load predicted by the FEM compared well with in vitro results. The current model showed that compressive follower pre-load decreased total as well as segmental ROM in flexion-extension by up to 18%, lateral bending by up to 42%, and torsion by up to 26%.     

Selective thymus settling regulated by cytokine and chemokine receptors

To generate T cells throughout adult life, the thymus must import hemopoietic progenitors from the bone marrow via the blood. In this study, we establish that thymus settling is selective. Using nonirradiated recipient mice, we found that hemopoietic stem cells were excluded from the thymus, whereas downstream multipotent progenitors (MPP) and common lymphoid progenitors rapidly generated T cells following i.v. transfer. This cellular specificity correlated with the expression of the chemokine receptor CCR9 by a subset of MPP and common lymphoid progenitors but not hemopoietic stem cells. Furthermore, CCR9 expression was required for efficient thymus settling. Finally, we demonstrate that a prethymic signal through the cytokine receptor fms-like tyrosine kinase receptor-3 was required for the generation of CCR9-expressing early lymphoid progenitors, which were the most efficient progenitors of T cells within the MPP population. We conclude that fms-like tyrosine kinase receptor-3 signaling is required for the generation of T lineage-competent progenitors, which selectively express molecules, including CCR9, that allow them to settle within the thymus.     

Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis

Cyclic nucleotide monophosphate (cNMP) hydrolysis in bacteria and eukaryotes is brought about by distinct cNMP phosphodiesterases (PDEs). Since these enzymes differ in amino acid sequence and properties, they have evolved by convergent evolution. Cyclic NMP PDEs cleave cNMPs to NMPs, and the Rv0805 gene product is, to date, the only identifiable cNMP PDE in the genome of Mycobacterium tuberculosis. We have shown that Rv0805 is a cAMP/cGMP dual specificity PDE, and is unrelated in amino acid sequence to the mammalian cNMP PDEs. Rv0805 is a dimeric, Fe(3+)-Mn(2+) binuclear PDE, and mutational analysis demonstrated that the active site metals are co-ordinated by conserved aspartate, histidine and asparagine residues. We report here the structure of the catalytic core of Rv0805, which is distantly related to the calcineurin-like phosphatases. The crystal structure of the Rv0805 dimer shows that the active site metals contribute to dimerization and thus play an additional structural role apart from their involvement in catalysis. We also present the crystal structures of the Asn97Ala mutant protein that lacks one of the Mn(2+) co-ordinating residues as well as the Asp66Ala mutant that has a compromised cAMP hydrolytic activity, providing a structural basis for the catalytic properties of these mutant proteins. A molecule of phosphate is bound in a bidentate manner at the active site of the Rv0805 wild-type protein, and cacodylate occupies a similar position in the crystal structure of the Asp66Ala mutant protein. A unique substrate binding pocket in Rv0805 was identified by computational docking studies, and the role of the His140 residue in interacting with cAMP was validated through mutational analysis. This report on the first structure of a bacterial cNMP PDE thus significantly extends our molecular understanding of cAMP hydrolysis in class III PDEs.     

Rational Design of Graphene‐Supported Single Atom Catalysts for Hydrogen Evolution Reaction

The proper choice of nonprecious transition metals as single atom catalysts (SACs) remains unclear for designing highly efficient electrocatalysts for hydrogen evolution reaction (HER). Herein, reported is an activity correlation with catalysts, electronic structure, in order to clarify the origin of reactivity for a series of transition metals supported on nitrogen‐doped graphene as SACs for HER by a combination of density functional theory calculations and electrochemical measurements. Only few of the transition metals (e.g., Co, Cr, Fe, Rh, and V) as SACs show good catalytic activity toward HER as their Gibbs free energies are varied between the range of –0.20 to 0.30 eV but among which Co‐SAC exhibits the highest electrochemical activity at 0.13 eV. Electronic structure studies show that the energy states of active valence d_z² orbitals and their resulting antibonding state determine the catalytic activity for HER. The fact that the antibonding state orbital is neither completely empty nor fully filled in the case of Co‐SAC is the main reason for its ideal hydrogen adsorption energy. Moreover, the electrochemical measurement shows that Co‐SAC exhibits a superior hydrogen evolution activity over Ni‐SAC and W‐SAC, confirming the theoretical calculation. This systematic study gives a fundamental understanding about the design of highly efficient SACs for HER.     

Engineering Proteins for Thermostability with iRDP Web Server

Engineering protein molecules with desired structure and biological functions has been an elusive goal. Development of industrially viable proteins with improved properties such as stability, catalytic activity and altered specificity by modifying the structure of an existing protein has widely been targeted through rational protein engineering. Although a range of factors contributing to thermal stability have been identified and widely researched, the in silico implementation of these as strategies directed towards enhancement of protein stability has not yet been explored extensively. A wide range of structural analysis tools is currently available for in silico protein engineering. However these tools concentrate on only a limited number of factors or individual protein structures, resulting in cumbersome and time-consuming analysis. The iRDP web server presented here provides a unified platform comprising of iCAPS, iStability and iMutants modules. Each module addresses different facets of effective rational engineering of proteins aiming towards enhanced stability. While iCAPS aids in selection of target protein based on factors contributing to structural stability, iStability uniquely offers in silico implementation of known thermostabilization strategies in proteins for identification and stability prediction of potential stabilizing mutation sites. iMutants aims to assess mutants based on changes in local interaction network and degree of residue conservation at the mutation sites. Each module was validated using an extensively diverse dataset. The server is freely accessible at http://irdp.ncl.res.in and has no login requirements.