Characterization of a dual-active enzyme, DcpA, involved in cyclic diguanosine monophosphate turnover in Mycobacterium smegmatis

We have reported previously that the long-term survival of Mycobacterium smegmatis is facilitated by a dual-active enzyme MSDGC-1 (renamed DcpA), which controls the cellular turnover of cyclic diguanosine monophosphate (c-di-GMP). Most mycobacterial species possess at least a single copy of a DcpA orthologue that is highly conserved in terms of sequence similarity and domain architecture. Here, we show that DcpA exists in monomeric and dimeric forms. The dimerization of DcpA is due to non-covalent interactions between two protomers that are arranged in a parallel orientation. The dimer shows both synthesis and hydrolysis activities, whereas the monomer shows only hydrolysis activity. In addition, we have shown that DcpA is associated with the cytoplasmic membrane and exhibits heterogeneous cellular localization with a predominance at the cell poles. Finally, we have also shown that DcpA is involved in the change in cell length and colony morphology of M. smegmatis. Taken together, our study provides additional evidence about the role of the bifunctional protein involved in c-di-GMP signalling in M. smegmatis.

Boundary Tracking using a Hybrid Cyclic Pursuit Scheme

There has been a lot of work in the literature, related to the mapping of boundaries of regions, using multiple agents. Most of these are based on optimization techniques or rely on potential fields to drive the agents towards the boundary and then retain them there while they space out evenly along the perimeter or surface (in two-dimensional and three-dimensional cases, respectively). In this paper an algorithm to track the boundary of a region in space is provided based on the cyclic pursuit scheme. This enables the agents to constantly move along the perimeter in a cluster, thereby tracking a dynamically changing boundary. The trajectories of the agents provide a sketch of the boundary. The use of multiple agents may facilitate minimization of tracking error by providing accurate estimates of points on the boundary, besides providing redundancy. Simulation results are provided to highlight the performance of the proposed scheme.

Effects of temperature and clay content on water absorption characteristics of modified MMT clay/cyclic olefin copolymer nanocomposite films: Permeability, dynamic mechanical properties and the encapsulated organic device performance

In the present study, amino-silane modified layered organosilicates were used to reinforce cyclic olefin copolymer to enhance the thermal, mechanical and moisture impermeable barrier properties. The optimum clay loading (4%) in the nanocomposite increases the thermal stability of the film while further loading decreases film stability. Water absorption behavior at 62 degrees C was carried out and compared with the behavior at room temperature and 48 degrees C. The stiffness of the matrix increases with clay content and the recorded strain to failure for the composite films was lower than the neat film. Dynamic mechanical analysis show higher storage modulus and low loss modulus for 2.5-4 wt% clay loading. Calcium degradation test and device encapsulation also show the evidence of optimum clay loading of 4 wt% for improved low water vapor transmission rates compared to other nanocomposite films. (C) 2014 Elsevier Ltd. All rights reserved.

Performance Analysis of Heat Sinks With Phase-Change Materials Subjected to Transient and Cyclic Heating

Phase-change cooling technique is a suitable method for thermal management of electronic equipment subjected to transient or cyclic heat loads. The thermal performance of a phase-change based heat sink under cyclic heat load depends on several design parameters, namely, applied heat flux, cooling heat transfer coefficient, thermophysical properties of phase-change materials (PCMs), and physical dimensions of phase-change storage system during melting and freezing processes. A one-dimensional conduction heat transfer model is formulated to evaluate the effectiveness of preliminary design of practical PCM-based energy storage units. In this model, the phase-change process of the PCM is divided into melting and solidification subprocesses, for which separate equations are written. The equations are solved sequentially and an explicit closed-form solution is obtained. The efficacy of analytical model is estimated by comparing with a finite-volume-based numerical solution for both transient and cyclic heat loads.

Novel Functions of (p)ppGpp and Cyclic di-GMP in Mycobacterial Physiology Revealed by Phenotype Microarray Analysis of Wild-Type and Isogenic Strains of Mycobacterium smegmatis

The bacterial second messengers (p)ppGpp and bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulate important functions, such as transcription, virulence, biofilm formation, and quorum sensing. In mycobacteria, they regulate long-term survival during starvation, pathogenicity, and dormancy. Recently, a Pseudomonas aeruginosa strain lacking (p) ppGpp was shown to be sensitive to multiple classes of antibiotics and defective in biofilm formation. We were interested to find out whether Mycobacterium smegmatis strains lacking the gene for either (p)ppGpp synthesis (Delta rel(Msm)) or c-di-GMP synthesis (Delta dcpA) would display similar phenotypes. We used phenotype microarray technology to compare the growth of the wild-type and the knockout strains in the presence of several antibiotics. Surprisingly, the Delta rel(Msm) and Delta dcpA strains showed enhanced survival in the presence of many antibiotics, but they were defective in biofilm formation. These strains also displayed altered surface properties, like impaired sliding motility, rough colony morphology, and increased aggregation in liquid cultures. Biofilm formation and surface properties are associated with the presence of glycopeptidolipids (GPLs) in the cell walls of M. smegmatis. Thin-layer chromatography analysis of various cell wall fractions revealed that the levels of GPLs and polar lipids were reduced in the knockout strains. As a result, the cell walls of the knockout strains were significantly more hydrophobic than those of the wild type and the complemented strains. We hypothesize that reduced levels of GPLs and polar lipids may contribute to the antibiotic resistance shown by the knockout strains. Altogether, our data suggest that (p)ppGpp and c-di-GMP may be involved in the metabolism of glycopeptidolipids and polar lipids in M. smegmatis.

Cyclic nucleotide binding and structural changes in the isolated GAF domain of Anabaena adenylyl cyclase, CyaB2

GAF domains are a large family of regulatory domains, and a subset are found associated with enzymes involved in cyclic nucleotide (cNMP) metabolism such as adenylyl cyclases and phosphodiesterases. CyaB2, an adenylyl cyclase from Anabaena, contains two GAF domains in tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both the GAFa and the GAFb domains are present in tandem(GAFab domain). In silico docking and mutational analysis identified distinct residues important for interaction with either cAMP or cGMP in the GAFb domain. Structural changes associated with ligand binding to the GAF domains could not be detected by bioluminescence resonance energy transfer (BRET) experiments. However, amide hydrogen-deuterium exchange mass spectrometry (HDXMS) experiments provided insights into the structural basis for cAMP-induced allosteric regulation of the GAF domains, and differences in the changes induced by cAMP and cGMP binding to the GAF domain. Thus, our findings could allow the development of molecules that modulate the allosteric regulation by GAF domains present in pharmacologically relevant proteins.

One-step synthesis of a cyclic 2,17-dioxo3,3](4,4 `)biphenylophane and first preparation of a microporous polymer network from a macrocyclic precursor by cyclotrimerization

One-step synthesis of a cyclic 2,17-dioxo3,3](4,4') biphenylophane (MC) was achieved in high yield; its structure was verified by single crystal X-ray analysis. As a first example, a microporous polymer network was formed from macrocycle MC via acid-catalysed cyclotrimerization yielding a BET surface area of ca. 570 m(2) g(-1).

Construction of Singer Subgroup Orbit Codes Based on Cyclic Difference Sets

A recent approach for the construction of constant dimension subspace codes, designed for error correction in random networks, is to consider the codes as orbits of suitable subgroups of the general linear group. In particular, a cyclic orbit code is the orbit of a cyclic subgroup. Hence a possible method to construct large cyclic orbit codes with a given minimum subspace distance is to select a subspace such that the orbit of the Singer subgroup satisfies the distance constraint. In this paper we propose a method where some basic properties of difference sets are employed to select such a subspace, thereby providing a systematic way of constructing cyclic orbit codes with specified parameters. We also present an explicit example of such a construction.

Cyclic Cationic Peptides Containing Sugar Amino Acids Selectively Distinguishes and Inhibits Maturation of Pre-miRNAs of the Same Family

The discovery of microRNAs (miRNAs) has added a new dimension to the gene regulatory networks, making aberrantly expressed miRNAs as therapeutically important targets. Small molecules that can selectively target and modulate miRNA levels can thus serve as lead structures. Cationic cyclic peptides containing sugar amino acids represent a new class of small molecules that can target miRNA selectively. Upon treatment of these small molecules in breast cancer cell line, we profiled 96 therapeutically important miRNAs associated with cancer and observed that these peptides can selectively target paralogous miRNAs of the same seed family. This selective inhibition is of prime significance in cases when miRNAs of the same family have tissue-specific expression and perform different functions. During these conditions, targeting an entire miRNA family could lead to undesired adverse effects. The selective targeting is attributable to the difference in the three-dimensional structures of precursor miRNAs. Hence, the core structure of these peptides can be used as a scaffold for designing more potent inhibitors of miRNA maturation and hence function.

Deviated linear cyclic pursuit

This paper analyses deviated linear cyclic pursuit in which an agent pursues its leader with an angle of deviation in both the continuous- and discrete-time domains, while admitting heterogeneous gains and deviations for the agents. Sufficient conditions for the stability of such systems, in both the domains, are presented in this paper along with the derivation of the reachable set, which is a set of points where the agents may converge asymptotically. The stability conditions are derived based on Gershgorin's theorem. Simulations validating the theoretical results presented in this paper are provided.

Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis

The alarmone (p)ppGpp regulates transcription, translation, replication, virulence, lipid synthesis, antibiotic sensitivity, biofilm formation, and other functions in bacteria. Signaling nucleotide cyclic di-GMP (c-di-GMP) regulates biofilm formation, motility, virulence, the cell cycle, and other functions. In Mycobacterium smegmatis, both (p) ppGpp and c-di-GMP are synthesized and degraded by bifunctional proteins Rel(Msm) and DcpA, encoded by rel(Msm) and dcpA genes, respectively. We have previously shown that the Delta rel(Msm) and Delta dcpA knockout strains are antibiotic resistant and defective in biofilm formation, show altered cell surface properties, and have reduced levels of glycopeptidolipids and polar lipids in their cell wall (K. R. Gupta, S. Kasetty, and D. Chatterji, Appl Environ Microbiol 81:2571-2578, 2015, http://dx.doi.org/10.1128/AEM.03999-14). In this work, we have explored the phenotypes that are affected by both (p) ppGpp and c-di-GMP in mycobacteria. We have shown that both (p) ppGpp and c-di-GMP are needed to maintain the proper growth rate under stress conditions such as carbon deprivation and cold shock. Scanning electron microscopy showed that low levels of these second messengers result in elongated cells, while high levels reduce the cell length and embed the cells in a biofilm-like matrix. Fluorescence microscopy revealed that the elongated Delta rel(Msm) and Delta dcpA cells are multinucleate, while transmission electron microscopy showed that the elongated cells are multiseptate. Gene expression analysis also showed that genes belonging to functional categories such as virulence, detoxification, lipid metabolism, and cell-wall-related processes were differentially expressed. Our results suggests that both (p) ppGpp and c-di-GMP affect some common phenotypes in M. smegmatis, thus raising a possibility of cross talk between these two second messengers in mycobacteria. IMPORTANCE Our work has expanded the horizon of (p) ppGpp and c-di-GMP signaling in Gram-positive bacteria. We have come across a novel observation that M. smegmatis needs (p) ppGpp and c-di-GMP for cold tolerance. We had previously shown that the Delta rel(Msm) and Delta dcpA strains are defective in biofilm formation. In this work, the overproduction of (p) ppGpp and c-di-GMP encased M. smegmatis in a biofilm-like matrix, which shows that both (p) ppGpp and c-di-GMP are needed for biofilm formation. The regulation of cell length and cell division by (p) ppGpp was known in mycobacteria, but our work shows that c-di-GMP also affects the cell size and cell division in mycobacteria. This is perhaps the first report of c-di-GMP regulating cell division in mycobacteria.

A Study of Composite Beam with Shape Memory Alloy Arbitrarily Embedded under Thermal and Mechanical Loadings

The constitutive relations and kinematic assumptions on the composite beam with shape memory alloy (SMA) arbitrarily embedded are discussed and the results related to the different kinematic assumptions are compared. As the approach of mechanics of materials is to study the composite beam with the SMA layer embedded, the kinematic assumption is vital. In this paper, we systematically study the kinematic assumptions influence on the composite beam deflection and vibration characteristics. Based on the different kinematic assumptions, the equations of equilibrium/motion are different. Here three widely used kinematic assumptions are presented and the equations of equilibrium/motion are derived accordingly. As the three kinematic assumptions change from the simple to the complex one, the governing equations evolve from the linear to the nonlinear ones. For the nonlinear equations of equilibrium, the numerical solution is obtained by using Galerkin discretization method and Newton-Rhapson iteration method. The analysis on the numerical difficulty of using Galerkin method on the post-buckling analysis is presented. For the post-buckling analysis, finite element method is applied to avoid the difficulty due to the singularity occurred in Galerkin method. The natural frequencies of the composite beam with the nonlinear governing equation, which are obtained by directly linearizing the equations and locally linearizing the equations around each equilibrium, are compared. The influences of the SMA layer thickness and the shift from neutral axis on the deflection, buckling and post-buckling are also investigated. This paper presents a very general way to treat thermo-mechanical properties of the composite beam with SMA arbitrarily embedded. The governing equations for each kinematic assumption consist of a third order and a fourth order differential equation with a total of seven boundary conditions. Some previous studies on the SMA layer either ignore the thermal constraint effect or implicitly assume that the SMA is symmetrically embedded. The composite beam with the SMA layer asymmetrically embedded is studied here, in which symmetric embedding is a special case. Based on the different kinematic assumptions, the results are different depending on the deflection magnitude because of the nonlinear hardening effect due to the (large) deflection. And this difference is systematically compared for both the deflection and the natural frequencies. For simple kinematic assumption, the governing equations are linear and analytical solution is available. But as the deflection increases to the large magnitude, the simple kinematic assumption does not really reflect the structural deflection and the complex one must be used. During the systematic comparison of computational results due to the different kinematic assumptions, the application range of the simple kinematic assumption is also evaluated. Besides the equilibrium study of the composite laminate with SMA embedded, the buckling, post-buckling, free and forced vibrations of the composite beam with the different configurations are also studied and compared.