Cyclic AMP-induced Conformational Changes in Mycobacterial Protein Acetyltransferases

The activities of a number of proteins are regulated by the binding of cAMP and cGMP to cyclic nucleotide binding (CNB) domains that are found associated with one or more effector domains with diverse functions. Although the conserved architecture of CNB domains has been extensively studied by x-ray crystallography, the key to unraveling the mechanisms of cAMP action has been protein dynamics analyses. Recently, we have identified a novel cAMP-binding protein from mycobacteria, where cAMP regulates the activity of an associated protein acetyltransferase domain. In the current study, we have monitored the conformational changes that occur upon cAMP binding to the CNB domain in these proteins, using a combination of bioluminescence resonance energy transfer and amide hydrogen/deuterium exchange mass spectrometry. Coupled with mutational analyses, our studies reveal the critical role of the linker region (positioned between the CNB domain and the acetyltransferase domain) in allosteric coupling of cAMP binding to activation of acetyltransferase catalysis. Importantly, major differences in conformational change upon cAMP binding were accompanied by stabilization of the CNB and linker domain alone. This is in contrast to other cAMP-binding proteins, where cyclic nucleotide binding has been shown to involve intricate and parallel allosteric relays. Finally, this powerful convergence of results from bioluminescence resonance energy transfer and hydrogen/deuterium exchange mass spectrometry reaffirms the power of solution biophysical tools in unraveling mechanistic bases of regulation of proteins in the absence of high resolution structural information.

Nonlocal continuum mechanics formulation for axial, flexural, shear and contraction coupled wave propagation in single walled carbon nanotubes

This paper presents the effect of nonlocal scaling parameter on the coupled i.e., axial, flexural, shear and contraction, wave propagation in single-walled carbon nanotubes (SWCNTs). The axial and transverse motion of SWCNT is modeled based on first order shear deformation theory (FSDT) and thickness contraction. The governing equations are derived based on nonlocal constitutive relations and the wave dispersion analysis is also carried out. The studies shows that the nonlocal scale parameter introduces certain band gap region in all wave modes where no wave propagation occurs. This is manifested in the wavenumber plots as the region where the wavenumber tends to infinite or wave speed tends to zero. The frequency at which this phenomenon occurs is called the escape frequency. Explicit expressions are derived for cut-off and escape frequencies of all waves in SWCNT. It is also shown that the cut-off frequencies of shear and contraction mode are independent of the nonlocal scale parameter. The results provided in this article are new and are useful guidance for the study and design of the next generation of nanodevices that make use of the coupled wave propagation properties of single-walled carbon nanotubes.

Conservative Failure Criteria for Optimal Design of Composite Structures Including Effect of Shear Loading

A minimum weight design of laminated composite structures is carried out for different loading conditions and failure criteria using genetic algorithm. The phenomenological maximum stress (MS) and Tsai-Wu (TW) criteria and the micro-mechanism-based failure mechanism based (FMB) failure criteria are considered. A new failure envelope called the Most Conservative Failure Envelope (MCFE) is proposed by combining the three failure envelopes based on the lowest absolute values of the strengths predicted. The effect of shear loading on the MCFE is investigated. The interaction between the loading conditions, failure criteria, and strength-based optimal design is brought out.

Cumulus clouds and convective boundary layers: a tropical perspective on two turbulent shear flows

This paper is a review prepared for the second Marseille Colloquium on the mechanics of turbulence, held in 2011, 50 years after the first. The review covers recent developments in our understanding of the large-scale dynamics of cumulus cloud flows and of the atmospheric boundary layer in the low-wind convective regime that is often encountered in the tropics. It has recently been shown that a variety of cumulus cloud forms and life cycles can be experimentally realized in the laboratory, with the transient diabatic plume taken as the flow model for a cumulus cloud. The plume is subjected to diabatic heating scaled to be dynamically similar to heat release from phase changes in clouds. The experiments are complemented by exact numerical solutions of the Navier-Stokes-Boussinesq equations for plumes with scaled off-source heating. The results show that the Taylor entrainment coefficient first increases with heating, reaches a positive maximum and then drops rapidly to zero or even negative values. This reduction in entrainment is a consequence of structural changes in the flow, smoothing out the convoluted boundaries in the non-diabatic plume, including the tongues engulfing the ambient flow. This is accompanied by a greater degree of mixedness in the core flow because of lower dilution by the ambient fluid. The cloud forms generated depend strongly on the history of the diabatic heating profile in the vertical direction. The striking effects of heating on the flow are attributable to the operation of the baroclinic torque due to the temperature field. The mean baroclinic torque is shown to peak around a quasi-cylindrical sheet situated midway between the axis of the flow and the edges. This torque is shear-enhancing and folds down the engulfment tongues. The increase in mixedness can be traced to an explosive growth in the enstrophy, triggered by a strong fluctuating baroclinic torque that acts as a source, especially at the higher wave numbers, thus enhancing the mixedness. In convective boundary layers field measurements show that, under conditions prevailing in the tropics, the eddy fluxes of momentum and energy do not follow the Monin-Obukhov similarity. Instead, the eddy momentum flux is found to be linear in the wind speed at low winds; and the eddy heat flux is, to a first approximation, governed by free convection laws, with wind acting as a small perturbation on a regime of free convection. A new boundary layer code, based on heat flux scaling rather than wall-stress scaling, shows promising improvements in predictive skills of a general circulation model.

Damage limit states of reinforced concrete beams subjected to incremental cyclic loading using relaxation ratio analysis of AE parameters

This paper presents an experimental study on damage assessment of reinforced concrete (RC) beams subjected to incremental cyclic loading. During testing acoustic emissions (AEs) were recorded. The analysis of the AE released was carried out by using parameters relaxation ratio, load ratio and calm ratio. Digital image correlation (DIC) technique and tracking with available MATLAB program were used to measure the displacement and surface strains in concrete. Earlier researchers classified the damage in RC beams using Kaiser effect, crack mouth opening displacement and proposed a standard. In general (or in practical situations), multiple cracks occur in reinforced concrete beams. In the present study damage assessment in RC beams was studied according to different limit states specified by the code of practice IS-456:2000 and AE technique. Based on the two ratios namely load ratio and calm ratio and when the deflection reached approximately 85% of the maximum allowable deflection it was observed that the RC beams were heavily damaged. The combination of AE and DIC techniques has the potential to provide the state of damage in RC structures.

Indentation size effect and shear transformation zone size in a bulk metallic glass in two different structural states

The existence of an indentation size effect (ISE) in the onset of yield in a Zr-based bulk metallic glass (BMG) is investigated by employing spherical-tip nanoindentation experiments. Statistically significant data on the load at which the first pop-in in the displacement occurs were obtained for three different tip radii and in two different structural states (as-cast and structurally relaxed) of the BMG. Hertzian contact mechanics were employed to convert the pop-in loads to the maximum shear stress underneath the indenter. Results establish the existence of an ISE in the BMG of both structural states, with shear yield stress increasing with decreasing tip radius. Structural relaxation was found to increase the yield stress and decrease the variability in the data, indicating ``structural homogenization'' with annealing. Statistical analysis of the data was employed to estimate the shear transformation zone (STZ) size. Results of this analysis indicate an STZ size of similar to 25 atoms, which increases to similar to 34 atoms upon annealing. These observations are discussed in terms of internal structure changes that occur during structural relaxation and their interaction with the stressed volumes in spherical indentation of a metallic glass. (C) 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Stochastically driven instability in rotating shear flows

The origin of hydrodynamic turbulence in rotating shear flows is investigated, with particular emphasis on the flows whose angular velocity decreases but whose specific angular momentum increases with the increasing radial coordinate. Such flows are Rayleigh stable, but must be turbulent in order to explain the observed data. Such a mismatch between the linear theory and the observations/experiments is more severe when any hydromagnetic/magnetohydrodynamic instability and then the corresponding turbulence therein is ruled out. This work explores the effect of stochastic noise on such hydrodynamic flows. We essentially concentrate on a small section of such a flow, which is nothing but a plane shear flow supplemented by the Coriolis effect. This also mimics a small section of an astrophysical accretion disc. It is found that such stochastically driven flows exhibit large temporal and spatial correlations of perturbation velocities and hence large energy dissipations of perturbation, which presumably generate the instability. A range of angular velocity (Omega) profiles of the background flow, starting from that of a constant specific angular momentum (lambda = Omega r(2); r being the radial coordinate) to a constant circular velocity (v(phi) = Omega r), is explored. However, all the background angular velocities exhibit identical growth and roughness exponents of their perturbations, revealing a unique universality class for the stochastically forced hydrodynamics of rotating shear flows. This work, to the best of our knowledge, is the first attempt to understand the origin of instability and turbulence in three-dimensional Rayleigh stable rotating shear flows by introducing additive noise to the underlying linearized governing equations. This has important implications to resolve the turbulence problem in astrophysical hydrodynamic flows such as accretion discs.

Effect of compressibility on the velocity gradient tensor at free shear flow boundaries

Analyses of the invariants of the velocity gradient ten- sor were performed on flow fields obtained by DNS of compressible plane mixing layers at convective Mach num- bers Mc=0:15 and 1.1. Joint pdfs of the 2nd and 3rd invariants were examined at turbulent/nonturbulent (T/NT) boundaries—defined as surfaces where the local vorticity first exceeds a threshold fraction of the maximum of the mean vorticity. By increasing the threshold from very small lev-els, the boundary points were moved closer into the turbulent region, and the effects on the pdfs of the invariants were ob-served. Generally, T/NT boundaries are in sheet-like regions at both Mach numbers. At the higher Mach number a distinct lobe appears in the joint pdf isolines which has not been ob-served/reported before. A connection to the delayed entrain-ment and reduced growth rate of the higher Mach number flow is proposed.

Shear bands mediate cavitation in brittle metallic glasses

Recent experimental studies have revealed nanoscale cavities and periodic corrugations on the fracture surfaces of brittle metallic glasses. How such cavitation in these materials leads to brittle failure remains unclear. Here we show, using atomistic and continuum finite element simulations, that a shear band can mediate cavity nucleation and coalescence owing to plastic flow confinement caused by material softening. This leads to brittle fracture as cavities nucleate and coalesce within a shear band, causing the crack to extend along it. (c) 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Seismic site classification and correlation between standard penetration test N value and shear wave velocity for Lucknow city in Indo-Gangetic basin

Subsurface lithology and seismic site classification of Lucknow urban center located in the central part of the Indo-Gangetic Basin (IGB) are presented based on detailed shallow subsurface investigations and borehole analysis. These are done by carrying out 47 seismic surface wave tests using multichannel analysis of surface waves (MASW) and 23 boreholes drilled up to 30 m with standard penetration test (SPT) N values. Subsurface lithology profiles drawn from the drilled boreholes show low- to medium-compressibility clay and silty to poorly graded sand available till depth of 30 m. In addition, deeper boreholes (depth >150 m) were collected from the Lucknow Jal Nigam (Water Corporation), Government of Uttar Pradesh to understand deeper subsoil stratification. Deeper boreholes in this paper refer to those with depth over 150 m. These reports show the presence of clay mix with sand and Kankar at some locations till a depth of 150 m, followed by layers of sand, clay, and Kankar up to 400 m. Based on the available details, shallow and deeper cross-sections through Lucknow are presented. Shear wave velocity (SWV) and N-SPT values were measured for the study area using MASW and SPT testing. Measured SWV and N-SPT values for the same locations were found to be comparable. These values were used to estimate 30 m average values of N-SPT (N-30) and SWV (V-s(30)) for seismic site classification of the study area as per the National Earthquake Hazards Reduction Program (NEHRP) soil classification system. Based on the NEHRP classification, the entire study area is classified into site class C and D based on V-s(30) and site class D and E based on N-30. The issue of larger amplification during future seismic events is highlighted for a major part of the study area which comes under site class D and E. Also, the mismatch of site classes based on N-30 and V-s(30) raises the question of the suitability of the NEHRP classification system for the study region. Further, 17 sets of SPT and SWV data are used to develop a correlation between N-SPT and SWV. This represents a first attempt of seismic site classification and correlation between N-SPT and SWV in the Indo-Gangetic Basin.

A cyclic peptide mimic of an RNA recognition motif of human La protein is a potent inhibitor of hepatitis C virus

Due to limited available therapeutic options, developing new lead compounds against hepatitis C virus is an urgent need. Human La protein stimulates hepatitis C virus translation through interaction with the hepatitis C viral RNA. A cyclic peptide mimicking the beta-turn of the human La protein that interacts with the viral RNA was synthesized. It inhibits hepatitis C viral RNA translation significantly better than the corresponding linear peptide at longer post-treatment times. The cyclic peptide also inhibited replication as measured by replicon RNA levels using real time RT-PCR. The cyclic peptide emerges as a promising lead compound against hepatitis C.

The precision with which single cells of Dictyostelium discoideum can locate a source of cyclic AMP

When stimulated by a point source of cyclic AMP, a starved amoeba of Dictyostelium discoideum responds by putting out a hollow balloon-like membrane extension followed by a pseudopod. The effect of the stimulus is to influence the position where either of these protrusions is made on the cell rather than to cause them to be made. Because the pseudopod forms perpendicular to the cell surface, its location is a measure of the precision with which the cell can locate the cAMP source. Cells beyond 1 h of starvation respond non-randomly with a precision that improves steadily thereafter. A cell that is starved for 1-2 h can locate the source accurately 43% of the time; and if starved for 6-7 h, 87% of the time. The response always has a high scatter; population-level heterogeneity reflects stochasticity in single cell behaviour. From the angular distribution of the response its maximum information content is estimated to be 2-3 bits. In summary, we quantitatively demonstrate the stochastic nature of the directional response and the increase in its accuracy over time.

Cyclic AMP-dependent protein lysine acylation in mycobacteria regulates fatty acid and propionate metabolism

Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guerin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host.

Yield stress, thixotropy and shear banding in a dilute aqueous suspension of few layer graphene oxide platelets

We demonstrate a rigidity percolation transition and the onset of yield stress in a dilute aqueous dispersion of graphene oxide platelets (aspect ratio similar to 5000) above a critical volume fraction of 3.75 x 10(-4) with a percolation exponent of 2.4 +/- 0.1. The viscoelastic moduli of the gel at rest measured as a function of time indicate the absence of structural evolution of the 3D percolated network of disks. However a shear-induced aging giving rise to a compact jammed state and shear rejuvenation indicating a homogenous flow is observed when a steady shear stress (sigma) is imposed in creep experiments. We construct a shear diagram (sigma vs. volume fraction phi) and the critical stress above which shear rejuvenation occurs is identified as the yield stress sigma(y) of the gel. The minimum steady state shear rate (gamma) over dot(m) obtained from creep experiments agrees well with the end of the plateau region in a controlled shear rate flow curve, indicating a shear localization below (gamma) over dot(m). A steady state shear banding in the plateau region of the flow curve observed in particle velocimetry measurements in a Couette geometry confirms that the dilute suspensions of GO platelets form a thixotropic yield stress fluid.