Autoinhibitory mechanism and activity-related structural changes in a mycobacterial adenylyl cyclase

An adenylyl cyclase from Mycobacterium avium, Mal 120, is a functional orthologue of a pseudogene Rv1120c from Mycobacterium tuberculosis. We report the crystal structure of Mal 120 in a monomeric form and its truncated construct as a dimer. Mal 120 exists as a monomer in solution and crystallized as a monomer in the absence of substrate or inhibitor. An additional alpha-helix present at the N-terminus of the monomeric structure blocks the active site by interacting with the substrate binding residues and occupying the dimer interface region. However, the enzyme has been found to be active in solution, indicating the movement of the helix away from the interface to facilitate the formation of active dimers in conditions favourable for catalysis. Thus, the N-terminal helix of Ma1120 keeps the enzyme in an autoinhibited state when it is not active. Deletion of this helix enabled us to crystallize the molecule as an active homodimer in the presence of a P-site inhibitor 2',5'-dideoxy-3'-ATP, or pyrophosphate along with metal ions. The substrate specifying lysine residue plays a dual role of interacting with the substrate and stabilizing the dimer. The dimerization loop region harbouring the second substrate specifying residue, an aspartate, shows significant differences in conformation and position between the monomeric and dimeric structures. Thus, this study has not only revealed that significant structural transitions are required for the interconversion of the inactive and the active forms of the enzyme, but also provided precise nature of these transitions. (C) 2015 Elsevier Inc. All rights reserved.

Smaller is Plastic: Polymorphic Structures and Mechanism of Deformation in Nanoscale hcp Metals

Using first-principles calculations, we establish the existence of highly-stable polymorphs of hcp metals (Ti, Mg, Be, La and Y) with nanoscale structural periodicity. They arise from heterogeneous deformation of the hcp structure occurring in response to large shear stresses localized at the basal planes separated by a few nanometers. Through Landau theoretical analysis, we show that their stability derives from nonlinear coupling between strains at different length scales. Such multiscale hyperelasticity and long-period structures constitute a new mechanism of size-dependent plasticity and its enhancement in nanoscale hcp metals.

Development of cup shaped microneedle array for transdermal drug delivery

Microneedle technology is one of the attractive methods in transdermal drug delivery. However, the clinical applications of this method are limited owing to: complexity in the preparation of multiple coating solutions, drug leakage while inserting the microneedles into the skin and the outer walls of the solid microneedle can hold limited quantity of drug. Here, the authors present the fabrication of an array of rectangular cup shaped silicon microneedles, which provide for reduced drug leakage resulting in improvement of efficiency of drug delivery and possibility of introducing multiple drugs. The fabricated solid microneedles with rectangular cup shaped tip have a total height of 200 mu m. These cup shaped tips have dimensions: 60 x 60 mu m (length x breadth) with a depth of 60 mu m. The cups are filled with drug using a novel in-house built drop coating system. Successful drug dissolution was observed when the coated microneedle was used on mice. Also, using the above method, it is possible to fill the cups selectively with different drugs, which enables simultaneous multiple drug delivery. (C) 2015 American Vacuum Society.

Identification of key amino acid residues in the catalytic mechanism of diaminopropionate ammonialyase from Salmonella typhimurium

Diaminopropionate ammonialyase (DAPAL), a fold-typeII pyridoxal 5-phosphate-dependent enzyme, catalyzes the ,-elimination of diaminopropionate (DAP) to pyruvate and ammonia. DAPAL was able to utilize both d- and l-DAP as substrates with almost equal efficiency. Mutational analysis of functionally important residues such as Thr385, Asp125 and Asp194 was carried out to understand the mechanism by which the isomers are hydrolyzed. Further, the putative residues involved in the formation of disulfide bond Cys271 and Cys299 were also mutated. T385S, T385D sDAPAL were as active with dl-DAP as substrate as sDAPAL, whereas the later exhibited a threefold increase in catalytic efficiency with d-Ser as substrate. Further analysis of these mutants suggested that DAPAL might follow an anti-E-2 mechanism of catalysis that does not involve the formation of a quinonoid intermediate. Of the two mutants of Asp125, D125E showed complete loss of activity with d-DAP as substrate, whereas the reaction with l-DAP was not affected significantly, demonstrating that Asp125 was essential for abstraction of protons from the d-isomer. By contrast, mutational analysis of Asp194 showed that the residue may not be directly involved in proton abstraction from l-DAP. sDAPAL does not form a disulfide bond in solution, although the position of Cys299 and Cys271 in the modeled structure of sDAPAL favored the formation of a disulfide bond. Further, unlike eDAPAL, sDAPAL could be activated by monovalent cations. Mutation of the cysteine residues showed that Cys271 may be involved in coordinating the monovalent cation, as observed in the case of other fold-typeII enzymes.

Chitosan-dextran sulphate nanocapsule drug delivery system as an effective therapeutic against intraphagosomal pathogen Salmonella

Objectives: The ability to target conventional drugs efficiently inside cells to kill intraphagosomal bacteria has been a major hurdle in treatment of infective diseases. We aimed to develop an efficient drug delivery system for combating infection caused by Salmonella, a well-known intracellular and intraphagosomal pathogen. Chitosan dextran sulphate (CD) nanocapsules were assessed for their efficiency in delivering drugs against Salmonella. Methods: The CD nanocapsules were prepared using the layer-by-layer method and loaded with ciprofloxacin or ceftriaxone. Antibiotic-loaded nanocapsules were analysed in vitro for their ability to enter epithelial and macrophage cells to kill Salmonella. In vivo pharmacokinetics and organ distribution studies were performed to check the efficiency of the delivery system. The in vivo antibacterial activity of free antibiotic and antibiotic loaded into nanocapsules was tested in a murine salmonellosis model. Results: In vitro and in vivo experiments showed that this delivery system can be used effectively to clear Salmonella infection, CD nanocapsules were successfully employed for efficient targeting and killing of the intracellular pathogen at a dosage significantly lower than that of the free antibiotic. The increased retention time of ciprofloxacin in the blood and organs when it was delivered by CD nanocapsules compared with the conventional routes of administration may be the reason underlying the requirement for a reduced dosage and frequency of antibiotic administration. Conclusions: CD nanocapsules can be used as an efficient drug delivery system to treat intraphagosomal pathogens, especially Salmonella infection, This delivery system might be used effectively for other vacuolar pathogens including Mycobacteria, Brucella and Legionella.

Broadcast Control Mechanism for Positional Consensus in Multiagent Systems

In this paper, a strategy for controlling a group of agents to achieve positional consensus is presented. The problem is constrained by the requirement that every agent must be given the same control input through a broadcast communication mechanism. Although the control command is computed using state information in a global framework, the control input is implemented by the agents in a local coordinate frame. We propose a novel linear programming (LP) formulation that is computationally less intensive than earlier proposed methods. Moreover, a random perturbation input in the control command that helps the agents to come close to each other even for a large number of agents, which was not possible with an existing strategy in the literature, is introduced. The method is extended to achieve positional consensus at a prespecified location. The effectiveness of the approach is illustrated through simulation results. A comparison between the LP approach and the existing second-order cone programming-based approach is also presented. The algorithm was successfully implemented on a robotic platform with three robots.

Molecular mechanism of water permeation in a helium impermeable graphene and graphene oxide membrane

Layers of graphene oxide (GO) are found to be good for the permeation of water but not for helium (Science, 2012, 335(6067), 442-444) suggesting that the GO layers are dynamic in the formation of a permeation route depending on the environment they are in (i.e., water or helium). To probe the microscopic origin of this observation we calculate the potential of mean force (PMF) of GO sheets (with oxidized and reduced parts), with the inter-planar distance as a reaction coordinate in helium and water. Our PMF calculation shows that the equilibrium interlayer distance between the oxidized part of the GO sheets in helium is at 4.8 angstrom leaving no space for helium permeation. In contrast, the PMF of the oxidized part of the GO in water shows two minima, one at 4.8 angstrom and another at 6.8 angstrom, corresponding to no water and a water filled region, thus giving rise to a permeation path. The increased electrostatic interaction between water with the oxidized part of the sheet helps the sheet open up and pushes water inside. Based on the entropy calculations for water trapped between graphene sheets and oxidized graphene sheets at different inter-sheet spacings, we also show the thermodynamics of filling.

Structures of substrate- and nucleotide-bound propionate kinase from Salmonella typhimurium: substrate specificity and phosphate-transfer mechanism

Kinases are ubiquitous enzymes that are pivotal to many biochemical processes. There are contrasting views on the phosphoryl-transfer mechanism in propionate kinase, an enzyme that reversibly transfers a phosphoryl group from propionyl phosphate to ADP in the final step of non-oxidative catabolism of L-threonine to propionate. Here, X-ray crystal structures of propionate- and nucleotide-bound Salmonella typhimurium propionate kinase are reported at 1.8-2.0 angstrom resolution. Although the mode of nucleotide binding is comparable to those of other members of the ASKHA superfamily, propionate is bound at a distinct site deeper in the hydrophobic pocket defining the active site. The propionate carboxyl is at a distance of approximate to 5 angstrom from the -phosphate of the nucleotide, supporting a direct in-line transfer mechanism. The phosphoryl-transfer reaction is likely to occur via an associative S(N)2-like transition state that involves a pentagonal bipyramidal structure with the axial positions occupied by the nucleophile of the substrate and the O atom between the - and the -phosphates, respectively. The proximity of the strictly conserved His175 and Arg236 to the carboxyl group of the propionate and the -phosphate of ATP suggests their involvement in catalysis. Moreover, ligand binding does not induce global domain movement as reported in some other members of the ASKHA superfamily. Instead, residues Arg86, Asp143 and Pro116-Leu117-His118 that define the active-site pocket move towards the substrate and expel water molecules from the active site. The role of Ala88, previously proposed to be the residue determining substrate specificity, was examined by determining the crystal structures of the propionate-bound Ala88 mutants A88V and A88G. Kinetic analysis and structural data are consistent with a significant role of Ala88 in substrate-specificity determination. The active-site pocket-defining residues Arg86, Asp143 and the Pro116-Leu117-His118 segment are also likely to contribute to substrate specificity.

Exploring the potential of adjunct therapy in tuberculosis

A critical unmet need for treatment of drug-resistant tuberculosis (TB) is to find novel therapies that are efficacious, safe, and shorten the duration of treatment. Drug discovery approaches for TB primarily target essential genes of the pathogen Mycobacterium tuberculosis (Mtb) but novel strategies such as host-directed therapies and nonmicrobicidal targets are necessary to bring about a paradigm shift in treatment. Drugs targeting the host pathways and nonmicrobicidal proteins can be used only in conjunction with existing drugs as adjunct therapies. Significantly, host-directed adjunct therapies have the potential to decrease duration of treatment, as they are less prone to drug resistance, target the immune responses, and act via novel mechanism of action. Recent advances in targeting host-pathogen interactions have implicated pathways such as eicosanoid regulation and angiogenesis. Furthermore, several approved drugs such as metformin and verapamil have been identified that appear suitable for repurposing for the treatment of TB. These findings and the challenges in the area of host- and/or pathogen-directed adjunct therapies and their implications for TB therapy are discussed.

Synthesis of Novel Benzofuran-Gathered C-2,4,6-substituted Pyrimidine Derivatives Conjugated by Sulfonyl Chlorides: Orally Bioavailable, Selective, Effective Antioxidants and Antimicrobials Drug Candidates

In the present study, we have made an effort to develop the novel synthetic antioxidants and antimicrobials with improved potency. The novel benzofuran-gathered C-2,4,6-substituted pyrimidine derivatives 5a, 5b, 5c, 5d, 5e, 5f, 6a, 6b, 6c, 6d, 6e, 6f, 7a, 7b, 7c, 7d, 7e, 7f, 8a, 8b, 8c, 8d, 8e, 8f, 9a, 9b, 9c, 9d, 9e, 9f were synthesized by simple and efficient four-step reaction pathway. Initially, o-alkyl derivative of salicylaldehyde readily furnish corresponding 2-acetyl benzofuran 2 in good yield, upon the treatment with potassium tertiary butoxide in the presence of molecular sieves. Further, Claisen-Schmidt condensation with aromatic aldehydes via treatment with thiourea followed by coupling reaction with different sulfonyl chlorides afforded target compounds. The structures of newly synthesized compounds were confirmed by IR, H-1 NMR, C-13 NMR, mass, and elemental analysis and further screened for their antioxidant and antimicrobial activities. The results showed that the synthesized compounds 8b, 8e, 9b, and 9e produced significant antioxidant activity with 50% inhibitory concentration higher than that of reference, whereas compounds 7d and 7c produced dominant antimicrobial activity at concentrations 1.0 and 0.5mg/mL compared with standard Gentamicin and Nystatin, respectively.

ABSOLUTE/CONVECTIVE INSTABILITY TRANSITION IN A BACKWARD FACING STEP COMBUSTOR: FUNDAMENTAL MECHANISM AND INFLUENCE OF DENSITY GRADIENT

Hydrodynamic instabilities of the flow field in lean premixed gas turbine combustors can generate velocity perturbations that wrinkle and distort the flame sheet over length scales that are smaller than the flame length. The resultant heat release oscillations can then potentially result in combustion instability. Thus, it is essential to understand the hydrodynamic instability characteristics of the combustor flow field in order to understand its overall influence on combustion instability characteristics. To this end, this paper elucidates the role of fluctuating vorticity production from a linear hydrodynamic stability analysis as the key mechanism promoting absolute/convective instability transitions in shear layers occurring in the flow behind a backward facing step. These results are obtained within the framework of an inviscid, incompressible, local temporal and spatio-temporal stability analysis. Vorticity fluctuations in this limit result from interaction between two competing mechanisms - (1) production from interaction between velocity perturbations and the base flow vorticity gradient and (2) baroclinic torque in the presence of base flow density gradients. This interaction has a significant effect on hydrodynamic instability characteristics when the base flow density and velocity gradients are co-located. Regions in the space of parameters characterizing the base flow velocity profile, i.e. shear layer thickness and ratio of forward to reverse flow velocity, corresponding to convective and absolute instability are identified. The implications of the present results on prior observations of flow instability in other flows such as heated jets and bluff-body stabilized flames is discussed.

Detailed mechanism and kinetic study of CO oxidation on cobalt oxide surfaces

Co3O4 catalysts were prepared by combustion synthesis using different fuels glycine (G), ODH (O) and urea (U). Morphological changes of the materials were observed by using different fuels. The prepared catalysts were characterized by XRD, XPS, SEM, TEM, BET and DRIFTS analysis. All compounds showed 100% conversion of CO below 175C. The prepared catalysts exhibited very high stability and conversions did not decrease even after 50 h of continuous operation. The oxygen storage capacity (OSC) of materials was measured by H-2-TPR analysis. Co3O4-O is having high OSC among the synthesized catalysts. The activation energies of these catalysts were found to be in the range of 42.3-64.8 kJ mol(-1). With DRIFTS analysis, the surface carbonates, superoxide anions, adsorbed CO, O-2 species on the catalyst surface were found and this information was used to develop a detailed reaction pathway. A kinetic model was developed with the help of proposed mechanism and used to fit the data. (C) 2014 Elsevier B.V. All rights reserved.

Insights into the catalytic mechanism of synthetic glutathione peroxidase mimetics

Glutathione Peroxidase (GPx) is a key selenoenzyme that protects biomolecules from oxidative damage. Extensive research has been carried out to design and synthesize small organoselenium compounds as functional mimics of GPx. While the catalytic mechanism of the native enzyme itself is poorly understood, the synthetic mimics follow different catalytic pathways depending upon the structures and reactivities of various intermediates formed in the catalytic cycle. The steric as well as electronic environments around the selenium atom not only modulate the reactivity of these synthetic mimics towards peroxides and thiols, but also the catalytic mechanisms. The catalytic cycle of small GPx mimics is also dependent on the nature of peroxides and thiols used in the study. In this review, we discuss how the catalytic mechanism varies with the substituents attached to the selenium atom.

Stimuli-responsive weak polyelectrolyte multilayer films: A thin film platform for self triggered multi-drug delivery

Polyelectrolyte multilayer (PEM) thin film composed of weak polyelectrolytes was designed by layer-by-layer (LbL) assembly of poly(allylamine hydrochloride) (PAH) and poly(methacrylic acid) (PMA) for multi-drug delivery applications. Environmental stimuli such as pH and ionic strength showed significant influence in changing the film morphology from pore-free smooth structure to porous structure and favored triggered release of loaded molecules. The film was successfully loaded with bovine serum albumin (BSA) and ciprofloxacin hydrochloride (CH) by modulating the porous polymeric network of the film. Release studies showed that the amount of release could be easily controlled by changing the environmental conditions such as pH and ionic strength. Sustained release of loaded molecules was observed up to 8 h. The fabricated films were found to be biocompatible with epithelial cells during in-vitro cell culture studies. PEM film reported here not only has the potential to be used as self-responding thin film platform for transdermal drug delivery, but also has the potential for further development in antimicrobial or anti-inflammatory coatings on implants and drug-releasing coatings for stents. (C) 2015 Elsevier B.V. All rights reserved.