Structure-Based Design of DevR Inhibitor Active against Nonreplicating Mycobacterium tuberculosis

Antitubercular treatment is directed against actively replicating organisms. There is an urgent need to develop drugs targeting persistent subpopulations of Mycobacterium tuberculosis. The DevR response regulator is believed to play a key role in bacterial dormancy adaptation during hypoxia. We developed a homology-based model of DevR and used it for the rational design of inhibitors. A phenylcoumarin derivative (compound 10) identified by in silico pharmacophore-based screening of 2.5 million compounds employing protocols with some novel features including a water-based pharmacophore query, was characterized further. Compound 10 inhibited DevR binding to target DNA, down-regulated dormancy genes transcription, and drastically reduced survival of hypoxic but not nutrient-starved dormant bacteria or actively growing organ ` isms. Our findings suggest that compound 10 ``locks'' DevR in an inactive conformation that is unable to bind cognate DNA and induce the dormancy regulon. These results provide proof-of-concept for DevR as a novel target to develop molecules with sterilizing activity against tubercle bacilli.

Oxidation of bis-(2-hydroxy-1-naphthyl)methane with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. X-Ray crystal structure of a novel product

A novel compound obtained by the oxidation of the title compound with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone has been assigned structure (5) on the basis of spectral data and X-ray crystal structure analysis.

Structure of disodium deoxyuridine 5'-phosphate pentahydrate

Disodium deoxyuridine 5'-nhosDhate pentahvdrate, Na2(C9H l INEOsP). 5 H20, Call 11N208 P2-. 2Na +. 5 H20, crystallizes in the monoclinic space group P2: with a = 7.250 (4), b = 35.45 (2), c = 7.132 (4)/~, fl = 102.2 (4) °, Z = 4. The Cu Ka intensity data were collected photographically and estimated visually. The structure was obtained by the minimum-function method and difference syntheses and refined to an R of 0.089. In both molecules the uracil base has an anti conformation (2cN = 57.1 and 59.9 °) with respect to the sugar. The deoxyribose moiety of molecule B shows a typical C(l')-exo puckering, with C(I') displaced by 0.52 /k from the best plane. The furanose ring conformation of molecule A can be described as C(2')-endo,C(l')-exo. Both the molecules have an unusual trans-gauche conformation about the exocyclic C(4')-C(5') bond with (~0oo = 171.1, 172.2°; ~0oc = -64.7, -65.9°).

Conformational studies of the triphenylphosphazenyl side chain in cyclophosphazenes. II. Crystal and molecular structure of 2,4,4,6,6,8,8-heptachloro-2-triphenylphosphazenylcyclotetra(phosphazene), N4P4Cl7(NPPh3)

Abstract is not available.

Acoustic emission source location and damage detection in a metallic structure using a graph-theory-based geodesic pproach

A geodesic-based approach using Lamb waves is proposed to locate the acoustic emission (AE) source and damage in an isotropic metallic structure. In the case of the AE (passive) technique, the elastic waves take the shortest path from the source to the sensor array distributed in the structure. The geodesics are computed on the meshed surface of the structure using graph theory based on Dijkstra's algorithm. By propagating the waves in reverse virtually from these sensors along the geodesic path and by locating the first intersection point of these waves, one can get the AE source location. The same approach is extended for detection of damage in a structure. The wave response matrix of the given sensor configuration for the healthy and the damaged structure is obtained experimentally. The healthy and damage response matrix is compared and their difference gives the information about the reflection of waves from the damage. These waves are backpropagated from the sensors and the above method is used to locate the damage by finding the point where intersection of geodesics occurs. In this work, the geodesic approach is shown to be suitable to obtain a practicable source location solution in a more general set-up on any arbitrary surface containing finite discontinuities. Experiments were conducted on aluminum specimens of simple and complex geometry to validate this new method.

Structure and Function of Hemoglobin Confined Inside Silica Nanotubes

Investigations on the structure and function of hemoglobin (Hb) confined inside sol-gel template synthesized silica nanotubes (SNTs) have been discussed here. Immobilization of hemoglobin inside SNTs resulted in the enhancement of direct electron transfer during an electrochemical reaction. Extent of influence of nanoconfinement on protein activity is further probed via ligand binding and thermal stability studies. Electrochemical investigations show reversible binding of n-donor liquid ligands, such as pyridine and its derivatives, and predictive variation in their redox potentials suggests an absence of any adverse effect on the structure and function of Hb confined inside nanometer-sized channels of SNTs. Immobilization also resulted in enhanced thermal stability of Hb. The melting or denaturation temperature of Hb immobilized inside SNTs increase by approximately 4 degrees C as compared with that of free Hb in solution.

Structure, function and intracellular dynamics of alphavirus replication complexes

Intracellular membrane alterations are hallmarks of positive-sense RNA (+RNA) virus replication. Strong evidence indicates that within these exotic compartments, viral replicase proteins engage in RNA genome replication and transcription. To date, fundamental questions such as the origin of altered membranes, mechanisms of membrane deformation and topological distribution and function of viral components, are still waiting for comprehensive answers. This study addressed some of the above mentioned questions for the membrane alterations induced during Semliki Forest virus (SFV) infection of mammalian cells. With the aid of electron and fluorescence microscopy coupled with radioactive labelling and immuno-cytochemistry techniques, our group and others showed that few hours after infection the four non structural proteins (nsP1-4) and newly synthesized RNAs of SFV colocalized in close proximity of small membrane invaginations, designated as spherules . These 50-70 nm structures were mainly detected in the perinuclear area, at the limiting membrane of modified endosomes and lysosomes, named CPV-I (cytopathic vacuoles type I). More rarely, spherules were also found at the plasma membrane (PM). In the first part of this study I present the first three-dimensional reconstruction of the CPV-I and the spherules, obtained by electron tomography after chemical or cryo-fixation. Different approaches for imaging these macromolecular assemblies to obtain better structure preservation and higher resolution are presented as unpublished data. This study provides insights into spherule organization and distribution of viral components. The results of this and other experiments presented in this thesis will challenge currently accepted models for virus replication complex formation and function. In a revisitation of our previous models, the second part of this work provides the first complete description of the biogenesis of the CPV-I. The results demonstrate that these virus-induced vacuoles, where hundreds of spherules accumulate at late stages during infection, represent the final phase of a journey initiated at the PM, which apparently serves as a platform for spherule formation. From the PM spherules were internalized by an endocytic event that required the activity of the class I PI3K, caveolin-1, cellular cholesterol and functional actin-myosin network. The resulting neutral endocytic carrier vesicle delivered the spherules to the membrane of pre-existing acidic endosomes via multiple fusion events. Microtubule based transport supported the vectorial transfer of these intermediates to the pericentriolar area where further fusions generated the CPV-I. A signal for spherule internalization was identified in one of the replicase proteins, nsP3. Infections of cells with viruses harbouring a deletion in a highly phosphorylated region of nsP3 did not result in the formation of CPV-Is. Instead, thousands of spherules remained at the PM throughout the infection cycle. Finally, the role of the replicase protein nsP2 during viral RNA replication and transcription was investigated. Three enzymatic activities, protease, NTPase and RNA-triphosphatase were studied with the aid of temperature sensitive mutants in vitro and, when possible, in vivo. The results highlighted the interplay of the different nsP2 functions during different steps of RNA replication and sub-genomic promoter regulation, and suggest that the protein could have different activities when participating in the replication complex or as a free enzyme.

Population structure and evolution in the ant Plagiolepis pygmaea and its two social parasites Plagiolepis xene and Plagiolepis grassei

Social behaviour affects dispersal of animals and is an important modifier of genetic population structures. The female sex is often philopatric, which maintains coancestry within the breeding groups and promotes cooperative behaviours. This enables also inclusive fitness returns from altruism and explains why some individuals sacrifice personal reproduction for the good of others in social insects such as ants. However, reduced dispersal and population substructuring at the level of colonies may also entail inbreeding, loss of genetic diversity, and vulnerability. In addition, the most vulnerable ants are species that are evolved to parasitize colonies of other ants, and which compromise between abilities to disperse and the efficiency to parasitize the host. On the other hand, certain social organisations of ant colonies may facilitate a species to disperse outside its natural range and become a pest. Altogether, knowledge on genetic structuring of ant populations, as well as the evolution of their life histories can contribute to conservation biology and population management. The aim of this thesis was to investigate population structures and phylogenetic evolution of the ant Plagiolepis pygmaea and its two obligatory, workerless social parasites (inquilines) P. xene and P. grassei with genetic markers and DNA sequence data. The results support the general assumption that populations of inquiline parasites are highly fragmented and genetically vulnerable. Comparison of the two parasites suggests that differences in their relative abundance may follow from their interaction with the host, i.e. how well the species is adapted to reproduce in the host colonies. The results also indicate that the most recent free living ancestor to these two parasite species is their common host. This is considered to provide evidence for the controversial issue of sympatric speciation. Further, given that the level of adaptations to parasitic life history depends on the evolutionary time since the free-living ancestor, the results establish a link between species rarity and its evolutionary age. The populations of the host species P. pygmaea displayed significantly reduced dispersal both among the females (queens) and males, and high levels of inbreeding which may enhance worker altruism. In addition, the queens were found to mate with multiple males. Given the high relatedness between the queens and their mates, this occurs probably for non-genetic reasons, e.g. without benefits associated in genetically more diverse offspring. The results hence caution that the contribution of non-genetic factors to the prevailing mating patterns and genetic population structures should not be underestimated.

Ruthenium(II)-CO complexes of N-[(2-pyridyl)methyliden]-alpha(or beta)-aminonaphthalene: Synthesis, spectral studies, crystal structure, redox properties and DFT calculation

The characterization and properties of trans-(X)-[RuX2(CO)(2)(alpha/beta-NaiPy)] (1, 2) (alpha-NaiPy (a), beta-NaiPy (b); X = Cl (1), I (2)) are described in this work. The structures are confirmed by single crystal X-ray diffraction studies. Reaction of these compounds with Me3NO in MeCN has isolated monocarbonyl trans-(X)-RuX2(CO)(MeCN)(alpha/beta-NaiPy)] (3, 4). The complexes show intense emission properties. Quantum yields of 1 and 2 (phi= 0.02-0.08) are higher than 3 and 4 (phi = 0.006-0.015). Voltammogram shows higher Ru(III)/Ru(II) (1.3-1.5 V) potential of 1 and 2 than that of 3 and 4 (0.8-0.9 V) that may be due to coordination of two pi-acidic CO groups in former. The electronic spectra and redox properties of the complexes are compared with the results obtained by density functional theory (DFT) and time-dependent density functional theory (TD-DFT) using polarizable continuum model (CPCM).

Structure and phase transition of the (1-x)PbTiO3-(x)BiAlO3 system

The system (1 - x)PbTiO3 - (x)BiAlO3 has been studied with regard to its structure and phase transition behaviour using x-ray, neutron and dielectric measurements. The structure is tetragonal within the solid solubility limit (x < 0.25). Interatomic distance analysis revealed that the Pb-O bond lengths remain unaffected and the only the Ti-O lengths are altered by BiAlO3 substitution. The results are suggestive of a crossover from and A-site & B site driven ferroelectric system for x = 0 to a dominant A-site driven ferroelectric system for higher x. This cross-over is brought about by (i) a reduction in the contribution to the ferroelectric stability from B-site cations due to dilution of the Ti-sublattice by Al and (ii) a reinforcement to the stability of the ferroelectric state by the A-site cations by the Bi+3 cations..

Structure, dynamics, and interactions of jacalin. Insights from molecular dynamics simulations examined in conjunction with results of X-ray studies

Molecular dynamics simulations have been carried out on all the jacalin-carbohydrate complexes of known structure, models of unliganded molecules derived from the complexes and also models of relevant complexes where X-ray structures are not available. Results of the simulations and the available crystal structures involving jacalin permit delineation of the relatively rigid and flexible regions of the molecule and the dynamical variability of the hydrogen bonds involved in stabilizing the structure. Local flexibility appears to be related to solvent accessibility. Hydrogen bonds involving side chains and water bridges involving buried water molecules appear to be important in the stabilization of loop structures. The lectin-carbohydrate interactions observed in crystal structures, the average parameters pertaining to them derived from simulations, energetic contribution of the stacking residue estimated from quantum mechanical calculations, and the scatter of the locations of carbohydrate and carbohydrate-binding residues are consistent with the known thermodynamic parameters of jacalin-carbohydrate interactions. The simulations, along with X-ray results, provide a fuller picture of carbohydrate binding by jacalin than provided by crystallographic analysis alone. The simulations confirm that in the unliganded structures water molecules tend to occupy the positions occupied by carbohydrate oxygens in the lectin-carbohydrate complexes. Population distributions in simulations of the free lectin, the ligands, and the complexes indicate a combination of conformational selection and induced fit. Proteins 2009; 77:760-777.