Deorphanization of Malonyl CoA:ACP Transacylase Drug Target in Plasmodium falciparum (PfFabD) Using Bacterial Antagonists: A Piggyback' Approach for Antimalarial Drug Discovery

Quest for new drug targets in Plasmodium sp. has underscored malonyl CoA:ACP transacylase (PfFabD) of fatty acid biosynthetic pathway in apicoplast. In this study, a piggyback approach was employed for the receptor deorphanization using inhibitors of bacterial FabD enzymes. Due to the lack of crystal structure, theoretical model was constructed using the structural details of homologous enzymes. Sequence and structure analysis has localized the presence of two conserved pentapeptide motifs: GQGXG and GXSXG and five key invariant residues viz., Gln109, Ser193, Arg218, His305 and Gln354 characteristic of FabD enzyme. Active site mapping of PfFabD using substrate molecules has disclosed the spatial arrangement of key residues in the cavity. As structurally similar molecules exhibit similar biological activities, signature pharmacophore fingerprints of FabD antagonists were generated using 0D-3D descriptors for molecular similarity-based cluster analysis and to correlate with their binding profiles. It was observed that antagonists showing good geometrical fitness score were grouped in cluster-1, whereas those exhibiting high binding affinities in cluster-2. This study proves important to shed light on the active site environment to reveal the hotspot for binding with higher affinity and to narrow down the virtual screening process by searching for close neighbors of the active compounds.

Mechanism of Fragility at BCL2 Gene Minor Breakpoint Cluster Region during t(14;18) Chromosomal Translocation

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Breaks in chromosome 18 are localized at the 3'-UTR of BCL2 gene or downstream and are mainly clustered in either the major breakpoint region or the minor breakpoint cluster region (mcr). The recombination activating gene (RAG) complex induces breaks at IgH locus of chromosome 14, whereas the mechanism of fragility at BCL2 mcr remains unclear. Here, for the first time, we show that RAGs can nick mcr; however, the mechanism is unique. Three independent nicks of equal efficiency are generated, when both Mg2+ and Mn2+ are present, unlike a single nick during V(D)J recombination. Further, we demonstrate that RAG binding and nicking at the mcr are independent of nonamer, whereas a CCACCTCT motif plays a critical role in its fragility, as shown by sequential mutagenesis. More importantly, we recapitulate the BCL2 mcr translocation and find that mcr can undergo synapsis with a standard recombination signal sequence within the cells, in a RAG-dependent manner. Further, mutation to the CCACCTCT motif abolishes recombination within the cells, indicating its vital role. Hence, our data suggest a novel, physiologically relevant, nonamer-independent mechanism of RAG nicking at mcr, which may be important for generation of chromosomal translocations in humans.

Facile synthesis of large area porous Cu2O as super hydrophobic yellow-red phosphors

By employing a thermal oxidation strategy, we have grown large area porous Cu2O from Cu foil. CuO nanorods are grown by heating Cu which were in turn heated in an argon atmosphere to obtain a porous Cu2O layer. The porous Cu2O layer is superhydrophobic and exhibits red luminescence. In contrast, Cu2O obtained by direct heating, is hydrophobic and exhibits yellow luminescence. Two more luminescence bands are observed in addition to red and yellow luminescence, corresponding to the recombination of free and bound excitons. Over all, the porous Cu2O obtained from Cu via CuO nanorods, can serve as a superhydrophobic luminescence/phosphor material.

Functional characterization of UvrD helicases from Haemophilus influenzae and Helicobacter pylori

Haemophilus influenzae and Helicobacter pylori are major bacterial pathogens that face high levels of genotoxic stress within their host. UvrD, a ubiquitous bacterial helicase that plays important roles in multiple DNA metabolic pathways, is essential for genome stability and might, therefore, be crucial in bacterial physiology and pathogenesis. In this study, the functional characterization of UvrD helicase from Haemophilus influenzae and Helicobacter pylori is reported. UvrD from Haemophilus influenzae (HiUvrD) and Helicobacter pylori (HpUvrD) exhibit strong single-stranded DNA-specific ATPase and 3'5' helicase activities. Mutation of highly conserved arginine (R288) in HiUvrD and glutamate (E206) in HpUvrD abrogated their activities. Both the proteins were able to bind and unwind a variety of DNA structures including duplexes with strand discontinuities and branches, three- and four-way junctions that underpin their role in DNA replication, repair and recombination. HiUvrD required a minimum of 12 nucleotides, whereas HpUvrD preferred 20 or more nucleotides of 3'-single-stranded DNA tail for efficient unwinding of duplex DNA. Interestingly, HpUvrD was able to hydrolyze and utilize GTP for its helicase activity although not as effectively as ATP, which has not been reported to date for UvrD characterized from other organisms. HiUvrD and HpUvrD were found to exist predominantly as monomers in solution together with multimeric forms. Noticeably, deletion of distal C-terminal 48 amino acid residues disrupted the oligomerization of HiUvrD, whereas deletion of 63 amino acids from C-terminus of HpUvrD had no effect on its oligomerization. This study presents the characteristic features and comparative analysis of Haemophilus influenzae and Helicobacter pylori UvrD, and constitutes the basis for understanding the role of UvrD in the biology and virulence of these pathogens.

Cascaded walks in protein sequence space: use of artificial sequences in remote homology detection between natural proteins

Over the past two decades, many ingenious efforts have been made in protein remote homology detection. Because homologous proteins often diversify extensively in sequence, it is challenging to demonstrate such relatedness through entirely sequence-driven searches. Here, we describe a computational method for the generation of `protein-like' sequences that serves to bridge gaps in protein sequence space. Sequence profile information, as embodied in a position-specific scoring matrix of multiply aligned sequences of bona fide family members, serves as the starting point in this algorithm. The observed amino acid propensity and the selection of a random number dictate the selection of a residue for each position in the sequence. In a systematic manner, and by applying a `roulette-wheel' selection approach at each position, we generate parent family-like sequences and thus facilitate an enlargement of sequence space around the family. When generated for a large number of families, we demonstrate that they expand the utility of natural intermediately related sequences in linking distant proteins. In 91% of the assessed examples, inclusion of designed sequences improved fold coverage by 5-10% over searches made in their absence. Furthermore, with several examples from proteins adopting folds such as TIM, globin, lipocalin and others, we demonstrate that the success of including designed sequences in a database positively sensitized methods such as PSI-BLAST and Cascade PSI-BLAST and is a promising opportunity for enormously improved remote homology recognition using sequence information alone.

Analysis of current voltage characteristics of MWIR homojunction photodiodes for uncooled operation

Dark currents n(+)/v/p(+) Hg0.69Cd0.Te-31 mid wave infrared photodiodes were measured at room temperature. The diodes exhibited negative differential resistance at room-temperature, but with increasing leakage currents as a function of reverse bias. The current-voltage characteristics were simulated and fitted by incorporating trap assisted tunneling via traps and Shockley-Read-Hall generation recombination process due to dislocations in the carrier transport equations. The thermal suppression of carriers was simulated by taking energy level of trap (E-t), trap density (N-t) and the doping concentrations of n(+) and v regions as fitting parameters. Values of E-t and N-t were 0.78E(g) and similar to 6-9 x 10(14) cm(-3) respectively for most of the diodes. Variable temperature current voltage measurements on variable area diode array (VADA) structures confirmed the fact that variation in zero bias resistance area product (R(0)A) is related to g-r processes originating from variation in concentration and kind of defects that intersect a junction area. (C) 2012 Elsevier B.V. All rights reserved.