The Mode of Inhibitor Binding to Peptidyl-tRNA Hydrolase: Binding Studies and Structure Determination of Unbound and Bound Peptidyl-tRNA Hydrolase from Acinetobacter baumannii

The incidences of infections caused by an aerobic Gram-negative bacterium, Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth) is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design.     

Substitution of Glutamate Residue by Lysine in the Dimerization Domain Affects DNA Binding Ability of HapR by Inducing Structural Deformity in the DNA Binding Domain

HapR has been given the status of a high cell density master regulatory protein in Vibrio cholerae. Though many facts are known regarding its structural and functional aspects, much still can be learnt from natural variants of the wild type protein. This work aims at investigating the nature of functional inertness of a HapR natural variant harboring a substitution of a conserved glutamate residue at position 117 which participates in forming a salt bridge by lysine (HapR_V2G-E¹¹⁷K). Experimental evidence presented here reveals the inability of this variant to interact with various cognate promoters by in vitro gel shift assay. Furthermore, the elution profiles of HapR_V2G-E¹¹⁷K protein along with the wild type functional HapR_V2G in size-exclusion chromatography as well as circular dichroism spectra did not reflect any significant differences in its structure, thereby indicating the intactness of dimer in the variant protein. To gain further insight into the global shape of the proteins, small angle X-ray scattering analysis (SAXS) was performed. Intriguingly, increased radius of gyration of HapR_V2G-E¹¹⁷K of 27.5 Å in comparison to the wild type protein from SAXS data analyses implied a significant alteration in the global shape of the dimeric HapR_V2G-E¹¹⁷K protein. Structure reconstruction brought forth that the DNA binding domains were substantially “parted away” in this variant. Taken together, our data illustrates that substitution of the conserved glutamate residue by lysine in the dimerization domain induces separation of the two DNA binding domains from their native-like positioning without altering the dimeric status of HapR variant.     

LED-Fluorescence Microscopy for Diagnosis of Pulmonary Tuberculosis under Programmatic Conditions in India

Background

Light-emitting diode fluorescence microscopy (LED-FM) has been shown to be more sensitive than conventional bright field microscopy using Ziehl-Neelsen (ZN) stain in detecting sputum smear positive tuberculosis in controlled laboratory conditions. In 2012, Auramine O staining based LED-FM replaced conventional ZN microscopy in 200 designated microscopy centres (DMC) of medical colleges operating in collaboration with India’s Revised National Tuberculosis Control Programme. We aimed to assess the impact of introduction of LED-FM services on sputum smear positive case detection under program conditions.

Methods

This was a before and after comparison study. In 15 randomly selected medical college DMCs, all presumptive TB patients who underwent sputum smear examination in the years 2011 (before LED-FM) and 2012 (after LED-FM) were compared. An additional 15 comparable DMCs that implemented conventional ZN sputum smear microscopy were also selected for comparison between 2011 and 2012.

Results

The proportion of presumptive TB patients (PTP)found sputum smear positive increased by 30%- from 13.6% (3432/25159) in 2011 to 17.8% (4706/26426) in 2012 (P value <0.01) in the sites that implemented LED-FM microscopy, whereas in DMCs where the ZN staining procedure is followed the proportion of sputum smear positive had remained unchanged (13.0%versus 12.6%;P value0.31).

Conclusion

Use of LED-FM significantly increased the proportion of smear positive cases among presumptive TB patients under routine program conditions in high workload laboratories. The study provides operational evidence needed to scale-up the use of LED-FM in similar settings in India and beyond.     

The First Report on Excellent Cycling Stability and Superior Rate Capability of Na3V2(PO4)3 for Sodium Ion Batteries

Sodium ion batteries are attractive for the rapidly emerging large‐scale energy storage market for intermittent renewable resources. Currently a viable cathode material does not exist for practical non‐aqueous sodium ion battery applications. Here we disclose a high performance, durable electrode material based on the 3D NASICON framework. Porous Na₃V₂(PO₄)₃/C was synthesized using a novel solution‐based approach. This material, as a cathode, is capable of delivering an energy storage capacity of ～400 mWh/g vs. sodium metal. Furthermore, at high current rates (10, 20 and 40 C), it displayed remarkable capacity retention. Equally impressive is the long term cycle life. Nearly 50% of the initial capacity was retained after 30,000 charge/discharge cycles at 40 C (4.7 A/g). Notably, coulombic efficiency was 99.68% (average) over the course of cycling. To the best of our knowledge, the combination of high energy density, high power density and ultra long cycle life demonstrated here has never been reported before for sodium ion batteries. We believe our findings will have profound implications for developing large‐scale energy storage systems for renewable energy sources.     

Promising Polyphenols in Parkinson’s Disease Therapeutics

Neurochemical Research - Parkinson’s disease (PD) is a slow progressive, second most common neurodegenerative disease characterized by the loss of dopaminergic neurons from the nigrostriatal...     

Isolation and physico-chemical characterisation of extracellular polymeric substances produced by the marine bacterium Vibrio parahaemolyticus

A marine bacterial strain identified as Vibrio parahaemolyticus by 16S rRNA gene (HM355955) sequencing and gas chromatography (GC) coupled with MIDI was selected from a natural biofilm by its capability to produce extracellular polymeric substances (EPS). The EPS had an average molecule size of 15.278 μm and exhibited characteristic diffraction peaks at 5.985°, 9.150° and 22.823°, with d-spacings of 14.76661, 9.29989 and 3.89650 Å, respectively. The Fourier-transform infrared spectroscopy (FTIR) spectrum revealed aliphatic methyl, primary amine, halide groups, uronic acid and saccharides. Gas chromatography mass spectrometry (GCMS) confirmed the presence of arabinose, galactose, glucose and mannose. ¹HNMR (nuclear magnetic resonance) revealed functional groups characteristic of polysaccharides. The EPS were amorphous in nature (CI_xrd 0.092), with a 67.37% emulsifying activity, thermostable up to 250°C and displayed pseudoplastic rheology. MALDI-TOF–TOF analysis revealed a series of masses, exhibiting low-mass peaks (m/z) corresponding to oligosaccharides and higher-mass peaks for polysaccharides consisting of different ratios of pentose and hexose moieties. This is the first report of a detailed characterisation of the EPS produced by V. parahaemolyticus, which could be further explored for biotechnological and industrial use.     

DFT study of structure and vibrational spectra of ceramide 3: comparison to experimental data

The Fourier transform Raman and infrared (IR) spectra of the Ceramide 3 (CER3) have been recorded in the regions 200–3500 cm^? 1 and 680–4000 cm^? 1, respectively. We have calculated the equilibrium geometry, harmonic vibrational wavenumbers, electrostatic potential surfaces, absolute Raman scattering activities and IR absorption intensities by the density functional theory with B3LYP functionals having extended basis set 6-311G. This work is undertaken to study the vibrational spectra of CER3 completely and to identify the various normal modes with better wavenumber accuracy. Good consistency is found between the calculated results and experimental data for the IR and Raman spectra.     

172 Role of conserved water molecular triad in the recognition of IMP,NAD+ with Asp 274, Asn 303, Arg 322, and Asp 364 in both the isoform of hIMPDH

Inosine monophosphate dehydrogenase (IMPDH) plays an important role in the Guanosine monophosphate (GMP) biosynthesis pathway. As hIMPDH-II is involved in CML-Cancer, it is thought to be an active target for leukemic drug design. The importance of conserved water molecules in the salt-bridge-mediated interdomain recognition and loop-flap recognition of hIMPDH has already been indicated in some simulation studies (Bairagya et al., 2009, 2011a, 2011b, 2012; Mishra et al., 2012). In this work, the role of conserved water molecules in the recognition of Inosine monophosphate (IMP) and NAD⁺ (co-factor) to active site residues of both the isoforms has been investigated by all atoms MD-Simulation studies. During 25-ns dynamics of the solvated hIMPDH-II and I (1B3O and 1JCN PDB structures), the involvement of conserved water molecular triad (W _M, W _L and W _C) in the recognition of active site residues (Asp 274, Asn 303, Arg 322, and Asp 364), IMP and NAD⁺ has been observed (Figure 1). The H-bonding co-ordination of all three conserved water molecular centers is within 4–7 and their occupation frequency is 1.0. The H-bonding geometry and the electronic consequences of the water molecular interaction at the different residues (and also IMP and NAD⁺) may put forward some rational clues on antileukemic agent design.     

181 Integrative analysis of gene expression and protein–protein interaction networks in Glioblastoma

Glioblastoma multiforme (GBM) is the most malignant of all the brain tumors with very low median survival time of one year, as per Central Brain Tumor Registry of the USA, 2001. Efforts are ongoing to understand this disease pathogenesis in complete details. Global gene expression changes in GBM pathogenesis have been studied by several groups using microarray technology (e.g. Carro et al., 2010). One of the many approaches to ‘understand the control mechanisms underlying the observed changes in the activity of a biological process’ (Cline et al., 2007) is integration of gene expression and protein–protein interactions (PPI) datasets. Among several examples, aberrant activation of Wnt/β-catenin signaling pathway as well as sonic hedgehog (SHH) signaling pathway is reported in GBMs (Klaus & Birchmeier, 2008). Further, these two pathways are also involved in proliferation and clonogenicity of glioma cancer stem cells (Li et al., 2009), which are thought to play a role in glioma initiation, proliferation, and invasion, and are one of the important points of intervention. Hedgehog–Gli1 signaling is also found to regulate the expression of stemness genes. In this paper, analyses of the relationship between the significant differential expression of these and other genes and the connectivity as well as topological features of a PPI network would be discussed. This way, genes potentially overlooked when relying solely on expression profiles may be identified which can be biologically relevant as possible drug target/s or disease biomarker/s.