Targeting RNA Polymerase Primary σ70 as a Therapeutic Strategy against Methicillin-Resistant Staphylococcus aureus by Antisense Peptide Nucleic Acid

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) causes threatening infection-related mortality worldwide. Currently, spread of multi-drug resistance (MDR) MRSA limits therapeutic options and requires new approaches to "druggable" target discovery, as well as development of novel MRSA-active antibiotics. RNA polymerase primary σ⁷⁰ (encoded by gene rpoD) is a highly conserved prokaryotic factor essential for transcription initiation in exponentially growing cells of diverse S. aureus, implying potential for antisense inhibition. METHODOLOGY/PRINCIPAL FINDINGS: By synthesizing a serial of cell penetrating peptide conjugated peptide nucleic acids (PPNAs) based on software predicted parameters and further design optimization, we identified a target sequence (234 to 243 nt) within rpoD mRNA conserved region 3.0 being more sensitive to antisense inhibition. A (KFF)₃K peptide conjugated 10-mer complementary PNA (PPNA2332) was developed for potent micromolar-range growth inhibitory effects against four pathogenic S. aureus strains with different resistance phenotypes, including clinical vancomycin-intermediate resistance S. aureus and MDR-MRSA isolates. PPNA2332 showed bacteriocidal antisense effect at 3.2 fold of MIC value against MRSA/VISA Mu50, and its sequence specificity was demonstrated in that PPNA with scrambled PNA sequence (Scr PPNA2332) exhibited no growth inhibitory effect at higher concentrations. Also, PPNA2332 specifically interferes with rpoD mRNA, inhibiting translation of its protein product σ⁷⁰ in a concentration-dependent manner. Full decay of mRNA and suppressed expression of σ⁷⁰ were observed for 40 µM or 12.5 µM PPNA2332 treatment, respectively, but not for 40 µM Scr PPNA2332 treatment in pure culture of MRSA/VISA Mu50 strain. PPNA2332 (≥1 µM) essentially cleared lethal MRSA/VISA Mu50 infection in epithelial cell cultures, and eliminated viable bacterial cells in a time- and concentration- dependent manner, without showing any apparent toxicity at 10 µM. CONCLUSIONS: The present result suggested that RNAP primary σ⁷⁰ is a very promising candidate target for developing novel antisense antibiotic to treat severe MRSA infections

Common and Distant Structural Characteristics of Feruloyl Esterase Families from Aspergillus oryzae

Background: Feruloyl esterases (FAEs) are important biomass degrading accessory enzymes due to their capability of cleaving the ester links between hemicellulose and pectin to aromatic compounds of lignin, thus enhancing the accessibility of plant tissues to cellulolytic and hemicellulolytic enzymes. FAEs have gained increased attention in the area of biocatalytic transformations for the synthesis of value added compounds with medicinal and nutritional applications. Following the increasing attention on these enzymes, a novel descriptor based classification system has been proposed for FAEs resulting into 12 distinct families and pharmacophore models for three FAE sub-families have been developed. Methodology/Principal Findings: The feruloylome of Aspergillus oryzae contains 13 predicted FAEs belonging to six sub-families based on our recently developed descriptor-based classification system. The three-dimensional structures of the 13 FAEs were modeled for structural analysis of the feruloylome. The three genes coding for three enzymes, viz., A.O.2, A.O.8 and A.O.10 from the feruloylome of A. oryzae, representing sub-families with unknown functional features, were heterologously expressed in Pichia pastoris, characterized for substrate specificity and structural characterization through CD spectroscopy. Common feature-based pharamacophore models were developed according to substrate specificity characteristics of the three enzymes. The active site residues were identified for the three expressed FAEs by determining the titration curves of amino acid residues as a function of the pH by applying molecular simulations. Conclusions/Significance: Our findings on the structure-function relationships and substrate specificity of the FAEs of A. oryzae will be instrumental for further understanding of the FAE families in the novel classification system. The developed pharmacophore models could be applied for virtual screening of compound databases for short listing the putative substrates prior to docking studies or for post-processing docking results to remove false positives. Our study exemplifies how computational predictions can complement to the information obtained through experimental methods. © 2012 Udatha et al.published_or_final_versio

A novel method for calculating the energy cost of turning during running

Yoichi Hatamoto,1 Yosuke Yamada,2 Tatsuya Fujii,3 Yasuki Higaki,3 Akira Kiyonaga,3 Hiroaki Tanaka31Graduate School of Sports and Health Science, Fukuoka University, Nanakuma Jonan-ku Fukuoka, Japan; 2The Fukuoka University Institute for Physical Activity, Nanakuma Jonan-ku Fukuoka, Japan; 3Faculty of Sports and Health Science, Fukuoka University, Nanakuma Jonan-ku Fukuoka, JapanAbstract: Although changes of direction are one of the essential locomotor patterns in ball sports, the physiological demand of turning during running has not been previously investigated. We proposed a novel approach by which to evaluate the physiological demand of turning. The purposes of this study were to establish a method of measuring the energy expenditure (EE) of a 180&deg; turn during running and to investigate the effect of two different running speeds on the EE of a 180&deg; turn. Eleven young, male participants performed measurement sessions at two different running speeds (4.3 and 5.4 km/hour). Each measurement session consisted of five trials, and each trial had a different frequency of turns. At both running speeds, as the turn frequency increased the gross oxygen consumption (V &middot; O2) also increased linearly (4.3 km/hour, r = 0.973; 5.4 km/hour, r = 0.996). The V &middot; O2 of a turn at 5.4 km/hour (0.55 [SD 0.09] mL/kg) was higher than at 4.3 km/hour (0.34 [SD 0.13] mL/kg) (P < 0.001). We conclude that the gross V &middot; O2 of running at a fixed speed with turns is proportional to turn frequency and that the EE of a turn is different at different running speeds. The Different Frequency Accumulation Method is a useful tool for assessing the physiological demands of complex locomotor activity.Keywords: energy expenditure, turning, turn frequency, running speed, V &middot; O2, heart rat