Background. Infections with extended-spectrum β-lactamase–producing Escherichia coli (ESBL-EC) have developed resistance to current therapies. Therefore, the underlying mechanisms of in vivo and in vitro activity of C-terminal–amidated thanatin (A-thanatin) against clinical isolates of ESBL-EC were studied in an attempt to resolve this problem.

Methods. A-thanatin was synthesized to determine its minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and kill curve for ESBL-EC. The hemolytic toxicity, stability, and resistance induction of A-thanatin were determined. ESBL-EC–infected mice were used to determine the in vivo activity of A-thanatin. Scanning and transmission electron microscopy and fluorescence microscopy were used to study the underlying mechanism of A-thanatin.

Results. A-thanatin is highly effective against ESBL-EC in vitro, with MIC values ≤4 μg/mL. It has been confirmed that A-thanatin has little hemolysis and relative high stability in plasma. Excellent in vivo therapeutic effects were also observed in a septicemic animal model, with survival rates of 50.0%, 66.7%, and 91.7% in the low-dose, middle-dose, and high-dose groups, respectively. Membrane permeabilization may be a major biological action of A-thanatin.

Conclusions. Because the development of multidrug resistance limits the available therapeutic options, A-thanatin may provide a novel strategy for treating ESBL-EC infection and other infections due to multidrug-resistant bacteria.

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.

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is caused by the production of low-affinity penicillin-binding protein 2a and β-lactamases, which are encoded by mecA and blaZ, respectively. Expressions of the two key genes are mutually regulated by MecI and BlaI. The aim of this study was to design specific anti-mecR1 and anti-blaR1 deoxyribozymes and identify the restoration of susceptibility in MRSA isolates with mecI or blaI or no deletions by interfering with the mutual regulation of mecA and blaZ.

Material and methods

Specific deoxyribozymes were designed by using the program RNA structure 4.6. RNA substrates were obtained by transcription in vitro and used to assess the target cleavage of DNAzymes. Transcription of mecR1-mecA and blaR1-blaZ was analysed by real time RT-PCR. The susceptibility of MRSA was tested.

Results

Specific deoxyribozymes showed efficient catalytic activity to each own substrate mecR1 or blaR1 in vitro and caused the reduction of mecR1 and blaR1 transcription in vivo. Furthermore, simultaneous administration of two DNAzymes to knockdown mecR1 and blaR1 resulted in increased susceptibility of all MRSA strains tested in this study.

Conclusions

These results demonstrated that combined use of the two specific phosphorothioate deoxyribozymes could be a viable and promising strategy to restore the susceptibility of almost all MRSA clinical isolates.

Studies of rice protein expression have increased considerably with the development of rice functional genomics. In order to obtain reliable expression results in western blotting, information on appropriate reference proteins is necessary for data normalization. To date, no published study has identified and systematically validated reference proteins suitable for the investigation of rice protein expression. In this study, nine candidate proteins were selected and their specific antibodies were obtained through immunization of rabbits with either recombinant proteins expressed in Escherichia coli or synthesized peptides. Western blotting was carried out to detect the expression of target proteins in a set of 10 rice samples representing different rice tissues/organs at different developmental stages. The expression stability of the proteins was analysed using geNorm and Microcal Origin 6.0 software. The results indicated that heat shock protein (HSP) and elongation factor 1-α (eEF-1α) were the most constantly expressed among all rice proteins tested throughout all developmental stages, while the proteins encoded by conventional internal reference genes fluctuated in amount. Comparison among the profiling of translation and transcription [expressed sequence tags (EST) and massively parallel signature sequencing (MPSS)] revealed that a correlation existed. Based on the standard curves derived from the antigen–antibody reaction, the concentrations of HSP and eEF-1α proteins in rice leaves were ∼0.12%. Under the present experimental conditions, the lower limits of detection for HSP and eEF-1α proteins in rice were 0.24 ng and 0.06 ng, respectively. In conclusion, the reference proteins selected in this study, and the corresponding antibodies, can be used in qualitative and quantitative analysis of rice proteins.

The asymmetric unit of the title compound, [Zn(C12H12N2)(H2O)4]SO4, consists of a ZnII complex cation, a sulfate anion and four mol­ecules of water coordinated to the ZnII atom. The ZnII complex cation, with approximate twofold symmetry, displays a slightly distorted octa­hedral geometry around the ZnII atom, which is coordinated by two N atoms from a 5,5′-dimethyl-2,2′-bipyridine ligand and by the O atoms of four water mol­ecules. In the crystal, O—H⋯O hydrogen bonds help to establish the packing.

Cytokines secreted by infiltrating immune cells during atherogenesis modulate vascular remodeling. One exemplary event is the antagonism between transformed growth factor (TGF-β) and interferon gamma (IFN-γ) on the transcriptional control of type I collagen gene (COL1A2). Previously we have reported that IFN-γ up-regulates regulatory factor for X-box B (RFXB) to repress collagen transcription while down-regulates the expression of RFXBSV, a splice variant of RFXB that blocks collagen repression in fibroblasts. Here we demonstrate that TGF-β abrogated COL1A2 repression by IFN-γ through altering the relative expression of RFXB and RFXBSV. Unlike RFXB, RFXBSV did not bind to the collagen promoter and competed with RFXB for the co-repressor histone deacetylase 2 (HDAC2), limiting HDAC2 recruitment to the collagen transcription start site as evidenced by chromatin immunoprecipitation assays. Over-expression of RFXB by lentiviral infection in HASMCs enhanced HDAC2 enlistment, promoted histone deacetylation surrounding the collagen site by IFN-γ, and blocked the TGF-β antagonism, a pattern reversed by RFXBSV infection. On the contrary, silencing of RFXB, but not both RFXB and RFXBSV, expression promoted the TGF-β antagonism. Thus, we have identified a novel mechanism whereby TGF-β antagonizes the IFN-γ repression of collagen transcription in HASMCs and as such provided new insights into antiatherogenic strategies.

The title compound, {[Tb(C7H3NO4)(C7H4NO4)(H2O)2]·2H2O}n, is isotypic with the analogous TmIII compound [Li, Zhang, Wang & Bai (2009). Acta Cryst. E65, m411]. The TbIII atom is octa­coordinated by two water mol­ecules and by four carboxyl­ate O atoms and two pyridyl N atoms from two pyridine-2,5-dicarboxyl­ate (2,5-pydc) and two 6-carboxy­nicotinate (2,5-Hpydc) ligands. The 2,5-pydc and 2,5-Hpydc ligands bridge TbIII atoms, generating helical coordination polymers along [001]. An extensive network of O—H⋯O hydrogen bonds is formed between the coordination polymers and the uncoordinated water mol­ecules. The refined Flack parameter of 0.54 (2) suggests inversion twinning.