Synthesis and antibacterial property of quinolines with potent DNA gyrase activity

Synthesis of a series of novel tetrahydroquinoline annulated heterocycles has been accomplished by intramolecular imino and bisimino Diels–Alder reaction. These compounds were evaluated for their antibacterial activity. All the synthetic compounds, exhibited good antibacterial activity against microorganisms of which one of them 7 was found to be as active as the antibiotic ciplofloxacin and is found to have MIC value of 2.5 mg/mL against Escherichia coli.     

Impact of DNA gyrase inhibition by antisense ribozymes on rec A in <Emphasis Type="Italic">E. coli</Emphasis>

The chromosome of E. coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. Regulation of DNA supercoiling yields a complex spectrum of effects on the E. coli recA system. Previous studies indicated that inhibition of DNA gyrase by antibiotics that act on the DNA gyrase A subunit results in turning on the recA system. Here we show that antisense ribozymes that act on the DNA gyrase A subunit can also induce recA. We used real time PCR and immunoblot to analyze the impact of DNA gyrase A inhibition by antisense ribozymes on recA expression. When gyrase A was inhibited by the RNase P mediated antisense ribozymes the expression of recA was induced around 130-fold as seen by real time PCR analysis. This suggests that repair pathway is induced by antisense ribozymes against DNA gyrase A and the damage produced by these ribozymes may be similar to that produced by fluroquinolones.     

Gamma-phage lysin PlyG sequence-based synthetic peptides coupled with Qdot-nanocrystals are useful for developing detection methods for Bacillus anthracis by using its surrogates, B. anthracis-Sterne and B. cereus-4342

Background  

Previous reports of site-directed deletion analysis on gamma (γ)-phage lysin protein (PlyG) have demonstrated that removal of a short amino acid sequence in the C-terminal region encompassing a 10-amino acid motif (190LKMTADFILQ199) abrogates its binding activity specific to the cell wall of Bacillus anthracis. Whether short synthetic peptides representing the10-amino acid PlyG putative binding motif flanked by surrounding N- and C-terminal residues also selectively bind to the bacterial cell wall has not been evaluated. If such peptides do demonstrate selective binding to the cell wall, they could serve as bio-probes towards developing detection technologies for B. anthracis. Furthermore, by using B. anthracis (Sterne, 34F2), an animal vaccine and B. cereus-4342, a γ-phage susceptible rare strain as surrogates of B. anthracis, development of proof-of-concepts for B. anthracis are feasible.     

Detection technologies for Bacillus anthracis: Prospects and challenges

Bacillus anthracis is a Gram-positive, spore-forming bacterium representing the etiological agent of acute infectious disease anthrax, a lethal but rare disease of animals and humans in nature. With recent use of anthrax as a bioweapon, a number of techniques have been recently developed and evaluated to facilitate its rapid detection of B. anthracis in the environment as well as in point-of-care settings for humans suspected of exposure to the pathogen. Complex laboratory methods for B. anthracis identification are required since B. anthracis has similarities with other Bacillus species and its existence in both spore and vegetative forms. This review discusses current challenges and various improvements associated with anthrax agent detection.     

Peptides panned from a phage-displayed random peptide library are useful for the detection of Bacillus anthracis surrogates B. cereus 4342 and B. anthracis Sterne

Recent use of Bacillus anthracis as a bioweapon has highlighted the need for a sensitive monitoring system. Current bacterial detection tests use antibodies as bio-molecular recognition elements which have limitations with regard to time, specificity and sensitivity, creating the need for new and improved cost-effective high-affinity detection probes. In this study, we screened a commercially available bacteriophage-displayed random peptide library using Bacillus cereus 4342 cells as bait to identify peptides that could be used for detection of Bacillus. The method enabled us to identify two 12-amino acid consensus peptide sequences that specifically bind to B. cereus 4342 and B. anthracis Sterne, the nonpathogenic surrogates of B. anthracis strain. The two Bacillus-binding peptides (named BBP-1 and BBP-2) were synthesized with biotin tag to confirm their binding by four independent detection assays. Dot-blot analysis revealed that the peptides bind specifically to B. cereus 4342 and B. anthracis Sterne. Quantitative analysis of this interaction by ELISA and fluorometry demonstrated a detection sensitivity of 10² colony forming U/ml (CFU/ml) by both assays. When the peptides were used in combination with Qdots, the sensitivity was enhanced further by enabling detection of even a single bacterium by fluorescence microscopy. Immunoblot analysis and protein sequencing showed that BBP-1 and BBP-2 bound to the S-layer protein of B. anthracis Sterne. Overall, our findings validate the usefulness of synthetic versions of phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity bioprobes for various microbial detection platforms.     

Identification of XMRV infection-associated microRNAs in four cell types in culture

Introduction

XMRV is a gammaretrovirus that was thought to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS) in humans until recently. The virus is culturable in various cells of human origin like the lymphocytes, NK cells, neuronal cells, and prostate cell lines. MicroRNAs (miRNA), which regulate gene expression, were so far not identified in cells infected with XMRV in culture.

Methods

Two prostate cell lines (LNCaP and DU145) and two primary cells, Peripheral Blood Lymphocytes [PBL] and Monocyte-derived Macrophages [MDM] were infected with XMRV. Total mRNA was extracted from mock- and virus-infected cells at 6, 24 and 48 hours post infection and evaluated for microRNA profile in a microarray.

Results

MicroRNA expression profiles of XMRV-infected continuous prostate cancer cell lines differ from that of virus-infected primary cells (PBL and MDMs). miR-193a-3p and miRPlus-E1245 observed to be specific to XMRV infection in all 4 cell types. While miR-193a-3p levels were down regulated miRPlus-E1245 on the other hand exhibited varied expression profile between the 4 cell types.

Discussion

The present study clearly demonstrates that cellular microRNAs are expressed during XMRV infection of human cells and this is the first report demonstrating the regulation of miR193a-3p and miRPlus-E1245 during XMRV infection in four different human cell types.     

Down regulation of gyrase A gene expression in E. coli by antisense ribozymes using RT-PCR

Nucleic acid-based gene interference technologies represent promising strategies for specific inhibition of mRNA sequences of choice. Recently, small interfering RNAs have been implicated in inducing endogenous RNase of the RNA-induced silencing complex in the RNA interference pathway to inhibit gene expression and growth of several human viruses. We report down regulation of gene expression of E. coli gyrase A, an essential gene for DNA supercoiling and antibiotic susceptibility in BL21 (DE3) strain of E. coli, using Ribonuclease P based external guide sequence (EGS) technique. EGS directed against gyrase A gene that was cloned into pUC vector, which contains the ampicillin (Amp) resistance gene. The recombinant plasmid pT7EGyrA was transformed into BL21 (DE3) and inductions were performed using IPTG. RT-PCR experiment was done to investigate the down regulation of gyrase A gene. RT-PCR results demonstrated a significant decrease of gyrase A gene after 18 h of induction of the transformants. These experiments showed that the down regulation of the gene was seen after 18 h of induction than earlier hours of induction with IPTG suggesting inhibition of gyrase A gene with profound effect on cell viability. These results demonstrate the utility of EGS RNAs in gene therapy applications, by inhibiting the expression of essential proteins.     

Identification and evaluation of a novel peptide binding to the cell surface of Staphylococcus aureus

Identification of short peptides that serve as specific ligands to biological materials such as microbial cell surfaces has major implications in better understanding the molecular recognition of cell surfaces. In this study we screened a commercially available random phage-display library against Staphylococcus aureus cells and identified peptides specifically binding to the bacteria. A synthetic peptide (SA5-1) representing the consensus sequence (VPHNPGLISLQG) of the bacteria-binding peptide was evaluated for its binding potential against S. aureus. Dot-blot, immunoblot assay and ELISA results revealed the SA5-1 peptide to be highly specific to S. aureus. The SA5-1 peptide binding was optimal between pH 6.0 and 8.0. Nanogold Transmission Electron Microscopy demonstrated that the SA5-1 binds to the outer membrane surface of S. aureus. Diagnostic potential of the SA5-1 peptide was evaluated in human platelet samples spiked with S. aureus and specific detection of the bacteria by biotinylated-SA5-1 and streptavidin-conjugated fluorescent quantum dots. Fluorometry results indicated that the peptide was able to detect ～100 organisms per ml in a spiked biological sample providing a proof-of-concept towards potential of this peptide as a S. aureus diagnostic tool that can be of use in different detection platforms.