A reliable, quick and universally applicable method for monitoring aptamer SELEX progress.

Nucleic acid aptamers for biosensing are developed from a complex ssDNA library through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. Monitoring of SELEX process is crucial for generating high-affinity aptamers. Extant methods for monitoring aptamer selection are either arduous or give false-positive signals, which adversely impact the outcome of selection. We describe a colorimetric, simple and cost-effective, novel method to monitor the progress of in vitro selections. The power of rolling circle amplification (RCA) and inherent Horse Radish Peroxidase (HRP)-mimicking activity of G-quadruplex/hemin DNAzyme were employed to produce a colorimetric signal. A unique extension of DNA population at 3'-OH end by PCR generated concatenated repeats by rolling circle amplification (RCA) reaction. Oxidation of substrate ABTS (2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) in presence of H2O2 and hemin cofactor produced colorimetric signal. Analysis of the signal generated by the DNA pool bound to their target provided a quantitative measurement of SELEX. We demonstrate the reproducibility and accuracy of the method by evaluating the progress of two discrete selections.

Antibacterial properties and in vivo efficacy of a novel nitrofuran, IITR06144, against MDR pathogens.

OBJECTIVES: The emergence of MDR Gram-negative pathogens and increasing prevalence of chronic infections presents an unmet need for the discovery of novel antibacterial agents. The aim of this study was to evaluate the biological properties of a small molecule, IITR06144, identified in a phenotypic screen against the Gram-negative model organism Escherichia coli. METHODS: A small-molecule library of 10956 compounds was screened for growth inhibition against E. coli ATCC 25922 at concentration 50 µM. MICs of lead compounds were determined by the broth microdilution method. Time-kill kinetics, anti-persister activity, spontaneous frequency of resistance, biofilm inhibition and disruption were assessed by standard protocols. Resistant mutants were generated by serial passaging followed by WGS. In vitro toxicity studies were carried out via the MTT assay. In vivo toxicity and efficacy in a mouse model were also evaluated. RESULTS: IITR06144 was identified as the most promising candidate amongst 29 other potential antibacterial leads, exhibiting the lowest MIC, 0.5 mg/L. IITR06144 belongs to the nitrofuran class and exhibited broad-spectrum bactericidal activity against most MDR bacteria, including the 'priority pathogen', carbapenem-resistant Acinetobacter baumannii. IITR06144 retained its potency against nitrofurantoin-resistant clinical isolates. It displayed anti-persister, anti-biofilm activity and lack of spontaneous resistance development. IITR06144 demonstrated a large therapeutic index with no associated in vitro and in vivo toxicity. CONCLUSIONS: In the light of excellent in vitro properties displayed by IITR06144 coupled with its considerable in vivo efficacy, further evaluation of IITR06144 as a therapeutic lead against antibiotic-resistant infections is warranted.

Characterization of difference in structure and function of fresh and mastitic bovine milk fat globules.

Characterization of milk fat globule (MFG) was performed to investigate the difference in MFG membrane (MFGM) between fresh and mastitis Holstein Friesian cow milk. Lipid distribution investigated by exogenous phospholipids using microscopy showed higher phospholipid content in fresh compared to mastitic MFGM. Xanthine oxidase assay indicative of membrane impairment revealed lower activity in mastitic samples compared to fresh globules. Of note, significantly higher roughness of globule surface and zeta potential was observed in mastitis compared to fresh globules. Influence of globule membrane on the interaction with L. fermentum demonstrated preferential adhesion of bacteria to fresh compared to mastitic globules including enhanced extent of binding. Results of the present study provides an insight of the interfacial changes occurring at the globule surface as well as highlighting the importance of selective bacterial interaction with milk components for the potential development of functional food with relevance to human health.

A study on metabolic prowess of Pseudomonas sp. RPT 52 to degrade imidacloprid, endosulfan and coragen.

A bacterial strain identified as Pseudomonas sp. RPT 52, was isolated from an agricultural field by soil enrichment technique. The bacterial strain was able to metabolize three different chlorinated pesticides; imidacloprid, endosulfan and coragen (belonging to neonicotinoid, organochlorine and anthranillic diamide categories, respectively). RPT 52 was able to degrade 46.5%, 96.6%, 92.7% and 80.16% of 0.5 mM of imidacloprid, endosulfan α, endosulfan ß and coragen, respectively, in minimal medium over a period of 40 h, when provided as sole source of carbon and energy. Degradation kinetics showed that imidacloprid, endosulfan α and endosulfan ß followed first order kinetics whereas coragen followed zero order kinetics. Toxicity studies show reduction in toxicity of the parent compound when degraded by RPT 52. Laboratory scale, soil microcosm studies showed that strain RPT 52 is a suitable candidate for bioremediation of endosulfan and coragen contaminated sites. Thus, RPT 52 holds potential for toxicity reduction in the affected environment.

Synthesis, characterization and bactericidal activity of silica/silver core-shell nanoparticles.

Silica/silver core-shell nanoparticles (NPs) were synthesized by coating silver NPs on silica core particles (size ~300 ± 10 nm) via electro less reduction method. The core-shell NPs were characterized for their structural, morphological, compositional and optical behavior using X-ray diffraction, scanning electron microscopy, energy dispersive X-ray analysis and UV-Visible spectroscopy, respectively. The size (16-35 nm) and loaded amount of silver NPs on the silica core were found to be dependent upon reaction time and activation method of silica. The bactericidal activity of the NPs was tested by broth micro dilution method against both Bacillus subtilis (gram positive) and Escherichia coli ATCC25922 (gram negative) bacterium. The bactericidal activity of silica/silver core-shell NPS is more against E. coli ATCC25922, when compared to B. subtilis. The minimal inhibitory concentration of the core-shell NPs ranged from 7.8 to 250 µg/mL and is found to be dependent upon the amount of silver on silica, the core. These results suggest that silica/silver core-shell NPs can be utilized as a strong substitutional candidate to control pathogenic bacterium, which are otherwise resistant to antibiotics, making them applicable in diverse medical devices.

In vitro selection of protein-binding DNA aptamers as ligands for biosensing applications.

Aptamers are single-stranded functional nucleic acids that possess cognate ligand recognition capability. These functional nucleic acids have been used for biosensing of a variety of ligands. Aptamers are isolated by "in vitro selection" or SELEX from random-sequence nucleic acid pools. For example, DNA aptamers that recognize a protein can be generated by applying a DNA library to an affinity column containing the protein target and retrieving the bound sequences after wash. These sequences are amplified and used for a new round of binding and amplification. The identity of enriched sequences are subsequently revealed by cloning and sequencing. The binding of individual aptamers to the protein can be confirmed by techniques such as gel mobility shift. This chapter will provide a detailed protocol for isolating protein-binding DNA aptamers.

Nucleic acid aptamers and enzymes as sensors.

The function of nucleic acids has been an endless source of discovery and invention that has drastically enhanced our appreciation of DNA and RNA as multifaceted polymers. It is now widely known that nucleic acids can act as enzymes (deoxyribozymes and ribozymes) and as receptors (aptamers), and that these functional nucleic acids (FNAs) can either be found in nature or isolated from pools of random nucleic acids. The availability of many natural and artificial FNAs has opened a new horizon for the development of 'smart' molecules for a variety of chemical and biological applications. This review provides a snapshot of recent progress in the application of FNAs as novel sensors for biomolecular detection, drug discovery and nanotechnology.

Nitric oxide scavenging and detoxification by the Mycobacterium tuberculosis haemoglobin, HbN in Escherichia coli.

Nitric oxide (NO), generated in large amounts within the macrophages, controls and restricts the growth of internalized human pathogen, Mycobacterium tuberculosis H37Rv. The molecular mechanism by which tubercle bacilli survive within macrophages is currently of intense interest. In this work, we have demonstrated that dimeric haemoglobin, HbN, from M. tuberculosis exhibits distinct nitric oxide dioxygenase (NOD) activity and protects growth and cellular respiration of heterologous hosts, Escherichia coli and Mycobacterium smegmatis, from the toxic effect of exogenous NO and the NO-releasing compounds. A flavohaemoglobin (HMP)-deficient mutant of E. coli, unable to metabolize NO, acquired an oxygen-dependent NO consumption activity in the presence of HbN. On the basis of cellular haem content, the specific NOD activity of HbN was nearly 35-fold higher than the single-domain Vitreoscilla haemoglobin (VHb) but was sevenfold lower than the two-domain flavohaemoglobin. HbN-dependent NO consumption was sustained with repeated addition of NO, demonstrating that HbN is catalytically reduced within E. coli. Aerobic growth and respiration of a flavohaemoglobin (HMP) mutant of E. coli was inhibited in the presence of exogenous NO but remained insensitive to NO inhibition when these cells produced HbN, VHb or flavohaemoglobin. M. smegmatis, carrying a native HbN very similar to M. tuberculosis HbN, exhibited a 7.5-fold increase in NO uptake when exposed to gaseous NO, suggesting NO-induced NOD activity in these cells. In addition, expression of plasmid-encoded HbN of M. tuberculosis in M. smegmatis resulted in 100-fold higher NO consumption activity than the isogenic control cells. These results provide strong experimental evidence in support of NO scavenging and detoxification function for the M. tuberculosis HbN. The catalytic NO scavenging by HbN may be highly advantageous for the survival of tubercle bacilli during infection and pathogenesis.

Mycobacterium tuberculosis hemoglobin HbO associates with membranes and stimulates cellular respiration of recombinant Escherichia coli.

The truncated hemoglobins HbN and HbO of Mycobacterium tuberculosis H37Rv share little sequence similarity and display structural differences in their EF-loop regions, suggesting distinct function(s) for these hemoglobins. HbO of M. tuberculosis was expressed in Escherichia coli and Mycobacterium smegmatis as a 14.5-kDa homodimeric heme protein exhibiting nearly 50-fold (P(50) approximately 0.51) lower oxygen affinity than HbN. 40-50% of HbO remained associated with the cell membranes and significantly enhanced its respiration in comparison with the membrane fractions of control cells or cells overproducing HbN. Oxygen uptake of HbO-associated membranes was decreased by washing and restored by adding HbO. Additionally, membrane vesicles prepared from terminal oxidase-deficient (cyo(-), cyd(-)) mutants of E. coli did not exhibit significant enhancement in oxygen uptake in the presence of HbO, suggesting its interaction(s) with the electron transport chain. Expression of HbO in Mycobacterium bovis bacillus Calmette-Guérin, an experimental model of M. tuberculosis, was observed (0.2-0.5% of total cellular proteins) throughout its aerobic growth. These results provided evidence for the involvement of HbO with the component of aerobic electron transport chain, suggesting that its function may be related to the facilitation of oxygen transfer during aerobic metabolism of M. tuberculosis. Membrane association properties of HbO may thus play a crucial role in sequestering oxygen and facilitating its availability to internalized M. tuberculosis (an obligate aerobe) under the hypoxic conditions of its intracellular habitat.