Impact of HMGB1 binding on the structural alterations of platinum drug-treated single dsDNA molecule.

High mobility group B1 (HMGB1) is an architectural protein that recognizes the DNA damage sites formed by the platinum anticancer drugs. However, the impact of HMGB1 binding on the structural alterations of the platinum drug-treated single dsDNA molecules has remained largely unknown. Herein, the structural alterations induced by the platinum drugs, the mononuclear cisplatin and it's analog the trinuclear BBR3464, have been probed in presence of HMGB1, by atomic force microscopy (AFM) and AFM-based force spectroscopy. It is observed that the drug-induced DNA loop formation enhanced upon HMGB1 binding, most likely as a result of HMGB1-induced increase in DNA conformational flexibility that allowed the drug-binding sites to come close and form double adducts, thereby resulting in enhanced loop formation via inter-helix cross-linking. Since HMGB1 enhances DNA flexibility, the near-reversible structural transitions as observed in the force-extension curves (for 1 h drug treatment), generally occurred at lower forces in presence of HMGB1. The DNA structural integrity was largely lost after 24 h drug treatment as no reversible transition could be observed. The Young's modulus of the dsDNA molecules, as estimated from the force-extension analysis, increased upon drug treatment, due to formation of the drug-induced covalent cross-links and consequent reduction in DNA flexibility. The Young's modulus increased further in presence of HMGB1 due to HMGB1-induced enhancement in DNA flexibility that could ease formation of the drug-induced covalent cross-links. To our knowledge, this is the first report that shows an increase in the stiffness of the platinum drug-treated DNA molecules in presence of HMGB1.

Searching for Blockers of Dengue and West Nile Virus Viroporins.

Flavivirus infections, such as those caused by dengue and West Nile viruses, emerge as new challenges for the global healthcare sector. It has been found that these two viruses encode ion channels collectively termed viroporins. Therefore, drug molecules that block such ion-channel activity can serve as potential antiviral agents and may play a primary role in therapeutic purposes. We screened 2839 FDA-approved drugs and compounds in advanced experimental phases using three bacteria-based channel assays to identify such ion channel blockers. We primarily followed a negative genetic screen in which the channel is harmful to the bacteria due to excessive membrane permeabilization that can be relieved by a blocker. Subsequently, we cross-checked the outcome with a positive genetic screen and a pH-dependent assay. The following drugs exhibited potential blocker activities: plerixafor, streptomycin, tranexamic acid, CI-1040, glecaprevir, kasugamycin, and mesna were effective against dengue virus DP1. In contrast, idasanutlin, benzbromarone, 5-azacytidine, and plerixafor were effective against West Nile Virus MgM. These drugs can serve as future antiviral therapeutic agents following subsequent in vitro and in vivo efficacy studies.

XNAs: A Troubleshooter for Nucleic Acid Sensing.

The strategies for nucleic acid sensing based on nucleic acid hybridization between the target sequence and the capture probe sequence are considered to be largely successful as far as detection of a specific target of known sequence is concerned. However, when compared with other complementary methods, like direct sequencing, a number of results are still found to be either "false positives" or "false negatives". This suggests that modifications in these strategies are necessary to make them more accurate. In this minireview, we propose that one way toward improvement could be replacement of the DNA capture probes with the xeno nucleic acid or XNA capture probes. This is because the XNAs, especially the locked nucleic acid, the peptide nucleic acid, and the morpholino, have shown better single nucleobase mismatch discrimination capacity than the DNA capture probes, indicating their capacity for more precise detection of nucleic acid sequences, which is beneficial for detection of gene stretches having point mutations. Keeping the current trend in mind, this minireview will include the recent developments in nanoscale, fluorescent label-free applications, and present the cases where the XNA probes show clear advantages over the DNA probes.

Molecularly resolved, label-free nucleic acid sensing at solid-liquid interface using non-ionic DNA analogues.

Nucleic acid-based biosensors, where the capture probe is a nucleic acid, e.g., DNA or its synthetic analogue xeno nucleic acid (XNA), offer interesting ways of eliciting clinically relevant information from hybridization/dehybridization signals. In this respect, the application of XNA probes is attractive since the drawbacks of DNA probes might be overcome. Within the XNA probe repertoire, peptide nucleic acid (PNA) and morpholino (MO) are promising since their backbones are non-ionic. Therefore, in the absence of electrostatic charge repulsion between the capture probe and the target nucleic acid, a stable duplex can be formed. In addition, these are nuclease-resistant probes. Herein, we have tested the molecularly resolved nucleic acid sensing capacity of PNA and MO capture probes using a fluorescent label-free single molecule force spectroscopy approach. As far as single nucleobase mismatch discrimination is concerned, both PNA and MO performed better than DNA, while the performance of the MO probe was the best. We propose that the conformationally more rigid backbone of MO, compared to the conformationally flexible PNA, is an advantage for MO, since the probe orientation can be made more upright on the surface and therefore MO can be more effectively accessed by the target sequences. The performance of the XNA probes has been compared to that of the DNA probe, using fixed nucleobase sequences, so that the effect of backbone variation could be investigated. To our knowledge, this is the first report on molecularly resolved nucleic acid sensing by non-ionic capture probes, here, MO and PNA.

Free-Energy-Based Gene Mutation Detection Using LNA Probes.

We have developed a label-free approach for direct detection of gene mutations using free-energy values that are derived from single-molecule force spectroscopy (SMFS)-based nucleic acid unbinding experiments. From the duplex unbinding force values acquired by SMFS, the force-loading-rate-independent Gibbs free-energy values were derived using Jarzinsky's equality treatment. Because it provides molecule-by-molecule information, this approach is a major shift compared to the earlier reports on label-free detection of DNA sequences, which are mostly based on ensemble level data. We tested our approach in the disease model framework of multiple drug-resistant tuberculosis using the nuclease-resistant and conformationally rigid locked nucleic acid probes that are a robust and efficient alternative to the DNA probes. All of the major mutations in Mycobacterium tuberculosis (MTB), as relevant to MTB's resistance to the first-line anti-TB drugs rifampicin and isoniazid, could be identified, and the wild type could be discriminated from the most prevalent mutation and the most prevalent mutation from the less occurring ones. Our approach could also identify DNA sequences (45 mer), having overhang stretches at different positions with respect to the complementary stretch. Probably for the first time, the findings show that free-energy-based detection of gene mutations is possible at molecular resolution.

Nanoscale Nucleic Acid Recognition at the Solid-Liquid Interface Using Xeno Nucleic Acid Probes.

Challenges in reliable nucleic acid detection are manifold. The major ones are related to false positive or negative signals due to a lack of target specificity in detection and to low sensitivity, especially when a plethora of background sequences are present that can mask the specific recognition signal. Utilizing designed synthetic nucleic acids that are commonly called xeno nucleic acids could offer potential routes to meeting such challenges. In this article, we present the general framework of nucleic acid detection, especially for nanoscale applications, and discuss how and why the xeno nucleic acids could be truly an alternative to the DNA probes. Two specific cases, locked nucleic acid (LNA) and peptide nucleic acid (PNA), which are nuclease-resistant and can form thermally stable duplexes with DNA, are addressed. It is shown that the relative ease of the conformationally rigid LNA probe to be oriented upright on the substrate surface and of the nonionic PNA probe to result into high probe density assists in their use in nanoscale nucleic acid recognition. It is anticipated that success with these probes may lead to important developments such as PCR-independent approaches where the major aim is to detect a small number of target sequences present in the analyte medium.

Discriminating unalike single nucleobase mismatches using a molecularly resolved, label-free, interfacial LNA-based assay.

A number of reports have been made in recent times on label-free detection of nucleic acid sequences. However, most of these studies deal with ensemble measurements, therefore lacking in molecular level resolution. These assays have usually employed ssDNA sensor probes, and often suffered from problems of irreproducibility and poor sequence-selectivity. Herein, the applicability of surface-anchored single stranded locked nucleic acid (ssLNA) probes has been assessed in the detection of target DNA sequences, as an alternative to the DNA-based assay. Importantly, the effectiveness of the LNA-based assay in identifying different types of single nucleobase mismatches has been tested. Since the duplex melting temperature is an indicator of duplex stability, the ensemble on-surface Tm values of the surface-confined LNA-DNA duplexes have been compared to the duplex unbinding force values obtained from atomic force spectroscopy (AFS) experiments. A common mismatch discrimination pattern elicited by both the ensemble and the molecular level AFS approach could be identified. Apart from quantitative delineation of the different types of mismatches, the label-free AFS analysis confirms different degrees of efficiency of the purine and pyrimidine bases, present on the LNA backbone, in discriminating different nucleobase mismatch types. Importantly, the LNA-based AFS analysis can distinguish between the disease-relevant gene fragments, e.g., multidrug-resistant Mycobacterium tuberculosis (MTB) mutation, and the wild type. Since LNA probes are nuclease-resistant, these findings could potentially pave way to diagnostic applications of the LNA-based AFS assay.

Molecularly resolved label-free sensing of single nucleobase mismatches by interfacial LNA probes.

So far, there has been no report on molecularly resolved discrimination of single nucleobase mismatches using surface-confined single stranded locked nucleic acid (ssLNA) probes. Herein, it is exemplified using a label-independent force-sensing approach that an optimal coverage of 12-mer ssLNA sensor probes formed onto gold(111) surface allows recognition of ssDNA targets with twice stronger force sensitivity than 12-mer ssDNA sensor probes. The force distributions are reproducible and the molecule-by-molecule force measurements are largely in agreement with ensemble on-surface melting temperature data. Importantly, the molecularly resolved detection is responsive to the presence of single nucleobase mismatches in target sequences. Since the labelling steps can be eliminated from protocol, and each force-based detection event occurs within milliseconds' time scale, the force-sensing assay is potentially capable of rapid detection. The LNA probe performance is indicative of versatility in terms of substrate choice - be it gold (for basic research and array-based applications) or silicon (for 'lab-on-a-chip' type devices). The nucleic acid microarray technologies could therefore be generally benefited by adopting the LNA films, in place of DNA. Since LNA is nuclease-resistant, unlike DNA, and the LNA-based assay is sensitive to single nucleobase mismatches, the possibilities for label-free in vitro rapid diagnostics based on the LNA probes may be explored.

Protective effect of antioxidant rich aqueous curry leaf (Murraya koenigii) extract against gastro-toxic effects of piroxicam in male Wistar rats.

Piroxicam (chemically 4-hydroxy-2-methyl-N-2-pyridinyl-2H-1,2-benzothiazine-3-carboxamide), a classical non-steroidal anti-inflammatory drug (NSAID) is orally administered to arthritic patients. Inhibition of prostaglandin E2 (PGE2) synthesis and subsequent free hydroxyl radical generation in vivo exert gastro-toxic side effects on piroxicam treatment. Leaves of curry plant are rich in antioxidants with prolific free radical scavenging activities. This led us to investigate the efficiency of the use of curry leaves in ameliorating piroxicam induced gastric damage. Piroxicam was orally (30 mg per kg body weight) administered in male albino Wistar rats to generate gastric ulcers. These rats were orally fed with graded doses of aqueous extract of curry or Murraya koenigii leaves (Cu LE) prior to piroxicam administration. Oxidative stress biomarkers, activities of antioxidant and pro-oxidant enzymes, mucin content and nature, PGE2 level, activities of mitochondrial enzymes and histomorphology of gastric tissues were studied. Piroxicam treatment altered all the above mentioned parameters whereas, curry leaf extract pre-treated animals were protected against piroxicam induced alterations. Hence, the protective action of the antioxidant rich Cu LE was investigated to propose a new combination therapy or dietary management to arthritic patients using piroxicam.