Design and development of topoisomerase inhibitors using molecular modelling studies

Topoisomerase inhibitors are used as anticancer and antibacterial agents. A series of novel 2,4,6-tri-substituted pyridine derivatives reported as topoisomerase inhibitors were used for quantitative structure–activity relationship (QSAR) study. In order to understand the structural requirement of these topoisomerase inhibitors, a ligand-based pharmacophore and atom-based 3D-QSAR model have been developed. A five-point pharmacophore with one hydrophobic group (H4), four aromatic rings (R5, R6, R7 and R8) was obtained. The pharmacophore hypothesis yielded a 3D-QSAR model with good partial least-square (PLS) statistic results. The training set correlation is characterized by PLS factors (r² = 0.7892, SD = 0.2948, F = 49.9, P = 1.379). The test set correlation is characterized by PLS factors (q² = 0.7776, root mean squared error = 0.2764, Pearson R = 0.8926). The docking study revealed the binding orientations of these inhibitors at active site amino acid residues of topoisomerases enzyme. The results of pharmacophore hypothesis and 3D-QSAR provided the detail structural insights as well as highlighted the important binding features of novel 2,4,6-tri-substituted pyridine derivatives and can be developed as potent topoisomerase inhibitors.

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Open in a separate windowKey structural requirement for topoisomerase activity     

Comprehensive Mutation Analysis for Congenital Muscular Dystrophy: A Clinical PCR-Based Enrichment and Next-Generation Sequencing Panel

The congenital muscular dystrophies (CMDs) comprise a heterogeneous group of heritable muscle disorders with often difficult to interpret muscle pathology, making them challenging to diagnose. Serial Sanger sequencing of suspected CMD genes, while the current molecular diagnostic method of choice, can be slow and expensive. A comprehensive panel test for simultaneous screening of mutations in all known CMD-associated genes would be a more effective diagnostic strategy. Thus, the CMDs are a model disorder group for development and validation of next-generation sequencing (NGS) strategies for diagnostic and clinical care applications. Using a highly multiplexed PCR-based target enrichment method (RainDance) in conjunction with NGS, we performed mutation detection in all CMD genes of 26 samples and compared the results with Sanger sequencing. The RainDance NGS panel showed great consistency in coverage depth, on-target efficiency, versatility of mutation detection, and genotype concordance with Sanger sequencing, demonstrating the test''s appropriateness for clinical use. Compared to single tests, a higher diagnostic yield was observed by panel implementation. The panel''s limitation is the amplification failure of select gene-specific exons which require Sanger sequencing for test completion. Successful validation and application of the CMD NGS panel to improve the diagnostic yield in a clinical laboratory was shown.     

LASIK following Small Incision Lenticule Extraction (SMILE) Lenticule Re-Implantation: A Feasibility Study of a Novel Method for Treatment of Presbyopia

Presbyopia remains a major visual impairment for patients, who have previously undergone laser refractive correction and enjoyed unaided distance vision prior to the onset of presbyopia. Corneal stromal volume restoration through small incision lenticule extraction (SMILE) lenticule re-implantation presents an opportunity for restoring the patients’ non-dominant eye to previous low myopia to achieve a monovision. In this study, we investigated the feasibility of performing LASIK after lenticule re-implantation as a method to create presbyopic monovision. A -6.00D SMILE correction was performed in 9 rabbit eyes. The lenticules were cryopreserved for 14 days and re-implanted. Five weeks later, 3 of these eyes underwent LASIK for -5.00D correction (RL group); 3 underwent LASIK flap creation, which was not lifted (RN); and no further procedures were performed on the remaining 3 eyes. These groups were compared with 3 eyes that underwent standard LASIK for a -5.00D correction (LO); 3 that underwent creation of non-lifted flap (LN); and 3 non-operated eyes. Rabbits were euthanized 1 day post-surgery. Tissue responses were analyzed by immunohistochemistry, slit lamp and in vivo confocal microscopy (IVCM). Intrastromal irregularities and elevated reflectivity levels of the excimer-ablated plane were observed on slit lamp and IVCM, respectively in the RL group. The results were comparable (P = 0.310) to IVCM findings in the LO group. RL and LO groups showed similar fibronectin expression levels, number of CD11b-positive cells (P = 0.304) and apoptotic cells (P = 0.198). There was no difference between the RN and LN groups in reflectivity levels (P = 0.627), fibronectin expression levels, CD11b-positive cells (P = 0.135) and apoptotic cells (P = 0.128). LASIK can be performed following lenticule re-implantation to create presbyopic monovision. The tissue responses elicited after performing LASIK on corneas that have undergone SMILE and subsequent lenticule re-implantation are similar to primary procedure.     

Does soil organic carbon quality or quantity govern relative temperature sensitivity in soil aggregates?

Soil aggregates govern soil organic carbon (SOC) sequestration. But, sparse understanding about the process leads to inaccuracy in predicting potential of soil to stabilize C in warming world. We appraised effects of 43 years of fertilization on relative temperature sensitivity of SOC decomposition (Q₁₀) in soil aggregates to know whether SOC quality or quantity governs Q₁₀. Treatments were: fallow, control, 100% recommended dose of nitrogen (N), N and phosphorus (NP), N, P and potassium (NPK), and NPK + farmyard manure (FYM) (NPK + FYM). Macroaggregates, microaggregates and silt + clay (s + c) fractions were incubated for 16 weeks at 25, 35 and 45 °C, SOC quality (R₀) and Q₁₀ were computed. SOC mineralization from macro- and micro- aggregates were 34 and 28% higher than s + c across the treatments. The s + c fraction of NPK + FYM had ~ 41, 40 and 24% higher C decay rate than NPK plots at 25, 35 and 45 °C, respectively. For s + c fraction Q₁₀ increased over other aggregates. Mean Q₁₀ of s + c fraction was ~ 18.3 and 17.5% higher than macro and micro-aggregate-C, respectively. R₀ was the lowest for NPK + FYM, suggesting long-term manuring with balanced NPK significantly enhance recalcitrance of C. We observed Q₁₀ of macroaggregates and s + c fraction is controlled by C quality but C quantity governs Q₁₀ of microaggregates in Vertisol. Specifically, microaggregates of NPK + FYM were more temperature sensitive, and could be vulnerable to C loss. Hence, practices facilitating microaggregate formation should be avoided. Thus, we recommend manure application for facilitating C sequestration.

     

Isolation and characterization of rhizobacteria associated with coastal agricultural ecosystem of rhizosphere soils of cultivated vegetable crops

In this study, a total of 80 rhizobacteria was isolated from coastal agricultural ecosystem of cultivated vegetable rhizosphere soils. The isolates were screened for antagonistic activity against Sclerotium rolfsii and Colletotrichum capsici and plant growth promoting traits. The results revealed that 15.0 and 43.7% isolates showed statistically significant inhibition of mycelial growth of S. rolfsii and C. capsici respectively, while 48.7% isolates produced siderophore, 57.5% isolates solubilized phosphate and 21.1% isolates produced indole-3-acetic acid more than 20 μg/mL. However, only three isolates PfS1, PfR2 and BL5 were found positive to all properties tested. The identification of potential bacterial isolates through Microbial Identification System (BIOLOG) and 16S rDNA sequencing of the isolates revealed Bacillus species were dominant in the cultivated vegetable rhizosphere soil of Neil and Havelock Islands, India.     

Exoribonuclease R in Pseudomonas syringae is essential for growth at low temperature and plays a novel role in the 3' end processing of 16 and 5 S ribosomal RNA

The (3'-->5') exoribonuclease RNase R interacts with the endoribonuclease RNase E in the degradosome of the cold-adapted bacterium Pseudomonas syringae Lz4W. We now present evidence that the RNase R is essential for growth of the organism at low temperature (4 degrees C). Mutants of P. syringae with inactivated rnr gene (encoding RNase R) are cold-sensitive and die upon incubation at 4 degrees C, a phenotype that can be complemented by expressing RNase R in trans. Overexpressing polyribonucleotide phosphorylase in the rnr mutant does not rescue the cold sensitivity. This is different from the situation in Escherichia coli, where rnr mutants show normal growth, but pnp (encoding polyribonucleotide phosphorylase) and rnr double mutants are nonviable. Interestingly, RNase R is not cold-inducible in P. syringae. Remarkably, however, rnr mutants of P. syringae at low temperature (4 degrees C) accumulate 16 and 5 S ribosomal RNA (rRNA) that contain untrimmed extra ribonucleotide residues at the 3' ends. This suggests a novel role for RNase R in the rRNA 3' end processing. Unprocessed 16 S rRNA accumulates in the polysome population, which correlates with the inefficient protein synthesis ability of mutant. An additional role of RNase R in the turnover of transfer-messenger RNA was identified from our observation that the rnr mutant accumulates transfer-messenger RNA fragments in the bacterium at 4 degrees C. Taken together our results establish that the processive RNase R is crucial for RNA metabolism at low temperature in the cold-adapted Antarctic P. syringae.     

Antiproliferative and apoptosis inducing properties of pyrano[3,2-c]pyridones accessible by a one-step multicomponent synthesis

4-Arylpyrano-[3,2-c]-pyridones have been prepared by a one-step cyclocondensation of 4-hydroxy-1,6-dimethylpyridin-2(1H)-one with various substituted benzaldehydes and malononitrile. These heterocycles exhibit micromolar and submicromolar antiproliferative activity in HeLa and induce apoptosis in Jurkat cell lines. Structure-activity studies performed on a small library of these compounds show a pronounced cytotoxicity enhancing effect of the bromo substituent at the meta position of the C4 aromatic moiety.     

H2O2 Inhibits ABA-Signaling Protein Phosphatase HAB1

Due to its ability to be rapidly generated and propagated over long distances, H₂O₂ is an important second messenger for biotic and abiotic stress signaling in plants. In response to low water potential and high salt concentrations sensed in the roots of plants, the stress hormone abscisic acid (ABA) activates NADPH oxidase to generate H₂O₂, which is propagated in guard cells in leaves to induce stomatal closure and prevent water loss from transpiration. Using a reconstituted system, we demonstrate that H₂O₂ reversibly prevents the protein phosphatase HAB1, a key component of the core ABA-signaling pathway, from inhibiting its main target in guard cells, SnRK2.6/OST1 kinase. We have identified HAB1 C186 and C274 as H₂O₂-sensitive thiols and demonstrate that their oxidation inhibits both HAB1 catalytic activity and its ability to physically associate with SnRK2.6 by formation of intermolecular dimers.