Optimum DNA Curvature Using a Hybrid Approach Involving an Artificial Neural Network and Genetic Algorithm

Abstract

In the present paper, a hybrid technique involving artificial neural network (ANN) and genetic algorithm (GA) has been proposed for performing modeling and optimization of complex biological systems. In this approach, first an ANN approximates (models) the nonlinear relationship(s) existing between its input and output example data sets. Next, the GA, which is a stochastic optimization technique, searches the input space of the ANN with a view to optimize the ANN output. The efficacy of this formalism has been tested by conducting a case study involving optimization of DNA curvature characterized in terms of the R_L value. Using the ANN-GA methodology, a number of sequences possessing high R_L values have been obtained and analyzed to verify the existence of features known to be responsible for the occurrence of curvature. A couple of sequences have also been tested experimentally. The experimental results validate qualitatively and also near-quantitatively, the solutions obtained using the hybrid formalism. The ANN-GA technique is a useful tool to obtain, ahead of experimentation, sequences that yield high R_L values. The methodology is a general one and can be suitably employed for optimizing any other biological feature.     

Structure-activity studies of a macrocyclic peptide inhibitor of histone lysine demethylase 4A

The combination of genetic code reprogramming and mRNA display is a powerful approach for the identification of macrocyclic peptides with high affinities to a target of interest. We have previously used such an approach to identify a potent inhibitor (CP2) of the human KDM4A and KDM4C lysine demethylases; important regulators of gene expression. In the present study, we have used genetic code reprogramming to synthesise very high diversity focused libraries (>10¹² compounds) based on CP2 and, through affinity screening, used these to delineate the structure activity relationship of CP2 binding to KDM4A. In the course of these experiments we identified a CP2 analogue (CP2f-7) with ～4-fold greater activity than CP2 in in vitro inhibition assays. This work will facilitate the development of more potent, selective inhibitors of lysine demethylases.     

Biofilm inhibition and anti-Candida activity of a cationic lipo-benzamide molecule with twin-nonyl chain

A series of cationic lipo-benzamide compounds with varying lengths of hydrocarbon chains (C2M–C18M) were evaluated for anti-Candida activity. Four compounds harbouring 8–11 hydrocarbon chains demonstrated concentration-dependent inhibition of fungal cell growth with Minimum Inhibitory Concentration (MIC) of ≤6.2?µg?ml^?1. The most active compound (C9M) inhibited growth of both Candida albicans and non-albicans strains and is equally active against pairs of azole sensitive and resistant clinical isolates of C. albicans. Compound C9M also inhibited different stages of Candida biofilms. Scanning Electron Microscopy (SEM) of Candida cells after C9M treatment was also done and no significant cell lysis was observed. Hemolysis assay was performed and only 2.5% haemolysis was observed at MIC concentration.     

Molecular docking,molecular modeling,and molecular dynamics studies of azaisoflavone as dual COX-2 inhibitors and TP receptor antagonists

Designed multi-target ligand (DML) is an emerging strategy for the development of new drugs and involves the engagement of multiple targets with the same moiety. In the context of NSAIDs it has been suggested that targeting the thromboxane prostanoid (TP) receptor along with cyclooxygenase-2 (COX-2) may help to overcome cardiovascular (CVS) complications associated with COXIBs. In the present work, azaisoflavones were studied for their COX-2 and TP receptor binding activities using structure based drug design (SBDD) techniques. Flavonoids were selected as a starting point based on their known COX-2 inhibitory and TP receptor antagonist activity. Iterative design and docking studies resulted in the evolution of a new class scaffold replacing the benzopyran-4-one ring of flavonoids with quinolin-4-one. The docking and binding parameters of these new compounds are found to be promising in comparison to those of selective COX-2 inhibitors, such as SC-558 and celecoxib. Owing to the lack of structural information, a model for the TP receptor was generated using a threading base alignment method with loop optimization performed using an ab initio method. The model generated was validated against known antagonists for TP receptor using docking/MMGBSA. Finally, the molecules that were designed for selective COX-2 inhibition were docked into the active site of the TP receptor. Iterative structural modifications and docking on these molecules generated a series which displays optimum docking scores and binding interaction for both targets. Molecular dynamics studies on a known TP receptor antagonist and a designed molecule show that both molecules remain in contact with protein throughout the simulation and interact in similar binding modes.

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In vitro leishmanicidal effects of the anti-fungal drug natamycin are mediated through disruption of calcium homeostasis and mitochondrial dysfunction

Natamycin, a Food and Drug Administration approved anti-fungal drug, and also used as a food additive was evaluated for anti-leishmanial activity since it is known to specifically bind to ergosterol, which is essential to these parasites but absent in mammals. Promising anti-proliferative activity was observed in both promastigote and amastigote forms of the parasite with IC₅₀ values of 15 and 8 µM respectively and a selective index of 12.5. The ultrastructural effects of natamycin on both forms of the parasite and physiological effects on promastigotes were studied in detail for the first time. Electron microscopic observations in treated cells revealed sub-cellular changes like plasma membrane alterations, accumulation of vesicles in the flagellar pocket and extensive mitochondrial damage. Natamycin treatment in promastigotes resulted in elevation of cytosolic calcium (Ca²⁺) levels which caused irreversible loss of mitochondrial membrane potential. This resulted in depletion of cellular ATP levels along with ROS generation finally leading to apoptosis-like and necrotic cell death. In view of our observations along with the safety profile of an existing anti-fungal drug, natamycin may be further investigated for repurposing it as a promising drug candidate against Leishmaniasis.     

Enterocyte viability and mitochondrial function after graded intestinal ischemia and reperfusion in rats

Ischemia/reperfusion of the small intestine can lead to metabolic and structural alterations in the mucosa. Cellular dysfunction occurs when mitochondrial metabolism is compromised, which may ultimately lead to impaired organ function. The aims of this study were to assess the suppression of cellular and mitochondrial oxidative metabolism and involvement of mitochondria in the ischemia/reperfusion injury. The mitochondria were prepared from isolated enterocytes obtained from the small intestine of anesthetized adult rats following different time periods of ischemia and ischemia followed by 5 min reperfusion. Cellular and mitochondrial function were assessed using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assay. Ischemia of increasing time periods caused a progressive decrease in cellular and mitochondrial MTT reduction in enterocytes and reperfusion showed further decrease of MTT formazan formation. Inclusion of 1 mM succinate, as respiratory subs trate, showed reversal of suppression of mitochondrial function in 30-60 min ischemia whereas 90 min ischemia or short time period ischemia followed by 5 min reperfusion indicated an irreversible damage to mitochondria. This study indicated that mitochondria are a sensitive target of damage due to oxygen deficiency and possibly due to sudden burst of oxygen free radicals. Mitochondria can withstand short periods of ischemia whereas long duration ischemia or reperfusion results in irreversible damage to mitochondrial function. (Mol Cell Biochem 167: 81-87, 1997)     

Cloning and characterization of ovine alphaS1-casein gene promoter: a transfection study in rat mammary gland cell line.

Promoter regions of milk protein genes are frequently used to produce pharmaceutically and medically important proteins in the mammary gland of transgenic animals and also can be used for the construction of an inducible eukaryotic expression vector. The aim of the present study was to clone, sequence and characterize the regulatory elements in ovine alphaS1-CSNGP. For the first time we have cloned and sequenced region extending from - 2136 to +49 bp containing 5'-flanking region and exon I. Computational analysis of the sequence showed presence of core promoter elements viz., TATA box, CAAT box and initiator sequence. Mammary gland specific sequences included MGF/STAT 5, MPBF, Yu Lee 2, 4 and 5, Oka box C and hormone responsive elements (HRE) viz., GRE, PRE, PRL, IRE and also Polyoma enhancer 3 sequences. Computational analysis data is validated by following the reporter gene expression studies in rat breast cell line. Six reporter gene constructs under the control of full length, proximal, distal, minimal and proximal-distal fused promoter segments were constructed to assess the effect of presence or absence of few selected regulatory elements on expression ability of the promoter. Based on qualitative evaluation of fluorescence, the pGFP-F/VspI showed highest fluorescence followed by pGFP-P, pGFP-F/SpeI, pGFPminimal and pGFP-D.     

Decline in photosynthesis as related to alterations in chloroplast ultrastructure of a cotton leaf during ontogeny

Field studies were conducted to investigate ontogenic changes in leaf photosynthesis and chloroplast ultrastructure of a single cotton (Gossypium hirsutum L.) leaf subtending the fruit. A 20-d old leaf was the most physiologically active with net photosynthetic rate (PN) of 16.5 mol m-2 s-1 and nitrogen (N) concentration of 168 mmol m-2. These values declined with leaf age and a close relationship existed between them. Concurrent with declines in PN, ultrastructural alterations occurred in the chloroplast: the 20-d old leaf had increased grana number and thylakoids per granum and a few plastoglobuli. Afterwards, the grana number and thylakoids per granum declined with leaf age indicating disintegrated grana and stroma lamellae. Concomitant with disintegrated membrane system was the presence of numerous large plastoglobuli. The PN was closely related to grana number and thylakoids per granum suggesting that the decline in PN with leaf age was associated with ultrastructural changes in the chloroplast.