Identification of promoter PcadR, in silico characterization of cadmium resistant gene cadR and molecular cloning of promoter PcadR from Pseudomonas aeruginosa BC15

2018 ◽  
Vol 34 (12) ◽  
pp. 819-833 ◽  
Author(s):  
Rajkumar Prabhakaran ◽  
Sebastin Nirmal Rajkumar ◽  
Tharmarajan Ramprasath ◽  
Govindan Sadasivam Selvam
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Cloning ◽  
Binding Affinity ◽  
In Silico ◽  
Dependent Manner ◽  
Atomic Contact ◽  
In Silico Studies ◽  
Metal Sensing

Cadmium (Cd) remediation in Pseudomonas aeruginosa is achieved through the function of two vital genes, cadA and cadR, that code for P-type ATPase (CadA) and transcription regulatory protein (CadR), respectively. Although numerous studies are available on these metal-sensing and regulatory proteins, the promoter of these genes, metal sensing and binding ability, are poorly understood. The present work is aimed at the characterization of the CadR protein, identification of the P cadR promoter and protein–promoter–metal binding affinity using bioinformatics and to validate the results by cloning the P cadR promoter in Escherichia coli DH5α. The promoter regions and its curvature were identified and analysed using PePPER software (University of Groningen, The Netherland) and the Bendit program (Version: v.1.0), respectively. Using Phyre, the three-dimensional structure of CadR was modelled, and the structure was validated by Ramachandran plots. The DNA-binding domain was present in the N-terminal region of CadR. A dimeric interface was observed in helix-turn-helix and metal ion-binding sites at the C-terminal. Docking studies showed higher affinity of Cd to both CadR (Atomic contact energy = −15.04 kcal/Mol) and P cadR (Atomic contact energy = −40.18 kcal/Mol) when compared to other metal ions. CadR with P cadR showed the highest binding affinity (Atomic contact energy= −250.40 kcal/Mol) when compared with P cadA. In vitro studies using green fluorescent protein tagged with P cadR ( gfp-P cadR) cloned in E. coli-expressed gfp protein in a concentration-dependent manner upon Cd exposure. Based on our in silico studies and in vitro molecular cloning analysis, we conclude that P cadR and CadR are active only in the presence of Cd. The CadR protein has the highest binding affinity with P cadR. As it became apparent that the cadR gene regulates the P cadR activity in the presence of Cd with high specificity, and the cadR and P cadR can be used as a biological tool for development of a microbial biosensor.

scholarly journals Preclinical Techniques to Investigate Exercise Training in Vascular Pathophysiology

2021 ◽  
Author(s):  
Gurneet Singh Sangha ◽  
Craig J. Goergen ◽  
Sushant M. Ranadive ◽  
Steven J. Prior ◽  
Alisa M Clyne
Keyword(s):  
In Silico ◽  
Cardiovascular Health ◽  
Dependent Manner ◽  
Preclinical Research ◽  
High Intensity Training ◽  
In Silico Studies ◽  
Tissue Systems ◽  
Arterial Plaques

Atherosclerosis is a dynamic process starting with endothelial dysfunction and inflammation and eventually leading to life-threatening arterial plaques. Exercise generally improves endothelial function in a dose-dependent manner by altering hemodynamics, specifically by increased arterial pressure, pulsatility, and shear stress. However, athletes who regularly participate in high-intensity training can develop arterial plaques, suggesting alternative mechanisms through which excessive exercise promotes vascular disease. Understanding the mechanisms that drive atherosclerosis in sedentary versus exercise states may lead to novel rehabilitative methods aimed at improving exercise compliance and physical activity. Preclinical tools, including in vitro cell assays, in vivo animal models, and in silico computational methods, broaden our capabilities to study the mechanisms through which exercise impacts atherogenesis, from molecular maladaptation to vascular remodeling. Here, we describe how preclinical research tools have and can be used to study exercise effects on atherosclerosis. We then propose how advanced bioengineering techniques can be used to address gaps in our current understanding of vascular pathophysiology, including integrating in vitro, in vivo, and in silico studies across multiple tissue systems and size scales. Improving our understanding of the anti-atherogenic exercise effects will enable engaging, targeted, and individualized exercise recommendations to promote cardiovascular health rather than treating cardiovascular disease that results from a sedentary lifestyle.

Anti-quorum sensing and anti-biofilm activity of 5-hydroxymethylfurfural against Pseudomonas aeruginosa PAO1: Insights from in vitro, in vivo and in silico studies

2019 ◽  
Vol 226 ◽  
pp. 19-26 ◽  
Author(s):  
Jobina Rajkumari ◽  
Subhomoi Borkotoky ◽  
Dhanasekhar Reddy ◽  
Saswat Kumar Mohanty ◽  
Ranjith Kumavath ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
In Silico ◽  
Pseudomonas Aeruginosa Pao1 ◽  
In Silico Studies

Synthesis and characterization of new thiosemicarbazones, as potent urease inhibitors: In vitro and in silico studies

2019 ◽  
Vol 87 ◽  
pp. 155-162 ◽  
Author(s):  
Muhammad Islam ◽  
Ajmal Khan ◽  
Muhammad Tariq Shehzad ◽  
Abdul Hameed ◽  
Nadeem Ahmed ◽  
...  
Keyword(s):  
In Silico ◽  
Synthesis And Characterization ◽  
Urease Inhibitors ◽  
In Silico Studies

scholarly journals Copper(II) and silver(I)-1,10-phenanthroline-5,6-dione complexes interact with double-stranded DNA: further evidence of their apparent multi-modal activity towards Pseudomonas aeruginosa

2022 ◽  
Author(s):  
Anna Clara Milesi Galdino ◽  
Lívia Viganor ◽  
Matheus Mendonça Pereira ◽  
Michael Devereux ◽  
Malachy McCann ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Affinity ◽  
Ethidium Bromide ◽  
Topoisomerase I ◽  
Electrostatic Interactions ◽  
Multidrug Resistant ◽  
Free Radical Scavengers ◽  
Dependent Manner ◽  
Double Stranded Dna

AbstractTackling microbial resistance requires continuous efforts for the development of new molecules with novel mechanisms of action and potent antimicrobial activity. Our group has previously identified metal-based compounds, [Ag(1,10-phenanthroline-5,6-dione)2]ClO4 (Ag-phendione) and [Cu(1,10-phenanthroline-5,6-dione)3](ClO4)2.4H2O (Cu-phendione), with efficient antimicrobial action against multidrug-resistant species. Herein, we investigated the ability of Ag-phendione and Cu-phendione to bind with double-stranded DNA using a combination of in silico and in vitro approaches. Molecular docking revealed that both phendione derivatives can interact with the DNA by hydrogen bonding, hydrophobic and electrostatic interactions. Cu-phendione exhibited the highest binding affinity to either major (− 7.9 kcal/mol) or minor (− 7.2 kcal/mol) DNA grooves. In vitro competitive quenching assays involving duplex DNA with Hoechst 33258 or ethidium bromide demonstrated that Ag-phendione and Cu-phendione preferentially bind DNA in the minor grooves. The competitive ethidium bromide displacement technique revealed Cu-phendione has a higher binding affinity to DNA (Kapp = 2.55 × 106 M−1) than Ag-phendione (Kapp = 2.79 × 105 M−1) and phendione (Kapp = 1.33 × 105 M−1). Cu-phendione induced topoisomerase I-mediated DNA relaxation of supercoiled plasmid DNA. Moreover, Cu-phendione was able to induce oxidative DNA injuries with the addition of free radical scavengers inhibiting DNA damage. Ag-phendione and Cu-phendione avidly displaced propidium iodide bound to DNA in permeabilized Pseudomonas aeruginosa cells in a dose-dependent manner as judged by flow cytometry. The treatment of P. aeruginosa with bactericidal concentrations of Cu-phendione (15 µM) induced DNA fragmentation as visualized by either agarose gel or TUNEL assays. Altogether, these results highlight a possible novel DNA-targeted mechanism by which phendione-containing complexes, in part, elicit toxicity toward the multidrug-resistant pathogen P. aeruginosa. Graphical abstract

scholarly journals Comparative Effects of Di-(2-ethylhexyl)phthalate and Di-(2-ethylhexyl)terephthalate Metabolites on Thyroid Receptors: In Vitro and In Silico Studies

Metabolites ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 94
Author(s):  
Nicolas Kambia ◽  
Isabelle Séverin ◽  
Amaury Farce ◽  
Laurence Dahbi ◽  
Thierry Dine ◽  
...  
Keyword(s):  
In Silico ◽  
Hormone Receptors ◽  
Partial Agonist ◽  
Biological Fluids ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
In Silico Studies ◽  
Ethylhexyl Phthalate ◽  
T3 Receptors

Plasticizers added to polyvinylchloride (PVC) used in medical devices can be released into patients’ biological fluids. Di-(2-ethylhexyl)phthalate (DEHP), a well-known reprotoxic and endocrine disruptor, must be replaced by alternative compounds. Di-(2-ethylhexyl) terephthalate (DEHT) is an interesting candidate due to its lower migration from PVC and its lack of reprotoxicity. However, there is still a lack of data to support the safety of its human metabolites with regard to their hormonal properties in the thyroid system. The effects of DEHT metabolites on thyroid/hormone receptors (TRs) were compared in vitro and in silico to those of DEHP. The oxidized metabolites of DEHT had no effect on T3 receptors whereas 5-hydroxy-mono-(ethylhexyl)phthalate (5-OH-MEHP) appeared to be primarily an agonist for TRs above 0.2 µg/mL with a synergistic effect on T3. Monoesters (MEHP and mono-(2-ethylhexyl)terephthalate, MEHT) were also active on T3 receptors. In vitro, MEHP was a partial agonist between 10 and 20 µg/mL. MEHT was an antagonist at non-cytotoxic concentrations (2–5 µg/mL) in a concentration-dependent manner. The results obtained with docking were consistent with those of the T-screen and provide additional information on the preferential affinity of monoesters and 5-OH-MEHP for TRs. This study highlights a lack of interactions between oxidized metabolites and TRs, confirming the interest of DEHT.

IN VITRO/IN SILICO CHARACTERIZATION OF ACTIVE AND PASSIVE STRESSES IN CARDIAC MUSCLE

2012 ◽  
Vol 10 (2) ◽  
pp. 171-188 ◽  
Author(s):  
Markus Boel ◽  
Oscar J. Abilez ◽  
Ahmed N Assar ◽  
Christopher K. Zarins ◽  
Ellen Kuhl
Keyword(s):  
Cardiac Muscle ◽  
In Silico ◽  
In Silico Characterization

Recent In Vitro and In Silico Advances in the Understanding of Intranasal Drug Delivery

2020 ◽  
Vol 26 ◽  
Author(s):  
John Chen ◽  
Andrew Martin ◽  
Warren H. Finlay
Keyword(s):  
Drug Delivery ◽  
In Silico ◽  
Local Drug Delivery ◽  
In Vivo Studies ◽  
Intranasal Drug Delivery ◽  
Nasal Sprays ◽  
In Silico Studies ◽  
Drug Delivery Devices

Background: Many drugs are delivered intranasally for local or systemic effect, typically in the form of droplets or aerosols. Because of the high cost of in vivo studies, drug developers and researchers often turn to in vitro or in silico testing when first evaluating the behavior and properties of intranasal drug delivery devices and formulations. Recent advances in manufacturing and computer technologies have allowed for increasingly realistic and sophisticated in vitro and in silico reconstructions of the human nasal airways. Objective: To perform a summary of advances in understanding of intranasal drug delivery based on recent in vitro and in silico studies. Conclusion: The turbinates are a common target for local drug delivery applications, and while nasal sprays are able to reach this region, there is currently no broad consensus across the in vitro and in silico literature concerning optimal parameters for device design, formulation properties and patient technique which would maximize turbinate deposition. Nebulizers are able to more easily target the turbinates, but come with the disadvantage of significant lung deposition. Targeting of the olfactory region of the nasal cavity has been explored for potential treatment of central nervous system conditions. Conventional intranasal devices, such as nasal sprays and nebulizers, deliver very little dose to the olfactory region. Recent progress in our understanding of intranasal delivery will be useful in the development of the next generation of intranasal drug delivery devices.

In-silico Studies of Isolated Phytoalkaloid Against Lipoxygenase: Study Based on Possible Correlation

2018 ◽  
Vol 21 (3) ◽  
pp. 215-221
Author(s):  
Haroon Khan ◽  
Muhammad Zafar ◽  
Helena Den-Haan ◽  
Horacio Perez-Sanchez ◽  
Mohammad Amjad Kamal
Keyword(s):  
In Silico ◽  
Docking Studies ◽  
In Vivo Studies ◽  
In Vitro Assays ◽  
Chronic Obstructive ◽  
Lipoxygenase Inhibitors ◽  
Lipoxygenase Inhibition ◽  
In Silico Studies

Aim and Objective: Lipoxygenase (LOX) enzymes play an important role in the pathophysiology of several inflammatory and allergic diseases including bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, rheumatoid arthritis and chronic obstructive pulmonary disease. Inhibitors of the LOX are believed to be an ideal approach in the treatment of diseases caused by its over-expression. In this regard, several synthetic and natural agents are under investigation worldwide. Alkaloids are the most thoroughly investigated class of natural compounds with outstanding past in clinically useful drugs. In this article, we have discussed various alkaloids of plant origin that have already shown lipoxygenase inhibition in-vitro with possible correlation in in silico studies. Materials and Methods: Molecular docking studies were performed using MOE (Molecular Operating Environment) software. Among the ten reported LOX alkaloids inhibitors, derived from plant, compounds 4, 2, 3 and 1 showed excellent docking scores and receptor sensitivity. Result and Conclusion: These compounds already exhibited in vitro lipoxygenase inhibition and the MOE results strongly correlated with the experimental results. On the basis of these in vitro assays and computer aided results, we suggest that these compounds need further detail in vivo studies and clinical trial for the discovery of new more effective and safe lipoxygenase inhibitors. In conclusion, these results might be useful in the design of new and potential lipoxygenase (LOX) inhibitors.

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