Intracellular ROS level in OM3 and WT with cells treated with or without 4 mM H₂O₂.

<p>Mid exponential phase grown cells (OD₆₀₀ 0.6) were incubated with 10 µm H₂DCFDA (dissolved in dimethyl sulfoxide) at 30°C, 200 rpm. The oxidized fluorophore was quantified using excitation wavelength 485 nm and emission wavelength 528 nm. Each data point is the mean of five independent observations.</p

DNA microarray data of certain genes in OM3 after H₂O₂ treatment (<i>p</i><0.05, Log₂ Fold Change>2.0).

*<p>- Analyzed by qRT-PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051179#pone.0051179.s011" target="_blank">Table S4</a>).</p

Cell growth profile after the introduction of 12 mM H₂O₂ during mid log phase (OD₆₀₀ 0.65).

<p>Each data point is the mean of three replicates.</p

OM3 and WT growth in cumene hydroperoxide or at high temperature (A) 0.3 mM cumene hydroperoxide, (B) 48°C.

<p>Cells were grown in LB-kanamycin at 37°C, 200 rpm under above stressors. Each data point is the mean of three replicates.</p

Cell growth in the absence or presence of H₂O₂ (A) 0 mM H₂O₂, (B) 8 mM H₂O₂, (C) 12 mM H₂O₂,

<p>Cells were cultured in LB-kanamycin medium at 37°C, 200 rpm. Each data point is the mean of three replicates.</p

Amino acid mutations in OM3.

<p>The main carbonyl of F69 interacts with the amine group of R123. The guanidium group of R82 has the electrostatic interaction with the phosphate group of cAMP. V139 is in the hinge region that participates in the inter-domain interaction between N-terminal cAMP binding domain and the C-terminal DNA binding domain. The structural stereoview was prepared by PyMOL using native CRP structure as template (PDB: 1G6N).</p

Amino acid substitutions in OM1∼OM3.

<p>Amino acid substitutions in OM1∼OM3.</p

Enhancing <em>E. coli</em> Tolerance towards Oxidative Stress via Engineering Its Global Regulator cAMP Receptor Protein (CRP)

<div><p>Oxidative damage to microbial hosts often occurs under stressful conditions during bioprocessing. Classical strain engineering approaches are usually both time-consuming and labor intensive. Here, we aim to improve <em>E. coli</em> performance under oxidative stress <em>via</em> engineering its global regulator cAMP receptor protein (CRP), which can directly or indirectly regulate redox-sensing regulators SoxR and OxyR, and other ∼400 genes in <em>E. coli</em>. Error-prone PCR technique was employed to introduce modifications to CRP, and three mutants (OM1∼OM3) were identified with improved tolerance <em>via</em> H₂O₂ enrichment selection. The best mutant OM3 could grow in 12 mM H₂O₂ with the growth rate of 0.6 h⁻¹, whereas the growth of wild type was completely inhibited at this H₂O₂ concentration. OM3 also elicited enhanced thermotolerance at 48°C as well as resistance against cumene hydroperoxide. The investigation about intracellular reactive oxygen species (ROS), which determines cell viability, indicated that the accumulation of ROS in OM3 was always lower than in WT with or without H₂O₂ treatment. Genome-wide DNA microarray analysis has shown not only CRP-regulated genes have demonstrated great transcriptional level changes (up to 8.9-fold), but also RpoS- and OxyR-regulated genes (up to 7.7-fold). qRT-PCR data and enzyme activity assay suggested that catalase (<em>katE</em>) could be a major antioxidant enzyme in OM3 instead of alkyl hydroperoxide reductase or superoxide dismutase. To our knowledge, this is the first work on improving <em>E. coli</em> oxidative stress resistance by reframing its transcription machinery through its native global regulator. The positive outcome of this approach may suggest that engineering CRP can be successfully implemented as an efficient strain engineering alternative for <em>E. coli</em>.</p> </div

DNA microarray data of certain endogenous genes in OM3 (<i>p</i><0.05, Log₂ Fold Change>2.0).

*<p>- Analyzed by qRT-PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051179#pone.0051179.s011" target="_blank">Table S4</a>).</p

Genetic algorithm-<i>de novo</i>, molecular dynamics and MMGBSA based modelling of a novel Benz-pyrazole based anticancer ligand to functionally revert mutant P53 into wild type P53

Mutations in P53 cause a loop unfolding, resulting in loss of activity and finally leading to cancer. One strategically reported way to arrest such oncogenesis is the restoration of tertiary structure as well as the function of mutant P53. In this attempt, we have designed a benzo-pyrazole-based novel ligand starting from a carbazole compound (EYB or PK9324) reported earlier to reinstate such function in mutant P53 (Y220C mutant, PDB: 6GGD). Assuming PK9324 as the template scaffold, de novo technique (Genetic algorithm, eLEA3D) was adopted within the binding pocket of 6GGD and our ligand DLIG1 was designed after several rounds of mutations. Docking and molecular dynamics (MD) simulation revealed significant interactions with key amino acid residues such as Cys220, Asp228, Leu145, Trp146, Val147, Thr150, Pro151, Pro152, Pro222, Pro223, Asp228, and Thr230. Along with sufficient binding stability, the MMGBSA analysis revealed its comparable binding free energy with other reported reference ligands (i.e. PK9324 and PK9318). Similar to these reference ligands, DLIG1 exhibited specificity in binding towards the Y220C mutant rather than towards wild-type P53. Finally, DLIG1 displayed a reorientation of a hydrophobic cavity in Y220C that hinted restoration of electrostatic interactions within the key loops of P53 favoring regain of its function.</p