Free energy profiles determined for the binding of GTP with WT (blue), G12D (red) and G13D (green).

<p>The reaction coordinate is defined as the distance between the mass center of the GTP and the mass center of the WT, G12D, and G13D KRAS proteins, respectively.</p

Computational Analysis of KRAS Mutations: Implications for Different Effects on the KRAS p.G12D and p.G13D Mutations

<div><p>Background</p><p>The issue of whether patients diagnosed with metastatic colorectal cancer who harbor KRAS codon 13 mutations could benefit from the addition of anti-epidermal growth factor receptor therapy remains under debate. The aim of the current study was to perform computational analysis to investigate the structural implications of the underlying mutations caused by c.38G>A (p.G13D) on protein conformation.</p> <p>Methods</p><p>Molecular dynamics (MD) simulations were performed to understand the plausible structural and dynamical implications caused by c.35G>A (p.G12D) and c.38G>A (p.G13D). The potential of mean force (PMF) simulations were carried out to determine the free energy profiles of the binding processes of GTP interacting with wild-type (WT) KRAS and its mutants (MT).</p> <p>Results</p><p>Using MD simulations, we observed that the root mean square deviation (RMSD) increased as a function of time for the MT c.35G>A (p.G12D) and MT c.38G>A (p.G13D) when compared with the WT. We also observed that the GTP-binding pocket in the c.35G>A (p.G12D) mutant is more open than that of the WT and the c.38G>A (p.G13D) proteins. Intriguingly, the analysis of atomic fluctuations and free energy profiles revealed that the mutation of c.35G>A (p.G12D) may induce additional fluctuations in the sensitive sites (P-loop, switch I and II regions). Such fluctuations may promote instability in these protein regions and hamper GTP binding.</p> <p>Conclusions</p><p>Taken together with the results obtained from MD and PMF simulations, the present findings implicate fluctuations at the sensitive sites (P-loop, switch I and II regions). Our findings revealed that KRAS mutations in codon 13 have similar behavior as KRAS WT. To gain a better insight into why patients with metastatic colorectal cancer (mCRC) and the KRAS c.38G>A (p.G13D) mutation appear to benefit from anti-EGFR therapy, the role of the KRAS c.38G>A (p.G13D) mutation in mCRC needs to be further investigated.</p> </div

Distributions of docking scores.

<p>The docking scores for WT (blue), G12D (red) and G13D (green) KRAS proteins docked with GTP are shown in different colors.</p

Analysis of atomic fluctuations.

<p>The structures of (A) WT, (B) G12D and, (C) G13D KRAS proteins are drawn in cartoon putty representations at the P-loop, switch I and II regions; blue represents the lowest and red the highest B-factor value. In addition, the size of the tube reflects the value of the B-factor, in that the larger the B-factor, the thicker the tube. The structures in the other regions are colored in white and displayed in cartoon tube representation, where the size of the tube is independent of the B-factors.</p

Figure 2

<p>The molecular dynamics trajectories for: (A) Comparison of the RMSD plots of the sensitive sites (P-loop, switch I and II regions) of WT, G12D and G13D structures with respect to the initial conformation during the course of the simulation; (B) the pocket distances between the mass center of residues 12–13 and the mass center of residues 32–34 for WT, G12D, and G13D, respectively.</p

Molecular modeling of Human KRAS.

<p>The structure contains three sensitive sites: the P-loop (green), the switch I region (blue) and the switch II (red). The GTP and the Mg²⁺ ion are shown by ball-and-stick representations.</p

Evaluation of Alpha 1-Antitrypsin and the Levels of mRNA Expression of Matrix Metalloproteinase 7, Urokinase Type Plasminogen Activator Receptor and COX-2 for the Diagnosis of Colorectal Cancer

<div><h3>Background</h3><p>Colorectal cancer (CRC) is the second most common cause of death from cancer in both men and women in the majority of developed countries. Molecular tests of blood could potentially provide this ideal screening tool.</p> <h3>Aim</h3><p>Our objective was to assess the usefulness of serum markers and mRNA expression levels in the diagnosis of CRC.</p> <h3>Methods</h3><p>In a prospective study, we measured mRNA expression levels of 13 markers (carbonic anhydrase, guanylyl cyclase C, plasminogen activator inhibitor, matrix metalloproteinase 7 (MMP7), urokinase-type plasminogen activator receptor (uPAR), urokinase-type plasminogen activator, survivin, tetranectin, vascular endothelial growth factor (VEGF), cytokeratin 20, thymidylate synthase, cyclooxygenase 2 (COX-2), and CD44) and three proteins in serum (alpha 1 antitrypsin, carcinoembryonic antigen (CEA) and activated C3 in 42 patients with CRC and 33 with normal colonoscopy results.</p> <h3>Results</h3><p>Alpha 1-antitrypsin was the serum marker that was most useful for CRC diagnosis (1.79±0.25 in the CRC group vs 1.27±0.25 in the control group, P<0.0005). The area under the ROC curve for alpha 1-antitrypsin was 0.88 (0.79–0.96). The mRNA expression levels of five markers were statistically different between CRC cases and controls: those for which the ROC area was over 75% were MMP7 (0.81) and tetranectin (0.80), COX-2 (0.78), uPAR (0.78) and carbonic anhydrase (0.77). The markers which identified early stage CRC (Stages I and II) were alpha 1-antitrypsin, uPAR, COX-2 and MMP7.</p> <h3>Conclusions</h3><p>Serum alpha 1-antitrypsin and the levels of mRNA expression of MMP7, COX-2 and uPAR have good diagnostic accuracy for CRC, even in the early stages.</p> </div

ROC curves comparing carcinoembrionic antigen (CEA) in serum and MMP7 RNA expression.

<p>The areas under ROC curve for CEA and matrix metalloproteinase 7 (MMP7) were 0.84 and 0.81, respectively.</p

The levels of mRNA expression of the proteins analysed and the statistical significance of differences in the values between cancer tissue and healthy tissue in patients with CRC.

<p>SD; standard deviation PAI; plasminogen activator inhibitor MMP7;matrix metalloproteinase 7 uPAR; urokinase-type plasminogen activator receptor uPA;urokinase-type plasminogen activator VEGF; vascular endothelial growth factor COX-2; cyclooxygenase 2.</p

Oligonucleotide primer sequences generated used for the detection of mRNA for CRC markers.

<p>Oligonucleotide primer sequences generated used for the detection of mRNA for CRC markers.</p