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    ABSTRACT: Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.
    Full-text · Article · Dec 2014 · PLoS ONE
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    ABSTRACT: p38α is a significant target for drug designing against cancer. The overproduction of p38α MAPK promotes tumorigenesis in head and neck squamous cell carcinoma (HNSCC). The ATP binding and an allosteric site referred as DFG are the key sites of the p38α mitogen activated protein kinase (MAPK) exploited for the design of inhibitors. This study demonstrated design of peptide inhibitor on the basis of allosteric site using Glide molecular docking software and the biochemical analysis of the best modeled peptide. The best fitted tetrapeptide (FWCS) in the allosteric site inhibited the pure recombinant and serum p38α of HNSCC patients by 74 and 72%, respectively. The potency of the peptide was demonstrated by its IC50 (4.6 nM) and KD (3.41×10-10 M) values, determined by ELISA and by surface plasmon resonance (SPR) technology, respectively. The cell viability of oral cancer i.e. KB cell line was reduced in dose dependent manner by 60 and 97% by the treatment of peptide and the IC50 was 600 and 210 µM after 24 and 72 h incubation, respectively. Our result provides an insight for the development of a proficient small peptide as a promising anticancer agent targeting DFG site of p38α kinase.
    Full-text · Article · Jul 2014 · PLoS ONE
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    ABSTRACT: Aim The aim of the present study was to measure the accuracy and reproducibility of probe forces in simulated assessments of periodontal pocket depth. The study included experienced and inexperienced examiners and used manual and pressure-sensitive probes. Materials and Methods Sixty-one participants were divided into seven groups and asked to probe selected anterior and posterior sites with three different probes (Williams 14W, Chapple UB-CF-15, and Vivacare TPS probes). The model was positioned on a digital electronic balance to measure force, which was recorded initially and after 15 minutes. Probe preferences were recorded. Accuracy was measured by comparing to a standardized 25 g force, and reproducibility was calculated for all duplicate measurements. Results The Vivacare probe produced the most accurate and most reproducible forces, whereas the Williams probe produced the least accurate and least reproducible forces. Probe forces were lighter at anterior sites compared to posterior sites at baseline. Probe forces were reduced at both sites after 15 minutes compared to baseline. Conclusions Vivacare TPS periodontal probes are more accurate and reproducible than Chapple and Williams probes. Many clinicians in this study preferred the Chapple probe.
    Full-text · Article · Apr 2014 · Saudi Dental Journal
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