Insights into cellular adhesion: reducing agent and thiol blocker treatments
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Citation
Abstract
Expected trace or low-level amounts of deoxyribonucleic acid (DNA) swabbed or cut from evidence prove crucial in the removal of as much of that DNA as possible from the substrate in order to obtain a probative profile. While cotton substrates contain polar side groups aiding in the retrieval of biological matter, purifying DNA further from that fabric proves difficult. Failure to remove the cells from the substrate before amplification could result in the presence of unwanted inhibitors from the substrate, inhibiting the polymerase chain reaction (PCR).
Cellular adhesion molecules (CAMs), specifically integrin, are known to play a role in cellular adhesion to a variety of matrices. Through reduction and oxidation (redox) reactions, integrin is able to shuffle its allosteric disulfide bonds, exposing free thiols that may be necessary in promoting adhesion.
Through the preparation of epithelial cells placed onto a cotton fabric swatch or a cotton swab, different variables were able to be tested including the use of disaccharides to reduce cell adhesion, the use of reducing agents to strengthen cell adhesion, and the use of thiol blockers to inhibit the effects of the reducing agents. Through this experimentation, 0.75 M D-(+)- Trehalose Dihydrate showed to improve cellular recovery from the cotton fabric, while reducing agents such as Dithiothreitol
(DTT) and Tris(2-carboxyethyl)phosphine Hydrochloride (TCEP) impeded cellular recovery. However, unexpected results occurred with the use of N-ethylmaleimide (NEM) thiol blocker, leading to ambiguous results. Through these experiments, insights into the cause of cellular adhesion has been revealed.