Aire-dependent genes undergo Clp1-mediated 3'UTR shortening associated with higher transcript stability in the thymus.

The ability of the immune system to avoid autoimmune disease relies on tolerization of thymocytes to self-antigens whose expression and presentation by thymic medullary epithelial cells (mTECs) is controlled predominantly by Aire at the transcriptional level and possibly regulated at other unrecognized levels. Aire-sensitive gene expression is influenced by several molecular factors, some of which belong to the 3'end processing complex, suggesting they might impact transcript stability and levels through an effect on 3'UTR shortening. We discovered that Aire-sensitive genes display a pronounced preference for short-3'UTR transcript isoforms in mTECs, a feature preceding Aire's expression and correlated with the preferential selection of proximal polyA sites by the 3'end processing complex. Through an RNAi screen and generation of a lentigenic mouse, we found that one factor, Clp1, promotes 3'UTR shortening associated with higher transcript stability and expression of Aire-sensitive genes, revealing a post-transcriptional level of control of Aire-activated expression in mTECs.

Effect of PEGylation on drug entry into lipid bilayer.

Poly(ethylene glycol) (PEG) is a polymer commonly used for functionalization of drug molecules to increase their bloodstream lifetime, hence efficacy. However, the interactions between the PEGylated drugs and biomembranes are not clearly understood. In this study, we employed atomic-scale molecular dynamics (MD) simulations to consider the behavior of two drug molecules functionalized with PEG (tetraphenylporphyrin used in cancer phototherapy and biochanin A belonging to the isoflavone family) in the presence of a lipid bilayer. The commonly held view is that functionalization of a drug molecule with a polymer acts as an entropic barrier, inhibiting the penetration of the drug molecule through a cell membrane. Our results indicate that in the bloodstream there is an additional source of electrostatic repulsive interactions between the PEGylated drugs and the lipid bilayer. Both the PEG chain and lipids can bind Na(+) ions, thus effectively becoming positively charged molecules. This leads to an extra repulsive effect resulting from the presence of salt in the bloodstream. Thus, our study sheds further light on the role of PEG in drug delivery.

Study of interaction between PEG carrier and three relevant drug molecules: piroxicam, paclitaxel, and hematoporphyrin.

Molecular dynamics simulation has been used to study the specific interactions between poly(ethylene glycol) (PEG) and three drug molecules for which PEG is used to aid delivery: paclitaxel and piroxicam, where PEG is a carrier agent, and hematoporphyrin, where PEG is covalently attached to form a "stealth shield". Simulating at physiological salt concentration, we found no evidence of any specific interaction between paclitaxel or piroxicam with PEG, but found a strong interaction for the case of hematoporphyrin. This interaction is lipophilic in nature, between the nonpolar (CH(2))(2) groups of the PEG and the porphin ring of the hematoporphyrin. This interaction was found to be strong enough that the PEG aggregated to the hematoporphyrin, independent of whether or not it was covalently bound. Interestingly, when the simulation was repeated in absence of salt we found evidence of this interaction being weakened. This led us to hypothesize a previously unforeseen mechanism: interaction with salt cations cause the PEG to coil around the salt ions, each ion binding to many PEG oxygens, increasing the exposure of the nonpolar ethylene groups, thus increasing the effective hydrophobicity of PEG. The Hydrophobic ethylene groups of the PEG chains adhere strongly to the hydrophobic porphin ring. Experiments involving absorption spectra measurements were conducted, and these results also indicated that presence of salt at physiological level increases the effective attractive interaction between PEG and hematoporphyrin. Taken together, our results demonstrate that while PEG, due to its solubility in both polar and nonpolar solvents, may act as a dissolution aid for paclitaxel and piroxicam, of the three drug molecules studied it will only have a protective role for the case of the hematoporphyrin.