Modified DNA bases are widespread in biology. 5-Methylcytosine (mC) is a predominant epigenetic marker in higher eukaryotes involved in gene regulation, development, aging, cancer, and disease. Recently, 5-hydroxymethylcytosine (hmC) was identified in mammalian brain tissue and stem cells. However, most of the currently available assays cannot distinguish mC from hmC in DNA fragments. We investigate here the physical properties of DNA with modified cytosines, in efforts to develop a physical tool that distinguishes mC from hmC in DNA fragments. Molecular dynamics simulations reveal that polar cytosine modifications affect internal base pair dynamics, while experimental evidence suggest a correlation between the modified cytosine’s polarity, DNA flexibility, and duplex stability. Based on these physical differences, solid-state nanopores can rapidly discriminate among DNA fragments with mC or hmC modification by sampling a few hundred molecules in the solution. Further, the relative proportion of hmC in the sample can be determined from the electronic signature of the intact DNA fragment.

A proteomic analysis was performed on spent fermentation medium following bioreactor propagation of a wild-type industrial strain to identify proteins naturally secreted by Kluyveromyces lactis cells. Here, we report changes detected in the K. lactis secretome as a result of growth in three different carbon sources: glucose, galactose and glycerol. A total of 151 secreted proteins were detected by multi-dimensional separations and reversed-phase online nanoESI-MS/MS analysis. From these, we were able to identify 63 proteins (termed the "base secretome") that were common to all three fermentation conditions. The majority of base secretome proteins, 79%, possessed general secretory pathway (GSP) sequences and were involved with cell wall structure, glycosylation, carbohydrate metabolism and proteolysis. There was little variation in the functional groupings of base secretome GSP proteins and GSP proteins that were not part of the base secretome. In contrast, the majority of non-GSP proteins detected were not part of the base secretome and the functions of these proteins varied significantly. Finally, through further identification of non-GSP proteins in carbon sources not originally tested, we have gained further evidence of a protein export mechanism separate from the GSP in K. lactis.