It is vitally important that further research into epileptogenesis builds on prior knowledge and pitfalls. It is the opinion of our laboratory that the entire process of molecular transcription and translation be interrogated with validation of global expression profiling results utilizing qRT-PCR followed by proteomic profiling using any combination of Western blotting, immunoperoxidase staining and in situ hybridization. Focusing genomic profiles into specific anatomical regions may enable causative molecular pathways to be identified and allow greater understanding of the histological changes, particular in the situation of HS associated MTLE whereby disagreement remains over the cause or effect of the pathological changes. Fluorescence Competition Assay Measurements of Free Energy Changes for RNA Pseudoknots [Image: see text] RNA pseudoknots have important functions, and thermodynamic stability is a key to predicting pseudoknots in RNA sequences and to understanding their functions. Traditional methods, such as UV melting and differential scanning calorimetry, for measuring RNA thermodynamics are restricted to temperature ranges around the melting temperature for a pseudoknot. Here, we report RNA pseudoknot free energy changes at 37 °C measured by fluorescence competition assays. Sequence-dependent studies for the loop 1−stem 2 region reveal (1) the individual nearest-neighbor hydrogen bonding (INN-HB) model provides a reasonable estimate for the free energy change when a Watson−Crick base pair in stem 2 is changed, (2) the loop entropy can be estimated by a statistical polymer model, although some penalty for certain loop sequences is necessary, and (3) tertiary interactions can significantly stabilize pseudoknots and extending the length of stem 2 may alter tertiary interactions such that the INN-HB model does not predict the net effect of adding a base pair. The results can inform writing of algorithms for predicting and/or designing RNA secondary structures. In duplex part, optical melting result (-12.9 kcal/mol) of model duplex (5΄GAACAAACGG3΄/3΄AACUUGUUUGF5΄ in Table 1 ) made less favorable by 1.9 kcal/mol for 3΄ dangling AA (62) and 2.7 (=0.82+1.9) kcal/mol for 5΄FG/3΄GGC terminal overhangs, made more favorable by 2.36 kcal/mol for 5΄CG/3΄GC nearest neighbor parameter (39) and 2.1 (=0.82+1.3+0.0) kcal/mol for 5΄FC/3΄GGG terminal overhangs. Here 5΄FC/3΄G is approximated as the 0.82 kcal/mol of 5΄FG/3΄C (Table 1) Plots of reported vs T m for the BWYV pseudoknot (45) reveal the possibility that the transitions F→PK and PK→HP may have non-zero . Reported errors in values are ±15% (45), which does not allow a reliable estimate of . The data, however, indicates the possibility that for transitions F→PK, PK→HP, and HP→U can be on the order of -700, -330, and -20 calK -1 mol -1 , respectively. Points shown as diamonds are excluded from linear fit. Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells Adenoviruses replicate primarily in the host cell nucleus, and it is well established that adenovirus infection affects the structure and function of host cell nucleoli in addition to coding for a number of nucleolar targeted viral proteins. Here we used unbiased proteomics methods, including high throughput mass spectrometry coupled with stable isotope labeling by amino acids in cell culture (SILAC) and traditional two-dimensional gel electrophoresis, to identify quantitative changes in the protein composition of the nucleolus during adenovirus infection. Two-dimensional gel analysis revealed changes in six proteins. By contrast, SILAC-based approaches identified 351 proteins with 24 proteins showing at least a 2-fold change after infection. Of those, four were previously reported to have aberrant localization and/or functional relevance during adenovirus infection. In total, 15 proteins identified as changing in amount by proteomics methods were examined in infected cells using confocal microscopy. Eleven of these proteins showed altered patterns of localization in adenovirus-infected cells. Comparing our data with the effects of actinomycin D on the nucleolar proteome revealed that adenovirus infection apparently specifically targets a relatively small subset of nucleolar antigens at the time point examined. nucleus are inhibited, and a number of viral nucleolar antigens accumulate in the nucleoli of infected cells (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) . Moreover, two cellular nucleolar antigens, UBF 1 and B23.1, have been shown to be sequestered into viral DNA replication centers where they affect viral DNA replication (11) (12) (13) . Thus, there is clear evidence that adenovirus infection has a substantial impact on the composition and function of the nucleolus. What is not clear is whether the sequestration of cellular nucleolar antigens is limited to a small number of proteins or widespread. In addition, it is unclear whether adenovirus primarily modulates the nucleolus for replicative advantage or whether the effects on the nucleolus are a side effect of cellular proteins being sequestered into virally induced structures.