Genome-wide expression profiling is a powerful tool for implicating novel gene ensembles in cellular mechanisms of health and disease. The most popular platform for genome-wide expression profiling is the Affymetrix GeneChip. However, its selection of probes relied on earlier genome and transcriptome annotation which is significantly different from current knowledge. The resultant informatics problems have a profound impact on analysis and interpretation the data. Here, we address these critical issues and offer a solution. We identified several classes of problems at the individual probe level in the existing annotation, under the assumption that current genome and transcriptome databases are more accurate than those used for GeneChip design. We then reorganized probes on more than a dozen popular GeneChips into gene-, transcript- and exon-specific probe sets in light of up-to-date genome, cDNA/EST clustering and single nucleotide polymorphism information. Comparing analysis results between the original and the redefined probe sets reveals ∼30–50% discrepancy in the genes previously identified as differentially expressed, regardless of analysis method. Our results demonstrate that the original Affymetrix probe set definitions are inaccurate, and many conclusions derived from past GeneChip analyses may be significantly flawed. It will be beneficial to re-analyze existing GeneChip data with updated probe set definitions.

MicroRNAs (miRNAs) are known to post-transcriptionally regulate target mRNAs through the 39-UTR, which interacts mainly with the 59-end of miRNA in animals. Here we identify many endogenous motifs within human 59-UTRs specific to the 39-ends of miRNAs. The 39-end of conserved miRNAs in particular has significant interaction sites in the humanenriched, less conserved 59-UTR miRNA motifs, while human-specific miRNAs have significant interaction sites only in the conserved 59-UTR motifs, implying both miRNA and 59-UTR are actively evolving in response to each other. Additionally, many miRNAs with their 39-end interaction sites in the 59-UTRs turn out to simultaneously contain 59-end interaction sites in the 39-UTRs. Based on these findings we demonstrate combinatory interactions between a single miRNA and both end regions of an mRNA using model systems. We further show that genes exhibiting large-scale protein changes due to miRNA overexpression or deletion contain both UTR interaction sites predicted. We provide the predicted targets of this new miRNA target class, miBridge, as an efficient way to screen potential targets, especially for nonconserved miRNAs, since the target search space is reduced by an order of magnitude compared with the 39-UTR alone. Efficacy is confirmed by showing SEC24D regulation with hsa-miR-605, a miRNA identified only in primate, opening the door to the study of nonconserved miRNAs. Finally, miRNAs (and associated proteins) involved in this new targeting class may prevent 40S ribosome scanning through the 59-UTR and keep it from reaching the start-codon, preventing 60S association.

Extensive simulations of PAMAM dendrimer generation 2 were performed at several pH conditions with explicit water molecules, to obtain proper conditions and validity for additional simulations without explicit water. Within the range of validity, simulation without water greatly extends the size and duration of practical simulations. We investigated the effects of long-range interaction parameters such as interaction distance and dielectric constant for molecular dynamics simulations of PAMAM dendrimer without water, concluding that charged dendrimer simulation with distance-dependent dielectric constant but without cutoff distance best mimics explicit water results. Structural variations of PAMAM dendrimers were analyzed as a function of pH and dendrimer generation using MD simulations with these long-range interaction parameters. Globular and loosely compact structures at high pH (g10) show conservation of atom density distribution across dendrimer generations. Highly ordered extended structures at low pH (e4) present an increasingly hollow interior as dendrimer generation grows, resulting in more open structure which provides easier access by chemical agents. By contrast, significant branch back-folding occurred at neutral pH in addition to major peripheral distribution of the terminal groups, yielding higher interior density in the intermediate radial region between the center and the maximum radius as the generation grows. Higher generation dendrimers provide a cavity surrounded by dense atom populations, producing a more stable agent carrier. Transition to high-density packing occurs between generations 4 and 5. Volume differences between neutral and low pH calculated from R G show a dramatic increase beginning at generation 5.