Surface immobilization of DNA aptamers for biosensing and protein interaction analysis

To utilize aptamers as molecular recognition agents in biosensors and biodiagnostics, it is important to develop strategies for reliable immobilization of aptamers so that they retain their biophysical characteristics and binding abilities. Here we report on quartz crystal microbalance (QCM) measurements and atomic force microscope (AFM)-based force spectroscopy studies to evaluate aptasensors fabricated by different modification strategies. Gold surfaces were modified with mixed self assembled monolayers (SAMs) of aptamer and oligoethylene glycol (OEG) thiols (HS-C(11)-(EG)(n)OH, n=3 or 6) to impart resistance to nonspecific protein adsorption. By affinity analysis, we show that short OEG thiols have less impact on aptamer accessibility than longer chain thiols. Backfilling with OEG as a step subsequent to aptamer immobilization provides greater surface coverage than using aptamer and OEG thiol to form a mixed SAM in one-step. Immunoglobulin E and vascular endothelial growth factor (VEGF) were studied as target proteins in these experiments. Binding forces obtained by these strategies are similar, demonstrating that the biophysical properties of the aptamer on the sensors are independent from the immobilization strategy. The results present mixed SAMs with aptamers and co-adsorbents as a versatile strategy for aptamer sensor platforms including ultrasensitive biosensor design.     

Secondary Structure,a Missing Component of Sequence-Based Minimotif Definitions

Minimotifs are short contiguous segments of proteins that have a known biological function. The hundreds of thousands of minimotifs discovered thus far are an important part of the theoretical understanding of the specificity of protein-protein interactions, posttranslational modifications, and signal transduction that occur in cells. However, a longstanding problem is that the different abstractions of the sequence definitions do not accurately capture the specificity, despite decades of effort by many labs. We present evidence that structure is an essential component of minimotif specificity, yet is not used in minimotif definitions. Our analysis of several known minimotifs as case studies, analysis of occurrences of minimotifs in structured and disordered regions of proteins, and review of the literature support a new model for minimotif definitions that includes sequence, structure, and function.     

Prdx1 deficiency in mice promotes tissue specific loss of heterozygosity mediated by deficiency in DNA repair and increased oxidative stress

The loss of the H(2)O(2) scavenger protein encoded by Prdx1 in mice leads to an elevation of reactive oxygen species (ROS) and tumorigenesis of different tissues. Loss of heterozygosity (LOH) mutations could initiate tumorigenesis through loss of tumor suppressor gene function in heterozygous somatic cells. A connection between the severity of ROS and the frequency of LOH mutations in vivo has not been established. Therefore, in this study, we characterized in vivo LOH in ear fibroblasts and splenic T cells of 3-4 month old Prdx1 deficient mice. We found that the loss of Prdx1 significantly elevates ROS amounts in T cells and fibroblasts. The basal amounts of ROS were higher in fibroblasts than in T cells, probably due to a less robust Prdx1 peroxidase activity in the former. Using Aprt as a LOH reporter, we observed an elevation in LOH mutation frequency in fibroblasts, but not in T cells, of Prdx1(-/-) mice compared to Prdx1(+/+) mice. The majority of the LOH mutations in both cell types were derived from mitotic recombination (MR) events. Interestingly, Mlh1, which is known to suppress MR between divergent sequences, was found to be significantly down-regulated in fibroblasts of Prdx1(-/-) mice. Therefore, the combination of elevated ROS amounts and down-regulation of Mlh1 may have contributed to the elevation of MR in fibroblasts of Prdx1(-/-) mice. We conclude that each tissue may have a distinct mechanism through which Prdx1 deficiency promotes tumorigenesis.     

Salicylic acid regulates basal resistance to Fusarium head blight in wheat

Fusarium head blight (FHB) is a destructive disease of cereal crops such as wheat and barley. Previously, expression in wheat of the Arabidopsis NPR1 gene (AtNPR1), which encodes a key regulator of salicylic acid (SA) signaling, was shown to reduce severity of FHB caused by Fusarium graminearum. It was hypothesized that SA signaling contributes to wheat defense against F. graminearum. Here, we show that increased accumulation of SA in fungus-infected spikes correlated with elevated expression of the SA-inducible pathogenesis-related 1 (PR1) gene and FHB resistance. In addition, FHB severity and mycotoxin accumulation were curtailed in wheat plants treated with SA and in AtNPR1 wheat, which is hyper-responsive to SA. In support of a critical role for SA in basal resistance to FHB, disease severity was higher in wheat expressing the NahG-encoded salicylate hydroxylase, which metabolizes SA. The FHB-promoting effect of NahG was overcome by application of benzo (1,2,3), thiadiazole-7 carbothioic acid S-methyl ester, a synthetic functional analog of SA, thus confirming an important role for SA signaling in basal resistance to FHB. We further demonstrate that jasmonate signaling has a dichotomous role in wheat interaction with F. graminearum, constraining activation of SA signaling during early stages of infection and promoting resistance during the later stages of infection.     

Independent Component Analysis for Brain fMRI Does Indeed Select for Maximal Independence

A recent paper by Daubechies et al. claims that two independent component analysis (ICA) algorithms, Infomax and FastICA, which are widely used for functional magnetic resonance imaging (fMRI) analysis, select for sparsity rather than independence. The argument was supported by a series of experiments on synthetic data. We show that these experiments fall short of proving this claim and that the ICA algorithms are indeed doing what they are designed to do: identify maximally independent sources.