13 research outputs found
pH-Controlled Resettable Modular DNA Strand-Displacement Circuits
Sophisticated
dynamic molecular systems with diverse functions
have been fabricated by using the fundamental tool of toehold-mediated
strand displacement (TMSD) in the field of dynamic DNA nanotechnology.
However, simple approaches to reset these TMSD-based dynamic systems
are lacking due to the difficulty in creating kinetically favored
pathways to implement the backward resetting reactions. Here, we develop
a facile proton-driven strategy to achieve complete resetting of a
modular DNA circuit by integrating a pH-responsive intermolecular
CG-C+ triplex DNA and an i-motif DNA into the conventional
DNA substrate. The pH-programmed strategy allows modular DNA components
to specifically associate/dissociate to promote the forward/backward
TMSD reactions, thereby enabling the modular DNA circuit to be repeatedly
operated at a constant temperature without generating any DNA waste
products. Leveraging this tractable approach, we further constructed
two resettable DNA logic gates used for logical computation and two
resettable catalytic DNA systems with good performance in signal transduction
and amplification
Model suggests plausible scenarios for Hsp72 regulation require both reduction of p65 and IKK inhibition.
<p>A. Dynamic simulations of model with p65 protein reduced to 70% (p65(t0)) show that reduction in total NF-κB partially accounts for the attenuated dynamics observed in Hsp72 expressing cells, but completely fails to account for reduction in IKK activity. B. Combining reduced p65 protein with a reduction in total IKK protein (to 5.5% of nominal levels) reduces peak IKK and NF-κB activation levels similar to experiments, suggesting the combined effect is one plausible scenario of interaction. C. The model with reduced p65 protein and interactions upstream (US, increasing inactivation rate <i>kiA20</i> 10-fold) and downstream (DS, simulated by decreasing IκBα phosphorylation rate, <i>kc2a</i>, by 6-fold) also match many qualitative features of the model. Gray lines are data renormalized such that basal IKK and NF-κB activity in BV2 cells is 10% of maximum.</p
Modifications to model parameters to simulate upstream and downstream regulatory interactions in the NF-κB signaling network.
<p>Hsp72 interactions with the NF-κB signaling network are interpreted as constant perturbations to rates corresponding to the relevant biological reaction. Equation numbers reference the complete model equations shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003471#pcbi-1003471-g002" target="_blank">Figure 2</a>, with altered parameters colored in red (inhibition) or blue (enhancement).</p
NF-κB signaling in response to TNFα stimulus is attenuated in BV2 cells overexpressing Hsp72 cells.
<p>A. Western blot assays of BV2 cells stably overexpressing Hsp72 at high levels (Hsp72) and low levels (Hsp72-L) compared with recombinant Hsp72 (Re-Hsp72), control cells (BV2), and control cells transfected with LacZ vector (LacZ). B. NF-κB p65/RelA DNA binding activity is significantly reduced in Hsp72 cells at 15, 20, 30, 105 min time points compared to other cell types. Concentrations were measured with ELISA and normalized with respect to the average measured p65 activity in BV2 cells at 20 min (n = 4 at 150 min for LacZ, n = 6 for all other time points and cell types). C. IKKβ kinase activity following treatment with 10 ng/ml TNFα at 0 min is significantly lower in Hsp72 cells at 0, 2.5, 5, 10 and 20 min. Concentrations were measured with ELISA and normalized with respect to average IKK activation in BV2 cells at 5 min (n = 6 at each time point). D. Total IκBα protein is reduced in Hsp72 cells compared to other cell types at 0, 60 and 90 min. Total IκBα levels were measured using ELISA optical density (OD) readings and normalized with respect to the average OD in BV2 cells at 0 min (n = 9 at each time point). E. Ser-32 phosphorylated IκBα is lower in Hsp72 cells than other cell types at 5, 20, 60 and 90 min. Concentrations were measured with ELISA and normalized with respect to the average level in BV2 cells at 5 min (n = 9 at each time point). F. Western blot assays of total and Ser-32 phosphorylated IκBα protein from whole cell extracts.</p
Modeling suggests Hsp72 attenuation of IKK is essential for differential IKK and NF-κB activation.
<p>A. Model simulations show expected dynamics of IKK, NF-κB and total IκBα protein when Hsp72 inhibits TNF-induced IKK activation. Each curve corresponds to simulations with rate <i>ka</i> multiplied by the factor indicated. B. Model simulations when Hsp72 is assumed to inhibit phosphorylation of IκBα (rate <i>kc2a</i>). When phosphorylation is sufficiently attenuated, peak NF-κB activation drops similar to levels observed experimentally, but IKK activation at early times is largely unaffected.</p
Hsp72 overexpression decreases total NF-κB-p65 in resting cells and reduces steady state IκBα protein levels.
<p>A. Model simulations directly reducing IκBα protein synthesis suggest mechanism inconsistent with data. Simulations were obtained from the model with nominal parameters (control, blue lines) and from the model with the rate of IκBα proteins translation (<i>c2a</i>) reduced to 1/30 the nominal level (red). Gray data points are rescaled from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003471#pcbi-1003471-g001" target="_blank">Fig. 1B</a>. B. Representative western blot assay of p65 NF-κB in BV2, LacZ, and Hsp72 cells show that p65 levels are decreased in Hsp72 overexpressing cells compared to controls. C. Quantification of p65 western blot assays from multiple experiments compared with initial total IκBα protein levels as assessed by ELISA assay in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003471#pcbi-1003471-g001" target="_blank">Fig. 1F</a> are shown (left panel) and compared with simulated initial concentrations (right panel). Data points for p65 are the averages from n = 5 independent measurements, each normalized with respect to the initial amount of p65 in untreated BV2 cells. IκBα data points are averages from (n = 9) independent measurements and normalized with respect to the mean initial measurement in untreated BV2 cells. Mathematical model simulated with 70% initial total NF-κB correctly accounts for the reduction in steady state IκBα levels observed from experiments. The mean protein levels in Hsp72 cells are significantly different (P<0.05) from BV2 cells as determined by t-test (p65) and different from both BV2 and LacZ cells as determined by ANOVA and Newman-Keuls <i>post hoc</i> test (IκBα).</p
Updated mechanistic diagram for Hsp72 regulation of NF-κB signaling in microglia.
<p>Hsp72 regulation at the upstream level inhibiting IKK is an essential component in microglia, although the precise biomolecular mechanism cannot be discriminated. Hsp72 also plays a critical role in reducing synthesis of the p65 NF-κB subunit. Additionally, Hsp72 appears to increase the induction of IκBα transcript. Downstream of IKK, Hsp72 regulation may act to decrease phosphorylation and/or other steps in the stimulus-induced degradation of IκBα, though it appears less essential than the other two levels of regulation in this particular cell type and context.</p
Modified model parameters to simulate possible regulation of steady state protein levels in the NF-κB signaling network.
<p>Hsp72 interactions with the NF-κB signaling network are interpreted as constant perturbations to rates or initial species corresponding to the relevant biological reaction. Equation numbers reference the complete model equations shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003471#pcbi-1003471-g002" target="_blank">Figure 2</a>, with altered parameters colored in red (inhibition) or blue (enhancement).</p
Additional file 1: Table S1. of RNA sequencing from human neutrophils reveals distinct transcriptional differences associated with chronic inflammatory states
Genes identified as expressed in neutrophils in all 9 subjects. (XLSX 893 kb
Hsp72 overexpression differentially regulates IκBα transcription.
<p>A. Model simulations with the nominal parameters predict that A20 and IκBα mRNA are similarly induced (blue solid lines). The model with modified parameters (<i>kiA20</i> set to 10-fold the nominal value, <i>kc2a</i> 1/6 the nominal value, and initial p65 70% of the nominal value) was simulated to find the transcript levels assuming that Hsp72 regulates the network by reducing total p65 and by inhibiting both upstream IKK activation and IκBα phosphorylation (red dashed lines). mRNA levels for IκBα and A20 transcripts are normalized with respect to their initial levels. B. IκBα and A20 mRNA transcript levels (fold change) in all three cell types were measured using qPCR at the indicated times following TNFα addition. IκBα and A20 transcripts are expressed similarly in BV2 and LacZ cells, while relative A20 is decreased in Hsp72 cells. Mean levels in Hsp72 cells are significantly different (P<0.05) from both BV2 and LacZ cells determined by ANOVA and Newman-Keuls <i>post hoc</i> tests at 30, 60 and 120 min (n = 3).</p