Coordinated regulation of acid resistance in Escherichia coli.

BACKGROUND: Enteric Escherichia coli survives the highly acidic environment of the stomach through multiple acid resistance (AR) mechanisms. The most effective system, AR2, decarboxylates externally-derived glutamate to remove cytoplasmic protons and excrete GABA. The first described system, AR1, does not require an external amino acid. Its mechanism has not been determined. The regulation of the multiple AR systems and their coordination with broader cellular metabolism has not been fully explored. RESULTS: We utilized a combination of ChIP-Seq and gene expression analysis to experimentally map the regulatory interactions of four TFs: nac, ntrC, ompR, and csiR. Our data identified all previously in vivo confirmed direct interactions and revealed several others previously inferred from gene expression data. Our data demonstrate that nac and csiR directly modulate AR, and leads to a regulatory network model in which all four TFs participate in coordinating acid resistance, glutamate metabolism, and nitrogen metabolism. This model predicts a novel mechanism for AR1 by which the decarboxylation enzymes of AR2 are used with internally derived glutamate. This hypothesis makes several testable predictions that we confirmed experimentally. CONCLUSIONS: Our data suggest that the regulatory network underlying AR is complex and deeply interconnected with the regulation of GABA and glutamate metabolism, nitrogen metabolism. These connections underlie and experimentally validated model of AR1 in which the decarboxylation enzymes of AR2 are used with internally derived glutamate.

Light-Triggered Release of Bioactive Molecules from DNA Nanostructures.

Recent innovations in DNA nanofabrication allow the creation of intricately shaped nanostructures ideally suited for many biological applications. To advance the use of DNA nanotechnology for the controlled release of bioactive molecules, we report a general strategy that uses light to liberate encapsulated cargoes from DNA nanostructures with high spatiotemporal precision. Through the incorporation of a custom, photolabile cross-linker, we encapsulated cargoes ranging in size from small molecules to full-sized proteins within DNA nanocages and then released such cargoes upon brief exposure to light. This novel molecular uncaging technique offers a general approach for precisely releasing a large variety of bioactive molecules, allowing investigation into their mechanism of action, or finely tuned delivery with high temporal precision for broad biomedical and materials applications.

Rationally Designed MicroRNA-Based Genetic Classifiers Target Specific Neurons in the Brain.

Targeting transgene expression to specific cell types in vivo has proven instrumental in characterizing the functional role of defined cell populations. Genetic classifiers, synthetic transgene constructs designed to restrict expression to particular classes of cells, commonly rely on transcriptional promoters to define cellular specificity. However, the large size of many natural promoters complicates their use in viral vectors, an important mode of transgene delivery in the brain and in human gene therapy. Here, we expanded upon an emerging classifier platform, orthogonal to promoter-based strategies, that exploits endogenous microRNA regulation to target gene expression. Such classifiers have been extensively explored in other tissues; however, their use in the nervous system has thus far been limited to targeting gene expression between neurons and supporting cells. Here, we tested the possibility of using combinatory microRNA regulation to specify gene targeting between neuronal subtypes, and successfully targeted inhibitory cells in the neocortex. These classifiers demonstrate the feasibility of designing a new generation of microRNA-based neuron-type- and brain-region-specific gene expression targeting neurotechnologies.

Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro.

Angiogenesis is a complex morphogenetic process whereby endothelial cells from existing vessels invade as multicellular sprouts to form new vessels. Here, we have engineered a unique organotypic model of angiogenic sprouting and neovessel formation that originates from preformed artificial vessels fully encapsulated within a 3D extracellular matrix. Using this model, we screened the effects of angiogenic factors and identified two distinct cocktails that promoted robust multicellular endothelial sprouting. The angiogenic sprouts in our system exhibited hallmark structural features of in vivo angiogenesis, including directed invasion of leading cells that developed filopodia-like protrusions characteristic of tip cells, following stalk cells exhibiting apical-basal polarity, and lumens and branches connecting back to the parent vessels. Ultimately, sprouts bridged between preformed channels and formed perfusable neovessels. Using this model, we investigated the effects of angiogenic inhibitors on sprouting morphogenesis. Interestingly, the ability of VEGF receptor 2 inhibition to antagonize filopodia formation in tip cells was context-dependent, suggesting a mechanism by which vessels might be able to toggle between VEGF-dependent and VEGF-independent modes of angiogenesis. Like VEGF, sphingosine-1-phosphate also seemed to exert its proangiogenic effects by stimulating directional filopodial extension, whereas matrix metalloproteinase inhibitors prevented sprout extension but had no impact on filopodial formation. Together, these results demonstrate an in vitro 3D biomimetic model that reconstitutes the morphogenetic steps of angiogenic sprouting and highlight the potential utility of the model to elucidate the molecular mechanisms that coordinate the complex series of events involved in neovascularization.