Light-Controlled Release of Therapeutic Proteins from Red Blood Cells.

Protein therapeutics are a powerful class of drugs known for their selectivity and potency. However, the potential efficacy of these therapeutics is commonly offset by short circulatory half-lives and undesired action at otherwise healthy tissue. We describe herein a targeted protein delivery system that employs engineered red blood cells (RBCs) as carriers and light as the external trigger that promotes hemolysis and drug release.

Fluorescent sensors for neuronal signaling.

Dissecting neuronal structure and function in relation to behavior is an immense undertaking. Researchers require imaging tools to study neuronal activity and biochemical signaling in situ in order to study the roles of neuronal and biochemical activity in information processing. A large number of genetically encoded fluorescent biosensors have been reported in the literature over the past few years as there is a push to develop new technology in neuroscience.

Engineered BRET-Based Biologic Light Sources Enable Spatiotemporal Control over Diverse Optogenetic Systems.

Light-inducible optogenetic systems offer precise spatiotemporal control over a myriad of biologic processes. Unfortunately, current systems are inherently limited by their dependence on external light sources for their activation. Further, the utility of laser/LED-based illumination strategies are often constrained by the need for invasive surgical procedures to deliver such devices and local heat production, photobleaching and phototoxicity that compromises cell and tissue viability.

Compartmentalized cAMP Generation by Engineered Photoactivated Adenylyl Cyclases.

Because small-molecule activators of adenylyl cyclases (AC) affect ACs cell-wide, it is challenging to explore the signaling consequences of AC activity emanating from specific intracellular compartments. We explored this issue using a series of engineered, optogenetic, spatially restricted, photoactivable adenylyl cyclases (PACs) positioned at the plasma membrane (PM), the outer mitochondrial membrane (OMM), and the nucleus (Nu). The biochemical consequences of brief photostimulation of PAC is primarily limited to the intracellular site occupied by the PAC.

Design, construction, and validation of optogenetic proteins.

Cellular optogenetics employs light-regulated, genetically encoded protein actuators to perturb cellular signaling with unprecedented spatial and temporal control. Here, we present a potentially generalized approach for transforming a given protein of interest (POI) into an optogenetic species. We describe the rational and methods by which we developed three different optogenetic POIs utilizing the Cry2-Cib photodimerizing pair.

Optogenetics: A Primer for Chemists.

The field of optogenetics uses genetically encoded, lightresponsive proteins to control physiological processes. This technology has been hailed as the one of the ten big ideas in brain science in the past decade, the breakthrough of the decade, and the method of the year in 2010 and again in 2014. The excitement evidenced by these proclamations is confirmed by a couple of impressive numbers.

Cellular Cyborgs: On the Precipice of a Drug Delivery Revolution.

Cell-based drug delivery systems offer the prospect of biocompatibility, large-loading capacity, long in vivo lifespan, and active targeting of diseased sites. However, these opportunities are offset by an array of challenges, including safeguarding the integrity of the drug cargo and the cellular host, as well as ensuring that drug release occurs at the appropriate time and place. Emerging strategies that address these, and related, issues, are described herein.

Chemotext: A Publicly Available Web Server for Mining Drug-Target-Disease Relationships in PubMed.

Elucidation of the mechanistic relationships between drugs, their targets, and diseases is at the core of modern drug discovery research. Thousands of studies relevant to the drug-target-disease (DTD) triangle have been published and annotated in the Medline/PubMed database. Mining this database affords rapid identification of all published studies that confirm connections between vertices of this triangle or enable new inferences of such connections.

Design and Profiling of a Subcellular Targeted Optogenetic cAMP-Dependent Protein Kinase.

Although the cAMP-dependent protein kinase (PKA) is ubiquitously expressed, it is sequestered at specific subcellular locations throughout the cell, thereby resulting in compartmentalized cellular signaling that triggers site-specific behavioral phenotypes. We developed a three-step engineering strategy to construct an optogenetic PKA (optoPKA) and demonstrated that, upon illumination, optoPKA migrates to specified intracellular sites. Furthermore, we designed intracellular spatially segregated reporters of PKA activity and confirmed that optoPKA phosphorylates these reporters in a light-dependent fashion.

The Plasma Membrane as a Reservoir, Protective Shield, and Light-Triggered Launch Pad for Peptide Therapeutics.

Although peptide-based therapeutics are finding increasing application in the clinic, extensive structural modification is typically required to prevent their rapid degradation by proteases in the blood. We have evaluated the ability of erythrocytes to serve as reservoirs, protective shields (against proteases), and light-triggered launch pads for peptides. We designed lipidated peptides that are anchored to the surface of red blood cells, which furnishes a protease-resistant environment.

Reduction in expression of the astrocyte glutamate transporter, GLT1, worsens functional and histological outcomes following traumatic spinal cord injury.

The astrocyte glutamate transporter, GLT1, is responsible for the vast majority of glutamate uptake in the adult central nervous system (CNS), thereby regulating extracellular glutamate homeostasis and preventing excitotoxicity. Glutamate dysregulation plays a central role in outcome following traumatic spinal cord injury (SCI). To determine the role of GLT1 in secondary cell loss following SCI, mice heterozygous for the GLT1 astrocyte glutamate transporter (GLT1+/-) and wild-type mice received thoracic crush SCI.