Unexpected Multivalent Display of Proteins by Temperature Triggered Self-Assembly of Elastin-like Polypeptide Block Copolymers

We report herein the unexpected temperature triggered self-assembly of proteins fused to thermally responsive elastin-like polypeptides (ELPs) into spherical micelles. Six ELP block copolymers (ELPBC) with different hydrophilic and hydrophobic block lengths were genetically fused to two single domain proteins, thioredoxin (Trx) and a fibronectin type III domain (Fn3) that binds the αvβ3 integrin. The self-assembly of these protein-ELPBC fusions as a function of temperature was investigated by UV spectroscopy, light scattering, and cryo-TEM. Self-assembly of the ELPBC was –unexpectedly- retained upon fusion to the two proteins, resulting in the formation of spherical micelles with a hydrodynamic radius that ranged from 24–37 nm, depending on the protein and ELPBC. Cryo-TEM images confirmed the formation of spherical particles with a size that was consistent with that measured by light scattering. The bioactivity of Fn3 was retained when presented by the ELPBC micelles as indicated by the enhanced uptake of the Fn3-decorated ELPBC micelles in comparison to the unimer by cells that overexpress the αvβ3 integrin. The fusion of single domain proteins to ELPBCs may provide a ubiquitous platform for the multivalent presentation of proteins

GPR56/ADGRG1 regulates development and maintenance of peripheral myelin

Myelin is a multilamellar sheath generated by specialized glia called Schwann cells (SCs) in the peripheral nervous system (PNS), which serves to protect and insulate axons for rapid neuronal signaling. In zebrafish and rodent models, we identify GPR56/ADGRG1 as a conserved regulator of PNS development and health. We demonstrate that, during SC development, GPR56-dependent RhoA signaling promotes timely radial sorting of axons. In the mature PNS, GPR56 is localized to distinct SC cytoplasmic domains, is required to establish proper myelin thickness, and facilitates organization of the myelin sheath. Furthermore, we define plectin-a scaffolding protein previously linked to SC domain organization, myelin maintenance, and a series of disorders termed "plectinopathies"-as a novel interacting partner of GPR56. Finally, we show that Gpr56 mutants develop progressive neuropathy-like symptoms, suggesting an underlying mechanism for peripheral defects in some human patients with GPR56 mutations. In sum, we define Gpr56 as a new regulator in the development and maintenance of peripheral myelin

Digital Switching of Local Arginine Density in a Genetically Encoded Self-Assembled Polypeptide Nanoparticle Controls Cellular Uptake

Cell-penetrating peptides (CPPs) are a class of molecules that enable efficient internalization of a wide variety of cargo in diverse cell types, making them desirable for delivery of anticancer drugs to solid tumors. For CPPs to be useful, it is important to be able to turn their function on in response to an external trigger that can be spatially localized in vivo. Here we describe an approach to turning on CPP function by modulation of the local density of arginine (Arg) residues by temperature-triggered micelle assembly of diblock copolymer elastin-like polypeptides (ELP_BCs). A greater than 8-fold increase in cellular uptake occurs when Arg residues are presented on the corona of ELP_BC micelles, as compared to the same ELP_BC at a temperature in which it is a soluble unimer. This approach is the first to demonstrate digital ‘off-on’ control of CPP activity by an extrinsic thermal trigger in a clinically relevant temperature range by modulation of the interfacial density of Arg residues on the exterior of a nanoparticle

Controlled Apoptosis by a Thermally Toggled Nanoscale Amplifier of Cellular Uptake

Internalization into cancer cells is a significant challenge in the delivery of many anticancer therapeutics. Drug carriers can address this challenge by facilitating cellular uptake of cytotoxic cargo in the tumor, while preventing cellular uptake in healthy tissues. Here we describe an extrinsically controlled drug carrier, a nanopeptifier, that amplifies cellular uptake by modulating the activity of cell-penetrating peptides with thermally toggled self-assembly of a genetically encoded polypeptide nanoparticle. When appended with a proapoptotic peptide, the nanopeptifier creates a cytotoxic switch, inducing apoptosis only in its self-assembled state. The nanopeptifier provides a new approach to tune the cellular uptake and activity of anticancer therapeutics by an extrinsic thermal trigger

Quantitative Mapping of the Spatial Distribution of Nanoparticles in Endo-Lysosomes by Local pH

Understanding the intracellular distribution and trafficking of nanoparticle drug carriers is necessary to elucidate their mechanisms of drug delivery and is helpful in the rational design of novel nanoparticle drug delivery systems. The traditional immunofluorescence method to study intracellular distribution of nanoparticles using organelle-specific antibodies is laborious and subject to artifacts. As an alternative, we developed a new method that exploits ratiometric fluorescence imaging of a pH-sensitive Lysosensor dye to visualize and quantify the spatial distribution of nanoparticles in the endosomes and lysosomes of live cells. Using this method, we compared the endolysosomal distribution of cell-penetrating peptide (CPP)-functionalized micelles to unfunctionalized micelles and found that CPP-functionalized micelles exhibited faster endosome-to-lysosome trafficking than unfunctionalized micelles. Ratiometric fluorescence imaging of pH-sensitive Lysosensor dye allows rapid quantitative mapping of nanoparticle distribution in endolysosomes in live cells while minimizing artifacts caused by extensive sample manipulation typical of alternative approaches. This new method can thus serve as an alternative to traditional immunofluorescence approaches to study the intracellular distribution and trafficking of nanoparticles within endosomes and lysosomes

Perspectives of Volunteer Firefighters during the COVID-19 Pandemic: Stumbling Blocks and Silver Linings

The COVID-19 pandemic has profoundly affected the lives of almost every individual in every nation, with numbers of infections continuing to grow. Across these nations, first responders are essential in their roles addressing emergencies, despite their risk of exposure to COVID-19 in the course of their work. We sought to understand the impacts of the COVID-19 pandemic on the lives of volunteer firefighters in the United States, an understudied group of these first responders. Interviews were conducted with volunteer firefighters between September and November 2021. Interviews were analyzed using deductive dominant thematic analysis. Thirty-three firefighters were interviewed who had an average of 22 years of service and a mean age of 52 years. Interviewees described pandemic-related challenges including the fear of COVID exposure and frustrations with work and personal relationships. They also identified unexpected work-related benefits including a deepened commitment to serve and improvements to training and safety. Further, some volunteers noted personal benefits such as developing stronger connections with others, having a new outlook on life, and observing goodwill. Our findings provide insight into the multifaceted and complex impact of the COVID-19 pandemic on volunteer firefighters