Ferromagnetism in Laves-phase WFe\u3csub\u3e2\u3c/sub\u3e nanoparticles

While rare-earth based Laves phases are known to exhibit large magnetostriction, the magnetic properties of some binary Laves phases containing transition metals alone are not well known. This is because many of these compounds contain refractory elements that complicate melt processing due to high melting temperatures and extensive phase separation. Here, phase-pure WFe2 nanoclusters, with the hexagonal C14 Laves structure, were deposited via inert gas condensation, allowing for the first known measurement of ferromagnetism in this phase, with MS of 26.4 emu/g (346 emu/cm3) and a KU of 286 kerg/cm3, at 10 K, and a TC of 550 K

Radiochemical Approaches to Imaging Bacterial Infections: Intracellular versus Extracellular Targets

The discovery of penicillin began the age of antibiotics, which was a turning point in human healthcare. However, to this day, microbial infections are still a concern throughout the world, and the rise of multidrug-resistant organisms is an increasing challenge. To combat this threat, diagnostic imaging tools could be used to verify the causative organism and curb inappropriate use of antimicrobial drugs. Nuclear imaging offers the sensitivity needed to detect small numbers of bacteria in situ. Among nuclear imaging tools, radiolabeled antibiotics traditionally have lacked the sensitivity or specificity necessary to diagnose bacterial infections accurately. One reason for the lack of success is that the antibiotics were often chelated to a radiometal. This was done without addressing the ramifications of how the radiolabeling would impact probe entry to the bacterial cell, or the mechanism of binding to an intracellular target. In this review, we approach bacterial infection imaging through the lens of bacterial specific molecular targets, their intracellular or extracellular location, and discuss radiochemistry strategies to guide future probe development

Intracellular Context Affects Levels of a Chemically Dependent Destabilizing Domain

<div><p>The ability to regulate protein levels in live cells is crucial to understanding protein function. In the interest of advancing the tool set for protein perturbation, we developed a protein destabilizing domain (DD) that can confer its instability to a fused protein of interest. This destabilization and consequent degradation can be rescued in a reversible and dose-dependent manner with the addition of a small molecule that is specific for the DD, Shield-1. Proteins encounter different local protein quality control (QC) machinery when targeted to cellular compartments such as the mitochondrial matrix or endoplasmic reticulum (ER). These varied environments could have profound effects on the levels and regulation of the cytoplasmically derived DD. Here we show that DD fusions in the cytoplasm or nucleus can be efficiently degraded in mammalian cells; however, targeting fusions to the mitochondrial matrix or ER lumen leads to accumulation even in the absence of Shield-1. Additionally, we characterize the behavior of the DD with perturbants that modulate protein production, degradation, and local protein QC machinery. Chemical induction of the unfolded protein response in the ER results in decreased levels of an ER-targeted DD indicating the sensitivity of the DD to the degradation environment. These data reinforce that DD is an effective tool for protein perturbation, show that the local QC machinery affects levels of the DD, and suggest that the DD may be a useful probe for monitoring protein quality control machinery.</p> </div

N- and C-terminal DDs targeted to the mitochondria.

<p>(A) Fluorescence micrographs of mDDn cells in the presence and absence of Shield-1 (1 µM). The overlay image shows mDD (XFP, green), Mitotracker Orange (red), and Hoechst stain (blue). (B) Fluorescence micrographs of mDDc cells as in (A). (C) Flow cytometry of mDDn cells with Shield-1 (2 µM) or vehicle control after a 6 hour incubation. Cells were co-treated with cycloheximide (CHX, 5 µg/mL), MG132 (5 µM), or both CHX and MG132. (D) Flow cytometry of mDDn cells exposed to small molecules as in (C). Micrograph scale bars indicate 10 microns. Error bars represent ± S.E.M. (n = 3).</p

ER and secreted destabilizing domains.

<p>(A) Fluorescence micrographs of eDD cells. The overlay image shows eDD (green), ER-Tracker Red (red), and Hoechst stain (blue). (B) Flow cytometry of eDD cells with Shield-1 (2 µM) or vehicle control after a 6 hour incubation. Cells were co-treated with cycloheximide (CHX, 5 µg/mL), MG132 (5 µM), or brefeldin-A (BFA, 2.5 µg/mL). * P-value<0.05, ** P-value<0.005. (C) Bioluminescence quantification of media from eDDs cells after exposure to vehicle control, Shield-1 (1 µM), CHX (1 µg/mL), or co-treatment with both Shield-1 and CHX. (D) Bioluminescence quantification of media from eDDs cells after exposure to vehicle control, Shield-1 (1 µM), MG132 (1 µM), or co-treatment with both. Error bars represent ± S.E.M. (n = 3).</p

Regulation of eDHFR-tagged proteins with trimethoprim PROTACs

Abstract Temporal control of protein levels in cells and living animals can be used to improve our understanding of protein function. In addition, control of engineered proteins could be used in therapeutic applications. PRoteolysis-TArgeting Chimeras (PROTACs) have emerged as a small-molecule-driven strategy to achieve rapid, post-translational regulation of protein abundance via recruitment of an E3 ligase to the target protein of interest. Here, we develop several PROTAC molecules by covalently linking the antibiotic trimethoprim (TMP) to pomalidomide, a ligand for the E3 ligase, Cereblon. These molecules induce degradation of proteins of interest (POIs) genetically fused to a small protein domain, E. coli dihydrofolate reductase (eDHFR), the molecular target of TMP. We show that various eDHFR-tagged proteins can be robustly degraded to 95% of maximum expression with PROTAC molecule 7c. Moreover, TMP-based PROTACs minimally affect the expression of immunomodulatory imide drug (IMiD)-sensitive neosubstrates using proteomic and biochemical assays. Finally, we show multiplexed regulation with another known degron-PROTAC pair, as well as reversible protein regulation in a rodent model of metastatic cancer, demonstrating the formidable strength of this system. Altogether, TMP PROTACs are a robust approach for selective and reversible degradation of eDHFR-tagged proteins in vitro and in vivo