Oxidative stress is a central part of innate-immune induced neurodegeneration. However, the transcriptomic landscape of the central nervous system (CNS) innate immune cells contributing to oxidative stress is unknown, and therapies to target their neurotoxic functions are not widely available. Here, we provide the oxidative stress innate immune cell atlas in neuroinflammatory disease, and report the discovery of new druggable pathways. Transcriptional profiling of oxidative stress-producing CNS innate immune cells (Tox-seq) identified a core oxidative stress gene signature coupled to coagulation and glutathione pathway genes shared between a microglia cluster and infiltrating macrophages. Tox-seq followed by a microglia high-throughput screen (HTS) and oxidative stress gene network analysis, identified the glutathione regulating compound acivicin with potent therapeutic effects decreasing oxidative stress and axonal damage in chronic and relapsing multiple sclerosis (MS) models. Thus, oxidative stress transcriptomics identified neurotoxic CNS innate immune populations and may enable the discovery of selective neuroprotective strategies.
Much research aimed at discovering the genetic bases of longevity focuses on the budding yeast Saccharomyces cerevisiae. Unfortunately, yeast researchers use a definition of longevity not applied to other species. We propose here a method that makes it possible to estimate for yeast the same measures of longevity calculated for other species. We also show that the conventional method (equating longevity with the number of offspring) is only an approximate measure of true chronological lifespan. Our method will allow results for yeast to be compared more correctly with those for other species.
Production of E1-deleted adenovirus (rAd) vectors requires complementation by E1A and E1B functions provided by the production cell line. The two cell lines most commonly used for production of rAd vectors, 293 and Per.C6, were derived from human primary cells and contain contiguous E1A and E1B sequences from the Ad genome. As an alternative system, we tested complementation of rAd vectors using sequential transfection of individual E1A and E1B expression cassettes into A549 human lung tumor cells, which support highly efficient replication of wild type adenovirus. We found that E1A function could be complemented in A549 cells by the mutant E1Adl01/07, and that E1B function could be provided in such cells using only the 55K E1B gene. Production yields in the resulting producer cell line, designated SL0003, were similar to those obtained from 293 cells without generation of detectable recombinant replication competent adenovirus.
Prothymosin-α is a small, multifunctional intrinsically disordered protein associated with cell survival and proliferation which binds multiple Zn ions and undergoes partial folding. The interaction between prothymosin-α and at least two of its protein targets is significantly enhanced in the presence of Zn ions, suggesting that Zn binding plays a role in the protein's function. The primary sequence of prothymosin-α is highly acidic, with almost 50% comprised of Asp and Glu, and is unusual for a Zn-binding protein as it lacks Cys and His residues. To gain a better understanding of the nature of the Zn-prothymosin-α interactions and the protein's ability to discriminate Zn over other divalent cations (e.g., Ca, Co, Mg) we synthesized a set of three model peptides and characterized the effect of metal binding using electrospray ionization mass spectrometry (ESI MS) and circular dichroism (CD) spectroscopy. ESI MS data reveal that the native peptide model of the glutamic acid rich region binds 4 Zn ions with apparent, stepwise K values that are, at highest, in the tens of micromolar range. A peptide model with the same amino acid composition as the native sequence, but with the residues arranged randomly, showed no evidence of structural change by CD upon introduction of Zn. These results suggest that the high net negative charge of the glutamic acid-rich region of prothymosin-α is not a sufficient criterion for Zn to induce a structural change; rather, Zn binding to prothymosin-α is sequence specific, providing important insight into the behavior of intrinsically disordered proteins.
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