Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first expansion microscopy method was unable to retain native proteins in the gel and used custom made reagents not widely available. Here, we describe protein retention ExM (proExM), a variant of ExM that anchors proteins to the swellable gel allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validate and demonstrate utility of proExM for multi-color super-resolution (~70 nm) imaging of cells and mammalian tissues on conventional microscopes.

PF-06651600 was developed as an irreversible inhibitor of JAK3 with selectivity over the other three JAK isoforms. A high level of selectivity toward JAK3 is achieved by the covalent interaction of PF-06651600 with a unique cysteine residue (Cys-909) in the catalytic domain of JAK3, which is replaced by a serine residue in the other JAK isoforms. Importantly, 10 other kinases in the kinome have a cysteine at the equivalent position of Cys-909 in JAK3. Five of those kinases belong to the TEC kinase family including BTK, BMX, ITK, RLK, and TEC and are also inhibited by PF-06651600. Preclinical data demonstrate that inhibition of the cytolytic function of CD8+ T cells and NK cells by PF-06651600 is driven by the inhibition of TEC kinases. On the basis of the underlying pathophysiology of inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, alopecia areata, and vitiligo, the dual activity of PF-06651600 toward JAK3 and the TEC kinase family may provide a beneficial inhibitory profile for therapeutic intervention.

Protein S-nitrosation (SNO-protein), the nitric oxide-mediated posttranslational modification of cysteine thiols, is an important regulatory mechanism of protein function in both physiological and pathological pathways. A key first step toward elucidating the mechanism by which S-nitrosation modulates a protein's function is identification of the targeted cysteine residues. Here, we present a strategy for the simultaneous identification of SNO-cysteine sites and their cognate proteins to profile the brain of the CK-p25-inducible mouse model of Alzheimer's disease-like neurodegeneration. The approach-SNOTRAP (SNO trapping by triaryl phosphine)-is a direct tagging strategy that uses phosphine-based chemical probes, allowing enrichment of SNO-peptides and their identification by liquid chromatography tandem mass spectrometry. SNOTRAP identified 313 endogenous SNO-sites in 251 proteins in the mouse brain, of which 135 SNO-proteins were detected only during neurodegeneration. S-nitrosation in the brain shows regional differences and becomes elevated during early stages of neurodegeneration in the CK-p25 mouse. The SNO-proteome during early neurodegeneration identified increased S-nitrosation of proteins important for synapse function, metabolism, and Alzheimer's disease pathology. In the latter case, proteins related to amyloid precursor protein processing and secretion are S-nitrosated, correlating with increased amyloid formation. Sequence analysis of SNO-cysteine sites identified potential linear motifs that are altered under pathological conditions. Collectively, SNOTRAP is a direct tagging tool for global elucidation of the SNO-proteome, providing functional insights of endogenous SNO proteins in the brain and its dysregulation during neurodegeneration.S-nitrosation | Alzheimer's disease | secretase pathway | presenilin pathway | neurodegeneration P rotein S-nitrosation (SNO-protein), in which a cysteine (Cys) thiol is converted to a nitrosothiol (RSNO), is an important posttranslational modification (PTM). Cys residues targeted for S-nitrosation often impact enzyme activity, protein localization, and protein-protein interactions (1). SNO begins with the production of nitric oxide radicals (NO • ) via conversion of L-arginine to L-citrulline by nitric oxide synthase 1 (NOS1) (neuronal), NOS2 (inducible), and NOS3 (endothelial). NO-mediated SNO PTMs are thought to occur in vivo predominantly through radical recombination between NO • and a thiyl radical, transnitrosation by low-molecular weight NO carriers such as S-nitrosoglutathione (GSNO), or protein-assisted transnitrosation (2-8). In the healthy brain, low levels of NO and normal SNO PTMs play important roles in regulating synaptic plasticity, gene expression, and neuronal survival. In contrast, elevated NO levels associated with aging and environmental stress have been linked to neurological pathologies, including Alzheimer's (AD), Parkinson's, and Huntington's disease (9). AD is the most prevalent form of human dementia, with a frequency that progressiv...

Abstract1,2,3,4-Diepoxybutane (DEB)1 is considered the ultimate carcinogenic metabolite of 1,3-butadiene, an important industrial chemical and environmental pollutant present in urban air. Although it preferentially modifies guanine within DNA, DEB induces a large number of A → T transversions, suggesting that it forms strongly mispairing lesions at adenine nucleobases. We now report the discovery of three potentially mispairing exocyclic adenine lesions of DEB:. The structures and stereochemistry of the novel DEB-dA adducts were determined by a combination of UV and NMR spectroscopy, tandem mass spectrometry, and independent synthesis. We found that synthetic N 6 -(2-hydroxy-3,4-epoxybut-1-yl)-2′-deoxyadenosine (compound 1) representing the product of N 6 -adenine alkylation by DEB spontaneously cyclizes to form 3 under aqueous conditions or 2 under anhydrous conditions in the presence of organic base. Compound 3 can be interconverted with 4 by a reversible unimolecular pericyclic reaction favoring 4 as a more thermodynamically stable product. Both 3 and 4 are present in double stranded DNA treated with DEB in vitro and in liver DNA of laboratory mice exposed to 1,3-butadiene by inhalation. We propose that in DNA under physiological conditions, DEB alkylates the N-1 position of adenine in DNA to form N1- (2-hydroxy-3,4-epoxybut-1-yl)adenine adducts, which undergo an S N 2-type intramolecular nucleophilic substitution and rearrangement to give 3 (minor) and 4 (major). Formation of exocyclic DEB-adenine lesions following exposure to 1,3-butadiene provides a possible mechanism of mutagenesis at the A:T base pairs.

adducts are formed in DNA by 1,2,3,4-diepoxybutane (metabolite of human carcinogen 1,3-butadiene). Results: hpols and carry out translesion synthesis, incorporating T, G, or A opposite the 1,N