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  1. Abstract The vertebrate brain consists of diverse neuronal types, classified by distinct anatomy and function, along with divergent transcriptomes and proteomes. Defining the cell-type specific neuroproteomes is important for understanding the development and functional organization of neural circuits. This task remains challenging in complex tissue, due to suboptimal protein isolation techniques that often result in loss of cell-type specific information and incomplete capture of subcellular compartments. Here, we develop a genetically targeted proximity labeling approach to identify cell-type specific subcellular proteomes in the mouse brain, confirmed by imaging, electron microscopy, and mass spectrometry. We virally express subcellular-localized APEX2 to mapmore »the proteome of direct and indirect pathway spiny projection neurons in the striatum. The workflow provides sufficient depth to uncover changes in the proteome of striatal neurons following chemogenetic activation of Gα q -coupled signaling cascades. This method enables flexible, cell-type specific quantitative profiling of subcellular proteome snapshots in the mouse brain.« less
    Free, publicly-accessible full text available December 1, 2022
  2. Free, publicly-accessible full text available September 1, 2022
  3. Deformation twinning is a prevalent mode of plastic deformation in hexagonal close packed (HCP) magnesium. Twin domains are associated with significant lattice reorientation and localized shear. The theoretical misorientation angle for the most common 1012 tensile twin in magnesium is 86.3°. Through electron backscatter diffraction characterization of twinning microstructure, we show that the twin boundary misorientation at the twin tips is approximately 85°, and it is close to the theoretical value only along the central part of the twin. The variations in twin/matrix misorientation along the twin boundary control the twin thickening process by affecting the nucleation, glide of twinningmore »partials, and migration of twinning facets. To understand this observation, we employ a 3D crystal plasticity model with explicit twinning. The model successfully captures the experimentally observed misorientation variation, and it reveals that the twin boundary misorientation variations are governed by the local plasticity that accommodates the characteristic twin shear.« less
  4. Gram-negative bacteria expressing class A β-lactamases pose a serious health threat due to their ability to inactivate all β-lactam antibiotics. The acyl–enzyme intermediate is a central milestone in the hydrolysis reaction catalyzed by these enzymes. However, the protonation states of the catalytic residues in this complex have never been fully analyzed experimentally due to inherent difficulties. To help unravel the ambiguity surrounding class A β-lactamase catalysis, we have used ultrahigh-resolution X-ray crystallography and the recently approved β-lactamase inhibitor avibactam to trap the acyl–enzyme complex of class A β-lactamase CTX-M-14 at varying pHs. A 0.83-Å-resolution CTX-M-14 complex structure at pH 7.9more »revealed a neutral state for both Lys73 and Glu166. Furthermore, the avibactam hydroxylamine-O-sulfonate group conformation varied according to pH, and this conformational switch appeared to correspond to a change in the Lys73 protonation state at low pH. In conjunction with computational analyses, our structures suggest that Lys73 has a perturbed acid dissociation constant (pKa) compared with acyl–enzyme complexes with β-lactams, hindering its function to deprotonate Glu166 and the initiation of the deacylation reaction. Further NMR analysis demonstrated Lys73 pKato be ∼5.2 to 5.6. Together with previous ultrahigh-resolution crystal structures, these findings enable us to follow the proton transfer process of the entire acylation reaction and reveal the critical role of Lys73. They also shed light on the stability and reversibility of the avibactam carbamoyl acyl–enzyme complex, highlighting the effect of substrate functional groups in influencing the protonation states of catalytic residues and subsequently the progression of the reaction.

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  5. Twins in hexagonal close-packed polycrystals, most often nucleate at grain-boundaries (GBs), propagate into the grain and terminate at opposing GBs. Regularly, multiple parallel twins of the same variant form inside the same grain. When twins terminate inside the grains, rather than the grain boundary, they tend to form a staggered structure. Whether a staggered twin structure or the more common grain spanning twin structure forms can greatly affect mechanical behavior. In this work, the underlying mechanism for the formation of staggered twins is studied using an elasto-visco-plastic fast Fourier transform model, which quantifies the local stresses associated with 1012-type staggeredmore »twins in magnesium for different configurations. The model results suggest that when a twin tip is close to the lateral side of another twin, the driving force for twin propagation is significantly reduced. As a result, the staggered twin structure forms.« less