Trends in Racial and Ethnic Diversity in Vascular Neurology Fellowships From 2006 to 2018: A Cross-Sectional Analysis.

BACKGROUND: Workforce diversity in vascular neurology is a crucial component of reducing disparities in stroke care and outcomes. The objective of this study is to describe trends in the racial and ethnic diversity of neurology residents pursuing vascular neurology fellowship and propose an actionable plan for improvement. METHODS: This was a cross-sectional study of race/ethnicity of neurology residents and vascular neurology fellows using published Graduate Medical Education census reports from 2006, when race/ethnicity data were first included, to 2018. Percentage of trainees underrepresented in medicine are reported for 3-year epochs and were analyzed using the Cochran-Armitage test (χ2 test for trend). RESULTS: Across the study period, underrepresented in medicine representation has not changed significantly among all neurology residents and subspecialty fellows (11.9% in 2006-2009; 12.5% in 2015-2018, P=0.82) nor among neurology residents alone (12.0% in 2006-2009; 12.6% in 2015-2018, P=0.81). Among vascular neurology fellows, however, there was a significant downtrend of underrepresented in medicine representation from 16.9% in 2006 to 2009 to 9.3% in 2015 to 2018 (P=0.013). CONCLUSIONS: Racial/ethnic underrepresentation among all neurology residents as well as those pursuing vascular neurology fellowship has persisted across the study period. Concerted efforts should be pursued to increase diversity in neurology residents and vascular neurology fellowship training.

Approach to Loss of Consciousness: Distinguishing Epileptic Seizures, Psychogenic Nonepileptic Seizures, and Syncope.

Transient loss of consciousness (TLOC) is a common emergent neurological issue, which can be attributed to syncope, epileptic seizures, and psychogenic nonepileptic seizures. The purpose of this article is to outline an approach to diagnosing the most common etiologies of TLOC by focusing on the importance of the history and physical examination, as well as targeted diagnostic tests.

Developing the Neurology Diversity Officer: A Roadmap for Academic Neurology Departments.

Academic Neurology Departments must confront the challenges of developing a diverse workforce, reducing inequity and discrimination within academia, and providing neurologic care for an increasingly diverse society. A neurology diversity officer should have a specific role and associated title within a neurology department as well as a mandate to focus their efforts on issues of equity, diversity and inclusion that affect staff, trainees and faculty. This role is expansive and works across departmental missions but it has many challenges related to structural intolerance and cultural gaps. In this review, we describe the many challenges that diversity officers face and how they might confront them. We delineate the role and duties of the neurology diversity officer and provide a guide to departmental leaders on how to assess qualifications and evaluate progress. Finally, we describe the elements necessary for success. A neurology diversity officer should have the financial, administrative and emotional support of leadership in order for them to carry out their mission and to truly have a positive influence.

Architecture of TAF11/TAF13/TBP complex suggests novel regulation properties of general transcription factor TFIID.

General transcription factor TFIID is a key component of RNA polymerase II transcription initiation. Human TFIID is a megadalton-sized complex comprising TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). TBP binds to core promoter DNA, recognizing the TATA-box. We identified a ternary complex formed by TBP and the histone fold (HF) domain-containing TFIID subunits TAF11 and TAF13. We demonstrate that TAF11/TAF13 competes for TBP binding with TATA-box DNA, and also with the N-terminal domain of TAF1 previously implicated in TATA-box mimicry. In an integrative approach combining crystal coordinates, biochemical analyses and data from cross-linking mass-spectrometry (CLMS), we determine the architecture of the TAF11/TAF13/TBP complex, revealing TAF11/TAF13 interaction with the DNA binding surface of TBP. We identify a highly conserved C-terminal TBP-interaction domain (CTID) in TAF13, which is essential for supporting cell growth. Our results thus have implications for cellular TFIID assembly and suggest a novel regulatory state for TFIID function.

The relationship between mood disorder and insomnia depends on race in US veterans with epilepsy.

PURPOSE: Few data exist on race, medical/psychiatric comorbidities, and insomnia symptoms in US veterans with epilepsy. Our aims were to examine 1) whether insomnia symptom prevalence was different between Black and White veterans and 2) whether predictors of insomnia symptoms varied by race. METHODS: This retrospective, cross-sectional study included veterans evaluated in an epilepsy clinic over the course of 1.5years. Individuals completed standardized assessments for epilepsy and sleep complaints. Insomnia criteria were met by 1) report of difficulty with sleep initiation, maintenance, or premature awakenings accompanied by daytime impairment or 2) sedative-hypnotic use on most days of the month. Demographics, medical/psychiatric comorbidities, and medications were determined per electronic medical record review. Hierarchical multivariable logistic regression analyses were performed to determine if race, medical/mental health comorbidities, and the potential interaction of race with each comorbid condition were associated with insomnia. RESULTS: Our sample consisted of 165 veterans (32% Black). The unadjusted prevalence of insomnia was not different between Black and White veterans (42% vs 39%, p=0.68). In adjusted analyses, the association between mood disorder and insomnia varied by race. Depressed White veterans had over 11-times higher predicted odds of insomnia (OR 11.4, p<0.001) than non-depressed White veterans, while depressed Black veterans had 4-times higher predicted odds of insomnia (OR 4.1, p=0.06) than non-depressed Black veterans. Although mood disorder diagnosis was associated with insomnia for both racial groups, White veterans had a stronger association between mood disorder diagnosis and insomnia than Black veterans. CONCLUSIONS: The relationship between mood disorder diagnosis and insomnia was stronger for White than Black veterans with epilepsy. Future studies are needed to explore mental health symptoms and psychosocial determinants of insomnia with larger samples of minority individuals with epilepsy.

The interchange of immunophilins leads to parallel pathways and different intermediates in the assembly of Hsp90 glucocorticoid receptor complexes.

Hormone receptors require participation of the chaperones Hsp40/Hsp70 to form client-transfer complexes with Hsp90/Hop. Interaction with the co-chaperone p23 releases Hop and Hsp70, and the immunophilin FKBP52 mediates transfer of the Hsp90-receptor complex to the nucleus. Inhibition of glucocorticoid receptor (GR) transport by FKBP51, but not by FKBP52, has been observed at the cellular level, but the subunit composition of the intermediates involved has not been deduced. Here we use mass spectrometry to show that FKBP51/52 form analogous complexes with GR/Hsp90/Hop/Hsp70/ATP, but differences emerge upon addition of p23 to client-transfer complexes. When FKBP51 is present, a stable intermediate is formed (FKBP51)1(GR)1(Hsp90)2(p23)2 by expulsion of Hsp70 and Hop. By contrast, in the presence of FKBP52, ejection of p23 also takes place to form the nuclear transfer complex (FKBP52)1(GR)1(Hsp90)2. Our results are therefore consistent with pathways in which FKBP51/52 are interchangeable during the early assembly reactions. Following interaction with p23, however, the pathways diverge with FKBP51 sequestering GR in a stable intermediate complex with p23. By contrast, binding of FKBP52 occurs almost concomitantly with release of p23 to form a highly dynamic transfer complex, primed for interaction with the dynactin transport machinery.

Structural evidence for Nap1-dependent H2A-H2B deposition and nucleosome assembly.

Nap1 is a histone chaperone involved in the nuclear import of H2A-H2B and nucleosome assembly. Here, we report the crystal structure of Nap1 bound to H2A-H2B together with in vitro and in vivo functional studies that elucidate the principles underlying Nap1-mediated H2A-H2B chaperoning and nucleosome assembly. A Nap1 dimer provides an acidic binding surface and asymmetrically engages a single H2A-H2B heterodimer. Oligomerization of the Nap1-H2A-H2B complex results in burial of surfaces required for deposition of H2A-H2B into nucleosomes. Chromatin immunoprecipitation-exonuclease (ChIP-exo) analysis shows that Nap1 is required for H2A-H2B deposition across the genome. Mutants that interfere with Nap1 oligomerization exhibit severe nucleosome assembly defects showing that oligomerization is essential for the chaperone function. These findings establish the molecular basis for Nap1-mediated H2A-H2B deposition and nucleosome assembly.

Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90.

Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90.

Cytoplasmic TAF2-TAF8-TAF10 complex provides evidence for nuclear holo-TFIID assembly from preformed submodules.

General transcription factor TFIID is a cornerstone of RNA polymerase II transcription initiation in eukaryotic cells. How human TFIID-a megadalton-sized multiprotein complex composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs)-assembles into a functional transcription factor is poorly understood. Here we describe a heterotrimeric TFIID subcomplex consisting of the TAF2, TAF8 and TAF10 proteins, which assembles in the cytoplasm. Using native mass spectrometry, we define the interactions between the TAFs and uncover a central role for TAF8 in nucleating the complex. X-ray crystallography reveals a non-canonical arrangement of the TAF8-TAF10 histone fold domains. TAF2 binds to multiple motifs within the TAF8 C-terminal region, and these interactions dictate TAF2 incorporation into a core-TFIID complex that exists in the nucleus. Our results provide evidence for a stepwise assembly pathway of nuclear holo-TFIID, regulated by nuclear import of preformed cytoplasmic submodules.

The p97-FAF1 protein complex reveals a common mode of p97 adaptor binding.

p97, also known as valosin-containing protein, is a versatile participant in the ubiquitin-proteasome system. p97 interacts with a large network of adaptor proteins to process ubiquitylated substrates in different cellular pathways, including endoplasmic reticulum-associated degradation and transcription factor activation. p97 and its adaptor Fas-associated factor-1 (FAF1) both have roles in the ubiquitin-proteasome system during NF-κB activation, although the mechanisms are unknown. FAF1 itself also has emerging roles in other cell-cycle pathways and displays altered expression levels in various cancer cell lines. We have performed a detailed study the p97-FAF1 interaction. We show that FAF1 binds p97 stably and in a stoichiometry of 3 to 6. Cryo-EM analysis of p97-FAF1 yielded a 17 Å reconstruction of the complex with FAF1 above the p97 ring. Characteristics of p97-FAF1 uncovered in this study reveal common features in the interactions of p97, providing mechanistic insight into how p97 mediates diverse functionalities.

The N-terminal region of the human autophagy protein ATG16L1 contains a domain that folds into a helical structure consistent with formation of a coiled-coil.

Autophagy is a fundamental cellular process required for organelle degradation and removal of invasive pathogens. Autophagosome formation involves the recruitment of, and interaction between, multiple proteins produced from autophagy-related (ATG) genes. One of the key complexes in autophagosome formation is the ATG12-ATG5-ATG16L1 complex. ATG16L1 functions as a molecular scaffold mediating protein-protein interactions necessary for formation of the autophagosome in response to both classical and pathogen-related autophagy stimuli. The coiled-coil domain of the yeast ortholog, ATG16, exists as a homodimer both in solution and in the crystal form. The yeast and human orthologs show poor sequence identity. Here we have sought to determine the minimal boundaries of the human ATG16L1 coiled-coil domain and ascertain its oligomeric status in solution. Using a range of biochemical and biophysical techniques we show that the secondary structure of the human ATG16L1 coiled-coil has the expected helical composition and that the domain forms a homodimer in solution. We also observe extensive sequence conservation across vertebrates providing strong support for the crucial functional role of the ATG16L1 coiled-coil.

Structural basis for DNA recognition and loading into a viral packaging motor.

Genome packaging into preformed viral procapsids is driven by powerful molecular motors. The small terminase protein is essential for the initial recognition of viral DNA and regulates the motor's ATPase and nuclease activities during DNA translocation. The crystal structure of a full-length small terminase protein from the Siphoviridae bacteriophage SF6, comprising the N-terminal DNA binding, the oligomerization core, and the C-terminal ß-barrel domains, reveals a nine-subunit circular assembly in which the DNA-binding domains are arranged around the oligomerization core in a highly flexible manner. Mass spectrometry analysis and four further crystal structures show that, although the full-length protein exclusively forms nine-subunit assemblies, protein constructs missing the C-terminal ß-barrel form both nine-subunit and ten-subunit assemblies, indicating the importance of the C terminus for defining the oligomeric state. The mechanism by which a ring-shaped small terminase oligomer binds viral DNA has not previously been elucidated. Here, we probed binding in vitro by using EPR and surface plasmon resonance experiments, which indicated that interaction with DNA is mediated exclusively by the DNA-binding domains and suggested a nucleosome-like model in which DNA binds around the outside of the protein oligomer.

Heterogeneity and dynamics in the assembly of the heat shock protein 90 chaperone complexes.

The Hsp90 cycle depends on the coordinated activity of a range of cochaperones, including Hop, Hsp70 and peptidyl-prolyl isomerases such as FKBP52. Using mass spectrometry, we investigate the order of addition of these cochaperones and their effects on the stoichiometry and composition of the resulting Hsp90-containing complexes. Our results show that monomeric Hop binds specifically to the Hsp90 dimer whereas FKBP52 binds to both monomeric and dimeric forms of Hsp90. By preforming Hsp90 complexes with either Hop, followed by addition of FKBP52, or with FKBP52 and subsequent addition of Hop, we monitor the formation of a predominant asymmetric ternary complex containing both cochaperones. This asymmetric complex is subsequently able to interact with the chaperone Hsp70 to form quaternary complexes containing all four proteins. Monitoring the population of these complexes during their formation and at equilibrium allows us to model the complex formation and to extract 14 different K(D) values. This simultaneous calculation of the K(D)s from a complex system with the same method, from eight deferent datasets under the same buffer conditions delivers a self-consistent set of values. In this case, the K(D) values afford insights into the assembly of ten Hsp90-containing complexes and provide a rationale for the cellular heterogeneity and prevalence of intermediates in the Hsp90 chaperone cycle.

Structures of SAS-6 suggest its organization in centrioles.

Centrioles are cylindrical, ninefold symmetrical structures with peripheral triplet microtubules strictly required to template cilia and flagella. The highly conserved protein SAS-6 constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. We determined the x-ray structure of the amino-terminal domain of SAS-6 from zebrafish, and we show that recombinant SAS-6 self-associates in vitro into assemblies that resemble cartwheel centers. Point mutations are consistent with the notion that centriole formation in vivo depends on the interactions that define the self-assemblies observed here. Thus, these interactions are probably essential to the structural organization of cartwheel centers.