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Phelps JS, Hildebrand DGC, Graham BJ, Kuan AT, Thomas LA, Nguyen TM, Buhmann J, Azevedo AW, Sustar A, Agrawal S, Liu MG, Shanny BL, Funke J, Tuthill JC, Lee WCA
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Reconstruction of motor control circuits in adult Drosophila using automated transmission electron microscopy (opens in new window)

CELL 2021 FEB 4; 184(3):759-774.e18
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To investigate circuit mechanisms underlying locomotor behavior, we used serial-section electron microscopy (EM) to acquire a synapse-resolution dataset containing the ventral nerve cord (VNC) of an adult female Drosophila melanogaster. To generate this dataset, we developed GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM. Using this dataset, we studied neuronal networks that control leg and wing movements by reconstructing all 507 motor neurons that control the limbs. We show that a specific class of leg sensory neurons synapses directly onto motor neurons with the largest-caliber axons on both sides of the body, representing a unique pathway for fast limb control. We provide open access to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. We also provide GridTape instrumentation designs and software to make large-scale EM more accessible and affordable to the scientific community.
Li L, Liu HW, Krout M, Richmond JE, Wang Y, Bai JH, Weeratunga S, Collins BM, Ventimiglia D, Yu Y, Xia JY, Tang J, Liu J, Hu ZT
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A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release (opens in new window)

JOURNAL OF CELL BIOLOGY 2021 FEB 11; 220(4):? Article e202008121
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Ca2+-dependent neurotransmitter release requires synaptotagmins as Ca2+ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains-C2A and C2B-to Ca2+. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca2+ sensor in Caenorhabditis elegans. Here, we report a new Ca2+ sensor, SNT-3, which triggers delayed Ca2+-dependent neurotransmitter release. snt 1;snt 3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NM)s use a dual Ca2+ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca2+ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca2+. entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini-mediated membrane tethering. Our findings therefore reveal a novel dual Ca2+ sensor system in C. elegans and provide significant insights into Ca2+-regulated exocytosis.
Glickman JW, Dubin C, Renert-Yuval Y, Dahabreh D, Kimmel GW, Auyeung K, Estrada YD, Singer G, Krueger JG, Pavel AB, Guttman-Yassky E
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Cross-sectional study of blood biomarkers of patients with moderate to severe alopecia areata reveals systemic immune and cardiovascular biomarker dysregulation (opens in new window)

JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY 2021 FEB; 84(2):370-380
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Background: Although there is increased understanding of the alopecia areata (AA) pathogenesis based on studies in scalp tissues, little is known about its systemic profile. Objective: To evaluate the blood proteomic signature of AA and determine biomarkers associated with increased disease severity. Methods: In a cross-sectional study, we assessed 350 inflammatory and cardiovascular proteins using OLINK high-throughput proteomics in patients with moderate to severe AA (n = 35), as compared with healthy individuals (n = 36), patients with moderate to severe psoriasis (n = 19), and those with atopic dermatitis (n = 49). Results: Seventy-four proteins were significantly differentially expressed between AA and control individuals (false discovery rate, <.05) including innate immunity (interleukin [IL] 6/IL-8), T helper (Th) type 1 (interferon [IFN] gamma/CXCL9/CNCL10/CNCL11), Th2 (CCL13/CCL17/CCL7), Th17 (CCL20/P13/ S100Al2), and cardiovascular-risk proteins (OLR1/OSM/MPO/PRTN3). Eighty-six biomarkers correlated with AA clinical severity (P < .05), including Th1/Th2, and cardiovascular/atherosclerosis-related proteins, including SELP/PGLYRP1/MPO/IL-18/OSM (P < .05). Patients with AA totalis/universalis showed the highest systemic inflammatory tone, including cardiovascular risk biomarkers, compared to control individuals and even to patients with atopic dermatitis and those with psoriasis. The AA profile showed some Th1/Th2 differences in the setting of concomitant atopy. Limitations: Our analysis was limited to 350 proteins. Conclusion: This study defined the abnormalities of moderate to severe AA and associated circulatory biomarkers. It shows that AA has systemic immune, cardiovascular, and atherosclerosis biomarker dysregulation, suggesting the need for systemic treatment approaches.
van Straalen KR, Frew JW
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The Importance of Methodological Rigor in Proof-of-Concept Clinical Trials: A Lesson from Hidradenitis Suppurativa (opens in new window)

JOURNAL OF INVESTIGATIVE DERMATOLOGY 2021 FEB; 141(2):429-431
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Huang H, Zhang D, Weng YJ, Delaney K, Tang ZY, Yan C, Qi SK, Peng C, Cole PA, Roeder RG, Zhao YM
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The regulatory enzymes and protein substrates for the lysine beta-hydroxybutyrylation pathway (opens in new window)

SCIENCE ADVANCES 2021 FEB; 7(9):? Article eabe2771
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Metabolism-mediated epigenetic changes represent an adapted mechanism for cellular signaling, in which lysine acetylation and methylation have been the historical focus of interest. We recently discovered a beta-hydroxybutyrate-mediated epigenetic pathway that couples metabolism to gene expression. However, its regulatory enzymes and substrate proteins remain unknown, hindering its functional study. Here, we report that the acyltransferase p300 can catalyze the enzymatic addition of beta-hydroxybutyrate to lysine (Kbhb), while histone deacetylase 1 (HDAC1) and HDAC2 enzymatically remove Kbhb. We demonstrate that p300-dependent histone Kbhb can directly mediate in vitro transcription. Moreover, a comprehensive analysis of Kbhb substrates in mammalian cells has identified 3248 Kbhb sites on 1397 substrate proteins. The dependence of histone Kbhb on p300 argues that enzyme-catalyzed acylation is the major mechanism for nuclear Kbhb. Our study thus reveals key regulatory elements for the Kbhb pathway, laying a foundation for studying its roles in diverse cellular processes.
Lee YL, Ito K, Pi WC, Lin IH, Chu CS, Malik S, Cheng IH, Chen WY, Roeder RG
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Mediator subunit MED1 is required for E2A-PBX1-mediated oncogenic transcription and leukemic cell growth (opens in new window)

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2021 FEB 9; 118(6):? Article e1922864118
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The chimeric transcription factor E2A-PBX1, containing the N-terminal activation domains of E2A fused to the C-terminal DNA-binding domain of PBX1, results in 5% of pediatric acute lymphoblastic leukemias (ALL). We recently have reported a mechanism for RUNX1-dependent recruitment of E2A-PBX1 to chromatin in pre-B leukemic cells; but the subsequent E2A-PBX1 functions through various coactivators and the general transcriptional machinery remain unclear. The Mediator complex plays a critical role in cell-specific gene activation by serving as a key coactivator for gene-specific transcription factors that facilitates their function through the RNA polymerase II transcriptional machinery, but whether Mediator contributes to aberrant expression of E2A-PBX1 target genes remains largely unexplored. Here we show that Mediator interacts directly with E2A-PBX1 through an interaction of the MED1 subunit with an E2A activation domain. Results of MED1 depletion by CRISPR/Cas9 further indicate that MED1 is specifically required for E2A-PBX1-dependent gene activation and leukemic cell growth. Integrated transcriptome and cistrome analyses identify pre-B cell receptor and cell cycle regulatory genes as direct cotargets of MED1 and E2A-PBX1. Notably, complementary biochemical analyses also demonstrate that recruitment of E2A-PBX1 to a target DNA template involves a direct interaction with DNA-bound RUNX1 that can be further stabilized by EBF1. These findings suggest that E2A-PBX1 interactions with RUNX1 and MED1/Mediator are of functional importance for both gene-specific transcriptional activation and maintenance of E2A-PBX1-driven leukemia. The MED1 dependency for E2A-PBX1-mediated gene activation and leukemogenesis may provide a potential therapeutic opportunity by targeting MED1 in E2A-PBX1(+) pre-B leukemia.
Galea S, Vaughan R
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The Public Health Penalty of Attending to the Proximal (opens in new window)

AMERICAN JOURNAL OF PUBLIC HEALTH 2021 FEB; 111(2):193-194
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Lagresle-Peyrou C, Olichon A, Sadek H, Roche P, Tardy C, Da Silva C, Garrigue A, Fischer A, Moshous D, Collette Y, Picard C, Casanova JL, Andre I, Cavazzana M
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A gain-of-function RAC2 mutation is associated with bone marrow hypoplasia and an autosomal dominant form of severe combined immunodeficiency (opens in new window)

HAEMATOLOGICA 2021 FEB; 106(2):404-411
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Svere combined immunodeficiencies (SCID) constitute a heterogeneous group of life-threatening genetic disorders that typically present in the first year of life. They are defined by the absence of autologous T cells and the presence of an intrinsic or extrinsic defect in the B-cell compartment. In three newborns presenting with frequent infections and profound leukopenia, we identified a private, heterozygous mutation in the RAC2 gene (p.G12R). This mutation was de novo in the index case, who had been cured by hematopoietic stem cell transplantation but had transmitted the mutation to her sick daughter. Biochemical assays showed that the mutation was associated with a gain of function. The results of in vitro differentiation assays showed that RAC2 is essential for the survival and differentiation of hematopoietic stem/progenitor cells. Therefore, screening for RAC2 gain-of-function mutations should be considered in patients with a SCID phenotype and who lack a molecular diagnosis.
Wang ZJ, Schmidt F, Weisblum Y, Muecksch F, Barnes CO, Finkin S, Schaefer-Babajew D, Cipolla M, Gaebler C, Lieberman JA, Oliveira TY, Yang Z, Abernathy ME, Huey-Tubman KE, Hurley A, Turroja M, West KA, Gordon K, Millard KG, Ramos V, Da Silva J, Xu JL, Colbert RA, Patel R, Dizon J, Unson-O'Brien C, Shimeliovich I, Gazumyan A, Caskey M, Bjorkman PJ, Casellas R, Hatziioannou T, Bieniasz PD, Nussenzweig MC
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mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants (opens in new window)

NATURE 2021; 592(7855):616-622
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Here we report on the antibody and memory B cell responses of a cohort of 20 volunteers who received the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccine against SARS-CoV-2(1-4). Eight weeks after the second injection of vaccine, volunteers showed high levels of IgM and IgG anti-SARS-CoV-2 spike protein (S) and receptor-binding-domain (RBD) binding titre. Moreover, the plasma neutralizing activity and relative numbers of RBD-specific memory B cells of vaccinated volunteers were equivalent to those of individuals who had recovered from natural infection(5,6). However, activity against SARS-CoV-2 variants that encode E484K-, N501Y- or K417N/E484K/N501-mutant S was reduced by a small-but significant-margin. The monoclonal antibodies elicited by the vaccines potently neutralize SARS-CoV-2, and target a number of different RBD epitopes in common with monoclonal antibodies isolated from infected donors(5-8). However, neutralization by 14 of the 17 most-potent monoclonal antibodies that we tested was reduced or abolished by the K417N, E484K or N501Y mutation. Notably, these mutations were selected when we cultured recombinant vesicular stomatitis virus expressing SARS-CoV-2 S in the presence of the monoclonal antibodies elicited by the vaccines. Together, these results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid a potential loss of clinical efficacy.
Showalter K, Spiera R, Magro C, Agius P, Martyanov V, Franks JM, Sharma R, Geiger H, Wood TA, Zhang YX, Hale CR, Finik J, Whitfield ML, Orange DE, Gordon JK
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Machine learning integration of scleroderma histology and gene expression identifies fibroblast polarisation as a hallmark of clinical severity and improvement (opens in new window)

ANNALS OF THE RHEUMATIC DISEASES 2021 FEB; 80(2):228-237
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Objective We sought to determine histologic and gene expression features of clinical improvement in early diffuse cutaneous systemic sclerosis (dcSSc; scleroderma). Methods Fifty-eight forearm biopsies were evaluated from 26 individuals with dcSSc in two clinical trials. Histologic/immunophenotypic assessments of global severity, alpha-smooth muscle actin (aSMA), CD34, collagen, inflammatory infiltrate, follicles and thickness were compared with gene expression and clinical data. Support vector machine learning was performed using scleroderma gene expression subset (normal-like, fibroproliferative, inflammatory) as classifiers and histology scores as inputs. Comparison of w-vector mean absolute weights was used to identify histologic features most predictive of gene expression subset. We then tested for differential gene expression according to histologic severity and compared those with clinical improvement (according to the Combined Response Index in Systemic Sclerosis). Results aSMA was highest and CD34 lowest in samples with highest local Modified Rodnan Skin Score. CD34 and aSMA changed significantly from baseline to 52 weeks in clinical improvers. CD34 and aSMA were the strongest predictors of gene expression subset, with highest CD34 staining in the normal-like subset (p<0.001) and highest aSMA staining in the inflammatory subset (p=0.016). Analysis of gene expression according to CD34 and aSMA binarised scores identified a 47-gene fibroblast polarisation signature that decreases over time only in improvers (vs non-improvers). Pathway analysis of these genes identified gene expression signatures of inflammatory fibroblasts. Conclusion CD34 and aSMA stains describe distinct fibroblast polarisation states, are associated with gene expression subsets and clinical assessments, and may be useful biomarkers of clinical severity and improvement in dcSSc.