ERAP2 increases the abundance of a peptide submotif highly selective for the Birdshot Uveitis-associated HLA-A29

Birdshot Uveitis (BU) is a blinding inflammatory eye condition that only affects HLA-A29-positive individuals. Genetic association studies linked ERAP2 with BU, an aminopeptidase which trims peptides before their presentation by HLA class I at the cell surface, which suggests that ERAP2-dependent peptide presentation by HLA-A29 drives the pathogenesis of BU. However, it remains poorly understood whether the effects of ERAP2 on the HLA-A29 peptidome are distinct from its effect on other HLA allotypes. To address this, we focused on the effects of ERAP2 on the immunopeptidome in patient-derived antigen presenting cells. Using complementary HLA-A29-based and pan-class I immunopurifications, isotope-labeled naturally processed and presented HLA-bound peptides were sequenced by mass spectrometry. We show that the effects of ERAP2 on the N-terminus of ligands of HLA-A29 are shared across endogenous HLA allotypes, but discover and replicate that one peptide motif generated in the presence of ERAP2 is specifically bound by HLA-A29. This motif can be found in the amino acid sequence of putative autoantigens. We further show evidence for internal sequence specificity for ERAP2 imprinted in the immunopeptidome. These results reveal that ERAP2 can generate an HLA-A29-specific antigen repertoire, which supports that antigen presentation is a key disease pathway in BU.Proteomic

THE S. FYODOROV EYE MICROSURGERY COMPLEX–FEDERAL STATE INSTITUTIO – THE INNOVATIVE VECTOR OF DEVELOPMENT IN MICROSURGICAL TECHNOLOGIES

The S . Fyodorov Eye Microsurgery Federal State Institution  - 30 years

Features of the structure of ophthalmopathology in the vector of development and implementation of innovative technologies

One of the key points discussed in this article is the structure of ophthalmopathology. According to the statistical data of the S. Fyodorov Eye Microsurgery Federal State Institution, 567799 patients were registered for the first time in 2018. The distribution of ophthalmic pathology among these patients was as follows: cataract – 24.2% including complicated cataract 15.8%; glaucoma – 3.2%; vitreoretinal pathology – 8.8%; diabetes – 2.9%; oculoplastic pathology – 3.5%; oncology – 1.1%; eye diseases in children – 11.9%; refractive errors – 16.6%; fundus diseases – 15.9%; corneal pathology was 0.8%; inflammatory diseases of 8.6%; other diseases – 2.5%. The article describes in detail the innovative technologies and the main trends that determine the overall development of such areas of ophthalmic surgery as: cataract surgery, glaucoma, cornea, vitreoretinal surgery, laser refractive surgery, laser surgery of the retina and vitreous body, operations in children, oculoplasty, ophthalmic oncology

To the 35th anniversary of the Fyodorov Eye Microsurgery Federal State Institution. New frontiers of health management

The article describes the history of the creation of the Fyodorov Eye Microsurgery Federal State Institution from a problem laboratory to the present day, namely, functioning as a national medical research center. The final figures for medical and scientific work, educational activities are presented. The Fyodorov Eye Microsurgery Federal State Institution as an NMRC closely interacts with medical institutions of the assigned territories. cooperates closely with medical institutions in the assigned territories. Active benchmarking, continuous monitoring of ophthalmic care, analytical, organizational, methodological work, development of telemedicine. Each year, the Fyodorov Eye Microsurgery Federal State Institution performs more than 320 thousand operations and more than 1.5 million consultations. Over 35 years, about 25 million consultations and more than 7.5 million operations have been performed and more than 8 million patients have been treated. The share of the Fyodorov Eye Microsurgery Federal State Institution in the volume of ophthalmic surgical care provided in Russia is 37%. More than 15 thousand children undergo surgery in the centers for children of the Fyodorov Eye Microsurgery Federal State Institution every year. 35 years: 400 thousand foreign patients from 120 countries. A surge in online conferences. 2019: about four thousand conference participants. 2020: more than ten thousand. Institute of Continuing Professional Education with wellequipped WetLab simulation operating rooms. Every year, 150 residents and postgraduates; training cycles – 600 ophthalmologists

ERAP2 Increases the Abundance of a Peptide Submotif Highly Selective for the Birdshot Uveitis-Associated HLA-A29

Contains fulltext : 231666.pdf (publisher's version ) (Open Access)Birdshot Uveitis (BU) is a blinding inflammatory eye condition that only affects HLA-A29-positive individuals. Genetic association studies linked ERAP2 with BU, an aminopeptidase which trims peptides before their presentation by HLA class I at the cell surface, which suggests that ERAP2-dependent peptide presentation by HLA-A29 drives the pathogenesis of BU. However, it remains poorly understood whether the effects of ERAP2 on the HLA-A29 peptidome are distinct from its effect on other HLA allotypes. To address this, we focused on the effects of ERAP2 on the immunopeptidome in patient-derived antigen presenting cells. Using complementary HLA-A29-based and pan-class I immunopurifications, isotope-labeled naturally processed and presented HLA-bound peptides were sequenced by mass spectrometry. We show that the effects of ERAP2 on the N-terminus of ligands of HLA-A29 are shared across endogenous HLA allotypes, but discover and replicate that one peptide motif generated in the presence of ERAP2 is specifically bound by HLA-A29. This motif can be found in the amino acid sequence of putative autoantigens. We further show evidence for internal sequence specificity for ERAP2 imprinted in the immunopeptidome. These results reveal that ERAP2 can generate an HLA-A29-specific antigen repertoire, which supports that antigen presentation is a key disease pathway in BU

Cumulus provides cloud-based data analysis for large-scale single-cell and single-nucleus RNA-seq

© 2020, The Author(s), under exclusive licence to Springer Nature America, Inc. Massively parallel single-cell and single-nucleus RNA sequencing has opened the way to systematic tissue atlases in health and disease, but as the scale of data generation is growing, so is the need for computational pipelines for scaled analysis. Here we developed Cumulus—a cloud-based framework for analyzing large-scale single-cell and single-nucleus RNA sequencing datasets. Cumulus combines the power of cloud computing with improvements in algorithm and implementation to achieve high scalability, low cost, user-friendliness and integrated support for a comprehensive set of features. We benchmark Cumulus on the Human Cell Atlas Census of Immune Cells dataset of bone marrow cells and show that it substantially improves efficiency over conventional frameworks, while maintaining or improving the quality of results, enabling large-scale studies

Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Harbors Frequent Splicesosome Mutations That Cause Aberrant RNA Splicing Affecting Genes Critical in pDC Differentiation and Function

Abstract Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive malignancy thought to result from transformation of plasmacytoid dendritic cells (pDCs). Clinical outcomes are poor and pathogenesis is unclear. To better understand BPDCN genomics and disease mechanisms, we performed whole exome- (12 BPDCNs), targeted DNA- (additional 12 BPDCNs), bulk whole transcriptome RNA- (12 BPDCNs and 6 BPDCN patient-derived xenografts [PDXs]), and single cell RNA-sequencing (scRNA-seq) compared to normal DCs. We observed RNA splicing factor mutations in 16/24 cases (7 ZRSR2, 6 SRSF2, 1 each SF3B1, U2AF1, SF3A2, SF3B4). Additional recurrent alterations were in genes known to be mutated in other blood cancers: TET2, ASXL1, TP53, GNB1, NRAS, IDH2, ETV6, DNMT3A, and RUNX1. From exome sequencing we also discovered recurrent mutations in CRIPAK (6/12 cases), NEFH (4/12), HNF1A (2/12), PAX3 (2/12), and SSC5D (2/12) that may be unique to BPDCN. ZRSR2 is notable among the recurrently mutated splicing factors in hematologic malignancies in that all mutations are loss-of-function (e.g., nonsense, frameshift). Of note, BPDCN is very male predominant, ZRSR2 is located on chrX and all mutations are in males. ZRSR2 plays a critical role in "minor" or U12-type intron splicing (only 0.3% of all introns). Thus, we hypothesized that mis-splicing, possibly of U12 genes, contributes to BPDCN pathogenesis. Using RNA-seq, we measured aberrant splicing in BPDCN. Intron retention was the most frequent abnormality in ZRSR2 mutant BPDCNs and PDXs compared to non-mutant cases. ZRSR2 mutant intron retention predominantly affected U12 introns (patients: 29.4% of retained introns, P<0.0001; PDX: 94%, P<0.0001). To test if ZRSR2 loss directly causes U12 intron retention in otherwise isogenic cells, we performed ZRSR2 knockdown using doxycycline-inducible shRNAs in the BPDCN cell line, CAL1, which has no known splicing factor mutation. RNA-seq was performed 0, 2, and 7 days after addition of doxycycline in 3 independent clones each of control or ZRSR2 knockdown. Consistent with what we observed in primary BPDCN, intron retention events were higher in ZRSR2 compared to control shRNA cells after 7 days of doxycycline (mean 885.7 vs 122.7 events, P=0.041). Aberrant intron retention after ZRSR2 knockdown largely involved U12 introns (30/732 U12 vs 37/207,344 U2 introns, P<0.0001). SRSF2 and SF3B1 mutations in BPDCN were at hotspots seen in other cancers: SRSF2 P95H/L/R and SF3B1 K666N, mutants that induce specific types of aberrant splicing (Kim, Ca Cell 2015; Darman, Cell Rep 2015). Mutant BPDCNs demonstrated the same aberrations: SRSF2, exon inclusion/exclusion based on CCNG/GGNG exonic splicing enhancer motifs; SF3B1, aberrant 3' splice site recognition. We hypothesized that aberrant splicing may affect RNAs important for pDC development or function. To further define genes uniquely important in BPDCN, we performed scRNA-seq on 4 BPDCNs and on DCs from healthy donors. By principal component analysis, BPDCNs were more similar to pDCs than to conventional DCs (cDCs) or other HLA-DR+ cells. However, several critical genes for pDC function had markedly lower expression in BPDCN including the transcription factors IRF4 and IRF7. Next we determined which genes were commonly mis-spliced in splicing factor mutant BPDCNs. Strikingly, this list included genes already known to be important in driving DC biology or identified in our scRNA-seq as being differentially expressed between BPDCN and healthy pDCs, including IRF7, IRF8, IKZF1, FLT3, and DERL3. To determine if splicing factor mutations affect DC function, we stimulated ZRSR2 knockdown or control CAL1 cells with Toll-like receptor (TLR) 7, 8, and 9 agonists (R848 or CpG oligo). ZRSR2 knockdown inhibited upregulation of the CD80 costimulatory molecule and aggregation of CAL1 cells, suggesting impairment in activation. Using mouse conditional knock-in bone marrow in ex vivo multipotent progenitor assays, DC differentiation induced by FLT3 ligand was biased toward pDCs and away from cDCs in SRSF2 P95H mutant compared to wild-type cells. However, cDC and monocyte differentiation in the presence of GM-CSF was not affected. In conclusion, splicing factors are frequently mutated in BPDCN and lead to specific splicing defects. Splicing factor mutations may promote BPDCN by affecting pathways important in DC maturation or activation, which could contribute to transformation. Disclosures Seiler: H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. Lane:Stemline Therapeutics: Research Funding; N-of-1: Consultancy