Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance

Introduction Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology. Methods Human fibrinogen was citrullinated in vitro by peptidylarginine deiminases (PAD), subjected to proteolysis and the resulting peptides were fractionated by ion exchange chromatography. The peptide composition of the citrullinated peptide-containing fractions was determined by high resolution tandem mass spectrometry. The recognition of these fractions by patient sera was subsequently analyzed by imaging surface plasmon resonance on microarrays. Results In total about two-thirds of the 81 arginines of human fibrinogen were found to be susceptible to citrullination by the human PAD2, the human PAD4 or the rabbit PAD2 enzymes. Citrullination sites were found in all three polypeptide chains of fibrinogen, although the α-chain appeared to contain most of them. The analysis of 98 anti-citrullinated protein antibody-positive RA sera using the new methodology allowed the identification of three major citrullinated epitope regions in human fibrinogen, two in the α- and one in the β-chain. Conclusions A comprehensive overview of citrullination sites in human fibrinogen was generated. The multiplex analysis of peptide fractions derived from a post-translationally modified protein, characterized by mass spectrometry, with patient sera provides a versatile system for mapping modified amino acid-containing epitopes. The citrullinated epitopes of human fibrinogen most efficiently recognized by RA autoantibodies are confined to three regions of its polypeptides

The ABC130 barrel module prototyping programme for the ATLAS strip tracker

For the Phase-II Upgrade of the ATLAS Detector, its Inner Detector, consisting of silicon pixel, silicon strip and transition radiation sub-detectors, will be replaced with an all new 100 % silicon tracker, composed of a pixel tracker at inner radii and a strip tracker at outer radii. The future ATLAS strip tracker will include 11,000 silicon sensor modules in the central region (barrel) and 7,000 modules in the forward region (end-caps), which are foreseen to be constructed over a period of 3.5 years. The construction of each module consists of a series of assembly and quality control steps, which were engineered to be identical for all production sites. In order to develop the tooling and procedures for assembly and testing of these modules, two series of major prototyping programs were conducted: an early program using readout chips designed using a 250 nm fabrication process (ABCN-25) and a subsequent program using a follow-up chip set made using 130 nm processing (ABC130 and HCC130 chips). This second generation of readout chips was used for an extensive prototyping program that produced around 100 barrel-type modules and contributed significantly to the development of the final module layout. This paper gives an overview of the components used in ABC130 barrel modules, their assembly procedure and findings resulting from their tests.Comment: 82 pages, 66 figure

Characterization of the Transcriptional Elongation Factor ELL3 in B cells and Its Role in B-cell Lymphoma Proliferation and Survival

The studies presented in this dissertation establish the dynamics of Eleven nineteen Lysine-rich leukemia (ELL) family of elongation factors during B cell differentiation and provide a description of ELL3 function in B cells. The transition from a mature naïve B cells into an activated B cell is dependent on a large increase in transcriptional output, which is followed by focused expression on secreted immunoglobulin upon terminal differentiation into plasma cell. While ELL family members have previously been implicated in alternative splicing at the immunoglobulin heavy chain locus in plasma cells, their presence and function prior to differentiation is currently not known. However, the use of elongation factors has been implied by the finding of mostly paused RNA polymerase II in the genome of naïve B cells. In the first study, the expression of transcriptional elongation factor ELL3 is shown to be restricted to activated B cells and B cell lymphomas. All three family members were characterized in B cell lymphoma cell lines, genome wide expression, microarray analysis and primary B cell stimulus. The expression of ELL3 was induced upon activation of B cells concurrently with family member ELL. In addition, the abundant expression of ELL3 was restricted to GC derived B cell lymphoma cell lines. While the expression of ELL is maintained, the expression of ELL3 is diminished and ELL2 is up-regulated in terminally differentiated plasma cells. The expression of master regulator of terminal plasma cell differentiation PRDM1 was inverse correlated with that of ELL3. To further establish PRDM1s role in regulating the ELL family member dynamics, global binding was assessed in plasma cell lines. Chromatin immunoprecipitation followed by quantitative PCR was utilized to identify direct association of PRDM1 at exclusively the ELL3 loci. Ectopic expression of PRDM1 in B cells down regulated the expression of ELL3. Furthermore, two consensus PRDM1 binding sites were defined at the ELL3 loci, which mediate significant repression of the promoter activity. Collectively, these experiments indicate that PRDM1 mediates the switch from ELL3 in B cells to ELL2 in plasma cells. The data presented in the final chapter aimed at defining a function for ELL3 in the cells that express it most abundantly, which are B cell lymphoma cell lines. Transient depletion of ELL3 in a Burkitt’s lymphoma cell line resulted in a diminished proliferation rate due to a severe disruption of DNA replication and its regulators minichromosome maintenance proteins. Additionally, compromised cell division and mitotic regulators were observed along with increased DNA damage and cell death. The data presented here demonstrate a key role for ELL3 in the proliferation and survival of B cell lymphomas and positions ELL3 as an attractive therapeutic target against B cell lymphoma’s with a germinal center origin

Characterization of the Transcriptional Elongation Factor ELL3 in B cells and Its Role in B-cell Lymphoma Proliferation and Survival

The studies presented in this dissertation establish the dynamics of Eleven nineteen Lysine-rich leukemia (ELL) family of elongation factors during B cell differentiation and provide a description of ELL3 function in B cells. The transition from a mature naïve B cells into an activated B cell is dependent on a large increase in transcriptional output, which is followed by focused expression on secreted immunoglobulin upon terminal differentiation into plasma cell. While ELL family members have previously been implicated in alternative splicing at the immunoglobulin heavy chain locus in plasma cells, their presence and function prior to differentiation is currently not known. However, the use of elongation factors has been implied by the finding of mostly paused RNA polymerase II in the genome of naïve B cells. In the first study, the expression of transcriptional elongation factor ELL3 is shown to be restricted to activated B cells and B cell lymphomas. All three family members were characterized in B cell lymphoma cell lines, genome wide expression, microarray analysis and primary B cell stimulus. The expression of ELL3 was induced upon activation of B cells concurrently with family member ELL. In addition, the abundant expression of ELL3 was restricted to GC derived B cell lymphoma cell lines. While the expression of ELL is maintained, the expression of ELL3 is diminished and ELL2 is up-regulated in terminally differentiated plasma cells. The expression of master regulator of terminal plasma cell differentiation PRDM1 was inverse correlated with that of ELL3. To further establish PRDM1s role in regulating the ELL family member dynamics, global binding was assessed in plasma cell lines. Chromatin immunoprecipitation followed by quantitative PCR was utilized to identify direct association of PRDM1 at exclusively the ELL3 loci. Ectopic expression of PRDM1 in B cells down regulated the expression of ELL3. Furthermore, two consensus PRDM1 binding sites were defined at the ELL3 loci, which mediate significant repression of the promoter activity. Collectively, these experiments indicate that PRDM1 mediates the switch from ELL3 in B cells to ELL2 in plasma cells. The data presented in the final chapter aimed at defining a function for ELL3 in the cells that express it most abundantly, which are B cell lymphoma cell lines. Transient depletion of ELL3 in a Burkitt’s lymphoma cell line resulted in a diminished proliferation rate due to a severe disruption of DNA replication and its regulators minichromosome maintenance proteins. Additionally, compromised cell division and mitotic regulators were observed along with increased DNA damage and cell death. The data presented here demonstrate a key role for ELL3 in the proliferation and survival of B cell lymphomas and positions ELL3 as an attractive therapeutic target against B cell lymphoma’s with a germinal center origin

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance

Introduction Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology. Methods Human fibrinogen was citrullinated in vitro by peptidylarginine deiminases (PAD), subjected to proteolysis and the resulting peptides were fractionated by ion exchange chromatography. The peptide composition of the citrullinated peptide-containing fractions was determined by high resolution tandem mass spectrometry. The recognition of these fractions by patient sera was subsequently analyzed by imaging surface plasmon resonance on microarrays. Results In total about two-thirds of the 81 arginines of human fibrinogen were found to be susceptible to citrullination by the human PAD2, the human PAD4 or the rabbit PAD2 enzymes. Citrullination sites were found in all three polypeptide chains of fibrinogen, although the α-chain appeared to contain most of them. The analysis of 98 anti-citrullinated protein antibody-positive RA sera using the new methodology allowed the identification of three major citrullinated epitope regions in human fibrinogen, two in the α- and one in the β-chain. Conclusions A comprehensive overview of citrullination sites in human fibrinogen was generated. The multiplex analysis of peptide fractions derived from a post-translationally modified protein, characterized by mass spectrometry, with patient sera provides a versatile system for mapping modified amino acid-containing epitopes. The citrullinated epitopes of human fibrinogen most efficiently recognized by RA autoantibodies are confined to three regions of its polypeptides

In Vitro Optimization of Non-Small Cell Lung Cancer Activity with Troxacitabine,l-1,3-Dioxolane-cytidine, Prodrugs

L-1,3-Dioxolane-cytidine, a potent anticancer agent against leukemia, has limited efficacy against solid tumors, perhaps due to its hydrophilicity. Herein, a library of prodrugs were synthesized to optimize in vitro antitumor activity against non-small cell lung cancer. N4-Substituted fatty acid amide prodrugs of 10-16 carbon chain length demonstrated significantly improved antitumor activity over L-1,3-dioxolane-cytidine. These in vitro results suggest that the in vivo therapeutic efficacy of L-1,3-dioxolane- cytidine against solid tumors may be improved with prodrug strategies

Data supporting the functional role of Eleven-nineteen Lysine-rich Leukemia 3 (ELL3) in B cell lymphoma cell line cells

The data presented here are related to the research article entitled âSelective expression of the transcription elongation factor ELL3 in B cells prior to ELL2 drives proliferation and survivalâ (Alexander et al., 2017) [1]. The cited research article characterizes Eleven-nineteen Lysine-rich Leukemia 3 (ELL3) expression in the B cell compartment and functional dependence in B lymphoma cell lines. This data report describes the mRNA expression pattern in a panel of cell lines representing the B cell compartment, supplementing the protein expression data presented in the associated research report. In addition, a reanalysis is presented of publicly available mRNA expression data from primary murine B cells to reveal dynamic regulation of the ELL family members post LPS stimulation (Barwick et al., 2016) [2]. The effect of ELL3 depletion on cell morphology, latent Epstein Barr Virus (EBV) lytic replication and differentiation markers in a Burkitt's lymphoma (BL) cell line cells are presented. Keywords: ELL3, Transcription elongation, B-cell Lymphoma, Cell division, EB

Cross-ancestry atlas of gene, isoform, and splicing regulation in the developing human brain

INTRODUCTION Genome-wide association studies (GWASs) have identified thousands of loci associated with neurodevelopmental and psychiatric disorders, yet our lack of understanding of the target genes and biological mechanisms underlying these associations remains a major challenge. GWAS signals for many neuropsychiatric disorders, including autism spectrum disorder, schizophrenia, and bipolar disorder, are particularly enriched for gene-regulatory elements active during human brain development. However, the lack of a unified population-scale, ancestrally diverse gene-regulatory atlas of human brain development has been a major obstacle for the functional assessment of top loci and post-GWAS integrative analyses. RATIONALE To address this critical gap in knowledge, we have uniformly processed and systematically characterized gene, isoform, and splicing quantitative trait loci (cumulatively referred to as xQTLs) in the developing human brain across 672 unique samples from 4 to 39 postconception weeks spanning European, African-American, and Latino/admixed American ancestries). With this expanded atlas, we sought to specifically localize the timing and molecular features mediating the greatest proportion of neuropsychiatric GWAS heritability, to prioritize candidate risk genes and mechanisms for top loci, and to compare with analogous results using larger adult brain functional genomic reference panels. RESULTS In total, we identified 15,752 genes harboring a gene, isoform and/or splicing cis-xQTL, including 49 genes associated with four large, recurrent inversions. Highly concordant effect sizes were observed across populations, and our diverse reference panel improved resolution to fine-map underlying candidate causal regulatory variants. Substantially more genes were found to harbor QTLs in the first versus second trimester of brain development, with a notable drop in gene expression and splicing heritability observed from 10 to 18 weeks coinciding with a period of rapidly increasing cellular heterogeneity in the developing brain. Isoform-level regulation, particularly in the second trimester, mediated a greater proportion of heritability across multiple psychiatric GWASs compared with gene expression regulation. Through colocalization and transcriptome-wide association studies, we prioritized biological mechanisms for ~60% of GWAS loci across five neuropsychiatric disorders, with >2-fold more colocalizations observed compared with larger adult brain functional genomic reference panels. We observed convergence between common and rare-variant associations, including a cryptic splicing event in the high-confidence schizophrenia risk gene SP4. Finally, we constructed a comprehensive set of developmentally regulated gene and isoform coexpression networks harboring unique cell-type specificity and genetic enrichments. Leveraging this cell-type specificity, we identified >8000 module interaction QTLs, many of which exhibited additional GWAS colocalizations. Overall, neuropsychiatric GWASs and rare variant signals localized more strongly within maturing excitatory- and interneuron-associated modules compared with those enriched for neural progenitor cell types. Results can be visualized at devbrainhub.gandallab.org. CONCLUSION We have generated a large-scale, cross-population resource of gene, isoform, and splicing regulation in the developing human brain, providing comprehensive developmental and cell-type-informed mechanistic insights into the genetic underpinnings of complex neurodevelopmental and psychiatric disorders

Timing of surgery following SARS-CoV-2 infection: an international prospective cohort study

Peri-operative SARS-CoV-2 infection increases postoperative mortality. The aim of this study was to determine the optimal duration of planned delay before surgery in patients who have had SARS-CoV-2 infection. This international, multicentre, prospective cohort study included patients undergoing elective or emergency surgery during October 2020. Surgical patients with pre-operative SARS-CoV-2 infection were compared with those without previous SARS-CoV-2 infection. The primary outcome measure was 30-day postoperative mortality. Logistic regression models were used to calculate adjusted 30-day mortality rates stratified by time from diagnosis of SARS-CoV-2 infection to surgery. Among 140,231 patients (116 countries), 3127 patients (2.2%) had a pre-operative SARS-CoV-2 diagnosis. Adjusted 30-day mortality in patients without SARS-CoV-2 infection was 1.5% (95%CI 1.4-1.5). In patients with a pre-operative SARS-CoV-2 diagnosis, mortality was increased in patients having surgery within 0-2 weeks, 3-4 weeks and 5-6 weeks of the diagnosis (odds ratio (95%CI) 4.1% (3.3-4.8), 3.9% (2.6-5.1) and 3.6% (2.0-5.2), respectively). Surgery performed >= 7 weeks after SARS-CoV-2 diagnosis was associated with a similar mortality risk to baseline (odds ratio (95%CI) 1.5% (0.9-2.1%)). After a >= 7 week delay in undertaking surgery following SARS-CoV-2 infection, patients with ongoing symptoms had a higher mortality than patients whose symptoms had resolved or who had been asymptomatic (6.0% (95%CI 3.2-8.7) vs. 2.4% (95%CI 1.4-3.4) vs. 1.3% (95%CI 0.6-2.0%), respectively). Where possible, surgery should be delayed for at least 7 weeks following SARS-CoV-2 infection. Patients with ongoing symptoms >= 7 weeks from diagnosis may benefit from further delay