Robustness of randomized rumour spreading

In this work we consider three well-studied broadcast protocols: push, pull and push&pull. A key property of all these models, which is also an important reason for their popularity, is that they are presumed to be very robust, since they are simple, randomized and, crucially, do not utilize explicitly the global structure of the underlying graph. While sporadic results exist, there has been no systematic theoretical treatment quantifying the robustness of these models. Here we investigate this question with respect to two orthogonal aspects: (adversarial) modifications of the underlying graph and message transmission failures. We explore in particular the following notion of local resilience: beginning with a graph, we investigate up to which fraction of the edges an adversary may delete at each vertex, so that the protocols need significantly more rounds to broadcast the information. Our main findings establish a separation among the three models. On one hand, pull is robust with respect to all parameters that we consider. On the other hand, push may slow down significantly, even if the adversary may modify the degrees of the vertices by an arbitrarily small positive fraction only. Finally, push&pull is robust when no message transmission failures are considered, otherwise it may be slowed down. On the technical side, we develop two novel methods for the analysis of randomized rumour-spreading protocols. First, we exploit the notion of self-bounding functions to facilitate significantly the round-based analysis: we show that for any graph the variance of the growth of informed vertices is bounded by its expectation, so that concentration results follow immediately. Second, in order to control adversarial modifications of the graph we make use of a powerful tool from extremal graph theory, namely Szemerédi’s Regularity Lemma.

OP0118 DECIPHERING THE ANTI-PROTEIN-ARGININE DEIMINASE (PAD) RESPONSE IDENTIFIES PAD1 AND PAD6 AS NOVEL AUTOANTIGENS IN RHEUMATOID ARTHRITIS

Background:Protein-arginine deiminase (PAD) 4 enzymes play a central role in the pathogenesis of rheumatoid arthritis (RA) and represents an antigenic target. Among the five known family members (PAD1, PAD2, PAD3, PAD4 and PAD6), only PAD2, PAD3 and PAD4 have been described to have autoantigenic properties. Furtheremore, very little is known on the the isotype usage of these autoantibodies. Understanding the molecular basis of the anti-PAD antibody reponse has the potential to open novel approaches for precision medicine in RA.Objectives:The objectives of this study were to screen for the presence of antibodies to the five PAD family members and to evaluate the isotype usage of the anti-PAD4 response in RA.Methods:First, we developed a panel for the detection of anti-PAD IgG based on a particle-based multi-analyte technology (PMAT), that utilized paramagnetic particles coupled with the different human recombinant PAD proteins (PAD1, PAD2, PAD3, PAD4 and PAD6) and anti-human IgG conjugate. This panel was used to test sera from RA patients (n=33) and non-RA controls (n=36). The controls were comprised of apparently healthy individuals (n=10), and patients with infectious diseases (n=10), systemic lupus erythematosus (n=7), systemic sclerosis (n=9) and Sjogren’s syndrome (n=1). Next, the PAD4-coupled beads were tested with anti-human IgM, IgA and IgG conjugates on an extended cohort of RA patients (n=62) and the same non-RA controls.Results:All five anti-PAD IgG (Figure 1) demonstrated the ability to discriminate between RA patients and controls. At greater than 90% specificity, anti-PAD4 IgG, followed by anti-PAD3 IgG, showed the best diagnostic performance. Significantly higher levels of the five antibodies were observed in RAvs.controls (p-values of 0.0041, <0.0001, 0.0014, 0.0039, and 0.0140 for anti-PAD1, 2, 3, 4 and 6, respectively). Significant correlation was observed between all the antibodies, with the highest between anti-PAD1 and anti-PAD4 (Spearman´srho=0.87,p<0.0001) and the lowest between anti-PAD4 and anti-PAD2 (Spearman’srho=0.38,p=0.0015) and anti-PAD4 and anti-PAD6 (Spearman’srho=0.38,p=0.0011). While principal component analysis (PCA) (Figure 2) showed an association between all anti-PAD antibodies, there was further discrimination that displayed closer association between anti-PAD1, 3 and 4 on one hand, and between anti-PAD2 and 6. For the extended testing of anti-PAD4 with IgG, IgA and IgM, all three isotypes were identified in the sera of RA patients. Higher levels of the three isotypes were observed in RA patients with erosive disease when compared with the patients without erosion, but this association was only significant for anti-PAD4 IgA (p=0.0086).Figure 1.Receiver operating characteristics (ROC) analysis of the discrimination between rheumatoid arthritis (RA) and controls of IgG to protein-arginine deiminase (PAD) 1, PAD2, PAD3, PAD4 and PAD6. The area under the curve (AUC) values are shown in brackets for each biomarker.Abbreviations:TPF: true positive fraction; FPF: false positive fractionFigure 2.Two dimensional principal component analysis (PCA) plot of the anti-PAD levels in RA patients (n=33) and controls (n=36). Anti-PAD1, 3 and 4 have the main contribution to PC1, which explains 51.7% of the variance, and anti-PAD2 and 6 to PC2, that represents 20.8% of it.Abbreviations:PC: principal componentConclusion:Our study is the first to describe PAD1 and PAD6 as novel antigenic targets in RA and to demostrate that the anti-PAD4 B-cell immune response uses all three isotypes (IgG, IgA and IgM). The strong and significant association between anti-PAD4 IgA and joint erosion is of particular clinical relevance.Disclosure of Interests:Laura Martinez-Prat Employee of: I am an employee of Inova Diagnostics, an in vitro diagnostics company., Mary Ann Aure Employee of: I am an employee of Inova Diagnostics, an in vitro diagnostics company., Chelsea Bentow Employee of: I am an employee of Inova Diagnostics, an in vitro diagnostics company., David Lucia Employee of: I am an employee of Inova Diagnostics, an in vitro diagnostics company., Marcos Lopez-Hoyos Consultant of: Inova Diagnostics, an in vitro diagnostics company., Michael Mahler Employee of: I am an employee of Inova Diagnostics, an in vitro diagnostics company.

Enrichment of hematopoietic stem/progenitor cells in the zebrafish kidney

Hematopoietic stem cells (HSCs) maintain the entire blood system throughout life and are utilized in therapeutic approaches for blood diseases. Prospective isolation of highly purified HSCs is crucial to understand the molecular mechanisms underlying regulation of HSCs. The zebrafish is an elegant genetic model for the study of hematopoiesis due to its many unique advantages. It has not yet been possible, however, to purify HSCs in adult zebrafish due to a lack of specific HSC markers. Here we show the enrichment of zebrafish HSCs by a combination of two HSC-related transgenes, gata2a:GFP and runx1:mCherry. The double-positive fraction of gata2a:GFP and runx1:mCherry (gata2a+runx1+) was detected at approximately 0.16% in the kidney, the main hematopoietic organ in teleosts. Transcriptome analysis revealed that gata2a+runx1+ cells showed typical molecular signatures of HSCs, including upregulation of gata2b, gfi1aa, runx1t1, pbx1b, and meis1b. Transplantation assays demonstrated that long-term repopulating HSCs were highly enriched within the gata2a+runx1+ fraction. In contrast, colony-forming assays showed that gata2a−runx1+ cells abundantly contain erythroid- and/or myeloid-primed progenitors. Thus, our purification method of HSCs in the zebrafish kidney is useful to identify molecular cues needed to regulate self-renewal and differentiation of HSCs.

Isolation and Testing of Bacteria from Steroid Compounds obtained from Anting-anting Leaf (Acalypha indica L.)

Isolation of steroid compounds from the leaves of the earrings (Acalypha indica L.) and the antibacterial test has been performed. This study aims to obtain information about secondary metabolite compound leaves of Anting-anting, obtaining and identifying steroid isolates from the leaves of Anting-anting and knowing the antibacterial activity of the positive fraction of steroid compounds. The research stages include sample preparation, phytochemical test, isolation, separation, purification of steroid compounds, identification of steroid isolates using LC-MS/MS, and antibacterial test by paper disc method. The results of phytochemical screening show that the leaves of the earrings contain alkaloid compounds, flavonoids, steroids, saponins, tannins, and quinones. From the results of steroid isolation, we found steroid isolates weighing 0.0065 grams (0.0058%). Identification of steroid isolates using LC-MS/MS at a retention time of 7.49 min with [M+H]+ 399 m/z indicated the presence of a brassicasterol compound. The results of antibacterial test of fraction A from chloroform extract containing steroid compound showed antibacterial activity against Staphylococcus aureus bacteria and Escherichia coli bacteria.

Clonal Hierarchy of Piga Mutant Cells Assessed By Next Generation Sequencing Is More Complex Than Previously Recognized in Paroxysmal Noctural Hemoglobinuria

Since the pivotal revelation of the PIGA gene mutations responsible for glycosylphosphatidylinositol (GPI) anchor deficiency over 20 years ago, molecular and clinical research into the evolution of Paroxysmal Nocturnal Hemoglobinuria (PNH) has significantly advanced the current understanding of the disease, expanding upon the foundational work by numerous investigators over the previous two centuries. The discovery of multiple PIGA mutations in normal individuals using the bacterial toxin aerolysin as well as with florescent activated cell sorting (FACS) clearly demonstrated that PIGA mutations are common in normal hematopoiesis. A strong association of PNH with Aplastic Anemia (AA) and the failure of PIGA clones to expand in animal models argued for the necessity of permissive conditions, largely understood to be immune mediated bone marrow failure. While GPI anchor deficiency may lead to escape of the PNH clone from autoimmunity, recent research has added to the body of knowledge by demonstrating that the PNH clone may acquire additional mutations in other genes that promote clonal expansion in the absence of competition from normal hematopoiesis as found in AA. Sequencing studies of PIGA in PNH patients suggested that one, two, or at most three hematopoietic stem cells were sufficient to supply the necessary blood cells for survival. Furthermore, specific monoclonal antibodies combined with the fluorescently labeled inactive proaerolysin variant (FLAER) currently used to perform PNH diagnostic assays visualized the relatively frequent occurrence of both Type II and Type III PNH cells, suggesting the presence of two PIGA mutations in a significant number of patients. Using a combination of multiparameter FACS and a custom designed multiamplicon next generation sequencing (NGS) assay targeting PIGA, our results suggest that this may be an underestimate. 17 sequential patients with PNH (N=7, 3 Male, 4 Female) or AA/PNH (N=10, 5 Male, 5 Female) were enrolled in this study. A flow cytometry panel consisting of CD235a/CD59 for RBCs and FLAER/CD24/CD15/CD45 for granulocytes was used to assess PNH clone size and to sort WBCs into PNH positive and negative fractions. Mean RBC clone size was 31.6% (range 1.1-63.5%); mean WBC clone size was 53.2% (range 0.17-99.7%). Sort purity was confirmed at &gt;98% in both fractions, DNA was extracted and subjected to analysis using an NGS assay and a stringent bioanalytic pipeline with an average depth of 18,000 reads. At least one PIGA mutation was detected in the PNH positive fraction of every patient. A total of 68 PIGA mutations were observed, consisting of 31 nonsynonymous SNVs, 16 frameshift insertion/deletions, 12 stopgains, 7 splice site, and 2 nonframeshift deletions. 13/17 (76%) had more than one mutation, and 12/17 (70%) had 3 or more mutations (range 3-14). Analysis of variant allelic frequency (VAF) indicated that multiple clones with distinct PIGA mutations greater than 5% VAF of the PNH positive fraction were found in 9/17 (53%) patients with a median VAF of 11% (range 5-86%) and 5/9 demonstrating 3 mutations &gt;5%. Repeat identical experiments from three patients were performed on samples obtained roughly one month apart with concordant results. Overall, our results suggest that a complex clonal hierarchy with multiple dominant and/or subdominant yet expanded clones is relatively common in PNH. The clonal hierarchy in PNH patients can include up to 14 PNH clones with distinct frequencies and mutations. In addition, it is a widely held notion that PIGA mutations occur only in a hematopoietic stem cell, thus affecting all lineages, yet anomalous cases of PNH have been reported where the RBC PNH clone size is markedly higher than that of the granulocyte clone, and comparison between monocyte and granulocyte clone size is significantly different. We have identified two such cases in this cohort, with flow cytometry revealing RBC PNH clones of 18.63% and 35.61%, while granulocyte clones were 0.17% and 8.11%, and monocyte clones were 53.12% and 50.35%, respectively. Current experiments isolating and sequencing both sorted PNH positive cell fractions as well as hematopoietic precursors for PIGA and other commonly mutated genes found in hematologic malignancies are underway to confirm and elucidate the complex clonal hierarchy these results suggest. Disclosures No relevant conflicts of interest to declare.