Drosophila Argonaute-1 is critical for transcriptional cosuppression and heterochromatin formation

Argonaute-1 (Ago-1) plays a crucial role in gene regulation and genome stability via biogenesis of small non-coding RNAs. Two “Argonaute” family genes, piwi and Ago-2 in Drosophila are involved in multiple silencing mechanisms in the nucleus, transgene cosuppression, long-distant chromosome interaction, nuclear organization and heterochromatin formation. To investigate whether Ago-1 also plays a similar role, we have generated a series of Ago-1 mutations by excising P element, inserted in the Ago-1 promoter (Ago-1k08121). AGO-1 protein is distributed uniformly in the nucleus and cytosol in early embryos but accumulated predominantly in the cytoplasm during the gastrulation stage. Repeat induced silencing produced by the mini-white (mw) array and transcriptional cosuppression of non-homologous transgenes Adh-w/w-Adh was disrupted by Ago-1 mutation. These effects of Ago-1 are distict from its role in microRNA processing because Dicer-1, a critical enzyme for miRNA biogenesis, has no role on the above silencing. Reduction of AGO-1 protein dislodged the POLYCOMB, EZ (enhancer of zeste) and H3me3K27 binding at the cosuppressed Adh-w transgene insertion sites suggesting its role in Polycomb dependent cosuppression. An overall reduction of methylated histone H3me2K9 and H3me3K27 from the polytene nuclei precisely from the mw promoters was also found that leads to concomitant changes in the chromatin structure. These results suggest a prominent role of Ago-1 in chromatin organization and transgene silencing and demonstrate a critical link between transcriptional transgene cosuppression, heterochromatin formation and chromatin organization. We propose Drosophila Ago-1 as a multifunctional RNAi component that interconnects at least two unrelated events, chromatin organization in the nucleus and microRNA processing in the cytoplasm, which may be extended to the other systems

A Pre-mRNA–Associating Factor Links Endogenous siRNAs to Chromatin Regulation

In plants and fungi, small RNAs silence gene expression in the nucleus by establishing repressive chromatin states. The role of endogenous small RNAs in metazoan nuclei is largely unknown. Here we show that endogenous small interfering RNAs (endo-siRNAs) direct Histone H3 Lysine 9 methylation (H3K9me) in Caenorhabditis elegans. In addition, we report the identification and characterization of nuclear RNAi defective (nrde)-1 and nrde-4. Endo-siRNA–driven H3K9me requires the nuclear RNAi pathway including the Argonaute (Ago) NRDE-3, the conserved nuclear RNAi factor NRDE-2, as well as NRDE-1 and NRDE-4. Small RNAs direct NRDE-1 to associate with the pre-mRNA and chromatin of genes, which have been targeted by RNAi. NRDE-3 and NRDE-2 are required for the association of NRDE-1 with pre-mRNA and chromatin. NRDE-4 is required for NRDE-1/chromatin association, but not NRDE-1/pre-mRNA association. These data establish that NRDE-1 is a novel pre-mRNA and chromatin-associating factor that links small RNAs to H3K9 methylation. In addition, these results demonstrate that endo-siRNAs direct chromatin modifications via the Nrde pathway in C. elegans

HP1 Recruitment in the Absence of Argonaute Proteins in Drosophila

Highly repetitive and transposable element rich regions of the genome must be stabilized by the presence of heterochromatin. A direct role for RNA interference in the establishment of heterochromatin has been demonstrated in fission yeast. In metazoans, which possess multiple RNA–silencing pathways that are both functionally distinct and spatially restricted, whether RNA silencing contributes directly to heterochromatin formation is not clear. Previous studies in Drosophila melanogaster have suggested the involvement of both the AGO2-dependent endogenous small interfering RNA (endo-siRNA) as well as Piwi-interacting RNA (piRNA) silencing pathways. In order to determine if these Argonaute genes are required for heterochromatin formation, we utilized transcriptional reporters and chromatin immunoprecipitation of the critical factor Heterochromatin Protein 1 (HP1) to monitor the heterochromatic state of piRNA clusters, which generate both endo-siRNAs and the bulk of piRNAs. Surprisingly, we find that mutation of AGO2 or piwi increases silencing at piRNA clusters corresponding to an increase of HP1 association. Furthermore, loss of piRNA production from a single piRNA cluster results in genome-wide redistribution of HP1 and reduction of silencing at a distant heterochromatic site, suggesting indirect effects on HP1 recruitment. Taken together, these results indicate that heterochromatin forms independently of endo-siRNA and piRNA pathways

P517 A randomised, placebo-controlled pilot trial of faecal microbiota transplantation for paediatric Crohn’s disease

Abstract Background The role of faecal microbiota transplant (FMT) in Crohn’s disease (CD) remains unclear. Small, open-label case series have shown high rates of clinical remission but protocols have varied across studies, and no randomised controlled trials (RCT) have been performed. We present a protocol for the first pilot RCT of FMT in paediatric CD patients, using a novel colonoscopic + oral capsular intervention that uses fresh-frozen and prepared, lyophilised donor stool. We will measure improvements in clinical disease activity, inflammatory biomarkers, endoscopic markers of mucosal inflammation, as well as, assess key aspects of trial feasibility. Methods Patients 3–17 years with active CD, on stable medication doses for 4 weeks are eligible. Patients will have an initial colonoscopy during which they will receive an infusion into the terminal ileum of normal saline (placebo) or prepared healthy donor stool (RBX2660; Rebiotix, USA) (active). This will be followed by 6-weeks of bi-weekly oral capsular therapy, containing methylcellulose (placebo) or lyophilised healthy adult donor stool (RBX7455; Rebiotix, USA). Randomisation is 2:1 to active and placebo arms (n = 45). Patients will be followed over 24 weeks. (Figure 1) Results Study feasibility will be assessed by: rate of recruitment, completion of sample collections, and frequency and type of adverse events. Clinical outcomes will be measured serially using: Paediatric Crohn’s Disease Activity Index (PCDAI), faecal calprotectin, blood inflammatory markers, and Simple Endoscopic Score for Crohn’s Disease (SES-CD) at baseline and end-of-study. Stool 16s rRNA, metagenomics, and urine metabolomics profiles will be performed on collected stool and urine samples. Patients who complete the placebo arm will be eligible to enter an open-label extension study. Results will be measured through ITT and PP analyses. Proportions and percentages will be reported on feasibility outcomes; odds ratios, mean differences and 95% confidence intervals will be reported on clinical outcomes as preliminary estimates of efficacy. Conclusion This is the first pilot RCT of FMT in the treatment of CD. Recruitment commenced April 2019 and 13 patients have met screening criteria, with 5 enrolled thus far. This study is novel for its focus on paediatric CD patients, and its use of a combined oral + colonoscopic FMT delivery method. The results of this trial will offer preliminary estimates of efficacy for FMT-based therapies in CD, and may support expansion to a future larger multicenter paediatric RCT using our validated study protocol. </jats:sec

ARACE – A New Method for Verbal Decision Analysis

ARACE is a new method developed within the framework of Verbal Decision Analysis (VDA). VDA methods work with verbal form of preference elicitation and evaluation of alternatives without resorting to numbers. ARACE is based on the ideas of the VDA method ZAPROS but uses a more flexible approach to inconsistency of the decision-maker's preferences by introducing a special construct of a "quasi-expert." Mismatched preferences of the decision-maker are viewed as preferences of several quasi-experts. The preference system for each quasi-expert is transitive, leading to consistent decision rules formed for each quasi-expert separately. These rules are used to compare alternatives. Differences in possible comparison of alternatives based on different quasi-experts are resolved through a Clustered Rankings Method for rankings with ties. </jats:p

Characteristics of laser-driven electron acceleration in vacuum

The interaction of free electrons with intense laser beams in vacuum is studied using a 3D test particle simulation model that solves the relativistic Newton-Lorentz equations of motion in analytically specified laser fields. Recently, a group of solutions was found for very intense laser fields that show interesting and unusual characteristics. In particular, it was found that an electron can be captured within the high-intensity laser region, rather than expelled from it, and the captured electron can be accelerated to GeV energies with acceleration gradients on the order of tens of GeV/cm. This phenomenon is termed the capture and acceleration scenario (CAS) and is studied in detail in this paper. The maximum net energy exchange by the CAS mechanism is found to be approximately proportional to a 2_o, in the regime where a_o &gt; 100, where a_o = eE_o/m_ewc is a dimensionless parameter specifying the magnitude of the laser field. The accelerated GeV electron bunch is a macro-pulse, with duration equal or less than that of the laser pulse, which is composed of many micro-pulses that are periodic at the laser frequency. The energy spectrum of the CAS electron bunch is presented. The dependence of the energy exchange in the CAS on various parameters, e.g., a 2_o (laser intensity), w_o (laser radius at focus), tao (laser pulse duration), b_o (the impact parameter), and theta_i (the injection angle with respect to the laser propagation direction), are explored in detail. A comparison with diverse theoretical models is also presented, including a classical model based on phase velocities and a quantum model based on nonlinear Compton scattering

Characteristics of laser-driven electron acceleration in vacuum

The interaction of free electrons with intense laser beams in vacuum is studied using a 3D test particle simulation model that solves the relativistic Newton-Lorentz equations of motion in analytically specified laser fields. Recently, a group of solutions was found for very intense laser fields that show interesting and unusual characteristics. In particular, it was found that an electron can be captured within the high-intensity laser region, rather than expelled from it, and the captured electron can be accelerated to GeV energies with acceleration gradients on the order of tens of GeV/cm. This phenomenon is termed the capture and acceleration scenario (CAS) and is studied in detail in this paper. The maximum net energy exchange by the CAS mechanism is found to be approximately proportional to a 2_o, in the regime where a_o &gt; 100, where a_o = eE_o/m_ewc is a dimensionless parameter specifying the magnitude of the laser field. The accelerated GeV electron bunch is a macro-pulse, with duration equal or less than that of the laser pulse, which is composed of many micro-pulses that are periodic at the laser frequency. The energy spectrum of the CAS electron bunch is presented. The dependence of the energy exchange in the CAS on various parameters, e.g., a 2_o (laser intensity), w_o (laser radius at focus), tao (laser pulse duration), b_o (the impact parameter), and theta_i (the injection angle with respect to the laser propagation direction), are explored in detail. A comparison with diverse theoretical models is also presented, including a classical model based on phase velocities and a quantum model based on nonlinear Compton scattering