Traffic Jam activates the Flamenco piRNA cluster locus and the Piwi pathway to ensure transposon silencing and Drosophila fertility.

Flamenco (Flam) is the most prominent piRNA cluster locus expressed in Drosophila ovarian follicle cells, and it is required for female fertility to silence gypsy/mdg4 transposons. To determine how Flam is regulated, we used promoter-bashing reporter assays in OSS cells to uncover novel enhancer sequences within the first exons of Flam . We confirmed the enhancer sequence relevance in vivo with new Drosophila Flam deletion mutants of these regions that compromised Flam piRNA expression and lowered female fertility from activated transposons. Our proteomic analysis of proteins associated with these enhancer sequences discovered the transcription factor Traffic Jam (TJ). Tj knockdowns in OSS cells caused a decrease in Flam transcripts, Flam piRNAs, and multiple Piwi pathway genes. A TJ ChIP-seq analysis from whole flies and OSS cells confirmed TJ binding exactly at the enhancer that was deleted in the new Flam mutant as well as at multiple Piwi pathway gene enhancers. Interestingly, TJ also bound the Long Terminal Repeats of transposons that had decreased expression after Tj knockdowns in OSS cells. Our study reveals the integral role TJ plays in the on-going arms race between selfish transposons and their suppression by the host Piwi pathway and the Flam piRNA cluster locus.

Rotational culture and integration with amniotic stem cells reduce porcine islet immunoreactivity in vitro and slow xeno-rejection in a murine model of islet transplantation.

BACKGROUND: Pre-transplant modification of porcine islets may improve their suitability for clinical use in diabetes management by supporting graft function and reducing the potential for xeno-rejection. The present study investigates intra-graft incorporation of stem cells that secrete beta (ß)-cell trophic and immunomodulatory factors to preserve function and alter immune cell responsiveness to porcine islets. METHODS: Isolated porcine islets were maintained in a three-dimensional rotational cell culture system (RCCS) to facilitate aggregation with human amniotic epithelial cells (AECs). Assembled islet constructs were assessed for functional integrity and ability to avoid xeno-recognition by CD4+ T-cells using mixed islet:lymphocyte reaction assays. To determine whether stem cell-mediated modification of porcine islets provided a survival advantage over native islets, structural integrity was examined in a pig-to-mouse islet transplant model. RESULTS: Rotational cell culture system supported the formation of porcine islet:AEC aggregates with improved insulin-secretory capacity compared to unmodified islets, whilst the xeno-response of purified CD4+ T-cells to AEC-bearing grafts was significantly (P < 0.05) attenuated. Transplanted AEC-bearing grafts demonstrated slower rejection in immune-competent recipients compared to unmodified islets. CONCLUSIONS/INTERPRETATION: Rotational culture enables pre-transplant modification of porcine islets by integration with immunomodulatory stem cells capable of subduing xeno-reactivity to CD4+ T-cells. This reduces islet rejection and offers translational potential to widen availability and improve the clinical effectiveness of islet transplantation.

Subtropical adaptation of a temperate plant (Brassica oleracea var. italica) utilizes non-vernalization-responsive QTLs.

While many tropical plants have been adapted to temperate cultivation, few temperate plants have been adapted to the tropics. Originating in Western Europe, Brassica oleracea vernalization requires a period of low temperature and BoFLC2 regulates the transition to floral development. In B. oleracea germplasm selected in Taiwan, a non-vernalization pathway involving BoFLC3 rather than BoFLC2 regulates curd induction. In 112 subtropical breeding lines, specific haplotype combinations of BoFLC3 and PAN (involved in floral organ identity and a positional candidate for additional curd induction variation) adapt B. oleracea to high ambient temperature and short daylength. Duplicated genes permitted evolution of alternative pathways for control of flowering in temperate and tropical environments, a principle that might be utilized via natural or engineered approaches in other plants. New insight into regulation of Brassica flowering exemplifies translational agriculture, tapping knowledge of botanical models to improve food security under projected climate change scenarios.

PGE2 production at sites of tissue injury promotes an anti-inflammatory neutrophil phenotype and determines the outcome of inflammation resolution in vivo.

Neutrophils are the first immune cells recruited to a site of injury or infection, where they perform many functions. Having completed their role, neutrophils must be removed from the inflammatory site-either by apoptosis and efferocytosis or by reverse migration away from the wound-for restoration of normal tissue homeostasis. Disruption of these tightly controlled physiological processes of neutrophil removal can lead to a range of inflammatory diseases. We used an in vivo zebrafish model to understand the role of lipid mediator production in neutrophil removal. Following tailfin amputation in the absence of macrophages, neutrophillic inflammation does not resolve, due to loss of macrophage-dependent handling of eicosanoid prostaglandin E2 (PGE2) that drives neutrophil removal via promotion of reverse migration. Knockdown of endogenous PGE synthase gene reveals PGE2 as essential for neutrophil inflammation resolution. Furthermore, PGE2 is able to signal through EP4 receptors during injury, causing an increase in Alox12 production and switching toward anti-inflammatory eicosanoid signaling. Our data confirm regulation of neutrophil migration by PGE2 and LXA4 (lipoxin A4) in an in vivo model of inflammation resolution. This pathway may contain therapeutic targets for driving inflammation resolution in chronic inflammatory disease.

The triune of intestinal microbiome, genetics and inflammatory status and its impact on the healing of lower gastrointestinal anastomoses.

Gastrointestinal resections are a common operation and most involve an anastomosis to rejoin the ends of the remaining bowel to restore gastrointestinal (GIT) continuity. While most joins heal uneventfully, in up to 26% of patients healing fails and an anastomotic leak (AL) develops. Despite advances in surgical technology and techniques, the rate of anastomotic leaks has not decreased over the last few decades raising the possibility that perhaps we do not yet fully understand the phenomenon of AL and are thus ill-equipped to prevent it. As in all complex conditions, it is necessary to isolate each different aspect of disease for interrogation of its specific role, but, as we hope to demonstrate in this article, it is a dangerous oversimplification to consider any single aspect as the full answer to the problem. Instead, consideration of important individual observations in parallel could illuminate the way forward towards a possibly simple solution amidst the complexity. This article details three aspects that we believe intertwine, and therefore should be considered together in wound healing within the GIT during postsurgical recovery: the microbiome, the host genetic make-up and their relationship to the perioperative inflammatory status. Each of these, alone or in combination, has been linked with various states of health and disease, and in combining these three aspects in the case of postoperative recovery from bowel resection, we may be nearer an answer to preventing anastomotic leaks than might have been thought just a few years ago.

Zebrafish screens for new colitis treatments - a bottom-up approach.

Many factors interact in the development and progression of inflammatory bowel disease. This complexity demands a combination of methods to disentangle the pathology of this disease. As reported by Oehlers et al. in this issue, the zebrafish drug screen is one simple approach that could enable the elucidation of the pathways involved in IBD and ultimately the discovery of new therapeutics.

Low gravity rotational culture and the integration of immunomodulatory stem cells reduce human islet allo-reactivity.

Modification of human islets prior to transplantation may improve long-term clinical outcome in terms of diabetes management, by supporting graft function and reducing the potential for allo-rejection. Intragraft incorporation of stem cells secreting beta (ß)-cell trophic and immunomodulatory factors represents a credible approach, but requires suitable culture methods to facilitate islet alteration without compromising integrity. This study employed a three-dimensional rotational cell culture system (RCCS) to achieve modification, preserve function, and ultimately influence immune cell responsiveness to human islets. Islets underwent intentional dispersal and rotational culture-assisted aggregation with amniotic epithelial cells (AEC) exhibiting intrinsic immunomodulatory potential. Reassembled islet constructs were assessed for functional integrity, and their ability to induce an allo-response in discrete T-cell subsets determined using mixed islet:lymphocyte reaction assays. RCCS supported the formation of islet:AEC aggregates with improved insulin secretory capacity compared to unmodified islets. Further, the allo-response of peripheral blood mononuclear cell (PBMC) and purified CD4+ and CD8+ T-cell subsets to AEC-bearing grafts was significantly (p < 0.05) attenuated. Rotational culture enables pre-transplant islet modification involving their integration with immunomodulatory stem cells capable of subduing the allo-reactivity of T cells relevant to islet rejection. The approach may play a role in achieving acute and long-term graft survival in islet transplantation.