Mitochondrial OMA1 and OPA1 as Gatekeepers of Organellar Structure/Function and Cellular Stress Response

Mammalian mitochondria are emerging as a critical stress-responsive contributor to cellular life/death and developmental outcomes. Maintained as an organellar network distributed throughout the cell, mitochondria respond to cellular stimuli and stresses through highly sensitive structural dynamics, particularly in energetically demanding cell settings such as cardiac and muscle tissues. Fusion allows individual mitochondria to form an interconnected reticular network, while fission divides the network into a collection of vesicular organelles. Crucially, optic atrophy-1 (OPA1) directly links mitochondrial structure and bioenergetic function: when the transmembrane potential across the inner membrane (ΔΨm) is intact, long L-OPA1 isoforms carry out fusion of the mitochondrial inner membrane. When ΔΨm is lost, L-OPA1 is cleaved to short, fusion-inactive S-OPA1 isoforms by the stress-sensitive OMA1 metalloprotease, causing the mitochondrial network to collapse to a fragmented population of organelles. This proteolytic mechanism provides sensitive regulation of organellar structure/function but also engages directly with apoptotic factors as a major mechanism of mitochondrial participation in cellular stress response. Furthermore, emerging evidence suggests that this proteolytic mechanism may have critical importance for cell developmental programs, particularly in cardiac, neuronal, and stem cell settings. OMA1\u27s role as a key mitochondrial stress-sensitive protease motivates exciting new questions regarding its mechanistic regulation and interactions, as well as its broader importance through involvement in apoptotic, stress response, and developmental pathways

Mitochondrial OPA1 cleavage is reversibly activated by differentiation of H9c2 cardiomyoblasts

Optic atrophy-1 (OPA1) is a dynamin-like GTPase localized to the mitochondrial inner membrane, playing key roles in inner membrane fusion and cristae maintenance. OPA1 is regulated by the mitochondrial transmembrane potential (Δψm): when Δψm is intact, long OPA1 isoforms (L-OPA1) carry out inner membrane fusion. Upon loss of Δψm, L-OPA1 isoforms are proteolytically cleaved to short (S-OPA1) isoforms by the stress-inducible OMA1 metalloprotease, causing collapse of the mitochondrial network and promoting apoptosis. Here, we show that L-OPA1 isoforms of H9c2 cardiomyoblasts are retained under loss of Δψm, despite the presence of OMA1. However, when H9c2s are differentiated to a more cardiac-like phenotype via treatment with retinoic acid (RA) in low serum media, loss of Δ ψm induces robust, and reversible, cleavage of L-OPA1 and subsequent OMA1 degradation. These findings indicate that a potent developmental switch regulates Δ ψm-sensitive OPA1 cleavage, suggesting novel developmental and regulatory mechanisms for OPA1 homeostasis

Single-Cell Sequencing of Developing Human Gut Reveals Transcriptional Links to Childhood Crohn's Disease.

Human gut development requires the orchestrated interaction of differentiating cell types. Here, we generate an in-depth single-cell map of the developing human intestine at 6-10 weeks post-conception. Our analysis reveals the transcriptional profile of cycling epithelial precursor cells; distinct from LGR5-expressing cells. We propose that these cells may contribute to differentiated cell subsets via the generation of LGR5-expressing stem cells and receive signals from surrounding mesenchymal cells. Furthermore, we draw parallels between the transcriptomes of ex vivo tissues and in vitro fetal organoids, revealing the maturation of organoid cultures in a dish. Lastly, we compare scRNA-seq profiles from pediatric Crohn's disease epithelium alongside matched healthy controls to reveal disease-associated changes in the epithelial composition. Contrasting these with the fetal profiles reveals the re-activation of fetal transcription factors in Crohn's disease. Our study provides a resource available at www.gutcellatlas.org, and underscores the importance of unraveling fetal development in understanding disease

On the motivations for Merleau-Ponty’s ontological research

This paper attempts to clarify Merleau-Ponty’s later work by tracing a hitherto overlooked set of concerns that were of key consequence for the formulation of his ontological research. I argue that his ontology can be understood as a response to a set of problems originating in reflections on the intersubjective use of language in dialogue, undertaken in the early 1950s. His study of dialogue disclosed a structure of meaning-formation and pointed towards a theory of truth (both recurring ontological topics) that post-Phenomenology premises could not account for. A study of dialogue shows that speakers’ positions are interchangeable, that speaking subjects are active and passive in varying degrees, and that the intentional roles of subjects and objects are liable to shift or ‘transgress’ themselves. These observations anticipate the concepts of ‘reversibility’ and ‘narcissism’, his later view of activity and passivity, and his later view of intentionality, and sharpened the need to adopt an intersubjective focus in ontological research

DNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development.

OBJECTIVE: Human intestinal epithelial organoids (IEOs) are increasingly being recognised as a highly promising translational research tool. However, our understanding of their epigenetic molecular characteristics and behaviour in culture remains limited. DESIGN: We performed genome-wide DNA methylation and transcriptomic profiling of human IEOs derived from paediatric/adult and fetal small and large bowel as well as matching purified human gut epithelium. Furthermore, organoids were subjected to in vitro differentiation and genome editing using CRISPR/Cas9 technology. RESULTS: We discovered stable epigenetic signatures which define regional differences in gut epithelial function, including induction of segment-specific genes during cellular differentiation. Established DNA methylation profiles were independent of cellular environment since organoids retained their regional DNA methylation over prolonged culture periods. In contrast to paediatric and adult organoids, fetal gut-derived organoids showed distinct dynamic changes of DNA methylation and gene expression in culture, indicative of an in vitro maturation. By applying CRISPR/Cas9 genome editing to fetal organoids, we demonstrate that this process is partly regulated by TET1, an enzyme involved in the DNA demethylation process. Lastly, generating IEOs from a child diagnosed with gastric heterotopia revealed persistent and distinct disease-associated DNA methylation differences, highlighting the use of organoids as disease-specific research models. CONCLUSIONS: Our study demonstrates striking similarities of epigenetic signatures in mucosa-derived IEOs with matching primary epithelium. Moreover, these results suggest that intestinal stem cell-intrinsic DNA methylation patterns establish and maintain regional gut specification and are involved in early epithelial development and disease.This work was supported by funding from the following charitable organizations: Crohn’s in Childhood Research Association (CICRA), the Evelyn Trust, Crohn’s and Colitis in Childhood (“3Cs”), Addenbrooke’s Charitable Trust (ACT), and the Newlife Foundation for Disabled Children. J.K. was funded by a CICRA PhD studentship, K.H. was funded by an EBPOD EMBL-EBI/Cambridge Computational Biomedical Postdoctoral Fellowship. B-K.K. was supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society [101241/Z/13/Z] and received core support from the Wellcome Trust and MRC to the WT - MRC Cambridge Stem Cell Institute. GD and JF received support from the Wellcome Trust. J.F. was supported by a studentship from the MRC. P.R was supported by the Deutsche Forschungsgemeinschaft EXC306 Cluster “Inflammation at Interfaces” and BMBF IHEC DEEP TP5.2

Macrophage IL-1β-positive microvesicles exhibit thrombo-inflammatory properties and are detectable in patients with active juvenile idiopathic arthritis

ObjectiveIL-1β is a leaderless cytokine with poorly known secretory mechanisms that is barely detectable in serum of patients, including those with an IL-1β-mediated disease such as systemic juvenile idiopathic arthritis (sJIA). Leukocyte microvesicles (MVs) may be a mechanism of IL-1β secretion. The first objective of our study was to characterize IL-1β-positive MVs obtained from macrophage cell culture supernatants and to investigate their biological functions in vitro and in vivo. The second objective was to detect circulating IL-1β-positive MVs in JIA patients.MethodsMVs were purified by serial centrifugations from PBMCs, or THP-1 differentiated into macrophages, then stimulated with LPS ± ATP. MV content was analyzed for the presence of IL-1β, NLRP3 inflammasome, caspase-1, P2X7 receptor, and tissue factor (TF) using ELISA, Western blot, or flow cytometry. MV biological properties were studied in vitro by measuring VCAM-1, ICAM-1, and E-selectin expression after HUVEC co-culture and factor-Xa generation test was realized. In vivo, MVs’ ability to recruit leukocytes in a murine model of peritonitis was evaluated. Plasmatic IL-1β-positive MVs were studied ex vivo in 10 active JIA patients using flow cytometry.ResultsTHP-1-derived macrophages stimulated with LPS and ATP released MVs, which contained NLRP3, caspase-1, and the 33-kDa precursor and 17-kDa mature forms of IL-1β and bioactive TF. IL-1β-positive MVs expressed P2X7 receptor and released soluble IL-1β in response to ATP stimulation in vitro. In mice, MVs induced a leukocyte peritoneal infiltrate, which was reduced by treatment with the IL-1 receptor antagonist. Finally, IL-1β-positive MVs were detectable in plasma from 10 active JIA patients.ConclusionMVs shed from activated macrophages contain IL-1β, NLRP3 inflammasome components, and TF, and constitute thrombo-inflammatory vectors that can be detected in the plasma from active JIA patients

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Les droits disciplinaires des fonctions publiques : « unification », « harmonisation » ou « distanciation ». A propos de la loi du 26 avril 2016 relative à la déontologie et aux droits et obligations des fonctionnaires

The production of tt‾ , W+bb‾ and W+cc‾ is studied in the forward region of proton–proton collisions collected at a centre-of-mass energy of 8 TeV by the LHCb experiment, corresponding to an integrated luminosity of 1.98±0.02 fb−1 . The W bosons are reconstructed in the decays W→ℓν , where ℓ denotes muon or electron, while the b and c quarks are reconstructed as jets. All measured cross-sections are in agreement with next-to-leading-order Standard Model predictions.The production of $t\overline{t}$, $W+b\overline{b}$ and $W+c\overline{c}$ is studied in the forward region of proton-proton collisions collected at a centre-of-mass energy of 8 TeV by the LHCb experiment, corresponding to an integrated luminosity of 1.98 $\pm$ 0.02 \mbox{fb}^{-1}. The $W$ bosons are reconstructed in the decays $W\rightarrow\ell\nu$, where $\ell$ denotes muon or electron, while the $b$ and $c$ quarks are reconstructed as jets. All measured cross-sections are in agreement with next-to-leading-order Standard Model predictions

The Human Cell Atlas White Paper

The Human Cell Atlas (HCA) will be made up of comprehensive reference maps of all human cells - the fundamental units of life - as a basis for understanding fundamental human biological processes and diagnosing, monitoring, and treating disease. It will help scientists understand how genetic variants impact disease risk, define drug toxicities, discover better therapies, and advance regenerative medicine. A resource of such ambition and scale should be built in stages, increasing in size, breadth, and resolution as technologies develop and understanding deepens. We will therefore pursue Phase 1 as a suite of flagship projects in key tissues, systems, and organs. We will bring together experts in biology, medicine, genomics, technology development and computation (including data analysis, software engineering, and visualization). We will also need standardized experimental and computational methods that will allow us to compare diverse cell and tissue types - and samples across human communities - in consistent ways, ensuring that the resulting resource is truly global. This document, the first version of the HCA White Paper, was written by experts in the field with feedback and suggestions from the HCA community, gathered during recent international meetings. The White Paper, released at the close of this yearlong planning process, will be a living document that evolves as the HCA community provides additional feedback, as technological and computational advances are made, and as lessons are learned during the construction of the atlas