Inflammatory caspases (caspase-1, -4, -5 and -11) are critical for innate defences. Caspase-1 is activated by ligands of various canonical inflammasomes, and caspase-4, -5 and -11 directly recognize bacterial lipopolysaccharide, both of which trigger pyroptosis. Despite the crucial role in immunity and endotoxic shock, the mechanism for pyroptosis induction by inflammatory caspases is unknown. Here we identify gasdermin D (Gsdmd) by genome-wide clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 nuclease screens of caspase-11- and caspase-1-mediated pyroptosis in mouse bone marrow macrophages. GSDMD-deficient cells resisted the induction of pyroptosis by cytosolic lipopolysaccharide and known canonical inflammasome ligands. Interleukin-1β release was also diminished in Gsdmd(-/-) cells, despite intact processing by caspase-1. Caspase-1 and caspase-4/5/11 specifically cleaved the linker between the amino-terminal gasdermin-N and carboxy-terminal gasdermin-C domains in GSDMD, which was required and sufficient for pyroptosis. The cleavage released the intramolecular inhibition on the gasdermin-N domain that showed intrinsic pyroptosis-inducing activity. Other gasdermin family members were not cleaved by inflammatory caspases but shared the autoinhibition; gain-of-function mutations in Gsdma3 that cause alopecia and skin defects disrupted the autoinhibition, allowing its gasdermin-N domain to trigger pyroptosis. These findings offer insight into inflammasome-mediated immunity/diseases and also change our understanding of pyroptosis and programmed necrosis.

Cytosolic inflammasome complexes mediated by a pattern recognition receptor (PRR) defend against pathogen infection by activating caspase 1. Pyrin, a candidate PRR, can bind to the inflammasome adaptor ASC to form a caspase 1-activating complex. Mutations in the Pyrin-encoding gene, MEFV, cause a human autoinflammatory disease known as familial Mediterranean fever. Despite important roles in immunity and disease, the physiological function of Pyrin remains unknown. Here we show that Pyrin mediates caspase 1 inflammasome activation in response to Rho-glucosylation activity of cytotoxin TcdB, a major virulence factor of Clostridium difficile, which causes most cases of nosocomial diarrhoea. The glucosyltransferase-inactive TcdB mutant loses the inflammasome-stimulating activity. Other Rho-inactivating toxins, including FIC-domain adenylyltransferases (Vibrio parahaemolyticus VopS and Histophilus somni IbpA) and Clostridium botulinum ADP-ribosylating C3 toxin, can also biochemically activate the Pyrin inflammasome in their enzymatic activity-dependent manner. These toxins all target the Rho subfamily and modify a switch-I residue. We further demonstrate that Burkholderia cenocepacia inactivates RHOA by deamidating Asn 41, also in the switch-I region, and thereby triggers Pyrin inflammasome activation, both of which require the bacterial type VI secretion system (T6SS). Loss of the Pyrin inflammasome causes elevated intra-macrophage growth of B. cenocepacia and diminished lung inflammation in mice. Thus, Pyrin functions to sense pathogen modification and inactivation of Rho GTPases, representing a new paradigm in mammalian innate immunity.

SUMMARY Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprise a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.

While N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNA, is linked to cell differentiation and tissue development, the biological significance of m6A modification in mammalian glial development remains unknown. Here, we identify a novel m6A reader, Prrc2a (Proline rich coiled-coil 2 A), which controls oligodendrocyte specification and myelination. Nestin-Cre-mediated knockout of Prrc2a induces significant hypomyelination, decreased lifespan, as well as locomotive and cognitive defects in a mouse model. Further analyses reveal that Prrc2a is involved in oligodendrocyte progenitor cells (OPCs) proliferation and oligodendrocyte fate determination. Accordingly, oligodendroglial-lineage specific deletion of Prrc2a causes a similar phenotype of Nestin-Cre-mediated deletion. Combining transcriptome-wide RNA-seq, m6A-RIP-seq and Prrc2a RIP-seq analysis, we find that Olig2 is a critical downstream target gene of Prrc2a in oligodendrocyte development. Furthermore, Prrc2a stabilizes Olig2 mRNA through binding to a consensus GGACU motif in the Olig2 CDS (coding sequence) in an m6A-dependent manner. Interestingly, we also find that the m6A demethylase, Fto, erases the m6A modification of Olig2 mRNA and promotes its degradation. Together, our results indicate that Prrc2a plays an important role in oligodendrocyte specification through functioning as a novel m6A reader. These findings suggest a new avenue for the development of therapeutic strategies for hypomyelination-related neurological diseases.

Despite being only one-atom thick, defect-free graphene is considered to be completely impermeable to all gases and liquids [1][2][3][4][5][6][7][8][9][10] . This conclusion is based on theory 3-8 and supported by experiments 1,9,10 that could not detect gas permeation through micrometre-size membranes within a detection limit of 10 5 to 10 6 atoms per second. Here, using small monocrystalline containers tightly sealed with graphene, we show that defect-free graphene is impermeable with an accuracy of eight to nine orders of magnitude higher than in the previous experiments. We could discern permeation of just a few helium atoms per hour, and this detection limit is also valid for all other tested gases (neon, nitrogen, oxygen, argon, krypton and xenon), except for hydrogen. Hydrogen shows noticeable permeation, even though its molecule is larger than helium and should experience a higher energy barrier. The puzzling observation is attributed to a two-stage process that involves dissociation of molecular hydrogen at catalytically active graphene ripples, followed by adsorbed atoms flipping to the other side of the graphene sheet with a relatively low activation energy of about 1.0 electronvolt, a value close to that previously reported for proton transport 11,12 . Our work provides a key reference for the impermeability of two-dimensional materials and is important from a fundamental perspective and for their potential applications.From a theory standpoint, monolayer graphene imposes a very high energy barrier for penetration of atoms and molecules. Density-functional-theory calculations predict that the barrier E is at least several eV 2-6 , which should prohibit any gas permeation under ambient conditions. Indeed, one can estimate that at room temperature T it would take longer than the lifetime of the universe to find an atom energetic enough to pierce a defect-free membrane of any realistic size. These expectations agree with experiments that reported no detectable gas permeation through mechanically-exfoliated graphene. The highest sensitivity was achieved using micrometersize wells etched in oxidized silicon wafers, which were sealed with graphene 1,9,10 . In those measurements, a pressurized gas (e.g., helium) could permeate along the SiO 2 layer and gradually fill the microcontainers making so-called 'nanoballoons'. Their consecutive deflation in air could be monitored using atomic force microscopy (AFM), and it was shown that the leakage occurred only along the SiO 2 surface, within several minutes but independently of the number of graphene layers used for the sealing 1 . These studies allowed a conclusion that graphene membranes were impermeable to all gases, at least with the achieved accuracy of 10 5 -10 6 atoms s -1 . This was further corroborated by creating individual atomic-scale defects in graphene nanoballoons, which resulted in their relatively fast deflation/inflation and confirmed the high sensitivity of the method 9,10 .

Interferon-inducible guanylate-binding proteins (GBPs) mediate cell-autonomous antimicrobial defences. Shigella flexneri, a Gram-negative cytoplasmic free-living bacterium that causes bacillary dysentery, encodes a repertoire of highly similar type III secretion system effectors called invasion plasmid antigen Hs (IpaHs). IpaHs represent a large family of bacterial ubiquitin-ligases, but their function is poorly understood. Here we show that S. flexneri infection induces rapid proteasomal degradation of human guanylate binding protein-1 (hGBP1). We performed a transposon screen to identify a mutation in the S. flexneri gene ipaH9.8 that prevented hGBP1 degradation. IpaH9.8 targets hGBP1 for degradation via Lys48-linked ubiquitination. IpaH9.8 targets multiple GBPs in the cytoplasm independently of their nucleotide-bound states and their differential function in antibacterial defence, promoting S. flexneri replication and resulting in the death of infected mice. In the absence of IpaH9.8, or when binding of GBPs to IpaH9.8 was disrupted, GBPs such as hGBP1 and mouse GBP2 (mGBP2) translocated to intracellular S. flexneri and inhibited bacterial replication. Like wild-type mice, mutant mice deficient in GBP1-3, 5 and 7 succumbed to S. flexneri infection, but unlike wild-type mice, mice deficient in these GBPs were also susceptible to S. flexneri lacking ipaH9.8. The mode of IpaH9.8 action highlights the functional importance of GBPs in antibacterial defences. IpaH9.8 and S. flexneri provide a unique system for dissecting GBP-mediated immunity.

No abstract

The Cre-loxP recombination system is the most widely used technology for in vivo tracing of stem or progenitor cell lineages. The precision of this genetic system largely depends on the specificity of Cre recombinase expression in targeted stem or progenitor cells. However, Cre expression in nontargeted cell types can complicate the interpretation of lineage-tracing studies and has caused controversy in many previous studies. Here we describe a new genetic lineage tracing system that incorporates the Dre-rox recombination system to enhance the precision of conventional Cre-loxP-mediated lineage tracing. The Dre-rox system permits rigorous control of Cre-loxP recombination in lineage tracing, effectively circumventing potential uncertainty of the cell-type specificity of Cre expression. Using this new system we investigated two topics of recent debates-the contribution of c-Kit cardiac stem cells to cardiomyocytes in the heart and the contribution of Sox9 hepatic progenitor cells to hepatocytes in the liver. By overcoming the technical hurdle of nonspecific Cre-loxP-mediated recombination, this new technology provides more precise analysis of cell lineage and fate decisions and facilitates the in vivo study of stem and progenitor cell plasticity in disease and regeneration.