Small-molecule-induced epigenetic rejuvenation promotes SREBP condensation and overcomes barriers to CNS myelin regeneration.

Remyelination failure in diseases like multiple sclerosis (MS) was thought to involve suppressed maturation of oligodendrocyte precursors; however, oligodendrocytes are present in MS lesions yet lack myelin production. We found that oligodendrocytes in the lesions are epigenetically silenced. Developing a transgenic reporter labeling differentiated oligodendrocytes for phenotypic screening, we identified a small-molecule epigenetic-silencing-inhibitor (ESI1) that enhances myelin production and ensheathment. ESI1 promotes remyelination in animal models of demyelination and enables de novo myelinogenesis on regenerated CNS axons. ESI1 treatment lengthened myelin sheaths in human iPSC-derived organoids and augmented (re)myelination in aged mice while reversing age-related cognitive decline. Multi-omics revealed that ESI1 induces an active chromatin landscape that activates myelinogenic pathways and reprograms metabolism. Notably, ESI1 triggered nuclear condensate formation of master lipid-metabolic regulators SREBP1/2, concentrating transcriptional co-activators to drive lipid/cholesterol biosynthesis. Our study highlights the potential of targeting epigenetic silencing to enable CNS myelin regeneration in demyelinating diseases and aging.

The choroid plexus links innate immunity to CSF dysregulation in hydrocephalus.

The choroid plexus (ChP) is the blood-cerebrospinal fluid (CSF) barrier and the primary source of CSF. Acquired hydrocephalus, caused by brain infection or hemorrhage, lacks drug treatments due to obscure pathobiology. Our integrated, multi-omic investigation of post-infectious hydrocephalus (PIH) and post-hemorrhagic hydrocephalus (PHH) models revealed that lipopolysaccharide and blood breakdown products trigger highly similar TLR4-dependent immune responses at the ChP-CSF interface. The resulting CSF "cytokine storm", elicited from peripherally derived and border-associated ChP macrophages, causes increased CSF production from ChP epithelial cells via phospho-activation of the TNF-receptor-associated kinase SPAK, which serves as a regulatory scaffold of a multi-ion transporter protein complex. Genetic or pharmacological immunomodulation prevents PIH and PHH by antagonizing SPAK-dependent CSF hypersecretion. These results reveal the ChP as a dynamic, cellularly heterogeneous tissue with highly regulated immune-secretory capacity, expand our understanding of ChP immune-epithelial cell cross talk, and reframe PIH and PHH as related neuroimmune disorders vulnerable to small molecule pharmacotherapy.

Alternative platelet differentiation pathways initiated by nonhierarchically related hematopoietic stem cells.

Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.

Infodemics: A new challenge for public health.

The COVID-19 information epidemic, or "infodemic," demonstrates how unlimited access to information may confuse and influence behaviors during a health emergency. However, the study of infodemics is relatively new, and little is known about their relationship with epidemics management. Here, we discuss unresolved issues and propose research directions to enhance preparedness for future health crises.

Multimodal Analysis of Composition and Spatial Architecture in Human Squamous Cell Carcinoma.

To define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication. Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma. Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis. These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer.

Coupled Single-Cell CRISPR Screening and Epigenomic Profiling Reveals Causal Gene Regulatory Networks.

Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ∼4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.

A Mechanosensitive GPCR that Detects the Bloody Force.

Mechanoreceptors mediate a wide variety of physiological processes, such as hearing, touch, proprioception, and blood flow regulation. It is generally believed that mechanoreceptors are force-gated ion channels. Now, Xu et al. uncover a GPCR that is activated by shear force in endothelial cells of blood vessels.

Unveiling the Role of the Most Impactful Cardiovascular Risk Locus through Haplotype Editing.

The 9p21.3 cardiovascular disease locus is the most influential common genetic risk factor for coronary artery disease (CAD), accounting for ∼10%-15% of disease in non-African populations. The ∼60 kb risk haplotype is human-specific and lacks coding genes, hindering efforts to decipher its function. Here, we produce induced pluripotent stem cells (iPSCs) from risk and non-risk individuals, delete each haplotype using genome editing, and generate vascular smooth muscle cells (VSMCs). Risk VSMCs exhibit globally altered transcriptional networks that intersect with previously identified CAD risk genes and pathways, concomitant with aberrant adhesion, contraction, and proliferation. Unexpectedly, deleting the risk haplotype rescues VSMC stability, while expressing the 9p21.3-associated long non-coding RNA ANRIL induces risk phenotypes in non-risk VSMCs. This study shows that the risk haplotype selectively predisposes VSMCs to adopt a cell state associated with CAD phenotypes, defines new VSMC-based networks of CAD risk genes, and establishes haplotype-edited iPSCs as powerful tools for functionally annotating the human genome.

The Egyptian Rousette Genome Reveals Unexpected Features of Bat Antiviral Immunity.

Bats harbor many viruses asymptomatically, including several notorious for causing extreme virulence in humans. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We found an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons, which dramatically differ from their functional counterparts in other mammals. Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense. An evaluation of the theoretical function of these genes suggests that an inhibitory immune state may exist in bats. Based on our findings, we hypothesize that tolerance of viral infection, rather than enhanced potency of antiviral defenses, may be a key mechanism by which bats asymptomatically host viruses that are pathogenic in humans.

Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer's disease.

Genetic association studies have demonstrated the critical involvement of the microglial immune response in Alzheimer's disease (AD) pathogenesis. Phospholipase C-gamma-2 (PLCG2) is selectively expressed by microglia and functions in many immune receptor signaling pathways. In AD, PLCG2 is induced uniquely in plaque-associated microglia. A genetic variant of PLCG2, PLCG2P522R, is a mild hypermorph that attenuates AD risk. Here, we identified a loss-of-function PLCG2 variant, PLCG2M28L, that confers an increased AD risk. PLCG2P522R attenuated disease in an amyloidogenic murine AD model, whereas PLCG2M28L exacerbated the plaque burden associated with altered phagocytosis and Aß clearance. The variants bidirectionally modulated disease pathology by inducing distinct transcriptional programs that identified microglial subpopulations associated with protective or detrimental phenotypes. These findings identify PLCG2M28L as a potential AD risk variant and demonstrate that PLCG2 variants can differentially orchestrate microglial responses in AD pathogenesis that can be therapeutically targeted.

3D RNA and Functional Interactions from Evolutionary Couplings.

Non-coding RNAs are ubiquitous, but the discovery of new RNA gene sequences far outpaces the research on the structure and functional interactions of these RNA gene sequences. We mine the evolutionary sequence record to derive precise information about the function and structure of RNAs and RNA-protein complexes. As in protein structure prediction, we use maximum entropy global probability models of sequence co-variation to infer evolutionarily constrained nucleotide-nucleotide interactions within RNA molecules and nucleotide-amino acid interactions in RNA-protein complexes. The predicted contacts allow all-atom blinded 3D structure prediction at good accuracy for several known RNA structures and RNA-protein complexes. For unknown structures, we predict contacts in 160 non-coding RNA families. Beyond 3D structure prediction, evolutionary couplings help identify important functional interactions-e.g., at switch points in riboswitches and at a complex nucleation site in HIV. Aided by increasing sequence accumulation, evolutionary coupling analysis can accelerate the discovery of functional interactions and 3D structures involving RNA.

Mapping model units to visual neurons reveals population code for social behaviour.

The rich variety of behaviours observed in animals arises through the interplay between sensory processing and motor control. To understand these sensorimotor transformations, it is useful to build models that predict not only neural responses to sensory input1-5 but also how each neuron causally contributes to behaviour6,7. Here we demonstrate a novel modelling approach to identify a one-to-one mapping between internal units in a deep neural network and real neurons by predicting the behavioural changes that arise from systematic perturbations of more than a dozen neuronal cell types. A key ingredient that we introduce is 'knockout training', which involves perturbing the network during training to match the perturbations of the real neurons during behavioural experiments. We apply this approach to model the sensorimotor transformations of Drosophila melanogaster males during a complex, visually guided social behaviour8-11. The visual projection neurons at the interface between the optic lobe and central brain form a set of discrete channels12, and prior work indicates that each channel encodes a specific visual feature to drive a particular behaviour13,14. Our model reaches a different conclusion: combinations of visual projection neurons, including those involved in non-social behaviours, drive male interactions with the female, forming a rich population code for behaviour. Overall, our framework consolidates behavioural effects elicited from various neural perturbations into a single, unified model, providing a map from stimulus to neuronal cell type to behaviour, and enabling future incorporation of wiring diagrams of the brain15 into the model.

SlyB encapsulates outer membrane proteins in stress-induced lipid nanodomains.

The outer membrane in Gram-negative bacteria consists of an asymmetric phospholipid-lipopolysaccharide bilayer that is densely packed with outer-membrane ß-barrel proteins (OMPs) and lipoproteins1. The architecture and composition of this bilayer is closely monitored and is essential to cell integrity and survival2-4. Here we find that SlyB, a lipoprotein in the PhoPQ stress regulon, forms stable stress-induced complexes with the outer-membrane proteome. SlyB comprises a 10 kDa periplasmic ß-sandwich domain and a glycine zipper domain that forms a transmembrane α-helical hairpin with discrete phospholipid- and lipopolysaccharide-binding sites. After loss in lipid asymmetry, SlyB oligomerizes into ring-shaped transmembrane complexes that encapsulate ß-barrel proteins into lipid nanodomains of variable size. We find that the formation of SlyB nanodomains is essential during lipopolysaccharide destabilization by antimicrobial peptides or acute cation shortage, conditions that result in a loss of OMPs and compromised outer-membrane barrier function in the absence of a functional SlyB. Our data reveal that SlyB is a compartmentalizing transmembrane guard protein that is involved in cell-envelope proteostasis and integrity, and suggest that SlyB represents a larger family of broadly conserved lipoproteins with 2TM glycine zipper domains with the ability to form lipid nanodomains.

Reproducible graphene synthesis by oxygen-free chemical vapour deposition.

Chemical vapour deposition (CVD) synthesis of graphene on copper has been broadly adopted since the first demonstration of this process1. However, widespread use of CVD-grown graphene for basic science and applications has been hindered by challenges with reproducibility2 and quality3. Here we identify trace oxygen as a key factor determining the growth trajectory and quality for graphene grown by low-pressure CVD. Oxygen-free chemical vapour deposition (OF-CVD) synthesis is fast and highly reproducible, with kinetics that can be described by a compact model, whereas adding trace oxygen leads to suppressed nucleation and slower/incomplete growth. Oxygen affects graphene quality as assessed by surface contamination, emergence of the Raman D peak and decrease in electrical conductivity. Epitaxial graphene grown in oxygen-free conditions is contamination-free and shows no detectable D peak. After dry transfer and boron nitride encapsulation, it shows room-temperature electrical-transport behaviour close to that of exfoliated graphene. A graphite-gated device shows well-developed integer and fractional quantum Hall effects. By highlighting the importance of eliminating trace oxygen, this work provides guidance for future CVD system design and operation. The increased reproducibility and quality afforded by OF-CVD synthesis will broadly influence basic research and applications of graphene.

Transcription factor-mediated intestinal metaplasia and the role of a shadow enhancer.

Barrett's esophagus (BE) and gastric intestinal metaplasia are related premalignant conditions in which areas of human stomach epithelium express mixed gastric and intestinal features. Intestinal transcription factors (TFs) are expressed in both conditions, with unclear causal roles and cis-regulatory mechanisms. Ectopic CDX2 reprogrammed isogenic mouse stomach organoid lines to a hybrid stomach-intestinal state transcriptionally similar to clinical metaplasia; squamous esophageal organoids resisted this CDX2-mediated effect. Reprogramming was associated with induced activity at thousands of previously inaccessible intestine-restricted enhancers, where CDX2 occupied DNA directly. HNF4A, a TF recently implicated in BE pathogenesis, induced weaker intestinalization by binding a novel shadow Cdx2 enhancer and hence activating Cdx2 expression. CRISPR/Cas9-mediated germline deletion of that cis-element demonstrated its requirement in Cdx2 induction and in the resulting activation of intestinal genes in stomach cells. dCas9-conjugated KRAB repression mapped this activity to the shadow enhancer's HNF4A binding site. Altogether, we show extensive but selective recruitment of intestinal enhancers by CDX2 in gastric cells and that HNF4A-mediated ectopic CDX2 expression in the stomach occurs through a conserved shadow cis-element. These findings identify mechanisms for TF-driven intestinal metaplasia and a likely pathogenic TF hierarchy.

Programming multicellular assembly with synthetic cell adhesion molecules.

Cell adhesion molecules are ubiquitous in multicellular organisms, specifying precise cell-cell interactions in processes as diverse as tissue development, immune cell trafficking and the wiring of the nervous system1-4. Here we show that a wide array of synthetic cell adhesion molecules can be generated by combining orthogonal extracellular interactions with intracellular domains from native adhesion molecules, such as cadherins and integrins. The resulting molecules yield customized cell-cell interactions with adhesion properties that are similar to native interactions. The identity of the intracellular domain of the synthetic cell adhesion molecules specifies interface morphology and mechanics, whereas diverse homotypic or heterotypic extracellular interaction domains independently specify the connectivity between cells. This toolkit of orthogonal adhesion molecules enables the rationally programmed assembly of multicellular architectures, as well as systematic remodelling of native tissues. The modularity of synthetic cell adhesion molecules provides fundamental insights into how distinct classes of cell-cell interfaces may have evolved. Overall, these tools offer powerful abilities for cell and tissue engineering and for systematically studying multicellular organization.

Non-viral precision T cell receptor replacement for personalized cell therapy.

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Mutant p53 regulates Survivin to foster lung metastasis.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers worldwide and evolves often to lung metastasis. P53R175H (homologous to Trp53R172H in mice) is a common hot spot mutation. How metastasis is regulated by p53R175H in ESCC remains to be investigated. To investigate p53R175H-mediated molecular mechanisms, we used a carcinogen-induced approach in Trp53R172H/- mice to model ESCC. In the primary Trp53R172H/- tumor cell lines, we depleted Trp53R172H (shTrp53) and observed a marked reduction in cell invasion in vitro and lung metastasis burden in a tail-vein injection model in comparing isogenic cells (shCtrl). Furthermore, we performed bulk RNA-seq to compare gene expression profiles of metastatic and primary shCtrl and shTrp53 cells. We identified the YAP-BIRC5 axis as a potential mediator of Trp53R172H -mediated metastasis. We demonstrate that expression of Survivin, an antiapoptotic protein encoded by BIRC5, increases in the presence of Trp53R172H Furthermore, depletion of Survivin specifically decreases Trp53R172H-driven lung metastasis. Mechanistically, Trp53R172H but not wild-type Trp53, binds with YAP in ESCC cells, suggesting their cooperation to induce Survivin expression. Furthermore, Survivin high expression level is associated with increased metastasis in several GI cancers. Taken together, this study unravels new insights into how mutant p53 mediates metastasis.

Initial seeding of the embryonic thymus by immune-restricted lympho-myeloid progenitors.

The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling. T-IPs did not include multipotent stem cells or molecular evidence of T cell-restricted progenitors. Instead, single-cell molecular and functional analysis demonstrated that most fetal T-IPs expressed genes of and had the potential to develop into lymphoid as well as myeloid components of the immune system. Moreover, studies of embryos deficient in the transcriptional regulator RBPJ demonstrated that canonical Notch signaling was not involved in pre-thymic restriction to the T cell lineage or the migration of T-IPs.