The TMEM132B-GABAA receptor complex controls alcohol actions in the brain.

Alcohol is the most consumed and abused psychoactive drug globally, but the molecular mechanisms driving alcohol action and its associated behaviors in the brain remain enigmatic. Here, we have discovered a transmembrane protein TMEM132B that is a GABAA receptor (GABAAR) auxiliary subunit. Functionally, TMEM132B promotes GABAAR expression at the cell surface, slows receptor deactivation, and enhances the allosteric effects of alcohol on the receptor. In TMEM132B knockout (KO) mice or TMEM132B I499A knockin (KI) mice in which the TMEM132B-GABAAR interaction is specifically abolished, GABAergic transmission is decreased and alcohol-induced potentiation of GABAAR-mediated currents is diminished in hippocampal neurons. Behaviorally, the anxiolytic and sedative/hypnotic effects of alcohol are markedly reduced, and compulsive, binge-like alcohol consumption is significantly increased. Taken together, these data reveal a GABAAR auxiliary subunit, identify the TMEM132B-GABAAR complex as a major alcohol target in the brain, and provide mechanistic insights into alcohol-related behaviors.

Anteromedial thalamus gates the selection and stabilization of long-term memories.

Memories initially formed in hippocampus gradually stabilize to cortex over weeks-to-months for long-term storage. The mechanistic details of this brain re-organization remain poorly understood. We recorded bulk neural activity in circuits that link hippocampus and cortex as mice performed a memory-guided virtual-reality task over weeks. We identified a prominent and sustained neural correlate of memory in anterior thalamus, whose inhibition substantially disrupted memory consolidation. More strikingly, gain amplification enhanced consolidation of otherwise unconsolidated memories. To gain mechanistic insights, we developed a technology for simultaneous cellular-resolution imaging of hippocampus, thalamus, and cortex throughout consolidation. We found that whereas hippocampus equally encodes multiple memories, the anteromedial thalamus preferentially encodes salient memories, and gradually increases correlations with cortex to facilitate tuning and synchronization of cortical ensembles. We thus identify a thalamo-cortical circuit that gates memory consolidation and propose a mechanism suitable for the selection and stabilization of hippocampal memories into longer-term cortical storage.

Live birth of chimeric monkey with high contribution from embryonic stem cells.

A formal demonstration that mammalian pluripotent stem cells possess preimplantation embryonic cell-like (naive) pluripotency is the generation of chimeric animals through early embryo complementation with homologous cells. Whereas such naive pluripotency has been well demonstrated in rodents, poor chimerism has been achieved in other species including non-human primates due to the inability of the donor cells to match the developmental state of the host embryos. Here, we have systematically tested various culture conditions for establishing monkey naive embryonic stem cells and optimized the procedures for chimeric embryo culture. This approach generated an aborted fetus and a live chimeric monkey with high donor cell contribution. A stringent characterization pipeline demonstrated that donor cells efficiently (up to 90%) incorporated into various tissues (including the gonads and placenta) of the chimeric monkeys. Our results have major implications for the study of primate naive pluripotency and genetic engineering of non-human primates.

High-throughput sequencing of single neuron projections reveals spatial organization in the olfactory cortex.

In most sensory modalities, neuronal connectivity reflects behaviorally relevant stimulus features, such as spatial location, orientation, and sound frequency. By contrast, the prevailing view in the olfactory cortex, based on the reconstruction of dozens of neurons, is that connectivity is random. Here, we used high-throughput sequencing-based neuroanatomical techniques to analyze the projections of 5,309 mouse olfactory bulb and 30,433 piriform cortex output neurons at single-cell resolution. Surprisingly, statistical analysis of this much larger dataset revealed that the olfactory cortex connectivity is spatially structured. Single olfactory bulb neurons targeting a particular location along the anterior-posterior axis of piriform cortex also project to matched, functionally distinct, extra-piriform targets. Moreover, single neurons from the targeted piriform locus also project to the same matched extra-piriform targets, forming triadic circuit motifs. Thus, as in other sensory modalities, olfactory information is routed at early stages of processing to functionally diverse targets in a coordinated manner.

Multi-organ proteomic landscape of COVID-19 autopsies.

The molecular pathology of multi-organ injuries in COVID-19 patients remains unclear, preventing effective therapeutics development. Here, we report a proteomic analysis of 144 autopsy samples from seven organs in 19 COVID-19 patients. We quantified 11,394 proteins in these samples, in which 5,336 were perturbed in the COVID-19 patients compared to controls. Our data showed that cathepsin L1, rather than ACE2, was significantly upregulated in the lung from the COVID-19 patients. Systemic hyperinflammation and dysregulation of glucose and fatty acid metabolism were detected in multiple organs. We also observed dysregulation of key factors involved in hypoxia, angiogenesis, blood coagulation, and fibrosis in multiple organs from the COVID-19 patients. Evidence for testicular injuries includes reduced Leydig cells, suppressed cholesterol biosynthesis, and sperm mobility. In summary, this study depicts a multi-organ proteomic landscape of COVID-19 autopsies that furthers our understanding of the biological basis of COVID-19 pathology.

Pan-genome analysis of 33 genetically diverse rice accessions reveals hidden genomic variations.

Structural variations (SVs) and gene copy number variations (gCNVs) have contributed to crop evolution, domestication, and improvement. Here, we assembled 31 high-quality genomes of genetically diverse rice accessions. Coupling with two existing assemblies, we developed pan-genome-scale genomic resources including a graph-based genome, providing access to rice genomic variations. Specifically, we discovered 171,072 SVs and 25,549 gCNVs and used an Oryza glaberrima assembly to infer the derived states of SVs in the Oryza sativa population. Our analyses of SV formation mechanisms, impacts on gene expression, and distributions among subpopulations illustrate the utility of these resources for understanding how SVs and gCNVs shaped rice environmental adaptation and domestication. Our graph-based genome enabled genome-wide association study (GWAS)-based identification of phenotype-associated genetic variations undetectable when using only SNPs and a single reference assembly. Our work provides rich population-scale resources paired with easy-to-access tools to facilitate rice breeding as well as plant functional genomics and evolutionary biology research.

A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS.

Vaccines are urgently needed to control the ongoing pandemic COVID-19 and previously emerging MERS/SARS caused by coronavirus (CoV) infections. The CoV spike receptor-binding domain (RBD) is an attractive vaccine target but is undermined by limited immunogenicity. We describe a dimeric form of MERS-CoV RBD that overcomes this limitation. The RBD-dimer significantly increased neutralizing antibody (NAb) titers compared to conventional monomeric form and protected mice against MERS-CoV infection. Crystal structure showed RBD-dimer fully exposed dual receptor-binding motifs, the major target for NAbs. Structure-guided design further yielded a stable version of RBD-dimer as a tandem repeat single-chain (RBD-sc-dimer) which retained the vaccine potency. We generalized this strategy to design vaccines against COVID-19 and SARS, achieving 10- to 100-fold enhancement of NAb titers. RBD-sc-dimers in pilot scale production yielded high yields, supporting their scalability for further clinical development. The framework of immunogen design can be universally applied to other beta-CoV vaccines to counter emerging threats.

Pan-Genome of Wild and Cultivated Soybeans.

Soybean is one of the most important vegetable oil and protein feed crops. To capture the entire genomic diversity, it is needed to construct a complete high-quality pan-genome from diverse soybean accessions. In this study, we performed individual de novo genome assemblies for 26 representative soybeans that were selected from 2,898 deeply sequenced accessions. Using these assembled genomes together with three previously reported genomes, we constructed a graph-based genome and performed pan-genome analysis, which identified numerous genetic variations that cannot be detected by direct mapping of short sequence reads onto a single reference genome. The structural variations from the 2,898 accessions that were genotyped based on the graph-based genome and the RNA sequencing (RNA-seq) data from the representative 26 accessions helped to link genetic variations to candidate genes that are responsible for important traits. This pan-genome resource will promote evolutionary and functional genomics studies in soybean.

Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B.

Enterovirus B (EV-B), a major proportion of the genus Enterovirus in the family Picornaviridae, is the causative agent of severe human infectious diseases. Although cellular receptors for coxsackievirus B in EV-B have been identified, receptors mediating virus entry, especially the uncoating process of echovirus and other EV-B remain obscure. Here, we found that human neonatal Fc receptor (FcRn) is the uncoating receptor for major EV-B. FcRn binds to the virus particles in the "canyon" through its FCGRT subunit. By obtaining multiple cryo-electron microscopy structures at different stages of virus entry at atomic or near-atomic resolution, we deciphered the underlying mechanisms of enterovirus attachment and uncoating. These structures revealed that different from the attachment receptor CD55, binding of FcRn to the virions induces efficient release of "pocket factor" under acidic conditions and initiates the conformational changes in viral particle, providing a structural basis for understanding the mechanisms of enterovirus entry.

Alarmin-loaded extracellular lipid droplets induce airway neutrophil infiltration during type 2 inflammation.

Group 2 innate lymphoid cells (ILC2s) play a crucial role in allergic diseases by coordinating a complex network of various effector cell lineages involved in type 2 inflammation. However, their function in regulating airway neutrophil infiltration, a deleterious symptom of severe asthma, remains unknown. Here, we observed ILC2-dependent neutrophil accumulation in the bronchoalveolar lavage fluid (BALF) of allergic mouse models. Chromatography followed by proteomics analysis identified the alarmin high mobility group box-1 (HMGB1) in the supernatant of lung ILC2s initiated neutrophil chemotaxis. Genetic perturbation of Hmgb1 in ILC2s reduced BALF neutrophil numbers and alleviated airway inflammation. HMGB1 was loaded onto the membrane of lipid droplets (LDs) released from activated lung ILC2s. Genetic inhibition of LD accumulation in ILC2s significantly decreased extracellular HMGB1 abundance and BALF neutrophil infiltration. These findings unveil a previously uncharacterized extracellular LD-mediated immune signaling delivery pathway by which ILC2s regulate airway neutrophil infiltration during allergic inflammation.

Systemic Human ILC Precursors Provide a Substrate for Tissue ILC Differentiation.

Innate lymphoid cells (ILCs) represent innate versions of T helper and cytotoxic T cells that differentiate from committed ILC precursors (ILCPs). How ILCPs give rise to mature tissue-resident ILCs remains unclear. Here, we identify circulating and tissue ILCPs in humans that fail to express the transcription factors and cytokine outputs of mature ILCs but have these signature loci in an epigenetically poised configuration. Human ILCPs robustly generate all ILC subsets in vitro and in vivo. While human ILCPs express low levels of retinoic acid receptor (RAR)-related orphan receptor C (RORC) transcripts, these cells are found in RORC-deficient patients and retain potential for EOMES+ natural killer (NK) cells, interferon gamma-positive (IFN-γ+) ILC1s, interleukin (IL)-13+ ILC2s, and for IL-22+, but not for IL-17A+ ILC3s. Our results support a model of tissue ILC differentiation ("ILC-poiesis"), whereby diverse ILC subsets are generated in situ from systemically distributed ILCPs in response to local environmental signals.

Structural basis for plasmid restriction by SMC JET nuclease.

DNA loop-extruding SMC complexes play crucial roles in chromosome folding and DNA immunity. Prokaryotic SMC Wadjet (JET) complexes limit the spread of plasmids through DNA cleavage, yet the mechanisms for plasmid recognition are unresolved. We show that artificial DNA circularization renders linear DNA susceptible to JET nuclease cleavage. Unlike free DNA, JET cleaves immobilized plasmid DNA at a specific site, the plasmid-anchoring point, showing that the anchor hinders DNA extrusion but not DNA cleavage. Structures of plasmid-bound JetABC reveal two presumably stalled SMC motor units that are drastically rearranged from the resting state, together entrapping a U-shaped DNA segment, which is further converted to kinked V-shaped cleavage substrate by JetD nuclease binding. Our findings uncover mechanical bending of residual unextruded DNA as molecular signature for plasmid recognition and non-self DNA elimination. We moreover elucidate key elements of SMC loop extrusion, including the motor direction and the structure of a DNA-holding state.

A transient transcriptional activation governs unpolarized-to-polarized morphogenesis during embryo implantation.

During implantation, embryos undergo an unpolarized-to-polarized transition to initiate postimplantation morphogenesis. However, the underlying molecular mechanism is unknown. Here, we identify a transient transcriptional activation governing embryonic morphogenesis and pluripotency transition during implantation. In naive pluripotent embryonic stem cells (ESCs), which represent preimplantation embryos, we find that the microprocessor component DGCR8 can recognize stem-loop structures within nascent mRNAs to sequester transcriptional coactivator FLII to suppress transcription directly. When mESCs exit from naive pluripotency, the ERK/RSK/P70S6K pathway rapidly activates, leading to FLII phosphorylation and disruption of DGCR8/FLII interaction. Phosphorylated FLII can bind to transcription factor JUN, activating cell migration-related genes to establish poised pluripotency akin to implanting embryos. Resequestration of FLII by DGCR8 drives poised ESCs into formative pluripotency. In summary, we identify a DGCR8/FLII/JUN-mediated transient transcriptional activation mechanism. Disruption of this mechanism inhibits naive-poised-formative pluripotency transition and the corresponding unpolarized-to-polarized transition during embryo implantation, which are conserved in mice and humans.

A novel pathogenic mutation of MeCP2 impairs chromatin association independent of protein levels.

Loss-of-function mutations in MECP2 cause Rett syndrome (RTT), a severe neurological disorder that mainly affects girls. Mutations in MECP2 do occur in males occasionally and typically cause severe encephalopathy and premature lethality. Recently, we identified a missense mutation (c.353G>A, p.Gly118Glu [G118E]), which has never been seen before in MECP2, in a young boy who suffered from progressive motor dysfunction and developmental delay. To determine whether this variant caused the clinical symptoms and study its functional consequences, we established two disease models, including human neurons from patient-derived iPSCs and a knock-in mouse line. G118E mutation partially reduces MeCP2 abundance and its DNA binding, and G118E mice manifest RTT-like symptoms seen in the patient, affirming the pathogenicity of this mutation. Using live-cell and single-molecule imaging, we found that G118E mutation alters MeCP2's chromatin interaction properties in live neurons independently of its effect on protein levels. Here we report the generation and characterization of RTT models of a male hypomorphic variant and reveal new insight into the mechanism by which this pathological mutation affects MeCP2's chromatin dynamics. Our ability to quantify protein dynamics in disease models lays the foundation for harnessing high-resolution single-molecule imaging as the next frontier for developing innovative therapies for RTT and other diseases.

Electrocatalytic reductive deuteration of arenes and heteroarenes.

The incorporation of deuterium in organic molecules has widespread applications in medicinal chemistry and materials science1,2. For example, the deuterated drugs austedo3, donafenib4 and sotyktu5 have been recently approved. There are various methods for the synthesis of deuterated compounds with high deuterium incorporation6. However, the reductive deuteration of aromatic hydrocarbons-ubiquitous chemical feedstocks-to saturated cyclic compounds has rarely been achieved. Here we describe a scalable and general electrocatalytic method for the reductive deuteration and deuterodefluorination of (hetero)arenes using a prepared nitrogen-doped electrode and deuterium oxide (D2O), giving perdeuterated and saturated deuterocarbon products. This protocol has been successfully applied to the synthesis of 13 highly deuterated drug molecules. Mechanistic investigations suggest that the ruthenium-deuterium species, generated by electrolysis of D2O in the presence of a nitrogen-doped ruthenium electrode, are key intermediates that directly reduce aromatic compounds. This quick and cost-effective methodology for the preparation of highly deuterium-labelled saturated (hetero)cyclic compounds could be applied in drug development and metabolism studies.

Bitter taste TAS2R14 activation by intracellular tastants and cholesterol.

Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents1,2. TAS2R14 is also widely expressed in extragustatory tissues, suggesting its extra roles in diverse physiological processes and potential therapeutic applications3. Here we present cryogenic electron microscopy structures of TAS2R14 in complex with aristolochic acid, flufenamic acid and compound 28.1, coupling with different G-protein subtypes. Uniquely, a cholesterol molecule is observed occupying what is typically an orthosteric site in class A G-protein-coupled receptors. The three potent agonists bind, individually, to the intracellular pockets, suggesting a distinct activation mechanism for this receptor. Comprehensive structural analysis, combined with mutagenesis and molecular dynamic simulation studies, elucidate the broad-spectrum ligand recognition and activation of the receptor by means of intricate multiple ligand-binding sites. Our study also uncovers the specific coupling modes of TAS2R14 with gustducin and Gi1 proteins. These findings should be instrumental in advancing knowledge of bitter taste perception and its broader implications in sensory biology and drug discovery.

DNA-measuring Wadjet SMC ATPases restrict smaller circular plasmids by DNA cleavage.

Structural maintenance of chromosome (SMC) complexes fold DNA by loop extrusion to support chromosome segregation and genome maintenance. Wadjet systems (JetABCD/MksBEFG/EptABCD) are derivative SMC complexes with roles in bacterial immunity against selfish DNA. Here, we show that JetABCD restricts circular plasmids with an upper size limit of about 100 kb, whereas a linear plasmid evades restriction. Purified JetABCD complexes cleave circular DNA molecules, regardless of the DNA helical topology; cleavage is DNA sequence nonspecific and depends on the SMC ATPase. A cryo-EM structure reveals a distinct JetABC dimer-of-dimers geometry, with the two SMC dimers facing in opposite direction-rather than the same as observed with MukBEF. We hypothesize that JetABCD is a DNA-shape-specific endonuclease and propose the "total extrusion model" for DNA cleavage exclusively when extrusion of an entire plasmid has been completed by a JetABCD complex. Total extrusion cannot be achieved on the larger chromosome, explaining how self-DNA may evade processing.

Interferon-γ induces tumor resistance to anti-PD-1 immunotherapy by promoting YAP phase separation.

Interferon-γ (IFN-γ)-mediated adaptive resistance is one major barrier to improving immunotherapy in solid tumors. However, the mechanisms are not completely understood. Here, we report that IFN-γ promotes nuclear translocation and phase separation of YAP after anti-PD-1 therapy in tumor cells. Hydrophobic interactions of the YAP coiled-coil domain mediate droplet initiation, and weak interactions of the intrinsically disordered region in the C terminus promote droplet formation. YAP partitions with the transcription factor TEAD4, the histone acetyltransferase EP300, and Mediator1 and forms transcriptional hubs for maximizing target gene transcriptions, independent of the canonical STAT1-IRF1 transcription program. Disruption of YAP phase separation reduced tumor growth, enhanced immune response, and sensitized tumor cells to anti-PD-1 therapy. YAP activity is negatively correlated with patient outcome. Our study indicates that YAP mediates the IFN-γ pro-tumor effect through its nuclear phase separation and suggests that YAP can be used as a predictive biomarker and target of anti-PD-1 combination therapy.

DELE1 maintains muscle proteostasis to promote growth and survival in mitochondrial myopathy.

Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy, but how muscle senses and adapts to mitochondrial dysfunction is not well understood. Here, we used diverse mouse models of mitochondrial myopathy to show that the signal for mitochondrial dysfunction originates within mitochondria. The mitochondrial proteins OMA1 and DELE1 sensed disruption of the inner mitochondrial membrane and, in response, activated the mitochondrial integrated stress response (mt-ISR) to increase the building blocks for protein synthesis. In the absence of the mt-ISR, protein synthesis in muscle was dysregulated causing protein misfolding, and mice with early-onset mitochondrial myopathy failed to grow and survive. The mt-ISR was similar following disruptions in mtDNA maintenance (Tfam knockout) and mitochondrial protein misfolding (CHCHD10 G58R and S59L knockin) but heterogenous among mitochondria-rich tissues, with broad gene expression changes observed in heart and skeletal muscle and limited changes observed in liver and brown adipose tissue. Taken together, our findings identify that the DELE1 mt-ISR mediates a similar response to diverse forms of mitochondrial stress and is critical for maintaining growth and survival in early-onset mitochondrial myopathy.

Epigenomic analysis of multilineage differentiation of human embryonic stem cells.

Epigenetic mechanisms have been proposed to play crucial roles in mammalian development, but their precise functions are only partially understood. To investigate epigenetic regulation of embryonic development, we differentiated human embryonic stem cells into mesendoderm, neural progenitor cells, trophoblast-like cells, and mesenchymal stem cells and systematically characterized DNA methylation, chromatin modifications, and the transcriptome in each lineage. We found that promoters that are active in early developmental stages tend to be CG rich and mainly engage H3K27me3 upon silencing in nonexpressing lineages. By contrast, promoters for genes expressed preferentially at later stages are often CG poor and primarily employ DNA methylation upon repression. Interestingly, the early developmental regulatory genes are often located in large genomic domains that are generally devoid of DNA methylation in most lineages, which we termed DNA methylation valleys (DMVs). Our results suggest that distinct epigenetic mechanisms regulate early and late stages of ES cell differentiation.