Realizing the full utility of brain organoids to study human development requires understanding whether organoids precisely replicate endogenous cellular and molecular events, particularly since acquisition of cell identity in organoids can be impaired by abnormal metabolic states. We present a comprehensive single-cell transcriptomic, epigenetic, and spatial atlas of human cortical organoid development, comprising over 610,000 cells, from generation of neural progenitors through production of differentiated neuronal and glial subtypes. We show that processes of cellular diversification correlate closely to endogenous ones, irrespective of metabolic state, empowering the use of this atlas to study human fate specification. We define longitudinal molecular trajectories of cortical cell types during organoid development, identify genes with predicted human-specific roles in lineage establishment, and uncover early transcriptional diversity of human callosal neurons. The findings validate this comprehensive atlas of human corticogenesis in vitro as a resource to prime investigation into the mechanisms of human cortical development.

Comment in Cell. 2022 Sep 29;185(20):3648-3651. Nat Rev Cancer. 2022 Dec;22(12):659.

Epstein-Barr virus (EBV) is a ubiquitous, oncogenic virus that is associated with a number of different human malignancies as well as autoimmune disorders. The expression of EBV viral proteins and non-coding RNAs contribute to EBV-mediated disease pathologies. The virus establishes life-long latency in the human host and is adept at evading host innate and adaptive immune responses. In this review, we discuss the life cycle of EBV, the various functions of EBV-encoded proteins and RNAs, the ability of the virus to activate and evade immune responses, as well as the neoplastic and autoimmune diseases that are associated with EBV infection in the human population.

Necrosis of macrophages in the granuloma, the hallmark immunological structure of tuberculosis, is a major pathogenic event that increases host susceptibility. Through a zebrafish forward genetic screen, we identified the mTOR kinase, a master regulator of metabolism, as an early host resistance factor in tuberculosis. We found that mTOR complex 1 protects macrophages from mycobacterium-induced death by enabling infection-induced increases in mitochondrial energy metabolism fueled by glycolysis. These metabolic adaptations are required to prevent mitochondrial damage and death caused by the secreted mycobacterial virulence determinant ESAT-6. Thus, the host can effectively counter this early critical mycobacterial virulence mechanism simply by regulating energy metabolism, thereby allowing pathogen-specific immune mechanisms time to develop. Our findings may explain why Mycobacterium tuberculosis, albeit humanity's most lethal pathogen, is successful in only a minority of infected individuals.

Comment in Cell. 2022 Sep 29;185(20):3645-3647.

Comment in Cell. 2022 Sep 29;185(20):3648-3651. Nat Rev Cancer. 2022 Dec;22(12):659.

The intestinal microbiota is an important modulator of graft-versus-host disease (GVHD), which often complicates allogeneic hematopoietic stem cell transplantation (allo-HSCT). Broad-spectrum antibiotics such as carbapenems increase the risk for intestinal GVHD, but mechanisms are not well understood. In this study, we found that treatment with meropenem, a commonly used carbapenem, aggravates colonic GVHD in mice via the expansion of Bacteroides thetaiotaomicron (BT). BT has a broad ability to degrade dietary polysaccharides and host mucin glycans. BT in meropenem-treated allogeneic mice demonstrated upregulated expression of enzymes involved in the degradation of mucin glycans. These mice also had thinning of the colonic mucus layer and decreased levels of xylose in colonic luminal contents. Interestingly, oral xylose supplementation significantly prevented thinning of the colonic mucus layer in meropenem-treated mice. Specific nutritional supplementation strategies, including xylose supplementation, may combat antibiotic-mediated microbiome injury to reduce the risk for intestinal GVHD in allo-HSCT patients.

Comment in Nat Rev Genet. 2022 Dec;23(12):714.

Biochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here, we demonstrate synthetic membraneless organelles in Escherichia coli termed transcriptionally engineered addressable RNA solvent droplets (TEARS). TEARS are assembled from RNA-binding protein recruiting domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures with composition and reaction robustness governed by non-equilibrium dynamics. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates, and scaffolding protein-protein interactions. We anticipate TEARS to be a simple and versatile tool for spatially controlling E. coli biochemistry. Particularly, the modular design of TEARS enables applications without expression fine-tuning, simplifying the design-build-test cycle of bioengineering.

Comment on Cell. 2022 Sep 29;185(20):3753-3769.e18.

Comment on Cell. 2022 Sep 29;185(20):3789-3806.e17. Cell. 2022 Sep 29;185(20):3807-3822.e12.

Comment on Cell. 2022 Sep 29;185(20):3671-3688.e23.

Comment in Cell. 2022 Sep 29;185(20):3643-3645.

Lysosomal amino acid efflux by proton-driven transporters is essential for lysosomal homeostasis, amino acid recycling, mTOR signaling, and maintaining lysosomal pH. To unravel the mechanisms of these transporters, we focus on cystinosin, a prototypical lysosomal amino acid transporter that exports cystine to the cytosol, where its reduction to cysteine supplies this limiting amino acid for diverse fundamental processes and controlling nutrient adaptation. Cystinosin mutations cause cystinosis, a devastating lysosomal storage disease. Here, we present structures of human cystinosin in lumen-open, cytosol-open, and cystine-bound states, which uncover the cystine recognition mechanism and capture the key conformational states of the transport cycle. Our structures, along with functional studies and double electron-electron resonance spectroscopic investigations, reveal the molecular basis for the transporter's conformational transitions and protonation switch, show conformation-dependent Ragulator-Rag complex engagement, and demonstrate an unexpected activation mechanism. These findings provide molecular insights into lysosomal amino acid efflux and a potential therapeutic strategy.

Computational analysis of cellular activity has developed largely independently of modern transcriptomic cell typology, but integrating these approaches may be essential for full insight into cellular-level mechanisms underlying brain function and dysfunction. Applying this approach to the habenula (a structure with diverse, intermingled molecular, anatomical, and computational features), we identified encoding of reward-predictive cues and reward outcomes in distinct genetically defined neural populations, including TH+ cells and Tac1+ cells. Data from genetically targeted recordings were used to train an optimized nonlinear dynamical systems model and revealed activity dynamics consistent with a line attractor. High-density, cell-type-specific electrophysiological recordings and optogenetic perturbation provided supporting evidence for this model. Reverse-engineering predicted how Tac1+ cells might integrate reward history, which was complemented by in vivo experimentation. This integrated approach describes a process by which data-driven computational models of population activity can generate and frame actionable hypotheses for cell-type-specific investigation in biological systems.

Comment in Nat Rev Immunol. 2022 Nov;22(11):654-655.

Efforts to model the human gut microbiome in mice have led to important insights into the mechanisms of host-microbe interactions. However, the model communities studied to date have been defined or complex, but not both, limiting their utility. Here, we construct and characterize in vitro a defined community of 104 bacterial species composed of the most common taxa from the human gut microbiota (hCom1). We then used an iterative experimental process to fill open niches: germ-free mice were colonized with hCom1 and then challenged with a human fecal sample. We identified new species that engrafted following fecal challenge and added them to hCom1, yielding hCom2. In gnotobiotic mice, hCom2 exhibited increased stability to fecal challenge and robust colonization resistance against pathogenic Escherichia coli. Mice colonized by either hCom2 or a human fecal community are phenotypically similar, suggesting that this consortium will enable a mechanistic interrogation of species and genes on microbiome-associated phenotypes.

Comment in Cell. 2022 Sep 15;185(19):3459-3461.

Cells are continuously exposed to tissue-specific extrinsic forces that are counteracted by cell-intrinsic force generation through the actomyosin cytoskeleton and alterations in the material properties of various cellular components, including the nucleus. Forces impact nuclei both directly through inducing deformation, which is sensed by various mechanosensitive components of the nucleus, as well as indirectly through the actomyosin cytoskeleton and mechanosensitive pathways activated in the cytoplasm. To view this SnapShot, open or download the PDF.

Comment on Cell. 2022 Sep 15;185(19):3487-3500.e14.

Interactions between cells are indispensable for signaling and creating structure. The ability to direct precise cell-cell interactions would be powerful for engineering tissues, understanding signaling pathways, and directing immune cell targeting. In humans, intercellular interactions are mediated by cell adhesion molecules (CAMs). However, endogenous CAMs are natively expressed by many cells and tend to have cross-reactivity, making them unsuitable for programming specific interactions. Here, we showcase "helixCAM," a platform for engineering synthetic CAMs by presenting coiled-coil peptides on the cell surface. helixCAMs were able to create specific cell-cell interactions and direct patterned aggregate formation in bacteria and human cells. Based on coiled-coil interaction principles, we built a set of rationally designed helixCAM libraries, which led to the discovery of additional high-performance helixCAM pairs. We applied this helixCAM toolkit for various multicellular engineering applications, such as spherical layering, adherent cell targeting, and surface patterning.

We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6-12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6-12 residue size range cross membranes with an apparent permeability greater than 1 × 10-6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.

Comment in Cell. 2022 Sep 15;185(19):3461-3463.

Comment in Cell. 2022 Sep 15;185(19):3463-3466.

Comment on Cell. 2022 Sep 15;185(19):3467-3486.e16.

The effects of mutations in continuously emerging variants of SARS-CoV-2 are a major concern for the performance of rapid antigen tests. To evaluate the impact of mutations on 17 antibodies used in 11 commercially available antigen tests with emergency use authorization, we measured antibody binding for all possible Nucleocapsid point mutations using a mammalian surface-display platform and deep mutational scanning. The results provide a complete map of the antibodies' epitopes and their susceptibility to mutational escape. Our data predict no vulnerabilities for detection of mutations found in variants of concern. We confirm this using the commercial tests and sequence-confirmed COVID-19 patient samples. The antibody escape mutational profiles generated here serve as a valuable resource for predicting the performance of rapid antigen tests against past, current, as well as any possible future variants of SARS-CoV-2, establishing the direct clinical and public health utility of our system.

Comment on Cell. 2022 Sep 15;185(19):3533-3550.e27.

Comment in Nat Rev Microbiol. 2022 Nov;20(11):637. Cell Metab. 2022 Oct 4;34(10):1422-1424.

The extracellular pH is a vital regulator of various biological processes in plants. However, how plants perceive extracellular pH remains obscure. Here, we report that plant cell-surface peptide-receptor complexes can function as extracellular pH sensors. We found that pattern-triggered immunity (PTI) dramatically alkalinizes the acidic extracellular pH in root apical meristem (RAM) region, which is essential for root meristem growth factor 1 (RGF1)-mediated RAM growth. The extracellular alkalinization progressively inhibits the acidic-dependent interaction between RGF1 and its receptors (RGFRs) through the pH sensor sulfotyrosine. Conversely, extracellular alkalinization promotes the alkaline-dependent binding of plant elicitor peptides (Peps) to its receptors (PEPRs) through the pH sensor Glu/Asp, thereby promoting immunity. A domain swap between RGFR and PEPR switches the pH dependency of RAM growth. Thus, our results reveal a mechanism of extracellular pH sensing by plant peptide-receptor complexes and provide insights into the extracellular pH-mediated regulation of growth and immunity in the RAM.

Comment in Nat Rev Mol Cell Biol. 2022 Nov;23(11):697.

Comment in Cell. 2022 Sep 1;185(18):3282-3284. Nat Rev Endocrinol. 2022 Dec;18(12):719-720. Nat Med. 2022 Nov;28(11):2259-2260.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease involving multiple immune cells. To elucidate SLE pathogenesis, it is essential to understand the dysregulated gene expression pattern linked to various clinical statuses with a high cellular resolution. Here, we conducted a large-scale transcriptome study with 6,386 RNA sequencing data covering 27 immune cell types from 136 SLE and 89 healthy donors. We profiled two distinct cell-type-specific transcriptomic signatures: disease-state and disease-activity signatures, reflecting disease establishment and exacerbation, respectively. We then identified candidate biological processes unique to each signature. This study suggested the clinical value of disease-activity signatures, which were associated with organ involvement and therapeutic responses. However, disease-activity signatures were less enriched around SLE risk variants than disease-state signatures, suggesting that current genetic studies may not well capture clinically vital biology. Together, we identified comprehensive gene signatures of SLE, which will provide essential foundations for future genomic and genetic studies.

Comment in Nature. 2022 Sep;609(7926):230-231. Nat Methods. 2022 Sep;19(9):1033. Nature. 2022 Oct;610(7930):39-40. Cell Stem Cell. 2022 Oct 6;29(10):1423-1425.

Comment in Nat Rev Gastroenterol Hepatol. 2022 Nov;19(11):687.

Comment in Cell. 2022 Sep 1;185(18):3284-3286. Nat Rev Neurosci. 2022 Nov;23(11):644-645.

Copyright © 2022. Published by Elsevier Inc.

Author information: (1)New York Genome Center, New York, NY 10013, USA. Electronic address: mbyrska-bishop@nygenome.org. (2)New York Genome Center, New York, NY 10013, USA. (3)Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA. (4)McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA; Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA. (5)New York Genome Center, New York, NY 10013, USA; Outlier Informatics Inc., Saskatoon, SK S7H 1L4, Canada. (6)European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK. (7)Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. (8)McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA; Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA; Center for Genomic Health, Yale University School of Medicine, New Haven, CT 06510, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA. (9)New York Genome Center, New York, NY 10013, USA. Electronic address: mczody@nygenome.org.

The Mediator complex controls RNA polymerase II transcription genome-wide. In humans, Mediator consists of 26 subunits; furthermore, a four-subunit "Mediator kinase module" can reversibly associate with the complex. Mediator structure is generally conserved from yeast to humans, although the human complex is larger, more structurally disordered, and contains metazoan-specific subunits. To view this SnapShot, open or download the PDF.

Comment on Cell. 2022 Sep 1;185(18):3390-3407.e18.

Comment on Cell. 2022 Sep 1;185(18):3307-3328.e19.

Comment on Cell. 2022 Sep 1;185(18):3426-3440.e19.

Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 μm and report voltage correlations in pairs of neurons.

Type 1 secretion systems (T1SSs) are widespread in pathogenic Gram-negative bacteria, extruding protein substrates following synthesis of the entire polypeptide. The Escherichia coli hemolysin A secretion system has long been considered a prototype in structural and mechanistic studies of T1SSs. Three membrane proteins-an inner membrane ABC transporter HlyB, an adaptor protein HlyD, and an outer membrane porin TolC-are required for secretion. However, the stoichiometry and structure of the complex are unknown. Here, cryo-electron microscopy (cryo-EM) structures determined in two conformations reveal that the inner membrane complex is a hetero-dodecameric assembly comprising three HlyB homodimers and six HlyD subunits. Functional studies indicate that oligomerization of HlyB and HlyD is essential for protein secretion and that polypeptides translocate through a canonical ABC transporter pathway in HlyB. Our data suggest that T1SSs entail three ABC transporters, one that functions as a protein channel and two that allosterically power the translocation process.