Time-series reconstruction of the molecular architecture of human centriole assembly.

Centriole biogenesis, as in most organelle assemblies, involves the sequential recruitment of sub-structural elements that will support its function. To uncover this process, we correlated the spatial location of 24 centriolar proteins with structural features using expansion microscopy. A time-series reconstruction of protein distributions throughout human procentriole assembly unveiled the molecular architecture of the centriole biogenesis steps. We found that the process initiates with the formation of a naked cartwheel devoid of microtubules. Next, the bloom phase progresses with microtubule blade assembly, concomitantly with radial separation and rapid cartwheel growth. In the subsequent elongation phase, the tubulin backbone grows linearly with the recruitment of the A-C linker, followed by proteins of the inner scaffold (IS). By following six structural modules, we modeled 4D assembly of the human centriole. Collectively, this work provides a framework to investigate the spatial and temporal assembly of large macromolecules.

Therapeutic potential of co-signaling receptor modulation in hepatitis B.

Reversing CD8+ T cell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ T cells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ T cells. Targeting all dysfunctional T cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.

Human prefrontal cortex gene regulatory dynamics from gestation to adulthood at single-cell resolution.

Human brain development is underpinned by cellular and molecular reconfigurations continuing into the third decade of life. To reveal cell dynamics orchestrating neural maturation, we profiled human prefrontal cortex gene expression and chromatin accessibility at single-cell resolution from gestation to adulthood. Integrative analyses define the dynamic trajectories of each cell type, revealing major gene expression reconfiguration at the prenatal-to-postnatal transition in all cell types followed by continuous reconfiguration into adulthood and identifying regulatory networks guiding cellular developmental programs, states, and functions. We uncover links between expression dynamics and developmental milestones, characterize the diverse timing of when cells acquire adult-like states, and identify molecular convergence from distinct developmental origins. We further reveal cellular dynamics and their regulators implicated in neurological disorders. Finally, using this reference, we benchmark cell identities and maturation states in organoid models. Together, this captures the dynamic regulatory landscape of human cortical development.

Co-option of Neutrophil Fates by Tissue Environments.

Classically considered short-lived and purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional, and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion of neutrophils compromised angiogenesis during early age, genotoxic injury, and viral infection, and impaired hematopoietic recovery after irradiation. Neutrophils acquired these properties in target tissues, a process that, in the lungs, occurred in CXCL12-rich areas and relied on CXCR4. Our results reveal that tissues co-opt neutrophils en route for elimination to induce programs that support their physiological demands.

Cancer Burden Is Controlled by Mural Cell-ß3-Integrin Regulated Crosstalk with Tumor Cells.

Enhanced blood vessel (BV) formation is thought to drive tumor growth through elevated nutrient delivery. However, this observation has overlooked potential roles for mural cells in directly affecting tumor growth independent of BV function. Here we provide clinical data correlating high percentages of mural-ß3-integrin-negative tumor BVs with increased tumor sizes but no effect on BV numbers. Mural-ß3-integrin loss also enhances tumor growth in implanted and autochthonous mouse tumor models with no detectable effects on BV numbers or function. At a molecular level, mural-cell ß3-integrin loss enhances signaling via FAK-p-HGFR-p-Akt-p-p65, driving CXCL1, CCL2, and TIMP-1 production. In particular, mural-cell-derived CCL2 stimulates tumor cell MEK1-ERK1/2-ROCK2-dependent signaling and enhances tumor cell survival and tumor growth. Overall, our data indicate that mural cells can control tumor growth via paracrine signals regulated by ß3-integrin, providing a previously unrecognized mechanism of cancer growth control.

A Paleogenomic Reconstruction of the Deep Population History of the Andes.

There are many unanswered questions about the population history of the Central and South Central Andes, particularly regarding the impact of large-scale societies, such as the Moche, Wari, Tiwanaku, and Inca. We assembled genome-wide data on 89 individuals dating from ∼9,000-500 years ago (BP), with a particular focus on the period of the rise and fall of state societies. Today's genetic structure began to develop by 5,800 BP, followed by bi-directional gene flow between the North and South Highlands, and between the Highlands and Coast. We detect minimal admixture among neighboring groups between ∼2,000-500 BP, although we do detect cosmopolitanism (people of diverse ancestries living side-by-side) in the heartlands of the Tiwanaku and Inca polities. We also highlight cases of long-range mobility connecting the Andes to Argentina and the Northwest Andes to the Amazon Basin. VIDEO ABSTRACT.

Characterizing Mutational Signatures in Human Cancer Cell Lines Reveals Episodic APOBEC Mutagenesis.

Multiple signatures of somatic mutations have been identified in cancer genomes. Exome sequences of 1,001 human cancer cell lines and 577 xenografts revealed most common mutational signatures, indicating past activity of the underlying processes, usually in appropriate cancer types. To investigate ongoing patterns of mutational-signature generation, cell lines were cultured for extended periods and subsequently DNA sequenced. Signatures of discontinued exposures, including tobacco smoke and ultraviolet light, were not generated in vitro. Signatures of normal and defective DNA repair and replication continued to be generated at roughly stable mutation rates. Signatures of APOBEC cytidine deaminase DNA-editing exhibited substantial fluctuations in mutation rate over time with episodic bursts of mutations. The initiating factors for the bursts are unclear, although retrotransposon mobilization may contribute. The examined cell lines constitute a resource of live experimental models of mutational processes, which potentially retain patterns of activity and regulation operative in primary human cancers.

Programmed 'disarming' of the neutrophil proteome reduces the magnitude of inflammation.

The antimicrobial functions of neutrophils are facilitated by a defensive armamentarium of proteins stored in granules, and by the formation of neutrophil extracellular traps (NETs). However, the toxic nature of these structures poses a threat to highly vascularized tissues, such as the lungs. Here, we identified a cell-intrinsic program that modified the neutrophil proteome in the circulation and caused the progressive loss of granule content and reduction of the NET-forming capacity. This program was driven by the receptor CXCR2 and by regulators of circadian cycles. As a consequence, lungs were protected from inflammatory injury at times of day or in mouse mutants in which granule content was low. Changes in the proteome, granule content and NET formation also occurred in human neutrophils, and correlated with the incidence and severity of respiratory distress in pneumonia patients. Our findings unveil a 'disarming' strategy of neutrophils that depletes protein stores to reduce the magnitude of inflammation.

Bedside Back to Bench: Building Bridges between Basic and Clinical Genomic Research.

Genome sequencing has revolutionized the diagnosis of genetic diseases. Close collaborations between basic scientists and clinical genomicists are now needed to link genetic variants with disease causation. To facilitate such collaborations, we recommend prioritizing clinically relevant genes for functional studies, developing reference variant-phenotype databases, adopting phenotype description standards, and promoting data sharing.

Metabolic Control of Longevity.

Several metabolic alterations accumulate over time along with a reduction in biological fitness, suggesting the existence of a "metabolic clock" that controls aging. Multiple inborn defects in metabolic circuitries accelerate aging, whereas genetic loci linked to exceptional longevity influence metabolism. Each of the nine hallmarks of aging is connected to undesirable metabolic alterations. The main features of the "westernized" lifestyle, including hypercaloric nutrition and sedentariness, can accelerate aging as they have detrimental metabolic consequences. Conversely, lifespan-extending maneuvers including caloric restriction impose beneficial pleiotropic effects on metabolism. The introduction of strategies that promote metabolic fitness may extend healthspan in humans.

Identification of a Kupffer cell subset capable of reverting the T cell dysfunction induced by hepatocellular priming.

Kupffer cells (KCs) are highly abundant, intravascular, liver-resident macrophages known for their scavenger and phagocytic functions. KCs can also present antigens to CD8+ T cells and promote either tolerance or effector differentiation, but the mechanisms underlying these discrepant outcomes are poorly understood. Here, we used a mouse model of hepatitis B virus (HBV) infection, in which HBV-specific naive CD8+ T cells recognizing hepatocellular antigens are driven into a state of immune dysfunction, to identify a subset of KCs (referred to as KC2) that cross-presents hepatocellular antigens upon interleukin-2 (IL-2) administration, thus improving the antiviral function of T cells. Removing MHC-I from all KCs, including KC2, or selectively depleting KC2 impaired the capacity of IL-2 to revert the T cell dysfunction induced by intrahepatic priming. In summary, by sensing IL-2 and cross-presenting hepatocellular antigens, KC2 overcome the tolerogenic potential of the hepatic microenvironment, suggesting new strategies for boosting hepatic T cell immunity.

Neutralization potency of monoclonal antibodies recognizing dominant and subdominant epitopes on SARS-CoV-2 Spike is impacted by the B.1.1.7 variant.

Interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the receptor ACE2 on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies, and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, N-terminal domain (NTD) and S2 subunits of Spike. To understand how these mutations affect Spike antigenicity, we isolated and characterized >100 monoclonal antibodies targeting epitopes on RBD, NTD, and S2 from SARS-CoV-2-infected individuals. Approximately 45% showed neutralizing activity, of which ∼20% were NTD specific. NTD-specific antibodies formed two distinct groups: the first was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Mutations present in B.1.1.7 Spike frequently conferred neutralization resistance to NTD-specific antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes should be considered when investigating antigenic drift in emerging variants.

Gene-specific translation regulation mediated by the hormone-signaling molecule EIN2.

The central role of translation in modulating gene activity has long been recognized, yet the systematic exploration of quantitative changes in translation at a genome-wide scale in response to a specific stimulus has only recently become technically feasible. Using the well-characterized signaling pathway of the phytohormone ethylene and plant-optimized genome-wide ribosome footprinting, we have uncovered a molecular mechanism linking this hormone's perception to the activation of a gene-specific translational control mechanism. Characterization of one of the targets of this translation regulatory machinery, the ethylene signaling component EBF2, indicates that the signaling molecule EIN2 and the nonsense-mediated decay proteins UPFs play a central role in this ethylene-induced translational response. Furthermore, the 3'UTR of EBF2 is sufficient to confer translational regulation and required for the proper activation of ethylene responses. These findings represent a mechanistic paradigm of gene-specific regulation of translation in response to a key growth regulator.

Arresting failure propagation in buildings through collapse isolation.

Several catastrophic building collapses1-5 occur because of the propagation of local-initial failures6,7. Current design methods attempt to completely prevent collapse after initial failures by improving connectivity between building components. These measures ensure that the loads supported by the failed components are redistributed to the rest of the structural system8,9. However, increased connectivity can contribute to collapsing elements pulling down parts of a building that would otherwise be unaffected10. This risk is particularly important when large initial failures occur, as tends to be the case in the most disastrous collapses6. Here we present an original design approach to arrest collapse propagation after major initial failures. When a collapse initiates, the approach ensures that specific elements fail before the failure of the most critical components for global stability. The structural system thus separates into different parts and isolates collapse when its propagation would otherwise be inevitable. The effectiveness of the approach is proved through unique experimental tests on a purposely built full-scale building. We also demonstrate that large initial failures would lead to total collapse of the test building if increased connectivity was implemented as recommended by present guidelines. Our proposed approach enables incorporating a last line of defence for more resilient buildings.

Bound star clusters observed in a lensed galaxy 460 Myr after the Big Bang.

The Cosmic Gems arc is among the brightest and highly magnified galaxies observed at redshift z ∼ 10.21. However, it is an intrinsically UV faint galaxy, in the range of those now thought to drive the reionization of the universe2-4. Hitherto the smallest features resolved in a galaxy at a comparable redshift are between a few hundreds and a few tens of parsecs5,6. Here we report JWST observations of the Cosmic Gems. The light of the galaxy is resolved into five star clusters located in a region smaller than 70 parsec. They exhibit minimal dust attenuation and low metallicity, ages younger than 50 Myr and intrinsic masses of ∼ 106 M⊙. Their lensing-corrected sizes are approximately 1 pc, resulting in stellar surface densities near 105 M⊙ /pc2, three orders of magnitude higher than typical young star clusters in the local universe7. Despite the uncertainties inherent to the lensing model, they are consistent with being gravitationally bound stellar systems, i.e., proto-globular clusters (proto-GCs). We conclude that star cluster formation and feedback likely contributed to 3 shape the properties of galaxies during the epoch of reionization.

The intrinsic substrate specificity of the human tyrosine kinome.

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.

A high-resolution map of human RNA translation.

Translated small open reading frames (smORFs) can have important regulatory roles and encode microproteins, yet their genome-wide identification has been challenging. We determined the ribosome locations across six primary human cell types and five tissues and detected 7,767 smORFs with translational profiles matching those of known proteins. The human genome was found to contain highly cell-type- and tissue-specific smORFs and a subset that encodes highly conserved amino acid sequences. Changes in the translational efficiency of upstream-encoded smORFs (uORFs) and the corresponding main ORFs predominantly occur in the same direction. Integration with 456 mass-spectrometry datasets confirms the presence of 603 small peptides at the protein level in humans and provides insights into the subcellular localization of these small proteins. This study provides a comprehensive atlas of high-confidence translated smORFs derived from primary human cells and tissues in order to provide a more complete understanding of the translated human genome.

Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

Gut dysbiosis might underlie the pathogenesis of type 1 diabetes. In mice of the non-obese diabetic (NOD) strain, we found that key features of disease correlated inversely with blood and fecal concentrations of the microbial metabolites acetate and butyrate. We therefore fed NOD mice specialized diets designed to release large amounts of acetate or butyrate after bacterial fermentation in the colon. Each diet provided a high degree of protection from diabetes, even when administered after breakdown of immunotolerance. Feeding mice a combined acetate- and butyrate-yielding diet provided complete protection, which suggested that acetate and butyrate might operate through distinct mechanisms. Acetate markedly decreased the frequency of autoreactive T cells in lymphoid tissues, through effects on B cells and their ability to expand populations of autoreactive T cells. A diet containing butyrate boosted the number and function of regulatory T cells, whereas acetate- and butyrate-yielding diets enhanced gut integrity and decreased serum concentration of diabetogenic cytokines such as IL-21. Medicinal foods or metabolites might represent an effective and natural approach for countering the numerous immunological defects that contribute to T cell-dependent autoimmune diseases.

Erratum: Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes.

This corrects the article DOI: 10.1038/ni.3713.

Integration of genomic data enables selective discovery of breast cancer drivers.

Identifying driver genes in cancer remains a crucial bottleneck in therapeutic development and basic understanding of the disease. We developed Helios, an algorithm that integrates genomic data from primary tumors with data from functional RNAi screens to pinpoint driver genes within large recurrently amplified regions of DNA. Applying Helios to breast cancer data identified a set of candidate drivers highly enriched with known drivers (p < 10(-14)). Nine of ten top-scoring Helios genes are known drivers of breast cancer, and in vitro validation of 12 candidates predicted by Helios found ten conferred enhanced anchorage-independent growth, demonstrating Helios's exquisite sensitivity and specificity. We extensively characterized RSF-1, a driver identified by Helios whose amplification correlates with poor prognosis, and found increased tumorigenesis and metastasis in mouse models. We have demonstrated a powerful approach for identifying driver genes and how it can yield important insights into cancer.