Seven days in the life cycle of Homo sapiens.

The second week of embryonic development is a critical phase of the human life cycle and one that has been largely inaccessible to scientific investigation. Recent studies of human embryo models built from stem cells promise to yield dramatic insights into the key events of cell specification and morphogenesis that occur during this brief window of embryogenesis.

An ethical framework for human embryology with embryo models.

A human embryo's legal definition and its entitlement to protection vary greatly worldwide. Recently, human pluripotent stem cells have been used to form in vitro models of early embryos that have challenged legal definitions and raised questions regarding their usage. In this light, we propose a refined legal definition of an embryo, suggest "tipping points" for when human embryo models could eventually be afforded similar protection to that of embryos, and then revisit basic ethical principles that might help to draft a roadmap for the gradual, justified usage of embryo models in a manner that aims to maximize benefits to society.

A Reverse Ecology Approach Based on a Biological Definition of Microbial Populations.

Delineating ecologically meaningful populations among microbes is important for identifying their roles in environmental and host-associated microbiomes. Here, we introduce a metric of recent gene flow, which when applied to co-existing microbes, identifies congruent genetic and ecological units separated by strong gene flow discontinuities from their next of kin. We then develop a pipeline to identify genome regions within these units that show differential adaptation and allow mapping of populations onto environmental variables or host associations. Using this reverse ecology approach, we show that the human commensal bacterium Ruminococcus gnavus breaks up into sharply delineated populations that show different associations with health and disease. Defining populations by recent gene flow in this way will facilitate the analysis of bacterial and archaeal genomes using ecological and evolutionary theory developed for plants and animals, thus allowing for testing unifying principles across all biology.

Author Correction: STEEP mediates STING ER exit and activation of signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

STEEP mediates STING ER exit and activation of signaling.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.

Secondary influenza challenge triggers resident memory B cell migration and rapid relocation to boost antibody secretion at infected sites.

Resident memory B (BRM) cells develop and persist in the lungs of influenza-infected mice and humans; however, their contribution to recall responses has not been defined. Here, we used two-photon microscopy to visualize BRM cells within the lungs of influenza -virus immune and reinfected mice. Prior to re-exposure, BRM cells were sparsely scattered throughout the tissue, displaying limited motility. Within 24 h of rechallenge, these cells increased their migratory capacity, localized to infected sites, and subsequently differentiated into plasma cells. Alveolar macrophages mediated this process, in part by inducing expression of chemokines CXCL9 and CXCL10 from infiltrating inflammatory cells. This led to the recruitment of chemokine receptor CXCR3-expressing BRM cells to infected regions and increased local antibody concentrations. Our study uncovers spatiotemporal mechanisms that regulate lung BRM cell reactivation and demonstrates their capacity to rapidly deliver antibodies in a highly localized manner to sites of viral replication.

Evidence of G.O.D.'s Miracle: Unearthing a RAG Transposon.

Diversity of antibodies and T cell receptors is generated by gene rearrangement dependent on RAG1 and RAG2, enzymes predicted to have been derived from a transposable element (TE) that invaded an immunoglobulin superfamily gene early in the evolution of jawed vertebrates. Now, Huang et al. report the discovery of ProtoRAG in the lower chordate Amphioxus, the long-anticipated TE related to the RAG transposon.

Emergence and Evolution of Secondary Lymphoid Organs.

For effective adaptive immunity to foreign antigens (Ag), secondary lymphoid organs (SLO) provide the confined environment in which Ag-restricted lymphocytes, with very low precursor frequencies, interact with Ag on Ag-presenting cells (APC). The spleen is the primordial SLO, arising in conjunction with adaptive immunity in early jawed vertebrates. The spleen, especially the spleen's lymphoid compartment, the white pulp (WP), has undergone numerous modifications over evolutionary time. We describe the progressive advancement of splenic WP complexity, which evolved in parallel with the increasing functionality of adaptive immunity. The Ag-presenting function of follicular dendritic cells (FDC) also likely emerged at the inception of adaptive immunity, and we propose that a single type of hematopoietically derived APC displayed Ag to both T and B cells. A dedicated FDC, derived from a vascular precursor, is a recent evolutionary innovation that likely permitted the robust affinity maturation found in mammals.

Oncogenic chimeric transcription factors drive tumor-specific transcription, processing, and translation of silent genomic regions.

Many cancers are characterized by gene fusions encoding oncogenic chimeric transcription factors (TFs) such as EWS::FLI1 in Ewing sarcoma (EwS). Here, we find that EWS::FLI1 induces the robust expression of a specific set of novel spliced and polyadenylated transcripts within otherwise transcriptionally silent regions of the genome. These neogenes (NGs) are virtually undetectable in large collections of normal tissues or non-EwS tumors and can be silenced by CRISPR interference at regulatory EWS::FLI1-bound microsatellites. Ribosome profiling and proteomics further show that some NGs are translated into highly EwS-specific peptides. More generally, we show that hundreds of NGs can be detected in diverse cancers characterized by chimeric TFs. Altogether, this study identifies the transcription, processing, and translation of novel, specific, highly expressed multi-exonic transcripts from otherwise silent regions of the genome as a new activity of aberrant TFs in cancer.

Structures of a sperm-specific solute carrier gated by voltage and cAMP.

The newly characterized sperm-specific Na+/H+ exchanger stands out by its unique tripartite domain composition1,2. It unites a classical solute carrier unit with regulatory domains usually found in ion channels, namely, a voltage-sensing domain and a cyclic-nucleotide binding domain1,3, which makes it a mechanistic chimera and a secondary-active transporter activated strictly by membrane voltage. Our structures of the sea urchin SpSLC9C1 in the absence and presence of ligands reveal the overall domain arrangement and new structural coupling elements. They allow us to propose a gating model, where movements in the voltage sensor indirectly cause the release of the exchanging unit from a locked state through long-distance allosteric effects transmitted by the newly characterized coupling helices. We further propose that modulation by its ligand cyclic AMP occurs by means of disruption of the cytosolic dimer interface, which lowers the energy barrier for S4 movements in the voltage-sensing domain. As SLC9C1 members have been shown to be essential for male fertility, including in mammals2,4,5, our structure represents a potential new platform for the development of new on-demand contraceptives.

Antiviral signalling by a cyclic nucleotide activated CRISPR protease.

CRISPR defence systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter orchestrates a complex antiviral response that is initiated through the synthesis of cyclic oligoadenylates after recognition of foreign RNA3-5. Among the large set of proteins that are linked to type III systems and predicted to bind cyclic oligoadenylates6,7, a CRISPR-associated Lon protease (CalpL) stood out to us. CalpL contains a sensor domain of the SAVED family7 fused to a Lon protease effector domain. However, the mode of action of this effector is unknown. Here we report the structure and function of CalpL and show that this soluble protein forms a stable tripartite complex with two other proteins, CalpT and CalpS, that are encoded on the same operon. After activation by cyclic tetra-adenylate (cA4), CalpL oligomerizes and specifically cleaves the MazF homologue CalpT, which releases the extracytoplasmic function σ factor CalpS from the complex. Our data provide a direct connection between CRISPR-based detection of foreign nucleic acids and transcriptional regulation. Furthermore, the presence of a SAVED domain that binds cyclic tetra-adenylate in a CRISPR effector reveals a link to the cyclic-oligonucleotide-based antiphage signalling system.

Adenylate cyclase activity of TIR1/AFB auxin receptors in plants.

The phytohormone auxin is the major coordinative signal in plant development1, mediating transcriptional reprogramming by a well-established canonical signalling pathway. TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) auxin receptors are F-box subunits of ubiquitin ligase complexes. In response to auxin, they associate with Aux/IAA transcriptional repressors and target them for degradation via ubiquitination2,3. Here we identify adenylate cyclase (AC) activity as an additional function of TIR1/AFB receptors across land plants. Auxin, together with Aux/IAAs, stimulates cAMP production. Three separate mutations in the AC motif of the TIR1 C-terminal region, all of which abolish the AC activity, each render TIR1 ineffective in mediating gravitropism and sustained auxin-induced root growth inhibition, and also affect auxin-induced transcriptional regulation. These results highlight the importance of TIR1/AFB AC activity in canonical auxin signalling. They also identify a unique phytohormone receptor cassette combining F-box and AC motifs, and the role of cAMP as a second messenger in plants.

PHGDH heterogeneity potentiates cancer cell dissemination and metastasis.

Cancer metastasis requires the transient activation of cellular programs enabling dissemination and seeding in distant organs1. Genetic, transcriptional and translational heterogeneity contributes to this dynamic process2,3. Metabolic heterogeneity has also been observed4, yet its role in cancer progression is less explored. Here we find that the loss of phosphoglycerate dehydrogenase (PHGDH) potentiates metastatic dissemination. Specifically, we find that heterogeneous or low PHGDH expression in primary tumours of patients with breast cancer is associated with decreased metastasis-free survival time. In mice, circulating tumour cells and early metastatic lesions are enriched with Phgdhlow cancer cells, and silencing Phgdh in primary tumours increases metastasis formation. Mechanistically, Phgdh interacts with the glycolytic enzyme phosphofructokinase, and the loss of this interaction activates the hexosamine-sialic acid pathway, which provides precursors for protein glycosylation. As a consequence, aberrant protein glycosylation occurs, including increased sialylation of integrin αvß3, which potentiates cell migration and invasion. Inhibition of sialylation counteracts the metastatic ability of Phgdhlow cancer cells. In conclusion, although the catalytic activity of PHGDH supports cancer cell proliferation, low PHGDH protein expression non-catalytically potentiates cancer dissemination and metastasis formation. Thus, the presence of PHDGH heterogeneity in primary tumours could be considered a sign of tumour aggressiveness.

Senataxin suppresses the antiviral transcriptional response and controls viral biogenesis.

The human helicase senataxin (SETX) has been linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS4) and ataxia with oculomotor apraxia (AOA2). Here we identified a role for SETX in controlling the antiviral response. Cells that had undergone depletion of SETX and SETX-deficient cells derived from patients with AOA2 had higher expression of antiviral mediators in response to infection than did wild-type cells. Mechanistically, we propose a model whereby SETX attenuates the activity of RNA polymerase II (RNAPII) at genes stimulated after a virus is sensed and thus controls the magnitude of the host response to pathogens and the biogenesis of various RNA viruses (e.g., influenza A virus and West Nile virus). Our data indicate a potentially causal link among inborn errors in SETX, susceptibility to infection and the development of neurologic disorders.

Opportunities and challenges of macrogenetic studies.

The rapidly emerging field of macrogenetics focuses on analysing publicly accessible genetic datasets from thousands of species to explore large-scale patterns and predictors of intraspecific genetic variation. Facilitated by advances in evolutionary biology, technology, data infrastructure, statistics and open science, macrogenetics addresses core evolutionary hypotheses (such as disentangling environmental and life-history effects on genetic variation) with a global focus. Yet, there are important, often overlooked, limitations to this approach and best practices need to be considered and adopted if macrogenetics is to continue its exciting trajectory and reach its full potential in fields such as biodiversity monitoring and conservation. Here, we review the history of this rapidly growing field, highlight knowledge gaps and future directions, and provide guidelines for further research.

The immunoglobulin J chain is an evolutionarily co-opted chemokine.

The joining (J) chain regulates polymerization of multimeric Immunoglobulin(Ig)M and IgA, forming a disulfide bond to the C termini of their Ig heavy chains, and it controls IgM/IgA transport across mucosal epithelia. Like Ig itself and human-like adaptive immunity, J chain emerged in jawed vertebrates (gnathostomes), but its origin has remained mysterious since its discovery over 50 y ago. Here, we show unexpectedly that J chain is a member of the CXCL chemokine family. The J chain gene (JCHAIN) is linked to clustered CXCL chemokine loci in all gnathostomes except actinopterygians that lost JCHAIN. JCHAIN and most CXCL genes have four exons with the same intron phases, including the same cleavage site for the signal peptide/mature protein. The second exon of both genes encodes a CXC motif at the same position, and the lengths of exons 1 to 3 are similar. No other gene in the human secretome shares all of these characteristics. In contrast, intrachain disulfide bonds of the two proteins are completely different, likely due to modifications in J chain to direct Ig polymerization and mucosal transport. Crystal structures of CXCL8 and J chain share a conserved beta-strand core but diverge otherwise due to different intrachain disulfide bonds and extension of the J chain C terminus. Identification of this ancestral affiliation between J chain and CXCL chemokines addresses an age-old problem in immunology.

Point mutation in a virus-like capsid drives symmetry reduction to form tetrahedral cages.

Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of unique symmetry-reduced structures. Starting with the encapsulin from Myxococcus xanthus, a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryoelectron microscopy, we determine the structures of a precedented 60-mer icosahedral assembly and an unexpected 36-mer tetrahedron that features significant geometric rearrangements around a new interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple-point mutation to various amino acids and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent a unique example of tetrahedral geometry when surveying all characterized encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in the protein sequence.

Author Correction: Elephant shark genome provides unique insights into gnathostome evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Biophysical and structural characterization of a multifunctional viral genome packaging motor.

The large dsDNA viruses replicate their DNA as concatemers consisting of multiple covalently linked genomes. Genome packaging is catalyzed by a terminase enzyme that excises individual genomes from concatemers and packages them into preassembled procapsids. These disparate tasks are catalyzed by terminase alternating between two distinct states-a stable nuclease that excises individual genomes and a dynamic motor that translocates DNA into the procapsid. It was proposed that bacteriophage λ terminase assembles as an anti-parallel dimer-of-dimers nuclease complex at the packaging initiation site. In contrast, all characterized packaging motors are composed of five terminase subunits bound to the procapsid in a parallel orientation. Here, we describe biophysical and structural characterization of the λ holoenzyme complex assembled in solution. Analytical ultracentrifugation, small angle X-ray scattering, and native mass spectrometry indicate that 5 subunits assemble a cone-shaped terminase complex. Classification of cryoEM images reveals starfish-like rings with skewed pentameric symmetry and one special subunit. We propose a model wherein nuclease domains of two subunits alternate between a dimeric head-to-head arrangement for genome maturation and a fully parallel arrangement during genome packaging. Given that genome packaging is strongly conserved in both prokaryotic and eukaryotic viruses, the results have broad biological implications.

Final Analysis of Efficacy and Safety of Single-Dose Ad26.COV2.S.

BACKGROUND: The Ad26.COV2.S vaccine was highly effective against severe-critical coronavirus disease 2019 (Covid-19), hospitalization, and death in the primary phase 3 efficacy analysis. METHODS: We conducted the final analysis in the double-blind phase of our multinational, randomized, placebo-controlled trial, in which adults were assigned in a 1:1 ratio to receive single-dose Ad26.COV2.S (5×1010 viral particles) or placebo. The primary end points were vaccine efficacy against moderate to severe-critical Covid-19 with onset at least 14 days after administration and at least 28 days after administration in the per-protocol population. Safety and key secondary and exploratory end points were also assessed. RESULTS: Median follow-up in this analysis was 4 months; 8940 participants had at least 6 months of follow-up. In the per-protocol population (39,185 participants), vaccine efficacy against moderate to severe-critical Covid-19 at least 14 days after administration was 56.3% (95% confidence interval [CI], 51.3 to 60.8; 484 cases in the vaccine group vs. 1067 in the placebo group); at least 28 days after administration, vaccine efficacy was 52.9% (95% CI, 47.1 to 58.1; 433 cases in the vaccine group vs. 883 in the placebo group). Efficacy in the United States, primarily against the reference strain (B.1.D614G) and the B.1.1.7 (alpha) variant, was 69.7% (95% CI, 60.7 to 76.9); efficacy was reduced elsewhere against the P.1 (gamma), C.37 (lambda), and B.1.621 (mu) variants. Efficacy was 74.6% (95% CI, 64.7 to 82.1) against severe-critical Covid-19 (with only 4 severe-critical cases caused by the B.1.617.2 [delta] variant), 75.6% (95% CI, 54.3 to 88.0) against Covid-19 leading to medical intervention (including hospitalization), and 82.8% (95% CI, 40.5 to 96.8) against Covid-19-related death, with protection lasting 6 months or longer. Efficacy against any severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was 41.7% (95% CI, 36.3 to 46.7). Ad26.COV2.S was associated with mainly mild-to-moderate adverse events, and no new safety concerns were identified. CONCLUSIONS: A single dose of Ad26.COV2.S provided 52.9% protection against moderate to severe-critical Covid-19. Protection varied according to variant; higher protection was observed against severe Covid-19, medical intervention, and death than against other end points and lasted for 6 months or longer. (Funded by Janssen Research and Development and others; ENSEMBLE ClinicalTrials.gov number, NCT04505722.).