Ex utero monkey embryogenesis from blastocyst to early organogenesis.

The third and fourth weeks of gestation in primates are marked by several developmental milestones, including gastrulation and the formation of organ primordia. However, our understanding of this period is limited due to restricted access to in vivo embryos. To address this gap, we developed an embedded 3D culture system that allows for the extended ex utero culture of cynomolgus monkey embryos for up to 25 days post-fertilization. Morphological, histological, and single-cell RNA-sequencing analyses demonstrate that ex utero cultured monkey embryos largely recapitulated key events of in vivo development. With this platform, we were able to delineate lineage trajectories and genetic programs involved in neural induction, lateral plate mesoderm differentiation, yolk sac hematopoiesis, primitive gut, and primordial germ-cell-like cell development in monkeys. Our embedded 3D culture system provides a robust and reproducible platform for growing monkey embryos from blastocysts to early organogenesis and studying primate embryogenesis ex utero.

Single-cell transcriptomic analysis identifies an immune-prone population in erythroid precursors during human ontogenesis.

Nonimmune cells can have immunomodulatory roles that contribute to healthy development. However, the molecular and cellular mechanisms underlying the immunomodulatory functions of erythroid cells during human ontogenesis remain elusive. Here, integrated, single-cell transcriptomic studies of erythroid cells from the human yolk sac, fetal liver, preterm umbilical cord blood (UCB), term UCB and adult bone marrow (BM) identified classical and immune subsets of erythroid precursors with divergent differentiation trajectories. Immune-erythroid cells were present from the yolk sac to the adult BM throughout human ontogenesis but failed to be generated in vitro from human embryonic stem cells. Compared with classical-erythroid precursors, these immune-erythroid cells possessed dual erythroid and immune regulatory networks, showed immunomodulatory functions and interacted more frequently with various innate and adaptive immune cells. Our findings provide important insights into the nature of immune-erythroid cells and their roles during development and diseases.

Phosphorylation-Mediated IFN-γR2 Membrane Translocation Is Required to Activate Macrophage Innate Response.

As a critical step during innate response, the cytoplasmic ß subunit (IFN-γR2) of interferon-γ receptor (IFN-γR) is induced and translocates to plasma membrane to join α subunit to form functional IFN-γR to mediate IFN-γ signaling. However, the mechanism driving membrane translocation and its significance remain largely unknown. We found, unexpectedly, that mice deficient in E-selectin, an endothelial cell-specific adhesion molecule, displayed impaired innate activation of macrophages upon Listeria monocytogenes infection yet had increased circulating IFN-γ. Inflammatory macrophages from E-selectin-deficient mice had less surface IFN-γR2 and impaired IFN-γ signaling. BTK elicited by extrinsic E-selectin engagement phosphorylates cytoplasmic IFN-γR2, facilitating EFhd2 binding and promoting IFN-γR2 trafficking from Golgi to cell membrane. Our findings demonstrate that membrane translocation of cytoplasmic IFN-γR2 is required to activate macrophage innate response against intracellular bacterial infection, identifying the assembly of functional cytokine receptors on cell membrane as an important layer in innate activation and cytokine signaling.

Transcription factor Hoxb5 reprograms B cells into functional T lymphocytes.

Deletion of master regulators of the B cell lineage reprograms B cells into T cells. Here we found that the transcription factor Hoxb5, which is expressed in uncommitted hematopoietic progenitor cells but is not present in cells committed to the B cell or T cell lineage, was able to reprogram pro-pre-B cells into functional early T cell lineage progenitors. This reprogramming started in the bone marrow and was completed in the thymus and gave rise to T lymphocytes with transcriptomes, hierarchical differentiation, tissue distribution and immunological functions that closely resembled those of their natural counterparts. Hoxb5 repressed B cell 'master genes', activated regulators of T cells and regulated crucial chromatin modifiers in pro-pre-B cells and ultimately drove the B cell fate-to-T cell fate conversion. Our results provide a de novo paradigm for the generation of functional T cells through reprogramming in vivo.

Publisher Correction: Transcription factor Hoxb5 reprograms B cells into functional T lymphocytes.

In the version of this article initially published, some identification of the supplementary information was incorrect. The items originally called Supplementary Tables 1, 2, 3, 4 and 5 should be Source Data Figures 1, 2, 4, 5 and 7, respectively; those originally called Supplementary Tables 6, 7 and 8 should be Supplementary Tables 1, 2 and 3, respectively; and those originally called Source Data Figures 1, 2, 4, 5 and 7 should be Supplementary Tables 4, 5, 6, 7 and 8, respectively. The errors have been corrected in the HTML version of the article.

Combinatorial Single-Cell Analyses of Granulocyte-Monocyte Progenitor Heterogeneity Reveals an Early Uni-potent Neutrophil Progenitor.

Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs.

Single-Cell RNA Sequencing Resolves Spatiotemporal Development of Pre-thymic Lymphoid Progenitors and Thymus Organogenesis in Human Embryos.

Generation of the first T lymphocytes in the human embryo involves the emergence, migration, and thymus seeding of lymphoid progenitors together with concomitant thymus organogenesis, which is the initial step to establish the entire adaptive immune system. However, the cellular and molecular programs regulating this process remain unclear. We constructed a single-cell transcriptional landscape of human early T lymphopoiesis by using cells from multiple hemogenic and hematopoietic sites spanning embryonic and fetal stages. Among heterogenous early thymic progenitors, one subtype shared common features with a subset of lymphoid progenitors in fetal liver that are known as thymus-seeding progenitors. Unbiased bioinformatics analysis identified a distinct type of pre-thymic lymphoid progenitors in the aorta-gonad-mesonephros (AGM) region. In parallel, we investigated thymic epithelial cell development and potential cell-cell interactions during thymus organogenesis. Together, our data provide insights into human early T lymphopoiesis that prospectively direct T lymphocyte regeneration, which might lead to development of clinical applications.

Coherent interfaces govern direct transformation from graphite to diamond.

Understanding the direct transformation from graphite to diamond has been a long-standing challenge with great scientific and practical importance. Previously proposed transformation mechanisms1-3, based on traditional experimental observations that lacked atomistic resolution, cannot account for the complex nanostructures occurring at graphite-diamond interfaces during the transformation4,5. Here we report the identification of coherent graphite-diamond interfaces, which consist of four basic structural motifs, in partially transformed graphite samples recovered from static compression, using high-angle annular dark-field scanning transmission electron microscopy. These observations provide insight into possible pathways of the transformation. Theoretical calculations confirm that transformation through these coherent interfaces is energetically favoured compared with those through other paths previously proposed1-3. The graphite-to-diamond transformation is governed by the formation of nanoscale coherent interfaces (diamond nucleation), which, under static compression, advance to consume the remaining graphite (diamond growth). These results may also shed light on transformation mechanisms of other carbon materials and boron nitride under different synthetic conditions.

The H3K4 demethylase JMJ1 is required for proper timing of flowering in Brachypodium distachyon.

Flowering is a key developmental transition in the plant life cycle. In temperate climates, flowering often occurs in response to the perception of seasonal cues such as changes in day-length and temperature. However, the mechanisms that have evolved to control the timing of flowering in temperate grasses are not fully understood. We identified a Brachypodium distachyon mutant whose flowering is delayed under inductive long-day conditions due to a mutation in the JMJ1 gene, which encodes a Jumonji domain-containing protein. JMJ1 is a histone demethylase that mainly demethylates H3K4me2 and H3K4me3 in vitro and in vivo. Analysis of the genome-wide distribution of H3K4me1, H3K4me2, and H3K4me3 in wild-type plants by chromatin immunoprecipitation and sequencing (ChIP-seq) combined with RNA sequencing (RNA-seq) revealed that H3K4m1 and H3K4me3 are positively associated with gene transcript levels, whereas H3K4me2 is negatively correlated with transcript levels. Furthermore, JMJ1 directly binds to the chromatin of the flowering regulator genes VRN1 and ID1 and affects their transcription by modifying their H3K4me2 and H3K4me3 levels. Genetic analyses indicated that JMJ1 promotes flowering by activating VRN1 expression. Our study reveals a role for JMJ1-mediated chromatin modification in the proper timing of flowering in B. distachyon.

Rickettsia symbionts spread via mixed mode transmission, increasing female fecundity and sex ratio shift by host hormone modulating.

Heritable symbionts are common among animals in nature, but the molecular mechanisms underpinning symbiont invasions of host populations have been elusive. In this study, we demonstrate the spread of Rickettsia in an invasive agricultural pest, the whitefly Bemisia tabaci Mediterranean (MED), across northeastern China from 2018 to 2023. Here, we show that the beneficial symbiont Rickettsia spreads by manipulating host hormone signals. Our analyses suggest that Rickettsia have been horizontally acquired by B. tabaci MED from another invasive whitefly B. tabaci Middle East-Asia Minor 1 during periods of coexistence. Rickettsia is transmitted maternally and horizontally from female B. tabaci MED individuals. Rickettsia infection enhances fecundity and results in female bias among whiteflies. Our findings reveal that Rickettsia infection stimulates juvenile hormone (JH) synthesis, in turn enhancing fecundity, copulation events, and the female ratio of the offspring. Consequently, Rickettsia infection results in increased whitefly fecundity and female bias by modulating the JH pathway. More female progeny facilitates the transmission of Rickettsia. This study illustrates that the spread of Rickettsia among invasive whiteflies in northeastern China is propelled by host hormone regulation. Such symbiont invasions lead to rapid physiological and molecular evolution in the host, influencing the biology and ecology of an invasive species.

Genetic and phenotypic profiling of single living circulating tumor cells from patients with microfluidics.

Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non-small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction.

Cathelicidin peptide analogues inhibit EV71 infection through blocking viral entry and uncoating.

Given the serious neurological complications and deaths associated with enterovirus 71 (EV71) infection, there is an urgent need to develop effective antivirals against this viral infection. In this study, we demonstrated that two Cathelicidin-derived peptides, LL-18 and FF-18 were more potent against EV71 infection than the parent peptide LL-37, which is the mature and processed form of Cathelicidin. These peptides could directly bind to the EV71 virus particles, but not to coxsackievirus, indicative of their high specificity. The binding of peptides with the virus surface occupied the viral canyon region in a way that could block virus-receptor interactions and inhibit viral uncoating. In addition, these peptide analogues could also relieve the deleterious effect of EV71 infection in vivo. Therefore, Cathelicidin-derived peptides might be excellent candidates for further development of antivirals to treat EV71 infection.

Deciphering human macrophage development at single-cell resolution.

Macrophages are the first cells of the nascent immune system to emerge during embryonic development. In mice, embryonic macrophages infiltrate developing organs, where they differentiate symbiotically into tissue-resident macrophages (TRMs)1. However, our understanding of the origins and specialization of macrophages in human embryos is limited. Here we isolated CD45+ haematopoietic cells from human embryos at Carnegie stages 11 to 23 and subjected them to transcriptomic profiling by single-cell RNA sequencing, followed by functional characterization of a population of CD45+CD34+CD44+ yolk sac-derived myeloid-biased progenitors (YSMPs) by single-cell culture. We also mapped macrophage heterogeneity across multiple anatomical sites and identified diverse subsets, including various types of embryonic TRM (in the head, liver, lung and skin). We further traced the specification trajectories of TRMs from either yolk sac-derived primitive macrophages or YSMP-derived embryonic liver monocytes using both transcriptomic and developmental staging information, with a focus on microglia. Finally, we evaluated the molecular similarities between embryonic TRMs and their adult counterparts. Our data represent a comprehensive characterization of the spatiotemporal dynamics of early macrophage development during human embryogenesis, providing a reference for future studies of the development and function of human TRMs.

Kaposi's sarcoma herpesvirus exploits the DNA damage response to circularize its genome.

To establish lifelong, latent infection, herpesviruses circularize their linear, double-stranded, DNA genomes through an unknown mechanism. Kaposi's sarcoma (KS) herpesvirus (KSHV), a gamma herpesvirus, is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman's disease. KSHV persists in latently infected cells as a multi-copy, extrachromosomal episome. Here, we show the KSHV genome rapidly circularizes following infection, and viral protein expression is unnecessary for this process. The DNA damage response (DDR) kinases, ATM and DNA-PKcs, each exert roles, and absence of both severely compromises circularization and latency. These deficiencies were rescued by expression of ATM and DNA-PKcs, but not catalytically inactive mutants. In contrast, γH2AX did not function in KSHV circularization. The linear viral genomic ends resemble a DNA double strand break, and non-homologous DNA end joining (NHEJ) and homologous recombination (HR) reporters indicate both NHEJ and HR contribute to KSHV circularization. Last, we show, similar to KSHV, ATM and DNA-PKcs have roles in circularization of the alpha herpesvirus, herpes simplex virus-1 (HSV-1), while γH2AX does not. Therefore, the DDR mediates KSHV and HSV-1 circularization. This strategy may serve as a general herpesvirus mechanism to initiate latency, and its disruption may provide new opportunities for prevention of herpesvirus disease.

INDETERMINATE1-mediated expression of FT family genes is required for proper timing of flowering in Brachypodium distachyon.

The transition to flowering is a major developmental switch in plants. In many temperate grasses, perception of indicators of seasonal change, such as changing day-length and temperature, leads to expression of FLOWERING LOCUS T1 (FT1) and FT-Like (FTL) genes that are essential for promoting the transition to flowering. However, little is known about the upstream regulators of FT1 and FTL genes in temperate grasses. Here, we characterize the monocot-specific gene INDETERMINATE1 (BdID1) in Brachypodium distachyon and demonstrate that BdID1 is a regulator of FT family genes. Mutations in ID1 impact the ability of the short-day (SD) vernalization, cold vernalization, and long-day (LD) photoperiod pathways to induce certain FTL genes. BdID1 is required for upregulation of FTL9 (FT-LIKE9) expression by the SD vernalization pathway, and overexpression of FTL9 in an id1 background can partially restore the delayed flowering phenotype of id1. We show that BdID1 binds in vitro to the promoter region of FTL genes suggesting that ID1 directly activates FTL expression. Transcriptome analysis shows that BdID1 is required for FT1, FT2, FTL12, and FTL13 expression under inductive LD photoperiods, indicating that BdID1 is a regulator of the FT gene family. Moreover, overexpression of FT1 in the id1 background results in rapid flowering similar to overexpressing FT1 in the wild type, demonstrating that BdID1 is upstream of FT family genes. Interestingly, ID1 negatively regulates a previously uncharacterized FTL gene, FTL4, and we show that FTL4 is a repressor of flowering. Thus, BdID1 is critical for proper timing of flowering in temperate grasses.

Co-representation of Functional Brain Networks Is Shaped by Cortical Myeloarchitecture and Reveals Individual Behavioral Ability.

Large-scale functional networks are spatially distributed in the human brain. Despite recent progress in differentiating their functional roles, how the brain navigates the spatial coordination among them and the biological relevance of this coordination is still not fully understood. Capitalizing on canonical individualized networks derived from functional MRI data, we proposed a new concept, that is, co-representation of functional brain networks, to delineate the spatial coordination among them. To further quantify the co-representation pattern, we defined two indexes, that is, the co-representation specificity (CoRS) and intensity (CoRI), for separately measuring the extent of specific and average expression of functional networks at each brain location by using the data from both sexes. We found that the identified pattern of co-representation was anchored by cortical regions with three types of cytoarchitectural classes along a sensory-fugal axis, including, at the first end, primary (idiotypic) regions showing high CoRS, at the second end, heteromodal regions showing low CoRS and high CoRI, at the third end, paralimbic regions showing low CoRI. Importantly, we demonstrated the critical role of myeloarchitecture in sculpting the spatial distribution of co-representation by assessing the association with the myelin-related neuroanatomical and transcriptomic profiles. Furthermore, the significance of manifesting the co-representation was revealed in its prediction of individual behavioral ability. Our findings indicated that the spatial coordination among functional networks was built upon an anatomically configured blueprint to facilitate neural information processing, while advancing our understanding of the topographical organization of the brain by emphasizing the assembly of functional networks.

The Raf-like MAPKKKs STY8, STY17, and STY46 negatively regulate Botrytis cinerea resistance by limiting MKK7 protein accumulation in Arabidopsis.

Plant diseases, which seriously damage crop production, are in most cases caused by fungal pathogens. In this study, we found that the Raf-like MAPKKKs STY8 (SERINE/THREONINE/TYROSINE KINASE 8), STY17, and STY46 negatively regulate resistance to the fungal pathogen Botrytis cinerea through jasmonate response in Arabidopsis. Moreover, STY8/STY17/STY46 homologs negatively contribute to chitin signaling. We further identified MKK7 as the MAPKK component interacting with STY8/STY17/STY46 homologs. MKK7 positively contributes to resistance to B. cinerea and chitin signaling. Furthermore, we found that STY8/STY17/STY46 homologs negatively affect the accumulation of MKK7, in accordance with the opposite roles of MKK7 and STY8/STY17/STY46 homologs in defense against B. cinerea. These results provide new insights into the mechanisms precisely regulating plant immunity via Raf-like MAPKKKs.

Tirzepatide Once Weekly for the Treatment of Obesity.

BACKGROUND: Obesity is a chronic disease that results in substantial global morbidity and mortality. The efficacy and safety of tirzepatide, a novel glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 receptor agonist, in people with obesity are not known. METHODS: In this phase 3 double-blind, randomized, controlled trial, we assigned 2539 adults with a body-mass index (BMI; the weight in kilograms divided by the square of the height in meters) of 30 or more, or 27 or more and at least one weight-related complication, excluding diabetes, in a 1:1:1:1 ratio to receive once-weekly, subcutaneous tirzepatide (5 mg, 10 mg, or 15 mg) or placebo for 72 weeks, including a 20-week dose-escalation period. Coprimary end points were the percentage change in weight from baseline and a weight reduction of 5% or more. The treatment-regimen estimand assessed effects regardless of treatment discontinuation in the intention-to-treat population. RESULTS: At baseline, the mean body weight was 104.8 kg, the mean BMI was 38.0, and 94.5% of participants had a BMI of 30 or higher. The mean percentage change in weight at week 72 was -15.0% (95% confidence interval [CI], -15.9 to -14.2) with 5-mg weekly doses of tirzepatide, -19.5% (95% CI, -20.4 to -18.5) with 10-mg doses, and -20.9% (95% CI, -21.8 to -19.9) with 15-mg doses and -3.1% (95% CI, -4.3 to -1.9) with placebo (P<0.001 for all comparisons with placebo). The percentage of participants who had weight reduction of 5% or more was 85% (95% CI, 82 to 89), 89% (95% CI, 86 to 92), and 91% (95% CI, 88 to 94) with 5 mg, 10 mg, and 15 mg of tirzepatide, respectively, and 35% (95% CI, 30 to 39) with placebo; 50% (95% CI, 46 to 54) and 57% (95% CI, 53 to 61) of participants in the 10-mg and 15-mg groups had a reduction in body weight of 20% or more, as compared with 3% (95% CI, 1 to 5) in the placebo group (P<0.001 for all comparisons with placebo). Improvements in all prespecified cardiometabolic measures were observed with tirzepatide. The most common adverse events with tirzepatide were gastrointestinal, and most were mild to moderate in severity, occurring primarily during dose escalation. Adverse events caused treatment discontinuation in 4.3%, 7.1%, 6.2%, and 2.6% of participants receiving 5-mg, 10-mg, and 15-mg tirzepatide doses and placebo, respectively. CONCLUSIONS: In this 72-week trial in participants with obesity, 5 mg, 10 mg, or 15 mg of tirzepatide once weekly provided substantial and sustained reductions in body weight. (Supported by Eli Lilly; SURMOUNT-1 ClinicalTrials.gov number, NCT04184622.).

Temperature-dependent carrier state mediated by H-NS promotes the long-term coexistence of Y. pestis and a phage in soil.

The study of carrier state phages challenged the canonical lytic-lysogenic binary, and carrier state appears to be ubiquitous and ecologically important. However, the mechanisms of the carrier state are not well elucidated due to the limited phage models. Herein, we reported phage HQ103, similar to Escherichia coli phage P2. In contrast to the temperate P2 phage, the HQ103 phage does not insert its genome into the bacterial chromosome and displays a dual behavior depending on the temperature. At 37°C, HQ103 lyses the host and forms clear plaques due to the truncation of repressor CI and mutation of promoter Pc. In contrast, HQ103 maintains a carrier state lifestyle with Y. pestis at an environmental temperature (21°C). Mechanistically, we found that the host-encoded histone-like nucleoid-structuring protein H-NS, which is highly expressed at 21°C to silence the Cox promoter Pe and inhibits the phage lytic cycle. Subsequently, the HQ103 carrier state Y. pestis could grow and co-exist with the phage in the soil at 21°C for one month. Thus, this study reveals a novel carrier state lifestyle of phage HQ103 due to the H-NS mediated xenogeneic silencing and demonstrates that the carrier state lifestyle could promote long-term phage-host coexist in nature.