Few studies have addressed the impact of maternal mild/asymptomatic SARS-CoV-2 infection on the developing neonatal immune system. In this study, we analyzed umbilical cord blood and placental chorionic villi from newborns of unvaccinated mothers with mild/asymptomatic SARSCoV-2 infection during pregnancy using flow cytometry, single-cell transcriptomics, and functional assays. Despite the lack of vertical transmission, levels of inflammatory mediators were altered in cord blood. Maternal infection was also associated with increased memory T, B cells, and non-classical monocytes as well as increased activation. However, ex vivo responses to stimulation were attenuated. Finally, within the placental villi, we report an expansion of fetal Hofbauer cells and infiltrating maternal macrophages and rewiring towards a heightened inflammatory state. In contrast to cord blood monocytes, placental myeloid cells were primed for heightened antiviral responses. Taken together, this study highlights dysregulated fetal immune cell responses in response to mild maternal SARS-CoV-2 infection during pregnancy.

Pregnant women are an at-risk group for severe COVID-19, though the majority experience mild/asymptomatic disease. Although severe COVID-19 has been shown to be associated with immune activation at the maternal-fetal interface even in the absence of active viral replication, the immune response to asymptomatic/mild COVID-19 remains unknown. Here, we assessed immunological adaptations in both blood and term decidua from 9 SARS-exposed pregnant women with asymptomatic/mild disease and 15 pregnant SARS-naive women. In addition to selective loss of tissue-resident decidual macrophages, we report attenuation of antigen presentation and type I IFN signaling but upregulation of inflammatory cytokines and chemokines in blood monocyte derived decidual macrophages. On the other hand, infection was associated with remodeling of the T cell compartment with increased frequencies of activated CD69+ tissue-resident T cells and decreased abundance of Tregs. Interestingly, frequencies of cytotoxic CD4 and CD8 T cells increased only in the blood, while CD8 effector memory T cells were expanded in the decidua. In contrast to decidual macrophages, signatures of type I IFN signaling were increased in decidual T cells. Finally, T cell receptor diversity was significantly reduced with infection in both compartments, albeit to a much greater extent in the blood. The resulting aberrant immune activation in the placenta, even with asymptomatic disease may alter the exquisitely sensitive developing fetal immune system, leading to long-term adverse outcomes for offspring.

Decoding the gene regulatory mechanisms mediating self-renewal of hematopoietic stem cells (HSCs) during their amplification in the fetal liver (FL) is relevant for advancing therapeutic applications aiming to expand transplantable HSCs, a long-standing challenge. Here, to explore intrinsic and extrinsic regulation of self-renewal in FL-HSCs at the single cell level, we engineered a culture platform designed to recapitulate the FL endothelial niche, which supports the amplification of serially engraftable HSCs ex vivo. Leveraging this platform in combination with single cell index flow cytometry, serial transplantation assays, and single cell RNA-sequencing, we elucidated previously unrecognized heterogeneity in immunophenotypically defined FL-HSCs and demonstrated that differentiation latency and transcriptional signatures of biosynthetic dormancy are distinguishing properties of self-renewing FL-HSCs with capacity for serial, long-term multilineage hematopoietic reconstitution. Altogether, our findings provide key insights into HSC expansion and generate a novel resource for future exploration of the intrinsic and niche-derived signaling pathways that support FL-HSC self-renewal.

Knowledge of locations and activities of cis -regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult across species. In contrast, we conducted a cross-species study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable across species. This study used integrative modeling of eight epigenetic features jointly in human and mouse in our V al i dated S ystematic I ntegrati on (VISION) Project. The contribution of each epigenetic state in cCREs to gene regulation was estimated from a multivariate regression against gene expression across cell types. We used these values to estimate epigenetic state Regulatory Potential (esRP) scores for each cCRE in each cell type, which are useful for visualizing and categorizing dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbored distinctive transcription factor binding motifs that were similar across species. Genetic variants associated with blood cell phenotypes were highly and specifically enriched in the catalog of human VISION cCREs, supporting its utility for understanding impacts of noncoding genetic variants on blood cell-related traits. A cross-species comparison of cCREs, based on the joint modeling, revealed both conserved and lineage-specific patterns of epigenetic evolution, even in the absence of genomic sequence alignment. We provide these resources through tools and browsers at http://usevision.org .

Recent advances in protein structure prediction using AlphaFold2, known for its high efficiency and accuracy, have opened new avenues for comprehensive analysis of all structures within a single protein family. In this study, we evaluated the capabilities of AphaFold2 in analyzing integrin structures. Integrins are heterodimeric cell surface receptors composed of a combination of 18 α and 8 β subunits, resulting in a family of 24 different members. Both α and β subunits consist of a large extracellular domain, a short transmembrane domain, and typically, a short cytoplasmic tail. Integrins play a pivotal role in a wide range of cellular functions by recognizing diverse ligands. Despite significant advances in integrin structural studies in recent decades, high-resolution structures have only been determined for a limited subsets of integrin members, thus limiting our understanding of the entire integrin family. Here, we first analyzed the single-chain structures of 18 α and 8 β integrins in the AlphaFold2 protein structure database. We then employed the newly developed AlphaFold2-multimer program to predict the α/β heterodimer structures of all 24 human integrins. The predicted structures show a high level of accuracy for the subdomains of both α and β subunits, offering high-resolution structure insights for all integrin heterodimers. Our comprehensive structural analysis of the entire integrin family unveils a potentially diverse range of conformations among the 24 members, providing a valuable structure database for studies related to integrin structure and function. We further discussed the potential applications and limitations of the AlphaFold2-derived integrin structures.

Mitochondrial genome encodes handful genes of respiratory chain complexes, whereas all the remaining mitochondrial proteins are encoded on the nuclear genome. However, the mechanisms coordinating these two genomes to control mitochondrial biogenesis remain largely unknown. To identify transcription circuits involved in these processes, we performed a candidate RNAi screen in developing eyes that had reduced mitochondrial DNA contents. We reasoned that impaired mitochondrial biogenesis would synergistically interact with mtDNA deficiency in disrupting tissue development. Over 638 transcription factors annotated in the fly genome, we identified 77 transcription factors that may be involved in mitochondrial genome maintenance and gene expression. Additional genetic and genomic analyses revealed that a novel transcription factor, CG1603, and its upstream factor YL-1 are essential for mitochondrial biogenesis. We constructed a regulator network among positive hits using the published CHIP-seq data. The network analysis revealed extensive connections, and complex hierarchical organization underlying the transcription regulation of mitochondrial biogenesis.

Immunization in pregnancy is a critical tool that can be leveraged to protect the infant with an immature immune system but how vaccine-induced antibodies transfer to the placenta and protect the maternal-fetal dyad remains unclear. Here, we compare matched maternal-infant cord blood from individuals who in pregnancy received mRNA COVID-19 vaccine, were infected by SARS-CoV-2, or had the combination of these two immune exposures. We find that some but not all antibody neutralizing activities and Fc effector functions are enriched with vaccination compared to infection. Preferential transport to the fetus of Fc functions and not neutralization is observed. Immunization compared to infection enriches IgG1-mediated antibody functions with changes in antibody post-translational sialylation and fucosylation that impact fetal more than maternal antibody functional potency. Thus, vaccine enhanced antibody functional magnitude, potency and breadth in the fetus are driven more by antibody glycosylation and Fc effector functions compared to maternal responses, highlighting prenatal opportunities to safeguard newborns as SARS-CoV-2 becomes endemic.

Scanning electron microscopy (SEM) offers an unparalleled view of the membrane topography of mammalian cells by using a conventional osmium (OsO4) and ethanol-based tissue preparation. However, conventional SEM methods limit optimal resolution due to ethanol and lipid interactions and interfere with visualization of fluorescent reporter proteins. Therefore, SEM correlative light and electron microscopy (CLEM) has been hindered by the adverse effects of ethanol and OsO4 on retention of fluorescence signals. To overcome this technological gap in achieving high-resolution SEM and retain fluorescent reporter signals, we developed a freeze-drying method with gaseous nitrogen (FDGN). We demonstrate that FDGN preserves cyto-architecture to allow visualization of detailed membrane topography while retaining fluorescent signals and that FDGN processing can be used in conjunction with a variety of high-resolution imaging systems to enable collection and validation of unique, high-quality data from these approaches. In particular, we show that FDGN coupled with high resolution microscopy provided detailed insight into viral or tumor-derived extracellular vesicle (TEV)-host cell interactions and may aid in designing new approaches to intervene during viral infection or to harness TEVs as therapeutic agents.

Hematopoietic stem cells are cells that differentiate into all blood cell types. Although the placenta secretes hormones, proteins and other factors important for maternal and fetal health, cross-talk between placental cells and hematopoietic stem cells is poorly understood. Moreover, toxicant impacts on placental-hematopoietic stem cell communication is understudied. The goals of this study were to determine if factors secreted from placental cells alter transcriptomic responses in hematopoietic stem cells and if monoethylhexyl phthalate (MEHP), the bioactive metabolite of the pollutant diethylhexyl phthalate, modifies these effects.

Influenza A (IAV) and SARS-CoV-2 (SCV2) viruses represent an ongoing threat to public health. Both viruses target the respiratory tract, which consists of a gradient of cell types, receptor expression, and temperature. Environmental temperature has been an un-derstudied contributor to infection susceptibility and understanding its impact on host responses to infection could help uncover new insights into severe disease risk factors. As the nasal passageways are the initial site of respiratory virus infection, in this study we investigated the effect of temperature on host responses in human nasal epithelial cells (hNECs) utilizing IAV and SCV2 in vitro infection models. We demonstrate that temperature affects SCV2, but not IAV, viral replicative fitness and that SCV2 infected cultures are slower to mount an infection-induced response, likely due to suppression by the virus. Additionally, we show that that temperature not only changes the basal transcriptomic landscape of epithelial cells, but that it also impacts the response to infection. The induction of interferon and other innate immune responses were not drastically affected by temperature, suggesting that while the baseline antiviral response at different temperatures remains consistent, there may be metabolic or signaling changes that affect how well the cultures are able to adapt to new pressures such as infection. Finally, we show that hNECs respond differently to IAV and SCV2 infection in ways that give insight into how the virus is able to manipulate the cell to allow for replication and release. Taken together, these data give new insight into the innate immune response to respiratory infections and can assist in identifying new treatment strategies for respiratory infections.

Hematopoietic stem and progenitor cell (HSPC) transplantation is an essential therapy for hematological conditions, but finer definitions of human HSPC subsets with associated function could enable better tuning of grafts and more routine, lower-risk application. To deeply phenotype HSPCs, following a screen of 328 antigens, we quantified 41 surface proteins and functional regulators on millions of CD34+ and CD34- cells, spanning four primary human hematopoietic tissues: bone marrow, mobilized peripheral blood, cord blood, and fetal liver. We propose more granular definitions of HSPC subsets and provide new, detailed differentiation trajectories of erythroid and myeloid lineages. These aspects of our revised human hematopoietic model were validated with corresponding epigenetic analysis and in vitro clonal differentiation assays. Overall, we demonstrate the utility of using molecular regulators as surrogates for cellular identity and functional potential, providing a framework for description, prospective isolation, and cross-tissue comparison of HSPCs in humans.

Primary differentiated human epithelial cell cultures have been widely used by researchers to study viral fitness and virus-host interactions, especially during the COVID19 pandemic. These cultures recapitulate important characteristics of the respiratory epithelium such as diverse cell type composition, polarization, and innate immune responses. However, standardization and validation of these cultures remains an open issue. In this study, two different expansion medias were evaluated and the impact on the resulting differentiated culture was determined. Use of both Airway and Ex Plus media types resulted in high quality, consistent cultures that were able to be used for these studies. Upon histological evaluation, Airway-grown cultures were more organized and had a higher proportion of basal progenitor cells while Ex Plus- grown cultures had a higher proportion terminally differentiated cell types. In addition to having different cell type proportions and organization, the two different growth medias led to cultures with altered susceptibility to infection with SARS-CoV-2 but not Influenza A virus. RNAseq comparing cultures grown in different growth medias prior to differentiation uncovered a high degree of differentially expressed genes in cultures from the same donor. RNAseq on differentiated cultures showed less variation between growth medias but alterations in pathways that control the expression of human transmembrane proteases including TMPRSS11 and TMPRSS2 were documented. Enhanced susceptibility to SARS-CoV-2 cannot be explained by altered cell type proportions alone, rather serine protease cofactor expression also contributes to the enhanced replication of SARS-CoV-2 as inhibition with camostat affected replication of an early SARS-CoV-2 variant and a Delta, but not Omicron, variant showed difference in replication efficiency between culture types. Therefore, it is important for the research community to standardize cell culture protocols particularly when characterizing novel viruses.

Bradykinin is a peptide implicated in inflammatory pain in both humans and rodents. In rodent sensory neurons, activation of B1 and B2 bradykinin receptors induces neuronal hyperexcitability. Recent evidence suggests that human and rodent dorsal root ganglia (DRG), which contain the cell bodies of sensory neurons, differ in the expression and function of key GPCRs and ion channels; whether BK receptor expression and function are conserved across species has not been studied in depth. In this study, we used human DRG tissue from organ donors to provide a detailed characterization of bradykinin receptor expression and bradykinin-induced changes in the excitability of human sensory neurons. We found that B2 and, to a lesser extent, B1 receptors are expressed by human DRG neurons and satellite glial cells. B2 receptors were enriched in the nociceptor subpopulation. Using patch-clamp electrophysiology, we found that acute bradykinin increases the excitability of human sensory neurons, while prolonged exposure to bradykinin decreases neuronal excitability in a subpopulation of human DRG neurons. Finally, our analyses suggest that donor’s history of chronic pain and age may be predictors of higher B1 receptor expression in human DRG neurons. Together, these results indicate that acute BK-induced hyperexcitability, first identified in rodents, is conserved in humans and provide further evidence supporting BK signaling as a potential therapeutic target for treating pain in humans.

Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a novel series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking out viral receptor ACE2. The lead drug possessing an Fc tag ( Nanosota-1C-Fc ) bound to SARS-CoV-2 RBD with a K d of 15.7picomolar (∼3000 times more tightly than ACE2 did) and inhibited SARS-CoV-2 infection with an ND 50 of 0.16microgram/milliliter (∼6000 times more potently than ACE2 did). Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy in hamsters subjected to SARS-CoV-2 infection. Unlike conventional antibody drugs, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-F c documented a greater than 10-day in vivo half-life efficacy and high tissue bioavailability. Nanosota-1C-Fc is a potentially effective and realistic solution to the COVID-19 pandemic.

Human pregnancy is frequently accompanied by nausea and vomiting that may become severe and life-threatening, as in hyperemesis gravidarum (HG), the cause of which is unknown. Growth Differentiation Factor-15 (GDF15), a hormone known to act on the hindbrain to cause emesis, is highly expressed in the placenta and its levels in maternal blood rise rapidly in pregnancy. Variants in the maternal GDF15 gene are associated with HG. Here we report that fetal production of GDF15, and maternal sensitivity to it, both contribute substantially to the risk of HG. We found that the great majority of GDF15 in maternal circulation is derived from the feto-placental unit and that higher GDF15 levels in maternal blood are associated with vomiting and are further elevated in patients with HG. Conversely, we found that lower levels of GDF15 in the non-pregnant state predispose women to HG. A rare C211G variant in GDF15 which strongly predisposes mothers to HG, particularly when the fetus is wild-type, was found to markedly impair cellular secretion of GDF15 and associate with low circulating levels of GDF15 in the non-pregnant state. Consistent with this, two common GDF15 haplotypes which predispose to HG were associated with lower circulating levels outside pregnancy. The administration of a long-acting form of GDF15 to wild-type mice markedly reduced subsequent responses to an acute dose, establishing that desensitisation is a feature of this system. GDF15 levels are known to be highly and chronically elevated in patients with beta thalassemia. In women with this disorder, reports of symptoms of nausea or vomiting in pregnancy were strikingly diminished. Our findings support a causal role for fetal derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by pre-pregnancy exposure to GDF15, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.

Human neural organoid models offer an exciting opportunity for studying often inaccessible human-specific brain development; however, it remains unclear how precisely organoids recapitulate fetal/primary tissue biology. Here, we characterize field-wide replicability and biological fidelity through a meta-analysis of single-cell RNA-sequencing data for first and second trimester human primary brain (2.95 million cells, 51 datasets) and neural organoids (1.63 million cells, 130 datasets). We quantify the degree to which primary tissue cell-type marker expression and co-expression are recapitulated in organoids across 12 different protocol types. By quantifying gene-level preservation of primary tissue co-expression, we show neural organoids lie on a spectrum ranging from virtually no signal to co-expression near indistinguishable from primary tissue data, demonstrating high fidelity is within the scope of current methods. Additionally, we show neural organoids preserve the cell-type specific co-expression of developing rather than adult cells, confirming organoids are an appropriate model for primary tissue development. Overall, quantifying the preservation of primary tissue co-expression is a powerful tool for uncovering unifying axes of variation across heterogeneous neural organoid experiments.

SARS-CoV-2 infection during pregnancy leads to an increased risk of adverse pregnancy outcomes. Although the placenta itself can be a target of virus infection, most neonates are virus free and are born healthy or recover quickly. Here, we investigated the impact of SARS-CoV-2 infection on the placenta from a cohort of women who were infected late during pregnancy and had tested nasal swab positive for SARS-CoV-2 by qRT-PCR at delivery. SARS-CoV-2 genomic and subgenomic RNA was detected in 23 out of 54 placentas. Two placentas with high virus content were obtained from mothers who presented with severe COVID-19 and whose pregnancies resulted in adverse outcomes for the fetuses, including intrauterine fetal demise and a preterm delivered baby still in newborn intensive care. Examination of the placental samples with high virus content showed efficient SARS-CoV-2 infection, using RNA in situ hybridization to detect genomic and replicating viral RNA, and immunohistochemistry to detect SARS-CoV-2 nucleocapsid protein. Infection was restricted to syncytiotrophoblast cells that envelope the fetal chorionic villi and are in direct contact with maternal blood. The infected placentas displayed massive infiltration of maternal immune cells including macrophages into intervillous spaces, potentially contributing to inflammation of the tissue. Ex vivo infection of placental cultures with SARS-CoV-2 or with SARS-CoV-2 spike (S) protein pseudotyped lentivirus targeted mostly syncytiotrophoblast and in rare events endothelial cells. Infection was reduced by using blocking antibodies against ACE2 and against Neuropilin 1, suggesting that SARS-CoV-2 may utilize alternative receptors for entry into placental cells.

In response to changes in activity induced by environmental cues, neurons in the central nervous system undergo homeostatic plasticity to sustain overall network function during abrupt changes in synaptic strengths. Homeostatic plasticity involves changes in synaptic scaling and regulation of intrinsic excitability. Increases in spontaneous firing and excitability of sensory neurons are evident in some forms of chronic pain in animal models and human patients. However, whether mechanisms of homeostatic plasticity are engaged in sensory neurons under normal conditions or altered after chronic pain is unknown. Here, we showed that sustained depolarization induced by 30mM KCl induces a compensatory decrease in the excitability in mouse and human sensory neurons. Moreover, voltage-gated sodium currents are robustly reduced in mouse sensory neurons contributing to the overall decrease in neuronal excitability. Decreased efficacy of these homeostatic mechanisms could potentially contribute to the development of the pathophysiology of chronic pain.

Macrophages play a crucial role in eliminating respiratory pathogens. Both pulmonary resident alveolar macrophages (AMs) and recruited macrophages contribute to detecting, responding to, and resolving infections in the lungs. Despite their distinct functions, it remains unclear how these macrophage subsets regulate their responses to infection, including how activation by the cytokine IFNγ is regulated. This shortcoming prevents the development of therapeutics that effectively target distinct lung macrophage populations without exacerbating inflammation. We aimed to better understand the transcriptional regulation of resting and IFNγ-activated cells using a new ex vivo model of AMs from mice, fetal liver-derived alveolar-like macrophages (FLAMs), and immortalized bone marrow-derived macrophages (iBMDMs). Our findings reveal that IFNγ robustly activates both macrophage types; however, the profile of activated IFNγ-stimulated genes varies greatly between these cell types. Notably, FLAMs show limited expression of costimulatory markers essential for T cell activation upon stimulation with only IFNγ. To understand cell type-specific differences, we examined how the inhibition of the regulatory kinases GSK3α/β alters the IFNγ response. GSK3α/β controlled distinct IFNγ responses, and in AM-like cells, we found GSK3α/β restrained the induction of type I IFN and TNF, thus preventing the robust expression of costimulatory molecules and limiting CD4+ T cell activation. Together, these data suggest that the capacity of AMs to respond to IFNγ is restricted in a GSK3α/β-dependent manner and that IFNγ responses differ across distinct macrophage populations. These findings lay the groundwork to identify new therapeutic targets that activate protective pulmonary responses without driving deleterious inflammation.

Mental imagery has been proposed to play a critical role in the amplification of cravings. Here we tested whether olfactory imagery drives food cue reactivity strength to promote adiposity in 45 healthy individuals. We measured odor perception, odor imagery ability, and food cue reactivity using self-report, perceptual testing, and neuroimaging. Adiposity was assessed at baseline and one year later. Brain responses to real and imagined odors were analyzed with univariate and multivariate decoding methods to identify pattern-based olfactory codes. We found that the accuracy of decoding imagined, but not real, odor quality correlated with a perceptual measure of odor imagery ability and with greater adiposity changes. This latter relationship was mediated by cue-potentiated craving and intake. Collectively, these findings establish odor imagery ability as a risk factor for weight gain and more specifically as a mechanism by which exposure to food cues promotes craving and overeating.