In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies.

SARS-CoV-2-neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques.

Runx3 drives a CD8+ T cell tissue residency program that is absent in CD4+ T cells.

Tissue-resident memory T cells (TRM cells) provide rapid and superior control of localized infections. While the transcription factor Runx3 is a critical regulator of CD8+ T cell tissue residency, its expression is repressed in CD4+ T cells. Here, we show that, as a direct consequence of this Runx3-deficiency, CD4+ TRM cells lacked the transforming growth factor (TGF)-ß-responsive transcriptional network that underpins the tissue residency of epithelial CD8+ TRM cells. While CD4+ TRM cell formation required Runx1, this, along with the modest expression of Runx3 in CD4+ TRM cells, was insufficient to engage the TGF-ß-driven residency program. Ectopic expression of Runx3 in CD4+ T cells incited this TGF-ß-transcriptional network to promote prolonged survival, decreased tissue egress, a microanatomical redistribution towards epithelial layers and enhanced effector functionality. Thus, our results reveal distinct programming of tissue residency in CD8+ and CD4+ TRM cell subsets that is attributable to divergent Runx3 activity.

Discrete tissue microenvironments instruct diversity in resident memory T cell function and plasticity.

Tissue-resident memory T (TRM) cells are non-recirculating cells that exist throughout the body. Although TRM cells in various organs rely on common transcriptional networks to establish tissue residency, location-specific factors adapt these cells to their tissue of lodgment. Here we analyze TRM cell heterogeneity between organs and find that the different environments in which these cells differentiate dictate TRM cell function, durability and malleability. We find that unequal responsiveness to TGFß is a major driver of this diversity. Notably, dampened TGFß signaling results in CD103- TRM cells with increased proliferative potential, enhanced function and reduced longevity compared with their TGFß-responsive CD103+ TRM counterparts. Furthermore, whereas CD103- TRM cells readily modified their phenotype upon relocation, CD103+ TRM cells were comparatively resistant to transdifferentiation. Thus, despite common requirements for TRM cell development, tissue adaptation of these cells confers discrete functional properties such that TRM cells exist along a spectrum of differentiation potential that is governed by their local tissue microenvironment.

Single-cell protein expression profiling resolves circulating and resident memory T cell diversity across tissues and infection contexts.

Memory CD8+ T cells can be broadly divided into circulating (TCIRCM) and tissue-resident memory T (TRM) populations. Despite well-defined migratory and transcriptional differences, the phenotypic and functional delineation of TCIRCM and TRM cells, particularly across tissues, remains elusive. Here, we utilized an antibody screening platform and machine learning prediction pipeline (InfinityFlow) to profile >200 proteins in TCIRCM and TRM cells in solid organs and barrier locations. High-dimensional analyses revealed unappreciated heterogeneity within TCIRCM and TRM cell lineages across nine different organs after either local or systemic murine infection models. Additionally, we demonstrated the relative effectiveness of strategies allowing for the selective ablation of TCIRCM or TRM populations across organs and identified CD55, KLRG1, CXCR6, and CD38 as stable markers for characterizing memory T cell function during inflammation. Together, these data and analytical framework provide an in-depth resource for memory T cell classification in both steady-state and inflammatory conditions.

Neurons limit angiogenesis by titrating VEGF in retina.

Vascular and nervous systems, two major networks in mammalian bodies, show a high degree of anatomical parallelism and functional crosstalk. During development, neurons guide and attract blood vessels, and consequently this parallelism is established. Here, we identified a noncanonical neurovascular interaction in eye development and disease. VEGFR2, a critical endothelial receptor for VEGF, was more abundantly expressed in retinal neurons than in endothelial cells, including endothelial tip cells. Genetic deletion of VEGFR2 in neurons caused misdirected angiogenesis toward neurons, resulting in abnormally increased vascular density around neurons. Further genetic experiments revealed that this misdirected angiogenesis was attributable to an excessive amount of VEGF protein around neurons caused by insufficient engulfment of VEGF by VEGFR2-deficient neurons. Moreover, absence of neuronal VEGFR2 caused misdirected regenerative angiogenesis in ischemic retinopathy. Thus, this study revealed neurovascular crosstalk and unprecedented cellular regulation of VEGF: retinal neurons titrate VEGF to limit neuronal vascularization. PAPERFLICK:

Human SNP links differential outcomes in inflammatory and infectious disease to a FOXO3-regulated pathway.

The clinical course and eventual outcome, or prognosis, of complex diseases varies enormously between affected individuals. This variability critically determines the impact a disease has on a patient's life but is very poorly understood. Here, we exploit existing genome-wide association study data to gain insight into the role of genetics in prognosis. We identify a noncoding polymorphism in FOXO3A (rs12212067: T > G) at which the minor (G) allele, despite not being associated with disease susceptibility, is associated with a milder course of Crohn's disease and rheumatoid arthritis and with increased risk of severe malaria. Minor allele carriage is shown to limit inflammatory responses in monocytes via a FOXO3-driven pathway, which through TGFß1 reduces production of proinflammatory cytokines, including TNFα, and increases production of anti-inflammatory cytokines, including IL-10. Thus, we uncover a shared genetic contribution to prognosis in distinct diseases that operates via a FOXO3-driven pathway modulating inflammatory responses.

Malaria protection due to sickle haemoglobin depends on parasite genotype.

Host genetic factors can confer resistance against malaria1, raising the question of whether this has led to evolutionary adaptation of parasite populations. Here we searched for association between candidate host and parasite genetic variants in 3,346 Gambian and Kenyan children with severe malaria caused by Plasmodium falciparum. We identified a strong association between sickle haemoglobin (HbS) in the host and three regions of the parasite genome, which is not explained by population structure or other covariates, and which is replicated in additional samples. The HbS-associated alleles include nonsynonymous variants in the gene for the acyl-CoA synthetase family member2-4 PfACS8 on chromosome 2, in a second region of chromosome 2, and in a region containing structural variation on chromosome 11. The alleles are in strong linkage disequilibrium and have frequencies that covary with the frequency of HbS across populations, in particular being much more common in Africa than other parts of the world. The estimated protective effect of HbS against severe malaria, as determined by comparison of cases with population controls, varies greatly according to the parasite genotype at these three loci. These findings open up a new avenue of enquiry into the biological and epidemiological significance of the HbS-associated polymorphisms in the parasite genome and the evolutionary forces that have led to their high frequency and strong linkage disequilibrium in African P. falciparum populations.

Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses.

Betacoronaviruses caused the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome, as well as the current pandemic of SARS coronavirus 2 (SARS-CoV-2)1-4. Vaccines that elicit protective immunity against SARS-CoV-2 and betacoronaviruses that circulate in animals have the potential to prevent future pandemics. Here we show that the immunization of macaques with nanoparticles conjugated with the receptor-binding domain of SARS-CoV-2, and adjuvanted with 3M-052 and alum, elicits cross-neutralizing antibody responses against bat coronaviruses, SARS-CoV and SARS-CoV-2 (including the B.1.1.7, P.1 and B.1.351 variants). Vaccination of macaques with these nanoparticles resulted in a 50% inhibitory reciprocal serum dilution (ID50) neutralization titre of 47,216 (geometric mean) for SARS-CoV-2, as well as in protection against SARS-CoV-2 in the upper and lower respiratory tracts. Nucleoside-modified mRNAs that encode a stabilized transmembrane spike or monomeric receptor-binding domain also induced cross-neutralizing antibody responses against SARS-CoV and bat coronaviruses, albeit at lower titres than achieved with the nanoparticles. These results demonstrate that current mRNA-based vaccines may provide some protection from future outbreaks of zoonotic betacoronaviruses, and provide a multimeric protein platform for the further development of vaccines against multiple (or all) betacoronaviruses.

Comprehensive genetic and phenotype analysis of 95 individuals with mosaic tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a neurogenetic disorder due to loss-of-function TSC1 or TSC2 variants, characterized by tumors affecting multiple organs, including skin, brain, heart, lung, and kidney. Mosaicism for TSC1 or TSC2 variants occurs in 10%-15% of individuals diagnosed with TSC. Here, we report comprehensive characterization of TSC mosaicism by using massively parallel sequencing (MPS) of 330 TSC samples from a variety of tissues and fluids from a cohort of 95 individuals with mosaic TSC. TSC1 variants in individuals with mosaic TSC are much less common (9%) than in germline TSC overall (26%) (p < 0.0001). The mosaic variant allele frequency (VAF) is significantly higher in TSC1 than in TSC2, in both blood and saliva (median VAF: TSC1, 4.91%; TSC2, 1.93%; p = 0.036) and facial angiofibromas (median VAF: TSC1, 7.7%; TSC2 3.7%; p = 0.004), while the number of TSC clinical features in individuals with TSC1 and TSC2 mosaicism was similar. The distribution of mosaic variants across TSC1 and TSC2 is similar to that for pathogenic germline variants in general TSC. The systemic mosaic variant was not present in blood in 14 of 76 (18%) individuals with TSC, highlighting the value of analysis of multiple samples from each individual. A detailed comparison revealed that nearly all TSC clinical features are less common in individuals with mosaic versus germline TSC. A large number of previously unreported TSC1 and TSC2 variants, including intronic and large rearrangements (n = 11), were also identified.

Burning questions for a warming and changing world: 15 unknowns in plant abiotic stress.

We present unresolved questions in plant abiotic stress biology as posed by 15 research groups with expertise spanning eco-physiology to cell and molecular biology. Common themes of these questions include the need to better understand how plants detect water availability, temperature, salinity, and rising carbon dioxide (CO2) levels; how environmental signals interface with endogenous signaling and development (e.g. circadian clock and flowering time); and how this integrated signaling controls downstream responses (e.g. stomatal regulation, proline metabolism, and growth versus defense balance). The plasma membrane comes up frequently as a site of key signaling and transport events (e.g. mechanosensing and lipid-derived signaling, aquaporins). Adaptation to water extremes and rising CO2 affects hydraulic architecture and transpiration, as well as root and shoot growth and morphology, in ways not fully understood. Environmental adaptation involves tradeoffs that limit ecological distribution and crop resilience in the face of changing and increasingly unpredictable environments. Exploration of plant diversity within and among species can help us know which of these tradeoffs represent fundamental limits and which ones can be circumvented by bringing new trait combinations together. Better defining what constitutes beneficial stress resistance in different contexts and making connections between genes and phenotypes, and between laboratory and field observations, are overarching challenges.

A chemical probe to modulate human GID4 Pro/N-degron interactions.

The C-terminal to LisH (CTLH) complex is a ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via its substrate receptor Glucose-Induced Degradation 4 (GID4), but its function and substrates in humans remain unclear. Here, we report PFI-7, a potent, selective and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation and quantitative proteomics enabled the identification of GID4 interactors and GID4-regulated proteins. GID4 interactors are enriched for nucleolar proteins, including the Pro/N-degron-containing RNA helicases DDX21 and DDX50. We also identified a distinct subset of proteins whose cellular levels are regulated by GID4 including HMGCS1, a Pro/N-degron-containing metabolic enzyme. These data reveal human GID4 Pro/N-degron targets regulated through a combination of degradative and nondegradative functions. Going forward, PFI-7 will be a valuable research tool for investigating CTLH complex biology and facilitating development of targeted protein degradation strategies that highjack CTLH E3 ligase activity.

Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization.

To identify pathways involved in adult lung regeneration, we employ a unilateral pneumonectomy (PNX) model that promotes regenerative alveolarization in the remaining intact lung. We show that PNX stimulates pulmonary capillary endothelial cells (PCECs) to produce angiocrine growth factors that induce proliferation of epithelial progenitor cells supporting alveologenesis. Endothelial cells trigger expansion of cocultured epithelial cells, forming three-dimensional angiospheres reminiscent of alveolar-capillary sacs. After PNX, endothelial-specific inducible genetic ablation of Vegfr2 and Fgfr1 in mice inhibits production of MMP14, impairing alveolarization. MMP14 promotes expansion of epithelial progenitor cells by unmasking cryptic EGF-like ectodomains that activate the EGF receptor (EGFR). Consistent with this, neutralization of MMP14 impairs EGFR-mediated alveolar regeneration, whereas administration of EGF or intravascular transplantation of MMP14(+) PCECs into pneumonectomized Vegfr2/Fgfr1-deficient mice restores alveologenesis and lung inspiratory volume and compliance function. VEGFR2 and FGFR1 activation in PCECs therefore increases MMP14-dependent bioavailability of EGFR ligands to initiate and sustain alveologenesis.

Red blood cell tension protects against severe malaria in the Dantu blood group.

Malaria has had a major effect on the human genome, with many protective polymorphisms-such as the sickle-cell trait-having been selected to high frequencies in malaria-endemic regions1,2. The blood group variant Dantu provides 74% protection against all forms of severe malaria in homozygous individuals3-5, a similar degree of protection to that afforded by the sickle-cell trait and considerably greater than that offered by the best malaria vaccine. Until now, however, the protective mechanism has been unknown. Here we demonstrate the effect of Dantu on the ability of the merozoite form of the malaria parasite Plasmodium falciparum to invade red blood cells (RBCs). We find that Dantu is associated with extensive changes to the repertoire of proteins found on the RBC surface, but, unexpectedly, inhibition of invasion does not correlate with specific RBC-parasite receptor-ligand interactions. By following invasion using video microscopy, we find a strong link between RBC tension and merozoite invasion, and identify a tension threshold above which invasion rarely occurs, even in non-Dantu RBCs. Dantu RBCs have higher average tension than non-Dantu RBCs, meaning that a greater proportion resist invasion. These findings provide both an explanation for the protective effect of Dantu, and fresh insight into why the efficiency of P. falciparum invasion might vary across the heterogenous populations of RBCs found both within and between individuals.

Publisher Correction: Exuberant fibroblast activity compromises lung function via ADAMTS4.

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

Exuberant fibroblast activity compromises lung function via ADAMTS4.

Severe respiratory infections can result in acute respiratory distress syndrome (ARDS)1. There are no effective pharmacological therapies that have been shown to improve outcomes for patients with ARDS. Although the host inflammatory response limits spread of and eventually clears the pathogen, immunopathology is a major contributor to tissue damage and ARDS1,2. Here we demonstrate that respiratory viral infection induces distinct fibroblast activation states, which we term extracellular matrix (ECM)-synthesizing, damage-responsive and interferon-responsive states. We provide evidence that excess activity of damage-responsive lung fibroblasts drives lethal immunopathology during severe influenza virus infection. By producing ECM-remodelling enzymes-in particular the ECM protease ADAMTS4-and inflammatory cytokines, damage-responsive fibroblasts modify the lung microenvironment to promote robust immune cell infiltration at the expense of lung function. In three cohorts of human participants, the levels of ADAMTS4 in the lower respiratory tract were associated with the severity of infection with seasonal or avian influenza virus. A therapeutic agent that targets the ECM protease activity of damage-responsive lung fibroblasts could provide a promising approach to preserving lung function and improving clinical outcomes following severe respiratory infections.

Non-O ABO blood group genotypes differ in their associations with Plasmodium falciparum rosetting and severe malaria.

Blood group O is associated with protection against severe malaria and reduced size and stability of P. falciparum-host red blood cell (RBC) rosettes compared to non-O blood groups. Whether the non-O blood groups encoded by the specific ABO genotypes AO, BO, AA, BB and AB differ in their associations with severe malaria and rosetting is unknown. The A and B antigens are host RBC receptors for rosetting, hence we hypothesized that the higher levels of A and/or B antigen on RBCs from AA, BB and AB genotypes compared to AO/BO genotypes could lead to larger rosettes, increased microvascular obstruction and higher risk of malaria pathology. We used a case-control study of Kenyan children and in vitro adhesion assays to test the hypothesis that "double dose" non-O genotypes (AA, BB, AB) are associated with increased risk of severe malaria and larger rosettes than "single dose" heterozygotes (AO, BO). In the case-control study, compared to OO, the double dose genotypes consistently had higher odds ratios (OR) for severe malaria than single dose genotypes, with AB (OR 1.93) and AO (OR 1.27) showing most marked difference (p = 0.02, Wald test). In vitro experiments with blood group A-preferring P. falciparum parasites showed that significantly larger rosettes were formed with AA and AB host RBCs compared to OO, whereas AO and BO genotypes rosettes were indistinguishable from OO. Overall, the data show that ABO genotype influences P. falciparum rosetting and support the hypothesis that double dose non-O genotypes confer a greater risk of severe malaria than AO/BO heterozygosity.

Taste and pheromonal inputs govern the regulation of time investment for mating by sexual experience in male Drosophila melanogaster.

Males have finite resources to spend on reproduction. Thus, males rely on a 'time investment strategy' to maximize their reproductive success. For example, male Drosophila melanogaster extends their mating duration when surrounded by conditions enriched with rivals. Here we report a different form of behavioral plasticity whereby male fruit flies exhibit a shortened duration of mating when they are sexually experienced; we refer to this plasticity as 'shorter-mating-duration (SMD)'. SMD is a plastic behavior and requires sexually dimorphic taste neurons. We identified several neurons in the male foreleg and midleg that express specific sugar and pheromone receptors. Using a cost-benefit model and behavioral experiments, we further show that SMD behavior exhibits adaptive behavioral plasticity in male flies. Thus, our study delineates the molecular and cellular basis of the sensory inputs required for SMD; this represents a plastic interval timing behavior that could serve as a model system to study how multisensory inputs converge to modify interval timing behavior for improved adaptation.

ARGprofiler-a pipeline for large-scale analysis of antimicrobial resistance genes and their flanking regions in metagenomic datasets.

MOTIVATION: Analyzing metagenomic data can be highly valuable for understanding the function and distribution of antimicrobial resistance genes (ARGs). However, there is a need for standardized and reproducible workflows to ensure the comparability of studies, as the current options involve various tools and reference databases, each designed with a specific purpose in mind. RESULTS: In this work, we have created the workflow ARGprofiler to process large amounts of raw sequencing reads for studying the composition, distribution, and function of ARGs. ARGprofiler tackles the challenge of deciding which reference database to use by providing the PanRes database of 14 078 unique ARGs that combines several existing collections into one. Our pipeline is designed to not only produce abundance tables of genes and microbes but also to reconstruct the flanking regions of ARGs with ARGextender. ARGextender is a bioinformatic approach combining KMA and SPAdes to recruit reads for a targeted de novo assembly. While our aim is on ARGs, the pipeline also creates Mash sketches for fast searching and comparisons of sequencing runs. AVAILABILITY AND IMPLEMENTATION: The ARGprofiler pipeline is a Snakemake workflow that supports the reuse of metagenomic sequencing data and is easily installable and maintained at https://github.com/genomicepidemiology/ARGprofiler.

Pinpointing the causal influences of stomatal anatomy and behavior on minimum, operational, and maximum leaf surface conductance.

Leaf surface conductance to water vapor and CO2 across the epidermis (gleaf) strongly determines rates of gas exchange. Thus, clarifying the drivers of gleaf has important implications for resolving mechanisms of photosynthetic productivity and leaf and plant responses and tolerance to drought. It is well recognized that gleaf is a function of the conductances of the stomata (gs) and of the epidermis + cuticle (gec). Yet, controversies have arisen around the relative roles of stomatal density (d) and size (s), fractional stomatal opening (α; aperture relative to maximum) and gec in determining gleaf. Resolving the importance of these drivers is critical across the range of leaf surface conductances, from strong stomatal closure under drought (gleaf, min), to typical opening for photosynthesis (gleaf, op), to maximum achievable opening (gleaf, max). We derived equations and analyzed a compiled database of published and measured data for approximately 200 species and genotypes. On average, within and across species, higher gleaf, min was determined ten times more strongly by α and gec than by d, and negligibly by s; higher gleaf, op was determined approximately equally by α (47%) than by stomatal anatomy (45% by d, and 8% by s), and negligibly by gec; and higher gleaf, max was determined entirely by d. These findings clarify how diversity in stomatal functioning arises from multiple structural and physiological causes with importance shifting with context. The rising importance of d relative to α, from gleaf, min to gleaf, op, enables even species with low gleaf, min, which can retain leaves through drought, to possess high d and thereby achieve rapid gas exchange in periods of high water availability.

Hydroxyurea treatment is associated with lower malaria incidence in children with sickle cell anemia in sub-Saharan Africa.

Realizing Effectiveness Across Continents with Hydroxyurea (REACH, NCT01966731) provides hydroxyurea at maximum tolerated dose (MTD) for children with sickle cell anemia (SCA) in sub-Saharan Africa. Beyond reducing SCA-related clinical events, documented treatment benefits include ∼50% reduction in malaria incidence. To identify associations and propose mechanisms by which hydroxyurea could be associated with lower malaria rates, infections were recorded across all clinical sites (Angola, Democratic Republic of Congo, Kenya, and Uganda). Hazard ratios (HR) with 95% confidence intervals (CIs) for baseline demographics, and time-varying laboratory and clinical parameters were estimated in a modified Cox gap-time model for repeated events. Over 3387 patient-years of hydroxyurea treatment, 717 clinical malaria episodes occurred in 336 of 606 study participants; over half were confirmed by blood smear and/or rapid diagnostic testing with 97.8% Plasmodium falciparum. In univariate analysis limited to 4 confirmed infections per child, malaria risk was significantly associated with absolute neutrophil count (ANC), splenomegaly, hemoglobin, and achieving MTD; age, malaria season, MTD dose, fetal hemoglobin, α-thalassemia, and glucose-6-phosphate dehydrogenase deficiency had no effect. In multivariable regression of confirmed infections, ANC was significant (HR, 1.37 per doubled value; 95% CI, 1.10-1.70; P = .0052), and ANC values <3.0 × 109/L were associated with lower malaria incidence. Compared with nonpalpable spleen, 1- to 4-cm splenomegaly also was associated with higher malaria risk (HR, 2.01; 95% CI, 1.41-2.85; P = .0001). Hydroxyurea at MTD is associated with lower malaria incidence in SCA through incompletely defined mechanisms, but treatment-associated mild myelosuppression with ANC <3.0 × 109/L is salutary. Splenomegaly is an unexplained risk factor for malaria infections among children with SCA in Africa.