Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease.

The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications.

Local and systemic responses to SARS-CoV-2 infection in children and adults.

It is not fully understood why COVID-19 is typically milder in children1-3. Here, to examine the differences between children and adults in their response to SARS-CoV-2 infection, we analysed paediatric and adult patients with COVID-19 as well as healthy control individuals (total n = 93) using single-cell multi-omic profiling of matched nasal, tracheal, bronchial and blood samples. In the airways of healthy paediatric individuals, we observed cells that were already in an interferon-activated state, which after SARS-CoV-2 infection was further induced especially in airway immune cells. We postulate that higher paediatric innate interferon responses restrict viral replication and disease progression. The systemic response in children was characterized by increases in naive lymphocytes and a depletion of natural killer cells, whereas, in adults, cytotoxic T cells and interferon-stimulated subpopulations were significantly increased. We provide evidence that dendritic cells initiate interferon signalling in early infection, and identify epithelial cell states associated with COVID-19 and age. Our matching nasal and blood data show a strong interferon response in the airways with the induction of systemic interferon-stimulated populations, which were substantially reduced in paediatric patients. Together, we provide several mechanisms that explain the milder clinical syndrome observed in children.

Tracking early mammalian organogenesis - prediction and validation of differentiation trajectories at whole organism scale.

Early organogenesis represents a key step in animal development, during which pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000 single-cell transcriptomes from mouse embryos between E8.5 and E9.5 in 6-h intervals and combined this new dataset with our previous atlas (E6.5-E8.5) to produce a densely sampled timecourse of >400,000 cells from early gastrulation to organogenesis. Computational lineage reconstruction identified complex waves of blood and endothelial development, including a new programme for somite-derived endothelium. We also dissected the E7.5 primitive streak into four adjacent regions, performed scRNA-seq and predicted cell fates computationally. Finally, we defined developmental state/fate relationships by combining orthotopic grafting, microscopic analysis and scRNA-seq to transcriptionally determine cell fates of grafted primitive streak regions after 24 h of in vitro embryo culture. Experimentally determined fate outcomes were in good agreement with computationally predicted fates, demonstrating how classical grafting experiments can be revisited to establish high-resolution cell state/fate relationships. Such interdisciplinary approaches will benefit future studies in developmental biology and guide the in vitro production of cells for organ regeneration and repair.

Single-cell multi-omics analysis of COVID-19 patients with pre-existing autoimmune diseases shows aberrant immune responses to infection.

In COVID-19, hyperinflammatory and dysregulated immune responses contribute to severity. Patients with pre-existing autoimmune conditions can therefore be at increased risk of severe COVID-19 and/or associated sequelae, yet SARS-CoV-2 infection in this group has been little studied. Here, we performed single-cell analysis of peripheral blood mononuclear cells from patients with three major autoimmune diseases (rheumatoid arthritis, psoriasis, or multiple sclerosis) during SARS-CoV-2 infection. We observed compositional differences between the autoimmune disease groups coupled with altered patterns of gene expression, transcription factor activity, and cell-cell communication that substantially shape the immune response under SARS-CoV-2 infection. While enrichment of HLA-DRlow CD14+ monocytes was observed in all three autoimmune disease groups, type-I interferon signaling as well as inflammatory T cell and monocyte responses varied widely between the three groups of patients. Our results reveal disturbed immune responses to SARS-CoV-2 in patients with pre-existing autoimmunity, highlighting important considerations for disease treatment and follow-up.

A single-cell molecular map of mouse gastrulation and early organogenesis.

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.

Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in cluster structures that protrude into the embryonic aortic lumen. Although much is known about the molecular characteristics of the developing hematopoietic cells, we lack a complete understanding of their origin and the three-dimensional organization of the niche. Here, we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modeling to show the two-step process of intra-aortic hematopoietic cluster (IACH) formation. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the IAHC. Next, surrounding hemogenic cells are recruited into the IAHC, increasing their size and heterogeneity. We identified the Notch ligand Dll4 as a negative regulator of the recruitment phase of IAHC. Blocking of Dll4 promotes the entrance of new hemogenic Gfi1+ cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modeling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions.

Myeloid progenitor cluster formation drives emergency and leukaemic myelopoiesis.

Although many aspects of blood production are well understood, the spatial organization of myeloid differentiation in the bone marrow remains unknown. Here we use imaging to track granulocyte/macrophage progenitor (GMP) behaviour in mice during emergency and leukaemic myelopoiesis. In the steady state, we find individual GMPs scattered throughout the bone marrow. During regeneration, we observe expanding GMP patches forming defined GMP clusters, which, in turn, locally differentiate into granulocytes. The timed release of important bone marrow niche signals (SCF, IL-1ß, G-CSF, TGFß and CXCL4) and activation of an inducible Irf8 and ß-catenin progenitor self-renewal network control the transient formation of regenerating GMP clusters. In leukaemia, we show that GMP clusters are constantly produced owing to persistent activation of the self-renewal network and a lack of termination cytokines that normally restore haematopoietic stem-cell quiescence. Our results uncover a previously unrecognized dynamic behaviour of GMPs in situ, which tunes emergency myelopoiesis and is hijacked in leukaemia.

CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation.

Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation.

Thioguanine restoration through type I photosensitization-superoxide oxidation-glutathione reduction cycles.

UVA-induced deleterious effect of thiopurine prodrugs including azathioprine, 6-mercaptopurine and 6-thioguanine (6-TG) increases the risk of cancer development due to the incorporation of 6-TG in patients' DNA. The catalytic mechanism by which thiobases act as a sustained oxidant producer has yet to be explored, especially through the Type I electron transfer pathway that produces superoxide radicals (O2˙-). Under Fenton-like conditions O2˙- radicals convert to extremely reactive hydroxyl radicals (˙OH), thus carrying even higher risk of biological damage than that induced by the well-studied type II reaction. By monitoring 6-TG/UVA-induced photochemistry in mass spectra and superoxide radicals (O2˙-) via nitro blue tetrazolium (NBT) reduction, this work provides two new findings: (1) in the presence of reduced glutathione (GSH), the production of O2˙-via the type I reaction is enhanced 10-fold. 6-TG thiyl radicals are identified as the primary intermediate formed in the reaction of 6-TG with O2˙-. The restoration of 6-TG and concurrent generation of O2˙- occur via a 3-step-cycle: 6-TG type I photosensitization, O2˙- oxidation and GSH reduction. (2) In the absence of GSH, 6-TG thiyl radicals undergo oxygen addition and sulfur dioxide removal to form carbon radicals (C6) which further convert to thioether by reacting with 6-TG molecules. These findings help explain not only thiol-regulation in a biological system but chemoprevention of cancer.

Mechanisms for Pd-Au enrichment in porphyry-epithermal ores of the Elatsite deposit, Bulgaria.

Porphyry Cu can contain significant concentrations of platinum-group elements (PGE: Os, Ir, Ru, Rh, Pt, Pd). In this study, we provide a comprehensive in situ analysis of noble metals (PGE, Au, Ag) for (Cu-Fe)-rich sulfides from the Elatsite, one of the world's PGE-richest porphyry Cu deposits. These data, acquired using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), indicate that Pd was concentrated in all the (Cu-Fe)-rich sulfides at ppm-levels, with higher values in pyrite (~6 ppm) formed at the latest epithermal stage (i.e., quartz-galena-sphalerite assemblage) than in bornite and chalcopyrite (<5 ppm) from the hypogene quartz-magnetite-bornite-chalcopyrite ores. Likewise, Au is significantly more concentrated in pyrite (~5 ppm) than in the (Cu-Fe)-rich sulfides (≤0.08 ppm). In contrast, Ag reaches hundreds of ppm in pyrite and bornite (~240 ppm) but is in much lesser amounts in chalcopyrite (<25 ppm). The inspection of the time-resolved spectra collected during LA-IPC-MS analyses indicates that noble metals are present in the sulfides in two forms: (1) structurally bound (i.e., solid solution) in the lattice of sulfides and, (2) as nano- to micron-sized inclusions (Pd-Te and Au). These observations are further confirmed by careful investigations of the PGE-rich (Cu-Fe)-rich sulfides by combining high-spatial resolution of field emission scanning electron microscope (FESEM) and focused ion beam and high-resolution transmission electron microscopy (FIB/HRTEM). A typical Pd-bearing mineral includes the composition PdTe2 close to the ideal merenskyite but with a distinct crystallographic structure, whereas Au is mainly found as native element. Our detailed mineralogical study coupled with previous knowledge on noble-metal inclusions in the studied ores reveals that noble metal enrichment in the Elatsite porphyry ores was mainly precipitated from droplets of Au-Pd-Ag telluride melt (s) entrained in the high-temperature hydrothermal fluid. These telluride melts could separate at the time of fluid unmixing from the silicate magma or already be present in the latter either derived from deep-seated crustal or mantle sources. Significant enrichment in Pd and Au (the latter correlated with As) in low-temperature pyrite is interpreted as remobilization of these noble metals from pre-existing hypogene ores during the epithermal overprinting.

Assessing the reliability of spike-in normalization for analyses of single-cell RNA sequencing data.

By profiling the transcriptomes of individual cells, single-cell RNA sequencing provides unparalleled resolution to study cellular heterogeneity. However, this comes at the cost of high technical noise, including cell-specific biases in capture efficiency and library generation. One strategy for removing these biases is to add a constant amount of spike-in RNA to each cell and to scale the observed expression values so that the coverage of spike-in transcripts is constant across cells. This approach has previously been criticized as its accuracy depends on the precise addition of spike-in RNA to each sample. Here, we perform mixture experiments using two different sets of spike-in RNA to quantify the variance in the amount of spike-in RNA added to each well in a plate-based protocol. We also obtain an upper bound on the variance due to differences in behavior between the two spike-in sets. We demonstrate that both factors are small contributors to the total technical variance and have only minor effects on downstream analyses, such as detection of highly variable genes and clustering. Our results suggest that scaling normalization using spike-in transcripts is reliable enough for routine use in single-cell RNA sequencing data analyses.

Enhanced hemato-endothelial specification during human embryonic differentiation through developmental cooperation between AF4-MLL and MLL-AF4 fusions.

The t(4;11)(q21;q23) translocation is associated with high-risk infant pro-B-cell acute lymphoblastic leukemia and arises prenatally during embryonic/fetal hematopoiesis. The developmental/pathogenic contribution of the t(4;11)-resulting MLL-AF4 (MA4) and AF4-MLL (A4M) fusions remains unclear; MA4 is always expressed in patients with t(4;11)+ B-cell acute lymphoblastic leukemia, but the reciprocal fusion A4M is expressed in only half of the patients. Because prenatal leukemogenesis manifests as impaired early hematopoietic differentiation, we took advantage of well-established human embryonic stem cell-based hematopoietic differentiation models to study whether the A4M fusion cooperates with MA4 during early human hematopoietic development. Co-expression of A4M and MA4 strongly promoted the emergence of hemato-endothelial precursors, both endothelial- and hemogenic-primed. Double fusion-expressing hemato-endothelial precursors specified into significantly higher numbers of both hematopoietic and endothelial-committed cells, irrespective of the differentiation protocol used and without hijacking survival/proliferation. Functional analysis of differentially expressed genes and differentially enriched H3K79me3 genomic regions by RNA-sequencing and H3K79me3 chromatin immunoprecipitation-sequencing, respectively, confirmed a hematopoietic/endothelial cell differentiation signature in double fusion-expressing hemato-endothelial precursors. Importantly, chromatin immunoprecipitation-sequencing analysis revealed a significant enrichment of H3K79 methylated regions specifically associated with HOX-A cluster genes in double fusion-expressing differentiating hematopoietic cells. Overall, these results establish a functional and molecular cooperation between MA4 and A4M fusions during human hematopoietic development.

Key regulators control distinct transcriptional programmes in blood progenitor and mast cells.

Despite major advances in the generation of genome-wide binding maps, the mechanisms by which transcription factors (TFs) regulate cell type identity have remained largely obscure. Through comparative analysis of 10 key haematopoietic TFs in both mast cells and blood progenitors, we demonstrate that the largely cell type-specific binding profiles are not opportunistic, but instead contribute to cell type-specific transcriptional control, because (i) mathematical modelling of differential binding of shared TFs can explain differential gene expression, (ii) consensus binding sites are important for cell type-specific binding and (iii) knock-down of blood stem cell regulators in mast cells reveals mast cell-specific genes as direct targets. Finally, we show that the known mast cell regulators Mitf and c-fos likely contribute to the global reorganisation of TF binding profiles. Taken together therefore, our study elucidates how key regulatory TFs contribute to transcriptional programmes in several distinct mammalian cell types.

Integrated genome-scale analysis of the transcriptional regulatory landscape in a blood stem/progenitor cell model.

Comprehensive study of transcriptional control processes will be required to enhance our understanding of both normal and malignant hematopoiesis. Modern sequencing technologies have revolutionized our ability to generate genome-scale expression and histone modification profiles, transcription factor (TF)-binding maps, and also comprehensive chromatin-looping information. Many of these technologies, however, require large numbers of cells, and therefore cannot be applied to rare hematopoietic stem/progenitor cell (HSPC) populations. The stem cell factor-dependent multipotent progenitor cell line HPC-7 represents a well-recognized cell line model for HSPCs. Here we report genome-wide maps for 17 TFs, 3 histone modifications, DNase I hypersensitive sites, and high-resolution promoter-enhancer interactomes in HPC-7 cells. Integrated analysis of these complementary data sets revealed TF occupancy patterns of genomic regions involved in promoter-anchored loops. Moreover, preferential associations between pairs of TFs bound at either ends of chromatin loops led to the identification of 4 previously unrecognized protein-protein interactions between key blood stem cell regulators. All HPC-7 data sets are freely available both through standard repositories and a user-friendly Web interface. Together with previously generated genome-wide data sets, this study integrates HPC-7 data into a genomic resource on par with ENCODE tier 1 cell lines and, importantly, is the only current model with comprehensive genome-scale data that is relevant to HSPC biology.

α-Methyl phenylglycines by asymmetric α-arylation of alanine and their effect on the conformational preference of helical Aib foldamers.

α-Arylated alanine derivatives were made enantioselectively by migratory rearrangement of a urea derivative using (R,R)-pseudoephedrine as a chiral auxiliary. Incorporation of a single residue of the product α-methyl phenylglycine into an otherwise achiral oligomer of aminoisobutyric acid oligomer induced a preferred screw sense, detectable by a NMR reporter located at the remote terminus of the oligomer. The magnitude of the screw sense induction was greater when the chiral residue was located at the N-terminus of the foldamer, and in some cases the sense of induction was opposite to that of related α-methylated amino acids with α-substituents other than aryl.

Computer-aided structure-based design of multitarget leads for Alzheimer's disease.

Alzheimer's disease is a neurodegenerative pathology with unmet clinical needs. A highly desirable approach to this syndrome would be to find a single lead that could bind to some or all of the selected biomolecules that participate in the amyloid cascade, the most accepted route for Alzheimer disease genesis. In order to circumvent the challenge posed by the sizable differences in the binding sites of the molecular targets, we propose a computer-assisted protocol based on a pharmacophore and a set of required interactions with the targets that allows for the automated screening of candidates. We used a combination of docking and molecular dynamics protocols in order to discard nonbinders, optimize the best candidates, and provide a rationale for their potential as inhibitors. To provide a proof of concept, we proceeded to screen the literature and databases, a task that allowed us to identify a set of carbazole-containing compounds that initially showed affinity only for the cholinergic targets in our experimental assays. Two cycles of design based on our protocol led to a new set of analogues that were synthesized and assayed. The assay results revealed that the designed inhibitors had improved affinities for BACE-1 by more than 3 orders of magnitude and also displayed amyloid aggregation inhibition and affinity for AChE and BuChE, a result that led us to a group of multitarget amyloid cascade inhibitors that also could have a positive effect at the cholinergic level.

Pseudoephedrine-Directed Asymmetric α-Arylation of α-Amino Acid Derivatives.

Available α-amino acids undergo arylation at their α position in an enantioselective manner on treatment with base of N'-aryl urea derivatives ligated to pseudoephedrine as a chiral auxiliary. In situ silylation and enolization induces diastereoselective migration of the N'-aryl group to the α position of the amino acid, followed by ring closure to a hydantoin with concomitant explulsion of the recyclable auxiliary. The hydrolysis of the hydantoin products provides derivatives of quaternary amino acids. The arylation avoids the use of heavy-metal additives, and is successful with a range of amino acids and with aryl rings of varying electronic character.

The human GFI136N variant induces epigenetic changes at the Hoxa9 locus and accelerates K-RAS driven myeloproliferative disorder in mice.

The coding single nucleotide polymorphism GFI136N in the human gene growth factor independence 1 (GFI1) is present in 3%-7% of whites and increases the risk for acute myeloid leukemia (AML) by 60%. We show here that GFI136N, in contrast to GFI136S, lacks the ability to bind to the Gfi1 target gene that encodes the leukemia-associated transcription factor Hoxa9 and fails to initiate histone modifications that regulate HoxA9 expression. Consistent with this, AML patients heterozygous for the GFI136N variant show increased HOXA9 expression compared with normal controls. Using ChipSeq, we demonstrate that GFI136N specific epigenetic changes are also present in other genes involved in the development of AML. Moreover, granulomonocytic progenitors, a bone marrow subset from which AML can arise in humans and mice, show a proliferative expansion in the presence of the GFI136N variant. In addition, granulomonocytic progenitors carrying the GFI136N variant allele have altered gene expression patterns and differ in their ability to grow after transplantation. Finally, GFI136N can accelerate a K-RAS driven fatal myeloproliferative disease in mice. Our data suggest that the presence of a GFI136N variant allele induces a preleukemic state in myeloid precursors by deregulating the expression of Hoxa9 and other AML-related genes.

Host-guest chemistry of a water-soluble pillar[5]arene: evidence for an ionic-exchange recognition process and different complexation modes.

The complexation of an anionic guest by a cationic water-soluble pillararene is reported. Isothermal titration calorimetry (ITC), (1)H NMR, (1)H and (19)F DOSY, and STD NMR experiments were performed to characterize the complex formed under aqueous neutral conditions. The results of ITC and (1)H NMR analyses showed the inclusion of the guest inside the cavity of the pillar[5]arene, with the binding constant and thermodynamic parameters influenced by the counter ion of the macrocycle. NMR diffusion experiments showed that although a fraction of the counter ions are expelled from the host cavity by exchange with the guest, a complex with both counter ions and the guest inside the pillararene is formed. The results also showed that at higher concentrations of guest in solution, in addition to the inclusion of one guest molecule in the cavity, the pillararene can also form an external complex with a second guest molecule.

Autophagy counters inflammation-driven glycolytic impairment in aging hematopoietic stem cells.

Autophagy is central to the benefits of longevity signaling programs and to hematopoietic stem cell (HSC) response to nutrient stress. With age, a subset of HSCs increases autophagy flux and preserves regenerative capacity, but the signals triggering autophagy and maintaining the functionality of autophagy-activated old HSCs (oHSCs) remain unknown. Here, we demonstrate that autophagy is an adaptive cytoprotective response to chronic inflammation in the aging murine bone marrow (BM) niche. We find that inflammation impairs glucose uptake and suppresses glycolysis in oHSCs through Socs3-mediated inhibition of AKT/FoxO-dependent signaling, with inflammation-mediated autophagy engagement preserving functional quiescence by enabling metabolic adaptation to glycolytic impairment. Moreover, we show that transient autophagy induction via a short-term fasting/refeeding paradigm normalizes glycolytic flux and significantly boosts oHSC regenerative potential. Our results identify inflammation-driven glucose hypometabolism as a key driver of HSC dysfunction with age and establish autophagy as a targetable node to reset oHSC regenerative capacity.