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Systemic autoimmune and autoinflammatory diseases are characterized by genetic and cellular heterogeneity. While current single-cell genomics methods provide insights into known disease subtypes, these analysis methods do not readily reveal novel cell-type perturbation programs shared among distinct patient subsets. Here, we performed single-cell RNA-Seq of PBMCs of patients with systemic juvenile idiopathic arthritis (SJIA) with diverse clinical manifestations, including macrophage activation syndrome (MAS) and lung disease (LD). We introduced two new computational frameworks called UDON and SATAY-UDON, which define patient subtypes based on their underlying disrupted cellular programs as well as associated biomarkers or clinical features. Among twelve independently identified subtypes, this analysis uncovered what we believe to be a novel complement and interferon activation program identified in SJIA-LD monocytes. Extending these analyses to adult and pediatric lupus patients found new but also shared disease programs with SJIA, including interferon and complement activation. Finally, supervised comparison of these programs in a compiled single-cell pan-immune atlas of over 1,000 healthy donors found a handful of normal healthy donors with evidence of early inflammatory activation in subsets of monocytes and platelets, nominating possible biomarkers for early disease detection. Thus, integrative pan-immune single-cell analysis resolved what we believe to be new conserved gene programs underlying inflammatory disease pathogenesis and associated complications.

How two-chambered hearts in basal vertebrates have evolved from single-chamber hearts found in ancestral chordates remains unclear. Here, we show that the teleost sinus venosus (SV) is a chamber-like vessel comprised of an outer layer of smooth muscle cells. We find that in adult zebrafish nr2f1a mutants, which lack atria, the SV comes to physically resemble the thicker bulbus arteriosus (BA) at the arterial pole of the heart through an adaptive, hypertensive response involving smooth muscle proliferation due to aberrant hemodynamic flow. Single cell transcriptomics show that smooth muscle and endothelial cell populations within the adapting SV also take on arterial signatures. Bulk transcriptomics of the blood sinuses flanking the tunicate heart reinforce a model of greater equivalency in ancestral chordate BA and SV precursors. Our data simultaneously reveal that secondary complications from congenital heart defects can develop in adult zebrafish similar to those in humans and that the foundation of equivalency between flanking auxiliary vessels may remain latent within basal vertebrate hearts. Nr2fs are conserved transcription factors that regulate atrial chamber and venous development. Here, the authors use adult zebrafish nr2f1a mutants to investigate compensatory remodeling of the inflow tract and hypotheses of cardiac evolution.

Macrophage activation syndrome (MAS) is a life-threatening cytokine storm complicating systemic juvenile idiopathic arthritis (SJIA) driven by IFN-γ. SJIA and MAS are also associated with an unexplained emerging inflammatory lung disease (SJIA-LD), with our recent work supporting pulmonary activation of IFN-γ pathways pathologically linking SJIA-LD and MAS. Our objective was to mechanistically define the potentially novel observation of pulmonary inflammation in the TLR9 mouse model of MAS. In acute MAS, lungs exhibit mild but diffuse CD4-predominant, perivascular interstitial inflammation with elevated IFN-γ, IFN-induced chemokines, and alveolar macrophage (AMϕ) expression of IFN-γ-induced genes. Single-cell RNA sequencing confirmed IFN-driven transcriptional changes across lung cell types with myeloid expansion and detection of MAS-specific macrophage populations. Systemic MAS resolution was associated with increased AMϕ and interstitial lymphocytic infiltration. AMϕ transcriptomic analysis confirmed IFN-γ-induced proinflammatory polarization during acute MAS, which switches toward an antiinflammatory phenotype after systemic MAS resolution. Interestingly, recurrent MAS led to increased alveolar inflammation and lung injury, and it reset AMϕ polarization toward a proinflammatory state. Furthermore, in mice bearing macrophages insensitive to IFN-γ, both systemic features of MAS and pulmonary inflammation were attenuated. These findings demonstrate that experimental MAS induces IFN-γ-driven pulmonary inflammation replicating key features of SJIA-LD and provides a model system for testing potentially novel treatments directed toward SJIA-LD.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease. In this Resource article, the authors integrate genomic, bioinformatic and flow cytometric data from human bone marrow to provide an atlas of hematopoietic progenitor cell states in health and disease.

Affinity-matured plasma cells (PCs) of varying lifespans are generated through a germinal center (GC) response. The developmental dynamics and genomic programs of antigen-specific PC precursors remain to be elucidated. Here, using a model antigen in mice, we demonstrate biphasic generation of PC precursors, with those generating long-lived bone marrow PCs preferentially produced in the late phase of GC response. Clonal tracing using single-cell RNA sequencing and B cell antigen receptor sequencing in spleen and bone marrow compartments, coupled with adoptive transfer experiments, reveals a new PC transition state that gives rise to functionally competent PC precursors. The latter undergo clonal expansion, dependent on inducible expression of TIGIT. We propose a model for the proliferation and programming of precursors of long-lived PCs, based on extended antigen encounters in the GC. Singh and colleagues show Tigit controls the generation of germinal center-derived plasma cell precursors that give rise to long-lived differentiated progeny in the bone marrow.

Circadian rhythms are biological clocks featured in a variety of living organisms ranging from unicellular organisms to mammals. These clocks have a period of approximately 24 hours in humans. Understanding the circadian rhythm is quite significant because of its importance in understanding sleep disorders like insomnia and other psychological illnesses. This report provides two mathematical models that describe circadian clocks in Neurospora crassa, a filamentous fungus. Mathematical models, in this research, are representations of molecular mechanisms of core clock components in the form of equations (ordinary differential equations, in this case). These two mathematical models have been compared and analyzed based on the numerical solutions and parameter analysis. Two types of degradation of messenger RNA were considered - linear type degradation and Hill type degradation. The circadian clock that has been studied in this research is that of Neurospora crassa because of its simple clock model and its similarity with the circadian oscillators of humans and other mammals. The mathematical models were simulated using the software MATLAB and the detailed analysis of each parameter in the ordinary differential equations of mathematical models was done by using the program XPP. Based on the results obtained from XPP and comparing both models, it is observed that Hill type degradation of messenger RNA keeps the circadian rhythm more stable than the linear type degradation of messenger RNA.

Electroencephalogram (EEG) records the time-varying electrical activity of the brain. EEG can carry non-uniform noise, thereby making it a complex signal to analyze. In this thesis, we consider a set of two discrete, time-varying EEG vectors of the left and right hemispheres of brain. We develop dynamic models of EEG vectors based on different assumptions that pertain to the properties of a recorded EEG signal. These assumptions are primarily related to the amplitude, frequency, phase shift, and noise content of the two EEG vectors. For every model, we analyze this two-dimensional EEG vector in its phase plane based on the defined geometric properties of the phase plane portrait. For all models in this thesis, we show that the geometric properties are functions of the phase shift between two given signals. Moreover, by analyzing the geometric properties of two given EEG vectors with unknown amplitude and frequency, we can extract a relative phase shift between the given EEG vectors. A limitation in this study is an extracted value of phase shift between two signals is not highly accurate. One of the factors contributing to the low accuracy of an extracted phase shift is the minimizing numerical algorithm that is implemented to compute the geometric properties of the phase plane portrait of two EEG vectors. Other factors may include high noise content in EEG signals. An estimation of a phase shift between two EEG signals by geometrically analyzing their phase plane portrait can allow us to infer about the synchronization of two regions of brain from where the EEG signals are recorded.

Prematurity is associated with low nephron endowment and an increased risk of chronic kidney disease. Human nephrogenesis is complete at 34-36 weeks gestation, with 60% of nephrons forming during the third trimester through lateral branch nephrogenesis (LBN). We hypothesized that a differentiated but dividing population of nephron progenitor cells (NPCs) would contribute to the amplification of nephrons in late gestation. Methods: Single-cell RNA-sequencing (scRNA-Seq) was performed on cortically-enriched fetal rhesus kidneys (n=9) from late second trimester and third trimester during LBN. This data was integrated with publicly available human scRNA-seq datasets from 8-18 weeks gestation kidneys (n=8) using state-of-the-art bioinformatics pipelines. Differentially expressed genes and ligand-receptor interactions were assessed and validated using RNAScope on human and rhesus archival tissue. scRNA-Seq of 64,782 rhesus cells revealed 37 transcriptionally distinct cell populations, including 7,879 rhesus NPCs. Pseudotime analyses identified a late gestation-specific lineage branch of differentiated NPC in rhesus that was not observed in mid-gestation humans. Differential expression analyses identified increased , and and decreased , , , and within the late-gestation rhesus NPC compared to mid-gestation human NPC and increased SEMA3D within the rhesus UB tip, suggesting a compositional shift in WNT and SEMA signaling components within the naive NPC population during LBN. The rhesus macaque uniquely enables molecular studies of late-gestation primate nephrogenesis. Our study suggests the hypothesis that a transitional state of self-renewing NPC supported by compositional shifts in key pathways may underlie the switch from branching phase nephrogenesis to lateral branch nephrogenesis and support ongoing nephron formation in late gestation.

Germinal-center (GC) B-cell responses are defined by many positive regulators of affinity maturation, but few components that restrain clonal dominance, notably , are known. We reveal an unsuspected role for PRDM1 (BLIMP1), a plasma-cell determinant, as a feedback regulator of affinity maturation. Single cell RNA-seq and BCR-seq showed that B- cell-specific loss drives an exaggerated GC reaction with larger clones, increased somatic hypermutation, and greater clonal dominance, independent of . Single cell chromatin profiling with base-resolution modelling indicated that PRDM1 represses expression of BCR-signaling genes and gates chromatin accessibility at ISRE, EICE, NF-κB, and POU (Oct) motifs. In the absence of PRDM1, enhanced engagement of signaling-inducible transcription factors promotes G1-S transition during light-zone (LZ) selection and fuels dark-zone (DZ) expansion. Thus, PRDM1 attenuates BCR signaling and constrains the LZ→DZ transition, fine-tuning clonal competition thereby maintaining repertoire diversity. The chromatin-encoded checkpoint could be leveraged to modulate vaccine responses.

Affinity-matured plasma cells (PCs) of varying lifespans are generated through a germinal center (GC) response. The developmental dynamics and genomic programs of antigen-specific PC precursors remain to be elucidated. Using a model antigen, we demonstrate biphasic generation of PC precursors, with those generating long-lived bone marrow PCs preferentially produced in the late phase of GC response. Clonal tracing using scRNA-seq+BCR-seq in spleen and bone marrow compartments, coupled with adoptive transfer experiments, reveal a novel PC transition state that gives rise to functionally competent PC precursors. The latter undergo clonal expansion, dependent on inducible expression of TIGIT. We propose a model for the proliferation and programming of precursors of long-lived PCs, based on extended antigen encounters followed by reduced antigen availability.