We employed single cell RNA sequencing (scRNA-seq) of fine needle aspirates (FNAs) to describe the cells and communication networks characterizing granulomatous lymph nodes of TB patients. We uniformly identified several cell types known to characterize granulomas. Overall we found the T cell cluster to be the most abundant. Other cell clusters that were uniformly detected, but that varied in abundance amongst the individual patient samples, were the B cell, plasma cell and macrophage/dendritic and NK cell clusters. When we combined all our scRNA-seq data from our current 19 patients, we distinguished T, B, macrophage, dendritic and plasma cell subclusters. The sizes of these subclusters also varied dramatically amongst the individual patients. In comparing FNA composition we noted trends in which T cell populations were negatively correlated with NK cell populations and with macrophage/dendritic cell populations. In addition, we discovered that the scRNA-seq pipeline detects Mtb RNA transcripts and associates them with their host cell’s transcriptome, thus identifying individual infected cells. The number of infected cells also varies in abundance amongst the patient samples. CellChat analysis identified predominating signaling pathways amongst the cells comprising the various granulomatous lymph nodes, identifying several pathways involved in immune cell maturation, migration and adhesion.

Background/Objectives: Upon systemic delivery, macrophages take up a significant portion of nanoparticles and may become saturated. The saturation of macrophages may pose risks to overall immune function and signaling pathways. While some information is available on the survival and functionality of macrophages upon saturation with nanoparticles, there is limited understanding of the molecular-level changes that can occur and their corresponding influences on macrophage phenotypes, gene expression, and immune signaling pathways. Methods: In this study, RAW 264.7 macrophages were saturated with silica nanoparticles (SNPs) of different sizes (50 and 100 nm), porosities (nonporous, mesoporous), densities (solid, mesoporous, and hollow), and surface compositions (hydrophobicity) at their maximum non-toxic concentrations. The saturated macrophages were evaluated for changes in gene expression and immune signaling pathways by RNA sequencing, weighted gene co-expression network analysis (WGCNA), and Hallmark and KEGG pathway analyses. Results: Our results show that in the range studied, the particle size did not have a significant effect on the gene expression profile. Porous SNPs of comparable sizes resulted in increased and unique changes in the gene expression profile compared to nonporous SNPs. Major immune signaling pathways, including TNF-alpha signaling via NF-κB pathways, mTORC1 signaling, and p53 pathways, were modulated in SNP-saturated macrophages. This modulation depended on the physicochemical properties of the particles. The Th1/Th2 multiplex immunoassay revealed that the uptake of SNPs increases the amount of the TNF-alpha cytokine compared to the nontreated controls, whereas no changes in IL-6 and IL-12p70 pro-inflammatory cytokines were observed. Conclusions: Our results demonstrate that physicochemical properties of SNPs, such as porosity, size, surface functionality, and density, influence the modulation of gene expression and macrophage immune signaling pathways. These results, along with others, can provide guidance on the selection of silica nanoparticles for the safe and effective systemic delivery of bioactive agents.

Exposure to excess UVR is associated with approximately three-quarters of the global burden of melanoma. Yet, much of what is known about the effects of UV on melanocytes comes from studies of cell lines, animal models, and ex vivo-cultured human skin. In this in vivo study of human melanocytic nevi, we describe a protocol that facilitates the characterization of the effects of simulated solar radiation on a wide array of cell types using RNA sequencing of single nuclei isolated from optimal cutting temperature-embedded frozen tissue. With no enrichment for rare cell types, our RNA sequencing of single nuclei data reveal the transcriptional diversity of melanocytes, keratinocytes of the epidermis and adnexa, lymphatic and vascular endothelium, fibroblasts, and immune cells of the skin. Differences in gene expression between interfollicular basilar and nevomelanocytes, including those involved in senescence, are delineated. Co-expression analysis identified changes elicited by simulated solar radiation in gene modules containing SERPINE2, and components of the IGF-1 signaling axis, in nevus cells, fibroblasts, and endothelial cells. These findings were corroborated by RNAscope analysis of formalin-fixed parafin-embedded samples, thereby providing valuable pharmacologic targets and biomarkers for clinical trials of melanoma prevention agents.

Tourette disorder (TD) is a neurodevelopmental condition with a robust genetic basis, characterized by multiple motor and vocal tics. Tics arise from dysfunction within cortico-striatal-thalamo-cortical circuits, with pathological evidence largely implicating striatal interneuron deficits and microglial activation. Recently, the largest genome-wide association study (GWAS) meta-analysis of TD identified polygenic risk enrichment in Brodmann area 9 (BA9), corresponding to the dorsolateral prefrontal cortex (DLPFC), a region implicated in executive control and tic suppression. However, the molecular landscape of BA9 in TD remains unexplored. Here, we performed single-nucleus RNA sequencing of postmortem BA9 from five males with TD and five matched controls, yielding 72,340 nuclei across neuronal and glial populations. While cell-type proportions were preserved, transcriptional remodeling was pervasive. In particular, biosynthetic and translational programs were upregulated across microglia, interneurons, oligodendrocytes, and superficial- and middle-layer excitatory neurons. Most cell types showed enrichment for glucocorticoid-responsive and immediate early gene modules consistent with stress-associated transcriptional activation. Finally, cross-regional comparison with striatal datasets revealed conservation of microglial and oligodendrocyte programs. These findings point to extensive transcriptional reprogramming in the DLPFC of individuals with TD, characterized by stress-associated activation, most strongly in oligodendrocytes and neurons.

Tics are sudden, partially controllable motor or vocal events that arise from disruptions within cortico–striatal–thalamo–cortical circuits. Within this network, converging evidence implicates the dorsolateral prefrontal cortex (DLPFC) as an important cortical node supporting voluntary tic control. This region is critical for top-down regulation and is particularly sensitive to stress, raising the possibility that molecular vulnerabilities within the DLPFC may influence the capacity to suppress tics. However, the underlying cellular and molecular architecture remains poorly defined. To address this gap, we conducted the first single-nucleus RNA sequencing analysis of postmortem DLPFC tissue from men with Tourette disorder (TD) and age-matched neurotypical controls. Overall cell-type proportions did not differ significantly between groups. In contrast, gene ontology analyses revealed broad upregulation of transcripts involved in protein synthesis, most prominently in microglia, oligodendrocytes, and interneurons. Among neuronal lineages, these changes were most pronounced in superficial and middle-layer pyramidal neurons and vasointestinal peptide-positive interneurons. To determine whether these alterations reflect engagement of stress-related transcriptional programs, we compared TD-associated differentially expressed genes with published single-nucleus datasets from major depressive disorder and posttraumatic stress disorder. Across DLPFC cell populations, and especially within pyramidal neurons and interneurons, we observed significant enrichment for stress-associated gene signatures, including glucocorticoid-responsive transcripts and immediate early genes. Together, these findings identify the DLPFC in TD as a region of heightened stress responsivity and altered excitatory–inhibitory dynamics, offering new insight into cortical mechanisms that may constrain tic suppression.

A key response to acute stress is the increased brain synthesis of the neurosteroid allopregnanolone (AP). Although the rate-limiting step of this reaction is catalyzed by 5α-reductase (5αR), the role of its two primary isoenzymes, 5αR1 and 5αR2, in stress reactivity remains unclear. Here, we found that acute stress led to increased levels of 5αR2, but not 5αR1, in the medial prefrontal cortex (mPFC) of male, but not female, rats. Down-regulation of 5αR2 in the mPFC significantly reduced stress response in males, and similar sexual dimorphic effects were observed in a novel line of 5αR2 knockout rats. Notably, 5αR1 regulated baseline AP synthesis, whereas 5αR2 enabled AP production under stress. Acute AP administration restored stress response in 5αR2 knockdown rats. Single-nucleus transcriptomics showed that 5αR2 enabled stress-induced protein translation in neurons and glia. These results highlight the crucial role of 5αR2 in mediating sex-specific differences in acute stress reactivity.

Dravet Syndrome (DS) is a severe genetic epileptic encephalopathy caused by mutations in the SCN1A gene that encodes the voltage-gated sodium channel (NaV1.1) subunit alpha. DS is characterized by intractable seizures, progressive developmental delay, cognitive impairment, and high mortality due to sudden unexpected death in epilepsy (SUDEP). SUDEP is mediated by respiratory dysfunction, but the exact molecular underpinnings are unclear. Though hippocampal metabolic alterations have been reported in DS mice, such changes in brain regions controlling breathing have not been studied. We used Scn1aA1783V/WT DS mice to study temporal alterations in the brain metabolome, including analysis of brainstem and forebrain regions. Glycolytic and pentose phosphate pathway intermediates were significantly elevated in the brainstem of DS mice during the period of enhanced susceptibility to mortality (post-natal days P20-30). In older P40-P50 mice, mitochondrial aconitate and the antioxidant glutathione were significantly elevated in the brainstem. Single-nuclei RNA sequencing (snRNA seq) and proteomic analyses revealed alterations in genes associated with neurotransmission, cellular respiration, and protein translation, as well as reorganization of protein kinase-mediated pathways that are specific to the brainstem. These findings suggest that there are widespread metabolic changes in the brainstem of DS mice.

The enteric nervous system (ENS) is a complex network of neurons and glial cells. Hirschsprung's disease (HSCR) is a congenital condition characterized by the absence of ganglion cells in the distal colon, leading to functional bowel obstruction. In this study, we used single-cell RNA sequencing (scRNA-seq) and whole genome sequencing (WGS) to analyze healthy and aganglionic colon segments from HSCR patients. Using scRNA-seq, we identified 13 major cell types in patient samples and observed that neural progenitor cells were present in both healthy and aganglionic colon regions, while mature neurons were absent from aganglionic colon. In these progenitor cells, critical differentiation pathway genes displayed reduced expression in the aganglionic colon, suggesting a disruption in their transition to mature neuronal cell types. Furthermore, transcriptomic analysis revealed significant alterations in gene expression across several stromal cell types. These transcriptomic shifts, particularly in mast cells, support the hypothesis that altered gene expression in the microenvironment of neural progenitor cells contributes to impaired differentiation. Our findings support the hypothesis that neural precursors in HSCR are capable of migration, but they are defective in their differentiation to mature cell types. Our analysis provides insights into potential therapeutic targets to stimulate neurogenesis in the aganglionic colon.

The gene CELSR3 (Cadherin EGF LAG Seven-pass-G-type Receptor 3) has been recently recognized as a high-confidence risk factor for Tourette syndrome (TS). Additionally, Celsr3 mutant mice have been reported to exhibit TS-related behaviors and increased dopamine release in the striatum. Building on these findings, we further characterized the neurobehavioral and molecular profile of Celsr3 mutant mice to understand better the biological mechanisms connecting the deficiency of this gene and TS-related phenotypes. Our analyses confirmed that Celsr3 mutant mice displayed grooming stereotypies and tic-like jerks, as well as sensorimotor gating deficits, which were opposed by TS therapies. Spatial transcriptomic analyses revealed widespread extracellular matrix abnormalities in the striatum of Celsr3 mutants. Single-nucleus transcriptomics also showed significant upregulation of the Drd3 gene, encoding the dopamine D3 receptor, in striosomal D1-positive neurons. In situ hybridization and immunofluorescence confirmed dysregulated D3 receptor expression, with lower levels in presynaptic striatal fibers and higher levels in striatal D1-positive neurons. Activating and blocking D3 receptors amplified or decreased tic-like jerks and stereotypies in Celsr3-deficient mice, respectively. These findings suggest that modifications of D3 receptor distribution contribute to the tic-like responses associated with Celsr3 deficiency.

Bruton's tyrosine kinase (BTK) inhibitors are effective for the treatment of chronic lymphocytic leukemia (CLL) due to BTK's role in B cell survival and proliferation. Treatment resistance is most commonly caused by the emergence of the hallmark BTKC481S mutation that inhibits drug binding. In this study, we aimed to investigate whether the presence of additional CLL driver mutations in cancer subclones harboring a BTKC481S mutation accelerates subclone expansion. In addition, we sought to determine whether BTK-mutated subclones exhibit distinct transcriptomic behavior when compared to other cancer subclones. To achieve these goals, we employ our recently published method (Qiao et al. 2024) that combines bulk DNA sequencing and single-cell RNA sequencing (scRNA-seq) data to genotype individual cells for the presence or absence of subclone-defining mutations. While the most common approach for scRNA-seq includes short-read sequencing, transcript coverage is limited due to the vast majority of the reads being concentrated at the priming end of the transcript. Here, we utilized MAS-seq, a long-read scRNAseq technology, to substantially increase transcript coverage across the entire length of the transcripts and expand the set of informative mutations to link cells to cancer subclones in six CLL patients who acquired BTKC481S mutations during BTK inhibitor treatment. We found that BTK-mutated subclones often acquire additional mutations in CLL driver genes, leading to faster subclone proliferation. When examining subclone-specific gene expression, we found that in one patient, BTK-mutated subclones are transcriptionally distinct from the rest of the malignant B cell population with an overexpression of CLL-relevant genes.

The lung is constantly exposed to a myriad of exogenous stressors. Ground-level ozone represents a ubiquitous and extremely reactive anthropogenic toxicant, impacting the health of millions across the globe. While abundant, epidemiological, in vivo, and in vitro data focuses the ozone toxicity in individual cell types (e.g. epithelial type II, alveolar macrophages) or signaling pathways involved in the injury (e.g., akt, glutathione). When appropriately used, bulk and single cell RNA sequencing techniques have the potential to provide complete, and in certain cases unbiased, information of the molecular events taking place in the steady state and injured lung, and even capture the phenotypic diversity of neighboring cells. To this end, this review compiles information pertaining to the latest understanding of lung cell identity and activation in the steady state and ozone exposed lung. In addition, it discusses the value and benefits of multi-omics approaches and other tools developed to predict cell-cell communication and dissect spatial heterogeneity.

IntroductionGenetic mutations in critical nodes of pulmonary epithelial function are linked to the pathogenesis of pulmonary fibrosis (PF) and other interstitial lung diseases. The slow progression of these pathologies is often intermitted and accelerated by acute exacerbations, complex non-resolving cycles of inflammation and parenchymal damage, resulting in lung function decline and death. Excess monocyte mobilization during the initial phase of an acute exacerbation, and their long-term persistence in the lung, is linked to poor disease outcome.MethodsThe present work leverages a clinical idiopathic PF dataset and a murine model of acute inflammatory exacerbations triggered by mutation in the alveolar type-2 cell-restricted Surfactant Protein-C [SP-C] gene to spatially and phenotypically define monocyte/macrophage changes in the fibrosing lung.ResultsSP-C mutation triggered heterogeneous CD68+ macrophage activation, with highly active peri-injured cells relative to those sampled from fully remodeled and healthy regions. Ingenuity pathway analysis of sorted CD11b-SigF+CD11c+ alveolar macrophages defined asynchronous activation of extracellular matrix re-organization, cellular mobilization, and Apolipoprotein E (Apoe) signaling in the fibrosing lung. Cell-cell communication analysis of single cell sequencing datasets predicted pro-fibrogenic signaling (fibronectin/Fn1, osteopontin/Spp1, and Tgfb1) emanating from Trem2/TREM2+ interstitial macrophages. These cells also produced a distinct lipid signature from alveolar macrophages and monocytes, characterized by Apoe expression. Mono- and di-allelic genetic deletion of ApoE in SP-C mutant mice had limited impact on inflammation and mortality up to 42 day after injury.DiscussionTogether, these results provide a detailed spatio-temporal picture of resident, interstitial, and monocyte-derived macrophages during SP-C induced inflammatory exacerbations and end-stage clinical PF, and propose ApoE as a biomarker to identify activated macrophages involved in tissue remodeling.

Genetic and gene expression heterogeneity is an essential hallmark of many tumors, allowing the cancer to evolve and to develop resistance to treatment. Currently, the most commonly used data types for studying such heterogeneity are bulk tumor/normal whole-genome or whole-exome sequencing (WGS, WES); and single-cell RNA sequencing (scRNA-seq), respectively. However, tools are currently lacking to link genomic tumor subclonality with transcriptomic heterogeneity by integrating genomic and single-cell transcriptomic data collected from the same tumor. To address this gap, we developed scBayes, a Bayesian probabilistic framework that uses tumor subclonal structure inferred from bulk DNA sequencing data to determine the subclonal identity of cells from single-cell gene expression (scRNA-seq) measurements. Grouping together cells representing the same genetically defined tumor subclones allows comparison of gene expression across different subclones, or investigation of gene expression changes within the same subclone across time (i.e., progression, treatment response, or relapse) or space (i.e., at multiple metastatic sites and organs). We used simulated data sets, in silico synthetic data sets, as well as biological data sets generated from cancer samples to extensively characterize and validate the performance of our method, as well as to show improvements over existing methods. We show the validity and utility of our approach by applying it to published data sets and recapitulating the findings, as well as arriving at novel insights into cancer subclonal expression behavior in our own data sets. We further show that our method is applicable to a wide range of single-cell sequencing technologies including single-cell DNA sequencing as well as Smart-seq and 10x Genomics scRNA-seq protocols.

Acute stress triggers rapid brain responses, including increased allopregnanolone (AP) synthesis via 5α-reductase (5αR). However, the involvement of this enzyme in stress response remains elusive. Here, we investigated the roles of the two major 5αR isoenzymes in acute stress. Acute stress increased mRNA and protein levels of 5αR2, but not 5αR1, in the medial prefrontal cortex (mPFC) of male, but not female, rats. Targeted mPFC downregulation of 5αR2, but not 5αR1, markedly reduced the responsiveness of males, but not females, to both stressful and arousing stimuli. Similar sex differences were observed in 5αR2 knockout rats. While 5αR1 regulated AP synthesis under baseline conditions, 5αR2 enabled this process in response to acute stress. Single-nucleus transcriptomic analyses revealed that 5αR2 enabled stress-induced protein synthesis in pyramidal neurons and glia of the mPFC. These findings underscore the pivotal role of 5αR2 in shaping sex-related differences in acute stress reactivity. ### Competing Interest Statement MB consults for Asarina Pharmaceuticals and receives research funding from Asarina and Lundbeck Pharmaceuticals. The other authors declare no conflict of interest.

Mycobacterium tuberculosis (Mtb) remains a global human health threat. However, understanding effects of the microbe on cellular interactions in infected tissue has been hindered by inability to discriminate between infected versus un-infected cells. We included the H37Rv Mtb reference genome when assembling scRNA seq libraries from fine needle aspirate samples of peripheral nodal TB patients. Using the 10X Genomics Cell Ranger tool to align sequencing reads, we consistently detected bacterial small and large ribosomal subunit RNA sequences. We interpret Mtb reads associated with a cell's UMI and transcriptome to indicate infection of that individual host cell. This provides a new window into the status of infected cells in the context of the bystander cells in the infected tissue. We investigated these Mtb transcripts to explore their clinical utility. The Mtb transcripts showed frequent sequence variation from the reference genome, with greater than 90% of the rrs or rrl reads from many clinical samples having at least one sequence difference. The highly conserved nature of the rrs and rrl gene sequences limited the ability to assign bacterial lineage based solely transcriptome analysis. However, rapid improvements in sequencing depth may soon allow transcriptome analysis of infecting microbes and improved certainty regarding their lineage, drug resistance, and virulence factors.

We successfully employed a single cell RNA sequencing (scRNA-seq) approach to describe the cells and the communication networks characterizing granulomatous lymph nodes of TB patients. When mapping cells from individual patient samples, clustered based on their transcriptome similarities, we uniformly identify several cell types that known to characterize human and non-human primate granulomas. Whether high or low Mtb burden, we find the T cell cluster to be one of the most abundant. Many cells expressing T cell markers are clearly quantifiable within this CD3 expressing cluster. Other cell clusters that are uniformly detected, but that vary dramatically in abundance amongst the individual patient samples, are the B cell, plasma cell and macrophage/dendrocyte and NK cell clusters. When we combine all our scRNA-seq data from our current 23 patients (in order to add power to cell cluster identification in patient samples with fewer cells), we distinguish T, macrophage, dendrocyte and plasma cell subclusters, each with distinct signaling activities. The sizes of these subclusters also varies dramatically amongst the individual patients. In comparing FNA composition we noted trends in which T cell populations and macrophage/dendrocyte populations were negatively correlated with NK cell populations. In addition, we also discovered that the scRNA-seq pipeline, designed for quantification of human cell mRNA, also detects Mtb RNA transcripts and associates them with their host cell's transcriptome, thus identifying individual infected cells. We hypothesize that the number of detected bacterial transcript reads provides a measure of Mtb burden, as does the number of Mtb-infected cells. The number of infected cells also varies dramatically in abundance amongst the patient samples. CellChat analysis identified predominating signaling pathways amongst the cells comprising the various granulomas, including many interactions between stromal or endothelial cells and the other component cells, such as Collagen, FN1 and Laminin,. In addition, other more selective communications pathways, including MIF, MHC-1, MHC-2, APP, CD 22, CD45, and others, are identified as originating or being received by individual immune cell components.

The gene CELSR3 (cadherin EGF LAG seven-pass-G-type receptor 3) has been recently recognized as a high-confidence risk factor for Tourette syndrome (TS) Here, we characterized the behavioral phenotypes of Celsr3 mutant mice to verify whether the deficiency of this gene is associated with TS predisposition. Celsr3 mutant mice displayed tic-like grooming stereotypies and jerks, as well as sensorimotor gating deficits, which were opposed by TS therapies. Single-nucleus transcriptomic analyses revealed that Celsr3 mutants featured a unique group of eccentric striatal projection neurons. Notably, the Drd3 gene, encoding the dopamine D3 receptor, was significantly upregulated in these cells as well as striosomal D1-positive neurons, while it was reduced in calretinin-positive GABAergic interneurons. Activating and blocking D3 receptors amplified or decreased tic-like jerks and stereotypies in Celsr3 -deficient mice, respectively. These findings suggest that modifications of D3 receptor distribution across various striatal cell populations contribute to the tic-like responses associated with Celsr3 deficiency.### Competing Interest StatementThe authors have declared no competing interest.

This study tested whether a medicinal plant, Vasaka, typically consumed as a tea to treat respiratory malaise, could protect airway epithelial cells (AECs) from wood smoke particle-induced damage and prevent pathological mucus production. Wood/biomass smoke is a pneumotoxic air pollutant. Mucus normally protects the airways, but excessive production can obstruct airflow and cause respiratory distress. Vasaka tea pre- and co-treatment dose-dependently inhibited mucin 5AC (MUC5AC) mRNA induction by AECs treated with wood smoke particles. This correlated with transient receptor potential ankyrin-1 (TRPA1) inhibition, an attenuation of endoplasmic reticulum (ER) stress, and AEC damage/death. Induction of mRNA for anterior gradient 2, an ER chaperone/disulfide isomerase required for MUC5AC production, and TRP vanilloid-3, a gene that suppresses ER stress and wood smoke particle-induced cell death, was also attenuated. Variable inhibition of TRPA1, ER stress, and MUC5AC mRNA induction was observed using selected chemicals identified in Vasaka tea including vasicine, vasicinone, apigenin, vitexin, isovitexin, isoorientin, 9-oxoODE, and 9,10-EpOME. Apigenin and 9,10-EpOME were the most cytoprotective and mucosuppressive. Cytochrome P450 1A1 (CYP1A1) mRNA was also induced by Vasaka tea and wood smoke particles. Inhibition of CYP1A1 enhanced ER stress and MUC5AC mRNA expression, suggesting a possible role in producing protective oxylipins in stressed cells. The results provide mechanistic insights and support for the purported benefits of Vasaka tea in treating lung inflammatory conditions, raising the possibility of further development as a preventative and/or restorative therapy.

Introduction: BTK inhibition is an effective frontline therapy for patients with CLL. However, patients who develop BTK C481S mutations relapse quickly. We recently published a longitudinal study investigating subclonal evolution in 38 patients receiving BTKi treatment for CLL. Of the 38 patients, 6 developed a subclone containing a BTK C481S mutation identified in the whole-exome sequencing (WES) data at the time of relapse. To understand whether a BTK C481S mutation alone can drive relapse or if an additional CLL-relevant mutation is required, we investigate these subclones on a single-cell level using PacBio Multiplexed Arrays Sequencing (MAS-seq). MAS-seq generates single-cell, long-read RNA sequencing data, which is highly accurate and provides coverage across the entire mRNA transcript. Methods: For each patient, B cells were isolated from a pre-BTKi treatment sample and a sample taken at the time of relapse. Single-cell cDNA library preps were generated using the 10X Genomics Chromium platform. The cDNA was then sequenced using MAS-Seq. After processing the MAS-seq data using PacBio's Isoseq pipeline, Seurat was used to perform a single-cell analysis for each sample. We used scBayes, a Bayesian-statistical approach developed in our lab (Qiao et al., Genome Research, in review), to assign subclone identity to individual cells based on the expression of previously identified somatic mutations. The transcript-wide coverage of MAS-seq increases the likelihood of coverage at the somatic mutation sites compared to traditional single-cell RNA sequencing methods. Results: We have sequenced and analyzed samples from 4 patients in this study (pt 1-4). In the pretreatment sample of pt 1, two subclones containing different TP53 mutations were present. At the time of relapse, a BTK C481S subclone evolved from one of these TP53 subclones, becoming the dominant clonal population. No other CLL-relevant mutations were detected in this patient. Pts 2-4 had an additional CLL-relevant mutation evolve with the BTK C481S mutation in the dominant subclone at the time of relapse. In pt 2, a subclone with an NFKBIE and the BTK mutation evolved from a cell population where BRAF, BCOR, and EGFR2 mutations were present. The BTK C481S subclone in Pt 3 also had new KMT2D and CREBBP mutations. This subclone evolved from a cell population with an ASXL1 and SF3B1 mutation. In pts 1-3, the BTK subclone was only seen at the time of relapse. Pt 4 did not have any CLL-relevant mutations identified before treatment. In a sample taken 6 months before relapse, a BTK C481S subclone is visible at a low frequency with no other CLL-relevant mutations. At the time of relapse, a DICER1 variant, a gene recently implicated in CLL (Knisbacher et al., Nature Genetics, 2022), evolved from the BTK subclone and is seen at the time of relapse as the dominant subclone. Furthermore, the full-transcript sequencing at the single-cell level enabled us to identify a second BTK C481S subclone present at the time of relapse and determine that it was independent of the subclone containing both the BTK C481S and DICER1 mutations. This second BTK subclone had no other CLL-relevant mutations and remained at a very low frequency. Conclusion: Single-cell long-read RNA sequencing via MAS-seq provides transcript-wide coverage, which enables genotyping at somatic mutation sites. Using this method, we found that the dominant relapse clones all contained a BTK C481S mutation, which either co-evolved with additional CLL-relevant mutations (3/4 patients) or occurred on the background of existing TP53 mutations (1/4 patients).

Acute inflammatory exacerbations (AIE) represent precipitous deteriorations of a number of chronic lung conditions, including pulmonary fibrosis (PF), COPD, and asthma. AIEs are marked by diffuse and persistent polycellular alveolitis that profoundly accelerate lung function decline and mortality. Excess monocyte mobilization during AIE, and their persistence in the lung are linked to poor disease outcome. Guided by clinical evidence, we have developed an inducible model of pulmonary fibrosis leveraging the PF-linked missense isoleucine to threonine mutation at position 73 [I73T] in the alveolar type-2 cell-restricted Surfactant Protein-C [SP-C] gene [SFTPC]. We have previously shown that ablation of CCR2+ monocytes was protective with respect to lung histopathology, mouse survival, and overall inflammation following SP-C mutant induction. Here we identify and phenotypically characterize resident and recruited monocytes/macrophages populations intervening during the initiation and progression of AIE-PF. Single cell sequencing shows pro-inflammatory and pro-fibrotic signatures originating from immature recruited clusters (itgax-itgam+ ccr2+ cx3cr1+)as well as activated macrophages (cd68+ itgax+ itgam+ arg1+)14 d after SP-C I73Tinduction. Using CellChat Explorer database, these cells were predicted to coordinate intense tgfb1, fn1/fibronectinand spp1/osteopontinsignaling. Together, these results provide a clear picture of the fibrogenic role of specific activated monocyte/macrophage subsets responding to alveolar epithelial stress. NIEHS R01ES032553 (AV); ALSAM Foundation for Research Initiatives Grant (AV)