Spatial transcriptomics reveals segregation of tumor cell states in glioblastoma and marked immunosuppression within the perinecrotic niche.

Glioblastoma (GBM) remains an untreatable malignant tumor with poor patient outcomes, characterized by palisading necrosis and microvascular proliferation. While single-cell technology made it possible to characterize different lineage of glioma cells into neural progenitor-like (NPC-like), oligodendrocyte-progenitor-like (OPC-like), astrocyte-like (AC-like) and mesenchymal like (MES-like) states, it does not capture the spatial localization of these tumor cell states. Spatial transcriptomics empowers the study of the spatial organization of different cell types and tumor cell states and allows for the selection of regions of interest to investigate region-specific and cell-type-specific pathways. Here, we obtained paired 10x Chromium single-nuclei RNA-sequencing (snRNA-seq) and 10x Visium spatial transcriptomics data from three GBM patients to interrogate the GBM microenvironment. Integration of the snRNA-seq and spatial transcriptomics data reveals patterns of segregation of tumor cell states. For instance, OPC-like tumor and NPC-like tumor significantly segregate in two of the three samples. Our differentially expressed gene and pathway analyses uncovered significant pathways in functionally relevant niches. Specifically, perinecrotic regions were more immunosuppressive than the endogenous GBM microenvironment, and perivascular regions were more pro-inflammatory. Our gradient analysis suggests that OPC-like tumor cells tend to reside in areas closer to the tumor vasculature compared to tumor necrosis, which may reflect increased oxygen requirements for OPC-like cells. In summary, we characterized the localization of cell types and tumor cell states, the gene expression patterns, and pathways in different niches within the GBM microenvironment. Our results provide further evidence of the segregation of tumor cell states and highlight the immunosuppressive nature of the necrotic and perinecrotic niches in GBM.

Accelerated epigenetic age is associated with whole-brain functional connectivity and impaired cognitive performance in older adults.

While chronological age is a strong predictor for health-related risk factors, it is an incomplete metric that fails to fully characterize the unique aging process of individuals with different genetic makeup, neurodevelopment, and environmental experiences. Recent advances in epigenomic array technologies have made it possible to generate DNA methylation-based biomarkers of biological aging, which may be useful in predicting a myriad of cognitive abilities and functional brain network organization across older individuals. It is currently unclear which cognitive domains are negatively correlated with epigenetic age above and beyond chronological age, and it is unknown if functional brain organization is an important mechanism for explaining these associations. In this study, individuals with accelerated epigenetic age (i.e. AgeAccelGrim) performed worse on tasks that spanned a wide variety of cognitive faculties including both fluid and crystallized intelligence (N = 103, average age = 68.98 years, 73 females, 30 males). Additionally, fMRI connectome-based predictive models suggested a mediating mechanism of functional connectivity on epigenetic age acceleration-cognition associations primarily in medial temporal lobe and limbic structures. This research highlights the important role of epigenetic aging processes on the development and maintenance of healthy cognitive capacities and function of the aging brain.

Subventricular zone stem cell niche injury is associated with intestinal perforation in preterm infants and predicts future motor impairment.

Brain injury is highly associated with preterm birth. Complications of prematurity, including spontaneous or necrotizing enterocolitis (NEC)-associated intestinal perforations, are linked to lifelong neurologic impairment, yet the mechanisms are poorly understood. Early diagnosis of preterm brain injuries remains a significant challenge. Here, we identified subventricular zone echogenicity (SVE) on cranial ultrasound in preterm infants following intestinal perforations. The development of SVE was significantly associated with motor impairment at 2 years. SVE was replicated in a neonatal mouse model of intestinal perforation. Examination of the murine echogenic subventricular zone (SVZ) revealed NLRP3-inflammasome assembly in multiciliated FoxJ1+ ependymal cells and a loss of the ependymal border in this postnatal stem cell niche. These data suggest a mechanism of preterm brain injury localized to the SVZ that has not been adequately considered. Ultrasound detection of SVE may serve as an early biomarker for neurodevelopmental impairment after inflammatory disease in preterm infants.

Transcriptome Analysis Identifies An ASD-Like Phenotype In Oligodendrocytes And Microglia From C58/J Amygdala That Is Dependent On Sex and Sociability.

Background: Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with higher incidence in males and is characterized by atypical verbal/nonverbal communication, restricted interests that can be accompanied by repetitive behavior, and disturbances in social behavior. This study investigated brain mechanisms that contribute to sociability deficits and sex differences in an ASD animal model. Methods: Sociability was measured in C58/J and C57BL/6J mice using the 3-chamber social choice test. Bulk RNA-Seq and snRNA-Seq identified transcriptional changes in C58/J and C57BL/6J amygdala within which DMRseq was used to measure differentially methylated regions in amygdala. Results: C58/J mice displayed divergent social strata in the 3-chamber test. Transcriptional and pathway signatures revealed immune-related biological processes differ between C58/J and C57BL/6J amygdala. Hypermethylated and hypomethylated genes were identified in C58/J versus C57BL/6J amygdala. snRNA-Seq data in C58/J amygdala identified differential transcriptional signatures within oligodendrocytes and microglia characterized by increased ASD risk gene expression and predicted impaired myelination that was dependent on sex and sociability. RNA velocity, gene regulatory network, and cell communication analysis showed diminished oligodendrocyte/microglia differentiation. Findings were verified using bulk RNA-Seq and demonstrated oxytocin's beneficial effects on myelin gene expression. Limitations: Our findings are significant. However, limitations can be noted. The cellular mechanisms linking reduced oligodendrocyte differentiation and reduced myelination to an ASD phenotype in C58/J mice need further investigation. Additional snRNA-Seq and spatial studies would determine if effects in oligodendrocytes/microglia are unique to amygdala or if this occurs in other brain regions. Oxytocin's effects need further examination to understand its potential as an ASD therapeutic. Conclusions: Our work demonstrates the C58/J mouse model's utility in evaluating the influence of sex and sociability on the transcriptome in concomitant brain regions involved in ASD. Our single-nucleus transcriptome analysis elucidates potential pathological roles of oligodendrocytes and microglia in ASD. This investigation provides details regarding regulatory features disrupted in these cell types, including transcriptional gene dysregulation, aberrant cell differentiation, altered gene regulatory networks, and changes to key pathways that promote microglia/oligodendrocyte differentiation. Our studies provide insight into interactions between genetic risk and epigenetic processes associated with divergent affiliative behavior and lack of positive sociability.

UBE2N is essential for maintenance of skin homeostasis and suppression of inflammation.

UBE2N, a Lys63-ubiquitin conjugating enzyme, plays critical roles in embryogenesis and immune system development and function. However, its roles in adult epithelial tissue homeostasis and pathogenesis are unclear. We generated conditional mouse models that deleted Ube2n in skin cells in a temporally and spatially controlled manner. We found that Ube2n-knockout (KO) in the adult skin keratinocytes induced a range of inflammatory skin defects characteristic of psoriatic and actinic keratosis. These included eczematous inflammation, epidermal and dermal thickening, parakeratosis, and increased immune cell infiltration, as well as signs of edema and blistering. Single cell transcriptomic analyses and RT-qPCR showed that Ube2n KO keratinocytes expressed elevated myeloid cell chemo-attractants such as Cxcl1 and Cxcl2 and decreased the homeostatic T lymphocyte chemo-attractant, Ccl27a. Consistently, the infiltrating immune cells of Ube2n-KO skin were predominantly myeloid-derived cells including neutrophils and M1-like macrophages that were highly inflammatory, as indicated by expression of Il1ß and Il24. Pharmacological blockade of the IL-1 receptor associated kinases (IRAK1/4) alleviated eczema, epidermal and dermal thickening, and immune infiltration of the Ube2n mutant skin. Together, these findings highlight a key role of keratinocyte-UBE2N in maintenance of epidermal homeostasis and skin immunity and identify IRAK1/4 as potential therapeutic target for inflammatory skin disorders.

Ataxia-telangiectasia mutated ( Atm ) disruption sensitizes spatially-directed H3.3K27M/TP53 diffuse midline gliomas to radiation therapy.

Diffuse midline gliomas (DMGs) are lethal brain tumors characterized by p53-inactivating mutations and oncohistone H3.3K27M mutations that rewire the cellular response to genotoxic stress, which presents therapeutic opportunities. We used RCAS/tv-a retroviruses and Cre recombinase to inactivate p53 and induce K27M in the native H3f3a allele in a lineage- and spatially-directed manner, yielding primary mouse DMGs. Genetic or pharmacologic disruption of the DNA damage response kinase Ataxia-telangiectasia mutated (ATM) enhanced the efficacy of focal brain irradiation, extending mouse survival. This finding suggests that targeting ATM will enhance the efficacy of radiation therapy for p53-mutant DMG but not p53-wildtype DMG. We used spatial in situ transcriptomics and an allelic series of primary murine DMG models with different p53 mutations to identify transactivation-independent p53 activity as a key mediator of such radiosensitivity. These studies deeply profile a genetically faithful and versatile model of a lethal brain tumor to identify resistance mechanisms for a therapeutic strategy currently in clinical trials.

20-αHydroxycholesterol, an oxysterol in human breast milk, reverses mouse neonatal white matter injury through Gli-dependent oligodendrogenesis.

White matter injuries (WMIs) are the leading cause of neurologic impairment in infants born premature. There are no treatment options available. The most common forms of WMIs in infants occur prior to the onset of normal myelination, making its pathophysiology distinctive, thus requiring a tailored approach to treatment. Neonates present a unique opportunity to repair WMIs due to a transient abundance of neural stem/progenitor cells (NSPCs) present in the germinal matrix with oligodendrogenic potential. We identified an endogenous oxysterol, 20-αHydroxycholesterol (20HC), in human maternal breast milk that induces oligodendrogenesis through a sonic hedgehog (shh), Gli-dependent mechanism. Following WMI in neonatal mice, injection of 20HC induced subventricular zone-derived oligodendrogenesis and improved myelination in the periventricular white matter, resulting in improved motor outcomes. Targeting the oligodendrogenic potential of postnatal NSPCs in neonates with WMIs may be further developed into a novel approach to mitigate this devastating complication of preterm birth.

The RNA helicase DDX39B activates FOXP3 RNA splicing to control T regulatory cell fate.

Genes associated with increased susceptibility to multiple sclerosis (MS) have been identified, but their functions are incompletely understood. One of these genes codes for the RNA helicase DExD/H-Box Polypeptide 39B (DDX39B), which shows genetic and functional epistasis with interleukin-7 receptor-α gene (IL7R) in MS-risk. Based on evolutionary and functional arguments, we postulated that DDX39B enhances immune tolerance thereby decreasing MS risk. Consistent with such a role we show that DDX39B controls the expression of many MS susceptibility genes and important immune-related genes. Among these we identified Forkhead Box P3 (FOXP3), which codes for the master transcriptional factor in CD4+/CD25+ T regulatory cells. DDX39B knockdown led to loss of immune-regulatory and gain of immune-effector expression signatures. Splicing of FOXP3 introns, which belong to a previously unrecognized type of introns with C-rich polypyrimidine tracts, was exquisitely sensitive to DDX39B levels. Given the importance of FOXP3 in autoimmunity, this work cements DDX39B as an important guardian of immune tolerance.

Ganglioglioma deep transcriptomics reveals primitive neuroectoderm neural precursor-like population.

Gangliogliomas are brain tumors composed of neuron-like and macroglia-like components that occur in children and young adults. Gangliogliomas are often characterized by a rare population of immature astrocyte-appearing cells expressing CD34, a marker expressed in the neuroectoderm (neural precursor cells) during embryogenesis. New insights are needed to refine tumor classification and to identify therapeutic approaches. We evaluated five gangliogliomas with single nucleus RNA-seq, cellular indexing of transcriptomes and epitopes by sequencing, and/or spatially-resolved RNA-seq. We uncovered a population of CD34+ neoplastic cells with mixed neuroectodermal, immature astrocyte, and neuronal markers. Gene regulatory network interrogation in these neuroectoderm-like cells revealed control of transcriptional programming by TCF7L2/MEIS1-PAX6 and SOX2, similar to that found during neuroectodermal/neural development. Developmental trajectory analyses place neuroectoderm-like tumor cells as precursor cells that give rise to neuron-like and macroglia-like neoplastic cells. Spatially-resolved transcriptomics revealed a neuroectoderm-like tumor cell niche with relative lack of vascular and immune cells. We used these high resolution results to deconvolute clinically-annotated transcriptomic data, confirming that CD34+ cell-associated gene programs associate with gangliogliomas compared to other glial brain tumors. Together, these deep transcriptomic approaches characterized a ganglioglioma cellular hierarchy-confirming CD34+ neuroectoderm-like tumor precursor cells, controlling transcription programs, cell signaling, and associated immune cell states. These findings may guide tumor classification, diagnosis, prognostication, and therapeutic investigations.

JAK-STAT activation contributes to cytotoxic T cell-mediated basal cell death in human chronic lung allograft dysfunction.

Chronic lung allograft dysfunction (CLAD) is the leading cause of death in lung transplant recipients. CLAD is characterized clinically by a persistent decline in pulmonary function and histologically by the development of airway-centered fibrosis known as bronchiolitis obliterans. There are no approved therapies to treat CLAD, and the mechanisms underlying its development remain poorly understood. We performed single-cell RNA-Seq and spatial transcriptomic analysis of explanted tissues from human lung recipients with CLAD, and we performed independent validation studies to identify an important role of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling in airway epithelial cells that contributes to airway-specific alloimmune injury. Specifically, we established that activation of JAK-STAT signaling leads to upregulation of major histocompatibility complex 1 (MHC-I) in airway basal cells, an important airway epithelial progenitor population, which leads to cytotoxic T cell-mediated basal cell death. This study provides mechanistic insight into the cell-to-cell interactions driving airway-centric alloimmune injury in CLAD, suggesting a potentially novel therapeutic strategy for CLAD prevention or treatment.

Aging and obesity prime the methylome and transcriptome of adipose stem cells for disease and dysfunction.

The epigenome of stem cells occupies a critical interface between genes and environment, serving to regulate expression through modification by intrinsic and extrinsic factors. We hypothesized that aging and obesity, which represent major risk factors for a variety of diseases, synergistically modify the epigenome of adult adipose stem cells (ASCs). Using integrated RNA- and targeted bisulfite-sequencing in murine ASCs from lean and obese mice at 5- and 12-months of age, we identified global DNA hypomethylation with either aging or obesity, and a synergistic effect of aging combined with obesity. The transcriptome of ASCs in lean mice was relatively stable to the effects of age, but this was not true in obese mice. Functional pathway analyses identified a subset of genes with critical roles in progenitors and in diseases of obesity and aging. Specifically, Mapt, Nr3c2, App, and Ctnnb1 emerged as potential hypomethylated upstream regulators in both aging and obesity (AL vs. YL and AO vs. YO), and App, Ctnnb1, Hipk2, Id2, and Tp53 exhibited additional effects of aging in obese animals. Furthermore, Foxo3 and Ccnd1 were potential hypermethylated upstream regulators of healthy aging (AL vs. YL), and of the effects of obesity in young animals (YO vs. YL), suggesting that these factors could play a role in accelerated aging with obesity. Finally, we identified candidate driver genes that appeared recurrently in all analyses and comparisons undertaken. Further mechanistic studies are needed to validate the roles of these genes capable of priming ASCs for dysfunction in aging- and obesity-associated pathologies.

Genetic and epigenetic signatures associated with plasma oxytocin levels in children and adolescents with autism spectrum disorder.

Oxytocin (OT), the brain's most abundant neuropeptide, plays an important role in social salience and motivation. Clinical trials of the efficacy of OT in autism spectrum disorder (ASD) have reported mixed results due in part to ASD's complex etiology. We investigated whether genetic and epigenetic variation contribute to variable endogenous OT levels that modulate sensitivity to OT therapy. To carry out this analysis, we integrated genome-wide profiles of DNA-methylation, transcriptional activity, and genetic variation with plasma OT levels in 290 participants with ASD enrolled in a randomized controlled trial of OT. Our analysis identified genetic variants with novel association with plasma OT, several of which reside in known ASD risk genes. We also show subtle but statistically significant association of plasma OT levels with peripheral transcriptional activity and DNA-methylation profiles across several annotated gene sets. These findings broaden our understanding of the effects of the peripheral oxytocin system and provide novel genetic candidates for future studies to decode the complex etiology of ASD and its interaction with OT signaling and OT-based interventions. LAY SUMMARY: Oxytocin (OT) is an abundant chemical produced by neurons that plays an important role in social interaction and motivation. We investigated whether genetic and epigenetic factors contribute to variable OT levels in the blood. To this, we integrated genetic, gene expression, and non-DNA regulated (epigenetic) signatures with blood OT levels in 290 participants with autism enrolled in an OT clinical trial. We identified genetic association with plasma OT, several of which reside in known autism risk genes. We also show statistically significant association of plasma OT levels with gene expression and epigenetic across several gene pathways. These findings broaden our understanding of the factors that influence OT levels in the blood for future studies to decode the complex presentation of autism and its interaction with OT and OT-based treatment.

The effect of oxytocin nasal spray on social interaction in young children with autism: a randomized clinical trial.

Early supports to enhance social development in children with autism are widely promoted. While oxytocin has a crucial role in mammalian social development, its potential role as a medication to enhance social development in humans remains unclear. We investigated the efficacy, tolerability, and safety of intranasal oxytocin in young children with autism using a double-blind, randomized, placebo-controlled, clinical trial, following a placebo lead-in phase. A total of 87 children (aged between 3 and 12 years) with autism received 16 International Units (IU) of oxytocin (n = 45) or placebo (n = 42) nasal spray, morning and night (32 IU per day) for twelve weeks, following a 3-week placebo lead-in phase. Overall, there was no effect of oxytocin treatment over time on the caregiver-rated Social Responsiveness Scale (SRS-2) (p = 0.686). However, a significant interaction with age (p = 0.028) showed that for younger children, aged 3-5 years, there was some indication of a treatment effect. Younger children who received oxytocin showed improvement on caregiver-rated social responsiveness ( SRS-2). There was no other evidence of benefit in the sample as a whole, or in the younger age group, on the clinician-rated Clinical Global Improvement Scale (CGI-S), or any secondary measure. Importantly, placebo effects in the lead-in phase were evident and there was support for washout of the placebo response in the randomised phase. Oxytocin was well tolerated, with more adverse side effects reported in the placebo group. This study suggests the need for further clinical trials to test the benefits of oxytocin treatment in younger populations with autism.Trial registration www.anzctr.org.au (ACTRN12617000441314).

Adolescent peer struggles predict accelerated epigenetic aging in midlife.

This study examined struggles to establish autonomy and relatedness with peers in adolescence and early adulthood as predictors of advanced epigenetic aging assessed at age 30. Participants (N = 154; 67 male and 87 female) were observed repeatedly, along with close friends and romantic partners, from ages 13 through 29. Observed difficulty establishing close friendships characterized by mutual autonomy and relatedness from ages 13 to 18, an interview-assessed attachment state of mind lacking autonomy and valuing of attachment at 24, and self-reported difficulties in social integration across adolescence and adulthood were all linked to greater epigenetic age at 30, after accounting for chronological age, gender, race, and income. Analyses assessing the unique and combined effects of these factors, along with lifetime history of cigarette smoking, indicated that each of these factors, except for adult social integration, contributed uniquely to explaining epigenetic age acceleration. Results are interpreted as evidence that the adolescent preoccupation with peer relationships may be highly functional given the relevance of such relationships to long-term physical outcomes.

Single-cell genome-wide association reveals that a nonsynonymous variant in ERAP1 confers increased susceptibility to influenza virus.

During pandemics, individuals exhibit differences in risk and clinical outcomes. Here, we developed single-cell high-throughput human in vitro susceptibility testing (scHi-HOST), a method for rapidly identifying genetic variants that confer resistance and susceptibility. We applied this method to influenza A virus (IAV), the cause of four pandemics since the start of the 20th century. scHi-HOST leverages single-cell RNA sequencing (scRNA-seq) to simultaneously assign genetic identity to cells in mixed infections of cell lines of European, African, and Asian origin, reveal associated genetic variants for viral burden, and identify expression quantitative trait loci. Integration of scHi-HOST with human challenge and experimental validation demonstrated that a missense variant in endoplasmic reticulum aminopeptidase 1 (ERAP1; rs27895) increased IAV burden in cells and human volunteers. rs27895 exhibits population differentiation, likely contributing to greater permissivity of cells from African populations to IAV. scHi-HOST is a broadly applicable method and resource for decoding infectious-disease genetics.

Sex-dimorphic gene effects on survival outcomes in people with coronary artery disease.

Background: Ischemic coronary heart disease (IHD) is the leading cause of death worldwide. Genetic variation is presumed to be a major factor underlying sex differences for IHD events, including mortality. The purpose of this study was to identify sex-specific candidate genes associated with all-cause mortality among people diagnosed with coronary artery disease (CAD). Methods: We performed a sex-stratified, exploratory genome-wide association (GWAS) screen using existing data from CAD-diagnosed males (n = 510) and females (n = 174) who reported European ancestry from the Duke Catheterization Genetics biorepository. Extant genotype data for 785,945 autosomal SNPs generated with the Human Omni1-Quad BeadChip (Illumina, CA, USA) were analyzed using an additive inheritance model. We estimated instantaneous risk of all-cause mortality by genotype groups across the 11-year follow-up using Cox multivariate regression, covarying for age and genomic ancestry. Results: The top GWAS hits associated with all-cause mortality among people with CAD included 8 SNPs among males and 15 among females (p = 1 × 10-6 or 10-7), adjusted for covariates. Cross-sex comparisons revealed distinct candidate genes. Biologically relevant candidates included rs9932462 (EMP2/TEKT5) and rs2835913 (KCNJ6) among males and rs7217169 (RAP1GAP2), rs8021816 (PRKD1), rs8133010 (PDE9A), and rs12145981 (LPGAT1) among females. Conclusions: We report 20 sex-specific candidate genes having suggestive association with all-cause mortality among CAD-diagnosed subjects. Findings demonstrate proof of principle for identifying sex-associated genetic factors that may help explain differential mortality risk in people with CAD. Replication and meta-analyses in larger studies with more diverse samples will strengthen future work in this area.

Resistance of mitochondrial DNA to cadmium and Aflatoxin B1 damage-induced germline mutation accumulation in C. elegans.

Mitochondrial DNA (mtDNA) is prone to mutation in aging and over evolutionary time, yet the processes that regulate the accumulation of de novo mtDNA mutations and modulate mtDNA heteroplasmy are not fully elucidated. Mitochondria lack certain DNA repair processes, which could contribute to polymerase error-induced mutations and increase susceptibility to chemical-induced mtDNA mutagenesis. We conducted error-corrected, ultra-sensitive Duplex Sequencing to investigate the effects of two known nuclear genome mutagens, cadmium and Aflatoxin B1, on germline mtDNA mutagenesis in Caenorhabditis elegans. Detection of thousands of mtDNA mutations revealed pervasive heteroplasmy in C. elegans and that mtDNA mutagenesis is dominated by C:G → A:T mutations generally attributed to oxidative damage. However, there was no effect of either exposure on mtDNA mutation frequency, spectrum, or trinucleotide context signature despite a significant increase in nuclear mutation rate after aflatoxin B1 exposure. Mitophagy-deficient mutants pink-1 and dct-1 accumulated significantly higher levels of mtDNA damage compared to wild-type C. elegans after exposures. However, there were only small differences in mtDNA mutation frequency, spectrum, or trinucleotide context signature compared to wild-type after 3050 generations, across all treatments. These findings suggest mitochondria harbor additional previously uncharacterized mechanisms that regulate mtDNA mutational processes across generations.

Single Cell RNA-Seq Analysis of Human Red Cells.

Human red blood cells (RBCs), or erythrocytes, are the most abundant blood cells responsible for gas exchange. RBC diseases affect hundreds of millions of people and impose enormous financial and personal burdens. One well-recognized, but poorly understood feature of RBC populations within the same individual are their phenotypic heterogeneity. The granular characterization of phenotypic RBC variation in normative and disease states may allow us to identify the genetic determinants of red cell diseases and reveal novel therapeutic approaches for their treatment. Previously, we discovered diverse RNA transcripts in RBCs that has allowed us to dissect the phenotypic heterogeneity and malaria resistance of sickle red cells. However, these analyses failed to capture the heterogeneity found in RBC sub-populations. To overcome this limitation, we have performed single cell RNA-Seq to analyze the transcriptional heterogeneity of RBCs from three adult healthy donors which have been stored in the blood bank conditions and assayed at day 1 and day 15. The expression pattern clearly separated RBCs into seven distinct clusters that include one RBC cluster that expresses HBG2 and a small population of RBCs that express fetal hemoglobin (HbF) that we annotated as F cells. Almost all HBG2-expessing cells also express HBB, suggesting bi-allelic expression in single RBC from the HBG2/HBB loci, and we annotated another cluster as reticulocytes based on canonical gene expression. Additional RBC clusters were also annotated based on the enriched expression of NIX, ACVR2B and HEMGN, previously shown to be involved in erythropoiesis. Finally, we found the storage of RBC was associated with an increase in the ACVR2B and F-cell clusters. Collectively, these data indicate the power of single RBC RNA-Seq to capture and discover known and unexpected heterogeneity of RBC population.

Lifetime marijuana use and epigenetic age acceleration: A 17-year prospective examination.

AIMS: This study was designed to assess links between lifetime levels of marijuana use and accelerated epigenetic aging. DESIGN: Prospective longitudinal study, following participants annually from age 13 to age 30. SETTING AND PARTICIPANTS: A community sample of 154 participants recruited from a small city in the Southeastern United States. MEASUREMENTS: Participants completed annual assessments of marijuana use from age 13 to age 29 and provided blood samples that yielded two indices of epigenetic aging (DNAmGrimAge and DunedinPoAm) at age 30. Additional covariates examined included history of cigarette smoking, anxiety and depressive symptoms, childhood illness, gender, adolescent-era family income, and racial/ethnic minority status. FINDINGS: Lifetime marijuana use predicted accelerated epigenetic aging, with effects remaining even after covarying cell counts, demographic factors and chronological age (ß's = 0.32 & 0.27, p's < 0.001, 95% CI's = 0.21-0.43 & 0.16-0.39 for DNAmGrimAge and DunedinPoAm, respectively). Predictions remained after accounting for cigarette smoking (ß's = 0.25 & 0.21, respectively, p's < 0.001, 95% CI's = 0.14-0.37 & 0.09-0.32 for DNAmGrimAge and DunedinPoAm, respectively). A dose-response effect was observed and there was also evidence that effects were dependent upon recency of use. Effects of marijuana use appeared to be fully mediated by hypomethylation of a site linked to effects of hydrocarbon inhalation (cg05575921). CONCLUSIONS: Marijuana use predicted epigenetic changes linked to accelerated aging, with evidence suggesting that effects may be primarily due to hydrocarbon inhalation among marijuana smokers. Further research is warranted to explore mechanisms underlying this linkage.