Distinct Gene Expression Signatures Research Articles

The cortical amygdala consolidates a socially transmitted long-term memory.

Social communication guides decision-making, which is essential for survival. Social transmission of food preference (STFP) is an ecologically relevant memory paradigm in which an animal learns a desirable food odour from another animal in a social context, creating a long-term memory1,2. How food-preference memory is acquired, consolidated and stored is unclear. Here we show that the posteromedial nucleus of the cortical amygdala (COApm) serves as a computational centre in long-term STFP memory consolidation by integrating social and sensory olfactory inputs. Blocking synaptic signalling by the COApm-based circuit selectively abolished STFP memory consolidation without impairing memory acquisition, storage or recall. COApm-mediated STFP memory consolidation depends on synaptic inputs from the accessory olfactory bulb and on synaptic outputs to the anterior olfactory nucleus. STFP memory consolidation requires protein synthesis, suggesting a gene-expression mechanism. Deep single-cell and spatially resolved transcriptomics revealed robust but distinct gene-expression signatures induced by STFP memory formation in the COApm that are consistent with synapse restructuring. Our data thus define a neural circuit for the consolidation of a socially communicated long-term memory, thereby mechanistically distinguishing protein-synthesis-dependent memory consolidation from memory acquisition, storage or retrieval.

METB-01. EFFECTS OF H3.3G34V MUTATION ON GENOMIC H3K36 AND H3K27 METHYLATION PATTERNS IN ISOGENIC PEDIATRIC GLIOMA CELLS

Abstract BACKGROUND Histone H3.3 mutation (H3F3A) occurs in 50% of cortical pediatric high-grade gliomas (pHGGs). This mutation replaces glycine 34 with arginine or valine (G34R/V), impairing SETD2 activity (H3K36-specific trimethyltransferase). Consequently, reduced H3K36me3 is observed on H3.3G34V nucleosomes relative to wild-type, contributing to genomic instability and driving a distinct gene expression signature associated with tumorigenesis. However, it is not known if this differential H3K36me3 enrichment is due to H3.3G34V mutant protein alone. Therefore, we set to elucidate the effect of H3.3G34V mutant protein in pediatric glioma on H3K36me3, H3K27me3 and H3.3 enrichment in vitro. METHODS Experiments were conducted using an established H3.3G34V mutant patient-derived pediatric high-grade glioma cell line (KNS42), and Histone H3.3 wild-type human astrocytes (NHAs). Lentiviral delivery of a doxycycline-inducible RNAi vector targeted against H3F3A was transduced to KNS42 cells to knock down H3.3G34V protein expression. Cell lines were then compared via ChIP-Seq, Western Blot, proliferation and viability studies. RESULTS We found that the doxycycline-inducible shRNA knockdown of mutant H3F3A encoding the H3.3G34V protein resulted in loss of H3.3G34V enrichment and increased H3K36me3 enrichment throughout the genome. After knockdown, H3.3G34V enrichment was preserved at loci observed to have the greatest H3.3G34V and H3K36me3 enrichment prior to knockdown. Induced expression of mutant H3.3G34V protein in vitro was insufficient to induce genomic H3K36me3 enrichment patterns observed in H3.3G34V mutant glioma cells. We also observed strong co-enrichment of H3.3G34V and wild-type H3.3 protein, as well as greater H3K27me3 enrichment, in cells expressing H3.3G34V. CONCLUSIONS Our study demonstrates the effects of H3.3G34V mutant protein on genomic H3K36me3, H3K27me3 and H3.3 enrichment patterns in isogenic cell lines. Further studies examining the impact of theses findings are therefore underway.

Right ventricular outflow tract cardiomyocytes possess distinct calcium handling and contractile properties

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): Rembrandt Research Grant 2021 Background The right ventricular outflow tract (RVOT) is the predominant site of origin of idiopathic ventricular tachycardia. Idiopathic ventricular tachycardias are typically sensitive to adenosine, consistent with triggered activity caused by delayed afterdepolarizations due to intracellular Ca2+ overload. The distinct properties of RVOT myocytes explaining increased susceptibility to Ca2+ overload and triggered activity are unknown. Purpose To identify distinct RVOT electrophysiological properties including Ca2+ handling and its molecular underpinnings. Methods and Results Experiments were performed using freshly isolated cardiomyocytes from the RVOT and right ventricular (RV) apex of adult rabbit hearts. To identify distinct RVOT electrophysiological properties, we employed the perforated patch clamp methodology and measured action potentials and L-type Ca2+ current. Action potentials of RVOT myocytes were significantly shorter compared to RV myocytes, particularly at lower pacing frequency. L-type Ca2+ current peak density was significantly lower in RVOT myocytes compared to RV myocytes (34.0 ± 9.1% at 0 mV). However, voltage-dependencies of L-type Ca2+ current activation and inactivation were not statistically different. Next we measured intracellular Ca2+ transients using the fluorescent Ca2+ indicator indo-1 and found that Ca2+ transient amplitudes were significantly lower in RVOT myocytes compared to RV myocytes. To investigate a relationship between RVOT myocyte Ca2+ handling and contractility, we performed unloaded sarcomere shortening measurements using the Cytocypher Multicell system. Shortening of relaxation time following increased pacing frequency (1-2 Hz) was significantly larger for RVOT myocytes compared to RV myocytes. To identify molecular factors explaining the distinct properties of RVOT myocytes, we performed RNA-seq analysis of differential gene expression using the DESeq2 method. RVOT and RV transcriptomic profiles consisted of 30 differentially expressed genes (adjusted p ≤ 0.05). One of genes significantly higher expressed by the RV was synaptotagmin (SYT1), a Ca2+ sensor and positive regulator of the cardiac L-type Ca2+ channel through inhibition of syntaxin 1A [Ref. 1, 2]. Conclusion RVOT cardiomyocytes possess distinct electrophysiological and contractile properties, reflected by shorter action potentials, lower Ca2+ transient amplitudes, and larger shortening of relaxation time upon increasing pacing frequency. The RVOT and RV have similar but distinct gene expression signatures. Future experiments will focus on determining the role of SYT1 and other genes in regulation of the L-type Ca2+ current and Ca2+ handling in RVOT myocytes.Distinct properties of RVOT myocytes.Transcriptomic profile of the RVOT.

Whole-blood transcriptome analysis reveals distinct gene expression signatures in paediatric patients with sickle cell anaemia before and after exercise.

Sickle cell anaemia (SCA) patients display elevated levels of circulating pro-inflammatory cytokines and endothelial activation markers compared to healthy peers. The impact of exercise on the pro-inflammatory state in SCA remains unclear. This study aimed to characterize the whole-blood transcriptome profile in response to an acute bout of exercise in paediatric SCA patients. Twenty-three SCA participants (13 ± 3 years, 52% girls) and 17 healthy controls (14 ± 3 years, 29% girls) performed eight 2-min bouts of cycle ergometry interspersed with 1-min rest intervals. Whole-blood transcriptome profile (RNA-seq) was performed before and after exercise. At baseline, gene pathways associated with gas transport in erythrocytes were up-regulated in SCA patients compared to controls. Following exercise, gene pathways associated with innate immunity were altered in both groups. Interaction analyses revealed 160 annotated genes (101 up- and 59 down-regulated) that differentially altered by exercise in SCA patients. Moreover, genes that exhibited a blunted response to exercise in SCA patients were enriched in the IL-17 signalling pathway, suggesting an impaired innate immune response to exercise. This data will contribute to the development of evidence-based exercise prescription guidelines for this patient population.

Single-Cell Transcriptomic Profiling Unveils Dynamic Immune Cell Responses during Haemonchus contortus Infection.

Haemonchus contortus is a parasite widely distributed in tropical, subtropical, and warm temperate regions, causing significant economic losses in the livestock industry worldwide. However, little is known about the genetics of H. contortus resistance in livestock. In this study, we monitor the dynamic immune cell responses in diverse peripheral blood mononuclear cells (PBMCs) during H. contortus infection in goats through single-cell RNA sequencing (scRNA-Seq) analysis. A total of four Boer goats, two goats with oral infection with the L3 larvae of H. contortus and two healthy goats as controls, were used in the animal test. The infection model in goats was established and validated by the fecal egg count (FEC) test and qPCR analysis of the gene expression of IL-5 and IL-6. Using scRNA-Seq, we identified seven cell types, including T cells, monocytes, natural killer cells, B cells, and dendritic cells with distinct gene expression signatures. After identifying cell subpopulations of differentially expressed genes (DEGs) in the case and control groups, we observed the upregulation of multiple inflammation-associated genes, including NFKBIA and NFKBID. Kyoto Encyclopedia of the Genome (KEGG) enrichment analysis revealed significant enrichment of NOD-like receptor pathways and Th1/Th2 cell differentiation signaling pathways in CD4 T cells DEGs. Furthermore, the analysis of ligand-receptor interaction networks showed a more active state of cellular communication in the PBMCs from the case group, and the inflammatory response associated MIF-(CD74 + CXCR4) ligand receptor complex was significantly more activated in the case group, suggesting a potential inflammatory response. Our study preliminarily revealed transcriptomic profiling characterizing the cell type specific mechanisms in host PBMCs at the single-cell level during H. contortus infection.

Comprehensive meta-analysis reveals distinct gene expression signatures of MASLD progression.

Metabolic dysfunction-associated steatotic liver disease (MASLD) and its progressive form, metabolic dysfunction-associated steatohepatitis (MASH), pose significant risks of severe fibrosis, cirrhosis, and hepatocellular carcinoma. Despite their widespread prevalence, the molecular mechanisms underlying the development and progression of these common chronic hepatic conditions are not fully understood. Here, we conducted the most extensive meta-analysis of hepatic gene expression datasets from liver biopsy samples to date, integrating 10 RNA-sequencing and microarray datasets (1,058 samples). Using a random-effects meta-analysis model, we compared over 12,000 shared genes across datasets. We identified 685 genes differentially expressed in MASLD versus normal liver, 1,870 in MASH versus normal liver, and 3,284 in MASLD versus MASH. Integrating these results with genome-wide association studies and coexpression networks, we identified two functionally relevant, validated coexpression modules mainly driven by SMOC2, ITGBL1, LOXL1, MGP, SOD3, and TAT, HGD, SLC25A15, respectively, the latter not previously associated with MASLD and MASH. Our findings provide a comprehensive and robust analysis of hepatic gene expression alterations associated with MASLD and MASH and identify novel key drivers of MASLD progression.

A single-cell atlas enables mapping of homeostatic cellular shifts in the adult human breast

Here we use single-cell RNA sequencing to compile a human breast cell atlas assembled from 55 donors that had undergone reduction mammoplasties or risk reduction mastectomies. From more than 800,000 cells we identified 41 cell subclusters across the epithelial, immune and stromal compartments. The contribution of these different clusters varied according to the natural history of the tissue. Age, parity and germline mutations, known to modulate the risk of developing breast cancer, affected the homeostatic cellular state of the breast in different ways. We found that immune cells from BRCA1 or BRCA2 carriers had a distinct gene expression signature indicative of potential immune exhaustion, which was validated by immunohistochemistry. This suggests that immune-escape mechanisms could manifest in non-cancerous tissues very early during tumor initiation. This atlas is a rich resource that can be used to inform novel approaches for early detection and prevention of breast cancer.

Abstract 1998: ABCB1 promotes uveal melanoma cell resistance to FR900359 and identifies a tumor cell subpopulation with a distinct gene expression signature

Abstract Uveal melanoma (UM) is the leading form of intraocular cancer in adults and the second most common form of melanoma. Although rare, UM has a median survival rate of ~1 year after metastasis occurs. Metastatic UM is largely refractory to treatment and there are no effective pharmacological therapies, resulting in poor overall survival. In >90% UM cases, the oncogenic initiator proteins are constitutively active mutant GNAQ or GNA11 proteins (GNAQ/11). FR900359 (FR) is a natural compound that inhibits GNAQ/11 and has anti-proliferative effects on UM cells. To identify key oncogenic mechanisms downstream of mutant GNAQ/11, we performed a Sleeping Beauty (SB) transposon forward genetic screen to identify drivers of FR resistance. The SB screen identified ATP-binding cassette sub-family B member 1 (ABCB1) as one of the top genes upregulated by SB in FR-resistant UM cell populations. We determined that although ABCB1 does not provide immediate resistance to FR, the overexpression of ABCB1 leads to emergence of resistant colonies at a much higher rate than spontaneous resistance, validating ABCB1 as contributors to FR resistance. Unexpectedly, immunofluorescence revealed that a relatively small fraction of UM cells strongly expresses the transgenes after stable transfection. Immunoblotting confirmed that ABCB1 expression was initially modest in ABCB1-transfected populations but increased in the cells that developed resistance to FR. To examine the kinetics of FR resistance, population doubling versus time (PDVT) assays were performed in UM cells expressing ABCB1 or empty vector control treated with FR or vehicle. FR potently suppressed the proliferation of vector control cells. Drug-naïve ABCB1-transfected cells developed resistance to FR over time but accomplished fewer population doublings than vehicle-treated cells in the same time interval. Once transgenic populations emerged as resistant populations in FR, they grew similarly in drug or vehicle. Ongoing RNA-sequencing analysis from PDVT samples has identified distinct gene expression signatures associated with FR treatment and ABCB1 expression. These findings are expected to illuminate tumor cell heterogeneity amongst UM cell populations that may be related to drug resistance and metastasis. Citation Format: Sarina Dion Murray, Jesse D. Riordan, Eric R. Anderson, Michael D. Onken, Kendall J. Blumer, Adam J. Dupuy, Christopher S. Stipp. ABCB1 promotes uveal melanoma cell resistance to FR900359 and identifies a tumor cell subpopulation with a distinct gene expression signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1998.

Abstract 5599: Role of HOX gene expression in differentiation of colon cancer stem cells

Abstract HOX genes encode a highly conserved set of transcription factors critical for stem cell (SC) function and embryonic development. Several HOX genes have also been implicated in a myriad of cancers, including colorectal cancer (CRC). However, how HOX genes play a role in SC regulation and contributes to cancer development is unclear. HOX expression is regulated by WNT, FGF, and retinoic acid (RA) signaling - pathways which also become dysregulated in cancer. Goal: To determine how regulation of HOX genes specifies differentiation of colonic SCs into specialized cell lineages and how dysregulation of HOX genes contributes to CRC development. Hypotheses: i) Each of the different cell types within human colonic epithelium has a distinct HOX gene expression signature; ii) All-trans retinoic acid (ATRA) induced differentiation of CRC cells changes HOX expression that correlates with specific HOX expression signatures in normal colon and CRC cells. Results: Immunostaining shows that specific HOX proteins (e.g., HOXA9 & HOXC9) are selectively expressed in ALDH-positive SCs and expression of these HOX genes is increased in CRC tissues compared to normal epithelium. Bioinformatics analysis predicted that these HOX genes (plus HOXA4, HOXA10, HOXC8) are regulated by RA signaling. Accordingly, we studied how ATRA influences HOX expression in CRC cells (HT29 & SW480). We found ATRA treatment: 1) Decreases proliferation and increases neuroendocrine cell (NEC) differentiation; and 2). Leads to specific changes in HOX gene expression patterns. Using NanoString profiling, we also discovered that LGR5+ SCs, ALDH+ SCs and GLP2R+ NECs have unique HOX signatures. By defining how these HOX gene expression changes in response to ATRA treatment in comparison to the HOX gene signatures of different colonic cell lineages will help us understand the role that HOX genes play in SC differentiation and how dysregulation of HOX gene expression contributes to CRC development. Citation Format: Brian T. Osmond, Caroline O. Facey, Lynn O. Opdenaker, Chi Zhang, Bruce M. Boman. Role of HOX gene expression in differentiation of colon cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5599.

Abstract 2139: Identifying molecular mechanisms in triple negative breast cancer disparities: Unveiling the role of Hedgehog signaling in non-Hispanic Black women

Abstract Triple negative breast cancer (TNBC) represents an aggressive and therapeutically challenging breast cancer subtype with higher incidence and mortality rates in non-Hispanic Black (NHB) women. This disparity persists even after adjusting for socioeconomic factors, tumor biology, and quality of care. The study aims to unravel specific molecular mechanisms underlying this disparity, with a focus on Hedgehog signaling pathways in premenopausal NHB women. Understanding these mechanisms is vital for developing targeted therapies and personalized medicine approaches to address these health disparities. Our research utilized a unique cohort of NHB and non-Hispanic White (NHW) women who underwent breast reduction surgery at the Mayo Clinic and donated breast tissue samples for our study. We isolated RNA from whole tissue sections from a cohort of 12 NHB and 40 NHW women who were parous and premenopausal at time of surgery. We performed gene expression profiling using NanoString BC360 and IO360 assays. This allowed us to identify a distinct gene expression signature characteristic of basal-like/TNBC and indicative of heightened activity in the Hedgehog signaling pathway. We also employed advanced cell culture techniques to generate tissue organoids and subculture human mammary epithelial cells (HMECs) that preserve the intrinsic characteristics of the original tissue samples. We found that significantly differential expression of IL20RA and SOX10, principal factors in the gene expression signature, were preserved in both the derived organoids and the HMECs. These results suggest a potential intrinsic difference in the activation of the Hedgehog pathway in the breast epithelial cells of NHB women. This activation may contribute to increased proliferation and transformation of basal stem cells within the breast, possibly fueling the development and progression of TNBC. The implication of activated Hedgehog signaling in NHB women opens new avenues for understanding TNBC disparities. IL20RA and SOX10 represent potential biomarkers for TNBC risk that may serve as a stratification tool for NHB women. The results of this study have the potential to improve assessment and management of TNBC risk among NHB women, thereby contributing significantly to reducing health disparities in breast cancer outcomes. Citation Format: Jennifer Vanessa Cabezas, Savanna Touré, Matthew Wilson, Melody L. Stallings Mann, Laura M. Pacheco-Spann, Joshua W. Ogony, Mark E. Sherman, Derek C. Radisky. Identifying molecular mechanisms in triple negative breast cancer disparities: Unveiling the role of Hedgehog signaling in non-Hispanic Black women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2139.

A Single-Cell Landscape of Spermioteleosis in Mice and Pigs.

(1) Background: Spermatozoa acquired motility and matured in epididymis after production in the testis. However, there is still limited understanding of the specific characteristics of sperm development across different species. In this study, we employed a comprehensive approach to analyze cell compositions in both testicular and epididymal tissues, providing valuable insights into the changes occurring during meiosis and spermiogenesis in mouse and pig models. Additionally, we identified distinct gene expression signatures associated with various spermatogenic cell types. (2) Methods: To investigate the differences in spermatogenesis between mice and pigs, we constructed a single-cell RNA dataset. (3) Results: Our findings revealed notable differences in testicular cell clusters between these two species. Furthermore, distinct gene expression patterns were observed among epithelial cells from different regions of the epididymis. Interestingly, regional gene expression patterns were also identified within principal cell clusters of the mouse epididymis. Moreover, through analysing differentially expressed genes related to the epididymis in both mouse and pig models, we successfully identified potential marker genes associated with sperm development and maturation for each species studied. (4) Conclusions: This research presented a comprehensive single-cell landscape analysis of both testicular and epididymal tissues, shedding light on the intricate processes involved in spermatogenesis and sperm maturation, specifically within mouse and pig models.

Abstract 6245: Missed opportunity at the subcellular level: Enhancing the utility of cellular imaging modality in spatial transcriptomic profiling of tumor tissues

Abstract Mini-bulk or single-spot spatial transcriptomic technologies, such as GeoMX Digital Spatial Profiler (DSP), have revolutionized our ability to probe spatial heterogeneity and examine transcripts at a subcellular level. In cancer research, this level of granularity is crucial for uncovering the distinct gene expression signatures within specific compartments or structures of interest such as tumor, immune, stroma, and tertiary lymphoid structures. However, in most GeoMX DSP studies, the spatial information obtained from multiplex immunofluorescence imaging has been primarily used for identifying the regions of interest (ROI), rather than as an integral part of the downstream transcriptomic data interpretation. To fix this missed opportunity, we developed a machine learning based analytical framework to fully leverage the rich spatial context provided by the in situ imaging modality. The framework has two main functionalities. (1) ROI cropping and cell segmentation: our framework enables the automated cropping of ROI images, followed by a cell segmentation process utilizing a multi-color-optimized deep learning model pre-trained based on the TissueNet. (2) Cell typing and integrative spatial analysis: the pipeline extracts cell-level morphological features (e.g. cell size and circularity), and AI-driven characteristics. These features are subsequently employed for cell typing and the quantification of cell mixture. The framework is equipped to perform a series of advanced spatial analyses, such as spatial metrics calculation and consensus clustering analysis with the matched gene expression data. To validate the framework, we conducted GeoMX DSP analyses on samples obtained from bladder cancer and upper tract urothelial carcinoma, encompassing 56 ROIs annotated by pathologists. Our results demonstrated high accuracy, with the calculated tumor-immune proportions aligning precisely with the original annotations. We further compared the cell deconvolution results of our framework with those obtained using SpatialDecon. Consistency was noted across both methods, with a high correlation reaching 0.90. However, we observed that SpatialDecon tends to underestimate tumor purity in tumor core regions, with discrepancies as large as 0.30, and overestimating tumor purity in stromal regions. Additionally, analysis of ROIs in tumor margins revealed that SpatialDecon consistently overestimates the overall proportions of immune cells. In conclusion, our results underscore the importance of integrating in situ imaging with subcellular-level spatial transcriptomics for a more accurate and reliable analysis of tumor tissues. Our approach provides critical insights into the tumor microenvironment and cellular interactions, with significant implications for both research and clinical applications in oncology. Citation Format: Xiaofei Song, Xiaoqing Yu, Carlos M. Moran-Segura, G Daniel Grass, Roger Li, Xuefeng Wang. Missed opportunity at the subcellular level: Enhancing the utility of cellular imaging modality in spatial transcriptomic profiling of tumor tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6245.

Sexually dimorphic control of affective state processing and empathic behaviors

Recognizing the affective states of social counterparts and responding appropriately fosters successful social interactions. However, little is known about how the affective states are expressed and perceived and how they influence social decisions. Here, we show that male and female mice emit distinct olfactory cues after experiencing distress. These cues activate distinct neural circuits in the piriform cortex (PiC) and evoke sexually dimorphic empathic behaviors in observers. Specifically, the PiC → PrL pathway is activated in female observers, inducing a social preference for the distressed counterpart. Conversely, the PiC → MeA pathway is activated in male observers, evoking excessive self-grooming behaviors. These pathways originate from non-overlapping PiC neuron populations with distinct gene expression signatures regulated by transcription factors and sex hormones. Our study unveils how internal states of social counterparts are processed through sexually dimorphic mechanisms at the molecular, cellular, and circuit levels and offers insights into the neural mechanisms underpinning sex differences in higher brain functions.

HHV-6B detection and host gene expression implicate HHV-6B as pulmonary pathogen after hematopoietic cell transplant

Limited understanding of the immunopathogenesis of human herpesvirus 6B (HHV-6B) has prevented its acceptance as a pulmonary pathogen after hematopoietic cell transplant (HCT). In this prospective multicenter study of patients undergoing bronchoalveolar lavage (BAL) for pneumonia after allogeneic HCT, we test blood and BAL fluid (BALF) for HHV-6B DNA and mRNA transcripts associated with lytic infection and perform RNA-seq on paired blood. Among 116 participants, HHV-6B DNA is detected in 37% of BALs, 49% of which also have HHV-6B mRNA detection. We establish HHV-6B DNA viral load thresholds in BALF that are highly predictive of HHV-6B mRNA detection and associated with increased risk for overall mortality and death from respiratory failure. Participants with HHV-6B DNA in BALF exhibit distinct host gene expression signatures, notable for enriched interferon signaling pathways in participants clinically diagnosed with idiopathic pneumonia. These data implicate HHV-6B as a pulmonary pathogen after allogeneic HCT.

Host and Microbe Blood Metagenomics Reveals Key Pathways Characterizing Critical Illness Phenotypes.

Rationale: Two molecular phenotypes of sepsis and acute respiratory distress syndrome, termed hyperinflammatory and hypoinflammatory, have been consistently identified by latent class analysis in numerous cohorts, with widely divergent clinical outcomes and differential responses to some treatments; however, the key biological differences between these phenotypes remain poorly understood.Objectives: We used host and microbe metagenomic sequencing data from blood to deepen our understanding of biological differences between latent class analysis-derived phenotypes and to assess concordance between the latent class analysis-derived phenotypes and phenotypes reported by other investigative groups (e.g., Sepsis Response Signature [SRS1-2], molecular diagnosis and risk stratification of sepsis [MARS1-4], reactive and uninflamed).Methods: We analyzed data from 113 patients with hypoinflammatory sepsis and 76 patients with hyperinflammatory sepsis enrolled in a two-hospital prospective cohort study. Molecular phenotypes had been previously assigned using latent class analysis.Measurements and Main Results: The hyperinflammatory and hypoinflammatory phenotypes of sepsis had distinct gene expression signatures, with 5,755 genes (31%) differentially expressed. The hyperinflammatory phenotype was associated with elevated expression of innate immune response genes, whereas the hypoinflammatory phenotype was associated with elevated expression of adaptive immune response genes and, notably, T cell response genes. Plasma metagenomic analysis identified differences in prevalence of bacteremia, bacterial DNA abundance, and composition between the phenotypes, with an increased presence and abundance of Enterobacteriaceae in the hyperinflammatory phenotype. Significant overlap was observed between these phenotypes and previously identified transcriptional subtypes of acute respiratory distress syndrome (reactive and uninflamed) and sepsis (SRS1-2). Analysis of data from the VANISH trial indicated that corticosteroids might have a detrimental effect in patients with the hypoinflammatory phenotype.Conclusions: The hyperinflammatory and hypoinflammatory phenotypes have distinct transcriptional and metagenomic features that could be leveraged for precision treatment strategies.

Distinct gene expression signatures comparing latent tuberculosis infection with different routes of Bacillus Calmette-Guérin vaccination

Tuberculosis remains an international health threat partly because of limited protection from pulmonary tuberculosis provided by standard intradermal vaccination with Bacillus of Calmette and Guérin (BCG); this may reflect the inability of intradermal vaccination to optimally induce pulmonary immunity. In contrast, respiratory Mycobacterium tuberculosis infection usually results in the immune-mediated bacillary containment of latent tuberculosis infection (LTBI). Here we present RNA-Seq-based assessments of systemic and pulmonary immune cells from LTBI participants and recipients of intradermal and oral BCG. LTBI individuals uniquely display ongoing immune activation and robust CD4 T cell recall responses in blood and lung. Intradermal BCG is associated with robust systemic immunity but only limited pulmonary immunity. Conversely, oral BCG induces limited systemic immunity but distinct pulmonary responses including enhanced inflammasome activation potentially associated with mucosal-associated invariant T cells. Further, IL-9 is identified as a component of systemic immunity in LTBI and intradermal BCG, and pulmonary immunity following oral BCG.

Single-cell transcriptomic atlas of goat ovarian aging

BackgroundThe ovaries are one of the first organs that undergo degenerative changes earlier in the aging process, and ovarian aging is shown by a decrease in the number and quality of oocytes. However, little is known about the molecular mechanisms of female age-related fertility decline in different types of ovarian cells during aging, especially in goats. Therefore, the aim of this study was to reveal the mechanisms driving ovarian aging in goats at single-cell resolution.ResultsFor the first time, we surveyed the single-cell transcriptomic landscape of over 27,000 ovarian cells from newborn, young and aging goats, and identified nine ovarian cell types with distinct gene-expression signatures. Functional enrichment analysis showed that ovarian cell types were involved in their own unique biological processes, such as Wnt beta-catenin signalling was enriched in germ cells, whereas ovarian steroidogenesis was enriched in granulosa cells (GCs). Further analysis showed that ovarian aging was linked to GCs-specific changes in the antioxidant system, oxidative phosphorylation, and apoptosis. Subsequently, we identified a series of dynamic genes, such as AMH, CRABP2, THBS1 and TIMP1, which determined the fate of GCs. Additionally, FOXO1, SOX4, and HIF1A were identified as significant regulons that instructed the differentiation of GCs in a distinct manner during ovarian aging.ConclusionsThis study revealed a comprehensive aging-associated transcriptomic atlas characterizing the cell type-specific mechanisms during ovarian aging at the single-cell level and offers new diagnostic biomarkers and potential therapeutic targets for age-related goat ovarian diseases.

Clinical and Immunological Factors Associated with Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation in Myelofibrosis Patients

Allogeneic hematopoietic stem cell transplantation (HSCT) is widely recognized as the sole curative treatment for myelofibrosis (MF). However, patients with MF often experience graft function impairment despite successful engraftment and absence of relapse signs. Although it has been reported that poor graft function can have a negative impact on the outcomes of patients with MF after HSCT, the specific factors that can influence poor graft function are not yet clearly understood. In this background, we analyzed various clinical and immunological factors that may contribute to poor graft function after transplantation. The study included 93 patients who underwent HSCT for the treatment of MF at Seoul St. Mary's Hospital between the years 2000 and 2022. By cumulative incidence analysis of poor graft function (death, relapse, graft failure, and loss of chimerism as competing risks), we evaluated which pre-HSCT factors has significant impact on poor graft function after HSCT. The definition of poor graft function and graft failure in this study followed the criteria set by the European Society for Blood and Marrow Transplantation (EBMT). We measured the longitudinal diameter of spleen up to 2 months prior to HSCT, and classified the patients into pre-HSCT splenomegaly (≥ 14cm) or not. Next, we investigated the association of immune-related gene expression signature in bone marrow immune-microenvironment with the occurrence of poor graft function, by performing targeted gene expression profiling and gene set enrichment analysis using a custom NanoString panel composed of 579 immune-related genes in pre- and post-HSCT BM samples from patients with poor graft function and good graft function (total 24 samples). A median age of 93 patients was 57 years, and 55.9% were male. Secondary MF was observed in 30.1% of the cases. Fifty-three (57.0%) received graft from a matched sibling donor graft and 40 (43.0%) from a matched unrelated donor. Sixty-three (67.7%) patients were classified to the pre-HSCT splenomegaly group. In terms of ABO match, 45 (48.4%) patients were matched, 10 (10.8%) had bi-directional mismatch, 17 (18.3%) had major mismatch, and 19 (20.4%) had minor mismatch. The median infused CD34 (+) cell dose was 6.4 x 10^6/kg (Interquartile range 5.3 x 10^6/kg-8.3 x 10^6/kg). In our patients, cumulative incidence of poor graft function after 5 years from HSCT was 32.0% (95% CI: 23.4-42.7). Infused CD34 (+) cell dose, the presence of splenomegaly before HSCT were revealed to have an impact on the occurrence of poor graft function after HSCT. The other significant factor associated with poor graft function was ABO mismatch, in particular, with a substantial difference between matched patients and patients with bi-directional ABO mismatch. (Figure A). In NanoString panel analysis, although the expression of immune-related genes mostly upregulated after HSCT, genes related to processes such as complement system, inflammasomes, phagocytosis and degradation, and MHC class II antigen presentation pathways were significantly downregulated in PGF. Whereas, oxidative stress, inflammasomes, and autophagy pathways were downregulated in GGF (Figure B). In this study, we could observe that a significant proportion of MF patients undergoing HSCT experienced poor graft function. Factors that significantly associated with the occurrence of poor graft function included the CD34 (+) cell dose at the time of HSCT, the presence of splenomegaly before HSCT, and ABO mismatch. Interestingly, distinct gene expression signatures present in IME between pre- and post-SCT in both PGF and GGF. Further research is warranted to investigate the impact of poor graft function caused by these factors on outcomes such as transfusion requirements and quality of life.

EPCO-23. EFFECTS OF H3.3G34V MUTATION ON GENOMIC H3K36 AND H3K27 METHYLATION PATTERNS IN ISOGENIC PEDIATRIC GLIOMA CELLS

Abstract BACKGROUND Histone H3.3 mutation (H3F3A) occurs in 50% of cortical pediatric high-grade gliomas (pHGGs). This mutation replaces glycine 34 with arginine or valine (G34R/V), impairing SETD2 activity (H3K36-specific trimethyltransferase). Consequently, reduced H3K36me3 is observed on H3.3G34V nucleosomes relative to wild-type, contributing to genomic instability and driving a distinct gene expression signature associated with tumorigenesis. However, it is not known if this differential H3K36me3 enrichment is due to H3.3G34V mutant protein alone. Therefore, we set to elucidate the effect of H3.3G34V mutant protein in pediatric glioma on H3K36me3, H3K27me3 and H3.3 enrichment in vitro. METHODS Experiments were conducted using an established H3.3G34V mutant patient-derived pediatric high-grade glioma cell line (KNS42), and Histone H3.3 wild-type human astrocytes (NHAs). Lentiviral delivery of a doxycycline-inducible RNAi vector targeted against H3F3A was transduced to KNS42 cells to knock down H3.3G34V protein expression. Cell lines were then compared via ChIP-Seq, Western Blot, proliferation and viability studies. RESULTS We found that the doxycycline-inducible shRNA knockdown of mutant H3F3A encoding the H3.3G34V protein resulted in loss of H3.3G34V enrichment and increased H3K36me3 enrichment throughout the genome. After knockdown, H3.3G34V enrichment was preserved at loci observed to have the greatest H3.3G34V and H3K36me3 enrichment prior to knockdown. Induced expression of mutant H3.3G34V protein in vitro was insufficient to induce genomic H3K36me3 enrichment patterns observed in H3.3G34V mutant glioma cells. We also observed strong co-enrichment of H3.3G34V and wild-type H3.3 protein, as well as greater H3K27me3 enrichment, in cells expressing H3.3G34V. CONCLUSION Our study demonstrates the effects of H3.3G34V mutant protein on genomic H3K36me3, H3K27me3 and H3.3 enrichment patterns in isogenic cell lines.

Post-zygotic rescue of meiotic errors causes brain mosaicism and focal epilepsy.

Somatic mosaicism is a known cause of neurological disorders, including developmental brain malformations and epilepsy. Brain mosaicism is traditionally attributed to post-zygotic genetic alterations arising in fetal development. Here we describe post-zygotic rescue of meiotic errors as an alternate origin of brain mosaicism in patients with focal epilepsy who have mosaic chromosome 1q copy number gains. Genomic analysis showed evidence of an extra parentally derived chromosome 1q allele in the resected brain tissue from five of six patients. This copy number gain is observed only in patient brain tissue, but not in blood or buccal cells, and is strongly enriched in astrocytes. Astrocytes carrying chromosome 1q gains exhibit distinct gene expression signatures and hyaline inclusions, supporting a novel genetic association for astrocytic inclusions in epilepsy. Further, these data demonstrate an alternate mechanism of brain chromosomal mosaicism, with parentally derived copy number gain isolated to brain, reflecting rescue in other tissues during development.