Seq Data Research Articles

Detecting Rhythmic Gene Expression in Single-cell Transcriptomics.

An autonomous, environmentally synchronizable circadian rhythm is a ubiquitous feature of life on Earth. In multicellular organisms, this rhythm is generated by a transcription-translation feedback loop present in nearly every cell that drives daily expression of thousands of genes in a tissue-dependent manner. Identifying the genes that are under circadian control can elucidate the mechanisms by which physiological processes are coordinated in multicellular organisms. Today, transcriptomic profiling at the single-cell level provides an unprecedented opportunity to understand the function of cell-level clocks. However, while many cycling detection algorithms have been developed to identify genes under circadian control in bulk transcriptomic data, it is not known how best to adapt these algorithms to single-cell RNA seq data. Here, we benchmark commonly used circadian detection methods on their reliability and efficiency when applied to single-cell RNA seq data. Our results provide guidance on adapting existing cycling detection methods to the single-cell domain and elucidate opportunities for more robust and efficient rhythm detection in single-cell data. We also propose a subsampling procedure combined with harmonic regression as an efficient strategy to detect circadian genes in the single-cell setting.

Functional metagenomics highlights varied infection states with dynamics of pathogens and antibiotic resistance in lower respiratory tract infections

BackgroundAntimicrobial resistance (AMR) amongst pathogenic bacterial species poses significant challenges in treating infections of the lower respiratory tract (LRT), leading to higher hospitalization and mortality rates. MethodsBronchoalveolar lavage fluid (BALF) from 84 clinically adjudicated LRTI patients were subjected to respiratory pathogen ID/AMR (RPIP) enrichment and sequencing followed by Explify and CZID seq data analysis to identify potential LRTI pathogens and associated AMR genes. Patients were categorized as LRTI-WP (with pneumonia) and LRTI-WoP (without pneumonia). FindingsmNGS achieved 100 % pathogen detection compared to 73 % through clinician-used BioFire panel. Predominant pathogens included Acinetobacter baumannii, Klebsiella pneumoniae along with detection of Aspergillus versicolor and Herpes simplex virus. Double and polymicrobial infections were captured, involving non-respiratory pathogens like Rothia mucilaginosa, Escherichia coli, and Moraxella osloensis. AMR detection highlighted macrolide (MPH; ERM) and Sulfonamide (SUL) rich resistome in 60 % of patients followed by extended spectrum beta lactamase (OXA) and tetracycline (TET). LRTI-WP showed high abundance of A. baumannii, majorly associated with MPH whereas K. pneumoniae with beta-lactams was comparable in both groups. Differences in clinical severity may stem from non-respiratory pathogens, newly recognized via mNGS. CZID seq pipeline validated and revealed additional microbes and AMR genes in the cohort. InterpretationThe prevalence of common pathogens like A. baumannii and K. pneumoniae along with the non-respiratory pathogens identified by RPIP-Explify and CZID seq provided an understanding to evaluate the LRTI. mNGS is crucial for precise pathogen and antibiotic resistance detection, vital for combating antibiotic resistance.

Association of COL4A2 indel polymorphism with the development of stomach adenocarcinoma in Chinese populations

Objective The objective of the study was to assess the potential association between the indel polymorphism (rs34802628) located within the intron of the collagen type ⅳ alpha 2 gene (COL4A2) and the susceptibility to stomach adenocarcinoma (STAD) within a Chinese population. Methods Peripheral venous blood samples were collected from a total of 497 STAD patients and 804 healthy control individuals to extract genomic DNA. The genotyping of the COL4A2 rs34802628 polymorphism was carried out using a polymerase chain reaction assay. Additionally, statistical analyses were conducted on the expression levels of COL4A2 mRNA using the GEPIA database. Meanwhile, the expression of COL4A2 mRNA was also validated by Real-time PCR using STAD tissue samples. Then, based on an analysis of patient tumor RNA seq data available from the Cancer Genome Atlas (TCGA), we assessed the prognostic value of mRNA expression of the COL4A2 gene in STAD patients using K–M plotter. Results The study presented compelling evidence supporting an association between the rs34802628 polymorphism in the COL4A2 gene and susceptibility to STAD. Logistic regression analysis revealed that both the heterozygote and homozygote 4-bp del/del genotypes were significantly associated with a decreased risk of STAD, even after controlling for other variables (adjusted odds ratio [OR] = 0.663, 95% confidence interval [CI] 0.519–0.848, p = 0.037; OR = 0.422, 95% CI 0.290–0.614, p = 0.000005, respectively). Importantly, individuals carrying the 4-bp deletion allele demonstrated a notably lower risk of developing the disease (OR = 0.696, 95% CI 0.591–0.820, p = 0.000014). Furthermore, Genotype-phenotype correlation studies in human STAD tissue samples demonstrated that the higher mRNA expression levels of COL4A2 were associated with the ins allele of rs34802628. Bioinformatics analysis revealed that higher expression of the COL4A2 gene was significant with development and poor prognosis of STAD. Conclusion The results of our study provide strong evidence indicating a potential involvement of genetic variants in the COL4A2 gene in the development of STAD. Nonetheless, to validate and consolidate these findings, additional investigations incorporating larger sample sizes and functional experiments are necessary.

On discovery of novel hub genes for ER+ and TN breast cancer types through RNA seq data analyses and classification models

Breast cancer (BC) is a malignant neoplasm which is classified into various types defined by underlying molecular factors such as estrogen receptor positive (ER+), progesterone receptor positive (PR+), human epidermal growth factor positive (HER2+) and triple negative (TNBC). Early detection of ER+ and TNBC is crucial in the choice of diagnosis and appropriate treatment strategy. Here we report the key genes associated to ER+ and TNBC using RNA-Seq analysis and machine learning models. Three ER+ and TNBC RNA seq datasets comprising 164 patients in-toto were selected for standard NGS hierarchical data processing and data analyses protocols. Enrichment pathway analysis and network analysis was done and finally top hub genes were identified. To come with a reliable classifier which could distinguish the distinct transcriptome patterns associated to ER+ and TNBC, ML models were built employing Naïve Bayes, SVM and kNN. 1730 common DEG’s exhibiting significant logFC values with 0.05 p-value threshold were identified. A list of top ten hub genes were screened on the basis of maximal clique centrality (MCC) which included CDC20, CDK1, BUB1, AURKA, CDCA8, RRM2, TTK, CENPF, CEP55 and NDC80.These genes were found to be involved in crucial cell cycle pathways. k-Nearest Neighbor (kNN) model was observed to be best classifier with accuracy 84%, specificity 66% and sensitivity 95% to differentiate between ER+ and TNBC RNA-Seq transcriptomes. Our screened list of 10 hub genes can thus help unearth novel molecular signatures implicated in ER+ and TNBC onset, prognosis and design of novel protocols for breast cancer diagnostics and therapeutics.

Epsins oversee smooth muscle cell reprograming by influencing master regulators KLF4 and OCT4.

Smooth muscle cells in major arteries play a crucial role in regulating coronary artery disease. Conversion of smooth muscle cells into other adverse cell types in the artery propels the pathogenesis of the disease. Curtailing artery plaque buildup by modulating smooth muscle cell reprograming presents us a new opportunity to thwart coronary artery disease. Here, our report how Epsins, a family of endocytic adaptor proteins oversee the smooth muscle cell reprograming by influencing master regulators OCT4 and KLF4. Using single-cell RNA sequencing, we characterized the phenotype of modulated smooth muscle cells in mouse atherosclerotic plaque and found that smooth muscle cells lacking epsins undergo profound reprogramming into not only beneficial myofibroblasts but also endothelial cells for injury repair of diseased endothelium. Our work lays concrete groundwork to explore an uncharted territory as we show that depleting Epsins bolsters smooth muscle cells reprograming to endothelial cells by augmenting OCT4 activity but restrain them from reprograming to harmful foam cells by destabilizing KLF4, a master regulator of adverse reprograming of smooth muscle cells. Moreover, the expression of Epsins in smooth muscle cells positively correlates with the severity of both human and mouse coronary artery disease. Integrating our scRNA-seq data with human Genome-Wide Association Studies (GWAS) identifies pivotal roles Epsins play in smooth muscle cells in the pathological process leading to coronary artery disease. Our findings reveal a previously unexplored direction for smooth muscle cell phenotypic modulation in the development and progression of coronary artery disease and unveil Epsins and their downstream new targets as promising novel therapeutic targets for mitigating metabolic disorders.

Knockout of the WD40 domain of ATG16L1 enhances foot and mouth disease virus replication

The WD40 domain is one of the most abundant domains and is among the top interacting domains in eukaryotic genomes. The WD40 domain of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Canonical autophagy was utilized by FMDV, while the relationship between FMDV and non-canonical autophagy is still elusive. In the present study, WD40 knockout (KO) PK15 cells were successfully generated via CRISPR/cas9 technology as a tool for studying the effect of non-canonical autophagy on FMDV replication. The results of growth curve analysis, morphological observation and karyotype analysis showed that the WD40 knockout cell line was stable in terms of growth and morphological characteristics. After infection with FMDV, the expression of viral protein, viral titers, and the number of copies of viral RNA in the WD40-KO cells were significantly greater than those in the wild-type PK15 cells. Moreover, RNA‒seq technology was used to sequence WD40-KO cells and wild-type cells infected or uninfected with FMDV. Differentially expressed factors such as Mx1, RSAD2, IFIT1, IRF9, IFITM3, GBP1, CXCL8, CCL5, TNFRSF17 were significantly enriched in the autophagy, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and TNF signaling pathway, etc. The expression levels of differentially expressed genes were detected via qRT‒PCR, which was consistent with the RNA‒seq data. Here, we experimentally demonstrate for the first time that knockout of the WD40 domain of ATG16L1 enhances FMDV replication by downregulation innate immune factors. In addition, this result also indicates non-canonical autophagy inhibits FMDV replication. In total, our results play an essential role in regulating the replication level of FMDV and providing new insights into virus–host interactions and potential antiviral strategies.

The de novo assembly and characterization of the complete mitochondrial genome of bottle gourd (Lagenaria siceraria) reveals the presence of homologous conformations produced by repeat-mediated recombination.

Bottle gourd is an annual herbaceous plant that not only has high nutritional value and many medicinal applications but is also used as a rootstock for the grafting of cucurbit crops such as watermelon, cucumber and melon. Organellar genomes provide valuable resources for genetic breeding. A hybrid strategy with Illumina and Oxford Nanopore Technology sequencing data was used to assemble bottle gourd mitochondrial and chloroplast genomes. The length of the bottle gourd mitochondrial genome was 357547 bp, and that of the chloroplast genome was 157121 bp. These genomes had 27 homologous fragments, accounting for 6.50% of the total length of the bottle gourd mitochondrial genome. In the mitochondrial genome, 101 simple sequence repeats (SSRs) and 10 tandem repeats were identified. Moreover, 1 pair of repeats was shown to mediate homologous recombination into 1 major conformation and 1 minor conformation. The existence of these conformations was verified via PCR amplification and Sanger sequencing. Evolutionary analysis revealed that the mitochondrial genome sequence of bottle gourd was highly conserved. Furthermore, collinearity analysis revealed many rearrangements between the homologous fragments of Cucurbita and its relatives. The Ka/Ks values for most genes were between 0.3~0.9, which means that most of the genes in the bottle gourd mitochondrial genome are under purifying selection. We also identified a total of 589 potential RNA editing sites on 38 mitochondrial protein-coding genes (PCGs) on the basis of long noncoding RNA (lncRNA)-seq data. The RNA editing sites of nad1-2, nad4L-2, atp6-718, atp9-223 and rps10-391 were successfully verified via PCR amplification and Sanger sequencing. In conclusion, we assembled and annotated bottle gourd mitochondrial and chloroplast genomes to provide a theoretical basis for similar organelle genomic studies.

Abstract Mo120: Overexpression of Prostaglandin E2 EP3 Receptor Subtype Alters Calcium-Handling Proteins in Mouse Hearts

Prostaglandin E2 (PGE2) is an autacoid that acts through G-protein coupled receptors (EP1-EP4) in various cell types within the heart, including cardiomyocytes in which EP3 and EP4 predominate. We previously reported that mice with cardiomyocyte-specific overexpression of the EP3 receptor (EP3 TG) demonstrate reduced cardiac function and diminished cardiomyocyte contractility in isolated cardiomyocytes. We therefore hypothesized that PGE2 via the EP3 receptor regulates calcium handling proteins to reduce contractility. To test our hypothesis, we performed RNA sequencing (RNA seq) on the left ventricle of fifteen-week-old male EP3 TG mice along with their WT controls. Western blots were used to confirm changes in the expression of calcium-handling proteins such as Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), Ryanodine receptor 2 (RYR2), Phospholamban (PLB), Troponins (C, I, T) and voltage-gated calcium channel (CACNB2). For RNA seq, all data is reported as Log2Fold change, adjusted P value, and western blot data is reported as mean ± standard error (SE) of the mean. Additionally, isolated cardiomyocyte contractility was assessed using the IonOptix system, and data is reported as the mean ± SE. RNA seq data shows that SERCA2a and RYR2 expression were significantly downregulated in TG mice (SERCA2a -0.44, p=4.72 x 10 -6 ; RYR2: -0.86, p=4.08 x 10 -5 ) in addition to significant reductions in Troponin I (-0.43, p=0.038) and the Voltage-gated calcium channel (CACNB2: -0.71,p=0.0055). Western blot analyses confirmed the reductions in SERCA2a, RYR2, and CACNB2 although these changes were only significant for CACNB2 (1.0 ± 0.03 for WT vs 0.59 ± 0.04 for TG, p = 0.016). Expression of Troponins did not change in TG mice. Consistent with our previous data, IonOptix analysis shows reduced contractility of myocytes from EP3 TG mice (bl% peak h is 3.47 ± 0.20 for WT vs 1.0 ± 0.14 for TG, p<0.0001) despite a similar increase in the Ca 2+ transients (Ca 2+ peak h for WT= 0.86 ± 0.04 and TG=0.86 ± 0.07) suggesting potential changes in the sensitivity of the myofilament to Ca 2+ . In conclusion, the reduced contractility observed in EP3 TG mice may be due to alterations in calcium handling proteins and/or changes in calcium sensitivity at the level of the myofilament.

Mesenchymal stromal/stem cell tissue source and in vitro expansion impact extracellular vesicle protein and miRNA compositions as well as angiogenic and immunomodulatory capacities.

Recently, therapies utilizing extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) have begun to show promise in clinical trials. However, EV therapeutic potential varies with MSC tissue source and in vitro expansion through passaging. To find the optimal MSC source for clinically translatable EV-derived therapies, this study aims to compare the angiogenic and immunomodulatory potentials and the protein and miRNA cargo compositions of EVs isolated from the two most common clinical sources of adult MSCs, bone marrow and adipose tissue, across different passage numbers. Primary bone marrow-derived MSCs (BMSCs) and adipose-derived MSCs (ASCs) were isolated from adult female Lewis rats and expanded in vitro to the indicated passage numbers (P2, P4, and P8). EVs were isolated from the culture medium of P2, P4, and P8 BMSCs and ASCs and characterized for EV size, number, surface markers, protein content, and morphology. EVs isolated from different tissue sources showed different EV yields per cell, EV sizes, and protein yield per EV. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of proteomics data and miRNA seq data identified key proteins and pathways associated with differences between BMSC-EVs and ASC-EVs, as well as differences due to passage number. In vitro tube formation assays employing human umbilical vein endothelial cells suggested that both tissue source and passage number had significant effects on the angiogenic capacity of EVs. With or without lipopolysaccharide (LPS) stimulation, EVs more significantly impacted expression of M2-macrophage genes (IL-10, Arg1, TGFβ) than M1-macrophage genes (IL-6, NOS2, TNFα). By correlating the proteomics analyses with the miRNA seq analysis and differences observed in our in vitro immunomodulatory, angiogenic, and proliferation assays, this study highlights the trade-offs that may be necessary in selecting the optimal MSC source for development of clinical EV therapies.

Identification of novel immune-related signatures for keloid diagnosis and treatment: insights from integrated bulk RNA-seq and scRNA-seq analysis

BackgroundKeloid is a disease characterized by proliferation of fibrous tissue after the healing of skin tissue, which seriously affects the daily life of patients. However, the clinical treatment of keloids still has limitations, that is, it is not effective in controlling keloids, resulting in a high recurrence rate. Thus, it is urgent to identify new signatures to improve the diagnosis and treatment of keloids.MethodBulk RNA seq and scRNA seq data were downloaded from the GEO database. First, we used WGCNA and MEGENA to co-identify keloid/immune-related DEGs. Subsequently, we used three machine learning algorithms (Randomforest, SVM-RFE, and LASSO) to identify hub immune-related genes of keloid (KHIGs) and investigated the heterogeneous expression of KHIGs during fibroblast subpopulation differentiation using scRNA-seq. Finally, we used HE and Masson staining, quantitative reverse transcription-PCR, western blotting, immunohistochemical, and Immunofluorescent assay to investigate the dysregulated expression and the mechanism of retinoic acid in keloids.ResultsIn the present study, we identified PTGFR, RBP5, and LIF as KHIGs and validated their diagnostic performance. Subsequently, we constructed a novel artificial neural network molecular diagnostic model based on the transcriptome pattern of KHIGs, which is expected to break through the current dilemma faced by molecular diagnosis of keloids in the clinic. Meanwhile, the constructed IG score can also effectively predict keloid risk, which provides a new strategy for keloid prevention. Additionally, we observed that KHIGs were also heterogeneously expressed in the constructed differentiation trajectories of fibroblast subtypes, which may affect the differentiation of fibroblast subtypes and thus lead to dysregulation of the immune microenvironment in keloids. Finally, we found that retinoic acid may treat or alleviate keloids by inhibiting RBP5 to differentiate pro-inflammatory fibroblasts (PIF) to mesenchymal fibroblasts (MF), which further reduces collagen secretion.ConclusionIn summary, the present study provides novel immune signatures (PTGFR, RBP5, and LIF) for keloid diagnosis and treatment, and identifies retinoic acid as potential anti-keloid drugs. More importantly, we provide a new perspective for understanding the interactions between different fibroblast subtypes in keloids and the remodeling of their immune microenvironment.

VlMYB4 and VlCDF3 co-targeted the VlLOG11 promoter to regulate fruit setting in grape (Vitis vinifera L).

The VlLOG11 mediates the cytokinin signaling pathway to regulate grape fruit setting. Fruit set, as an accepted agronomic trait, is inextricably linked with fruit quality and yield. Previous studies have demonstrated that exogenous treatment with the synthetic cytokinin analog, forchlorfenuron (CPPU), significantly enhances fruit set. In this study, a significant reduction in endogenous cytokinins was found by measuring the content of cytokinins in young grape berries after CPPU treatment. LONELY GUYs (VlLOGs), a key cytokinin-activating enzyme working in the biosynthesis pathway of cytokinins, exhibited differential expression. Some differentially expressed VlLOGs genes were presented by RNA seq data and their functions and regulation patterns were further investigated. The results showed that VlLOG11 was differentially expressed in young grape berries after CPPU treatment. Overexpression of VlLOG11 in tomato increases the amount of fruit set, and upregulated the expression of genes associated with cytokinin signaling including SlHK4, SlHK5, SlHP3, SlHP4, SlPHP1, SlPHP2. VlMYB4 and VlCDF3 could regulate the expression of VlLOG11 by directly binding to its promoter in young grape berries during fruit set. These results strongly demonstrated that VlMYB4/VlCDF3-VlLOG11 regulatory module plays a key role in the process of fruit setting in grape. This provided a basis for the molecular mechanism of VlLOG11-mediated cytokinin biosynthesis in young grape fruit set.

CRISPR-GEM: A Novel Machine Learning Model for CRISPR Genetic Target Discovery and Evaluation.

CRISPR gene editing strategies are shaping cell therapies through precise and tunable control over gene expression. However, achieving reliable therapeutic effects with improved safety and efficacy requires informed target gene selection. This depends on a thorough understanding of the involvement of target genes in gene regulatory networks (GRNs) that regulate cell phenotype and function. Machine learning models have been previously used for GRN reconstruction using RNA-seq data, but current techniques are limited to single cell types and focus mainly on transcription factors. This restriction overlooks many potential CRISPR target genes, such as those encoding extracellular matrix components, growth factors, and signaling molecules, thus limiting the applicability of these models for CRISPR strategies. To address these limitations, we have developed CRISPR-GEM, a multi-layer perceptron (MLP)-based synthetic GRN constructed to accurately predict the downstream effects of CRISPR gene editing. First, input and output nodes are identified as differentially expressed genes between defined experimental and target cell/tissue types respectively. Then, MLP training learns regulatory relationships in a black-box approach allowing accurate prediction of output gene expression using only input gene expression. Finally, CRISPR-mimetic perturbations are made to each input gene individually and the resulting model predictions are compared to those for the target group to score and assess each input gene as a CRISPR candidate. The top scoring genes provided by CRISPR-GEM therefore best modulate experimental group GRNs to motivate transcriptomic shifts towards a target group phenotype. This machine learning model is the first of its kind for predicting optimal CRISPR target genes and serves as a powerful tool for enhanced CRISPR strategies across a range of cell therapies.

SMRT sequencing of a full-length transcriptome reveals cold induced alternative splicing in Vitis amurensis root

Alternative splicing enhances diversity at the transcriptional and protein levels that widely involved in plant response to biotic and abiotic stresses. V. amurensis is an extremely cold-tolerant wild grape variety, however, studies on alternative splicing (AS) in amur grape at low temperatures are currently poorly understood. In this study, we analyzed full-length transcriptome and RNA seq data at 0, 2, and 24 h after cold stress in V. amurensis roots. Following quality control and correction, 221,170 high-quality full-length non-concatemer (FLNC) reads were identified. A total of 16,181 loci and 30,733 isoforms were identified. These included 22,868 novel isoforms from annotated genes and 2815 isoforms from 2389 novel genes. Among the distinguished novel isoforms, 673 Long non-coding RNAs (LncRNAs) and 18,164 novel isoforms open reading frame (ORF) region were found. A total of 2958 genes produced 8797 AS events, of which 189 genes were involved in the low-temperature response. Twelve transcription factors show AS during cold treatment and VaMYB108 was selected for initial exploration. Two transcripts, Chr05.63.1 (VaMYB108short) and Chr05.63.2 (VaMYB108normal) of VaMYB108, display up-regulated expression after cold treatment in amur grape roots and are both localized in the nucleus. Only VaMYB108normal exhibits transcriptional activation activity. Overexpression of either VaMYB108short or VaMYB108normal in grape roots leads to increased expression of the other transcript and both increased chilling resistance of amur grape roots. The results improve and supplement the genome annotations and provide insights for further investigation into AS mechanisms during cold stress in V. amurensis.

Retinoic acid receptor alpha inhibits ferroptosis by promoting thioredoxin and protein phosphatase 1F in lung adenocarcinoma

Ferroptosis is a recently discovered form of cell death that plays an important role in tumor growth and holds promise as a target for antitumor therapy. However, evidence in the regulation of ferroptosis in lung adenocarcinoma (LUAD) remains elusive. Here, we show that retinoic acid receptor alpha (RARA) is upregulated with the treatment of ferroptosis inducers (FINs). Pharmacological activation of RARA increases the resistance of LUAD to ferroptosis according to cell viability and lipid peroxidation assays, while RARA inhibitor or knockdown (KD) does the opposite. Through transcriptome sequencing in RARA-KD cells and chromatin immunoprecipitation (CHIP)-Seq data, we identify thioredoxin (TXN) and protein phosphatase 1 F (PPM1F) as downstream targets of RARA, both of which inhibit ferroptosis. We confirm that RARA binds to the promotor region of TXN and PPM1F and promotes their transcription by CHIP-qPCR and dual-luciferase assays. Overexpression of TXN and PPM1F reverses the effects of RARA knockdown on ferroptosis in vitro and vivo. Clinically, RARA knockdown or inhibitor increases cells’ sensitivity to pemetrexed and cisplatin (CDDP). Immunohistochemistry (IHC) of LUAD from our cohort shows the same expression tendency of RARA and the downstream targets. Our study uncovers that RARA inhibits ferroptosis in LUAD by promoting TXN and PPM1F, and inhibiting RARA-TXN/PPM1F axis represents a promising strategy for improving the efficacy of FINs or chemotherapy in the treatment of LUAD patients.

DIPG-55. DISSECTING THE ECOSYSTEM OF PEDIATRIC DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Pediatric gliomas are heterogeneous tumors encompassing high grade (pHGG) and low grade (pLGG) subtypes. For tumors not amenable to surgery, in particular high grade H3K27 mutant diffuse midline glioma (DMG), no curative options exist. DMG is characterized by 1) extensive heterogeneity of tumor cells that can exist in different interchangeable cellular states mimicking normal development and 2) extensive heterogeneity of the tumor microenvironment (TME). A better understanding of the DMG ecosystem is needed for therapeutic target identification. METHODS Publicly available single cell RNA seq data of DMG (n=17, Jessa et al. Nat. Genet. 2022), high grade pediatric glioblastoma (GBM, n=16, scPCA) and low grade tumors (ganglioglioma, n=5; pilocytic astrocytoma, n=14, scPCA) were analyzed to define key molecular characteristics and differences between these pediatric brain tumors. Doublet cells were removed from each dataset and tumor and TME cells were discriminated based on marker gene expression and inferred CNVs. RESULTS In DMG, glioma cellular states and TME heterogeneity differed depending on the tumor location. DMG arising in the pons harbored a bigger fraction of astrocyte-like glioma cells whereas oligodendrocyte-like cells were more abundant in thalamic tumors. In line with this observation, Liana cellular communication analysis inferred more intratumoral (autocrine) signaling involving oligodendrocyte-like glioma cells in thalamic tumors (e.g. NCAM1-PTPRZ1, NLGN4X-NRXN1, NTN1-DSCAM) than in pons tumors. Notably, the TME of thalamic tumors contained a bigger fraction of immunotolerant macrophages expressing CD163 or VSIG4. In contrast, pons tumors harbored a unique cluster of VEGFA expressing astrocytes, not found in GBM or pLGG. Liana analysis inferred COPA/APP-CD74 interaction between VEGFA+ astrocytes and CD163/VSIG4+ macrophages and microglia, potentially contributing to an immunotolerant TME in pons DMG. CONCLUSIONS In DMG, patterns of tumor and TME composition associated with tumor location were uncovered. Furthermore, unique TME celltypes could contribute to the particularly aggressive nature of these tumors.

On the limits of inferring biophysical parameters of RBP-RNA interactions from in vitro RNA Bind’n Seq data

We develop a thermodynamic model describing the binding of RNA binding proteins (RBP) to oligomers in vitro. We apply expectation-maximization to infer the specificity of RBPs, represented as position-specific weight matrices (PWMs), by maximizing the likelihood of RNA Bind’n Seq data from the ENCODE project. Analyzing these public data we find sequence motifs that can partly explain the data for more than half of the studied 111 RBPs, and for 48 of the proteins these motifs are consistent with the known specificity. Our code is publicly available, facilitating analysis of RBP binding data.

Abstract PO3-25-02: Circadian Rhythm Disruption Accelerates Aggressive Mammary Tumorigenesis by Altering Tumor Microenvironment

Abstract \Breast cancer is the most common cancer in women worldwide, leading to over 650,000 deaths worldwide. The circadian rhythm is a 24-hour biological rhythm that controls most cellular, hormonal, and behavioral processes in living organisms, which is why perturbation of these rhythms is linked to several devastating diseases such as cardiovascular diseases, diabetes, and cancer. In the mammary gland, circadian clocks regulate the rhythmic expression of numerous genes. Circadian rhythm disruption (CRD), which has been termed a "carcinogen" by the International Agency for Research in Cancer, is caused by different factors, one of them being shift work. Several epidemiological studies have revealed that women who work night shifts are predisposed to breast cancer. The aim of this study is to investigate the effect of circadian rhythm disruption on aggressive mammary tumorigenesis. To do this, we used genetically engineered mouse models of mammary tumors (HER2-tumor and triple-negative breast tumor, or TNBC). Chronic jetlag, which mimics shift work in mice and is known to cause CRD, was introduced in one group of mice by perturbing the normal light and dark cycles with an 8-hour advance every three days. Wheel-running, which is represented by an actogram, was used to confirm the circadian disruption in the CRD mice. CRD was found to accelerate tumor initiation with a significant increase in weight in the CRD group of mice. CRD enhanced tumor burden significantly compared to LD mice. Our results also showed high metastatic foci in the lungs of CRD mice compared to LD mice. A flow cytometry study showed a higher cancer stem cell population in CRD tumors. Mammosphere assays with tumor-derived cells showed that CRD increased the number and size of the mammospheres compared to the control tumors. The tumor cells were analyzed using single-cell RNA sequencing, which showed that CRD alters the tumor microenvironment by developing more immunosuppressive and "cold" tumors. The tumor-derived organoids (tumoroid) showed resistance to chemotherapeutics. Finally, bioinformatics analysis using single-cell RNA seq data from human breast tumors (TNBC, ER/PR, and HER2) showed that the malignant cells in TNBC tumors had a significantly higher circadian rhythm disrupted score (CRD score) compared to ER/PR tumors. Taken together, our data showed how circadian rhythm disruption affects TNBC tumorigenesis and drug response by altering the tumor microenvironment. Citation Format: Olajumoke Ogunlusi, Mrinmoy Sarkar, Tristan Nguyen, Devon Boland, James Cai, Arhit Chakrabarti, Bani Mallick, Alex Keene, Tapasree Roy Sarkar. Circadian Rhythm Disruption Accelerates Aggressive Mammary Tumorigenesis by Altering Tumor Microenvironment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-02.

Mitochondrial Turnover Regulates Ischemic Endothelial Angiogenic Capacity in Peripheral Artery Disease

Background: Atherosclerotic occlusions in lower limbs cause ischemia that results in Peripheral artery disease. Despite functional mitochondria, normal endothelial cells use Glycolysis as the primary metabolic pathway for cellular maintenance. While mitochondrial anomalies are extensively studied in skeletal muscle myocytes in PAD, the role of mitochondria in regulating ischemic endothelial (EC) function is largely unknown. Rationale: Based on a recent study that showed Mitochondrial OxPhos is required for EC migration, we aimed to understand the role of EC Mitochondrial OxPhos in regulating ischemic EC angiogenic capacity in PAD. Methods: To study the role of EC-Mitochondrial OxPhos, we developed an EC-specific (using Cdh5-Cre) mitochondrial Transcription factor-A gene (TFAM, shown to be a critical regulator of mitochondrial OxPhos) deficient mice. Since complete deletion of TFAM in ECs resulted in embryonic lethality, male and female EC-TFAM+/− mice were used to study the role of mitochondrial metabolism in regulating EC angiogenic capacity and perfusion recovery in preclinical PAD model (hind limb ischemia (HLI) by femoral artery ligation and resection). Results: HLI significantly decreased perfusion recovery and significantly increased necrosis in both male and female EC-TFAM+/− mice compared to WT controls. Consistent with impaired perfusion recovery, microvascular density was also significantly decreased in EC-TFAM+/− mice vs. WT controls. To understand the mechanism by which EC-TFAM deficiency results in impaired perfusion recovery, we isolated ECs from EC-TFAM+/− and WT mice ischemic muscle at day-7 post HLI, and performed bulk RNA-Seq. GeneOntology Pathway analysis of RNA Seq data showed that Autophagy/mitophagy and OxPhos as the top 2 pathways that were significantly inhibited in EC-TFAM+/− ischemic ECs vs. WT ischemic ECs. Hypothesizing 'accumulation of non-functional mitochondria due to impaired mitophagy inhibits the angiogenic capacity of ischemic ECs in PAD' we systematically examined the expression of key mitophagy gene expression in EC-TFAM+/− ischemic ECs vs. WT ischemic ECs. This mitophagy gene screening showed a significant decrease in Parkin expression in EC-TFAM+/− ischemic ECs vs. WT ischemic ECs. Despite higher mitochondrial DNA content in hypoxia serum starved (HSS, in vitro model for PAD), TFAM+/− ECs vs. WT, seahorse Mitostress assays showed a significant decrease in the OxPhos of HSS-TFAM+/− ECs vs. WT. Accordingly, flow cytometry analysis showed a significant decrease in mitochondrial membrane potential (using Mito-ID) in HSS-TFAM+/− ECs vs. WT. Loss of mitochondrial membrane potential and OxPhos in HSS-TFAM+/− ECs resulted in a significant loss of HSS-TFAM+/− EC angiogenic capacity on growth factor reduced matrigel compared to HSS-WT ECs. Conclusion: These data presented that 'impaired mitophagy results in the accumulation of non-functional mitochondria that inhibits the angiogenic capacity of ischemic ECs resulting in impaired perfusion recovery in PAD. Ongoing experiments are aimed at understanding the causal role of Parkin-Mitophagy in regulating ischemic EC angiogenic capacity and perfusion recovery in PAD. R01HL146673-02. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Differential Cell Types in the Thick Ascending Limb of the Kidney: Insights into Functionally Distinct TAL Claudin 10 and TAL Claudin 16 Cells

Study objective, hypothesis: The Thick Ascending Limb (TAL) in the kidney is crucial for reabsorbing Na+ and divalent cations like Ca2+ and Mg2+. This reabsorption depends on the apical cotransporter NKCC2, encoded by the Slc12a1 gene. The TAL extends from the outer medulla to the cortex. While typically considered a single functional cell type, recent evidence suggests the presence of two distinct TAL cell types, distinguished by the expression of Claudin 10 and Claudin 16 (PMID: 28028216). Despite these insights, the lack of correlation with functional and morphological data and the absence of a comprehensive model for the cooperative regulation of NaCl and Ca2+/Mg2+ transport remain significant knowledge gaps. Our objective is to couple immunohistochemistry with single nuclei (sn) RNA sequencing technology to uncover unique cell features that are obscure, when a single approach is employed. Methodology: Immunofluorescence analysis was performed on samples from mice, rats, and humans. Additionally, Slc12a1-positive cells were analyzed using sn-RNA sequencing datasets that contained spatial information about cell localization. These datasets were sourced from published studies on mouse (PMID: 31689386) and human samples (PMID: 37468583). Data Immunofluorescence images from mice, rats, and humans revealed mutually exclusive expression patterns of Kir4.1 and ROMK. Apical ROMK was observed only in cells that express Claudin 10, while Kir4.1 was expressed only in cells that express Claudin 16. phospho NKCC2 and CaSR displayed a mosaic pattern, with expression in the Claudin 16 cells throughout the entire TAL and, in both Claudin 10 and Claudin 16 cells only in the cortex. Unsupervised clustering of mouse sn-RNA seq data identified two distinct clusters, TAL Cldn10 and TAL Cldn16, present in both the cortex and medulla. Key Differentially Expressed Genes (DEG) for TAL Cld10 included Avp2r, Wnk4, Spak, Kcnt1 (encoding KCa4.1 or Slo2.2), and genes related to arachidonic acid signaling such as Ptger3 (encoding Ep3), Pla2g7, Cox7a1. Kcnj1 (encoding ROMK) transcripts were found in both clusters, even though ROMK is located apically only in TAL Claudin 10 cells. TAL Claudin 16 cells showed a higher expression of calcium-related genes, including Casr, Pth1r, Vdr, Kl (encoding Klotho), as well as transcripts for Kcnj10 (encoding Kir4.1), Kcnj16 (encoding Kir5.1), Wnk1, Clcnkb, and Bsnd (encoding Barttin). This transcriptional heterogeneity was also observed in human TAL cells. Summary of results Combining morphological and transcriptomic analyses identified two functionally distinct cell types within the TAL, present in both cortex and medulla. Each cell type appears to be associated with specific transport pathways: TAL Claudin 10 cells, express ROMK apically and are thus responsible for generating the transepithelial voltage and for paracellular Na+ reabsorption. They also express more Na+ transport-regulatory genes, including Avp2r and Wnk4, along with genes related to arachidonic acid signaling. TAL Claudin 16 cells mediate Ca2+ & Mg2+ transport via the paracellular pathway, driven by the voltage generated by claudin 10 cells; they express calcium-regulatory genes, including Casr, Pth1r, and Vdr. Interestingly, they also express genes associated with transcellular Na+, K+, and Cl− transport, including Kcnj10 (Kir4.1), Kcnj16 (Kir5.1), Wnk1, and Clcnkb. Together, these results present a new model of discrete but coupled ion transport pathways in the TAL. NIH DK51496 | NIH DK054983 | VA 1I01BX002228 | Global Research Network of Excellence, Leducq Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification of key TE associated with myocarditis based on RNA and single-cell sequencing data mining

Cardiomyopathy is a severe cardiac condition characterized by complex immune regulatory mechanisms. While the role of immune genes is recognized, the specifics of their regulation in cardiomyopathy are not fully understood. Recent studies highlight the significance of transposable elements (TEs) in various diseases, particularly their potential to modulate immune responses. This paper utilizes publicly available databases to explore the role of TEs in myocarditis: RNA Seq data and single-cell sequencing data were analyzed, with a focus on the mouse model of experimental autoimmune myocarditis (EAM). The RNA-Seq analysis revealed substantial upregulation of a range of immune genes in cardiac tissue. Further investigation using single-cell sequencing of cardiac immune cells identified specific expression of certain transposable elements (TEs) across different types of immune cells in the heart. Additionally, there was an overall increase in the expression of the ERVB7-1. LTR-MM transposon across various cells in the EAM model, suggesting a widespread impact of this transposon on the immune response in this disease context. The findings of this study highlight the intricate interplay between transposable elements and the immune system in cardiomyopathy, providing new insights into the molecular mechanisms underlying this condition. The discovery of specific TEs expression in cardiac immune cells and the overall increase in ERVB7-1. LTR-MM expression across the EAM model underscore the potential of these elements in modulating immune responses and contribute to our understanding of cardiomyopathy's pathogenesis. These observations open avenues for further research into the role of TEs in cardiac disases and may lead to novel therapeutic strategies.