Differential Expression Research Articles

The Clinical and Molecular Response of Pyoderma Gangrenosum to Interleukin 23 Blockade: Result from a proof-of-concept open-label clinical trial.

Pyoderma Gangrenosum is a severe ulcerative disease with a great need for novel therapies. A major barrier to the development of novel therapies is a lack of understanding of disease pathogenesis. We present the results of a proof-of-concept open label clinical trial of IL-23p19 antagonism with Tildrakizumab ion Pyoderma Gangrenosum. Gene expression analysis identified pro-inflammatory genes associated with interferon responses and dendritic cell activity including IFI27, XBP1, SAA1 LGALS3 and STAT3 significantly downregulated in lesional tissue after 12 weeks of therapy. Immunohistochemistry confirmed reduction in IL-17A and IL-17 F positive cells as well as reduction in TNF-a, C5a and IL-1B positive cells in week 12 samples compared to baseline. Significant reduction in serum inflammation was observed via serum proteomics, with IL-8, IL-6, and CASP-8 levels reduced comparable to healthy controls at Week 12. Clinical outcomes demonstrated significant reduction in ulcer size, pain, itch and quality of life outcomes in line with the molecular findings. Differential expression of key inflammatory cytokines such as IL-8, CXCL5, PD-L1, SPP1 and MMP1 were observed in tissue and serum when stratified by clinical responders and non-responders. This data provides insights into the clinical relevance of alterations in molecular markers in pyoderma gangrenosum and the potential for the identification of clinically relevant biomarkers of disease activity.

Pan-genome wide identification and analysis of the SAMS gene family in sunflowers (Helianthus annuus L.) revealed their intraspecies diversity and potential roles in abiotic stress tolerance

IntroductionS-adenosylmethionine (SAM), a key molecule in plant biology, plays an essential role in stress response and growth regulation. Despite its importance, the SAM synthetase (SAMS) gene family in sunflowers (Helianthus annuus L.) remains poorly understood.MethodsIn this study, the SAMS genes were identified from the sunflower genome. Subsequently, the protein properties, gene structure, chromosomal location, cis-acting elements, collinearity, and phylogeny of the SAMS gene family were analyzed by bioinformatic methods. Finally, the expression patterns of SAMS genes in different tissues, under different hormonal treatment and abiotic stress were analyzed based on transcriptome data and qRT-PCR.ResultsThis study identified 58 SAMS genes across nine cultivated sunflower species, which were phylogenetically classified into seven distinct subgroups. Physicochemical properties and gene structure analysis showed that the SAMS genes are tightly conserved between cultivars. Collinearity analysis revealed segmental duplications as the primary driver of gene family expansion. The codon usage bias analysis suggested that natural selection substantially shapes the codon usage patterns of sunflower SAMS genes, with a bias for G/C-ending high-frequency codons, particularly encoding glycine, leucine, and arginine. Analysis of the cis-regulatory elements in promoter regions, implied their potential roles in stress responsiveness. Differential expression patterns for HanSAMS genes were observed in different tissues as well as under hormone treatment or abiotic stress conditions by analyzing RNA-seq data from previous studies and qRT-PCR data in our current study. The majority of genes demonstrated a robust response to BRA and IAA treatments in leaf tissues, with no significant expression change observed in roots, suggesting the response of HanSAMS genes to hormones is tissue-specific. Expression analyses under abiotic stresses demonstrated diverse expression profiles of HanSAMS genes, with HanSAMS5 showing significant upregulation in response to both drought and salt stresses.DiscussionThis comprehensive genomic and expression analysis provides valuable insights into the SAMS gene family in sunflowers, laying a robust foundation for future functional studies and applications in crop improvement for stress resilience.

PP2 gene family in Phyllostachys edulis: identification, characterization, and expression profiles.

Phloem protein 2 (PP2), a dimeric lectin, is known for its involvement in plant responses to biotic and abiotic stresses. However, research on PP2 proteins in Moso bamboo is lacking. In this study, comprehensive genome-wide analysis of the PP2-like gene family was conducted in Moso bamboo (Phyllostachys edulis), which has a significant economic and ecological value. Using HMMER3 search and InterPro domain analysis, 23 PP2-like genes (PhePP2-1 to PhePP2-23) were identified in the P. edulis genome. These genes were distributed across 12 chromosomal scaffolds, with proteins ranging from 216 to 556 amino acids in length. Phylogenetic analysis, including 163 PP2 proteins from eight plant species, revealed six distinct groups, with Group III and Group V being the largest. Gene structure and motif analyses indicated conserved domains across the PhePP2 proteins. In addition, Cis-element analysis of the promoter regions highlighted their potential regulatory roles in hormone, stress, and light responses. Expression pattern analysis using RNA-seq data showed differential expression of PhePP2 genes under drought, salt, salicylic acid, and abscisic acid treatments, indicating their involvement in stress response pathways. Furthermore, qPCR validation in different tissues and organs of Moso bamboo confirmed the expression profiles of the selected PhePP2 genes. This study provides a comprehensive understanding of the functional roles of PP2-like genes in Moso bamboo and insights into their potential applications in enhancing stress tolerance and growth in plants.

Elucidating common biomarkers and pathways of osteoporosis and aortic valve calcification: insights into new therapeutic targets.

Osteoporosis and aortic valve calcification, prevalent in the elderly, have unclear common mechanisms. This study aims to uncover them through bioinformatics analysis. Microarray data from GEO was analyzed for osteoporosis and aortic valve calcification. Differential expression analysis identified co-expressed genes. SVM-RFE and random forest selected key genes. GO and KEGG enrichment analyses were performed. Immunoinfiltration and GSEA analyses were subsequently performed. NetworkAnalyst analyzed microRNAs/TFs. HERB predicted drugs, and molecular docking assessed targeting potential. Thirteen genes linked to osteoporosis and aortic valve calcification were identified. TNFSF11, KYNU, and HLA-DMB emerged as key genes. miRNAs, TFs, and drug predictions offered therapeutic insights. Molecular docking suggested 17-beta-estradiol and vitamin D3 as potential treatments. The study clarifies shared mechanisms of osteoporosis and aortic valve calcification, identifies biomarkers, and highlights TNFSF11, KYNU, and HLA-DMB. It also suggests 17-beta-estradiol and vitamin D3 as potential effective treatments.

LiaR-dependent gene expression contributes to antimicrobial responses in group A Streptococcus.

The ability to sense and respond to host defenses is essential for pathogen survival. Some mechanisms involve two-component systems (TCSs) that respond to host molecules, such as antimicrobial peptides (AMPs), and activate specific gene regulatory pathways to aid in survival. Alongside TCSs, bacteria coordinate cell division proteins, chaperones, cell wall sortases, and secretory translocons at discrete locations within the cytoplasmic membrane, referred to as functional membrane microdomains (FMMs). In group A Streptococcus (GAS), the FMM or "ExPortal" coordinates protein secretion, cell wall synthesis, and sensing of AMP-mediated cell envelope stress via the LiaFSR three-component system. Previously, we showed that GAS exposure to a subset of AMPs (α-defensins) activates the LiaFSR system by disrupting LiaF and LiaS co-localization in the ExPortal, leading to increased LiaR phosphorylation, expression of the transcriptional regulator SpxA2, and altered GAS virulence gene expression. The mechanisms by which LiaFSR integrates cell envelope stress with responses to AMP activity and virulence are not fully elucidated. Here, we show the LiaFSR regulon is comprised of genes encoding SpxA2 and three membrane-associated proteins: a PspC domain-containing protein (PCP), the lipoteichoic acid-modifying protein LafB, and the membrane protein insertase YidC2. Our data support that phosphorylated LiaR induces transcription of these genes via a conserved operator, whose disruption attenuates GAS virulence and increases susceptibility to AMPs in a manner primarily dependent on differential expression of SpxA2. Our work expands our understanding of the LiaFSR regulatory network in GAS and identifies targets for further investigation of mechanisms of cell envelope stress tolerance contributing to GAS pathogenesis.

Comparative meta-analysis of transcriptomic studies in spinal muscular atrophy: comparison between tissues and mouse models.

Spinal Muscular Atrophy (SMA), a neuromuscular disorder that leads to weakness in the muscles due to degeneration of motor neurons. Mutations in the survival motor neuron 1 (SMN1) gene leads to the deficiency of SMN protein that causes SMA. The molecular alterations associated with SMA extends across the transcriptome and proteome. Although several studies have examined the transcriptomic profile of SMA, the difference in experimental settings across these studies highlight the need for a comparative meta-analysis to better understand these differences. We conducted a systematic comparative meta-analysis of publicly available gene expression data from six selected studies to elucidate variations in the transcriptomic landscape across different experimental conditions, including tissue types and mouse models. We used both microarray and RNA-seq datasets, retrieved from Gene Expression Omnibus (GEO) and ArrayExpress (AE). Methods included normalization, differential expression analysis, gene-set enrichment analysis (GSEA), network reconstruction and co-expression analysis. Differential expression analysis revealed varying numbers of differentially expressed genes ranging between zero and 1,655 across the selected studies. Notably, the Metallothionein gene Mt2 was common in several of the eight comparisons. This highlights its role in oxidative stress and detoxification. Additionally, genes such as Hspb1, St14 and Sult1a1 were among the top ten differentially expressed genes in more than one comparison. The Snrpa1 gene, involved in pre-mRNA splicing, was upregulated in the spinal cord and has a strong correlation with other differentially expressed genes from other comparisons in our network reconstruction analysis. Gene-set enrichment analysis identified significant GO terms such as contractile fibers and myosin complexes in more than one comparison which highlights its significant role in SMA. Our comparative meta-analysis identified only few genes and pathways that were consistently dysregulated in SMA across different tissues and experimental settings. Conversely, many genes and pathways appeared to play a tissue-specific role in SMA. In comparison with the original studies, reproducibility was rather weak.

A pyroptosis-related lncRNA risk model for the prediction of prognosis and immunotherapy response in head and neck squamous cell carcinoma

BackgroundRecent research has highlighted pyroptosis as a key factor in cancer progression. This study aims to explore the association between pyroptosis-related signatures and overall survival (OS) in head and neck squamous cell carcinoma (HNSC) and develop a pyroptosis-related long non-coding RNA (lncRNA) risk model to predict prognosis and response to immunotherapy in HNSC.MethodsWe extracted expression data for 18 pyroptosis-related genes and identified lncRNA probes specific to HNSC by using datasets from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA). Consensus clustering was performed to categorize HNSC patients into distinct subtypes. A six-lncRNA risk score model was constructed through univariate and least absolute shrinkage and selection operator (LASSO) Cox regression analyses. We evaluated the predictive ability of the lncRNA model for patients’ survival and immunotherapy response. Gene expression was evaluated using immunohistochemistry (IHC) and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR).ResultsOur analysis revealed two distinct pyroptosis-related subtypes in HNSC patients, Cluster A and Cluster B. Notably, patients in Cluster B exhibited significantly poorer overall survival compared to those in Cluster A. Through differential expression analysis, we identified six lncRNAs (AC002331.1, CTA-384D8.35, RP11-291B21.2, AC006262.5, RP1-27K12.2, and RP11-54H7.4) that were differentially expressed between these clusters. A 6-lncRNA risk score model was developed, which successfully stratified patients into high- and low-risk groups with distinct overall survival outcomes. Validation using RT-qPCR confirmed the differential expression of these six lncRNAs in HNSC tumor tissues compared to adjacent normal tissues, we found that the expression of CTA-384D8.35 was significantly increased in the tumor group (t=-6.203, P<0.001). Furthermore, the 6-lncRNA risk score model demonstrated a significant association with patient response to immunotherapy, with the low-risk group exhibiting a higher objective response rate to immune checkpoint blockade (ICB) therapy and longer survival compared to the high-risk group.ConclusionOur study underscores the role of pyroptosis signatures in HNSC prognosis and identifies two distinct pyroptosis subtypes with differing survival outcomes. The six-lncRNA risk score model offers a valuable tool for predicting patient prognosis and potential benefits from ICB therapy. These findings highlight the importance of pyroptosis and associated lncRNAs in the tumor microenvironment, paving the way for novel targeted therapies in HNSC.

Abstract 4125893: Deciphering the Mechanism of Action of Drug Combinations by Boolean Modeling of Transcriptomes: a Case Study of Atorvastatin/Simvastatin and Ezetimibe

Introduction: Drug combinations offer increased therapeutic efficacy and reduced toxicity and plays a vital role in treating chronic complex diseases. Understanding a drug combination's mechanism of action (MoA) can provide important insights into its therapeutic efficacy. The MoA of many FDA-approved drugs, however, often remains unclear. Methods: In this study, we investigated the combination of a statin [atorvastatin (ATO) or simvastatin (SIM) plus ezetimibe (EZE)] and utilized drug-treated RNA-seq transcriptome data from SOAT2-only-HepG2 cells (treated with ATO 5 umol/L, EZE, 25 umol/L, their combination or vehicle) and from liver biopsies of patients with uncomplicated cholesterol gallstone disease in the Stockholm Study (a single-blind, randomized trial with SIM 80 mg daily, EZE 10 mg daily, their combination, or placebo) to decipher the underlying molecular mechanisms of the drug combination. We proposed a Boolean logical modeling framework to simulate the MoA of a drug combination. Specifically, fourteen two-variable Boolean models were used to describe the combinatory relationships of ATO/SIM and EZE. Thereafter, a pattern matching approach was applied to associate drug-induced differentially expressed genes with the idealized differential expression templates derived from Boolean models. Results: We found 1,560 and 594 genes differentially expressed in at least one treatment condition in SOAT2-only-HepG2 cells and liver biopsies, respectively. Our analysis revealed both expected and novel combinatorial modes of ATO/SIM and EZE. For example, ATO independently activates downstream genes (i.e. B 4 (ATO,EZE)=ATO), and ATO and EZE synergistically inhibits downstream genes (i.e. B 15 (ATO,EZE)=NOT (ATO AND EZE)) , as the two most prevalent MoAs in SOAT2-only-HepG2 cells (Figure 1). Similarly, the combination of SIM and EZE synergistically inhibiting downstream genes was also the most prevalent MoA in the liver biopsies. We mapped the downstream genes of each combinatorial mode to the human interactome and obtained underlying subnetworks, which are important for understanding the therapeutic effects of the drug combination. Functional enrichment and disease-association analyses of the downstream suggest the additional therapeutic indications of the drugs. Conclusion: Drug-induced transcriptomes are informative in deciphering the MoA of drug combinations using Boolean logical modeling. This framework can be easily extended to the combinations of three drugs.

Pan-cancer landscape of DCTPP1 and preliminary exploration of DCTPP1 in renal clear cell carcinoma.

dCTP pyrophosphatase 1 (DCTPP1) is widely expressed in tumors and the immune system and plays a critical role in human cancer progression. However, multi-omics characterization of DCTPP1 and its role in prognosis and immune microenvironment of tumor patients has not been explored. We collected data from 33 cancers and comprehensively analyzed the aberrant expression, prognostic role, pathway enrichment, immune microenvironment, and association with drug sensitivity of DCTPP1 in cancers, as well as the prediction of patients' immunotherapeutic response to ICIs and targeted small molecule drugs. Finally, we experimentally confirmed the role of DCTPP1 in renal clear cell carcinoma. We discovered that DCTPP1 has a differentiable expression and a diagnostic biomarker significance in cancer. Additionally, we discovered that DCTPP1 is important for the tumor microenvironment and pan-cancer. TMB and MSI are frequent immunological checkpoints that are significantly correlated with DCTPP1 expression. Patients who express high levels of DCTPP1 have greater rates of survival and therapeutic response following immunotherapy. Ultimately, it was discovered that renal clear cell carcinoma cells' invasion and proliferation were suppressed by DCTPP1 knockdown. Our findings demonstrate the significant potential of DCTPP1 as a biomarker for prognosis and immunotherapeutic response, which may pave the way for more investigation into the mechanism of tumor infiltration and DCTPP1's potential for cancer therapy.

Molecular mechanisms underlying floral fragrance in Camellia japonica ‘High Fragrance’: a time-course assessment

Camellia japonica ‘High Fragrance’ is a camellia hybrid known for its unique and intense floral scent. The current understanding of the dynamic changes in its fragrance and the underlying mechanisms are still limited. This study employed a combination of metabolomic and transcriptomic approaches to reveal the characteristics of the metabolites involved in the remarkable fragrance of this camellia and their biosynthetic mechanisms along three flower developmental stages (flower bud, initial bloom, and full bloom). Among the 349 detected volatile organic compounds (VOCs), the majority were terpenes (57, 16.33%) and esters (53, 15.19%). Of these, 136 VOCs exhibited differential accumulation over time. Transcriptomic data from floral organs at different flowering stages identified 56,303 genes, with 13,793 showing significant differential expression. KEGG enrichment analysis revealed 57, 91, and 33 candidate differential genes related to the biosynthesis of terpenes, phenylpropanoids, and fatty acid derivatives, respectively. This indicates that terpenes, esters, and their related synthetic genes might play a crucial role in the formation of ‘High Fragrance’ characteristics. During the entire flowering process, the majority of genes exhibited an elevated expression pattern, which correlated with the progressive accumulation of VOCs. Interestingly, the expression patterns of the differentially expressed genes in the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, associated with terpene synthesis, showed opposite trends. A transcriptional-metabolic regulatory network linking terpenoid compounds, related synthetic enzymes, and potential transcription factors could be outlined for ‘High Fragrance’ camellia, thus providing a theoretical basis for further exploring these events and breeding more fragrant camellias.

Abstract 4136692: Spatial Transcriptomics Identifies Novel Clinical Markers to Classify the Etiology of Rare and Aggressive Cardiac Sarcomas

Background: Cardiac sarcomas, such as leiomyosarcoma (LMS), synovial sarcoma (SS), and undifferentiated pleomorphic sarcoma (UPS), lack sufficient diagnostic markers for early diagnosis and tailored therapeutic interventions. Understanding their molecular signatures with spatial-temporal resolution compared to normal heart tissue is crucial. Hypothesis: Spatial transcriptomics will reveal a comprehensive set of novel clinical markers, including changes in cellular composition, stemness, and metabolic profiles unique for each cardiac sarcoma subtypes. Aims: Identify novel molecular markers for the accurate diagnosis of cardiac sarcoma subtypes. Methods: Using 10X Visium spatial transcriptomics, we analyzed three cardiac sarcoma subtypes (LMS, SS, UPS) and compared them with normal heart tissue. Differential expression analysis identified novel target genes suitable for clinical disease stratification. Subsequently, machine learning models reveal an unbiased transcriptomic profile to distinguish normal from sarcoma tissues. Finally, immunohistochemistry analysis validated the differential marker gene expression. Results: We identify several novel clinical markers, such as IGKC, PCOLCE, NET1, TLE2, MDFI, BGN, TNNC1, and CNN3, that classify normal heart tissue from sarcoma subtypes. Machine learning models and immunostaining analysis confirm the transcriptomic signature in each sarcoma subtype. Pathway enrichment analysis show LMS enriched in antigen processing and B cell-mediated immunity, SS in protein folding and response to incorrect proteins, and UPS in axon development and Wnt signaling regulation. Trajectory Reconstruction Analysis indicate higher stemness scores in all sarcomas, with SS highest, followed by UPS and LMS. Additional cell cycling analysis show LMS cells in G2M phase, SS in G1, and UPS in S phase. SS express a high number of cancer stem markers (EZH2, BMI1, KDM5B, LGR5, HIF1A), while UPS express KDM5B and CTNNB1, and LMS show fewer stem-related marker genes (EZH2, BMI1, KDM5B, CTNNB1). Both SS and LMS express CD44. Glucose and oxidative pathways are also differentially regulated in each sarcoma subtype. Conclusion: Spatial transcriptomics uncovers distinct clinical markers, stemness characteristics, and metabolic activity profiles in cardiac sarcomas, offering insights for fast, accurate and improved diagnostics and therapeutics.

Abstract 4141486: Site-Specific Transcriptomic Patterns and Cell Types Associated with Hypertension

Introduction: The biology of early human hypertensive heart disease (HHD) is largely unexplored due to limited cardiac tissue access. Site-specific transcriptomics of human cardiac tissue before development of overt HHD offers a unique vantage to understand the biology of HHD in human cardiac tissue. Hypothesis: We hypothesize that transcriptomic signatures and cell subtype proportions differ in human heart tissue from HTN donors compared to normotensive (NTN) donors in a site-specific manner. Methods: We performed bulk RNA-seq on rRNA-depleted libraries from left (LV) and right (RV) mid-ventricular-wall tissue (average depth 114 million reads/sample) collected postmortem from HTN (n=3, 67% Black/Latinx) and NTN (n=2, 50% Black/Latinx) donors. We performed single-nucleus RNA-seq (snRNA-Seq) on cardiac tissue from the same donors (average of 4,062 LV nuclei). Differential gene expression, lineage identification, and Gene Set Enrichment Analysis (GSEA) were performed with publicly available software. Results: All donors were female with matched postmortem intervals (time from death to tissue preservation; HTN 19±9 hours, NTN 18±6 hours). HTN donors were older (65±7 years) than NTN donors (36±16 years). A total of 416 genes were associated with HTN (Wald test, FDR q <0.05) across both ventricles (correcting for site). When tested separately, 72 (LV) or 83 (RV) genes were associated with HTN (FDR q <0.05), with 13 common genes, indicating site-specific differences. GSEA of genes ranked by Wald statistic computed across both ventricles showed that transcripts involved in single-stranded DNA helicase activity, branched-chain amino acid catabolism, and NAD-dependent deacetylase activity were coordinately associated with HTN (FDR q <0.05). snRNA-seq revealed 9 cell types in these samples, including immune cells and chamber-specific cardiomyocytes (CMs) with higher proportions of LV CMs and lymphocytes associated with HTN compared to NTN. MYH6 and MYL7 expression was unchanged with respect to HTN by RNA-seq but lower in LV HTN CMs compared to NTN by snRNA-seq, suggesting dysregulation of contractile programming in HTN LV CMs. Conclusion: HTN is associated with altered cardiomyocyte/lymphocyte ratios, and site-specific differential expression of cardiac contractile elements. Further study is required to validate these findings with additional samples and elucidate mechanisms driving transcriptional program differences in the hypertensive human heart.

Abstract 4137829: Comprehensive Single-Cell Atlas of the Human Lung in Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a complex cardiopulmonary disease characterized by progressive vascular remodeling. Although various immune and vascular cell populations have been implicated in the pathobiology of PAH, a comprehensive assessment of the various cell types in human PAH lungs is still lacking. Methods: Single-nucleus RNA sequencing was performed on lung samples from the Pulmonary Hypertension Breakthrough Initiative using the 10x Genomics Chromium Fixed RNA Profiling workflow. Rigorous quality control measures were implemented, including the detection and removal of ambient RNA using CellBender and doublets using scDblFinder. Data normalization and dimensionality reduction were performed using Seurat, and alignment by sample and sequencing batch was conducted using Harmony. Cells were annotated by mapping onto the Human Lung Cell Atlas using Azimuth. Differential gene expression analysis was conducted using a robust pseudobulk approach with edgeR to limit false positive findings. Gene set enrichment analysis was performed using the Hallmark pathways. Results: Lung samples from a total of 67 individuals were profiled, including 42 PAH and 25 age- and sex-matched control lungs. After quality control filtering, over 800,000 cells were captured, with a median of 12,000 cells per sample. Clustering uncovered over 30 cell types, representing endothelial, stromal, immune, and epithelial lineages of the lung. A median of 2,500 transcripts and 1,500 genes were detected per cell. Differentially expressed genes between PAH and control were detected in all major cell populations (FDR < 0.05). Comparative analysis across all cell types revealed interferon response in monocyte subpopulations and endothelial-to-mesenchymal transition in endothelial subpopulations as top upregulated pathways. Conclusion: Comprehensive single-nucleus profiling of human lungs in PAH revealed extensive gene and pathway dysregulation across diverse cell types. This rich dataset provides a valuable resource for the research community, offering opportunities to advance and refine our understanding of the cellular pathobiology of PAH.

Novel Differentially Expressed LncRNAs Regulate Artemisinin Biosynthesis in Artemisia annua

Long non-coding RNAs (lncRNAs) are crucial in regulating secondary metabolite production in plants, but their role in artemisinin (ART) biosynthesis, a key anti-malarial compound from Artemisia annua, remains unclear. Here, by investigating high-artemisinin-producing (HAP) and lowartemisinin-producing (LAP) genotypes, we found that the final artemisinin content in A. annua is influenced by the quantity of the precursor compounds. We report on RNA deep sequencing in HAP and LAP genotypes. Based on the application of a stringent pipeline, 1419 novel lncRNAs were identified. Moreover, we identified 256 differentially expressed lncRNAs between HAP and LAP. We then established correlations between lncRNAs and artemisinin biosynthesis genes in order to identify a molecular framework for the differential expression of the pathway between the two genotypes. Three potential lncRNAs (MSTRG.33718.2, MSTRG.30396.1 and MSTRG.2697.4) linked to the key artemisinin biosynthetic genes (ADS: Amorpha-4,11-diene synthase, DXS: 1-deoxy-D-xylulose-5-phosphate synthase, and HMGS: 3-hydroxyl-3-methyglutaryl CoA synthase) were detected. Importantly, we observed that up-regulation of these lncRNAs positively modulates the target artemisinin biosynthetic genes, potentially leading to high artemisinin biosynthesis in HAP. In contrast, BAS (beta-amyrin synthase), which is involved in the artemisinin competing pathway, was strongly down-regulated in HAP compared to LAP, in line with the expression pattern of the linked lncRNA MSTRG.30396.1. By identifying and characterizing lncRNAs that are potentially linked to the regulation of key biosynthetic genes, this work provides new insights into the complex regulatory networks governing artemisinin production in A. annua. Such findings could pave the way for innovative approaches in metabolic engineering, potentially enhancing artemisinin yields and addressing challenges in sustainable production.

Dependence of eigenvalues for higher odd-order differential operator with general transmission conditions

In this paper, we consider the dependence of eigenvalues on the parameter for higher odd-order differential operator with general boundary conditions and general transmission conditions. By defining an appropriate Hilbert space and its inner product, we prove that the self-adjointness of the operator associated with the problem. In addition, we show that the eigenvalues are not only continuously but also smoothly dependent on the parameters of the problem, and give the corresponding differential expressions. In particular, giving the general formula for derivative formulas of the eigenvalues with respect to the left and right sides of the inner discontinuity point.

Abstract 4118949: Factors Influencing Left Ventricular Function Recovery Following Left Ventricular Assist Device Implantation in Pediatric Dilated Cardiomyopathy: Insights from Single-Cell RNA Sequencing Analysis

Background and Objective: Heart transplantation is a recognized treatment for pediatric severe heart failure, but long-term issues necessitate alternative treatments. Some dilated cardiomyopathy (DCM) cases show left ventricular function recovery after LVAD implantation. This occurrence could lead to new treatments, but mechanisms are unclear. This study explores preoperative molecular factors interacting with LVAD's effects using single-cell transcriptomics on clinical samples. Methods: Twenty-five pediatric DCM patients underwent Berlin Heart EXCOR (BHE) implantation at our institution since 2013. Those with left ventricular ejection fraction (LVEF) improving by >20% were in the Recovery group (R), others in Non-Recovery (NR). Single-cell RNA-sequence was performed on myocardial tissue from six patients (three per group). Differential gene expression and pathway analysis were conducted for each cell type. Findings were validated by comparing them with histological evaluation or clinical examination in all 25 cases. Results: R: 10 patients; NR: 15. R's weight was 6.3 kg, NR's 9.9 kg (p=0.049); LVEF: R 23%, NR 16.5% (p=0.61). In pseudobulk analysis of cardiomyocytes, upregulated genes in R included NPPB, MYL7, PCDH9. KEGG analysis indicated activation of Tight Junction and ECM receptor pathways in R. In fibroblasts, major extracellular matrix proteins were higher in R, GSL gene expression in NR. Plasma Brain Natriuretic Peptide (BNP) at BHE implantation was significantly higher in R; 2913 [1836-3990 (95% CI)] vs 1278 [667-1888] pg/ml (Image1,2). No differences in tissue fibrosis rates were found between the groups. Conclusion: Alterations in gene expression patterns in some cell clusters, including cardiomyocytes, may contribute to left ventricular function recovery post-BHE implantation in pediatric DCM patients. Plasma BNP levels correlated with gene expression alterations, suggesting both a potentially protective effect on cardiomyocytes and a useful marker of LV functional recovery. Further validation of these candidate genes may elucidate intracellular molecular mechanisms.

Transcriptomic Changes During the Replicative Senescence of Human Articular Chondrocytes

Aging is a major risk factor for osteoarthritis (OA), but the specific mechanisms connecting aging and OA remain unclear. Although chondrocytes rarely divide in adult articular cartilage, they undergo replicative senescence in vitro, offering a model to study aging-related changes under controlled conditions. OA cartilage was obtained from an 80-year-old male and a 72-year-old female, while normal cartilage was sourced from a 26-year-old male. Chondrocyte cultures were established and sub-cultured to their Hayflick limit. Bulk RNA sequencing on early- and late-passage human articular chondrocytes identified transcriptomic changes associated with cellular aging. Early-passage OA chondrocytes already showed senescent phenotypes, unlike normal chondrocytes. All three cultures underwent 30 population doublings before replicative exhaustion, at which point all cells displayed senescence. During this process, cells lost their ability to form cartilaginous pellets. Differential gene expression analysis revealed distinct transcriptomic profiles between early- and late-passage chondrocytes and between normal and OA-derived cells. Genes related to matrix synthesis, degradation, inflammation, and the senescence-associated secretory phenotype (SASP) showed significant expression changes. Despite being a small pilot study, these findings suggest that further research into the molecular and metabolic changes during chondrocyte senescence could provide valuable insights into OA pathobiology.

Abstract 4119210: Differential Proteomic Expression of IGFßP-1 and IGFßP-2 in Primary Angioplasty for Acute Myocardial Infarction

Introduction: Insulin growth factor (IGF) binding protein-1 and 2 (IGFBP-1 and 2) are implicated in acute myocardial infarction (AMI) and have been suggested as predictors of mortality and development of heart failure. The IGF axis has been shown to play roles in cell proliferation and apoptosis. We analysed the serum IGFBP-1 and 2 levels in AMI before and after percutaneous coronary intervention (PCI) and correlated this with cardiac magnetic resonance (CMR) imaging to better establish their significance as biomarkers in AMI. Methods: Patients with AMI who presented to Royal Free Hospital, London, were recruited for proteomic analysis (O link Proximity Extension Immunoassay) at presentation, 24h and 72h following primary PCI. All underwent both admission and follow-up CMR at 6 months to assess for myocardial oedema, left ventricular volumes and function, and assessment of scar. Results: Proteomic evaluation in 20 AMI patients (male 95%; median age 59) revealed significant reduction in the average IGFBP-1 values at discharge compared with presentation, from 5.93 to 4.52 (24% reduction, p=0.003). Conversely, the average IGFBP-2 values at discharge significantly increased from 7.66 to 8.29 (8% increase, p=0.001). Higher discharge IGFBP-2 value was also correlated with increased indexed left ventricle end diastolic volume (LVEDV) at 6-month follow-up (R=0.54, p=0.032). Discussion: IGFBP-1 and 2 showed significant change following intervention for AMI. IGFBP-1 is a stress hormone, which is directly and indirectly activated by cytokines, dysglycaemia and vasopressin activation. Its subsequent reduction following PCI in AMI indicates relative resolution of inflammation, but this was not correlated with changes in infarct size or LV function at follow-up. IGFBP-2 showed significant upregulation, and higher values at discharge were significantly correlated with increased LDEDV at follow-up, suggesting adverse clinical outcomes within this setting. This data suggests differential roles of IGFBP-1 and 2 in the pathogenesis of AMI.

Investigating bacteria-induced inflammatory responses using novel endometrial epithelial gland organoid models

IntroductionThe endometrium plays a crucial role in early human pregnancy, particularly in embryo implantation, survival, and growth. However, invasion and infection by pathogens can lead to endometritis, infertility, and poor reproductive outcomes. Understanding the mechanisms of endometritis and its impact on fertility remains limited. An infection model using patient-derived endometrial epithelial gland organoids (EEGOs) was established to advance in vitro studies on endometritis and related infertility.MethodsAn EEGOs infection model was constructed and characterized from human endometrium, treating the organoids with estrogen and progesterone to observe changes in the proliferative and secretory phases. The organoids were infected with E. coli, and the release of inflammatory cytokines in the supernatant was detected using ELISA. RNA-seq was employed to analyze the differences before and after E. coli treatment, and differential gene mRNA expression was validated using real-time quantitative PCR. Additionally, the effect of E2 in alleviating inflammation was assessed through markers of receptivity (PAEP, LIF, ITGβ), proliferation (Ki67), and barrier repair (ZO-1).ResultsThe constructed human EEGOs exhibited long-term expansion capability, genetic stability, and characteristic hormonal responses, strongly expressing epithelial markers (MUC1, E-Cadherin). After E. coli infection, the expression levels of inflammatory cytokines TNF-α, IL-8, and IFN-γ increased significantly (P < 0.05). RNA-seq indicated that the MAPK signaling pathway was activated post-infection, with increased expression levels of heat shock proteins and transcription factor mRNA. E2 treatment post-infection significantly decreased the mRNA expression of inflammatory genes IL-1β, IL8, IL6 and TNF-α compared to the E. coli infected group (P < 0.05). Additionally, the expression of genes related to receptivity, proliferation, and barrier repair was enhanced in the E2-treated organoids.ConclusionsOur findings demonstrate that patient-derived EEGOs are responsive to bacterial infection and are effective models for studying host-pathogen interactions in bacterial infections. These organoids revealed the anti-inflammatory potential of E2 in alleviating E. coli-induced inflammation, providing insights into the mechanisms of endometritis and its impact on infertility. The study supports the use of EEGOs as valuable tools for understanding endometrial health and developing targeted treatments.

Abstract 4134900: Cellular Crosstalk in Endocardial Fibroelastosis: NRG Signaling Dysregulation Contributes to Fibrosis and Myocardial Dysfunction in Hypoplastic Left Heart Complex

Background: Endocardial fibroelastosis (EFE) is marked by a thickened fibro-elastic layer derived by endothelial-to-mesenchymal transition (EndMT), leading to restrictive myocardial function and poor cardiac growth, complicating biventricular repair in hypoplastic left heart complex (HLHC) patients. Neuregulin (NRG) signaling, crucial in EndMT and myocardial development, is implicated in various cardiomyopathies and heart failure, and its dysregulation may drive EFE pathogenesis. Hypothesis: NRG signaling dysregulation influences the cellular and molecular mechanisms of EFE. Methods: Single nucleus RNA sequencing (snRNA-seq) was conducted on human left ventricular (LV) tissue resected from 7 EFE patients (0.4-15 years) and 4 non-congenital heart disease (CHD) postmortem human controls (0.02-16 years). Differential gene expression was analyzed using the Wilcoxon Rank Sum test. We used CellChat, a computational tool for analyzing intercellular communication networks, to identify signaling changes via quantitative contrasts and joint manifold learning. Results: We identified 11 cell types in 64,963 nuclei. In EFE samples compared to non-CHD samples, NRG1-expressing fibroblasts increased from 3.4% to 21.2% (P<0.0001), and NRG2-expressing cardiomyocytes from 11.9% to 45.9% (P<0.001). CellChat analysis revealed a denser NRG signaling network in EFE, with enhanced interactions among cardiomyocytes, endocardial cells, fibroblasts, mast cells, and smooth muscle cells (Fig. 1A-B). Cardiomyocytes emerged as significant senders of NRG signaling in EFE, while endocardial cells, though still key senders, showed reduced influence. Fibroblasts became strong mediators, receivers, and influencers, smooth muscle cells emerged as prominent receivers, and mast cells gained roles as mediators and influencers (Fig. 1 C-D). These changes indicate a complex NRG signaling network in EFE, highlighting altered cellular interactions driving disease progression. Conclusion: Dysregulated NRG signaling in EFE contributes to pathological cellular crosstalk, fibrosis, and myocardial dysfunction, potentially hindering successful biventricular repair in HLHC patients. Targeting NRG pathways could represent a novel therapeutic approach. These findings offer promising implications for affected patients, potentially improving outcomes. Future research should explore additional molecular pathways involved in EFE and assess the therapeutic efficacy of targeting NRG signaling.