Marker Genes Research Articles

A putative new Besnoitia species in the southern black-eared opossum Didelphis aurita

Besnoitia spp. are cyst-forming coccidian parasites with a broad host range, infecting various wild and domestic animal species. Northamerican opossums (Didelphis virginiana) are severely affected by the infection with B. darlingi. This study presents a case of infection with Besnoitia in a road-killed female southern black-eared opossum (Didelphis aurita) in Misiones, Argentina. Many 0.5 to 1 mm cysts were observed in several muscles and visceral organs and were microscopically identified in skeletal muscles, tongue, and heart. Histological analysis disclosed multiple spherical cysts with a myriad of bradyzoites like-cells and a well-defined cyst wall. A small number of degenerate and ruptured cysts, surrounded by mild to moderate inflammation were observed. Genomic DNA from an individual cyst and muscle was extracted and ITS1 marker and 18S rRNA gene fragments from sarcocystid protozoa were successfully amplified by PCR and sequenced. The 18S sequence exhibited 100% identity with sequences of B. darlingi and B. oryctofelisi. Comparison of the complete ITS1 sequence (259 bp) revealed an identity of 99.2% with B. oryctofelisi and 97.7% with B. darlingi. This result together with the phylogeny positioning, suggest that the Besnoitia sp. in the present case differ from B. darlingi, being closely related with B. oryctofelisi.

Probe set selection for targeted spatial transcriptomics

AbstractTargeted spatial transcriptomic methods capture the topology of cell types and states in tissues at single-cell and subcellular resolution by measuring the expression of a predefined set of genes. The selection of an optimal set of probed genes is crucial for capturing the spatial signals present in a tissue. This requires selecting the most informative, yet minimal, set of genes to profile (gene set selection) for which it is possible to build probes (probe design). However, current selections often rely on marker genes, precluding them from detecting continuous spatial signals or new states. We present Spapros, an end-to-end probe set selection pipeline that optimizes both gene set specificity for cell type identification and within-cell type expression variation to resolve spatially distinct populations while considering prior knowledge as well as probe design and expression constraints. We evaluated Spapros and show that it outperforms other selection approaches in both cell type recovery and recovering expression variation beyond cell types. Furthermore, we used Spapros to design a single-cell resolution in situ hybridization on tissues (SCRINSHOT) experiment of adult lung tissue to demonstrate how probes selected with Spapros identify cell types of interest and detect spatial variation even within cell types.

Single-cell data revealed the regulatory mechanism of TNK cell heterogeneity in liver metastasis from gastric cancer.

The present work set out to classify cell subpopulations related to liver metastasis from gastric cancer (GC) and the mechanisms of their interactions with other immune cell subpopulations. GC is characterized by a high degree of heterogeneity and liver metastasis. Exploring the mechanism of liver metastasis of GC from the perspective of heterogeneity of the tumor microenvironment (TME) might help improve the efficacy of GC treatment. Based on the cellular subpopulation characteristics of GC with liver metastasis, the regulatory mechanisms contributing to GC progression were analyzed, with special focuses on the roles of signaling pathways, transcription factors (TFs) and ligand-receptor pairs. The GSE163558 dataset was downloaded from the Gene Expression Omnibus (GEO) database to collect single-cell transcriptomic data of GC patients and their metastasis groups for cell clustering and relevant analyses. Differentially expressed genes (DEGs) in the GC and GC liver metastasis groups were screened and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. SCENIC analysis was used to mine TFs that affected cellular subpopulations during liver metastasis from GC. The relative expression levels of TFs in GC were determined using qRT-PCR. Transwell and wound healing assays were utilized to verify the regulation of the TFs on the migration and invasion of GC cells. Interaction network between the cellular subpopulations was developed applying CellChat. Single-cell clustering was performed to group six major cell subpopulations, namely, Myeloid cells, B cells, Mast cells, Epithelial cells, Fibroblasts, and TNK cells, among which the number of TNK cells was significantly increased in the GC liver metastasis group. Differentially enriched pathways of TNK cells between GC and GC liver metastasis groups mainly included IL-17 and Pi3k-Akt signaling pathways. TNK cell subsets could be further categorized into CD8 T cells, Exhausted T cells, NK cells, NKT cells, and Treg cells, with the GC liver metastasis group showing significantly more CD8 T cells and NKT cells. FOS and JUNB were the TFs of TNK cell marker genes that contributed to liver metastasis from GC and the invasion and migration of GC cell lines. Significant differences in immune cell communication ligand-receptor pairs existed between the GC and GC liver metastasis groups. This study revealed the critical role of TNK cell subsets in GC with liver metastasis applying single-cell transcriptomics analysis. The findings provided an important theoretical basis for developing novel therapies to inhibit liver metastasis from GC.

Identification of apelin/APJ signaling dysregulation in a human iPSC-derived granulosa cell model of Turner syndrome.

The interaction between germ cells and somatic cells in the ovaries plays a crucial role in establishing the follicle reserve in mammals. Turner syndrome (TS) predominantly affects females who have a partial or complete loss of one X chromosome. Our understanding of the role that granulosa cells (GCs) play in TS disease progression and pathogenesis remains limited. In this study, we achieved GC differentiation efficiency of up to 80% from iPSCs. When attempting to replicate the differentiation process of embryonic granulosa cells, we observed the downregulation of specific genes-GATA4, FOXL2, AMHR2, CYP19A1, and FSH-in Turner syndrome-derived granulosa cells (TS-GCs). Additionally, we identified dysregulation of the cell cycle in TS-GCs. To uncover the endogenous defects in TS-GCs, we compared global transcriptome patterns between iPSC-derived granulosa cells from healthy individuals and those with Turner syndrome. The apelin/APJ pathway exhibited differential signaling between the healthy and TS groups. Supplementation with apelin ligands and activation of apelin/APJ downstream signaling via Akt/PKB restored cell cycle progression and marker gene expression. We hypothesize that during early embryonic development, failures in apelin/APJ signaling in GCs of Turner syndrome patients lead to abnormalities in ovarian development, ultimately resulting in early oocyte loss and infertility.

Multi-omics with dynamic network biomarker algorithm prefigures organ-specific metastasis of lung adenocarcinoma.

Efficacious strategies for early detection of lung cancer metastasis are of significance for improving the survival of lung cancer patients. Here we show the marker genes and serum secretome foreshadowing the lung cancer site-specific metastasis through dynamic network biomarker (DNB) algorithm, utilizing two clinical cohorts of four major types of lung cancer distant metastases, with single-cell RNA sequencing (scRNA-seq) of primary lesions and liquid chromatography-mass spectrometry data of sera. Also, we locate the intermediate status of cancer cells, along with its gene signatures, in each metastatic state trajectory that cancer cells at this stage still have no specific organotropism. Furthermore, an integrated neural network model based on the filtered scRNA-seq data is successfully constructed and validated to predict the metastatic state trajectory of cancer cells. Overall, our study provides an insight to locate the pre-metastasis status of lung cancer and primarily examines its clinical application value, contributing to the early detection of lung cancer metastasis in a more feasible and efficacious way.

LncRNA CASC11 regulates the progress of delayed fracture healing via sponging miR-150-3p.

Long non-coding RNA (lncRNA) plays a pivotal role in bone regeneration by interaction with microRNAs (miRNAs) and constructing a lncRNA-miRNA regulatory network. This research aimed to elucidate the role of lncRNA CASC11 in the delayed healing process of tibial fractures and to explore its potential regulatory mechanisms. The expression levels of CASC11 and miR-150-3p in serum samples were detected and the predictive capability of CASC11 regarding delayed healing in fracture patients. Furthermore, the study confirmed the accuracy of the binding sites between CASC11 and miR-150-3p. Subsequently, overexpression/interference plasmids of CASC11, along with overexpression plasmids co-transfected with both CASC11 and miR-150-3p, were systematically introduced into MC3T3-E1 cells to investigate their effects on the expression of osteogenic marker genes, as well as their influence on cellular proliferation and apoptosis. The expression levels of CASC11 were significantly elevated, while miR-150-3p levels were markedly decreased in individuals exhibiting delayed fracture healing (P < 0.001). CASC11 was observed to suppress the expression of osteogenic marker genes, inhibit the proliferation of MC3T3-E1 cells, and promote cell apoptosis (P < 0.05). Furthermore, the overexpression of miR-150-3p effectively countered the inhibitory impact of CASC11 on osteogenic differentiation and the promoting effect on cell apoptosis (P < 0.05). The sponging effect of CASC11 on miR-150-3p led to delayed fracture healing. CASC11 emerges as a potential target for treating delayed fracture healing.

Gene expression screening and cell factory engineering for enhancing echinocandin B production in Aspergillus nidulans NRRL8112.

Echinocandin B (ECB) is a key precursor of the antifungal drug anidulafungin and its biosynthesis occurs via ani gene cluster in Aspergillus nidulans NRRL8112. Strain improvement for industrial ECB production has mainly relied on mutation breeding due to the lack of genetic tools. Here, a CRISPR-base-editing tool was developed in A. nidulans NRRL8112 for simultaneous inactivation of the nkuA gene and two marker genes, pryoA and riboB, which enabled efficient genetic manipulation. Then, in-vivo plasmid assembly was harnessed for ani gene expression screening, identifying the rate-limiting enzyme AniA and a pathway-specific transcription factor AniJ. Stepwise titer enhancement was achieved by overexpressing aniA and/or aniJ, and ECB production reached 1.5g/L during 5-L fed-batch fermentation, an increase of ~ 30-fold compared with the parent strain. This study, for the first time, revealed the regulatory mechanism of ECB biosynthesis and harnessed genetic engineering for the development of an efficient ECB-producing strain.

Wolbachia in Antarctic terrestrial invertebrates: Absent or undiscovered?

Interactions between a host organism and its associated microbiota, including symbiotic bacteria, play a crucial role in host adaptation to changing environmental conditions. Antarctica provides a unique environment for the establishment and maintenance of symbiotic relationships. One of the most extensively studied symbiotic bacteria in invertebrates is Wolbachia pipientis, which is associated with a wide variety of invertebrates. Wolbachia is known for manipulating host reproduction and having obligate or facultative mutualistic relationships with various hosts. However, there is a lack of clear understanding of the prevalence of Wolbachia in terrestrial invertebrates in Antarctica. We present the outcomes of a literature search for information on the occurrence of Wolbachia in each of the major taxonomic groups of terrestrial invertebrates (Acari, Collembola, Diptera, Rotifera, Nematoda, Tardigrada). We also performed profiling of prokaryotes based on three marker genes and Kraken2 in available whole genome sequence data obtained from Antarctic invertebrate samples. We found no reports or molecular evidence of Wolbachia in these invertebrate groups in Antarctica. We discuss possible reasons underlying this apparent absence and suggest opportunities for more targeted future research to confirm bacteria's presence or absence.

Ferroptosis-related biomarkers for adamantinomatous craniopharyngioma treatment: conclusions from machine learning techniques

IntroductionAdamantinomatous craniopharyngioma (ACP) is difficult to cure completely and prone to recurrence after surgery. Ferroptosis as an iron-dependent programmed cell death, may be a critical process in ACP. The study aimed to screen diagnostic markers related to ferroptosis in ACP to improve diagnostic accuracy.MethodsGene expression profiles of ACP were obtained from the gene expression omnibus (GEO) database. Limma package was used to analyze the differently expressed genes (DEGs). The intersection of DEGs and ferroptosis-related factors was obtained as differently expressed ferroptosis-related genes (DEFRGs). Enrichment analysis was processed, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), disease ontology (DO), gene set enrichment analysis (GSEA), and Gene Set Variation Analysis (GSVA) analysis. Machine learning algorithms were undertaken for screening diagnostic markers associated with ferroptosis in ACP. The levels of DEFRGs were verified in ACP patients. A nomogram was drawn to predict the relationship between key DEFRG expression and risk of disease. The disease groups were then clustered by consensus clustering analysis.ResultsDEGs were screened between ACP and normal samples. Ferroptosis-related factors were obtained from the FerrDb V2 and GeneCard databases. The correlation between DEFRGs and ferroptosis markers was also confirmed. A total of 6 overlapped DEFRGs were obtained. Based on the results of the nomogram, CASP8, KRT16, KRT19, and TP63 were the protective factors of the risk of disease, while GOT1 and TFAP2C were the risk factors. According to screened DEFRGs, the consensus clustering matrix was differentiated, and the number of clusters was stable. CASP8, KRT16, KRT19, and TP63, were upregulated in ACP patients, while GOT1 was downregulated. CASP8, KRT16, KRT19, TP63, CASP8, and GOT1 affect multiple ferroptosis marker genes. The combination of these genes might be the biomarker for ACP diagnosis via participating ferroptosis process.DiscussionFerroptosis-related genes, including CASP8, KRT16, KRT19, TP63, and GOT1 were the potential markers for ACP, which lays the theoretical foundation for ACP diagnosis.

Arabidopsis GLYI4 Reveals Intriguing Insights into the JA Signaling Pathway and Plant Defense

Plant hormones play a central role in various physiological functions and mediate defense responses against (a)biotic stresses. Jasmonic acid (JA) has emerged as one of the key phytohormones involved in the response to necrotrophic pathogens. Under stressful conditions, plants can also produce small molecules, such as methylglyoxal (MG), a cytotoxic aldehyde. The enzymes glyoxalase I (GLYI) and glyoxalase II primarily detoxify MG. In Arabidopsis thaliana, GLYI4 has been recently characterized as having a crucial role in MG detoxification and emerging involvement in the JA pathway. Here, we investigated the impact of a GLYI4 loss-of-function on the Arabidopsis JA pathway and how MG affects it. The results showed that the glyI4 mutant plant had stunted growth, a smaller rosette diameter, reduced leaf size, and an altered pigment concentration. A gene expression analysis of the JA marker genes showed significant changes in the JA biosynthetic and signaling pathway genes in the glyI4 mutant. Disease resistance bioassays against the necrotroph Botrytis cinerea revealed altered patterns in the glyI4 mutant, likely due to increased oxidative stress. The MG effect has a further negative impact on plant performance. Collectively, these results contribute to clarifying the intricate interconnections between the GLYI4, MG, and JA pathways, opening up new avenues for further explorations of the intricate molecular mechanisms controlling plant stress responses.

Abstract PR011: Advance prostate cancer detection through epigenomic profiling of cell-free DNA

Abstract Introduction: Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease which can be classified into clinically relevant subtypes based on the expression of genes, such as the androgen receptor (AR) and neuroendocrine markers. Neuroendocrine prostate cancer (NEPC), characterized by gain of stem-like and neuroendocrine features and lack of AR expression is a clinically aggressive variant. Due to the lack of adequate biomarkers, NEPC is usually detected at a very advanced stage. There is mounting evidence that molecular subtype changes seen in NEPC are enforced by widespread epigenetic alterations, in particular DNA methylation changes. In this study, we aim to devise a novel DNA methylation-based assay for molecular subtyping and disease monitoring from cell-free DNA (cfDNA). Methods: We analyzed genome wide methylation patterns in 56 prostate cancer patient-derived xenograft (PDX) and 128 mCRPC tumors using array- and sequencing-based assays. We integrated DNA methylation at promoters, gene bodies and transcription factor binding site (TFBS) to determine the landscape of methylation alterations at key lineage specific genes. Using whole genome methylation derived from tissue with matched expression data we developed a deep learning framework to predict gene expression directly from tissue or cfDNA. Using key marker genes, the model was used to discern tumor molecular phenotypes from tissue and cfDNA in three independent cohorts of mCRPC patients using whole genome bisulfite sequencing and low-pass Enzymatic Methyl-Seq (EM-seq). Results: We observed a tight association between promoter, gene body and TFBS methylation with gene expression. Inferring gene expression from methylation for lineage specific markers such as AR, KLK3, ASCL1, INSM1, SRRM4 and DLL3 we classified molecular subtypes from both tissue and cfDNA. Additionally, for AR and ASCL1, we identified core sets of TFBSs whose differential methylation allowed for accurate assay-independent molecular subtype quantification. Applying the optimized quantitative model to mCRPC patients who underwent comprehensive tissue sampling by rapid autopsy we observed accurate subtype classification from both tissue samples and cfDNA for all cases. A similar analytical performance was observed in additional clinical mCRPC cohorts with cfDNA. Conclusion: Whole-genome methylation analysis of cfDNA allows for the prediction of gene expression patterns in tumor tissues, enabling non-invasive tumor subclassification and assessment of therapeutic targets. Citation Format: Mohamed Adil, Brian Hanratty, Pallabi Mustafi, Chitvan Mittal, Helen Richards, Ilsa Coleman, Radhika Patel, Anna-Lisa Doebley, Robert Patton, Eden Cruikshank, Patricia Galipeau, Ruth Dumpit, Martine Roudier, Jin-Yih Low, Navonil Sarkar, Robert Montgomery, Eva Corey, Colm Morrissey, Peter Nelson, Gavin Ha, Michael Haffner. Advance prostate cancer detection through epigenomic profiling of cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR011.

Cas9/guide RNA-based gene-drive dynamics following introduction and introgression into diverse anopheline mosquito genetic backgrounds.

Novel technologies are needed to combat anopheline vectors of malaria parasites as the reductions in worldwide disease incidence has stalled in recent years. Gene drive-based approaches utilizing Cas9/guide RNA (gRNA) systems are being developed to suppress anopheline populations or modify them by increasing their refractoriness to the parasites. These systems rely on the successful cleavage of a chromosomal DNA target site followed by homology-directed repair (HDR) in germline cells to bias inheritance of the drive system. An optimal drive system should be highly efficient for HDR-mediated gene conversion with minimal error rates. A gene-drive system, AgNosCd-1, with these attributes has been developed in the Anopheles gambiae G3 strain and serves as a framework for further development of population modification strains. To validate AgNosCd-1 as a versatile platform, it must perform well in a variety of genetic backgrounds. We introduced or introgressed AgNosCd-1 into different genetic backgrounds, three in geographically-diverse Anopheles gambiae strains, and one each in an An. coluzzii and An. arabiensis strain. The overall drive inheritance, determined by presence of a dominant marker gene in the F2 hybrids, far exceeded Mendelian inheritance ratios in all genetic backgrounds that produced viable progeny. Haldane's rule was confirmed for AgNosCd-1 introgression into the An. arabiensis Dongola strain and sterility of the F1 hybrid males prevented production of F2 hybrid offspring. Back-crosses of F1 hybrid females were not performed to keep the experimental design consistent across all the genetic backgrounds and to avoid maternally-generated mutant alleles that might confound the drive dynamics. DNA sequencing of the target site in F1 and F2 mosquitoes with exceptional phenotypes revealed drive system-generated mutations resulting from non-homologous end joining events (NHEJ), which formed at rates similar to AgNosCd-1 in the G3 genetic background and were generated via the same maternal-effect mechanism. These findings support the conclusion that the AgNosCd-1 drive system is robust and has high drive inheritance and gene conversion efficiency accompanied by low NHEJ mutation rates in diverse An. gambiae s.l. laboratory strains.

The establishment of the anther somatic niche with single cell sequencing.

The anther is the developmental housing of pollen and therefore the male gametes of flowering plants. The meiotic cells from which pollen are derived must differentiate de novo from somatic anther cells and synchronously develop with the rest of the anther. Anthropogenic control over anther development has become crucial for global agriculture so as to maintain inbred lines and generate hybrid seeds of many crops. Understanding the genes that underlie the proper differentiation, developmental landmarks, and functions of each anther cell type is thus fundamental to both basic and applied plant sciences. We investigated the development of the somatic niche of the maize (Zea mays) anther using single-cell RNA-seq (scRNA-seq). Extensive background knowledge on the birth then pace and pattern of cell division of the maize anther cell types and published examples of cell-type gene expression from in situ hybridization allowed us to identify the primary cell types within the anther lobe, as well as the connective cells between the four lobes. We established the developmental trajectories of somatic cell types from pre-meiosis to post-meiosis, identified putative marker genes for the somatic cell types that previously lacked any known specific functions, and addressed the possibility that tapetal cells sequentially differentiate. This comprehensive scRNA-seq dataset of the somatic niche of the maize anther will serve as a baseline for future analyses investigating male-sterile genotypes and the impact of environmental conditions on male fertility in flowering plants.

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.

A mechanistic integration of hypoxia signaling with energy, redox and hormonal cues.

Oxygen deficiency (hypoxia) occurs naturally in many developing plant tissues but can become a major threat during acute flooding stress. Consequently, plants as aerobic organisms must rapidly acclimate to hypoxia and the associated energy crisis to ensure cellular and ultimately organismal survival. In plants, oxygen sensing is tightly linked with oxygen-controlled protein stability of group VII ETHYLENE-RESPONSE FACTORs (ERFVII) which, when stabilized under hypoxia, act as key transcriptional regulators of hypoxia-responsive genes (HRGs). Multiple signaling pathways feed into hypoxia signaling to fine-tune cellular decision making under stress. First, ATP shortage upon hypoxia directly affects the energy status and adjusts anaerobic metabolism. Secondly, altered redox homeostasis leads to reactive oxygen and nitrogen species (ROS and RNS) accumulation, evoking signaling and oxidative stress acclimation. Finally, the phytohormone ethylene promotes hypoxia signaling to improve acute stress acclimation, while hypoxia signaling in turn can alter ethylene, auxin, abscisic acid, salicylic acid and jasmonate signaling to guide development and stress responses. In this Update, we summarize the current knowledge on how energy, redox and hormone signaling pathways are induced under hypoxia and subsequently integrated at the molecular level to ensure stress-tailored cellular responses. We show that some HRGs are responsive to changes in redox, energy and ethylene independently of the oxygen status, and propose an updated HRG list that is more representative for hypoxia marker gene expression. We discuss the synergistic effects of hypoxia, energy, redox and hormone signaling and their phenotypic consequences in the context of both environmental and developmental hypoxia.

Abstract 4113617: Single-cell Analysis Reveals Endothelial Cell CD45 Deficiency Prevents Endothelial-to-Mesenchymal Transition (EndMT) in Atherosclerosis

Introduction: Protein-tyrosine-phosphatase CD45 is exclusively expressed in all nucleated cells of the hematopoietic system but is rarely expressed in endothelial cells (ECs). However, a recent study indicated that activation of the endogenous CD45 promoter in human endothelial colony forming cells induced expression of EndMT marker genes. Also, the CD45 phosphatase inhibitor blocked EndMT activities in TGFβ1-treated ovine mitral valve endothelial cells. EndMT contributes to atherosclerotic pathobiology and is associated with more complex plaques. We identified CD45-positive ECs undergoing EndMT in atherosclerotic ApoE-deficient (ApoE -/- ) mice. Here, we aim to investigate whether endothelial CD45-specific deletion can prevent EndMT in a mouse model of atherosclerosis. Methods and Results: We generated a tamoxifen-inducible EC-specific CD45-deficient mouse strain (EC-iCD45KO) on an ApoE -/- background (C57BL/6) and fed these mice a Western diet for 16 weeks. We isolated and enriched mouse aortic endothelial cells with CD31 beads to perform single-cell RNA-seq. Cells populated 15 major clusters after integrating the single-cell transcriptomic profiles. In the EC-iCD45KO group, the population of endothelial, resident macrophage, and mesenchymal-endothelial transition cells increased while vascular smooth muscle, EndMT, and fibroblast cells decreased. In EndMT cells, EC markers (Cdh5, Cldn5, Pecam1) increased, but SMC markers (Acta2) and EndMT markers (Col1a2 and Col3a1）decreased. In addition, we characterized EndMT cells into two sub-clusters: 1 and 2. EC-specific CD45 deletions reduced the cell numbers in the EndMT2 sub-cluster in atherosclerotic mice but had less impact on the EndMT1 sub-cluster. FGFR pathways were potentiated while TGFβ1 and Tgfbr2 decreased in EndMT1 sub-cluster， and the genes controlling endothelial cell development were upregulated but the genes controlling actin cytoskeleton organization were downregulated in both EndMT1 and 2 sub-clusters. Conclusion: Our single-cell analysis indicates that the loss of endothelial CD45 protects against EndMT-driven atherosclerosis, inhibiting TGFβ and EndoMT marker expression while stimulating FGFR pathways. Consistently, EC-iCD45KO/ApoE -/- mice showed reduced plaque macrophages, expression of cell adhesion molecules, foam cell formation, and lesion development when compared to ApoE -/- controls. Thus, targeting endothelial CD45 may be a novel therapeutic strategy for EndMT and atherosclerosis.

Identification of CAPG as a potential prognostic biomarker associated with immune cell infiltration and ferroptosis in uterine corpus endometrial carcinoma

IntroductionCapping actin protein, gelsolin-like (CAPG) is a potential therapeutic target in various cancers. However, the potential immunotherapeutic effects and prognostic value of CAPG in uterine corpus endometrial carcinoma (UCEC) remain unclear.MethodsThe characterization, methylation effects, prognostic value, targeted miRNAs of CAPG, and the correlation of CAPG with immune cell infiltration and ferroptosis in UCEC were investigated using multiple public databases and online tools. Furtherly, we explored the potential physiological function of CAPG using EdU and Transwell migration assays, identified the cell localization and expression of CAPG and GPX4 by immunofluorescence, and detected the intracellular Fe2+ levels using a FerroOrange fluorescent probe in Ishikawa cells. Additionally, the OncoPredict package was used to analyze the potential chemotherapeutic drugs for UCEC.ResultsCAPG showed generally high expression in tumor group. The overall survival rate of the high-risk group was significantly lower than that of the low-risk group. Enrichment analysis indicated that CAPG is involved in immune-related pathways and is closely associated with the tumor microenvironment. CAPG expression levels were affected by abnormal DNA methylation and/or targeted miRNAs, infiltration levels and marker genes of various immune cells, thereby impacting immune response, ferroptosis, and patient prognosis. Ferroptosis analysis indicated that ALOX5 and VLDLR were the top CAPG-related ferroptosis markers; glutathione metabolism levels in tumor group were generally high, and decitabine was a ferroptosis inducer. CAPG-siRNA suppressed the cell proliferation and invasion, and markedly elevated the expression levels of immune-related genes IL8, TNF, TLR4 and the intracellular Fe2+ levels. CAPG co-located with GPX4 in nucleus and co-regulated ferroptosis and metabolism in Ishikawa cells. Moreover, four chemotherapy drugs showed better sensitivity to UCEC patients in the low-risk cohort.ConclusionsCAPG may serve as a potential biomarker of UCEC owing to its role in modulating the immune response and ferroptosis, providing novel perspectives for combined immunotherapy of UCEC.

Abstract 4120919: Cell physiology and multi-omics analysis revealed disordered embryogenesis as early as mesoderm specification in models of Holt-Oram Syndrome

Introduction: Congenital heart diseases (CHDs) are the leading cause of childhood morbidity and mortality. The dysregulation of several cardiac transcription factors (TFs) leads to CHD. The coordination of several cardiac TFs is required and essential for cardiogenesis. However, the mechanisms to acquire its cardiac cell identity remain unclear. Hypothesis: Mutant Tbx5 dysregulates embryogenesis during mesoderm specification, prior to its expression in the tissues in Holt-Oram syndrome. Methods: Using cellular physiology and multiomics analysis in both human ES cells and a zebrafish model of TBX5 germline mutation. we evaluated embryonic structure and function, prior to the onset of gastrulation in the context of both heterozygous and homozygous TBX5 mutation. Results: Zebrafish time course transcriptome profiles over gastrulation revealed that loss of Tbx5 impacted transcriptional profiles at the blastula stage. Single cell RNA sequencing (scRNA-seq) on 16243 zebrafish blastula stage cells showed loss of Tbx5 impacted transcription noise at the blastula stage prior to the expression of zygotic Tbx5 with associated effects on chromatin accessibility using omni-assay for transposase-accessible chromatin (ATAC) and evidence of aberrant Wnt signaling even at the blastula stage. Embryo-wide cell structure and function were abnormal in both Tbx5 mutant zebrafish heterozygotes or homozygotes. To validate Undifferentiated human ES cell H3K4me3 Cleavage Under Targets&amp;Release Using Nuclease (CUT&amp;RUN) also showed abnormal Wnt signaling in TBX5 homozygous mutation and TBX5 CUT&amp;RUN showed aberrant mesoderm pathway. Calcium imaging analysis demonstrated the lowest excitation frequency in TBX5 homozygous mutation prior to mesoderm specification. TBX5 homozygous human ES derived mesoderm cells exhibited low expression levels of mesoderm marker genes compared to TBX5 wild type mesoderm. Conclusion: Integrating single cell physiology and multi-omics technologies, we idneifified fundamental dysregulation of embryogenesis in mutant cardiac-restricted gene disorder prior to mesoderm specification or the zygotic expression of the mutant gene. These findings suggest that Tbx5 started to determine cell fate prior to mesoderm specification by altering chromatin accessibilities and histone modification. Tbx5 affected mesoderm differentiation by influencing Wnt signaling and cell physiology.

Abstract 4116949: Specialized Pericyte Subtypes in the Pulmonary Capillary

Introduction: Lung pericytes (PCs) are mural cells in close contact with endothelial cells (ECs) in the microvasculature. A significant challenge in investigating PC biology is largely due to the absence of a unique cell marker, making it difficult to distinguish them from other mural cell populations. The identification of such a marker would allow investigators to describe the role of PCs in various diseases including pulmonary arterial hypertension. Hypothesis: We hypothesize that HIG1 hypoxia-inducible domain family member 1B ( Higd1b ) is exclusively expressed in lung PCs, which contributes to hypoxia (Hx)-induced vascular remodeling. Methods: We utilized single-cell RNA sequence (scRNA-seq) databases, spatial transcriptomics, and RNAscope from human and murine lungs to identify a PC-specific cell marker and compare the gene expression between PC subtypes. We utilized Cre-LoxP and CRISPR technology to construct a novel tamoxifen-inducible Higd1b-CreERT2 knock-in mouse model and performed lineage-tracing studies to describe the role of PC subtypes in Hx-induced pulmonary hypertension (PH). Results: ScRNA-seq analysis from the lungs of humans and mice identified Higd1b as a specific gene marker for PCs whose expression is absent in other mural cell populations. Validation with a reporter mouse line Higd1b-CreERT2::R26-tdT confirmed Higd1b-Cre+ cells specifically label PCs and no other mural cells ( Fig 1A ). Lineage tracing in the Hx-induced murine model of PH demonstrated the accumulation of PCs in the muscularized distal arterioles ( Fig 1B ). Through scRNA-seq and immunofluorescence validation, we identified two HIGD1B + PC subtypes that exist in the pulmonary capillary: Type 1 PCs, which are quiescent in the capillaries, and Type 2 PCs exhibit multipotent cell-like properties and accumulate in the arterioles after exposure to Hx. Furthermore, we found Type 2 PCs transited into SMC-like cells via the upregulation of Vimentin, contributing to vascular remodeling in Hx-induced PH. Conclusion: We identified Higd1b as a unique marker for PCs and generated a novel Higd1b-CreERT2 mouse that specifically labels PCs in the lungs. The discovery of PC-subtype specialization advances our understanding of lung pericyte biology and PC’s contribution to capillary remodeling under pathological conditions.

Efficient DNA-free co-targeting of nuclear genes in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii, a model organism for unicellular green microalgae, is widely used in basic and applied research. Nonetheless, proceeding towards synthetic biology requires a full set of manipulation techniques for inserting, removing, or editing genes. Despite recent advancements in CRISPR/Cas9, still significant limitations in producing gene knock-outs are standing, including (i) unsatisfactory genome editing (GE) efficiency and (ii) uncontrolled DNA random insertion of antibiotic resistance markers. Thus, obtaining efficient gene targeting without using marker genes is instrumental in developing a pipeline for efficient engineering of strains for biotechnological applications. We developed an efficient DNA-free gene disruption strategy, relying on phenotypical identification of mutants, to (i) precisely determine its efficiency compared to marker-relying approaches and (ii) establish a new DNA-free editing tool. This study found that classical CRISPR Cas9-based GE for gene disruption in Chlamydomonas reinhardtii is mainly limited by DNA integration. With respect to previous results achieved on synchronized cell populations, we succeeded in increasing the GE efficiency of single gene targeting by about 200 times and up to 270 times by applying phosphate starvation. Moreover, we determined the efficiency of multiplex simultaneous gene disruption by using an additional gene target whose knock-out did not lead to a visible phenotype, achieving a co-targeting efficiency of 22%. These results expand the toolset of GE techniques and, additionally, lead the way to future strategies to generate complex genotypes or to functionally investigate gene families. Furthermore, the approach provides new perspectives on how GE can be applied to (non-) model microalgae species, targeting groups of candidate genes of high interest for basic research and biotechnological applications.