Key Molecular Events Research Articles

Single-Cell Analysis Reveals the Cellular and Molecular Changes of Liver Injury and Fibrosis in Mice During the Progression of Schistosoma japonicum Infection

Schistosomiasis is a parasitic disease that poses a serious threat to human health. However, the pathogenic mechanism during the progression of Schistosoma japonicum infection remains unclear. In order to elucidate this mechanism, we used single-cell RNA sequencing (scRNA-seq) to investigate the transcriptome characteristics of the cellular (single-cell) landscape in the livers of mice infected with Schistosoma japonicum, which were divided into three groups: uninfected mice (0 week (w)), infected mice at 6 w post-infection (the acute phase), and infected mice at 10 w post-infection (the chronic phase). A total of 31,847 liver cells were included and clustered into 21 groups. The cells and T-cells had high heterogeneity in the liver during the progression of schistosome infection. The number and intensity of the intercellular interactions significantly increased at 6 w after infection but decreased at 10 w. The inflammatory signaling pathways chemoattractant cytokine ligand (CCL)5-chemokine C-C-motif receptor (CCR)5 between macrophages and T-cells were predominant at 6 w post-infection; the CCL6-CCR2 signaling pathway between macrophages was predominant at 10 w. The CD80 signaling pathway related to T-cell activation was increased at 6 w after infection, and increased expression of its receptor CD28 on the surfaces of CD4+ and CD8+ T-cells was confirmed by flow cytometry, suggesting an increase in their activation. In addition, scRNA-seq and quantitative reverse transcription polymerase chain reaction (qRT-PCR) confirmed that the intercellular communication between secretory phosphoprotein 1 (SPP1)-cluster of differentiation (CD44), insulin-like growth factor (IGF)-1-IGF1r and visfatin-insulin receptor (Insr) associated with bone metabolism and insulin metabolism was increased and enhanced in the liver at 6 w post-infection. Overall, we provide the comprehensive single-cell transcriptome landscape of the liver in mice during the progression of schistosome infection and delineate the key cellular and molecular events involved in schistosome infection-induced liver injury and fibrosis. The elevated CCL5-CCR5 and CCL6-CCR2 signaling pathways in the liver may be a drug target for liver injury and fibrosis caused by schistosome infection, respectively.

Identification of Necroptosis-related Molecular Subtypes and Construction of Necroptosis-related Gene Signature for Glioblastoma Multiforme.

Necroptosis is a highly regulated and genetically controlled process, and therefore, attention has been paid to the exact effects of this disorder on a variety of diseases, including cancer. An in-depth understanding of the key regulatory factors and molecular events that trigger necroptosis can not only identify patients at risk of cancer development but can also help to develop new treatment strategies. This study aimed to increase understanding of the complex role of necroptosis in glioblastoma multiforme (GBM) and provide a new perspective and reference for accurate prediction of clinical outcomes and gene-targeted therapy in patients with GBM. The objective of this study was to analyze the gene expression profile of necroptosis regulatory factors in glioblastoma multiforme (GBM) and establish a necroptosis regulatory factor-based GBM classification and prognostic gene signature to recognize the multifaceted impact of necroptosis on GBM. The necroptosis score of the glioblastoma multiforme (GBM) sample in TCGA was calculated by ssGSEA, and the correlation between each gene and the necroptosis score was calculated. Based on necroptosis score-related genes, unsupervised consensus clustering was employed to classify patients. The prognosis, tumor microenvironment (TME), genomic changes, biological signal pathways and gene expression differences among clusters were analyzed. The gene signature of GBM was constructed by Cox and LASSO regression analysis of differentially expressed genes (DEGs). Based on 34 necroptosis score-related genes, GBM was divided into two clusters with different overall survival (OS) and TME. A necroptosis-related gene signature (NRGS) containing 8 genes was developed, which could stratify the risk of GBM in both the training set and verification set and had good prognostic value. NRGS and age were both independent prognostic indicators of GBM, and a nomogram developed by the integration of both of them showed a better predictive effect than traditional clinical features. In this study, patients from public data sets were divided into two clusters and the unique TME and molecular characteristics of each cluster were described. Furthermore, an NRGS was constructed to effectively and independently predict the survival outcome of GBM, which provides some insights for the implementation of personalized precision medicine in clinical practice.

Dissection of Key Molecular Events in Podocyte Response to Hyperinsulinemia and Its Implications in Prediabetic Microalbuminuria

Dissection of Key Molecular Events in Podocyte Response to Hyperinsulinemia and Its Implications in Prediabetic Microalbuminuria

TSC/mTORC1 mediates mTORC2/AKT1 signaling in c-MYC-induced murine hepatocarcinogenesis via centromere protein M.

Activated mTORC2/AKT signaling plays a role in hepatocellular carcinoma (HCC). Research has shown that TSC/mTORC1 and FOXO1 are distinct downstream effectors of AKT signaling in liver regeneration and metabolism. However, the mechanisms by which these pathways mediate mTORC2/AKT activation in HCC are not yet fully understood. Amplification and activation of c-MYC is a key molecular event in HCC. In this study, we explored the roles of TSC/mTORC1 and FOXO1 as downstream effectors of mTORC2/AKT1 in c-MYC-induced hepatocarcinogenesis. Using various genetic approaches in mice, we found that manipulating the FOXO pathway had minimal impact on c-MYC-induced HCC. In contrast, loss of mTORC2 inhibited c-MYC-induced HCC, an effect that was completely reversed by ablating TSC2, which activated mTORC1. Additionally, we discovered that p70/RPS6 and 4EBP1/eIF4E act downstream of mTORC1, regulating distinct molecular pathways. Notably, the 4EBP1/eIF4E cascade is crucial for cell proliferation and glycolysis in c-MYC-induced HCC. We also identified centromere protein M (CENPM) as a downstream target of the TSC2/mTORC1 pathway in c-MYC-driven hepatocarcinogenesis, and its ablation entirely inhibited c-MYC-dependent HCC formation. Our findings demonstrate that the TSC/mTORC1/CENPM pathway, rather than the FOXO cascade, is the primary signaling pathway regulating c-MYC-driven hepatocarcinogenesis. Targeting CENPM holds therapeutic potential for treating c-MYC-driven HCC.

Unraveling the key molecular events of Pinot noir berry ripening under varying crop load

Aligned to exploring the physiological and molecular complexity of grape berry development, there is a need to characterize the influence of the source:sink relationships on the genetic regulation of fruit composition. Crop load, as defined by the amount of fruit produced per unit vegetative growth at dormancy, is a common measure of source:sink relationships used to evaluate vineyard production efficiency. We studied the impact of varying crop load on the transcriptome and metabolome of Pinot noir grape berries by comparing the development and ripening of fruit grown on vines with either 50 % or 75 % of their grape clusters removed immediately following fruit set compared to unthinned vines for three consecutive vintages. A clear impact on the general phenylpropanoid pathway resulting in a redistribution between stilbenes and anthocyanins was revealed under varying crop loads and consistent with the transcriptomic profiles of the corresponding branches. Moreover, we identified genes, such as LBDIa3 and AG2, modulated by crop load around veraison, representing putative transcriptional key triggers of the berry ripening phase responding to differences in the vine source:sink ratio generated by the application of cluster thinning. Genes, specifically EXPA1 and EXPA18, involved in softening and other crucial events of ripening initiation responded to crop load and likely influenced the progression of the ripening process. Beyond the major impacts represented by a shift of the onset and completion of ripening, we were able to highlight more subtle effects of the crop load, related to the rate at which the molecular and metabolic changes occur. This study asserts that grape metabolism and transcriptome are remarkably flexible, and that manipulations such as cluster thinning induce extensive, genome-wide changes in expression during berry development. The insights gained here pave the way to progress towards the construction of robust models depicting the molecular network that characterizes berry development and the impact of crop load on its molecular regulation.

Mll4 regulates postnatal palate growth and midpalatal suture development.

MLL4, also known as KMT2D, is a histone methyltransferase that acts as an important epigenetic regulator during various organogenesis programs. Mutations in the MLL4 gene are the major cause for Kabuki syndrome, a human developmental disorder that involves craniofacial birth defects, including anomalies in the palate. The purpose of this study was to investigate the role of Mll4 and the underlying mechanisms in the development and growth of the palate. We generated a novel conditional knockout (cKO) mouse model with tissue-specific deletion of Mll4 in the palatal mesenchyme. By using micro-computed tomography (CT), histology, cell mechanism assays, and gene expression analysis approaches, we examined the development and growth of the palate in the Mll4 -cKO mice. Gross intra-oral examination at adult stages showed that Mll4 -cKO mice had defects along the midline of the palate, which included disrupted rugae pattern and widened midpalatal suture. Micro-CT-based skeletal analysis in the adult mice revealed that the overall palate width was decreased in the Mll4 -cKO mice. By using whole-mount and histological staining approaches at perinatal stages, we identified that the midline defects started to appear as early as 1 day prior to birth, manifesting initially as a widened midpalatal suture, accompanied by increased cell apoptosis in the suture mesenchyme cells. Genome-wide analysis of mRNA expression in the midpalatal suture tissue showed that Mll4 is essential for timely expression of major genes for cartilage development, such as Col2a1 and Acan , at birth. These results were validated through immunofluorescence staining, confirming that the expression of chondrogenic markers Sox9 and Col2a1 were markedly decreased, whereas that of the osteogenic marker Runx2 remained unchanged, in the midpalatal suture of the Mll4 -cKO mice. Indeed, time-course histological analysis during postnatal palate growth revealed retardation in the development of the suture cartilage in the Mll4 -cKO mice. In parallel, time-course micro-CT analysis during postnatal palatogenesis confirmed a transverse growth deficit in the palate of the Mll4 -cKO mice. Taken together, our results show that Mll4 is essential for timely occurrence of key cellular and molecular events that lead to proper midpalatal suture development and palate growth.

Deciphering the Relationship between Cell Growth and Cell Cycle in Individual Escherichia coli Cells by Flow Cytometry.

Accurate coordination of chromosome replication and cell division is essential for cellular processes, yet the regulatory mechanisms governing the bacterial cell cycle remain contentious. The lack of quantitative data connecting key cell cycle players at the single-cell level across large samples hinders consensus. Employing high-throughput flow cytometry, we quantitatively correlated the expression levels of key cell cycle proteins (FtsZ, MreB, and DnaA) with DNA content in individual bacteria. Our findings reveal distinct correlations depending on the chromosome number (CN), specifically whether CN ≤2 or ≥4, unveiling a mixed regulatory scenario in populations where CN of 2 or 4 coexist. We observed function-dependent regulations for these key proteins across nonoverlapping division cycles and various nutrient conditions. Notably, a logarithmic relationship between total protein content and replication origin number across nutrient conditions suggests a unified mechanism governing cell cycle progression, confirming the applicability of Schaechter's growth law to cells with CN ≥4. For the first time, we established a proportional relationship between the synthesis rates of key cell cycle proteins and chromosome dynamics in cells with CN ≥4. Drug experiments highlighted CN 2 and 4 as pivotal turning points influencing cellular resource allocation. This high-throughput, single-cell analysis provides interconnected quantitative insights into key molecular events, facilitating a predictive understanding of the relationship between cell growth and cell cycle.

Key molecular DNA damage responses of human cells to radiation.

Our understanding of the DNA damage responses of human cells to radiation has increased remarkably over the recent years although some notable signaling events remain to be discovered. Here we provide a brief account of the key molecular events of the responses to reflect the current understanding of the key underlying mechanisms involved.

Status and development of research on clear cell carcinoma of the ovary-a visualization-based bibliometric analysis.

Clear cell carcinoma of the ovary (CCCO) is a relatively rare type of epithelial ovarian cancer (EOC) that has unique biological characteristics and clinical features. Researchers have paid less attention to this disease than to other types of EOCs. However, in recent years, research in this area has still progressed. In this paper, a bibliometric analysis is used to integrate and analyse the literature in the field of CCCO in the past 20 years to determine research development, better understand the current status of research, and provide a reference for future study directions in this field. With CCCO as the research subject, relevant publications indexed in the Web of Science (WOS) core dataset from September 2003 to September 2023 were retrieved. After screening the publications, we used EXCEL, VOSviewer, CiteSpace, Charticulator, Gephi, OriginPro and other tools to perform in-depth analyses of and to visualize the data. Through a comprehensive analysis of the literature in this field, we found that research on CCCO experienced a relatively rapid increase in 2006 and is now in a period of relatively high fluctuation. The quality of the literature in this field is generally high. In this field, countries in East Asia and North America play core roles, with Japan accounting for the most studies. A stable research group has been formed in this field, and extensive collaboration has occurred among the various research groups. In the past 20 years, basic research and clinical research in the field of CCCO have developed together, and a healthy development model in which basic and clinical research promote each other has formed. Research in this field has been continuously developed from a preliminary understanding of clinical features to in-depth explorations of the pathogenesis and the continuous optimization of treatment methods. The key molecular events in the pathogenesis and development of this disease and the application of novel antitumour drugs for this disease are the current research focuses and the future development direction in this field. Research on CCCO has progressed significantly in the past 20 years, but there are still many important issues regarding its pathogenesis and treatment that need to be addressed, and therefore, more research in this area should be conducted in the future. The study of key molecular events and the use of novel antitumour drugs are future development directions in this field.

P53 Expression in Sporadic Colorectal Carcinoma

Objective: To determine expression of p53 in colorectal carcinoma (CRC) tissues and to find the frequency of p53 over-expression in various stages and grades of tumor. Methodology: A prospective observational design study was conducted at BMSI JPMC between January 2023 till October 2023, after obtaining approval from ethics review committee. Fifty-six patients of CRC were recruited and the tumor tissue was subjected to H&E staining. The extent of nuclear immunoreactivity of p53 was scored from 0-3. The intensity was score from mild to strong, with score zero for no intensity. The results of immunohistochemistry were used to determine frequency of p53 over-expression with Astler and Coller grading and staging system and TNM staging. Results: There were 30 cases (53.5 %) cases which showed p53 over-expression. The Astler and Coller grading and staging system showed that the majority of cases were in stage B2 (44.6%) followed by C2. As compared to other stages, TNM stage 3 showed higher frequency of p53 overexpression. Conclusion: there is high frequency of p53 over-expression which highlights its significance. The over-expression has been found in higher stage and grade which indicates that it may form a key molecular event that may act as a prognostic as well as possible therapeutic marker.

Prolonged Cadmium Exposure and Osteoclastogenesis: A Mechanistic Mouse and in Vitro Study.

Cadmium (Cd) is a highly toxic and widespread environmental oxidative stressor that causes a myriad of health problems, including osteoporosis and bone damage. Although nuclear factor erythroid 2-related factor 2 (NRF2) and its Cap 'n' Collar and basic region Leucine Zipper (CNC-bZIP) family member nuclear factor erythroid 2-related factor 1 (NRF1) coordinate various stress responses by regulating the transcription of a variety of antioxidant and cytoprotective genes, they play distinct roles in bone metabolism and remodeling. However, the precise roles of both transcription factors in bone loss induced by prolonged Cd exposure remain unclear. We aimed to understand the molecular mechanisms underlying Cd-induced bone loss, focusing mainly on the roles of NRF2 and NRF1 in osteoclastogenesis provoked by Cd. Male wild-type (WT), global Nrf2-knockout () and myeloid-specific Nrf2 knockout [Nrf2(M)-KO] mice were administered Cd (50 or ) via drinking water for 8 or 16 wk, followed by micro-computed tomography, histological analyses, and plasma biochemical testing. Osteoclastogenesis was evaluated using bone marrow-derived osteoclast progenitor cells (BM-OPCs) and RAW 264.7 cells in the presence of Cd (10 or ) with a combination of genetic and chemical modulations targeting NRF2 and NRF1. Compared with relevant control mice, global or Nrf2(M)-KO mice showed exacerbated bone loss and augmented osteoclast activity following exposure to Cd in drinking water for up to 16 wk. In vitro osteoclastogenic analyses suggested that Nrf2-deficient BM-OPCs and RAW 264.7 cells responded more robustly to low levels of Cd (up to ) with regard to osteoclast differentiation compared with WT cells. Further mechanistic studies supported a compensatory up-regulation of long isoform of NRF1 (L-NRF1) and subsequent induction of nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1 (NFATc1) as the key molecular events in the Nrf2 deficiency-worsened and Cd-provoked osteoclastogenesis. L-Nrf1 silenced (via lentiviral means) Nrf2-knockdown (KD) RAW cells exposed to Cd showed dramatically different NFATc1 and subsequent osteoclastogenesis outcomes compared with the cells of Nrf2-KD alone exposed to Cd, suggesting a mitigating effect of the Nrf1 silencing. In addition, suppression of reactive oxygen species by exogenous antioxidants -acetyl-l-cysteine () and mitoquinone mesylate (MitoQ; ) mitigated the L-NRF1-associated effects on NFATc1-driven osteoclastogenesis outcomes in Cd-exposed Nrf2-KD cells. This invivo and in vitro study supported the authors' hypothesis that Cd exposure caused bone loss, in which NRF2 and L-NRF1 responded to Cd and osteoclastogenic stimuli in a cooperative, but contradictive, manner to coordinate Nfatc1 expression, osteoclastogenesis and thus bone homeostasis. Our study suggests a novel strategy targeting NRF2 and L-NRF1 to prevent and treat the bone toxicity of Cd. https://doi.org/10.1289/EHP13849.

Oncofetal SNRPE promotes HCC tumorigenesis by regulating the FGFR4 expression through alternative splicing.

Due to insufficient knowledge about key molecular events, Hepatocellular carcinoma (HCC) lacks effective treatment targets. Spliceosome-related genes were significantly altered in HCC. Oncofetal proteins are ideal tumor therapeutic targets. Screening of differentially expressed Spliceosome-related oncofetal protein in embryonic liver development and HCC helps discover effective therapeutic targets for HCC. Differentially expressed spliceosome genes were analysis in fetal liver and HCC through bioinformatics analysis. Small nuclear ribonucleoprotein polypeptide E (SNRPE) expression was detected in fetal liver, adult liver and HCC tissues. The role of SNRPE in HCC was performed multiple assays in vitro and in vivo. SNRPE-regulated alternative splicing was recognized by RNA-Seq and confirmed by multiple assays. We herein identified SNRPE as a crucial oncofetal splicing factor, significantly associated with the adverse prognosis of HCC. SOX2 was identified as the activator for SNRPE reactivation. Efficient knockdown of SNRPE resulted in the complete cessation of HCC tumorigenesis and progression. Mechanistically, SNRPE knockdown reduced FGFR4 mRNA expression by triggering nonsense-mediated RNA decay. A partial inhibition of SNRPE-induced malignant progression of HCC cells was observed upon FGFR4 knockdown. Our findings highlight SNRPE as a novel oncofetal splicing factor and shed light on the intricate relationship between oncofetal splicing factors, splicing events, and carcinogenesis. Consequently, SNRPE emerges as a potential therapeutic target for HCC treatment. Model of oncofetal SNRPE promotes HCC tumorigenesis by regulating the AS of FGFR4 pre-mRNA.

A Novel Frameshift Variant of the ELF4 Gene in a Patient with Autoinflammatory Disease: Clinical Features, Transcriptomic Profiling and Functional Studies.

We described the diagnosis and treatment of a patient with autoinflammatory disease, named "Deficiency in ELF4, X-linked (DEX)". A novel ELF4 variant was discovered and its pathogenic mechanism was elucidated. The data about clinical, laboratory and endoscopic features, treatment, and follow-up of a patient with DEX were analyzed. Whole exome sequencing and Sanger sequencing were performed to identify potential pathogenic variants. The mRNA and protein levels of ELF4 were analyzed by qPCR and Western blotting, respectively. The association of ELF4 frameshift variant with nonsense-mediated mRNA decay (NMD) in the pathogenesis DEX was examined. Moreover, RNA-seq was performed to identify the key molecular events triggered by ELF4 variant. The relationship between ELF4 and IFN-β activity was validated using a dual-luciferase reporter assay and a ChIP-qPCR assay. An 11-year-old boy presented with a Behçet's-like phenotype. The laboratory abnormality was the most obvious in elevated inflammatory indicators. Endoscopy revealed multiple ileocecal ulcers. Intestinal histopathology showed inflammatory cell infiltrations. The patient was treated with long-term immunosuppressant and TNF-α blocker (adalimumab), which reaped an excellent response over 16 months of follow-up. Genetic analysis identified a maternal hemizygote frameshift variant (c.1022del, p.Q341Rfs*30) in ELF4 gene in the proband. The novel variant decreased the mRNA level of ELF4 via the NMD pathway. Mechanistically, insufficient expression of ELF4 disturbed the immune system, leading to immunological disorders and pathogen susceptibility, and disrupted ELF4-activating IFN-βresponses. This analysis detailed the clinical characteristics of a Chinese patient with DEX who harbored a novel ELF4 frameshift variant. For the first time, we used patient-derived cells and carried out transcriptomic analysis to delve into the mechanism of ELF4 variant in DEX.

Dermal Dive: An Overview of Cutaneous Wounding Techniques in Zebrafish

Cutaneous wounds are common injuries that affect millions of people around the world. In vulnerable populations such as the elderly and those with diabetes, defects in wound healing can lead to the development of chronic open wounds. Although mammalian models are commonly used to study cutaneous wound healing, the challenges of invivo imaging in mammals have hampered detailed observation of cell coordination and cell signaling during wound healing. The zebrafish is becoming increasingly popular for studying cutaneous wound healing owing to its genetic accessibility, suitability for experimental manipulation, and the ability to perform live, invivo imaging with cellular or even subcellular resolution. In this paper, we review some of the techniques that have been developed for eliciting cutaneous wounds in the zebrafish, including an economical method we recently developed using a rotary tool that generates consistent and reproducible full-thickness wounds. Combined with the thousands of transgenic lines and experimental assays available in zebrafish, the ability to generate reproducible cutaneous wounds makes it possible to study key cellular and molecular events during wound healing using this powerful experimental model organism.

Single-cell transcriptome sequencing analysis reveals intra-tumor heterogeneity in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a prevalent malignant tumor of the digestive system that poses a significant threat to human life and health. It is crucial to thoroughly investigate the mechanisms of esophageal carcinogenesis and identify potential key molecular events in its carcinogenesis. Single-cell transcriptome sequencing is an emerging technology that has gained prominence in recent years for studying molecular mechanisms, which may help to further explore the underlying mechanisms of the ESCC tumor microenvironment in depth. The single-cell dataset was obtained from GSE160269 in the Gene Expression Omnibus database, including 60 tumor samples and four paracancer samples. The single-cell data underwent dimensional reduction clustering analysis to identify clusters and annotate expression profiles. Subcluster analysis was conducted for each cellular taxon. Copy number variation analysis of tumor cell subpopulations was performed to primarily identify malignant cells within them. A proposed chronological analysis was performed to obtain the process of cell differentiation. In addition, cell communication, transcription factor analysis, and tumor pathway analysis were also performed. Relevant risk models and key genes were established by univariate COX regression and LASSO analysis. The key genes obtained from the screen were subjected to appropriate silencing and cellular assays, including CCK-8, 5-ethynyl-2'-deoxyuridine, colony formation, and western blot. Single-cell analysis revealed that normal samples contained a large number of fibroblasts, T cells, and B cells, with fewer other cell types, whereas tumor samples exhibited a relatively balanced distribution of cell types. Subclassification analysis of immune cells, fibroblasts, endothelial cells, and epithelial cells revealed their specific spatial characteristics. The prognostic risk model, we constructed successfully, achieved accurate prognostic stratification for ESCC patients. The screened key gene, UPF3A, was found to be significantly associated with the development of ESCC by cellular assays. This process might be linked to the phosphorylation of ERK and P38. Single-cell transcriptome analysis successfully revealed the distribution of cell types and major expressed factors in ESCC patients, which could facilitate future in-depth studies on the therapeutic mechanisms of ESCC.

Abstract B093: Investigating early events of ovarian cancer transcoelomic metastasis identifies the small Rho-GTPase RHOV as an essential gene for cellular detachment and aggregate formation

Abstract B093: Investigating early events of ovarian cancer transcoelomic metastasis identifies the small Rho-GTPase RHOV as an essential gene for cellular detachment and aggregate formation

The molecular and metabolic events behind different germination stages of rice seeds: A metabolomics perspective

AbstractSeed germination is a crucial stage for the completion of plants' life cycle. The process of seed germination is a very important phenomenon for agricultural food and crop production and to understand the underlying food chemistry. Rice (Oryza sativa L.) being easy to grow and cheap source of phytonutrients is a fundamental food crop across the world. Several researches are going on for years to improve the production of rice, ameliorate the deadly bacterial or fungal diseases utilizing genetics, transcriptomics, and proteomics aspects. Metabolomics is a very new arena of research in this field. With the help of modern‐day metabolomic tools and technologies, researchers are incessantly trying to unveil the metabolic construction of rice seeds, and new to this topic is seed germination. The principal aim of this review on metabolomics of germinating rice seeds is to have a big picture on the key molecular, cellular, and metabolic events of germination. This review tries to summarize all the available data to fulfill the aforesaid aim.

Abstract B035: Evidence for CD73 loss promoting cancer cell stemness via metabolic reprogramming in endometrial cancer

Abstract Cancer stem cells (CSCs) are a subpopulation of cancer cells that are responsible for disease recurrence and chemoresistance. The mechanisms by which CSCs arise in endometrial cancer are not fully understood and elucidating key molecular events may provide insight into therapeutic targets. CD73, a cell surface 5’nucleotidase that generates adenosine, is frequently downregulated in endometrial tumorigenesis, and CD73 loss is associated with aggressive disease and poor patient outcomes. In developing and characterizing CRISPR/Cas-9 models for CD73, we discovered CD73 deletion (CD73−/−) in HEC-1-A cells causes growth arrest and prolonged survival which are features suggestive of a stemness phenotype. Thus, we hypothesized that loss of CD73 is critical for promoting CSCs in endometrial cancer. To evaluate CSC characteristics, we performed MTT and spheroid assays and interrogated CSC marker protein expression (e.g., CD44 and NOTCH3). CD73−/− HEC-1-A cells formed larger spheroids and expressed high levels of CD44 and NOTCH3 compared to wild-type (CD73+/+) cells. To assess whether loss of CD73 enzymatic activity is responsible for the enrichment of CSCs, CD73+/+ HEC-1-B and HEC-50 cells were treated with AoPCP, a catalytic inhibitor of CD73. Similar to genetic deletion of CD73 in HEC-1-A cells, AoPCP treatment resulted in HEC-1-B and HEC-50 cells forming larger spheroids and expressing high CD44 and NOTCH3. These data indicate that loss of CD73 enzyme activity is important to the enrichment of CSCs. In contrast, HEYA8 ovarian cancer cells showed the opposite effects, wherein treatment with AoPCP decreased spheroid size. These differences support the emerging paradigm that CD73 has tissue- and cancer-specific roles. As CD73 is a major node in purine metabolic signaling, we further hypothesized that loss of CD73 activity may be promoting CSC enrichment via changes to metabolic pathways. Mass spectrometry metabolomics revealed significantly different profiles of CD73+/+ and CD73−/− HEC-1-A cells. Conditioned media experiments showed CD73−/−-conditioned media induces growth arrest in HEC-1-A wild-type cells, whereas the growth arrest and CSC marker expression enrichment of CD73−/− cells was abrogated by CD73+/+-conditioned media or daily media replenishment. Metabolomic profiling of CD73+/+ and CD73−/− HEC-1-A cells with/without daily media replenishment revealed metabolic pathways, such as NAD+ biosynthesis, lipid metabolism, and sugar alcohol synthesis, are up-regulated by CD73 deficiency and parallels the enrichment of CSCs. Current studies are dissecting these metabolic dependencies in supporting endometrial cancer cell stemness. Altogether, these studies provide novel insight into how CSCs arise in endometrial cancer and why CD73 loss is detrimental in this disease. Citation Format: Emily M. Rabjohns, Blake R. Rushing, Sayali Joseph, Cyrus Vaziri, Jessica L. Bowser. Evidence for CD73 loss promoting cancer cell stemness via metabolic reprogramming in endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B035.

Abstract B017: VCP extracts the chromatin remodeler SNF2H from nascent DNA to stabilize stressed replication forks

Abstract Valosin-containing protein (VCP) is an evolutionarily conserved AAA+ ATPase which plays pleiotropic roles in global proteostasis by extracting polyubiquitinated proteins from various cellular organelles and structures and facilitating their turnover. Our recent work demonstrated that nuclear VCP activity during DNA damage response specifically depends on ATM/ATR/DNA-PK-mediated Ser784 phosphorylation. Ser784phosphorylation increases VCP ability to facilitate chromatin-associated protein degradation and is required for DNA repair, checkpoint signaling, and cell survival. Clinically, high nuclear pSer784-VCP levels are significantly associated with poor survival outcome among chemotherapy-treated breast and pancreatic cancer patients. Thus, pSer784-VCP is a broadly important and therapeutically relevant genome stabilizer. Here, we focused specifically on the role of pSer784-VCP in DNA replication fork stability during stress, a known source of DNA damage if not properly regulated. By performing single-molecule DNA fiber analysis using human cell lines with genetic gain and loss-of-function of VCP, we observed that VCP protects nascent DNA from hydroxyurea (HU)-induced over-resection and this activity requires Ser784 phosphorylation. Using proximity ligation assay (PLA), we detected HU-induced pSer784-VCP on nascent DNA, suggesting that it may protect stressed forks by extracting functionally important regulators. Consistent with this theory, we detected an interaction of pSer784-VCP with SNF2H, a nucleosome-sliding enzyme which we recently found essential for nascent DNA over-resection at unprotected forks. In support of the hypothesis that SNF2H is a novel substrate of pSer784-VCP at stressed forks, we observed SNF2H retention on nascent DNA upon VCP knockdown in HU-treated cells, and the functional rescue of SNF2H retention by VCP re-expression requires Ser784phosphorylation. In support of their epistatic relationship, SNF2H depletion fully rescued fork over-resection caused by VCP loss. Similarly, blocking fork reversal by depleting SMARCAL1 or FBH1 also rescued fork over-resection caused by VCP loss. Importantly, mutating the ATPase activity of SNF2H abolished its ability to promote fork over-resection, suggesting that nucleosome remodeling by SNF2H underlies its effect on fork stability and may be a key molecular event enabling fork reversal upstream of fork degradation. Collectively, our data suggest that pSer784-VCP is a previously unrecognized stabilizer of stressed DNA replication forks, and it does so at least in part via its physical extraction of SNF2H from nascent DNA and the consequent attenuation of nucleosome remodeling needed for fork reversal and over-resection. More experiments are currently underway to further dissect the mechanistic aspects of this working model as well as to evaluate the potential of developing pSer784-VCP into a clinically relevant chemo-predictive biomarker and chemo-sensitizing target for different types of cancer. Citation Format: Jieya Shao, Mari Iwase, Rong Xu, Shuyang Lin. VCP extracts the chromatin remodeler SNF2H from nascent DNA to stabilize stressed replication forks [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B017.

Mitochondrial fusion-fission dynamics and its involvement in colorectal cancer.

The incidence and mortality rates of colorectal cancer have elevated its status as a significant public health concern. Recent research has elucidated the crucial role of mitochondrial fusion-fission dynamics in the initiation and progression of colorectal cancer. Elevated mitochondrial fission or fusion activity can contribute to the metabolic reprogramming of tumor cells, thereby activating oncogenic pathways that drive cell proliferation, invasion, migration, and drug resistance. Nevertheless, excessive mitochondrial fission can induce apoptosis, whereas moderate mitochondrial fusion can protect cells from oxidative stress. This imbalance in mitochondrial dynamics can exert dual roles as both promoters and inhibitors of colorectal cancer progression. This review provides an in-depth analysis of the fusion-fission dynamics and the underlying pathological mechanisms in colorectal cancer cells. Additionally, it offers partial insights into the mitochondrial kinetics in colorectal cancer-associated cells, such as immune and endothelial cells. This review is aimed at identifying key molecular events involved in colorectal cancer progression and highlighting the potential of mitochondrial dynamic proteins as emerging targets for pharmacological intervention.