Hallmark Gene Research Articles

Single-cell transcriptomics reveals over-activated reactive oxygen species pathway in hepatocytes in the development of hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is a highly heterogeneous tumor and a primary cause of cancer-relevant deaths worldwide. The role of reactive oxygen species (ROS) in HCC development is less studied. Methods: Seurat package and CellMarker database were employed for single-cell RNA sequencing (scRNA-seq) analysis based on the GSE189175 dataset from Gene Expression Omnibus (GEO). DAVID and MsigDB database were utilized for pathway analysis. SCENIC analysis was performed to map a transcription factors (TFs) regulatory network. CellChat was used for cellular communication analysis. Results: Six major cell subpopulations were identified, among which hepatocytes accounted for the highest proportion in both cancer and adjacent tissues. The enrichment scores of the 50 hallmark gene sets showed that the ROS pathway was abnormally activated in HCC hepatocytes and positively correlated with energy metabolism-related pathways (glucose metabolism, lipid metabolism, amino acid metabolism, etc.). Then, the hepatocytes were divided into four subgroups. Noticeably, GPX4+ hepatocytes with the highest activity of the ROS pathway was related to a worse prognosis of HCC. Mechanism analysis revealed that JUND was involved in the positive regulation of the ROS pathway in GPX4+ hepatocytes. It was found that the interdependent ligand-receptor interaction between GPX4+ liver cells and immune cells facilitated the malignant development of HCC. Conclusion: ROS pathway was over-activated in the hepatocytes of HCC tissues. GPX4+ hepatocytes having the highest activity of the ROS pathway closely interacted with T cells and M2 macrophage cells. Molecular subtypes and risk score signature based on the ROS pathway and its potential target gene JUND are encouraged to be developed for improving the precision treatment of HCC.

Rare germline variants in cancer-relevant genes are associated with breast cancer risk in young women with high-risk family history.

It is incompletely understood why some women develop breast cancer 15-20years earlier than the majority of women. We hypothesize that women with early-onset breast cancer and high-risk family history without germline pathogenic variants in cancer-predisposing genes (CPGs, n = 83) have a higher load of germline high functional impact variants (gHFI) in cancer hallmark genes (n = 1508) compared to healthy controls. Germline whole exome sequencing data were analyzed from 5818 breast cancer cases from UKBiobank and from 94 young women with breast cancer without CPG pathogenic variants from the Yale Cancer Prevention Clinic, and compared to 149 controls from Yale GENERATIONS project and 56,917 controls from UKBiobank. Rare gHFI variants were compared between cases and controls using the burden test and the optimal unified SNP-set Kernel Association Test (SKAT-O). We assessed germline versus somatic origins of pathway level alterations in the TCGA and Yale data. In UKBiobank, higher gHFI variant load was seen in CPGs in cancer cases regardless of family history (p < 8.78 × 10-8), and in hallmark genes in cases with family history vs controls (p = 0.0093). Similarly, numerically higher rare gHFI burden was seen in hallmark genes in the Yale cohort vs controls, but this difference was not statistically significant. Pathway level alterations were dominated by somatic events. These results suggest that rare germline variants in cancer biology-related genes partly mediate the contribution of family history to cancer risk in individuals without germline alterations in high penetrance CPGs.

Abstract B038: Establishing a screening platform to identify key signaling molecules regulating cold tumor formation through immune evasion and exclusion

Abstract Many trial data have shown that cancer immunotherapy can successfully induce long lasting anti-cancer immune responses and increased the survival rate of cancer patients. One of the key challenges facing cancer immunotherapy is the low response rate of cancer patients to immunotherapy, suggesting that these cancer patients may have acquired resistance toward immunotherapy before treatments. Therefore, there is an unmet need for identifying the underlying mechanisms that promote resistance. One important hypothesis is that immune-excluded tumors (so called cold tumors) that lack of functional T cells have been often found in cancer patients who cannot respond well to cancer immunotherapy. To address this, we have successfully established a preclinical model for cold lung tumors, conducted RNA-seq, performed hallmark gene set enrichment analysis (GSEA) on cold tumor cells sorted in vivo, and identified several candidates associated with epithelial-mesenchymal transition, Wnt signaling, type I and II interferon (IFN) signaling, and p53 signaling. We hypothesize that their roles may either promote or inhibit the formation of cold tumors. One of the molecular candidates we identified, a molecule designated as IRC001, represents a crucial proof of concept for our experimental tumor model. We have unveiled a novel role for IRC001 in enhancing anti-tumor immunity, leading to the transformation of cold tumors into hot tumors and facilitating swift tumor regression through pathways reliant on T cells and type I IFN signaling. Our mechanistic studies have shown that IRC001 can trigger type I IFN signaling by engaging STING/TBK-1/IRF3-dependent pathways. To delve deeper into the spatial and molecular distinctions in mouse tumors (± IRC001), we initiated a preliminary study utilizing 10x Genomics/Visium to examine the spatial variations between these tumor types. Significantly, our spatial transcriptomic analysis revealed consistent immune-excluded and immune-enriched characteristics compared to traditional RNA sequencing methods. We conducted an analysis of TCGA datasets (The Cancer Genome Atlas) and observed that elevated IRC001 expression is associated with improved prognosis in SKCM (Skin Cutaneous Melanoma). Furthermore, our bioinformatics analysis revealed that melanoma patients exhibiting heightened IRC001 expression displayed positive responses to immune checkpoint blockade therapies. Therefore, we believe that our experimental tumor model can provide vital insights into the cold tumor microenvironment, shaping future therapeutic strategies, and pinpointing molecular candidates that can assist in selecting patients for successful immunotherapy. Citation Format: Yi-Ting Liao, Li-Mei Chen, Wen-Jye Lin. Establishing a screening platform to identify key signaling molecules regulating cold tumor formation through immune evasion and exclusion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B038.

Modulation of SRC by SNTB1 activates the Hippo-YAP pathway during colon adenocarcinoma metastasis

BackgroundColon adenocarcinoma (COAD) ranks as the third most prevalent and lethal cancer in 2020, with metastasis being the primary cause of cancer-related mortality. A comprehensive understanding of the mechanism underlying distant metastasis is imperative for enhancing the prognosis and quality of life of patients with COAD.MethodsThis study employed gene set enrichment analysis (GSEA) on RNA-sequencing data from 408 patients with COAD in The Cancer Genome Atlas (TCGA) database. GSEA analysis was applied to find the significant hallmark gene set, and genes in the significant hallmark gene set was performed by univariate Cox regression to select the key gene. Then, multivariate Cox regression model was constructed. And various databases were utilized to validate and find the significant oncoprotein and hallmark gene set of the key gene. In addition, the expression of key regulators in para-carcinoma tissue, colon cancer and distant metastases samples were detected by real-time PCR, Immunohistochemistry and Western blot. Additionally, the biological functions were examined by in vitro and in vivo experiments. The scRNA-seq and CellphoneDB were performed to explore cell characters in COAD and the potential mechanism of metastasis.ResultsThe regulatory network analysis revealed SRC/YAP1 as the most significant oncoprotein and signature gene set associated with SNTB1. Moreover, significant SNTB1 overexpression in COAD metastatic tissues was observed compared to para-carcinoma and primary COAD tissues. Co-immunoprecipitation assays demonstrated the formation of a complex between SNTB1 and SRC proteins. Furthermore, the overexpression of SNTB1 enhanced the proliferation, migration and invasion capacities of COAD cell lines. Caudal vein injection of COAD cells overexpressing SNTB1 in nude mice resulted in increased tumour growth and metastasis to the lung, liver and bone. Finally, single cell RNA-seq revealed alterations in the cellular subtypes of COAD, and CellphoneDB indicated that the interaction between cancer cells exhibiting high SNTB1 expression and enterocytes promoted EMT through cellular communications involving TGF-β, accelerating metastasis in COAD.ConclusionThis study postulates that SNTB1 interacts with SRC to activate the Hippo-YAP pathway, thereby promoting COAD metastasis. Furthermore, cellular communication with enterocytes promotes EMT, facilitating metastasis. These findings propose novel therapeutic targets for preventing or treating metastatic COAD.

IMMU-16. ANTI-VEGF TREATMENT EFFECT AGAINST GLIOMA IS IMMUNE DEPENDENT AND MEDIATED BY ENHANCED T CELL ACTIVITY

Abstract Immunotherapy trials in glioblastoma patients increasingly avoid immunosuppressive steroids and consider bevacizumab as alternative anti-edema treatment. Since VEGF is also a potent immunosuppressive factor, we explored the dimension and mechanisms of immune-mediated effects of anti-VEGF treatment. Murine glioma cells were injected intracerebrally into C57BL/6 wild-type mice or immunodeficient Pfp/Rag2 knockout mice and animals were treated with B20 (anti-murine VEGF) or dexamethasone. Immune cells were profiled by flow cytometry, and RNAseq was performed on sorted tumor and immune cells at different timepoints. B20 prolonged the survival of immunocompetent mice but not of Pfp/Rag2 knockout mice, indicating that the immune system is essential for its effect. At an early timepoint when controls became symptomatic, B20 “early” tumors (B20-ea) showed increased infiltration with T cells and myeloid cells, and decreased vascularization, whereas B20 tumors at the survival endpoint resembled IgG-controls. Gene expression in B20-ea tumor cells was dominated by hypoxia, neural development, invasion, TGF-β, Notch and Wnt signatures as well as concerted upregulation of Kmt2 H3K4 methyltransferases. In CD11b+ myeloid cells from B20-ea tumors, M1 hallmark genes and T cell activation genes were upregulated, while M2 genes were downregulated. Deconvolution revealed a transient shift towards pro-immune microglia/macrophages as well as increased NK cells and DCs. In CD8+ and CD4+ T cells, B20 increased the expression of S1pr1 and α/β TCR genes. The proportion of progenitor-like exhausted CD8+ cells, which are responsive to PD-1 blockade, was increased in B20-ea. In CD4+ cells, B20 caused skewing towards TFH, while dexamethasone increased TH2 cytokine expression. Depletion experiments demonstrated that CD8+ and CD4+ T cells but not NK cells were necessary for the survival-prolonging effect of B20. To conclude, anti-VEGF therapy has significant immunostimulatory effects which are, however, largely transient, so that an early treatment window of opportunity may exist during which it could actively support immunotherapy.

V- and VL-Scores Uncover Viral Signatures and Origins of Protein Families.

Viruses are key drivers of microbial diversity, nutrient cycling, and co-evolution in ecosystems, yet their study is hindered due to challenges in culturing. Traditional gene-centric methods, which focus on a few hallmark genes like for capsids, miss much of the viral genome, leaving key viral proteins and functions undiscovered. Here, we introduce two powerful annotation-free metrics, V-score and VL-score, designed to quantify the "virus-likeness" of protein families and genomes and create an open-access searchable database, 'V-Score-Search'. By applying V- and VL-scores to public databases (KEGG, Pfam, and eggNOG), we link 38-77% of protein families with viruses, a 9-16x increase over current estimates. These metrics outperform existing approaches, enabling precise detection of viral genomes, prophages, and host-derived auxiliary viral genes (AVGs) from fragmented sequences, and significantly improving genome binning. Remarkably, we identify up to 17x more AVGs, dominated by non-metabolic proteins of unknown function. This innovation unlocks new insights into virus signatures and host interactions, with wide-ranging implications from genomics to biotechnology.

RKER-012, a novel modified ActRIIB ligand trap, attenuated right ventricular cardiomyopathy in a preclinical model of pulmonary arterial hypertension

Abstract Background The primary cause of death in pulmonary arterial hypertension (PAH) is right ventricular (RV) failure. Initially, the RV adapts to heightened pressure through adaptive remodeling, but prolonged pressure overload triggers maladaptive remodeling, culminating in failure. Overactive activin/growth differentiation factor (GDF) signaling has been implicated in cardiomyopathy and heart failure. RKER-012, a research version of KER-012, is an investigational modified activin receptor type IIB (ActRIIB) ligand trap designed to specifically bind and inhibit select TGF-β ligands, including activins A and B and GDFs 8 and 11. RKER-012 previously exhibited a cardioprotective effect in a pulmonary arterial banding (PAB) model of RV dysfunction. To determine the effect of RKER-012 on afterload-induced RV cardiomyopathy caused by increased pulmonary artery pressure, we utilized an angio-obliterative Sugen/hypoxia (SH) rat model of PAH that closely mimics human PAH pathology. Methods Male Sprague Dawley rats were subcutaneously (s.c.) injected with one dose of Sugen 5416 and exposed to 10% hypoxia for 3 weeks during which they received either vehicle (SH-Veh; s.c; BIW) or RKER-012 (10 mg/kg; s.c; BIW). Normoxic (Nx) control rats were maintained in room air for 3 weeks with BIW s.c. vehicle treatment. After 3 weeks of treatment, systolic pulmonary arterial pressure (sPAP) and Fulton index (FI) were assessed. Hallmark genes of inflammatory process, endothelial/platelet activation, and fibrosis were evaluated in the RV by qPCR. Results Increases in FI (+99.4%;p&amp;lt;0.0001) and sPAP (+250.9%;p&amp;lt;0.0001) were observed in SH-Veh rats vs. Nx rats, consistent with the development of pulmonary and cardiac impairment. In contrast, treatment with RKER-012 reduced FI (-28.0%;p&amp;lt;0.001) and sPAP (-44.5%;p&amp;lt;0.001). In the RV, SH-Veh rats had increased expression of TGF-ß pathway genes involved in inflammation, endothelial/platelet activation, and fibrosis. RKER-012 treatment reduced expression of these genes (Tgf-β1, -44.7%;p=0.003; Fstl3, -43.5%;p=0.0162; Mcp1, -22.5%;p=0.535; Cd68, -60.3%;p=0.0048; Ctgf, -57.1%;p=0.0139; Col1a1, -47.2%, p=0.0255 ;Col3a1, -69.5%;p&amp;lt;0.0001; Lox, -61.4%;p=0.0054; P-selectin, -63.9%;p=0.018) in comparison to SH-Veh treatment. Conclusion Consistent with previous findings in a PAB mouse model, RKER-012 treatment protected against increased sPAP and maladaptive cardiac remodeling in a SH rat model of PAH. Additionally, RKER-012 attenuated RV cardiomyopathy by reducing the expression of genes involved in inflammatory and fibrotic processes. These preclinical findings in the RV paralleled the changes in circulating cardiovascular health biomarkers, including reduced NT-proBNP, observed in a Phase 1 trial of KER-012 in healthy volunteers. These findings, along with the safety and tolerability observed in the Phase 1 trial, provided rationale for the ongoing Phase 2 TROPOS trial of KER-012 in patients with PAH (NCT05975905).

The Role of BAG3 Protein Interactions in Cardiomyopathies.

Bcl-2-associated athanogene 3 (BAG3) plays an important function in cellular protein quality control (PQC) maintaining proteome stability. Mutations in the BAG3 gene result in cardiomyopathies. Due to its roles in cardiomyopathies and the complexity of BAG3-protein interactions, it is important to understand these protein interactions given the importance of the multifunctional cochaperone BAG3 in cardiomyocytes, using an in vitro cardiomyocyte model. The experimental assay was conducted using high pressure liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in the human AC16 cardiomyocyte cell line with BioID technology. Proteins with BAG3-interaction were identified in all the 28 hallmark gene sets enriched in idiopathic cardiomyopathies and/or ischemic disease. Among the 24 hallmark gene sets enriched in both idiopathic cardiomyopathies and ischemic disease, 15 gene sets had at least 3 proteins with BAG3-interaction. This study highlights BAG3 protein interactions, unveiling the key gene sets affected in cardiomyopathies, which help to explain the molecular mechanisms of the cardioprotective effects of BAG3. In addition, this study also highlighted the complexity of proteins with BAG3 interactions, implying unwanted effects of BAG3.

Sex specific gene expression is present prior to metamorphosis in the sea urchin

A profound collaboration between the germline and somatic cells of an organism is the creation of a functional gonad. Here we establish a foundation for studying molecular gonadogenesis in the sea urchin by use of RNA-seq, quantitative mRNA measurements, and in-situ hybridizations throughout the life cycle of the variegated sea urchin, Lytechinus variegatus (Lv). We found through three distinct analyses that the ovary and testis of this echinoderm expresses unique transcripts involved in gametogenesis, and also discovered uncharacterized gene products unique to each gonad. We further developed a pipeline integrating timepoint RNA-seq data throughout development to identify hallmark gene expression in gonads. We found that meiotic and candidate genes involved in sex determination are first expressed surprisingly early during larval growth, and well before metamorphosis. We further discovered that individual larvae express varying amounts of male- or female-hallmarks before metamorphosis, including germline, oocyte, sperm, and meiotic related genes. These distinct male- or female-gonad gene profiles may indicate the onset of, and commitment to, development of a bipotential gonad primordium, and may include metabolic differences, supported by the observation that transcripts involved in glycolysis are highly enriched in the ovary compared to the testis. Together these data support a hypothesis that sex determination is initiated prior to metamorphosis in the sea urchin and that the many uncharacterized genes unique to each gonad type characterized herein may reveal unique pathways and mechanisms in echinoderm reproduction.

5-hydroxymethylcytosine sequencing in plasma cell-free DNA identifies unique epigenomic features in prostate cancer patients resistant to androgen deprivation therapies.

Currently, no biomarkers are available to identify resistance to androgen-deprivation therapies (ADT) in men with hormone-naive prostate cancer. Since 5-hydroxymethylcytosines (5hmC) in gene body are associated with gene activation, in this study, we evaluated whether 5hmC signatures in cell-free DNA (cfDNA) predicts early resistance to ADT. We collected a total of 139 serial plasma samples from 55 prostate cancer patients receiving ADT at three time points including baseline (prior to initiating ADT, N=55), 3-month (after initiating ADT, N=55), and disease progression (N=15) within 24 months or 24-month if no progression was detected (N=14). To quantify 5hmC abundance across the genome, we used selective chemical labeling sequencing and mapped sequence reads to individual genes. Differential methylation analysis in baseline samples identified significant 5hmC difference in 1,642 of 23,433 genes between patients with and without progression (false discovery rate, FDR<0.1). Patients with disease progression showed significant 5hmC enrichments in multiple hallmark gene sets with androgen responses as top enriched gene set (FDR=1.19E-13). Interestingly, this enrichment was driven by a subgroup of patients featuring a significant 5hmC hypermethylation in the gene sets involving AR , FOXA1 and GRHL2 . To quantify overall activities of these gene sets, we developed a gene set activity scoring algorithm and observed significant association of high activity scores with poor progression-free survival (P<0.05). Longitudinal analysis showed that the high activity scores were significantly reduced after 3-months of initiating ADT (P<0.0001) but returned to higher levels when the disease was progressed (P<0.05). This study demonstrates that 5hmC-based activity scores from gene sets involved in AR , FOXA1 and GRHL2 may be used as biomarkers to determine early treatment resistance, monitor disease progression, and potentially identify patients who would benefit from upfront treatment intensification.

Establishment of the Prognostic Signature with Genes Related to G2/M Checkpoint in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality in the world. Prognostic indicators such as clinicopathological characteristics and single molecular signature are far from satisfactory in clinical practice. More and more researches have suggested that polygenic prognostic features could predict the prognosis of cancer more precisely than single genes nowadays. In this study, we performed gene set enrichment analysis (GSEA) to identify the sets of TCGA hallmark genes. Univariate Cox regression analysis was used to select preliminary genes, and then multivariate Cox regression analysis was used to identify genes associated with overall survival (OS). We also used Kaplan-Meier analysis and receiver operating characteristic (ROC) analysis to validate the prognostic gene signature. Lastly, qRT-PCR was used to evaluate the expression of these genes in clinical samples, and immunohistochemical staining was performed to confirm the signature. A 12-gene signature was finally built and the risk score was significantly associated with the survival of the patients. Subsequent validation by qRT-PCR and immunohistochemical staining in clinical specimens confirmed the value of the risk score in predicting the prognosis. We developed a 12-gene signature that could predict the prognosis of HCC patients. This signature is of high precision and would help identifying subgroups of HCC patients with high or low risk of unfavorable survival.

Genomics and evolutionary analysis of Chlorella variabilis-infecting viruses demarcate criteria for defining species of giant viruses.

Chloroviruses exhibit a close relationship with their hosts with the phenotypic aspect of their ability to form lytic plaques having primarily guided the taxonomy. However, with the isolation of viruses that are only able to complete their replication cycle in one strain of Chlorella variabilis, systematic challenges emerged. In this study, we described the genomic features of 53 new chlorovirus isolates and used them to elucidate part of the evolutionary history and taxonomy of this clade. Our analysis revealed new chloroviruses with the largest genomes to date (>400 kbp) and indicated that four genomic features are statistically different in the viruses that only infect the Syngen 2-3 strain of C. variabilis (OSy viruses). We found large regions of dissimilarity in the genomes of viruses PBCV-1 and OSy-NE5 when compared with the other genomes. These regions contained genes related to the interaction with the host cell machinery and viral capsid proteins, which provided insights into the evolution of the replicative and structural modules in these giant viruses. Phylogenetic analysis using hallmark genes of Nucleocytoviricota revealed that OSy-viruses evolved from the NC64A-viruses, possibly emerging as a result of the strict relationship with their hosts. Merging phylogenetics and nucleotide identity analyses, we propose strategies to demarcate viral species, resulting in seven new species of chloroviruses. Collectively, our results show how genomic data can be used as lines of evidence to demarcate viral species. Using the chloroviruses as a case study, we expect that similar initiatives will emerge using the basis exhibited here.IMPORTANCEChloroviruses are a group of giant viruses with long dsDNA genomes that infect different species of Chlorella-like green algae. They are host-specific, and some isolates can only replicate within a single strain of Chlorella variabilis. The genomics of these viruses is still poorly explored, and the characterization of new isolates provides important data on their genetic diversity and evolution. In this work, we describe 53 new chlorovirus genomes, including many isolated from alkaline lakes for the first time. Through comparative genomics and molecular phylogeny, we provide evidence of genomic gigantism in chloroviruses and show that a subset of viruses became highly specific for their hosts at a particular point in evolutionary history. We propose criteria to demarcate species of chloroviruses, paving the way for an update in the taxonomy of other groups of viruses. This study is a new and important piece in the complex puzzle of giant algal viruses.

Investigating the p21 Ubiquitin-Independent Degron Reveals a Dual Degron Module Regulating p21 Degradation and Function.

A group of intrinsically disordered proteins (IDPs) are subject to 20S proteasomal degradation in a ubiquitin-independent manner. Recently, we have reported that many IDPs/IDRs are targeted to the 20S proteasome via interaction with the C-terminus of the PSMA3 subunit, termed the PSMA3 Trapper. In this study, we investigated the biological significance of the IDP-Trapper interaction using the IDP p21. Using a split luciferase reporter assay and conducting detailed p21 mutagenesis, we first identified the p21 RRLIF box, localized at the C-terminus, as mediating the Trapper interaction in cells. To demonstrate the role of this box in p21 degradation, we edited the genome of HEK293 and HeLa cell lines using a CRISPR strategy. We found that the p21 half-life increased in cells with either a deleted or mutated p21 RRLIF box. The edited cell lines displayed an aberrant cell cycle pattern under normal conditions and in response to DNA damage. Remarkably, these cells highly expressed senescence hallmark genes in response to DNA damage, highlighting that the increased p21 half-life, not its actual level, regulates senescence. Our findings suggest that the p21 RRLIF box, which mediates interactions with the PSMA3 Trapper, acts as a ubiquitin-independent degron. This degron is positioned adjacent to the previously identified ubiquitin-dependent degron, forming a dual degron module that functionally regulates p21 degradation and its physiological outcomes.

8433 Elucidation of the Molecular Basis of Human and Mouse Pancreatic β-Cell Senescence Induced by Hyperglycemia

Abstract Disclosure: K. Iwasaki: None. P. Carapeto: None. C. Aguayo-Mazzucato: None. Background and AIM: Senescent cells accumulate with age and contribute to the development of chronic diseases, including Type 2 Diabetes (T2D). Whereas senescence is an important pathogenic mechanism in diabetes, the role of hyperglycemia as a metabolic driver of pancreatic β-cells has not been clarified. The aim of this study is to elucidate the molecular basis of hyperglycemia-induced pancreatic β-cell senescence using human and rodent islets. Methods: 1) Human islets were cultured for 2 weeks in 20.2 mM high (HG) or 2.8mM low glucose (LG), and analyzed by qPCR and glucose-responsive insulin secretion (GSIS) to explore the molecular basis of senescence in ex vivo. To test the effects of senescent cell removal, senolytics Dasatinib (1μM) and Quercetin (20μM) (DQ) were used. 2) To evaluate the effects of hyperglycemia in vivo, we transplanted mouse and human islets under the kidney capsule of normoglycemic (NG) or streptozocin-induced hyperglycemic (Stz-Db) immunodeficient mice for two weeks. Graft function was evaluated by intraperitoneal glucose tolerance test and cellular identity using qPCR or immunohistochemistry. DQ (5mg/kg/day + 50mg/kg/day) was used to evaluate the effects of removing senescent cells in transplantation (Tx). To investigate the effect of specific removal of p16-positive senescent β-cells in transplanted islets, INK-ATTAC mice islets (p16Ink4a-mediated apoptosis by targeted activation of caspases) were transplanted into Stz-Db and treated with BB homodimer (BB) to remove p16+ cells or vehicle. Results: (1) Human islets from 5 healthy donors cultured in HG had an increased senescence index (mean of senescence markers p16, p21 and HMGB1 mRNA) when compared to LG islets (p&amp;lt;0.05) and lost glucose responsiveness as evaluated by ex vivo GSIS compared to LG (p&amp;lt;0.05). 2) Human islets (1000IEQ) were transplanted to Stz-Db or NG mice. In Stz-Db graft, P21 mRNA significantly increased with decreased expression of β-cell genes when compared to NG. Stz-Db mice treated with DQ (Db-DQ) for 5 days after Tx, significantly decreased expression of senescence index (p&amp;lt;0.05) and increased in β-cell index (mean of β-cell hallmark genes) (p&amp;lt;0.05) at mRNA levels. Stz-Db mice transplanted with INK-ATTAC mice islets had lower fed glucose levels, decreased p16 mRNA, increased expression β-cell hallmark genes and improved insulin secretion ex vivo after removal of p16+ cells. Conclusion: Sustained exposure of human islets to hyperglycemia induced cellular senescence, leading to pancreatic β-cell dysfunction and loss of cellular identity. Furthermore, elimination of senescent cells may protect pancreatic β-cells from hyperglycemia-induced functional decline, which may have potential clinical application in both Type 1 Diabetes and T2D. Presentation: 6/2/2024

Effect of aberrant fructose metabolism following SARS-CoV-2 infection on colorectal cancer patients' poor prognosis.

Most COVID-19 patients have a positive prognosis, but patients with additional underlying diseases are more likely to have severe illness and increased fatality rates. Numerous studies indicate that cancer patients are more prone to contract SARS-CoV-2 and develop severe COVID-19 or even dying. In the recent transcriptome investigations, it is demonstrated that the fructose metabolism is altered in patients with SARS-CoV-2 infection. However, cancer cells can use fructose as an extra source of energy for growth and metastasis. Furthermore, enhanced living conditions have resulted in a notable rise in fructose consumption in individuals' daily dietary habits. We therefore hypothesize that the poor prognosis of cancer patients caused by SARS-CoV-2 may therefore be mediated through fructose metabolism. Using CRC cases from four distinct cohorts, we built and validated a predictive model based on SARS-CoV-2 producing fructose metabolic anomalies by coupling Cox univariate regression and lasso regression feature selection algorithms to identify hallmark genes in colorectal cancer. We also developed a composite prognostic nomogram to improve clinical practice by integrating the characteristics of aberrant fructose metabolism produced by this novel coronavirus with age and tumor stage. To obtain the genes with the greatest potential prognostic values, LASSO regression analysis was performed, In the TCGA training cohort, patients were randomly separated into training and validation sets in the ratio of 4: 1, and the best risk score value for each sample was acquired by lasso regression analysis for further analysis, and the fifteen genes CLEC4A, FDFT1, CTNNB1, GPI, PMM2, PTPRD, IL7, ALDH3B1, AASS, AOC3, SEPINE1, PFKFB1, FTCD, TIMP1 and GATM were finally selected. In order to validate the model's accuracy, ROC curve analysis was performed on an external dataset, and the results indicated that the model had a high predictive power for the prognosis prediction of patients. Our study provides a theoretical foundation for the future targeted regulation of fructose metabolism in colorectal cancer patients, while simultaneously optimizing dietary guidance and therapeutic care for colorectal cancer patients in the context of the COVID-19 pandemic.

Long non-coding RNA FAM87A is associated with overall survival and promotes cell migration and invasion in gastric cancer.

The role of long non-coding RNAs (lncRNAs) in the invasion and metastasis of gastric cancer remains largely unclear. Integrating transcriptome data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, differentially expressed genes were identified in gastric cancer. Using the Catalogue of Somatic Mutations in Cancer (COSMIC) database-curated gene set, lncRNAs associated with invasion and metastasis were identified. The Cox analyses were performed to identify prognostic lncRNAs. The competing endogenous RNA (ceRNA) regulation network was constructed to identify hub lncRNAs in gastric cancer. Functional and pathway analyses were used to investigate the function of identified lncRNAs. RT-qPCR and Transwell assays were used to investigate the expression in gastric cancer tissues and functions in gastric cancer cell lines. Based on GEO and TCGA databases, 111 differentially expressed lncRNAs were identified between gastric cancer and normal samples. A total of 43 lncRNAs were significantly correlated with hallmark genes of cancer invasion and metastasis. Among them, as a hub lncRNA in the invasion-related ceRNA regulation network, FAM87A showed potential regulation on MAPK signaling and transforming growth factor (TGF) signaling cascade, such as TGFB2, TGFBR1, and TGFBR2. Furthermore, FAM87A also showed a significant correlation with cell adhesion molecules, such as Integrin alpha 6 (ITGA6) and Contactin-1 (CNTN1). RT-qPCR experiments showed that FAM87A expression was upregulated in gastric cancer tissues compared to normal samples (n = 30). Transwell assays showed that FAM87A knockdown inhibited the migration and invasion abilities of gastric cancer cells in vitro. Notably, clinical data analysis showed that lncRNA FAM87A could be an independent factor for the overall survival of patients with gastric cancer. LncRNA FAM87A may play a pivotal role in regulating migration and invasion of gastric cancer cells. FAM87A could be a potential biomarker for the overall survival of patients with gastric cancer.

Beyond the Spectrum: Subtype-Specific Molecular Insights into Autism Spectrum Disorder Via Multimodal Data Integration.

Some toddlers with autism spectrum disorder (ASD) have mild social symptoms and developmental improvement in skills, but for others, symptoms and abilities are moderately or even severely affected. Those with profound autism have the most severe social, language, and cognitive symptoms and are at the greatest risk of having a poor developmental outcome. The little that is known about the underlying biology of this important profound autism subtype, points clearly to embryonic dysregulation of proliferation, differentiation and neurogenesis. Because it is essential to gain foundational knowledge of the molecular biology associated with profound, moderate, and mild autism clinical subtypes, we used well-validated, data-driven patient subtyping methods to integrate clinical and molecular data at 1 to 3 years of age in a cohort of 363 ASD and controls representative of the general pediatric population in San Diego County. Clinical data were diagnostic, language, cognitive and adaptive ability scores. Molecular measures were 50 MSigDB Hallmark gene pathway activity scores derived from RNAseq gene expression. Subtyping identified four ASD, typical and mixed diagnostic clusters. 93% of subjects in one cluster were profound autism and 93% in a different cluster were control toddlers; a third cluster was 76% moderate ability ASD; and the last cluster was a mix of mild ASD and control toddlers. Among the four clusters, the profound autism subtype had the most severe social symptoms, language, cognitive, adaptive, social attention eye tracking, social fMRI activation, and age-related decline in abilities, while mild autism toddlers mixed within typical and delayed clusters had mild social symptoms, and neurotypical language, cognitive and adaptive scores that improved with age compared with profound and moderate autism toddlers in other clusters. In profound autism, 7 subtype-specific dysregulated gene pathways were found; they control embryonic proliferation, differentiation, neurogenesis, and DNA repair. To find subtype-common dysregulated pathways, we compared all ASD vs TD and found 17 ASD subtype-common dysregulated pathways. These common pathways showed a severity gradient with the greatest dysregulation in profound and least in mild. Collectively, results raise the new hypothesis that the continuum of ASD heterogeneity is moderated by subtype-common pathways and the distinctive nature of profound autism is driven by the differentially added profound subtype-specific embryonic pathways.

Immune and inflammatory insights in atherosclerosis: development of a risk prediction model through single-cell and bulk transcriptomic analyses.

Investigation into the immune heterogeneity linked with atherosclerosis remains understudied. This knowledge gap hinders the creation of a robust theoretical framework essential for devising personalized immunotherapies aimed at combating this disease. Single-cell RNA sequencing (scRNA-seq) analysis was employed to delineate the immune cell-type landscape within atherosclerotic plaques, followed by assessments of cell-cell interactions and phenotype characteristics using scRNA-seq datasets. Subsequently, pseudotime trajectory analysis was utilized to elucidate the heterogeneity in cell fate and differentiation among macrophages. Through integrated approaches, including single-cell sequencing, Weighted Gene Co-expression Network Analysis (WGCNA), and machine learning techniques, we identified hallmark genes. A risk score model and a corresponding nomogram were developed and validated using these genes, confirmed through Receiver Operating Characteristic (ROC) curve analysis. Additionally, enrichment and immune characteristic analyses were conducted based on the risk score model. The model's applicability was further corroborated by in vitro and in vivo validation of specific genes implicated in atherosclerosis. This comprehensive scRNA-seq analysis has shed new light on the intricate immune landscape and the role of macrophages in atherosclerotic plaques. The presence of diverse immune cell populations, with a particularly enriched macrophage population, was highlighted by the results. Macrophage heterogeneity was intricately characterized, revealing four distinct subtypes with varying functional attributes that underscore their complex roles in atherosclerotic pathology. Intercellular communication analysis revealed robust macrophage interactions with multiple cell types and detailed pathways differing between proximal adjacent and atherosclerotic core groups. Furthermore, pseudotime trajectories charted the developmental course of macrophage subpopulations, offering insights into their differentiation fates within the plaque microenvironment. The use of machine learning identified potential diagnostic markers, culminating in the identification of RNASE1 and CD14. The risk score model based on these biomarkers exhibited high accuracy in diagnosing atherosclerosis. Immune characteristic analysis validated the risk score model's efficacy in defining patient profiles, distinguishing high-risk individuals with pronounced immune cell activities. Finally, experimental validation affirmed RNASE1's involvement in atherosclerotic progression, suggesting its potential as a therapeutic target. Our findings have advanced our understanding of atherosclerosis immunopathology and paved the way for novel diagnostic and therapeutic strategies.

Analysis of the Mechanism Underlying Radiotherapy Resistance Caused by Oligodendroglia Cells in Glioblastoma by Applying the Single-cell RNA Sequencing Technology.

Glioblastoma (GBM) is an aggressive malignancy. The inherent resistance of GBM to radiotherapy poses great challenges for clinical treatment. The primary objective of this study is to explore the molecular mechanisms of radiotherapy resistance in GBM and identify the key influencing factors that contribute to this phenomenon. The single-cell RNA sequencing (scRNA-seq) data of GBM were downloaded from the Gene Expression Omnibus (GEO) database. Cells were clustered using the Seurat R package, and the clusters were annotated using the CellMarker database. Pseudotime analysis was conducted using Monocle2. Marker scores were calculated based on the RNA-seq data of GBM from the UCSC database, and the enrichment of Hallmark gene sets was measured with the AUCell package. Furthermore, the most frequently mutated genes were identified using the simple nucleotide variation data from The Cancer Genome Atlas (TCGA) applying the maftools package. This study identified two oligodendrocyte subsets (ODC3 and ODC4) as radiotherapy-resistant groups in GBM. Enrichment and Pseudotime analysis revealed that the inflammatory response and immune activation pathways were enriched in ODC3, while the cell division and interferon response pathways were enriched in ODC4. The enrichment scores of hallmark gene sets further confirmed that ODC3 and ODC4 subpopulations developed radiotherapy resistance via distinct molecular mechanisms. Analysis of gene mutation frequencies showed that TP53 exhibited the most significant change in mutation frequency, indicating that it was an important risk factor involved in radiotherapy resistance in GBM. We identified two ODC subpopulations that exhibited resistance to radiotherapy, providing a new perspective and potential targets for personalized treatment strategies for GBM.

ThymoSpheres culture: A model to study human polyclonal unconventional T cells.

In vitro cultures remain crucial for studying the fundamental mechanisms of human T-cell development. Here, we introduce a novel in vitro cultivation system based on ThymoSpheres (TS): dense spheroids consisting of DLL4-expressing stromal cells and human hematopoietic precursor cells, in the absence of thymic epithelial cells. These spheroids are subsequently cultured at the air-liquid interphase. TS generate large numbers of mature T cells, are easy to manipulate, scalable, and can be repeatably sampled to monitor T-cell differentiation. The mature T cells generated from primary human hematopoietic precursor cells were extensively characterized using single-cell RNA and combined T-cell receptor (TCR) sequencing. These predominantly CD8α T cells exhibit transcriptional and TCR CDR3 characteristics similar to the recently described human polyclonal αβ unconventional T cell (UTC) lineage. This includes the expression of hallmark genes associated with agonist selection, such as IKZF2 (Helios), and the expression of various natural killer receptors. The TCR repertoire of these UTCs is polyclonal and enriched for CDR3-associated autoreactive features and early rearrangements of the TCR-α chain. In conclusion, TS cultures offer an intriguing platform to study the development of this human polyclonal UTC lineage and its inducing selection mechanisms.