title: N1-SRC regulates mRNA splicing during neural development

city: York

first: harry

middle: victor

last: isaacs

state: North Yorkshire

address: wentworth way

department: biology

institute: university of york

summary: Alternative mRNA splicing generates transcriptomic diversity to direct tissue-specific functions. There is a high level of alternative splicing in the brain, particularly in development, but the master regulators are poorly understood. A key splicing event early in neuronal differentiation is the inclusion of a microexon in the SH3 domain of the ubiquitous tyrosine kinase, C-SRC, to yield the constitutively active, neural-specific N1-SRC kinase. We previously demonstrated that specific inhibition of N1-SRC in developing Xenopus embryos inhibits neurogenesis, but the targets and mode of action of N1-SRC are unknown. In the current study we screened for N1-SRC SH3 domain interactors, surprisingly finding no unique targets compared to C-SRC, but rather a subset of low affinity binders, enriched in splicing regulators. Analysis of public phosphoproteomic data revealed that SRC-dependent phosphorylation of the splicing machinery is widespread and enriched in RNA binding proteins. To investigate whether N1-SRC-dependent regulation of splicing underpins its role in neurogenesis, we undertook long and short read RNAseq analysis of N1-SRC knockdown Xenopus embryos. We observed an upregulation of splicing factor expression and aberrant splicing of splicing regulators, principally HNRNPA1 and TRA2A. The affected splice junctions in both genes were in their glycine rich C-termini and enriched in SFPQ/NONO and FUS binding sites. These RNA binding proteins are SRC substrates and suggest a mechanism by which N1-SRC knockdown leads to mis-splicing of HNRNPA1 and TRA2A. Thus, the neuronal splicing of C-SRC to generate N1-SRC regulates the alternative splicing landscape during neurogenesis.

overall_design: We undertook triplicate RNA-seq analysis on standard control morpholino and n1-src splice blocking slice blocking morpholino injected Xenopus laevis embryos at neurula stage 16.

first: Harry

middle: V

last: Isaacs


title: Heterogeneity of CD8αα intraepithelial lymphocytes is transcriptionally conserved between TCRαβ and TCRγδ cells

status: Public on Aug 27 2025

city: Amherst

first: Leonid

last: Pobezinsky

state: MA

address: 661 N Pleasant St

department: Veterinary and Animal Sciences

country: USA

institute: University of Massachusetts Amherst

type: Expression profiling by high throughput sequencing

summary: Intestinal intraepithelial lymphocytes (IELs) are a versatile population of immune cells with both effector and regulatory roles in gut immunity. Although this functional diversity is thought to arise from distinct IEL subpopulations, the heterogeneity of TCRαβ+ and TCRγδ+ IELs have not been well-characterized. Here, we sorted small intestinal IELs from Vil1 Cre+ R26 ZsGreen+ mice into four groups based on expression of either TCR or TCR and fluorescence intensity of ZsGreen and performed scRNAseq. We identified CD8αα+ T cell subsets with memory-like (Tcf7⁺) and effector-like (Prdm1⁺) profiles in both TCRαβ+ and TCRγδ+ IELs. Using CD160 and CD122 as markers of memory-like and effector-like cells, respectively, we found that while effector-like cells dominated the small intestine, memory-like IELs were more prevalent in the large intestine, suggesting a functional specialization of immune responses along the gut. Further transcriptional analysis revealed shared profiles between TCRαβ+ and TCRγδ+ small intestinal IEL subsets, suggesting conserved functional roles across these populations. Finally, our analysis indicated that TCRαβ+ memory-like IELs arise from Tcf7⁺ double-negative (DN) precursors, and that effector-like IELs subsequently differentiate from the memory-like population. In contrast, TCRγδ+ IELs appear to originate from two distinct precursor populations, one expressing Tcf7 and the other Zeb2, indicating the presence of parallel developmental pathways within this lineage. Overall, our findings reveal that both TCRαβ+ and TCRγδ+ cells contain memory-like and effector-like subsets, which may contribute to the functional heterogeneity of IELs.

overall_design: Single cell RNA sequencing of Intraepithelial lymphocytes, collected from the small intestine of Vil1 Cre+ R26 ZsG+ mice and sorted into "TCRαβ+ ZsG-", "TCRαβ+ ZsG+", "TCRγδ+ ZsG-", "TCRγδ+ ZsG+" groups.

first: Kaito

middle: A

last: Hioki

first: Elena

middle: L

last: Pobezinskaya


title: JNK pathway suppression drives resistance to combination endocrine therapy and CDK4/6 inhibition in ER+ breast cancer

city: Darlinghurst

first: Leila

last: Eshraghi

state: NSW

address: 384 Victoria Street

country: Australia

institute: Garvan Institute of Medical Research

summary: Endocrine therapy in combination with CDK4/6 inhibition doubles the progression-free survival of patients with advanced ER+ breast cancer, but resistance is inevitable, leaving patients with limited treatment options. Here, we performed unbiased genome-wide CRISPR/Cas9 knockout screens using ER+ breast cancer cells to identify novel drivers of resistance to combination endocrine therapy (tamoxifen) and CDK4/6 inhibitor (palbociclib) treatment. Our screens identified the inactivation of JNK signalling, including loss of the kinase MAP2K7, as a key driver of combination resistance. We developed multiple CRISPR/Cas9 knockout ER+ breast cancer cell lines (MCF-7 and T-47D) to investigate the effects of MAP2K7 and downstream MAPK8 and MAPK9 loss. MAP2K7 knockout increased metastatic burden in vivo and led to impaired JNK-mediated stress responses, as well as promoting cell survival and reducing senescence entry following endocrine therapy and CDK4/6 inhibitor treatment. Mechanistically, this occurred via loss of the AP-1 transcription factor c-JUN, leading to an attenuated response to combination endocrine therapy plus CDK4/6 inhibition. Furthermore, we analysed ER+ advanced breast cancer patient cohorts and found that inactivation of the JNK pathway was associated with increased metastatic burden, and low pJNKT183/Y185 activity correlated with a poorer response to systemic endocrine and CDK4/6 inhibitor therapies. Overall, we demonstrate that suppression of JNK signalling enables persistent growth during combined endocrine therapy and CDK4/6 inhibition. Our data provide a pre-clinical rationale to screen patients’ tumours for JNK signalling deficiency prior to receiving combined endocrine therapy and CDK4/6 inhibition.

overall_design: RNAseq analysis of ER+ breast cancer cells lines (MCF-7 and T-47D) with MAP2K7 knockout or pLenti wild-type treated for 48 hours with tamoxifen + palbociclib or fulvestrant + palbociclib.

first: Sarah

last: Alexandrou

first: Christine

middle: S

last: Lee

first: Kristine

middle: J

last: Fernandez

first: Celine

middle: E

last: Wiharja

first: John

last: Reeves

first: Daniel

last: Reed

first: Neil

last: Portman

first: Zoe

last: Phan

first: Heloisa

middle: H

last: Milioli

first: Iva

last: Nikolic

first: Antonia

last: Cadell

first: David

middle: R

last: Croucher

first: Kaylene

last: Simpson

first: Elgene

last: Lim

first: Theresa

last: Hickey

first: Ewan

middle: K

last: Millar

first: Carla

last: Alves

first: Henrik

last: Ditzel

first: Catherine

last: Caldon


title: Telmisartan Modulates Exercise Responses in Peripheral Artery Disease: Analyses of Skeletal Muscle from the TELEX Trial

city: Lexington

first: Ahmed

last: Ismaeel

state: Kentucky

address: 900 S Limestone St

department: Physiology

institute: University of Kentucky

summary: Background: In people with peripheral artery disease (PAD), the TELEX randomized clinical trial tested whether telmisartan (TEL), with or without exercise, significantly improved 6-minute walk distance at 6-month follow-up, compared to placebo (PLA). Objectives: The present study analyzed the effects of TEL on exploratory outcomes of muscle cellular and molecular characteristics. Methods: Baseline and 6-month follow-up muscle biopsies were obtained from 13 participants with PAD in the TELEX trial randomized to exercise + TEL or exercise + PLA. Immunohistochemistry was used to assess measures including myofiber cross-sectional area and satellite cell content, and the GeoMx digital spatial profiling system was used for transcriptomic analyses of alpha-smooth muscle actin (α-SMA)-positive and α-SMA-negative cells (primarily myofibers). Results: Myofiber cross-sectional area (CSA) and the number of satellite cells associated with Type II myofibers were significantly increased in the exercise + TEL group relative to exercise + PLA (p = 0.038 and p = 0.031, respectively). Among participants undergoing supervised exercise, those randomized to TEL had up-regulated N-linked glycosylation and down-regulated Ras signaling pathways in α-SMA-positive cells, compared to PLA. In α-SMA-negative cells, participants randomized to TEL showed up-regulated PPARγ activation-related pathways, including NO-cGMP-PKG signaling, and fatty acid oxidation. TEL also significantly reduced myostatin expression (adj. p = 0.0097) relative to PLA in α-SMA-negative cells. Conclusions: Overall, these findings suggest that TEL may regulate the effects of exercise on muscle in individuals with PAD by reducing myostatin expression, increasing myofiber size, and increasing activation of PPARγ.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: Muscle tissue samples were stained with a fluorescent-conjugated antibody against α-SMA (1:400, Abcam, Cambridge, United Kingdom, #ab202368, RRID: AB_2924381) and a nucleic acid stain (SYTO 83, Thermo Fisher Scientific, #S11364), as well as GeoMX DSP oligo-conjugated RNA detection probes (GeoMx Human Whole Transcriptome Atlas [WTA] Human RNA for Illumina Systems, NanoString Technologies, Seattle, WA, #GMX-RNA-NGSHuWTA). Following region on interest (ROI) selection (660 µm x 785 µm), DSP barcodes were UV-cleaved, collected, and oligos were dispensed into 96-well plates prior to library preparation. Sequencing was performed using a NovaSeq 6000 S4 flow cell (Illumina, San Diego, CA). FASTQ files were processed via the GeoMX NGS Pipeline on DRAGEN via BaseSpace Sequence Hub (Illumina), and counts data were uploaded to the GeoMx DSP Analysis Suite (NanoString Technologies) for analyses. For quality control (QC) and filtering, probes with low counts (<5 reads in more than 2 ROIs) and probes failing global outlier (ratio of geometric mean over all ROIs to geometric mean of individual target over all ROIs <0.1) and Grubbs outlier (outlier probes in >20% of ROIs) tests were excluded. A total of 45 ROIs were analyzed for α-SMA+ cells, and 44 ROIs were analyzed for α-SMA- cells

first: Ismaeel

last: Ahmed


title: A phosphorylation switch within the Mediator subunit MED15 controls cellular senescence and cognitive decline [ChIP-seq]

city: Shanghai

first: Yuanming

last: Zheng

address: 220 Handan Rd., Shanghai

country: China

institute: fudan university

type: Genome binding/occupancy profiling by high throughput sequencing

summary: A hallmark of aging is chronic systemic inflammation, which is exacerbated by the hypersecretory aging phenotype known as the senescence-associated secretory phenotype (SASP). How the SASP is initiated to accelerate tissue inflammation and aging is an outstanding question in ageing biology. Here, we showed that phosphorylation of the mediator subunit MED15 at T603 is able to control the SASP and aging. Transforming growth factor-β (TGFβ) selectively induces CDK1-mediated MED15 T603 phosphorylation to control SASP gene expression. The MED15 T603 dephosphorylated mutant (T603A) inhibits the SASP and cell senescence, whereas the T603 phosphorylation-mimicking mutant (T603D) has the opposite effect. Mechanistically, forkhead box protein A1 (FOXA1) preferentially binds to unphosphorylated but not phosphorylated MED15 at T603 to suppress SASP gene expression. Notably, aging mice harboring dephosphorylated mutation in this phosphosite exhibit improved learning and memory through the attenuation of the SASP across tissues. Overall, our study indicates that MED15 T603 phosphorylation serves as a control switch for SASP production, which underlies tissue aging and cognitive decline and provides a novel target for age-related pathology.

overall_design: To investigate the function of MED15 T603 phosphorylation, we generated MED15 T603A HaCaT cell line and T603D MCF7 cell line and performed RNA-seq analysis. The WT and  MED15 T603A HaCaT were treated with or with TGFβ for 3 hours. We further performed RNA-seq analysis of hippocampus in aging WT and Med15 T604A mice (17-20M).

first: Haozheng

last: Li

first: Gang

last: Wang


title: Multiomics analysis of lung adenocarcinoma manifesting as radiological part-solid features in female never-smokers

city: Seoul

first: Sang-Won

last: Um

address: 81 Irwon-ro

department: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Samsung Medical Center

country: South Korea

institute: Sungkyunkwan University School of Medicine

summary: Lung adenocarcinomas (LUADs) with part-solid radiological features exhibit slow progression to invasive malignancy; however, the key genomic alterations driving this progression remain poorly understood. We conducted a multiomics analysis of four components (distant and adjacent normal lung and solid [S] and ground-glass [GG] components) of part-solid-type LUADs in female never-smokers. Although GG and S components displayed similar mutational profiles, epithelial–mesenchymal transition and hypoxia pathways were significantly upregulated in the S component. Using single-cell RNA sequencing, we identified a novel cancer-associated secretory (CAS) cell population (SCGB3A2/CEACAM6+). The proportion of CAS cells and interleukin (IL)-1β+ macrophages showed a significant upward trend toward the S component. Spatial transcriptomic analysis revealed that IL-1β+ macrophages and CAS cells co-localized in invasive regions of the tumor. In conclusion, CAS cells and IL-1β+ macrophages play crucial roles in the progression from preinvasive to invasive LUADs, positioning them as promising targets for cancer prevention and therapeutic strategies.

overall_design:
Whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) were performed on lung adenocarcinoma (LUAD) samples from seven female, never-smoker patients with part-solid radiological features. For each patient, three distinct regions were analyzed: distant normal lung (DN), ground-glass component (GG), and solid component (S), resulting in a total of 18 samples for bulk sequencing analysis.In addition, single-cell RNA sequencing (scRNA-seq) was conducted on four female, never-smoker patients using the 10X Genomics Chromium platform. Four regions were analyzed for each patient: distant normal lung (DN), adjacent normal lung (AN), ground-glass component (GG), and solid component (S), leading to a total of 16 single-cell RNA-seq samples.

***************************************************************
Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access.
***************************************************************

first: Junsu

last: Choe

first: Jinyeong

first: Noeul

last: Kang

first: Eun Seop

last: Seo

first: Mina

last: Hwang

first: Se-Hoon

last: Lee

first: Myung-ju

last: Ahn

first: Yoon-La

last: Choi

first: Hong Kwan

last: Kim

first: Woong-Yang

last: Park


title: Next Generation Sequencing of Wild Type and Gata2; Asxl1; Vav-iCre Heterozygote HSCs

city: CARDIFF

first: Neil

last: Rodrigues

address: Maindy Road

department: The European Cancer Stem Cell Research Institute

country: United Kingdom

institute: Cardiff University

summary: Clinical GATA2 deficiency syndromes arise from germline haploinsufficiency inducing mutations in GATA2, resulting in immunodeficiency that evolves to myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). How GATA2 haploinsufficiency disrupts the function and transcriptional network of hematopoietic stem/progenitors (HSCs/HSPCs) to facilitate the shift from immunodeficiency sequalae to pre-leukemia is poorly characterised. Using a conditional mouse model harboring a single allele deletion of Gata2 when HSCs emerge in utero, we identify pervasive defects in HSPC differentiation from young adult Gata2 haploinsufficient mice during B-cell maturation, early erythroid specification, megakaryocyte maturation to platelets and inflammatory cell generation. Gata2 haploinsufficiency abolishes HSC self-renewal and multi-lineage differentiation capacity following transplantation. These alterations closely associate with deregulated DNA damage responses and inflammatory signalling conveyed from Gata2 haploinsufficient HSCs. We also identify functional interplay between Gata2 and Asxl1, a driver of DNA damage and inflammation and, notably, a recurrent secondary mutation found in GATA2 haploinsufficiency disease progression to MDS/AML. shRNA mediated knockdown of Asxl1 in Gata2 haploinsufficient HSPCs led to a differentiation block in committed progenitor formation beyond that in Gata2 haploinsufficient HSPCs. By analysis of HSCs from young adult compound Gata2/Asxl1 haploinsufficient mice, we discover hyperproliferation of double haploinsufficent HSCs, which are also functionally compromised in transplantation compared to their single haploinsufficient counterparts. Through both Gata2/Asxl1 dependent and unique transcriptional programs, HSCs from young adult compound Gata2/Asxl1 haploinsufficient mice fortify deregulated DNA damage responses and inflammatory signalling in HSCs initiated in Gata2 haploinsufficient mice and establish a broad pre-leukemic program. Our data reveal how Gata2 haploinsufficiency initially drives deregulation of HSC genome integrity and suggest the mechanisms of how secondary mutations like ASXL1 take advantage of HSC genomic instability to nurture a pre-leukemic state in GATA2 haploinsufficiency syndromes.

overall_design:
Wild Type and Gata2; Asxl1; Vav-iCre Heterozygote HSCs were isolated from 8-to-12-week old mice (4 mice per genotype)
Please note that the WT data (from GSE283398 records) have been re-used in the current study.

** Submitters state that raw data files are not retained and thus not provided******

first: Ali

last: Abdelfattah

first: Peter

last: Giles


title: Lamp2-Flotillin-2 interaction enhances autophagosome-lysosome fusion to protect the septic heart in response to ILC2

first: Rongjiao

last: Shao

state: Shanghai

address: Department of Critical Care Medicine, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai

institute: Shanghai Jiao Tong University School of Medicine

summary: Cardiac dysfunction is a serious complication of sepsis-induced multiorgan failure in intensive care units and is characterized by an uncontrolled immune response to overwhelming infection. Type 2 innate lymphoid cells (ILC2s), as a part of the innate immune system, play a crucial role in the inflammatory process of heterogeneous cardiac disorders. However, the role of ILC2 in regulating sepsis-induced cardiac dysfunction and its underlying mechanism remain unknown. The present study demonstrated that autophagic flux blockage exacerbated inflammatory response and cardiac dysfunction, which was associated with mortality of sepsis. Using a cecal ligation and puncture (CLP) mouse sepsis model, we observed an expansion of ILC2s in the septic heart. Furthermore, IL-4 derived from ILC2 mitigated cardiac inflammatory responses and improved cardiac function during sepsis. Additionally, IL-4 restored the impaired autophagic flux and enhanced lysosomal-associated membrane protein 2 (LAMP2) expression to stabilize lysosomal homeostasis during sepsis. Notably, LAMP2 preferentially bound to Flotillin2 (FLOT2) after IL-4 exposure and the interaction enhanced autophagosome-lysosome fusion in cardiac endothelial cells. Loss of FLOT2 reversed the regulatory effects of LAMP2 on autophagy mediated by IL-4, leading to autophagosome accumulation and suppressed autophagosome clearance. Conclusively, these findings provide novel insights that ILC2 regulates incomplete autophagic flux to protect septic heart and expand our understanding of immunoregulation for sepsis.

overall_design: the underlying mechanisms of IL-4 in septic hearts remain unknown. Therefore, heart tissues of control and CLP-induced mice with or without IL-4 treatment were collected for RNA sequencing (RNA-seq) to explore the underlying mechanisms.


title: IGFBP3-mediated effects of an effective combination therapy on HCC

city: Beijing

first: Lin

last: Chen

address: Chuiyangliunan Road 2

institute: Chuiyangliu Hospital

summary: As all known, hepatocellular carcinoma (HCC) accounts for the majority of cases of liver cancer, which is the third leading cause of cancer mortality globally. Moreover, HCC is always accompanied with HBV infection. Here, we used CMAP, a systematic approach for the discovery of functional connections among diseases and drug actions, to identify quercetin as an effective compound to potentially treat HCC. Furthermore, we proved the inhibitory effects of quercetin on HCC cells, shown as decreased cell viability in HCCLM3 and HepG2 cells. In addition, quercetin disturbed the migration of HCC cells in a dose-dependent manner. Furthermore, quercetin treatments effectively elevated the activities of caspase-3 as well as caspase-9 and increased the Bax expression in HCC cells accompanied with decreased levels of p53 and BCL-2, indicating an enhancement of apoptosis induced by quercetin. Notably, quercetin depressed the activities of antioxidant enzymes, including SOD, GST, GPx and CAT, leading to an increase of ROS accumulation. Additionally, quercetin also exhibited an obvious inhibition of tumor growth of HCC in vivo. Through RNA-seq, results showed that genes related to regulation of cell proliferations were enriched, in which IGFBP3 played a critical role in mediating the effects of quercetin on HCC cells by reducing PI3K-mTOR activation. After silencing IGFBP3 in HCCLM3 cells, quercetin exhibited weaken effects on cell proliferation and apoptosis. Notably, IGFBP3 promotor strengthened the suppressed effects induced by single quercetin administration, indicating a potential drug combination for treatments of HCC. Collectively, this study clarified a novel mechanism underlying the inhibitory effects of quercetin on HCC, providing a potential approach for HCC treatment in clinic.

overall_design: To investigate the effects of quercetin on HCC, we established HCCLM3 cell lines in which were treated with quercetin in different concentrations.

first: Lei

last: Zhao

first: Guozhi

last: Wu

first: Tiantian

last: Zhang

first: Zhiwu

last: Liu

first: David

last: Fisher

first: Nguten

last: Thi Thu Hien

first: Erkin

last: Musabaev


title: Ropinirole improves the amyotrophic lateral sclerosis phenotype of TDP43-mutant iPSC-derived motor neurons via a D2R-independent mechanism

city: fujisawa-shi

first: Hirotsugu

last: Asano

state: kanagawa

address: 2-26-1, Muraokahigashi

country: Japan

institute: K Pharma

summary: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron (MN) degeneration. The dopamine D2 receptor (D2R, DRD2) agonist ropinirole hydrochloride (ROPI) constitutes a potential therapeutic candidate for ALS through phenotypic screening of disease models of MNs that use patient-derived induced pluripotent stem cells (iPSCs). The ROPALS trial, a phase I/II trial in patients with ALS, demonstrated the safety and efficacy of ROPI. However, the D2R antagonist and modulator only partially mitigated the suppressive effect of ROPI on the ALS phenotype, and the detailed mechanism of action of ROPI has not been fully elucidated. In this study, we investigated whether the therapeutic effects of ROPI in ALS depend on D2R. Using D2R-deficient iPSCs, we demonstrated that ROPI effectively mitigated neuronal cell death, reactive oxygen species (ROS) production, and neuronal hyperexcitation in a D2R-independent manner. Further analyses revealed that ROPI rescued aberrant RNA splicing and restored mRNA expression of mitochondrial proteins in a D2R-independent manner. Our data suggest that ROPI has not only a canonical D2R agonist effect, but also multifunctional effects that are mediated in a D2R-independent manner. Therefore, our findings may open new avenues for the development of more effective therapeutic approaches for ALS.

overall_design: isogenic iPSCs with the TARDBP M337V mutation and DRD2 gene knockout were generated using the CRISPR-Cas9 genome editing technology. Using iPS cell-derived motor neurons, we examined DRD2-dependent and -independent changes in gene expression upon addition of ROPI.

first: Tetsuya

last: Kawaguchi

first: Chris

last: Kato

first: Satoru

last: Morimoto

first: Daisuke

last: Yasuda

first: Komei

last: Fukushima

first: Hideyuki

last: Okano


title: Genome-wide DNA replication profiling and full-length total RNA sequencing from the same single cell [02_Mouse8cell_embryos]

city: Tsu-shi

first: Rawin

last: Poonperm

state: Mie

address: 1577 Kurimamachiya

department: Graduate School of Bioresources

institute: Mie University

summary: Single-cell sequencing has advanced our understanding of cell-type diversity and heterogeneity. However, existing single-cell multi-omics methods lack DNA data resolution and full-length total RNA detection. Here, we introduce single-cell (sc)Repli-RamDA-seq (scRR-seq), a novel multi-omics method that enables high-resolution DNA replication profiling and full-length total RNA sequencing from the same single cell, made possible by the clean separation of DNA and RNA. scRR-seq generates DNA replication and RNA sequencing data comparable to individually obtained scRepli-seq and scRamDA-seq data, respectively. scRR-seq also allows one to tell the cell-cycle stage of a given S-phase cell and detects copy-number variation (CNV) in non-S-phase cells. scRR-seq outperforms other scRNA-seq methods in the number of transcripts identified and full-length total RNA detection. Furthermore, scRR-seq allowed haplotype-specific analysis and the identification of novel S-phase markers. Taken together, scRR-seq is a robust single-cell multi-omics method with promising potential for comprehensive genome and transcriptome analysis.

overall_design: We performed simultaneous DNA-seq and RNA-seq on the same cells using the scRR-seq protocols (scRR1 and scRR3). The following cell types were used for the experiments: (1) hTERT-RPE1 cells collected from mid-S phase, (2) mouse 8-cell embryos, (3) CBMS1 mouse embryonic stem cells collected from the entire S phase, (4) hTERT-RPE1 cells collected from the entire S phase, (5) HAP1 cells collected from mid-S phase, and (6) IMR-90 cells treated with or without 0.4 µM aphidicoline, collected from the G1 phase.  Single cells were collected into 0.2 ml PCR tubes by cell sorter or manually picked and lysed in RamDA-seq lysis buffer (TOYOBO, #RMD-101) containing 2 mg/ml Dynabeads™ MyOne™ Carboxylic Acid (Invitrogen, #65011). Genomic DNA was trapped on the beads, while RNA was released into the lysis buffer. The isolated DNA on magnetic beads was subjected to whole-genome amplification (WGA) and Next-Generation Sequencing (NGS) library preparation, as described in scRepli-seq (Takahashi et al., Nat Genet 2019). The DNA-seq (scRepli-seq) data was used to assess genome-wide DNA copy numbers, karyotype G1-phase cells, and analyze the replication state of S-phase cells. In parallel, the RNA isolated in the supernatant was subjected to scRamDA-seq (Hayashi et al., Nat Commun 2018) using the GenNext®RamDA-seq™ Single Cell Kit (TOYOBO, #RMD-101) according to the manufacturer’s instructions with modifications.

first: Shin-ichiro

last: Takebayashi


title: Activation and exhaustion of CD8 T cells in patients with chronic lymphocytic leukemia treated with ibrutinib and pembrolizumab [scRNA-seq, scTCR-seq and bulk RNA-seq]

city: Bethesda

first: Adrian

last: Wiestner

state: MD

address: 10 Center Drive

department: NHLBI

institute: National Institutes of Health

summary: Single-agent anti-PD-1 antibodies lack clinical efficacy in patients with chronic lymphocytic leukemia (CLL). Here, we study the peripheral blood samples from high-risk or relapsed/refractory CLL before and after combination therapy of ibrutinib, fludarabine, and pembrolizumab (IFP). We performed bulk RNA-sequencing (bulk RNA-seq) to detect the transcriptomic changes of CLL and T cells during treatment, and demonstrated the immune responses of CD8 T cells to pembrolizumab by single-cell RNA sequencing (scRNA-Seq).

overall_design: Multiomics scRNA-seq libraries (GEX, ADT, TCR) libraries were prepared using 10X Genomics platform using CD8+ T cells harvested from peripheral blood with negative selection using Miltenyi beads. Libraries from C1D1, C2D1 (after 1 cycle of pembrolizumab), and C9D1 (after 6 months of pembrolizumab) were marked with HTO hashtags and multiplexed prior to sequencing.

first: Rui

last: Mu

first: Katherine

middle: M

last: Hornick

first: Neelam

last: Redekar


title: Next Generation Sequencing of Wild Type and Asxl1; Vav-iCre Heterozygote HSCs

first: Neil

last: Rodrigues

overall_design:
Wild Type and Asxl1; Vav-iCre Heterozygote HSCs were isolated from 8-to-12-week old mice (4 mice per genotype)

** Submitters state that raw data files are not retained and thus not provided******

first: Ali

last: Abdelfattah

first: Peter

last: Giles


title: Genome-wide DNA replication profiling and full-length total RNA sequencing from the same single cell [HAP1 mid-S]


title: Nuclease CAD restrains cGAS-STING signaling to inhibit radiosensitivity of colorectal cancer

city: Tianjin

first: Zhengkun

last: Cai

address: Tianjin, State

institute: Tianjin Medical University Cancer Institute and Hospital

summary: Targeting CAD enhances radiosensitivity by modulating cGAS-STING pathway activation, highlighting the significance of CAD as a potential therapeutic target for CRC.

overall_design: To investigate the role of CAD, we performed RNA-seq analysis on control and CAD knockout (KO) HCT116 cell lines for subsequent enrichment analysis.


title: Genome-wide DNA replication profiling and full-length total RNA sequencing from the same single cell [03_CBMS1_mESC]


title: BAG2 inhibits the proliferation and migration of cervical cancer by slowing the degradation of STING

city: Wuhan

first: Jie

middle: Shi

last: Yao

state: Hubei

address: 169 Donghu Road, Wuchang District, Wuhan City, Hubei Province

department: Department of Gynecological Oncology

institute: Wuhan University

summary: The fourth most prevalent illness in the world among women, cervical cancer has significant negative effects on both health and the economy in addition to high rates of morbidity and death. Understanding its molecular basis to guide clinical management is critical. STING, an innate immune sensor that induces type I interferon, plays a key role in enhancing anti-tumor activity. Despite the growing interest in the role played by STING in cervical cancer, the homeostatic regulatory mechanisms of STING in cervical cancer remain unknown. Here, we found that the BAG2-STUB1 complex regulates ubiquitin proteasomal degradation of STING, which affects the development of cervical cancer. Mechanistically, BAG2 inhibits ubiquitination of STING and stabilizes it by interacting with STING. Specifically, BAG2 inhibits STUB1 from linking the K48-conjugated ubiquitin chains at K338 and K370 of STING by forming a complex with STUB1. Functionally, upregulation of BAG2 expression could inhibit cervical cancer progression by activating the type I interferon response in a STING-dependent manner. Importantly, clinical cervical cancer samples showed a positive correlation between BAG2 and STING expression, and low BAG2 expression was strongly associated with clinical progression and poor prognosis in cervical cancer. Together, these findings describe the molecular mechanism by which the BAG2-STUB1 complex mediates the homeostatic regulation of STING, highlighting the important potential role of BAG2 in the diagnosis and treatment of cervical cancer.

overall_design: To investigate the downstream mechanism of BAG2 regulation of cervical cancer,  we performed RNA sequencing on three repeated Vector and BAG2 overexpression-treated Siha cells.

first: Shijie

first: Hongbing


title: Integrative Single-Cell Analysis Reveals Iron Overload-Induced Senescence and Metabolic Reprogramming in Ovarian Endometriosis-Associated Infertility

first: Yangshuo

last: Li

address: 168 Changhai Road

institute: Navy Medical University

summary: Endometriosis, particularly ovarian endometriosis (OE), significantly impacts fertility by often leading to decreased oocyte quality and impaired ovarian function. Iron overload has been identified as a critical factor driving disease progression in endometriosis lesions. This study aims to investigate the effects of iron overload on follicular function in OE-associated infertility (OEI). Using single-cell sequencing technologies, we first identified oocyte dysfunction in OEI patients and established a single-cell atlas of iron overload in follicular fluid prior to ovulation in OEI patients. This atlas revealed dynamic changes in iron metabolism and identified iron-induced senescence phenotypes. We also examined the differentiation trajectories of granulosa cells and the relationship between macrophage M1/M2 phenotypes and iron metabolism. Additionally, we utilized Stereo-seq (spatiotemporal enhanced resolution omics-sequencing) to construct a spatial transcriptomic map of iron-overloaded mouse ovaries, uncovering various enriched senescence phenotypes. Finally, we extended our findings by exploring the dynamics of iron metabolism imbalance in aging human ovaries. Overall, these findings enhance our understanding of iron overload-related cellular interactions and molecular characteristics in ovarian pathology, providing new therapeutic targets for improving oocyte quality in OEI patients.

overall_design: Bulk RNA-sequencing was performed on granulosa cells from infertile patients' follicular fluid. Follicular fluid was collected separately from patients with EMS-related infertility and male factor infertility, as described previously. Cells were pelleted by centrifugation at 4°C, 2000 rpm for 10 minutes, and resuspended in 10 ml PBS. The cell suspension was carefully layered over lymphocyte separation medium (1:1 volume) along the tube wall and centrifuged at 4°C, 2000 rpm for 20 minutes. The cloudy white layer was gently aspirated into a 15 ml centrifuge tube. After addition of red blood cell lysis buffer and gentle agitation to lyse red blood cells, the suspension was centrifuged at 4°C, 2000 rpm for 10 minutes. The resulting cell pellet was washed twice with PBS and RNA was extracted from granulosa cells using TRIzol reagent (Invitrogen) for subsequent Bulk RNA-seq.

last: Ding

first: Yalun

last: He

first: Di

last: Song

first: Chaoqin

last: Yu


title: Genome-wide DNA replication profiling and full-length total RNA sequencing from the same single cell [IMR-90 G1]

first: Rawin


title: Genome-wide DNA replication profiling and full-length total RNA sequencing from the same single cell [hTERT-RPE1 Whole-S]

first: Shin-ichiro


title: The effect of extracellular microenvironment stiffness on the differentiation of dental pulp stem cells into glial cells and its impact on osteogenesis

city: chengdu

first: zhaowei

last: zhang

state: sichuan

address: No. 14, Section 3, Renmin South Road

institute: sichuan university

summary: Inoculating human dental pulp stem cells on soft and sitff substrates to investigate the effect of substrate hardness on gene expression of dental pulp stem cells

overall_design: To investigate the protective effect of substrate hardness on dental pulp stem cells, we cultured cells using two different hardness substrates (stiff and soft) and analyzed gene expression profiles based on RNA seq data obtained from dental pulp stem cells.

first: Mingru

last: Bai


title: Molecular signatures underlying heterogeneous hypertrophy responsiveness to resistance training in older men and women: a within-subject design

city: Aurora

first: Manoel

middle: Emilio

last: Lixandrao

state: Colorado

institute: University of Colorado Anschutz Medical Campus

summary: We showed higher resistance training (RT) volume (4-sets) offsets muscle hypertrophy non-responsiveness to single-set RT (1-set), while enhancing hypertrophic response among responders. Here, we investigated molecular traits underpinning RT-related hypertrophic variability via targeted (ribosome density and biogenesis) and untargeted approaches (RNAseq gene-ontology (GO) and Pathway-Level Information ExtractoR [PLIER]). Twenty-seven older (693 years) participants in the parental trial had one leg randomly allocated to 1-set and the contralateral leg allocated to 4-sets of RT for 10-wk, 2x/week. Pre- and postintervention, participants underwent magnetic resonance imaging (MRI) and muscle biopsies. We selected participants based on quadriceps muscle cross-sectional area (qCSA) changes and used the MRI typical error (2xTE=3.27%) to allocate those in distinct hypertrophy phenotype: low-responders: blunted hypertrophy, regardless of RT-volume; medium-responders: hypertrophy only following the higher RT-volume; and high-responders: hypertrophy following both RT-volume. The qCSA was higher for 4-sets compared with 1-set for medium- and high-responders only. Higher RT-volume, regardless of responsiveness groups, was followed by increased Frizzled-1 protein expression. RNAseq showed that high-responder’s robust hypertrophy was followed by up-regulation of pathways linked with protein turnover metabolism, RT-related metabolic response, and with protein-folding biological processes. Hypertrophy in medium-responders was marked by down-regulation of pathways linked with muscle catabolism (Proteasome and Spliceosome), modulations on distinct RT-related metabolic response, and biological processes associated with hormone- and steroid-related pathways. Low-responders, on the other hand, did not show measurable changes in any molecular pathways, regardless of RT volume manipulation. Overall, these finding reveal differential molecular signatures across the spectrum of RT responsiveness among older individuals.

overall_design: Before (baseline) and after the 10-week RT intervention, we assessed participants’ bilateral qCSA via MRI. At baseline, participants underwent a vastus lateralis muscle biopsy in one of the legs in a randomized manner in a fasted condition. Forty-eight hours after the last training session, subjects underwent two muscle biopsies (one biopsy from each leg) in a similar fasted condition. Each subject had one leg randomly allocated in a counter-balanced fashion to one set (1 set) and the contralateral leg allocated to four sets (4 sets) of unilateral knee-extension resistance exercise at 8-15RM for 10 weeks, 2x/week.

first: Marcas

last: Bammam

first: Kaleen

last: Lavin

first: Igor

last: Longobardi

first: Guilherme

last: Telles

first: Felipe

middle: C

last: Vechin

last: Damas

first: Devin

last: Drummer

first: Jeremy

last: McAdam

first: Luiz

last: Riani

first: Cleiton

last: Libardi

first: Bruno

last: Gualano

first: Hamilton

last: Roschel


title: Effects of Dll1 blocking antibody on gene expression profiles of the brachiocephalic arteries in ApoE-deficient mice fed a high-fat diet

city: Kitakyushu

first: Jun-ichiro

last: Koga

state: --- Select One ---

address: 1-1, Iseigaoka, Yahatanishi-ku

institute: University of Occupatinal and Environmental Health, Japan

summary: To examine the effects of Dll1 blocking antibody (Dll1 Ab) on gene expression profiles of the brachiocephalic arteries, ApoE-deficient mice were fed a high-fat diet from 12 weeks of age. Dll1 Ab or control non-immune IgG was intraperitoneally injected from 20 weeks of age. At 32 weeks of age, brachiocephalic arteries were excised, and gene expressions were evaluated.

overall_design: To examine the effects of Dll1 Ab, 250 micrograms of the antibody (clone HMD1-5) were intraperitoneally injected twice a week. In the control group, an equivalent dose of non-immune IgG was administered similarly.

first: Nasanbadrakh

last: Orkhonselenge


title: Genetic Characterization of the Cell Types of in Developing Feathers, and the Evolution of Feather Complexity

city: New Haven

first: Cody

last: Limber

state: CT

address: 165 Prospect St

department: Ecology and Evolutionary Biology

institute: Yale University

summary: Feathers are the most complex and diverse epidermal appendages found in vertebrates. Their unique hierarchical organization and development is based on a diversity of cell types and morphologies. Despite being well characterized morphologically and extensive molecular developmental research focusing on candidate genes, little is known about the gene regulatory identities of these presumptive feather cell types. Here, we use single cell and single nuclear RNA sequencing with in situ hybridization to identify and characterize cells types in embryonic chicken feathers. We show that the distinct cell morphologies correspond to feather cell types with distinct gene expression profiles. We also describe a previously unidentified cell type, the basal barb ridge epithelium, which appears to play a role in signaling necessary for barb ridge differentiation and pulp cap production. We also analyze RNA velocity trajectories of developing feather cells, and find distinct developmental trajectories for epidermal cells that constitute the mature feather and those that function only in feather development. Finally, we produce an evolutionary tree of feather cell types based on transcription factor expression in order to test prior developmental hypotheses about feather evolution. Our tree is consistent with the developmental model of feather evolution, and sheds light on the influence of ancestral epidermal stratification on feather cell evolution. This transcriptomic approach to study feather cell types helps lay the ground work for understanding the developmental evolutionary complexity and diversity of feathers.

overall_design: We dissected feathers from H&H stage 38, 39, and 40 white leghorn chicken embryos. For the sample labeled, Feathers in Skin, we dissected off a tract of epidermis with feathers embedded in it from the dorsal midline of the embryo. The “Plucked Feather” samples were plucked from the dorsal midline as well. Cells were isolated from these tissues and analyzed using scRNAseq for stage 38 embryos, and snRNAseq for stage 39 and 40 embryos.

first: Günter

middle: P

last: Wagner

first: Richard

middle: O

last: Prum


title: DNAPKCs as a therapeutic target in small cell lung cancer

city: New York

first: BiNGS

last: Core

address: 1470 Madison Ave

institute: Icahn School of Medicine at Mount Sinai

summary: Small cell lung cancer is the most aggressive form of lung cancer. Current frontline treatment of anti-PD-L1 with chemotherapy only results in a modest increase in overall survival and progression-free survival due to the poor immunogenic nature of SCLC. In this investigation, we report DNAPKcs inhibition sensitizes SCLC by inducing apoptosis and DNA damage leading to micro-nuclei formation. These aberrant cytosolic DNA activates cGAS-STING pathway resulting in induction of type-I/II interferon and MHC class-I. This study provides evidence of proteasomal degradation of cMYC in a GSK3B dependent manner in response to DNAPKcs inhibition further enhancing pro-inflammatory immune pathways. Using two unique immune-competent SCLC GEM models, we show STING mediated modulation of tumor immune microenvironment by increasing CD8+ T cells, MHC-I+ cells, and M1 macrophages in response to DNAPKcs inhibition with anti-PD-L1 therapy. We further provide strong evidence of significant tumor regression in immune-competent GEMMs by treatment with DNAPKcs inhibitor alone and in combination DNAPKcsi and anti-PD-L1. Taken together this study reports induction of DNAPKcs inhibitor increases the efficacy of immune checkpoint blockade therapy remarkably by activating immune microenvironment in a STING-MYC dependent manner which is of significant translational value.

overall_design: RNA.seq analysis data of small cell lung cancer cells, DMS114, H146, H196, H446, H720 and H1341  comparing only DMSO treated (control) and NU7441 treated cells.

first: Triparna

last: Sen

first: Dan

last: Hasson

first: Subhamoy

last: Chakraborty

first: Charles

last: Coleman


title: RNA-Seq experiment of peritoneal macrophgae or IC-21 cell line treated with LPS

first: Haiming

last: Cao

address: 9000 Rockville Pike

institute: NIH

summary: A growing number of micro-peptides (miPs) have been identified and annotated in recent years. However, the biological significance of these hidden genomic products remains unclear. In this study, we established a novel strategy to screen human miPs with metabolic relevance by identifying miPs linked to metabolic traits in GWAS datasets. The selected miP, SMIM30, represents a promising drug target due to its capacity to regulate both adipose tissue and systemic insulin sensitivity. We demonstrated that SMIM30 functions as a modulator of inflammatory responses in ex vivo/in vitro macrophage systems. Specifically, isolated mouse peritoneal macrophage and mouse macrophage cell line, IC-21, were treated with LPS with/without SMIM30 expression. Differentially expressed genes can be obtained from RNAseq results, which may guide the characterization of SMIM30-related biological pathways.

overall_design: (1) Peritoneal macrophages were isolated from wild-type or SMIM30-Mut mice, with 2´106 cells seeded on the 24-well-plates. Cells were treated with 5ng/ml LPS for 6 hours before collection for RNA isolation. Data shown is triplicates of wild-type or SMIM-30 Mut macrophages. (2) Peritoneal macrophages were isolated from SMIM30-Mut mice, with 2´106 cells seeded on the 24-well-plates. Six hours after seeding, SMIM30-overexpression adenovirus or control adenovirus was added to the wells. Cells were treated with adenovirus for 24h. Cells were then treated with 5ng/ml LPS for 6 hours before collection for RNA isolation. Data shown is triplicates of SMIIM30 overexpression or SMIM-30 Mut macrophages. (3) IC-21 was obtained from ATCC and cultured following manufacturer’s instructions. SMIM30 loss-of-function was accomplished by transfection of IC-21 cells with SMIM30 siRNA for 24 hours. Cells were then treated with 5ng/ml LPS for 6 hours before collection for RNA isolation. Data shown is triplicates of wild-type or SMIM-30 knockdown IC-21 cells.

first: Yonghe

last: Ma


title: Identification of RNAs that can accept assembly of an Sm-protein ring

city: Cleveland

first: Anton

middle: James

last: Blatnik III

state: Ohio

address: 2109 Adelbert Road

institute: Case Western Reserve University

summary: Sm-ring assembly is important for the biogenesis, stability, and function of uridine-rich small nuclear RNAs (U snRNAs) involved in pre-mRNA splicing and histone pre-mRNA 3’ end processing. Assembly of Sm-rings occurs in the cytoplasm and is dependent on a specific sequence and structure motif (Sm-site), ATP, and the Survival motor neuron (SMN) protein complex. The following study informatically investigates the occurence of Sm-sites within the mouse and human transcriptomes and biochemically assesses whether these sites can accept Sm-rings. Sm-sites were found on snRNAs, but are highly prevalent in the 3’ untranslated regions (3’UTR) of long mRNAs. RNA immunoprecipitation experiments confirm that Sm-site containing mRNAs associate with Sm-proteins in the cytoplasm. Established Sm-ring assembly assays were modified to identify polyA-RNAs that specifically associate with Sm-proteins in an ATP-dependent manner, modeling newly assembled Sm-rings. Sm-rings were then specifically assembled onto candidate Sm-site containing mRNAs in an ATP and Sm-site dependent manner. mRNAs containing Sm-sites are down-regulated in models of SMA, suggesting reduced Sm-ring assembly on these mRNAs may contribute to the mechanism of SMA pathogenesis. Together, this study establishes that Sm-site containing mRNAs can accept Sm-rings and identifies a novel mechanism for Sm-proteins in regulation of cytoplasmic mRNAs.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: To identify human and mouse RNAs that can accept Sm-rings, four RNA-sequencing library conditions were compared. 1) a polyA-RNA sequencing library obtained by oligo-dT enrichment from human S3-iNPC or mouse NSC-34 cell extracts. 2) an anti-Sm RNA immunoprecipitation from cytoplasmic cell extracts isolated from human S3-iNPC or mouse NSC-34 cells. 3) a modified Sm-ring assembly condition in which an anti-Sm-RIP was performed following incubation of human polyA-RNA within a mouse cytoplasmic cell extract, but in the absence of ATP (also performed in the opposite direction). 4) lastly, a modified Sm-ring assembly condition in which an anti-Sm-RIP was performed following incubation of human polyA-RNA within a mouse cytoplasmic cell extract, but in the presence of ATP (also performed in the opposite direction). Each condition was performed in triplicate and sequencing libraries were generated from isolated RNA following Takara SMARTer Stranded Total RNA-Seq Kit v3-Pico Input Mammalian (Takara 634451).

first: Manu

last: Sanjeev

first: Jacob

last: Slivka

first: Benjamin

last: Pastore

first: Caleb

last: Embree

first: Wen

last: Tang

first: Guramrit

last: Singh

first: Arthur H

last: Burghes


title: BAT and SAT Treg cells and of BAT tissue with or without Treg cells under different experimental conditions [scRNA-seq]

city: Boston

first: CBDM

last: Lab

address: 77 Avenue Louis Pasteur

department: Microbiology and Immunobiology

institute: Harvard Medical School

summary: Profiling Treg cells from BAT,SAT,VAT,SPLN

overall_design: CD4+ T cells (DAPI-TCRb+CD3+CD4+) were double sorted, biological duplicate samples hashed and encapsulated using Chromium Single Cell 3' v3 platform.

first: Nathan

last: Zammit

first: Diane

last: Mathis

first: Christophe

last: Benoist


title: Impaired nutrient absorption and alterations in gut microbiome contribute to reduced postnatal growth in Zeb2+/- mice

first: Yang

middle: yang

last: Ge

state: Beijing

address: No. 2 Yabao Road

department: Department of Medical Genetics

institute: Capital Institute of Pediatrics

summary: As a member of the ZFH1 family of two-handed zinc finger/homeodomain transcription factors, ZEB2 plays a pivotal role in regulating cell differentiation and early fetal development. Loss-of-function mutations lead to Mowat-Wilson syndrome (MWS), a rare genetic disorder associated with significant growth retardation for reasons that remain unknown. In this study, we established a global Zeb2 heterozygous knockout mouse model and observed reduced postnatal growth closely resembling the delayed physical development seen in patients with MWS. Both the daily and cumulative food intake of Zeb2+/- mice were significantly lower compared to their wild-type counterparts. This was accompanied by lower fat mass, reduced weights of epididymal,perigonadal, and mesenteric white adipose tissues, and reduced bone mass. H&E staining revealed a significant reduction in villi height. Downregulated genes in duodenum were enriched in fat and protein digestion and absorption pathways.Additionally, we observed a shift in the gut microbiome of Zeb2+/- mice, with multiple microbiota showing significant and positive correlations with postnatal growth-related indicators. Our findings underscore the critical role of the Zeb2 gene in physical development, its direct regulation of small intestine function, and its influence on the composition of the gut microbiome. The globally Zeb2 heterozygous knockout mouse model we established also provides a robust platform for gaining deeper insights into the physiological and pathophysiological impacts on various organs affected by MWS.

overall_design: we established a global Zeb2 heterozygous knockout mouse model to investigate the molecular underpinnings of the growth retardation observed in MWS.

first: Yangyang

first: Lingya

last: Liu

first: Lihua

last: Wu

first: Jianxin

first: Guangxu

last: Ren

first: Qian

last: Jiang


title: BAT and SAT Treg cells and of BAT tissue with or without Treg cells under different experimental conditions [RNA-seq]

summary: We profiled the transcriptome of sorted BAT and SAT Tregs and the transcriptional effects of BAT activating factors on whole-BAT transcriptomes when Tregs are present or absent.

overall_design: RNA-seq of BAT and SAT Treg cells and of BAT tissue with or without Treg cells under different experimental conditions.


title: TFAP2A mediates HIF-1α dependent activation of hypoxia-inducible genes via interaction with Ku70/80 complex in a SUMO-dependent manner

city: Larissa

first: Antonis

last: Giakountis

address: Biopolis, Mezourlo

department: Biochemistry and Biotechnology

country: Greece

institute: University of Thessaly

summary: Hypoxia can be established under pathological conditions, such as cancer, due to the imbalance between oxygen supply and consumption. Hypoxia Inducible Transcription Factor HIF-1 mediates the physiological response to hypoxia but also regulates multiple steps of carcinogenesis. Despite its well-defined oxygen-dependent activation, many aspects of HIF-1 transcriptional activity as well as interaction with chromatin remain elusive. We have recently shown that TFAP2A physically interacts with HIF-1 and hypoxia-dependent deSUMOylation of TFAP2A positively affects HIF-1 activity. We now present ChIP-seq analysis showing that TFAP2A resides together with HIF-1α on the promoters of a subset of hypoxia-regulated genes, the mRNA expression of which is downregulated by silencing of TFAP2A. Interestingly, CRISPR-mediated knockdown of TFAP2A expression under hypoxia decreased the occupancy of HIF-1α on these promoters and affected chromatin accessibility. Mechanistically, we reveal that the Ku70/Ku80 protein complex interacts with TFAP2A in a SUMO-dependent manner under hypoxia and participates in HIF-dependent gene expression. Moreover, using stable expression of TFAP2A forms that either lack or constitutively carry a SUMO modification, we could show that SUMOylation affects binding of TFAP2A to chromatin. Overall, our data suggest that TFAP2A is an important co-regulator of the HIF-1-dependent transcriptional response to hypoxia and SUMOylation fine-tunes this regulation. As both TFAP2A and HIF-1 play critical roles in cancer progression, a detailed characterization of their crosstalk could lead to novel therapeutic strategies for targeting and killing cancer cells in hypoxic tumors

overall_design: Chromatin Immunoprecipitation of TFAP2A under hypoxic and normoxic conditions in HeLa cells

first: Amalia

last: Kanoura

first: Georgia

last: Chachami


title: BAT and SAT Treg cells and of BAT tissue with or without Treg cells under different experimental conditions

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Effect of LINC01871 silencing on CD4+ T cell activation.

city: Turku

first: Riitta

last: Lahesmaa

address: Tykistökatu 6A

department: Turku Bioscience

country: Finland

institute: University of Turku

summary: Long intergenic noncoding RNAs (lincRNAs) play crucial roles in regulating biological processes in health and disease. However, little is known about the contribution of lincRNAs in T-cell activation. Here, we identified a lincRNA, LINC01871, which is highly induced upon T-cell activation and is predominantly located in the cytoplasm. The anti-inflammatory cytokine TGF-β was found to suppress its expression. Silencing LINC01871 led to a modest decrease in IL-2 secretion. RNA-seq and proteomic analyses of LINC01871-deficient CD4+ T cells revealed several targets, including genes associated with autophagy and membrane organization such as ATG2B; TRIM5; SNX30; TIMM8B; and ATP10A. Notably, LINC01871 expression was highly specific to T cells in several cross tissue single cell RNA-seq atlases. Furthermore, ex-vivo CD4+ T cells from children progressing to beta-cell autoimmunity showed higher LINC01871 expression as compared to their age, sex and HLA-risk matched controls. These data suggest that LINC01871 has an in-vivo function in T-cell-mediated immunity.

overall_design: CD4 T cells were isolated using magnetic bead-based positive selection kit from Dynal. Cells were nucleofected with the three antisense locked nuelceic acid (LNAs) targeting the lincRNA LINC01871 or non targeting control LNAs (NT). After 24 hours of rest, cells were activated with CD3/CD28 for 48 hours. The cells were then reactivated for 30 minutes and RNA samples were extracted. There were three biological replicates* three LNAs  =9 samples.

first: Kedar

last: Batkulwar

first: Ubaid Ullah

last: Kalim

first: Tommi

last: Välikangas

first: Omid

last: Rasool


title: Genome-wide DNA replication profiling and full-length total RNA sequencing from the same single cell [01_RPE1_midS]

first: Rawin

last: Poonperm

summary: Single-cell sequencing has advanced our understanding of cell-type diversity and heterogeneity. However, existing single-cell multi-omics methods lack DNA data resolution and full-length total RNA detection. Here, we introduce single-cell (sc)Repli-RamDA-seq (scRR-seq), a novel multi-omics method that enables high-resolution DNA replication profiling and full-length total RNA sequencing from the same single cell, made possible by the clean separation of DNA and RNA. scRR-seq generates DNA replication and RNA sequencing data comparable to individually obtained scRepli-seq and scRamDA-seq data, respectively. scRR-seq also allows one to tell the cell-cycle stage of a given S-phase cell and detects copy-number variation (CNV) in non-S-phase cells. scRR-seq outperforms other scRNA-seq methods in the number of transcripts identified and full-length total RNA detection. Furthermore, scRR-seq allowed haplotype-specific analysis and the identification of novel S-phase markers. Taken together, scRR-seq is a robust single-cell multi-omics method with promising potential for comprehensive genome and transcriptome analysis.

overall_design:
We performed simultaneous DNA-seq and RNA-seq on the same cells using the scRR-seq protocols (scRR1 and scRR3). The following cell types were used for the experiments: (1) hTERT-RPE1 cells collected from mid-S phase, (2) mouse 8-cell embryos, (3) CBMS1 mouse embryonic stem cells collected from the entire S phase, (4) hTERT-RPE1 cells collected from the entire S phase, (5) HAP1 cells collected from mid-S phase, and (6) IMR-90 cells treated with or without 0.4 µM aphidicoline, collected from the G1 phase.  Single cells were collected into 0.2 ml PCR tubes by cell sorter or manually picked and lysed in RamDA-seq lysis buffer (TOYOBO, #RMD-101) containing 2 mg/ml Dynabeads™ MyOne™ Carboxylic Acid (Invitrogen, #65011). Genomic DNA was trapped on the beads, while RNA was released into the lysis buffer. The isolated DNA on magnetic beads was subjected to whole-genome amplification (WGA) and Next-Generation Sequencing (NGS) library preparation, as described in scRepli-seq (Takahashi et al., Nat Genet 2019). The DNA-seq (scRepli-seq) data was used to assess genome-wide DNA copy numbers, karyotype G1-phase cells, and analyze the replication state of S-phase cells. In parallel, the RNA isolated in the supernatant was subjected to scRamDA-seq (Hayashi et al., Nat Commun 2018) using the GenNext®RamDA-seq™ Single Cell Kit (TOYOBO, #RMD-101) according to the manufacturer’s instructions with modifications.

***************************************************************
The table below lists GEO accessions reused/reanalyzed for this study.
***************************************************************

first: Shin-ichiro

last: Takebayashi


title: Spatial transcriptome profile of EGFR-TKI resistant non-small cell lung cancer

city: Goyang-si

first: Sehwa

last: Hong

state: Gyeonggi-do

address: 323, Ilsan-ro, Ilsandong-gu

department: Research Institute

institute: National cancer center

summary: Spatial transcriptome profile was assessed from patients' tumor tissues to receive Osimertinib. We uncovered the mechanism of EGFR-TKI resistance, and tumor progression to develop thepeutic strategy.

overall_design: Lung cancer FFPE tissue slides were acquired from patient to be resistant to EGFR-TKI Osimertinib.

first: Charny

last: Park

first: Ji-youn

last: Han


title: The mRNA-Seq data of human-Zic3 overexpression in chicken limbs

city: Shenyang

first: Bai

last: Shibin

address: 120 Dongling Road

institute: Shenyang Agriculture University

summary: We observed Zic3 highly expressed in the anterior regions of limb buds in all investigated tetrapods. To investigate the function of the Zic3 in limbs, we overexpressed the human-Zic3 gene in the lateral plate mesoderm (LPM) of developing chicken hindlimbs at HH10 using the RCAS avian retrovirus. Subsequently, the embryos were allowed to incubate until HH30. Autopod and zeugopod regions were dissected and subjected to transcriptome sequencing (mRNA-Seq), with the corresponding positions from individuals injected with the empty RCAS vector serving as controls. The results of the differential analysis and enrichment analysis revealed the regulatory network of Zic3 and various regulatory pathways that may be affected.

overall_design: After 5 days of infection (HH30), the entire autopod and zeugopod regions of individuals injected with RCAS-Zic3 were collected under a microscope. The same procedure was applied to the control group, where the corresponding areas of individuals injected with RCAS were collected. Each group included five biological replicates.

first: Shanshan

last: Li

first: Shibin

last: Bai

first: Zhe

last: Wang


title: NSUN2 Facilitates Dicer Cleavage of DNA Damage-Associated R-Loops to Promote Repair

city: Oxford

first: Monika

last: Gullerova

address: S Parks Rd

department: Sir William Dunn School of Pathology

institute: University of Oxford

summary: DNA integrity is constantly challenged by both endogenous and exogenous damaging agents, resulting in various forms of damage. Failure to repair DNA accurately leads to genomic instability, a hallmark of cancer. Distinct pathways exist to repair different types of DNA damage. Double-strand breaks (DSBs) represent particularly severe form of damage, due to physical separation of DNA strands. Repair of DSBs requires activity of RNA Polymerase II (RNAPII) and generation of Damage-associated transcripts (DARTs).  Here we show that the RNA m5C-methyltransferase NSUN2 localizes to DSBs in transcription-dependent manner, where it binds to and methylates DARTs. Depletion of NSUN2 results in an accumulation of nascent DARTs around DSBs, specifically of the de novo primary DARTs. Furthermore, we detected an RNA-dependent interaction between NSUN2 and DICER, which was stimulated by DNA damage. NSUN2 activity promoted Dicer cleavage of DARTs associated R-loops, which is required for efficient DNA repair. We report a previously unrecognized role of the RNA m5C-methyltransferase NSUN2 within RNA-dependent DNA damage response, highlighting its function as a DICER chaperone for the clearance of non-canonical substrates such as DARTs, thereby contributing to genomic integrity.

overall_design: To investigate the role of NSUN2 in regulating processing of damage associated transcripts at genome-wide level, we emplyed the chromatin associated RNA sequencing to detect the nascent transcripts around DSBs in both mock and NSUN2-depleted cells.

first: Adele

last: Alagia

first: Kamal

last: Ajit


title: A role of p53 in mediating epigenetic stress (WGBS)

city: Cambridge

first: Arndt

last: Wallmann

address: Tennis Ct Rd

institute: MRC Toxicology Unit

type: Methylation profiling by high throughput sequencing

summary: 5-Azacytidine (Aza) and 5-Aza-2'-deoxycytidine (Dac) are the only epigenome modifying drugs in widespread clinical use, and are some of the primary therapeutics for Acute Myeloid Leukaemia treatment. The two are frequently used interchangeably, surprisingly as their selectivity of de-methylation is unique from the other, with to date no identified predictors of response or known biomarkers to indicate drug preference. Using these drugs to induce de-methylating conditions, we combine data from DRIPc-Seq, Immunostaining, RNA-Seq and Mass spectrometry with epigenetic analysis to uncover unique cellular responses following Aza and Dac treatment.

overall_design: HEK293 cells were treated with 1.5µM of Azacitidine or Decitabine or solvent for 48 hours and Whole genome bisulphite (WGBS) was performed in two replicates to assess the level of DNA methylation.

first: Emma

last: Hands

first: Mathew

last: Van de Pette


title: The p75 neurotrophin receptor regulates the skeletal stem cell niche through sensory innervation

city: xiamen

first: zuoxing

last: wu

address: No. 4221, Xiangan Road, Xiangan District, Xiamen

institute: xiamen university

summary: Low bone mass is strongly associated with multiple neurologic diseases such as Alzheimer’s disease (AD), however it remains unclear if this represents direct specific biologic sequalae of the primary disease process or rather a non-specific finding attributable to alterations in behavior and activity. The recent discovery of skeletal stem cells (SSCs) generating bone forming osteoblasts offers a new opportunity to understand both AD effects on bone and broader neural effects on bone by determining whether there are neural contributions to the SSC niche. Deletion of p75NTR (an essential neurotrophin receptor) was used to probe the contributions of peripheral innervation to the SSC niche. p75NTR deletion either broadly in neurons (p75NTRsyn1 mice) or more specifically in sensory nerves (p75NTRadv mice) but not osteogenic cells (p75NTRocn and p75NTRprx1 mice) or sympathetic nerves (p75NTRth mice), led to decrease in sensory innervation, impaired SSC homeostasis, and bone loss. Consistent with this, pharmacological sensory denervation resulted in a decrease in SSC numbers. More directly, wild-type SSCs transplanted orthotopically into the bones of p75NTRadv host mice with reduced sensory innervation displayed an impaired osteogenic capability, indicating that sensory nerves comprise part of the SSC niche. Decreased sensory innervation in p75NTRadv mice further impaired fracture healing by suppressing SSC expansion. Transcriptomic profiling to identify sensory nerve derived mediators of the SSC niche effect identified that p75NTR regulates expression of Osteopontin (SPP1) in neurons of the dorsal root ganglion (DRG), and SPP1 in turn acts as to promote SSC self-renewal and osteogenic capacity. Lastly, this p75NTR-SPP1 axis is impaired in AD mice, providing a new, direct mechanism for osteopenia in AD both impacting basal bone turnover and fracture repair. Altogether, our findings newly establish sensory nerves as a key component of SSC niche that is disrupted in AD, providing new therapeutic opportunities to augment SSC function to treat bone disorders such as Alzheimer’s associated osteopenia.

overall_design: We next focused on identifying the mechanism by which p75NTR-mediated sensory innervation constitutes part of the SSC niche regulating osteogenesis, we performed RNA sequencing (RNA-seq) and filtered for secreted factors the list of genes exhibiting lower expression in the absence of p75NTR.


title: A role of p53 in mediating epigenetic stress (RNA-Seq)

overall_design: HEK293 cells were treated with 1.5µM of Azacitidine or Decitabine or solvent for 48 hours and RNA-seq was performed in three replicates to assess the change to the transcriptome.


title: A role of p53 in mediating epigenetic stress (DRIPc-seq)

overall_design: HEK293 (WT)  and p53 KO HEK293 (p53KO) cells were treated with 1.5 µM of Azacitidine or Decitabine or solvent for 48 hours and DRIPc-seq was performed in two replicates to assess the change to the formation of R-loops. Two additional HEK293 (WT) control samples were pre-treated with RNaseH, which specifically removes R-loops.


title: Skin expression data from psoriasis patients treated with IL-17A inhibitor

city: Shanghai

first: Jie

last: Zheng

address: No.197, Rui Jin Er Road

department: Department of Dermatology

institute: Ruijin Hospital, School of Medicine, Shang Jiao Tong University

summary: Psoriasis is a chronic, recurrent, inflammatory systemic disease that affects approximately 2-3% of the population worldwide. IL-17A plays a key pathogenic role in psoriasis, and IL-17A antigen have emerged as first-line agents for the treatment of moderate-to-severe plaque psoriasis.However, the associated mechanism underlying treatment-associated adverse and psoriasis relapse has not been reported

overall_design: The study samples are from psoriasis patients treated with anti-IL-17A inhibitor. Skin samples consist of baseline, post-treatment, and recurrent, resulting in a total of 40 samples. Gene expression profiling analysis of RNA-seq data for different time points.

first: Lihong

last: Chen

first: Li

last: Zhang


title: Dual role of ZIC2 during neural induction: from pioneer transcription factor to enhancer activator [Multiome]

status: Public on Aug 26 2025

city: Santander

first: Alvaro

last: Rada-Iglesias

state: Cantabria

address: Albert Einstein 22, PCTCAN

country: Spain

institute: Institute of Biomedicine and Biotechnology of Cantabria

summary: ZIC2, a member of the Zinc Finger of the Cerebellum family of transcription factors (TFs), plays crucial roles during neural development. In humans, defects in ZIC2 cause holoprosencephaly, a congenital brain malformation characterized by the defective cleavage of cerebral hemispheres due to problems in midline patterning. However, the gene regulatory network (GRN) controlled by ZIC2 and the regulatory mechanisms it employs during neural development remain largely unexplored. Here, we combined a mouse embryonic stem cell (mESC) in vitro differentiation model towards anterior neural progenitors (AntNPCs) with genome editing approaches, bulk and single cell (i.e. Multiome scATAC + scRNAseq) genomic methods to elucidate the precise GRN controlled by ZIC2 and the underlying mechanisms. We found that ZIC2 shows widespread binding throughout the genome already in mESC as well as upon pluripotency exit and neural induction. Despite its extensive binding in mESC, ZIC2 function is dispensable in pluripotent cells due to compensation by ZIC3. In contrast, ZIC2 plays a major regulatory function during neural induction, directly controlling the expression of master regulators implicated in the patterning and morphogenesis of specific brain regions, such as the midbrain (e.g., En1, Lmx1b, Pax2, Wnt1) and the roof plate (e.g., Lmx1a, Wnt3a). Mechanistically, ZIC2 plays a dual role in neural differentiation: (i) during pluripotency exit, ZIC2 acts as a pioneer TF, binding de novo to distal enhancers and promoting their chromatin accessibility; (ii) during neural induction, ZIC2 is essential for the activation of a subset of the previously primed enhancers, which in turn control the expression of major neural patterning regulators and signaling pathways (i.e. WNT) that prevent the premature differentiation of neural progenitors. Overall, our work shows that, by sequentially acting as a promiscuous pioneer and selective activator of enhancer elements, ZIC2 canalizes pluripotent cells towards neural progenitors with rostro-dorsal identities.

overall_design: Multiome 10X experiment (snRNA-seq and snATAC-seq) was performed using WT and Zic2-/- (Z2KO) day 3 (D3) progenitor cells and day 6 (D6) AntNPCs, obtained from a 6 days in-vitro differentiation from mESCs (D0) towards AntNPCs (D6). To do so, the initial nuclei isolation started by washing cells twice with PBS before detaching them from the tissue culture plate with Stempro™ Accutase™ Cell Dissociation Reagent (Thermo Scientific, A1110501). The Accutase incubation times were adjusted to the cell type (6 min for D3 progenitors and 10 min for D6 AntNPCs). Next, Accutase was inactivated with BSA 0.04% in PBS. Cell clumps were disrupted by gently pipetting up and down the cell suspension in the collection tubes. Cell suspension was then centrifuged for 5 min at 300 rcf. The supernatant was aspirated without disrupting the cell pellet and cells were washed twice in BSA 0.04% in PBS. Then, cells were diluted 1/10 in BSA 0.04% in PBS and counted. Next, 106 cells were transferred to a DNA LoBind® 1.5 ml microtube (Eppendorf, 0030108051). Cells were centrifuged again at 300 rcf for 5 min at 4ºC. Next, the supernatant was carefully aspirated and cells were lysed by adding 175 µl of pre-chilled Lysis Buffer (10X Genomics) to the pellet while smoothly pipetting up and down 10 times.After incubation for 4 min on ice, suspension was centrifuged at 500 rcf for 5 min at 4º C. Then, supernatant was completely removed without disrupting the nuclei pellet. Nuclei suspension was cleaned up by adding Wash Buffer (10X Genomics) followed by centrifugation (500 rcf, 5 min at 4ºC). Assuming ~50% nuclei loss during cell lysis, cells were resuspended in chilled Diluted Nuclei Buffer (10X Genomics) to achieve the desired final nuclei concentration. The rest of the steps of the scATAC-seq and scRNA-seq coupled protocol were performed exactly as indicated in the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide CG000338 Rev F (https://cdn.10xgenomics.com/image/upload/v1666737555/support-documents/CG00033_ChromiumNextGEM_Multiome_ATAC_GEX_User_Guide_RevF.pdf).

first: Sara

last: Lobato-Moreno

first: Karin

last: Prummel

first: Víctor

last: Sánchez-Gaya

first: Álvaro


title: Dual role of ZIC2 during neural induction: from pioneer transcription factor to enhancer activator [RNA-Seq]

overall_design: RNA-seq profiling of WT and Zic2-/- (Z2KO)  mESC–derived neural progenitors (AntNPCs) collected at Day 0 (mESC, D0), Day 3 (D3), Day 5 (AntNPCs, D5), and Day 6 (AntNPCs, D6) of differentiation. RNA-seq profiling of Zic3-/Y (Z3KO) mESC (D0) is also included. Additional samples include RNA-seq from Zic2FKBPV36 (Z2-FKBP) AntNPCs (D6) treated with DMSO or d-TAG13 during: the whole in vitro differentiation (total) and the last 48h or 24h. An independent dataset comprising WT, Zic2-/- (Z2KO), and Zic2+/- (Z2HET) AntNPCs (D6) from a separate differentiation experiment is also included. These samples are tagged as _D6HetDif_.

first: María

last: Mariner-Faulí

first: Sarah

last: M. Robert


title: Dual role of ZIC2 during neural induction: from pioneer transcription factor to enhancer activator [ATAC-Seq]

summary: ZIC2, a member of the Zinc Finger of the Cerebellum family of transcription factors (TFs), plays crucial roles during neural development. In humans, defects in ZIC2 cause holoprosencephaly, a congenital brain malformation characterized by the defective cleavage of cerebral hemispheres due to problems in midline patterning. However, the gene regulatory network (GRN) controlled by ZIC2 and the regulatory mechanisms it employs during neural development remain largely unexplored. Here, we combined a mouse embryonic stem cell (mESC, D0) in vitro differentiation model towards anterior neural progenitors (AntNPCs, D6) with genome editing approaches, bulk and single cell (i.e. Multiome scATAC + scRNAseq) genomic methods to elucidate the precise GRN controlled by ZIC2 and the underlying mechanisms. We found that ZIC2 shows widespread binding throughout the genome already in mESC as well as upon pluripotency exit and neural induction. Despite its extensive binding in mESC, ZIC2 function is dispensable in pluripotent cells due to compensation by ZIC3. In contrast, ZIC2 plays a major regulatory function during neural induction, directly controlling the expression of master regulators implicated in the patterning and morphogenesis of specific brain regions, such as the midbrain (e.g., En1, Lmx1b, Pax2, Wnt1) and the roof plate (e.g., Lmx1a, Wnt3a). Mechanistically, ZIC2 plays a dual role in neural differentiation: (i) during pluripotency exit, ZIC2 acts as a pioneer TF, binding de novo to distal enhancers and promoting their chromatin accessibility; (ii) during neural induction, ZIC2 is essential for the activation of a subset of the previously primed enhancers, which in turn control the expression of major neural patterning regulators and signaling pathways (i.e. WNT) that prevent the premature differentiation of neural progenitors. Overall, our work shows that, by sequentially acting as a promiscuous pioneer and selective activator of enhancer elements, ZIC2 canalizes pluripotent cells towards neural progenitors with rostro-dorsal identities.

overall_design: Bulk ATAC-seq profiling of WT and Zic2-/- mESCs (day 0) differentiated into anterior neural progenitors (AntNPCs, day 6). Samples for ATAC-seq were collected at differentiation day 0 (D0), day 3 (D3) and day 6 (D6).

first: Matteo

last: Trovato


title: CellCousin2: An Optimized System for Partial Ablation and Tracing of Regenerative Lineages

city: Greater Noida

first: Sumeet

middle: Pal

last: Singh

state: Uttar Pradesh

address: NH91, Tehsil Dadri

department: Life Sciences

country: India

institute: Shiv Nadar Institution of Eminence

summary: Regeneration can rely on multiple cellular sources, including stem cells, self-duplicating cells, and transdifferentiating cells. A central question in regenerative biology is how these distinct lineages contribute to repair and interact within a functional regenerate. We previously developed the CellCousin system to study hcellular plasticity using inducible recombination and nitroreductase-mediated ablation in zebrafish. Here, we present CellCousin2, which introduces two key improvements for long-term tracking of spared and regenerating cells. First, to reduce background recombination, we developed a Dihydrofolate Reductase (DHFR)-CreER system with dual control: DHFR- mediated degradation in the absence of trimethoprim, and tamoxifen-dependent activation. This combination minimizes leakiness while maintaining high recombination efficiency. Second, we replaced the original nitroreductase with NTR2.0, enabling effective ablation with tenfold lower metronidazole concentration, reducing off-target effects on the liver. Together, these enhancements make CellCousin2 a robust platform for dissecting the dynamics and interactions of regenerative lineages.

overall_design: Bulk RNA-Seq of liver collected from zebrafish larvae treated with Vehicle, 1 mM Metronidazole, 10 mM Metronidazole.

first: Gabriel

middle: G

last: Hovhannisyan


title: Endothelial plasticity in atrial fibrosis by integrating single-cell sequencing and genetic lineage tracing

city: Beijing

first: Xiaoyi

last: Wang

address: No.167, Beilishi Road, Xicheng District, 100037

department: Department of Cardiovascular Surgery, Fuwai Hospital

institute: Fuwai Hospital, National Center for Cardiovascular Diseases

summary:
Background and aims: Atrial fibrosis represents a critical determinant in atrial fibrillation (AF) pathogenesis. Although endothelial dysfunction is a hallmark feature of AF, the precise mechanisms by which endothelial cells (ECs) contribute to atrial fibrosis remain incompletely understood.

Methods: This study employed single-cell RNA sequencing (scRNA-seq) to analyze intercellular communication networks in atrial tissues from both sinus rhythm (SR) and AF patients. Cdh5-CreERT2;RFP mice were generated to track endothelial plasticity following transverse aortic constriction (TAC). Cell-cell interactions were investigated using isolated human primary atrial ECs and fibroblasts (FBs) in vitro. To elucidate the regulatory role of endothelial-derived TGF-β1 on FB function, endothelial-specific Tgf-β1 knockout (Tgf-β1ECKO) mice were generated.

Results: scRNA-seq analysis identified ECs as predominant signal-sending cells with extensive FB connectivity in both SR and AF atrial tissues. During fibrosis progression, ECs displayed significant mesenchymal activation at the transcriptional level. However, immunofluorescence and high-content screening revealed minimal complete endothelial-to-FB transition. Cell-cell communication analysis and in vitro studies identified TGF-β1 as the key mediator through which mesenchymal-activated ECs (EndoMA) promoted FB proliferation and collagen production. Notably, endothelial-specific Tgf-β1 deletion attenuated TAC-induced atrial fibrosis and reduced AF susceptibility.

Conclusions: Our findings demonstrate that EndoMA-derived TGF-β1 critically regulates FB function and drives atrial fibrosis progression. Targeting endothelial-specific pathways represents a promising therapeutic strategy for attenuating atrial fibrosis in AF pathogenesis.

overall_design:
Human: Single cell sequencing from heart donors with sinus rhythm and patients with atrial fibrillation;  Mice: Single cell sequencing from C57BL/6 J Cdh5-CreERT2/RFP  mice performed transverse aortic constriction or sham surgery.

***Raw data for human samples will be submitted to the Genome Sequence Archive for Human due to patient privacy concerns***

first: Zhiwei

last: Zeng

first: Jianqiu

last: Pei

first: Peng

first: Jun

last: Li

first: Fangzhou

first: Juncheng

first: Yan

last: Zhao

first: Chunyu

last: Yu

first: Hanning

last: Liu

first: Zhe


title: IRS2 Promotes Proliferation and Drives Invasion-Migration in SI-NETs.

city: Shijiazhuang

first: Meng

last: Zhang

state: Hebei

address: Changan district No.12 Jiankang Road Shijiazhuang, 050031, Hebei Province China

department: Gastroenterology

institute: The Fourth Hospital of Hebei Medical University

summary: I investigated the role of insulin receptor substrate 2 (IRS2) in small-intestinal neuroendocrine tumours (SI-NETs). Using SI-NET cell lines (STC-1 and Glu-tag), I found that IRS2 was significantly upregulated. IRS2 knockdown inhibited cellular proliferation, migration, and invasion, while its overexpression promoted these processes. For in vivo validation, I established stable IRS2-knockdown cell lines and performed xenograft experiments in nude mice. Transcriptomic profiling by RNA sequencing was conducted to identify downstream targets of IRS2 signaling.

overall_design: RNA sequencing was performed on control and stable IRS2-knockdown SI-NET cell lines (Glu-tag) in biological triplicate to identify transcriptomic changes induced by IRS2 silencing.

first: Xuemei

last: Sun

first: Shengmian


title: A role of p53 in mediating epigenetic stress

first: Arndt

last: Wallmann

overall_design: Refer to individual Series


title: Oscillatory shear stress induced extracellular vesicles mediate valvular endothelial-interstitial cell crosstalk exacerbating aortic valve calcification [RNA-Seq]

city: changsha

first: ruijiang

address: No.139 Renmin Road Central

institute: The Second Xiangya Hospital of Central South University

summary: Calcific aortic valve disease (CAVD) involves pathological crosstalk between human valvular endothelial cells (hVECs) and interstitial cells (hVICs), yet the role of this communication in pathogenesis remains poorly defined. Here, we identify a novel extracellular vesicle-derived circRNA, CircILRUN, originating from hVECs and demonstrate its critical function in promoting hVIC osteogenesis and CAVD progression in vitro and in vivo. CircILRUN expression is significantly elevated in extracellular vesicles from hVECs under oscillatory shear stress. Its deletion markedly attenuates aortic valve calcification (AVC) in mice, reducing valve thickness, calcium deposition, and improving echocardiographic parameters, findings corroborated in osteogenic medium-induced hVICs. Through unbiased protein-RNA screening and biochemical validation, we identify N-acetyltransferase 10 (NAT10) as a key target of CircILRUN. Nat10 depletion also effectively reduces AVC in vivo. Mechanistically, CircILRUN acts as a molecular scaffold facilitating the interaction between NAT10 and the deubiquitinase USP11, thereby stabilizing NAT10 protein. Integrated transcriptomic and ac4C epitranscriptomic profiling reveals that CircILRUN promotes NAT10-mediated acetylation of CD36 mRNA, stabilizing CD36 and accelerating AVC progression. Collectively, our findings establish an extracellular vesicle-mediated circILRUN–NAT10–CD36 axis driving CAVD pathogenesis and identify both circILRUN and NAT10 as promising therapeutic targets

overall_design: CircRNAs expression in human valvular endothelial cell-derived exosomes (hVECs-Exos) were measured after exposure to oscillatory shear stress (OSS) and laminar shear stress (LSS), respectively. Three independent experiments were performed at each group (OSS and LSS) using different donors for each experiment.

first: Tinwen

last: Zhou


title: mrRNA-directed DNA methylation enables selective mutant oncogene silencing therapy without exogenous proteins [RRBS]

first: Yifan

last: Guo

address: Dongan Road No.150

institute: Fudan university, Shanghai, China

summary: The outcome of recent demethylation therapy is limited by the unspecific hypomethylation of oncogenes. The mechanism underlying differential DNA methylation distribution in the same topological domain remains unclear. Here, we established an efficient framework based on heterogeneity across pan-cancers to systemically characterize DNA methylation regulatory boxes (mrBoxes). Through  plasmid-based functional motif interactome analysis assay, the role of mrBoxes on DNA methylation was validated. Moreover, the U1 small nuclear ribonucleoprotein A (SNPRA) was found to regulate the chromatin localization of DNMT1 to mrBox. In this process, long noncoding RNAs were identified to act as a bridge, connecting the targeted mrBox element and SNRPA/DNMT1 complex. Based on this regulatory model, we synthesized exogenous methylation-regulating RNA (mrRNA), which contains only the mrBox-recognizing and SNRPA/DNMT1 complex-binding motif. Furthermore, we developed a methylation-regulating therapy called “OncoMutation Silence”, which regulates the DNA methylation of mutated oncogenes through mrRNA. The efficacy and specificity of this therapy have been verified in tumors with KrasG12D mutation. Collectively, this study reveals mrBox-related regulatory model of DNA methylation, and provides mrRNA-based tumor therapy for silencing specific mutated oncogenes.

overall_design: Bisulfite-Seq of DNMT1 dynamics in HEK293T cell lines treated with siZFPM2AS1 and siSNRPA

first: Yvan

first: Jie

last: Gu

first: Di

last: Ge

first: Ronghua


title: AmeloD regulates cell proliferation and differentiation process of ameloblasts through modulation of Sonic hedgehog (Shh) signaling pathway

city: Fukuoka

first: Sae

last: Oka

address: 3-1-1 Maidashi Higashi-ku

institute: Kyushu University

summary: Tooth morphogenesis is regulated by various molecules, with the enamel knot playing a particularly important role as a signaling center during early development. AmeloD, a basic-helix-loop-helix transcription factor, is expressed in the enamel knot. Cell proliferation was suppressed in AmeloD-overexpressing SF2 cells, consistent with its function in the cell proliferation arrest region of the enamel knot. RNA sequencing analysis also revealed decreased expression of proliferation-related molecules, such as Mki67, in AmeloD-overexpressing cells. Shh is also known to be expressed in the enamel knot, and AmeloD-overexpressing cells showed increased expression of ameloblastin and Sox21 upon shh stimulation. Indeed, AmeloD-deficient mice exhibited reduced tooth cusp distance, reduced mesio-distal and buccal-lingual tooth diameters, and enamel hypoplasia. To clarify how AmeloD functions in cells, we used a yeast two-hybrid system to identify AmeloD-binding proteins. Odam, Selenof, Kct2, and TCF4 were identified as AmeloD-binding molecules. AmeloD and Odam were expressed in similar regions in mouse postnatal day 1 molar tooth germs, and their expression was confirmed in SF2 cells. To clarify the mechanism by which Odam regulates AmeloD, we transfected AmeloD-overexpressing cells with the Odam gene, and found that AmeloD-mediated cell growth inhibition was reversed. Furthermore, cell scratch assays showed enhanced migration, which was suppressed by overexpression of AmeloD. These results suggest that AmeloD enhances cell arrest at the enamel knot and the stimulation of Shh signaling, and that Odam binds to AmeloD, regulating AmeloD function through these interactions.

overall_design:
Examination of 4 different method in 2 cell types.
RNAseq

first: Yuta

last: Chiba

first: Hiroshi

last: Sato

first: Mitsuki

first: Itaru

last: Komine

first: Aya

last: Yamada

first: Satoshi

last: Fukumoto


title: Single-cell RNA-seq of traumatic brain injury samples

city: Tianjin

first: Jianhao

last: Wang

address: Anshan Road No.154

institute: Tianjin Medical University General Hospital

summary: The pathological process of traumatic brain injury (TBI) is complicated, and endothelial cells (ECs) is critical in these pathological and physiological processes. In this study, ECs from surgically removed injury tissues were isolated, and then subjected to single-cell RNAseq to explore their transcriptomic profiles.

overall_design: We profiled isolated cells from injury center and injury periphery tissues of three TBI patients. CD45-CD31+ cell suspensions were generated using magnetic-activated cell sorting (MACS), which were then subjected to scRNA-seq performing a 10X Genomics protocol.

first: Fan

last: Yang

first: Yuan

last: Xie

first: Lei


title: GPRC5B preserves a mature β-cell state in obesity by controlling MafA expression [dof92_RNA-seq]

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

country: Germany

institute: MPI for heart and lung research

summary: In vitro studies have implicated orphan receptor GPRC5B in β-cell survival, proliferation and insulin secretion, but its relevance for glucose homeostasis in vivo is largely unknown. Using tamoxifen-inducible, β-cell-specific GPRC5B knockout mice (Ins-G5b-KOs) we show here that loss of GPRC5B does not affect β-cell function in the lean state, but results in strongly reduced insulin secretion and disturbed glucose tolerance in mice subjected to high fat diet for 16 weeks. Flow cytometry and single-cell expression analyses in islets from obese mice show a reduced β-cell abundance and a less mature β-cell phenotype in Ins-G5b-KOs. Expression of β-cell-specific transcription factor MafA is reduced both on the RNA and protein level, as are transcripts of MafA target genes. Mechanistically, we show that phosphorylation of cAMP response element-binding protein (CREB), a major regulator of MafA expression, is reduced in islets of obese Ins-G5b-KOs, and that this phenotype precedes the downregulation of MafA and MafA target genes. Taken together, GPRC5B helps to maintain mature β-cell function in obesity through cAMP/CREB-dependent regulation of MafA expression.

overall_design: mRNA sequencing in isolated pancreatic beta cells from control mice and mice with beta cell-specific GPRC5B deficiency

first: Tianpeng

first: Nina

last: Wettschureck

last: Guenther


title: The single-cell level molecular characteristics according to combination immunotherapy response of non-small cell lung cancer patients.

first: Sehwa

last: Hong

summary: Non-small cell lung cancer patients finally acquire EGFR-TKI resistance. For these patients, combination immunotherapy, atezolizumab plus bevacizumab and carboplatin/paclitaxel (ABCP), was proposed as a therapy based on targetting both cancer and stromal cells. Despite of relatively good therapeutic outcome, precise mechanism was not fully understood. To dissect tumor microenvironment and regulatory mechanism involved in ABCP response, we performed scRNA-seq analysis from patients to receive ABCP.

overall_design: Single-cell transcriptome analysis was performed using malignant cells acquired from pleural effusion samples of seven patients before treatment. Cellular characteristics according to therapeutic response were dissected.

first: Charny

last: Park

first: Ji-youn

last: Han

first: Sunshin

last: Kim


title: GPRC5B preserves a mature β-cell state in obesity by controlling MafA expression

summary: This SuperSeries is composed of the SubSeries listed below.


title: Distinct immunometabolic signatures of type 1 versus type 2 diabetes in a murine model of myocardial infarction

city: Jackson

first: Alan

last: Mouton

state: Mississippi

address: 2500 North State Street

department: Physiology and Biophysics

institute: University of Mississippi Medical Center

summary: Background: Diabetes mellitus (DM), which consists of type I and type 2 diabetes (T1D and T2D), is a known risk factor for myocardial infarction (MI) and negatively impacts post-MI outcomes. However, the mechanisms by which DM exacerbates cardiac remodeling in T1D versus T2D have not been well defined. Here, we assessed acute and chronic post-MI outcomes in T1D and T2D mice, focusing on immune and metabolic pathways. Methods: T1D was induced in adult male mice by a single high dose of streptozotocin (STZ), and T2D induced by high fat/fructose feeding and multiple low STZ doses. Two weeks following STZ administration, MI was induced by permanent coronary artery ligation, and mice were studied at days (D) 3, 7, and 28 post-MI. Cardiac function was assessed by echocardiography. Results: Compared to non-diabetic mice, T1D and T2D mice had worse cardiac dysfunction after MI, including increased wall thinning and decreased ejection fraction, despite similar infarct sizes. T1D mice also displayed acute pulmonary congestion. By RNA-sequencing analysis, T1D and T2D mice displayed upregulation of genes associated with canonical chemokine/monocyte-mediated inflammatory pathways, and downregulation of genes associated with extracellular matrix remodeling. T1D and T2D delayed activation of M2-like (CD206+) macrophages in the heart, and impaired normal collagen and elastin deposition after MI. T2D also increased expression of genes associated with T cell activation, and increased CD8+ T cells in the infarct. T1D and T2D hearts showed signs of impaired glucose and ketone oxidation, and T1D hearts had increased markers of fatty acid oxidation. Extracted D3 cardiac macrophages from T1D and T2D mice exhibited higher basal oxygen consumption, and increased M1 markers and chemokine expression. Plasma from T1D and T2D mice increased chemokine expression (Ccl2, Ccl7, Cxcl1) in cultured bone marrow macrophages, and T2D plasma impaired mitochondrial function. Conclusions: DM promotes adverse cardiac remodeling, which is associated with activation of overlapping and unique inflammatory pathways, impaired ECM remodeling, remote metabolic remodeling, and alterations in macrophage metabolism. Our results provide novel insights into potential therapeutic pathways for DM patients suffering from MI.

overall_design: Mouse left ventricular infarct tissue collected at day 7 after myocardial infarction induced by permanent ligation of the coronary artery

first: A


title: Multi-omics analysis identifies progenitors of CD4-CTLs [invitrodiff_n_scm_scRNA]

city: New Delhi

first: Veena

middle: S.

last: Patil

state: Delhi

address: Aruna Asaf Ali Marg

institute: National Institute of Immunology

summary: The CD4+ T cells expressing cytolytic molecules such as granzymes and perforins have been detected in many diseases and are shown to be enriched in the effector memory expressing CD45RA (TEMRA) in humans, but their origin remains elusive. However, their gene expression profile in comparison to classically defined cytotoxic T lymphocytes (CTLs), the CD8-CTLs has not been well demonstrated, hence their relevance remains debatable.  Thus in this study by parallelly analysing the CD4-CTLs and CD8-CTLs, we demonstrate that they are indistinguishable for the cytolytic program with both showing similar gene expression profile and T cell antigen-receptor (TCR) clonal expansion. Further using an integrative multi-omics approach combining the transcriptome, TCR repertoire, and open chromatin profile of CD4+ naïve (CD4-TN) and memory subsets, we discovered a stem-cell memory subset that is pre-committed to CTL program within the CD4+ T cell lineage. Through an in vitro differentiation model we developed CD4+ T cells with cytolytic potential from CD4-TN cells. The in vitro differentiated cells followed the trajectory of different developmental memory subsets from ex vivo CD4+ T cells for transcriptomic patterns as well as open-chromatin landscape. Thus, through this model, we deciphered the molecular signatures of early commitment of CD4-TN cells to cytotoxicity program. Of particular interest was the expression of both longevity as well as cytotoxicity associated gene sets by the in vitro differentiated CD4-CTLs, hence generating long-lived CD4-CTL effectors. This specific property can be further explored for vaccine development as well as testing the efficacy and cell based therapies for precision medicine.

overall_design: CD4-Naive (CD3+CD4+CD45RA+CCR7+CD95-) and CD4-TSCM (CD3+CD4+CD45RA+CCR7+CD95+) were isolated from PBMCs of healthy human donors and activated for 48 hrs using aCD3/aCD28 Dynabeads in a Th1 polarizing milieu of cytokines and antibodies. The cells were then cultured in the Th1 polarizing condition for 8 days (2+8 day cycle) before doing scRNA-Seq (Day 10).

first: Raunak

last: Kar

first: Shreya

last: Sinha

first: Zainab

last: Khatun

first: Anjali

last: Sharma