title: Fibroblast-Derived Thrombospondin 2 Shapes the Immune-Exclusionary Environment at Invasive Fronts in Colorectal Cancer [Visium

city: Kyoto-City

first: Kosuke

last: Iwane

address: 54 Shogoin-Kawahara-Cho, Sakyo-Ku

department: Department of Gastroenterology and Hepatology

institute: Kyoto University Graduated School of Medicine

summary: Fibrotic colorectal cancers (CRC) are characterized by immune exclusion driven by activated stroma, particularly by cancer-associated fibroblasts (CAFs). This study aims to describe characteristics of spatial immune landscape of CRCs, which are subtyped by transcriptomic signatures and microsatellite instability.

overall_design: Four slides from FFPE blocks of surgically resected CRC specimens, which were determined as CMS1/MSI, CMS2, or CMS4 using bulk-RNA-seq data, were analyzed using spatial transcriptomics (ST).

first: Yuki

last: Nakanishi

first: Yu

last: Muta

last: Fukui

first: Hiroaki

last: Kasashima


title: Arabidopsis DXO1 is an evolutionarily diverged homolog that impacts ribosome loading and mRNA surveillance

status: Public on Oct 28 2025

city: Philadelphia

first: Diep

middle: R

last: Ganguly

state: PA

address: 433 S University Ave

department: Department of Biology

country: USA

institute: University of Pennsylvania

summary: The Rai1/Dxo1/DXO protein family are involved in eukaryotic 5′-end RNA quality control by hydrolyzing non-canonical mRNA cap structures, such as the NAD+ 5′-cap. The plant DXO1 ortholog shows distinct biochemical properties, compared to its fungal and mammalian counterparts, and only minor phenotypic traits can be attributed to its NAD+-decapping (deNADding) activity. On the other hand, the chloroplast and growth defects observed in dxo1 null mutants appear linked to the presence of a serine-rich domain at its N-terminus. Here, we report that plant DXO1 is a distinct ortholog that likely diverged earlier than its fungal and mammalian counterparts. We attribute this to the presence of a serine-rich N-terminal domain, which is found almost exclusively in plants and likely arose during the emergence of angiosperms. Degradome profiling revealed that Arabidopsis DXO1 contributes to mRNA surveillance, via examination of exon junction complex and ribosome footprints, and this activity is reliant on the presence of the serine-rich N-terminal domain in Arabidopsis. Furthermore, polysome profiling revealed that plants lacking DXO1 have severe ribosome loading defects, which may explain the loss of mRNA surveillance and alterations in pre-rRNA processing. Taken together, our data leads us to propose a model whereby Arabidopsis DXO1 is involved in 5′-end RNA quality control, which is critical for ribosome loading that impacts translational fidelity and translation-dependent mRNA surveillance.

overall_design: GMUCT was performed on two biological replicates of 12-day-old transgenic Arabidopsis seedlings expressing DXO1 variants fused to GFP under the control of the 35S promoter from the cauliflower mosaic virus (Kwasnik et al 2019 Nucleic Acids Research).

first: Xiang

last: Yu

first: Brian

middle: D

last: Gregory


title: Fibroblast-Derived Thrombospondin 2 Shapes the Immune-Exclusionary Environment at Invasive Fronts in Colorectal Cancer [RNA-seq_Human_CRC]

country: Japan

type: Expression profiling by high throughput sequencing

summary: Fibrotic colorectal cancers (CRC) are characterized by immune exclusion driven by activated stroma, particularly by cancer-associated fibroblasts (CAFs). We identified that the matricellular protein thrombospondin-2 (THBS2) is highly expressed in the stroma of fibrotic CRCs. We performed orthotopic implantation of CMS4-like mouse tumor organoid (MTO) into WT and Thbs2 KO mice. Tumors developed in Thbs2 KO mice were considerably smaller and exhibited increased CD8 T cell infiltration and higher numbers of apoptotic tumor cells compared with WT mice. This study aims to elucidate the interaction between fibrotic CRCs and CAFs by comparing the phenotypic characteristics of bulk tumor tissues and their corresponding CAFs.

overall_design: RNA-seq profiling of fresh samples of surgically resected human colorectal carcinoma

first: Yasuhiro


title: Fibroblast-Derived Thrombospondin 2 Shapes the Immune-Exclusionary Environment at Invasive Fronts in Colorectal Cancer [RNA-seq_Mouse_Tumor]

summary: Fibrotic colorectal cancers (CRC) are characterized by immune exclusion driven by activated stroma, particularly by cancer-associated fibroblasts (CAFs). We identified that the matricellular protein thrombospondin-2 (THBS2) is highly expressed in the stroma of fibrotic CRCs. We performed orthotopic implantation of CMS4-like mouse tumor organoid (MTO) into WT and Thbs2 KO mice. Tumors developed in Thbs2 KO mice were considerably smaller and exhibited increased CD8 T cell infiltration and higher numbers of apoptotic tumor cells compared with WT mice. Our aim is to elucidate how Thbs2 in tumor microenvironment influences tumor growth.

overall_design: RNA-seq profiling of bulk-tumors derived from orthotopic implanted mouse tumor organoids (MTOs) harboring mutations in Apc, Trp53, Kras, and Tgfbr2 in the rectum of wild-type (WT) and Thbs2KO mice


title: Transcriptomic Analysis of Zebrafish Larvae Exposed to Tire Wear Particle Leachates

city: Busan

first: Tae Hwan

last: Shin

address: Nakdong-daero 550 beon-gil

department: Health Sciences

country: South Korea

institute: Dong-A University

summary: For transcriptomic analysis, total RNA was extracted from pooled zebrafish larvae (80 per sample, three biological replicates per group) preserved in RNAlater™. High-quality RNA (RIN > 7.0), isolated using the RNeasy Mini Kit, was quantified and assessed for integrity. Ribosomal RNA was depleted prior to cDNA library construction using the TruSeq Stranded Total RNA Library Prep Gold Kit. The resulting libraries were quantified, size-validated, and sequenced using the Illumina NovaSeqX platform with 2×100 bp paired-end reads.

overall_design: Adult wild-type zebrafish were acclimated under controlled laboratory conditions for three months prior to breeding. Fertilized embryos were collected and exposed to vehicle control or one of three concentrations of tire wear particle (TWP) leachates (TWP 250, 350, and 700) from 4 to 168 hours post-fertilization (hpf). Each group included 200 embryos and was tested in triplicate. At 72 hpf, larvae were transferred to clean medium with adjusted water quality parameters while maintaining consistent temperature and photoperiod. For transcriptomic analysis, 80 larvae per replicate were pooled, and total RNA was extracted from three biological replicates per group. RNA samples with RIN > 7.0 were used for cDNA library construction, followed by high-throughput sequencing using the Illumina NovaSeqX platform.

first: Beomsu

last: Kim

first: Songyeon

first: Yunwi

last: Heo

first: Jung-Man

last: Jo

first: Soyoung

last: Ahn

first: Soyeon

last: Jeon

first: Sang-Hee

last: Woo

first: Seokhwan

last: Lee

first: Jae-Jun

first: June-Woo

last: Park

first: Wan-Seob

last: Cho


title: In Vitro Modeling of and Automated Drug Screening in Embryonal Brain Tumors Using Tumor-Brain Organoids

city: Münster

first: Kornelius

last: Kerl

address: Albert-Schweitzer-Campus 1

department: Department of Pediatric Hematology and Oncology

country: Germany

institute: University Children’s Hospital Münster

summary: Embryonal brain tumors (EBTs), arising during early brain development, present significant challenges in understanding pathogenesis and developing of treatments. This study explores the efficacy of forebrain organoid (FBO) models in replicating the complexities of the EBT microenvironment. We established an EBT-forebrain-organoid (TBO) model, incorporating embryonal tumor with multilayered rosettes (ETMR) and atypical teratoid and rhabdoid tumor (ATRT), using a scalable liquid-handling-assisted coaggregation method. Comprehensive characterization, including wholemount immunostaining, immunohisto-chemistry, single-cell RNA sequencing, integration with existing datasets, and automated cell type-specific drug screening, validated the TBO model. TBOs closely mimicked mid-gestational fetal brain environments, and EBTs in FBOs better approximated native intratumoral heterogeneity at both histological and transcriptomic levels than tumor-only 3D approaches. Drug screening in ETMR-FBOs identified promising treatment options, including anthracyclins and triptolide, demonstrating anti-tumoral effects with minimal neurotoxicity. Scalable TBO models that incorporate immature neural microenvironments better recapitulate individual tumor heterogeneity and mark significant progress towards targeted therapy and personalized precision medicine.

overall_design: scRNA-seq (10xGenomics) of fresh tumor forbrain organoid samples (n = 2) and fixed snRNA-seq samples from tumors (n = 17) and cell lines (n = 10).

first: Riedel

last: Nicole C.

first: Walter

last: Carolin

first: de Faria

last: Flavia W.

first: Atendorf

last: Lea

first: Aust

last: Paula

first: Göbel

first: Verma

last: Archana

first: Ballast

last: Annika

first: Bedzhov

last: Ivan

first: Roy

last: Rajanya

first: Münter

last: Daniel

first: Albert

last: Thomas K.

first: Rössig

last: Claudia

first: Johann

last: Pascal

first: von Zezschwitz

last: Barbara

first: Sandmann

last: Sarah

first: Varghese

last: Julian

first: Thomas

last: Christian

first: Schüller

last: Ulrich

first: Bruder

last: Jan M.

first: Kerl

last: Kornelius


title: Targeting IGF2BP1 Enhances Immune Checkpoint Therapy in High-grade Serous Ovarian Cancer [mouse]

city: Halle

first: Markus

last: Glaß

address: Kurt-Mothes-Str. 3a

department: Institute of Molecular Medicine

institute: Martin Luther University Halle-Wittenberg

summary: High-grade serous ovarian cancer (HGSC) accounts for the vast majority (>70%) of ovarian cancer-associated deaths with minor therapeutic improvements. Among four proposed molecular subtypes of HGSC, the C5 subtype is distinguished by high proliferative potential and elevated immune evasion which is indicated by an unfavorable MHCI/PD-L1 ratio. However, the underlying key drivers of the C5 subtype remained elusive. Here we identify oncofetal RNA-binding proteins (RBPs) promoting the immune evasion of C5-HGSC. The IGF2 mRNA binding protein 1 (IGF2BP1) enhances expression of the E3 ligase MDM2, which induces degradation of IRF1 resulting in reduced MHCI presentation. Concomitantly, IGF2BP1 elevates PD-L1 synthesis by impairing its microRNA-directed silencing. This shifts the intra-tumoral MHCI/PD-L1 ratio, limits immune cell infiltration, and promotes evasion of tumor cells from cytotoxic T cells (CTLs) in human and syngeneic mouse models of ovarian cancer. The small molecule inhibitor BTYNB impairs IGF2BP1-directed regulation of MHCI/PD-L1 ratios, promotes CTL-directed killing as well as activation, and strongly synergizes with immune checkpoint inhibition (ICI) by Nivolumab in vitro and in vivo.

overall_design: Intraperitoneal syngeneic mouse model using the IGF2BP1-directed small molecule inhibitor BTYNB (closed conformation) with or without PD-1 blocking antibodies. Ascites was analyzed by scRNA-seq.

first: Nadine

last: Bley

first: Alexander

last: Rausch

first: Robin

last: Rolnik


title: EZH2 alters aggressive variant prostate cancer subtype evolution, but is not required for neuroendocrine prostate cancer. [DNA methylation]

city: Buffalo

first: David

last: Goodrich

state: NY

address: Elm and Carlton Streets

department: Department of Pharmacology & Therapeutics

institute: Roswell Park Comprehensive Cancer Center

type: Methylation profiling by genome tiling array

summary: Advanced prostate cancer (PrCa) remains a leading cause of cancer-related death among men because nearly all patients progress on standard therapy that targets androgen receptor (AR) signaling. An important mechanism driving therapeutic resistance is lineage plasticity, the ability of PrCa cells to reprogram into AR independent lineage variants like neuroendocrine (NE) PrCa. The histone methyltransferase EZH2 has been implicated in PrCa lineage plasticity and EZH2 inhibitors are being evaluated clinically for the treatment of PrCa. Here we test how Ezh2 impacts the evolution of PrCa lineage plasticity using genetically engineered mice and human patient data. Ezh2 deficiency in mice diversifies the evolution of PrCa lineage variants, including a newly discovered variant with high expression of neurofilament genes. Analogous PrCa lineage variants are detectable in human specimens where they similarly correlate with EZH2 expression. Lineage variant diversification is associated with activation of transcription factor programs and AR cistrome reprogramming that is normally repressed by Ezh2. These findings advance the understanding of PrCa lineage plasticity and have implications for targeting EZH2 as a PrCa therapy.

overall_design: Intact (non-castrated) mouse endstage prostate tumor tissue from GEMMs (SKO, DKO, TKO) or with Ezh2 deletions (DKO_EP and DKO_EE) were profiled using the Infinium Mouse Methylation MM285 BeadChip array.

first: Justine

last: Jacobi


title: Targeting IGF2BP1 Enhances Immune Checkpoint Therapy in High-grade Serous Ovarian Cancer [HGSC]

overall_design: HGSC patient samples were analyzed by scRNA-seq.


title: Targeting IGF2BP1 Enhances Immune Checkpoint Therapy in High-grade Serous Ovarian Cancer [polyA RNA-seq, unsorted co-cultures]

city: Halle (Saale)

first: Danny

last: Misiak

department: Molecular Cell Biology

overall_design: PolyA-RNA sequencing of unsorted co-cultures from ES-2 cells and PBMCs, treated with different conditions (3x DMSO, 3x BTYNB 10µM, 3x CUB (cucurbitacine B, 10 nM)).


title: RNA-sequencing of SVF-derived preadipocytes from inguinal adipose tissue of male mice differentiating under control conditions or treated with CGRP

city: ANN ARBOR

first: Jorge

middle: L

last: Ruas

state: Michigan

address: 2800 Plymouth RD, B025-2681 NCRC - Ruas Lab NCRC B025

institute: University of Michigan

summary: To investigate the effects of CGRP on adipocyte differentiation, we treated stromal vascular fraction (SVF)-derived preadipocytes from inguinal white adipose tissue (iWAT) of male mice with CGRP during differentiation. RNA-sequencing was performed across a full time course of normal differentiation to identify transcriptional changes induced by CGRP treatment compared to control conditions.

overall_design: RNA sequencing analysis of stromal vascular fraction (SVF)-derived preadipocytes from inguinal white adipose tissue (iWAT) of male mice (n = 3 per group, C57BL/6N) undergoing differentiation under control conditions or with CGRP treatment. Total RNA was extracted using TRI reagent (Sigma-Aldrich, T9424) according to the manufacturer's instructions. RNA was further purified, and DNase treated (Zymo, R1013), and integrity confirmed prior to sequencing (Agilent Technologies, 2100 Bioanalyzer), with an RNA integrity number (RIN) greater than 7 as a threshold. Samples were purified by poly-A enrichment before library preparation using the Illumina NovaSeq X Plus Series platform with a paired-end 150 base pair strategy.

first: Kyle

last: Dumont


title: Developing exocrine glands harbour a distinct macrophage population that is maintained by ILC2 derived GM-CSF [bulk RNA-seq]

city: Zurich

first: Burkhard

last: Becher

address: Winterthurerstrasse 190

country: Switzerland

institute: University of Zurich

summary: Granulocyte-macrophage colony-stimulating factor (GM-CSF) has a non-redundant role in the emergence and maintenance of alveolar macrophages (AMs). However, its role in steady-state myelopoiesis outside the lung is largely unexplored. We discovered a strong signal of GM-CSF production by type 2 innate lymphoid cells (ILC2s) in the developing salivary glands (SG). ILC2 derived GM-CSF fosters the development of a hitherto undescribed phagocyte subset, which we named adenophages. Detailed analysis focusing on phenotypic and transcriptional profiling revealed that adenophages represent a non-canonical macrophage subset displaying shared aspects of both, macrophages and dendritic cells. We found them to be homogenously distributed across the SG, but always forming a niche-triad with GM-CSF producing ILC2s and myoepithelial cells. Importantly, adenophages were present throughout all analyzed exocrine glands such as lacrimal glands and mammary glands, and were also identified in human SG, indicating a conserved role in exocrine glands across species.

overall_design: Myoepithelial cells were facs sorted from the salivary glands of 1 month old Csf2-KO (N=4) or Control (N=4) mice (male and female) and RNA-seq was performed

first: Frederike

last: Westermann

first: Selma

last: Tuzlak

first: Victor

last: Kreiner


title: Extracellular vesicles from obese visceral adipose promote pancreatic cancer development and resistance to immune checkpoint blockade therapy.

city: Guangzhou

first: Zhou

last: yifan

state: 周一帆

address: 74 Zhongshan Second Road

country: China

institute: Sun Yat-sen University Cancer Center

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Obesity is correlated with multitype of cancer development, and pancreatic ductal adenocarcinoma (PDAC) in obese patients shows dismal prognosis and resistance to immune checkpoint blockade (ICB) therapy. The molecular mechanism is largely unknown. Here we show that obese visceral adipose tissues (VAT) can communicate with distant PDAC by delivering extracellular vesicle (EV) carrying signal molecules. We reveal that PDAC cells can take in VAT-EVs to their lysosomes where EVs-delivered Cathepsin A (Ctsa) stabilize ribonuclease Rnaset2b to produce free pseudouridines from RNA cleavage. Pseudouridines further activate mast cells via mediating an increase in reactive oxygen species (ROS) generation, which leads to a decrease in the distribution of H3K27me3 at the gene promoter, ultimately inhibiting CD8+ T cell activity, forming an immunosuppressive tumor microenvironment which enhances cancer progression and ICB resistance. We also demonstrate harmful effects of VAT-EV CTSA-pseudouridine-mast cell axis for obesity-related PDAC. Animal experiments indicate that Ctsa knockdown effectively enhances ICB efficacy on PDAC. Our study uncovers a mechanism connecting obesity and cancer which holds promise for developing new therapeutic strategy for obesity-related cancers.

overall_design: CUT&Tag-seq (Cleavage Under Targets and Tagmentation sequencing) for H3K27me3 in MC9 cells

first: Jian

last: Zheng

first: Chunling

last: Xue


title: EZH2 alters aggressive variant prostate cancer subtype evolution, but is not required for neuroendocrine prostate cancer [ChIP-seq]

overall_design: ChIP-seq profiling of prostate tumor tissue from mouse models (SKO,DKO, TKO) and DKO mouse models with Ezh2 deletions (DKO_EP and DKO_EE) for androgen receptor (Ar) or histone marks (H3K27ac, H3K27me3, or H3K4me3). Paired-end sequencing reads were mapped to mm10 using bwa and deduplicated with Picard. deeptools was used to generate bigwig files.


title: Distinct Molecular Responses of Human Intestinal Organoids to Proton and Photon Radiation

city: Houston

first: Sarah

middle: Elizabeth

last: Blutt

state: Texas

address: One Baylor Plaza

department: Molecular Virology and Microbiology

institute: Baylor College of Medicine

summary: Radiation exposure causes damage to tissues with high numbers of rapidly dividing cells such as the intestine. Mouse models have been widely used to understand the effects of radiation injury, repair following exposure, and to develop and test potential therapies for radiation-induced damage. However, although the mouse intestine shares physiological and molecular similarities with human intestine, translation and validation of findings in mouse radiation models to the human intestine have been limited by the lack of models. The development of ex vivo human intestinal organoids provides a new model in which the effects of radiation on the human intestinal epithelium can be examined and compared to in vivo animal models. Using human small intestinal jejunal organoids, multi-omic profiles were analyzed using bioinformatic pipelines to explore the response to a dose of proton and photon radiation exposure which may be encountered during a radiotherapy treatment or over time in high background radiation environments. Transcriptional signatures following radiation exposure were indicative of reprogramming of cells to a more primitive state that has been associated with regeneration and repair. Integrative analyses of transcriptomic and metabolomic datasets revealed enrichment of amino acid synthesis and metabolism pathways. Human intestinal organoids can complement and validate findings from traditional animal models and are emerging as a powerful tool to study responses to radiation exposure.

overall_design: RNAseq analysis of crypt-like or villus-like  human intestinal organoids (HIO) exposed to 1Gy gamma irradiation, 1Gy LET proton irradiation or left unirradiated.  Three technical replicates for each donor crypt and villus HIO +/- radiation exposure were pooled prior to RNA extraction.

first: Victoria

last: Poplaski

first: Tajhal D.

last: Patel

first: Mary K.

last: Estes

first: Cristian

last: Coarfa

first: Sarah E.


title: dHyperCas12a enables multiplexed CRISPRi screens

city: Durham

first: Schuyler

last: Melore

state: North Carolina

address: 101 Science Dr

institute: Duke University

summary: Interactions between multiple genes or cis-regulatory elements (CREs) underlie a wide range of biological processes in both health and disease. Here, we combine a hyper-efficient version of Lachnospiraceae bacterium dCas12a (dHyperLbCas12a) with RNA Polymerase II expression of long CRISPR RNA (crRNA) arrays to enable efficient highly-multiplexed epigenome editing. We demonstrate that the dCas12a system is highly effective for high-throughput screens. We use four different dCas12a repressors and a ~900-member library of crRNA arrays containing four crRNAs each to target the promoters of essential genes or cell surface negative controls. This fitness screen allows us to compare the activity of different dCas12a repressors across many genomic loci.

overall_design:
To perform a combinatorial screen at the PER1 locus, we created two crRNA array plasmid libraries, with each construct containing six crRNAs and a unique barcode. The PER1-targeting library contains ~9,000 crRNA arrays and the Non-targteing library contains ~10,000 crRNA arrays. Each plasmid library was sequenced twice. These libraries were then transduced into cells at low MOI and selected with puromycin for ten days. On day ten they were rreated with different concentrations of dexamethasone, and sorted on PER1 expression. Top and bottom 12% PER1-expressing cells were sorted and sequenced, as well as bulk unsorted cells (Input).
To perform a fitness screen in A549 cells, we created two crRNA array plasmid libraries, containing the same spacers but either the As or Lb Cas12a direct repeat sequence, with each array construct containing four crRNAs. Each library contained ~900 crRNA arrays. These libraries were then transduced into A549 cells that stably express either dHyperLbCas12a-KRAB, dHyperLbCas12a-4xSID, dEnAsCas12a-KRAB, or dEnAsCas12a-4xSID repressors at low MOI (~0.2) and selected with puromycin. Three independent transductions were performed for each repressor cell line. Cells were passaged for three weeks post-transduction and cell pellets were collected at days 5, 7, 14, and 21 (post-transduction). crRNA array sequences were amplified from these cell pellets as well as the plasmid libraries and sequenced on the NextSeq2000.

first: Timothy

middle: E

last: Reddy

middle: M

first: Marisa

middle: C

last: Hamilton

first: Christian

last: McRoberts Amador

first: Charles

middle: A

last: Gersbach


title: EZH2 alters aggressive variant prostate cancer subtype evolution, but is not required for neuroendocrine prostate cancer. [scRNA-seq]

first: David

last: Goodrich

overall_design: scRNA-seq profiling of prostate tumor tissue from mouse models (SKO,DKO, TKO) and DKO mouse models with Ezh2 deletions (DKO_EP and DKO_EE).

first: Justine

last: Jacobi


title: In vivo intratumoral Heterogeneity in a dish: Scalable Forebrain Organoid Models of Embryonal Brain Tumors for High-Throughput Personalized Drug Discovery

first: Carolin

last: Walter

address: Domagkstraße 9

department: Medical Faculty of the WWU Münster

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

overall_design: Refer to individual Series


title: Myeloid lineage (CD45+ CD11b+ cells) analysis sorted from stromal vascular fraction of visceral and subcutaneous adiposetissue in mice in different diet conditions or after rosiglitazone treatment

city: La Jolla

first: Yun Sok

last: Lee

state: California

address: 9500 Gilman Dr MC0673

department: Medicine

institute: University of California San Diego

summary: Unlike visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT) can play a protective role against the development of insulin resistance and metabolic dysfunction in obesity. These changes are associated with higher induction of pro-inflammatory programs and macrophage infiltration in VAT compared with SAT in obesity. Moreover, our results suggest that subcutaneous adipose tissue macrophages (ATMs) release small extracellular vesicles (sEVs) that can improve insulin sensitivity, opposite to the effect of visceral ATM sEVs in obesity. This functional difference was associated with changes in the proportion of resident ATMs, as well as proinflammatory, recruited ATMs. To study the mechanism by which adipose tissue inflammation and macrophage infiltration are differentially regulated in SAT vs VAT, we performed single cell RNA sequencing (scRNA-seq) analysis in sorted live CD45+ CD11b+ myeloid cells from stromal vascular fraction (SVCs) of SAT and VAT of mice fed normal chow diet, high fat diet, or SVCs of HFD mice after the diet switch from high fat diet to normal chow diet, or treatment with insulin sensitizing, rosiglitazone.

overall_design: Four different groups of C57BL6 mice were used. Group 1 mice fed normal chow diet for 15 weeks. Group2 mice were fed a high fat diet (HFD) for 15 weeks. Group3 mice were fed a HFD for 12 weeks, followed by the diet switch from HFD to normal chow diet for additional 3 weeks. Group4 mice were fed a HFD for 12 weeks, followed by switch diet to rosiglitazone-containing HFD for additional 3 weeks. At sacrifice, all mice were in the same age. Subcutaneous (inguinal) and visceral (epididymal) adipose tissues taken from these mice were chopped and digested in collagenase buffer. Stromal vascular cells (SVCs) were separated from adipocytes using centrifugation, and pelleted SVCs were used to sort live CD45+ CD11b+ myeloid lineage cells. Sorted cells were, then, subjected to scRNA-seq.

first: Yun


title: Extracellular vesicles from obese visceral adipose promote pancreatic cancer development and resistance to immune checkpoint blockade therapy [ATAC-seq]

first: Zhou

last: yifan

summary: Obesity is correlated with multitype of cancer development, and pancreatic ductal adenocarcinoma (PDAC) in obese patients often shows dismal prognosis and resistance to immune checkpoint blockade (ICB) therapy. The molecular mechanism for these phenomena is largely unknown. Here we show that obese visceral adipose tissues (VAT) can talk to distant PDAC by delivering their extracellular vesicle (EV) carrying signal molecules. We reveal that PDAC cells can take in VAT-EVs to their lysosomes where EVs-delivered Cathepsin A (Ctsa) stabilize ribonuclease Rnaset2b to produce extra pseudouridines from RNA cleavage. Pseudouridines released from cancer cells activate mast cells which inhibit CD8+ T cell activity, forming an immunosuppressive tumor microenvironment  which enhances cancer progression and resistance to ICB. We also demonstrate the harmful effects of VAT-EV CTSA-pseudouridine-mast cell axis for PDAC in obese patients. Animal experiments indicate that Ctsa knockdown effectively enhances the ICB efficacy on PDAC. Our study uncovers a mechanism connecting obesity and cancer which holds promise for developing new therapeutic strategy for obesity related cancers.

overall_design: We performed ATAC sequencing (ATAC-seq) of MC9 cells, KPC cells and CD8T cells to explore the epigenetic regulation caused by PseudoU

first: Jian

first: Chunling


title: Extracellular vesicles from obese visceral adipose promote pancreatic cancer development and resistance to immune checkpoint blockade therapy [RNA-seq]

overall_design: We also extracted RNA from MC9 cells with Vehicle and PseudoU treatment and performed RNA sequencing to evaluate the effects on transcriptional profiles of PseudoU.

last: Zheng


title: Cell-free fat extract promotes cornea epithelial repair and restores neurodegeneration via an anti-inflammation pathway

city: HONGKONG

first: TSE

last: YUENWAH

state: CHINA

address: JIULONG

institute: Xiangya Hospital of Central South University

summary: Corneal homeostasis is a complex process associated with various ocular pathologies, and the development of therapeutic strategies to promote neuroprotection and tissue regeneration is of great challenge. This study investigated the potential benefits of a cell-free fat extract in ameliorating neurodegenerative changes in laser-induced cornea neurodegeneration and alkali burn-induced epithelial injury mouse models. The cell-free fat extract, rich in bioactive compounds, was administered topically, and its effects on neuro regeneration and epithelial cell proliferation were evaluated. Our results exhibited a robust pro-proliferative effect on corneal epithelial cells, contributing to enhanced tissue regeneration in epithelial repair and significant improvement in neuroprotection, characterized by preserving corneal nerve density and branching complexity. These findings suggest that the application of cell-free fat extract holds promise as a novel therapeutic approach for addressing cornea neurodegeneration, offering neuroprotective effects, and promoting tissue repair. Further investigations into the underlying molecular mechanisms and long-term efficacy are warranted to advance the translational potential of this innovative intervention for ocular health.

overall_design: RNA seq of  Human Corneal Epithelial Cells (HCE-Ts)  in control and experiment CEFFE group

first: Yuanhua

last: Xie


title: RNA-sequencing of SVF-derived preadipocytes and adipocytes from inguinal white adipose tissue of male mice treated with vehicle, CGRPa, NPY, NE, or SP

first: Jorge

last: Ruas

summary: To investigate the effects of sensory neuropeptides on preadipocyte and adipocyte function, we treated cultures with vehicle, calcitonin gene-related peptide-alpha (CGRPa), neuropeptide y (NPY), norepinephrine (NE), or substance P (SP) for 10 hours and collected RNA for RNA-sequencing.

overall_design: RNA sequencing analysis of stromal vascular fraction (SVF)-derived preadipocytes from inguinal white adipose tissue (iWAT) of male mice (n = 3 per group, C57BL/6N). Preadipocyte or adipocytes were treated with vehicle, CGRPa, NPY, or SP for 10 hours. Total RNA was extracted using TRI reagent (Sigma-Aldrich, T9424) according to the manufacturer's instructions. RNA was further purified, and DNase treated (Zymo, R1013), and integrity confirmed prior to sequencing (Agilent Technologies, 2100 Bioanalyzer), with an RNA integrity number (RIN) greater than 7 as a threshold. Samples were purified by poly-A enrichment before library preparation using the Illumina NovaSeq X Plus Series platform with a paired-end 150 base pair strategy.

first: Kyle

last: Dumont


title: Cellular and molecular mediators of thymic dendritic cell homeostasis

city: Nashville

first: Bryan

middle: Robert

last: Helm

state: Tennessee

address: 2525 West End Avenue, Suite 1100

department: Center for Quantitative Science

institute: Vanderbilt University Medical Center

summary: To determine changes in cellular composition and gene signatures of hematopoietic antigen-presenting cells (APCs) in thymi harvested from mice where T-cell development is blocked at different maturation stages.

overall_design: 1) CD45+ MHC-II lo to + hematopoietic APCs were FACS-isolated from WT (C57BL6/J), Rag2-/-, B2m-/- and MHC-II-/- mice to determine the role of thymocytes in regulating DC homeostasis and activation (N=2 experimental repeats per genotype). We used scRNA-seq to determine the transcriptional heterogeneity of DCs across the four genotypes. 2) To determine if our flow-validated DC subsets correlate with transcriptional DC clusters, we FACS-isolated 7 DC subsets and labeled each DC subset with a unique TotalSeqBTM hashtag antibody (BioLegend), prior to pooling all subsets for scRNA-seq and hashtag-derived oligonucleotide (HTO) amplification.

first: Ehrlich

last: Lauren

first: Srinivisan

last: Jayashree

first: Liu

last: Qi

first: Lau

last: Ken

first: Helm

last: Bryan


title: FeaSion Decodes the Regulatory Landscape and Functional Diversity of RNA Polymerase II CTD Phosphorylation

city: Beijing

first: Junyi

last: Zhu

address: Peking University, No.5 YiHeYuan Road, Haidian District

department: School of Life sciences

institute: Peking University

summary: We utilize the FeaSion (Feature-Screening-Function) strategy to investigate five known phosphorylation states (pY1, pS2, pT4, pS5, pS7) of RNAPII. Our study demonstrates that mutations at these phosphorylation sites broadly influence transcriptional and co-transcriptional processes. And regulators of these sites involve various biological processes.

overall_design:
To investigate the levels of nascent transcripts of each RNAPII CTD mutants, we performed TT-seq in each mutant cells line before and after treatment of Dox for 24 hours followed with dTAGV-1 for 4 hours. Then we comapred the gene expression profiling in the untreated cells and treated cells.
Please note that each processed data was generated from both replicates and is linked to the corresponding rep1 sample records.

first: Lijun

last: Bao


title: Targeting IGF2BP1 Enhances Immune Checkpoint Therapy in High-grade Serous Ovarian Cancer

first: Danny

last: Misiak

type: Non-coding RNA profiling by high throughput sequencing


title: Targeting IGF2BP1 Enhances Immune Checkpoint Therapy in High-grade Serous Ovarian Cancer [polyA RNA-seq, sorted co-cultures]

overall_design: Co-culture of ES-2 cells with PBMCs with BTYNB treatment ES-2 cells (2.5x105) we treated with 10 µM BTYNB 24h before HLA-matched human PBMCs in a 1:2 ratio together with CD3/CD28 activation beads (Thermo Fisher) were added for additional 36h. ES-2 cells and PBMCs were separated by FACS sorting before RNA was isolated by TRIZOL. RNA integrity was examined using a BioAnalyzer 2100 (Agilent) before poly(A) RNA enrichment was performed with the poly(A) RNA Selection kit V1.5 (Lexogen). The CORALL Total RNA-Seq V1 (Lexogen) was used for strand-specific library generation. Library preparation and sequencing of paired-end reads was performed on an Illumina NextSeq 1000 platform at the Core Facility Imaging (MLU Halle).

first: Nadine

last: Bley

first: Alexander

last: Rausch


title: Digital transformation of herbal medicine: Conversion to biological entity data using digestive herbal medicine-induced transcriptome sequencing_second_HepG2

city: Daejeon

first: Musun

last: Park

address: 1672 Yuseong-daero, Yuseong-gu

institute: Korean Institute of Oriental Medicine


title: Fibroblast-Derived Thrombospondin 2 Shapes the Immune-Exclusionary Environment at Invasive Fronts in Colorectal Cancer [scRNA-seq]

city: Kyoto-City

first: Kosuke

last: Iwane

address: 54 Shogoin-Kawahara-Cho, Sakyo-Ku

summary: Fibrotic colorectal cancers (CRC) are characterized by immune exclusion driven by activated stroma, particularly by cancer-associated fibroblasts (CAFs). We identified that the matricellular protein thrombospondin-2 (THBS2) is highly expressed in the stroma of fibrotic CRCs. We performed orthotopic implantation of CMS4-like mouse tumor organoid (MTO) into WT and Thbs2 KO mice. Tumors developed in Thbs2 KO mice were considerably smaller and exhibited increased CD8 T cell infiltration and higher numbers of apoptotic tumor cells compared with WT mice. Our aim is to elucidate how Thbs2 in tumor microenvironment influences tumor growth.

overall_design: Tumors derived from orthotopic implanted mouse tumor organoids (MTOs) harboring mutations in Apc, Trp53, Kras, and Tgfbr2 in the rectum of wild-type, Thbs1KO and Thbs2KO mice were processed into single-cell suspension and analyzed using scRNA-seq

first: Yuki

last: Nakanishi

first: Mayuki

last: Omatsu

first: Nobuyuki

last: Kakiuchi

first: Masahiro

last: Nakagawa

first: Yu

last: Muta


title: Fibroblast Derived Thrombospondin-2 Shapes the Immune-Exclusionary Environment at Invasive Fronts in Colorectal Cancer [RNA-seq_Mouse_CAF]

overall_design: RNA-seq profiling of CAFs isolated from tumors derived from orthotopic implanted mouse tumor organoids (MTOs) in the rectum of wild-type (WT) and Thbs2KO mice


overall_design: To investigate the gene expression changes of each RNAPII CTD mutants, we performed Poly(A) RNA-seq in each mutant cells line before and after treatment of Dox for 24 hours followed with dTAGV-1 for 12 hours. Then we comapred the gene expression profiling in the untreated cells and treated cells.


overall_design:
To investigate the the chromatin binding  the chromatin binding of each CTD phosphorylated RNAPII, the chromatin binding of RNAPII CTD mutants, and the changes of H3K36me3, H2A.ZAc, H2A.Z after mutation of each RNAPII CTD phosphorylation sites, we performed ChIP-seq and CUT&Tag in each mutant cells line before and after treatment of Dox for 24 hours followed with dTAGV-1 for 4 hours. Then we comapred the gene expression profiling in the untreated cells and treated cells.
To investigate the the chromatin binding of CLK1, CLK4 and YES1 as well as the alterations of RNAPII binding after their knock out, we performed ChIP-seq of CLK1, CLK4 and YES1 in wild-type A549 cells, and ChIP-seq of RNAPII and pS7 in CLK1, CLK4 knock-out cells ,and ChIP-seq of RNAPII and pY1 in YES1 knock-out cells.
To investigate the chromatin binding of CLK1, CLK4 and YES1 after their knock-out as the negtive control, we performed ChIP-seq of CLK1, CLK4 and YES1 in their knock-out A549 cells. Also, to test whether low-concentration of TG003 and CH6953755 reduces levels of pS7 and pY1, we performed pS7 and pY1 ChIP-seq after treatment of 1uM.
Please note that each *merged.bw was generated from both replicates and is linked to the corresponding rep1 sample records.


title: Comprehensive analysis of transcriptome-wide N6-methyladenosine methylomes in the rat Barrett’s esophagus

city: Jinan

first: Ke

last: Zou

address: No. 105, Jiefang Road, Jinan, Shandong 250013, China

institute: Shandong university

summary: To investigate the mRNA m6A modification pattern in Barrett’s esophagus,we performed transcriptome-wide maps of m6A methylation，foreseeing a molecular mechanism of m6A-mediated gene in Barrett’s esophagus.

overall_design: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-sequencing (RNA-seq) for Barrett’s esophagus tissues and normal esophagus tissue.

first: Hui

last: Dong

first: Meng-Meng

last: Li

first: Ying

last: Zhang


title: MATRIN3 deficiency leads to cGAS-STING activation (PAR-CLIP)

city: Bethesda

first: Stefan

last: Muljo

state: MD

address: 10 Center Drive

institute: NIH, NIAID

summary: To explore the role of MATR3 as an RNA-binding protein, we performed Photoactivatable-Ribonucleotide-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) of MATR3 in HAP1 cells, together with a MATR3 KO negative control.

overall_design: Transcriptome binding profile analysis of two independent replicates of MATR3 PAR-CLIP, together with a MATR3 KO as a negative control

first: Zohir

last: Islam

first: Bryan

last: Chim

first: Amir

middle: K

last: Foroushani


first: Markus

last: Glaß

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Genome-wide transcriptional profiling of Bacillus subtilis in response to Hermetia illucens larvae extract

city: Dresden

first: Thorsten

last: Mascher

address: Zellescher Weg 20b

department: Institute of Microbiology

institute: Technische Universität Dresden

summary: The black soldier fly, Hermetia illucens, is a promising source for sustainable production of proteins, lipids and bioactive substances. Feeding on a variety of decomposing organic substrates which are typically inhabited by a range of different microorganisms like bacteria and fungi, H. illucens larvae is expected to be able to strongly produce antimicrobial peptides (AMPs) and other substances possessing antibiotic activity. It has been shown that H. illucens larvae extract exhibits antimicrobial activity against some microorganisms, such as Escherichia coli and Staphylococcus aureus, and antimicrobial effect on bacterial biofilm. Numbers of AMPs produced by H. illucens larvae have also been identified so far. Moreover, H. illucens larvae extract was recently observed with significant induction of cell envelop stress response (CESR) in B. subtilis, indicative of potential production of antimicrobial compounds. B. subtilis as a well-established Gram‑positive model strain is widely used in scientific research and an arguably robust platform to investigate antimicrobial compounds at the respect of, such as the mode of reaction of and the stress response elicited by corresponding compounds. In this study, we aimed to characterize the stress responses in B. subtilis induced by H. illucens larvae extract by employing transcriptomic study via RNA sequencing technique. With transcriptional profiling, we are expecting to be able to give some hints of potential antimicrobial compounds produced by H. illucens larvae by referring to the transcriptomic researches on well-studied antimicrobial substances.

overall_design: Wild type Bacillus subtilis W168 cells at exponential phase of growth were treated with H. illucens larvae extract for 10 min. Non-treatment samples were prepared under the same condition serving as controls. After that, all samples were processed to undergo total RNA extraction, cDNA library preparation and then high-throughput RNA sequencing. The resulting sequencing raw data were analyzed in silico to reveal the differential gene expression in the treatment relative to the non-treatment control.

first: Qian

first: Diana

last: Wolf


title: Transcriptomic study of Bacillus subtilis lacking seven extracytoplasmic function (ECF) σ factors

summary: Bacillus subtilis encodes seven extracytoplasmic function (ECF) σ factors, σM, σW, σX, σY, σZ, σV and σYlaC, that mediate response to cell envelop-active compounds. In this study, we aimed to define the transcriptomic differences between B. subtilis W168 wild type and the derivative 168 Δ7ECF mutant lacking seven ECF sigma factors under unstressed condition, which allows to identify the genes under control of these seven ECF sigma factors. 168 Δ7ECF mutant was as well treated with cell wall-targeting antibiotics, vancomycin and tunicamycin, in order to unravel the genes that respond to these two compounds, however are not under the seven ECF sigma regulon.

overall_design: B. subtilis W168 Δ7ECF mutant at exponential phase of growth were treated with cell envelop-targeting compounds, vancomycin and tunicamycin, respectively, or remained untreated. The wild type B. subtilis W168 untreated undergoing the same growth procedure was used as control. With 10 min of induction, all samples were processed to undergo total RNA extraction, cDNA library preparation and then high-throughput RNA sequencing. The resulting sequencing raw data were analyzed in silico to reveal the differential gene expression in different treatments relative to non-treatment samples and control samples.


title: Genome-wide Transcriptional Profiling of Bacillus subtilis in response to valinomycin and CCCP

summary: Valinomycin is a cyclic depsipeptide antibiotic produced by Streptomyces fulvissimus, and also known as K+ ion-selective ionophore, that has been studied and used in research for over half of century. The broad antimicrobial activity of valinomycin is reported as an effect of the disturbance of ionic equilibrium and transmembrane potential that may influence the whole organism and then cause fatal consequences. CCCP, carbonyl cyanide 3-chlorophenylhydrazone, is a member of the lipophilic weak acid class and acts essentially as an ionophore and can reduce the overall metabolic activity of the cell, causing severe defects of cell growth.  In this study, we investigated the global cellular response of Gram-positive model bacterium Bacillus subtilis upon exposure to valinomycin as well as CCCP by genome-wide transcriptional profiling using RNA sequencing technique, aimed to unveil the underlying regulatory network of the stress response.

overall_design: Wild type Bacillus subtilis W168 cells at exponential phase of growth were treated with cell membrane‑disturbing compounds, valinomycin and CCCP (carbonyl cyanide 3-chlorophenylhydrazone), respectively. With 10 min of induction, all samples were processed to undergo total RNA extraction, cDNA library preparation and then high-throughput RNA sequencing. The resulting sequencing raw data were analyzed in silico to reveal the differential gene expression in different treatments relative to non-treatment control.


title: Developing exocrine glands harbour a distinct macrophage population that is maintained by ILC2 derived GM-CSF [CD45 scRNA-seq]

first: Burkhard

last: Becher

summary: Granulocyte-macrophage colony-stimulating factor (GM-CSF) has a non-redundant role in the emergence and maintenance of alveolar macrophages (AMs). However, its role in steady-state myelopoiesis outside the lung is largely unexplored. We discovered a strong signal of GM-CSF production by type 2 innate lymphoid cells (ILC2s) in the developing salivary glands (SG). ILC2 derived GM-CSF fosters the development of a hitherto undescribed phagocyte subset, which we named adenophages. Detailed analysis focusing on phenotypic and transcriptional profiling revealed that adenophages represent a non-canonical macrophage subset displaying shared aspects of both, macrophages and dendritic cells. We found them to be homogenously distributed across the SG, but always forming a niche-triad with GM-CSF producing ILC2s and myoepithelial cells. Importantly, adenophages were present throughout all analyzed exocrine glands such as lacrimal glands and mammary glands, and were also identified in human SG, indicating a conserved role in exocrine glands across species.

overall_design: Cells from salivary glands of 20 day old mice were released by digestion. Total CD45+ cells were pre-selected using CD45 MicroBeads (Milteny Biotec, #130-052-301) on LS columns and further facs sort-purified for CD45+ cells. Each sample was pooled from two individual P20 mice.

first: Frederike

last: Westermann

first: Selma

last: Tuzlak

first: Victor

last: Kreiner


title: TNFR2 blockade promotes anti-tumoral immune response in PDAC by targeting activated Treg and reducing T cell exhaustion

city: Creteil

first: Caroline

last: Pilon

address: 51 avenue du Mal de Lattre de Tassigny

country: France

institute: INSERM U955 Eq21, Hopital Henri Mondor

summary: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, highly resistant to standard chemotherapy and immunotherapy. Regulatory T cells (Tregs) expressing TNFα receptor 2 (TNFR2) contribute to immunosuppression in PDAC. Treg infiltration correlates with poor survival and tumor progression in PDAC patients. We hypothesized that TNFR2 inhibition using a blocking monoclonal antibody (mAb) could shift the Treg-effector T cell balance in PDAC, thus enhancing anti-tumoral responses. In orthotopic and immunocompetent mouse models of PDAC, we described the immune environment of PDAC after immune cell sorting and single-cell analysis. By flow cytometry and single cell RNAseq analysis, we identified two Treg populations in orthotopic mouse models: resting and activated Tregs. The anti-TNFR2 mAb selectively targeted activated tumor-infiltrating Tregs, reducing T cell exhaustion markers in CD8+ T cells. However, anti-TNFR2 treatment alone had limited efficacy in activating CD8+ T cells and only slightly reduced the tumor growth.

overall_design: Tumor infiltrating T cells from PDAC bearing mice (control-untreated or anti-TNFR2 treated) were cell-sorted by flow cytometry and scRNA-seq and TCR VDJ analysis were done.

first: Wilfrid

last: Richer

first: José

last: Cohen

first: Ilaria

last: Cascone


title: Developing exocrine glands harbour a distinct macrophage population that is maintained by ILC2 derived GM-CSF [scRNA-seq]

overall_design: Cells from salivary glands of 20 day old mice were released by digestion. Nuclei were isolate ….


title: Identification of ALKBH5-interacting RNAs in lung cancer cells by RNA immunoprecipitation sequencing (RIP-Seq)

status: Public on Oct 27 2025

first: tianyu

last: tao

state: Guangdong

address: No.74, Zhongshan Er Road

department: Zhongshan School of Medicine

institute: Sun Yat-sen University

summary: This experiment was performed to identify the RNAs, particularly lncRNAs, that bind to the m6A demethylase ALKBH5 under nutrient stress.  Lysates from 95D cells cultured under glucose- or glutamine-deficient conditions were subjected to immunoprecipitation using an antibody against ALKBH5. Co-precipitated RNAs were isolated, used to construct sequencing libraries, and analyzed to identify enriched transcripts.

overall_design: Immunoprecipitation using an anti-ALKBH5 antibody compared to a total RNA input control. 95D cells were cultured under nutrient deprivation for 48 hours to induce the relevant cellular state.

first: Tianyu

last: Tao

first: Yi

last: Yang

first: Xianming

last: Feng

first: Bangdong

last: Liu

first: Junchao

last: Cai

first: Mengfeng

last: Li


title: Loss of METTL3 potentiates transposable element expression and drives inflammation-induced cell plasticity in the mammary epithelium

city: Boston

first: Xintao

last: Qiu

state: MA

address: 440 brookline ave

institute: Dana Faber Cancer Institute

overall_design: Refer to individual Series


title: Activin-A production by recruited alveolar macrophages exacerbates pulmonary fibrosis following acute lung injury

city: Shanghai

first: Jian

last: Xu

address: 180 Fenglin Road

institute: Zhongshan hospital

summary:
Background: Acute respiratory distress syndrome (ARDS) is a complicated pathological cascade process of excessive pulmonary inflammation and alveolar epithelial cells apoptosis which results in respiratory dysfunction and failure. Some ARDS can result in more severe state of pulmonary fibrosis, referred to as post-injury lung fibrosis. The mortality and incidence rate of ARDS are particularly high when it leads to continuing alveolar and interstitial fibrosis, which requires urgent treatment and appropriate management. Lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model has been widely implemented for studying ARDS in human. Here, we found alterations in the alveolar macrophage (AM) profile in such a mouse model. Specifically, activin-A produced by dominantly recruited AMs (recAMs), was noted to be implicated in the processing of post-injury lung fibrosis.
Methods: ALI animal model in C57BL/6 mice was established via 3.5mg/kg LPS intra-tracheal administration. Single-cell RNA (scRNA) sequencing was used for detailed classification and functional characterization of lung macrophages. in vivo experiments, we evaluated the role that activin-A plays in post-injury lung fibrosis in an ALI mouse model using ELISA (Enzyme-linked immunosorbent assay), histological staining methods and immunofluorescence. in vitro experiments, we analyzed the effect of activin-A on MLE-12 cells and bone marrow-derived macrophages (BMDMs) using western blot (WB), qPCR (quantitative real-time PCR), RNA-seq (RNA sequencing) and immunofluorescence.
Results: Our findings revealed that recAMs replaced tissue resident alveolar macrophages (TRAMs) as a dominant macrophage population in the setting of ALI. The result of GO (gene ontology) analysis suggested that activin-A was associated with wound healing and SMAD (suppressor of mothers against decapentaplegic) protein signaling pathways. Immunofluorescence results revealed that the receptor of activin-A mainly localized to alveolar epithelial cells and macrophages. Subsequently, activin-A was specifically found to drive MLE-12 to mesenchymal cell transformation via the TGF-β (transforming growth factor-β)/SMAD signaling. Moreover, the results of transcriptome analysis and WB confirmed that activin-A could enhance the concerted activity of Hippo and TGF-β/SMAD pathways in BMDMs, leading to an increased expression of pro-fibrotic mediator. At the same time, yes-associated protein (YAP) and transcriptional coactivated with PDZ-binding motif (TAZ) proteins were found to drive BMDM activin-A expression, which could generate a positive feedback mechanism that perpetuates fibrosis.
Conclusion: Our findings revealed that activin-A is involved in the pathological mechanisms in post-injury lung fibrosis by promoting EMT (epithelial–mesenchymal transition) and the formation of an underlying profibrotic positive feedback loop in recAMs. Activin-A is thus a potential therapeutic target for developing ALI and ALI-associated pulmonary fibrosis therapeutics.

overall_design: Mouse lung tissue samples from two ALI and control mice each were minced and digested by collagenase II and trypsin for single cell suspensions.

first: Ting

last: Pan


title: Early female germline development in Xenopus laevis: Stem cells, nurse cells and germline cysts

city: Baltimore

first: Asya

last: Davidian

state: MARYLAND

address: 3520 San Martin Dr

department: Embryology

institute: Carnegie Institution for Science

summary: In Drosophila, germline cysts arise through synchronous mitotic divisions and acquire a polarized architecture organized by the fusome, which guides oocyte specification and supports meiotic progression. Similar cyst structures exist in non-mammalian vertebrate ovaries, but their polarity and function have remained uncertain. Using single-cell RNA sequencing and high-resolution imaging, we reconstructed the germ cell differentiation trajectory in Xenopus laevis and uncovered striking parallels with invertebrate and mouse cyst development. We identified a distinct germline stem cell (GSC) population marked by piwil4, low translational activity, and expression of neuronal-specific and transposon-silencing genes. Downstream from GSCs, during cyst development, an asymmetric fusome-like structure (FLS) composed of stable microtubules forms a rosette-like connection between cystocytes and co-localizes with Golgi vesicles and ER, suggesting polarized trafficking. In contrast to previous claims, ∼80% of EdU-prelabeled cyst cells turned over rather than forming oocytes, consistent with a nurse cell fate. The striking parallels described here between cyst and fusome formation, polarization, cyst breakdown, and nurse cell turn over to produce relatively few oocytes, argue that amphibian cysts have important functions in female gametogenesis.

overall_design: We performed single-cell RNA sequencing on juvenile Xenopus laevis ovaries collected at developmental stages NF62–66 to characterize germline populations during early ovarian development. Dissected ovaries were enzymatically dissociated, and approximately 10,000 viable cells per sample were loaded onto the 10X Genomics Chromium platform. Libraries were generated from three biological replicates and sequenced on an Illumina NextSeq 500. Raw reads were processed using Cell Ranger v8.0.1, and the resulting count matrices were used for downstream analysis.

first: Allan

last: Spradling


title: Identification of DAMER-interacting RNAs by RNA-RNA pulldown assay and sequencing [RNA-Seq]

summary: This experiment aimed to identify all transcripts that directly interact with the lncRNA DAMER. Full-length DAMER was transcribed in vitro and biotinylated at the 3' end. The biotinylated DAMER was used as a probe to capture interacting RNAs from 95D cell lysates. The captured RNAs were then eluted and subjected to high-throughput sequencing to identify transcripts specifically enriched by DAMER.

overall_design: 95D cells were cultured under nutrient deprivation for 48 hours to induce the relevant cellular state. Full-length DAMER was transcribed in vitro and biotinylated at the 3' end. The biotinylated DAMER was used as a probe to capture interacting RNAs from 95D cell lysates. The captured RNAs were then eluted and subjected to high-throughput sequencing.


title: EZH2 directs breast cancer progression through the modulation of epithelial cellular plasticity [ChIP-Seq]

city: Montreal

first: William

last: Muller

state: Quebec

address: 1160 Av des Pins O

department: Biochemistry

country: Canada

institute: McGill University

summary: Breast cancer remains a leading cause of death among women, encompassing diverse molecular subtypes with distinct clinical outcomes. The HER2+ subtype is particularly aggressive, with many patients eventually developing resistance to HER2-targeted therapies, underscoring the urgent need for alternative treatment strategies. Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of Polycomb Repressive Complex 2, represses the expression of genetic programs crucial for differentiation, control of proliferation, and apoptosis through histone H3 lysine 27 trimethylation (H3K27me3). To investigate the role of EZH2 in HER2+ tumor progression, we crossed a genetically engineered mouse model (GEMM) of HER2-driven breast cancer with a conditional Ezh2 knockout strain. The results showed that Ezh2 is essential for accelerating tumor initiation and metastatic dissemination in HER2-driven breast cancer. Combined bulk and single cell RNA sequencing analyses revealed a significant downregulation of basal cell populations in the absence of Ezh2, characterized by the loss of Tp63 expression, and an upregulation of luminal progenitor cell populations, driven by crucial transcription factors such as Esr1, thereby driving luminal lineage commitment. Further, inhibition of EZH2 in vitro resulted in increased expression of ER in HER2+ human breast cancer cell lines and rendered them susceptible to tamoxifen. Altogether, these findings demonstrate that EZH2 dictates breast cancer plasticity through control of cellular identity and provides rationale for combining EZH2 inhibitors with endocrine therapies to improve outcomes in HER2+ breast cancer.

overall_design:
1) ChIP-seq was performed on wild-type EIC/EZH2 and EIC/EZH2 knockout tumor cells, collected at endpoint from mice; two biological replicates were included per group.
2) ChIP-seq was performed on SKBR3 cell lines treated with either DMSO or EPZ6438 for 7 days; two technical replicates were included per condition.

first: Linshan

first: Ellie

last: Massey

first: Dongmei

last: Zuo

first: Alain

last: Pacis

first: Mert

last: Demirdizen

first: Jessica

last: Cinkornpumin

first: Yu

last: Gu

first: Elizabeth

last: Podleszanski

first: Hailey

last: Proud

first: Virginie

last: Sanguin-Gendreau

first: Vasilios

last: Papavasiliou

first: Zhengze

last: Jiang

first: Harvey W.

last: Smith

last: Pastor

first: Paolo

last: Ceppi

first: William J.


title: Single cell RNA sequencing analysis of native fat tissue, RMF pallets, and expanded ADSCs

first: Rulin

last: Huang

state: Shanghai

address: No.639, Manufacturing Bureau Road

department: Department of plastic and reconstructive surgery

institute: Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine

summary:
Objectives: 1. To explore cell population changes after a 4-week in vitro self-reaggregation culture, comparing RMF pellets, native fat tissue, and expanded ADSCs.
2. To analysis the cellular composition and maturity of RMF-islet organoids before and post transplantation.
Methods: Sc-Seq was performed on cells from RMF pellets (n = 3), native fat (n = 3), expanded ADSCs (n = 3), in vitro RMF-islet organoids (n = 2), and in vivo RMF-islet organoid grafts (n = 2) using the 10× Genomics platform.
Results: A total of 65501 cells were clustered from the RMF pellets, native fat, and expanded ADSC samples and visualized using UMAP. Cells were categorized into main cell types based on their spatial distribution on the UMAP plots: adipose-derived stem and progenitor cell (ASPC) clusters (ASPC-1, ASPC-2, ASPC-3, and ASPC-4), preadipocyte cluster, endothelial cell cluster, smooth muscle cell cluster (SMC), and immune cell cluster. Following a 4-week in vitro culture, the percentage of ASPCs increased from 24.57% in native fat to 97.17% in RMF pellets and further to 99.9% in expanded ADSCs. Pseudotime analyses the ASPC-1 cluster, predominantly derived from native fat samples (96.04%), occupied the initial position on the developmental trajectory, followed by ASPC-2 and ASPC-3 clusters primarily originating from RMF pellet samples (94.91% and 96.55%, respectively). Conversely, the ASPC-4 cluster mainly derived from expanded ADSC samples were positioned at the terminal end of the trajectory. Furthermore, pseudotime values indicated that both ASPC-2 and ASPC-3 clusters from RMF pellet samples exhibited an intermediate differentiation state between those observed in native fat and expanded ADSC samples.
For RMF-islet organoids, a total of 8715 cells and 5328 cells were analyzied from the in vitro and in vivo RMF-islet organoid samples, respectively. Compared these data with published transcriptomes of human islet cells, we identified four distinct populations of pancreatic endocrine cells, including β-, α-, δ-, or γ-like cells, within the two in vitro RMF-islet organoid samples and the presence of all four major pancreatic endocrine cell types within the organoid grafts and displayed a similar cellular composition before and after transplantation.
Conclusion: 1. ASPC clusters from RMF pellet samples possess an intermediary differentiated phenotype compared to their counterparts derived from native fat and expanded ADSC samples.
2. The RMF-islet organoids harbor a population of functionally matured β-like cells exhibiting transcriptional similarities to native human islet β-cells.

overall_design:
We performed single-cell RNA sequencing (sc-Seq) on cells derived from RMF pellets (4 mm), native adipose tissue, and expanded ADSCs using the 10× Genomics platform (n = 3). A total of 84,993 cells were analyzed, clustered based on the expression of cell-type-specific marker genes, and visualized via UMAP.

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Submitters state "all relevant processed data to the study are included in the article. However, due to Chinese policy on human genome data, the raw data for single-cell RNA sequencing reported in this publication can only be accessed under the Genome Sequence Archive (https://ngdc.cncb.ac.cn) after the uploading process has finished."
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first: Ru-Lin

first: Jing

last: Yang

first: Xiya

last: Yin

first: Kang

last: Zhang

first: Qingfeng

last: Li


title: Deletion of Ptpn2 in B cells contributes to development of autoimmunity

city: Aurora

first: Kristen

last: Wells

state: CO

address: 1775 Aurora Ct,

department: Barbara Davis Center

institute: University of Colorado Anschutz

summary: Autoimmunity arises when self-reactive B and T cells target the body’s own tissues, with B cells contributing through antigen presentation to T cells and production of autoantibodies and pro-inflammatory cytokines. Genome wide association studies (GWAS) have suggested that single nucleotide polymorphisms (SNPs) in protein tyrosine phosphatase non-receptor type 2 (PTPN2), which is ubiquitously expressed throughout the body, are associated with development of autoimmune diseases, such as rheumatoid arthritis (RA), Crohn’s disease, and type 1 diabetes (T1D). While prior studies have focused on the role of Ptpn2 in T cells and other cell types, its function in B cells remains unknown. To test the B cell-intrinsic role of Ptpn2, we generated a B cell-specific deletion of Ptpn2 in mice (Mb1-Cre;Ptpn2fl/fl). We found that loss of Ptpn2 in B cells promotes organ inflammation, increases the frequency of autoimmune-associated B cells (ABCs) and plasmablasts in the periphery, and increases autoantibody levels in the serum. Moreover, we found that Ptpn2 acts as a negative regulator of the JAK/STAT and TLR7 pathways in B cells. In line with this, treatment of B cells from Mb1-Cre;PTPN2fl/fl mice with IFNy and TLR7 agonist leads to enhanced differentiation into ABCs. These findings highlight the critical role of Ptpn2 in B cell function and its potential as a key regulator in preventing B cell associated autoimmunity.

overall_design: B cells were isolated from spleen from 5 control and 6 PTPN2 B cell knock out mice.

first: Bridget

last: Alexander

first: Soojin

last: Kim

first: Kevin

middle: P

last: Toole

first: Maya

middle: J

last: Hundter

first: Scott

last: Wemlinger

first: Daniel

last: Regan

first: Andrew

last: Getahun

first: Mia


title: Patient-derived Induced Pluripotent Stem Cells with a C9orf72 Expansion as a Model to Study Frontotemporal Dementia Pathologies

city: San Francisco

first: Sonia

last: Infante

state: CA

address: 513 Parnassus Ave

institute: University of California San Francisco

summary: The neurodegenerative disorder Frontotemporal Dementia (FTD) can be caused by a repeat expansion (GGGGCC; G4C2) in C9orf72. The function of wild-type C9orf72 and the mechanism by which the C9orf72-G4C2 mutation alteration causes FTD, however, remain unresolved. Diverse disease models including human brain samples and differentiated neurons from patient-derived induced pluripotent stem cells (iPSCs) identified some hallmarks associated with FTD, but these models have limitations, including biopsies capturing only a static snapshot of dynamic processes and differentiated neurons being labor-intensive, costly, and post-mitotic. We find that patient-derived iPSCs, without being differentiated into neurons, exhibit established FTD hallmarks, including increased lysosome pH, decreased lysosomal cathepsin activity, cytosolic TDP-43 proteinopathy, and increased nuclear TFEB. Moreover, lowering lysosome pH in FTD iPSCs mitigates TDP-43 proteinopathy, suggesting a key role for lysosome dysfunction. RNA-seq reveals dysregulated transcripts in FTD iPSCs affecting calcium signaling, cell death, synaptic function, and neuronal development. We confirm differences in protein expression for some dysregulated genes not previously linked to FTD, including CNTFR (neuronal survival), Annexin A2 (anti-apoptotic), NANOG (neuronal development), and moesin (cytoskeletal dynamics). Our findings underscore the potential of FTD iPSCs as a model for studying FTD cellular pathology and for drug screening to identify therapeutics

overall_design: Comparative gene expression profiling has been conducted using RNA-seq data from two sets of iPSCs: one derived from a healthy 64-year-old male (WT) and the other from a 48-year-old male affected by frontotemporal dementia with a C9orf72 alteration (FTD).

last: Infante-Tadeo

first: Diane

last: Barber


title: Effects of dexamethasone stimulation on gene expression in human trabecular meshwork cells

first: Yang

address: 197 Ruijin Er Road

institute: Ruijin Hospital Affiliated Medical School, Shanghai Jiaotong University

summary: The changes in the trabecular meshwork in steroid-induced glaucoma are similar to those in human primary open-angle glaucoma. To explore the changes in the trabecular meshwork in POAG, we extracted RNA from human trabecular meshwork cells with or without dexamethasone, followed by next-generation transcriptome sequencing to observe changes in gene expression in trabecular meshwork cells, thereby better understanding the mechanism of increased IOP.

overall_design: To explore the changes in the trabecular meshwork in glaucomatous eyes, we performed next-generation sequencing on human trabecular meshwork cells stimulated with vehicle DMSO and human trabecular meshwork cells stimulated with 100 nM dexamethasone.

first: Ruiqi

last: Han

first: Shushu

last: Xu

first: Bingqiao

last: Shen

first: Huan

last: Yu

first: Junjue

last: chen

first: Huiping

last: Yao

first: Shouyue

first: Yisheng

last: Zhong


title: Extracellular vesicles from obese visceral adipose promote pancreatic cancer development and resistance to immune checkpoint blockade therapy [scRNA-seq]

city: Guangzhou

first: Zhou

last: yifan

address: 74 Zhongshan Second Road

institute: Sun Yat-sen University Cancer Center

summary: Obesity is correlated with multitype of cancer development, and pancreatic ductal adenocarcinoma (PDAC) in obese patients often shows dismal prognosis and resistance to immune checkpoint blockade (ICB) therapy. The molecular mechanism for these phenomena is largely unknown. Here we show that obese visceral adipose tissues (VAT) can talk to distant PDAC by delivering their extracellular vesicle (EV) carrying signal molecules. We reveal that PDAC cells can take in VAT-EVs to their lysosomes where EVs-delivered Cathepsin A (Ctsa) stabilize ribonuclease Rnaset2b to produce extra pseudouridines from RNA cleavage. Pseudouridines released from cancer cells activate mast cells which inhibit CD8+ T cell activity, forming an immunosuppressive tumor microenvironment  which enhances cancer progression and resistance to ICB. We also demonstrate the harmful effects of VAT-EV CTSA-pseudouridine-mast cell axis for PDAC in obese patients. Animal experiments indicate that Ctsa knockdown effectively enhances the ICB efficacy on PDAC. Our study uncovers a mechanism connecting obesity and cancer which holds promise for developing new therapeutic strategy for obesity related cancers.

overall_design: To evaluate the effects of Obese VAT-EVs on tumor microenvironment, we performed scRNA sequencing of tumor tissue from the KPC mice with Normal VAT-EVs, Obese VAT-EVs and Ctsa KD obese VAT-EVs treatment.

first: Jian

last: Zheng

first: Chunling

last: Xue


title: Small molecule inhibition rescues the skeletal dysplasia phenotype of Trpv4 mutant mice

city: Los Angeles

first: Jennifer

last: Zieba

address: 615 Charles E. Young Dr.

institute: University of California, Los Angeles

summary: The TRPV4 skeletal disorders are characterized by short stature and scoliosis but the cellular mechanisms behind this phenotype are unclear. We isolated tibial and femoral cartilage growth plate chondrocytes from postnatal day 3 wild type and Col2a1-Cre driven Trpv4 p.R594H conditional knock-in mice, which models the phenotype, and analyzed differential expression patterns using single cell RNA-seq.

overall_design: Growth plate chondrocytes from wild type and Col2a1-Cre induced Trpv4 p.R594H conditional knock-in mice were isolated from femoral and tibial cartilage tissue at postnatal day 3 and analyzed using scRNAseq.

first: Lisette

last: Nevarez

first: Taylor

last: Ismaili

first: Jorge

last: Martin

first: Derick

last: Diaz

middle: H

first: Jocelyn

last: Ramirez

first: David

last: Goldstein

first: Sunil

last: Sahdeo

first: Deborah

last: Krakow

first: Daniel

last: Cohn


title: Regulated TRESLIN-MTBP loading governs initiation zones and replication timing in human DNA replication [ChIP-Seq]

city: Mishima

first: Masato

last: Kanemaki

state: Shizuoka

address: Yata 1111

department: Department of Chromosome Science

institute: National Institute of Genetics

summary: Replication origins in human cells form clusters ranging from tens to hundreds of kilobases called initiation zones (IZs), typically located in intergenic regions between active genes. On a larger megabase scale, chromosomes replicate in a temporally defined replication timing (RT) pattern, where euchromatic regions replicate early, and heterochromatic regions replicate late. Although the stochastic model of IZ firing with a temporally regulated limiting factor can explain RT formation, this limiting factor in human cells remains unclear. To investigate the relationship between IZ and RT, we mapped the temporal firing pattern of IZs and examined the genome-wide distributions of replication licensing and firing factors in human cells. We identified TRESLIN-MTBP as the key limiting firing factor for replication initiation. Its loading onto phosphorylated MCM2–7 double hexamer (MCM-DH) is controlled by the opposing phosphorylation events on MCM-DH by Dbf4-dependent kinase and RIF1-Protein Phosphatase 1, which ultimately determine IZs and establish RT.

overall_design: Proteins of interest were CRISPR-edited and tagged with 3xFLAG. All ChIP-seq experiments were conducted using anti-FLAG M2 antibody. Control experiments were performed using cell line without any FLAG-tagged proteins. Cells with different genetic backgrounds were used to explore target enrichments at different conditions.


title: Effects of Normoxia, Hypoxia, Oscillatory Hypoxia, and Lactate on Wild-Type, NOTCH1-Knockdown, and TP53-Knockdown MDA-MB-231 Cells

city: Farmington

first: Xihua

last: Qiu

state: CT

address: 263 Farmington Ave

department: Biomedical Engineering

institute: University of Connecticut

summary: Wild-type, NOTCH1-knockdown, and TP53-knockdown MDA-MB-231 cells were cultured under normoxia, hypoxia, oscillatory hypoxia, and lactate treatment, and subjected to RNA sequencing to assess transcriptional changes.

overall_design: Three replicates were collected for each condition (normoxia, hypoxia, oscillatory hypoxia, and lactate treatment) in wild-type, NOTCH1-knockdown, and TP53-knockdown MDA-MB-231 cells.

last: Kshitiz

first: Liu

last: Yamin

first: Suhail

last: Yasir

first: Qiu

last: Xihua


title: Digital transformation of herbal medicine: Conversion to biological entity data using digestive herbal medicine-induced transcriptome sequencing_second_SW1783

first: Musun

last: Park

address: 1672 Yuseong-daero, Yuseong-gu

institute: Korean Institute of Oriental Medicine

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


title: Inhibition of CDK10 triggers nucleic acid sensors-mediated anti-tumor immunity [scRNA-Seq]

city: wuhan

first: Gaoshan

last: Xu

state: -- Please Select --

address: wuhan

institute: wuhan university

summary: Cdk10 knockout significantly reduced CT26 tumor growth in immunocompetent BALB/c WT mice. To investigate how Cdk10 influences the tumor immune microenvironment in vivo, we performed single-cell RNA sequencing (scRNA-seq) on WT and Cdk10 KO CT26 tumors derived from immunocompetent BALB/c mice.

overall_design: We subcutaneously inoculated sgControl and sgCdk10 CT26 cells into immunocompetent BALB/c WT mice,and single-cell sequencing was performed


title: Digital transformation of herbal medicine: Conversion to biological entity data using digestive herbal medicine-induced transcriptome sequencing_second_HT29

city: Daejeon


title: Digital transformation of herbal medicine: Conversion to biological entity data using digestive herbal medicine-induced transcriptome sequencing_second_A549


title: Nutrient stress diverts RRN3 from rRNA transcription to alternative polyadenylation of autophagy mRNAs in ovarian cancer [PAR-CLIP]

city: Tianjin

first: Jianying

last: Lv

state: Tianjin

address: weijin Road No.94

department: School of medicine

institute: Nankai University

summary: Stress-induced alternative processing of mRNA is emerging as an essential mechanism to drive almost every hallmark of cancer. Through a genome-wide screening based on an abnormal transcriptional readthrough event favoring the malignant progression of ovarian carcinoma (OC), we identified novel mRNA processing regulators including RRN3, an essential factor for the transcriptional initiation of rRNA. The long-read RNA sequencing and PAR-CLIP analyses revealed that RRN3 was involved in the usage of alternative polyadenylation (APA) sites, resulting in the altered stability of autophagy-related mRNAs. More interestingly, we discovered that nutrient-deprivation-induced phosphorylation of RRN3 at serine 199 was sufficient to divert RRN3 out of the nucleolus to the nuclear plasma, where RRN3 regulated the APA of autophagy mRNAs, such as OPTN, to enhance their stability and eventually promoted autophagy. Further in vivo experiments showed that nutrient-stress-triggered switch of RRN3 from rRNA transcription to APA regulation was essential for the growth and dissemination of OC in mice.

overall_design: V5-RRN3-expressing SK-OV-3 cells were grown in its complete medium supplemented with 100 μM 4-thiouridine (4-SU) for 14 h before crosslinking.Ten percent of cleared cell lysates were used as the input and the rest were subjected to immunoprecipitation with anti-V5 antibody (Thermo-Fisher Scientific), followed by the elution with V5 peptide (MedChemExpress, Shanghai, China).


title: Endothelial SRSF1 directly regulates ATF3-KLF2 pathway to promote ischemia-induced angiogenesis [ALP_RNA-seq]

city: Beijing

first: Wenting

last: Zhu

address: No.95 Yong'an Road, Xicheng District

institute: Beijing Friendship Hospital, Capital Medical University

summary: Mechanistically, SRSF1 modulated ATF3 alternative splicing, wherein SRSF1 directly bound to ATF3 pre-mRNA and elevated full-length ATF3 transcript at the expense of truncated ATF3Δzip2 transcript, and in turn, ATF3 directly bound to the KLF2 promoter and suppressed KLF2-S1PR1 signaling, through which mediated the SRSF1-promoted endothelial tube formation and angiogenesis. Additionally, alprostadil, the prostaglandin E1 analog, could activate the SRSF1 signaling to improve endothelial angiogenesis in vitro and in vivo. Our findings identified SRSF1 as a novel regulator of ischemia-induced angiogenesis that enhances endothelial angiogenic functions by regulating the ATF3-KLF2/SIPR1 pathway. These results suggest that modulation of endothelial SRSF1 may represent a promising therapeutic approach for treating ischemic vascular diseases.

overall_design: To investigate the mechanisms contributing to alprostadil-promoted SRSF1 upregulation in HUVECs, we treated HUVECs with alprostadil (50 nM) for 24 h, while using DMSO-treated HUVECs as controls. We then performed gene expression profiling analysis using data obtained from RNA-seq of HUVECs treated with alprostadil and DMSO.

first: Ning

last: Xie

first: Chun-Mei

last: Cao


title: Multidimensional analysis reveals innate and adaptive immune features associated with toxicities in patients receiving checkpoint inhibitor therapy

city: dallas

first: Shaheen

last: Khan

state: texas

address: 5323 Harry Hines Blvd

institute: UT Southwestern Medical Center

summary: Analysis of 162 patients revealed that individuals who developed clinically significant irAEs exhibited a baseline proinflammatory, autoimmune-like state characterized by a significantly higher abundance of CD57+ T and natural killer (NK) T cells, plasmablasts, proliferating and activated CXCR3+ lymphocytes, CD8+ effector and terminal effector memory T cells, along with reduced NK cells and elevated plasma ANA levels. In irAE cases, we identified distinct baseline proinflammatory gene signatures, including markedly higher expression of IL1B and CXCL8 in monocytes and CXCR3, TNF, and IFNG in T/NK cells. TNF signaling was the most enriched pathway, while immunosuppressive genes SIGLEC7 and CXCR4 were downregulated. Following ICI initiation, these patients exhibited an enhanced shift toward an activated and inflammatory immune phenotype, including monocyte reprogramming characterized by upregulation of IL18 and elevated gene expression levels of CXCL10. Conversely, post-treatment levels of CXCL8 were decreased in irAE patients. Notably, in patients who did not develop clinically significant irAE, we identified increased baseline abundance of a TGFBIhigh myeloid cluster enriched in immunosuppressive markers such as STAB1. In addition, patients without irAE exhibited upregulation of TNF and AIRE, accompanied by distinct myeloid protumorigenic reprogramming.

overall_design: We used single-cell RNA sequencing to assess PBMCs from cancer patients receiving immune checkpoint therapy at baseline and posttherapy

last: Gerber


title: Targeting IGF2BP1 Enhances Immune Checkpoint Therapy in High-grade Serous Ovarian Cancer [small RNA-seq]

city: Halle (Saale)

first: Danny

last: Misiak

address: Kurt-Mothes-Str. 3a

institute: Martin Luther University Halle-Wittenberg

summary: High-grade serous ovarian cancer (HGSC) accounts for the vast majority (>70%) of ovarian cancer-associated deaths with minor therapeutic improvements. Among four proposed molecular subtypes of HGSC, the C5 subtype is distinguished by high proliferative potential and elevated immune evasion which is indicated by an unfavorable MHCI/PD-L1 ratio. However, the underlying key drivers of the C5 subtype remained elusive. Here we identify oncofetal RNA-binding proteins (RBPs) promoting the immune evasion of C5-HGSC. The IGF2 mRNA binding protein 1 (IGF2BP1) enhances expression of the E3 ligase MDM2, which induces degradation of IRF1 resulting in reduced MHCI presentation. Concomitantly, IGF2BP1 elevates PD-L1 synthesis by impairing its microRNA-directed silencing. This shifts the intra-tumoral MHCI/PD-L1 ratio, limits immune cell infiltration, and promotes evasion of tumor cells from cytotoxic T cells (CTLs) in human and syngeneic mouse models of ovarian cancer. The small molecule inhibitor BTYNB impairs IGF2BP1-directed regulation of MHCI/PD-L1 ratios, promotes CTL-directed killing as well as activation, and strongly synergizes with immune checkpoint inhibition (ICI) by Nivolumab in vitro and in vivo.

overall_design: MiTRAP experiments using 3´UTR of PD-L1 or MS2 control RNA were essentially performed as described recently (Busch et al. 2016). MBP-MS2BP (maltose-binding protein – MS2 binding protein) was coupled to amylose resin. Bait RNA was in vitro transcribed form linearized templates containing MS2 loops and coupled to the pre-coupled MBP-MS2BP. Cell-free extracts of 5x106 ES-2 cells per condition generated in binding buffer (20mM Tris, pH 7.5, 150mM NaCl, 1.5mM MgCl2, 8.6% glycerol and 0.05% NP40) supplemented with protease inhibitor cocktail (1:200; Sigma Aldrich) were cleared by centrifugation and incubated with bait-coupled resin and supplements (11 mg/ml heparin, 1mM DTT and 400 U/ml RNAsin (Promega)) for 30 min at room temperature. After three washing steps with heparin-supplemented binding buffer, protein–RNA complexes were eluted twice in 150 μl binding buffer supplemented with 15mM maltose. Protein enrichment was tested by Western blotting. To identify associated microRNAs, RNA was isolated by phenol-chloroform extraction and quality checked by a Bioanalyzer.  Library preparation and single-end short-read RNA sequencing was performed on Illumina HiSeq 1500 platform at Novogene (Hong Kong).

first: Nadine

last: Bley

first: Alexander

last: Rausch


title: Single cell transcriptomes of genetic landscape from actinic keratosis to cutaneous squamous cell carcinoma

first: Tao

address: Fengyang road

department: dermatology

institute: Changzheng hospital

summary: Here, we performed scRNA-seq to compare the transcriptomes of individual cells from three groups: (1) healthy sun-exposed skin; (2) AK samples; (3) cSCC samples. A total of 81792 cells from 8 AK samples, 3 cSCC samples and 3 control skin tissue were characterized, which comprising representatives from each of the major dermal cell types. Our results demonstrate the heterogeneity within tissues/patients and corroborated the effect of the tumor environment during carcinogenesis, especially immune cell populations. Additionally, we identified a list of gene expression changes that affected several known cancerous pathways, with potential evolution trajectory from a subset of AK premalignant cells to cSCC cells. Our findings will improve current understanding about the mechanism of AK-cSCC progression, and are potentially valuable in providing novel precautionary/ therapeutic markers.

overall_design: Single-cell sequencing has been performed on three kinds of human skin samples: normal sun-exposed skin, actinic keratosis and cutaneous squamous cell carcinoma samples.

first: Chen

last: Jianghan

first: Wang

last: Zhongzhi

first: Xu

last: Tao

first: Li

last: Shengli


title: Transcriptome profiling of 95D cells in response to glucose or glutamine deprivation.

first: tianyu

last: tao

address: No.74, Zhongshan Er Road

institute: Sun Yat-sen University

summary: This experiment was designed to identify genes and long non-coding RNAs transcriptionally upregulated in response to metabolic stress. Human lung cancer cell line 95D was cultured in complete medium, glucose-free medium, or glutamine-free medium. Total RNA was then extracted for library preparation and sequencing to compare the transcriptomic profiles across these conditions. The data were used to identify robustly induced genes under nutrient stress.

overall_design: 95D cells were subjected to three different nutrient conditions for 48 hours: (1) Control (complete DMEM), (2) Glucose deprivation (-Glc), and (3) Glutamine deprivation (-Gln).

first: Tianyu

first: Yi

last: Yang

first: Xianming

last: Feng

first: Bangdong

last: Liu

first: Junchao

last: Cai

first: Mengfeng

last: Li


title: Probing the structure of the lncRNA DAMER and its interaction with ATF4 mRNA using SHAPE-MaP

summary: This experiment was conducted to obtain direct structural evidence for the base-pairing interaction between DAMER and ATF4 mRNA. In vitro transcribed RNAs (DAMER-WT, ATF4 mRNA, DAMER mutants) were folded either alone or together and subsequently modified with the SHAPE reagent 1-methyl-7-nitroisatoic anhydride (1M7). A DMSO-only reaction served as the negative control. Sites of modification, which indicate flexible nucleotides, were identified as mutations during reverse transcription. The resulting cDNA was used for library construction and deep sequencing. The data were processed using ShapeMapper2 to calculate reactivity profiles and model RNA secondary structures.

overall_design: In vitro transcribed RNA: DAMER (wild-type and mutants ΔA4B, ΔA5B) and ATF4 mRNA 5' UTR. RNA samples were prepared under different conditions: (1) DAMER alone, (2) ATF4 mRNA alone, (3) DAMER and ATF4 mRNA co-incubated. Each condition was treated with either the SHAPE reagent 1M7 or DMSO as a negative control.


title: Nutrient stress diverts RRN3 from rRNA transcription to alternative polyadenylation of autophagy mRNAs in ovarian cancer

first: Jianying

last: Lv

overall_design: RNA polymerase II ChIP-seq  in SK-OV-3 cells(shLacZ/shRRN3) to investigate the influence of RRN3 on Pol II transcription process.


title: A catecholamine-independent pathway controlling adaptive adipocyte lipolysis

city: Saint Louis

first: Erica

last: Scheller

state: MO

address: 660 S Euclid Ave

institute: Washington University

summary: Several adipose depots, including constitutive bone marrow adipose tissue (cBMAT), resist conventional lipolytic cues. However, under starvation, wasting, or cachexia, the body eventually catabolizes stable adipocytes through unknown mechanisms. Here, we developed a mouse model of brain-evoked depletion of all fat, including stable cBMAT, independent of food intake, to study this phenomenon. Genetic, surgical, and chemical approaches demonstrated that catabolism of stable adipocytes required adipose triglyceride lipase-dependent lipolysis but was independent of local nerves, the sympathetic nervous system, and catecholamines. Instead, concurrent hypoglycemia and hypoinsulinemia activated a potent catabolic state by suppressing lipid storage and increasing catecholamine-independent lipolysis via downregulation of cell-autonomous lipolytic inhibitors including G0s2. This was also sufficient to delipidate classical adipose depots and was recapitulated in tumor-associated cachexic mice. Overall, this defines unique adaptations of stable adipocytes to resist lipolysis in healthy states while isolating a potent catecholamine-independent neurosystemic pathway by which the body can rapidly catabolize all adipose tissues.

overall_design: To identify candidate mechanisms of stable fat lipolysis, we performed RNAseq on caudal vertebrae (CV, stable cBMAT-enriched) from 4- to 5-month-old male and female BMAd-Pnpla2+/+ (WT) mice treated for 9-days with either ICV PBS or 100 ng/hr ICV leptin. Inguinal white adipose tissue (iWAT, responsive fat control) and lumbar vertebrae (LV, no fat control) from the same WT females were included to identify cBMAT-enriched genes. CV samples from age- and sex-matched BMAd-Pnpla2-/- (cKO) mice were also included to isolate the effect of ATGL-mediated fat depletion. To determine which of the differentially expressed lipolysis genes by ICV leptin treatment were reversed with insulin supplementation in vivo, we also included CV samples from age-matched WT males treated with either ICV PBS or 100 ng/hr leptin along with subcutaneous insulin for 9-days.

first: Xiao

last: Zhang

first: Sreejith

middle: S

last: Panicker

first: Jordan

last: Bollinger

first: Anurag

last: Majumdar

first: Rami

last: Kheireddine

first: Lila

last: Dabill

first: Clara

last: Kim

first: Brian

last: Kleiboeker

first: Fengrui

first: Yongbin

last: Chen

first: Kristann

last: Magee

last: Learman

first: Adam

last: Kepecs

first: Gretchen

last: Meyer

first: Jun

first: Steven

last: Thomas

first: Irfan

last: Lodhi

first: Ormond

last: MacDougald


title: DNMT3A:R882H Is Not Required for Disease Maintenance in Primary Human AML, but Is Associated With Increased Leukemia Stem Cell Frequency [scRNA-Seq]

city: Stanford

first: Thomas

last: Koehnke

address: 265 Campus Drive

institute: Stanford University

summary: Genetic mutations are being thoroughly mapped in human cancers, yet a fundamental question in cancer biology is whether such mutations are functionally required for cancer initiation, maintenance of established cancer, or both. Here, we study this question in the context of human acute myeloid leukemia (AML), where DNMT3A:R882 missense mutations often arise early, in pre-leukemic clonal hematopoiesis, and corrupt the DNA methylation landscape to initiate leukemia. We developed CRISPR-based methods to directly correct DNMT3A:R882 mutations in leukemic cells obtained from patients. Surprisingly, DNMT3A:R882 mutations were largely dispensable for disease maintenance. Replacing DNMT3A:R882 mutants with wild-type DNMT3A did not impair the ability of AML cells to engraft in vivo, and minimally altered DNA methylation. Taken together, DNMT3A:R882 mutations are initially necessary for AML initiation, but are largely dispensable for disease maintenance. The notion that initiating oncogenes differ from those that maintain cancer has important implications for cancer evolution and therapy.

overall_design: Single-cell RNA-Seq of human pre-leukemic HSPCs differentiated in vitro for 14 days after re-mutation or correction of DNMT3A:R882H

last: Köhnke

first: Daiki

last: Karigane

first: Ravindra

last: Majeti


title: Effect of nucleolin-binding aptamer AS1411 on gene expression in 3T3 cells

city: Tallinn

first: Indrek

last: Koppel

address: Akadeemia tee 15-241

department: Department of Chemistry and Biotechnology

country: Estonia

institute: Tallinn University of Technology

summary: AS1411 is a G-rich DNA aptamer that targets the multifunctional RNA-binding protein nucleolin. AS1411 has both antiproliferative and cell size-regulating activities and has been evaluated for clinical utility, reaching phase II trials as an anticancer agent. The mechanisms underlying the different activities of AS1411 are not completely understood and broad characterization of its molecular effects is lacking. Here, we used a multi-omics approach to profile transcriptome, proteome and lipidome changes in AS1411-treated NIH-3T3 cells, which increase in size in response to the aptamer. We found that AS1411 caused downregulation of cholesterol biosynthesis pathway enzymes at both mRNA and protein levels, without an accompanying drop in cellular cholesterol levels. In addition, AS1411 induced changes in several lipid classes, including increases in phospatidylethanolamine levels. Thus, nucleolin regulates lipid biosynthesis and homeostasis, potentially underlying its roles in cell proliferation and size control.

overall_design: NIH-3T3 cells were treated with 10 µM nucleolin-binding aptamer AS1411 (AS) or 10 µM control aptamer (CTR) for 24-48h and profiled gene expression changes using bulk RNA-seq


title: Transcriptomic analysis of mouse heart after a minimally invasive mitral regurgitation surgery (MiMRS)

first: Xiang

last: Li

address: 11 fengxin road, guangzhou science city

institute: Guangdong Laboratory Animals Monitoring Institute

summary: Mitral valve was damaged to cause regurgitation induced by a novel and minimally invasive surgery  in mice, and hearts were collected for high-throughput sequencing at week 2 (compensatory) after surgery.

overall_design: To explore the signaling pathways involved in volume overload cardiac hypertrophy induced by MR, and to discover the characteristic genes closely related to MR.

first: Jian

last: Wu

first: Yunzeng

last: Zou

first: Xuan


title: Lack of single amino acids transcriptionally tunes sensory systems to enhance microbiota intake

city: Lisbon

first: Gili

last: Ezra

address: Av. Brasília

country: Portugal

institute: Champalimaud foundation

summary: Maintaining sufficient levels of Amino acids (AAs) from the diet is essential for the organism to maintain protein translation and other metabolic processes. Amazingly, the removal of even a single eAA from the diet is sufficient to induce an increase of proteinaceous food intake in Drosophila melanogaster. This process of turning a physiological “need” for any eAA into a psychological “want” driving proteinaceous-food search and consumption still remains poorly understood. Moreover, dietary manipulations of different eAAs have varying effects on metabolism and physiology which depend on the specific eAA depleted. Thus, a systemic analysis of single eAAs effects on the organism is needed. To achieve that, we sequenced RNA from fly heads following exposure to diets depleted of single AAs. We found that depletion of eAAs had different impacts on transcription, however a significant number of regulated transcripts were shared across deprivations, indicating common mechanisms of response to deprivation. Surprisingly chemosensory receptor genes were found amongst the consistently diet-modulated genes, in spite of minimal, if any, change in sensory environment. Genetic manipulations of Or92a confirm its importance for the acceptance of the appropriate food sources, namely , yeast. While Ir76a governs approaches to specific gut bacteria. These results expand the idea that metabolic-internal state can alter sensory processing  already at the transcriptional level, regardless of the sensory environment. Our work uncovers common and unique transcriptional changes induced by all eAA deprivations and reveals novel mechanisms underlying the organism’s behavioral and physiological response to dietary challenges.

overall_design: Each sample is obtained from 10 fly-heads following 3 days on full holidic medium diet or a diet lacking a single eAA

last: Ezra-Nevo

first: Silvia

last: Henriques

first: Daniel

last: Münch

first: Francisco

last: Patricia

first: Celia

last: Baltazar

first: Ana-Paula

last: Elias

first: Bart

last: Deplancke

first: Carlos

last: Ribeiro


title: C44H9.4/HZL-1 AMPK mutant dauer transcriptome

city: Montreal

first: Sabih

last: Rashid

address: 3611 Rue Sainte Famille, Unit 11

department: Biology

institute: McGill University

summary: C. elegans lacking AMPK do not exhibit reproductive defects during normal growth, but when passaged through dauer, exhibit germline hyperplasia and premature spermatogenesis, followed by complete sterility post-dauer (Narbonne and Roy, Development, 2006). Loss of RNAi pathway  components suppresses several of these phenotypes (Kadekar and Roy, PLoS Biol, 2019). We sought to carry out a transcriptomic analysis of control and AMPK mutant animals in the dauer and post-dauer stage to understand what gene expression changes occur due to the absence of AMPK, and how RNAi pathway components may be affected. The daf-2 mutation was used to induce dauer by a temperature shift to 25 °C.

overall_design: RNA-Seq of dauer (96 hours post-hatching at restricted temperature) and post-dauer (96 hours after shift to permissive temperature) in the daf-2 and daf-2; aak-1; aak-2 (aak(0)) genetic background. Standard Trizol RNA extraction of collected samples.

first: Rashid

last: Sabih

first: Roy

last: Richard