title: Selective RNA sequestration in biomolecular condensates directs cell fate transitions

summary: Recent studies have emphasized the significance of biomolecular condensates in modulating gene expression through RNA processing and translational control. However, the functional roles of RNA condensates in cell fate specification remains poorly understood. Here, we profiled the coding and non-coding transcriptome within intact biomolecular condensates, specifically P-bodies, in diverse developmental contexts, spanning multiple vertebrate species. Our analyses revealed the conserved, cell type-specific sequestration of untranslated RNAs encoding key cell fate regulators. Notably, P-body contents did not directly reflect active gene expression profiles for a given cell type, but rather were enriched for translationally repressed transcripts characteristic of the preceding developmental stage. Mechanistically, microRNAs (miRNAs) direct the selective sequestration of RNAs into P-bodies in a context-dependent manner, and perturbing AGO2 or alternative polyadenylation profoundly reshapes P-body RNA content. Building on these mechanistic insights, we demonstrate that modulating P-body assembly or miRNA activity dramatically enhances both activation of a totipotency transcriptional program in naïve pluripotent stem cells as well as the programming of primed human embryonic cells towards the germ cell lineage. Collectively, our findings establish a direct link between biomolecular condensates and cell fate decisions across vertebrate species and provide a novel framework for harnessing condensate biology to expand clinically relevant cell populations.

overall_design: For m2c cell sequencing, mES cells harboring 2C-specific reporters, Mervl-tdTomato and Zscan4-GFP were sorted based on reporter expression. Mervl/Zscan4 double-positive cells or negative cells with or without Ddx6 knockdown were sequenced. For hESC samples, a naive ES cell line carrying the totipotency specific reporter TPRX1-GFP was sorted based on reporter expression. TPRX1 positive cells and TPRX1 negative cells were sorted and sequenced in DDX6 KD or Ctrl KD conditions. This was repeated in primed conditions. For germ cell differentiations, human BTAG iPSCs harboring PGC specific reporters for TFAP2C-GFP and BLIMP1-TOMATO were used. TFAP2C/BLIMP1 double positive and negative cells were sorted and sequenced in DDX6 and Ctrl KD conditions, in naive conditions, and then after differentiation in iMELCs, and then PGCs.

first: Patrizia

last: Pessina

first: Bruno

last: Di Stefano

first: Justin

last: Brumbaugh


title: Transcriptional and functional architecture of the whole neutrophil compartment [RNA-seq IPSCs]

status: Public on Sep 30 2025

city: New Haven

first: Andrés

last: Hidalgo

address: 10 Amistad Street

country: USA

institute: Yale University

type: Expression profiling by high throughput sequencing

summary: In this study, we present NeuMAP, a comprehensive single-cell analysis of neutrophils spanning over 40 anatomical, physiological, and pathological contexts in mice. NeuMAP confirms and expands previous models of neutrophil diversity, revealing the organization of neutrophils into distinct functional hubs under both normal and pathological conditions. Furthermore, we delineate prototypical trajectories as key organizers of granulopoiesis and examine neutrophil dynamics along these trajectories during acute inflammation and cancer. Integrating insights from fate mapping, mutant mouse models, and in vitro experiments, we identified immunological signals guiding neutrophil through these typical trajectories of granulopoiesis, that balance immune protection, tissue homeostasis and repair. Specifically, we identified IFNB, GMCSF, and TGFB as drivers of pro-inflammatory, cancer-associated, and mature neutrophil states, respectively and found that the transcription factor JUNB drives the angiogenic and immunosuppressive function of neutrophils. Additionally, we uncover conserved transcriptional signatures for human neutrophil states, validate their prognostic significance in cancer patients, and introduce a proof-of-concept strategy for exploring the diagnostic potential of neutrophil functional diversity across various diseases using single-cell transcriptomics of blood neutrophils. Our study provides a model that delineates the global architecture of the neutrophil compartment in mammals, and stablishes a robust framework for further exploration of neutrophil biology.

overall_design: Human iPS cells (SCTi003-A; STEMCELL Technologies) were maintained in 6-well sterile tissue culture plates (ThermoFisher) coated with Matrigel (Corning), using mTeSR™ Plus serum-free medium (STEMCELL Technologies). The medium was replaced daily. iPS cell colonies were cultured at a density of fewer than 5 colonies per well in a 6-well tissue culture plate coated with Matrigel (Corning). When individual colonies reached approximately 500 um of diameter, mTeSR™ Plus medium was replace with Stemline II hematopoietic stem cell expansion medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with insulin-transferrin-selenium (ITS, Invitrogen) and cytokines as follows: bone morphogenetic protein (BMP) 4 (20 ng/mL, R&D Systems) was added for four days and then replaced with vascular endothelial growth factor (VEGF) 165 (40 ng/mL, R&D Systems) on Day 4. On Day 6, VEGF 165 was replaced with a combination of stem cell factors (SCF, 50 ng/mL, R&D Systems), interleukin-3 (IL-3, 50 ng/mL, R&D Systems), thrombopoietin (TPO, 5 ng/mL, Merck), and granulocyte colony-stimulating factor (G-CSF, 50 ng/mL; Merck). Thereafter, the medium was replaced every five days. Neutrophils were collected every five days starting on day 11 of differentiation. Neutrophils were collected after 24h of treatment with different cytokines: TGFβ, IFNβ and GM-CSF. Total RNA was prepared with the RNA Extraction RNeasy Plus Mini-kit (QIAGEN)

first: Daniela

last: Cerezo-Wallis

first: Andrea

last: Rubio-Ponce

first: Ivan

last: Ballesteros


title: The FERONIA receptor kinase is required for high humidity responses in Arabidopsis

city: Durham

first: Sheng Yang

last: He

address: 124 Science Drive

institute: Duke University

summary: High humidity greatly influences plant growth and development by triggering adaptive physiological responses such as leaf hyponasty. Recent studies identified CNGC2/4-mediated Ca2+ influx and CAMTA2/3-mediated transcription as essential for this process, but the upstream regulators that control these pathways remain unknown. Here, we show that the receptor-like kinase FERONIA (FER) is required for Arabidopsis adaptation to high humidity. Transcriptomic profiling revealed rapid induction of CNGC2, CAMTA targets, and pectin remodeling genes in wild-type plants but not in fer-4 loss of function mutants. A targeted mutant screen confirmed that FER and its co-receptor LORELEI-LIKE GPI-ANCHORED PROTEIN1 (LLG1) are essential for humidity-induced hyponasty. Whole-plant Ca²⁺ imaging demonstrated that high humidity triggers conserved cytosolic Ca²⁺ elevations across species, including petiole-localized Ca²⁺ waves in Arabidopsis that precede hyponastic movement by ~2 h. These signals were significantly attenuated in fer-4, providing a mechanistic explanation for its transcriptional and physiological defects. Together, our findings identify FERONIA as a key upstream component of a sensing mechanism linking extracellular high humidity to Ca²⁺ signaling, cell wall remodeling, and adaptive leaf movement.

overall_design: RNA-seq profliing of Arabidopsis plants (Col-0 and the fer-4 mutant) exposed to the high humidity conditions after 0 min, 15 min, 60 min, 24 hours and 6 days of ambient and high humidity conditions,

first: Comzit

last: Opachaloemphan

first: Richard

last: Hilleary

last: Wu

first: Chi

last: Kuan

first: Kinya

last: Nomura


title: Architecture of the neutrophil compartment in mice [scRNA-seq germ_free]

overall_design: Germ-free C57BL/6 mice were bred and maintained under sterile conditions in flexible film isolators (Class Biologically Clean) in the Palm laboratory Gnotobiotic Facility at Yale School of Medicine. Mice were housed in a temperature- and humidity-controlled room under a dark–light cycle of 12 h–12 h. C5BL6 SPF and Germ Free mice were sacrificed via CO2, and blood, spleen, bone marrow, and lung were collected. 1ml of blood was collected in BD Microtainer tubes containing EDTA (365974, BD), and red blood cell were lysed in 10ml of 1X ACK buffer for 10min on ice. The spleen, lung and tumors  were enzymatically digested to generate cell suspensions that were  washed, resuspended in 1ml of 1X ACK buffer and incubated for 5min on ice to get rid of the red blood cells. The cell suspension was washed, resuspended in cold staining buffer, and kept on ice. The bone marrow was harvested by flushing the femur’s shaft with cold staining buffer and filtered to obtain a single cell suspension. The cells were washed and red blood cell were lysed using 1ml of 1X ACK buffer (5min on ice). After lysis, the cells were further washed, resuspended in cold staining buffer, and kept on ice. Cell suspensions  were incubated with Fc block (TruStain FcX PLUS – 156603 – Biolegend) for 10min on ice, and for another 30min with an antibody cocktail containing: anti-Ly6G_PE (127608 - Biolegend), anti-CD11b_APC (101211 - Biolegend). Before acquisition and sorting, single cell suspensions were incubated for 15min with Dapi (NBP2311561, Novus Biologicals). Live-CD11b+-Ly6G+ neutrophils were sorted out. After sorting, the  neutrophil suspensions were Taged (BD Multiplexing Kit)  and pooled in equal proportion. For the generation of single-cell whole-transcriptomes we used a BD Rhapsody system according to the manufacturer instructions. Briefly, cell suspensions from each condition were incubated with Sample Tags (BD) for 20 minutes at RT. Cells were then washed three times and pooled in a single tube. Viability and cell concentration using the Countess III cell counter (Thermofisher). 60,000 cells were loaded into a Rhapsody Single Cell Analysis System cartridge. Cell capture and cDNA synthesis were performed according to manufacturer’s instructions; cells were isolated into nanowells by gravity, then cells lysed and mRNAs together with sample tags oligos were released and captured by beads present in the nanowells. Each bead contained a unique oligo named “cell label” to identify each individual bead. All beads present in the cartridge were collected and cDNA synthesis took place in a single reaction. At this point each cDNA and Sample Tag oligo were attached to its corresponding “cell label” oligo. Two separated indexed libraries were prepared for whole transcriptome analysis and Sample Tag demultiplexing following manufacturer’s instructions. The average size of the libraries was calculated using the 2100 Bioanalyzer (Agilent) and the concentration was determined using the Qubit fluorometer (Thermofisher). Finally, libraries were combined and sequenced together in a paired-end run (60x42) using a NextSeq 2000 system (Illumina) and a P2 flow cell. Output files were processed with NextSeq 1000/2000 Control Software Suite v1.4.1. FastQ files for each sample were obtained using BCL Convert v3.6.3 software (Illumina).

first: Iván


title: Architecture of the neutrophil compartment in mice [scRNA-seq clonal]

summary: In this study, we present NeuMAP, a comprehensive single-cell analysis of neutrophils spanning over 40 anatomical, physiological, and pathological contexts in mice. NeuMAP confirms and expands previous models of neutrophil diversity, revealing the organization of neutrophils into distinct functional hubs under both normal and pathological conditions. Furthermore, we delineate prototypical trajectories as key organizers of granulopoiesis and examine neutrophil dynamics along these trajectories during acute inflammation and cancer. Integrating insights from fate mapping, mutant mouse models, and in vitro experiments, we identified immunological signals guiding neutrophil through these typical trajectories of granulopoiesis, that balance immune protection, tissue homeostasis and repair. Specifically, we identified IFNB, GMCSF, and TGFB as drivers of pro-inflammatory, cancer-associated, and mature neutrophil states, respectively and found that the transcription factor JUNB drives the angiogenic and immunosuppressive function of neutrophils. Additionally, we uncover conserved transcriptional signatures for human neutrophil states, validate their prognostic significance in cancer patients, and introduce a proof-of-concept strategy for exploring the diagnostic potential of neutrophil functional diversity across various diseases using single-cell transcriptomics of blood neutrophils.

overall_design: Model of Clonal Hematopoiesis and PCSK9-Induced Hypercholesterolemia: To model TET2 loss-of-function-driven clonal hematopoiesis, we performed an adoptive bone marrow transfer (ADT) without pre-conditioning. Ten-week-old unirradiated JAXBoy (PtprcK302E) recipient mice were intravenously injected with a total of 1.5 × 10⁷ unfractionated CD45.2⁺ Tet2–/– bone marrow (BM) cells, administered as three consecutive daily doses of 5 × 10⁶ cells. Donor cells were harvested from age-matched littermate Tet2–/– mice (8–10 weeks old) by flushing femurs and tibias following euthanasia. To induce hypercholesterolemia, a recombinant AAV vector encoding a gain-of-function form of murine or PCSK9 (pAAV/D377Y-mPCSK9) was delivered via a single tail vein injection. One week later, mice were placed on either a high-cholesterol Western diet (Envigo, TD.88137; 42% calories from fat, 0.2% cholesterol) or a matched control diet for 13 weeks. At endpoint, ADT mice were euthanized and Tet2–/– (CD45.2⁺) or WT (CD45.1⁺) neutrophils were isolated from peripheral blood, lung and bone marrow by cell sorting. Live CD45.1+CD11b+-Ly6G+ and CD45.2+CD11b+-Ly6G+ cells were sorted out. After sorting, the  neutrophil suspensions were Taged (BD Multiplexing Kit)  and pooled in equal proportion. For the generation of single-cell whole-transcriptomes we used a BD Rhapsody system according to the manufacturer instructions. Briefly, cell suspensions from each condition were incubated with Sample Tags (BD) for 20 minutes at RT. Cells were then washed three times and pooled in a single tube. Viability and cell concentration using the Countess III cell counter (Thermofisher). 60,000 cells were loaded into a Rhapsody Single Cell Analysis System cartridge. Cell capture and cDNA synthesis were performed according to manufacturer’s instructions; cells were isolated into nanowells by gravity, then cells lysed and mRNAs together with sample tags oligos were released and captured by beads present in the nanowells. Each bead contained a unique oligo named “cell label” to identify each individual bead. All beads present in the cartridge were collected and cDNA synthesis took place in a single reaction. At this point each cDNA and Sample Tag oligo were attached to its corresponding “cell label” oligo. Two separated indexed libraries were prepared for whole transcriptome analysis and Sample Tag demultiplexing following manufacturer’s instructions. The average size of the libraries was calculated using the 2100 Bioanalyzer (Agilent) and the concentration was determined using the Qubit fluorometer (Thermofisher). Finally, libraries were combined and sequenced together in a paired-end run (60x42) using a NextSeq 2000 system (Illumina) and a P2 flow cell. Output files were processed with NextSeq 1000/2000 Control Software Suite v1.4.1. FastQ files for each sample were obtained using BCL Convert v3.6.3 software (Illumina).

first: Cerezo-Wallis

last: Daniela

first: Rubio-Ponce

last: Andrea

first: Ballesteros

last: Iván

first: Hidalgo

last: Andrés


title: Effect of IL-6 Trans-Signaling Blockade by sgp130 overexpression on hepatic gene expression in the context of mature-onset obesity in mice

city: Jerusalem

first: Jonathan

middle: H.

last: Axelrod

state: n

address: Ein Kerem

department: Goldyne Savad Institute of Gene Therapy

country: Israel

institute: Hadassah Hebrew University Hospital

summary: Interleukin-6 (IL-6) performs multiple roles in regulating metabolic pathways in both mice and man. Here, we examined the age-dependent metabolic phenotype of mice overexpressing sgp130 (SGP mice), which specifically blocks IL-6 trans-signaling. Transgenic SGP mice engineered to block IL-6 trans-signaling and wild-type (WT) littermates were raised to 6 and 14 months of age. RNA-seq analyses were performed on liver samples as a function of age and genotype. At ~6 months of age, weight gain, body fat accumulation, hepatosteatosis, hyperglycemia, and macrophage recruitment to adipose tissue emerged and progressed with age in SGP mice. IL-6/sIL-6R blockade strongly reduced STAT3 phosphorylation in the liver. RNA-seq analyses of mouse livers revealed genotype-related changes in gene expression profiles attributable to bacterial byproducts. Depletion of the gut microbiota by antibiotic treatment from the age of 6 months reversed the obese phenotype in transgenic mice, confirming the crucial role of the microbiome in the phenotype. These findings reveal the crucial functions of IL-6 trans-signaling in preventing mature-onset body fat accumulation induced by certain intestinal microbiota.

overall_design:
RNA-seq profiling of total RNA extracted from liver samples from male littermates of wildtype (C57BL6) and sgp130Fc (SGP) transgenic (C57BL6) mice at 6 and 14 months-of-age.
Please note that WT-replica1 (C7_BL47BL51) and SGP-replica2 C11_SL29SL30) appeared to be exceptional from their group (i.e. exceptionally large variance) and thus were excluded fron the processed data analysis.

middle: H

first: Tali

last: Lanton

first: Eithan

last: Galun


title: Ribonucleoprotein (RNP) condensates modulate survival in response to Mars-like stress conditions

city: Bangalore

first: Purusharth

middle: I

last: Rajyaguru

state: Karnataka

address: CV Raman road

department: Biochemistry

country: India

institute: Indian Institute of Science Bangalore

summary: Scientific advances have emboldened human efforts toward exploring the potential of extinct, extant, or future life on Mars. An important aspect of this endeavor is understanding how an organism adapts to stress-inducing environmental conditions on Mars such as radiations, shock waves, extreme temperatures, and chaotropic stress due to higher levels of perchlorates. A conserved approach organisms use across evolutionary scales to adapt and overcome stress conditions is the assembly of ribonucleoprotein (RNP) condensates. In this study, we employ a multidisciplinary approach to understand yeast survivability and adaptation under Mars-like stress conditions specifically shock waves and perchlorate by focusing on RNP condensates. Our study reveals that yeast survives 5.6 M intensity shock waves. Exposure to either shock waves or sodium perchlorate induces the formation of P-bodies, a conserved stress-induced condensate. Yeast mutants defective in P-body assembly show defective growth in response to perchlorate stress. Transcriptome analysis followed by validation identified several relevant transcripts whose levels are perturbed in response to Mars-like conditions. Finally, identification of several transcripts whose abundance is altered in the P-body assembly mutant upon stress highlights new connection between response to Martian stress conditions and RNP condensates. This study, a first of its kind, highlights the importance of RNP condensates in understanding the impact of Martian conditions on life in general. This study paves the way for using RNP condensates as a biomarker for assessing the health of life forms during space explorations.

overall_design: RNA-seq profilling in wild-type and edc3∆lsm4∆C mutant cells of S.cerevisiae in response to sodium perchlorate and in wildtype cells recovering from exposure to 5.6 Mach intensity shock wave. RNA seq experiments were carried out for untreated cells, sodium perchlorate treated and shocked cells. Two replicates were used for each sample. Validation of RNA seq results using Real time quantitative PCR (RT-qPCR) indicated that the results obtained by RNA sequencing were reproducible and the extent of changes were comparable.

first: Riya

last: Dhage

first: Purusharth I


title: An RNA sponge directs the transition from feast to famine in Caulobacter crescentus

city: Würzburg

first: Manuel

last: Velasco Gomariz

address: Josef-Schneider-Straße 2/D15

country: Germany

institute: Helmholtz Institute for RNA-based Infection Research (HIRI)

summary:
In bacteria, regulatory networks controlling the adaptation of gene expression in response to stress are frequently complemented by base-pairing small regulatory RNAs (sRNAs) that act at the post-transcriptional level. While many regulatory circuits governing stress resilience have been studied in the model bacterium Caulobacter crescentus, only a small fraction of its diverse sRNA repertoire has been characterized.
In this study, we globally identify interacting RNA-RNA pairs associated with the major RNA-binding protein Hfq in C. crescentus. In addition to numerous connections between sRNAs and mRNAs, we also recover RNA-RNA pairs consisting of two non-coding transcripts. Our results indicate that the sRNA CrfA acts as a sponge to inactivate a family of four conserved sRNAs, SisA-D. When induced by carbon starvation, CrfA redirects gene expression towards the utilization of distinct energy sources, and loss of the RNA sponge is linked to a severe growth defect in environments with fluctuating nutrient availability.

overall_design: RIL-seq of a Wild-type and a Hfq-3xFLAG strain under rich media condition. Samples were collected at a OD660nm=1.

first: Velasco Gomariz

last: Manuel

first: Kathrin

middle: S

last: Fröhlich

first: Vogt

last: Laura


title: RNA sequestration regulates cell fate [RNA-Seq]

city: Boulder

first: Mika

last: Nevo

state: Colorado

address: 2315 Walnut St

institute: CU Boulder

summary: Recent studies have highlighted the significance of RNA condensates in governing various aspects of cellular RNA processing. Nonetheless, the functional roles and composition of RNA condensates in the context of cell fate specification have remained elusive. In this study, we conducted a comprehensive characterization of the coding and non-coding transcriptome within cytoplasmic condensates known as P-bodies across diverse developmental contexts and vertebrate species. Notably, our investigation unveiled that the dissolution of RNA condensates reprograms both human and mouse pluripotent stem cells into a blastomere-like state. Mechanistically, we observed a high enrichment of untranslated fate-instructive RNAs within P-bodies, subject to cell type-specific sequestration. Employing degron systems, we demonstrated that the acute loss of P-bodies facilitates the translation of stored mRNAs. Furthermore, our research identifies the AGO-miRNAs molecular node as central in orchestrating RNA sequestration in P-bodies in a context-dependent manner. Perturbing AGO proteins, RNA 3’ UTR length, or expression of selected miRNAs, profoundly reshapes the localization of RNAs within cells. Collectively, our findings illuminate how RNA processing within condensates drives essential aspects of lineage-specific cell fate trajectories across vertebrate species.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: human and mouse embryonic stem cells were differentiated into each of the cell types below, and then pbody contents were sorted and sequenced, and cytoplasmic fractions were sequenced seperately. Two replicates were sequenced for each sample. In treatment or knockout conditions, each treatment condition was sequenced alongside a control cell line, two replicates of each for pbody and cytoplasm.

first: Eduard

last: Casas

first: Patrizia

last: Pessenia

first: Bruno

last: Di Stefano

first: Justin

last: Brumbaugh


title: Effect of DEHP on piRNA expression in rat testis

city: Yichang

first: Guoqing

last: Fu

address: 8 University Road

country: China

institute: China Three Gorges University

summary: To explore the effect of DEHP on piRNA expression in rat testes, we have employed rat piRNA microarray expression profiling as a discovery platform to identify piRNAs with the potential to study the role of DEHP exposure on piRNA expression in rat testes to provide new insight into DEHP-induced male reproductive injury.

overall_design: 6-week Sprague-Dawley (SD) male rats were randomly divided into control group and DEHP-treated group (1000 mg/kg/d) after 1 week of acclimatization. Rats in control and DEHP groups were orally administrated with 1000 mg/kg/d DEHP (dissolved in corn oil, gavage volume of 0.2 mL/10 g body weight) and corn oil for 28 consecutive days respectively. Three testes were collected from three rats in each group for RNA extraction and subsequent piRNA detection.

first: Yuqin

last: Shi


title: Cleavage under targets & tagmentation (CUT&Tag) sequencing of mouse utricle explants following BMP4 treatment

city: SHANGHAI

first: yunzhong

last: zhang

state: SHANGHAI

address: 83 fenyang road, xuhui district

department: Department of ENT Institute and Otorhinolaryngology

institute: Eye & ENT Hospital of Fudan University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We performed the Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing assay to identify proteins that interact with c-Fos in order to validate the microenvironment stimulation by BMP4.

overall_design: Comparative CUT&Tag analysis was performed by pulling down with an anti-c-Fos antibody and comparing this with a nonspecific negative control IgG.


title: Transcriptional and functional architecture of the whole neutrophil compartment [III]

first: Andrés

last: Hidalgo

summary: In this study, we present NeuMAP, a comprehensive single-cell analysis of neutrophils spanning over 40 anatomical, physiological, and pathological contexts in mice. NeuMAP confirms and expands previous models of neutrophil diversity, revealing the organization of neutrophils into distinct functional hubs under both normal and pathological conditions. Furthermore, we delineate prototypical trajectories as key organizers of granulopoiesis and examine neutrophil dynamics along these trajectories during acute inflammation and cancer. Integrating insights from fate mapping, mutant mouse models, and in vitro experiments, we identified immunological signals guiding neutrophil through these typical trajectories of granulopoiesis, that balance immune protection, tissue homeostasis and repair. Specifically, we identified IFNB, GMCSF, and TGFB as drivers of pro-inflammatory, cancer-associated, and mature neutrophil states, respectively and found that the transcription factor JUNB drives the angiogenic and immunosuppressive function of neutrophils. Additionally, we uncover conserved transcriptional signatures for human neutrophil states, validate their prognostic significance in cancer patients, and introduce a proof-of-concept strategy for exploring the diagnostic potential of neutrophil functional diversity across various diseases using single-cell transcriptomics of blood neutrophils.  Our study provides a model that delineates the global architecture of the neutrophil compartment in mammals, and stablishes a robust framework for further exploration of neutrophil biology.

overall_design: 8 weeks old Male C57Bl6 mice were sacrificed via CO2, and blood, spleen, bone marrow, and lung were collected. 1ml of blood was collected in BD Microtainer tubes containing EDTA (365974, BD), and red blood cell were lysed in 10ml of 1X ACK buffer for 10min on ice. The spleen and lung  cell suspensions were washed, resuspended in 1ml of 1X ACK buffer and incubated for 5min on ice to get rid of the red blood cells. The cell suspension was washed, resuspended in cold staining buffer, and kept on ice. The bone marrow was harvested by flushing the femur’s shaft with cold staining buffer and filtered to obtain a single cell suspension. The cells were washed and red blood cell were lysed using 1ml of 1X ACK buffer (5min on ice). After lysis, the cells were further washed, resuspended in cold staining buffer, and kept on ice. Cell suspensions  were incubated with Fc block (TruStain FcX PLUS – 156603 – Biolegend) for 10min on ice, and for another 30min with an antibody cocktail containing: anti-Ly6G_PE (127608 - Biolegend), anti-CD11b_APC (101211 - Biolegend), TotalSeq_A Hashtag antibodies cocktail (Biolegend). The hashtag antibodies used were: TotalSeq_A0301 (155801, Biolegend – spleen), TotalSeq_A0302 (155803, Biolegend – bone marrow), TotalSeq_A0303 (155805, Biolegend – blood), TotalSeq_A0310 (155819, Biolegend – lung). Before acquisition and sorting, single cell suspensions were incubated for 15min with Dapi (NBP2311561, Novus Biologicals). Live-CD11b+-Ly6G+ neutrophils were sorted out. After sorting, the 4 neutrophil suspensions were enumerated and pooled in equal proportion. After that, we proceded with the library preparation.

first: Daniela

last: Cerezo-Wallis

first: Ivan

last: Ballesteros

first: Andrea

last: Rubio-Ponce

first: Samuel

last: Ovadia


title: Transcriptional and functional architecture of the whole neutrophil compartment I

overall_design: To elucidate the transcriptional landscape of neutrophil heterogeneity in mice, we conducted single-cell RNA sequencing of Lin- (B220, CD3, CD19, NK1.1, Ter119) cells isolated from the bone marrow, along with CD11b+Ly6G+ cells obtained from blood and various tissues across different physiological and pathological contexts

first: Iván


title: RNA sequestration in cytoplasmic condensates regulates cell identity (ncRNA-Seq)

type: Non-coding RNA profiling by high throughput sequencing

summary: Post-transcriptional regulation is increasingly recognized as pivotal in guiding the gene expression programs that instruct cell fate decisions. Recent studies have highlighted the significance of RNA condensates in modulating gene expression through RNA processing and translational control. Yet, the functional roles and composition of RNA condensates in the context of cell fate specification remain unclear. In this study, we leveraged Fluorescence-Activated Particle Sorting to profile the coding and non-coding transcriptome within cytoplasmic condensates known as P-bodies across diverse developmental contexts and vertebrate species. Our analyses revealed the highly conserved, cell type-specific sequestration of fate-instructive RNAs, which are translationally suppressed and stored within P-bodies. Using degron systems in both human and mouse pluripotent cells, we demonstrate that the acute dissolution of P-bodies facilitates the translation of mRNAs otherwise suppressed in condensates, including genes associated with zygotic genome activation and totipotency. Consistently, loss of RNA sequestration awakens the totipotency transcriptional program in human and mouse pluripotent cells. Mechanistically, we provide evidence that miRNAs play a pivotal role in orchestrating the sequestration of specific RNAs into P-bodies in a context-dependent manner. Accordingly, perturbing AGO2, alternative polyadenylation, and miRNA function profoundly reshapes the sequestration of RNAs into P-bodies and alters cell fate. We further identify mir-300/381 as a key repressor of the totipotent transcriptional state. Collectively, our findings connect RNA processing within condensates to cell fate decisions across vertebrate species.

overall_design: The hESC and mESC were differentiated into each of the cell types below, and then pbody contents were sorted and sequence, and cytoplasmic fractions were sequenced seperately. Two replicates were sequenced for each sample. In treatment or knockout conditions, each treatment condition was sequenced alongside a control cell line, two replicates of each for pbody and cytoplasm.


title: RNA sequestration regulates cell fate

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

sample_organism:
Gallus gallus
Homo sapiens
Mus musculus

platform_organism:
Gallus gallus
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Transcriptional and functional architecture of the whole neutrophil compartment

overall_design: To elucidate the effect of JunB deletion in neutrophils, we conducted Bulk RNA sequencing of CD11b+Ly6G+ cells obtained from the bone marrow,  blood and lungs in wild type (C57BL6) or JunB-defficient neutrophils during homeostasis

last: Rubio


title: Architecture of the neutrophil compartment in mice [scRNA-seq balbc]

overall_design: 8 weeks old Male BALB/C and C57Bl6 mice were sacrificed via CO2, and blood, spleen, bone marrow, and lung were collected. 1ml of blood was collected in BD Microtainer tubes containing EDTA (365974, BD), and red blood cell were lysed in 10ml of 1X ACK buffer for 10min on ice. The spleen, lung and tumors  were enzymatically digested to generate cell suspensions that were  washed, resuspended in 1ml of 1X ACK buffer and incubated for 5min on ice to get rid of the red blood cells. The cell suspension was washed, resuspended in cold staining buffer, and kept on ice. The bone marrow was harvested by flushing the femur’s shaft with cold staining buffer and filtered to obtain a single cell suspension. The cells were washed and red blood cell were lysed using 1ml of 1X ACK buffer (5min on ice). After lysis, the cells were further washed, resuspended in cold staining buffer, and kept on ice. Cell suspensions  were incubated with Fc block (TruStain FcX PLUS – 156603 – Biolegend) for 10min on ice, and for another 30min with an antibody cocktail containing: anti-Ly6G_PE (127608 - Biolegend), anti-CD11b_APC (101211 - Biolegend). Before acquisition and sorting, single cell suspensions were incubated for 15min with Dapi (NBP2311561, Novus Biologicals). Live-CD11b+-Ly6G+ neutrophils were sorted out. After sorting, the  neutrophil suspensions were Taged (BD Multiplexing Kit)  and pooled in equal proportion. For the generation of single-cell whole-transcriptomes we used a BD Rhapsody system according to the manufacturer instructions. Briefly, cell suspensions from each condition were incubated with Sample Tags (BD) for 20 minutes at RT. Cells were then washed three times and pooled in a single tube. Viability and cell concentration using the Countess III cell counter (Thermofisher). 60,000 cells were loaded into a Rhapsody Single Cell Analysis System cartridge. Cell capture and cDNA synthesis were performed according to manufacturer’s instructions; cells were isolated into nanowells by gravity, then cells lysed and mRNAs together with sample tags oligos were released and captured by beads present in the nanowells. Each bead contained a unique oligo named “cell label” to identify each individual bead. All beads present in the cartridge were collected and cDNA synthesis took place in a single reaction. At this point each cDNA and Sample Tag oligo were attached to its corresponding “cell label” oligo. Two separated indexed libraries were prepared for whole transcriptome analysis and Sample Tag demultiplexing following manufacturer’s instructions. The average size of the libraries was calculated using the 2100 Bioanalyzer (Agilent) and the concentration was determined using the Qubit fluorometer (Thermofisher). Finally, libraries were combined and sequenced together in a paired-end run (60x42) using a NextSeq 2000 system (Illumina) and a P2 flow cell. Output files were processed with NextSeq 1000/2000 Control Software Suite v1.4.1. FastQ files for each sample were obtained using BCL Convert v3.6.3 software (Illumina).

first: Cerezo-Wallis

last: Daniela

first: Rubio-Ponce

last: Andrea

first: Ballesteros

last: Iván

first: Hidalgo

last: Andrés


title: Novel gene therapy CM-YAPon protects the mouse heart from myocardial infarction - MI

city: Cincinnati

first: Jeffrey

middle: D

last: Steimle

email: jeffrey.steimle@cchmc.org, jeffrey.steimle@bcm.edu, jsteimle@uchicago.edu, jsteimle@alumni.nd.edu

state: OH

address: 240 Albert Sabin Way

department: Molecular Cardiovascular Biology

institute: Cincinnati Children's Medical Hospital Center

summary: Myocardial infarction (MI), which affects about 3 million people globally each year, permanently damages the heart and reduces cardiac function1. Recent studies have indicated that activating YAP in cardiomyocytes (CMs) promotes cardiac regeneration and mitigates pathological remodeling in mouse and pig MI models2,3. To precisely control YAP activity in vivo and encourage its clinical application, we developed an adeno-associated virus 9 (AAV9)-based therapy, termed CM-YAPon, which triggers YAP activation in CMs upon exposure to a small molecule LMI070. One dose of LMI070 in mice induced a transient expression of active YAP (YAP5SA), which was subsequently degraded within a week. Consistent with earlier findings, YAP activation after injury improved cardiac function. Interestingly, administering a single LMI070 injection two weeks before MI provided lasting cardioprotection, which diminished by four weeks after the transient YAP activation, by reducing non-CMs cell death and enhancing cardiac function. Taken together, our novel gene therapy CM-YAPon presents a promising avenue for developing protective strategies against MI-induced cardiac injury.

overall_design: AAV9 virus expressing YAPon was administrated to P7 ICR mice by intraperitoneal injection. At 6 weeks of age, animals were injected with DMSO or LMI070. Two weeks later, left anterior descending (LAD) ligation was performed ("MI") or sham surgery ("Sham"). Ventricular tissue below the suture was separated from the heart 7 days following surgery. Nuclei were collected using a Chromium Nuclei Isolation Kit and single nuclei RNA-sequencing was performed using the 10x Single Cell 3′ Reagents.

first: Fansen

last: Meng

first: James

middle: F

last: Martin


title: Novel gene therapy CM-YAPon protects the mouse heart from myocardial infarction - 3 day post-LMI

overall_design: AAV9 virus expressing YAPon or saline was administrated to 6 week old adult mice by retro-orbital injection. Two weeks later, animals were injected with DMSO or LMI070. The apex of the ventricle was separated from the heart 3 days later. Nuclei were isolated from the apex tissue we collected for single nucleus RNA sequencing. Nuclei were collected using a Chromium Nuclei Isolation Kit (10x Genomics, PN-1000493). Single nuclei RNA-seq was performed using Single Cell 3′ Reagents Kit (10x Genomics, Chromium Next GEM Single Cell 3′ Kit version 3.1 or GEM-X Single Cell 3' Kit v4).


title: Architecture of the neutrophil compartment in mice


title: Novel gene therapy CM-YAPon protects the mouse heart from myocardial infarction


title: RNA sequestration regulates cell fate (Human)

first: Mika

last: Nevo

overall_design: Human hESC were differentiated into the following cell types (excluding HEK cells), and the pbody and cytoplasm fractions were collected and sequenced, with two replicates of each.

first: Patrizia

last: Pessina

first: Eduard

last: Casas


title: scRNA-seq analysis of mouse tumor-infiltrating immune cells

city: Paris

first: Elodie

last: Segura

address: 156 rue de Vaugirard

department: INSERM U1151

country: France

institute: INSERM

summary: Cancer immuno-surveillance and response to checkpoint blockade therapy can be affected by multiple environmental factors, including nutrition. However, the direct effects of individual nutrients on anti-tumoral immune responses remain poorly understood. Here we addressed the impact of dietary ligands of Aryl Hydrocarbon receptor (AhR), a transcription factor activated by indoles and tryptophan catabolites generated through food digestion and microbiota metabolism. To this aim, we analyzed pre-clinical tumor models in mice fed on a diet naturally poor in AhR ligands, or the same diet enriched for Indole-3-carbinol, a phytonutrient cleaved by stomach enzymes into AhR agonists.

overall_design: Mice were fed with a diet poor in AhR ligands (AhR-poor diet) or the same diet supplemented in indole-3-carbinaol (I3C diet). Mice were inoculated with MCA-101-OVA tumor cells. Tumor infiltrating cells were isolated as CD45+CD11b+ or CD45+TCRb+ and analyzed by scRNA-seq.

first: Alice

last: Coillard


title: Human iPSC-liver organoid transplant reduces fibrosis by immunomodulation

city: Yokohama

first: Tomomi

last: Tadokoro

state: Kanagawa

address: 3-9 Fukuura

department: Graduate School of Medicine

country: Japan

institute: Yokohama City University

summary: Donor organ shortage for transplant is a serious problem worldwide. Organoid culture technology, which allows the generation of complex organs from stem cells, provides a potential solution. We previously reported in vivo vascularization of human induced pluripotent stem cell (hiPSC)-derived liver buds (LBs) and subsequent improved survival of recipient mice with acute liver failure1,2. Here, we show that mid-gestational fetal liver tissue can generate de novo liver when grafted onto the surface of host livers and improve survival and liver functions in a model of liver failure. Next, we created complex fetal liver-like organoids (LOs) by a fusion of hiPSC-LBs to induce static cell-cell interactions, and show that these contain hepatocytes, vasculature, and bile duct after transplantation. Fused hiPSC-LOs show superior liver functions compared to hiPSC-LBs and improve survival and functions of liver failure model upon transplantation. Finally, transplant of fused hiPSC-LOs ameliorates liver fibrosis, a symptom of liver cirrhosis which leads to organ dysfunction, through their immunomodulatory effects especially on macrophage polarization that plays an important role in the regulation of liver fibrosis3–5. Taken together, our results suggest that hiPSC-LO transplant could be a promising therapeutic option for liver failure patients in the near future

overall_design: Transcriptome profiling of human iPSC-derived liver buds (hiPSC-LB) and hiPSC-liver organoids

first: T

first: H

last: Taniguchi


title: single cell RNA sequencing of thoracic aorta from Angiotensin II infused control and Sting whole body knockout mice

city: Houston

first: Yanming

last: Li

state: TX

address: One Baylor Plaza

department: Michael E. DeBakey Department of Surgery

institute: Baylor College of Medicine

summary: Background: Sporadic aortic aneurysm and dissection (AAD), caused by progressive aortic smooth muscle cell (SMC) loss and extracellular matrix degradation, is a highly lethal condition. Identifying mechanisms that drive aortic degeneration is a crucial step in developing an effective pharmacologic treatment to prevent disease progression. Recent evidence has indicated that cytosolic DNA and abnormal activation of the cytosolic DNA sensing adaptor STING (stimulator of interferon genes) play a critical role in vascular inflammation and destruction. Here, we examined the involvement of this mechanism in aortic degeneration and sporadic AAD formation. Methods: The presence of cytosolic DNA in aortic cells and activation of the STING pathway were examined in aortic tissues from patients with sporadic ascending thoracic AAD. The role of STING in AAD development was evaluated in Sting-deficient (Stinggt/gt) mice in a sporadic AAD model induced by challenging mice with a combination of a high-fat diet and angiotensin II. We also examined the direct effects of STING on SMC death and macrophage activation in vitro. Results: In human sporadic AAD tissues, we observed the presence of cytosolic DNA in SMCs and macrophages and significant activation of the STING pathway. In the sporadic AAD model, Stinggt/gt mice showed significant reductions in challenge-induced aortic enlargement, dissection, and rupture in both the thoracic and abdominal aortic regions. Single-cell transcriptome analysis revealed that aortic challenge in wild-type mice induced the DNA damage response, the inflammatory response, dedifferentiation and cell death in SMCs, and matrix metalloproteinase expression in macrophages. These changes were attenuated in challenged Stinggt/gt mice. Mechanistically, nuclear and mitochondrial DNA damage in SMCs and the subsequent leak of DNA to the cytosol activated STING signaling, which induced cell death through apoptosis and necroptosis. In addition, DNA from damaged SMCs was engulfed by macrophages in which it activated STING and its target interferon regulatory factor 3, which directly induced matrix metalloproteinase-9 expression. We also found that pharmacologically inhibiting STING activation partially prevented AAD development. Conclusions: Our findings indicate that the presence of cytosolic DNA and subsequent activation of cytosolic DNA sensing adaptor STING signaling represent a key mechanism in aortic degeneration and that targeting STING may prevent sporadic AAD development.

overall_design: To understand the role of STING in aorta, we performed single cell RNAseq of thoracic aortic tissues from wild type mice, AngII infused wild type mice, Sting KO mice, AngII infused Sting KO mice, and AngII infusion was applied for 7 days.

first: Chen

last: Zhang

first: Scott

middle: A

last: LeMaire

first: Ying

last: Shen


title: Schizophrenia risk gene ZNF536 modulates retinoic acid response and neuronal gene networks in SH-SY5Y cells

first: Artemiy

last: Kurishev

address: Kashirskoe Sh., 34

country: Russia

institute: Menatal Health National Center

summary: ZNF536, a brain-specific transcriptional repressor, has recently emerged as a candidate risk gene for schizophrenia (SZ), yet its functional role in human neurodevelopment remains poorly understood. In this study, we used CRISPR/Cas9 genome editing to generate a dual-allelic ZNF536 knockout model in SH-SY5Y cells, combining a 103 kb deletion encompassing SZ-associated intronic regions with a disruption of zinc finger domains in exon 2. Transcriptome profiling of mutant cells undergoing all-trans retinoic acid (ATRA)-induced differentiation revealed impaired activation of retinoic acid receptor (RAR) target genes, reduced neurite outgrowth, and failure of neuronal maturation. Notably, gene set enrichment analysis uncovered dysregulation of E2F4-mediated cell cycle pathways, suggesting that ZNF536 modulates neurogenic commitment through coordinated control of transcriptional repression and cell proliferation. Moreover, the targeted intronic deletion altered the expression of multiple SZ-associated genes, supporting the functional importance of cis-regulatory elements within ZNF536. Together, our results identify ZNF536 as a critical regulator of RA-responsive gene networks and neuronal differentiation, offering new mechanistic insights into its contribution to schizophrenia (SZ) pathogenesis.

overall_design: SH-SY5Y neuroblastoma cells of three genotypes (NTC control; ZNF536 homozygous knock-in, KI/KI; ZNF536 compound heterozygous KI/del) were profiled by paired-end RNA-seq under two conditions: basal growth medium or 10 µM all-trans retinoic acid (ATRA) for 7 days. Each genotype×condition was performed in biological triplicate (3 genotypes × 2 conditions × 3 replicates = 18 samples) to assess the transcriptomic impact of ZNF536 disruption on RA-induced neuronal differentiation.

middle: O

first: Dmitrii

last: Abashkin

first: Dmitry

last: Karpov

first: Ekaterina

middle: V

last: Marilovtseva

first: Yulia

last: Chaika

last: Semina

first: Vera

middle: E

last: Golimbet


title: Cell therapy using ex vivo reprogrammed macrophages enhances antitumor immune responses in melanoma

city: Washington

first: Anelia

last: Horvath

state: DC

address: 2300 I St NW

department: Biochemistry and Molecular Medicine

institute: George Washington University

summary: Background: Macrophage-based cell therapies have shown modest success in clinical trials, which can be attributed to their phenotypic plasticity, where transplanted macrophages get reprogrammed towards a pro-tumor phenotype. In most tumor types, including melanoma, the balance between antitumor M1-like and tumor-promoting M2-like macrophages is critical in defining the local immune response with a higher M1/M2 ratio favoring antitumor immunity. Therefore, designing novel strategies to increase the M1/M2 ratio in the TME has high clinical significance and benefits macrophage-based cell therapies. Methods: In this study, we reprogrammed antitumor and proinflammatory macrophages ex-vivo with HDAC6 inhibitors (HDAC6i). We administered the reprogrammed macrophages intratumorally as an adoptive cell therapy (ACT) in the syngeneic SM1 murine melanoma model and patient-derived xenograft bearing NSG-SGM3 humanized mouse models. We phenotyped the tumor-infiltrated immune cells by flow cytometry and histological analysis of tumor sections for macrophage markers. We performed bulk RNA-seq profiling of murine bone marrow-derived macrophages treated with vehicle or HDAC6i and single-cell RNA-seq profiling of SM1 tumor-infiltrated immune cells to determine the effect of intratumor macrophage ACT on the tumor microenvironment (TME). We further analyzed the single-cell data to identify key cell-cell interactions and trajectory analysis to determine the fate of tumor-associated macrophages post-ACT. Results: Macrophage ACT resulted in diminished tumor growth in both mouse models. We also demonstrated that HDAC6 inhibition in macrophages suppressed the polarization toward tumor-promoting phenotype by attenuating STAT3-mediated M2 reprogramming. Two weeks post-transplantation, ACT macrophages were viable, and inhibition of HDAC6 rendered intratumor transplanted M1 macrophages resistant to repolarization towards protumor M2 phenotype in-vivo. Further characterization of tumors by flow cytometry, single-cell transcriptomics, and single-cell secretome analyses revealed a significant enrichment of antitumor M1-like macrophages, resulting in increased M1/M2 ratio and infiltration of CD8 effector T-cells. Computational analysis of single-cell RNA-seq data for cell-cell interactions and trajectory analyses indicated activation of monocytes and T-cells in the TME. Conclusions: In summary, for the first time, we demonstrated the potential of reprogramming macrophages ex-vivo with HDAC6 inhibitors as a viable macrophage cell therapy to treat solid tumors.

first: Satish kumar

last: Reddy Noonepalle

first: Maria

last: Gracia-Hernandez

first: Nima

last: Aghdam

first: Michael

last: Berrigan

first: Hawa

last: Coulibaly

first: Xintang

first: Christian

last: Zevallos-Delgado

first: Andrew

last: Pletcher

first: Bryan

last: Weselman

first: Erica

last: Palmer

first: Tessa

last: Knox

first: Eduardo

last: Sotomayor

first: Katherine

middle: B

last: Chiappinelli

first: Duncan

last: Wardop

first: Brett

last: Shook

first: Norman

last: Lee

first: Anatoly

last: Dritschilo

first: Rohan

last: Fernandes

first: Karthik

last: Musunuri

first: Maho

last: Shibata

first: Alejandro

last: Villagra


title: A plastic EMP1⁺ to LGR5⁺ cell state conversion as a therapeutic bypass to KRAS-G12D inhibition in metastatic colorectal cancer

status: Public on Sep 29 2025

city: Barcelona

first: Adria-Jaume

last: Roura

address: Carrer de Baldiri Reixac, 10

country: Spain

institute: IRB Barcelona

summary: Most patients with metastatic colorectal cancer (mCRC) ultimately succumb to the disease due to a lack of effective therapeutic options. A new generation of inhibitors targeting the oncogene KRAS holds promise for treating mCRC and are currently under clinical investigation. In this study, we explore the activity of a small molecule KRAS inhibitor, RM-044, which covalently binds to the G12D mutation in the active (ON) conformation of KRAS. We found that despite robust inhibition of the KRAS signaling pathway, RM-044 exhibited limited therapeutic efficacy in patient-derived models of mCRC. Instead, liver metastases treated with RM-044 transitioned from a transcriptionally poor-prognosis-associated Emp1⁺ cell state to a WNT-driven Lgr5⁺ stem cell-like state that supported tumor growth even in the absence of KRAS-G12D activity. This plastic conversion occurred within hours of treatment, involved a switch in transcription factor usage, and did not require extensive chromatin remodeling. To explore vulnerabilities of RM-044–treated metastases, we engineered mCRC models to express the diphtheria toxin receptor under control of the Lgr5 locus. Enforced conversion of CRC cells to the Lgr5⁺ state via KRAS-G12D inhibition, followed by targeted ablation of this population using diphtheria toxin, produced strong therapeutic effects in the liver metastatic context.

overall_design: CUT&RUN was performed to assess the impact of RM-044 on AKTP MTO. Treatments with either DMSO or RM-044 were administered two days post-seeding, and MTOs were harvested 48 hours later, with a treatment refresh at the 24-hour mark. CUT&RUN was performed in biological duplicates based on previously described protocols (Skene & Henikoff, 2017) with minor modifications.

first: Alessia

last: Centonze

first: Adrià-Jaume

first: Meritxell

last: Novillo-Font

first: Cristina

last: Giordano

first: Xavier

last: Hernando

first: Montserrat

last: Llanses

first: Paula

last: Prats

first: Marta

last: Sevillano

first: Gabriel

last: Pabst

first: Florian

last: Andersch

first: Adrià

last: Cañellas-Socias

first: Chong

last: Zhang

first: Hugh

last: Sparks

first: Chris

last: Dunsby

first: Julien

last: Colombelli

first: Asunción

last: Fernández-Barral

first: Elena

last: Sancho

first: Camille

last: Stephan-Otto Attolini

first: Alberto

last: Muñoz

first: Antonio

last: Barbachano

first: Johannes

last: Zuber

last: Blaj

first: Elsa

last: Quintana

first: Carme

last: Cortina

first: Marc

last: Martí-Renom

first: Eduard

last: Batlle


title: Macrophages Promote Pre-Metastatic Niche Formation of Breast Cancer through AHR Activity

city: Shanghai

first: Xu

last: Jiang

address: 319 Yueyang St

institute: Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences

summary: Macrophages that acquire an immunosuppressive phenotype are crucial for creating the pre-metastatic niche (PMN), which is essential for facilitating breast cancer metastasis to the distant organ. Our study showed that increased aryl hydrocarbon receptor (AHR) activity in lung macrophages is vital for establishing the immunosuppressive PMN in breast cancer. AHR activation led to higher PD-L1 expression on macrophages through directly binding to the promoter of Pdl1, subsequently, promoting Treg cell differentiation in the PMN. Mice with Ahr conditional deletion in macrophages had reduced lung metastasis of breast cancer. The elevated AHR level in PMN macrophages was induced by GM-CSF released from breast cancer cells, which activated STAT5 and prevented AHR ubiquitination in macrophages. In breast cancer patients, the expression of AHR and PD-L1 correlated with Treg cell infiltration, and high AHR expression was associated with a poor prognosis. Our findings uncover a previously unknown cellular and molecular mechanism through which macrophages establish the lung PMN in breast cancer.

overall_design: To investigate the role of increased aryl hydrocarbon receptor (AHR) activity in macrophages in the pre-metastasis niche of breast cancer, we used normal medium or 4T1 conditioned medium with or without AHR inhibitor-CH223191 (10 µM) to treat peritoneal macrophages. No replicates. Then, we performed gene expression profiling analysis using data obtained from RNA seq of these four samples.

first: Jiaqi

last: Wang


title: Parental noncoding RNAs choreograph pre and post fertilization events

city: Kolkata

first: ZHUMUR

last: GHOSH

address: EN 80, Sector V, Bidhan Nagar

institute: Bose Institute

summary: The fusion of two terminally differentiated gametes, a sperm and an oocyte, results in the formation of a totipotent zygote, which is the first step in the development of a mammalian embryo. As the embryo progresses from pre- to post-implantation stage, significant structural and transcriptional changes take place within the embryonic lineage to ensure the success of a series of crucial biological events, such as oocyte activation and maternal-to-zygotic transition coordinated with zygotic gene activation, followed by the first cell-fate decision and lineage-specific differentiation, laying the foundation for the subsequent phase of gastrulation. LncRNAs are mostly transcribed by RNA polymerase II and are longer than 200 nts in length. They can interact with multiple molecules, including DNA, RNA, and protein, for regulating transcription, inducing epigenetic and translational modifications. miRNAs control the regulation of mRNAs by targeting it post transcriptionally. The importance of lncRNAs and miRNAs in germ cell development leads to successful fertilization and subsequently towards post fertilization events. The detail contribution of the maternal and paternal ncRNA profile towards fine tuning these events remain to be deciphered. All these motivated us to look into the parental ncRNA profile within mature fertile sperm and oocyte as well as zygote in mouse system, followed by an in-depth analysis to understand their modes of regulation which finetunes fertilization as well as post fertilization events prior to ZGA.

overall_design: To elucidate the parental contribuiton of long and small non coding RNA signatures during fertilization, we performed long and small RNA expression profiling within mature fertile sperm and oocyte as well as zygote in mouse system

first: Zhumur

last: Ghosh

first: Troyee

last: Das

first: Byapti

first: Arpana

last: Mukherjee

first: Aritra

last: Deb

first: Ayan


title: Hypoxia induces histone clipping and H3K4me3 loss in neutrophil progenitors resulting in long-term impairment of neutrophil immunity [CUT&Run]

city: Edinburgh

first: Manuel

middle: Alejandro

last: Sanchez Garcia

state: Midlothian

address: 4 Little France Drive

country: United Kingdom

institute: University of Edinburgh

summary: A deeper understanding of the processes that sustain long-term reprogramming of rapidly turned-over cells in humans can offer unprecedented therapeutic potential, especially for immune cells like neutrophils, given their short lifespan in circulation and importance in both acute and chronic diseases. Hypoxia is a hallmark of a plethora of disease states and significantly shapes neutrophil effector functions. However, its potential contribution to long-term neutrophil reprogramming remains largely unexplored. We have observed persistent perturbations in newly formed circulating neutrophils’ phenotype and function in patients 3-6 months following hospitalisation with acute respiratory distress syndrome (ARDS), where poor oxygenation takes place.  These lasting changes are associated with widespread loss of H3K4me3 in genomic regions of relevance to neutrophil activities. Notably, the replication of these findings in healthy volunteers months following altitude-induced hypoxaemia attributes a role to the oxygen deprivation as a driver for long-term neutrophil reprogramming. Mechanistically, mouse models of systemic hypoxia reveal deleterious outcomes for infection and show that persistent loss of H3K4me3 originates in proNeu and preNeu populations within the bone marrow and is linked to hypoxia-driven n-terminal histone 3 clipping. Thus, we present novel evidence that acute adaptations to systemic hypoxia trigger central changes in newly formed neutrophil populations, resulting in long-term impairment of effector functions and placing hypoxia as a crucial orchestrator of sustained neutrophil reprogramming.

overall_design: We used Cut&Run to determine H3K4me3 abundance in granulocyte monocyte progenitors (GMP) from mice exposed to hypoxia only (H), LPS nebulization and hypoxia (HLPS) or normoxia (N) for 1 week followed by a recovery period of 5 weeks in normoxia.

last: Sanchez-Garcia

first: Yutaka

last: Negishi

first: Janine

last: Mengede

first: Moira

middle: K

last: Whyte

first: Musa

middle: M

last: Mhalanga

first: Sarah

middle: R

last: Walmsley


title: Transcriptional and functional architecture of the whole neutrophil compartment II

city: New Haven

first: Andrés

last: Hidalgo

address: 10 Amistad Street

institute: Yale University

overall_design: To elucidate the effect of JunB deletion in neutrophils, we conducted Bulk RNA sequencing of CD11b+Ly6G+ cells obtained from the bone marrow,  blood and lungs in wild type (C57BL6) or JunB-defficient nwutrophils during homeostasis

first: Daniela

last: Cerezo-Wallis

first: Ivan

last: Ballesteros

first: Andrea

last: Rubio


title: Novel gene therapy CM-YAPon protects the mouse heart from myocardial infarction - Virus Test

first: Jeffrey

last: Steimle

overall_design: AAV9 virus expressing GFP and YAPon was administrated to P7 mice by intraperitoneal injection. Two weeks later, we collected mouse heart for single nucleus RNA sequencing. Nuclei were collected from the entire heart using a Chromium Nuclei Isolation Kit (10x Genomics, PN-1000493). In total, 20,000 nuclei per sample were loaded onto the 10x Genomics Chromium Controller to obtain the gel beads in emulsion. The sequencing libraries were then prepared according to the manufacturer’s protocols for the Single Cell 3′ Reagents Kit (10x Genomics, Chromium Next GEM Single Cell 3′ Kit version 3.1, 1000269). Sequencing was performed by using the NovaSeq XPlus instrument.

first: Fansen

last: Meng

first: James

last: Martin


title: RNAseq analysis of mouse tumor-infiltrating CD8 T cells WT or KO for AhR

first: Elodie

last: Segura

summary: Cancer immuno-surveillance and response to checkpoint blockade therapy can be affected by multiple environmental factors, including nutrition. However, the direct effects of individual nutrients on anti-tumoral immune responses remain poorly understood. Here we addressed the impact of dietary ligands of Aryl Hydrocarbon receptor (AhR), a transcription factor activated by indoles and tryptophan catabolites generated through food digestion and microbiota metabolism. We show that T cells are the direct cellular targets of dietary AhR ligands. Transcriptomic analysis showed that tonic activation of AhR promotes anti-PD1-mediated reinvigoration of progenitor exhausted CD8 T cells by controlling their metabolic fitness, and licences the functional program of effector CD8 T cells.

overall_design: For RNA-seq analysis of T cells, samples were prepared by pooling tumors from 4 individual mice in each group. Cells were sorted on a FACSAria instrument. Effector CD8 T cells were gated as live CD11b- CD3+ CD4- CD8+ PD1- CD62L- and 'progenitor exhausted' CD8 T cells were gated as live CD11b- CD3+ CD4- CD8+ PD1+ CD62L- Slamf6+.

first: Alba

last: De Juan

first: Lamine

last: Alaoui


title: Hippocampal single-cell RNA Atlas of chronic methamphetamine abuse-induced cognitive decline in mice

city: Guangzhou

first: Hai

last: Qiu

state: Guangdong province

address: 1023 Satai South Road, Jingxi Street

department: Institute of forensic medicine

institute: southern medical university

summary: Chronic methamphetamine abuse induced cognitive decline is a frequent neuropsychiatric disorder with few effective treatments, who threatens human health and social stability and brings huge economic burden to society. We analyzed heterogeneity of mouse hippocampal cells under chronic methamphetamine treatment by scRNAseq to provide new molecular mechanisms of methamphetamine neurotoxicity and reliable, effective and accurate therapeutic targets.

overall_design: Mice were treated by saline or methamphetamine for 4 weeks and joined in cognitive-related behavioral tests. After tests, hippocampal tissues were harvested integrally and dissociated rapidly for scRNAseq.


title: RNA sequestration regulates cell fate (Mouse)

first: Mika

last: Nevo

summary: Recent studies have highlighted the significance of RNA condensates in governing various aspects of cellular RNA processing. Nonetheless, the functional roles and composition of RNA condensates in the context of cell fate specification have remained elusive. In this study, we conducted a comprehensive characterization of the coding and non-coding transcriptome within cytoplasmic condensates known as P-bodies across diverse developmental contexts and vertebrate species. Notably, our investigation unveiled that the dissolution of RNA condensates reprograms both human and mouse pluripotent stem cells into a blastomere-like state. Mechanistically, we observed a high enrichment of untranslated fate-instructive RNAs within P-bodies, subject to cell type-specific sequestration. Employing degron systems, we demonstrated that the acute loss of P-bodies facilitates the translation of stored mRNAs. Furthermore, our research identifies the AGO-miRNAs molecular node as central in orchestrating RNA sequestration in P-bodies in a context-dependent manner. Perturbing AGO proteins, RNA 3’ UTR length, or expression of selected miRNAs, profoundly reshapes the localization of RNAs within cells. Collectively, our findings illuminate how RNA processing within condensates drives essential aspects of lineage-specific cell fate trajectories across vertebrate species.

overall_design: mESC were differentiated into each of the cell types below, and then pbody contents were sorted and sequence, and cytoplasmic fractions were sequenced seperately. Two replicates were sequenced for each sample. In treatment or knockout conditions, each treatment condition was sequenced alongside a control cell line, two replicates of each for pbody and cytoplasm.

first: Patrizia

last: Pessina

first: Eduard

last: Casas


title: A plastic EMP1⁺ to LGR5⁺ cell state conversion as a therapeutic bypass to KRAS-G12D inhibition in metastatic colorectal cancer [Hi-C]

first: Adria-Jaume

last: Roura

summary: Inhibitors of the oncogene KRAS holds promise for treating metastatic CRC (mCRC). Here we show that the activity of a small molecule RAS inhibitor, RM-044, which covalently binds to the G12D mutation in the active (ON) conformation of RAS, demonstrated strong curative effects in CRC models of early liver metastases, but its therapeutic activity was diminished in the advanced metastatic disease. RM-044-treated metastases underwent a fast transition from a poor-prognosis-associated Emp1⁺ transcriptional cell state to a WNT-driven Lgr5⁺ stem cell-like state that supported tumor growth in the absence of RAS G12D activity. This plastic conversion involved a switch in transcription factor usage, and did not require extensive chromatin remodeling. Enforced conversion of metastatic cells to the Lgr5⁺ state via RAS G12D inhibition, followed by genetic ablation of this population, produced strong therapeutic effects. Overall, these findings demonstrate a central role for oncogenic KRAS in governing cellular plasticity in mCRC.

overall_design: Hi-C was performed to assess the impact of RM-044 on AKTP MTO. Treatments with either DMSO or RM-044 were administered two days post-seeding, and MTOs were harvested 48 hours later, with a treatment refresh at the 24-hour mark. In situ Hi-C was performed based on the previously described protocol Rao et al., 2014.

first: Alessia

last: Centonze

first: Adrià-Jaume

first: Meritxell

last: Novillo-Font

first: Cristina

last: Giordano

first: Xavier

last: Hernando

first: Montserrat

last: Llanses

first: Paula

last: Prats

first: Marta

last: Sevillano

first: Mireia

last: Novell

first: Gabriel

last: Pabst

first: Florian

last: Andersch

first: Adrià

last: Cañellas-Socias

first: Chong

last: Zhang

first: Hugh

last: Sparks

first: Chris

last: Dunsby

first: Julien

last: Colombelli

first: Asunción

last: Fernández-Barral

first: Elena

last: Sancho

first: Camille

last: Stephan-Otto Attolini

first: Alberto

last: Muñoz

first: Antonio

last: Barbachano

first: Johannes

last: Zuber

last: Blaj

first: Elsa

last: Quintana

first: Carme

last: Cortina

first: Marc

last: Martí-Renom

last: Batlle


title: RNA sequestration regulates cell fate (Chicken)

first: Mika

last: Nevo

overall_design: The pbody contents of chicken ES cells were sorted using FAPs of LSM14a-GFP expressing cells. Two replicates of p-body contents and two replicates of cytoplasm fractions were sequenced.

first: Patrizia

last: Pessina

first: Eduard

last: Casas

first: Justin

last: Brumbaugh

first: Bruno

last: Di Stefano


title: The Effects of miR-526b, miR655 and COX-2 Overexpression on Oxidative and Genotoxic Stress in MCF7 Breast Cancer Cells

city: Brandon

first: Mousumi

last: Majumder

state: Manitoba

address: 270 18th Street

department: Biology

country: Canada

institute: Brandon University

summary: Breast cancer progression and therapeutic resistance are influenced by the tumor’s ability to adapt to cellular stress, including oxidative damage and chemotherapeutic pressure. Two microRNAs, miR-526b and miR-655, have been shown to promote aggressive breast cancer phenotypes, such as enhanced proliferation, migration, invasion, cancer stem cell characteristics, and metastatic potential, through the activation of the COX-2/EP4/PI3K/Akt signaling pathway. Notably, overexpression of these miRNAs increases total intracellular reactive oxygen species (ROS) production. At the same time, exposure to modest levels of hydrogen peroxide (H₂O₂) further enhances their expression, suggesting a positive feedback loop between oxidative stress and miRNA activity. While their role in driving tumor aggressiveness is established, their influence on therapeutic outcomes remains underexplored. This project investigates how the overexpression of miR-526b, miR-655, and COX-2 in breast cancer cells alters cellular responses to both oxidative stress and the chemotherapeutic agent doxorubicin. By examining how these factors contribute to stress adaptation and drug resistance, the study aims to uncover key mechanisms underlying tumor progression and chemoresistance, with potential implications for improving treatment strategies.

overall_design: The MCF7 breast cancer cell line, as well as three modified MCF7 cell lines (MCF7-miR526b, MCF7-miR655, and MCF7-COX2), were treated with either H2O2 (216 μM), DOX (0.8 μM), or DMSO for 48 hours. Analyzed via paired end sequencing. PolyA-Enriched Illumina NovaSeq PE100 2500M reads.

first: Reid

last: Opperman

first: Sujit

last: Maiti


title: Hypoxia induces histone clipping and H3K4me3 loss in neutrophil progenitors resulting in long-term impairment of neutrophil immunity [ChIP-seq]

first: Manuel

last: Sanchez Garcia

overall_design: We used ChIP-seq to determine locus-specific changes in H3K4me3 levels across the genome in circulating neutrophils from healthy donors and survivors from acute respiratory distress syndrome (ARDS) 3-6 months after hospital admission

last: Sanchez-Garcia

first: Brian

last: Ortmann

first: Niek

last: Wit

first: Jessica

last: Kindrick

first: David

last: Mole

first: Moira

last: Whyte

first: James

last: Nathan

first: Sarah

last: Walmsley


title: Lithium partially rescues gene expression and enhancer activity from heterozygous knockout of AKAP11 while inducing novel differential changes [ChIP-Seq]

city: Montreal

first: Alain

last: Pacis

state: QC

address: 1010 Sherbrooke #1800

institute: Canadian Centre for Computational Genomics (C3G)

summary: Bipolar disorder (BD) is a complex psychiatric condition usually requiring long-term treatment. Lithium (Li) remains the most effective mood stabilizer for BD, yet it benefits only a subset of patients, and its precise mechanism of action remains elusive. Exome sequencing has identified AKAP11 (A-kinase anchoring protein 11) as a shared risk gene for BD and schizophrenia (SCZ). Given that both the AKAP11-Protein Kinase A (PKA) complex and Li target and inhibit Glycogen Synthase Kinase-3 beta (GSK3β), we hypothesize that Li may partially normalize the transcriptomic and/or epigenomic alterations observed in heterozygous AKAP11-knockout (Het-AKAP11-KO) iPSC-derived neurons. In this study, we employed genome-wide approaches to assess the effects of Li on the transcriptome and epigenome of human iPSC-derived Het-AKAP11-KO neuronal culture. We show that chronic Li treatment in this cellular model upregulates key pathways that were initially downregulated by Het-AKAP11-KO, several of which have also been reported as downregulated in synapses of BD and SCZ post-mortem brain tissues. Moreover, we demonstrated that Li treatment partially rescues certain transcriptomic alterations resulting from Het-AKAP11-KO, bringing them closer to the WT state. We suggest two possible mechanisms underlying these transcriptomic effects: (1) Li modulates histone H3K27ac levels at intergenic and intronic enhancers, influencing enhancer activity and transcription factor binding, and (2) Li enhances GSK3β serine 9 phosphorylation, impacting WNT/β-catenin signaling and downstream transcription. These findings underscore Li's potential as a therapeutic agent for BD and SCZ patients carrying AKAP11 loss-of-function variants or exhibiting similar pathway alterations to those observed in Het-AKAP11-KO models.

overall_design: We performed H3K27ac profiling (ChIP-seq) on three AKAP11-KO clones and their three WT counterpart replicates, both untreated and treated with Li (chronic treatment, 11 days, 1mM), to analyze differential acetylation patterns. Specifically, we aimed to (1) identify differentially acetylated peaks in AKAP11-KO-Li compared to AKAP11-KO-UNT, (2) detect peaks in AKAP11-KO-UNT vs. WT-UNT that lost their differential status in AKAP11-KO-Li vs. WT-UNT, and (3) identify new differential peaks in AKAP11-KO-Li vs. WT-UNT that were not differential in AKAP11-KO-UNT vs. WT-UNT.

first: Nargess

last: Farhangdoost

first: Alessia

last: Pietrantonio

first: Yumin

last: Liu

first: Calwing

last: Liao

first: Daniel

last: Rochefort

first: Martin

last: Alda

first: Patrick

last: Dion

first: Boris

last: Chaumette

first: Anouar

last: Khayachi

first: Guy

last: Rouleau


title: Effect of Gum Arabic supplement on Zebrafish gene expression in brain tissues

city: Norwich

first: Justin

last: Abi Assaf

address: Research Park

department: School of Biological Sciences

institute: University of East Anglia

summary: Gum Arabic supplementation in the zebrafish differentially regulates genes related to appetite control and brain function when compared to the control group

overall_design: Fish were either fed a control diet or an experimental diet for 2 weeks before running a RNA-seq experiment on their brain tissues


title: Single cell profiling reveals three endothelial to hematopoietic transitions with divergent isoform expression landscapes [RNA-seq]

city: Manchester

first: Georges

last: Lacaud

state: England

address: Wilmslow Raod

department: Stem Cell Biology

institute: Cancer Research UK Manchester Institute

summary: Hemogenic endothelium (HE) is recognized as the origin of all definitive blood cells including hematopoietic stem cells (HSC). However, the mechanisms governing the hematopoietic progenitor versus HSC fate choice within the HE remain unknown. To explore this, we combined differentiation assays with full-length single-cell transcriptome data for extra-embryonic yolk sac (YS) and intra-embryonic aorta–gonad–mesonephros (AGM) region HE populations. Here we identify and localized three differentiation trajectories, each containing a distinct HE subset: erythro-myeloid progenitor-primed HE in the YS plexus, lympho-myeloid progenitor-primed HE in large YS arteries, and HSPC-primed HE in the AGM. Chromatin modifiers and spliceosome components were enriched in AGM HE. This correlated with a higher isoform complexity of the AGM HE transcriptome. Distinct HEAGM specific isoform expression patterns were observed for a broad range of genes, including stemness-associated factors like Runx1. Our data forms a unique resource for studying cell fate decisions in different HE populations.

overall_design: Long Read single cell RNA-seq of cells undergoing hemogenic to endothelial transition in AGM at E9.5 and E10.5 and Yolk Sac (YS) at E9, E9.5, E10.5. To study the HE populations present at different anatomical locations and time points during the mouse development, we used transgenic reporter mouse lines GFI1-GFP and RUNX1B-RFP and performed Oxford Nanopore Long Read Sequencing (single cell RNA seq) on FACS isolated E9.5 and E10.5 AGM HE cells (RUNX1+, KIT-,CDH5+, GFI1+/-, CD41-, CD45-), and E9, E9.5, E10.5 YS HE cells (RUNX1+, KIT+, GFI1+/-, CD31+, CD41-, CD45-). Based on analyses of GEO accession GSE274544 cells selected for nanopore sequencing came from HEAGM (clusters AGMc3 andAGMc4), HEYSA (cluster YSm1 and YSm2), HEYSP (clusters YSb2 and YSb3) early yolk sac hematopoietic progenitor cluster p1 and p2

first: Wen Hao

last: Neo

first: Muhammad Zaki Hidayatullah

last: Fadlullah

first: Harshangda

last: Bhatnagar

first: Cristiana

last: Barone

first: Giulia

last: Quattrini

first: Filipa

last: Timóteo-Ferreira

first: Joana

last: Carrelha

first: Gianluca

last: Sala

first: Robert

last: Sellers

first: John

last: Weightman

first: Wolfgang

last: Breitwieser

first: Natalia

last: Moncaut

first: Roshana

last: Thambyrajah

first: Sten Eirik

middle: W

last: Jacobsen

first: Mudassar

last: Iqbal

first: Syed

last: Baker

first: Emanuele

last: Azzoni

first: Michael

last: Lie-A-Ling


title: A neuroepithelial rheostat controls enteric type 1 and type 2 immunity [bulk RNA-seq]

city: Lisbon

first: Eric

last: de Sousa

address: Champalimaud Centre for the Unknown, Avenida Brasília

department: Champalimaud Research

country: Portugal

institute: Champalimaud Foundation

summary: The nervous and immune systems cooperate to regulate mucosal barrier integrity and host defence. Nevertheless, whether enteric neurons establish neuroepithelial interactions to coordinate immunity remains elusive. Here, we identified a neuroepithelial rheostat that differentially controls type 1 and type 2 immunity. Gut epithelial cells displayed type 1 and type 2 alarmins in distinct circadian cycles, and co-expressed vasoactive intestinal peptide (VIP) receptor 1 (VIPR1), a molecule that integrates diurnal neural-derived VIP signals. Chemogenetic modulation of enteric VIPergic neurons led to altered epithelial-derived cytokines and tuft cells. Epithelial-intrinsic deletion of Vipr1 resulted in diminished type 1 immunity signatures, including reduced type 1 alarmins and intra-epithelial lymphocytes (IEL). In contrast, Vipr1 deficiency in epithelial cells led to increased intestinal type 2 immunity, comprising type 2 alarmins, tuft cells and activated group 2 innate lymphoid cells (ILC2). Disruption of these neuroepithelial interactions led to increased susceptibility to bacterial infection, which greatly contrasted with an increased resistance to parasite infection. Our work identifies a neuroepithelial hub that distinctively controls enteric type 1 and type 2 immunity, deciphering a multi-tissue hub that integrates diurnal physiological cues to balance gut immunity and host defence.

overall_design: Intestinal epithelial cells from the duodenum and ileum of C57BL/6J mice were isolated by FACS

first: Roksana

last: Pirzgalska

first: Beatriz

last: Henriques-Alves

first: Bruno

last: Raposo

first: Júlio

last: Torres

first: Jaechan

last: Ryu

first: Cristina

last: Godinho-Silva

first: Miguel

last: Rendas

first: Madalena

last: Pereira

first: Inês

last: Godinho

first: Hélder

last: Ribeiro

first: Vasco

last: Correia

first: Manuel

last: Jakob

first: Patrycja

last: Forster

first: Christoph

last: Wilhelm

last: Klose

first: Ricardo

last: Rio-Tinto

first: Verena

last: Link

first: Tânia

last: Carvalho

first: Carlos

last: Minutti

first: Manuela

last: Ferreira

first: Henrique

last: Veiga-Fernandes


title: A neuroepithelial rheostat controls enteric type 1 and type 2 immunity [scRNA-seq]

first: Eric

last: de Sousa

overall_design: Intestinal segments (duodenum and ileum; ~50mg) from C57BL/6J, Vipr1fl and Vipr1∆Villin mice were analyzed using scRNA-Seq

first: Roksana

last: Pirzgalska

first: Beatriz

last: Henriques-Alves

first: Bruno

last: Raposo

first: Júlio

last: Torres

last: Ryu


title: RNA-seq profiling of A375 melanoma cells treated with Salvianolic Acid B

city: Beijing

first: Yongli

last: Zhang

address: Sunny South Street, Fangshan District

institute: Beijing University of Chinese Medicine

summary: This dataset describes the transcriptomic profiling of A375 human melanoma cells treated with Salvianolic Acid B (Sal B), a bioactive compound derived from Salvia miltiorrhiza. A375 cells were cultured under standard conditions and treated with Sal B at concentrations of 100 µM and 200 µM for 48 hours, with an untreated control group included for comparison. Total RNA was extracted from each group and subjected to RNA-seq analysis to investigate global gene expression changes induced by Sal B. The goal of this study was to identify differentially expressed genes and pathways related to glycolysis inhibition and immune modulation, providing insight into the potential anti-tumor mechanisms of Sal B in melanoma. This dataset may serve as a resource for exploring the metabolic and immunological effects of Salvianolic Acid B in melanoma cells.

overall_design: A total of nine samples were collected from A375 human melanoma cells divided into three groups: untreated control (Con), Salvianolic Acid B low-dose treatment (SalB 100 µM), and high-dose treatment (SalB 200 µM). Each group included three biological replicates. Cells were treated for 48 hours under standard culture conditions (37°C, 5% CO₂). Total RNA was extracted using TRIzol reagent, and RNA quality was verified by Bioanalyzer. Libraries were prepared using a standard mRNA-seq protocol and sequenced on an Illumina platform to generate paired-end reads.

first: Cang

last: Li

first: Jianxin

last: Chen


title: A plastic EMP1⁺ to LGR5⁺ cell state conversion as a therapeutic bypass to KRAS-G12D inhibition in metastatic colorectal cancer [RNA-Seq human]

first: Adria-Jaume

last: Roura

overall_design: PDOs were cultured in 70% cold BME/medium drops and complete organoid CRC tumor medium (CTM) containing AdvancedDMEM/F12, Hepes, Glutamax, B27, Nicotinamide 10mM, N-Acetyl- cystein 1.25nM, 50 ng/ml EGF, R-spondin1 1ug/ml, Noggin 100 ng/ml, SB202190 10 uM, Gastrin 10nM, A83-01 500nM, ProstaglandinE2 2.5 uM and Normocin 100 ug/ml. For RM-044 experiments, 7-day grown organoids were disaggregated with TrypLE™, counted and plated in 70% BME at a density of 750 cell/µl for P26 and PDO131 and 250 cell/µl for C31M in complete CTM with rock inhibitor Y27632 10uM. After three days, media was changed to CTM without rock inhibitor and cells were treated with RM-044 100nM or DMSO with a daily media change. Samples for RNA-sequencing were collected at day 4 after treatment and growth curves were measured during 10 days from treatment start.

first: Alessia

last: Centonze

first: Adrià-Jaume

first: Meritxell

last: Novillo

first: Cristina

last: Giordano

first: Xavier

last: Hernando

first: Montserrat

last: Llanses

first: Paula

last: Prats

first: Marta

last: Sevillano

first: Mireia

last: Novell

first: Gabriel

last: Pabst

first: Florian

last: Andersch

first: Adrià

last: Cañellas-Socias

first: Chong

first: Hugh

last: Sparks

first: Chris

last: Dunsby

first: Julien

last: Colombelli

first: Asunción

last: Fernández-Barral

first: Elena

last: Sancho

first: Camille

last: Stephan-Otto Attolini

first: Alberto

last: Muñoz

first: Antonio

last: Barbachano

first: Johannes

last: Zuber

last: Blaj

first: Elsa

last: Quintana

first: Carme

last: Cortina

first: Marc

last: Martí-Renom

first: Eduard

last: Batlle


title: A plastic EMP1⁺ to LGR5⁺ cell state conversion as a therapeutic bypass to KRAS-G12D inhibition in metastatic colorectal cancer [RNA-Seq mouse]

first: Adria-Jaume

last: Roura

overall_design: RNA sequencing was performed to assess the impact of RM-044 on AKTP MTO and APK CTO. Treatments with either DMSO or RM-044 were administered two days post-seeding, and MTOs were harvested 48 hours later, with a treatment refresh at the 24-hour mark. RNA extraction was carried out using the PureLink™ RNA Mini Kit (Life Technologies, 12183025) according to the manufacturer’s instructions. Elution was performed with distilled water (Invitrogen, 10977-035), and RNA concentrations were measured using a Nanodrop 1000 spectrophotometer (ThermoFisher). Library preparation was conducted using the RNA-seq Stranded mRNA Library Illumina kit, followed by sequencing on the Illumina NextSeq 2000 P3 platform.

first: Alessia

last: Centonze

first: Adrià-Jaume

first: Meritxell

last: Novillo

first: Cristina

last: Giordano

first: Xavier

last: Hernando

first: Montserrat

last: Llanses

first: Paula

last: Prats

first: Marta

last: Sevillano

first: Mireia

last: Novell

first: Gabriel

last: Pabst

last: Zhang


title: A plastic EMP1⁺ to LGR5⁺ cell state conversion as a therapeutic bypass to KRAS-G12D inhibition in metastatic colorectal cancer [RNA-Seq]

overall_design: RNA sequencing was performed to assess the impact of RM-044 or MRTX on AKTP MTO and APK CTO. Treatments with either DMSO or RM-044 were administered two days post-seeding, and MTOs were harvested 48 hours later, with a treatment refresh at the 24-hour mark. RNA extraction was carried out using the PureLink™ RNA Mini Kit (Life Technologies, 12183025) according to the manufacturer’s instructions. Elution was performed with distilled water (Invitrogen, 10977-035), and RNA concentrations were measured using a Nanodrop 1000 spectrophotometer (ThermoFisher). Library preparation was conducted using the RNA-seq Stranded mRNA Library Illumina kit, followed by sequencing on the Illumina NextSeq 2000 P3 platform.

first: Florian

last: Andersch

first: Adrià

last: Cañellas-Socias

first: Chong

first: Hugh

last: Sparks

first: Chris

last: Dunsby

first: Julien

last: Colombelli

first: Asunción

last: Fernández-Barral

first: Elena

last: Sancho

first: Camille

last: Stephan-Otto Attolini

first: Alberto

last: Muñoz

first: Antonio

last: Barbachano

last: Zuber


title: Single-Cell Splicing Isoform Atlas of the Adult Human Heart and Heart Failure

city: Evanston

first: Timothy

last: Pan

state: IL

address: 633 Clark St

institute: Northwestern University

summary:
BACKGROUND:
Alternative splicing plays crucial roles in normal heart development and cardiac disease by influencing protein-coding sequences, functional domains, and molecular networks. However, a detailed characterization of the human heart isoform landscape remains incomplete.
METHODS:
Leveraging long-read single-nucleus RNA sequencing and computational analysis, we dissected full-length isoform heterogeneities, expression patterns, and usage shifts across cell types, cell states, and cardiac conditions of the adult left ventricle. We applied in silico approaches to assess the functional relevance of identified isoforms; validated isoform compositions of representative cardiac genes using reverse transcription quantitative polymerase chain reaction and targeted amplicon sequencing; and developed a web server for interactive navigation of our results.
RESULTS:
The data revealed that isoform heterogeneity is widespread in the cardiac cellular system, serving as a posttranscriptional buffer system that calibrates the molecule reservoirs in human hearts. In healthy left ventricles, ≈30% of cell type–specific genes were polyform, using multiple isoforms tailored to cell type–specific programs. Among ubiquitously expressed genes, >300 showed differential isoform usage with cell type specificity. Compared with heart failure, 379 genes in cardiomyocytes demonstrated marked isoform usage shifts, most of which are predicted to change protein coding outcomes through direct changes in protein coding sequences and switches between intron retention and non–protein-coding biotypes. In contrast, cell state–specific programs tend to operate on monoform genes associated with changes among cell states. In addition, our data revealed heart failure–associated differential isoform usage events in stromal and immune cell types in the cardiac microenvironment.
CONCLUSIONS:
We present a comprehensive atlas of splicing isoforms in the normal adult heart and heart failure through long-read single-nucleus RNA sequencing and comprehensive computational analyses. The results suggest crucial roles of isoforms in buffering core cellular programs and contributing to disease-associated cell states. The full-length details of these cell-specific isoforms serve as an important reference for downstream translational and mechanistic studies and are available on our online data portal at https://github.com/gaolabtools/heart-isoform-atlas.

Github repo: https://github.com/gaolabtools/heart-isoform-atlas
doi: 10.1161/CIRCULATIONAHA.125.074959

overall_design: Single-nucleus Nanopore RNA-sequencing of 12 frozen human left ventricle samples (6 non-diseased, 6 ischemic cardiomyopathy)

first: Lina

last: Lu

first: Keith

last: Youker

first: Cheng-Kai

last: Shiau

first: Minhua

last: Wang

first: Hsiao-Yun

last: Lin

first: Anh

last: Nguyen

first: Yueying

last: He

first: Eric

last: Tong

first: Rajul

last: Ranka

first: Yuanqing

last: Yan

first: Arjun

last: Sinha

first: Ankit

last: Bharat

first: Todd

last: Eagar

first: Jane

last: Wilcox

first: Arvind

last: Bhimaraj

first: Ruli

last: Gao


title: A plastic EMP1⁺ to LGR5⁺ cell state conversion as a therapeutic bypass to KRAS-G12D inhibition in metastatic colorectal cancer [scRNA-Seq]

first: Adria-Jaume

last: Roura

overall_design: FACS-sorted Epcam+/CD45- epithelial cells were concentrated by centrifugation (500g for 7 min at 4ºC) and counted. Cells were partitioned into Gel Bead In Emulsions (GEMs) with a target of 5.000 cells per sample using the Chromium Controller system (10X Genomics). cDNA sequencing libraries were prepared using the Chromium Single Cell 3ʹ Kit v3 (PN-1000075). cDNA was amplified, quality checked, and quantified on an Agilent Bioanalyzer High Sensitivity chip (Agilent Technologies), then used for Gene Expression (GEX) library preparation (PN-220103 Chromiumi7 Sample Index Plate). Final libraries were indexed by PCR and verified for size and concentration. Sequencing was performed on an Illumina NovaSeq 6000 to obtain approximately 40.000 reads per cell.

first: Alessia

last: Centonze

first: Adrià-Jaume

first: Meritxell

last: Novillo

first: Cristina

last: Giordano

first: Xavier

last: Hernando

first: Montserrat

last: Llanses

first: Paula

last: Prats

first: Marta

last: Sevillano

first: Mireia

last: Novell

first: Gabriel

last: Pabst

first: Florian

last: Andersch

first: Adrià

last: Cañellas-Socias

first: Chong

last: Zhang

first: Hugh

last: Sparks

first: Chris

last: Dunsby

first: Julien

last: Colombelli

first: Asunción

last: Fernández-Barral

first: Elena

last: Sancho

first: Camille

last: Stephan-Otto Attolini

first: Alberto

last: Muñoz

first: Antonio

last: Barbachano

first: Johannes

last: Zuber

last: Blaj

first: Elsa

last: Quintana

first: Carme

last: Cortina

first: Marc

last: Martí-Renom

first: Eduard

last: Batlle


title: Progesterone receptor and the RNA polymerase III subunit POLR3A co-occupy tRNAs in breast cancer cells

city: Aurora

first: Jessica

last: Finlay-Schultz

state: CO

department: Pathology

institute: University of Colorado Denver Anschutz Medical Campus

summary: Progesterone receptors (PR) can regulate transcription by RNA Polymerase III (Pol III), which transcribes small non-coding RNAs, including all transfer RNAs (tRNAs). This and previous work demonstrated that PR associates with the Pol III complex at tRNA genes and that progestins downregulate tRNA transcripts in breast tumor models.

overall_design: ChIP-seq for progesterone receptor (PR) and RNA Polymerase III subunit POLR3A in T47D breast cancer cells with 1h treatment with ethanol vehicle (Veh) or progesterone (P4).

first: Kiran

last: Paul

first: Lynsey

last: Fettig

first: Peter

last: Kabos

first: Carol

last: Sartorius


title: Allogeneic iPSC-derived iNKT cells in recurrent head and neck cancer: a phase 1 trial

status: Public on Sep 28 2025

city: Chiba

first: Takahiro

last: Aoki

state: Chiba

address: 1-8-1 Inohana Chuo-ku

institute: Chiba University

summary: Invariant Natural killer T (iNKT) cells exhibit cytotoxic activity and immunomodulatory functions and have gained interest in cancer immunotherapy. We conducted a phase 1, first-in human clinical trial to evaluate the safety and efficacy of clinical-grade allogeneic iNKT cells generated from induced pluripotent stem cells (iPSC-iNKT cells) in patients with recurrent head and neck cancer (jRCT2033200116). The primary endpoint was the incidence of dose-limiting toxicity (DLT). The secondary endpoints were to assess safety and efficacy, as well as to evaluate immunological dynamics. iPSC-iNKT cells were administered intra-arterially to 10 patients.  One subject developed grade 3 skin rash at the second dose, identified as DLT. No other severe adverse events were observed in any patients. Tumor progression was suppressed in two patients, in whom clonal expansion of memory- and effector-phenotype CD8+ T cells was observed, along with activation of the IFN-γ signaling pathway. Here, we show that iPSC-iNKT cells are safe and possess therapeutic potential as an immunotherapy for solid tumors.

overall_design: scRNA-seq and scVDJ-seq of 9 PBMC samples collected from 3 patients before treatment and 3 days and 21 days after treatment

first: Tomohisa

last: Iinuma

first: Haruhiko

last: Koseki

first: Shinichiro

last: Motohashi


title: A plastic EMP1⁺ to LGR5⁺ cell state conversion as a therapeutic bypass to KRAS-G12D inhibition in metastatic colorectal cancer [ATAC-Seq]

first: Adria-Jaume

last: Roura

overall_design: ATAC-seq was performed to assess the impact of RM-044 on AKTP MTO. Treatments with either DMSO or RM-044 were administered two days post-seeding, and MTOs were harvested 48 hours later, with a treatment refresh at the 24-hour mark. ATAC-seq was performed based on previously described protocols (Buenrostro et al., 2015 and Corces et al., 2017).

first: Alessia

last: Centonze

first: Adrià-Jaume

first: Meritxell

last: Novillo-Font

first: Cristina

last: Giordano

first: Xavier

last: Hernando

first: Montserrat

last: Llanses

first: Paula

last: Prats

first: Marta

last: Sevillano

first: Mireia

last: Novell

first: Gabriel

last: Pabst

first: Florian

last: Andersch

first: Adrià

last: Cañellas-Socias

first: Chong

last: Zhang

first: Hugh

last: Sparks

first: Chris

last: Dunsby

first: Julien

last: Colombelli

first: Asunción

last: Fernández-Barral

first: Elena

last: Sancho

first: Camille

last: Stephan-Otto Attolini

last: Muñoz


title: Spatial Transcriptomics of Controlled vs Uncontrolled Tuberculosis in a Mouse Model

city: Mountain View

first: Lester

last: Kobzik

state: California

address: 320 Logue Avenue

institute: Cellecta, Inc

summary: To further characterize gene expression in controlled vs uncontrolled tuberculosis lung lesions, we performed spatial transcriptomics analysis using the NanostringGeoMX Digital Spatial Profiler (DSP) system.

overall_design: Based on routine hematoxyline and eosin histopathology, we selected lungs from 2 mice with paucibacillary controlled lesions and 2 mice with advanced uncontrolled multibacillary lesions with necrotic areas. Additional FFPE sections were stained with fluorescent CD45-, pan-keratin-specific antibodies and DAPI. Diseased regions of interest (ROI) were selected to focus on myeloid-rich areas avoiding necrosis and tertiary lymphoid tissue . Eight ROI each of contolled and uncontrolled lesions (respectively) were studied. The profiling used the Mouse Whole Transcriptome Atlas (WTA) panel which targets ~21,000+ transcripts for mouse protein coding genes

first: Igor

last: Kramnik


title: Aging associated dysfunction and disease mediated by epigenetic dysbiosis

city: Kiel

first: Seyed Mohammd Mahdi

last: Rasa

state: Schleswig-Holstein

address: Michaelisstrasse 5

department: Institut for Immunology

institute: Universitätsklinikum Schleswig-Holstein

type: Methylation profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Effect of conditional triple-knockout of Jmjd2a/Kdm4a, Jmjd2b/Kdm4b and Jmjd2c/Kdm4c or overexpression of KDM4C/JMJD2C mutants with different phase separation ability on gene expression in mouse embryonic stem cells. [RNA-Seq]

first: Xinyi

last: Liu

state: Guangdong

address: No. 74, 2nd Zhongshan Road

institute: Sun Yat-Sen University

summary: To investigate the functions of JMJD2/KDM4 family in mouse embryonic stem cells (mESCs), we treat the the conditional triple-knockout  mESCs of Jmjd2a/Kdm4a, Jmjd2b/Kdm4b and Jmjd2c/Kdm4c with 4-hydroxytamoxifen (4-OHT) for 72h. Further, to investigate whether JMJD2/KDM4 phase separation affects 2-Cell-like cells (2CLCs) induction, we generated IDR-truncated JMJD2C/KDM4C (Mut) to disrupt its phase separation ability, and rescued it by by fusing two IDRs in other species (hIDR1 and hIDR2). We overexpressed those phase separation mutants in the Zscan4-GFP::Mervl-tdTomoto mESCs separately.

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of 8 different cells (Ctrl (WT mESC), JMJD2 TKO, JMJD2C FL (Full length), JMJD2C Mut (IDR truncated), Mut-hIDR1 (rescued by hIDR1, from human HNRNPA), hIDR1, Mut-hIDR2 (rescued by hIDR2, from human FUS), hIDR2.

first: Shaoshuai

last: Jiang


title: A plastic EMP1⁺ to LGR5⁺ cell state conversion as a therapeutic bypass to KRAS-G12D inhibition in metastatic colorectal cancer

city: Barcelona

first: Adria-Jaume

last: Roura

address: Carrer de Baldiri Reixac, 10

institute: IRB Barcelona