title: Limited Immune Perturbations in Mice Exposed to Sustained Low-Dose Ionizing Radiation

city: Ottawa

first: Seung-Hwan

last: Lee

state: Ontario

address: 451 Smyth Road

department: Biochemistry, Immunology and Microbiology

institute: University of Ottawa

summary: The effects of high-dose ionizing radiation (HDIR) exposure on the immune system are largely understood with consensus, yet there remains a fragmented understanding of the impact of low-dose ionizing radiation (LDIR) on immune homeostasis, especially in sustained exposure conditions. This study investigates the effects of continuous LDIR exposure on the murine immune system, focusing on transcriptomic responses and cellular perturbations following low-dose-rate whole-body 𝛾-radiation. Female 18-week-old C57BL/6 mice were continuously exposed to low-dose-rate 60Co radiation over a period of 7 days, resulting in cumulative absorbed doses of 10 mGy and 100 mGy. Our findings indicate that the LDIR exposure induced, at most, only minimal transcriptomic perturbations to the immune system in C57BL/6 mice. These results suggest a preservation of immune cell homeostasis under the sustained low-dose-rate exposure conditions studied. It contributes to a broader understanding of radiation biology, emphasizing that the effects of LDIR on the immune system can be limited at low-dose-rates in mice.

overall_design:
Eighteen-week-old female C57BL/6 mice (Jackson Laboratories) were housed and irradiated at the CNL (Chalk River, Ontario, Canada). Whole-body gamma irradiation was administered using a Nordion (Ottawa, Ontario, Canada) Gammabeam 150C irradiator equipped with a Cobalt-60 source. The irradiation was continuous for 7 days in the pathogen-free irradiation hall of the CNL. Treatment groups were positioned at varying distances from the irradiator to achieve different dose rate exposures. Mice receiving a cumulative dose of 10 mGy (n=6) received an average dose rate of 0.0614mGy/hr. Mice receiving a cumulative dose of 100 mGy (n=6) received an average dose rate of 0.625mGy/hr. The positioning ensured the beam targeted the mid-level of each cage, centered between the two cages. Dosimetry was performed using an Exradin A8 ion chamber and a Supermax electrometer. Control mice (n=6) were housed separately in a conventional animal room.
***Note: Raw FASTQ files for two samples (10 mGy biological replicates 1 and 5 (B1 & B6)) were irretrievably lost due to a storage system failure at the Digital Research Alliance of Canada HPC. Processed TPM values for these samples are retained in the submitted matrix and were generated prior to the data loss. Furtermore, Kallisto abuindances.tsv were retained and submitted for all files.

first: Bryan

last: Marr

first: Holly

last: Laakso

first: Melinda

last: Blimkie

first: Andrew

last: Cao

first: Abrar Ul Haq

last: Khan


title: Molecular disruptions to the future fetal tissue lineage are associated with delayed euploid blastulation

status: Public on Apr 14 2026

city: Lone Tree

first: Mary

middle: E

last: Haywood

state: CO

address: 10290 RidgeGate Circle

department: Genetics

country: USA

institute: CCRM

type: Expression profiling by high throughput sequencing

summary: Objective: To study the underlying molecular activity driving the reduced reproductive potential of Day 7 euploid blastocysts. Design: Euploid inner cell mass and trophectoderm transcriptome analysis examined in association with timing of blastulation. Subjects: Equivalent grade and maternal age-matched Day 5 (n=20) and Day 7 (n=20) surplus cryopreserved euploid blastocysts donated with informed patient consent. Intervention: None Main Outcome Measures: RNA sequencing was used for transcriptome analysis, followed by enriched pathways and gene ontology terms to determine functional relevance. Quantitative real-time PCR was performed for targeted gene expression analysis and validation of differentially expressed genes identified by RNA sequencing. Results: Timing of blastocyst development leads to significant differences in transcription for equivalent grade, maternal age-matched euploid blastocysts. Controlling for cellular lineage, 2880 differentially expressed genes were identified in the Day 7 inner cell mass (padj≤0.05) and 2057 genes in the Day 7 trophectoderm (padj≤0.05), compared to their equivalent grade Day 5 counterparts. However, pathway enrichment was predominantly driven by downregulated gene expression in the inner cell mass of Day 7 blastocysts (padj≤0.05) impacting signal transduction pathways and genes involved in primitive endoderm development. Slower developing blastocysts appear to face metabolic and endoplasmic reticulum stress, leading to increased protein degradation in the inner cell mass, irregular apoptotic activity, as well as compromised trophectoderm epigenetic regulation. Conclusions: The Day 7 inner cell mass transcriptome revealed disproportionate perturbations involving the downregulation of many critical networks and genes required for embryogenesis, providing an array of molecular mechanisms responsible for developmental delay. With no significant impact on Day 7 trophectoderm pathways, reproductive potential of the blastocyst may be primarily driven by the molecular activity of the future fetal tissue lineage, thereby explaining the halving of live birth rates following euploid Day 7 blastocyst transfers. Defining the network of embryonic pathways essential for developmental competence will allow for the future development of culture systems to support optimal extended culture for poor-prognosis patients.

overall_design: RNA-seq profiling of euploid Day 5 and Day 7 blastocysts separated into inner cell mass (ICM, n = 20 D5, n = 20 D7) and trophectoderm (TE, n = 20 D5, n = 20 D7)

first: Blair

middle: R

last: McCallie

first: Michelle

middle: M

last: Denomme

first: Kalyn

last: Trowbridge

first: Jennifer

last: Hamm

first: Keelee

middle: J

last: McCarty

first: William

middle: B

last: Schoolcraft

first: Mandy

middle: G

last: Katz-Jaffe


title: Reduction of SynGAP-γ, disrupted splicing of Agap3, and oligodendrocyte deficits in Srrm2 mice, a genetic model of schizophrenia and neurodevelopmental disorder

city: Cambridge

first: Sameer

last: Aryal

state: Massachusetts

address: 75 Ames St

institute: Broad Institute of MIT and Harvard

summary: To investigate the effect of loss-of-function of the schizophrenia and DD/ID risk gene SRRM2, we performed single nucelus RNA-Sequencing in the PFC and the striatum of 1-month-old Srrm2 +/- (heterozygous) mutant mice and wild-type littermate controls.

overall_design:
Bulk RNA-Seq of eight brain regions—cerebellar cortex (CC), cerebellar vermis (CV), prefrontal cortex (PFC), striatum (ST), hippocampus (HP), substantia nigra (SN), somatosensory cortex (SSC), and thalamus (TH)—from Srrm2 heterozygous mutant mice and wild-type littermate controls at 1 and 3 months of age.
Single nucleus RNA-Seq of two brain regions—prefrontal cortex (PFC), striatum (STR)—from Srrm2 heterozygous mutant mice and wild-type littermate controls at 1 month of age.


title: Generation of Stable Chambered Cardioids from human Pluripotent Stem Cells [scRNA-seq]

city: Hangzhou

first: XINLE

last: ZOU

address: No 600 Dunyun Road

country: China

institute: WESTLAKE UNIVERSITY

summary: The human heart is a vital organ under intense investigation for the hope to improve health and longevity. Recent advances to generate cardiac organoids (cardioids) provide unprecedented opportunities to cardiac research. However, achieving stable chambered cardioids structures remains a challenge, thus limiting its application in disease modeling and drug screening. In this study, we address this challenge by orchestrating cardiac signaling pathways, including synergistic modulation of FGF and Wnt signaling via the HAND1 transcription factor. We have successfully demonstrated the formation of stable chambered cardioids structured with endocardium, myocardium, and epicardium, mimicking early cardiac development with well-organized structural components. Furthermore, we demonstrate the feasibility to leverage this platform for drug toxicity screening. Our findings provide a system to understand human heart development and generate models for cardiac tissue engineering and regenerative medicine.

overall_design: There are three groups in this study: 2D cultured tissue, stable chambered cardioids with PD173074 treatment, and stable chambered cardioids without PD173074 treatment. All samples were collected for single-cell sequencing on day 42.5.

first: FANWEN

last: WANG

first: HUILING

last: ZHENG


title: Mitochondrial metabolism regulates the immunogenic responsiveness of dendritic cells [RNA-seq]

city: MADRID

first: Ignacio

last: Heras-Murillo

state: MADRID

address: c/Melchor Fernandez Almagro, 3

country: Spain

institute: CNIC

summary: Activation of dendritic cells (DCs) is linked to increased glycolysis. However, the role of the mitochondrial electron transport chain (ETC) in conventional DC subset function is poorly defined. Here, we revealed that immunogenic activation elevates late oxygen consumption in cDC1s, but not cDC2s. To investigate the relevance of the ETC in DC immunogenicity, we generated mice with impaired ETC complex III function in DCs. ETC complex III impairment attenuated poised or adjuvant-triggered cDC1 activation, migration and capability to prime T cells for anti-cancer immunity, while it had a mild effect on cDC2s. Mechanistically, loss of complex III in cDC1s led to a deregulated redox and metabolite balance causing DNA hypermethylation of PU.1-binding regions. This DNA hypermethylation diminished early stimulus-induced gene expression linked to immunogenic responsiveness of cDC1s, which was rescued via ectopic expression of alternative oxidase. Our findings show how the ETC differentially regulates the immunogenic function of cDC subsets.

overall_design: 50 ug poly(I:C) LMW and 50 ug R848 or the control PBS were intravenously administered into Uqcrqf/f or Itgax-Uqcrqf/f mice and, after 1.5 hours, the spleen  was harvested and processed. CD8+ cDC1 and CD11b+ cDC2 were FACS sorted, the RNA extracted and used for RNA sequencing.

first: Stefanie

middle: K

last: Wculek

first: David

last: Sancho


title: Age influences CMV-specific NK and CD8 T cell memory fates (NK cells)

city: New York

first: Isaac

last: Jensen

state: NY

address: 701 West 168th St

institute: Columbia University

summary: Cytomegalovirus (CMV) infection is ubiquitous; though often unnoticed in healthy adults it is a leading cause of neurodisability in neonates/ infants. NK and CD8 T cell cytolysis control CMV; yet their coordinate response to CMV remains uncharacterized, particularly in infants. We interrogated how NK and CD8 T cell responses differed between adults and infants in a murine CMV (MCMV) infection model. NK and CD8 T cells both limited infant MCMV infection morbidity. Notably, adult and infant NK cells effectuate discrete responses to MCMV infection with the latter preferentially forming memory associated with transcriptional distinctions prior to infection. Adoptive transfer studies further revealed that infant T cells are effector-biased but acquire the capacity to form memory in an age-dependent manner. Infant T cells' restricted ability to form memory was associated with a unique transcriptional state before and after infection. Further, acquisition of T cell memory capacity coincided with an age-dependent waning in the induction of adaptive NK cells. Our data suggests non-redundant roles for NK and CD8 T cells in MCMV infection during infancy, as NK cells compensate for the limited establishment of memory T cell subsets.

overall_design: Adult (8wk old) and Infant (10dpn) mice were infected with MCMV-K181 by i.p. injection. Splenic NK cells were enriched prior to infection (D0), 7 days post-infection (D7), 15 days post-infection (D15). Cells stained with CITE-Seq antibodies and sequenced. Infant cells are CD45.1, Adult cells are CD45.2, individual mice are for adult and infant mice are identified through hashtag ADTs

first: Donna

middle: L

last: Farber


title: RNA-seq of DLX6 KD A549 cells vs. control A549 cells.

city: JIANGSU

first: J

last: ff

address: JIANGSU NANJING

institute: Jiangsu Cancer Hospital

summary: To investigate the gene expression characteristics of the transcription factor DLX6, we performed RNA-seq analysis in A549 cells to examine changes in the gene expression profile following DLX6 knockdown.

overall_design:
Comparative gene expression profiling analysis of RNA-seq data of DLX6 KD A549 cells versus control A549 cells.

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Submitter states that missing raw files are due to file loss.
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first: Sun

last: Zedong

first: Ji

last: Feifan

first: Chen

last: Jiayu

first: Song

last: Ningdan

first: Liu

last: Mingrui

first: Zhou

last: Minmin

first: Wang

last: Can


title: Characterization of a panel of mouse primary cell lines from Myc-driven mammary gland tumours and lung metastasis derived from them

city: London

first: Josep

last: Tarrago-Celada

address: 1 Midland Road

country: United Kingdom

institute: The Francis Crick Institute

summary: Triple negative breast cancer (TNBC) represents 15% of all breast cancer subtypes. It is the most aggressive type, with limited treatment options and is often associated with metastasis. TNBC are associated with high expression of an oncogene, c-Myc, driving metabolic to promote tumour growth and metastasis. To study the metabolic changes that allow cancer cells to become metastatic, we used MMTV-Myc mouse model (mouse mammary tumour virus – Myc) of mammary gland tumorigenesis to generate tumour cell lines either from primary mammary gland tumours or lung metastasis. “MYAZ16.1e”, “MYAZ6.6g”, and “MYAZ12.3i” are cell lines generated from primary MMTV-Myc mammary gland tumours (the number represents the I.D. of a tumour-bearing mouse). These cells have low metastatic capacity. “Myc310” is also a tumour cell line generated from one of the MMTV-Myc tumours, but which has higher metastatic capacity and forms visible metastatic lesions in the lung. To generate models with high metastatic capacity, tumour cells were isolated from the lungs of mice bearing mammary gland tumours generated by ectopic injection of Myc310 cells into the fat pad. These cell lines were named “LM1”, “LM2” and “LM3”. LMs cells were further injected in the fat pads of mice to generate mammary gland tumours. LMs cells showed higher metastatic capacity than Myc310 cells. Tumour cell lines isolated from the lungs of mice bearing LM1, LM2 or LM3 mammary gland tumours were named “LM1-LM”, “LM2-LM”, “LM3-LM”. We have demonstrated that that the metastatic cell lines, Myc310, LM and LM-LM are more dependent on extracellular cysteine/cystine to survive than non-metastatic MYAZ cell lines. We therefore performed transcriptomics of some of the generated cell lines in control and cysteine/cystine deprived conditions. To generate a non-tumourigenic control for in vitro experiments and analysis, normal mammary gland epithelial cells were immortalized by ectopic expression of p53DN and E1A and named “iMMEC” cell line (immortalized mouse mammary epithelial cells). To model the event during acute induction of c-MYC overexpression, we generated the “iMMEC+MYC”, iMMECs via transducing with a doxycycline inducible c-MYC construct.

overall_design: All cells were seeded at the same density (250,000 cells per well in 6-well plates) in DMEM:F12 (supplemented with 10% FBS, 1% P/S, 1% HEPES, insulin, hydrocortisone and EGF). For iMMECs+MYC, 500 ng/mL doxycycline was added to the media to induce MYC. After 24 hours media was changed for some of the cell lines (MYAZs, Myc310, LMs and LM1-LM models), to either control or cysteine/cystine deprived media for 6h. Then cells were collected and RNA extraction was performed with RNAeasy kit (QIAGEN 74104). Three technical replicates were done for each cell lines, and the rest of the cell lines have also three biological replicates except Myc310, iMMECs, and iMMEC+MYC.

first: Michael

last: Kossifos

first: Ersa

last: Gjelaj

first: Yulia

last: Panina

first: Tegan

last: Gilmore

first: Mariia

last: Yuneva


title: Adaptor protein CIN85 potentiates migration of osteosarcoma cells

city: Brno

first: Lucia

last: Knopfova

address: Kamenice 5

department: Department of Experimental Biology

country: Czech Republic

institute: Faculty of Science, Masaryk University

summary: The adaptor protein CIN85, encoded by the SH3KBP1 gene, is a member of the CIN85/CMS family of adaptor molecules involved in many signaling pathways. CIN85 functionally contributes to different biological processes in normal and cancer cells, such as vesicle-mediated transport, cell adhesion, migration, and invasiveness. In this study, we found that the expression of CIN85 was elevated in osteosarcoma compared to normal bone, and in metastases compared to primary bone tumors. Overexpression of CIN85 in osteosarcoma cell lines enhanced their migration and collagen invasion while silencing CIN85 suppressed cell migration. We used human osteosarcoma HOS cells with the overexpression of CIN85 or its knockdown to study the transcriptomic changes induced by this protein. Differentially expressed genes were enriched in cell proliferation, apoptosis, cell differentiation, extracellular matrix, adhesion, focal adhesion, tight junctions, and cell migration. Several abundant signaling pathways involved in tumorigenesis were identified, including NF-κB, TNF, MAPK, Wnt, PI3K/Akt/mTOR, TGF, and VEGF.

overall_design: RNA-seq analysis of the human osteosarcoma HOS cells with overexpression of knockdown of the adaptor protein CIN85. CIN85-overexpressing cells were obtained by the transfection with pRc/CMV2-Rukl plasmid (https://doi.org/10.1093/carcin/bgs228), mock-transfected cells were used as a control. CIN85 was silenced by the specific siRNA, and non-targeting siRNA was used as a control.

first: Iryna

last: Horak

last: Knopfová


title: Pilot NanoString targeted mRNA profiling of neonatal saliva following prenatal opioid exposure

status: Public on Apr 13 2026

city: Boston

first: Tomoko

last: Kaneko-Tarui

address: 800 Washington St., Box #394

department: Women, Mother + Baby Research Institute

institute: Tufts Medical Center

summary: This study profiles targeted mRNA expression in neonatal saliva to investigate the sex-specific effects of prenatal opioid exposure. Saliva samples collected within 48 hours of birth from opioid-exposed and non-exposed neonates were analyzed using the NanoString nCounter Analysis System with a custom CodeSet targeting 72 genes, including housekeeping genes. Raw RCC files are provided for all assay instances, and processed data are provided as normalized, log2-transformed expression values for the QC-passed samples included in the final analysis.

overall_design: Neonatal saliva samples were collected within 48 hours after birth from opioid-exposed and non-exposed neonates born at >=34 weeks gestation. Total RNA was extracted and profiled using the NanoString nCounter Analysis System with a custom CodeSet targeting 72 genes, including housekeeping genes. A total of 36 assay instances were generated. Most assay instances represented independent neonatal saliva samples from different patients, while two biological samples were assayed more than once using newly extracted RNA from the same sample. Eighteen unique neonatal saliva samples passed quality control and were included in the final analysis. Raw RCC files for all assay instances are provided as supplementary files. The processed expression matrix contains normalized, log2-transformed expression values for the 18 QC-passed samples only.

first: Elizabeth

last: Yen


title: Loop extrusion by cohesin plays a role in enhancer-activated gene expression early in differentiation (ATAC-seq)

city: Oxford

first: Felice

middle: Hoching

last: Tsang

state: Oxfordshire

address: John Radcliffe Hospital/Headley Way

department: Weatherall Institute of Molecular Medicine

institute: University of Oxford

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Enhancers and their target promoters often come into close physical proximity when activated. This may be explained by a variety of mechanisms, including cohesin-mediated chromatin loop extrusion. However, acute depletion of cohesin does not cause widespread changes in gene expression. We have tested the role of cohesin-mediated loop extrusion in gene expression at the mouse alpha-globin locus during erythropoiesis. Acute depletion of cohesin disrupts alpha-globin expression at early but not late stages of differentiation. Furthermore, when single or multiple CTCF sites, known to block cohesin, are placed between the alpha-globin enhancers and promoters, alpha-gene expression is disrupted. Importantly, the CTCF site’s orientation is critical, suggesting that within this activated domain, in definitive erythroid cells cohesin predominantly but not exclusively, translocates from the enhancers to the promoters. We find that loop extrusion does play an important role in establishing enhancer-promoter proximity and consequent expression of inducible genes during differentiation.

overall_design: ATAC-seq on chromatin isolated from fetal liver cells (Ter119) and EB-derived erthroid cells (CD71) with the CTCF insertion in forward (EB clones D2H3, D3F3, F2D3) or reverse (EB clones D2D6, D3H2, F2G6) orientation, or wild type. Biological triplicates, for EB independent clones or independent differentiation (WT only).

first: Rosa

last: Stolper


title: Loop extrusion by cohesin plays a role in enhancer-activated gene expression early in differentiation  (NG Capture-C)

overall_design: NG CaptureC was performed for EB derived erythroid (CD71) cells, either in triplicate (Forward CTCF insertion (clones D2H3, D3F3, F2D3) and wild type) or duplicate (Reverse CTCF insertion(clones F2G6 and D3H2). Capture was performed with biotinylated oligonucleotides targeting sequences adjacent to DpnII sites. Probes were divided into pools targeting upstream CTCF sites (HS38/HS39), the main enhancers (R1/R2) and downstream CTCF sites (HS44/48), except for one reverse sample (F2G6) which was probed with a combined upstream CTCF/enhancer pool and a seperate downstream CTCF pool.

middle: H


title: Neuronal activation triggers rapid biphasic FMRP-mediated translational control linking synaptic and nuclear regulation

first: Ruth

middle: A

last: Singer

address: 1230 York Avenue, TSH 314

institute: The Rockefeller University

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

overall_design: Refer to individual Series


title: Reconstructing signaling histories of single cells via perturbation screens and transfer learning [scRNA]

first: Nicholas

middle: T

last: Hutchins

state: MA

address: 455 Main St.

institute: Massachusetts Institute of Technology

summary: Manipulating the signaling environment is an effective approach to alter cellular states for broad-ranging applications, from engineering tissues to treating diseases. Such manipulation requires knowing the signaling states and histories of the cells in situ, for which high-throughput discovery methods are lacking. Here, we present an integrated experimental-computational framework that learns signaling response signatures from high-throughput in vitro perturbation atlas and infers combinatorial signaling activities in in vivo cell types with high accuracy and temporal resolution. Specifically, we generated signaling perturbation atlas across diverse cell types/states through multiplexed sequential combinatorial screens on human pluripotent stem cells. Using the atlas to train IRIS, a neural network-based model, and predicting on mouse embryo scRNAseq atlas, we discovered global features of combinatorial signaling code usage over time, identified biologically meaningful heterogeneity of signaling states within each cell type, and reconstructed signaling histories along diverse cell lineages. We further demonstrated that IRIS greatly accelerates the optimization of stem cell differentiation protocols by drastically reducing the combinatorial space that needs to be tested. This framework leads to the revelation that different cell types share robust signal response signatures, and provides a scalable solution for mapping complex signaling interactions in vivo to guide targeted interventions.

overall_design: hESC derived mesenchymal cells and hESC derived endodermal cells

first: Miram

last: Meziane

first: Claire

last: Lu

first: Maisam

last: Mitalipova

first: David

last: Fischer

first: Pulin

last: Li


title: Loop extrusion by cohesin plays a role in enhancer-activated gene expression early in differentiation


title: Human breast milk-derived extracellular vesicles alleviate Alzheimer’s disease phenotypes through delivering CD36

city: Shanghai

first: Zhanping

last: Liang

state: Shanghai

address: zhennan Rd 500 tongji n=university

institute: Tongji Hospital Affiliated to Tongji University

summary: Alzheimer's disease (AD) is an age-related progressive neurodegenerative disease that is the most common cause of dementia globally. To date, multiple types of extracellular vesicles (EVs), particularly those derived from stem cells, have been implicated to improve AD pathology, but the clinical applications of stem cell-derived EVs are restricted due to ethical/religious concerns, supply constraints, and other challenges. Here we found that human breast milk-derived extracellular vesicles (HM-EVs) mitigated AD pathology and rescued cognitive decline of 5xFAD mice, a preclinical mouse model of AD. We identified CD36, a microglial anti-Aβ receptor as the key cargo to mediate the therapeutic effects of HM-EVs by enhancing microglial phagocytosis and Aβ clearance. Moreover, blockage of CD36 using a monoclonal antibody abolished HM-EVs-driven Aβ phagocytosis of microglia, therefore aggravating cognitive performance and pathological changes of 5xFAD mice. Together, our findings indicate HM-EVs as a novel intervention strategy of AD through delivering CD36.

overall_design: RNA-seq profiling of the prefrontal cortex and hippocampus of male wildtype C57BL6J mice and 5xFAD mice intravenously injected with PBS, or 5xFAD mice intravenously injected with human breast milk-derived extracellular vesicles from 5-month-age to 6-month-age. Injections were conducted every 3 days for 10 times.

first: Xiaohuan

last: Xia

first: Shaomin

last: Wang

first: Jiangqin

last: Liu

first: Xuanran

last: Feng

first: Guangzhi

last: Shi

first: Mingyuan

first: Wanting

last: Xu

first: Yi

first: Jialin

middle: C

last: Zheng


title: Single-cell spatial transcriptomic profiling of 3xTg-AD mouse brains following fecal microbiota transplantation from stroke and healthy human donors

city: Columbia

first: Ai-Ling

last: Lin

state: MO

address: 1101 Hospital Drive

department: Radiology

institute: University of Missouri

summary: Stroke-induced gut dysbiosis has been linked to accelerated neurodegeneration, but whether stroke-altered microbiota alone can initiate Alzheimer's disease (AD) pathology remains unclear. We performed fecal microbiota transplantation (FMT) from human stroke and healthy donors into pre-symptomatic 3xTg-AD mice and profiled brain-wide gene expression using the NanoString CosMx Spatial Molecular Imager. Single-cell spatial transcriptomics revealed extensive cell-type-specific transcriptional remodeling in Stroke-FMT mice, including upregulation of genes involved in amyloid processing, neuroinflammation, and lipid metabolism, alongside downregulation of autophagy and calcium-signaling pathways. Region-specific analyses of cortex and hippocampus demonstrated convergent upregulation of AD-relevant drivers including Apoe, App, and Grin2b. These transcriptomic changes were accompanied by elevated phospho-Tau, GFAP, and IBA1 immunoreactivity and disrupted gut-brain redox and energy metabolism.

overall_design: 3xTg-AD transgenic mice received FMT from human stroke donors (Stroke-FMT) or healthy donors (Healthy-FMT), with naive controls. At 6.5 months of age, sagittal brain sections were profiled using the CosMx Spatial Molecular Imager with a high-plex RNA panel. Nine samples across five CosMx slides were analyzed. Samples represent three treatment groups: Stroke-FMT, Healthy-FMT, and untreated controls.

first: Chetan

last: Aware

first: Carter

last: Woods

first: Pavlo

last: Khodakivskyi

first: Alok

last: Dwivedi

first: Amitai

last: Zuckerman

first: Maalavika

last: Govindarajan

first: Kira

last: Ivanich

first: Wenyan

last: Yu

first: Jiankun

last: Cui

first: Zezong

last: Gu

first: Elena

last: Goun

first: Aaron

last: Ericsson

first: Priti

last: Balchandani

first: Ai-ling


title: Mitochondrial metabolism regulates the immunogenic responsiveness of dendritic cells [ATAC-seq]

first: Ignacio

last: Heras-Murillo

overall_design: cDC1s or cDC2s from Uqcrqf/f or Itgax-Uqcrqf/f mice were FACS sorted and used for ATAC sequencing.

first: Diego

last: Mañanes

first: Stefanie

last: Wculek

first: David

last: Sancho


title: Age influences CMV-specific NK and CD8 T cell memory fates(OT-Is)

first: Isaac

last: Jensen

overall_design: OT-I CD8 T cells were enriched from adult (8wk old) and infant (10dpn) mice and submitted for sequencing or transferred at a 1:1 ratio into congenically distinct recipient mice. Recipient mice were then infected with MCMV-K181-OVA. Splenic CD8 T cells were enriched7 days post-infection (D7). Cells stained with CITE-Seq antibodies and sequenced. Infant OT-I cells are CD45.2+Thy1.1+, Adult OT-I cells are CD45.2+Thy1.2+, recipient mice are CD45.1+Thy1.2+, individual mice are for adult and infant mice are identified through hashtag ADTs.

first: Donna

last: Farber


title: RNA-Seq of paired diagnosis and relapse AML specimens with healthy controls

city: Charlottesville

first: Subhash

last: Prajapati

address: 1340 Jefferson Park Avenue

department: Biochemistry and Molecular Genetics

institute: University of Virginia

summary: Despite therapeutic advances, acute myeloid leukemia (AML) patients remains a clinically challenging malignancy, highlighting the continued need for identification of previously unrecognized targetable drivers of the disease to further improve current treatment options. Aberrantly expressed genes are associated with AML pathogenesis and can function as disease drivers. Targeting of their function or related pathways has shown promising results in identifying novel therapeutic targets. We performed RNA sequencing (RNA-Seq) of AML patients specimens to identify aberrantly expressed AML genes associated with AML progression. The dataset comprised of two cohorts (I: n = 29 and II: n = 30), each cohort containing matched diagnosis-relapse pairs along with corresponding healthy control specimens (cohort I: n = 3 and cohort II: n = 5) derived from CD34+ normal bone marrow cells. All the diagnosis patients received combination intensive chemotherapy (7+3: continuous infusion of cytarabine for 7 days combined with anthracycline on days 1-3) during their induction treatment and all patients achieved remission after induction treatment. This sumission provides raw gene-level read counts to facilitate differential gene expression analyses between diagnosis and relapse specimens and relative to healthy controls. The fastq files for the data can be accessed through dbGap accession number phs001027.

overall_design:
Bulk RNA-Sequencing of paired diagnosis and relapse specimens from AML patients and healthy controls. Data processing is described in PMIDs 41608320 and 41844049.

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Raw data have been deposited under dbGap accession number phs001027.v4
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first: Garrett-Bakelman

last: Francine

first: Neelamraju

last: Yaseswini

first: Dunham

last: Nicholas

first: Prajapati

last: Subhash


title: Generation of Stable Chambered Cardioids from human Pluripotent Stem Cells [bulk RNA-seq]

first: XINLE

last: ZOU

overall_design: This study investigates the effects of PD173074 on samples across different time points. Samples were divided into two groups: with PD173074 treatment (SCPD) and without PD173074 treatment (SC without PD). Each group was assessed at multiple time points: Day 0, Day 1.5, Day 3.5, Day 6.5, Day 10.5, Day 15.5, and Day 25.5. For each condition and time point, two biological replicates were used (rep1 and rep2), resulting in a comprehensive dataset to analyze the impact of PD173074 over time.

first: FANWEN

last: WANG

first: HUILING

last: ZHENG


title: Air pollutant multiomics improves functional annotation of SNPs associated with lung disease

city: Denver

first: Arnav

last: Gupta

address: 1400 Jackson St. K720

department: Pulmonary and Critical Care

institute: National Jewish Health

summary: Asthma and chronic obstructive pulmonary disease (COPD) are lung diseases strongly influenced by interactions between genetic background and environmental exposures, particularly air pollutants. However, the biological mechanisms by which genetic variation and pollutant exposure impact disease remain poorly understood. Interpretation of genome-wide association studies is limited because most disease-associated variants occur in noncoding regions and are difficult to connect to functional regulatory mechanisms and downstream gene targets. Here, we integrate nascent RNA run-on sequencing with regulatory network inference to identify pollutant-responsive transcriptional regulatory elements and their upstream regulators in lung cells. Using newly generated and published datasets, we analyze multiomics responses to multiple air pollutants, including wood smoke particles, urban particulate matter, and Afghan dust particles. These analyses provide the first characterization of the nascent transcriptional response to urban particulate matter in primary lung cells and reveal both shared and pollutant-specific regulatory dynamics. We then integrate pollutant-responsive regulatory networks with genetic associations for asthma and COPD to prioritize noncoding variants for experimental validation. By linking these variants to candidate transcription factors and target genes, we identify mechanisms through which environmental exposures may interact with noncoding genetic variants to influence disease risk. This framework enables functional interpretation of noncoding variants through environmentally responsive transcriptional networks.

overall_design: This submission includes all ATAC-seq (Assay for Transposase Accessible Chromatin with sequencing) libraries used in the chromatin accessibility quantitative trait locus (ca-qtl) analysis section of the manuscript. Briefly, raw sequencing files were mapped to the human genome (hg38). After removal of duplicate reads, mapped read files were analyzed individually to count alleles at single nucleotide polymorphisms across the genome. The resulting allele frequencies in ATAC-seq read files were defined as ca-qtls.

first: Hope

last: Townsend

first: Sarah

last: Sasse

first: Shu Yi

last: Liao

first: Anthony

middle: N

last: Gerber

first: Robin

middle: D

last: Dowell


title: PrPC-facilitated cell signaling activates phospholipase Cɣ1 and triggers an Arc/Arg3.1 response in mouse neurons and human iPSC-derived neurons

city: La Jolla

first: Daniel

last: Ojeda-Juarez

state: CA

address: 9500 Gilman Dr. MC 0612

institute: UC San Diego

summary: Synapse loss is an early feature of prion disease, yet the underlying drivers are poorly understood. We recently found evidence of neuronal hyperactivity and synaptic loss in prion-infected mice. Herein we identified increased Arc/Arg3.1 in patients with prion disease, suggesting heightened neuronal activity also occurs in human prion-affected brain. To determine the signaling events initiated by prion aggregates (PrPSc), we developed a disease model in which human iPSC-derived excitatory neurons are stimulated with a PrPSc-mimetic antibody that binds cellular prion protein (PrPC). Within two hours, we detected an Arc/Arg3.1 response together with transcriptomic changes previously reported in prion-infected mice. We identified altered phosphorylation of PLC-γ1, ERK1/2, and EGFR as PrPC-triggered cell signaling events that accompany the Arc/Arg3.1. These results suggest that PrPC ligands, including PrPSc, trigger rapid signaling events linked to neuronal hyperactivity in human neurons, and indicate PLC-γ1 as a potential therapeutic target.

overall_design: 6 - 8 week-old iNs were treated with PBS, POM1 (33 nM), or IgG (33 nM) for 2 hours. Samples were lysed in RLT buffer and RNA isolated using the RNeasy Micro kit (Cat# 74004; Qiagen) following the manufacturer’s protocol. Total RNA was assessed for quality using an Agilent Tapestation 4200, and samples with an RNA Integrity Number (RIN) greater than 8.5 were used to generate RNA sequencing libraries using the TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA). Samples were processed following the manufacturer's instructions. Resulting libraries were multiplexed and sequenced with 100 base pair (bp) single reads (SR100) to a depth of approximately 25 million reads per sample on an Illumina NovaSeq X Plus. Samples were demultiplexed using bcl2fastq Conversion Software (Illumina, San Diego, CA). QC and RNA-seq were conducted at the IGM Genomics Center, University of California, San Diego, La Jolla, CA.

first: Gail

last: Funk

last: McClatchy

first: Emily

last: Richards

first: Alexander

last: Rajic

first: Katrin

last: Soldau

first: Michael

last: Geschwind

first: Xu

last: Chen

first: John

last: Yates III

first: Steven

last: Gonias

first: Christina

last: Sigurdson


title: Dual regulation of chemical stress-induced DDI2/3 expression by a transcription factor Fzf1 and nucleosome in Saccharomyces cerevisiae

city: Vancouver

first: LeAnn

last: Howe

state: BC

address: 2350 Health Sciences Mall

department: Biochemistry and Molecular Biology

country: Canada

institute: University Of British Columbia

summary: DDI2 and DDI3 (DDI2/3) are duplicated genes in Saccharomyces cerevisiae that exhibit strong induction by a transcription factor Fzf1 in response to chemical treatments like cyanamide (CY) and methyl methanesulfonate (MMS). Although, like DDI2/3, SSU1, YHB1 and YNR064C also contain an Fzf1-binding consensus sequence CS2 and are coordinately regulated by Fzf1, these genes are only modestly induced by CY and MMS. To identify additional cis-acting elements in the DDI2/3 promoter, we made DDI2/3 promoter deletions in a reporter system and identified upstream repressing sequences (URS) spanning 480 nucleotides. To test a hypothesis that the chromatin structure constitutes the URS, we utilized a yeast strain capable of histone H3/H4 depletion by shifting carbon sources. Following histone depletion, DDI2/3 were strongly induced in an Fzf1 dependent manner, while YHB1 was repressed. Interestingly, under histone depletion conditions, CY or MMS treatment further increased expression of all Fzf1-regulated genes to comparable levels in an Fzf1 dependent manner. A genome-wide MNase-seq analysis showed that CY treatment reduced the nucleosome occupancy at the mapped DDI2/3 URS region in wild-type cells, but not in in fzf1Δ cells. These findings collectively indicate that Fzf1 plays dual roles in regulating the DDI2/3 response to CY. Firstly, it binds CS2 and serves as a transcription activator. Secondly, it is required for the chromatin remodeling at URS. This two-tier regulation at the DDI2/3 promoter helps to explain why DDI2/3 achieve much higher fold induction by CY and MMS than other Fzf1-regulated genes, suggesting Fzf1 to be a candidate pioneer transcription factor.

overall_design: Micrococcal nuclease-resistant fragments were subjected to paired-end sequencing.to examine nucleosome occupancy over genes impacted by cyanamide treatment,


title: Loop extrusion by cohesin plays a role in enhancer-activated gene expression early in differentiation (ChIP-seq)

first: Felice

last: Tsang

overall_design: To confirm binding of CTCF to the newly inserted site and confirm recruitment to existing sites, we performed ChIP-seq in erythroid (Ter119) fetal liver cells and EB derived erythroid (CD71) cells. Fetal liver data is biological triplicates and EB data is biological duplicates (independent ES clones (Fwd: D2H3, F2D3. Rev: D2D6, D3H2)/independent differentiations for WT).

first: Rosa

last: Stolper


title: Distantly metastatic differentiated thyroid carcinoma is   kinase-driven and enriched for DNA repair and DNA   methylation gene alterations

city: Qingdao

first: liu

last: yanjun

state: shangdong

address: No. 1677 Wutaishan Road

department: Department of Pathology

institute: Qingdao University

type: Methylation profiling by genome tiling array

summary: Although distant metastasis is uncommon in differentiated thyroid carcinoma (DTC),   it remains the leading cause of thyroid cancer-related mortality. The genetic landscape   of distantly metastatic DTC (DMDTC) has not been well characterized in large   cohorts. This study aimed to identify functional genetic alterations in DMDTC and   validate their biological significance. We included 78 patients with DMDTC and   performed DNA-based next-generation sequencing (NGS) in all cases, followed by   RNA-based NGS for fusion gene detection, along with a review of previously   reported isolated cases. Plasmids harbouring novel variants, including SPON1::ALK  and RFTN1::BRAF fusions, and mutations in PTEN (c.322_345del, c.740del,   c.968dup), STK11 (c.842C>T, c.1225C>T), and DNMT3A (c.891G>A, c.2312G>A,   c.2606G>A), were constructed and transfected into TPC-1 and HEK293T cells to   investigate downstream signalling. Methylation status of differentially methylated   genes (DMGs) associated with DNMT3A mutations was analysed using the Infinium   Methylation EPIC v2.0 BeadChip, with several DMGs validated by quantitative real-time PCR. The cohort consisted of 25 males and 53 females, with a mean age of 60.3   years at the diagnosis of metastasis. Histologic types included papillary carcinoma (31   cases), follicular carcinoma (44 cases), and oncocytic carcinoma (3 cases). The lung   and bone were the most common metastatic sites. Multiple metastases and older age   were associated with metastasis-free and overall survival. Genetic alterations   involving phosphorylation signalling pathways were identified in 61 cases, among   which pathologic alterations of DNA damage repair (DDR)-related gene were   detected in 10 cases. Novel RFTN1::BRAF and SPON1::ALK fusions, along with   PTEN (c.740del, c.968dup) and STK11 (c.842C>T) mutations, could enhance down   streaming phosphorylation levels. DNMT3A mutations (c.891G>A, c.2312G>A,   c.2595A>T, c.2606G>A) induced genome-wide methylation dysregulation, with   altered expressions of SLC12A7, FLUC, HMGB2, BNC2, and DAPK1. This study   shows that DMDTCs are characterized by dysregulated phosphorylation signalling,   accompanied by chromosomal instability and aberrant methylation, thus underscoring   DDR gene-targeted therapy as a promising strategy.

overall_design: This is a comparative methylation profiling study. The study includes three groups: 1) Empty vector control group (pcDNA3.1(+)-Flag); 2) DNMT3A wild-type group; 3) DNMT3A mutant group (four mutations: c.891G>A (p.W297*), c.2312G>A (p.R771Q), c.2595A>T (p.E865D), c.2606G>A (p.G869D)). Each group has two biological replicates. TPC-1 cells were transfected with corresponding plasmids using Lipofectamine™ 3000, and genomic DNA was extracted 48h post-transfection for methylation array analysis.

first: Weimao

last: Kong

first: Longnv

last: Bao


title: Long telomeres induce a genome-wide transcriptional response in budding yeast

city: Newcastle upon Tyne

first: David

last: Lydall

address: Cookson Building, Medical School, Framlington Place

institute: Newcastle University

summary: Eukaryotes maintain telomere length within a defined range. While short telomeres are known to activate DNA damage responses and limit cell proliferation, long telomeres are associated with extended proliferative capacity, but their broader cellular consequences remain less well understood. In budding yeast Saccharomyces cerevisiae, long telomeres have been shown to influence gene expression at specific loci, but whether long telomeres affect transcription genome-wide has not been reported. Here, we analysed the transcriptomes of diploid yeast strains in a single lineage in which long telomeres were initially generated by a rif2delta mutation and subsequently inherited through successive rounds of mitosis and meiosis in the absence of this mutation. We show that strains with long telomeres exhibit a distinct gene expression profile, enriched in transcripts encoding membrane transporters. Both up- and down-regulated genes were distributed across the genome, arguing against a purely telomere-proximal effect on gene expression. Affected genes were enriched for Rap1 binding sites, consistent with a model in which long telomeres alter the availability of telomere-associated transcriptional regulators such as Rap1 and thereby affect gene expression at non-telomeric binding sites for these regulators. The magnitude of transcriptional changes was greatest in strains with the longest telomeres. Together, our findings demonstrate that long telomeres induce a genome-wide transcriptional response that accompanies inherited long telomeres across generations but diminishes as telomeres shorten.  These types of transcriptional changes may also occur in other eukaryotes, including humans, where long telomeres can be inherited and are associated with disease.

overall_design: RNA-seq profiling of Saccharomyces cerevisiae strains with different telomere lengths: wild-type (WT/WT) with normal telomeres, rif2delta heterozygous diploid with elongated telomeres (WT/rif2delta), and two successive generations of wild-type descendants of the WT/rif2delta diploid that inherited long telomeres (WT/WT_long1, WT/WT_long2). Three biological replicates per genotype (12 samples total).

first: Vasilisa

last: Sidarava


title: Immunological tolerance is maintained by self-reactive CD8 T cells

city: hangzhou

first: wang

last: pengfei

address: yuhangtang road 866

institute: zhejiang university

summary: CD8 T cells play a critical role in immune tolerance maintenance after immune activation. They have recently been reported to target activated CD4 T cells in an MHC-Ia–restricted manner. However, the specific peptides and the corresponding reactive CD8 TCRs responsible for this targeting process remain unknown. In this study, we identified the self-peptides on activated CD4 T cells, cloned the corresponding CD8 TCRs, and validated the in vitro and in vivo immunosuppressive function of CD8 T cells carrying these self-reactive TCRs. The therapeutic potential of peptide vaccination and self-reactive CD8 T cells   was confirmed in a mouse model of experimental autoimmune encephalitis (EAE). This study consequently redefines the nature of CD8 regulatory T (Treg) cells as self-reactive CD8 T cells.

overall_design: RNAseq profiling of total CD8 T cells purified from lymph nodes and spleens of WT or TCR4 mice, each group has three biological replicates

first: Yadan

last: Bai

first: Pengfei

last: Wang

first: Yuzhen

last: Liu

first: Yuwen

last: Shao

first: Di

last: Sun

first: Yu

last: Zheng

first: Linrong

last: Lu


title: Bacteria-mimicking cancer cells reprogram macrophages via multiple PRR pathways for cancer immunotherapy

city: Incheon

first: Dongjun

last: Yu

address: 100, Inha-ro, Michuhol-gu, Building 4, Room 105

department: Department of Biological Sciences and Bioengineering

country: South Korea

institute: Inha University

summary: Although macrophages are a powerful cell-based platform for cancer immunotherapy, their antitumor functions, such as phagocytosis and inflammatory responses, are limited by the immunosuppressive tumor microenvironment. Here, we show that decorating cancer cell membranes with bacteria-derived pathogen-associated molecular patterns (PAMPs) initiates phagocytosis and inflammatory responses of macrophages toward cancer cells involving various pattern recognition receptor signaling. Membrane-attached PAMP nanoparticles maintained their immunostimulatory functions. The phagocytic macrophage fraction increased when coincubated with membrane-decorated cancer cells, along with an increased secretion of inflammatory cytokines. Transcriptome analysis revealed that the response of macrophages to PAMP-decorated cancer cells resembled their response to bacteria. In a mouse model, locally injected membrane-decorating PAMP nanoparticles suppressed tumor growth, and the effect was enhanced in combination with the chemotherapeutic drug doxorubicin. Our findings provide insights into the use of macrophages as a cancer immunotherapy modality.

overall_design: RNA-seq profiling of macrophages isolated from mice subcutaneous CT-26 tumors intratumorally treated with PBS or BMEplus to characterize transcriptional reprogramming induced by PAMP-mediated activation. BMEplus denotes nanoparticles prepared from bacterial membrane extracts from E. coli (ATCC 25922) and S. aureus (ATCC 29213).

first: Seoyoon

last: Song

first: Haneul

last: Kang

first: Deborah

last: Lee

first: Jiye

first: Sungjun

last: Kwak

first: Jinhee

last: Park

first: Lifeng

first: Stanislav Y

last: Emelianov

first: Jong-Ho

last: Cha

first: Yun Suk

last: Huh

first: Sun Min

last: Kim

first: Tae-Joon

last: Jeon


title: Spatiotemporal transcriptomics characterizes immune microenvironment during mouse liver aging [snRNA-Seq]

city: Wuhan

first: Jie

last: Cheng

state: Hubei

address: 13 Hangkong Road, Qiaokou District

department: School of Basic Medicine

institute: Huazhong University of Science and Technology

summary: The liver is a major metabolic organ, responsible for synthesizing and breaking down diverse metabolites. Recently, the liver's immunological functions have gradually been unveiled: combating pathogens and maintaining tissue homeostasis. Age-related functional alterations in these immune cells emerge as potential drivers of hepatic dysfunction and age-associated pathologies. However, systematic investigations into spatiotemporal immune cell dynamics during liver aging remain limited. To address this gap, we analyzed young and old mouse livers using single-cell/nuclei and spatial transcriptomics, revealing T cells as the immune cell population with the most pronounced transcriptomic alterations, marked by enrichment of exhausted CD8+ T cells in aged livers. Spatial mapping showed exhausted CD8+ T cells accumulating in portal vein (PV) zone, co-localizing with periportal hepatocytes (PP hepatocytes). Up-regulation of LPIN1 in PP hepatocyte promoted T cell exhaustion. CD8+ T cell exhaustion was tightly associated with disease progression. Therefore, our findings suggest that targeting LPIN1 may alleviate T cell exhaustion, offering potential therapeutic strategies for age-related liver diseases.

overall_design: We collected livers from young and aged mice and performed single-cell transcriptomics (on CD45+ cells only), single-nucleus transcriptomics (on all liver cells), and spatial transcriptomics to investigate changes in the hepatic immune microenvironment during aging.

first: Jiahua

first: Yuqian

last: Wang

first: Wenxue

last: Zhao

first: Zihao

first: Zhaoya

last: Gao

first: Jin

last: Gu

first: Cheng

last: Li


title: Spatiotemporal transcriptomics characterizes immune microenvironment during mouse liver aging

last: Lu


title: Transcriptomic  characterization of Wild type mouse Embryonic Stem Cells (mESCs),  β-actin Knock out mESC and upon rescue with a nuclear localization signal  β-actin

city: Abu Dhabi

first: Piergiorgio

last: Percipalle

state: abu dhabi

address: NYUAD Saadiyat Island

country: United Arab Emirates

institute: Biology

summary: Pluripotency and lineage commitment in embryonic stem cells depend on coordinated regulation of chromatin architecture and extracellular matrix (ECM) signaling. Here, we identify nuclear β-actin as a key regulator linking these processes in mouse embryonic stem cells. Loss of β-actin disrupts core pluripotency factors, including Oct4 and Sox2, and causes broad transcriptional changes, while nuclear re-expression rescues these defects. Chromatin accessibility analysis revealed reduced accessibility at regulatory regions of pluripotency genes, consistent with impaired chromatin remodeling. β-actin depletion also altered ECM-related gene expression, matrix properties, and cellular biomechanics, leading to impaired self-renewal, skewed lineage specification, and defective differentiation, particularly reduced neuronal potential and increased mesodermal-like fate bias. In vivo, β-actin loss restricted teratoma growth and compromised tri-lineage differentiation. Together, these results define nuclear β-actin as an important regulator of chromatin accessibility, ECM-dependent signaling, and stem cell fate.

overall_design: RNA-seq Profiling of mESC Wild type, Knock out β-actin, and Nuclear Localization signal β-actin rescue

first: Nadine

last: Hosny El Said

first: Colin

last: Campbell

first: Amani

last: Nuairi

first: Sabrina

last: Desbordes


title: RNAseq analysis of peritoneal macrophages from ID8 tumor-bearing high fat diet-fed or control-fed mice after chemotherapy

city: Guangzhou

first: Chaoyun

last: Pan

address: 74 Zhongshan Er Rd.

institute: Sun Yat-sen University

summary: Overweight/obesity is an established risk factor for ovarian cancer onset, yet its impact on relapses after first-line chemotherapy remains unclear, complicating therapeutic strategies. Peritoneal macrophages, as the most abundant immune population in the peritoneal cavity, serve as the first line of host defense, eliminating pathogens or disseminating tumor cells through phagocytosis in the peritoneal cave. In this study, we found that Impaired peritoneal macrophage phagocytosis correlates with tumor recurrence in the peritoneal cavity of obese mice or overweight patients. To investigate how HFD affects the anti-tumor capacity of peritoneal macrophages, we isolated peritoneal macrophages by flow cytometry from ID8 tumor-bearing mice one week after chemotherapy, when tumor burdens were comparable between groups and performed RNA sequencing to systemic address the underlying mechanisms.

overall_design: Peritoneal macrophage from the ID8 tumor-bearing mice one week after chemotherapy on HFD or CD conditions were harvested and analyzed uisng RNAseq

first: Jingyi


title: Fra2 role in PDAC response under MRTX-1133 treatment

city: Columbus

first: Gian Luca

last: Rampioni Vinciguerra

state: OH

address: 460W 12th Avenue

department: Cancer Biology and Genetics

institute: The Ohio State University

summary:
Fra2 is involved in the stress adaptivity of PDAC cells. We explored its possible role under the administration of the KRAS-inhibitor MRTX-1133
We used Affymetrix analysis to identify the downstream targets of Fra2 transcriptional activity under MRTX-1133 treatment in AsPC-1 cells

overall_design: We compared the transcriptome of the following AsPC-1 cells: wild-type untreated, wild-type resistant (obatined with continuous administration of escalating doses of MRTX-1133), FRA2KO untreated and FRA2KO (50nM) MRTX-1133 treated.

first: Giovanni

last: Nigita


title: Multiomic profiling reveals the regulation of important im-mune genes by PU.1 in porcine alveolar macrophages [CUT&Tag]

city: Yangzhou

first: Juan

last: Yang

address: 大学南路88号

department: College of Veterinary Medicine

institute: Yangzhou University

summary: Based on the siRNA-mediated knockdown experiments of SPI1, we further applied CUT&Tag to delineate the genome-wide binding landscape of PU.1 along with the histone modification H3K27ac, which primarily occurs at active promoters and enhancers.

overall_design: PAMs were isolated from healthy piglets by bronchoalveolar lavage and cryopreserved. Upon revival, cells were cultured in RPMI-1640 containing 10% FBS and 1% P/S at 37°C with 5% CO₂. SPI1 knockdown was achieved using siRNA transfection, with scrambled siRNA serving as the negative control. Two replicates per sample were used for CUT&Tag.

first: Jiayao

last: Jiang

first: Liangliang

last: Zhang

first: Yanhua

first: Cui

last: Du

first: Mingan

last: Sun


title: Dietary omega-6 lipids promote post-injury aberrant bone formation in obesity

city: Dallas

first: Sneha

last: Korlakunta

state: TX

address: 6000 Harry Hines Blvd

department: Surgery

institute: UT Southwestern Medical Center

summary: Obesity is associated with impaired wound healing, but the mechanisms linking excess adiposity to aberrant tissue repair remain unresolved. Heterotopic ossification (HO) is a severe example of pathologic tissue repair in which mesenchymal progenitor cells (MPCs) undergo aberrant osteochondral differentiation within soft tissue, leading to joint contractures and pain. Here, we show that injury site accumulation of dietary omega-6 (ω-6) lipids is a key mechanism linking obesity to heterotopic ossification. Specifically, in mice fed a high-fat diet (HFD), injured tissues are enriched in linoleic and arachidonic acids, providing substrate for myeloid cyclooxygenase-2 (COX-2)-dependent prostaglandin E2 (PGE2) production. PGE2 then drives a transcriptional program in mesenchymal progenitor cells that promotes osteochondral differentiation. An isocaloric, low linoleic acid HFD reduces HO despite comparable obesity, demonstrating that dietary lipid composition, rather than adiposity alone, drives pathological repair. Clinical data mirror these findings, showing that obesity confers increased HO risk, and COX-2 inhibition reduces HO exclusively in obese patients. Together, these findings identify injury site ω-6 lipid enrichment as the key signal linking the diet to MPC reprogramming, pointing to dietary lipid modulation as an actionable strategy to limit HO in obesity.

overall_design: scRNA-seq of cells isolated from the injury site at 0, 3, and 7 days after B/T injury was performed on C57BL/6 mice maintained on a high-fat diet, along with age-matched C57BL/6 high-fat diet controls, following previously published protocols.

first: Stephanie

last: Moye

first: Benjamin

last: Levi


title: Pediatric Cancer Dependencies Accelerator [ATAC-Seq]

city: Memphis

first: Pediatric Cancer Dependencies

last: Acclerator

state: TN

address: 262 Danny Thomas Blvd

summary: Datasets characterizing epigenomic aspects of pediatric cancer cell lines as part of the Pediatric Cancer Dependencies Accelerator

overall_design: ATAC-seq in pediatric cancer cell lines used in the DepMap/PedDep Consortium


title: Carbohydrate responsive element binding protein promotes colorectal carcinogenesis by regulating the Wnt/beta-catenin pathway

city: Shanghai

first: Ming

last: Feng

address: 280 S. Chongqing Road

department: Department of Biochemistry and Molecular Cell Biology

institute: Shanghai Jiao Tong University School of Medicine

summary: ChREBP is a glucose-responsive transcription factor involved in glycolysis and de novo lipogenesis. Elevated ChREBP levels were observed in human colorectal cancer (CRC) samples and correlated with poor 5-year survival rates in patients. To investigate the role and mechanism of ChREBP in colorectal carcinogenesis in vivo, we used ChREBP knockout mice, which were intraperitoneally injected with azoxymethane (AOM) followed by dextran sulfate sodium (DSS) in drinking water. In the AOM/DSS-induced colorectal cancer model, carcinogenesis was reduced in ChREBP null mice. In the initial phases of colorectal carcinogenesis, ChREBP deficiency was associated with diminished epithelial cell proliferation and a lower number of aberrant crypt foci (ACF), but it had no impact on DNA damage or the severity of colitis. The key transcription factor beta-catenin and Wnt target gene expression were both decreased in the colons of ChREBP null mice and in ChREBP-knockdown Caco-2 colorectal cancer cells. In vitro studies showed that ChREBP knockdown promoted beta-catenin phosphorylation and reduced beta-catenin levels and nuclear localization in Caco-2 cells. Conversely, ChREBP overexpression was sufficient to promote beta-catenin accumulation, nuclear localization, and transcriptional activity in human HEK293T cells, thereby contributing to compensatory proliferation and tumorigenesis. Given its elevated expression in human colorectal cancer, ChREBP may represent a potential therapeutic target. The correlation between ChREBP-mediated Wnt signaling activation and colorectal carcinogenesis would provide deeper insights into the link between metabolic dysfunction and colorectal cancer.

overall_design: RNA-seq profiling of distal colon tissues from wildtype and ChREBP KO mice (n=3 per group) at Day17 of AOM/DSS treatment.

first: Wenrui

last: He

first: Guoxiao

last: Ji

first: Xuemei

last: Tong

first: Jian

last: Meng

first: Lifang

last: Wu


title: iCNG99: a validated genome-scale metabolic model of Cryptococcus neoformans strain H99

city: Shenzhen

first: feng

last: chun

institute: SUSTech

summary: Cryptococcus neoformans is a ubiquitous environmental fungus that can also cause life-threatening infections in immunocompromised individuals. As a competent pathogen, Cryptococcus needs to reprogram its metabolism to adapt the drastic differences between environmental niches and host niches. A well-curated genome-scale metabolic model (GEM) is a powerful tool to facilitate the investigation of the metabolic resilience of an organism Here we reconstructed and validated iCNG99, a GEM for C. neoformans reference strain H99, and evaluated its predictive performance across 43 growth conditions and gene essentiality benchmarks. The model achieved high confidence essential gene prediction (precision = 0.77) and recapitulated pathways targeted by clinically available antifungals. Integration with transcriptomic and metabolomic data enabled iCNG99 to capture condition-specific metabolic adaptations and to identify candidate vulnerabilities in drug tolerance, revealing metabolic adaptations associated with survival within host conditions and drug susceptibility. Together, iCNG99 provides a systems-level computational platform for investigating C. neoformans metabolism and for prioritizing antifungal vulnerabilities.

sample_organism:
Cryptococcus neoformans
Mus musculus

platform_organism:
Cryptococcus neoformans
Mus musculus

overall_design: Wild-type C. neoformans H99 strain at stationary phase were generated by transferring colonies grown on YPD agar for 24 h into 3 mL liquid YPD and incubating at 30 °C on a slanted shaker set at a 45° angle for 12 h. Cultures were then re inoculated into 4.0 mL fresh liquid YPD at an initial density of 1 × 107 cells/mL and incubated under the same conditions for 72 h to obtain stationary phase cells. For infection, cells were washed twice with phosphate buffered saline by centrifugation at 4000 rpm for 2 min, resuspended in 1 mL sterile phosphate buffered saline, and counted using a hemocytometer. The inoculum was adjusted to 2 × 108 cells/mL, and mice were anesthetized with isoflurane and intranasally inoculated with 50 μL of the suspension, corresponding to 1.0 × 107 cells per mouse. Animals were maintained on a warming pad until recovery and then returned to their cages. At 7 days post infection, lungs were harvested, snap frozen in liquid nitrogen, and processed for total RNA extraction. RNA sequencing was performed by Novogene (Beijing, China).

first: Chun

first: Pengjie

last: Hu

first: Yin

last: Zhu

first: Weixin

last: Ke

first: Xindi

last: Gao

first: Chen

last: Ding

first: Bing

last: Zhai

first: Linqi

last: Wang

first: Ziwei

last: Dai


title: Functional Genomic Profiling of Schizophrenia-Associated Genes Reveals Key Microglial Regulators

city: Boston

first: Liam

last: McCrea

address: 185 Cambridge Street

department: Center for Genomic Medicine

institute: Massachusetts General Hospital

summary: Microglia are increasingly recognized as key regulators of neural circuit development and putative contributors to the pathophysiology of neuropsychiatric disorders such as schizophrenia (SCZ). However, the functional impact of SCZ-associated genes in microglia remains largely unexplored. Here, we performed an arrayed CRISPR targeting screen of 30 schizophrenia-associated genes predicted to be differentially expressed in human microglia-like cells. Target genes were prioritized based on post-mortem transcriptomic relevance and predicted ontology-based roles in phagocytosis pathways. We quantified phagocytic activity and morphological changes following gene targeting using high-content confocal imaging. Key targets, including CYFIP1, MSR1, TREM2, SYK, ITGB2, ITGAM and IRF8, modulated phagocytosis and altered morphological properties consistent with activation states, validating their functional roles in microglia. To elucidate transcriptional impact, we further applied a multiplexed RNA sequencing platform across gene targets. These analyses revealed gene-specific transcriptional signatures, implicating divergent pathways related to phagocytic, activation, cytoskeletal, and lysosomal function. Together, these findings demonstrate the utility of CRISPR-based functional genomics in characterizing microglia function and identifying new target genes and mechanisms that may underlie their contributions to schizophrenia pathophysiology.

overall_design: RNA-seq profiling of PBMC-derived human microglia-like cell response to knockout of schizophrenia-associated genes using an arrayed CRISPR targeting screen.

first: Joy

last: Horng

first: Rebecca

last: Batorsky

first: Joshua

last: Bowen

first: Camilla

last: Boschian

first: Yoonjae

last: Song

first: Roy

last: Perlis

first: Steven

last: Sheridan


title: Enforced ZFP281 expression delays breast cancer initiation and can provide lifelong protection against breast cancer metastasis

city: Bronx

first: Julio

last: Aguirre-Ghiso

address: 1301 Morris Park Ave

department: Cell Biology

institute: Albert Einstein College of Medicine

summary: Breast cancer metastatic reactivation and its links to mammary development are largely unknown. Here, using conditional knockout and overexpression in normal and HER2+ mammary glands, we show that the dormancy regulator ZFP281 promotes branching and dissemination while suppressing growth, and its overexpression can even override HER2-driven cancer initiation. Notably, while ZFP281 does not limit HER2-driven early dissemination, it constrains DCC plasticity, confining cells to a dormant mesenchymal/hybrid-like state and effectively suppressing metastasis throughout the mouse lifespan. ZFP281 is induced by estrogen, progesterone, and glucocorticoid signaling, and RNA sequencing of early lesions revealed that it regulates glutathione metabolism and ferroptosis, potentially supporting fitness during dormancy, while repressing angiogenesis, Th17-like inflammation, innate immune genes, and pro-inflammatory programs that might otherwise trigger awakening. Integrating these findings with human data, we show that DCIS and IBC lesions that later relapse are selectively enriched for ZFP281-regulated M-like and dormancy signatures and, after pregnancy, depleted for a ZFP281-induced stress-autophagy module, indicating that erosion or imbalance of these programs marks lesions that seed DCCs with higher reactivation potential. We propose that ZFP281 acts as a hormone-regulated dormancy gatekeeper that uncouples dissemination from growth, enforcing a metabolically fit, angiogenesis-low, immune-evasive dormant state in breast DCCs that shapes the timing of metastatic relapse and may be exploitable for durable metastasis prevention.

overall_design: RNAseq analysis of mammary glands of MMTV-HER2 mice (ZFP281 WT, KO and OE) in virgin (10 weeks old) and post-pregnant stage (16-18 weeks old).

first: Deepak

last: Singh


title: eIF4G2-dependent translation restrains pancreatic cancer progression

city: New York

first: Chritine

last: Chio

department: ICG

institute: Columbia Unievristy

summary: Pancreatic ductal adenocarcinoma (PDA) is among the most lethal cancers, driven by cellular plasticity that fuels therapeutic resistance and early dissemination. The contribution of translational control to this plasticity remains poorly understood. Through an in vivo CRISPR/Cas9 screen, we identify the non-canonical initiation factor eIF4G2 (DAP5/NAT1) as a translational checkpoint restraining PDA progression. Loss of eIF4G2 accelerated tumor growth, induced poorly differentiated, basal-like histology, and triggered widespread metastatic spread. Ribosome profiling revealed that eIF4G2 loss did not increase bulk protein synthesis but instead suppressed translation of a selective regulon, including tumor suppressors such as PTEN and CREBBP. Functional studies confirmed that PTEN loss was sufficient to drive dedifferentiation but insufficient to promote metastasis, implicating the broader eIF4G2-dependent program—including translational control of transcriptional regulators like CREBBP—in limiting dissemination. Consistently, eIF4G2-deficient tumors showed transcriptomic enrichment of migratory and stemness programs. Computational inference from human PDA datasets demonstrated reduced eIF4G2 activity in metastases, aligning with basal-like features and predicting poorer survival. These results define eIF4G2 as a translational regulator that maintains epithelial identity and restrains metastatic potential, highlighting selective translation as a determinant of PDA subtype and clinical outcome.

overall_design: To define the selective translational program regulated by eIF4G2, we performed ribosome profiling (Ribo-seq) and RNA-seq in three independent murine PDA cell lines (ccmT1, ccmT2, and ccmT4) transduced with sgRosa or sgEif4g2

first: Justin

last: Powers

first: Wei

last: Lai

first: Hiroki

last: Kobayashi

first: Pardis

last: Ahmadi

first: Alvaro

last: Curiel-Garcia

first: Elizabeth

last: Valenzuela

first: Marko

last: Jovanovic

first: Alejandro

last: Chavez


title: Multiomic profiling reveals the regulation of important im-mune genes by PU.1 in porcine alveolar macrophages [RNA-Seq]

first: Juan

last: Yang

overall_design: PAMs were isolated from healthy piglets by bronchoalveolar lavage and cryopreserved. Upon revival, cells were cultured in RPMI-1640 containing 10% FBS and 1% P/S at 37°C with 5% CO₂. SPI1 knockdown was achieved using siRNA transfection, with scrambled siRNA serving as the negative control. Two biological replicates per condition were prepared for RNA-seq.

first: Jiayao

last: Jiang

first: Liangliang

last: Zhang

first: Yanhua

last: Li

first: Cui

last: Du

first: Mingan

last: Sun


title: Pediatric Cancer Dependencies Accelerator [HiChIP]

first: Pediatric Cancer Dependencies

last: Acclerator

overall_design: H3K27ac HiChIP in human medulloblastoma cell lines used in the DepMap/PedDep Consortium


title: Pediatric Cancer Dependencies Accelerator [Hi-C]

overall_design: HiC in human pediatric cancer cell lines used in the DepMap/PedDep Consortium


title: H3K9me3 controls epidermis development by repressing RNA Pol II activity on key promoters and enhancers [CUT&Run - Temporal]

city: Ithaca

first: Chris

middle: Ke

last: Bai

address: 526 Campus Rd

department: MBG

institute: Cornell University

summary: Histone H3 lysine 9 trimethylation (H3K9me3) is a heterochromatin mark important for silencing repetitive DNA elements and for repressing lineage-inappropriate genes during early murine development, but its role in later organogenesis remains understudied. Here, we simultaneously target three histone methylases, Suv39h1, Suv39h2 and Setdb1, to deplete H3K9me3 specifically in developing epidermis. H3K9me3 loss blocks epidermis morphogenesis, resulting in defective cell differentiation, impaired barrier function, disrupted hair follicle formation, and failed Merkel cell specification. Single cell transcriptomic analysis reveals epidermal cells with mixed lineage identity and perturbed cell-type specific gene programs, including cell cycle, extracellular matrix, cytoskeleton, and metabolism. Using our recently developed approach, PReCIS-seq, we present the first cell-type specific, genome-wide landscape of transcriptionally engaged RNA polymerase II (RNA Pol II) in intact tissue following H3K9me3 depletion. We find that H3K9me3-enriched islands restrict inappropriate RNA Pol II activity at select developmentally regulated enhancers and promoters. This work highlights the indispensable role of H3K9me3, mediated by its three major histone methyltransferases, in regulating lineage diversification and developmental progression during mouse organogenesis.

overall_design: Epcam+α6+ epidermal cells were sorted from E12.5, E14.5 and E16.5 wild type mouse embryos for CUT&RUN profiling. Anti-H3K9me3 antibody was used for experimental group and anti-IgG antibody was used for control group.

first: Tudorita

last: Tumbar


title: MAIT cells at different developmental stages egress the thymus to colonize specific tissues and sense increases in peripheral ligands

city: PARIS

first: Anne-laure

last: Le gac

address: 26 rue d'ulm

country: France

institute: Institut Curie

summary: MAIT but not iNKT cells can be selected by thymic epithelial cells, while both require CD4+CD8+ thymocytes for maturation. MAIT and iNKT cells have similar developmental dynamics and thymic-residency properties but proliferate at different developmental stages.MAIT cells exit the thymus in an S1PR-dependent manner at different maturation stages to colonize specific peripheral organs.PLZF+Tbet-RORgt- MAIT cells preferentially colonize the intestine in a CCR9-dependent manner.Thymic output contributes to peripheral MAIT and iNKT cell numbers at steady state. Thymic MAIT17 cells are activated and expand in colitis and after bacterial exposure.

overall_design: For Thy_FTY dataset bone marrow chimeras were generated (WT into MR1KO) and, after 7 weeks, treated with Vehicle or FTY720 for 1 week. For Thy_Mr1fl, MR1fl, Foxn1-cre+Mr1fl, Vav1-icre+Mr1fl and Cd11c-creMr1fl mice were analysed. In both case thymi were mechanically dissociated, MAIT cell were magenicatically enriched with MR1:5-OP-RU-PE tetramer and anti-PE beads and total MAIT cells (Tcrblo/+ and tetramer+) were FACS sorted.

first: Rafael

last: Almeida Paiva

first: Hugues

last: Briere

first: Anne-Laure

last: Le Gac

first: Aurélie

last: Darbois

first: Laetitia

last: Perrin

first: Anya

last: Belmellat

first: Chaimae

last: Saji

first: Mariela

last: Furstenheim

first: Lea

last: Guyonnet

first: François

last: Legoux

first: Bernard

last: Malissen

first: Marion

last: Salou

first: Olivier

last: Lantz


title: Ki-67 promotes circulating tumor cell intravasation and metastasis in breast cancer

city: Basel

first: Yongzhan

last: Zhang

address: Otto-Stern-Weg 7 HPL H17.2

country: Switzerland

institute: ETH Zurich

summary: Hematogenous dissemination by circulating tumor cells (CTCs) drives metastatic spread. However, the molecular features that empower these cells to colonize distant organs remain unclear. We profiled single CTCs and CTC clusters from three breast cancer xenograft models by single-cell RNA sequencing and uncovered a shared transcriptional program marked by broad upregulation of cell-cycle genes. A targeted CRISPR loss-of-function screen identified MKI67 as an essential regulator of CTC abundance, particularly of cluster abundance, and metastatic progression. MKI67 disruption markedly reduced CTC numbers and decreased the fraction ofCTC clusters, leading to diminished lung metastases. CRISPRa-mediated re-expression of downstream adhesion genes CD47 and KLF4 restored both cluster prevalence and metastatic burden. Our findings link cell-cycle upregulation to enhanced metastatic capacity and position MKI67-dependent adhesion signals as tractable targpendent adhesion signals as tractable targets against metastatic spread.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: MKI67 KO cells were injected to corresponding mice and at day 20 post-injection, tumor tissues were harvested and processed into single-cell suspensions following established protocols and were isolated using a BD FACSAria Fusion cell sorter.

first: Jianwen

last: Zhou

first: Karin

last: Strittmatter

first: Yu-Wei

first: Maria

last: Waldmeier

first: Selina

last: Budinjas

first: Massimo

last: Saini

first: Ece-Su

last: Ildiz

first: Ana

last: Gvozdenovic

first: Werner

last: Kovacs

first: Francesc

last: Castro-Giner

first: Nicola

last: Aceto


title: Mapping CCR4-NOT recruitment elements at high resolution reveals transcriptome-wide determinants of mRNA decay [miRNA-Seq]

city: Montreal

first: Marc

last: Fabian

state: Quebec

address: 3755 Côte Ste-Catherine Road

department: Department of Oncology

institute: McGill

type: Non-coding RNA profiling by high throughput sequencing

summary: The CCR4-NOT complex is a central regulator of gene expression, orchestrating mRNA destabilization and decay through interactions with RNA-binding proteins (RBPs) and the miRNA-induced silencing complex (miRISC). However, identifying which RBP and miRNA target sites actively recruit this complex to engender mRNA turnover remains challenging, due in part to the multiple modes by which the CCR4-NOT can be recruited to targets. Here, to address this, we developed TRACER (Targeted RNA Association of CCR4-NOT and  Element Recovery), a crosslinking approach that enables high-throughput mapping of cis-acting RNA elements that recruit the CCR4-NOT complex to target RNAs. TRACER analysis of a human epithelial cell line uncovers thousands of CCR4-NOT target elements, including many of which map to known and/or predicted RBP binding motifs, as well as miRNA target sites. Importantly, we show that CCR4-NOT recruitment elements identified by TRACER play important roles in promoting mRNA decay. Taken together, our approach provides the first high-resolution transcriptome-wide map of CCR4-NOT targeting elements in human cells that engender mRNA decay.

overall_design: miRNA-seq of HEK293 cells to identify cis-acting RNA elements that associate with the CCR4-NOT complex

first: Zijun

last: Luo

first: William

last: Brothers

first: Aldo

last: Hernandez-Corchado

first: Justin

last: Zhao

first: Zara

last: Aryampour

first: Connor

last: Campbell-Smith

first: Hamed

last: Najafabadi


title: Mitochondrial metabolism regulates the immunogenic responsiveness of dendritic cells [methylation]

city: MADRID

first: Ignacio

last: Heras-Murillo

address: c/Melchor Fernandez Almagro, 3

institute: CNIC

summary: Activation of dendritic cells (DCs) is linked to increased glycolysis. However, the role of the mitochondrial electron transport chain (ETC) in conventional DC subset function is poorly defined. Here, we revealed that immunogenic activation elevates late oxygen consumption in cDC1s, but not cDC2s. To investigate the relevance of the ETC in DC immunogenicity, we generated mice with impaired ETC complex III function in DCs. ETC complex III impairment attenuated poised or adjuvant-triggered cDC1 activation, migration and capability to prime T cells for anti-cancer immunity, while it had a mild effect on cDC2s. Mechanistically, loss of complex III in cDC1s led to a deregulated redox and metabolite balance causing DNA hypermethylation of PU.1-binding regions. This DNA hypermethylation diminished early stimulus-induced gene expression linked to immunogenic responsiveness of cDC1s, which was rescued via ectopic expression of alternative oxidase. Our findings show how the ETC differentially regulates the immunogenic function of cDC subsets.

overall_design: cDC1s or cDC2s from Uqcrqf/f or Itgax-Uqcrqf/f mice were FACS sorted, the  DNA purified and bisulfite converted. Infinium Methylation BeadChip arrays (Illumina) were used to analyze DNA methylation.

first: Stefanie

last: Wculek

first: Josep

last: Calafell-Segura

first: Esteban

last: Ballestar

first: David

last: Sancho


title: Mapping CCR4-NOT recruitment elements at high resolution reveals transcriptome-wide determinants of mRNA decay

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

overall_design: Refer to individual Series


title: Transcriptome sequencing of Hodgkin lymphoma Hodgkin and Reed-Stenberg cells reveals escape from NK cell recognition and an unfolded protein response

first: Isabella

last: Kong

state: New York

address: 1300 York Ave

institute: Weill Cornell Medicine

summary: The mutational profile of classic Hodgkin lymphoma (cHL) overlaps with that of related B cell lymphomas, including primary mediastinal B cell lymphoma (PMBL), and yet these are different histologically and clinically. To discover the molecular features that distinguish cHL, we deployed flow cytometric cell sorting and low-input RNA sequencing to generate full transcriptome data from viable, isolated Hodgkin and Red-Sternberg (HRS) cells from eighteen primary tumors, alongside matched intra-tumoral non-neoplastic B cells and four cell lines.  Comparison of HRS cells to normal cellular subsets revealed evidence of abortive plasma cell differentiation, with an unfolded protein response signature, shared with plasma cell neoplasms, but not other B-cell lymphoma types. Comparison of cHL to PMBL revealed similarities but also key differences in B cell differentiation programs accompanied by upregulation of genes involved in microtubule cytoskeleton organization in cHL, which may be related to the unique multinucleated nature of HRS cells. In HRS cells, we also observed a downregulation of SLAM family receptors, which are crucial for NK cell activation, providing a potential mechanism for immune evasion from NK-mediated killing. New fusion transcripts were seen, including a recurrent transcript predicted to result in an in-frame fusion protein involving TRAF1 and PHF19.

overall_design: Bulk RNA-seq profiling the transcriptome differences between HRS cells and intra-tumoral B cells following flow-cytometry sorting.

first: Mikhail

last: Roshal

middle: Y

first: Wikum

last: Dinalankara

first: Lisa

last: Giulino-Roth

first: Ethel

last: Cesarman


title: Effector differentiation by stem-like intraepithelial γδ T cells is required for host defense against infection

city: Hangzhou

first: Shiyuan

last: Xie

address: Shilongshan Street

institute: Westlake University

summary: Intraepithelial γδ T cells (γδIELs) are crucial for maintaining intestinal homeostasis, yet their functional regulation remains incompletely understood. Here, we identified two distinct γδIEL subsets: CD160+TCF1+BCL6+ stem-like and granzymeB+BLIMP1+ effector-like cells; the latter exhibited cytotoxic activity. Stem-like γδIELs differentiated into effector-like cells in response to microbiota and pathogen infection. IL-12, in cooperation with IL-15 and TCR signaling, activated a STAT4-BLIMP1 pathway to induce effector differentiation, and IL-12 signaling was required for their cytotoxic function. Tcf7 deletion significantly reduced stem-like but not effector-like γδIELs, while Bcl6 deficiency increased effector-like γδIELs selectively. In contrast, the absence of Prdm1 not only abolished the development of effector-like γδIELs but also impaired the clearance of enterogenous pathogens. Taken together, these findings define two functionally distinct γδIEL subsets with a hierarchical regulatory program linking microbial signals and cytokine pathways to their cytotoxic function in the gut.

overall_design: For RNA sequencing of γδIEL subsets from BCL6-RFP reporter mice, CD160+ BCL6-RFP+ and CD160– BCL6-RFP– γδIELs from the small or large intestine were FACS-sorted under steady-state conditions. Total RNA was extracted for RNA-seq. For sequencing of target gene knockout (KO) γδIELs, CD160+ CD122– CD8αα γδIELs from the small intestine of Bcl6ΔCd8a (CD8a-cre Bcl6-flox/flox) or Tcf7ΔCd8a (CD8a-cre Tcf7-flox/flox) mice, and total CD8αα γδIELs from the small intestine of Prdm1ΔCd8a (CD8a-cre Prdm1-flox/flox) mice and their respective littermate controls, were FACS-sorted under steady-state conditions. Total RNA was extracted for RNA-seq. For sequencing of the epithelial cell layer, mice were pretreated with streptomycin and infected with 10^8 CFU of Salmonella typhimurium SL3261. Fourteen days post-infection, the entire large intestinal epithelium from S. Tm. (SL3261)-infected or uninfected mice was isolated, and total RNA was extracted for RNA-seq.

first: Chen

last: Dong


title: Trem2 activation by renal tubular debris sustains Arg1+ macrophage survival and promotes tubular epithelial repair in renal ischemia–reperfusion injury

city: Beijing

first: Jiayu

last: Wang

state: Beijing

address: Beijing Chao-Yang Hospital, 8 Gongren Tiyuchang Nanlu, Beijing, China

institute: Beijing Chao-Yang Hospital, Capital Medical University

summary: Renal ischemia–reperfusion injury (RIRI) profoundly impairs the function of both native and transplanted kidneys. Arg1⁺ macrophages play a pivotal role in tubular regeneration and repair. The myeloid receptor triggering receptor expressed on myeloid cells 2 (Trem2) mediates the clearance of cellular debris by recognizing lipid components from injured cells. This study aimed to investigate whether Trem2 not only facilitates the debris clearance activity of Arg1⁺ macrophages in renal tubules but also sustains their survival, thereby providing a potential therapeutic target for the prevention and treatment of RIRI. To this end, we established stable RAW264.7 cell lines with Trem2 knockdown using lentivirus-mediated shRNA, followed by IL-4–induced polarization. Tubular epithelial fragments were prepared from TCMK-1 cells subjected to hypoxia/reoxygenation (H/R) and subsequently co-cultured with macrophages. RNA sequencing was performed to compare transcriptional profiles between normal Arg1⁺ macrophages and Trem2-deficient Arg1⁺ macrophages under tubular debris stimulation.

overall_design: Tubular epithelial cell (TCMK-1) fragments were co-cultured with Arg1⁺ macrophages transduced with either an empty vector (shNC) or with Trem2 knockdown (shTrem2) for 24 hours. After co-culture, cells were collected, RNA was extracted, and RNA sequencing was performed to assess transcriptional profile changes.

first: Zhiyi

first: Runyi

first: Xin

last: Zheng

first: Xiaopeng

last: Hu


title: Organ-specific regulatory logic orchestrates gene expression in the coelomic mesothelium

city: Oxford

first: Quang

middle: Minh

last: Dang

state: Please select region, state or province

address: IDRM, Old Road Campus, Roosevelt Drive

department: Institute of Developmental and Regenerative Medicine (IDRM)

institute: University of Oxford

summary: The embryonic (coelomic) mesothelium undergoes epithelial-to-mesenchymal transition (EMT) to promote vascular growth and parenchymal development. A prominent example is the epicardium, which plays an essential role during heart development. Little is known about the mechanisms behind gene regulation in the coelomic mesothelium, and organ-specific enhancer logic that endows specialization. Using gene regulatory network inference via single-cell multi-omics, our study reveals trans- and cis-regulatory elements (CREs) that regulate mesothelial gene expression in three organs: heart, lung, and pancreas. Here, we expand on pivotal transcription factors (TFs) and CREs specific to the epicardium. In contrast, we find that the TF MAF may orchestrate pan-mesothelial gene expression via conserved CREs, which are absent from non-mesothelial lineages. MAF may preserve mesothelial identity, evidenced by negative correlation with EMT and conservation in mouse and human epicardial cells. Collectively, our work sheds light on the regulatory logic behind cell type identity and leverages single-cell integration to gain insights into mammalian organ development.

overall_design: Whole embryonic hearts were isolated from Rosa26tdTomato;Wt1CreERT2/+ embryos at E11.5 and E17.5, to define the chromatin landscape in FACS sorted epicardial cells (tdTomato labelled) at those developmental stages using bulk ATAC-seq. Epicardial lineage labelling was achieved in embryos by administering tamoxifen to pregnant females by oral gavage at E9.5. Live epicardial cells (CD31-tdTomato+PDPN+) isolated by FACS were subjected to the ATAC-seq protocol as previously described. At E12.5 and E13.5, we additionally isolate epicardial-derived cells (EPDCs) (CD31-tdTomato+PDPN-) and subject these cells to bulk ATAC-seq for comparison between epicardial cells and their mesenchymal derivatives.

first: Nicola

last: Smart

first: Andia

last: Redpath

first: Joaquim

last: Vieira


title: Physiologic variation in sperm miRNAs tunes embryonic gene regulatory programs and developmental outcomes

city: Philadelphia

first: Colin

last: Conine

state: Pennsylvania

address: 421 Curie Blvd BRB 1213

department: Genetics and Neonatology

institute: UPenn Perelman SOM and CHOP

summary: Small RNAs delivered by sperm can transmit environmentally regulated, epigenetically inherited phenotypes to offspring, yet the mechanisms by which modest changes in sperm microRNA (miRNA) abundance overcome dilution within the much larger egg to influence embryonic development remain unresolved. Here, we show that physiologically relevant variation in individual sperm miRNAs is sufficient to quantitatively program embryonic gene expression and developmental outcomes. Using parthenogenetic and fertilized embryos, we show that as few as 200 molecules of miR-200c-3p or miR-465c-3p induces reproducible, dose-dependent gene expression responses that are restricted to defined developmental windows. Parthenogenetic embryos faithfully recapitulate early miRNA-driven gene expression changes observed in fertilized embryos, validating their use for isolating early regulatory mechanisms. We further developed Ago-REMORA, an RNA adenosine base editor fused to Argonaute 2 to map miRNA–mRNA interactions in embryos, revealing that early mRNA repression reflects direct miRNA targeting, while transcriptional changes at later stages arise indirectly from propagation of early regulatory events. Furthermore, we show that modest elevation of miR-200c-3p during early development is sufficient to induce transcriptional alterations through early development and produce craniofacial phenotypes in late-stage embryos, recapitulating features of fetal alcohol syndrome associated with paternal alcohol consumption. Together, these findings establish a generalizable framework by which small perturbations in sperm miRNA content quantitatively modulate early gene regulatory programs, triggering cascades that persist throughout development and influence offspring phenotype.

overall_design: We microinjected defined quantities of synthetic miR-200c-3p or miR-465c-3p (including a physiologically relevant low dose of ~200 molecules) into mouse zygotes, using GFP-injected embryos as controls. Embryos were generated either by parthenogenetic activation or by sperm fertilization and collected at late 2-cell, 4-cell, and morula stages. Single-embryo RNA-seq was performed to quantify dose-dependent and stage-specific transcriptional responses to miRNA perturbation. For mechanistic validation, AGO2-based RNA editing (Ago-REMORA) was used at the 2-cell stage to identify direct miRNA target engagement.

first: Lee

last: Grace

first: Conine

last: Colin


title: H3K9me3 controls epidermis development by repressing RNA Pol II initiation on key promoters and enhancers [PReCISseq]

first: Chris

last: Bai

state: NY

overall_design: K14Cre+,Suv39h1 f/f, Suv39h2 -/-,Setdb1 f/f triple knockout line was crossed to Polr2b-GFP f/f line to tag RNA Pol II specifically in skin epithelium. PReCIS-seq was then performed on dissected embryo dorsal skin. Embryos heterozygous for Suv39h1, Suv39h2, Setdb1 were used for control. PReCIS-seq was performed following steps described in original paper.

first: Tudorita

last: Tumbar


title: Molecular phenotypes in early gallbladder and liver disease in the CFTR-/- sheep highlight aspects of CF-relevant hepatobiliary disease

city: Cleveland

first: Katreya

last: Lovrenert

address: 10900 Euclid Avenue

department: Department of Genetics and Genome Sciences

institute: Case Western Reserve University

summary: The initiating cellular events in cystic fibrosis (CF) hepatobiliary disease are not well characterized, in part due to lack of accessibility of primary tissues. However, enhanced longevity due to highly effective modulator therapies has generated renewed interest in key aspects of liver and gallbladder disease, and how these might be treated in people with CF (pwCF).  To extend the CF hepatobiliary knowledgebase, we performed a transcriptomic analysis of liver and gallbladder development in the wild type (WT) and CFTR-/- sheep. Bulk RNA was extracted from each tissue at specific timepoints through gestation (from 50 days to term) and used for RNA-seq. Differentially expressed genes between the timepoints within each genotype and between WT and CFTR-/- sheep at each timepoint were identified, and then used in gene ontology process enrichment analysis to reveal altered biological processes.  We find that at the molecular level, the gallbladder in the CFTR-/- animals is both structurally and functionally compromised by mid-gestation, consistent with the observed microgallbladder phenotype. In the liver, many aspects of differentiation are apparently well-established early in gestation. However, we find functional immaturity in the CFTR-/- liver at term, where genes associated with many key metabolic processes do not show the upregulation seen at term in WT liver.  We also show that the regulatory mechanisms for the CFTR gene in ovine gallbladder cells are highly conserved with those elucidated at the human CFTR locus, further enhancing the relevance of these data to advance understanding of hepatobiliary disease in pwCF.

overall_design:
RNA-seq of sheep (ovis aries) gallbladder epithelial cells and pancreatic organoids
ATAC-seq of sheep (ovis aries) gallbladder epithelial cells and pancreatic organoids

first: Frederick

last: Meckler

first: Ayushi

last: Umrigar

first: Shih-Hsing

last: Leir

first: Ann

last: Harris


title: B12 promotes gut dysbiosis and an inflammatory microenvironment that potentiates myeloid biased Tet2-deficient hematopoiesis [dataset 2]

city: Miami

first: Luisa

last: Cimmino

state: Florida

address: 1501 NW 10th Ave, BRB 711

institute: University of Miami

summary: Recent studies have linked elevated vitamin B12 serum levels with the presence of Clonal Hematopoiesis (CH) and an increased risk of developing myeloid malignancy. High B12 supplementation increases serum levels, alters gut microbial composition, and reduces the production of short-chain fatty acids (SCFAs), which help maintain gut barrier function and mucosal integrity. TET2 mutation is a frequent driver of CH that progresses in a positive feedback loop in response to microbial signals suggesting that B12 may influence CH via the gut microbiome. We evaluated the microenvironmental effects of B12 supplementation in a Tet2-deficient model of CH and found that B12 enhances myelopoiesis, heightens the responses of myeloid cells to bacterial stimuli, and increases the levels of circulating inflammatory cytokines. B12 supplementation also induced gut dysbiosis and reduced the levels of SCFA-producing bacteria in both wild-type and Tet2-deficient mice. Importantly, the effects of excess B12 were reversible upon oral supplementation with the SCFA butyrate. These findings suggest that B12 may promote CH progression by disrupting microbiome-derived SCFA metabolism, highlighting a potential therapeutic role for SCFA supplementation in mitigating CH.

overall_design: Single cell RNA-sequencing of murine bone marrow from Tet2+/- competitive transplant mice treated with high B12 +/- Butyrate

first: Peter

last: Lyon

first: John

middle: P

last: Brabson

first: Molly

last: Dalzell

first: Luis

last: Nivelo

first: Alejandro

last: Villarino


title: Microsomal triglyceride transfer protein restricts progesterone production in Leydig Cells by regulating SREBP2 activity [ATAC-Seq]

city: Mineola

first: M. Mahmood

last: Hussain

address: 101 Mineola Blvd.

department: Foundations of Medicine

institute: NYU Grossman Long Island School of Medicine

summary: Reduced serum testosterone or hypogonadism diminishes bone density, muscle mass, and sexual functions and increases obesity and CVD. Microsomal triglyceride transfer protein (MTP) is a lipid-transfer protein, primarily expressed in the liver and intestine, and is critical for apoB-containing lipoprotein assembly and secretion. MTP mRNA has been reported in the testis, but its function is unknown. Here, we report that MTP protein is present in the Leydig cells of human and mouse testes and plays a regulatory role in cholesterol metabolism and sex hormone production. Inhibition of the MTP activity by a specific inhibitor, knockdown, and gene ablation significantly increased progesterone secretion in mouse Leydig MA10 cells after stimulation with cyclic AMP. Molecular studies revealed that MTP enhanced the expression of genes in the uptake of lipoproteins, and in the synthesis and intracellular trafficking of cholesterol. Moreover, it enhanced the Lhcgr signaling as well as the expression of genes in the biosynthesis of progesterone. Transcriptome analysis confirmed increased expression of cholesterol and steroid biosynthesis pathways in MTP deficient clones. To understand how MTP deficiency increases expression of genes in cholesterol and steroid biosynthesis, we looked for changes in transcription factors. MTP deficiency enhanced the expression and activity of Srebp2. ATAC sequencing revealed enhanced binding of Srebp2 to the promoters of several enzymes in the biosynthesis of cholesterol and sex hormones. Furthermore, cholesterol and progesterone biosynthesis pathways were significantly attenuated after Srebp2 knockdown in MTP-deficient cells to the levels seen in wildtype cells. In short, these studies showed that MTP regulates cholesterol and steroid biosynthesis in Leydig cells by modulating the activity of Srebp2. Inhibition of testicular MTP might be useful to treat hypogonadism in some patients.

overall_design: RNA sequencing of 48h old culture of wildtype and MTP knockout MA10 cells after stimulation with 8-BrcAMP for 3h for steroidogenesis

first: Atrayee

last: Chattopadhyay

first: Tirumalasetty

last: Munichandra

first: Thomas

last: Palaia

first: Bhargavi

last: Gangula

first: Qing

last: Miao

first: Jacques

last: Tremblay

first: Mahmood

middle: M