title: Sex-specific effects of exogenous asparagine on colorectal tumor growth, 17β-estradiol levels, and aromatase

city: NEW HAVEN

first: Oladimeji

last: Aladelokun

state: Connecticut

department: Environmental Health Sciences

institute: YALE UNIVERSITY

summary: Sex-related differences in asparagine metabolism are associated with cancer prognosis. However, the effect of exogenous asparagine on colorectal cancer (CRC) growth in men and women remain unclear. This study aims to understand the relationship between exogenous asparagine supplementation and 17β-estradiol levels and to elucidate mechanisms underlying sex-dependent signaling during CRC development. We administered asparagine intraperitoneally to tumor-bearing immunodeficient male and female Rag2/Il2RG (R2G2) mice. Asparagine supplementation caused a significant increase in tumor asparagine levels in both the tumor-bearing male and female R2G2 mice but increased serum estradiol levels and suppressed tumor growth in female R2G2 mice only. Additionally, we combined transcriptome, metabolome and immunochemical analyses and found that intraperitoneal asparagine treatment induced sex-dependent intra-tumoral metabolic changes to asparagine, aspartate, glutamine and glutamate levels in CRC cells. We observed that in females, exogenous asparagine exerts a negative feed-back effect on de novo asparagine synthesis and is associated with the activation of a sub-population of macrophages that may secrete 17β-estradiol via an aromatase or cytochrome P450 family 19 (CYP19)-dependent mechanism, resulting in improved tumor-specific survival. Conversely, in male mice, asparagine treatment augments tumor growth and is related to decreased numbers of macrophages, reduction in CYP19-mediated 17β-estradiol secretion leading to worsened tumor-specific survival. Overall, our results reveal a novel and sex-specific role for exogenous asparagine during cancer progression and underscores the importance of understanding mechanisms that control asparagine biosynthesis.

overall_design: Thirty (30) R2G2 (n = 15 female, n = 15 male) mice were randomly separated into 6 groups of 5 for a tumor-specific survival experiment. HCT116 cancer xenograft establishment was confirmed as palpable tumors in all 30 mice. In group 1, female R2G2 mice received vehicle or control: PBS solution. Groups 2 and 3 represent female mice that were administered 1 mg/kg and 10 mg/kg asparagine respectively. In group 4, male R2G2 mice were administered PBS solution. In groups 5 and 6, male mice received 1 mg/kg and 10 mg/kg asparagine respectively. One mouse in the female PBS control group became severely moribund before study endpoint. RNA-sequencing was conducted on xenografts from n = 4 tumors/sex/group. Libraries were prepared with total RNA using the Total RNA TruSeq mRNA Stranded Library Prep Kit (Illumina, San Diego, CA, USA) as previously described [20]. Briefly, mRNA pulldown was performed using an oligodT primer attached to magnetic beads. The libraries were quantified using the Qubit dsDNA HS assay kit (Thermofisher Scientific, Waltham, MA, USA). Pooled library was sequenced on an efficient ultra-high-throughput sequencer using the NovaSeq 6000, an instrument with a patterned flow cell technology which provides the highest throughput across multiple applications (Illumina) according to the manufacturer’s protocol.

first: Katherine

last: Benitez

first: Yuying

last: Wang

first: Abhishek

last: Jain

first: Domenica

last: Berardi

first: Georgio

last: Maroun

first: Xinyi

last: Shen

first: Jatin

last: Roper

first: Joanna

last: Gibson

first: Kaelyn

last: Sumigray

first: Sajid

last: Khan

first: Caroline

middle: H

last: Johnson


title: Adipogenic Transcriptional Regulation Mediated by PARP7 Through NAD+ Sensing Mechanism [CUT&RUN]

status: Public on Dec 24 2025

city: Dallas

first: W. Lee

last: Kraus

state: TX

address: 5323 Harry Hines Blvd.

country: USA

institute: UT Southwestern Medical Center

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The process of differentiating from a preadipocyte into a mature, fat storing, adipocyte (adipogenesis) is a complex and tightly regulated process vital to human health. The molecular mechanisms regulating adipogenesis are incompletely understood, although key facets of the signaling and regulatory pathways have been defined. Here we have identified a mono(ADP-ribosyl)transferase (MART), PARP7, that plays a pivotal role in adipogenesis. ADP-ribosylation – the process whereby specific members of the poly(ADP-ribosyl)polymerase (PARP) family facilitate the covalent transfer of  ADP-ribose moieties from NAD+ to substrate proteins – has been shown to control multiple components of the adipogenic regulatory machinery. Herein we have found that PARP7 is required for modulation of the adipogenic transcriptional program during adipogenesis, and that the loss of PARP7 results in a decrease of the adipogenic process. Contrary to previous studies, the ability to modulate the adipogenic transcriptional program is independent of PARP7 catalytic activity. However, the catalytic activity of PARP7 plays a vital role in regulating protein stability, in a time and space specific manner, which is required for modulation of the adipogenic transcriptional program. This study has found a novel pathway which utilizes NAD+ compartmentalization to stabilize PARP7 at a specific time during the adipogenic process allowing for the regulation of adipogenic genes, through modulation of the well-known adipogenic transcription factor C/EBPß, in a time sensitive manner. Therefore, acting as another check and balance to the adipogenic differentiation process.

overall_design: 3T3-L1 cells were cultured and treated as described above in 15 cm diameter plates.  The cells were transfected with siRNAs targeting Parp7 or a universal control 48hrs before the addition of the differentiation cocktail. The cells were then differentiated for 24hrs

first: MiKayla

middle: S

last: Stokes

first: Yoon Jung

last: Kim

first: Sneh

last: Koul

first: Josue

last: Zuniga

first: Shu-Ping

last: Chiu

first: Tulip

last: Nandu

first: Kraus

last: W Lee


title: Adult leptomeningeal vestigial neural crest-derived multipotent cells promote vascular repair after stroke.

city: Warsaw

first: Marcin

last: Tabaka

address: Kasprzaka 44/52

country: Poland

institute: International Centre for Translational Eye Research

type: Expression profiling by high throughput sequencing

summary: We collected cerebral cortex at days 3, 7, 14, 28 after dMCAO (distal middle cerebral artery occlusion) and sham.

overall_design: Cerebral cortex 17 samples (sham n=3, D3 n=3, D7 n=4, D14 n=3, D28 n=4)

first: Ji

last: Park

first: Wilber

last: Quispe


title: cMaf supports Zeb2 expression for primitive hematopoiesis (Multiome)

status: Public on Dec 26 2025

city: Saint Louis

first: Jing

last: Chen

state: MO

address: 600 S Euclid Ave

institute: Washington University in St. Louis

summary: Tissue-resident macrophages (TRMs) are essential for tissue homeostasis and play a key role in disease and pathology. They originate from distinct waves of hematopoietic progenitors, including primitive, transient-definitive, and definitive hematopoiesis. We found that loss of the Zeb2 ‒165kb enhancer impairs  monocyte development and alters TRM transcriptional landscapes, with only HSC-derived monocytes requiring Zeb2 for their development. In Zeb2∆‒165kb mice, primitive TRMs persist in tissues due to the lack of replacement by later hematopoietic waves, and Maf is required for maintaining Zeb2 expression in primitive macrophages.

overall_design: Fetal Liver CD45.2+CD64+ cells were isolated from E12.5 wildtype and KO embryos. Single nuclei were extracted and submitted for snRNA- and scATAC-seq sequencing using 10X genomics multiomic platform.

first: Theresa

middle: L

last: Murphy

first: Kenneth

middle: M


title: Serine inhibits granulosa cell ferroptosis to maintain ovarian function

status: Public on Dec 23 2025

city: Nanchang

first: Haocheng

last: Gu

state: Jiangxi Province

address: Hongguantang Sub-district

country: China

institute: Nanchang University

summary: Cyclophosphamide (CTX) is a primary medicine for curing breast cancer which often caused premature ovarian insufficiency (POI). Our recent publication revealed that CTX induced POI by promoting the expression of SLC1A4, a transporter of serine efflux, in ovarian granulosa cells (GCs). Here, we reported that there was a closed connection between the reduction of serum serine and ovarian hypofunction in the breast cancer patients treated with CTX or women of childbearing age who were suffered from the staying-up-late. Additionally, we observed that dietary serine supplementation protected mice from CTX-induced POI without altering its anti-breast cancer. Furthermore, we demonstrated that the elevated serine promoted S1P synthesis, and in turn, inhibited the nuclear translocation of Nrf2 and consequent HO-1 expression, to suppress ferroptosis in GCs. Our study revealed that the chemotherapy-induced or idiopathic POI shared the same mechanisms, indicating that serine was a critical factor for maintaining ovarian function.

overall_design: Mouse ovarian granulosa cells were stimulated with cyclophosphamide for 24 hours, after which RNA was extracted to analyze gene expression differences between unstimulated and cyclophosphamide-stimulated ovarian granulosa cells.

first: Hongbo

last: Xin


title: Integrative omics analysis elucidates the role of ANKRD9 in modulating chicken primary myoblast IMP metabolism via the purine metabolic pathway

city: Zhengzhou

first: Li

last: Yuanfang

address: No. 26 Longyuan East 7th Street, Zhengdong New District

institute: Harbin Institute of Technology Zhengzhou Research Institute

summary: With the continuous improvement of human dietary structure, the demand for high-quality chicken meat is steadily increasing. It is therefore crucial for breeders to enhance meat quality while maintaining muscle yield in broilers. Inosine monophosphate (IMP) is a key compound influencing meat flavour and quality. In this study, we used the Chinese local breed Gushi chicken breed as the research model and observed dynamic expression changes of the ankyrin repeat domain 9 (ANKRD9) gene in breast muscle tissues. By integrating transcriptomic and metabolomic analyses, we systematically investigated the role of ANKRD9 in chicken primary myoblasts. Our findings indicate that ANKRD9 may affect cell adhesion and IMP metabolism through regulation of the purine metabolic pathway. Further cellular experiments confirmed that ANKRD9 inhibits IMP metabolism in myoblasts. These results highlight the critical role of ANKRD9 in regulating IMP, a key flavour-related metabolite, and provide a potential molecular target and theoretical foundation for the breeding of high-quality broilers.

overall_design: RNA-seq expression profile of ANKRD9 overexpression and pcDNA3.1 in chicken primary myoblasts

first: Yuanfang

last: Li

first: Guoxi


title: Targeting mTORC2-Dependent AKT/FOXO1/RNF125 Signaling Exploits a Therapeutic Vulnerability in c-MET-Activated and β-catenin-Mutated Hepatocellular Carcinoma

city: Honolulu

first: Xue

last: Wang

state: HI

address: 701 Ilalo Street

department: Cancer Biology Program

institute: University of Hawaii Cancer Center

summary: Background and Aims: Approximately 10% of human hepatocellular carcinomas (HCC) exhibit concurrent c-MET activation and β-catenin gain-of-function mutations, representing a clinically relevant HCC subtype. This study aimed to investigate the role of mTORC2/AKT signaling in this subtype and identify potential therapeutic targets. Approach and Results: The mTORC2/AKT cascade was activated in c-Met/β-cateninΔ90 HCC lesions. Genetic ablation of Rictor, the essential mTORC2 subunit, strongly suppressed c-Met/β-cateninΔ90-dependent hepatocarcinogenesis. Mechanistically, both the TSC2/mTORC1 axis and FOXO1 transcription factors functioned as critical downstream effectors of mTORC2/AKT in this model. We further identified RNF125 as a direct transcriptional target of FOXO1. RNF125 overexpression significantly inhibited tumorigenesis in the c-Met/β-cateninΔ90 model and suppressed liver cancer cell growth in vitro. Notably, using an in vivo doxycycline-inducible system, we found that inducing RNF125 expression in established c-Met/β-cateninΔ90 HCC suppressed tumor progression, suggesting that activation of RNF125 may have translational implications for HCC treatment. Conclusions: Our study, for the first time, established the mTORC2/AKT/FOXO1/RNF125 axis as a critical driver and therapeutic vulnerability in c-MET-activated/β-catenin-mutated HCC. Our study filled a critical gap by defining the tumor-suppressive role of FOXO1 specifically in this HCC subtype. Furthermore, our results positioned RNF125 as a promising therapeutic target for this aggressive HCC subtype.

overall_design: RNA-seq profiling was performed on pInducer20-RNF125 HepG2 cells treated with or without 2 μM doxycycline hydrochloride (Dox) for 48 hours. The experiment aimed to assess gene expression changes upon RNF125 induction.

first: Xin


title: cMaf supports Zeb2 expression for primitive hematopoiesis (CUT&RUN)

summary: Microglia arise exclusively from yolk sac (YS) progenitors during primitive hematopoiesis, while other tissue resident macrophages (TRMs)originate during transient-definitive and definitive hematopoiesis. Macrophages require the transcriptional repressor Zeb2 for development, and macrophages developing during definitive hematopoiesis require the ‒165 kb Zeb2 enhancer. However, microglia develop normally in Zeb2 ‒165kb mice lacking this enhancer, indicating that a distinct genetic program supports macrophage development in primitive hematopoiesis. Here, we identify the molecular basis that distinguishes macrophage development in primitive vs. definitive hematopoiesis. We show that all tissue resident macrophages (TRMs) that develop in Zeb2 ‒165kb mice arise exclusively during primitive hematopoiesis, showing that even erythro-myeloid progenitors (EMPs) from the transient-definitive stage rely on the ‒165 Zeb2 enhancer. Next, we identified the transcription factors Maf and Jun as being expressed by yolk sac (YS) progenitors during primitive hematopoiesis, but not by progenitors during definitive hematopoiesis. Further, we find that Maf and Jun together induce Zeb2 expression independently of the ‒165 kb Zeb2 enhancer and restore macrophage development from adult bone marrow progenitors in Zeb2 ‒165kb mice.  Finally, Maf-deficient embryos show severe ablation of microglia development, confirming the in vivo requirement for Maf in supporting Zeb2 during primitive hematopoiesis. These results illustrate distinct transcriptional pathways controlling macrophage development during primitive and subsequent waves of hematopoiesis.

overall_design: Lin- bone marrow cells were isolated from Zeb2 ‒165kb mice and overexpressed with Maf-hCD4 and Jun-Thy1.1 or empty vector controls (hCD4 and Thy1.1). 250k single and double infected cells were sorted and cut&run was performed with antibody against Maf or Jun.

first: Chen

last: Jing

first: Murphy

last: Theresa

last: Kenneth


title: Adipogenic Transcriptional Regulation Mediated by PARP7 Through NAD+ Sensing Mechanism [ChIP-Seq]

first: W. Lee

last: Kraus

overall_design: 3T3-L1 cells were exposed to various treatments and culture conditions for the experiments described herein.  For treatment with MG-132 (10 μM, Sigma, M7449), the cells were grown as described above, cells were then treated with 10 µM MG-132 for 6 hours before collection.  For treatment with RBN2397, the cells were grown as described above upon differentiation of 3T3-L1 cells using differentiation cocktail the cells were treated with 400 nM RBN2397. The treatment continued every other day with media changes until specified collection time.  For PARP7 MAR immunoprecipitation assays, the cells were treated with RBN 2397 for 16hrs

first: MiKayla

last: Stokes

first: Yoon Jung

last: Kim

first: Sneh

last: Koul

first: Josue

last: Zuniga

first: Shu-Ping

first: Tulip


title: Antibiotic-induced microbiota depletion impairs the pro-regenerative response to a biological scaffold

city: Baltimore

first: Christopher

last: Cherry

state: MD

address: 1800 Orleans St

institute: Johns Hopkins University

summary: Therapeutic biological scaffolds promote tissue repair primarily through the induction of type 2 immunity. However, systemic immunological factors, including aging, sex, and previous infections, can modulate this response. The gut microbiota is a well-established modulator of immune function across organ systems, yet its influence on type 2-mediated repair remains underexplored. Here, we establish a bidirectional relationship between the gut microbiota and biological scaffold-mediated tissue repair. Utilizing a conventionalized germ-free mouse, we demonstrate that scaffold implantation induces compositional and functional changes in the gut microbiome, particularly affecting amino acid biosynthesis. Additionally, in a model of antibiotic-induced microbiota depletion (AIMD), we show that dysbiosis disrupts key immune regulators of type 2 immunity, including reductions in eosinophils, pro-regenerative macrophages, and IL-4-producing CD4+ T cells. At 6 weeks post-scaffold implantation, we observed a significant decrease in myocytes with centrally located nuclei alongside an upregulation in pro-fibrotic gene expression with antibiotic treatment. These findings provide insights into the influence of the gut microbiota on type 2-mediated tissue repair.

overall_design: Mice were randomly assigned to either antibiotic treated or control groups, six mice per group. Each mouse was then given VML surgery treated with ECM with or without antibiotic treatment as described in the protocols sections.

first: Natalie

last: Rutkowski


title: Aromatic Microbial Metabolite Hippuric Acid Potentiates Pro-Inflammatory Responses in Macrophages through TLR-MyD88 Signaling and Lipid Remodeling

city: Philadelphia

first: Priyankara

middle: J

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: The gut microbiome generates a diverse array of metabolites that actively shape host immunity, yet the pro-inflammatory potential of microbial metabolites remains incompletely understood. Using a non-targeted, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics, we identified hippuric acid, an aromatic gut microbe-derived metabolite, as a potent enhancer of pro-inflammatory responses in Escherichia coli infection model. Intraperitoneal administration of hippuric acid significantly heightened pro-inflammatory responses, promoted innate immune cell activation, and reduced survival in infected mice. Similar pro-inflammatory effects were observed in an LPS-induced inflammation model.  In vitro, hippuric acid selectively potentiated M1-like macrophage polarization (LPS + IFNγ) but had no effect on M2-like  polarization (IL-4). Hippuric acid further augmented responses to multiple myeloid differentiation primary response 88 (MyD88)-dependent toll-like receptor (TLR) ligands, but not to TRIF-dependent TLR3, or to cytosolic innate immune stimuli such as STING and NOD2 agonists, implicating TLR-MyD88 signaling as a likely mechanism of action. Genetic deletion of MyD88 abrogated the pro-inflammatory effects of hippuric acid both in vitro and in vivo, confirming its dependence on the MyD88 pathway. Transcriptomic and lipidomic analyses revealed that hippuric acid upregulated cholesterol biosynthesis and induced lipid accumulation. Pharmacological reduction of cellular cholesterol using fluvastatin or 25-hydroxycholesterol attenuated its pro-inflammatory effects. Notably, hippuric acid also enhanced pro-inflammatory responses in human macrophages, and its elevated levels correlated with increased sepsis mortality, underscoring its clinical relevance. Together, these findings identify hippuric acid as a previously unrecognized microbial-derived pro-inflammatory modulator that links gut microbial metabolism, lipid remodeling, and innate immune signaling, and offer new insights into its role in infection and inflammation.

overall_design: RNAseq for control and hippuric acid treatment, under untreated and LPS+IFNg treated conditions

first: Gauri

last: Mirji

first: Rahul

last: Shinde


title: Single-cell RNA sequencing of neurogenic bladder resulting from pelvic nerve injury in rats

city: Wuhan

first: Jiaxin

address: No. 1037, Luoyu Road, Hongshan District, Wuhan City, Hubei Province

institute: Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology

summary: Neurogenic bladder (NB)-induced fibrosis is the major cause of irreversible bladder dysfunction, yet the underlying mechanisms remain undefined. Here, we performed single-cell RNA sequencing on bladder tissues collected from rats following bilateral pelvic nerve injury, and characterized the alterations in the bladder microenvironment post-injury.

overall_design: A rat model of neurogenic bladder was established via bilateral pelvic nerve injury (BPNI). Bladder tissues from control (sham-operated, n=3), 3-day post-injury (n=3), and 28-day post-injury groups (n=2) were processed for single-cell RNA sequencing using the 10X Genomics Chromium platform.

first: Lei

last: Zhang

first: Xinqi

last: Liu

first: Shuaixiang

last: Zheng

first: Lida

last: Ren

first: Jihong

first: Qing

last: Ling


title: Bradyzoite subtypes rule the crossroads of Toxoplasma development

city: Riverside

first: Emma

middle: H.

last: Wilson

state: California

address: 169 Aberdeen Dr, Multidisciplinary Research Building

department: Division of Biomedical Sciences

institute: University of California, Riverside School of Medicine

summary: Reactivation of toxoplasmosis is a significant health threat to chronically infected individuals, especially those who are or become immunocompromised.  An estimated one-third of the world population is infected with Toxoplasma, placing millions at risk.  The Toxoplasma cyst is the foundation of disease with its ingestion leading to infection and its reactivation, from slow replicating bradyzoites to fast replicating tachyzoites, leading to cell lysis in tissues such as the brain. There are no treatments that prevent or eliminate cysts in part due to our poor understanding of the mechanisms that underlie cyst formation and recrudescence.  In this study, we aimed to understand the biology of bradyzoites prior to recrudescence and the developmental pathways they initiate. We have discovered ME49EW cysts from infected mice harbor multiple bradyzoite subtypes that can be identified by their expression of distinct proteins. Sorting of these subtypes revealed they initiate distinct developmental pathways in animals and in primary astrocyte cell cultures.  Single bradyzoite RNA sequencing indicates 5 major bradyzoite subtypes occur within these cysts.  We further show that a crucial subtype comprising the majority of bradyzoites in chronically infected mice is absent from conventional in vitro models of bradyzoite development. Altogether this work establishes new foundational principles of Toxoplasma cyst development and reactivation that operate during the intermediate life cycle of Toxoplasma.

overall_design: Chronically infected mouse brains were harvested and bradyzoites were purified and subjected to scRNA-seq using 10X Genomics Chromium Next GEM platform.

first: Michael

middle: W

last: White

first: Arzu

last: Ulu

first: Sandeep

last: Srivastava

first: Nala

last: Kachour

first: Brandon

last: Le


title: Nitroparaffin Transcriptional Profiles and Toxicokinetics Support Chemical-Specific Carcinogenicity Assessment

city: Durham

first: Rasim

last: Barutcu

state: NC

address: 6 Davis Drive

institute: ScitoVation

summary: The toxicological profiles of nitroparaffins—nitromethane (NM), nitroethane (NE), and 1-nitropropane (1NP)—remain incompletely characterized. This has led to reliance on read-across approaches, extrapolating carcinogenicity data from NM to NE and 1NP, though the validity of such extrapolations is uncertain. In this study, we examined the concentration-dependent transcriptional effects of NM, NE, and 1NP in primary rat hepatocytes to clarify their cellular impact and mechanisms of toxicity. We assessed cytotoxicity, differential gene expression, and pathway enrichment, and applied in vitro to in vivo extrapolation (IVIVE) to estimate systemic bioavailability and clearance rates. Cytotoxicity was observed at 3 mM for NM and NE but not up to 10 mM for 1NP. Transcriptomic profiling revealed minimal overlap in dysregulated genes and enriched pathways among the three compounds. NE elicited the broadest transcriptional response, whereas NM and 1NP showed more limited effects. IVIVE further indicated distinct systemic bioavailability and clearance, suggesting toxicokinetic differences. Taken together, these results demonstrate that structural similarity alone does not predict comparable toxicological behavior. Each nitroparaffin exhibited unique molecular activity and toxicokinetics, indicating that read-across within this class requires demonstrated concordance in both bioactivity and toxicokinetic profiles, which are currently absent for NE, NM and 1NP. Thus, reliance on read-across from NM to NE or 1NP may misrepresent health risks and carries significant regulatory implications.

overall_design: Transcriptional profiling of 1-nitropropane, nitromethane and nitroethane on primary rat hepatocytes.

first: A Rasim

first: Jamie

last: Scaglione

first: Marjory

last: Moreau

first: Elizabeth

last: Hofstetter

first: Todor

last: Antonijevic

first: Andrew

last: Maier

first: Pierre

last: Serfass

first: Leslie

last: Recio

first: Pamela

last: Spencer


title: Single-Cell Multiomic Atlas of Human Cortical Development in Down Syndrome - iPSC-derived Neural Xenografts Multiome Dataset

city: London

last: Lattke

address: Hammersmith Hospital Campus, Du Cane Road

department: Brain Sciences

country: United Kingdom

institute: Imperial College London

summary: Down syndrome, caused by an extra copy of chromosome 21, is the most common genetic form of intellectual disability affecting up to 1 in 700 live births1-3. Yet, it remains unclear how the increased dosage of ~200 protein-coding genes on chromosome 21 affects brain development, particularly in the cerebral cortex—the area central to higher-level cognitive functions4-7. Here we generated a single-cell transcriptome and chromatin accessibility atlas from 30 human fetal cortical samples at mid gestation (10-20 weeks after conception), a critical period of cortical development8. We discovered an early global transcriptional network disruption, subtly altering ~600 genes involved in neural development and function, mostly in excitatory neurons accompanied by a significant reduction in RORB/FOXP1 expressing excitatory neurons. Multimodal network analyses predicted the chromosome 21 transcription factors BACH1, PKNOX1, and GABPA as key regulatory hubs controlling dozens of genes genetically linked to intellectual disability. Antisense-mediated normalization of the increased activity of these transcription factors in stem-cell-derived neural cells in vitro, which partially recapitulated molecular changes in the Down syndrome cortex, rescued expression of several of these predicted intellectual disability-associated targets. Finally, a humanized in vivo model replicates key molecular features of Down syndrome not recapitulated in neural cells in vitro. This resource defines the gene-regulatory landscape of the developing human cortex in Down syndrome, revealing early molecular and cellular signatures and candidate therapeutic targets, along with a human in vivo experimental platform for their preclinical testing.

overall_design: We performed combined single-cell transcriptional and chromatin accessibility profiling (10X Genomics Multiome technology) of iPSC-derived neural grafts (generated with approach from Real et al., 2018) from an individual with DS and from isogenic control iPSCs (CON, normal diploid karyotype; iPSC lines DS and DS2U from Weick et al., 2013, aquired from WiCell, UWWC1-DS1 and UWWC1-DS2U). We combined graft libraries with snRNA-seq libraries generated by Singleron and compared grafts to fetal tissue and neural cells in vitro, derived from the same iPSC line.

first: Vincenzo

last: De Paola


title: Single-Cell Multiomic Atlas of Human Cortical Development in Down Syndrome - Fetal Cortex Bulk RNA-seq Dataset

summary: Down syndrome, caused by an extra copy of chromosome 21, is the most common genetic form of intellectual disability, which affects up to 1 in 700 live births. Yet, how increased dosage of the ~200 protein-coding genes on human chromosome 21 affects cortical development and function remains unclear. Here we generated a single-cell transcriptome and chromatin accessibility atlas of the human foetal cortex at mid gestation (11-20 weeks after conception), a critical period of cortical development. We uncovered an early global transcriptional network disruption, subtly altering ~1000 genes involved in neural development and function primarily in excitatory neurons. These changes reflected clinical phenotypes like intellectual disability and epilepsy, were accompanied by a significant reduction in layer 4 neurons, and were driven by a network of transcription factors including chromosome 21 genes BACH1, PKNOX1, and GABPA. Finally, a xenograft model replicates key molecular features, offering an experimental platform for validating new therapeutic targets. This resource defines the gene-regulatory landscape of the developing human cortex in Down syndrome, revealing the earliest known molecular and cellular signatures of its neurological manifestations and novel candidate targets for therapeutic interventions.

overall_design: We performed bulk RNA-seq of  human brain samples from each 6 fetuses with DS and controls (CON, normal diploid karyotype), from surgical terminations of pregnancy spanning PCW 11 to 14, to complement our single nucleus Multiome (RNA/ATAC sequencing) dataset. One DS sample was excluded from the final analysis, as the data strongly diverged from all other samples, probably due to poor library quality.


title: Single-Cell Multiomic Atlas of Human Cortical Development in Down Syndrome

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

overall_design: Refer to individual Series


title: Inosine monophosphate dehydrogenases are key players for functional embryo and seedling development in Arabidopsis

city: Kaiserslautern

first: Torsten

last: Möhlmann

address: Paul-Ehrlich-Str

department: Biology

country: Germany

institute: University of Kaiserslautern

summary: In plants, nucleotide de novo synthesis is required throughout development to energize biochemical reactions, provide DNA for cell division and RNAs for gene expression and translation. Inosine Monophosphate Dehydrogenases (IMPDH) are key enzymes in guanylate synthesis. We identify IMPDH2 from Arabidopsis as the major isoform, being enzymatically active in the plant cytosol and catalyzing the key enzymatic step in CTP biosynthesis. BIFC analysis suggests that IMPDH 1 nd 2 interact with thenmselves and with Cytidine Triphosphate Synthetase (CTPS) isoforms, by this connecting purone and pyrimidine metabolsim. Nucleotide quantification identifies IMPDH2 as bottleneck in guanylate biosynthesis, leading to nucleotide imbalance and limitation, causing reductions in ribosomal RNAs by reducing Target of Rapamycin (TOR) activity. Impaired photosynthesis and growth are consequences thereof. In contrast, overexpression of IMPDH supported growth and in strong lines lead to altered organ development presumably due to altered auxin metabolism during embryo development

overall_design: To analyze the lack of IMPDH2 on global gene expression , Col-0 (wild-type control) plants were compared to IMPDH2 T-DNA insertion line imphh2-0 at 4 and 8 days after germination. Plants were grown on soil at 120 µmol photons m-2 s-1, and samples were harvested after 3 hours of light  in three biological replicates.

first: Dörfer

last: Eva

first: Möhlmann

last: Torsten


title: Single-Cell Multiomic Atlas of Human Cortical Development in Down Syndrome - Neural Progenitors in vitro - Treatment with Antisense Oligonucleotides for PKNOX1, BACH1 and GABPA (Bulk RNA-seq Dataset)

first: Michael

overall_design: We performed bulk RNA-seq of from iPSC-derived human neural progenitors (iNPCs) in vitro from an individual with DS and from isogenic control iPSCs (CON, normal diploid karyotype; C13(DS) and C9 (CON) from Murray et al., 2015 and Alic et al. 2021, generated and provided by Dean Nizetic and Ivan Alic). Progenitors were treated with antisense oligonucleotides (ASOs) to downregulate the transcription factors PKNOX1, BACH1 and GABPA, to assess consequences for the regulation of predicted downstream target genes. For ASO designs see related manuscript.


title: Iron deficiency induces maturation-dependent loss of pancreatic β-cells.

city: Jette

first: Annelore

last: Van Mulders

state: Brussels

address: Laarbeeklaan 103

department: Faculty Medicine & Pharmacy

country: Belgium

institute: Vrije Universiteit Brussel (VUB)

summary: Pancreatic β-cells maintain glucose homeostasis by secreting insulin in response to rising blood glucose, a process fueled by mitochondrial ATP production. Iron, a core cofactor in the electron transport chain, is essential for this metabolic coupling. While the cytotoxic effects of iron overload are well known, the role of iron sufficiency during β-cell development remains unclear. Here, we identify a maturation-dependent requirement for iron in mouse and human β-cells. Using chemical chelation and genetic disruption of transferrin receptor (TFRC)-mediated uptake, we show that immature β-cells depend on iron during metabolic transition to functional maturity. Iron restriction at this stage impairs oxidative metabolism and compromises survival. In contrast, mature β-cells remain resilient to iron depletion, revealing a developmental switch in iron dependency. These findings establish iron as a key metabolic cue in β-cell development and suggest strategies to generate fully functional stem cell-derived β-cells for diabetes modeling and cell replacement therapy.

overall_design: To investigate the effect of Tfrc knockout in beta cells on the transcriptome pancreatic islet cells, islets from 3-week-old control and β-Tfrc-KO mice were isolated, dissociated, and subjected to single-cell RNA sequencing.

first: Willem

last: Staels


title: Revealing an unconventional JAK1/2-STAT3 branch in macrophage IFNγ signaling [ChIP-Seq]

city: Amsterdam

first: Josje

last: Huisman

state: Noord-Holland

address: Meibergdreef 9

department: Medical Biochemistry

country: Netherlands

institute: Amsterdam UMC

summary: Interferon-γ (IFNγ) is a key cytokine in immune activation and anti-viral responses that classically signals via JAK1/2-mediated STAT1 homodimers to drive macrophage activation. Here, we identify a non-canonical signaling component in which IFNγ also activates STAT3. Our results show that IFNγ activates STAT3 rapidly and directly through JAK1 and JAK2. We provide the first evidence that STAT3 can form heterodimers with STAT1 in this context and demonstrate that STAT3 is co-recruited to a subset of IFNγ-induced, STAT1-bound regulatory elements. While IFNγ directly activates STAT3, our results reveal that its contribution to gene regulation is limited, indicating that STAT1 easily substitutes the STAT1–STAT3 heterodimer for STAT1 homodimers when STAT3 is absent. These findings uncover STAT3 as a new unconventional player in macrophage IFNγ signaling, underscoring the complex and context-dependent nature of cytokine signaling networks.

overall_design: ChIP-seq of BMDMs stimulated with IFNγ, IFNy-IL6 or control conditions, targeting pSTAT1, pSTAT3, or Pol2.

first: Mees

last: Botman

first: Marten

middle: A

last: Hoeksema


title: Single-cell RNA sequencing profiles of schistosoma japonicum during reproductive development

city: Shanghai

first: SHAOYUN

last: CHENG

address: Songhu Road 2005

department: Department of Microbiology and Microbial Engineering, School of Life Science

institute: Fudan University

summary: Schistosomes are parasitic flatworms that require male-female pairing for sexual maturation and egg production, driving schistosomiasis pathology. However, the cellular dynamics underlying this process remain poorly understood. Here, we performed single-cell RNA sequencing on 104,671 cells from male and female Schistosoma japonicum across four developmental stages, identifying 76 distinct cell populations. Our transcriptomic atlas revealed developmental trajectories of multiple cell types, particularly within the reproductive and nervous systems. Notably, we discovered three male-specific neuron populations, including delta-3 neurons located in the gynecophoric canal. We identified the lsamp gene, co-expressed with nrps in delta-3 neurons, is essential for female sexual development. Loss of lsamp impaired axonal growth and disrupted the male ventral nerve network, reducing the targeted delivery of pheromone BATT precursors to delta-3 neurons and hindering female maturation. This work provides a high-resolution cellular landscape of schistosome sexual development and suggests new strategies to disrupt parasite reproduction and control schistosomiasis.

overall_design: scRNA-seq profiling of male and female S. japonicum across 4 time points (14dpi, 18dpi, 22dpi and 26dpi) throughout the reproductive development during their interplay.

first: Yanmin

last: You

first: Shaoyun

last: Cheng

first: Xu

last: Chen

first: Xi

first: Jipeng

last: Wang

first: Wei

last: Hu


title: Revealing an unconventional JAK1/2-STAT3 branch in macrophage IFNγ signaling [RNA-Seq]

overall_design: RNA-seq of STAT1 knockout (STAT1KO), STAT3 knockout (STAT3KO), and wild-type (WT) BMDMs under IFNγ, IL-6, IFNy-IL6-costimulation or no stimulation


title: In Vivo Dual RNA-Seq identifies a novel AB Toxin-like as a key effector of Leptospira interrogans barrier disruption (or host colonization)

city: Paris

first: Marc

last: Monot

address: 28 rue du docteur roux

country: France

institute: Institut Pasteur

summary: The ability to evade host immune responses and disrupt epithelial barriers is essential for microbial pathogenesis, yet the underlying mechanisms of these processes in extracellular pathogens remain poorly understood. Through in vivo dual RNA-Seq profiling, we reveal how Leptospira interrogans compromises cell barrier integrity and disseminates within its host. Unlike most pathogens that trigger a rapid immune response, L. interrogans employs a stealth strategy, delaying host recognition even as it establishes tissue colonization. Through our analysis, we identify a novel family of bacterial effectors, the Virulence-Modifying (VM) proteins, as key mediators of barrier disruption. These AB toxin-like proteins, also identified in other extracellular pathogens, induce intracellular calcium influx in epithelial cells, activating calmodulin and myosin light chain kinase to disrupt tight junctions. Mutants deficient in VM proteins show a loss of virulence in animals, underscoring their essential role in pathogenicity. This study uncovers a unique strategy by which an extracellular pathogen exploits host calcium signaling to breach epithelial barriers through a novel class of AB toxin-like proteins. These findings offer new possibilities for therapeutic targeting and our understanding of microbial pathogenesis.

sample_organism:
Leptospira interrogans
Mesocricetus auratus

overall_design: Here, we developed a dedicated protocol for dual RNA-Seq profiling of L. interrogans-infected hamsters, a model of human acute leptospirosis, to elucidate how this pathogen spreads and compromises cell barrier integrity. Golden Syrian hamsters were infected with 108 L. interrogans by intraperitoneal route.

first: Alexandre

last: Giraud-Gatineau

first: Georges

last: Haustant

first: Mathieu

last: Picardeau

first: Nadia

last: Benaroudj


title: Single-Cell Multiomic Atlas of Human Cortical Development in Down Syndrome - iPSC-derived Neural Xenografts snRNA-seq Dataset

first: Michael

last: Lattke

overall_design: We performed  single-cell transcriptional profiling (Singleron Celescope technology) of iPSC-derived neural grafts (generated with approach from Real et al., 2018) from an individual with DS and from isogenic control iPSCs (CON, normal diploid karyotype; iPSC lines DS1 and DS2U from Weick et al., 2013, aquired from WiCell, UWWC1-DS1 and UWWC1-DS2U). We combined graft libraries generated with snRNA-seq with ibraries generated with the 10X-Multiome technology and compared grafts to fetal tissue and neural cells in vitro derived from the same iPSC line.

first: Vincenzo

last: De Paola


title: A streamlined nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing (Fig2_s5PSeq)

city: Solna

first: Honglian

last: Liu

address: Tomtebodavägen 23

department: Department of Microbiology, Tumor and Cell Biology

country: Sweden

institute: Karolinska Institutet

summary: Antimicrobial resistance (AMR) poses a significant threat to public health. Rapid and accurate antimicrobial sensitivity testing is essential to guide effective treatment. Here, we present “simplified 5PSeq” (s5PSeq), a streamlined protocol for profiling 5’ monophosphorylated (5’P) mRNA degradation intermediates that reflect ribosome dynamics in vivo. By capturing antibiotic-induced, context-specific ribosome stalling events, s5PSeq provides a molecular proxy for bacterial growth inhibition—offering a molecular phenotypic readout without the need for culturing. s5PSeq reduces library preparation time to under four hours and incorporates a novel rRNA blocking strategy. We demonstrated its clinical utility by identifying erythromycin-resistant and sensitive Clostridioides difficile clinical isolates. Combining s5PSeq with real-time nanopore sequencing enables fast AMR diagnosis with as few as 3000 reads. In addition to simplifying the study of 5’P co-translational mRNA decay, our work suggests that utilizing information-rich phenotypic molecular readouts can significantly improve AMR diagnostics.

sample_organism:
Staphylococcus aureus
Enterococcus faecalis
Bacillus subtilis
Clostridioides difficile

overall_design: s5PSeq library using 200ng of total RNA extracted from exponetially growing (OD600=0.6-0.8)B.subtilis, C.difficile, S.aureus, E.faecalis

first: Susanne

last: Huch

first: Ryan

last: Hull

first: Fabricio

middle: R

last: Garcia

first: Lilit

last: Nersisyan

first: Wei-Hua

first: Juan

last: Du

first: Vicent

last: Pelechano


title: A streamlined nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing (Fig.4_MinION)

overall_design: s5PSeq library were prepared using 200ng extracted total RNA from exponetially growing (OD600=0.6-0.8) C.difficile clinical isolates that are resistant or sensitive to erythromycin. Bacteria cells are treated with erythromycin(1ug/ml) for 10min or without treatment.

last: Chen


title: A streamlined nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing (Fig4_PromethION)


title: A New Form of Insulin Mutation Diabetes Defined by Zygosity, Stem Cell Models, and Population Data

city: Brussels

first: Yue

last: Tong

address: Route de Lennik, 808

institute: Université Libre de Bruxelles

summary: The heterozygous INS c.16C>T (insulin p.Arg6Cys, R6C) mutation has been described to cause monogenic diabetes, but its pathogenicity has recently been questioned. R6C preproinsulin exhibits impaired translocation into the endoplasmic reticulum (ER), potentially affecting insulin secretion. We explored why "one copy isn’t enough" for the R6C pathogenicity using integrative clinical, genetic, and functional approaches. Homozygous INS R6C individuals presented early-onset insulin-dependent diabetes, whereas heterozygous carriers showed variable or absent glycemic phenotypes. Population-level analysis revealed no significant enrichment of diabetes among heterozygous carriers. Heterozygous R6C induced pluripotent stem cell (iPSC)-derived β cells exhibited minimal defects, while homozygous R6C cells displayed preproinsulin accumulation and reduced insulin content and secretion. In vivo, homozygous R6C β cell transplants recapitulated human insulin deficiency and responded poorly to GLP-1 receptor agonist therapy. Transcriptionally, homozygous R6C β cells presented repression of translation, translocation and ER related pathways. Our findings establish R6C as a recessive pathogenic mutation, prompting a reassessment of the clinical interpretation of heterozygous R6C carriers. This study highlights the power of iPSC-based disease modeling and multi-modal variant classification frameworks for dissecting the functional consequences of rare genetic variants in human monogenic diabetes.

overall_design: We performed bulk-RNA sequencing on magnetic-activated cell sorting (MACS)-purified iPSC-derived stage 7 β-like cells.

first: Xiaoyan

last: Yi

first: Miriam

last: Cnop


title: RBPscan: A Quantitative, In Vivo Tool for Profiling RNA-Binding Protein Interactions II

city: Québec

first: Dmitry

last: Kretov

state: Québec

department: Oncology Division

country: Canada

institute: CRCHU de Québec - Université Laval

summary: RNA-binding proteins (RBPs) are essential regulators of gene expression at post-transcriptional level, yet obtaining quantitative insights into RBP–RNA interactions in vivo remains a challenge. Here we developed RBPscan, a method that integrates RNA editing with massively parallel reporter assays (MPRAs) to profile RBP binding in vivo. RBPscan fuses the catalytic domain of ADAR to the RBP of interest, using RNA editing of a recorder mRNA as a readout of binding events. We demonstrate its utility in zebrafish embryos, human cells, and yeast, where it quantifies binding strength, resolves dissociation constants, identifies high-specificity motifs for a variety of RBPs, and links binding affinities to their impact on mRNA stability. RBPscan also provide positional information of conserved and novel Pumilio-binding sites in lncRNA NORAD. With its simplicity, scalability, and compatibility across systems, RBPscan offers a versatile tool for investigating RBP–RNA interactions and complements established methods for studying post-transcriptional regulatory networks.

overall_design: RBPscan analysis of drePum1 binding to different PREs was performed in zebrafish embryos. Recorder mRNA contained 1,000 different PREs as well as other sequences. The experiment was performed in three biological replicates for each concentration of drePum1.

first: Owen

last: Sanborn

first: Thora

last: McIssac

first: Elaine

last: Park

first: Imrat

last: Imrat

first: Samuel

last: Wu

first: Daniel

last: Cifuentes


title: Neurons undergo IFNγ-driven persistent epigenetic shifts and synaptic remodeling in the inflamed brain [snRNA-Seq]

city: Geneva

first: Sylvain

last: LEMEILLE

address: 1 rue Michel-Servet

department: Department of Pathology and Immunology

country: Switzerland

institute: University of Geneva Medical School

summary: Our study provides novel evidence that neuronal intrinsic epigenetic changes are established as a consequence of cytotoxic T cell-mediated immune attacks and persist long after the resolution of the inflammatory process and associate to long lasting synaptic loss

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Single nuclear transcriptomes of neurons derived from  hippocampus, from sequential timepoints of viral déjà vu disease and corresponding control in a genetic background of NuTRAP transgenic mice

first: Shammas

last: Ghazal

first: Merkler

last: Doron


title: A streamlined nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing(Fig1_HT-5PSeq)

first: Honglian

last: Liu

sample_organism:
Bacillus subtilis
Lactiplantibacillus plantarum

overall_design: HT-5PSeq library of exponetially growing (OD600=0.6-0.8)b.subtilis and l.plantarum treated with different concentration of chloramphenicol(untreated, 1.6ug/ml, 8ug/ml, 40ug/ml) or erythromycin (untreated, 0.1ug/ml, 0.5ug/ml, 2.5ug/ml) for 10min.

first: Susanne

last: Huch

first: Ryan

last: Hull

first: Fabricio

last: Garcia

first: Lilit

last: Nersisyan


title: Neurons undergo IFNγ-driven persistent epigenetic shifts and synaptic remodeling in the inflamed brain [RNA-Seq]

overall_design: Single nuclear transcriptomes of sorted nuclei derived from  Brain hippocampal resections of Rasmussen encephalitis patients

first: Egarvari

last: Kristof


title: RBPscan: A Quantitative, In Vivo Tool for Profiling RNA-Binding Protein Interactions I

overall_design: RBPscan analysis of the binding of various zebrafish and human RBPs to randomized 7-mer sequence motifs. The recorder mRNA contained a fully randomized 7-mer sequence in its 3'UTR. The experiment was performed in three biological replicates for each RBP.


title: IFITM3 Deficiency Drives SARS-CoV-2 Adaptation While Preserving Variant-Specific Traits

city: Columbus

first: Jacob

last: Yount

state: OH

address: 460 W 12th Ave, BRT 790

department: Microbial Infection and Immunity

institute: The Ohio State University

summary: Here we investigated whether interferon-induced transmembrane protein 3 (IFITM3), a key antiviral protein deficient in certain human populations, affects interspecies adaptation of SARS-CoV-2. We found that SARS-CoV-2 Beta and Omicron variants passaged through IFITM3-deficient versus wild type mice exhibit enhanced replication and pathogenesis in this new host species. Enhancements were associated with amino acid substitutions in the viral genome, suggesting that IFITM3 limits accumulation of adaptive mutations. Mouse-adapted viruses enabled comparative studies of variants in mice. Beta caused lung dysfunction and altered cilia-associated gene programs, consistent with broad viral antigen distribution in the lungs. Omicron, which shows low pathogenicity and upper respiratory tract preference in humans, replicated to high nasal titers while exhibiting limited lung spread and reduced inflammatory responses compared to Beta. Our findings demonstrate that IFITM3 deficiency accelerates SARS-CoV-2 adaptation while preserving pre-existing variant-specific properties, highlighting that host adaptation enhances viral fitness without necessarily overriding intrinsic viral characteristics.

overall_design: RNA-seq profiling of WT (C57BL/6) mice infected with either MA10, mouse adapted Beta, or Omicron BA.4 SARS-CoV-2 strains (10^5 TCID50 instranasal) with mock animals serving as controls. RNA was extracted from lung homogenates at day 2 post infection.


title: RBPscan: A Quantitative, In Vivo Tool for Profiling RNA-Binding Protein Interactions IV

first: Dmitry

last: Kretov

sample_organism:
Danio rerio
Homo sapiens

platform_organism:
Danio rerio
Homo sapiens

overall_design: RBPscan analysis of drePum1 and hsaPum2 binding to various PREs was conducted in zebrafish embryos and HEK293T cells. Recorder mRNA contained 1,000 distinct PREs and other sequences. Each experiment was performed in three biological replicates.


title: RBPscan: A Quantitative, In Vivo Tool for Profiling RNA-Binding Protein Interactions.

overall_design: RBPscan analysis of drePum1 and hsaPum2 binding to NORAD RNA was performed in zebrafish embryos and HEK293T cells. Recorder mRNA contained tiled fragments spanning the sequence of human NORAD RNA.

first: Owen

last: Sanborn


title: Neurons undergo IFNγ-driven persistent epigenetic shifts and synaptic remodeling in the inflamed brain [ChIP-Seq]

overall_design: Histone mark deposition of neurons derived from two neuroanatomical structures, namely hippocampus and brainstem, from control conditions and sequential disease timepoints of CD8 T cell-mediated encephalitis diseas.


title: A streamlined nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing

sample_organism:
Staphylococcus aureus
Enterococcus faecalis
Bacillus subtilis
Clostridioides difficile
Lactiplantibacillus plantarum

overall_design: Refer to individual Series


title: Kupffer cells control neonatal hepatic glucose metabolism via Igf1 signaling [3' scRNA-seq]

city: Bonn

first: Joachim

last: Schultze

state: NRW

address: Carl-Troll-Strasse 31

department: Genomics and Immunoregulation

institute: LIMES (Life and Medical Sciences Center Genomics and Immunoregulation)

summary: During perinatal development, liver metabolism is tightly regulated to ensure energy supply for the newborn. Before birth, glycogen is stored in hepatocytes and later metabolized to glucose, meeting the energy demands of the neonate. Shortly after birth, lipogenesis begins, driven by the transcriptional activation of enzymes involved in fatty acid oxidation. These processes are thought to be largely regulated by systemic insulin and glucagon levels. However, the role of liver-derived local factors in neonatal hepatocyte metabolism remains unexplored. Kupffer cells (KCs), the liver’s resident macrophages, colonize the fetal liver early in embryogenesis and support liver metabolism in adulthood. Yet, whether KCs influence neonatal hepatocyte metabolism is unknown. Here, using conditional knockout mouse models targeting macrophages, we demonstrate that yolk sac-derived KCs play a critical role in hepatocyte glycogen storage and function by regulating the TCA cycle - a role that monocyte-derived KC-like cells cannot substitute. Newborn pups lacking KCs mobilize glycogen more rapidly, a process regulated by insulin-like growth factor 1 (Igf1) production. Our findings reveal that macrophages are a major source of Igf1 at birth and that local Igf1 production by KCs is essential for balanced hepatocyte metabolism.

overall_design: P0 livers from the Spiflox Tnfrsf11aeGFPCre mouse model were digested to form single cell suspension then 50g spin step was berformed to enrich hepatocytes cells in the pellet and liver immune cells in the supernatant.the pups were WT(Spi1f/f Tnfrsf11aeGFP+/+) and KO (Spif/f Tnfrsf11aeGFPCre/+) .  3' counting scRNAseq was performed on the cells using SeqWell ^s3. ).  Full length scRNAseq libraries were generated via SmartSeq2.

first: Nikola

last: Makdissi

first: Daria

last: Hirschmann

first: Aleksej

last: Frolov

first: Nelli

last: Blank-Stein

first: Inaam

last: Sado

first: Fabian

last: Nikolka

first: Jingyuan

last: Cheng

first: Mohamed

last: Yaghmour

first: Philipp

last: Arnold

first: Christoph

last: Thiele

first: Felix

last: Meissner

first: Karsten

last: Hiller

first: Marc

last: Beyer

first: Elvira

last: Mass


title: Kupffer cells control neonatal hepatic glucose metabolism via Igf1 signaling


title: Neurons undergo IFNγ-driven persistent epigenetic shifts and synaptic remodeling in the inflamed brain [ATAC-Seq]

first: Sylvain

last: LEMEILLE

overall_design: Chromatin accessibility of neurons derived from two neuroanatomical structures, namely hippocampus and brainstem, from control conditions and sequential disease timepoints of CD8 T cell-mediated encephalitis disease in a genetic background of NuTRAP transgenic and NuTRAP transgenic and IFNGR floxed mice

first: Shammas

last: Ghazal

first: Merkler

last: Doron


title: RBPscan: A Quantitative, In Vivo Tool for Profiling RNA-Binding Protein Interactions III

first: Dmitry

last: Kretov

overall_design: RBPscan analysis of the specificity and dynamics (dose- and time-dependency) of N22 peptide binding to the recorder mRNA, which contains three BoxB elements in its 3' UTR. Recorder mRNAs without BoxB sequences were used as controls. Each experiment was performed in three biological replicates.

first: Owen

last: Sanborn

first: Thora

last: McIssac

first: Elaine

last: Park

first: Imrat

last: Imrat

first: Samuel

last: Wu

first: Daniel

last: Cifuentes


title: A streamlined nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing (Fig3_Illumina)

first: Honglian

last: Liu

first: Susanne

last: Huch

first: Ryan

last: Hull

first: Fabricio

last: Garcia

first: Lilit

last: Nersisyan

first: Wei-Hua

last: Chen

first: Juan

last: Du

first: Vicent

last: Pelechano


title: Pre-cancerous Niche Remodelling Dictates Nascent Tumour Persistence

city: CAMBRIDGE

first: Maria

middle: Pilar

last: Alcolea

state: Cambridgeshire

address: Jeffrey Cheah Biomedical centre, Puddicombe way

department: Cambridge Stem Cell Institute

institute: University of Cambridge

summary: Interactions between mutant cells and their environment play a key role in determining cancer susceptibility. However, our understanding of how the pre-cancerous microenvironment contributes to early tumorigenesis remains limited. Here, we show that newly emerging tumours at their most incipient stages shape their microenvironment in a critical process that determines their survival. Analysis of nascent squamous tumours in the upper gastrointestinal tract of the mouse reveals that the stress response of early tumour cells instructs the underlying mesenchyme to form a supportive “pre-cancerous niche”, which dictates the long-term outcome of epithelial lesions. Stimulated fibroblasts beneath emerging tumours activate a wound healing response that triggers a dramatic remodelling of the underlying extracellular matrix, resulting in the formation of a fibronectin-rich stromal scaffold that promotes tumour growth. Functional heterotypic 3D culture assays and in vivo grafting experiments, combining carcinogen-free healthy epithelium and tumour derived stroma, demonstrate that the pre-cancerous niche alone is sufficient to confer tumour properties to normal epithelial cells. We propose a model where both mutations and the stromal response to genetic stress together define the likelihood of early tumours to persist and progress towards more advanced disease stages.

overall_design: scRNAseq of murine upper gastro-intestinal tract epithelial and stromal cells from early squamous tumours, morphologically normal (internal control) areas, and areas from age-matched (external control) animals.

first: Greta

last: Skrupskelyte

first: Jesus

middle: E

last: Rojo Arias

first: Yiteng

last: Dang

first: Seungmin

last: Han

first: Harikrishnan

last: Ajith

first: Steffen

last: Rulands

first: Ben

middle: D

last: Simons

middle: P


title: Stepwise evolution of root nodule symbiosis [RNA-seq]

city: Kunming

first: Dingjie

last: Wang

state: Yunnan

address: NO. 132 Lanhei Road

institute: Kunming Institute of Botany, Chinese Academy of Sciences

summary: Root nodule symbiosis (RNS) represents a significant phenotypic adaptation in plants to thrive in nitrogen-deficient environments. Recent findings propose a single gain of RNS at the crown of the nitrogen-fixing clade (NF clade). However, the genetic mechanisms underlying the origin and subsequent evolution of RNS remain largely unexplored. Here, we newly sequenced and assembled eleven genomes from the NF clade and studied genetic change along the evolution of RNS. Our results elucidated three key evolutionary stages leading to the formation of stable RNS between plants and symbiotic nitrogen-fixing bacteria.

sample_organism: Populus deltoides x Populus x canadensis

overall_design: For cross species RNA-Seq analysis, we newly conducted RNA-Seq of the roots and nodules of five species (Coriaria nepalensis, Elaeagnus umbellata, Alnus trabeculosa, Chamaecrista pumila, Mimosa pudica), as well as the mycorrhizal and non-mycorrhizal roots of Populus deltoides x Populus euramericana cv. "Nanlin895". DNAaffinitypurificationsequencing (DAP-Seq) for transcription factor MtNIN and INPUT100 in Medicago truncatula.


title: Stepwise evolution of root nodule symbiosis

platform_organism: Populus deltoides x Populus x canadensis


title: TrAEL-seq captures DNA replication

status: Public on Dec 22 2025

city: Cambridge

first: Laura

last: Biggins

address: Babraham Research Campus

institute: The Babraham Institute


title: Single-cell and isoform-specific translational profiling of the mouse brain with long-read sequencing [scRNA-Seq, longread]

city: La Jolla

first: Brian

last: Yee

state: CA

address: 9500 Gilman Dr

department: Health

institute: UCSD

summary: The brain displays the richest repertoire of post-transcriptional mechanisms regulating mRNA translation. Among these, alternative splicing has been shown to drive cell type specificity and, when disrupted, is strongly linked to neurological disorders. However, genome-wide measurements of mRNA translation with isoform-sensitivity at single-cell (sc) resolution have not been achieved. To address this, we deployed Ribo-STAMP (Surveying Ribosomal Targets by APOBEC-Mediated Profiling) coupled with long-read scRNA-sequencing (scRNA-seq) in the brain. We generated the first isoform-sensitive sc translatomes of the mouse hippocampus at postnatal day 25, discovering cell type-specific translation of 3,857 alternative transcripts across 1,641 genes, and identifying isoforms of the same genes undergoing differential translation within and across eight different cell types. We defined high and low translational states in CA1 and CA3 neurons, with synaptic and metabolic genes enriched in high states. Surprisingly, we found that CA3 exhibited higher basal translation compared to CA1, as confirmed by metabolic labeling of newly synthesized proteins and immunohistochemistry of translational machinery components. This accessible platform will expand our understanding of how cell type- and isoform-specific translation drives brain physiology and disease.

overall_design: Single-cell suspensions from hippocampi were obtained from three male P22 C57BL/6J mice which were stereotactically injected in the dHC with 800nl EF1a-Ribo-STAMP virus. Results were analyzed using long-read scRNA-seq on PacBio Revio.

first: Samantha

last: Sison

first: Federico

last: Zampa

first: Eric

last: Kofman

first: Su Yeun

last: Choi

first: Pratibha

last: Jagannatha

first: Gene

last: Yeo

first: Giordano

last: Lippi


title: Epigenetic Signatures of Brain Aging by Single Nucleus Methylation Sequencing [FC-18mo]

first: Joseph

last: Ecker

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

institute: HHMI-Salk-Institute

type: Methylation profiling by high throughput sequencing

summary: Illustrating aging effects on the epigenomic features of brain is critical to understand brain functional decline and neurodegeneration. Single nucleus methylation sequencing was applied to brain regions of both male and female mouse at the age of 2, 9 and 18 months. We use these data to identify the epigenomic changes with aging in mouse brains.

overall_design:
Apply snmC-seq3 to mouse brain regions to identify aging effects on epigenomics

Processed data for these samples are available at:

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE313770

first: Q

last: Zeng

first: Wei

last: Tian

first: Anna

last: Bartlett

first: J

last: Nery

first: Rosa

last: Gomez Castanon

first: Julia

middle: K

last: Osteen

first: Jordan

last: Altshul

first: N

last: Johnson

first: M

last: Kenworthy

first: C

last: Valadon

first: Huaming

last: Chen

last: Behrens


title: Single-cell and isoform-specific translational profiling of the mouse brain [scRNA-seq]

first: Brian

last: Yee

summary: The brain displays the richest repertoire of post-transcriptional mechanisms regulating mRNA translation. Among these, alternative splicing has been shown to drive cell type specificity and, when disrupted, is strongly linked to neurological disorders. However, genome-wide measurements of mRNA translation with isoform-sensitivity at single-cell (sc) resolution have not been achieved. To address this, we deployed Ribo-STAMP (Surveying Ribosomal Targets by APOBEC-Mediated Profiling) coupled with short- and long-read scRNA-sequencing (scRNA-seq) in the brain20. We generated the first isoform-sensitive sc translatomes of the mouse hippocampus at postnatal day 25, discovering cell type-specific translation of 3,857 alternative transcripts across 1,641 genes, and identifying isoforms of the same genes undergoing differential translation within and across eight different cell types. We defined high and low translational states in CA1 and CA3 neurons, with synaptic and metabolic genes enriched in high states. Surprisingly, we found that CA3 exhibited higher basal translation compared to CA1, as confirmed by metabolic labeling of newly synthesized proteins and immunohistochemistry of translational machinery components. This accessible platform will expand our understanding of how cell type- and isoform-specific translation drives brain physiology and disease.

overall_design: Single-cell suspensions from hippocampi were obtained from three male P22 C57BL/6J mice which were stereotactically injected in the dHC with 800nl EF1a-Ribo-STAMP virus. Results were analyzed using scRNA-seq.

first: Samantha

last: Sison

first: Federico

last: Zampa

first: Eric

last: Kofman

last: Choi


title: Functional and molecular characterization of exosomes secreted from mesenchymal stromal cells exposed to IFNg in the presence of autophagy modulators tamoxifen and chloroquine

city: Fort Lauderdale

first: Vladimir

last: Beljanski

state: FL

address: 3300 S. University Drive

department: NSU MD

institute: Nova Southeastern Uni

type: Non-coding RNA profiling by high throughput sequencing

summary: Bone marrow mesenchymal stromal cells (MSCs) are therapeutic cells which adopt an immunomodulatory phenotype when exposed to pro-inflammatory cytokines and thus can be used for treatment of conditions associated with excessive inflammation. It was suggested recently that many therapeutic benefits of MSCs can be attributed to the secretion of exosomes, small membrane vesicles of endocytic origin. Exosome formation and release are impacted by the autophagy pathway, a homeostatic quality control mechanism that recycles proteins and organelles via lysosomal degradation. We hypothesized that the exposure of MSCs to autophagy modulators in the presence of a pro-inflammatory cytokine IFN may impact on exosome RNA content and immunomodulatory properties. To address this hypothesis, we treated MSCs with either tamoxifen (TX) or chloroquine (CQ) to stimulate or inhibit autophagy, respectively, in the presence of pro-inflammatory cytokine, IFN. Subsequently, we characterized exosomes from such cells and determined their functional and molecular properties. Exosomes’ immunoregulatory properties were evaluated ex-vivo using activated CD4 T cells and subsequent flow cytometry analysis to assess the effects of exosomes on CD4 T cell proliferation. We also performed small RNA sequencing to determine exosomal microRNA content. Subsequently, we performed functional analysis to determine potential effects of exosomal microRNAs on the expression of genes in target cells. Our data indicate that the exosomes isolated from MSCs treated with CQ+IFN possess higher capacity to decrease T cell proliferation. Small RNA sequencing of exosomal RNA revealed that, although similar microRNAs were found to be expressed in exosomes isolated from all treated cells, the treatments did affect the levels of multiple microRNAs. Analysis of putative gene targets revealed that most microRNA-target genes belong to either cancer or inflammation-related biological pathways. Overall, we conclude that the pre-treatment of MSCs with small molecules, in addition to pro-inflammatory cytokines, has a potential to additionally modulate biological targets of exosomes and thus biological effects of the latter.

overall_design: MSCs were grown in serum-free media and treated with IFNgamma in the presence or absence of tamoxifen (5 microM) or chloroquine (10 microM) for 48 h. Subsequently serum-free media was used to harvest MSC-secreted exosomes which were used to extract microRNAs which were subjected to deep sequencing. microRNA were then counted and following contrasts were created: 1. untreated vs. IFNgamma treated; 2. untreated vs. tamoxifene treated; 3. untreated vs chloroquine treated; 4. untreated vs. IFNgamma+tamoxifene treated; 5. untreated vs. IFNgamma+chloroquine treated.

last: Hollweg

first: Renee

last: Potens

first: Tanner

last: Blaylock

first: Andres

middle: B

last: Irausquin

first: Nikhila

last: Paleati

first: Lubov

last: Nathanson


title: Epigenetic Signatures of Brain Aging by Single Nucleus Methylation Sequencing [FC-8wk-m-018]

first: Joseph

last: Ecker

summary: Illustrating aging effects on the epigenomic features of brain is critical to understand brain functional decline and neurodegeneration. Single nucleus methylation sequencing was applied to brain regions of mouse at the age of 2, 9 and 18 months. We use these data to identify the epigenomic changes with aging in mouse brains.

first: Q

last: Zeng

first: Wei

last: Tian

first: Anna

last: Bartlett

first: J

last: Nery

first: Rosa

last: Gomez Castanon

last: Osteen

last: Altshul

last: Johnson


title: RUVBL1 and RUVBL2 are druggable MYCN regulators in neuroblastoma

city: Ghent

first: Sarah-Lee

last: Bekaert

state: No state

address: Corneel Heymanslaan 10

institute: UGent

summary: High-risk neuroblastoma (NB), accounting for approximately 50% of all cases, exhibits high relapse rates and life-threatening toxicities despite intensive therapies, highlighting the need for safer and more effective treatments. Analysis of publicly available NB transcriptomic datasets identified RUVBL2 as a key node in the MYC(N) regulatory network. High MYC signature scores were observed in high-risk NB cohorts, and multivariate Cox regression analysis showed that RUVBL1/2 expression is an independent predictor of both overall and event-free survival. Experimental validation through cell culture, western blotting, qPCR, and RNA-seq demonstrated that knockdown or pharmacological inhibition of RUVBL1/2 using CB-6644 induced replication stress, leading to cell cycle arrest, DNA damage, and apoptosis. Mechanistically, RUVBL1/2 regulated ATR and ATM protein stability and transcriptionally controlled MYC(N) expression. These findings indicate that RUVBL1 and RUVBL2 are novel regulators of the DNA damage response with both therapeutic and prognostic potential in high-risk NB.

overall_design: Identification of MYCN, RUVBL1 and RUVBL2  and various histone mark bindingsites through CUT&RUN sequencing in the neuroblastoma cell lines CLB-Bar

first: Joachim

last: Siaw

first: Kaat

last: Durinck

first: Jimmy

last: Van den Eynden


title: Single-Cell Multiomic Atlas of Human Cortical Development in Down Syndrome - Fetal Cortex Multiome Dataset

city: London

first: Michael

last: Lattke

address: Hammersmith Hospital Campus, Du Cane Road

institute: Imperial College London

summary: Down syndrome, caused by an extra copy of chromosome 21, is the most common genetic form of intellectual disability affecting up to 1 in 700 live births1-3. Yet, it remains unclear how the increased dosage of ~200 protein-coding genes on chromosome 21 affects brain development, particularly in the cerebral cortex—the area central to higher-level cognitive functions4-7. Here we generated a single-cell transcriptome and chromatin accessibility atlas from 30 human fetal cortical samples at mid gestation (10-20 weeks after conception), a critical period of cortical development8. We discovered an early global transcriptional network disruption, subtly altering ~600 genes involved in neural development and function, mostly in excitatory neurons accompanied by a significant reduction in RORB/FOXP1 expressing excitatory neurons. Multimodal network analyses predicted the chromosome 21 transcription factors BACH1, PKNOX1, and GABPA as key regulatory hubs controlling dozens of genes genetically linked to intellectual disability. Antisense-mediated normalization of the increased activity of these transcription factors in stem-cell-derived neural cells in vitro, which partially recapitulated molecular changes in the Down syndrome cortex, rescued expression of several of these predicted intellectual disability-associated targets. Finally, a humanized in vivo model replicates key molecular features of Down syndrome not recapitulated in neural cells in vitro. This resource defines the gene-regulatory landscape of the developing human cortex in Down syndrome, revealing early molecular and cellular signatures and candidate therapeutic targets, along with a human in vivo experimental platform for their preclinical testing.

overall_design: We performed combined single-cell transcriptional and chromatin accessibility profiling (10X Genomics Multiome technology) of human brain samples from fetuses with DS and controls (CON, normal diploid karyotype). We acquired 20 CON and 19 DS foetal brain samples from surgical terminations of pregnancy spanning PCW 10 to 20. After excluding two poorly preserved samples, we performed single nucleus RNA/ATAC sequencing. After excluding 3 samples with a large fraction of non-cortical cells and stringent quality controls, we retained 248,998 high-quality cells from 15 CON and 15 DS samples for final analyses.

first: Vincenzo

last: De Paola


title: Peptidyl-tRNA hydrolase 2 is an essential negative regulator of peripartum cardiomyopathy with maternal heart failure (human and mouse RNA-seq)

city: Torrance

first: Michelle

last: Matter

address: 1124 West Carson Street

department: Genomics

institute: The lundquist Institute

summary: The peptidyl-tRNA hydrolase 2 (PTRH2, Bit-1; BIT1) gene plays a pro-survival role during development with loss of function gene mutations causing congenital infantile multisystem disease (IMNEPD). In wild-type female mice hearts, ptrh2 protein levels significantly increase during pregnancy and decrease postpartum, demonstrating a protective role in response to pregnancy-initiated cardiac stresses. Peripartum cardiomyopathy (PPCM) is due to dysregulated protective signaling in the pregnant heart. The genetic and molecular mechanisms underlying PPCM remain poorly defined with no specific therapies. Here, we engineered a cardiac-specific Ptrh2 knockout (Ptrh2-CKO) mouse and show these maternal mice develop left ventricular systolic dysfunction, exhibit high rates of postpartum heart failure, and model key features of human PPCM. Infusion of a caspase 3-specific inhibitor attenuated the PPCM phenotype. Collectively, our findings demonstrate Ptrh2 is a negative regulator of pregnancy-induced cardiac stresses by activating pro-survival signals and blocking apoptotic signals, suggesting Ptrh2 may be a therapeutic target for the treatment of PPCM

overall_design: RNA-seq of LV tissue from donor and PPCM patients. The hypothesis tested in the present study was that pregnancy induced stress alters gene expression that may lead to maternal heart failure. Results identify up-and down-regulated genes that are altered in PPCM/DCM (humans and mice).

first: Vanessa

last: Montoya-Uribe

first: Pooja

last: Choubey


title: Stepwise evolution of root nodule symbiosis [DAP-seq]

first: Dingjie

last: Wang


title: Functional correction and genome integrity with duplex base editing of β-thalassemic hematopoietic stem cells

city: Nicosia

first: Nikoletta

middle: Y.

last: Papaioannou

address: Iroon Avenue

department: Molecular Genetics Thalassemia Department

country: Cyprus

institute: The Cyprus Institute of Neurology and Genetics

summary: Background: Beta-thalassemia is among the commonest monogenic disorders, posing a major global health challenge. Editing of genetic modifiers of β-thalassemia, such as BCL11A erythroid enhancer and HBG promoters, enhances fetal hemoglobin (HbF) expression and confers major therapeutic potential. Double-strand-break (DSB)-independent genome editing tools, such as base editors, are potentially safer and better suited for multiplexed application than traditional DSB-dependent CRISPR/Cas technology. However, harmful inadvertent on- and off-target events remain a concern and must be excluded before clinical application, including chromosomal rearrangements, which are invisible to standard detection technologies. Results: Using primary patient-derived CD34+ cells from three donors, we investigated simplex and duplex BE-based disruption of the BCL11A erythroid enhancer and the BCL11A binding site (-115bp) on the HBG promoter for DNA-level events and functional studies at the RNA, protein, and morphological level. Analyses included direct comparison to DSB-based editing, as the current clinically applied standard, and analysis of DNA recombination events by CAST-seq to allow wider inferences for relative safety of DSB-, BE- and duplex BE-based editing. Our study reveals the effectiveness of duplex base editing, with robust γ-globin and HbF induction and significantly improved functional correction over simplex editing. Moreover, duplex editing resulted in low incidence of simple and complex genomic alterations in both therapeutically relevant target loci. Conclusions: Here we display simultaneous duplex base editing by targeting both BCL11A erythroid enhancer and HBG promoter for functional correction and genome integrity. Our study highlights the efficacy, safety, and therapeutic potential of the present duplex BE approach

overall_design: After the collection of BFU-E colonies and the investigation of editing levels, three colonies from each treatment were selected for RNA sequencing by Genewiz (Germany). For RNA sequencing workflow, the ultra-low Input RNA-seq assay was performed. The RNA was enriched for full-length transcripts by poly(A) selection and was then processed for library preparation including cDNA synthesis and adapter ligation. Samples were sequenced on the Illumina HiSeq or NovaSeq platforms with 2× 150 bp read length. Raw RNA-Seq data in FASTQ format were trimmed to remove adapter sequences and low-quality nucleotides using Trimmomatic v0.36. The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using the STAR aligner v2.5.2b, a splice-aware aligner that detects splice junctions and incorporates them into the alignment to improve mapping accuracy, generating BAM files. Unique gene hit counts were then obtained for each sample, and using DESeq2, a comparison of gene expression between the CONTROL vs. DSB-Cas9, CONTROL vs. BE-BCL11A, CONTROL vs. HBG and CONTROL vs. 2xBE groups of samples was performed. The Wald test was applied to generate p-values and log2 fold change values, producing tables of differential expression for all genes as well as significantly differentially expressed genes.

middle: Y

first: Petros

last: Patsali

first: Julia

last: Klermund

first: Panayiota

last: Papasavva

first: Geoffroy

last: Andrieux

first: Lola

last: Koniali

first: Basma

last: Naiisseh

first: Soteroula

last: Christou

first: Maria

last: Sitarou

first: Marina

last: Kleanthous

first: Toni

last: Cathomen

first: Carsten

last: Lederer


title: Combined epigenetic therapy with FACT and BET inhibitors remodels chromatin and disrupts oncogenic transcription in Diffuse Midline Glioma [Nascent RNA-Seq]

city: Kensington

first: Holly

last: Holliday

address: Lowy Cancer Research Centre

country: Australia

institute: Children's Cancer Institute

summary: Aberrant epigenetic regulation is a hallmark of Diffuse Midline Glioma (DMG), an incurable pediatric brain tumor. The H3K27M driver histone mutation leads to transcriptional dysregulation, indicating that targeting the epigenome and transcription may be key therapeutic strategies against this highly aggressive cancer. One such target is the Facilitates Chromatin Transcription (FACT) histone chaperone. We found FACT to be enriched at developmental gene promoters, coinciding with regions of open chromatin and binding motifs of core DMG regulatory transcription factors. Furthermore, FACT interacted and co-localized with the Bromodomain and Extra-Terminal Domain (BET) protein BRD4 at promoters and enhancers, suggesting functional cooperation between FACT and BRD4 in DMG. In vitro, a combinatorial therapeutic approach using the FACT inhibitor CBL0137, coupled with BET inhibition revealed potent and synergistic cytotoxicity across a range of DMG cultures. These results were recapitulated in vivo, significantly extending survival in three independent orthotopic PDX models of DMG. Mechanistically, we show that CBL0137 treatment decreased chromatin accessibility, synergizing with BET inhibition to cause broad transcriptional collapse, silencing several key oncogenes including MYC, PDGFRA, MDM4 and SOX2, as well as causing alterations to the splicing landscape. Notably, this combination also elicited immune-related effects, including activation of the interferon response and antigen presentation mechanisms in DMG cells and induction of an activated state in macrophages and T cells, as demonstrated in an immunocompetent setting with spatial transcriptomics. Altogether, our data highlights the therapeutic promise of simultaneously targeting FACT and BET proteins in DMG, offering a dual tumor-intrinsic and immune-mediated strategy for combating this devastating pediatric brain tumor.

overall_design: H3.3 K27M DMG cells (HSJD-DIPG007 and SU-DIPGVI) were treated with 100µM 4SU to label nascent transcripts, and with CBL0137 (1µM for DIPG7 and 2µM for DIPGVI) or JQ1 (1µM) as single agents and in combination for 2h. Vehicle cotrol cells were treated with DMSO at the same concentration. RNA was then extracted and sequenced.

first: Chelsea

last: Mayoh


title: Different modes of engagement with the nucleosome acidic patch yield distinct functional outcomes

city: Durham

first: Michael-Christopher

last: Keogh

address: 6 Davis Dr

institute: EpiCypher, Inc

summary: The nucleosome acidic patch is a hub of coordinated engagement by proteins that regulate genomic function. Here we report that S. cerevisiae Dot5 contains an arginine-rich HMGN-like motif that mediates nucleosome acidic patch binding and is required for the cell growth, DNA repair and heterochromatin defects that are exhibited when the protein is overexpressed. The heterologous expression of single chain antibodies to the nucleosome acidic patch confers a similar range of phenotypes, with the most severe observed when an ‘arginine-anchor’ mode of binding analogous to many endogenous factors is employed. This highlights a delicate balance between nucleosome acidic patch interactors that are critical for normal cellular functions and dysregulated in disease.

overall_design:
Genome binding/occupancy profiling by high throughput sequencing
CUT&RUN reactions performed in yeast (S. cerivisiae) cells

first: Ujani

last: Chakraborty

first: Emma

middle: C

last: Saccone

first: Grisel

last: Cruz-Becerra

first: Laiba

middle: F

last: Khan

first: Nina

last: Arslanovic

first: Rhiannon

last: Aguilar

first: Susan

last: Gloor

first: Sabrina

last: Hunt

first: Heather

last: Folkwein

first: Natalia

last: Ledo Husby

first: Keith

last: Maier

first: Matthew

last: Marunde

first: Noah

last: Schomburg

first: Anup

last: Vaidya

first: Martis

last: Cowles

first: Bryan

last: Venters

first: George

last: Kassavetis

first: Zu-Wen

last: Sun

first: James

middle: T

last: Kadonaga

first: Jean-Paul

last: Armache

first: Jessica

last: Tyler


title: Maintenance of Embryonic Positional Identity Programs in the Adult Sheep Tail: Evidence from HOXB13 Spatial RNA Expression Gradients

city: München

first: Caroline

last: Friedel

address: Amalienstr. 17

department: Institut für Informatik

institute: Ludwig-Maximilians-Universität München

summary: Body axis patterning in vertebrates is controlled by HOX genes during embryogenesis, with their expression gradients defining spatial identity along the anterior–posterior axis. While their developmental roles are well known, less is understood about their persistence and function in adult tissues. Tail length in sheep provides a model for studying the phenotypic effects of HOX gene variants. We used Improved Jezersko–Solčava sheep, which segregate for both tail length and HOXB13 promoter variants, to examine genotype–phenotype associations. The HOXB13 genotype was the main determinant of adult tail length, acting primarily by modulating caudal vertebral number. RNA‑seq and qPCR analyses revealed a clear anterior–posterior HOXB13 expression gradient in adult tail skin and bones, with stronger distal expression in short‑tailed animals. These results provide in vivo evidence that spatially patterned HOX gene expression persists in adulthood, suggesting that residual developmental programs may maintain regional identity in adult tissues.

overall_design: RNA-seq profiling was performed on tail skin samples collected post-mortem from adult Improved Jezersko–Solčava rams with homozygous short-tail (A/A) or long-tail (D/D) HOXB13 genotypes. For each genotype (A/A: N=4; D/D: N=3), tissue was sampled at three defined anatomical positions along the anterior–posterior tail axis (base, mid-tail, tip) to investigate spatial expression gradients and genotype-specific transcriptional differences.

first: Simon

last: Horvat

first: Stefan

last: Kreb

first: Ivica

last: Medugorac


title: Spatio-temporal chromosomal arrangements by late-replicating heterochromatin

city: Bangalore

first: Kaustuv

last: Sanyal

state: Karnataka

address: Jakkur

department: Molecular Biology and Genetics Unit

country: India

institute: Jawaharlal Nehru Center for Advanced Scientific Research

summary: Centromere clustering orchestrates the three-dimensional genome organization and, in turn, impacts genome stability. In this study, we report the principles governing the chromosome-scale architectural changes associated with centromere clustering and declustering in Cryptococcus neoformans, an opportunistic human fungal pathogen. We demonstrate that centromeres and telomeres in C. neoformans are scattered around the nuclear periphery during interphaseG1, resembling metazoans rather than model budding yeasts. Hi-C data of the synchronised cells reveal centromere and telomere-associated heterochromatic regions are organized as separate compartments at the nuclear periphery, distinct from the euchromatic regions, that eventually shape the global organization of interphaseG1 chromosomes. Polymer modeling of the Hi-C data at interphase and anaphase stages strongly supports a global reorganization of chromosomes from the non-Rabl to the Rabl-like chromosomal arrangement. Most strikingly, while centromeres are clustered and replicate early in most yeasts, our sort-seq studies provide evidence that C. neoformans centromeres replicate late during the S-phase.  In summary, we uncover yet unknown principles governing the chromosome-scale architecture that link centromere DNA replication timing, spatial centromere positioning, functional compartmentalization of chromosomes, and dynamic changes in the global three-dimensional genome architecture in a species that shared a common ancestor with metazoans more than a billion years ago.

overall_design: Hi-C of synchronized Cryptococcus neoformans cells at G1, metaphase and anaphase stages of cell cycle.

first: Satya Dev

last: Polisetty

first: Shuvadip

last: Dutta

first: Rakesh

middle: N

last: Vadnala

first: Dimple

last: Notani

first: Ranjith

last: Padinhateeri


title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

city: Seattle

first: Emma

last: Wrenn

state: WA

address: 1920 Terry Ave

institute: Seattle Children's Research Institute

summary: Sub-populations of cancer-associated fibroblast (CAF)-like tumor cells deposit extracellular matrix (ECM) proteins that support Ewing sarcoma (EwS) progression and metastasis. We previously showed a hallmark of CAF-like EwS cells is their hybrid transcriptional state wherein the driver fusion oncogene, EWS::FLI1, maintains activation of proliferative programs but loses capacity to repress mesenchymal genes. Here, we studied primary patient tumors and cell line models to identify molecular drivers of this hybrid state. Our data reveal that hybrid EwS cells are induced and maintained by a TGFβ signaling positive feedback loop. Hybrid cells de-repress TGFBR2 and upregulate expression and secretion of TGFβ2 to sustain pathway activation and ECM deposition. While TGFβ ligands can potently induce growth arrest in cells of epithelial origin, we show that TGFβ1 and TGFβ2 have no impact on EwS proliferation but instead promote cell invasion. Thus, stroma and tumor-derived TGFβ ligands induce and maintain hybrid EwS cells to promote metastatic phenotypes.

overall_design: Poly(A)-capture RNA-seq was performed on RNA (purified using Qiagen RNeasy kit) from TC71, A673, CHLA10, and PDX305 cells treated with TGFβ1 (10 ng/mL), TGFβ2 (10 ng/mL), vactosertib (1uM), or vehicle controls for 24hrs. Paired end 150 bp sequencing was performed on a NovaSeq X Plus.

first: Beth

last: Lawlor


title: IFNγ-induced memory in human macrophages is not sustained by epigenetic changes but the durability of the cytokine itself [RNA-Seq]

city: Los Angeles

first: Aleksandr

last: Gorin

address: 611 Charles E Young Dr E 570C Boyer Hall

institute: UCLA

summary: Macrophages, as key sentinel cells of the innate immune system, can retain memory of prior stimulus exposure. Interferon gamma (IFNγ) plays a central role in maintaining trained immunity in vivo and can induce potent memory in macrophages. Such memory is associated with the formation of de novo enhancers that alter gene expression responses to subsequent stimuli. However, how such enhancers are maintained after cytokine exposure remains unclear. We report that durable IFNγ-induced enhancers can last for days after cytokine washout, yet the underlying persistence mechanism is not cell-intrinsic. IFNγ-treated macrophages continue to exhibit JAK/STAT signaling days after cytokine removal. Blocking IFNγ signaling with a JAK inhibitor or anti-IFNγ neutralizing antibodies after cytokine removal is sufficient to reverse IFNγ-induced enhancers and erase the potentiated state of the treated macrophages. Our findings suggest that epigenetic changes in macrophages do not inherently encode innate immune memory or a “potentiated” macrophage state, but that rather epigenetic changes are themselves dependent on ongoing cytokine signaling. These findings suggest new possibilities for pharmacologic interventions to reverse aberrantly trained immune states associated with pathology.

overall_design: RNAseq to identify LPS-induced genes potentiated by IFNy preatreatment and determine effect of continued JAK/STAT signaling on potentiated gene expression.

first: Vyas

last: Koduvayur

first: Noa

last: Harriott

first: Quen

last: Cheng

first: Alexander

last: Hoffmann


title: Iron deficiency induces maturation-dependent loss of pancreatic β-cells

city: Brussels

first: Yue

last: Tong

address: Route de Lennik, 808

institute: Université Libre de Bruxelles

summary: Pancreatic β-cells maintain glucose homeostasis by secreting insulin in response to rising blood glucose, a process fueled by mitochondrial ATP production. Iron, a core cofactor in the electron transport chain, is essential for this metabolic coupling. While the cytotoxic effects of iron overload are well known, the role of iron sufficiency during β-cell development remains unclear. Here, we identify a maturation-dependent requirement for iron in mouse and human β-cells. Using chemical chelation and genetic disruption of transferrin receptor (TFRC)-mediated uptake, we show that immature β-cells depend on iron during metabolic transition to functional maturity. Iron restriction at this stage impairs oxidative metabolism and compromises survival. In contrast, mature β-cells remain resilient to iron depletion, revealing a developmental switch in iron dependency. These findings establish iron as a key metabolic cue in β-cell development and suggest strategies to generate fully functional stem cell-derived β-cells for diabetes modeling and cell replacement therapy.

overall_design: We performed bulk-RNA sequencing on magnetic-activated cell sorting (MACS)-purified iPSC-derived stage 7 β-like cells exposed to DFO (100 μM, Vehicle H2O) with a composition of 75% INS single positive cells quantified by immunocytochemistry staining, n=4 independent differentiations.

first: Annelore

last: Van Mulders

first: Xiaoyan

last: Yi

first: Miriam

last: Cnop

first: Willem

last: Staels


title: ADH1C downregulation is a key hypoxia response in colon epithelium

city: Baltimore

first: Maged

last: Zeineldin

address: 1812 Ashland Ave

institute: Johns Hopkins University School of Medicine

summary: Defining how normal epithelial cells adapt to microenvironmental perturbations may reveal molecular vulnerabilities obscured later in carcinogenesis. Although the colon is susceptible to hypoxic injury and hypoxia is common in colorectal cancer (CRC), the insight into how non-neoplastic colon epithelium responds to hypoxia is lacking. To discover these mechanisms, human colon organoids (colonoids) were subjected to sustained hypoxia in vitro with characterization of consequent phenotypes and transcriptional changes. Hypoxia tolerance in human colonoids resulted in robust downregulation of alcohol dehydrogenase 1C (ADH1C), previously associated with poor prognosis and metabolic reprogramming in CRC. In normal crypts, ADH1C transcripts localized to transit amplifying (TA) and progenitor epithelial cells, and colon epithelial ADH1C downregulation was validated in archival tissue from patients with acute ischemic colitis, a form of hypoxic injury. Hypoxia induced PHGDH (the first and rate-limiting step in the serine synthesis pathway, SSP) in colonoids in an ADH1C-dependent manner, and the SSP contributed to neutralization of hypoxia-induced reactive oxygen species (ROS) in colonoids. Hypoxia-induced ADH1C downregulation was also associated with reduced TA cell signatures and increased expression of regeneration-associated stem cell marker FGFBP1. Finally, ADH1C-low CRC showed significant enrichment for hypoxia-associated colon epithelial signatures. Taken together, these results establish ADH1C as a mediator of colon epithelial hypoxia responses, metabolic reprogramming, and epithelial identity with relevance to human CRC.

overall_design:
RNA-seq for 4 differed colon epithelial organoid lines grown under either abient oxygen concentration or at 0.2% oxygene for one week. RNA-seq as paired end sequencing.
RNA-seq for 3samples of ischemic colitis. Regions with ischemia compared to normal regions in the same patients.

first: Tianhao

last: Bi

first: Varuni

last: Rastogi

first: Yi

last: Dong

first: Reem

last: Abu-Shamma

first: Tatianna

last: Larman