title: Non-coding structural variants identify a commonly affected regulatory region steering FOXG1 transcription in early neurodevelopment [CUT&Run]

city: Gent

first: Eva

last: D'haene

address: Corneel Heymanslaan 10

department: Department of Biomolecular Medicine

country: Belgium

institute: Ghent University

summary: The FOXG1 transcription factor is a crucial regulator of embryonic brain development. Pathogenic FOXG1 variants cause FOXG1 syndrome. Although structural variants (SVs) in the non-coding region downstream of FOXG1 have been reported in 38 individuals with similar characteristics, the regulatory pathomechanisms remain unknown.  We identified a de novo non-coding deletion in an individual with FOXG1 syndrome-like disorder, allowing us to delineate a ~124 kb commonly affected regulatory region (CARR). By integrating epigenomics data, 3D chromatin interaction profiles (Hi-C, UMI-4C), and in vivo enhancer assays in zebrafish, we uncovered multiple regulatory elements within this CARR, including a neuronal enhancer cluster and a conserved boundary of the FOXG1-containing topologically associating domain (TAD). Hi-C analysis on case lymphoblastoid cells revealed increased interactions with the adjacent TAD. Moreover, sequential UMI-4C and CUT&RUN assays during neural progenitor cell (NPC) differentiation demonstrated dynamic activation of, and interaction with the enhancer cluster. Finally, CRISPR-Cas9 deletion of the enhancer cluster and TAD boundary in NPCs resulted in decreased FOXG1 transcription.  We identified and characterized enhancer and architectural elements essential for proper FOXG1 transcription. Our findings provide new insights into chromatin architecture and gene regulation at the FOXG1 locus, improving SV interpretation in individuals with FOXG1 syndrome-like disorder.

overall_design: CUT&RUN assays for epigenomic markers H3K4m3, H3K4me1 and H3K27ac were performed during an 8-day differentiation of human iPSCs to NPCs using dual SMAD inhibition at day 0, 4, 6 and 8 of differentiation (n = 2).

first: Lisa

last: Hamerlinck

first: Sarah

last: Vergult


title: Single cell RNA sequencing of bone marrow cells in patients post allogenic stem cell transplantation

status: Public on Mar 16 2026

city: Bethesda

first: Shouguo

last: Gao

state: MD

address: 9000 Rockville Pike

department: NHLBI

country: USA

institute: National Institutes of Health

type: Expression profiling by high throughput sequencing

summary: We performed single cell RNA sequencing, and VDJ sequencing of TCR and BCR of bone marrow samples (BMMMNCs) from 11 patients who received allogenic stem cell transplantation, among them, 5 with good graft function and 6 with poor graft function, to understand hematopoietic and immune reconstitution after stem cell transplantation, and potential mechanisms of poor graft function.

overall_design: RNA profiles of single bone marrow hematopoietic and immune cells of 11 patients were generated by deep sequencing using Illumina NovaSeq.

first: Zhijie

last: Wu


title: Programmable RNA-triggered counterselection in eukaryotic cells using CRISPR-Cas12a2

city: Salt Lake City

first: Emily

middle: Rose

last: Wilson

state: UT

address: 2000 Circle of Hope

department: Oncological Sciences

institute: Huntsman Cancer Institute

summary: To identify upregulated gene sets and pathways enriched by GeCas12a2 activation.

overall_design: RNA-seq in HeLa cervical cancer cells expressing GFP, treated with GeCas12a2 in complex with either a GFP targeting gRNA (gGFP), a non-targeting gRNA (gNT), or treated with Nigericin as a positive control or phosphate buffered saline (PBS) as a vehicle control.

first: Jared

last: Thompson

middle: R

first: Xiaoyang

last: Zhang

first: Yang

last: Liu


title: Antigen cross-presentation at single-cell resolution

city: Kensington

first: Fabio

last: Zanini

state: NSW

address: High and Botany St

country: Australia

institute: University of New South Wales

summary: Antigen cross-presentation by dendritic cells (DCs) to CD8+ cytotoxic T cells (CTLs) is a key step in the immunological cascade. Although the overall sequence of events at DC synapses has been studied at the level of entire tissues, the molecular dynamics of cross-presentation, CTL activation and reprogramming at the level of individual cellular interactions remain incompletely understood. Studying direct physical interactions between cells presents greater challenges than analysing isolated individual cells separately. Previous approaches have relied on antibody-based methods, limiting the discovery of novel ligand-receptor interactions at the transcriptome-wide level. Here, we investigate the molecular and temporal changes characteristic of DC maturation and synapses in two steps: i) We first analyse the transcriptomes of single DCs over time as they uptake ovalbumin peptide, and discovered that foreign antigen uptake increases DC susceptibility to viral infection. ii) We then coculture these primed DCs with ovalbumin-specific OT1 CTLs and capture real-time transcriptomic profiles of single DCs, CTLs, and intentional DC-CTL doublets. Comparative analyses highlight the role of Rab and SNARE proteins in antigen cross-presentation. The methodology developed in this study provides a high-throughput, low-barrier approach to investigate the dynamic molecular basis in various cellular communication systems.

overall_design: Culture media for primary immune cells is RPMI (Gibco, Carlsbad CA, USA) supplemented with 10 % FBS (Bovogen, Sydney, Australia), 2 mM L-glutamine (Thermo Fisher Scientific, Waltham MA, USA), 1 % penstrep (Sigma-Aldrich, St. Louis MO, USA) and 50 μM 2-mercaptoethanol (2-ME, Thermo Fisher Scientific, Waltham MA, USA) at 37 °C in a humidified atmosphere with 5 % CO2. All mice used in this study were maintained on C57BL/6 (RRID:MGI:5656552) background and housed in specific-pathogen-free conditions at Australian BioResources (Moss Vale, NSW, Australia). The OT-1-Thy1.1 mice were created by crossing mice from the OT-1 line and Thy1.1 line (Jackson Laboratories, Bar Harbor Maine, USA). All animal experiments and procedures were approved by the Garvan Institute of Medical Research/St Vincent’s Hospital Animal Ethics Committee. Spleen and lymph nodes from OT-1-Thy1.1 mice were collected and placed into cold PBS on ice. The tissues were torn apart into ~ 0.2 mm3 pieces with forceps and scissors and next incubated in the Tissue Digestion Buffer at 37 °C, 110 g for 30 min in an Eppendorf ThermoMixer (Eppendorf, Hamburg, Germany). Cell suspensions were transferred into a 50 mL tube through a 100 μm cell strainer (Corning Inc., Corning NY, USA) with a 1 mL syringe plunger. Lymph nodes were washed through the cell strainer using cold PBS (Thermo Fisher Scientific, Waltham MA, USA), while the spleen was washed through the cell strainer using the Red Blood Cell Lysis Solution and then washed twice using cold PBS. Cell counting was done on a hemocytometer by adding 0.4 % Trypan Blue solution (Thermo Fisher Scientific, Waltham MA, USA) to cell suspensions. CD8+ T cells and dendritic cells (DCs) were isolated from lymph nodes and spleen cell suspensions via commercially available kits (EasySep Mouse CD8+ T Cell Isolation Kit and EasySep Mouse Pan-DC Enrichment Kit II, STEMCELL Technologies, Vancouver, Canada), separately. The recommended medium was PBS free from Ca2+ and Mg2+ (Gibco, Waltham MA, USA) containing 2 % FBS and 1 mM EDTA (Sigma-Aldrich, St. Louis MO, USA). The isolation was carried out following the manufacturer's protocols with multiple cycles of mixing and incubation with the provided RapidSpheresTM and EasySep™ isolation cocktail. Isolated leukocytes were collected in a 5 mL polystyrene round-bottom tube (Thermo Fisher Scientific, Waltham MA, USA), centrifuged at 400 g for 5 min, and resuspended for further use. To avoid mRNA degradation, 1 % RNAsin (Promega, WI, USA) was added in steps lasting more than 5 min. Enriched DCs were incubated with 5 ug/mL OVA257-263 (AnaSpec, Fremont CA, USA) inside a humidified 5 % CO2 incubator maintained at 37 °C for 30 min, and then pelleted for fluorescent labelling. Enriched DCs were incubated with 5 ug/mL OVA257-263 (AnaSpec, Fremont CA, USA) inside a humidified 5 % CO2 incubator maintained at 37 °C for 0, 5, 10, 20, and 30 min (OVA257-263 was added at different times to ensure the incubation for each group ended at the same time). Culture media was added to achieve a 1 in 5 dilution of OVA257-263, after which the DCs were pelleted and resuspended in FACS buffer. 12 single cells from each group were sorted by BD LSRFortessa to a 384-well Frame-Star plate containing 1.2 μL lysis buffer. The plate was stored at −80 °C for subsequent procedures. CD8+ T cells and DCs pellets were suspended and incubated with 1.25 μM CellTrace CFSE and CellTrace Yellow (Thermo Fisher Scientific, Waltham MA, USA) working solutions in a 37 °C water bath for 20 min, separately. The working solutions were prepared by diluting 5 mM DMSO stock solutions with pre-warmed (37 °C) PBS. Samples were quenched with a 1 in 5 dilution in culture media for 5 min, then washed twice with PBS. Cells were counted before further processing. U40 UFO nanowell slides (size of 40 µm, depth of 40 µm, 50,000 nanowells/well, 50 pL/nanowell) (Sartorius AG, Niedersachsen, Germany) were pre-wetted with 100 % ethanol (Sigma-Aldrich, St. Louis MO, USA) and washed five times with culture media. 0.5 mL media was left in each well before seeding cells. 25,000 DCs and 25,000 CD8+ T cells labelled with CellTrace were dispensed in a ‘Z’ shape across the well sequentially. The slide was centrifuged at 300 g for 1 min to seed cells. Antigen presenting time was counted from the moment CD8+ T cells were added into the Dcs. After seeding into U40 nanowell slides, CD8+ T cells, DCs, and doublets were picked by the 30 µm glass capillary under the automatic picking mode of CellCelector (Sartorius AG, Niedersachsen, Germany). Three channels (i.e. brightfield, FITC for CD8+ T cells labelled with CellTrace CFSE, and TRITC for DCs labelled with CellTrace Yellow) were selected for scanning to validate cell type. Captured cells with 0.1 uL DNase/RNase-Free Water were dispensed into a 384-well Frame-Star plate containing 1.1 μL lysis buffer. The lysis plate was cooled down to 4 °C at the rack tray during cell capture and was stored at −80 °C for subsequent procedures. The library was prepared following Smart-seq2 protocol 58. Briefly, cells were picked into 384 well lysis plates followed by reverse transcription, template switching and 23 cycles of PCR to generate and amplify the cDNA. MANTIS automated liquid handler (Tecan, Zürich, Switzerland) was used during the process. cDNA was normalised to 0.1-1 ng/µl according to the qualification by Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermo Fisher Scientific, Waltham MA, USA) via automated liquid handling robot Mosquito (SPT Labtech, Melbourn, Cambridgeshire, UK). TruePrep DNA Library Prep Kit V2 (Vazyme #TD503, Nanjing, China) with 15 cycles of amplification were used for tagmenting immune cells. Purification was conducted to remove primer by Agencourt Ampure XP (Beckman Coulter, Brea CA, USA) magnetic beads at a ratio of 0.7x two times. Libraries were quantified using Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany). Libraries were sequenced on NextSeq 500 (Illumina, San Diego, CA, USA) using 75 base paired-end reads at Ramaciotti Centre for Genomics (UNSW). Sequencing coverage was around 500,000 to 5,000,000 read pairs per cell.

first: Yike

last: Xie

first: Jacqueline

last: Bailey

first: Francis

last: Lin

first: Emma M V

last: Johansson

first: Tatyana

last: Chtanova


title: Non-coding structural variants identify a commonly affected regulatory region steering FOXG1 transcription in early neurodevelopment [UMI-4C]

overall_design: UMI-4C interaction mapping was performed with the FOXG1 promoter as bait in human induced pluripotent stem cells (iPSCs, n = 3), neural stem cells (NSCs, n = 2), neural progenitor cells (NPCs, n = 3) and induced neurons (iNs, n = 3), as well as during a 12-day differentiation of human iPSCs to NPCs at days 0, 4, 6, 8 and 12 of differentiation. UMI-4C interaction mapping with the PRKD1 promoter as bait was performed in human induced pluripotent stem cells (iPSCs, n = 2), neural stem cells (NSCs, n = 2) and neural progenitor cells (NPCs, n = 2).


title: Intracranial mesenchymal tumor, FET-CREB fusion-positive: an integrative analysis of 81 cases

city: BETHESDA

first: Zhichao

address: BG 10 RM 4A31 MSC 1500, 10 CENTER DR

department: Center for Cancer Research

institute: NIH/NCI

type: Methylation profiling by genome tiling array

summary: Intracranial mesenchymal tumors, FET-CREB fusion-positive (ICMT ) show fusions involving FET RNA-binding protein family gene (EWSR1 or FUS) and CREB family of transcription factors (ATF1, CREB1 or CREM).  The methylation characteristics and clinical behavior of this important tumor type require further characterization. We study the methylation profiles of 81 ICMT cases (61 newly profiled cases and 20 cases from publicly available sources). ICMT showed a relatively distinct methylation signature compared to related tumors. We confirmed the prior description of two distinct subgroups of ICMT (subclasses A and B).

overall_design: Bisulfite converted genomic DNA extracted from tumor samples was hybridized to Illumina Infinium EPIC (n = 41) or EPICv2 (n = 20) Human DNA Methylation Beadchips. Public collected tumor samples (n = 20) were bisulfite converted genomic DNA extracted from tumor samples was hybridized to Illumina Infinium EPIC Human DNA Methylation Beadchips. The 20 samples are from PMID: 34821426 and available in GEO  GSE164994

first: Kenneth

last: Aldape


title: HBV cccDNA Generated Using Single-Stranded M13 Phage DNA for Authentic Minichromosome Functionality

city: Shanghai

first: Yumeng

last: Li

address: 131 Dong'an Road

country: China

institute: Fudan University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) persists as minichromosomes in the nuclei of hepatocytes and serves as the transcriptional template for viral RNAs. The cccDNA pool, characterized by a low copy number, poses challenges for cccDNA research. To accurately mimic the full-length sequences and structure of HBV cccDNA, we developed a novel method using an M13 phage-based system and deoxyribozyme to produce full-length single-strand DNA (ssDNA) corresponding to both the positive and negative strands of cccDNA. This method involves the cyclization of the ssDNA followed by the addition of T4 DNA ligase, thereby producing the cccDNA. The in vitro cyclized cccDNA, formed by M13 phage-derived single-stranded DNA (McccDNA), allows the formation of minichromosomes in transiently transfected hepatic cells, recapitulates key stages of the HBV life cycle, and effectively responds to multiple antiviral drugs. Additionally, McccDNA was observed to more accurately transcribe major viral RNAs than recombinant cccDNAs containing exogenous sequences, and was also able to organize nucleosomes in a sequence-specific manner, resembling those events during natural viral infection. In conclusion, McccDNA generated from single-stranded M13 phage DNA recapitulates key natural properties of HBV cccDNA, and serves as an authentic and promising model to study cccDNA minichromosomes functionality.

overall_design: Nucleosome postioning of HepG2-NTCP cells transfected with McccDNA was profiled by MNase-seq.

first: Ting

last: Hua


title: A flexible enrichment strategy for the isolation and single-cell characterization of disease-specific circulating tumor cells

city: Portland

first: Rosalyn

middle: M

last: Fey

state: OR

address: 3181 S.W. Sam Jackson Park Road

institute: Oregon Health & Science University

summary: Background: Circulating tumor cells (CTCs) are cells that have broken off from a primary solid tumor, entered the blood stream, and have the potential to metastasize to nearby tissues. Thus, CTCs represent an important biomarker to monitor cancer progression and patient outcome. The isolation, identification and characterization of CTCs are critical for this goal. We previously developed a method for the detection and isolation of melanoma CTCs from peripheral blood through negative and positive antibody enrichment. Methods: Here we show that this protocol is easily applied to other cancers; in this case we expand and validate this method to include prostate cancer.  Results: We show that this combined negative and positive enrichment protocol successfully isolates CTCs for characterization using single cell RNA sequencing and have updated the protocol to include a newer version of single cell library preparation, Smart-seq3. In our analysis, cells prepared for sequencing with Smart-seq3, which uses unique molecular identifiers (UMIs), showed no considerable difference compared to those prepared using the previous iteration of the protocol, Smart-seq2. Conclusion: This study demonstrates a robust and flexible method for the isolation and characterization of CTCs from various cancers for potential use as biomarkers for disease management and for characterization with single cell RNA-seq.

overall_design: Melanoma A375 or prostate 22Rv1 cells were spiked into healthy human blood, isolated using a combination of negative and positive enrichment strategies, and analyzed using scRNA-seq.

first: Zachary

last: Garrison

first: Terri

last: Clister

first: Connor

last: Hall

first: Khanh

middle: L

last: Doan

first: Rajan

middle: P

last: Kulkarni


title: Gene expression profile in Shh-expressing cells of hair follicles in mouse during CIA

city: Fuzhou

first: Guixuan

last: Zhou

state: Fujian

address: No.2 Xueyuan Road,Minhou

institute: Fuzhou University

summary: Chemotherapy-induced alopecia (CIA) is a major unsolved adverse effect in clinical oncology. In previous studies, we proved the Sonic hedgehog(Shh) signaling pathway is a major target of cyclophosphamide (CYP). However, how CYP suppresses Shh gene remains unknown. In this study, we isolated Shh-expressing cells in hair follicles of Shh-GFP+/- mice treated with CYP and tested the effect of CYP on these cells. The result showed that Shh-expressing cells decreased significantly post-CYP treatment. RNA sequencing data showed that compared with the control group, 536 and 2,439 genes expressed differentially at 12hrs and 24hrs respectively post-CYP treatment. KEGG enrichment analysis revealed the p53 and the apoptosis signaling pathway were significantly enriched at 12hrs and 24hrs after CYP injection. The immunofluorescence staining of active Caspase 3 confirmed the apoptosis of Shh-expressing cells at 12 hours post-CYP treatment. GSEA analysis, ChIP assay, and AG-490 rescue assay revealed the IFN/JAK/STAT1 signaling pathway participated in regulating Shh during CIA. The results of this study demonstrated the decrease of Shh during CIA not only because of the loss of Shh-expressing cells but also because of the down expression of Shh in Shh-expressing cells. Moreover, this study also proved that apoptosis is the main reason for the loss of Shh-expressing cells, while the IFN/JAK/STAT1 signaling pathway may play an important role in the downregulation of Shh.

overall_design: Hairs in the back skin of 7-8 weeks-old mice were removed by wax to induce anagen. CYP was intraperitoneally injected in mice 9 days after depilation at 150mg/kg to cause hair loss. Shh-expressing cells were isolated from hair follicles in mice before or 12 and 24 hours post CYP treatment using flow cytometry. A pool of 9 mice were used for RNA sequencing, and divided into the control group (SGC001~SGC003), the CYP12h group (SGC121~SGC123) and the CYP24h group (SGC241~SGC243), among which SGC002, SGC241 and SGC243 were male mice, while the other 6 mice were females. Total RNAs of the Shh-GFP cells were isolated using a RNA isolater Reagent (Vazyme, Nanjing, China), and quality was monitored by Agilent 2100 analysis and proceeded for sequencing by an Illumina NovaSeq 6000 RNA sequencing platform (Novogene, Beijing, China). The RNA sequencing data were mapped to the mouse genome assembly GRCm39 using the HISAT2 program.

first: Ruifang

last: Fan

first: Xiang

first: Jingling

last: Huang

first: Tingjiao

last: Lan

first: Yuli

last: Tang


title: UV irradiation alters TFAM binding specificity and compaction of DNA

city: Durham

first: Raluca

last: Gordan

state: NC

address: 101 Science Dr, CIEMAS 2179

department: Center for Genomic and Computational Biology

institute: Duke University

summary: Mitochondria lack nucleotide excision repair; however, mitochondrial DNA (mtDNA) is resistant to mutation accumulation following DNA damage. These observations suggest additional damage sensing or protection mechanisms. Transcription Factor A, Mitochondrial (TFAM) compacts mtDNA into nucleoids and binds differentially to certain forms of DNA damage. As such, TFAM has emerged as a candidate for protecting mtDNA or sensing damage. To examine the possibilities that TFAM might protect DNA from damage or act as a damage sensing protein for irreparable forms of mtDNA damage, we used live-cell imaging and HeLa cell-based assays, atomic force microscopy (AFM), and high-throughput protein-DNA binding assays to characterize the binding properties of human TFAM to ultraviolet-C (UVC) irradiated DNA and the cellular consequences of UVC irradiation. Our cell data show increased TFAM mRNA after exposure and suggest an increase in mtDNA degradation without a loss in mitochondrial membrane potential that might trigger mitophagy. Our protein-DNA binding assays indicate a reduction in sequence specificity of TFAM following UVC irradiation and a redistribution of TFAM binding throughout the mitochondrial genome. Our AFM data show increased compaction of DNA by TFAM in the presence of damage. Despite the TFAM-mediated compaction of mtDNA in vitro, we do not observe any protective effect of increased TFAM protein on DNA damage formation in cells or in vitro. Increased TFAM protein did not alter levels of mtDNA damage over time after UVC exposure in vivo, but knockdown of TFAM did alter mtDNA damage levels in HeLa cells both at baseline and after UVC exposure. Taken together, these studies indicate that UVC-induced DNA damage alters TFAM binding and promotes compaction by TFAM in vitro. We hypothesize that that TFAM may act as a damage sensing protein in vivo, sequestering damaged genomes to prevent mutagenesis by facilitating removal or suppression of replication.

overall_design: We designed a custom library to cover the entirety of the human mitochondrial genome using a sliding 33-nucleotide window width with a 2-nucleotide shift for each consecutive probe. A subset of the chambers were irradiated with UVC to induce photolesions on the DNAs. We then incubated the chambers with either 30 nM or 300 nM TFAM. The concentrations of TFAM were selected based on preliminary experiments to ensure fluorescence intensity values were well within the range of detection. We were able to identify locations in the mitochondrial genome at which TFAM exhibits high occupancy and observed changes in sequence specificity of TFAM binding in the context of UVC-irritated DNA.


title: Single nucleus chromatin accessibility Profiling on the human metabolic dysfunction associated steatotic liver disease samples

city: Guangzhou

last: He

state: Guangdong

address: No. 9 XingDaoHuanBei Road, Guangzhou International Bio Island, Guangzhou 510005, Guangdong Province, China

department: Department of Basic Research

institute: Guangzhou National Laboratory

summary: Single-nucleus Assay for Transposase-Accessible Chromatin with sequencing (snATAC-seq) in human mapping of chromatin accessibility in specific cell clusters profiled alterations in Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) samples compared to normal controls, providing insights into cell-type-specific epigenetic changes associated with MASLD.

overall_design: Normal and MASH human livers samples (each have 6 replicates) were analyzed with snATAC-seq (10x Genomics platform)

first: Biying

last: Zhu

first: Yang

last: Xiao

first: Bing

last: Chen

first: Chen


title: Integrative functional genomics reveals transcriptional regulatory function of risk alleles for metabolic liver disease

summary: Genome-wide association studies (GWAS) have identified nearly 100 loci associated with metabolic dysfunction-associated steatotic liver disease (MASLD), but the molecular functions of these variant alleles remain elusive, particularly when they occur in non-coding regions. Here we profiled the chromatin landscape of liver nuclei from 12 individuals with varying stages of metabolic liver disease, which provide a global regulatory program in liver. Then we used massively parallel reporter screen to identify differential activity variants (DAVs). The target genes of these DAVs were delineated by expression quantitative trait loci (eQTLs), chromatin loops and single-cell CRISPRi screening. These multimodal integration analyses provide insights into the regulatory mechanisms of MASLD progression driven by genetic risks.

sample_organism:
Escherichia coli
Homo sapiens

platform_organism:
Escherichia coli
Homo sapiens

overall_design: MASLD-associated variants were obtained from the GWAS Catalog and relevant publications. SNPs in LD, with MAF > 5% and R² > 0.8 were also collected.  Finally, 126 bp sequence centered on 5,369 variants was synthesized and inserted into MPAR plasmid library, following by transfecting into HepG2 and LX-2 cells for MPRA experiment. Differential activity variants were detected from MPRA data by compare the MPRA activity between reference allele and alternative allele.

last: Li


title: Widespread non-Mendelian inheritance of DNA methylation patterns in mice [ONT Liver]

city: Baltimore

first: Adam

last: Davidovich

state: Maryland

address: 855 N. Wolfe Street

institute: Johns Hopkins University

summary: We performed long-read ONT sequencing and matched RNA sequencing on two Collaborative Cross mouse strains and their F1 crosses. Allele-specific methylation and expression analyses revealed widespread and diverse patterns of intergenerational epigenetic inheritance. Included among these are a large number of non-Mendelian inheritance patterns, many of which have not previously been observed in naturally occurring mammalian genomes. Targeted ONT sequencing of F2 crosses of the same Collaborative Cross strains enabled further characterization and validation of these epigenetic inheritance patterns, including distinguishing those patterns mediated by cis- or trans-acting regulatory factors.

overall_design: ONT and RNA sequencing were performed on liver DNA and RNA, respectively, of two Collaborative Cross mouse strains and their F1 crosses. Subsequent targeted ONT sequencing was performed on F2 crosses of the same Collaborative Cross strains.

first: Danila

last: Cuomo

first: Hang

last: Su

first: Sandeep

last: Kambhampati

first: Qingqing

last: Gong

first: Alexandra

last: Naron

first: Rakel

last: Tryggvadottir

first: Leonard

last: McMillan

first: Kasper

middle: D

last: Hansen

first: David

middle: W

last: Threadgill

first: Andrew

last: Feinberg


title: Widespread non-Mendelian inheritance of DNA methylation patterns in mice [RNA-Seq Liver]


title: Relevance of distinct skin-resident cell populations in human contact allergy

city: Wuerzburg

first: Marc

last: Schmidt

address: Josef Schneider Str. 2

department: Dept. of Dermatology

country: Germany

institute: University Hospital Wuerzburg

summary: Metal allergies are prime examples of delayed hypersensitivity divided in two phases: sensitization and elicitation. Sensitization occurs upon initial contact with the allergen and leads to activation of skin resident cells and formation of metal-reactive T cells. During the elicitation phase, these T cells mount an immune response causing symptoms such as itchy eczema approximately 72 hours after exposure. Research has identified two main mechanisms involved in the initiation of contact hypersensitivity: direct activation of innate immune receptors such as Toll-like receptor 4 by the respective allergen, and conditional innate immune activation via the NLRP3 inflammasome. Elicitation is dominated by the activity of cytotoxic T cells, but it is unclear if only metal-responsive circulating T cells or also tissue-resident T cells contribute. To identify the relevant cell types and signals mediating human nickel hypersensitivity, we used single-cell RNA sequencing of cells from a donor sensitized to nickel (but not chromium) who was epicutaneously exposed to either diluent or both metals for 8 or 72 hours. Our results suggest that nickel exposure specifically activates certain populations of endothelial cells, suprabasal keratinocytes, and CCR7+ dendritic cells within 8 hours, a response not observed with chromium exposure. Skin resident T cells were not involved in the early hypersensitivity response as their gene expression remained unaltered 8 hours after nickel exposure. However, substantial changes in the cutaneous T cell compartments were observed after 72 hours of nickel exposure, including infiltration of SELL+ central memory T cells, effector Tc cells, TH cells, and Treg cells.

overall_design:
To study immune responses and their dynamics during initiation (8h after exposure), or elicitation of a full-blown nickel-induced contact hypersensitivity (72h post exposure), a human donor previously confirmed to be sensitized to Ni2+ but not to Cr2O72 (Ni2+ pos, Cr2O72- neg) was epicutaneously exposed on the volar forearm to  a) diluent (vaseline) 8h (as negative control) b) 5 % NiSO4 for 8h  c) 0.5 % K2Cr2O4 for 8h d) 5 % NiSO4 for 72h or  e) 0.5 % K2Cr2O4 Exposure was performed by using occlusive Finn Chambers employed for standard patch testing and the expected reactivity monitored by visual inspection of the 72h time points. Samples of the 72h time points were stimulated 64 h before the 8h time points so that samples could be taken simultaneously (to minimize batch effects) and visual confirmations of the desired reactivities could be performed prior to sample acquisition. For each condition and time point, 5mm (8h timepoints) or 8 mm (72h time points) punch biopsies were obtained and samples freshly processed for single cell preparation followed by single cell RNA sequencing . A minimum of about 6000 cells was used for sequencing and sequencings performed individually for the respective samples.

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

first: Matthias

last: Goebeler

first: Pierre

last: Khoueiry


title: Amygdala engram ensembles mediate differential susceptibility to short-term negative stress-induced remote depression in adolescent and adult mice

status: Public on Mar 15 2026

city: Xuzhou

first: Gao

last: can

state: Jiangsu

address: 209 Tongshan Road

institute: Jiangsu Province Key Laboratory of Anesthesiology Xuzhou Medical University

summary: This study aims to investigate genes associated with stress during adolescence and adulthood.

overall_design: Total RNA was extracted from the basolateral amygdala (BLA) tissues of 6-week-old and 9-week-old control (CON) and social defeat stress (SDS) mice, respectively, for RNA-seq.

first: Yue

last: You

first: Changting

last: Lv

first: Pengtao

last: Ni

first: Shijie

last: Wang


title: Fis suppresses late-stage virulence gene expression in Yersinia pseudotuberculosis at environmental temperatures

city: Bochum

first: Franz

last: Narberhaus

address: Universitätsstraße 150

department: Microbial biology

institute: Ruhr University Bochum

summary: The nucleoid-associated protein Fis is a key transcriptional regulator in Gram-negative bacteria that supports rapid adaptation to environmental changes. In Yersinia pseudotuberculosis, Fis plays a critical, yet poorly understood role in virulence. Here, we present a comparative transcriptomic analysis of Y. pseudotuberculosis wild type and fis deletion mutant at environmental (25°C) and host-relevant (37°C) temperatures. Our data show that Fis modulates the expression of more than 600 genes across 16 functional categories. Notably, Fis exerts reciprocal, temperature-dependent control over virulence genes, including those encoding the type III secretion system (T3SS) and Yersinia effector proteins (Yops), flagella biosynthesis, and cell adherence/invasion factors. Functional assays revealed that fis deletion disrupts this regulatory balance, producing a host-defense-like state at 25°C characterized by complete loss of motility, upregulation of the virulence master regulator LcrF, aberrant Yop secretion, impaired phagocytosis by host cells, and increased pathogenicity in the Galleria mellonella infection model. These findings establish Fis as a central regulator that coordinates motility and early host-cell engagement while preventing premature activation of antiphagocytic defenses, thereby optimizing the initial stages of infection.

overall_design: Illumina RNA sequencing of the transcriptomes of Yersinia pseudotuberculosis wild type and fis deletion mutant (dfis) at different growth phases (OD600 0.5 & 1.5) and different temperatures (25°C & 37°C)

first: Soheila

last: Javadi

first: Stephan

last: Pienkoß

first: Dominik

last: Meggers

first: Andrea

last: Wimbert

first: Vivian B.

last: Brandenburg

first: Pascal

last: Dietze

first: Sina

last: Schäkermann

first: Lilo

last: Greune

first: Petra

last: Dersch


title: RNA-Seq data for PyMT mammary tumor cells with or without LSD1-loss

city: Boston

first: Zhe

last: Li

state: MA

address: 77 Avenue Louis Pasteur, Room 466

department: Medicine, Genetics Division

institute: Brigham and Women's Hospital

summary: LSD1 (KDM1A) is a histone demethylase.  In human cancers, genetic abnormalities of LSD1 mainly include deletions and mutations; importantly, such deletions/mutations have also been found in metastatic breast cancers, raising a possibility that LSD1 is a breast cancer metastasis suppressor.  We found induced loss of LSD1 in luminal mammary tumor cells in the MMTV-PyMT model led to a profound increase in lung metastasis.  Intriguingly, PyMT tumor cells with LSD1-loss also exhibited a profound change in immune-related genes, suggesting that LSD1 may suppress breast cancer metastasis in part via a cell-extrinsic, immune mechanism.  In particular, LSD1 disruption led to upregulation of expression of most MHC class I genes, which might cause reduced number, maturation and activation of NK cells.  Reduced clearance of PyMT tumor cells with LSD1 disruption by NK cells might lead to their increased metastasis.

overall_design: Lsd1-null or wild-type (WT) YFP+ PyMT tumor cells were sorted from MMTV-PyMT female mice that carried a Rosa26-Stop-YFP (R26Y) reporter, as well as the Lsd1 conditional knockout alleles (Lsd1L/L) (experimental)  or not (Lsd1+/+) (control) ~2-3 months after intraductal induction of Cre-expressing adenovirus under the control of the Krt8 promoter (Ad-K8-Cre), respectively; total RNAs were prepared from each sorted cell populations and subjected to RNA-seq expression profiling.

first: Dongxi

last: Xiang


title: Transcriptomic profiling of mouse colorectal organoids following NAP treatment.

city: Beijing

first: Qingtao

last: Meng

address: No.10, West Tou Tiao, Youan Men Wai, Fengtai District

institute: capital Medical University

summary: Bisphenol F (BPF) is a widely used substitute for bisphenol A (BPA) in plastic manufacturing; however, its potential toxicity remains insufficiently characterized. In this study, we investigated the impact of BPF exposure on the intestinal barrier and cardiovascular system. Based on prior untargeted metabolomic analysis, we revealed that BPF can be converted into N-acetylputrescine (NAP) through a microbiota-associated metabolic process. Further experiments demonstrated that BPF exposure stimulated intestinal epithelial cells to secrete spermidine/spermine N1-acetyltransferase 1 (Sat1), an enzyme involved in this conversion. To explore the molecular mechanisms underlying NAP-induced effects, we performed RNA sequencing on mouse colorectal organoids treated with NAP. Transcriptomic analysis was conducted to identify gene expression changes and signaling pathways associated with NAP exposure. The raw sequencing data and processed expression matrices are provided in this submission.

overall_design: RNA-seq of mouse colorectal organoids treated with NAP (n=6) and control (n=5).

first: Jing

first: Jie

last: Xu

first: Huanzhuo

last: Mai

first: Guolei

last: Niu

first: Shenshen

last: Wu

first: Xianan

last: Zhang

first: Jiahao

last: Zhu

first: Michael

last: Aschner

first: Rui

last: Chen


title: Anti-asthma drug Montelukast induces autistic behaviors via disrupting neuronal retinoic acid signaling [scRNA-Seq]

city: Shanghai City

first: Yali

address: No.320, Yueyang Avenue

institute: Center for Excellence in Molecular Cell Science

summary: Globally, autism spectrum disorder (ASD) affects approximately 1.0-2.5% of human newborns, with non-genetic risk factors estimated to contribute to over 50% ASD cases through yet elusive mechanisms. Montelukast (MTK), a leukotriene receptor antagonist and FDA-approved anti-asthma drug, was found to potently disrupt retinoic acid (RA) signaling, and substantially increase the risk of ASD in humans when used during pregnancy or in early childhood, according to prospective and retrospective cohort studies. Both maternal and postnatal exposure to montelukast caused autistic-like behaviors in rat offspring. Montelukast also altered brain regional patterning through disrupting RA signaling in human cerebral organoids, similar to DEAB, an established inhibitor of RA-synthesizing ALDH1As. Mechanistically, montelukast was found to directly bind to retinoic acid receptor alpha (RARα) and impair RARα heterodimer formation to disrupt RA signaling. Altogether, this work highlighted not only the developmental origin of ASD, but the utmost importance in cautioned use of medicinal chemicals during maternity or early childhood to prevent psychiatric disorders, e.g. ASD.

overall_design: Cerebral organoids were treated with different compounds (DMSO,DEAB, and MTK) and analyzed using scRNAseq.

first: Rong-Gui

last: Hu

first: Shan

last: Bian

first: Ya-Li

first: Meng

first: Meng-Xia

first: Zhen-Ming

last: Guo

first: Min


title: Root Internalization and Temperature-Dependent Regulation of Persistence and Virulence Genes in Pseudomonas aeruginosa PAO1 on Oak Leaf Lettuce

city: Hamburg

first: Tim

last: Hoffmann

state: Hamburg

address: Ohnhorststraße 18

institute: Universität Hamburg

summary: Increasing evidence of Pseudomonas aeruginosa on fresh plant-based foods raises food safety concerns. While internalization of pathogens such as Salmonella enterica in vegetables such as lettuce is well documented, corresponding data for P. aeruginosa are lacking. Moreover, climate change-associated temperature shifts may influence the plant microbiota and the presence of human pathogens. This study investigates the internalization and temperature-dependent gene expression of P. aeruginosa PAO1 on green oak leaf lettuce as a model system. For this purpose, oak leaf lettuce was cultivated in soil inoculated with P. aeruginosa PAO1_sfGFP_UHH07, and internalization was analyzed using confocal laser scanning microscopy. Temperature-dependent transcriptomic changes of P. aeruginosa PAO1 were assessed by analyzing differentially expressed genes following plant inoculation and incubation at 18 and 22 °C, respectively. P. aeruginosa PAO1 is capable of internalizing into the roots of oak leaf lettuce, but a translocation into leaves was not detected. Transcriptomic analyses showed that a moderate temperature increase shifts bacterial gene expression, with virulence genes upregulated at 22 °C and persistence-associated genes predominating at 18 °C. These results show that temperature influences the persistence and pathogenic potential of P. aeruginosa present on oak leaf lettuce, highlighting potential impacts of climate change on food safety.

overall_design: Prokaryotes RNA-seq Analysis of Pseudomonas aeruginosa PAO1 incubated on oak leaf lettuce (Lactuca sativa var. crispa) at 18 °C and 22 °C for 2, 5 and 7 days.

first: Jennifer

last: Griese


title: CROP-seq of 20 MASLD-associated differential activity variants from HepG2 MPRA screen

last: He

summary: We performed CROP-seq to 20 MASLD-associated differential activity variants from HepG2 MPRA screen. This provides a powerful strategy for accurately locating the target gene pool associated with functional variants.

overall_design: We extended 150 bp upstream and downstream of plausibly causal noncoding variants from HepG2 MPRA-screen, then used FlashFry v1.15 and CRISPick to detect potential gRNAs. Top two gRNAs were synthesized and transfected to CRISPRi HepG2 cells. Then whole-transcriptome scRNA-Seq libraries and the sgRNA-enrichment libraries were separately sequenced.

first: Biying

first: Yang

last: Xiao

first: Bing

first: Chen


title: Anti-asthma drug Montelukast induces autistic behaviors via disrupting neuronal retinoic acid signaling [RNA-seq and Ribo-seq]

last: Li

overall_design: Comparative gene expression profiling analysis of RNA-seq and Ribo-seq data for cerebral organoids treated with compounds (DMSO,DEAB, and MTK).

first: Meng-Huan


title: Single-cell epigenetic landscape, microenvironment interactions, and gene regulatory modules of non-functioning pituitary adenomas

city: Los Angeles

first: Frederique

last: Ruf-Zamojski

state: CA

address: 8700 Beverly Blvd

institute: Cedars-Sinai Medical Center

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

overall_design: Refer to individual Series


title: Anti-leukemic effect of MAR-3.6.2, a novel RAN inhibitor, on AML-LSCs by targeting RAN.

city: Duarte

first: Le Xuan Truong

last: Nguyen

address: 1500 E. Duarte Road

department: Hematology/Bone Marrow T MC

institute: City of Hope

summary: The small GTPase RAN plays a role in the biogenesis of mature miR-126, which is supplied by the bone marrow arterioles to leukemic stem cells (LSCs). MiR-126 supports the homeostasis of LSCs that initiate and maintain acute myeloid leukemia (AML). While therapeutic targeting of RAN has been difficult due to its structural features, through molecular dynamics simulations and docking studies, we have identified MAR-3.6.2 as a novel allosteric inhibitor that binds in a cryptic pocket in the C-terminal domain of RAN. We showed that MAR-3.6.2 disrupted RAN interaction with its guanine nucleotide exchange factor RCC1 and prevented the nuclear switch of RAN-GDP to RAN-GTP. This in turn led to RAN nuclear retention and reduced the RAN/XPO5-mediated export of pre-miR-126, thereby limiting mature miR-126 biogenesis in endothelial cells and their exogenous supply of mature miR-126 to LSCs. In a MllPTD/WT/Flt3ITD/ITD AML murine model, MAR-3.6.2 reduced leukemia burden, prolonged survival, and decreased LSC frequency in secondary transplants. These findings highlight MAR-3.6.2 and future, potential derivates as a promising small molecule-based approach to eradicate AML LSCs via inhibition of RAN/XPO5 trafficking and block of miR-126 biogenesis.

overall_design: Transcriptome profiling of patient-drived primary AML cells trreated with vehicle vs MAR-3.6.2.

first: Truong


title: Widespread non-Mendelian inheritance of DNA methylation patterns in mice [ONT Liver - Targeted]

first: Adam

last: Davidovich

first: Danila

last: Cuomo

first: Hang

last: Su

first: Sandeep

last: Kambhampati

first: Qingqing

last: Gong

first: Alexandra

last: Naron

first: Rakel

last: Tryggvadottir

first: Leonard

last: McMillan

first: Kasper

last: Hansen

first: David

last: Threadgill

first: Andrew

last: Feinberg


title: Effect of Gnmt overexpression on rhythmic gene expression in Drosophila melanogaster photoreceptor cells

city: West Lafayette

first: Seth

last: Lammert

state: IN

address: 175 South University Street

department: Biochemistry

institute: Purdue University

summary: Nearly half of the genes in the eye are regulated through the molecular circadian clock, a transcription-translation feedback loop that is governed by environmental stimuli. In aging, vision loss correlates with perturbations to the circadian clock. Rhythmic gene expression is substantially altered in the aging Drosophila melanogaster eye in that over 1/3 of genes have differential rhythmic expression. H3K4me3 is important for transcriptional activation of genes regulated by the circadian clock and is globally decreased in aging photoreceptors. It is unknown what mechanism underlies decreased H3K4me3. Glycine-N-methyltransferase (Gnmt) regulates the availability of the universal methyl donor S-adenosyl methionine (SAM) by methylating glycine to produce sarcosine and S-adenosylhomocysteine (SAH), a potent inhibitor of histone methyltransferase activity. Both Gnmt and SAH are increased in the aging eye, therefore we hypothesize that an age-dependent increase of Gnmt contributes to disruption of rhythmic gene expression. To test this hypothesis, nuclear RNA-seq was performed on photoreceptor nuclei that were collected every four hours from flies outcrossed to wild type or with overexpression of either active or partially active Gnmt

overall_design: Drosophila were maintained in population cages in a 25°C incubator with a 12:12 hour light:dark cycle. 100 male flies in triplicate were collected at 10 days post-eclosion (D10) and at the indicated Zeitgeber times (ZT0, ZT4, ZT8, ZT12, ZT16, ZT20). GFP-positive photoreceptor nuclei were immunoprecipitated using the nuclei immunoenrichment protocol (dx.doi.org/10.17504/protocols.io.buiqnudw), followed by RNA isolation and purification. Glycine-N-methyltransferase (Gnmt) was overexpressed in Drosophila eyes using an eye-specific Gal4 driver: LongGMR-Gal4 (derived from Bloomington Drosophila Stock Center 8121) crossed with a line with UAS-Gnmt (https://doi.org/10.1038/ncomms9332) - samples 37-54. As a negative control, the driver line was outcrossed to w[1118] - samples 1-18. A partially active mutant of Gnmt was also overexpressed, UAS-GnmtS145A (https://doi.org/10.1038/ncomms9332) - samples 19-36.

first: Vikki

last: Weake


title: Anti-asthma drug Montelukast induces autistic behaviors via disrupting neuronal retinoic acid signaling

first: Yali


title: Targeting E-Proteins Licenses cDC1 Immunogenicity for Potent Anti-Tumor Vaccination [ChIP-seq]

city: Nanning

first: Wang

last: Xi

state: Guangxi

address: 100 University East Road, Nanning City, Guangxi

department: School of Medicine

institute: Guangxi University

summary: Type 1 conventional dendritic cells (cDC1s) are essential for priming anti-tumor CD8⁺ T cells, yet the intrinsic pathways limiting their function remain unclear. Here we identify E-proteins (E2A/HEB) as a master checkpoint that actively suppresses cDC1 immunogenicity by directly transactivating PTEN, thereby restraining PI3K-AKT-mTOR signaling, FLT3L-driven proliferation, and NF-κB-mediated IL-12 production. Genetic ablation of E-proteins in DCs unleashes a hyperfunctional program—expanded cDC1 populations, enhanced antigen presentation, and superior CD8⁺ T cell priming—while deletion of the E-protein antagonist Id2 impairs cDC1 function, establishing a bidirectional rheostat. This axis is clinically relevant, as dynamic E-protein activity in human tumor DCs correlates with patient survival and response to immune checkpoint blockade. Finally, vaccination with E-protein-deficient DCs elicits potent antigen-specific T cell expansion and achieves robust therapeutic efficacy across multiple preclinical tumor models, defining a targetable checkpoint and establishing E-protein inactivation as a platform for next-generation cancer immunotherapies.

overall_design: Splenocytes from wild-type mice were cross-linked, and chromatin was sheared to 200-500 bp. ChIP was performed with anti-E47 antibody or IgG control. Libraries were sequenced on an Illumina NovaSeq 6000 (150 bp PE). Reads were aligned to mm10 using Bowtie2, and peaks were called with MACS2 (q < 0.01) and annotated using ChIPseeker.

first: Xiaoyu

last: Zhai

first: Ping

last: Gao

first: Xiaojuan

last: Han

first: Xi

last: Wang

first: Xiangwen

last: Liu

first: Chao

first: Yuanyuan

last: Shi

first: Baidong

last: Hou

first: Xuyu

last: Zhou

first: Jianwei

first: Jian

last: Song

first: Fuping

last: Zhang


title: Comprehensive stage- and tissue-specific transcriptome of the global ecosystem service insect, marmalade hoverfly Episyrphus balteatus

city: Baoding

first: He

last: Yuan

address: Dongguan Street, number 437

institute: Hebei Academy of Agriculture and Forestry Sciences

summary: Hoverflies (Diptera: Syrphidae) are globally beneficial insects that provide key ecological services, such as pollination and biological pest control. Importantly, extensive research has revealed that numerous species within this group engage in long-distance migrations, thereby enabling these services to operate across broad spatial scales. Hence, the conservation and sustainable use of this functionally important group is crucial for maintaining ecosystem balance and stability, especially in the face of ongoing losses of ecosystem functioning. However, limited genomic and transcriptomic resources hinder the advancement of research on this significant group. To address this gap, we generated a comprehensive developmental- and tissue-resolved transcriptome for Episyrphus balteatus, the dominant and most extensively researched migratory hoverfly species. We sequenced 30 RNA-seq libraries across three life stages (egg, larva, and pupa) and seven adult tissues (antenna, proboscis, head, thorax, body, leg, and wing), yielding 133.87 Gb of clean reads. The subsequent de novo assembly yielded 85,676 unigenes (N50 = 1,028 bp) exhibiting high completeness (97.9% BUSCO), and 45,479 unigenes (53.08%) were functionally annotated. Notably, differential expression analysis identified key gene sets and enriched pathways linked to development, sensory perception, and environmental responsiveness, which are molecular characteristics that may facilitate long-distance migration and ecosystem service delivery. Together, this dataset serves as a valuable community resource for ecological, evolutionary, and comparative research on hoverflies and other beneficial insects.

overall_design: For trancriptome sequencing, different developmental stages of E. balteatus, including egg (1d after oviposition, n = 100), larva (mixed at four stages, n = 10) and pupa (2d after pupation, n = 10), and different tissues of adults, including antenna (n = 100), body (abdomen, n = 30), head (without antennae or proboscis) (n = 30), leg (n = 100), proboscis (n = 100), thorax (without legs, n = 30) and wing (n = 100) were collected.The adults consist of unmated female and male individuals, which were sampled 1d after eclosion. Three replicates were prepared for each sample.

first: Huiru

last: Jia

first: Xianyong

first: Hui

last: Li

first: Kongming

last: Wu


title: Targeting E-Proteins Licenses cDC1 Immunogenicity for Potent Anti-Tumor Vaccination [RNA-seq]

first: Wang

overall_design: Splenic cDC1s were sorted by FACS from WT, E2A/HEB ΔDC, and Id2 ΔDC mice under steady-state or after HKLM-OVA stimulation. RNA was extracted and sequenced on an Illumina NovaSeq 6000 (150 bp PE, ~35 million reads/sample). Reads were aligned to mm10 using HISAT2, and differential expression analysis was performed with DESeq2 (adjusted P < 0.05, |log2FC| > 1). GSEA was conducted using MSigDB Hallmark gene sets.

first: Xiaoyu


title: Molecularly distinct subtypes of Lhx6-positive neurons of the zona incerta differentially regulate sleep pressure and recovery sleep

first: Seth

last: Blackshaw

address: 725 North Wolfe St

department: The Solomon H. Snyder Department of Neuroscience

institute: Johns Hopkins University School of Medicine

summary: Sleep pressure is regulated not only by circadian rhythms, but also by sleep homeostasis, an activity-dependent process that dissipates during sleep. Recent work implicates Lhx6-positive GABAergic neurons of the zona incerta (ZI) in regulating sleep pressure, but their precise role remains unclear. Using sleep deprivation and HiPlex single-molecule fISH, we show that Lhx6-positive ZI neurons are broadly activated by both natural and induced increases in sleep pressure and remain active for more than three hours into recovery sleep. Anterior Lhx6-positive neurons showed stronger activation. Fos induction differed across molecularly distinct subpopulations, with Nkx2-2-positive cells showing robust responses and Calb2-positive cells showing reduced activation. We also identified distinct sleep pressure-responsive Lhx6-negative Slc32a1-positive GABAergic ZI subpopulations. Finally, intersectional genetic loss of Nkx2-2 reduced and redistributed Lhx6-positive neurons, blunted their activation, and increased total sleep time. These findings reveal a central, heterogeneous role for Lhx6-positive ZI neurons in sleep homeostasis.

overall_design: Mouse was housed in a climate-controlled pathogen-free facility and allowed food, water, and sleep ad libitum during lights on (6:30am-12:30pm). Euthanized after 6 hours (12:30pm).

first: Parris

last: Washington Chandler

first: Soo Lee

last: Sang

first: Leighton H.

last: Duncan

first: Dong Won

last: Kim

first: Mark


title: A Comparative Analysis of the Methylation Status of Non-Coding RNA promoters in Fibroid and Matched Myometrium

city: torrance

first: Omid

last: Khorram

address: 1124  w carson st

institute: The lundquist Institute at UCLA medical center

type:
Non-coding RNA profiling by array
Methylation profiling by array

summary: Genomic DNA from 8 paired uterine leiomyoma (fibroid) and matched myometrial tissues was analyzed using the Arraystar Human ncRNA Promoter Array (4×180K) combined with methylated DNA immunoprecipitation (MeDIP-chip). This platform interrogates promoter regions of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) to identify DNA methylation enrichment patterns. Differential promoter methylation analysis identified 538 lncRNAs and 61 miRNAs with significant differences between fibroid and matched myometrium. Among these, 300 lncRNAs and 47 miRNAs showed increased promoter methylation, whereas 238 lncRNAs and 14 miRNAs showed decreased promoter methylation in fibroid tissues.

overall_design: Genome-wide promoter methylation profiling of non-coding RNAs was performed using MeDIP-chip on the Arraystar Human ncRNA Promoter Array (4×180K). Genomic DNA from eight uterine leiomyoma (fibroid) tissues and their matched myometrial controls from the same patients were subjected to methylated DNA immunoprecipitation followed by microarray hybridization. Differential methylation enrichment peaks between fibroid and matched myometrium were identified to characterize epigenetic alterations in ncRNA promoter regions.

first: Tsai-Der

last: Chuang

first: Shawn

last: Rysling

first: Abigail

last: Wiseman

first: Gabriela

last: Alfaro

first: Sayna

last: Jahromi

first: Sepideh


title: Gene expression profiling of lung organoids at CSD5, CSD14, and ND14 stages.

city: Kepala Batas

first: Syahidatulamali

last: Che Shaffi

address: Bandar Putra Bertam

country: Malaysia

institute: Universiti Sains Malaysia

summary:
Lung organoid culture in both normal and COPD-derived from cigarette smoke exposure of COPD mice model.
We used microarrays to analyse the molecular mechanisms and pathways involved in regulating the regenerative potential of lung stem cells in organoid culture development, both normal and COPD derived 3D-organoid models.

overall_design: Lung organoid were harvested in different time point and groups. Cigarette Smoke Day 5 (CSD5), Cigarette Smoke Day 14 (CSD14) and Naive Day 14 (ND14) for RNA extraction and hybridization on Affymetrix microarrays. We sought to understanding the molecular mechanisms behind AT2 cell dysfunction and their contribution to COPD.

middle: C

last: Shaffi

first: Badrul

last: Hisham


title: Deciphering the PURA syndrome: loss of PURA alters neural cell fate decisions and impairs neuronal homeostasis

city: Neuherberg

first: Dierk

last: Niessing

address: Ingolstädter Landstraße 1

department: Institute of Structural Biology

institute: Helmholtz Zentrum München

summary: PURA syndrome is a rare neurodevelopmental disorder caused by heterozygous de novo mutations in the PURA gene, leading to intellectual disability, hypotonia, and epilepsy. While the molecular functions of PURA have been partially elucidated, its role in human neural development remains unclear. We used PURA-knockout (KO) human induced pluripotent stem cells (iPSCs) and isogenic controls to generate 2D neural progenitor cultures and 3D cerebral organoids.Transcriptomic profiling identified PURA-regulated RNAs critical for early neuronal development.

overall_design: Wildtype and PURA knockout iPSCs and NPCs cultivated in adhesion were suspended, RNA was extracted and used for library preparation.

first: Sabrina

last: Bacher


title: Expression of cFLIP is necessary for diffuse large B cell lymphoma pathogenesis

city: Cologne

first: Alessandro

last: Annibaldi

state: North-Rhine-Westphalia

address: Robert-Koch-Strasse 21

institute: Center for Molecular Medicine Cologne

summary: Diffuse large B cell lymphoma (DLBCL) is a highly heterogenous malignant disease that remains a major clinical challenge as relapsed and refractory disease is difficult to treat. Apoptosis evasion is a major feature of DLBCL. However, while the suppression of intrinsic apoptosis has long been recognized as a lymphoma-promoting event, the role of extrinsic apoptosis has remained poorly defined. Here, we demonstrated at the genetic level that expression of cFLIP, the most crucial, non-redundant inhibitor of extrinsic apoptosis, in B cells is necessary for the development of diffuse large B cell lymphoma (DLBCL) in an autochthonous murine model. Indeed, B cell-specific deletion of Cflar, the gene encoding for cFLIP, prevented lymphomagenesis mediated by oncogenic Myd88 and overexpression of BCL2. In human lymphoma cells, we showed that the absence of cFLIP sensitizes ABC- but not GCB DLBCL subtype cells to TRAIL- or LPS-induced, Caspase-8-mediated apoptosis. Furthermore, we unveiled a cell death-independent role of cFLIP in the suppression of pro-inflammatory cytokines at the transcriptional level, selectively in the ABC subtype. These results indicate that the suppression of intrinsic apoptosis can support lymphomagenesis only if extrinsic apoptosis is properly controlled. Moreover, licensing extrinsic apoptosis via cFLIP deletion can efficiently promote the death of DLBCL cells despite the suppression of the intrinsic pathway. Overall, these data provide the rationale for the development of cFLIP inhibitors for the treatment of ABC DLBCL and possibly other haematological cancers.

overall_design: RNA-Seq profiling of untreated CFLAR wildtype and CFLAR knockout TMD8 and SU-DHL-4 human DLBCL cell lines.

first: Kristie

last: Bariboloka

first: Justinas

last: Valiulis

first: Martin

last: Peifer


title: Convergent Transcriptomic Signatures in Blood Link Lumbar Disc Herniation and Cervical Artery Dissection in Young Adults

city: Moscow

first: Polina

middle: Sergeevna

last: Shlapakova

address: Volokolamskoye Shosse, 80

country: Russia

institute: Research Center of Neurology

summary: Recurrent lumbar disc herniation (rLDH) and cervical artery dissection (CeAD) are significant neurological manifestations of undifferentiated connective tissue dysplasia (UCTD) in young adults. Molecular and histopathological evidence points to impaired cellular differentiation and aberrant extracellular matrix organization in these patients. Nevertheless, the pathogenic mechanisms of UCTD and the basis for its clinical heterogeneity remain largely unknown. Here, we conducted the first comparative transcriptome-wide analysis of peripheral blood mRNA in two UCTD cohorts—19 patients with CeAD and 12 with rLDH—alongside 18 healthy volunteers. Both UCTD cohorts exhibited common transcriptomic signatures indicating suppressed translation and RNA processing, mitochondrial dysfunction, and premature cellular senescence. Notably, the activated compensatory pathways, functionally annotated to small GTPase signaling, diverged between the cohorts. The rLDH group displayed a transcriptional signature of generalized extracellular matrix reorganization, whereas the CeAD cohort showed alterations primarily involved in cellular differentiation, adhesion, migration, and intercellular communication. Further differential analysis of upstream regulatory genes and splicing variants, complemented by single-cell RNA sequencing of tissue biopsies, is warranted to elucidate the precise mechanisms that drive the clinical heterogeneity of UCTD within the context of a potential sporadic ribosomopathy.

overall_design: Comparative bulk RNA-seq profiling of peripheral blood in two UCTD cohorts—19 patients with CeAD and 12 with rLDH—alongside 18 healthy volunteers, with adjustment for age, sex, estimated cell type proportions (from CIBERSORT), and sample preparation batch

middle: S

first: Larisa

middle: A

last: Dobrynina

first: Andrey

last: Manakhov

first: Fedor

middle: E

last: Gusev

first: Evgeny

middle: I

last: Rogaev


title: Preliminary Study on the Role of lncRNA ENST00000608721 in Mycobacterium tuberculosis Infection of Macrophages

first: Yuehan

last: Zhao

address: No. 97, Beiguan Street, Tongzhou District

institute: Beijing Tuberculosis and Thoracic Tumor Research Institute

summary: To identify long non-coding RNAs (lncRNAs) associated with Mycobacterium tuberculosis (M.tb) infection, and preliminarily investigate the role of lncRNA ENST00000608721 in macrophages during M.tb infection. Methods: Total RNA was extracted from M.tb-infected and uninfected THP-1 cells, and high-throughput sequencing was performed on the Illumina platform to obtain the transcriptional profile. The differential lncRNAs were subjected to Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, respectively. Furthermore, the differential lncRNAs were verified by real-time quantitative PCR (qPCR). A THP-1 cell model with silenced target lncRNA was constructed using siRNA chemical transfection, and the bacterial load after M.tb infection was determined by colony-forming units (CFU) assay.

overall_design: In this study, four THP-1 macrophage samples (parallel wells) from the same batch, simultaneously seeded and either infected with H37Rv or left uninfected, were collected. Total RNA was extracted from the samples following the procedure outlined in the miRNeasy Mini Kit manual. The collected RNA samples were sent to Shanghai Biotechnology Corporation for analysis. A lncRNA-Seq library was constructed using the HieffNGS® MaxUp Human rRNA Depletion Kit, and high-throughput sequencing was performed on the Illumina NovaSeq 6000 platform. The quality, concentration, and sequencing volume of all RNA samples in this study met the required standards for lncRNA sequencing. Differential expression analysis of lncRNA sequencing data was conducted using the screening criteria of q < 0.05 and FC > 2 or FC < 0.5, identifying lncRNAs that were differentially expressed following M.tb infection.

first: Jing

last: Dong

first: Xuetian

last: Shang

first: Xinyu

last: Wang

first: Liping

last: Pan

first: Zongde

last: Zhang

first: Qiuyue

last: Liu


title: PPP1CA and RPSA dual inhibition suppresses castration resistance and bone metastasis for prostate cancer by disrupting AR-V7/RANKL signaling

city: Shenyang

first: Wensu

address: 77 Puhe Road, Shenbei District

institute: China Medical University

summary: Androgen is important in the formation, development and progress of prostate cancer, and deprivation of androgen, which is also named castration, is an effective way in prostate cancer therapies. For patients undergoing castration therapies, castration was gradually becoming ineffective in prostate cancer treatment and therefore resulting in castration-resistance for prostate cancer. Co-IP mass spectrometry, Ribo-seq, RNA-seq and system biology methods were applied to detect castration resistance drivers. Ribosomal drivers were found to be highly expressed and promoting invasion and migration in castration resistant prostate cancer cells and also in bone metastasis prostate cancer cells. Within these ribosomal drivers, RPSA played central roles in promoting castration resistance in assistance of RPL28, RPL4X, RPL11 and RPL23A. RPSA promotes castration resistance through interacting with PPP1CA and AR-V7 to form PPP1CA-RPSA-AR-V7 axis. This axis is bind by RPL23A and RPL28 and assisted by RPL4X. IGF-1 is also engaged in the process of promoting castration resistance and bone metastasis by PPP1CA-RPSA-AR-V7 axis. RPSA inhibitors plus PPP1CA inhibitors have also been proven to significantly restore castration resistance and bone metastasis for prostate cancer. This effect can be reversed by IGF-1 and retinoid acid. For these reasons, we propose that dual inhibition of PPP1CA and RPSA significantly inhibit castration resistance and bone metastasis for prostate cancer.

overall_design: Ribo-seq on castration-resistant prostate cancer and bone metastasis prostate cancer cells

first: Wenshuo

first: Wei

last: Wan

first: Tingting

last: Zhou

first: Renlong

last: Zou

first: Xiaobei


title: Transcriptome profiling of human dermal fibroblasts transfected with tRF-34 mimics or negative control under UVA1 exposure

city: Nanjing

first: XIAOXI

last: DAI

address: 12 Jiangwangmiao Street

department: Department of Physiotherapy

institute: Hospital for Skin Diseases, Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College

summary: tRNA-derived fragments (tRFs) are emerging regulators of cellular senescence and stress responses. In this study, we profiled transcriptomic changes in primary human dermal fibroblasts (HDFs) transfected with tRF-34 mimics or negative control (NC), followed by UVA1 irradiation. High-throughput RNA sequencing was performed to identify differentially expressed genes.

overall_design: RNA-seq profiling of primary human dermal fibroblasts (HDFs) transfected with tRF-34 mimics or negative control. Cells were exposed to UVA1 (15 J/cm²) daily for three consecutive days. RNA was extracted 24 hours after the final irradiation. Each group included biological replicates (n=3). Libraries were prepared and sequenced on an Illumina NovaSeq platform.

first: Xiaoxi

last: Dai

first: Dan

last: Huang

first: Zhuohong

last: Xu

first: Lihao

first: Pianpian

last: Hong

first: Yu

last: Hu

first: Kun

last: Chen

first: Jiaan


title: RNA-seq data of PEPD over-expressed vs NC GBM cells

city: Wuhan

first: Lei

last: Shen

state: Hubei

address: Donghu Road No.169

institute: Department of Neurosurgery, Zhongnan Hospital of Wuhan University

summary: Peptidase D (PEPD) is a cytosolic metallopeptidase previously reported to interact with nuclear p53, suppressing its phosphorylation and transcriptional activity to facilitate tumor progression in several cancer types, including bladder and colorectal cancer. However, the role of PEPD in glioma has not been described. Our preliminary proteomic analysis revealed that PEPD is markedly upregulated in glioma, with increased expression observed in both tumor tissues and tumor-supplying arterial blood, suggesting a potential involvement in glioma pathogenesis. To characterize the transcriptomic impact of PEPD in glioma, we conducted strand-specific RNA sequencing on two human glioblastoma cell lines following PEPD overexpression, comparing PEPD-overexpressing cells to their respective negative controls in both T98G and U251 models. Differential expression analysis was performed to identify PEPD-associated transcriptional alterations and to explore downstream signaling pathways and regulatory networks. These data provide a foundational resource for understanding PEPD-mediated molecular mechanisms and support further studies into its potential function as a previously unreported regulator in glioblastoma.

overall_design: PEPD-OE vs NC, two cell lines, three replicates

first: Jingyi

last: Yang

first: Nanxiang

last: Xiong


title: Mathematical modelling reveals a ribosome level-induced survival checkpoint in drug-tolerant cancer persister cells

city: Chengdu

first: Xinpu

last: Tang

state: Sichuan

address: Guoxue Alley, Chengdu

institute: West China Hospital

summary: Systemic treatments for advanced cancers initially provide benefits but rarely lead to a cure due to acquired resistance. Drug-tolerant persister (DTP) cells, devoid of new mutations, can temporarily tolerate treatment. In previous study, we observed that melanoma DTP cells downregulated mRNA translation activity while enhancing the selective translation of a subset of mRNAs. However, the mechanisms governing the selective translation in DTP cells remain unclear. In this study, we have established a mathematical framework to investigate selective mRNA translation in DTP cells, employing a combination of stochastic mathematical modelling and experimental characterization.  Our findings indicate that changes in mRNA translation are a common occurrence in DTP cells across various cancer types and treatments. The diminished activity of Myc-mediated ribosome biogenesis leads to a restricted resource for active mRNA translation in DTP cells. This bottleneck effect, resulting from reduced ribosome levels, initiates a competition among mRNAs, as demonstrated through stochastic mathematical modelling. Utilizing this mathematical model, we have explored potential therapeutic strategies to delay the onset of drug resistance.  Hence, our theoretical framework exposes a ribosome-induced survival checkpoint in DTP cells, driven by limited ribosome levels. This discovery suggests therapeutic potential based on ribosome thresholds, providing a foundation for developing strategies to counteract drug resistance.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: We performed polysome profiling on the parental cells and drug-tolerant persister (DTP) cells derived from the human melanoma cell line A375 and the mouse melanoma cell line Yumm1.7, respectively. Following sucrose density gradient centrifugation, the various fractions were divided into the monosome fraction (the 80S peak and the several fractions preceding it) and the polysome fraction (the 4–5 fractions at the very bottom of the sucrose gradient). Finally, RNA was extracted from the input, monosome, and polysome fractions of each group, and these RNAs were subjected to sequencing.

first: Yi

last: Pu

first: Yuqing

last: Wang

first: Shensi


title: Ferroptotic tumor cells revamp tumor-associated macrophage antigen presentation to unleash immune checkpoint blockade efficacy

city: Guangzhou

first: dingdang

last: Yu

address: No. 106, Zhongshan 2nd Road, Yuexiu District

institute: Guangdong Provincial People's Hospital

summary: Activation of antigen presentation has been shown to contribute to improved immunotherapy outcomes. Herein, we show that inducing tumor cell ferroptosis boosts anti-tumor immunity by potentiating major histocompatibility complex II (MHC-II)- dependent antigen presentation in tumor-infiltrating macrophages. Supporting our findings with multi-omics data, we reported that ferroptotic tumor-derived all-trans retinoic acid (ATRA) is a novel regulator of MHC-II in macrophages. ATRA directly targets CD38 through transcriptional factor RARα and activates master regulator TFEB to control MHC-II expression by inducing autophagy. Clinically, a ferroptosis signature is associated with better immunotherapy response. We also innovatively develop a drug-free nano-redox lever (DFNRL) to circumvent the non-specific limitations of conventional drugs, which specifically targets and disrupts glutathione metabolism in tumor cells by accepting electrons, thereby boosting ferroptosis-mediated immune stimulation. This synergizes with anti–PD-1 immunotherapy across preclinical models. Our study answers an unidentified role for ferroptosis in modulating anti-tumor immunity and provides a clinically translatable approach to enhance immunotherapy efficacy.

overall_design:
ATAC-seq profiling of BMDM cells treated with 200 nM ATRA or DMSO for 24hr;
TT-seq profiling of BMDM cells treated with 200 nM ATRA or DMSO for 24hr;
Cut&Tag for RPB1, H3K4me3 and H3K27ac using BMDM cells treated with 200 nM ATRA or DMSO for 24hr;
RNA-seq profiling of BMDM cells treated with 200 nM ATRA or DMSO for 24hr.

first: Jia lei

last: Sun

first: Dingdang

first: Shibin

first: Jimin

last: Zhu


title: Structural Basis of a Novel Heme Binding Bacterial One-Component Switch

city: Boston

first: Stephen

last: Gisselbrecht

address: 77 Avenue Louis Pasteur, Rm 468

department: Division of Genetics, Department of Medicine

institute: Brigham and Women's Hospital

type: Genome binding/occupancy profiling by array

summary: One-component systems (OCSs) integrate sensory and effector functions within a single protein, enabling rapid gene expression changes in response to environmental cues. Here, we characterized FG214, a heme binding OCS transcription factor from Fimbriimonas ginsengisoli. The Per-ARNT-Sim (PAS) domain of FG214 binds a hexacoordinate heme b in oxidized conditions and undergoes redox and ligand-dependent conformational changes, transitioning from a monomeric state to an active homodimer. Spectroscopic and structural data reveal that oxidation stabilizes the Helix-Turn-Helix (HTH)-PAS intramolecular domain interface, while reduction of the heme iron promotes HTH dissociation. Furthermore, addition of imidazole as a ferric heme ligand drives homodimerization and DNA binding. A 1.47 Å crystal structure of an imidazole-bound truncated construct captured an activated dimer, revealing heme coordination sites and homodimerization interfaces. Using DNA-binding assays, we identified an artificial promoter sequence and demonstrated ligand-enhanced protein-DNA binding. Additionally, we performed proof of concept experiments to showcase the ability of FG214 to homodimerize in vivo, setting the stage for a redox or gas sensitive biosensor. Together, these findings define FG214 as a novel heme-binding PAS transcription factor and establish a model for a redox-regulated molecular switches.

overall_design: Binding of purified FG214 in vitro to all 8mers is assessed by protein binding microarray. Wild type protein is compared to a designed point mutant (H175I), and binding is compared in the presence and absence of 500 mM imidazole. Each condition is assayed in parallel in one chamber of an 8-chamber Agilent microarray slide. Each 8mer is represented at least 16 times on the array.

first: James

middle: J

last: Siclari

first: Malvin

last: Forson

first: Cullen

last: Roeder

first: Eta

last: Isiorho

first: Denize

last: Favaro

first: Rinat

last: Abzalimov

first: Alec

last: Follmer

first: Martha

last: Bulyk

first: Kevin

middle: H

last: Gardner


title: Modeling human enterovirus A71 infection using an intestinal microphysiological system

city: Bunkyo-ku

first: Kazuo

last: Takayama

state: Tokyo

address: Yushima 1-5-45

country: Japan

institute: Institute of Science Tokyo

summary: Enterovirus A71 (EV-A71), a causative virus of hand, foot, and mouth diseases, primarily infects and replicates in the intestine and, in severe cases, spreads to the central nervous system (CNS), leading to neurological complications. Therefore, suppressing viral replication in the intestine is important to prevent severe complications. However, the intestinal pathophysiological changes in EV-A71-infected patients remain poorly understood. In this study, we aimed to examine the intestinal response to EV-A71 infection using the intestinal microphysiological system (MPS) we previously developed using human pluripotent stem cells and microfluidic devices. The viral RNA was detectable in the cell culture supernatant of the intestinal MPS for 14 days after the viral infection. Despite this, EV-A71 infection did not induce significant morphological changes in the intestinal MPS and alter the expression of epithelial cell markers, suggesting that the virus can infect the intestinal MPS without causing intestinal epithelial damage. In addition, we found that the secretion of interferons (IFNs) in the cell culture supernatant was not increased by viral infection. Interestingly, the treatment with recombinant IFNs increased the expression of innate immune response-related genes and reduced viral mRNA levels. A strong association was observed between EV-A71 infection and IFN signaling in the intestinal MPS. We believe that the intestinal MPS would be a valuable platform for studying EV-A71 infection and evaluating antiviral strategies.

overall_design: Total RNA was collected from the EV-A71-infected intestinal MPS, and then RNA-seq was performed.


title: Evaluation of the immortalized primary human hepatocyte cell line Fa2N-4 as a model for MASLD

city: Groningen

first: Jingyuan

last: Fu

address: Hanzeplein 1

department: Department of Genetics

country: Netherlands

institute: University Medical Center Groningen

summary: Background and aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a leading cause of chronic liver disease worldwide, yet preclinical in vitro models remain suboptimal. MASLD studies predominantly rely on hepatocellular carcinoma–derived cell lines, such as HepG2, which are poorly differentiated and exhibit cancer-associated metabolic reprogramming that suppresses key adult hepatic functions. Primary human hepatocytes, on the other hand, offer greater physiological relevance, but their use is limited by cost, availability, donor variability, and rapid dedifferentiation. Here, we evaluated the immortalized primary human hepatocyte cell line Fa2N-4 as an alternative in vitro model for MASLD. Methods: We performed a comparative analysis of Fa2N-4 and HepG2 cells, assessing genomic architecture, lipid-induced steatosis, and pharmacological responsiveness. Results: The two models exhibited pronounced differences, including distinct karyotypes, divergent MASLD-associated genetic risk variant profiles, and markedly different transcriptional responses to lipid loading and drug treatment. We further examined the hepatic response to resmetirom, the first U.S. Food and Drug Administration (FDA) approved medication specifically for treating MASH. Resmetirom, a thyroid hormone receptor-β agonist, reduced intracellular triglyceride accumulation in Fa2N-4 cells, but not in HepG2 cells, and this was accompanied by induction of mitochondrial glycolysis and oxidative phosphorylation pathways. Conclusions: Together, these findings demonstrate that hepatocyte model selection critically influences experimental outcomes in MASLD research and highlight Fa2N-4 cells as a physiologically relevant platform for mechanistic and translational studies of MASLD therapeutics.

overall_design: HepG2 or Fa2N-4 hepatocyte cell lines were exposed to negative control (.6% BSA), free fatty acids (mix oleic to palmitic 2:1 of total 600 uM) with fructose (1mM), with or without resmetirom (200 uM).

first: Victoria

last: E.J.M. Palasantzas

first: Dicky

last: Struik

first: Trijnie

last: Bos

first: Mirjam

last: Koster

first: Jody

last: Gelderloos-Adders

first: Ellen

last: Vos

first: Krista

last: van Dijk-Bos

first: Sebo

last: Withoff

first: Joanne

last: Hoogerland

first: Johan

last: Jonker


title: A biohybrid chiral hydrogel enhances preclinical postoperative glioblastoma therapy by multi-pronged inhibition of tumor stemness

city: Singapore

first: Tingting

last: Cui

address: 4 Science Drive 2

department: Department of Diagnostic Radiology

country: Singapore

institute: National University of Singapore

summary: Glioblastoma stem cells (GSCs), which exhibit resistance to multiple treatments, are a prominent driver of postoperative glioblastoma (GBM) relapse. Declining the GSC population holds promise in GBM therapy but remains challenging, due to the difficulty in orchestrating the complex cytokine signaling programs and extracellular matrix features that induce GSC expansion. Here, we develop a biohybrid chiral hydrogel that allows intracavity implantation after GBM surgical debulking to comprehensively regulate GSC stemness, enhancing postoperative therapy. The hydrogel encapsulates GSC membrane-coated nanoparticles (GSNP), which serve as potent decoys to broadly neutralizing GSC-targeted pro-stemness and chemotaxis cytokines, allowing functional blocking and hydrogel infiltration of GSCs. Moreover, we showed that the D-chiral biohybrid hydrogel (D-gel@GSNP), in contrast to its L- and DL-chiral counterparts, further diminishes GSC stemness phenotype via D-chiral geometry-regulated mechanotransduction pathways. In three orthotopic intracranial GBM models, multi-pronged inhibition of GSC stemness enhances the gold nanoclusters-based hydrogel scaffold-sensitized radioimmunotherapy, enabling suppression of GBM relapse post-resection. This integrated regulation of biochemical and biophysical cues shows potential for treating cancer stem cell (CSC)-enriched malignancies.

overall_design: RNA-seq profiling of non-SP Luci+ GL261 cells and Hoechst-CD133+ GL261 cells at Day 6 of different treatments.


title: DSS chronic colitis induces osteoporosis by changing osteal macrophages to an inflammatory phenotype.

city: Sapporo

first: Mohamad Alaa

last: Terkawi

state: Hokkaido

address: Kita-15, Nish-7, Kita-ku

department: Department of Orthopedic Surgery

institute: Hokkaido University

summary: Osteal macrophages are bone-resident macrophages.In this study, we investigated the functional properties of Osteal macrophages in osteoporosis caused by chronic colitis. In this study, we investigated the functional properties of Osteal macrophage in osteoporosis caused by chronic colitis.We found that Osteal macrophages are altered in an inflammatory manner and affect osteoblast and osteoclast differentiation. To understand this mechanism, we isolated and collected Osteal macrophages from mice with DSS-induced colitis and comprehensively analyzed changes in gene expression by bulk RNA analysis

overall_design: One and three weeks after the 3% DSS water feeding, as well as three days after LPS administration and in the control group, F4/80-positive osteomacrophages were isolated and changes in gene expression were examined by RNA-seq.

first: Ryota

last: Suzuki

first: Mohamad

first: Norimasa

last: Iwasaki


title: The m5C orchestrator NSUN7 drives SPARC/HMGB1 axis–mediated inflammation to exacerbate kidney injury [RNA-Seq]

city: Hefei

first: Yuhang

last: Dong

address: Meishan Road NO.81

institute: Anhui Medical University

summary: Growing evidence indicates that kidney inflammation is a major contributor to the pathogenesis of various renal diseases, including acute kidney injury (AKI). Although RNA modifications have been implicated in regulating kidney inflammation, their precise roles remain largely unknown. Here, we show that kidney inflammation and injury were associated with elevated RNA 5-methylcytosine (m5C) modifications, primarily driven by the m5C “writer” NOP2/Sun RNA methyltransferase family member 7 (NSUN7). Both global and kidney-specific deletion of Nsun7 in mice reduced m5C abundance, attenuated inflammatory responses, and decreased macrophage infiltration, underscoring its proinflammatory role in the kidney. Mechanistically, we identified secreted protein acidic and cysteine-rich (SPARC) as a major downstream effector of NSUN7. SPARC upregulation amplified inflammatory responses in renal tubular epithelial cells by interacting with high mobility group box 1 and further promoted proinflammatory macrophage infiltration via tubular-macrophage crosstalk. Notably, therapeutic silencing of Nsun7 using a kidney-specific DNA tetrahedral molecular carrier developed for this study effectively alleviated inflammation and improved renal outcomes in AKI models. Collectively, our findings identify NSUN7 as a key driver of renal inflammation via SPARC regulation and underscore its potential as a therapeutic target in inflammatory kidney diseases.

overall_design: To identify specific targets of NSUN7 in renal inflammation, we conducted RNA-seq and m5C MeRIP-seq analyses in cisplatin-stimulated HK2 cells with or without silencing of NSUN7

first: Xiaoming

last: Meng


title: The m5C orchestrator NSUN7 drives SPARC/HMGB1 axis–mediated inflammation to exacerbate kidney injury [MeRIP-Seq]

type: Methylation profiling by high throughput sequencing

overall_design: To identify specific targets of NSUN7 in renal inflammation, we conducted RNA-seq and MeRIP-seq analyses in cisplatin-stimulated HK2 cells with or without silencing of NSUN7


title: Age-related gene expression alteration responding to mechanical force and TGFβ signaling

city: Villigen

last: Shivashankar

state: Aargau

address: Forschungsstrasse 111

department: LNB

country: Switzerland

institute: Paul Scherrer Institute

summary: The aging process significantly alters the biomechanical properties of fibroblasts, impacting their cytoskeletal and chromatin organization. These changes are pivotal in modulating cellular responses to mechanical and biochemical stimuli. This study investigates the hypothesis that aging leads to fibroblasts’ divergent adaptation mechanisms to external stimuli. Herein, fibroblasts from young and old cohorts were embedded in 3D collagen gels and subjected to tension force alone (tension stimulus) or tension +/- Transforming Growth Factor beta (TGFb) (tension/TGFb stimuli). The study revealed that mechanical forces and aging exert profound effects on chromatin architecture. Young cells adapted more rapidly to mechanical stress and showed a heightened responsiveness to TGFβ compared to older cells. The identification of key transcription factors implicated in age-dependent responses was further examined, assessing their potential in cellular rejuvenation.

overall_design: RNA-seq profiling of primary dermal fibroblasts from two age groups (old: 75 years old; young: 10 years old) seeded in 3D collagen gel exposed to tension force and TGFβ stimulation

first: Yawen

last: Liao

first: Luezhen

last: Yuan

first: Trinadha

last: Sornapudi

first: G

middle: V


title: Single-cell multiomic integration identifies widespread, cell-type resolved fetal reactivation in the diseased human heart

city: Philadelphia

first: William

last: Gao

state: PA

address: 415 Curie Blvd

department: Genetics

institute: UNIVERSITY OF PENNSYLVANIA

summary:
Background:
As the first organ to develop in utero, the human heart undergoes extensive molecular, structural and metabolic remodeling during development and must sustain its function throughout life.

Results:
We generate an integrated multiomic atlas of human cardiac cells, combining newly generated and publicly available single-nucleus RNA sequencing datasets from 299 donors and single nucleus ATAC-seq datasets from 106 donors. Developmental and disease-associated processes drive far more extensive molecular remodeling than sex-associated or aging-dependent effects. Across nearly all cardiac cell types, developmental and disease-driven changes exhibit strong overlap at both the transcriptomic and epigenomic levels, revealing widespread reactivation of fetal-associated gene programs beyond cardiomyocytes. Both cardiac development and disease show convergent shifts in intercellular communication, including increased TGFβ signaling. Integration of gene expression and chromatin accessibility data reveals putative cell-type–specific transcriptional factors driving fetal reactivation in major cardiac diseases. Spatial transcriptomics data orthogonally identifies localization of this fetal reactivation signature within spatially distinct niches in ischemic and fibrotic zones of acute myocardial infarction. Finally, we construct a cell-type–resolved enhancer-to-gene linkage map that refines the association of dilated and hypertrophic cardiomyopathy genetic risk loci to downstream target genes.

Conclusions:
This study presents a comprehensive multimodal, cell-type–resolved atlas of the human heart, providing a foundation for understanding human cardiac gene regulation across the human lifespan and in cardiac diseases.

overall_design:
New datasets were generated for this study. snRNA-seq (Drop-seq platform) was generated for 17 donors. snATAC-seq using 10X Genomics ATAC.

As part of this study, we also integrated additional datasets across previously published studies. The table below lists reanalyzed samples, and the link to the raw data sources (fragment files or BAM files, depending on the study).

The final processed ATAC dataset is GSE290367_integrated_ATAC_adata.h5ad.gz, which includes log normalized counts in adata.X and raw counts in adata.layers['counts']. The final processed RNA dataset is GSE290367_integrated_RNA_adata.h5ad.gz, with the raw counts in adata.layers['counts'] and the log normalized counts in adata.X.

first: Peng

last: Hu

first: Qi

last: Qiu

first: Kenneth

last: Bedi

first: Kotaro

last: Sasaki

last: Margulies

first: Hao

last: Wu


title: FOXM1 drives metabolic adaptations in colorectal cancer brain metastases and represents a potential therapeutic target

city: Tel Aviv

first: Guy

last: Shapira

address: Chaim Levanon 30

country: Israel

institute: Tel Aviv University

summary: Colorectal cancer (CRC) is a leading cause of cancer-related mortality in the Western world. While brain metastasis (BMs) are relatively uncommon in CRC, they represent the fourth most frequent cause of BMs overall and their occurrence is increasingly recognized as a complication in advanced CRC, associated with poor prognosis and limited treatment options. The brain microenvironment presents unique metabolic challenges, including low oxygen levels and restricted lipid availability. In order to survive in this hostile environment, CRC cells must acquire metabolic adaptations allowing them to survive and proliferate. To identify key drivers that enable CRC cells to metastasize to and survive within the brain, we conducted a transcriptomic screen of CRC BMs. Among the upregulated genes, FOXM1 was identified, a transcription factor critical for tumor progression. This was further validated by immunohistochemistry in human samples. To study the role of brain microenvironment in mediating FOXM1 upregulation, CRC cells were cultured in either astrocyte-conditioned media (A-CM) or compared to hepatocyte-conditioned media (H-CM). CRC cells exposed to A-CM exhibited markedly higher FOXM1 expression. Moreover, using an in vivo model of intracranial CRC BM mouse model demonstrated significant FOXM1 overexpression in CRC cells following brain injection. And notably, our study revealed a significant correlation between FOXM1 and fatty acid synthase (FASN) in the brain microenvironment. These findings suggest that FOXM1 plays a key role in CRC brain metastasis and may serve as a promising therapeutic target. Targeting FOXM1 could provide novel treatment strategies for CRC patients with BMs, potentially improving outcomes in this challenging disease setting.

overall_design: RNAseq of FFPE samples from KRAS-mutated colorectal cancer brain metastasis n=7 and liver metastasis n=10

first: Fayhaa

last: Khair-Dabour

first: Inbal

last: Greenberg

first: Sivan

last: Fuchs

first: Asia

last: Zubkov

first: Ido

last: Wolf

first: Tami

last: Rubinek


title: Mechanisms by which whole genome doubling drives breast tumor evolution [scMultiomic-seq]

first: Kornelia

last: Polyak

address: 450 Brookline Ave. S1070

institute: Dana-Farber Cancer Institute

summary: Whole genome doubling (WGD) is one of the most common genetic alterations in human cancer generally associated with poor clinical outcome, yet the underlying mechanisms have not been delineated. Here we developed an experimental model of WGD using murine mammary tumor cell lines in immunocompetent mice and found that WGD triggers immune evasion characterized by reduced infiltration of CD8+ T cells. Via comprehensive cellular and molecular characterization of WGD+ and WGD- tumors at the single cell level, we determined that WGD+ cancer cells have diminished antigen presentation due to muted response to IFN stimulation in part due to epigenetic silencing of transcriptional regulators of MHCI genes via the PRC2 complex. WGD+ tumors also have lower alpha-ketoglutarate/succinate ratio that might lead to the observed increase in histone H3 lysine 27 trimethyl levels associated with repressive chromatin. Lastly, WGD+ tumors are more responsive to anti-PD-L1 blockade and to EED inhibitors and this is associated with increased antigen presentation and CD8+ T cell infiltration. Our results have important implications for the treatment of patients with WGD+ breast cancer.

overall_design: TA3 and 67NR WGD- and WGD+ cell models are directly preocessed for scMultiomic-seq library prep and sequecning for scATAC-seq and scRNA-seq.

first: Pierre

last: Foidart

first: Zheqi

last: Li

first: Marco

last: Seehawer


title: Uhrf1 loss alters Ctcf-mediated topological organization during early mouse embryogenesis

city: New York City

first: Bohan

last: Zhu

state: NY

address: 1425 Madison Ave

department: Genetics and Genomic Sciences

institute: Icahn School of Medicine at Mount Sinai

summary: UHRF1 is a chromatin-binding protein essential for maintaining DNA methylation and histone modification states, yet its integrated role in vivo remains incompletely understood. To define its function, we generated conditional Uhrf1 knockout embryonic stem cells (ESCs) and embryos. Uhrf1⁻/⁻ ESCs exhibited near-complete loss of 5mC and 5hmC but maintained pluripotency, whereas Uhrf1-null embryos developed normally until E8.5 and then failed to further develop by E9.5, phenocopying Dnmt1 loss. Single-cell multi-omic (ME-seq) profiling of E8.5 embryos revealed impaired lineage stabilization, widespread hypomethylation, and disrupted chromatin architecture. Uhrf1 loss predicted altered CTCF occupancy, accompanied with disorganized enhancer-promoter interactions, and defective Bmp signaling, particularly within neural crest populations. These findings identify Uhrf1 as a central regulator tightly coupling DNA methylation maintenance to 3D genome organization during gastrulation to direct early lineage specification, positioning Uhrf1 as a pivotal mediator of epigenetic information transfer during early embryogenesis.

overall_design: E8.5 embryos were dissected from the uterus in PBS/FBS and extra-embryonic tissue was removed. The placenta was used for genotyping for SRY to determine the sex. The embryos were washed in PBS without Ca2+/Mg2+ and each was put in a 3 cm dish with 1 ml TrypLE, chopped into small pieces, and incubated at 37°C for 20 min. 300 μl serum was added and pipetted up and down until tissue was dissociated in single cells. Joint scRNA-seq, scATAC-seq, and sc-methyl-seq libraries were prepared using the ME-seq protocol.

first: Agata

last: Kurowski

first: Yifei

last: Sun

first: Nimisha

last: Jain

first: Ashley

last: Chan

first: Martin J.

last: Walsh

first: Sai

last: Ma


title: CITE-Seq analysis of intratumoral immune cells from YUMM1.7 melanoma treated with anti-PD-1 therapy.

city: Wuerzburg

first: Julia

last: Krug

email: ag.schmieder@gmail.com, krug_j@ukw.de

state: Bavaria

address: Josef-Schneider-Straße 2

department: Dermatology

institute: University Hospital Wuerzburg

summary:
Metastatic melanoma remains a disease difficult to treat, as 50% of the patients are resistant to immunotherapies or experience high toxicities. Investigations to identify underlying mechanisms focus on T cells or the immunosuppressive tumormicroenvironment (TME).  Tumor-associated macrophages (TAMs) make up a large fraction of the TME and high levels of TAM have been associated with poor survival of cancer patients.
In this study, the subcutaneous YUMM1.7 melanoma model was used to investigate the responsiveness to anti-PD-1 therapy. The tumor cell line was injected into GRf/f and GRf/f-LysMCre mice to enable insights into glucocorticoid-dependent effects within the myeloid compartment and how they contribute to the responsiveness to anti-PD-1 immunotherapy. Therefore, we profiled sorted CD45+ immune cells from tumors of GRf/f and GRf/f-LysMCre treated with anti-PD-1 therapy or an isotype control antibody via the cellular indexing of transcriptomes and epitomes-method (CITE-Seq).

overall_design: YUMM1.7 tumors were grown subcutaneously in eight week old female GRf/f and GRf/f-LysMCre mice were treated with anti-PD-1 immunotherapy or isotype control antibody (four mice per condition). The subcutaneous tumors were dissociated and sorted for CD45-positive immune cells. In addition, the 16 samples were labelled with hashtag antibodies to allow multiplexing of eight samples per library (resulting in two libraries in total). Subsequently, the libraries were processed using the Chromium Single Cell 3' Reagents Kit and the final sequencing was performed on a Novaseq 6000 Platform.

first: Iris

last: Mueller

first: Nazia

last: Afrin

first: Fabian

last: Imdahl

first: Matthias

last: Goebeler

first: Emmanuel

last: Saliba

first: Clément

last: Cochain

first: Angela

last: Riedel

first: Astrid

last: Schmieder


title: Mechanisms by which whole genome doubling drives breast tumor evolution [scATAC-seq]

first: Kornelia

last: Polyak

overall_design: TA3 cell lines that engineered for WGD- and WGD+ was used for mouse orthgotopic injection into the ingunal mammar gland of NSG and A/J mice. And the tumors were collected at the desired time points for single cell ATAC-seq.

first: Pierre

last: Foidart

first: Zheqi

last: Li

first: Marco

last: Seehawer