title: Spatial transcriptomics data of the wild-type and App NL-G-F mouse posterior cortex

city: London

first: Samuel

last: Barnes

address: Hammersmith Hospital Campus, Du Cane Road

country: United Kingdom

institute: Imperial College London

summary: Our in vivo imaging data suggest that GABAergic microcircuitry is dysfunctional in the App NL-G-F mouse.   To determine the relative contribution of different GABAergic subclasses to abnormal synaptic processing,  we tested for cell-type-specific molecular processes associated with early-stage amyloidosis.   To do this, we used the CosMx (Nanostring/Bruker) spatial transcriptomic system to investigate   cellular changes in posterior cortical regions (visual and retrosplenial cortices) identified as expressing  dysregulated resting-state activity in 3-4 months and 6-8 months App NL-G-F mice. Our results suggest  that parvalbumin interneurons show some of the earliest transcriptomic changes in posterior cortical regions,   with dysregulated activity in App NL-G-F mice, which are linked to a downregulation of GABAergic synaptic transmission.   Both parvalbumin and somatostatin interneurons show the highest number of transcripts that covary with local plaque load,   which are associated with synaptic processes.

overall_design: CosMx technology [CosMx Mouse Neuroscience Panel (950-plex); Bruker/Nanostring, USA]   was applied to the posterior cortex and adjacent regions of AppNL-G-F mice and wild-type controls   at 3-4 months and 6-8 months of age. [Platform: GPL36279 NanoString CosMx SMI (Mus musculus)]

first: Leire

last: Melgosa-Ecenarro

first: Carola

middle: I

last: Radulescu

first: Nazanin

last: Doostdar

first: Anna

last: Mallach


title: Lineage-Specific Oncogenic Reprogramming of COL17A1 Orchestrates a Pro-Invasive and Hypoxia-Adapted Niche in HNSC

status: Public on Feb 11 2026

city: Guangzhou

first: Xiaoxia

last: Wang

state: Guangdong

address: No. 280, Waihuan East Road, Xiaoguwei Sub-district, Panyu District

country: China

institute: Guangdong Pharmaceutical University

type: Expression profiling by high throughput sequencing

summary: Head and Neck Squamous Cell Carcinoma (HNSC) originates from specialized epithelium, and identifying lineage-specific oncogenes is crucial for understanding tumor progression. In this study, we identified COL17A1 as a potential core regulator in HNSC. To characterize the transcriptional landscape and molecular mechanisms regulated by COL17A1, we performed bulk RNA-sequencing on the human oral squamous cell carcinoma cell line, SAS. We compared the gene expression profiles of control cells (transfected with negative control siRNA) against COL17A1-depleted cells (transfected with COL17A1-specific siRNAs). This dataset reveals that depletion of COL17A1 leads to the dismantlement of a specific "Partial-EMT" gene module and induces a metabolic shift, highlighting its role in maintaining the malignant phenotype of HNSC cells.

overall_design: SAS cells were cultured and transfected with either a negative control siRNA (NC) or a specific siRNA targeting COL17A1 (siRNA sequence #2). Total RNA was extracted post-transfection. Messenger RNA libraries were prepared and sequenced on the Illumina NovaSeq platform to generate paired-end reads. The experiment consists of 2 samples: one negative control sample and one COL17A1-knockdown sample.

first: Zhengyan

last: Liang

first: Weixun

last: Fang

first: Yin

last: Zhang

first: Size

last: Chen

first: Zhiquan

last: Huang


title: Spatial whole transcriptome analysis (GeoMx; Nanostring) of cervical lymph nodes (cLNs) from C57BL/6 mice infected intradermally (i.d.) with Mycobacterium tuberculosis (Mtb) reveals transcriptional changes at the edge of lesions.

city: Cairns

first: Andreas

last: Kupz

state: Queensland

address: McGregor Road, Smithfield

department: Australian Institute of Tropical Health and Medicine

country: Australia

institute: James Cook University

summary: Here, we used a mouse model of latent lymphatic M. tuberculosis (Mtb) infection (LTBI) to dissect the immunological mechanisms underlying LTBI containment versus reactivation. We show that immunosuppression-mediated reactivation of lymphatic LTBI and the subsequent induction of progressive disease can be prevented by vaccination with BCG or recombinant BCG (BCG::ESAT-6-PE25SS (abbreviated to PE25) even in the absence of CD4+ T cells. Spatial transcriptomics, network analysis and bioinformatic reveal that anti-CD4-mediated immunosuppression is associated with and enhanced immune response at the edge of TB lesions within the infection-draining cervical lymph nodes.

overall_design: 30 Regions of Interest (ROI) including 3 different tissue lesions of cLNs were analysed. Treament groups included are 1) untreated mice (Normal); 2) unvaccinated and Mtb-infected (Mtb); 3)  unvaccinated, immunosuppressed with anti-CD4 mAb and Mtb-infected (Mtb+aCD4); 4) vaccinated with BCG,  immunosuppressed with anti-CD4 mAb and Mtb-infected (BCG+aCD4+Mtb); 5) vaccinated with PE25,  immunosuppressed with anti-CD4 mAb and Mtb-infected (PE25+aCD4+Mtb). Each treated group included different ROI types (Normal in naive or normal in disease, Lesion and Edge of Lesion).

first: Matt

middle: A

last: Field

first: Manoharan

last: Kumar


title: Lamin A/C maintains genome topology and regulates transcriptional programs essential for virus-driven B cell activation.

city: Philadelphia

first: Priyankara

middle: J

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

country: USA

institute: The Wistar Institute

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Genome-wide DNA methylation analysis of placentas from pregnancies complicated by early-onset fetal growth restriction

city: Szczecin

first: Magdalena

last: Bednarek-Jedrzejek

address: Rybacka 1

country: Poland

institute: Pomeranian Medical University in Szczecin

summary: Fetal growth restriction (FGR) affects approximately 5–10% of pregnancies and remains a leading cause of perinatal morbidity and mortality. The condition is characterized by the failure of the fetus to achieve its genetically determined growth potential, most commonly as a consequence of placental dysfunction. Despite extensive research, the molecular mechanisms underlying FGR remain incompletely understood. In this study, we investigated whether alterations in placental DNA methylation are associated with early-onset FGR and may contribute to its pathophysiology.

overall_design: Placentas were collected from 11 pregnancies complicated by early-onset FGR and from 5 uncomplicated control pregnancies. Genomic DNA was extracted from four pooled, spatially independent placental fragments per sample. Genome-wide DNA methylation profiling was performed using the Illumina Infinium MethylationEPIC BeadChip array. Raw data were processed using the ChAMP pipeline with BMIQ normalization. Differentially methylated probes (DMPs) were defined as CpG sites showing an absolute mean β-value difference ≥0.05 between groups and an adjusted p-value ≤0.05. To validate the findings, the same analytical pipeline was applied to an independent cohort comprising 11 FGR placentas and 43 controls from a publicly available dataset (GSE100197).

last: Bednarek-Jędrzejek

first: Olga

last: Taryma-Leśniak

first: Małgorzata

last: Poniatowska

first: Mateusz

last: Cejko

first: Katarzyna

last: Maksym

first: Sylwia

last: Dzidek

last: Blatkiewicz

first: Ewa

last: Kwiatkowska

first: Andrzej

last: Torbé

first: Sebastian

last: Kwiatkowski


title: Activated Interferon Signaling Suppresses Age-Dependent Liver Cancer [RNAseq_WholeLiver]

city: La Jolla

first: Kathryn

last: Lande

state: CA

address: 10010 N Torrey Pines Rd

department: IGC

institute: Salk Institute

summary: Age is a major risk factor for liver cancer, as is the case for most adult human cancers. However, the underlying mechanisms are not well defined. A better understanding of the role of aging in liver and other cancers can facilitate approaches for risk assessment, early detection and prevention. We hypothesize that age-driven changes render aged liver more sensitive to oncogenic stress and hence tumorigenesis. To investigate how the liver changes with age, we documented the immune profile, transcriptome and epigenome of healthy livers from both young and aged mice, revealing pronounced alterations with aging. Notably, in aged hepatocytes, we identified heightened interferon (IFN) signaling, as well as simultaneous tumor suppressor and oncogene signaling at both bulk and single cell level, suggestive of an aged liver that is poised for neoplasia. To challenge this seemingly poised state, we employed adeno-associated virus (AAV)-mediated expression of a c-Myc oncogene in young and aged mouse liver hepatocytes in vivo. Analysis of aged hepatocytes expressing c-Myc revealed further elevated expression of IFN Stimulated Genes (ISGs). This ISG upregulation was evident in multiple models of oncogenic stress and transformation in older mice and also observed in aged humans with Metabolic dysfunction-Associated Steatohepatitis (MASH). We determined that Stat1 is both necessary and sufficient for the age specific elevated ISG expression in old wild type mice. Remarkably, inhibiting Jak/Stat signaling alongside ectopic c-Myc expression led to high-grade hepatocyte dysplasia and tumor formation, selectively in aged mice. Together, these results suggest that an aged liver is in a state of “precarious balance”, due to concurrent activation of oncogenic and tumor suppressor pathways, but protected against neoplastic progression by IFN-signaling. Age-dependent activation of IFN signaling has been observed in many tissues and recent studies have demonstrated its detrimental consequences on aging, raising the question as to why IFN-signaling is activated during aging. We propose that aged tissues are intrinsically at higher risk of cancer and age-dependent activation of IFN-signaling is an adaptive process to protect from tumorigenesis, but one that also has maladaptive consequences.

overall_design: RNAseq of whole livers in young and old mice fed high fat (58Y1) and normal diets for 9-weeks. normal diet is Teklad Gloabal 18% Protein Rodent diet from Envigo, cat no 2018 and high fat diet is cat no 58Y1 from Test-Diet

first: Aaron

last: Havas

first: Peter

middle: D

last: Adams


title: Parenteral Vaccination with an Adjuvanted mRNA Vaccine Induces Protective Mucosal Immunity Against Rotavirus in neonatal mice

city: Beijing

first: Li

last: Jingjiao

address: Zhongguancun North First Street 2

institute: Institute of Chemistry, Chinese Academy of Sciences

summary: Gastrointestinal viruses such as rotavirus remain a major cause of childhood gastroenteritis and mortality worldwide. Although current live-attenuated rotavirus vaccines are effective, they face challenges including production, reduced efficacy in low- and middle-income countries, and rare adverse events, highlighting the need for vaccines that can induce strong gut mucosal immunity. Herein, we introduce a lipid nanoparticle (LNP) platform that co-delivers messenger RNA (mRNA) and the retinoic acid receptor agonist Am80 (Am80-LNP), enabling antigen-specific mucosal immune responses in the gut via parenteral intramuscular vaccination. Am80 incorporation preserved the vaccine’s ability to imprint expression of the gut-homing receptors CCR9 and α4β7 on T and B cells, improved mRNA delivery, enhanced lymph node accumulation, and mitigated injection-site inflammation driven by the LNP. In mice and Bama miniature pigs, Am80-LNP induced antigen-specific serum antibody titers, cellular immune responses, and intestinal IgA production. Importantly, neonatal mice vaccinated with Am80-LNP exhibited reduced incidence and duration of diarrhea following live rotavirus challenge, whereas LNPs without Am80 conferred negligible protection. These findings highlight the importance of gut mucosal immunity in mediating protection against rotavirus and suggest that Am80-LNP may offer a versatile mRNA vaccine platform against gastrointestinal viruses.

overall_design: RNA-seq profiling of small intestinal tissue from wild-type C57BL/6 mice immunized with PBS, SM102-LNP, or Am80-LNP at day 14 after booster immunization

first: Jingjiao

last: Li

first: Xueguang

last: Lu

first: Haowei

last: Zu


title: Lamin A/C maintains genome topology and regulates transcriptional programs essential for virus-driven B cell activation. [RNA-Seq]

first: Priyankara

last: Wickramasinghe

summary: Lamin A/C is a crucial structural component of the nuclear lamina that influences chromatin organization and gene regulation. In this study, we demonstrate that lamin A/C is vital for maintaining higher-order genome organization and transcriptional programs that support EBV-driven B-cell activation. Loss of lamin A/C in a B-lymphoblastoid cell line caused significant three-dimensional reorganization of the genome, evidenced by the loss of long-range chromatin loops, an increase in short-range contacts, and redistribution of H3K9me2- marked heterochromatin. These structural disruptions were linked to widespread changes in gene expression affecting metabolic, signaling, and differentiation pathways. Mechanistically, lamin A/C influences the nuclear positioning and transcription of CTCF-bound loci by preventing their relocation to the periphery and their association with lamin B1. Blocking H3K9me2 deposition mimicked the transcriptional effects of lamin A/C depletion and revealed increased sensitivity to PI3K inhibitors. Overall, our results identify lamin A/C as a key organizer of genome structure and epigenetic regulation in EBV-infected B cells, uncovering a lamin-dependent pathway that connects nuclear architecture, metabolism, and viral disease processes.

overall_design: RNA-seq of WT and LMNA/C depleted cells

first: Lisa

middle: B

last: Caruso

first: Italo

last: Tempera


title: Lamin A/C maintains genome topology and regulates transcriptional programs essential for virus-driven B cell activation. [Hi-C]

overall_design: HiC of WT and LMNA/C depleted cells


title: A GCLC Inhibitor Enhances the Antitumor Efficacy of Glutathione Metabolic Pathway Inhibition in SMARCB1-Deficient Rhabdoid Tumors

city: Chuo-ku

first: Hideaki

last: Ogiwara

state: Tokyo

address: 5-1-1, Tsukiji

department: Division of Cancer Therapeutics

country: Japan

institute: National Cancer Center Research Institute

summary: Malignant rhabdoid tumors and epithelioid sarcomas, characterized by SMARCB1 deficiency, are aggressive cancers with limited effective treatments, necessitating development of therapeutic strategies. This study investigated the therapeutic efficacy and mechanism of action of inhibitors targeting glutamylcysteine ligase catalytic subunit (GCLC) in these intractable malignancies. The GCLC inhibitors GCLCi0 (ONO-6428513) and GCLCi1 (ONO-7068506) demonstrated high selectivity and potent anti-tumor effects in SMARCB1-deficient cancer cells in mouse tumor xenograft models, surpassing the efficacy of existing drugs. GCLC inhibition led to the depletion of intracellular glutathione (GSH), an increase in reactive oxygen species (ROS), and elevated lipid peroxidation, ultimately inducing ferroptotic cell death. SMARCB1-deficient cells exhibited reduced expression of SLC7A11, which led to low basal GSH levels and sensitivity to GCLC inhibition. Significant synergistic effects were observed when GCLC inhibitors were combined with agents targeting the GSH synthesis pathway, specifically SLC7A11 inhibitors and the glutaminase inhibitor telaglenastat. In a mouse tumor xenograft model, the combination of a GCLC inhibitor and telaglenastat showed superior anti-tumor efficacy compared to monotherapy, with good tolerability. These findings highlight the vulnerability of glutathione metabolism in SMARCB1-deficient cancers, suggesting that a GCLC inhibitor may be a promising therapeutic option. This study provides a preclinical foundation for the development of effective treatment strategies for SMARCB1-deficient cancers, including combination therapies, and supports further investigation toward future translational applications.

overall_design: JMU-RTK-2 -SMARCB1 and +SMARCB1 cells (n=1 sample per condition) were seeded in 6-well plates at 5 × 10⁵ cells per well and cultured for 24 h.


title: NSD2 is upregulated by high-risk HPV E6/E7 and modulates epithelial differentiation in HPV-associated Head and Neck Cancer

city: Milano

first: Susanna

last: Chiocca

address: Via Adamello 16

country: Italy

institute: European Institute of Oncology, IEO

summary: Epigenetic alterations are a key hallmark of cancer and are increasingly recognized as promising clinical biomarkers and therapeutic targets. In recent years Head and Neck Squamous Cell Carcinomas (HNSCC) have been classified into two subtypes: HPV-positive (HPV+), driven by human papillomavirus (HPV) infections, and the HPV-negative (HPV-), associated with environmental risk factors. Although exhibiting distinct molecular and clinicopathological features, both subtypes lack effective, tailored therapies. Thus, profiling their epigenetic landscape could thus have important clinical implications. In this study, we employed a quantitative mass spectrometry approach to profile histone post-translational modification (hPTMs) in HPV+ and HPV- HNSCC. We found that the histone mark H3K36me2 is significantly enriched in the HPV+ subtype and that this alteration is driven by hr-HPV E6/E7 oncoproteins which also upregulate NSD2, a histone methyltransferase specific for H3K36 dimethylation. Both H3K36me2 and NSD2 are upregulated in several cancers. Given the NSD2’s context and cell-type-dependent functions, we investigated its role in HPV+ and HPV- HNSCC. We observed that NSD2 regulates cell proliferation and migration in both the subtypes. Importantly, we identified a novel role for NSD2 in regulating epithelial cell differentiation, particularly in HPV+ HNSCC, where it appears to modulate the E6/E7-induced differentiation blockade. Overall, our findings highlight H3K36me2 as a potential biomarker for patient stratification and position NSD2 as a promising therapeutic target across HNSCC subtypes, regulating both shared and subtype-specific oncogenic pathways. Specifically, NSD2 inhibition in HPV+ tumors may restore epithelial differentiation and arrest tumor progression.

overall_design: Four HPV- (UM-SCC-4, UM-SCC-6, UM-SCC-18, UM-SCC-19) and three HPV+ (UD-SCC-2, UM-SCC-104, UPCI:SCC-152) HNSCC cell lines were transduced with shNSD2 and scrambled (scr) as a control. To assess wheter common and/or different pathways are regulated in HPV+ and HPV- HNSCC subtypes, DEGs between shNSD2 and scr control were calculated for each subgroup.

first: Lavinia

last: Ghiani

first: Ottavio

last: Croci


title: Independent control of neurogenesis and dorsoventral patterning by NKX2-2

city: New York

first: Sumin

last: Jang

state: NY

institute: Columbia University

summary: Human neurogenesis is disproportionately protracted, lasting >10 times longer than in mouse, allowing neural progenitors to undergo more rounds of self-renewing cell divisions and generate larger neuronal populations. In the human spinal cord, expansion of the motor neuron lineage is achieved through a newly evolved progenitor domain called vpMN (ventral motor neuron progenitor) that uniquely delays and expands motor neurogenesis. This behavior of vpMNs is controlled by transcription factor NKX2-2, which in vpMNs is co-expressed with classical motor neuron progenitor (pMN) marker OLIG2. In this study, we sought to determine the molecular basis of NKX2-2-mediated extension and expansion of motor neurogenesis. We found that, unlike in mouse or chick, NKX2-2 in the human spinal cord does not repress dorsoventral patterning genes like OLIG2. However, it retains its ability to repress NEUROG2, a proneural gene that promotes exit from cell cycle and motor neurogenesis. Interestingly, we found that ectopic expression of Tinman-mutant Nkx2-2 in mouse pMNs phenocopies human vpMNs, repressing Neurog2 but not Olig2, resulting in delayed motor neurogenesis. Thus, our studies reveal that the classical patterning function of NKX2-2 that depends on its Tinman repressive domain is dissociated from NKX2-2’s ability to repress NEUROG2 to control the onset and duration of motor neurogenesis in human ventral motor neuron progenitors.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Mouse ES cell- or human iPS cell-derived motor neuron organoids were used to generate high-throughput sequencing data. Human organoids were dissociated on day 11 of motor neuron differentiation following established protocols (Maury et al., Nat. Biotech.), followed by fixation, immunolabeling for NKX2-2 and OLIG2, then FACS-purified for NKX2-2+/OLIG2+ or NKX2-2-/OLIG2+ populations. RNA was extracted from purified populations and sequenced. Mouse cultures (following established motor neuron differentiation conditions; Wichterle et al., Cell) were exposed to doxycycline on day 3.5 to overexpress WT, TN domain mutant or SD mutant Nkx2-2 for 24 hours (control population was cultured in the absence of doxycycline). These populations were then used for RNA-seq and ChIP-seq against NKX2-2 or H3K27 acetylation.

first: Elena

middle: V

last: Abarinov

first: Julie

last: Dobkin

first: Erica

middle: C

last: Hurley

first: Michael

last: Closser

first: Lori

last: Sussel

first: Hynek

last: Wichterle


title: Age-stratified transcriptomic profiling reveals biologically distinct molecular phenotypes across pediatric, adolescent, and adult osteosarcoma

first: Jichuan

last: Wang

state: Beijing

address: No. 11 Xizhimen South St.

institute: Peking University People's Hospital

summary: Osteosarcoma exhibits a bimodal age distribution with distinct clinical behaviors between pediatric and adult patients. In this study, bulk RNA sequencing was performed on tumor specimens from 70 osteosarcoma patients stratified into pediatric (14 years and younger), adolescent (15 to 18 years), and adult (19 years and older) groups. Differential expression, functional enrichment, and immune infiltration analyses were conducted to characterize age-associated transcriptional differences. Pediatric tumors exhibited a hyperproliferative phenotype, adolescent tumors showed enhanced immune-related signatures, and adult tumors displayed activation of osteogenic differentiation programs. A characteristic pediatric-specific gene signature was further identified, providing insights into age-related molecular heterogeneity in osteosarcoma.

overall_design:
Tumor specimens from 70 osteosarcoma patients were analyzed by bulk RNA sequencing to investigate age-associated transcriptional differences among pediatric, adolescent, and adult groups.

***************************************************************
Due to limitations in data archiving and long-term storage, the raw sequencing data are currently unavailable.
***************************************************************

first: Li

last: Hu

first: Feiyang

last: Qi

first: Huimin

last: Liu


title: Epigenetic plasticity and regulatory remodeling of metabolism and cell wall dynamics drive genotype-specific embryogenesis in wheat

city: Saskatoon

first: Morgan

middle: W

last: Kirzinger

state: Saskatchewan

address: 110 Gymnasium Place

country: Canada

institute: National Research Council Canada

summary: Microspore embryogenesis is a process in which immature male gametophytes are induced to form embryo-like structures that can regenerate into doubled haploid (DH) plants following chromosome doubling. By producing complete homozygosity in a single generation, DH technology accelerates cultivar development and is particularly valuable for breeding resilient crops under increasing biotic and abiotic stresses. However, bread wheat remains largely recalcitrant to microspore embryogenesis, with strong genotype dependence limiting its broad application in breeding programs. To investigate the molecular basis of this variation, we performed a comparative transcriptomic time-course in two spring wheat cultivars with contrasting embryogenic responses: Nanda (highly responsive) and Sadash (recalcitrant). Dynamic gene expression reprogramming was observed during embryogenesis, with Nanda showing distinct enrichment of biological processes linked to epigenetic regulation, including nucleosome assembly, chromatin remodeling, and chromatin organization, while the integration of stress perception, hormonal signaling, cytoskeletal and cell wall remodeling, and targeted metabolic shifts collectively establishes the cellular environment necessary for successful microspore embryogenesis. Differentially expressed gene (DEG) hotspots suggested possible structural variation underlying the divergent responses between genotypes. Machine learning highlighted potential biomarkers, notably a histone deacetylase gene TRAESCS1D02G454400 located within a DEG-enriched region. Subgenome-specific analysis further revealed pronounced suppression of B-subgenome homoeologs in Sadash, 65% of which overlapped with DEGs from the genotype comparison. These findings highlight the central role of epigenetic regulation, homoeolog expression bias, and genotype-specific genomic variation in determining embryogenic efficiency. These insights also provide new molecular targets and biomarkers to overcome recalcitrance and enhance the utility of microspore embryogenesis in wheat DH breeding pipelines.

overall_design: RNAseq profiling of microspore cultures from three main stages of androgenesis – microspores at Day 0, proembryos at Day 7, and embryo-like structures at Day 21, using Nanda and Sadash, two spring wheat genotypes with contrasting embryogenic responses.

first: Hai Ying

last: Yuan

first: Yunfei

last: Jiang

first: Palak

last: Kathiria

first: Venkatesh

last: Bollina

first: Yifang

last: Tan

first: Jean

middle: L

last: Enns

first: Alison

middle: M

last: Ferrie

first: Sateesh

last: Kagale


title: Multi-omics Analysis Revealed Unique Features of Age-associated Type2 CD8 Memory T cells [CUT&Run]

city: Kyoto

first: Hiroyuki

last: Matsui

state: Kyoto

address: 54 Shogoin-Kawahara-cho

department: Hematology

institute: Kyoto University Graduate School of Medicine

summary: Aging impacts immune function, yet how and why various subsets of immune cells change during aging is still incompletely understood. Here, we analyzed peripheral blood mononuclear cells (PBMCs) from a cohort of healthy donors aged 20–82 years to uncover previously unrecognized age-dependent changes, using a 36-color spectral flow cytometry panel focused on T cells. We found that CXCR3- memory CD8 T cells, which produce a Th2-like cytokine response, accumulate with age. Comprehensive multi-omics analysis revealed transcriptional and epigenetic signatures that support Th2-like function. Importantly, we also identified an age-dependent bias towards the production of Th2 cytokines after activation in naive CD8 T cells, along with transcriptional and epigenetic changes supporting this outcome. Moreover, health outcome association analysis demonstrated a pathogenic feature of CXCR3- CM accumulation. Together, our results suggest that age-dependent corruption of epigenetic programming may cause the aberrant emergence of this potentially pathogenic Th2-like immune population

overall_design: Cut&Run with H3K4me3 and H3K27me3 of FACS sorted 4 subsets of human CD8 T cells (CXCR3- CM, CXCR3+ CM, CD28+CD57+ EM and CD57+ EMRA).

first: Marlene

last: Cervantes

first: Jorge

last: Landgrave-Gomez

first: Eric

last: Verdin


title: Proteomic Identification of Plasma Biomarkers of Response to IL-5 Inhibitor Biologics in Healthy Subjects.

city: Silver Spring

first: Paula

last: Hyland

state: MD

address: 10903 New Hampshire Avenue

department: CDER

institute: FDA

summary: The current project was designed to evaluate the utility of proteomics in identifying pharmacodynamic (PD) biomarkers for IL-5 inhibitors mepolizumab and reslizumab. We profiled over 7,000 plasma proteins from longitudinal samples collected from healthy participants treated with mepolizumab (24 mg, n=8), reslizumab (0.8 mg/kg, n=8), or placebo (n=8), and validated findings at lower doses of mepolizumab (12 mg, n=8) and reslizumab (0.4 mg/kg, n=8). We identified eosinophil major basic protein (EMBP) and proteoglycan-3 (PRG3) as promising plasma PD biomarkers, with EMBP responding to mepolizumab and PRG3 to reslizumab treatment. Both biomarkers demonstrated >20% fold change differences versus placebo, achieved statistical significance, and showed dose-response trends and comparable variability to placebo. Our study identified EMBP and PRG3 as promising plasma PD biomarkers for IL-5 inhibitors, warranting further validation for early phase trials and biosimilar development programs.

overall_design: The study includes data on >7000 proteins from 8 subjects treated with mepolizumab (24 mg), 8 subjects treated with reslizumab (0.8 mg/kg), and 8 subjects treated with placebo.  Plasma samples were selected for proteomics testing at pre-dose (referred to as baseline sample or day 1) and at 2, 3, 7, 14, 21, 28, 49, 63, 93, and 123 days. Replicates for 3 random samples were included on the array along with two covid convalescent plasma samples. Raw data was normalized using specific plasma standards by the manufacturer. The raw data can not be used as is.  Of note, aptamer seq.4148.49 was reassigned to EMBP as the target protein and not PAPP-A circa November 2024.

first: Lakshmi

last: Manasa Sakuntala Chekka


title: Transcriptional Response to Combination Antiretroviral Therapy Predicts Side Effects and Novel Targets

first: Avi

last: Ma'ayan

address: One Gustave L. Levy Place, Box 1603

department: Pharmacological Sciences

institute: Icahn School of Medicine at Mount Sinai

summary:
Antiretroviral therapy (ART) has revolutionized the clinical management of people with human immunodeficiency virus (HIV), transforming HIV infection into a chronic condition. Yet, the mechanisms of action and off-target effects of modern combination ART regimens versus individual ART medications are not fully understood.
We found that ART regimens affected transcriptional signatures of genes involved in HIV-host PPIs and were less tied to common ART-related side effects. Our findings support the use of high-throughput transcriptomics to detect specific mechanisms of ART on- and off-target effects to help prioritize new drug targets and compounds in future development and optimization of safer and more efficient ART.

overall_design: Using the L1000 assay, we profiled transcriptional responses to 11 single ART drugs and 6 ART combination regimens across three human cell lines, HepPG2 (liver), HK2 (kidney), and THP-1 (monocyte). Differentially expressed genes were analyzed against host-HIV protein-protein interactions (PPIs) and genes implicated in ART-associated side effects.

first: Alexander

last: Lachmann

first: Letizia

last: Amadori

first: Paola

last: Nicoletti

first: Heidi

last: Crane

first: Chiara

last: Giannarelli

last: Ma’ayan

first: Inga

last: Peter


title: Lamin A/C maintains genome topology and regulates transcriptional programs essential for virus-driven B cell activation [ChiP-seq]

first: Priyankara

last: Wickramasinghe

overall_design: RNA-seq, CHIP-seq and HiC of WT and LMNA/C depleted cells

first: Lisa

last: Caruso

first: Italo

last: Tempera


title: A reversible broad-spectrum antiviral targets the human V-ATPase VO domain

city: Vancouver

first: Diana

last: Bautista-Sánchez

state: British Columbia

address: 2350 Health Sciences Mall

department: Microbiology & Immunology

institute: University of British Columbia

summary: Increasing threats of viral disease underscore the urgent need for broad-spectrum antiviral drugs (BSADs). Host proteins utilized by human pathogenic viruses are key BSAD targets. The vacuolar-type H⁺-ATPase (V-ATPase) has been identified as a proviral factor for most pH-dependent enveloped viruses classified as pandemic threats. We report here the discovery of cladoniamide A (CA)—a V-ATPase inhibitor with single-digit nanomolar antiviral activity and a high selectivity index (SI: 103-104) against human enveloped viruses [e.g., SARS-CoV-2 variants, influenza A viruses (H1N1, H5N1), respiratory syncytial virus, dengue serotypes 1–4, and Zika virus]. Transcriptome profiling, pH estimation assays, and V-ATPase bioassays indicate that CA interferes with V-ATPase-dependent acidification of the host endolysosomal network thus preventing viral entry. Using pseudoviruses derived from five pathogenic virus families, we confirmed that CA is a BSAD acting as an entry inhibitor. CryoEM revealed that CA inhibits the V-ATPase rotary motor by occupying unique binding sites in the membrane-embedded Vo motor. Importantly, intranasal CA treatment in mice infected with influenza A H1N1 significantly reduced viral load in the lung by four log orders. Together, these findings pave the way for developing next-generation BSADs targeted at unique druggable pockets that enable the reversible pharmacological modulation of the human V-ATPase.

overall_design: To investigate the cellular mechanism of cladoniamide A (CA), we performed RNA-sequencing on Calu-3 cells pretreated for 3 h with the natural compounds [CA (250 nM), cladoniamide D (CD, 250 nM), bafilomycin A (BafA1, 100 nM), and DMSO (0.1%)] and collected at the designated time points (3, 6, 12, and 24 h post-treatment).

first: Jimena

last: Pérez-Vargas

first: Rebecca

last: Oot

first: Connor

last: Thompson

first: David

middle: E

last: Williams

first: Lianne

last: Presley

first: Danielle

middle: G

last: Gordon

first: Annika

last: Schulz

first: Kassidy

last: Knight

first: Siobhan

last: Ennis

last: Scheibner

first: Nidhi

last: Kaushik

first: Syan

last: Olver

first: Guang

last: Gao

first: Jun Bae

last: Park

first: Ivan

last: Villanueva

first: Kéryanne

last: Gagnon

first: Luka

last: Kramptert

first: Steven

last: McArthur

last: Nowakowski

first: Claire

last: Brown

first: Leon

middle: H

last: Chew

first: Erin

last: Knock

first: Brian

last: Hetrick

first: Sandra

last: Diederich

first: Soung-Hun

last: Roh

first: Masahiro

last: Nikura

first: Bryce

last: Warner

first: Stephan

last: Wilkens

first: Raymond

last: Andersen

first: François

last: Jean


title: Development of a human induced pluripotent stem cell-derived ovarian support cell clinical grade product for in vitro fertilization

first: Christian

last: Kramme

state: New York

address: 450 E 29th Street, Fl 5, Suite 506

institute: Gameto, Inc.

summary: Human induced pluripotent stem cells (hiPSCs) show promise in the development of novel strategies to alleviate reproductive diseases and improve reproductive outcomes. Here, we detail the clinical development and application of an ovarian support cell (OSC) product, Fertilo, to improve the in vitro maturation (IVM) of human oocytes. First, we demonstrate that transcription factor-mediated hiPSC differentiation produces OSCs that improve the oocyte MII maturation rate. To support clinical application, we describe raw material upgrades and the generation of clinically-suitable hiPSC Seed and Master Cell Banks, with transcriptomic analysis of resultant OSCs showing consistent and reproducible outcomes. Next, we detail analytical release testing and development of a murine oocyte maturation assay to establish product potency. Finally, application of Fertilo in a longitudinal cohort analysis shows improvement in key outcomes compared to traditional IVM. Our findings demonstrate the first-time clinical development and application of an hiPSC-derived product to promote successful reproductive outcomes.

overall_design: Following batch production and cryopreservation, OSCs (final product) are analyzed by scRNA-seq

first: Bruna

last: Paulsen

first: Ferran

last: Barrachina

first: Sabrina

last: Piechota

first: Alexander

last: Noblett

first: Mark

last: Johnson

first: Simone

last: Kats

first: Cassandra

last: Lew

first: Maria

last: Marchante

first: Alexandra

last: Figueroa

first: Itzel

last: Garcia Granada

first: Elizabeth

last: Ingalls Lopez

first: Erick

last: Martinez Martinez

first: Paula

last: Ricra

first: Camila

last: Carlos

first: Jazmin

last: Meza

first: Wendy

last: Montanchez

first: Pilar

last: Pino

first: Cesar

last: Reategui

first: Enrique

last: Noriega

first: Alicia

last: Elias

first: Luis

last: Noriega-Portella

first: Gus

last: Haddad

first: Dina

last: Radenkovic

first: Eugenia

last: Moran

first: Pamela

first: Jose

last: Guiterrez

last: Guzman

first: Pietro

last: Bortolleto

first: David

middle: F

last: Albertini

first: Michel

last: De Vos


title: Stress-related prefrontal activation in Tourette disorder

city: Salt Lake City

first: Philip

last: Moos

state: UT

department: Pharmacology & Toxicology

institute: University of Utah

summary: We performed the first single-nucleus transcriptomic analysis of postmortem DLPFC tissue from individuals with TD and age-matched neurotypical controls. No significant differences in cell numbers were observed between groups. Gene ontology analyses revealed broad upregulation of transcripts related to protein synthesis, most prominently in microglia, oligodendrocytes, and interneurons. Within neuronal clusters, these changes were most pronounced in superficial and middle-layer pyramidal neurons and vasointestinal peptide (VIP)-positive interneurons. Differential expression analyses showed widespread increases in immediate early genes (IEGs) and glucocorticoid-responsive transcripts across all cell types in TD. Furthermore, all cell populations in TD samples exhibited enrichment for genes associated with stress-related psychopathology. These findings implicate the DLPFC as a locus of heightened stress responsivity and disrupted excitatory–inhibitory development in TD.

overall_design: Brain tissue samples from the superior DLPFC (Brodmann area 9, BA9) were obtained from the NIH NeuroBioBank (NBB) Brain Tissue Resource Center (BTR) at Harvard University and nuclei were processed for snRNA-seq using a 10X genomics Chromium pipeline followed by analysis using Seurat.

first: Marco

last: Bortolato


title: LsrL modulates Lsr2-induced chromatin structure to tune biosynthetic gene cluster regulation in Streptomyces venezuelae [RNA-seq]

city: Ann Arbor

first: Vivian

last: Ramirez

state: MI

address: 1150 W. Medical Center Dr

department: Department of Computational Medicine and Bioinformatics

institute: University of Michigan

summary: Specialized biosynthetic gene clusters in Streptomyces are subject to complex regulation involving both transcriptional control and chromosome organization. The nucleoid-associated protein Lsr2 silences many of these clusters, yet how it shapes the global chromatin structure and how its conserved paralog LsrL contributes to this process remain poorly understood. In this study, we applied a multi-omics approach, combining transcriptional activity, genome-wide protein-DNA binding profiles, and three-dimensional chromosome conformation to characterize the coordination of Lsr2 and LsrL in exerting transcriptional control and genome architecture in Streptomyces venezuelae. In line with established Lsr2 functions, we find that Lsr2 sets broad transcriptional boundaries, while LsrL acts in a more context-specific manner that depends on the presence of Lsr2 and may function to reinforce or modulate Lsr2-mediated silencing. Loss of Lsr2 reshaped the chromatin landscape genome-wide, relieving its restriction on short-range contacts, triggering strong transcriptional changes and new domain boundaries near de-repressed BGCs. These findings establish Lsr2 as a dominant but contextually modulated regulator whose interplay with LsrL coordinates specialized metabolism with higher-order chromosome organization.

overall_design: RNA-seq was performed to characterize genome-wide transcriptional changes resulting from deletion of lsr2, lsrL, and/or lsr2/lsrL in Streptomyces venezuelae. Wild-type, Δlsr2, ΔlsrL, and Δlsr2ΔlsrL cultures were grown in MYM medium at 30 °C to 16h. Total RNA was isolated. Three independent biological replicates were included for Wild-type, Δlsr2, and Δlsr2ΔlsrL strains, and two independent biological replicates were included for ΔlsrL strain. Differential expression analysis was performed using Kallisto and Sleuth for each mutant strain relative to the Wild-type control.

first: Lauren

last: Tiller

first: Marcin Jan

last: Szafran

first: Xiafei

last: Zhang

first: Jakimowicz

last: Dagmara

first: Marie

last: Elliot

first: Lydia

last: Freddolino


title: LsrL modulates Lsr2-induced chromatin structure to tune biosynthetic gene cluster regulation in Streptomyces venezuelae [IPOD-HR]

overall_design: IPOD-HR was performed to characterize RNA polymerase protein occupancy changes resulting from deletion of lsr2, lsrL, and/or lsr2/lsrL in Streptomyces venezuelae. Wild-type, Δlsr2, ΔlsrL, and Δlsr2ΔlsrL cultures were grown in MYM medium at 30 °C to 16h. Three independent biological replicates were included for Wild-type, Δlsr2, and Δlsr2ΔlsrL strains, and two independent biological replicates were included for ΔlsrL strain.


title: Multi-omics Approaches Revealing Bile Acid Metabolism Dysregulation in Abdominal Aortic Aneurysm Pathogenesis

city: Zhengzhou

first: Shuai

last: Cheng

address: No.1, East Jian She Road

institute: The First Affiliated Hospital of Zhengzhou University

summary: Transcriptional profiling of infrarenal abdominal aortic tissue from male 8-week-old C57BL/6J mice after AAA induction via elastase adventitial incubation combined with β-aminopropionitrile administration, compared with sham-operated mice.

overall_design: Two condition experiment. Sham vs. AAA at day 28 post-operatively. Total 13 samples: 7 sham, 6 AAA.

first: Xinyu

last: Hao

first: Xu

last: Li

first: Linfeng

first: Wei

last: Wang

first: Junfeng

last: Cui

first: Shijie

last: Xin

first: Zhen

first: Lei

first: Hui

last: Cao


title: Glucocorticoid transcriptome in macrophages upon activation [RNA-seq]

city: RTP

first: Thomas

last: Randall

state: NC

address: 101 TW Alexander

institute: NIEHS

summary: Glucocorticoids (GCs) are lifesaving medicines prescribed to treat inflammatory diseases. GCs act on numerous cell types and tissue of the body, however, one of the most important effects are their ability to suppress the immune system. Synthetic glucocorticoids, exemplified by dexamethasone (Dex) and prednisone, rank among the most widely prescribed anti-inflammatory and immunomodulatory drugs globally. The therapeutic efficacy of glucocorticoids extents across a broad spectrum of immune diseases, including conditions like acute and chronic inflammation. The action of the glucocorticoids in immune cells is mediated by the glucocorticoid receptor (here after GR; encoded by Nr3c1 gene) that is a member of the nuclear receptor superfamily of ligand dependent transcription factors. Macrophages emerge as central targets for the anti-inflammatory actions of glucocorticoids during periods of inflammation. Macrophages play a pivotal role during hyperinflammation and cytokine storm, two processes intricately linked to NLRP3 inflammasome activation in human diseases. Upon activation macrophages undergo a process of transcriptional reprogramming to resolve inflammation and restore homeostasis. To elucidate how glucocorticoids impact the transcriptome of macrophages upon activation, RNA-seq was performed on RNA isolated from macrophages unpolarized (M0) and polarized to M1 and M2 profile from WT and GRKO treated with vehicle and Dex. In addition, M0 and LPS M1-like macrophages were treated with vehicle and Dex and then analyzed by Cut&Tag.

overall_design: Bone marrow derived macrophages (BMDMs) were obtained from 8- to 12-wk-old WT (GR-Flox) and GRKO mice. For polarization into M1-like and M2-like, BMDMs unpolarized (M0) were stimulated for 24 hours with 100 ng/mL of LPS for M1-like, and 10 ng/mL of murine recombinant IL-4 for M2-like, all in 10% charcoal-stripped serum medium. M0, M1-like and M2-like macrophages from WT and GRKO were stimulated with vehicle or 100nM Dex for 4 hours for gene expression. Total RNA was isolated from macrophages using Qiagen RNeasy Mini kit. A TruSeq RNA kit (Illumina, San Diego, CA) was utilized to prepare the poly(A)-enriched RNA-seq libraries that were sequenced on the Illumina NovaSeq 6000 in a 75-base paired-end mode according to the manufacturer’s protocol. For Cut&Tag  experiments, BMDM from vehicle, LPS treated for 4 hours; 4 hours Dex after LPS-primed and 4 hours LPS and Dex as a co-treatment were harvested. Cells were treated, harvested, and subjected to CUT&TAG analysis using a modified published protocol [Kaya-Okur et al., 2019].  Trypsinized cells were washed once with PBS and incubated with NE nuclear extraction buffer (20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol, 0.5 mM Spermidine, 100 uM PMSF, and 1x EDTA-free protease inhibitors (Roche cOmplete) for 10 min on ice.  Samples were centrifuged for 3 min at 600g, nuclei pellet resuspended in NE buffer supplemented with 0.1 % formaldehyde and incubated at room temperature for 2 minutes followed by quench with 0.125 M glycine.  Following formaldehyde cross-link, nuclei were re-centrifuged, resuspended in fresh NE buffer and counted with trypan blue dye using Bio-Rad TC20 Automated Cell Counter. 100,000 total nuclei [90,000 nuclei from experimental sample with 10,000 human spike-in nuclei] were incubated with activated concanavalin A-coated (ConA) magnetic beads (EpiCypher) in 0.2 ml thin-walled tubes for 10 min at room temperature.  Nuclei-bound ConA beads were resuspended in antibody incubation buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.01% Digitonin, 2 mM EDTA, 0.5 mM Spermidine, 100 uM PMSF, and 1x EDTA-free protease inhibitor), 0.5 ug primary antibody added and samples incubated overnight at 4C with gently rotation. Samples were incubated with 0.5 ug species specific secondary antibody for 30 min at room temperature with gentle rotation followed by two washes with Digitonin150 wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.01% Digitonin, 0.5 mM Spermidine, 100 uM PMSF, and 1x EDTA-free protease inhibitor) then incubation with pAG-Tn5 in Digitonin300-wash buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.01% Digitonin, 0.5 mM Spermidine, 100 uM PMSF, and 1x EDTA-free protease inhibitor) for 1h at room temperature.   After pAG-Tn5 incubation, bound ConA beads were washed twice with Digitonin300-wash buffer then resuspended in Tagmentation buffer (Digitonin300 wash buffer containing 10 mM MgCl2) and incubated for 1h at 37C.  Beads were then pelleted, resuspended in TAPS buffer (10 mM TAPS pH 8.5, 0.2 mM EDTA).  TAPS buffer removed and beads carefully resuspended in SDS Release buffer (10 mM TAPS pH 8.5, 0.1% SDS) and incubated for 1 h at 58 C.  Following incubation, SDS Quench buffer (0.67% Triton X-100) was added to each sample along with 2 μl of a universal i5 and a uniquely barcoded i7 primers (from 10 μM stocks).  Equal volume (25 ul) NEBNext High-Fidelity 2x PCR Master mix (M0541, New England Biolabs) was added to each sample and mixed by gentle pipetting.  Samples subjected to DNA amplification using a thermocycler and the CUT&TAG-specific PCR cycling parameters: 58C for 5 min; 72C for 5 min; 98C for 45 sec; with 19 cycles of 98C for 15 sec and 60C for 10 sec; final extension at 72 °C for 1 min; and hold at 4C.  DNA clean-up performed by adding 1.3x AMPure XP beads (A63880, Beckman Coulter) to each sample, allowing libraries to incubated with beads for 10 min at room temperature, followed by two washes with 80% ethanol, then elution in 15 μl of 10 mM Tris pH 8.0.  While on magnetic stand, supernatant was carefully taken and transferred to a fresh tube.  Library size distribution determined by Agilent TapeStation 4200 (Agilent Technologies) and libraries mixed to yield equal representation prior to paired-end (2 x 50 bp) Illumina sequencing by NIEHS The Epigenomics and DNA Sequencing Core Facility.

first: David

last: Diaz-Jimenez

first: Jackson

last: Hoffman

first: Jianying

first: John

last: Cidlowski


title: A precision medicine approach to interpret a GATA4 genetic variant in a paediatric patient with congenital heart disease [RNA-seq]

city: Nedlands

first: Kevin

middle: Guangxing

last: Chen

address: Hospital Avenue

institute: The Kids Research Institute Australia

summary:
Background. Patients with congenital heart disease are identified in 1% of live births, and with improved surgical intervention many patients now survive to adulthood, the corollary of which is increased mortality in the over-65-year-old CHD population. In the clinic, genetic sequencing increasingly identifies novel genetic variants in genes related to congenital heart disease (CHD). Traditional assays for interpreting novel genetic variants are often limited by gene-specificity, whereas animal models are cumbersome and may not accurately reflect human disease. This study investigates CRISPR gene editing in induced pluripotent stem cells and cardiomyocyte-directed differentiation as a human disease model to investigate novel genetic variants identified in association with CHD.
Methods and Results. The GATA4 p.Arg284His genetic variant was introduced into iPSCs using high efficiency CRISPR gene editing with homology-directed repair. GATA4 genetic variant and isogenic control cell pairs were selected and differentiated into cardiomyocytes. Expression of the GATA4 p.Arg284His variant resulted in altered calcium transient’s indicative of CHD and consistent with the patient’s clinical phenotype. Transcriptomics revealed molecular and cellular pathway changes in cardiac development, calcium handling, and energy metabolism that contribute to disease aetiology and mechanism, and identified potential treatments that could restore normal cell function.
Conclusion. Directed differentiation of iPSCs harbouring the GATA4 p.Arg284His genetic variant recapitulated the CHD phenotype, indicated disease mechanisms, and pointed to potential treatments. The study highlights the utility of transcriptomics for the functional interpretation of cardiac genetic variants and is an exemplar for precision medicine approaches for the investigation of CHD.

overall_design: To investigate the functional consequences of a variant of unknown significance (VUS), we used CRISPR gene editing to induce the GATA4 VUS into iPSCs, and differentiated these into cardiomyocytes. We compared the transcriptomic profile of the VUS lineage with a WT lineage.

first: Catherine

last: Forbes

first: Nicole

last: Shaw

first: Mitchell

last: Hedges

first: Teagen

middle: S

last: Er

first: Livia

last: Hool

first: Michelle

last: Ward

first: Cathryn

last: Poulton

first: Gareth

last: Baynam

first: Timo

last: Lassmann

first: Vanessa

last: Fear


title: Targeting metabolic vulnerabilities: REV-ERB agonist SR9009 potentiates sorafenib efficacy in liver cancer

city: Ferrara

first: Cristian

last: Bassi

state: Ferrara

address: Via Fossato di Mortara 70

department: Medicina Traslazionale e per la Romagna

institute: Università di Ferrara

summary: Hepatocellular carcinoma (HCC) is one of the most common cancers and the third leading cause of cancer-related death worldwide. The prognosis is poor, with a median survival of 12–15 months in patients with advanced-stage disease. Early diagnosis and the development of new, more effective therapeutic strategies are needed to address the challenges posed by this malignancy. Although immune checkpoint inhibitors have replaced multikinase inhibitors as first-line therapy, sorafenib continues to represent a valuable option for patients with contraindications to newer treatments. Based on genome-wide RNA-seq analysis, which identified mitochondrial oxidative phosphorylation (OxPhos) and HMOX1 upregulation as potential pro-survival mechanisms in sorafenib-resistant cells, we investigated whether SR9009, a synthetic agonist of the nuclear receptor REV-ERBα/β, heme competitor, and inhibitor of mitochondrial respiration, could enhance the antitumor efficacy of sorafenib in liver cancer models. Co-treatment with SR9009 and sorafenib significantly enhanced cytotoxic effects in both mouse and human liver cancer cells. This synergistic activity was associated with increased levels of free heme and a complete inhibition of mitochondrial OxPhos. In vivo xenograft studies confirmed that the combination was effective even in sorafenib-resistant tumors. Furthermore, in a N-Nitrosodiethylamine (DEN)-induced HCC model, the combination therapy led to a reduction in size in over 90% of tumor nodules, representing a significant improvement over sorafenib alone. The combination was well tolerated, with no evident signs of acute toxicity. These findings support the concept that the efficacy of anticancer therapies can be enhanced by targeting the metabolic adaptations that tumor cells rely on for survival. Combining sorafenib with agents like SR9009, that disrupt metabolic homeostasis, may offer a promising strategy for treating advanced HCC.

overall_design: RNA sequencing was conducted on 5 samples derived from H55-RES sorafenib resistant cell-lines, 2 samples derived from H55.1C wild-type sorafenib sensitive cells, 7 samples derived from H55.1C wild-type cells exposed to sorafenib at different time-points (0-24h), 1 sample derived from H55.1C wild-type cells exposed to sorafenib and SR9009


title: Effect of SMURF2 depletion on M-CSF-treated BMDM.

city: Hangzhou City

first: Xiang

last: An

state: Zhejiang Province

address: 866 Yuhangtang Road

institute: Zhejiang University

summary: Dysregulated tissue-resident macrophages (TRMs) contribute to pathogenesis of inflammatory bowel disease (IBD) and multiple sclerosis (MS), yet the molecular pathways that regulates TRMs remain unclear. Smurf2 deficiency significantly exacerbates the TRM proliferation in dextran sulfate sodium (DSS)-induced colitis and experimental autoimmune encephalomyelitis (EAE), leading to augmented autoimmune inflammation.

overall_design: RNA-seq profiling of M-CSF-treated bone marrow-derived macrophages (BMDMs) from SMURF2f/f (WT) and LysM-SMURF2f/f (MKO) mice.

first: Wenlong

last: Lin

first: Xiaojian

last: Wang


title: Time-Series Gene Expression of Drosophila melanogaster under Different Temperatures

city: College Station

first: Wanhe

last: Li

state: Texas

address: 424 Nagle St

department: Biology

institute: Texas A&M University

summary: This project investigates rhythmic gene expression in Drosophila melanogaster under different temperature and light conditions. RNA sequencing was performed on adult male fly heads collected every 4 hours over two consecutive days. Samples were collected under constant dark (DD) or light/dark (LD) cycles at 18C, 25C, and 29C. The dataset enables analysis of circadian and temperature-dependent transcriptional regulation in Drosophila.

overall_design: RNA-Seq was performed on adult male fly heads collected every 4 hours for two consecutive days under different environmental conditions: constant darkness (DD) at 18C, 25C, and 29C, and a 12:12 light/dark cycle (LD) at 25C. Two biological replicates were collected per timepoint. PolyA-selected RNA libraries were sequenced using paired-end Illumina NovaSeq.


title: Developmental Stage-Dependent Transcriptomic Responses to Neonatal Intraventricular Hemorrhage

city: Charleston

first: Stefano

last: Berto

state: SC

address: 68 President Street, BE208

institute: MUSC

summary: Neonatal intraventricular hemorrhage (IVH) is a major complication of preterm birth, yet how developmental stage influences the brain’s response to injury remains unclear. We performed single-nucleus RNA sequencing on rat brains 24 hours after IVH at postnatal day 2 (PND2) or day 5 (PND5) to define transcriptional responses across cell types. We identified 42 distinct cell populations and found that PND5 brains exhibited a markedly stronger immune and inflammatory response to IVH, with a threefold increase in differentially expressed genes compared to PND2. Microglia were the most perturbed cell type at both stages, showing increased oxidative stress and polarization toward both pro- and anti-inflammatory phenotypes at PND5. Ligand-receptor and regulon analysis revealed a shift from reparative IGF2 and TGF-β signaling at PND2 to proinflammatory Wnt signaling and activation of Runx1 and Stat5 at PND5. These findings highlight the importance of developmental timing in shaping the neuroimmune response to IVH and identify potential stage-specific therapeutic targets.

overall_design: Using the single-nucleus RNA sequencing (snRNA-seq) platform from 10x Genomics, we obtained transcriptomic data from 161,933 nuclei from 32 rat brains. Rats underwent IVH at postnatal day 2 and postnatal day 5, and their brains were harvested for analysis 24 hours later. The experiment used 32 rats overall, with 16 for each timepoint. Of these 16, 8 were control and 8 underwent IVH at each timepoint. Four of each set were male and the other 4 were female.

first: Elizabeth

last: Wallace-Anthony

first: Miriam

last: Zamorano

first: Hemendra

last: Vekaria

first: Braden

last: Oldham

first: Kiara

last: Umpornpun

first: Scott

last: Olson

first: Brandon

last: Miller


title: A precision medicine approach to interpret a GATA4 genetic variant in a paediatric patient with congenital heart disease [AmpSeq]

first: Kevin

last: Chen

summary: Background. Patients with congenital heart disease are identified in 1% of live births, and with improved surgical intervention many patients now survive to adulthood, the corollary of which is increased mortality in the over-65-year-old CHD population. In the clinic, genetic sequencing increasingly identifies novel genetic variants in genes related to congenital heart disease (CHD). Traditional assays for interpreting novel genetic variants are often limited by gene-specificity, whereas animal models are cumbersome and may not accurately reflect human disease. This study investigates CRISPR gene editing in induced pluripotent stem cells and cardiomyocyte-directed differentiation as a human disease model to investigate novel genetic variants identified in association with CHD. Methods and Results. The GATA4 p.Arg284His genetic variant was introduced into iPSCs using high efficiency CRISPR gene editing with homology-directed repair. GATA4 genetic variant and isogenic control cell pairs were selected and differentiated into cardiomyocytes. Expression of the GATA4 p.Arg284His variant resulted in altered calcium transient’s indicative of CHD and consistent with the patient’s clinical phenotype. Transcriptomics revealed molecular and cellular pathway changes in cardiac development, calcium handling, and energy metabolism that contribute to disease aetiology and mechanism, and identified potential treatments that could restore normal cell function. Conclusion. Directed differentiation of iPSCs harbouring the GATA4 p.Arg284His genetic variant recapitulated the CHD phenotype, indicated disease mechanisms, and pointed to potential treatments. The study highlights the utility of transcriptomics for the functional interpretation of cardiac genetic variants and is an exemplar for precision medicine approaches for the investigation of CHD.

first: Catherine

last: Forbes

first: Nicole

last: Shaw

first: Mitchell

last: Hedges

first: Teagen

last: Er

first: Livia

last: Hool

first: Michelle

last: Ward

first: Cathryn

last: Poulton


title: Innate lymphoid cells in unaffected colon, colorectal cancer and peritoneal metastasis

city: Huddinge

first: Anne

last: Marchalot

address: Alfred Nobel Allé 8, Center for Infectious Medicine

country: Sweden

institute: Karolinska Institutet

summary: 10X Next GEM Single Cell 5’ Kit v2 scRNAseq of maximum 15 000 sorted CD127+ ILCs (CD45+, CD3-, CD19-, Lin- and CD127+), NK cells (CD45+, CD3-, CD19-, Lin-, CD94+/- and CD56+) and non-conventional (nc)ILCs (CD45+, CD3-, CD19-, Lin-, CD94+/-, CD56- and CD7+) per sample from 11 patients. Total of 4 paired healthy colon and colorectal cancer tumours, 2 paired healthy colon, colorectal cancer tumours and peritoneal metastasis and 5 unpaired peritoneal metastasis.

overall_design:
scRNA-seq profiling of ILC and NK cells from primary colorectal tumors, peritoneal metastasis and unafected colon

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

first: Jenny

last: Mjösberg


title: INO80 regulates promoter-associated R-loops to coordinate transcription and maintain genome stability in embryonic stem cells

city: Seoul

first: Kwonho

last: Hong

address: 120 Neungdong

country: South Korea

institute: Konkuk university

summary: Embryonic stem cells (ESCs) possess a distinctive cell cycle structure and chromatin landscape that necessitate precise coordination between transcription and DNA replication. The chromatin remodeler INO80 regulates both transcription and genome stability; however, its role in R-loop homeostasis in ESCs remains incompletely understood. R-loops, which are formed by RNA–DNA hybrids, play regulatory roles but can also induce replication stress and genomic instability if unregulated. We investigated the effects of INO80 loss on R-loop dynamics, replication stress responses, and transcriptional regulation in mouse ESCs. INO80-deficient cells exhibited a global reduction in steady-state R-loops under normal conditions, with the most pronounced loss at promoters enriched for pluripotency-associated transcription factors. R-loop levels were restored under hydroxyurea-induced replication stress, indicating that INO80 is dispensable for stress-induced R-loop accumulation but is required for maintaining physiological R-loops. Cleavage Under Targets and Tagmentation and chromatin immunoprecipitation sequencing revealed that several R-loop regions overlap with INO80-binding sites and are linked to transcriptional activation and genome maintenance. Transcriptome analysis showed that INO80-stabilized R-loops are associated with genes involved in cell cycle progression, DNA repair, and chromatin organization, whereas INO80 loss results in altered transcriptional programs, G1 arrest, and increased apoptosis. These findings indicate that INO80 stabilizes functional R-loops that support transcriptional regulation and genomic integrity. Our study identifies a previously unrecognized role for INO80 in preserving functional R-loops in ESCs. By stabilizing promoter-associated R-loops and coordinating transcription during cell cycle progression, INO80 maintains both transcriptional regulation and genome stability. These findings underscore the importance of chromatin remodeling in maintaining R-loop homeostasis and suggest that INO80 dysfunction may have implications for stem cell identity and genomic integrity.

overall_design: R-loop CUT&Tag libraries generated from mouse embryonic stem cells (Mus musculus). Two experimental conditions were examined: control cells harboring floxed alleles of Ino80 (CON) and knockout cells with Ino80 deletion (KO). Each condition was analyzed in two biological replicates, resulting in a total of four samples. The study was designed to compare R-loop landscapes between control and INO80-deficient embryonic stem cells, with the control group serving as a reference for the knockout condition.

first: Hoseong

last: Lim

first: Ohbeom

last: Kwon


title: LsrL modulates Lsr2-induced chromatin structure to tune biosynthetic gene cluster regulation in Streptomyces venezuelae [ChIP-seq]

first: Vivian

last: Ramirez

overall_design: RNA polymerase ChIP-seq was performed to characterize RNA polymerase protein occupancy changes resulting from deletion of lsr2, lsrL, and/or lsr2/lsrL in Streptomyces venezuelae. Wild-type, Δlsr2, ΔlsrL, and Δlsr2ΔlsrL cultures were grown in MYM medium at 30 °C to 16h. Three independent biological replicates were included for Wild-type, Δlsr2, and Δlsr2ΔlsrL strains, and two independent biological replicates were included for ΔlsrL strain.

first: Lauren

last: Tiller

first: Marcin Jan

last: Szafran

first: Xiafei

last: Zhang

first: Jakimowicz

last: Dagmara

first: Marie

last: Elliot

first: Lydia

last: Freddolino


title: Effect of depletion of CYP4F11 on proliferation and migration in lung cancer cell lines NCI-H460 cell line.

city: Pittsburgh

first: Simone

last: Brixius-Anderko

state: PA

address: 335 Sutherland Dr

department: Pharmaceutical Sciences

institute: University of Pittsburgh

summary: Lung cancer remains the leading cause of cancer deaths worldwide. Drug resistance in lung cancer patients often limits treatment effectiveness, underscoring the need for novel therapeutic targets. In our recent work, we identified the cytochrome P450 4F11 (CYP4F11) as a promising therapeutic target for lung cancer. CYP4F11 belongs to the CYP4F family of fatty acid omega-hydroxylases catalyzing the conversion of arachidonic acid to the potent lipid mediator 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is a long-known key player in cancer cell proliferation and migration. CYP4F11 is highly overexpressed in patients with lung cancer. Through genetic modification studies in lung cancer cell lines, we found that CYP4F11 indeed mediates the proliferation and migration through 20-HETE production. However, it is currently unknown how CYP4F11 contributes to lung cancer progression and what the underlying mechanism is. Our data identify differentially expressed genes in CYP4F11-knockdown lung cancer cells compared to control cells via RNA sequencing. We provide better understanding on the cellular mechanism involving CYP4F11 in lung cancer.

overall_design: RNA-seq profiling of wildtype NCI-H469 cells and ther knockdown derivatives (siCYP4F11) 24h post transfection.

first: Huiting

last: Jia


title: Sepsis–induced skeletal muscle wasting is ameliorated by the pharmacological inhibition of STAT3 signaling in mice [RNA-seq]

city: Kobe

first: Yuko

last: Ono

state: Hyogo

address: Kusunoki-cho 7-5-2

department: Department of Disaster and Emergency Medicine

institute: Kobe University

summary: Septic patients frequently develop skeletal muscle wasting and weakness, which adversely affects survival and functional outcomes. Currently, no drugs are available for the treatment of sepsis-associated skeletal muscle wasting. Here, we tested the effects of C188-9, a STAT3-specific signaling inhibitor, on muscle wasting in vivo and myotube atrophy in vitro induced by sepsis. In mice, sepsis severity-dependently phosphorylated STAT3, which subsequently activated the ubiquitin–proteasome and autophagy proteolytic pathways, resulting in skeletal muscle atrophy. Similar dose-dependent responses were observed in lipopolysaccharide-treated C2C12 myotubes. In human septic patients, plasma interleukin-6 levels positively correlated with the extent of sepsis-associated skeletal muscle wasting. STAT3 inhibition in mice or cells treated with C188-9 suppressed activation of the ubiquitin–proteasome degradation pathway—but not autophagy pathways—and alleviated sepsis-associated skeletal muscle wasting. Overall, our results indicate that pharmacological inhibition of STAT3 signaling may represent a novel therapeutic strategy for sepsis-associated skeletal muscle wasting.

overall_design: RNA sequencing profiling was performed on wild-type male sham control and septic C57BL/6 mice (8–10 weeks old, weighing 22–26 g). To induce polymicrobial sepsis, mice received an intraperitoneal (i.p.) injection of cecal slurry (CS) at 1.0 mg/g body weight using a 1-mL syringe and a 27G needle (septic mice). Control mice were injected with an equal volume of vehicle (7.5% glycerol-PBS). After 24 hours of treatment, tibialis anterior muscles were removed and subjected to RNA sequencing analysis.

first: Masafumi

last: Saito

first: Ikumi

last: Yoshihara

first: Yutaka

last: Kondo

first: Kazuho

last: Sakamoto

first: Jun

last: Sugiyama

first: Nobuto

last: Nakanishi

first: Shigeaki

last: Inoue

first: Joji

last: Kotani


title: A single-cell CRISPR screen defines a gene regulatory network governing human pluripotency in primed and naïve cells

city: Jerusalem

first: Gal

last: Keshet

state: Yerushalayim

address: Institute of Life Sciences, Givat Ram

department: Genetics

country: Israel

institute: The Hebrew Univerisy

summary: Human pluripotent stem cells (hPSCs) can give rise to the three embryonic germ layers and possess a limitless propagation potential. We have previously defined the essentialome of hPSCs through a genome-wide CRISPR screen, but the function of each essential gene is still obscure. Here, we uncovered the role of pluripotent-specific transcription factors (TFs) that are essential in hPSCs, using a pooled CRISPR screen combined with single-cell transcriptomics. We show that most of these TFs form a remarkably interconnected gene regulatory network (GRN), controlling multiple aspects of pluripotency, spanning regulation of differentiation, self-renewal, cell survival, and expression of transposable elements. Interestingly, various TFs function as positive regulators of pluripotency, serving as gatekeepers from differentiation, and preventing transition into different cell lineages in a TF-specific manner. Surprisingly, other TFs inhibit the pluripotency network, potentially balancing its maintenance with responsiveness to differentiation cues, which we could validate through individual gene knockouts. Finally, by perturbing the TFs network in naïve cells, we reveal both shared and distinct roles for the GRN between the two pluripotency states. In summary, this analysis enabled the construction of an extended network for human pluripotency. This study sheds new light on principles in early human development and can hopefully be used to enhance the use of hPSCs for disease modelling or cell therapy.

overall_design: For the primed perturb-seq experiment, Cas9-EGFP hPSCs cells were dissociated using TrypLE Select (Thermo Fisher Scientific), centrifuged, and resuspended in mTeSR1 medium (STEMCELL Technologies) supplemented with 10 μM ROCK inhibitor (Y27632) and 8 μg/ml polybrene (Sigma). A total 3.6X106 cells were transduced in suspension with the virus library at a multiplicity of infection (MOI) of 0.3 and the cells were plated on 3 Matrigel coated 6-well plates (Corning). 24h after transduction, the virus containing medium was replaced with puromycin-containing mTeSR1 medium (0.3 mg ml−1, Sigma). The medium was subsequently replaced daily with puromycin-containing medium until day 6 post transduction, when cells were harvested for 10X genomics. For harvesting, cells were dissociated using TrypLE Select (Thermo Fisher Scientific), and after centrifugation were re-suspended in PBS containing 0.002% Trypan Blue to stain dead cells. Cells were again centrifuged and resuspended in PBS containing 10% KSR and 10 µM ROCK inhibitor Y-27632, filtered through a 70 µm cell strainer (Corning) and sorted using BD FACSAria III (BD Biosciences). Specifically, cells which were GFP+ (positive for Cas9-EGFP), BFP+ (positive for the gRNA library) and far-red negative (Trypan Blue+ cells emit far red signal, enabling the isolation of live cells) were sorted with 488 nm, 405 nm, and 650 nm lasers, and subjected to scRNA-seq using 10X genomics. For the naïve condition, the experimental design of the experiment was adjusted as naïve cells under the 5i/L/A condition proved to be highly resistant to lenti-viral infection. Specifically, primed hPSCs were first infected with the viral library as mentioned above and kept under puromycin selection for 7 days. Then, naïve conversion was performed as mentioned above. On day 18 of the conversion, naïve cells were dissociated using TrypLE Select (Thermo Fisher Scientific) and split into 6 6-well plates with a density of 1.5X105 cells/well. After 24h, the cells were transfected with a pSpCas9(BB)-2A-GFP (PX458) construct (a gift from Feng Zhang; addgene plasmid #48138) in the presence of Lipofectamine Stem Transfection Reagent (Invitrogen; STEM00003). 5 days after the transfection, cells were sorted based on the same criteria as in the primed experiment and subjected to scRNA-seq using 10X genomics.

first: Nissim

last: Benvenisty


title: Single-molecule tracking in living cells reveals dynamics of DNMT1 through the cell cycle and its redistribution upon drug treatment

city: Boulder

first: Thomas

last: Cech

state: CO

address: 3415 Colorado Avenue

department: Biochemistry

institute: University of Colorado Boulder

type: Methylation profiling by high throughput sequencing

summary: DNMT1 is a methyltransferase that restores 5-methylcytidine marks on newly replicated DNA and is required for maintaining epigenetic inheritance. Using Halo-tagged DNMT1 and highly inclined thin illumination (HiLo) microscopy, we show that DNMT1 mobility in living human cells changes under a variety of conditions. Consistent with its role in methylating DNA, DNMT1 molecules become increasingly bound to chromatin in the S-phase of the cell cycle, but surprisingly a 4% increase in chromatin bound DNMT1 is sufficient to maintain DNA methylation. In G2 phase, however, DNMT1 is released from DNA and becomes even more dynamic. Upon treatment with small molecule inhibitors, GSK-3484862 (GSK) and 5-azacytidine (5-azaC), the in vivo DNMT1 dynamics are greatly altered. Unexpectedly, treatment of cells with GSK, a non-covalent inhibitor, causes binding of DNMT1 to chromatin similar to that observed upon treatment with 5-azaC, a covalent inhibitor. 5-azaC inhibition of DNMT1 dynamics occurs during the S phase of the cell cycle. Collectively, our work using live cell single molecule imaging quantifies the molecular dynamics of DNMT1 and how this relates to its function under physiological (different phases of the cell cycle) and non-physiological (drug inhibition) conditions. Understanding the dynamics of DNMT1 in vivo provides a framework for developing better therapeutics that target DNMT1.

overall_design: Nanopore sequencing genome skimming was used to monitor DNMT1 inhibition in U2OS cells following drug treatment with either DMSO (vehicle), 5-azacytidine, or GSK-3484862 . Following drug treatment, cells were trypsinized and genomic DNA was extracted. Genomic DNA was quantified using ThermoFisher Qubit 1xdsDNA High Sensitivity Assay Kit per manufacturer’s instructions. DNA quality was measured by A260/280 and A260/230 ratios using a ThermoScientific NanoDrop ONEc. DNA size distribution was measured using Agilent Bioanalyzer 2100 DNA 12000 chip per manufacturer’s instructions. 400 ng genomic DNA was carried forward to library preparation. Libraries were prepared using Oxford Nanopore Technologies Native Barcoding Kit 24 V14 (SQK-NBD114.24) per manufacturer’s instructions. Libraries were barcoded, pooled, and loaded onto an Oxford Nanopore Technologies MinION Flow Cell (R10.4.1, FLO-MIN114). Libraries were sequenced for 48 h at a rate of 400-450 bases per second with live basecalling off. Approximately 500 Mb of data per genome were collected, representing ~0.1X genome coverage for an accurate and precise measure of global DNA methylation. For post-sequencing analysis, data were basecalled on the command-line interface of the CU Boulder BioFrontiers Institute FIJI Compute Cluster. Basecalling was performed using Dorado v0.7.4. Reads were aligned to the GRCh38 human genome assembly using Samtools v1.3.1. Methylation data were extracted using the Dorado v0.7.4 modkit pileup command. Downstream analyses were conducted in R version 2024.12.0+467.

first: Eliza

last: Lee

first: Ella

middle: R

last: Tommer

first: Paul

last: Rothman

first: Sarah

last: Middleton

first: Daniel

middle: T

last: Youmans


title: Extracellular vesicles-derived miR-760 and miR-98-5p as differentiating biomarkers for MS phenotypes

city: Wroclaw

first: Lukasz

last: Laczmanski

address: Weigla 12

institute: Hirszfeld Institute of Immunology and Experimental Therapy

type: Non-coding RNA profiling by high throughput sequencing

summary:
Objective.
Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system with heterogenous clinical course, lacking non-invasive biomarkers for early phenotype differentiation. This study aimed to identi-fy circulating extracellular vesicle (EV)-derived miRNA signatures and as-sociated disease-specific molecular markers capable of distinguishing stable relapsing-remitting MS (RRMS) from secondary progressive MS (SPMS).
Materials and methods.
Plasma samples were collected from patients with stable RRMS, SPMS, and healthy controls (HC), followed by EVs isolation and characterization using transmission electron microscopy, dynamic light scattering, and flow cytometry. RNA was extracted from EVs, and miRNA profiles were ana-lyzed via RNA sequencing and RT-qPCR. Cytokine and neurodegeneration marker levels were quantified using the BioPlex® multiplex system and ELISA. Functional enrichment and network analyses of miRNA targets were performed, alongside logistic regression modeling to assess diagnostic utility.
Results.
Four EV-derived miRNAs (miR-760, miR-98-5p, miR-301a-3p, and miR-223-3p) showed significant differences between stable RRMS and SPMS. The integrative model incorporating these miRNAs with FGF basic protein achieved excellent discrimination between MS phenotypes (AUC = 0.97). Moreover, miR-760 emerged as the most robust predictive biomarker for stable RRMS classification. In turn, miR-98-5p showed the highest up-regulation in both stable RRMS and SPMS relative to HC. miRNA targets network analysis further revealed distinct immunoregulatory patterns for stable RRMS and SPMS.
Conclusions.
Plasma-derived EV-miRNAs, particularly miR-760 and miR-98-5p, showed strong potential as non-invasive biomarkers for stable RRMS and SPMS phenotypes. Integrating EV-miRNA profiling with protein bi-omarkers enhances diagnostic precision and may support personalized management strategies in MS.

first: Karina

last: Maciak

first: Angela

last: Dziedzic

first: Elżbieta

last: Miller

first: Łukasz

last: Łaczmański

first: Radosław

last: Zajdel

first: Sylwia

last: Michlewska

first: Dorota

last: Kujawa

first: Marta

last: Gancarek

first: Patrycja

last: Nowak

first: Joanna

last: Saluk


title: Transcriptional levels of immune activation genes were altered in the tumor post circRNACRT-E7E6 treatment

city: Beijing

first: Rong

last: Zhou

state: Beijing

address: No. 9 North Yongteng Road

institute: Therorna

summary: Immunization with circRNACRT-E7E6 vaccines have proved delayed tumors growth in mice and promoted a strong infiltration of antigen-specific T cells into the tumor site. We therefore sought to verify whether these efficacies were corroborated at the transcriptomic level within bulk tumor samples. Accordingly, RNA sequencing (RNA-seq) was conducted on TC-1 tumor samples from mice administered circRNACRT-E7E6 and from empty LNP controls. Gene Ontology (GO) analysis and Gene set enrichment analysis (GSEA) suggested enhanced immune activation and anti-tumor T cell response in circRNACRT-E7E6 administrated mice.

overall_design: C57BL/6 mice were subcutaneously inoculated with 2.5×10^⁵ TC-1 tumor cells. Twelve days post-inoculation, when average tumor volume reached approximately 100 mm³, mice were randomized into two groups (n=5 per group). The negative control group received intramuscular (IM) injections of empty LNP on Days 0, 3, and 7. The experimental group received IM injections of circRNACRT-E7E6 (2 µg) on the same schedule. Five days after the final treatment, tumor samples were collected, processed according to controlled standard operating procedures, and subsequently submitted for RNA sequencing (RNA-seq) to determine whole tumor transcriptomes.

first: Jing

last: Zeng

first: Kunlun

last: Xiang

first: Chonghui

last: Li

first: Minliang

last: Zhu

first: Lu

last: Gao


title: LsrL modulates Lsr2-induced chromatin structure to tune biosynthetic gene cluster regulation in Streptomyces venezuelae

first: Vivian

last: Ramirez

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

overall_design: Refer to individual Series


title: gene expression profiling identifies ferroptosis-related genes in human colon cancer cell lines

first: NIEHS

last: Microarray Core

address: 111 T.W. Alexander Drive

department: DIR

summary: We used microarray gene expression profiling to identify ferroptosis related biomarker genes in colon cancer cells and several uniquely expressed gene were identified that were highly expressed during ferroptosis induced by Erastin.

overall_design: Various colon cancer cells were treated with Erastin for 4h and 24h and RNA was isolated and purified. Gene microarray were carried out and analysis were performed for identifications of gene expression.

first: B

middle: K

last: Sinha

first: Erik

last: Tokar


title: ATGL suppresses ferroptosis in acute myeloid leukemia cells via modulating the CEBPα/SCD1 axis and induces gilteritinib resistance.

city: Chongqing

first: x

last: q

address: 76 Linjiang Road, Chongqing

institute: Department of Hematology, The Second  Affiliated Hospital, Chongqing Medical  University

summary: Metabolic reprogramming disrupts energy homeostasis and promotes tumor cell proliferation.  We here report PNPLA2/ATGL as a key regulator of lipid metabolism in progress of AML.The depletion of ATGL will slow the malignant progression of AM L through the plasma metabolic pathway.Our data provide transcriptional data from an AML in which ATGL is missing from the mass metabolism direction.

overall_design: RNA-seq profiling of knockdown of ATGL on gene expression  of MV4-11 cells (shNC, shATGL)


title: Multi-omics Analysis Revealed Unique Features of Age-associated Type2 CD8 Memory T cells [array]

city: Kyoto

first: Hiroyuki

last: Matsui

address: 54 Shogoin-Kawahara-cho

institute: Kyoto University Graduate School of Medicine

summary: Aging impacts immune function, yet how and why various subsets of immune cells change during aging is still incompletely understood. Here, we analyzed peripheral blood mononuclear cells (PBMCs) from a cohort of healthy donors aged 20–82 years to uncover previously unrecognized age-dependent changes, using a 36-color spectral flow cytometry panel focused on T cells. We found that CXCR3- memory CD8 T cells, which produce a Th2-like cytokine response, accumulate with age. Comprehensive multi-omics analysis revealed transcriptional and epigenetic signatures that support Th2-like function. Importantly, we also identified an age-dependent bias towards the production of Th2 cytokines after activation in naive CD8 T cells, along with transcriptional and epigenetic changes supporting this outcome. Moreover, health outcome association analysis demonstrated a pathogenic feature of CXCR3- CM accumulation. Together, our results suggest that age-dependent corruption of epigenetic programming may cause the aberrant emergence of this potentially pathogenic Th2-like immune population

overall_design: This is a part of the multi-omics data set to comprehensively characterize age-related CD8 T cell subsets. DNA methylation array of FACS sorted 2 subsets of human CD8 T cells (CXCR3- CM, CXCR3+ CM) was performed.

first: Alan

last: Tomusiak

first: Varun

last: Dwaraka

first: Eric

last: Verdin


title: Dissecting the development of bovine testicular tissue using spatial transcriptomics

city: Yinchuan

first: Jin

last: Haoyan

state: NingXia

address: No. 489, West Helanshan Road

department: colllege of animal science and technology

institute: NingXia University

summary: Mammalian spermatogenesis is critical for the transmission of male genetic information, and single-cell sequencing technology can reveal its complex process. However, at present, there is no research on the dynamic transcription of bovine germ cell population. In this study, we used Stereo-seq to construct a spatial transcription map of bovine testicular tissue at two ages. Four germ cell groups and five somatic cell groups were determined, and functional enrichment characterized their different biological functions and the differences between calves and adult bovines. At the same time, we also defined the subpopulations of cells and marker genes, then, clarified the communications between germ cells. These data laid the foundation for the study of spermatogenesis in large mammals and elucidated the process of transcriptional changes for the mechanism of male germ cells.

overall_design: In this study, the testicular tissues of calves and adult cattle were used for analysis. The researchers studied the development of germ cells in the testes of calves and adult cattle by spatial transcriptome sequencing technology.

first: Haoyan

last: Jin

first: Lingkai

last: Zhang


title: Circulating microRNA signatures for diagnosis and prediction of progression in pediatric idiopathic scoliosis

city: Brno

first: Robin

last: Jugas

address: Kamenice 5

country: Czech Republic

institute: CEITEC MU

summary: Introduction: Idiopathic scoliosis (IS) is the most common pediatric spinal deformity, yet no biomarker currently enables early diagnosis or reliable prediction of progression to guide individualized treatment. Circulating microRNAs (miRNAs) are promising non-invasive biomarkers reflecting multifactorial disease mechanisms. Methods: We conducted a prospective study in a Czech cohort comprising 114 pediatric IS patients at the time of diagnosis (82 females, 32 males; median age 13 years, range 7–17) and 89 age-matched healthy controls (60 females, 29 males; median age 13 years, range 7–18). Plasma miRNA profiles were obtained by next-generation sequencing, corrected for batch effect, and analyzed for differential expression. Logistic regression models were used to construct miRNA diagnostic and prognostic signatures, validated by leave-one-out cross-validation (LOOCV). Differential expression analysis identified multiple miRNAs with significant diagnostic potential. A 28-miRNA diagnostic signature distinguished IS patients from controls with AUC = 0.95 and LOOCV accuracy = 0.85, with high PPV and NPV. For prognosis, comparison of high-risk versus low/medium-risk patients revealed a 7-miRNA prognostic signature, achieving AUC = 0.83 and LOOCV accuracy = 0.81. Notably, the incorporation of clinical variables such as age or sex did not improve model performance. Our study demonstrates that circulating miRNA signatures have potential as diagnostic markers and, importantly, as predictors of disease progression in pediatric idiopathic scoliosis. These findings highlight the clinical utility of miRNA-based models for individualized patient management and support further validation in larger, independent cohorts.

overall_design: All patients included in our study were both females or males diagnosed with juvenile or adolescent idiopathic scoliosis.. The blood plasma samples of 114 IS patients and 89 healthy controls were analysed. IS patients were divided into three subgroups according to the risk of progression.

first: Jana

last: Orlickova

first: Ondrej

last: Slaby


title: Improved RNA preparation for RNA-seq of the intracellular bacterium Wolbachia wAlbB

city: Tübingen

first: Kay

last: Nieselt

state: DE

address: Sand 14

department: Department for Computer Science

country: Germany

institute: Institute for Bioinformatics and Medical Informatics

summary: Despite advances in RNA-seq, investigating the transcriptome of intracellular bacteria remains challenging due to the substantial presence of host RNA. In the case of Wolbachia spp. that are propagated in insect cell lines, commercially available rRNA depletion kits are often not suitable. Here, we describe a method to study the transcriptome of Wolbachia wAlbB in the Aedes albopictus cell line C6/36. To enrich the bacterial mRNA, eukaryotic mRNA was depleted by Dynabeads (Thermo Fisher Scientific). Then custom-designed riboPOOLs (siTOOLs Biotech) were used to remove both prokaryotic and eukaryotic rRNA. Compared to RNA prepared using the Illumina Ribo-Zero Plus rRNA Depletion Kit alone, additional depletion of eukaryotic mRNA increased wolbachial reads 7-fold to 0.7% of all reads. After removing eukaryotic and prokaryotic rRNAs with custom-designed riboPOOLs, there was a 300-fold increase of reads that mapped to Wolbachia (30.2%). Combining customized rRNA depletion from both organisms with eukaryotic mRNA depletion was more cost-effective than simply increasing the number of sequencing reads. This method can potentially be used for the enrichment of bacterial mRNA in studies of intracellular bacteria that cannot be propagated in standard cell lines.

overall_design: RNA-seq of Wolbachia bacteria infecting Aedes albopictus. Different methods were tested for rRNA depletion. In the first pilot run the Illumina Ribo-Zero Plus Depletion Kit was used to deplete rRNA from total RNA or poly(A)-depleted RNA, following the manufacturer/s protocol. In the second pilot and third pilot, the rRNA depletion using riboPOOLs (siTOOLs Biotech, Planegg, Germany) was carried out on total RNA according to the manufacturer's instructions, using the maximum input of 5 µg RNA. Four different riboPOOLs were employed: Pan-Bacteria (dp-K012-26), A. albopictus (dp-K012-47), and custom-designed riboPOOLs for Wolbachia and A. albopictus.

first: Lara

last: Behrmann

first: Theresa

middle: A

last: Harbig

first: Achim

last: Hoerauf

first: Kenneth

last: Pfarr


title: Pooled CRISPRi screen with scRNA-seq to knockdown 7 transcription factors in k562 cells

city: Rotterdam

first: Eskeatnaf

last: Mulugeta

address: Wytemaweg 80

department: Cell Biology

country: Netherlands

institute: Erasmus University Medical Center Rotterdam (Erasmus MC)

summary: To systematically investigate the gene regulatory networks in K562 cells, we performed CROP-seq—a pooled CRISPRi screen combined with single-cell RNA-seq—targeting seven transcription factors. To enhance gRNA recovery, we incorporated the Single Cell 5′ CRISPR Screening Assay, which enables direct capture of gRNA sequences on the beads and generates a gRNA count matrix.

overall_design: After designing and ordering 26 gRNAs (three targeting each gene plus five non-targeting controls) in a pooled format, we generated the gRNA library following the CROP-seq protocol. Next, we performed next-generation sequencing (NGS) to assess the quality of both the plasmid and lentiviral gRNA libraries. Upon confirming that both libraries passed quality control, we separately transduced wild-type K562 cells with dCas9 and the gRNA lentiviral library. Following FACS sorting and subsequent culturing, the cells were processed for single-cell RNA-seq.

first: Hong

first: Peifen

last: Bindels


title: Intronic Polyadenylation Derived Long Non-coding RNA modulates Nucleolar Integrity and Function

city: Bryan

first: Irtisha

last: Singh

address: 8447 John Sharp Pkwy MREB 2

summary: Intronic polyadenylation (IPA) events occurring near the transcription start site or within early introns can generate numerous non-coding RNAs derived from protein-coding loci (ncpRNAs). Despite their abundance, the functional roles of ncpRNAs remain largely unexplored. In this study, we characterized one such long non-coding RNA, ncpCUL1, transcribed from the CUL1 gene locus. Our results revealed that ncpCUL1 is a polyadenylated, RNA polymerase II dependent IPA isoform that is stable and translocates to the nucleolus. Functional characterization demonstrated that ncpCUL1 plays a critical role in maintaining nucleolar integrity. RNA-protein interaction assays identified GPATCH4 and NOP58, two nucleolar proteins involved in rRNA processing, as binding partners of ncpCUL1. Consistent with its localization and interactions, loss of ncpCUL1 led to a reduction in rRNA levels and consequent decrease in protein synthesis. This effect on rRNA levels could be reversed by reintroducing ncpCUL1, confirming its functional importance. Furthermore, as nucleolar stress is known to affect cell cycle progression, we found that ncpCUL1 depletion resulted in G2 phase cell cycle arrest. Together, our findings demonstrate that ncpCUL1, an IPA-derived lncRNA from the CUL1 locus, forms an RNA-protein complex in the nucleolus to support nucleolar structure and function.

overall_design: K562/KMS34/ HEK293 cells were treated with 12 micro Molar (uM) of µeither scramble (Non-targeting) Anti-sense oligo (ASO) or the ncpCUL1- targeting-ASO for 44hr. The Total RNA was isolated using trizol method.

first: Sumana

last: Mallick

first: Pranita

last: Borkar

first: Jaspreet

last: Thind

first: Daniel

last: Chung

first: Taylor

last: Hubbs


title: Multi-omics Analysis Revealed Unique Features of Age-associated Type2 CD8 Memory T cells

first: Hiroyuki

last: Matsui

state: Kyoto

overall_design: RNA-seq profiling of FACS sorted 5 subsets of human CD8 T cells (Naïve, CXCR3- CM, CXCR3+ CM, CD28+CD57+ EM and CD57+ EMRA).

first: Marlene

last: Cervantes

first: Eric

last: Verdin


title: Single-cell RNA sequencing of non-cardiomyocytes obtained from wild-type mouse hearts 20 days after transverse aortic constriction or sham surgery

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

institute: MPI for heart and lung research

overall_design: The non-cardiomyocyte population was isolated from mouse hearts obtained 20 days after sham ("Sham") or transverse aortic constriction ("TAC")

first: Haruya

last: Kawase

first: Nina

last: Wettschureck

last: Guenther


title: Role lymphatic-derived neurotensin in pressure overload-induced cardiac damage

summary: Neuropeptide neurotensin (NTS) was found to be upregulated after induction of cardiac injury in cardiac lymphatic endothelial cells (LEC). We generated tamoxifen-inducible, LEC-specific NTS knockout mice and used scRNA-seq to understand the consequences of LEC-specific NTS deletion on cardiac remodeling 7 days after induction of pressure overload by transverse aortic constriction.

overall_design: Non-cardiomyocyte population was isolated from control (3) and Nts KO (4) mice after 7 days of pressure overload (TAC model).

first: Niharika

last: Shiva


title: Mirdametinib treatment for the osteofibrous dysplasia RASopathy

city: Dallas

first: Jonathan

last: Rios

state: TX

address: 2222 Welborn Street

institute: Scottish Rite for Children

summary: The goal of this study was to investigate the molecular response of primary cells cultured from the pseudarthrosis sites of Osteofibrous Dysplasia patients following 24h treatment with vehicle (DMSO), capmatinib (10uM, Catalog# S2788, Selleck Chemicals, USA), selumetinib (5uM, Catalog# S1008, Selleck Chemicals, USA), or mirdametinib (100nM, Catalog# S1036, Selleck Chemicals, USA). Transcriptional changes were

overall_design: Primary cells were cultured from n=5 subjects in two different experiments. In experiment 1, cells were treated with vehicle, capmatinib, or selumetinib. In experiment 2, cells were treated with vehicle or mirdmetinib. Data from both experiments are provided. Gene expression was evaluated by RNA-sequencing with data analyzed using Partek Flow.

first: Rios

last: Jonathan

first: Paria

last: Nandina


title: CTCF (CCCTC-binding factor) binding patterns in the human genome following exposures to Clostridioides difficile TcdB.

city: Oklahoma City

first: Jason

last: Larabee

state: OK

address: 940 Stanton L. Young Blvd, BMSB 1035

department: Microbiology and Immunology

institute: The University of Oklahoma Health Science Center

summary: This experiment uses a CUT&RUN approach to determine if the CCCTC-binding factor (CTCF) is altered in its ability to bind the human genome following exposure to TcdB. TcdB is a bacterial toxin and primary virulence factor produced by the enteric pathogen C. difficile. CTCF is a DNA binding protein that regulates gene transcription in the host.

overall_design: HeLa cells  exposed to 1 ng/ml of TcdB2 for 24 h were compared to untreated controls. Following treatment, CUT&RUN was used to map genomic sites binding CTCF and H3K4me3.

first: Jimmy

middle: D

last: Ballard


title: A precision medicine approach to interpret a GATA4 genetic variant in a paediatric patient with congenital heart disease

city: Nedlands

first: Kevin

last: Chen

address: Hospital Avenue

institute: The Kids Research Institute Australia