title: Evolutionary guided transcription factor design programs novel T cell states [ChIP-Seq]

city: Stanford

first: Oliver

last: Takacsi-Nagy

address: 234 Pasteur Dr. (BMI 4600)

department: Pathology

institute: Stanford University School of Medicine

summary: Human protein-coding genes evolved via modular rearrangement of domains from ancestral genes. Here, we develop a scalable, evolutionarily guided method to assemble novel protein-coding genes from constituent domains within a protein family, termed DESynR (Domain Engineered via Synthesis and Recombination) genes. Using primary human chimeric antigen receptor T cells as a model, we find that the expression of DESynR Activator Protein-1 (AP-1) transcription factors (TFs) significantly outperforms the overexpression of natural AP-1 TFs in multiple functional assays in vitro and in vivo. Top DESynR AP-1 TFs exhibit non-intuitive domain architectures, including from TFs not canonically expressed in T cells. DESynR AP-1 TFs induce broad transcriptional and epigenetic reprogramming and establish non-natural T cell states that optimize features of exhaustion, effector and cytotoxic function, and persistence— in some cases co-opting gene expression modules from disparate cell types. We show that this reprogramming is primarily driven by differential regulation of established AP-1-bound regulatory elements, rather than unique binding. Finally, we extend the DESynR methodology to Erythroblast Transformation Specific (ETS) and Forkhead box (FOX) TFs to support the generalizability across protein families. Overall, we demonstrate that novel configurations of existing protein domains may uncover non-evolved genes that program cell states with therapeutic relevance.

overall_design: Live-sorted human CD19-28z CAR T cells overexpressing various natural and engineered AP-1 TFs were analyzed by ChIP-seq following a single stimulation with Nalm6 targets in vitro.

first: Sivakanthan

last: Kasinathan

first: Austin

last: Hartman

first: Theo

middle: L

last: Roth

first: Ansuman

middle: T

last: Satpathy


title: 10x Genomics Xenium analysis of adult and aged mouse brain control and heat stress cohorts

status: Public on Jun 17 2026

city: Irvine

first: Saurabh

last: Chatterjee

state: California

address: 847 Health Sciences Road

department: Department of Environmental and Occupational Health

country: USA

institute: University of California, Irvine

summary: 10x Genomics Xenium in situ targeted gene expression was performed on FFPE mouse brain tissue using the Xenium Mouse Brain Gene Expression panel (mBrain_v1.1). The staged dataset contains adult and aged cohorts with control and heat-stress sample annotations. Heat-stress mice received periodic heat exposure at approximately 40 deg C for 3 hours/day for 14-15 days. Raw files include high-resolution morphology images and decoded transcripts; processed files include transcript count matrices, cell summaries, and cell and nucleus segmentation boundaries.

overall_design: Two Xenium regions/cohorts (Adult_cohort and Aged_cohort) from run ChatS_10032025 were analyzed. Each cohort includes control_1-control_4 and heatstress_1-heatstress_4 sample annotations across four areas as recorded in sample_map.csv. Heat-stress animals underwent periodic heat exposure at approximately 40 deg C for 3 hours/day for 14-15 days.

first: Roy

last: Subhajit

first: Trivedi

last: Ayushi

first: Sarkar

last: Subham

first: Kumar

last: Ram

first: Chatterjee

last: Saurabh


title: Evolutionary guided transcription factor design programs novel T cell states [RNAseq_HA]

state: CA

type: Expression profiling by high throughput sequencing

overall_design: Live-sorted human “High Affinity” (HA) GD2-28z CAR T cells overexpressing various natural and engineered AP-1 TFs were analyzed by RNA-seq following two stimulations with Nalm6-GD2 targets in vitro.


title: Evolutionary guided transcription factor design programs novel T cell states [RNAseq_Paired]

overall_design: Live-sorted human CD19-28z CAR T cells overexpressing various natural and engineered AP-1 TFs were analyzed by RNA-seq following a single stimulation with Nalm6 targets in vitro.


title: Functional characterization of the orphan hypothetical protein TGGT1_245740, a transmembrane protein required for Toxoplasma gondii growth and virulence II

city: China

first: Jing

last: Li

address: No. 182, Tianmu Mountain Road, Xihu District, Hangzhou City, Zhejiang Province, Hangzhou Medical College

country: China

institute: Laboratory of Pathogenic Biology, School of Basic Medicine and Forensic Medicine, Hangzhou Medical College, China.

summary: Numerous hypothetical proteins in Toxoplasma gondii (T. gondii) are predicted to participate in diverse biological processes. TGGT1_245740, characterized in this study, is annotated as a hypothetical protein lacking identifiable orthologs, conserved domains, and intrinsically disordered regions (IDRs), suggesting that it represents an orphan protein in T. gondii, a class that remains largely unexplored. In this study, the biological function of TGGT1_245740 was investigated through bioinformatic analysis, monoclonal antibody production, expression profiling, gene tagging, gene knockout, phenotypic assays, and transcriptomic analysis. TGGT1_245740 was found to be diffusely distributed in the inner membrane complex (IMC). The knockout of TGGT1_245740 gene could reduce the intracellular proliferation and virulence of the parasite. The host transcriptome suggested that infection with the knockout strain enhanced the host immune response, and the two sets of data together further revealed the potential role of the protein in regulating parasite metabolic homeostasis and host immune recognition. This study provides the first functional characterization of this orphan protein, highlighting its potential role in the physiological metabolism and host–parasite interaction of T. gondii.

overall_design: RNA-seq of human HFF-1 foreskin fibroblasts: uninfected negative control, cells infected with WT RH tachyzoites, cells infected with TGGT1_245740 knockout tachyzoites. Three biological replicates per group to compare host transcriptomic responses to different parasite strains.

first: Bin

last: Zheng


title: Pulmonary mRNA-LNP Vaccines for Rapid and Durable Protection Against Bacterial Infection

city: Shanghai

first: Xiaoli

last: Wei

address: Dongan Road 130

institute: Fudan University

summary: Pulmonary bacterial infections remain a major clinical challenge. Although vaccination reduces infection rates and mortality, the vulnerable post-vaccination immunity gap can still result in infection and vaccine failure. In addition, effective vaccines are unavailable for many clinically important bacterial pathogens. Here, we report a pulmonary mRNA-lipid nanoparticle (mRNA-LNP) vaccine incorporating a novel ionizable lipid engineered for localized high-level expression, which elicits both rapid and durable protections against bacterial lung infections, effectively bridging this critical window of vulnerability. Intratracheal delivery of mRNA-LNP rapidly primes lung neutrophils and macrophages into a transcriptionally pre-activated state, enhancing their phagocytic activity and enabling rapid, antigen-independent bacterial clearance during the early post-vaccination period (approximately 1-7 days). Subsequently, vaccination induces potent antigen-specific adaptive responses, conferring sustained protection against both laboratory and clinical drug-resistant Pseudomonas aeruginosa strains. Single-cell transcriptomics and immune profiling reveal coordinated activation of innate and adaptive immune programs. This dual-phase immune response exemplifies a paradigm-shifting vaccine design that integrates innate and adaptive immunity to confer both immediate and long-term protection. Our findings establish a mechanistic basis for rapid antibacterial defense and highlight pulmonary mRNA-LNP vaccination as a promising strategy for combating respiratory infections.

overall_design: Female BALB/c mice were anesthetized and intratracheally nebulized with PcrV mRNA-loaded LNPCY7 (2 μg mRNA per mouse) using a microsprayer. Three days post-immunization, mice were euthanized, and lungs were collected along with control lungs.


title: Neuroprotective Effect of Mesenchymal Stem Cell-Derived miR-143-3p on Dopaminergic Neurons via the TLR3/NF-κB/p53 Signaling Pathway

city: boston

last: Song

address: 55 Fruit Street

institute: Massachusetts General Hospital

type: Non-coding RNA profiling by high throughput sequencing

summary: Background Transplantation of midbrain dopaminergic neurons (mDANs) derived from human pluripotent stem cells (hPSCs) represents a promising therapeutic approach for Parkinson’s disease (PD), with several clinical trials currently in progress. However, the poor survival of grafted mDANs, in part due to inflammation triggered by surgical injury, poses significant challenges to both therapeutic efficacy and safety. In recent years, mesenchymal stem cells (MSCs) and their derivatives have played an important role in the field of stem cell therapy due to their easy accessibility and immunomodulatory properties. However, their potential role as an adjunct in cell therapy remains unclear. Therefore, this study aims to investigate the protective effects of conditioned medium (CM) derived from MSCs and the mechanisms of action by which it attenuates surgery-induced neuroinflammation and enhances the survival of transplanted mDANs.  Methods: To mimic the neuroinflammatory response caused by the surgical aspect of cell transplantation, transplantation medium alone (i.e. without cells) was injected into the striatum of male NOD SCID mice. The anti-inflammatory effects of MSC-CM in vivo were evaluated by assessing the activation of microglia and expression levels of pro-inflammatory cytokines. Next, an in vitro cell death model was established using IFN-γ and TNF-α stimulation. The protective effects of MSC-CM against IFN-γ and TNF-α-induced inflammatory injury in mDANs were assessed using immunofluorescence staining and TUNEL assay. To identify the active components and underlying mechanisms for the protective effects of MSC-CM, inhibitory treatments, miRNA sequencing, dual-luciferase reporter assay, and subsequent validation by qPCR and Western blotting were performed. Finally, MSC-CM was administered intranasally into male NOD SCID mice following mDAN transplantation to evaluate its protective effects on the grafted mDANs in vivo.  Results: MSC-CM suppressed neuroinflammation and significantly protected mDANs from inflammation-induced cell death via inhibition of TLR3/NF-κB/p53 signaling pathway in this model system. This protective effect was largely reproduced by treatment with a specific component of the MSC-CM, miR-143-3p. Overexpression of miR-143-3p further enhanced the protective effects and mechanisms of action of MSC-CM.  Conclusions: This study demonstrates that MSC-CM or its component miR-143-3p exert protective effects on mDANs against inflammatory injury, primarily by inhibiting the TLR3/NF-κB/p53 signaling pathway.

overall_design: miRNAs derived from MSC-CM were analyzed, with 293T-CM-derived miRNAs serving as controls. Three biological replicates were performed for each group.

last: Sun

first: Tian

last: Wang

first: Chen

last: Qiu

first: Tianwen

first: Peng

first: Tatyana

last: Matveeva

first: Tanner

last: Boyd

first: Qingyuan

last: Tang

first: Jun

last: Xue

first: Lu

last: Su

first: Hyemyung

last: Seo

first: Yi Eve

first: Tian-Lin

last: Cheng

first: Jeffrey

last: Schweitzer


title: Functional characterization of the orphan hypothetical protein TGGT1_245740, a transmembrane protein required for Toxoplasma gondii growth and virulence.

overall_design: RNA-seq of purified extracellular Toxoplasma gondii tachyzoites, including wild-type RH strain and one single-gene knockout strain (TGGT1_245740). Three independent biological replicates were prepared for each strain to detect differential gene expression caused by gene deletion.


title: Human HSPCs clones balance stochastic diversification with cytokine-induced differentiation

city: Rehovot

first: Liran

last: Shlush

address: 234 Herzl Street

country: Israel

institute: Weizmann Institute of Science

summary: Hematopoietic stem and progenitor cells (HSPCs) balance self-renewal with on-demand differentiation of blood lineages. Modern bone marrow atlases specify static transcriptional states, but not HSPC dynamics, which are only partly understood, particularly in humans. Here, we introduce cHSPCTrack (clonal HSPC tracker), an in vitro framework for serially tracking clonal differentiation and fate acquisition from human HSPCs extracted from circulation. By following thousands of single-cell-derived clones stimulated by different cytokine combinations, cHSPCTrack reconstructs the balance between stochasticity and directed differentiation in human HSPCs. Single HSPCs stochastically yield multiple lineages under a uniform cytokine environment, with stimulatory inputs modulating differentiation probabilities by introducing biases toward preferred fates. In parallel, differentiation rates vary stochastically across and within clones. Clonal memory stabilizes the induction of a few genes per clone over otherwise archetypical differentiation trajectories. Within clones, multiple fates can interact with each other, as shown for clones mixing basophils, eosinophils, and mast cells with other fates. cHSPCTrack opens avenues for understanding hematopoietic clonal dynamics in health and disease.

overall_design:
CD34⁺ HSPCs were enriched from PBMCs of healthy donors by magnetic selection and transduced with a high-complexity lentiviral CellTag library. Single GFP⁺ cells were sorted into 384 well plates and cultured under defined cytokine combinations. To capture clonal dynamics, we employed a serial sampling strategy: half of each well was harvested at multiple time points for 10x Genomics 3’ scRNA-seq (V3.1), while the remaining half continued to mature. Barcode identity of cells and wells was recovered by combining targeted genomic DNA sequencing and targeted Genotyping of Transcriptomes (GoT), inferring clones to their culture condition using a dedicated graph algorithm for imputing missing data. scRNA-seq data were organized into metacells and annotated into cell types and states as previously described. Differentiation trajectories were modeled using de-novo inference of gene programs and re-clustering of single cells given inferred program activities.

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

first: Elia

last: Colin

first: Dror

last: Brook

first: Jonathan

last: Izraeli

first: Oren

last: Milman

first: Aviezer

last: Lifshitz

first: Tal

last: Bachrach

first: Noa

last: Chapal-Ilani

first: Amos

last: Tanay


title: ACKR2 aids the regulation of bone marrow eosinophils to promote monocyte differentiation and anti-bacterial immunity

city: Glasgow

first: Heather

last: Mathie

state: --None--

address: Sir Graeme Davies Building, 126 University Place

country: United Kingdom

institute: University of Glasgow

summary: Chemokine receptors control cell migration within the body. Here we reveal interactions between eosinophils and monocytes in the bone marrow, indirectly controlled by the atypical chemokine receptor ACKR2. In the absence of ACKR2, there are decreased numbers of eosinophils in the bone marrow. As a result, eosinophil and monocyte interactions are reduced within the bone marrow niche, and are associated with changes in monocyte gene expression. Monocytes from ACKR2-/- mice are recruited to the tissues but are fundamentally altered in their ability to differentiate into macrophages, in the lung, peritoneal cavity and cavity wall. Bacterial elimination is impaired in ACKR2-/- mice during peritoneal infection. ACKR2 is therefore a key regulator of eosinophil-driven monocyte education in the bone marrow, required for full monocyte differentiation and macrophage function within the tissues.

overall_design: FACS sorted monocytes from the bone marrow, MHCII negative macrophages from the lungs, and SPMs from the peritoneal cavity of WT and ACKR2-/- mice were isolated. RNA extraction was performed on sorted samples for RNA sequencing. RNA was extracted using the RNeasy Microkit with on-column DNAse I digestion (Qiagen), following manufacturer’s instructions. RNA samples were then sent to a commercial sequencing provider (Novogene), for RNA QC, mRNA library preparation (poly-A enrichment) and Illumina Sequencing

first: Gillian

middle: J

last: Wilson

first: Lily

last: Koumbas Foley

first: Zuzanna

last: Pocalun

first: Elise

last: Pitmon

first: Ayumi

last: Fukuoka

first: Kit

middle: M

last: Lee

first: Lauren

last: Fernandez

first: Robin

last: Bartolini

first: Laura

last: Medina-Ruiz

first: John

last: Cole

first: Gerard

last: Graham


title: Transcriptomic profiling of morphine and LPS exposure across mouse brain regions

city: la jolla

first: Yanmiao

last: Du

address: 4254 Urey Hall, 9500 Gilman Drive

department: Department of Chemistry and Biochemistry/Cellular and Molecular Medicine

institute: UCSD

summary: We profiled genome-wide gene expression across four brain regions (hippocampus, prefrontal cortex, striatum, and hypothalamus) in male and female C57BL/6NTac mice using a perinatal morphine exposure model of Neonatal Opioid Withdrawal Syndrome (NOWS). Mice received morphine or saline perinatally, then were challenged with LPS or saline in adulthood, yielding four groups (SAL-SAL, SAL-LPS, MOR-SAL, MOR-LPS). NeuN+ neuronal nuclei (~5,000 per sample) were isolated by FACS from PFC, hippocampus, hypothalamus, and striatum and profiled by RNA-seq (Illumina NovaSeq X Plus, paired-end 150 bp). RNA-seq identified differentially expressed genes enriched in immune and metabolic pathways, with the hypothalamus displaying the most pronounced transcriptional response to perinatal morphine exposure.

overall_design: C57BL/6NTac mice received perinatal morphine (osmotic minipump during gestation plus 10 mg/kg SC twice daily PND1-14) or saline. Adults were challenged with LPS or saline, yielding four groups: SAL-SAL, SAL-LPS, MOR-SAL, MOR-LPS. Brain tissues (PFC, hippocampus, hypothalamus, striatum) were dissected 2 h post-challenge. NeuN+ nuclei (~5,000 per sample) isolated by FACS. RNA-seq paired-end 150 bp on Illumina NovaSeq X Plus, ~15M reads per library. Males and females combined for profiling, analyzed separately within each brain region.

first: Julia R.

last: Ferrante

first: Xin

last: Zhang

first: Jacob D.

last: Neice

first: Toby K.

last: Eisenstein

first: Wei

last: Wang

first: Chang

last: Lu

first: Julie A.

last: Blendy


title: Epigenomic profiling of H3K27ac in mouse brain regions under morphine and LPS exposure

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We profiled genome-wide H3K27ac chromatin modifications across four brain regions (hippocampus, prefrontal cortex, striatum, and hypothalamus) in male and female C57BL/6NTac mice using a perinatal morphine exposure model of Neonatal Opioid Withdrawal Syndrome (NOWS). Mice received morphine or saline perinatally, then were challenged with LPS or saline in adulthood, yielding four groups (SAL-SAL, SAL-LPS, MOR-SAL, MOR-LPS). NeuN+ neuronal nuclei (~10,000 per replicate) were isolated by FACS and profiled by MOWChIP-seq (Illumina NovaSeq X Plus, paired-end 150 nt). H3K27ac ChIP-seq identified differentially acetylated enhancers and promoters enriched in immune and metabolic pathways, with the hypothalamus exhibiting the most pronounced chromatin remodeling.

overall_design: C57BL/6NTac mice received perinatal morphine (osmotic minipump during gestation plus 10 mg/kg SC twice daily PND1-14) or saline. Adults were challenged with LPS or saline, yielding four groups: SAL-SAL, SAL-LPS, MOR-SAL, MOR-LPS. Brain tissues (PFC, hippocampus, hypothalamus, striatum) dissected 2 h post-challenge. NeuN+ nuclei (~10,000 per replicate) isolated by FACS. H3K27ac MOWChIP-seq with anti-H3K27ac (39135, Active Motif) on Illumina NovaSeq X Plus (paired-end 150 nt, ~15M reads/library). Input controls pooled per treatment-sex group. Males and females combined for profiling.


title: Non-human primate LIBRA-Seq accelerates neutralizing antibody discovery in RM vaccinated against HIV-1

city: Atlanta

first: Gregory

middle: K

last: Tharp

state: GA

address: 954 Gatewood Dr

department: Developmental and Cognitive Neuroscience

institute: Yerkes National Primate Research Center

summary: Broadly neutralizing antibodies (bNAbs) exhibit protective efficacy against HIV-1 infection making them an ideal archetype for HIV-1 vaccine design. Presently, no vaccine candidate has induced antibody responses capable of meaningful protection against the swathe of circulating, difficult to neutralize tier 2 HIV-1 viruses. However, the development of stabilized, native-like envelope (Env) trimers such as BG505.SOSIP.664.T332N (BG505 SOSIP) has marked a significant advancement in vaccine design, due to their ability to elicit NAbs that neutralize tier 2 viruses in rhesus macaques (RM). NAb development following envelope trimer immunization in RM remains poorly understood, with hypothesized contributions from genetic variation at the IG loci, naive B cell repertoire, and differential gene expression in B cell lineages. To address these knowledge gaps, we have developed a set of BG505 SOSIP probes capable of recovering paired clonotype identity, antigen specificity, and gene expression of B cells in a high throughput fashion. These probes were constructed by conjugating biotinylated BG505 SOSIP to streptavidin covalently linked to both sc-RNA-Seq compatible DNA oligonucleotides and flow cytometry compatible fluorophores. Using these reagents, we isolated and sequenced BG505 SOSIP specific memory B cells from the PBMCs of an RM that developed high titers of neutralizing antibodies. To benchmark the accuracy of our technology, we compared our recovered heavy and light chain sequences to those identified from the same animal using conventional methodology and recovered 100% of previously identified NAbs. We then applied this technology to recover BG505 SOSIP specific memory B cells from five additional vaccinated RMs, cloned 34 antibodies for functional characterization, and identified ten antibodies with autologous neutralizing activity.

sample_organism:
Macaca mulatta
Homo sapiens

platform_organism:
Macaca mulatta
Homo sapiens

overall_design: 10x feature barcode and VDJ libraries were generated from sorted and captured BG505.SOSIP specific memory B cells isolated from RM vaccinated with BG505.SOSIP.664.

first: Christopher

last: Edwards

first: Aaron

middle: D

last: Silva-Trenkle

first: Anusmita

last: Sahoo

first: Kendra

last: Cruickshank

first: Sydney

last: Nelson

first: Stacey

middle: A

last: Lapp

first: Nagarajan

last: Raju

first: Thang

last: Ton

first: Amanda

last: Metz

first: Emily

last: McGhee

first: Faith

last: Mbadugha

first: Tysheena

middle: P

last: Charles

first: Ankur

last: Saini

first: Kiran

last: Gill

first: Kathryn

last: Pellegrini

first: Rui

last: Kong

first: Jens

last: Wrammert

first: Amit

last: Upadhyay

first: Cynthia

last: Derdeyn

first: Rama Rao

last: Amara

first: Gabriel

last: Kwong

first: Steven

middle: E

last: Bosinger


title: Replicative senescence in neural progenitors induces senescence-associated markers in midbrain organoids

city: Maastricht

first: Silvia

last: Bolognin

address: Universiteitssingel 40

country: Netherlands

institute: Maastricht University

summary: Aging is the primary risk factor for Parkinson’s disease (PD), and astrocyte senescence, or astrosenescence, has emerged as a potential mechanism through which aging may progressively impair glial homeostatic neuronal support and render the brain more vulnerable to neurodegeneration. Yet human experimental models that specifically recapitulate astrosenescence within a PD-relevant genetic background are lacking. Here, we applied passage-induced replicative exhaustion to human iPSC-derived neuroepithelial stem cells (NESCs) from healthy donors and LRRK2 G2019S PD patients to establish an in vitro midbrain aging model. Extended passaging induced senescence-associated changes in NESCs, including reduced proliferative capacity, apoptosis resistance, reduction in telomerase activity, and sustained DNA damage response (DDR) activation, while preserving ventral midbrain identity and differentiation potential. Astrocytes derived from aged progenitors recapitulated multiple senescence hallmarks, including increase in SA-β-gal activity, nuclear lamina disruption, persistent DDR activation, and alteration in mitochondrial homeostasis. In human midbrain organoids (hMOs), passage-induced aging was associated with astrocyte-specific DDR activation, widespread lipidomic remodeling, and a selective reduction of dopaminergic neuron content in LRRK2 G2019S organoids compared to healthy donor-derived organoids. Multi-modal analysis of NESCs, astrocytes, and hMOs further revealed that the LRRK2 G2019S genetic background not only shifts baseline cellular states toward an aged-like profile but actively reshapes aging trajectories in a cell-type- and feature-dependent manner. Together, this work establishes a human iPSC-based midbrain model recapitulating key features of cellular senescence in both healthy and LRRK2 G2019S contexts and provides an in vitro platform to investigate the contribution of astrosenescence to PD-related neurodegeneration.

overall_design: Neural Epithelial Stem Cells (NESCs) derived from iPSCs of one healthy donor (HT), two Parkinson’s disease donors carrying the LRRK2-G2019S mutation (PD1 and PD2), and one isogenic corrected line (PD1 GC) were profiled by RNA-sequencing. Cells were analyzed at early (P < 8) and high passage number (P > 16) stages, with three independent batches per condition.

first: Virginia

last: Cora

first: Daniele

last: Ferrante

first: Nika

last: Lu Yang

first: Javier

last: Jarazo

first: Alise

last: Zagare

middle: C

last: Schwamborn


title: Gene expression profile of human endometrial primary culture with added  exosomes obtained from the conditioned medium of multipotent mesenchymal stromal cells (MMSCs) at a concentration of 50 ng/ml

city: Moscow

first: Polina

last: Vishnyakova

address: Oparina 4

country: Russia

institute: National Medical Research Center for Obstetrics, Gynecology and Perinatology

summary:
The aim of the work was to examine gene expression profile of human endometrial primary culture with added exosomes obtained from the conditioned medium of  mesenchymal stromal cells (MSCs) at a concentration of 50 ng/ml.
Exosomes obtained from the conditioned medium of  mesenchymal stromal cells (MSCs) were added to the primary endometrial cell culture at a concentration of 50 ng/ml. Samples were collected and lysed in MagZol reagent for subsequent RNA extraction.

overall_design: Exosomes obtained from the conditioned medium of MSCs  were added to primary endometrial cell cultures at a concentration of 50 ng/ml. The cells with exosomes were incubated for 24 hours. Primary endometrial culture cells without the addition of exosomes served as a control. Samples were collected and lysed in MagZol reagent for subsequent RNA extraction.

first: Maria

last: Kuznetsova


title: Inhibition of Salt-Inducible Kinases 2 and 3 reduces inflammatory signature in Psoriasis

city: Munich

first: Stefanie

last: Eyerich

address: Biedersteinerstr. 29

country: Germany

institute: TUM

summary: Psoriasis is a highly prevalent inflammatory skin disease for which good systemic therapeutic agents are available. However, there still is a great need for new treatment options, especially oral ones. Salt-inducible kinases (SIKs) are emerging as new druggable targets in immune diseases and the SIK2/SIK3 inhibitor GLPG3970 recently proved to efficiently reduce psoriasis lesions in a phase Ib trial. This study assesses molecular events underlying beneficial effects of SIK2/SIK3 inhibition via GLPG3970 in psoriasis. Using spatial transcriptomics and single cell sequencing, abundant expression of SIK2 and SIK3, but not of SIK1, was observed in psoriatic skin. In vitro, GLPG3970 reduced psoriasis marker cytokines and chemokines (TNF, IFN-G, IL-1B, IL-6, CXCL8, CXCL10) in peripheral mononuclear cells, T cells and fibroblasts, but not in keratinocytes. Bulk RNA sequencing of lesional psoriasis biopsies treated ex vivo with GLPG3970 and of biopsies received from responders vs. placebo-treated patients of the clinical trial showed downregulation of inflammatory pathways with suppression of IL1B, CXCL1, CXCL8, CEBPB, CD177, and CLEC5A in both settings. In summary, this study suggests that specific inhibition of SIK2/SIK3 reduces acute phase immunity and neutrophil recruitment in psoriasis.

overall_design: Bulk RNA sequencing of punch biopsies of psoriasis lesions that were treated ex vivo with 100 µM GLPG3970  or vehicle.

first: Anna

last: Pilz

first: Eva

last: Briem

first: Maikel

last: Colli

first: Christina

last: Hillig

first: Stephanie

last: Lavazais

first: Michael

last: Menden

first: Tilo

last: Biedermann

first: Felix

last: Lauffer

first: Kilian

first: Steve

last: De Vos

first: Manja

last: Jargosch


title: SMD2 reads pseudouridines to regulate mRNA splicing and promote tumorigenesis [RNA-seq]

city: Shanghai, P.R. China

first: weiying

last: meng

address: 1239 Siping Road, Shanghai, P.R. China

institute: Tongji University

summary: Pseudouridines (Ψ) in mRNA are linked to alternative splicing, but their regulatory mechanisms remain unclear due to the lack of identified reader proteins. Here, we identify SMD2, a core spliceosomal component, as a direct Ψ reader. Using in vitro and ex vivo assays, we show that SMD2 preferentially binds Ψ over unmodified uridines. SMD2 collaborates with PUS family enzymes to regulate alternative splicing by binding Ψ near exon-intron boundaries, modulating the splicing of numerous pre-mRNAs. Notably, the gene encoding SMD2, SNRPD2, is overexpressed across multiple cancers and is essential for tumor cell proliferation through the maturation of key transcripts. These findings uncover a direct mechanistic link between Ψ and spliceosomal function, establishing SMD2 as a critical regulator of Ψ-mediated splicing and a potential cancer therapeutic target.

overall_design: To investigate the specific role of the SMD2 protein in splicing, we performed a rescue experiment following SNRPD2 knockout. Specifically, we rescued the knockout cells with either wild-type (WT) or mutant SMD2 constructs and analyzed changes in downstream alternative splicing events. This approach allowed us to assess the functional importance of the specific mutation sites for the protein's activity and, concurrently, to demonstrate that this protein is directly involved in splicing.

first: Weiying

last: Meng

first: Moping

last: Xu

first: Shuxia

last: Sun

first: Zhiyan

first: Jiejun

last: Shi


title: Aging disrupts spatiotemporal coordination in the cycling murine ovary

city: New Haven

first: Hattie

last: Chung

state: CT

address: 300 George St

institute: Yale University

summary: Throughout the female reproductive lifespan, the ovary completes hundreds of cycles of follicle development, ovulation, and tissue regeneration. These processes rely on the precisely coordinated intricate multicellular interactions across time and space. How aging disrupts these interactions, leading to an overall decline in reproductive and endocrine functions, remains understudied. To understand the multicellular dynamics that underlie ovarian function and their changes with age, here we use Slide-seq, a near-cellular spatial transcriptomics method, to profile 22 mouse ovaries across the reproductive cycle and chronological age, representing 610,620 near-cellular spots across 69 spatial transcriptomic profiles. We develop a segmentation analysis to identify spatial niches that capture different states of folliculogenesis from static snapshots in situ, allowing us to examine the multicellular dynamics of 358 oocytes, 668 follicles, and 236 corpora lutea. We find that aging disrupts both the spatial organization and temporal coordination of folliculogenesis before the cessation of cycling, which may contribute to the dysregulation of hormone production and signaling. These disruptions are marked by altered immune cell dynamics, inflammatory signaling, and global tissue disorganization that impair the cyclic remodeling required for ovarian function. Our findings reveal how multicellular niches orchestrate ovarian function and demonstrate how age-related breakdown of tissue organization across time and space precedes reproductive decline.

overall_design: C56BL/6 mice across three age groups (young 10-12 weeks, middle 36-40 weeks, old 52-54 weeks) were staged for their estrous cycle and harvested at either estrus or metetrus. After intracardial perfusion with PBS, ovaries were dissected and fresh frozen embedded in OCT. Frozen tissue sections cut at 10 µm thickness were profiled using Slide-seqV2 for spatial transcriptomics.

first: Jennifer

last: Garrison

first: David

last: Fischer

first: Tammy

last: Lan

first: Alison

last: Kochersberger


title: Transcriptomic profiling of NK cells in CHB and CHB-MASLD

city: Hefei

first: Zhang

last: luyuan

state: Anhui

address: Anhui Medical University, Meishan Road, Hefei, Anhui, China

institute: The First Affiliated Hospital of Anhui Medical University

summary: Clinical evidence suggests that coexistence of metabolic dysfunction-associated steatotic liver disease (MASLD) and chronic hepatitis B (CHB) is associated with more severe liver disease progression, but the underlying immune mechanisms remain incompletely understood. In this study, RNA sequencing was performed on peripheral natural killer (NK) cells isolated from patients with CHB and CHB-MASLD to characterize transcriptomic alterations associated with metabolic dysfunction. Differential expression and pathway analyses were used to identify immune activation programs and candidate signaling pathways linked to NK-cell remodeling in CHB-MASLD.

overall_design: Peripheral blood NK cells were isolated from patients with CHB and CHB-MASLD and subjected to bulk RNA sequencing. The study included biological replicates from each group. Differential gene expression analysis was performed to compare transcriptomic profiles between CHB and CHB-MASLD, followed by pathway enrichment analysis to identify immune and inflammatory programs associated with metabolic dysfunction.

first: Luyuan

last: Zhang


title: Restraint of Powassan virus replication by TRIM5a facilitates viral avoidance of antiviral immunity

city: Bethesda

first: Skyler

middle: Alexander

last: Kuhn

state: Maryland

address: 9000 Rockville Pike, BG 29B RM 1NN20

department: National Institute of Allergy and Infectious Diseases

institute: National Institutes of Health

summary: TRIpartite Motif (TRIM) protein 5 alpha (TRIM5a) is a well characterized cellular inhibitor of lentivirus replication that limits transmission of related viruses between primates. We previously reported that TRIM5a derived from humans and rhesus macaques inhibits replication of orthoflaviviruses belonging to the tick-borne encephalitis virus (TBEV) serocomplex, including TBEV, Kyasanur forest disease virus and Langat virus (LGTV), but interestingly not the tick-borne Powassan virus (POWV). To further characterize the primate TRIM5a and orthoflavivirus interface, we screened TRIM5a variants from representative old- and new-world primates for restriction capacity. TRIM5a from old-world African green monkey, De Brazza’s monkey and chimpanzee demonstrated virus-specific restriction of tick-borne orthoflaviviruses. Efforts to determine why TRIM5a fails to inhibit POWV revealed that our lab stock had acquired a non-synonymous mutation in NS3 that, when introduced into a POWV molecular clone, facilitated virus replication in the presence of all inhibitory primate TRIM5a proteins. Infection of human dendritic cells with TRIM5a-resistant POWV resulted in high early replication and  strong induction of interferon responses that limited replication compared with the wild-type virus. Thus, primate TRIM5a functions as a potent cellular barrier to infection with tick-borne orthoflaviviruses that restrains replication to a level that may help avoid early innate immune recognition.

overall_design: RNA-seq profiling of human monocyte-derived dendritic cells (MDDCs) after mock-infection and tick-borne infection of powassan virus (POWV).

first: Broeckel

last: Rebecca

first: Fitzmeyer

last: Emily

first: Chebishev

last: Eva

first: Shannon

last: Jeffrey

first: Shue

last: Byron

first: Hage

last: Adam

first: Spada

last: Stephanie

first: Artikis

last: Efrosini

first: Chiramel

last: Abhilash

first: McNally

last: Kristin

first: Larson

last: Charles

first: Kutzleben

last: Maarit

first: Robertson

last: Shelly

first: Offerdahl

last: Danielle

first: Lakin

last: Hilary

first: Bloom

last: Marshall

first: Nair

last: Vinod

first: Eyre

last: Nicholas

first: Beard

last: Michael

first: Grigg

first: Bouamr

last: Fadila

first: Kuhn

last: Skyler

first: Best

last: Sonja


title: 10X Genomics Multiome Assay (GEX + ATAC) of hypothalamic preoptic area in prenatally androgenized and control prepubertal female mice.

city: Ann Arbor

first: Bioinformatics

last: Core

state: MI

address: BRCF Bioinformatics Core, 2800 Plymouth Rd

institute: University of Michigan

summary: Polycystic ovary syndrome (PCOS) is the most common cause of infertility in women. The etiology of PCOS is not well understood. Both genetics and early life exposures are considered contributing factors. Women with PCOS have elevated androgens, potentially exposing female offspring to high androgens in utero. Daughters of women with PCOS are at increased risk of developing this disorder. Prenatal androgenization (PNA) of several species, including mice, recapitulates many phenotypes observed in PCOS. Adult female PNA mice exhibit elevated luteinizing hormone (LH) levels and increased gonadotropin-releasing hormone (GnRH) neuron pulse frequency. These changes in GnRH neurons emerge during the pubertal transition. We utilized the 10X Genomics Multiome (gene expression and ATAC-seq) assay to investigate changes in both gene expression and chromatin accessibility in the hypothalamic preoptic area of prepubertal (postnatal day 21) control and PNA females. Through analysis of ~51,000 cells across 34 cell populations, we identified cell-type-specific chromatin remodeling in response to prenatal androgen exposure, despite the absence of coordinated transcriptional changes at postnatal day 21, suggesting that critical developmental programming occurs earlier with persistent epigenetic consequences.

overall_design: Experimental design: Timed pregnant dams were treated with either vehicle (VEH, 90% oil, 10% EtOH) or 225µg dihydrotestosterone on gestational days 16-18. On postnatal days 18-21, female offspring were sacrificed and hypothalamic tissue was collected using 1.2mm Palkovits punches of the preoptic area (2mm rostral from the caudal extent of the optic chiasm, ventral midline). Tissue from all females within a litter (2-5 pups) was pooled to constitute one biological sample, flash frozen, and stored at -80°C until nuclear isolation. The study contains 4 VEH and 4 PNA pooled litter samples.

first: Laura L

last: Burger

first: Rujuta M

last: Chikodikar

first: Suzanne M

last: Moenter


title: Beyond motif recognition: Specificity of human transcription factors in yeast

first: Naama

last: Barkai

department: Molecular Genetics

summary: Transcription factors (TFs) bind DNA through sequence-specific DNA-binding domains (DBDs), yet genome-wide analyses show that TFs occupy only a small fraction of their motif occurrences. This raises the question of how TFs distinguish specific targets from the many potential sites in the genome. To investigate determinants of binding specificity beyond the cognate motif and cofactor influences, we measured the binding of 60 human TFs across the budding yeast genome. Although human TFs robustly recognized their motifs, they displayed strong selectivity in site occupancy. Nucleosome abundance explained this selectivity only in part: among the 5-20% of motif sites that were bound, a substantial fraction remained nucleosome covered. Furthermore, TFs recognizing similar motif sequences independently localized to distinct subsets of sites within different promoters. Despite the absence of human-specific cofactors in yeast, both binding stability and genomic preferences depended on largely disordered non-DBD regions. These findings suggest intrinsically disordered regions (IDRs) may therefore direct genome binding TF target recognition across evolutionarily distant genomes.

overall_design: We integrated 60 human TFs from 10 distinct DBD families into the budding yeast genome, fusing them to yellow fluorescent protein (YFP) to monitor their abundance and nuclear localization, and to MNase to profile their genomic binding by ChEC-seq.

first: Joshua

last: Bugis

first: Dan

middle: R

last: Zlotnik-Weinberg

first: Wajd

last: Manadre

first: Vladimir

last: Mindel

first: Yunwei

last: Lu

first: Juan

middle: I

last: Fuxman Bass


title: Ezh2 Control of Bivalent Genes Fine-Tunes Developmental Competence During Retinogenesis

city: Pittsburgh

first: Issam

last: Al Diri

state: PA

address: 1622 Locust St

department: Ophthalmology

institute: University of Pittsburgh School of Medicine

summary: Polycomb group proteins play central roles in developmental gene regulation, yet how their genome-wide chromatin occupancy relates to transcriptional control in the developing retina remains incompletely defined. Here, we characterize the genomic distribution of EZH2, the catalytic subunit of the Polycomb repressive complex 2 (PRC2), and examine its relationship to chromatin state and gene expression in the developing mouse retina. EZH2 is enriched at promoters and regulatory regions marked by H3K27me3, consistent with its role in transcriptional repression. Integrated analysis of EZH2 binding, H3K27me3 deposition, and transcriptomic changes upon Ezh2 loss revealed that bivalent genes marked by Ezh2 are particularly susceptible to derepression, highlighting a poised yet transcriptionally restrained state maintained by EZH2 in RPCs. We provide evidence for coordinated activity between retinal progenitor cell transcription factors and PRC2 in fine-tuning developmental gene-expression programs and controlling retinal cell-fate decisions. Notably, PRC2 restrains a subset of non-neuronal developmental regulators by repressing transcriptionally competent enhancer–promoter circuitries already primed by lineage-specific transcription factors. Loss of EZH2 unlocks these poised regulatory elements, driving aberrant gene activation and revealing how PRC2 prevents activation of non-retinal developmental gene programs. Collectively, these results highlight EZH2’s critical role in controlling developmental gene expression and preserving lineage fidelity during retinogenesis.

overall_design: ChIP-seq EZH2 and H3K27me3 from mouse E14.5 WT retina. Two antibodies for EZH2 were used (CST and AM) and one for H3K27me3 (AM), each with two replicates. Input used as control.

first: Emily

last: Davis

first: Abdullah

last: Khan


title: Modeling reptile virus infection in vitro using Python regius airway organoids II

city: Utrecht

first: Gijs

middle: Jacob Fransiscus

last: van Son

address: Heidelberglaan 25

institute: Prinses maxima centrum

summary: Zoonoses pose substantial global health risks, highlighting the need to better understand animal-to-human transmission. Reptiles are increasingly recognized as hosts of diverse pathogens, including many viruses. Despite this, reptile pathogens remain poorly understood in terms of their diversity, prevalence, and potential risk to humans. Yet human–reptile contact is increasing, driven in part by the global wildlife trade and pet industry. This underscores the need to better understand reptile-associated pathogens; however, models to study reptile viruses remain scarce. Here, we establish and characterize airway organoids derived from Python regius, providing an in vitro model of the reptile airway. Through de novo assembly of a Python regius reference genome, we characterized airway organoids at single-cell resolution, revealing diverse cell populations including ionocytes, ciliated, goblet, club, endocrine, tuft, and basal cells. The organoids supported productive infection with ball python nidovirus (BPNV) and mounted a robust epithelial antiviral response through induction of interferon-stimulated genes, cytokines and genes involved in chemical defense. As a proof-of-concept, antiviral drug treatment reduced BPNV levels, highlighting the model's utility for drug testing. By providing a reductionist system of the serpentine airway, these organoids constitute a physiologically relevant in vitro model to study reptile viruses and host–pathogen interactions in the native host. This system has potential veterinary applications, ecology of serpentes, and zoonotic disease research.

overall_design: Python Regius organoids in expansion and differentiation medium

first: Sam

last: Willemsen

first: Melanie

last: Rissmann

first: Ninouk

last: Akkerman

first: Hans

last: Clevers


title: Modeling reptile virus infection in vitro using Python regius airway organoids

overall_design: Python Regius organoids are infected with nidovirus


title: Mitochondrial PEP import controls amino acid catabolism and muscle integrity through SLC25A34

city: Boston

first: Masanori

last: Fujimoto

state: MA

address: Center for Life Sciences 3 Blackfan Circle, Room CLS730

department: Division of Endocrinology

institute: Beth Israel Deaconess Medical Center, Harvard Medical School

summary: Metabolic flexibility enables eukaryotic cells to adapt to stress by dynamically reallocating fuels among distinct metabolite pools. In skeletal muscle, prolonged fasting promotes a shift in substrate utilization from glucose toward amino acids, yet the molecular mechanisms governing this fuel switching remain incompletely defined. Mitochondria are central to this process by coordinating oxidative metabolism and importing/exporting metabolites through inner-membrane transporters, including members of the SLC25 carrier family. Notably, many SLC25 transporters remain “orphan carriers,” with unknown substrates and functions. Here, we identify SLC25A34 as a fasting-inducible orphan mitochondrial carrier that is highly enriched in the slow-twitch skeletal muscles. Muscle-specific Slc25a34 deficiency results in muscle hypertrophy accompanied by myopathic features, implicating SLC25A34 in muscle homeostasis. Additionally, we reconstituted SLC25A34 into proteoliposomes and directly quantified transport activity, revealing that SLC25A34 functions as a phosphoenolpyruvate (PEP) transporter. Together, these results identified SLC25A34 is a PEP transporter that regulates amino acid catabolism and muscle integrity.

overall_design: Soleus muscles were isolated from 19-week-old male mice under normal chow and ad libitum–fed conditions. Tissues were collected from control and muscle-specific Slc25a34 knockout mice, and total RNA was extracted using the RNeasy Fibrous Tissue Mini Kit (Qiagen).

first: Satoshi

last: Oikawa

first: Shingo

last: Kajimura


title: Quiescent tumor cells shape the immunosuppressive microenvironment in pancreatic ductal adenocarcinoma [RNA-Seq]

city: Austin

first: Bryan

last: McClellan

state: TX

address: 1601B Trinity St, Hdb 6.302

institute: University of Texas at Austin

summary: Immunotherapy, including chimeric antigen receptor (CAR) T-cell therapy, has limited activity in pancreatic ductal adenocarcinoma (PDAC). Using orthotopic PDAC mouse models, we identified a rare population of quiescent PDAC cells that increases after CAR-T cell therapy and exhibits relatively higher clonogenic growth and self-renewal potential than bulk tumor cells. These quiescent cells express high levels of Epiregulin (EREG), a secreted ligand for EGFR and ErbB4, and induce an immunosuppressive tumor microenvironment by increasing the frequency of ErbB4-expressing tumor-associated macrophages. Silencing EREG expression increased the sensitivity of both quiescent cells and tumors to CAR-T cells and improved relapse rates and overall survival. These findings demonstrate that rare quiescent tumor cells can modulate the tumor microenvironment in PDAC and suggest that EREG inhibition may enhance the efficacy of adoptive immunotherapeutic approaches in this disease.

overall_design: Ultra-low input RNA-seq profiling of FACS isolated cancer cells with high or negative expression of the p27k-mVenus reporter. (mVenus high vs mVenus negative). mVenus High cells were isolated from tumors expressing the target CAR T-cell antigen (human CD19, hCD19 Positive) , while mVenus Negative were isolated from tumors without the CAR T-cell antigen (hCD19 Negative). Tumors were bilaterally implanted in the left and right flank of the same animal prior to the adoptive transfer of anti-human CD19 murine CAR T-cells. Two-weeks after adoptive transfer, RNA was harvested sorted tumor cells.

first: Quiju

last: Wang

first: William

last: Matsui


title: Quiescent tumor cells shape the immunosuppressive microenvironment in pancreatic ductal adenocarcinoma [Spatial Transcriptomics]

overall_design: Orthotopic pancreas tumors were treated with five million anti-mouse mesothlin CAR T-cells. Three days later, tumors frozen as fresh tissue and sectioned at 10uM for Visium 3' HD spatial transcriptomics.


title: FABP4 binding cancer cells exhibit an invasive phenotype via PKC-mitoROS signaling

city: IOWA CITY

first: Shanshan

last: Liu

state: IA

address: 431 Newton Road

institute: The University of Iowa

summary: Using several high-fat diet-induced murine obesity models, we demonstrate that obesity promotes breast cancer lung metastasis through elevated levels of adipocyte-derived circulating FABP4. Circulating FABP4 directly binds to a subset of breast cancer cells via the membrane lipid PIP2. This FABP4/PIP2 interaction activates PKC signaling and enhances mitochondrial ROS production, driving cancer cell migration. these findings identify circulating FABP4 as a critical mediator of obesity-driven metastasis and highlight it as a promising therapeutic target for treating metastatic breast cancer.

overall_design: To separate the FABP4 binding (MDA-F4-1, MDA-F4-2, MDA-F4-3) and FABP4 non-binding (MDA-1, MDA-2, MDA-3) subpopulation, MDA-MB-231 was stained by FABP4 (human recombinant) FITC conjugated. Cell sorting was performed using a BD FACSAria Fusion flow cytometer.

first: Jiaqing

last: Hao

first: Jianyu

last: Yu

first: Bing

last: Li


title: Single-cell RNA sequencing reveals chondrocyte heterogeneity and fibrocartilage transition in osteonecrosis of the femoral head

city: Xi'an

first: Jidong

last: Song

state: Shaanxi

address: NO.157, Xiwu Road

institute: Xi'an Jiaotong University

summary: This study investigates the role of chondrocyte heterogeneity in osteonecrosis of the femoral head (ONFH) through single-cell RNA sequencing of 50,851 chondrocytes from 10 ONFH patients and 10 controls.

overall_design: We performed single-cell RNA sequencing on 50,851 chondrocytes from 10 ONFH patients and 10 normal controls.  Differential gene expression, pseudotemporal trajectory inference, and intercellular communication networks were investigated to delineate chondrocyte subpopulations and their roles in ONFH progression.

first: Tun

first: Kai

first: Xiaoqian

last: Dang

first: Wei


title: Transient YAP/TAZ inhibition exposes therapeutic vulnerabilities in advanced cancers [ATAC-seq]

city: Jena

first: Bjoern

last: von Eyss

address: Beutenbergstr. 11

institute: Leibniz Institute on Aging

summary: YAP and TAZ, key effectors of the Hippo pathway, are frequently hyperactivated in cancer, where they drive tumor progression and resistance to therapy. Their oncogenic activity relies on interaction with TEAD transcription factors, making the TEAD-YAP/TAZ complex an attractive therapeutic target. Using translational mouse models, we demonstrate that sustained systemic YAP/TAZ depletion leads to severe side effects. However, even transient YAP/TAZ inhibition alone is sufficient to suppress tumor growth in advanced stages. Mechanistically, YAP/TAZ activity promotes T cell exclusion from the tumors by inducing target genes involved tissue remodelling. Consequentially, YAP/TAZ inhibition induces immune infiltration, but the infiltrating T cells rapidly become exhausted. Combining YAP/TAZ inhibition with immune checkpoint blockade (ICB) overcomes this exhaustion and sensitizes previously resistant tumors to immunotherapy.

overall_design: Cholangiocarcinomas (CCAs) were induced in mice using hydrodynamic tail vein injection (HDTVI). The driving oncogenes used here, were Notch intracellular domain (NICD) and myristoylated AKT (myr-AKT). The tumors were induced in mice expressing a doxycycline-inducible shRNA construct which is expressed ubiquitously using the CAG-rtTA3 driver. As control, an shRNA targeting Renilla (shRenilla) was used, and two independent lines targeting YAP and TAZ (shYT#1 and shYT#2) were used. The shRNA expression was induced at day 25 after HDTVI and the tumors were analyzed at day 33.

first: Marco

last: Jessen

first: Björn


title: Identification of Targets of Clinical FOXA1 Mutants Involved in EMT and Immune Suppression in Prostate Cancer

city: Valhalla

first: Shuai

last: Gao

state: NY

address: 40 Sunshine Cottage Rd

institute: New York Medical College

summary: FOXA1 is a critical transcription factor in prostate cancer (PCa) progression, functioning as a pioneer factor for the androgen receptor (AR). Its expression is elevated in advanced stages of PCa and plays a central role in oncogenic reprogramming. Recurrent coding mutations in the forkhead DNA-binding domain (FKHD) of FOXA1 become increasingly prevalent in drug-resistant metastatic PCa, particularly clustering in the Wing2 region of FKHD. While recent studies demonstrated that these mutations promote PCa progression, the downstream targets of FKHD mutants remain poorly understood. In this study, we employed three analytical approaches to elucidate these targets: (i) traditional transcriptomic analyses, (ii) chromatin structure–based super-enhancer (SE) profiling, and (iii) immune deconvolution. We identified HPSE, an enzyme that degrades heparan sulfate, as a druggable target activated by FKHD mutants both in vitro and in vivo. SE profiling further revealed that FKHD mutants can reprogram the SE landscape, with FOXP1 emerging as a consistently SE-driven and upregulated gene across multiple FOXA1 mutants. Additionally, FOXA1 mutations were associated with immune suppression in PCa patient samples, and B2M, a key gene in antigen processing and presentation, was notably downregulated in FKHD-mutant tumors. Together, our findings provide new insights into how FOXA1 FKHD mutations drive PCa progression and highlight potential therapeutic targets in tumors harboring these mutations.

overall_design: RNA-seq profiling of 22Rv1 xenograft with wild-type FOXA1 overexpression and FKHD mutant overexpression

first: Mingyu


title: Transient YAP/TAZ inhibition exposes therapeutic vulnerabilities in advanced cancers [CUT&Run]

overall_design: Cholangiocarcinomas (CCAs) were induced in mice using hydrodynamic tail vein injection (HDTVI). The driving oncogenes used here, were Notch intracellular domain (NICD) and myristoylated AKT (myr-AKT). The tumors were induced in mice expressing a doxycycline-inducible shRNA construct which is expressed ubiquitously using the CAG-rtTA3 driver.Here, a mouse shRNA line targeting Renilla (shRenilla) was used.


title: Transient YAP/TAZ inhibition exposes therapeutic vulnerabilities in advanced cancers [scRNA-Seq 2]

overall_design: Cholangiocarcinomas (CCAs) were induced in mice using hydrodynamic tail vein injection (HDTVI). The driving oncogenes used here, were Notch intracellular domain (NICD) and myristoylated AKT (myr-AKT). The tumors were induced in mice expressing a doxycycline-inducible shRNA construct which is expressed ubiquitously using the CAG-rtTA3 driver. As control, an shRNA targeting Renilla (shRenilla) was used, and two independent lines targeting YAP and TAZ (shYT#1 and shYT#2) were used. The shRNA expression was induced at day 25 after HDTVI, anti-PD1 antibody was i.p. injected at day 26,29,32, and the tumors were analyzed at day 33.


title: Transient YAP/TAZ inhibition exposes therapeutic vulnerabilities in advanced cancers [scRNA-Seq 1]


title: Transient YAP/TAZ inhibition exposes therapeutic vulnerabilities in advanced cancers [RNA-Seq]


title: Processing and release of the maize phytocytokine Zip1

city: Köln

first: Gunther

last: Doehlemann

address: Zülpicherstrasse 47a

department: Institute for Plant Sciences

institute: University of Cologne

summary: Phytocytokines are peptide signaling molecules in plant immunity. Several extracellular phytocytokines and their cognate membrane-receptors have been described in various species; however, processing and release of these signals remains largely unknown. The Zea mays immune peptide 1 (Zip1) is a maize-specific phytocytokine, which is associated to the salicylic acid (SA) signaling pathway. Zip1 resides central in its precursor PROZIP1, thus C- and N-terminal cleavage is required to release bioactive Zip1 peptide. Two maize PLCPs, CP1 and CP2, were previously shown to cleave PROZIP1 in the apoplast. We investigated the localization and processing steps of PROZIP1 leading to Zip1 release and found that PROZIP1 undergoes processing in the endoplasmic reticulum (ER) and cytoplasm, where the N‑terminal PROZIP1 domain is cleaved. C-terminal PROZIP1 translocates to the apoplast via an unconventional secretion pathway, likely involving exocyst-positive organelles (EXPO). The combination of antibody detection, protease cleavage assays and mass spectrometry provided evidence for a proteolytic cascade, in which intracellular processing of PROZIP1 is executed by the calcium-dependent metacaspases ZmMC9 through arginine-dependent cleavage. After secretion, C-terminal PROZIP is processed by apoplastic PLCPs, which release, but also degrade the Zip1 peptide. Together, these findings reveal a two-step mechanism of phytocytokine processing, translocation, activation and clearance.

overall_design: Total RNA was extracted from maize leaves vacuum infiltrated with 2mM salicylic acid, 5µM peptide or water with 0.1% DMSO (mock) at 6 hpi.

first: Maurice

last: König

first: Zarah

last: Sorger

first: Melissa

last: Mantz

first: Andreas

last: Perrar

first: Carmen

last: Castell

first: Pitter

last: Huesgen

first: Simon

last: Stael

first: Johana

last: Misas-Villamil


title: From Traditional Decoction to Refined Formulation: Network Pharmacology Unlocks Qingdai Decoction’s Anti-Tumor Network and Enables Molecularly Compatible sTCM Design

city: Shanghai

first: Fuwen

last: Yuan

state: 上海

address: Cailun Road #1200, Pudong New area

institute: Shanghai University of Traditional Chinese Medicine

summary: Traditional Chinese medicine (TCM) formulations like Qingdai Decoction (QDT) offer multi-target therapeutic potential for cancer but suffer from compositional complexity and undefined mechanisms. Integrating computational and experimental approaches is critical to bridge this gap and enable rational optimization. We established an integrative network pharmacology (NP) framework to decode QDT’s anti-tumor mechanisms and design a refined formulation. LC-MS/MS identified 25 bioactive compounds targeting 520 proteins enriched in cancer pathways. Using multi-omics signatures, including transcriptomics, protein-protein interaction networks, and pan-cancer clinical prognosis, we engineered a simplified QDT (sQDT) comprising 15 compounds targeting 75 core targets. In vitro and patient-derived organoid validation demonstrated sQDT’s superior anti-tumor efficacy over traditional QDT (tQDT), with enhanced suppression of proliferation, invasion, and senescence-associated pathways. sQDT also exhibited heightened cancer specificity and minimal toxicity in normal cells. Our study pioneers a "molecular compatibility" strategy for TCM optimization, transforming complex decoctions into target-defined formulations. The NP-driven framework, uniting chemical profiling, multi-omics integration, and clinical bioinformatics, provides a blueprint for elucidating and refining multi-target therapeutics.

overall_design: mRNA profiles of C4-2B cells treated with vehicle, QDT were generated by deep sequencing, in triplicate, using Illumina HiSeq6000

first: Xuehui


title: Checkpoint control of NOTCH via K27-linked ubiquitination by the UVRAG-ITCH-ESCRT pathway

city: Philadelphia

first: Priyankara

last: Wickramasinghe

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: NOTCH signaling is a highly conserved intercellular communication pathway essential for cell fate determination and tissue homeostasis, and its dysregulation contributes to diverse human cancers, including T-cell acute lymphoblastic leukemia (T-ALL)1,2. Although the proteolytic activation of the NOTCH1 receptor is well characterized1, little is known about the mechanisms that regulate the signaling amplitude and response dynamics. Here, we defined the molecular basis by which the activated membrane-tethered NOTCH1 intermediate (NOTCH1ΔE) undergoes stringent checkpoint control mediated by UV radiation resistance-associated gene (UVRAG). We demonstrate that UVRAG acts in concert with the E3 ubiquitin ligase ITCH to specifically bind NOTCH1ΔE and facilitate K27-linked ubiquitination of NOTCH1ΔE at a conserved lysine residue. This ubiquitination directs NOTCH1ΔE toward degradation via the ESCRT-lysosome pathway, limiting the availability of NOTCH1ΔE for subsequent γ-secretase cleavage and nuclear signaling activation. Disruption of the UVRAG-ITCH-ESCRT axis stabilizes NOTCH1ΔE and exacerbated oncogenic signaling in T-ALL with activating NOTCH1 mutations. Re-expression of  UVRAG, suppressed in T-ALL, leads to attenuated NOTCH signaling, inhibited leukemia progression, diminished leukemia-initiating cell populations, and enhanced sensitivity to γ-secretase inhibitor therapies across xenograft, patient-derived, and NOTCH1-drivien murine models of T-ALL. Collectively, our findings uncover a previously unappreciated endo-lysosomal control mechanism governing NOTCH1 signaling activation, and highlight the UVRAG–ITCH–ESCRT axis as a promising therapeutic target in NOTCH-dependent malignancies

overall_design: RNA-seq of mouse spleen under control and Dox treatment

first: Behzad

last: Mansoori

first: Chengyu

last: Liang


title: SUV39H1/2-mediated H3K9me3 restricts TE-induced immune response in Acute Myeloid Leukemia (ATAC-Seq)

city: Chicago

first: Feng

last: Yue

address: 420 E. Superior St.

institute: Northwestern University

summary: Histone 3 Lysine 9 tri-methylation (H3K9me3) is a heterochromatin-associated histone mark deposited broadly across the genome and its regulation is required in normal hematopoiesis. Furthermore, H3K9me3 has been linked with 3D genome organization. However, in the context of acute myeloid leukemia (AML), the regulation of H3K9me3 and its function in 3D genome architecture remained understudied. In this study, through systematic Knockdowns and Knockouts, we identified SUV39H1 and by part its paralogue SUV39H2 as the major H3K9me3 depositors in AML. We demonstrated the role of SUV39H1/2 in establishing global H3K9me3 levels in AML cells and showed H3K9me3 depletion upon SUV39H1/2 Knockout preferentially occurred at sub-megabase level broad peaks and in GC poor regions. Loss of SUV39H1/2-mediated H3K9me3 poised AML cells towards monocyte/macrophage differentiation, induced TE re-activation and interferon response activation, and reduced AML cell growth and clonogenicity in vitro. Analysis of higher-order chromatin architecture reveals that while SUV39H1/2-mediated H3K9me3 is associated with a small fraction of larger chromatin structures, loss of SUV39H1/2 drives concomitant increase of H3K27me3 at B compartments. Taken together, our findings characterize the regulation of SUV39H1/2-mediated H3K9me3 and reveal its role in leukemogenesis and higher-order chromatin structure.

overall_design: ATAC-Seq profiling of U937 derivatives: sgConntrol, SUV39H1 SKO and SUV39H1/2 DKO.

first: Josiah Hiu-yuen

last: Wong

first: Shaojun

last: Yu

first: Yang

last: Cheng

first: Hengqiang

last: Zhao

first: Xintong

last: Chen

first: Yihao

last: Fu

first: Ping

last: Wang

first: Huijue

last: Lyu

first: Alok

last: Swaroop


title: Sequencing analysis of artificial decidual uterus in mice

city: Guangzhou

first: Jinwen

last: Kang

state: Guangdong

address: 483 Wushan Road, Tianhe District

institute: South China Agricultural University

summary: By inducing uterus decidualization in control mice and uterine PIEZO1 knockout mice, the regulation of PIEZO1 ion channel on uterine decidualization was analyzed

overall_design: Using the artificially induced mouse uterine decidualization model, the 2 Days decidual uteri of control mice and knockout mice were collected from the surgical side and non-surgical side, creating a total of 4 groups for in-depth analysis.

first: Renwei

last: Su

first: Yao

last: Wu

first: Xiaozheng

last: Liu

first: Shanshan

last: Song

first: Shuwen

last: Wei


title: Mutant KRAS-Driven Selective mRNA Translation Reveals Mechanisms and Therapeutic Vulnerabilities in Cancer

city: Bronx

first: Kamini

last: Singh

address: 1300 Morris Park Ave, Golding 203

department: Molecular Pharmacology

institute: Albert Einstein College of Medicine

summary: mRNA translation is highly regulated and rapidly controls the functional proteome in response to physiological cues in health and disease. Mutant KRAS signals to ERKs intersect with the mTOR and EIF4E-EIF4F complex, thereby influencing cap-dependent mRNA translation to promote tumorigenesis. With recent clinical advances in KRAS-targeted therapies, it is important to understand the changes in the mRNA translational landscape and its role in cellular adaptations in response to mutant KRAS-targeted therapies. We discover that mutant KRAS controls the translation of a subset of mRNAs and affects the protein production of the translation apparatus. Interestingly, these specific subsets of mRNAs have short, weakly folded 5’UTRs, or harbor low folding energy consensus RNA sequences. This mutant KRAS-dependent mechanism differs from mTOR and EIF4E-EIF4F regulation, which governs cap-dependent translation of mRNAs with highly structured, GC-rich 5' UTRs or TOP motifs. Our findings clarify the unique and indispensable role of mutant KRAS in regulating mRNA translation, distinctly setting it apart from the mTOR and EIF4E-EIF4A-dependent mechanism. Our findings uncover a novel mechanism by which mutant KRAS selectively uncouples the mRNA translation of the translation machinery from the broader mRNA pool, redefining our understanding of the oncogenic regulation of mRNA translation in cancer.

overall_design: We performed ribosome footprinting and RNA sequencing on twelve samples of MiaPaca2 cells and twelve samples of Panc10.05 cells. For MiaPaca2 cells, there are three control DMSO-treated biological replicates (labeled ‘MiaPaca2 DMSO’), three biological replicates (labeled ‘MiaPaca2 Harr’) collected after incubation with harringtonine, three biological replicates collected 2 hours after 10 µM Sotorasib treatment (labeled ‘MiaPaca2 Soto’) and three biological replicates collected 2 hours after 10 µM Sotorasib treatment and incubation with harringtonine (labeled ‘MiaPaca2 Harr’). For Panc10.05 cells ribosome footprinting and RNA sequencing, there are three control DMSO-treated biological replicates (labeled ‘Panc10.05 DMSO-1-x’) paired with three biological replicates (labeled ‘Panc10.05 MRTX1133-1-x’) and another set of three DMSO-treated biological replicates (labeled ‘Panc10.05 DMSO-2-x’) paired with three harringtonine-treated biological replicates (labeled ‘Panc10.05 Harr-2-x’). Data from ribosome protected RNA are labeled as 'RF', and from total RNA as 'RNA'.

first: Ankita

last: Shrivastava

first: Eric

middle: N

last: Pederson

first: Trang

middle: U

last: Nguyen

first: Jacky

last: Chuen

first: Prathibha

last: Mohan

first: Shivangi

last: Pande

first: Ana

last: Toribio

first: Nicolas

last: Lecomte

first: Jerry

last: Melchor

first: John

last: McAuliffe

first: Paul

last: Grandgenett

first: Vinagolu

last: Rajasekhar

first: Kaila

last: Nishikawa

first: Anna

last: Knörlein

first: Yael

last: David

first: Ian

last: Willis

first: Christine

last: Iacobuzio-Donahue

first: Zhengqing

last: Ouyang


title: Sequencing analysis of decidual uterus in mice

first: Jinwen

last: Kang

summary: By inducing uterus decidualization in control mice and uterine PIEZO1 knockout mice, the regulation of PIEZO1 ion channel on decidualization was analyzed

overall_design: Using the artificially induced mouse uterine decidualization model, the uteri of control mice and knockout mice were collected from the surgical side and non-surgical side, creating a total of 4 groups for in-depth analysis.

first: Renwei

last: Su

first: Yao

last: Wu

first: Xiaozheng

last: Liu

first: Shanshan

last: Song

first: Shuwen

last: Wei


title: SUV39H1/2-mediated H3K9me3 restricts TE-induced immune response in Acute Myeloid Leukemia [HiC]

first: Feng

last: Yue

overall_design: RNA-Seq profiling of AML cell line (U937 and THP-1) derivatives: sgConntrol, SUV39H1 SKO and SUV39H1/2 DKO.

first: Josiah Hiu-yuen

last: Wong

first: Shaojun

last: Yu

first: Yang

last: Cheng

first: Hengqiang

last: Zhao

first: Xintong

last: Chen

first: Yihao

last: Fu

first: Ping

last: Wang

first: Huijue

last: Lyu

first: Alok

last: Swaroop


title: SUV39H1/2-mediated H3K9me3 restricts TE-induced immune response in Acute Myeloid Leukemia [RNA-Seq]

first: Yue

last: Feng

first: Wong

last: Josiah Hiu-yuen

first: Yu

last: Shaojun

first: Cheng

last: Yang

first: Zhao

last: Hengqiang

first: Chen

last: Xintong

first: Fu

last: Yihao

first: Wang

last: Ping

first: Lyu

last: Huijue

first: Swaroop

last: Alok


title: Emergency Myelopoiesis in Murine Arthritis Results in Cell-Intrinsic Changes in Dendritic Cell Development and Function

city: MONTREAL

first: David

last: Langlais

state: QC

address: 740 Ave Dr Penfield, rm 4203, McGill Genome Centre

department: Human Genetics

country: Canada

institute: McGill University

summary: Rheumatoid arthritis (RA) is a chronic autoimmune disease that affects up to 0.5-1% of the adult global population.. Immune and inflammatory tissue damage is the main pathogenic mechanism in RA, and involves a hyperactivation of both innate and adaptive immune systems. Trained immunity,  an important feature of the innate immune system,  allows the host to mount functionally altered immune responses based on their previous history of immune exposures, independently of the classical adaptive immunological memory. However, the role of trained immunity in systemic autoimmune and inflammatory disorders remains poorly understood, with the effects on monocyte and macrophage differentiation and function being most widely studied. We demonstrate that emergency myelopoiesis is induced in chronic rheumatoid arthritis in murine models and acts not only to enhance the counts but also to produce functionally altered innate immune cells. Such effects are cell-intrinsic affecting hematopoietic stem and progenitor cells (HSPCs) and persist independently of the inflammatory disease milieu. Importantly, these trained immunity mechanisms impact not only macrophages but also dendritic cells, which are the major professional antigen presenting cells, bridging the innate and adaptive immune responses. Such mechanisms result in changes in the global transcriptional profiles of dendritic cells, and significantly alter their responses to recall immune stimulation and capacity for T cell activation. This study therefore represents the first demonstration of ‘dendritic cell trained immunity’ in rheumatoid arthritis.

overall_design: SGK mice were treated with intraperitoneal zymosan to induce rheumatoid arthritis disease, or with intraperitoneal PBS for controls. Bone marrow-derived dendritic cells (BMDC) were generated from these mice, and stimulated with (1>3)-beta-D-glucan, Poly(I:C), or PBS (unstimulated) for 12 h and processed for bulk RNA-seq to measure the effect of trained immunity  on the transcriptome.

first: Lin Tze

last: Tung

first: Roselyn

last: Jiang

first: Bianca

last: Pozzebon

first: Mathieu

last: Mancini

first: Lucie

last: Droin

first: Mitra

last: Yousefi

first: Joo Eun (June)

last: Kim

first: Marwah

last: Moussa

last: Liang

first: Connie

last: Krawczyk

first: Ines

last: Colmegna

first: Danielle

last: Malo

first: Silvia

last: Vidal

first: Anastasia

last: Nijnik


title: SUV39H1/2-mediated H3K9me3 restricts TE-induced immune response in Acute Myeloid Leukemia [ChIP-Seq]

first: Feng

last: Yue

overall_design: ChIP-Seq profiling of AML cell line U937 derivatives: sgControl, SUV39H1 SKO and SUV39H1/2 DKO.

first: Josiah Hiu-yuen

last: Wong

first: Shaojun

last: Yu

first: Yang

last: Cheng

first: Hengqiang

last: Zhao

first: Xintong

last: Chen

first: Yihao

last: Fu

first: Ping

last: Wang

first: Huijue

last: Lyu

first: Alok

last: Swaroop


title: RNA-sequencing data from mouse kidneys to identify candidate players in kidneys responding to VNS and/or macrophage-specific deletion of Notch2.

city: Charlottesville

first: Vikram

last: Sabapathy

state: Virginia

address: 1340 Jefferson Park Ave., Pinn Hall, Room No: 4043

department: Center for Immunity, Inflammation and Regenerative Medicine

institute: University of Virginia

summary: The cholinergic anti-inflammatory pathway (CAP) plays a central role in neuroimmunomodulation, and its activation by vagus netve stimulation (VNS) protects the kidneys against acute kidney injury (AKI). The molecular mechanisms in immune system underlying anti-inflammatory and tissue-protective effects of the CAP are emerging, and we have found that Notch2 in macrophages mediates those effects of VNS-mediated CAP activation in the context of sepsis-associated AKI. However, the mechanisms in the kidneys have not been well investigated. To identify key players in tissue protection by VNS in the kidneys, we report here the gene expression changes in the kidneys of mice that underwent VNS during LPS-induced AKI. We also used macrophage-specific Notch2 knockout mice for this analysis to examine the relationship between VNS-responsive factors in the kidneys and Notch2 in macrophages.

overall_design: RNA-seq profiling of the kidneys of wild-type (Notch2flox/flox) mice or macrophage-specific Notch2 knockout (Cx3cr1-Cre; Notch2flox/flox) mice subjected to cervical electrical VNS (or sham VNS) and subsequent LPS (5 mg/kg) (or saline) challenge. The experiment consisted of six conditions: 1, Notch2 WT + sham + saline; 2, Notch2 WT + VNS + saline; 3, Notch2 WT + sham + LPS; 4, Notch2 KO + sham + LPS; 5, Notch2 WT + VNS + LPS; 6, Notch2 KO + VNS + LPS. Each condition consisted of three biological replicates.

first: Shuhei

last: Kuwabara

first: Hiro

last: Inoue

first: Mark

last: Okusa


title: RNA-seq of bnp Cheek injection model with skin tbx20 knockout mouse

city: Henan

first: xiaolong

last: Dai

address: kai feng

institute: Henan University

summary: Differential gene expression of skin tbx20 knockout in bnp Cheek injection induced acute pruritus model

overall_design: RNA sequencing of 8  samples was performed. They are 4 samples of skin tbx20 knockout mouse, and 4 samples from wide type

first: Xiaolong

first: Jianghui

last: Meng


title: In vitro drug screening of RAF/MEK/ERK Inhibitors in combination with Autophagy inhibition in the context of two PDA Cell Lines

city: New York

first: Lorenzo

last: Tomassoni

address: 1130 St Nicholas Avenue

institute: Columbia University

summary: In this study 14 different RAF, MEK, or ERK inhibitors were tested in ASPC1 and PANC1 PDA cell lines in combination with two Autophagy Inhibitors

overall_design: Panc-1 and AsPC-1 pancreatic cancer cells were cultured in 96-well tissue culture-treated plates at optimized density, in 100 μl of their optimal media. After 24 h of incubation, the plates were treated with inhibitors for 24h.

first: Urszula

last: Wasko

first: Andrea

last: Califano

first: Kenneth

last: Olive


title: Deletion of Egfr in myeloid cells counteracts an immuno-suppressive tumor microenvironment in CRC

city: Vienna

first: Iros

last: Barozzi

address: Borschkegasse 8a

country: Austria

institute: Medical University Vienna

summary: Analysis of Tumor-promoting role of Egfr+ myeloid cells in CRC by scRNAseq revealed that Egfr signaling in myeloid cells promote CRC development via increased T cell exhaustion.

overall_design: Single-cell RNA-seq of intratumoral immune (CD45+) and non-immune (CD45-) cells, in inflammation-driven (AOM/DSS-induced) murine colorectal cancers (CRC), in mice with Egfr deletion specific for the myeloid lineage, and control mice, generated using 10x Chromium and subjected to high-throughput sequencing.

first: Ourania

last: Fari

first: Angelika

last: Neuhofer

first: Martin

last: Holcmannn

first: Tatiana

last: Chontorotzea

first: Thomas

last: Mohr

first: Bernadette

last: Blauensteiner

first: Dana

last: Krauss

first: Dario

last: Frey

first: Michaela

last: Lang

first: Barbara

last: Helm

first: Ursula

last: Klingmüller

first: Matthias

last: Farlik

first: Marina

last: Sibilia


title: Combination of MEK and Autophagy Inhibition in the preclinical GEMM model of PDAC (KPC mice)

first: Lorenzo

last: Tomassoni

summary: In order to investigate the mechiansim of action and the effect of the combination of MEK/Autophagy Inhibition on pancreatic tumors, KPC mice were treated with Vehicle or combination of Trametinib and Hydroxychloroquine for 24 hours. PDAC tumors were collected and single cell suspension was subjected to single-cell RNA-Sequencing analysis.

overall_design: A total number of six KPC mice were enrolled in the study. Three of them were treated with Vehicle and three of them were treated with the combination of Trametinib and Hydroxychloroquine for 24 hours before being collected for single-cell RNA-Sequencing.

first: Urszula

last: Wasko


title: In vivo double knockout CAR-T screen identifies synergistic gene pairs that enhance anti-tumor immunity.

city: West Haven

first: Sidi

last: Chen

address: 850 West Campus Drive

institute: Yale University

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

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design:
Refer to individual Series.
Note: Donor IDs were changed in the publication as follows: Donor3 -> D1; Donor5 -> D2; Donor6 -> D3.

first: Paul

last: Renauer


title: In vivo double knockout CAR-T screen identifies synergistic gene pairs that enhance anti-tumor immunity [scRNA-seq]

summary: Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of hematological malignancies. However, its efficacy against solid tumors is hindered by multifaceted negative regulatory mechanisms intrinsic to T cells. Although single gene screens have been performed in T cells, such studies cannot provide causality for the complex genetic interactions governing T cell function. To systematically discover genetic interactions that are critical for CAR-T cell’s anti-tumor immunity, here, we perform a high-throughput in vivo double-knockout (DKO) CRISPR screen in human CAR-T cells and identify multiple DKO gene pairs that are both effective and synergistic. All five top-scoring DKO hits are validated to promote CAR-T cell activation, cytotoxicity, degranulation, effector cytokine production, and memory formation, and reduce exhaustion. Mechanistically, these DKOs enhance calcium influx, phospho-ERK, NF-κB and NFAT/AP-1 signaling. Among these hits, NR4A1_SOCS3 DKO has multiple favorable immunological features and showcases the most robust phenotypes. NR4A1_SOCS3 DKO CAR-T cells show potent in vivo anti-tumor efficacy and markedly enhanced infiltration as compared to both single gene knockouts, without increase in safety risk. Whole-transcriptome profiling and single cell RNA sequencing of tumor-infiltrating CAR-T cells reveal that NR4A1_SOCS3 DKO specifically enhanced metabolic fitness of CAR-T cells, rendering them exceptionally potent in long-term tumor control in an orthotopic model, superior to both single gene knockouts and the PD1_CTLA4 dual-checkpoint DKO benchmark. These data together demonstrate the efficacy of high-throughput DKO screening to rapidly uncover critical genetic interactions within T cells, and discover NR4A1_SOCS3 as promising joint intracellular checkpoints to engineer high-performance CAR-T therapies against solid tumors.

overall_design: To examine how NR4A1_SOCS3 DKO, NR4A1 SKO, SOCS3 SKO, and GTC affect the function and differentiation of CAR-T cells inside tumor, we performed single-cell RNA-sequencing (scRNA-seq) on the tumor-infiltrating CAR-T cells isolated from U87-GL-EGFRvIII tumors in all four groups (NR4A1_SOCS3 DKO, NR4A1 SKO, SOCS3 SKO, and GTC).

first: Liqun

last: Zhou


title: In vivo double knockout CAR-T screen identifies synergistic gene pairs that enhance anti-tumor immunity [bulk RNA-seq]

overall_design: To understand the mechanisms by which NR4A1 and SOCS3 deficiency and their interactions directly contribute to CAR-T cells in a cell-intrinsic manner, we profiled the full transcriptomic changes in CAR-T cells before and during chronic stimulation with a time-course bulk mRNA-seq analysis.


title: In vivo double knockout CAR-T screen identifies synergistic gene pairs that enhance anti-tumor immunity [crispr_screen]

overall_design:
We established an in vivo DKO CAR-T screening system using a U87-EGFRvIII solid tumor model. We generated a construct encoding the 2nd generation CAR (anti-EGFRvIII- 4-1BB-CD3ζ). We also utilized a highly immune suppressive U87 glioblastoma cell line engineered to express GFP-luciferase-EGFRvIII (U87-GL-EGFRvIII), which endogenously expresses PD-L1. We then performed an in vivo DKO CAR-T screen by subcutaneously injecting 3 million U87-GL-EGFRvIII cells into NSG mice. After the tumor volumes reached to approximately 100 mm3, we infused 20 million DKO library transduced CAR-T cells (aiming for a library coverage of 400×) into those tumor-bearing mice. We then performed next-generation sequencing (NGS) readout of the DKO screen samples.
Note: Donor IDs were changed in the publication as follows: Donor3 -> D1; Donor5 -> D2; Donor6 -> D3.


title: Chromatin accessibility and transcriptomic changes underlying progression from islet autoantibody positivity to type 1 diabetes

city: Parkville

first: Alexandra

last: Garnham

state: Victoria

address: 1G Royal Parade

country: Australia

institute: Walter and Eliza Hall Institute of Medical Research

overall_design: Refer to individual Series


title: Chromatin accessibility changes underlying progression from islet autoantibody positivity to type 1 diabetes

summary: Type 1 diabetes (T1D) usually has a preclinical phase identified by the presence of circulating autoantibodies to pancreatic islet antigens, and most young children who have multiple autoantibodies progress to diabetes within 10 years. While autoantibodies denote underlying islet autoimmunity, how this process is initiated and then progresses to clinical diabetes on a background of genetic susceptibility is not clearly understood. Only one study has thus far reported changes in gene expression in isolated immune cells associated with progression to islet autoimmunity. We analysed gene expression and chromatin accessibility by ATAC-seq in CD4+ T cells in four genetically at-risk children with islet autoantibodies who progressed to diabetes in ≤ 3 years (‘progressors’), compared to five at-risk children matched for sex, age and HLA who had not progressed (‘non-progressors). In progressors, differentially expressed genes (DEGs) were enriched for cytotoxic genes/pathways and CD4+ TEMRA-like expression signatures. From ATAC-seq data, we generated the first map of chromatin accessibility in T cells of islet autoantibody-positive children. Progression was associated with a subtle reconfiguration of regulatory chromatin regions, and some of the chromatin conformation changes could be linked to progression associated expression changes at cytotoxic genes. In summary, our findings highlight a role for cytotoxic CD4+ T cells in progressive islet immunopathology, and suggest that some of the alterations in chromatin accessibility could underly progression associated gene expression changes

overall_design:
Nine samples of CD4+ T cells were analysed. The samples were obtained from children who are genetically at-risk of developing T1D. Of these children, 5 did not develop T1D while 4 progressed to clinical T1D. Differential accessibility analyses were conducted, comparing samples from those children who progressed to T1D to those that did not.

>>>Submitter states: We are unable to upload raw data files because of privacy issues with human samples. Raw data will be made available on request, subject to approval by our institutional Data Access Committee (dataaccess@wehi.edu.au).<<<

first: Naiara

middle: G

last: Bediaga

first: Lenoard

last: Harrison


title: Transcriptomic changes underlying progression from islet autoantibody positivity to type 1 diabetes

summary: In this study, we we collected RNA-seq data from four different human immune cells (CD4+ T cells, CD8+ T cells,  B cells, NK ) to  assess gene expression changes during type 1 diabetes (T1D) disease progression

overall_design:
To define gene expression changes, we performed RNA-seq analysis of CD4+ T cells, CD8+ T cells, NK cells and B cells in a screening cohort of 10 genetically at-risk children. All children had autoantibodies to two or more islet antigens, five of whom progressed to clinical T1D within 36 months. Sampling was performed 36 months before and at diabetes onset in the progressors. Progressors and non-progressors were matched for age, sex and HLA genotype (DR3,4).  Note that the data for the CD4+ Tcells was generated separately to all other cell types, and that there are multiple sequencing runs within each data set. Differential expression between progressors and non-progressors at each time point was performed independently for each cell type.

>>>Submitter states: We are unable to upload raw data files because of privacy issues with human samples. Raw data will be made available on request, subject to approval by our institutional Data Access Committee (dataaccess@wehi.edu.au).<<<

middle: L


title: Evaluating the cis-regulatory potential of full-length LINE-1 elements

city: Paris

first: Yuvia

middle: Alheli

last: Perez Rico

address: 26 rue d'Ulm

country: France

institute: Institut Curie

summary: Long interspersed element-1 (L1) retrotransposons constitute the largest transposable element (TE) family in mammalian genomes and contribute prominently to inter- and intra-individual genetic variation. Although most L1 elements are inactive, some evolutionary younger insertions remain intact and genetically competent for transcription and occasionally retrotransposition. Despite being generally more abundant in gene-poor regions, intact or full-length L1s (FL-L1) are also enriched around specific classes of genes and on the X chromosome. How proximal FL-L1 may affect nearby gene expression remains unclear. Here, we examine this systematically using engineered mouse embryonic stem cells (ESCs) in which expression of one active L1 subfamily is perturbed. We found that FL-L1 activation leads to the misregulation of ~1,024 genes, whereas FL-L1 repression affects ~81 genes. In most cases, misexpressed genes contain an intronic FL-L1 or lie near a FL-L1 (<260 kb). Gene ontology analysis shows that upon L1 activation, upregulated genes are enriched for neuronal function-related terms, suggesting that some L1 elements may have evolved to control neuronal gene networks. These results illustrate the cis-regulatory potential of FL-L1 elements and suggest a broader role for L1s than originally anticipated.

overall_design:
Gene and repeat expression analysis of total RNA-seq data for hybrid PGK12.1 mouse embryonic stem cells upon repression or activation of L1MdTf repeats.
ChIP-seq of Cas9 in mouse embryonic stem cells with and without DOX-induction of dCas9-VPR effector. Cells were collected from 3 independent clones.

first: Anne-Valerie

last: Gendrel

last: Pérez-Rico


title: Synergistic activation of gingival fibroblasts by Fusobacterium nucleatum and IFNγ is driven by autocrine mechanisms and enhances inflammatory responses of immune cells

status: Public on Jun 16 2026

city: Kraków

first: Mariia

last: Melnykova

address: ul. Gołębia 24

department: Microbiology Department

country: Poland

institute: Uniwersytet Jagielloński

summary: Gingival fibroblasts (GFs) are important sentinel cells which modulate immune responses within inflamed gingival tissue in response to oral bacteria and inflammatory mediators during periodontitis. Yet, the mechanisms governing GF-immune cell cross-talk remain poorly defined. Here, we investigated how the interaction between the oral pathobiont Fusobacterium nucleatum and the proinflammatory cytokine interferon-γ (IFNγ) modulates primary human GF inflammatory responses and interactions with immune cells. Transcriptomic analysis using RNA-seq revealed a unique transcriptional program induced in GFs infected with F. nucleatum in the presence of IFNγ. This program was characterized by synergistic upregulation of multiple genes involved in immune cells recruitment and activation, including CXC and CC family chemokines, colony stimulating factors, and components of the TNF pathway. The synergistic effect was specific to GFs and was not observed in keratinocytes or macrophages. However, it was not restricted to F. nucleatum, as Porphyromonas gingivalis and Filifactor alocis also synergized with IFNγ to induce CXCL9, CXCL10, and CXCL11 expression in GFs. These transcriptional changes had pronounced functional consequences: conditioned media from GFs exposed to both F. nucleatum and IFNγ enhanced neutrophil viability and activation, as shown by increased CD11b surface expression, and promoted a proinflammatory macrophage phenotype, characterized by reduced CD163 and increased CD80 expression. Mechanistically, the synergistic activation of GFs by F. nucleatum and IFNγ was driven by an autocrine TNF-dependent loop that promoted cross-talk between STAT1, c-Jun N-terminal kinase (JNK), and NFκB signaling pathways, leading to amplification of inflammatory gene expression. Collectively, our findings identify an autocrine TNF-dependent signaling circuit that integrates bacterial and IFNγ-derived signals in GFs to orchestrate stromal–immune cell communication, which may contribute to excessive activation of neutrophils and macrophages during periodontitis.

overall_design: RNA-seq profiling of primary gingival fibroblasts isolated from healthy donors, cultured and infected with *Fusobacterium nucleatum*, stimulated with interferon-gamma, or treated with both simultaneously, to assess transcriptional differences.

first: Aureliusz

last: Schuster

first: Aleksander

last: Grabiec


title: A Pseudomonas aeruginosa LysR-type transcriptional regulator, PA3321, essential for productive biofilm and influences virulence and pathogenesis

city: Honolulu

first: Hung

last: Vo

state: Hawaii

address: 1800 East-West Road Life Sciences Building, Rm 318

department: Microbiology

institute: University of Hawaii at Manoa

summary: The transposon insertion mutation of regulator PA3321 (PA3321∷Tn) significantly reduced biofilm formation, while enhancing virulence in Drosophila melanogaster fruit fly and BALB/C mouse infection models. This regulator influenced bacterial motility, c-di-GMP signaling, and the RhlI-RhlR and Pseudomonas quinolone signal (PQS) quorum-sensing (QS) systems. We used  RNA-seq assays to identify the regulatory mechanisms of PA3321. We observed that 391 genes were up-regulated and 114 genes were down-regulated in the absence of PA3321, many of which are involved in rhamnolipid synthesis, type II, type III, and type VI secretion systems, and QS systems.

overall_design: The mutant PA3321::Tn and the wt PA01 strain were grown overnight in LB broth, diluted 100-fold into fresh LB broth, and grown in the same biofilm growth condition (30oC, no shake, 48 hours), as described above. Bacteria were harvested, and total RNA was isolated using the Qiagen RNeasy Mini Kit (Qiagen), with on-column DNase I treatment; rRNA was depleted using RiboZero (Epicentre®); and Illumina® NGS libraries were created and sequenced at TUCF Genomics (http://tucf-genomics.tufts.edu/). All transcriptomic analyses were done in biological triplicate. The transcriptional profiles of the mutant PA3321∷Tn were compared with those of wt PA01 to identify the specific sets of genes that the regulator PA3321 controls. RNA-seq results were analyzed using software Rockhopper (https://cs.wellesley.edu/~btjaden/Rockhopper/), which was designed for analysis of bacterial RNA-seq data

first: Yun

last: Heacock-Kang

first: Jan

last: Zarzycki-Siek

first: Ian

last: A. McMillan

first: Tung

middle: T

last: Hoang


title: Single-cell transcriptomic profiling of 4-NQO-induced oral squamous cell carcinoma in a mouse model

city: Hung Hom

first: Xiaofan

last: Xu

address: 11 Yuk Choi Rd

country: Hong Kong

institute: The Hong Kong Polytechnic University

summary: To investigate the cellular heterogeneity and molecular drivers of oral squamous cell carcinoma (OSCC), we performed single-cell RNA sequencing (scRNA-seq) on tongue tissues from a 4-nitroquinoline 1-oxide (4-NQO)-induced mouse model. This dataset revealed eight distinct cell populations, with a significant reduction in epithelial cells and an increase in fibroblasts in OSCC compared to controls. Notably, a distinct Col1a1⁺ epithelial subpopulation was identified that exhibited the highest epithelial-mesenchymal transition (EMT) signature and was significantly expanded in OSCC. This study provides a valuable resource for understanding the transcriptional landscape and cellular dynamics underlying OSCC progression.

overall_design: Healthy control group (n=3 mice) and 4-NQO-treated OSCC group (n=3 mice). Tongues were harvested after 16 weeks of treatment.

first: Xing

last: Liu

first: Yalun

last: Li

first: Lanlan

last: Gao

first: Rihui

last: Lu

first: Yongjin

last: Fang

first: Xiao

last: Yang


title: Integrative single-cell multi-omics profiling of human pancreatic islets identifies T1D-associated genes and regulatory signals  [single cell]

city: St Louis

first: Ricardo

last: D'Oliveira Albanus

state: MO

address: 4370 Duncan Avenue, Suite 6306D, Jeffrey T. Fort Neuroscience Bdg.

department: Neurology

institute: Washington University School of Medicine

summary: Genome-wide association studies (GWAS) have identified over 100 signals associated with type 1 diabetes (T1D). However, it has been challenging to translate any given T1D GWAS signal into mechanistic insights, such as causal variants, their target genes, and the specific cell types involved. Here, we present a comprehensive multi-omic integrative analysis of single-cell/nucleus resolution profiles of gene expression and chromatin accessibility in human pancreatic islets under baseline and T1D-stimulating conditions. We nominate effector cell types for all T1D GWAS signals and the regulatory elements and genes for three independent T1D signals acting through beta cells at the DLK1/MEG3, RASGRP1, and TOX loci. We validated the functional impact of these genes and regulatory regions using isogenic human embryonic stem cells (hESCs). Loss of RASGRP1 or DLK1, as well as disruption of their corresponding regulatory regions, led to increased beta cell apoptosis, and beta cells derived from isogenic hESCs carrying the T1D risk allele of rs3783355 showed elevated beta cell death. Through additional RNA-seq and ATAC-seq analyses, we identified five genes upregulated in both RASGRP1-/- and DLK1-/- beta-like cells, four of which are near T1D GWAS signals.

overall_design: Single-cell RNA-seq and single-nucleus ATAC-seq were performed on human pancreatic islets from 8 donors using the 10X Genomics Chromium platform. For three donors (HPAP059, ICRH134, ICRH135), islets were cultured under three conditions: baseline/mock, cytokine stimulation, and Coxsackievirus B4 (CVB4) infection, to model T1D-stimulating conditions. Remaining donors were profiled under baseline conditions only. Libraries were prepared using 10X Genomics scRNA-seq (3' v3) and scATAC-seq (v1) chemistries and sequenced on Illumina NovaSeq instruments.

first: Xiaoshan

last: Zhang

first: Zeping

last: Zhao

first: Stephen

last: Parker

first: Shuibing

last: Chen