title: RBM39 Modulates UPR Signaling Through Alternative Splicing of IRE1α/ERN1 [siERN1_Rescue_RNA-Seq]

city: Frederick

first: Thomas

last: Gonatopoulos-Pournatzis

state: Maryland

address: 1050 Boyles Street

institute: National Cancer Institute

summary: The unfolded protein response (UPR) preserves endoplasmic reticulum proteostasis through coordinated signaling pathways, including the IRE1α-XBP1 axis, which promotes adaptive transcriptional programs via non-canonical XBP1 mRNA splicing. However, upstream mechanisms regulating this pathway remain incompletely defined. Here, we apply CRASP-Seq, a scalable RNA-coupled CRISPR screening platform, to systematically identify regulators of XBP1 splicing. We uncover the U2 snRNP auxiliary factor RBM39 as a critical positive regulator of this process. Perturbation of RBM39 or U2 snRNP components induces alternative splicing of ERN1, leading to exon-18 skipping and the production of an unstable transcript subject to nonsense-mediated decay, as well as a truncated IRE1α isoform that acts in a dominant-negative manner to suppress XBP1 splicing. Mechanistically, we show that heat shock reduces RBM39 functional activity and promotes ERN1 exon-18 skipping, thereby attenuating IRE1α–XBP1 signaling. Functionally, hyperactivation of this pathway is detrimental under proteotoxic stress, suggesting that exon-18 skipping serves as a stress-adaptive mechanism to limit UPR output. Together, our findings reveal a previously unrecognized regulatory axis linking the canonical splicing machinery to UPR signaling and establish alternative splicing of ERN1 as a key modulator of cellular stress responses.

overall_design: mRNA-Seq experiments were performed in HEK293T cells treated with siERN1 for 48h with stable overexpression of either ERN1-FL or an ERN1-ΔE18 followed byTunicamycin treatment to study the regulatory mechanisms underlying unfolded protein response (UPR) in cell.

first: Jeongjin

last: Kim

first: Amit

middle: K

last: Behera

first: Arun

last: Damodaran

first: Shreya

last: Kordale


title: RBM39 Modulates UPR Signaling Through Alternative Splicing of IRE1α/ERN1 [Spliceosome_Knockdown_RNA-Seq]

status: Public on Apr 27 2026

country: USA

type: Expression profiling by high throughput sequencing

overall_design: mRNA-Seq experiments were performed in HAP1 cells with knockdown of different splicesomal components with siRNAs along with Tunicamycin treatments to study gene regulations mediated by splicing factors in the context of unfolded protein response (UPR) in cell.

middle: J

middle: P


title: Molecular Drivers of Neuroinflammation in Amyotrophic Lateral Sclerosis

status: Public on Apr 28 2026

city: Chicago

first: David

last: Gate

state: IL

address: 745 N Fairbanks Ct

department: Neurology

institute: Northwestern University

summary: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder marked by progressive motor neuron (MN) degeneration in the brain and spinal cord. Although neuroinflammation is increasingly recognized as a hallmark of ALS, the precise molecular programs linking immune responses to MN pathology remain poorly defined. Using an integrated approach that combines single-cell and bulk RNA sequencing with spatial proteogenomics, we characterized both shared and distinct immune dynamics in peripheral blood and spinal cord tissues from sporadic ALS patients and patients carrying C9orf72 repeat expansions. Our analysis revealed broad immune remodeling in C9orf72-ALS, ALS subtype-specific and progression-associated differences in monocyte activation, and antigen-experienced CD8 effector memory T cells with clonal features consistent with antigen-driven responses. Spatial mapping revealed complement activation and lipid-programmed myeloid states converging at sites of MN loss and TDP-43 pathology. Together, these findings connect peripheral and central immune alterations to ALS heterogeneity and highlight stratified immunomodulation as a potential therapeutic strategy.

overall_design: 18 sections of cervical spinal cords from 9 controls, 4 sporadic cases and 5 C9orf72 cases.

first: Zhang

last: Ziyang

first: Gate

last: David


title: RBM39 Modulates UPR Signaling Through Alternative Splicing of IRE1α/ERN1 [CRASP-Seq]

overall_design: CRASP-Seq assays of XBP1 splicing minigene reporter was performed in three different cell lines (HAP1, RPE1, and HepG2) with Tunicamycin treatment condition and untreated control condition to identify splicing regulators relevant to UPR (unfolded protein response) signaling.


title: Spatiotemporal regulation of arbuscular mycorrhizal symbiosis at cellular resolution [MC]

city: Cambridge

first: Gabriel

last: Ferreras-Garrucho

state: Cambridgeshire

address: 93 Lawrence Weaver Rd

department: Crop Science Centre, Department of Plant Sciences

country: United Kingdom

institute: University of Cambridge

summary: Arbuscular mycorrhizal (AM) symbiosis develops through successive colonization of root epidermal and cortical cells, culminating in the formation of arbuscules, tree-like intracellular structures that are transient yet essential sites of nutrient exchange. To dissect the cellular and structural complexity of AM establishment in rice roots colonized by Rhizophagus irregularis, we applied dual-species spatial transcriptomics to simultaneously monitor plant and fungal gene expression at single-cell resolution. This approach revealed surprising differences in transcriptional activity between fungal structures and showed that morphologically similar arbuscules can be transcriptionally distinct. These findings suggest hidden functional diversity among arbuscules, indicating that nutrient exchange is not uniform but finely modulated across individual structures. Because arbuscules form and degenerate within only a few days, we further sought to capture translational regulation across their life span. We pioneered AM-inducible TRAP-seq (Translating Ribosome Affinity Purification followed by RNA-seq) using stage-specific promoters, enabling cell-type- and stage-resolved profiling for the first time in AM symbiosis. This revealed extensive spatiotemporal reprogramming of nutrient transport and signaling, with distinct sets of phosphate, nitrogen, and carbon transporters and regulators upregulated or repressed at different stages of arbuscule development, suggesting that nutrient exchange is dynamically regulated across the arbuscule life cycle. More broadly, cell wall biosynthesis genes and key defence markers were suppressed during arbuscule formation, whereas upon collapse, defence markers were strongly upregulated, suggesting a host-driven shift towards symbiosis termination. Together, these findings highlight the nuanced and dynamic regulation of AM symbiosis at cellular resolution, refining our understanding of how nutrient exchange and fungal development are coordinated in space and time, and providing a framework for further functional exploration.

sample_organism:
Oryza sativa
Rhizophagus irregularis

overall_design: Molecular Cartography (Resolve Biosciences), using a panel of 100 probes against transcripts of Oryza sativa (rice) and the arbuscular mycorrhizal fungus Rhizophagus irregularis, for crown roots (CR) and large lateral roots (LLR) of rice either colonised (+Ri) or not colonised (-Ri) by R. irregularis at 6 weeks post-inoculation.

first: Tania

last: Chancellor

first: Garo

last: Akmakjian

first: Héctor

last: Montero

first: Emma

last: Wallington

first: Samik

last: Bhattacharya

first: Christian

last: Korfhage

first: Julia

last: Bailey-Serres

first: Uta

last: Paszkowski


title: Pioneer-factor activity requires stable chromatin occupancy mediated by both sequence-specific binding and disordered protein domains [ChIP-seq]

city: Madison

first: Melissa

middle: M

last: Harrison

state: WI

address: 420 Henry Mall

department: Biomolecular Chemistry

institute: University of Wisconsin Madison

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Pioneer transcription factors overcome the restrictive barrier imposed by chromatin to drive cell-fate specification, yet how their domains collectively support this activity remains unclear. Here, we use the deeply conserved pioneer factor Grainy head to define the protein-intrinsic features that govern pioneering activity. By integrating biochemistry, genomics and quantitative live-cell imaging, we determined that both the conserved DNA-binding domain and the extended, intrinsically disordered N-terminus are required for the stable chromatin occupancy that supports access to closed chromatin and the induction of chromatin accessibility. The disordered N-terminus supports pioneer activity through interactions that do not rely on strict amino acid sequence but instead overall composition. While our results show that pioneering activity depends on the combinatorial contributions of structured and disordered domains, mitotic retention depends solely on sequence-specific DNA binding. These results support stable chromatin occupancy mediated by multiple protein domains as necessary for pioneering function and that this is separable from the mechanisms required for mitotic retention.

overall_design: This data set contains ChIP-seq samples from S2 cells. Stable cell lines containing transgenes under a copper inducible promoter were induced with CuSO4. ChIP-seq was done using antibodies that recognize Grh and HA. Stable cell lines contained various Grh transgenes including wt Grh, HA-Grh, HA-GRHL2, HA-GRHL2.Grh, HA-Grh.GRHL2, Grh_Scrambled, Grh_ScrambledwSD, GrhC800AK807A, or GrhR806A.

first: Meghan

last: Freund

first: Javier

last: deHaro-Arbona

first: Sarah

last: Baloul

first: Ali

last: Torhorst

first: Charalambos

last: Roussos

first: Abby

last: Ruffridge

first: Andrew

middle: Q

last: Rashoff

first: Ryen

last: Hazzard

first: Peter

middle: W

last: Lewis


title: RBM39 Modulates UPR Signaling Through Alternative Splicing of IRE1α/ERN1 [Spliceosome_Inhibition_RNA-Seq]

first: Thomas

last: Gonatopoulos-Pournatzis

overall_design: mRNA-Seq screens were performed in HAP1 cells with inhibition of different splicesomal components in addition to Tunicamycin treatments to study effect of unfolded protein response (UPR).

first: Jeongjin

last: Kim

first: Amit

last: Behera

first: Arun

last: Damodaran

first: Shreya

last: Kordale


title: Pioneer-factor activity requires stable chromatin occupancy mediated by both sequence-specific binding and disordered protein domains [ATAC-seq]

overall_design: This data set contains ATAC-seq samples from S2 cells. Stable cell lines containing transgenes under a copper inducible promoter were induced with CuSO4. Uniduced cells were harvested as a control. Stable cell lines contained various Grh transgenes including wt Grh, HA-Grh, HA-GRHL2, HA-GRHL2.Grh, HA-Grh.GRHL2, Grh_Scrambled, Grh_ScrambledwSD, GrhC800AK807A, or GrhR806A.


title: Chromosomal instability drives ECM remodelling and drug vulnerabilities in the first Afro-Caribbean breast cancer cell lines

city: Kingston

first: Simone

last: Badal

address: Mona, Kingston 7

department: Department of Basic Medical Sciences

country: Jamaica

institute: University of the West Indies

summary: Women of African ancestry develop more aggressive breast cancer (BCa) with poorer survival outcomes, yet only 8% of available cell lines represent this population, impeding targeted treatment development. Here, we aimed to establish and characterize new cell lines from an Afro-Caribbean patient to better understand population-specific BCa biology. We developed three lines (ACRJ-BC24 parent, α, and β) from a patient with 100% African ancestry. Karyotype analysis revealed progressive chromosomal instability, with the β-clone showing X-11 translocations correlating with higher Ki-67 expression and enhanced tumorigenic capacity. Immunohistochemistry and immunoblotting demonstrated their transition from hormone-positive to triple-negative phenotypes. Transcriptional profiling identified significant enrichment in extracellular matrix organization pathways mechanistically linked to chromosomal instability, explaining their distinct drug responses. The β-clone's enhanced sensitivity to PARP inhibitors correlates with its chromosomal abnormalities, while the parent line's sensitivity to gemcitabine possibly relates to ECM-mediated nucleoside transporter regulation. These lines provide valuable tools for studying BCa disparities.

overall_design: RNA-seq profiling of breast cancer cell lines from a Jamaican population. This included the original samples and two clones (alpha and beta)

first: Camille

last: Ragin

first: Albert

last: Doughan

first: Shannique

last: Clarke

first: Isabella

last: Allen

first: Jovanka

first: Sophia

last: George


title: Elimination of myotonia improves myopathy in a muscleblind knockout model of myotonic dystrophy: Deep RNAseq of quadriceps muscle to assess myotonia dependent changes in transcript expression and splicing.

city: Rochester

first: John

middle: D

last: Lueck

state: NY

address: 601 Elmwood Ave

department: Pharmacology and Physiology

institute: University of Rochester

summary: A cardinal sign of myotonic dystrophy type 1 (DM1) is myotonia, slow muscle relaxation after voluntary contraction. Myotonia results from mis-regulated splicing of chloride channel 1 (ClC-1), leading to loss of channel function and runs of involuntary action potentials in muscle fibers. Heralding the onset of weakness, myotonia is often the first symptom of DM1, and thus this raises the possibility that muscle hyperexcitability promotes the subsequent development of myopathy. We used genome editing to test this possibility by deleting the alternatively spliced and frameshift inducing ClC-1 exon 7a (E7a) in the Mbnl1 knockout model of DM1. Although several ClC-1 exons exhibit mis-regulated splicing in DM1, deletion of this single cryptic exon was sufficient to restore ClC-1 function and eliminate myotonia systemically and permanently. Through direct comparison of quadriceps muscle from myotonic (Mbnl1 knockout) and non-myotonic (Mbnl1 knockout; Clcn1 delta E7a) DM1 model mice, we were able to observe significant changes in transcript expression and splicing regulated by muscle hyperexcibility.

overall_design: Total RNA was isolated from quadriceps muscle from 4-6 month-old WT, Clcn1 delta E7a, Mbnl1 knockout, and Mbnl1 knockout; Clcn1 delta E7a mice with 4 biological replicates per group. This allowed for assessment of transcript expression and splicing changes between myotonic Mbnl1 KO and non-myotonic Mbnl1 KO; Clcn1 delta E7a with WT and Clcn1 delta E7a controls.

first: Matthew

middle: T

last: Sipple

first: Vanessa

last: Todorow

last: Meinke

first: Charles

middle: A

last: Thornton


title: Elimination of myotonia improves myopathy in a muscleblind knockout model of myotonic dystrophy: longread sequencing of Clcn1 transcripts in muscle from Mbnl1 knockout, Exon 7a deletion, and ADR mice.

middle: Thomas

state: New York

summary: A cardinal sign of myotonic dystrophy type 1 (DM1) is myotonia, slow muscle relaxation after voluntary contraction. Myotonia results from mis-regulated splicing of chloride channel 1 (ClC-1), leading to loss of channel function and runs of involuntary action potentials in muscle fibers. Heralding the onset of weakness, myotonia is often the first symptom of DM1, and thus this raises the possibility that muscle hyperexcitability promotes the subsequent development of myopathy. We used genome editing to test this possibility by deleting the alternatively spliced and frameshift inducing ClC-1 exon 7a (E7a) in the Mbnl1 knockout model of DM1. Although several ClC-1 exons exhibit mis-regulated splicing in DM1, deletion of this single cryptic exon was sufficient to restore ClC-1 function and eliminate myotonia systemically and permanently. We utilized long-read sequencing to assess transcript isoform utilization in mice with Mbnl1 knockout, deletion of E7a reduced the frequency of other splicing defects in ClC-1 transcripts, likely as a passive consequence of restoring reading frame and nonsense surveillance.

overall_design: To assess the impact of Mbnl1 knockout, deletion of Clcn1 exon 7a, and constitutive NMD on Clcn1 transcript splicing and isoform utilization targeted long-read (PacBio) sequencing of libraries of Clcn1 transcripts amplified from exon 1 to exon 23 isolated from quadriceps muscle from early and late postnatal mice of WT, Clcn1 delta E7a, Mbnl1 knockout, Mbnl1 knockout; Clcn1 delta E7a, and adr-mto2J mice was completed. Experiments were conducted at P7 and P18 with each timepoint/genotype in biological triplicate. Results were analyzed for changes in individual alternative splice events in Clcn1 as well as transcript isoform utilization.


title: Bulk RNA-seq Toxic Lipid-induced Epigenetic Activation of ICAM1 in Liver Sinusoidal Endothelium Regulates Myeloid-Driven Fibro-inflammatory Response in MASH

first: Alexandra

last: Krivonos

state: MN

address: 200 1st St SW

department: Quantitative Health Sciences

institute: Mayo Clinic

summary: Background: Liver sinusoidal endothelial cells (LSECs) acquire a proinflammatory phenotype in metabolic dysfunction-associated steatohepatitis (MASH), characterized by elevated adhesion molecules and inflammatory mediators—a process termed lipotoxic endotheliopathy. However, the molecular drivers of this transformation remain incompletely defined. Methods: We employed complementary models of lipotoxicity and MASH, including palmitate-treated primary human LSECs (in vitro), cultured precision-cut liver slices from normal human and mice liver with MASH-inducing media as well as MASH mice (ex vivo), and diet-induced MASH models (in vivo). Results: GeoMx digital spatial profiling of human MASH livers revealed enriched proinflammatory and fibrogenic signaling between LSECs and myeloid cells. Among the top upregulated genes and ligand-receptor pairs was ICAM1, whose expression correlated with disease severity and was increased in human MASH by immunostaining. ATAC-seq on primary mouse LSECs showed enhanced chromatin accessibility at the Icam1 promoter in MASH. Chromatin immunoprecipitation confirmed enrichment of the active epigenetic mark H3K27ac and BRD4 binding at the ICAM1 promoter in human LSECs under lipotoxic stress. Mechanistically, we identified the GSK3β/c-Jun/BRD4 axis as a key regulator of ICAM1 induction in LSECs. In vivo, ICAM1-neutralizing antibody or endothelial-specific epigenetic suppression reduced hepatic inflammation, injury, and fibrosis in MASH mice. Conclusion: ICAM1 is epigenetically upregulated in LSECs during lipotoxic stress and promotes myeloid cell recruitment in MASH. Targeting the epigenetic regulation of ICAM1 may offer a novel therapeutic strategy for treating human MASH.

overall_design: Primary human LSECs (HHSEC) treated with vehicle, 500µM PA, 800µM PA or 800 µM PA+LY, overnight. Total RNA was prepared for bulk RNA-sequencing.

first: Qianqian

last: Guo

first: Nantawat

last: Satthawiwat

first: Akitoshi

last: Sano

first: Chady

last: Meroueh

first: Asma

last: Hamdi

first: Xin

last: Dai

first: Khaled

last: Warasnhe

first: Alexander

last: Washington

first: Patrick

last: Starlinger

first: Alex

first: Sheng

last: Cao

first: Enis

last: Kostallari

first: Johan

last: Jonker

first: Bart

last: van de Sluis

first: Feda

middle: H

last: Hamdan

first: Samar

last: Ibahim


title: RNA-seq of SW620 colorectal cancer cells: control (sh-control) versus ANO1 (TMEM16A) knockdown (sh-ANO1)

city: Changchun

first: Sui

last: Yujie

address: 265 Ziqiang Street

country: China

institute: The Second Hospital of Jilin University

summary:
The calcium-activated chloride channel ANO1 (TMEM16A) is overexpressed in colorectal cancer (CRC) and associated with poor prognosis, but its role in regulating metastatic plasticity is not fully understood. To elucidate the transcriptomic networks governed by ANO1, we performed RNA sequencing on SW620 CRC cells stably expressing sh-control or sh-ANO1. Total RNA was extracted using Trizol reagent. Sequencing libraries were constructed with the NEBNext® Ultra RNA Library Prep Kit for Illumina® and sequenced on an Illumina Novaseq platform, generating 125-150 bp paired-end reads. Bioinformatics analysis included read alignment with HISAT2 (v2.0.5) and differential expression analysis using DESeq2 (v1.20.0) with a threshold of |log2(Fold Change)| ≥ 1 and adjusted p-value < 0.05. Functional enrichment analysis for Gene Ontology (GO) and KEGG pathways was performed with clusterProfiler (v3.8.1). Gene Set Enrichment Analysis (GSEA) was conducted using gene sets from MSigDB.
Transcriptomic profiling identified 1,894 differentially expressed genes (DEGs) upon ANO1 knockdown, with 1,523 downregulated and 371 upregulated. Downregulated genes included key EMT and signaling components (e.g., PIK3C2A, PIK3CA, KRAS, ZEB1). GO analysis revealed enrichment in processes related to genomic integrity and cell division. KEGG pathway analysis highlighted significant enrichment in “Homologous recombination” and “Platinum drug resistance”. A trend-level downregulation was observed for the “mTOR signaling pathway”. This dataset illustrates that ANO1 knockdown drives broad changes in a pro-metastatic transcriptome network and implicates mTOR signaling as a potential downstream mechanism, providing a resource for understanding how ANO1 promotes CRC progression.

overall_design: RNA-seq profiling of SW620 cells stably transfected with sh-control and their knockdown derivatives (sh-ANO1)

first: Fei

last: Wu

first: Kun

last: Zhang

first: Zhenwu

last: Du

last: Li

first: Longfei

last: Yang

first: Linxian

last: Zhao

first: Bo

last: Cheng

first: Lili

last: Nie

first: Qiwei

first: Yujie

last: Sui


title: Deciphering the Single-Cell Molecular Landscape of Ampullary Cancer: A Rare Gastrointestinal malignancy

city: Toulouse

first: Juan Pablo

last: Cerapio

address: Av 2 Hubert Curien

country: France

institute: CRCT

summary: Ampullary carcinoma (AC) is a rare epithelial cancer, representing around 0.2% of all gastrointestinal malignancies worldwide, with an estimated incidence of 0.73 per 100,000 people. The rarity of AC, combined with the intricate anatomy of its site of origin, often complicates histopathological diagnosis. Recent advances in molecular research, however, have begun to illuminate its underlying biological complexity. In this study, we present the first comprehensive single-cell transcriptomic atlas of AC, encompassing both malignant epithelial populations and the tumor immune microenvironment (TIME). We identified upregulation of oncogenic drivers previously linked to AC, along with an AC-specific transcriptional program featuring potential biomarkers for minimally invasive diagnostics. Furthermore, we characterized an immunosuppressive TIME, enriched in anti-inflammatory-like TAMs and T cell subsets with limited antitumor activity. Collectively, these findings provide a foundational resource for the identification and validation of candidate biomarkers for early detection and targeted therapies in AC.

overall_design: Fresh samples of ampullary carcinama from three patients, and two fresh samples of ampulla of vater from healthy donors were used to perform a 10X genomics single cell RNA-Sequencing 3' assay.

last: Cerapio Arroyo

first: Karina

last: Cancino Maldonado

first: Stéphane

last: Bertani


title: Post-Hoc Trial and Preclinical Data Identify IL1RAP as a Tumor Microenvironment Vulnerability to Improve Chemoimmunotherapy Sensitivity in Pancreatic Cancer

city: Miami

first: Jashodeep

last: Datta

state: FL

address: 1425 NW 10th Ave

department: Surgery

institute: University of Miami Miller School of Medicine

summary: INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) remains highly resistant to chemotherapy and immunotherapy due to a tumor microenvironment (TME) dominated by myeloid-stromal immunosuppressive circuitries and T-cell dysfunction. Key mediators of this chemoimmunoresistance are interleukin-1 (IL-1) family cytokines (IL-1α/β, IL-33, IL-36), which converge on IL-1 receptor accessory protein (IL1RAP) signaling to reinforce a tolerogenic, pro-inflammatory TME. We hypothesized that IL1RAP-expressing myeloid-stromal compartments sustain a therapeutic barrier in PDAC, and that disrupting this via pharmacologic IL1RAP inhibition could reprogram the TME to enhance chemoimmunotherapy efficacy. METHODS: Chemoresistance in PDAC samples from Human Tumor Atlas Network (HTAN) was stratified by cell-type IL1RAP expression. Clinical outcomes in patients treated with anti-IL1RAP Ab nadunolimab (Nadu)+gemcitabine+nab-paclitaxel (GnP) in Phase II CANFOUR trial (NCT03267316) were stratified by immune and stromal IL1RAP expression; paired PDAC biopsies pre-/post-treatment were analyzed by mIF. Preclinical studies were conducted in the IL1RAPhi stromatogenic Ptf1aCre/+;LSL-KrasG12D;Tgfbr2fl/fl (PKT) murine model. Nadu surrogate Ab-treated tumors were analyzed by scRNAseq, flow cytometry, and histology. Combination Nadu, GnP, and anti-PD1 therapy was tested in survival \ studies. RESULTS: In HTAN dataset, IL1RAP expression was elevated across multiple TME compartments—myeloid, cancer-associated fibroblast (CAF), and tumor cells; notably, IL1RAP was selectively enriched in chemotherapy-resistant (vs sensitive) samples, predominantly in myeloid and CAF/stromal transcriptomes. Stratification of evaluable specimens from CANFOUR trial revealed correlation between high stromal/CAF (p=0.0058) and myeloid (p=0.014) IL1RAP expression and prolonged duration of response to Nadu+GnP. In PKT mice, Nadu treatment reduced tumor volume (p=0.019) and stromal fibrosis. Flow cytometry showed reduced trafficking/persistence (p=0.0077) and increased T-cell abundance (p=0.0033), which were corroborated by mIF in paired human PDAC biopsies pre/post Nadu treatment, showing diminished CD11b+CD14+/CD15+ myeloid infiltration and expansion of GzmB+Ki67+CD8+ T-cells. scRNAseq in PKT tumors revealed reprogramming in myeloid (↑antigen presentation and Type IIFN response; ↓IL-1/TNF signaling) and T-cell (↑activation/memory, TCR engagement, IL-2 signatures; ↓regulatory/exhausted states) subsets. Phenotypic skewing toward memory progenitor-exhausted CD8+ subsets (Ly108+CD69+/-) supported enhanced immune responsiveness. Triple therapy with Nadu, GnP, and anti-PD-1 significantly prolonged survival versus controls (p<0.001). CONCLUSIONS: IL1RAP-expressing myeloid-stromal networks may represent a distinct therapeutic barrier in PDAC, which can be disrupted pharmacologically to invigorate immunotherapy-permissive CD8+ T-cell subsets and improve chemoimmunotherapy sensitivity. These findings support an upcoming neoadjuvant trial combining nadunolimab with chemoimmunotherapy in patients with operable PDAC.

overall_design: RNA-seq of Ptf1aCre/+;LSL-KrasG12D;Tgfbr2fl/fl (PKT) murine tumors treated with mNadunolimab (Cantargia AB; anti-mouse surrogate of human IgG1 IL1RAP inhibitor; 10 mg/kg7) or isotype control, twice on the first day of treatment (at 4-4.5 weeks of age), followed by 3x weekly dosing for two weeks, after which mice were sacrificed and tumors were processed for scRNA-seq.

first: Erin

last: Dickey

first: Harper

last: Marsh

first: Camilla

last: Rydberg Millrud

first: Haleh

last: Amirian

first: Karthik

last: Rajkumar

first: Manan

last: Patel

first: Andrew

last: Adams

first: Nipun

middle: B

last: Merchant

first: Anna

last: Bianchi

first: Peter

last: Hosein

first: David

last: Lidberg


title: PU.1 is a mediator of the reactive stroma response associated with castration-resistant prostate cancer

city: Bern

first: Panagiotis

last: Chouvardas

address: Murtenstrasse 24

country: Switzerland

institute: University of Bern

summary: Emerging evidence demonstrates the pivotal role played by the tumor microenvironment, particularly cancer-associated fibroblasts, during prostate cancer tumorigenesis and the development of castration-resistant prostate cancer. In this study, we aimed to characterize the stromal gene expression profile of advanced stages of prostate cancer to identify targetable factors associated with castration-resistant prostate cancer progression. Transcriptomic and histological analysis of the mouse stromal component of androgen-sensitive (PNPCa, BM18) and castration-resistant (LAPC9) patient-derived xenograft models representative of advanced prostate cancer was performed. Master regulator analysis of the patient-derived xenograft stromal gene expression profile was performed to identify targetable stromal-associated transcription factors. Patient-derived xenograft collagen-based organoid tumor-fibroblast 3D co-cultures were established to study tumor-fibroblast interactions and to test the effect of an identified fibroblast-targeting compound. Transcriptomic and histological analysis determined the presence of a “reactive” pro-fibrotic stroma in the castration-resistant (LAPC9) versus the castration-sensitive (PNPCa, BM18) xenograft models, characterized by alpha-smooth muscle actin-positive “myofibroblast-like” cancer-associated fibroblasts, high levels of collagen and tenascin C deposition, and upregulation of myofibroblast and inflammatory markers. Intra-tumoral collagen- and tenascin-positive stromal areas specifically correlate with higher tumor invasiveness. Master regulator analysis identified the transcription factor PU.1 as a mediator of the LAPC9 pro-fibrotic phenotype, whose transcriptional activity can be specifically inhibited by a small molecule (DB1976) that prevents its DNA binding. In LAPC9 collagen-based tumor-fibroblast 3D co-cultures, inhibition of stromal PU.1 activity reverted the fibroblast-associated myofibroblast phenotype and, in turn, reduced tumor organoid growth. In this study, we identified the transcription factor PU.1 as a novel targetable molecular player of the pro-fibrotic, cancer-associated fibroblast phenotype in PCa, and we highlighted the applicability of 3D tumor organoid-fibroblast co-cultures as in vitro tools to test the effect of stromal-targeting compounds.

overall_design: The mouse fraction of 3 prostate PDX models was isolated and profiled with BRB-seq (p0 and p2 culture passages).

first: Francesco

last: Bonollo

first: Daniel

last: Hanhart

first: Simone

last: De Brot

first: Wanli

last: Cheng

first: Beat

last: Roth

first: George N.

last: Thalmann

first: Natalie

last: Sampson

first: Marianna

last: Kruithof-de Julio

first: Sofia

last: Karkampouna


title: Transcriptomic insights into a GalNAc/Man-Specific lectin in yeast fermentation

city: Dalian

first: Changqing

last: Tong

state: Liaoning

address: Heishijiao Str.

department: College of Food science and Engineering

institute: Dalian Ocean University

summary: This study aimed to investigate the regulatory effects of Cyclina sinensis lectin (CSL) on ethanol production in Saccharomyces cerevisiae. An integrated analysis of transcriptomic and metabolomic changes was conducted to explore how CSL treatment influences yeast metabolism. Total RNA was extracted from yeast cells with and without CSL treatment. High-throughput RNA-Seq was performed using the Illumina platform. Differential gene expression analysis was conducted using DESeq2, and functional enrichment was performed through Gene Ontology (GO) and KEGG pathway analysis. Results revealed significant changes in genes involved in energy metabolism, stress response, and biosynthesis pathways. This study provides insight into the molecular mechanisms underlying CSL's enhancement of ethanol production in yeast and highlights potential metabolic targets for industrial fermentation optimization.

overall_design: The experimental design included 12 yeast transcriptome samples, consisting of control and lectin-treated groups. Each group included biological replicates to ensure statistical robustness. Total RNA was extracted using TRIzol reagent, and RNA quality was assessed with Agilent 2100 and Nanodrop. After quality filtering, mRNA was enriched using poly-A selection and used to construct strand-specific libraries. Libraries were sequenced on an Illumina platform to generate paired-end reads.Raw reads were trimmed to remove adapters and low-quality sequences. Clean reads were aligned to the reference Saccharomyces cerevisiae genome using HISAT2. Gene expression levels were calculated as FPKM using featureCounts, and differentially expressed genes were identified using DESeq2 with thresholds of |log2FC| ≥ 1 and adjusted p-value < 0.05. Identified genes were subjected to functional enrichmentusing GO and KEGG databases.

first: Hongxin

last: Niu

first: Wei

last: Li

first: Linge


title: Copper-sensitive SPL9 TF regulates expression of indica rice domestication associated miRNAs and phenotypes [RNA-seq]

city: Bangalore

first: Shivaprasad

last: Padubidri V.

address: UAS-GKVK Campus

country: India

institute: National Centre for Biological Sciences - TIFR

summary: Domestication of indica rice has facilitated better harvest and yield, however the molecular mechanisms that drove multitude of phenotypes is not fully understood. A few genetic mechanisms such as sequence variations as well as small (s)RNAs and epigenetics have been attributed to indica rice domestication, but upstream regulators of these variations are unknown. Here we identified a copper (Cu) dependent regulatory module involved in the regulation of a SPL family transcription factor (TF) as well as two classes of RNAs under its direct control. Differential accumulation of Cu-associated micro (mi)RNAs and Cu-associated protein-coding RNAs were a major portion of the differences between wild relative of indica rice (Oryza nivara) and cultivated rice lines. We identified OsSPL9 as an upstream regulator of these changes through genetic and molecular analysis as well as by using Cu limited and excess conditions. OsSPL9 bound to the promoters of these genes through a conserved GTAC enriched motif. OsSPL9, Cu-associated miRNAs and their targets were in a regulatory loop, since mis-expression of SPL9 alone, or any individual Cu-associated miRNA also altered levels of other Cu-associated miRNAs and their cognate targets. OsSPL9 mediated regulation was closely linked to Cu accumulation and metabolism, indicating previously unappreciated roles of metal ions in mediating domestication-associated phenotypes. Our study has the potential to facilitate a better understanding of the crosstalk between genetic and epigenetic regulation that contributed to rice domestication connected to altered environment and growth conditions.

overall_design: We have performed RNA sequencing to understand transcriptomic status of wild, cultivated lines and SPL9 perturbation upon varying copper concentrations

first: Padubidri

middle: V

last: Shivaprasad

first: Raju

last: Steffi


title: Copper-sensitive SPL9 TF regulates expression of indica rice domestication associated miRNAs and phenotypes [ChIP-seq]

overall_design: We have performed ChIP-seq with N-terminal 3XFlag tagged SPL9 transgenic plants - 21 day whole seedlings under different copper deficient (0mg/l), copper sufficient (0.025 mg/l), copper excess (2.5 mg/l). ChIP was performed using Anti-FLAG M2 Magnetic Beads-M8823.


title: Single cell RNA sequencing analysis following Kmt2c/Kmt2d and Pten knockout in mouse stomach mucosa.

city: New York

first: Naitao

last: Wang

address: 1275 York Avenue

department: HOPP

institute: Memorial Sloan Kettering Cancer Center

summary: KMT2C, KMT2D and genes in PI3K pathway are frequently mutated in gastric cancer. In this study, we developed genetically engineered mouse models to knock out these genes in stomach mucosa and observed development of pre-cancerous and cancerous lesions. To investigate cellular differentiation and transcriptional alterations, we performed single cell RNA sequencing three weeks post gene knockout.

overall_design: Comparision of single cell transcriptomes in Control (TY), Pten KO (TP), Kmt2c/Kmt2d dKO (TCD), and Kmt2c/Kmt2d/Pten tKO (TPCD) groups.

first: Wang

last: Naitao

first: Chen

last: Yu


title: Altered primary somatosensory neuron development in a Pten heterozygous model for autism spectrum disorder

city: Portland

first: Kevin

last: Wright

state: Oregon

address: 3181 SW Sam Jackson Rd.

department: Vollum Institute

institute: Oregon Health & Science University

summary: Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by deficits in social interactions, repetitive behaviors, and hyper- or hyposensitivity to sensory stimuli. The cellular mechanisms underlying the emergence of abnormal sensory sensitivity in ASD are not fully understood. Recent studies in rodent models of ASD identified differences in dorsal root ganglia (DRG) neurons that convey somatosensory information to the central nervous system. However, it is unknown how ASD-associated alterations in DRG neurons emerge during development and if these phenotypes are conserved across ASD models. We examined Pten (phosphatase and tensin homolog) heterozygous mice (PtenHet) as a model for syndromic ASD and identified altered responses to sensory stimuli. Transcriptomic and in vivo analysis identified alterations in subtype-specific markers of DRG neurons in PtenHet mice that emerged during early DRG development and involved dysregulation of signaling pathways downstream of PTEN. Finally, we show that mice harboring an ASD-associated mutation (PtenY69H) show nearly identical alterations in expression of somatosensory neuron subtype-specific markers. These results show that precise levels of PTEN are required for proper somatosensory development and provide insight into the molecular and cellular basis of sensory abnormalities in a model for syndromic ASD.

overall_design: RNAseq profiling of adult DRG of wild type and Pten heterozygous mutant mice

first: Alejandra

last: Fernandez

first: Nick

last: Sarn

first: Charis

last: Eng

first: Kevin M


title: Clusterin marks maladaptive regenerative responses throughout the kidney following ischemia reperfusion.

city: Montreal

first: Alex

last: Gregorieff

state: Quebec

country: Canada

institute: Research Institute of the McGill University Health Centre (RI-MUHC)

summary: The proliferative capacity of the kidney declines after birth due to loss of its reserves of nephrogenic progenitors. Nevertheless, kidney tubules maintain a limited capacity to regenerate following various insults, through transient de-differentiation of the tubular epithelium into a progenitor phenotype. Recent studies describing the transcriptional changes underlying this process have primarily focused on proximal tubules with few studies examining the regenerative potential of other renal segments. To address this knowledge gap, we employed single cell RNA sequencing (scRNAseq) and lineage tracing experiments to dissect the transcriptional changes in cells expressing the secreted factor Clusterin (Clu), a known biomarker and potential regulator of acute kidney injury (AKI) in mice and humans. Our data indicates that de novo induction of Clu mRNA serves initially as a robust marker of injured distal tubules and collecting duct cells following ischemia reperfusion injury (IRI). Fate mapping revealed that while Clu+ nephron progenitors were multipotent, Clu+ collecting duct cells in the adult initially activated a transient proliferative program but ultimately persisted in a de-differentiated state or were lost. By scRNAseq, we also identified delayed and maladaptive responses in injured proximal tubules and the loop of Henle that are marked by Clu. Furthermore, we uncovered a “mixed lineage” gene signature comprised partly of podocyte and glomerular parietal epithelial cell identity markers that is activated in converging epithelial, mesenchymal and macrophages within the medulla. Together these data indicate that Clu marks maladaptive responses in the kidney and highlights the medulla as a particularly vulnerable site to the fibro-inflammatory cascade triggered by ischemic injury.

overall_design: BAC-CluGFP mice subjected to 35 min IRI treatment. Kidneys were harvested at days 2, 7, or 30. GFP+ cells isolated by FACS for RNA-seq.

first: Agnieszka

last: Masztalerz

first: Loïck

last: Joumier

first: Sunny

last: Zhen

first: Tomoko

last: Takano

first: Serge

last: Lemay

first: Mohan

last: Malleshaiah


title: Spatial transcriptomic sequencing of fresh frozen canine osteosarcoma tumor samples

city: Gainesville

first: Rebecca

last: Nance

address: 2015 SW 16th Ave

department: Small Animal Clinical Sciences

institute: University of Florida

summary: Osteosarcoma (OSA) is a highly aggressive and heterogeneous bone cancer in dogs and humans, with limited treatment advances over decades. Using the 10x Genomics Visium spatial transcriptomics platform, this study analyzed 14 canine OSA tumors to map gene expression patterns within the tumor microenvironment and correlate them with patient outcomes. Spatial and integrated analyses revealed conserved transcriptional clusters and immune cell infiltration patterns, including macrophage-associated regions in long-term survivors. Deconvolution with single-cell RNA-seq data identified co-localizing immune cell populations such as mast cells with osteoclasts and NKT cells with regulatory dendritic cells. These findings provide insight into tumor heterogeneity and immune landscapes, offering potential targets for future therapies in both species.

overall_design: Samples for spatial transcriptional profiling were taken from 11 canine osteosarcoma patients and included 11 primary tumor samples, 2 patient-matched metastatic samples, and 1 patient-matched recurrent sample.

last: Nance-Richey

first: Rowan

last: Milner

first: Leah

last: Ackerman


title: Assessing the impact of microplastics in inflammatory liver conditions using spatial single cell transcriptomics

city: Oklahoma City

first: Tae

middle: Gyu

last: Oh

state: Oklahoma

address: 940 Stanton L Young Blvd

department: Oncology Science

institute: University of Oklahoma Health Science Center

summary: Recent evidence highlights the multisystem effects of microplastics. We investigated the effect of the most common microplastic, polyethylene, on liver conditions. We included 4 experimental groups of mice; chow diet, chow diet and microplastics, high-fat/fructose/cholesterol diet, high-fat/fructose/cholesterol diet and microplastics. Following tissue collection after treatment, bulk RNA sequencing was carried out to profile transcriptomic activity of each group.

overall_design: We included 4 distinct experimental mice groups: 1) Chow Diet (n = 4), 2) Chow Diet and Microplastics (n = 5), 3) High-fat/fructose/cholesterol diet (n = 5), 4) High-fat/fructose/cholesterol diet and Microplastics (n = 5)


title: Longitudinal changes in DNA methylation in IDH-mutant glioma fuel disease progression through altered cell state differentiation [Smart-seq2]

city: Charlestown

first: Masashi

last: Nomura

state: MA

address: 149 13th street

department: Pathology

institute: Massachusetts General Hospital

summary: IDH-mutant gliomas are initially slow-growing but invariably progress to fatal disease. Major gaps remain in our understanding of the molecular underpinnings and cellular dynamics that drive IDH-mutant evolution. To address this, we integrated joint-capture multi-omic single-cell profiling of DNA methylation, transcriptome and genotype in a longitudinal cohort of 35 IDH-mutant gliomas. Transcriptional and epigenetic comparisons of cell states during glioma progression revealed increased stem-like states, decreased differentiation and identified potential cell states regulators. Single-cell DNA methylation analysis demonstrated methylation loss in progressed tumors, however global DNA methylation was similar between different malignant cells states within individual tumors. This suggests that lower DNA methylation is not due to a change in cell state composition, but rather that it may alter cell state landscapes. To address whether decreased methylation in progressed tumors affects cellular dynamics, we applied a quantitative framework to directly measure cell state heritability and plasticity based on transcriptional annotation of high-resolution phylogenetic trees in clinical samples. Our analysis suggested that decreased methylation reshapes cellular architecture to increased heritability of stem-like states and decreased differentiation. Our study provides insight into the functional impact of DNA methylation on glioma evolution, integrating transcriptional cancer cell states with epigenetic encoding and cell state transition dynamics during cancer progression.

overall_design:
Nuclei from frozen tumor tissue were isolated. Nucleus suspensions were inspected by microscope, counted using a hemocytometer, and sorted into 96 well plates by FACS. The single nucleus plates were processed by dual Smart-seq2 and DNA methylation protocol.

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

last: Ravirum

first: Joshua S.

last: Schiffman

first: Dan A.

last: Landau

first: Mario L.

last: Suvà


title: Longitudinal changes in DNA methylation in IDH-mutant glioma fuel disease progression through altered cell state differentiation

type: Methylation profiling by high throughput sequencing

overall_design:
Nuclei from frozen tumor tissue were isolated. Nucleus suspensions were inspected by microscope, counted using a hemocytometer, and sorted into 96 well plates by FACS. The single nucleus plates were processed by dual XRBS and Smart-seq2 protocol.

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


title: ROS-responsive OMV-liposome hybrid targets macrophages and annulus fibrosus cells to modulate macrophage polarization and alleviate disc degeneration

city: Hangzhou

first: Zilong

last: Li

address: The Second Affiliated Hospital of Zhejiang University Jiefangyuan District

institute: the second Affiliated Hospital of Zhejiang University

summary: Intervertebral disc degeneration (IVDD) is a leading cause of chronic low back pain, contributing significantly to disability and imposing substantial economic burdens. Despite its prevalence, therapeutic strategies targeting IVDD remain limited. Recent advances in single-cell sequencing have highlighted the pivotal role of macrophages in driving the inflammatory microenvironment of degenerating intervertebral discs, particularly in the annulus fibrosus (AF). We demonstrate that macrophages infiltrating degenerated discs predominantly adopt the pro-inflammatory M1 phenotype, driving extracellular matrix degradation and AF degeneration.Specifically, we identify the glycoprotein SPP1 (osteopontin) as a key regulator of macrophage activation and M1 polarization within the disc, driving the expression of matrix metalloproteinases (MMPs) and exacerbating tissue damage. To modulate macrophage polarization and attenuate AF degeneration, we developed a novel targeted delivery system—@LEV@SPP1—by fusing siRNA-loaded liposomes with bacterial exosomes to achieve efficient macrophage targeting. However, the clinical translation of this system is challenged by the avascular nature and unique microenvironment of the intervertebral disc. To overcome these challenges, we developed a novel ROS-responsive, macrophage-targeted delivery system, AF-PTK@LEV@SPP1, by conjugating an AF-binding peptide (AF-P) to @LEV@SPP1 exosomes using a thioketal linker. This composite system leverages both macrophage specificity and ROS responsiveness to release the therapeutic payload upon elevated oxidative stress in the degenerate disc. In vivo experiments demonstrated that AF-PTK@LEV@SPP1 significantly reduced M1 macrophage polarization, alleviated AF degeneration, and promoted extracellular matrix repair, offering a promising strategy for long-term, targeted treatment of IVDD. These findings suggest that precisely modulating macrophage activity via AF-PTK@LEV@SPP1 holds substantial therapeutic potential for IVDD, with implications for regenerative strategies in other inflammatory disorders.

overall_design: To investigate transcriptional changes in Intervertebral disc tissue comes from Rats treated with AF-PTK@LEV@SPPP1 compared to untreated controls, providing insights into the therapeutic effects of the treatment.Two groups of mice, with 3 biological replicates per group:Treated Group: Intervertebral disc tissue comes from untreated rats treated withAF-PTK@LEV@SPP1.Control Group: Intervertebral disc tissue comes from untreated rats.

first: Yucheng

last: Xue

first: Zhenyu

first: Yiwen

last: Xu

first: Miaojie

last: Fang

first: Jianbin

first: Ning

last: Zhang

first: Kelei

last: Wang


title: Longitudinal changes in DNA methylation in IDH-mutant glioma fuel disease progression through altered cell state differentiation [scRNA-seq]

overall_design:
Nuclei from frozen tumor tissue were isolated. The nuclear suspensions were inspected by microscope, counted using a hemocytometer, and used for 10x Genomics workflow.

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


title: 20S proteasomes are required for ELVAs-mediated protein degradation during oocyte-to-embryo transition and aging

first: Chen

last: Yingyan

address: No. 3 Qingchundong Road

institute: Zhejiang University

summary: The degradation of maternal factors is essential for oocyte-to-embryo transition (OET). While extensive research has focused on maternal mRNA degradation, the mechanisms underlying maternal protein degradation remain poorly understood. ELVAs, the oocyte-specific membraneless structure which comprises proteasomes, endolysosomes and autophagosomes, are required for protein degradation in maturing oocytes and zygotes. In this study, we embloyed oocyte-specific Psma7 knockout, targeting the α4 subunit of 20S proteasome, to investigate the physiological function of proteasomes in ELVAs-mediated protein degradation. PSMA7-deficiency destabilizes 20S proteasome components and disrupts ELVAs assembly and spatial organization, resulting in the accumulation of ubiquitinated proteins in both oocytes and zygotes. Consequently, maternal PSMA7-deletion results in female infertility, manifested by impaired oocyte maturation and developmental arrest at the 1- to 2-cell stage. Furthermore, in oocytes from both aged mice and women, reduced proteasome abundance and aberrations in ELVAs distribution and activity were found, providing a mechanistic explanation for the diminished developmental competence aged oocytes. Taken together, our findings elucidate the critical function of proteasome-regulated proteostasis in oocyte quality control during OET and reproductive aging in females.

overall_design: RNA-seq profiling of oocytes from wildtype and oocyte-specific Psma7 conditional knockout (Zp3-Cre; Psma7 flox/flox, termed Psma7 cKO) mice at different stages of oocyte maturation (germinal vesicle [GV] and metaphase II [MII])

first: Yan

last: Rong

first: Songying

first: Chao

last: Yu


title: Single-cell gene expression and TCR profiling reveal age-related differences in recent thymic emigrants

city: Ithaca

first: Connor

last: Kean

address: 215 Tower Rd

institute: Cornell University

summary: Following thymic egress, CD8+ T cells must undergo a post-thymic maturation process to transition from a recent thymic emigrant (RTE) to a mature naïve T cell.  Since neonatal animals are comprised of significantly more RTEs, there is a prevailing notion that neonatal CD8+ T cells behave differently than their adult counterparts simply because they have undergone less post-thymic maturation.  To test this theory, we leveraged a fate mapping mouse model and single cell sequencing to compare neonatal and adult CD8+ RTEs that have undergone that same amount of post-thymic maturation.  Interestingly, we found neonatal and adult CD8+ RTEs exhibit distinct phenotypes, gene expression profiles, TCR usage and functions.  These data suggest that neonatal CD8+ T cells are not simply ‘young’ adult CD8+ T cells and that age-related changes in CD8+ T cell functions in early life cannot be solely attributed to differences in the amounts of post-thymic maturation.

overall_design: Paired scRNA-seq and scTCR-seq: Tamoxifen was administered to neonatal mice (n = 4) at 1d of age and adult mice (n = 4) at 28d of age to generate timestamped CD8+ T cells. Two weeks post timestamp, ZsGreen+Va2+CD8+ T cells were sorted by FACS.

first: Cybelle

last: Tabilas

first: Vanessa

last: Venturi

first: Anastasia

last: Minervina

first: Paul

middle: G

last: Thomas

first: Andrew

last: Grimson

first: Jennifer

last: Grenier

first: Miles

last: Davenport

first: Norah

middle: L

last: Smith

first: Brian

last: Rudd


title: Spatiotemporal regulation of arbuscular mycorrhizal symbiosis at cellular resolution [RNA-seq]

city: Cambridge

first: Gabriel

last: Ferreras-Garrucho

address: 93 Lawrence Weaver Rd

institute: University of Cambridge

summary: Arbuscular mycorrhizal (AM) symbiosis develops through successive colonization of root epidermal and cortical cells, culminating in the formation of arbuscules, tree-like intracellular structures that are transient yet essential sites of nutrient exchange. To dissect the cellular and structural complexity of AM establishment in rice roots colonized by Rhizophagus irregularis, we applied dual-species spatial transcriptomics to simultaneously monitor plant and fungal gene expression at single-cell resolution. This approach revealed surprising differences in transcriptional activity between fungal structures and showed that morphologically similar arbuscules can be transcriptionally distinct. These findings suggest hidden functional diversity among arbuscules, indicating that nutrient exchange is not uniform but finely modulated across individual structures. Because arbuscules form and degenerate within only a few days, we further sought to capture translational regulation across their life span. We pioneered AM-inducible TRAP-seq (Translating Ribosome Affinity Purification followed by RNA-seq) using stage-specific promoters, enabling cell-type- and stage-resolved profiling for the first time in AM symbiosis. This revealed extensive spatiotemporal reprogramming of nutrient transport and signaling, with distinct sets of phosphate, nitrogen, and carbon transporters and regulators upregulated or repressed at different stages of arbuscule development, suggesting that nutrient exchange is dynamically regulated across the arbuscule life cycle. More broadly, cell wall biosynthesis genes and key defence markers were suppressed during arbuscule formation, whereas upon collapse, defence markers were strongly upregulated, suggesting a host-driven shift towards symbiosis termination. Together, these findings highlight the nuanced and dynamic regulation of AM symbiosis at cellular resolution, refining our understanding of how nutrient exchange and fungal development are coordinated in space and time, and providing a framework for further functional exploration.

overall_design: Gene expression profiling analysis by RNA-seq data of rice (Oryza sativa subsp. japonica cv. Nipponbare) roots at 3 weeks post transfer to sand either mock-inoculated (-Ri) or inoculated with Rhizophagus irregularis (+Ri), of total polyA or TRAP (Translating Ribosome Affinity Purification, that is, after polysomal pull-down with specific antibodies for tagged ribosomal proteins) mRNA pools, for a selection of transgenic TRAP lines under different promoters, either p35S as ubiquitous/constitutive control, or AM-inducible promoters active at distinct developmental stages of the interaction: pAM1 as early, pPT11 as mid, and pARK1 as late colonisation marker.

first: Tania

last: Chancellor

first: Garo

last: Akmakjian

first: Héctor

last: Montero

first: Emma

last: Wallington

first: Samik

last: Bhattacharya

first: Christian

last: Korfhage

first: Julia

last: Bailey-Serres

first: Uta

last: Paszkowski


title: Proteasome inhibition alleviates proteinuria in Lmx1b knock-in mice with dysfunctional LIM domains

city: Regensburg

first: Uwe

last: Schwartz

address: Universitätstraße 32

department: NGS Analysis Center

country: Germany

institute: University of Regensburg

summary: LMX1B codes for a transcription factor of the LIM-homeodomain family, mutations in this gene cause the autosomal-dominant hereditary disease nail-patella syndrome.  We have generated two knock-in mouse lines which carry missense mutations in the LIM domains of LMX1B and thus mimic the situation in patients.  Surprisingly, both mouse lines only developed a phenotype in the recessive state.  This is due to the misfolding of the respective LIM domains, in which a zinc-chelating amino acid was exchanged by an alternative amino acid, and the subsequent ubiquitin-dependent proteasomal degradation of mutant LMX1B.  The proteasomal inhibitors bortezomib and MG132 stabilized the mutant proteins in vitro.  A therapeutic study demonstrated that bortezomib markedly reduced podocyte damage and alleviated proteinuria in adult mice with a floxed and a knock-in Lmx1b allele.  RNA sequencing of isolated podocytes from those mice revealed a number of genes which were up- or down-regulated in both knock-in mouse lines but also led to the identification to genes whose regulation specifically depended on the LIM-B domain.  Our data provide proof of principle for a therapeutic strategy in patients with nail-patella syndrome suffering from missense mutations in the LIM domains of LMX1B

overall_design: RNA-seq profiling of podocytes isolated from either DMSO or bortezomib (Btz.) treated Lmx1b+/lox and Lmx1bH77L/lox mice

first: Joshua

last: Hermens

first: Melanie

last: Zaparty

first: Ralph

last: Witzgall


title: Transient SP140 inhibition unlocks hematopoietic stem cell fate from human pluripotent stem cells [scRNA-seq]

city: Shanghai

first: Cui

last: Xinyu

address: Siping Road

institute: Tongji University

summary: Efficient derivation of transplantable hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is constrained by epigenetic silencing. Through a CRISPR/Cas9 screen with a BCL11A-eGFP reporter, we identified epigenetic reader SP140 suppressing hematopoiesis. Transient genetic or pharmacologic inhibition of SP140 in hPSC-derived teratoma  and embryoid body cultures promoted robust multilineage hematopoiesis and accelerated production of HSCs with serial transplantability and durable reconstitution in immunodeficient mice. Mechanistically,  SP140 blockade unlocked transcription at endothelial-to-hematopoietic transition (EHT) loci through topoisomerase 1-dependent chromatin remodeling, activating key hematopoietic and stem cell programs. Transcriptomic analysis showed activation of these regulators upon SP140 inhibition, which was prevented by topoisomerase 1 blockade. CUT&Tag profiling identified SP140 binding at EHT and HSC-specification gene loci. SP140’s function as an epigenetic gatekeeper was conserved in diverse hPSCs and murine embryo models, where its downregulation enhanced physiological HSC emergence. Importantly, selective SP140 inhibition in a chemically defined, scalable protocol enabled rapid in vitro generation of bona fide human HSCs suitable for transplantation. These findings identify transient SP140 inhibition as an effective strategy to overcome epigenetic barriers and unlock clinically relevant HSC specification from hPSCs, advancing regenerative hematopoiesis and cell therapy.

overall_design:
To analyze the influence of SP140 on embryoid body (EB) modal, we conducted scRNA seq during the whole differentiation days (day1 to day3). We collected EBs on different days with SP140knock-out or not (e.g. Day1_WT represents EB without SP140 knock-out on day 1 from the beginning of differentiation, Day1_SP140LSLrepresents EB with SP140 knock-out on day 1 from the beginning of differentiation), Then EBs were dissociated in 3000IU/m collagenase type II (Gibco) for 2-5 min at 37°C. Next, cells were treated with 0.25% TrypLE Express (Gibco) for 3 min and digested by gentle mechanical shaking to make single-cell suspensions for scRNA seq by10x genomics.
To analyze the generation of blood cells and HSCs in teratomas, we collected teratomas from mice and dissociated them in 0.88 mg/mL collagenase/dispase(Roche) for 15 minutes at 37°C. After incubation, teratomas were mechanically disaggregated and filtered through 70uM membranes into Single-cell suspensions. CD45+(2replicates)and CD45+Lin- cells were sorted for scRNA seq by 10x genomics.


title: Klinefelter Syndrome: a neurodevelopmental disease of the synapse

city: La Jolla

last: Haddad

department: Pediatrics

institute: University of California, San Diego

summary: Klinefelter syndrome (KS; 47, XXY) is the most common sex chromosome disorder, affecting approximately 1 in every 500 to 650 newborn males. Children with KS display a spectrum of phenotypic manifestations, including abnormal neurocognitive phenotypes. However, due to the limited research focusing on the central nervous system (CNS), our understanding of the neurobiology of KS at the cellular and molecular levels remains largely unclear. In this study, we utilized brain organoids derived from pluripotent stem cells to explore the mechanisms underlying early brain developmental defects in KS patients. We demonstrate that KS organoids display altered neurogenesis, gliogenesis, and glutamate signaling pathways. We believe these early alterations contribute to the abnormal brain development and later cognitive phenotypes in KS patients.

overall_design: RNA-seq profiling of control and KS pluripotent stem cell-derived brain organoids


title: Transient SP140 inhibition unlocks hematopoietic stem cell fate from human pluripotent stem cells [CUT&Tag]

overall_design:
To find the histone modifications and binding genes of SP140 and TOP1, Day1 EBs were dissociated to form single-cell suspensions and CD31hi cells were collected for CUT&Tag. Three independent replicates were set for SP140, H3K27me3 and H3K4me3 group (R1, R2, R3). Two independent replicates were set for TOP1 group (R1 and R2).
To analyze the impact of SP140i treatment on H3K4me3 modification in CD34+ hemogenic endothelial cells during hematopoietic transformation, CD34+ cells were isolated individually and subjected to EHT culture in vitro (with DMSO or SP140i added to the medium). All cells were collected 24 hours later for CUT&Tag experiments.


title: A viral noncoding RNA functions as master regulator of gene expression in latently infected cells

city: Salt Lake City

first: Demian

middle: E

last: Cazalla

state: UT

address: 15 N Medical Drive East, Rm 4100

department: Biochemistry

institute: University of Utah

summary: HSUR1 is a viral small nuclear noncoding RNA of the Sm class that is abundantly expressed by the oncogenic Herpesvirus saimiri. HSUR1 binds a host microRNA, miR-142-3p, and AU-rich element-binding proteins (ARE-BPs). The functional significance of these interactions, and how HSUR1 functions as an Sm-class RNA, remain unclear. Here we show that HSUR1 is a master regulatory noncoding RNA that interacts through base pairing with hundreds of host mRNAs in latently infected cells. HSUR1 target mRNAs that encode proteins with roles in several gene expression processes, T cell differentiation and apoptosis. Consistently, HSUR1 expression changes the transcriptomic profile and identity of the infected T cell and inhibits apoptosis. Mechanistically, we show that HSUR1 recruits miR-142-3p and ARE-BPs to the 3¢ untranslated regions of target mRNAs to destabilize them. These results describe an indirect mechanism by which microRNAs and ARE-BPs can regulate gene expression.

overall_design: For iRICC experiments (experiment 15955R), polyadenylated (polyA+) RNA was selected and hybridized to an antisense DNA oligonucleotide complementary to full-length HSUR1 (anti-H1 DNA) followed by limited single-strand S1 endonuclease digestion to fragment polyA+ RNA while maintaining HSUR1 integrity. After removal of anti-H1 DNA by DNAse I digestion, samples were split into two samples and hybridization with anti-H1 DNA was repeated in one (Control) sample, followed by digestion of both samples with RNAse H, which resulted in removal of HSUR1 only from the Control sample. Following a second digestion with DNAse I, HSUR1 and crosslinked RNAs were purified in both samples under stringent conditions with a biotinylated RNA oligonucleotide antisense to HSUR1. High-throughput sequencing libraries were prepared for both samples using random primers. HSUR1 target mRNAs were identified by comparison of pull downs from HSUR1 samples with Control samples in three independent replicate experiments. For RNA-seq experiment of cj38637-WT and cj38637-deltaHSUR1 cells (experiment 22466R), libraries were prepared from total RNA of these cell lines with NEBNext Ultra II Directional RNA Library Prep with poly(A) mRNA Isolation.

first: Demián

first: Carlos

last: Gorbea

first: Xavier

last: Roca

first: Donald Yuhui

last: Sim


title: Transient SP140 inhibition unlocks hematopoietic stem cell fate from human pluripotent stem cells [bulk RNA-seq]

overall_design: To clarify the mechanism of gene expression change during EHT in in vitro modal, day 2 EBs, treated with DMSO or SP140i on Day 1, were dissociated and CD31hi cells were sorted for bulk RNA seq. CD34+ cells treated with DMSO, SP140i, SP140i and TOP1 inhibitor(TOP1i) combined after EHT were obtained for bulk RNA seq.Three independent replicates were set for each group (R1-R3).


title: Transient SP140 inhibition unlocks hematopoietic stem cell fate from human pluripotent stem cells [ATAC-seq]

overall_design: To clarify the mechanism of gene expression change during EHT in in vitro modal, day 2 EBs, treated with DMSO or SP140i on Day 1, were dissociated and sorted CD31hi cells for ATAC seq. CD34+ cells treated with DMSO, SP140i, SP140i and TOP1 inhibitor combined after EHT were obtained for ATAC seq. Three independent replicates were set for each group (R1, R2, R3)


title: Progressive intestinal tumor cell plasticity, Myc activation and loss of Lgr5+ tumor stem cell lineage commitment upon Wnt depletion

city: Helsinki

first: Marika

last: Lassila

address: Haartmaninkatu 8

country: Finland

institute: University of Helsinki

summary: ScRNA-seq and ATAC-seq was used to investigate the role of Tcf7 and Lef1 in intestinal adenomas

overall_design: Tumors from the small intestines of Apc flox mice were dissociated into single-cells and subjected to scRNA-seq and ATAC-seq.

first: Fatemeh

last: Seyednasrollah

first: Cinzia

last: Bessone

first: Maritta

last: Räisänen

first: Inkeri

last: Vuori

first: Davide

last: Berta

first: Johanna

last: Aspholm

first: Tatiana

last: Petrova

first: Lauri

last: Aaltonen

first: Pauliina

last: Kallio

first: Kari

last: Alitalo


title: scRNA-seq analyses of cerebrospinal fluid (CSF) cells in older adults with or without Alzheimer's disease after Bacillus Calmette Guerin (BCG) vaccination.

first: Mahesh Chandra

last: Kodali

institute: Massachusetts General Hospital / Harvard Medical School

summary: Cerebrospinal Fluid cells collected from older adults before and after BCG vaccination were studied. This database is part of the following. NCT04507126 - A Pilot Study of the Effects of Bacillus Calmette-Guérin (BCG) Immunization on Cerebrospinal Fluid and Blood-based Biomarkers in Older Adults - ClinicalTrials.gov. https://clinicaltrials.gov/ct2/show/NCT04507126 NCT05004688 - A Trial to Evaluate the Effects of BCG in Adults With MCI and Mild-to-Moderate AD - - ClinicalTrials.gov. https://clinicaltrials.gov/ct2/show/NCT05004688

overall_design: Briefly, In the present study, Cerebrospinal Fluid (CSF) Cells were collected and cryopreseved from subjects before (Baseline, 0 months or BL) and after (3 months and 12 months) BCG Vaccination. The cells at each of the time points (i.e. BL, 3mo and 12mo) were thawed followed by scRNA-seq on 10x.

first: Marc

middle: S

last: Weinberg

first: Steven

last: Arnold


title: Functional characterization of Central Nervous System (CNS) and peripheral immune responses in Alzheimer's Disease after Bacillus Calmette Guerin (BCG) vaccination in older adults.

summary: Cerebrospinal Fluid cells collected from older adults before and after BCG vaccination were studied for evaluating celltype specific functional responsivity to stimulation with antigens. Peripheral blood mononuclear cells were also studied to compare the peripheral and central nervous system (CNS) responsivities.This datase is part of the following. NCT04507126 - A Pilot Study of the Effects of Bacillus Calmette-Guérin (BCG) Immunization on Cerebrospinal Fluid and Blood-based Biomarkers in Older Adults - ClinicalTrials.gov. https://clinicaltrials.gov/ct2/show/NCT04507126 NCT05004688 - A Trial to Evaluate the Effects of BCG in Adults With MCI and Mild-to-Moderate AD - - ClinicalTrials.gov. https://clinicaltrials.gov/ct2/show/NCT05004688

overall_design: Briefly, In the present study, Cerebrospinal Fluid (CSF) Cells were collected and cryopreseved from four subjects before (Baseline, BL) and after (3months and 1year) BCG Vaccination. The cells at each of the time points (i.e. BL, 3mo and 1yr) were stimulated with either X-Vivo i.e. control media, or lipopolysaccharide (LPS, 10ng/mL) or heat killed bacillus calmette guerin (HK-BCG, 1e5 cfu/mL) for four hours, immediately followed by  scRNA-seq on 10x. Additionally, peripheral blood mononuclear cells (PBMCs) from three subjects (Paired subjects to the CSF Cell samples)) before (Baseline, BL) and after (3months and 1year) BCG Vaccination were also stimulated with the same conditions followed by scRNA-seq using 10X.


title: Single-cell RNA sequencing of hyaloid blood vessels from endothelial-specific Gpr124 knockout mice and control littermates

city: Würzburg

first: Mario

last: Vallon

address: Koellikerstr. 6

department: Institute of Anatomy and Cell Biology

institute: University of Würzburg

summary: To explore the molecular mechanisms of GPR124-mediated hyaloid blood vessel regression, single-cell RNA sequencing of hyaloid vessels from P6 endothelial-specific Gpr124 knockout mice and control littermates was performed. The sequenced cells clustered into two major populations: endothelial cells and pericytes. Differentially expressed genes in hyaloid endothelial cells included known WNT signaling regulators, along with evidence for endothelial-mesenchymal transition (EndMT). Although not directly affected by the Gpr124 knockout, hyaloid pericytes displayed drastic transcriptional changes in genes regulating autophagy and apoptosis as well as TOR and Hippo signaling.

overall_design: Hyaloid blood vessels were isolated from P6 endothelial-specific Gpr124 knockout mice and control littermates. Single-cell suspensions were generated using Subtilisin A and cells were labeled with 7-AAD as well as CD31 and PDGFRB antibodies. Live cells positive for CD31 or PDGFRB were sorted into 384-well plates, one cell per well, two plates per genotype. Cells were processed and sequenced using the SORT-seq PLUS protocol (Single Cell Discoveries).

first: Laura

last: Hannig


title: Protective effect of energized structured water on bioenergetic function and oxidative stress in H9c2 cells: An explorative study

first: Andrei

last: Chernov

state: CA

address: 9500 Gilman Dr

department: Anesthesiology

institute: University of California San Diego

summary: The energized structured water (ESW) concept supposes water can be imbued with energetic frequencies or vibrations, improving its quality and providing various health benefits. This study investigates the impact of ESW on cellular bioenergetic function and oxidative stress in H9c2 cardiomyocytes. H9c2 cells were pretreated with ESW or control water (CW) at equivalent dilutions. Mitochondrial function was assessed using the Agilent Seahorse XF Pro Analyzer. Results demonstrated that ESW pretreatment significantly increased maximum and spare respiratory capacity in H9c2 cells. In addition, ESW treatment increased the glycolytic ATP production without affecting mitochondrial and total ATP production. ESW treatment enhanced cell viability and reduced ROS production in cells exposed to H2O2-induced oxidative stress. Based on differential gene expression analysis, 717 genes including Myh1, Akr1b8, Hmox1, Kcng1, Ugt1a6, Clu, Gaa, Ftl1, Hspb7, and Gba1 were up-regulated and 422 genes including Hist1h2an, Slfn4, Rpl22l1, Polr2k, Il1rl1, Ndufb1, Atp5mkl1 were down-regulated by ESW compared to the controls. Gene Ontology (GO) analysis indicated that ESW treatment significantly affects biological processes related to cellular stress response pathways. These findings suggest that ESW treatment may enhance cellular bioenergetics and stress resistance in H9c2 cells, potentially through modulation of gene expression related to stress responses and energy metabolism in H9c2 cells.

overall_design: To identify the effects of ESW on gene expression levels, H9c2 cells were pre-treated with either ESW or CW for 72 hours. Untreated H9c2 cells served as a control group. RNA-seq analysis was then performed on all three groups (ESW-treated, CW-treated, and untreated), with three replicates per group. Subsequently, we compared the gene expression levels of the ESW-treated group to both the untreated control group and the CW-treated group

first: Sachithra

last: Ranaweera

first: Juan

last: Zuniga-Hertz

first: Ramamurthy

last: Chitteti

first: Hemal

last: Patel


title: Age- and Tissue-dependent Diversity of Human Plasmacytoid Dendritic Cells Uncovers a Cycling Subset Dominant in Early Life and Cancer

city: Saint Louis

first: Alina

last: Ulezko Antonova

state: MO

address: 660 S. Euclid Ave

department: Path/Immuno

institute: WUSM

summary: Plasmacytoid dendritic cells (pDCs) are innate sentinels that produce type I interferons (IFN-I) during infection. Here, we asked how developmental stage and tissue context shape human pDC transcriptional states. Single-cell RNA sequencing of pDCs from blood, thymus, lymph nodes, and tonsils across fetal, infant, and pediatric stages revealed tissue-enriched programs, including IFN-I-imprinted pDCs in the thymus, NFκB-imprinted pDCs in tonsils, and resting pDCs in blood and lymph nodes. Across tissues, we identified a pDC subset characterized by prostaglandin D2 production and dopamine responsiveness, indicating a potential neuromodulatory axis. Cycling pDCs were abundant in fetal and infant lymphoid tissues and declined with age, while remaining enriched in bone marrow (BM) throughout life. Blastic pDC neoplasm associated with enrichment of cycling, mutation-bearing pDCs in the BM, suggesting that this niche serves as a reservoir for malignant pDCs. Thus, early in life, various lymphoid tissues can generate pDCs. This tissue-specific hematopoiesis may result in distinct cellular and functional outputs.

overall_design: Plasmacytoid dendritic cells were isolated via magnet assisted cell sorting (MACS) and flow assisted cell sorting (FACS) from tonsil, thymus, perithymic lymph node and blood of children (n=21) and adults (n=2).

first: Marco

last: Colonna


title: LPS-induced lncRNA expression profiling in human PBMC-derived macrophages

status: Public on Apr 26 2026

city: Boston

first: DAIKI

last: HOSOKAWA

address: 3 Blackfan Street, 17th Floor

department: Center for Interdisciplinary Cardiovascular Sciences

institute: Brigham and Women's Hospital

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

summary: Introduction: Macrophages play a key role in inflammatory diseases. We aimed to identify long noncoding RNAs that regulate pro-inflammatory activation of human macrophages.  Methods: We performed human lncRNA microarray analysis in LPS-stimulated primary human macrophages. We then carried out loss-of-function and gain-of-function experiments, luciferase reporter assays, RNA pulldown, RNA immunoprecipitation, and mouse endotoxemia studies, including humanized mouse models.  Results: We identified 11 lncRNAs that were significantly increased by LPS. Among them, lnc-FAM164A1 was selected for further study. Silencing of lnc-FAM164A1 by antisense oligonucleotides or siRNA reduced the LPS-induced expression of pro-inflammatory cytokines, including CCL2, IL-6, and TNF-a. In contrast, enforced expression of lnc FAM164A1 enhanced inflammatory responses in human and mouse macrophages. lnc FAM164A1 also promoted NF-kB-related signaling. RNA pulldown and RNA immunoprecipitation identified ACLY as an lnc-FAM164A1-  associated protein. ACLY silencing reduced the inflammatory effects induced by lnc-FAM164A1.  Discussion: These findings support that human lnc-FAM164A1 promotes pro-inflammatory activation of macrophages through its interaction with ACLY and NF-kB-related signaling.

overall_design: 8 RNA samples from human PBMC-derived macrophages were analyzed. Four samples were untreated control samples (CtrlA, CtrlB, CtrlC, and CtrlD), and four samples were treated with 10 ng/mL LPS for 3 hours (TreatE, TreatF, TreatG, and TreatH).

first: Masanori

last: Aikawa

first: Daiki

last: Hosokawa


title: Development and characterization of 3D spinal cord organoids to advance the study of amyotrophic lateral sclerosis

city: Pavia

first: Matteo

last: Bordoni

address: Via Mondino 2

country: Italy

institute: IRCCS Mondino Foundation

summary: ALS is a non-cell autonomous disorder as many cell types contribute to motor neurons death. The lack of effective treatments is probably due to the absence of a realistic model. Organoids are pluripotent stem cell-derived self-organizing structures that allow in vitro generation of the tissues. We developed a new method for the generation of spinal cord organoids (SCOs). Aim of the work was to characterize a 3D organoid model for the study of ALS pathogenesis. We started from iPSCs obtained from healthy controls and sALS patients. We differentiated iPSCs into neural stem cells (NSCs). NSCs were plated on low-attachment plates and were cultured in floating conditions using an orbital shaker. NSCs were differentiated in these conditions into motor neuron progenitors, immature motor neurons (MNs) and finally mature MNs. We then characterized cells by phase-contrast and confocal microscopy. We found that SCOs derived from sALS patients were smaller and with irregular morphology compared to healthy controls. Using the GFAP marker, we found that sALS SCOs have a thicker glial layer compared to healthy controls. We also found that healthy controls show longer neurites compared to sALS SCOs. By RNAseq, we found a ten-fold increase of deregulated gene in SCOs respect to 2D cell models. Moreover, in ALS SCOs we found an extensive deregulation of genes involved in extracellular matrix organization when compared to control. By deconvolution analysis we found that healthy controls SCOs show a higher amount of differentiated cells compared to sALS SCOs. Our data suggest that brain organoids represent a promising tool for the investigation of pathogenic mechanisms of ALS. In fact, we found typical pathological hallmarks of the pathology, such as the presence of gliosis, the smaller length of neurites, decreased level of mature MNs, and deregulation of genes implicated in ALS. In conclusion, SCOs represent a promising tool for the investigation of pathogenic mechanisms of ALS.

overall_design: SCOs were characterized by morphological, immunofluorescence, and bulk RNAseq analysis

first: Eveljn

last: Scarian

first: Letizia

last: Messa

first: Maria

last: Garofalo

first: Emanuela

last: Jacchetti

first: Manuela

last: Raimondi

first: Luca

last: Diamanti

first: Stella

last: Gagliardi

first: Stephana

last: Carelli

first: Cristina

last: Cereda

first: Orietta

last: Pansarasa


title: Undernutrition during late gestation disrupts placental metabolism, transport, and immune function in a sheep model

city: Hefei

first: weibin

last: wu

address: Changjiang Western Road 130

institute: Anhui Agricultural University

summary: In the current study, five late gestation multiparous ewes were restricted to a 30% feeding level to create a model of malnutrition, while five other ewes were fed normally as controls. All ewes were sacrificed and placental samples were collected for transcriptome sequencing to study metabolic changes.

overall_design: Compare the differences in transcriptome profiles between the placental sample from CON and TR ovis aries.

first: Weibin

last: Wu

first: Pengpeng

last: Liu

first: Huizhen

last: Lu

first: Yanfeng

last: Xue


title: Cell autonomous control of CAR signaling and receptor shedding via ADAM17-mediated proteolysis

city: san francisco

first: Jeremy

last: Bjelajac

address: 2190 grove st

institute: Stanford University

summary: We sought to endow T cell autonomous regulation of cell surface protein expression by exploiting the conditional proteolytic activity of ADAM17 following T cell activation.  Screening of canonical ADAM17 substrates yielded a minimal 15-aa CD62L-derived motif that confers rapid and reversible cleavage of a receptor following T cell activation—termed Activation-Induced Release (AIR). Embedding AIR into tonic-signaling CARs reduced basal CAR expression proportional to the degree of tonic signaling induced, curtailing exhaustion and improving antitumor potency. In non-tonic signaling CARs, AIR decreased activation-induced cell death and enhanced T cell expansion after stimulation. AIR’s modularity supports higher-order logic-gating; AIR-regulated peptide masks enable antigen-dependent unmasking of an EGFR-targeting CAR. Finally, CRISPR knock-in of AIR into endogenous FAS or TGFBR2 endowed them with activation-induced shedding, which enhanced tumor clearance, while preserving signaling in non-activating conditions. AIR is a compact switch that provides fast, autonomous regulation of surface proteins for next-generation cell therapies.

overall_design: RNAseq profiling the transcriptomic differences between HA-GD2 CAR v. AIR-HA-GD2 CAR v. Mock T cells made from 3 different healthy donors following treatment with either DMSO or Dasatinib

middle: R

first: Adrià

middle: C

last: Socias

first: Preeti

last: Nehra

first: Kevin

last: Reynolds

first: Meena

last: Malipatlolla

first: Naiara

last: Martinez-Velez

first: Diane

last: Manjarrez

first: Katie

last: Ho

first: Peng

last: Xu

first: Jennifer

last: Hamad

first: Sean

last: Yamada-Hunter

first: Louai

last: Labanieh

first: Elena

last: Sotillo

first: Crystal

last: Mackall


title: XG knockout and transcriptome perturbations in Ewing's sarcoma cell line TC-71

city: Lund

first: Martin

last: Olsson

address: Baravägen 18

department: Department of Laboratory Medicine

country: Sweden

institute: Lund University

summary: In order to assess the effect of XG knockout, CRISPR-Cas9 targeting exon 2 of XG was applied to TC-71 cells. Knockout cells were collected by fluorescence-activated cell sorting and validated by immunoblotting. Differential expression analysis was performed on wildtype versus knockout cells.

overall_design: Gene expression analysis of RNA-seq data for TC-71 cell line with and without XG gene knockout

first: Yan

last: Lee


title: Lineage 2-Beijing Mycobacterium tuberculosis strains suppress BCG-trained innate immunity early after infection

city: Nijmegen

first: Todia Pediatama

last: Setiabudiawan

state: Gelderland

address: Geert Grooteplein Zuid 10, 6525 GA Nijmegen, Netherlands

department: Internal Medicine

country: Netherlands

institute: Radboudumc

summary: Genetically distinct lineages of Mycobacterium tuberculosis differ in their virulence, transmissibility, and immune evasion capacity. The modern Lineage 2-Beijing (L2-B) clade of Mtb, which is highly prevalent in Asia and now globally distributed, is associated with resistance to the bacille Calmette Guerin (BCG) vaccine. Using a murine BCG vaccination model followed by aerosol challenge with geographically matched L2-B or Lineage 4 clinical isolates, we examined how L2-B circumvents vaccine-induced immunity. BCG-trained alveolar macrophages showed impaired control of L2-B growth in vitro. Spatial transcriptomic profiling of infected lungs revealed broad transcriptional reprogramming, including selective suppression of pathways essential for macrophage activation and trained innate immunity. Similar immune dysregulation was observed in peripheral blood from BCG-vaccinated household contacts of L2-B smear-positive TB patients. Together, these findings define mechanisms by which L2-B evades immune control, highlight key components of BCG-induced protection, and support re-evaluation of current TB vaccine strategies.

overall_design: Human whole blood RNA-seq from household contacts of smear-positive TB patients in Bandung, Indonesia (INFECT cohort). Contacts were classified by the Mtb lineage of their index case: L2-Beijing (n=15) or L4 (n=24). Blood was collected in PAXgene tubes. Globin genes were excluded. DESeq2 was used with age and IGRA status as covariates.

first: Naomi

last: Daniels

first: Todia

first: Taru

last: Dutt

first: Philip

last: Hill

first: Marcela

last: Henao-Tamayo

first: Reinout

last: van Crevel

first: Joanna

last: Kirman


title: Targeting Cyclin K-CDK12 Synergizes with ATR Inhibition by Limiting RPA Chromatin Loading in Triple-Negative Breast Cancer

first: Eunbee

last: Choi

address: 440 Brookline Ave, Mayer-434

institute: Dana farber cancer institute

summary: Cyclin K-CDK12 inhibition has emerged as a promising therapeutic strategy to induce homologous recombination deficiency (HRD). However, clinical and genomic studies have not clearly linked CDK12 inactivation to HRD. Here, we demonstrate that cyclin K-CDK12 depletion drives a replication stress (RS)-associated vulnerability through RPA regulation. A DNA damage response-focused CRISPR screen identified DNA replication factors and ATR activators as top sensitizers to cyclin K degradation. Consistently, cyclin K-CDK12 depletion synergized with ATR inhibition in triple-negative breast cancer (TNBC) models. Cyclin K degradation reduces RPA chromatin loading and impairs ATR activation, compromising cellular tolerance to RS and increasing susceptibility to ATR inhibition. We show that CDK12-mediated phosphorylation of CDC5L is required for RPA chromatin loading and mediates resistance to combined cyclin K and ATR depletion. Our findings identify cyclin K-CDK12 as a critical regulator of RPA dynamics and support its inhibition as rational therapy for RS-high tumors, including TNBC.

overall_design: RNA-seq profiling of parental MDA-MB-231 cells (Par) and their derivatives resistant to CT7311, a cyclin K protein degrader (CTR200). Each group was analyzed in triplicate to compare the transcriptomic landscape associated with acquired drug resistance to cyclin K protein degradation.

first: Eun-Bee

first: Sophia

last: Podeszwa

first: Geoffrey

middle: I

last: Shapiro


title: A Druggable TGF-β-Mediated Stromal Programming Axis in ATM-Deficient Pancreatic Cancer

first: Kyle

last: Gaulton

summary: The tumor microenvironment (TME) actively contributes to pancreatic ductal adenocarcinoma (PDAC) pathogenesis via a dynamic bidirectional tumor–stroma dialog. Here, we show that homologous recombination-defective (HRD) neoplastic epithelium reprograms its TME in a genotype-specific manner to promote cancer aggressiveness. Autochthonous mouse models, co-culture systems, single-nucleus multiomics investigations and human PDAC specimens revealed that tumoral ATM serine/threonine kinase status impacts cancer-associated fibroblast fate towards αSMA+ myofibroblastic (myCAF) differentiation, independently of P53 loss-of-function. Vice versa, myCAFs foster cancer aggressiveness and specific chemoresistance patterns. Specifically, ATM deficiency is associated with the activation of reactive oxygen species and actomyosin signaling axis, increased contractility, and a greater TGF-β1 release. Pharmacological interference with TGF-β signaling reverts myofibroblast differentiation, chemoresistance, and tumor promotion in various ATM-deficient PDAC models. Overall, our findings demonstrate that both mouse and human HRD-PDACs reprogram their TME towards a cancer-promoting fate, making them suitable for combinatorial therapies targeting intrinsic vulnerabilities and extrinsic tumor–stroma crosstalks.

overall_design: Pancreatic ductal adenocarcinoma tumors were extracted from two female KPC (Atm+/+; Trp53fl/fl; Kras+; p48Cre+) and two female AKPC (Atmfl/fl; Trp53fl/fl; Kras+; p48Cre+) mice after 8-11 weeks of life. Nuclei from both KPC and from both AKPC tumors were pooled together and then two runs (one KPC, one AKPC) of paired snRNA-seq and snATAC-seq were performed.

first: Hannah

middle: M

last: Mummey

first: Elodie

last: Roger

first: Yun

middle: J


title: Early-life nutritional imbalance impairs colonic epithelial regeneration through gut microbiota dysbiosis and metabolic suppression

city: Yangzhou

first: Yimin

last: Zhuang

state: Jiangsu

address: No. 88, Daxue South Road

institute: Yangzhou University

summary: Childhood malnutrition represents a major global health burden that extends beyond caloric deficiency to include dietary imbalance. The early-life gut microbiome functions as a dynamic ecosystem whose structure and metabolic output are intimately linked to host development, yet how nutritional imbalance disrupts this ecosystem and its integration with the host remains poorly understood.

overall_design: A total of 30 three-week-old female specific pathogen-free (SPF) C57BL/6J mice (Sibeifu Biotechnology Co., Ltd., China) were used in the experiment and underwent a 7-day acclimation period before treatment.


title: Reprogramming of epidermal keratinocytes by PITX1 transforms the cutaneous cellular landscape and promotes wound healing [CUT&Tag]

city: Bethesda

first: Stephen

last: Brooks

state: MD

address: 50 SOUTH DR, RM 1140

department: Biodata Mining and Discovery Section

institute: NIAMS/NIH

summary: Skin injuries heal slowly, compared to oral epithelial tissues, and often do not regenerate lost adnexa. Using a murine mouse model expressing the oral epithelial transcription factor PITX1 in the skin, we performed CUT&Tag-seq analysis in healthy skin to delinate PITX1 and histone occupancies in the skin.

overall_design: Whole murine dorsal skin (healthy, Control and PITX1+) was dissociated into single cells and processed for CUT&Tag-seq.

first: Overmiller

last: Andrew Michael

first: Jiang

last: Kan

first: Morasso

last: Maria Irene


title: Image Tracing of Inflammatory Intestinal Organoids via Computational Clearing

city: Daegu

first: Pacian

middle: Sung-Wook

last: Nam

state: Daegu

address: 90 Chilgokjoongangdaero-136-gil

department: School of Medicine

country: South Korea

institute: Kyungpook National University

summary: Gene expression of dextran sulfate sodium (DSS)-treated mouse intestinal organoids (mIOs) was examined by RNA-seq to characterize the inflammatory response. Gene set enrichment analysis (GSEA) revealed the upregulation of inflammatory pathways, including TNF-α signaling via NF-κB and IL-6/JAK/STAT3 signaling, alongside the downregulation of stem cell maintenance and proliferation pathways, including WNT/β-catenin signaling and PI3K/AKT/mTOR signaling.

overall_design: Mouse intestinal crypts were embedded in Matrigel and cultured in EGF, Noggin, and R-spondin (ENR) medium to induce mIOs. The mIOs were treated with dextran sulfate sodium (DSS) to induce inflammation in vitro.


title: Stromal and Endothelial Transcriptional Changes during Progression from MGUS to Myeloma and after Treatment Response [RNA-Seq]

city: Pamplona

first: Itziar

last: Cenzano

state: Navarra

address: Avenida de Pio XII, 55

country: Spain

institute: CIMA Universidad de Navarra

summary: The role of the non-immune bone marrow microenvironment (BME) in the transition from monoclonal gammopathy of undetermined significance (MGUS) into clinically active multiple myeloma (MM) remains incompletely defined. To address this, we transcriptionally profiled endothelial cells (EC), mesenchymal stem cells (MSC), and MM cells at single-cell resolution from genetically engineered mouse models (BIc1 and MIc1) that recapitulate MGUS to MM progression. Our analysis of the BIc1 model revealed distinct transcriptional alterations in EC and MSC, uncovering stage-specific BME-PC interactions shaping disease progression. Interestingly, we identified an interferon (IFN)-related myeloma signature in both EC and MSC that was reversed after treatment with bortezomib (Velcade), lenalidomide (Revlimid), and dexamethasone (VRd).

overall_design:
We performed bulk RNA-seq on prospectively isolated BM mesenchymal stem cells (MSC) from healthy donors (HD; n=8, bone marrow samples from orthopedic surgery, 50–80 years), MGUS (n=5), SMM (n=2), and MM (n=10) patients. Samples from both sexes were collected with informed consent and ethic approval. Total RNA was extracted, and full-length cDNA libraries were prepared with SMART-Seq v4 plus (Takara), enzymatically fragmented, indexed with unique dual indexes, and sequenced on an Illumina NextSeq 2000 using paired-end.
HR stands for High risk.

first: Felipe

last: Prosper


title: Single cell RNA-seq characterization of non-fibrotic stromal wound repopulation in the rabbit

city: Dallas

first: Katherine

last: Borner

address: 5323 Harry Hines Blvd.

department: Ophthalmology

institute: UT Southwestern Medical Center

summary: Following injury or surgery, quiescent stromal keratocytes can transition into fibroblasts or myofibroblasts leading to either transient or protracted corneal haze. In this study, we investigate the transcriptional changes associated with non-fibrotic wound healing using a transcorneal freeze injury (FI) in the rabbit, which induces full-thickness stromal cell loss without inducing keratocyte-myofibroblast transformation. In control corneas, scRNA-seq revealed multiple clusters expressing markers associated with keratocyte identity (e.g. KERA, LUM, DCN, and ALDH1A1), suggesting heterogeneity in stromal keratocytes in the uninjured stroma. On day 7 after FI, in vivo imaging revealed elongated cells with increased backscatter, consistent with fibroblast migration into the wounded region. Using scRNA-seq, two additional clusters expressing fibroblast markers were also identified. These clusters retained many markers consistent with keratocyte identity, and trajectory analysis demonstrated a continuous progression from quiescent keratocytes to fibroblasts. Both fibroblast clusters had elevated expression genes encoding tenascin C (TNC), claudin 5 (CLDN5), developmental proteoglycans (e.g., BGN, ASPN, VCAN), and cytoskeletal genes (MYL9, MYH10, CDH11), but did not express markers of myofibroblast transformation. Together these genes suggest a mechanically active but non-fibrotic phenotype. One of the two fibroblast clusters also expressed genes related to cell proliferation. By day 28, fibroblastic gene expression was reduced, consistent with resolution of wound healing. These findings define the transcriptional dynamics of intrastromal cell migration following FI and reveal a transient fibroblastic state that supports wound repopulation without fibrosis. Understanding this non-fibrotic repair mechanism could inform strategies to prevent scarring following corneal surgery or injury.

overall_design: A transcorneal freeze injury was performed on one eye of four New Zealand White rabbits. Two rabbits were sacrificed at 7 and 28 days, respectively. Full thickness central corneas from both the procedure eye and the contralateral control eye were collected and analyzed using scRNA-seq.

first: Borner

last: Katherine

first: Yu

last: Ze

first: Xing

last: Chao

first: Petroll

last: W. Matthew


title: Transcriptomic profiling of mouse subgranular zone-derived neural stem cells exposed to cadmium

city: Shanghai

first: Xiuli

last: Chang

address: No. 130, Dong'an Road, Shanghai, 200032, China

institute: Fudan University

summary: We performed RNA-seq to investigate the effects of cadmium (Cd) exposure on the transcriptome of primary mouse neural stem cells (NSCs) isolated from the subgranular zone (SGZ) of the hippocampus. NSCs at passage 3 were exposed to 0.8 μM CdCl₂ for 72 hours, and total RNA was extracted for sequencing on an Illumina platform. Differential expression analysis identified 684 differentially expressed genes (334 downregulated, 350 upregulated). Gene Ontology enrichment analysis of downregulated genes revealed impairment of neurogenesis-related processes, and WikiPathways analysis indicated enrichment of Wnt signaling pathway components. These transcriptomic data support the finding that Cd disrupts non-canonical Wnt5a signaling and impairs neuronal differentiation of SGZ-derived NSCs.

overall_design: RNA-seq of primary mouse SGZ-derived neural stem cells treated with 0.8 μM CdCl₂ (n=3) versus untreated control (n=3) for 72 hours.

first: Xutong

last: Qin

first: Yuwei

last: Zhang

first: Longfei

last: Feng