title: Mapping TRMT61B-mediated m¹A methylation of a synthetic RNA pool using uMRT

summary: The transcriptome-wide distribution of N¹-methyladenosine (m¹A) in mRNA remains contested, hindering a clear understanding of regulation of mRNA metabolism by m¹A. Additionally, the enzyme(s) that installs the majority of reported mRNA m¹A sites has yet to be identified. In this study, we characterized TRMT61B, an m¹A methyltransferase known to methylate mitochondrial RNAs, but whose exact sequence preferences have been underexplored. We in vitro methylated a synthetic pool of 5UTR and CDS sequences with TRMT61B, and mapped m¹A sites using mutation signatures installed by uMRT.

overall_design: An RNA pool containing >35,000 human and >500 viral 5′UTR and CDS regions (300 nt total length, including adapter sequences, full 5'UTRs, and truncated endogenous CDS was generated through in vitro transcription of a synthetic DNA oligonucleotide library. RNA was thenused for in vitro methylation reactions with TRMT61B, with either no (NE),  low (LE) or high enzyme (HE) amounts. Methylation was carried out in triplicate, with two small (40 uL) and one large (640-680 uL) reaction, with equal ratios of all reaction components. The large reactions were pooled for sequencing and deconvoluted during analysis. Sequencing was carried out using ultraMarathonRT to generate cDNA. Three rounds of sequencing were carried out on each of three replicate samples (2x150 on NovaSeq, 2x250 twice on NextSeq 2000) to get sufficient coverage depth for mutational analysis. After alignment, bam files from the three rounds of sequencing were merged by sample and then run through an ad-hoc mutational analysis pipeline to look for increased mutation rates in LE and HE enzymes vs. NE.


title: Comprehensive single-nucleus molecular and cell profiling features for cardiac tissues across platforms [ICELL8_snRNAseq]

status: Public on Mar 10 2026

city: Saint Louis

first: Haewon

last: Shin

state: MO

address: 660 S Euclid

country: USA

institute: Washington University School of Medicine in St. Louis

type: Expression profiling by high throughput sequencing

summary: Single-nucleus RNA sequencing (snRNA-seq) enables transcriptomic profiling of complex tissues such as the heart, where dissociation into intact single cells is challenging. Among commercially available snRNA-seq platforms, 10x Genomics Chromium and Takara Bio’s ICELL8 cx represent technically distinct approaches, yet systematic comparisons in cardiac tissue are limited. Here, we benchmarked the 3′ Chromium, 5′ Chromium, and ICELL8 cx platforms using nuclei isolated from mouse and human heart tissues, including healthy donors and patients with non-ischemic cardiomyopathy. We assessed nuclei capture efficiency, library complexity, transcript detection sensitivity, RNA biotype coverage, ambient RNA contamination, and cell-type resolution. The Chromium platforms achieved higher nuclear throughput with robust capture efficiency and low ambient RNA contamination, whereas ICELL8 cx provided deeper transcriptomic coverage per nucleus, detecting a broader range of RNA biotypes, including low-abundance non-coding RNAs, albeit with lower nuclei yield. All platforms consistently resolved major cardiac cell populations. Together, these results inform selection of snRNA-seq workflows for cardiac tissue studies, highlighting trade-offs between cellular throughput and transcriptomic depth.

overall_design: Ethical Approval for Human Specimens The study is compliant with all relevant ethical regulations and has been approved by the Washington University School of Medicine Institutional Review Board (IRB #201104172). Informed consent was obtained from each patient prior to tissue collection by Washington University School of Medicine, and no compensation was provided in exchange for subject participation in the study. All demographic and clinical data have been de-identified.  Inclusion Criteria Strict inclusion criteria were employed before tissue collection and any patients with human immunodeficiency virus or hepatitis were excluded. Healthy donor heart tissue samples were collected from patients with stable ejection fraction with no known history of cardiovascular disease and with a non-cardiac cause of death/transplant. NICM heart tissue samples were collected from patients with HF independent of ischemia and no known history of myocardial infarction.  Human single cell isolation for snRNA-seq Frozen myocardial tissue from left ventricular assist device (LVAD) cores or the corresponding region from the apex of explanted donors were minced with a razor blade and dissociated with a plastic pestle in Chromium Nuclei Isolation Kit Lysis Buffer (10X Genomics, Pleasanton, CA). Samples were incubated on ice for 10 minutes, and the tissue suspension was pipetted onto the Nuclei Isolation Column and Collection Tube and centrifuged at 16,000 rcf for 20 seconds at 4°C. Flow-through in the Collection Tube was centrifuged again at 1,000 rcf for 3 minutes to pellet the nuclei and resuspended in 300 μl of Wash and Resuspension Buffer before staining with 1 μl  DRAQ5 (5 mM solution, ThermoFisher Scientific) and incubating on ice for 10 minutes. DRAQ5+ nuclei were sorted into Nuclei Wash Buffer on a BD FACS Melody (BD Biosciences) using a 100 μm nozzle. Recovered nuclei were centrifuged again under the above parameters and were gently resuspended in Nuclei Wash Buffer to a target concentration of 1,000 nuclei μl−1. Nuclei were counted on a hemocytometer and concentration was adjusted as necessary. Following flow cytometry, sorted DRAQ5+ nuclei were centrifuged at 2,000 rcf for 5 minutes at 4°C, and nuclei were resuspended in 100 µl 1X PBS, prior to loading into the Chromium or ICELL8 cx systems as described below.  After manually counting nuclei using Trypan Blue, samples were loaded at a concentration of 40,000 nuclei ml-1. Libraries were prepared using the manufacturers’ instructions- SMART-Seq Pro (ICELL8 cx Single-Cell System User Manual kit (Takara Bio, San Jose, CA)-and sequenced on a NovaSeqX (Illumina) instrument following manufacturers’ guidelines: 151 X 8 X 8 X 151 [read1 X i7 index X i5 index X read2].

first: Urvi

middle: N

last: Shroff

first: Xianglong

last: Zhang

first: Tracy

last: Yamawaki

first: Daniel

middle: R

last: Lu

first: Junedh

middle: M

last: Amrute

first: Andrea

last: Bredemeyer

first: Amrita

last: Das

first: Brandon

last: Ason

first: Simon

last: Jackson

first: Chi-Ming

last: Li

first: Kory

middle: J

last: Lavine


title: Immunoprecipitation of m¹A methylated mRNA upon TRMT61B overexpression

city: New Haven

first: Sigrid

last: Nachtergaele

state: CT

address: 260 Whitney Ave

department: Molecular, Cellular, and Developmental Biology

institute: Yale University

summary: The transcriptome-wide distribution of N¹-methyladenosine (m¹A) in mRNA remains contested, hindering a clear understanding of regulation of mRNA metabolism by m¹A. Additionally, the enzyme(s) that installs the majority of reported mRNA m¹A sites has yet to be identified. In this study, we characterized TRMT61B, an m¹A methyltransferase known to methylate mitochondrial RNAs, but whose exact sequence preferences have been underexplored. We integrated in vivo overexpression of TRMT61B and in vitro methylation of a synthetic pool of diverse human RNA sequences to characterize the sequence preferences of TRMT61B target RNAs.

overall_design: RNA-seq profiling of U2OS cells was collected for transiently overexpressing TRMT61B or a control plasmid, with and without m1A-IP treatment. Normalized counts tables were generated using DESeq2, and mutational calling was carried out using bakR.

first: Dorthy

last: Fang

first: John

last: Babich

first: Ryan

last: Stanton

first: Isaac

last: Vock

first: Kyrillos

last: Abdallah

first: Mingyi

last: Zhu

first: Richard

first: Matthew

last: Simon

first: Wendy V

last: Gilbert


title: A personalized therapeutic approach for cancer containing the African-centric P47S variant of TP53

city: Philadelphia

first: Priyankara

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: We performed RNA-sequencing comparing WT and P47S HepG2 tumor cells (P47S clone is 5B4).  We previously performed a drug screen in WT and P47S HepG2, and we found that Lexibulin (an anti-mitotic compound) preferentially decreases cell viability in the P47S mutant compared to WT.  Our preliminary data show that Lexibulin induces p53 target gene expression but no apparent differences in cell death between WT and P47S HepG2.  We performed RNA-seq to capture the whole transcriptional profile, comparing our vehicle controls to Lexibulin-treated cells, and comparing Lexibulin-treated WT to Lexibulin-treated P47S cells to identify key genes that contribute to preferential targeting of P47S HepG2 in an unbiased manner.

overall_design: RNA-seq profiling of WT and P47S HepG2 treated with vehicle / DMSO (0 hours Lexibulin) and 10 µM Lexibulin for 8 and 24 hours.

first: Maya

last: Foster

first: Kaitlyn

last: Casey

first: Joel

last: Cassel

first: Ying

last: Ye

first: Jason

last: Diaz

first: Krist

middle: S

last: McGuire

first: Thibaut

last: Barnoud

first: Maureen

middle: E

last: Murph


title: RNA-seq data for P. aeruginosa PAO1 under diverse conditions for regulatory network analysis

city: La Jolla

first: Heera

last: Bajpe

state: CA

address: 9500 Gilman Dr.

institute: UC San Diego

summary: This dataset consists of RNA-seq data for P. aeruginosa PAO1 to enable comparison of transcriptional regulatory networks across Pseudomonas strains.

overall_design: Expression profiling data for P. aeruginosa PAO1 under a diverse set of conditions (different media types, osmotic stress, oxidative stress, nitrogen starvation, iron starvation, copper ion stress, antibiotic treatment, mixed carbon source treatment) were generated through RNA sequencing.

last: Hefner

last: Szubin

first: Jaemin

last: Sung

first: Bernhard

middle: O

last: Palsson


title: An In Vitro Functional Characterization of the Cholesterol-Transforming Blautia hominis Strain HA2291 Isolated from the Human Gut

city: 青岛 Qingdao

first: Warren

last: Chanda

state: Shandong Province

address: No. 72 Beihai road, Jimo District

department: State Key Laboratory of Microbial Technology

country: China

institute: Shandong University

summary: Cholesterol is an essential lipid required for membrane structure and normal physiological functions. However, dysregulation of cholesterol homeostasis, manifesting as hypercholesterolemia, can precipitate a range of metabolic and cardiovascular diseases. Blautia species are important gut commensals, but their role in cholesterol metabolism remains poorly defined. Methods: A total of 63 Blautia strains isolated from human fecal samples were screened for cholesterol conversion using the o-phthalaldehyde colorimetric assay in cholesterol-containing media with or without oxgall. Cholesterol removal by live and heat-inactivated cells was compared. Metabolomic, transcriptomic, and proteomic analyses were employed to investigate molecular mechanisms and involved genes. Results: Nine strains significantly lowered cholesterol levels (live cells: 31–78%; heat-inactivated cells: 8–64%), with the B. hominis strain HA2291, the Blautia sp. strain HA3515, and the B. coccoides strain HA4419 showing the strongest activity. Oxgall increased cholesterol removal by live cells to 74–83%, indicating bile-tolerant metabolism activity. Metabolomic profiling revealed that B. hominis HA2291 transformed cholesterol into cholest-4-en-3-one and epicholestanol. An SCP2-like protein, RS03310, was identified as a candidate cholesterol-interacting factor; its recombinant form catalyzed measurable NAD+-dependent cholesterol oxidation in vitro. Conclusions: Blautia hominis HA2291 may employ multiple in vitro strategies for cholesterol-lowering, including cell-surface adsorption (heat-inactivated cells), bile-enhanced removal (oxgall effect), and enzymatic transformation, with the gene RS03310 implicated as the main contributor. These findings provide in vitro mechanistic insights into Blautia-mediated cholesterol metabolism, highlight RS03310 as a candidate gene associated with cholesterol biotransformation, and advance our understanding of the potential role of Blautia in host cholesterol homeostasis.

overall_design: RNA-seq profiling of B. hominis HA2291 cells after 48h growth (OD600=0.8) in mGAM media supplemented with (BHC)and without (BH) cholesterol (100µg/mL).

first: He

last: Jiang

first: Shuang-Jiang

last: Liu


title: Transcriptome profiling of B-cell lymphoma cells with pharmacological inhibition of KDM5A/B by using the inhibitor JQKD82

city: Columbus

first: Dawei

last: Zhou

state: OH

address: 400 W 12th Ave, Wiseman Hall 351C

department: Pathology

institute: The Ohio State University Wexner Medical Center

summary: Histone methylation critically controls the progression of human diseases, including infections and cancers. Our study reported that histone lysine demethylases (KDMs) in the KDM5 family KDM5A/B play profound roles in suppressing lytic reactivation of oncogenic human herpesvirus 8 (HHV-8), i.e., Kaposi's sarcoma-associated herpesvirus (KSHV), a human gamma-herpesvirus and an etiological agent of multiple malignancies. During KSHV lytic reactivation from latency, host innate immune responses are also activated, which is expected to favor the tumor cell killing by generating the additional oncolytic effects. To further study the systemic effect of KSHV on host immune response under the regulation of KDM5A/B, we performed the transcriptome profiling analysis for both KSHV-infected BCBL-1 vs KSHV-negative BJAB cells with or without JQKD82 treatment by RNA-seq. Our RNA-seq analysis and further experimental validation suggested that the KDM5 inhibitor JQKD82 is capable of inducing antiviral/antitumor innate immune responses in KSHV-negative B-cell lymphomas, but such effects are more striking in KSHV-positive B-cell lymphomas likely due to KSHV lytic reactivation that further triggers the innate immune activation in tumor cells.

overall_design: KSHV-infected BCBL-1 and KSHV-negative BJAB cells were treated with either DMSO as a control, or JQKD82 (20 µM) for 4 days. Total RNA was extracted and submitted to Novogene for QC, polyA selection, multiplexing, library preparation and sequencing. Three independent experiments were performed. Totally 12 samples were subjected to RNA-seq assay.

first: Zhenyu

last: Wu

first: Jian

last: Zhu


title: Spatial transcriptomic profiling reveals body site-specific inflammatory differences in psoriasis lesions

city: Aarhus

first: Morten

middle: Muhlig

last: Nielsen

address: Palle Juul-Jensens Bouleward 99

department: Department of Molecular Medicine

country: Denmark

institute: Aarhus University

summary: Psoriasis is a common chronic inflammatory skin disease. Treatments lead to substantial improvement of most psoriasis plaques. However, it can be challenging to reach disease resolution in certain hard to treat areas such as scalp, and lower extremity. Here we map histologic and spatial transcriptomic differences between psoriasis lesions across different anatomical locations, to understand if differences can be linked to plaque-site specific treatment resistance. Quantitative immunohistochemical analysis and transcriptomic digital spatial profiling were performed on skin punch biopsies obtained from unaffected areas on the trunk, lesional (LS) areas of the scalp, upper extremity and lower extremity of 12 patients with psoriasis. Histological analysis showed no significant differences in epidermal thickness among LS skin from different body locations. Immunohistochemical markers (CD3, CD4, CD8, CD103, CD207, IL-12RB1, IL-17A, IL-23R, RORγt, FOXP3, and MPO) did not differ significantly between LS sites. Whole transcriptome spatial RNA profiling identified several differentially expressed genes that revealed site-specific transcriptomic differences. Notably, IL-23 signaling was significantly enriched in the lower extremity epidermis, and IL-17 signaling was more pronounced in the epidermis of LS samples. These findings highlight minimal histological and immunohistochemical variation, yet significant transcriptomic and pathway differences between psoriasis body locations, suggesting potential targets for site-specific therapeutic strategies.

first: Thomas

last: Emmanuel

first: Hakim

middle: B

last: Abdallah

first: Claus

last: Johansen


title: 4D force patterning enables spatial control of angiogenesis

city: Cambridge

first: Jessica

last: Shah

state: MA

address: 77 Massachusetts Ave

institute: Massachusetts Institute of Technology

summary: Engineering organized microvascular networks remains a critical challenge in tissue engineering and regenerative medicine. While biochemical approaches for patterning angiogenesis via growth factor delivery have shown promise, their inability to pattern sustained growth factor gradients with precise spatiotemporal control limit their effectiveness. Here, we demonstrate that dynamically patterned mechanical forces can provide precise spatiotemporal control over angiogenic sprouting in 3D microvascular networks. We developed a magnetically actuated 3D human vessel-on-a-chip platform that integrates a perfusable endothelialized microchannel within a collagen matrix and allows non-invasive, tunable, multi-axial mechanical stimulation across all three spatial dimensions and time (4D). Using an automated 3-axis actuator, we systematically investigated how strain magnitude, frequency, and direction modulate endothelial cell behavior and vessel morphogenesis. Dynamic mechanical stimulation at physiological strain magnitudes (5–15%) enhanced endothelial alignment and barrier function while promoting angiogenesis in a strain-magnitude–dependent manner: lower dynamic strain (5%) maximized sprout initiation, whereas higher dynamic strain (15%) promoted elongation of individual sprouts. Sequential reorientation of strain direction further reprogrammed sprouting trajectories along X, Y, and Z directions, generating complex 3D microvascular geometries such as L-shaped branches. RNA sequencing revealed mechanically induced transcriptional profiles distinct from unstimulated controls, characterized by coordinated upregulation of angiogenic (VEGFA, FGFR1, PDGFA), mechanotransduction (ANKRD1, ZYX, FOSB), extracellular matrix remodeling (ABI3BP, SPOCK1, COL8A1), and barrier-associated (SERPINE2, P2RY1) genes and pathways. Functional perturbation of the mechanosensitive ion channel Piezo1 attenuated strain-induced sprouting without altering barrier stabilization, indicating that dynamic mechanical stimulation engages multiple mechanotransduction pathways to regulate angiogenic growth. Collectively, these findings establish a strategy for spatiotemporally controlled angiogenesis through engineered mechanical cues and demonstrate that 4D force patterning can program vascular morphogenesis while preserving function. This approach provides a foundation for engineering hierarchically organized vascular networks for tissue regeneration and disease modeling.

overall_design: HUVECs were cultured as 3D vessels in collagen-based extracellular matrix within a microfluidic “vessel-on-a-chip” device. After lumen formation and stabilization, devices were assigned to one of three mechanical conditions: (1) control (C), (2) 5% “low” strain (L), and (3) 15% “high” strain (H). Mechanical stimulation was applied using an external magnetic actuation system that deforms the surrounding matrix and vessel wall, thereby transmitting strain to the 3D endothelial vessel. At the end of the stimulation period, HUVECs were harvested from the 3D vessels for downstream profiling. Each RNA sample consisted of cells pooled from two independent devices under the same mechanical condition.

first: Sina

last: Kheiri

first: Ritu

last: Raman


title: A Neutrophil–Mesangial Cell Axis Promotes Glomerular Injury in Lupus Nephritis

city: Bethesda

first: Kan

state: MD

address: 9000 Rockville Pike

institute: NIAMS/NIH

summary: Neutrophil extracellular trap (NET)-producing neutrophils infiltrate the kidney early in lupus nephritis (LN). NET-derived citrullinated histone H3 activates mesangial cells (MCs) through TLR4, promoting inflammation and fibrosis, establishing a novel NET–MC axis that may represent a mechanistic driver and therapeutic target in LN.

overall_design: Primary human MCs were seeded at a density of 5 × 10⁴ cells per well in 12-well plates and cultured in RPMI-1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS for 24 hours at 37 °C. The medium was then replaced with RPMI-1640 medium containing 10% KnockOut Serum Replacement (Thermo Fisher Scientific) and the cells were cultured for an additional 24 hours. After washing with PBS, the cells were stimulated with either 50 µg spontaneously generated NETs for 72 hours or 5 µg recombinant citH3 for 48 hours at 37 °C. Following stimulation, the cells were washed with PBS and lysed in 500 µL of TRIzol Reagent (Thermo Fisher Scientific) for RNA extraction. Total RNA was isolated followed by column-based purification with RNA Clean & Concentrator-5 Kit (Zymo Research). RNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). cDNA concentrations were quantified using the PicoGreen dsDNA Assay (Thermo Fisher Scientific), normalized to 3 nM, pooled, and sequenced on the Illumina NovaSeq X Plus platform.

first: Norio

last: Hanata

first: Carmelo

last: Carmona-Rivera

first: Victoria

last: Hoffmann

first: Zerai

last: Manna

first: Davide

last: Randazzo

first: Meryl

last: Waldman

first: Sarfaraz

last: Hasni

first: Mariana

last: Kaplan


title: KLF2 overrides the resident memory CD8 T cell differentiation program, in opposition to KLF3

city: Chicago

first: Weiguo

last: Cui

state: IL

address: 303 E Chicago Ave

institute: Northwestern University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Numerous transcriptional regulators have been associated with the differentiation pathways that lead to recirculating versus tissue-resident memory T cells. However, it is unclear whether independent, coordinated expression of these regulators is required to determine residency versus recirculation, or whether there is a hierarchy, with some factors playing a dominant role in controlling T cell trafficking. We report that ablation of the gene encoding Kruppel-like factor 2 (KLF2) during CD8+ T cell activation leads to rapid transcriptional reprograming, such that effector T cells fail to recirculate and prematurely acquire canonical phenotypic and transcriptional characteristics of resident memory cells (TRM). Klf2-deficient memory CD8+ T cells retained the capacity to undergo recall responses, including in vivo pathogen control. These data suggest KLF2 diverts CD8+ T cells from the TRM differentiation program. In contrast, ablation of another member of the KLF family, KLF3, enhanced differentiation of some recirculating T cell subsets and limited production of TRM in lymphoid tissues. However, both KLF2 and KLF3 were required for differentiation of long-lived effector cells, suggesting cooperation between these factors in some situations. These findings indicate KLFs occupy a central nexus in coordinating activated CD8+ T cell differentiation and trafficking.

overall_design: KLF2 CUT&Tag assay on P14 cells isolated from naïve mice or LCMV Armstrong infected mice on day 8 post-infection.

first: Kexin

last: Gai


title: Transcriptome profiling of WERI-Rb1 retinoblastoma cells to elucidate ELF2-driven mechanisms of topotecan resistance

city: Guangzhou

first: Jing

last: Zhuang

address: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, No. 54 South Xianlie Road

summary: To investigate the role of ELF2 in topotecan resistance, we performed RNA-seq on WERI-Rb1 retinoblastoma cells. Control (NTC) and ELF2-knockout (sgELF2) cells were treated with either vehicle or 30 nM topotecan for 96 hours. Differential gene expression analysis revealed transcriptomic changes associated with ELF2 loss and its impact on chemoresistance, providing insights into potential mechanisms and therapeutic targets.

overall_design: A 2x2 factorial design: WERI-Rb1 cells with two genetic backgrounds (Non-Targeting Control vs. ELF2-knockout) were subjected to two treatment conditions (Vehicle vs. 30 nM Topotecan for 96 hours). Biological replicates were included for each condition (n=3).

first: Guei-Sheung

last: Liu

first: Shuilian

last: Chen

first: Jingyi

last: Jiang


title: Loss of KNTC1 Triggers a 53BP1 and USP28 Dependent Cell Cycle Arrest Without Prolonged Mitotic Delays

city: New York

first: Bic

last: MSKCC

state: NY

address: 1275 York Ave.

institute: Memorial SLoan-Kettering Cancer Center

summary: Accurate and efficient mitosis is the hallmark of animal cycling cells. While the spindle assembly checkpoint (SAC) ensures mitotic fidelity by delaying mitotic progression, a SAC-independent surveillance program is known to monitor the duration – without affecting the progression – of mitosis. This pathway, mediated by 53BP1, USP28, and p53, induces post-mitotic arrest in daughter cells following mitoses that exceed a defined temporal threshold. To uncover additional inputs into this pathway, we performed genome-wide CRISPR-Cas9 screens in wild-type, p53-/-, and USP28-/- human cells. Among genes selectively required in wild-type cells, we identified two components of the RZZ kinetochore complex—KNTC1 (ROD) and ZWILCH—as high-confidence hits. Remarkably, depleting KNTC1, which impaired SAC maintenance, triggered a robust 53BP1, USP28, and p53-dependent arrest without the characteristic mitotic delay. KNTC1-deficent cells could arrest following multiple, consecutive divisions with normal durations, and the last mitosis prior to arrest progressed no differently from the previous. Instead, loss of KNTC1 was found to trigger 53BP1-USP28-p53 complex formation soon after mitotic entry, an indication of pathway activation without prolonged mitosis. These findings suggest that the 53BP1–USP28–p53 surveillance pathway can monitor various mitotic anomalies to safeguard cell fitness.

overall_design: Full genome CRISPR screens were undertaken in hTERT-RPE1 cells WT, p53 knockout, and USP28 knockout backgrounds, using the BRUNELLO library. Cells were grown for four weeks after puromycin selection, with no additional selection pressure. Genomic DNA was isolated immediately following selection as T0, and at the end of four weeks as T4, and read counts were analyzed to identify genes selectively required for growth in WT but not p53 knockout and USP28 knockout cells.

first: Avital

last: Shulman

first: Kanako

last: Ozaki

first: Lukas

last: Cheng

first: Meng-Fu

last: Tsou


title: Single cell transcriptomics of retinal organoids reveal effects of PRPF31 mutations on Mueller glial activation and progressive photoreceptor degeneration

city: Linz

first: Jingjing

last: Qi

address: Altenberger Straße 69

department: Zentrum für Medizinische Forschung

country: Austria

institute: Johannes Kepler University Linz

summary: Retinitis pigmentosa (RP) encompasses a group of inherited retinal disorders characterized by progressive degeneration of rod and cone photoreceptors, leading to vision loss. Among RP subtypes, RP11 is linked to mutations in PRPF31, a key spliceosome component, resulting in retinal cell dysfunction. Although PRPF31 is ubiquitously expressed, its mutations predominantly impact retinal cells, leading to the progressive loss of photoreceptors. Human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) offer a powerful in vitro model for studying RP pathophysiology and therapeutic development. Despite significant progress, studies focused on photoreceptor and retinal pigment epithelium dysfunction in late disease stages, leaving early molecular events and the involvement of other retinal cell types unresolved. Moreover, comprehensive single-cell analyses capturing dynamic transcriptional changes across all retinal populations at early and late differentiation stages are still lacking. Using patient-derived ROs, this study investigates the developmental trajectory of PRPF31-RP11 mutation through single-cell RNA sequencing, highlighting early Müller glial expansion, retinal ganglion cell stress, and progressive photoreceptor degeneration. Findings identify dysregulated molecular pathways associated with phototransduction, oxidative stress, and inflammation, providing insights into RP11 pathogenesis and potential therapeutic targets.

overall_design: Retinal organoids were generated from human iPSCs of wild-type and PRPF31-mutant lines.

first: For each genotype

last: single-cell RNA-seq was performed on organoids collected at day 85 (early/RC2 phase) and day 285 (mature/RC1 phase).

first: Alessandro

last: Bellapianta

first: Ahmad

last: Salti


title: Cooperation between architectural C2H2 proteins in CP190 recruitment to Drosophila regulatory elements [ChIP-Seq]

city: Moscow

first: Natalia

last: Klimenko

address: 34/5 Vavilova Street

country: Russia

institute: Institute of Gene Biology (IGB) of the Russian Academy of Sciences

summary: CTCF is the best-studied architectural protein that is highly conserved among animals, including Drosophila and mammals. In Drosophila, CTCF is involved in the organization of functional promoters and insulators, in cooperation with many other architectural proteins, including Su(Hw) and Pita. These proteins, like many other architectural proteins, interact with CP190, which, along with its partners, recruits transcriptional complexes to target promoters. This study was conducted to investigate the cooperation between architectural proteins in the recruitment of CP190 to regulatory elements. We generated transgenic flies expressing double and triple mutants of dCTCF, Su(Hw), and Pita that cannot interact with CP190. The single mutants are fully viable; however, few triple mutants develop into adults. In the triple mutant, although the CP190 concentration is reduced, the level of gene transcription remains unaltered, suggesting that co-expression of the three mutant proteins is responsible for CP190 instability. ChIP-seq analysis showed that CP190 is required for dCTCF–chromatin binding. In the triple mutant, CP190 demonstrates an almost complete loss of association with the promoters and insulators to which the tested architectural proteins bind; however, these regulatory elements were found to retain their activity. Architectural proteins cooperate to recruit CP190 to regulatory elements and determine its stability. Despite the important role of CP190 in transcriptional regulation, its functions may be partially performed by partner proteins.

overall_design: ChIP-seq signal of the CTCF and CP190 in whole bodies of wt flies (larvae stage) and flies with mutation in CTCF, Pita and Su(Hw) domains interacting with CP190

first: Oksana

last: Maksimenko


title: Cooperation between architectural C2H2 proteins in CP190 recruitment to Drosophila regulatory elements [RNA-Seq]

overall_design: RNA-Seq in whole bodies of wt flies (larvae stage) and flies with mutation in CTCF, Pita and Su(Hw) domains interacting with CP190


title: Phase separation of endothelial PML drives inflammatory macrophage infiltration during liver fibrosis [scRNA-Seq 1]

city: Chengdu

first: Enjiang

last: Lai

address: 37 Guoxue Alley

institute: Sichuan University

summary: PML directly binds with BRD4 in the nucleus and facilitates BRD4 protein accumulation to amplify pro-inflammatory gene expression through phase separation-dependent SE-activation via PML/BRD4 condensate formation. In the preclinical mouse models, LSEC-specific deletion of PML/BRD4 complex mitigates liver inflammation and fibrosis. Utilizing single-cell RNA-sequencing, we show that epigenetically aberrant LSECs exhibit a reprogramed pro-inflammatory angiocrine landscape in mouse fibrotic livers and establish that TIMP1+ LSECs promote the recruitment of CD63+ monocyte-derived macrophages (MoMFs) during the progression of liver fibrosis. Thereby, we demonstrate that PML/BRD4 in LSECs govern inflammatory immune cell recruitment in liver fibrosis, and that pharmacological BRD4 inhibition or epigenetic repression of PML SE alleviates liver inflammation and fibrosis. In conclusion, we highlight in this study that PML/BRD4-mediated SE activation via phase separation drives pro-inflammatory angiocrine signaling in LSECs, initiating the inflammatory cascade and subsequent immune cell recruitment during liver fibrosis.

overall_design: Single cell RNA-seq was performed on liver tissue extracted from normal and LSEC specific Brd4 knockout CCl4 induced liver fibrosis model mice.

first: Jinhang

last: Gao

first: Can

last: Gan

first: Yang

last: Tai


title: CDK16 and CDK18 inhibition suppresses nuclear pore-mediated DNA repair and hepatocellular carcinoma [liver tumor]

city: Barcelona

first: Agustín

middle: Sánchez

last: Belmonte

state: Barcelona

address: Nazaret 115 -117

department: Systems Oncology Program

country: Spain

institute: Vall d ́Hebron Institute of Oncology (VHIO)

summary: Inhibition of cyclin-dependent kinases (CDK) 4 and 6 has become part of the current standard-of-care treatment in advanced estrogen receptor-positive breast cancer. However, many tumor types are resistant to current CDK inhibitors and the therapeutic value of other CDK family members remains to be validated. By using gene-editing techniques in human cancer cells and genetically-modified mouse models we report here that MYC and beta-catenin driven hepatocellular carcinoma (HCC) is sensitive to the inactivation of CDK16 and CDK18. Whereas individual ablation of CDK14-18 subfamily members displays little effect, concomitant genetic ablation or pharmacological inhibition of CDK16 and CDK18 results in proliferative arrest accompanied by DNA damage and apoptotic cell death. Mechanistically, these defects are associated with a novel role of these proteins in maintaining nuclear pore integrity and the regulation of nucleocytoplasmatic transport. Cyclin Y-CDK16/18 complexes directly phosphorylate the nuclear pore complex (NPC) components NUP35 and NUP214. CDK16/18 inhibition leads to defective nuclear accumulation of the DNA recombinase RAD51, altering DNA repair and inducing lethal levels of DNA damage. Genetic ablation of Cdk16 and Cdk18 in vivo triggers DNA damage and apoptotic cell death in murine liver tumors induced by MYC and -catenin, resulting in tumor regression. Collectively, these data support the development of CDK16/18-targeted therapies for the treatment of HCC.

overall_design: A total of 14 tumor samples underwent RNA-seq: 5 biological replicates from wild-type (WT) mice, 5 from Cdk18 knockout (CDK18 KO) mice, and 4 from Cdk16/Cdk18 double knockout (DKO) mice. Mice were randomly selected within genotype groups for RNA-seq analysis. All tumors were processed uniformly: snap-frozen tissue was mechanically lysed in RLT buffer, followed by total RNA extraction using the QIAGEN RNeasy Mini Kit. The primary experimental variable is genotype (WT vs. CDK18 KO vs. DKO), enabling comparative transcriptomic analysis of CDK family roles in Myc-driven liver tumors.

first: Diego

last: Martínez-Alonso

first: Marcos

last: Malumbres

last: Sánchez-Belmonte


title: Motor learning drives region-specific transcriptomic remodeling in the motor cortex and dorsal striatum

city: Stanford

first: Yue

last: Sun

address: 290 Jane Stanford Way

institute: Stanford University

summary: Motor learning depends on coordinated activity across the motor cortex (M1) and dorsal striatum (dSTR), yet the molecular mechanisms driving learning-related synaptic and circuit remodeling remain unclear. Here, we combine activity-dependent genetic labeling (TRAP) with single-cell RNA sequencing to generate an unbiased, cell type-resolved transcriptional atlas of behaviorally engaged populations during a forelimb reaching task.

overall_design: TRAP mice (Fos-CreER:: Ai9 or Arc-CreER:: Ai9) were trained on a forelimb reaching task for either 2 days (early TRAP) or 8 days (late TRAP) and 4-hydroxytamoxifen (4-OHT) was administered to label behaviorally activated neurons. Control mice received 4-OHT but were not trained. Fos-TRAP mice were used to label active neurons in the motor cortex, and Arc-TRAP mice for the dorsal striatum, due to region-specific TRAP efficiency. Three days after labeling, brain tissues were collected for single-cell RNA sequencing.

first: Sun

last: Yue

first: Ding

last: Jun


title: Detection of statistically robust interactions from diverse RNA-DNA ligation data

city: Frankfurt

first: Simonida

last: Zehr

state: Hesse

address: Theodor-Stern-Kai 7

department: Institute for Cardiovascular Physiology

country: Germany

institute: Goethe University, Frankfurt am Main, Germany,

summary: Chromatin-localized RNAs play diverse roles in gene regulation and nuclear architecture. Mapping genome-wide RNA-DNA interactions is possible using a variety of molecular methods, including using bridging oligonucleotides to ligate RNA and DNA in proximity. While molecular methods have progressed, a robust computational method for calling biologically meaningful RNA-DNA interactions from these data is lacking. Herein, we present RADIAnT, a reads-to-interactions pipeline for analyzing RNA-DNA ligation data. RADIAnT calls interactions against a dataset-specific, unified background which considers RNA binding site-TSS distance and genomic region bias. By scaling the background with RNA abundance, RADIAnT is sensitive enough to detect specific interactions of lowly expressed transcripts, while remaining specific enough to discount false positive interactions of highly abundant RNAs. RADIAnT outperforms previously proposed methods in the accurate recall of genome-wide Malat1-DNA interactions, and in a use-case, was utilized to identify dynamic chromatin-associated RNAs in the physiologically- and pathlologically-relevant process of endothelial-to-mesenchymal transition, whose gene regulatory behaviour was validated experimentally.

overall_design: Total RNA-sequencing was performed on 3 HUVEC replicates, once in wildtype (endothelial phenotype), and once after siRNA-mediated knockdown of GAS5, MIR222hG and NUTM2BAS1, respectively.

first: Sandra

last: Seredinski

first: Katalin

last: Pálfi

first: James

middle: A

last: Oo

first: Emma

middle: C

last: Walsh

last: Bonetti

first: Matthias

last: Leisegang

first: Ralf

middle: P

last: Brandes

first: Marcel

middle: H

last: Schulz

first: Timothy

last: Warwick


title: Phase separation of endothelial PML drives inflammatory macrophage infiltration during liver fibrosis [ChIP-Seq 3]

first: Enjiang

last: Lai

overall_design: ChIP-seq of PML was performed on PML shRNA knockdown human liver sinusoidal endothelial cells treated with human recombinant TNFa.

first: Jinhang

last: Gao

first: Can

last: Gan

last: Tai


title: Phase separation of endothelial PML drives inflammatory macrophage infiltration during liver fibrosis [scRNA-Seq 2]

overall_design: Single cell RNA-seq was performed on liver tissue extracted from normal and LSEC specific Pml knockout CCl4 induced liver fibrosis model mice.

first: Yang


title: Early transcriptional divergence underlies cell fate bias in bovine embryos

city: Fuchu

first: Satoshi

last: Sugimura

state: Tokyo

address: 3-5-8 Saiwai-cho

country: Japan

institute: Tokyo Uiniversity of Agriculture and Technology

summary: We performed single blastomere RNA-seq on individual cells from 1-cell to 8-cell stage bovine embryos to investigate transcriptional heterogeneity during early embryonic development. A total of 90 blastomeres were collected from in vitro-produced embryos. The results provide insights into cell-to-cell variability during early cleavage stages in cattle.

overall_design: In vitro-produced bovine embryos at 1-cell, 2-cell, 4-cell, and 8-cell stages were individually disaggregated to isolate single blastomeres. RNA was extracted and sequenced using paired-end 150 bp reads on the Illumina NovaSeq 6000 platform. Transcript abundance was quantified using STAR and RSEM, and data were analyzed with PCA and clustering.

first: Hinata

last: Koyama

first: Daisuke

last: Mashiko

first: Masahiro

last: Kaneda

first: Atchalalt

last: Khurchabilig


title: Phase separation of endothelial PML drives inflammatory macrophage infiltration during liver fibrosis [ChIP-Seq 2]

overall_design: ChIP-seq of BRD4 was performed on human liver sinusoidal endothelial cells treated with BRD4 inhibitor iBET151, and then treated with human recombinant TNFa.


title: Phase separation of endothelial PML drives inflammatory macrophage infiltration during liver fibrosis

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: COMPARATIVE RNAseq ANALYSIS OF THE IRIDOCORNEAL REGION OF DBA/2J AND DBA/2J-Gpnmb+/SjJ MICE

city: Durham

first: Paloma

last: Liton

state: NC

address: DUMC Box 3802

institute: Duke University

summary: We performed RNA sequencing analysis of the iridocorneal region in DBA/2J and Gpnmb+ mice to investigate alterations in gene expression and gain deeper insight into the molecular mechanisms underlying IOP elevation following iris pigment dispersion.

overall_design: Iridocorneal angles from nine DBA/2J mice and six Gpnmb+ mice (males and females, 12 month old) were dissected and total RNA extracted. RNA from three individual mice were pooled, resulting in three biological replicates for the DBA/2J group and two replicates for the Gpnmb+ group.

first: Liton

last: Paloma

first: Shim

last: Myoung Sup

first: Grimsrud

last: Aleks

first: Desikan

last: Vaihav

first: Sung

last: Mi Sun


title: scRNAseq of retinal cells in experimental autoimmune uveitis comparing young vs. old mice

city: Saint Louis

first: Joseph

last: Lin

address: 660 South Euclid Ave, MSC-8096-06-07

department: Ophthalmology & Visual Sciences

institute: Washington University School of Medicine

summary: We profiled using single cell RNA sequencing the cellular heterogeneity of the aging retina in a mouse model of uveitis.

overall_design: We used young (6-7 weeks old) and aged (92-93 weeks old) male C57BL/6J mice for the experimental autoimmune mouse model of uveitis (EAU) as described previously. Briefly, we administered to each mouse by subcutaneous injection 300 micrograms human interphotoreceptor retinoid-binding protein peptide (sequence 651-670, LAQGAYRTAVDLESLASQLT) emulsified in Complete Freund's adjuvant containing 5 mg/ml M. tuberculosis strain H37Ra. We also administered intraperitoneally to each mouse 0.5 ug Bordetella pertussis toxin (5 ug/ml in PBS). At 2- and 5-weeks after immunization, we sacrificed mice and dissected eyes to obtain neural retinas. We pooled retinas and digested tissue in papain solution (125 units/ml) containing protease & phosphatase inhibitor and DNAse I (5,000 units/ml). After 40 min digestion at 4C, we neutralized the reaction using ovomucoid solution. Then, we enriched the single cell suspension for CD45-positive cells using the EasySep Mouse CD45 Positive Selection Kit following the manufacturer's recommended protocol. Finally, we washed and re-suspended the cells in DMEM media containing 10% fetal bovine serum and used them as input for single cell RNA sequencing on the 10x Genomics platform.

first: Taku

last: Yamamoto

first: Keitaro

last: Hase

first: Rajendra

last: Apte


title: Phase separation of endothelial PML drives inflammatory macrophage infiltration during liver fibrosis [RNA-Seq 1]

first: Enjiang

last: Lai

overall_design: Bulk RNAseq was performed on human liver sinusoidal endothelial cells treated with BRD4 inhibitor iBET151, and then treated with human recombinant TNFa.

first: Jinhang

last: Gao

first: Can

last: Gan


title: Phase separation of endothelial PML drives inflammatory macrophage infiltration during liver fibrosis [ChIP-Seq 1]

overall_design: ChIP-seq of H3K27ac was performed on human liver sinusoidal endothelial cells treated with BRD4 inhibitor iBET151, and then treated with human recombinant TNFa.

first: Yang

last: Tai


title: Phase separation of endothelial PML drives inflammatory macrophage infiltration during liver fibrosis [ATAC-seq]

overall_design: ATAC-seq was performed on human liver sinusoidal endothelial cells treated with BRD4 inhibitor iBET151, and then treated with human recombinant TNFa.


title: Distinct phenotypes and repertoires of bronchoalveolar and airway mucosal T cells in health and allergic asthma

city: Charlestown

first: Neal

middle: Patrick

last: Smith

address: 149 13th Street

institute: Massachusetts General Hospital

summary: T cells play a central role in host protection against respiratory pathogens, but a maladaptive T cell response can lead to pulmonary diseases. Defining the biology of protective versus pathogenic T cell responses in the lungs of humans will be critical to nominate novel approaches to improve respiratory health. Previous studies have examined T cells from the lungs captured via bronchoalveolar lavage (BAL), endobronchial brushings, or biopsies. However, whether these different approaches are capturing distinct T cell phenotypes and/or clonotypes remains unclear. We aimed to evaluate and compare the transcriptional signatures of T cells isolated via BAL versus endobronchial brushings in healthy controls (HCs) and allergic asthmatics (AAs). The most significant difference in T cell subsets abundance between AAs and HCs was the enrichment of CD4 T helper type 2 (Th2) cells when comparing endobronchial brush samples (OR=26.2, P=0.002), but not when examining BAL (OR=1.7, P=0.46), indicating differences in the T cell subsets captured from the BAL versus airway mucosa specimen processing. TCR repertoire analysis revealed that brush T cells contained dramatically expanded TCR clones. Expanded T cell clones from the brush expressed high levels of resident-memory markers, suggesting the airway mucosa is enriched for TRM cells with unique TCR specificity.

overall_design:
Flexible bronchoscopy was performed to obtain BAL and endobronchial brushings from 3 HC and 3 AA subjects. CD3+ T cells were sorted and single cell RNA- and T cell receptor (TCR)-sequencing was performed using the 10X Chromium platform. Unbiased clustering, differential gene expression analysis and TCR repertoire analysis was performed.

***************************************************************
Raw files will be uploaded to dbGaP due to patient privacy concerns.
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first: Rod

last: Rahimi

first: Andrew

middle: D

last: Luster

first: Alexandra-Chloe

last: Villani


title: Phase separation of endothelial PML drives inflammatory macrophage infiltration during liver fibrosis [Hi-C]

overall_design: Hi-C sequencing was performed on human liver sinusoidal endothelial cells treated with BRD4 inhibitor iBET151, and then treated with human recombinant TNFa.


title: Myosin VB is Critical for Progenitor Cell Function in the intestine

city: Nashville

first: Izumi

last: Kaji

state: TN

address: Rm 10465H, MRB IV

department: Surgery

institute: Vanderbilt University Medical Center

summary: Microvillus inclusion disease (MVID) is a congenital diarrheal disorder, caused by inactivating mutations in myosin Vb (MYO5B). MYO5B deficient mice and cell lines have demonstrated the importance of MYO5B in enterocyte brush border development; however, the previous MYO5B knockout models lacked specificity to the stem cell function. To address the direct impact of MYO5B loss on the epithelial stemness, we investigated the effects of progressive MYO5B deficiency originating in mouse intestinal crypt cells. Our transcriptomic and multi-plex immunostaining datasets demonstrate that MYO5B is critical for intestinal stem cell identity. MYO5B-deficient crypts acquire a hyperproliferative phenotype and lack the ability of proper cell lineage differentiation. An evident disruption in mitochondrial structure and fatty acid metabolism likely underlies these crypt deficits. Consistent with mouse models, MVID patient biopsies demonstrate abnormal expansion of proliferative zone besides villus blunting. These data reveal the direct impacts of MYO5B loss on intestinal epithelial cell progenitor functions.

overall_design: Jejunal epithelial cells and lamina proprial cells were isolated from the mice of crypt-specific MYO5B knockout (Day 3 Lrig1-M5B), crypt-specific MYO5B mutation (Day 3 Lrig1-G519R), whole epithelial MYO5B knockout (Day 5 Lrig1-M5B), whole epithelial MYO5B mutant (Day 5 Lrig1-G519R), and littermate controls with intact MYO5B. Each group has two replicates.

first: Andreanna

last: Burman

first: Monica

last: Brown

first: Yilin

last: Yang

first: Ken

last: Lau

first: Michael

last: Momoh


title: Effects of 500nm polystyrene nanoplastic exposure on immortlized GnRH neurons

city: Milan

first: Anna

last: Cariboni

address: Via Balzaretti 9

department: Disfeb

country: Italy

institute: Università degli Studi di Milano

summary: Plastic pollution, especially from mismanaged waste degrading into nanoplastics (NPs), poses a growing threat to environmental and human health. Polystyrene NPs (PS-NPs) are particularly prevalent, infiltrating ecosystems and accumulating in organs, including the brain. While NPs are linked to behavioral, metabolic, and developmental effects, their impact on reproduction and fertility remains poorly understood. Declining fertility rates suggest environmental toxicants like NPs may contribute to adverse reproductive outcomes. Reproductive function relies on the hypothalamus-pituitary-gonad (HPG) axis, centrally regulated by gonadotropin-releasing hormone (GnRH) neurons. These neurons originate in the nasal placode and migrate to the hypothalamus during embryogenesis. Altered GnRH neuron development or release can lead to absent puberty and infertility, whose genetic causes explain only 50% of known cases. In this context, environmental factors including NPs might contribute to the etiology of these disorders. In this work, we take advantage of an established model of migrating GnRH neurons (GN11) to assess PS-NPs effects and to provide the first evidence that these environmental factors interfere with cellular and molecular processes of GnRH neurons, thus suggesting a novel mechanism contributing to idiopathic infertility.

overall_design: RNA-seq profiling of immortalized GnRH neurons cell line GN11 untreated or treted with 50ug/mL 500nm polystyrene nanoplastic for 24 hours

first: Roberto

last: Oleari

first: Federica

last: Amoruso

first: Alyssa

last: Paganoni


title: CDK16 and CDK18 inhibition suppresses nuclear pore-mediated DNA repair and hepatocellular carcinoma

first: Agustín

last: Belmonte


title: Blood vessel-resident macrophages safeguard blood and vessel integrity in zebrafish

city: Utrecht

first: Catherine

last: Robin

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

summary: Macrophages are essential in immune defence, tissue development and maintenance, and are involved in disease onset and progression. Their function is influenced by their microenvironment and origin. While tissue-resident macrophages originate from yolk sac progenitors and persist in some adult tissues through self-renewal, no dedicated macrophage population is known to function within the vast network of blood vessels, despite the importance of maintaining a healthy vascular and blood system. Here, using high spatiotemporal live imaging of zebrafish embryos, we identified a distinctive population of macrophages residing within blood vessels, which we term blood vessel-resident macrophages (bMs). bMs actively survey the bloodstream, clearing foreign particles and unfit cells, and are the first responders to endothelial damage. bMs emerge directly from axial vessels through an atypical endothelial-to-macrophage transition (EMacT), independent of Runx1 and Csf1r, both essential for hematopoiesis and myelopoiesis, respectively. We further demonstrate that bMs are evolutionary conserved in mice. Our data thus unveil the existence of a specialized population of macrophages that function within the vasculature to safeguard blood from foreign threats and maintain blood vessel integrity. These discoveries broaden our understanding of immune surveillance, opening new avenues for treating bloodborne and vascular diseases.

overall_design: scRNA-sequence blood vessel-resident macrophages, tissue macrophages and neutrophils from the trunk of 3 dpf wildtype zebrafish embryos and blood vessel-resident macrophages and metaphocytes from  3 dpf csf1ra-/- csf1rb-/- zebrafish embryos

first: Bart

last: Weijts


title: Single-cell transcriptomic profiling of the developing zebrafish heart.

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

institute: MPI for heart and lung research

summary: Cardiac development is a complex process where bilateral precursors undergo migration, proliferation, and differentiation to form a multi-chambered organ that sustains life.  While scRNA-seq technology has advanced significantly, a comprehensive analysis of vertebrate heart development from early to late developmental stages is still lacking.  We profiled over 34,000 cells from resected zebrafish hearts spanning 10 developmental stages from 25 hours to 60 days post-fertilization.  We identified and sub-clustered all known cardiac cell types, revealing distinct proliferation dynamics.  We also analysed the cardiac interactome, focusing on the ligand-receptor interactions between myocardial and endocardial cells.  By investigating later stages, we discovered novel marker genes, which appears to be enriched in trabecular cardiomyocytes. This zebrafish heart atlas enhances our understanding of vertebrate heart development and represents a valuable resource for future studies.

overall_design: For each condition, hearts (number depending on developmental stage) were dissected, dissociated and analyzed by scRNA-seq.

first: Marco

last: Tarasco

first: Thomas

last: Juan

first: Yousef

last: Alayoubi

first: Agatha

last: Ribeiro da Silva

first: Radha

last: Kulkarni

first: Marga

last: Albu

first: Annika

last: Nürnberger

last: Guenther

first: Mario

last: Looso

first: Didier

last: Stainier


title: In vivo single-cell CRISPR screen for microproteins identifies a critical ribosome component

city: Schlieren

first: Umesh

last: Ghoshdastider

address: Wagistrasse 12

country: Switzerland

institute: University of Zurich

summary: The dark proteome includes a rapidly expanding catalog of microproteins with unknown functions that have been historically ignored in genome annotations. Here, we exploit an in vivo single-cell CRISPR screening strategy in the mouse epidermis to systematically investigate the tissue-wide function of microproteins. We document global and cell-type-specific roles of microproteins during epidermal development and homeostasis at single-cell transcriptomic resolution. Focusing on select candidates, we identify a novel microprotein on Gm10076, identical to the ribosomal intersubunit bridge protein RPL41, whose perturbation strongly impairs proliferation and protein synthesis. Employing ribosome profiling and RNA sequencing, we show that Gm10076 perturbation profoundly reshapes the translational landscape. Contrary to its prior classification as nonessential, we find that the ribosomal protein RPL41 is critical for cellular proliferation, warranting further investigation into its role as an intersubunit bridge in the translational machinery. Collectively, our study comprehensively charts the tissue-wide functional landscape of the dark proteome, uncovers a second Rpl41 gene critical for ribosome function and establishes a basis for exploring the impact of microproteins on disease pathogenesis.

overall_design: We use the mouse epidermis as a model to systematically dissect the tissue wide function of microproteins at single-cell transcriptomic resolution. We employ ultrasound-guided in utero injections of lentiviral libraries with 228 microproteins into embryonic stage E9.5 and combine it with a CRISPR CROP-seq strategy to document cell-type specific function of microproteins. We accessed a library of 720 sgRNAs targeting 155 uORFs and 65 new ORFs. We later validate a subset of 29 top altered microproteins in vivo and in vitro. Together our results reveal a second Rpl41 gene enconded in a long noncoding RNA which is characterized using ribosome profiling.

first: Fabiola

last: Valdivia-Francia

first: Peter

middle: F

last: Renz

first: Daniel

last: Spies

first: Mark

last: Ormiston

first: Katie

last: Hyams

first: Chenke

last: Shi

first: Clara

last: Duré

first: Merve

last: Yigit

first: David

last: Taborsky

first: Ameya

last: Khandekar

first: Ramona

last: Weber

first: Homare

last: Yamahachi

first: Stephanie

last: Ellis

first: Ataman

last: Sendoel


title: The study investigates the function of the nphp4 gene in the testes of medaka fish (Oryzias latipes)

city: Xiamen

first: Tiansheng

last: Chen

address: No. 43, Yindou Road, Jimei District, Fisheries College of Jimei University

institute: Jimei University

summary: In this study, we employed CRISPR-Cas9 genome editing to generate nphp4 knockout medaka fish. By comparing the transcriptomic profiles of the testes from wild-type (WT) and nphp4-/- mutants, we conducted a comprehensive analysis of gene expression to elucidate the role of the nphp4 gene in medaka testis development and function.

overall_design: This study aims to compare the gene expression differences between wild-type (WT) and nphp4 gene knockout mutant (nphp4-/-) medaka fish testes. The experiment included two groups, WT and nphp4-/-, each with three biological replicates, and each replicate consisted of testes from two  individuals. Total RNA was extracted and sequencing libraries were constructed under identical conditions for all samples. Six RNA libraries were constructed using Illumina RNA-seq and sequenced for gene expression analysis.

first: Jingjie

last: Liang

first: Jiangling

last: Li


title: Co-targeting CDK2 and CDK9 inhibits MYC oncogenic programs and has anti-tumor effects in CIC-rearranged sarcoma

city: Cambridge

first: Nicolas

last: Fernandez

address: 71 Ames St, 8th floor

institute: Broad Institute of MIT and Harvard

summary: CIC-rearranged sarcoma is an aggressive mesenchymal neoplasm characterized by capicua (CIC) gene rearrangement and molecular dependence on the CDK2-cyclin E complex. Given its high metastatic potential and poor clinical outcomes, there is an urgent need to identify biologic vulnerabilities for therapeutic targeting. Here, we determined the efficacy of CDK2-targeted therapies in preclinical models of CIC- rearranged sarcoma including two new cell lines and xenografts driven by CIC::DUX4 and CIC::NUTM1 fusion, respectively. Functional analyses revealed that CIC- rearranged sarcomas are MYC-driven cancers and can be targeted with the selective CDK2 inhibitor tagtociclib (PF-07104091) in vitro and in vivo. Concomitant with RB1- mediated proliferative arrest, tagtociclib inhibited MYC oncogenic programs. Moreover, combination of tagtociclib with the CDK9 inhibitors KB-0742 or AZD4573 was synergistic and enhanced apoptotic cell death in vitro and in vivo. These results demonstrate anti-tumor effects of concurrent inhibition of CDK2 and CDK9 through MYC transcriptional repression and provide a strong rationale for clinical evaluation of CDK2 and CDK9-targeted combination therapies in patients with CIC-rearranged sarcoma.

overall_design: RNA-seq profiling of Tagtociclib treated sarcoma cell lines.

first: Schaefer

last: Inga-Marie


title: Phase separation of endothelial PML drives inflammatory macrophage infiltration during liver fibrosis [RNA-Seq 2]

first: Enjiang

last: Lai

first: Jinhang

last: Gao

first: Can

last: Gan

first: Yang

last: Tai


title: Inhibition of NFAT after human uterus transplant promotes loss of tissue-resident NK cells and attendant pregnancy complications [scRNA-seq]

city: Birmingham

first: Rebecca

last: Asiimwe

state: Alabama

address: 1720 2nd Ave South

institute: The University of Alabama at Birmingham

summary: Uterine natural killer (uNK) cells are a tissue-resident lymphocyte population critical for pregnancy success. Although mouse models have demonstrated that uNK cell deficiency results in abnormal placentation and poor pregnancy outcomes, the generalizability of this knowledge to humans remains unclear. Here, we compared tissue samples collected from a cohort of human recipients of uterus transplant (UTx) at high risk for pregnancy complications with healthy controls using flow cytometry, immunofluorescence microscopy, and single-cell RNA sequencing (scRNA-seq). Subsets of tissue-resident uNK cells were reduced in endometrial and decidual samples from recipients of UTx compared with healthy control samples. Loss of tissue-resident uNK cells was associated with histopathologic evidence of maternal vascular malperfusion in placentas from recipients of UTx and related pregnancy complications including preeclampsia. scRNA-seq of UTx endometrial biopsies and deciduae further revealed that the NK cell reduction in recipients of UTx correlated with impaired transcriptional programming of NK tissue residency arising from the inhibition of signaling by nuclear factor of activated T cells (NFAT). In vitro culture of uNK cells from healthy controls with the NFAT inhibitor tacrolimus resulted in down-regulation of adhesion molecules. Together, these experiments suggested that NFAT-dependent genes modulate multiple molecular tissue residency programs in uNK cells, including early residency programs involving activator protein-1 (AP-1) family transcription factors and later residency programs characterized by up-regulation of surface integrins by transforming growth factor–β (TGF-β). Collectively, these data identify a previously undescribed role for NFAT in uNK tissue residency and provide mechanistic insights into the biologic basis of pregnancy complications due to alteration of tissue-resident NK cell subsets in humans.

overall_design: Human endometrium was sampled from 1) healthy control participants, 2) uterus transplant recipients, and 3) brain-dead deceased donors for this study. Endometrial dating was known for all healthy control volunteers and uterus transplant recipients but not for any deceased donor sample, as healthy control individuals and UTx recipients used ovulation predictor kits. Endometrial samples were analyzed with single cell approaches (i.e., scRNA-seq, CITE-seq, flow cytometry) as well as imaging (i.e., immunofluorescence microscopy). Single cell suspensions of endometrium were cultured with IL-15 and FK506 for 3-7 days.

first: Brittney

last: Knott

first: Morgan

last: Greene

first: Emma

last: Wright

first: Markayla

last: Bell

first: Daniel

last: Epstein

first: Stefani

last: Yates

first: Michael

middle: V

last: Gonzalez

first: Samantha

last: Fry

first: Emily

last: Boydston

last: Clevenger

first: Jayme

last: Locke

first: Brian

last: Brocato

first: Constantine

last: Burgan

first: Richard

last: Burney

first: Nitin

last: Arora

first: Virginia

last: Duncan

first: Holly

last: Richter

first: Deidre

last: Gunn

first: Aharon

middle: G

last: Freud

first: Shawn

last: Little

first: Paige

last: Porrett


title: Acute intervention reveals critical roles of BAF, KMT2D and p300 in MyoD binding on enhancers

city: Bethesda

first: Kai

last: Ge

address: 50 South Dr Rm 4154

department: NIDDK

institute: NIH

summary: Previous studies using constitutive expression of transcription factors (TFs) and genetic knockout of cofactors have revealed important roles of chromatin remodeler BAF, H3K4me1 methyltransferase KMT2D, and H3K27 acetyltransferase p300 in enhancer regulation. However, binding dynamics of these cofactors and TFs on enhancers remain unclear. Here we show that within one hour of its nuclear translocation, the master myogenic TF MyoD is co-localized with BAF, KMT2D, and p300 on over half of MyoD+ enhancers. Acute depletion of KMT2D protein disrupts de novo binding of MyoD, BAF, and p300 on enhancers. Short-term inhibition of BAF or p300 enzymatic activity dramatically reduces de novo binding of MyoD, BAF, KMT2D, and p300 on enhancers. Together, these results highlight the interdependency of MyoD and chromatin regulators BAF, KMT2D and p300 in their binding to enhancers. Our data also suggest that p300 may hold a more prevailing role than previously reported.

overall_design: Chromatin accessibility profiling by ATAC-seq in controls and BRM014-treated preadipocytes, and CUT&RUN profiling of MyoD, enhancer regulators (ARID1A, KMT2D, and p300), histone modifications (H3K4me1 and H3K27ac) in preadipocytes

first: Hieu

middle: T

last: Van

first: Young-Kwon

last: Park

first: Ji-Eun

last: Lee


title: Rhinovirus triggers distinct host responses through differential engagement of epithelial innate immune signaling

city: New Haven

first: Bao

last: Wang

state: Connecticut

address: 333 Cedar Street

department: Deparment of Laboratory Medicine

institute: Yale University

sample_organism:
Homo sapiens
Human rhinovirus 1A

platform_organism:
Homo sapiens
Human rhinovirus 1A

overall_design: Refer to individual Series


title: Rhinovirus triggers distinct host responses through differential engagement of epithelial innate immune signaling [RNA-Seq]

summary: Rhinovirus (RV) is the most frequent cause of common colds, but also a major cause of serious respiratory illness in high-risk groups (e.g. smokers and asthmatics). The molecular mechanisms leading to disparate outcomes of infection are not fully understood. Variation in the host epithelial interferon response has been implicated susceptibly to RV illness. We performed single cell sequencing air-liquid interface (ALI) cultured human nasal epithelial cells (HNECs) with infection of HRV-01A in the presence of IRF3 activation inhibitor BX795. We showed that the epithelial interferon response restricts RV infection to <2% of cells, however, inhibiting IRF3 activation increases the proportion of infected cells to >30%, and leads to a distinct NFKB-dependent pro-inflammatory response, with production of neutrophil-attracting chemokines, increased mucus, and IL-1b. Bulk RNA sequencing further revealed that inhibiting both IRF3 and NFKB induce ER stress and squamous metaplasia genes. Overall, our study demonstrates how distinct forms of epithelial defense responses impact RV-induced inflammation.

overall_design: Bulk RNA sequencing of ALI cultured human nasal epithelial cells infected with mock, RV1A, RV1A+BX795 (IRF3 activation inhibitor), RV1A+TPCA1 (NFkB activation inhibitor) or RV1A+BX795+TPCA1. Samples are collected on day 5 post infection.

first: Ellen

last: Foxman


title: Rhinovirus triggers distinct host responses through differential engagement of epithelial innate immune signaling [scRNA-Seq]

overall_design: Split-seq single cell RNA sequencing (Parse Biosciences) of ALI cultured human nasal epithelial cells infected with mock, RV1A or RV1A+BX795 (IRF3 activation inhibitor). Samples are collected on day 1, 2 and 5 post infection.

first: Foxman

last: Ellen

first: Wang

last: Bao


title: Kdm6a ChIP-seq of Sv129 mouse embryonic stem cells with MTX

city: Shanxi

first: wenbin

last: ren

address: 56 Xinjian South Road, Yingze District, Taiyuan City

institute: Shanxi Medical University

summary: In order to investigate whether folate deficiency affects kdm6a enrichment and then affecting the double-strand breaks, we performed ChIP-seq of kdm6a on  embryonic stem cells  with MTX and normal embryonic stem cells, respectively. Our sequencing data provides a new theoretical basis for the effect of folate on histone modification.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for Kdm6a

first: Wenbin

last: Ren

first: Jianting

last: Li

first: Lu

first: Yuan

last: Fu

first: Zhihua

last: Cao


title: CDK16 and CDK18 inhibition suppresses nuclear pore-mediated DNA repair and hepatocellular carcinoma [Huh7]

city: Barcelona

first: Agustín

last: Belmonte

address: Nazaret 115 -117

institute: Vall d ́Hebron Institute of Oncology (VHIO)

summary: Inhibition of cyclin-dependent kinases (CDK) 4 and 6 has become part of the current standard-of-care treatment in advanced estrogen receptor-positive breast cancer. However, many tumor types are resistant to current CDK inhibitors and the therapeutic value of other CDK family members remains to be validated. By using gene-editing techniques in human cancer cells and genetically-modified mouse models we report here that MYC and -catenin driven hepatocellular carcinoma (HCC) is sensitive to the inactivation of CDK16 and CDK18. Whereas individual ablation of CDK14-18 subfamily members displays little effect, concomitant genetic ablation or pharmacological inhibition of CDK16 and CDK18 results in proliferative arrest accompanied by DNA damage and apoptotic cell death. Mechanistically, these defects are associated with a novel role of these proteins in maintaining nuclear pore integrity and the regulation of nucleocytoplasmatic transport. Cyclin Y-CDK16/18 complexes directly phosphorylate the nuclear pore complex (NPC) components NUP35 and NUP214. CDK16/18 inhibition leads to defective nuclear accumulation of the DNA recombinase RAD51, altering DNA repair and inducing lethal levels of DNA damage. Genetic ablation of Cdk16 and Cdk18 in vivo triggers DNA damage and apoptotic cell death in murine liver tumors induced by MYC and -catenin, resulting in tumor regression. Collectively, these data support the development of CDK16/18-targeted therapies for the treatment of HCC.

overall_design: RNA-seq

first: Diego

last: Martínez-Alonso

first: Marcos

last: Malumbres

last: Sánchez-Belmonte


title: Consensus molecular classification of triple-negative breast cancers

city: Saint Herblain

first: Wilfried

last: Gouraud

address: bd Jacques Monod

department: Integrated Center of Oncology René Gauducheau

country: France

institute: ICO (Integrated Center of Oncology Nantes)

summary: The goal of this project was to establish a consensus molecular classification of triple-negative breast cancers (TNBC), ie. to identify TNBC robust subtypes. In order to optimize this process, we retrieved as much transcriptomic data as possible, measured by Affymetrix® microarrays (n = 886) and RNAseq (n = 699). In this last group, we included 48 new TNBC patients followed in our Cancer Centre. To our knowledge, this study is the largest one which aims at subtyping TNBC; it exploits a total of 1585 samples. Pooled data were standardized across different public datasets for both gene expression profiling technologies. Subsequently, clustering analysis revealed four robust subtypes.

overall_design:
48 new TNBC patients followed in our Cancer Centre have been RNA-sequenced.
*** Raw data not provided.  Due to patient consent and the possibility that the sequencing data may contain personally identifiable information, the submitter may not make the raw sequencing data available in a public repository.. ***

first: Pascal

last: Jézéquel

first: Hamza

last: Lasla

first: Bertrand

last: Michel

first: Jonathan

last: Dauvé

first: Alain

last: Morel

first: Fadoua

last: Ben Azzouz

first: Mario

last: Campone


title: Dietary Restriction Impacts Peripheral Circadian Clock Output Important for Longevity in Drosophila

city: Chicago

first: Marta

last: Iwanaszko

address: 303 E. Superior Street, Simpson Querrey 7th Floor,

department: Biochemistry and Molecular Genetics

institute: Northwestern University Feinberg School of Medicine

summary:
Circadian clocks may mediate lifespan extension by caloric or dietary restriction (DR). We find that the core clock transcription factor Clock is crucial for a robust longevity and fecundity response to DR in Drosophila. To identify clock-controlled mediators, we performed RNA-sequencing from abdominal fat bodies across the 24 h day after just 5 days under control or DR diets. In contrast to more chronic DR regimens, we did not detect significant changes in the rhythmic expression of core clock genes. Yet we discovered that DR induced de novo rhythmicity or increased expression of rhythmic clock output genes. Network analysis revealed that DR increased network connectivity in one module comprised of genes encoding proteasome subunits. Adult, fat body specific RNAi knockdown demonstrated that proteasome subunits contribute to DR-mediated lifespan extension. Thus, clock control of output links DR-mediated changes in rhythmic transcription to lifespan extension.
Funding: DARPA D12AP00023

overall_design: Drosophila melanogaster fat body transcriptional profiles under two conditions: control diet (15% Sucrose/Yeast, 15SY) and dietary restriction (5% Sucrose/Yeast, 5SY). Young mated female flies (~ 3 day old) were entrained under either DR diet (5SY) or control diet (15SY) for 5 days in 12L:12D cycles at 25°C. After 5 days flies were directly dissected to harvest fat tissues attached to the epidermis in the abdomen. Fat body from ~ 10 flies was harvested within 10 minutes for each time point of RNA-Seq analysis. Tissue was collected every 2hr over 24hr (12 time points), 2 biological replicates per time point, 2 conditions, yielding total of 48 samples.

first: Dae-Sung

last: Hwangbo

first: Yong-Jae

last: Kwon

first: Peng

last: Jiang

first: Alan

middle: L

last: Hutchison

first: Aaron

middle: R

last: Dinner

first: Rosemary

last: Braun

first: Ravi

last: Allada


title: Single cell sequencing of BMSCs

city: Qujing

first: Dayuan

last: Tang

state: Yunnan

address: No. 1 , Qilin Road, Qujing, Yunnan

institute: Qujing Hospital Affiliated of Kunming Medical University

summary: Regulation of osteoimmune microenvironment and osteogenesis by electrospun poly-l-lactic acid membranes for bone regeneration

overall_design: In this experiment, 6 cell samples were included for single-cell sequencing analysis

first: Daiyuan

first: Bing

last: Han

first: Yunrong

last: Xu

first: Fei

last: He


title: Silencing of PARN in NCI-H520 cells induces changes in the miRNome

city: Trento

first: Michela

middle: A.

last: Denti

address: Via Sommarive 9

department: Department of Cellular, Computational and Integrative Biology

institute: University of Trento

summary: To understand the role of PARN  we have employed miRNAs microarray expression profiling as a discovery platform to identify miRNAs whose expression is regulated by PARN

overall_design: NCI-H520 cells were silenced with shRNAs against PARN or control shRNAs, in six replicas, as previously described (Maragozidis, P., Papanastasi, E., Scutelnic, D., Totomi, A., Kokkori, I., Zarogiannis, S.G., Kerenidi, T., Gourgoulianis, K.I., and Balatsos, N.A. (2015). Poly(A)-specific ribonuclease and Nocturnin in squamous cell lung cancer: prognostic value and impact on gene expression. Mol Cancer 14, 187. PMID: 26541675).  RNA was collected 72 hours after transfection and knock-down of PARN mRNA was validated by RT-PCR on each independent replicate sample.

first: Nikolaos

last: Balatsos


title: Mitochondrial DNA heteroplasmy drives cortical neuronal disturbances in human organoids harbouring the common m.3243A>G mutation

status: Public on Mar 09 2026

first: Denisa

last: Hathazi

address: Robinson Way

department: Clinical Neurosciences

country: United Kingdom

institute: University of Cambridge

summary: Mitochondrial diseases frequently affect the brain leading to severe and disabling neurological symptoms. The heteroplasmic m.3243A>G mutation in MT-TL1, encoding mt-tRNALeu, is responsible for ~80% of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), which is one of the most characteristic mitochondrial syndromes, leading to disability and early death. There are no animal models harbouring this mutation to provide precise mechanistic insights informing therapeutic interventions. Here, we generated a human iPSC-derived cerebral organoid slice model that recapitulates cortical architecture and mitochondrial pathology. Using biological assays and single-cell RNA sequencing, we uncovered heteroplasmy-dependent transcriptional shifts and changes in key cellular processes in cortical neurons. Organoids with high heteroplasmy showed a predominant impairment of deep-layer neurons triggered by mitochondrial stress, leading to axonal degeneration and apoptosis, similar to brain autopsy of a MELAS patient. Our findings provide insights into the vulnerability of long-range projection neurons in mitochondrial diseases, advancing our understanding of disease mechanisms with a view to potential therapeutic strategies.

overall_design: Two patient lines with m.3243A>G mutation. For each patient we have an isogenic control and aditional cell lines with low medium and high heteroplasmy. For patient 1, we have three clones with heteroplasmy levels of 12%, 42%, and 72%. For patient 2, we have organoids with 0%, 30%, 49%, and 70% heteroplasmy

first: Camilla

last: Lyons

first: Daniel

last: Lagos

first: Oliver

last: Podmanicky

first: Mariana

last: Zarate-Mendez

first: Yu

last: Nie

first: Julianne

middle: S

last: Mueller

first: Kieren

last: Allinson

first: Huw

last: Naylor

first: Majlinda

last: Lako

first: Ibrahim

last: Elsharkawi

first: Irena

last: Muffels

first: Eva

last: Morava

first: Tamas

last: Kozicz

first: Patrick

last: Chinnery

first: Andras

last: Lakatos

first: Rita

last: Horvath


title: Single cell transcriptional profile of third instar larval wing discs in Drosophila using single cell RNA-seq (Chromium 10x)

first: Maria

middle: Paula

last: Zappia

address: 900 S Ashland Ave

institute: University of Illinos at Chicago

summary: Our study focuses on understanding the early transcriptional changes taking place during the divergence of the adult muscle precursors that give rise to indirect flight muscles and direct flight muscles in Drosophila. Here, we studied the functional relevance of Arginine Kinase in flight muscle development by depleting Argk expression specifically in the adult muscle precursors. We determined the transcriptional changes and perturbations in AMP cell identity upon Argk knockdown.

overall_design: 10X scRNA-seq on the single cell suspension from third instar larval wing discs (hinge and notum, pouch was manually removed during dissection).

first: Maxim

last: Frolov

first: Abul

last: Islam


title: Unveiling the Developmental and Tumor Suppressive Roles of the p53 variant, p53psi

city: Toronto

first: Liam

last: Hendrikse

state: Ontario

address: 610 University Ave

department: Princess Margaret Cancer Centre

country: Canada

institute: University Health Network

summary: Through alternative splicing, the TP53 gene can generate multiple protein isoforms with distinct biochemical properties. Among the thirteen splicing isoforms described to date, the p53psi isoform has been identified as a variant that is shorter than full-length p53 as it lacks nuclear localization, oligomerization and part of the DNA binding domains due to the use of an alternative 3' splice site in intron 6. Several TP53-truncating mutations, including those producing p53psi, have been detected in a significant proportion of human tumors. However, the mechanistic roles of these truncated p53 proteins remain poorly understood. Here, we describe the generation and analysis of a genetically engineered mouse model that expresses the p53psi protein in place of the full-length p53 protein. In the C57/BL6J genetic background, mice heterozygous for the targeted p53psi allele (p53KI/+) appear phenotypically normal, survive to adulthood, and reproduce. However, heterozygote mating fails to yield viable p53psi homozygote knock-in (p53KI/KI) pups, indicating that forced p53psi expression disrupts embryogenesis. Timed mating studies revealed that homozygous p53psi expression is embryonically lethal at day E16.5. Embryos at E14.5-16.5 were pale, reduced in size, and exhibited exencephaly, a defect typically associated with a failure in neural tube closure. Mouse embryonic fibroblasts (MEFs) derived from p53psi embryos and transformed with the E1A and H-RasV12 oncogenes formed tumors with a decreased growth rate compared to their p53null counterparts, suggesting that p53psi retains at least some tumor-suppressive functions. Our mechanistic studies suggest that p53psi modulates tumorigenesis by triggering senescence. These findings provide new insights into the role of the p53psi variant, paving the way for a better interpretation of TP53 mutational patterns in human cancers.

overall_design: Comparative RNA-seq profiling of E1a/RASV12-transformed murine embryonic fibroblasts (MEFs) derived from p53psi and p53null background embryos.

first: Chiara

last: Gorrini

first: Parameswaran

last: Pamachandran

first: Tak

middle: W

last: Mak


title: Single-nucleus RNA sequencing of human trophoblast organoids and endometrial organoids co-culture model

city: Sendai

first: Shun

last: Shibata

address: 2-1 Seiryo-chou

department: Graduate School of Medicine

institute: Tohoku University

summary: To model the maternal–fetal interface, we co-cultured rotation-derived human trophoblast organoids (hTOr) with human endometrial organoids (hEMO). Here, we performed single-nucleus RNA sequencing of hEMO mono-culture, hTOr mono-culture, and their co-culture to dissect the transcriptomic landscape of this interaction. Our analysis reveals that hTOr-derived signals drive a metabolic and secretory activation in the endometrial epithelium. We identified a unique 'Activated' hEMO population emerging specifically upon co-culture, distinct from the LGR5+ homeostatic state. This transition was characterized by a robust upregulation of oxidative phosphorylation and glycolysis, concurrent with sustained proliferation, demonstrating that the trophoblast actively reprograms the endometrium into a hyper-metabolic state.

overall_design: Single-nucleus RNA sequencing of day 8 hEMO mono-culture, day 11 hTOr mono-culture, and hEMO-hTOr co-culture (maintained for 8 days). Libraries were prepared using 10x Genomics Chromium Single Cell 3' v3.1 with dual indexing and sequenced on NovaSeq X Plus. Raw data processed using Cell Ranger v8.0.1.

first: Yuki

last: Ishikawa


title: Bulk RNA-seq of human trophoblast organoids under four culture conditions

summary: Bulk RNA-seq was performed to compare transcriptomic profiles of human trophoblast stem cell (hTSC)-derived organoids cultured under four conditions: rotation, floating (static suspension), shaking (horizontal agitation), and Matrigel embedding. Two independent hTSC lines (B31 and CT27) were used. Total RNA was extracted, and strand-specific poly(A)-selected libraries were sequenced on Illumina NovaSeq X Plus (PE150).

overall_design: 8 samples total: 2 hTSC lines (B31, CT27) x 4 culture conditions (rotation, floating, shaking, Matrigel). Libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit with NEBNext Poly(A) mRNA Magnetic Isolation Module and sequenced on Illumina NovaSeq X Plus (150 bp paired-end).

first: Marina

last: Kosaka


title: Single-cell RNA sequencing of bone marrow celld from patients with paroxysmal nocturnal hemoglobinuria.

first: Shouguo

last: Gao

address: 9000 Rockville Pike

department: NHLBI

institute: National Institutes of Health

summary: We performed single cell RNA sequencing of bone marrow samples (sorted Lineage+ cells, and sorted Lin-CD34+ HSPCs) from 8 patients with paroxysmal nocturnal hemoglobinuria (PNH) to understand PNH pathophysiology at high multi-dimensional resolution.

overall_design: RNA profiles of single bone marrow hematopoietic cells of 8 PNH patients and 5 healthy donors were generated by deep sequencing using Illumina NovaSeq.

first: Hiroki

last: Mizumaki


title: Oxygen as a limiting ecological variable for invertebrate tissue maintenance

city: Lund

first: Paula

last: Martos-Salvo

address: Sölvegatan 19

country: Sweden

institute: Lund University

summary: Ambient oxygen shapes tissue physiology, yet whether elevated oxygen directly perturbs stem cell fate and regeneration in invertebrates remains unclear. Here we test how chronic oxygen exposure influences intestinal stem cell (ISC) behaviour, epithelial homeostasis, and survival in Drosophila melanogaster, an organism lacking vertebrate-style stemness buffering via HIF-2a-driven pseudohypoxia. Adult flies were maintained under an atmosphere with moderate hypoxia (10% oxygen), normoxia (20% oxygen), and hyperoxia (40% oxygen) and assessed by lifespan analysis, midgut immunostaining, and intestinal transcriptomics. Hyperoxia markedly shortened lifespan, revealing high oxygen as a strong constraint on adult Drosophila viability. In the midgut, elevated O2 increased apoptosis and altered lineage outputs, with evidence of impaired fate segregation (mixed stem/differentiation marker expression) and a shift toward enteroendocrine differentiation. Consistent with these cellular phenotypes, transcriptomic profiling of the intestines at the onset of elevated mortality indicated enrichment of inflammatory signalling that compromised epithelial homeostasis. Together, our data support a model in which hyperoxia induces a ‘non-productive regenerative state’, where ISCs persist but their fate regulation and progeny survival are disrupted, leading to epithelial degeneration and reduced organismal viability. These findings position ambient oxygen as a key environmental variable that can destabilize stem cell-driven renewal in invertebrate tissues and motivate comparative studies on how oxygen-sensing strategies shape regenerative capacity across animal lineages.

overall_design: RNAseq of dissected Drosophila melanogaster midgut growth under 10, 20, or 40% oxygen for 53 days.

first: Kai

last: Aragaki

first: Hwei-yen

last: Chen

last: Mazariegos

first: Mazharul

first: Udo

last: Häcker

first: Sofie

last: Mohlin

first: Emma

middle: U

last: Hammarlund


title: Inhibition of NFAT after human uterus transplant promotes loss of tissue-resident NK cells and attendant pregnancy complications [CITE-seq]

first: Rebecca

last: Asiimwe

overall_design: Human endometrium was sampled from 1) healthy control participants, 2) uterus transplant recipients, and 3) brain-dead deceased donors for this study. Endometrial dating was known for all healthy control volunteers and uterus transplant recipients but not for any deceased donor sample, as healthy control individuals and UTx recipients used ovulation predictor kits. Endometrial samples were analyzed with single cell approaches (i.e., scRNA-seq, CITE-seq, flow cytometry) as well as imaging (i.e., immunofluorescence microscopy). Single cell suspensions of endometrium were cultured with IL-15 and FK506 for 3-7 days.

first: Brittney

last: Knott

first: Morgan

middle: E

last: Greene

last: Wright

first: Markayla

last: Bell

first: Daniel

last: Epstein

first: Stefani

last: Yates

first: Michael

last: Gonzalez

first: Samantha

last: Fry

first: Emily

last: Boydston

first: Stephanie

last: Clevenger

first: Jayme

last: Locke

first: Brian

last: Brocato

first: Constantine

middle: M

last: Burgan

first: Richard

last: Burney

first: Nitin

last: Arora

first: Virginia

last: Duncan

first: Holly

last: Richter

first: Deidre

last: Gunn

first: Aharon

last: Freud

first: Shawn

last: Little

first: Paige

last: Porrett


title: A Role for Rapid Transcription Dynamics in Cytarabine Resistance in Acute Myeloid Leukemia [ATAC-seq]

city: Bronx

first: Russell

last: Scharf

state: New York

address: 1945 Eastchester Rd

institute: Albert Einstein College of Medicine

summary: Chemotherapy resistance remains a critical challenge in the treatment of patients with cancer including acute myeloid leukemia (AML). While genetic alterations contribute to chemotherapy resistance, rapid-adaptive non-genetic mechanisms, particularly transcription dynamics, remain poorly understood. Given the role of cell-intrinsic transcriptional variability in cell fate decisions, understanding and targeting chemotherapy-induced changes in transcription dynamics could offer new strategies to prevent chemotherapy resistance. In this study, we demonstrate that short-term treatment with the widely used chemotherapeutic cytarabine (AraC) leads to the rapid emergence of RNA-induced AML cells with increased AraC resistance in both cell lines as well as primary patient samples. Mechanistically, through global transcriptomic analyses and targeted high-resolution analysis of transcription dynamics using single-molecule RNA FISH, we found rapid induction of transcriptional dynamics and upregulation of key transcription factors (TFs) - which we term “AraC rapid response TFs (ARR-TFs)” and which include PU.1 and GATA1 – following chemotherapy treatment. At a functional level, short-term pre- and co-treatment with RNA transcription inhibitors effectively suppressed chemotherapy-induced RNA induction and prevented resistance acquisition, in both in vitro and in vivo models. Furthermore, we found that CRISPR-mediated suppression of PU.1 and GATA1 induction significantly attenuated AraC resistance. In summary, our findings reveal a previously unrecognized role of rapid and early adaptive transcriptional dynamics in AML chemotherapy resistance, highlighting master TFs as key regulators of drug resistance. These insights offer a novel, pharmacologically targetable and accessible approach to alleviate chemotherapy resistance, and encourage further testing with the ultimate goal to improve AML treatment outcomes.

overall_design: HL60 cells were treated with 250nM AraC for 1 day (Day 1 sample) or 3 days followed by cuture in non-drug media for another 9 days (Day 14 sample). Controls were treated with the AraC solvent DMSO.

first: Goichi

last: Tatsumi

first: Rajni

last: Kumari

first: Yutaro

last: Suzuku

first: Shuting

last: Li

first: Samuel

last: Taylor

first: Sriram

last: Sundaravel

first: Amit

last: Verma

first: Justin

last: Wheat

first: Ulrich

last: Steidl


title: Genomic language models improve cross-species gene expression prediction and accurately capture regulatory variant effects in Brachypodium mutant lines

city: Aarhus

first: Guillaume

last: Ramstein

address: Moellers Alle

institute: Aarhus University

summary: Predicting gene expression from cis-regulatory DNA sequences is a central challenge in plant genomics. Here, we developed deep learning sequence-to-expression (S2E) models that leverage high-dimensional representations from auxiliary foundational models (genomic language model PlantCaduceus, chromatin accessibility model a2z) instead of one-hot encoding of sequences, to predict gene expression across 17 plant species. We first evaluated our models to predict gene expression on unseen gene families via cross-validation, demonstrating our model’s prediction accuracy across all species outperforms PhytoExpr, a state-of-the-art (SOTA) S2E model trained on the same dataset (Pearson R=0.82 vs. R=0.74). We then validated variant effect predictions using an experimental dataset across 796 Brachypodium mutant lines, specifically designed to test predictions at single-base resolution. Our models outperformed the SOTA models in predicting between-gene expression differences (regression coefficient β=0.78 vs. β=0.57). Remarkably, they also accurately predicted the effects of single-nucleotide mutations on within-gene expression, while SOTA models showed only weak associations (regression coefficient β=0.38 vs. β=0.08). Our results demonstrate the value of context-aware DNA sequence embeddings for predicting regulatory variant effects in plants. They also reveal a persistent accuracy gap in S2E models when moving from between-gene to allelic variation, a challenge that needs to be addressed in future studies.

overall_design: RNA-seq profiling of independent lines in Brachypodium distachyon, derived from a single genotype (Bd21-3): 769 mutant lines, 27 control lines. Mutant lines were generated by chemical mutagenesis: seed treatment by 7 mM of sodium azide. Control lines were generated by a neutral treatment: seed treatment by 0 mM of sodium azide. RNA-seq measurements were taken at the M6 generation (six generations after mutagenesis) under controlled conditions in growth chambers.

middle: P

first: Camous

last: Moslemi

first: Behrooz

last: Vahedi Torghabeh


title: A Role for Rapid Transcription Dynamics in Cytarabine Resistance in Acute Myeloid Leukemia

overall_design: HL60 cells were treated with 250nM AraC for 1 day. Cells were then stained for Pyronin Y and Hoechst and Pyronin Y low and high fraction within G0/G1 gated cells were sorted using flow cytometry for RNA seq analysis

last: Suzuki


title: Pseudomonas isolates reconfigure gene expression in walnut seedlings exposed to salt stress

city: Beijing

first: Gaoya

last: Su

address: Research Institute of Forestry Chinese Academy of Forestry No 1, Dong Xiao Fu Beijing , China

institute: Chinese Academy of Forestry

summary: To understand the potential mechanisms underlying the effects of the four Pseudomonas strains P1, P2, P3, and P4 on the growth and salt tolerance of walnut trees, the transcriptomic responses of walnut seedlings inoculated with these isolates under conditions of salt stress were further evaluated using RNA-seq analysis.

overall_design: Transcriptomic analysis of walnut seedlings under control (mock) and salt stress (100 mM NaCl) conditions, with or without inoculation of four Pseudomonas bacterial strains (P1–P4), profiled at the end of treatment period.


title: Phage-encoded CasPRs transcriptionally silence diverse CRISPR-Cas systems [ChIP-seq]

city: Seattle

first: Alexander

last: Meeske

state: WA

address: 750 Republican St

institute: University of Washington

summary: Anti-CRISPRs (Acrs) are diverse proteins or RNAs that protect invading phages and plasmids from host CRISPR-Cas immunity. Most Acrs neutralize their cognate Cas proteins via direct physical interaction. Here we describe CasPRs, a particularly widespread family of DNA-binding Acrs that recognize specific sequence motifs within cas gene coding regions, thereby blocking RNA polymerase and silencing transcription. We demonstrate that eight diverse CasPRs bind to the cas8b gene to repress the type I-B CRISPR-Cas system in its native host, Listeria seeligeri. Meanwhile, a CasPR from Streptococcus dysgalactiae silences type II-A CRISPR-Cas immunity by binding to the cas9 coding sequence. We found that one CasPR is required to inhibit CRISPR immunity during lysogeny by its host prophage. Taken together, our results indicate that members of the CasPR family have diverged to silence completely unrelated CRISPR types, and suggest transcriptional repression is a common mode of phage-mediated immune antagonism.

overall_design:
Chip-seq of his6-tagged CasPR proteins expressed in Listeria seeligeri (and no tag controls)
Chip-seq of his10-tagged RpoC (RNA polymerase) in the presence and absence of CasPR expression in Listeria seeligeri.

first: Edith

last: Sanderson

first: Julieta

last: Peralta

first: Sam

last: Nouwens

first: Luke

last: Oriolt

first: Victoria

last: Hayes

first: Brett

middle: K

last: Kaiser

middle: J


title: IRE1 drives a homeostatic response to reduced protein influx into the endoplasmic reticulum (Translation initiation interruption)

city: Redwood City

first: Altos Labs

last: Bioinformatics Hub

state: CA

address: 2600 Island Dr.

department: Institute of Technology

institute: Altos Labs

summary: IRE1, alongside ATF6 and PERK, orchestrates the unfolded protein response, a network of signaling pathways that maintains endoplasmic reticulum (ER) homeostasis. Two modes of IRE1 activation are known: i) in response to an accumulation of unfolded proteins in the ER lumen and ii) in response to compositional changes to the ER membrane that alter its physical properties. Here, we identify a third, independent mode of IRE1 activation: ER co-translational translocation deficits activate IRE1 through a mechanism that relies on the release of IRE1 molecules from unoccupied translocons. We define this mechanism as TRES for “TRanslocon Engagement Surveillance”. TRES leads to spontaneous activation of IRE1 and bypasses its unfolded protein- and ER membrane composition-sensing functions. Inhibiting translation initiation similarly activates IRE1 by TRES, as it leads to a decline in ER protein import, thus linking the integrated stress response (ISR) to IRE1 signaling. Surprisingly, TRES drives IRE1 activation without activating ATF6 or PERK, resulting in a distinct gene expression program that feeds back into the co-translational translocation pathway to rebalance the ER protein load. Our findings demonstrate that monitoring and adjusting the rates of protein translocation are critical for maintaining ER homeostasis.

overall_design: RNA-seq analysis of wild-type, IRE1-knockout, and IRE1-rescued U-2 OS cells treated with DMSO, Rocaglamide (100 nM) for 16 hours, or Tunicamycin (5 μg/ml) for 4 hours. Doxycycline (DOX) was added 24 hours before drug treartment.

first: Francesca

last: Zappa

first: Advait

last: Subramanian

first: Benjamin

middle: A

last: Yang

first: Julia

last: Conrad

first: Tsan-Wen

last: Lu

first: Jing

last: Wang

first: Nicolas

last: Debeaubien

first: Rose

last: Yan

first: Renato

last: Minopoli

first: Tristan

last: Croll

first: Stefka

last: Tyanova

first: Mauro

last: Costa-Mattioli

first: Peter

last: Walter

first: Diego

last: Acosta-Alvear


title: IRE1 drives a homeostatic response to reduced protein influx into the endoplasmic reticulum (ISR Timecourse)

overall_design: Time-course RNA-sequencing analysis of H4 cells expressing FKBP-PKR fusion constructs (active, inactive T446A mutant, or FKBP-only control) treated with chemical dimerizer with or without IRE1 inhibitor 4μ8C, sampled at 0, 2, 4, 8, 16, and 24 hours post-treatment (n=3 per condition) to dissect PKR-mediated integrated stress response signaling.