title: Intestinal Macrophages Modulate Brain Synucleinopathy in Parkinson’s Disease

city: London

first: Daria

last: Gavriouchkina

state: London

address: UCL Cruciform Building, Gower Street

department: UK Dementia Research Institute at UCL

institute: University College London

summary: Emerging evidence suggests that Parkinson’s disease (PD) may have its origin in the enteric nervous system (ENS), from where alpha-synuclein (αS) pathology spreads to the brain. Decades before the onset of motor symptoms, PD patients suffer from constipation and present with circulating T cells responsive to αS, suggesting that peripheral immune responses initiated in the ENS may be involved in the early stages of PD. However, cellular mechanisms that trigger αS pathology in the ENS and its spread along the gut-brain axis remain elusive. Here, we demonstrate that muscularis macrophages (ME-Macs), housekeepers of ENS integrity and intestinal homeostasis, modulate αS pathology and neurodegeneration in models of PD. ME-Macs contain misfolded αS, adopt a signature reflecting endolysosomal dysfunction, and modulate the expansion of T cells that travel from the ENS to the brain via the dura mater as αS pathology progresses. Directed ME-Mac depletion leads to reduced αS pathology in the ENS and CNS, prevents T cell expansion, and mitigates neurodegeneration and motor dysfunction, suggesting a role for ME-Macs as early cellular initiators of αS pathology along the gut-brain axis. Understanding these mechanisms could pave the way for early-stage biomarkers in PD.

overall_design: In this study, single-cell RNA sequencing was performed on duodenal muscularis externa from wild-type and 3KL α-synuclein transgenic mice to capture transcriptional changes associated with α-synuclein pathology. 4 biologically independent samples were analyzed per genotype, each consisting of pooled tissue from 4 mice to ensure reproducibility. Macrophages and T cells were isolated from the muscularis externa and profiled in parallel to assess their transcriptional states and potential interactions. The design allowed direct comparison between disease and control conditions, with quality control and clustering used to define distinct cell populations. Main variable: genotype (WT vs 3KL).

first: Sebastiaan

last: De Schepper

first: Viktoras

last: Konstantellos

first: James A

last: Conway

first: Dimitra

last: Sokolova

first: Ludovica

last: Zaccagnini

first: Matthew V

last: Cowley

first: Annerieke

last: Sierksma

first: Maria

last: Yudina

first: Marisa

last: Edmonds

first: Bethany

last: Geary

first: Amber

last: Wallis

first: Meral

last: Celikag

first: Zeynep

last: Baykam

first: Monica

last: Vara-Perez

first: Gerard

last: Crowley

first: Fabian Tobias

last: Hager

first: Mitchell

last: Bijnen

first: David

last: Posner

first: Kelvin

last: Luk

first: Vuk

last: Cerovic

first: Menna

last: Clatworthy

first: Elizabeth J

last: Videlock

first: Zane

last: Jaunmuktane

first: Kiavash

last: Movahedi

first: Melanie

last: Greter

first: Benny

last: Chain

first: Dario R

last: Alessi

first: Soyon

last: Hong

first: Tim

last: Bartels


title: PAXT/NEXT targeting of RNA exosome regulates antisense transcripts through cohesin-mediated long-range contacts [ChIP-seq]

status: Public on Nov 20 2025

city: Montpellier

first: Rosemary

last: Kiernan

address: 141 rue de la cardonille

country: France

institute: UMR9002 CNRS-UM

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Here, we mapped the binding sites of PAXT/ZFC3H1, NEXT/ZCCHC8 and their shared MTR4 helicase subunit. Our data highlight a key interplay between cohesin-mediated long-range interactions in 3D and ncRNA regulation by MTR4 helicase and nuclear RNA exosome.

overall_design: Examination of ZFC3H1, ZCCHC8 and MTR4 binding sites on chromatin by ChIP-seq in HeLa cells. Examination of Rad21 binding sites after depletion of MTR4 and ZFC3H1.

first: Xavier

last: Contreras

first: Alexandre

last: Heurteau

first: Olivier

last: Cuvier


title: Comparative analysis of shared and unique mechanisms important for diverse strains of Pasteurella multocida to cause systemic infection in mice

status: Public on Nov 19 2025

city: Clayton

first: Thomas

middle: R

last: Smallman

state: Victoria

address: 19 Innovation walk

department: Microbiology

country: Australia

institute: Monash University

summary: Pasteurella multocida is a pathogen that causes a range of distinct diseases in livestock animals. Different P. multocida strains produce different capsule and lipopolysaccharide (LPS) structures. Different P. multocida diseases are associated with different capsule and LPS types, and little is known about what underpins this disease specificity. In this study, we utilised transposon-directed insertion site sequencing (TraDIS, also called Tn-Seq) to identify genes required for growth in rich media, and genes required for survival during systemic infections in BALB/c mice, for two diverse P. multocida strains, strain VP161 (capsule type A and LPS type L1) and strain M1404 (capsule type B and LPS type L2). Rich media analysis showed that both VP161 and M1404 shared 461 genes essential for growth in rich media, with comparison to the entire species showing that 95% of these rich media essential genes present in all publicly available closed P. multocida genomes. In vivo fitness analysis identified 63 and 94 genes important for VP161 and M1404 survival in BALB/c mice, respectively. Only 35 homologs were identified in both strains as important for survival, showing that conserved biological systems can be differentially important for different P. multocida strains. Investigation of proteins involved in the catabolite response showed that an active cyclic-adenosine monophosphate (cAMP) receptor protein (CRP) was required for maximal fitness in M1404.  Furthermore, disrupting CRP or cAMP production also reduced capsule production in M1404, but increased capsule production in VP161, showing different strains of P. multocida have different regulatory systems for crucial virulence factors.

overall_design: This study used transposon-directed insertion site sequencing (TraDIS, also called Tn-Seq) to investigate genes required for growth in rich media, and genes important for in vivo fitness, of two Pasteurella multocida strains, strain VP161 and strain M1404. A large VP161 Himar1 mutant library was grown in heart infusion broth, used to inject six-to-eight-week-old BALB/c mice via intraperitoneal injection with ~1 x 107 colony forming units. At the humane end point, surviving mutants were recovered from the bloodstream, liver and spleen, then plated on heart infusion agar and incubated overnight at 37ºC. The input library was also plated onto heart infusion agar and incubated overnight. The M1404 Himar1 mutant library was also used in the same way. Cells were recovered from the overnight growths, genomic DNA extracted and used to produce TraDIS libraries. TraDIS libraries were generated by shearing DNA to ~300 bp using Covaris sonication, using NEBNext kits to repair the ends of the DNA, A-tail, and then ligate adapters on the end. Specific amplification of transposon-chromosome junctions was performed using a Himar1-specific primer, and an adapter-specific primer. The adapter-specific primer contained index sequences specific for each dataset, and the primers contained Illumina P5 and P7 sequences to allow for Illumina sequencing. The TraDIS libraries were sequenced using an Illumina MiSeq, and the data analysed using the Bio-TraDIS toolkit. The data uploaded are the reads generated from the Illumina MiSeq, and the excel table that contains the analysed data shows the number of reads per gene, the number of unique Himar1 insertion sites per gene, and whether they were called as essential for growth in rich media or important for in vivo fitness in different tissues recovered from BALB/c mice.

first: Xiaochu

last: Wang

first: Xenia

last: Kostoulias

first: Amy

last: Wright

first: Faye

middle: C

last: Morris

first: Marina

last: Harper

first: John

middle: D

last: Boyce


title: Stc1-expressing myofibroblasts are a developmentally distinct lineage cleared through apoptosis in the neonatal lung

city: Philadelphia

first: Sylvia

middle: N

last: Michki

state: PA

address: 3615 Civic Center Blvd

department: Pulmonary & Sleep Medicine

country: USA

institute: Children's Hospital of Philadelphia

type: Expression profiling by high throughput sequencing

summary: Lung myofibroblasts are necessary for early postnatal alveolar growth. The unique contributions of individual myofibroblast lineages to alveolar development is unresolved by existing genetic tools. We generated a Stc1CreERT2 mouse line that labels the developmentally transient secondary crest myofibroblasts (SCMFs), distinguishing them from alveolar duct myofibroblasts (DMF) and smooth muscle. SCMFs expand through clonal proliferation of Stc1-expressing progenitors and are cleared by apoptosis. Deleting the apoptosis effectors Bax and Bak1 in the Stc1-lineage prevented SCMF clearance during alveologenesis. Single-cell RNA sequencing showed that surviving Stc1-lineage cells lose myofibroblast identity while coexpressing SCMF and DMF markers. Embryonic lineage tracing identified distinct progenitors for SCMFs and DMFs, and genetic activation of Hedgehog (Hh) or Wnt signaling pathways failed to interconvert these lineages. These findings establish Stc1-lineage SCMFs as a discrete, developmentally divergent population, and define their life cycle independent of other myofibroblast lineages.

overall_design: Stc1-CreERT2 conditional apoptosis knockout (Stc1-BB) mice were injected with tamoxifen at P1 and sacrificed at P30 for lung cell harvest and scRNA-seq. Cells from each animal were labelled with cell multiplexing oligos (CMOs) prior to FACS in order to assign single cells to animals (biological replicates) within the joined scRNA-seq data. Cell suspensions were not combined across experimental conditions (i.e. HET/KO libraries were generated separately).

first: Melinda

middle: E

last: Snitow

first: Jarod

middle: A

last: Zepp


title: A tumor-associated macrophage-targeted immunocytokine leveraging T and NK cell synergy for potent cancer immunotherapy [scRNA-seq]

city: Rehovot

first: Ido

last: Amit

address: 234 Herzl st.

department: Immunology

country: Israel

institute: Weizmann Institute of Science

summary: Tumor-associated macrophages (TAMs) expressing the myeloid checkpoint TREM2 are key immunosuppressive cells in the tumor microenvironment (TME), driving tumor progression and contributing to poor prognosis in cancer patients. Due to their pivotal role, TAMs have emerged as a promising target for immunotherapies. However, current TAM-targeting monotherapies show only modest efficacy in clinical trials in solid tumors . Here, we have developed a new class of cancer immunotherapies: myeloid-targeted immunocytokines and NK-T cell enhancers (MiTEs). MiTEs are trans-acting immunocytokines with tumor-specific activation, allowing dual targeting of TAMs and lymphocytes by TREM2 antagonism and cytotoxic effector cell activation through IL-2. To avoid off-target toxicities, MiTEs contain an IL-2 masking moiety, which is cleaved by a TAM-specific protease. MiTEs demonstrate high efficacy in preclinical tumor models through extensive immune reprogramming spanning TAM, T and NK cell compartments. MiTEs show transformative potential for treating solid-cancers by inducing potent multi-arm anti-tumor immunity and minimizing toxicities.

overall_design: hTREM2 mice were s.c. injected with 1M MC38 tumor cells (day 0). Mice were intravenously treated with 200 µg per dose of anti-RSV, anti-TREM2, anti-PD-1, anti-TREM2 + anti-PD1, anti-CTLA-4, anti-TREM2 + anti-CTLA-4, MiTE-144, MiTE-144 + anti-PD1, or MiTE-144 + anti-CTLA-4 on days 8 and 10 (n = 5). Immune cells were isolated on day 11, FACS sorted (total CD45+ and T/NK cell enrichment) and underwent scRNAseq employing SPID-seq resulting in a total of 45,505 high-quality cells from 45 mice.

first: Michelle

last: von Locquenghien

first: Pascale

last: Zwicky

first: Ken

last: Xie

first: Diego

last: Jaitin

first: Fadi

last: Sheban

first: Florian

last: Uhlitz

first: Chamutal

last: Gur

first: Reut

last: Sharet Eshed

first: Eyal

last: David

first: Kfir

last: Mazuz

first: Jahan

last: Adeeb Rahman

first: Noam

last: Solomon

first: Roberto

last: Avellino

first: Assaf

last: Weiner


title: WDR5 Remodels NANOG Condensates to Drive Transcriptional Programs and Sustain Stem Cell Identity [ChIP-seq]

city: Shanghai

first: jiaying

last: xie

address: Dongfang Road

country: China

institute: Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine

summary: Stem cell pluripotency relies on a finely tuned interplay between transcription factors and epigenetic regulators. Here, we identify a direct interaction between NANOG, a master pluripotency transcription factor, and WDR5, a core component of the SET1/MLL complex that is essential for maintaining stem cell identity. Mechanistically, WDR5 reshapes the prion-like structures of NANOG into dynamic, liquid-liquid phase-separated (LLPS) condensates, facilitating its recruitment to pluripotency-associated promoters and activating target gene expression. X-ray crystallography and NMR spectroscopy reveal that the NANOG homeodomain engages WDR5 through an extensive structural interface distinct from previously characterized short linear motifs. The NANOGR153A mutation disrupts its interaction with WDR5, leading to impaired LLPS formation, reduced chromatin co-occupancy, and diminished levels of active histone marks (H3K4me3 and H4K16ac), ultimately compromising the pluripotency of embryonic stem cells. Furthermore, pharmacological inhibition of the WDR5-NANOG interaction using a WIN motif inhibitor robustly suppresses leukemia stem cell expansion in vivo, highlighting its therapeutic potential. Collectively, this study uncovers an LLPS-mediated regulatory mechanism whereby WDR5 modulates NANOG condensate dynamics to sustain stem cell identity in both physiological and pathological contexts.

overall_design: Chromatin immunoprecipitation followed by sequencing (ChIP–seq) for H3K4me3/H4K16ac/Nanog/Wdr5 in mouse embryonic stem cells

first: Xianle

last: Shi

first: Yanjing

last: Li


title: Molecular and spatial integration of algal endosymbionts of the freshwater sponge, Ephydatia muelleri, throughout development in light and dark conditions

city: Lewiston

first: April

middle: L

last: Horton

email: ahorton@bates.edu, ahill5@bates.edu

state: Maine

address: 44 Campus Avenue, Carnegie Science Hall

department: Biology

institute: Bates College

summary: Animal-algal photosymbioses are a unique group of symbiotic relationships in which animals harbor photosynthetic algae within their cells and tissues. Both marine and freshwater sponges host algal endosymbionts. In previous work, we demonstrated that freshwater sponges can acquire these endosymbionts horizontally through algal infection and that potentially conserved evolutionary pathways may lead to the establishment of the endosymbioses including those involved in endocytosis, ion transport, vesicle-mediated transport, innate immunity, redox regulation, and metabolic processes.  Here, we show that algal symbionts can be transferred vertically from algal-bearing overwintering gemmules to adult sponges, and that their proliferation is enhanced by light. Sponges grown under light conditions harbored higher algal loads than those in the dark; however, algae were still able to proliferate and persist in sponges reared in the dark, occupying similar spatial locations to those grown in light. RNA-Seq analysis of algal-bearing sponges across developmental stages in light and dark conditions revealed genetic regulatory pathways involved in the transmission and establishment of the endosymbiosis. Differential expression analysis indicated that the endocytosis and SNARE pathways regulate the internalization and transport of algae  at the earliest stage of hatching under light conditions and later in development under dark conditions, potentially contributing to the recruitment of endosymbiotic algae. In sponges hatched in the dark, genes involved in vesicle acidification are regulated, alongside changes in the expression of genes in the pentose phosphate pathway - a key metabolic route involved in redox homeostasis and circadian rhythm regulation via NADPH metabolism, is observed. E. muelleri serves as a versatile model system, supported by robust genomic and transcriptomic resources, for studying host-symbiont interactions. It offers a unique opportunity to investigate the molecular signaling and environmental factors that shape symbiosis in a system where the host can exist with or without algal endosymbionts, symbionts can be acquired either horizontally or vertically, and proliferation of the algae can occur with or without photosynthesis.

overall_design: RNASeq profiling of freshwater sponges with algal endosymbionts at four developmental stages and grown under light:dark conditions.

first: April L

first: Sally P

last: Leys

first: Ana

last: Riesgo


title: A tumor-associated macrophage-targeted immunocytokine leveraging T and NK cell synergy for potent cancer immunotherapy [CITE-seq]

first: Ido

last: Amit

summary: Tumor-associated macrophages (TAMs) expressing the myeloid checkpoint TREM2 are key immunosuppressive cells in the tumor microenvironment (TME), driving tumor progression and contributing to poor prognosis in cancer patients. Due to their pivotal role, TAMs have emerged as a promising target for immunotherapies. However, current TAM-targeting monotherapies show limited efficacy, highlighting the need for strategies engaging multiple immune modalities. Here, we developed a new class of cancer immunotherapies: myeloid-targeted immunocytokines and NK-T cell enhancers (MiTEs). MiTEs are trans-acting immunocytokines with tumor-specific activation, allowing dual targeting of TAMs and lymphocytes by TREM2 antagonism and cytotoxic effector cell activation through IL-2. To avoid off-target toxicities, MiTEs contain an IL-2 masking moiety, which is cleaved by a TAM-specific protease. MiTEs demonstrate high efficacy in preclinical tumor models through extensive immune reprogramming spanning TAM, T and NK cell compartments. MiTEs show transformative potential for treating solid-cancers by inducing potent multi-arm anti-tumor immunity and minimizing toxicities.

overall_design:
Fresh tumor samples were obtained from discarded surgical resection specimens collected from consented patients under a Cooperative Human Tissue Network-CHTN IRB approved protocol.  Tumor samples were cut into 1-2mm3 Patient-Derived Tumor Fragments (PDTFs) and cryopreserved in 1ml CryoStor CS10).
Cryopreserved PDTFs from five distinct renal cell carcinoma patient tumors were rapidly thawed in a 37°C water bath and immediately placed in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS). Following thawing, individual tumor fragments were cultured for 48 hours as previously detailed73. For each donor, a total of 16 fragments per treatment condition were allocated to each of the four experimental arms: Isotype Control: anti-Respiratory Syncytial Virus (RSV) IgG1 (10 nM) and anti-Hen Egg Lysozyme (HEL) IgG4 (10 µg/mL), anti-PD-1 Monotherapy: anti-PD-1 (10 µg/mL; Cat: SIM0010) and anti-RSV-IgG1 (10 nM), MiTE144 Monotherapy: MITE144 (1 0nM) and anti-HEL-IgG4 (10 µg/mL), Combination Therapy: MITE144 (10 nM) and anti-PD-1 (10 µg/mL).
Upon completion of the 48-hour culture period, 90ul of supernatants were collected and frozen for secreted protein analysis. Next, all fragments from the same donor and treatment arm were pooled and digested into single cell suspensions. Immune cells were enriched from the digested fragments using CD45 magnetic-activated cell sorting (MACS). The resulting CD45-enriched cell fractions were subsequently stained with a custom Immunai CITE-seq antibody panel (see STAR methods) and subjected to fluorescence-activated cell sorting (FACS) to isolate viable CD45 cells.
Single-cell gene expression (GEX) and feature (CITE-seq) libraries were prepared from the sorted cells using the 10x Genomics Chromium Next GEM Single Cell 5' Kit v2. Libraries were sequenced on a Novaseq X Plus sequencer using a 300 cycle kit.

first: Michelle

last: von Locquenghien

first: Pascale

last: Zwicky

first: Ken

last: Xie

first: Diego

last: Jaitin

first: Fadi


title: Maintenance of gene expression homeostasis by systems-level feedback loops in response to changes in RNA polymerase II dosage [RNAPII_levels_CUT&RUN]

city: Copenhagen

first: Jesper

middle: Q

last: Svejstrup

address: Blegdamsvej 3B

department: ICMM

country: Denmark

institute: University of Copenhagen

summary: Transcription is regulated by sequence-specific transcription factors and enzymes allowing access to genes in chromatin. However, recent data indicate that the abundance of RNA polymerase II (RNAPII) may itself under certain circumstances represent an additional, crucial determinant of transcription regulation. Here, we titrated the cellular dosage of human RPB1, the largest RNAPII subunit, using the dTAG system to more generally assess the importance of RNAPII levels. Unexpectedly, cells are extremely sensitive to RPB1 dosage, with a mere 30% reduction sufficient to perturb cell proliferation, cell cycle progression, and global transcription. Using a combination of sequencing and proteomic approaches, we uncover the existence of multiple feedback loops between transcriptional initiation, promotor-proximal pause release, transcript elongation, splicing, and mRNA half-life, which facilitate the maintenance of gene expression homeostasis. Together, our data suggests that gene expression is highly sensitive to RPB1 dosage and that the cellular levels of RNAPII must be tightly regulated.

overall_design: To investigate the effect of reduced cellular RPB1 abundance on the loading of RNAPII on chromatin, we modulated RPB1 levels by treating RPB1 degron cells with 4 concentrations of dTAGv-1 for 16 hours, followed by CUT&RUN of total RNAPII and hyperphosphorylated RNAPII using an antibody against the HA-tag and Ser5+Ser2 phosphorylated RNAPII CTD respectively.

first: Anouk

middle: M

last: Olthof

first: Melvin

middle: J

last: Noe Gonzalez

first: Nanna

middle: B

last: Poulsen

first: Nicolas

last: Nieto Moreno

first: Theodora

last: Bautu

first: Simona

last: Cugusi


title: 4EHP and NELF-E regulate factors required for ATF4 expression in the Drosophila fat body

city: New York

first: Hyung Don

last: Ryoo

state: NY

address: 550 First Avenue, MSB 497

department: Cell Biology

institute: NYU School of Medicine

summary: Cells adapt to proteostatic and metabolic stresses, in part, by triggering the phosphorylation of eIF2alpha and the subsequent synthesis of ATF4. eIF2alpha phosphorylation facilitates translational induction of ATF4 by regulating reinitiation of the scanning ribosomes at the ATF4 mRNA’s 5’ leader. In addition to eIF2alpha, ATF4 induction requires other regulators that remain poorly understood. Here, we report an ATF4 regulatory network consisting of eIF4E-Homologous Protein (4EHP), NELF-E, the 40S ribosome, and eIF3 subunits. Specifically, 4EHP was required for ATF4 signaling in the Drosophila larval fat body and in disease models associated with abnormal ATF4 signaling. 4EHP’s mRNA cap-binding domain specifically interacted with NELF-E mRNA, which encodes a regulator of pol II-mediated transcription. Both NELF-E and 4EHP were required for the expression of ATF4 and enzymes that mediate the serine-one-carbon pathway. Knockdown of NELF-E also reduced several subunits of the 40S ribosome and the eIF3 translation initiation factor complex. Reduction of Rps12 and eif3h suppressed physiological ATF4 induction. These results uncover a previously unrecognized ATF4 regulatory network that impacts stress adaptation and pathological outcomes.

overall_design: We knocked down 4EHP in the Drosophila fat body using the dcg-Gal4 driver that drived 4EHP RNAi (VDRC #38399). NELF-E was knocked down by the expression of NELF-E RNAi (VDRC #21009) with the dcg-Gal4 driver. To identify 4EHP binding mRNAs, we expressed 4EHP fused to ADAR in the fat body (using dcg-Gal4) and compared the extent of mRNA editing with a negative control 4EHP-ADAR that has an impaired mRNA cap-binding domain. The fat bodies were dissected from late third instar male larvae and the RNAs were extracted using Trizol. PolyA mRNA was sequenced through Novaseq 6000.

first: Kristoffer

last: Walsh

first: Christoph

last: Dieterich


title: Quantifying Transcription Factor Specificity with Advanced DNA Universal Microarrays Featuring Long and Modified Binding Sites [420k]

first: Yuval

last: Bayer

address: Herzl St 234

summary: In the study, we have created new de Bruijn sequences for universal Protein Binding Microarrays with varying orders and DNA letter sets.

overall_design: The design is of probes which include three de Bruijn sequences of order 9, two of order 10, and two of order 11.  The layout is two chambers, each of 796x528 probes.  We used part of the entire design that includes a single de Bruijn of order 9, where each of all 9-mers is contained in 2 probes on average.  The rest of the de Bruijn sequences are denoted as PBM, where each of the 9-mers is contained in 105 probes on average.

first: Ariel

last: Afek


title: Maintenance of gene expression homeostasis by systems-level feedback loops in response to changes in RNA polymerase II dosage [RNAPII_levels_DRB-TTseq]

overall_design: To investigate the effect of RPB1 abundance on transcript elongation rates, we modulated RPB1 levels by treating RPB1 degron cells with 4 concentrations of dTAGv-1. After 12.5 hours, transcription was synchronized at the promoter proximal pause site through the addition of DRB. Afterwards, RNA polymerases were released for different times by washing out DRB, and nascent RNA was metabolically labeled with a short pulse of the uridine analog 4-thiouridine (4sU). Transient transcriptome chem-sequencing (TTchem-seq) was then used to fragment, affinity purify and sequence 4sU-labelled RNA for high-resolution profiles of nascent transcription. Finally, the transcription wave peak was measured at different time points to estimate elongation speed.


title: Maintenance of gene expression homeostasis by systems-level feedback loops in response to changes in RNA polymerase II dosage [RNAPII_levels_mRNAseq]

overall_design: To investigate the effect of RPB1 abundance on the steady-state transcriptome, we modulated RPB1 levels by treating RPB1 degron cells with 12 concentrations of dTAGv-1. In addition, an RPB1 transgene expressed from the UBC promoter was flipped into the FRT locus to study the effects of overexpression. After 16 hours, all cells were harvested and used for mRNA-seq.


title: WDR5 Remodels NANOG Condensates to Drive Transcriptional Programs and Sustain Stem Cell Identity [Nanog_WT_R153A_RNAseq]

first: jiaying

last: xie

overall_design: Comparative gene expression profiling analysis of RNA-seq data for mESCs in Nanog WT and Nanog R153A mutant cells

first: Xianle

last: Shi

first: Yanjing

last: Li


title: Quantifying Transcription Factor Specificity with Advanced DNA Universal Microarrays Featuring Long and Modified Binding Sites [48k]

overall_design: The design comprises probes with a single de Bruijn of order seven over seven letters. The layout is one chamber of 320x150 probes. Each non-palindromic 6-mers over {A, C, M, W, G, T} is contained in at least 33 probes. M represents 5-methylcytosine (5mC) and W represents guanine paired with 5mC.

first: Irina

last: Miodownik


title: Single-cell transcriptomic profiles of metastatic lesion biopsies and PBMCs from M1-stage prostate cancer patients

city: Seoul

first: Baek Gil

last: Kim

address: 50-1 Yonsei-Ro

country: South Korea

institute: Yonsei University

summary: This dataset contains single-cell RNA sequencing (scRNA-seq) data generated from patients with M1-stage metastatic prostate cancer, comprising two complementary sample types: metastatic lesion biopsies from five patients and peripheral blood mononuclear cells (PBMCs) from seven patients. Together, these datasets support integrated profiling of the metastatic tumor microenvironment and the systemic immune landscape. The processed data include log-normalized gene expression matrices and detailed cell-level metadata, enabling analyses of tumor–immune interactions, circulating immune phenotypes, and molecular features associated with metastatic disease progression. A related bulk RNA-seq dataset from primary tumor biopsies is available (see Related Series: GSE297742), providing opportunities for comparative transcriptomic analyses between primary and metastatic disease states.

overall_design: This Series contains single-cell RNA sequencing (scRNA-seq) data generated from metastatic biopsy samples (n = 5) and peripheral blood mononuclear cells (PBMCs; n = 7) collected from patients with M1-stage metastatic prostate adenocarcinoma. Metastatic lesion biopsies were enzymatically dissociated into single-cell suspensions, while PBMCs were isolated using density gradient centrifugation. All samples were processed using the 10x Genomics Chromium Single Cell 3’ v3 platform for single-cell library construction and sequenced on the Illumina NovaSeq 6000 with paired-end reads (2 × 101 bp). Raw sequencing data were processed using the Cell Ranger pipeline (10x Genomics) for alignment to the GRCh38 reference genome and generation of gene-by-cell count matrices, followed by downstream analyses in Seurat for quality control, normalization, dimensionality reduction, clustering, and cell-type annotation. The resulting dataset includes high-quality log-normalized gene expression matrices and comprehensive cell-level metadata across both biopsy- and PBMC-derived single-cell populations, enabling integrated analysis of metastatic tumor biology and systemic immune remodeling in advanced prostate cancer.

first: Hyunho

last: Han

first: Jongsoo

last: Lee

first: Jihee

last: Jung

first: Ayoung

last: Yoo

first: Jungchan

last: Jun

first: Dongwook

last: Song

first: Yeonsue

last: Jang

first: Baek

middle: G


title: WDR5 Remodels NANOG Condensates to Drive Transcriptional Programs and Sustain Stem Cell Identity

overall_design: To elucidate the mechanisms underlying the enhanced self-renewal capacity of AE9a hematopoietic stem and progenitor cells (HSPCs), potentially mediated through elevated NANOG expression and its functional interplay with WDR5, we conducted transcriptome-wide RNA sequencing (RNA-seq) analyses to systematically characterize transcriptomic alterations in both wild-type and AE9a murine HSPCs following pharmacological inhibition of WDR5 and MLL1.

first: Duo

last: Wang

first: Jiaying

last: Xie

first: Lijie

last: Zhao

first: Bin

last: Wu


title: Maintenance of gene expression homeostasis by systems-level feedback loops in response to changes in RNA polymerase II dosage [RNAPII_levels_TTseq]

first: Jesper

last: Svejstrup

overall_design: To investigate the effect of RPB1 abundance on nascent transcription, we modulated RPB1 levels by treating RPB1 degron cells with 12 concentrations of dTAGv-1. In addition, an RPB1 transgene expressed from the UBC promoter was flipped into the FRT locus to study the effects of overexpression. After 16 hours, nascent RNA was metabolically labeled with a short pulse of the uridine analog 4-thiouridine (4sU). Transient transcriptome chem-sequencing (TTchem-seq) was then used to fragment, affinity purify and sequence 4sU-labelled RNA for high-resolution profiles of nascent transcription.

first: Anouk

last: Olthof

first: Melvin

last: Noe Gonzalez

first: Nanna

last: Poulsen

first: Nicolas

last: Nieto Moreno

first: Theodora

last: Bautu

first: Simona

last: Cugusi


title: A tumor-associated macrophage-targeted immunocytokine leveraging T and NK cell synergy for potent cancer immunotherapy

first: Ido

last: Amit

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: Dextran-based T-cell expansion nanoparticles for manufacturing CAR T cells with augmented efficacy

city: Kgs. Lyngby

first: Kamilla Kjærgaard

last: Munk

address: Henrik Dams Allé, 204

department: Department of Health Technology

institute: Technical University of Denmark

summary: Adoptive T cell therapy (ACT) using chimeric antigen receptor (CAR) engineered T cells is currently being explored in multiple cancer types beyond leukemia/lymphoma. A key step in CAR-T cell manufacturing is the activation and expansion of T cells, which facilitates viral transduction, however, may hamper T cell fitness and reduce in vivo persistence. We developed “T-Expand” for T cell activation and expansion, comprising dextran-based nanoparticles (NPs) conjugated with anti-CD3 and anti-CD28 antibodies. The NPs triggered robust polyclonal expansion of human T cells with efficiency in the range of commercial microbeads (Dynabeads™). Engineered in the presence of T-Expand, CD19 CAR T cells exhibited enhanced proliferative capacity, cytotoxicity and persistence in vitro, and furthermore, showed superior anti-lymphoma activity in mouse models, resulting in complete tumor clearance at one fourth of the CAR T cell dose. Importantly, T-Expand is biocompatible with no observed toxicity, circumventing removal steps after T cell expansion compared to DynabeadsTM. As a biocompatible T cell expansion platform, T-Expand simplifies the manufacturing process while enhancing T cell persistence and functionality, and thereby holds promise for increasing clinical efficacy of CAR T cell therapy.

overall_design: RNA-seq profiling of CAR T cells expanded with T-Expand or Dynabeads in the presence of 20 IU/ml IL-2 for 10 days.

first: Tao

last: Zheng

first: Keerthana

last: Ramanathan

first: Maria

last: Ormhøj

first: Mikkel

last: Hansen

first: Hólmfridur

last: Halldórsdóttir

first: Hanxi

last: Li

first: Kamilla

middle: K

first: Carlos

last: Rodriguez-Pardo

first: Rasmus

last: Friis

first: Seder

last: Islam

first: Peter

last: Heegaard

first: Klaus

last: Qvortrup

first: Hinrich

last: Abken

first: Yi

last: Sun

first: Sine

last: Hardup


title: Menin inhibitor DS-1594b drives differentiation and induces synergistic lethality in combination with Venetoclax in acute myeloid leukemia cells with rearranged MLL1 and NPM1 mutations

city: Oxford

first: Catherine

last: Chahrour

address: MRC Weatherall Institute of Molecular Medicine

country: United Kingdom

institute: University of Oxford

summary: MLL1 (KMT2A) rearrangements and NPM1 mutations are associated with acute leukemias whose pathogenesis is critically influenced by protein-protein interactions between menin and MLL1. We hypothesized that targeting the menin-MLL interaction using DS-1594b and targeting the antiapoptotic factor BCL-2 using venetoclax (ABT-199) may promote differentiation and enhance lethality in patients with MLL1-rearranged and NPM1-mutated leukemias. We treated acute myeloid leukemia (AML) cell lines with MLL1 rearrangements, NPM1 mutations, and no mutations and samples from patients with primary AML with ABT-199 alone, DS-1594b alone, and their combination. We measured proliferation, viability, apoptosis, and differentiation using several assays, Western blotting, and BH3 profiling. Treatment with DS-1594b and ABT-199 exerted significant synergy, resulting in enhanced differentiation and inhibited proliferation across several cell lines. In the PDX model, DS-1594b single-agent treatment significantly extended survival (pv=***). Compared with DS-1594b monotherapy, the combination of DS-1594b and ABT-199 more profoundly reduced leukemic burden and prolonged mouse survival (pv=***). Overall, we observed synergistic effects on differentiation induction and proliferation inhibition, both in vitro and in vivo. Menin inhibition is the primary driver of transcription changes in this model, and impact on the expression of antiapoptotic regulators provide mechanistic explanation for the synergy observed between these drugs. Together, this underscores the promise of this combination strategy as a novel therapeutic approach for improving treatment outcomes in patients with these specific genetic alterations.

overall_design: RNA-seq profiling of PDX spleen cells harboring NPM1c, DNMT3A and FLT3-ITD mutations treated with vehicle control, ABT-199, DS-1594b, and ABT-199 plus DS-1594b.

first: Valerio

last: Ciaurro

first: Vassilena

last: Sharlandjieva

first: Anna

last: Skwarska

first: Natalia

last: Baran

first: Zeng

last: Zhihong

first: Cassandra

last: Ramage

first: Naval

last: Daver

first: Maria Paola

last: Martelli

first: Thomas

last: Milne

first: Marina

last: Konopleva


title: Establishment of a simple, efficient, and scalable method to generate oocyte-like cells in vitro

city: Kofu

first: Mizuki

last: Sakamoto

country: Japan

institute: University of Yamanashi

summary: Understanding the molecular basis of oocyte identity and function is essential not only for basic biology but also for clinical applications, as it is closely linked to female infertility. However, technical challenges remain in advancing this understanding, mainly due to the difficulty of obtaining a sufficient number of oocytes. In this study, we established a two-dimensional culture method that efficiently generates oocyte-like cells, referred to as mini-oocytes, from mouse embryonic stem cells. This method requires minimal labor and leverages a transcription factor-mediated approach for oocyte-like cell generation. Our analyses revealed significant similarities between in vitro-derived mini-oocytes and in vivo oocytes. Furthermore, we demonstrated the utility of mini-oocytes for investigating oocyte-specific molecular features through a small-scale knockout screen targeting the subcortical maternal complex. Given the simplicity, efficiency, and scalability of the mini-oocyte induction method, it offers a practical alternative for conducting experiments that are otherwise challenging with in vivo oocytes.

overall_design: Total RNA-seq were performed using mini-oocytes.

first: Takashi

last: Ishiuchi

first: Ami

last: Banno


title: Gasdermin E is Dispensable for H1N1 Influenza Virus Pathogenesis in Mice

city: Columbus

first: Jacob

last: Yount

state: OH

address: 460 w 12th Ave.

department: Department of Microbial Infection and Immunity

institute: The Ohio State University College of Medicine

summary: Targeting cell death pathways, including pyroptosis and necroptosis, has been shown to mitigate influenza virus infection severity. Here, we examined whether pyroptosis specifically driven by the pore-forming protein gasdermin E (GSDME) is involved in regulating influenza virus infection outcomes. We found that Gsdme-/- mice showed similar weight loss and survival in severe A/PR/8/34 (H1N1) virus infections compared to WT counterparts. Likewise, lung dysfunction, histopathological damage, viral titers, and inflammatory cytokine levels were similar in the two groups. Global transcriptomic analysis also revealed similar gene expression programs in WT versus Gsdme-/- mouse lungs at baseline and in response to infection. To confirm the generality of these findings, we infected mice with 2009 pandemic H1N1 virus and again observed similar weight loss, mortality, and lung dysfunction in WT and Gsdme-/- mice. Our results overall demonstrate that GSDME contributes negligibly to the host response against H1N1 influenza virus, refining our understanding of cell death pathways in influenza pathogenesis.

overall_design: Bulk RNAseq performed on the lungs of WT and Gsdme-/- mice at day 7 post infection with 50 TCID50 PR8 (H1N1) intranasally.

first: Speaks

last: Samuel

first: McFadden

last: Matthew

first: Forero

last: Adriana

first: Yount

last: Jacob


title: Quantifying Transcription Factor Specificity with Advanced DNA Universal Microarrays Featuring Long and Modified Binding Sites [974k]

first: Yuval

last: Bayer

overall_design: The design is of probes which include a single de Bruijn of order 12. Layout is one chamber of 1824x534 probes. Each of all non-palindromic 10-mers is contained in at least 35 probes.

first: Ariel

last: Afek


title: Smart RNA Sequencing of hepatic Regulatory T Cells from mice with autoimmune hepatitis

city: Wuhan

first: Han

last: Wang

state: Hubei

address: 1095 Jiefang Avenue

department: Department of Gastroenterology,

institute: Tongji Hospital, Tongji Medical College,

summary: Autoimmune hepatitis (AIH) is characterized by progressive liver inflammation, often associated with the dysfunction of regulatory T (Treg) cells within the liver microenvironment. To investigate the potential role of Treg dysregulation in AIH progression, we established an AIH mouse model and utilized Foxp3-GFP reporter mice to isolate liver-resident Treg cells through flow cytometric sorting. Subsequently, we performed Smart RNA sequencing to comprehensively profile the transcriptomic landscape of these cells. Our analysis provides novel insights into the molecular mechanisms underlying Treg dysfunction in AIH and offers potential targets for therapeutic intervention.

overall_design: To explore the involvement of Treg cells in the progression of autoimmune hepatitis (AIH), we developed a murine AIH model using well-established protocols. Foxp3-GFP reporter mice were employed to enable the precise identification and sorting of liver-resident Treg cells via fluorescence-activated cell sorting (FACS). Sorted Treg cells were subjected to Smart RNA sequencing, a method optimized for low-input RNA samples, to obtain high-resolution transcriptomic data. This approach facilitated the identification of key genes, pathways, and molecular signatures associated with Treg dysfunction in the context of AIH.

first: Dean

last: Tian

first: Mei

last: Liu


title: Single-nucleus multiomics reveals the regulatory programs in three brain regions of sporadic early-onset Alzheimer's Disease

city: Houston

first: Zhongming

last: Zhao

state: Texas

address: 7000 Fannin St.

institute: The University of Texas Health Science Center at Houston

summary: Sporadic early-onset Alzheimer’s Disease (sEOAD) represents a significant but less-studied subtype of Alzheimer’s Disease. In this study, we generated a single-nucleus multiome atlas from over 70,000 nuclei derived from the postmortem prefrontal cortex, entorhinal cortex, and hippocampus of four sEOAD patients and five control donors. Comprehensive analyses were conducted to delineate cell type-specific transcriptomic changes and linked candidate cis-regulatory elements (cCREs) across these brain regions. We identified and prioritized seven conservative transcription factors in glial cells and neurons in multiple brain regions, including RFX4 in astrocytes and IKZF1 in microglia, which are implicated in the regulation of sEOAD-associated genes. Moreover, we investigated altered intercellular signaling between glial cells and neurons, highlighting their regulatory potential on gene expression in receiver cells. Finally, we reported an enrichment of late-onset Alzheimer’s Disease risk loci within microglial cCREs linked to sEOAD-associated genes. This atlas enhances our understanding of the transcriptional and chromatin dynamics in sEOAD and provides a foundational resource for further pathological investigations.

overall_design: Nuclei were isolated from postmortem prefrontal cortex (PFC,n=9), entorhinal cortex (EC, n=6), and hippocampus (HIP, n=5) of sprodic early-onset Alzheimer's Disease patient (n=4) and matched controls (n=5). The isolated nuclei were then subjected to single-nuclei multiome sequcing. ll tissues were de-identified under The Health Insurance Portability and Accountability Act of 1996 (HIPAA) privacy rules. The tissue donation consent was obtained from all participants by UTHealth.


title: Bulk RNA-seq of primary tumor biopsy samples from prostate adenocarcinoma patients

first: Baek Gil

last: Kim

summary: This dataset contains bulk RNA sequencing data from primary tumor biopsy samples of patients with prostate adenocarcinoma. TPM-normalized expression values are provided along with sample metadata. The data were generated to support transcriptomic profiling of human prostate tumors and are related to a companion single-cell RNA-seq dataset from the same cohort.

overall_design:
RNA was extracted from fresh primary tumor biopsy samples of prostate cancer patients using the Maxwell CSC RNA FFPE kit. cDNA libraries were prepared using the SureSelect XT HS2 RNA kit and sequenced on the Illumina NovaSeq 6000 platform in paired-end mode (2×101 bp). After quality control (FASTQC), trimming (Trimmomatic), alignment (STAR, GRCh38), and quantification (RSEM), TPM-normalized expression values were generated.

***************************************************************
No raw data files: RNA sequencing data derived from human prostate biopsy samples would be submitted, due to patient privacy concerns.
***************************************************************

first: Hyunho

last: Han

first: Jongsoo

last: Lee

first: Jihee

last: Jung

first: Ayoung

last: Yoo

first: Jungchan

last: Jun

first: Dongwook

last: Song

first: Yeonsue

last: Jang

first: Baek


title: BAF60A KO RNA seq (primary cells)-Highly discriminative globin gene activation by the non-canonical BAF chromatin remodeling complex

status: Public on Nov 18 2025

first: Eugene

last: Khandros

address: 3615 Civic Center Blvd.

department: Hematology

institute: The Children's Hospital of Philadelphia

summary: The regulation of beta-like globin genes, including the switch from fetal (HBG) to adult (HBB and HBD) genes has served as a paradigm of developmental transcriptional control. While HBG repressors such as BCL11A and LRF have been well characterized, recent discoveries of HIF1a and BGLT3 as HBG-specific activators have raised interest in finding new activators as potential therapeutic targets. The ATP-dependent chromosome remodeler BRG1 has previously been demonstrated as necessary for hemoglobin gene transcription. In loss of function studies in adult-like HUDEP2 cells, we found that BRG1 depletion preferentially decreased fetal HBG and the minor adult HBD. BRG1 forms multisubunit complexes with combinations of 27 subunits in 3 families - canonical BAF, polybromo BAF, and non-canonical BAF (ncBAF) - to regulate chromatin accessibility and gene transcription in a cell and condition-specific manner. Using CRISPR loss of function studies in HUDEP2 cells and primary adult erythroid cells, we identified the non-canonical BAF complex as a positive regulator of HBG and HBD. Loss of the non-canonical BAF complex subunits BRD9 and BAF60A/SMARCD1 preferentially decreased HBG and HBD in HUDEP2 cells and primary adult erythroid cultures, while accelerating erythroid differentiation and hemoglobinization. Pharmacological depletion of BRD9 led to acute decreases in chromatin accessibility at HBD and HBG promoters and reduced their primary transcripts, while longer treatment recapitulated  genetic ablation effects. This surprising demonstration of BAF complex selectivity within a multi-gene cluster extends our understanding of globin gene transcriptional regulation and identifies a potential target for therapeutic manipulation of beta-like globins.

overall_design: We performed RNA seq analysis in 3 healthy donor erythroid cells derived from CD34+ HSPC after BAF60A knockout. Total RNA was isolated form erythoid cells after 10 days of erythroid differentiation targetted with CRISPR control sgRNA (AAVS1 targetting) or BAF60A targetting. Transcriptome changes were analysed by bulk RNA-seq after ribosomal RNA removal.

first: Abhirup

last: Bagchi

first: Sindhu

last: Billakanti

first: Vanessa

last: Shehu

first: Kathryn

last: Farrell

first: Sarah

last: Cotteta

first: Bailey

last: Godfrey

first: Micaela

last: Alpert

first: Jacob

middle: F

last: Zeranski

first: Krista

last: Budinich

first: Zhuangzhuang

last: Geng

first: Osheiza

last: Abdulmalik

first: Belinda

last: Giardine

first: Cheryl

last: Keller

first: Ross

last: Hardison

first: Junwei

last: Shi


title: SCOPE-C Reveals the Key Role of Long-Range Loop Extrusion in Dynamic Enhancer Networks for Robust Gene Activation in Human Fetal Cortical Neurogenesis

city: Beijing

first: Zejia

last: Cui

address: Yiheyuan Road

institute: Peking University

summary: The chromatin in the human brain cortex exhibits a higher number of enhancer-enhancer (E-E) contacts compared to macaques and mice. However, the specific organization of these E-E contacts across different cellular states, as well as the mechanisms for associating these dynamic enhancer interaction networks with particular genes, remain poorly understood. Here, we present SCOPE-C, a cost-effective technique designed for mapping DNase I Hypersensitive Sites (DHSs) and enriching their spatial interactions, with a remarkable capacity to handle samples as small as 1 to 1,000 cells. Employing SCOPE-C, we generated a comprehensive chromatin map detailing cis-regulatory elements (CREs) in the developing fetal cortex across human, macaque, and mouse species, utilizing data from Radial Glia (RG), Intermediate Progenitor Cells (IPCs), Excitatory Neurons (ENs), and Interneurons (INs) collected at similar mid-gestation stages. This map reveals dynamic enhancer interaction hubs and their target genes during cortical neurogenesis. We find that loop extrusion profoundly shapes these dynamic interaction networks, forming 'stripes' that can extend up to 10Mb in ENs and exhibit species-specific dynamics. These EN-gained interaction networks are abundantly enriched with human-specific enhancers and SNPs associated with neuropsychiatric disorders, predominantly regulating marker genes of ENs, suggesting a dual role for these enhancers: they not only upregulate gene expression but also play a crucial role in reinforcing the robustness of gene activation. Our findings introduce a dynamic model of gene activation specific to human neurogenesis, orchestrated by loop extrusion and involving species-specific enhancers, providing new perspectives on human brain development, evolution, and neurodevelopmental disorders.

sample_organism:
Macaca mulatta
Homo sapiens
Mus musculus

overall_design: SCOPE-C (simultaneously capture open-chromatin and conformation) by high throughput sequencing

first: Lumeng

last: Jia

first: Yingping

last: Hou


title: Next Generation Sequencing Facilitates Quantitative Analysis of B73 and Zmcryq mutant in dark or blue light treat Transcriptomes

city: Shenzhen

first: Zhao

last: Zhiwei

state: Guangdong

address: 1066, Xueyuan Rd

department: College of Life Sciences and Oceanography

institute: Shenzhen univerisity

summary: Analysis of differential gene expression regulated by Blue light. The hypothesis tested in the present study was that ZmCRYs mediate mostly Blue light regulated genes' expression

overall_design: mRNA profiles of 7 days of B73 and Zmcryq seedlings  grown in longday white light, pretreated with 48h dark, then moved to blue light for 1 hour or kept in dark were generated by deep sequencing, in triplicate, using Illumina Hiseq6000

first: Zhiwei

last: Zhao

first: Hongtao

last: Liu


title: Differentiation-induced reduction in functional diversity restricts the ability of cytomegalovirus-specific CD8 T cells to eliminate virus-infected cells

city: Hannover

first: Ahmed

last: Hassan

address: Carl-Neuberg-Straße 1

country: Germany

institute: Hannover Medical School

summary: Individuals with expanded CD8 T cells recognizing the pp65-HLA-A*02:01–restricted viral epitope NLVPMVATV (NLV-T cells) have weakened immune control of human cytomegalovirus (HCMV) reactivation. Here, we characterized NLV-T cells from healthy HCMV-positive donors, dividing them into two groups: those with low and those with high NLV-T cell frequencies (LF and HF, respectively). Our comprehensive multimodal analysis revealed that NLV-T cells from HF donors preferentially exhibit a phenotype of advanced differentiation, characterized by granzyme B and perforin expression. Although these cells were more efficient in eliminating peptide-loaded target cells than NLV-T cells from LF donors, the latter were more potent in cytokine secretion and the elimination of HCMV-infected cells with virus-induced HLA class-I downregulation. Overall, these findings suggest that HCMV exploits CD8 T cell differentiation to evade immune protection and explain the previously observed decline in HCMV reactivation control in individuals with NLV-T cell accumulation.

overall_design:
For single cell RNA seq: PBMCs were thawed and either used for staining directly (HF donors) or CD8+ T cells were enriched using magnetic activated cell sorting (MACS) (LF donor samples). The PBMCs from HF donors and the enriched CD8+ T cells from LF donors were stained with NLV-peptide loaded MHC class I tetramers for 15 minutes at 37°C, followed by staining for extracellular markers, Totalseq antibodies, and cell-hashing antibodies (Table 4) for 25 minutes at RT. The samples were washed and up to 8 samples were pooled and cells were sorted for CD19-, CD14-, CD16-, CD56-, CD3+, CD8+, NLV-tetramer+ cells on a Becton-Dickinson FACSAria III Fusion or Becton-Dickinson FACSAria IIu cell sorter.
For bulk RNA and TCR sequencing: The NLV-T cells were expanded via NLV-peptide stimulation for 9 days and frozen. For sequencing, the cells were thawed and the NLV-T cells were isolated with NLV-loaded MHC class I streptamers.

first: Lea

last: Fritz

first: Berislav

last: Čuvalo

first: Bibiana

last: Costa

first: Britta

last: Wieland

last: Eiz-Vesper

first: Christine

last: Falk

first: Lennart M

last: Roesner

first: Thomas

last: Werfel

first: Thilo

last: Dörk-Bousset

first: Ulrich

last: Kalinke

first: Hristo

last: Georgiev

first: Reinhold

last: Förster

last: Bošnjak


title: Bi-directional regulation between NAD/NAMPT and IFN-γ/PD-L1 axes via BRD4/IRF1 and mitochondrial respiration in metastatic cutaneous melanoma

city: Alessandria

first: Valentina

last: Audrito

address: Viale Teresa Michel 11

department: DIST

country: Italy

institute: University of Eastern Piedmont

summary: Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme in NAD biosynthesis and is frequently upregulated in tumors, including melanoma, where it drives disease progression and resistance to targeted therapies. Beyond its metabolic role, NAMPT also exerts immune-modulatory functions, acting as a cytokine-like mediator that shapes tumor–host interactions and influences the efficacy of immunotherapy. Building on recent evidence indicating a connection between the pro-inflammatory cytokine interferon-gamma (IFN-γ) signaling and NAD metabolism, we sought to explore this poorly understood relationship in melanoma context. Analysis of the TCGA melanoma cohort revealed a significant correlation between NAMPT expression and IFN-γ signaling, including specific genes belonging to this pathway, such as IRF1, STAT1, and CD274/PD-L1. The correlation between NAMPT and PD-L1 was also confirmed at the protein level in more than 100 cases included in a melanoma tissue microarray (TMA). Functionally, IFN-γ treatment (100ng/ml for 24h) of three MM cell lines led to increased NAMPT transcription and intracellular expression, as well as increased release. Molecularly, as previously demonstrated for IFN-γ-inducible PD-L1, also NAMPT expression upon IFN-γ signaling activation is regulated at epigenetic/transcriptional level via bromodomain protein BRD4/IRF1 complex, as revealed using BRD4 inhibitors and chromatin immunoprecipitation (ChIP)-RT-PCR experiments. In the second part of our results, we showed a bidirectional regulation between NAD/NAMPT axis and IFN-γ signaling. NAMPT inhibition, as well as genetic silencing, reduced IFN-γ signaling activation, as highlighted by RNAseq data and confirmed by biochemical validation. This reciprocal regulation involved the modulation of mitochondrial respiration, specifically mitochondrial Complex I. Analysis of the melanoma TCGA cohort revealed a significant correlation between NAMPT expression, IFN signaling and several mitochondrial Complex I subunit genes. Consistently, activation of IFN-γ signaling in melanoma cell lines induced an increase in oxygen consumption rate, which was completely abrogated by NAD/NAMPT-dependent inhibition of complex I activity, indicating a direct metabolic crosstalk.

overall_design: Overall, these findings highlight a novel reciprocal regulatory loop between NAMPT/NAD axis and IFN γ signaling, linking NAMPT-dependent metabolic reprogramming and immune regulation.

first: Irene

last: Fiorilla

first: Lidia

last: Avalle


title: Inhibition of Acetyl-CoA metabolic enzymes by EVT0185 impairs hepatic stellate cell activation and reverses MASH and fibrosis in mouse models [nanostring]

city: Hamilton

first: Russta

last: Fayyazi

state: ON

address: 1280 Main St W

department: Medicine

country: Canada

institute: McMaster University

summary: Metabolic Dysfunction-Associated Steatohepatitis (MASH) is characterized by liver steatosis and inflammation which lead to fibrosis following hepatic stellate cell (HSC) activation. Acetyl-CoA is fundamental for de novo lipogenesis (DNL) and cholesterol synthesis and can be derived from citrate via ATP citrate lyase (ACLY), or from acetate via acetyl-CoA synthetase (ACSS2). Here, we find that EVT0185, an ACLY and ACSS2 inhibitor, leads to resolution of MASH and fibrosis while lowering insulin and glucose in four distinct mouse models. EVT0185 also reduces fibrosis alone and in combination with semaglutide and more effectively than bempedoic acid in a model not characterized by steatosis or insulin resistance. Spatial transcriptomic and scRNAseq reveal marked downregulation of cholesterol synthesis in HSCs with EVT0185 treatment in vivo and in vitro. These data indicate a critical role for ACLY and ACSS2 in HSCs and suggest further clinical evaluation of EVT0185 for reducing MASH and fibrosis may be warranted.

overall_design: Liver RNA from Vehicle (n = 8) and EVT0185-treated (100 mg/kg; n = 8) MASH mice was analyzed using the NanoString nCounter Fibrosis Panel (760 genes). Data were normalized in nSolver 4.0 using global geometric mean normalization, with pathway scores computed from normalized expression values. The dataset therefore includes 16 total samples (8 biological replicates per group) with one treatment group and one vehicle control group for comparative analysis.

first: Fiorella

last: Di Pastena

first: Jaya

last: Gautam

first: James

last: Lally

first: Gregory

last: Steinberg


title: Biologic roles of mRNA N6-methyladenosine (m6A) in high L-2-hydroxyglutarate renal cancer

city: Birmingham

first: Chen

last: Huang

state: AL

address: 720 20th St S

institute: University of Alabama at Birmingham

summary: The oncometabolite L-2-hydroxyglutarate (L-2HG) is commonly elevated in the most prevalent form of renal cancer.  Oncometabolites can alter the epigenome by disrupting the activities of enzymes that mediate both DNA and histone demethylation.  L-2HG has also been shown to inhibit the enzymatic activity of the RNA demethylases ALKBH5 and FTO.  Both enzymes demethylate the N6-methyladenosine (m6A) mark, the most prevalent RNA modification within mRNA.  We performed an integrative analysis on the role of m6A in high L-2HG renal carcinoma.  On a global level, L-2HG-induced m6A promotes reduced transcript levels consistent with the well-established role of m6A in reducing mRNA stability.  Ablation of METTL3, a key component of the m6A methyltransferase complex, led to bimodal effects on the transcriptome.  METTL3 activity promoted expression of genes related to interferon signalling.  In contrast, METTL3 suppressed the expression of genes related to cell cycle, specifically the G2-M phase, with concomitant effects on proliferation and cell cycle progression.  Furthermore, m6A is associated with splicing events including retained introns, not previously implicated in tumor cells.  Our data provide new insights into the functional role of m6A in renal cancer which have implications for both intrinsic tumor cell biology and the immune response.

overall_design: RNA-seq profiling of control 786-O cell lines and METTL3 shRNA knockdown cell lines.

first: Madhuparna

last: Pandit

first: Lakshmi Bhargavi

last: Paruchuru

first: David

last: Crossman

first: Han-Fei

last: Ding

first: Anirban

last: Kundu

first: Sunil

last: Sudarshan


title: Transcriptomic analysis of SW620-conditioned medium-treated THP-1 macrophages

city: Taipei

first: Yung-Chi

last: Chang

address: Rm 736, No. 1, Sec. 1, Jei-Ai Rd

country: Taiwan

institute: National Taiwan University, College of Medicine

summary: Siglec-5 and Siglec-14, expressed on myeloid cells, share ligand-binding domains but have opposing signaling functions. While these paired receptors regulate macrophage immune responses against bacterial infection, their role in the tumor microenvironment remains unclear. We found that Siglec-14 expression in THP-1 macrophages, when exposed to CRC tumor-conditioned medium, induces a protumoral SPP1+ tumor-associated macrophage phenotype, with enrichment of angiogenic pathways.

overall_design: RNA-seq profiling of human monocytic THP-1 cells engineered to express either an empty vector (EV/THP-1), human Siglec-5 (S5/THP-1), or human Siglec-14 (S14/THP-1) were differentiated with PMA and then treated with SW620-conditioned medium for 48 h

first: Lin

last: Kuan-Yu

first: Chang

last: Yung-Chi


title: The Underlying Dermal Scaffold Affects Molecular Differentiation Markers in Three-Dimensional Human Epidermal Equivalents

city: New Haven

first: Ryland

last: Mortlock

state: Connecticut

address: 15 York Street, HRT 618C

institute: Yale University

summary: We undertook a study to compare two commonly used dermal scaffolds for three-dimensional human epidermal equivalents (HEEs): contracted collagen (CC) and acellular dermis (ACD). Using the same population of primary keratinocytes grown at an air-liquid interface in each model, we demonstrated improved basal keratinocyte morphology and compartmentalization of basal and suprabasal keratin markers in the ACD model. Using whole-transcriptome sequencing, we found that proliferation markers were more highly expressed in the ACD model at day seven and this may be explained by increased expression of key transcription factors which govern epidermal self-renewal,  DNMT1, TP63, and CEBPD.

overall_design: RNAseq of human keratinocytes grown at an air-liquid interface on contracted collagen matrix at day 3, 7, and 14 (in duplicate) and grown on acellular dermis at day 3 and 7 (in triplicate).

first: Michael

last: Zhao

first: Lilianne

last: Sutton

first: Keith

last: Choate


title: BRD9 inhibition as potential treatment option for testicular germ cell tumors

city: Bonn

first: Aylin

last: Hansen

address: Venusberg-Campus 1

department: Pathology, Developmental Pathology

institute: University Hospital Bonn

summary: Testicular germ cell tumors (TGCT) are the predominant tumor in younger males. Usually, curation rates are quite high, but 15-20 % of patients with metastatic non-seminomas are resistant to standard cisplatin-based therapy. Interfering with the epigenetic landscape has already been shown to be effective in prostate cancer. BRD9 is an epigenetic reader which is part of the SWI-SNF chromatin-remodeling complex involved in regulation of gene expression. Alternative treatment options for therapy-resistant TGCT patients need to be investigated. BRD9 seems to be a promising target for analyzing the effect of BRD9 inhibition in TGCT cell lines. BRD9 expression was analyzed by meta-analysis of microarray data as well as by Western Blot and immunohistochemistry of tissue microarrays in TGCT cell lines and TGCT tissues. Viability was assessed by performing XTT-assay to determine the effect of BRD9 inhibition in TGCT cell lines. FACS analysis was used to display changes in cell cycle distribution as well as apoptosis. The impact on transcriptome level of BRD9 inhibition was analyzed by 3’mRNA-sequencing. BRD9 was heterogeneously expressed in TGCT cell lines and tissues. Nevertheless, inhibition of BRD9 led to strong decrease in viability. I-BRD9 induced apoptosis as well as cell cycle arrest in G1-phase. On transcriptome level, prominent downregulation of pluripotency markers (NANOG, PRMD14 and KLF4) and upregulation of genes involved in epithelium development were detected. I-BRD9 treatment of GCT cell lines reduced viability, induced apoptosis and cell cycle arrest while control cells remain mainly unaffected. Transcriptomic data indicate exit of pluripotency and differentiation towards the epithelial fate. In fact, loss of pluripotency and differentiation seems to be a common aspect of GCT reacting to drug application. The data implicate I-BRD9 as a possible treatment alternative for TGCTs.

overall_design: Total RNA was isolated from cell lines (2102 EP and TCam2) treated for 24 hours with I-BRD9 or DMSO as control. In total 12 samples with n=3 for each condition were obtained.

first: Kai

last: Funke

first: Christine

last: Sanders

first: Florian

last: Fronhoffs

first: Glen

last: Kristiansen

first: Hubert

last: Schorle


title: Resident mesenchymal progenitor cells require autocrine IGF-I for  function in homeostatic and regenerating skeletal muscle

city: GAINESVILLE

first: FAHONG

last: YU

state: FL

address: 2033 Morway RD

department: ICBR

institute: UNIVERSITY OF FLORIDA

summary: Fibro-Adipogenic Progenitors (FAPs) are mesenchymal progenitor cells vital for muscle homeostasis and regeneration, but they also produce fibrosis and differentiate into intramuscular fat under pathological conditions. Insulin-like Growth Factor-I (IGF-I), a key regulator of muscle repair, influences satellite cell activity, macrophage polarization, and extracellular matrix (ECM) remodeling. Although FAPs are a major source of IGF-I in muscle, its specific role remains unclear. Thus, we generated inducible FAP-specific Igf1 knockout mice (FID mice) to determine the necessity of the FAP IGF-I source. After acute BaCl2 injury, FID mice exhibited impaired muscle regeneration, characterized by fewer Pax7+ cells, increased CD68+ macrophage accumulation, smaller fibers with lower proportions of EmbMHC+ fibers, and reduced ECM content compared to controls. FAP proliferation was impaired, with fewer total and Ki67+ FAPs in regenerating muscles from FID mice. Following glycerol injury, muscles from FID mice exhibited reduced adipocyte accumulation. To probe the autocrine role of FAP IGF-I, we isolated FAPs from muscles 3 days post-injury. Primary FAPs isolated from FID mice had blunted growth, accompanied by upregulation of immune-regulatory genes and downregulation of ECM and cell proliferation genes by RNASeq. Additionally, cultured FID FAPs had delayed differentiation and sustained PDGFRα expression in response to TGF-β, as well as impaired adipocyte differentiation in adipogenic media. Finally, FID FAPs entered a senescent state shortly after Cre recombination, showing high β-Gal expression in vivo and in vitro, and suppression of IGF-IR and IGF-I-dependent pathways. Taken together, our findings demonstrate that IGF-I derived from FAPs is a critical autocrine factor, with its absence leading to impaired function of FAPs for maintenance of muscle regenerative capacity

overall_design: In this study, we hypothesized that FAP IGF-I is a necessary autocrine factor for FAP behavior.  We investigated the significance of IGF-I secreted from FAPs on skeletal muscle properties, including regenerative capacity, as well as FAP proliferative and differentiation capacity, using a mouse model that afforded FAP specific deletion of Igf1

first: Yangyi

last: Luo

first: Zoe

last: Abe-The

first: T

middle: Y

last: Alsaghir

first: Liying

last: Kuo

first: Fahong

last: Yu

first: Bradley

last: Ambili Bai Appu

first: Daniel

last: Kopinke

first: Elisabeth

last: Barton


title: Secretory kinase FAM20C triggers adipocyte dysfunction inciting insulin resistance and inflammation in obesity

city: New York

first: Ankit

last: Gilani

state: New York

address: 413E 69th Street

institute: Weill Cornell Medical College

summary: Obesity is a major driver of type 2 diabetes (T2D) and related metabolic disorders, characterized by chronic inflammation and adipocyte dysfunction. However, the molecular triggers initiating these processes remain poorly understood. We identify FAM20C, a serine/threonine kinase, as an early obesity-induced mediator of adipocyte dysfunction. Fam20c expression is substantially upregulated in adipocytes in response to obesity, correlating with a proinflammatory transcriptional signature. Forced expression of Fam20c in adipocytes promotes robust upregulation of proinflammatory cytokines and induces insulin resistance that is dependent on its kinase activity. Conversely, deletion of adipocyte Fam20c after established obesity and hyperglycemia improves glucose tolerance, augments insulin sensitivity, and reduces visceral adiposity, without altering body weight. Phosphoproteomic studies reveal that FAM20C regulates phosphorylation of intracellular and secreted proteins, modulating pathways critical to inflammation, metabolism, and extracellular matrix remodeling. We identify FAM20C-dependent substrates, such as CNPY4, whose phosphorylation contributes to proinflammatory adipocyte signaling. Of translational relevance, we show that in humans visceral adipose FAM20C expression positively correlates with insulin resistance. Our findings establish FAM20C as an early regulator of obesity-induced adipocyte dysfunction and systemic metabolic impairment. Our studies provide proof of concept that inhibition of FAM20C may serve as a potential therapy for T2D by restoring adipocyte health.

overall_design: RNA-Seq profiling of primary differentiated adipocytes from control and adipocyte-specific Fam20c KO mice.


title: Nox4 inhibitor treatment ameliorates cardiac function in mouse models of Duchenne Muscular Dystrophy

city: Tampa

first: Da-Zhi

last: Wang

address: 560 Channelside Dr.

institute: University of South Florida

summary: Duchenne Muscular Dystrophy (DMD) is caused by mutations in dystrophin, leading to degeneration and weakness of the skeletal and cardiac muscles. Despite numerous efforts in developing therapies for this devastating disease, DMD remains a fatal disease. Our recent study showed that loss of the cardiac Isl1-interacting protein (CIP), which interacts with dystrophin in sarcolemma of cardiomyocytes, accelerates the progression of dystrophic cardiomyopathy. We identified Nox4 as one of the downstream mediators of this process. Here, we report that Setanaxib, a Nox1/4 inhibitor, protected the heart of CIP/Mdx double knockout (dKO) mice from heart failure and reduced cardiac fibrosis. Similarly, Setanaxib treatment exhibited lower cardiac fibrosis and protection against heart failure in Mdx/Utrn dKO mouse, another murine model for dystrophic cardiomyopathy. Additionally, treatment with Nox4 inhibitor reduced the expression of cardiomyopathy genes in dystrophic mice. Interestingly, transcriptomic analysis of the hearts from Mdx/Utrn dKO mice treated with Nox4 inhibitor reveals an enrichment of genes associated with fatty acid metabolism, oxidative phosphorylation, and protein binding, while genes related to inflammation and epithelial-mesenchymal transition were downregulated. Consistent with these results, we found increased expression of Pgc1a and Tcap in the hearts of Mdx/Utrn dKO mice treated with Nox 4 inhibitor, and reduced expression of Mcp1. Collectively, these findings suggest that Nox4 plays a key role in development of myocardial fibrosis and heart failure caused by dystrophin deficiency, and suggest that Nox4 inhibition could be a potential novel therapy to alleviate cardiomyopathy in DMD patients.

overall_design: In order to characterize the role of Nox4 in dystrophic cardiomyopathy, Mdx/Utrn dKO male mice (2 to 3-month-old) were daily treated with Setanaxib (60 mg/kg/day) or vehicle (corn oil) via oral gavage for 12 weeks. After the treatment, mice were euthanized and the hearts were collected for transcriptomic analysis.

first: Rui

last: Deng

first: Tabatha

last: De Oliveira Silva

first: Xiang

last: Liu

first: Cheng

last: Fan

first: Hee

last: Young Seok

first: Jingjing

last: Wu

first: Haipeng

last: Guo

first: Tian

last: Lin

first: Shiju

last: Zhang

first: Nihan

last: Gunay

first: Yao Wei

last: Lu

first: Zhan-Peng

last: Huang

first: John

last: Mably

first: Mingxiang

last: Teng

first: Hong

last: Chen

first: Gabriela

middle: P

last: Diniz


title: Effect of neuropeptide substance P on gene expression in the hippocampal tissue of mice after fecal microbiota transplantation

first: Jing

last: Lan

address: No. 2 Yuanmingyuan West Road, Haidian District

institute: China Agricultural University

summary: To explore how SP affects the function of the hippocampus mediated via intestinal flora, we performed RNA-seq analysis on the hippocampus of mice subjected to FMT treatment.

overall_design: The mice were divided into the FMT-CON group, the FMT-DSS group, and the FMT-DSS+SP group. After the fecal microbiota transplantation treatment, we used RNA-seq to detect gene expression in hippocampal tissue of each group.


title: APOBEC3B enhances the efficacy of PARP inhibitors in elimination of ovarian cancer stem cell

city: LA JOLLA

first: QINGFEI

last: JIANG

state: CA

address: 2880 TORREY PINES SCENIC DR

institute: UNIVERSITY OF CALIFORNIA SAN DIEGO

summary: Late detection and tumor recurrence are major factors driving the lethality of high-grade serous ovarian carcinoma (HGSOC). PARP inhibitors (PARPi) have achieved significant clinical efficacy by selectively targeting DNA repair deficiencies in HGSOC patients with BRCA mutations and homologous recombination deficiency (HRD). However, a subset of patients ultimately develops resistance to PARPi, necessitating alternative effective treatment options. The mutational signatures of APOBEC3 family of DNA deaminases are widespread across a broad array of cancer types. Here, we report that cancer stem cell (CSC)-like tumorspheres exhibit reduced A3B expression compared to non-CSC adherent counterparts. Importantly, inhibition of A3B leads to PARPi resistance, elevated frequency of CSCs, and enhanced expression of stemness factors. In addition, we found that high A3B-expressing cells are under strong replication stress and thus synchronize efficiently with PARPi. In this study we knocked down of A3B in A2780 3D tumorsphere by lentiviral tranduction, followed by treated of DMSO control or Olaparib, and performed bulk RNA-sequencing to compare gene expression changes (n=3 for each condition).

overall_design: RNA-sequecning of A2780 following lentiviral knockdown of A3B and the backbone control, followed by treatment of DMSO or Olaparib (n=3 for each conditon, total n=12).

first: Maria

last: Rivera

first: Lucy

first: Sabina

last: Enlund

first: Chae-Eun

last: Lim

first: Haoran

first: Kaifu

last: Yang

first: Roman

last: Sasik

first: Leslie

last: Crews

first: Kathleen

last: Fisch

first: Ramez

last: Eskander

first: Frida

last: Holm

first: Qingfei

last: Jiang


title: Nascent strand sequencing of U2OS cells

city: La Jolla

first: Xiaohua

last: Wu

address: 10550 N Torrey Pines Rd

institute: Scripps Research

summary: In order to analyze the relationship between the replication fork direction and the repair products at a specific genomic sites, we performed nascent strand sequencing of U2OS cells to mapping strong origins in genome, which can indicate the replication fork direction of a specific site adjacent to the origin.

overall_design: U2OS cells were synchronized with double thymidine block at G1/S. 2 hours after removing thymidine, nascent strand DNA was isolated from cells released into S phase, size fractionated, and subjected to lambda exunuclease digestion and library constrction.


title: Complex interplay between FMRP and DHX9 during DNA replication stress

city: Syracuse

first: Wenyi

last: Feng

address: 750 East Adams Street

department: Biochemistry and Molecular Biology

institute: SUNY Upstate Medical University

summary: Mutations in, or deficiency of, fragile X messenger ribonucleoprotein (FMRP) is responsible for the Fragile X syndrome (FXS), the most common cause for inherited intellectual disability. FMRP is a nucleocytoplasmic protein, primarily characterized as a translation repressor with poorly understood nuclear function(s). We recently reported that FXS patient cells lacking FMRP sustain higher level of DNA double-strand breaks (DSBs) than normal cells, specifically at sequences prone to forming R-loops, a phenotype further exacerbated by DNA replication stress. Moreover, expression of FMRP, and not an FMRPI304N mutant known to cause FXS, reduced R-loop-associated DSBs. We subsequently reported that recombinant FMRP directly binds R-loops, primarily through the carboxyl terminal intrinsically disordered region. Here, we show that FMRP directly interacts with an RNA helicase, DHX9. This interaction, which is mediated by the amino terminal structured domain of FMRP, is reduced with FMRPI304N. We also show that FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids and the inhibition is also dependent on the amino terminus. Furthermore, the FMRPI304N mutation causes both FMRP and DHX9 to persist on the chromatin in replication stress. These results suggest an antagonistic relationship between FMRP and DHX9 at the chromatin, where their proper interaction leads to dissociation of both proteins from the fully resolved R-loop. We propose that the absence or the loss of function of FMRP leads to persistent presence of DHX9 or both proteins, respectively, on the unresolved R-loop, ultimately leading to DSBs. Our study sheds new light on our understanding of the genome functions of FMRP.

overall_design: HEK293T cells containing FMR1 KO and expressing CMV-EGFP-FMRP (wild type or I304N mutant) were used for ChIP-seq for FMRP or DHX9. Before chromatin immunoprecipitation,, cells were treated with 0.6 or 1 micromolar aphidicolin (APH), with DMSO or no treatment as controls.  Input DNA (whole genomic DNA) for each sample was used as control for library construction.

first: Rosemarie

last: Daoud


title: Single-cell RNA-seq of undirected human cerebral organoids (HuCOs) infected with MV1 or MV2 compared with uninfected controls at 120 and 180 days post-infection

first: cathryn

last: haigh

state: MT

address: 903 S 4th St

institute: NIAID

summary: Undirected human cerebral organoids (HuCOs) derived from induced pluripotent stem cells (hiPSCs) were used to model neural development and infection response in vitro. Organoids were maintained in spinning bioreactors and divided into three experimental groups: MV1-infected, MV2-infected, and uninfected control (NBH). Each infection group was harvested at two developmental stages, approximately 120 and 180 days post-infection (dpi), following five months of infection. Single-cell RNA-seq libraries were prepared using the 10x Genomics Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 and sequenced on an Illumina NovaSeq 6000 platform. The study aimed to characterize infection-induced transcriptional and cellular changes across time points and between infection subtypes. This dataset provides a high-resolution single-cell transcriptomic resource to explore cellular heterogeneity and host–pathogen interactions in undirected human cerebral organoids.

overall_design: Human cerebral organoids (HuCOs) derived from hiPSCs were generated under undirected differentiation conditions and maintained in spinning bioreactors. Organoids were assigned to three experimental groups: MV1-infected, MV2-infected, and uninfected control (NBH). Samples were collected at two time points, 120 and 180 days post-infection (dpi), yielding a total of 16 single-cell RNA-seq libraries (8 per time point). Single-cell suspensions were prepared from each organoid and processed with the 10x Genomics Chromium Next GEM Single Cell 3′ Reagent Kits v3.1. Libraries were sequenced on an Illumina NovaSeq 6000 to generate paired-end reads. Data were processed with Cell Ranger v7.1 (10x Genomics) using the GRCh38 reference genome and analyzed with the Loupe Browser for clustering and differential expression. Comparative analyses included: (1) NBH vs MV1 vs MV2 at 120 dpi; (2) NBH vs MV1 vs MV2 at 180 dpi; (3) 120 vs 180 dpi within NBH, MV1, and MV2 groups.

first: Catheryn

last: Haigh

first: Bradley

last: Groveman

first: James

last: Carroll

first: Craig

last: Martens

first: Justin

last: Lacks

first: Amirhossein

last: Shamsaddini

first: Paul

last: Gardina


title: An immunocompetent human iPSC-derived multicellular Alveolus-on-Chip reveals early pathological events of M. tuberculosis infection

city: London

first: Maximiliano

last: Gutierrez

state: --- Select One ---

address: 1 Midland Road

institute: Francis Crick Institute

summary: Immunocompetent and experimentally accessible alveolar systems to study human respiratory diseases are lacking. Here, we combined cells derived from a single induced pluripotent stem cell (iPSC) source with microfluidic devices that mimic lung 3D mechanical stretching and air-liquid interface. This single donor iPSC-derived Lung-on-Chip (iLoC) combines four alveolar cell types, including alveolar epithelial cells Type II and I, vascular endothelial cells, and macrophages. A combination of imaging and scRNA-seq analysis revealed that the iLoC recapitulated cellular profiles of distal lung cells present in the human lung including a range of AT2-to-AT1 cellular states and alveolar macrophages. We show that the presence of an endothelial barrier in the iLoC impacted tissue immunity in both the epithelium and macrophages. Different from 2D cellular models, infection of iLoC with the human pathogen Mycobacterium tuberculosis (Mtb) showed that both infected macrophages and epithelial cells were infected but not permissive to bacterial replication. Stochastically, large macrophage clusters containing necrotic core-like structure and Mtb replication were observed. Building a genetically engineered autophagy deficient iLoC (GE-iLoC), we found that after Mtb infection, macrophage cell death was elevated upon ATG14 deficiency with a reduced endothelial barrier integrity. Altogether, we report the first of its kind genetically tractable human alveolar model to study infections in human distal lungs.

overall_design: Human iPSC were differentiated into alveolar epithelial cells, vascular endothelial cells and alveolar macrophages and assemble on a microfluidic device to build a lung on chip. The experiment was design to test the significance of vascular endothelial cells and mechanical stretch, therefore the conditions: alveolar epithelium + alveolar macrophage_static (AM_S); alveolar epithelium + alveolar macrophage_breathing (AM_B);  alveolar epithelium + vascular endothelial cells + alveolar macrophage_static (LOC_S) and alveolar epithelium + vascular endothelial cells + alveolar macrophage_breathing (LOC_B)

first: Chak

middle: H

last: Luk

first: Gabriel

last: Conway

first: Irene

last: Rodriguez-Hernandez


title: Small Extracellular Vesicles From Platelet-Rich and Platelet-Poor Plasma Are Primarily From Platelets and Protect Synoviocytes From IL-1β–Induced Inflammation

city: Naples

first: Logan

last: Piening

address: 1370 Creekside Blvd

department: Orthobiologics

institute: Arthrex Inc.

type: Non-coding RNA profiling by high throughput sequencing

summary: This study sought to investigate the miRNA content of various extracellular vesicles derived from plasma fluids in order to better understand their functions in musculoskeletal injuries.

overall_design: To determine the miRNA content of extracellular vesicles derived from various plasma based devices. Compare the miRNA content between each of the groups.

first: Preston

last: Wolfe

first: David

last: Shepard

first: Lisa

last: Fortier

first: Michael

last: Baria


title: BAF60A KO ATAC seq (primary cells)-Highly discriminative globin gene activation by the non-canonical BAF chromatin remodeling complex

city: Philadelphia

first: Eugene

last: Khandros

address: 3615 Civic Center Blvd.

institute: The Children's Hospital of Philadelphia

summary: The regulation of beta-like globin genes, including the switch from fetal (HBG) to adult (HBB and HBD) genes has served as a paradigm of developmental transcriptional control. While HBG repressors such as BCL11A and LRF have been well characterized, recent discoveries of HIF1a and BGLT3 as HBG-specific activators have raised interest in finding new activators as potential therapeutic targets. The ATP-dependent chromosome remodeler BRG1 has previously been demonstrated as necessary for hemoglobin gene transcription. In loss of function studies in adult-like HUDEP2 cells, we found that BRG1 depletion preferentially decreased fetal HBG and the minor adult HBD. BRG1 forms multisubunit complexes with combinations of 27 subunits in 3 families - canonical BAF, polybromo BAF, and non-canonical BAF (ncBAF) - to regulate chromatin accessibility and gene transcription in a cell and condition-specific manner. Using CRISPR loss of function studies in HUDEP2 cells and primary adult erythroid cells, we identified the non-canonical BAF complex as a positive regulator of HBG and HBD. Loss of the non-canonical BAF complex subunits BRD9 and BAF60A/SMARCD1 preferentially decreased HBG and HBD in HUDEP2 cells and primary adult erythroid cultures, while accelerating erythroid differentiation and hemoglobinization. Pharmacological depletion of BRD9 led to acute decreases in chromatin accessibility at HBD and HBG promoters and reduced their primary transcripts, while longer treatment recapitulated  genetic ablation effects. This surprising demonstration of BAF complex selectivity within a multi-gene cluster extends our understanding of globin gene transcriptional regulation and identifies a potential target for therapeutic manipulation of beta-like globins.

overall_design: We performed ATAC seq analysis in 4 HUDEP-2 clonal lines generated using single cell cloning of a pool of HUDEP-2 cells. ATAC-seq was conducted following the Omni-ATAC protocol (https://www.nature.com/articles/nmeth.4396). In brief, 100,000 cells underwent lysis using a buffer containing 0.1% NP-40, 0.1% Tween-20, and 0.01% digitonin, followed by incubation with Tagment DNA Enzyme (Illumina cat # 20034197) at 37°C for 30 minutes. DNA was subsequently purified using the QIAGEN MinElute Reaction Cleanup Kit. For library preparation, fragmented DNA was amplified with Phusion High-Fidelity PCR Master Mix supplemented with HF Buffer (Thermo Fisher Scientific, catalog no. F531S) and indexed primers featuring unique dual barcodes. Libraries were purified with AMPure XP beads (Beckman Coulter, catalog no. A63881) at a 1:1 ratio, adhering to the manufacturer’s guidelines.

first: Abhirup

last: Bagchi

first: Sindhu

last: Billakanti

first: Vanessa

last: Shehu

first: Kathryn

last: Farrell

first: Sarah

last: Cotteta

first: Bailey

last: Godfrey

first: Micaela

last: Alpert

first: Jacob

last: Zeranski

first: Krista

last: Budinich

first: Zhuangzhuang

last: Geng

first: Osheiza

last: Abdulmalik

first: Belinda

last: Giardine

first: Cheryl

last: Keller

first: Ross

last: Hardison

first: Junwei

last: Shi


title: Epigenetic changes by EZH2 inhibition increase translocations in B cells with high AID activity or DNA repair deficiency [LAM-HTGTS for MEC-1 cell]

city: Boston

first: Roberto

last: Chiarle

state: Ma

address: Longwood 333

institute: Boston Childrens Hospital

summary: The EZH2 histone methyltransferase inhibitors tazemetostat and valemetostat have recently received approval for clinical use in follicular lymphoma and adult T-cell leukemia/lymphoma, respectively. In follicular lymphoma, the expression of AID is responsible for increased mutational signatures and genomic instability. Because EZH2 inhibitors induce epigenetic and transcriptional changes in normal and lymphoma B cells, we studied whether these inhibitors could alter the pattern of AID-mediated chromosomal translocations. Here we show that treatment with EZH2 inhibitors did not significantly change AID expression or AID-dependent chromosomal translocation frequency when used as monotherapy in either CH12F3 mouse B cells or MEC-1 human B cells. In contrast, when combined with PI3Kδ inhibition, which enhances AID expression, EZH2 inhibition significantly increased the frequency of chromosomal translocations compared to either EZH2 or PI3Kδ inhibition alone both in mouse CH12F3 cells and human MEC-1 cells. EZH2 inhibition also further enhanced translocation formation in mouse B cells that were Ligase4 deficient. Mechanistically, EZH2 inhibition in B cells depletes the repressive histone modification H3K27me3 while concurrently enhancing the active histone modification H3K27ac, thereby selectively increasing transcriptional activity and facilitating chromosomal translocation formation in the presence of high AID activity or Ligase4 deficiency. These findings highlight the impact of drugs that induce epigenetic changes to influence chromosomal translocations and demonstrate the genetic safety of EZH2 inhibitors as monotherapy while highlighting the increased risk of genomic instability when used in cells prone to translocations, such as B cells with high AID levels or DNA-repair deficiency.

overall_design: We performed LAM-HTGTS to assess chromosomal translocation alterations following treatment with tazemetostat or valemetostat in MEC-1 cells.

first: Jianli

last: Tao

first: Luca

last: Alessandri


title: Deoxyguanosine Kinase Deficiency Couples Purine Metabolism to Innate Immune Activation and Lipid Accumulation in Hepatocytes

first: Sonia

last: Sharma

address: 9420 Athena Cir

department: Center for Autoimmunity and Inflammation

institute: La Jolla Institute of Immunology

summary: Mitochondrial DNA depletion syndromes (MDS) encompass a heterogeneous set of metabolic disorders caused by defects in enzymes responsible for maintaining mitochondrial nucleotide pools and genome integrity. Among these, deoxyguanosine kinase (DGUOK) acts within the mitochondrial purine-salvage pathway and loss-of-function mutations give rise to DGUOK deficiency, a severe hepatocerebral form of MDS marked by liver failure, neurodevelopmental impairment, and systemic metabolic inflammation. Although the clinical manifestations of DGUOK deficiency have been primarily ascribed to defective mitochondrial DNA (mtDNA) replication, some patients exhibit hepatic steatosis and inflammation despite preserved mtDNA content, suggesting that DGUOK deficiency may deregulate additional metabolic and immune pathways. Here we show that DGUOK depletion reprograms hepatocellular metabolism and innate immune signaling through a purine-dependent mechanism operating independently of mtDNA depletion. In Human hepatocellular carcinoma (HEPG2) hepatocytes subjected to siRNA-mediated DGUOK silencing, mitochondrial architecture and respiration remained intact but cells exhibited pronounced lipid droplet accumulation and a robust cell-intrinsic innate immune type I interferon response. Bulk RNA sequencing revealed widespread transcriptional reprogramming, including upregulation of human endogenous retroviruses (HERVs) and interferon-stimulated genes (ISGs), suppression of lipid metabolic pathways, and changes in purine, methionine, and methylation-associated gene networks. Perturbing purine homeostasis through deoxyadenosine (dAdo) supplementation in wild-type cells phenocopied DGUOK disruption, causing cellular DNA hypomethylation and activation of viral mimicry pathways. Together, these findings identify DGUOK as a central regulator of the purine-regulated lipid-immune axis in hepatocytes, demonstrating that mitochondrial nucleotide salvage preserves hepatic immune and metabolic homeostasis beyond its canonical role in mtDNA synthesis. By linking purine imbalances to steatosis and type I interferon activation, this study establishes a mechanistic framework for immunometabolic pathology in DGUOK deficiency.

overall_design: Two conditions: siControl and siDGUOK. n = 3 independent biological replicates per condition (total 6 libraries). Cells were cultured under identical conditions and processed in parallel. RNA was collected 48 h after transfection. Libraries were prepared and sequenced together to minimize batch effects. Differential expression contrasts were pre-specified (siDGUOK vs siControl).

first: Maija

last: Corey

first: Jeamin

last: Jung


title: Transcriptomic response of a genetically modifed Escherichia coli strain at higher growth temperatures

city: Cuernavaca

first: Gilberto

last: Pérez Morales

state: Morelos

address: Avenida Universidad 2001, Colonia Chamilpa

department: Cellular Engineering Department

country: Mexico

institute: Instituto de Biotecnología - Universidad Nacional Autónoma de México

summary: These transcriptomic data are part of the proteomic analysis of thermotolerant strains, undertaken to deepen understanding of the changes acquired at the multi-omics level during adaptation to high temperatures.

overall_design: The analysis compared the transcriptomic response of two strains (a parental and thermal adapted strain) at diffent growth temperatures in mid-exponential phase culture.

last: Pérez-Morales

first: Alfredo

last: Martínez


title: JNK knockdown enhances CAR-T cell cytotoxicity through NFATc1 in preclinical models of ovarian cancer

first: Charles

middle: Joseph

last: Kuhlmann

address: 845 19th St S

department: Microbiology

institute: University of Alabama at Birmingham

summary: Background: Boosting the performance of chimeric antigen receptor T (CAR-T) cell therapy in solid tumors may provide a substantial advantage for cancer patients. Recognizing the vital role of the nuclear factor of activated T cells (NFAT) in T cell function, we hypothesized the strategic regulation of NFAT activity by targeting c-Jun N-terminal Kinases (JNK) can bolster the tumor-eradicating potential of CAR-T cells. Methods: We developed a lentivirally encoded short-hairpin RNA (shRNA) for stable knockdown of JNK in CAR-T cells. CAR-T cells targeting human epidermal growth factor receptor 2 (HER2) were produced from human peripheral blood. Functionality was tested in vitro and in two xenograft models of human ovarian cancer. Results: JNK knockdown in CAR-T cells suppressed antigen-induced stimulation and helper T cell cytokine production, while enhancing anti-tumor cytotoxicity in vitro and in ovarian cancer xenograft experiments. Mechanistically, JNK knockdown altered NFAT signaling to facilitate NFATc1-dependent transcription, leading to elevated levels of granzyme B expression. Conclusions: JNK signaling is a significant regulator of CAR-T cell cytotoxicity, offering a potential strategy to directly enhance CAR-T effectiveness in human cancer therapies.

overall_design: CAR-T cells were manufactured from peripherial blood T cells of healthy human donors. CAR vectors encoded shRNA targeting JNK1/2 or a scrambled sequence. CD8+ CAR-T cells were purified, then stimulated with plate-bound HER2-Fc protein for 16 hours. RNA was isolated using Direct Zol RNA Miniprep Plus kit (Zymogen #R2050). RNA was sequenced after poly-A enrichment on the NovaSeq X plus platform (6 gigabytes per sample, paired end reads) through Novogene corporation (Sacramento, California).

first: Chloe

last: Jepson

first: Madison

middle: T

last: Blucas

first: Fatema

last: Suleiman

first: Anusha

last: Manda

first: Yoshiko

last: Kamata

first: Masakazu


title: Antibody-Mediated Targeting of Secretory Protein SCUBE3 Suppresses Cancer Progression by Inhibiting Oncogenic Signaling and Inducing Anti-tumor Immunity

city: San Antonio

first: Yidong

last: Chen

address: 8403 Floyd Curl Drive, MSC 7784

department: Population Health Sciences

institute: UT Health Science Center at San Antonio

summary: Through a loss-of-function genomic screening approach, we identified Signal peptide CUB domain EGF-like 3 (SCUBE3) as a critical player for the survival and therapy resistance of cancer cells, particularly in triple negative breast cancer (TNBC).  To further understand the mechanism by which SCUBE3 may support tumor growth and metastasis, we performed gene expression analysis on MDA-MB-231 cells stably silenced for SCUBE3. Pathway analysis reveled that Myc signaling, G2M checkpoint and DNA repair-associated genes were highly downregulated, while IFN gamma, inflammatory response and immunity-associated pathways were highly upregulated in SCUBE3-silenced TNBC cells.

overall_design: MDA-MB-231 cells were transfected with lentivirus containing human shRNA (or scrambled shRNA) plasmids for SCUBE3. Transfected cells were then selected with Puromycin for a week and checked for expression of SCUBE3 by western blots. Three replicates for each group were used for RNA-seq library construction and sequenced with Illumina HiSeq 3000 system.

first: Deepika

last: Singh

first: Benjamin

middle: C

last: Onyeagucha

first: Manjeet

last: Rao

first: Zhao

last: Lai