title: Secretome analysis during human induced pluripotent stem cell-derived cardiomyocyte differentiation

city: Suita

first: Yuki

last: Saito

state: Osaka

address: Life System Building, 1-3 Yamadaoka

department: Department of Future Medicine

country: Japan

institute: Osaka Univ.

summary: Introduction: Cell transplantation therapy using human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) is a promising treatment for ischemic heart disease. While paracrine effects mediated by the secretome are recognized as a key therapeutic mechanism, the dynamic changes in the secretome profile during the manufacturing process remain poorly understood. This study aimed to comprehensively elucidate the secretome dynamics of clinical-grade hiPSC-CM to identify their "Mode of Action" (MoA) and candidate quality attributes (CQAs). Methods: We integrated multi-omics profiling-including total RNA-seq, proteome analysis, and extracellular vesicle (EV)-derived miRNA-seq-with functional assays (proliferation, cell migration, and tube formation) using samples obtained across time points of the hiPSC-CM manufacturing process (Day 4-25). Results: High-purity hiPSC-CM (>95% cTnT positive) exhibited a distinct transcriptomic maturation between Day 16 and Day 25, characterized by the upregulation of cardiac maker genes. Proteomic clustering revealed four distinct stages, showing a functional transition from proliferation-centric signaling to tissue-repair signaling. Notably, the purified Day 25 secretome showed a qualitative shift toward a PDGF and SDF-1 rich profile. Simultaneously, EVs from Day 25 were enriched with "myomiRs" (miR-133b, -208b, -499b) and multiple anti-proliferative miRNAs (let-7e-5p, miR-145-5p). Functionally, the Day 25 secretome significantly enhanced mesenchymal stem/stromal cell (MSC) migration and promoted mature, highly branched endothelial tube formation compared to earlier stages. Conclusions: The hiPSC-CM secretome undergoes a programmed evolution during manufacturing, shifting from promoting undifferentiated growth to orchestrating complex tissue repair through stable angiogenesis and MSC recruitment. These findings establish a molecular foundation for the MoA of hiPSC-CM therapy and provide critical CQAs for ensuring the potency and consistency of clinical-grade cardiac products.

overall_design: The analyzed samples consisted of total RNA extracted from cells at three stages of iPS cell–derived cardiomyocyte differentiation: the undifferentiated stage, Day 16, and Day 25.

first: Kazuaki

last: Takemura

first: Junya

last: Aoyama

first: Sawako

last: Hibino

first: Kouichi

last: Hasegawa

first: Yoshiki

last: Sawa


title: Microbiota-Derived Isovalerate Ameliorates Sex-Specific Gut Barrier Dysfunction in Malnutrition [RNA-seq colon epithelium]

status: Public on Jul 24 2026

city: Houston

first: Mary

last: Estes

state: TX

address: 1 Baylor Plaza

department: Microbiology and Virology

country: USA

institute: Baylor College of Medicine

type: Expression profiling by high throughput sequencing

summary: Malnutrition is a major global health challenge that increases intestinal permeability and susceptibility to sepsis, yet the mechanisms driving barrier dysfunction remain poorly defined. We aimed to identify how the gut microbiome and microbiota-derived metabolites regulate intestinal barrier integrity during malnutrition. We used a low-protein, low-fat diet (LPLFD) to induce malnutrition in specific pathogen-free (SPF) and germ-free (GF) mice. Colonic permeability and mucus thickness were quantified. Metabolomics identified microbiota-derived metabolites altered by malnutrition. Human colonoids were used to test mechanistic effects of candidate metabolites. Barrier restoration was evaluated following colonic administration of isovalerate or oral supplementation with its precursor amino acid, leucine. LPLFD-induced malnutrition increased colonic permeability and reduced mucus thickness in male, but not female, SPF mice. These defects were absent in malnourished GF mice, indicating a microbiota-dependent and sexually dimorphic mechanism of barrier disruption. Metabolomic analysis revealed reduced colonic levels of branched-chain fatty acids (BCFAs) in malnourished mice. Supplementation of human colonoids with the BCFA isovalerate improved barrier function and altered expression of genes associated with epithelial junctional complexes. Restoring isovalerate levels, either directly via colonic administration or indirectly through oral leucine supplementation, partially rescued barrier defects in malnourished male mice. These findings identify BCFAs, particularly isovalerate, as essential microbiota-derived regulators of intestinal barrier integrity during malnutrition. This work reveals a sex-specific, microbiota-dependent pathway of barrier dysfunction and highlights microbial metabolites as promising therapeutic targets for mitigating sepsis risk in undernourished populations.

overall_design: EXP 1: Mouse colon epithelium from control or LPLFD mice gavaged with either (1) vehicle (2) 114 mM alanine or (3) 114 mM leucine 2X/d for 2.5 d were analyzed via RNA-seq for transcriptional differences. EXP 2: Mouse colon epithelium from control or LPLFD mice gavaged with either (1) vehicle (2) 100 mM sodium isovalerate were analyzed via RNA-seq for transcriptional differences.

first: Lauren

middle: E

last: Lynch

first: Krishnakant

middle: G

last: Soni

first: Jennifer

middle: K

last: Spinler

first: Abu Hena Mostafa

last: Kamal

first: Nagireddy

last: Putluri

first: Stephanie

middle: W

last: Fowler

first: Margaret

last: Conner

first: Hoa

last: Nguyen-Phuc

first: Xi-Lei

last: Zeng

first: Sarah

last: Blutt

first: Geoffrey

middle: A

last: Preidis


title: RNA sequencing for gastric-type endocervical adenocarcinoma (GAS), usual-type endocervical adenocarcinoma (UEA), and lobular endocervical glandular hyperplasia (LEGH)

city: Nagoya

first: Kosuke

last: Yoshida

state: Aichi

address: 65 Tsuruma-cho

institute: Nagoya University

summary: Cervical cancer is the fourth most common malignancy among women worldwide. While the majority of cases are human papillomavirus (HPV)-associated squamous cell carcinomas, a subset consists of adenocarcinomas. Uterine cervical adenocarcinoma is further classified into gastric-type adenocarcinoma (GAS), usual-type endocervical adenocarcinoma (UEA), and other subtypes. Lobular endocervical glandular hyperplasia (LEGH), a non-neoplastic glandular lesion, has been proposed as a potential precursor of GAS. In this study, we investigated the gene expression profiles of GAS, UEA, and LEGH using RNA sequencing.

overall_design: Formalin-fixed, paraffin-embedded (FFPE) tissue samples collected between 2005 and 2024 at Kurume University Hospital were analyzed. Total RNA was extracted from FFPE sections, and RNA sequencing was performed by Rhelixa (Tokyo, Japan).

first: Hiroaki

last: Yamada

first: Kazuhiro

last: Suzuki

first: Akira

last: Yokoi


title: Microbiota-Derived Isovalerate Ameliorates Sex-Specific Gut Barrier Dysfunction in Malnutrition

overall_design: Human intestinal organoids (HIOs) from adult male ascending and transverse colon were treated with 10 mM isovaleric acid and were analyzed via RNA-seq for transcriptional differences.


title: Integrative spatial profiling of 3D genome organization and gene expression in complex tissue

city: philadelphia

first: Pengfei

last: Guo

state: PA

address: 422 Curie Boulevard, Stellar Chance Labs, 711-712 (lab), philadelphia

institute: University of Pennsylvania

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Three-dimensional genome organization shapes transcriptional regulation, yet measuring its spatial coordination in situ within intact tissues remains challenging. We present Spatial Hi-C-RNA, a multimodal platform that simultaneously maps genome-wide chromatin contacts and transcriptomes from the same tissue section at near single-cell resolution. Across mouse brain, developing embryos, and human melanoma, Spatial Hi-C-RNA generated multimodal maps that aligned with tissue anatomy while revealing complementary chromatin- and RNA-defined spatial patterns. Multiscale features, including A/B compartments, topologically associating domains, and chromatin loops, were associated with region- and cell-type-specific transcriptional programs. In mouse embryos, Spatial Hi-C-RNA resolved coordinated chromatin and transcriptional remodeling during neuronal maturation across developmental stages. In human melanoma, chromatin architecture delineated intratumoral subregions not detected by RNA alone and linked tumor-state transitions to changes in compartments, domain boundaries, and regulatory programs. Spatial Hi-C-RNA thus provides a broadly applicable framework for investigating genome structure–function relationships in development and disease within native tissue environments.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Spatial joint profiling of Hi-C and RNA in complex tissues

first: Yan

last: Cui

first: Yanxiang

last: Deng


title: DPDH prevents cerebral cavernous malformation progression

city: Nanjing

first: weiwei

last: Zheng

state: Jiangsu

address: Xianlin Road 138

country: China

institute: Nanjing University of Chinese Medicine

summary: Cerebral cavernous malformations (CCMs) are deemed to be common neurovascular dysplasias that are prone to hemorrhage characterized by abnormal activation of MEKK3. Current understanding of precise effects of MEKK3 on governing the transformation from initial lesions into mature and active lesions in CCMs is far from complete and whether MEKK3 could serve as a reliable target for hindering the development of CCMs still remain unclarified. In this study, we demonstrate a prominent reduction in light of the number and size of vascular lesions in the hindbrains of iECre;Krit1fl/fl;Mekk3fl/+ mice compared to that of iECre;Krit1fl/fl mice, indicating that activation of MEKK3 signaling in the endothelium yields CCM lesions, which is consistent with previous findings. More interestingly, our data further unveil that MEKK3 signaling in the microglial cells that are recruited by fibrinogen exacerbates the lesions at the late stage of CCM progression. Mechanistically, aberrant activation of MEKK3 on endothelial cells (ECs) is inclined to disrupt the endothelial junctions via regulating the MEKK3-KLF2/4 signaling axis. Meanwhile, the recruited microglial cells can phagocytize ECs by virtue of the MEKK3-NFκB signaling cascade. Therefore, manipulation of MEKK3 on both ECs and microglial cells may emerge as an effective and efficient therapeutic strategy for retarding the progression of CCMs. Leveraging artificial intelligence-assisted drug design and structural modification, we identified (R)-1,6-dimethyl-11-phenyl-1,10-dihydro-2H-furo[2',3':1,2]phenanthrol[3,4-d]imidazole (DPDH) as an optimal small-molecule inhibitor of MEKK3 enzyme activity. To better explore  the underlying mechanisms of DPDH retarding the progression of CCM lesions, we thus carried out the RNA-seq to examine the transcriptome of ECs isolated from the cerebella of P19 Krit1iECKO mice in the absence or presence of DPDH treatment

overall_design: RNA-seq profiling of ECs isolated from the cerebella of P19 Krit1iECKO mice in the absence or presence of DPDH treatment

first: Yang

last: Zhao

first: Yin

last: Lu

first: Weiwei

first: Suyun

last: Yu

first: Liwenyu

last: Chen

first: Jing

last: Ma

first: Chang


title: RNA-seq of Burkitt lymphoma cell lines Ramos and ST14 knockout Ramos

city: Taipei

first: Yi-Lin

last: Chiu

address: No. 161, Sec. 6, Minquan E. Rd., Neihu Dist.

country: Taiwan

institute: National Defense Medical Center

summary: This dataset presents RNA sequencing (RNA-seq) data from human Burkitt lymphoma cell lines, Ramos and ST14 knockout Ramos. The study aimed to investigate transcriptomic changes between parental cells and ST14 knockout cells.

overall_design: A total of 10^7 cells were incubated in RPMI medium at a concentration of 10^6 cells/mL at 37°C, and collected after 24 hours. Then cells were lysed and total RNA was extracted with QIAzol/RNeasy. Strand-specific libraries were prepared by Genewiz and and sequenced on an Illumina NovaSeq 6000 platform using 2×150 bp paired-end mode. FastQC was used for read-quality assessment, adapters and low-quality bases were trimmed, and transcript abundance was quantified against the Ensembl GRCh38 (release 114) transcriptome using Salmon v1.10.0 and kallisto v0.46.1.

first: Xin-Jie

first: Hsing-Fan

last: Lai


title: Komagataella phaffii encodes two Pho4 transcription factors: a comparative functional study under phosphate starvation and high pH stress

city: CERDANYOLA DEL VALLES

first: JOAQUIN

last: ARINO

state: Barcelona

address: ED. IBB

department: Biochemistry & Mol. Biol.

country: Spain

institute: UNIVERSITAT AUTONOMA BARCELONA

summary: The transcription factor Pho4 plays a crucial role in the response to phosphate starvation in many fungi, and it has been linked to tolerance to alkalinization of the medium. The vast majority of fungi encode a single Pho4-encoding gene. However, the genome of the industrially relevant yeast Komagataella phaffii appears to encode two possible Pho4 proteins, namely PAS-chr1-1_0265 and PASchr2-1_0177, designated here PHO4(A) and PHO4(B), respectively, which have never been functionally characterized. Phenotypic analysis of single and double mutants suggests that Pho4(B) plays a major role in adaptation to Pi scarcity. The phenotypes of single mutants do not overlap, but the double mutant is sensitive to multiple types of stress, including alkaline pH. Transcriptomic analysis under Pi starvation confirms that Pho4(B) is crucial for the typical transcriptional response (induction of PHO5, PHO89, VTC1, etc.). However, under high pH stress, induction of many of these genes becomes also PHO4(A) dependent, at least in the short-time response. Notably, expression of both PHO4(A) and PHO4(B) allows growth of a S. cerevisiae pho4 deletion mutant in the absence of phosphate. These results suggest that both transcription factors are functional in K. phaffii, although their functions are only partially overlapping, that these genes can respond differently to diverse stimuli, and that together are a key component in the adaptation to a variety of stresses.

overall_design: There are two kind of experiments reported here: 1) Comparison of mRNA levels by RNA-Seq between Komagataella phaffii yeast cells grown on YP-glycerol at pH 5.5 (reference) and 8.0 (alkaline pH) for wild-type  strain and cell mutated for PAS-chr1-1_0265 and PASchr2-1_0177 (designated here PHO4(A) and PHO4(B)) transcription factors, as well as for the double pho4(A) pho4(B) mutant. Samples were taken after 0, 15, 30 and 60 min upon alkalinization of the medium and total RNA was prepared. 2) The same wild-type and mutant cells were grown for 24 h in synthetic medium with glycerol as carbon source and with or without 7.35 mM KH2PO4 and total RNA was prepared (phosphate starvation).

first: Marcel

last: Albacar

first: Antonio

last: Casamayor

first: Asier

last: González

first: Joaquín

last: Ariño


title: CDK8/19 inhibition suppresses adaptive resistance to CDK4/6 inhibitors in vitro and in vivo

city: COLUMBIA

first: HAO

last: JI

state: SC

address: 715 SUMTER ST CLS RM 109

department: College of Pharmacy

institute: University of South Carolina

summary: CDK4/6 inhibitors have become a standard of care for estrogen receptor-positive breast cancer and are being developed for other malignancies. However, resistance to these drugs readily develops, limiting their impact on patient survival. Mechanisms of resistance to CDK4/6 inhibition involve multiple changes in gene expression, which may be followed by genetic alterations. We have investigated the process of tumor cell adaptation to CDK4/6 inhibitors and the impact of selective inhibitors of CDK8/19 Mediator kinases, broad-spectrum regulators of transcriptional reprogramming, on such adaptation. The addition of CDK8/19 inhibitors (SNX631/SNX631-6) prevented the development of CDK4/6 inhibitor resistance in different cell lines and in vivo tumor models with varying levels of CDK4/6 inhibitor sensitivity. RNA-seq analysis revealed specific transcriptomic changes associated with adaptation to palbociclib in vitro and in vitro. Combining palbociclib with CDK8/19 inhibitors suppressed many of the adaptation-associated changes in gene expression. The findings indicate a potential therapeutic benefit of combining CDK4/6 and CDK8/19 inhibitors.

overall_design: To investigate the transcriptomic changes underlying adaptation to palbociclib and its prevention by CDK8/19 inhibition, three separate RNA-Seq experiments were conducted using MCF-7 cells: a short-term (ST) in vitro study, a long-term (LT) in vitro study, and an in vivo (IV) study using tumors excised at the end of the in vivo experiment. In the ST study, RNA was collected after 72 hours of treatment with 0.1% DMSO (vehicle control), 500 nM SNX631, 500 nM palbociclib, or a combination of 500 nM SNX631 and 500 nM palbociclib. This experiment aimed to characterize the early transcriptional effects of palbociclib and how these are modulated by CDK8/19 inhibition. The LT study analyzed RNA from ten independently adapted cell populations treated with 500 nM palbociclib for 28 days, along with five replicates of 0.1% DMSO-treated controls.  The IV study allowed for the assessment of long-term effects in tumors (4-5 per treatment group), particularly those associated with the development of in vivo palbociclib resistance and its prevention by SNX631-6 after nearly 80 days of treatment.        MCF-7 cells (5x10^6 cells in 0.1 mL 50% Matrigel per animal) were inoculated in female NSG mice subcutaneously in the side flank. Once tumors reached ~150 mm^3, mice were randomized into groups with similar-size tumors for treatment with SNX631-6 alone, palbociclib alone, SNX631-6 and palbociclib combination, or equivalent vehicle control. 50 mg/kg palbociclib isethionate in H2O was administered p.o. daily for 5 days, followed by a 2-day drug holiday. 30 mg/kg SNX631-6 in 70% PEG400 / 30% PG was administered p.o. q.d. At the endpoint for each tumor, tumors were dissected from euthanized animals using sterilized surgical tools. Pieces of non-necrotic tumor tissues 50~100mm^3 were excised at the margin near midline section for RNA extraction. At least 4 biological replicates (xenograft tumors from different animals) per study group. Data were analyzed separately for tumor (human) and stromal (mouse) genes.

first: Zachary

middle: T

last: Mack

first: Hao

last: Ji

first: Mengqian

first: Igor

middle: B

last: Roninson

first: Eugenia

middle: V

last: Broude


title: Transcriptional activity during seed imbibition is conserved, robust and required for germination. [imb_temp]

city: Warsaw

first: Michal

last: Krzyszton

address: Pawinskiego 5a

country: Poland

institute: Institute of Biochemistry and Biophysics, Polish Academy of Sciences

summary: In most plants, germination begins with the reactivation of metabolism during water uptake by the desiccated seed. Notably, Pol II transcription is hypothesised not to be required for early germination, as seeds appear to initially rely on stored mRNAs produced during their maturation. Here, we show that efficient blocking of Pol II transcription completely stops germination in Arabidopsis seeds. Pol II activity is initiated early and is widespread, following expression and Pol II binding patterns established during seed drying. Our findings suggest that the earliest transcriptional phase relies on pre-existing protein factors. The transcriptional programme initiated during water uptake is robust across diverse imbibition conditions and in seeds matured at different temperatures, and is conserved in rapeseed (Brassica napus). Only after the successful completion of this initial phase and the passing of the translation-dependent checkpoint do seeds activate genes that lead to germination. Our results redefine the role of transcription in seed germination and highlight a conserved pattern of early transcriptional activation.

overall_design: mRNAs were labelled with 4-SU during 3h seed imbibition at different temperatures. 3'RNA-seq was performed for enriched labelled and input samples.

first: Szymon

last: Swiezewski


title: The U1 snRNP protein U1C and Helix H of U1 snRNA are critical for small molecule splicing modulator function

city: Bridgewater

first: Wencheng

last: Li

state: NJ - New Jersey

institute: PTC Therapeutics

summary: Risdiplam and branaplam are two small molecule splicing modulators that enhance the ability of U1 snRNP to recognize non-canonical GA/GU-containing 5’ splice sites (ss). We demonstrated that branaplam improves the recognition of these 5’ ss by reconstituted U1 snRNP in vitro and that this effect is dependent on the ZnF domain of U1C and Helix H of U1 snRNA but not U1A or U1-70K. We then showed in vivo depletion of U1C decreases the potency of most compound-induced cassette exons. However, many cassette exons only become responsive to compound when U1C is depleted, leading to a model where U1C stabilizes different context-dependent structural conformations of U1 snRNP which either promotes or hinders compound binding and its effect on splicing. Surprisingly, risdiplam has no effect on the recognition of these 5’ ss in vitro, suggesting additional cellular factors are required.

overall_design: Two RNAseq datasets in HEK293 cells. (1) U1C siRNA and Negative control siRNA treated cells with compound branaplam or risdiplam at 5 doses each (duplicates) or DMSO (n=4). (2) U1-GA variant, 4 different U1-GA helixH variants treatment and mock control treatment.

first: Lanqing

last: Ying

first: Brian

last: Kosmyna

first: Kerstin

last: Effenberger

first: Jairo

last: Sierra

first: Scott

middle: J

last: Barraza

first: Christopher

middle: R

last: Trotta


title: Transcriptional activity during seed imbibition is conserved, robust and required for germination. [CHX]

overall_design: mRNAs were labelled with 4-SU during 3h and 6h seed imbibition in the presence of cycloheximide or mock. 3'RNA-seq was performed for enriched labelled and input samples.


title: Transcriptional activity during seed imbibition is conserved, robust and required for germination. [alpha]

overall_design: Time course of Arabidopsis seeds imbibition in the presence of mock or alpha-amanitin analysed using 3'RNA-seq.


title: Transcriptional activity during seed imbibition is conserved, robust and required for germination. [maturation_temp]

overall_design: mRNAs were labelled with 4-SU during 3h imbibition of seeds that had matured at different temperatures. 3'RNA-seq was performed for enriched labelled RNAs, input samples, and dry seed samples.


title: Transcriptional activity during seed imbibition is conserved, robust and required for germination.  [napus]

overall_design: mRNAs were labelled with 4-SU during 4 and 8h imbibition of Brassica napus seeds. 3'RNA-seq was performed for enriched labelled RNAs, input samples, and dry seed samples.


title: Transcriptional activity during seed imbibition is conserved, robust and required for germination. [ABA and ANT]

overall_design: mRNAs were labelled with 4-SU during 6h seed imbibition in the presence of ABA, antabactin or mock. 3'RNA-seq was performed for enriched labelled and input samples.


title: Proteolytic cleavage of Tie1 governs vascular response to inflammatory stimulations

city: Mannheim

first: Lorna

last: Wessels

address: Ludolf-Krehl-Straße 13-17

country: Germany

institute: European Center for Angioscience

summary: We here show that proteolytic cleavage of the Tie1 receptor evokes a vascular response to inflammatory stimulations such as TNFalpha and LPS. Resisting Tie1 cleavage reprogrammed EC gene networks, rendering them non-responsive to inflammatory stimulations.

overall_design: RNA-seq profiling of lung endothelial cells isolated from Tie1-CR (cleavage resistant) mice, in which Tie1 is resistant to proteolytic cleavage upon inflammatory insults, and control Tie1-WT (wild type) mice

first: Miki

last: Kamiyama

first: Jingjing

last: Shi

first: Yanzhu

last: Lin

first: Yifang

last: Mao

first: Xiaowen

last: Zhang

first: Ashik Ahmed

last: Abdul Pari

first: Stephanie

middle: F

last: Preuss

first: Marlene

last: Hoffarth

first: Asmaa

last: Zahran

first: Carleen

last: Spegg

first: Maria

last: Riedel

first: Donato

last: Inverso

first: Roxana

last: Ola

first: Junhao

last: Hu

first: Julio

last: Saez-Rodriguez

first: Kari

last: Alitalo

first: Hellmut

last: Augustin

first: Mahak

last: Singhal


title: RNA profiles in extracellular vesicles from severe sepsis patients reveal pathogen-specific immune signatures in meningococcal versus pneumococcal infections

city: Oslo

first: Berit

middle: Sletbakk

last: Brusletto

address: Kirkeveien 166

department: Oslo University Hospital

country: Norway

institute: Medical Biochemisty

summary: This study is the first to investigate and compare RNA profiles in plasma extracellular vesicles (EVs) isolated from patients with severe sepsis caused by Neisseria meningitidis and patients with systemic infections due to Streptococcus pneumoniae. In some cases, invasive pneumococcal disease can resemble meningococcal infections at the time of hospital admission. Blood samples from a 1980s epidemic in Norway were used to isolate EVs and characterize their RNA content by microarray technology. The results revealed both shared and distinct RNA characteristics between the two patient groups, with 1,798 shared transcripts. Key findings included significant changes in mRNA transcripts associated with the S100 family signaling pathway and the AQP9 gene across all patient groups.  RNA types varied across patient groups based on filtering criteria. The levels of RNA subtypes such as mRNA, piRNA, lncRNA, lincRNA, tRNA, miRNA, YRNA, and snoRNA differed among the groups. Notably, the meningococcal septic shock group exhibited the highest transcript levels (5,927), significantly surpassing both the pneumococcal disease group (2,341) and the meningococcal meningitis group (390). Specific piRNA transcripts were identified in both meningococcal and pneumococcal patient groups, suggesting potential roles in immune modulation. Additionally, long non-coding RNAs (lncRNAs) exhibited expression patterns that may indicate immunosuppression, particularly in systemic pneumococcal disease.  The study also compared plasma EV-RNA profiles from patients with meningococcal sepsis at hospital admission to cellular RNA profiles from post-mortem large organ tissue samples of patients with lethal Neisseria meningitidis septic shock, revealing a striking similarity in RNA profiles. This highlights the role of EVs in modulating immune responses during severe sepsis and indeed pave the way for extracellular vesicles´ role in communication during sepsis. The differences and similarities in RNA profiles suggest that EVs may reflect different on going systemic pathophysiological states, providing new insights for personalized treatment strategies for sepsis in the future.

overall_design: Plasma extracellular vesicles (Evs) from plasma samples from : Meningococcal septic shock (MSS) patients (N= 6). Meningococcal meningitis (MM) (N=6). Systemic pneumococcal disease patients (SPD) (N=6). Healty Controls ( N=3) from blood donors

middle: S

last: Haug

first: Mari

last: Kaarbø

first: Ole

last: Olstad

first: Trude

last: Aspelin

first: Erik

last: Amundsen

first: Petter

last: Brandtzaeg

first: Reidun

last: Øvstebø


title: Comprehensive transcriptomic profiling of cortical and striatal regions in a postnatal mouse model of attention-deficit/hyperactivity disorder

city: Charleston

first: Stefano

last: Berto

address: 68 President Street, BE208

institute: MUSC

summary: Attention-deficit/hyperactivity disorder (ADHD) is a complex neurodevelopmental disorder characterized by attentional deficits, hyperactivity, and impulsivity. Despite advances in understanding its genetic and neurobiological underpinnings, comprehensive molecular characterization, particularly at early developmental stages, remains underexplored. This study investigates transcriptomic alterations in the prefrontal cortex (PFC) and striatum (STR) of a postnatal ADHD mouse model induced by 6-hydroxydopamine (6-OHDA), which selectively eliminates dopaminergic neurons. Employing bulk RNA sequencing (RNA-seq) coupled with weighted gene co-expression network analysis (WGCNA), we identified 369 DEGs in the PFC, 493 DEGs in the STR, and 32 modules in WGCNA, uncovering distinct expression profiles associated with ADHD. Functional enrichment analyses identified that PFC-DEGs were involved in PFC development, plasma membrane signaling, and transcriptional regulation. STR-DEGs were associated with STR development, locomotion, extracellular matrix, and response to amphetamine. Notably, we identified significant overlaps between PFC- and STR-DEGs and genes linked to ADHD, autism spectrum disorders (ASD), schizophrenia (SCZ), and bipolar disorder (BD). Co-expression network analyses identified module-specific enrichments related to neurodevelopmental and cognitive functions, highlighting the roles of development, synaptic signaling, metabolism, immunity, and transcription in the pathophysiology of ADHD. These findings demonstrate the intricate gene expressions and co-expression networks underlying ADHD, providing deeper insights into the molecular mechanisms.

overall_design: We investigated transcriptomic alterations in the prefrontal cortex (PFC) and striatum (STR) of a postnatal ADHD mouse model induced by 6-hydroxydopamine (6-OHDA), which selectively eliminates dopaminergic neurons. We conducted bulk RNA-seq and analyzed the data to identify DEGs and WGCNA modules, aiming to uncover the molecular mechanisms of ADHD.

first: Noriyoshi

last: Usui


title: Bulk liver RNA-sequencing of mice co-exposed to simvastatin and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

city: East Lansing

first: Tim

last: Zacharewski

state: Michigan

address: 1129 Farm Ln

department: Biochemistry and Molecular Biology, Integrative Toxicological Sciences

institute: Michigan State University

summary: Bulk RNA-sequencing of 60 liver samples from C57Bl/6NCrl males gavaged every 4 days for 84 days with 0.1 to 10 µg/kg TCDD or sesame oil vehicle, half of whom were exposed to ~70 mg/kg/day of simvastatin (n=5 per dose). Gavaging and tissue collection occurred between ZT0-3.

overall_design: Beginning on post-natal day (PND) 30, male C57BL/6 mice were repeatedly gavaged every 4 days for 84 days with 2,3,7,8-tetrachlorodibenzo-p-dioxin (0.1, 0.3, 1, 3, or 10 µg/kg TCDD) or sesame oil vehicle, half of whom were exposed to ~70 mg/kg/day of simvastatin supplemented in their chow (n = 5 per dose). Mice were sacrificed on PND 114 with CO2. Gavaging and tissue collection occurred between ZT0 and ZT3. Tissues were flash frozen in liquid nitrogen and stored at -80°C. For total RNA extraction using trizol, 100 mg was used per liver sample. Only samples which passed Nanodrop and RIN quality control were used. 150 bp paired-end RNA reads were sequenced using a NovoSeq 6000.

first: Warren

last: Sink

first: Jesmin

last: Ara

first: Rance

last: Nault

first: John

last: Lapres


title: Single cell profiling of peripheral blood mononuclear cells in diverse men with prostate cancer reveals lethal disease-associated immune signatures

city: Bethesda

first: Huaitian

last: Liu

state: MD

address: Building 37, Room 3050

department: CCR

institute: NCI

summary:
Purpose: Prostate cancer disproportionately affects men of African ancestry (AA), who also exhibit a distinct immunobiological profile. We aimed to investigate the composition and gene expression patterns of circulating peripheral blood mononuclear cells (PBMCs) in AA and European American (EA) prostate cancer patients, and to evaluate their association with ancestry and lethal prostate cancer outcomes.
Experimental Design: We performed single-cell RNA sequencing (scRNA-seq) on 273,229 high-quality PBMCs isolated from 59 prostate cancer patients and healthy controls, along with single-cell TCR and BCR sequencing (scTCR-seq and scBCR-seq) in 53 individuals of African American (AA) and European American (EA) ancestry. Immune cell subpopulations were characterized, and differential gene expression and pathway analyses were conducted to identify ancestry-related differences and signatures associated with lethal disease.
Results: Prostate cancer patients showed increased CD4⁺ cytotoxic and CD8⁺ effector memory T cells, with reduced CD4⁺ naïve and CD56 bright NK cells, indicating immune activation and dysregulation. AA patients had elevated CD4⁺ CTLs and cDC1s and fewer CD56 bright NK cells compared to EA. Lethal cases showed reduced cDC2s, suggestive of impaired immune priming.
CD16⁺ monocytes shared 98 genes and 9 pathways across race and lethality contrasts, including enhanced interferon signaling in AA patients. Inflammatory and cytokine pathways—especially TNFα and NF-κB—were enriched in AA and in lethal disease, indicating shared immune dysregulation.
TCR profiling revealed more large and hyperexpanded clonotypes in lethal cases, reflecting antigen-driven T cell responses and potential exhaustion. BCR sequencing showed reduced repertoire diversity and greater clonal dominance in lethal and EA patients, consistent with skewed humoral responses.
Conclusions: Our findings reveal ancestry- and lethality-associated immune differences in prostate cancer. Elevated T cell exhaustion and inflammatory signaling in AA patients correlated with poor outcomes, highlighting potential ancestry-informed biomarkers and targets. Expanded T and B cell clones in lethal cases suggest antigen-driven immune activation with possible dysfunction.

overall_design: Peripheral Blood Mononuclear Cells (PBMCs) were collected from prostate cancer patients from a diverse population of African American (AA) and European American (EA). IN this study we utilized single cell RNA sequencing (scRNA-seq)  to analyze differential charteristics of immune cells lanscape in those populations and their relation to develop lethal prostate cancer.

first: Alexandra

last: Harris

first: Tiffany

last: Dorsey

first: Shakir

middle: U

last: Ahmed

first: Ashlie

last: Casiano

first: Michael

last: Kelly

first: Clayton

middle: C

last: Yates

first: Stefan

last: Ambs


title: MDA5 regulates bleomycin-induced proinflammatory response of macrophages

city: Tokyo

first: Takashi

last: Ishii

address: 7-3-1, Hongo, BUnkyo-ku

institute: The University of Tokyo

summary: The role of innate immune receptors recognizing RNA and self RNA in lung inflammation and fibrosis formation is not well understood. Here, we demonstrate melanoma differentiation-associated gene 5 (MDA5) regulates bleomycin-induced lung fibrosis, accompanied by increased IL-17+ γδ T cells and neutrophils compared to wild-type (WT) mice. Lung CD11b+ macrophages, with similar characteristics of human Spp1+ macrophages, displayed unique gene expression profiles, marked by elevated levels of ligands for CCR2 and collagen production. Moreover, MDA5 was found to regulate these ligands in bleomycin-treated bone marrow-derived macrophages and U937 cells, contributing to the clearance of extracellular RNA. In conclusion, our findings suggest that MDA5 plays a pivotal role in regulating bleomycin-induced lung fibrosis through influencing the interplay among macrophages, lymphocytes, and neutrophils.

overall_design: (1) WT or MDA5 KO mice were subject to bleomycin treatemt and lung CD11b+ macrophages  were sorted after two weeks. Their RNA was extracted with Nucleospin® RNA plus kit for subsequent RNA-seq analysis. (2) Bone marrow cells derived from WT or MDA5 KO mice were collected and incubated with DMEM/F-12 containing 10% FBS, antibiotic–antimycotic mixed stock solution, and 2 mg/ml mouse recombinant M-CSF. On day 7, cells were treated with 1 μg/ml bleomycin and cells were harvested on day 9 for RNA-seq analysis.

first: Ishii

last: Takashi


title: Multi-omics analysis of FOXO3 pioneer factor activity reveals distinct networks in proliferating and DNA damage induced senescent cells

city: Providence

first: Abigail

last: Brown

state: RI

address: 70 Ship Street

department: Molecular Biology, Cell Biology, Biochemistry

institute: Brown University

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

overall_design: Refer to individual Series


title: Gene expression profile at single cell level of type 2 innate lymphoid cells (ILC2s) from human lungs

city: Beijing

first: Hongjie

last: Chen

address: No.5 Yiheyuan Road

institute: Peking University

summary: Human lung ILC2s are a rare cell population. We used single cell RNA-sequencing (scRNA-seq) to analyze gene expressions of human lung ILC2s, as the number of cells was not enough to conduct bulk RNA-seq

overall_design: Human lung ILC2s were isolated by fluorescence-activated cell sorting (FACS) according to certain markers and analyzed using scRNA-seq

first: Jing

last: Yang


title: Multi-omics analysis of FOXO3 pioneer factor activity reveals distinct networks in proliferating and DNA damage induced senescent cells [RNA-Seq]

summary: Determine how FOXO3 functions at the chromatin level to modulate aging and promote longevity.

overall_design: We performed RNA-seq analysis on the transcriptomic changes following FOXO3 activation, knockdown and induction of senescence in U87MG cells.

first: Ashley

last: Webb


title: Multi-omics analysis of FOXO3 pioneer factor activity reveals distinct networks in proliferating and DNA damage induced senescent cells [ATAC-Seq]

summary: FOXO transcription factors are critical for cellular homeostasis and have been implicated in longevity in several species. Yet how these factors directly affect aging, particularly in humans, is poorly understood. Here, we take an integrated multi-omics approach to identify the chromatin-level mechanisms by which FOXO3 coordinates transcriptional programs in replicating cells and in an aging model of senescence.

overall_design: ATAC-seq used to define accessible regions of the genome when FOXO3 is activated specifically through induction, knocked down, or activated under senescent condtions.

first: Sun

middle: Y

last: Maybury-Lewis


title: Therapy Induced Senescence Promotes Immunogenicity in Acute Myeloid Leukemia through Reduced EZH2 Activity (RNA-seq AraC)

city: Milan

first: Raffaella

last: Di Micco

address: Via Olgettina, 60

country: Italy

institute: San Raffaele Telethon Institute for Gene Therapy

summary: Chemotherapy resistance and disease relapse are major determinants of treatment failure in acute myeloid leukemia (AML). Therapy-induced senescence (TIS) is one outcome of chemotherapy, but its immunological consequences in AML remain unclear. Here we show that ex vivo chemotherapy induces senescence in a subset of therapy-naïve AML samples. TIS is marked by elevated interferon signaling, upregulation of human leukocyte antigen (HLA) class I and II molecules, and increased presentation of leukemia- and senescence-associated peptides, conferring AML cells antigen-presenting cell-like features. These changes enhance autologous CD4+ and CD8+ T cell responses against AML, both ex vivo and in patient-derived xenograft models. TIS also restores AML sensitivity to immune checkpoint blockade therapy. Mechanistically, we identify reduced Polycomb Repressive Complex 2 (PRC2) activity as central to TIS induction and its immunogenicity. PRC2 inhibition reactivates senescence-related genes and HLA expression in non-senescent AML cells, enabling T cell activation. These findings uncover a senescence-driven immune mechanism with potential to improve therapy outcomes in AML.

overall_design: RNA-seq analysis of primary AML blast treated with ARA-C collected at day 5, and compared with their paired untreated sample

first: Diego

last: Gilioli

first: Simona

last: Fusco

first: Teresa

last: Tavella


title: Multi-omics analysis of FOXO3 pioneer factor activity reveals distinct networks in proliferating and DNA damage induced senescent cells [CETCh-Seq]

summary: Determining the direct target genes of FOXO3 in human cells has been challenging. We employed the newly developed CETCh-seq method to determine the endogenous target genes of FOXO3.

overall_design: CRISPR epitope tagging ChIP-seq (CETCh-seq) of FOXO3 in U87MG cells


title: 3D epigenomic landscape of human retinal pigment epithelium

status: Public on Jul 23 2026

city: Hangzhou

first: tang

middle: dong

last: mei

address: 866 Yuhangtang Road

institute: Zhejiang University

summary:
This SuperSeries integrates transcriptomic and epigenomic data generated from human retinal pigment epithelium.

This SuperSeries is composed of the SubSeries listed below.


title: Systematic identification of age-related microbiome metabolites that impact the brain - hCMEC screen

city: Stanford

first: Meenakshi

last: Chakraborty

state: CA

address: 269 Campus Drive

institute: Stanford University

summary: These RNA-seq data were generated from immortalized hCMEC/D3 cells treated with either: (1) one of 30 blood metabolites associated with aging and the microbiome, (2) vehicle control, or (3) lipopolysaccharide (LPS), a positive control with well-characterized transcriptomic effects. For more details, please see our preprint at https://doi.org/10.1101/2025.10.03.680371.

overall_design: RNA-seq profiling of hCMEC/D3 cells following 3-hour treatment with each of 30 metabolites individually, vehicle controls, or LPS

first: Ami

last: Bhatt


title: Protein microarray analysis of ACAP1 interacting proteins

city: Shanghai

first: Jiwei

address: 130 DongAn Road

institute: Fudan University

summary: Purified ACAP1 protein was assayed on the ProtoArray Human Protein Microarray (Invitrogen) to identify potential interacting proteins.

overall_design: Flag- and V5-double-tagged ACAP1 (Flag-ACAP1-V5) fusion protein was purified from lysates of transfected HEK293T cells and applied to the ProtoArray Human Protein Microarray (v5.1, Invitrogen) according to the manufacturer's instructions. Bound ACAP1 was detected using an Alexa Fluor 647-conjugated anti-V5 antibody, and the microarrays were scanned with a GenePix microarray scanner (Molecular Devices).

first: Duanwu

last: Zhang

first: Bruce

last: Beutler


title: Distinct activation programs in naïve and memory CD8 T cells govern progeny stemness and effector persistence (single cell experiment, NupHox mice, Lm-OVA infection, Day 3 and Day 5)

city: Prague

first: Juraj

last: Michalik

address: Vídeňská 1083

country: Czech Republic

institute: Institute of Molecular Genetics of the Czech Academy of Sciences

summary: We longitudinally profiled gene expression in naive and central and effector memory CD8+ T cells across multiple models of acute cognate infection to reveal subset-specific activation programs. However, how prior antigen experience alters T-cell activation programs remains poorly understood. We longitudinally profiled gene expression in naive, central memory, and effector memory CD8+ T cells in response to cognate antigen across multiple models of acute infection. Naive T cells engaged TOX- and TCF7-centered programs and generated early stem-like central memory precursors. Their initially slow proliferation was followed by rapid expansion and the production of large numbers of effector cells. Central memory T cells rapidly triggered effector and proliferation programs while maintaining a smaller self-renewing population. Effector memory T cells expanded poorly and generated almost exclusively effector progeny. Effector progeny derived from both memory subsets survived contraction more efficiently than naive T-cell-derived progeny and established persistent effector memory populations. These data show that prior antigen experience does not simply accelerate CD8+ T-cell activation but redirects cell-intrinsic programs governing progeny fate and persistence. These distinct programs may reflect adaptation to different histories and anticipated patterns of antigen exposure.

overall_design: Here we compared different cell types (Tm, Taim and Tn) from multiple samples using single-cell analysis.

first: Eva

last: Salyova

first: Darina

last: Paprckova

first: Veronika

last: Niederlova

first: Ondrej

last: Stepanek


title: Transcriptomic reprogramming driven by nuclear and cell size perturbations via compartment-specific protein accumulation and G1 arrest in yeast and human cells

city: Zurich

first: Shin

last: Ohsawa

address: Otto-Stern-Weg 3

department: Institute of Biochemistry

country: Switzerland

institute: ETH Zurich

summary: While nuclear and cell sizes are critical determinants of cellular function, their specific impacts on gene expression remain poorly understood. To dissect the direct transcriptomic consequences of nuclear size alterations, we engineered inducible systems in budding yeast (Saccharomyces cerevisiae) that accumulate massive proteins specifically within the nucleus (3xFP-NLS) or the cytosol (3xFP-NES), alongside corresponding control systems (empty vector, 3xFP, and 1xFP-NLS). Using these systems, we profiled global transcript abundance and RNA polymerase II (Pol II) occupancy via high-throughput mRNA-seq and ChIP-seq. Furthermore, to uncouple the transcriptomic effects of general cell volume expansion from nuclear size changes, we performed mRNA-seq on a temperature-sensitive G1-arrest yeast mutant (cdc28-13) expressing the cytosolic accumulation system (3xFP-NES) under restricted temperature (37°C). Finally, to examine the evolutionarily conserved impact of excessive cell expansion in mammalian systems, we performed mRNA-seq on human retinal pigment epithelial (RPE1) cells undergoing palbociclib-induced G1 arrest over an extended time course. Collectively, our integrated cross-species multi-omics analyses uncover a fundamental, nuclear size-dependent transcriptome reprogramming mechanism.

sample_organism:
Saccharomyces cerevisiae
Homo sapiens

platform_organism:
Saccharomyces cerevisiae
Homo sapiens

overall_design: This SuperSeries encompasses multi-assay (mRNA-seq and ChIP-seq) and multi-organism (Saccharomyces cerevisiae and Homo sapiens) high-throughput sequencing datasets designed to investigate the effects of nuclear-to-cytosolic (N/C) ratio and cell volume perturbations on chromatin occupancy and gene expression. The study is structured into four overarching experimental modules: 1. Yeast mRNA-seq (Compartment-specific protein accumulation): Transcriptomic profiling of S. cerevisiae expressing inducible 3xFP-NLS, 3xFP-NES, and control constructs (empty vector, 3xFP, 1xFP-NLS) under uninduced (mock/ethanol) and induced (estradiol) conditions after 6 hours. Biological replicates: n=3 independent biological replicates per condition. 2. Yeast Pol II ChIP-seq: Genome-wide RNA Polymerase II occupancy profiling using an anti-Pol II antibody (clone 8WG16) in S. cerevisiae expressing 3xFP-NLS, 3xFP-NES, and control constructs under mock and induced conditions. Biological replicates: n=3 independent biological replicates per condition. 3. Yeast mRNA-seq (G1 arrest coupled with nuclear compression): Transcriptomic profiling of the temperature-sensitive cdc28-13 mutant carrying the inducible 3xFP-NES system across a 6-hour time course (2, 4, and 6 hours) at the restrictive temperature (37°C) under mock and induced conditions to evaluate the mitigation of excessive cell expansion-induced transcriptomic changes. Biological replicates: n=3 independent biological replicates per condition. 4. Human mRNA-seq (Palbociclib-induced G1 arrest): Transcriptomic profiling of human RPE1 cells cultured in the presence of the CDK4/6 inhibitor palbociclib to induce excessive cell volume expansion, comparing early arrest (Day 2) versus extended arrest (Day 7). Biological replicates: n=3 independent biological replicates per time point.

first: Gabriel

last: Neurohr


title: Rare variants in a PCSK9 distal-enhancer as drivers of LDL-cholesterol levels

city: Nantes

first: Antoine

last: Rimbert

address: 8 quai moncousu

country: France

institute: institut du thorax

summary: BACKGROUND. Discoveries of genetic causes of dyslipidemia have significantly advanced our understanding of lipoprotein metabolism and its implications in cardiometabolic diseases. However, a significant number of patients with dyslipidemia still have unidentified genetic causes. Leveraging insights from genome-wide association studies, where most common associated variants are located in non-coding regions, we propose that rare non-coding variants located in genomic regulatory regions may be responsible for dyslipidemia. METHODS. Using whole-genome sequencing data from the UK Biobank (n > 200,000), we performed region-based rare-variant association analyses, focusing on liver specific genomic regulatory regions (assessed by transposase-accessible chromatin assay coupled with sequencing (ATAC-seq)), to assess whether the cumulative burden of rare genetic variants (allele frequency < 0.01) in these genomic regulatory regions was associated with LDL-cholesterol levels. To validate our findings, we used an advanced cellular model of human induced pluripotent stem cells (hiPSCs) stably expressing a CRISPR-interference system. This system enables, when combined with guide-RNAs, to target regions of interest and silence regulated genes. RESULTS. We found that the cumulative burden of rare genetic variants in 18 among 53,039 ATAC-seq peaks in human liver, is significantly associated with LDL-cholesterol. We focus our proof-of-concept study on a peak located ~17 kb upstream PCSK9, strongly associated with LDL-cholesterol (p=5.85e-52), apolipoprotein-B (p=3.82e-49) and PCSK9 plasma levels (p=1.15e-67). We showed that this regulatory region is also active in hiPSCs and hiPSC-derived liver organoids. When transfected with guide-RNAs targeting this region, we showed that CRISPRi-hiPSCs present a hypermethylation of the targeted region which in terms leads to a substantial decrease in PCSK9 gene expression (-48%), intracellular protein (-65%) and secretion (-36%) (p<0.001). CONCLUSIONS. This study identifies a non-coding genetic region upstream PCSK9 where rare variants lead to decreased PCSK9 expression and lowered LDL-cholesterol. Such findings advocate for the genetic screening of non-coding regulatory regions in patients with unidentified genetic causes of extreme dyslipidemia and open novel perspectives for genetic regulatory therapeutic intervention.

overall_design: transposase-accessible chromatin assay coupled with sequencing of human induced pluripotent stem cells (hiPSCs) and of hiPSC-derived liver organoids.

first: Amandine

last: Caillaud

last: Lainé

first: Zoé

last: Begué-Racapé

first: Aurore

last: Girardeau

first: Laurine

last: Zielinski

first: Myriam

last: Dugast

first: Alice

last: Gosset

first: Estelle

last: Baron

first: Lise

last: Bray

first: Pierre

last: Lindenbaum

last: Boisseau

first: Adeline

last: Goudal

first: Julien

last: Barc

first: Mathilde

last: Di Filippo

first: Cédric

last: Le May

first: Bertrand

last: Cariou


title: MYCN sustains a progenitor-stage differentiation block in human neuroepithelial stem cells to drive ETMR-like tumourigenesis.

city: London

first: Diego

last: Chillon Pino

address: Great Ormond Street Hospital for Children NHS

department: Cancer development and genomics

country: United Kingdom

institute: UCL


title: MYCN sustains a progenitor-stage differentiation block in human neuroepithelial stem cells to drive ETMR-like tumourigenesis [NanoString miRNA]

summary: Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric brain tumours driven in most cases by amplification of the chromosome 19 microRNA cluster (C19MC), which upregulates the oncogene MYCN. Whether MYCN dysregulation alone is sufficient to produce ETMR-like tumours, independent of the broader C19MC machinery, has not been tested. Here, we show that sustained MYCN expression in human neuroepithelial stem (NES) cells, i.e. primitive neural progenitors resembling the proposed cell of origin of ETMR, is sufficient to generate ETMR-like brain tumours in vivo. NanoString nCounter miRNA profiling was used to assess microRNA expression in this MYCN-only model, which lacks C19MC amplification.

overall_design: NanoString nCounter Human v3 miRNA expression profiling (827 miRNAs, 5 mRNAs and 25 internal reference controls; CodeSet CSO-MIR3-12) of 12 samples: two NESt xenograft tumours (353486, 353490) generated by intracranial implantation of Sai2 NES cells carrying doxycycline-inducible MYCN(T58A), and NES cells cultured with doxycycline (MYCN-induced, NES ON) or without doxycycline (uninduced, NES CTRL). Each sample was profiled in three replicates (A-C). Approximately 100 ng total RNA input per reaction; endpoint digital counts.

first: Sumana

last: Shrestha

last: Chillón Pino

first: Colin

last: Kwok

first: Yura

last: Grabovska

first: Barbara

last: Martins da Costa

first: Kevin

last: Greenslade

first: Karen

last: Barker

first: Yann

last: Jamin

first: Angela

last: Hayes

first: Maximilian

last: Crane

first: Laurie

last: Liu

first: Fupan

last: Yao

first: Otis

last: Agbaimoni

first: Claire

last: Lynn

first: Thomas

last: Jacques

first: Ulrich

last: Schüller

first: Annie

last: Huang

first: Laura

last: Donovan

first: Louis

last: Chesler


title: Silver ions and nanoparticles elicit distinct patterns of stress compartmentalization in Pseudomonas aeruginosa

city: Austin

first: Jeffrey

middle: E.

last: Barrick

address: 2500 Speedway, Stop A4800

department: Molecular Biosciences

institute: The University of Texas at Austin

summary: Silver nanoparticles (AgNPs) and dissolved silver ions (Ag(aq)) both exhibit antimicrobial activity, yet the subcellular mechanisms by which bacteria respond to each form remain poorly characterized. Using whole-transcriptome analysis of sublethal silver exposures under controlled water chemistry conditions, we compared the effects of intact AgNPs (i.e., not prone to substantial bulk shedding of Ag(aq)) to Ag(aq) on Pseudomonas aeruginosa.  The results show that AgNP stress is primarily exerted in the bacterial periplasm, while Ag(aq) stress is primarily exerted intracellularly. Thus, AgNPs and Ag(aq) elicit distinct, spatially segregated stress response programs in P. aeruginosa.

overall_design: RNA-seq profilng of the effects of sublethal exposure to silver nanoparticles (AgNPs) or dissolved silver ions (ag(aq)) on Pseudomonas aeruginosa PAO1 compared to controls with no silver present (no-Ag) under controlled water chemistry conditions.

first: Kirisits

last: Mary Jo

first: Dowdell

last: Katherine

first: Barrick

last: Jeffrey


title: MYCN sustains a progenitor-stage differentiation block in human neuroepithelial stem cells to drive ETMR-like tumourigenesis [RNA-seq]

first: Diego

last: Chillon Pino

summary: Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric brain tumours driven in most cases by amplification of the chromosome 19 microRNA cluster (C19MC), which upregulates the oncogene MYCN. Whether MYCN dysregulation alone is sufficient to produce ETMR-like tumours, independent of the broader C19MC machinery, has not been tested. Here, we show that sustained MYCN expression in human neuroepithelial stem (NES) cells, i.e. primitive neural progenitors resembling the proposed cell of origin of ETMR, is sufficient to generate ETMR-like brain tumours in vivo. These tumours share methylation and transcriptional signatures with ETMR and reproduce its primitive, stem-like cellular hierarchy. MYCN-expressing tumour cells are arrested at an early neural progenitor stage, and MYCN withdrawal arrests growth and engages neuronal differentiation. Our findings identify sustained MYCN as sufficient to drive an ETMR-like developmental arrest in primitive human neural cells.

overall_design: Bulk stranded mRNA sequencing of human embryonic hindbrain-derived neuroepithelial stem (NES) cells (Sai2 line) lentivirally transduced with a doxycycline-inducible MYCN^T58A construct. Libraries were generated for the doxycycline-induced condition (NES ON) and the control condition (NES CTRL), with 3 biological replicates per condition. Bulk mRNA-seq was additionally performed on snap-frozen NESt xenograft tumours, n=5. Libraries were prepared and sequenced by BGI using DNBSEQ technology (PE100, ~30M reads/sample).

first: Sumana

last: Shrestha

last: Chillón Pino

first: Colin

last: Kwok

first: Yura

last: Grabovska

first: Barbara

last: Martins da Costa

first: Kevin

last: Greenslade

first: Karen

last: Barker


title: Defining nature, responses and drivers of regulatory- and conventional CD4+ T cells in the human term decidua

city: Leiden

first: Jannie

last: Borst

address: Albinusdreef 2

country: Netherlands

institute: Leiden University Medical Center (LUMC)

summary: To understand the nature and response of maternal CD4+ Tregs and conventional CD4+ T-cells (Tconvs) in healthy human pregnancy, we analyzed these cells in decidua and blood at term, caesarean delivery by single-cell transcriptomics and TCR sequencing.

overall_design: CD4+ T cells were isolated from decidua basalis, decidua parietalis (from placenta), maternal peripheral blood and umbilical cord blood after delivery (n=3). CD4+ T cells from different donors were pooled per tissue type. Tregs and Tconvs were flow cytometrically sorted based on cell surface phenotype per tissue type and combined in a 1 : 1 ratio. Cells from different tissues were labeled with distinct hashtag oligonucleotides to enable tracing of tissue origin and next pooled. The resulting total cell pool was subjected to scRNAseq and TCRseq.

first: Lotte

last: Verleng

first: Julia

last: Busselaar

first: Mark

last: Mensink

first: Ellen

last: Schrama

first: Sander

last: de Kivit

first: Xin

last: Lei

first: Yanling

last: Xiao


title: SLC7A5 Promotes Lung Adenocarcinoma Metastasis by Reprogramming Fatty Acid Metabolism via PPARA

city: Nanchang

first: Penghui

last: Liu

state: Jiangxi

address: No. 17 Yongwaizheng Street

institute: First Affiliated Hospital of Nanchang University

summary: Metastasis remains the leading cause of mortality in lung adenocarcinoma (LUAD), yet the molecular mechanisms driving metastatic progression, particularly to the liver, remain incompletely understood. Metabolic reprogramming has emerged as a hallmark of cancer, but how amino acid transport interfaces with lipid metabolism to regulate metastasis is poorly defined. We integrated bioinformatic analysis of The Cancer Genome Atlas (TCGA) LUAD cohort with in vitro and in vivo functional studies. Weighted gene co-expression network analysis (WGCNA) was performed to identify metastasis-associated modules. SLC7A5 function was evaluated using knockdown approaches in LUAD cell lines, assessed by Western blot, wound healing, Transwell migration assays, and an orthotopic lung cancer mouse model. Mechanistic studies included RNA-seq, targeted fatty acid profiling, BODIPY FL C16 uptake assays, Seahorse FAO analysis, and PPARA luciferase reporter assays. WGCNA identified SLC7A5 as a hub gene within a metastasis-associated transcriptional network, with high expression predicting poor overall survival, disease-free survival, and liver metastasis risk in LUAD patients. SLC7A5 knockdown reversed epithelial-mesenchymal transition (EMT), suppressed cell migration in vitro, and significantly reduced intrapulmonary tumor growth in an orthotopic mouse model. Mechanistically, RNA-seq and metabolomic profiling revealed that SLC7A5 deficiency induced profound reprogramming of fatty acid metabolism, characterized by reduced exogenous fatty acid uptake, paradoxically enhanced fatty acid oxidation (FAO), and consequent intracellular fatty acid depletion. Palmitic acid supplementation rescued the migratory defects caused by SLC7A5 knockdown. Transcription factor enrichment analysis identified PPARA as a downstream effector, and SLC7A5 silencing significantly reduced PPARA expression, transcriptional activity, and the expression of its target genes governing fatty acid uptake, synthesis, and oxidation. CPT1 enzymatic activity was correspondingly diminished. Crucially, PPARA overexpression rescued the migratory defects induced by SLC7A5 knockdown, establishing PPARA as a critical functional mediator. Our study identifies SLC7A5 as a key regulator of LUAD metastasis through PPARA-mediated control of fatty acid metabolism, revealing an unexpected metabolic axis linking amino acid transport to lipid homeostasis. These findings establish SLC7A5 as a promising prognostic biomarker and therapeutic target for metastatic LUAD, particularly for patients at high risk of liver metastasis.

overall_design: Two groups of A549 human lung adenocarcinoma cells were generated via siRNA transfection: negative control with scrambled siRNA, and SLC7A5 knockdown with target-specific siRNA. Oligo-dT-based mRNA libraries were built and subjected to paired-end RNA-seq. Transcriptional differences between the two groups were analyzed to explore SLC7A5-mediated lipid metabolic pathways related to lung adenocarcinoma metastasis.

first: Penghui Liu

last: Feng Qiu


title: Detection of early relapse in multiple myeloma patients

city: Brno

first: Sabina

last: Ševčíková

address: Kamenice 5

institute: Masaryk University

type: Non-coding RNA profiling by high throughput sequencing

summary: Background: Multiple myeloma (MM) represents the second most common hematological malignancy characterized by the infiltration of the bone marrow by plasma cells that produce monoclonal immunoglobulin. While the quality and length of life of MM patients have significantly increased, MM remains a hard-to-treat disease; almost all patients relapse. As MM is highly heterogenous, patients relapse at different times. It is currently not possible to predict when relapse will occur; numerous studies investigating the dysregulation of non-coding RNA molecules in cancer suggest that microRNAs could be good markers of relapse.  Results: Using small RNA sequencing, we profiled microRNA expression in peripheral blood in three groups of MM patients who relapsed at different intervals. In total, 24 microRNAs were significantly dysregulated among analyzed subgroups. Independent validation by RT-qPCR confirmed changed levels of miR-598-3p in MM patients with different times to relapse. At the same time, differences in the mass spectra between groups were identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry. All results were analyzed by machine learning.  Conclusion: Mass spectrometry coupled with machine learning shows potential as a reliable, rapid, and cost-effective preliminary screening technique to supplement current diagnostics.

overall_design:
Expression miRNA profile of 24 relapsed multiple myeloma patients, 8 from each subgroup: group A (relapse within 6 months), group B (relapse between 12 and 30 months), and group C (relapse after more than 5 years).

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

first: Tereza

last: Růžičková

first: Monika

last: Vlachová

first: Lukáš

last: Pečinka

last: Brychtová

first: Marek

last: Večeřa

first: Lenka

last: Radová

first: Simona

first: Marie

last: Jarošová

first: Josef

last: Havel

first: Luděk

last: Pour


title: Molecular characterization of the uterotrophic assay: Temporal gene expression changes in uterus and pituitary gland following ethinylestradiol

city: Wuppertal

first: Mira

last: Pavkovic

address: Aprather Weg 18a

institute: Bayer AG

summary:
an OECD-compliant uterotrophic assay was performed using ethinyl estradiol (EE) in juvenile female rats, followed by an analysis of the temporal gene expression profiles and associated pathways in uterus and pituitary gland in comparison to the standard parameters like uterine weight and histopathology. Additionally, published gene and protein expression analyses of uteri were included for evaluation of overall common pathways.
Immature, 15-day old female Wistar rats (strain Hsd/Cpb:WU; Harlan) were maintained under controlled conditions of temperature, relative humidity, and illumination. Animals had free access rat pellet diet and drinking water ad libitum. At the age of 19 days, rats were randomly assigned to groups (n=3-6) and were subcutaneously dosed with either 3 µg/kg ethinyl estradiol (EE, Sigma) or control (corn oil,) at 4 ml/kg body weight every 24h for three consecutive days. The animal experimental procedure was identical to the OECD uterotrophic assay (OECD 2000). Two, four, eight, and 24h after the first, four and 24h after the second, and 24h after the last application, animals were sacrificed in deep anesthesia (see Fig.1A) and uteri, pituitary glands and vaginas were rapidly removed. Uterine wet weight was determined, then uteri were longitudinally cut in halves. The right uterine halves and the pituitary glands for RNA isolation were shock frosted with liquid nitrogen and stored at −80°C.

overall_design: n=5 rat uterus samples per group (control and EE-treated, each at 2,4,8,24,28,48,72h) and n=3 rat pituitary gland samples per group (control and EE-treated, each at 4,8,24,48,72h)

first: Heidrun

last: Ellinger-Ziegelbauer


title: ERK overstimulation leads to cell hyperproliferation in hereditary hemorrhagic telangiectasia landscape.

city: L'Hospitalet de Llobregat

first: Jose Luis

last: Rocamora

address: Avinguda de la Granvia de l’Hospitalet, 199

institute: IDIBELL

summary: Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disorder caused by pathogenic variants in members of the BMP9/ALK1 signaling hub. In the present study we show that, regardless of whether the alterations are caused by reduced BMP9/ALK1 signaling (pathogenic variants in the ENG or ALK1 genes) or by overactivation of this pathway (such as the SMAD6 pathogenic variants), all are associated with increased endothelial cell (EC) proliferation and high levels of ERK MAPK activation in patient biopsies. We reproduced this phenotype in vitro in ECs lacking SMAD6 or after SMAD1 knockdown using siRNA. Loss of SMAD6 leads to dysregulation of the Notch pathway, with downregulation of phosphatases and consequent overstimulation of ERK. In normal ECs, BMP9 and Notch signaling inhibit ERK activity by upregulating PPP1R3C, a regulatory subunit of the PP1 phosphatase. Notably, BMP9-mediated inhibition of ERK is abolished when cells are transfected with siRNA targeting PPP1R3C. ERK hyperactivation was also observed in an HHT2 mouse model (ALK1-2loxP;Cdh5-CreERT2). Loss of both ALK1 alleles in adult mice leads to vascular failure and hemorrhages in the lung and intestine; these injuries are significantly reduced by treatment with the MEK/ERK inhibitor selumetinib. Overall, our work identifies a key role for ERK activation involved in HHT pathogenesis, suggesting that ERK inhibition may represent a promising therapeutic strategy for these patients.

overall_design: Homo sapiens whole transcriptome sequencing was performed in order to examine the different gene expression profiles, and to perform gene annotation on set of useful genes based on gene ontology pathway information. RNA from control HDMECs and SMAD6KO HDMEC (4 replicates each) was successfully analyzed by Macrogen company. The high-quality trimmed reads were mapped to the reference genome using HISAT2, a highly efficient splice-aware aligner designed to accurately capture exon-exon junctions. Following alignment, transcript assembly was performed with StringTie, which reconstructs full-length transcripts from the aligned reads, enabling a comprehensive representation of transcriptomic structures. Gene expression profiles were then generated, represented both as raw read counts and as normalization values, taking into account transcript length and sequencing depth. Specifically, normalization metrics such as FPKM (Fragments Per Kilobase of transcript per Million mapped reads), RPKM (Reads Per Kilobase of transcript per Million mapped reads), and TPM (Transcripts Per Kilobase Million) were calculated to allow for robust comparisons across samples. Finally, for comparative analyses between the different cell lines, genes or transcripts exhibiting significant differential expression were identified through rigorous statistical hypothesis testing, ensuring a high level of confidence in the observed biological differences.

first: Anna

last: Casellas

first: Agnes

last: Figueras

first: Pau

last: Cerda

first: Ferran

last: Medina-Jover

first: Raquel

last: Torres-Iglesias

first: Sandra

last: Castillo

first: Mariona

last: Graupera

first: Roxana

last: Ola

first: Antoni

last: Riera-Mestre

first: Francesc

last: Vinyals


title: Y chromosome gene KDM5D coordinates epigenetic and metabolic control of sex differences in CD8⁺ T cell immunity [CUT&Run]

city: Houston

first: Jiexi

last: Li

address: 1881 East Road

institute: The University of Texas MD Anderson Cancer Center

summary: Sex differences in anti-tumor immunity remain incompletely understood yet have important implications for tumor biology and immunotherapy. Here, we combine murine colorectal cancer (CRC) models, primary CD8+ T-cell perturbation studies, and single-cell transcriptomic analyses of human CRC to define a male-biased regulatory axis that limits CD8+ T-cell effector function. Male CD8+ T cells displayed reduced cytokine production, proliferation, cytotoxicity, TCR abundance, and TCR signaling relative to female CD8+ T-cells. Mechanistically, depletion of histone demethylase KDM5D, encoded by the Y chromosome, enhanced TCR expression and clustering, increased proximal TCR signaling, reduced cholesterol biosynthetic programs, and lowered expression of exhaustion-associated genes. In human CRC single-cell datasets, male tumor-infiltrating CD8+ T-cells showed enrichment for exhaustion and cholesterol-associated programs, supporting the translational relevance of this axis. Consistent with the known role of cholesterol in TCR clustering and activation, lovastatin reduced exhaustion-associated marker expression in male CD8+ T-cells and delayed CRC tumor growth in vivo – findings which align with the male-specific CRC risk reduction role of statins. Together, these findings identify KDM5D as a sex-chromosome-linked regulator of male CD8+ T-cell dysfunction and validate cholesterol metabolism as an actionable target for sex-specific immunotherapeutic modulation in cancer.

overall_design: Male and female OT-1 CD8+ T cells were isolated from mouse spleen and stimulated with anti-CD3/CD28 beads and mouse recombinant IL2 for 3 days before collection. A separate batch of male CD8+ T cells underwent electroporation to introduce Cas9-gRNA to knock down KDM5D, or gRNA alone as a control, and were cultured for 3 days before cell collection.


title: ERVmancer: A phylogeny-guided framework for decoding mechanisms of human endogenous retrovirus regulation in health and disease

city: Philadelphia

first: Priyankara

last: Wickramasinghe

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: Human endogenous retroviruses (HERVs), remnants of ancient infections, comprise ~8% of the human genome and influence development, immunity, and cancer. Studying them is limited by short-read sequencing, which cannot uniquely assign reads to repetitive elements. We present ERVmancer, a phylogeny-informed method that resolves mapping ambiguity and quantifies HERV expression from individual loci to entire clades. Benchmarking with matched long- and short-read data shows ERVmancer surpasses existing methods in sensitivity and specificity. Application recapitulates long-read observed HERV patterns in multiple sclerosis and reveals novel biology in breast cancer, including p53-mediated suppression of HERVH-LTR7, enabling scalable study of retroviral mechanisms.

overall_design: Spontaneous lymphoblastoid cell lines (SLCLs) were generated ex vivo from MS patients and healthy controls (HC) as described previously (37562974). Importantly, SLCLs are distinct from traditional LCLs because they are transformed by the individual’s endogenous EBV, rather than using lab strain virus (e.g. B95.8). For this study, a total of ten SLCL lines were characterized; these lines were obtained from three healthy blood donors (HC1-3), three MS patients with active disease as determined by magnetic resonance imaging (AMS 2,3, and 5), and four patients with stable disease (SMS 2, 3, 5, and 6) (Supplemental Table 2).  Notably, two pairs of cell lines were generated from the same individuals during different phases of their disease (AMS2= SMS6 and AMS5=SMS2).  This cohort was used as a pseudo-gold standard to benchmark method’s ability to detect and quantify patterns of HERV expression within these cells. SLCLs were maintained in growth medium [RPMI-1640 medium (Gibco, Gaithersburg, MD) with 10% fetal bovine serum (FBS; Gibco, Gaithersburg, MD), 1% gentamicin (50mg/ml; Quality Biological, Gaithersburg, MD), and 1% L-Glutamine (200mM; Quality Biological)]. Total RNA was isolated from 2 × 10^6 cells using the RNeasy Mini Kit (Qiagen, Germany) and treated with deoxyribonuclease (Qiagen, Hilden, Germany), following the manufacturer’s protocol. An RNA integrity number (RIN) value greater than 9.0 was determined by TapeStation (Agilent Technologies) for all samples prior to short paired-end and long-read RNA-seq.  For paired-end RNA-seq, sequencing library preparation for SLCLs was completed using the True-seq library preparation kit (Illumina, San Diego). Sequencing was performed with an Illumina NextSeq 500 system in high-output mode to generate ∼15-20 × 106 reads (2 × 150 bp) across three multiplexed and pooled samples. For long-read RNA-seq isoform libraries were prepared with the PacBio Iso-Seq Express kit according to the manufacturer’s protocol, which included oligo(dT)-primed cDNA synthesis and SMRTbell adapter ligation. Barcoded libraries were pooled equimolarly and sequenced on the PacBio Revio platform (Mount Sinai, NY), targeting ~4–5 million HiFi reads per sample.

first: Noam

last: Auslander

first: Samantha

last: Soldan

first: Paul

last: Lieberman

first: Andrew

last: Patterson

first: Jayamanna


title: High and intermittent hydrostatic pressure promotes maturation of engineered cardiac tissues derived from human pluripotent stem cells

city: Kyoto

first: Hidetoshi

last: Masumoto

state: Japan

address: 54 Shogoin Kawahara-cho, Sakyo-ku

institute: KYOTO UNIVERSITY

summary: The immaturity of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and engineered cardiac tissues (ECTs) limits their use in regenerative therapies. Ventricular loading pressure plays a vital role in cardiac repair, prompting the hypothesis that hydrostatic pressure could enhance ECT maturation. ECTs were created by co-culturing hPSC-derived cardiovascular cells with extracellular matrix proteins and subjecting them to intermittent hydrostatic pressure (1 hour/day at 50 kPa for 3 days after 2 weeks of pre-culture). This protocol improved cardiomyocyte alignment, mitochondrial content, and metabolic and functional maturation, evidenced by enhanced contractility, calcium flux, and single-cell RNA sequencing. Endothelial cells were critical for these effects, as their absence prevented maturation. Hydrostatic pressure combined with dynamic culture also promoted vascular network formation. Transplanted trained ECTs significantly improved ejection fraction in a rat myocardial infarction model. These findings demonstrate the potential of hydrostatic pressure training to advance ECT maturation and therapeutic application.

overall_design: Single-cell RNA sequencing was performed to investigate the cellular and molecular effects of hydrostatic pressure training on engineered cardiac tissues (ECTs) derived from human pluripotent stem cells. ECTs cultured under standard conditions were compared with ECTs subjected to hydrostatic pressure training (50 kPa, 1 hour/day for 3 days following 2 weeks of pre-culture). The study examined pressure-induced changes in cellular composition, transcriptional profiles, and signaling pathways across cardiomyocytes, vascular endothelial cells, and vascular mural cells.

first: Wusiman

last: Maihemuti

first: Kozue

last: Murata

first: Takahiro

last: Jimba

first: Jun

last: Iida

first: Keisuke

last: Hakamada

first: Akane

last: Sakaguchi

first: Mosha

last: Abulaiti

first: Yuichi

last: Saito

first: Laura Yuriko

last: González-Teshima

first: Satoshi

last: Takatori

last: Kenmotsu

first: Kenichi

last: Yoshikawa

first: Kenji

last: Minatoya

first: Seitaro

last: Nomura

first: Wataru

last: Kimura


title: Synergistic Inhibition of PI3K and HSP90 Enhanced Antitumorigenic Efficacy in Adrenocortical Carcinoma

city: Bethesda

first: Naris

last: Nilubol

address: Building 10 - Room 45952

department: Surgical Oncology Program

institute: National Cancer Institute

summary: Adrenocortical cancer (ACC) is a rare and aggressive malignancy with poor survival due to a lack of effective treatments; therefore, it is important to identify therapies that can be readily studied in clinical trials. Quantitative high-throughput drug combination screening identified potent synergy between phosphatidylinositol-3-kinase (PI3K) inhibition and heat shock protein 90 (HSP90) inhibition. This GEO submission contains the 18 bulk RNA-seq samples from NCI-H295R ACC cells that were used in the publication to evaluate transcriptional responses to PI3K and HSP90 inhibitors alone and in combination. The submitted samples include untreated controls and cells treated with STA9090, PIK75, BGT226, STA9090+PIK75, or STA9090+BGT226, with three biological replicates per condition. These RNA-seq data supported the comparison of molecular responses induced by PI3K/HSP90 inhibitor combinations in ACC cells, including distinct responses associated with PIK75+STA9090 and BGT226+STA9090 treatment.

overall_design: Gene expression profiling was performed by bulk RNA sequencing on 18 NCI-H295R adrenocortical carcinoma cell samples used in the publication. Cells were treated for 48 hours with 20 nM inhibitor conditions comprising untreated control, STA9090, PIK75, BGT226, STA9090+PIK75, and STA9090+BGT226, with three biological replicates per condition. RNA was extracted using the RNeasy Mini Kit (74104, Thermofisher Scientific), quantified by Nanodrop 1000 (Thermofisher Scientific), and RNA integrity was evaluated with the Agilent 2100 Bioanalyzer (Agilent Technologies). RNA sequencing was performed at the Sequencing Facility, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research.


title: TGFβ-Dependent Epithelial–Mesenchymal Plasticity in Immortalized Human Atrial Epicardial Cells: An mRNA Profiling Study

city: Bielefeld

first: Katja

last: Nowak

address: Universitätstraße 25

department: Faculty of Biology, Cell Biology

institute: University of Bielefeld

summary: The adult mammalian heart exhibits limited regenerative capacity, and a stable adult human in vitro model for investigating epicardial activation remain limited. Here, we isolated human epicardium-derived cells from the adult heart auricle. Successful immortalization led to the generation of a stable immortalized epicardium-derived cell (iEPDC) population, allowing the investigation of intercellular dynamics upon EMT activation. TGFβ signalling was modulated using SB431542 or TGFβ3.

overall_design: RNAseq profiling of iEPDCs with SB431542-, NoAdditive- and TGFB3- treatment after 5 days

first: Christian

last: Kaltschmidt

first: Cornelius

last: Knabbe

first: Anna L.

last: Höving


title: Y chromosome gene KDM5D coordinates epigenetic and metabolic control of sex differences in CD8⁺ T cell immunity [RNA-seq]

first: Jiexi

last: Li


title: Methylation Profiling and Alternative Classification Approaches in a Glioma-Enriched FFPE Stereotaxic Biopsy Cohort

city: Frankfurt

first: Katharina

last: Weber

address: Heinrich-Hoffmann-Straße 7

department: Institute of Neurology

institute: University Hospital Frankfurt

type: Methylation profiling by genome tiling array

summary: DNA methylation profiling of CNS tumors supports and refines histological diagnoses. In cases with limited tissue like in stereotaxic (STX) biopsies, there is a need for informed triage to the most promising analysis techniques. Diagnostic potential and accuracy of DNA methylation profiling and molecular grading in STX samples is investigated in 237 FFPE samples of brain tumor patients.

overall_design: DNA methylation profiling of 237 FFPE stereotaxic brain tumor biopsy samples using Illumina Infinium MethylationEPIC BeadChip (v1). Samples were analyzed with the Heidelberg (v11b4), Bethesda (v2) and EpiDiP brain tumor classifiers. Copy number profiling was performed using the Heidelberg brain tumor classifier. Reference-based tumor deconvolution was conducted using MethylCIBERSORT and the LUMP algorithm.

first: Katharina Johanna

first: Jürgen

last: Hench

first: Markus

last: Zumpt

first: Thomas

last: Eska

first: Moritz

last: Armbrust

first: Pia Susann

last: Zeiner

first: Tabea Isabell

last: Hartung

first: Viktoria

last: Ruf

first: Eike

last: Steidl

first: Mareike

last: Dettki

first: Tim

last: Fenton

first: Joachim Peter

last: Steinbach

first: Marcus

last: Czabanka

first: David

last: Capper

first: Stephan

last: Frank

first: Karl-Heinz

last: Plate

first: Marie-Thérèse

last: Forster

first: Patrick Nikolaus

last: Harter


title: Targeting ABL kinases to overcome melanoma immune evasion during MAPKi resistance

city: Lexington

first: Rina

last: Plattner

state: KY

address: 760 Press Ave

department: Pharmacology & Nutritional Science

institute: University of Kentucky

summary: We utilized single-cell sequencing to determine the mechanism by which the ABL1/2 inhibitor, nilotinib, prevents the onset of BRAF/MEK inhibitor resistance. Mice harboring mouse melanomas were treated with BRAF/MEK inhibitors or BRAF/MEK inhibitors plus nilotinib for 4 days, and the resulting tumors were subjected to single-cell RNA-sequencing in order to assess changes in the melanoma and immune populations.

overall_design: Male C57BL/6 mice were injected with YUMM5.2 melanoma cells, and when tumors were evident (100-150mm3), mice were randomized and treated with dabrafenib (20mg/kg/d) and trametinib (0.15mg/kg) by oral gavage (D/T) once daily or treated with D/T+nilotinib (42mg/kg; DTnilo) twice daily. Drugs were dissolved in DMSO:PEG400:Vitamin E TPGS: Poloxamer 407 (Pluronic F127); PBS-1X at a 5:25:10:1:59 ratio. After 4 days of treatment, mice were euthanized, tumors removed, dissociated, and live cells subjected to single-cell sequencing.

first: Jinpeng

last: Liu

first: Chi

last: Wang

first: Rakshamani

last: Tripathi


title: A Receptor-Clamped IL-2 Uncouples Treg Expansion from Injection Site Reactions

city: South San Francisco

first: Weiwen

last: Deng

address: 750 Gateway Blvd

institute: Amgen

summary: Low-dose interleukin-2 (IL-2) and IL-2 muteins are being developed to expand regulatory T cells (Tregs) as therapies for autoimmune diseases, but IL-2 mediated injection site reactions (ISRs) remain frequent and poorly understood. Here we show IL2Mut24, a murine surrogate of IL-2 mutein with reduced CD122 binding and extended half-life, robustly promotes Treg expansion yet causes more severe ISRs than wild-type IL-2. Through studies using immune-deficient mice, antibody blockade, and adoptive transfers, we identify CD25⁺ group 2 innate lymphoid cells (ILC2s) as key ISR mediators and sufficient to trigger ISR. To address safety, we engineered anti-CD25–IL2Mut24 fusion proteins (“receptor clamps”) with graded CD25 antagonism. This approach reveals an optimal window that preserves Treg selectivity, suppresses ISRs, and outperforms IL2Mut24 in experimental autoimmune encephalomyelitis. These findings uncover cellular basis of IL-2-induced ISRs and support the development of receptor-tuned IL-2 fusion proteins as a next-generation therapeutic strategy for autoimmune diseases.

overall_design: BALB/c.Foxp3-GFP mice were treated with a single dose of Fc-IL2WT, Fc-IL2Mut24  or PBS via subcutaneous (s.c.) injection. Skin samples from mice within the same treatment group were incubated with a cocktail of TotalSeq™-C Mouse Universal Cocktail, V1.0 plus one TotalSeq™-C Hashtag antibodies (Hashtags C0306, C0307, C0308, C0309, and C0310). Samples were pooled and sorted on a FACS Aria into three single cell fractions: CD45+ TCRβ+ GFP+, CD45+ TCRβ + GFP-, CD45+ TCRβ-.

first: Anupama

last: Sahoo

first: Cody

last: Moorman

first: Mina

last: Tsenkova

first: Yi

last: Jing

first: Alexis

last: Valdovinos

first: Xin

last: Luo

first: Shiping

last: Lu

first: Songyu

first: Renee

last: Hukkanen

first: Helen

last: Tang

first: Ronya

last: Primack

first: Shweta

last: Mandavalli


title: Spatial Transcriptomic Profiling Reveals Joint Inflammation Signatures in Borrelia burgdorferi–Infected C3H Mice

city: Grand Forks

first: Archana

last: Dhasarathy

state: ND

address: 1301 N. Columbia Road, STOP 9061, STOP 9037

department: Biomedical Sciences

institute: UNIVERSITY OF NORTH DAKOTA

summary: Lyme arthritis, a manifestation of Lyme disease, is triggered by the spirochetal bacterium Borrelia burgdorferi (Bb), which is transmitted through the bite of the Ixodes tick. Although multiple studies have been performed on the complex host immune response in Lyme arthritis, the spatial gene expression environment in the joint tissue remains unexplored. Here, we applied spatial transcriptomics to ankle joints of C3H mice infected with B. burgdorferi, profiling tissues at peak inflammation (2 weeks post infection) and after antibiotics (4 weeks post-infection) during inflammation resolution. Analysis revealed spatially restricted signatures: pro-inflammatory responses dominated synovial and fibroblast populations 2 weeks post-infection, with elevated levels of Vimentin and I-Ek related gene and protein expression localized to these regions. In contrast, 4 weeks post-infection during the inflammation resolution phase, levels of Vimentin and I-Ek related gene and protein expression are reduced drastically despite the persistent pathology seen by H&E staining. Notably, fibroblasts and synoviocytes in the medial humeroulnar joint regions adopted immune-like phenotypes during peak inflammation, while the same cell types in the lateral humeroradial joint displayed a more infection-resilient phenotype. Comparison with scRNA-seq synoviocyte cluster revealed little overlap, but concordant genes that were upregulated were involved in ECM remodeling (Col1a1, Col3a1, Mmp2, Timp1), fibroblast activation (Postn, Runx1, Ctnnb1), and inflammation (Saa3, Ccl7, Tnfaip6). By week 4, concordant downregulation of genes coding for ribosomal proteins, mitochondrial complex subunits, and Jun (an immediate-early inflammatory gene) was noted. These spatially resolved maps demonstrate that joint microenvironments play a crucial role in pathogenesis, offering unique insights into Lyme arthritis pathology.

overall_design: B. burgdorferi-infected murine joints were sectionaed and stained with Vimentin and IEK, and ROIs identified. Probes were added, and sequenced to characterize spatial gene expression patterns and cellular organization during peak inflammation (2 weeks post-infection) and resolution following antibiotic treatment (4 weeks, post-infection). Control animals at both time points were used.

first: Kaitlyn

last: Gura

first: Mollie

last: Hostetter

first: Veena

last: Potluri

first: Sarah

last: Johnson

first: Michael

last: Hill

first: Yuelin

last: Zhong

first: Eleanor

middle: L

last: Astley

first: Tanja

last: Petnicki-Ocwieja

first: Suba

last: Nookala

first: Catherine

last: Brissette


title: Secretome analysis during human induced pluripotent stem cell-derived cardiomyocyte differentiation [miRNA-Seq]

city: Suita

first: Yuki

last: Saito

address: Life System Building, 1-3 Yamadaoka

department: Department of Future Medicine

institute: Osaka Univ.

summary: Introduction: Cell transplantation therapy using human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) is a promising treatment for ischemic heart disease. While paracrine effects mediated by the secretome are recognized as a key therapeutic mechanism, the dynamic changes in the secretome profile during the manufacturing process remain poorly understood. This study aimed to comprehensively elucidate the secretome dynamics of clinical-grade hiPSC-CM to identify their "Mode of Action" (MoA) and candidate quality attributes (CQAs). Methods: We integrated multi-omics profiling-including total RNA-seq, proteome analysis, and extracellular vesicle (EV)-derived miRNA-seq-with functional assays (proliferation, cell migration, and tube formation) using samples obtained across time points of the hiPSC-CM manufacturing process (Day 4-25). Results: High-purity hiPSC-CM (>95% cTnT positive) exhibited a distinct transcriptomic maturation between Day 16 and Day 25, characterized by the upregulation of cardiac maker genes. Proteomic clustering revealed four distinct stages, showing a functional transition from proliferation-centric signaling to tissue-repair signaling. Notably, the purified Day 25 secretome showed a qualitative shift toward a PDGF and SDF-1 rich profile. Simultaneously, EVs from Day 25 were enriched with "myomiRs" (miR-133b, -208b, -499b) and multiple anti-proliferative miRNAs (let-7e-5p, miR-145-5p). Functionally, the Day 25 secretome significantly enhanced mesenchymal stem/stromal cell (MSC) migration and promoted mature, highly branched endothelial tube formation compared to earlier stages. Conclusions: The hiPSC-CM secretome undergoes a programmed evolution during manufacturing, shifting from promoting undifferentiated growth to orchestrating complex tissue repair through stable angiogenesis and MSC recruitment. These findings establish a molecular foundation for the MoA of hiPSC-CM therapy and provide critical CQAs for ensuring the potency and consistency of clinical-grade cardiac products.

overall_design: The analyzed samples consisted of miRNA isolated from extracellular vesicles at three stages of iPS cell–derived cardiomyocyte differentiation: the undifferentiated stage, Day 16, and Day 25.

first: Kazuaki

last: Takemura

first: Junya

last: Aoyama

first: Sawako

last: Hibino

first: Kouichi

last: Hasegawa

first: Yoshiki

last: Sawa


title: Gene expression data of mouse small intestine organoids treated with pegtarazimod

city: Freiburg

first: Geoffroy

last: Andrieux

address: Breisacherstr 153

institute: University clinics Freiburg

summary: Small intestine organoids were derived from BALB/c mice, challenged with TNF and treated with pegtarazimod or vehicle. Influence of pegtarazimod treatment on gene expression was evaluated by microarray.

overall_design: 5 biological replicates per group were analyzed. Pegtarazimod treatment was compared to control group receiving vehicle treatment.

first: Melanie

last: Boerries

first: Verena

last: Holzmüller

first: Robert

last: Zeiser


title: Divergent SAGA Complexes Shape the Toxoplasma Transcriptome across Host-Responsive and Core Gene Networks [ChIP-Seq]

city: La Tronche

first: Christopher

last: Swale

address: Batiment Jean Roger

department: U1209 - Institute of Advanced Biosciences

institute: INSERM

summary: GCN5 (General Control Non-repressed protein 5) plays a central role in the regulation of gene expression through its histone acetyltransferase (HAT) activity. Within the SAGA complex (Spt-Ada-Gcn5 acetyltransferase), GCN5 regulates transcription by modifying the chromatin structure and thus influencing the accessibility of gene promoters. Its conservation in all eukaryotes underscores its essential role in cell biology. Remarkably, among invertebrates, Toxoplasma possesses two HAT variants of the GCN5 family. GCN5a plays an important role in parasite gene expression under alkaline conditions, but is not critical for growth. In contrast, GCN5b is essential for parasite survival. Previous pioneering studies have begun to elucidate the unique molecular properties of GCN5b HAT activity, but also its partnership. However, these studies were performed on zoites still expressing the native GCN5b gene, which may lead to potentially misleading or incomplete conclusions. Here, we re-examine the unique interactome associated with the native GCN5b protein and we investigate the transcriptional consequences of depleting the protein using auxin-inducible degron technology. We are also investigating the interplay between GCN5b and its counterpart HDAC3 at the chromatin level as the parasite transitions from the tachyzoite to the merozoite stage. We will analyze their selective engagement as activator, repressor or chromatin insulator using ChIP-seq and try to decipher the ‘histone code’ that ensures precise spatial and temporal regulation of stage-specific genes. We are also comparing the dynamics of the Toxoplasma SAGA complex between the two different zoite populations, focusing on the DNA-targeting AP2 transcription factors, but also on the PHD-containing histone PTM reader identified in the interactome of GCN5b.

overall_design: We report genome-wide GCN5b, PHD1, MORC, HDAC3 and histone marks, profiles in Toxoplasma gondii using ChIP-seq in the context of GCN5b protein knock-down

first: Dominique

last: Cannella

first: Belen

last: Pachano

first: Martina

last: Shahinas

first: Jon

last: deVries

first: Charlotte

last: Corrao

first: Léa

last: Pounot

first: Lucid

last: Belmudes

first: Anne-Marie

last: Hesse

first: Yohann

last: Couté

first: Alexandre

last: Bougdour

first: Mohamed-Ali

last: Hakimi


title: Divergent SAGA Complexes Shape the Toxoplasma Transcriptome across Host-Responsive and Core Gene Networks [RNAseq]

overall_design: We report transcriptome of RH Ku80 GCN5b KD and  RH Ku80 PHD1 KD in Toxoplasma gondii tachyzoites left untreated, after 24 h or after 48h of IAA treatment using Illumina RNA sequencing.


title: Divergent SAGA Complexes Shape the Toxoplasma Transcriptome across Host-Responsive and Core Gene Networks

summary: Histone acetylation, a fundamental epigenetic mechanism that controls gene activity, is essential for the developmental plasticity and virulence of the parasite Toxoplasma gondii. However, how this parasite organizes and deploys its acetyltransferase machinery has remained unclear. Here, we show that T. gondii rewired this process using a plant-like system built around the acetyltransferase TgGCN5b, which differs from the typical SAGA complex found in other eukaryotes. Proteomic and structural analyses reveal a modular assembly that integrates multiple acetyltransferase (GNAT) enzymes and chromatin-reader proteins carrying PHD and PZP domains and Apetala-related transcription factors, an organization unique to apicomplexan parasites. TgGCN5b catalyzes a selective tri-site acetylation pattern on histone H3 at lysines 9, 14, and 18 that maintains open chromatin and sustains transcription of core metabolic, invasion, and virulence genes. Its conditional depletion disrupts these post-translational modifications, silencing key promoters and decoupling transcription from histone methylation. Genome-wide analyses further show that TgGCN5b functions independently of the MORC/HDAC3 repressive pathway, defining a distinct regulatory circuit that connects chromatin acetylation with gene expression and developmental transitions. These findings reveal that the SAGA complex has been evolutionarily reconfigured in T. gondii into a plant-like, yet featuring divergent and apicomplexan-specific feature, positioning TgGCN5b as a central regulator that links epigenetic control to parasite growth, adaptation, and virulence.

overall_design: We report Tn5 transposase accessiblity data (ATAC-Sequencing) on the RHK80 GCN5b KD strain in an untreated condition (UT) and induced knockdown (IAA 24h). Intracellular tachyzoites (non-treated or IAA treated for 24h) were prepared using HFF cells monolayers in a T175 format, which was freshly scrapped, gently homogenised by pipetting and centrifuged at 500*g. Prior to initiating the transposition protocol, the pellet was gently washed with warm DPBS (Life technologies) and resuspended in 500 μl of cold PBS + protease inhibitor (Diagenode kit). Nuclei preparation, permeabilization, Tn5 transposition and library preparation was prepared following precisely the Diagenode ATAC-SEQ kit protocol (C01080002). Nuclei permeabilization was performed on an estimated 100,000 tachyzoites by diluting 10 μl of DPBS cell suspension (from one T175 resuspended in 500 μl) in 240 of DPSB + protease inhibitor (1/25 dilution). From this dilution, 50 μl were then taken to perform the transposition reaction. Of note, the permeabilization protocol used a 3 min 0,02% digitonin (Promega) treatment. Following the Tn5 reaction, libraries were amplified using the Diagenode 24 UDI kit 1 (ref 01011034) following standard protocol procedures. Libraries were multiplexed and sequenced on a single Novaseq6000 lane by Fasteris (Genesupport SA) using 2*50 cycles, generating on average 27 million reads. Following the demultiplexing of raw reads by bcl2fastq V3, trimming and quality control, reads were aligned to T. gondii reference genome (TGME49) using BOWTIE V2.