title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2–FASN axis [RNA-Seq TRAF2-KD]

city: Wuxi, Jiangsu 214023, China

first: Maorong

last: Cai

address: Wuxi, Jiangsu, China

institute: Department of Neurosurgery, Wuxi People’s Hospital Affiliated to Nanjing Medical University

summary: Lipid metabolism plays a critical role in the progression of cancers, including glioblastoma (GBM). Tumor necrosis factor-α (TNF-α) exhibits a dual role in the tumor microenvironment: at high concentrations, it induces cell death and exerts anti-tumor effects, while at low doses, it demonstrates pro-tumorigenic activity. This study investigates the regulatory effects of low-dose TNF-α on the malignant behavior and lipid metabolism of GBM. The results show that low-dose TNF-α (10 ng/mL) significantly promotes GBM cell Malignant phenotypes and lipid droplet accumulation via the TNFR signaling pathway, a process dependent on its downstream adaptor protein, Tumor necrosis factor receptor-associated factor 2 (TRAF2). Mechanistically, TRAF2 interacts with Fatty acid synthase (FASN) through its TRAF domain and, serving as an E3 ubiquitin ligase, leverages its RING domain to mediate K63-linked polyubiquitination of FASN. This enhances FASN protein stability, promotes lipid synthesis, and ultimately drives tumor progression. Furthermore, through virtual screening, the small-molecule compound Jionoside B1 was identified to target the RING domain of TRAF2, effectively inhibiting K63-linked ubiquitination of FASN and disrupting the TRAF2–FASN interaction and protein accumulation. In both in vitro and in vivo experiments, Jionoside B1 significantly suppressed lipid synthesis and attenuated tumor growth. This study systematically elucidates the mechanism by which low-dose TNF-α regulates lipid metabolism and promotes GBM malignant progression via the TRAF2–FASN axis, providing not only new insights into the "double-edged sword" role of TNF-α in the tumor microenvironment but also a potential novel therapeutic strategy for targeting this pathway in GBM treatment.

overall_design: U251 cells transfected with a non-targeting control lentivirus served as the negative control group, while U251 cells transfected with a TRAF2‑specific knockdown lentivirus constituted the experimental group. After successful establishment of the stable cell lines, RNA sequencing (RNA‑seq) was performed on both groups, with three biological replicates included per group.

first: Yang

first: Xinyu

last: Mao

first: Jiantong

last: Jiao


title: Low-dose TNF-α drives malignant progression and lipid metabolism in glioblastoma through the TRAF2–FASN axis.

status: Public on Feb 04 2026

country: China

type: Expression profiling by high throughput sequencing

overall_design: In this study, RNA sequencing (RNA-seq) was performed to analyze two groups of cells: the negative control group (U251 cells treated with deionized water) and the experimental group (U251 cells treated with 10 ng/mL TNF‑α for 24 hours). Each group included three biological replicates.

last: Liu


title: Transcriptional profiles of individuals with KDM5A variants

city: Dallas

first: Maria

middle: H

last: Chahrour

state: Texas

address: 6000 Harry Hines Boulevard, NB10.104

country: USA

institute: UT Southwestern Medical Center

summary: KDM5A is an ASD gene and chromatin regulator. We identified a cohort of 22 individuals harboring monoallelic and biallelic KDM5A variants. We cultured lymphoblastoid cell lines from a subset of these individuals and performed RNAseq.

overall_design: RNA was extracted from LCL pellets derived from patients, parents, and siblings, when available, using TRIzol reagent (Invitrogen), and purified with the RNeasy Mini Kit (Qiagen). Genomic DNA was removed using the RNase-free DNase kit (Qiagen). RNA quality was assessed using the Agilent TapeStation 4200, and all samples had a RIN score of ≥ 8. RNA concentrations were measured using a Qubit fluorometer, and 1 μg of DNase-treated total RNA was used for library preparation with the TruSeq Stranded mRNA Library Prep Kit (Illumina). Poly-A RNA was purified and fragmented, and strand-specific libraries were amplified and purified using AmpureXP beads. Samples were sequenced on the Illumina NextSeq 500 using V2.5 reagents.

first: Lauretta

last: ElHayek

first: Ashlesha

last: Gogate


title: Characterizing the metabolomes of microglia, astrocytes, and neurons in the brain across states of health, aging, and disease

city: Beijing

first: fengzhi

last: li

address: Bldg.3, NO.9, YIKE Rd, Zhongguancun Life Science Park, Changping District,

institute: Chinese Institute for Brain Research, Beijing

summary: Neurons and glia are distinct in their morphology, development, and function. They have unique transcriptomes and proteomes, but little is known about their metabolomes. The challenge of brain cell metabolic profiling is to obtain a large number of pure cells for reliable analysis. Here, we purify microglia, astrocytes, and neurons from the genetically labeled-brain. We identified >70 metabolites in them with targeted metabolomics and 9,854 metabolite features with untargeted metabolomics. We systematically characterized cell type–enriched metabolites and metabolic pathways. The enrichment of glutathione (GSH) metabolism in microglia was further validated in vivo. A significant decrease in GSH levels and GSH metabolism observed in microglia in aging and Alzheimer's disease (AD) models. Disrupting GSH metabolism in microglia results in aberrant morphogenesis, upregulation of mitophagy-related genes, and the deposition of β-Amyloid. Our results provide a valuable resource for metabolic studies related to aging, AD and other neurological diseases.

overall_design: Three different transgenic mouse strains were used, which result in neurons and glial cells that are labeled with EGFP or EYFP. We then used these strains to isolate neurons and glial cells from the cerebral cortex and hippocampus by flow cytometry. We further simultaneously performed targeted metabolomics, untargeted metabolomics, and bulk RNA-seq for metabolome and transcriptome analyses

first: Jie

last: Yu

first: Fengzhi

last: Li

first: Woo-ping

last: Ge


title: Multiscale analysis and functional validation of the cellular and genetic determinants of skeletal disease [Human scRNA-seq]

city: SYDNEY

first: Peter

last: Croucher

state: NSW

address: 384 VICTORIA ST, DARLINGHURST NSW 2010

country: Australia

institute: Garvan Institute of Medical Research

summary: Musculoskeletal diseases are a major health burden. Development of bone-active therapies has been hindered by limited understanding of the cells and genes that regulate the skeleton. We exploited the value of cross-species analysis and developed single-cell methodologies in skeletal tissues to define the critical endosteal compartment that regulates bone turnover. Thirty-four distinct cell types were identified, and disease-relevant cells prioritised by enrichment for rare skeletal disorder genes and bone mineral density-associated genes in an extended UK Biobank GWAS.  Functional validation was undertaken in over one thousand genetically modified mouse models. Endothelial and vascular smooth muscle cells were identified as novel skeletal disease-relevant cells alongside osteoblast, chondrocyte and osteoclast cell lineages. Hundreds of cell-specific genes with unappreciated roles in skeletal pathophysiology were identified. This comprehensive cellular and molecular framework underpins skeletal physiology and disease, and will help prioritise new therapeutic targets to accelerate development of novel therapies to treat musculoskeletal disease.

overall_design: scRNA seq of 31 samples, cells isolated from femoral head of 10 adults undergoing hip replacement surgery.

first: Ryan

middle: C

last: Chai

first: Mischa

last: Lundberg

first: Bernard

last: Freudenthal

first: James

middle: T

last: Smith

first: Andrew

middle: P

last: Boughton

first: Yuandan

last: Zhang

first: Kaitlyn

middle: A

last: Flynn

first: Monika

last: Frysz

first: Alexander

last: Corr

first: Weng Hua

last: Khoo

first: Davide

last: Komla-Ebri

first: Michael

middle: R

last: Dack

first: Siobhan

middle: E

last: Guilfoyle

first: John

middle: G

last: Logan

first: Natalie

last: Butterfield

first: Victoria

middle: D

last: Leitch

first: Andrea

middle: S

last: Pollard

first: Riikka

last: Mäkitie

first: Nathaniel

last: Bradford

first: Lorenzo

last: Ramos-Mucci

first: Amaia

last: Vilas-Zornoza

first: Yunshun

last: Chen

first: Raymond

middle: K

last: Yip

first: Jeremy

last: Er

first: Siew Zhuan

last: Tan

first: Michelle

middle: M

last: McDonald

first: Scott

last: Youlten

first: Marcelo

last: Sergio

first: Ariel

last: Castro-Martinez

first: Shelley

last: Young

first: Elena

last: Skorokhodova

first: David

last: Evans

first: Joseph

last: Powell

first: Christiaan

last: de Leeuw

first: Adam

last: Ewing


title: Identification of Potential NR3C2 Targets by RNA‑Seq in HK2 Cells

city: WUHAN

first: heng

middle: zhi

last: du

address: 1095 Jiefang Dadao, Wuhan City, Hubei Province, China

institute: Wuhan Tongji Hospital

summary: To identify potential targets of NR3C2 overexpression, RNA sequencing was performed to compare the transcriptomes of control and NR3C2-overexpressing (NR3C2-OE) HK2 cells.

overall_design: We report the application of sequencing technology for high-throughput profiling of mRNA expressions in NR3C2-OE and Control cells. Total RNA was extracted from both control and NR3C2-OE HK2 cells (1×106 cells per sample) using Trizol reagent(Takara, Cat# 9109). RNA concentration and purity were meas assessed by agarose gel electrophoresis and the Agilent 2100 Bioanalyzer system for RIN determination. All RNA samples used for library preparation met the following quality control thresholds: total amount ≥1 μg, concentration ≥35 ng/μL, OD260/280 ≥1.8, OD260/230 ≥1.0, and RIN >8.0. In this project, rRNA was removed from the total RNA by ribosomal RNA removal kit, and the way of ion breaking were used to break the RNA into 200-300bp fragments. By using RNA as template, the first strand of cDNA was synthesized with 6 base random primers and reverse transcriptase. The first strand cDNA was used as the template to synthesize the second strand cDNA. When the second strand cDNA is synthesized, the base T is replaced by U to achieve the goal of a chain specific library. All of our RNA-seq projects used strand specific kits to construct the libraries. Finally, we show that mRNA different expression in HK2 cells. This study provides abundance of mRNA that may predict kidney diseases.

first: hairui

last: zhao


title: Generational Stability of Environmentally Induced Epigenetic Transgenerational Inheritance of Adult-Onset Disease

city: Pullman

first: Michael

last: Skinner

state: WA

address: Abelson 507

department: SBS

institute: WSU

type: Methylation profiling by high throughput sequencing

summary: In traditional models of Drosophila flies and C. Elegans worms have shown epigenetic transgenerational inheritance has persisted and remained stable for hundreds of generations. Mammalian studies, however, have primarily investigated up to three or four generations. Environmental exposures to toxicants or other stressors have been linked to having a role in epigenetic alterations and epigenetic transgenerational inheritance. Changes within the epigenome have the potential to change gene expression and genetic processes, as well as potentially alter phenotypes and evolution in all organisms. In this study, an outcrossed mammalian colony of outbred rats was bred for twenty three successive generations and assessed for epigenetic alterations and incidence of pathological abnormalities. Sperm from male rats were analyzed for DNA methylation and pathology abnormalities in the female and male rats in successive generations. Observations showed an increase in disease incidence within the ten generations (F13) and twenty generations (F23) compared to the control lineage. Our results indicate that ten generations of transgenerational exposure led to an increase in epigenetic alterations and a higher incidence of pathological abnormalities. Ancestrally exposed rats to vinclozolin showed significant parturition abnormalities in both the maternal and paternal lineages after 16 generations. This included maternal deaths during labor and stillbirths. Pathological assessments revealed abnormalities across multiple tissue types and an increased incidence of disease. This suggests the physiological consequences of the generational stability of epigenetic inheritance. Observations establish the generational stability of epigenetic inheritance over twenty generations in a mammalian model system, however, new pathology in later generations involving parturition abnormalities were also observed.

overall_design: The experimental design involved exposing the F0 generation gestating female rats to the toxicant vinclozolin through intraperitoneal injection.  The control lineage female rats were exposed to DMSO alone through intraperitoneal injection. Each generation had a maternal outcross (MOC) and paternal lineage outcross (POC) that were outbred with wild-type non-exposed Sprague Dawley rats. This generated a female lineage maternal outcross (MOC) and male lineage paternal outcross (POC) that were both investigated for generational pathology. Although sperm collection can provide millions of cells and sufficient DNA for epigenetic analysis, the very limited number of mature oocytes does not allow the female germline epigenetic analysis. Therefore, only the male sperm were used for epigenetic analysis for both the male (POC) and female (MOC) lineages for epigenetic analysis. All rats were euthanized and dissected at one year of age where tissues and epididymal sperm were collected. Further analysis was done on the collected tissue and sperm for generational stability of transgenerational epigenetic inheritance effects in the F13 and F23 generations.

first: Alexandra

last: Korolenko

first: Eric

last: Nilsson

first: Sarah

last: De Santos


title: Multiscale analysis and functional validation of the cellular and genetic determinants of skeletal disease [snATAC-seq]

first: Peter

last: Croucher

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: snATACseq of nuclei isolated from endsoteal cells from femora and tibiae of 9-week old male C57BL/6J mice

first: Ryan

last: Chai

first: Mischa

last: Lundberg

first: Bernard

last: Freudenthal

first: James

last: Smith

first: Andrew

last: Boughton

first: Yuandan

last: Zhang

first: Kaitlyn

last: Flynn

first: Monika

last: Frysz

first: Alexander

last: Corr

first: Weng Hua

last: Khoo

first: Davide

last: Komla-Ebri

last: Dack

first: Siobhan

last: Guilfoyle

first: John

last: Logan

first: Natalie

last: Butterfield

first: Victoria

last: Leitch

first: Andrea

last: Pollard

first: Riikka

last: Mäkitie

first: Nathaniel

last: Bradford

first: Lorenzo

last: Ramos-Mucci

first: Amaia

last: Vilas-Zornoza

first: Yunshun

last: Chen

first: Raymond

last: Yip

first: Jeremy

last: Er

first: Siew Zhuan

last: Tan


title: Spatial analysis of IPMNs defines a paradoxical KRT17-positive, low-grade epithelial population harboring malignant features [scRNA-seq]

city: Ann Arbor

first: Eileen

last: Carpenter

state: MI

address: 1137 E Catherine St Med Sci II R 7712

department: Internal Medicine

institute: University of Michigan

summary:
Background & Aims:
Intraductal papillary mucinous neoplasms (IPMNs) are pancreatic cysts that represent one of the few radiologically identifiable precursors to pancreatic ductal adenocarcinoma (PDAC). Though the IPMN-bearing patient population represents a unique opportunity for early detection and interception, current guidelines provide insufficient accuracy in determining which patients should undergo resection versus surveillance, resulting in a sizable fraction of resected IPMNs only harboring low-grade dysplasia, suggesting that there may be overtreatment of this clinical entity.
Methods:
To investigate the transcriptional changes that occur during IPMN progression, we performed spatial transcriptomics using the Nanostring GeoMx on patient samples containing the entire spectrum of IPMN disease including low-grade dysplasia, high-grade dysplasia, and IPMN-derived carcinoma.  Single cell RNA sequencing was performed on side branch and main duct IPMN biospecimens.
Results:
We identified a subpopulation of histologically low-grade IPMN epithelial cells that express malignant transcriptional features including  KRT17, S100A10 and CEACAM5,  markers that are enriched in PDAC. We validated this high-risk gene signature in both single-cell RNA sequenced samples and an external ST dataset containing a larger number of IPMN samples including non-tumor bearing IPMN (i.e. low-grade IPMN in isolation). Immunofluorescence staining of a large cohort of patient tissues confirmed the presence of KRT17-positive cells, which were found to comprise a small subset of epithelial cells within histologically low-grade IPMN in a patchy distribution.
Conclusions:
Our study demonstrates that KRT17 marks a distinct transcriptional signature in a subpopulation of epithelial cells within histologically low-grade IPMN.  This population of cells likely represents a transitional state of histologically low-grade epithelial cells undergoing progression to a higher grade of dysplasia and thus may represent a higher risk of progression to carcinoma.

overall_design:
We obtained two freshly acquired IPMN samples in the following manner: Sample 1556 was a side branch IPMN with a mural nodule within the cyst; sample was obtained via endoscopic fine needle biopsy performed during endoscopic ultrasound evaluation. Sample 1593 was acquired from a total pancreatectomy of a patient with main duct IPMN; sample was acquired by longitudinally bisecting and shaving off a portion of the dilated main duct.

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Raw files for human/patient samples are being made available in dbGaP (https://www.ncbi.nlm.nih.gov/gap/) for controlled access to the personally identifiable sequence data.
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title: IAP retrotransposons contribute to the transcriptional diversity of the murine placenta [RNA-Seq]

city: London

first: Miguel

last: Branco

address: 4 Newark Street

country: United Kingdom

institute: Blizard Institute

summary: Transposable elements (TEs) have made important contributions to the evolution of the placenta, and are argued to have played a role in the wide inter-species diversification of this critical developmental organ. Co-option of TEs by host genomes has led to the genesis of important placental genes, as well as trophoblast-specific gene regulatory elements. In mice, past work has demonstrated how multiple species-specific TE subfamilies are used as transcriptional enhancers in trophoblast stem cells. However, the involvement of TEs in the regulation of mouse placental gene expression in vivo remains unclear. Here, we characterised the TE regulatory and transcriptional landscape in mouse placenta and gauged their evolutionary dynamics through a comparative approach. We found that overall, TE cis-regulatory activity is greatly diminished in differentiated mouse trophoblast when compared to their stem cell counterpart. On the other hand, evolutionarily young IAP elements are highly expressed in the placenta and create several alternative, placenta-specific transcriptional start sites for protein-coding genes. Placenta-expressed IAP elements are genetically polymorphic between mouse strains and drive species-specific expression of associated genes. These putative co-option events are therefore recent and may represent a prime example of how TE activity can drive fast placental evolution.

sample_organism:
Mus musculus
Mus pahari

platform_organism:
Mus musculus
Mus pahari

overall_design: RNA-seq profiling of Mus musculus placenta and trophoblast stem cells, and Mus pahari placenta and selected adult tissues. CRISPR interference of IAP endogenous retroviruses in mouse trophoblast stem cells.

first: Samuele

last: Amante

first: Maria

middle: L

last: Vignola

first: Cyril

last: Pulver

first: Tessa

last: Bertozzi

first: Anne

last: Ferguson-Smith

first: Marika

last: Charalambous


title: IAP retrotransposons contribute to the transcriptional diversity of the murine placenta [ATAC-Seq]

overall_design: ATAC-seq profiling of trophoblast and extraembryonic mesoderm sorted from mouse placentas using fluorescent reporters.


title: Stage-specific transcriptomics of a leader cell reveals cell machineries driving collective invasion

city: Tel Aviv

first: Hila

last: Gingold

address: 30 Haim Levanon Street

country: Israel

institute: Tel-Aviv University

summary: Collective cell invasion underlies organ development, epithelial repair, and cancer metastasis. “Leader cells” remodel extracellular matrix, sense guidance cues, reorganize their cytoskeleton, and coordinate follower cells, but the molecular programs enabling these functions remain unclear. Here, we present a stage-specific transcriptomic dataset of the C. elegans gonadal leader cell, the distal tip cell (DTC), which invades basement membrane and guides germ cells to form U-shaped gonadal arms. Comparing invasive larval-stage DTCs with non-invasive adult-stage DTCs defines the molecular signature of an actively invading leader cell in vivo. Our dataset recapitulates known regulators of gonad morphogenesis and reveals numerous uncharacterized genes with potential roles in leader cell activity. Demonstrating dataset utility, we identify vesicular trafficking proteins enriched in invading DTCs, and demonstrate their importance for gonad development using endogenous tagging and DTC-specific RNAi. We also catalog diverse DTC-specific knockdown phenotypes. This resource establishes a molecular framework for leader cell activity and a platform to investigate conserved mechanisms of invasive migration.

overall_design: RNA-seq profiling of the Venus-positive DTCs and Venus-negative other tissues isolated from late L3, early, L4, late L4, and adult stage wild-type hermaphrodite

first: Priti

last: Agarwal

first: Iska

last: Maimon

first: Victor

last: Stolzenbach

first: Sarit

last: Anava

first: Olga

last: Antonova

first: Erin

last: Cram

first: Oded

last: Rechavi

first: Ronen

last: Zaidel-Bar


title: Ontogeny and transcriptional regulation of Thetis cells [Smart-seq3]

city: New York

first: Chrysothemis

last: Brown

state: NY

address: 417 East 68th Street

department: Immunology Program, Sloan Kettering Institute

institute: Memorial Sloan Kettering Cancer Center

summary: Thetis cells (TCs), a recently identified family of RORgt+ antigen-presenting cells (APCs), comprise four subsets, including the TC IV subset that instructs tolerance to gut microbiota and food antigens. A developmental wave of TCs creates a window of opportunity for establishing intestinal tolerance during early life. However, the ontogeny of TCs and the cues that determine their abundance and heterogeneity are not known, limiting our ability to interrogate their functional roles and exploit their therapeutic potential. Here, we identify a RORgt+ TC and lymphoid tissue inducer (LTi) cell progenitor (TLP) that differentiates into the immediate RORgt+ TC progenitor (TCP) and the LTi progenitor (LTiP), with the transcription factor PU.1 determining TC fate. Despite their similarity to myeloid-derived classical dendritic cells (cDCs), we found that TCs were descended from the common lymphoid progenitor (CLP) with deletion of the plasmacytoid DC (pDC) lineage-determining transcription factor TCF4 leading to expansion of TLP and TCs, suggesting a shared developmental precursor with pDCs. TLP is enriched in mouse fetal liver, but in contrast to LTi cells, TCs emerge postnatally, pointing to a role for developmentally-timed environmental cues in promoting TCP differentiation. We identify one such cue–provision of RANKL by lymphoid tissue organizer cells–which was essential for TC I differentiation. Together, these findings identify the ontogeny of TCs and define the transcription factors that promote TC differentiation and heterogeneity. These insights will facilitate future investigations of these enigmatic cells, which have potential therapeutic applications in promoting tolerance.

overall_design: SMART3 sequencing of mesenteric lymph nodes (mLN), peripheral lymph nodes (pLN) and large intestine (LI)  of 2-week-old Rorc-Venus mice. Cells were enriched using the strategies outlined below.

first: Yoselin

last: Paucar Iza

first: Tyler

last: Park


title: Ontogeny and transcriptional regulation of Thetis cells [CITE-Seq]

summary: Thetis cells (TCs), a recently identified family of RORgt+ antigen-presenting cells (APCs), comprise four subsets, including the TC IV subset that instructs tolerance to gut microbiota and food antigens1–6. A developmental wave of TCs creates a window of opportunity for establishing intestinal tolerance during early life1,5. However, the ontogeny of TCs and the cues that determine their abundance and heterogeneity are not known, limiting our ability to interrogate their functional roles and exploit their therapeutic potential. Here, we identify a RORgt+ TC and lymphoid tissue inducer (LTi) cell progenitor (TLP) that differentiates into the immediate RORgt+ TC progenitor (TCP) and the LTi progenitor (LTiP), with the transcription factor PU1 determining TC fate. Despite their similarity to myeloid-derived classical dendritic cells (cDCs), we found that TCs were descended from the common lymphoid progenitor (CLP) with deletion of the plasmacytoid DC (pDC) lineage-determining transcription factor TCF4 leading to expansion of TLP and TCs, suggesting a shared developmental precursor with pDCs. TLP is enriched in mouse fetal liver, but in contrast to LTi cells, TCs emerge postnatally, pointing to a role for developmentally-timed environmental cues in promoting TCP differentiation. We identify one such cue–provision of RANKL by lymphoid tissue organizer cells–which was essential for TC I differentiation. Together, these findings identify the ontogeny of TCs and define the transcription factors that promote TC differentiation and heterogeneity. These insights will facilitate future investigations of these enigmatic cells, which have potential therapeutic applications in promoting tolerance.

overall_design: scRNA-seq analysis of TCs isolated from mesenteric lymph nodes of 2-week-old  RorcVenusTnfrsf11afl/fl (n = 4 biological replicates) and RorcVenusRorgtcreTnfrsf11afl/fl mice (n = 6 biological replicates).

first: Anushka

last: Yadav


title: Large adipocytes increase vesicle-mediated lipid release and promote breast cancer malignancy

city: Ithaca

first: Claudia

last: Fischbach

address: 157 Weill Hall

institute: Cornell University

summary: Primary adipocytes exhibit striking variability in size, yet the functional consequences of adipocyte hypertrophy remain unclear due to a lack of experimental approaches to directly manipulate cell size. Here, we establish methods to isolate and culture large and small primary adipocytes from the same adipose tissue, enabling size-resolved analyses independent of systemic obesity. Using transcriptomic, lipidomic, and functional profiling across two mouse models of obesity, as well as human clinical samples, we show that adipocyte size—rather than body weight—drives distinct phenotypic cell states. Notably, large adipocytes increase lipid release mediated by elevated secretion of extracellular vesicles. In coculture assays, this shift enhances lipid uptake, migration, and proliferation of neighboring breast cancer cells through fatty acid oxidation. Consistent with these findings, individuals with larger mammary adipocytes exhibit elevated fasting triglycerides and dyslipidemia independent of body mass index. Together, our results identify adipocyte size as a key determinant of adipose tissue function with implications for both metabolic disease and cancer progression.

overall_design: RNA sequencing of size-sorted mammary adipocytes isolated from both diet-induced and genetic mouse models of obesity. Four to five biological replicates are included per condition.

first: Garrett

middle: F

last: Beeghly


title: Spatial analysis of IPMNs defines a paradoxical KRT17-positive, low-grade epithelial population harboring malignant features

first: Eileen

last: Carpenter

summary: Background & Aims: Intraductal papillary mucinous neoplasms (IPMNs) are pancreatic cysts that represent one of the few radiologically identifiable precursors to pancreatic ductal adenocarcinoma (PDAC). Though the IPMN-bearing patient population represents a unique opportunity for early detection and interception, current guidelines provide insufficient accuracy in determining which patients should undergo resection versus surveillance, resulting in a sizable fraction of resected IPMNs only harboring low-grade dysplasia, suggesting that there may be overtreatment of this clinical entity. Methods: To investigate the transcriptional changes that occur during IPMN progression, we performed spatial transcriptomics using the Nanostring GeoMx on patient samples containing the entire spectrum of IPMN disease including low-grade dysplasia, high-grade dysplasia, and IPMN-derived carcinoma.  Single cell RNA sequencing was performed on side branch and main duct IPMN biospecimens. Results: We identified a subpopulation of histologically low-grade IPMN epithelial cells that express malignant transcriptional features including  KRT17, S100A10 and CEACAM5,  markers that are enriched in PDAC. We validated this high-risk gene signature in both single-cell RNA sequenced samples and an external ST dataset containing a larger number of IPMN samples including non-tumor bearing IPMN (i.e. low-grade IPMN in isolation). Immunofluorescence staining of a large cohort of patient tissues confirmed the presence of KRT17-positive cells, which were found to comprise a small subset of epithelial cells within histologically low-grade IPMN in a patchy distribution. Conclusions: Our study demonstrates that KRT17 marks a distinct transcriptional signature in a subpopulation of epithelial cells within histologically low-grade IPMN.  This population of cells likely represents a transitional state of histologically low-grade epithelial cells undergoing progression to a higher grade of dysplasia and thus may represent a higher risk of progression to carcinoma.

overall_design: A total of 3 slides containing tissue sections from pancreatic cysts, associated malignancy, and/or adjacent normal tissue was submitted for spatial transcriptomics.  The Solid Tumor TME Morphology Kit was utilized according to manufacturer instructions to detect epithelial cells (pan-cytokeratin [CK]) together with DAPI for nuclei detection. 310 ROIs were captured for whole transcriptome profiling.


title: Identifying expression and DNA methylation biomarkers for lung adenocarcinoma risk in East Asia [RNA-seq]

city: Miaoli County

first: Shih-Sheng

last: Jiang

address: 35, Keyan Road, Zhunan Town

department: National Institute of Cancer Research

country: Taiwan

institute: National Health Research Institutes

summary: Lung adenocarcinoma (LUAD) is the most common type of lung cancer. A recent genome-wide association study (GWAS) of LUAD in East Asia reported 28 independent susceptibility variants across 25 loci and identified 2 genes whose genetically predicted expression levels are associated with LUAD risk, using an ancestry-matched lung tissue expression quantitative trait loci (eQTL) dataset. It is desirable to identify additional susceptibility loci and to understand their underlying biological mechanisms. Using an expanded GWAS of LUAD in East Asia and ancestry-matched lung tissue eQTL and DNA methylation QTL datasets, we performed transcriptome-wide association studies and DNA methylome-wide association studies simultaneously and examined the association between measured expression of genes and DNA methylation of nearby CpGs (eQTM). Genes and nearby CpGs are termed CpG–gene–LUAD trios if these three associations hold simultaneously. At Bonferroni-corrected P<0.05, we identified a new susceptibility locus (6p21.31; lead SNP rs7772643), 10 LUAD-associated genes and 86 LUAD-associated CpGs. At false discovery rate q<0.05, we identified 28 LUAD-associated genes, 220 LUAD-associated CpGs, and 45 CpG–gene–LUAD trios; among them, 43 were direction-matched regarding these three associations. These show that 23 of the known 28 susceptibility variants for LUAD in East Asia are near these genes or CpGs and MARCH3, ELF5, IKZF3, GSDMB, CCDC116, and DSP are putative novel susceptibility loci. Few of them was reported in LUAD in European populations. This study substantially advances our understanding of the etiology of LUAD in East Asia and could be useful in developing translational applications.

overall_design: From the Lung Cancer Tissue Core, 61 non-smoking subjects of lung adenocarcinoma were selected, after data preprocessing, to have obtained the gene expression profiles of the normal lung tissues adjacent to tumor, using Illumina RNA Access library kit.

first: Chao

last: Hsiung

first: I-Shou

last: Chang

first: Shih Sheng


title: Effects of maternal early gestational weight gain on skeletal muscle gene expression profile in F1 and F2 beef cattle offspring

city: Auburn

first: Wellison

middle: J. S.

last: Diniz

state: Alabama

address: CASIC Building 559 Devall Drive Auburn University, 205

department: Animal Sciences

institute: Auburn University

summary: This study evaluated how the rate of maternal body weight gain in F0 dams during early gestation affects gene expression in the skeletal muscle of F1 offspring and their F2 fetuses. During the first 84 days of gestation, we applied a nutritional plan to the F0 dams to promote two different rates of body weight gain (0.28 kg/day and 0.79 kg/day). We tested the hypothesis that maternal nutrition aiming at two rates of body weight gain (low [LG] or moderate [MG] gain) during early gestation would affect the gene expression of skeletal muscle in both the F1 offspring and the subsequent F2 generation. Thus, our aim was to measure the gene expression profiles and identify differentially expressed genes (DEG), metabolic pathways (KEGG), and biological processes (GO BP) in the skeletal muscle of F1 heifers at birth, as well as during the early gestation phase, applying the same approach to F2 fetuses.

overall_design: Sixteen crossbred Angus heifers (F0) were assigned to a low gain (LG; 0.28 kg/day) or moderate gain (MG; 0.79 kg/day). The treatments were randomly assigned during the AI process during the first 84 days of gestation. After day 84 of gestation, F0 heifers were managed as a single group until parturition, and F0 dam/F1 offspring pairs were collectively raised on pasture until weaning of F1 heifers. F1 heifers were managed similarly regardless of the F0 maternal treatments to investigate potential multigeneration effects. For newborn F1 heifers, a sample of approximately 1g of Longissimus thoracis muscle (LT) between the 12th and 13th thoracic vertebrae was collected immediately after calving and before suckling. For F2 fetuses, the whole fetus was collected, and the Longissimus dorsi was dissected. All samples collected were placed immediately on dry ice and then transferred to an ultra-freezer at −80°C for storage.

first: Carl

last: Dahlen

middle: J


title: Three Muscle-Specific DAF-16/FOXO Transcriptional Targets Activated by Reduced Insulin/IGF-1 Signaling

city: Toronto

first: Shifei

last: Wu

state: Ontario

address: 1 King's College Cir

country: Canada

institute: University of Toronto

summary: C. elegans insulin/insulin-like growth factor 1 signaling, IIS, affects diverse physiological processes through the DAF-16/FOXO transcription factor. Despite its ubiquitous presence in somatic cells, DAF-16’s effects exhibit prevalent tissue specificity as well as tissue crosstalk. This implies that tissue-specific DAF-16 transcriptional programs contribute to functional diversity of IIS. To further investigate this possibility, we sought muscle-cell-specific DAF-16 transcriptional targets. Using fluorescence-activated cell sorting to enrich for body wall muscle cells from young hermaphroditic adults, we compared the muscle cell mRNA transcriptomes under conditions of high and low IIS activity, with and without DAF-16. We further analyzed DAF-16a’s binding sites in muscle and intestine cells by chromatin-immunoprecipitation sequencing. Combined output of these analyses is 12 candidate DAF-16 targets enriched for muscle cells. Transcriptional and translational reporters for three out of the four top candidates - a secreted protein C54F6.5, a calcium-binding protein CEX-1/calexcitin, and a fatty acid metabolic enzyme MLCD-1/MCD - showed DAF-16-dependent activation specifically in body wall muscle cells. Notably, reporters for C54F6.5 and cex-1 exhibit DAF-16-independent, constitutive expression in non-muscle cells, explaining their low rank or absence from the DAF-16 target lists generated by whole-animal microarray or mRNA-sequencing analyses. These results highlight the need to examine FOXO targets in a cell-type-specific manner.

overall_design: C. elegans of different genotypes (wild-type, daf-16(lf), daf-2(lf), daf-2(lf); daf-16(lf)) expressing GFP (green fluorescent protein) specifically in the body wall muscle cells were sorted with fluorescence-activated cell sorting. GFP+; PI- (propidium iodide negative) cells were collected as muscle cells. PI- cells were collected as all cells. mRNA-seq were performed on samples from four genotypes, all with both sorted ‘muscle cell’ and ‘all cell’ samples.

first: Jinghua

last: Gao

first: Yan

last: Li

first: Charline

last: Roy

first: Ying

last: Wang

first: Ben

last: Mulcahy

first: William

first: Sruthy

last: Ravivarma

first: John

last: Calarco

first: Wesley

last: Hung

first: Mei

last: Zhen


title: singIST: an integrative method for comparative single-cell transcriptomics between disease models and humans

first: Poulami

last: Dey

address: 1301 Catherine St

institute: Michigan Medicine

summary: Disease models are fundamental tools in drug discovery and early-stage drug development, but they only approximate human disease, and selecting a suitable model is challenging. Quantitative computational methods exist to assess molecular resemblance to human conditions, but approaching that work at single-cell resolution, and doing so in an explainable and generalizable way, remain very limited. We present singIST, a computational method for comparative single-cell transcriptomics analysis between disease models and human conditions. singIST provides explainable quantitative measures on disease model similarity to the human reference at the pathway, cell type and gene levels. These measures jointly account for gene orthology, cell type presence in the model, cell type and gene importance in the human condition, and gene level fold changes in the model, within a unifying framework that controls for the intrinsic complexities of single-cell data. We first test singIST in three well-characterized murine models against moderate-to-severe Atopic Dermatitis, showing that it recapitulates established biology while generating new hypotheses. We then apply it to Hidradenitis Suppurativa, comparing in vivo human lesions with ex vivo skin explants with and without CD3/CD28 stimulation, and show that stimulation selectively improves pathways that already recapitulate the human signal. Finally, we perform simulation studies that: (i) unit-test the implementation and behaviour of the algorithm under controlled scenarios and (ii) compare singIST against a na¨ıve baseline based on overlapping differentially expressed genes.

overall_design: Human skin biopsies from affected individuals were collected and processed for library prep and next-generation sequencing at the Advanced Genomics Core at the University of Michigan

first: Johann

last: Gudjonsson

first: Lam

last: Tsoi


title: Identifying expression and DNA methylation biomarkers for lung adenocarcinoma risk in East Asia [DNA Methylation Array]

first: Shih-Sheng

last: Jiang

type: Methylation profiling by genome tiling array

overall_design: From the Lung Cancer Tissue Core, 161 non-smoking subjects with lung adenocarcinoma were selected, after data preprocessing, to have obtained DNA methylation profiles of their adjacent normal lung tissues. Of these, 114 samples were profiled using Illumina 450K chips, 31 using EPIC, and 16 using EPICv2.

first: Chao

last: Hsiung

first: I-Shou

first: Shih Sheng


title: IAP retrotransposons contribute to the transcriptional diversity of the murine placenta [Cut & Tag]

first: Miguel

last: Branco

overall_design: CUT&Tag for H3K27ac, H3K4me1 and H3K4me3 in Mus musculus placenta, trophoblast, extraembryonic mesoderm and trophoblast stem cells, and in Mus pahari placenta and selected adult tissues.

first: Samuele

last: Amante

first: Maria

last: Vignola

first: Cyril

last: Pulver

first: Tessa

last: Bertozzi

first: Anne

last: Ferguson-Smith

first: Marika

last: Charalambous


title: Mitochondrial transfer to granulocytic myeloid-derived suppressor cells augments immunosuppressive activity [scRNA-seq]

city: Omaha

first: Prabhakar

last: Arumugam

state: Nebraska

address: 985900 Nebraska Medical Center

department: Department of Pathology, Microbiology and Immunology

institute: University of Nebraska Medical Center

summary: The anti-inflammatory properties of granulocytic myeloid-derived suppressor cells (G-MDSCs) promote Staphylococcus aureus (S. aureus) biofilm persistence. Evidence suggests that G-MDSC activity is not only shaped by S. aureus products but also by intrinsic metabolic programs. This study explored whether G-MDSC activity could be modulated by increasing mitochondrial abundance using a co-culture paradigm with macrophages as a mitochondrial donor. Macrophages transferred mitochondria directly to G-MDSCs via tunneling nanotubes, which enhanced G-MDSC respiration as reflected by increased basal, maximal, and spare respiratory capacity. Augmenting mitochondrial abundance in G-MDSCs enhanced T cell suppressive activity concomitant with decreased TNF and IL-6 production. Adoptive transfer of macrophages in a mouse model of S. aureus prosthetic joint infection revealed mitochondrial transfer to G-MDSCs in vivo that enhanced their suppressive activity, leading to increased bacterial burden, which was reversed when mitochondrial DNA-depleted macrophages were introduced. Mitochondrial transfer in vivo was also demonstrated using transgenic mice where macrophage mitochondria were tagged with GFP. These findings support that G-MDSCs can exploit mitochondria to augment their anti-inflammatory properties in response to S. aureus biofilm.

overall_design: At day 3 post-infection, untreated and EtBr-treated macrophages were adoptively transferred into mice with Staphylococcus aureus prosthetic joint infection. Phosphate-buffered saline (PBS) was administered to both wild-type and interferon gamma knockout (IFNG KO) mice. Twenty-four hours following adoptive transfer, infected tissue was harvested, and single-cell suspensions were prepared. Live CD45⁺ cells were isolated by fluorescence-activated cell sorting (FACS) and subjected to single-cell RNA sequencing analysis.

first: Tammy

last: Kielian


title: Prehabilitation as a biologically active intervention reprograms pancreatic tumor-immune microenvironment to enhance antitumor immunity

city: HOUSTON

first: HONG

last: ZHAO

address: 6670 Bertner Ave

institute: HOUSTON METHODIST HOSPITAL

summary: This dataset contains NanoString GeoMx Digital Spatial Profiling data generated from surgically resected pancreatic ductal adenocarcinoma (PDAC) specimens collected from patients who underwent a multimodal prehabilitation program and matched untreated controls. Spatial transcriptomic profiling was performed across immune (CD45 positive), tumor (PanCK positive), and stromal regions of interest to investigate the biological effects of prehabilitation on the PDAC tumor microenvironment. Differential expression, pathway enrichment, and immune deconvolution analyses were used to evaluate transcriptional changes associated with cytotoxic immune activation, stromal remodeling, and oncogenic signaling. The goal of this exploratory dataset is to provide an initial resource for investigating how prehabilitation may influence immune–tumor interactions in PDAC and to support future studies integrating biological and clinical endpoints.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: Formalin-fixed paraffin-embedded pancreatic ductal adenocarcinoma (PDAC) tissues were obtained from patients who participated in a multimodal prehabilitation program prior to surgical resection and from matched control patients who underwent standard management. Spatial transcriptomic profiling was performed using the NanoString GeoMx Digital Spatial Profiler with whole-transcriptome probes. Regions of interest were selected based on immunofluorescent segmentation markers to capture immune (CD45 positive), tumor (PanCK positive), and stromal compartments within each specimen. Raw DSP count matrices were generated for each compartment and used for downstream differential expression, pathway enrichment, and immune deconvolution analyses. The dataset was generated as part of an exploratory pilot study to examine the biological effects of prehabilitation on the tumor microenvironment in PDAC.

first: Hong

last: Zhao

first: Renee

last: Stubbins


title: DNA Methylation-based Stratification of Pediatric Thyroid Carcinoma Invasiveness

city: Philadelphia

first: Wanding

last: Zhou

state: PA

address: 3501 Civic Center Blvd

department: Pathology and Lab Medicine

institute: Children's Hospital of Philadelphia

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

overall_design: Refer to individual Series


title: Ontogeny and transcriptional regulation of Thetis cells [Flex]

first: Chrysothemis

last: Brown

overall_design: Lin– IL7R fate-mapped and Lin–RORgt(Venus)+ cells were isolated from E16.5-18.5 FL, BM, and mesenteric anlagen, and P7 liver, BM, and mLN of  Il7rcreR26lsl-tdTomato and RorcVenus mice.

first: Yoselin

last: Paucar Iza

first: Tyler

last: Park


title: E96A baseline transcriptional changes

city: Indianapolis

first: Kelley

last: Mark

state: Indiana

institute: Herman B Wells Center for Pediatric Research

summary: APE1/Ref-1 is the primary apurinic/apyrimidinic endonuclease in the base excision repair pathway. To define baseline transcriptional changes resulting from loss of APE1 endonuclease activity, RNA-seq was performed on Pa03C pancreatic ductal adenocarcinoma cell lines engineered to carry a homozygous E96A mutation and on matched Cas9 controls. Cells were cultured under untreated conditions. This dataset provides gene expression profiles associated with impaired APE1 endonuclease function in PDAC cells.

overall_design: Pa03C pancreatic ductal adenocarcinoma (PDAC) cell lines were engineered to carry a homozygous APE1 E96A endonuclease-deficient mutation or a matched Cas9 control allele. Cells were cultured under standard growth conditions without drug treatment. RNA-seq was then performed on Pa03C Cas9 and Pa03C E96A PDAC cell lines under untreated conditions. The study aims to capture baseline transcriptional consequences of impaired APE1 endonuclease activity

first: Mahmut

last: Mijit

first: Eyram

last: Kpenu

first: Mark

last: Kelley


title: Alzheimer’s Disease-Associated circPDE4B Interacts with GEMIN5 to Regulate Translation

city: Boston

first: Junming

last: Hu

state: MA

address: 75 E Concord St

institute: Boston University

type: Non-coding RNA profiling by high throughput sequencing

summary: In the intricate landscape of Alzheimer’s disease (AD) pathology, circular RNAs (circRNA) are emerging as influential players, orchestrating crucial aspects of gene expression, synaptic plasticity, and neuronal function. In this study, we characterize the biological role of circPDE4B, a highly abundant circRNA that is markedly downregulated in AD across brain regions. Unlike most circRNAs, circPDE4B is expressed at substantially higher levels than its linear PDE4B transcript, suggesting a distinctive regulatory significance. Functional analyses in neuronal progenitor cells revealed that circPDE4B reduction triggers a translational stress response, indicating its critical role in maintaining protein synthesis homeostasis. Pulldown of circPDE4B identified the translation regulatory factor GEMIN5 as a highly specific, major interactor, and GEMIN5 knockdown showed it as a key mediator of actions of circPDE4B on translation. Loss of circPDE4B also reduced mTOR levels and subsequently enhanced autophagic flux. Notably, circPDE4B downregulation was sufficient to mitigate tau pathology in a 3D human assembloid model of tauopathy. We also identified a subset of AD-associated miRNAs interacting with circPDE4B, implicating additional layers of post-transcriptional regulation. Collectively, these findings define circPDE4B as a multifunctional regulator of neuronal homeostasis that integrates translational control, autophagy, and miRNA-mediated pathways, illuminating its potential role in the stress response and pathophysiology of AD.

overall_design: To identify potential miRNA partners of circPDE4B, we performed RNA immunoprecipitation (RIP) targeting circPDE4B in neuronal progenitor cells (NPCs). Small RNAs were isolated from the immunoprecipitates and subjected to small RNA sequencing (smRNA-seq). The experiment included five biological replicates for the circPDE4B IP and five for the scrambled control IP.

first: Sambhavi

last: Puri

first: Mintao

last: Lin

first: Xiaoling

last: Zhang

first: Benjamin

last: Wolozin


title: Single-cell Spatial Transcriptomics Reveals Hepatocyte Metabolic Reprogramming in Fontan Associated Liver Disease

city: Pittsburgh

first: Shuchang

last: Liu

address: 203 Lothrop Street

institute: University of Pittsburgh

summary: Background: Fontan-associated liver disease (FALD) is a common sequelae of single-ventricle patients palliated with the Fontan operation. FALD severity can impact clinical decisions; however, the pathophysiology of FALD progression is unknown.   Methods: Single-cell spatial transcriptomics (ST) was performed on liver explant tissue sections from FALD patients with early and advanced fibrosis using CosMxTM Spatial Molecular Imaging with in-situ hybridization of 6000 genes (n=2). Immunofluorescence for hepatic zonation and cellular stress markers was used to confirm protein expression based on ST analysis in biopsy and explant FALD tissues (n=17).   Results: Unbiased clustering yielded 12 liver cell types, comprising five subtypes of hepatocytes. FALD with advanced fibrosis demonstrated the expansion of mid-zonal hepatocytes, accompanied by the loss of metabolic markers associated with canonical pericentral and periportal hepatocytes. Advanced FALD hepatocytes uniquely demonstrated increased cellular stress and a redundant metabolic phenotype.  Advanced FALD hepatocytes changed into a senescent-like state, which we have termed hepatocyte metabolic exhaustion. Protein expression analysis confirmed metabolic exhaustion within hepatocytes and upregulation of cellular stress markers in advanced FALD tissue specimens.  Immunofluorescence staining of FALD samples demonstrated a disruption of zonation and a significant increase in heat shock protein 70 (HSP70). HSP70 expression strongly correlated with the congestive hepatic fibrosis (CHF) score.  Conclusions: Single-cell ST has identified a population of hepatocytes with features of metabolic exhaustion and cellular stress unique to advanced FALD.

overall_design: Single-cell spatial transcriptomics (ST) was performed on liver explant tissue sections from FALD patients with early and advanced fibrosis using CosMxTM Spatial Molecular Imaging with in-situ hybridization of 6000 genes.

first: Brandon

last: Lehrich

first: Jordann

middle: N

last: Lewis

first: Vik

last: Meadows

first: Lori

last: Schmitt

first: Mylarappa

middle: B

last: Ningappa

first: Jia-Jun

first: Silvia

first: Catherine

last: Gestrich

first: Victor

middle: O

last: Morell

first: Rakesh

last: Sindhi

first: Satdarshan

last: Monga

first: Anita

last: Saraf


title: E96A TMZ Treatment Study

first: Kelley

last: Mark

summary: Temozolomide (TMZ) induces DNA alkylation lesions repaired primarily through base excision repair. To characterize transcriptional responses to TMZ in the context of impaired APE1 endonuclease activity, RNA-seq was performed on Pa03C Cas9 and E96A mutant cell lines following treatment. Cells were treated with TMZ for 4 hours or 8 hours, along with untreated controls. This dataset captures genotype-specific and time-dependent transcriptional responses to alkylation stress in PDAC cells.

overall_design: Pa03C PDAC cell lines carrying either a Cas9 control allele or a homozygous APE1 E96A endonuclease-deficient mutation were treated with temozolomide (TMZ) or left untreated. For each genotype, cells were collected at baseline (untreated), 4 hours after TMZ treatment, and 8 hours after TMZ treatment. Total RNA was isolated each individual sample. This design enables comparison of genotype-specific and time-dependent transcriptional responses to TMZ-induced DNA damage

first: Eyram

last: Kpenu

first: Mark

last: Kelley


title: Functional characterization of BAP1 mutations in genome edited cholangiocarcinoma organoids: role in cell death and drug responses

city: Rotterdam

first: Henk

middle: P.

last: Roest

state: Choose State

address: Doctor Molewaterplein 40

department: Surgery

country: Netherlands

institute: Erasmus MC

summary: Cholangiocarcinoma (CCA) is a genetically heterogeneous malignancy of the bile ducts with limited effective treatments and variable chemotherapeutic responses. BRCA1-associated protein 1 (BAP1), a tumor suppressor gene frequently mutated in CCA, encodes a nuclear deubiquitinating enzyme involved in chromatin remodeling and cell death regulation. In this study, we investigated the role of BAP1 mutations in programmed cell death and drug response using patient-derived and prime-edited CCA organoids (CCAOs). BAP1-mutant organoids exhibited impaired activation of apoptosis and necroptosis, as evidenced by reduced cleaved caspase-3 and pMLKL expression. Transcriptomic analysis revealed BAP1-dependent gene expression changes including enrichment of pathways related to stress response, ion transport, and metabolic detoxification. Interesting, BAP1 mutant CCAOs showed enhanced sensitivity to sorafenib, a multikinase inhibitor commonly used in biliary tract cancer. These findings highlight BAP1 as a modulator of cell death and a potential predictive biomarker for sorafenib response in CCA, with implications for personalized therapy design.

overall_design: investigate how BAP1 influences cell death pathways in prime-edited CCAOs. CCAOBAP1-WT was prime edited, FACSorted and clonally expended. Clones were screened by Western blot for BAP1 expression and one clone was selected based on substantial reduction of BAP1 protein level. Three technical replicates of targeted and control were sequenced

first: Wunan

last: Mi

first: Shaojun

last: Shi

first: Imre

last: Schene

first: Indi

middle: P

last: Joore

first: Titia

last: Sixma

first: Sabine

last: Fuchs

first: Luc

last: van der Laan

first: Monique

last: Verstegen


title: V gamma 1 gamma-delta T cells steer airway macrophages towards a pro-fibrotic response in an autochthonous lung cancer mouse model. [RNA-Seq]

city: Glasgow

first: Robin

last: Shaw

address: Switchback Road

institute: CRUK Scotland Institute

summary: Gamma-delta T cells are important for host defence at the respiratory mucosa, acting directly or through interactions with other cells. However, how Gamma-Delta T cells influence other immune cells in the lung remains unclear. Using a genetically engineered mouse model of lung cancer, we show that tumours drive expansion of both CD27+ and CD27— Gamma-Delta T cells. Advanced microscopy techniques indicated that CD27— Gamma-Delta T cells are enriched in tumours, while CD27+ Gamma-Delta T cells are more prone to interact with macrophages in tumour-associated adventitial cuffs. SiglecFlow pro-fibrotic airway macrophages were more prevalent in lung tumour-bearing mice than tumour-free mice. This pro-fibrotic subset was reduced in lungs when the cancer model was crossed to Tcrd knockout mice or treated with V Gamma1-depleting antibodies, but not in TcrgV4/6 knockout mice. Thus, our findings implicate V Gamma1 Gamma-Delta T cells in driving tumour-associated airway macrophage functional imprinting. Determining the translatability to human health may offer new avenues for refining patient management and immunotherapeutic strategies.

overall_design: Bulk RNA-seq was performed on FACS-isolated alveolar macrophage subsets from: 1) Tumour-bearing KM lungs from Tcrd knockout mice vs wild type to identify differentially expressed genes. These genes were used to create a module score for assessing scRNA-seq clusters. 2) Tumour-bearing KM lungs, comparing SiglecF low and SiglecF hi AMs to define transcriptional programmes associated with profibrotic versus immunoreactive states; these signatures were then related back to scRNA-seq clusters.

first: Ximena L.

last: Raffo-Iraolagoitia

first: Amanda J.

last: McFarlane

first: Sarah

last: Laing

first: Ryan

last: Corbyn

first: Lindsey W. G.

last: Arnott

first: Frédéric

last: Fercoq

first: Lynn

last: McGarry

first: Judith

last: Secklehner

first: Marco

last: De Donatis

first: John B. G.

last: Mackey

first: Bjorn

last: Kruspig

first: Robert

last: Wiesheu

first: Ya-Ching

last: Hsieh

first: Kai

last: Rakovic

first: John

last: Le Quesne

first: Graeme

last: Clark

first: Colin

last: Nixon

first: Crispin

last: Miller

first: Kristina

last: Kirschner

first: Calum C.

last: Bain

first: Daniel J.

last: Murphy

first: Seth B.

last: Coffelt

first: Leo M.

last: Carlin


title: DNA Methylation-based Stratification of Pediatric Thyroid Carcinoma Invasiveness [2nd batch]

first: Wanding

last: Zhou

summary: Accurate triage of pediatric thyroid carcinomas requires reliable biomarkers of tumor invasiveness. DNA methylation, a proven molecular analyte for cancer classification, offers potential for stratifying thyroid carcinoma behavior. In a cohort of 88 patients, we identified distinct methylation profiles associated with both tumor invasiveness and driver mutations, such as BRAF p.V600E, Ras-like, kinase fusions, and DICER1 mutations. Differentially methylated regions implicate immune infiltration and disrupted thyroid tissue development and function, implicating nuclear receptor, Hippo, and AP1 signaling pathways. Using these features, we developed accurate classifiers for tumor invasiveness and driver mutation groups. Our findings highlight the clinical utility of DNA methylation profiling for risk stratification and prognostic assessment in pediatric thyroid cancers.

overall_design: This is an analysis of a cohort of children with thyroid carcinoma from the Children’s Hospital of Philadelphia (CHOP) after obtaining prospective consent from caregivers. In this study, we selected 88 primary tissue and 10 matched lymph node metastasis samples for DNA methylation profiling. Patients with regional LN metastasis were subgrouped into high-invasive tumors (N1a and N1b, n=55) and low-invasive tumors (N0, n=33). 16 high-invasive cases (14 N1a, 2 N1b) with less than five LN positive for the disease were considered highly invasive with low confidence. 11 N1b cases are associated with distant metastasis (M1). We isolated and quantified DNA from tissue, and performed methylation analysis using the Infinium MethylationEPIC v2.0 BeadChip. Processing IDAT files generated from Infinium BeadChip-based data was performed using the openSesame workflow within the SeSAMe package. This workflow effectively addresses critical processing challenges, including signal background correction, dye bias adjustment, inference of color channel swaps, masking of suboptimal signals, and precise extraction of DNA modification levels (beta values).

first: Jenny

middle: Z

last: Li

first: Julio

last: R Filho

first: Andrew

last: Bauer

first: Aime

middle: T

last: Franco


title: CPSF73 activation and 3’ Pol II pausing are lost during read-through transcription after heat shock  [RNA-seq]

city: Boulder

first: Kaitlyn

last: Walsh

state: CO

address: 3415 Colorado Ave

institute: CU Boulder

summary: Multiple types of cellular stress induce RNA polymerase II (Pol II) to read through normal termination sites at mRNA genes, causing long downstream regions to be transcribed. We observed strong read-through transcription downstream of thousands of genes after heat shock. Additionally, the 3’ Pol II pause that normally occurs during termination was globally lost. Heat shock also increased phosphorylation of Tyr1 and Ser2 residues in the Pol II C-terminal domain (CTD) at the 3’ ends of genes, with smaller increases observed at genes with read-through transcription. We found that 3’ RNA cleavage, a key step of transcription termination, was defective at read-through genes after heat shock; however, the CPSF73 endonuclease responsible for cleavage remained at the 3’ ends of genes. Over-expressing an activator of CPSF73 activity, RBBP6, during heat shock rescued the loss of cleavage and attenuated read-through transcription. Our data suggest that heat shock alters Pol II speed, CTD phosphorylation, and CPSF73 activity via RBBP6, contributing to a multifaceted mechanism that enables transcription to continue far downstream of normal termination sites during cellular stress.

overall_design: 4sU RNA-seq, ctl and heat shocked HEK293 cells, basal and N-RBBP6 expressing cell line

middle: E

first: James

middle: A

last: Goodrich

first: Jennifer

last: Kugel


title: CPSF73 activation and 3’ Pol II pausing are lost during read-through transcription after heat shock  [ChIP-seq]

overall_design: ChIP-seq, ctl and heat shocked HEK293 cells, ChIP for total RNA Pol II, Tyr1P RNA Pol II, Ser2P RNA Pol II


title: Human intranasal olfactory biopsy analysis of Alzheimer's Disease subjects

city: Durham

first: Bradley

last: Goldstein

state: NC

address: 40 Duke Medicine Cir

institute: Duke University

summary: Single-cell RNA sequencing was performed on brush biopsy samples from the human olfactory epithelium (n = 22 donors) spanning control (n = 6), pre-clinical (n = 9) and clinical (n = 7) Alzheimer’s disease stages. The dataset comprises ~200,000 high-quality live cells encompassing neuronal, epithelial and immune lineages. These profiles enable comparative analyses of early neuroimmune changes associated with Alzheimer’s progression and provide a resource for identifying peripheral biomarkers of disease.

overall_design: Single cell 3' and 5' RNAseq of live olfactory epithelium from 22 adult human Control (n=6), Pre-Clinical Alzheimer's (n=9) or Clinical Alzheimer's (n=7) (based on cognitive function and CSF Aβ42/Aβ40 ratio) using the 10X Genomics Chromium platform and sequenced on Illumina NovaSeqX. Control Subject 20, Clinical AD Subject 21, and Clinical AD Subject 22 were 5' scRNAseq, rest are 3' scRNAseq.

first: Vincent

last: D'Anniballe


title: V gamma 1  gamma-delta T cells steer airway macrophages towards a pro-fibrotic response in an autochthonous lung cancer mouse model  [scRNA-Seq]

first: Robin

last: Shaw

overall_design: Lung tissue from Mus musculus was extracted, and three distinct regions were sampled: tumour tissue, tumour-distant tissue, and normal tissue. Macrophages were isolated from each sample using flow cytometry. The sorted cell populations were then sequenced using the 10X Genomics Chromium Single Cell 3′ protocol.

first: Ximena L.

last: Raffo-Iraolagoitia

first: Amanda J.

last: McFarlane

first: Sarah

last: Laing

first: Ryan

last: Corbyn

first: Lindsey W. G.

last: Arnott

first: Frédéric

last: Fercoq

first: Lynn

last: McGarry

first: Judith

last: Secklehner

first: Marco

last: De Donatis

first: John B. G.

last: Mackey

first: Bjorn

last: Kruspig

first: Robert

last: Wiesheu

first: Ya-Ching

last: Hsieh

first: Kai

last: Rakovic

first: John

last: Le Quesne

first: Graeme

last: Clark

first: Colin

last: Nixon


title: Transcriptional competence defines the heterochromatin nucleating potential of isolated MSR copies [HP1a_ChIP]

city: Freiburg im Breisgau

first: Galina

last: Erikson

address: Stübeweg 51

country: Germany

institute: Max Planck Institute of Immunobiology and Epigenetics

summary: Heterochromatin defines a transcription-repressive chromatin state. In mouse cells, heterochromatin is associated with underlying arrays of A/T-rich, 234 bp DNA repeat elements, called the major satellite repeats (MaSat or MSR). We have examined > 18,000 copies of MSR repeat units in mouse ES cells and identified that heterochromatin is only formed at transcriptionally competent MSR variants. To directly dissect the function of MSR DNA, we inserted isolated MSR copies into an inert genomic region that is repeat- and gene-free. Insertion of three or more intact MSR units, but not one MSR unit, induced heterochromatic histone marks, recruitment of HP1 and incorporation of histone H1. Only transcriptionally competent MSR copies, but not permutated MSR variants or other DNA repeats, such as LINE1 5’UTR elements can form de novo heterochromatin. MSR-derived transcription is bi-directional and MSR driven transcripts are attenuated by the RNA polymerase II (RNAPII) associated Integrator complex. This study resolves a long-standing paradox between heterochromatic and euchromatic transcriptional activity and uncovers a DNA/RNA based logic for the establishment of heterochromatin in the mouse epigenome.

overall_design: To investigate whether HP1⍺ is altered at endogenous MSR variants upon INTS11 depletion for 24 hrs. INTS11-degron mESCs (E14) were fixed by double cross-linking (2 mM DSG and 1% formaldehyde) and proccessed using RELACS nuclei barcoding protocol (Arrigoni et al., 2018).

first: Yi-Hsuan

last: Lo

first: Thomas

last: Jenuwein


title: Transcriptional competence defines the heterochromatin nucleating potential of isolated MSR copies [H3K9me3_ChIP]

overall_design: To investigate whether H3K9me3 is altered at endogenous MSR variants upon INTS11 depletion for 24 hrs. INTS11-degron mESCs (E14) were fixed by double cross-linking (2 mM DSG and 1% formaldehyde) and proccessed using RELACS nuclei barcoding protocol (Arrigoni et al., 2018).


title: Transcriptional competence defines the heterochromatin nucleating potential of isolated MSR copies. [RNA-Seq]

overall_design: To investigate the effect of INTS11 on regulating transcription of MSR repeats.

first: Lo

last: Yi-Hsuan

first: Erikson

last: Galina

first: Jenuwein

last: Thomas


title: DNA Methylation-based Stratification of Pediatric Thyroid Carcinoma Invasiveness [1st batch]

first: Wanding

last: Zhou

first: Jenny

last: Li

first: Julio

last: R Filho

first: Andrew

last: Bauer

first: Aime

last: Franco


title: Mitochondrial transfer to granulocytic myeloid-derived suppressor cells augments immunosuppressive activity

first: Prabhakar

last: Arumugam

summary: The anti-inflammatory properties of granulocytic myeloid-derived suppressor cells (G-MDSCs) promote Staphylococcus aureus (S. aureus) biofilm persistence. Evidence suggests that G-MDSC activity is not only shaped by S. aureus products but also by intrinsic metabolic programs. This study explored whether G-MDSC activity could be modulated by increasing mitochondrial abundance using a co-culture paradigm with macrophages as a mitochondrial donor. Macrophages transferred mitochondria directly to G-MDSCs via tunneling nanotubes, which enhanced G-MDSC respiration as reflected by increased basal, maximal, and spare respiratory capacity. Augmenting mitochondrial abundance in G-MDSCs enhanced T cell suppressive activity concomitant with decreased cytokine production. Adoptive transfer of macrophages in a mouse model of S. aureus prosthetic joint infection revealed mitochondrial transfer to G-MDSCs in vivo that enhanced their suppressive activity, leading to increased bacterial burden. These findings support that G-MDSCs can exploit mitochondria to augment their anti-inflammatory properties in response to S. aureus biofilm.

overall_design: Bulk RNA sequencing control and mitochondria-transferred G-MDSCs that are either unstimulated or stimulated with S. aureus biofilm for 2 h.

first: Tammy

last: Kielian


title: RNA-seq of PBMCs of patients with sepsis and nonseptic controls

city: Shanghai

first: wenchen

last: luo

address: Dongan Road

institute: Fudan University

first: Wenchen

last: Luo

first: Jing

last: Zhong

first: Zhixin

last: Qiu


title: Luteolin inhibits Th17 cell differentiation and spinal infiltration in RA [RNA-seq]

city: Hangzhou

first: Yuepeng

last: Jiang

address: binwen road

institute: zhejiang chinese medical university

summary: RNA-seq was used to explore the DEGs from control vs. CIA and CIA+LUT vs. CIA CD4+ T cell

overall_design: Total RNA was extracted from CD4+ T cells by using TRIzol Reagent (Invitrogen, Cat NO. 15596026). The RNA purity, concentration, and integrity were evaluated with an Agilent TapeStation 4200 (Agilent Technologies). RNA sequencing library was prepared by mRNA Library Prep Kit for Illumina®, and the indexed libraries were subjected to sequencing on DNBSEQ-T7 sequencer with PE150 model. Read mapping was performed via STRA software (version 2.5.3a) using CHOK1GS from Ensembl as a reference genome. Afterwards, high-quality reads mapped to the exon regions of each gene were counted by featureCounts (Subread-1.5.1; Bioconductor) and then RPKMs were calculated. Differential expression analysis was identified using the edgeR package (version 3.12.1).


title: Discovery and Evaluation of Biomarkers for Triple-Negative Breast Cancer Subtypes Uncovers Patient Stratification and Targeted Therapeutic Strategies

city: Badalona

first: David

last: Olivares

state: Select One

address: Ctra de Can Ruti, Camí de les Escoles s/n 08916 Badalona, Barcelona

country: Spain

institute: Josep Carreras Research Instiute

summary: Triple-negative breast cancer (TNBC) is the most heterogeneous and aggressive subtype of breast carcinoma, defined by the absence of clinical biomarkers and the lack of targeted therapies. Despite numerous clinical trials, patient stratification remains suboptimal, limiting the identification of effective treatment strategies. In this study, we aimed to identify biomarkers exclusively expressed in the basal mammary epithelial compartment to refine TNBC subclassification. Through computational analysis of single-cell RNA sequencing data, we defined a set of basal identity genes, which were subsequently validated by immunohistochemistry in two independent TNBC cohorts. This approach enabled the identification of a novel TNBC subgroup, termed true basal TNBC (tB-TNBC), associated with poorer prognosis and distinct molecular features. To uncover therapeutic vulnerabilities in this subgroup, we conducted a high-throughput screen of 3,200 FDA-approved compounds in breast cancer cell lines classified by basal marker expression. This analysis identified dasatinib as a promising candidate with selective activity against tB-TNBC models. Furthermore, TAGLN emerged as a strong predictive biomarker of dasatinib response, with functional studies confirming its role in modulating drug sensitivity. Altogether, these findings support the clinical utility of basal markers for TNBC stratification and highlight a targeted treatment opportunity for tB-TNBC patients.

overall_design: Total RNA was extracted using the NZY miRNA Isolation & RNA Clean-up Kit (MB13402, NZYtech) following the manufacturer’s protocol. The purity and concentration of the RNA from each sample were assessed using an Agilent TapeStation with an RNA ScreenTape Analysis Kit. mRNA enrichment was subsequently performed using polyA-tail-connected magnetic beads and oligos. This was followed by double-stranded DNA synthesis and polymerase chain reaction (PCR) amplification using specific primers. The PCR products were subjected to thermal denaturation to produce single-stranded DNA, which was cyclized into a circular DNA library using bridge primers. Sequencing was conducted using the DNBSEQ platform (BGI Genomics Co.). Raw sequencing data were preprocessed using SOAPnuke software (BGI Genomics Co.). This involved removing reads with adaptor contamination, more than 5% N content, and low-quality reads (where more than 20% of the bases had a quality score below 15). The resulting "Clean Reads" were saved in FASTQ format. RNA-seq data were analyzed using the Galaxy workbench platform [33], adhering to specific recommendations [34]. Quality control and trimming of the reads were performed using MultiQC [35] and Cutadpt [36]. The reads were then mapped to the reference genome (Hg38, human genome build 38) using STAR [37]. From the mapped sequences, the number of reads per annotated gene was counted using featureCounts [38]. Subsequently, DESeq2 [39] was used to normalize the read counts and identify differentially expressed genes. Functional enrichment analysis of differentially expressed genes was performed using GSEA [40]

first: D

last: Ortega-Álvarez

last: Olivares-Osuna

first: V

last: Rodilla


title: TGFβ signaling promotes cell cycle progression and resistance to the CDK4/6 inhibitor palbociclib through SOX4 transcriptional modulation in breast cancer cells

city: Uppsala

first: Mohamad

middle: Moustafa

last: Ali

state: Uppsala län

address: husargatan 3, Biomedical Center

department: IMBIM

country: Sweden

institute: Uppsala University

summary: During pre-malignant hyperplastic growth, TGFβ restricts cell proliferation and inflammation, while on the other hand, TGFβ promotes migration and distal metastasis of cancer cells. To dissect the temporal chromatin-based transcriptional response to TGFβ, we employed 3D culture models of isogenic human breast epithelial cells, exemplified by non-oncogenic MCF-10A (MI) and its HRAS-transformed counterpart (MII). Genome-wide chromatin accessibility profiling revealed the extensive chromatin opening induced by TGFβ at transcription start sites and enhancer elements in both models, with a marked enrichment of SOX4 binding motifs in oncogenic cells.

overall_design: Comparative gene expression and chromatin landscape profiling using RNA-seq and ATAC-seq in normal-like and RAS-transformed breast epithelial cells growing in 3D conditions.

first: Carl-Henrik

last: Heldin

first: Aristidis

last: Moustakas


title: Luteolin inhibits Th17 cell differentiation and spinal infiltration in RA

summary: Cut&tag was applied to explore the mechanism of NFATC2 confers Th17 cell differentiation and migration in CIA.

overall_design: CD4+T cells from CIA and control mice were harvested, counted, and centrifuged at 600g for 3min at room temperature.  A total of 50,000 cells were subject to the CUT&Tag assay performed by SeqHealth (Wuhan, China) using a commercial kit (Vazyme, China, TD903) according to the manufacturer's protocol.  Briefly, cells were incubated with Concanavalin A-coated magnetic beads.  Subsequently, anti-NFATC2 or IgG control antibody were added, and the cells were incubated on a nutator overnight at 4℃.  After that, secondary antibody was performed on a nutator for 1h at room temperature followed by incubation with hyperactive pG-Tn5 transposonase.  After transposon activation and tagmentation, DNA was extracted, amplified, and purified to construct library.  Sequencing was performed in the Novaseq 6000 sequencer (Illumina) with PE150 model.


title: Isolated DNA repeat units nucleate heterochromatin through Integrator-coupled transcriptional attenuation

first: Galina

last: Erikson

summary: Heterochromatin defines a transcription-repressive chromatin state. In mouse cells, heterochromatin is associated with underlying arrays of A/T-rich, 234 bp DNA repeat elements, called the major satellite repeats (MaSat or MSR). We have examined > 18,000 copies of MSR repeat units in mouse ES cells and identified that heterochromatin is only formed at transcriptionally competent MSR variants. To directly dissect the function of MSR DNA, we inserted isolated MSR copies into an inert genomic region that is repeat- and gene-free. Insertion of three or more intact MSR units, but not one MSR unit, induced heterochromatic histone marks, recruitment of HP1 and incorporation of histone H1. Only transcriptionally competent MSR copies, but not permutated MSR variants or other DNA repeats, such as LINE1 5’UTR elements can form de novo heterochromatin. MSR-derived transcription is bi-directional and MSR driven transcripts are attenuated by the RNA polymerase II (RNAPII) associated Integrator complex. These data link RNA processing of MSR transcripts by the Integrator with heterochromatin formation. This study resolves a long-standing paradox between heterochromatic and euchromatic transcriptional activity and uncovers a DNA/RNA based logic for the establishment of heterochromatin in the mouse epigenome.

overall_design: To investigate whether Integrator complex is presented at endogenous MSR. Wild type mESCs (WT26) are fixed by double cross-linking (2 mM DSG and 1% formaldehyde) and proccessed using RELACS nuclei barcoding protocol (Arrigoni et al., 2018).

first: Yi-Hsuan

last: Lo

first: Thomas

last: Jenuwein


title: tRNA-derived RNA processing in sperm transmits non-genetically inherited phenotypes to offspring in C. elegans

first: Colin

last: Conine

state: Pennsylvania

address: 421 Curie Blvd BRB 1213

department: Genetics and Neonatology

institute: UPenn Perelman SOM and CHOP

summary: The environment encountered by an organism can modulate epigenetic information in the gametes to transmit non-genetically inherited phenotypes to offspring. In mouse models, the diet of males regulates specific tRNA-derived RNAs (tDRs) in sperm. After fertilization, tDRs regulate embryonic gene expression and generate metabolic phenotypes in adult offspring through uncharacterized changes during development. Here we demonstrate that in C. elegans tDRs also accumulate in sperm and can similarly transmit epigenetically inherited phenotypes to offspring. We identify the RNaseT2 enzyme, rnst-2, as a regulator of C. elegans’ tDR accumulation. RNST-2 processes or degrades tRNA-halves, leading to the accumulation of shorter <30 nt fragments. This rnst-2 dependent regulation of tDR length distribution modulates specific tDRs in sperm which, after fertilization, regulate early embryonic and developmental gene expression, leading to adaptive phenotypes in progeny. Our findings establish tDRs as a deeply conserved carrier of intergenerational epigenetic information and position the worm as a model for dissecting paternal non-genetic inheritance mechanistically.

overall_design: Small RNA-seq on adult C. elegans males and females, as well as sperm. mRNA-seq on adult C. elegans males and females. Single embryo RNA-seq on C.elegans 2- and 8-cell embryos.

middle: C

first: Nicholas

middle: S

last: Galambos


title: Whole transcriptome analysis of unmutated sporadic ALS patients’ peripheral blood reveals phenotype-specific gene expression signature

city: Pavia

first: Francesca

last: Dragoni

state: Italy

address: Via Mondino, 2

country: Italy

institute: IRCCS Mondino Foundation

summary: Amyotrophic lateral sclerosis (ALS) is an adult neurodegenerative disorder. According to clinical criteria, ALS patients can be classified into eight subgroups: classic, bulbar, pyramidal, pure lower motor neuron, flail arm, pure upper motor neuron, flail leg, and respiratory. There are no well-established molecular biomarkers for early diagnosis, prognosis, and progression monitoring of this fatal disease. Classification based on clinical phenotypes could be associated with peculiar gene expression patterns shaped during lifespan, allowing the identification of specific sporadic ALS (sALS) subtypes with less heterogeneous clinical and biological features. Our objective was to define a phenotype-specific transcriptomic signature of distinct ALS phenotypes, and lay the foundation for biomarkers development. We characterized 48 sALS patients by clinical and paraclinical parameters, and subdivided them in “Classic” (n=12), “Bulbar” (n=10), “Flail Arm” (n=7), “Flail Leg” (n=10) and “Pyramidal” (n=9) phenotypes. RNAs extracted from patients’ PBMCs and 19 controls were sequenced. Our analysis allowed the visualization of gene expression differential clusters between patients and controls. Interestingly, only one gene (Y3_RNA, a misc_RNA component of the Ro60 ribonucleoprotein involved in cellular response to interferon-alpha) was upregulated at different levels across all phenotypes, whereas other genes appeared phenotype-specific. The work proposed stress the innovative view of ALS as a multi-systemic disorder rather than a pure motor neuron-associated and ‘neurocentric’ pathology. The possibility to cluster ALS patients based on their molecular signature pave the way for future personalized clinical trials and early diagnosis.

overall_design: We performed RNA-seq profiling of 48 sALS patients classified on their phenotypes and 19 healthy controls. We used RNA extracted from PBMCs.

first: Maria

last: Garofalo

first: Bartolo

last: Rizzo

first: Rosalinda

last: Di Gerlando

first: Matteo

last: Bordoni

first: Eveljn

last: Scarian

first: Veronica

last: Bettoni

first: Orietta

last: Pansarasa

first: Luca

last: Diamanti

first: Stella

last: Gagliardi


title: Early cAMP signaling orchestrates single-cell synchronicity throughout Dictyostelium development

city: Ljubljana

first: Lena

last: Trnovec

address: Vecna pot 113

department: Faculty of Computer and Information Science

country: Slovenia

institute: University of Ljubljana

summary: Synchronicity is a fascinating biological property observed across many organizational levels, from physiological functions such as heartbeat, breathing, and brain activity, to circadian rhythms, and even population dynamics in insects and plankton. It is also a hallmark of developmental biology, evident in the coordinated formation of the vertebrate body axis, the fly eye, and the aggregative development of the social amoeba Dictyostelium discoideum. Despite its prevalence, quantitative and mechanistic analyses of synchronicity at the single-cell level remain rare. Here, we show that developmental synchronicity in D. discoideum is mediated by pulsatile cyclic adenosine monophosphate (cAMP) signaling. Using single-cell RNA sequencing (scRNA-seq) and the Universal Cell Embedding (UCE) model, we quantified transcriptome similarity between individual cells across developmental stages. We found that synchronicity initially declined upon starvation but increased markedly with the onset of cAMP-pulse signaling during aggregation. Synchronicity remained stable throughout development and differed between prespore and prestalk cells, with prespore cells exhibiting greater transcriptome homogeneity. Genetic perturbations in cAMP production and response revealed that cAMP-pulse signaling is essential for establishing and maintaining synchronicity, as its absence led to highly asynchronous development even when morphogenesis was restored. Interestingly, starvation alone induced a modest synchronizing effect in the absence of development in a strain unable to produce cAMP. These findings highlight the central role of early cAMP-pulse signaling in coordinating transcriptomic and morphological synchronicity, and establish scRNA-seq as a powerful tool for quantitative analysis of developmental synchronicity at single-cell resolution.

overall_design: A total of 30 samples were prepared for single-cell RNA sequencing using the 10x Genomics Chromium X system. Wild-type AX4, acaA⁻, and acaA⁻ PkaCoe strains were generated and collected at six time points, spaced four hours apart, up to 20 hours. The AX4 strain expressed three distinct fluorescent proteins, while the acaA⁻ strain expressed four. Each fluorescently tagged strain was cultured and processed independently, then pooled prior to loading onto the Chromium X platform. Fluorescent tag expression was used to distinguish biological replicates. The acaA⁻ PkaCoe strain was analyzed in three biological replicates.

first: Mariko

last: Katoh-Kurasawa

first: Gad

last: Shaulsky

first: Blaz

last: Zupan


title: Ventricles of wild type and TRPC6 knockout hearts

city: Kamakura

first: Satoshi

last: Kondo

state: Kanagawa

address: Tebiro 6-10-1

department: New Projects Development Division

country: Japan

institute: Toray Industries,Inc.

summary: To determine the effect of genetic deletion of TRPC6 in the cardiomyocytes, we compared the mRNA expression levels in the ventricle of a wild-type mouse and a TRPC6 genetic deletion mouse.

overall_design: We used wild-type mouse heart, 2 replicates, and TRPC6 genetic deletion, 2 replicates.

first: Yohei

last: Yamaguchi

first: Toshiyuki

last: Kaneko


title: RNAseq of activated CD8+ T cells with and without Glutamine Synthetase

city: Nashville

first: John

last: Karijolich

state: TN

address: 1211 Medical Center Drive

department: PMI

institute: Vanderbilt University Medical Center

summary: Cellular immunotherapy has revolutionized the treatment of hematologic malignancies yet has limited success in the solid tumor microenvironment (TME). While insufficient nutrients can lead to T cell metabolic stress in the TME, the glutamine antagonist 6-Diazo-5-oxo-L-norleucine (DON) can paradoxically enhance anti-tumor immunity. Because DON inhibits a range of glutamine metabolizing enzymes, including some that are essential and may contribute to dose-limiting toxicities, mechanisms underlying DON-induced anti-tumor activity have remained unclear. Here we aimed to identify specific DON targets responsible for increased T cell anti-tumor activity and test if more selective inhibition of glutamine metabolism could replicate the effects of DON with reduced toxicity. CRISPR screening in the TME of DON-relevant glutamine metabolizing enzymes identified some targets that were essential in tumor infiltrating CD8 T cells (TILs), but that TILs lacking the DON target glutamine synthetase (GS) were enriched. Upon adoptive T cell transfers, GS-deficient CD8+ T cells displayed improved survival, a more stem-like phenotype, and greater anti-tumor function. GS converts glutamate to glutamine and GS-deficient cells exhibited increased intracellular glutamate and reduced glutathione levels, which correlated with enhanced mitochondrial respiration and resistance to reactive oxygen species (ROS). Pharmacological inhibition of GS reduced tumor burden in multiple orthotopic murine tumor models in a manner dependent on adaptive immunity. Our findings establish GS as a key metabolic regulator of CD8+ T cells in the TME. By preserving intracellular glutamate, GS inhibition reprograms T cells for improved survival and function, offering a promising therapeutic strategy to enhance immune-based cancer treatments.

overall_design: RNA-Sequencing performed on activated CD8+ T cells from WT or Glutamine Synthetase (GS) knockout mice

first: Emilie

last: Fisher-Gupta

first: Jeffrey

last: Rathmell


title: Expression of synaptic function genes in iPSC-derived induced human neurons

city: Piscataway

first: Ronald

last: Hart

state: NJ

address: 604 Allison Rd Rm B430

department: Cell Biology & Neuroscience

institute: Rutgers University

summary: To determine expression levels of neuron-associated genes in induced human neurons, cultures of Ngn2-induced neurons grown on mouse glia were dissociated and assayed for nuclear RNA using the 10X Multiome kits.  Associated ATAC data were not evaluated in this study.  A mixture of 10 iPSC cell lines, 6 from diagnosed participants with alcohol use disorder (AUD) and 4 unaffected controls, were infected with lentiviral vectors expressing doxycycline-inducible Ngn2.  Following drug selection for successful transduction, cells were plated onto primary mouse glial cells and allowed to mature for approximately 4 weeks.  Cells were harvested with Accutase and Papain, then processed using standard 10X Genomics protocols using the Multiome kit.  After importing into Seurat and clustering, excitatory neuron cells were selected and deconvolved to identify the source participant by matching SNPs from earlier GWAS studies.  Tables of genes x barcode were then aggregated into genes x cell line matrices, which were loaded into DESeq2 for scaling and normalization.  For this study, only processed data from 3 unaffected control subjects are reported.  Results demonstrate robust expression of a series of synaptic receptor, channel, and associated signaling genes.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: The goal was to evaluate expression levels of selected synaptic genes to confirm that the cultured induced neurons were likely to respond to stimuli and fire action potentials, as shown in our study.

first: Dina

last: Popova

first: Petronio

last: Zalamea

first: Jaime

last: Messenger


title: Comprehensive single-cell analysis deciphered the immunoregulatory mechanism of TPPU in alleviating sepsis-related acute liver injury

first: Chen

last: Xue

state: Zhejiang

address: 866 Yuhangtang Rd, Hangzhou

institute: Zhejiang University

summary: Introduction: Sepsis-related acute liver injury involves complex immune dysfunctions. Epoxyeicosatrienoic acids (EETs), bioactive molecules derived from arachidonic acid (AA) via cytochrome P450 (CYP450) and rapidly hydrolyzed by soluble epoxide hydrolase (sEH), possess anti-inflammatory properties. Nevertheless, the impact of the sEH inhibitor TPPU on sepsis-related acute liver injury remains uncertain. Objectives: This study utilized comprehensive single-cell analysis to investigate the immunoregulatory mechanism of TPPU in alleviating sepsis-related acute liver injury. Methods: Hepatic bulk RNA sequencing and proteomics analyses were employed to investigate the mechanisms underlying sepsis-related acute liver injury induced by cecal ligation and puncture in mice. Cytometry by time-of-flight and single-cell RNA sequencing were conducted to thoroughly examine the immunoregulatory role of TPPU at single-cell resolution. Results: Downregulation of AA metabolism and the CYP450 pathway was observed during sepsis-related acute liver injury, and TPPU treatment reduced inflammatory cytokine production and mitigated sepsis-related hepatic inflammatory injury. Comprehensive single-cell analysis revealed that TPPU promotes the expansion of anti-inflammatory CD206+CD73+ M2-like macrophages and PDL1-CD39-CCR2+ neutrophils, reprogramming liver neutrophils to an anti-inflammatory CAMP+NGP+CD177+ phenotype. Additionally, TPPU inhibits the CCL6-CCR1 signaling mediated by M2-like macrophages and CAMP+NGP+CD177+ neutrophils, altering intercellular communication within the septic liver immune microenvironment. Conclusion: This study demonstrated TPPU's protective efficacy against sepsis-related acute liver injury, underscoring its vital role in modulating liver macrophages and neutrophils and enhancing prospects for personalized immunomodulatory therapy.

overall_design: CLP mice (CLP group) and sham mice (Sham group) were used to investigate liver transcriptome and proteome alterations during sepsis-related acute liver injury. To evaluate TPPU's effects on sepsis-related acute liver injury, CLP mice were randomly assigned to two groups: a control group (Ctrl group) receiving 0.9% sterile saline via oral gavage and a treatment group (TPPU group) receiving TPPU (3 mg/kg per day) orally for three consecutive days before CLP. Mice were euthanized for analysis 24 hours after CLP. A cytometry by time-of-flight (CyTOF) panel consisting of 42 metal-labeled antibodies was developed for this study. Single-cell sequencing (scRNA-seq) libraries were prepared as per the manufacturer's instructions. CyTOF and scRNA-seq were conducted to thoroughly examine the immunoregulatory role of TPPU at single-cell resolution.


title: Activation of sympathetic nervous system drives dry eye onset via norepinephrine-β2-adrenergic receptor signaling in mice

city: Qingdao

first: xiaoyu

last: li

state: Shandong

address: NO.5 Yanerdao Road

institute: Qingdao Eye Hospital of Shandong First Medical University

summary: Dry eye disease (DED) and sympathetic nervous system (SNS) activation have clear association with chronic environmental and psychogenic stress. However, the relationship and mechanism of SNS activation in dry eye pathogenesis remains elusive. Here we first found that the DED mice induced by chronic desiccating stress and scopolamine exhibited the SNS activation and elevated corneal norepinephrine (NE) contents. However, systemic SNS ablation with 6-OHDA and local NE depletion with DSP-4 treatment markedly alleviated the dry eye severity. More directly, topical application of NE phenocopied the dry eye syndrome in healthy mice, but not in mice with Adrb2 gene knockout, the dominant adrenergic receptor in cornea. In contrast, topical administration of selective Adrb2 antagonist ICI 118551 significantly attenuated the dry eye severity, including the increased tear secretion, improved corneal epithelial barrier function, and the reduced expressions of matrix-metalloproteinases, chemokines and inflammatory cytokines. Transcriptomic analysis and experimental validations underscored the strong links of top 10 downregulated pathways and dry eye-related inflammatory and immune responses, including TNF, NF-κB, chemokines and IL-17 signaling. Taken together, these results provide direct evidence of SNS activation in driving dry eye onset through NA-Adrb2 signaling pathway, which offers potential preventative and therapeutic approach for dry eye diseases.

overall_design: RNA-seq profiling of corneas from dry eye mice, with or without ICI 118551 treatment (four corneas pooled per sample) at day14 of scopolamine injection.


title: Multiple defects in mouse pseudo-embryos lacking all Hox function

city: Paris

first: Hocine

last: Rekaik

address: 11, Place Marcelin Berthelot

department: CIRB

country: France

institute: College de France

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

overall_design: Refer to individual Series


title: RNA-seq of Dpep2 knockdown in RAW264.7

first: wenchen

last: luo

summary: RNA-seq of Dpep2 knockdown in RAW264.7

overall_design: RNA-seq analysis of Dpep2 knockdown in RAW264.7.

first: Wenchen

last: Luo

first: Jing

last: Zhong

first: Zhixin

last: Qiu


title: Multiple defects in mouse pseudo-embryos lacking all Hox function [ATAC-seq]

summary: Mammals have four genomic copies of an ancestral Hox cluster, with a total of 39 genes. While gene inactivation approaches and full cluster deletions have revealed their critical functions during early development, the effect of removing all Hox function in the embryo has remained elusive due to both biological and technological challenges. We have used gastruloids, an embryo model where Hox genes are properly activated in time and space, to assess the effect of removing all four clusters together. We report that gastruloids lacking Hox function can still elongate and reach a general shape resembling their control counterparts, with a well-established AP polarity. However, unlike controls, they fail to produce any endoderm and are unable to properly segment their presomitic mesoderm into persistent somite-like structures when cultivated into permissive conditions. Instead, they produce a type of mesoderm with a more anterior identity, which aborts segmentation early on. Multiomics analysis revealed range of modifications in chromatin accessibility in the absence of any HOX proteins, involving in particular their PBX and MEIS co-factors. We conclude that, similar to Drosophila, mammalian HOX proteins are necessary to posteriorize an existing ‘ground-state’ structure by strongly interacting with PBX and MEIS cofactors, either to form instructive complexes, or to sequester the later factors to prevent their actions. In this later view, mammalian HOX proteins may be considered as a successive series of repressors, as proposed by Ed Lewis in 1978.

overall_design: ATAC-seq of wt and Hox-/- mES cells cultured under standard conditions.

first: Lucille

last: Lopez-Delisle

first: Alexandre

last: Mayran

first: Yuliia

last: Romaniuk

first: Cynthia

last: Diabanza

first: Anne-Catherine

last: Cossy

first: Denis

last: Duboule


title: Multiple defects in mouse pseudo-embryos lacking all Hox function [multiome]

overall_design: scMultiome of wt and Hox-/- gastruloids at 96h


title: Multiple defects in mouse pseudo-embryos lacking all Hox function [RNA-seq]

overall_design: RNA-seq of wt and Hox-/- mES cells cultured under standard conditions, and subjected to an endoderm differentiation protocol.


title: Multiple defects in mouse pseudo-embryos lacking all Hox function [scRNA-seq]

overall_design: scRNA-seq of wt and Hox-/- gastruloids at 72, 96 and 120h


title: Detecting Circadian Rhythmicity By Single Cell Multiomics

city: College Station

first: Chun Yip

last: Tong

state: Tx

address: 400 Bizzell St

department: Biology

institute: Texas A&M University

summary: This study employs advanced single cell multiomics to map the transcriptomic and epigenomic landscapes of liver peripheral clocks. By integrating these data, the research identifies core circadian genes, such as Bmal1, Clock, Per1, and Per2, exhibiting cyclic patterns across liver cell populations. Additionally, the study uncovers cell-type- and cell-state-specific cyclic genes, providing new insights into the circadian regulation tailored to the liver’s diverse metabolic functions.

overall_design: Sequence mouse liver for six time points under light dark condition by the 10X Genomics Multiome ATAC + Gene Expression protocol.

first: Jerome

last: Menet

first: Yuchao

last: Jiang