title: Rapid short- and long-range synaptic remodeling in hyperacute ischemic stroke

city: London

first: Luminita

last: Ruje

address: Imperial London Hammersmith Campus, Du Cane Road

institute: LMS MRC

summary: Physiological mechanisms of the pivotal hyperacute (0-24 hours) stage of stroke remain incompletely understood. Synaptic plasticity has been strongly implicated in early stages of neurodegenerative and neurodevelopmental disorders. Here, we describe distinct region-specific patterns of synaptic remodeling across the brain, arising within 1-4 hours of stroke onset. As synapses in the ischemic core decline profoundly, mildly ischemic penumbra retains synaptic structure albeit with decreased function, whereas the contralateral hemisphere shows an increase in synaptic size and function. Mechanistically, stroke leads to synaptic recruitment of NMDA-type glutamate receptors (NMDARs) in ischemic core and penumbra, and NMDAR signaling blockade triggers synaptic loss in the penumbra and abolishes contralateral synaptic strengthening. Proteomic analysis confirms synaptic enhancement in the contralateral hemisphere and reveals rapid onset of metabolic rearrangement in the penumbra, while RNAseq shows decline of synaptic gene expression in the penumbra. These findings identify brain-wide synaptic rebalancing as a potential mechanism for rapid-response homeostatic compensation in stroke, highlighting an extent of brain resilience to acute functional perturbation.

overall_design: RNAseq profiling of whole-brain tissues (brain) and biochemically purified synaptosomes (syn) from frontal cortex of 3 rats (rep 1, 2, 3) subjected to middle cerebral artery occlusion for 4 hours. For each brain, 3 regions were analysed, namely ischemic core (4c), surrounding penumbra (4p), and contralateral hemisphere (4z). For control (CA), 3 rats (rep 1, 2, 3) were subjected to sham surgery without occlusion, and whole-brain tissues and synaptosome samples were prepared as described above, with 1 region from each brain.

first: Ye

last: Wei

first: Oleg

middle: O

last: Glebov

first: Luminiţa

first: Mikhail

last: Spivakov


title: Autophagy is required for the development and functionality of lacrimal gland-like organoids

status: Public on Dec 18 2025

city: Birmingham

first: Gamze

last: Kocak

address: Edgbaston

country: United Kingdom

institute: University of Birminghham

type: Expression profiling by high throughput sequencing

summary: Lacrimal glands (LGs) serve as pivotal exocrine glands crucial for protecting the ocular surface. Dysfunction in LG cell composition or secretion is implicated in dry eye disease (DED). While autophagy plays a vital role in tissue. homeostasis in many organs, how it affects LG development and secretory function is not known. Here we have undertaken a genetic study by utilizing autophagy-deficient human embryonic stem cells and differentiating them into LG-like organoids. Autophagy-deficient LG-like organoids exhibited improper development and secretion, along with increased protein aggregation, proliferation, and cell death. These phenotypes were associated with an accumulation of PAX6, a transcription factor crucial for brain and eye development, which we identified as an autophagy substrate. Pharmacological interventions with nicotinamide mononucleotide and melatonin were able to rescue the cellular dysfunction in autophagy-deficient LG-like organoids. Together, our study highlights the role of autophagy in LG along with potential therapeutic interventions for DED.

overall_design:
To investigate the role of autophagy in LG-like organoids, we used multi-zonal eye cells (SEAM) differentiation from ATG5 WT and KO hESCs.
We then performed gene expression profiling analysis using data obtained from bulk RNA-seq of  ATG5 WT and KO hESC-derived SEAM.
Comperative gene expressing profiling of RNA-seq data for  ATG5 WT and KO hESC-derived SEAM.


title: Neuronal APOE4-induced Early Hippocampal Network Hyperexcitability in Alzheimer’s Disease Pathogenesis

city: San Francisco

first: Ayushi

last: Agrawal

state: CA

address: 1650 Owens St

country: USA

institute: Gladstone Institutes

summary: The full impact of apolipoprotein E4 (APOE4), the strongest genetic risk factor for Alzheimer's disease (AD), on neuronal and network function remains unclear. We found hippocampal region-specific network hyperexcitability in young APOE4 knock-in (E4-KI) mice which predicted cognitive deficits at old age. Network hyperexcitability in young E4-KI mice was mediated by hippocampal region-specific subpopulations of smaller and hyperexcitable neurons that were eliminated by selective removal of neuronal APOE4. Aged E4-KI mice exhibited hyperexcitable granule cells, a progressive inhibitory deficit, and E/I imbalance in the dentate gyrus, exacerbating hippocampal hyperexcitability. Single-nucleus RNA-sequencing revealed neuronal cell type-specific and age-dependent transcriptomic changes, including Nell2 overexpression in E4-KI mice. Reducing Nell2 expression in specific neuronal types of E4-KI mice with CRISPRi rescued their abnormal excitability phenotypes, implicating Nell2 overexpression as a cause of APOE4-induced hyperexcitability. These findings highlight the early transcriptomic and electrophysiological alterations underlying APOE4-induced hippocampal network dysfunction and its contribution to AD pathogenesis with aging.

overall_design: Single-nucleus RNA sequencing on isolated hippocampi from 5- and 10-month-old fE4-KI and fE3-KI mice with or without Syn1-Cre.

first: Dennis

middle: R

last: Tabuena

first: Sung-Soo

last: Jang

first: Brian

last: Grone

first: Oscar

last: Yip

first: Emily

middle: A

last: Aery Jones

first: Jessica

last: Blumenfeld

first: Zherui

last: Liang

first: Nicole

last: Koutsodendris

first: Antara

last: Rao

first: Leonardo

last: Ding

first: Alex

last: Zhang

first: Yanxia

last: Hao

first: Qin

last: Xu

first: Seo

last: Yeon Yoon

first: Samuel

last: De Leon

first: Yadong

last: Huang

first: Misha

last: Zilberter


title: Phage-encoded small RNA hijacks host replication machinery to support the phage lytic cycle

city: Jerusalem

first: Reut

last: Bruner

address: Hadassah Ein Kerem Campus

country: Israel

institute: The Hebrew University of Jerusalem

summary: Bacteriophages (phages) significantly influence bacterial populations in their natural environment. However, one aspect that has not been thoroughly explored in the context of phage-bacteria interactions is the post-transcriptional regulation of gene expression, despite the growing attention it has received for bacterial physiology over the last two decades. Important players in this process are small RNAs (sRNAs) that regulate target mRNAs via base-pairing, typically using RNA chaperones like Hfq to facilitate this regulation. Here, we apply RIL-seq, to map in-vivo the sRNA-RNA network in Escherichia coli upon lambda phage infection. We highlight changes in the bacterial transcriptome and sRNA interactome while uncovering a novel phage-encoded sRNA that regulates key genes in E. coli. We decipher the molecular mechanism of the sRNA-mediated regulation and illustrate how it hijacks the host replication machinery and helps the infection cycle. Overall, we uncover an RNA-level regulatory layer that shapes the E. coli - lambda interactions.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: RIL-seq and Total RNA-seq of E. coli MG1655 WT carrying a chromosomal FLAG-tagged Hfq, infected and non-infected with phage lambda, at 30 and 60 minutes after infection. As a control, libraries were similarly generated for WT cells not encoding tagged proteins. Two biological replicates were done for each sample.

first: Aviezer

last: Silverman

first: Raneem

last: Nashef

last: Wasserman

first: Tamar

last: Noy

first: Sahar

last: Melamed


title: Multiomic profiling of pediatric solid organ transplant recipients and healthy children

city: Boston

first: Johannes

last: Wedel

state: MA

address: 300 Longwood Ave

institute: Boston Children's Hospital

summary: PBMC of 12 pediatric solid organ transplant recipients (SOTR) and 8 healthy children (HC) were phenotyped using multiomic scRNAseq.

overall_design: Multiomic scRNAseq was performed in two independent library preparations (run1 and run2). PBMC were stained with hashtag and CITE-antibodies, pooled and loaded onto 5' Chromium (v2) channels. Libraries for gene expression, hashtags, CITE-antibodies and TCR/BCR immune cell repertoire were constructed and pooled prior to sequencing.

first: Vicki

last: Do

first: Marc

last: Schwartz

first: Ying

last: Tang

first: Kenneth D.

last: Mandl

first: Leslie S.

last: Kean

first: Scott B.

last: Snapper

first: David M.

last: Briscoe

first: Bruce H.

last: Horwitz


title: Sarcopenia promotes tumorigenesis by disrupting Notch-SDC2-regulated biogenesis of muscle-derived extracellular vesicles

city: Cardiff

first: NATHAN

last: HARMSTON

address: Museum Avenue

institute: Cardiff University

type: Non-coding RNA profiling by high throughput sequencing

summary: Studies using mouse xenograft and APCMin/+ models found that muscle acts as an anti-tumor organ by inhibiting tumor growth through muscle EVs. However, sarcopenic muscles exhibit reduced EV secretion and altered EV cargoes, promoting cancer progression. Our miRNA sequencing identified miR-7a-5p, a tumor-suppressor microRNA highly enriched in healthy muscle EVs but diminished in aging muscle EVs. miR-7a-5p targets TEAD1, suppressing its activity in tumors and inhibiting tumor growth.

overall_design: miRNA sequencing to identify differences in miRNA cargoes of EVs secreted by healthy muscle compared to damaged muscle (using hydrogen peroxide)

first: Hong-Wen

first: QianHui

last: Tan

first: Nathan

last: Harmston


title: The ADAR G1007R mutation in Aicardi-Goutières Syndrome causes neuroinflammation through RNA sensing signaling pathways in mice

city: Pittsburgh

first: Shuchang

last: Liu

state: PA

address: 203 Lothrop Street

institute: University of Pittsburgh

summary: The ADARG1007R mutation is one of the most frequent mutations found in type six Aicardi-Goutières Syndrome (AGS), a progressive inflammatory encephalopathy in pediatric patients, typically begins to manifest  in newborns or infants. Due to the rarity of this disease and the limited accessibility of the human brain tissues, the pathologic features and the underlying mechanism of this mutation has not been well understood.  In this study, we generated a genetic mutant mouse model which successfully recapitulated the genetic and pathologic features of human ADARG1007R AGS, including the early-onset brain inflammatory reaction in heterozygous mutant mice and the deep gray matter area ISG expression. Through analysis of the homozygous ADARG1007R/G1007 mice, we found that the G1007R mutation interrupts the RNA splicing donor site in the RNA transcripts that alters ADAR1 RNA splicing, leading to depletion of ADAR1 protein and embryonic death. Furthermore, we demonstrated that the brain inflammation could be reversed by deletion of the cellular RNA receptor MDA5 which blocks the cellular RNA sensing signaling pathway. Collectively, this study generated a clinically relevant AGS animal model, revealed the molecular mechanism of the ADARG1007R mutation in AGS, and demonstrated that the MDA5-dependent RNA sensing signaling pathway plays a critical role in neuroinflammation.

overall_design: All mice used in this study were kept in a specific pathogen-free animal facility and all studies were carried out following protocols approved by our Institutional Animal Care and Use Committee. The G1007R mutation was introduced into the mouse genome using our CRISPR/Cas9 protocol as described previously. Considering the genetic diversity of human diseases of ADAR1 mutations, mice used in this study, including AdarG1007R/G1007R and AdarG1007R/G1007R; Ifih1-/- mice, were purposely maintained in C57/BL6J and DBA2 mixed background. Three repeats for wild-type (WT), three repeats for G1007R mutant, and three repeats for G1007R mutatant + MDA5 retreat

first: XinFeng

last: Guo

first: Jia-Jun

first: Chaowei

last: Shang

first: Christopher

middle: J

last: Guerriero

first: Clayton

last: Wiley

first: Richard

last: Steinman

first: Yi

last: Sheng

first: Jeffrey

middle: L

last: Brodsky

first: Timothey

last: Billiar

first: Silvia

first: Qingde

last: Wang


title: NFκB1-deficiency associated with Flt3-myeloid leukemia enhances the self-renewal capacity via dysregulated RelA:p52 and SOD2 expression

city: Los Angeles

first: Benancio

last: Rodriguez

address: 611 Charles E Young Dr.

department: MIMG

institute: UC Los Angeles

summary: In acute myeloid leukemia (AML), abnormal expression of NFκB and Flt3 are frequently detected, and dual IKK2/Flt3 inhibitor regimes are used for treatment. However, how NFκB is involved in Flt3-driven myelopoiesis remains unknown. We report that AML is associated with reduced expression of NFKB1, and that Nfkb1 deficiency conveys poor survival in a mouse Flt3-ITD-driven myeloid leukemia model. Interpreting myelopoiesis dynamics with mathematical modeling suggests that Nfkb1-/- hematopoietic stem and progenitor cells possess enhanced self-renewal and are skewed towards plasmacytoid dendritic cells. Single-cell RNA-seq confirmed this interpretation via pseudotime analysis and gene signatures indicating stemness and pDC priming. Biochemical analysis and genetic rescues demonstrate how an imbalanced NFκB signaling system elevates basal nuclear NFκB, where RelA:p52 drives Sod2 expression and enables prolonged progenitor proliferation. Our work reveals how the inflammation response NFκB system controls in stress-responsive myelopoiesis, and how system imbalances can contribute to the pathogenesis of myeloid neoplasms.

overall_design: To investigate the interplay between RelA and p50 during BMDC differentation, we generated RNA-seq in Flt3 differeniating BMDCs in both wild-type and Nfkb1-/- cells.

first: Daly

last: Allisoj

first: Liu

last: Yi

first: Rodriguez

last: Benancio


title: MicroRNA-127-3p inhibits the in vitro osteogenic differentiation of bone marrow-derived mesenchymal stromal cells

city: Araçatuba

first: Victor Gustavo Balera

last: Brito

email: vgb.brito@unesp.br, brito@wustl.edu

state: SP

address: Marechal Rondon, km 527/8

department: Basic sciences

country: Brazil

institute: São Paulo State University

summary: MicroRNAs (miRNAs) are small non-coding RNAs that play a crucial role in post-transcriptional gene regulation. They are key regulators in various biological processes, including skeletal development and homeostasis. Disruptions in osteogenic differentiation pathways can lead to skeletal disorders such as osteoporosis and delayed fracture healing. MiR-127, initially identified for its tumor suppressor functions, has been already associated with skeletal disorders, but its direct role in regulating osteogenesis is still not fully understood. In this study, we demonstrated that overexpression of miR-127-3p using a miRNA mimics, significantly inhibits the in vitro osteogenesis of human bone marrow-derived mesenchymal stromal cells (hBMSCs). We observed reduced alkaline phosphatase activity and mineralization, along with decreased expression of bone marker genes. Additionally, pathway enrichment analysis from RNA sequencing at day 7 of osteogenesis indicated downregulation of cell cycle- and proliferation-related processes, which was confirmed by an BrdU incorporation assay. Also, upregulation of inflammation- and cell adhesion-related pathways was observed, and we were able to confirm the upregulation of Notch pathway by RT-qPCR. In conclusion, miR-127-5p inhibits the osteogenic differentiation and mineralization of hBMSCs.

overall_design: To investigate the role of hsa-miR-127-3p in osteogenic differentiation in vitro, human bone marrow-derived mesenchymal stromal cells (hBMSCs) were transfected with a miR-127-3p mimic or the appropriate negative control. A non-transfected group served as an additional control. Osteogenic differentiation was induced, and total RNA was obtained on day 7.

first: Audrey

last: McAlinden

first: Victor


title: Effect of depletion of Rictor on gene expression during 3% DSS treatment of colonic epithelial cells.

city: Beijing

first: Meng

middle: zhen

last: Li

address: 2 West Yuanmingyuan Road

department: College of biological science

country: China

institute: China Agricultural University

summary: mTORC2 signaling is critical for maintaining cellular metabolic hemostasis. We here report mTORC2 as a key regulator of mitochondrial oxidative phosphorylation in colonic epithelial cells. The depletion of Rictor results in serious colitis after 5 days of DSS treatment. In addition, loss of Rictor impaired OXPHOS activity and ATP production. Taken together, our data provide a molecular framework for mTORC2 signaling in colonic inflammation, in which mTORC2 positively regulate mitochondrial OXPHOS activity.

overall_design:
To investigate mTORC2 signaling in the regulation of colitis, we generated Villin-Cre driven colonic epithelial conditional knockout Rictor mice (cKO), and induced colitis in both control and cKO mice by administering DSS in drinking water.
We then performed gene expression profiling analysis using data obtained from RNA sequencing of colonic epithelial cells from control and cKO mice with or without DSS treatment.
Comparation gene expression profiling analysis of RNA sequencing data for epithelial cells from control and cKO mice, epithelial cells from control and cKO mice after 3%DSS treatment, epithelial cells from normal water and 3% DSS treated mice.

first: Mengzhen

first: Zhengquan

last: Yu


title: Endothelial transcription factor EB protects against doxorubicin-induced endothelial toxicity and cardiac dysfunction

city: Columbus

first: Wa

last: Du

state: OH

address: 460 West 12th Avenue, Biomedical Research Tower, Rm 360A

department: Department of Surgery, Division of Vascular Diseases and Surgery

institute: The Ohio State University

summary: Doxorubicin (DOX), an effective chemotherapeutic drug for various cancers, has been demonstrated to induce cardiovascular toxicity in cancer survivors. Endothelial cell (EC) dysfunction is recognized to play a critical role in the onset and severity of cardiotoxicity associated with DOX. Transcription factor EB (TFEB), a master autophagy regulator, regulates cardiovascular homeostasis. In the present study, we aimed to test whether endothelial TFEB protects against EC damage after DOX treatment.

overall_design: Total RNA was extracted from HUVECs stably overexpressing sfGFP-TFEB or empty vector after treatment with vehicle or DOX (1 uM) for 24 hours. RNA sequencing analysis was performed in empty vector and TFEB stable expressing ECs with or without DOX treatment.

first: Yanbo

last: Fan


title: Microarray gene expression data from MPFR cells treated with inhibitors of the PI3K/AKT/mTOR pathway

city: Odense M

first: Carla

last: Alves

address: Fioniavej 36, 2. sal

country: Denmark

institute: SDU

summary:
CDK4/6i palbociclib in combination with fulvestrant is currently FDA approved for the treatment of advanced ER+ breast cancer following progression on endocrine therapy. It is unclear which treatment strategy is beneficial following progression on this combination, and therefore, identification of novel treatmenmts is needed.
We used microarrays to detail the gene expression in cells resistant to the combination of CDK4/6i and fulvestrant (MPFR) treated with various inhibitors of the PI3K pathway. The gene expression analysis revealed a significant number of genes altered in MPFR cells following different treatments.

overall_design: The combined palbociclib and fulvestrant- resistant cell line is derived from MCF-7 and was established through long-term treatment with fulvestrant and high concentrations of CDK4/6. Total RNA from MPFR cells treated with palbociclib, fulvestrant and an inhibitor of the PI3K pathway (alpelisib, gedatolisib or sapanisertib) was extracted and arrayed separately in Human Transcriptome Array 2.0 (HTA, Affymetrix).

first: Alves

last: Carla

first: Karimi

last: Leena


title: Transcriptome of Hepato-Hematopoietic Lineages in Fetal Liver Organoids from Human Pluripotent Stem Cells

city: Cincinnati

first: Kentaro

last: Iwasawa

address: 3333 BURNET AVENUE

department: Division of Gastroenterology, Hepatology and Nutrition

institute: Cincinnati Children's Hospital Medical Center

summary: To investigate the functional difference between different cell lineages in highly hematopoietic fetal liver organoids (hFLOs) from induced pluripotent stem cell, we FACS-sorted cell lineages from hFLOs and compared them to conventional iPSC-monocellular or primary cell controls by bulk RNA-sequencing.

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of FACS-isolated lineages of hematopoietic fetal liver organoids (HLOs) from two different iPSC lines, and controls (PBMC-derived macrophages, primary hepatocytes, primary postnatal liver macrophages, iPSC-derived monocytes and -macrophages from a third iPSC-line

first: Milad

last: Rezvani

first: Kyle

last: Lewis

first: Konrad

last: Thorner

first: Takanori

last: Takebe


title: E. coli CFT073 gene expression at t= 4 hours after a temperature upshift from 23˚C to 37˚C (M9 minimal glycerol medium, exponential phase growth)

city: Northampton

first: Christine

middle: Ann

last: White-Ziegler

address: Clark Science Center

department: Biological Sciences

institute: Smith College

summary: We investigated the effect of a temperature shift from 23˚C to 37˚C, mimicking the temperature transition experienced by a microbe as it enters a human host.  This strategy allowed the exploration of gene expression changes in a short term response to a temperature upshift and to characterize the suite of genes required to respond to 37˚C in the first few hours.  We demonstrate that a significant number of genes are rapidly altered in expression within hours after a temperature shift.   Many of these genes are related to adhesion, immune evasion, and stress responses, suggesting that temperature may serve as a sentinel cue to predict and prepare for niche adaptation in the host.

overall_design:
An aerobic, mid-exponential phase (A600= 0.2-0.6) culture was grown at 23˚C in M9 minimal medium containing glycerol as a carbon source.  Aliquots of the initial culture were shifted to 37˚C to allow harvest of cells at time points after the shift.  RNA-Seq results compare the T=4 hours timepoint after the shift to 37˚C to a culture retained at 23˚C, T=4 hours.
Analysis used 23˚C (T=4 hours) RNA as control for comparison to the experimental samples harvested at T=4 hours after a temperature upshift to 37˚C

first: Daniel

last: Stoebel


title: Effect of microplastic particles on gene expression in Daphnia magna

city: Bayreuth

first: Alfons

last: Weig

address: Universitaetsstrasse 30

department: Genomics and Bioinformatics

country: Germany

institute: University of Bayreuth

summary: Transcriptional profiling of the waterflea Daphnia magna, when exposed to microplastic particles made of polyvinylchloride (PVC) and the incorporated plasticizer diisononyl phtalate (DINP)

overall_design: Three experimental conditions: control (no particles), microplastics (PVC) and microplatics/plasticizer(PVC+DINP)

first: Benjamin

last: Trotter


title: Esomeprazole Inhibits Proliferation of Scleroderma Fibroblasts via Cell Cycle Regulation

status: Public on Dec 17 2025

city: San Antonio

first: Shaheer

last: Koniyan

state: TX

address: 4939 Charles Katz Dr

department: Radiation Oncology

institute: UT San Antonio

summary: Scleroderma is a complex autoimmune disease characterized by abnormal fibroblast proliferation and excessive collagen deposits in the skin and internal organs. We previously showed that esomeprazole, an FDA-approved drug for gastric disorders, may prevent dermal fibrosis. Here, we demonstrate that esomeprazole exerts anti-proliferative effects on scleroderma fibroblasts by interfering with cell cycle regulation. To test this, we evaluated the antiproliferative effect of esomeprazole and the underlying molecular mechanisms using primary fibroblasts derived from patients with scleroderma. BrdU incorporation, flow cytometry, immunofluorescence, Western blot analysis, RNA sequencing, and functional enrichment analysis all showed a decrease in fibroblast proliferation. In addition, esomeprazole inhibited the proliferation of scleroderma fibroblasts in a dose-dependent manner, as measured by the Ki-67 marker.  Intriguingly, esomeprazole arrested fibroblasts in the G1 phase of the cell cycle, resulting in a reduction of cells in the S phase. Expression of p21, a known inhibitor of cyclin-dependent kinases (CDKs), was elevated, while CDK1 and CDK2 levels decreased following esomeprazole treatment. These data provide important insights into how esomeprazole regulates fibroblast proliferation in scleroderma and suggest that it may represent a potential therapeutic intervention for scleroderma.

overall_design: Primary dermal fibroblasts from two limited scleroderma male donors (SScF_P01and P02) were cultured under identical conditions and treated with esomeprazole (0, 10, 20, 50, 100 uM) for 24h. RNA Seq profiling was done on extracted mRNA. All patient samples were gifted by our collaborator Maureen D Mayes, Department of Internal Medicine, The University of Texas Health Science Center at Houston.

first: Yohannes

middle: T

last: Ghebre

first: Mohammad

last: Aasif Khan

first: Anil

middle: G

last: Jegga


title: RAB3GAP2 is a Regulator of Skeletal Muscle Endothelial Cell Proliferation and Associated with Capillary Density

city: Malmo

first: Dmytro

last: Kryvokhyzha

address: Jan Waldenströms gata 35

department: Clinical Sciences

country: Sweden

institute: Lund University

summary: Skeletal muscle fiber composition and capillary density influence physical performance and whole-body metabolic properties. ~45% of the variance in fiber type is heritable, which motivated us to perform a genome-wide association study of skeletal muscle histology from 656 Swedish men. Four independent variants were associated (p < 5x10− 8) with proportion of type IIx fibers or capillary-to-fiber ratio (C:F). The strongest signal localized to the rs115660502 variant, where the G-allele corresponded with increased C:F and reduced skeletal muscle expression of the proximal gene, RAB3 GTPase Activating Non-Catalytic Protein Subunit 2 (RAB3GAP2). The G-allele was less frequent in elite short-track sprinters and more frequent in endurance athletes than in matched non-athlete (population) controls; RAB3GAP2 expression was reduced by high-intensity intermittent training. RAB3GAP2 protein was not uniformly expressed in muscle tissue but localized to the endothelium and capillaries. Experimental reduction of RAB3GAP2 in human endothelial cells led to increased tube formation in vitro, to regulation of secreted factors promoting angiogenesis and T-cell activation, to reduced intracellular levels of von Willebrand factor (VWF) and, post-implantation, to increased endothelial cell density in vivo in mice. The amount of RAB3GAP2 in skeletal muscle was positively associated with exercise-induced release of VWF in vivo in humans. By regulating the release of protein factors (VWF, CD70, TNC, TNXB, MCP1, IGFBP3, COL1A1, TFPI2 and tPA), RAB3GAP2 influences fitness adaptation after exercise by improving muscle healing and promotion of capillary formation.

overall_design: HMEC cells were sequenced with RNAseq to identify an angiogenic factor that is secreted more from the KD-cells vs controls.


title: Uncoupling the two distinct roles of targeting BDCA2 in treating autoimmune diseases: direct inhibition of BDCA2-positive B cells and normalization of proinflammatory monocyte-derived macrophages via the global suppression of type I IFN

city: Shanghai

first: Haomin

last: Huang

address: 399 Libing Road

institute: 3SBio Inc.

summary: Systemic lupus erythematosus (SLE) is a devastating autoimmune disease with two hallmarks: elevated levels of type I interferon (IFN) secreted by Plasmacytoid Dendritic Cells (pDCs) and autoantibodies produced by malfunctioning B cells. Indeed, the two marketed monoclonal antibodies for treating SLE are belimumab that targets B cells or anifrolumab that targets type I IFN. In this study, we developed a novel anti-BDCA2 monoclonal antibody, 230V5, with an enhanced interaction with Fc receptors. The antibody exhibited superior inhibitory effects on IFN production and IgM production. Further study using scRNA-seq revealed that CpG induced a group of proinflammatory macrophages whose expression could be suppressed by 230V5 in a BDCA2-targeting and FcR-dependent manner. More importantly, we showed for the first time that BDCA2 is also expressed in B cells. BDCA2 expression in B cells can be induced by CpG and BAFF. Treatment with 230V5 effectively inhibited IgM production by PBMCs in a CD32B-dependent manner and reduced the number of BDCA2-expressing B cells. Thus, targeting BDCA2 is a promising therapeutic method for treating lupus-related diseases by targeting both B cells and pDCs

overall_design: PBMC from human were treated with different conditions, inclduing CpG-A or different Antibody drugs, then the cells were prepared for scRNA-seq analysis to investigate how the treatment changes the cell composizition and expression profiles.

first: Lan

last: Deng

first: Lihua

last: Wang

first: Xiaoqi

last: Li

first: Yun

last: Meng

first: Jing

last: Lou


title: Immune Gene Diversity and STING1 Variants in Shaping Cancer Immunity Across Different Genetic Ancestry Populations

city: Philadelphia

first: Lin

last: Zhang

address: 421 Curie Blvd

institute: Univerisity of Pennsylvania

summary: As human populations migrated to diverse geographical regions, they encountered varying pathogens, leading to pronounced natural selection pressures on the immune system. Analysis of nonsynonymous single nucleotide polymorphisms (nsSNPs) across major geographically structured populations showed greater variation in immune-related genes than in non-immune genes, consistent with pathogen-driven selection, whereas cancer-related genes exhibited lower variation, reflecting evolutionary conservation of critical cellular functions. We prioritized nsSNPs in pattern recognition receptor genes based on population diversity and their association with type I interferon activity. Among the top-ranked variants were rs11554776, rs78233829, and rs7380824 in STING1, which demonstrated functional impacts on intrinsic cGAS-STING1-IFN signaling in cancer cells and potential influences on tumor immunity. We further conducted a genome-wide characterization of nsSNPs in immune-related genes across genetic ancestry populations and established a publicly accessible database. Our study suggests that genetic ancestry-related germline variations may influence cancer immunity and treatment, supporting their consideration in personalized medicine.

overall_design: The SK-OV-3 parent cells and clones obtained from the dual CRISPR/Cas9 experiment were treated with 2’3’-cGAMP for 30 minutes and then allowed to rest for 4 hours. Subsequently, cells were harvested immediately and extracted using TRIzol Reagent. Illumina mRNA TruSeq library preparation was employed, followed by sequencing on an Illumina NovaSeq platform with paired-end 150 bp reads.

first: Xiaowen

last: Hu

first: Jie

first: Zhongyi


title: Sequence and chemical specificity define the functional landscape of intrinsically disordered regions [RNA-seq]

city: Garching

first: Frauke

last: Huth

address: James-Franck Str. 1

department: Department of Biosciences

institute: Technical University Munich

summary: Intrinsically disordered regions (IDRs) pervasively engage in essential molecular functions, yet they are often poorly conserved as assessed by sequence alignment. To explore the seeming paradox of how sequence variability is compatible with persistent function, we examined the functional determinants for a poorly conserved but essential IDR. We show that IDR function depends on two distinct but related properties: sequence and chemical specificity. While sequence-specificity operates via binding motifs and depends on the precise order and identity of residues, chemical specificity reflects the sequence-encoded chemistry of multivalent interactions across an IDR and depends on local and global chemical properties. Unexpectedly, a binding motif essential in the wild- type IDR can be removed when compensatory changes to the sequence chemistry are introduced, highlighting the orthogonality and interoperability of these properties and expanding the sequence space compatible with function. Our results provide a general framework for the functional constraints on IDR evolution.

overall_design: The effects of WT Abf1, Abf1 variants and no Abf1 on nucleosome positioning and gene regulation were investigated through an Anchor-Away approach in vivo, read out via methylation-aware Nanopore-Sequencing or total RNA-Sequencing as well as through an in vitro Remodeller assay, read out using MNase-Sequencing.

first: Iris

last: Langstein-Skora

first: Andrea

last: Schmid

first: Drin

last: Shabani

first: Lorenz

last: Spechtenhauser

first: Mariia

last: Likhodeeva

first: Franziska

last: Kunert

first: Felix

last: Metzner

first: Ryan

last: Emenecker

first: Mary

last: Richardson

first: Wasim

last: Aftab

first: Maximilian

last: Götz

first: Sarah

middle: K

last: Payer

first: Niccoló

last: Pietrantoni

first: Valentina

last: Sjeničić

first: Sakthi

last: Ravichandran

first: Till

last: Bartke

first: Karl-Peter

last: Hopfner

first: Ulrich

last: Gerland

first: Philipp

last: Korber

first: Alex

middle: S

last: Holehouse


title: Sequence and chemical specificity define the functional landscape of intrinsically disordered regions [MNase-seq]

first: Frauke

last: Huth

type: Genome binding/occupancy profiling by high throughput sequencing

first: Iris

last: Langstein-Skora

first: Andrea

last: Schmid

first: Drin

last: Shabani

first: Lorenz

last: Spechtenhauser


title: Sequence and chemical specificity define the functional landscape of intrinsically disordered regions [ONT]

type: Methylation profiling by high throughput sequencing

first: Mariia

last: Likhodeeva

first: Franziska

last: Kunert

first: Felix

last: Metzner

first: Ryan

last: Emenecker

first: Mary

last: Richardson


title: Non-hematopoietic erythropoietin splice variants are produced in the diseased human brain and confer neuroprotection

city: Berlin

first: January

last: Weiner

address: Charitéplatz 1

department: CUBI

institute: Berlin Institute of Health, Charité Medical University of Berlin

summary: Erythropoietin (EPO) is a pleiotropic cytokine with important functions in neuronal development and neuroprotection, but hematopoietic effects limit the therapeutic application of EPO in neurological diseases. We discovered human endogenous EPO splice variants that are non-hematopoietic but cytoprotective. Here, we demonstrate at the single-cell level that an alternative splice variant lacking exon 3 (hS3) is expressed in the human brain and is upregulated above EPO mRNA levels in ischemic and inflammatory neurological diseases. Conversely, hS3 mRNA expression is reduced below EPO levels in neurodegenerative disease. In an oxygen-glucose deprivation (OGD) model of ischemia, a single dose of cell-free synthesized constant glycosylated active hS3 protects neuronal cultures derived from human induced pluripotent stem cells (hiPSC) and human embryonic stem cells (hESC) more effectively than EPO. We identify the D-helix as a key functional domain of hS3 and demonstrate that the neuroprotective effect is enhanced by PD29, a novel small peptide derived from the D-helix of hS3. Long-term hS3 administration increases the neuroprotective effects in the OGD model by dose-dependent differential expression of apoptosis-related protein-coding genes and long non-coding RNAs (lncRNAs). In addition, our results suggest that hS3 induces early cell cycle inhibition without impairing differentiation of hiPSC and hESC into neuronal subtypes. In conclusion, EPO splice variants are part of the endogenous neuroprotective system in the human brain with significant therapeutic potential.

overall_design: Two cell lines (MDC_P4_H1 and BIH005_A) were subjected to two types of treatment (hS31 or hS32) or left untreated. RNA was collected at one of four different time points.

first: Theresa

last: Hartung

first: Andreas

last: Meisel

first: Josef

last: Priller


title: Responders to low-dose ATG induce CD4+ T cell exhaustion in type 1 diabetes

city: Seattle,

first: Stephanie

last: Osmond

state: WA

address: 1201 9th Ave

institute: Benaroya Research Institute

summary: Low-dose anti-thymocyte globulin (ATG) transiently preserves C-peptide and lowers HbA1c in individuals with recent-onset type 1 diabetes (T1D); however, the mechanisms of action and features of the response remain unclear. Here, we characterized the post hoc immunological outcomes of ATG administration and their potential use as biomarkers of metabolic response to therapy (i.e., improved preservation of endogenous insulin production).METHODSWe assessed gene and protein expression, targeted gene methylation, and cytokine concentrations in peripheral blood following treatment with ATG (n = 29), ATG plus granulocyte colony-stimulating factor (ATG/G-CSF, n = 28), or placebo (n = 31).RESULTSTreatment with low-dose ATG preserved regulatory T cells (Tregs), as measured by stable methylation of FOXP3 Treg-specific demethylation region (TSDR) and increased proportions of CD4+FOXP3+ Tregs (P < 0.001) identified by flow cytometry. While treatment effects were consistent across participants, not all maintained C-peptide. Responders exhibited a transient rise in IL-6, IP-10, and TNF-α (P < 0.05 for all) 2 weeks after treatment and a durable CD4+ exhaustion phenotype (increased PD-1+KLRG1+CD57- on CD4+ T cells [P = 0.011] and PD1+CD4+ Temra MFI [P < 0.001] at 12 weeks, following ATG and ATG/G-CSF, respectively). ATG nonresponders displayed higher proportions of senescent T cells (at baseline and after treatment) and increased methylation of EOMES (i.e., less expression of this exhaustion marker).CONCLUSIONAltogether in these exploratory analyses, Th1 inflammation-associated serum and CD4+ exhaustion transcript and cellular phenotyping profiles may be useful for identifying signatures of clinical response to ATG in T1D.TRIAL REGISTRATIONClinicalTrials.gov NCT02215200.FUNDINGThe Leona M. and Harry B. Helmsley Charitable Trust (2019PG-T1D011), the NIH (R01 DK106191 Supplement, K08 DK128628), NIH TrialNet (U01 DK085461), and the NIH NIAID (P01 AI042288).

overall_design: RNA-sequencing was performed on whole blood samples from 88 participants in the trial (31 placebo treated, 29 ATG treated, and 28 ATG-GCSF) treated. A total of 496 samples were profiled. Samples for each donor were collected at up to six visits measured in weeks following the start of therapy (visits 0, 2, 12, 24, 48, and 96).

first: Laura

middle: M

last: Jacobsen

first: Kirsten

last: Diggins

first: Lori

last: Blanchfield

first: James

last: McNichols

first: Daniel

last: Perry

first: Jason

last: Brant

first: Xiaoru

last: Dong

first: Rhonda

last: Bacher

first: Vivian

middle: H

last: Gersuk

first: Desmond

last: Schatz

first: Mark

last: Atkinson

first: Clayton

middle: E

last: Mathews

first: Michael

last: Haller

first: S

middle: Alice

first: Peter

last: Linsley

first: Todd

last: Brusko


title: Epigenetic Signatures of Brain Aging by Single Nucleus Methylation Sequencing [DHC-9mo-m-001]

city: La Jolla

first: Joseph

last: Ecker

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

institute: HHMI-Salk-Institute

summary: Illustrating aging effects on the epigenomic features of brain is critical to understand brain functional decline and neurodegeneration. Single nucleus methylation sequencing was applied to brain regions of mouse at the age of 2, 9 and 18 months. We use these data to identify the epigenomic changes with aging in mouse brains.

overall_design: Apply snmC-seq3 to mouse brain regions to identify aging effects on epigenomics

first: Wei

last: Tian

first: Anna

last: Bartlett

first: Jordan

last: Altshul

first: Rosa

last: Gomez Castanon

last: Nery

first: Jacinta

middle: D

last: Lucero

first: Julia

last: Osteen

first: Huaming

last: Chen

first: M

last: Behrens


title: Joint single-cell profiling of Cas9 edits and transcriptomes reveals on- and off-target effects on gene expression (RNA-seq)

first: Graham

last: McVicker

address: 10010 N Torrey Pines Rd

institute: Salk Institute for Biological Studies

summary: A longstanding barrier in genome engineering with CRISPR-Cas9 has been the inability to characterize editing outcomes at single-cell resolution. We present "Superb-seq", a new scRNA-seq method that measures on-target and off-target genome edits and associated transcriptomes. In contrast to Perturb-seq that captures single-cell guide information, Superb-seq directly captures single-cell edit profiles by T7 in situ transcription. We demonstrate and validate this method through two Superb-seq experiments in 30,000 cells of three cell types, amplicon sequencing (rhAmpSeq), and bulk RNA-seq. Superb-seq uses off-the-shelf kits, standard laboratory equipment, and requires no virus, which will improve CRISPR screens in diverse tissues and functional characterization of CRISPR therapeutics.

overall_design: RNA-seq was performed on two sets of three human nuclei samples, one set per target site and cell type pair (CTLA4 in Jurkat, B2M in K562). For each set, first was a control sample with mock in situ transcription (no T7 RNA polymerase). Second was another control sample with CRISPR-Cas9 RNP only (no T7 promoter) and in situ transcription. Third was a test sample with both CRISPR-Cas9 RNP and T7 promoter, and in situ transcription. Two technical replicate libraries per sample.

last: Lorenzini

first: Brad

last: Balderson


title: Wildtype granuloma cells from M. marinum-infected zebrafish

city: Durham

first: David

last: Tobin

state: North Carolina

address: 207 Research Drive

institute: Duke University

summary: We use our previously published single-cell RNA sequencing protocol to interrogate the transcriptional nature of granuloma structures extracted from Mycobacterium marinum-infected zebrafish (Danio rerio).

overall_design: Granulomas were dissected from M. marinum-infected zebrafish adults. Single-cell suspensions were generated by brief digestion with trypsin and sent for 10X Genomics scRNAseq processing.

first: Jacob

middle: P

last: Lowy

first: Gopinath

last: Viswanathan

first: Erika

last: Hughes

first: Charlie

last: Pyle

first: Simon

last: Gregory


title: Effect of B cell receptor class-switching on lymphoma cells

city: London

first: Pavel

last: Tolar

address: Institute of Immunity and Transplantation, Rowland Hill St

institute: University College London

summary: Although immunoglobulin class-switching from IgM to IgG enhances B cell receptor (BCR) signalling and antibody responses, certain subsets of B cell lymphomas retain unswitched IgM BCRs despite signs of class-switching in the immunoglobulin heavy chain loci. This suggests that expression of the IgG BCR suppresses progression of specific lymphoma subsets. We found that inducing BCR class switching from IgM to IgG1 or IgG4 inhibits proliferation of EZB DLBCL lymphoma cell lines. To understand changes associated with class-switching we compared transcritional profiles of isogenic WSU-FSCCL and NU-DHL1 cell lines artificially class-switched to express IgM and IgG1 BCRs. In addition, we analysed transcriptional profiles of IgG1+ cell lines into which we introduced the IgG1 W375* mutation that truncates the intracellular IgG tail.

overall_design: RNASeq was performed on NU-DHL1 IgM+, NU-DHL1 IgG1+ wildtype, NU-DHL1 IgG1+ W375*, WSU-FSCCL IgG1+  wildtype, and WSU-FSCCL IgG1+ W375*cells

first: Tooki

last: Chu


title: Rational redesign of antigen binding domain improves in vivo efficacy of the CD22-CAR

city: Aurora

first: Terry

last: Fry

state: CO

institute: University of of Colorado Denver and Children's Hospital Col

summary: Chimeric antigen receptor (CAR) T cells targeting CD19 are highly effective against B-lineage malignancies. However, about half of patients either fail to achieve complete remission (in the case of lymphoma) or relapse (in the case of acute lymphoblastic leukemia). CD22 represents an alternative highly B-lineage-restricted target. Although CD22-targeted CAR T cells are clinically active, targeting this antigen has proven difficult relative to CD19, attributable, in part, to lower expression levels. Commonly, patients relapse or progress with reduced CD22 expression compared to pre-treatment, contrasting with the loss of CD19 expression typically observed after CD19-CAR therapy. Prior work demonstrated that an antigen-independent “tonic” signaling CD22-CAR has enhanced clinical efficacy relative to other CD22-CARs tested. However, tonic signaling has been shown to be detrimental to long-term CAR T cell function. Here, we demonstrate a balance between binding affinity and antigen-independent signaling (determined by length of the linker between fragment variable regions) in determining CAR function against CD22. We show that CAR function against both CD22Lo and WT leukemia can be augmented by boosting binding affinity without shortening the linker to induce tonic signaling, establishing rational combinatorial modification of antigen binding domain as an important approach for modulating the function of cellular therapeutics.

overall_design: Human donor PBMC-derived CD3+ T cells were activated and virally transduced with unique chimeric antigen receptor (CAR) constructs targeting the CD22 antigen, enriched for CAR+ cells, then cryopreserved. Following thaw, cells either stimulated on plate-bound CD22 or left at rest for 24 hours, followed by dead cell depletion and analysis by scRNA-seq.

first: Kole

last: DeGolier

first: Catherine

last: Pham-Danis

first: Samuel

last: Burciaga

first: Zachary

last: Walsh

first: Christine

last: Brzezinski

first: Amanda

last: Novak

first: Lillie

last: Leach

first: Jennifer

last: Cimons

first: Wei

last: Li

first: Zhu

last: Zhongyu

first: Dimiter

last: Dimitrov

first: James

last: Scott-Browne

first: Mark

last: Kohler


title: Single cell characterization of the gastrointestinal HIV reservoir reveals heterogeneous cellular phenotypes.

city: Chapel Hill

first: Edward

middle: Patrick

last: Browne

state: NC

address: 120 Mason Farm Road

institute: UNC-Chapel Hill

summary: Human gastrointestinal (GI) tissues are a major site of HIV-1 viral persistence, but the nature of the GI reservoir remains poorly described.  To characterize the GI HIV reservoir, we profiled cells from GI tissue and matched peripheral blood mononuclear cells from ten people with HIV (PWH) on antiretroviral therapy (ART) using single cell RNA sequencing.  We identified distinct compartment-specific patterns of gene expression, highlighting key differences between blood and colon CD4 T cell populations.  vRNA+ cells from both blood and GI tissue were heterogeneous and found in multiple subtypes of CD4 T cells, although vRNA+ cells were particularly enriched in cells with Th17 or Treg17 phenotypes.  Transcriptomic comparison of HIV vRNA+ and vRNA- T cells revealed 116 differentially expressed genes that were associated with HIV infection including ZBED2, MAF and IL17F.  These data provide novel information regarding the GI-resident HIV reservoir and suggest that compartment-specific patterns of gene expression are associated with HIV infection.

overall_design: Single cell RNA sequencing of samples derived from 10 HIV seropositive donors and 1 HIV seronegative donor (Donor 11). HIV seropositive participants had been durably suppressed on ART for a median of 12.8 years (IQR 11.2-19.8)  . Samples were derived from the peripheral blood and from the sigmoid region of the colon, optionally isolated with CD4 positive selection, and treated with PMA ionomycin or control conditions. CITE seq using TotalSeqB panel with hash tag oligo (HTO) indexing was performed on peripheral blood samples from three donors (Donor 9:HTO_2 , Donor 10:HTO_1, and Donor 11:HTO_3).

first: Jackson

last: Peterson

first: Nancie

last: Archin


title: Circulating angiogenic profiles in ischemic stroke and intracranial atherosclerosis (ICAD)

city: Los Angeles

first: Nestor R.

last: Gonzalez

state: California

address: 127 S San Vicente Blvd

department: Neurosurgery

institute: Cedars Sinai Medical Center

summary: Background and Aims: Intracranial atherosclerotic disease (ICAD) carries high recurrence rates despite intensive medical management. Prior studies suggest anti-angiogenic circulating profiles may contribute to treatment failure. This exploratory study evaluated associations between pro- and anti-angiogenic factors and stroke patterns in ICAD, specifically comparing hemodynamic (borderzone) versus embolic presentations.  Methods: We conducted a cross-sectional analysis of 110 patients with severe ICAD (≥70% stenosis) from four U.S. centers (2019-2023). Five circulating factors were measured: VEGF, VEGF-165b, HGF (pro-angiogenic), and endostatin, angiostatin (anti-angiogenic). Stroke patterns were classified as hemodynamic (borderzone/watershed, n=28) or non-hemodynamic (embolic/perforator, n=82) based on MRI. A Random Forest classifier was developed to identify biomarker patterns associated with stroke type, with feature importance analysis to determine relative contributions.  Results: The Random Forest model achieved 85% accuracy (AUC 0.87) in discriminating hemodynamic from non-hemodynamic strokes using 5-fold cross-validation. Feature importance analysis revealed anti-angiogenic factors as the strongest predictors: angiostatin (25.6% importance) and endostatin (21.5%), followed by pro-angiogenic factors VEGF (18.3%) and HGF (18.1%). Secondary analyses showed moderate associations with functional independence (AUC 0.71) but not cognitive scores. Exosomal microRNA pathway analysis revealed enrichment in pathways common to both neurovascular insufficiency and neurodegenerative processes, suggesting potential shared mechanisms between vascular and degenerative pathologies.  Conclusions: This hypothesis-generating study identifies differential expression of angiogenic factors associated with hemodynamic versus embolic stroke patterns in ICAD. The predominance of anti-angiogenic factors in predicting hemodynamic strokes suggests impaired collateral formation may contribute to watershed ischemia. These preliminary associations warrant prospective validation to determine whether anti-angiogenic profiles could identify ICAD patients at risk for hemodynamic compromise.

overall_design: Blood samples were collected from patients with ICAD ischemia. Levels of five angiogenic factors were measured by ELISA and recorded. Exosomal mi-RNA expression profiles were measured and compared between patients with hemodynamic and non-hemodynamic patterns of stroke.

first: Nestor

middle: R

first: Sofia

last: Ramirez-Guerrero

first: Maria Paula

last: Aguilera-Pena

first: Santiago

last: Mendoza-Ayus

first: Miguel David

last: Quintero-Consuegra

first: Hongyu

last: Liu

first: Nicholas

last: Tatonetti

last: Balanay

first: Shlee

last: Song

first: Konrad

last: Schlick

first: Jeffrey

last: Saver

last: Harris

first: Alexis

middle: N

last: Simpkins

first: Parneet

last: Grewal

first: Kwan Leung

last: Ng

first: Jose

last: Gutierrez


title: Depletion of individual dietary amino acids induces distinct metabolic and chromatin states

city: Madison

first: Cara

last: Green

state: WI

address: 2500 Overlook Terrace

institute: University of Wisconsin-Madison

summary: Reducing dietary levels of protein or specific essential amino acids (EAAs) promotes favorable metabolic reprogramming, including improved glucose tolerance, increased insulin sensitivity and reduced fat mass. However, the extent to which shared or EAA-specific mechanisms facilitate diet-associated phenotypes remains unclear. Here, we compared the physiological and molecular responses to dietary levels of methionine, leucine, and isoleucine by feeding C57BL/6J mice diets in which each of these specific AAs is depleted. Dietary depletion of Met, Leu, or Ile (Met-D, Leu-D, or Ile-D) elicited distinct, AA-specific physiological and hepatic molecular (transcriptome, metabolome, histone proteome) responses that were not phenocopied by mTORC1 inhibition via rapamycin treatment. Ile-D yielded the most distinct and dramatic responses, highlighted by expression of select chromatin modifying and metabolic enzymes that led to a prominent epigenetic state of histone H2A/H4 hypoacetylation and maintained hepatic acetyl-CoA levels despite downregulated β-oxidation. Multi-Omics Factor Analysis of 14,139 data points objectively affirmed Ile-D phenotypes are distinct from Met-D or Leu-D and identified metabolic and chromatin features as primary discriminators. We further demonstrated the metabolic and epigenetic responses to Ile-D can be recapitulated in vitro, suggesting that these responses are cell intrinsic. Together, these results demonstrate that dietary depletion of EAAs induce unique phenotypes and highlight distinct molecular mechanisms by which individual EAAs may control metabolic health.

overall_design: All mice were housed three per cage, maintained at a temperature of approximately 22°C, and health checks were completed on all mice daily. Studies were commenced when the mice were 14 weeks of age. Mice were then randomized to receive the indicated diets from Inotiv either the Control (TD.01084), Met-D (TD.140119), Leu-D (TD.180789) or Ile-D (TD.190879) diets, or the Control diet plus rapamycin (delivered I.P. in 5% Tween 20, 5% PEG40, 0.9% NaCl, 3% ethanol at 4 mg/kg/day). The randomization of mice was performed at the cage level to ensure that all groups had approximately the same initial starting weight and body composition. Mice were housed in a SPF mouse facility in static microisolator cages, except when temporarily housed in a Columbus Instruments Oxymax/CLAMS-HC metabolic chamber system. Mice were housed under a 12:12 h light/dark cycle with free access to food and water. R, L or N indicate mouse ID (within the same cage).

first: Spencer

last: Haws

first: Yang

first: Krittisak

last: Chaiyakul

first: Pragyan

last: Mishra

first: Reji

last: Babygirija

first: Eric

last: Armstrong

first: Anusha

last: Mehendale

first: Irene

last: Ong

first: Dudley

last: Lamming

first: John

last: Denu


title: Lung derived extracellular vesicles have distinct pro-repair effects, depending on the age of their source tissue

first: Sally

middle: Yunsun

last: Kim

address: Franklin-Wilkins Building, 150 Stamford Street

department: Institute of Pharmaceutical Sciences

institute: King's College London

summary: Many age-related lung diseases encompass aberrant repair mechanisms. To develop novel treatments for these diseases, it is critical to understand how biology and repair signals are altered in aged lungs. Extracellular vesicles (EVs) carry bioactive signals reflecting their source tissue; therefore, we used EVs derived from precision-cut lung slices (PCLS) as tools to investigate differences in lung biology and repair signals that occur upon ageing and injury. We compared the physicochemical properties and biological content of four groups of EVs, obtained from uninjured and injured, young and aged PCLS. Treatment with EVs obtained from young, uninjured (YU) PCLS decreased apoptotic cells in both young and aged spatially injured (AIR)-PCLS. YU EVs also increased the percentage of alveolar progenitor cells in aged AIR-PCLS, indicating that aged lungs retain the capacity to mount a repair response, if provided with the right biological cues. Small RNA sequencing revealed the miRNA content of each EV group is altered, depending on the physiological conditions of their source tissue. Bioinformatic analysis of the target genes regulated by differentially expressed miRNAs identified enrichment of genes required for lung development and repair. These findings pave the way for future therapeutic strategies to repair or regenerate aged lungs.

overall_design: Small RNA-sequencing of total RNA content of extracellular vesicles (EVs) derived from uninjured and acid-injured, aged and young adult mouse precision-cut lung slices (PCLS).

first: Qi

last: Chen

middle: Y

first: Charlotte

last: Dean

first: Paulson

last: Tseng

first: Celia

last: Nicieza

first: Fiona

middle: J

last: Culley


title: Joint single-cell profiling of Cas9 edits and transcriptomes reveals on- and off-target effects on gene expression (rhAmpSeq)

first: Graham

last: McVicker

overall_design: RhAmpseq was performed on two sets of eight genomic DNA samples. The first sample of each set was a control sample of unedited (electroporation only) K562 cells. The subsequent seven test samples were K562 cells edited with CRISPR-Cas9 and T7 promoter, using seven guides targeting chromatin remodeler genes ARID1A, SMARCA4, CHD3, and CHD4, one guide per sample. These eight samples were processed into rhAmpSeq libraries using one of two primer sets: either a multiplex panel of 52 primer pairs targeting Superb-seq detected and in silico predicted edit sites, or a singleplex primer pair targeting ADSS1. Sixteen total libraries, one replicate per sample and primer set.

first: Michael

last: Lorenzini

first: Brad

last: Balderson


city: Hangzhou

first: Da

last: Qian

state: Zhejiang

address: No. 158 Shangtang Road

department: Breast Surgery

institute: Zhejiang Provincial People's Hospital

summary: Lcariin Ameliorates Exercise-Induced Fatigue by Promoting TFEB-Dependent Mitochondrial Clearance and Metabolic Re-programming, which reveals a novel molecular mechanism underlying icariin’s anti-fatigue efficacy and highlights its potential as a natural agent for enhancing exercise capacity.

overall_design: Total RNA sequencing was performed on primary vascular smooth muscle cells isolated from wild-type rats. The cells were first subjected to serum starvation and then treated with Icariin. In contrast, control cells were cultured in fresh serum-free medium without the addition of any exogenous growth factors. Total RNA was extracted from the control group at 24 h post-treatment and from the Icariin-treated group at 48 h post-treatment, with three biological replicates set for each group.

first: Liang

last: Zheng


title: Restoring lysosomal homeostasis in microglia via a catch-and-patch nanomedicine for Alzheimer’s disease

city: Nanjing

first: Guangyu

last: Long

state: Jiangsu

address: #639 longmian avenue

institute: China Pharmaceutical University

summary: Lysosomal homeostasis acts as the tipping point at Aβ deposition and clearance, critically dictating the trajectory of Alzheimer’s disease (AD) pathology. While monoclonal antibodies have shown promise in reducing extracellular Aβ burden, they inadvertently induce a massive Aβ influx into microglia that overwhelms the lysosomal degradation capacity. The Aβ overload results in toxic intracellular Aβ spillover and increases the risk of inflammatory side effects. We show that the AMPK-TFEB axis is essential for maintaining microglial function by regulating lysosome activity and Aβ clearance. Building on this insight, we develop a polymeric micelle capable of removing both extracellular and intracellular Aβ while preserving intracerebral homeostasis. This nanomedicine facilitates microglial endocytosis of Aβ deposits and stimulates TFEB-mediated lysosome biogenesis in a pH-responsive manner, thereby expanding the lysosomal capacity to manage the Aβ influx. Through this “catch-and-patch” mechanism, it functions as an Aβ scavenger and stabilizes microglial activity at a steady-state setpoint. Intravenous administration of the micelle significantly reduces Aβ burden and mitigates cognitive decline in 5xFAD mice. Minimal cerebral amyloid angiopathy or inflammation is observed compared to anti-Aβ antibody therapy. We provide a proof of principle for safe and effective AD treatment by synergistically targeting the extracellular-intracellular Aβ cascade.

overall_design: Hippocampus from 5xFAD mice treated with saline, Aducanumab, or ACLP-NT were collected and immediately immersed in RNA preservation solution for storage at -80°C. Total RNA was extracted from hippocampus using Trizol reagent, and mRNA sequencing was performed.

first: Yuelong

last: Wang

first: Siqi

last: Zhu

first: Limin

last: Tian

first: Yi-an

last: Zou

first: Zhirong

last: He

first: Junmei

last: Zhang


title: Multi-omics Analysis of Metabolic Characteristics and Validation of HBEGF-Mediated Regulatory Mechanisms of Metabolism during Catch-Up Growth in Small-for-Gestational-Age Infants

city: Dalian

first: Li

address: 222 Zhongshan Road, Xigang District

institute: The First Affiliated Hospital of Dalian Medical University

summary: Small-for-gestational-age (SGA) is a globally recognized public health concern. Infants born SGA may experience metabolic disturbances. This study elucidated the key regulatory factors and mechanisms underlying catch-up growth (CUG) and metabolic homeostasis in SGA infants. High-throughput targeted metabolomics were employed to compare the serum profiles of SGA and appropriate-for-gestational-age (AGA) neonates, identifying key metabolic pathways and associated regulatory signaling pathways enriched with differentially expressed metabolites. Animal experiments were conducted, and liver tissue samples were collected from neonatal SGA and AGA rats. Subsequently, mRNA sequencing to identify and analyze differentially expressed genes. HBEGF—a gene critically involved in metabolic processes—was significantly upregulated in SGA rat liver tissues. HBEGF silencing can suppress CUG in SGA rats and influence carbohydrate and lipid metabolism. Furthermore, metabolomic profiling was performed to investigate the role of HBEGF in metabolic regulation during CUG in individuals with SGA. Finally, the impact of HBEGF on the AKT/GSK-3β (protein kinase B/glycogen synthase kinase 3 beta) signaling pathway was explored in both in vivo and in vitro settings. This study confirms metabolic differences between SGA and AGA individuals are evident at early-life. HBEGF may be crucial in CUG progression in SGA by modulating metabolic processes via AKT/GSK-3β signaling pathway.

overall_design: The RNAs were isolated from rat liver tissues using TRIpure (BioTeke). RNA samples were evaluated using the A260/A280 absorbance ratio with a Nanodrop system, and only qualified samples were utilized to construct the library. Library quality was further evaluated using an Agilent 2100 Bioanalyzer system. Sequencing was performed on an Illumina NovaSeq 6000 platform. Q30 was chosen to filter the original data, and the clean reading was compared with the rat reference genome by HISAT2. Differentially expressed genes (DEGs) were identifined based on criteria of p < 0.05 and |log2FC| > 1. KEGG pathway enrichment analysis was conducted to investigate biological pathways significantly associated with the identified DEGs.

first: Jingfei

last: Liu

first: Yingwei

last: Lin

first: Zhihe

last: Ren

first: Dianyong

last: Gao

first: Yufan

last: Yao

first: Xuanzhe

last: Zhan

first: Tian

first: Shichao

first: Dong

last: Li


title: Immune imbalance in the human hippocampus in PTSD revealed by single-nucleus transcriptomics

city: Charleston

first: Liu

state: South Carolina

address: 173 Ashley Ave. CRI 610

institute: Medical University of South Carolina

summary: Post-traumatic stress disorder (PTSD) is increasingly recognized as a neuroimmune disorder in which disrupted neuron–glia interactions contribute to long-term cognitive and emotional dysfunction. However, the cellular and molecular basis of immune imbalance in the human hippocampus remains unclear.We performed single-nucleus RNA sequencing on postmortem hippocampal tissues from donors with PTSD and matched controls, identifying 10 major cell types, with particular emphasis on neurovascular and glial populations. Differential expression, pathway enrichment, pseudotime trajectory, and cell–cell communication analyses were applied to characterize cellular and molecular alterations.PTSD samples showed prominent activation of stress-response and inflammatory signaling across astrocytes, microglia, endothelial cells, and mural cells. Microglia and astrocytes underwent robust transcriptional reprogramming with enrichment of immune-related pathways, while endothelial and mural cells exhibited inflammation and impaired blood–brain barrier homeostasis. Trajectory analyses revealed altered state transitions in astrocytes and microglia, indicating dysregulated responses to stress. Furthermore, cell–cell communication analysis uncovered markedly reduced interactions between astrocytes or microglia with excitatory neurons and oligodendrocyte lineage cells, particularly involving stress- and inflammation-related ligand–receptor pairs.These findings demonstrate that PTSD is characterized by immune imbalance at both cellular and intercellular levels, driven by maladaptive glial activation and disrupted neuron–glia communication. Our study provides a comprehensive single-cell atlas of the hippocampal neuroimmune landscape in PTSD and highlights dysfunctional glial–neuronal interactions as a central mechanism underlying disease pathogenesis.

overall_design: Frozen hippocampal tissue samples from three PTSD patients and three age-matched controls were processed for single-nucleus RNA sequencing. Nuclei were isolated at Novogene (San Jose, CA) by tissue homogenization, debris removal, and centrifugation. Single-nucleus 3′ RNA libraries were prepared using the 10x Genomics Chromium Single Cell 3′ v3.1 platform and sequenced on an Illumina NovaSeq X Plus system at a target depth of ~50,000 read pairs per nucleus. Sequencing reads were aligned to the human reference genome (GRCh38), and gene expression matrices were generated using Cell Ranger (10x Genomics) with default parameters.

first: Pengfei

first: Hongkuan

last: Fan


title: Expression data from rat heart tissue ischemia/reperfusion (I/R) treated for 12,24 hours

city: Beijing

first: 唐

last: 伟评

address: 2 Anzhen Rd, Chaoyang District,

institute: Beijing Institute of Heart Lung and Blood Vessel Diseases

summary:
Acute pancreatitis (AP), a common inflammatory disorder, can lead to systemic complications such as myocardial injury. However, the dynamic transcriptomic changes and key molecular mechanisms behind these pancreatic-associated cardiac complications remain unclear.
We investigated SAP-mediated gene expression alteration associated with the development of cardiac injury by microarray in a rat model.

overall_design: All rats were euthanized by an overdose of pentobarbital on hours 12 and 24 of  SAP treated. Total RNA was isolated with TRIzol (Invitrogen) from heart organs (n=5 per group) at each time point according to manufacturer’s instructions. Gene expression profiling was performed using Affymetrix GeneChip mouse Genome 430 2.0 array according to the manufacturer’s instructions (Affymetrix, Inc., Santa Clara, CA). On the GeneChip Mouse Genome 430 2.0 Array, over 45,000 probe sets analyze the expression level of over 39,000 transcripts and variants from over 34,000 well characterized mouse genes.

first: Yun-Long

first: Xiao

last: Han


title: Epigenetic Signatures of Brain Aging by Single Nucleus Methylation Sequencing [CP-9mo-m-023]

first: Joseph

last: Ecker

summary: Illustrating aging effects on the epigenomic features of brain is critical to understand brain functional decline and neurodegeneration. Single nucleus methylation sequencing was applied to brain regions of both male and female mouse at the age of 2, 9 and 18 months. We use these data to identify the epigenomic changes with aging in mouse brains.

first: Wei

first: Anna

last: Bartlett

first: Jordan

last: Altshul

first: Rosa

last: Gomez Castanon

last: Nery

first: Jacinta

last: Lucero

first: Julia

last: Osteen

first: Huaming

last: Chen

first: M

last: Behrens


title: A Glucocorticoid-Fas axis controls immune evasion during metastatic seeding [CUT&Run_4T1-Dex]

city: Boston

first: Judith

last: Agudo

address: 1 Jimmy fund way

institute: Dana Farber Cancer Institute

summary: Metastasis is the major cause of death for patients with Triple Negative Breast Cancer (TNBC) and other solid malignancies. Metastases arise from cancer cells that disseminate from the original tumour, survive systemic immune surveillance, and colonize new organs. Little is known about how initial disseminated tumour cells (DTCs) overcome anti-tumour immunity upon seeding a new organ. Here we used a visible antigen in a model of TNBC with cognate CD8+ T cells to interrogate mechanisms of immune evasion in early metastatic seeding. Analysis of surviving DTCs revealed Glucocorticoid Receptor (GR) activation as a key driver of resistance to both CD8+ T cells and Natural Killer (NK) cells. Niche profiling using an optimized labelling tool uncovered Fas-FasL as a key pan-cytotoxic pathway against DTCs, which is repressed by GR activation. Pharmacologic inhibition of GR in combination with immunotherapy reduced metastatic burden and expanded lifespan in mice. Thus, we discovered a novel mechanism of immune evasion that operates specifically in DTCs, illustrating the unique immune-cancer interactions at this stage in the metastatic cascade. Our findings suggest that there are therapeutic opportunities to eliminate DTCs, separately from treatments aimed at primary tumours, with GR inhibition as one promising target.

overall_design: 4T1 breast tumour cells were treated with Dexamethasone or vehicle control for one week in vitro. Tumour cells were collected and ChIP was performed using a Glucocorticoid Receptor (GR) antibody. CUT&Run was performed on the resulting DNA. Details are included under Fig. 4.

first: Monica

last: Cassandras

first: Adam

last: Getzler


title: Adult Mouse Controlled Cortical Impact Visium Spatial Transcriptomics

city: Bethesda

first: Martin

middle: L

last: Doughty

state: MD

address: 4301 Jones Bridge Road

department: APG

institute: USU

summary: Here we investigated the topographical relationship of early transcriptional responses to a single, focal TBI in mice by controlled cortical impact (CCI). Guided by the presence of the anterior commissure (AC) in coronal sections at the rostro-caudal point of impact, we compared gene expression changes in the neocortex (CTX) and corpus callosum-external capsule (CC-EC), striatum (STR) and AC. Injury-induced gene expression changes were detected in the CTX, CC-EC and STR but not AC and were principally segregated based on cytoarchitecture, and secondarily by proximity to the site of impact. Additionally, unbiased spatial clustering revealed a positive relationship between proximity to the impact and the number of acutely differentially expressed genes within the laminar CTX. Next, we examined the effects of systemic depletion of neutrophils and monocytes on spatial gene expression changes in the injured brain.

overall_design: Visium (10X Genomics) spatial transcriptiomics prolfiling of the contused left hemisphere of the forebrains of mice given a single controlled cortical impact (CCI). Coronal sections at the focal point of contusion (at the level of the anterior commissure) were profiled from 15-20 weeks-old C57BL/6J mice given a CCI or Sham surgery (no craniectomy or impact). Experiment 1: CCI versus Sham mice at 2 days post-injury (DPI) and 7 DPI. Experiment 2: CCI mice treated with anti-GR-1 antibodies (to deplete circulating neutrophils and monocytes) versus CCI mice treated with control antibodies at 2 DPI and 7 DPI.

first: Savannah

last: Kounelis-Wuillaume

first: Andrew

last: Frank

first: Camille

last: Alba

first: Gauthaman

last: Sukumar

first: Matthew

last: Wilkerson

first: Clifton

last: Dalgard


title: A Glucocorticoid-Fas axis controls immune evasion during metastatic seeding [RNAseq_4T1-tdTomato]

overall_design: 4T1-tdTomato breast tumour cells were injected into BALB/c or NSG female mice. Tumour cells were FACS-sorted (DAPI-/CD45-/tdTomato+) from the mammary tumor or lungs of mice at indicated tmepoints. Details are included under Fig. 1a.


title: A Glucocorticoid-Fas axis controls immune evasion during metastatic seeding [scRNAseq_4T1-tdTomato-cTAT-zsGreen]

overall_design: 4T1-shGR (Glucocorticoid Receptor, GR, knockdown) and 4T1-shControl breast tumour cells were transduced to express tdTomato and secreted lipid-soluble cTAT-zsGreen for niche labelling. Tumour cells were injected into primary tumour-bearing BALB/c mice. Tumour cells and labelled niche cells were FACS-sorted from the lungs of mice at D7 post-injection. Details are included under Fig. 3.


title: A Glucocorticoid-Fas axis controls immune evasion during metastatic seeding [RNAseq_4T1-GFP]

overall_design: 4T1-GFP breast tumour cells were intravenously injected into Jedi or NSG female mice. Tumour cells were FACS-sorted (DAPI-/GFP+) from the lungs of mice at D7 post-injection. Details are included under Fig. 1e.


title: Pharmacologic reversion of Merkel cell carcinoma via CBP/p300 inhibition

city: Pittsburgh

first: Hyun Jung

last: Park

address: 1013 River Oaks Dr.

institute: University of Pittsburgh-Pittsburgh Campus

summary: Merkel cell polyomavirus (MCV) T antigen functions as an oncoprotein that drives the transformation of Merkel cell carcinoma (MCC) cells by activating transcription factors involved in cell proliferation. The viral T antigen promoter requires the activity of the cellular coactivator CREB-binding protein (CBP)/p300 for its expression. Inhibition of CBP/p300 with two distinct small-molecule inhibitors suppresses T antigen expression, leading to cell cycle arrest and upregulation of the cell cycle inhibitor p27Kip1. This shift promotes neuronal differentiation, associated with neurite outgrowth in MCC cells. RNA sequencing revealed downregulation of genes involved in E2F, Myc, mTORC1 oncogenic signaling, as well as markers of the Merkel cell lineage including Sox2 and Atoh1. Notably, a rare MCC case exhibiting a mixed cellular composition, with loss of T antigen expression and neuroblastic phenotype, showed a transcriptomic profile resembling that of MCC cells treated with CBP/p300 inhibitors. This suggests that similar differentiation processes may contribute to tumor heterogeneity in patients. This study presents the first model system enabling reversible switching between a transformed and differentiated cell state in a human cancer using small-molecule treatment.

overall_design: RNA-Seq profiling of CVG-1 cells under CBP/p300 inhibition (mock, A-485 [5 μM], and dCBP-1 [100 nM]) after 6 days of treatment

first: Masahiro

last: Shuda

first: Joseph

last: Collura


title: A Glucocorticoid-Fas axis controls immune evasion during metastatic seeding [scRNAseq_4T1-GFPThy11]

overall_design: A mix of 4T1-GFP and 4T1-Thy1.1 breast tumour cells were intravenously injected into BALB/c female mice following adoptive transfer of activated Jedi anti-GFP CD8+ T cells. Tumour cells were FACS-sorted (DAPI-/CD45-/GFP+ or Thy1.1+) from the lungs of mice at D7 post-injection. Details are included under Fig. 1g.


title: Granzyme B-mimicking nanozyme-containing nanovesicles for tumour-targeting delivery

city: tinajin

first: qiqi

last: liu

state: other

address: nankai university

institute: nankai university

summary: Cytotoxic T lymphocytes play a crucial role in anti-tumour immunity, with granzyme B (GrB) being a decisive factor in this process. Developing a GrB-based delivery system to mimic T cell-based immunotherapy holds promise but remains challenging. Here, we present an artificial metalloenzyme (nanozyme) with GrB-like protease activity capable of inducing caspase-dependent cell apoptosis. The nanozyme is based on the site-specific binding of Pd(II) ions to recombinant human heavy chain apo-ferritin nanocages to generate a binuclear catalytic centre consisting of two Pd atoms with bridging cysteine ligands, monodentate methionine and histidine residues, and two water molecules. We show that encapsulation of the Pd-ferritin complex within membrane-fused lipid nanoparticles comprising surface-displayed single-chain antibodies affords GrB-mimicking nanozyme-based nanovesicles capable of receptor-mediated delivery of the nanozyme into the cytoplasm of tumour cells and induction of caspase-dependent apoptosis. This study provides valuable insights into the construction of nano-delivery systems with artificial GrB activity and presents a promising therapeutic option for solid tumours.

overall_design: Tumour cells were treated with FNP-antiHER2 for 24 h. RNA was extracted and sequenced using the Illumina platform.

first: Xueyan

last: Hu

first: Qiqi

last: Liu

first: Helong

last: Kang

first: Ming

last: Zhao

first: Zheng

last: Zhou

first: Yuanke

last: Li

first: Weicheng

last: Peng

first: Tianyi

last: Qi

first: Ruming

first: Li

last: Jiao

first: Jie

last: Zhuang

first: Yijin

first: Stephen

last: Mann

first: Xinglu

last: Huang


title: Hypoxia-Enhanced Podoplanin-Defined Tumour Plasticity Drives CCR7-Mediated Lymphatic Metastasis in Breast Cancer

city: Huddinge

first: Jonas

last: Fuxe

address: Alfred Nobels Allé 8

department: Laboratory Medicine

institute: Karolinska Institutet

summary: Background: Lymphatic metastasis is strongly associated with poor prognosis. Although the chemokine receptor CCR7 is a well-established promoter of lymphatic dissemination, its prognostic relevance remains weak. We show that tumour cell plasticity, defined by upregulation of podoplanin (PDPN) and promoted by hypoxia, intersects with CCR7 function in triple-negative breast cancer (TNBC). Methods: In vivo and in vitro studies using a CCR7-expressing TNBC mouse model were combined with transcriptomic profiling. Human relevance was assessed using scRNA-seq datasets from cell lines and primary tumours, as well as the METABRIC breast cancer cohorts (n = 1784). Results:  A PDPN-defined epithelial–mesenchymal transition (EMT) state—promoted by hypoxia—was required for efficient CCR7-driven lymphatic metastasis and tumour progression. PDPN-expression was linked to elevated tumour cell collagen-expression and suppression of interferon-signalling, features associated with an immune-cold microenvironment. PDPN-expression with effects on interferon and collagen programs were observed across murine and human TNBC/basal-like cell lines. In primary TNBC, PDPN-expression correlated with EMT, collagen, and hypoxia signatures, mirroring murine findings. In METABRIC, high CCR7–PDPN co-expression predicted poor survival in lymph node–positive patients, whereas either marker alone lacked prognostic value. Conclusions: PDPN is a tumour-cell-associated biomarker of plasticity in TNBC, revealing synergy between specific hypoxia-induced EMT-states and CCR7 in promoting lymphatic dissemination and poor prognosis.

overall_design: Total RNA was extracted from four different subclones of EO771 mouse mammmary tumor cells (4 technical replicates/subclone), using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. Libraries were generated using the TruSeq Stranded mRNA kit (Illumina) and sequenced on an Illumina NextSeq 550 platform in single-end mode, producing 75 bp reads mapped to the mouse reference genome (GRCm38/mm10). Sequencing was run in two batches: the first batch included X2 (EO771 cells expressing vector control), CCR7 (EO771 cells overexpressing CCR7), CCR7-PDPN (CCR7-overexpressing EO771 cells sorted for high expression of PDPN); and the second batch included X2, X2-PDPN (vector control cells sorted for high PDPN expression), and CCR7-PDPN. All the data was analysed using Python (v.3.11.5). Normalization was performed using Transcripts Per Million (TPM) by rnanorm (v.1.5.1). TPM values below 1 were considered as noise.

first: Zhi

last: Wang

first: Lise

last: Ingebriktsen

first: Tove

last: Bekkhus

first: Lyusong

last: Ma

first: Wenyang

last: Shi

first: Anastasia

last: Bazioti

first: Michail

middle: A

last: Panagias

first: Martina

last: Vigorelli

first: Joakim

last: Lehrstrand

first: Sarah

last: Ring

first: Elisabeth

last: Wik

first: Erling

last: Hoivik

first: Maria

last: Ulvmar


title: Regulation of INSM1 Gene Expression and Neuroendocrine Differentiation in High-Risk Neuroblastoma

city: New Orleans

first: Kuan-Jui

last: Su

state: LA

address: 1440 Canal St,suite 1619G

department: Center for Biomedical Informatics and Genomics

institute: Tulane University

summary: Neuroblastoma (NB), a pediatric cancer of sympatho-adrenal (SA) lineage, is marked by disrupted differentiation and cellular heterogeneity. INSM1, a zinc-finger transcription factor, is highly expressed in NB and developing SA tissues, where it regulates neuroendocrine differentiation, especially in chromaffin cells. We investigated INSM1’s role in maintaining an undifferentiated, progenitor-like state in NB and its regulation via metabolic and epigenetic mechanisms. Transcriptomic profiling, promoter assays, and metabolic flux analysis revealed that INSM1 expression correlates with methionine cycle activity, particularly the S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) ratio. Disruption of SAM/SAH balance altered INSM1 promoter activity and histone methylation, implicating epigenetic control in NB cell fate. Retinoic acid-induced differentiation downregulated INSM1 and N-Myc, linking INSM1 to tumor cell immaturity. INSM1 overexpression in SH-SY-5Y cells upregulated neuroendocrine and thyroid hormone-related genes (CHGA, CHGB, DDC, NCAM1, DIO3, TH), while suppressing genes involved in cell cycle (MYBL2, RRM, CDC25A), methionine metabolism (AHCY, MAT2A), transcriptional regulation (EZH2), and oncogenic signaling (ALK, LINC011667). These findings suggest that INSM1 promotes NB aggressiveness by sustaining a neuroendocrine progenitor-like phenotype through metabolic-epigenetic coupling.

overall_design: SH-SY-5Y cells  transduced with INSM1 adenoviral vectors for INSM1 overexpression. The effects of INSM1 on neuroblastoma differentiation  and growth were analyzed by RNA-seq

first: Chiachen

last: Chen

first: Siyuan

last: Cheng

first: Xiuping

last: Yu

first: Michael

last: Lan


title: Epigenetic Regulation of Serine Biosynthesis by PHF8 During Neurogenesis [ChIP-Seq H3K9me3]

city: Barcelona

first: Marta

middle: Artés

last: Hernández

address: Baldiri Reixac 15

country: Spain

institute: IBMB-CSIC

summary: During neurodevelopment, progenitor proliferation is a highly orchestrated process, involving precise epigenetic modifications and immense metabolic demands. Yet, the intricate interplay between epigenetics and metabolism remains largely unexplored. To investigate this, we utilized neural stem cells to examine the role of PHF8, a histone demethylase whose mutations are linked to Siderius-Hamel syndrome, a rare neurodevelopmental disorder. Through an integrated multi- omics approach—combining transcriptomics, epigenomics, and metabolomics—we identify PHF8 as a key driver of the serine biosynthesis pathway, safeguarding the intracellular serine pool essential for neural progenitor proliferation. PHF8 fine- tunes chromatin accessibility at promoters of metabolic genes, ensuring their activation during development. Notably, loss of PHF8 not only disrupts amino acid metabolism and mTORC1 signaling, but also blocks autophagic flux, and impairs vesicle formation—ultimately stalling proliferation and triggering replicative defects and DNA damage. In vivo, PHF8 deficiency in mouse embryos halts neurogenesis, affecting progenitor expansion and neuron generation in the developing brain. Altogether, our work positions PHF8 as a pivotal molecular node linking chromatin dynamics, metabolic regulation, and neural development. These findings not only deepen our understanding of brain development but also open new avenues for exploring the epigenetic basis of metabolic and neurodevelopmental disorders.

overall_design: 1x 10^6 shCTR and shPHF8 NSCs were cross-linked with Cross-link Gold 0.4% (Diagenode, C01019027) in PBS 30 minutes, and then fixed with methanol-free formaldehyde 1% for 10 min and stopped with 0.125 mM glycine for 10 minutes. Sonication was performed using a Bioruptor sonicator. The libraries were prepared and amplified to make DNA NanoBall (DNB) Libraries were loaded into a nanoarray and single end 50 base pairs reads were generated by combinatorial Probe-Anchor synthesis (cPAS)

last: Artés

first: Simona

last: Iacobucci

first: María

last: Balladore

first: Paula

last: Caballeria

last: García

first: Alex

last: Pérez

first: Natalia

last: Padilla

first: Bárbara

last: Viegas

first: Aitana

last: Díaz

first: Silvina

last: Natch

first: Guillermo

last: Vicent

last: Vaquero

first: Mariona

last: Arbonés

first: Xavier

last: de la Cruz

last: Nieto

first: Neus

last: Agell

first: Caroline

last: Mauvezin

first: Marian

last: Martínez


title: Hv1 upregulation worsens spinal, spleen, and lung molecular pathology and impairs locomotion after spinal cord injury in aged male mice

city: Baltimore

first: Hangnoh

middle: Joseph

last: Lee

address: 670 W Baltimore St

institute: University of Maryland School of Medicine

summary: Spinal cord injury (SCI) induces both intraspinal damage and systemic organ pathology, with aging as a major determinant of outcome. However, how advanced age exacerbates SCI pathology remains unclear. The voltage-gated proton channel Hv1, a regulator of immune activation, increases with age and after SCI, and its deletion is neuroprotective in young mice.  Thus, we hypothesized that age-related Hv1 upregulation worsens spinal cord, spleen, and lung pathology and hinders locomotor recovery after SCI through immune modulation. Using aged Hv1 KO and WT male mice subjected to moderate SCI, we assessed behavioral and molecular outcomes. Transcriptomic analysis revealed that Hv1 mRNA expression was higher in the brains of aged sham mice and further upregulated in injured spinal cord tissues. Spinal cord RNA-seq showed acute innate immune and cytokine activation in both genotypes. Hv1 deletion enhanced chromatin remodeling, epigenetic and Wnt signaling, while suppressing NF-κB–driven inflammation and oxidative stress–induced apoptosis. In the spleen, Hv1 depletion enhanced immune responses while suppressing mitosis. T cell and leukocyte activation increased, whereas lung cytokine signaling decreased. At 6 weeks after SCI, Hv1 KO mice showed elevated expression of genes related to circadian rhythm and T cell proliferation in the spinal cord, increased mitotic gene expression but reduced adaptive immunity in the spleen, and enhanced immune activation with decreased cilium activity in the lungs.

overall_design: RNA-Seq gene expression profiling was performed on 18–22-month-old male C57BL/6 (WT)  and Hv1 KO mice subjected to either sham treatment or SCI. Total RNA was extracted from the spinal cord, lung, and spleen tissue samples at 7 days and 6 weeks post-injury.  Transcriptomic differences between Sham and Injury, WT and KO mice were analyzed.

first: Zihui

last: Wang

first: Yun

last: Li

first: Junfang

last: Wu


title: Altered MDC1 Interactions and Dysfunctional DNA Repair in Lobular Breast Cancer Confers Sensitivity to PARP Inhibition

city: AURORA

first: Etienne

last: Danis

department: Cancer Center

institute: University of Colorado, Denver - Anschutz Medical Campus

summary: Invasive lobular carcinoma of the breast (ILC) is typically estrogen receptor α (ER)-positive and presents with biomarkers of anti-estrogen sensitive disease. Unfortunately, patients with ILC face particularly poor long-term outcomes with high recurrence risk, suggesting a divergent endocrine response and ER function in ILC compared to other breast cancers. ER is co-regulated by the DNA repair protein MDC1 specifically in ILC cells, driving distinct ER activity. Here, we profiled the MDC1 interactome to examine how MDC1 regulates ER activity and DNA repair function in ILC. MDC1-associated proteins in ILC cells mirrored a “BRCA-like” state lacking key homologous recombination (HR) proteins, consistent with HR dysfunction but distinct from classic “BRCAness”. Single-cell transcriptome and DNA repair activity analyses, along with DNA repair signaling and functional data, substantiated dysfunctional induction and execution of HR in ILC cells. In parallel, ILC tumor data were consistent with a form of HR dysfunction distinct from overt HR deficiency, lacking BRCA-like genomic scarring but showing elevated signatures of PARP inhibitor sensitivity. Treatment with the PARP inhibitor talazoparib produced a durable growth suppression both in vitro and in multiple ILC xenografts in vivo. Together, these findings reveal that ILC-specific ER:MDC1 activity comes at the cost of DNA repair dysfunction, which may be therapeutically targetable.

overall_design: ILC cell line MDA MB 134VI (MM134) was hormone-deprived, then treated with vehicle (0.01% EtOH) or estradiol (E2, 100nM) for 24 hours prior to sample collection. After treatment, single cell suspensions were prepared according to 10X Genomics recommendations; Haircut libraries were prepared as described (PMID: 32286626 and bioRxiv 2019.10.29.820233), and separate targeted capture libraries were prepared according to the manufacturer’s instructions (https://www.10xgenomics.com/products/targeted-gene-expression).

first: Matthew

last: Sikora

first: Joseph

last: Sottnik

first: Amanda

last: Richer

first: Rui

last: Fu

first: Jay

last: Hesselberth


title: RNA-seq of human atherosclerotic carotid plaque tissue from diabetic patients

first: Thomas

middle: Cooper

last: Woods

address: 1430 Tulane Avenue

department: Physiology

institute: Tulan

summary: Diabetes Mellitus is associated with increased risk of myocardial infarctions and strokes due to accelerated atherosclerotic plaque development and rupture. The mechanisms driving plaque rupture in the diabetic setting remain unclear. We sequenced ribosome-depleted total RNA from carotid plaques obtained from diabetic subjects undergoing carotid endarterectomy with high-grade stenosis, who either recently experienced a carotid-related ischemic cerebrovascular event (Symptomatic, n=6) or had no such event (Asymptomatic, n=6). Principal component analysis of the most variable transcripts demonstrated that sex and insulin use, rather than plaque rupture status, were the major contributors to the variability in the dataset. Hierarchical clustering further highlighted the importance of these factors in the dataset but also included two clusters whose expression was elevated in the symptomatic group. These clusters were enriched with transcripts for immunoglobulins and matrix metalloproteinases, suggesting increased B-cell infiltration and extracellular matrix degradation in the symptomatic plaques. We identified only two transcripts, RNU2-1 and PI4KAP1, as significantly up-regulated in the symptomatic group. We identified 35 transcripts with altered expression in subjects on insulin therapy, including regulators of B-cells (MZB1), T cells (BTN3A1, BTN3A2, BTN3A3, CD96, and CTSW), and macrophages (MPEG1). Next, we used spatial deconvolution to focus on differentially expressed genes in the fibrous cap of the carotid plaques. In the symptomatic group, we identified elevated levels of five angiogenesis-associated transcripts (ALS2CL, EPHB4, SMAD1, PREX2, EFNA5), suggesting increased intraplaque neovascularization in ruptured plaques. Thus, insulin use significantly impacts the transcriptome of atherosclerotic plaques, promoting vascular inflammation.

overall_design: Single-end rRNA-depleted RNA-seq was performed on carotid plaque samples from diabetic patients to assess transcriptomic differences between symptomatic and asymptomatic lesions.

first: Woods

last: Thomas Cooper


title: Adipose tissue-derived microRNAs as epigenetic modulators of type 2 diabetes [DPDR_smallSeq]

city: Nuthetal

first: Heike

last: Vogel

state: BRB

address: Arthur-Scheunert-Allee 114-116

department: Department of Experimental Diabetology

institute: German Institute of Human Nutrition Potsdam-Rehbruecke

summary: Diabetes-resistant and diabetes-prone female New Zealand Obese mice were assessed based on liver fat content and early blood glucose concentrations at 10 weeks of age before the onset of T2D. Analysis of the microRNA profile in gonadal white adipose tissue revealed miRNA-335-5p, which could contribute to systemic metabolic changes associated with the risk of developing T2D.

overall_design: smallRNA-sequencing was performed in gonadal white adipose tissue of 14 diabetes resistant (DR) and 15 diabetes prone (DP) animals, DR animals are healthy animals and are considered as control group.

first: Annette

last: Schürmann

first: Meriem

last: Ouni

first: Ratika

last: Sehgal

first: Neele

last: Haacke

first: Pascal

last: Gottmann

first: Markus

last: Jähnert


title: Adipose tissue-derived microRNAs as epigenetic modulators of type 2 diabetes

summary: Diabetes-resistant and diabetes-prone female New Zealand Obese mice were assessed based on liver fat content and early blood glucose concentrations at 10 weeks of age before the onset of T2D. Analysis of the DNA methylation profile in gonadal white adipose tissue revealed that the imprinted region Dlk1-Gtl2 and Mest (miR-335) potentially regulated by DNA methylation.

overall_design: Analysis of DNA methylation was performed in gonadal white adipose tissue of 6 diabetes resistant (DR) and 6 diabetes prone (DP) animals, DR animals are healthy animals and are considered as control group.


title: SBFI inhibitors reprogram transcriptomic landscape of prostate cancer leading to cell death

city: Stony Brook

first: Agnieszka

middle: B.

last: Bialkowska

state: New York

address: HSC-T17 Room 090

department: Dept. of Medicine

institute: Stony Brook University

summary: Prostate cancer (PCa) remains the second leading cause of cancer-related deaths in the United States. Fatty acid-binding protein 5 (FABP5), a member of a class of intracellular lipid transporters, promotes PCa progression via enhanced lipid metabolism and trafficking of lipid ligands. Previous work from our group has shown that the small molecule FABP5 inhibitors based on the truxillic-acid monoester (TAMEs) scaffold reduce PCa growth. In the current study, we demonstrate that the third-generation FABP5 inhibitor SBFI-1143 significantly inhibited the viability and proliferation of PCa cells by arresting cells at the G0/G1 and G2/M phases of the cell cycle and inducing apoptosis and cell death compared to 1st and 2nd generation inhibitors. Stinkingly, SBFI-1143 efficiently inhibited the growth of PCa spheroids compared to its predecessor, SBFI-103. RNA sequencing and Gene Set Enrichment Analysis (GSEA) demonstrated that SBFI-1143 more effectively suppressed the activity of pathways involved in cell cycle progression, cell cycle division, and chromosome organization, while upregulated genes associated with the endoplasmic reticulum (ER) stress, responses to the topologically incorrect folded proteins, and regulating apoptosis compared to the SBFI-103. We further show that SBFI-1143 inhibits oxidative phosphorylation, a process on which PCa depends during cancer initiation and progression. Notably, SBFI-1143 treatment downregulated genes attributed to the subpopulation of PCa cells characterized by the lineage plasticity-related signature leading to trans-differentiation, recurrence, and poor cancer prognosis. Our findings demonstrate that SBFI-1143 significantly modifies the transcriptomic landscape of PCa and may serve as a potentially effective therapeutic option for PCa.

overall_design: To investigate the mechanism of action and efficacy of SBFI-103 in vitro, we treated the mouse prostate cancer cell line, RCaP, with vehicle control (DMSO) and 15uM of SBFI-103 for 24 hours. Total RNA was isolated and RNA-seq was conducted. We then performed differential gene expression analysis using RNA-seq data from three biological replicates of each treatment condition to characterize significant transcriptomic changes in response to treatments.

first: Shubhra

last: Rajput

first: Joseph F

last: LaComb

first: Chris

last: Gordon

first: Hehe

last: Wang

first: Martin

last: Kaczocha

first: Agnieszka B

first: Iwao

last: Ojima


title: OR7A10 GPCR engineering boosts CAR-NK therapy against solid tumors

city: West Haven

first: Sidi

last: Chen

state: CT

address: 850 West Campus Drive

institute: Yale University

summary: Chimeric antigen receptor (CAR)-natural killer (NK) cell therapies hold promise for solid tumors but remain limited by poor tumor infiltration, persistence, and resistance within the tumor microenvironment (TME). To identify gain-of-function (GOF) targets that enhance CAR-NK efficacy, we performed an unbiased in vivo Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) activation (CRISPRa) screen, followed by a barcoded targeted in vivo open reading frame (ORF) screen in primary human CAR-NK cells. We identified, and robustly validated OR7A10, a G protein-coupled receptor (GPCR), as the top candidate. Engineering CAR-NKs with OR7A10 cDNA, a CRISPR-independent method with simple manufacturing strategy, enhanced proliferation, activation, degranulation, cytokine production, death ligand expression, chemokine receptor expression, cytotoxicity, persistence, metabolic fitness, and TME resistance, while reducing exhaustion in primary human NK cells derived from multiple peripheral blood and cord blood donors. OR7A10-GOF CAR-NKs displayed robust in vivo efficacy across multiple solid tumor models, achieving a 100% complete response in an orthotopic breast cancer model with long term tumor control and survival benefit. These findings establish OR7A10-engineered CAR-NKs as a highly potent and scalable off-the-shelf therapeutic for solid tumors.

overall_design: Bulk RNA-seq profiling of NK cells transduced with different CAR constructs (OR7A10, OR7A10Stop, truncatedHER2CAR_OR7A10, truncatedHER2CAR_OR7A10Stop, HER2CAR_OR7A10, HER2CAR_OR7A10Stop) under 0hr and 24hrs stimulation conditions.

first: Luojia

last: Yang

first: Kaiyuan

last: Tang