title: NanoString profiling of IRF3 and IRF7 deficient macrophages and dendritic cells during influenza infection

city: Singapore

first: Lina

middle: H K

last: Lim

state: Singapore

address: 28 medical drive

department: Department of Physiology

country: Singapore

institute: National University of Singapore

summary:
Interferon regulatory factors IRF3 and IRF7 are key transcriptional regulators of antiviral immune responses. However, their cell type–specific roles in macrophages and dendritic cell subsets during influenza virus infection remain incompletely understood. In this study, NanoString nCounter gene expression profiling was performed to systematically characterize transcriptional responses in alveolar macrophages (AM), conventional dendritic cells type 1 (DC1), and type 2 (DC2).
Wild-type and IRF3- or IRF7-deficient cells were analyzed under uninfected and influenza-infected conditions. This dataset enables comparative analysis of IRF3- and IRF7-dependent gene expression programs across immune cell types and infection states, providing a resource for dissecting distinct antiviral and inflammatory regulatory mechanisms.

overall_design: Alveolar macrophages (AM), DC1, and DC2 were isolated from wild-type, IRF3 knockout, and IRF7 knockout mice. Cells were either left uninfected or infected with influenza virus. Gene expression was quantified using the NanoString nCounter platform. For each genotype and condition, multiple biological replicates were analyzed. Raw NanoString RCC files and processed gene expression matrices are provided for downstream analysis and reuse.

first: Jianzhou

last: Cui


title: Deregulated Myt3 translation predisposes islet β-cells to dysfunction under obesity-induced metabolic stress

status: Public on Feb 18 2026

city: Nashville

first: guoqiang

last: Gu

state: Tennessee

address: 9415D Medical Research Building IV

department: Department of Cell and Developmental Biology

country: USA

institute: Vanderbilt University Medical Center

type: Expression profiling by high throughput sequencing

summary: In response to obesity-related metabolic stress, islet beta-cells adapt (or compensate) by increasing their secretory function and mass. Yet, for unknown reasons, this compensation is reversed in some individuals at some point to induce beta-cell failure and overt type 2 diabetes. We have previously shown that transcription factor Myt3 (St18) and its paralogs, Myt1 and Myt2, prevent beta-cell failure. Myt3 was induced at post-transcriptional levels by obesity-related stress in both mouse and human beta cells and its downregulation accompanied beta-cell dysfunction during type 2 diabetes development. Single-nucleotide polymorphisms in MYT3 were associated with an increased risk of developing human diabetes. We now demonstrate that Myt3 translation is regulated by an upstream open-reading frame that overlaps with the main Myt3 open-reading frame in mice. Disrupting this overlap enhances Myt3 translation in mouse beta cells without metabolic stress but decreases it under high-fat-diet challenges. Consequently, this deregulation results in beta-cell dysfunction and glucose intolerance in mice, accompanied by compromised expression of several beta-cell function genes, demonstated by sc-RNAseq studies here. These findings suggest that stress-induced Myt3 translation is part of the compensation mechanism that prevents beta-cell failure.

overall_design: CRISPR-Cas9-based technique were used to insert 12 bps in one exon of Myt3 (verified by sequencing), which  disrupt an unstream open reading frame. The founder was outcrossed 3 rounds and then used for producing homozygous mice (for WT and mutant alleles). Islets were isolated from these mice for sc-RNAseq.

first: Ruiying

last: Hu

first: Yu

last: Wang

first: Qi

last: Liu

first: Lau

last: Ken

first: Xanwen

last: Xu

first: Alan J.

last: Simmons

first: Guoqiang


title: Spatial transcriptomics of primary cutaneous melanoma

city: Manchester

first: Amaya

last: Viros

address: Wilmslow Road

department: Skin Cancer Ageing

country: United Kingdom

institute: Cancer Research UK

summary: We performed spatial transcriptomics on primary cutaneous melanoma samples to characterise the tumour microenvironment

overall_design: Two primary cutaneous melanoma FFPE samples were analysed using spatial transcriptomics

last: Virós


title: METTL3 overexpression regulates transcriptomic programs in ovarian cancer cells

city: Chicago

first: Junzui

last: Li

state: IL

address: 303 E. Superior Street

department: OBGYN

institute: Northwestern University

summary: This study investigates transcriptomic changes induced by METTL3 overexpression in ovarian cancer cells. RNA-seq was performed on METTL3-overexpressing (MT) and control (P) samples to profile gene expression alterations.

overall_design: We generated RNA-seq data from six samples: three METTL3-overexpressing (MT1–3) and three control parental samples (P1–3). Paired-end sequencing (2 × 150 bp) was performed on Illumina NovaSeq.

first: JUNZUI

last: LI


title: METTL3 knockdown regulates transcriptomic programs in ovarian cancer cells

summary: This study investigates transcriptomic changes induced by METTL3 knockdown in ovarian cancer cells. RNA-seq was performed on METTL3-kd (SM) and control (NC) samples to profile gene expression alterations.

overall_design: We generated RNA-seq data from 9 samples: 6 METTL3-knockdown (SM2 and SM4) and three control parental samples (NC). Paired-end sequencing (2 × 150 bp) was performed on Illumina NovaSeq.


title: Comparison of the kidney transcriptome of 28 days old principal cell-specific Tsc1 knockout (Tsc1f/f/Aqp2 Cre) vs wild-type (WT) mice

city: Albuquerque

first: Marybeth

last: Brooks

state: New Mexico

address: 915 Camino de Salud

department: DOIM - Nephrology

institute: University of New Mexico

summary: Tuberous sclerosis complex is a genetic disorder that affects multiple organs. In the kidney, it presents as hamartomas and cysts. The epithelium of TSC renal cysts is primarily composed of A-intercalated cells. In these studies, we compared the renal transcriptomes of 28 days old principal cell-specific Tsc1 knockout (Tsc1f/f/Aqp2 Cre; Tsc1 KO) and WT mice.

overall_design: The kidney transcriptome of principal cell-specific C57BL/6 Tsc1 knockout mice (Tsc1f/f/Aqp2 Cre) vs WT C57BL/6 at 28 days of age were compared.

first: Manoocher

last: Soleimani

first: Kamyar

last: Zahedi

first: Sharon

last: Barone


title: Bulk RNA-sequencing of four soft tissue sarcoma cell lines treated in vitro for 6 hours with BO-112 or vehicle control

city: Stanford

first: Anusha

last: Kalbasi

state: CA

address: 269 Campus Drive

department: Radiation Oncology

institute: Stanford

summary: Impact of BO-112 on gene expression of sarcoma cell lines treated with  BO-112 or control

overall_design: Bulk RNA-sequencing was done on 4 sarcoma cell lines in two conditions (BO-112 for 6 hours versus vehicle control for 6 hours).

first: Kalbasi

last: Anusha

first: Tariveranmoshabad

last: Mito


title: Dynamics of the association between miRNA levels and PDAC risk through the years prior to pancreatic ductal adenocarcinoma (PDAC) diagnosis

first: Hui

last: Cai

state: TN

address: 2525 West End Ave.

institute: VUMC

summary: circulating levels of miRNAs may identify individuals at imminent risk of developing PDAC for a targeted screening and close surveillance

overall_design: This ia a 1:1 mateched case-control study nested in the five cohorts. Pre-diagnosed blood samples were collected for 1,307 cases and their matched controls. Their miRNA levels were measured using the NanoString technics


title: PRECISE: A prognostic thyroid follicular-cell derived gene signature for papillary thyroid carcinoma

city: Houston

first: Jennifer

last: Wang

state: TX

address: 1515 Holcombe Blvd

institute: University of Texas MD Anderson Cancer Center

summary: Papillary thyroid carcinoma (PTC) exhibits heterogeneous behavior. There is a need for effective biomarkers and improved risk stratification methods in PTC. Gene signatures derived from bulk RNA sequencing capture composite transcriptional profiles that are confounded by tumor microenvironment cells. We developed a thyroid follicular cell–enriched gene signature, Prognostic RNA Expression Cell-specific Integrated SignaturE (PRECISE), using single-cell RNA sequencing techniques and evaluated its prognostic utility across three independent cohorts with bulk RNA-seq data and survival outcomes.

overall_design: Primary tumor samples were from surgical resection and analyzed using bulk RNA sequencing.

first: Rui Jennifer

first: Chia-Chin

last: Wu


title: Herpesvirus-encoded lncRNA targets host splicing by sequestering splicing factors

city: Gainesville

first: Rolf

last: Renne

state: FL

address: 1200 Newell Dr.

institute: University of Florida

summary: Kaposi's sarcoma-associated herpesvirus (KSHV) encodes multiple short and long noncoding RNAs which contribute to viral latency, persistence, host gene regulation, and immune evasion. The Antisense-to-Latency Transcript (ALT) is a ~10 kb long noncoding RNA (lncRNA) located on the opposite strand of the major latency-associated region encoding the latency associated nuclear antigen, vCyclin, vFLIP, the Kaposin's and 12 microRNA genes. ALT is a nuclear lncRNA that is lowly expressed during latency, but strongly upregulated during lytic replication. Using RNA antisense purification and quantitative mass spectrometry (RAP-MS) in lytically induced primary effusion lymphoma cells, we identified 51 human and 3 viral proteins that directly interact with ALT. Of these enriched proteins, 48 are splicing factors, including core and alternative splicing proteins, such as U2AF2, PTBP1/2, SRSF1/3 and MBNL1. Interaction and co-localization of ALT was confirmed with various splicing factors in ribonucleoprotein complexes suggesting that ALT sequesters splicing factors in nuclear condensates.  We further identified that induction of lytic replication in lymphoid and epithelial cells leads to thousands of host gene splicing changes, which are partially restored upon perturbation of ALT expression.  Finally, transient knockdown of ALT strongly inhibits viral reactivation and virion production. Hence, sequestering of splicing factors by ALT, interferes with host gene expression. Our results uncover a novel mechanism that shifts gene expression from the host to the virus late during the viral replication cycle to efficiently produce progeny virus and potentially antagonize host immune defenses.

overall_design: mRNA splicing analysis of iSLK cells overexpressing ALT or control plasmid by performing RNA sequencing on Illumina

first: Ritu

last: Shekhar


title: CD4+ T cells' immune response during the early phase of infection with Schistosoma japonicum

city: Changsha

first: Nouhoum

last: Dibo

state: Hunan

address: No. South Lushan Road

country: China

institute: Central South University

summary:
Single-cell transcriptome analysis reveals that the CD4+ T cells' immune response was more intense in the non-permissive host Microtus fortis than in the permissive host Kunming mice during the early phase of infection with Schistosoma japonicum
We have employed a single-cell sequencing approach using Singleron scRNA-seq to explore the immune response of M. fortis and Kunming mice to S. japonicum. The study allowed us to decipher M. fortis PBMC heterogeneity and provide detailed information on the transcriptome profile of the different cell types following exposure to S. japonicum.

sample_organism:
Mus musculus
Alexandromys fortis

platform_organism:
Mus musculus
Alexandromys fortis

overall_design: The peripheral blood mononuclear cells were isolated from uninfected and infected animals by density gradient centrifugation and analysed by scRNA-seq.


title: Monocytes acquire a tumor-associated IL1B program upon encountering patient-derived colon cancer organoids

city: Mannheim

first: Volker

last: Ast

address: Theodor-Kutzer-Ufer 1-3

department: NGS Core Facility

country: Germany

institute: University Hospital Mannheim

summary: Tumor-associated monocytes and macrophages (TAM) accumulate in colorectal cancer (CRC) and play an active role in shaping the tumor microenvironment (TME). While they have been linked to both pro- and anti-tumor functions, understanding the cues that instruct the phenotype of individual subsets as well as their functional impact on the TME remains challenging. In this study, we established co-cultures comprising primary human monocytes and patient-derived organoids (PDOs) from CRC tumors to emulate myeloid cell/carcinoma interactions in vitro that drive tumor-associated monocyte  programming. Upon encountering PDOs, monocytes acquired a complex phenotype not reflected by classical TAM markers or polarization protocols. Single-cell RNA sequencing revealed that PDO-exposed monocyte transcriptionally resembled IL1B-programmed monocytes previously identified in CRC patients. This phenotype emerged independently of the tumors’ mutational profiles or CMS type. Mechanistically, soluble PDO-derived mediators induced the production of chemotactic CXCL2, CXCL5 and CXCL7, while phagocytic uptake of tumor debry limited the MHC class II-mediated antigen presentation capabilities of monocytes in co-culture. Additionally, our in vitro system allowed the functional assessment of PDO-exposed monocytes demonstrating a compromised capacity to mount an inflammatory response upon TLR stimulation. Together, our PDO–monocyte co-cultures offer a tractable, human-specific model system to study the interplay between epithelial cancer cells and monocytes, and advance our understanding of myeloid plasticity and function in cancer patients.

overall_design: Single-cell RNA sequencing of CD45+ sorted monocytes after four days of monoculture or co-culture with PDO19 or PDO30

first: Bianca

middle: M

last: Balzasch

first: Adelheid

last: Cerwenka


title: Autopolyploidization-induced chromatin remodeling regulates leaf size variation in Brassica rapa

city: Baoding

first: Haoyuan

last: Dong

state: Prof.

address: Lekai South Street

institute: Hebei Agricultural University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Whole-genome duplication (WGD) is an evolutionary event that impacts gene regulation and trait development, but its role in chromatin remodeling and heritable phenotypic changes remains unclear. In this study, employing ATAC-seq, ChIP-seq (H3K4me3, H3K27ac, H3K27me3), and RNA-seq, we examined the epigenomic and transcriptomic dynamics in monoploid, diploid, and autotetraploid B. rapa (Brassica rapa), which share a genome from the same source. Our findings demonstrate that autopolyploidization exerts ploidy-dependent effects on the epigenome and transcriptome, characterized by nonlinear patterns, particularly pronounced during the monoploid-to-diploid transition. Increased ploidy leads to reprogramming chromatin accessibility, characterized by reduced-proximal versus expanded-distal regions. H3K4me3 modifications near transcription start sites alter global gene expression. We identified numerous transcription factor genes, of which BrGRF13 and BrARF11 are crucial to regulate leaf size and polarity during head development. This study is the first to utilize a monoploid system to reveal the mechanisms by which autopolyploidization regulates epigenetic changes through chromatin remodeling, filling a critical knowledge gap and providing novel insights into how polyploidization drives phenotypic variation in plants.

overall_design: Leaf samples were collected during the heading transition stage from monoploid, diploid, and autotetraploid B. rapa plants derived from the same genome. Each ploidy group included biological replicates: three for RNA-seq and two for ChIP-seq (H3K4me3, H3K27ac, H3K27me3) and ATAC-seq. Monoploid samples served as the reference to assess the effects of increasing ploidy on chromatin accessibility, histone modifications, and gene expression.

first: Yanhong

last: Liu

first: Yuanming

first: Shuxin

last: Xuan

first: Huanhuan

last: Chen

first: Lai

last: Wei

first: Guibao

last: Zhang

first: Hongcui

last: Pei

first: Zilong

last: Dai

first: Yanhua

first: Jinzhu

last: Qiao

first: Shuangxia

last: Luo

first: Xueping

first: Yiguo

last: Hong

first: Jianjun

last: Zhao

first: Shuxing

last: Shen

first: Zefu

last: Lu

first: Aixia

last: Gu


title: Single-nucleus RNA sequencing Resolves Microenvironmental Dynamics in Brown/Beige Adipose Tissue after Bariatric Surgery

city: Hangzhou

first: wang

last: my

address: Cangqian Street, Cangxing Street

institute: BoYu Intelligent Health Innovation Laboratory, Hangzhou, 311100, P. R. China.

summary: Obesity, a chronic disease marked by pathological adipose expansion, drives systemic comorbidities such as type 2 diabetes, cardiovascular disease, and cancer, severely impairing quality of life. Bariatric surgery remains the most effective intervention for sustained weight loss and metabolic improvement, yet the mechanisms underlying adipose tissue remodeling post-surgery remain poorly understood. Using single-cell RNA sequencing, we generated high-resolution transcriptional maps of murine brown adipose tissue (BAT) and beige adipose tissue pre- and post-bariatric surgery, uncovering dynamic microenvironmental shifts. Following surgery, both BAT and beige adipose tissues displayed a notable expansion of myofibroblast (myoFB) populations and a parallel reduction in adipocytes (AP). Cell communication analysis via CellChat revealed enhanced AP-myoFB crosstalk mediated by IGF1 signaling activation, alongside suppression of VEGF pathway interactions between AP and endothelial cells (EC). Pseudotemporal trajectory analysis identified an AP subpopulation (AP6) enriched in fibrosis-associated genes (Zbtb16, Fkbp5) that progressively upregulated 11 genes during differentiation toward myoFB. Spatial correlation analysis further demonstrated co-localization of these genes with Igf1r in myoFBs. Collectively, this work delineates surgery-induced microenvironmental reprogramming in thermogenic adipose depots and proposes a novel mechanism whereby 11 potential transition regulators synergize with IGF1 signaling to drive AP6-myofibroblast transdifferentiation.

overall_design: A total of four samples were included in this study, with two derived from brown adipose tissue (BAT) and the other two from beige adipose tissue (BeAT). Within each adipose tissue group (BAT or BeAT), the two samples were further categorized into either a high-fat diet surgical intervention group (HFD-operated) or a high-fat diet sham surgery control group (HFD-sham).

first: Qiankun

last: Li

first: Mengyao

last: Wang

first: Jihong

last: Yang


title: Hepatocyte CD44 in aged mouse liver correlates with immune modulation and fibrotic priming

city: La Jolla

first: K. Garrett

last: Evensen

address: 10010 N Torrey Pines Rd

institute: The Salk Institute for Biological Studies

summary: Liver cancer incidences increase beyond 55 years of age suggesting that molecular and immunological changes accompanying aging contribute critically to tumor initiation. However, the mechanisms linking aging and liver cancer initiation are not well understood. Our study investigates the role of CD44, a transmembrane glycoprotein implicated in cancer development, in aging-associated liver pathophysiology. CD44 expression was analyzed in young and aged mouse livers, with aged livers showing an accumulation of CD44 expressing hepatocytes. Analyses by single nucleus RNA sequencing reveal that CD44 expressing hepatocytes in aged livers exhibit enrichment of immune modulatory genes and interferon associated pathways. Spatial analyses showed that CD44 expression in hepatocytes co-occurs with T-cell neighborhoods exhibiting reduced cytokine expression. Functional assays showed reduced antigen-specific IFN-γ production by adoptively transferred, ex vivo activated young CD8+ T cells in the aged liver environment. Finally, aged livers lacking hepatocyte CD44 showed attenuation in immune-associated and fibrosis-related gene signatures, consistent with a role for hepatocyte CD44 in age-associated inflammation and early fibrosis. Our findings identify an aging-associated hepatocyte CD44 state that co-occurs with immune modulation and early fibrotic cues, overall suggestive of an early cancer-initiating niche signature in aged livers.

overall_design: Animals were procured from NIA aging colony and housed at SBP Animal facility. After 2 weeks, recombinant AAV8-TBG-gp33DYDDK80 was used to express LCMV antigen Gp33 in a hepatocyte specific manner. Viral suspension was prepared at a dose of 1 million viral genomes per mouse in 100 microL and injected retro-orbitally. After 2 weeks of AAV8 infection, ex vivo activated (with gp33 peptide) Thy1.1+ P14 T cells from young mice were injected at 1 million cells per 100 microlitre sterile T cell media. After 5 days of adoptive T cell transfer, livers were collected and immune cells from 7 young and 7 old mice were combined to enrich for adoptively transferred P14 CD8+ T cells. Thy1.1+ CD8+ T cells were purified by flow sorting and loaded onto 10X scRNAseq chip. The samples included in this submission are Thy1.1+ CD8+ P14 T cells isolated from young and old Gp33-expressing mouse livers.

first: Garrett

first: Armin

last: Gandhi

first: Peter D.

last: Adams


title: The IL-25–ILC2–IL-13 axis improves traumatic brain injury by mediating CXCL-10-dependent regulation of Blood Brain Barrier integrity

city: Wuhu

first: Min

last: Peng

address: Zheshan West Road num2

institute: First Affiliated Hospital of Wannan Medical College (Yijishan Hospital)

summary: Traumatic brain injury (TBI) induces severe disruption of the blood brain barrier (BBB), a pivotal event that drives secondary neurological damage. Interleukin-25 (IL-25) has emerged as a key modulator of neuroinflammation, but its role and underlying mechanisms in BBB repair after TBI remain unclear. Here, we investigated the protective effects of IL-25 on BBB integrity and neurological function in mice following TBI. Using enzyme-linked immunosorbent assay, we found that IL-25 levels in brain tissue and serum were significantly elevated after TBI, peaking at day 3, with brain microvascular endothelial cells (BMECs) identified as the primary source through immunofluorescence staining. Exogenous IL-25 administration ameliorated BBB dysfunction, as shown by restored expression of tight junction proteins (ZO-1, occludin, claudin-5), reduced Evans Blue extravasation, and decreased cerebral edema on magnetic resonance imaging. Mechanistically, flow cytometry and in vitro oxygen glucose deprivation/reoxygenation models revealed that IL-25 exerted its effects indirectly by activating brain-resident group 2 innate lymphoid cells to secrete interleukin-13 (IL-13), rather than acting directly on BMECs. Cytokine array and Western blot analyses demonstrated that IL-13 preserved BBB integrity by suppressing C-X-C motif chemokine ligand 10 (CXCL-10) expression and inhibiting endothelial cell pyroptosis. Single-cell RNA sequencing further confirmed that IL-25 upregulated BBB-protective genes (e.g., tiam1, hsp90aa1) and activated tight junction and transforming growth factor-beta signaling pathways. Behaviorally, IL-25 treatment improved motor coordination (modified Neurological Severity Score, rotarod test) and cognitive performance (Morris water maze) after TBI. Collectively, our findings indicate that IL-25 promotes BBB repair and neurological recovery after TBI by activating ILC2-derived IL-13, which suppresses CXCL-10 and endothelial pyroptosis. This study highlights IL-25 as a promising therapeutic target for TBI-induced BBB disruption.

overall_design: Single-cell suspensions were prepared from the left perilesional brain tissue of male C57BL/6 mice selected from the control group, TBI+PBS group, and TBI+IL-25 group, and analyzed using scRNA-seq.

first: Daoxing

last: Shu

first: Zheng

last: Chen


title: Transcriptional programs dictating tumor cell fate decisions in neuroblastoma lymph node metastasis [bulk RNA-seq]

city: Cincinnati

first: Richard

state: Ohio

institute: CINCINNATI CHILDREN'S HOSPITAL MEDICAL CENTER

summary: Neuroblastoma, a deadly pediatric cancer from the sympathetic ganglia of the peripheral nervous system, frequently metastasizes, driving poor outcomes in high-risk cases. While primary tumors are well-characterized, the cellular and molecular dynamics of metastasis  remain poorly understood. Here, we employed  single-cell multi-omics and spatial transcriptomics to profile lymph node metastases in high-risk neuroblastoma compared to primary adrenal masses. We found that lymph node metastases displayed unique cellular heterogeneity and plasticity marked by a shift toward mesenchymal-like and cancer stem cell states, with enriched epithelial-to-mesenchymal transition (EMT) programs. Lymph node metastatic niche exhibited altered tumor microenvironment dynamics, characterized by increased immunosuppressive myeloid subsets, heightened immune checkpoint signaling and lymphocyte exhaustion, indicative of immune evasion and dysfunction. Our multi-omics studies reveal distinct features of high-risk neuroblastoma that contribute to metastasis and therapy resistance, pointing to potential therapeutic vulnerabilities of the aggressive metastatic disease.

overall_design: RNA-seq profiling of 10 neuroblastomas from adrenal masses and 8 from metastases in lymph nodes and chest wall.

first: Natalie

last: Wu

first: Brian

last: Weiss


title: GJB2 c.109G>A mutation causes hereditary non-syndromic deafness through the mitochondrial apoptosis pathway

city: Fuzhou

first: Yao

state: Fujian

address: Fujian Provincial Maternity and Children's Hospital, 18Daoshan Road, Gulou District

department: Medical Genetic Diagnosis and Therapy Center, Fujian Key Laboratory for Prenatal Diagnosis and Birth Defect

institute: Fujian Maternity and Child Hospital, Fujian Medical University

summary: To further investigate the regulatory pathway of GJB2 c.109G>A mutants in hereditary non-syndromic deafness, we performed a transcriptomic sequencing analysis. Differentially expressed genes between the Ctrl and MO groups were screened based on FDR-p<0.05 and |log2 Fold Change|>0.5. The results showed that 243 differentially expressed genes were screened in the Ctrl and MO groups, with 63 genes downregulated and 180 genes upregulated. These differentially expressed genes were visualized by drawing heatmaps  and volcano plots. GO analysis was performed on the differentially expressed genes. GO analysis results indicated that the main enrichment in the Biological Process category was secondary alcohol metabolic process, release of cytochrome c from mitochondria, and defense response to other organism .

overall_design: Collected 72 hpf wild-type AB strain zebrafish and GJB2-MO zebrafish for RNA-seq sequencing.

first: Peiran

first: Qingying

last: Lin

first: Yinglin

last: Zeng

first: Xiaolong

last: Qiu

first: Ting

last: Huang

first: Jianping

last: Tang

first: Liangpu

last: Xu


title: Single nucleus RNA-seq analysis of two-month old Csf1r+/- mouse brains

city: Bronx

first: Deyou

last: Zheng

state: NY

address: 1300 Morris Park Ave

institute: Albert Einstein College of Medicine

summary: Dominant inactivating mutations in the colony stimulating factor-1 receptor (CSF1R) cause an adult-onset neurodegenerative disease associated with white matter loss and axonal degeneration designated CSF-1R related leukoencephalopathy (CRL), that is modeled in the Csf1r+/- mouse. CRL is caused by microglial dysfunction. However, the primary microglial deficit, caused by insufficient CSF-1R signaling, is unknown. To address this question, we employed single-nucleus RNA sequencing of brains from young Csf1r+/- mice without pathological or behavioral alterations. Reduction of CSF-1R signaling caused defects in mitochondrial function and metal ion homeostasis in brain macrophages, with concomitant activation of cell death and stress response pathways in oligodendrocytes and neuronal subpopulations.

overall_design: To investigate how reduced CSF-1R signaling impacts microglia function and their communication with other brain cells, we performed single nuclei RNA-seq (snRNA-seq) of brains isolated from 2-month-old asymptomatic Csf1r+/- mice and wild-type (wt) controls (2 mice per genotype) .

first: Violeta

last: Chitu

first: Yang

last: Liu

last: Stanley


title: Novel humanized loss-of-function NF1 mouse model of juvenile myelomonocytic leukemia

city: Palo Alto

first: Roshani

last: Sinha

address: 240 Pasteur Drive BMI

department: Pediatrics

institute: Stanford University

summary: Juvenile myelomonocytic leukemia (JMML) is a fatal pediatric cancer characterized by classical features such as splenomegaly, monocytosis, and GM-CSF hypersensitivity, with RAS pathway mutations being the major drivers. Mutations causing loss-of-function of the Neurofibromin1 gene (NF1LOF) occur in ~20% of JMML patients.  NF1LOF drives upregulation of RAS/MAPK/PI3K pathways that lead to aggressive proliferation/differentiation of immature myeloid cells. Hematopoietic stem cell transplantation is the only curative option, but relapse occurs in ~50% of patients, indicating an urgent need for novel and targeted therapeutic strategies. However, low patient sample availability and a lack of reliable disease models have made it difficult to study and treat JMML.  Using CRISPR/Cas9, we have generated NF1LOF in human umbilical cord blood-derived hematopoietic stem and progenitor cells (HSPCs). We achieved a high gene knockout rate of ~89% and concomitant loss of NF1 protein in the modified HSPCs. Importantly, the NF1LOF cells displayed marked hypersensitivity to GM-CSF in in vitro colony-forming unit assays, mirroring a distinctive trait seen in JMML patient cells. Humanized NSG-SGM3 mouse neonates transplanted with NF1LOF HSPCs had a median survival of 32 days, and displayed abrogation of lymphocytes, accumulation of myeloid cells and extensive leukemia infiltration in spleen, liver, and lungs, consistent with those observed in human JMML patients. This is the first successful development of a humanized mouse model of NF1LOF that recapitulates many of the features of JMML thus providing a novel system to investigate JMML progression and interrogate targeted treatment strategies.

overall_design: Total RNA was extracted from isolated human CD45+ cells from bone marrow and spleen cells collected from control and NF1-KO HSPCs transplanted in NSG-SGM3 recipients at week 4.


title: transcriptome analysis of renal cell carcinoma tisues

city: Yokohama

first: Taku

last: Kouro

state: Kanagawa

address: 2-3-2 Nakao, Asahi-ku

department: Division of Cancer Immunotherapy

country: Japan

institute: Kanagawa Cancer Center Research Institute

summary: To elcidate tumor microenvironment and immune cell composition, total RNA from RCC tissues were sequenced.

overall_design: Resected tumor tissues from stage I, III, IV RCC patients were collected.

first: Kayoko

last: Tsuji

first: Tetsuro

last: Sasada


title: Transcriptional programs dictating tumor cell fate decisions in neuroblastoma lymph node metastasis [single-cell multiomics]

first: Richard

last: Lu

overall_design: Single nuclei were extracted from 6 high-risk neuroblastomas from primary adrenal masses and 5 high-risk neuroblastomas from lymph nodes. Joint snRNA- and scATAC libraries were prepared using the 10x Genomics platform.

first: Natalie


title: AF6 promotes lipid droplet fusion and aggravates MASH via C/EBPβ–CIDEA axis

city: Shanghai

first: Qian

last: Wen

address: 320 Yueyang Road, Xuhui District

institute: Shanghai Institute of Nutrition and Health

summary: Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease characterized by steatosis, inflammation, and fibrosis. However, the mechanisms linking lipid droplet (LD) metabolism to MASH progression remain poorly understood. AF6 is a polarity protein known to regulate metabolic homeostasis, but its role in the development of MASH remains largely undefined. Here, we identify AF6 as a critical regulator of hepatic lipid accumulation and inflammation in MASH. Hepatic AF6 expression is elevated in MASH patients and diet-induced mouse models. Liver-specific deletion of AF6 attenuated steatosis, inflammation, and fibrosis in both high-fat, high-cholesterol (HFHC) and choline-deficient, amino acid-defined high-fat diet (CDA-HFD)-induced MASH models. Mechanistically, lipid overload promotes nuclear translocation of AF6, which interacts with the transcription factor C/EBPβ and enhances its nuclear accumulation. This interaction increases transcription of the lipid droplet fusion protein CIDEA, promoting LD growth and lipid deposition. AF6 also exacerbates MASH by enhancing C/EBPβ-mediated proinflammatory signaling. Restoration of CIDEA expression in AF6-deficient mice reverses the protective effects. In human liver biopsies and iPSC-derived liver organoids, the AF6–C/EBPβ–CIDEA axis is upregulated and correlates with disease severity. Importantly, a CRISPR-Cas9–based lipid nanoparticle (LNP) therapy targeting AF6 effectively reduced lipid accumulation, inflammation, and fibrosis in MASH mice and organoids. AF6 promotes MASH progression by facilitating C/EBPβ nuclear localization and transcriptional activation of CIDEA. Targeting the AF6–C/EBPβ–CIDEA axis represents a promising therapeutic strategy for treating MASH.

overall_design: Liver-specific AF6 knockout mice (AF6fl/fl; AlbCre) and control littermates (AF6fl/fl) were used for in vivo studies. At 8 weeks of age, male mice were randomly assigned to receive HFHC diet containing 14% protein, 42% fat, 44% carbohydrates, and 0.2% cholesterol for 16 weeks to induce MASH. After the treatment, liver tissue samples from the mice were collected for RNA-seq sequencing analysis.

first: Lixing

last: Zhan


title: TLR8 agonists remodel the tumor immune microenvironment through PF4-dependent T cell recruitment and ancillary mechanisms

first: Qikai

last: Zhou

state: Shanghai

address: 1954 Huashan Road, Xuhui District, Shanghai

institute: Shanghai Jiao Tong University

summary: Pattern Recognition Receptors (PRRs) modulate tumor immune microenvironments (TIME), but their comparative efficacy remains unclear. Platelet factor 4 (PF4/CXCL4) exhibits paradoxical roles in cancer, and its regulation by PRRs remains unexplored. We systematically evaluated PRR agonists to identify optimal TIME modulators and elucidate PF4’s role. TLR8 agonists optimally remodel TIME by activating PF4-dependent T cell recruitment and engaging PF4-independent ancillary mechanisms. Context-dependent PF4 induction resolves its dual roles in cancer. These findings position TLR8 agonists as promising partners for immunotherapy.

overall_design: Mouse tumor tissues (5 biological replicates per group) were rinsed with ice-cold PBS, mechanically dissociated into small fragments, and enzymatically digested using collagenase D/DNase I (Roche) with gentleMACS Tissue Dissociator (Miltenyi Biotec). The resulting single-cell suspensions were filtered through 70-μm strainers and PBS-washed before immune cell enrichment through CD45+ selection using biotin-conjugated anti-mouse CD45 antibody (BioLegend) and anti-biotin MicroBeads (Miltenyi Biotec), yielding >95% pure and >85% viable populations.

first: Bin

last: Ma

first: Zhixue

last: Wang

first: Yixian

last: Cao

first: Wenqin

last: Luo

first: Zhengheng

first: Wen

last: Li

first: Zifeng

last: Fu


title: Single-cell RNA sequencing of Pacific white shrimp hepatopancreas infected with Vibrio parahaemolyticus

city: Auburn

first: Jason

last: Abernathy

state: AL

address: 990 Wire Road

institute: USDA-ARS

summary: The shrimp aquaculture industry is vulnerable to large losses due to acute hepatopancreatic necrosis disease (AHPND), caused by the bacterium Vibrio parahaemolyticus. The mechanism by which the pathogen causes disease, and the host immune response, is not completely understood. The shrimp hepatopancreas is a multi-functional organ with roles in digestion, immunity, molting and reproduction. Therefore, we set out to characterize the cells of the hepatopancreas and the host response to Vibrio parahaemolyticus infection at single-cell resolution. The hepatopancreas from three individual shrimp were processed to create a single-cell transcriptomic atlas. Then the hepatopancreas from three Vibrio parahaemolyticus infected and two mocked treated shrimp were sampled for infection study. All single-cell libraries were generated using the 10X Genomics platform and sequenced on an Illumina sequencer. Data were aligned to the Litopenaeus vannamei reference genome using Cell Ranger. Seurat and clusterProfiler were used for downstream analyses. Cells of the hepatopancreas were characterized and the transcriptomic response to AHPND-causing V. parahaemolyticus was examined. Data will inform further functional studies and has the potential to aid in the development of novel preventative measures or treatments.

overall_design: Atlas: shrimp (average 13 g) were housed in 50 L tanks (23°C, 16.5 ppt salinity). Prior to sampling, the shrimp were not fed for 18 hours. Infection: 70 shrimp (average 19 g) were dispersed evenly between seven 50 L tanks (28°C, 4 ppt salinity). The shrimp were not fed for 24 hours prior to the infection. Mock (n = 30) and infection (n = 40) treatments were randomly assigned to the tank groups. The control shrimp were treated via reverse gavage with 100 μl of media, while the shrimp in the treatment group were infected with 100 μl of V. parahaemolyticus (6.90 x 10^5 cfu/ml). A total of 2 mock and 3 infected shrimp hepatopancreas samples were collected one hour after treatment.

first: Johanna

middle: E

last: Aldersey

middle: W

first: Miles

middle: D

last: Lange

first: Julio

middle: C

last: Garcia

first: Craig

middle: A

last: Shoemaker

first: Benjamin

middle: H

last: Beck


title: Iron deficiency promotes bile acid deposition in an m6A-YTHDF2-CYP7A1 dependent mechanism

city: Nanjing

first: Xiang

last: Zhong

state: Jiangsu

address: Tongwei Road

institute: Nanjing agricultural university

summary: Background: Iron deficiency anemia is a major challenge in in both human health and animal husbandry. Disrupted bile acid metabolism is a key characteristic among people with anemia. However, effects and mechanisms of iron deficiency on bile acid metabolism remain unclear. N6-methyladenine (m6A) is the most abundant epigenetic modification, which can regulate various nutrient metabolism. This study aimed to investigate effect of iron deficiency on bile acid metabolism and elucidate its mechanism from the perspective of m6A methylation. Results: Iron deficiency (ID) destroyed liver morphology and ultrastructure in suckling piglet. Notably, ID induced moderate dilation of the endoplasmic reticulum and a reduction in mitochondrial number. At the metabolite level, ID significantly promoted bile acid deposition and reshaped bile acid profile. Specifically, ID decreased contents of GCA, CDCA, and 6-KetoLCA, while increasing levels of THDCA, TωMCA, DCA, GDCA, and TDCA (P < 0.05). At the molecular level, ID upregulated CYP7A1 expression to promoted bile acid deposition. Moreover, ID significantly decreased m6A content, and reduced expression of FTO and YTHDF2 (P < 0.05). M6A-seq data revealed an increase in m6A methylated peaks on CYP7A1 under ID condition. Importantly, RNA immunoprecipitation and stability experiments demonstrated YTHDF2 could bind and decrease stability of CYP7A1 (P < 0.05). Mechanistically, ID reduced YTHDF2 expression, thereby stabilizing CYP7A1 and driving bile acid accumulation. Conclusion: Iron deficiency induced bile acid deposition in an m6A-YTHDF2-CYP7A1 dependent manner, which further causes liver injury.

overall_design: Twenty newborn male piglets (Duroc× [Landrace × Yorkshire]), each with a birth weight of approximately 1.72 kg, were selected from Kangle Agriculture and Animal Husbandry (Nantong, Jiangsu, China). Their body weight was within ± 0.5 standard deviation (SD) of the population mean weight. All piglets were kept in the farrowing room for two days after birth, where they were nursed freely from their sows. Subsequently, the suckling piglets were transferred to the new room and divided into two groups: negative control (NC) and iron deficiency (ID) group

first: Yaxu

last: Liang

first: Anrui

last: Liu

first: Dongming

last: Yu

first: Xiaoyun


title: Hepatocyte CD44 drives IL6/STAT3-high neighborhoods linked to blunted T-cell effector function in the aged liver

first: Kathryn

last: Lande

department: IGC

institute: Salk Institute

summary: Liver cancer incidences increase beyond 55 years of age suggesting that molecular, cellular and tissue changes accompanying aging contribute critically to tumor initiation. However, the mechanisms linking aging and liver cancer initiation are not well understood. Our study investigates the role of CD44, a transmembrane glycoprotein and a known marker of tumor-initiating cells (TICs), in age-associated liver pathophysiology. Aged livers showed an accumulation of CD44 expressing hepatocytes. Single nucleus RNA sequencing revealed that CD44 expressing hepatocytes in aged livers exhibit enrichment of immune modulatory genes and activation of the IL6/JAK/Stat3 pathway. Spatial analyses confirmed upregulation of the IL6/JAK/Stat3 pathway and showed that CD44-expressing hepatocytes are proximal to T-cell neighborhoods exhibiting reduced cytokine expression. In adoptive transfer assays, ex vivo-activated CD8+ T cells mounted a lower antigen-specific IFNg response in aged livers than in young liver, consistent with an immune suppressive aged milieu. Hepatocyte-specific loss of Cd44 expression in aged livers compromised IL6/JAK/Stat3 activity and other immune/fibrotic gene signatures, supporting a contribution of hepatocyte Cd44 to age-associated immune dysregulation and early fibrosis. Our findings identify an aging-associated hepatocyte CD44 state with IL6/JAK/Stat3 activation, blunted T cell effector signals and early fibrotic cues.

overall_design: Hepatocyte-specific Cd44 knockout was achieved by AAV8-TBG-saCas9-U6-sgCd44 injection in male mice. AAV was delivered at 7 and 23 months of age. RNA-seq was performed on young (Rosa26 control, 9 mo), old (Rosa26 control, 25 mo), and old Cd44 knockout (25 mo) livers.

first: Armin

last: Gandhi

first: Peter

last: Adams


title: Technical and biological sources of noise confound multiplexed enhancer AAV screening

city: Seattle

first: John

middle: Kenneth

last: Mich

state: WA

address: 615 Westlake Ave N

institute: Allen Institute for Brain Science

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

overall_design: Refer to individual Series

first: Avery

last: Hunker

middle: K

first: Jean-Benoît

last: Lalanne


title: In Vivo Evolution of Antibody CR3022 Expands Cross-Neutralization of SARS-CoV-2 Variants and Informs Pan-sarbecovirus Immunity

city: New York

first: Patrick

last: Wilson

address: 413 E 69th St

department: Drukier Institute

institute: Weill Cornell Medicine

summary: Monoclonal antibody CR3022, isolated from a patient infected during the 2003 SARS-CoV outbreak, shows cross-reactive binding and, with targeted mutagenesis, neutralization of SARS-CoV-2. These findings suggest that targeting the CR3022 epitope may be a promising candidate for broadly protecting against a wide range of coronavirus variants and zoonotic strains that may pose future pandemic threats.  However, it is unclear how readily and broadly this epitope can be adapted against variant coronavirus strains in vivo. To address this issue, we generated immunoglobulin (Ig)-humanized mice expressing the predicted germline heavy chain of CR3022 for a direct evaluation of the epitopes’ in vivo adaptation potential. Following a SARS-CoV/SARS-CoV-2 sequential immunization regimen, we observed the convergent evolution of the germline CR3022 variable gene through somatic hypermutation to resemble that of the mutated WT counterpart. Most CR3022-like clones acquired cross-neutralizing activity against SARS-CoV-2, plus adaptation against key variants and more divergent Sarbecovirus strains. Notably, while simple prime-boost strategies drove CR3022-epitope targeting, an intensive vaccination protocol drove dominant responses to other epitopes even with a predominance of CR3022-like precursors.  X-ray crystallography revealed that SARS-CoV-2-neutralizing CR3022 antibodies readily exhibited enhanced affinity due to varied adaptive changes, resulting in significantly increased electrostatic interactions with the receptor-binding domain (RBD). Overall, these findings show ready adaptation of CR3022-like clones by minor shifts in affinity with appropriate vaccination strategies.

overall_design: This study includes B cells sorted from CR3022 knock-in mice following immunization with SARS-CoV or SARS-CoV-2 spike proteins under different regimens. Samples represent distinct time points post-immunization and include biological replicates.

first: Yanbin

last: Fu

first: Jordan

last: Chervin

first: Chloe

last: Troxell


title: Loss of ATM causes R-loop-associated transcriptional dysregulation and attenuates the related response to DNA damage [DRIP-Seq]

city: Atlanta

first: Bing

last: Yao

address: 615 Michael Street

department: Department of Human Genetics

institute: Emory University

summary: An early childhood onset neurodegenerative disorder, Ataxia Telangiectasia (AT) affects 1 in 40,000 to 100,000 individuals worldwide and is caused by mutations in the ataxia telangiectasia mutated (ATM) threonine-serine kinase, which regulates the DNA damage response (DDR). While the cause of AT has been known for years, the exact molecular mechanisms underlying disease progression, particularly at the transcriptomic level, remain poorly understood. Three stranded structures, known as R-loops, have recently emerged as important players in the DDR via regulating key gene expression. Here, we utilized neuronal progenitor cells (NPCs) derived from induced pluripotent stem cells reprogrammed from patient-derived somatic cells to identify how loss of ATM impacts R-loop and transcriptional dynamics, both at baseline and in response to acute DNA damage. AT-derived NPCs (AT-NPCs) exhibited elevated spontaneous R-loop levels compared to controls (C-NPCs), as well as a strong positive correlation between R-loop accumulation and gene expression on a subset of dysregulated genes. Upon acute damage, loss of ATM resulted in an attenuated response, characterized by the impaired R-loop and transcriptional response to irradiation. Both C- and AT-NPCs underwent a similar cell cycle arrest, but AT-NPCs displayed an attenuated R-loop and transcriptional response, failing to activate proper DDR response. Importantly, R-loop formation is required for many key genes to properly respond to DNA damage, supporting a direct and causal role in this process. Overall, our data reveal an underappreciated mechanistic link between ATM, R-loop regulation, and transcription, the disruption of which may contribute to the impaired DDR observed in AT-NPCs.

overall_design: DRIP-seq data of control and Ataxia Telangiectasia (AT) patient-derived neuronal progenitor cells either untreated or irradiated with 10Gy and incubated for 6h. DRIP-seq data of control neuronal progenitor cells treated with 100nM of ATM inhibitor AZD1390 for 12 hours. DRIP-seq data of control and AT patient-derived neuronal progenitor cells treated with 1mM of hydorgen peroxide for 6 hours. DRIP-seq data of control and AT patient-derived neuronal progenitor cells infected with a lentivirus overexpressing FLAG-tagged RNaseH1 which were then irradiated with 10Gy after 42 hours and placed in an incubator for 6 hours.

first: Katherine

last: Westover

first: Yingzi

last: Hou

first: Feng

last: Wang

first: Yilin

first: Yangping

last: Li

first: Rachel

last: Seong

first: Jie

last: Xu

first: Zhexing

last: Wen


title: Single cell transcriptome analysis of TIL for gene expression and TCR immunoprofiling in the renal cell carcinoma

first: Taku

last: Kouro

summary: Unlike other carcinomas, renal cell carcinoma is known to have an inverse correlation between the abundance of T cells in the tumor and prognosis. To elucidate the background, we performed single-cell gene expression analysis and TCR immunoprofiling of tumor-infiltrating T cells in early and advanced stages of renal cancer.

overall_design: Five stage I, seven stage III andthree stage IV RCC samples were included.

first: Rika

last: Kasajima

first: Yohei

last: Miyagi

first: Testuro

last: Sasada


title: Whole body MondoA  deletion protects against diet-induced obesity through uncontrolled multi-organ substrate utilization and futile cycling

city: Philadelphia

first: Daniel

last: Kelly

state: PA

address: 3400 Civic Center Blvd

department: Penn Cardiovascular Institute

institute: University of Pennsylvania

summary: Objective – Delineating the nodal control points that maintain whole-body energy homeostasis is critical for understanding potential treatments of obesity and cardiometabolic diseases. The nutrient-sensing transcription factor MondoA is a regulator of skeletal muscle fuel storage, where muscle-specific inhibition improves glucose tolerance and insulin sensitivity. However, the role of MondoA in whole body energy metabolic homeostasis is not understood. Methods – Generalized MondoA knockout (gKO) mice were generated and assessed for glucose tolerance and insulin sensitivity, body composition, energy expenditure, cold tolerance, and tissue specific transcriptional changes in response to high fat diet. Complementary studies in cultured human adipocytes assessed the impact of MondoA deficiency on substrate utilization and lipolysis. Results – gKO mice are protected from diet-induced obesity and insulin resistance, through increased whole body energy expenditure. gKO mice exhibit reduced brown and inguinal white adipose tissue mass, without evidence of beiging. The gKO mice are hyperlactemic and isolated MondoA-deficient adipocytes have increased 2-deoxyglucose uptake and glycolytic function. Lastly, gKO mice and KO adipocytes display increased circulating glycerol relative to free fatty acids in response to adrenergic stimulus consistent with elevated re-esterification. This phenotype is not recapitulated in adipose-specific KO mice. Conclusions – MondoA deficiency alters cellular sensing of nutrient availability and storage/utilization mechanisms. In the whole-body setting, this results in increased energy expenditure, potentially related to increased glucose uptake, glycolytic flux, and glycerol synthesis to supply high rates of lipolysis and lipid re-esterification. These results suggest that MondoA functions to maintain fuel storage and when lost, inter-organ futile fuel cycling ensues.

overall_design: Male WT and gKO mice age 8 weeks were fed chow or HFD for 16 weeks prior to tissue collection. BAT and iWAT were surgically excised and RNA isolated for RNAseq.

first: Justin

last: Berger

first: Allison

middle: N

last: Lau

first: Larry

last: James

first: Renee

last: Taing

first: Byungyong

last: Ahn

first: Xiaofei

last: Yin

first: Tomoya

last: Sakamoto

first: Kirill

last: Batmanov

first: Jiten

last: Patel

first: Jeffrey

last: Zhou

first: Brian

last: Finck

first: Paul

last: Titchenell

first: Gregory

middle: J

last: Tesz

middle: P


title: Comparative Transcriptomic Analysis of Embryonic Hearts in Atf4 Exon 2 and Exon 2–3 Conditional Knockout Mice

city: San Diego

first: Zeyu

last: Chen

address: 9500 Gilman Drive, BFRII 1217

department: School of Medicine

institute: UCSD

summary: This dataset comprises RNA sequencing (RNA-seq) profiles of E11.5 embryonic hearts from two Atf4 conditional knockout (cKO) mouse models, targeting different exons of the gene. In one model, exons 2 and 3 were floxed and excised in cardiomyocytes using the Xmlc2-Cre driver, while the second model involved deletion of exon 2 only. The aim of this study is to determine whether Atf4 exon-targeting strategy influences transcriptomic outcomes and to evaluate the impact of ATF4 deficiency on early cardiac development. RNA-seq was performed on ventricular tissues from both models and their respective littermate controls, each with four biological replicates. The dataset serves to clarify the necessity and function of ATF4 in the embryonic heart using refined conditional knockout strategies.

overall_design: To investigate the role of ATF4 in heart development, we generated two cardiomyocyte-specific Atf4 knockout mouse lines using Xmlc2-Cre. The first model floxed both exons 2 and 3 of Atf4 (Atf4 fl(e2/3)), while the second model floxed only exon 2 (Atf4 fl(e2)). Embryos were harvested at E11.5, and ventricular tissue was microdissected from each embryo. Total RNA was extracted and used for Illumina stranded mRNA library preparation. Libraries were sequenced on the Illumina HiSeq 6000 platform to a depth of 30-70 million paired-end reads per sample. Each genotype (control and cKO) was analyzed with four biological replicates.

first: Zengming

last: Zhang

first: Tongbin

last: Wu

first: Ju


title: RNA-Seq of Mouse Embryonic Heart Development in Atf4 Exon 3 Frameshift Knockout Mice

summary: This dataset contains RNA sequencing (RNA-seq) profiles from embryonic hearts of Atf4 del7/del7  mice, which harbor a 7-base-pair deletion in exon 3 of Atf4 resulting in complete loss of ATF4 protein expression. The purpose of this experiment is to evaluate the transcriptional consequences of ATF4 deficiency in embryonic cardiomyocytes, independent of transcriptional readthrough artifacts associated with an earlier widely used Atf4 floxed mouse model. RNA-seq was performed on ventricular tissue collected from E11.5 embryos of Atf4 del7/del7  mice and their wild-type littermates to assess gene expression changes associated with loss of ATF4 protein.

overall_design: The Atf4(del7/del7) model was generated by CRISPR/Cas9-mediated deletion of 7 base pairs within exon 3 of the Atf4 gene, introducing a frameshift and premature stop codon. This mutation abolishes ATF4 protein production without altering its polyadenylation signal. Embryos were harvested at embryonic day 11.5 (E11.5), and ventricular heart tissue was dissected for RNA extraction using TRIzol reagent. RNA quality was assessed with an Agilent Bioanalyzer. RNA-seq libraries were prepared using the Illumina TruSeq stranded mRNA kit and sequenced on an Illumina HiSeq 6000 platform. Four biological replicates were collected for each genotype.


title: Nuclease-NTPase systems use shared molecular features to control bacterial anti-phage defense

city: Boston

first: Amy

middle: Si-Ying

last: Lee

state: MA

address: 450 Brookline Ave SM520

department: Cancer Immunology and Virology

institute: DFCI

summary: Bacteria encode diverse defense systems including restriction-modification and CRISPR-Cas that cleave nucleic acid to protect against phage infection. Bioinformatic analyses demonstrate many recently identified anti-phage defense operons are comprised of a nuclease and NTPase protein, suggesting additional nucleic acid targeting systems remain to be understood. Here we develop large-scale comparative cell biology and biochemical approaches to analyze 16 nuclease-NTPase systems and define molecular features that control anti-phage defense. Purification, biochemical characterization, and in vitro reconstitution of nucleic acid degradation demonstrates protein–protein complex formation is a shared feature of multi-gene nuclease-NTPase systems. We show that AbpAB, Hachiman, and PD-T4-8 system nucleases use highly degenerate recognition site preferences to enable broad nucleic acid degradation, and the Azaca system exhibits specific phage targeting through the recognition of modified phage genomic DNA. Our results uncover principles of anti-phage defense system function and highlight the mechanistic diversity of nuclease-NTPase systems in bacterial immunity.

sample_organism:
Pseudomonas aeruginosa
Escherichia coli
Bacillus toyonensis
Bacillus massilioanorexius

overall_design: Cleavage sequencing analysis of DNA (Bacteriophage T4 and pGEM9z plasmid) incubated with Nuclease-NTPase proteins (PaAbpAB, BmAzaca, BtHachiman, EcPD-T4-8).

first: Adelyn

last: Ragucci

first: Sadie

last: Antine

first: Ethan

last: Leviss

first: Sarah

last: Mooney

first: Jasmine

last: Garcia

middle: S

first: Philip

last: Kranzusch


title: Rps19bp1 cKO Xmlc2Cre E11.5 mouse heart RNAseq

summary: This study investigates the transcriptomic consequences of cardiomyocyte-specific deletion of Rps19bp1 during mouse embryonic development. Rps19bp1 encodes AROS (Active Regulator of SIRT1), a known negative regulator of p53. To understand its role in cardiac development and cell cycle regulation, we performed RNA sequencing (RNA-seq) on ventricular heart tissues from E11.5 embryos of Rps19bp1 conditional knockout (cKO) mice and littermate controls. The data reveal widespread gene expression changes and activation of p53 signaling in Rps19bp1-deficient hearts, providing mechanistic insights into how Rps19bp1 modulates cardiomyocyte proliferation and embryonic heart morphogenesis.

overall_design: Rps19bp1 floxed mice (Rps19bp1 fl/fl) were crossed with Xmlc2-Cre mice to generate cardiomyocyte-specific knockout animals (Rps19bp1 fl/fl; Xmlc2-Cre +/−). Embryos were harvested at embryonic day 11.5 (E11.5). Ventricular heart tissues were dissected under a stereomicroscope, and total RNA was extracted using TRIzol reagent. RNA integrity was confirmed using Agilent Bioanalyzer prior to library preparation. Four biological replicates were collected for each genotype (cKO and control). Libraries were prepared using the Illumina TruSeq stranded mRNA kit and sequenced on the Illumina HiSeq 6000 platform to a depth of ~30 million paired-end reads per sample.


title: Simvastatin alleviates ConA-induced autoimmune hepatitis by inhibiting CD4+T cell activation via calcium-NFATC3 pathway

city: Beijing

first: Xiyu

last: Wang

address: No. 8 Gongren Tiyuchang South Road

institute: Beijing Chaoyang Hospital, Capital Medical University

summary: The mevalonate pathway plays a crucial role in various immune disease and Hmgcr is the key enzyme of this pathway. In addition, simvastatin, as an inhibitor of Hmgcr , also exhibits therapeutic potential for acute liver injury such as APAP-induced toxicity and hepatic ischemia-reperfusion injury (HIRI). However,its effect in ConA-induced acute liver injury remains unclear.

overall_design: To investigate the role of simvastatin on CD4+ T cells in the progression of ConA-induced liver injury,  CD4+ T cells from liver tissue of the mice which were treated with or without simvastatin were performed mRNA transcriptome sequencing.

first: Hua

last: Jin

first: Dong


title: Atomistic TCR-ligand interactions instruct memory T-cell differentiation and crossreactivity

first: Michael

last: Tolstorukov

address: 360 Longwood Avenue

department: Informatics

institute: Dana Farber Cancer Institute

summary: While memory T-cells provide protective immunity against specific pathogens, their post-encounter differentiation into central (TCM) and/or effector (TEM) subpopulations remains enigmatic. We thus explored the CD8 T-cell receptor (TCR) repertoire of 242 murine TCR clonotypes directed against an immunodominant influenza A virus (IAV) peptide/major histocompatibility complex (pMHC) ligand, the nucleoprotein NP366-374/Db, leveraging single-cell transcriptomics with paired TCR sequencing, mechanosensing metrics, and in vivo memory development plus TCR-pMHC structural analyses. Polar TEM and more variegated TCM repertoires as well as bipotential “bipolar” clonotypes (TBP) revealed weak force-dependent bonding parameters associated with heterosubtypic IAV crossreactivities. TCM and TEM polarities manifest pMHC binding skewed to TCR- versus TCR-subunit mechanotransduction, respectively, unlike the more signaling-balanced TBP TCRs. In sum, TCR diversity anticipates pathogen evolution whereas divergent signaling regulates memory fate.

overall_design:
NP1_GEX, NP1_TCR, NP2_GEX, and NP2_TCR were derived from NP366-374-Db tetramer+ tetramer⁺ Zombie Aqua⁻ CD8β⁺ CD44⁺ T cells sorted from pooled mesenteric lymph nodes (mLNs) of 10 C57BL/6 (B6) mice at day 34 following primary infection with the PR8 strain of influenza A virus (IAV). NP1 and NP2 originated from the same pooled sample but were separated during barcoding.
DN2_LN_GEX and DN2_LN_TCR were obtained from Zombie Aqua⁻ CD8β⁺ CD44⁺ T cells isolated from pooled mLNs of 2 B6 mice undergoing a memory recall response, 2 days after heterologous challenge with the X31 strain. DN4_LN_GEX and DN4_LN_TCR were similarly derived from pooled mLNs of 2 B6 mice, 4 days after X31 challenge.
NS_LN_GEX and NS_LN_TCR were isolated from Zombie Aqua⁻ CD44⁺ CD8β⁺ T cells sorted from pooled mLNs of four B6 mice at day 33 post-infection.

first: Aoi

last: Akitsu

first: Ellis

last: Reinherz

first: Jonathan

last: Duke-Cohan

first: Matthew

last: Booker


title: Loss of ATM causes R-loop-associated transcriptional dysregulation and attenuates the related response to DNA damage [RNA-Seq]

first: Bing

last: Yao

overall_design: RNA-seq data of control and Ataxia Telangiectasia (AT) patient-derived neuronal progenitor cells either untreated or irradiated with 10Gy and given 6h recovery. RNA-seq data of control and AT patient-derived neuronal progenitor cells infected with a lentivirus overexpressing FLAG-tagged RNaseH1 which were then irradiated with 10Gy after 42 hours and placed in an incubator for 6 hours.

first: Katherine

last: Westover

first: Yingzi

last: Hou

first: Feng

first: Yilin

first: Yangping

last: Li

first: Rachel

last: Seong

first: Jie

last: Xu

first: Zhexing

last: Wen


title: Depletion of Peripheral Monocytes Alters Long-Term Gene Expression in Microglia in a Murine Model of Traumatic Brain Injury

city: Chicago

first: Mecca

middle: Bennta Abour Rahim

last: Islam

state: IL

address: 240 E Huron St., M410

institute: Northwestern University

summary: Background: Traumatic brain injury (TBI) is a growing and under-recognized public health threat with significant long-term complications suffered by its survivors. Monocytes are amongst the first immune cells to infiltrate the injured brain and have the ability to both foster wound repair and promote inflammation. Microglia, the resident innate immune cells in the brain, drive the long-term direction of the inflammatory response within the injured brain. Previously published data from our laboratory has shown that a brief course of peri-injury monocyte depletion attenuates long-term neurocognitive deficits and preserves white matter connectivity post-injury. Nonetheless, the role that monocytes play in directing the local inflammatory response to injury via crosstalk with microglia remains unknown. To this end, we hypothesized that infiltrating monocytes shape the long-term transcriptional response of microglia to TBI. Methods: We employed a 2x2 study design consisting of four experimental groups—TBI, sham, TBI with monocyte depletion, and TBI with sham monocyte depletion. Male C57BL/6 mice were randomly assigned to groups. Monocyte depletion and sham depletion were induced via intravenous injection of liposome-encapsulated clodronate versus naked liposomes 24 hour prior to TBI. Depletion was maintained via repeat injections on days 2 and 5.  Monocyte depletion was confirmed via flow cytometry. TBI was induced using our established model of controlled cortical impact. BMice were euthanized on post-injury days 1, 7, 14, 30, and 60. Brains were harvested and microglia sorted via flow cytometry. The transcriptional response of microglia across groups and timepoints was assess via bulk RNA sequencing. Conclusion:These data shows that short-course of peri-injury depletion of peripheral monocytes may have a neuroprotective effect after TBI. While the mechanisms of this protection are multifactorial, alteration of the long-term transcriptional profile of microglia may, in part, be responsible for the observed improvements in motor coordination, learning, and memory. Identified pathways include inflammation, neuroplasticity and regeneration, and a neuroprotective heat shock response. These data warrant further investigation into possible therapeutic benefits of peri-injury immune modulation.Results:Monocyte-depleted mice demonstrated improvement in motor coordination, contextual and associative learning, and memory. These neurocognitive differences were associated with distinctly different microglial transcriptional profiles evident within the first 1-2 weeks post injury. In particular, microglia within the monocyte-depleted group showed distinct upregulation of pathways including synaptic signaling, regulation of neuron differentiation, and myeloid leukocyte activation as compared to those from Vehicle TBI groups. By 60 days post injury, microglia from the monocyte-depleted TBI group had upregulation of the heat shock protein transcripts as compared to microglia from the Vehicle TBI group.

overall_design: RNA-seq profiling of microglia after depleting periphel monocytes with clodornate encapsultated lipsomes or vehicle lipsomes following severe Traumatic Brain Injury at time points (1, 7, 14, 30, and 60 days) post-injury. Sham (no injury) and TBI only were included as controls

middle: B

first: Zhanying

last: Chen

first: Talia

last: Just

first: Gaurav

last: Gadhvi

first: Kacie

last: Ford

first: Booker

middle: T

last: Davis

last: Dapas

first: Hiam

last: Abdala Valencia

first: Hadijat

last: Makinde

first: Steven

last: Schwulst


title: Effect of ARSI cell line or IL-10 on macrophage polarization

city: Bordeaux

first: Laurence

last: Bresson-Bepoldin

address: 146 rue de saignat

country: France

institute: INSERM

summary: Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT) is a rare subset of diffuse large B-cell lymphoma (DLBCL) exhibiting genetic features shared with LBCL of immune-privileged sites (notably the MYD88L265P mutation) and enriched in tumor associated macrophages (TAM) expressing M2 markers. Using a unique PCDLBCL-LT cell line (ARSI cell line) developed in our institute, we sought to mechanistically decipher the interplay between lymphoma cells and macrophages. We demonstrate that ARSI cells induce phenotypic, transcriptional and functional changes in macrophages obtained from primary monocytes . These changes do not require cell-cell contact, and proteome analysis of ARSI secretome reveals high concentration of several cytokines and chemokines known to affect macrophages, including IL-10. We then demonstrate that IL-10/IL-10RA interaction blockade inhibits macrophage polarization induced by tumor cells. In conclusion, these results confirm the robustness of this model to study lymphoma cell and macrophage interplays, underline the critical role of IL-10 in lymphoma microenvironment modeling, and may contribute to better define its specificities in an era of rising microenvironment-targeted therapies.

overall_design: Monocytes were exposed for 24 hours to one of the following conditions: untreated (control), co-culture with ARSI cell line at 75,000 cells per well, or recombinant human IL-10 at a final concentration of 5 ng/mL. CD14⁺ monocytes were subsequently isolated from all experimental groups using magnetic-activated cell sorting (MACS), according to the manufacturer’s instructions, to ensure consistency across conditions. Only samples achieving a minimum post-sort purity of 99.7% were selected for downstream analysis. Purity was assessed by confirming the absence of contaminating ARSI cells, identified by CD20⁺ or CD19⁺ expression. The purified CD14⁺ cells were then cryopreserved

first: Rémi

last: Peanne

first: Audrey

last: Gros


title: Clonal dynamics reveal cancer resistance arises from adaptive programs [RNA-Seq]

first: Yeqing

address: 3601 Spruce Street

institute: The Wistar Institute

summary: Most advanced cancers initially respond to targeted therapies but eventually relapse1. Rather than acquiring new mutations, resistance is driven by drug-tolerant persister cells (DTP) that enter a reversible drug-refractory state and sustain minimal residual disease2. Here, we developed MeRLin, a high-resolution barcoding platform combining single-cell RNA sequencing, RNA fluorescence in situ hybridization, and computational analyses to track clonal and transcriptional dynamics of melanoma cells during targeted therapy. Clonal tracking reveals that dominant resistant clones arise from minor pre-treatment subpopulations. The pre-treatment melanoma populations diversify into phenotypically distinct DTP subpopulations, marked by stress-like, lipid metabolism, PI3K signaling, and extracellular matrix remodeling programs associated with adaptive resistance. Spatial transcriptomics revealed the co-localization of lipid metabolism and PI3K signaling programs near the tumor boundaries, and a complex network of autocrine and paracrine interactions among DTP subpopulations. Using barcoded RNA fluorescence in situ hybridization, we identified a dominant persister subpopulation in resistant tumors marked by SLC2A1 expression. Thus, MeRLin provides a robust framework to dissect melanoma heterogeneity and uncover vulnerabilities in persister populations to improve long-term treatment efficacy.

overall_design: WM4237-1 cells were spinfected with a lentiviral barcode library in the presence of polybrene (8 μg/mL). mNeptune2.5-positive cells were sorted by flow cytometry to ensure a multiplicity of infection (MOI) below 40%. Barcoded cells were then expanded and split into replicate populations, with one aliquot preserved as the original control. Replicates of barcoded WM4237-1 cells were subcutaneously injected into immunodeficient mice and monitored for tumor growth. Upon establishment, tumors were treated with BRAFi/MEKi, and samples were collected longitudinally throughout treatment until relapse.

first: Haiyin

last: Li

first: Jessica

last: Kaster

first: Maggie

last: Dunne

first: Min

last: Xiao

first: Ling

first: Monzy

last: Thomas

first: Nazifa

last: Promi

first: Dylan

last: Fingerman

first: Gregory

last: Brown

first: Xingyue

last: Zhu

first: Meenhard

last: Herlyn


title: Multifunctional exosomes inhibiting autophagy ameliorate immune tolerance in non-small cell lung cancer II

city: Changchun

first: Simiao

last: Wang

state: other

address: Jilin university

institute: Simiao Wang

summary: The modulation of tumor autophagy to enhance antitumor immunity has garnered significant attention, underscoring its critical role in cancer immunotherapy. However, advanced strategies for precise autophagy-regulating drug delivery remain a pressing need. Here, we introduce a targeted exosome-based drug delivery system capable of simultaneously loading antibodies and nucleic acid drugs while ensuring their accurate release in the tumor microenvironment (TME). We developed a dual-stimulation electroporation system integrating nanosecond electric pulses and ultrasound to enhance exosome production, yielding IL-7 mRNA-enriched exosomes overexpressing CD64 receptors for efficient anti-PD-L1 antibody capture. These multifunctional autophagy-inhibiting and immunomodulatory exosomes (AI-Exos) are designed to inhibit autophagy and modulate immune responses in lung cancer. Upon delivery to the tumor site, AI-Exos mediate the enzymatic cleavage of peptide bonds to release IL-7 mRNA. This process induces autophagy suppression and restores MHC-I expression, which synergizes with anti-PD-L1 immune checkpoint inhibition to overcome acquired drug resistance and enhance antitumor efficacy. In conclusion, this study proposes an innovative methodology utilizing engineering exosomes for the co-delivery of protein antibodies and genetic materials. This approach establishes a promising strategy to advance cancer immunotherapy through targeted autophagy modulation.

overall_design: RNA sequencing of paraffin-embedded tumor biopsy specimens from six NSCLC patients who subsequently underwent anti-PD-L1 neoadjuvant immunotherapy


title: DICER1 dysregulation triggers miRNA reprogramming, promotes mesenchymal cell fates and slows TNBC tumorigenic phenotypes

city: Columbus

first: Ganesh

middle: Ram

last: Koshre

state: OH

address: 460 W 12th Ave

institute: The Ohio State University

summary: DICER1 is a key processing enzyme required for the generation of mature and active miRNAs. Mutations that diminish DICER1 function result in widespread changes in miRNA levels, resulting in changes in the transcriptome and cellular phenotypes. Previously, we have found that mutations within the 3’UTR of the Dicer1 mRNA greatly diminish DICER1 protein levels and miRNA production. Triple negative breast cancer cells that contain these mutations have slower growth and migration and diminished tumorigenic potential. By comparing the transcriptome and miRNA profile of these cells, we find that miR30d-5p are significantly reduced in DICER1 mutant cells. This reduction in miR30d-5p results in increased mRNA stability and protein levels of the miR30d-5p target, SNAIL, a transcription factor that promotes the transcription of genes required for mesenchymal cell fates. We show that elevated SNAIL protein levels drive an epithelial- mesenchymal transition in these cells that can be partially rescued by the addition of miR30d-5p.Our results highlight the important role for DICER1 and miRNA levels in modulating cell fate in breast cancer.

overall_design: RNA-Seq, miRNA-Seq, qRT-PCR, RNA Stability assay, Luciferase assay, Western blot, Migration assay, In vivo mice study

middle: R

first: Misbah

last: Khan

first: Shweta

last: Rajasekharan

first: Amity

middle: L

last: Mannings

first: James

last: Walker

first: Wayne

last: Miles


title: Maternal gut microbial legacy shapes intestinal health and colitis susceptibility of offspring

city: Daegu

first: Ji-Min

last: Lee

address: 680, Gukchaebosang-ro

department: Cell & Matrix Research Institute

country: South Korea

institute: Kyungpook National University

summary: Offspring born to mothers with colitis exhibited gut microbial dysbiosis, impaired barrier function, low-grade intestinal inflammation, compromised Wnt signaling and reduced crypt cell proliferation, which collectively increased their susceptibility to colitis in adulthood.

overall_design: 1.5% dextral sulfate sodium (DSS) was treated to induce colitis for 5 days at 2nd trimester of pregnant mice.  We examined their offspring's intestinal conditions and collected fecal samples at 3-week after birth.

first: Hoyul

first: Eun Soo

last: Kim


title: Effect of cardiomyocyte-specific activation of nuclear receptor subfamily 4 group A member 2 (NR4A2) on poly(A) RNA expression profile in the mouse heart [RNA-seq]

city: Houston

first: Romain

last: Harmancey

state: Texas

address: 1881 East Rd.

department: Internal Medicine - Division of Cardiology

institute: The University of Texas Health Science Center at Houston

summary: Nuclear receptor subfamily 4 group A member 2 (NR4A2) is a widely expressed immediate early gene induced by a host of physiological signals such as growth factors, cytokines, and neurotransmitters. In the adult mammalian heart, including in cardiomyocytes, NR4A2 mRNAs increase more than 60-fold in response to beta-adrenergic stimulation. However, the nature of the transcriptional remodeling induced by NR4A2 in the stressed heart has remained poorly investigated. We show here that NR4A2 activation in mouse cardiomyocytes profoundly alters the sub-transcriptomes controlling metabolic pathways and calcium transients in the heart. At the metabolic level, NR4A2 induced the expression of glycolytic enzymes while simultaneously down-regulating fatty acid oxidation. Increased NR4A2 expression was also associated with the repression of several genes critical for t-tubules formation, dyads organization, and calcium flux. The findings shed light on a novel regulatory mechanism controlling cardiac homeostasis, and establish a framework to investigate further the roles played by NR4A2 in fetal reprogramming of the adult mammalian heart under various stress conditions.

overall_design: Cardiac poly(A) RNA-seq profiling of mice with cardiomyocyte-specific, tamoxifen-inducible expression of the nuclear receptor NR4A2 and of their Cre recombinase expression control littermates before (Day 0) and at 3 days, 6 days, and 12 days  after tamoxifen treatment.

first: Harmancey

last: Romain

first: Ashraf

last: Sadia


title: Effect of cardiomyocyte-specific activation of nuclear receptor subfamily 4 group A member 2 (NR4A2) on the regulation of gene expression in the mouse heart [ChIP-Seq]

summary: Nuclear receptor subfamily 4 group A member 2 (NR4A2) is a widely expressed immediate early gene induced by a host of physiological signals such as growth factors, cytokines, and neurotransmitters. In the adult mammalian heart, including in cardiomyocytes, NR4A2 mRNAs increase more than 60-fold in response to beta-adrenergic stimulation. However, the identity of the genes directly regulated by NR4A2 in the stressed heart has remained poorly investigated. Using quantitative ChIP-seq data, we show here that activation of glycolysis in the mouse heart upon NR4A2 induction is directly linked to increased binding of the nuclear receptor to the promoter and enhancers of genes encoding glycolytic enzymes. Increased binding of NR4A2 to enhancers located within the imprinted Dlk1-Dio3 locus was also associated with the up-regulation of several dozen non-coding RNAs (lncRNAs and miRNAs) known for coordinating critical steps of cardiac development and for inhibiting mitochondrial function in myocytes. The findings shed light on a novel regulatory mechanism controlling cardiac homeostasis, and establish a framework to investigate further the roles played by NR4A2 in cardiac metabolic remodeling in the face of various acute and pathological stress conditions.

overall_design: Comparative analysis of ChIP-seq profiles of NR4A2 obtained in mice with cardiomyocyte-specific, tamoxifen-inducible expression of the nuclear receptor NR4A2 (Nr4a2-icTg) and that of their Cre recombinase expression control littermates at 3 days after tamoxifen treatment. Validation of the NR4A2 antibody for ChIP-seq analyses and generation of normalized ChIP-seq data were carried out by Active Motif’s Epigenetic Services team.


title: Ufd2p promotes meiotic crossover formation  by destabilizing Top2p in budding yeast

city: Jinan

first: cong

middle: Tai

last: Tan

state: Shandong

address: Jingshi Street

department: State Key Laboratory of Microbial Technology

institute: Shandong University

summary: Meiotic crossover (CO) serves dual essential roles in ensuring accurate chromosome segregation and generating genetic diversity. While protein ubiquitination has been implicated in meiotic CO formation, the specific E3 ligases involved and their molecular mechanisms remain largely characterized. Through systematic screening, we identified Ufd2p as a key meiotic CO regulator. Depletion of Ufd2p severely impaired meiotic recombination by reducing CO numbers and enhancing CO interference strength. Multi-omics analysis identified topoisomerase II (Top2p) as a specific substrate of Ufd2p, and Ufd2p could catalyze Top2p ubiquitination to promote its degradation through ubiquitin-proteasome pathway. Accumulated Top2p in Ufd2p depletion strains resolves DNA negative supercoils, which in turn enhances CO interference strength and reduces CO numbers. Remarkably, expression of either mouse or human UBE4B in yeast effectively rescued the meiotic and recombination defects caused by Ufd2p depletion. These findings provide important insights into the ubiquitin-mediated regulatory network governing chromatin structure and meiotic recombination pattern

overall_design: Wild type(UFD2-9MYC) cells were induced to synchronously enter meiosis and samples were collected at 4h in SPM and fixed with 1% formaldehyde for chromatin crosslinking and mechanically lysed using glass beads. Chromatin fragments were sonicated to 200-500 bp, followed by overnight incubation with mouse monoclonal anti-Myc antibodies and Protein A/G magnetic beadsl After sequential washing, immunoprecipitated complexes were eluted and reverse-crosslinked. DNA purification was conducted using the QIAquick PCR Purification Kit. Libraries were prepared and sequenced in paired-end 150-bp (PE150) mode on an Illumina platform.

first: Taicong

first: Yanan

last: Zhao

first: Yali

last: Mi


title: Endothelial Prolyl Hydroxylase 3 Mitigates Maladaptive Inflammation to Promote Post-Ischemic Kidney Repair

first: Pinelopi

last: Kapitsinou

state: Il

address: 303 East Superior Street

department: Medicine/Nephrology

institute: Northwestern University, Feinberg School of Medicine

summary: Acute kidney injury (AKI) is a major health concern and well-established risk factor for the development of chronic kidney disease (CKD). Tissue hypoxia is a prominent feature of the injured kidney that shapes the biological behavior of parenchymal and immune cells. Endothelial cells have important immunomodulatory roles, but the impact of dysregulated oxygen sensing on their responses remains poorly understood. By leveraging a combination of conditional mouse strains, single-cell analyses of mouse and human samples, and in vitro experiments, we demonstrate that the oxygen sensor PHD3 in the endothelium suppresses maladaptive inflammatory responses, thereby promoting kidney repair. Specifically, post-ischemic inactivation of endothelial PHD3, but not PHD1, leads to maladaptive kidney repair, characterized by increased fibrosis and inflammation. scRNA-seq analysis of the postischemic endothelial PHD3-haplodeficient kidney shows an endothelial IFN-γ gene signature, mirroring the responses observed in patients with severe AKI. By performing loss- and gain-of-function experiments in vitro, we demonstrate that PHD3 regulates IFN-γ responsive pro-inflammatory signatures in a HIF-dependent manner. Consistent with this, simultaneous deletion of ARNT in endothelial PHD3-deficient mice restored kidney repair. Thus, our findings provide novel mechanistic insights into endothelial oxygen sensing and the AKI-to-CKD transition, highlighting potential therapeutic avenues for mitigating disease progression.

overall_design: Injured kidneys from Cdh5-CreERT2;Phd3f/+ and Cre- mice were collected on day 14 post unilateral IRI


title: Immunosuppressive myeloid signaling and distinct malignant cell states in pancreatic neuroendocrine tumors revealed by single-nucleus RNA-seq

first: William

last: Hwang

state: Massachusetts

address: 185 Cambridge St

department: Center for Systems Biology

institute: Massachusetts General Hospital

summary: Pancreatic neuroendocrine tumors (PNET) are rare and heterogeneous neoplasms with increasing incidence worldwide. Currently, there is no clinically-relevant molecular framework for risk-stratifying patients with PNET. In this study, we used single-nucleus RNA-sequencing to uncover the spectrum of malignant cell states and interactions within the tumor microenvironment.  We uncovered a gene expression program enriched in neural/synaptic signaling genes associated with aggressive clinical behavior, altered telomeres, and broad chromosomal loss of heterozygosity. Moreover, it is associated with worse overall survival in independent cohorts. Another novel malignant cell state was enriched for VEGF-signaling and found to interact with macrophages via glutamate, promoting an immunosuppressive phenotype in macrophages and a more invasive phenotype in malignant cells. This study provides insight into the heterogeneity of malignant cells in PNET, and these diverse cell states and intercellular interactions that may be further explored for prognostication and therapeutic targeting.

overall_design:
We performed single-nucleus RNA sequencing on resected primary and metastatic PNETs (n = 20).

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

first: Jeanna

middle: M

last: Arbesfeld-Qiu

first: Jae-Won

last: Cho

first: Jennifer

last: Su

first: Jimmy

last: Guo

first: Hannah

last: Hoffman

first: Carina

last: Shiau

first: Steven

first: Martin

last: Hemberg


title: METTL5 Blockade Enhances Anti-Tumor Immune Response via Inducing Neoantigen Generation

city: Shenzhen

first: Hao

last: Chen

address: 1088 Xueyuan Avenue

institute: Southern University of Science and Technology (SUSTech)

summary: Tumor neoantigens play a pivotal role in immunotherapy through eliciting tumor-specific immune responses. However, their clinical application is limited by the restricted repertoire of naturally occurring neoantigens, which undergoes continuous immune editing within the tumor microenvironment. To address this critical challenge, we explored translational regulation as a reversible and safer strategy to augment neoantigen production. Through systematic analysis of translational regulatory factors associated with immunotherapy response, we identified METTL5 as a key modulator of anti-tumor immunity. METTL5 depletion in syngeneic tumor models demonstrated significant enhancement of endogenous anti-tumor immunity across distinct MHC-I haplotypes, with synergistic effects observed when combined with immune checkpoint blockade. Based on evolutionary conservation of ribosomal decoding center modifications and established mechanisms from prokaryotic studies, we propose that METTL5-mediated m6A modification at the small ribosomal subunit decoding center may contribute to translational fidelity regulation. Mechanistically, integrated MHC-I immunoprecipitation-mass spectrometry and ribosome profiling revealed the identification of non-canonical peptides, including those derived from novel unannotated open reading frames (nuORFs), which provide previously absent neoantigen sources for tumor cells. T-cell receptor sequencing further demonstrated significantly enhanced clonal diversity and tumor-specific reactivity of infiltrating T cells in METTL5-deficient tumors. Therapeutic vaccination strategies targeting METTL5-deficiency-induced neoantigens, including both peptide-based and mRNA vaccines, exhibited partial anti-tumor efficacy in murine tumor models. This study establishes METTL5 as a critical translational regulator that restricts neoantigen diversity and proposes a novel combinatorial strategy to enhance immunotherapy through translational control.

overall_design: To identify the mRNAs translationally regulated by METTL5, Ribo-seq were carried out in wild-type and Mettl5-KO cell lines. Input mRNAs and RPFs were isolated, ribosomal RNAs were depleted and cDNA libraries were prepared and sequenced according to the TruSeq Ribo Profile pipeline (Illumina). To investigate whether METTL5 deletion enhances antitumor immunity through T-cell repertoire remodeling, we performed TCRβ sequencing on peripheral blood from Mettl5-KO versus control (SCR) B16-F10 melanoma-bearing mice. Since METTL5-deficient tumors exhibit elevated neoantigen loads, we hypothesized this would drive broader TCR clonality and diversity in hosts. Comparative analysis revealed conserved CDR3 length distributions (dominant 10–15 aa peaks) but significant diversity differences between groups. This study directly links METTL5 loss to adaptive immune responses by quantifying TCR repertoire expansion, supporting its role in immunogenic tumor evolution. To investigate transcriptional alterations in Mettl5-KO tumors, we performed single-cell RNA sequencing (scRNA-seq) on freshly isolated tumor tissues

first: Yangyi

last: Zhang

first: Xiaoyan

last: Shi


first: Jonathan

middle: S.

last: Duke-Cohan

address: 450 Brookline Avenue JF517

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary: While memory T-cells provide protective immunity against specific pathogens, their post-encounter differentiation into central (TCM) and/or effector (TEM) subpopulations remains enigmatic. We thus explored the CD8 T-cell receptor (TCR) repertoire of 242 murine TCRαβ clonotypes directed against an immunodominant influenza A virus (IAV) peptide/major histocompatibility complex (pMHC) ligand, the nucleoprotein NP366-374/Db, leveraging single-cell transcriptomics with paired TCR sequencing, mechanosensing metrics, and in vivo memory development plus TCR-pMHC structural analyses. Polar TEM and more variegated TCM repertoires as well as bipotential “bipolar” clonotypes (TBP) revealed weak force-dependent bonding parameters associated with heterosubtypic IAV crossreactivities. TCM and TEM polarities manifest pMHC binding skewed to TCRβ- versus TCRα-subunit mechanotransduction, respectively, unlike the more signaling-balanced TBP TCRs. In sum, TCR diversity anticipates pathogen evolution whereas divergent signaling regulates memory fate.

overall_design: RNA-Seq profiling of retrogenic T cells, each variant expressing a unique influenza-specific T cell receptor, adoptively transferred to normal mice and then isolated from mediastinal lymph nodes following resolution of influenza infection.

first: Aoi

last: Akitsu

first: Ellis

last: Reinherz


title: Single nucleus transcriptomics to study sex differences during murine E. coli pyelonephritis

city: Cambridge

first: Ashlee

last: Earl

address: 415 Main Street

department: Infectious Disease and Microbiome Program

institute: Broad Institute

summary: This project employs models of urinary tract infection (UTI) in female, androgen-exposed female, and male mice to determine molecular mechanisms by which host androgen exposure promotes Escherichia coli pyelonephritis as well as renal scarring, a common sequela of upper-tract UTI. Our prior findings indicated that the influence of sex, including androgen exposure, on these common bacterial infections is more complex than previously appreciated. Specifically, male sex was associated with increased risk for chronic cystitis and high-titer pyelonephritis, as well as formation of renal abscesses. These snRNA-seq experiments were designed to interrogate the fundamental sex differences impacting the host-pathogen interaction and cellular responses in E. coli pyelonephritis, thereby influencing pathogenicity and outcome of infection.

overall_design: single nucleus RNA-seq investigation of male, female, and androgen-explosed female C3H/HeN mice in a model of urinary tract infection (UTI).

first: Teri

last: Hreha

first: Abigail

last: Manson

first: Christina

last: Collins

first: Haojia

last: Wu

first: Christophe

last: Georgescu

first: Benjamin

last: Humphreys

first: David

last: Hunstad


title: Unfolded protein response signaling promotes myeloid cell production  and cooperates with oncogenic mutation

city: St. Louis

first: Yoona

last: Kang

state: MO

address: Washington University in St. Louis

department: Department of Medicine

institute: Washington University

summary: Unfolded protein response (UPR) is an evolutionally conserved adaptive mechanism that promotes protein homeostasis under endoplasmic reticulum (ER) stress. UPR signaling has numerous functions in metabolism, cancer, immunology, and neurodegenerative diseases. Recent studies also showed that UPR signaling has important roles in hematopoietic stem and progenitor cell biology. However, whether UPR signaling regulates hematopoietic lineage fate decision remains elusive. Here, we found that FcgR- MPP3 generates erythroid lineage and Jak2V617F mutation leads to overproduction of erythroid cells by expanding FcgR- MPP3. We showed that UPR signaling increases myeloid cell production through promoting FcgR- MPP3 transition to granulocyte/macrophage progenitor (GMP) producing FcgR+ MPP3 at the expense of erythroid lineage via the XBP1 pathway. Under a disease condition, UPR signaling cooperates with Jak2V617F mutation and exacerbates disease phenotype as increasing red blood cells in a mouse model of polycythemia vera (PV) through the ATF4 pathway. Activation of UPR signaling also increased myeloid output in healthy donor bone marrow MPP cells while skewing the output towards erythroid lineage in PV patient bone marrow MPP cells. Together, our results identify a novel function of UPR signaling in hematopoietic lineage specification and provide critical insights into targeting UPR signaling in hematological malignancies.

overall_design: RNA was isolated from 7,000 to 10,000 cells per sample using RNeasy Plus Micro Kit (Qiagen, 74034). RNA samples were submitted to the Genome Access Technology Center at the McDonnell Genome Institute for low input RNA sequencing. RNA was isolated from 7,000 to 10,000 cells per sample using RNeasy Plus Micro Kit (Qiagen, 74034). RNA samples were submitted to the Genome Access Technology Center at the McDonnell Genome Institute for low input RNA sequencing.

first: Hyunjoo

last: Choi

first: Sang-Eun

last: Jung

first: Hyojung

last: Paik

first: Yesenia

last: Earth

first: Jiye

last: Yi

first: Maggie

last: Cox

first: Stephen

last: Oh

first: Yoon-A


title: Spatiotemporal transcriptomic mapping reveals region-specific glial activation and astrocyte subtype shifts in epileptogenesis beyond the hippocampus

city: Paris

first: Adrien

last: Dufour

address: 48, boulevard serurier

summary:
Temporal lobe epilepsy (TLE) is a prevalent neurological disorder often initiated by status epilepticus (SE), followed by a latent phase that culminates in chronic epilepsy. The molecular and cellular mechanisms driving this transformation remain incompletely understood. Here, we applied Visium-based spatial transcriptomics to coronal brain sections from lithium-pilocarpine–induced SE rats and controls (n = 16) to map transcriptional dynamics across epileptogenesis.
Spatial clustering accurately defined anatomically relevant regions and canonical markers in controls. Comparative analyses revealed extensive SE-associated transcriptional alterations spanning latent and chronic phases across all examined regions. Notably, spatial profiling demonstrated that microglial activation and reactive astrogliosis extended well beyond the hippocampus, encompassing white matter tracts and multiple thalamic nuclei during the latent phase. Cell-type deconvolution further identified pronounced regional shifts in astrocyte functional subtypes within these reactive zones.
These findings uncover the spatial heterogeneity of epileptogenic processes, highlighting previously underappreciated thalamic and white matter involvement. The identification of region-specific glial responses and astrocyte subtype transitions provides new mechanistic insights into epileptogenesis and underscores the need for region- and cell-type–targeted strategies to inform therapeutic interventions in TLE.

overall_design: We based our study on the lithium-pilocarpine model of TLE introduced before and selected four time points to conduct a comprehensive study of epileptogenesis: 5, 10, 20 and 40 days after SE induction (D5, D10, D20 and D40). The latent period is supposed to start as soon as SE ends, either by itself or because of drug injection. Nonetheless, acute SE effects seem to appear around D2 in hippocampus and can be observed until D3. Latent period total duration in itself is highly dependent on SE duration, SE arresting method, strain, age and gender. A mean duration for TLE models is estimated to be 15 days. So D10 and D20 were selected to encompass the medium to late latent period. Finally, considering the variability of latent/chronic switch, D40 was chosen to represent the chronic period but without being too far into it.

last: Le Priol

first: Baptiste

last: Porte

first: Andrée

last: Delahaye-Duriez


title: Multiomic single nuclei profiling of a mouse model of Alzheimer’s disease reveals that ACSS2 confers neuronal resilience to tauopathy [RNA-Seq]

first: Gregory

last: Donahue

address: 3400 Civic Center Blvd, Bldg 421

department: Cell & Developmental Biology

institute: The University of Pennsylvania

summary: Loss of cell identity and global epigenomic dysregulation are emerging as key contributors to Alzheimer’s disease (AD). The mechanisms by which protective or risk-conferring epigenetic marks are established and maintained are under intense investigation. ACSS2 (Acetyl-CoA Synthetase 2) is a key metabolic enzyme that is nuclear-localized in neurons. In healthy brains, ACSS2 fuels histone acetylation and drives expression of neuronal genes that regulate learning and memory. Here, we examine how loss of ACSS2 contributes to AD-associated cellular, genomic and behavioral outcomes, focusing on long-term steady state changes. Using a mouse model of human pathological AD-Tau injection, we show that loss of ACSS2 exacerbates Tau-related memory impairments, while dietary supplementation of acetate rescues learning in an ACSS2-dependent manner. Combining state-of-the-art proteomic and genomic approaches, we demonstrate that this effect is accompanied by ACSS2-dependent incorporation of acetate into hippocampal histone acetylation, which facilitates gene expression programs related to learning. We identify the most severely affected hippocampal neuronal populations, including pyramidal cells of the perforant pathway and Cajal-Retzius cells. Overall, these results reveal ACSS2 as a neuroprotective metabolic enzyme in key hippocampal neuronal populations, and dysregulation of which may play an important role in the etiology of AD. These findings may guide development of future therapies for AD, other tauopathies and related dementia.

overall_design: Contains RNA-seq data obtained from male and female mice (dissected dorsal hippocampus) treated intracranially with either PBS or purified tau protein from human AD patient brains. Mice have either WT or ACSS2KO genetic background. The purpose of the study is to examine the interaction between the ACSS2 absence and the presence of pathological Tau, with a focus on memory. Six study groups are provided: male-PBS-WT, male-PBS-KO, male-Tau-WT, male-Tau-KO, female-Tau-WT, and female-Tau-KO; note that we do not have PBS-treated females) with three biological replicates each except for female-Tau-WT which has two. For purposes of downstream analysis, only the male samples were used.

first: Gabor

last: Egervari

first: Desi

last: Alexander

first: Hua

last: Huang

first: Greg

first: Connor

last: Hogan

first: Mariel

last: Mendoza

first: Hong

last: Xu

first: Virginia

last: Lee

first: Ben

middle: A

last: Garcia

first: Nancy

last: Bonini

first: Shelley

last: Berger