title: In vivo BMAL1 occupancy mapping using MACS-Calling Cards reveals disease-associated retargeting in Cln3Δex7/8 astrocytes

city: St. Louis

first: India

last: Reiss

state: Missouri

address: 660 S. Euclid Ave.

department: Genetics

institute: Washington University in St. Louis

summary: Astrocytes maintain neuronal homeostasis through cellular programs that are frequently disrupted early in neurodegenerative disease, many of which are regulated by the circadian clock. The core clock transcription factor BMAL1 is required for normal astrocyte function, yet whether its regulatory activity is altered during neurodegeneration remains unclear. Moreover, astrocyte circadian gene expression patterns are profoundly reprogrammed in disease, yet the underlying mechanisms remain unclear, primarily due to a lack of methods for identifying DNA binding sites of BMAL1 in specific cell types in the brain. Here, we have developed an in vivo strategy to record transcription factor genomic occupancy specifically in astrocytes (MACS-Calling Cards, MACS-CC), and used it to map BMAL1 binding. We applied this strategy to the Cln3Δex7/8 mouse model of CLN3 disease, a fatal neurodegenerative disorder, which exhibits early astrocyte dysfunction prior to widespread neuronal loss as well as circadian dysregulation. This allowed us to test how the progressive pathological cascade caused by CLN3 deficiency reprograms BMAL1 binding in astrocytes. BMAL1 binding was extensively redistributed, with comparable numbers of wild-type-specific and disease-specific sites. Wild-type-specific BMAL1 binding was enriched near genes involved in circadian regulation and astrocyte differentiation, whereas disease-specific sites lacked coherent functional enrichment. Consistent with these changes, RNA-seq of sorted astrocytes revealed reduced expression of mature astrocyte markers in Cln3Δex7/8 mice. To define the mechanisms underlying BMAL1 retargeting in Cln3Δex7/8  astrocytes, we tested whether changes in chromatin accessibility could explain disease-associated gains and losses of BMAL1 binding. Although chromatin accessibility was broadly remodeled, differential accessibility did not predict BMAL1 binding gains or losses. Instead, motif analyses supported a model in which loss of normal cooperative transcription factor partnerships contributes to BMAL1 retargeting. Together, these findings demonstrate that MACS-CC enables in vivo transcription factor occupancy mapping in astrocytes and reveals the mechanisms behind early rewiring of circadian regulatory programs within a model of progressive pathology leading to neurodegeneration. Our data also shed new light on astrocyte dysfunction in CLN3 disease.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: BMAL1 Calling Card, Mnase-seq, and RNA-seq datasets from astrocytes (enriched from MACS), as well as BMAL1 Calling Card datasets from HepG2 cells.

first: Reiss

last: India


title: The FGF4–integrin β1 axis orchestrates diabetic wound regeneration by restoring directional collective motility [ChIP-Seq]

status: Public on May 01 2026

city: Wenzhou

first: Zhiming

last: Zhang

address: Chashan Campus of Wenzhou Medical University

country: China

institute: Department of Cardiology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

type: Genome binding/occupancy profiling by high throughput sequencing

summary: This dataset was generated to investigate the mechanism by which Fibroblast Growth Factor 4 (FGF4) regulates the binding of C-FOS to the ITGB1 promoter. ChIP sequencing was performed on high glucose-induced HacaT cells, which were divided into low glucose, high glucose, and high glucose plus FGF4 treatment groups.

overall_design: To investigate the effects of high glucose and FGF4 treatment, this study employed ChIP-seq to analyze the binding of C-FOS to the ITGB1 promoter in HacaT cells under low glucose, high glucose, and high glucose combined with FGF4 treatment conditions.

first: Jian

last: Sun

first: Zhifeng

last: Huang

first: Jie

last: Zhou

first: Shuodan

last: Ding


title: Combination of a CCL21-gene modified dendritic cell vaccine and pembrolizumab induces immune responses in non-small cell lung cancer

city: Los Angeles

first: Linh

middle: My

last: Tran

state: California

department: Medicine - Pulmonary & Critical Care

country: USA

institute: University of Los Angeles

type: Expression profiling by high throughput sequencing

summary: Immune checkpoint inhibitors (ICIs) have transformed treatment for non-small cell lung cancer (NSCLC), but resistance to these therapies is common. We report results from a phase I trial combining intratumoral administration of a CCL21-gene modified dendritic cell (CCL21-DC) vaccine with pembrolizumab in patients with advanced NSCLC. Among 23 patients that received trial therapy, there were no dose-limiting toxicities and a low incidence of treatment-related adverse events. Although no objective responses were observed, 36.8% of patients had a best response of stable disease (SD). Correlative analyses of longitudinal tumor biopsies revealed that disease stability was associated with reduced tumor mutational heterogeneity and increased intratumoral T cell receptor diversity following therapy. Post-treatment tumor biopsies exhibited increased CD4+ T cell infiltration and the presence of novel T cell clones that predominantly possessed CD4+ memory T cell phenotypes. These novel clones experienced greater clonal expansion and a transition towards exhausted phenotypes in SD samples. However, many novel clones failed to persist over time, and immunosuppressive myeloid signaling signatures were identified especially in progressive disease samples. Our findings demonstrated that CCL21-DC vaccination combined with pembrolizumab is safe and can promote antitumor immune responses in a subset of patients. However, efficacy may have been constrained due to limited tumor specificity of the T cell response and an inability to fully reprogram the immunosuppressive tumor microenvironment.

overall_design: Core needle biopsies were obtained at the time of each CCL21-DC vaccine injection on days 0, 21, and 42 of the clinical trial. Combined scRNA (5’ GEX) and VDJ (TCR) sequencing of these tumor biopsies was performed. Tumor samples were mixed with the KPL mouse lung cancer cell line to function as carrier cells prior to library construction. PBMCs were also obtained on days 0, 21, 42, and 63 of the trial. ScRNA-seq (3' GEX) was performed on pooled PBMCs from each patient, with each time point tagged using the CellPlex kit. Bulk TCR-seq was performed on PBMC samples separately for each time point and after flow sorting for CD4+ and CD8+ T cell populations.

first: Michael

middle: S

last: Oh

first: Aaron

last: Lisberg

first: Camelia

last: Dumitras

first: Ramin

last: Salehi-Rad

middle: M

first: Kostyantyn

last: Krysan

first: Edward

middle: B

last: Garon

first: Bin

last: Liu

first: Steven

last: Dubinett


title: Multi-omic analysis reveals molecular subtypes with distinct immunological phenotypes in SLE

city: Indianapolis

first: Helen

last: Masson

address: 893 S Delaware St.

institute: Eli Lilly and Company

summary: Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by variability in clinical manifestations and underlying molecular mechanisms. To investigate this heterogeneity, we performed integrative multi-omic profiling—including epigenomic (EM-seq), transcriptomic (RNA-seq), and proteomic (Olink) analyses—on whole blood samples from over 700 SLE patients enrolled in two Phase III clinical trials. Using this approach, we identified three stable molecular endotypes of SLE, providing a framework for understanding disease subtypes and informing precision medicine strategies.

overall_design: Olink proteomic profiling was performed on whole blood samples from patients with systemic lupus erythematosus (SLE) enrolled in two Phase III clinical trials: BRAVE-I (NCT03616912) and BRAVE-II (NCT03616964), as well as from 84 healthy control subjects.

first: Christoph

last: Preuss

first: Ernst

last: Dow


title: Multiple Mechanisms Contribute to Robust Type 1 Diabetes Protection in Nfkbid Overexpressing NOD mice

city: Bar Harbor

first: Dave

last: Serreze

state: ME

address: 600 Main Street

institute: The Jackson Laboratory

summary: Transgenic overexpression of Nfkbid in NOD mice causes robust type 1 diabetes protection associated with expansion of Tregs with increased suppressive capacity. Complementarily, Nfkbid ablation leads to decreased Treg numbers, decreased suppressive capacity and accelerated diabetes.The goal of this experiment was to further identify molecular mechanisms underlying strong protection and susceptibility from type 1 diabetes imbued by modifying Nfkbid expression. mRNA sequencing analysis of Tregs from NOD mice with absent or overexpressed Nfkbid was performed. Differential gene expression was calculated and Ingenuity Pathway analyses showed enrichment of cellular replication pathways.  Understanding the mechanisms underlying protection from Nfkbid overexpression may lead to novel therapeutic avenues.

overall_design: CD4+ T-cells were enriched from NOD.Foxp3-GFP, NOD-Nfkbid-KO.Foxp3-GFP and NOD-60A.Foxp3-GFP spleen prior to FACS for GFP+ Tregs.  Tregs were sorted directly into RLT buffer containing beta-mercaptoethanol and RNA was isloated. RNA was extracted by RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions and RNA Integrity Number was measured by Pico Assay using a 2100 Bioanalyzer (Agilent Technologies). Control and Knockout samples were pooled from n=2 samples, while individual transgenic samples had ample RNA for library creation. Libraries were constructed using a KAPA mRNA HyperPrep Kit (Roche sequencing and Life Science). Briefly, polyA containing RNA was isolated using oligo-dT magnetic bead separation followed by RNA fragmentation, first and second strand synthesis, ligation of adapter sequences (including unique molecular identifiers for each library) and PCR amplification. Library quality and concentration were measured using a D5000 ScreenTape assay (Agilent Technologies) and Qubit dsDNA HS assay (ThermoFisher) respectively according to maker’s protocols. Approximately 27 million reads per library were sequenced on an Illumina NextSeq 500 using the High Output Reagent Kit v2.5 using a 67bp read with a 9bp unique molecular identifier. RNA sequencing reads were aligned to mouse genome GRCm38 using bowtie2 (https://pubmed.ncbi.nlm.nih.gov/22388286/) and Ensembl 102 transcripts. Differential gene expression for Nfkbid-ablated and Nfkbid overexpressing GFP+ Tregs compared to control NOD GFP+ Tregs were analyzed using the Bioconductor R package edgeR (https//pubmed.ncbi.nlm.nih.gov/19910308/).

first: Jennifer

middle: R

last: Dwyer

first: Jeremy

middle: J

last: Racine

first: Harold

middle: D

last: Chapman

first: Amy

last: Bell

first: Grace

middle: A

last: Stafford

first: David

middle: V


title: CYD-4-61 Suppresses Gastric Tumor Growth by Targeting SOX9–CDK4–Driven Oncogenic Programs and Enhances Anti-PD-1 Therapy

city: houston

first: Xueshuai

last: Han

state: texas

address: 1881 East Rd

institute: MD anderson cancer center

summary: Gastric adenocarcinoma (GAC), particularly with peritoneal carcinomatosis, remains highly lethal and therapeutically refractory. We investigated the anti-tumor activity and mechanisms of CYD-4-61, a small-molecule BAX activator, with emphasis on the SOX9–CDK4 oncogenic axis and tumor immune microenvironment. CYD-4-61 efficacy was evaluated using in vitro GAC models and in vivo xenograft, genetically engineered, and syngeneic mouse models. Bulk, single-cell, and spatial transcriptomics, along with molecular and functional assays, were used to define apoptotic, cell-cycle, and immune-related mechanisms. CYD-4-61 induced BAX-dependent apoptosis and suppressed proliferation, stem-like properties, and tumor growth across multiple GAC models. Transcriptomic analyses revealed inhibition of SOX9-dependent programs, including CDK4, and activation of apoptotic and inflammatory pathways. CYD-4-61 destabilized SOX9 via caspase-dependent cleavage, inhibited cell-cycle progression, activated STING signaling, and enhanced response to PD-1 blockade in immunocompetent models. CYD-4-61 exerts potent anti-tumor effects in GAC by targeting SOX9–CDK4–driven oncogenic programs and modulating immune signaling, supporting its development as a therapeutic agent alone or in combination with immunotherapy.

overall_design: RNA-seq profiling of CYD-4-61–treated AGS vs. control AGS; RNA-seq profiling of CYD-4-61–treated GA0518 vs. control GA0518; RNA-seq profiling of CYD-4-61–treated GA0518 SOX9 KO vs. CYD-4-61–treated GA0518

first: Gengyi

last: Zou

first: Katsuhiro

last: Yoshimura

first: Yibo

last: Fan


title: MCM10 and RECQL4 have cooperative and redundant roles in activating the CMG helicase during the replication initiation

city: Mishima

first: Masato

last: Kanemaki

state: Shizuoka

address: Yata 1111

department: Department of Chromosome Science

country: Japan

institute: National Institute of Genetics

summary: DNA replication initiation requires activation of the CMG helicase to establish the replisome. This process involves the extrusion of single-stranded DNA (ssDNA) from the central channel of MCM double hexamers, allowing the two CMG helicases to pass each other; however, the factors that mediate this process in human cells remain unclear. We show that degron-mediated depletion of either MCM10 or RECQL4 alone causes only mild replication defects, whereas simultaneous depletion of both proteins completely blocks CMG activation. ChIP-seq analyses demonstrate that RECQL4 localises to replication initiation zones (IZs) independently of MCM10, whereas MCM10 recruitment to IZs is enhanced upon RECQL4 depletion, suggesting RECQL4 primarily functions in CMG activation, and MCM10 acts as a backup or supporting factor. Rescue experiments further indicate that RECQL4 cooperates with MCM10 through direct interaction, and that their ssDNA-binding activity underlies their functional overlap. We propose MCM10 and RECQL4 act cooperatively and redundantly to promote CMG activation.

overall_design: For both MCM10 and RECQL4 ChIP-seq experiments, the endogenous proteins were individually labeled with a 3xFLAG tag, and immunoprecipitation was performed using an anti-FLAG antibody. To enable conditional depletion, the MCM10-FLAG cell line was engineered to express RECQL4 fused to an mAID-BromoTag (mAB) double-degron system; conversely, in the RECQL4-FLAG cell line, MCM10 was tagged with the same mAB system. Cells were synchronized with nocodazole and released into M phase. Upon release, the cells were treated with either DMSO (control) or the appropriate ligands to induce depletion of the target proteins.


title: Effects of birth weight and milk replacer protein content on growth and transcriptomic profiles of the small intestine, liver, and muscle in complete artificially reared piglets

city: Frederiksberg C

first: Andrew

last: Williams

address: Dyrlægevej 100

country: Denmark

institute: University of Copenhagen

summary: Large litters of modern sows have complicated the rearing of piglets during the suckling period. The subpopulation of piglets with low birth weight and reduced growth potential has increased and has contributed to increasing the mortality rate from birth to weaning. Two of the most important measures to reduce mortality rate is to ensure sufficient energy and nutrient intake, while creating a safe rearing environment for vulnerable piglets. Use of complete artificial rearing with physical absence of the sow, with inclusion of unlimited access to a milk replacer high in fat and protein might potentially improve growth of low-birth-weight piglets.

overall_design: The effects of low and high protein levels in milk replacer (MR) and birth weight class (BWC), on the growth performance of suckling piglets reared under fully artificial on-farm conditions were investigated across two experiments (Exp.1 and Exp.2) using a 2 x 2 factorial design. In each round, 40 piglets ((Yorkshire x Landrace) x Duroc) were removed from the sow no later than 12 hours post-partum and evenly distributed between four rescue decks (RD) (RescueDeck complete NT, VDL Agrotech, Eindhoven, The Netherlands). Twenty piglets were classified as low-birth-weight (LBW, < 1000 g BtW), and twenty as normal-birth-weight (NBW, ≥ 1000 g BtW). From the day of birth, all RD piglets were fed MR ad libitum according to their treatment group. The two MR used in this experiment were optimized to resemble sow milk based on findings from Hojgaard et al. (2020), and were mixed so that DM content was 20% of prepared MR. To mimic sow milk energy level, fat constituted 32% of MR DM (non-mixed) and 6.4% in prepared milk for both MR. The MR differed in protein content and were named High-Fat-Low-Protein (HFLP) and High-Fat-High-Protein (HFHP). Unmixed HFHP contained 22.2% crude protein and 4.4% in prepared milk which resembles sow milk (Hojgaard et al., 2020). Comparatively, HFLP contained 18.0% crude protein on DM basis and 3.6% in prepared milk.


title: Comprehensive Serum Proteomic Profiling Identifies Biomarkers for Pediatric Inflammatory Bowel Diseases and Discriminates between Ulcerative Colitis and Crohn's Disease

city: Boston

first: Simon

middle: T

last: Dillon

state: Massachusetts

address: 99 Brookline Ave, Research North Bldg, Room 107

department: IMBIO

institute: BIDMC RESEARCH

summary: Background and Aims:  The diagnosis of Inflammatory Bowel Diseases (IBD), ulcerative colitis (UC), and Crohn's disease (CD), relies on clinical and pathologic criteria. Non-invasive precision medicine tools to diagnose IBD and discriminate between UC and CD are needed to personalize management. Serum proteomics identified protein biomarkers capable of diagnosing IBD and differentiating Crohn’s disease from ulcerative colitis subtypes.  Methods:  We obtained serum samples from 47 IBD and non-IBD patients seen in a tertiary care pediatric gastroenterology clinic and applied SomaScan proteomics to measure 1,305 proteins to discriminate between IBD and non-IBD and UC and CD. Four proteins were further validated by immunoassays in two cohorts of 295 and 105 individuals and multi-protein predictors were developed using Support Vector Machines (SVM). Findings:  The SomaScan discovery phase identified 95 serum protein biomarkers (BH p<0.01) that differentiated IBD from non-IBD and 70 proteins (p<0.01) that distinguished UC from CD. Pathway analysis linked specific inflammatory processes and vascular functions to IBD and UC versus CD. An 8-protein classifier achieved an AUC of 0.95 for identifying IBD. Significant elevation of four key predictor proteins (MMP1, MMP3, Resistin, Haptoglobin) in IBD was validated by ELISA in the expanded cohort (N=295). The 4-protein SVM predictor achieved an AUC of 0.86 and 0.90 for IBD discrimination in two independent cohorts. A separate 4-protein SVM predictor for differentiating UC from CD achieved an AUC of 0.93 in independent validation.  Interpretation:  Patients with pediatric-onset IBD have a unique serum protein signature associated with pro-inflammatory and vascular pathways. Additional studies are needed to determine whether these dysregulated proteins can be used in conjunction with traditional risk factors to support non-invasive biomarkers that identify IBD and discriminate between its subtypes.  The diagnosis of Inflammatory Bowel Diseases (IBD), ulcerative colitis (UC), and Crohn's disease (CD), relies on clinical and pathologic criteria. Non-invasive precision medicine tools to diagnose IBD and discriminate between UC and CD are needed to personalize management. Serum proteomics identified protein biomarkers capable of diagnosing IBD and differentiating Crohn’s disease from ulcerative colitis subtypes.    Methods  We obtained serum samples from 47 IBD and non-IBD patients seen in a tertiary care pediatric gastroenterology clinic and applied SomaScan proteomics to measure 1,305 proteins to discriminate between IBD and non-IBD and UC and CD. Four proteins were further validated by immunoassays in two cohorts of 295 and 105 individuals and multi-protein predictors were developed using Support Vector Machines (SVM).     Findings  Som The SomaScan discovery phase identified 95 serum protein biomarkers (BH p<0.01) that differentiated IBD from non-IBD and 70 proteins (p<0.01) that distinguished UC from CD. Pathway analysis linked specific inflammatory processes and vascular functions to IBD and UC versus CD. An 8-protein classifier achieved an AUC of 0.95 for identifying IBD. Significant elevation of four key predictor proteins (MMP1, MMP3, Resistin, Haptoglobin) in IBD was validated by ELISA in the expanded cohort (N=295). The 4-protein SVM predictor achieved an AUC of 0.86 and 0.90 for IBD discrimination in two independent cohorts. A separate 4-protein SVM predictor for differentiating UC from CD achieved an AUC of 0.93 in independent validation.    Interpretation  Patients with pediatric-onset IBD have a unique serum protein signature associated with pro-inflammatory and vascular pathways. Additional studies are needed to determine whether these dysregulated proteins can be used in conjunction with traditional risk factors to support non-invasive biomarkers that identify IBD and discriminate between its subtypes.

overall_design: 47 samples analyzed; 24 non-IBD, 12 CD, 11 UC.  1.3k SomaScan manual kit format used for running all samples and kit controls.

first: Towia

last: Libermann


title: p53 maintains healthy aging [P53KO_HFD_RNAseq]

city: La Jolla

first: Kathryn

last: Lande

state: CA

address: 10010 N Torrey Pines Rd

department: IGC

institute: Salk Institute

summary: The tumor suppressor p53 is a master regulator of cellar stress responses implicated in the aging process. As a tumor suppressor, the antagonistic pleiotropy (AP) theory predicts a beneficial cancer suppression function for p53 early in life that becomes detrimental late in life, for example by promoting accumulation of senescent cells, an inflammatory cell fate driven by tumor suppressors and linked to age-associated disease.  Although p53 signaling is generally activated in aging and disease, p53 is also known to suppress some phenotypes of cellular senescence, and the function of p53 in aged tissues, as well as its causal role in aging, is not well-defined. Here we show, contrary to the prediction of AP, that p53 promotes healthy aging in mice by suppressing inflammation and maintaining metabolic function. Multimodal RNA, protein, epigenetic, and spatial profiling at the single-cell level in aged mouse liver confirmed the expected age-dependent increase of immune infiltration, accumulation of senescent cells, and p53 pathway activation. Knockout of p53 in vivo in hepatocytes of aged, but not young, mice further increased inflammation and immune infiltration. Mechanistically, p53 suppressed DNA replication stress and damage, a precursor to inflammatory signaling, in senescent cells and in aged liver. In aged hepatocytes, p53 directly targeted and maintained expression of metabolic genes, and knockout of p53 sensitized aged mice to metabolic stress-induced liver damage. Conversely, pharmacological activation of p53 with a small molecule MDM2 inhibitor in aged mice reversed age-associated suppression of metabolic genes, decreased age-associated inflammation, delayed onset of frailty, (and extended lifespan) of aged mice. These data suggest that in mice, p53 delays phenotypes of aging, and that this function is targetable, potentially for the benefit of healthy aging in humans.

overall_design: RNA-seq profiling of livers from 24mo P53-KO and Rosa26-KO mice fed either a control or high fat diet.

first: Marcos

last: Garcia Teneche

first: Peter

last: Adams


title: p53 maintains healthy aging [P53KO_PTENKO_RNAseq]

overall_design: RNA-seq profiling of livers from young and old P53-KO, PTEN-KO, and Rosa26-KO mice.


title: LRMDA suppresses colonic inflammation, colorectal cancer and bacterial infection [CRC_RNA-seq]

city: wuhan

first: yang

last: liping

address: 115 East Lake Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University School of Medicine

institute: Wuhan University

summary: The leucine-rich melanocyte differentiation-associated protein (LRMDA), a recently characterized intracellular trafficking regulator, plays a critical yet unexplored role in intestinal homeostasis 1. Here, we show that LRMDA expression is downregulated in the inflamed mucosa of patients with inflammatory bowel disease (IBD) and in colorectal tumor tissues. Similarly, we demonstrate that LRMDA deficiency exacerbates dextran sulfate sodium (DSS)-induced colitis, promotes tumorigenesis in azoxymethane (AOM)/DSS and AOM/Vil-Cre;Trp53fl/fl (VP) models and aggravates enteric pathogen infection. Mechanistically, LRMDA may positively regulate the normal exocytosis of mucins from intestinal goblet cells into the intestinal lumen via the dynein-dynactin-microtubule pathway by interacting with the ICD domain of DCTN1. These findings identify LRMDA as a novel regulator of intestinal mucosal barrier function.

overall_design: RNA-seq of tumors of WT (n=4) and Lrmda-/- (n=4) AOM/DSS-induced CRC mice.

first: Liping

last: Yang

first: Yuyao

last: Guo

first: Bikun

last: Xiao


title: LRMDA suppresses colonic inflammation, colorectal cancer and bacterial infection [Colitis_RNA-seq]

overall_design: RNA-seq of intestinal tissue of WT (n=3) and Lrmda-/- (n=3) DSS-induced colitis mice.


title: p53 maintains healthy aging

summary: The tumor suppressor p53 is a master regulator of cellar stress responses implicated in the aging process. As a tumor suppressor, the antagonistic pleiotropy (AP) theory predicts a beneficial cancer suppression function for p53 early in life that becomes detrimental late in life, for example by promoting accumulation of senescent cells, an inflammatory cell fate driven by tumor suppressors and linked to age-associated disease.  Although p53 signaling is generally activated in aging and disease, p53 is also known to suppress some phenotypes of cellular senescence, and the function of p53 in aged tissues, as well as its causal role in aging, is not well-defined. Here we show, contrary to the prediction of AP, that p53 promotes healthy aging in mice by suppressing inflammation and maintaining metabolic function. Multimodal RNA, protein, epigenetic, and spatial profiling at the single-cell level in aged mouse liver confirmed the expected age-dependent increase of immune infiltration, accumulation of senescent cells, and p53 pathway activation. Knockout of p53 in vivo in hepatocytes of aged, but not young, mice further increased inflammation and immune infiltration. Mechanistically, p53 suppressed DNA replication stress and damage, a precursor to inflammatory signaling, in senescent cells and in aged liver. In aged hepatocytes, p53 directly targeted and maintained expression of metabolic genes, and knockout of p53 sensitized aged mice to metabolic stress-induced liver damage. Conversely, pharmacological activation of p53 with a small molecule MDM2 inhibitor in aged mice reversed age-associated suppression of metabolic genes, decreased age-associated inflammation, delayed onset of frailty, (and extended lifespan) of aged mice. These data suggest that in mice, p53 delays phenotypes of aging, and that this function is targetable, potentially for the benefit of healthy aging in humans.

overall_design: CosMx data from C57BL/6J young and old WT mouse livers treated with HDM or a vehicle control, or untreated Pten-KO, P53-KO, Rosa26-KO young and old mouse livers. This experiment uses a standard 1000-gene panel.


title: Artificial intelligence-assisted prediction of PANoptosis related molecular targets and precise screening of neuroprotective drugs for spinal cord injury

city: Guangzhou

first: Ge

last: Li

state: Guangdong

address: No.106 Zhongshan Rd.2

institute: Guangdong Provincial People's Hospital(Guangdong Academy of Medical Sciences),  Southern Medical University

summary: Abstract: Spinal cord injury (SCI) is a highly disabling central nervous system disease with complex pathology, and targeted neuroprotective drugs remain clinically lacking. However, traditional molecular target screening and drug prediction methods are inefficient, costly, and poorly targeted, failing to meet clinical precision treatment needs. To address this, we introduced artificial intelligence to construct a multi-dimensional data integration framework. First, we established normal, acute- and subacute-phase SCI mouse complete transection models, and RNA-seq combined with single-cell sequencing confirmed acute-phase extensive neuronal PANoptosis. Using WGCNA and MCC algorithms, 25 candidate genes for extensive neuronal PANoptosis in the acute phase were screened out. Then, we comprehensively applied machine learning algorithms including Random Forest, Support Vector Machine, and Elastic Net Model to predict and prioritize potential molecular targets, identifying 13 core genes for extensive neuronal PANoptosis, including Tacc3, Aurka, Mcm6, Mcm5, Ripk1, etc. With the help of the Connectivity Map, drug prediction was performed on these 13 genes, and the 8 candidate drugs with neuroprotective effects were screened out. Through protein domain screening, it was verified via proof-by-contradiction assays that the drug Xaliproden can establish robust interactions with the 7XMK, 7FCZ and 7FD0 domains of Ripk1, a core molecule of the PANoptosome, via a network of multiple hydrogen bonds. This finding provides a novel screening strategy for neuroprotective drugs for spinal cord injury and is of great significance for promoting the establishment of a precision treatment system for the acute phase of injury.

overall_design: RNA sequencing was performed on the spinal cord tissue samples of mice with complete T9 spinal cord transection injury at 0 days, 3 days, and 21 days post-injury.


title: Activated Interferon Signaling Suppresses Age-Dependent Liver Cancer [methylation_all]

type: Methylation profiling by genome tiling array

summary: Age is a major risk factor for liver cancer, as is the case for most adult human cancers. However, the underlying mechanisms are not well defined. A better understanding of the role of aging in liver and other cancers can facilitate approaches for risk assessment, early detection and prevention. We hypothesize that age-driven changes render aged liver more sensitive to oncogenic stress and hence tumorigenesis. To investigate how the liver changes with age, we documented the immune profile, transcriptome and epigenome of healthy livers from both young and aged mice, revealing pronounced alterations with aging. Notably, in aged hepatocytes, we identified heightened interferon (IFN) signaling, as well as simultaneous tumor suppressor and oncogene signaling at both bulk and single cell level, suggestive of an aged liver that is poised for neoplasia. To challenge this seemingly poised state, we employed adeno-associated virus (AAV)-mediated expression of a c-Myc oncogene in young and aged mouse liver hepatocytes in vivo. Analysis of aged hepatocytes expressing c-Myc revealed further elevated expression of IFN Stimulated Genes (ISGs). This ISG upregulation was evident in multiple models of oncogenic stress and transformation in older mice and also observed in aged humans with Metabolic dysfunction-Associated Steatohepatitis (MASH). We determined that Stat1 is both necessary and sufficient for the age specific elevated ISG expression in old wild type mice. Remarkably, inhibiting Jak/Stat signaling alongside ectopic c-Myc expression led to high-grade hepatocyte dysplasia and tumor formation, selectively in aged mice. Together, these results suggest that an aged liver is in a state of “precarious balance”, due to concurrent activation of oncogenic and tumor suppressor pathways, but protected against neoplastic progression by IFN-signaling. Age-dependent activation of IFN signaling has been observed in many tissues and recent studies have demonstrated its detrimental consequences on aging, raising the question as to why IFN-signaling is activated during aging. We propose that aged tissues are intrinsically at higher risk of cancer and age-dependent activation of IFN-signaling is an adaptive process to protect from tumorigenesis, but one that also has maladaptive consequences.

overall_design: Methylation array on young and old WT mouse hepatocytes, performed in two batches.

first: Aaron

last: Havas


title: Activation of TWEAK/Fn14 is required for TLR4-mediated antifungal immunity during Candida albicans intradermal infection

city: nanjing

first: x

last: wang

address: jiefang road 76,nanjing

institute: jinling hospital

summary: Candida albicans (C. albicans) is a common fungal opportunist that colonizes the skin and mucosal surfaces of most healthy individuals which can cause mucocutaneous candidiasis when barrier immunity fails. Keratinocytes act as frontline sentinels by sensing pathogenic organisms and initiate local inflammation via pattern recognition receptors on cellular surface. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) exerts its biological effects primarily via the unique known receptor, fibroblast growth factor-inducible 14 (Fn14), and regulated various physiological processes in keratinocytes. Here, we identify the TWEAK/Fn14 axis as a previously underappreciated epithelial pathway engaged during cutaneous C. albicans infection. We demonstrated that intradermal infection of C. albicans induced significant TWEAK and Fn14 expression in vivo, and fungal exposure rapidly increased Fn14 abundance and recruited Fn14 to fungal–keratinocyte contact sites in HaCaT cells. Conserved fungal cell-wall components including zymosan, laminaran, mannan and chitin induced significant expression of Fn14. Functionally, TWEAK/Fn14 promoted toll-like receptor 4 (TLR4) -associated mitogen-activated protein kinases (MAPK) signaling and amplified keratinocyte production of interleukin-1beta (IL-1β), IL-8, IL-17A and the antimicrobial peptide human beta-Defensin 2 (HBD-2), enhancing leukocyte chemotactic activity. Depletion of Fn14 attenuated TLR4 associated MAPK activation and reduced inflammatory and antimicrobial production. Importantly, intradermal administration of TWEAK improved early control of cutaneous infection by increasing local cytokines expression and reducing fungal burden. Collectively, our findings uncover a TWEAK/Fn14-dependent keratinocyte response axis that promotes host defense against cutaneous C. albicans infection.

overall_design: Examination of gene expression in HaCaT cells after infected with  C.alibicans SC5314 (MOI of 10) for 1h.

first: Xiaoyu

last: Wang

first: Yubing

last: Wu

first: Xiaoke

last: Zang

first: Qing

last: Zhong

first: Mei

last: Lu

first: Yingjun

last: Gao

first: Binyi

last: An

first: Yingying

first: Hong

last: Sang

first: Qingtao

last: Kong


title: Single-cell V(D)J-seq of tumors from Eif3e cg1cre KO mice

city: xiamen

first: Lianghua

last: Lin

state: Fujian

address: Xiang An Nan Lu 4221

institute: 生命科学学院

summary: white nodules collected from tissues with evident tumor development in organs of cKO mice, such as liver, lung, and spleen were predominantly enriched with B cells or CD4+ T cells, and the lymphocytes within these tissues exhibited highly monoclonal enrichment (in one case, oligoclonal enrichment was observed), indicating that lymphocytes in the tumor tissues of cKO mice display characteristics of monoclonal expansion.

overall_design: White nodules were collected from tissues with evident tumor development in cKO mice, such as liver, lung, and spleen covered with white nodules. The nodules were preserved in tissue storage solution and sent to SeekGene for tissue dissociation. After dead cell removal, both single-cell RNA-seq and single-cell V(D)J-seq were performed simultaneously to assess clonal expansion of cells within the tissues.

first: Jiazhen

first: Yunjie

last: Zhang

first: Huilin

last: Song


title: Activated Interferon Signaling Suppresses Age-Dependent Liver Cancer [MERFISH]

first: Kathryn

last: Lande

summary: MERFISH data from two young and two old WT C57BL/6J mouse livers, using a custom 500-gene panel targeted at aging and cancer-associated genes.

overall_design: Age is a major risk factor for liver cancer, as is the case for most adult human cancers. However, the underlying mechanisms are not well defined. A better understanding of the role of aging in liver and other cancers can facilitate approaches for risk assessment, early detection and prevention. We hypothesize that age-driven changes render aged liver more sensitive to oncogenic stress and hence tumorigenesis. To investigate how the liver changes with age, we documented the immune profile, transcriptome and epigenome of healthy livers from both young and aged mice, revealing pronounced alterations with aging. Notably, in aged hepatocytes, we identified heightened interferon (IFN) signaling, as well as simultaneous tumor suppressor and oncogene signaling at both bulk and single cell level, suggestive of an aged liver that is poised for neoplasia. To challenge this seemingly poised state, we employed adeno-associated virus (AAV)-mediated expression of a c-Myc oncogene in young and aged mouse liver hepatocytes in vivo. Analysis of aged hepatocytes expressing c-Myc revealed further elevated expression of IFN Stimulated Genes (ISGs). This ISG upregulation was evident in multiple models of oncogenic stress and transformation in older mice and also observed in aged humans with Metabolic dysfunction-Associated Steatohepatitis (MASH). We determined that Stat1 is both necessary and sufficient for the age specific elevated ISG expression in old wild type mice. Remarkably, inhibiting Jak/Stat signaling alongside ectopic c-Myc expression led to high-grade hepatocyte dysplasia and tumor formation, selectively in aged mice. Together, these results suggest that an aged liver is in a state of “precarious balance”, due to concurrent activation of oncogenic and tumor suppressor pathways, but protected against neoplastic progression by IFN-signaling. Age-dependent activation of IFN signaling has been observed in many tissues and recent studies have demonstrated its detrimental consequences on aging, raising the question as to why IFN-signaling is activated during aging. We propose that aged tissues are intrinsically at higher risk of cancer and age-dependent activation of IFN-signaling is an adaptive process to protect from tumorigenesis, but one that also has maladaptive consequences.

first: Peter

last: Adams


title: Activated Interferon Signaling Suppresses Age-Dependent Liver Cancer [snRNAseq]

overall_design: snRNAseq on whole livers of young and old mice.

first: Aaron


title: Activated Interferon Signaling Suppresses Age-Dependent Liver Cancer [MYC_WL_MethylArray]

overall_design: Methylation array on young and old Myc-AAV or mApple-AAV mouse livers.


title: Activated Interferon Signaling Suppresses Age-Dependent Liver Cancer [CosMx_Stat1KO]

summary: CosMx data from Rosa26KO+mApple and Stat1KO+mApple C57BL/6J aged mouse livers, using a standard 1000-gene panel.


title: Activated Interferon Signaling Suppresses Age-Dependent Liver Cancer [WGBS]

type: Methylation profiling by high throughput sequencing

overall_design: WGBS profiling of hepatocytes isolated from young and old mice.


title: scRNA-seq revealed Ad5-HA-VLP-driven remodeling of the pulmonary immune landscape

city: Wuhan

first: YUAN

last: ZHANG

state: Hubei

address: Shizishan Street, Hongshan District, Wuhan City, Hubei Province

institute: Huazhong Agricultural University

summary: To delineate VLPs-induced remodeling of the pulmonary immune landscape, we performed single-cell RNA sequencing (scRNA-seq) on lung immune cells at 0 and 7 dpi with Ad5-HA-VLP and Ad5-HA

overall_design: The experimental design involved performing single-cell RNA sequencing on CD45+ immune cells isolated from the lungs of C57BL/6 mice at Day 0 (naive baseline) and Day 7 post intranasal immunization with either Ad5-HA or Ad5-HA-VLP

first: Yuan

first: Caiqian

first: Yanhong

last: Zheng

first: Ling

last: Zhao


title: Activated Interferon Signaling Suppresses Age-Dependent Liver Cancer [RNAseq_IFNblockade]

overall_design: RNAseq profiling of whole mouse livers isolated from old mice treated with either IFN-blockade or IgG in old samples for 21-days, each antibody provided 10mg/kg Monday, Wednesday and Friday IP.

last: Havas


title: A Dual Approach to Evaluate the Performance of RNA-Seq Data Analysis Pipelines with Weak Signals

city: Fontenay-aux-Roses

first: Imène

last: GARALI

address: 31 Av. de la Division Leclerc

country: France

institute: ASNR

summary: RNA-Seq data analysis involves several critical steps, from quality control to differential expression analysis, each offering multiple algorithmic options. Selecting the optimal pipeline is particularly challenging in complex scenarios with weak signals.This study highlights the significant impact of pipeline selection on RNA-Seq data analysis, particularly in contexts characterized by weak transcriptional signals, as in this study for low-dose metallic particle exposure. By evaluating 90 bioinformatics pipelines using two complementary approaches — correlation with qRT-PCR data and classification performance — we were able to identify optimal configurations that ensure both analysis robustness for biological relevant observation.

overall_design: The data were obtained from an in vivo experiment conducted on an adult male Sprague-Dawley rat model, which was exposed via inhalation of a particulate aerosol at different doses (low and high) of an environmental chemical pollutant (tungsten) to assess its biological effects on the brain. This project is the subject of an experimental study in our laboratory. The animal ethics committee approved the study protocol, which has been published. More details are available in Exposure to tungsten particles via inhalation triggers early toxicity marker expression in the rat brain. Inhalation Toxicology, 36(4):261–274, 2024. "Leo Mace, Chloe Brizais, Florence Bachelot, Annabelle Manoury , Sébastien Thomé , Celine Gloaguen, Imene Garali Zineddine, Victor Magneron, V. Monceau, Amandine Sache, Frederic Voyer, Christelle Elie, Laurence Roy, Francois Gensdarmes, Dmitry Klokov , Michelle L Block, Chrystelle Ibanez".

first: Chrystelle

last: IBANEZ


title: Activated Interferon Signaling Suppresses Age-Dependent Liver Cancer [RNA-Seq]

overall_design: Young and old mice over-expressing oncogenes (Ccnd1, Myc) or an mApple control, AAV for 30-days.


title: CSF-1R inhibition and lenalidomide synergize to promote myeloma control after autologous stem cell transplantation

city: Seattle

first: Julie

last: Boiko

state: Washington

address: 1101 Fairview Ave N

institute: Fred Hutchinson Cancer Center

summary: Autologous stem cell transplantation (ASCT) with maintenance lenalidomide remains the mainstay of consolidation therapy for eligible multiple myeloma (MM) patients but preventing disease relapse remains a critical unmet need. Here we investigated whether immunosuppressive myeloid populations in bone marrow (BM) correlated with ASCT outcomes. Using a preclinical ASCT model with suboptimal endogenous anti-myeloma activity, we demonstrated that while neither CSF-1R inhibition nor lenalidomide monotherapy significantly improved outcomes, their combination synergistically attenuated disease progression and prolonged survival. Single-cell RNA sequencing revealed that lenalidomide expanded NK-like CD8+ T-cells but paradoxically also increased the frequency of Csf1r+ macrophages. Cell-cell communication analyses identified Csf1r+ macrophages as suppressors of these NK-like and effector-like exhausted (Tphex) CD8 T-cell populations through CD94/NKG2A and PD-L1/PD-1, respectively. CSF-1R blockade depleted these immunosuppressive macrophages, which correlated with decreased expression of inhibitory receptors and enhanced expression of activation markers in Tphex.

overall_design: Vk12653-bearing recipients were transplanted with 10 x 106 BM from naïve donors and 2 x 106 T-cells from concurrent donor cohorts injected with myeloma at the same time as the recipients. Therapies were administered starting 7 days post-transplant. CRBN transgenic mice were used as donors and recipients in lenalidomide-treated groups (50mg/kg for 21 days by daily oral gavage). Anti-CSF-1R (𝛼CSF-1R) or control IgG (IgG) was administered twice weekly at a dose of 400µg per mouse for 4 weeks.

first: Simone

last: Minnie

first: Kenneth

last: Ho

first: Geoffrey

last: Hill


title: High throughput sequencing of T cells from a patient undergoing immune checkpoint inhibitor therapy

city: Seattle,

first: Stephanie

last: Osmond

state: WA

address: 1201 9th Ave

institute: Benaroya Research Institute

summary: This is part of a prospective longitudinal study designed to identify biomarkers predictive of the onset of irAEs associated with ICI therapy and determine whether these biomarkers are further related to treatment efficacy. This experiment was a pilot study of T-cells from three visits of a single donor.

overall_design: Cells were collected from a single donor  across collected across three visits (pre-treatment, 20 days post therapy, and 62 days post therapy) for profiliing using 10X sequencing. Samples from the three visits were hashtagged and pooled and prepared across three 10X channels for a total of three gene expression sequencing libraries, 3 TCR libraries, and 3 feature barcode libraries.

first: Maochang

last: Liu

first: Hannah

last: DeBerg

last: Linsley

first: Erik

last: Wambre


title: High throughput sequencing of T cells from a patient undergoing immune checkpoint inhibitor therapy [P365-5]

summary: This is part of a prospective longitudinal study designed to identify biomarkers predictive of the onset of irAEs associated with ICI therapy and determine whether these biomarkers are further related to treatment efficacy. This experiment was a pilot study of CD8 T-cells from three visits of a single donor.

overall_design: Cells were collected from a single donor  across collected across three visits (pre-treatment, 20 days post therapy, and 62 days post therapy) for profiliing using 10X sequencing. Samples from the three visits were prepared across three 10X channels for a total of three gene expression sequencing libraries, 3 TCR libraries, and 3 feature barcode libraries.

first: Peter


title: Lipid-Associated Macrophage Polarization Drives Chronic Xenograft Fibrosis via APOE-SDC2 Signaling

city: Seoul

first: Jaeseok

last: Yang

address: 50, Yonsei-ro, Seodaemun-gu, Seoul, Republic of Korea

country: South Korea

institute: Yonsei University

summary: Chronic injury of xenografted kidneys, characterized by interstitial fibrosis and tubular atrophy, remains a critical barrier to clinical translation. We employed integrated single-nucleus RNA sequencing, spatial transcriptomics, and plasma proteomics on pig-to-cynomolgus monkey kidney xenografts. We identified lipid-associated macrophage polarization and macrophage-fibroblast crosstalk via APOE-SDC2 signaling as central mechanisms driving fibrosis. Combined immunosuppression suppressed this pathway and prevented injury progression.

sample_organism:
Macaca fascicularis
Sus scrofa

platform_organism:
Macaca fascicularis
Sus scrofa

overall_design: Twelve cynomolgus monkeys underwent kidney xenotransplantation from genetically engineered pigs under five immunosuppressive protocols. Biopsies were collected at multiple timepoints.

first: Gaeun

last: Byeon

first: Minsun

last: Jung

first: Joon-Yong

last: An

first: Kyu-Hyun

last: Han


title: Activated Interferon Signaling Suppresses Age-Dependent Liver Cancer [RNAseq_MycRux]

first: Kathryn

last: Lande

overall_design: Bulk RNAseq on young/old whole liver, myc/mapple, rux/veh mice. Young and old mice were provided AAV8-TBG-mApple or AAV8-TBG-Myc and oral gavage of either vehicle or Ruxolitinib (45mg/kg) oral gavage 2x daily (2-day break over weekend) for 30-days. Sequenced in two batches.

first: Aaron

last: Havas

last: Adams


title: Engineered Senescence-Targeting Apoptotic Extracellular Vesicles-Laden Injectable Hydrogel Microspheres Mitigating Nucleus Pulposus Cell Ferroptosis against Intervertebral Disc Degeneration

first: Zhuo-ran

address: No. 1037 Luoyu Road, Hongshan District

department: Hubei Engineering Research Center for Biomaterials and Medical Protective Materials, School of Chemistry and Chemical Engineering

institute: Huazhong University of Science and Technology (HUST)

summary: As the rapid growth of aging population, intervertebral disc degeneration (IVDD) becomes a prevalent degenerative disorder in clinical practice. Ferroptosis-induced senescence in nucleus pulposus cells (NPCs) represents a primary pathological mechanism underlying IVDD. Strategies for reducing ferroptosis to delay NPC senescence hold prospects for therapeutic advances. According to previously reported proteomic profiles of ApoEVs, we noticed that most key ferroptosis-regulating proteins were identified in the ApoEVs proteome, including a marked upregulation of GPX4. In this study, we developed injectable hydrogel microspheres integrating with senescent NPC-targeting apoptotic extracellular vesicles (ApoEVs) as GPX4 delivery vehicle and anti-inflammatory diclofenac sodium (DFS) for IVDD treatment. The effect of ApoEVs on reducing NPC ferroptosis and reversing NPC senescence-related metabolic dysfunction and damage was reinforced through modification of reactive oxygen species responsive and senescent NPC-targeting peptide. In vivo experiments and single cell RNA sequencing analysis elucidated that the senescence-targeted system significantly inhibited ferroptosis and inflammatory pathways, and preventedthe transformation of inflammatory NPC phenotypes, thereby offering opportunitiesfor rescuing senescent NPCs from ferroptosis and mitigating IVDD progression.

overall_design: Upon completion of the in vivo experiments, intervertebral disc tissues were harvested from both the degenerative group and the B2M-ApoEV/DFS@HMs-treated group, followed by meticulous isolation of NP tissues. The NP specimens were uniformly minced into approximately 0.5 mm³ fragments and subsequently subjected to enzymatic digestion to obtain single-cell suspensions. Cell concentration and viability were quantitatively assessed using a Luna automated cell counter. Single cell RNAsequencing was conducted by Shanghai OE Biotech Co., Ltd. utilizing the BD Rhapsody™ platform. Following the manufacturer’s recommendations, the single cell suspension was adjusted to the appropriate volume for loading and capture using the BD Rhapsody™ Enhanced Cartridge Reagent Kit and BD Rhapsody™ Cartridge Kit. Reverse transcription was performed using the BD Rhapsody™ cDNA kit. BD Rhapsody™ WTA Amplification Kit was used for DNA library construction. High-throughput sequencing was performed with the PE-150 mode.

first: Zhuo-Ran

first: Pengzhi

last: Shi


title: The RNA binding protein LIN28A mediates chromatin dynamics during neuronal differentiation

city: Rome

first: Guido

last: Leoni

address: Via Pontina

department: Bioinformatics

country: Italy

institute: IRBM

summary: The transition of embryonic stem cells (ESCs) from pluripotency to lineage commitment involves complex regulatory mechanisms, including chromatin dynamics and transcriptional/post- transcriptional processes. These mechanisms often interact within intricate networks that require thorough investigation. In this study, we highlight the critical role of the mouse RNA-binding protein LIN28A in neuronal differentiation. LIN28A mediates RNA-dependent interactions with thePolycomb repressive complex 2 (PRC2), leading to the eviction of PRC2 from chromatin andactivation of a neuronal lineage-specific transcriptional program. Proteomic analyses revealed thatthe LIN28A interactome undergoes substantial remodeling during differentiation, corresponding to changes in LIN28A localization. In undifferentiated ESCs, LIN28A primarily resides in the nucleus,interacting with PRC2 components in an RNA-dependent manner, assisting in chromatin dynamics. The absence of LIN28A results in persistent PRC2 association with chromatin, impairing theexpression of genes critical for neuronal differentiation. Chromatin immunoprecipitationsequencing further confirmed that loss of LIN28A results in preferential PRC2 occupancy at thepromoters of differentiation-associated genes. These findings reveal a novel role of LIN28A inepigenetic remodeling, crucial for proper neuronal differentiation of ESCs.

overall_design: Chip-Seq of H3K27ME3 Mouse Embrional Stem Cells (ESC) and serum-free embryoid bodies (sFEBs)  KO/Wt  of LIN28A

first: Silvia

last: Piscitelli

first: Emanuela

last: Cascone

first: Chiara

last: D’Ambrosio

first: Giuseppina

last: Divisato

first: Emilia

last: Giannino

first: Laura

last: De Lisio

first: Daniel

last: D'Andrea

first: Danilo

last: Swann Matassa

last: Lanzuolo

first: Valentina

last: Rosti

first: Maria Chiara

last: Zizolfi

first: Monica

last: Matuozzo

first: Emanuele

last: di Patrizio Soldateschi

first: Paolo

last: Maiuri

first: Andrea

last: Scaloni

first: Fabiana

last: Passaro

last: Parisi


title: In vivo antimicrobial therapeutic efficacy and immune homeostasis analysis of S. aureus peritonitis

city: Xiamen

first: Bin

last: Fang

address: Xiang'an South Road

institute: Xiamen University

summary: Type-I photosensitization addresses the dilemma of intracellular bacterial hypoxia-induced photodynamic therapeutic (PDT) inefficacy, yet precise targeting and eradicating sequestered pathogens remains a formidable challenge. Herein, we engineer an albumin-based photodynamic conversion nanomedicine integrating target-specific recognition with controlled antibacterial functions to precisely eliminate intracellular pathogens and restore infected cell activity. Mechanistic studies reveal a vertically-crossed conformation that promotes intramolecular electron transfer and charge separation, redirecting photosensitizer (PS) reactivity toward type-I reactive oxygen species (ROS) generation. Electron-rich albumin further enhances cationic PS+• recycling, photostability and O2-• generation, while minimizing off-target organelle effects. We demonstrate application in S. aureus-infected sepsis showing high type-I ROS levels for antibacterial function from the nanomedicine to restore redox and immune homeostasis without noticeable toxicity. This work pioneers a strategy to convert ROS generation from type-II to type-I pathways for hypoxia-tolerant bacterial elimination, merging mechanistic precision with translational potential for clinical sepsis management.

overall_design: Female BALB/c mice (6-8 weeks of age, each weighing about 25 g) were inoculated with 100 mL of the S. aureus-infected RAW 264.7 cells (1 × 108 CFU/mL) via subcutaneous injection in the dorsal region.

first: Wenxuan

last: Wang

first: Hua

last: Bai

first: Xi

last: Chen

first: Xuekang

last: Yang

first: Lin

last: Li

first: Wei

last: Huang


title: Cellular and molecular signatures of the vascular microenvironment define the pre-metastatic brain and meninges

city: Melbourne

first: Lucas

last: Tobar

state: VIC

address: 305 Grattan Street

country: Australia

institute: Peter MacCallum Cancer Centre

summary: The contribution of resident brain and meningeal cells in facilitating brain metastasis remains underappreciated. Here, we characterise the pre-metastatic molecular changes that occur in brain and meningeal resident cells in mouse models of breast cancer using single-cell and spatial transcriptomics. Increased inflammatory alterations preceding metastasis were identified in bridging veins spanning the leptomeninges, forming adhesion hotspots by upregulating Icam1 and Vcam1. Furthermore, bridging veins and encapsulating leptomeningeal fibroblasts upregulate cell trafficking ligands, including chemokines. Brain metastasising tumour cells correspondingly upregulate adhesion counter-receptor LFA-1 and chemokine receptors, demonstrating an enhanced capacity to traffic towards altered brain vessels and stroma. Moreover, PD-L1+ immunosuppressive neutrophils infiltrate the dura and brain, coupled with upregulation of checkpoint molecule PD-1 in CD8+ T cells. Neutrophils furthermore upregulate VEGFA, driving angiogenesis and vascular remodelling. These results indicate that breast cancers can drive multiple alterations in the leptomeningeal and dural vasculature that collectively facilitate formation of brain metastases.

overall_design: For single cell RNA-sequencing experiments (chemistry: 10X Genomics Single Cell 5' R2-only), brain and dura captures were sorted into CD45+ and CD45- fractions. Calvarium was not sorted. Each capture contains samples from n=9 mice, that each have been individually barcoded with either TotalSeq-C HTOs or MULTI-seq lipid binding oligos. Brain samples are barcoded with both MULTI-seq and TotalSeq-C, dura and Calvarium are only barcoded with MULTI-seq. Of the n = 9 mice, n=3 mice are non-tumour bearing controls, n = 3 mice are day 17 post-tumour inoculation (4T1Br4 in mammary fat pad), and n = 3 mice are day 23 post-tumour inoculation (4T1Br4 in mammary fat pad).

first: Oliver

last: Cucanic

first: Aadya

last: Nagpal

first: Niko

last: Thio

first: Timothy

last: Semple

first: George

last: Howitt

first: Jovana

last: Maksimovic

first: Raymond

last: Yip

first: Leon

last: Smyth

first: Normand

last: Pouliot

first: Arian

last: Lasocki

first: Alicia

last: Oshlack

first: Rae

last: Farnsworth

first: Steven

last: Stacker


title: Dual Orexin Receptor Antagonism with Lemborexant Enhances Microglial Clearance of β-Amyloid in Mice

city: St. Louis

first: Eric

last: Tycksen

address: 4444 Forest Park Avenue

department: McDonnell Genome Institute

institute: Washington University in St. Louis School of Medicine

summary: Background: Sleep regulates brain amyloid-beta (Aβ) levels, and disruption of sleep is a risk factor for Alzheimer’s Disease (AD) and can exacerbate amyloid pathology. The orexin/hypocretin system, a key sleep-wake regulator, represents an emerging therapeutic target in AD, though the effects of FDA-approved dual orexin receptor antagonist drugs (DORAs) on amyloid pathology are unknown. Here, we assessed and compared the effects of lemborexant (LEM), an FDA-approved DORA, and doxepin, an anti-histaminergic sleep drug, on amyloid pathology and glial responses in the APP/PS1 mouse model of AD. Methods: APP/PS1 mice were treated with lemborexant (10 or 30mg/kg daily) or doxepin (35mg/kg daily) for 3 months, starting just before the onset of amyloid plaque accumulation, or for 1 month after plaque pathology was established. Sleep was measured using a piezoelectric system, and amyloid pathology and glial responses were quantified by immunohistochemistry and confocal microscopy, as well as single cell RNAseq of isolated microglia. Amyloid plaques were labeled with methoxy-X04 to monitor plaque growth, and microglial plaque phagocytosis was assessed by flow cytometry. Microglial elimination was performed using the CSF1r inhibitor PLX3397. Results: Despite equivalent total sleep enhancement, LEM more effectively prevented formation of and slowed growth of both fibrillar and diffuse Aβ plaques. LEM enhanced peri-plaque microglial activation and enhanced microglial amyloid phagocytosis in vivo. Single-cell microglial transcriptomics showed a LEM-mediated shift toward pro-phagocytic and antigen-presentation gene signatures. Depleting microglia via CSF1R inhibition abolished LEM’s anti-amyloid effects, underscoring microglia as essential mediators. Conclusions: These results demonstrate that dual orexin receptor antagonism with lemborexant can augment microglial function to mitigate AD pathology.

overall_design: Microglia from APPswe/PS1δE9 mouse brains were labeled with CD45 and CD11b antibodies and isolated by fluorescence-activated cell sorting (FACS). Microglia from five mice per group (VEH and LEM) were pooled into a single sample per condition and processed for scRNA-seq.

first: Sharma

last: Ashish

first: Segawa

last: Emiko

first: Chen

last: Xiaoying

first: Park

last: Sohui

first: Wang

last: Shoutang

first: Irmen

middle: E

last: Riley

first: Constantino

last: Nicholas

last: Chanung

first: Kanan

middle: F

last: Michael

first: Macauley

middle: L

last: Shannon

first: Colonna

last: Marco

first: Hatanaka

last: Ken

first: Cheng

middle: Y

last: Jocelyn

first: Moline

last: Margaret

first: Musiek

last: Erik


title: Exercise sculpting of the tumor microenvironment and cancer cell phenotypes in breast cancer

city: Burnaby

first: Graeme

middle: J.

last: Koelwyn

state: BC

address: 8888 University Drive

department: Faculty of Health Sciences

country: Canada

institute: Simon Fraser University

summary: Exercise links with improved cancer outcomes following a diagnosis of primary breast cancer but experimental evidence from preclinical studies and molecular mechanistic insights are limited. Here, we show that exercise inhibits breast cancer growth and metastases across multiple syngeneic mouse models compared to sham control. Utilizing the Met1-iRFP model to interrogate tumor microenvironmental (TME) and tumor-specific effects, exercise decreased intratumoral immune cell content, particularly tumor-associated macrophages, and prompted an activated anticancer innate immune response. Cancer cell-intrinsic effects were characterized by broad transcriptomic reprogramming including downregulation of metabolic pathways and upregulation of pathways regulating proliferation and apoptosis. Metabolomic analyses unveiled broad shifts in tumor metabolism including decreased NAD+ and lactate, as well as availability of biosynthetic precursors. Finally, in silico analyses identified TME ligands such as Hmgb2 as drivers of downstream gene expression changes in cancer cells. Collectively, our findings show that exercise suppresses breast cancer progression and does so via broad reprogramming of TME-cancer components and their interaction.

overall_design: RNA-Seq profiling of Met1-iRFP tumor cancer (iRFP+) and tumor microenviroment (TME, iRFP-) cells from exercised and sedentary mice.

first: Sayyed Hamed

last: Shahoei

first: Courtenay

last: Graham

first: Claudia

last: Siguenza

first: Geoffrey

last: Nonis

first: Seyed Milad

last: Vahedi

first: Puneet

last: Sidhu

first: Emily

last: Brown

first: Juan

last: Liu

first: Elisa De

last: Stanchina

first: Kathryn

last: Moore

first: Jason

middle: W

last: Locasale

first: Erik

last: Nelson

first: Lee

last: Jones


title: Gonadotrope Remodeling in Sustained Low Estrogen States: Single-Cell Transcriptomic Analysis Reveals Gonadotrope Subtypes and Activation of Stem Cell Populations.

city: LITTLE ROCK

first: Melanie

last: MacNicol

state: Arkansas

address: 4301 W Markham St

department: Neuroscience

institute: UAMS

summary: The 4-vinylcyclohexene diepoxide (VCD)-induced ovarian failure model provides a physiologically relevant alternative to ovariectomy to gradually reduce serum estradiol (E2) for studying tissue responses to a menopause-like state. This study utilized single-cell sequencing transcriptomics analyses (scRNA-seq) to determine how reduced E2 levels influence pituitary gonadotrope gene expression and cell state, with a focus on potential transcriptomic contributions from stem cells to gonadotrope population growth. VCD-treated mice were acyclic and exhibited 8–15-fold increases in serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) gonadotropin levels. E2 levels were 2X lower than those of normally cycling proestrous females. scRNA-seq identified two gonadotrope clusters based upon distinct gene expression signatures (designated GON1 and GON2). The GON1 population increased in number significantly in VCD-treated mice, expressing markers of progenitor cells. Both gonadotrope clusters showed upregulated genes for canonical pathways supporting mRNA translation and secretion, which correlated well with the high serum gonadotropin levels. However, the LH beta subunit (Lhb) transcript was unchanged and FSH beta subunit (Fshb) was significantly reduced, suggesting that the high gonadotropin secretion may be supported by post-transcriptional regulators. The pituitary Sox2-positive (Sox2) stem cell cluster exhibited increased expression of Sox9, a marker for more mature transitional progenitors, and showed upregulation of pathways involved in stem cell development, mitotic activity, and mRNA translation. This model provides insight into the cellular and molecular adaptations of the gonadotropes and their progenitor cells to the low estrogen/menopausal state, with potential relevance for understanding human perimenopausal and menopausal physiology.

overall_design: Pituitary single cell transcriptome was compared between adult (3-4 mth) female mice treated with 4-vincyclohexene (VCD), which results in a menopausal-like reduction of estradiol, and Control, vehicle-treated (sesame oil) adult female mice.

first: Angela

last: Odle

first: Tiffany

last: Miles

first: Ashley

last: Herdman

first: Stephanie

last: Byrum

first: Alex

last: Lagasse

first: Anessa

last: Haney

first: Angus

first: Gwen

last: Childs


title: The FGF4–integrin β1 axis orchestrates diabetic wound regeneration by restoring directional collective motility

city: Wenzhou

first: Zhiming

last: Zhang

address: Chashan Campus of Wenzhou Medical University

institute: Department of Cardiology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

summary: This dataset was generated to investigate the mechanisms underlying the pro-healing effects of Fibroblast Growth Factor 4 (FGF4) on diabetic wounds. We performed RNA sequencing on wound tissues from streptozotocin (STZ)-induced diabetic mice. The mice received daily topical applications of either recombinant FGF4 (rFGF4) or PBS vehicle control. Wound tissues were harvested on day 7 post-wounding for transcriptomic analysis.

overall_design: On day 7 following skin wounding, transcriptomic analysis via RNA sequencing was carried out on cutaneous tissues from streptozotocin (STZ)-induced diabetic mice that received either PBS (control) or recombinant FGF4 (rFGF4) treatment.

first: Jian

last: Sun

first: Zhifeng

last: Huang

first: Jie

last: Zhou

first: Shuodan

last: Ding


title: HMGB2 Promotes Cardiomyocyte Proliferation and Heart Regeneration Through MTA2-driven Metabolic Reprogramming

city: Nanjing

first: LIUHUA

last: ZHOU

address: 101Longmian Avenue, Jiangning District

institute: Nanjing medical University

summary: Neonatal heart possesses the unique ability to regenerate post-injury. Underlying related mechanisms and reactivation of this process are crucial for regeneration medicine. Using quantitative proteomics with tandem mass tag labeling, RNA-sequencing (RNA-seq) and single-nucleus RNA-seq dataset analyses, high mobility group box 2 (HMGB2) was identified as a key regulator of cardiomyocyte proliferation, whose expression declines during postnatal heart development and increases in the high regenerative potential cardiomyocyte populations in hearts post-injury. Cardiomyocyte-specific HMGB2 knockdown curtails cardiomyocyte proliferation and impairs heart regeneration following apical resection (AR) in neonatal mice, while cardiomyocyte-specific HMGB2 overexpression enhances cardiomyocyte proliferation and facilitates cardiac regeneration and repair in adult mice post-myocardial infarction (MI). Mechanistically, RNA-seq analysis revealed that HMGB2 promotes cardiomyocyte proliferation via activating hypoxia inducible factor 1ɑ (HIF-1α)-mediated glycolysis. This study further found HMGB2 can directly interact with metastasis-associated protein 2 (MTA2) and inhibit its ubiquitination degradation to stabilize HIF-1α protein through immunoprecipitation-mass spectrometry (IP-MS) analysis. Finally, activating HIF-1α or MTA2 could also promote cardiomyocyte proliferation and cardiac repair in adult mice following MI. Taken together, these findings highlight HMGB2 plays a crucial role in promoting heart regeneration through regulating glycolysis. Activating the HMGB2-MTA2-HIF-1α axis might serve as potential therapeutic options for regenerative therapies post-myocardial injury.

overall_design: RNA-seq was performed on NMCMs transfected with Ad5-cTNT-HMGB2 or Ad5-cTNT vector.

first: Liuhua


title: Selective reduction of Nav1.7 with divalent small interfering RNA delivers strong and durable analgesia in thermal and pinprick models of acute pain

city: Yes

first: Wimalanathan

last: Kokulapalan

address: 51 SLEEPER ST FL 7

department: Neuroscience Biology

institute: Atalanta Therapeutics

summary: Humans missing the SCN9A gene encoding the Nav1.7 sodium ion channel are wholly insensitive to pain, but inhibitors of Nav1.7 tested clinically have not produced strong analgesia. Here we investigated whether pharmacological challenges of selectivity, CNS distribution, and target engagement might be addressed by transcript knockdown with divalent small interfering RNA (di-siRNA), a modality with efficient distribution in the brain following dosing into cerebrospinal fluid. SCN9A_2544 di-siRNA sequence catalyzing SCN9A transcript cleavage was identified by in-vitro screening. Following intrathecal dosing in rats, di-siRNA was internalized into over 90% of neuronal cell bodies within the dorsal root ganglion, knocked down cytoplasmic transcript with no detectable off-target knockdown, and reduced Nav1.7 protein up to 75% in DRG and up to 85% in spinal cord. Results show that SCN9A_2544 di-siRNA reduces Nav1.7 function in vivo selectively and to a level that produces powerful analgesia.

overall_design: We investigated the specificity of siRNA SCN9A_2544 using RNA-seq that compared the SCN9A_2544 treated rats to PBS treated rats.

first: Corrie

last: Gallant-Behm

first: Kokulapalan

last: Wimalanathan

first: Stefan

middle: I

last: McDonough


title: Analysis by RNA-seq of the transcriptional profile of left ventricles of WT, CST-KO and CST-KO+CST treated mice

city: La Jolla

first: Debashis

last: Sahoo

address: 9500 Gillman Drive

department: Pediatrics

institute: UCSD

summary: Hypertension is a critical risk factor for heart failure (HF), with metabolic inflexibility playing a pivotal role. Chromogranin A (CgA)-derived peptide Catestatin (CST: hCgA352-372) exerts hypotensive and cardioprotective effects, nonetheless CST’s effect on cardiac metabolism remains unexplored. Our transcriptomic approach coupled with filtering through Boolean implication relationships identified a set of gene signatures (fibroblast, cardiomyocyte, and macrophage) that were altered in CST knockout (CST-KO) mice and restored after CST supplementation. These gene signatures obtained from our in vivo experiments corroborated with publicly available HF patient datasets.

overall_design: Total RNA was isolated from left ventricle of C57BL6 WT (4), CST-KO (4) and CST-KO+CST mice (4).

first: Sushil

middle: K

last: Mahata


title: RNA-seq of PRDX6 Knockdown and Control A223 Squamous Cell Carcinoma Cells Treated With HDAC Inhibitor Largazole

city: Boulder

first: Zeyu

last: Liu

address: 3415 Colorado Ave

institute: University of Colorado Boulder

summary: This dataset comprises RNA sequencing data generated from A223 squamous cell carcinoma cells with stable knockdown of PRDX6 or parental control, treated with either DMSO or 100 nM largazole (a class I histone deacetylase inhibitor) for 18 hours. The study investigates the role of PRDX6 in modulating ferroptosis susceptibility and inflammatory responses upon HDAC inhibition. Transcriptomic profiling was performed to evaluate pathway enrichment and gene expression signatures associated with ferroptosis, redox homeostasis, and immune activation. Results demonstrate that PRDX6 knockdown enhances largazole-induced ferroptotic and inflammatory transcriptional programs, implicating PRDX6 as a redox rheostat suppressing immunogenic stress responses in squamous carcinoma.

overall_design: RNA-seq profiling of wild-type and PRDX6 knockdown A223 cells at 0 hour and 18 hours of 100 nM largazole treatment.


title: Cellular and molecular signatures of the vascular microenvironment define the pre-metastatic brain and meninges [MERFISH]

first: Lucas

last: Tobar

overall_design: For MERFISH experiments, a total of 3 slides were imaged on the Vizgen MERSCOPE (v1 chemistry) using a custom 500 gene-panel with cell boundary stains. Two slides were run for PFA-fixed dura whole-mount samples, with each slide containing a representative control (non-tumour bearing) sample, and either a day 17 or day 23 post-tumour inoculation (4T1Br4) mouse sample. One slide was used for FFPE brain sample, with a representative coronal section of one hemisphere of the brain for each of control (non-tumour bearing) mouse, day17 and day 23 4T1Br4 bearing mice, and day 23 TBCP-1 bearing mouse.

first: Oliver

last: Cucanic

first: Aadya

last: Nagpal

first: Niko

last: Thio

first: Timothy

last: Semple

first: George

last: Howitt

first: Jovana

last: Maksimovic

first: Raymond

last: Yip

first: Leon

last: Smyth

first: Normand

last: Pouliot

first: Arian

last: Lasocki

first: Alicia

last: Oshlack

first: Rae

last: Farnsworth

first: Steven

last: Stacker


title: Effect of mitochondrial fusion and fission on gene expresssion in early Drosophila embryos

city: Pune

first: Richa

last: Rikhy

state: Maharashtra

address: IISER, Dr. Homi Bhabha Road, Pashan

department: Biology

country: India

institute: Indian Institute of Science Education and Research (IISER) Pune, India

summary: Mitochondria are fragmented and dispersed in early blastoderm Drosophila embryos. Expression of mutant of mitochondrial fission protein, Drp1 (Drp1SG), leads to larger mitochondria with aberrant distribution. Increasing ROS genetically by knocking down SOD2 in Drp1SG embryos (Drp1SG;SOD2i) rescues the defects of mitochondrial distribution.

overall_design: Differential gene expression analysis of cellularizing and gastrulating (ventral furrow formation stage) control, Drp1SG, SOD2i and Drp1SG;SOD2i Drosophila embryos (3 biological replicates each)

first: Somya

last: Madan

first: Sakshi

last: Phalke


title: Snhg18 promotes hypoxic pulmonary hypertension by enhancing glycolysis

first: Ruifeng

last: Zhang

address: 87 Dingjiaqiao

institute: Zhongda Hospital of Southeast University

summary: Pulmonary hypertension (PH) is a life-threatening vascular disorder characterized by progressive pulmonary vascular remodeling. In this study, we aim to clarify the lncRNA small nucleolar RNA host gene 18 (Snhg18) implicated in pulmonary vascular remodeling and investigate its underlying mechanisms. Snhg18 is upregulated in pulmonary artery smooth muscle cells (PASMCs) and lung tissues under hypoxic conditions. Inhibition of Snhg18 attenuates cell proliferation in vitro and in vivo. Snhg18 interacts with the m6A “reader” protein heterogeneous nuclear ribonucleoprotein A2/B1 (Hnrnpa2b1), thus increasing enolase 3 (Eno3) mRNA stability in an m6A-dependent manner. The elevation of Eno3 augments glycolysis of PASMCs, thus promoting cell proliferation and vascular remodeling. Our study demonstrates the important role of Snhg18/Hnrnpa2b1/Eno3 axis in pulmonary vascular remodeling, and provide a potential novel therapeutic target for PH.

overall_design: RNA-seq analysis was performed after knockdown of Snhg18 in PASMCs.

first: Tianyu

last: Qu

first: Kai

last: Ma

first: Qiang

last: Du

first: Yingying

last: Liu

first: Chun

first: Ziling

last: Shen

first: Enze

last: Wang

first: Zhixuan

last: Chen

first: Pingsheng


title: Transcriptome sequencing of hepatocytes (with or without GFP) during liver development in zebrafish

city: chengdu

first: Zhilin

last: Deng

state: sichuan

address: sichuan chengdu chengdu medical college

department: School of Basic Medical Sciences

institute: Chengdu medical college

summary: This study presents a comprehensive transcriptomic analysis of hepatocytes and non-hepatocytes during zebrafish liver development at 3 days post-fertilization (dpf), 7 dpf, and adult stages.  Utilizing GFP-labeled hepatocytes from Tg(fabp10:GFP) zebrafish, cells were isolated via flow cytometry, followed by RNA sequencing to profile gene expression dynamics.  High-quality sequencing data (Q30 > 90%) revealed distinct transcriptional patterns, with hepatocytes at 3 dpf exhibiting significant differences compared to 7 dpf and adult stages.  Differential expression analysis identified over 10,000 genes per comparison, highlighting key regulators of liver growth.  Functional enrichment analyses (GO and KEGG) underscored the importance of ATP metabolism, DNA replication, and cell cycle pathways, particularly in early developmental stages.  Notably, genes such as TFDP1, SKP2, and CCNB1 were consistently differentially expressed across stages, suggesting their pivotal roles in hepatocyte proliferation and liver maturation.  These findings provide a foundational resource for understanding the molecular mechanisms driving liver development and regeneration, with potential implications for liver disease research and therapeutic strategies.

overall_design: The hepatocytes and non-hepatocytes within the livers of 3dpf, 7 dpf, and adult transgenic Tg(fabp10:GFP) zebrafish were utilized for experimentation. Upon cessation of opercular movements, animals were euthanized via immersion in ice-cold water for ten minutes. In this transgenic line, hepatocytes in the liver express GFP. Subsequently, liver tissues were subjected to enzymatic digestion using 1 mg Collagenase IV dissolved in 1 ml L-15 medium, incubated at room temperature for 20 minutes. Liver tissues were then transferred to Leibovitz L-15 medium with an osmolarity of 300 mOsm and pH adjusted to 7.35. Hepatocytes and non- hepatocytes were isolated through gentle trituration. The chamber was mounted onto the stage of an Olympus inverted microscope equipped with fluorescence capabilities, perfused with fresh L-15 medium for 5 minutes to rinse off debris. Calibrate flow cytometer (Moflo XDP, Beckman), ensuring that the instrument settings (such as voltage thresholds and compensation matrices) are properly configured for GFP detection. Use beads calibrated for green fluorescent protein to adjust the photomultiplier tube voltages and other parameters. Prepare sterile sorting tubes filled with a suitable buffer to collect sorted cells. Pre-chill if necessary. Use an argon laser typically at 488 nm for GFP excitation. Set appropriate filters to detect GFP emission, usually centred around 530 nm. Discriminate intact cells from cell fragments based on size and internal complexity. Balance purity and yield by adjusting sort speed and stringency of gating parameters. Follow strict cleaning protocols after sorting to avoid contamination. Isolated cells were collected utilizing flow cytometry, enabling precise sorting of GFP-positive hepatocytes from GFP-negative non-hepatocytes. Approximately 400 hepatocytes and non-hepatocytes were harvested from around 12 zebrafish, with 3 replicates per sample. The careful isolation procedures and subsequent sorting by flow cytometry facilitated acquisition of pure populations of hepatocytes and non-hepatocytes, ensuring gene expression profiles and reliable downstream analyses of cellular composition.

first: Long

first: Sizhou

last: Huang


title: Transcriptomic profiling of human CD34⁺/CD45RA-/CD90+ HSCs folowing lncRNA CRISPR/Cas9 editing and cytosine base editing [bulk_RNAseq]

city: Boston

first: Peng

last: Lyu

address: 1 Blackfan Street

institute: Boston Children's Hospital

summary: The HOXA gene locus coordinates body patterning, hematopoiesis, and differentiation. While studying blood phenotype-associated variation within the HOXA locus, we identified a genetic variant, rs17437411, associated with globally reduced blood counts, protection from blood cancers, and variation in anthropometric phenotypes. We find that this variant disrupts the activity of a previously unstudied antisense long non-coding RNA (lncRNA) located between HOXA7 and HOXA9, which we have named HOTSCRAMBL. The HOTSCRAMBL variant disrupts lncRNA function and reduces human hematopoietic stem cell (HSC) self-renewal. Mechanistically, HOTSCRAMBL enables appropriate expression and splicing of HOXA genes in HSCs, most notably HOXA9, in an SRSF2-dependent manner. Given the critical role of HOXA gene expression in some blood cancers, we also demonstrate that HOTSCRAMBL variation or deletion compromises HOXA-dependent acute myeloid leukemias. Collectively, we show how insights from human genetic variation can uncover critical regulatory processes required for effective developmental gene expression.

overall_design: RNA profiling of human peripherial blood derived CD34⁺CD45RA-CD90+ HSCs under long non-coding RNA HOTSCRAMBL knockout and CBE condition. AAVS1 KO and CBE as CRISPR/Cas9 and cytosine base editing controls, respectively.

first: Vijay

middle: G

last: Sankaran


title: Population cohort-validated PM2.5-induced gene signatures: A Machine Learning Approach to Individual Exposure Prediction

city: Hualien

first: Hung-Jung

address: 701 Zhongyang Rd., Sec. 3

department: Institute of Medical Sciences

country: Taiwan

institute: Tzu Chi University

summary: PM2.5, a prominent air pollutant, has been extensively studied through gene expression profiling, demonstrating its activity to modulate gene expressions linked to adverse health outcomes and diseases. Despite the studies, population-based cohort investigations exploring the relationship between PM2.5 exposure and specific gene sets remain limited. In this study, we conducted an unbiased transcriptomic profiling examined gene expression in a PM2.5-exposed mouse model to identify PM2.5-responsive genes. The genes were validated in human cell lines and population-based cohort investigation. Two cohorts comprising healthy older adults aged 65 and above, residing in the regions with contrasting PM2.5 levels, were used to assess the expression of the genes. These gene expression profiles were further used to develop predictive models for PM2.5 exposure by employing logistic regression and decision tree algorithms. Our results showed that five genes (FAM102B, PPP2R1B, OXR1, ITGAM, and PRP38B) increased with PM2.5 exposure in both cell-based assays and population-based cohort studies. The predictive models exhibited high accuracy in assigning high-low PM2.5 exposure, enabling the integration of gene biomarkers into public health practices. This approach may transform air pollution management, shifting from reliance solely on environmental monitoring to incorporating biological and genetic monitoring that reflects individual-level health risks.

overall_design: RNA-seq profiling of the PM2.5-exposed mice (buffy coats) and the controls for 24 weeks.

first: Wen-Chi

last: Cheng

first: Pinpin

last: Lin


title: Clonal memory of cell division diverges between human healthy haematopoiesis and acute myeloid leukaemia

city: Paris

first: Alessandro

last: Donada

address: 11 rue Pierre et Marie Curie, Batiment Curie, RDC, R12, UMR168 - Equipe Perie

institute: Institut Curie

summary: Clonal memory, a cellular property inherited across at least two divisions, has emerged as a key driver of cell heterogeneity.  To uncover its roles in human haematopoiesis, we developed high-resolution ex vivo tools to track both division and fate commitment of individual primary human haematopoietic stem and progenitor cells (HSPCs). We show that human HSPCs display a clonal memory of division, as cells descending from the same ancestor cell divide synchronously over multiple generations. In parallel, HSPCs inherit a clonal memory of fate commitment, independently of lineage identity. Both forms of clonal memory persist over at least two divisions, across different HSPC commitment stages and cell culture conditions. In contrast, malignant haematopoiesis exhibits lower synchronicity, revealing a disruption of clonal memory in leukemic cells. Epigenetic remodelling using a bromodomain inhibitor partially restore the clonal memory in division in leukemic HSPCs, highlighting the plasticity of this trait and its potential for therapeutic modulation. Our findings position clonal memory as a key regulator of human haematopoietic stem cell behaviour. Demonstrating that clonal memory can be modulated opens new avenues for tuning cell heterogeneity in healthy and pathological tissues.

overall_design: Following cell culture, haematopoietic stem and progenitor cells (HSPCs) derived from human cord blood have been isolated through low intensity, image-activated cell sorting or underwent CITE-seq sequencing


title: Chromatin Isolation by RNA purification (ChIRP)-followed RNA sequencing of human leukemia cell lines, MOLMO13 and OCI-AML4, folowing lncRNA CRISPR/Cas9 editing and cytosine base editing

overall_design: RNA profiling of human leukemia cell lines, MOLMO13 and OCI-AML4 under long non-coding RNA HOTSCRAMBL knockout and cytosine base edited conditons. AAVS1 KO and CBE serve as CRISPR/Cas9 and cytosine base editing controls, respectively.


title: Histone lactylation enhances m6A-mediated PDK4 stabilization to drive osteogenic differentiation in calcific aortic valve disease

first: Song

state: Jiangsu province

address: Guangzhou Road

institute: The First Affiliated Hospital of Nanjing Medical University

summary: Calcific aortic valvular disease (CAVD) is characterized by progressive thickening and calcification of the valvular leaflets. Emerging evidence suggests that glycolysis, particularly regulated by pyruvate dehydrogenase kinase 4 (PDK4), plays a significant role in calcification-related diseases. Nevertheless, the mechanisms by which PDK4 affects glycolysis and influences CAVD remain unexplored. This study investigated the contribution of PDK4 to glycolytic reprogramming and osteogenic differentiation in CAVD. Osteogenic differentiation of human valvular interstitial cells (VICs) and CAVD valves was associated with enhanced glycolysis, leading to increased lactate production that further promoted VICs' calcification. PDK4 expression was significantly upregulated in both osteogenically differentiated VICs and CAVD valves. Silencing PDK4 reduced osteogenic differentiation in VICs, whereas PDK4 overexpression aggravated differentiation and increased glycolytic activity. Mechanistically, PDK4 facilitated nuclear translocation of Yes-associated protein (YAP), a key regulator of the Hippo signaling pathway, thereby enhancing RUNX2 transcription and promoting osteogenic differentiation. Nuclear accumulation of lactate increased H3K18 lactylation, which stabilized PDK4 mRNA through the METTL3-m6A-YTHDF1 pathway, establishing a positive feedback loop. This study identifies mechanisms by which glycolysis drives CAVD progression and highlights PDK4 as a potential molecular target for therapeutic intervention.

overall_design: RNA-seq profiling of human aortic valves.Calcified valves were collected from patients undergoing surgical replacement for clinically diagnosed CAVD, and non-calcified control valves were obtained from age-matched individuals undergoing Bentall procedures for acute aortic dissection.

first: Ying

first: Xiufan

last: Xu

first: Yuqiu

last: He

first: Yefan

last: Jiang

first: Yongfeng

last: Shao


title: Single-Nucleus RNA Sequencing Reveals Molecular Determinants of Functional and Physiological Differences Between Extraocular and Quadriceps Muscles in Macaca fascicularis

city: Shanghai

first: Peipei

state: Shanghai

address: No. 115 Jinzun Road, Pudong New Area, Shanghai

institute: Shanghai Jiao Tong University

summary:
Purpose: Extraocular muscles (EOMs) display unique physiological and pathological features distinct from limb skeletal muscles. To elucidate the molecular and cellular basis of these differences, we performed a comparative single-nucleus transcriptomic analysis of macaque EOM and quadriceps femoris muscle (QFM).
Methods: EOM and QFM tissues from Macaca fascicularis were subjected to single-nucleus RNA sequencing (snRNA-seq). Unsupervised clustering, cell-type annotation, differential gene expression, pathway enrichment, and intercellular communication analyses were performed to compare cellular composition, gene expression, and signaling patterns between the two muscles.
Results: We constructed a high-resolution single-nucleus transcriptomic atlas of EOM, identifying major cell types including multiple myofiber subtypes, fibro-adipogenic progenitors (FAPs), Schwann cells, astrocytes, and immune cells. Compared to QFM, EOM exhibited a higher proportion of fast-twitch fibers (2A/2X), FAPs, and neural-associated cells. EOM myonuclei showed enriched expression of genes related to synaptic transmission and axon development, while QFM fibers expressed genes involved in oxidative phosphorylation and glycolysis. EOM uniquely expressed diverse myosin isoforms, including MYH13 and MYH4, supporting its specialized contractile properties. Disease-associated gene profiling revealed that EOM had lower expression of limb muscle disease genes but was susceptible to mitochondrial dysfunction. Cell-cell communication analysis showed that EOM relied more on neuromuscular and regenerative signaling (e.g., WNT, nWNT), while QFM favored immune and metabolic pathways.
Conclusions: Our study reveals the unique cellular composition, gene expression patterns, and intercellular signaling features of EOM compared to QFM. These findings provide a comprehensive molecular framework for understanding the specialized function and disease susceptibility of EOM.

overall_design: Total RNA was extracted from EOM and QFM tissues of macaca fascicularis, with RNA quality assessed using the RNA Nano 6000 Assay Kit and Agilent 2100 Bioanalyzer. For scRNA-seq, mRNA was isolated via poly-T oligo magnetic beads, fragmented, and first-strand cDNA synthesized. For snRNA-seq, nuclei were isolated, and RNA extraction followed similar procedures. cDNA preparations underwent end repair, adaptor ligation, and size selection (~370–420 bp) using AMPure XP beads, followed by PCR amplification. Libraries were assessed for quality using the Qubit 2.0 Fluorometer and Bioanalyzer 2100. Sequencing was performed on the Illumina NovaSeq 6000 platform with 150 bp paired-end reads.

first: Moxin

first: Qian

last: Bian

first: Lin

last: Li


title: Cepharanthine Triggers Immunogenic Cell Death in Solid Tumors by Suppressing PKCζ/NF-κB-Driven PARP1 Expression and Synergizes with Immunotherapy

city: shanghai

first: Chen

last: kaiwen

address: shanghai jiaotong university

institute: shanghai jiaotong university

summary: Induction of immunogenic cell death (ICD) represents a promising strategy to suppress tumor growth and enhance antitumor immunity, highlighting the urgent need to develop more safe and effective ICD inducers. Through screening of a panel of natural alkaloids, we identified cepharanthine (CEP) as a potent ICD inducer in solid tumors. CEP treatment promotes damage-associated molecular pattern (DAMP) release, enhances antigen-presenting cell phagocytosis, and subsequently activates T cell-mediated immunity. CEP-induced ICD is featured by intracellular reactive oxygen species (ROS) accumulation, DNA damage, and endoplasmic reticulum (ER) stress. Mechanistically, CEP directly binds to protein kinase C zeta (PKCζ), and inhibits the NF-κB signaling pathway, leading to attenuated nuclear translocation of p65 and subsequent poly ADP-ribose polymerase 1 (PARP1) transcription. The ICD-inducing effect of CEP depends on PARP1 downregulation. Furthermore, CEP significantly enhances the therapeutic efficacy of PD-1 antibody and OX40 agonist antibody in syngeneic mouse tumor models. Hematological and biochemical analyses confirm the favorable biosafety profile of CEP. These findings demonstrate the clinical translatable potential of CEP as a novel ICD inducer for cancer 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: Kaiwen


title: Single-cell RNA-sequencing profiles reveal the developmental landscape of hawthorn leaves.

city: Kunming

first: Guigang

last: Zhao

state: Yunnan

address: #21 Qingsong Road

institute: Kunming Institute of Zoology

summary: Hawthorn (Crataegus spp.) is a valuable genus of medicinal and edible plants in the Rosaceae family that are rich in bioactive compounds. We applied complementary protoplast‑based single‑cell RNA sequencing (scRNA‑seq) and nucleus‑based snRNA‑seq to construct the first single‑cell atlas of hawthorn leaves. We optimized isolation protocols for two Crataegus species (C. pinnatifida var. major and C. scabrifolia), generated four single‑cell transcriptomic libraries across platforms, and profiled 32,292 high‑quality cells to explore the developmental landscape of hawthorn leaves. The cells were clustered into sixteen groups that we annotated into nine major leaf cell types (mesophyll, pavement, guard, xylem, metaxylem, phloem parenchyma, companion, sieve element, and meristematic cells), revealing extensive cellular heterogeneity and candidate marker genes. Pseudotime reconstruction revealed branched developmental trajectories, and cell‑type‑resolved profiling revealed the cell‑specific expression of flavonoid biosynthetic genes. This single‑cell atlas lays a foundation for mechanistic investigations of tissue‑specific metabolite biosynthesis and offers a cellular framework to guide future functional studies and trait improvement breeding in hawthorn.

sample_organism:
Crataegus pinnatifida
Crataegus scabrifolia

overall_design: The collected leaf material was cut into approximately 1 mm fragments and placed in an ice-cold dissociation pretreatment buffer (20 mM KCl, 10 mM CaCl₂, 0.1% bovine serum albumin, 20 mM MES, 0.6 M mannitol).

first: GuiGang

first: Xien

last: Wu

first: Baozheng

last: Wang

first: Xiaolu

first: Junjuan

last: Zheng

first: Zhaoli

last: Ding

first: Guodong

first: Ticao

last: Zhang


title: RNA-seq and Ribo-seq analysis of Arabidopsis thaliana immune response

city: Beijing

first: Qiangsheng

last: Yu

address: Tsinghua university

department: School of life sciences

institute: Tsinghua university

summary: In land plants, guard cells sense pathogens and close stomata to resist their entry. Pathogen-infected local leaves transmit the danger status to uninfected distal systemic leaves and trigger their stomatal closure as a global defense termed systemic stomatal immunity. However, the corresponding mobile signals remain unknown. Here, we report that an upstream open reading frame (uORF) encodes a long-distance mobile peptide inducing systemic stomatal closure and named uORF-encoded systemic stomatal immune conductor (USIC). In local leaves, USIC expression is upregulated upon pathogen/pattern signals. In systemic leaves, the SIRK1 receptor and KIN7 coreceptor perceives USIC, and induces KIN7 cleavage by MC4. KIN7 translocates to tonoplasts and mediates stomatal closure by associating with H+-ATPases and aquaporins. This study reveals a systemic signaling mechanism whereby an uORF-encoded mobile peptide and its receptor pathway activating systemic stomatal immunity.

overall_design: In this study, transgenic Dex::AvrRps4 plants, in which the Pseudomonas syringae effector AvrRps4 was controlled by a dexamethasone (Dex)-inducible system, was generated. On liquid-cultured Arabidopsis seedlings, pattern-triggered immunity (PTI) was induced by flg22 application (1 μM), and effector-triggered immunity (ETI) was induced by Dex application (10 μM). To screen for ORFs that potentially encode the immune-responsive mobile peptides, we collected 4-day-old Col-0 wild-type seedlings under basal condition, and under immune activation of both PTI and ETI for RNA-seq and Ribo-seq analysis. The functions of SIRK1 and KIN7 were also validated by RNA-seq analysis of Col-0 and the SIRK1/KIN7 knockout strains upon mock and USIC treatment.

first: Changzhen

last: Liu


title: ChAHP Silences SINE Retrotransposons by Inhibiting TFIIIB Recruitment [ChIP-seq]

city: Basel

first: Fabio

last: Mohn

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

summary: Short interspersed nuclear elements (SINEs) are abundant non-autonomous transposable elements (TEs) derived from RNA polymerase III (Pol III)-transcribed short non-coding RNAs. SINEs retain sequence features recognized by the Pol III machinery and constitute a substantial portion of vertebrate genomes. Despite their impact on genome stability and evolution, the mechanisms governing SINE transcription remain poorly understood. Although DNA methylation and heterochromatin formation have been implicated in their repression, we find these pathways play only a minor role in mouse embryonic stem cells. Instead, we identify the ChAHP complex as a key repressor of SINE B2 elements. ChAHP directly inhibits Pol III transcription by blocking TFIIIB recruitment without affecting TFIIIC binding. This selective interference prevents transcription initiation and highlights a distinct regulatory mechanism. Our findings establish ChAHP as a non-canonical repressor of Pol III-dependent SINE transcription, offering new insights into the control of this pervasive class of non-coding genomic elements.

overall_design: Analysis of RNA Polymerase III transcribed SINEs in mouse embryonic stem cells (mESC) in  wild-type and under accute ADNP- and GTF3C1-depletion conditions. ChIP-seq for RNA Pol III, TFIIIB, TFIIIC, TFIIIA and ADNP were in general performed in biologically indepdenden duplicates. For ADNP ChIP in wt conditions, we included 6 replicates. All ChIPs except for Pol III were performed using endogenously tagged proteins.

first: Jakob

last: Schnabl

first: Michaela

last: Schwaiger

first: Marc

last: Bühler