title: Generation and Characterization of Lung Cancer PDX Models from Human Specimens Across Serial Passages

city: Taipei

first: Bing-Ying

last: Ho

address: Rm. 102, No.81, Changxing St.

department: Molecular Imaging Center

institute: National Taiwan University

summary: This study aims to investigate the regulatory influence of the host microenvironment on tumor growth during the establishment of patient-derived xenograft (PDX) models of human lung cancer in athymic nude mice. By serially passaging tumor tissues from primary clinical specimens through generations P0 to P3, we examined how the immunodeficient but NK cell-competent microenvironment of athymic nude mice affects tumor adaptation and progression. Whole transcriptome (RNA-seq) profiling was used to identify changes in gene expression related to immune signaling and stromal interaction across passages. This project provides important insights into the role of the murine host microenvironment, particularly innate immune responses, in shaping the development and evolution of human lung cancer in PDX models, and supports future translational studies using PDX systems as clinically relevant tools for cancer research.

overall_design: Fresh human lung tumor specimens were cut into 3-5 mm3 fragments, coated in Matrigel, and implanted subcutaneously into the lower backs of 6-8 week old athymic nude mice. Tumors were harvested when they reached approximately 2000 mm3. Excised tumors were re-implanted into new mice to establish serial passages from P0 to P3.

middle: Y

first: Jong-Kai

middle: K

last: Hsiao


title: A population of  Regulatory T cells controls nociception through enkephalins to restrain skin inflammation [fixed scRNA-seq]

status: Public on Jul 09 2025

city: La Jolla

first: Stanislav

last: Dikiy

state: CA

address: 10550 North Torrey Pines Road

department: Immunology

country: USA

institute: Scripps Research

type: Expression profiling by high throughput sequencing

summary: The skin integrates diverse signals discerned by sensory neurons and immune cells to elicit adaptive responses to a range of stresses. Considering interactions between nervous and immune systems, we questioned whether regulatory T (Treg ) cells, that suppresses systemic and local inflammation, can modulate activation of peripheral neurons. Acute Treg cell “loss-of-function” increased neuronal activation to noxious stimuli independently from immunosuppressive function. We find that a population of activated skin Treg cells is highly enriched for Penk expression, a precursor for endogenous opioid enkephalins. Punctual selective depletion of Penk-expressing Treg cells, or specific ablation of Penk in Treg cells increases neuronal activation in response to noxious stimuli and associated inflammation. Our study indicates that a population of tissue Treg cells exhibits neuromodulatory activity to restrain local inflammation.

overall_design: Cervical DRGs were isolated from Penkfl/fl;Foxp3creERT2 and littermate control mice treated following 3 day treatment with imiquimod and fixed in 4% PFA. Fixed DRG were dissociated and analyzed by snRNAseq.

first: Juan Sebastian

last: Jara

first: Alejandra

last: Mendoza

first: Regina

last: Bou Puerto

first: Paolo

last: Giovanelli

first: Alexander

last: Rudensky


title: RNA-Seq Time-Course of Rat Bladder Transcriptomes Following Spinal Cord Injury

city: Boston

first: Ali

last: Hashemi Gheinani

address: 300 Longwood

department: Urology

institute: Boston Children's Hospital

summary: Spinal cord injury (SCI) evokes profound dysfunction in hollow organs such as the urinary bladder and gut. Current treatments are limited by a lack of molecular data to inform novel therapeutic avenues. Previously, we showed systemic treatment with the neuroprotective agent inosine improved bladder function following SCI in rats. This RNA-seq dataset captures global transcriptomic changes in the rat bladder at multiple time points following spinal cord injury (SCI). Full-thickness bladder tissues were collected from male Sprague Dawley rats at 2, 8, and 16 weeks post-complete T8 spinal cord transection, alongside non-injured controls.

overall_design: RNA-seq was performed on bladder tissues harvested from male Sprague Dawley rats (6–7 weeks old) subjected to complete T8 spinal cord transection, with matched non-injured controls. At 2, 8, and 16 weeks post-injury, full-thickness bladders were isolated, snap-frozen, and processed for RNA extraction using TRIzol and miRNeasy columns. RNA quality was assessed using NanoDrop, Qubit, and Bioanalyzer systems. Libraries were prepared using the KAPA Total RNA-Seq RiboErase Kit (ribodepletion protocol), and paired-end 51 bp reads were generated using an Illumina HiSeq 2500 platform. Raw read data were converted from BCL to FASTQ using Bcl2fastq. A total of 18 samples were sequenced (n=3 per group), enabling differential gene expression and pathway enrichment analyses across the SCI time course.

first: Bryan

middle: S

last: Sack

first: Alex

last: Bigger-Allen

first: Hatim

last: Thaker

first: Hussein

last: Atta

first: George

last: Lambrinos

first: Kyle

last: Costa

first: Claire

last: Doyle

first: Mehrnaz

last: Gharaee-Kermani

first: Susan

last: Patalano

first: Mary

last: Piper

first: Justin

middle: F

last: Cotellessa

first: Dijana

last: Vitko

first: Haiying

last: Li

first: Manubhai Kadayil

last: Prabhakaran

first: Vivian

last: Cristofaro

first: John

last: Froehlich

first: Richard

last: Lee

first: Wei

last: Yang

first: Maryrose

middle: P

last: Sullivan

first: Jill

middle: A

last: Macoska

first: Rosalyn

middle: M

last: Adam


title: Transcriptomic Remodeling of the Spinal Cord-Injured Bladder and Its Modulation by Inosine Treatment

first: Ali

last: Hashemi Gheinani

summary: Neurogenic detrusor overactivity (NDO) is a frequent complication of spinal cord injury (SCI), often accompanied by profound structural and functional changes in the bladder. While inosine has previously been shown to improve bladder function in SCI models, its molecular effects on the bladder remain largely unexplored. In this study, we performed transcriptomic profiling of bladder tissues from SCI rats treated with inosine or vehicle. Bladder compartments were microdissected into detrusor and mucosa to capture compartment-specific changes. SCI induced widespread transcriptional alterations in both compartments, with inosine treatment partially restoring gene expression patterns toward those observed in non-injured controls. Unsupervised clustering and principal component analyses confirmed distinct transcriptional profiles associated with injury and treatment. Notably, inosine influenced a set of genes consistently altered by SCI, suggesting a protective or regulatory role at the molecular level. These findings reveal a compartment-specific transcriptomic response to spinal cord injury and provide insight into the mechanisms by which inosine may exert its therapeutic effects on the injured bladder.

overall_design: Complete spinal cord transection was performed at the T8 vertebral level in male Sprague Dawley rats (6–7 weeks old, approximately 250 grams; Charles River Laboratories, Wilmington, MA) under isoflurane anesthesia, as previously described. The spinal cord was exposed between T8–T10 using bony and vascular landmarks and transected completely at T8. Following injury, rats received daily intraperitoneal injections of either inosine (225 mg/kg/day) or vehicle control (12 mM sodium bicarbonate buffer, pH 9.2) for 8 weeks. At the end of the treatment period, animals were euthanized, bladders were harvested, weighed, and microdissected into two distinct tissue compartments: detrusor (smooth muscle layer) and mucosa (urothelium and lamina propria). Tissues were flash-frozen in liquid nitrogen and stored at -80°C until RNA extraction and sequencing. RNA-seq was performed to assess transcriptomic changes in response to spinal cord injury and inosine treatment across bladder compartments.

first: Bryan

last: Sack

first: Alex

last: Bigger-Allen


title: Diabetic foot ulcers debridement SCRNA-seq showed healing association with inflammatory fibroblasts in minorities

status: Public on Jul 10 2025

city: Atlanta

first: Manoj

last: Bhasin

state: GA

address: 101 Woodruff Cir

institute: Emory University

summary: In this pilot study, we optimized diabetic foot ulcers (DFUs) single-cell profiling from debrided DFUs of underrepresented African American (AA) patients. The healing-DFUs were significantly enriched (P=0.03) with unique heterogenous Healing-associated Fibroblasts (HE-Fibro), expressing inflammation (CHI3L1), ECM-remodeling (MMP13, ASPN) and wound healing (NRG1, COL7A1) associated genes, validating our previous findings with surgically resected healer-DFUs. HE-Fibro/Fibro cells depicted significantly lower expression of key healing-associated CHIL3L1, and TIMP1 genes in AAs compared to Whites, signifying race-associated heterogeneity. Reparative M2 macrophages were enriched in Healing-DFUs (P=0.05), while inflammatory M1 macrophages displayed distinct transcriptome differences in Nonhealing-DFUs and Healing-DFUs. Cellular communication analysis revealed a pivotal role for HE-Fibro/Fibroblasts (signal-senders) and M1 (signal-receivers). HE-Fibro’s signaling significantly upregulated wound-resolving WNT, and ECM-remodeling CXCL pathways in Healing-DFUs, while M1 signaling upregulated healing-disruptive, and inflammation-associated, SPP1 pathways, in Nonhealing-DFUs. This study justifies using debrided tissues for single-cell assays and highlights the need for in-depth investigations into dysregulated wound healing microenvironments in AAs.

overall_design:
DFU debrided samples were collected from patients with Healing- and Nonhealing DFUs and transported to lab from clinic on ice. Tissue was dissociated into single cells using standardized method and frozen viably. Viably thawed cells were used for preparing single cell RNA sequencing libraries using 3'v3.1 10x genomics kits. The libraries were then sequenced using Novaseq S4 and NextSeq 500 platforms.

>>>Raw data for human samples not available due to patient privacy concerns<<<

first: Dahim

last: Choi


title: RNA sequence analysis of STYK1 knockdown in pancreatic cancer PANC-1 cells

city: WuHan

first: Cefan

last: Zhou

state: HuBei

address: Hubei University of Technology, 28 NanLi Road, Wuhan, Hubei, China

country: China

institute: Hubei University of Technology

summary: Serine/threonine/tyrosine kinase 1 (STYK1), a proto-oncogenic transmembrane receptor alternatively termed NOK which consists of a kinase domain, intracellular domain, and transmembrane domain, was identified as an oncogene with high transformation potential in multiple cancer types. Recently, our group recognized that STYK1 depletion disrupts autophagosome biogenesis, establishing it as a critical modulator of autophagic flux. STYK1 was also reported as an oncogenic amplifier that hijacks KRAS effector networks, specifically the PI3K/AKT and MEK/ERK signaling axes, to drive tumor progression across malignancies. This molecular paradigm gains particular relevance in PDAC, where somatic KRAS mutations dominate (~95% prevalence) and functionally orchestrate pancreatic carcinogenesis from its earliest premalignant stages, notably during pancreatic intraepithelial neoplasia (PanIN) precursor lesions. This prompted a focused investigation into STYK1's functional implications within pancreatic cancer. By using multiple in vitro and in vivo assays, we figured out that higher STYK1 expression is related to poor pancreatic cancer survival and that STYK1 depletion suppresses pancreatic cancer cell development.To determine the molecular mechanism by which STYK1 promotes pancreatic cancer development, we performed RNA sequencing and found that STYK1 is involved in regulating the Wnt/β-Catenin signaling pathway. We also elucidate a novel STYK1-driven mechanism for Wnt/β-catenin activation in APC-independent pancreatic cancer and provide preclinical evidence for targeting STYK1-mediated signaling as a therapeutic strategy.

overall_design: To investigate the molecular mechanism of STYK1 regulation in pancreatic cancer, we established the PANC-1 cell line of shSTYK1 using lentivirus. Total RNA was extracted for RNA sequencing.

first: Xueying

last: Dong

first: Shi


title: Differential gene expression in mouse liver with lamin A/C KO in adipocytes

city: Ann Arbor

first: Bioinformatics

last: Core

state: MI

address: BRCF Bioinformatics Core, 2800 Plymouth Rd

institute: University of Michigan

summary: Lmna mutations can result in lipodystrophic syndromes including fat atrophy (lipoatrophy) and metabolic alterations resulting from insulin resistance (glucose tolerance abnormalities, dyslipidemia, non-alcoholic fatty liver disease).

overall_design: RNA-seq profiling of female mouse liver with Lmna-KO genotype treated with vehicle (6 replicates), Lmna-KO genotype treated with rescue (8 replicates),  Lmna-WT genotype treated with vehicle (7 replicates), Lmna-WT genotype treated with rescue (3 replicates)

first: Ormond

last: MacDougald

first: Jessica

middle: N

last: Maung


title: Examining the transcriptional changes thar result from loss of p53 in mutant p53 A431 cells

city: Durham

first: Patricia

last: Muller

address: Stockton Road

country: United Kingdom

institute: Durham University

summary: A431 mutant p53 cells are able to engulf neighbouring cancer cells. Loss of p53 in these cells impairs  this behaviour. To determine if transcriptional changes could underlie these phenotypic changes we compared A431 cells in which p53 was deleted using CRISPR (p53 KO) to parental cells (P) or cells that were transfected with a CRISP control construct (p53 ctr). Three indivicual clones were derived from single cells and compared to each other.

overall_design: RNA-seq profiling of A431 p53 KO, p53 ctr cells and parental cells grown under normal conditions of DMEM, 10% FBS at 37 degrees and 5% CO2.

first: Mary

last: Patterson

first: Lobsang

last: Dolma

first: Antonio

last: Banyard

first: Callum

last: Hall

first: Steven

last: Bell

first: Wolfgang

last: Breitweiser

first: Sudhakar

last: Sahoo

first: John

last: Weightman

first: Maria

last: Gil

first: Gary

last: Ashton

first: Caron

last: Behan

first: Nicola

last: Fullard

first: Lewis

last: Williams


title: Uncovering common transcriptional features shared by mature PMN-MDSCs and tumor-associated neutrophils in humans

city: Verona

first: Patrizia

last: Scapini

state: VR

address: strada le grazie,8

department: Medicine

country: Italy

institute: University of Verona

summary: In this study, we performed single-cell transcriptomic profiling of mature PMN-MDSCs (mPMN-MDSCs) from non-small cell lung cancer (NSCLC) patients and identified a distinct cell cluster (NSCLC c6) exhibiting immunosuppressive and protumor features. Comparative analysis with publicly available single-cell RNA sequencing (scRNA-seq) datasets of tumor-associated neutrophils (TANs) revealed shared transcriptomic features between the identified NSCLC mPMN-MDSC and TAN clusters. These transcriptomic features included the expression of common genes, activation of the hypoxia signaling pathway, and metabolic reprogramming. Additionally, scRNA-seq analysis of mature immunosuppressive neutrophils from G-CSF-treated donors (GDs) – which are a reliable model of circulating mPMN-MDSCs - demonstrated similar dysregulation of hypoxia and metabolic pathways, further reinforcing the existence of transcriptomic similarities between circulating mPMN-MDSCs and TANs.

overall_design: Total CD66b+ low-density neutrophils (LDNs) and normal-density neutrophils (NDNs) from the peripheral blood of NSCLC patients (n =6) and G-CSF-treated donors (GDs; n=6), as well as total CD66b+ NDNs from the peripheral blood of healthy donors (HDs; n=8), were isolated by magnetic bead selection with fluorescence-conjugated anti-CD66b mAbs and processed with BD Rhapsody Single-Cell Analysis System for scRNA-seq. Publicly available single-cell RNA sequencing (scRNA-seq) datasets of TANs were utilized for comparative analyses.

first: Chiara

last: Lattanzi


title: Impacts of Inorganic Arsenic Exposure on Genetic Stability of Human Mesenchymal Stromal Cells (hMSCs)

city: Ottawa

first: Matthew

last: Meier

state: Ontario

address: 251 Sir Frederick Banting Driveway

department: Health Canada

country: Canada

institute: Government of Canada

summary: Human mesenchymal stem/stromal cells (hMSCs) can differentiate into mesoderm-type cells, making them suitable candidates for tissue repair therapies. However, their relatively low frequency in adult tissue necessitates ex vivo expansion prior to regenerative medicine applications. Expansion can lead to selection of chromosomally abnormal clones and therefore, long-term hMSC stability should be studied. hMSCs applications in regenerative medicine ensures commercial availability of normal karyotype human primary cells for toxicity assessment and hMSCs could serve as alternatives to immortalized human cell models. In this work, we evaluated the potential of hMSCs in toxicity testing using inorganic arsenic (iAs) as a case study. hMSCs were exposed to iAs at different durations to track cellular aging and study long-term genetic stability. iAs exposures (48 hours) resulted in micronuclei induction. hMSCs were also exposed to iAs for 6 days to determine if hMSCs would become more susceptible to chromosomal damage following exposure to the model genotoxicant, mitomycin C (MMC). The culture duration and iAs exposure did not alter MMC potency, indicating that the hMSCs susceptibility to chromosomal damage remained unchanged. We also used gene expression analysis to investigate the molecular impacts of iAs on hMSCs over the course of short (3 days total) and long (30 days total) experiments. Both iAs exposures activated biomarkers associated with oxidative stress, but not biomarkers for direct DNA damage, providing support for an indirect mode of action for iAs genotoxicity. Overall, this study establishes the utility of hMSCs as a new model for toxicity screening and provides mechanistic information underlying iAs toxicity.

overall_design: Gene expression profiles were compared for hMSCs treated for 3 and 30 days with doses of sodium arsenite versus a solvent control (water).

first: Matt

first: Marc

last: Beal

first: Tanvi

last: Sharma

first: Cathy

last: Cummings-Lorbetskie

first: Andrea

last: Rowan-Carroll


title: Splicing regulatory dynamics for precision analysis and treatment of heterogenous leukemias

city: Cincinnati

first: Nathan

last: Salomonis

state: OH

address: 3333 Burnet Avenue

department: Biomedical Informatics

institute: Cincinnati Children's Hospital

summary: The role of splicing dysregulation in cancer is underscored by splicing factor mutations, however, its impact in the absence of such rare mutations remains poorly understood. Prompted by the finding that splicing uniquely resolved genetic subtypes of cancer, we developed an unsupervised computational workflow called OncoSplice to comprehensively define tumor molecular landscapes. In adult and pediatric Acute Myeloid Leukemia (AML), OncoSplice identified the spectrum of driver genetics from splicing profiles alone, defined over a dozen new molecular subtypes recurrent across AML cohorts and discovered a dominant splicing subtype that partially phenocopies U2AF1-mutant splicing. Although pediatric leukemias lack splicing factor mutations, this U2AF1-like subtype similarly spanned pediatric and adult AML genetics and consistently predicted poor prognosis. Using long-read single-cell RNA-Seq, we confirmed discovered U2AF1-like splicing was shared across cell states, co-opts a healthy circadian gene program, was stable through relapse and induced a leukemia stem cell (LSC) program. Pharmacological inhibition of the OncoSplice implicated U2AF1-like splicing regulator, PRMT5, rescued leukemia mis-splicing and inhibited leukemic cell growth. Finally, genetic deletion of IRAK4, a common target of U2AF1-like and PRMT5 treatment, blocked leukemia development in xenograft models and induced differentiation. This work suggests broad splicing dysregulation, in the absence of select mutations, is a therapeutic target in heterogenous leukemias.

overall_design: Bulk paired-end RNA-Seq was performed on the MDS-L cell line with PRMT5-inhibitor (PRT543, Prelude Therapeutics) or DMSO (control) in biological duplicate. Single-cell long-read RNA-Seq using the Kinnex (PacBio) protocol with Revio sequencing for the bone marrow of patients with AML. The AML samples have matching CITE-Seq libraries in GSE245108 (AML-7, AML-12, AML-14) with reverse complemented cell barcodes.

first: Meenakshi

last: Venkatasubramanian

first: Leya

last: Schwartz

first: Nandini

last: Ramachandra

first: H. Leighton

last: Grimes

first: Daniel T.

last: Starczynowski

first: Amit

last: Verma


title: N6-methyladenosine RNA landscape in the aged mouse hearts

city: Dongguan

first: xia

last: jing

state: None Selected

address: Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Institute of Aging Research, Guangdong Medical University, Xin Cheng Avenue 1#, Songshan Lake, Dongguan 523808, P. R. China

institute: Guangdong Medical College - Dongguan Campus

summary: Objective: Cardiac aging is a major risk factor for the development of cardiovascular diseases. Although evidence suggests an association between N6-methyladenosine (m6A) modification and numerous cardiovascular diseases, its role in cardiac aging remains unclear. This study was conducted to elucidate the role of m6A modification in cardiac aging and the molecular mechanisms involved. Methods: Global methylation levels and the expression of major m6A regulators were compared between young and aged hearts. Transcriptome-wide m6A landscape analysis was conducted using methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to identify aberrant m6A peaks. Furthermore, gene set enrichment analysis (GSEA) was performed to identify gene sets associated with cardiac aging. Functional validation of key molecules was carried out through in vitro experiments. Results: The overall m6A level remained constant; however, the expression of the methyltransferase METTL14 and the demethyltransferase FTO were significantly upregulated in aged hearts. Knockdown of METTL14 alleviated H2O2-induced senescence phenotypes, as reflected by a reduction in the number of SA-β-gal positive cells and a decrease in p21 expression. Compared with young hearts, the dysregulated m6A peaks were significantly enriched in genes associated with dilated cardiomyopathy, hypertrophic cardiomyopathy, and the PI3K-Akt signaling pathway. GSEA showed that these genes were enriched in the aging of heart and aorta cardiomyocytes. Additionally, 255 genes with siginificantly changed of both m6A peaks and RNA expression were identified by combining MeRIP-seq and RNA-seq data. Among these genes, EFEMP1 was significantly upregulated in aged hearts, accompanied by enhanced m6A modification. Treatment with the methyltransferase inhibitor cycloleucine significantly suppressed the expression level of EFEMP1. In AC16 cells, silencing EFEMP1 suppressed H2O2-induced cell senescence. Furthermore, we found a positive correlation between METTL14 and EFEMP1 in multiple datasets related to cardiac aging. Conclusion: Our findings indicate that m6A modification plays an essential role in the process of cardiac aging. EFEMP1 may serve as a potential new therapeutic target for age-related cardiac diseases.

overall_design: To explore the role of m6A RNA methylation in cardiac aging, we utilized C57BL/6 mice at young (6–7 weeks) and advanced (26–27 months) ages. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was used to perform genome-wide profiling of methylation modified mRNA and lncRNA in the hearts of young and aged mice. Each of the two groups include three mice.

first: Xia

last: Jing

first: Yingming

last: Li

first: Zhaofu

last: Liao

first: Jing-Yu

last: Wu

first: Zi-Yang

last: Xu

first: Zhi-Peng

last: Yang

first: Hai-Liang

last: Mo

first: Shun

first: Xinguang

last: Liu

first: Zhuguo

first: Jun

last: Tao

first: Xing-Dong

last: Xiong


title: Region-specific roles of stress-activated protein kinase MKK7 in the developing and maturing murine brain

city: Bunkyo-ku

first: Satoshi

last: Kofuji

state: Tokyo

address: 1-5-45 Yushima

country: Japan

institute: Institute of Science Tokyo

summary: The stress-responsive kinase MKK7 controls various brain functions. Neural stem cell-specific deletion of the Mkk7 gene caused abnormal brain development and death immediately after birth (perinatal lethality). However, the region-specific roles of MKK7 during embryonic brain development remain unclear. We have found that the mice with loss of MKK7 in the cerebrum were born at the expected Mendelian ratio but died within five weeks of birth. To investigate its molecular mechanism, we performed RNA-sequencing analysis in the brain.

overall_design: RNA-seq data for the cerebral cortex of Mkk7flox/flox and Mkk7flox/flox Emx1-Cre mice at P1.

first: Hiroshi

last: Nishina


title: Rapid DNA and RNA isolation from few or single cells using low-cost NAxtra magnetic nanoparticles

city: Trondheim

first: Magnar

last: Bjørås

address: Erling Skjalgsons gate 1

country: Norway

institute: NTNU

summary: A novel, cost-effective nucleic acid (NA) isolation method for purifying total NA, DNA, or RNA from both two- and three-dimensional cell cultures has been developed at the Norwegian University of Science and Technology utilizing NAxtra magnetic nanoparticles. This method achieves comparable yields to existing isolation kits while offering significant improvements in cost and processing speed. However, the original protocol was not optimized for small-cell numbers or single-cell applications. Given the growing interest in single-cell and rare-cell population studies, there is a critical need for more sensitive isolation techniques. In this study, we have enhanced the sensitivity of the NAxtra-based isolation method to facilitate mid- to high-throughput purification from as few as 10,000 cells down to single cells. Automated processing using KingFisher systems enables the rapid handling of 96 samples within 12-18 minutes. Our findings indicate that this method not only matches but can exceed the performance of existing alternatives in (RT)-qPCR detection while being significantly more economical and efficient. Additionally, it enables the extraction of high-quality RNA suitable for transcriptomics analyses from limited cell quantities, including single cells, as indicated by comparison with the Evercode Whole Transcritome v2 kit. This advancement holds substantial promise for improving the accessibility and efficiency of nucleic acid research, particularly in studies involving scarce cellular materials.

overall_design: Single-cell RNA sequencing of HAP1 cells using the Evercode Whole Transcriptome v2 kit (Parse Biosciences), for comparison with the RNA-seq data generated for cells from the same population using the novel NAxtra-based method (GEO accession number: GSE284534).

first: Eirin

middle: J

last: Starheim

first: Niklas

last: Andersen

first: Vidar

middle: L

last: Saasen

first: Erlend

last: Ravlo

first: Wei

last: Wang

first: Adnan

last: Hashim


title: Genomic editing of a pathogenic mutation in ACTA2 rescues multisystemic smooth muscle dysfunction syndrome

city: Dallas

first: Lei

last: Guo

state: TX

address: 5323 Harry Hines Blvd

department: Department of Population and Data Sciences

institute: University of Texas Southwestern Medical Center

summary: BACKGROUND: Vascular smooth muscle cells (vSMCs), the predominant cell type in the aortic wall, play a crucial role in maintaining aortic integrity, blood pressure, and cardiovascular function. vSMC contractility and function depend on smooth muscle alpha-actin 2 (ACTA2). The pathogenic variant ACTA2 c.536G>A (p. R179H) causes multisystemic smooth muscle dysfunction syndrome (MSMDS), a severe disorder marked by widespread smooth muscle abnormalities, resulting in life-threatening aortic disease and high risk early mortality from aneurysms or stroke. No effective treatments exist for MSMDS. METHODS: To develop a comprehensive therapy for MSMDS, we utilized CRISPR-Cas9 adenine base editing to correct the ACTA2 R179H mutation. We generated isogenic human induced pluripotent stem cell (iPSC) lines and humanized mice carrying this pathogenic missense mutation. iPSC-SMCs were evaluated for key functional characteristics, including proliferation, migration, and contractility. The adenine base editor (ABE) ABE8e-SpCas9-VRQR under control of either a SMC-specific promoter or a CMV promoter, and an optimized single guide RNA (sgRNA) under control of U6 promoter were delivered intravenously to humanized R179H mice using adeno-associated virus serotype 9 (AAV9) and phenotypic outcomes were evaluated. RESULTS: The R179H mutation causes a dramatic phenotypic switch in human iPSC-SMCs from a contractile to a synthetic state, a transition associated with aneurysm formation. Base editing prevented this pathogenic phenotypic switch and restored normal SMC function. In humanized mice, the ACTA2R179H/+ mutation caused widespread smooth muscle dysfunction, manifesting as decreased blood pressure, aortic dilation and dissection, bladder enlargement, gut dilation, and hydronephrosis. In vivo base editing rescued these pathological abnormalities, normalizing smooth muscle function. CONCLUSIONS: This study demonstrates the effectiveness of adenine base editing to treat MSMDS and restore aortic smooth muscle function. By correcting the ACTA2 R179H mutation, the pathogenic phenotypic shift in SMCs was prevented, key aortic smooth muscle functions were restored, and life-threatening aortic dilation and dissection were mitigated in humanized mice. These findings underscore the promise of gene-editing therapies in addressing the underlying genetic causes of smooth muscle disorders and offer a potential transformative treatment for patients facing severe vascular complications.

overall_design: RNA-seq profiling of wildtype, ACTA2R179H/+, ACTA2R179H/R179H, and ABE-corrected human iPSC-derived smooth muscle cells

first: Qianqian

last: Ding

first: Peiheng

last: Gan

first: Zhisheng

first: Hui

last: Li

first: Ning

first: Eric

last: Olson


title: Transcriptomics of salinomycin's molecular toxicity in turkey heart and liver

city: Balice

first: Kacper

last: Zukowski

address: Krakowska 1 Street

country: Poland

institute: National Research Institute of Animal Production

summary: Salinomycin (Sal) is used as a coccidiostat to treat coccidiosis disease caused by Eimeria protozoa invasion in avian. Generally, salinomycin has been considered a safe feed additive if the recommended dosage is properly used depending on the animal species. However, in some cases, it has been reported that the accidental use, misuse, or interaction of salinomycin with other drugs generates toxicity in the organism. Yet, the exact molecular mechanisms of salinomycin intoxication have not been fully understood. To understand the salinomycin toxicity mechanism at the cellular level in turkey species we performed high-throughput sequencing using heart and liver samples.

overall_design:
The experimental group received a diet with salinomycin (0.9 mg/kg b.w daily dose) whereas the control group received a diet without salinomycin for 2 weeks.
Heart and liver samples were collected from turkey at week 13.
RNA sequencing was carried out with 12 pe samples (6 samples from each tissue).

first: Berfin

middle: I

last: Ekinci

last: Żukowski

first: Anna

last: Sławińska

first: Małgorzata

last: Olejnik


title: Circular RNA expression in ALS is progressively deregulated and tissue-dependent

city: Zaragoza

first: Janne

last: Toivonen

address: Miguel servet 177

department: Departamento de Anatomía, Embriología y Genética Animal

country: Spain

institute: Universidad de Zaragoza

summary: There is increasing evidence on the role of circular RNAs (circRNAs) in neuronal and muscular processes. Accordingly, their dysregulation is associated with neurodegenerative diseases and myopathies. We investigated circRNA expression in the central nervous system (CNS) and skeletal muscle, the two main tissues affected in amyotrophic lateral sclerosis (ALS). Based on circRNA sequencing analysis in spinal cord from ALS mice (SOD1G93A) followed by a literature search, 30 circRNAs potentially involved in ALS were tested. All selected circRNAs were downregulated in the SOD1G93A spinal cord, whereas only half of these were quantifiable and were generally upregulated in quadriceps muscle of SOD1G93A mice. Such tissue-dependent expression pattern was observed in both sexes and circRNA abundance in the spinal cord was higher than in the muscle, both in wild type and in SOD1G93A mice. Finally, we assessed the 18 circRNAs with the largest expression differences and the highest degree of interspecies conservation in brain samples from sporadic ALS (sALS) patients and healthy controls. Similar to the mouse model, circRNA levels tended to decrease in the CNS of sALS patients.  Expression of circRNAs may be systematically altered in the two tissues most affected by ALS in a progressive and opposed manner. Although more detailed studies are warranted, circRNAs are potentially related to ALS etiopathogenesis and could possibly serve as future biomarkers, therapeutic targets, or customized therapeutic tools to modulate the pathology.

overall_design: Circular RNA-Seq profiling of spinal cord from 50-day-old male wild type (n=8) and SOD1-G93A (n=8)mice.

first: Rosario

last: Osta

first: Ana Cristina

last: Calvo

first: Leticia

last: Moreno-García


title: Methylseq, Single-nuclei RNAseq, and Discovery Proteomics Identifies Pathways Associated with Nephron Deficit CKD in the HSRA Rat Model [scRNA-Seq]

city: Jackson

first: Bioinformatics

last: Core

state: MISSISSIPPI

address: 2500 N state street

institute: Molecular and Genomics Core Facility

summary: Low nephron numbers are associated with increased risk of developing chronic kidney disease (CKD) and hypertension, both significant global health problems.  To investigate the impact of nephron deficiency, our lab developed a novel inbred rat model (HSRA rat). In this model, approximately 75% of offspring are born with a single kidney (HSRA-S), compared to two-kidney littermates (HSRA-C). HSRA-S rats show impaired kidney development, resulting in ~20% fewer nephrons. Our previous data and current findings demonstrate nephron deficit (failure of one kidney to form and altered development in the remaining kidney) predispose HSRA-S to CKD late in life (increased proteinuria by 18 months of age in HSRA-S=51± 3.4 versus HSRA-C = 8± 1.5 mg/24hours). To understand early molecular mechanisms contributing to the increased predisposition to CKD, Methylseq using reduced representation bisulfite sequencing, single nuclei (sn)RNAseq, and discovery proteomics were performed in kidneys of 4-week-old HSRA rats. Methylation analysis revealed a small number of differences, including 5 differentially methylated cytosines and 6 differentially methylated regions between groups. The snRNAseq analysis identified differentially expressed genes in most kidney cell types, with several hundred genes dysregulated depending on the analysis method (Seurat vs DESeq2). Notably, many genes are involved in kidney development. Discovery proteomic analysis identified 366 differentially expressed proteins. A key finding was dysregulation of Deptor/DEPTOR and Amdhd2/AMDHD2 across omics layers, suggesting a potential role in compensatory mechanisms or the genetic basis of altered kidney development.  Further understanding of these mechanisms may guide interventions to preserve nephron health and slow kidney disease progression.

overall_design: snRNASeq: Nuclei were isolated from archived kidneys using a central section/biopsy (cortex and medulla were included) collected at 4 weeks of age from HSRA-S (n=3) and HSRA-C (n=4) per 10X Chromium Nuclei Isolation Kit (PN-1000493). Single nuclei samples were processed through the 10X Genomics Chromium Single Cell 3’ protocol v3.1 per manufacturers’ instruction to capture 5000 cells per sample.

first: Andrew

middle: R

last: Milner

first: Ashley

middle: C

last: Johnson

first: Esinam

last: Attipoe

first: Wenjie

last: Wu

first: Lavanya

last: Challagundla

first: Michael

last: Garrett


title: Spatial transcriptomic and morpho-functional information derived from single mouse FFPE slides allows in-depth fingerprinting of lung fibrosis

city: Parma

first: Erica

last: Ferrini

address: Via Largo Belloli

institute: Chiesi Farmaceutici

summary: RNA sequencing can provide insightful information on the molecular processes underlying development and progression of multiple diseases, including idiopathic pulmonary fibrosis (IPF) and its bleomycin (BLM)-induced preclinical model. We investigated the feasibility of applying RNAseq analysis to formalin-fixed and paraffin-embedded (FFPE) lung slides from control and BLM-treated mice. The results were validated by comparison with public bulk data from fresh-frozen mouse tissue and human IPF biopsies. Unsupervised cluster analysis on Differentially Expressed Genes revealed gene clusters associated with extracellular matrix organization, tissue remodeling and inflammatory response pathways, distinguishing untreated and BLM-treated fibrotic lung samples. For each sample, expression levels of individual gene clusters were highly correlated with 2D histology readouts and aeration compartments determined on matched 2D coronal slices by micro-CT imaging. This combined approach can produce a precise, spatially oriented picture of pulmonary disease development, paving the way to the identification of novel translationally relevant biomarkers and therapeutic targets.

overall_design: RNAseq profiling of FFPE lung slides from control and bleomycin-treated C57Bl/6 mice, 7-8 weeks old, at Day 21 after BLM administration.

first: Ferrini

last: Erica

first: Bonfini

last: Costanza

first: Marchese

last: Giovanna

first: Ravo

last: Maria

first: Stellari

last: Franco Fabio


title: Long-term resident adipose-derived stromal stem cells in the microenvironment remodeling BLCA cell stemness and EMT promotes bladder cancer progression

city: Haikou

first: Denggao

last: Huang

state: Hainan

address: No.43 avenue Renmin Road

institute: Haikou people hospital

summary: The role of adipose-derived stromal stem cells (ADSCs) in BLCA progression is unclear. We investigated the effects of invasion, stemness, Epithelial-mesenchymal transition (EMT), and drug resistance of BLCA cells co-cultured with ADSCs for a long period of time. Cells were divided into six groups: ADSCs group, ADSCs:T24 group (10:1, 3:1 and 1:1 groups), ADSCs-derived conditioned medium group (CM) and T24 cell group (T24), and cells in each group were cultured to 14 days, and puromycin (puro) was added to the co-cultured cell to remove ADSCs cells without puro resistance , and then the function of T24 cells (10:1-COC, 3:1-COC and 1:1-COC) after co-culture was studied; CCK-8 assay, Transwell, Wound healing, Flow cytometry, RNA-sequencing, qRT-PCR and Western Bloting assay were used to detect cell proliferation, invasion, migration, apoptosis and cellular mRNA and protein expression levels, respectively.  We unexpectedly found enhanced stemness and drug resistance of BLCA cells after prolonged contact culture with ADSCs. T24 cells after co-culture mediated cell proliferation, invasion, EMT, stemness, drug resistance and immune escape by up-regulating MDM2, mt-P53 and PD-L1, compared to CM and T24 groups. The inhibitor Atezo and CP-31398 eliminated mt-P53 and PD-L1-mediated T24 cell drug resistance and stemness, respectively. This study demonstrated that after prolonged co-culture of BLCA cells with ADSCs, the stemness, drug resistance, and immune evasion of T24 cells were dramatically enhanced.

overall_design: RNA-seq profiling of the T24 alone culture group (T24), the ADSC-sourced CM group, and the ADSC:T24 coculture groups at ratios of 10:1, in addition to the ADSC-only culture group.


title: ZFHX3 modulates the androgen/AR signaling activity in prostate cancer cells

city: Shenzhen

first: Jin-Tang

last: Dong

state: Guangdong

address: 1088 Xueyuan Blvd.

institute: Southern University of Science  and Technology

summary: The androgen/androgen receptor (AR) signaling drives prostate development and prostatic carcinogenesis, whereas loss of the zinc finger homeobox 3 (ZFHX3) transcription factor attenuates prostate development and promotes prostatic tumorigenesis. The androgen/AR signaling upregulates the transcription of ZFHX3 in prostate cancer cells. However, whether and how ZFHX3 is involved in the function of AR signaling in prostate cancer cells is unknown. In this study, we first carried out RNA-seq analysis in C4-2B prostate cancer cells to detect what genes and signaling pathways are caused by the deletion of ZFHX3. Gene set enrichment analysis (GSEA) revealed that among the top altered hallmark gene sets after ZFHX3 deletion, only the one for androgen response was decreased, whereas those for TNFα, interferon γ, and inflammatory response were enriched. For the 27 genes indicative AR activities, as defined in a previous study, expression levels for 18 of the 27 genes were significantly changed by ZFHX3 loss. Fifteen of these 18 genes were downregulated, including classical AR target genes KLK3, FKBP5, and TMPRSS2.

overall_design: Prostate cancer mRNA profiles of Wt and KO8 cells. Wt is the vector control, while KO8 is a ZFHX3-null clone of C4-2B cells generated using the CRISPR/Cas9 system.

first: Xing

last: Fu

first: Zhiqian

last: Zhang


title: Methylseq, Single-nuclei RNAseq, and Discovery Proteomics Identifies Pathways Associated with Nephron Deficit CKD in the HSRA Rat Model [Methyl-Seq]

first: Bioinformatics

last: Core

type: Methylation profiling by high throughput sequencing

overall_design: MethylSeq: DNA was isolated from 4-week-old male HSRA-C (n=6) and HSRA-S (n=6) kidney samples using the PureLink Genomic DNA Mini Kit (Invitrogen)


title: RNA sequencing data showing influence of p62 mRNA knockout on gene and miRNA expression

city: Schlieren

first: Hans-Dietmar

last: Beer

state: ZH

address: Wagistrasse 18

department: Dermatology

country: Switzerland

institute: USZ

type: Non-coding RNA profiling by high throughput sequencing

summary: The inflammasome sensor NLRP1 is mainly expressed by epithelial cells including keratinocytes of human skin. Germline gain-of-function mutations in NLRP1 cause inflammatory skin syndromes and predispose patients to the development of cutaneous squamous cell carcinomas (cSCCs), a major type of skin cancer originating from keratinocytes. However, expression of NLRP1 is strongly reduced in cSCCs suggesting a complex role of the NLRP1 inflammasome in the development of this type of skin cancer. Suppression of NLRP1 expression in SCC cells is partially caused by an increase in p62 (SQSTM1), a cargo receptor for autophagy-dependent protein degradation. p62 is upregulated in numerous types of cancer and plays key roles in tumor development by activating different pathways. Here, we characterized the molecular mechanisms underlying suppression of NLRP1 expression by p62 in cSCCs. In SCC cells, NLRP1 activation is rescued by a knockdown or knockout of p62 mRNA and, consequently, protein expression, rather than by a knockout of p62 protein expression only. As these experiments suggest a regulation of NLRP1 by the p62 mRNA, we characterized p62 mRNA-regulated gene expression in SCC cells through RNA sequencing. In addition to mRNAs, we identified several differentially regulated microRNAs (miRs), including miR-34a-5p. These short non-coding RNAs regulate the stability or translation of mRNAs in a dynamic manner and a single miR can target multiple mRNAs. miR-34a-5p is an established tumor suppressor in different types of cancer and its expression is also downregulated in cSCCs. Although miR-34a-5p seems to bind neither p62 nor NLRP1 mRNA directly, it increases NLRP1 expression, most likely through a complex mechanism at the RNA level. In summary, our findings revealed a novel pathway regulating suppression of the inflammasome sensor NLRP1 in SCC cells by p62, which occurs at the mRNA level and is mediated by miRs, including the tumor suppressive miR-34a-5p. p62 mRNA, but not protein, regulates expression of other mRNAs and miRNAs. Differential expression analysis of RNA-seq data from SCC12 cells with a protein (p62.1) or mRNA (p62.2K) knockout, and control cells were performed. CRISPR/Cas9 knockout (p62.1 - protein only) or CRISPR/dCas9-KRAB knockout (p62.2K - mRNA and protein) of p62 in SCC12 cells

overall_design: We performed an RNA sequencing experiment in SCC12 cells. Upon lentiviral transduction and selection with antibiotics, we generated stable polyclonal dCas9 control (transduction with non-targeting empty vector), p62 protein (by CRISPR/Cas9) and p62 mRNA/protein (by CRISPR/dCas9-KRAB) knockout SCC12 cells. Total RNA was isolated (quadruplet) and analyzed by RNA sequencing. We identified 156 or 106 genes (mRNAs), which were up- or down-regulated by p62 mRNA, respectively. Furthermore, 30 miRs were regulated by p62 mRNA, from which 18 were up- and 12 downregulated. Interestingly, several miRs were suppressed by a p62 mRNA knockout and, therefore, their expression is positively correlated with NLRP1 expression. This experiment indeed demonstrates that p62 mRNA is able to regulate mRNA and miR expression in a positive as well as in a negative manner. N=4. Total RNA was isolated using the miRNeasy Kit (217004, Qiagen) according to the manufacturer’s instruction. For mRNA sequencing, libraries were prepared with TruSeq RNA (Illumina), while miRNA libraries utilized RealSeq®-AC (somagenics), following standard protocols at the Functional Genomics Centre Zurich (FGCZ). High Throughput Sequencing (NGS) was performed on Illumina Novaseq 6000. Raw reads were quality-checked using FastQC. mRNA reads were mapped to the human transcriptome using salmon version 1.9.0. The quantified transcripts were imported into R and collapsed to the gene level using the R package “tximport”. Mature and hairpin microRNA quantification was performed using the nf-core/smrnaseq pipeline v2.3.0 (10.5281/zenodo.3456879). Differential expression analysis of mRNA and miRNA was performed using the R package DESeq2

first: Paulina

last: Henng

first: Patrick

last: Turko


title: A Single-Cell Atlas of the Upper Respiratory Epithelium Reveals Heterogeneity in Cell Types and Patterning Strategies

city: San Diego

first: Alexander

last: Foote

address: 3381 Lebon Drive

department: Pediatrics

institute: UCSD

summary: The upper respiratory tract, organized along the pharyngolaryngeal-to-tracheobronchial axis, is essential for homeostatic functions such as breathing and vocalization. The upper respiratory epithelium is frequently exposed to pollutants and pathogens, making this an area of first-line defense against respiratory injury and infection. The respiratory epithelium is composed of a rich array of specialized cell types, each with unique capabilities in immune defense and injury repair. However, the precise transcriptomic signature and spatial distribution of these cell populations, as well as potential cell subpopulations, have not been well defined. Here, using single cell RNAseq combined with spatial validation, we present a comprehensive atlas of the mouse upper respiratory epithelium. We systematically analyzed our rich RNAseq dataset of the upper respiratory epithelium to reveal 17 cell types, which we further organized into three spatially distinct compartments: the Tmprss11a+ pharyngolaryngeal, the Nkx2-1+ tracheobronchial, and the Dmbt1+ submucosal gland epithelium. We profiled/analyzed the pharyngolaryngeal epithelium, composed of stratified squamous epithelium, and identified distinct regional signatures, including a Keratin gene expression code. In profiling the tracheobronchial epithelium, which is composed of a pseudostratified epithelium-with the exception of the hillock structure-we identified that regional luminal cells, such as club cells and basal cells, show varying gradients of marker expression along the proximal-distal and/or dorsal-ventral axis. Lastly, our analysis of the submucosal gland epithelium, composed of an array of cell types, such as the unique myoepithelial cells, revealed the colorful diversity of between and within cell populations. Our single-cell atlas with spatial validation highlights the distinct transcriptional programs of the upper respiratory epithelium and serves as a valuable resource for future investigations to address how cells behave in homeostasis and pathogenesis.

overall_design: Control WT tissue was digested to dissociate cells, FAC sorted and analyzed uding scRNA-seq.

first: Xin

last: Sun


title: Profiling the endothelial translatome in vivo using ‘AngioTag’ zebrafish (reanalysis)

city: Bethesda

first: Derek

last: Gildea

state: MD

address: 12 South Drive Rm 4009-24

institute: NHGRI/NIH

summary: We profiled the gene expression patterns of undisturbed endothelial cells in living animals using a novel ‘AngioTag’ zebrafish transgenic line that permits isolation of actively translating mRNAs from endothelial cells in their native environment. This transgenic line uses the endothelial cell-specific kdrl promoter to drive expression of an epitope tagged Rpl10a 60S ribosomal subunit protein, allowing for Translating Ribosome Affinity Purification (TRAP) of actively translating endothelial cell mRNAs. We also collected the whole embryo translatome using the TRAP protocol and a ubiquitously expressed tagged Rpl10a, and the endothelial transcritome by collecting the GFP+ endothelial cells that had the kdrl promoter driving GFP.

relation:
Reanalysis of: GSM3452116
Reanalysis of: GSM3452117
Reanalysis of: GSM3452118
Reanalysis of: GSM3452119
Reanalysis of: GSM3452120
Reanalysis of: GSM3452121
Reanalysis of: GSM3452122
Reanalysis of: GSM3452123
Reanalysis of: GSM3452124
Reanalysis of: GSM3452125
Reanalysis of: GSM3452126
Reanalysis of: GSM3452127
Reanalysis of: GSM3452128
Reanalysis of: GSM3452129
Reanalysis of: GSM3452130
Reanalysis of: GSM3452131
Reanalysis of: GSM3452132
Reanalysis of: GSM3452133

overall_design:
Examination of 24hpf zebrafish whole embryo translatome, 24hpf zebrafish endothelial translatome, and 24hpf zebrafish endothelial transciptome mRNA levels. RNAseq analysis was redone using GRCz11 zebrafish reference genome and Ensembl version 99 models for protein-coding genes on the following GEO sample accessions: GSM3452116, GSM3452117, GSM3452118, GSM3452119, GSM3452120, GSM3452121, GSM3452122, GSM3452123, GSM3452124, GSM3452125, GSM3452126, GSM3452127, GSM3452128, GSM3452129, GSM3452130, GSM3452131, GSM3452132, GSM3452133.

***************************************************************
The table below lists GEO accessions reused/reanalyzed for this study.
***************************************************************

first: Mayumi

last: Miller

first: Leah

last: Greenspan

middle: E

first: Kathryn

last: Monzo

first: Gennady

last: Margolin

first: Van

last: Pham

first: Keith

last: Ameyaw

first: Lisa

last: Price

first: Natalie

last: Aloi

first: Amber

last: Stratman

first: Andrew

last: Davis

first: Isabella

last: Cisneros

first: Caleb

last: Mertus

first: Ryan

last: Dale

first: Andreas

middle: D

last: Baxevanis

first: Brant

last: Weinstein


title: Comparative analysis of the cell transcriptome of primary B lymphocytes infected with the Epstein-Barr virus (EBV), its EBNA2 or EBNALP KO derivatives, or activated by CD40L and IL4 at 48 hours post-infection or treatment.

city: Corbeil-Essonnes

first: Hélène

last: Polvèche

address: 28, Rue Henri Desbruères

country: France

institute: I-Stem

summary: Epstein-Barr virus (EBV) is a human herpesvirus that has been associated with the development of human cancers across the globe. In laboratory settings, the virus efficiently infects resting human B lymphocytes, inducing their growth transformation and leading to the establishment of lymphoblastoid cell lines (LCLs) within a few weeks. This process is accompanied by a global reprogramming of cellular gene transcription. A systematic time-resolved analysis of cellular mRNA splice variant expression during EBV infection of resting B lymphocytes has previously been performed. The results demonstrated that significant alterations in alternative splice variant expression occur as early as day 1 post-infection, indicating that splicing regulation, in addition to transcription, represents an additional mechanism of gene expression regulation at the onset of B cell activation and proliferation. The present study had two principal objectives. The first was to identify which transcriptional and splice events are specific to the EBV infection, in comparison to the activation of primary B lymphocytes by a combination of CD40 ligand (CD40L) and interleukin 4 (IL4). The second was to ascertain which events are dependent on the EBNA2 and EBNALP viral factors. In order to achieve these objectives, a comparative RNA-seq analysis of the transcriptome of the infected cells or those treated with CD40L/IL4, as well as LCLs derived from the wild-type and EBNALP KO EBV-infected cells, was conducted.

overall_design: RNA-seq profiling of human primary B lymphocytes infected or not by wild-type Epstein-Barr virus (EBV) or its knockdown derivatives (EBNA2 or EBNALP KO) at day 2 post-infection, or treated by a combination of CD40 ligand (CD40L) and interleukin 4 (IL4) at day 2 post-treatment. RNA-seq profiling was also conducted on the lymphoblastoid cell lines (LCLs) derived from the wild-type EBV and EBNALP KO infections at 2 to 3 weeks post-infection.

first: Sylvain

last: de Breyne

first: Didier

last: Auboeuf

first: Théophile

last: Ohlmann

first: Henri

last: Gruffat

first: Evelyne

last: Manet


title: Putrescine treatment has a higher effect on 5mC DNA methylation profile of wheat leaves under white than under blue light conditions

city: Budapest

first: István

last: Likó

address: Üllői út 26.

country: Hungary

institute: Semmelweis University

summary: Polyamines play a pivotal role in plant growth and development by modulating metabolite levels and even regulating DNA methylation. To reveal novel insights into the intricate relationship among polyamines, metabolites and DNA methylation, the impact of exogenous putrescine on wheat was investigated at several levels. Since the outcome of putrescine treatment may vary depending on the light quality, the experiment was performed under both white and blue light. Metabolite profiling revealed distinct changes induced by blue light and/or putrescine. Blue light alone had increasing effects on several metabolites, but the effect of putrescine was dominant under blue light in most cases. Putrescine had a decreasing effect on some amino acids and the γ-glutamyl-cycle, while increasing effect on the TCA-cycle. Distinct changes in DNA methylation patterns were detected in response to blue light, putrescine or their combination. Blue light exerted strong effects on methylation, and putrescine application slightly could induce further changes. Putrescine had a higher impact under white as increased the leaf spermidine level, and parallel with this, a lower level of up-methylated genes involved in cellular processes, but a higher level of down-methylated genes involved in molecular function and biological processes occurred compared to the blue light.

overall_design: MeDIP DNA sequencing on the population of winter wheat (Triticum aestivum L. variety ‘Mv Béres’) grown on modified Hoagland solution in growth chambers under 16/8-h light/dark periodicity and 22/20 °C day/night temperature with and 75% relative humidity [26], and under two different light spectrums. Under white (W) light, 32.32% blue, 45.71% green, 20.71% red and 1.25% far-red composition, while under blue (B) light, 82.14% blue, 1% green, 16.7% red and 0.17% far-red composition was applied at 250 µmol m-2 s−1 photosynthetic photon flux density. Seven days after the germination, half of the plants grown under either W or B light conditions were further grown without any treatment (WC or BC), while the other part of them got 0.5 mM PUT supplementation in the hydroponic solution (WP or BP) in the next 7 days.

first: Magda

last: Pál

first: Imre

last: Majláth


title: The effect of enriched environment and resocialization on the social  and cognitive behaviors in social isolated 5×FAD mice

city: Nanjing

first: Ming

last: Xiao

address: 101 Longmian Avenue

institute: NMU

summary: 4-week old 5xFAD mice were isolated housed for 3 weeks and then treated by enriched environment (EE) and resocialization with normal wildtype mice (WT) for 3 weeks

overall_design: The medial prefrontal cortex were isolated for RNA sequencing

first: Yimiao

last: Wang

first: Yue

last: Li

first: Min

last: Cao

first: Shixin

last: Ding

first: Yanli

last: Zhang


title: Unveiling Immune Metabolic Mechanisms in Bladder Cancer: Spatial Profiling Identifies ZFP36 as a Therapeutic Target

first: Xuedan

last: Han

address: School of Life Science and Technology, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 639 Longmian Road. Nanjing , China.

institute: China Pharmaceutical University

summary: Treatment of bladder cancer (BLCA) is currently facing formidable challenges due to its high heterogeneity and immune evasion. In this study, we utilize spatial metabolomics (SM) and spatial transcriptomics (ST) to systematically analyze and visualize metabolic and transcriptional landscapes of BLCA. Correlation analysis reveals significant metabolic and transcriptional differences among tumor regions, shedding light on the heterogeneity and immune-related metabolic reprogramming mechanisms in BLCA. Further studies identify ZFP36 as a potential immunotherapeutic marker, with its higher expression correlating with poor prognosis in BLCA patients. Mechanistically, ZFP36 mediates the mRNA decay of key immune regulatory genes, including C1QBP, thereby suppressing T-cell activation and cytotoxicity. Notably, the combination of ZFP36 knockout and anti-PD-1 therapy demonstrated synergistic antitumor effects, suggesting that targeting ZFP36 may offer a promising therapeutic strategy for BLCA treatment. In summary, this study, through integrated multi-omics analysis, unveils the immune metabolic regulatory mechanisms of BLCA and provides crucial molecular targets for immunotherapy.

overall_design: Validation of immunometabolic reprogramming in bladder cancer by association analysis of spatial transcriptomics and spatial metabolomics

first: Lufeng

last: Zheng

first: Fangdie

last: Ye


title: TEAD promotes lineage progression of subpallial neural progenitor cells independent of YAP/TAZ

city: Memphis

first: Xinwei

state: TN

address: 62 Danny Thomas Pl

department: Developmental Neurobiology

institute: St. Jude Children's Research Hospital

summary: The embryonic ventral telencephalon (subpallium) gives rise to extremely diverse types of cells. Although it is known that subpallial neural progenitors possess characteristics distinct from their cortical counterparts, the mechanisms governing their lineage progression are poorly understood. Here we show that TEAD, the key DNA-binding factor of the Hippo pathway, promotes lineage progression of subpallial neural progenitors; the loss of TEAD1 and TEAD2 (TEAD1/2) during mammalian brain development expands early progenitors and reduces late progenitors uniquely in the subpallium, but not in the cortex. Importantly, this function of TEAD1/2 is independent of their canonical partners in the Hippo pathway, transcriptional coactivators YAP and TAZ (YAP/TAZ), as the loss of YAP/TAZ leads to the opposite phenotype—a depletion of early progenitors and increase in late progenitors. We further show that TEAD1/2 act at least in part by inhibiting Notch signaling and by cooperating with INSM1. It has been shown that orthologs of TEAD and INSM1 cooperate to regulate neuronal cell fate decisions in worms and flies. Our study reveals a remarkable evolutionary conservation of the function of this transcription factor complex during metazoan neural development.

overall_design: The MGEs from wild-type mice at E12.5 and E15.5, as well as Tead1/2 and Yap/Taz knockout mutant mice and appropriate controls at E14.5, were collected for scRNASeq analysis

first: Charles

middle: H

last: Perry

first: Alfonso

last: Lavado

first: Venkata

last: Thulabandu

first: Cody

last: Ramirez

first: Joshua

last: Pare

first: Rajiv

last: Dixit

first: Akhilesh

last: Mishra

first: Jiyuan

last: Yang

first: Jiyang

last: Yu


title: Mitigation of Doxorubicin Cardiotoxicity with Synergistic miRNA Combinations Identified Using Combinatorial Genetics en Masse (CombiGEM)

city: Ann Arbor

first: Anthony

last: Rosenzweig

state: Michigan

address: 2800 Plymouth Rd, NCRC Building 25

department: Stanley and Judith Frankel Institute for Heart and Brain Health

institute: University of Michigan Medical Center

summary: Background: Cardiomyocyte loss occurs in acute and chronic cardiac injury, including cardiotoxicity due to chemotherapeutic agents such as doxorubicin, and contributes to development of heart failure. There is a pressing need for cardiac-specific therapeutics that prevent the cardiac complications of chemotherapy without compromising efficacy by directly targeting cardiomyocyte loss. Objectives: We employed massively parallel combinatorial genetic screening to search for miRNA combinations that promote cardiomyocyte survival. Methods: Combinatorial genetics en masse (CombiGEM) screening in a cardiomyocyte cell line was followed by validation studies in the original cell type as well as primary cardiomyocytes to search for miRNA combinations that promote survival under various stress conditions. The top candidate combination was tested human iPSC-derived cardiomyocyte and in vivo mouse and developing zebrafish models of doxorubicin cardiotoxicity. RNA sequencing and in silico miRNA target prediction provided insight into possible mechanisms. Results: CombiGEM screens and validation experiments identified multiple miRNA combinations that protected cardiomyocytes from doxorubicin in vitro. The most effective miRNA combination (miR-222 + miR-455) appeared to act synergistically and had protective effects in both murine and zebrafish in vivo doxorubicin cardiotoxicity models. RNA sequencing data revealed synergistic and additive regulation of mechanistically relevant genes and biological processes, including mitochondrial homeostasis, cellular respiration, DNA replication/damage response, oxidative and ER stress, angiogenesis, muscle contraction/relaxation, and others. Conclusions: We identified miR-222 and miR-455 as a combination with potential therapeutic applications for cardioprotection. The results of this study further our knowledge of the cardiac effects of individual miRNAs and their combinations and demonstrate the value of the CombiGEM approach for discovery of cardioprotective combinatorial therapeutic candidates.

overall_design: To gain insight into potentially synergistic protective effects of miR-222 and miR-455 against doxorubicin cardiotoxicity, we transfected cultured neonatal rat ventricular myocytes with Ambion Pre-miR miRNA mimics of miR-222-3p (Thermofisher Scientific assay ID PM11376), miR-455-3p (assay ID PM12568), both miRNA mimics combined, or a negative control mimic (Cat # AM17110) at 5nM each. At 48h after transfection, cells were treated with 4mM doxorubicin or vehicle. RNA was extracted 24h later for RNA sequencing followed by differential expression and gene ontology analysis.

first: Yasutomi

last: Higashikuni

first: Colin

last: Platt

first: Margaret

last: Hastings


title: Injured tubule-derived CCN1 exacerbates renal congestion-mediated acute kidney injury and fibrosis

city: Kyoto

first: Tetsuro

last: Kusaba

state: Kyoto

address: 465 Kajii-cho, Kamigyo-ku

department: Department of Nephrology

institute: Graduate School of Medical Science, Kyoto Prefectural University of Medicine

summary: Epidemiological studies showed that patients with heart failure frequently develop kidney dysfunction, called cardio-renal syndrome (CRS). Elevated central venous pressure rather than low cardiac output strongly correlates with worsening renal function, and is being increasingly recognized as the pathophysiology responsible for CRS. However, due to the lack of appropriate animal models, the molecular mechanisms underlying the congestion-mediated acceleration of kidney injury, called renal congestion, remain unclear. In the present study, using a novel mouse renal congestion model, we detected injured tubule-specific cell-cell interactions in congestive kidneys and showed that Cellular Communication Network Factor 1 (CCN1) played an important role in this process. Transcriptomics of kidneys with ischemia-reperfusion injury (IRI) and renal congestion revealed the up-regulation of paracrine chemokine-related pathways. The up-regulation of CCN1 was observed in the acute phase after kidney injury with renal congestion. Positive staining for phosphorylated focal adhesion kinase (pFAK), a downstream signaling molecule of CCN1, was noted in fibroblasts at injury sites in congestion-IRI kidneys. CCN1 activated the phosphorylation of FAK and ERK in vitro, which accelerated the migration of fibroblasts and macrophages. We then examined the effects of CCN1 deletion in tubular epithelia on congestion-mediated kidney injury in vivo. pFAK expression in injury sites was down-regulated in CCN1-KO mice, and the congestion-mediated worsening of tissue fibrosis was significantly ameliorated. In conclusion, we herein demonstrated the important role of injured tubule-derived CCN1 on pFAK-mediated fibroblast migration in congestive kidneys. The inhibition of CCN1 has potential as a therapeutic candidate for preventing the transition of renal congestion-mediated kidney injury to fibrosis.

overall_design: RNA-seq profiling of IRI kidney with or without renal congestion at 1 day after surgery

first: Atsushi

last: Minamida

first: Yuhei

last: Kirita


title: Transcriptomics of salinomycin's molecular toxicity in chicken heart and liver

first: Kacper

last: Zukowski

summary: Salinomycin (Sal) is used as a coccidiostat to treat coccidiosis disease caused by Eimeria protozoa invasion in avian. Generally, salinomycin has been considered a safe feed additive if the recommended dosage is properly used depending on the animal species. However, in some cases, it has been reported that the accidental use, misuse, or interaction of salinomycin with other drugs generates toxicity in the organism. Yet, the exact molecular mechanisms of salinomycin intoxication have not been fully understood. To understand the salinomycin toxicity mechanism at the cellular level in chicken species we performed high-throughput sequencing using heart and liver samples.

overall_design:
The experimental group received a diet with salinomycin (0.9 mg/kg b.w daily dose) whereas the control group received a diet without salinomycin for 2 weeks.
Heart and liver samples were collected from chicken at week 5.
RNA sequencing was carried out with 12 pe samples (6 samples from each tissue).

first: Berfin

last: Ekinci

last: Żukowski

first: Anna

last: Sławińska

first: Małgorzata

last: Olejnik


title: Rapid DNA and RNA isolation from few or single cells using low-cost NAxtra magnetic nanoparticles

city: Oslo

first: Adnan

last: Hashim

state: Oslo

address: NCMM - Forskningsparken, House 1, 3rd Floor - Gaustadalléen 21

department: NCMM (Centre for Molecular Medicine Norway)

institute: UiO (University of Oslo)

summary: A low-cost nucleic acid (NA) isolation method for purification of total NA, DNA or RNA from two- or three-dimensional cell culture was recently developed at the Norwegian University of Science and Technology (NTNU) based on NAxtra magnetic nanoparticles. The method displayed similar yields to alternative isolation kits, as well as significant improvement in cost and speed. In molecular research, NA isolation from rare or single cells is of increasing interest. Here, we improved the sensitivity of the NAxtra-based NA isolation method to allow mid- to high-throughput purification from 10 000 cells down to single cells. Automated processing on KingFisher (Thermo Scientific) robot systems can be performed in 12-18 minutes for 96 samples. This method is considerably cheaper and faster than similar purification alternatives, while achieving comparable or superior detection by (RT)-qPCR. In addition, high-quality transcriptomics data can be obtained from RNA extracted from few or single cells.

overall_design: RNA sequencing of total RNA extracted from 1, 10, 100 and 1000 sorted cells (HAP1) using a novel, rapid, low-cost isolation method based on NAxtra technology.

first: Magnar

last: Bjørås

first: Eirin

last: Starheim

first: Adeel

last: Manaf


title: IL-18 promotes surgical recovery by mobilizing monocytes and promoting their differentiation while avoiding inflammation [RNA-seq]

city: Brno

first: Ioanna

last: Papatheodorou

address: Studentska

department: Center for Translational Medicine

country: Czech Republic

institute: International Clinical Research Center (ICRC), St.Anne’s Hospital

summary: Innate immune activation upon surgery serves as a protective mechanism, its orchestration and timely resolution is essential for patient recovery. We investigated the determinants associated with recovery after orthopedic surgery including monocyte and cytokine signature. Blood samples from orthopedic surgery (ORT) patients were collected before (T0), and 24 h (T1) and 3-5 days (T2) after surgery. Bulk RNAseq of peripheral blood monocyte samples was performed in order to delinate the profile of monocytes in each of the time points. The bulk RNAseq data evealed upregulated genes controlling immune cell migration and differentiation, but not of pro-inflammatory genes in T2.

overall_design:
In order to investigate the determinants of patient recovery, peripheral blood monocytes were isolated from ORT patients at T0, T1 and T2.
Differential gene expression analysis from RNAseq data from ORT patient monocytes, isolated in T0, T1 and T2

first: Gabriela

last: Blažková

first: Veronika

last: Bosáková

first: Zuzana

last: Tomášiková

first: Erin

last: Spearing

first: Robin

last: Kleiber

first: Pavel

last: Ostašov

first: Julie

last: Štíchová

first: Martina

last: Dvončová

first: Alexandra

last: Mýtniková

first: Jan

last: Emmer

first: Tomáš

last: Tomáš

first: Vladimír

last: Šrámek

first: Petros

last: Kolovos

first: Monika

last: Holubová

first: Martin

last: Helán

first: Marcela

last: Vlková

last: Frič

last: Hortová Kohoutková


title: IL-18 promotes surgical recovery by mobilizing monocytes and promoting their differentiation while avoiding inflammation [scRNA-seq]

first: Ioanna

last: Papatheodorou

summary: Innate immune activation is regulated by cytokines and serves as a protective mechanism, its orchestration and timely resolution is essential for patient recovery. In order to investigate the role of IL-18 in a relevant human model, we cocultivated healthy donor monocytes with human iPSC -derived intestinal organoids and stimulated them with IL-18. Single-cell RNAseq data revealed a prominent effect of the stimulations on organoid tissue remodeling.

overall_design:
In order to investigate the effect of IL-18 on monocyte mobility and differentiation we co-cultivated human primary monocytes with 3D human intestinal organoids (IOs) and stimulated them with IL-18.
Differential gene expression analysis from single-cell RNAseq data from (IOs) stimulated with IL-18.

last: Blažková

last: Bosáková


title: RNA-Seq analysis of mouse immortalized fibroblasts derived from WT or mir210lox/lox mice and cultured in low or high oxygen conditions

city: Valbonne - Sophia-Antipolis

first: Kevin

last: Lebrigand

address: 660 route des lucioles

institute: IPMC/CNRS

summary: To study the consequence of MIR210 locus knock-out,  mouse immortalized fibroblasts derived from WT or mir210lox/lox (KO) mice were cultured in hypoxic (1%, HX)) or normoxic oxygen (21%, NX) conditions for 24 hours. RNA samples were then harvested. Two independent experiments were carried out.

overall_design: 4 samples in duplicates: WT_NX, WT_HX, KO_NX, KO_HX

first: Bernard

last: Mari

first: Ivan

last: Mircea

first: Pascal

last: Barbry


title: Investigation of flow cytometry for analysis of mouse brain

city: Isehara

first: Takatoshi

last: Iijima

state: Kanagawa

address: 143 Shimokasuya

department: School of Medicine

institute: Tokai University

summary: The brain is difficult to analyze using flow cytometry because of its complex interactions of cells, high lipid content, and high autofluorescence. In this study, we investigated methods to isolate various types of brain cells with high yields and viability. The results showed that proteinase selection had a significant effect on the viability of various types of cells in the brain. The differences in the developmental stage also affected cell yield and viability. Moreover, the intensity of autofluorescence was found to differ greatly among the various regions of the brain. We also searched for neuronal markers that recognize a wide range of neurons. The ratios of the various exosomes contained by neurons differed depending on the type of neuronal marker. We also developed a method to quantify microglial phagocytosis in large quantities and in a short time using flow cytometry. These results have revealed critical factors that must be considered when analyzing various types of brain cells using flow cytometry.

overall_design: Treat mouse telencephalon with papain at P8, centrifuge at 24% SIP, and identify precipitated cell types.

first: Tetsushi

last: Sadakata

first: Mizuki

first: Ayumu

last: Konno

first: Akinori

last: Takase

first: Tetsuhiro

last: ]Kasamatsu

first: Hirokazu

last: Hirai


title: Effect of cation carriers on gene expression of Raji cell line

city: Warsaw

first: Beata

last: Pyrzynska

state: Mazowieckie

address: Zwirki i Wigury 61

department: Biochemistry

institute: Medical University of Warsaw

summary: Our investigation uncovers that nanomolar concentrations of salinomycin, monensin, nigericin, and narasin (a group of potassium/sodium cation carriers) robustly enhance surface expression of CD20 antigen in B-cell-derived tumor cells, including primary malignant cells of chronic lymphocytic leukemia and diffuse large B-cell lymphoma. Experiments in vitro, ex vivo, and animal model reveal a novel approach of combining salinomycin or monensin with therapeutic anti-CD20 monoclonal antibodies or anti-CD20 CAR-T cells, significantly improving non-Hodgkin lymphoma (NHL) therapy. The results of RNA-seq, genetic editing, and chemical inhibition delineate the molecular mechanism of CD20 upregulation, at least partially, to the downregulation of MYC, the transcriptional repressor of the MS4A1 gene encoding CD20. Our findings propose the cation carriers as compounds targeting MYC oncogene, which can be combined with anti-CD20 antibodies or adoptive cellular therapies to treat NHL and mitigate resistance, which frequently depends on the CD20 antigen loss, offering new solutions to improve patient outcomes.

overall_design: RNA-seq profiling of Raji cell line treated with monensin for 18 hours

first: Anna

last: Torun


title: Single cell analysis of an engineered organoid based model of pancreatic cancer identifies hypoxia as a contributing factor in the determination of transcriptional subtypes

city: Toronto

first: Alison

last: McGuigan

state: ON

address: 200 College St, Rm 338

institute: University of Toronto

summary: Pancreatic ductal adenocarcinoma is a high-mortality cancer with an aggressive, treatment resistant phenotype and a complex tumour microenvironment featuring significant hypoxia. We leveraged a unique 3D in vitro platform (TRACER) to perform single-cell transcriptome analysis of organoids cultured in a spatially-defined microenvironmental gradient to explore the effect of oxygen and other microenvironmental gradients on organoid heterogeneity.

overall_design: Pancreatic cancer patient-derived organoids (PDOs) were dissociated to single cells and cultured in the TRACER platform. After 24h of culture in the rolled configuration featuring microenvironmental gradients, cells from each of the 6 layers were isolated from the construct and analyzed by scRNA-seq.

first: Natalie

last: Landon-Brace

first: Simon

last: Latour

first: Brendan

middle: T

last: Innes

first: Michelle

last: Nurse

first: Jose

last: Cadavid

first: Ileana

last: Co

first: Cassidy

last: Tan

first: Ferris

last: Nowlan

first: Sibil

last: Drissler

first: Faiyaz

last: Notta

first: Hartland

middle: W

last: Jackson

first: Gary

last: Bader


title: miR-210 Locus Deletion Disrupts Cellular Homeostasis; An Integrated Genetic Study

city: Bucharest

first: Mihai Bogdan

last: Preda

state: - Select -

address: BP Hasdeu, Nr. 8, Sector 5

department: Stem Cell Biology

country: Romania

institute: Institute of Cellular Biology and Pathology "Nicolae Simionescu"

summary: MiR-210 is widely recognized as the quintessential hypoxia-responsive miRNA and thought to fine-tune various facets of cellular homeostasis. We hereby present an integrative appraisal of phenotypic and molecular repercussions of disrupting the corresponding locus in human and mouse cells using multiple genetic strategies. Briefly, MIR210 deletion led to decreased cellular fitness and suboptimal responses to several stress types. Transcriptomic comparisons using different profiling platforms, performed independently by members of this collaboration, revealed consistent deregulation of neighboring genes, in locus-disrupted cells. Interestingly, the anticipated enrichment in miR-210 targets failed to materialize in unbiased analyses. Our results point to the biological significance of unrecognized regulatory elements that overlap miRNA genes and should serve as note of caution for studies based for genetic disruption of such loci.

overall_design: mRNA-seq of WT, MIR210-KO, and MIR210HG-KO HEK cells cultured under normoxic (21% O₂) or hypoxic (1% O₂) conditions for 24 hours.

first: Mihai-Bogdan

first: Sergiu-Bogdan

last: Catrina

first: Alexandrina

last: Burlacu

first: Mircea

last: Ivan


title: Novel insights into Emx2 and Dmrta2 cooperation during cortex development and evidence for Dmrta2 function in choroid plexus

city: Goettingen

first: Gabriela

last: Salinas

state: Lower-Saxony

address: Kreuzbergring 57

department: Department of Pathology

country: Germany

institute: Universitaetsmedizin Goettingen

summary: To understand the genes deregulated in WT us Emx2 Knockout dorsal telencephalon at E12.5. We then performed gene expression profiling analysis using data obtained from RNA-seq of 5 different WT and Emx2KO dorsal telencephalon samples at E12.5

overall_design: Comparative gene expression profiling analysis of RNA-seq data for WT vs Emx2 KO dorsal telencephalon samples at E12.5

first: Jithu

last: Anirudhan

first: Maren

last: Sitte

last: Salinas-Riester

first: Eric

last: Bellefroid


title: Profiling the endothelial translatome in vivo using ‘AngioTag’ zebrafish

first: Gennady

last: Margolin

address: 9000 Rockville Pike

institute: National Institutes of Health

summary: Vascular endothelial cells in vivo are exquisitely regulated by their local environment, which is disrupted or absent when using methods such as FACS sorting of cells isolated from animals or in vitro cell culture.  Here, we profile the gene expression patterns of undisturbed endothelial cells in living animals using a novel “AngioTag” zebrafish transgenic line that permits isolation of actively translating mRNAs from endothelial cells in their native environment.  This transgenic line uses the endothelial cell-specific kdrl promoter to drive expression of an epitope tagged Rpl10a 60S ribosomal subunit protein, allowing for Translating Ribosome Affinity Purification (TRAP) of actively translating endothelial cell mRNAs.  By performing TRAP-RNAseq on AngioTag animals, we demonstrate strong enrichment of endothelial specific genes and uncover novel endothelial genes and unique endothelial gene expression signatures for different adult organs.  Finally, we generated a versatile “UAS:RiboTag” transgenic line to allow a widerarray of different zebrafish cell and tissue types to be examined using TRAP-RNAseq methods.  These new tools offer an unparalleled resource to study the molecular identity of cells in their normal in vivo context.

overall_design: We profiled the endothelial translatome of different vascular beds within organs of the adult zebrafish as well as the whole fish to determine endothelial genes shared among all organs and those unique to each vascular bed.

first: Leah

last: Greenspan

first: Van

last: Pham

first: Keith

last: Ameyaw

first: Brant

last: Weinstein


title: Multi-omics analysis reveals inhibition of osteosarcoma progression by sporoderm-removed ganoderma lucidum spores via targeting glycerophospholipid and fatty acid Metabolism

city: Hangzhou

first: wang

last: my

address: Cangqian Street, Cangxing Street

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

summary: Osteosarcoma (OS) is a severe malignancy affecting children and adolescents, with limited effective treatments leading to poor outcomes. This study explored the potential of sporoderm-removed Ganoderma lucidum spores (RGLS) as a novel therapeutic agent against OS and elucidated its mechanism of action. Our in vivo and in vitro experiments showed that RGLS significantly inhibited S-180 OS cell proliferation, colony formation, and migration, while simultaneously inducing apoptosis. Multi-omics analysis revealed that RGLS disrupted glycerophospholipid metabolism by upregulating lysophosphatidylcholine (LysoPC) levels through phospholipase A2 (PLA2)-mediated pathways. Co-culture assays further demonstrated that RGLS promoted the endocytosis of macrophage-derived Pla2g7 protein into S-180 cells, enhancing its anti-cancer effects. Additionally, RGLS modulated the cellular fatty acid profile and suppressed the β-oxidation of long-chain fatty acids, leading to energy depletion in OS cells. These findings provide a novel view of the multi-targeted mechanisms of RGLS, positioning it as a promising candidate for OS therapy.

overall_design: We administered RGLS prophylactically to BALB/c nude mice and then injected them subcutaneously with S-180 cells to evaluate RGLS's tumor-inhibiting effects. We then extracted tissues from three model samples and three RGLS processed samples for RNA-seq.

first: Mengyao

last: Wang

first: Jihong

last: Yang


title: Normoxia and Hypoxia transcriptomics

city: shanghai

first: Haoming

last: Zhao

address: zhizaoju road

department: Department of Oral and Maxillofacial-Head and Neck Oncology

institute: Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University

summary: Hypoxia leads to the aggregation of PD-L1 on the cell membrane without altering the overall protein expression level of PD-L1. Identify differentially expressed genes in the hypoxic transcriptome to reveal the key proteins involved in PD-L1 membrane trafficking, using samples of SCC25 cells treated with normoxia and hypoxia for 12 hours.

overall_design: There are a total of 6 samples, with the control group being the normoxic group and the experimental group being the hypoxic group. There are 3 normoxic samples and 3 hypoxic samples, all treated for 12 hours.


title: Transcriptomic alterations associated with the progression of metabolic dysfunction-associated steatotic liver disease in CC042 Collaborative Cross mice.

city: Jefferson

first: Volodymyr

last: Tryndyak

address: 3900 NCTR Rd

department: Division of Biochemical Toxicology

institute: US-FDA National Center for Toxicological Research

summary: Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as nonalcoholic fatty liver disease, has emerged as the most common chronic liver disease globally, affecting more than one-third of the world’s population. Its rate has increased by 13%, rising from 25% to 38% over the past three decades.  MASLD is characterized by excessive fat accumulation in hepatocytes and, if left untreated, progresses to its more severe form, metabolic dysfunction-associated steatohepatitis (MASH), which poses a significant global health concern as one of the leading causes of cirrhosis, cirrhotic complications, and hepatocellular carcinoma.  The molecular determinants of MASLD stratification are not clearly defined and require additional investigation.  In this study, we used male and female CC042 mice, a Collaborative Cross mouse strain susceptible to the development of MASLD, to analyze the global transcriptomic profile in liver and to clarify the molecular mechanisms involved in the progression of the disease.  Male and female CC042 mice were maintained on a high fat/high sucrose (HF/HS) diet for 20, 40, or 60 weeks, and hepatic gene expression profiles were determined by next-generation RNA sequencing.  Chronic feeding of the HF/HS diet induced profound alterations in gene expression at all time points.  The number of significantly differentially expressed genes (DEGs) in the livers of male mice was greater than female mice at 20 and 40 weeks and reached a maximum of 2764 genes at 40 weeks.  In contrast, the number of DEGs in the livers of female mice steadily increased with the time on diet reaching a maximum of 2974 DEGs at 60 weeks after HF/HS diet initiation.  The number of upregulated DEGs was higher than downregulated DEGs in both male and female mice at all time points.  Functional analysis of DEGs showed that in both sexes, the most dysregulated liver toxicity-related pathways were associated with liver fibrosis, liver necrosis/cell death, inflammation, steatosis, liver damage, and liver proliferation/hyperplasia.  In male mice, pathways associated with fibrosis, necrosis/cell death, inflammation, and steatosis remained constant from 20 to 40 weeks, and activation of liver hyperplasia pathway was constant from 40 and 60 weeks. In contrast, in female mice, dysregulation of genes associated with these pathways gradually increased from 20 to 60 weeks after HF/HS diet initiation.  Importantly, we found that the expression of 701 and 871 genes significantly correlated with total NASH pathology scores (Pearson r > |0.8|) in the livers of male and female mice, respectively.  These findings provide comprehensive insights into the dynamics of time- and sex-specific transcriptomic changes during NAFLD progression.  This knowledge can be helpful for identifying promising targets for prevention and treatment, as well as for determining key potential biomarkers of NAFLD to NASH progression.

overall_design: Liver transcriptomic profiles of male and females CC042 mouse strain fed a HF/HS diet for 20, 40 and 60 weeks were generated by total RNA sequencing using Illumina NextSeq 500 NGS system.

first: Rose

last: Willett

first: Frederick

last: Beland

first: Ivan

last: Rusyn

first: Igor

last: Pogribny


title: Effect of enhanced early life nutrition on anterior pituitary miRNA transcriptome in bull calves.

city: Co. Meath

first: Kate

last: Keogh

address: Dunsany

department: Animal and Bioscience Department

country: Ireland

institute: Teagasc Grange

summary: The aim of this study was to measure the impact of contrasting feeding regimes in the first 12 wk of life, on the molecular control of anterior pituitary tissue in bull calves. Holstein bull calves  were designated to high (HI; n=15) or moderate (MOD; n=12) dietary groups, with diets designed to provoke growth rates of 1.0 and 0.5 kg/day, respectively. At 12 wk of age, all calves were euthanized, and anterior pituitary tissue harvested. RNA was extracted from the anterior pituitary and used for miRNAseq.

overall_design: In total 30 RNA samples were analysed, 15 samples were derived from bull calves fed the High plane of nutrition (HI, n=15) and 15 samples were derived from the calves fed the Moderate plane of nutrition (MOD, n=15). From 2 weeks of age calves on the High-plane of nutrition received 1.5 kg of milk replacer (10 liters) per day and ad libitum concentrates, whilst  te MOD group received 0.5 kg of milk replacer (4 litres) per day and 0.5 kg of concentrates. Tissue samples were collected from all calves at 12 weeks of age.

first: Stephen

last: Coen

first: Pat

last: Lonergan

first: Sean

last: Fair

first: David

last: Kenny


title: Pdgfragene inactivation mediates tissue remodeling in hypomyelinated cerebral cortex

city: Toyama

first: Noriko

last: Okuno

state: Toyama

institute: University of Toyama

summary: Inactivation of the platelet-derived growth factor receptor-α gene in Nestin-positive cells induced not only hypomyelination, but also aberrant blood vessel formation and axonal degeneration in genetically engineered mice (N-PRα-KO mice). Here, the extended analyses, including histological study and single-cell RNA-sequencing, revealed extensive tissue remodeling in the meninges and cerebral cortex of N-PRα-KO mice. Activation of border-associated macrophages was followed by the recruitment of transcriptionally reprogrammed pial fibroblasts to new perivascular locations in the cerebral cortex. These new perivascular fibroblasts were found to be involved in self-sustaining chronic inflammation and play a critical role in tissue remodeling involving various resident cells of the meninges and cerebral cortex. Together with these facts, the heterogeneity of the fibroblasts identified here may contribute to the understanding of the pathological tissue remodeling including fibrotic process in chronic neurological diseases. Activation and mobilization of these fibroblasts may be an effective therapeutic target to prevent chronic progressive neurological diseases.

overall_design: To investigate the heterogeneity of the activated fibroblast in hypomeylinated cerebral cortex of conditional Pdgfra knock out mice(N-PRα-KO), we performed single cell RNA-seq. Cerebral cortex of N-PRα-KO mice and wild type mice as control were dissociated and PDGFRα positive cells were sorted using the CD140a antibody.  We performed gene expression profiling analysis using data obtained from RNA-seq.

first: Seiji

last: Yamamoto

first: Masakiyo

last: Sasahara


title: Reduced somatosensory innervation alters the skeletal transcriptome at a single cell level in a mouse model of type 2 diabetes

city: Baltimore

first: Aaron

last: James

address: 720 Rutland Ave

department: School of Medecine. Surgical Pathology

institute: Johns Hopkins University

summary: Small peripheral neuropathies (SPN) are a common complication in diabetes, affecting around 50% of the diabetic population. Co-occurrence of diabetic peripheral neuropathy (DPN) and diabetic bone disease has led to the hypothesis that DPN influences bone metabolism, although little experimental evidence has yet supported this premise. To investigate, high fat diet (HFD) feeding in mice was performed followed by phenotyping of skeletal-innervating neurons and bone architectural parameters. Results showed that HFD feeding conditions resulted in a marked decrease in skeletal innervation (69-76% reduction in Beta-III-Tubulin-stained nerves, 50-66% reduction in CGRP-stained nerves in long bone periosteum). These changes in skeletal innervation were associated with alterations in bone mass and in cortical and trabecular bone microarchitecture. Single cell RNA-sequencing of sensory neurons and bone tissue was next utilized to reconstruct potential nerve-to-bone signaling interactions, including implication of sensory nerve derived neurotrophins (Bdnf), neuropeptides (Gal, Calca and Calcb) and other morphogens (Vegfa, Pdgfa, and Angpt2). Moreover, scRNA-Seq identified marked shifts in periosteal cell transcriptional changes within HFD fed conditions, including a reduction in proliferation, osteogenic differentiation potency and reductions in WNT, TGFb and MAPK signaling activity. When isolated, periosteal cells from HFD fed mice showed deficits in proliferative and osteogenic differentiation potential. Indeed, these cellular changes in proliferation and differentiation capacity were restored by treatment of HFD exposed periosteal cells to sensory neuron conditioned medium. In sum, HFD modeling of Type 2 diabetes results in a skeletal polyneuropathy. Moreover, the combination of multi-tissue scRNA-Seq and isolated in vitro studies strengthen the case for altered nerve-to-bone signaling in diabetic bone disease.

overall_design: Four left femurs and tibias were dissected from ND and HFD groups at the experimental endpoint for scRNA-Seq. The bones were digested in a pre-warmed dissociation solution (3 mg ml−1 collagenase I (Worthington) and 4 mg ml−1 dispase II (Roche) in PBS) for 1.5 h min at 180 r.p.m. at 37 °C, run through a 40 μm filter (VWR) and rinsed with 3 ml medium (αMEM (Gibco) with 20% FBS, 1% penicillin/streptomycin). Periosteal cells were centrifuged at 300g for 30 min at 4 °C. The cell pellet was washed twice with PBS and spun for 10 min at 300g at 4 °C. Periosteal cells were then resuspended in HBSS at a concentration of ~1,000 cells/μl. Cell viability was assessed with Trypan blue exclusion on a Countess II (ThermoFisher Scientific) automated counter and showed >85% viability. Cells were sent to the JHMI Transcriptomics and Deep Sequencing Core. The library was generated using the 10X Genomics Chromium controller following the manufacturer's protocol

first: Masnsen

last: Cherief

first: Mario-Armando

last: Salazar-Gomez


title: The Role of LINC01235 in Regulating Chromatin Accessibility

city: Shanghai

first: Zhibo

last: Shao

institute: Fudan University Shanghai Cancer Center

type: Genome binding/occupancy profiling by high throughput sequencing

summary: LINC01235 plays a crucial role in HER2-positive breast cancer by regulating global histone modifications (H3K27ac, H3K9ac, H3K36me3) and promoting H2A.Z expression in its regulatory regions. It enhances the accessibility of DNA licensing factors at their promoter regions, leading to increased DNA replication and chromosomal instability. These mechanisms contribute to clonal expansion and resistance to anti-HER2 therapies, highlighting LINC01235 as a potential therapeutic target.

overall_design: ATAC-Seq was performed in ASO-NC and ASO-1235 HCC1954 cells .

first: Qi

last: Zhang

first: Xuliren

last: Wang

first: Bingqiu

last: Xiu


title: Small RNA-Seq analysis of mouse immortalized fibroblasts derived from WT or mir210lox/lox mice and cultured in low or high oxygen conditions

first: Kevin

last: Lebrigand

first: Bernard

last: Mari

first: Ivan

last: Mircea

first: Pascal

last: Barbry


title: Enrichment of ornithine decarboxylase degron transduced colorectal cancer cells for extended application of cancer stem cell models [colorectal cancer]

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

institute: NGS core facility, Research Institute for Microbial Diseases, Osaka University

summary: Cancer stem cells (CSCs) are present in small quantities in tumor populations. To permit various analyses of CSCs, we attempted to enrich and expand ornithine decarboxylase (ODC) degron-transduced colorectal cancer (CRC) cells, which retain low proteasome activity. ZsGreen fluorescence-positive (ZsGreen+) cells were collected by sorting the ODC degron-transduced HCT116, DLD1, and SW480 cells, which were defined as enriched ZsGreen+ cells. ZsGreen+ cells still maintained CSC properties. These cells had higher stem cell marker expression and increased resistance to chemotherapy with 5-fluorouracil and oxaliplatin. ZsGreen+ HCT116 and DLD1 cells had greater sphere-forming ability and enhanced tumorigenicity compared to ZsGreen- control cells. Time-lapse microscopy showed that a single enriched ZsGreen+ HCT116 cell had asymmetric cell division. Thus, model CSCs were acquired in sufficient quantity. Using these cells, we performed a comprehensive microRNA analysis; miR- 491-3p was a candidate to suppress cancer stemness. Finally, we found that up- regulated genes in the enriched HCT116 ZsGreen+ cells correlated with those up- regulated in human clinical spheroid samples established from patient-derived xenografts derived from CRC tissue samples, further supporting the acquisition of enriched model CSCs. These cells would be useful in identifying novel CSC markers and developing medicine for anti-CSC therapy.

overall_design: RNA-sequencing analyses were performed in colorectal cancers

first: Ryo

last: Ikeshima

last: Okuzaki

first: Hirofumi


title: Gene expression profile at single cell level of aorta from the Slc38a2flox/flox mice and Slc38a2△EC mice

city: Dalian

first: Chunxiu

last: Du

address: Lushunkou District

institute: Dalian medical university

summary: We used single cell RNA sequencing (scRNA-seq) to analyze the different of aotra cells between Slc38a2flox/flox and Slc38a2 △EC mice.

overall_design: We gave Slc38a2flox/flox and Slc38a2△EC mice a high-salt diet for 3 weeks and we collected the same part of aorta from Slc38a2flox/flox and Slc38a2△EC mice. we analyzed mRNA transcripts of single cells extracted from the aorta using the BD Rhapsody.

first: Xiaoyan

first: Youfei

last: Guan


title: LINC01235 regulate global histone modifications and enhances licensing factor accessibility

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChlP-seg) for histone variants H2AZ as well as the histone modifications H3K27ac, H3K9ac and H3K36me3 in HCC1954 cells


title: Role of LINC01235 on transcriptional output in HCC1954 HER2 positive breast cancer cells

overall_design: RNA-Seq was performed inASO-NC and ASO-1235 HCC1954 cells .