title: RNASeq from "a cell-nonautonomous heme acquisition pathway enables erythroid hemoglobinization under stress"

city: Baltimore

first: Iqbal

last: Hamza

state: MD

address: 670 W Baltimore St

department: Pediatrics

institute: University of Maryland Baltimore

summary: Heme is an essential iron-containing cofactor synthesized in mitochondria through a conserved eight-step enzymatic pathway. Cells have been thought to manage their own heme supply independently. However, over 1,000 proteins contribute to the production, transport, and regulation of the cofactor. During terminal erythroid differentiation, cells lose their mitochondria yet continue to make hemoglobin, implying a non-canonical, cell-nonautonomous heme source. We show that under stress, erythroid precursors import heme through the permease Heme Responsive Gene 1 (HRG1), which localizes to the plasma membrane and accumulates during stress erythropoiesis, an accelerated production of red blood cells outside the bone marrow in response to anemia. HRG1 loss impaired heme uptake, inhibited terminal erythroid differentiation, and caused anemia. With beta-thalassemia mice, partial loss of HRG1 improved ineffective erythropoiesis, underscoring the importance of balanced heme import. These findings reveal intercellular heme sharing and identify HRG1 as a potential therapeutic target in hemoglobinopathies.

overall_design: RNASeq of WT and HRG1-KO K562 cells treated or not treated with sodium butyrate at Day8 of differentiation

first: Audrey

last: Belot

first: Andrew

last: Rock

first: Sohini

last: Dutt

first: Gia

last: Haemmerle

first: Amaury

last: Maros

first: Xiaojing

last: Yuan

first: Satoru

last: Otsuru

first: David

last: Bodine


title: Minimally invasive 1 mm skin biopsies capture regional transcriptomic heterogeneity in vitiligo

status: Public on Jun 03 2026

city: Ghent

first: Yannick

last: Gansemans

address: Ottergemsesteenweg 460

department: Pharmaceutical Sciences

country: Belgium

institute: Ghent University

type: Expression profiling by high throughput sequencing

summary: Vitiligo is an autoimmune depigmentation disorder characterized by melanocyte loss and complex immune dysregulation. Despite therapeutic advances, variable efficacy and frequent relapse highlight the need for deeper insight into its pathophysiology. Conventional 3–5 mm skin biopsies are invasive and require suturing, limiting their use in clinical and translational research. Here, we demonstrate that minimally invasive 1 mm punch biopsies yield adequate RNA compatible with bulk transcriptomic profiling and enable comprehensive molecular characterization of vitiligo skin. RNA sequencing of 105 biopsies spanning lesional, perilesional, non-lesional, and control skin revealed distinct region-specific transcriptional signatures involving interferon signaling, immune activation, and metabolic reprogramming. Beyond canonical melanocyte and immune pathways, we identify underexplored dysregulation of cellular clearance and structural regulatory mechanisms, implicating ferroptosis, peroxisome, and efferocytosis pathways in lesion persistence. Connectivity Map analysis further predicted candidate compounds capable of reversing the lesional transcriptional signature, pointing to epigenetic regulators, tyrosine kinase inhibitors, and metabolic modulators. These findings establish 1 mm skin biopsies as a feasible and biologically informative tool for minimally invasive molecular profiling and highlight new therapeutic axes in vitiligo pathogenesis.

overall_design:
Gene expression profiling of lesional, perilesional and non-lesional skin punch biopsies of vitilago patients.

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

first: Danique

last: Berrevoet

first: Arno

last: Belpaire

first: Elise

last: Van Caelenberg

first: Barbara

last: Boone

first: Guillaume

last: Cattebeke

last: Callens

first: Koen

last: Deserranno

first: Dieter

last: Deforce

first: Reinhart

last: Speeckaert

first: Filip

last: Van Nieuwerburgh


title: A cell-nonautonomous heme acquisition pathway enables erythroid hemoglobinization under stress

first: Iqbal

last: Hamza

country: USA

overall_design: BM Ter119+ cells were counted and viability assessed using a Countess 3 (Invitrogen, Carlsbad,CA). Cell suspensions were loaded onto a 10x Genomics Chromium X (Pleasanton,CA) using the NextGEM 3 V3.1 Gel bead kit. Cell capture, GEM generation, cDNA amplification, and library preparation were performed according to manufacturer protocol. The resulting 3 gene expression libraries were assessed for concentration and fragment size using the DNA High Sensitivity Assay on a GX Touch (Revity, Waltham, MA). The libraries were pooled, assessed by qPCR using the KAPA Library Quantification Kit (Complete, Universal) (Kapa Biosystems, Woburn, MA), and sequenced on an Illumina NovaSeq 6000 using 100bp PE reads (Illumina, San Diego, CA). The resulting sequence data were demultiplexed using Illuminas bcl2fastq2 Conversion Software v2.20. Each sample was analyzed using 10x Genomics Cell Ranger v8.0.0. The reference for gene expression was refdata-gex-GRCh38-2024-A, available at https://www.10xgenomics.com/support/software/cell-ranger/downloads.

first: Audrey

last: Belot

first: Andrew

last: Rock

last: Dutt

last: Osturu


title: DNA methylation changes in PBMCs of multiple sclerosis patients on glatiramer acetate therapy

city: Moscow

first: Ivan

middle: S

last: Kiselev

address: Ostrovitianov st. 1

country: Russia

institute: Pirogov Russian National Research Medical Universitty

type: Methylation profiling by genome tiling array

summary: DNA methylation is a universal epigenetic mechanism involved in the regulation of gene expression in both normal physiological conditions and various pathologies. It has been demonstrated that the development of multiple sclerosis (MS) is accompanied by significant alterations in DNA methylation profiles in the blood immune cells of patients. Studies investigating genome-wide DNA methylation changes during therapy for MS patients using immunomodulatory drugs are limited, and for glatiramer acetate (GA), such investigations have not been conducted at all.

overall_design: We performed genome-wide DNA methylation profiling in PBMCs of four female MS patients before the first administration of GA (time point “0 months”), as well as 4.2 ± 0.5 and 7.9 ± 0.5 months after therapy initiation (time points “4 months”, and “8 months”, respectively).


title: Modulating MyoD1 dosage activates alternate cell fate beyond myogenic differentiation [RNA-seq]

city: Berkeley

first: Oscar

last: Whitney

address: 1951 Oxford St

institute: UC Berkeley

summary:
Transcription factor (TF) expression and dosage regulate developmental cell fate decisions. Increased TF dosage has been predicted to enhance expression of high-affinity target genes but also increase the binding of lower-affinity loci. The relative importance of high- versus lower-affinity TF binding in guiding cell fate decisions remains unclear.
To test the roles of TF dosage, we examined the effects of increasing the dosage of MyoD1, the ?master regulator of myogenesis?, on skeletal muscle differentiation. Unexpectedly, increased MyoD1 dosage inhibited canonical myogenesis and redirected myoblast differentiation towards forming spontaneously contracting myotubes. This novel phenotype was driven by the MyoD1-dose-dependent upregulation of non-myogenic genes, including cell adhesion genes whose ectopic expression also inhibited classical myogenic differentiation and enabled myotube contraction.
Live-cell single-molecule imaging showed that elevated MyoD1 dosage increased total chromatin binding and CUT&RUN profiling demonstrated that this increase occurred via preferential binding to lower-affinity loci. Integration of CUT&RUN, ATAC-seq and RNA-seq experiments revealed that increased MyoD1 binding correlated to the upregulation of otherwise unexpressed or lowly expressed genes. These findings suggest that increased MyoD1 dosage induced a selective gene regulatory expansion from high- to lower-affinity cis-regulatory elements, leading to activation of a broader ensemble of target genes, revealing a TF dose-dependent mechanism that can trigger distinct developmental programs. This submission contains additional RNA-seq samples generated that should be added to existing GEO Series GSE309864.

overall_design: RNA-seq profiling of C2C12-derived inducible MyoD1, Cdh23, and Mcam cell lines during serum-starvation-induced myogenic differentiation, with or without doxycycline induction. Additional 120 h differentiated C2C12 iMyoD1 cultures were profiled as bulk populations or after 40 um filtration into myoblast-enriched and myotube-enriched fractions.


title: Modulating MyoD1 dosage activates alternate cell fate beyond myogenic differentiation [CUT&Run]

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Transcription factor (TF) expression and dosage regulate developmental cell fate decisions. Increased TF dosage has been predicted to enhance expression of high-affinity target genes but also increase the binding of lower-affinity loci. The relative importance of high- versus lower-affinity TF binding in guiding cell fate decisions remains unclear. To test the roles of TF dosage, we examined the effects of increasing the dosage of MyoD1, the “master regulator of myogenesis”, on skeletal muscle differentiation. Unexpectedly, increased MyoD1 dosage inhibited canonical myogenesis and redirected myoblast differentiation towards forming spontaneously contracting myotubes. This novel phenotype was driven by the MyoD1-dose-dependent upregulation of non-myogenic genes, including cell adhesion genes whose ectopic expression also inhibited classical myogenic differentiation and enabled myotube contraction. Live-cell single-molecule imaging showed that elevated MyoD1 dosage increased total chromatin binding and CUT&RUN profiling demonstrated that this increase occurred via preferential binding to lower-affinity loci. Integration of CUT&RUN, ATAC-seq and RNA-seq experiments revealed that increased MyoD1 binding correlated to the upregulation of otherwise unexpressed or lowly expressed genes. These findings suggest that increased MyoD1 dosage induced a selective gene regulatory expansion from high- to lower-affinity cis-regulatory elements, leading to activation of a broader ensemble of target genes, revealing a TF dose-dependent mechanism that can trigger distinct developmental programs.

overall_design: RNA-seq profiling of C2C12 myoblasts expressing endogenous or induced levels of MyoD1, with simultaneous serum-starvation induced myogenic differentiation

first: Whitney

last: Oscar


title: Microglia in diffuse midline glioma contribute to extracellular matrix remodelling and cancer cell invasion

city: Stockholm

first: Martin

last: Skandik

address: Nobels vag 13

country: Sweden

institute: Karolinska Institutet

summary: Background: Diffuse midline glioma, H3K27-altered (DMG) are aggressive and universally fatal paediatric tumours that arise in midline structures of the brain. DMG tumours are characterized by a substantial infiltration of tissue-resident microglia. The aim of this study was to determine whether in response to stimulation by DMG cells, microglia could acquire a reactive state that contributes to the progression of DMG. Methods: The microglial transcriptomic response to DMG cells was analysed by RNA-sequencing of microglia exposed to DMG, H3K27M cells and of tumour-associated myeloid cells isolated from DMG tumour biopsies. Meta-analysis of human paediatric glioma cohorts (Kids First, PNOC, and CBTTC) was used to confirm upregulation of expression for selective genes in DMG patients. Immunofluorescence analysis performed on archival DMG biobank tissues was used to confirm the findings at protein level. Invasion assay’s using a panel of DMG, H3K27M cells was used to evaluate the contribution of microglia-derived fibronectin,to promote DMG cell invasion. Results: Microglia are enriched in human DMG, H3K27M tumours. Upon stimulation by DMG, H3K27M cells, microglia contribute to the expression of extracellular matrix (ECM) components. Microglia-derived fibronectin promotes tumour invasiveness, and its inhibition reduces DMG cell migration. Across patient cohorts, DMG, H3K27M tumours exhibit elevated expression of myeloid cell-derived ECM components, with fibronectin expression showing prognostic value. Conclusions: Microglia appear to actively contribute to the diffuse invasive nature of DMG by producing ECM components, particularly fibronectin, which enhances tumour invasiveness. These findings identify microglia as potential therapeutic targets in DMG.

overall_design: RNA-Seq profiling of in vitro BV-2 microglia exposed to patient-derived pediatric High Grade Glioma (pHGG) SF188 cells and patient-derived pediatric Diffuse Midline Glioma (DMG) SF8628 cells for 3 and 6 hours

first: Lily

last: Keane

last: Škandík

first: Mercedes

last: Posada-Perez

first: Bertrand

last: Joseph


title: Modulating MyoD1 dosage activates alternate cell fate beyond myogenic differentiation [ATAC-seq]

summary: The study investigates how MyoD1 dosage controls myogenic cell fate, employing bulk RNA-seq, ATAC-seq, and CUT&RUN. We determined genome-wide transcriptional, chromatin accessibility, and MyoD1 binding profiles in C2C12 myoblasts expressing endogenous Halo-tagged MyoD1 or doxycycline-inducible Halo–MyoD1, under growth and differentiation conditions with low or high MyoD1 dosage.

overall_design: RNA-seq and ATAC-seq were performed in doxycycline-inducible Halo–MyoD1 C2C12 myoblast, myoblast or myotube fractions, or C2C12 inducing adhesion genes. Cells were cultured in growth or differentiation medium with or without doxycycline to modulate inducible protein expression, using 2-4 biological replicates per matched conditions. MyoD1 CUT&RUN was performed in a C2C12 line homozygous for 3×FLAG–Halo–MyoD1 and carrying additional inducible 3xFLAG-Halo–MyoD1 alleles, under low and high MyoD1 induction, with no-antibody controls, using three biological replicates per matched condition.

middle: N

first: Gina

middle: M

last: Dailey

first: Joseph

middle: K

last: McKenna

first: Xavier

last: Darzacq

first: Robert

last: Tjian


title: A pathway from the anterior cingulate cortex to the lateral habenula controls chronic pain induced depression in male mice

city: Strasbourg

first: Pierre-Eric

last: Lutz

address: 8 allée du général Rouvillois

country: France

institute: Institut des Neurosciences Cellulaires et Intégratives, INCI CNRS UPR3212

summary: Chronic pain induces long-lasting changes in the anterior cingulate cortex (ACC) activity that contribute to the development of comorbid mood disorders. However, how such alterations propagate throughout the ACC connectome remains to be elucidated. Here, we aimed to study the role of the ACC neurons projecting to the lateral habenula (LHb) (ACC-->LHb) in chronic pain induced depression (CPID). CPID was induced using sciatic nerve cuffing in male C57BL/6J mice. Anxiodepressive-like behaviors were evaluated using a battery of behavioral tests. Fiber photometry was used to study the Ca2+ dynamics in the ACC, LHb and AC-->LHb neurons. We combined viral Translating Ribosome Affinity Purification (vTRAP) and RNA sequencing to study the molecular alterations in the ACC-->LHb neurons. Finally, we used an optogenetic approach to study the functional role of this pathway in CPID. Our results confirmed a functional connectivity between the ACC and LHb and demonstrated that this connection plays a critical role in emotional processing. Activation of ACCLHb neurons elicited Ca2+ responses in the LHb and induced anxiodepressive-like behaviors in naive mice. Cell-type specific transcriptomic analysis revealed that CPID altered the expression of genes involved in neuronal excitability such as genes related to sphingolipid metabolism, glycophospholipids and Ca2+ channels in ACCLHb neurons. Interestingly, inhibition of this hyperactivity alleviated chronic pain- but not stress-induced anxiodepressive-like behaviors, demonstrating that the ACCLHb pathway selectively contributed to pain-induced emotional dysregulation. These results reveal that hyperactivity of the neuronal pathway linking the ACC to the LHb is essential for CPID in male mice.

overall_design: We used viral translating ribosome affinity purification (vTRAP; see Nectow et al, Cell Reports 2017) to immuno-precipitate (IP) the RNA fraction being translated by glutamatergic neurons from the ACC projecting to the LHb, in mice.

first: Sarah

last: Journée

first: Ipek

last: Yalcin


title: Extracellular vesicle-derived circRNA profiles from cART suppressed pregnant women living with HIV identifies interactome networks key to inflammation and viral latency

city: Atlanta

first: Erica

middle: L

last: Johnson

state: GA

address: 720 Westview Dr SW

department: Department of Microbiology, Biochemistry, and Immunology

institute: Morehouse School of Medicine

summary: Pregnant people living with HIV (PLWH) experience a disparate risk of chronic non-AIDS-related morbidities (NAMs) during pregnancy compared to people without HIV infection. Extracellular vesicles (EV) are fundamental to both HIV pathogenesis and maternal-placental-fetal systemic intercellular networking. However, there is limited research addressing the intersection of EV cargo and HIV during pregnancy. In this study, plasma-derived EV small RNA (smRNA) in pregnant PLWH as compared to pregnant people living without HIV (PLWoH) was examined through low-input RNA sequencing (Illumina PE150). Differential expression analysis identified significant alterations of EV circRNA levels for pregnant PLWH as compared to pregnant PLWoH. Novel interactome networks (circRNA-miRNA-gene and circRNA-protein) were generated from these differentially expressed EV circRNAs. Gene set enrichment analysis revealed physiological pathways influential to HIV reservoir establishment, latency, replication, immune activation, immunosenescence, as well as maternal-placental-fetal immune cross talk. These findings suggest that EV circRNA-mediated regulatory mechanisms may contribute to HIV persistence, inflammation, and immune modulation in pregnancy.

overall_design: RNA-seq profiling of plasma-derived EVs from pregnant PLWH (n=8) as compared to pregnant PLWoH (n=10)

first: Brena

last: Dara

first: Schuch

last: Viviane

first: Johnson

last: Erica L.


title: Molecular and cellular mechanisms of IL-27-induced neuroprotection in a mouse model of inherited retinal degeneration

city: Miami

first: Ashley

last: Zuniga

state: Florida

address: 1501 nw 10th avenue

department: Ophthalmology

institute: University of Miami

summary: Retinal degenerations are associated with aberrant activation of the innate immune system and broad acting anti-inflammatory agents partially reduce the amount of degeneration in animal models. Interleukin-27 (IL-27) is a cytokine in the IL-12 family that has anti-inflammatory activity in the CNS. We previously demonstrated in the rd10 mouse model of inherited retinal degeneration that an intravitreal injection of recombinant IL-27 provided photoreceptor protection and vision rescue for 2 weeks after injection. However, the molecular and cellular mechanisms by which IL-27 mediates its neuroprotective effects and the duration of protection are unknown. In this study, we tested the hypothesis that reducing inflammation at the early stages of degeneration prior to substantial photoreceptor loss would prolong vision rescue. Rd10 mice received an intravitreal injection of 20 ng IL-27 or saline prior to degeneration, and photoreceptor survival was measured 4 and 7 weeks later by optomotor assay, ERG, TUNEL and cone photoreceptor density. Early molecular changes were assessed by snRNAseq, cytoplex and immunohistochemistry. We demonstrated that IL-27 induced sustained retina protection compared to saline at 4 but not 7 weeks after injection. Additionally,  snRNAseq analysis two days after injection demonstrated suppression of a unique Muller glia subpopulation in IL-27 treated mice and differential expression of distinct gene categories across glial and photoreceptor cell types, including genes involved in ATP synthesis, transcription and translation, apoptosis and extracellular matrix. Therefore, reducing reactive inflammation prior to the peak of photoreceptor degeneration using IL-27 leads to changes in numerous cellular pathways and sustained neuroprotection in rd10 mice. Additionally, these findings suggest that suppression of a reactive Muller glia subset and critical glial signaling pathways contribute to the neuroprotective effect of IL-27.

overall_design: Inherited retinal degeneration was modeled using rd10 mice. To test whether early delivery of IL-27 alters disease progression, mice received a single intravitreal injection of recombinant IL-27 (20 ng) or saline prior to onset of rapid degeneration. Retinal tissue was collected at early and late timepoints for functional, structural, and molecular assessment.

first: Laura

last: McGee

first: James

last: Dollar

first: Brandon

last: Bessen

first: Marco

last: Galizia

first: Saili

last: Khorjekar

first: Stefan

last: Kurtenbach

first: Abigail

last: Hackam


title: Single-cell RNA and BCR sequencing of age-associated B cells in mouse and human lupus

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

country: Japan

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

summary: To characterize age-associated B cells (ABCs), we performed single-cell RNA and BCR sequencing on splenic B-lineage cells from a murine lupus model induced by Toll-like receptor 7 (TLR7) stimulation and on peripheral blood B-lineage cells from a treatment-naïve patient with systemic lupus erythematosus (SLE). We identified a population of ABCs with features of plasmablast precursors (pre-PB ABCs). In the TLR7-induced lupus model, B cell-specific Commd3 deletion reduced the frequency of pre-PB ABCs and was associated with increased apoptosis in this population.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: For the F5581 dataset, follicular B cells, germinal center B cells, ABCs, and PBs were sorted from the spleens of wild-type mice treated with the TLR7 agonist resiquimod. Each cell population was labeled with a distinct cell-hashing antibody (TotalSeq-C0301, -C0302, -C0303, and -C0304) and pooled in equal numbers. For the human F9545 dataset, ABCs and PBs were sorted from the PBMCs of the SLE patient, labeled with TotalSeq-C0251 and -C0252, and pooled at a 4:1 ratio. For the F10020 dataset, ABCs were sorted from the spleens of control and B cell-specific Commd3-deficient mice treated with resiquimod, labeled with TotalSeq-C0301 and -C0303, and pooled in equal numbers.

first: Taiichiro

last: Shirai

first: Kentaro

last: Kuzuya

first: Yu-Chen

last: Liu

last: Okuzaki

first: Kazuhiro

last: Suzuki


title: The effect of SRSF3 on gene and splicing profile of Tregs in mouse

city: Wuhan

first: can

last: Lin

state: Hubei province

address: 237 Luoyu Road

department: School & Hospital of Stomatology

country: China

institute: Wuhan University

summary: We have performed a single cell third generation sequencing assay by using Oxford Nanopore Technologies(ONT) assay system in Tregs from a Srsf3fl/+Foxp3Cre-YFP mouse to understand the effect of SRSF3 on gene and splicing of Tregs. SRSF3 is a splicing factor and plays multiple roles in cells, such as RNA splicing, RNA stability, alternative polyadenylation, translation, and miRNA biogenesis. This study analyzed the gene expression and alternative splicing profile in Tregs.

overall_design: Srsf3flox/flox mice were crossed to Foxp3Cre-YFP mice to obtain Srsf3fl/+Foxp3Cre-YFP chimeric female mice. Regulatory T cells (Tregs) were isolated from Srsf3fl/+Foxp3Cre-YFP female mice spleen by MACS using CD4+CD25+ Regulatory T Cell Isolation Kit according to the manufacturer's instructions. Tregs were subjected to single cell full-length transcriptome sequencing (Oxford Nanopore Technologies). The gene expression and alternative splicing profile of Tregs was analyzed according to the scRNA-seq data.

first: Rong

last: Jia

first: Jihua

last: Guo

first: Wanjun

last: Chen

first: Can


title: Gene expression profile of intratumoral Tregs in a FOXP3 humanized mice

summary: We have performed a single cell sequencing assay by using DNBelab C high-throughput single-cell RNA-seq system to study intratumoral Treg cells. Treg cells promote tumorigenesis by suppressing immune responses against tumor. FOXP3 protein is the master controller of Treg function. In human, FOXP3 gene produces two isoforms by the alternative splicing of exon 2. However, mouse only produces full-length FOXP3. This study analyzed the gene expression profile of intracellular Treg cells from wild-type mice or Foxp3 humanized mice which produce both full-length and exon 2-skipped FOXP3 protein.

overall_design: MC38 cells were inoculated in wild-type mice and FOXP3 humanized mice. MC38 tumors were harvested from mice on the 20th day following tumor implantation, and dissociated into single-cells. Tumor-infiltrating immune cells were isolated and  subjected to single cell RNA-seq (scRNA-seq). The gene expression profile of intracellular Treg cells was analyzed according to the scRNA-seq data.


title: Spatial transcriptomic profiling of the human aortic valve reveals cellular sex differences near sites of calcification

city: La Jolla

first: Talia

last: Baddour

state: California

address: 9500 Gilman Dr.

department: Bioengineering

institute: University of California San Diego

summary: Sex differences in aortic valve stenosis (AVS) progression have been documented clinically, but the underlying cellular mechanisms that drive sex-dependent calcification in aortic valve tissue remain poorly understood. Here, we harnessed single cell and spatial transcriptomics to investigate mechanisms that drive sex dependent spatial organization of valvular interstitial cell (VIC) and macrophage gene expression near calcification sites in human male and female aortic valve tissue. Histological analyses of aortic valve tissues stratified into healthy and diseased cohorts based on degree of calcification reveal increased valve calcification area in diseased male aortic valves relative to female, and increased valve thickening in diseased female aortic valves. Single cell sequencing analysis of heterogeneous valvular interstitial cell (VIC) populations reveals male-dependent gene expression of the Activator Protein 1 (AP-1) transcription factor complex. Spatial transcriptomics and RNA-FISH analyses of VIC populations near sites of calcification revealed male-dependent gene expression localization of Cartilage Oligomeric Matrix Protein (COMP), as opposed to diffuse COMP expression in female VICs. Cell-cell communication analyses were used to determine female-specific macrophage-VIC interactions. Secreted phosphoprotein 1 (also known as osteopontin) expressed from macrophages interacts with the cell surface receptor CD44 expressed by VICs to drive a pro-fibrotic phenotype in female aortic valves. Together, our results reveal sex differences in VIC and macrophage heterogeneity and functions near sites of calcification in aortic valve tissue. Our results highlight the importance of sex-based transcriptomics analyses to understand the cellular phenotypes responsible for causing sex differences in aortic valve fibrosis calcification.

overall_design: Aortic valves were dissected and split in half for single cell and spatial trenacriptomics. Cells were isolated from human healthy and diseased aortic valve leaflets and run through 10X genomics single cell RNA sequencing workflows. For a subset of patients, tissues were cryopreserved and using for 10X Visium workflows.

first: Van

last: Ninh

first: Rayyan

last: Gorashi

first: Rene

last: Peña

first: Kevin

middle: R

last: King

first: Brian

middle: A

last: Aguado


title: TUT1-catalyzed U6 snRNA 3′-end maturation is required for mRNA splicing to maintain stem cell pools during embryogenesis [Nanopore long-read RNA-seq]

city: Chengdu

first: chao

last: yang

state: Sichuan

address: No.20, Section 3, Renmin South Road

department: West China Second University Hospital

institute: Sichuan University

summary: The post-transcriptional maturation of U6 snRNA 3′-end is important for spliceosomal assembly and RNA splicing, and is catalyzed by sequential enzymatic activities of TUT1 and USB1, that is, an oligo(U) tail is first added by TUT1 to the freshly transcribed U6 snRNA, and then trimmed by USB1 to generate mature U6 snRNA.  However, biallelic inactivation of TUT1 or USB1 is linked to distinct human developmental disorders, and their physiological functions remain unknown.  Here, using multiple genetically engineered mouse models, we show that Tut1, but unexpectedly not Usb1, is essential for the maintenance of the epiblast and neural stem cells in vivo.  Loss of Tut1 weakens the interactions between essential splicing factors and U6 snRNA, causes defective RNA splicing, and triggers massive DNA damage and subsequent cell death.  Importantly, cell death can be prevented by recombinant U6 snRNA containing an oligo(U) tail.  These results challenge the current model of U6 snRNA 3′-end maturation and function.  We propose that TUT1 is an essential U6 snRNA 3′-end maturation or repair factor, and discuss the implications of these results for understanding the etiologies of TUT1- and USB1-related human disorders.

overall_design: We performed Nanopore long-read RNA sequencing of neural stem cells to investigate whether Tut1 regulates mRNA splicing.  Neural stem cells were derived from Tut1-floxed mice (Tut1f/f), Trp53-floxed mice (Trp53f/f), and double-floxed mice (Tut1f/f;Trp53f/f), respectively.  Tut1f/f neural stem cells treated with LNP alone was used as a control.  Tut1f/f, Trp53f/f, and Tut1f/f;Trp53f/f neural stem cells were treated, respectively, with LNP/Cre-mRNA,  Tut1f/f neural stem cells treated with LNP/Cre-mRNA and U6 snRNA for 3 days.


title: Transcriptomic Characterization of Doxorubicin Resistance in Bone Sarcoma Organoids

city: Oviedo

last: Rodriguez

state: Asturias

address: Av. Hospital Universitario s/n

country: Spain

institute: Health Research Institute of Asturias (ISPA)

summary: Tumor organoids have emerged as the most promising avatar to model cancer in vitro and to be used as drug testing platform in personalized medicine protocols. The derivation of organoids from epithelial tumors is well established, and their use has proven useful for biomarker discovery, drug screening, and clinical decision support. However, the generation of organoids from sarcomas is much less developed and current protocols require further optimization to fully harness their research and clinical potential. In this study, we explored the challenges of generating basement membrane extract (BME)-based organoids from patient-derived sarcoma samples. Of the 19 samples processed, 3D structures formed in 7 (37%), yet none of these organoids could be propagated beyond the first passage. As an alternative approach, we successfully generated 3D organoids with long-term growth potential from sarcoma xenografts. Of the 14 xenograft samples tested, 7 (50%) could be maintained through at least three passages and were successfully biobanked. Specifically, organoids derived from xenografts grown from parental and doxorubicinresistant 143B osteosarcoma cells could be maintained as long-term cultures and used as a 3D platform to investigate drug resistance in osteosarcoma. Transcriptomic profiling and functional assays performed in these models identified the nonreceptor protein tyrosine kinase SRC as a key mediator of resistance to doxorubicin in osteosarcoma. Importantly, we demonstrated that inhibition of SRC signaling using dasatinib restores doxorubicin sensitivity in osteosarcoma cells by suppressing c-JUN–driven ABCB1 expression.

overall_design: RNA-seq profiling of 143B DX-R organoids in contrast with the 143B Parental organoids.

first: René

last: Rodríguez

first: Borja

last: Gallego


title: TUT1-catalyzed U6 snRNA 3′-end maturation is required for mRNA splicing to maintain stem cell pools during embryogenesis [RNA-Seq]


title: Senescent Eimeria acervulina oocysts maintain transcriptional activity during extended refrigerated storage and differerntially express characteristic genes

city: Bradenton

first: Matthew

last: Tucker

address: 5000 Lakewood Ranch Blvd.

institute: LECOM

summary: Enteric coccidian parasites harm agriculture and human health. Infectious, sporulated parasites eventually senesce. Here, we examined transcriptional changes in sporulated oocysts of Eimeria acervulina stored for 4-30 months at 4°C. Precipitous decline in RNA abundance and transcription followed an interval of stability. Sixty constitutively expressed genes each contributed >1,000 transcripts per million (TPM) throughout, including a serine protease inhibitor, surface antigen genes, a cation-transporting ATPase, an oocyst wall protein, a zinc finger DHHC domain-containing protein, and highly expressed hypothetical proteins with no known function. Strikingly, ~82% of 6,867 annotated genes underwent differential expression when comparing freshly sporulated parasites to those held for 30 months; nearly one-third of these underwent significant expression change. In freshly sporulated oocysts, 86 significantly DEGs exceeded 1,000 TPM; these encoded heat shock proteins, lactate dehydrogenase, glucose- 6 isomerase, and various hypothetical proteins. The oldest parasites expressed 66 DEGs, including many ribosomal subunits, a haloacid dehalogenase-like hydrolase domain-containing protein, and various hypothetical proteins. Taken together, we identified markers of mature parasites that remain relatively abundant in the transcript pool as oocysts age and identified other transcripts (e.g. ribosomal RNA) that increase in their relative abundance even as RNA abundance declines in senescent parasites.

overall_design: To investigate transcriptional changes during oocyst senescence that may help identify biomarkers of viable and non-viable E. acervulina, oocysts were harvested from chickens and then allowed to sporulate. Freshly unsporulated and sporulated oocysts were collected and sporulated oocysts were stored at 4C for periods of time out to 30 months. The freshly collected samples and those stored for 4, 8, 13, 18, 25, 29, and 30 months were processed for RNA. Illumina mRNA sequencing libraries were made from three technical replicates per each cohort RNA sample and then subjected to Illumina sequencing

first: Doaa

last: Naguib

first: Celia

last: O'Brien

first: Christy

last: Yeager

first: Asis

last: Khan

first: Benjamin

last: Rosenthal

first: Mark

middle: C

last: Jenkins


title: Single-nucleus multi-omics profiling reveals cell state transitions and epigenetic programs in lung adenocarcinoma

city: Seoul

first: Yeji

last: Kim

state: Seoul

address: 222, Banpo-daero, Seocho-gu, Seoul

country: South Korea

institute: The Catholic University of Korea

summary: Using single-nucleus multi-omic sequencing, we assessed transcriptional and epigenetic programs in lung adenocarcinoma (LUAD). Our results identified target genes and transcription factors associated with open chromatin regions in epithelial cells. Furthermore, ROS1 fusion was linked to activation of proliferative signaling pathways. These findings provide insight into LUAD progression and potential therapeutic targets.

overall_design: We performed multiplexed single-nucleus RNA-seq (snRNA-seq) and single-nucleus ATAC-seq (snATAC-seq) on lung adenocarcinoma samples collected from two patients (T255 and T264) using the 10x genomics Multiome protocol. Pooled nuclei were demultiplexed post hoc using Souporcell (https://github.com/wheaton5/souporcell) based on SNPs from donor-specific VCF files generated by whole exome sequencing.

first: Hae-Ock

last: Lee

first: Seung Joon


title: Epicardial-derived cells secrete extracellular vesicles that modulate gene expression in the cardiac response to ischemia-reperfusion injury [BW mRNAseq]

city: Toronto

first: Milica

last: Radisic

state: Ontario

address: 160 College St

country: Canada

institute: University of Toronto

summary: The epicardium is an essential regulator of cardiac development, homeostasis, and injury. While advanced 3D human cardiac tissue models now facilitate physiologically relevant exploration of epicardial–myocardial interactions, the composition and impacts of epicardial cell-secreted extracellular vesicles (EVs) in regulating cardiac physiology remain incompletely understood. Here we harnessed an “Epicardial Biowire” platform integrating human epicardial cells with functional myocardium, applying transcriptomics to reveal enriched vesicle transport in engineered tissues containing epicardial cells. Profiling epicardial-EVs identified key miRNAs in their cargo and indicated considerable conservation of miRNA cargo through stimulated epithelial‐to‐mesenchymal transition. Supplementation of epicardial-EVs to engineered tissues undergoing simulated ischemia–reperfusion injury (IRI) influenced gene expression associated with reduction of extracellular matrix remodeling, fibroblast modulation, and suppression of cell-ECM interactions. Correlation of EV‐miRNAs with mRNA targets highlighted implications of miR‐30d‐5p, miR‐9‐5p, miR‐16‐5p, and the let‐7 family, supporting a potential role for epicardial-EVs and their miRNA cargo in moderating deleterious fibrotic activation and matrix remodelling during myocardial injury in vitro. Collectively, these findings expand our understanding of epicardial-EV composition and function, highlighting epicardial signalling as a promising avenue to direct cardiac repair and inspiring the design of future EV‐based therapeutics.

overall_design: RNA-seq profiling of engineered human 'Epicardial Biowire' cardiac tissues. Tissue mRNA consisted of 4 groups: mRNA was isolated and sequenced from engineered tissues fabricated with human iCell cardiomyocytes and primary human cardiac fibroblasts with ('Epi') and without ('NoEpi') the addition of hES-derived epicardial cells to tissues, from engineered Epicardial Biowire tissues exposed to simulated ischemia-reperfusion injuiry ('EpiIRI'), and from injured Epicardial Biowire tissues supplemented with mature epicardial cell-derived EVs added to the culture media during and after injury ('EpiIRI_SBEV'). For all tissues, mRNA was isolated on day 15 after tissue seeding (and 7 days after injury, where appropriate).

first: Karl

middle: T

last: Wagner

first: Dawn

last: Bannerman

first: Qinghua

last: Wu

first: Simon

last: Pascual-Gil

first: Ian

last: Fernandes

first: Luis Felipe

last: Jiménez Vargas

first: Shuo

last: Yan

first: Ramak

last: Khosravi

first: Sargol

last: Okhovatian

first: Dhana

last: Abdo

first: Gordon

last: Keller


title: Single-cell analysis of human iPSC-derived midbrain organoids generated with triphasic Wnt modulation combined with a dynamic bioreactor

city: Paris

first: Michela

last: Deleidi

address: 24 boulevard du Montparnasse

institute: Institut Imagine, INSERM UMR1163

summary: Human midbrain organoids (hMOs) derived from induced pluripotent stem cells offer a promising platform to model Parkinson’s disease (PD), but current systems often fall short in generating mature, regionally specified dopaminergic (DA) neurons that reflect disease-relevant vulnerabilities. Here, we present a refined differentiation strategy that combines tri-phasic WNT signaling modulation with dynamic bioreactor cultivation to enhance DA neuron yield, specification, and maturation within hMOs. This approach generated DA neurons with enriched substantia nigra-like identity (GIRK2⁺, ALDH1A1⁺), elevated dopamine release, and robust functional activity. Single cell transcriptomic profiling revealed upregulation of synaptic, metabolic, and neuroprotective pathways within the DA neuron cluster, while stress-related and pro-apoptotic programs were suppressed. Importantly, these hMOs exhibited selective vulnerability upon challenge with α-synuclein preformed fibrils (aSyn-PFFs), modeling PD-relevant neurodegeneration. Together, our findings establish a scalable, physiologically relevant platform for investigating PD mechanisms, therapeutic screening and advancing the development of cell replacement strategies.

overall_design: We performed single-cell RNA sequencing (scRNA-seq) on organoids subjected to two primary protocols: a "Standard" protocol and a "Wnt-TriP" protocol, which incorporates a tri-phasic Wnt signalling modulation. Organoids from both protocols were cultured in parallel on either orbital shakers or in spinning bioreactors, creating four distinct experimental conditions. Samples were collected for scRNA-seq at multiple developmental time points (Day in vitro 25, 40 and 65), with multiple independent organoids serving as biological replicates for each condition. For sequencing, organoids were dissociated into single-cell suspensions with viability exceeding 95%. Libraries were prepared using the 10x Genomics Chromium Next GEM Single Cell 3' kit and sequenced on an Illumina NovaSeq X platform.

first: Hariam

last: Raji

first: Maria Jose

last: Perez J.


title: Epicardial-derived cells secrete extracellular vesicles that modulate gene expression in the cardiac response to ischemia-reperfusion injury [EV miRNAseq]

type: Non-coding RNA profiling by high throughput sequencing

summary: The epicardium is an essential regulator of cardiac development, homeostasis, and injury. While advanced 3D human cardiac tissue models now facilitate physiologically relevant exploration of epicardial–myocardial interactions, the composition and impacts of epicardial cell-secreted extracellular vesicles (EVs) in regulating cardiac physiology remain incompletely understood. Here we harnessed an “Epicardial Biowire” platform integrating human epicardial cells with functional myocardium, applying transcriptomics to reveal enriched vesicle transport in engineered tissues containing epicardial cells. Profiling epicardial-EVs identified key miRNAs in their cargo and indicated considerable conservation of miRNA cargo through stimulated epithelial‐to‐mesenchymal transition. Supplementation of epicardial-EVs to engineered tissues undergoing simulated ischemia–reperfusion injury influenced gene expression associated with reduction of extracellular matrix remodeling, fibroblast modulation, and suppression of cell-ECM interactions. Correlation of EV‐miRNAs with mRNA targets highlighted implications of miR‐30d‐5p, miR‐9‐5p, miR‐16‐5p, and the let‐7 family, supporting a potential role for epicardial-EVs and their miRNA cargo in moderating deleterious fibrotic activation and matrix remodelling during myocardial injury in vitro. Collectively, these findings expand our understanding of epicardial-EV composition and function, highlighting epicardial signalling as a promising avenue to direct cardiac repair and inspiring the design of future EV‐based therapeutics.

overall_design: miRNA-seq profiling of extracellular vesicles (EVs) isolated via PEG-based precipitation from conditioned media of human embryonic stem cell (hES)-derived epicardial cells. EV-miRNA sequencing consisted of 4 groups: EVs were isolated from the conditioned media of matured epicardial cells ('SBEVs'), unstimulated pro-epicardial cells ('NTEVs'), epicardial cells stimulated with TGF-β ('TGFbEVs'), and epicardial cells stimulated with FGF ('FGFEVs').


title: DNA methylation microarray analysis of human DNA samples contaminated with mouse DNA.

city: Shinagawa-ku

first: Takahiro

last: Ebata

state: Tokyo

department: Epigenomics

institute: Hoshi University

summary: This study investigated the impact of mouse DNA contamination for methylation analysis. We established a method to minimize the impact of mouse DNA contamination in human DNA samples, which will be useful for the analysis of human CDX and patient-derived xenograft (PDX) models

overall_design: This study analysed pure human and mouse DNA and human DNA mixed with mouse DNA. In addiditon, cancer cell lines and their xenograt samples were analysed.

first: Toshikazu

last: Ushijima


title: Distinct interactions between pioneer factor Ascl1/E12a and nucleosomes drive cell fate transitions [ChIP-seq]

city: Philadelphia

first: Arjun

last: Raj

state: PA

address: 415/420 Clinical Research Building (CRB)

institute: University of Pennsylvania

summary: Understanding how pioneer transcription factors target nucleosomal DNA and initiate chromatin accessibility provides insight into the earliest events in cell fate control. We used structural, biophysical, biochemical approaches to assess how the neural pioneer factor Ascl1 heterodimerizing with E12a binds nucleosomes in vitro. The nucleosomal binding, but not free DNA binding, by Ascl1/E12 is enhanced by two types of interactions with core histones. We performed ChIP-seq and ATAC-seq to investigate how histone interaction mutants of Ascl12 (called AAA, allRs, and QA respecrively) affect its global chromatin targeting and chromatin opening.

overall_design: hBJ fibroblasts were transduced with doxycycline inducible lentiviral vectors expressing E12a with Ascl1 WT or Ascl1 mutants, allRs, AAA and QA, at the MOI of 0.7 - 1.0. Cells with stable integration of the lentiviral vectors were selected with puromycin at final concentration of 1 μg/mL for 7 days, followed by expansion for at least 3 passages in puromycin. The puromycin resistant cells were pooled and plated at 2 million cells per 15cm dish, and induced with 0.5 μg/mL dox for 48 hrs for ChIP-seq analysis. All conditions were performed in three biological replicates.

first: Bing-Rui

last: Zhou

first: Edgar

last: Luzete-Monteiro

first: Jingchao

last: Zhang

first: Hsin-Yao

last: Tang

first: Meilin

last: Fernandez Garcia

first: Mariel

last: Coradin

first: Naomi

last: Takenaka

first: Megan

last: Frederick

first: Benjamin

last: Garcia

first: Yawen

last: Bai

first: Kenneth

last: Zaret


title: Transcriptomic Insights into Exogenous Putrescine-Mediated Cold Adaptation in Cannabis sativa L. [miRNA-Seq]

city: Chuncheon-si

first: Byeong-Ryeol

last: Ryu

state: Gangwon-state

address: 1, Kangwondaehak-gil

department: Department of Bio-Health Convergence

institute: Kangwon National Univsersity

summary: Low temperature is a major abiotic stress factor that promotes oxidative stress and disrupts cellular homeostasis, ultimately impairing plant physiological functions. This study investigated the effects of exogenous putrescine treatment on the cold stress response of Cannabis sativa L. This in silico transcriptome analysis demonstrated that putrescine mediates cold stress tolerance in C. sativa, providing valuable insight for future in vitro validation studies and a foundational framework for the development of cold-resistant hemp cultivars.

overall_design: Plants were exposed to cold stress in a growth chamber (HanKuk Scientific Co., Ltd., Hwaseong, Korea) at 5 ± 1°C for 168 h. Control plants were maintained under the same conditions at 23 ± 1°C. Putrescine (Duchefa Biochemie, Haarlem, Netherlands) was dissolved in ultrapure water and follar-sprayed at 5 mL per plant onto the shoots. To prevent cross-contamination, putrescine-treated plants were allowed to air-dry for 5 min after spraying and then grown in an independent chamber.

first: Min-Jun

last: Kim

first: Min-Ji

last: Kang

first: Sang-Hyuck

last: Park

first: Jung-Dae

last: Lim


title: Transcriptomic Insights into Exogenous Putrescine-Mediated Cold Adaptation in Cannabis sativa L. [RNA-Seq]


title: A WAT-to-BAT communication facilitates sustained activation of BAT thermogenesis during cold exposure [RNA-Seq]

city: Shanghai

first: Jieyuan

last: Xue

address: 280 South Chongqing Road

institute: Shanghai Jiao Tong University

summary: BAT activation for thermogenesis is a physiological mechanism that maintains body temperature during cold exposure. However, how BAT is dynamically activated and preserve its sustained activation in cold is not completely understood. In this study, we identified soluble ST2 (sST2) mediates a WAT-to-BAT endocrine mechanism, which is required for constant BAT activation during cold exposure. Specific depletion of sST2 isoform blocks the alternative thermogenesis upon BAT denervation and leads to cold sensitive in a prolong cold exposure. Mechanistically, sST2 is induced and secreted from eWAT by the Adrb1/2 signaling driven Creb1 activation. The secreted sST2 directly binds to the Adrb3 receptor in BAT and in synergy with NE to induce BAT thermogenesis, which is independent of IL33. Additionally, supplement of sST2 induces beige fat formation in mice and humans. Therefore, our study demonstrated eWAT derived adipokine sST2 functions on BAT activation as a novel mechanism in sustained activation of thermogenesis of BAT in cold exposure. More importantly, sST2 may serve as an emerging therapeutic approach in combine with b3 receptor agonist for obesity treatment.

overall_design: To investigate the gene expression change in  BAT (brown adipose tissue) of sST2-KO mice with BAT denervation exposed to cold sitimuli. We performed gene expression profile using data obtained from RNA-seq of BAT from wild type and sST2-KO mice.

first: Xuyun

last: Zhao


title: A WAT-to-BAT communication facilitates sustained activation of BAT thermogenesis during cold exposure [CUT&Tag]

overall_design: C3H 10T1/2 were harvested by 0.05% trypsin. 10000 cells per sample were used as starting material. Libraries were amplified using 13 cycles on the thermocycler. Post amplificationlibraries were size selected at 250-450bp in length using Agencourt AMPure XP beads fromBeckman Coulter.


title: Mouse uterine gene expressions during decidual processes [Visium]

city: Bunkyo-ku

first: Shizu

last: Aikawa

address: 7-3-1 Hongo

institute: The University of Tokyo

summary: Spatiotemporal uterine gene expressions during peri-implantation period at pregnancy day 4 and 6 was investigated by scRNA-seq and Visium spatial transcriptome.

overall_design: The day when the vaginal plug was detected was considered day 1 of pregnancy. Pregnant mice were euthanized by cervical dislocation on designated days of pregnancy for the evaluation of pregnancy phenotypes and sample collection. On day 4, one uterine horn was flushed with saline to confirm the presence of blastocysts. Embryo attachment sites were observed as blue bands soon after intravenous injection of 1% solution of Chicago blue dye (Sigma-Aldrich) in saline on day6. Collected tissues were snap-frozen. For scRNA-seq, frozen tessues were processed by gentleMACS for sample preparation at Takara Bio. The barcoded RNAs were subjected to RNA-sequencing using NovaSeq 6000. For 10x Visium analyses, frozen sections (10 μm) from control or TAZ-uKO were mounted on gene expression slides and sent to KOTAI Biotechnologies for subsequent procedures. After reaction with Proteinase K for 40 min, the sections were hybridized with spatial tags on the slides and reverse-transcribed in situ. The synthesized cDNAs were subjected to RNA-sequencing using DNBseq (MGI), producing 300 million reads/sample.

first: Xueting

last: He

first: Yasushi

last: Hirota


title: Effect of the cardiac long non-coding RNA Charme depletion on the maturation and paracrine signaling of resident cardiac fibroblasts [RNA-Seq 1]

city: Rome

first: Alessandro

last: Palma

address: Piazzale Aldo Moro 5

department: Biology and Biotechnologies "Charles Darwin"

country: Italy

institute: Sapienza University of Rome

summary: Charme is a murine long non-coding RNA necessary for the embryonic development of the heart. In vivo Charme knock-out causes prominent alterations of tissue structure, due to cardiac hyperplasia, and leads to the development of cardiac dysfunctions. Although its role in cardiomyocytes has been deeply investigated, no evidence has been collected about its function in other cardiac cell types. Cardiac fibroblasts (CFs) play pivotal roles in the development and homeostatic maintenance of the cardiac tissue. This study investigated the phenotype and function of resident CFs isolated from Charme knockout (CharmeKO) mice, and revealed impaired maturation and functionality. CharmeKO hearts show decreased levels of collagen I content in the extracellular matrix, associated with reduced extracellular matrix-related gene expression and matrix remodelling ability of the CFs. CharmeKO CFs show impaired phenotypic conversion into myofibroblasts and reduced responsiveness to activation stimuli, accompanied by the retention of features proper of unactivated mesenchymal cells. Charme depletion also affects CF paracrine function, determining an impoverishment in cardioprotective cytokines in their secretome, with consequent reduced ability to mediate PI3K/Akt pathway activation in cardiomyocytes and to induce the angiogenic process in endothelial cells. CharmeKO CFs also proved to be less supportive to cardiomyocyte maturation in an in vitro model of cardiac differentiation, thus indicating a potential contribution to the in vivo impairment of cardiomyocyte maturation. Overall, the evidence collected suggests that Charme depletion in the heart impacts CF maturation capacity, leading to their impaired function, which can potentially contribute to the alterations observed in the CharmeKO mice. These findings pave the way to deeper investigations of the intercellular signalling occurring in the heart upon Charme ablation with the aim of identifying how microenvironment homeostasis contributes to cardiomyocyte maturation, and is potentially involved in cardiac diseases.

overall_design: RNA-seq profiling of wildtype and CharmeKO cardiac fibroblasts (CFs) from murine hearts. Cardiac fibroblasts were isolated both through cardiac tissue dissociation coupled with hematopoietic cell lineage depletion (native CFs) and through the explant culture protocol (cultured CFs).

first: Erica

last: Floris

first: Claudia

last: Cozzolino

first: Giulia

last: Buonaiuto

first: Valeria

last: Taliani

first: Antonella

last: Bordin

first: Vittorio

last: Picchio

first: Xhulio

last: Dhori

first: Francesco

last: Nutile

first: Carmine

last: Nicoletti

first: Giacomo

last: Frati

first: Monica

last: Ballarino

first: Isotta

last: Chimenti

first: Francesca

last: Pagano


title: Effect of the cardiac long non-coding RNA Charme depletion on the maturation and paracrine signaling of resident cardiac fibroblasts [RNA-Seq 2]


title: RNA-seq of RSV-Infected THP-1 Cells Treated with Recombinant CTSC

city: Changsha

first: Xizi

last: Du

address: Xiangya Road 88

institute: Cenrral South University

summary: We investigated the regulatory role of exogenous cathepsin C (CTSC) in RSV-infected THP-1 cells and assessed whether the activation of downstream inflammatory pathways could be attenuated by the application of proteinase 3 (PRTN3) inhibitors.

overall_design: Differentiation of THP-1 monocytes to macrophages was induced by treating the THP-1 cell line (2 × 105 cells/ml) with 200 nM phorbol 12-myristate 13-acetate (PMA)  for 48 h. After PMA stimulation, differentiation was enhanced by removing the PMA-containing medium and then incubating the cells in fresh RPMI 1640 medium (10% FBS, 1% penicillin-streptomycin solution). Recombinant Human Cathepsin C (rhCTSC) and PR3 inhibitor Sivelestatwere incubated with differentiated THP-1 cell for 24h, then cells were infected with RSV (MOI 5) for 24h.

first: Ye

last: Yao

first: Xinyu

last: Wu


title: Mouse uterine gene expressions during decidual processes [scRNA-seq]

first: Shizu

last: Aikawa

first: Xueting

last: He

first: Yasushi

last: Hirota


title: The histone lysine-specific demethylase inhibitor JIB-04 sensitizes AML cells to venetoclax by inducing ferroptosis [RNA-seq]

city: Houston

first: Margaret

last: Goodell

state: TX

address: One Baylor Plaza, N1030

institute: Baylor College of Medicine

summary: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with various molecular and cytogenetic subtypes. Treatment options for elderly patients are limited due to high toxicity of conventional chemotherapy. The BCL-2 inhibitor venetoclax is effective in combination with hypomethylating agents or low-dose cytarabine, but about 30% of patients don’t respond to the initial combination treatment. Thus, alternative combinations are needed to sensitize AML cells to venetoclax and overcome resistance mechanisms. Here, we report that targeting histone lysine-specific demethylases (KDMs) induces ferroptosis in various AML subtypes. In both patient samples and cell lines, JIB04 increases the level of reactive oxygen species (ROS), ferrous iron, and lipid peroxidation, all signs of ferroptosis. The combination of JIB04 and venetoclax proved to be highly synergistic and was dependent on ferroptosis induction. Blocking ferroptosis by using the antioxidant N-Acetyl-L-cysteine (NAC) reverses the synergistic killing. On the molecular level, the ferroptosis inducers HMOX1, SAT1, and PTGS2 were found to be upregulated by JIB04. Collectively, these findings identify JIB04 as a new ferroptosis inducer in AML and highlight the potential of ferroptosis induction as a valuable strategy in combination with venetoclax to treat AML.

overall_design: RNA-Seq profiling of AML-OCI3 cells treated with DMSO or JIB-04 for 24 and 48 hours

first: Katharina

last: Wohlan

first: Dandan

last: Fan

first: Anna

middle: G

last: Guzman

first: Alexis

last: Quezada

first: Elmira

last: Khabusheva

first: Steven

last: Kornblau

first: Courtney

middle: D

last: DiNardo

first: Jianzhong

last: Su

first: Rachel

middle: E

last: Rau


title: The histone lysine-specific demethylase inhibitor JIB-04 sensitizes AML cells to venetoclax by inducing ferroptosis.

overall_design: ChIP-Seq of H3K36me for AML-OCI3 treated with DMSO or JIB-04 for 24 and 48h


title: Glial-specific and inducible full body C3 deficiency does not affect amyloid pathology in the AppNL−G−F mouse model of Alzheimer’s disease

city: Zwijnaarde

first: Roosmarijn

middle: E.

last: Vandenbroucke

state: Gent

address: Technologiepark-Zwijnaarde 71

department: VIB Center for Inflammation Research

institute: VIB-UGent

summary:
Background
Alzheimer's disease (AD) is intricately linked with neuroinflammation, with the complement system, particularly C3, emerging as a critical player. However, research has been hampered by the reliance on classical germline C3 knockout and APP overexpressing mouse models, which do not allow to study temporal and cell-specific C3 effects, and do not accurately reflect the complexity of AD pathology.
Methods
In this study, we investigated the impact of conditional C3 deficiency on neuroinflammation and AD pathology, by generating microglia-specific (C3mKO), astrocyte-specific (C3aKO), and inducible full-body (C3iKO) C3 knockout mice. To assess the role of C3 in both acute and chronic neuroinflammation, we employed an intracerebroventricular (ICV) LPS injection model in these mice alongside studies in the AppNL-G-F knock-in mouse model of AD upon aging and mild peripheral inflammation.
Results
Our results show that complement genes, including C3, are upregulated in microglia and astrocytes from 40 weeks old AppNL-G-F mice compared to their wildtype counterparts. Both microglia and astrocytes were shown to be significant sources of C3, as conditional C3 deficiency in either cell type led to decreased C3 expression and a dampened neuroinflammatory transcriptional response following ICV LPS injection. However, microglial- and astrocytic-specific C3 deficiency in AppNL-G-F mice did not affect total hippocampal C3 protein levels and amyloid plaque burden upon both 40 weeks of aging and in mild peripheral inflammation conditions. Also full-body C3 knockout, induced at the age of 8 weeks, did not alter Aβ pathology and glial activation, despite the complete removal of C3.
Conclusions
Our findings show that while C3 contributes to neuroinflammatory responses, its role in chronic AD-associated pathology is more complex than previously thought. Our study using novel cell-specific and inducible C3 knockout mice combined with the knock-in AppNL-G-F model provides new insights into the cell-specific roles of complement in AD, and highlights the need for further investigation into the complement system's involvement in neurodegenerative diseases.

overall_design: Comparative gene expression profiling analysis of RNA-seq data to study the astrocyte and microglial transcriptomic response to Alzheimer's disease

first: Pieter

last: Dujardin

first: Clint

last: De Nolf


title: Differential Gene Expression related to Telomere Length in Arterial Wall Tissues and Granulocytes of Patients with Coronary Artery Disease

city: Singapore

first: ZHI CHNG

last: LAU

address: 60 Biopolis Street

country: Singapore

institute: Genome Institute of Singapore

summary: Shortened telomere length (TL) in blood cells is associated with atherosclerotic coronary artery disease (CAD). However, the mechanistic pathways underlying TL attrition in arterial wall tissues for patients with CAD remain unclear. In this study, we evaluated TL in arterial wall tissues and granulocytes and correlated these measurements with data on gene expressions in the arterial wall tissues of patients with CAD.

overall_design: RNA sequencing was conducted in 34 patients to profile the arterial wall tissue transcriptome. These patients were dichotomized based on the median TL of arterial wall tissues and granulocytes and studied for their differential gene expression with respect to TL.

first: Zhi Chng

last: Lau

first: Rajkumar

last: Dorajoo

first: Adaikalavan

last: Ramasamy

first: Gokce

last: Oguz


title: In vitro PI3Kδ inhibition skews CD8 T cell differentiation towards progenitor exhaustion in vivo and reprograms tumor microenvironment metabolism, immune composition, and inflammation [Spatial Transcriptomics]

city: Lebanon

first: Edward

last: Usherwood

state: NH

address: Rubin Building, Level 7, 710-53

department: Microbiology and Immunology

institute: Geisel School of Medicine at Dartmouth College

summary: In vitro PI3Kδ inhibition using Idelalisib (CAL-101) drives CD8 T cells toward a progenitor exhausted (Tpex) phenotype while resisting terminal exhaustion in vitro. We performed single-cell RNA sequencing and spatial transcriptomics on B16-melanoma tumors and show that CAL-101-treated T cells enhance oxidative phosphorylation, proliferation, and Ifnγ responsiveness. The CAL-101 treated cells deeply infiltrate tumors and upregulate the CXCR3-CXCL10 axis, coinciding with reduced tumor-associated macrophage signatures and increased pro-inflammatory signaling in the tumor microenvironment. CAL-101 reprograms T cell fate and remodels the TME, offering insight into how to improve immunotherapeutic efficacy for solid tumors.

overall_design: Six RAG-KO mice bearing B16-F10 melanoma were given adoptively transferred Pmel Thy1.1 CD8+ T cells. Three mice each received T cells with in vitro CAL-101 treatment or control treatment. Half tumors were processed from each sample for spatial transcriptomics analysis.

first: Alexandrea

last: Turnquist

first: Owen

last: Wilkins


title: B cell CD19 is widely transferred between immune cells [CD11b+]

city: Mainz

first: Matthias

last: Klein

address: Langenbeckstrasse 1

department: Inst. of Immunology

country: Germany

institute: University Medical Center Mainz

summary: When immune cells interact, they frequently exchange membrane-bound antigens. Our evolving understanding of these processes challenges the cellular specificity of lineage markers and therapeutically applied monoclonal antibodies. We here report that CD19, an assumingly exclusive B cell marker, is transferred via trogocytosis when B cells activate T cells as antigen-presenting cells.  In a B cell-driven model of experimental autoimmune encephalomyelitis, CD19+ T cells accordingly expanded and showed enhanced features of activation, differentiation, and encephalitogenicity. A similar profile of augmented pathogenic properties of CD19+ T cells was detected in patients with chronic CNS demyelination. Here, CD19+ T cells were found to be concomitantly depleted by inebilizumab, an approved anti-CD19 antibody, which may support its effectiveness. Furthermore, we report that CD19 is found on myeloid cells after phagocytosis of apoptotic B cells. These results highlight the commonness of membrane and antigen transfer between interacting cells and provide instrumental considerations for monoclonal antibody therapies.

overall_design: To analyze whether murine CD11b+ cells can endogenously express CD19, we isolated them from the spleens of wildtype C57Bl6 mice, isolated the RNA via the RNeasy Plus Micro Kit and performed bulk RNA sequencing.

first: Ochs

last: Jasmin

first: Schweineberg

last: Pia

first: Thode

last: Jacqueline

first: Blenkle

last: Alica

first: Husseini

last: Leila

first: Klein

last: Matthias

first: Bopp

last: Tobias

first: Schindler

last: patrick

first: Paul

last: Friedemann

first: Weber

last: Martin


title: Dysregulation of bile acid metabolism exacerbates diet-induced MASLD development in HuR deficient male mouse livers.

city: Kansas City

first: Yuxia

last: Zhang

state: KS

department: Pharmacology, Toxicology, and Therapeutics

institute: The University of Kansas Medical Center

summary: Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is a leading cause of hepatocellular carcinoma and liver transplantation worldwide, making identification of intervention strategies and therapeutic treatments essential to reducing morbidity and mortality associated with this disease. RNA binding proteins, like human antigen R (HuR), are ubiquitous and multi-faceted cell-stress regulators. HuR’s role in MASLD development is incompletely understood. This study aims to determine how hepatocyte HuR deficiency drives MASLD progression to Metabolic dysfunction-associated steatohepatitis (MASH). To model MASLD, we fed male hepatocyte-specific HuR knockout mice (HuRHep-/-) and WT control mice with the normal chow diet or the MASLD-inducing high fat, cholesterol, and fructose (HFCF) diet for 16 weeks. The liver transcriptomic profile was mapped using bulk RNA sequencing (RNA seq). After MASLD-inducing diet feeding, bile acid metabolism was modulated, while liver injury and fibrosis markers were increased in male HuRHep-/- mice, relative to WT. Our data suggests that hepatocyte HuR deficiency dysregulates bile acid metabolism and exacerbates MASLD progression.

overall_design: RNA seq analysis of livers from WT and HuRHep-/- mice following both normal chow diet and 16-weeks HFCF diet feeding.

first: Natalie

last: Eppler

first: Nancy

last: Magee

first: Forkan

last: Ahamed

first: Elizabeth

last: Jones

first: Lily

last: He


title: TEX cell fate and differentiation are shaped by 3D genome reorganization and chromatin architecture integrity [D3]

city: Washington

first: Weiqun

last: Peng

state: District of Columbia

address: Suite 4000 SEH Science & Engineering Hall 800 22nd Street NW

department: Physics

institute: The George Washington University

summary: CTCF is recognized for its role in facilitating long-range chromatin interactions and 3D genome organization. In CD8+ T cells responding to chronic infection, CTCF undergoes redistribution to activate the exhausted cytotoxic effector (Tex-eff) program while simultaneously utilizing its insulation function to regulate the fate of precursors to stem-like exhausted T (Tex-prec) cells.

overall_design: Naive P14 TCR transgenic CD8+ T cells, carrying a Tcf7-GFP reporter to mark Tcf1-expressing cells, were adoptively transferred into CD45-disparate recipients and subsequently infected with either LCMV Armstrong or LCMV-Clone 13 to induce acute or chronic viral infection, respectively. On day 8 post-infection in the acute infection model, KLRG1+IL-7Rα- P14 cells were sorted and purified as effector CD8+ T (Teff) cells for ATAC-seq analysis along with naive P14 cells.  In the chronic infection model, on day 8 post-infection, Tim3+Slamf6-GFP- P14 cells were sorted and purified as effector exhausted CD8+ T (Tex-eff) cells, while Tim3−Slamf6+GFP+ P14 cells were sorted and purified as precursors to stem-like exhausted CD8+ T (Tex-prec) cells, both for ATAC-seq analysis. Additionally, hCD2-Cre, Rosa26-lsl-GFP, and CTCF wild-type or floxed CD8+ T cells were adoptively transferred into CD45-disparate recipients, followed by infection with LCMV-Clone 13. At 72 hours post-infection, GFP+ Tim3+ Tex-eff P14 cells and GFP+ Tim3− Tex-prec P14 cells from both the WT and CTCF-KO groups were sorted and purified for ATAC-seq analysis.

first: Hai-Hui

last: Xue

first: Wei

last: Hu

first: Qing

last: Chen


title: B cell CD19 is widely transferred between immune cells [M]

first: Matthias

last: Klein

overall_design: To analyze whether murine T cells can endogenously express CD19, we isolated murine T cells from from the spleens and inguinal lymph nodes of wildtype C57Bl6 mice, isolated the RNA via the RNeasy Plus Micro Kit and performed bulk RNA sequencing. B cells isolated from the same mice served as positive controls.

first: Ochs

last: Jasmin

first: Schweineberg

last: Pia

first: Thode

last: Jacqueline

first: Blenkle

last: Alica


title: Effect of MbovP475 on gene expression in BoMac cells [ChIP-seq]

city: Yinchuan

first: Gang

last: Zhao

address: 489 Helan West Road, Xixia District

institute: ningxia university

summary: MbovP475 of M. bovis is a nucleomodulin and has the ability to enter into cells nucleus during infection. Recently, we verifed that MbovP475 can regulate the hostone PTMs. We found MbovP475 promoted the H3K4me3 and H3K27ac. So we proposed MbovP475 regulated gene expression in BoMac cells by promoting H3K4me3 and H3K27a, we constructed BoMac cells stablely expressing MbovP475. Our data provide globaly genes marked by H3K4me3 and H3K27ac enhanced by MbovP475.

overall_design: ChIP-seq for histone H3K4me3 and H3K27ac in BoMacWT and BoMac475 cells.


title: MeRIP-seq study of Vaccinia virus-infected Vero cell

city: Los Alamos

first: Taehyung

last: Kwon

email: ted3466@gmail.com, tae_kwon@lanl.gov

state: New Mexico

address: Los Alamos National Laboratory

department: Bioscience Division

institute: Los Alamos National Laboratory

summary: We aimed to understand the dynamic interplay between host and virus by investigating transcriptomic and epitranscriptomic dynamics in the Vero E6 cells during Vaccinia Virus infection. In this study, we characterized N6-methyladenosine (m6A) modifications, pivotal regulators of transcript lifecycle, throughout three different post-infection time points using RNA-seq and m6A RNA immunoprecipitation sequencing (MeRIP-seq). We identified changes in host m6A epitranscriptome in the early phase of viral infection, which includes genes involved in host immunity, RNA biosynthesis, and protein ubiquitination. Leveraging temporal analysis of host transcriptomic and epitranscriptomic landscapes, this study offers a novel insight into the host-virus interaction, emphasizing the importance of RNA modifications in host transcripts as an early response to viral infection.

overall_design: This experiment consists of two fractions: total RNA-seq (input) and m6A-enrichment immunoprecipitation using anti-m6A antibody (IP). For each fraction, we generated three biological replicates for three Vaccinia virus-infected samples (2 hpi, 6 hpi, and 24 hpi) and one MOCK-infected sample (MOCK).

first: Cheryl

last: Gleasner

first: Sofiya

last: Micheva-Viteva


title: Histone H3 epigenetic modifications ChIPseq [men]

city: Gothenburg

first: Maria

middle: I

last: Bokarewa

state: VG

address: Guldhedagatan 10A

department: Rheumatology and Inflammation Research

institute: Gothenburg University

summary: ChIPseq analysis of histone H3 epigenetic modification - K27me3 - chromatin binding in activated CD4+ T cells

overall_design: 3 men, CD4 cells from PBMC isolated by positive selection on Dynabeads (cat.11331D),  ConA+LPS for 48h, pooled from 3 samples. ChIPseq using anti-histone H3 antibodiy (0.5 microg/ml,  H3K27me3 - C15410195-10, Diagenode) and anti-survivin antibody (1 microg/ml, ab192675, abcam). DNA purified by the MAFnify immmunoprecipitation system (Invitrogen, cat-no 49-2024).

first: Karin

last: Andersson

first: Malin

last: Erlandsson

first: Chandrasekaran

last: Venkataragavan


title: Effect of MbovP475 on gene expression in BoMac cells

summary: MbovP475 of M. bovis is a nucleomodulin and has the ability to enter into cells nucleus during infection. Recently, we verifed that MbovP475 can regulate the hostone PTMs. To detect the effect of MbovP475 of M. bovis on gene expression in BoMac cells, we constructed BoMac cells stablely expressing MbovP475. Our data provide globaly change of mRNA profile caused by MbovP475.

overall_design: We extracted the total RNA of Wild type BoMac cells (BoMacWT) and BoMac cells expressing MbovP475 (BoMac475).  We then performed gene expression profiling analysis using data obtained from RNA-seq of BoMacWT and BoMac475.


title: TUT1 and USB1, the biogenesis enzymes for 2′,3′-cyclic phosphate modification at U6 snRNA 3’ end, have distinct roles in mammalian development [Nanopore]

city: Chengdu

first: chao

last: yang

address: No.20, Section 3, Renmin South Road

institute: Sichuan University

summary: The 3’ end of most cellular U6 snRNA has a 2′,3′-cyclic phosphate group, a post-transcriptional modification thought to be essential for proper RNA splicing and cellular function. Recent studies have established that the biogenesis of 2′,3′-cyclic phosphate requires two enzymes: first TUT1 adds a poly(U) tail to the 3’ end of U6 snRNA, followed by USB1-catalyzed trimming of the poly(U) tail. Here, we use nine genetically engineered mouse models of Tut1 and Usb1 to show that, contrary to what was previously believed, Usb1 and thus 2′,3′-cyclic phosphate is dispensable for intra-embryonic cell lineages. We find that Tut1, but not Usb1, is essential for the maintenance of the epiblast and neural stem cells. Loss of Tut1 causes defective RNA splicing, triggering massive DNA damage and subsequent cell death. In contrast, loss of Usb1 primarily damages endothelial cells of the extra-embryonic vasculature in the yolk sac by both cell-intrinsic and -extrinsic mechanisms, leading to fetal growth restriction. These results demonstrate that TUT1-catalyzed U6 snRNA 3’ end oligouridylation, rather than 2′,3′-cyclic phosphate as previously thought, is required for proper RNA splicing. Further studies are warranted to elucidate how these different post-transcriptional modifications regulate the function of U6 snRNA in RNA splicing in development and disease.

overall_design: Micro-dissected dorsal and medial palliums from wild-type and Emx1-Tut1-KO E11.5 embryos were used for long-read RNA sequencing to analyze alternative splicing (AS) events.


title: T cell exhastion is programmed by dynamic 3D genome reorganization

city: washington dc

first: Shaoqi

last: Zhu

state: WASHINGTON

address: 725 21st st nw

institute: the george washington university

overall_design: hCD2-Cre, Rosa26-lsl-GFP, and CTCF wild-type or floxed P14 CD8+ T cells were adoptively transferred into CD45-disparate recipients followed by infection with LCMV-Clone 13. At 72 hours (day 3) post-infection, GFP+ P14 cells were sorted and purified for pSer5 RNA polymerase II (RNAPII) CUT&RUN analysis.

first: Hai-Hui

last: Xue

first: Weiqun

last: Peng


title: TUT1 and USB1, the biogenesis enzymes for 2′,3′-cyclic phosphate modification at U6 snRNA 3’ end, have distinct roles in mammalian development [RNA-seq]

overall_design: To examine whether Usb1 is required for pluripotency, we treated wild-type and Usb1 knockout cells with retinoic acid (RA) for three days. Transcriptome profiling using RNA-seq was carried out for cells without RA treatment (day 0), treated with RA for 1 day (day 1), and for 3 days (day 3).


title: TUT1 and USB1, the biogenesis enzymes for 2′,3′-cyclic phosphate modification at U6 snRNA 3’ end, have distinct roles in mammalian development [scRNA-seq]

overall_design: To probe potential defects in Usb1-KO embryos at a higher resolution, we carried out single-cell sequencing analysis of wild-type and Usb1-KO embryos.


title: Depletion of RNA-editing enzyme ADAR1 invigorates NK cells anti-tumor immunity

city: Guangzhou

first: Rukang

last: Liang

state: Guangdong

address: Shipai Street, Tianhe district

department: Biotherapy Center

institute: The Third Affiliated Hospital of Sun Yat-Sen University

summary: Function exhaustion and inefficient tumor infiltration rate limit NK cells based cancer immunotherapy. Even though the role of ADAR1 in immune cells and tumorigenesis is gradually gaining attention, its role on NK cells is elusive. In this study, we found ADAR1 expression level was upregulated in peripheral blood (PB)-NK cells from patients with melanoma. ADAR1 knockdown NK cells showed enhanced anti-tumor activity in vitro and in vivo. NK cells specific Adar1 deletion mice showed better tumor control and higher NK cells infiltration level. RNA-seq analysis revealed that NK shADAR1 cells exhibited an activation phenotype with highly cell migration potential. Higher tumor infiltration level of NK shADAR1 cells were verified in three-dimensional (3D) Matrigel-spheroid experiment. Mechanically, we also observed ADAR1 deficiency in NK cells accompanied by RIG-I signaling pathway inhibition and CD38 expression decline, resulting in higher cell mobility, proliferation, and tumor killing capacity. These data suggest ADAR1 may be an emerging therapeutic target for enhanced NK cell immunotherapy.

overall_design: To investigate the role of ADAR1 in NK cells anti-tumor capacity, we used a lentiviral shRNA technique to knockdown ADAR1 expression in NK-92 cells. A scrambled shRNA (shscramble) was used as a control. We then performed RNA-Sequencing on NK-92 from the shsramble group and shADAR1 group with GENE DENHOVO Biotech Inc. (Guangzhou, China) to analyze gene expression changes in NK-92 cells. For each sample, two independent biological replicates were performed.

first: Shuhan

last: Chen

first: Di

last: Lu

first: Le

last: Han

first: Qiuli

last: Liu


title: TEX cell fate and differentiation are shaped by 3D genome reorganization and chromatin architecture integrity  [CUT&RUN]

overall_design: Naive P14 TCR transgenic CD8+ T cells, carrying a Tcf7-GFP reporter to mark Tcf1-expressing cells, were adoptively transferred into CD45-disparate recipients and subsequently infected with either LCMV Armstrong or LCMV-Clone 13 to induce acute or chronic viral infection, respectively. On day 8 post-infection in the acute infection model, KLRG1+IL-7Rα- P14 cells were sorted and purified as effector CD8+ T (Teff) cells for CTCF CUT&RUN analysis in live cells along with naive cells. In the chronic infection model, on day 8 post-infection, Tim3+Slamf6-GFP- P14 cells were sorted and purified as effector exhausted CD8+ T (Tex-eff) cells, while Tim3−Slamf6+GFP+ P14 cells were sorted and purified as precursors to stem-like exhausted  CD8+ T (Tex-prec) cells, both for CTCF CUT&RUN analysis.

first: Wei

last: Hu

first: Qing


title: Egr2 inhibition of PDGFRα in oligodendrocytes is necessary for resocialization normalizing cooperation defects in early socially isolated mice

city: Nanjing

first: Ming

last: Xiao

address: 101 Longmian Avenue

institute: NMU

summary: Social neglect is a major contributing factor to social behavior impairment during adolescence, thus it is crucial for exploring its underlying mechanism and therapeutic targets. In this study, we found social isolation (SI) mice who resocialized with group housing (GH) mice improved their impairments in social cooperation and myelination in the medial prefrontal cortex (mPFC). However, resocialization between SI mice did not correct the above disorders. The partition model of behavioral interaction negated the behavioral and pathological improvements by resocialization with GH mice. Early growth response 2 (Egr2) is essential for establishment and maintenance of cooperative behavior. In this study, the regulating role of Egr2 was reconfirmed by specifically knockdown and overexpressed Egr2 in mPFC oligodendrocytes (OLs). mPFC RNA-sequencing (RNA-seq) analysis and enzyme-linked immunosorbent assay further showed that the level of serum corticosterone (Cort) was increased by SI. Additionally, we observed that SI mice’s myelination and social cooperation behavior were improved or inhibited when their companions received intraperitoneal injection of metyrapone or Cort during resocialization. RNA-seq analysis of cultured primary OLs combined with resocialization models further revealed that Egr2 participates in myelin formation by inhibiting platelet derived growth factor receptor alpha (PDGFRα). This finding revealed the neurobiological mechanism by which Egr2 participates in the regulation of impaired cooperative behavior in early SI mice through the inhibition of PDGFRα expression in OLs and the involvement of resocialization.

overall_design: siEgr2 or siNC treated primary oligodendrocytes for 48 hours

first: Sijia

first: Shixin

last: Ding

first: Min

last: Cao

first: Shuying

last: Zhang

first: Yue

last: Li

first: Yuxi

last: Jin

first: Yimiao

last: Wang

first: Ze

first: Yu

last: Wu

first: Junying

last: Gao

first: Yanli


title: TEX cell fate and differentiation are shaped by 3D genome reorganization and chromatin architecture integrity  [ATAC-seq]

first: Weiqun

overall_design: Naive P14 TCR transgenic CD8+ T cells, carrying a Tcf7-GFP reporter to mark Tcf1-expressing cells, were adoptively transferred into CD45-disparate recipients and subsequently infected with either LCMV Armstrong or LCMV-Clone 13 to induce acute or chronic viral infection, respectively. On day 8 post-infection in the acute infection model, KLRG1+IL-7Rα- P14 cells were sorted and purified as effector CD8+ T (Teff) cells for ATAC-seq analysis along with naive P14 cells.  In the chronic infection model, on day 8 post-infection, Tim3+Slamf6-GFP- P14 cells were sorted and purified as effector exhausted CD8+ T (Tex-eff) cells, while Tim3−Slamf6+GFP+ P14 cells were sorted and purified as precursors to stem-like exhausted CD8+ T (Tex-prec) cells, both for ATAC-seq analysis.

first: Hai-Hui

last: Xue

first: Shaoqi

last: Zhu


title: Mycobacterium abscessus infection in fetal liver derived alveolar-like macrophages activates distinct inflammatory responses compared to myeloid-derived macrophages

city: Lansing

first: Kayla

last: Conner

state: Michigan

address: 4125 Beaumont Rd

institute: Michigan State University

summary: Pulmonary infections caused by Mycobacterium abscessus (Mab), a rapidly growing nontuberculous mycobacterium (NTM), are on the rise in patients with chronic or acquired lung disease. In contrast to immunocompetent individuals, these patient cohorts exhibit abnormal pulmonary function that result from chronic inflammation and mucus build-up. Treatment regimens rely on multi-drug cocktails yet Mab’s natural recalcitrance to common antibiotics extends treatment timelines and increases the frequency of treatment failures. Thus, it is important to understand the mechanisms by which immunocompetent individuals clear Mab with relative ease while susceptible individuals do not, to identify new treatment options that may protect at-risk patients. In the lungs, macrophages are the first immune cell Mab encounters following infection, with both resident alveolar macrophages and recruited myeloid derived macrophages playing important roles during infection control. However, the specific role of these distinct macrophage populations in regulating control and inflammatory responses during Mab remains limited due to a lack of ex vivo models that recapitulate the functions of different macrophage subsets. Here, we leverage a fetal-liver derived alveolar macrophage (FLAM) model to define the mechanistic interactions occurring at the Mab-macrophage interface compared to bone-marrow derived macrophages (BMDMs). Even though both FLAMs and BMDMs similarly control intracellular Mab, the inflammatory response between these macrophage populations is significantly different. While BMDMs robustly activated NF-κB transcriptional targets that include important chemokines and inflammatory cytokines like TNF, FLAMs transiently induced these genes following Mab infection. While activation of FLAMs or BMDMs with IFNγ prior to Mab infection did not alter Mab intracellular dynamics, it did drive FLAMs to be more inflammatory. However, while we found IFNγ activated FLAMs more robustly activate NF-κB during Mab infection, there remain important differences compared to BMDMs. This includes lower expression of the inducible nitric oxide synthase, which we found was reversed with chemical activation of HIF1α. We conclude that FLAMs and BMDMs differentially respond to Mab infection due to differences in signaling networks activated following innate immune sensing, with FLAMs being more hypo-inflammatory than BMDMs. More broadly our results highlight a key need to better understand the initial interactions with Mab and distinct macrophage populations to define pathways that contribute to pulmonary protection or disease during respiratory infections.

overall_design: BMDMs and FLAMs (both resting and stimulated with IFNg) were infected with Mycobacterium abscessus ATCC 19977. Cells were lifted at 6H and 24H time points. RNA was extracted and libraries were prepared for bulk RNA sequencing by SeqCenter. Following sequencing, we carried out differential gene expression analysis to understand how infection with Mycobacterium abscessus alters the transcriptomes of these cells with and without the presence of IFNg.

first: Haleigh

last: Gilliland

first: Soledad

last: Soverina

first: Taryn

last: Vielma

first: Andrew

middle: J

last: Olive


title: Glycosylation Orchestrates Megakaryocytic Fate of Hematopoietic Stem Cells via Wnt-Muc13 Signaling [scRNA]

city: Milwaukee

first: Karin

last: Hoffmeister

state: WI

address: 8727 Watertown Plank Road

institute: Versiti Blood Research Institute

summary: The cell-intrinsic and extrinsic programs governing hematopoietic stem and progenitor cells (HSPCs) cell fate determination remain unresolved. Our data reveals that loss of B4GALT1 glycosyltransferase biosynthetic activity restricts HSPC N- and O-glycosylation and reprograms previously unrecognized N-glycan gradients in the bone marrow environment with high expression of complex N-glycans in HSPC-rich regions to accumulate aberrant, cancer-like N-glycan signatures. The loss of B4GALT1 increases the expression of aberrantly glycosylated intracellular oncogenic Mucin13, which is likely to disrupt the destruction complex and mediate Wnt/β-catenin hyperactivation. This enhances metabolic and cell cycle activity, expands the megakaryocyte-primed stem cell pool, and promotes emergence from a steady state, highlighting the essential role of B4GALT1 in modulating the BM glycosylation landscape and its significance in regulating the expansion and differentiation of HSPCs.

overall_design: Bone marrow LSK cells were sorted by FACS using the Lineage-Sca-1+, cKit+ strategy for wild type and B4galt1 full knockout mice and analized using scRNAseq

first: Alejandro

last: Roisman

first: Leonardo

last: Rivadeneyra

first: Lindsey

last: Conroy

first: Simon

last: Glabere

first: Melissa

last: Lee-Sundlov

first: Natalia

last: Weich

first: George

middle: F

last: Steinhardt IV

first: Shikan

last: Zheng

first: Robert

last: Burns

first: Anthony

last: Veltri

first: Joseph

first: Hartmut

last: Weiler

first: Ramon

last: Sun


title: Glycosylation Orchestrates Megakaryocytic Fate of Hematopoietic Stem Cells via Wnt-Muc13 Signaling [bulkRNA]

overall_design: Bone marrow LSK cells were sorted by FACS using the strategy: Lineage-Sca-1+, cKit+,CD150+,CD48- for wild type and B4galt1 full knockout mice, and analyzed using RNAseq


title: Spatially tuneable multi-omics sequencing using light-driven combinatorial barcoding of molecules in tissues / RNA_LCM

city: Cambridge

first: Dario

last: Bressan

state: Cambridgeshire

address: Li Ka Shing Centre, Robinson Way

department: CRUK Cambridge Institute

country: United Kingdom

institute: University of Cambridge

summary: Mapping the molecular identities and functions of cells alongside their spatial tissue context is key to understanding the complex interplay within and between their tissue neighbourhoods. A wide range of methods enable spatial profiling of regions, some down to the level of individual cells, in their anatomical context, via different barcoding schemes that encode either the location or the identity of target molecules. However, all these technologies face a trade-off between spatial resolution, depth of profiling, and scalability. Here, we present Barcoding by Activated Linkage of Indexes (BALI), a method that uses light to write combinatorial spatial molecular barcodes directly onto target molecules in situ, enabling multi-omics profiling by next generation sequencing. A unique feature of BALI is that the user can define the number, size, and shape of the spatial locations to be interrogated, with the potential to profile millions of distinct regions down to subcellular scale. As a proof of concept, we used BALI to capture the transcriptome, chromatin accessibility, or both, from distinct areas of the mouse brain in single tissue sections, demonstrating strong concordance with publicly available datasets. BALI therefore combines high spatial resolution, high throughput, histological adaptability, and workflow accessibility to enable powerful spatial multi-omics profiling.

overall_design:
Technology development project for a new spatial multi-omic method. One section from a mouse embryonic brain (e16.5) was subjected to the new protocol. Two areas were selected for spatial profiling: the sub-ventricular zone and the cortical plate.
(contributor) IMAXT consortium

first: Giorgia

last: Battistoni

first: Sito

last: Torres-Garcia

first: Chee-Ying

last: Sia

first: Silvia

last: Corriero

first: Carla

last: Boquetale

first: Elena

last: Williams

first: Martina

last: Alini

first: Nicole

last: Hemmer

first: Shankar

last: Balasubramanian

first: Benjamin

last: Czech Nicholson

first: Gregory

last: Hannon


title: Spatially tuneable multi-omics sequencing using light-driven combinatorial barcoding of molecules in tissues

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

sample_organism:
Mus musculus
synthetic construct

platform_organism:
Mus musculus
synthetic construct

overall_design: Refer to individual Series


title: Spatially tuneable multi-omics sequencing using light-driven combinatorial barcoding of molecules in tissues / overhang screen


title: Spatially tuneable multi-omics sequencing using light-driven combinatorial barcoding of molecules in tissues / RNA_BALI


title: Spatially tuneable multi-omics sequencing using light-driven combinatorial barcoding of molecules in tissues / Multiomic


title: Spatially tuneable multi-omics sequencing using light-driven combinatorial barcoding of molecules in tissues / ATAC


title: Multiple layers of innate immune response antagonism of SARS-CoV-2

city: New York

last: Zaslavsky

state: NY

address: 1 Gustave L. Levy Pl

institute: Icahn School of Medicine at Mount Sinai

summary: Analysis of immune cells in lungs from infected mice with either WT SARS-CoV-2, the NSP1 or the NSP15 mutant or mock-infection by single-cell RNA-seq identified 15 populations of major myeloid and lymphoid cells with changes in the pattern of their activation associated with viral infection. The effects of mutations in NSP1 or NSP15 on these responses suggested different immunosuppressive mechanisms of the two viral proteins.

overall_design: Single-cell RNA-seq of immune cells in lungs from infected mices with either WT SARS-CoV-2, the NSP1 or the NSP15 mutant or mock-infection


title: Transcriptomic profiling of human placenta in gestational diabetes mellitus

city: DAEJEON

first: SEONG EUN

last: LEE

address: 282 MUNHWA-RO

institute: CHUNGNAM NATIONAL UNIVERSITY

summary: We performed transcriptomics analysis on the placental tissues from gestational diabetes mellitus (GDM) patients and normal pregnant participants.

overall_design: Twelve GDM patients and Ten normal gregnant participant were recruited from Chungnam National University Hospital. All of the GDM patients were diagnosed according to the guideline of Korean Diabetes Association.

first: Seong

last: Lee

first: Yea

last: Kang

first: Hyun

last: Kim


title: Effects of Huaier of different TNBC cells on gene expression.

city: Chongqing

first: Ziwei

last: Wu

institute: Breast Disease Center, Southwest Hospital, Army Medical University

summary: To investigate the function Huaier in tnbc, we established 3 cell lines in which each target gene has been knocked down by shRNA.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for MDAMB231 cells, HCC1806 and HCC1937.

first: Linxi

last: Zhou

first: Minghao

last: Wang

first: Hong

last: Zheng


title: RNA sequencing of aortic tissues from Prdm16 smooth muscle-specific KO and control mice

city: Augusta

first: Kunzhe

last: Dong

state: GA

address: 1462 Laney Walker Blvd

department: Immunology Center of Georgia

institute: Augusta University

summary: To explore the function of Prdm16 gene in vascular smooth muscle cells (VSMCs) in vivo, we generated Prdm16 SMC-specific KO mouse model using tamoxifen-inducible Myh11-CreERT2 Cre-driver. We performed bulk RNA-sequencing on aortic tissues of both Prdm16 SMC-specific KO and control mice. Differential analysis revealed ablation of Prdm16 in SMCs disrupted the global transcriptome profile of aortic tissues. Expression of a list of genes implicated in cardiovascular disease development and SMC phenotypic modulation are significantly altered following deletion of Prdm16. We propose these transcriptome changes following loss of Prdm16 likely predispose the VSMC to a disease state.

overall_design: Prdm16 was knocked out in SMCs in mice using tamoxifen-inducible Myh11-Cre ERT2 Cre-driver. The WT littermates carrying Cre with the same tamoxifen schedule serve as control. Aortic tissues from Prdm16 SMC-specific KO (n=3, male) and WT mice (n=3, male) were harvested and cleaned by removing adventitial layer, and then subjected to bulk RNA-sequencing analysis.

first: Jiliang


title: Role of TAp63 in myogenesis [ChIP-Seq]

city: Nijmegen

first: Jo Huiqing

address: Geert Grooteplein 26/28

country: Netherlands

institute: Radboud University

summary: Myogenesis is an ordered process driven by myogenic regulatory factors (MyoD, MyoG, MRF4) culminating in the fusion of myoblasts in myotubes. p53 family members are involved in myoblasts differentiation, although the role of the specific isoforms has not been investigated. Previous experiments indicated that TAp63g isoform is the only member of the p53 family up-regulated during in vitro myogenesis, suggesting an important function for this specific isoform during this process. As experimental models we used stable modified murine myoblasts cell lines C2C7 and C2C12 (shp63 C2C7 and doxycyclin-inducible TAp63g C2C12).  Microarray analysis of shp63 C2C7 cells compared to control revealed changes in the expression of genes which codify for proteins involved in cellular proliferation and metabolic processes. We confirmed that shp63 clones had an increase in their proliferative potential, evaluated by clonogenicity assay and EdU incorporation assay, and a delay in differentiation, evaluated by WB analysis of specific differentiation markers. The same assays showed that TAp63g overexpression is able to decrease cell proliferation. Moreover, we observed an increase in mitochondrial ROS production evaluated by citofluorimetric techniques and a decrease in oxygen consumption, revealed by Seahorse analyser. RNA sequencing and ChIP sequencing for histone modification H3K27ac, performed during C2C7 differentiation, followed by microarray analysis carried out in shp63 cells revealed also several genes (coding and non coding) modulated during in vitro myoblasts differentiation. Among them, we found Igf2. Igf2 is involved in muscle development  and differentiation, both  in vivo and in vitro. We found that p63 binds its enhancer regions, indicating its involvement in regulating Ifg2 expression during differentiation. Interestingly, we found that the long non coding RNA, Airn, is down-regulated in absence of p63 during differentiation and its silencing led to a decrease in either mRNA and protein level of myogenic differentiation markers (MyoD and MyoG). Moreover, by luciferase activity assay, we found that p63 regulates Airn expression directly, binding specific enhancers. In summary, our data reveal a novel role for TAp63 in controlling myoblasts proliferation, energy metabolism and also differentiation through the expression of specific target protein coding genes and long non coding genes. Preliminary observations, using publicity available datasets, confirmed that TAp63 isoform is among the p53-family members the isoform more expressed in adult human normal skeletal muscle. Interestingly, a genome-wide gene expression profiling of skeletal muscle from early Duchenne muscular dystrophy (DMD) patients showed a significant reduction of the TAp63 isoform expression in the affected tissues. The latter observation indicate new possible scenario for studying TAp63 contribution to muscle degenerative pathologies.

overall_design: Comparative gene expression profiling analysis of RNA-seq data and ChIP-seq data of C2C12 cells.

first: Jieqiong

last: Qu

first: Huiqing

first: Eleonora

last: Candi


title: Role of TAp63 in myogenesis


title: Role of TAp63 in myogenesis [RNA-Seq]


title: Mitochondrial Ca2+ homeostasis controls inter-compartmental proteostasis in Arabidopsis plants

city: Kaifeng

first: Xiaohong

last: Zhu

address: Jinming Street

institute: Henan University