title: Erythropoietin receptor on cDC1s dictates immune tolerance

city: Palo Alto

first: Christopher

last: McGinnis

address: Biomedical Innovations Building 4600, 240 Pasteur Drive

department: Pathology

institute: Stanford University

summary: Immune tolerance is an active state of unresponsiveness of the immune system to antigens (Ags) that have the potential to induce an immune response. Such tolerance is beneficial in avoiding autoimmunity but detrimental in cancer. Type 1 conventional dendritic cells (cDC1s) are unique in their efferocytosis and cross-presenting abilities, resulting in T cell-mediated immunity or tolerance. However, the mechanisms underlying the tolerogenic function of cDC1s remain largely unknown. Here, we show that the erythropoietin receptor (EpoR) acts as a critical switch that determines the tolerogenic function of cDC1s and the threshold of Ag-specific T cell responses. In total lymphoid irradiation-induced allograft tolerance, cDC1s upregulate EpoR expression, and conditional knockout of EpoR in cDC1s diminishes Ag-specific FOXP3+ Treg induction and expansion, resulting in allograft rejection. Mechanistically, EpoR promotes efferocytosis-induced tolerogenic maturation of splenic cDC1s towards late-stage CCR7⁺ cDC1s characterized by elevated Itgb8 expression, and conditional knockout of ITGB8 in cDC1s impairs TLI/ATS-induced tolerance. In peripheral lymph nodes (pLNs), migratory cDC1s preferentially express EpoR and exogenous EPO administration enhances their Treg induction capacity. In contrast, EpoR deficiency promotes immunogenic maturation in both pLN migratory and splenic CCR7⁺ cDC1s by upregulating genes essential for MHC class II-associated Ag presentation and cross-presentation and costimulation. In cancer, loss of cDC1 EpoR leads to tumor reduction by enhancing tumor Ag-specific CD8+ T cell priming and generating more precursor exhausted T cells in tdLNs and reducing Tregs in the tumor. Targeting EpoR on cDC1s to either induce or inhibit immune tolerance could pave the way for novel treatments of a variety of diseases.

overall_design: MULTI-seq or Cell Hashing barcoded Lin(CD3e, NK1.1, CD19, B200)-SiglecH-PDCA-1-F4/80-CD11c+MHCII+XCR1+SIRPa- cDC1s mouse cDC1s isolated from EpoR-loxp/loxp, EpoR-loxp/loxp-Xcr1-Cre, or EpoR-TdTomato-Cre mice using FACS and analyzed using 3' scRNA-seq (10x Genomics).

first: Xiangyue

last: Zhang

first: Edgar

last: Engleman


title: Precursor-like T cells  maintain tissue resident memory T cell heterogeneity and restrain their effector differentiation during reactivation

status: Public on Sep 15 2025

city: Melbourne

first: Nikita

last: Potemkin

state: Vic

address: 792 Elizabeth St

department: The Peter Doherty Institute for Infection and Immunity

country: Australia

institute: University of Melbourne

type: Expression profiling by high throughput sequencing

summary: Tissue resident memory T (TRM) cells are non-recirculating lymphocytes that provide localized immunity against intracellular pathogens and cancer. Upon antigen reencounter, TRM cells can differentiate into effector cells and give rise to another generation of memory cells. While considerable heterogeneity of TRM cells has been described, it is unclear how distinct populations contribute to their maintenance or recall capacity. Here we show that TRM cells that express the transcriptional regulators HOBIT, TCF1 and ID3 exhibited precursor-like properties, contributing to the maintenance of the TRM cell pool over time and the reformation of CD103+ TRM cells after recall. TGF-beta (TGF-b) and retinoic acid signalling were required for inducing and maintaining TCF1 and ID3 expression and restrained secondary effector differentiation during reinfection. Finally, we show that TRM cells could differentiate into tissue confined and recirculating CX3CR1+ effector cells thereby contributing to protective immunity  during recall. Thus, distinct precursor-like TRM cells contribute to the maintenance of tissue memory and anamnestic  secondary memory responses.

overall_design: Adoptively co-transferred CD45.1+/CD45.2+ HobitTomCre P14 and CD45.2+ Tcf7flox/floxHobitTomCre P14 cells from the SI-IEL and SI-LP were harvested at day 35 post LCMV Armstrong infection.  Additional parallel cohorts of LCMV-immune recipient mice were treated with anti-Gr1 (NIMP-R14) to deplete recirculating antigen specific memory cells one week prior to intravenous secondary rechallenge with LM-gp33.  To minimize P2RX7 mediated cell death, all recipient mice received 50ug of Artc2.2 nanobody (BioLegend) intravenously 30min prior to euthanasia. Single cell suspensions from mouse tissues were prepared as mentioned above. Viable CD45.1+/CD45.2+ and  CD45.2+ TCRb+CD44+CD8+Va2+ cells were pooled from recipient mice and sorted for single cell RNA sequencing.

first: Kevin

last: Man

first: Vinicius

last: Alves Duarte Da Silva

first: Sarah

middle: S

last: Gabriel

first: Teisha

last: Mason

first: Tarek

last: Elmzzahi

first: Laura

last: Mackay

first: Marc

last: Beyer

first: Jan

last: Schröder

first: Georg

last: Gasteiger

first: Axel

last: Kallies


title: A tri-specific antibody engages T and myeloid cells to augment anti-tumor immunity [bulk RNA-seq]

city: Shanghai

first: Changrui

last: Yang

address: 160 Pu Jian Rd

country: China

institute: Shanghai Jiao Tong University School of Medicine

summary: Immunologically inert tumors often resist immune checkpoint inhibitors due to a scarcity of tumor-specific T cells and the immunosuppressive microenvironment, despite pronounced and functional bystander T cell infiltration. Here, we develop a novel tri-specific T cell engager (Tri-TCE) designed in a unique format to redirect T cells while mitigating immunosuppression. Our comprehensive studies utilizing co-culture systems, patient-derived tumor suspensions (PDTSs), patient-derived tumor fragments (PDTFs), and humanized mouse tumor models, demonstrate that Tri-TCE effectively activates T cells and induces tumor cell lysis.

overall_design: We collected and sequenced mutiple tumor samples for TME decoding to further identify the key determents for the prediction of Tri-TCE response.

first: Fan


title: JNK activation dynamics drive distinct gene expression patterns over time mediated by mRNA stability

city: Brookings

first: Ryan

last: Hanson

state: South Dakota

address: 1390 College Avenue, Rm 252 Box 2140D

country: USA

institute: South Dakota State University

summary: c-Jun N-terminal kinase (JNK) plays a major role in the regulation of cell death.  Numerous studies have highlighted how the dynamics of this kinase dictate whether cells survive in response to cellular stress or induce cell death mechanisms.  However, it is less clear how these dynamics potentially contribute to downstream gene expression patterns through regulated transcription factors like c-Jun.  To investigate this question, we used a treatment strategy with the JNK agonist anisomycin to drive specific dynamics; sustained, transient, or pulsed activation, and assessed the impact on downstream gene expression patterns.  We observed that multiple gene expression patterns emerged depending on the dynamics of JNK activation. Ordinary Differential Equation (ODE) models suggest that a subset of these clusters are mediated by mRNA stability and supported by measured mRNA decay rates.  Specific gene clusters also show enrichment in specific cellular pathways, including cell death and inflammatory signaling, suggesting these dynamics contribute to differential regulation of these pathways.  These findings highlight another contribution of JNK dynamics to the regulation of cellular responses to stress stimuli.

overall_design: To induce JNK activation, cells were treated with 50 ng/ml of anisomycin. For sustained activation of JNK, cells received a single treatment with anisomycin. To induce transient activation cells were treated with anisomycin for 1 hour followed by three washes with DMEM/F12 growth media and then placed into fresh media. Pulsed activation of JNK was induced by introducing a second anisomycin treatment at 4 hours followed by an additional washout 1 hour later as performed for transient conditions. JNK inhibitor conditions also received 10 uM tanzisertib to inhibit JNK activation. Total mRNA was isolated using the Qiagen RNeasy Mini Kit (Qiagen, 74106) at the indicated time points (0, 2, 4, 6, or 8-hours post-treatment).  RNA was provided to the SDSU Genomics Sequencing Core Facility for quality assessment, library preparation, and sequencing.  Libraries were prepared using the Illumina TrueSeq mRNA and 45 samples prepared for NExtSeq500 (1x75bp) High output reads.  Sequencing was performed using 3 runs to get sufficient reads for all samples and performed by the SDSU Genomics Sequencing Core Facility.  All bioinformatic analyses were done using  CLC Genomics Workbench (vs 24.0). Trimmed reads were mapped to the human genome vs hg38  as reference using a minimum length fraction of 0.8 and a minimum similarity fraction of 0.8. Differential expression was calculated on a gene basis using an absolute fold change cut-off of 2 and a FDR adjusted p-value of 0.05.

first: Abbas

last: Jedariforoughi

first: Rachel

last: Burke

first: Andrew

last: Chesak

first: Jose

middle: L

last: Gonzalez-Hernandez


title: Characterization of shear stress response of in vitro cultured human dermal lymphatic endothelial cells [RNA-Seq]

city: Zürich

first: Aline

last: Bauer

address: Vladimir-Prelog-Weg 1-5/10

department: D-CHAB

country: Switzerland

institute: ETH Zürich

summary: A hallmark of the endothelial cell phenotype is the response to shear stress. Lymphatic endothelial cells (LECs) lining lymphatic vessels (LVs) have been shown to respond to shear stress induced by the flow of lymph within LVs. Further, it has been shown that shear stress is important for the identity and specialization of the lymphatic vasculature, by inducing the expression of transcription factors and of several other molecules on LECs. Our aim was to build upon this current knowledge, and identify shear stress regulated genes in skin-derived LECs derived. We exposed LECs derived from the foreskin of 3 different human juvenile donors (commercially available) to laminar shear stress, oscillatory shear stress or static conditions. We identified several genes regulated by shear stress.

overall_design: To investigate the impact of shear stress on the gene expression of human dermal LECs, we subjected 3 donors of human dermal LECs to in vitro laminar shear stress (4 dyn/cm2), oscillatory shear stress (0.25 Hz, 4 dyn/cm2) or static condition for 48 hours. We then performed bulk RNA-seq on the samples and compared the impact of laminar or oscillatory shear stress to static conditions.

first: Ioannis

last: Kritikos

first: Cornelia

last: Halin


title: Expression Data from DLBCL Cell Line U2932 (Parental, CDC-Treated, and CDC-Resistant) and 8 DLBCL Patient Samples (5 High CDC-Sensitive, 3 Low CDC-Sensitive)

city: Epalinges

first: Hilma

last: van der Horst

address: Chemin des Boveresses 155

department: Oncology

institute: Univeresity of Lausanne

summary: Complement-dependent cytotoxicity (CDC) is an important effector function of various therapeutic antibodies. Cancer cells often develop resistance to CDC, mainly due to external factors. In this study, using models of diffuse large B-cell lymphoma (DLBCL), we compared gene expression levels between the parental U2932 and CDC-resistant U2932 DLBCL cell lines, as well as gene expression differences between highly CDC-sensitive and low CDC-sensitive DLBCL patient samples. Hereby, we aimed to identify intrinsic regulators of CDC.

overall_design: Gene expression levels were compared between the parental U2932 DLBCL cell line (U2932-WT), the U2932-Treated cells (U2932-Tr) that were exposed once to antibody-induced complement activation, and the U2932-Resistant (U2932-R) cells that were CDC-resistant by repeated exposure to antibody-induced complement. Gene expression levels were also compared between high CDC-sensitive (n=5) and low CDC-sensitive (n=3) cancer B-cells isolated and sorted (CD19+CD3-) from lymph nodes of DLBCL patients.

middle: J

first: Tamás

last: Csikós

first: Marjolein

last: Quik

first: Jort

last: van der Schans

first: Klaas

last: de Lint

first: Gonzalo

last: Nuñez Moreno

first: Meijian

last: Guan

first: Kubra

last: Karagoz

first: Esther C

middle: W

last: Breij

first: Kim C

middle: M

last: Santegoets

first: Martine E

middle: D

last: Chamuleau

first: M Guy

last: Roukens

first: Marije

middle: B

last: Overdijk

first: Tuna

last: Mutis


title: A tri-specific antibody engages T and myeloid cells to augment anti-tumor immunity

first: Changrui

last: Yang

overall_design: Human PBMCs were successfully reconstituted into engineered immunodeficient mice, followed by SKOV3 tumor implantation. Mice received two doses of control IgG, BiTE or Tri-TCE during the treatment course. We further conducted RNA sequencing of the isolated tumor tissues at the end of experiments.

first: Fan


title: Neuropathy-associated Tecpr2 mutation knock-in mice reveal endolysosomal loss of  function phenotypes in neurons and microglia

status: Public on Sep 16 2025

city: Neuherberg

first: Johannes

last: Beckers

address: Ingolstaedter Landstr. 1

department: Institute of Experimental Genetics

country: Germany

institute: Helmholtz Zentrum Muenchen

summary: Mutations in the gene encoding Tectonic b-propeller repeat-containing repeat protein 2 (TECPR2) cause hereditary sensory and autonomic neuropathy subtype 9 (HSAN9) which is a fatal neurodevelopmental and neurodegenerative disorder involving the sensory and peripheral nervous system. TECPR2 is ubiquitously expressed and linked to trafficking and sorting within the cell, however, its functional role remains poorly defined. Moreover, molecular insights into pathogenic mechanisms underlying HSAN9 are lacking. Here, we report a novel mouse model which harbors a HSAN9-associated nonsense mutation that causes loss of TECPR2 expression. Mice show altered gait, highly region-specific axonal dystrophy and extensive local gliosis. The affected medulla area prominently features swollen axons filled with amorphous protein aggregates, glycogen granules, single and double membrane vesicles as well as aberrant organelles including ER and mitochondria whose proteome is distinctly altered. Despite the locally restricted pathology the neuronal demise is detectable in the cerebrospinal fluid and responded to by damage-associated microglia. However, their capacity to clear neuronal debris seems attenuated. Overall, neuronal and microglia phenotypes point to a dysfunctional endolysosomal system when TECPR2 is missing. This was confirmed in TECPR2 knockout cells and linked to TECPR2’s interaction with the homotypic fusion and protein sorting (HOPS)–tethering complex. Collectively, we uncovered a role of TECPR2 in endolysosome maintenance which seems relevant for healthy neurons in a particular brain region.

overall_design: We analysed whole brain tissue from homozygous 5-month-old Tecpr2 L1152fs knock-in mice, and wt/wt controls

first: Debjani

last: Bhattacharya

first: Patricia

last: da Silva-Buttkus

first: Karsten

last: Nalbach

first: Lizhen

last: Cheng

first: Lillian

last: Garrett

first: Martin

last: Irmler

first: Georg

last: Kislinger

first: Ramona

last: Rodde

last: Werner

first: Christoph

last: Lengger

last: Hrabe de Angelis

first: Benedikt

last: Wefer

first: Annemarie

last: Zimprich

first: Sabine

last: Hölter

first: Valerie

last: Gailus-Durner

first: Helmut

last: Fuchs

first: Wolfgang

last: Wurst

first: Monika

last: Brill

first: Martina

last: Schifferer

first: Stefan

middle: F

last: Lichtenthaler

first: Christian

last: Behrends


title: Systematic characterization of the composition and dynamics of processing body-associated mRNAs [dataset 1]

city: Shenzhen

first: Wei

last: Chen

state: Guangdong

address: 1088 Xueyuan Avenue, Shenzhen 518055,P.R. China

department: Department of Systems Biology

institute: Southern University of Science and Technology

summary: Processing bodies (PBs) are dynamic, membraneless organelles consisting of RNAs and proteins. While PB proteins have been extensively characterized, the methods for systematically profiling PB-associated RNAs are limited. To address this, we developed PB-TRIBE-STAMP, a new tool based on two orthogonal RNA editing enzymes. Simultaneously applying APOBEC1-DDX6 and LSM14A-ADAR2dd, PB-TRIBE-STAMP identified 1,639 and 2,577 PB-associated mRNAs in HCT116 and HEK293T cells, respectively. Further genetic perturbation demonstrated that these transcripts were translationally repressed by PBs. Next, integration of PB-TRIBE-STAMP with long-read sequencing revealed that the PB-associated transcripts possessed shorter poly(A)-tails. Moreover, we established a TRIBE-ID-based tool to characterize the mRNA-PB association at high temporal resolution and unveiled a higher splicing efficiency of PB-associated XBP1 transcripts during unfolded protein response (UPR). Finally, based on sc-LSM14A-TRIBE-ID, we demonstrated the dynamic pattern of mRNA-PB interaction during cell cycle progression.

overall_design: LSM14A-TRIBE-ID cells with or without rapamycin treatment were processed for the single-cell RNA sequencing based on the Smart-seq3xpress protocol.


title: Systematic characterization of the composition and dynamics of processing body-associated mRNAs [dataset 3]

summary: Processing bodies (PBs) are dynamic, membraneless organelles consisting of RNAs and proteins. While PB proteins have been extensively characterized, the methods for systematically profiling PB-associated RNAs are limited. To address this, we developed PB-TRIBE-STAMP, a new tool based on two orthogonal RNA editing enzymes. Simultaneously applying APOBEC1-DDX6 and LSM14A-ADAR2dd, PB-TRIBE-STAMP identified 1,639 and 2,577 PB-associated mRNAs in HCT116 and HEK293T cells, respectively. Further biochemical isolation of PBs followed by RNA-seq validated that edited transcripts of these mRNAs were indeed enriched in PBs. Integration of PB-TRIBE-STAMP with long-read sequencing revealed that the PB-associated transcripts possessed shorter poly(A)-tails and mRNA isoforms with longer 3’ UTRs were more likely to be associated with PBs than those with shorter ones. Moreover, we established a TRIBE-ID-based tool to characterize the mRNA-PB association at high temporal resolution and unveiled a higher splicing efficiency of PB-associated XBP1 transcripts during unfolded protein response (UPR). Finally, based on single-cell LSM14A-TRIBE-ID (sc-LSM14A-TRIBE-ID), we demonstrated the dynamic pattern of mRNA-PB association during cell cycle progression.

overall_design: To identify PB-associated mRNAs and investigate the PB-mediated RNA regulation, we established STAMP, HyperTRIBE, PB-TRIBE-STAMP, LSM14A-TRIBE-ID, and DDX6-KO cells.  We then performed mRNA-seq, ONT long-read sequencing, and polysome-seq.


title: Single-cell RNA sequencing of human LECs derived from human skin and subcutaneous adipose tissue [CITE-Seq]

first: Aline

last: Bauer

summary: We performed single-cell RNA sequencing (10X) on human lymphatic endothelial cells (LECs) isolated from human skin and subcutaneous adipose tissue. The goal was to analyze the different sub-populations of LECs present in those tissues (e.g. capillary, pre-collector, collector), which would give new insights into the human biology of lymphatics vessels present in skin and adipose tissue.

overall_design: We isolated the stromal vascular fraction from healthy human skin and subcutaneous adipose tissue. Using FACS sorting, we then isolated human LECs and subjected them to single-cell RNA sequencing.

first: Cornelia

last: Halin


title: Coordinated control of genome-nuclear lamina interactions by Topoisomerase 2B and Lamin B receptor

city: Milano

first: Stefano

middle: Giustino

last: Manzo

state: MI

address: Via Celoria 26, Dipartimento Bioscienze, Torre A, Piano 4

department: Biosciences

country: Italy

institute: University of Milan

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

overall_design: Refer to individual Series


title: Dnmt1 determines bone length by regulating energy metabolism of growth plate chondrocytes [MBD-seq]

city: Toon

first: Yuta

last: Yanagihara

state: Ehime

address: Shitsukawa

department: Division of Integrative Pathophysiology, Proteo-Science Center

country: Japan

institute: Ehime university

type: Methylation profiling by high throughput sequencing

summary: Chondrocytes differentiated from mesenchymal stem cells play a role in determining skeletal patterns by ossification. However, it is still unclear how the maintenance DNA methylation in chondrocytes regulates differentiation and skeletal formation. In Musculoskeletal Knowledge Portal, Dnmt1 was significantly associated with “Height”. In fact, long bone of limb specific Dnmt1 deficient mice was significantly shortened due to decreased chondrocyte proliferation and accelerated differentiation. Furthermore, integrated analysis of RNA-Seq and MBD-Seq revealed that in Dnmt1ΔPrx1 chondrocytes, reduced DNA methylation resulted in an increase in energy metabolism-related genes in addition to ossification-related genes. Metabolomic analysis confirmed that almost all energy metabolites were increased in Dnmt1ΔPrx1 chondrocytes. These results indicate that Dnmt1 mediated maintenance DNA methylation governs chondrocyte differentiation by regulating energy metabolism through both gene expression and metabolite supply. Taken together, this study suggested that appropriate DNA methylation status in chondrocytes can orchestrate growth plate mineralization and subsequently determine bone length.

overall_design: To determine the effects of Dnmt1 on gene expression via DNA methylation, we performed MBD-Seq using primary cultured chondrocyte obtained from knee articular cartilage in postnatal day 3-5 both control and mutant mice.

first: Yuuki

last: Imai


title: Tumor-reactive heterotypic CD8 T cell clusters from clinical samples

city: Amsterdam

first: Austin

last: George

address: Plesmanlaan 121,

department: Division of Molecular Oncology and Immunology

country: Netherlands

institute: Netherlands Cancer Institute

summary: Emerging evidence suggests a correlation between CD8+ T cell-tumor cell proximity and immunotherapy response1–3. However, it is unknown whether these cells can be captured as functional clusters from clinical samples. In defined human co-cultures, tumor antigen-recognizing T cells outcompeted unmatched T cells in forming clusters with tumor cells, prompting us to investigate whether this feature could be used to isolate tumor-reactive T cells directly from cancer specimens. By conventional and imaging flow cytometry, we show here that from 21/21 human melanoma metastases, we were able to isolate heterotypic clusters, comprising CD8+ T cells interacting with one or more tumor cells and/or antigen-presenting cells (APCs). Single cell RNA-sequencing revealed that CD8+ T cells from clusters were enriched for tumor-reactive and exhausted gene signatures. Integration with T cell receptor (TCR)-sequencing showed increased clonality of clustered T cells, indicative of expansion. In-depth analyses revealed that these T cells had conjugated with tumor cells and various APCs, each exhibiting specific enriched cell states, which were linked to distinct patterns of cell-cell communication. CD8+ T cells expanded from clusters ex vivo exerted on average 9-fold increased killing activity towards autologous melanomas, accompanied by enhanced cytokine production. Also upon adoptive cell transfer (ACT) into mice, T cells from clusters showed superior patient-derived xenograft (PDX) killing associated with more T cell infiltration and activation. Together, these results demonstrate that tumor-reactive CD8+ T cells are enriched in functional clusters with tumor cells and/or APCs, and that they can be isolated and expanded from clinical samples. Typically excluded during single-cell sorting by flow cytometry, these distinct heterotypic CD8+ T cell clusters serve as a valuable source amenable to deciphering functional tumor-immune cell interactions, while they may also be therapeutically explored.

overall_design:
From human melanomalymph node metastases digests, CD8+ T cells in a cluster with tumor cells, CD8+ T cells in a cluster with APCs or CD8/tumor/APC singlets were isolated by fluorescence activated cell sorting (FACS) and analysed using single cell RNA and TCR sequencing. For two patients, single CD8+CD39+ T cells were sorted seperately from live cells

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Raw files for human/patient samples are being made available in EGA (https://www.ebi.ac.uk/ega/) for controlled access to the personally identifiable sequence data.
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first: Sofía

last: Ibáñez-Molero

first: Johanna

last: Veldman

first: Juan

last: Simon-Nieto

first: Joleen

last: Traets

first: Daniel

last: Peeper


title: Systematic characterization of the composition and dynamics of processing body-associated mRNAs


title: Systematic characterization of the composition and dynamics of processing body-associated mRNAs [dataset 2]


title: Dnmt1 determines bone length by regulating energy metabolism of growth plate chondrocytes [RNA-seq]

overall_design: To determine the effects of Dnmt1 on gene expression, we performed RNA-Seq using primary cultured chondrocyte and cartilage tissue obtained from knee articular cartilage in postnatal day 3-5 both control and mutant mice.


title: Optogenetics-integrated gut organ culture system connects enteric neurons dynamics and gut homeostasis [RNASeq supp]

status: Public on Sep 14 2025

city: Ramat Gan

first: Nissan

last: Yissachar

address: Bar-Ilan University

country: Israel

institute: Bar-Ilan University

summary: The enteric nervous system (ENS) senses microbiota-derived signals and orchestrates mucosal immunity and epithelial barrier functions, in health and disease. However, mechanistic dissections of intestinal neuro-immune-microbiota communications remain challenging and existing research methods limit experimental controllability and throughput. Here, we present a novel optogenetics-integrated gut organ culture system that enables real-time, whole-tissue stimulation of specific ENS lineages, allowing for detailed analysis of their functional impact. We demonstrate that optogenetic activation of enteric cholinergic neurons rapidly modulates intestinal physiology. Interestingly, distinct neuronal firing patterns differentially modulate neuro-immunological gene expression and epithelial barrier integrity. Furthermore, diverse enteric neuronal lineages exert distinct regulatory roles. While cholinergic activation promotes gene-sets associated with type-2 immunity, tachykininergic enteric neurons differentially control mucosal defense programs. Remarkably, luminal introduction of the immunomodulatory bacterium T. ramosa significantly remodeled cholinergic-induced neuro-immunological transcription. These findings suggest that complex combinatorial signals delivered by gut microbes and enteric neurons are locally integrated to fine-tune intestinal immunity and barrier defense. Collectively, we provide a powerful platform for systematic discovery and mechanistic exploration of functional neuroimmune connections, and their potential modulation by microbes, drugs or metabolites.

overall_design: Colonic tissues from ChAT-ChR2 mice were illuminated ex vivo with yellow light vs darkness (no light) (30 min stimulation; tissues collected 2 hr later) at 10 Hz.

first: Gitali

last: Naim

first: Hadar

last: Romano

first: Sivan

last: Amidror

first: David

last: Levy

first: Adva

last: Cohen

first: Carmel

last: Sochen

last: Gilberg

first: Nairouz

last: Farah

first: Vladislav

last: Rudenko

first: Yasmin

last: Yarden

first: Mengyang

last: Feng

first: Rotem

last: Tsentsarevsky

first: Ziv

last: Brodie

last: Reich

first: Ariel

last: Simon

first: Einat

last: Toister

first: Irit

last: Shoval

first: Leah

last: Armon

first: Maya

last: Schiller

first: Yossi

last: Mandel

first: Moshe

last: Biton


title: VSX2-eGFP reporter in hiPSCs dynamically monitors human retinal development in retinal organoids and reveals cell fate transition and gene networks

city: Guangzhou

first: Xiufeng

last: Zhong

address: 54 xianlie Rd

department: Zhongshan Ophthalmic Center

institute: Sun Yat-sen University

summary: The lack of understanding of the molecular and cellular characteristics of human retinal progenitor cells (RPCs) has hindered their application in cell therapy for retinal degenerative diseases. This study aims to employ retinal organoids (ROs) derived from a VSX2-eGFP fluorescence reporter human induced pluripotent stem cell (hiPSC) line for positive selection of human RPCs, investigate their features, and facilitate their applications. hiPSCs were differentiated into three-dimensional ROs following established protocols. The fidelity of the VSX2-eGFP reporter was confirmed through immunostaining. Fluorescence-activated cell sorting was employed to select VSX2-eGFP-positive (+) cells at distinct developmental stages, followed by bulk RNA sequencing (RNA-seq) analysis to assess their transcriptome profile. Immunostaining and flow cytometry were utilized to validate the identity of VSX2-eGFP+ cells and potential cluster of differentiation (CD) biomarkers for identifying human RPCs. hiPSCs were successfully differentiated into ROs containing abundant RPCs. The spatiotemporal activity of the VSX2-eGFP reporter recapitulates the dynamic expression of endogenous VSX2 protein. Compared to VSX2-eGFP- cells, VSX2-eGFP+ cells mainly exhibited characteristics of RPCs at early stages of retinal development and of bipolar cells at late stages. RNA-seq analysis revealed transcriptional heterogeneity within VSX2-eGFP+ cells across four distinct developmental stages. Moreover, the dynamic expression of 394 known CD biomarkers at distinct developmental stages was analyzed herein for the first time. One CD biomarker, TNFRSF1B, which has never been reported to be expressed in RPCs, was found to be highly expressed in RPCs at the early stages and might serve as a candidate CD biomarker for sorting RPCs. This study provides valuable insights into the molecular and cellular characteristics of human RPCs, especially their expression profiles of CD biomarkers, laying a foundation for research on retinal development and the clinical translation of hiPSC-derived RPCs.

overall_design: Retinal organoids derived from VSX2 repoter (VSX2-eGFP) hiPSCs and parental hiPSCs were collected at D60. Flow-sorted eGFP positive and eGFP negative cells at D28 were separately collected from VSX2 reprter hiPSC-derived cultures. Flow-sorted eGFP positive and eGFP negative cells at D55, D118, and D170 were separately collected from the neuralretinas of retinal organoids derived from VSX2 reporter hiPSCs. Total RNA from two independent experiments of purified cells and three independent experiments of retinal organoids were extracted using Trizol reagent kit (Invitrogen). RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies) and checked using RNase free agarose gel electrophoresis. The qualified total RNA was subjected to RNA-seq library construction. Paired-end sequencing was performed using Illumina Novaseq6000. Reads were further filtered by fastp (Chen et al., 2018) (version 0.18.0). Clean reads were mapped to the reference genome using HISAT2. 2.4 (Kim et al., 2015). For each transcription region, a FPKM (fragment per kilobase of transcript per million mapped reads) value was calculated to quantify its expression abundance and variations, using RSEM (Li and Dewey, 2011) software.

first: Dandan

last: Zheng

first: Yuan

last: Wang

first: Yuanyuan

last: Guan

first: Ping

last: Xu

first: Bingbing

last: Xie

first: Guanjie

last: Gao

first: Xiaoming

last: Yu

first: Jian

last: Ge

first: Mengqing

last: Xiang


title: Spliceosome inhibition induces Z-RNA and ZBP1-driven Cell Death in Small Cell Lung Cancer

city: Philadelphia

first: Yunyun

last: Zhou

state: Pennsylvania

address: 314 Chandler Street

institute: Fox Chase Cancer Center

summary: Spliceosome inhibitors emerged as promising anticancer agents. Recent studies have demonstrated that spliceosome targeted-therapies (STTs) trigger antitumor immune responses by inducing the accumulation of right-handed double-stranded (ds)RNA (A-RNA), resulting in activation of RIG-I-like receptors and Type I interferon-driven antiviral responses. Here, we show that pharmacological spliceosome inhibition or genetic neutralization of SF3B1 activity induces the accumulation of endogenous left-handed dsRNAs (Z-RNAs) derived from intron-retained RNAs. These Z-RNAs activate the Z-form nucleic acid-sensor ZBP1, which triggers cell death in mouse embryonic fibroblasts (MEFs) and small cell lung cancer (SCLC) cells. Spliceosome inhibition induced potent ZBP1-dependent cell death in cancer-associated fibroblasts (CAFs), which was essential for enhancing immunotherapy response in mouse models of SCLC. Collectively, these results demonstrate that spliceosome inhibitors or STTs, can be used to generate Z-RNA and trigger on-demand ZBP1-dependent cell death in cells of the tumor microenvironment (TME) as a therapeutic strategy to enhance immunotherapy responses in resistant-cancers.

overall_design: To elucidate the Z-RNA species accumulated post spliceosome perturbation, we treated EV MEFs with DMSO or Pladienolide B (PlaB) for 18 hours followed by Z22-antibody RNA immunoprecipitation.

first: Xinpei

last: Jiang

first: Israel

last: Cañadas


title: Optogenetics-integrated gut organ culture system connects enteric neurons dynamics and gut homeostasis [16S]

first: Nissan

last: Yissachar

overall_design: Luminal contents from ChAT-ChR2 colonic tissues were collected following 10Hz blue or yellow light stimulation in the ex vivo culture system. After two hours, microbial composition was profiled using 16S rDNA sequencing (paired littermates were used to minimize cage-related variability).

first: Gitali

last: Naim

first: Hadar

last: Romano

first: Sivan

last: Amidror

first: David

last: Levy

first: Adva

last: Cohen

first: Carmel

last: Sochen

last: Gilberg

first: Nairouz

last: Farah

first: Vladislav

last: Rudenko

first: Yasmin

last: Yarden

first: Mengyang

last: Feng

first: Rotem

last: Tsentsarevsky

first: Ziv

last: Brodie

last: Reich

first: Ariel

last: Simon

first: Einat

last: Toister

first: Irit

last: Shoval

first: Leah

last: Armon


title: CAR-SPLASH identifies nascent pre-mRNA structures implicated in kinetic coupling and alternative splicing [smallRNA-Seq]

city: Aurora

last: Bentley

state: Colorado

department: BMG

institute: UCD AMC

type: Non-coding RNA profiling by high throughput sequencing

summary: Transcription and co-transcriptional pre-mRNA splicing are kinetically coupled as shown by the widespread effects of transcription speed on alternative splicing. The molecular basis for kinetic coupling is incompletely understood, but one potential mechanism is through elongation rate dependent alternative folding pathways of the nascent pre-mRNA. To search for RNA structures in nascent pre-mRNA, we modified SPLASH (1) (Sequencing of Psoralen Crosslinked, Ligated And Selected Hybrids) for use with Chromatin Associated RNA. We applied this new method called CAR-SPLASH to cells expressing WT and slow mutant pol II and identified >3000 intragenic RNA structures of which  >400 are proximal to splice sites. ASO disruption of several structures identified by CAR-SPLASH that sequester splice sites changed alternative splicing outcomes in vivo. We identified three examples of novel regulatory elements we designate “RNA kinetic switches”. ASO disruption of these structures modified alternative splicing of NISCH Exon 18 GAK Exon 7 and MEGF8 Exon 24 in a way that depended on the rate of transcription elongation. These results demonstrate that individual RNA structures can mediate kinetic coupling between transcription and pre-mRNA splicing and that nascent RNA structures can serve as targets for splice modifying ASO’s.

overall_design: HEK293 Flp-in cells expressing WT or slow pol II mutant (Rpb1 R749H) a-amanitin resistant pol II were cross-linked with a biotinylated Psoralen analogue. Cross-linked chromatin associated RNA was purified on streptavidin beads and libraries made of proximity ligated RNA fragments.

first: Hossein

last: Shenasa

first: Benjamin

last: Erickson


title: Optogenetics-integrated gut organ culture system connects enteric neurons dynamics and gut homeostasis [RNASeq]

first: Nissan

last: Yissachar

overall_design: Colonic tissues from ChAT-ChR2 or Tac-ChR2 mice were illuminated ex vivo with blue or yellow light (30 min stimulation; tissues collected 2 hr later) at 2 Hz or 10 Hz, with or without C.ram. Tissues were subjected to multiple experimental conditions, as detailed in the accompanying design summary.

first: Gitali

last: Naim

first: Hadar

last: Romano

last: Amidror

last: Levy

last: Cohen

first: Maya

last: Schiller

first: Yossi

last: Mandel

first: Moshe

last: Biton


title: Surgery-induced Neutrophil Extracellular Traps lead to tumor metastasis by reprogramming cancer cell lipid metabolism

city: Pittsburgh

first: Shuchang

last: Liu

state: PA

address: 203 Lothrop Street

institute: University of Pittsburgh

summary: Although surgery is a crucial intervention to treat patients with solid tumors, the accompanying immune response, including the release of Neutrophil Extracellular Traps (NETs), may enhance tumor metastasis. In this study, we explored the effects of systemic surgical NETs in reprogramming cancer metabolism and thereby fostering metastatic tumor growth. To model the effect of surgery on tumor progression, C57BL/6 mice inoculated with subcutaneous MC-38 or subcutaneous LLC-1 cells underwent a midline laparotomy with mesenteric exploration for 30 mins. Mice subjected to surgery showed accelerated primary subcutaneous and lung metastatic tumor growth. Perioperative NETs inhibition utilizing DNAse, GSK484, or PAD4-KO mice prevented the surgically-induced tumor growth. Pre-treating cancer cells with NETs in vitro before inoculation also increased tumor burden. Transcriptomic analysis of MC-38 cancer cells exposed to surgical stress in vivo or treated with NETs in vitro showed activation of the MYC oncogenic pathway. This was associated with upregulation of fatty acid oxidation (FAO), shown with METAFlux analysis and Acetyl-CoA quantification. Blocking FAO with etomoxir (CPT1α inhibitor) prevented the cancer proliferation and metastatic tumor growth induced by surgical NETs. NETs also stimulated uptake of long-chain fatty acids (LCFA) and expression of CD36, the main LCFA transporter. FA metabolism was crucial for cancer cells under anoikis stress, allowing survival of circulating cancer cells exposed to NETs. These findings were corroborated in human tumors analyzed from the TCGA database. In conclusion, the immune response to surgery promotes tumor metastasis through NETs activating cancer cell lipid metabolism. Inhibiting this metabolic reprogramming offers a potential strategy to modulate the protumorigenic effects of surgical stress.

overall_design: To elucidate the transcriptomic changes in MC-38 cancer cells exposed to surgically-induced NETs, we performed bulk RNA-sequencing on MC-38 cancer cells cultured in vitro alone or with NET-chromatin collected fro neutrophils in vitro. Bulk RNA seq was also performed on MC-38 cancer cells expressing GFP isolated by flow sorting at day 30 from in vivo tumors in WT mice, WT mice exposed to laparotomy on days 0 and 10, PAD-4 KO mice or PAD-4 KO mice exposed to laparotomy on days 0 and 10.

first: Tony

last: Haykal

first: Ruiqi

last: Yang

first: Celine

last: Tohme

first: Silvia

first: Zheng

last: Songyang

first: Yu

last: Yan Ping

first: Luo

last: Jian-Hua

first: Hamza

last: Yazdani

first: Samer


title: The evolution of facultative symbiosis in corals

city: Barcelona

first: Arnau

last: Sebe-Pedros

address: Dr. Aiguader 88

department: Systems Biology

country: Spain

institute: Centre for Genomic Regulation

summary: We performed single-cell transcriptome analysis of three stony coral species: Oculina patagonica (Opat), Acropora millepora (Amil) and Stylophora pistillata (Spis)

sample_organism:
Acropora millepora
Stylophora pistillata
Oculina patagonica

overall_design: Adult specimens were fixed, dissociated and pooled before sorting single-cells for scRNAseq. Cell hashing was performed using clicktag barcodes in order to overload 10X runs. Instructions for demultiplexing cells are provided.

first: Shani

first: Xavier

last: Grau-Bove


title: YEATS4 Reads Histone Crotonylation to Promote Fatty Acid Metabolism and Cancer Cell Stemness [CUT&Tag]

city: Beijing

first: Zijun

last: Peng

address: Xueyuan road number 38

institute: Peking University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: How histone lysine crotonylation (Kcr) is read and interpreted remains to be elucidated. We report here that YEATS4, a potential breast cancer driver identified recently by two independent genome-wide association studies, is a reader of H3K14cr. Integrative metabolomic, epigenomic, and transcriptomic analyses reveal that H3K14cr reading by YEATS4 is associated with a shift of cellular metabolic profile and transcription activation of a cohort of genes, including CD36, CPT1A, and ACOX1, that are critically involved in the uptake and metabolism of fatty acids. High expression of YEATS4 fortifies fatty acid metabolism, enhances self-renewal and growth of ALDH+ breast cancer stem cells, and is correlated with poor prognosis of breast cancer patients, especially the ER+ subtype. Our work uncovers YEATS4 as an “amplifier” in the feedforward circuit of histone crotonylation and lipid metabolism underlying the stemness and cell proliferation, supporting the pursuit of YEATS4 as a potential target for breast cancer intervention.

overall_design: Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing for YEATS domain family protein YEATS4 and histone modifications H3K14cr and H3K27ac in MCF-7 cells.

first: Huajing

last: Yu


title: Functional genetics of rice PISTILLATA genes reveals new roles and target genes in flowering time, female fertility and parthenocarpy

city: Bengaluru

first: Usha

last: Vijayraghavan

state: Karnataka

address: New biological Sciences, Indian Institute of Sciences

department: MCB

country: India

institute: Indian Institute of Science

summary: Diversification of transcription factors and of their downstream targets can contribute to new organ morphologies. An example is rice lodicule, a small fleshy petal analogue that aids floret opening, thus facilitates pollination and fertility. To understand the mechanism underlying its specification, we investigated the developmental functions of the rice PISTILLATA (PI) paralogs, OsMADS2 and OsMADS4. Null osmads2 mutants reiterated OsMADS2 nonredundant lodicule specification roles and importantly revealed new roles in flowering time and floral organ number and fate in multiple whorls. Doubly perturbed osmads2d8/d8 osmads4kd florets had severe abnormalities and initiated parthenocarpy. Remarkably, ubiquitous OsMADS4 overexpression rescued osmads2 abnormalities, though impaired anther development and seed setting. Target genes whose (in)direct regulation can contribute to OsMADS2 functions were discovered by identifying its genome-wide binding regions and by transcriptome profiling. Several OsMADS2 targets are implicated in lodicule and stamen development, floral organ number, cell wall, cell shape and osmotic homeostasis processes. Altogether, we reveal novel roles for the rice PI paralogs, e.g. panicle exsertion and embryo sac differentiation and provide mechanistic insights on their diversification and functions in floral organ specification.

overall_design: Genome wide binding of OsMADS2 transcription factor in rice genome by high throughput sequencing and downstream analysis

first: Sandhan

last: Prakash

first: Sara

last: Simmonini

first: Raghavaram

last: Peesapati


title: Single-cell RNA sequencing of ovarian cells from mice aged 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks

city: Baltimore

first: Allan

last: Spradling

state: MD

address: 3520 San Martin Drive

department: Department of Embryology

institute: Carnegie Institution for Science

summary: There are at least two waves of follicle development in mammals. Wave 1 follicle development occurs as early as postnatal day 0 (P0) in mice, when follicles are formed. Wave 2 follicles become primordial follicles, which are gradually recruited in older mice and persist throughout the animal's reproductive life. The wave 1 follicles reach the antral stage by 3 weeks and can be ovulated when treated with hormones, although this does not normally occur in natural development. Despite this phenomenon being known for decades, the functions of wave 1 follicles remain unclear, particularly if they do not provide mature, healthy oocytes. Through single-cell RNA sequencing, we found that androgens, rather than estrogens, are the main hormones produced during these stages. We also observed that wave 1 follicles generate hormone producing theca cells and related interstitial gland cells to produce androgens, which may be important for puberty onset and the development of the ovary, brain, and other tissues. In this database, we present data from single-cell RNA sequencing of ovarian cells from mice aged 2 to 6 weeks, collected weekly.

overall_design: The ovaries were dissected from the mice and dissociated into single cells for twenty minutes at 37°C using 0.25% trypsin. Cells stained with SYTOX Orange Dead Cell Stain were subject to fluorescence-activated cell sorting (FACS) from which non-stained live cells were recovered. The cells were then subjected to library construction using the Chromium Next GEM Single Cell 3 Reagent Kits v3.1 from 10X Genomics. The library was subsequently sequenced on an Illumina NextSeq 500 platform.

first: Qi

last: Yin

middle: C


title: YEATS4 Reads Histone Crotonylation to Promote Fatty Acid Metabolism and Cancer Cell Stemness [RNA-seq]

overall_design: Examination of YEATS4 target genes in MCF-7 cell line. We constructed YEATS4 knockdown MCF-7 cells using shRNA and performed RNA-sequencing (RNA-seq) to examine the YEATS4-regulated transcriptome. Repeat three times for each group.


title: EPAS1 directs a network of genes implicated in mitochondrial dysfunction in arrhythmogenic cardiomyopathy

city: Leiden

first: Sebastiaan

middle: Johannes

last: van Kampen

address: Sylviusweg 62

institute: LUMC


title: A tri-specific antibody engages T and myeloid cells to augment anti-tumor immunity [TCE_scRNA-seq]

city: Shanghai

first: Changrui

last: Yang

address: 160 Pu Jian Rd

institute: Shanghai Jiao Tong University School of Medicine

summary: Immunologically inert tumors often resist immune checkpoint inhibitors due to a scarcity of tumor-specific T cells and the immunosuppressive microenvironment, despite pronounced and functional bystander T cell infiltration. Here, we develop a novel tri-specific T cell engager (Tri-TCE) designed in a unique format to redirect T cells while mitigating immunosuppression. Our comprehensive studies utilizing co-culture systems, patient-derived tumor suspensions (PDTSs), patient-derived tumor fragments (PDTFs), and humanized mouse tumor models, demonstrate that Tri-TCE effectively activates T cells and induces tumor cell lysis.

overall_design: To elucidate the mechanisms underlying Tri-TCE treatment, we employed a clinical PDTS system. Clinical tumor samples (ovarian cancer) were digested with collagenase-II and DNase I. After filtration through a mesh strainer with 70-μm pores, single-cell suspensions were prepared and cultured for 3days treated with control-IgG or Tri-TCE. Using fluorescence-activated cell sorting to isolate CD45+ immune cells, we conducted single-cell transcriptome and TCR sequencing.

first: Fan


title: EPAS1 directs a network of genes implicated in mitochondrial dysfunction in arrhythmogenic cardiomyopathy [Spatial Transcriptomics]

summary: Patients diagnosed with arrhythmogenic cardiomyopathy (ACM) often harbor desmosomal mutations, predisposing them to arrhythmia and sudden cardiac death. The molecular mechanisms underlying pathobiology remain incompletely understood, which is reflected by the limited number of therapeutic strategies. Here, we performed spatially resolved transcriptomics on an explanted heart isolated from a patient bearing a mutation in desmoplakin. Exploration of the transcriptional atlas uncovered endothelial PAS domain-containing protein 1 (EPAS1) as a potential regulator of mitochondrial homeostasis in stressed cardiomyocytes. By combining these data with multiple models of cardiac disease, we show that induced EPAS1 levels activate expression of genes involved in mitophagy and the antioxidant response. Findings were validated in additional explanted heart from ACM and dilated cardiomyopathy patients. Together, genetically impaired cardiomyocytes display mitochondrial dysfunction, consequently resulting in stabilization of EPAS1. In turn, this factor dictates a gene network involved in mitigating the adverse effects of oxidative stress.

overall_design: Spatial transcriptomics on an explanted heart collected from an ACM patient bearing a nonsense mutation in DSP.

first: Eva

last: van Rooij


title: EPAS1 directs a network of genes implicated in mitochondrial dysfunction in arrhythmogenic cardiomyopathy [RNA-seq]

overall_design: hiPSC-derived cardiomyocyte mRNA profiles for DSP p.Arg1113X/WT and control cells.


summary: Diversification of transcription factors and of their downstream targets can contribute to new organ morphologies. An example is rice lodicule, a small fleshy petal analogue that aids floret opening, thus facilitates pollination and fertility. To understand the mechanism underlying its specification, we investigated the developmental functions of the rice PISTILLATA (PI) paralogs, OsMADS2 and OsMADS4. Null osmads2 mutants reiterated OsMADS2 nonredundant lodicule specification roles and importantly revealed new roles in flowering time and floral organ number and fate in multiple whorls. Doubly perturbed osmads2d8/d8 27 osmads4kd florets had severe abnormalities and initiated parthenocarpy. Remarkably, ubiquitous OsMADS4 overexpression rescued osmads2 abnormalities, though impaired anther development and seed setting. Target genes whose (in)direct regulation can contribute to OsMADS2 functions were discovered by identifying its genome-wide binding regions and by transcriptome profiling. Several OsMADS2 targets are implicated in lodicule and stamen development, floral organ number, cell wall, cell shape and osmotic homeostasis processes. Altogether, we reveal novel roles for the rice PI paralogs, e.g. panicle exsertion and embryo sac differentiation and provide mechanistic insights on their diversification and functions in floral organ specification

overall_design: Expression profiling of wildtype and osmads2 mutant in rice inflorescence segregated into three devlopmental pools. The developmental pools are named as Early (E), Middle (M) and Late (L) that represents three developmental pools of inflorescence. Early (E) : 0.2-0.5 cm panicle, Middle (M) : 0.5-1.0cm panicle, Late (L) : 1.0 - 2.0 cm panicle

first: Mohamed

last: Zamzam


title: WDR5 serves in co-activation and influences genome targeting of KLF3

city: Sydney

first: Merlin

last: Crossley

state: NSW

address: UNSW

department: BABS

institute: University of New South Wales

summary: Krüppel-like Factor 3 (KLF3) is a member of the archetypal SP/KLF family of transcription factors that bind GC-rich elements and CACCC boxes in promoters and enhancers via three classical Zinc Fingers (ZFs) at or near their C-termini. KLF3 can both repress and activate transcription. It represses by recruiting CtBP co-repressors via its N-terminal domain but the mechanism by which it activates was unknown. Here we show KLF3 associates with WDR5 and this interaction is associated with gene activation. We also demonstrate that this interaction is required for proper genomic targeting of KLF3. This helps explain previous results indicating that both the C-terminal ZF DNA-binding domain and N-terminal functional domain are required for KLF3 to identify its target genes. This result adds to only a handful of examples that transcriptional co-regulators, in addition to facilitating activation and repression, can also influence target gene selection.

overall_design: RNA-seq, KLF3-V5, and WDR5 ChIP-seq in KLF3-null MEF cells rescued by overexpressing WT KLF3-V5 or R254A KLF3-V5

first: Lu

first: Manan

last: Shah

first: Tanit

last: Chavalit

first: Annalise

last: Spek

first: Wooi

last: Lim

first: Ling

last: Zhong

first: Vala

last: Nejad Safari

first: Mahdi

last: Haddad

first: Thu

middle: H

last: Vu

first: Joel

middle: P

last: Mackay

first: Mark

last: Raftery

first: Marc

middle: R

last: Wilkins

first: Jacqueline

last: Matthews

first: Kate

middle: G

last: Quinlan


title: Analysis of hybrid RNA/DNA profiles in blood from autistic patients reveals differences in mRNA and ncRNA regions

status: Public on Sep 13 2025

city: Nice

first: Minoo

last: Rassoulzadegan

address: parc Valrose

department: Institut de Biologie Valrose: iBV

country: France

institute: Nice Sophia Antipolis

summary:
Autism spectrum disorder (ASD) is a set of genetically heterogenous neurodevelopmental disorders characterized by core symptoms including impaired social interaction, communication deficits, and restricted or stereotyped behaviors. While a significant number of cases are not explained by Mendelian inheritance, there is growing evidence in implication of non-coding RNAs (ncRNAs) in development and inheritance of ASD. Transcriptional studies face challenges from patient-specific variations in gene expression and technical differences in preserving RNA integrity. We propose that isolating RNA from DNA/RNA hybrids provides a robust method to capture transcriptional information. We performed a whole transcriptome analysis on blood samples from ASD
patients and healthy controls to investigate transcripts associated with DNA/RNA hybrids. We identified 278,300 novel transcripts across 68,487 DNA/RNA hybrid loci, with significant enrichment in exonic and intronic regions. The novel long non-coding RNAs (lncRNAs) we found showed higher expression levels compared to known transcripts. Differential expression analysis revealed 301 significantly upregulated and 401 downregulated transcripts in ASD samples. Through qRT-PCR validation, we confirmed the significant upregulation of RN7SK and SMARCC2 associated with DNA/RNA hybrids in ASD patients. Pathway and enrichment analyses highlighted mitochondrial dysfunction and energy metabolism. Our results suggest that ncRNAs can form DNA/RNA hybrids that influence gene expression, providing preliminary insights into the mechanisms of transcriptional dysregulation in ASD.

overall_design: DNA-associated RNA molecules were purified form 12 blood samples (6 autism patients, 6 Controls). 10-100 ng of RNA after Dnase digestion were recovered and analyzed by high throughput sequencing on Illumina.

first: Leila

last: Kianmehr

first: Kasra

last: Mokhtarzadeh Azar

first: Zeynep

last: Yilmaz

first: Ecmel

last: Mehmetbeyoglu

first: Elif Funda

last: Sener

first: Serpil

last: Taheri


title: Perturb-seq experiment on patient-derived cancer-associated fibroblast in vitro cell line models from pancreatic ductal adenocarcinoma

status: Public on Sep 12 2025

city: South San Francisco

first: Elysia

middle: Cecilia

last: Saputra

state: CA

address: 213 E Grand Ave

department: Data, AI, and Genome Sciences

institute: Merck & Co., Inc.

summary: Cancer-associated fibroblasts (CAFs) are sought after as potential therapeutic targets due to their plethora of pro- and antitumorigenic functions. Recent findings attributed this functional heterogeneity to specializations in CAF subtypes. A precise targeting of specific subtypes would thus be required to design therapies that effectively modulate CAF phenotypes, but our knowledge of CAF heterogeneity in solid tumors, particularly pancreatic ductal adenocarcinoma (PDAC), is still lacking. Here, we use single-cell transcriptomics to characterize CAF subtype heterogeneity in in vitro CAF cell lines isolated from pancreatic tumor patients and investigate subtype-resolution modulations arising from perturbing potential stromal genes. We use the immortalized CAF cell lines to perform single-cell CRISPR perturbations of candidate stromal targets, revealing not only the subtype-specific effects of the perturbations, but also the impact of model-type selection on the translatability of insights.

overall_design: hTERT-immortalized cell lines of patient-derived pancreatic cancer associated fibroblasts were perturbed with sgRNAs targeting TGFBR1, AEBP1, SPATS2L, PTGS1, or RUNX1, and then were either co-cultured with BxPC3 cell lines or kept as mono-cultures. The cells were then profiled for scRNA-seq readouts.

first: Shamsudheen

middle: K

last: Vellarikkal

first: Lixia

last: Li

first: Hong

last: Sun

first: Suchitra

last: Natarajan

first: Alex

last: Tamburino

first: Xin

last: Yu

first: Aleksandra

last: Olow


title: ZAP327 signaling domain–driven chimeric antigen receptor generates robust and long-term antitumor immunity in mouse models

city: Los Angeles

first: Rongfu

last: Wang

address: 1441 Eastlake Ave., NRT5517

institute: Keck School of Medicine, USC

summary: Chimeric antigen receptor (CAR) T-cell therapy has shown impressive clinical responses in the treatment of blood cancers, but high percentages of disease relapse one year after T cell infusion and severe toxicities associated with CAR-T cell therapy remain major issues. Here, we report the construction of CARs with a ZAP-70-derived signaling domain (ZAP327) that enhances therapeutic antitumor activity with increasing in vivo T cell persistence. Importantly, ZAP327-driven CAR-T cells markedly reduced cytokine release and expression of T cell exhaustion markers. Mechanistically, we show that the ZAP327 domain tuned down TCR signaling, increased the pools of stem-like memory T cells, and exhibited metabolic features associated with memory T cells by utilizing the oxidative phosphorylation pathway. These results highlight the therapeutic potential of ZAP327-driven CAR-T cells to overcome the limitations of the current CAR-T cell therapies and enhance the potency and persistence of antitumor T cell responses in solid tumors as well.

overall_design: RNA-seq of human T cells transduced with 1928z or 1928ZAP327 CARs. Cells were either stimulated or untreated.

last: Liu


title: Promoter G-quadruplex structures regulate tissue-specific gene expression and functional differentiation in Bombyx mori [CUT&Tag]

first: Lijun

last: Xiang

address: No.55, West of Zhongshan Avenue

department: Guangzhou Key Laboratory of Insect Development Regulation and Application Research

institute: South China Normal University

summary: Cell differentiation, tissue development and functional formation rely on the precise activation of specific gene expression which regulated by the interaction of epigenetic modifications and transcription factors. DNA G-quadruplex structures (G4s) have emerged as important regulators of gene transcription. Here, by nuclear G4-CUT&Tag, ATAC-seq, and histone modification data, we demonstrate that G4s in Bombyx mori are primarily located in promoter regions and are associated with active chromatin marks. Comparative analyses of G4 distribution in the brain and fat body revealed that identical promoter sequences can adopt distinct G4 folding states in different tissues. Notably, the presence of folded G4s strongly correlated with high transcriptional activity of genes essential for tissue-specific functions. Furthermore, treatment with G4-stabilizing ligands disrupted energy metabolism and significantly delayed neurodevelopment in B.mori.. In summary, our findings emphasize G4s as critical epigenetic regulators in maintaining tissue development and functional formation in insects.

overall_design: G4-CUT&Tag to detect G4 structures in the brain, fat body, and ovarian cell line BmN of silkworms

first: Kangkang

last: Niu


title: Transcriptomics- and 3D imaging–based characterization of the lymphatic vasculature in human skin

city: Zürich

first: Aline

last: Bauer

address: Vladimir-Prelog-Weg 1-5/10

institute: ETH Zürich

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

overall_design: Refer to individual Series


title: Promoter G-quadruplex structures regulate tissue-specific gene expression and functional differentiation in Bombyx mori [RNA-seq]

summary: Cell differentiation, tissue development and functional formation rely on the precise activation of specific gene expression which regulated by the interaction of epigenetic modifications and transcription factors. DNA G-quadruplex structures (G4s) have emerged as important regulators of gene transcription. Here, by nuclear G4-CUT&Tag, ATAC-seq, and histone modification data, we demonstrate that G4s in Bombyx mori are primarily located in promoter regions and are associated with active chromatin marks. Comparative analyses of G4 distribution in the brain and fat body revealed that identical promoter sequences can adopt distinct G4 folding states in different tissues. Notably, the presence of folded G4s strongly correlated with high transcriptional activity of genes essential for tissue-specific functions. Furthermore, treatment with G4-stabilizing ligands disrupted energy metabolism and significantly delayed neurodevelopment in B. mori. In summary, our findings emphasize G4s as critical epigenetic regulators in maintaining tissue development and functional formation in insect.

overall_design: RNA-seq analysis of gene expression in the silkworm brain at the 3rd day of the 5th larval instar


title: Promoter G-quadruplex structures regulate tissue-specific gene expression and functional differentiation in Bombyx mori [ATAC-seq]

overall_design: ATAC-seq to detect chromatin accessible region in the brain, fat body of silkworms


title: Single nucleus transcriptomics of spinal ependymoma subtypes recognizes intratumoral heterogeneity [methylation]

city: Hamburg

first: Karoline

last: Hack

address: Martinistraße 52

department: Children's Cancer Center Hamburg

institute: University Medical Center Hamburg-Eppendorf

type: Methylation profiling by genome tiling array

summary: The two most prevalent types of spinal ependymal tumors are myxopapillary ependymomas (MPE) and spinal ependymomas (SP-EPN). Based on molecular data from bulk tumor samples, we previously identified clinically relevant subtypes with poor (MPE-A; SP-EPN-A) and favorable progression-free survival (MPE-B; SP-EPN-B). However, detailed cellular compositions, molecular heterogeneity, and features of tumor progression are largely unknown. We performed single nucleus transcriptomic sequencing of 25 formalin-fixed and paraffin-embedded spinal ependymal tumors including all MPE and SP-EPN subtypes as well as six paired primary and relapsed MPE-A. Ependymoma subtypes presented with a broad, but comparable tumor microenvironment. Furthermore, neoplastic cells demonstrated inter- and intratumoral heterogeneity regarding cancer cell state composition. Overall, MPE tumors exhibited a significantly higher abundance of the astrocytic state, whereas a ciliated state was more prevalent in SP-EPN. Also, transcriptome-inferred copy number profiles revealed tumor cell clusters with distinct chromosomal alterations across tumors, suggesting subclonal neoplastic growth. Compared to normal human spinal cord cell populations, MPE tumor cells displayed similarities to astrocytes and ependymal cells, whereas SP-EPN exclusively matched ependymal cells. MPE-A demonstrated a distinct profile, being similar to the roof and floor plate of the developing spinal cord. Paired primary and relapsed MPE-A revealed mostly similar histology, epigenetics, copy number profiles, and cellular composition of neoplastic cells. Single nucleus transcriptomic sequencing provides valuable insights into the molecular composition of spinal ependymoma types and subtypes and may pave the way for a better understanding of tumor evolution and identification of therapeutic targets.

overall_design: We compared DNA-methylation array data of paired primary and recurred MPE-A tumors.

first: Hack

last: Karoline

first: Schüller

last: Ulrich


title: Type I IFN impedes early bacterial control in TB-resistant and -susceptible mice and activates neutrophils to restrict lung macrophage-T cell interactions [scRNA-seq]

city: London

first: Anne

last: O'Garra

address: 1 Midland Rd

country: United Kingdom

institute: The Francis Crick Institute

summary: Only a minority of M. tuberculosis-infected individuals progress to tuberculosis, however the early immune mechanisms determining outcome are unclear. C3HeB/FeJ mice display high, partially type I IFN-dependent, tuberculosis susceptibility, compared to relatively tuberculosis-resistant C57BL/6 mice. Using bulk and scRNA-seq over the first weeks of M. tuberculosis infection, we describe an unexpected, higher early pulmonary type I IFN response in C57BL/6 than C3HeB/FeJ mice, accompanying more pronounced early monocyte-derived macrophage (MDM) accumulation and lesion formation. The C57BL/6 type I IFN response plateaued with formation of extensive CD4+ T cell-MDM interactions in lesions, accompanied by high expression of T cell-attractant chemokines by MDMs. Conversely, delayed immune initiation in C3HeB/FeJ mice preceded rapid domination of lesions by inflammatory neutrophils, depletion of which enhanced CD4+ T cell-MDM interactions. Type I IFNs unexpectedly promoted early bacterial replication and limited lesion CD4+ T cell numbers in both C57BL/6 and C3HeB/FeJ mice, while pronounced later effects on inflammatory neutrophil activation were observed in C3HeB/FeJ, suggestive of context-dependent effects of type I IFNs during M. tuberculosis infection.

overall_design: Single-cell RNA-seq of mouse lung from C57BL/6 and C3HeB/FeJ mice, uninfected and infected with HN878 Mycobacterium tuberculosis strain at day 14 and day 20 of infection, using 10X Genomics Flex kit.

first: William

last: Branchett

first: Evangelos

last: Stavropoulos

first: Jessica

last: Shields

first: Alaa

last: Al-Dibouni

first: Marcos

last: Cardoso

first: Ana

middle: I

last: Fernandes

first: Lúcia

last: Moreira-Teixeira

first: Hubert

last: Slawinski

first: Ania

last: Mikolajczak

first: Angela

last: Rodgers

first: Margarida

last: Saraiva


title: Single cell transcriptomics of partial hepatocyte reprogramming

first: Abhinav

last: Illendula

address: 333 Cottman Avenue

institute: Fox Chase Cancer Center

summary: This study analyzes hepatocytes with Notch-induced reprogramming by single cell RNAseq compared to normal hepatocytes and biliary epithelial cells (BECs) in the normal liver.

overall_design: Three-weeks post-hydrodynamic tail vein injection (HDTVi) of our TTR>NICD-CAG>tdT Piggy-Bac transposon in an 8-week-old C57Bl/6 mouse, liver parenchymal cells were isolated and sorted by Fluorescence Activated Cell Sorting (FACS). tdT- hepatocytes, tdT+ reprogramming cells, and EpCAM+ BECs were sorted and subsequently mixed in a 1:2:1 ratio, respectively. This process was repeated in a second mouse and both samples were analyzed for their transcriptional profile by scRNAseq.

first: Lauren

last: Strathearn

first: Joan

last: Font-Burgada


title: Functional evidence for early origin of tactile acuity in the vertebrate somatosensory system

city: New Haven

first: Sviatoslav

middle: N

last: Bagriantsev

state: Connecticut

address: 333 Cedar St

department: Cellular and Molecular Physiology

institute: Yale University

summary: Mammals and reptiles possess a sophisticated somatosensory system for precise tactile discrimination via mechanosensory end-organs, such as Meissner and Pacinian corpuscles, and others. These structures detect sustained pressure, velocity, and vibrations, thereby facilitating nuanced environmental interactions. Whether the ancestral anamniotic somatosensory system, typically lacking such structures, provides comparable tactile discrimination, is unknown. Here, we investigate the Schnauzenorgan, a specialized foraging chin appendage in Mormyrid fish Gnathonemus petersii and show that it detects touch via functionally distinct myelinated mechanosensory afferents. Although these afferents terminate in the skin as seemingly free nerve endings, they detect sustained pressure, transient touch, velocity, low- and high-frequency vibrations. Thus, despite lacking typical end-organs, the Schnauzenorgan enables tactile discrimination rivaling that of amniotic extremities. Our findings reveal a previously unrecognized functional complexity in the ancestral piscine somatosensory system, suggesting that the nuanced mechanosensory capacity of amniotes was inherited from anamniote predecessors.

overall_design: Gene expression analysis of trigeminal ganglia, brains (cerebella), and spinal cords from 6 individual elephantnose fish (Gnathonemus petersii)

first: Ke

last: Tan

first: Elena

middle: O

last: Gracheva


title: Craters on the melanoma surface serve as hubs for CD8+ T cells [ATAC-Seq]

city: Boston

first: Leonard

last: Zon

state: MA

address: 1 Blackfan Circle

department: Oncology/Hematology

institute: Boston Children's Hospital

summary: Immune cell interactions with tumor cells are critical for cancer therapeutic response, but it is unclear whether T cells take a direct or random path into a tumor. We performed 3D, long-term imaging of zebrafish melanomas and found that CD8+ T cells localize to craters on the tumor surface. Craters are the primary site of CD8 T cells accumulation and activation following CpG oligonucleotide injection, TLR agonists that elicit an immune response, or systemic TGF-β inhibition.  Human melanomas exhibit craters at the tumor surface and in perivascular spaces where they harbor dysfunctional, proliferative CD8+ T cells. In both zebrafish and human tumors, crater size and number positively correlate with CD8+ T cell infiltration. These results identify craters as hubs that provide a direct path for CD8+ T cells into tumors.

overall_design: ATAC Seq lck cells

first: Aya

last: Ludin

first: Georgia

middle: L

last: Stirtz


title: Ribosomal DNA Accessibility in Hematopoiesis

city: Philadelphia

first: Vikram

last: Paralkar

address: 421 Curie Boulevard

department: Division of Hematology and Oncology, Department of Medicine

institute: University of Pennsylvania Perelman School of Medicine

summary: Transcription of ribosomal RNAs (rRNAs) from rDNA repeats is the first step of ribosome biogenesis, accounting for a major portion of all cellular transcription. Often regarded as a housekeeping process, its cell-type-specific regulation is widely neglected. We profiled rRNA transcription rate and mature rRNA (ribosome subunit) levels in detail across mouse hematopoiesis, and observed that rRNA abundance is cell-type-specific and largely uncoupled from cell cycling and protein synthesis rates. We tested the longstanding assumption that rRNA levels are elevated in acute myeloid leukemia (AML) and found that leukemic progenitors showed notably higher nascent and mature rRNA levels than matched normal counterparts. Across contexts of hematopoiesis, trends in rRNA transcription paralleled changes in chromatin accessibility of rDNA repeats. Absolute quantification of rRNA molecules unexpectedly revealed that large ribosome subunits are in excess to small subunits in all cell types, most prominently in the normal myeloid lineage. Collectively, our work provides a detailed profile of the complex dynamics of rRNAs within and between normal and leukemic hematopoiesis.

relation:
Reanalysis of: GSM5222812
Reanalysis of: GSM5567440

sample_organism:
Homo sapiens
Mus musculus

overall_design:
Reanalysis of published scATAC-Seq datasets from GEO (GSM5222812/GSE171220; GSM5567440/GSE183675) and ENCODE (ENCSR217VXJ/ENCBS249EEJ; ENCSR332SXG/ENCBS991GWL; ENCSR308ZGJ/ENCBS337EJK). Raw FASTQ files were downloaded from GEO or ENCODE as applicable. Raw Reads were mapped using cellranger-atac. Peakcalling and motif analysis were done using ArchR. Cell type barcodes were assigned using ArchR and used to split the BAM files into subfiles per celltype. BigWig tracks were generated using bamCoverage from deepTools. custom genomes: https://github.com/vikramparalkar/rDNA-Mapping-Genomes/blob/main/Human_hg38-rDNA_genome_v1.0.tar.gz; https://github.com/vikramparalkar/rDNA-Mapping-Genomes/blob/main/Mouse_mm39-rDNA_genome_v1.0.tar.gz. Reanalysis processed data includes: rds files from ArchR and BigWig tracks.

***************************************************************
The table below lists GEO accessions reused/reanalyzed for this study.
The reanalysis_samples.xlsx file also lists accessions reused/reanalyzed for this study.
***************************************************************

first: Eleanor

last: Sams

first: Subin

middle: S

last: George


title: Epigenetic profiles of tissue informative CpGs inform ALS disease status and progression

status: Public on Sep 11 2025

first: Noah

last: Zaitlen

address: 675 Charles E Young Dr S

department: Neurology

institute: UCLA

summary: Background: Cell-free DNA (cfDNA), derived from dying cells, has demonstrated utility across multiple clinical applications. However, its potential in neurodegenerative diseases remains underexplored, with most existing cfDNA technologies tailored to specific disease contexts like cancer or non-invasive prenatal screening. Methods: To address this gap, we developed a novel approach to characterize epigenetic cfDNA profiles by identifying key regions of DNA methylation that reveal the tissues origins undergoing apoptosis or necrosis. We evaluated this method in the largest cfDNA study of amyotrophic lateral sclerosis (ALS) and other neurological diseases (OND) to date, encompassing two independent cohorts (n=192) from Australia  (UQ Ncases=48, Ncontrols=32, NOND=15) and the USA, (UCSF Ncases=50 , Ncontrols=45)).Results: Our approach accurately distinguished ALS patients from controls (UQ AUC=0.82, UCSF AUC=0.99) and from individuals with other neurological diseases (AUC=0.91). It also identified an asymptomatic carrier of a pathogenic C9orf72 variant, and strongly correlated with ALS disease progression measures (Pearson’s R=0.66, p=3.71×10⁻⁹). Conclusions: We identified DNA methylation signals from multiple tissue types in ALS cfDNA, highlighting diverse tissue involvement in ALS pathology. These findings promote epigenetic cfDNA analysis as a powerful tool for advancing our understanding of neurodegenerative disease.

overall_design: Cell-free DNA was extracted from 46 Controls and 50 ALS patients from the UCSF, and 48 controls and 48 ALS patients from the University of Queensland. Cell free DNA was extracted from 20mL of whole blood in control patients and 10mL of whole blood for cases. cfDNA was then bisulfite converted and underwent high throughput methylation sequencing

first: Christa

last: Caggiano

first: Marco

last: Morselli

first: Xiaoyu

last: Qian

first: Barbara

last: Celona

first: Mike

last: Thompson

first: Shivangi

last: Wani

first: Anela

last: Tosevska

first: Kodi

last: Taraszka

first: Galen

last: Heuer

first: Shyuan

last: Ngo

first: Frederick

last: Steyn

first: Peter

middle: J

last: Nestor

first: Leanne

last: Wallace

first: Pamela

last: McCombe

first: Susan

last: Heggie

first: Kathryn

last: Thorpe

first: Caitlin

last: McElligott

first: Gemyka

last: English

first: Anjali

last: Henders

first: Robert

last: Henderson

first: Catherine

last: Lomen-Hoerth

first: Naomi

last: Wray

first: Allan

last: McRae

first: Matteo

last: Pellegrini

first: Fleur

last: Garton


title: A SOX2-VSX2 axis modulates a core cis-regulatory circuitry to safeguard retinal neural competence [ChIP-Seq]

city: Pittsburgh

first: Shuchang

last: Liu

address: 203 Lothrop Street

institute: University of Pittsburgh

summary: Retinal neurogenesis is mediated by the coordinated activities of a complex gene regulatory network (GRN) of transcription factors (TFs) in multipotent retinal progenitor cells (RPCs). How this GRN mechanistically guides neural competence remains poorly understood. Here, we describe integrated transcriptional, genetic, and genomic analyses to identify the regulatory mechanisms of SOX2, a critical factor for RPC neural competency. We show that SOX2 is preferentially enriched in the RPC-specific enhancer landscape associated with essential regulators of retinogenesis. Perturbation of SOX2 expression leads to a failure in retinogenesis initiation as a result of selective loss of enhancer activity near key genes underlying proliferation and lineage specification. We identified the RPC transcription factor VSX2 as a binding partner for SOX2, and together they target a core retina-specific chromatin repertoire characterized by enhanced TF binding and robust chromatin accessibility. We delineate a SOX2-VSX2 common transcriptional code that promotes the expression of critical regulators of neurogenesis and suppresses the acquisition of alternative lineage cell fate. Our data illuminate fundamental biological insights on how transcription factors act in concert to drive chromatin-based genetic programs underlying retinal neural competence.

overall_design: Mouse retina ChIP-seq on Sox2 E14.5 with the corresponding input. Chromatin immunoprecipitation (ChIP) was performed on 30-40 μg of sheared chromatin using 6 μl of SOX2 antibody

first: Fuyun

last: Bian

first: Kimia

last: Golestaneh

first: Emily

last: Davis

first: Abdullah

last: Khan

first: Marwa

last: Daghsni

first: Brendan

last: Nicholson

first: Keevon

last: Flohr

first: Silvia

first: Susana

last: da Silva

first: Len

last: Pennacchio

first: Issam

last: Aldiri


title: Craters on the melanoma surface serve as hubs for CD8+ T cells

first: Leonard

last: Zon


title: Craters on the melanoma surface serve as hubs for CD8+ T cells [RNA-Seq]

overall_design: For RNA Seq- examined the identity of EGFP+ lymphoid and myeloid cells in cd8:EGFP transgenic zebrafish

first: Aya

last: Ludin

first: Georgia

last: Stirtz


title: Single nucleus transcriptomics of spinal ependymoma subtypes recognizes intratumoral heterogeneity [snRNA-seq]

first: Karoline

last: Hack

overall_design: We analyzed archival formalin-fixed, paraffin-embedded-tissues of 25 myxopapillary ependymomas (MPE) and spinal ependymomas (SP-EPN) using the 10x Genomics Flex Gene Expression workflow with the "Next GEM" chemistry. Clinically relevant subtypes MPE-A, MPE-B, SP-EPN-A and SP-EPN-B  as well as paired primary and recurred MPE-A tumors were compared using this data.

first: Ulrich

last: Schüller


title: Molecular interplay between peptidoglycan integrity and outer membrane asymmetry in maintaining cell envelope homeostasis

city: Richardson

first: Joseph

middle: Michael

last: Boll

state: TX

address: 800 W Campbell

department: Biological Sciences

institute: University of Texas at Dallas

summary: Loss of LdtJ in Acinetobacter baumannii disrupts cell morphology, downregulates peptidoglycan precursor genes (e.g., dadA, alr), and activates the stringent response, including elevated ppGpp levels and dksA upregulation. These defects are fully suppressed in a ∆ldtJ ∆mla double mutant, implicating the outer membrane lipid transport Mla pathway in compensatory regulation. RNA sequencing revealed that transcriptional changes in the ∆ldtJ mutant are reversed in the double mutant, highlighting a functional interplay between peptidoglycan remodeling and outer membrane lipid asymmetry.

overall_design: Differential gene expression in A. baumannii strain ATCC 17978 wild type,  ∆ldtJ, and  ∆ldtJ ∆mla strains

first: Sinjini

last: Nandy

first: Arshya

last: Tehrani

first: Augusto

last: Hunt-Serracin

first: Jacob

last: Biboy

first: Christine

last: Pybus

first: Waldemar

last: Vollmer

middle: M


title: Characterization of CIC binding to chromatin in murine KP and KPCic cell lines

city: Madrid

first: Tirso

last: Pons

state: Madrid

address: C/ Darwin 3

department: Inmunology and Oncology

institute: Centro Nacional de Biotecnologia

summary: CIC binding was determined in KP murine lung cancer cells in untreated cells as well as after treatment with trametinib to enhance CIC binding to DNA. As a negative control, CIC binding was also determined in KPCic cells that express a non-functional CIC incapable of DNA binding.

overall_design: Cells were grown in DMEM supplemeted with 10% FBS. Cells were fixed with 1% formaldehyde for 10 min. After quenching and cell lysis, chromatin was sonicated using a Bioruptor Plus Diagenode. Library construction and sequencing was carried out by HaploX Gene Tech Limited (Hong Kong)

first: Matthias

last: Drosten

first: Irene

last: Ballesteros-González


title: Single-nucleus transcriptomics reveals sepsis-related neurovascular dysfunction in the human hippocampus

city: Charleston

first: Liu

last: Liu

state: South Carolina

address: 173 Ashley Ave. CRI 610

institute: Medical University of South Carolina

summary: Sepsis is increasingly recognized as a major precipitant of long-term cognitive impairment, yet the cellular mechanisms underlying hippocampal vulnerability remain elusive. Using single-nucleus RNA sequencing of human hippocampal tissues from sepsis and control patients, we identied profound neurovascular alterations involving 21 distinct cell populations. Notably, astrocytes and microglia exhibited marked polarization: Astrocyte 2 showed simultaneous upregulation of neurotoxic A1 and neuroprotective A2 gene signatures in sepsis, whereas Astrocyte 1 displayed reduced A1 activity and a relatively quiescent pro le. In parallel, Microglia 2 demonstrated a prominent M1-like in ammatory signature, including elevated expression of HLA-DRA, IL1B, and TNF, while Microglia 1 downregulated both M1 and M2 markers, suggesting a hypo-responsive state. Intercellular communication analysis revealed intensi ed astrocyte–microglia interactions in the septic hippocampus, potentially amplifying in ammatory signaling loops. Concurrently, endothelial and mural cells exhibited transcriptional signatures of blood-brain barrier (BBB) disruption, oxidative stress, and compromised vascular homeostasis. Key molecular pathways associated with antigen presentation, cytokine signaling, and vascular permeability were selectively activated across neurovascular compartments. Together, our ndings uncover a coordinated glial and vascular response to systemic in ammation, driven in part by dysfunctional astrocyte–microglia crosstalk and pro-in ammatory polarization. These changes may underlie BBB breakdown and contribute to sustained neuroin ammation and cognitive decline in sepsis survivors. Targeting glial-vascular signaling axes and modulating astrocyte or microglial polarization states may offer promising avenues for therapeutic intervention in post-sepsis neurological sequelae.

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

first: Pengfei

last: Li

first: Hongkuan

last: Fan


title: Synergistic Effect of Nucleoside Modification and Ionizable Lipid Composition on Translation and Immune Responses to mRNA Vaccines

city: Waltham

first: Sudha

last: Chivukula

address: 200 West

department: mRNA Center of Excellence

institute: Sanofi

summary: Lipid nanoparticle (LNP) structural components can impact the safety and immunogenicity of mRNA vaccines. Here we examine the mechanisms contributing to the performance of mRNA-LNP vaccines by exploring the impact of nucleoside modifications and LNP components on translational efficiency, innate immune activation, and immunogenicity, by employing in vitro (cell culture) and in vivo (mice and cynomolgus macaques) models. Our data reveal several molecular and immunological parameters affected by nucleoside modification. These include: i) a sequence-dependent increase in antigen expression; ii) decreased induction of genes associated with antiviral activity and RNA degradation in modified mRNA compared with unmodified mRNA; iii) reduced innate immune recognition of nucleoside-modified mRNA, accompanied by lower levels of inflammatory cytokines and chemokines in non-human primates; and iv) a synergistic effect of the mRNA and ionizable lipid composition on the immune activation triggered by the mRNA-LNP formulation. Our data show that changes in the LNP composition, independent from whether the mRNA is modified or unmodified, caused differential expression of genes associated with innate and antiviral immunity. For instance, modification of mRNA was able to silence the innate immune signatures more efficiently for certain LNPs but not others. We believe these findings offer valuable insights into mRNA vaccine function and offer strategies for enhancing vaccine efficacy and reducing the reactogenicity of next generation mRNA vaccines.

sample_organism:
Macaca fascicularis
Homo sapiens

platform_organism:
Macaca fascicularis
Homo sapiens

overall_design: RNA-seq profiling both human skeletal muscle cells (HSKM) and non-human primate peripheral blood mononuclear cells (PBMC) following mRNA-LNP treatments.

first: Hillary

last: Danz

first: Allison

last: Dauner

first: Shraddha

last: Sharma

first: Mihaela

last: Babiceanu

first: Nicholas

last: Clark

first: Robert

last: Ontiveros

last: Amezquita

first: Alisa

last: Zhilin-Roth

first: Eric

last: Reyes

first: Catherine

last: Khoo

first: Janhavi

last: Nadkarni

first: Omkar

last: Chaudhary

first: Christina

last: Lee

first: Mona

last: Motwani

first: Bin

last: Lu

first: Wei

last: Zong

first: Andrew

last: Kettring

first: Alex

last: Shumate

first: Shrirang

last: Karve

first: Anusha

last: Dias

first: Monica

last: Wu

first: Xiaobo

last: Gu

first: Yanhua

last: Yan

first: Daniel

last: Anderson

first: Brian

last: Schanen

first: Frank

last: DeRosa


title: Identification of key molecules promoting lepidic–filigree micropapillary–conventional/overt micropapillary progression in lung adenocarcinoma: A comprehensive gene expression analysis study using the spatial gene expression solution methodology

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

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

summary: The micropapillary (mPAP) histological subtype is a high-grade element and a poor prognostic marker in lung adenocarcinoma (LUAD). This subtype develops through the lepidic–filigree mPAP–conventional/overt mPAP pathway. In the present study, we aimed to identify key molecules that promote this progression. To this end, we investigated gene expression profiles specific to lepidic, filigree mPAP (filigree), and conventional/overt mPAP (mPAP) elements in histological sections obtained from four different LUAD cases.

overall_design: We studied four cases of surgically resected lung adenocarcinoma (LUAD).  Eight tissue FFPE sections (n = 2 each from the four LUAD samples) that included lepidic, filigree and mPAP elements were analyzed using Visium Spatial Gene Expression Profiling System. RNA transcriptome data were obtained from individual spots corresponding to each histological element. The mRNA levels in the lepidic, filigree, and mPAP components were then compared and analyzed.

first: Mai

last: Matsumura

first: Tetsukan

last: Woo

first: Hiromasa

last: Arai

first: Chihiro

last: Koike

first: Toshiaki

last: Kataoka

first: Kiyoharu

last: Fukushima

first: Koji

last: Okudela


title: CELF2 regulation in multiple myeloma

city: St. Louis

first: Jessica

last: Silva-Fisher

address: 425 Euclid

institute: Washington University in St. Louis

summary: In this study, we analyzed transcriptome sequencing data to compare CELF2 RNA binding protein regulation in multiple myeloma cell line

overall_design: We transfected RPMI 8226 cell line with 5nM CELF2 LNA ASO (n =2) and compared to Control LNA ASO (n=2) for 72 hours. We isolated RNA with Takara RNA isolation kit. Total RNA was extracted, and RNA integrity was assessed using an Agilent Bioanalyzer. Poly(A)-selected libraries were prepared and sequenced on the Illumina NovaSeq X Plus platform, generating 150 bp paired-end reads to a depth of approximately 50 million reads per sample.


title: LINC01432 regulation in multiple myeloma

summary: In this study, we analyzed transcriptome sequencing data of LINC01432 RNA regulation in a multiple myeloma cell line

overall_design: We transfected RPMI 8226 cell line with 50nM LINC01432 LNA ASO (n =2) and compared to Control LNA ASO (n=2) for 72 hours. We isolated RNA with Takara RNA isolation kit. Total RNA was extracted, and RNA integrity was assessed using an Agilent Bioanalyzer. Poly(A)-selected libraries were prepared and sequenced on the Illumina NovaSeq X Plus platform, generating 150 bp paired-end reads to a depth of approximately 50 million reads per sample.


title: Investigating oncoprotein-mediated chromatin dysregulation in Drosophila melanogaster uncovers novel modifiers of the developmental impact of H3 K27M and EZHIP [CUT&Run]

city: Madison

first: Melissa

last: Harrison

email: mharrison3@wisc.edu, ykaur@wisc.edu, torreszelada@wisc.edu, mmfreund2@wisc.edu

state: WI

address: 420 Henry Mall

department: Biomolecular Chemistry

institute: University of Wisconsin Madison

summary: Polycomb Repressive Complex 2 (PRC2) maintains epigenetic repression through the catalysis of H3K27 trimethylation (H3K27me3), which restricts gene expression and preserves developmental gene- regulatory networks. The integrity of PRC2-mediated gene silencing depends critically on the ability of PRC2 to establish and propagate H3K27me3 beyond initial recruitment sites. The oncoproteins EZHIP and histone H3 K27M specifically inhibit this propagation by blocking the allosterically activated state of PRC2, leading to global disruption of H3K27me3 patterns and developmental abnormalities. To uncover chromatin-related pathways intersecting with PRC2 repression, we developed a Drosophila melanogaster model with tissue-specific expression of EZHIP and H3 K27M. A targeted RNAi screen of conserved chromatin regulators identified genetic modifiers that when knocked down either enhanced or suppressed developmental phenotypes driven by these PRC2 inhibitors. Strong suppressors, including the Trithorax-group proteins Ash1 and Trx, the PR-DUB complex member Asx, and the nucleoporin Nup153, restored normal development despite persistent depletion of global H3K27me3. Gene expression analyses revealed that suppression reflected reduced expression of genes aberrantly activated following PRC2 inhibition. Together, these findings highlight conserved chromatin-regulatory pathways that intersect with Polycomb to maintain transcriptional balance and support developmental homeostasis.

overall_design: This data set contains CUT&Run on H3K27me3  in wing imaginal discs of Drosophila melanogaster upon expression of the oncohistone H3 K27M or H3 K27R control. Two biological replicates were used to assay H3K27me3 for each genotype, and one replicate was used in IgG control. SNAP-CUTANA K-MetStat nucleosomes were included to provide a standard for spike-in normalization. Spike-in normalization was based on percentage of reads mapping to all barcoded nucleosomes.

first: Sammuel

middle: D

last: Krabbenhoft

first: Tyler

middle: E

last: Masuda

first: Yadwinder

last: Kaur

first: Truman

last: Do

first: Siddhant

middle: U

last: Jain

first: Peter

middle: W

last: Lewis


title: Transcriptomic analysis of lung adenocarcinomas from KP and KPCic mice

first: Tirso

last: Pons

summary: The transcriptional repressor Capicua (CIC) is negatively regulated by KRAS/MAPK signaling. To characterize the impact of CIC loss on KrasG12V/Trp53-KO-driven lung adenocarcinomas in mice, the transcriptome of tumors from Kras (+/LSLG12V); Trp53 (lox/lox) [KP] and Kras (+/LSLG12V); Trp53 (lox/lox); Cic (lox/lox) [KPCic] mice are compared.

overall_design: RNA-seq profiling of lung adenocarcinomas from KP and KPCic mice obtained 5 months after infection with Adeno-Cre.

first: Matthias

last: Drosten

first: Irene

last: Ballesteros-González


title: Mapping the Hemocyte Surfaceome of Apis mellifera by a Proteomic and Transcriptomic Approach

city: Ghent

first: Merel

last: Braeckman

address: Krijgslaan 281

country: Belgium

institute: Ghent University

summary: Transcriptomic analysis was performed on hemocytes from 1-day old nurse bees and L5-larvae.

overall_design: RNA-Seq of Apis mellifera hemocytes in different developmental stages: L5-larvae and (<1 day old) nurse bees.

first: Dirk

middle: C

last: de Graaf

first: Lina

last: De Smet


title: Type I IFN impedes early bacterial control in TB-resistant and -susceptible mice and activates neutrophils to restrict lung macrophage-T cell interactions [RNA-seq]

first: Anne

last: O'Garra

summary: Only a minority of M. tuberculosis-infected individuals progress to tuberculosis, however the early immune mechanisms determining outcome are unclear. C3HeB/FeJ mice display high, partially type I IFN-dependent, tuberculosis susceptibility, compared to relatively tuberculosis-resistant C57BL/6 mice. Using bulk and scRNA-seq over the first weeks of M. tuberculosis infection, we describe an unexpected, higher early pulmonary type I IFN response in C57BL/6 than C3HeB/FeJ mice, accompanying more pronounced early monocyte-derived macrophage (MDM) accumulation and lesion formation. The C57BL/6 type I IFN response plateaued with formation of extensive CD4+ T cell-MDM interactions in lesions, accompanied by high expression of T cell-attractant chemokines by MDMs. Conversely, delayed immune initiation in C3HeB/FeJ mice preceded rapid domination of lesions by inflammatory neutrophils, depletion of which enhanced CD4+ T cell-MDM interactions. Type I IFNs unexpectedly promoted early bacterial replication and limited lesion CD4+ T cell numbers in both C57BL/6 and C3HeB/FeJ mice, while pronounced later effects on inflammatory neutrophil activation were observed in C3HeB/FeJ, suggestive of context-dependent effects of type I IFNs during M. tuberculosis infection.

overall_design: RNA-seq profiling of bronchoalveolar lavage and lung from C57BL/6 and C3HeB/FeJ mice, uninfected and infected with HN878 Mycobacterium tuberculosis strain at day 14 and day 21 of infection. RNA-seq profiling of lung from C3HeB/FeJ mice, uninfected and infected with HN878 Mycobacterium tuberculosis strain at day 26 and treated with either isotype control or anti-IFNAR antibody

first: William

last: Branchett

first: Evangelos

last: Stavropoulos

last: Shields

first: Alaa

last: Al-Dibouni

first: Marcos

last: Cardoso

first: Ana

last: Fernandes

first: Lúcia

last: Moreira-Teixeira

first: Hubert

last: Slawinski

first: Ania

last: Mikolajczak

first: Angela

last: Rodgers

first: Margarida

last: Saraiva


title: Investigating oncoprotein-mediated chromatin dysregulation in Drosophila melanogaster uncovers novel modifiers of the developmental impact of H3 K27M and EZHIP [RNA-seq]

first: Melissa

last: Harrison

overall_design: These data include bulk RNA-seq to analyze gene expression in imaginal discs of the third instar Drosophila melanogaster larvae (wing and eye) upon expression of the oncohistone H3 K27M or the control H3K27R. To determine how various genetically identified suppressors of the H3 K27M wing phenotype modify gene expression, RNA-seq was also performed on wing discs in which Trx, Ash1, Nup153 or Asx were depleted by RNAi in the context of either H3 K27M or H3 K27R expression.

first: Sammuel

last: Krabbenhoft

first: Tyler

last: Masuda

first: Yadwinder

last: Kaur

first: Truman

last: Do

first: Siddhant

last: Jain

first: Peter

last: Lewis


title: Autocrine ECM molecules establish MSC quiescence during incisor development by disrupting WNT ligand trafficking process

city: Beijing

first: Hu

last: Zhao

address: Bldg.3, NO.9, YIKE Rd, Zhongguancun Life Science Park, Changping District

institute: Chinese institution for brain research

summary: We used single-cell sequencing to understand the development of incisor tissues from mice. We utilized the single-cell sequencing platform from SeekGene Biotechnology to study the heterogeneity of mesenchyme in incisors at both embryonic and adult stages of mice, and discovered that Smoc2 is a marker gene for mesenchymal stem cells in mouse incisors. We employed the 10X platform to sequence the incisor tissues of smoc1/2 knockout (ko) and control mice during the embryonic stage to investigate the molecular mechanisms underlying the developmental abnormalities in smoc1/2 ko mice.Our study provides a molecular mechanism explaining how stem cells form during development.

overall_design: Single-cell transcriptomes were obtained from the digestion of the incisor pulp at different stages (E13.5, E14.5, E15.5, E16.5, E18.5 and P56) and group( control and Smoc1/2 ko).

first: Zexi

last: Chen

first: Junjun

last: Jing