title: Discovery of Key Regulators in Classical Monocyte Phenotypes Linked to COVID-19 Severity using Single-cell Multi-omics Sequencing

city: Gwangju

first: Donggun

last: Kim

address: 123, Cheomdangwagi-ro

department: Life Science

country: South Korea

institute: Gwangju Institute of Science and Technology

summary: Dysregulated immune responses often accompany severe COVID-19, but the underlying epigenetic mechanisms driving monocyte heterogeneity and disease progression remain poorly understood. Here, we applied single-cell multi-omics profiling to peripheral blood mononuclear cells from individuals across five COVID-19 severity stages. We identified two severity-associated classical monocyte subtypes-IL7R+ and CD163+-characterized by distinct transcriptional and epigenetic landscapes. Using constructing of gene regulatory network and in silico TF perturbations, we revealed ETS1 as a key driver of IL7R+ monocytes with T cell-like signaling features, and JDP2 as a repressor maintaining the pro-fibrotic, anti-inflammatory identity of CD163+ monocytes via suppression of AP-1 activity. These subtypes were enriched in moderate-to-critical stages and exhibited signaling pathways associated with tissue remodeling and immune suppression. Our findings define monocyte heterogeneity linked to COVID-19 severity and identify ETS1 and JDP2 as central regulators, offering insight into immune dysregulation and potential therapeutic targets for fibrosis and long-term sequelae.

overall_design:
Whole bloods (4-6mL per sample) were collected from adult COVID-19 patients. Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation with Histopaque®-1077 (Sigma-Aldrich, Cat. No. 10771), frozen in cryopreservation medium, and stored under controlled freezing conditions for subsequent analysis. The Single-cell multi-omics libraries were generated using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kits (10x Genomics, Cat. No. PN-1000283) and sequenced by HiSeq X Ten platform.

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

last: Choi

first: Eui Tae

first: Jihwan

last: Park


title: Step-wise signal integration governs adaptive programming of lymphocytes [bulkRNA-seq and ATAC-seq 6hr Stim]

status: Public on Jan 22 2026

city: New York

first: Hyunu

state: NY

address: 417 E 68th St

department: Immunology

country: USA

institute: Memorial Sloan Kettering Cancer Center

type: Genome binding/occupancy profiling by high throughput sequencing

type: Expression profiling by high throughput sequencing

summary: Lymphocyte differentiation depends on activation via antigen and cytokines during the immune response to infection. How the timing and integration of these signals program the epigenetic and functional fate of these cells is not completely understood. In this study, we find that inflammatory cytokine signals received by innate and adaptive lymphocytes have a context-dependent role for immune memory formation. Without preceding and sufficient antigen receptor signaling, inflammatory cytokines drive terminal differentiation into short-lived effector cells. In contrast, sufficient antigen-receptor signaling redirects inflammatory cytokine signals to promote memory differentiation via cooperation of STAT and AP-1 transcription factors. By this crucial epigenetic mechanism, optimally equipped lymphocytes are selected for memory formation rather than a terminal effector cell fate. Whereas T cells are hardwired to be shielded from premature inflammatory signals, NK cells rely on coincidental early antigen receptor signaling for adaptive responses. Together, step-wise integration of antigen and cytokine signaling optimizes both effector and memory differentiation, allowing for promiscuous recruitment into the acute immune response while promoting avidity maturation in memory populations of both innate and adaptive lymphocytes.

overall_design: Bulk RNA-seq and ATAC-seq of in vitro stimulated NK cells (with the indicated stimulations) as well as NK cells isolated from MCMV-infected mice taken after 1 day post infection (2-4 replicates each).

first: Simon

last: Grassmann

first: Joseph

last: Sun


title: IRE1signaling in Osx1-cre expressing cells

city: College Station

first: Kartik

last: Shankar

email: kartik.shankar@cuanschutz.edu, kartikster@gmail.com, kartik.shankar@usda.gov, kartik.shankar@ag.tamu.edu

state: TX

address: 498 Olsen Blvd TAMU 2250 TAMU 2250

department: RAFSRU

institute: USDA ARS

summary: The Unfolded Protein Response (UPR) fine tunes the protein folding capacity of the endoplasmic reticulum (ER) in eukaryotic cells via the actions of PERK, IRE1 and ATF6 proteins. However, the specific contributions of these proteins to bone health have not been investigated. These studies evaluated the role of IRE1 signlaing in preosteoblasts by condcting studies in osteoprogenitors lacking IRE1 (generated via Osx1-cre). The studies invovled isolation and culture of IRE1KO and flox control osteoprogenitor cells that gene expression analysis. Our studies reveal a novel regulatory role in stimulating osteoprogenitor proliferation by stimulating β-catenin/TCF mediated transcription and subsequent bone formation

overall_design: Osx1-Cre cells were flow sorted from neonatal calvarial cells cultured in alpha-MEM medium containing 10% FBS and plated in a 6-well plate. RNA was isolated after cells attained 90% confluence. Osx1-Cre cells isolated from 3-5 neonatal pups (2-4 d old) as described constituted 1 biological replicate. Total of 9 libraries (n=5 IRE1 KO and n=4 WT) were generated.

first: Iyer

last: Srividhya

first: Shankar

last: Kartik


title: Next generation sequencing facilitates quantitative analysis of transcriptional profiling of  Candida albicans SC5314 with compound 20 treatment or not.

city: Wuhan

first: hongxuan

last: cao

address: 152 Luoyu Road, Hongshan District

country: China

institute: Central China Normal University

summary: mRNA profiles of Candida albicans SC5314 treated with compound 20 and untreated Candida albicans SC5314 were generated by deep sequencing, in triplicate, using Illumina HiSeq X Ten. The sequence reads that passed quality filters were analyzed at the transcript isoform level. qRT–PCR validation was performed using TB Green assay. We find that some genes in ribosomes pathaway such as YVH1, RPL12 and MRT4 were upregulated, which confirmed that Mrt4 is a valid target for cis-fumaramidmycin-derived compounds.

first: Jiahui

last: Chen

first: Wenqiang

last: Chang

first: Hongxuan

last: Cao

first: Yanliang

last: Ren


title: Comparison of CD74+ vs CD74- microglia/macrophages from the brain TME from 2 different type of tumors (B16/F10-BrM & H2030-BrM)

city: Madrid

first: Manuel

last: Valiente

address: Melchor Fernández Almagro, 3

country: Spain

institute: CNIO

summary: To investigate the properties of CD74+ microglia/macrophages compared to the CD74- microglia/macrophages in brain metastasis.

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of CD74+ microglia/macrophages compared to CD74- microglia/macrophages isolated from 4 experiments of B16/F10-BrM brain metastasis (pool of at least 3 brains each) and 4 experiments of H2030-BrM brain metastasis (pool of at least 3 brains)

first: L

last: Álvaro

first: C

last: Hernández Oliver

first: M


title: iCLIP analysis of full-length and deletion mutants of myc-LARP6

city: Oxford

first: Faraz

middle: K.

last: Mardakheh

address: South Parks Road

department: Department of Biochemistry

country: United Kingdom

institute: University of Oxford

summary: Intrinsically disordered regions (IDRs) are prevalent in RNA-binding proteins (RBPs), yet their roles in RNA interactions remain poorly defined. We examined the structured and disordered RNA-binding activities of LARP6, an RBP with a diverse RNA-binding repertoire. U87 glioblastoma cells stably expressing myc-tagged full-length or various deletion mutants of LARP6 under a doxycycline switch were induced to express myc-LARP6 variants at near endogenous levels, before individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) was performed to assess each variants' RNA-binding targets on the transcriptome. A total RNA-seq experiment was also performed on the cells in parallel to distinguish between changes in RNA bidning and those that are affected via expression level variations.

overall_design:
iCLIP of myc-LARP6 from full-length (FL), N-terminal Region deleted (deltaNTR), La-module deleted (delta LaMod), N-terminal domain deleted (deltaNTD),  and C-terminal region deleted (deltaCTR1) U87 cells, along with untransfected control cells was performed as indicated in (https://doi.org/10.1101/2024.09.20.614075).
To normalise iCLIP differences based on total RNA variations, an rRNA depletion whole transcriptome sequencing of each stably expressing U87 cell-line following LARP6 induction was also performed in parallel, using short single read (SE50) and long paired end read (PE150) sequencing. These were used as input control.

first: Federica

last: Capraro

middle: K

first: Maria

middle: R

last: Conte


title: Virus-specific effector T cells impair airway secretory cell-mediated alveolar regeneration

city: Shanghai

first: pei

last: wu

address: 393 Middle Huaxia Road

institute: ShanghaiTech University

summary: Severe respiratory viral infection leads to extensive damage to the alveolar epithelium and also induce a robust immune response. How immune response impacts alveolar regeneration, especially how immune microenvironment interacts with lung stem/progenitor cells is poorly understood. Here, we find that dysplastic Krt5+ basal-like cells, which emerge after severe viral infection, preferably recruit and maintain the tissue residence of both CD4+ effector T cells and CD8+ T cells in a CXCR3- and Integrin α4/β2-dependent manner after viral clearance. Persistent CD4+ and CD8+ T cells impair alveolar regeneration mediated by airway secretory cells, thus inhibiting lung functional repair. Mechanically, CD4+ and CD8+ T cells function by secreting IFNγ rather than direct interactions with epithelial cells. Importantly, anti-IFNγ treatment promotes alveolar regeneration and functional recovery in vivo. Overall, our study reveals the pathogenetic role of Krt5+ pods in lung regeneration, providing new insights into how dysplastic repair impairs functional regeneration of the alveolar epithelium via interactions with immune cells.

overall_design: Airway epithelial cells (CD45- EPCAM+ CD31- Integrin β4+) from the lungs of influenza-infected mice at 11 days post-infection (dpi) were isolated using FACS. These cells were then cultured in1:1 mixed SAGM and LPM medium without CHIR medium for 10 days before being digested into single cells for scRNA-seq analysis.

first: Ying

last: Xi

first: Tiantian

last: Lu

first: Li

last: Liu

first: Pei

last: Wu


title: CD74⁺ Myeloid Cells across Brain Pathologies

summary: In this study, we conducted a comprehensive analysis of single-cell RNA sequencing data from immune cells isolated from the brain tissue of mouse models of Alzheimer’s disease (AD), multiple sclerosis (MS), and brain metastasis (BrM). Our findings reveal a shared transcriptional signature among CD74⁺ myeloid cells across these conditions, indicating a conserved inflammatory response that may be amenable to therapeutic targeting. These results highlight the role of CD74⁺ myeloid cells as a unifying component of the immune landscape in diverse neurological disorders and lay the groundwork for future investigations into targeted interventions aimed at mitigating these pathologies.

overall_design: scRNA-seq from CD45+ immune cells isolated from brain tissue of mouse models for Alzheimer's Disease, Multiple Sclerosis, and Brain Metastasis.

first: Angel

last: Marquez-Galera

first: Laura

last: Alvaro-Espinosa

first: Jose

middle: P

last: Lopez-Atalaya

first: Carolina

last: Hernandez-Oliver


title: Transcriptional Heterogeneity of Progression in MYOD1-mutant spindle cell rhabdomyosarcoma

first: Cristina

last: Antonescu

address: 1275 York Avenue

summary: We perform single nuclei transcriptomics (snRNA-seq) analysis of patient-derived tumor samples to define the dynamic myogenic cell states and intratumoral transcriptional heterogeneity of MYOD1-mutant spindle cell/sclerosing rhabdomyosarcoma.

overall_design: Primary and recurrent/metastatic, snap-frozen patient-derived samples (n = 6) of MYOD1-mutant spRMS were profiled by snRNA-seq using the 10X Genomics platform.

first: Josephine Kam Tai

last: Dermawan

first: Fabio

last: Vanoli

first: Henry

last: De Traux

first: Leonard

last: Wexler

first: Jovana

last: Pavisic

first: Alice

last: Soragni

first: Filemon

last: Dela Cruz


title: Step-wise signal integration governs adaptive programming of lymphocytes [CUT&RUN STAT4]

first: Hyunu

last: Kim

overall_design: STAT4 CUT&RUN of stimulated NK and CD8+ T cells

first: Simon

last: Grassmann

first: Joseph

last: Sun


title: TP53-TAU axis regulates microtubule bundling to control alveolar stem cell mediated regeneration

city: Durham

first: Purushothama Rao

last: Tata

email: pt93@duke.edu, tata.lab@yahoo.com

state: NC

address: 303 Research Dr., Alexander Sands Building, Room- 451, BOX 3709

department: Department of Cell Biology

institute: Duke University School of Medicine

summary: To assess the morphological dynamics during the differentiation of alveolar epithelial type 2 cells (AT2s) into alveolar epithelial type 1 cells (AT1s), we established an ex vivo differentiation model in 2-dimensional cultures with defined conditions using purified AT2s from mouse lungs.

overall_design: Purified murine AT2s were seeded on collagen-1 or fibronectin with serum-free feeder-free medium. We performed bulk RNA sequencing on cells collected from collagen-1 (for AT2s maintenance) or fibronectin-coated plates harvested on days 5 (for alveolar transitional cell states induction) and days 9 (for AT1s induction).

first: Satoshi

last: Konishi

first: Aleksandra


title: Step-wise signal integration governs adaptive programming of lymphocytes [scRNA-seq]

overall_design: scRNA-seq of NK cells isolated from spleen of C57BL/6 mice upon MCMV infection on day 0-4.

first: Endi

last: Santosa


title: pTα enhances mRNA translation and potentiates CAR T cells for solid tumor eradication [scRNA-seq]

first: Yuzhe

last: Shi

address: 1250 First Avenue

department: Immunolgy

summary: Current chimeric antigen receptor (CAR) therapies are effective against a range of hematological malignancies and autoimmune disorders but have shown limited activity against solid tumors. In searching for effective means to enhance the functional persistence and potency of CAR T cells, we explored the potential of integrating pre-T cell features into canonical CD28-based CARs. Thymocytes undergo a proliferation burst during the β-selection developmental stage, which is driven by the pre-T cell receptor and its unique pTα chain. CARs harboring the pTα 1A domain imparted greater expansion, cytokine production, and in vivo persistence to T cells, accompanied by lowered exhaustion and greater long-term tumor control in multiple liquid and solid tumor models. CARs incorporating the 1A domain showed sustained phosphorylation of the mRNA translation master regulator YBX1, which was required for enhanced tumor eradication. The programing of mRNA translation in T cells opens another avenue for regulating and potentiating immunotherapy.

overall_design: To confirm a role for YBX1 in CAR T cells, we performed repetitive NALM6 stimulation and single-cell RNA-sequencing (scRNA-seq) in control and YBX1-knockout (KO) CAR T cells

first: Michel

last: Sadelain


title: Step-wise signal integration governs adaptive programming of lymphocytes [bulkRNA-seq and ATAC-seq]

overall_design: Bulk RNA-seq and ATAC-seq of in vitro stimulated NK cells (with the indicated stimulations) as well as NK cells isolated from MCMV-infected mice taken after 1 day post infection (2--4 replicates each).


title: Dysregulation of microtubule dynamics caused by LOXL3 knockout triggers mitosis catastrophe and senescence in U87MG and U251 cells

city: São Paulo

first: Talita

middle: Sousa

last: Laurentino

state: São Paulo

address: Av Dr Arnaldo, 455. Room 4110

department: Neurologia

country: Brazil

institute: Faculdade de Medicina da Universidade de S&#x00E3

summary: The amine oxidase LOXL3 has been implicated in tumor development and progression, with elevated expression in glioblastoma (GBM). This enzyme influences GBM cell adhesion, migration, and proliferation, and is demonstrated correlation with tubulin expression. Microtubules, as the major component of the cytoskeleton, are crucial for cell cycle and mitotic progression. In this study, LOXL3 knockout was performed using CRISPR-Cas9 in two GBM cell lines, U87MG and U251, to achieve high silencing efficiency. The knockdown resulted in decreased cell viability in both cell lines. Transcriptome enrichment analysis of U87MG cells revealed downregulation of genes associated to tubulin acetylation, which was subsequently confirmed by Western blot analysis. Both U87MG and U251 cells exhibited downregulation of cell cycle and proliferation-related genes. Functional assays confirmed that LOXL3-knockout cells exhibited delayed cell cycle progression and significant alterations in mitotic spindles during metaphase-anaphase transition. Nuclear analysis revealed morphological changes indicative of mitotic catastrophe in U87MG cells and senescence in U251 cells. Additionally, LOXL3-knockout clones displayed reduced cell adhesion and migration. Interestingly, LOXL3 knockdown in GBM cell lines reduces tubulin acetylation, leading to mitotic catastrophe and subsequent cell death in a TP53 mutation-dependent manner. These findings highlight the potential of targeting LOXL3 as a combinatory strategy for advancing research and developing novel therapeutic approaches in TP53-mutated GBM.

overall_design: RNAseq profiling of wilditype U87MG and U251 glioblastoma cells and their permanent LOXL3 knockdown (LOXL3-KD), through CRISPR-Cas9 system.

middle: S

first: Roseli

last: Soares

first: Antônio

middle: M

last: Lerario

first: Ricardo

middle: C

last: Cintra

first: Suely

last: Marie

first: Sueli

last: Oba-Shinjo


title: Effects of a fibrate drug, bezafibrate on ascites-mediated transcriptional changes in epithelial cancer cells

first: Jen-Tsan Ashley

last: Chi

address: 101 Science Dr

department: Molecular Genetics and Microbiology

institute: Duke University

summary: Ascites is an abnormal fluid that accumulates in the peritoneal cavity of more than 90% of patients with metastatic ovarian cancer. Despite its common occurance, little is known about its effects on detached and metastasizing ovarian cancer cells, which frequently metastasize to the peritoneum. This experiment aims to identify any transcriptional changes that occur in ovarian cancer cell lines as a result of exposure to ovarian cancer ascites and whether the fibrate drug, bezafibrate had any reversing effect on the ascites-mediated changes

overall_design: To investigate any transcriptional changes that occur in ovarian cancer cell lines as a result of exposure to ovarian cancer ascites and the fibrate drug, bezafibrate

first: Yasaman

last: Setayeshpour


title: Virus-specific effector T cells impair airway secretory cell-mediated alveolar regeneration [immune_scrnaseq]

first: pei

last: wu

overall_design: Tissue resident immune cells (CD45+ EPCAM- CD31- i.v.CD45-) from influenza 11 dpi and PBS (control) lungs were isolated by FACS and  analyzed using scRNAseq.

first: Ying

last: Xi

first: Tiantian

last: Lu

first: Zhe

last: Chen

first: Pei

last: Wu


title: Virus-specific effector T cells impair airway secretory cell-mediated alveolar regeneration []

overall_design: Distal airway epithelial cells (CD45- EPCAM+ Integrin β4+), alveolar epithelial cells (CD45- EPCAM+ Integrin β4-) and CD4+ T cells from influenza infected and PBS mouse lungs were sorted for RNA extraction and transcriptome analysis.


title: Effects of inhibiting stearoyl-CoA desaturase (SCD) on the spatial transcriptome in a mouse model of Alzheimer's disease (AD)

city: Montreal

first: Valerie

last: Mongrain

state: QC

address: 333 rue Saint-Antoine Est

department: Research center

country: Canada

institute: CRCHUM

summary: Here, the effects of a stearoyl-CoA desaturase inhibitor (SCDi) on the mouse brain spatial transcriptome was investigated. More precisely, wild-type and 3xTg-AD female mice were infused in the brain left lateral ventricle with control (DMSO) or SCDi during 28 days (after the implantation of electrodes for electrocorticography and electromyography). Brains were sampled on the morning of Day 31 after the beginning of treatment, and immediately frozen. Brains were treated and libraries were prepared according to 10x Genomics protocols for Visium Spatial Gene Expression. Sequencing was conducted by Genome Quebec (Montreal, QC, Canada).

overall_design: The mouse brain transcriptome was evaluated after a 28-day treatment with SDCi in female wild-type (WT) and 3xTg-AD mice that were kept under a 12-hour light:12-hour dark cycle. The same measurements were conducted in mice of the two genotypes receiving DMSO (controls/vehicule). The total number of samples for this project is thus 4, consisting of one WT female receiving SCDi, one WT female receiving the vehicle, one 3xTg-AD female receiving SCDi, and one 3xTg-AD female receiving the vehicle. The brain transcriptome was quantified using the Visium Spatial Gene Expression Slide & Reagent Kit (10x Genomics). The original fastq files are provided as raw data.

first: Audrey

last: Hector

first: Benoît

last: Delignat-Lavaud


title: Dependence of cell fate potential and cadherin switching on primitive streak coordinate during differentiation of human pluripotent stem cells

status: Public on Jan 21 2026

city: Cincinnati

first: Aryeh

last: Warmflash

state: Ohio

address: 3333 Burnet Avenue

department: Developmental Biology

institute: Cincinnati Children's Hospital

summary: During gastrulation, the primitive streak (PS) forms and begins to differentiate into mesendodermal subtypes. This process involves an epithelial-mesenchymal transition (EMT),which is marked by cadherin switching, where E-Cadherin is downregulated, and N-Cadherin is upregulated.To understand the relationships between differentiation, EMT, and cadherin switching, we made measurements of these processes during differentiation of human pluripotent stem cells (hPSCs) to PS and subsequently to mesendoderm subtypes using established protocols, as well as variants in which signaling through key pathways including Activin, BMP, and Wnt were modulated.We found that perturbing signaling so that cells acquired identities ranging from anterior to posterior PS had little impact on the subsequent differentiation potential of cells but strongly impacted the degree of cadherin switching. The degree of E-Cadherin downregulation and N-Cadherin upregulation were uncorrelated and had different dependence on signaling. The exception to the broad potential of cells throughout the PS was the loss of definitive endoderm potential in cells with mid to posterior PS identities. Thus, cells induced to different PS coordinates had similar potential within the mesoderm but differed in cadherin switching. Consistently, E-Cadherin knockout or overexpression did not alter cell fates outcomes during differentiation. Overall, although all processes are regulated by the same set of signaling pathways, the extent of cadherin switching and EMT can vary substantially within cells adopting the same cell fate.

overall_design: RNA-seq of human pluripotent stem cells (ESI017) directed for 24 h into four primitive streak populations - Definitive Endoderm(day1) (DE(a)), Anterior PS (APS), Mid PS (MPS), Posterior PS (PPS) - and of matched cultures further differentiated for an additional 24 h toward Definitive Endoderm(day2) (DE(b)), Paraxial Mesoderm (PM), or Lateral Mesoderm (LM). Each condition was collected in 2 biological replicates.

first: Ye

last: Zhu


title: pTα enhances mRNA translation and potentiates CAR T cells for solid tumor eradication [bulk RNA-seq]

first: Yuzhe

last: Shi

overall_design: The pre-infusion, one-week, and two-week post-infusion 19-28z, 19-28z-1A, and 19-1XX CAR T cells from leukemic mouse bone marrow were purified and sequenced for transcriptomic analysis

first: Michel

last: Sadelain


title: Single cell transcriptome analysis of Cyfip2 knockout mouse forebrain

city: Daejeon

first: Yukyung

last: Jun

address: 245 Daehak-ro, Yuseong-gu

department: Center for Biomedical Computing

institute: Korea Institute of Science and Technology Information

summary: To elucidate the intricate molecular alterations resulting from Cyfip2 knockout, we conducted single cell transcriptomic analysis on the forebrain tissues of E18.5 Cyfip2 knockout mice and their wild-type littermates. The E18.5 stage was selected due to the nonviability of Cyfip2 knockout mice during the perinatal period. Cyfip2 knockout and wild-type mice were obtained by breeding Cyfip2 heterozygous males and females. For each genotype, we combined the forebrains from two mice.

overall_design: Single cell transcriptome of embryonic day 18.5 wild-type and Cyfip2 knockout mouse forebrain

first: Kihoon

last: Han


title: Effects of ascites exposure from ovarian cancer on epithelial cancer cell lines

summary: Ascites is an abnormal fluid that accumulates in the peritoneal cavity of more than 90% of patients with metastatic ovarian cancer. Despite its common occurance, little is known about its effects on detached and metastasizing ovarian cancer cells, which frequently metastasize to the peritoneum. This experiment aims to identify any transcriptional changes that occur in ovarian cancer cell lines as a result of exposure to ovarian cancer ascites

overall_design: To investigate any transcriptional changes that occur in ovarian cancer cell lines as a result of exposure to ovarian cancer ascites

first: Chi

last: Jen-Tsan Ashley

first: Setayeshpour

last: Yasaman


title: Spatiotemporal molecular profiling of macrophage-fibroblast crosstalk defines checkpoints orchestrating onset and resolution of inflammation [RNA-seq]

city: Berlin

first: Gerhard

last: Krönke

address: Virchowweg 11

department: Rheumatology and clinical immunology

country: Germany

institute: Charite Berlin

summary: The molecular details of macrophage-fibroblast crosstalk during the onset and resolution of inflammatory disease remain incompletely understood. Here, we apply a scRNAseq-, scATACseq- and bulk RNAseq-based bioinformatic modelling approach to map heterocellular signaling circuits of synovial macrophage and synovial fibroblast (SF) subsets during various stages of inflammatory arthritis. While SFs function as key pacemakers of synovial inflammation, individual subsets of synovial macrophages support both the perpetuation and the resolution of arthritis. While pro-inflammatory Il1b+ macrophages dominate the early stages of inflammation, these cells also retain a substantial intrinsic plasticity that is characterized by chromatin remodeling and an eventual differentiation into Spp1+ macrophages. These cells display a terminally-differentiated phenotype, suppresses activation of pro-inflammatory SFs, and initiates the resolution of arthritis by secretion of regulatory mediators including osteopontin. Our data highlight the dichotomous character of macrophage-fibroblast crosstalk and define the cellular and molecular checkpoints that control the onset and resolution of immune-mediated inflammatory diseases.

overall_design: We generated RNAseq data of synovial fibroblasts, either wildtype or fibroblasts derived from K/BxN serum transfer mice, stimulated in vitro with either Fgf2, Opn or a combined treatment

first: Katharina

last: Weishaupt

first: David

last: Chambers

first: Maria

last: Dzamukova

first: Ivana

last: Androšević

first: Jean-Philippe

last: Auger

first: Darleen

last: Hüser

first: Sébastien

last: Trzebanski

first: Andreas

last: Ramming

first: Anika

last: Grüneboom

first: Georg

last: Schett

first: Steffen

last: Jung

first: Markus

middle: H

last: Hoffmann


title: Comparison of sorted TRM populations and TRM-like cells in vitro

city: Freiburg

first: Geoffroy

last: Andrieux

address: Breisacherstr 153

institute: University clinics Freiburg

summary: Tissue-resident macrophages (TRM) perform organ-specific functions that are dependent on factors such as hematopoietic origin, local environment and biological influences. A diverse range of in vitro culture systems have been developed to decipher TRM functions, including bone marrow-derived macrophages (BMDM), induced pluripotent stem cells (iPSC) or immortalized cell lines. However, despite the usefulness of such systems, there are notable limitations. Attempts to culture primary macrophages often require purification of cells, lack a high cell yield and consistent phenotype. Here, we aimed to address these practical limitations by establishing a new organotypic primary cell culture protocol. We obtained long-term monocultures of macrophages derived from distinct organs without FACS or MACS purification using specific growth factors and tissue normoxic conditions that largely conserved a TRM-like identity in vitro. Thus, this new organotypic system offers an ideal screening platform for primary macrophages from different organs which can be used for a wide range of assays and read-outs.

overall_design: We analyzed 8 sample groups in total with 3 biological replicates in each, including 4 groups of FACS sorted tissue resident macrophage populations (lung, brain, peritoneum and liver) and 4 groups of cultured TRM-liek macrophages (lung, brain, peritoneum and liver).

first: Melanie

last: Boerries

first: Katrin

last: Kierdorf

first: Marco

last: Prinz

first: Olaf

last: Groß


title: Single-Cell RNA Sequencing Reveals Distinct Cellular and Molecular Features of Steroid-Induced Osteonecrosis of the Femoral Head Compared to Alcohol-Induced Osteonecrosis and Hip Osteoarthritis

city: Los Angel

first: Arijita

last: Sarkar

state: California

address: 1450 Biggy Street

institute: University of Southern California

summary: Osteonecrosis of the femoral head (ONFH) remains a devastating complication of glucocorticoid therapy and alcohol abuse, yet the pathophysiology is poorly understood. Using single-cell RNA sequencing, we analyzed femoral head specimens from patients undergoing total hip arthroplasty due to steroid-induced ONFH, alcohol-induced ONFH, or hip osteoarthritis (OA). Our comprehensive analysis across conditions revealed etiology-specific cellular compositions and transcriptional profiles. Steroid-induced ONFH demonstrated a distinctive immunological signature characterized by a neutrophil-predominant, elevated innate-to-adaptive immune cell ratio compared to alcohol-induced ONFH or OA. T cell analysis revealed impaired maturation in steroid-induced ONFH, with abundant naïve T cells but reduced mature CD4+ T cells and CD4+ memory T cells compared to both alcohol-induced ONFH and OA. In addition, compared to alcohol-induced ONFH, steroid-induced ONFH was associated with an increase in exhausted/dysfunctional CD8+ T cells which suggests an impaired response to stress and reflects chronic immune dysfunction. Within stromal cell populations, steroid-induced ONFH demonstrated an increase in pericytes, endothelial cells, and mesenchymal cell with osteogenic features as compared to both alcohol-induced ONFH or OA. These findings collectively reveal novel cellular mechanisms underlying steroid-induced ONFH which may contribute to further investigation of etiology-specific pathogenesis and treatment strategies.

overall_design: Single-cell mRNA sequencing for steroid-induced osteonecrosis of the femoral head (ONFH) and  hip osteoarthritis (OA) in human using 10x Genomics.

first: Jay R.

last: Lieberman


title: Mapping the total transcriptome at near cellular resolution

city: Ithaca

first: Iwijn

last: De Vlaminck

state: New York

address: 237 Tower Road

department: Biomedical Engineering

institute: Cornell University

summary: Spatial transcriptomics provides context to gene expression information, however most methods are only sensitive to a subset of the RNA species. Many noncoding and microbial RNA molecules lack the poly(A) tail which is used to capture RNAs in sequencing-based methods. These transcripts represent valuable analytes for studying gene regulation and host-microbe interactions, respectively. We and others recently showed that in situ polyadenylation combined with poly(dT) capture enables broad analysis of coding, noncoding, and non-host RNAs. However, current implementations neither attain single-cell resolution or retain spatial information. Recent commercialization of spatial transcriptomics tools have enabled broad adoption of these technologies. Several new tools have been developed which can attain near cellular resolution, including SlideSeq (Seeker, Takara/Curio Bioscience) and StereoSeq (STOmics, BGI). Here we adapt in situ polyadenylation to two commerical spatial transcriptomics platforms with near cellular resolution and show the spatial heterogeneity of the total transcriptome in the infected murine heart and testis.

overall_design: Frozen testis from DBA/2J mice and frozen reovirus-induced myocarditic hearts collected at 7 days post infection (dpi) following oral gavage with Reovirus type 1 Lang (T1L) were processed for Spatial Total RNA Sequencing using a modified Stereoseq (STOmics) workflow. Tissues were cryosectioned at 10 µm thickness and mounted onto a 1 cm × 1 cm STOmics Stereoseq chip. Sections were fixed in ice-cold methanol for 30 min at −20°C, followed by staining and imaging according to the manufacturer’s Stereoseq/STOmics protocol. In situ polyadenylation was then performed using yeast poly(A) polymerase (yPAP; Thermo Scientific): capture areas were equilibrated with 1× wash buffer, incubated with yPAP enzyme mix at 37°C, and the enzyme was removed by washing. A 15-min enzymatic permeabilization step was subsequently performed prior to continuing with the standard STOmics library preparation workflow to generate cDNA and sequencing libraries.

first: Ioannis

last: Ntekas

first: David

middle: W

last: McKellar

first: Lena

last: Takayasu


title: Artesunate Protects against Sepsis-induced Cardiomyopathy

city: Chongqing

first: Zhiyu

last: Ling

address: No.288, Tianwen Avenue

institute: Chongqin medical university

summary: Sepsis-induced cardiomyopathy (SICM) is a life-threatening complication of sepsis characterized by acute and reversible myocardial dysfunction, for which effective targeted therapies remain limited. Artesunate (ART), a well-established first-line antimalarial agent, has attracted increasing attention for its anti-inflammatory, antioxidant, and cytoprotective properties. However, its role in SICM has not been fully elucidated. In this study, a murine SICM model was established using lipopolysaccharide  to evaluate the effects of ART on animal mortality, cardiac function, histopathology, and biomarkers of myocardial injury.

overall_design: C57BL/6 male mice (8 – 12 weeks old), weighing approximately 25 g, were randomly assigned to following groups: normal control (Con), 50 mg/kg artesunate-treated control (ART), LPS-induced SICM model (LPS), SICM model treated with 50 mg/kg artesunate (LPS+ART). Sepsis was induced by intraperitoneal injection of lipopolysaccharide (LPS). ART was administered intraperitoneally 10 minutes prior to LPS injection and again 4 hours post-injection. Twenty-four hours after LPS administration, cardiac function was assessed and myocardial injury markers were measured to determine the severity of septic cardiomyopathy. RNA sequencing (RNA-seq) of the control (LPS, n = 5) and treatment (LPS + ART, n = 5) groups was performed by Shanghai Applied Protein Technology Corporation (Shanghai, China).

first: Xi

last: Jiang

first: Yankun

last: Chen

first: Yalin

last: Zeng

first: Yanghao

first: Dan

last: Yang


title: Single-cell RNA sequencing of thioglycollate-elicited peritoneal cells from myeloid-specific S1pr1 gain- and loss-of-function mice

city: Boston

first: Yueh-Chien

last: Lin

state: Massachusetts

address: 1 Blackfan Circle, Karp Research Building, 12F

department: Vascular Biology Program

institute: Boston Children's Hospital

summary: Sphingosine-1-phosphate receptor 1 (S1pr1) plays an important role in myeloid cell trafficking and inflammatory responses. Here, single-cell RNA sequencing was performed on thioglycollate-elicited peritoneal cells isolated from mice with myeloid-specific S1pr1 gain-of-function or knockout, as well as their corresponding Cre-negative controls. This dataset enables interrogation of S1pr1-dependent transcriptional programs across inflammatory myeloid populations, with downstream analyses focusing on neutrophils.

overall_design: Thioglycollate was administered to elicit inflammatory peritoneal cells in mice. Single-cell suspensions were prepared from the peritoneal cavity and subjected to single-cell RNA sequencing. Mice included myeloid-specific S1pr1 gain-of-function and knockout models generated using LysM-Cre, along with Cre-negative littermate controls. Although multiple myeloid populations were captured, downstream analyses focused on neutrophil subsets identified based on canonical marker expression.

first: Michel

middle: V

last: Levesque

first: Reed

last: Crocker

first: Timothy

last: Hla


title: Lipid influx enforces liver regeneration through the recruitment of CD36+ macrophages (scRNA-Seq and RNA-Seq)

city: china

first: Yao

last: Tao

state: zhejiang

address: zhejiang province

institute: First Affiliated Hospital, Zhejiang University

summary: The liver possesses a remarkable regeneration capacity under a wide variety of injury conditions, like partial hepatectomy (PHx) and over-dose acetaminophen (APAP) treatment. Macrophages, multitask at the wound site by facilitating wound debridement and producing chemokines, metabolites and growth factors, and consequently determine the balance between ongoing damage and repair following tissue injury. CD36, which is universally expressed in a wide variety of cells (i.e., macrophages, adipocytes, myocytes, enterocytes and hepatocytes), and serves as the predominant fatty acid uptake facilitator of the cells to uptake microenvironmentally enriched lipids in various settings. However, the role for macrophage CD36 in mediating lipid accumulation as well as the regeneration of liver post physiological or pathophysiological injury still remains largely unknown. Herein, by utilizing multiple seq-based methods, we found the transient lipid accumulation in injured livers post PHx or APAP treatment exerted fundamental impacts on hepatic immune remodeling and induced the recruitment of macrophages with high level of CD36 expression and excessive lipid accumulation which facilitate the regeneration of livers. CD36-mediated lipid influx in macrophages triggers the activation of IRE1a signaling pathways via intracellular ceramide accumulation to promote high levels of IL6 production to accelerate liver regeneration.

overall_design: Our experimental design involved single cell RNA sequencing (scRNA-seq) to analyze liver cell dynamics post-PHx and sham operations, focusing on two groups with three mice each group. Fresh liver tissues were processed using the Miltenyi Liver Digestion Kit to ensure high-quality single-cell suspensions, with cell viability consistently above 90%. This preparation step was crucial for reliable downstream scRNA-seq analysis. We used the 10X Genomics platform from BGI for sequencing. The data analysis included Seurat-guided clustering and UMAP dimensionality reduction to identify and annotate nine distinct cell populations. To explore this, we employed a controlled experimental design to assess the impact of HFD induced lipid accumulation on liver regeneration and transcriptome changes. Our design included two distinct groups of C57BL/6 male mice of 8 week old, each consisting of three individuals. The experimental group was subjected to a high-fat diet (Research diet, #12492i) for one week prior to PHx to induce acute hepatic lipid accumulation. In contrast, the control group was maintained on a standard diet during the same period. All mice underwent PHx, and liver tissue samples were collected 40 hours post-surgery from both groups. This setup was specifically devised to evaluate the effects of HFD on the liver's regenerative capacity and associated transcriptomic alterations following PHx, providing insights into the influence of lipid metabolism on liver regeneration. Our study explored the potential distinct functions of liver macrophage subpopulations in liver regeneration, including Kupffer cells, LY6C+ MDMs, CD36+ MDMs retrived from C57BL/6 wild type mice, as well as CD36+ MDMs retrived MDM-Ern1KO mice. Each group was represented by three samples. Each samples were pooled of the cells from three or more mice.  The cells were isolated from the livers using FACS two days post-PHx and subjected to bulk RNA sequencing.


title: A TCER-1-siRNA Regulatory Axis Suppresses Innate Immunity in C. elegans

city: Fort Collins

first: Taiowa

middle: A

last: Montgomery

state: CO

address: 1878 Campus Delivery

department: Department of Biology

institute: Colorado State University

type: Non-coding RNA profiling by high throughput sequencing

summary: Non-coding small RNAs (sRNAs) are important regulators of gene expression with well-established roles in development and anti-viral defense. However, their role in antibacterial immunity is not well understood. Here, we identify a role for an endogenous small interfering RNA (siRNA) pathway in repressing innate immunity in Caenorhabditis elegans. We show that genes required for the biogenesis or function of WAGO Argonaute-associated siRNAs, called 22G-RNAs, function in a common genetic pathway with the immune-suppressive transcription elongation and splicing factor TCER-1. Loss of tcer-1 leads to reduced levels of 22G-RNAs from a subset of WAGO targets, while mutations in several WAGO 22G-RNA pathway genes phenocopies the enhanced immune-resistance of tcer-1 mutants, indicating that TCER-1 and the WAGO 22G-RNA pathway function within a shared regulatory module to limit antibacterial immunity. However, rather than mediating widespread gene silencing, this regulatory module targets a restricted set of potential immune-relevant effectors, including scrm-4, encoding a phospholipid translocase that promotes antibacterial resistance.

overall_design: High-throughput small RNA and mRNA sequencing was performed on wild-type and tcer-1 mutant strains. RNA was extracted from cell lysates from C. elegans cultured at 20°C and libraries were prepared using NEB kits. Small RNA data was analyzed using the tinyRNA pipeline. mRNA data was analyzed using a custom pipeline of fastp, RSEM, STAR, and DESeq2.


title: Spatiotemporal molecular profiling of macrophage-fibroblast crosstalk defines checkpoints orchestrating onset and resolution of inflammation [scATAC-Seq]

first: Gerhard

last: Krönke

overall_design: We generated scATACseq data of myeloid Cd45+Cd11b+Ly6G- cells isolated from murine paws at three timepoints during K/BxN serum transfer arthritis, at steady state, day5 and day9.

first: Katharina

last: Weishaupt

last: Chambers

first: Maria

last: Dzamukova

first: Ivana

last: Androšević

first: Jean-Philippe

last: Auger

first: Darleen

last: Hüser

first: Sébastien

last: Trzebanski

first: Andreas

last: Ramming

first: Anika

last: Grüneboom

first: Georg

last: Schett

first: Steffen

last: Jung

first: Markus

last: Hoffmann


title: Extracellular Vesicles from Cytokine-Treated Human Pancreatic Ductal Cells Enhance HLA Class I Expression in Beta Cells

city: Duarte

first: James

last: Figarola

state: CA

address: 1500 E. Duarte Road

department: Diabetes-Metabo Res Inst-BRI

institute: City of Hope

summary:
Introduction/Objective: Type 1 diabetes (T1D) is an autoimmune disease characterized by the loss of insulin-producing beta cells and has no cure. The role of cell-cell interactions in immune recognition of beta cells remains poorly understood. Beta cells develop adjacent to ductal cells, which undergo changes during T1D progression, suggesting crosstalk between the two cell types. Extracellular vesicles (EVs) mediate intercellular communication through their cargo, but whether human ductal cells secrete EVs that modulate beta cells is unknown. We hypothesize that exposure of human ductal cells to T1D-associated cytokines alters their EV cargo, with consequence in beta cells.
Methods: Human exocrine tissues from non-diabetic cadaveric donors were cultured in a 3D suspension to support ductal cell survival. Cells were treated with TNF-α, IL-1β, and IFN-γ or vehicle control for 48 hours and analyzed for proinflammatory-related gene expression and viability by qRT-PCR and Trypan blue exclusion, respectively. EVs from cytokine- and vehicle-treated ductal cells were isolated using size exclusion chromatography and characterized by dot blot, transmission electron microscopy, and nanoparticle tracking. The effects of EVs on EndoC-βH1 human beta cells were assessed with qRT-PCR, RNA-sequencing and flow cytometry, and EV cargo was analyzed by proteomics and transcriptomics.
Results: EVs from cytokine-treated ductal cells were internalized by EndoC-βH1 beta cells and, compared to control ductal EVs, increased HLA Class I gene and protein expression in EndoC-βH1 cells, as shown by qRT-PCR, bulk RNA sequencing, and flow cytometry. EV cargo from cytokine-treated versus control ductal cells, was enriched for transcripts associated with inflammatory and antigen-presentation-related pathways, including CXCL9, CXCL10, CXCL11, IDO1, and GBP4.
Conclusion: Primary human ductal cells secrete EVs that, under T1D-associated cytokine exposure, alter their cargo and upregulate HLA Class I gene and protein expression in human EndoC-βH1 beta cells. Because HLA Class I is critical for beta cell recognition by autoimmune cells, our results reveal a previously unknown ductal cell communication pathway with implication for modulating beta cells during T1D progression.

overall_design: Bulk mRNA-sequencing of EndoC-βH1 cells treated for 48h with EVs isolated from vehicle- or cytokine- treated primary ductal cells from human pancreas. Cytokine doses for ductal cells were 250 IU/ml TNF-α, 25 IU/ml IL-1β, and 250 IU/ml IFN-γ.

first: Neslihan

last: Erdem

first: Min-Hsuan

last: Chen

first: Hsun Teresa

last: Ku


title: P7C3 targets eIF4A1 to alleviate hepatic fibrosis by inhibiting protein translation and autophagy in hepatic stellate cells

first: Ailing

last: Liang

state: Chongqing

address: Yuanjiagang Campus, Chongqing Medical University, Shiyou Road, Yuzhong District, Chongqing

department: College of Traditional Chinese Medicine

institute: Chongqing Medical University

summary: P7C3, an aminopropyl carbazole compound with established neuroprotective properties and therapeutic potential in neurodegenerative disorders, has demonstrated broad pharmacological activity across multiple pathologies. However, the effect of P7C3 on hepatic fibrosis remains unexplored. This research applied in vitro and in vivo systems to evaluate P7C3's antifibrotic efficacy. The findings demonstrated that P7C3 notably inhibited the proliferation of LX-2 cells and decreased the levels of the fibrotic markers collagen type alpha 1 (COL1A1) and fibronectin (FN). Eukaryotic initiation factor 4A1 (eIF4A1) was identified as a direct target of P7C3 through the integration of cellular thermal shift assay (CETSA) coupled with mass spectrometry and human protein microarray data, and subsequently validated using CETSA, drug affinity responsive target stability, and molecular docking analysis. P7C3 treatment markedly inhibited eIF4A1 levels in LX-2 cells, while eIF4A1 knockdown attenuated this effect by suppressing COL1A1 and FN expression. Mechanistically, P7C3 impaired global protein synthesis in hepatic stellate cells, including c-Myc, consistent with the outcomes observed following eIF4A1 silencing. Both eIF4A1 knockdown and P7C3 treatment significantly downregulated ULK1, induced accumulation of autophagic substrate p62, and increased LC3B-II/LC3B-I ratio, indicating potent disruption of autophagic flux via eIF4A1 targeting. Histopathological assessment of the liver tissues revealed that P7C3 significantly attenuated collagen deposition and architectural distortion in fibrotic mice. Concomitant improvements were observed in the hepatic function biomarkers, including serum ALT and AST levels, as well as in fibrotic markers (hydroxyproline content). Collectively, these findings delineate eIF4A1 as the primary target through which P7C3 alleviates hepatic fibrosis by suppressing protein translation and autophagic flux, providing mechanistic validation for advancing P7C3 as a promising antifibrotic agent.

overall_design: RNA-seq patterns of LX-2 cells in treatment group treated with drug P7C3(20µM) for 2 days (n = 3) and control group treated with no drug for 2 days (n = 3).

first: Liang

last: Ailing

first: Yao

last: Ling

first: Liu

last: Yuanyuan


title: High Resolution Spatial Mapping of Microbiome-Host Interactions via in situ Polyadenylation and Spatial RNA Sequencing [Spatial transcriptomics]

first: Iwijn

last: De Vlaminck

summary: Inter-microbial and host-microbial interactions are critical for the functioning of the gut microbiome, but few tools are available to measure these interactions in situ. Here, we report a method for broad spatial sampling of microbiome-host interactions in the gut at high resolution (1 µm). This method combines enzymatic in situ polyadenylation of both bacterial and host RNA with spatial RNA-sequencing to increase bacterial RNA recovery and enable transcriptomic analysis of low-abundance and spatially restricted microbial taxa. We benchmark the method against existing spatial transcriptomic workflows, demonstrating improved sensitivity and resolution. Application of this method in a mouse model of intestinal neoplasia revealed the biogeography of the mouse gut microbiome as function of location in the intestine, frequent strong inter-microbial interactions at short length scales, and tumor-associated changes in the architecture of the host-microbiome interface. This method is compatible with widely available commercial platforms for spatial RNA-sequencing and can therefore be readily adopted to study the role of short-range, bidirectional host-microbe interactions in microbiome health and disease.

overall_design:
Spatial transcriptomics
Cryosections were obtained from the ileum of  two 13 weeks old mice (male and female). For spatial total RNA sequencing, tissue sections (10 µm thick) were processed with a modified Stereoseq (STOmics) protocol.

first: Ioannis

last: Ntekas

first: David

last: McKellar

first: Lena

last: Takayasu


title: RNA-seq analysis of MDA-MB-231 triple-negative breast cancer cells treated with CIr2 for 24 h

city: Guilin

first: Houmin

last: Lin

state: Guangxi

address: 15 Lequn Road, Guilin, 541001, Guangxi, China

institute: The First Affiliated Hospital of Guilin Medical University

summary: This study investigates the transcriptomic alterations induced by a novel cyclometalated iridium(III) compound, CIr2, in triple-negative breast cancer (TNBC) cells. MDA-MB-231 cells were treated with CIr2 for 24 h, followed by RNA sequencing to profile global gene expression changes. The RNA-seq data were integrated with functional assays and morphological analyses to explore cellular responses associated with mitochondrial dysfunction, reactive oxygen species production, endoplasmic reticulum stress, and MAPK signaling. This dataset provides a resource for examining CIr2-induced transcriptional programs related to non-apoptotic cell death pathways in TNBC cells.

overall_design: RNA-seq was performed to compare global gene expression profiles of MDA-MB-231 triple-negative breast cancer cells treated with vehicle control or 500 nM CIr2 for 24 h.

first: Jianhua

last: Wei

first: Wenmin

last: Yao

first: Qinqin

last: Zhang

first: Junfei

last: Jin


title: Transcriptional profiles of macrophages during wound healing in zebrafish

city: Montpellier

first: Mai-Eva

last: Nguyen-chi

address: Pl. Eugène Bataillon

country: France

institute: CNRS

summary:
To investigate the polarization status of macrophages during wound healing in zebrafish we performed fin fold amputation at 3 days-post-fertilization and isolated macrophages at different time after wounding.
The objective is to characterize the transcriptional profile of macrophages at different time points after wounding.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for macrophages CUT larvae at 2 hours post-amputation (4 replicates), 5 hours post-amputation (4 replicates) and 29 hours post-amputation (4 replicates). Controls are macrophages from non-amputated larvae at 2 hours post-infection (4 replicates), 5 hours post-amputation (4 replicates) and 29 hours post-amputation (4 replicates).

first: Christina

last: Begon-Pescia

first: Mai

last: Nguyen-Chi

first: Laurent

last: Manchon


title: High Resolution Spatial Mapping of Microbiome-Host Interactions via in situ Polyadenylation and Spatial RNA Sequencing [bulk RNA-Seq]

summary: Inter-microbial and host–microbial interactions are thought to be critical for the functioning of the gut microbiome, but few substantive tools are available to measure these interactions. Here, we report a method for unbiased spatial sampling of microbiome-host interactions in the gut at high spatial resolution. This method combines enzymatic in situ polyadenylation of both bacterial and host transcripts with spatial RNA-sequencing. Application of this method revealed the biogeography of the mouse gut microbiome as function of location in the intestine, short-range intermicrobial interaction, local shaping of the microbiome by the host, and tumor-associated changes in the architecture of the host-microbiome interface. This method is compatible with broadly available commercial platforms for spatial RNA-sequencing, and can therefore be readily adopted to broadly study the role of short-range, bidirectional host-microbe interactions in microbiome health and disease.

overall_design: bulk RNA-Seq of intestinal tissue for host gene expression and microbiome composition analysis


title: Spatiotemporal molecular profiling of macrophage-fibroblast crosstalk defines checkpoints orchestrating onset and resolution of inflammation [scRNA-seq]

first: Gerhard

last: Krönke

overall_design: To extend previously generated scRNAseq data in GSE134691 and GSE129087 we profiled myeloid cells and stromal cells from murine paws to have 3 timepoints during K/BxN serum transfer arthritis at days 0, 5 and 9. Specifically we sequenced Cd45+Cd11b+Ly6G- myeloid cells at day 9 and Cd45- stromal cells at day 0 and day5.

first: Katharina

last: Weishaupt

last: Chambers

first: Maria

last: Dzamukova

first: Ivana

last: Androšević

first: Jean-Philippe

last: Auger

first: Darleen

last: Hüser

first: Sébastien

last: Trzebanski

first: Andreas

last: Ramming

first: Anika

last: Grüneboom

first: Georg

last: Schett

first: Steffen

last: Jung

first: Markus

last: Hoffmann


title: Antagonism between blue and red light-signalling controls thallus flatness in Marchantia polymorpha

city: Rehovot

first: Zohar

last: Meir

state: Shfela

address: Herzl 234

department: Mathematics & CS

country: Israel

institute: Weizmann Institute of Science

summary: The growth orientation of the Marchantia polymorpha thallus – a system of dorsiventralized, indeterminate axes – is modulated by light. We show that red and blue light act antagonistically to control thallus flatness, with red light signalling promoting epinasty and blue light signalling promoting hyponasty. We found that loss-of-function mutations in the blue light receptor MpPHOT led to epinasty, while loss-of-function mutations in the red light receptor MpPHY resulted in hyponasty. We hypothesize that these antagonistic activities of blue and red light signalling are balanced in white light, resulting in the development of flat thalli. Using time-resolved transcriptomics, we identified genes that were rapidly induced upon light exposure. Among these genes were all six members of the M. polymorpha BBX gene family. Mutants harbouring loss-of-function mutations in two of the six MpBBX transcription factors developed defective thalli. Mpbbx1 loss-of-function mutants formed hyponastic thalli, while Mpbbx5 loss-of-function mutants developed epinastic thalli. Double mutants Mpbbx1 Mpbbx5 grew flat, supporting the hypothesis that they function antagonistically. Together, these data indicate that phototropin-mediated blue light and phytochrome-mediated red light signalling antagonistically modulate thallus flatness, and that BBX transcription factors also act antagonistically to regulate thallus flatness.

overall_design: To examine light-responsive gene expression in Marchantia polymorpha, wild-type was grown under white light conditions, followed by a dark adaptation period and subsequent exposure to distinct light qualities. Plants were subjected to white, blue, red, or far-red light, as well as continued darkness, and samples were collected across a time course to capture early and late transcriptional responses. Total RNA was isolated from biological replicates for each condition and time point, and transcriptome-wide gene expression was quantified by RNA sequencing. Sequencing data were processed using a bioinformatic pipeline to map reads to the M. polymorpha reference genome and quantify transcript abundance. Gene expression levels were compared across treatments relative to the dark reference to identify differentially expressed genes, enabling a systematic analysis of light-quality- and time-dependent transcriptional responses.

first: Johannes

last: Rötzer

first: Liam

last: Dolan


title: Hypoxia-induced CD147-enriched migrasomes promote sorafenib resistance in HCC by inducing vasculogenic mimicry via PI3K/AKT/TWIST1 signaling

city: Tianjin

first: YUTONG

last: LI

address: Weijin Road No.94

department: Nankai University School of Medicine

institute: Nankai University

summary: Conventional anti-angiogenic drugs (AADs) combined with sorafenib have little success in HCC patients, as the formation of hypoxia areas in tumors and vasculogenic mimicry’s (VM's) non-responsiveness to AADs. Migrasomes are recently discovered extracellular vesicles produced during cell migration. In this study, the results show that hypoxia-induced migrasomes induce the formation of VM and promote the sorafenib resistance. Further studies reveal that hypoxia-induced migrasomes can be taken up by HCC cells via macropinocytosis, which activates PI3K/AKT/TWIST1 signaling. Moreover, CD147 on the surface of hypoxia-induced migrasomes is identified as a key protein in regulating the signaling pathway. Overall, our findings uncover a previously unrecognized mechanism mediated by hypoxia-induced migrasomes and the formation of VM and provide a new method for preventing SFR.

overall_design: RNA-seq profiling of MHCC-97H cells treated for 24 hours with PBS, migrasomes from MHCC-97H cells cultured in normoxia (Normo-Migs), migrasomes from MHCC-97H cells cultured in hypoxia (Hypo-Migs), or migrasomes from CD147 knockout MHCC-97H cells cultured in hypoxia (CD147KO-Migs)

first: Luomeng

last: Qian

first: Sihe

last: Zhang

first: Yutong

last: Li


title: MiR-424-5p Acts as an Oncogene in Hep3B Cells by Activating the PI3K/AKT Signaling Pathway

city: suzhou

first: zhixian

last: ding

state: province

address: No. 125, West Huaihe Road, Yongqiao District

institute: Wanbei Coal Electric Group General Hospital,

summary: Hepatocellular carcinoma (HCC) remains a global health challenge with high morbidity and mortality. MicroRNAs (miRNAs) play pivotal roles in cancer progression, yet their context-dependent functions in HBV-HCC are unclear. This study demonstrates that miR-424-5p is significantly upregulated in HBV positive Hep3B cells, correlating with poor patient prognosis. Integrated bioinformatic analysis predicted that the target genes of miR-424-5p are significantly enriched in the PI3K/AKT signaling pathway. Functional experiments showed that knockdown of miR-424-5p suppressed cell proliferation, migration, and colony formation. Mechanistically, miR-424-5p knockdown led to the upregulation of PTEN and downregulation of phosphorylated PI3K/AKT, indicating inhibition of this pathway. These findings unveil an oncogenic role of miR-424-5p in HBV-HCC, suggesting its function is driven by viral specific dysregulation of the PI3K/AKT pathway, with PTEN involvement. Our study highlights miR-424-5p as a potential therapeutic target and provides insights into etiology-specific miRNA regulatory networks.

overall_design: This dataset supports comparative transcriptomic research between normal and cancerous human liver cells, which is fundamental for understanding hepatocellular carcinoma (HCC) pathogenesis. The project includes RNA-seq data from 6 cell line samples in total, comprising 3 biological replicates of the normal human hepatocyte cell line L02 and 3 biological replicates of the human hepatocellular carcinoma cell line Hep3B. All samples were processed under consistent baseline conditions, such as standard culture, and the dataset provides raw sequencing data in FASTQ files along with a processed gene expression matrix, for example, FPKM or TPM counts. This resource is suitable for applications like identifying differentially expressed genes and pathways, exploring oncogenic mechanisms, and serving as a control or reference in studies of liver disease models, drug screening, or functional genomics.

first: Zhixian

last: Ding

first: Shuaiyong

last: Qi

first: Lusheng

last: Wang

first: Yu

last: Tang

first: Jiting

last: Sun

first: Lijie

last: Zheng

first: Yafen

first: Lili

first: Quangen

last: Ma

first: Heng


title: SLC6A3 Dopaminergic Signaling Subverts Spliceosomal Fidelity to Promote Immune Checkpoint Inhibitor Resistance in Clear Cell Renal Cell Carcinoma [1]

city: Shanghai

first: Junhui

last: Su

address: zhangheng Street

department: Pharmacology

institute: Fudan University

summary: Clear cell renal cell carcinoma (ccRCC) remains refractory to immune checkpoint inhibitors (ICIs) despite its high tumor mutational burden, highlighting an urgent need to elucidate tumor-intrinsic mechanisms of immune escape. Here, leveraging lineage tracing and single-cell multi-omic profiling in immunocompetent models, we identify a pre-existing SLC6A3-positive tumor subpopulation that persists following anti-PD-1 therapy and orchestrates immune evasion by exploiting renal dopamine uptake. SLC6A3 overexpression is robustly associated with diminished infiltration of cytotoxic CD8⁺ T cells and myeloid dendritic cells, establishing it as a biomarker of ICI resistance in ccRCC. Mechanistically, SLC6A3-mediated dopamine import disrupts the MHC-I antigen presentation pathway through direct sequestration of the spliceosomal regulator USP39, resulting in aberrant splicing of B2m and Tap1/2 pre-mRNAs and consequent loss of antigenicity. This disconnects high mutational burden from effective immune surveillance, enabling sustained immune escape. Critically, both genetic and pharmacologic inhibition of SLC6A3 restore antigen presentation competency, potentiate CD8⁺ T cell cytotoxicity, and synergize with anti-PD-1 treatment to achieve durable tumor regression in vivo. Our findings redefine dopamine as a tumor-intrinsic immunosuppressant in ccRCC and nominate the SLC6A3-USP39 axis as a tractable target for reversing antigen presentation defects and overcoming microenvironment-driven ICI resistance.

overall_design: In order to evaluate mRNA abundance of KVTR cells, we utilize mRNA sequencing to obtain RNA reads. And  using replicate multivariate analysis of transcript splicing (rMATS) to  identified dopamine-induced alternative splicing events in KVTR cells.


title: A plant-derived antimicrobial peptide with multiple mechanisms of action exhibiting antibacterial and antibiofilm activities comparable to or superior to polymyxin B

city: Szeged

first: Rui

middle: M.

last: Lima

address: Temesvari krt. 62

country: Hungary

institute: HUN-REN BRC

summary: Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics in the fight against multidrug-resistant pathogens. Here, we characterize the antibacterial properties and molecular mode of action of the C-terminal fragment of the Medicago truncatula nodule-specific cysteine-rich peptide NCR169 (NCR169C17-38). This peptide exhibits strong bactericidal activity against a broad panel of Gram-positive and Gram-negative pathogens, including members of the ESKAPE group (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli). NCR169C17-38 acted rapidly by permeabilizing bacterial membranes and causing severe morphological damage, outperforming polymyxin B in the rate of membrane disruption. The peptide bound selectively to anionic bacterial lipids such as cardiolipin and displayed remarkable thermal and storage stability. Moreover, NCR169C17-38 effectively inhibited biofilm formation and eradicated pre-formed biofilms of Acinetobacter baumannii even at sub-MBC concentration (3.2 M), a feature not observed for polymyxin B. Transcriptomic analysis of Escherichia coli exposed to sublethal peptide doses revealed a global shutdown of bacterial metabolic and genetic functions, with 450 of 503 differentially expressed genes being downregulated. Strong repression of genes involved in translation, energy production, and cell envelope biosynthesis was accompanied by upregulation of stress- and rescue-related genes linked to membrane repair, ion transport, and oxidative stress responses. Together, these results demonstrate that NCR169C17-38 exerts multifaceted antimicrobial effects - disrupting membranes, binding nucleic acids, and inducing transcriptional collapse - while maintaining its stability and biocompatibility. Together with its previously established antifungal activity and lack of mammalian cytotoxicity, NCR169C17-38 represents a compelling candidate for development as a next-generation antimicrobial agent.

overall_design: Genome-wide transcriptome profiling was performed to examine early global gene expression changes in E. coli (ATCC 8739) exposed to a 1.6 µM (sub-MBC dose) of the NCR169C(17-38) peptide for 20 minutes in culture medium (LSM).

first: Mohamad Anas

last: Al Bouni

last: M. Lima

first: Sándor

last: Jenei

first: Hilda

last: Tiricz

first: Edit

last: Tímár

first: Ildikó

last: Domonkos

first: Éva

last: Kondorosi

first: Gabriella

last: Endre


title: 4-Phenylbutyric Acid Activates an NF-κB - Egr-1 Axis to Control Myoblast Proliferation and ECM Gene Expression Profiles

city: Yamaguchi

first: Kana

last: Tominaga

address: 1-1-1 Minami-kogushi, Ube

country: Japan

institute: Yamaguchi University Graduate School of Medicine

summary: Myoblasts are proliferative precursors derived from satellite cells and play critical roles in skeletal muscle growth, regeneration, and repair. The transition from myoblast proliferation to differentiation is tightly controlled by epigenetic mechanisms, particularly histone acetylation. During myogenesis, increased histone acetylation at muscle-specific gene loci promotes chromatin accessibility and activates myogenic transcription programs. 4-Phenylbutyric acid (4-PBA) is a clinically approved drug that functions as a chemical chaperone and a histone deacetylase (HDAC) inhibitor. Although 4-PBA has been shown to improve protein homeostasis in muscle-related disorders, its epigenetic role in myogenesis remains unclear. In this study, we demonstrated that 4-PBA regulates myoblast proliferation but not differentiation through epigenetic and transcriptional mechanisms in C2C12 cells. Chromatin immunoprecipitation assays revealed early growth response-1 (Egr-1) as a direct transcriptional target of NF-κB p65. 4-PBA increases histone H3 acetylation, particularly at lysines 18 and 27, and downregulates HDAC5, inducing NF-κB p65–dependent Egr-1 expression. Transcriptome analysis revealed that Egr-1 regulates extracellular matrix and myogenesis-related genes, including multiple collagen genes. Similar gene expression changes were observed following 4-PBA treatment, suggesting that 4-PBA promotes extracellular matrix gene expression in part through modulation of the NF-κB–Egr-1 axis. These findings highlight a previously unrecognized epigenetic function of 4-PBA in skeletal myogenesis.

overall_design: RNA-seq profiling of C2C12 cells treated with 1mM 4-Phenylbutyric Acid (PBA) or DMSO at Day0 (Baseline) and Day4 of myoblast differentiation.

first: Naoomi


title: Age-Related Decline in NCKX4-Mediated Calcium Clearance Accelerates Aortic Remodeling and Drives Early Vascular Aging

city: New York

first: Guilherme Henrique

last: Souza Bomfim

address: 1 Washington Square Village 2R

department: Department of Molecular Pathobiology

institute: New York University (NYU)

summary: Aging is the primary nonmodifiable risk factor for cardiovascular diseases (CVDs), with older women facing a greater risk of CVDs than age-matched men. Vascular smooth muscle cells (VSMCs) dysfunction and impaired calcium (Ca2+) handling are recognized as central contributors to arterial stiffening and calcification. However, the molecular and functional determinants of Ca2+ clearance that drive vascular aging remain poorly understood. We identify the (Na+)-sodium/Ca2+-calcium (K+)-potassium-dependent exchanger 4 (NCKX4) as a crucial functional regulator of VSMCs Ca2+ clearance and vascular integrity. NCKX4 (encoded by Slc24A4) expression was significantly reduced in aortas from aged (72-78 weeks) mice, with a pronounced decline in females. Aged-related loss of NCKX4 impaired Ca2+ clearance function and increased calcium-phosphate mineralization. Young (12-15 weeks) Nckx4⁻/⁻ mice revealed fragmentation of elastic fibers, collagen accumulation, wall thickening, and extracellular matrix (ECM) remodeling, all hallmarks of vascular aging that closely resembled those of aged wild-type mice. Transcriptomic profiling showed that loss of NCKX4 alters pathways linked to Ca2+-integrin signaling, ECM turnover and mineralization, including dysregulation of anchorage integrins, microfibril-stabilizing, osteogenic drivers and pro-fibrotic integrins. These findings reveals that an impaired Ca2+ clearance promotes maladaptive inside-out integrin signaling, disrupting VSMCs anchorage, ECM homeostasis and mineralization. Our results establish NCKX4 as a previously unrecognized determinant of vascular aging, whose decline accelerates premature arterial remodeling and calcification. This study therefore, positions NCKX4 as a potential mechanistic link between age, sex-dependent vulnerability, and vascular stiffening, with implications for novel therapeutic strategies targeting Ca2+ handling in CVDs prevention.

overall_design: RNA-seq profiling of aortic tissue from young (Y, 12-15 weeks) and aged (A, 72-78 weeks) female wild-type (WT) and their knockdown Slc24a4 (Nckx4-/-) mice.


title: Loss of cisPTase function in the liver promotes a highly penetrant form of fatty liver disease that rapidly transitions to hepatocellular carcinoma [RNAseq_ChowDiet_NgBR_Liver]

city: New Haven

first: Abhishek

last: Singh

state: CT

address: 10 Amistad Street

department: Pharmacology and VBT

institute: Yale University

summary: In developed countries, obesity-linked fatty liver is a significant risk factor for hepatocellular carcinoma (HCC); however, the molecular mechanisms underlying the transition from non-alcoholic fatty liver disease (NAFLD) to HCC remains unclear. The present study explores the role of the endoplasmic reticulum (ER)-associated protein NgBR, an essential component of the cis-prenyltransferases (cis-PTase) enzyme, in chronic liver disease. Here we show that genetic depletion of NgBR in hepatocytes ofmice (N-LKO) intensifies triacylglycerol (TAG) accumulation, inflammatory responses, ER/oxidative stress, and liver fibrosis, ultimately resulting in HCC development with 100% penetrance after four months on a high-fat diet. Comprehensive genomic and single cell transcriptomic atlas from affected livers provides a detailed molecular analysis of the transition from liver pathophysiology to HCC development. Importantly, pharmacological inhibition of diacylglycerol acyltransferase-2 (DGAT2), a key enzyme in hepatic TAG synthesis, abrogates diet-induced liver damage and HCC burden in N-LKO mice. Overall, our findings establish NgBR/cisPTase as a critical suppressor of NAFLD- HCC conversion and suggests that DGAT2 inhibition may serve as a promising therapeutic approach to delay HCC formation in patients with advanced non-alcoholic steatohepatitis (NASH).

overall_design: Comparative gene expression in Wild type (WT) and Liver specific NgBR KO mice fed with Chow diet (CD) for 8 weeks.

first: Balkrishna

last: Chaube

first: Kathryn

last: Citrin

first: Fowler

last: Joseph

first: Sessa

last: William


title: Loss of JAK1 Function Causes G2/M Cell Cycle Defects Vulnerable to KIF18A Inhibition [Detroit562 RNAseq]

city: Cambridge

first: Vanessa

middle: Martine

last: Kelley

state: MA

address: 300 Binney

department: Gerstner Center for Cancer Diagnostics

institute: The Broad Institute

summary: Therapeutic resistance to DNA damage is a significant challenge in oncology. To gain insight into biological mechanisms that cause DNA damage resistance and to inform strategies for achieving synergy with therapeutic radiation, we performed parallel pooled genetic CRISPR-Cas9 screening for survival in high-risk head and neck squamous cell carcinoma (HNSCC) subtypes. Surprisingly, in addition to known mediators of radiotherapy response, including ATM, DNAPK, and NF-κB signaling, loss of JAK1 was identified as a driver of tumor cell radioresistance. Knockout of JAK1 in HNSCC increased cell survival by enhancing the DNA damage-dependent G2/M cell cycle arrest and delaying progression to radiation-induced mitotic catastrophe. In line with this finding, both JAK1 knockout and kinase inhibition with abrocitinib prevented subsequent formation of radiation-induced micronuclei. Loss of JAK1 function did not affect canonical CDK1 signaling but instead reduced activation of PLK1 and AURKA, two kinases with auxiliary roles in the regulation of G2 and M phase progression. Correspondingly, using both EdU labelling and live cell imaging techniques, JAK1 loss was found to cause prolonged metaphase, mitotic slippage, and progression to tetraploidy. Targeting the mitotic kinesin KIF18A with the small molecule sovilnesib exacerbated mitotic stress and enhanced the efficacy of radiation. These studies establish KIF18A inhibition as a strategy to counteract the protective G2/M cell cycle arrest induced by DNA damage and to thus enhance tumor cell sensitivity to radiation therapy.

overall_design: We performed RNA Sequencing on Detroit562 HNSCC cell lines.  The NT2 cell line was transduced with a non-targeting control gRNA, and the JAK1 knockout (KO) cell line was transwduced with a JAK1-targeting gRNA.  JAK1 KO was confirmed via Western blot.  Three seperate experimental replicates of the Detroit562 NT2 and JAK1 KO cell lines were submitted for sequencing.

first: Marta

last: Baro

first: William

last: Gasperi

first: Nicholas

last: Ader

first: Hannah

last: Lea

first: Joseph

last: Contessa


title: SLC6A3 Dopaminergic Signaling Subverts Spliceosomal Fidelity to Promote Immune Checkpoint Inhibitor Resistance in Clear Cell Renal Cell Carcinoma [2]

first: Junhui

last: Su


title: Human liver-derived organoids recapitulate Oropouche virus infection and manifestation, enabling antiviral drug discovery

city: Rotterdam

first: Qiuwei

last: Pan

address: Wytemaweg 80

country: Netherlands

institute: Erasmus University Medical Center

summary: Oropouche virus (OROV) is a neglected, re-emerging arbovirus that typically causes self-limiting febrile illness but can also result in severe complications. Currently, there are no approved vaccines or treatments for OROV infection. We integrated clinical data from OROV-infected patients with human liver-derived organoid models to investigate the virus's impact on the liver and evaluate repurposed antivirals. Patient blood tests show elevated liver enzymes, suggesting OROV-associated hepatic dysfunction. OROV isolates productively infect liver organoids and induce severe cellular damage. Transcriptomic profiling reveals strong virus-host interactions, including activation of interferon-stimulated genes and cell death pathways. Pharmacological inhibition of the interferon pathway enhances OROV replication, whereas treatment with therapeutic interferon-α suppresses the infection. Molnupiravir, a clinically approved antiviral targeting viral RNA-dependent RNA polymerase, markedly inhibits OROV replication and mitigates virus-induced cytopathology. Combining molnupiravir with interferon-α resulted in synergistic antiviral activity, indicating the complementarity of virus-targeted and host-directed strategies.

overall_design: Organoids cultured from adult human liver tissue were infected with both OROV isolates, OROV-1967 (strain RIVM/OROV/TRVL9760) and OROV-2024 (strain IRCCS-SCDC_1/2024_OROV-2024). Infected organoids were harvested at 1- 48-, and 96-hours post-infection. A treatment group was established using organoids infected with the OROV-2024 isolate and treated with 1 μM NHC treatment from 1 hour to 96 hours post-inoculation. As negative controls, uninfected organoids were cultured under same conditions for 96 hours. Total RNA was extracted from all samples using the MachereyNagel NucleoSpin RNA II Kit (Bioke, Netherlands) and quantified with the Bioanalyzer RNA 6000 Picochip. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads for cDNA synthesis and library construction by Novogene. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. RNA sequencing was conducted using a paired-end 150 bp (PE 150) sequencing strategy via Illumina platforms.

first: Jiajing

last: Li

first: Xin

last: Wang


title: Multimodal single-cell and spatial profiling reveals altered T cell-mediated immunity and B-cell follicular architecture in non-metastatic lymph nodes of patients with aggressive non-small cell lung cancer

first: Joshua

middle: D.

last: Campbell

address: 72 East Concord Street

institute: Boston University Chobanian & Avedisian School of Medicine

summary: Regional lymph nodes (LNs) in the thoracic cavity serve as essential immunological hubs that coordinate humoral and cell-mediated responses against the development and progression of non-small cell lung cancer (NSCLC). To investigate immune dysregulation in the non-metastatic regional LNs of patients with aggressive NSCLC, we performed multimodal profiling on 36 LNs from 11 patients undergoing curative-intent resection including CITE-seq, scRNA-seq, and Imaging Mass Cytometry (IMC). Regional N1 LNs from patients with more aggressive disease (stage IB–IIIA) exhibited a significant enrichment of dysfunctional CD8⁺ T cells and regulatory T cells (Tregs) compared to N2 LNs and LNs from patients with less aggressive disease (stage IA). These immune subsets were spatially co-localized with mature regulatory dendritic cells (mregDCs; CD1c⁺, TIM3⁺, LAMP3⁺), forming an immunosuppressive niche uniquely enriched in the N1 LNs of higher-stage patients. Concurrently, higher-stage N1 LNs contained larger number of “decorticated” B-cell follicles characterized by decreased encapsulation of the mantle zone layer surrounding the germinal centers. This mantle zone disorganization was associated with increased spatial niches involving Tregs, CD68⁺ CD163⁺ TIM3⁺ Macrophages, CD163⁺ TIM3dim Monocytic-Myeloid Derived Suppressor Cells (M-MDSC), plasma B cells, and a decrease in spatial niches involving CD4⁺ T helper cells and fibroblastic reticular cells (FRCs). Together, our findings reveal parallel alterations in humoral and cell-mediated immunity within the regional LNs of patients with aggressive NSCLC.

overall_design: Fine needle aspirate samples of lymph nodes, primary tumors, and normal lung tissue were collected from surgical specimens of patients undergoing curative-intent resection at Boston Medical Center. Mediastinal LN dissection was performed as per the Commission on Cancer guideline, removing, when possible, 3 N2 station LNs and at least 1 N1 station LN. Pathologic stage was determined per 8th edition of TNM Staging.

first: Zhan Hao

last: Xi

first: Yusuke

last: Koga

first: Sarah

last: Mazzilli

first: Kei

last: Suzuki

middle: D


title: Loss of JAK1 Function Causes G2/M Cell Cycle Defects Vulnerable to KIF18A Inhibition [Cal27 CRISPR Screen]

overall_design: We performed a kinome-focused (Brunello gRNA library) CRISPR screen on the Cal27 HNSCC cell line with radiation as a selection pressure.  Three seperate experimental replicates of the Cal27 cells were either irradiated or treated with a control regimen.  Sequencing was then performed to determine gRNAs that were enriched or depeletd in the irradiated samples compared to unirradiated samples.


title: Loss of JAK1 Function Causes G2/M Cell Cycle Defects Vulnerable to KIF18A Inhibition

overall_design: We performed RNA Sequencing on Cal27 HNSCC cell lines.  The NT2 cell line was transduced with a non-targeting control gRNA, and the JAK1 knockout (KO) cell line was transwduced with a JAK1-targeting gRNA.  JAK1 KO was confirmed via Western blot.  Three seperate experimental replicates of the Cal27 NT2 and JAK1 KO cell lines were submitted for sequencing.


title: RNA-seq data for studying the influence of fallopian tube stromal cells and/or estrogen treatment on fallopian tube epithilial cells

first: Zhe

address: 77 Avenue Louis Pasteur, Room 466

department: Medicine, Genetics Division

institute: Brigham and Women's Hospital

summary: This study aims to investigate the role of fallopian tube (FT) stromal cells (SCs) in supporting the growth, proliferation and differentiation of fallopian tube epithelial (FTE) cells in response to estrogen signaling. Estrogen receptor alpha (encoded by Esr1) positive cells are present in both FTE and FT stromal compartments. Thus, estrogen exposure can affect FTE cells either directly or indirectly via Esr1+ SCs. Here we used RNA-seq expression profiling of murine FTE cells under various treatment conditions to demonstrate how estradiol (E2) treatment affected their transcriptomics directly, and indirectly via the co-cultured FT SCs, utilizing an organoid culture/co-culture system.

overall_design: Wild-type (WT) and p53/Brca1/Pten-null (PBP-null) FTE organoids were established via serial passages in BET (B27, EGF, A83-01) medium. They were then either cultured alone or co-cultured with MACS-sorted CD24- stromal cells from oviducts of WT donor mice, and treated with or without 1nM of E2. After 7 days, EpCAM+ FTE cells were sorted from each organoid samples before proceeding to bulk RNA-seq.

first: Xueqing

last: Chen


title: RNA-Seq data for fallopian tube stromal cells in mice with Pdgfra-Cre mediated conditional knockout of Esr1

summary: This is a loss of function study to investigate how conditional knockout of Esr1 in Pdgfra+ fallopian tube (oviductal) stromal cells in mice affects stromal cells directly and epithelial cells indirectly.

overall_design: The conditional knockout cells are genetically marked by a Rosa26-YFP (R26Y) reporter. FACS-sorted YFP+ stromal cells from fallopian tubes (oviducts) of young adult Pdgfra-Cre;Esr1(fl/fl);R26Y experimental mice and Pdgfra-Cre;R26Y control mice were proceeded to total RNA preparation, followed by bulk RNA sequencing.


title: Genome-wide analysis of High Energy pulse electron beam (HEPE)-induced gene expression in Streptomyces lividans 1326 strain

city: hangzhou

first: xinyuan

last: feng

address: xihu street yuhangtang road 866

institute: zhejiang university

summary: Analysis the influence of HEPE on the gene expression through RNA-Seq. The hypothesis tested in the present study was that HEPE-induced structure variations influence gene expression in the strain. Results provide important information of the response of strain to HEPE, such as genes up or down regulation responsible for  secondary metabolisn products.

overall_design: RNA-seq profiling of wildtype Streptomyces lividans 1326 strain and its mutants derived from HEPE treatment.

first: Xinyuan

last: Feng

first: Mohammad R

last: Seyedsayamdost

first: Weishan

first: Fei

last: Xu


title: Single-cell RNA sequencing of mouse brain immune cells following peripheral SARS-CoV-2 infection

city: Iowa City

first: Stanley

last: Perlman

state: Iowa

address: 51 Newton Road

department: Dept of Microbiology and Immunology

institute: University of Iowa

summary: Neurological and neuropsychiatric symptoms are among the most prevalent Post-Acute Sequelae of COVID-19 (PASC), collectively referred to as neuroPASC. Despite growing recognition, the mechanisms driving neuroPASC remain elusive, hindering therapeutic development. Brain immune cells - particularly microglia - have been implicated, yet current knowledge largely derives from post-mortem tissues with variable disease courses and comorbidities and limited spatial coverage. To address this, we established a PASC animal model by infecting C57BL/6 mice with a sublethal dose of mouse-adapted SARS-CoV-2. Infected mice exhibited persistent behavioural alterations and prolonged neuroinflammation in the absence of direct viral neuroinvasion. Single-cell RNA sequencing of brain immune cells collected at 0, 6, 30, and 100 days post-infection (dpi) revealed underlying dynamic, longitudinal immune responses. Microglia, the predominant brain-resident sentinel cells, displayed sustained expansion of subclusters characterized by inflammatory, stress response, and metabolic reprogramming signatures across all time points. Border-associated macrophages upregulated monocyte attractants during acute infection and were partially replaced by infiltrating monocytes. Concurrently, inflammatory monocytes and neutrophils showed maximal brain infiltration and antiviral activity at 6 dpi, potentially triggering long-term microglial activation. Together, these findings provide a high-resolution atlas of brain myeloid immune dynamics during neuroPASC and highlight a central role of microglia in sustaining chronic neuroinflammation, offering insight into potential therapeutic targets.

overall_design: C57BL/6 mice were intranasally infected with 1000 PFU of SARS-CoV-2 N501YMA30. Brain immune cells were isolated from mock-infected mice or at 6, 30, 100 days post-infection using CD45+ magnetic beads. 4-5 mice per group were pooled together for scRNA-seq library preparation.

first: Lu

last: Tan

first: Shea

last: Lowery

first: Andrew

last: Thurman


title: Engineering Human Bone Marrow-derived Mesenchymal Stromal Cell Aggregates for Enhanced Extracellular Vesicle Secretion in a Vertical-Wheel Bioreactor

city: Tallahassee

first: li

last: sun

state: FL

address: 1115 West Call st.

institute: FSU College of Medicine

summary: Human mesenchymal stem/stromal cells (hMSCs) hold significant potential for regenerative medicine due to their anti-inflammatory and pro-angiogenic secretome. In particular, 3D hMSC aggregates secrete EVs that have enhanced anti-inflammatory and immunomodulatory properties. However, safety and efficacy concerns associated with stem cell therapy have led to the discovery of cell-free approaches utilizing hMSC-derived extracellular vesicles (EVs). Despite their therapeutic promise, the clinical application of hMSC-EVs is limited by low production yield. This study investigates the scalable generation of hMSC-EVs from 3D aggregates in a novel Vertical-Wheel Bioreactor (VWBR) through shear stress-mediated biochemical cues, promoting cargo expression relevant to nerve regeneration and neuropathic treatment. Bone marrow-derived hMSCs were cultured as 3D aggregates in VWBRs and exposed to two different culture media—αMEM/FBS (serum-containing) and DMEM/F12/B27 (serum-free)—under three agitation speeds (25, 40, and 64 rpm). Metabolite and gene analyses were performed to assess EV biogenesis, focusing on ESCRT machinery markers. Results showed that hMSCs cultured in VWBR exhibited higher expression of EV biogenesis genes and glycolytic genes compared to static culture. αMEM/FBS (serum-containing) condition was more robust than DMEM/F12/B27 (serum-free) condition. EV yield (EV number per cell) increased by 3-10 fold (in serum-containing medium) in VWBR compared to static culture, with size of 120-180 nm and EV marker expression. microRNA-sequencing of the EVs shows the upregulation of miR-29a-3p, miR-451a, miR-224-5p, miR-16-5p, miR-133a-3p, and miR-143-3p, etc., reflecting a combination that enhances EV biogenesis, promotes metabolic reprogramming, and supports immunomodulatory behavior characteristics. Functional assays demonstrated that the generated EVs effectively modulated Schwann cell responses under neural inflammation, supporting their therapeutic potential. These findings highlight the role of VWBR-driven hydrodynamics in promoting EV production from 3D hMSC aggregates. This study advances the knowledge of dynamic aggregation and metabolic influence on 3D hMSC-EV production and fundamentally scale up the preconditioned techniques used to promote hMSC aggregation and the consequent EV secretion.

overall_design: Human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) were cultured in regular 2D or 3D conditions. EVs were collected and total RNA was isolated for sequencing.

first: Li

last: Sun


title: Loss of JAK1 Function Causes G2/M Cell Cycle Defects Vulnerable to KIF18A Inhibition [Detroit562 CRISPR Screen]

first: Vanessa

last: Kelley

overall_design: We performed a kinome-focused (Brunello gRNA library) CRISPR screen on the Detroit562 HNSCC cell line with radiation as a selection pressure.  Three seperate experimental replicates of the Detroit562 cells were either irradiated or treated with a control regimen.  Sequencing was then performed to determine gRNAs that were enriched or depeletd in the irradiated samples compared to unirradiated samples.

first: Marta

last: Baro

first: William

last: Gasperi

first: Nicholas

last: Ader

first: Hannah

last: Lea

first: Joseph

last: Contessa


title: Fbxw11 regulates macrophage polarization and enhances the anti-leukemia activity

city: Tianjin (天津)

first: Siqi

last: Zhang

state: Tianjin (天津市)

address: 劝业场街道

institute: Institute of Hematology & Blood Disease Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College

summary: investigate the effects of Fbxw11 on macrophage phenotype and function, providing experimental evidence for understanding the potential role of Fbxw11 in anti-leukemia immunity.

overall_design: Compare theTranscriptomes of different RAWs (RAW-Con and RAW-Fbxw11).

first: Guoguang

last: Zheng

first: Lina

last: Wang


title: HELZ2 regulates APOB mRNA stability to modulate fatty liver disease and atherosclerosis

city: Dallas

first: Zhao

state: Texas

address: 5323 Harry Hines Blvd.

department: Center for the Genetics of Host Defense

institute: The University of Texas Southwestern Medical Center

summary:
Background:
Apolipoprotein B (APOB) is essential for lipoprotein assembly and secretion and plays a central role in the development of cardiovascular disease and metabolic dysfunction-associated steatotic liver disease (MASLD). While APOB protein degradation during very low-density lipoprotein (VLDL) maturation has been extensively studied, the regulation of APOB mRNA stability under physiological and pathological conditions remains unexplored.
Methods:
A forward genetic screen in randomly mutagenized mice identified helicase with zinc finger 2 (HELZ2) as a critical regulator of lipid metabolism. The metabolic effects of HELZ2 mutations or deficiency were evaluated in mice maintained on a chow diet or a high-fat diet (HFD). We also employed a doxycycline-inducible, liver-specific HELZ2 overexpression model to test the sufficiency of hepatocyte Helz2 upregulation. Biochemical assays were used to assess HELZ2 binding to APOB mRNA and its role in APOB mRNA degradation, while the effect of HELZ2 modulation on atherosclerosis was examined in Apoe-/- and Ldlr-/- mouse models.
Results:
We discovered a unique gain-of-function mutation in HELZ2 (L1833P, termed Colby) that promotes hepatic lipid accumulation independently of changes in body weight on a standard chow diet. Mechanistically, HELZ2 binds APOB mRNA and degrades it via its helicase activity, ensuring tight control of hepatic APOB levels. The Colby mutation enhances HELZ2 helicase activity, resulting in marked reductions in Apob expression and increased hepatic lipid accumulation. Conversely, Helz2-deficient mice show increased Apob mRNA levels and reduced hepatic triglycerides on a HFD. Notably, modest liver-restricted induction of HELZ2 was sufficient to decrease hepatic Apob mRNA and alter lipid handling, phenocopying the Helz2Colby state and supporting the gain-of-function mechanism. A single copy of the Helz2Colby mutation confers protection against atherosclerosis in Apoe-/- and Ldlr-/- mice.
Conclusions:
HELZ2 is a key regulator of APOB mRNA stability and lipid metabolism. Genetic or pharmacological modulation of HELZ2 activity represents a promising therapeutic strategy for cardiovascular disease and MASLD.

overall_design: We performed RNA immunoprecipitation (RIP) with anti-Flag beads to pull down liver lysates from wild type (WT) and Helz2-3xFlag mice, and used RNA sequencing (RNA-seq) to identify HELZ2 interacting RNAs. There are two samples in total, rip_ctrl and rip_helz2.

first: Yiao

last: Jiang


title: Study the role of the hypusination pathway in the progression of prostate cancer

city: Nice

first: Michel

last: KAHI

address: 151 Route Saint Antoine

institute: INSERM U1065

summary: We studied the involvement of the hypusination pathway in prostate cancer progression using various models, including patient-derived tumor organoid cell lines and in vivo mouse models. We employed different omics techniques to identify the molecular mechanisms linking hypusination to the aggressiveness of prostate cancer.

overall_design: We performed RNA-seq on DU145 cells treated for 24 h with GC7 at a concentration of 40 µM. And we carried out polysome-profiling followed by sequencing of polysome-associated mRNAs (i.e. heavy ribosome-bound fractions), as well as sequencing of the cytoplasmic (total) mRNA fraction. This combined approach was designed to compare changes at the transcriptome level (via RNA-seq) and at the translatome level (via polysome-associated mRNAs), thus allowing us to assess how inhibition of hypusination affects not only mRNA abundance, but also mRNA translation (ribosome loading) in DU145 prostate cancer cells.

last: Kahi

first: Anne

last: Vincent

first: JeanJacques

last: Diaz

first: Frederic

last: Bost


title: High Resolution Spatial Mapping of Microbiome-Host Interactions via in situ Polyadenylation and Spatial RNA Sequencing (Stereoseq)

city: Ithaca

first: Iwijn

last: De Vlaminck

address: 237 Tower Road

institute: Cornell University

summary: Inter-microbial and host–microbial interactions are thought to be critical for the functioning of the gut microbiome, but few substantive tools are available to measure these interactions. Here, we report a method for unbiased spatial sampling of microbiome-host interactions in the gut at high spatial resolution. This method combines enzymatic in situ polyadenylation of both bacterial and host transcripts with spatial RNA-sequencing. Application of this method revealed the biogeography of the mouse gut microbiome as function of location in the intestine, short-range intermicrobial interaction, local shaping of the microbiome by the host, and tumor-associated changes in the architecture of the host-microbiome interface. This method is compatible with broadly available commercial platforms for spatial RNA-sequencing, and can therefore be readily adopted to broadly study the role of short-range, bidirectional host-microbe interactions in microbiome health and disease.

overall_design: Spatial transcriptomics (Stereoseq)

first: Ioannis

last: Ntekas

first: David

last: McKellar

first: Lena

last: Takayasu