title: Single-cell RNA sequencing of aortic cellular  in ApoE-/- mice and MSC_ITGB3-treated ApoE-/- mice

city: Shijiazhuang

first: Haijuan

last: Hu

address: Heping West Road

institute: The Second Hospital of Hebei Medical University

summary: We reports 33,782 individual cells from HFD-fed mouse aortas with or without MSC_ITGB3 treatment were subjected to single-cell RNA sequencing. We generated a compendium of 30 cell clusters, mainly smooth muscle cells (SMCs), endothelial cells (ECs), and immune cells. In the HFD group, genes associated with proliferation, migration, and collagen were highly expressed in the major SMC subpopulations. However, the expression of contraction-related genes in SMC subpopulations was significantly higher in the MSC_ITGB3 group than in the HFD group. After HFD consumption, EC subpopulations with active PI3K-Akt signaling pathways, extracellular matrix-receptor interaction, and contraction-related genes were significantly enriched. In the MSC_ITGB3 group, the number of dendritic cells positively correlated with atherosclerotic lesion progression and contributed to lipid accumulation, and inflammatory factor levels decreased. These data on the composition, signaling pathways, and cellular communication of the aorta cells following stem cell treatment and atherosclerotic disease progression may help improve atherosclerosis treatment.

overall_design: Detection of aortic cell heterogeneity in HFD-fed mouse models with or without MSC_ITGB3 treatment

first: Wei

last: Cui


title: Spatiotemporal transcriptome atlas of water lily floral organogenesis provide insight to the evolutionary origin of novel traits of angiosperms [Spatial Transcriptomics]

status: Public on Jul 10 2026

city: Shanghai

first: Chang

last: Liu

address: Xinjiangwancheng street, Yangpu District, Shanghai

country: China

institute: Fudan University

summary: Petals are a key evolutionary innovation of flowers that reshaped plant-pollinator interactions and underlie the dominance of angiosperms in terrestrial ecosystems, yet their evolutionary origin remains debated as the morphological inferences rarely connect directly to the regulatory programs inferred from extant flowering plants. We employed spatial transcriptome sequencing on serial floral buds of the basal angiosperm Nymphaea colorata, producing a spatiotemporal atlas of cell states during primordium initiation and the subsequent formation of tepals (sepals and petals), stamens and carpels. Our analyses identified multiple meristematic cell populations organized hierarchically at the floral base, with one population give rise to both initiating petals (or inner tepals) and stamens whereas a second population forms carpel primordia. Developmental trajectory reconstruction based on thousands of tissue-preferentially expressed genes uncovers a continuous fate transition from stamen primordia via developing stamens to inner tepals undergoing morphogenesis. Quantitative modeling of MADS-box tetramer composition reveals dosage-sensitive shifts that support the hypothesis that petaloid organs evolved from sterile stamens at the outer whorls. Trajectory modules further show stepwise incorporation of vegetative programs, particularly genes for polarity establishment and photosynthetic essential for leaf development into petal organogenesis. These findings support a model in which petal merger occurred through quantitative retuning and redeployment of ancestral regulatory networks that predate the origin of angiosperms, providing clues for understanding the evolutionary innovation and diversification of petaloid organs across flowering plants.

overall_design: We performed spatial transcriptome sequencing on five serial longitudinal sections of N. colorata floral buds across four developmental stages, with tissues from actively dividing floral meristems to nearly matured reproductive organs

first: Ge

last: Wang

first: Binbin

last: Cao

first: Yonglong

last: Li

first: Yamao

last: Chen

first: Chunce

last: Guo

first: Ji

last: Qi


title: Menin inhibition impairs metastatic colonization of Ewing sarcoma

city: Seattle

first: Research

middle: Scientific

last: Computing

state: WASHINGTON

address: 818 Stewart Street

department: Research Scientific Computing

country: USA

institute: Seattle Children's Research Institute

type: Expression profiling by high throughput sequencing

summary: Menin is a scaffolding protein that interacts with context-specific partners to regulate gene expression. In MLL-rearranged leukemias, Menin:MLL interactions drive leukemogenesis and Menin inhibitors have been FDA approved for these cancers. We previously reported that Menin promotes oncogenic phenotypes in Ewing sarcoma (EwS). Here, we sought to define EwS-specific functions of Menin and determine if Menin inhibitors could be therapeutically leveraged for these tumors. Genetic knockout of Menin had no impact on EwS cell proliferation in vitro, but metastatic potential of Menin-depleted cells in vivo was impaired. Transcriptional profiling of Menin knockout cells in vitro showed reproducible downregulation of MYC signature genes and upregulation of developmental programs. Conversely, transcriptional rewiring of developmental genes and restoration of MYC target gene expression were evident in tumors that arose from Menin knockout cells. Exposing EwS cells to the Menin inhibitor VTP50469 (revumenib) inhibited expression of MYC targets and co-immunoprecipitation studies detected Menin:MYC interactions that were partially disrupted by the drug. Metastatic colonization of disseminated EwS cells in vivo was significantly inhibited in mice fed VTP50469 chow. Together these findings implicate Menin as a mediator of EwS metastasis and suggest that Menin inhibitors warrant investigation as novel therapeutics for patients with high-risk disease.

overall_design: RNA-seq profile of A673 MEN1 knock out (A673 gMEN1 5-1) and TC32 MEN1 knock out (TC32 gMEN1 5-5) cell lines in comparison with the control cell lines (A673 gControl 1 and TC32 gControl 3). RNA-seq profile of A673 and TC32 cells treated with 10 µM VTP50469 or 0.1% DMSO (control) for 72 hours. RNA-seq profile of tumors in the liver from A673 and TC32 xenografts treated for 5 or 6 weeks respectively with 0.1% VTP50469 mouse chow compared to control mouse chow. RNA-seq profile of A673 gControl 1 and three A673 MEN1 knock out (A673 gMEN1 1-3, A673 gMEN1 5-1, A673 gMEN1 5-2) xenograft subcutaneous tumors.

first: Elizabeth

middle: R

last: Lawlor

first: Katherine

middle: A

last: Braun


title: Benchmarking cell-type deconvolution in cross-platform transcriptomic data [GeoMx]

city: Frankfurt am Main

first: Katharina

last: Imkeller

address: Heinrich-Hoffmann-Str. 7

country: Germany

institute: University Hospital Frankfurt

summary:
Background: Transcriptomic data from diverse measurement technologies are widely used to study tissue heterogeneity. Cell-type deconvolution, which resolves mixed transcriptomic signals into cellular components, is a key analytical approach. However, achieving accurate deconvolution across platforms remains challenging due to platform-specific experimental and technological biases.
Results: We systematically benchmarked deconvolution performance using real-world cross-platform datasets and simulated data modeling distinct technological features. SpatialDecon and cell2location demonstrated the most reliable and consistent performance across both simulated and experimental settings across a broad range of technological biases.
Conclusions: Our results highlight how the different deconvolution tools are affected by data properties that depend on technological differences between transcriptomic platforms. Moreover, we provide practical guidelines for selecting computational methods dependent on experimental design for robust deconvolution of cross-platform transcriptomic data.

overall_design:
To explore differences in the cellular composition of tissue profiled on two spatial transcriptomics platforms, Visium and Nanostring, we analyzed 4 brain tumor specimens from the Frankfurt University Cancer Center. Sections from the same tissue specimen were profiled simultaneously on both platforms. Transcriptome profiles of 9 immune aggregates and 4 corresponding tumor-rich regions of interest (ROIs) were measured using the Nanostring GeoMx® Digital Spatial Profiler (DSP) platform with the whole transcriptome atlas (WTA) gene panel. The same 4 tissue specimens were also profiled using the 10x Genomics Visium v2 FFPE WTA assay. Immune and tumor regions were selected based on CD20 gene expression.

***************************************************************
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: Aakanksha

last: Singh

first: Pinar

last: Cakmak

first: Jennifer

middle: H

last: Lun

first: Jadranka

last: Macas

first: Jonathan

last: Schupp

first: Karl

last: Plate

first: Yvonne

last: Reiss


title: The combination of nelfinavir and cisplatin drives lytic cell death through a caspase-8/caspase-3/GSDME axis in platinum-resistant ovarian cancer cells

city: Montreal

first: Benjamin

last: Forgie

state: Quebec

address: 3775 Rue University

country: Canada

institute: McGill University

summary: Cisplatin (CDDP) is one of the most active chemotherapeutic agents used for ovarian cancer; however, primary or acquired platinum resistance is associated with poor prognosis. Nelfinavir (NFV), an HIV protease inhibitor, has demonstrated anti-tumor activity in multiple cancer models, but its interaction with CDDP in ovarian cancer has yet to be demonstrated. In this study, we investigated whether combined CDDP and NFV treatment provides a therapeutic advantage in platinum-resistant ovarian cancer cells. Drug synergy between NFV and CDDP was assessed using cell viability assays and Loewe additivity modelling. Apoptotic and pyroptotic signaling were evaluated by immunoblotting, mitochondrial membrane potential analysis, lactate dehydrogenase release, and caspase inhibition. Transcriptomic changes were assessed by bulk mRNA sequencing followed by differential gene expression analysis and gene set enrichment analysis. NFV synergized with CDDP to reduce the viability of platinum-resistant ovarian cancer cells and promoted a regulated lytic cell death phenotype involving apoptotic and pyroptotic features. Combination treatment induced caspase-8 and caspase-3 activation, with downstream gasdermin E (GSDME) processing. Inhibition of caspase-3 significantly attenuated cell death, while caspase-8 inhibition rescued viability and prevented Bid cleavage, caspase-3 activation, and GSDME cleavage. These effects occurred in the context of enhanced endoplasmic reticulum stress, increased DNA damage with reduced DNA repair, and impaired Akt-driven survival signaling. Together, these findings establish that NFV synergizes with CDDP in platinum-resistant ovarian cancer cells by promoting a caspase-8-dependent apoptotic-to-secondary pyroptotic response, supporting further investigation of NFV as a candidate drug for repurposing to enhance platinum-based therapy.

overall_design: IGROV-1/CP platinum-resistant ovarian cancer cells were treated with vehicle, cisplatin, nelfinavir, or cisplatin followed by nelfinavir. Three independent biological replicates were included per treatment group, for a total of 12 samples. Bulk mRNA sequencing was performed to compare treatment-associated transcriptional changes across experimental conditions.

first: Carlos

last: Telleria

first: Julia

last: Burnier


title: The Z-granule scaffold LOTR-1 bridges piRNA recognition to small-RNA amplification in C. elegans

city: BAR HARBOR

first: CGDS

last: Data

state: ME

address: 159 OLD BAR HARBOR RD

institute: MDI Biological Laboratory

summary: LOTR-1 is a Caenorhabditis elegans member of the conserved LOTUS- and Tudor-domain (TDRD5/7-like) family that resides in a germ granule subcompartment (Z granule). To dissect how LOTR-1 and its individual domains shape gene expression and translation, we performed paired total-lysate and polysome-associated mRNA sequencing across three domain-specific alleles: a full in-frame deletion (delta-LOTR-1), a LOTUS-domain deletion (delta-LOTUS), and a Tudor-domain deletion (delta-Tudor), each compared to a wildtype control. Differential expression analysis reveals widespread transcriptomic remodeling in all three alleles, strongest in the LOTUS-domain deletion, that is extensively offset by genome-wide translational buffering, including a coordinated downregulation of ribosomal protein mRNAs. The MiDAC scaffold saeg-2 is robustly derepressed across all alleles. These paired lysate and polysome datasets provide a read-out of both transcript abundance and translational engagement in lotr-1 domain mutants.

overall_design: To dissect the contributions of LOTR-1 and its individual protein domains to gene expression and translational regulation, we performed paired total-lysate and polysome-associated mRNA sequencing on three lotr-1 mutant alleles compared to a wildtype control. The strains are a wildtype control carrying an N-terminal 3xFLAG::GFP tag (DUP167, sam85), a full in-frame deletion (DUP207, delta-LOTR-1, sam117), a LOTUS-domain deletion (DUP217, delta-LOTUS, sam113), and a Tudor-domain deletion (DUP185, delta-Tudor, sam102). Synchronized hermaphrodites from each strain were harvested on day 1 of adulthood in four biological replicates. For each replicate lysate, total mRNA was isolated from one half while the remaining half was fractionated by sucrose gradient ultracentrifugation to isolate polysome-associated mRNA, yielding paired lysate and polysome libraries for every replicate (4 strains x 2 fractions x 4 replicates = 32 libraries). Differential expression was analyzed with DESeq2, processing lysate and polysome fractions separately, with each mutant strain compared to the DUP167 wildtype reference; translational efficiency was estimated within DESeq2 using a classical interaction design (strain x fraction).

first: Kehinde

last: Abayomi

first: Noah

last: Lind

first: Heath

last: Fuqua

first: Joel

last: Graber

first: Dustin

last: Updike


title: Methylation Clocks Do Not Predict Age or Alzheimer's Disease Risk Across Genetically Admixed Individuals

city: San Francisco

first: Sebastián

last: Cruz-González

state: California

institute: UCSF

type: Methylation profiling by genome tiling array

summary: Epigenetic aging clocks based on DNA methylation patterns across the genome have emerged as a potential biomarker for risk of age-related diseases, like Alzheimer's disease (AD), and environmental and social stressors. However, methylation clocks have not been comprehensively validated in genetically diverse individuals. Here we evaluate a set of first-, second-, and third-generation methylation clocks in 621 AD patients and matched controls from African American, Hispanic, and White cohorts. The clocks are less accurate at predicting age in genetically admixed cohorts compared to the White cohort, especially for those with substantial African ancestry. This decreased accuracy holds in >2,500 individuals of European and African ancestry from three additional datasets. The clocks also fail to consistently identify age acceleration in admixed AD cases compared to controls. To explore potential causes for the lack of generalization of the clocks, we intersected clock CpGs with methylation, germline genetic variants, and methylation QTL (meQTL) data from global populations. We find differential methylation between African and European ancestry individuals is common for clock CpGs. Genetic variants rarely disrupt clock CpGs between populations, but a substantial fraction of clock CpGs have meQTL with significantly higher frequencies in African genetic ancestries. Our results demonstrate that methylation clocks often fail to predict age and AD risk when applied across populations and suggest avenues for improving their portability by considering differences in genetic and epigenetic patterns across human populations.

overall_design: Genome-wide DNA methylation profiling using Illumina Infinium MethylationEPIC BeadChip arrays on whole blood samples from 621 individuals across five cohorts (NHW, REAAADI, PRADI, CuADI, PERUVIAN), including 313 Alzheimer's disease cases and 308 age-matched controls of African American (n=205), Hispanic (n=283), and Non-Hispanic White (n=133) ancestry. Ages ranged from 53 to 98.

first: Ogechukwu

last: Okpala

first: Esther

last: Gu

first: Lissette

last: Gomez

first: Makaela

last: Mews

first: Jeffery

middle: M

last: Vance

first: Michael

middle: L

last: Cuccaro

first: Mario

last: Cornejo-Olivas

first: Briseida

middle: E

last: Feliciano-Astacio

first: Goldie

middle: S

last: Byrd

last: Haines

first: Margaret

last: Pericak-Vance

first: Anthony

middle: J

last: Griswold

first: William

last: Bush

first: John

last: Capra


title: Genome-wide RRBS methylation profiling identifies NLRP7 promoter hypermethylation associated with disease severity in epidermolysis bullosa simplex

city: Tel Aviv

first: Lubna

last: Khair

address: Weizmann 6

country: Israel

institute: Sourasky medical center

type: Methylation profiling by high throughput sequencing

summary: Reduced representation bisulfite sequencing (RRBS) was performed to investigate DNA methylation profiles in patients with epidermolysis bullosa (EB) exhibiting variable disease severity. Differential methylation analysis identified a differentially methylated region within the NLRP7 promoter and first exon. The dataset provides genome-wide DNA methylation profiles generated from peripheral blood leukocyte DNA and is intended to facilitate studies of epigenetic mechanisms associated with EBS disease severity.

overall_design: Genomic DNA was isolated from peripheral blood leukocytes obtained from patients with epidermolysis bullosa. DNA methylation profiles were generated using Reduced Representation Bisulfite Sequencing (RRBS). Differential methylation analysis was performed to compare patients with severe and mild disease phenotypes.

first: Ofer

last: Sarig

first: Rawaa

last: Ishtewy

first: Yoni

last: Moskovitz

first: Kiril

last: Malovitski

first: Angi

last: Zenab

first: Thomas

last: Kocher

first: Julia

last: Hummel

first: Josefina

last: Hofbauer

first: May

last: Arad

first: Eylon

last: Sharoni

first: Naama

last: Adam

first: Ben

last: Avshalom

first: Ulrich

last: Koller

first: Verena

last: Wally

first: Liat

last: Samuelov

first: Eli

last: Sprecher


title: Spatiotemporal transcriptome atlas of water lily floral organogenesis provide insight into the evolutionary origin of novel traits of angiosperms [RNA-Seq]

first: Chang

last: Liu

overall_design: RNA profiling of outer tepals, inner tepals and stamens from developing water lily floral buds at stage 3.

first: Ge

last: Wang

first: Binbin

last: Cao

first: Yonglong

last: Li

first: Yamao

last: Chen

first: Chunce

last: Guo

first: Ji

last: Qi


title: Single cell perturb seq of A549 cells

first: Hani

last: Goodarzi

state: CA

address: 600 16th street

summary: Single cell perturb seq of A549 cells from Myllia Biotechnology.

overall_design: A549 cells were perturbed with a dual guide library for 218 genes

first: Stefanie

last: Fellinger

first: Adam

last: Krejci

first: Tilmann

last: Burckstummer

first: Christopher

last: Carpenter


title: Iron-Catalyzed Oxidative Stress Reveals an Exposome-Related Ferroptosis-Resistant Karyomegalic Niche in BRCA1-Linked Renal Carcinogenesis

city: Nagoya

first: YINGYI

last: KONG

state: Aichi

address: 65 Tsurumai-cho, Showa-ku

department: Graduate School of Medicine

country: Japan

institute: NAGOYA UNIVERSITY

summary: Early cancer initiation under chronic oxidative stress requires the survival of epithelial cells that should otherwise be eliminated by regulated cell death. However, the spatial and morphological identity of such stress-adapted cells remains poorly defined. Here, we integrated Xenium single-cell spatial transcriptomics, quantitative nuclear morphometrics, mitochondrial analyses, and human cancer datasets to investigate iron-driven renal carcinogenesis in Brca1(L63X/+) rats. Ferric nitrilotriacetate exposure induced dynamic remodeling of proximal tubular epithelium, characterized by loss of homeostatic transport and mitochondrial programs, and activation of Myc, Met, Lcn2, Anxa2 and iron-handling genes. BRCA1 haploinsufficiency expanded a karyomegalic proximal tubular state associated with mitochondrial dysfunction, chromatin hyperchromasia, nuclear remodeling, and ferroptosis-resistant transcriptional features. Subclassification of karyomegalic cells identified distinct stress-adaptive populations, including a BRCA1-associated K2 state enriched for Myc/Met/Lcn2 signaling and preferentially embedded within a pro-oncogenic epithelial–stromal microenvironment. Of note, neighboring non-karyomegalic proximal tubular cells acquired related iron-handling and survival-biased programs, indicating that karyomegaly marks a broader pre-neoplastic field rather than an isolated abnormality. Human TCGA renal cancer analyses and morphometric assessment of BRCA1-mutant breast tissue further supported the clinical relevance of karyomegaly-associated gene modules and nuclear remodeling. These findings define a spatially organized, ferroptosis-resistant karyomegalic niche as an early lesion in iron-driven carcinogenesis and establish quantitative nuclear morphology as a functional readout of exposome-related oxidative stress adaptation in genetically susceptible tissues.

overall_design: Xenium in situ spatial transcriptomics was performed on formalin-fixed, paraffin-embedded (FFPE) kidney sections from wild-type and Brca1(L63X/+) rats under three conditions: untreated control, Fe-NTA administered intraperitoneally (i.p.) for 1 week, and Fe-NTA administered i.p. for 3 weeks. A custom 50-gene panel targeting renal cell type markers and ferroptosis-, carcinogenesis-, and mitochondria-associated genes was used. H&E staining was performed on the same sections following the Xenium run to enable histopathological annotation and correlation with spatial gene expression.

first: Yingyi

last: Kong

first: Yukihiro

last: Shiraki

first: Kazuhiro

last: Furuhashi

first: Shoichi

last: Maruyama

first: Tatsuhiko

last: Imaoka

first: Atsushi

last: Enomoto

first: Shinya

last: Toyokuni


title: Therapy-persistent leukemia is selectively vulnerable to SLC23A1 restoration via targeting KHSRP (AML patient samples)

city: Boston

first: Qingyu

last: Luo

state: MA

address: 450 Brookline Ave

institute: Dana-Farber Cancer Institute

summary: Acute myeloid leukemia (AML) remains highly prone to relapse driven by therapy-persistent residual cells. To discover specific vulnerabilities in this population, we performed genome-wide CRISPR interference screens in AML cells treated with multiple agents. KHSRP was the top hit, whose depletion sensitized AML cells to therapy and substantially prolonged survival in treated AML-bearing mice. Profiling in vivo residual disease after therapy identified downregulation of the vitamin C and uric acid transporter SLC23A1 as a resistance mechanism. KHSRP depletion restored SLC23A1 through a previously unrecognized function by preventing ZC3H4-mediated nuclear mRNA degradation. KHSRP depletion therefore enhanced the synergistic cytotoxicity of vitamin C and uric acid, particularly in therapy-persistent leukemia cells. Re-expression of TET2 overrode the chemosensitizing effect of KHSRP depletion in TET2-mutant leukemia, suggesting that KHSRP phenotypes were linked to vitamin C and uric acid-mediated TET activation. These findings nominate targeting KHSRP to enhance treatment efficacy and selectively eradicate residual AML.

overall_design: RNAseq was performed to evaluate gene expressions from four AML patients before and after therapies

first: Andrew

last: Lane


title: scRNA-seq data for nonunion fracture

city: Beijing

first: Junlin

last: Zhou

state: Chaoyang

address: 8 Gongti South Road

institute: Beijing Chaoyang hospital

summary: This single-cell RNA-seq dataset profiles clinical fracture-healing tissues from patients with fracture nonunion and normally healed fracture controls. Eight human specimens were initially collected, including five fracture nonunion samples and three normally healed control samples. After quality-control and integration assessment, two nonunion samples (NUBF01 and NUBF03) were excluded from the final integrated analysis because of marked batch-related interference; the final analyzed dataset comprised three nonunion samples (NUBF02, NUBF04, NUBF05) and three control samples (G0531, G0601, G0607). Cell-cell communication analysis prioritized fibroblast-to-osteoblast signaling and highlighted an enhanced LIF-(LIFR+IL6ST) interaction in nonunion tissue.

overall_design: Observational human clinical single-cell transcriptomic study comparing fracture nonunion lesions with normally healed fracture tissues. Fresh tissues were collected during surgery, dissociated into single-cell suspensions, and subjected to droplet-based 3-prime single-cell RNA sequencing. Nonunion tissues were collected from bone ends and fibrous tissue within the nonunion gap; control tissues were collected from callus tissue and adjacent fibrous tissue during elective implant removal after clinical union. Downstream analysis compared cell populations, osteoblast-lineage recipient states, and cell-cell communication between nonunion and control groups.

first: Tianchao

last: Lu


title: Joint analysis of multiply perturbed cells improves statistical power and cost efficiency in Perturb-seq screens

city: South San Francisco

first: Jake

last: Yeung

email: yeungj3@gene.com, jakeyeung@gmail.com

address: 1 DNA Way

department: Genentech Research & Early Development

institute: Genentech Inc.

summary: Perturb-seq measures transcriptomic responses to genetic perturbations at scale, but standard low-MOI designs remain resource-intensive. Conventional designs aim to recover one guide RNA per cell and often discard cells carrying multiple guides during analysis. Here, we characterize how overloading cells with guides affects signal recovery, information loss, and cost reduction. Increasing guide RNA dosage eventually induced cellular stress and suppressed cell-cycle progression. Among multiplets, cells carrying two or three guides recovered perturbation responses better than higher-order guide multiplets. In a 5000-gene Perturb-seq screen across MOI levels, high MOI reduced per-cell costs by 81% while keeping information loss within 1.5-fold of a low-MOI baseline. In existing genome-wide Perturb-seq data, incorporating previously discarded multiplets increased usable cell numbers and improved statistical power. Compared with a singlet-only holdout set, adding multiplets moved signal recovery closer to the theoretical reproducibility limit. Together, these results provide an experimental and computational framework for designing cost-efficient Perturb-seq screens at scale.

overall_design: CRISPRi Perturb-seq was performed in DLD-1 cells expressing inducible Zim3-dCas9 at multiple multiplicities of infection (MOI). A pilot experiment tested six MOI levels (0.1, 0.2, 0.5, 1.0, 3.0, 5.0) with a 35-gene library across 4 10x Chromium channels. A scaled experiment used a 5000-gene CRISPRi sub-library across three MOI levels (Low ~0.3, Medium ~3, High ~10) in 44 10x Chromium channels. Cells were treated with doxycycline and Shield-1 ligand for 5 days to induce CRISPRi activity, then multiplexed via MULTI-seq CMO hashing. Three library types (GEX, sgRNA, CMO) were generated per channel and sequenced on Illumina NovaSeq 6000 (pilot) or NovaSeq X Plus (scaled experiment).

first: Jenille

last: Tan

first: Liang

last: Wang

first: Diana

last: Wu

first: Sandra

last: Melo Carlos

first: Jorge

last: Kageyama

first: Jack

last: Kamm

first: Benjamin B

last: Chu

first: Oleg

last: Mayba

first: William F

last: Forrest

first: Shiqi

last: Xie


title: Single-cell transcriptomic analysis of Wfdc2-deficient mouse lungs and response to amiloride treatment

city: Seoul

first: SeJin

last: Kim

address: 50-1, Yonsei-ro

country: South Korea

institute: Yonsei university

summary: This study investigated the transcriptomic consequences of WFDC2 deficiency in mouse lungs and the response to ENaC inhibition. WFDC2 deficiency caused respiratory distress associated with epithelial defects, immune activation, and hyperactivation of the epithelial sodium channel (ENaC). Single-cell RNA sequencing was used to profile lung cells from wild-type, Wfdc2 knockout, Wfdc2 p.C147W knock-in, and amiloride-treated mice. The data reveal altered epithelial cell differentiation and increased immune cell populations in Wfdc2-deficient lungs, while amiloride treatment partially restored epithelial cell states and respiratory phenotypes.

overall_design: Single-cell RNA-seq was performed on mouse lung samples to compare wild-type, Wfdc2 knockout, Wfdc2 p.C147W knock-in, and amiloride-treated conditions. Neonatal lungs were collected at P0.5 to assess the effects of Wfdc2 deficiency, and additional P4 samples were analyzed to evaluate transcriptional responses after amiloride treatment. Samples included WT P0.5, KO P0.5, KI P0.5, amiloride-treated KO P0.5, WT P4, amiloride-treated WT P4, and amiloride-treated KI P4 groups. Single-cell transcriptomes were analyzed to characterize lung cell populations, epithelial differentiation states, immune cell infiltration, and treatment-associated rescue effects.

first: HeonYung

last: Gee


title: sRNA Seq NuRD subunit knockdown

city: Urbana

first: Ruiying

last: Cheng

state: IL

address: B520 Chemical & Life Sciences Laboratory

department: School of Molecular & Cellular Biology

institute: The University of Illinois Urbana-Champaign

type: Non-coding RNA profiling by high throughput sequencing

summary: RNA polymerase III (Pol III) synthesizes noncoding RNAs essential for translation, RNA processing, and transcription regulation. Its activity aligns with cellular growth and nutrient availability, and its dysregulation is linked to diseases like breast cancer and leukodystrophy. Here we test the effect of NuRD subunit MTA3, HDAC1 and HDAC2 knockdown on small RNA expression

overall_design: small RNA seq from HEK293T cell treated with control siRNA, siHDAC1, siHDAC2 and siMTA3

first: Kevin

last: Van Bortle


title: FastGro-seq: POLR3G and POLR3GL knockout and HEK293T cell treated with Pol III inhibitor ML60218

summary: RNA polymerase III (Pol III) synthesizes noncoding RNAs essential for translation, RNA processing, and transcription regulation. Its activity aligns with cellular growth and nutrient availability, and its dysregulation is linked to diseases like breast cancer and leukodystrophy. RNA polymerase III composition is shaped by the mutually exclusive incorporation of two paralogous subunits, RPC7α and RPC7β, encoded by genes POLR3G and POLR3GL in vertebrates. The expression of POLR3G and POLR3GL is spatiotemporally regulated during development, and multiple reports point to RPC7α-enhanced Pol III activity patterns, indicating that Pol III identity may underlie dynamic Pol III transcription patterns observed in higher eukaryotes. In cancer, upregulation of POLR3G, but not POLR3GL, is associated with poor survival outcomes among patients, suggesting differences between RPC7α and RPC7β further influence disease progression and may translate into future biomarkers and therapeutic strategies.

overall_design: Nuclei run-on (nascent RNA) profiling of wild type THP1 monocyte with or without Pol III inhibitor ML-60218 treatment


title: RNA seq(normal depth) RNA Polymerase III inhibition leads to changes in protein coding gene expression

summary: RNA polymerase III (Pol III) produces small noncoding RNAs essential for diverse cellular processes, including translation (tRNA, 5S rRNA, 7SL RNA), RNA processing (U6 snRNA, RPPH1, RMRP), and transcription regulation (7SK snRNA). Here we explore the unexpected effect of Pol III repression on protein coding gene expression.

overall_design: HEK 293T cells are treated with various ASOs or siRNA targeting the Pol III subunit POLR3G and POLR3GL, as well as with overexpression of the Pol III inhibitor MAF1; THP-1 cells are treated with Pol III inhibitor ML-60218, in combination with a protein synthesis inhibitor. Libraries were prepared with the Kapa Hyper Stranded mRNA library kit (Roche) and sequenced on two 10B lanes for 151 cycles from both ends of the fragments on a NovaSeq X Plus with V1.0 sequencing kits.

last: VanBortle


title: N6-methyladenosine circRNA Epitranscriptomic Microarray analysis in peripheral blood of patients with β-thalassemia intermedia and major_10 samples

city: Fuzhou

first: hailong

last: Huang

address: 18 Daoshan Road, Gulou District

department: Medical Genetic Diagnosis and Therapy Center

institute: Fujian Maternity and Child Health Hospital

summary: β-thalassemia is a group of bone marrow disorders characterized by defects in erythropoiesis. Circular RNAs (circRNAs) are functionally significant and hold tremendous potential as diagnostic biomarkers and therapeutic targets in numerous human diseases. Although the functional interplay between N6-methyladenosine (m6A) RNA modifications and circRNAs is becoming increasingly well understood, their specific cross-talk in β-thalassemia remains poorly characterized. Here, we aim to identify the differentially expressed m6A circRNAs in β-thalassemia and elucidate the function of m6A modification and the mechanism by which circRNAs regulate erythropoiesis.

overall_design: Peripheral blood samples were collected from β-thalassemia intermedia and major (n=5) and healthy controls (n=5). RNA was extracted and co-immunoprecipitated with anti-m6A antibody. The modified RNAs were eluted from the immunoprecipitated magnetic beads as the “IP”. The unmodified RNAs were recovered from the supernatant as “Sup”. The “IP” and “Sup” RNAs were treated with RNase R, and then labeled with Cy5 and Cy3, respectively as cRNAs in separate reactions. The cRNAs were combined and hybridized onto the Arraystar Human circRNA Epitranscriptomic Microarray. After washing the slides, the arrays were scanned in two-color channels by an Agilent Scanner G2505C.

first: Hailong

first: Meihuan

last: Chen


title: Long-read sequencing reveals transposable element-derived chimeric transcripts at zygotic genome activation in mammalian embryos

city: Kyoto

first: Shinnosuke

last: Honda

address: Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto-shi

institute: Kyoto University

summary: We performed long-read RNA-seq using mouse late 2-cell and bovine 8-16 cell embryos for constructing transposable element (TE)-driven chimeric mRNA library.

sample_organism:
Bos taurus
Mus musculus

platform_organism:
Bos taurus
Mus musculus

overall_design: In vitro produced mouse and bovine embryos were processed to long-read RNA-Seq. Approximately 200 embryos were used per group.

first: Saki

last: Kawakami

first: Koichi

last: Kitao

first: Shuntaro

last: Ikeda


title: Single cell sequencing data supporting "IL-4/C1q activated astrocyte-derived extracellular vesicles promote stroke infarct recovery by recruiting peripheral leukocytes"

city: Hangzhou

first: Mengying

last: An

state: Zhejiang

address: 西湖区浙大路38号

institute: Zhejiang University

summary: This study aims to engineer an injectable biomaterial platform to promote brain repair post-stroke. We used extracellular vesicles derived from reactive astrocytes and decorated them onto the surface of hydrogel microparticles. Then they were injected into stroke cavity and crosslinked into porous scaffold in situ, which enabled persistent and uniform distribution of astrocyte-derived extracellular vesicles. Extracellular vesicles from reactive astrocytes activated with different cytokines showed different brain regeneration outcomes. IL-4/C1q activated astrocyte extracellular vesicles promoted robust angiogenesis within the stroke infarct and improved functional recovery significantly. Single-cell RNA sequencing was performed to characterize cellular and transcriptional changes in the post-stroke brain following implantation of extracellular vesicle-functionalized hydrogel microparticles. Briefly, ischemic stroke was induced in mice, and different hydrogel microsphere formulations were implanted into the infarct cavity at 5 days post-stroke. Ipsilateral hemispheres were collected at day 14 and dissociated into single-cell suspensions for transcriptomic profiling. The dataset was used to evaluate treatment-associated differences in cell composition, immune infiltration, vascular remodeling, and regenerative responses in the injured brain. From these data, we showed that IL-4/C1q extracellular vesicle group recruited reparative macrophages and neutrophils to promote brain repair post-stroke.

overall_design: Mice subjected to ischemic stroke received implantation of different reactive astrocyte derived extracellular vesicle-functionalized hydrogel microparticles into the infarct cavity at 5 days post-stroke. Ipsilateral brain hemispheres were harvested at day 14 after stroke and processed for single-cell RNA sequencing. Comparative transcriptomic analysis was performed to evaluate treatment-associated changes in cellular heterogeneity, immune cell recruitment, vascular remodeling, and tissue repair within the post-stroke microenvironment.

first: Shangjing

last: Xin

first: Lucy

last: Zhang

first: Nhi

middle: V

last: Phan

first: Ligen

last: Shi

first: S. Thomas

last: Carmichael

first: Tatiana

last: Segura


title: miRNA profiling data supporting “IL-4/C1q activated astrocyte-derived extracellular vesicles promote stroke infarct recovery by recruiting peripheral leukocytes”

summary: This study aims to engineer an injectable biomaterial platform to promote brain repair post-stroke. We used extracellular vesicles derived from reactive astrocytes and decorated them onto the surface of hydrogel microparticles. Then they were injected into stroke cavity and crosslinked into porous scaffold in situ, which enabled persistent and uniform distribution of astrocyte-derived extracellular vesicles. Extracellular vesicles from reactive astrocytes activated with different cytokines showed different brain regeneration outcomes. IL-4/C1q activated astrocyte extracellular vesicles promoted robust angiogenesis within the stroke infarct and improved functional recovery significantly. These miRNA profiling data shows the different cargos of extracellular vesicles derived from untreated, IL-1α/TNFα/C1q, IL-4/C1q. We showed IL-4/C1q extracellular vesicles possessed adhesive miRNAs that recruited pro-reparative leukocytes and resulted in improved regeneration.

overall_design: Primary astrocytes were isolated from neonatal rats and cultured based on established protocol. On Day 6, they were activated by IL-1α/TNF-α/C1q or IL-4/C1q, and there was an untreated control. After 24 h of treatment, extracellular vesicles were isolated from conditioned medium. Total RNA enriched for small RNAs was extracted from purified extracellular vesicles and subjected to miRNA sequencing. Comparative analysis was performed to identify differentially enriched miRNAs among treatment groups.


title: Single-cell tracking reveals tumor-reactive T cell plasticity during melanoma TIL therapy

city: Basel

first: Michael

middle: Thomas

last: Sandholzer

address: Hebelstrasse 20

department: Department for Biomedicine

country: Switzerland

institute: University Hospital Basel

summary: Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) can induce durable responses in metastatic melanoma, yet the clonal and transcriptional dynamics that govern tumor-reactive T cell fate during ex vivo expansion and after transfer remain poorly understood. Here, we performed longitudinal single-cell RNA and T cell receptor (TCR) sequencing across five timepoints, from baseline tumors through a two-phase ex vivo expansion to post-infusion peripheral blood and tumor biopsies, in seven melanoma patients, generating a high-resolution map of both CD8+ and CD4+ tumor-reactive T cell plasticity. During early expansion (preREP), tumor-reactive CD8+ T cells were reinvigorated from an exhausted state and acquired distinct HLA-II-high or KLF2-high transcriptional profiles. In-depth characterization of the CD4+ compartment revealed lineage-dependent reinvigoration. After transfer, both CD8+ and CD4+ tumor-reactive clones acquired stem-like profiles and tissue-homing markers before re-infiltrating tumor lesions. Mechanistically, we identify two processes linked to treatment failure in non-responders: de novo expansion of immunosuppressive regulatory T cells, and co-transfer of Type 17 T cells. These data provide a comprehensive longitudinal framework of T cell plasticity during TIL-ACT.

overall_design:
Longitudinal 10x Genomics single-cell 5-prime gene expression and TCR (V(D)J) sequencing of FACS-sorted CD3+ T cells collected at five timepoints across TIL therapy: baseline tumor (pre-ACT), intermediate product (preREP), final TIL product (REP), peripheral blood 7 days post-transfer (PBMC 7dpt), and post-treatment tumor lesions (post-ACT), from seven metastatic melanoma patients in the phase I BaseTIL trial (NCT04165967), plus one control PBMC donor (UPN000). 38 libraries (GEM wells) in total; each library yielded paired 5-prime GEX and TCR libraries.
Raw FASTQ are deposited under controlled access at EGA (EGAP50000000107) for patient-privacy protection; this GEO record provides processed data only.

10x Genomics Chromium Single Cell V(D)J v2 (5-prime); paired 5-prime gene expression (GEX) and TCR V(D)J libraries from the same GEM well.

***************************************************************
Raw files for human/patient samples are being made available in EGA (EGAP50000000107; https://www.ebi.ac.uk/ega/) for controlled access to the personally identifiable sequence data.
***************************************************************

middle: T

first: Clara

last: Serger

first: Alessia

middle: G

last: Liner

first: Sarp

last: Uzun

first: David

last: Koenig

first: Helen

last: Thut

first: Reto

last: Ritschard

first: Jonas

middle: D

last: Fuerst

first: Andreas

last: Zingg

first: Natalia

last: Rodrigues Mantuano

first: Benjamin

last: Kasenda

first: Katharina

last: Glatz

first: Elisabeth

last: Kappos

first: Matthias

last: Matter

last: Holbro

first: Frank

last: Stenner

first: Jakob

last: Passweg

first: Nina

last: Khanna

first: Lukas

last: Jeker

first: Mascha

last: Binder

first: Alfred

last: Zippelius

first: Heinz

last: Laeubli


title: Genome-wide DNA methylation profiling identifies ionizing radiation-induced hypomethylated genes with clinical significance in oral squamous cell carcinoma

city: Cheonan

first: Jihye

last: Park

address: 119 Dan-daero, Dongnam-gu

institute: Dankook University

summary:
Background: The epigenetic consequences of ionizing radiation (IR), particularly its effects on DNA methylation, remain poorly understood. Here, we investigated whether IR induces genome-wide DNA methylation changes in OSCC cells and examined whether IR-induced hypomethylated candidate genes correspond to epigenetically silenced loci in oral cancer patient tumors, and whether their methylation status influences clinical outcomes.
Methods: OSCC cells were treated with IR or 5-aza-2'-deoxycytidine (5-aza-dC) and subjected to genome-wide DNA methylation profiling using the Illumina HumanMethylationEPIC BeadChip. Candidate hypomethylated genes were validated by qRT-PCR and quantitative methylation-specific PCR (qMSP). DNMT1 promoter occupancy was assessed by chromatin immunoprecipitation (ChIP). Clinical relevance was evaluated using the TCGA-HNSCC dataset.
Results: IR significantly reduced DNMT1 and DNMT3b protein levels in OSCC cells, indicating IR-induced global DNA hypomethylation. Genome-wide profiling identified three candidate genes, MLPH, BMP4, and MYO5A, showing concordant promoter demethylation and transcriptional upregulation following IR treatment, with ChIP analysis confirming reduced DNMT1 occupancy at their promoters. Importantly, TCGA-HNSCC analysis revealed that BMP4 and MLPH are hypermethylated and transcriptionally silenced in untreated HNSCC tumors, a finding mechanistically complementary to our experimental data, suggesting that IR reverses tumor-associated epigenetic silencing through DNMT1-mediated passive demethylation. Consistently, BMP4 hypermethylation was associated with poor overall survival, and BMP4-hypermethylated patients receiving radiotherapy survived longer than those receiving chemotherapy.
Conclusions: This study provides the first evidence that IR induces genome-wide DNA hypomethylation in OSCC through DNMT1 and DNMT3b downregulation, reactivating epigenetically silenced genes including MLPH, BMP4, and MYO5A. These findings suggest that IR-induced epigenetic reactivation of tumor-silenced genes may represent a clinically meaningful mechanism of radiotherapy response, and that BMP4 methylation status may serve as a potential predictive biomarker in oral cancer.

overall_design: The objective of this study was to identify genome-wide DNA methylation changes induced by ionizing radiation (IR) in oral squamous cell carcinoma (OSCC) cells. Genomic DNA was isolated from irradiated and non-irradiated control OSCC cells and analyzed using the Illumina HumanMethylationEPIC BeadChip. Biological replicates were included for each experimental condition. Untreated OSCC cells served as reference controls for comparison with irradiated cells. Differential DNA methylation analysis was performed to identify CpG sites and genes exhibiting IR-associated methylation changes across the genome.

first: Yu Kyeong

last: Han

first: Bomin

last: Seol

first: Hye Su

first: Ha Young

first: So Young

last: Jung

first: Ji Won

last: Hyun

first: Hae Ryoun

first: Keunsoo

last: Kang

first: Joo Mi

last: Yi


title: Generation of spermatogonia from human and non-human primate pluripotent stem cells

city: Bethesda

first: Astrid

last: Haase

state: MD

address: 50 South Dr

department: LCDB

institute: NIDDK

summary: Failures in germline development drive male infertility, but the lack of model systems that recapitulate human spermatogenesis hampers therapeutic development. Here, we develop a system to differentiate human induced pluripotent stem cells (iPSCs) into primordial germ cell-like cells that self-organize with mouse fetal testicular cells into seminiferous tubule–like structures within xenogeneic reconstituted testis (xrTestis). Subsequent transplantation of xrTestes into immunodeficient mice results in efficient generation of male germ cells up to meiotic onset, including spermatogonia with evidence of prepachytene piRNA biogenesis, as well as differentiating spermatogonia and rare preleptotene spermatocytes exhibiting transcriptomic and phenotypic similarity to their in vivo counterparts. As future clinical applications will require testing in non-human primates, we apply a similar strategy to differentiate rhesus iPSCs through fetal germ cell stages into spermatogonia and differentiating spermatogonia. Together, these platforms provide a foundation for studying primate germ cell lineages and represent a step toward in vitro gametogenesis.

overall_design: RNA-seq and small RNA-seq from induced Pluripotent Stem Cells (iPSC), primordial germ cell-like cells (PGCLC) and xenogeneic reconstituted testes (xrTestes), and FACS-sorted VT⁺ germ cells isolated from xrTestes (VT+ xrTestes).

first: Eoin

middle: C

last: Whelan

first: Mingyue

last: Guo

first: Ryo

last: Yokomizo

first: Yasunari

last: Seita

first: Young Sun

last: Hwang

first: Yuichiro

last: Shirafuta

first: N

last: Leu

first: Keren

last: Cheng

first: Franziska

last: Ahrend

first: Kotaro

last: Sasaki


title: Mapping vascular plasticity in liver fibrogenesis identifies novel fibrosis-associated endothelial cells in early-stage liver disease

city: Manchester

first: Karen

last: Piper Hanley

address: Oxford Road

country: United Kingdom

institute: University of Manchester

summary: Vascular plasticity is a crucial biological asset enabling our bodies to rapidly adapt to infections and acute inflammation. However, repeated insult during chronic disease can result in these vascular adaptations becoming irreversible, thereby driving disease progression and fibrosis. This study aimed to understand if phenotypic changes in endothelial cell (EC) identity could be indicative of progressive fibrosis and thereby offer new diagnostic and therapeutic opportunities for patients with metabolic dysfunction-associated steatotic liver disease (MASLD).

overall_design: Samples are clinical liver biopsies taken from patients at with MASLD (Metabolic Dysfunction-Associated Steatotic Liver Disease)

first: Elliot

last: Jokl

first: Neil

last: Dufton


title: Human metapneumovirus and mumps virus alter the m6A landscapes in THP-1-derived immature dendritic cells

first: Mijia

last: Lu

address: 180 Tianmushan Rd

institute: Hangzhou Medical College

summary: N6-methyladenosine (m6A) is a widespread mRNA modification that regulates RNA metabolism and influences virus-induced immune responses. We performed MeRIP-seq to profile m6A landscapes in THP-1-derived immature dendritic cells (THP-iDCs) before and after infection with Human metapneumovirus (hMPV, NL/00/1) or mumps virus (MuV, JL2). Integration of m6A modifications on key innate immune transcripts with transcriptomic data, together with m6A levels on cellular and viral RNA, revealed a dual role of m6A in viral replication and host immune regulation. These datasets provide a resource for exploring epitranscriptomic regulation during hMPV and MuV infection.

overall_design: THP1-iDC was infected with rhMPV or rMuV, with the control of DMEM (n=2) for 16 hours.

first: Xueping

last: Zeng

first: Yifei

last: Zhang

first: Shixuan

last: Wang


title: Epigenetic landscape, key transcriptional regulators, and in vivo identification of human Tr1 cells [ATAC-seq]

city: Stanford

first: Laura

last: Amaya

address: 269 Campus Drive

institute: Stanford University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Type 1 regulatory T (Tr1) cells are CD4+ T cells with immunoregulatory function, which are induced from mature peripheral CD4+ T cells in response to persistent extra-thymic antigens. The role of human Tr1 cells in disease is not completely understood, because transcription factors that regulate their antigen-driven differentiation are not known, and there is lack of consensus on how to best identify them in the tissue, where antigen interactions occur. Here, we leveraged comprehensive bulk and single-cell (sc) transcriptomic, epigenomic, and TCR repertoire profiling of human antigen-induced Tr1 cells, complemented with CRISPR-based functional genomics, to uncover the role of transcription factors IRF4, BATF and MAF in human Tr1 cell differentiation and function. In addition, we identify a Tr1 cell transcriptional signature that can reveal Tr1-like cells in sc-RNA-seq datasets of peripheral blood CD4+ T cells of patients treated with Tr1 therapy, and within tumor-resident CD4+ T cells in patients with clear-cell renal cell carcinoma, triple-negative breast cancer, and colorectal cancer. These data enable further development of Tr1 therapies and Tr1-targeting treatments, and provide new insights into human Tr1 cell biology.

overall_design: T-allo10 and control T-allo cells were generated from total CD4+ T cell / dendritic cell donor pairs generated from 5 healthy donors. Tr1 and non-Tr1 cells were isolated from T-allo10 cell products, and Teff isolated from T-allo cell products at day 10 of culture by FACS.

first: AlmaMartina

last: Cepika

first: Colin

last: Waichler


title: Epigenetic landscape, key transcriptional regulators, and in vivo identification of human Tr1 cells

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

overall_design: Refer to individual Series


title: m6A modification landscapes of human metapneumovirus, mumps virus, and host A549 cell RNAs revealed by single-base-resolution GLORI-seq [A549 GLORI]

summary: N6-methyladenosine (m6A) is a common modification on mRNA. Multiple viruses exploit host m6A machinery to modify their own genomic RNA and mRNA, thereby facilitating viral replication and evasion of host innate immune surveillance. In this study, we employed the single-base-resolution high-throughput sequencing technique glyoxal and nitrite-mediated deamination of unmethylated adenosines (GLORI) to identify m6A modification sites on the RNAs of Human metapneumovirus (hMPV, NL/1/00 strain) and Mumps virus (MuV, JL2 strain), as well as to characterize the m6A landscapes of the host cell transcriptome in A549 lung epithelial cells. Extensive m6A modifications on viral RNAs exhibited dynamic and gene-specific distributions, while viral infection significantly reprogrammed the m6A landscape of A549 cells, including increased m6A modification in multiple innate immune signaling pathways. Furthermore, through integrated transcriptomic analyses and modulation of host m6A machinery levels, we demonstrated the dual regulatory roles of m6A in both viral replication and host immune responses.

overall_design: A549 cells were infected with rhMPV or rMuV, with the control of DMEM (n=2) for 20 hours.

first: Tongyu

last: Ping

first: Wanqing


title: m6A modification landscapes of human metapneumovirus, mumps virus, and host A549 cell RNAs revealed by single-base-resolution GLORI-seq [A549 mRNA]

summary: We infected human lung adenocarcinoma A549 cells with multiple nonsegmented negative-sense (NNS) RNA viruses, including Human metapneumovirus (hMPV), Mumps virus (MuV), Vesicular stomatitis virus (VSV), and Sendai virus (SeV), and evaluated the differences among these viruses in activating innate immune signaling pathways in epithelial cells through transcriptomic analyses of infected cells. Furthermore, by integrating the effects of viral infection on the m6A landscape of A549 cell transcripts, we analyzed the correlation between m6A epitranscriptomic regulation and the activation of innate immunity–related transcripts, as well as the role of m6A in viral immune evasion.

overall_design: A549 cells were infected by the individual virus, with the control of DMEM (n=3) for 20 hours. Total RNA samples were then collected for RNA-seq and transcriptomic analysis.


title: Epigenetic landscape, key transcriptional regulators, and in vivo identification of human Tr1 cells [RNA-Seq]


title: miR-155-driven dysregulation of the PPARγ/PGC-1α/ERRα-Drp1 axis contributes to mitochondrial damage in hypoxic/ischemic neuronal cells

city: Shijiazhuang

first: Ya

last: Wen

state: Hebei

address: No. 215, Heping West Road, Xinhua District

department: Neurology

institute: The Second Hospital of Hebei Medical University

summary: Background: Mitochondrial dysfunction is closely related to ischemic brain injury. Increased expression of dynamin-related protein 1 (Drp1) in neuronal cells plays a crucial role in ischemia/hypoxia-induced mitochondrial damage, and dysregulation of miR-155 expression is implicated in cerebral ischemic injury. However, the mechanistic link between miR-155 dysregulation and Drp1-mediated mitochondrial damage in ischemic/hypoxic neuronal cells remains largely elusive. Methods: Oxygen-glucose deprivation (OGD)-treated Neuro-2a cells were employed to investigate the effects of hypoxia/ischemia on miR-155 expression. The cells transfected with miR-155 mimic and miR-155 inhibitor were used to clarify the the role of miR-155 in OGD-induced mitochondrial damage. The expression levels of PGC-1α, PPARα, PPARβ/δ, PPARγ, ERRα, and Drp1 were assessed using qRT-PCR, Western blotting, and immunofluorescence staining, and their interactions were identified by confocal colocalization and co-immunoprecipitation experiments. Mitochondrial functions were evaluated by measuring ATP content, ROS, MMP, and mPTP opening. Chromatin immunoprecipitation (ChIP) assay was performed to detect the binding of ERRα to the Drp1 promoter. Results: miR-155 expression in OGD-treated neuronal cells exhibited a significant upregulation in a time-dependent manner. Elevated miR-155 induced excessive mitochondrial fission and dysfunction, as evidenced by a decreased ATP content, increased ROS generation, depolarized MMP, and mPTP opening, which is accompanied by a markedly reduced expression of PPAR family members, particularly the PPARγ. Mechanistically, miR-155 weakened the interaction between PGC-1α and PPARγ by suppressing their expressions and thus downregulated ERRα expression, which prevents the binding of ERRα to the Drp1 promoter, thereby relieving the repression of the Drp1 promoter by ERRα. These findings suggest that PGC-1α and ERRα negatively regulate Drp1 expression in neuronal cells. Notably, inhibition of miR-155 could ameliorate mitochondrial dysfunction by improving the OGD-induced dysregulation of PPARγ/PGC-1α/ERRα-Drp1 axis in Neuro-2a cells, providing new insights into the mechanisms of ischemia/hypoxia-induced mitochondrial dysfunction.  Conclusion:  Our findings reveal a novel mechanism by which miR-155 contributes to mitochondrial damage by driving the ischemia/hypoxia-induced dysregulation of PPARγ/PGC-1α/ERRα-Drp1 axis in neuronal cells, identifying potential new therapeutic targets for the treatment of post-ischemic mitochondrial damage.

overall_design: Gene expression analysis of RNA sequencing data from mouse neuroblastoma cells transfected with NC, miR-155 mimic, and miR-155 inhibitor

first: Xiaotong

last: Yang

first: Haitao

first: Jiejie

first: Wenjie

last: Gao

first: Yi

last: Qiao

last: Liu

first: Ning

first: Hong

last: Jiang

first: Shan


title: Neurexin and neuroligin loss drives system-wide transcriptional reprogramming in Caenorhabditis elegans

city: Ondo

first: Omamuyovwi

middle: Meashack

last: Ijomone

state: Ondo

address: Medical Village, Laje Road

department: Anatomy

country: Nigeria

institute: University of Medical Sciences

summary: Neurexins and neuroligins are cell adhesion molecules at synapses implicated in neurodevelopmental disorders, but their roles beyond synaptic function remain unclear. Here, we show that loss of neurexin (nrx-1) or neuroligin (nlg-1) in Caenorhabditis elegans drives system-wide transcriptional reprogramming. Mutants exhibited impaired growth, altered locomotion, increased repetitive behavior, increased social aggregation, and reduced ventral nerve cord neuronal integrity. Transcriptome profiling revealed coordinated downregulation of genes involved in structural development, synaptic signaling, and protein homeostasis, alongside upregulation of stress-response, immune, and metabolic pathways. These changes define a shift from developmental and synaptic programs toward stress-adaptive states, accompanied by signatures of mitochondrial remodeling. Notably, nrx-1 mutants displayed more pronounced effects. Together, our findings demonstrate that synaptic adhesion loss drives organism-wide transcriptional reprogramming linking neuronal dysfunction to systemic stress adaptation

overall_design: RNA-seq profiling of both wildtype Caenorhabditis elegans and those with nrx-1 and nlg-1 loss-of-function mutation

first: Victor

last: Anadu

first: Toheeb

middle: O

last: Oyerinde

first: Olayemi

middle: K

first: David

last: Oyeniran

first: Michael

last: Aschner


title: CRISPR/Cas9-MEDIATED PHOX2B FUNCTIONAL KNOCK-OUT IN IMR32 NEUROBLASTOMA CELLS IMPAIRS NEURONAL EXCITABILITY THROUGH DYSREGULATION OF ION CHANNELS GENES

city: Segrate

first: Simona

last: Di Lascio

address: Via Fratelli Cervi 93

country: Italy

institute: University of Milan

summary:
Introduction: PHOX2B is a master transcriptional regulator of autonomic nervous system development whose mutations cause neurocristopathies including neuroblastoma and congenital central hypoventilation syndrome (CCHS). However, the identification and functional relevance of PHOX2B target genes remain incompletely defined, partly due to the lack of robust cellular models.
Methods: Through CRISPR/Cas9-engineering we established a PHOX2B knockout IMR32 neuroblastoma model, and combined transcriptomic, chromatin-binding, electrophysiological, and live-cell imaging analysis to investigate PHOX2B-dependent pathways. We then assessed the developmental relevance of the obtained results in iPSC‑derived sympathetic neurons from CCHS patients carrying different PHOX2B mutations.
Results: PHOX2B-knockout (PHOX2B-KO) cells exhibited morphological and molecular features indicative of enhanced neuronal maturation. RNA-seq and ChIP-seq analyses highlighted an unexpected direct role for PHOX2B in regulating the expression of ion channels, including KCNQ5, SCN3A and RYR2, primarily through transcriptional repression. Functionally, PHOX2B KO cells displayed significant alterations in intrinsic electrical properties, including depolarized resting membrane potential, reduced action potential amplitude, attenuated Ca2+ responses and profoundly altered K+ efflux dynamics. Functional rescue experiments confirmed that PHOX2B re-expression restores both transcriptional and electrophysiological phenotypes, thereby establishing a causal relationship between PHOX2B expression and the maintenance of intrinsic excitability homeostasis. Extending these findings to development, sympathetic neurons differentiated from CCHS patient-derived iPSCs displayed dysregulated maturation, transcriptional network rewiring, and altered expression of the same ion channel genes, supporting their relevance to disease.
Discussion: Together, our data establish the PHOX2B knockout neuroblastoma model as a valuable platform for functional genomics, enabling systematic analysis of PHOX2B targets and highlightening its critical role in coordinating transcriptional programs that shape neuronal excitability and maturation.

overall_design: RNA was isolated from IMR32 cells (IMR32_CTRL), a PHOX2B knockout IMR32 cell clone (IMR32_KO), two stable PHOX2B re-expressing lines (IMR32_KO_rescue_1 and IMR32_KO_rescue_2), and a IMR32 transfected with a scramble gRNA (IMR32_scramble)

first: Erika

last: Donà

first: Diego

last: Fornasari

first: Roberta

last: Benfante


title: Telomere variant sequences encode a genetic blueprint for allele-specific telomere length (dataset 2)

city: Singapore

first: XIAORAN

last: CHAI

address: 8 College Rd, Singapore 169857

country: Singapore

institute: Duke-NUS Medical School

summary: The ends of human chromosomes are capped by specialized nucleoprotein structures known as telomeres, which are essential for genome stability. Recent advances in long-read sequencing have enabled allele-specific telomere length measurements at nucleotide resolution, uncovering extreme heterogeneity in telomere length between alleles that is determined at birth and gradually shortens with age. The shortening over time makes steady-state telomere length in human somatic cells a promising biomarker for age-associated diseases. However, the mechanisms underlying allele-specific telomere maintenance and its stability remain poorly understood. Here, we developed a high-resolution workflow combining PacBio and Nanopore long-read sequencing platforms to map allele-specific telomere length in human blood samples as well as cultured cell lines. By tracing allele-specific telomeric sequence in family members across multiple generations, we show that the allele-specific telomeric variant sequences (TVSs) are heritable (with mean similarity score > 0.95) and underlie the extreme heterogeneity of telomere length between alleles. Continuous cell proliferation likely drives the slow but stochastic evolution of allele-specific TVSs distribution, resulting in asymmetry in telomere inheritance from father and mother (p-value = 2.354e-7). Targeted deletion of allele-specific TVSs using CRISPR-Cas9 resets telomere length, further confirming their causal role in the control of allele-specific telomere maintenance (p-value < 2e-6). These results indicated that TVSs encode a novel class of heritable genetic elements underlying the allele-specific telomere length.

overall_design: telomere sequencing of human cell line 293T, HeLa, IGROV-1, IMR90, Raji, RKO, SJSA-1, and WI38

first: Xiaoran

last: Chai

first: Shang

last: Li


title: Canonical and isomiR products of miR-142 enhance dendritic cell reprogramming [ATAC-seq]

city: Lund

first: Ilia

last: Kurochkin

state: Skane

address: BMC A12, Sölvegatan 17

country: Sweden

institute: Lund University

summary: MicroRNAs (miRNAs) regulate gene expression and generate isoforms (isomiRs) with distinct targeting properties, but their roles in cellular specification and reprogramming remain poorly understood. Here, we investigated miRNAs and isomiRs during direct reprogramming to conventional type 1 dendritic cells (cDC1s), identifying miR-124 and miR-142 as facilitators of the process. The canonical miR-124-3p strand induced a permissive chromatin landscape enriched for cooperative transcription factor motifs, thereby transiently increasing reprogramming efficiency. In contrast, miR-142 enhanced lineage fidelity by repressing fibroblast identity and activating cDC1-specific programs through cooperating isomiRs that drove XCR1 expression and increased type III interferon production. In addition, miR-142 mimics enhanced cancer cell reprogramming and anti-tumor immunity in vivo. Finally, we developed RNA-based cDC1 reprogramming using transcription factor mRNAs combined with miRNA mimics. Together, these findings uncover miRNA isoform diversity as a programmable regulatory layer in immune cell specification and establish RNA-driven reprogramming as a non-viral strategy for cancer immunotherapy.

overall_design: Human derman fibroblasts transduced with lentiviral vectors coding for PU.1, IRF8 and BATF3 (PIB) and for miR-124, miR-142 or a scrambled control hairpin, were sorted for reprogramming markers CD45 and HLA-DR on  days 3, 6 and 9 and processed according to the procedure described by Buenrostro et al. (2015). Empty vector-transduced cells and human peripheral blood dendritic cells were included as controls.

first: Nejc

last: Arh

first: Beatriz Lourenço

last: Vaz

first: Carlos-Filipe

last: Pereira


title: Canonical and isomiR products of miR-142 enhance dendritic cell reprogramming [lenti_hairpins]

overall_design: Human derman fibroblasts transduced with lentiviral vectors coding for PU.1, IRF8 and BATF3 (PIB) and for miR-124, miR-142 or a scrambled control hairpin, were sorted for reprogramming markers CD45 and HLA-DR on  days 3, 6 and 9 and processed using TRIzol according to the manufacturer's instructions. Empty vector-transduced cells and human peripheral blood dendritic cells were included as controls.


title: Neutrophils Orchestrate Osteoclastogenesis in Orthodontic Tooth Movement

city: Sendai

first: Fumitoshi

last: Ohori

address: 4-1 Seiryo-machi, Aoba-ku

department: Graduate School of Dentistry

institute: Tohoku University

summary: To elucidate the biological mechanisms of alveolar bone remodeling during orthodontic tooth movement (OTM), we performed single-cell RNA sequencing (scRNA-seq) on periodontal ligament (PDL) tissues from a mouse OTM model. We identified 11 distinct cell clusters and revealed that neutrophils are a primary source of pro-inflammatory cytokines such as TNF, IL-1β, and OSM. Our analysis identified a specific “Inflammatory-Neu” subset that expands during OTM and coordinates macrophage recruitment. This study demonstrates that neutrophils act as indispensable upstream regulators that drive osteoclastogenesis during mechanical loading.

overall_design: Periodontal ligament  was collected from 8-week-old C57BL/6J male mice. Two groups were compared: (1) OTM group (maxillary left first molar subjected to 10-g mesial force for 6 days, n=5 pooled) and (2) Control group (unloaded right side, n=5 pooled). Single-cell libraries were prepared using the 10x Genomics Chromium platform.

first: Fumitohsi

first: Hideki

last: Kitaura

first: Kohei

last: Narita

first: Aseel

last: Marahleh

first: Jinghan

last: Ma

first: Kayoko

last: Kanou

first: Ziqiu

last: Fan

first: Angyi

last: Lin

first: Kou

last: Murakami

first: Hiroyasu

last: Kanetaka


title: Canonical and isomiR products of miR-142 enhance dendritic cell reprogramming [bulk RNA-seq]

overall_design: PIB lentivirus-transduced cells and PIB mRNA-transfected cells with scrambled miRNAs or other miRNA mimics were sorted for CD45 on days 3 and 6 of reprogramming directly into RLT lysis buffer and profiled by bulk total RNA sequencing. Cells transfected with a GFP mRNA and the scrambled miRNA mimics (sorted for the live cell gate) were included as controls.


title: Canonical and isomiR products of miR-142 enhance dendritic cell reprogramming [smRNA]

overall_design: PIB and miRNA expressing lentivirus-transduced HDFs were sorted for CD45 on day 9 of reprogramming directly into RLT lysis buffer and profiled small RNA sequencing. Non-reprogrammed HDFs and freshly isolated human peripheral blood dendritic cells cDC1s (HLA-DR+ CD11c+ CD141+), cDC2s (HLA-DR+ CD11c+ CD1c+ CD141-), and pDCs (HLA-DR+ CD11c- CD123+) were included as controls.


title: TOP1MT rs2293925 is a regulatory SNP with enhancer function that shapes mitochondrial R-Loop dynamics

city: Debrecen

first: Zsolt

last: Karanyi

address: Nagyerdei krt.98

department: Internal Med.Dept.

country: Hungary

institute: University of Debrecen

summary: Mitochondrial topoisomerase 1 (TOP1MT) regulates mitochondrial DNA (mtDNA) topology during transcription and replication. Perturbed mtDNA maintenance and RNA metabolism are implicated in neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), which is characterized by mitochondrial impairment. Here we show that the common TOP1MT variant rs2293925 (R525W) has enhancer-like activity and associates with increased mitochondrial R-loops (RNA:DNA hybrids) in ALS-relevant contexts. Tissue-dependent expression quantitative trait locus analysis, chromatin state annotation, reporter assays, and allele-specific DNA–protein binding assays support a transcriptional regulatory role for rs2293925. In isogenic cell models, the rs2293925-linked allele increased TOP1MT mRNA and protein abundance and was accompanied by increased mitochondrial R-loop signal. Enhanced mitochondrial R-loop formation was also observed in a pilot cohort of sporadic ALS samples carrying rs2293925 and in a neural lineage model using neural stem cells derived from C9orf72-positive ALS patients. TOP1MT trapping with lamellarin D supported increased TOP1MT-R525W occupancy at mitochondrial control-region sites together with increased R-loop signal, consistent with altered TOP1MT–mtDNA interaction dynamics. These data support a dual-effect model in which rs2293925 increases TOP1MT expression and associates with altered TOP1MT binding and mitochondrial RNA:DNA hybrid homeostasis in ALS-relevant contexts, linking common genetic variation to R-loop–associated mitochondrial stress and disease susceptibility.

overall_design: iPSC-derived NSCs were generated from healthy controls and C9orf72-ALS patients. Neural induction (7 days; PSC Neural Induction Medium), NSC expansion, and downstream characterization (Nestin staining and TOP1MT genotype determination).  Mitochondrial DRIP-seq analysis was perfomed on gDNA isolated from the above samples.

first: Lóránt

last: Székvölgyi

first: Dóra

last: Varga

last: Ráduly

first: Beáta

last: Boros-Oláh

first: Éva

last: Nagy

last: Karányi

first: László

last: Halász

first: Bálint L

last: Bálint

first: György

last: Vámosi

first: Emese

last: Tóth

first: Péter

last: Bai

first: Serena

last: Santangelo

first: Patrizia

last: Bossolasco

first: Antonia

last: Ratti

first: Vincent

last: Géli

first: Yves

last: Pommier


title: Canonical and isomiR products of miR-142 enhance dendritic cell reprogramming [Cut&Tag]

first: Ilia

last: Kurochkin

overall_design: PIB lentivirus- and pri-miRNA-transduced cells were sorted for CD45 on day 3 directly into FBS, frozen and profiled by Cut&Tag against H3K27me3. Non-reprogrammed HDFs  were included as controls.

first: Nejc

last: Arh

first: Carlos-Filipe

last: Pereira


title: Omega-3 Fatty Acids Mitigate Neuropathic Pain by Modulating Ferroptosis, Glutamate and Lipid Remodeling

city: Loma Linda

first: Hoang

middle: Viet

last: Dinh

address: 11085 CAMPUS STREET, MTH-105

department: Center for Health Disparities

institute: Loma Linda university

summary: Neuropathic pain (NP) is a debilitating condition resulting from nerve injury and is a common co-morbidity of type 2 diabetes. Dietary omega-3 fatty acids (-3) have been shown to alleviate hyperalgesia and NP, likely by modulating key pathological mechanisms, including neuroinflammation, oxidative stress, mitochondrial dysfunction, and ion channel dysregulation. Understanding how -3 regulate NP could help identify molecular targets for therapy. This study aimed to elucidate the primary cellular pathways by which dietary -3 attenuate NP. We analyzed multi-omics data from both preclinical rodent models of chronic constriction injury (CCI) and human cohorts with painful diabetic neuropathy, all exposed to -3 rich diets. In the preclinical study, 40 rats were fed diets enriched with either fish oil (-3) or soy oil (control) for four weeks prior to sciatic nerve CCI. Pain behaviors were assessed using the Hargreaves and CatWalk tests, and dorsal root ganglia tissue was harvested for RNA sequencing. Quantitative Real-Time PCR was used to confirm expression of promising RNA-seq transcripts. The clinical study enrolled 40 patients with type 2 diabetes and neuropathy who received -3 supplementation for three months. Plasma samples were analyzed via metabolomics and lipidomics. In rats, -3 significantly reduced hyperalgesia and allodynia and improved locomotion by day 7 post-CCI (p < 0.05).  RNA-seq data revealed downregulation of pain-associated genes including Scn10a (log₂FC = -1.3, p = 0.006), Trpa1(log₂FC = -1.4, p < 0.001), and P2rx7 (log₂FC = -0.73, p = 0.056), alongside modulation of the ferroptosis pathway gene Gpx4 (log₂FC = 1.1, p = 0.01). Markers of satellite glial cells, such as Gfap (log₂FC = 1.64, p = 0.0015) and Glul(log₂FC = 0.90, p = 0.023), were upregulated, suggesting enhanced neuronal repair and glutamate regulation. Genes involved in lipid metabolism, including Fabp7 (log₂FC = 0.39) and Fasn (log₂FC = -0.45), also showed significant shifts. Activation of selenoamino acid metabolism in rats corresponded with metabolomics analysis results in human subjects, indicating enhanced antioxidant capacity and potential inhibition of ferroptosis. Lipidomics in the human cohort demonstrated increased triglyceride catabolism (DG→MG, z = 2.03, PNPLA2) and a reduction in peroxidation-prone lipids (DG[40:6] →PC[40:6], z = 6.36, CHPT1). Together, these findings indicate that -3 reduce NP through modulation of ferroptosis, glutamate homeostasis, lipid peroxidation, and glial function

overall_design: Female Sprague-Dawley rats were randomized into control or fish oil-enriched diet groups for 4 weeks, followed by chronic constriction injury (CCI) or sham surgery, with diets continued for 4 additional weeks. Dorsal root ganglia were collected for RNAseq analysis to study gene expression changes.

first: Viet Hoang

first: Magda

last: Descorbeth

first: Francis

last: Zamora

first: Alfonso

last: Duran

first: Jo-Wen

last: Liu

first: Johnny

last: Figueroa

first: Marino

last: DeLeon

first: Cono

last: Badalamenti

first: Salvador

last: Soriano


title: Heterobifunctional Protein Binders Enable Cell Type-Specific Killing Through In-cell Enrichment

city: Darmstadt

first: Alexander

last: Loewer

address: Schnittspahnstrasse 13

department: Biologie

institute: Technical University Darmstadt

summary: Non-catalytic heterobifunctional molecules promise to expand the range of therapeutic options by establishing complexes between key target proteins and accessory presenter proteins equipped with additional properties. Here, we systematically investigate the rational design of such molecules, explore the biochemical basis of complex formation and determine how they achieve cellular efficacy using the endogenously expressed immunophilin FKBP12 and the transcriptional regulator BRD4 as paradigms. We present classes of bifunctional molecules that enable selective, FKBP12-dependent killing of specific cell types at subnanomolar concentrations and allow to differentiate between closely similar bromodomains. We propose that the strongly potentiated efficacy of these bifunctional compounds is based on cellular enrichment through binding to the highly abundant presenter protein FKBP12, a mechanism we term “CellTrap”. Our findings substantiate the concept that highly expressed, non-essential proteins can be repurposed as selective recruiters to expand therapeutic windows of existing small-molecule inhibitors, opening new avenues for designing targeted drugs with improved cell-type specificity.

overall_design: RNAseq profiling of MV4-11 cells treated with bifunctional molecules binding FKBP12 and BRD4 with the BRD4 inhibitor JQ1 as comparison.

first: Bulldan

last: Ahmed

first: Zheng

last: Min

first: Meyners

last: Christian

first: Knapp

last: Stefan

first: Loewer

last: Alexander

first: Hausch

last: Felix


title: Telomere variant sequences encode a heritable genetic blueprint for allele-specific telomere length (dataset 1)

first: XIAORAN

last: CHAI

summary: The ends of human chromosomes are capped by specialized nucleoprotein structures known as telomeres, which are essential for genome stability. Recent advances in long-read sequencing have enabled allele-specific telomere length measurements at nucleotide resolution, uncovering extreme heterogeneity in telomere length between alleles that is determined at birth and gradually shortens with age. The shortening over time makes steady-state telomere length in human somatic cells a promising biomarker for age-associated diseases such as clonal hematopoiesis. However, the mechanisms underlying allele-specific telomere maintenance and its stability remain poorly understood. Here, we developed a high-resolution workflow combining PacBio and Nanopore long-read sequencing platforms to map allele-specific telomere length in human blood samples as well as telomerase-positive cancer cell lines. By tracing allele-specific telomeric sequence in family members across multiple generations, we show that telomeric variant sequences (TVSs) interspersed throughout the canonical repeat region are heritable (with mean similarity score > 0.95), allele-specific, and account for the extreme heterogeneity of telomere length between alleles. Targeted deletion of allele-specific TVSs using CRISPR-Cas9 resets telomere length, further confirming their causal role in the control of allele-specific telomere maintenance (p-value < 3e-29). Continuous cell proliferation likely drives the slow but stochastic evolution of allele-specific TVSs distribution, resulting in asymmetry in telomere inheritance from father and mother (p-value < 0.045). These results indicated that TVSs encode a novel class of heritable genetic elements underlying the allele-specific telomere length.

overall_design: telomere sequencing of human cell line HCT116 and three subclones of HT1080

first: Xiaoran

last: Chai

first: Shang

last: Li


title: SHIP1 is required for T cell surveillance of occult malignancies

city: Genova

first: Chiara

last: Pedicone

address: Largo Rosanna Benzi, 10

department: Neuroscienze Sperimentali

institute: IRCCS Azienda Ospedaliera Metropolitana Policlinico San Martino

summary: SHIP1-deficient mice develop severe, transmural Crohn's-like ileitis and die prematurely. Recent analysis of a human Crohn's disease (CD) cohort revealed a significant subset of patients have severely reduced levels of SHIP1 expression suggesting SHIP1 deficiency leads to CD in both rodents and humans. The mucosal inflammatory disease that causes the premature death of SHIP1-/- mice is a consequence of the requirement of SHIP1 expression for the survival of effector T cells in mucosal tissues. T cell specific ablation of SHIP1 expression in CD4CreSHIP1flox/flox mice also results in Crohn's disease-like ileitis, but less severe than in their germline SHIP1-/- counterparts due to the absence of a SHIP1 deficient neutrophil compartment. Consequently CD4CreSHIP1flox/flox mice live much longer than their SHIP1-/- counterparts. As Crohn's disease is associated with an increased risk of intestinal cancers in humans and that T cells are proposed to protect from occult malignancies, we hypothesized that CD4CreSHIP1flox/flox mice would develop malignancies originating in the gut. Here we show that CD4CreSHIP1flox/flox mice with ileitis develop spontaneous metastatic cancer originating in the small intestine, including widely disseminated histiocytic sarcoma and intestinal adenocarcinomas. Our findings demonstrate that T cells in the gut are essential to protect against occult intestinal cancers and that SHIP1 is essential for this function. These findings may aid our understanding of the increased susceptibility to intestinal cancer in IBD patients, and particularly since a significant subset of adult IBD patients have defective expression of SHIP1.

overall_design: Four spleen tumors, three mesenteric lymph node tumors, two payer's patch tumor and one salivary gland tumor from CD4CreSHIP1flox/flox mice. Two normal payer's patch tissue from control mice

first: Fernandes

last: Sandra

first: William

last: Kerr


title: Genotype-phenotype single-cell transcriptomics for massive parallel assessment of genetic variants [scRNA_P63noconv]

city: Pozzuoli

first: Davide

last: Cacchiarelli

state: Napoli

address: via campi flegrei,34

institute: TIGEM

summary: Predicting the pathogenic effect of rare genetic variants is hampered by the limited cohort of diagnosed patients and the difficulty in evaluating the effect of existing and novel variants. To address this issue, we leveraged a protein Multiplexed Assay of Variants Effect (MAVE) to functionally assess the effect of ~2,300 missense variants of the TP63 gene, some of which cause autosomal dominant developmental disorders. The activity of each variant was measured using an optimized fibroblast-to-keratinocyte conversion protocol, and a subset of mutants was validated using a wide range of functional tests. To expand MAVEs to any disease-driving gene without a specific predefined assay, we developed SCRAMseq (Single Cell RNAseq Associated with MAVEs by sequencing), which can retrieve each variant and characterize the functional consequences at the single-cell level through full-length scRNAseq. The dataset generated and validated here reclassified hundreds of variants present in the general population and definitively classified a Variant of Unknown Significance as pathogenic in a patient with ectodermal dysplasia. This work provides a robust and easy-to-use workflow to dissect the effect of gene variants for any possible disease-driving gene.

overall_design: To validate SCRAMseq, we performed Smartseq3Express on cells transduced with SAM pools of variants for full length scRNAseq out of conversion assay

first: Rosa

last: De Santis

first: Lorenzo

last: Vaccaro


title: Genotype-phenotype single-cell transcriptomics for massive parallel assessment of genetic variants [scRNA_P63conv]

overall_design: To validate SCRAMseq, we performed Smartseq3Express on cells transduced with SAM and DBD pools of variants for full length scRNAseq


title: Genotype-phenotype single-cell transcriptomics for massive parallel assessment of genetic variants [RNAseq_P63noconv]

overall_design: To identify the transcriptomic signature induced by WT TP63, we transduced human fibroblasts with wild type P63 or empty ctr and induced overexpresison.


title: Genotype-phenotype single-cell transcriptomics for massive parallel assessment of genetic variants [RNAseq_MYOD]

overall_design: To identify the transcriptomic signature induced by WT MYOD1, we transduced human fibroblasts with wild type MYOD1 or empty ctr and induced overexpresison.


title: Genotype-phenotype single-cell transcriptomics for massive parallel assessment of genetic variants [scRNA_MYOD1]

overall_design: To validate SCRAMseq, we performed Smartseq3Express on cells transduced with MYOD1 pools of variants for full length scRNAseq out of conversion assay


title: Genotype-phenotype single-cell transcriptomics for massive parallel assessment of genetic variants [RNAseq_mutants]

overall_design: Human fibroblasts were transduced with DBD and SAM mutants and induced to convert to iKCs


title: Genotype-phenotype single-cell transcriptomics for massive parallel assessment of genetic variants [MITE_SAM]

overall_design: Human fibroblasts were transduced with SAM domain varaints library and induced to convert to iKCs


title: Genotype-phenotype single-cell transcriptomics for massive parallel assessment of genetic variants

overall_design: Human fibroblasts were transduced with DBD domain varaints library and induced to convert to iKCs


title: Lactate-induced H3K4me3 histone mark promotes PLOD1-driven prostate cancer invasion

city: Florence

first: Paola

last: Chiarugi

state: Florence

address: Viale Morgagni 50

department: Department of Experimental and Clinical Biomedical Sciences

institute: University of Florence

summary: Prostate cancer (PCa) progression is strongly influenced by the metabolites available in the tumor microenvironment (TME), including lactic acid (LA), which is actively imported by PCa cells to boost mitochondrial metabolism and drive de novo collagen synthesis, sustaining survival and malignancy. LA exploitation promotes the unbalance of tricarboxylic acid (TCA) cycle intermediates, particularly succinate and fumarate, well-known epigenetic modifiers for histone (de)methylation. Here, we show that the LA-induced increase in succinate levels affects the transcriptionally activating H3K4me3 methylation mark in PCa cells, promoting a pro-invasive phenotype. Notably, pharmacological targeting of H3K4me3 using OICR-9429 reduces LA-enhanced PCa cell invasiveness. Moreover, LA-induced H3K4me3 enrichment regulates the expression of procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 1 (PLOD1), a key enzyme involved in collagen maturation. Genetic impairment of PLOD1 reduces the LA-driven invasive potential of PCa cells, thereby highlighting PLOD1 as a crucial epigenetically regulated mediator of tumor invasion. Overall, our findings uncover a LA-fuelled metabolic-epigenetic axis that promotes the H3K4me3-mediated PLOD1 upregulation, thereby enhancing PCa aggressiveness and revealing potential vulnerability that could be exploited for targeted therapy.

overall_design: RNA-seq profiling of 16 samples: 4 22Rv1 control cells, 4 22Rv1 treated with Lactate, 4 22Rv1 treated with OICR-9429; 4 22Rv1 trated with Lactate and OICR9429; at Day 2 of treatment


title: CDK8 inhibition sequesters Med1 at EWS-FLI enhancers [CRISPR]

city: Dallas

first: Jiwoong

last: Kim

state: Texas

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

summary: Ewing sarcoma is characterized by a chromosomal translocation resulting in the fusion protein EWSR1-FLI1.  To identify small molecules that selectively impair the oncogenic transcriptional program enacted by EWS-FLI1 we employed a novel phenotypic screen. Our screening hits were enriched for inhibitors of cyclin dependent kinase 8/19.  Inhibition of CDK8/19 resulted in a proliferation defect in Ewing sarcoma cells and a reduction in growth of Ewing sarcoma xenografts.  We performed a genome wide pooled CRISPR screen that suggested that small molecule inhibition of CDK8/19 acted in a gain-of-function manner that did not replicate the effects of CDK8/19 genetic knockout.  Through biochemical studies and chromatin profiling, we discovered that CDK8/19 inhibitors act through a novel trapping mechanism in which the inhibitor prevents dissociation of the CDK8 kinase from core Mediator.  This mechanism has broad implications for the development of small molecules to target transcriptional regulation.

overall_design: A673 cells expressing Cas9 were transduced at 0.3 MOI with the Brunello library lentiguide-puro.  After selection cells were treated with DMSO or SW393109 (100nM) and cultured until a difference of approximately 6 doublings between DMSO and Sw393109 treatment pools.

first: James

last: McGinnis

first: Yang

last: Xie

first: David

last: McFadden


title: ALKBH5 acts as a facilitator for hair follicle telogen to anagen transition

city: Guangzhou

first: Kechen

last: Chen

state: Guangdong

address: Shatai South Road

institute: Southern Medical University

summary: Hair cycle transitions play a pivotal role in sustaining hair growth, particularly the switch from telogen to anagen. During this process, hair follicle stem cells (HFSCs) are regulated by a complex and dynamic niche of signals that drive the cell state from quiescence to activation. However, how HFSCs adjust their state to respond to these signals remains unclear. Here, we show that Alkbh5 expression is downregulated during late telogen, and its deletion promotes HFSC activation and proliferation, accelerating the telogen–anagen transition in mice. In vitro, ALKBH5-deficient HFSCs display enhanced proliferation under 3D culture. Loss of ALKBH5 increases the m6A methylation of Mcam, which is subsequently recognized and bound by YTHDF1, thereby stabilizing Mcam mRNA. Mcam expression increases as Alkbh5 decreases during late telogen, and Mcam knockdown impairs HFSC proliferation, potentially through reduced Wnt responsiveness. Furthermore, Mettl3 is also involved in maintaining hair cycle progression. We conclude that ALKBH5, a facilitator for hair cycle transition, dynamically regulates Mcam expression mediated by m6A methylation, and that proper HFSC activation potentially requires a balanced m6A regulatory mechanism involving both ALKBH5 and METTL3.

overall_design: The RNA-seq datasets were generated from bulge hair follicle stem cells and hair germ stem cells isolated from postnatal day 69 (P69) wild-type and Alkbh5 conditional knockout (cKO) mice.And m6A-seq profiling of  bulge hair follicle stem cells from P69 wild-type and Alkbh5 conditional knockout (cKO) mice

first: Shijun

first: Yanqun

last: Zhang

first: Yuchen

last: Liu

first: Tao

last: Huang

first: Ke

last: Ding

first: Ying

last: Luo

first: Hongwei

last: Zhou

first: Zhili

last: Rong

last: Lin


title: CDK8 inhibition sequesters Med1 at EWS-FLI enhancers [RNA-Seq]

overall_design:
RNA-seq analysis in Ewing sarcoma cell lines.Cells were treated with CDK8 inhibitors SW393109 or CCT251921 to determine gene expression changes in EWS cells upon CDK8 inhibition; Ewing sarcoma cells engineered with an auxin-inducible degron in the EWSR1-FLI1 fusion were treated with 300nM IAA to induce depletion of the fusion and RNA-seq was performed.  A673 EWS-FLI-SMASh cells were treated with DMSO or 316nM danoprevir.
RNA-seq analysis in A-673 AIDCDK8; CDK19-KO cells. Cells were treated with CDK8 inhibitors SW393109, 5-ph-IAA or the combination to evaluate the difference of CDK8 inhibition and CDK8 depletion, and determine the dependency of CDK8/19 upon CDK8 Inhibition.

first: Shuai

last: Wang


title: The TENT5A–ATXN2 axis modulates cellular survival during heat stress.

city: Durham

first: Marcos

last: Morgan

address: 111 TW Alexander Dr

institute: NIEHS

summary: Stress conditions elicit the formation of different kinds of stress granules. The assembly and composition of heat-induced stress granules remain poorly characterized. Here, we show that the cytoplasmic RNA polyadenylases TENT5A regulate the formation and function of ATXN2-positive heat-induced stress granules. Tent5a mRNA expression increases when somatic and germ cells are exposed to temperatures between 40°C and 42°C, but not upon exposure to other stressors. TENT5A decreases ATXN2 levels by reducing Atxn2 mRNA translation and accelerating ATXN2 protein decay. Upon heat exposure of the testis, ATXN2-positive stress granules are formed in pre-meiotic and meiotic cells independently of the presence of G3BP1 and the heat stress response protein DAZL. Depletion of TENT5A leads to increased cell-type-specific formation of ATXN2-positive stress granules and differentially regulates cell survival. In sum, TENT5A inhibits the formation of ATXN2-positive granules to modulate the heat shock response in vivo.

overall_design:
17-CL1 cells were infected with lentivirus carrying shRNAs against Tent5a and Tent5b. An unspecific shRNA was used to infect control cells. Total mRNA samples were obtained from independently generated TENT5A- and TENT5B-depleted cells. A transcriptome-wide poly(A) tail profile was obtained for each cell line using Oxford Nanopore Technologies RNA sequencing. The poly(A) tail lengths of transcripts from TENT5A/B-depleted cells and control cells were then compared.
The previously uploaded samples (GSM6032864, GSM6032865, and GSM6032866) were used as controls in this study.

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The table below lists GEO accessions reused/reanalyzed for this study.
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first: Ru-Pin

middle: A

last: Chi

first: Ankit

last: Gupta

first: Yin

last: Li

first: Marine

last: Baptissart

first: Brian

last: Papas


title: Regulation of Splicing by mec-8

city: Riverside

first: Zachery

last: Wolfe

address: Boyce Hall 1463

department: Biochemistry

institute: University of California, Riverside

summary: MEC-8 splicing factor affects alternative splicing of many genes in neurons and other tissues of C. elegans. MEC-8 consists of two RRMs, and this experiment set out to test whether RRM1 and RRM2 are both required for regulation of MEC-8 targets, by comparing RRM-specific amino acid substitutions with null mec-8 mutants.

overall_design: L4 staged animals from wild-type and mutant conditions. RNA extracted, and selected for polyA+ mRNA prior to library generation and short-read sequencing.

first: Xiaoyu

last: Liang

first: Adam

last: Norris


title: Bidirectional control of a metabolic transition by the GID ubiquitin ligase

city: Mainz

first: Frank

last: Rühle

address: Ackermannweg 4

department: Bioinformatics Core Facility

institute: Institute of Molecular Biology (IMB)

summary: The GID/CTLH ubiquitin ligase is a multisubunit E3 conserved across eukaryotes. GID/CTLH has been implicated in a variety of processes, including metabolic regulation, cell proliferation, embryonic development and cell differentiation. However, our understanding of substrate recognition by GID/CTLH remains incomplete. Here, we characterize the regulation, specificity and functions of the putative substrate receptor Gid11 in budding yeast. We find that upon its expression during the switch of carbon source from glucose to ethanol, Gid11 associates with the core GID complex, likely forming a large ring assembly. Through systematic mutagenesis, we identify a negatively charged pocket on Gid11 that binds substrates carrying N-terminal degrons starting with threonine and define the Thr/N-degron motif. Such Thr/N-degrons are exposed upon co-translational removal of the initiator methionine, while escaping N-terminal acetylation. Finally, we show that during the switch to ethanol, GIDGid11 targets for degradation factors linked to glycolysis, opposing the established role of GID in the degradation of gluconeogenic enzymes during the reverse switch from ethanol to glucose. Taken together, our results establish Gid11 as a bona fide GID substrate receptor and highlight GID as a regulator of metabolic transitions.

overall_design: Multiplexed protein stability (MPS) profiling using DNA amplicon-seq data. For this, a library of peptide variants (based on inserted random or pre-determined DNA sequences) fused to a tandem fluorescent protein timer (tFT) is expressed from yeast plasmids and sorted into 8 stability bins using the tFT readout. The contained variable regions are sequenced and the occurrences of all observed DNA sequence variants are counted per stability bin. The distributions of read counts are summarized in the form of a protein stability index (PSI) for each peptide.

first: Ka-Yiu

last: Kong

first: Christian

last: Ochs

first: Cécile

last: Debarnot

first: Matti

last: Myllykoski

first: Denís

last: Blanco

first: Susmitha

last: Shankar

first: Vivien

last: Schoonenberg

first: Julia

last: Kończak

last: Varga

first: Ora

last: Schueler-Furman

first: Katja

last: Luck

first: Falk

last: Butter

first: Jia-Xuan

last: Chen

first: Lukas

last: Stelzl

first: Thomas

last: Arnesen

first: Anton

last: Khmelinskii