title: Retinoic acid drives cell fate specification, maturation and retinal regionality in human retinal organoids [Visium]

city: Sydney

first: Carissa

last: Chen

state: NSW

address: 214 Hawkesbury Rd, Westmead

institute: Children's Medical Research Institute

summary: Retinoic acid (RA) is a key morphogen in human retinal development, activating transcriptional programs that drive retinal progenitor differentiation and photoreceptor development, ensuring proper spatial organisation within the neural retina, essential for vision. Despite its well-established role in retinal patterning, the concentration-dependent effects of RA on human retinal cell fate specification and the regional definition of the primate macula and peripheral retina remain poorly understood. Here, we show that temporal and dosage-dependant modulation of RA during human retinal organoid differentiation induces distinct changes in retinal cell abundance, maturation, and organisation. Single-cell transcriptomics and protein analysis revealed that RA dosage influenced the relative abundance and maturation of photoreceptors and retinal interneurons. Spatial transcriptomics analyses demonstrated that low RA levels biased retinal organoids toward a macular-like regional identity, whereas high RA levels promoted peripheral-like development. Collectively, our findings emphasise the critical role of RA signalling in retinal maturation and regional specification. This study elucidates mechanisms involved in human retinal development and we anticipate that controlled RA modulation in retinal organoids provides a strategy to refine disease modelling of inherited retinal disorders and enhance the specific generation of photoreceptors suitable for transplantation and regenerative therapies.

overall_design: 6-15 retinal organoids differentiated from the H7 A81.BRN3b.mCherry and iPSC 149br.CRX.Cherry reporter lines (with 2 replicates each) were collected at 10 weeks. Slides were stained with DAPI to be analysed. Single organoids per slide were manually segmented and the corresponding gene expression data was concatenated into a SpatialExperiment object.

first: Benjamin

middle: Y

last: Lim

first: Anna

last: Fredericks

first: Elham

last: Nilli

first: Santiago

middle: M

last: Mora

first: Megan

last: Weatherstone

first: To Ha

last: Loi

first: Peter

last: Newman

first: Nader

last: Arymanesh

first: Hala

last: Zreiqat

first: Patrick

last: Tam

first: Pengyi

first: Anai

last: Gonzalez-Cordero


title: Genome-wide discovery of cis-regulatory elements in a large genome [bulk ATAC-seq]

status: Public on Mar 21 2026

city: Lyon

first: Mathilde

last: PARIS

address: 32-34 Av. Tony Garnier

country: France

institute: CNRS

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Identifying non-coding regulatory elements in the genome poses a challenge in most organisms. Classical methods rely on trial and error to test the regulatory activities of DNA fragments using reporter constructs. In large eukaryotic genomes, where cis-regulatory elements can spread over long distances, separated by large stretches of non-functional DNA, this trial and error approach is particularly challenging. Here, we generate two types of resources that can be used to narrow the search for such cis-regulatory elements in the 3.6 Gbp genome of Parhyale hawaiensis (comparable in size to the human genome). First, we use bulk ATACseq to uncover genome-wide patterns of chromatin accessibility in embryonic and adult tissues of Parhyale (whole embryos and legs), and single-nucleus ATACseq to identify regions of open chromatin in diverse cell types recovered from adult legs, including epidermal, neuronal, muscle and blood cells. Second, by sequencing the genomes of three congeneric species of Parhyale hawaiensis – P. darvishi, P. aquilina and P. plumicornis – we identify islands of sequence conservation across the genome, corresponding to DNA elements that are functionally constrained during evolution. We present an approach by which low-coverage (10-15x) short-read genome sequencing, without genome assembly, is sufficient to provide reliable maps of sequence conservation. This approach cuts the cost and labour required to generate these maps, making the identification of cis-regulatory elements more widely accessible. We demonstrate the utility of these resources by identifying cis-regulatory elements that drive robust expression of fluorescent reporters ubiquitously and in specific cell types.

overall_design: Bulk ATAC-seq profiles of embryos, as well as embryonic and adult legs from Parhyale hawaiensis

first: Gillian

last: Forbes

first: Emilia

last: Skafida

first: Çağrı

last: Çevrim

first: Michalis

last: Averof

last: Paris


title: Tumors Hijack Immune-Privileging Regulons via Distinct Cell Types to Confer T Cell Desertion and Immunotherapy Resistance Across Various Cancers

city: Pittsburgh

first: Xiaosong

last: Wang

state: Pennsylvania

address: 5150 Centre Ave

department: Pathology

country: USA

institute: University of Pittsburgh

type: Expression profiling by high throughput sequencing

summary: Immune checkpoint blockade (ICB) has transformed oncology, yet most patients fail to respond, suffer from hyper-progressive disease, or face severe immune-related toxicities, underscoring the urgent need for biomarkers identifying non-responders. Here we show that tumors co-opt an immune-privileging regulon signature (IMPREG) mirroring transcriptional programs of immune-privileged organs — to enforce T-cell desertion and ICB resistance across cancer types. Single-cell and spatial transcriptomic analyses reveal that tumors activate IMPREG through three distinct cellular routes — malignant cells adopting immature neuronal states, cancer-associated fibroblasts assuming myofibroblast identities, or endothelial cells — each creating localized niches of immune suppression and antigen-presentation collapse. Across 4 discovery and 36 validation clinical datasets, IMPREG consistently predicts immunotherapy resistance in 14 distinct cancer types, functioning as an orthogonal marker independent of established biomarkers. Crucially, IMPREG-expressing tumors show enhanced sensitivity to EGFR inhibitors or anti-angiogenic therapies in specific tumor entities. These findings suggest IMPREG as a dual-utility predictive biomarker for personalized treatment stratification.

overall_design: 72 tumor tissues were procured from the Health Sciences Tissue Bank of University of Pittsburgh.

first: Bashir

last: Lawal

first: Akshat

last: Gupta

first: Renu

last: Sharma

first: Rohit

last: Bhargava

first: Yue


title: ST8Sia6-dependent glycosylation restrains gut inflammation and pathogenic Th1 and Th17 programs

status: Public on Mar 22 2026

city: Rochester

first: Virginia

middle: Smith

last: Shapiro

state: MN

address: 200 First St SW

department: Immunology

institute: Mayo Clinic

summary: Single-cell suspensions of small bowel lamina propria, small bowel epithelium, and whole colon were purified from mice sufficient, deficient, and heterozygous for sialic acid transferase ST8Sia6. Cells were enriched for CD45 expression, hashtagged according to genotype, and analyzed by 10x scRNA sequencing using the Illumina Novaseq 6000 as well as by 10X CITEseq to identify hashtags.

overall_design: Small intestines and colons were isolated from 1 ST8Sia6 knockout mouse, 1 ST8Sia6 heterozygote mouse, and 2 ST8Sia6 sufficient wildtype mice. The cells from the small intestinal lamina propria, the small intestinal epithelium, and the whole colon (lamina propria and epiethelium combined) were subjected to a percoll gradient and enriched for immune cells using the StemCell Mouse CD45 Positive Selection Kit. The samples were tagged with Biolegend TotalSeq hashtags based on their genotype: KO=C0301, het=C0302, WT=C0303. This procedure was done 3 independent times for combined 10X scRNA sequencing and 10X CITEseq using the Illumina Novaseq 6000. Processed data was aligned using CellRanger, and cells with fewer than 200 genes or greater than 8000 genes, cells with 20% or greater mitochondrial gene counts, and cells with less than 300 or more than 50000 molecules detected were filtered out.

middle: S

first: Sydney

middle: B

last: Crotts


title: Co-mutations of CTNNB1 and PTEN Synergistically Promote EMT and Drive Aggressive Tumor Progression in Endometrial Cancer.

city: Columbia

first: Jae-Wook

last: Jeong

state: MO

address: Rutland Drive

department: Obstetrics, Gynecology and Women’s Health

institute: University of Missouri

summary: Endometrioid endometrial cancer (EEC) is the most common gynecologic malignancy, with poor outcomes in advanced stages. Co-mutations in PTEN and CTNNB1 are frequent and linked to poor prognosis, but their functional roles remain unclear. Using uterine-specific Pten knockout and stabilized β-catenin mutant mice (Pgrcre/+Ptenf/fCtnnb1f(ex3)/+; Ptend/dCtnnb1f(ex3)/+), we found markedly reduced survival, early-onset invasive adenocarcinoma, and aggressive metastatic disease compared to Pgrcre/+Ptenf/f; Ptend/d mice. Transcriptomic and histopathological analyses revealed activation of oncogenic pathways (WNT/β-catenin, PI3K/AKT, Hedgehog, EMT), with loss of E-cadherin and upregulation of SNAIL confirming EMT. These findings show that PTEN–CTNNB1 co-mutations synergistically drive EMT and tumor progression, defining a distinct aggressive subtype of EEC.

overall_design: Uterine samples were collected from Ptend/d and Ptend/dCtnnb1f(ex3)/+ mice at 2-weeks of age and total RNA was extracted for RNA-sequencing (n = 4/genotype).

first: TaeHoon

last: Kim

first: JaeWook


title: whole-genome bisulfite sequencing (WGBS) of wild-type and Uhrf1-/- ESCs

city: New York

first: Yifei

last: Sun

address: 1468 Madison Ave

institute: Icahn School of Medicine at Mount Sinai

type: Methylation profiling by high throughput sequencing

summary: UHRF1 is a chromatin-binding protein essential for maintaining DNA methylation and histone modification states, yet its integrated role in vivo remains incompletely understood. To define its function, we generated conditional Uhrf1 knockout embryonic stem cells (ESCs) and embryos. Uhrf1⁻/⁻ ESCs exhibited near-complete loss of 5mC and 5hmC but maintained pluripotency, whereas Uhrf1-null embryos developed normally until E8.5 and then failed to further develop by E9.5.

overall_design: We collected  Whole Genome Bisulfite data from ESCs for further studies of 5mC and 5hmC changes in wild type and Uhrf1-/- cells.

first: Agata

last: Kurowski

first: Martin

middle: J

last: Walsh


title: A nanovaccine targeting cancer stem cells and bulk cancer cells reduces post-surgical tumor relapse [RIP-Seq]

city: Beijing

last: Huo

state: Beijing

address: Dongcheng District, Dongdan 3 Tiao 5 Hao

department: State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences

country: China

institute: Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College

summary: Residual cancer stem-like cells (CSCs) can cause tumor recurrence within a narrow margin around the initial tumour resection lesion, increasing post-surgical risk of relapse and incurability. Currently there are no efficient strategies for tracking and eradicating CSCs. Here we propose a nanovaccine strategy, NICER, based on a nanovesicle system integrating CSC-specific antigen (CSA) display and epigenetic nano-regulator (ENR)-encapsulation with a dendritic cell (DC)-targeting aptamer, to simultaneously eradicate CSCs and bulk tumor cells. Specifically, nanovesicles derived from aldehyde dehydrogenase-overexpressing tumors could serve as integrated antigens carrying both CSA and tumor associated antigen (TAA). ENR targeting YTH N6-methyladenosine RNA binding protein 1 could restrict DC lysosomal protease activity to modulate the effective cross-presentation of integrated antigens via major histocompatibility complex class I (MHC I) for immune responses. Overall, NICER represents a broad-spectrum vaccine approach against both CSCs and bulk tumors that can significantly inhibit postoperative cancer recurrence and metastasis, prolonging survival rates.

overall_design: We performed the RNA immunoprecipitation (RIP)-sequencing (RIP-seq) of BMDCs to test the m6A-marked transcripts encoding lysosomal proteases are recognized by YTHDF1. BMDCs were harvested, dissolved in RIP 1ysis buffer, performed with RNA immunoprecipitation assay. Then the extracted co-immunoprecipitated and input RNAs tested for RIP-sequencing.

first: Qing

last: You


title: A nanovaccine targeting cancer stem cells and bulk cancer cells reduces post-surgical tumor relapse [meRIP-seq]

overall_design: To detect the presence of m6A modifications in BMDCs (bone marrow-derived dendritic cells), we performed the methylated RNA immunoprecipitation sequencing (MeRIP-seq). Total RNAs extracted from the BMDCs were purified, incubated with an m6A antibody for immunoprecipitation. Then the obtained m6A-containing mRNA was analyzed by high-throughput sequencing.


title: Retinoic acid drives cell fate specification, maturation and retinal regionality in human retinal organoids [Stereo-seq]

first: Carissa

last: Chen

country: Australia

overall_design: 4-21 retinal organoids differentiated from the H7 A81.BRN3b.mCherry reporter line were collected at 16 and 26 weeks. Organoids were sectioned onto BGI Stereo-seq transcriptomic slides, fixed and tissue was permeabilised. Slides were then stained with ssDNA to be analysed. Using the bioinformatic pipeline provided by BGI, gene expression was aggregated in square arrays of 30um x 30um (bin60) when analysing 16 week organoid samples and 50um x 50um (bin100) when analysing 26 week organoid samples

first: Benjamin

last: Lim

first: Anna

last: Fredericks

first: Elham

last: Nilli

first: Santiago

last: Mora

first: Megan

last: Weatherstone

first: To Ha

last: Loi

first: Peter

last: Newman

first: Nader

last: Arymanesh

first: Hal

last: Zreiqat

first: Patrick

last: Tam

first: Pengyi

last: Yang

first: Anai

last: Gonzalez-Cordero


title: Retinoic acid drives cell fate specification, maturation and retinal regionality in human retinal organoids [scRNA-seq]

overall_design: Five to six organoids differentiated from the H7 A81.BRN3b.mCherry reporter line were collected after treatment with 10uM, 1uM retinoic acid, no retinoic acid or 1uM BMS-493 and analysed with scRNA-seq

first: Hala


title: The ac4C acetyltransferase Nat10 promotes RNA stability to support hematopoietic stem cell fate [RNA-seq-mouse]

first: Yue

summary: The control of gene expression program is believed to underlie hematopoietic stem cell (HSC) homeostasis. However, a comprehensive molecular understanding of the RNA level regulation is currently lacking. Here we provide evidence that the HSC-associated transcripts have longer 3’UTRs and more enriched AU-rich elements (AREs), which were recognized by Nat10. Nat10 is highly expressed in HSC and its deficiency disrupts HSC self-renewal and long-term maintenance. In contrast to current model that invoke ac4C modification to mediate downstream effects, we find Nat10 recruits ribosomes to mRNA 3’UTRs and protects them from degradation by ZFP36-CNOT RNA decay complex. Nat10 depletion leads to reduction of mRNA hail-life, and both wild-type and catalytically inactive Nat10 promote the stability of mRNA with AREs. We present a model that Nat10 regulates mRNA stability as an ARE-binding protein rather than ac4C writer in HSC, a mechanism is required for hematopoietic homeostasis, highlighting the importance of transcript-specific characteristics determining the contribution of Nat10 in HSC fate.

overall_design: We investigated the influence of ac4C modification and NAT10 binding on mRNAs by employing RNA lifetime profiling to measure mRNA half-life in mouse LT-HSCs.

first: Weiqian

last: Li

first: Zhaoru

last: Zhang


title: Genome-wide discovery of cis-regulatory elements in a large genome [scATAC-seq]

first: Mathilde

last: PARIS

overall_design: snATAC-seq profiles of adult legs from Parhyale hawaiensis using the 10x genomics technology

first: Gillian

last: Forbes

first: Emilia

last: Skafida

first: Michalis

last: Averof

last: Paris


title: Pheno-transcriptomic characterization of hooked hairs in common bean

city: Tucson

first: Sergio Alan

last: Cervantes-Perez

state: Arizona

address: 1140 E South Campus Dr

department: School of Plant Sciences

institute: University of Arizona

summary: The environmental changes caused by climate change enhance the effects of land degradation. This faces a big challenge for agriculture to secure food production in the world. Because of the key role of plant roots in the soil and environment interactions, understanding the cell structures, genetic circuits, and mechanisms controlling the root architecture may help to mitigate these effects. However, the information about the role of the cell structures composing the root architecture, especially during early developmental stages, is very limited. Here we reported a high-dimensional imaging method to support the existence of a novel root hair-like structure in common bean (Phaseolus vulgaris), named ‘hooked hairs’, by combining with comparative RNA-sequencing analyses to other common bean hair-like structures confirming a distinct transcriptomic landscape with particular pathways enriched. We found a phenotypic response to phosphate and nitrate starvation, suggesting the role of these hooked hairs in nutrient uptake. Our results provided strong evidence for the importance of high-imaging phenotyping methods and transcriptomics to characterize complex root structures, confirming the plasticity of root epidermal cells to adapt to different environments during different developmental stages.

overall_design: To investigate the molecular differences among different hair-like structures of the common bean plant between trichomes and hooked hairs. We then performed a gene expression profiling using data obtained from RNA-seq of 6 different libraries from three different hair like structures: AG(trichomes), EPI (trichomes), and BG(hooked hairs). Comparative gene expression analyses of RNA-seq data for these libraries were performed.

first: Sergio

middle: A

first: Ankita

last: Roy

last: Piertzyk

first: Sandra

last: Thibivilliers

first: Marc

last: Libault

first: Alexander

last: Bucksch


title: Dual-Coupling Amplified Electrostimulaton Drives In Situ Bone Regeneration

city: Seoul

first: Seung Cheol

last: Lee

address: Gwanak ro

country: South Korea

institute: Seoul National University

summary: Electrical stimulation is known to promote bone regeneration, but conventional systems often require wired connections or implanted power sources and provide limited stimulation to deep tissues. To address this, a dual-amplified body-coupled (DAB) electrostimulation approach was developed, enabling wireless deep-tissue stimulation by harvesting ambient low-frequency electrical fields. A conductive membrane electrode was chosen to focus electrical potentials at the bone-defect site, and both simulations and ex vivo measurements confirmed enhanced electric field intensity within bone tissue under DAB stimulation. To figure out the mechanistic insights, bulk RNA-seq analysis was conducted on DAB-stimulated human bone marrow mesenchymal stromal cells (hBMSCs) and their corresponding controls across multiple time points (Days 2, 4, 7, and 14). Differentially expressed gene (DEG) analysis revealed that by Day 14 of DAB treatment, osteogenic markers including BMP2, BMP6, and SPP1 were significantly upregulated, suggesting enhanced osteogenic differentiation, as these genes are integral to bone formation and mineralization processes.

overall_design: A custom DAB-mimicking system was constructed using a function generator, power amplifier, and oscilloscope to simulate daily-life static electricity exposure. Electrical signals (18 Hz, 20 V) were generated and amplified to deliver controlled stimulation, applied 8 hours/day to hBMSCs. Bulk RNA-seq analyzed DAB-stimulated hBMSCs versus controls at Days 2, 4, 7, and 14.

first: Dong Hyun

first: Byeongsoo

last: Eom

first: Jinman

last: Kim


title: Simultaneous mapping of RNA secondary, tertiary, and quaternary structure in living cells by multi-site DMS probing I

city: Houston

first: Anthony

last: Mustoe

state: TX

address: 1 Baylor Plaza

institute: Baylor College of Medicine

summary: Identifying tertiary structures and protein binding sites on RNA molecules remains a key challenge in RNA biology. We report a new chemical probing strategy termed multi-site DMS-MaP (msDMS-MaP) that exploits base tautomerization during mutational profiling reverse-transcription to measure typically invisible DMS-induced modifications at the N7 position of G. These N7-G modifications are now resolved concurrently with conventional N1 and N3 modifications with only minor modifications to established protocols, providing a multi-dimensional, single-molecule readout of RNA structure. We show that N7-G reactivity specifically reports on RNA tertiary and quaternary structure, enabling identification of diverse, functionally significant motifs such as cooperatively folding tertiary domains and protein binding sites in living cells. We apply msDMS-MaP to obtain a map of the quaternary structural ensemble of the 7SK non-coding snRNP, revealing unique protein binding sites across three 7SK structural isoforms. msDMS-MaP represents a broadly applicable strategy for enhanced RNA functional motif discovery and characterization.

sample_organism:
Escherichia coli
Tetrahymena
Homo sapiens

overall_design: RNAs were folded under varied conditions, chemically probed using DMS, and read out using regular or msDMS-MaP RT conditions. Cell-free = protein-free.

first: Irfana

last: Saleem

first: Thomas

last: Miller

first: Lucas

last: Kearns

last: Hoang

first: Joshua

last: Meehan

first: Ritwika

last: Bose

first: David

last: Mitchell

middle: H

last: Price

first: Calla

last: Olson

first: Chase

last: Weidmann


title: The ac4C acetyltransferase Nat10 promotes RNA stability to support hematopoietic stem cell fate [RNA-seq-32D]

first: Yue

last: Huo

overall_design: We investigated the influence of ac4C modification and NAT10 binding on mRNAs by employing RNA lifetime profiling to measure mRNA half-life in 32D cells.

first: Weiqian

last: Li

first: Zhaoru

last: Zhang


title: The ac4C acetyltransferase Nat10 promotes RNA stability to support hematopoietic stem cell fate [eCLIP-seq]

overall_design: We employed eCLIP-seq to screen NAT10 direct binding sites on mRNA, investigating NAT10 mechanism in HSCs.


title: microRNA-132 attenuates inflammation in induced pluripotent stem-cell derived microglia from Alzheimer’s disease patients

city: Amsterdam

first: Dilara

last: Ayyildiz

address: Meibergdreef 47

country: Netherlands

institute: Netherlands Institute For Neuroscience

summary: Microglia, the resident immune cells of the brain, are increasingly recognized as key contributors to Alzheimer’s disease (AD) pathology. Multiple studies have identified microRNA-132 (miR-132) as one of the most significantly downregulated microRNAs in AD. Apart from well-established pleiotropic regulatory functions in neurons, previous evidence also suggested a role for miR-132 in regulating (neuro)inflammation. Yet, the precise mechanisms by which miR-132 impacts microglia remain unknown. In this study, we investigated the role of miR-132 in modulating microglial gene expression and function using gain- and loss-of-function approaches in human-induced pluripotent stem cell (iPSC)-derived microglia (iMGs) from both healthy controls and sporadic AD (sAD) patients. Our findings indicate that while miR-132 may not be indispensable for some baseline microglial functions, increasing its expression in sAD iMGs can reverse disease-associated gene expression changes and attenuate inflammatory responses. To further explore its therapeutic potential, we overexpressed miR-132 in hippocampal neurons of an AD mouse model, employing a clinically relevant adeno-associated viral (AAV) delivery method. miR-132 overexpression was well-tolerated and induced non-cell autonomous effects in microglia. This study sheds light into the regulatory role of miR-132 in microglia under both physiological and AD conditions, and emphasizes the importance of optimizing safe dosage parameters for future clinical applications.

overall_design: Bulk RNA sequencing data of iPSC-microglia from four comparisons: healthy control vs sAD microglia, healthy control microglia miR-132 overexpression vs control, healthy control microglia miR-132 knockdown vs control, sAD microglia miR-132 overexpression vs control

first: Amber

last: Penning

first: Sarah

last: Snoeck

first: Olmo

last: Ruiz Ormaechea

first: Oliver

last: Polzer

first: Martin

last: Buitrago-Arango

first: Raffaella

last: Copabianco

first: Fred

last: de Winter

first: Sriram

last: Balusu

first: Joost

last: Verhaagen

first: Carlos P.

last: Fitzsimons

first: Constantin

last: d'Ydewalle

first: Paul J.

last: Lucassen

first: Dieder

last: Moechars

first: Lujia

last: Zhou

first: Salta

last: Evgenia


title: Transcriptome profiling of Arabidopsis thaliana PsbS and β-carbonic anhydrase overexpression lines in response to bicarbonate fertilization

city: Warsaw

first: Piotr

last: Gawroński

address: Nowoursynowska Street 159

country: Poland

institute: Warsaw University of Life Sciences

summary: Photosynthetic efficiency and stress tolerance in plants are regulated by complex interactions between photoprotective mechanisms and carbon metabolism. The photosystem II subunit PsbS plays a central role in non-photochemical quenching, while β-carbonic anhydrases contribute to CO₂ conversion and pH homeostasis in chloroplasts. To investigate the potential crosstalk between these components, we performed RNA-seq analysis of Arabidopsis thaliana lines overexpressing PsbS alone or in combination with β-carbonic anhydrase genes (βCA1 and βCA2). Wild-type (Col-0), the npq4 mutant, and transgenic overexpression lines were grown under controlled conditions and treated either with water or bicarbonate fertilization.  Transcriptome profiling was performed using Illumina NovaSeq sequencing of poly(A)+ RNA libraries prepared from total RNA. The dataset contains three biological replicates for each genotype and treatment combination. These data provide insight into transcriptional responses associated with altered PsbS and β-carbonic anhydrase activity and bicarbonate-induced signaling, and can be used to explore regulatory networks linking photoprotection, carbon metabolism, and stress responses in plants.

overall_design: Arabidopsis thaliana plants representing five genotypes were analyzed: wild type (Col-0), the npq4 mutant lacking PsbS, a PsbS overexpression line (oePsbS), and transgenic lines overexpressing PsbS together with β-carbonic anhydrase genes (oePsbS_CA1 and oePsbS_CA1_CA2). Plants were grown under controlled conditions (16 h light/8 h dark photoperiod, 120 µE light intensity, 21 °C, 50–60% humidity). Two-week-old plants were treated every two days with either 10 ml of 3 mM NaHCO₃ solution (pH 7) or water as a control.  For transcriptome analysis, rosette tissue was collected and total RNA was extracted. RNA-seq libraries were prepared using the TruSeq Stranded Sample Prep Kit and sequenced on an Illumina NovaSeq platform (paired-end 2 × 150 bp). The experiment included five genotypes and two treatments (water or bicarbonate), with three independent biological replicates per condition, resulting in a total of 30 RNA-seq samples.

first: Alex

last: Białas

first: Joanna

last: Dąbrowska-Bronk

first: Stanisław

last: Karpiński


title: HSV-1 temporally orchestrates RAP80 ubiquitination to balance viral replication and evasion [Hi-C]

status: Public on Mar 20 2026

first: jie

last: Wang

address: 5 Summer Palace Road, Haidian District, Beijing

institute: school of Life science

summary: The mechanisms governing the complete replication cycle of herpes simplex virus type 1 (HSV-1) remain incompletely elucidated. Here, we showed that HSV-1 employs a biphasic strategy to dynamically regulate the host protein RAP80 through stage-specific ubiquitination, thereby coordinating its replication cycle. Upon entry, RAP80 undergoes phase separation to form antiviral condensates that encapsulate the viral genome and suppress viral transcription. The HSV-1-encoded E3 ubiquitin ligase ICP0 counteracts this antiviral mechanism by mediating RAP80 ubiquitination to dissolve these condensates, facilitating viral replication. In middle stage, RAP80 depletion leads to accumulation of R-loops and activation of the cGAS–STING–apoptosis axis, threatening viral persistence. In response, in middle and late stage, the viral deubiquitinase UL36USP deubiquitinates and stabilizes RAP80 to suppress R-loop accumulation and this immune-triggered apoptosis. This biphasic regulation of a single host factor represents a sophisticated viral strategy to ensure productive replication and dissemination. Notably, pharmacological disruption of RAP80 ubiquitination using engineered inhibitory peptides potently inhibits HSV-1 replication in vitro and in vivo. Furthermore, RAP80 inhibition enhances both chemosensitivity and oncolytic virotherapy in tumors. These findings reveal a ubiquitination-mediated host–pathogen equilibrium with therapeutic potential in antiviral and anticancer contexts.

overall_design: To investigate the interactions between HSV-1 and the host genome under different conditions using Hi-C sequencing.To explore the effects of HSV-1 infection on the host after RAP80 knockdown using RNA-seq.

first: Jie

first: Xiaofeng

last: Zheng

first: Yi

last: Chi

first: Yujing

last: Deng

first: Dong

last: xin


title: HSV-1 temporally orchestrates RAP80 ubiquitination to balance viral replication and evasion [RNA-seq]


title: Longitudinal Multiomic and Spatial Transcriptomic Profiling of Lupus Nephritis Progression in a Murine Model

city: Bethesda

first: Kan

last: Jiang

state: MD

address: 9000 Rockville Pike

institute: NIAMS/NIH

summary: Lupus nephritis (LN) is characterized by progressive renal inflammation and tissue injury. Bulk RNA sequencing was performed on kidneys from lupus-prone female NZB/W F1 mice at 10, 20, and 30 weeks of age to characterize transcriptional changes during disease progression. Minimal differences were observed at early stages, whereas advanced disease was associated with marked upregulation of genes involved in immune activation, cytokine signaling, fibrosis, and oxidative stress. Spatial transcriptomic profiling of 30-week kidneys further resolved the spatial organization of these transcriptional changes, identifying nine distinct spatial clusters with unique gene expression signatures. Spatial molecular signature analysis revealed localized inflammatory programs enriched for cytokine/chemokine and interferon signaling pathways.

overall_design:
Female wild type (WT) mice aFemale NZB/W F1 lupus-prone mice were obtained from The Jackson Laboratory and housed under specific pathogen-free conditions at the NIH animal facility. Mice were maintained under standard controlled conditions with ad libitum access to food and water, and all procedures were approved by the NIAMS Animal Care and Use Committee (protocol A019-05-03). Animals were euthanized at 10, 20, and 30 weeks of age, and tissues and blood were collected for downstream analyses.
Spatial transcriptomic profiling of 30-week mouse kidneys was performed to characterize regional gene expression heterogeneity.

first: Eduardo

last: Patino-Martinez

first: Morteza

last: Hajihossein

first: Saumyadipta

last: Pyne

first: Mariana J.

last: Kaplan


title: Adenovirus phagocytosis by neutrophils triggers a pro-inflammatory response

city: Montigny-Le-Bretonneux

first: Sophie

last: Dupré

address: 2 av. de la Source de la Bièvre

department: Versailles Saint Quentin University

institute: Paris Saclay Université

summary: To determine if  transcriptional changes also occur during human neutrophils adenoviruses interaction, a single-cell RNA sequencing study was performed on blood purified PMNs exposed to human serum (HS) alone or to HS-opsonized Ad5.

overall_design: Human Neutrophils (PMNs) were extracted from the blood obtained from healthy donors (EFS  agreements). PMNs (5 × 10^5) per condition were resuspended in HBSS. HBSS, HS or HS-opsonized Ad5 (MOI 10^4 vp/cell) were incubated with PMNs for 30 min at 4°C. After washing with HBSS, samples were incubated 90 min at 37°C then filtered to discard aggregates. PMNs were counted with Trypan Blue to assess cell viability (that was around 80%) then transferred in DNA LoBind tubes. RNA sequencing was performed with 10X Genomics Chromium 3’ single-cell RNAseq by Gustave Roussy Institute genomics facility

first: Laurans

last: Salomé

first: Remy

last: Jeulin

first: Benihoud

last: Karim

first: Dupré-Crochet

last: Sophie


title: Phosphorylation of serines 287/288 in DEK regulates cell-type-specific chromatin occupancy and compaction [ChIP-Seq]

city: Suzhou

first: Gongjie

last: Wu

address: Renai Road

institute: Xi'an Jiaotong-Liverpool University

summary: The conserved multifunctional chromatin modulator and oncogene DEK exhibits context-dependent genomic binding and function, but how these activities are regulated in cancer remains poorly understood. Using multi-omics and biochemical approaches, we find that while DEK predominantly occupies promoter-proximal regions in HeLa cells and primary melanocytes, its chromatin binding is dramatically reduced in melanoma cell lines—despite DEK overexpression. We attributed this to CK2-mediated phosphorylation, which governs DEK chromatin association and transcriptional output in a cell-type-specific manner. Phosphoproteomics identified 34 phosphorylation sites, including S287 and S288 within the DEK C-terminal DNA-binding domain. Strikingly, CK2 inhibition and concomitant loss of phosphorylation at S287/S288 triggered DEK redistribution to promoter regions, coinciding with transcriptional repression of oncogenic pathways and global chromatin compaction. Melanoma subtypes showed divergent responses: NRAS-mutant cells displayed dynamic, phosphorylation-dependent DEK redistribution, whereas BRAF-mutant cells lacked detectable DEK binding. Our work establishes DEK as a phosphorylation-sensitive regulator of chromatin states, with CK2-mediated modification orchestrating its tumor-specific regulatory functions. These findings nominate phospho-DEK as a potential biomarker and therapeutic target in melanoma and possibly other cancers.

overall_design:
This submission includes chromatin accessibility, chromatin occupancy, and gene expression profiling from human cancer and non-cancer cell models under defined genetic and pharmacological perturbations.
ChIP-seq samples were generated from HeLa S3 cells, primary human melanocytes, and SK-Mel-103 melanoma cells to profile chromatin binding under control conditions and following treatment with the CK2 inhibitor CX-4945.
Gene expression microarray samples were obtained from parental HeLa S3 cells and DEK knockout (DEK KO, clone 1E7) HeLa S3 cells, allowing comparison of transcriptomic changes associated with loss of DEK expression.

first: Ferdinand

last: Kappes


title: Multi-omics profiling reveals systemic rejuvenation of the aged kidney through senolytic therapy

city: Greifswald

first: Yao

last: Xu

state: State

address: Felix-Hausdorff-Straße 3

country: Germany

institute: University Medicine Greifswald

summary: Cellular senescence is a key driver of kidney aging, leading to functional decline and increased susceptibility to chronic kidney disease. While the senolytic combination of dasatinib and quercetin (D+Q) has shown promise in mitigating age-related pathologies, its long-term effects and underlying multi-level systemic mechanisms in the aging kidney remain poorly defined. Here, we systematically evaluated the long-term effects of D+Q in naturally aged mice using multi-omics approaches. We show that D+Q treatment reduces senescence markers (p16, p21, SA-β-gal), restores the anti-aging protein Klotho, and attenuates renal fibrosis and inflammation. Proteomic profiling reveals that D+Q enhances apoptotic clearance of senescent cells and promotes proliferative and regenerative pathways. Moreover, D+Q reactivates PPARα signaling, improves fatty acid oxidation, and reduces lipid accumulation in aged kidneys. Single-cell transcriptomics further demonstrates that D+Q reverses transcriptional aging signatures across multiple renal cell types and remodels cell-type-specific pathways associated with metabolism, inflammation, and fibrosis. Cell-cell communication analysis reveals that D+Q normalizes the hyperconnected intercellular network in aged kidneys, particularly by modulating inflammation-related signaling. Our findings offer a comprehensive, systems-level understanding of how senolytic therapy restores renal homeostasis, emphasizing its potential as a multifaceted intervention to combat kidney aging.

overall_design: This study included three groups of male mice: young (3/4-month), aged vehicle-treated (20-month), and aged mice receiving D+Q treatment (20-month). All animals were housed in individually ventilated cages in the Animal Center of Affiliated Nanshan Hospital of Shenzhen University under controlled environmental conditions, with a 12-h light–dark cycle and unrestricted access to standard chow and drinking water. Allocation to experimental groups was performed by random number tables, and investigators were kept blinded to group assignments during baseline characterization, administration of interventions and outcome assessments. At the experimental endpoint, mice were deeply anaesthetized by inhalation of isoflurane until breathing ceased and subsequently euthanized by cervical dislocation. Both kidneys were rapidly excised, decapsulated and either fixed or snap-frozen for histological, molecular, proteomic and single-cell RNA-sequencing analyses, as detailed in the following sections.


title: Mackerel muscle cell lines (Mack1 and Mack2) before and after immortalization and in different phenotypes

city: Medford

first: Michael

last: Saad

state: MA

address: 200 College Ave

institute: Tufts University

summary: Bulk RNA sequencing data was obtained from two Atlantic mackerel (Scomber scombrus) muscle cell lines, Mack1 and Mack2, at pre-immortalization and post-immortalization passages. Mack1 cells were at passages 27 and 77 and Mack2 cells at passages 2 and 180. This project investigates how these cells were able to spontaneously immortalize and what transcriptomic changes occur in vitro during different muscle cell phenotypes.

overall_design: The analysis focuses on four distinct cell states of Mack1 cells, namely proliferation (p), serum starvation muscle differentiation (s), serum reduction muscle differentiation (m), and adipogenic-like phenotype induction (f). Mack2 is just in proliferation.

first: Saad

last: Michael K

first: Bromberg

last: Benjamin H


title: The RNA-binding protein TRA2B promotes colon cancer progression by modulating Dicer-dependent miRNA biogenesis.

city: Tokushima

first: Yuki

last: Kuwano

country: Japan

institute: Tokushima University

summary: To identify TRA2B-related miRNAs in colon cancer cells, we examined the miRNA expression profiles of TRA2B-knockdown HCT116 cells

overall_design: To investigate the molecular mechanism underlying the influence of TRA2B in colon cancer cells, TRA2B was reduced by siRNA treatment and then miRNA profiles were measured using a Human miRNA Microarray (Agilent).


title: In vivo Perturb-seq uncovers key obstacles to heart repair and regeneration through direct reprogramming

first: Yang

last: Zhao

address: Peking University, No.5 Yiheyuan Road, Haidian District

institute: Peking University

summary: We investigated the mechanism underlying Calr suppression-enhanced cardiac reprogramming. As CALR functions as an ER-resident chaperone and Ca2+-binding protein, with Ca2+ acting as a second messenger in fate-determining pathways, particularly cardiogenesis we performed RNA-seq on MGTMyoS-induced MICFs transduced with shCalr or shNT at days 7 and 14 in vitro

overall_design: RNA-seq profiling on MICFs with Calr knock down or shNT at day0, day7, day14.

first: Yihong

last: Cai

last: Yang

first: Junbo

first: Ruohan

last: Ding


title: Identifying clones in myelodysplastic syndromes by using single-cell RNA sequencing

city: Be'er Sheva

first: Roy

last: Pollak

address: David Ben Gurion Blvd 1

department: Life sciences

country: Israel

institute: Ben Gurion University

summary: We employed single-cell RNA sequencing to sequence hematopoietic stem cells (HSC) derived from bone marrow aspirations of seven patients with myelodysplastic syndrome (MDS) and four control subjects. Based on gene expression and pathway-enrichment analyses, we inferred chromosomal Y deletion and characterized the transcriptome of chromosome Y-deleted HSC in comparison to chromosomally intact HSC. Our results support the premise that chromosome Y deletion is not a benign aging phenomenon and highlight the importance of upregulation of the anti-apoptotic pathway in the pathogenesis of MDS.

overall_design: Eleven subjects who were evaluated for MDS were recruited to the study. Fresh bone marrow aspirations were collected and sorted for HSC by using fluorescence-activated cell sorting (FACS). The Smart-Seq2 protocol was applied to sequence 1,150 HSC. Reads were mapped to the human genome, and gene expression levels were calculated. After application of quality control measures, the expression matrix included 1,101 cells and 26,199 genes.

first: Hadas

last: Ner-Gaon

first: Leonid

last: Olender

first: Itai

last: Levi

first: Roi

last: Gazit

first: Tal

last: Shay

first: Uri

last: Greenbaum


title: Iron Limitation-Induced Modulation of Transcription in Chlamydia trachomatis

city: Lausanne

first: Daniel

last: Rodriguez Rozo

address: Avenue de Cour 10

department: IMUL

country: Switzerland

institute: CHUV

summary: Chlamydia trachomatis (C. trachomatis) is an obligate intracellular bacterial pathogen responsible for trachoma and sexually transmitted infections. Like other Chlamydiota members, such as Waddlia chondrophila, C. trachomatis undergoes a biphasic developmental cycle alternating between infectious elementary bodies (EBs) and replicative reticulate bodies (RBs). Under stressful conditions, RBs differentiate into non-dividing aberrant bodies (ABs), a reversible state associated with persistence. Here, we investigate how early iron deprivation affects transcriptional regulation in C. trachomatis. We performed RNA sequencing to compare transcriptional profiles between EBs and RBs and between RBs and ABs induced by iron chelation with 2,2′-bipyridyl at 8 hpi. In EBs, 29% of genes were downregulated and 30% upregulated compared to RBs, revealing extensive transcriptional remodeling. At 24 hpi, ABs displayed downregulation of two-component system (TCS) genes (atoS, atoC, and chxR), while genes encoding inclusion membrane proteins (Incs) and the trpRBA operon were upregulated. These findings show how C. trachomatis adapts transcriptionally to iron deprivation, revealing stress- and time-dependent changes in metabolism, stress responses, and host–pathogen interactions. Persistence alters the developmental cycle while maintaining inclusion integrity and modulating host interactions, contributing to chronic infection. Uncovering the mechanisms driving persistence may offer crucial insights into chlamydial pathogenesis.

overall_design: RNA-seq was used to profile gene expression across the developmental stages of C. trachomatis. HEp-2 cells were infected and sampled at time points corresponding to reticulate bodies (24 hpi), elementary bodies (72 hpi), and aberrant bodies induced by BPDL-mediated iron limitation (added at 8 hpi and harvested at 24 hpi). Three biological replicates were generated for each condition.

first: Simone

middle: E

last: Adams

first: Carole

last: Kebbi-Beghdadi

first: Trestan

last: Pillonel

first: Sébastien

last: Aeby

first: Gilbert

last: Greub


title: Transcriptional mechanisms of sex-biased gene expression and their connections to disease-associated variation

city: Durham

first: Angela

middle: G

last: Jones

state: NC

address: 0048B CARL, Box 3053 DUMC

department: MGM

institute: Duke Unversity

summary: Humans display sexual dimorphism across many traits, but little is known about underlying genetic mechanisms and impacts on disease. We utilized single-cell RNA-seq of 480 lymphoblastoid cell lines (LCLs) to identify 1200 genes with significantly sex-biased expression. While reproducibility was highest among LCL datasets, 71% were found to be sex-biased in at least one GTEx tissue, with a core dataset of 21 genes displaying sex-biased expression across all datasets and tissues examined. While 7.7% of sex-biased genes can be directly explained by differences in the number of sex chromosomes, most sex-biased genes (79%) are targets of transcription factors that display sex-biased expression. FOSL1, ZNF730, ZFX, and ZNF726 appear to make the largest contribution to this based on machine learning and linear modeling approaches, and all four of these transcription factors are regulated by the number of X chromosomes. Further, by testing the difference in genetic effect size (β) of conditionally independent expression quantitative trait loci (eQTL) identified in each sex separately, we identified 2,390 sex-biased eQTL (sb-eQTL) across the genome, but evidence of replication in an independent dataset was modest. However, permutation analysis demonstrated that sb-eQTL identified using real sex was more likely to have concordant direction of effect. Further exploratory analysis revealed that these sb-eQTL are enriched in over 100 GWAS phenotypes, including many loci associated with female-biased autoimmune diseases such as multiple sclerosis. Our results demonstrate widespread genetic impacts on sexual dimorphism and identify possible mechanisms and clinical targets for sex differences in diverse diseases.

overall_design: 384 human lymphoblastoid cell lines (LCLs) from the 1000 Genomes project were pooled and analyzed using droplet-based single-cell RNA-seq in 4 samples.

first: Guinevere

last: Connelly

first: Dennis

middle: C

last: Ko


title: Simultaneous mapping of RNA secondary, tertiary, and quaternary structure in living cells by multi-site DMS probing II

first: Anthony

last: Mustoe

overall_design: To discover protein binding regions in the 7SK RNA and corroborate our DMS mapping, we performed RNP-MaP.

first: Irfana

last: Saleem

first: Thomas

last: Miller

first: Lucas

last: Kearns

first: David

last: Mitchell

first: Ritwika

last: Bose

first: Chase

last: Weidmann


title: The B cell receptor-mTOR signaling axis restricts the accumulation of lung tissue-resident memory B cells following influenza infection

city: St. Louis

first: Brian

last: Laidlaw

address: 425 S Euclid Ave

institute: Washington University in St. Louis

summary: Lung tissue-resident memory B (BRM) cells are important in establishing protective immunity against respiratory pathogens. However, the pathways regulating the accumulation of lung BRM cells are poorly understood. Here we show that the transcription factors NFATC1 (nuclear factor of activated T cells 1) and EGR2 (early growth response 2) repress lung BRM cell accumulation following influenza infection, whereas IKZF1 (IKAROS family zinc finger 1) enhances it. IKZF1 restrains B cell receptor (BCR) and mTOR (mechanistic target of rapamycin) signaling and is highly expressed in lung BRM cells during their development. Lung BRM cells display reduced expression of genes associated with BCR and mTOR signaling pathways. Overexpression of a degradation-resistant form of SYK downstream of the BCR restricted lung BRM cell accumulation. Conversely, inhibition of mTOR using rapamycin reduced NFATC1 and EGR2 expression and promoted lung BRM cell accumulation. These findings establish how the BCR–mTOR signaling axis regulates mucosal B cell responses following influenza infection.

overall_design: We performed gene expression profiling using data obtained from RNA-seq analysis of germinal center-derived memory B cell subsets defined based on their expression of CXCR3 and whether they were labeled by in vivo injection of CD45.2 on day 30 post influenza PR8 infection.

first: Laidlaw

last: Brian


title: Blood-flow restriction resistance training improves skeletal muscle mitochondrial oxidative capacity and cardiovascular risk factors in type 2 diabetes

city: Ithaca

first: Philip

last: Ruppert

state: NY

address: 244 Garden Avenue

department: Division of Nutritional Sciences

institute: Cornell University

summary: Impaired muscle strength and mitochondrial functionality are hallmarks of type 2 diabetes (T2D). Conventional combined resistance/endurance exercise training has limited efficacy to simultaneously improve muscle function and metabolism. We examined whether low-load blood flow restriction training (BFRT) increases both muscle strength and mitochondrial oxidative capacity in T2D. Over 12 weeks, BFRT and conventional resistance training (CREST) similarly improved muscle strength despite lower workload in BFRT. Uniquely, BFRT enhanced muscle and adipose tissue oxidative capacity and increased muscle mitochondrial content. Transcriptomic profiling revealed more pronounced changes, particularly in angiogenesis-linked pathways, upon BFRT. BFRT also preferentially led to reductions in visceral adipose tissue volume and waist circumference, whereas CREST more effectively decreased subcutaneous adipose tissue volume. Both interventions lowered resting heart rate and diastolic blood pressure. These findings position BFRT as promising low-load exercising strategy to simultaneously improve mitochondrial oxidative capacity, muscle strength and body composition in individuals with T2D.

overall_design: People with T2D  completed 12 weeks of either blood flow restriction training (BFRT) or classical resistance training (CREST); biopsies of the vastus lateralis have been obtained 24 h pre-intervention and 72 h post-intervention.

first: Nina

last: Trinks

first: Sofiya

last: Gancheva

first: Martin

last: Schön

first: Maximilian

last: Huttasch

first: Kalliopi

last: Pafili

first: Lucia

last: Mastrototaro

first: Bedair

last: Dewidar

first: Yuliya

last: Kupriyanova

first: Christian

last: Herder

first: Klaus

last: Strassburger

first: Oana

middle: P

last: Zaharia

first: Sander

last: Kersten

first: Joris

last: Hoeks

first: Sandra

last: Trenkamp

first: Vera

last: Schrauwen-Hinderling

first: Patrick

last: Schrauwen

first: Dominik

last: Pesta

first: Michael

last: Roden


title: Next Generation Sequencing Facilitates Quantitative Analysis of the Role of Ddx21 in hematopoietic stem cells [RNA-seq]

city: Chongqing

first: Yalan

last: Xiao

address: No. 1 Yixueyuan Road, Yuzhong District, Chongqing

institute: Chongqing Medical University

summary: Hematopoietic stem cell (HSC) homeostasis is critically dependent on precisely regulated protein synthesis, with the nucleolus serving as the principal orchestrator of ribosome biogenesis and cellular stress sensing. DDX21, a nucleolus-localized DEAD-box RNA helicase, serves as a master regulator in ribosomal DNA (rDNA) transcription and ribosome assembly. Nevertheless, its precise function in adult hematopoiesis remains incompletely understood. Here, we reveal that conditional knockout of Ddx21 in the murine hematopoietic system triggers collapse of HSC homeostasis, characterized by loss of quiescence, impaired self-renewal capacity, and raised apoptosis. Mechanistically, we demonstrate that DDX21 physically associates with both rDNA loci and promoters of ribosomal protein genes. Its loss disrupts ribosome production, causes nucleolar stress, culminates in p53-mediated cell cycle arrest and apoptosis in HSPCs. Taken together, our study establishes DDX21 as a master regulator in HSCs, integrating ribosome biogenesis with nucleolar stress induction and hematopoietic homeostasis.

overall_design: HSCs from Ddx211fl/fl and Ddx211fl/fl,CAG-CreERT2 mice were sorted and then subjected to RNA-seq

first: Zhigang

last: Li

first: Yu

last: Hou


title: Genome-wide Maps of Chromatin State in Control and DDX21-deficient Hematopoietic Stem Cells [CUT&Tag]

summary: To identify chromatin state involved in the DDX21 regulating gene expression of hematopoietic stem cells (HSC) in mice, we performed DDX21 CUT&Tag in HSCs freshly sorted from mice.

overall_design: About 10,000 HSCs (Lin-Sca-1+c-Kit+CD48-CD150+) in bone marrow were isolated by FACS from C57BL/6 mouse. Genomic DNA profiles of HSCs treated with DDX21 antibody were generated by deep sequencing, using Illumina NovaSeq 6000.


title: Maping the thm1 mutation via bulked segregation analysis (BSA)

city: Beijing

first: Lei

last: Gao

address: East Building of Resources, No. 5 Yiheyuan Road, Haidian District, Beijing

summary: To map the thm1 mutation via bulked segregation analysis (BSA), the thm1 mutant was backcrossed once with WT to generate the BC1F2 population. Phenotypes in the second filial generation (BC1F2) progeny were analyzed for their ability to trigger HSCE in response to heat shock. Three DNA pools, each consisting of 25 plants showing thm1‑like HSCE, 25 plants showing WT‑like HSCE, and 18 WT reference individuals, were sequenced.Approximately 50 BC1F2 lines exhibiting the lowest and highest HSCE phenotypes were selected and pooled to generate thm1-like and WT-like bulks, respectively. Genomic DNA was extracted from rosette leaves of the bulks and WT using a 2% cetyltrimethylammonium bromide (CTAB) method. The WT DNA was used as an internal reference.

overall_design: Whole-Genome Sequencing of Arabidopsis thaliana leaves from WT,thm1_like and WT_like, to map the thm1 mutation site via bulked segregation analysis

first: Wang

last: Tian


title: AN465: A Novel Therapeutic Targeting METTL3 Inhibition in Hand, Foot, and Mouth Disease

city: Guangxi

first: Jun

last: Meng

address: ShuangYong Road

institute: Guangxi Medical University

summary: Abstract: Hand, foot, and mouth disease (HFMD) is a widespread viral infection predominantly affecting young children, for which specific therapies remain elusive despite extensive research efforts. N6-methyladenosine (m6A) modifications, prevalent in eukaryotic mRNA and long non-coding RNAs, are also found in various viral RNAs. A nuclear methyltransferase complex, including METTL3 as the catalytic subunit, catalyzes these m6A modifications co-transcriptionally, functioning as m6A 'writers'. Notably, enterovirus 71 (EV71) RNA undergoes m6A modification during infection, mediated by METTL3, influencing virus replication. We hypothesized that inhibiting METTL3 could directly suppress EV71 replication through m6A modifications. Despite the renown of STM2457 as a METTL3 inhibitor, our preliminary experiments indicated its ineffectiveness against EV71. Leveraging a compound library tenfold larger than that in STM2457's discovery, we identified AN465 as a potent METTL3 inhibitor. Through rigorous experiments and analyses, we demonstrate that AN465 surpasses STM2457 in inhibiting EV71 replication by effectively targeting METTL3. Our findings propose AN465 as a promising therapeutic candidate for combating HFMD by curbing EV71 replication.

overall_design: The experimental design of this study involved an advanced high-throughput virtual screening approach to identify a novel METTL3 inhibitor capable of suppressing EV71 replication by targeting m6A modifications on EV71 RNA. Utilizing two large compound libraries, Specs and ChemDiv, the team screened a massive library of 2.6 million compounds, representing a significant quantitative and qualitative leap compared to previous efforts. This exhaustive and detailed search aimed to discover bioactive small molecules with specific biological activities, ultimately leading to the identification of AN-465/42162872 (AN465), a novel METTL3 inhibitor. Rigorous experiments confirmed that AN465 significantly outperformed STM2457 in inhibiting EV71 replication by effectively targeting METTL3, validating the study’s hypothesis and demonstrating the superiority of the screening method in drug discovery.


title: L-type voltage-gated Ca2+ channels control T cell killing via non-canonical Hedgehog signaling

city: Cambridge

first: Tim

last: Halim

address: Robinson Way

department: CRUK CI

country: United Kingdom

institute: University of Cambridge

summary: Cytotoxic CD8+ T lymphocytes (CTLs) efficiently eliminate infected and cancerous cells throughout the body. Hedgehog signaling is critical for CTL-mediated killing. Remarkably the pathway cannot be induced canonically by extracellular Hedgehog ligands but instead is activated by the T cell receptor (TCR). How the Hedgehog pathway is mechanistically activated downstream of the TCR is unknown. Here we show that L-type voltage gated Ca2+ (Cav1) channels at the plasma membrane downstream of the TCR control an extracellular Ca2+ influx leading to induction of the Hedgehog transcription factor Gli1 which is essential for efficient CTL killing in vitro and in vivo. This novel non-canonical Hedgehog pathway is independent of canonical signaling and represents a primary mechanism of Gli1 induction in naive CD8+ T cells, while CTLs can also activate Gli1 via MAPK. We show that Cav1 channel-controlled Gli1 induction is functionally required for CTL killing in mice and humans and other cytotoxic lymphocytes. Importantly, we demonstrate that killing capacity can be amplified using a small molecule Cav1 channel agonist or by overexpression of a gain-of-function Cav1 subunit. Our findings indicate that Gli inhibitors, currently in clinical trials as anti-cancer therapeutics, likely inhibit the cytotoxic anti-tumor response. On the other hand, our work opens up the possibility to enhance CTL performance in the clinic by selectively increasing Ca2+ flux via Cav1 channels, for example in the context of CAR T cell therapy.

overall_design: Bulk RNA-Seq of murine naive CD8+ T cells following 3h and 24h of plate-bound CD3/CD28 stimulation in the presence or absence of FPL 64176

first: Flavio

last: Beke

first: Joachim

last: Hanna

first: Chrysa

last: Kapeni

first: Louise

last: M. O'Brien

first: Valentina

last: Carbonaro

first: Nico

last: Mueller

first: Chandra

last: Chilamakuri

first: Maike

last: de la Roche


title: Astrocyte glucocorticoid receptor signaling restricts neuronal plasticity [RNA-seq]

city: Boston

first: Bruno

last: Gegenhuber

state: Massachusetts

address: 220 Longwood Ave

department: Neuroscience

institute: Harvard Medical School

summary: Sensory experience refines neural circuits during critical periods of postnatal development. Although neuronal activity is known to orchestrate the circuit wiring that underlies this process, the environmental cues that restrain developmental plasticity as animals mature are less clear. Here, we examine the experience-dependent maturation of the mouse primary visual cortex (V1) across postnatal development using paired single-cell transcriptomic and chromatin accessibility sequencing. In addition to identifying the activity-dependent gene programs that emerge within each cortical cell type, we find that light exposure drives astrocyte maturation through cell type-specific recruitment of the glucocorticoid receptor (Nr3c1/GR) to chromatin. Astrocyte GR signaling activates an extensive gene regulatory program that is partially conserved in human brain development and promotes maturation processes that may trigger critical period closure. Collectively, these findings reveal that astrocyte GR signaling restricts neuronal plasticity. Glucocorticoid regulation of astrocyte maturation may also contribute to the effects of early-life stress across the brain, and the disruption of this process may increase susceptibility to neuropsychiatric disease.

overall_design: RNA-seq was performed on V1 astrocyte nuclei isolated from P14, P21, and P28 DR and NR GRfl/fl animals that were injected at P0-P1 with AAV2/5-GfaABC1D-ΔCre-T2A-EGFP-KASH-4x6T (GR-Con) or AAV2/5-GfaABC1D-Cre-T2A-EGFP-KASH-4x6T (GR-KO).

first: Takuma

last: Sonoda

first: Lisa

last: Traunmüller

first: Christopher

last: Davis

first: Shon

last: Koren

first: Eric

last: Griffith

first: Chinfei

last: Chen

last: Greenberg


title: Promoter mutagenesis and a massively parallel reporter screen of the MAPT locus identifies cis-regulatory elements and genetic variation effects [BAC MPRA]

city: Huntsville

first: Jesse

middle: Nicholas

last: Cochran

state: AL

address: 601 Genome Way

institute: HudsonAlpha Institute for Biotechnology

summary: Tau neurofibrillary tangles are a hallmark of several neurodegenerative diseases called tauopathies, including frontotemporal dementia and Alzheimer's Disease. Ongoing clinical trials for tauopathies seek to reduce Tau in the brain through immunotherapy, antisense oligonucleotides, and siRNA. MAPT codes for Tau, therefore understanding how the MAPT gene is regulated and the effect of genetic variation at its regulatory elements is likely to have high relevance for tauopathies. We screened a ~3 Mb region including the MAPT locus using 2 different massively parallel reporter assay (MPRA) strategies in KOLF2.1J h-NGN2 neurons and HEK293FT cells, identifying previously unannotated cis-regulatory elements (CREs). Using CRISPR interference (CRISPRi) in mixed neuron cultures, we identified a new CRE for MAPT, as well as 2 CREs for another nearby gene of interest, KANSL1. Known genetic variation from the Alzheimer's Disease sequencing project was tested in a separate MPRA at the top CREs near the MAPT gene, identifying variants with altered regulatory effects including those at previously identified CREs for MAPT. Using a saturation mutagenesis screen of a 2,000 bp region encompassing the MAPT promoter, we assessed regulatory effects of each possible single nucleotide variant in this region. We identified several neuron-specific regulatory variant effects at this region, including a high confidence binding site for the transcription factors EGR2, ZBTB14 and TCLF5 at a region of high MPRA activity and genetic conservation.

overall_design: LentiMPRA in KOLF2.1h-NGN2 Neurons (transduced on day 14, collected on day 18) and HEK293FT cells using sheared BACs as test sequences

first: Rebecca

last: Hauser

first: Nicholas


title: The ac4C acetyltransferase Nat10 promotes RNA stability to support hematopoietic stem cell fate [acRIP-seq]

first: Yue

last: Huo

address: Dongcheng District, Dongdan 3 Tiao 5 Hao

institute: Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College

summary: The control of gene expression program is believed to underlie hematopoietic stem cell (HSC) homeostasis. However, a comprehensive molecular understanding of the RNA level regulation is currently lacking. Here we provide evidence that the HSC-associated transcripts have longer 3’UTRs and more enriched AU-rich elements (AREs), which were recognized by Nat10. Nat10 is highly expressed in HSC and its deficiency disrupts HSC self-renewal and long-term maintenance. In contrast to current model that invoke ac4C modification to mediate downstream effects, we find Nat10 recruits ribosomes to mRNA 3’UTRs and protects them from degradation by ZFP36-CNOT RNA decay complex. Nat10 depletion leads to reduction of mRNA hail-life, and both wild-type and catalytically inactive Nat10 promote the stability of mRNA with AREs. We present a model that Nat10 regulates mRNA stability as an ARE-binding protein rather than ac4C writer in HSC, a mechanism is required for hematopoietic homeostasis, highlighting the importance of transcript-specific characteristics determining the contribution of Nat10 in HSC fate.

overall_design: To investigate ac4C modification on mRNA, we performed the acRIP-seq experiment in 32D cells

first: Weiqian

last: Li

first: Zhaoru

last: Zhang


title: Cross-Platform Transcriptomic Validation Identifies SERPINB2 as a Chondrogenic Biomarker and Reveals Coordinated Suppression of DPP4 During Cartilage Lineage Commitment

city: Ciudad de México

first: Julio

last: Granados-Montiel

state: Ciudad de México

address: Las Fuentes 17

department: Investigación

country: Mexico

institute: Universidad La Salle

summary:
Human mesenchymal stem cells (hMSCs) are a promising source for regenerative medicine, especially mesodermal lineages. Clinical applications require an understanding of the mechanisms for transcriptional control to maintain the desired cell type. The aim of this study was to identify novel markers for differentiation of cartilage with the use of Kartogenin, by RNA analysis using microarray technology.
Results: We found genes that were either up- or downregulated on differentiation, when compared to PI hMSC (control) at day 0 of differentiation.  SERPINB2, was significantly upregulated in the cartilage groups,The most significant hits were validated by quantitative polymerase chain reaction

overall_design: Commercial human bone-marrow derived primary mesenchymal stem cells were purchased from SINREG laboratories. Cells were differentiated in vitro in 2-dimensional cultures using Kartogenin. After 21 days of culture, whole RNA was extracted and analyzed via Clariom D microarrays.


title: SlCAX3 drives the formation of crystal idioblasts for tomato ion compartmentalization under salt stress

city: Urumqi

first: Ruiqiang

last: Xu

state: Xinjiang Uygur

address: No.311, Nongda East Road, Shaibak District, Urumqi, Xinjiang

institute: Xinjiang Academy of Agricultural Sciences

summary: Soil salinization is a primary constraint on global agricultural productivity. While the mechanisms of root ion exclusion and whole-plant long-distance transport are well established, how photosynthetic leaf tissues complexly adapt to high-salinity microenvironments at the spatial and cellular level remains a critical blind spot in plant stress biology. We integrated spatial transcriptomics (ST-seq) and single-nuclei RNA sequencing (snRNA-seq) to construct a high-resolution spatiotemporal transcriptomic atlas of tomato leaves subjected to long-term salt stress (LTSS). We combined pseudotime trajectory inference, multi-omics deconvolution, elemental analysis, and CRISPR-Cas9-mediated genetic validation to elucidate cellular responses. LTSS induces heterogeneous transcriptional remodeling across leaf tissues, primarily reinforcing vascular structural stability while suppressing mesophyll photosynthesis. Concurrently, LTSS triggers the explosive proliferation and developmental reprogramming of palisade mesophyll cells into highly specialized crystal idioblasts (CIs). This cell fate transition is orchestrated by enhanced cell wall remodeling and intensive microenvironmental crosstalk. Genetic analyses establish that the vacuolar transporter SlCAX3 is essential for CI formation. Sequence and structural evaluations indicate that SlCAX3 evolutionarily lost its conserved autoinhibitory N-terminal domain, a structural specialization that may support high-flux ion transport. Although SlCAX3-driven CI formation does not reduce total foliar Na⁺ content, it facilitates localized ion sequestration within CI vacuoles, which is associated with mitigated oxidative damage and cytotoxicity in adjacent photosynthetic tissues. This study demonstrates that tissue-level salt adaptation in tomato involves spatially coordinated transcriptional responses and localized biomineralization. The identification of SlCAX3's atypical architecture provides a functional supplement to the conserved CAX1/3 transport system in land plants. Our findings highlight localized cell-state transitions and ion sequestration as an effective tissue-level strategy for mitigating salt stress, offering strategic genetic targets for breeding climate-resilient crops.

overall_design: snRNA-seq and spRNA-seq profiling of wildtype tomato (cv. M82) under salt stress over 30d.


title: Effect of L-PGDS loss on cellular response during cuprizone induced demyelination

city: Houston

first: Jian

last: Hu

state: Texas

address: 1881 East Road

department: Cancer Biology

institute: MD Anderson Cancer Center

summary: L-PGDS is a secretory oligodendrocyte protein; we aimed to determine how the loss of this protein affected the transcriptome of different cell populations during demyelination.  We found that very distinct changes in the microglial transcriptome following L-PGDS loss. Specifically, these microglia adopt an aged, dysfunctional gene signature characterized by the increase in ribosomal genes.

overall_design: 11  weeks old WT and L-PGDS KO mice were fed a 0.2% cuprizone diet for 5 weeks.  Mice were then perfused with PBS transcardially, the brain removed and sectioned to reveal corpus callosum. Brain sections were then flash frozen in liquid nitrogen. Corpus callosum section was homogenized to isolate nuclei. Homogenate was stained with DAPI and PI to enable fluorescence-activated nuclei sorting. Purified nuclei were used to generate 10X v3.1 dual index libraries for single nuclei RNA sequencing.

first: Takese

last: McKenzie


title: Astrocyte glucocorticoid receptor signaling restricts neuronal plasticity [CUT&RUN]

first: Bruno

last: Gegenhuber

overall_design: GR CUT&RUN was performed on V1 nuclei isolated at P14 from 1) normal-reared (NR) Olig2-Cre;Sun1-GFP animals (oligodendrocytes), 2) NR Vgat-Cre;Sun1-GFP animals (inhibitory neurons), 3) NR Vglut1-Cre;Sun1-GFP animals (excitatory neurons), 4) dark-reared and NR WT mice that were injected at P0-P1 with AAV2/5-GfaABC1D-ΔCre-T2A-EGFP-KASH-4x6T (astrocytes), and 5) NR GRfl/fl mice that were injected at P0-P1 with AAV2/5-GfaABC1D-ΔCre-T2A-EGFP-KASH-4x6T (GR-Con) or AAV2/5-GfaABC1D-Cre-T2A-EGFP-KASH-4x6T (GR-KO). NFIA CUT&RUN was performed on V1 nuclei isolated at P14 from 1) NR Olig2-Cre;Sun1-GFP animals (oligodendrocytes), 2) NR Vgat-Cre;Sun1-GFP animals (inhibitory neurons), 3) NR Vglut1-Cre;Sun1-GFP animals (excitatory neurons), and 4) NR WT mice that were injected at P0-P1 with AAV2/5-GfaABC1D-ΔCre-T2A-EGFP-KASH-4x6T (astrocytes). GR CUT&RUN was also performed on 50,000 or 250,000 3T3 cells that were stimulated for 2 hours with vehicle (0.01% ethanol) or 200 nM dexamethasone (DEX).

first: Michael


title: Collective unstructured interactions drive chromatin binding of transcription factors. [CUT&RUN]

city: Berkeley

first: Robert

last: Tjian

state: CA

address: University of California, Berkeley 450 Li Ka Shing #3370

department: MCB

institute: UC Berkeley

summary: Eukaryotic transcription factors (TFs) contain both structured DNA-binding domains (DBDs) and intrinsically disordered regions (IDRs). While the structures and sequence preferences of DBDs have been extensively characterized, the role of IDR-mediated interactions in chromatin binding and nuclear organization remains poorly understood, in part because these interactions have been difficult to measure in living cells. Here, we use a recently developed single-molecule technique, proximity-assisted photoactivation (PAPA), to investigate how IDRs influence TF associations with each other and with chromatin, focusing on the factors Sp1 and Klf1. We find that the number and patterning of aromatic and basic residues within IDRs govern both TF self-association and chromatin binding. Unexpectedly, the isolated DBD of Sp1 binds chromatin very weakly and non-specifically. The isolated IDR, by contrast, interacts poorly with chromatin-bound wild-type Sp1, yet this interaction is enhanced when even minimal DNA-binding capacity is restored. Strikingly, replacing Sp1's native DBD with those of heterologous TFs recovers both IDR-mediated interactions and chromatin association, despite divergent sequence preferences. PAPA measurements also reveal extensive heterotypic interactions between wild-type Sp1 and other TFs. Together, these results establish PAPA as a powerful method for studying unstructured interactions in their native context and suggest that IDRs participate in widespread cooperative associations scaffolded by transient DBD-DNA contacts, which concentrate disordered regions along chromatin. In contrast to classical models, we propose that TF specificity in vivo emerges not solely from DBD sequence preferences, but from a constellation of weak, dynamic, and diverse interactions mediated by IDRs.

overall_design: Cut and Run of V5-Halo-SP1 endogenous knock in U2-OS cells (parental cell line) and various inducible cell lines, using anti-FLAG and anti-V5 antibodies

first: Claudia

last: Cattoglio

first: Natalie

middle: T

last: Tang

first: Abrar

last: Abidi

first: Gina

last: Dailey

first: Thomas

last: Graham


title: GLS1 governs vascular smooth muscle cell phenotypic switching and aortic dissection via glutamate metabolism

city: Nanjing

first: Hailong

last: Cao

state: Jiangsu

address: 87 Dingjiaqiao

department: Department of Cardiac Surgery

institute: Zhongda hospital

summary: To evaluate the roles of glutaminase 1 (GLS1) in vascular smooth muscle cells (VSMCs) phenotypic switching and aortic dissection (AD). Integrative transcriptomic analyses were performed to identify the candidate genes involved in VSMC phenotypic switching in AD. The expression of GLS1 in VSMCs was assessed by qRT-PCR, Western blot and immunofluorescence. RNA-sequencing analysis was performed to recapitulate possible changes in the transcriptome profile of GLS1 in VSMCs. We identified GLS1 as a potential regulator in AD. GLS1 expression was significantly downregulated in VSMCs from both human AD aortic tissues and mouse models. Mechanistically, down-regulation of GLS1 impaired glutamate metabolism, leading to reduced levels of glutathione and α-ketoglutarate, thereby promoting mitochondrial dysfunction and accumulation of reactive oxygen species, which activated the PI3K/AKT pathway and ultimately triggered VSMC phenotypic switching. These findings revealed a critical role of GLS1-mediated glutamate metabolism in VSMC phenotypic switching and suggest a promising therapeutic target for AD.

overall_design: RNA-seq profiling of human aortic smooth muscle cells (HASMCs) infected with adenovirus containing empty vector (Ad-Vector) or GLS1-encoding plasmids (Ad-GLS1), followed by treatment with PDGF-BB or control (n = 3)

first: Wei

last: Xie


title: microRNA146a suppresses pancreatitis via cross-talk with NF-kB and ASC-containing inflammasomes

city: Adelaide

first: Kristen

last: Feher

address: North Terrace

institute: SAiGENCI

summary: Acute pancreatitis (AP) is a potentially lethal pancreatic inflammatory disease with no effective therapeutic options. Here, we identify microRNA-146a (miR-146a) as a targetable potent negative regulator of pancreatic inflammatory responses and associated tissue damage. Genetic ablation of miR-146a in mice exacerbated cerulein-induced AP, evidenced by elevated inflammatory cell infiltrates and acinar cell death, the former associated with activated macrophage infiltrates and upregulation of cytokines (e.g. IL-1b) and chemokines (e.g. CXCL2). RNA-seq analysis on cerulein-treated mouse pancreas tissues indicated that miR-146a deficiency upregulated molecular pathways for intrinsic apoptosis, actin cytoskeleton organisation and inflammation, while gene networks for mitochondrial function and cellular respiration were downregulated. .

overall_design: RNA transcriptome sequencing to profile the differential regulation of gene networks in pancreatic tissues of WT and miR146a knockout (KO) mice subjected to cerulein-induced acute pancreatitis (AP) and compared to PBS controls.

first: Mohamed

middle: I

last: Saad

first: Brendan

last: Jenkins


title: Single-cell Transcriptomics Reveal Signatures of Altered B cell Response and T cell Senescence in Geriatric Non-responders to COVID-19 mRNA Vaccination

city: Cleveland

first: Mark

last: Cameron

state: OH

address: 10900 Euclid Ave

department: Population and Quantitative Health Sciences

institute: Case Western Reserve University

summary: The immune response to COVID-19 vaccines is diminished in older individuals. To understand the underlying immunobiology, we analyzed single-cell RNA-seq data from PBMCs of SARS-CoV-2 naïve nursing home residents with varying humoral responses following BNT162b2 vaccination and validated via flow cytometry. Responders (>4500 AU/mL anti-spike titers) showed enrichment for naïve B cell (IGHD, BACH2, CD22) and naïve CD4 T cell and early Tfh-related genes (CCR7, TCF7, LEF1, IL6ST, TGFBR2). Non-responders (<20 AU/mL) displayed elevated markers of T cell senescence (KLRG1, CCL4, CCL5, IL32), immune exhaustion (PD-1), and inflammation (TNF-α, IFN-γ). Flow cytometry revealed reduced CD4 T and B cell frequencies but higher CD8 T and NK cells in non-responders. Despite reduced B cell frequency, non-responders upregulated plasma B cell genes (PRDM1, XPB1, IRF4), suggesting dysregulated B cell differentiation. Our findings point to impaired lymphocyte responses and increased immunosenescence in non-responders, emphasizing the need for enhanced vaccine strategies in aging populations.

overall_design: Peripheral blood mononuclear cells (PBMCs) were isolated from human donors two weeks after receiving the BNT162b2 mRNA COVID-19 vaccine. PBMCs were obtained from buffy coats using Ficoll-Hypaque gradient centrifugation, cryopreserved, and later thawed for analysis. Approximately 10,000 PBMCs per sample were processed using the 10x Genomics Chromium Single Cell 3’ v3.1 platform to generate single-cell transcriptomes. Libraries were sequenced on an Illumina NovaSeq 6000 using an S2 flow cell, targeting ~40,000 reads per cell.

first: Jeeda

last: Ismail

first: Banumathi

last: Tamilselvan

last: Rubsamen

first: Jilian

last: Petrosky

first: Brian

last: Richardson

first: Leah

last: Zagore

first: Oladayo

last: Oyebanji

first: Debbie

last: Keresztesy

last: Payne

first: Dennis

last: Wilk

first: Lenore

last: Carias

first: Htin

last: Aung

first: Kerri

last: St. Denis

first: Maeghanm

last: Sheehan

first: Brigid

last: Wilson

first: Alejandro

last: Balazs

first: Stefan

last: Gravenstein

first: Christopher

middle: L

last: King

first: David

last: Canaday

first: Cheryl


title: The ac4C acetyltransferase Nat10 promotes RNA stability to support hematopoietic stem cell fate [Ribo-seq]

first: Yue

last: Huo

overall_design: We investigated the influence of ac4C modification and NAT10 binding on mRNAs by employing Ribo-seq to evaluate mRNA translation in 32D cells.

first: Weiqian

last: Li

first: Zhaoru

last: Zhang


title: Collective unstructured interactions drive chromatin binding of transcription factors. [ChIP-Seq]

first: Robert

last: Tjian

overall_design: Two independent ChIP-seq replicates of endogenous CTCF in wild type U2-OS cells with relative input controls.

first: Claudia

last: Cattoglio

first: Natalie

last: Tang

first: Abrar

last: Abidi

first: Gina

last: Dailey

first: Thomas

last: Graham


title: miR-99b-5p inhibition drives apoptosis and tumor shrinkage in triple-negative breast cancer: Functional characterization through AGO2-RIP-seq and mechanistic insights

city: Ankara

first: Efe

last: Dalli

address: Dogol Street

country: Turkey

institute: Ankara University

summary: Background: Dysregulated microRNAs (miRNAs) are critical contributors to breast cancer biology, yet the functional roles of many remain incompletely understood. miR-99b-5p has been widely characterized as a tumor-suppressive miRNA in numerous cancer types, where its expression is consistently reduced in tumors compared with normal tissues. In contrast, our analyses of breast cancer datasets revealed a unique expression pattern: miR-99b-5p is significantly upregulated in breast tumors, suggesting a context-dependent oncogenic function. In this study, we identified miR-99b-5p as an oncogenic driver in triple-negative breast cancer (TNBC). Methods and Results: TCGA-based expression profiling confirmed its elevated levels in breast tumors. Functional assays demonstrated that downregulation of miR-99b-5p in TNBC cells inhibits proliferation and induces apoptosis, indicating a critical role in sustaining tumor cell survival. To elucidate the molecular mechanisms underlying this activity, we performed AGO2-RNA immunoprecipitation followed by high-throughput sequencing (AGO2-RIP-Seq), enabling unbiased identification of miR-99b-5p-associated transcripts. Pathway enrichment analyses revealed that its direct targets converge on apoptotic regulation, cell-cycle control, and ubiquitin-mediated protein degradation. Mechanistic validation through qRT-PCR, Western blotting, and luciferase assays confirmed that miR-99b-5p modulates the TRAIL-R signaling pathway via DR5 and BAK, attenuating apoptotic signaling. In vivo studies using xenograft models established with MDA-MB-231 cells stably expressing miR-99b-5p knockdown showed marked tumor regression, further supporting its oncogenic role. Conclusion: Collectively, these findings establish miR-99b-5p as a context-specific oncogenic miRNA in breast cancer and a promising therapeutic target, particularly for TNBC, where targeted treatment options remain limited.

overall_design: This study aims to identify the direct targets of miR-99b-5p in triple-negative breast cancer (TNBC) using Argonaute-2 based RNA-immunoprecipitation sequencing (AGO2-RIP-seq). We utilized the MDA-MB-231 cell line, which was transfected with either a miR-99b-5p mimic to overexpress the miRNA or a scrambled negative control. To capture the miRNA-target mRNA complexes, immunoprecipitation was performed using an anti-AGO2 antibody, with IgG used as a non-specific binding control. The AGO2-RIP-seq was conducted with two independent biological replicates for each condition (miR-99b-5p mimic vs. Scrambled Control). The enrichment of target mRNAs in the AGO2-IP fraction compared to the control was analyzed to map the miR-99b-5p-dependent targetome. Complementary gene expression analysis and functional assays (proliferation, apoptosis, and luciferase reporter assays) were integrated to validate the biological impact of the identified targets.

first: Senem

last: Noyan

first: Kubra

middle: N

last: Kaplan-Ilhan

first: Muge

middle: O

last: Demirtas

first: Begum

first: Bora

last: Ergin

first: Rabia

last: Sen

first: Hakan

last: Gurdal

first: Bala

last: Dedeoglu


title: Mapping Endothelial-Macrophage Interactions in Diabetic Vasculature: Role of TREM2 in Vascular Inflammation and Ischemic Response [scRNA-Seq]

city: DUARTE

first: Naseeb

middle: Kaur

last: Malhi

email: naseebmalhi@gmail.com, nmalhi@coh.org

address: 1500 E Duarte Road

department: DMRI

institute: City of Hope

summary: Diabetes mellitus (DM) significantly accelerates vascular diseases like peripheral arterial disease (PAD). Endothelial cells (ECs) and macrophages (MΦs) singularly and synergistically are important contributors to DM-associated vascular dysfunction. Single-cell (sc) profiling technologies are revealing the true heterogeneity of ECs and MΦs, but how this cellular diversity translates to cell-cell interactions, and consequentially vascular function, remains unknown. We leveraged scRNA sequencing and spatial transcriptome (ST) profiling to analyze human mesenteric arteries from non-diabetic (ND) and type 2 diabetic (T2D) donors. We generated a transcriptome and interactome map encompassing the major arterial cells and highlighted Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) as a top T2D-induced gene in mononuclear phagocytes (MPs), with concomitant increases of TREM2 ligands in ECs. We verified DM-associated TREM2 induction in cell and mouse models, and found that TREM2 inhibition decreases pro-inflammatory responses in MPs and ECs, as well as increases EC migration in vitro. Furthermore, TREM2 inhibition using a neutralizing antibody enhanced ischemic recovery and flow reperfusion in DM mice subjected to hindlimb ischemia, suggesting that TREM2 promotes ischemic injury in DM. Finally, in human PAD, co-existing DM was associated with greater expression of TREM2 and its interaction with ECs, with a further increase in ischemic tissue compared to patient-matched non-ischemic tissue. Collectively, our study presents the first atlas of human diabetic vessels with single cell and spatial resolution, and identifies TREM2-EC interaction as a key driver of diabetic vasculopathies, the targeting of which may offer an opportunity to ameliorate vascular dysfunction associated with DM-PAD.

overall_design:
We performed scRNA-seq on the intima and whole tissue of mesenteric arteries from non-diabetic and diabetic donors.
Raw data for GSM8881941, GSM8881942, GSM8881946-GSM8881950 previously uploaded in GSE135357. See individual GSM records for details.

first: Viola

last: Luo

first: Xiaofang

first: Zhen

last: Chen


title: Mapping Endothelial-Macrophage Interactions in Diabetic Vasculature: Role of TREM2 in Vascular Inflammation and Ischemic Response [bulk RNA-Seq]

overall_design: We performed bulk RNA-seq on THP-1-derived macrophages, with and without siRNA-mediated TREM2 knockdown (100nM),  then 25 mM D-glucose, 5 ng/mL TNF-α (HT) treated.

last: K Malhi

first: Dongqiang

last: Yuan

first: Yingjun

last: B Chen


title: Mapping Endothelial-Macrophage Interactions in Diabetic Vasculature: Role of TREM2 in Vascular Inflammation and Ischemic Response [Spatial Transcriptomics]

overall_design: We performed spatial transcriptomics profiling using 10x Genomics Visium on mesenteric arteries from non-diabetic and diabetic donors

last: Tang


title: Molecular and functional dissection using CaMPARI-seq reveals the neuronal organization for dissociating optic flow-dependent behaviors

city: Wako

first: Fumi

last: Kubo

address: 2-1 Hirosawa

department: Center for Brain Science

institute: RIKEN

summary: Optic flow processing is critical for the visual control of body and eye movements in many animals. Rotational and translational binocular optic flow patterns need to be clearly distinguished to induce different behavior outputs. However, the specific neuron types and their connectivity involved in this computation remain unclear. Here, we developed a method to link the functional labeling using a photoconvertible calcium indicator called CaMPARI2 and single-cell RNA-sequencing (CaMPARI-seq) to investigate the transcriptional profile of the pretectum, a center for processing optic flow in larval zebrafish. Using this technique, we identified a pretectal cluster expressing tcf7l2, which can be further classified into molecularly distinct subclusters. In vivo calcium imaging and cell ablation revealed that nkx1.2lb-positive pretectal neurons are commissural inhibitory neurons required for the optomotor response but not for the optokinetic response. Our genetic and functional dissection using CaMPARI-seq uncovered the neuronal organization essential for dissociating different optic flow-dependent behaviors.

overall_design: To identify genetic markers for optic flow responsive cells, we conducted single-cell RNA sequencing analysis. We used zebrafish larvae pan-neuronally expressing NLS-CaMPARI2 to label neurons during optic flow processing. CaMPARI2 labeling was performing by deliverying light as zebrafish larvave respond to either moving or stationary optic flow stimuli. After dissection and dissociation of the brain, only active neurons (CaMPARI, red fluorescent) were isolated for sequencing.

first: Koji

last: Matsuda

first: Atsushi

last: Toyoda


title: Human-induced pluripotent Stem Cell-Derived Hepatic Stellate Cells in Liver Organoids: Maturation Promotion and Essential Role in Vascularization [scRNA-Seq]

city: Tokyo

first: Yunzhong

last: Nie

state: Tokyo

address: Room 330, 3F west, 1st  Bldg, Shirokanedai 4-6-1, Minato

department: Department of Computational Biology and Medical Sciences

institute: The University of Tokyo

summary: Human-induced pluripotent stem cell (hiPSC)-derived liver organoids (LOs) are valuable for studying human liver organogenesis but face challenges in faithfully recapitulating certain processes, like vasculogenesis, due to the lack of specific cell components. Hepatic stellate cells (HSCs), which are liver-specific pericytes and might be crucial for liver vasculogenesis, remain underutilized in developmental studies because of their disease-related "activated" status and inefficient generation process. Here, we present an efficient method for generating hiPSC-derived HSCs (hiPSC-HSCs) resembling fetal HSCs’ transcriptomic profiles. These hiPSC-HSCs exhibit exceptional expandability (>105-fold) while maintaining essential cellular features. Additionally, in entirely hiPSC-derived LOs consisting of HSCs, hepatic endoderm, and endothelial cells, hiPSC-HSCs play a vital role in LO maturation and vascularization, both in vitro and in vivo. This work represents a significant advancement in understanding HSC roles in human liver development, and LOs containing non-activated hiPSC-HSCs hold potential in modeling congenital human liver diseases.

overall_design: Single cell RNA expression profiles of hiPSC-HSC_P0, Liver organoids（containing hiPSC-HE, hiPSC-ECs, and hiPSC-HSCs)_Day10 in_vitro

first: Xia

last: Yang

first: Hideki

last: Taniguchi


title: p53 safeguards chemical reprogramming of human somatic cells towards pluripotency [CRISPR]

first: Jingxiao

last: Cao

state: Beijing

address: No.5 Yiheyuan Road, Yanyuan Street, Haidian District, Beijing

department: Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.

institute: Peking University

summary: Cell fate manipulation is powerful for generating desired cell types through reprogramming, such as induced pluripotent stem cells (iPSCs), with broad biological applications. However, reprogramming can be risky due to dramatic changes in cell identity, necessitating strict regulation to ensure safety and efficacy. p53 is essential for maintaining genome stability to prevent abnormalities induced by changes of cell states, which however, functionally counters Yamanaka factors for efficient reprogramming. Thus, delicately balancing p53 activity for efficient reprogramming with highly quality has proven challenging. Here, we demonstrate that efficient human chemical reprogramming is safeguarded by p53, in distinction from Yamanaka factors-mediated reprogramming. Suppressing p53 activity unexpectedly and substantially hinders the generation of chemically induced pluripotent stem cells (CiPSCs). Notably, p53 precisely regulates the mesenchymalization occurred at the early reprogramming stage where cell plasticity is re-established and curbs excessive epithelial-mesenchymal transition (EMT). Furthermore, the retinoid acid receptor agonist, TTNPB, leverages p53’s anti-metastatic functions to facilitate CiPSC generation by activating BTG2. Our results demonstrate that p53 activity distinguishes chemical reprogramming from Yamanaka factors-mediated reprogramming, and offer a promising strategy that delicately balances p53 with reprogramming for both safe and efficient cell fate manipulation.

overall_design: For the functional verification of p53 downstream genes, somatic cells were transfected with lentiviral vectors containing Cas9-sgRNA libarary targeting p53 targeted genes and subjected to chemical reprogramming. Genes depleted in CiPSCs represented the p53 downstream genes that required for chemical reprogramming.


title: HER2-positive breast cancer

first: Kornelia

last: Polyak

address: 450 Brookline Ave. S1070

institute: Dana-Farber Cancer Institute

summary: Intratumor heterogeneity for HER2 expression and amplification, observed in up to 40% of HER2-positive breast cancer is a driver of resistance to HER2-targeted therapies. The advancement of treatment for HER2 heterogeneous tumors has been hindered by the lack of preclinical models that accurately replicate the human disease. Here we describe human HER2 heterogeneous breast cancer cell models composed of ERBB2 amplified (HER2hi) and non-amplified (HER2lo) cell populations derived from the same tumor. Utilizing these models and cellular barcoding technology, we provide evidence for subclonal cooperation between HER2hi and HER2lo subpopulations and that HER2lo cells drive resistance to HER2-targeting antibody-drug conjugates (ADC) such as T-DXd but are sensitive to HER2 kinase inhibitors. CRISPR screens in heterogeneous co-cultures identified sensitizers of HER2lo cells to T-DXd including the USP9X deubiquitinating enzyme. USP9X inhibition enhances the lysosomal targeting of HER2 to potentiate ADC payload release, diminishing tumor recurrence after T-DXd treatment. Our results elucidate the functional relevance of HER2 heterogeneity and propose improved therapies for these tumors.

overall_design:
(1) RNAseq: HER2 heterogeneous and HER2-targeted therapies resistant cells
HCC1954, 21NT, and 21PT cells were sorted into HER2hi and HER2lo populations according to their HER2 expression levels. Also, resistant cells for T-DXd, T-DM1, neratinib and tucatinib were generated from the same parental cell lines.
RNA from growing cells was extracted.

(2) scRNA-seq of two human HER2+ breast cancer primary tumors
Primary tumors were digested and stored frozen before performing 10X library preparation for scRNA-seq (20,000 read pairs/cell for 5' Gene Expression Dual Index library)

first: Marie-Anne

last: Goyette


title: Nrf1 coordinates proteasome and autophagy to maintain cardiac proteostasis

first: miao

last: cui

state: Please select region, state or province

address: 300 LONGWOOD AVE

institute: Boston Children's Hospital

summary: Proteolytic stress frequently arises during disease and aging, particularly in long-lived, post-mitotic cells such as cardiomyocytes. To maintain proteostasis, cardiomyocytes depend on coordinated protein quality control pathways, including the ubiquitin-proteasome system and autophagy. Mechanisms that activate these pathways hold therapeutic potential for heart disease. Here, we demonstrate that transient activation of nuclear factor erythroid 2–like 1 (Nfe2l1, also known as Nrf1), a transcriptional regulator of proteasome activity, specifically in cardiomyocytes during ischemia/reperfusion (I/R) injury improves cardiac function. In addition to regulating the proteasome, we identify a critical role for Nrf1 in activating autophagy, which is essential for its cardioprotective effects. Through multi-omics analyses, we define both transcriptional and post-transcriptional functions of Nrf1 that underlie its cardioprotective activity. Loss-of-function studies in mice demonstrate that Nrf1, but not its homolog Nrf2, is required for autophagy and baseline cardiac function. Together, our findings establish a dual function of Nrf1 in promoting cardiac proteostasis by regulating both proteasomal and autophagic protein quality control pathways. Activating Nrf1 thus offers a therapeutic strategy for treating ischemic heart disease.

overall_design: RNA-seq profling of neonatal rat ventricule cardiomyocytes overepressing LacZ as control and the transcription factor Nrf1 with or without H2O2 treatment.

first: Miao

last: Cui


title: Dynamin-related protein AoVps1 mediating mitochondrial fission in complex with AoMdv1 is necessary for mitochondrial morphology, mitophagy, conidiation, trap formation, and energy metabolism of Arthrobotrys oligospora

city: kunming

first: xuemei

last: li

state: yunnan

address: South Road of Dongwai Ring, University Town, Chenggong District, Kunming City

institute: yunnan university

summary: Mitochondrial fission is critical for organelle homeostasis and cellular differentiation. Here, we functionally characterized the mitochondrial fission-related proteins AoMdv1 and AoVps1 in the nematode-trapping fungus Arthrobotrys oligospora. Deletion of Aomdv1 or Aovps1 completely arrested mitochondrial division, generating hyperfused, giant mitochondria. Multi-omics and phenotypic analysis revealed that this disruption precipitates a systemic cellular collapse, characterized by severe oxidative stress, elevated apoptosis, and a severe metabolic bottleneck that forces unconsumable carbon flux into extreme lipid droplet hypertrophy. Crucially, our study reveals a previously unrecognized dual function for the dynamin-like protein AoVps1. Beyond interacting with the adaptor AoMdv1 to execute mitochondrial fission, AoVps1 physically binds the core autophagic effectors AoAtg8 and AoAtg20 to directly coordinate macroautophagy and selective mitophagy, respectively. Given that targeted disruption of these autophagic pathways perfectly phenocopies the severe developmental defects of the fission mutants-including abolished conidiation and trap morphogenesis-we define a novel “fission-autophagy” regulatory axis. Collectively, our findings demonstrate that AoVps1 acts as an indispensable molecular bridge, intimately coupling mitochondrial dynamics with autophagic flux to drive the morphological and pathogenic lifestyle transitions in predatory fungi.

overall_design: RNA-seq analysis was performed on three wild-type samples, three ΔAomdv1 mutant samples, and three ΔAovps1 mutant samples, with three biological replicates per sample, and prediction of multiple cytological processes that may affect trap formation and conidiation in Arthrobotrys oligospora.

first: Xuemei

last: Li

first: Le

first: Lirong

last: Zhu

first: Xuewei

first: Meichen

first: Jinkui


title: Single-nucleus RNA sequencing of human subcutaneous adipose tissue biopsies from 49 Mexican participants

city: Los Angeles

first: Asha

last: Kar

address: 695 Charles E. Young Drive South

department: Human Genetics

institute: University of California Los Angeles

summary: We performed single-nucleus RNA-seq on subcutaneous adipose tissue (SAT) biopsies from Mexicans from Mexico City. The purpose of this study was to understand cell-type and subcell-type level profiles of SAT in Mexicans.

overall_design:
We isolated nuclei from frozen subcutaneous adipose tissue from individuals independently or by pooling biopsy samples in batches of four or five samples. The libraries were constructed using the Chromium Single Cell 3’ v3.1 chemistry and sequenced on an Illumina NovaSeq S4 with a target sequencing depth of 60,000 reads per nucleus.

***************************************************************
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: Carlos

last: Aguilar-Salinas

first: Maria Teresa

last: Tusié-Luna

first: Paivi

last: Pajukanta