title: Expression data from transfected HEK cells

status: Public on Oct 02 2025

city: Heidelberg

first: Torsten

last: Schmenger

address: Im Neuenheimer Feld 267

institute: BioQuant Heidelberg

summary:
Many missense variants with associated phenotypes lie in regions of proteins that are not well understood. However, often a putative function can be derived through study of related sequences that have known functional information in the respective protein region.
We used a simple transfection & microarray gene expression analysis to study such a missense variant, finding that differentially regulated genes hint at an increased activity of the protein of interest.

overall_design: Cells were seeded into wells of a 6-well plate (Day 1). The next day cells were transfected with a pDEST30 vector (2.5ng) containing the coding sequence of the protein of interest (MAP2K3) as WT or mutant (A84T) (Day2). One day later we added selection pressure (350 µg/ml Geneticin) to the mixture (Day 3). Cells were selected and compared to the neg. control. Typically cells would be induced on Day 7 for 24 h with 1 µg/ml Tetracycline and harvested one day later for total RNA extraction. cDNA libraries were prepared and analyzed by the DKFZ Genomics core facility, according to the standard operating protocols.

first: Gurdeep

last: Singh

first: Juan-Carlos

last: Gonzalez-Sanchez

first: Anastasiia

last: Kutkina

first: Nina

last: Bremec

first: Gauraw

last: Diwan

first: Pablo

last: Mozas

first: Cristina

last: López

first: Reiner

last: Siebert

first: Rocio

last: Sotillo

first: Robert

middle: B

last: Russell


title: Systematic benchmarking of imaging spatial transcriptomics platforms in FFPE tissues - scRNA-seq Data

status: Public on Oct 03 2025

city: Cambridge

first: Samouil

middle: L

last: Farhi

state: MA

address: 75 Ames St

department: Spatial Technology Platform

country: USA

institute: Broad Institute of MIT and Harvard

type: Expression profiling by high throughput sequencing

summary: Emerging imaging spatial transcriptomics (iST) platforms and coupled analytical methods can recover cell-to-cell interactions, groups of spatially covarying genes, and gene signatures associated with pathological features, and are thus particularly well-suited for applications in formalin fixed paraffin embedded (FFPE) tissues. Here, we benchmark the performance of three commercial iST platforms—10X Xenium, Vizgen MERSCOPE, and Nanostring CosMx—on serial sections from tissue microarrays (TMAs) containing 17 tumor and 16 normal tissue types for both relative technical and biological performance. On matched genes, we find that Xenium consistently generates higher transcript counts per gene without sacrificing specificity. Xenium and CosMx measure RNA transcripts in concordance with orthogonal single-cell transcriptomics.  All three platforms can perform spatially resolved cell typing with varying degrees of sub-clustering capabilities, with Xenium and CosMx finding slightly more clusters than MERSCOPE, albeit with different false discovery rates and cell segmentation error frequencies. Taken together, our analyses provide a comprehensive benchmark to guide the choice of iST method as researchers design studies with precious samples in this rapidly evolving field.

overall_design: Tumor TMA 1 (tTMA1) consisted of one hundred and seventy 0.6 mm diameter cores (i.e. sampled regions) from seven different cancer types, with 3-6 patients per cancer type, and 3-6 cores per patient. Tumor TMA 2 (tTMA2) consisted of forty-eight 1.2 mm diameter cores from nineteen different cancer types, with each tissue type coming from one or two patients and represented in 2-3 cores.

first: Huan

last: Wang

first: Ruixu

last: Huang

first: Jack

last: Nelson

first: Ce

last: Gao

first: Miles

last: Tran

first: Anna

last: Yeaton

first: Sachi

last: Krishna

first: Kristen

last: Felt

first: Kathleen

last: Pfaff

first: Teri

last: Bowman

first: Scott

middle: J

last: Rodig

first: Kevin

last: Wei

first: Brittany

middle: A

last: Goods


title: Cranial neural crest shortage leads to extensive craniofacial anomalies in mice mutant for the NR2F1/2 nuclear receptors

status: Public on Oct 15 2025

city: Cincinnati

first: Lindsey

last: Barske

state: OH

address: 3333 Burnet Ave MLC 7016

department: Human Genetics

institute: Cincinnati Children's Hospital Medical Center

summary: Nuclear receptors are iteratively deployed during neural crest development, from pre-induction through differentiation stages. NR2F1 and NR2F2 in particular have been proposed as broad regulators of early neural crest gene expression in mammals, but the timing, extent, and redundancy of their developmental requirement has remained unclear, as Nr2f1 and Nr2f2 single mouse mutants present only minimal craniofacial phenotypes. Here we report the dynamic expression patterns of Nr2f1 and Nr2f2 in the mouse cranial neural crest from specification through post-migratory stages. Combined conditional knockout of both Nr2f1 and Nr2f2 in the neural crest with Wnt1-Cre or Pax3Cre caused severe midfacial clefting, loss of the maxilla and palate, and hypoplasticity of all other facial skeletal elements except the distal mandible. These perinatal phenotypes were rooted in a major shortage of pharyngeal arch mesenchyme at mid-gestation. This in turn traced to a deficiency of migrating neural crest cells, first evident in the trailing part of the first arch migratory stream at embryonic day 8.75. RNAseq at a slightly earlier stage revealed downregulation of many migratory neural crest genes, including a possible direct target, the phospholipase Plcg2. These findings reveal a vital requirement for NR2F1/2 within the later-forming cranial neural crest.

overall_design: Bulk RNAseq was performed on individual heads cut caudal to the first pharyngeal arch of control and Wnt1-Cre;Nr2f1fl/fl;Nr2f2fl/fl mouse embryos at the 8 somite (±1 somite) and 12 somite (±1 somite) stages. Total of 12 samples, 3 replicates each genotype and stage.

first: David

last: Paulding

first: Simon

last: Han

first: Jonathon

last: Timmons

first: Michelle

last: Caye

first: Alexa

last: Riedel

first: Samantha

last: Brugmann


title: Transcriptomic Analysis of the Adrenal Glands, Heart, and Pancreas in Rats Following Benzalkonium Chloride Inhalation

status: Public on Oct 19 2025

city: Busan

first: Tae Hwan

last: Shin

address: Nakdong-daero 550 beon-gil

department: Health Sciences

country: South Korea

institute: Dong-A University

summary: Transcriptomic profiling was conducted on the adrenal glands, heart, and pancreas 24 hours after the 10th intratracheal instillation of benzalkonium chloride in rats. Total RNA was extracted using TRIzol and purified with the RNeasy Mini kit. High-quality RNA (RIN > 9) was used to construct sequencing libraries with the TruSeq Stranded mRNA kit, followed by sequencing on a NovaSeq 6000 platform.

overall_design: Quantitative RNA sequencing was performed with adrenal glands, heart, and pancreas at 24 h after the 10th intratracheal instillation for benzalkonium bhloride (BKC). Collected organs were added 1 mL of TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) per 50 mg of organ tissues. The paired adrenal glands per animal were pooled, and the heart was dissected in the left ventricular area. Samples weighed approximately 50 mg and were stored in TRIzol at -80℃ in a deep freezer until RNA purification. Total RNA was isolated using a RNeasy Mini kit (Qiagen, Valencia, CA, USA), and the RNA integrity was analyzed using a Bioanalyzer (Agilent, Santa Clara, CA, USA). RNA samples with RNA integrity values greater than 9 were selected to prepare the mRNA sequencing library. The library was prepared using a TruSeq Stranded mRNA library kit (Illumina, San Diego, CA, USA), and sequencing was performed using a NovaSeq 6000 (Illumina).

first: Gyuri

last: Kim

first: Jiyoung

last: Jeong

first: Jung Eun

last: Park

first: Soyeon

last: Jeon

first: Jae-Jun

first: Se-hwan

last: Choi

first: Jaebaek

last: Jang

first: Mihye

last: Kwon

first: Min-Seok

first: Iljung

last: Lee

first: Dong Hyun

first: Jongho

first: Wan-Seob

last: Cho


title: ChIP-seq of Stra8-Bound Genomic Regions in Mouse Hippocampus

city: Kansas City

first: Ning

last: Wang

state: KS

address: 3901 Rainbow Boulevard, Kansas City, KS

department: Cell Biology and Physiology

institute: UNIVERSITY OF KANSAS MEDICAL CENT

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We performed ChIP-seq for Stra8 in the hippocampus of Stra8-AVI mice using the Illumina NovaSeq 6000 sequencing platform.

overall_design: We performed ChIP-seq on four samples using the Illumina NovaSeq 6000 sequencing platform. Two samples, one male and one female Stra8-AVI hippocampus, were immunoprecipitated with an anti-AVI antibody. A third sample was generated by pooling hippocampal tissue from one male and one female Stra8+/+ mouse and performing immunoprecipitation with the same antibody. The fourth sample was the corresponding input control from the pooled Stra8 AVI hippocampal tissue.

first: Ming

last: Huang

first: Xiaoyu

last: Zhang


title: Stra8, an Activity-Dependent Modulator of Neural Circuit, Safeguards Neuronal Integrity

summary: While neuronal activity is essential for brain function, it inherently threatens neurons and impairs neuronal integrity1. Here we show that activity-induced expression of Stra8 (stimulated by retinoic acid gene 8) plays a crucial role in protecting neuronal integrity. To date, Stra8 has been considered as germline-specific2, and its role in somatic cells has not been explored. Our results show that Stra8 is expressed in the adult mouse brain. Neuronal activity (i.e., exploration of a novel environment) induced Stra8 expression, and this process is mediated by calcium influx and depolarization through NMDA-type glutamate receptors. Importantly, neuron-specific knockout of Stra8 induced multifaceted features of neuronal degeneration in young adult mice, including accumulation of DNA damage, impaired proteostasis, heightened inflammation, nuclear membrane erosion, decreased dendritic plasticity, as well as impaired learning and memory and increased vulnerability to kainic acid-induced excitotoxicity. Additionally, electrophysiology studies show that loss of Stra8 compromises inhibitory neural circuity and function. Mechanistically, Stra8 binds to the regulatory sequences of neuromodulator genes and regulates their expression, among which loss of Stra8 results in an upregulated expression of Npas4; however, aberrantly expressed Npas4 protein is confined to the nuclear periphery in neurons. Together, these findings establish Stra8 as an activity-dependent modulator of neural circuitry that protects neuronal integrity.

overall_design: To investigate the mechanism of Stra8 action, we enriched Stra8-expressing (GFP-positive) cells by FACS from the hippocampi of wild-type and Stra8-KO mice carrying the pStra8-GFP reporter. We then compared their transcriptomes by mRNA-seq.

first: Sumedha

last: Gunewardena


title: CrebA regulation of secretory capacity: Genome-wide transcription profiling coupled with in vivo DNA binding studies

status: Public on Oct 01 2025

city: Baltimore

first: Da

last: Peng

state: Maryland

address: 733 North Broadway, MRB 653

institute: Johns Hopkins University

summary: DNA binding assays, expression analyses, and binding site mutagenesis revealed that the Drosophila CrebA transcription factor (TF) boosts secretory capacity in the embryonic salivary gland (SG) through direct regulation of secretory pathway component genes (SPCGs). The mammalian orthologues of CrebA, the Creb3L-family of leucine zipper TFs, not only activate SPCG expression in a variety of mammalian tissues but can also activate SPCG expression in Drosophila embryos, suggesting a highly conserved role for this family of proteins in boosting secretory capacity. However, in vivo assays reveal that CrebA binds far more genes than it regulates, and it remains unclear what distinguishes functional binding. It is also unclear if CrebA is the major factor driving SPCG gene expression in all Drosophila embryonic tissues and/or if CrebA also regulates other tissue-specific functions. Thus, we did single cell RNA sequencing (scRNA-seq) of wild-type (WT) and CrebA null embryos to explore the relationship between CrebA binding and gene regulation. We find that CrebA binds the proximal promoters of its targets, that SPCGs are the major class of genes regulated by CrebA across tissues, and that CrebA is sufficient to activate SPCG expression even in cells that do not normally express the protein. A comparison of scRNA-Seq to other methods for capturing regulated transcripts reveals that the different methodologies identify overlapping but distinct sets of CrebA targets.

overall_design: CrebA23w-/TM3, twi-GFP and CrebAR3576/TM3, twi-GFP were crossed to generate CrebA protein-null embryos for processing using the same single-cell RNA-sequencing pipeline previously established in the lab (PENG et al. 2024). Briefly, embryos were collected and aged to capture stages 10-12 of embryonic development (“early organogenesis”) or stages 13-16 (“late organogenesis”). Embryos were dechorionated in a 50% bleach solution and sorted using a COPAS Flow Pilot sorter to isolate GFP- negative (CrebA protein null) embryos. Live embryos were visually inspected under a fluorescent microscope to confirm both stage and absence of GFP-positive individuals. Samples were immediately prepared for single cell isolation and 10X Genomics single-cell RNA sequencing.

first: Dorian

last: Jackson

first: Sagarika

last: Shinde

first: Ankita

last: Holenarasipura

first: Vidya

last: Ajay

first: Patrick

last: Cahan

first: Deborah

last: Andrew


title: Catch and release of sialoglycoRNAs enables sequencing-based profiling across cellular and extracellular material

status: Public on Oct 05 2025

city: Boston

first: Ryan

middle: A.

last: Flynn

address: 1 Blackfan Circle

department: Department of Pediatrics

institute: Boston Children’s Hospital

summary: Glycosylated RNAs (glycoRNAs) represent a recently discovered class of small RNAs, but their systematic characterization has been limited by reliance on metabolic chemical reporters and high RNA input requirements. Here we present rPAL sequencing (rPAL-seq), a sensitive and selective platform for de novo discovery of sialoglycoRNAs. rPAL-seq combines enhanced periodate oxidation of sialic acids with a capture–release workflow and optimized library construction using poly(A) extension coupled with template-switching reverse transcription. The method enabled reproducible profiling from less than 100 ng of input RNA, corresponding to less than 2% of the material required by previous approaches. When applied across 13 human cell lines, rPAL-seq identified lineage-associated glycoRNA patterns alongside a conserved core dominated by uridine-rich snRNAs and snoRNAs, with modification signatures implicating glycosylation on acp³U or related uridine-based modifications. Extending to extracellular fractions, rPAL-seq revealed secreted glycoRNA profiles distinct from those of the cellular fraction. rPAL-seq provides a robust, scalable strategy for glycoRNA profiling, opening new avenues for studying this emerging biopolymer.

sample_organism:
Homo sapiens
Canis lupus familiaris
Mus musculus

overall_design: This study aimed to profile sialoglycoRNA from diverse human and animal cell lines using rPAL-seq, a sequencing method first introduced in this study that combines enhanced reductive periodate–aminoxy labeling with sialidase-specific elution. Cells (including HeLa, A549, 293T, Huh7, LPS853, LN308, OCI-AML2, OCI-AML3, Molm13, Jurkat, Jeko1, Nalm6,  DiFi, MDCK, and EMT6) were cultured under standard conditions. Extracellular vesicles (EVs) and non-vesicular extracellular nanoparticles (NVEPs) were isolated from conditioned medium of DiFi and MDCK using previously established protocols. For each biological sample, sialoglycoRNA was captured and processed in 3 parallel groups: IP (VC sialidase–eluted), Control (mock elution with heat-inactivated enzyme), and Input (1% reserved prior to elution). Libraries were prepared using the rPAL-seq workflow and sequenced on an Illumina NovaSeq X platform with single-end 100 bp reads.

first: Ruiqi

last: Ge

first: Dennis

middle: K

last: Jeppesen

first: Sandeep

last: Rai

first: Robert

last: Coffey


title: Mobile-CRISPRi as a tool for genetic manipulation in the intracellular pathogen Piscirickettsia salmonis

city: Santiago

first: Christian

middle: E

last: Hodar

state: Santiago

address: Av. El Libano 5524

department: INTA

country: Chile

institute: Universidad de Chile

summary: Piscirickettsia salmonis is the causative agent of Salmonid Rickettsial Septicemia (SRS), the main bacterial disease affecting the salmon industry in Chile. In this work, we implemented a Mobile-CRISPRi system to generate gene silencing using a catalytically inactive dCas9 protein and an IPTG-inducible single-guide RNA (sgRNA). We demonstrate the efficacy of the CRISPRi system in P. salmonis by silencing an exogenous gene encoding green fluorescent protein (sfGFP), and the endogenous homolog of the fur gene, whose gene product regulates intracellular iron homeostasis in bacteria. The inducible expression of dcas9 and the sfGFP-directed sgRNA caused a 98.7% decrease in fluorescence in the knockdown strain. This silencing system was effective in seven P. salmonis strains from both genogroups. Furthermore, the same system was used to construct fur knockdown strains. A 50-fold decrease in fur expression level was determined in these strains, when the expression of the fur gRNA was induced with IPTG. By RNA-seq we detected a significant increase in the expression of genes encoding the Fe+2 and Fe+3 acquisition systems and iron mobilization in the fur1 knockdown, after IPTG induction. All the genes with over two-fold increased expression in the RNA-seq presented the Fur box consensus sequence in their regulatory region.

overall_design: This study employed a Mobile-CRISPRi system to genetically manipulate Piscirickettsia salmonis, which was first cultured under specific conditions (18°C in NPB/NPA media) with purity confirmed by PCR-RFLP. The system was delivered via tri-parental conjugation, where E. coli SM10 donor strains—one carrying the CRISPRi plasmid (pJMP2754, pJMP2774, or a sgRNA-targeting plasmid) and another providing the Tn7 transposase (pTNS3)—were mixed with a stationary-phase P. salmonis recipient culture on a filter. After incubation, transconjugants were selected on antibiotic-containing plates, and successful genomic integration of the plasmid into the glmS locus was confirmed by PCR screening, yielding genetically modified P. salmonis strains for subsequent functional assays.

first: Javiera

last: Ortiz-Severin

first: Paulette

last: Geoffroy

first: Pamela

last: Aravena

first: Daniel E.

last: Palma

first: Mauricio

last: González

first: Verónica

last: Cambiazo


title: Actin Dysregulation Induces Neuroendocrine 1 Plasticity and 2 Immune Evasion: A Vulnerability of Small Cell Lung Cancer

status: Public on Oct 17 2025

city: Houston

first: Jinho

last: Jang

state: Texas

address: 1755 Wyndale Street

institute: MD Anderson Cancer Center

summary: Small cell lung cancer (SCLC) is aggressive with limited therapeutic options. Despite recent advances in targeted therapies and immunotherapies, therapy resistance is a recurring issue, which might be partly due to tumor cell plasticity, a change in cell fate. Nonetheless, the mechanisms underlying tumor cell plasticity and immune evasion in SCLC remain elusive. CRACD, a capping protein inhibitor that promotes actin polymerization, is frequently inactivated in SCLC. Cracd knockout (KO) transforms preneoplastic cells into SCLC tumor-like cells and promotes in vivo SCLC development driven by Rb1, Trp53, and Rbl2 triple KO. Cracd KO induces neuroendocrine (NE) plasticity and increases tumor cell heterogeneity of SCLC tumor cells via regulated NOTCH1 signaling by actin cytoskeleton disruption. CRACD depletion also reduces nuclear actin and induces EZH2-mediated H3K27 methylation. This nuclear event suppresses the MHC-I genes and thereby depletes intratumoral CD8+ T cells for accelerated SCLC tumorigenesis. Pharmacological blockade of EZH2 inhibits CRACD-negative SCLC tumorigenesis by restoring MHC-I expression and immune surveillance. Unsupervised single-cell transcriptomics identifies SCLC patient tumors with concomitant inactivation of CRACD and downregulated MHC-I pathway. This study defines CRACD, an actin regulator, as a tumor suppressor that limits cell plasticity and immune evasion and proposes EZH2 blockade as a viable therapeutic option for CRACD-negative SCLC.

overall_design: For the Xenium In Situ experiment, a single FFPE tissue block containing both RPR2 and CRPR2 samples was prepared and mounted onto a Xenium slide. RPR2 refers to a genetically engineered mouse model (GEMM) with Rb1, Trp53, and Rbl2 knockout (KO), while CRPR2 represents a GEMM with Cracd, Rb1, Trp53, and Rbl2 KO. Spatial transcriptomic profiling was performed using the 10x Genomics Mouse Tissue Atlassing 379-gene panel, supplemented with an additional 100 custom-selected genes for further analysis.

first: Yoojeong

last: Seo

first: Shengzhe

last: Zhang

first: Kyung-Pil

last: Ko

first: Kee-Beom

last: Kim

first: Yuanjian

last: Huang

first: Dong-Wook

first: Bongjun

first: Gengyi

last: Zou

first: Jie

first: Sohee

last: Jun

first: Wonhong

last: Chu

first: Nicole

last: Kirk

first: Ye Eun

last: Hwang

first: Young Ho

last: Ban

first: Shilpa

middle: S

last: Dhar

first: Joseph

middle: M

last: Chang

first: Min Gyu

last: Lee

first: Charles

last: Rudin

first: Kwon-Sik

last: Park

first: Jae-Il


title: Perturbational single-cell RNA sequencing of 104 compounds across six immortalized and four primary cell lines

status: Public on Oct 23 2025

city: Somerville

first: Mauricio

last: Cortes

address: 101 South Street

institute: Cellarity

summary: we introduce a new perturbational scRNA-seq dataset that includes six immortalized lines (A549, A375, H1AE, HEK293T, HEP2G, and PC3) and four primary lines: CD8+ T-cells (CD8+), CD34+ hematopoietic progenitors (CD34+), primary adipocytes (PAD), and bronchial epithelial cells (HBEC). 104 compounds, including 88 from CMap, were tested across the 10 cell types in duplicate with library-matched controls, resulting in 1,737 scRNA-seq samples comprising a total of 1.26 M cells.

overall_design: Hashed single-cell RNA sequencing of cancer and primary cell lines, performed 24 hours after perturbation by 104 small molecules, with library-matched controls. Each plate repesents a different cell line. Cell lines are profiled in at least two distinct replicate plates each, per compound.

first: Daniel

last: Treacy

first: John

last: Bradley

first: Laura

last: Isaaco

first: Kaylie

last: Schneider

first: Sakina

last: Saif

first: Scott

last: Steelman

first: Jonathan

last: Bloom

first: Benjamin

last: DeMeo

first: Corey

last: Hayford

first: Maximilian

last: Rogers-Grazado


title: Nutritional c-Fos Induction Rewires Hepatic Metabolism and Can Promote Obesity-Associated Hepatocellular Carcinoma

city: Brussels

first: Ao

last: Li

state: Brussels

address: 808 Route de Lennik

department: Faculty of Medicine

country: Belgium

institute: Université libre de Bruxelles (ULB)

summary: The transcription factor c-Fos plays an important role in hepatic metabolism. Previous studies implicated the c-Fos/AP-1 in steatohepatitis development and hepatocellular carcinoma formation. However, its role in metabolic dysfunction-associated steatotic liver disease and hepatocellular carcinoma remains to be illustrated. Here, we analyse the transcriptional consequences of adenovirus-mediated c-Fos silencing in the liver of young adult mice during 2 weeks by bulk RNA-seq.

overall_design: Murine (males, C57BL/6N) liver RNA was sequenced after 2 weeks of adenovirus-mediated c-Fos silencing (3 Adv-Control and 3 Adv-shRNA-Fos, littermates) with 12h-Fasting&12h-Feeding. Adenovirus was always injected at 6 weeks old.  Comparative expression profiling was conducted across groups (Adv-shRNA-Fos compared to Adv-Control).

first: Eduardo

last: H. Gilglioni

first: Wadsen

last: St-Pierre-Wijckmans

first: Leila

last: Hosseinzadeh

first: Christelle

last: Veyrat-Durebex

first: Sumeet

last: P. Singh

first: Roberto

last: Coppari

first: Latifa

last: Bakiri

first: Esteban

last: N. Gurzov


title: Effect of depletion of CNOT4 on gene transcription in human cells.

city: Dallas

first: Yi

last: Liu

state: TX

address: 5323 HARRY HINES BLVD

department: Physiology

institute: UT Southwestern Medical Center

summary: Codon usage bias of an organism has been reported to affect the level of gene expression by influencing this process at multiple steps. Here, we have identified a role of a subunit of CCR4-NOT complex, CNOT4, in the transcriptional regulation of genes in a codon usage dependent manner.

overall_design: RNA-seq analysis of nuclear run-on BrU-IP and input RNA samples from control and CNOT4 depleted HEK293 cells. Two biological replicates were used.

first: Renu

last: Garg


title: Transcriptional Profile in Allergic Contact Dermatitis to Methylisothiazolinone

status: Public on Oct 14 2025

city: São Paulo

first: Maria

middle: Notomi

last: Sato

state: São Paulo

address: Av. Dr. Eneas de Carvalho Aguia, 470

department: Dermatology

country: Brazil

institute: University of São Paulo

summary: This study aimed to investigate the signaling pathways induced by MI in the skin test of individuals with MI-sensitized ACD, comparing them to healthy controls. Individuals initially testing positive only for MI were recruited at the Contact Dermatitis Clinic at Hospital das Clínicas of São Paulo, re-exposed to MI or saline, and biopsied 48 hours after exposure. Negative controls for the patch test were also exposed to MI and saline for comparison. Histopathological and transcriptomic (RNAseq) analyses identified two groups among the ACD patients: Group A (GA), with more pronounced histopathological features such as spongiosis and microvesicles, and Group B (GB), which showed milder reactions and absence of spongiosis. In GA, a total of 1588 genes were upregulated and 2090 downregulated in response to MI, compared to GB. These differentially expressed genes (DEGs) were associated with inflammation and neurological signaling, such as IL-24, IL-9, IL-13, and NTRK1, while IL-37 and IL-18 were downregulated. Similarly, GA compared to the MI-negative ACD group showed 1169 upregulated and 321 downregulated genes. DEG validation in GA through qPCR confirmed increased expression of NTRK1, IL-9, IL-6, IL-13, and CXCL8, with a reduction in IL-18. Protein analysis of IL-24 and IL-9 revealed higher expression in the dermal layer of GA. These results indicate distinct transcriptional profiles in MI-sensitized individuals, despite positive patch tests in all cases. The observed heterogeneity, not previously described in ACD, points to new potential therapeutic targets, such as IL-9, IL-37, and NTRK1. Furthermore, the study suggests that inflammatory and neurological cytokines, such as IL-9 and NTRK1, play key roles in the pathogenesis of ACD.

overall_design: ACD patients as well as skin from health individuals undergoing the same the same conditions and exposion to MI and saline, for histopathological analysis, RNA sequencing, PCR, immunohistochemistry.

first: Taynah

middle: C

last: Melo

first: Iara

middle: G

last: Fernandes

first: Frederico

last: Ferreira

first: Emanuella

last: Souza

first: Gabriel

last: Simões

first: Mirian

middle: N

last: Sotto

first: Yasmim

last: Ramos

first: Vitor

last: Reis


title: Simulating cell-free chromatin using preclinical cancer models for liquid biopsy applications [xenograft plasma WGS]

city: Toronto

first: Scott

middle: Victor

last: Bratman

state: ON

address: 101 College Ave.

department: Medical Biophysics

country: Canada

institute: Princess Margaret Cancer Centre

summary: Cell-free DNA circulates in blood bound to nucleosomes, forming cell-free chromatin (cfChromatin) that retains epigenetic features, including nucleosome positioning and histone modifications. cfChromatin provides a rich source of cancer biomarkers; however, low abundance of tumor-derived cfChromatin and limited availability of clinical samples pose challenges for liquid biopsy research. To address this, we developed a framework to simulate cfChromatin nucleosomal distributions using nuclease-treated conditioned media from tissue cultures. Whole-genome sequencing confirmed that inferred nucleosome positioning reflected cell-type-specific gene expression and chromatin accessibility patterns, and comparisons with plasma cfChromatin from xenografted mice revealed concordant nucleosome profiles. Notably, simulated cfChromatin displayed stronger tumor-specific nucleosome profiles than patient plasma, where hematopoietic-derived cfChromatin dilutes signal. We further leveraged simulated cfChromatin to advance cell-free chromatin immunoprecipitation and sequencing methods, identifying repressive and bivalent chromatin domains predictive of transcriptional activity. Altogether, our results demonstrate utility of simulated cfChromatin as a scalable preclinical tool for liquid biopsy research.

overall_design: CAMA-1 cells in a 50% matrigel solution were injected into the mammary fat pad of NOD/SCID IL2R-gamma-null female mice, along with a 90-day slow-release estrogen pellet implanted between the ear and shoulder of the mouse. At endpoint, blood was collected by terminal cardiac exsanguination under anesthesia. cfDNA from the double-spun plasma was isolated and sequenced.

first: Sasha

last: Main

first: Steven

middle: D

last: De Michino

first: Lucas

last: Penny

first: Aleem

last: Aamir

first: Tina

last: Keshavarzian

first: Benjamin

middle: H

last: Lok

first: Robert

last: Kridel

first: David

middle: W

last: Cescon

first: Michael

last: Hoffman

first: Mathieu

last: Lupien

middle: V


city: Innsbruck

first: Markus

last: Reindl

address: Innrain 66/2

department: Clinical Department of Neurology

country: Austria

institute: Maedical University of Innsbruck

summary:
Neuromyelitis optica spectrum disorder (NMOSD) is a rare neurological autoimmune disease caused autoantibodies targeting the astrocytic water channel aquaporin-4 (AQP4). Binding to AQP4 initiates the activation of innate immune components, especially the complement system. Both in-vivo and in-vitro models have been developed to study the molecular pathophysiology of NMOSD. The aim of our study was to characterize the molecular response of four human cell lines (AQP4-ECFP expressing U-87MG glioblastoma cells, U-87MG expressing only ECFP, HEK293 cells expressing AQP4-EmGFP and human primary astrocytes) to a treatment with AQP4 antibody E5415A and human complement. Complement-dependent cytotoxicity was induced by this treatment in AQP4-expressing cells by the classical complement pathway. Transcriptomic profiles of the in-vitro U-87MG-AQP4-ECFP model and an in-vivo rat model shared a proinflammatory shift towards NF-κB and interleukin-6 pathways. These findings were confirmed on the mRNA and protein levels and treatment with serum samples from AQP4 antibody seropositive NMOSD patients resulted in a similar response. Additionally, NF-κB upregulation was shown by immunohistochemistry in medulla oblongata lesions of NMOSD patients. In conclusion, interleukin-6 and NF-κB pathways play a key role in inflammation caused by the activation of the classical complement pathway in a human cellular model of NMOSD using U-87MG-AQP4-ECFP cells.

Brandl, S., Yu, Q., Hagenbuchner, J. et al. Inflammatory transcriptomic signatures in a human cellular NMOSD model reveal upregulation of NF-κB and IL6 pathways. Sci Rep 15, 43346 (2025). https://doi.org/10.1038/s41598-025-27335-9

overall_design:
RNA-seq profiling of U-87MG-AQP4-ECFP, U-87MG-ECFP, HA and HEK293-AQP4-EmGFP cells treated with anti-AQP4 antibody E5415A and active and heat-inactivated human complement

***************************************************************
Submitter states that only one of the two paired-end raw files is provided for the following samples:
S10, S11, S13, S14 and S16

and no raw files are provided S15, due to file loss.
***************************************************************

first: Sarah

last: Brandl


title: Fasting boosts breast cancer therapy efficacy via glucocorticoid activation (ChIP-Seq)

city: Amsterdam

first: Wilbert

last: Zwart

state: Noord-Holland

address: Plesmanlaan 121

department: Oncogenomics

country: Netherlands

institute: Netherlands Cancer Institute

summary: The vast majority of all breast cancers are driven by oestrogen receptor alpha (ERα) activation, and endocrine therapy represents the mainstay treatment for these patients. However, resistance is common and tumours progress despite years of systemic endocrine suppression. Periodic fasting enhances the efficacy of standard endocrine drugs and delay acquired resistance to them, although the underlying mechanisms remain unclear. Here, we show that fasting, in combination with endocrine therapy, induces extensive epigenetic reprogramming in hormone receptor-positive (HR+) breast cancer xenografts, with large-scale activation of glucocorticoid receptor (GR) and progesterone receptor (PR) signalling, and impairment of activator protein-1 (AP-1) family activity. GR-driven gene programs were selectively activated after fasting, and GR knockout perturbed the beneficial effects of fasting in combination with tamoxifen, in vivo. Exogenous administration of GR-ligands fully recapitulated the observed fasting-enhanced anti-oestrogen action, resulting in tumour regression. Finally, elevated progesterone and cortisol levels were detected in the blood of breast cancer patients after fasting, thus providing clinical validation of our animal findings. Our results indicate GR activation to play a pivotal role in fasting’s ability to enhance endocrine drug activity against hormone-receptor positive breast cancer and suggest that corticosteroid administration should be evaluated as an adjuvant to endocrine therapy in this condition.

overall_design: ChIPs for Erα, H3K27ac, GR, PR and c-JUN in MCF7 xenografted in mice and treated with Control (no treatment), Tamoxifen (TMX), Fasting (48h water-only diet) and combination of tamoxifen and fasting (TMXplusFasting).

first: Nuno

last: Padrão

first: Tesa

last: Severson

first: Sebastian

last: Gregoricchio

first: Ana

last: Guijarro

first: Angelica

last: Persia

first: Francesa

last: Ligorio

first: Merel

last: Roest

first: Joyce

last: Sanders

first: Maria

last: Donaldson Collier

first: Claudio

last: Vernieri

first: Filippo

last: de Braud

first: Livia

last: Pisciotta

first: Alessio

last: Nencioni

first: Irene

last: Caffa


title: Human iPSCs-based modeling unveils chromatin remodeling induced by SETBP1 mutation as a potential initiating factor in GATA2 deficiency.

city: L'Hospitalet de Llobregat

first: Alessandra

last: Giorgetti

state: Barcelona

address: Avinguda de la Granvia de l’Hospitalet, 199

department: PCMRC

country: Spain

institute: IDIBELL

summary: Patients with GATA2 deficiency are predisposed to developing myelodysplastic syndrome (MDS), which can progress to acute myeloid leukemia (AML). This progression is often associated with the acquisition of additional cytogenetic and somatic alterations. Mutations in SETBP1 and ASXL1 genes are frequently observed in pediatric GATA2 patients, but their roles in disease progression remain poorly understood. Genome editing of induced pluripotent stem cells (iPSCs) enabled precise reconstruction of mutation combinations found in patients. Here we developed a human hiPSC-based model to study the impact of SETBP1 and ASXL1 mutations in context of GATA2 deficiency. We show that germline heterozygous GATA2 mutation alone showed no significant effect on myeloid development, while the addition of SETBP1 and ASXL1 mutations impaired myelopoiesis, resulting in monocytopenia. We identified a key role of the SETBP1 mutation in promoting chromatin remodeling near genes involved in myeloid neoplasms, which likely initiated the blockage of myeloid differentiation observed in vitro. Motif analysis of more accessible chromatin regions in the SETBP1 and SETBP1/ASXL1 mutant background highlighted an enrichment for MEIS1, PU.1, RUNX1, and HOXA9, implicating these factors in the disease progression. Our study establishes a novel humanized model system for studying GATA2 deficiency. We demonstrate that SETBP1 mutations act as a primary driver in hematopoietic impairment, providing insights that may inform future therapeutic strategies for patients progressing to MDS/AML.

overall_design: After EBs differentiation CD34/CD43/CD33/CD45+ cells were isolated by Fluoresnce-activated cell sorting (FACS) and sequenced to perform scATAC-seq by SHARED-seq method

first: Joan

last: Pera


title: Effective repair of endometrial injury in rats using enzyme cross-linked gelatin hydrogel with human menstrual blood-derived stem cells

city: Guangzhou

first: Peilin

last: Liao

address: No. 382, Waihuan East Road, University Town, Panyu District, Guangzhou, Guangdong Province

country: China

institute: South China University of Technology

summary: The intact endometrium is critical for zygote implant and embryonic development. We found that gelatin hydrogel loaded with menstrual blood-derived mesenchymal stem cell spheroids effectively repaired endometrial injury in a rat model and improved fertility. To investigate this further, RNA sequencing was used to examine the differences between hydrogel-only repair, hydrogel loaded with single cells, and hydrogel loaded with cell spheroids.

overall_design: RNA-seq analysis of uterine tissue after 14 days of treatment in the rat model of endometrial physical injury, comparing the sham surgery group and the groups treated with different repair methods (gelatin hydrogel, gelatin hydrogel loaded with menstrual blood-derived mesenchymal stem cells, and gelatin hydrogel loaded with menstrual blood-derived mesenchymal stem cell spheroids).

first: Yuyou

last: Duan

first: Jinghe

last: Xie


title: PRMT5 regulates alternative splicing of TCF3 under hypoxia to promote EMT and invasion in breast cancer

city: Bhopal

first: Sanjeev

last: Shukla

state: Madhya Prades

address: Bhopal Bypass Road, Bhauri Bhopal 462 066 Madhya Pradesh, INDIA

department: Biological Sciences

country: India

institute: Indian Institute of Science Education and Research Bhopal

summary: Tumor hypoxia induced alterations in the epigenetic landscape and alternative splicing influence cellular adaptations. PRMT5 is a type II protein arginine methyl transferase that regulates several tumorigenic events in many cancer types. However, the regulation of PRMT5 and its direct implication on aberrant alternative splicing under hypoxia remains unexplored. In this study, we observed hypoxia induced upregulation of PRMT5 via the CCCTC binding factor, CTCF. Further, PRMT5 mediated symmetric arginine dimethylation H4R3me2s and H3R8me2s directly regulated the alternative splicing of Transcription Factor 3 (TCF3). Under hypoxia, PRMT5 mediated histone dimethylation at the intronic conserved region (ICR) present between TCF3 exon 18a and exon 18b recruits DNA methyl transferase 3A (DNMT3A), resulting in DNA methylation. DNA methylation at the TCF3-ICR is recognized and bound by Methyl CpG binding protein (MECP2) and results in RNA-Pol II pausing, promoting the recruitment of the negative splicing factor PTBP1 to the splicing locus of TCF3 mRNA. PTBP1 promotes the exclusion of exon 18a and inclusion of exon 18b which results in the production of the pro-invasive TCF3-18B (E47) isoform which promotes EMT and invasion of breast cancer cells under hypoxia. Collectively, our results indicate that under hypoxia, PRMT5 mediated symmetric arginine dimethylation of histones regulates alternative splicing of TCF3 gene thereby enhancing EMT and invasion in breast cancer.

overall_design: MDA-MB-231 shControl or shPRMT5 cells were kept under Hypoxia (1% oxygen) before performing RNA-seq. RNA-Seq was performed using RNA extracted from shControl and shPRMT5 MDA-MB-231 cells kept under hypoxia

first: Abhishek

last: Mutnuru


title: Non-visual photoreceptive brain specification in sea urchin larvae

status: Public on Oct 08 2025

city: Chiba

first: Koki

last: Tsuyuzaki

address: 1-8-1, Inohana, Chuo-ku, Chiba-shi

country: Japan

institute: Chiba University

summary:
We employed the Western Pacific model sea urchin, Hemicentrotus pulcherrimus, as our experimental system to establish a comprehensive single-cell RNA sequencing (scRNA-seq) data atlas. Our primary objective was to document stage-specific gene expression profiles during the normal developmental progression, ranging from 24 to 96 hours post-fertilization. Our particular focus lay on neurogenesis, a pivotal process occurring in the latter stages of sea urchin development. To elucidate the regulatory mechanisms governing neurogenesis, we utilized pharmacological interventions targeting the Delta-Notch signaling pathway, a central regulator of this process. By perturbing the pathway's lateral inhibition from 16 hours post-fertilization, we aimed to identify gene sets typically suppressed during neurogenesis. Subsequent to the inhibition, we observed a notable upregulation of neurogenic genes and a concurrent expansion in the cellular population expressing these genes, starting from the 24-hour time point.
In addition, the processed Seurat objects (RDS files), stratified by developmental stage, experimental condition, and sample, are available through the HpHase portal (https://cell-innovation.nig.ac.jp/Hpul/).

overall_design: To construct a comprehensive single-cell RNA sequencing (scRNA-seq) atlas of sea urchin development, we acquired datasets from embryos/larvae at 24, 36, 48, 72, and 96-hour time points utilizing the 10xChromium methodology. Additionally, to enhance the resolution of neuronal gene expression patterns, we employed DAPT to inhibit lateral inhibition, thereby promoting neural fate determination during sea urchin development. Subsequently, scRNA-seq data were gathered from the DAPT-treated cohort.

first: Junko

last: Yaguchi

first: Ikutaro

last: Sawada

first: Atsushi

last: Horiuchi

first: Naoaki

last: Sakamoto

first: Takashi

last: Yamamoto

first: Takahiro

last: Yamashita

first: Shunsuke


title: mRNA expression profiles of human iPSC-derived sensory neurons treated with paclitaxel, vincristine, bortezomib or cisplatin in the presence and absence of SP600125

city: Berlin

first: Lois

last: Hew

address: Charitéplatz 1

country: Germany

institute: Charité – Universitätsmedizin Berlin

summary: For our investigation of the role of c-Jun in the underlying mechanisms of chemotherapy-induced peripheral neuropathy (CIPN), human iPSCs derived from patients were differentiated into sensory neurons and subsequently treated in vitro with four different chemotherapeutic drugs (paclitaxel, vincristine, bortezomib or cisplatin) in the presence and absence of small molecule inhibitor, SP600125. The transcriptomic analyses of such treated samples revealed upregulated transcription of c-Jun mRNA across the four drug types, which was reduced upon addition of SP600125. While this suggests the involvement of the JNK signalling pathway, detailed analyses of correlation coefficient revealed that a high correlation of c-Jun with neuronal injury, neuroinflammatory, metabolic and neuromodulation markers, thus identifying a central role of c-Jun in CIPN pathophysiology.

overall_design: Human iPSC-derived sensory neurons (iPSC-DSN) were treated with 100nM of paclitaxel for 48h, or 50nM of vincristine for 16h, or 10nM of bortezomib for 16h, or 10µM of cisplatin for 16h, in combination with 10µM of SP600125, followed by RNA extraction.

first: Smilla

last: Maierhof

first: Andranik

last: Ivanov

first: Dieter

last: Beule

first: Christian

last: Schinke


title: De novo mutations in the MiDAC HDAC complex cause a neurodevelopmental 2 syndrome resulting from increased deacetylase activity [qPRO-seq]

status: Public on Oct 13 2025

city: Leicester

first: John

last: Schwabe

state: Leicestershire

address: Lancaster Road

department: Molecular and Cell Biology

country: United Kingdom

institute: University of Leicester

summary: MIDEAS is a scaffold protein that, together with DNTTIP1, mediates assembly of the 36 MiDAC histone deacetylase complex. Mice lacking MiDAC die before birth suggesting 37 a key developmental function. Here, we report two unrelated individuals, with a 38 multisystem disorder characterized by delayed speech development, joint 39 contractures, dysmorphic features and dysmotility of the gut. Both individuals have the 40 same de novo heterozygous mis-sense variant in MIDEAS (Tyr654Ser). A cryoEM 41 structure of the MiDAC complex reveals that this amino acid is located in a conserved 42 auto-inhibitory loop that covers the active site of the deacetylase enzyme. We suggest 43 that the mutation results in loop displacement leading to elevated deacetylase activity. 44 In support, we observe reciprocal gene expression changes in patient fibroblasts 45 compared with a cell line following rapid MiDAC degradation. Our results establish 46 Mideas as a novel dominant monogenic disease gene and that hyperactivity of the 47 MiDAC complex results in a characteristic multisystem disorder.

overall_design: qPRO nascent transcription profiling of HCT 116 cells CRISPR-engineered to express endogenous DNTTIP1 with C-terminal FKBPF36V/FLAG to allow rapid degradation using dTAGv1 at 2h and 6h.

first: Robert

last: Turnbull

first: Kristupas

last: Sirvydis


title: Tri-snRNP activity modulates tauopathy phenotypes in C. elegans

city: Seattle

first: Brian

last: Kraemer

state: WA

address: 1660 S Columbian Way

department: Medicine

institute: University of Washington

summary: Alzheimer's Disease (AD) and other tauopathies are neurodegenerative disorders with devastating consequences for cognition and memory. Pathogenic accumulation of tau can be modeled in Caenorhabditis elegans, which recapitulate human neurodegeneration including aging-dependent accumulation of phosphorylated tau, tau aggregation, neuronal dysfunction, and neuron degeneration. Using forward genetic screens to identify genes modulating tau pathology, we identified a single point mutation in dib-1 that ameliorates tau-driven behavioral defects, prevents neurodegeneration, and decreases tau protein levels. The dib-1 gene encodes a small, highly conserved protein, known as TXNL4A in humans, and participates in mRNA splicing via the U4/U6.U5 tri-snRNP. Notably, heterozygous loss of prp-8, a neighboring protein within the tri-snRNP, also prevents tau-driven neurodegeneration. RNA sequencing of dib-1 mutants demonstrates widespread intron retention consistent with disruption of tri-snRNP functions. Disruption of nonsense-mediated decay further rescues tau-driven phenotypes only in the presence of the dib-1 mutation. TXNL4A levels are decreased in Alzheimer's disease in human frontal cortex, demonstrating the translational relevance of dib-1. Taken together these findings suggest pathological tau impacts splicing function, and spliceosomal activity modulation can ameliorate tauopathy.

overall_design: Biological replicates of wildtype, dib-1 mutant, tau transgenic, and tau transgenic with mutant dib-1.

first: Katherine

last: LeBlanc

first: Randall

last: Eck

first: Aleen

last: Saxton

first: Pamela

last: McMillan

first: Jeanna

last: Wheeler

first: Nicole

last: Liachko

first: C. Dirk

last: Keene

first: Caitlin

last: Latimer

first: Rebecca

last: Kow


title: Resistance to Radiation Enhances Metastasis by Altering RNA Metabolism

city: Worcester

first: Kensei

last: Kishimoto

address: 364 Plantation St

institute: UMass Chan Medical School

summary: The cellular programs that mediate therapy resistance are often important drivers of metastasis, a phenomenon that needs to be understood better to improve screening and treatment options for cancer patients. Although this issue has been studied extensively for chemotherapy, less is known about a causal link between resistance to radiation therapy and metastasis. We investigated this problem in triple-negative breast cancer (TNBC) and established that radiation resistant tumor cells have enhanced metastatic capacity, especially to bone. Resistance to radiation increases the expression of integrin b3 (ITGb3), which promotes enhanced migration and invasion. Bioinformatic analysis and subsequent experimentation revealed an enrichment of RNA metabolism pathways that stabilize ITGb3 transcripts. Specifically, the RNA binding protein heterogenous nuclear ribonucleoprotein L (HNRNPL), whose expression is regulated by Nrf2, mediates the formation of circular RNAs (circRNAs) that function as competing endogenous RNAs (ceRNAs) for the family of let-7 microRNAs that target ITGb3. Collectively, our findings identify a novel mechanism of radiation-induced metastasis that is driven by alterations in RNA metabolism.

overall_design: Two biological replicates of radiation resistant MDA-MB-468 human breast cancer cell line with and without stable HNRNPL knockdown were sequenced on Nanopore using Induro-mediated circular RNA-sequencing to identify HNRNPL-dependent circular RNA.

first: Ayush

last: Kumar

first: Hira Lal

last: Goel

first: Christi

last: Wisniewski

first: Rui

last: Li

first: Brendan

last: Pacheco

first: Lihua Julie

last: Zhu

first: William A.

last: Flavahan

first: Arthur M

last: Mercurio


title: Disrupted developmental signaling induces novel transcriptional states

city: Stanford

first: Aleena

last: Patel

state: CA

address: 279 Campus Dr

institute: Stanford University

summary: Single cell RNA sequencing was used to measure the transcriptional profiles of 50% epiboly zebrafish embryos treated with constitutive ERK signaling pathway activation by the optogenetic tool photoswitchable MEK or left untreated.

overall_design: All cells from dechorionated 50% epiboly zebrafish embryos that were injected with psMEK mRNA and exposed light at the 1-cell stage for the treated condition were dissociated by manual pipetting. The same was performed for wild type uninjected embryos used in the untreated condition. 7 samples were collected over 4 batches. 3 samples were treated embryos, and 4 samples were untreated embryos.

middle: L

first: Maria

last: Avdeeva


title: Combinatorial BMP4 and Activin direct choice between alternate routes to endoderm during human gastrulation [RNA-seq]

city: London

first: Silvia

middle: DM

last: Santos

address: 1 Midland Road

institute: The Francis Crick Institute

summary: Cellular differentiation requires the proper interpretation of multiple signalling cues which vary in concentration. How exactly the combination and history of signals a cell is exposed to influences a fate decision remains poorly understood. In this study we use hESCs as a tractable model system to explore how combinations of cues guide state transitions during gastrulation. Using single-cell transcriptomics and live-cell imaging of engineered hESCs expressing endogenous cell state reporters, we reconstructed developmental lineages and obtained single-cell measurements of fate specification dynamics during gastrulation. We found that definitive endoderm, one of the three germ layers, arises from two distinct developmental trajectories: a direct route from pluripotency, and an indirect route via a mesoderm progenitor state. Furthermore, by modulating the signalling input we found that the relative concentration of Activin and BMP4 controls the choice between alternate trajectories to endoderm. These findings reveal a lineage convergence event during human gastrulation with multiple routes existing to definitive endoderm dictated by the combination of signalling cues presented. This work shows that the combination cues a cell is exposed to not only directs the final fate outcome it assumes, but the developmental route taken.

overall_design: The goal of this study was to characterise the transcriptional changes that occur in human embryonic stem cells (hESCs) during differentiation in response to Activin 100 ng/mL and BMP4 10 ng/mL or BMP4 50 ng/mL. RNA-Seq of mRNA was performed on cultured hESCs treated with either Activin 100 ng/mL and BMP4 10 ng/mL or BMP4 50 ng/mL for up to 96 hours. Experiments were performed in triplicate.


title: Combinatorial BMP4 and Activin direct choice between alternate routes to endoderm during human gastrulation.

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

overall_design: Refer to individual Series


title: A microenvironment-driven HLA-II-associated insulin neoantigen elicits persistent memory T cell activation in diabetes

status: Public on Sep 30 2025

city: St Louis

first: Tiandao

state: MO

address: 4444 Forest Park Ave

institute: Washington University

summary: The antigenic landscape of autoimmune diabetes reflects a failure to preserve self-tolerance. Yet, how novel neoantigens emerge in humans remains incompletely understood. Here, we designed an immunopeptidomics-based approach to probe HLA-II-bound, islet-derived neoepitopes in patients with type 1 diabetes (T1D). We uncovered a Cys→Ser transformation, conserved between mice and humans, that reshapes autoreactivity to insulin at the single-residue level. This transformation, which we call “C19S,” arises from oxidative remodeling of insulin in stressed pancreatic islets and also occur in cytokine-activated antigen-presenting cells, contributing to a feed-forward loop of neoepitope formation and presentation. Despite involving just one amino acid, C19S is recognized by HLA-DQ8-restricted, register-specific CD4+ T cells that expand at diabetes onset. These neoepitope-specific CD4+ T cells lack regulatory potential but acquire a poised central memory phenotype that persists across disease progression. These findings reveal a distinct, microenvironment-driven route of neoantigen formation that fuels sustained autoreactivity in diabetes.

overall_design: The study aims to investigate transcriptional features of CD4 T cells specifically recognizing the InsB12-20(C19S) neoepitope. Two tetramer-binding CD4+ T cell populations (C19S and native), as well as tetramer-negative controls, were FACS-sorted from pooled spleens and lymph nodes of 8-week-old female NOD mice. The cells were sumitted for 10x scRNA-seq and TCR vdj library preparation and analyzed for single-cell gene expression and TCR sequencing. The experiments were performed in replicates.

first: Neetu

last: Srivastava

first: Bo

last: Zhang

first: Xiaoxiao

last: Wan


title: Simulating cell-free chromatin using preclinical cancer models for liquid biopsy applications

first: Scott

last: Bratman

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

Cell-free DNA circulates in blood bound to nucleosomes, forming cell-free chromatin (cfChromatin) that retains epigenetic features, including nucleosome positioning and histone modifications. cfChromatin provides a rich source of cancer biomarkers; however, low abundance of tumor-derived cfChromatin and limited availability of clinical samples pose challenges for liquid biopsy research. To address this, we developed a framework to simulate cfChromatin nucleosomal distributions using nuclease-treated conditioned media from tissue cultures. Whole-genome sequencing confirmed that inferred nucleosome positioning reflected cell-type-specific gene expression and chromatin accessibility patterns, and comparisons with plasma cfChromatin from xenografted mice revealed concordant nucleosome profiles. Notably, simulated cfChromatin displayed stronger tumor-specific nucleosome profiles than patient plasma, where hematopoietic-derived cfChromatin dilutes signal. We further leveraged simulated cfChromatin to advance cell-free chromatin immunoprecipitation and sequencing methods, identifying repressive and bivalent chromatin domains predictive of transcriptional activity. Altogether, our results demonstrate utility of simulated cfChromatin as a scalable preclinical tool for liquid biopsy research.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

first: Sasha

last: Main

first: Steven

last: De Michino

first: Lucas

last: Penny

first: Aleem

last: Aamir

first: Tina

last: Keshavarzian

first: Benjamin

last: Lok

first: Robert

last: Kridel

first: David

last: Cescon

first: Michael

last: Hoffman

first: Mathieu

last: Lupien


title: Secretome translation shaped by Lysosomes and Lunapark-marked ER Junctions

city: Ashburn

first: Jennifer

last: Lippincott-Schawrtz

state: VA

address: 19700 Helix Dr

institute: HHMI

summary: This study investigates how translation of mRNAs encoding membrane and secretory proteins (“secretome mRNAs”) is spatially regulated in human cells. Using live-cell imaging, proteomics, and RNA-sequencing, we show that Lunapark (LNPK)–enriched ER junctions and lysosomes form specialized hubs that enhance secretome mRNA translation. RNA-seq was performed on U-2 OS cells with control and Lunapark knockout conditions to assess transcriptome changes.

overall_design: RNA-seq was performed on U-2 OS control cells and Lunapark knockout cells in biological triplicates. Total RNA was extracted, converted to cDNA, libraries prepared with modified Nextera XT protocol, and sequenced on Illumina NextSeq 2000. Differential expression analysis was performed using STAR/STARsolo alignment and DESeq2 in MATLAB.

first: Heejun

last: Choi

first: Ya-Cheng

last: Liao

first: Young

middle: J

last: Yoon

first: Jonathan

last: Grimm

first: Luke

last: Lavis

last: Singer

last: Lippincott-Schwartz


title: Combinatorial BMP4 and Activin direct choice between alternate routes to endoderm during human gastrulation [RN25025_RNAseq]

first: Silvia

last: Santos

overall_design: The goal of this study was to characterise the transcriptional differences between definitive endoderm cells derived from two distinct developmental trajectories. This was achieved by ultilising a H1 hESCs (human embryonic stem cells) engineered with a FOXC1 Achilles transcriptional reporter. After 72 hours of differentiation in response to Activin 100 ng/mL and BMP4 10 ng/mL hESCs were fixed with 4% PFA, stained for SOX17 and sorted into four disitnct populations: SOX17+ Achilles+, SOX17+ Achilles-, SOX17- Achilles+ and SOX17+ Achilles+ and RNA extracted for sequencing. Experiments were performed in triplicate. Control cells were maintained in mTeSR1.


title: ATF-4 regulates C. elegans defense against pathogen infection

city: Spokane

first: Yiyong

last: Liu

address: 412 E Spokane Falls Blvd

institute: Washington State University

summary: To gain molecular insights into how ATF-4 regulates C. elegans defense against bacterial infection, we employed RNA-seq to profile gene expression in atf-4(ok576) and WT animals with or without S. enterica infection.

overall_design: Four biological replicates of RNA samples from each of four groups of worms (atf-4(ok576) and WT animals with or without exposure to S. enterica for 24 hours) were collected and subjected to RNA-seq analysis.

first: Jingru

last: Sun

first: Phillip

last: Wibisono


title: Strength of CAR signaling determines T cell versus ILC differentiation from pluripotent stem cells

city: Los Angeles

first: Gay

middle: Miriam

last: Crooks

state: California

address: 610 Charles E. Young Drive, East

department: Pathology & Laboratory Med

institute: University of California Los Angeles

summary: Generation of chimeric antigen receptor (CAR) T cells from pluripotent stem cells (PSCs) will enable advances in cancer immunotherapy. A detailed understanding of the effects of CARs on T cell differentiation from PSCs is important to this effort. The recently described artificial thymic organoid (ATO) system supports in vitro differentiation of PSCs to T cells. Unexpectedly, PSCs transduced with a CD19-targeted CAR resulted in diversion of T cell differentiation to the ILC2 lineage in ATOs. T cells and ILC2s are closely related lymphoid lineages which share certain developmental and transcriptional programs. Mechanistically, we show that antigen-independent CAR signaling during early lymphoid development was evident by single cell RNA-seq of ILC2-primed precursors. We applied this understanding to rationally modulate CAR signaling strength through changes to CAR expression level, structure, or expression of cognate antigen which demonstrate that the T-versus-ILC decision can be controlled in either direction, including restoration of normal CAR-T cell development.

overall_design: To further characterize the CAR ILC2-like cells, we sorted H1-CAR DN cells, containing ILC2s, as DAPI-eGFP+CD3-TCRαβ-CD8α-CD4- from week 6 ATOs in biological triplicates and compared them with the previously published ATO derived CD8SP T cell gene expression (GSE116015)

first: Suwen

last: Li

first: Chloe

last: Wang

first: Amélie

last: Montel-Hagen

first: Ho-Chung

last: Chen

first: Shawn

last: Lopez

first: Olivia

last: Zhou

first: William

last: Satyadi

first: Carlos

last: Botero

first: Steven

last: Tsai

first: Claudia

last: Wong

last: Casero

first: Christopher

last: Seet


title: Regulation of corneal stromal cell behavior by modulating curvature using a hydraulically-controlled organ chip array

city: Salt Lake City

first: Minju

last: Kim

state: Utah

department: Mechanical Engineering

institute: The University of Utah

summary: Cornea exhibits distinct curvature-dependent mechanical environments that influence stromal cell phenotype and extracellular matrix remodeling. In this study, we employed a hydraulically-controlled cornea-on-a-chip platform to systematically vary curvature and examine transcriptional and phenotypic responses of human corneal keratocytes. RNA sequencing revealed differential expression profiles associated with curvature states that mimic pathological conditions such as cornea plana and keratoconus. Curvature-dependent regulation of extracellular matrix genes, cytoskeletal organization, and signaling pathways was validated with qPCR and immunostaining. These findings provide transcriptomic insights into how corneal tissue geometry contributes to disease progression and may inform future therapeutic strategies for corneal ectatic disorders.

overall_design: RNA-seq profiling of primary human corneal keratocytes cultured on curvature-controlled chips under four conditions: flat (control), low curvature, medium curvature, and high curvature. Each sample was collected at Day 9 after applying curvature for 6 days.

first: Kanghoon

last: Choi

first: David

last: Krizaj

first: Jungkyu


title: CD94/NKG2A-Qa-1b Axis as a Key Modulator of Vaccine Responsiveness in Aging Populations

city: Chengdu

first: Li

state: None Selected

address: No. 17, Block 3rd, Renmin Road South,

institute: National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University

summary: Vaccine responses decline with age, yet underlying mechanisms remain unclear. Using an Alum-adjuvanted spike (S+Alum) vaccine, this study identifies an aging-linked pathway in which senescence-associated secretory phenotype cytokines (CCL2, IL-6, IFN-γ) at the injection site are upregulated and associate with increased H2-T23 (Qa-1b) expression on monocytes. These H2-T23high monocytes interact with MHCIIhigh cDC2 via IL-10/TGF-β, limiting dendritic-cell antigen presentation, and engage the CD94/NKG2A-Qa-1b checkpoint on T cells, further dampening immunity. In humans, HLA-E+ monocytes and NKG2A⁺ T cells are infrequent in young individuals but show higher prevalence in peripheral blood from older adults, highlighting this pathway as a regulatory target. Blocking the CD94/NKG2A-Qa-1b axis thus emerges as a potential strategy to enhance vaccine performance in aging populations. Overall, the findings define a mechanistic link between SASP-driven myeloid programming, impaired antigen presentation, and checkpoint-mediated T-cell restraint in aging, with implications for vaccine design tailored to older individuals.

overall_design: To delineate the molecular landscape of early immune responses in young and aged mice, single-cell RNA sequencing (scRNA-seq) analysis was performed on CD45+ immune cells isolated from skeletal muscle 24 hours after intramuscular injection.

first: Ziyi

last: Bai

first: Dandan

last: Wan

first: Wenyan

last: Ren

first: Haiying

last: Que

first: Jiayu

first: Yishan

last: Lu

first: Xuemei

last: He

first: Weiqi

last: Hong

first: Aqu

last: Alu

last: Peng

first: Minyang

last: Fu

first: Yuhe

last: Huang

first: Jiayuan

last: Ai

first: Qiaonan

first: Hantai

last: Tan

first: Zirui

last: Gao

first: Xinyu

last: Tian

first: Cong

last: Tang

first: Yanan

first: Shuaiyao

first: Xiaohe

first: Xiawei

last: Wei


title: Phospho-proteome profiling in human neurons reveals targets of TBK1 in ALS/FTD-Phospho-proteome profiling in human neurons reveals targets of TBK1 in ALS/FTD-associated autophagy networks [iNeurons]

city: San Francisco

first: Julie

last: Smeyers

address: 1651 4th street

department: IND

institute: UCSF

summary: Loss-of-function variants in TBK1, encoding a serine/threonine protein kinase, are strongly associated with sporadic and familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the targets of TBK1 in neurons are unknown, and how haploinsufficiency for TBK1 leads to age-related neurodegeneration remains unresolved. Here, we utilized sets of isogenic induced pluripotent stem cells (iiPSCs) with loss of TBK1 or loss of optineurin (OPTN) for quantitative proteomics and phospho-proteomics across tens of thousands of phospho-sites in proliferating stem cells and induced excitatory neurons. We found that TBK1 sustains the abundance and phosphorylation of its interacting adapter proteins, AZI2/NAP1, TBKBP1, and TANK1, in the cellular context of neurons. Moreover, TBK1 regulates the phosphorylation of selective autophagy proteins, particularly at novel phospho-serine residues within cargo receptors, and endo-lysosomal pathways, including the late-endosome GTPase RAB7A, in human neurons. Additionally, we observed that the disease-associated TBK1 G217R variant acts as a phospho-protein quantitative trait locus (ppQTL) for the phosphorylation status of familial ALS-associated proteins p62/SQSTM1 and OPTN, as well as GABARAPL2, an Atg8- family autophagy protein. Furthermore, we demonstrate that TBK1 is required for neurite outgrowth and lysosomal function. Finally, we provide a census of the phospho-proteome in nascent human neurons for further dissection of protein phosphorylation in neural-enriched proteins, such as tau, in neurological and psychiatric disorders. These studies provide a global view of TBK1 as a central point of convergence in ALS/FTD-linked endo-lysosomal networks that act in a cell-autonomous manner to maintain protein homeostasis in neurons.

overall_design: RNA-seq profiling of  control, TBK1 KO, OPTN KO, TBK1 E696KSNV/WT and TBK1 E696KSNV/SNV hiPSCs and iNeurons

first: Juan

middle: A

last: Oses-Prieto

first: Lin

last: Yadanar

first: Man

last: Wang

first: Serena

first: Karen

first: Marci

last: Iadarola

first: Taylor

last: Watanabe

first: Jayanta

last: Debnath

first: Alma

last: Burlingame

first: Daniel

last: Mordes


title: Transcriptomic analysis was conducted on Siha cells with the HPV 16E7 gene knocked down.

city: Hangzhou

first: Boya

last: Zhang

state: Zhejiang

address: Qingchun East Road

institute: Department of dermatology,Sir Run Run Shaw Hospital, Zhejiang University, School of Medicine

summary: RNA sequencing (RNA-seq) analysis demonstrated that the knockdown of HPV 16E7 in SiHa cells resulted in alterations in gene expression compared to the control group.

overall_design: Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The quantity and purity of the total RNA were analyzed using a Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA), with an RNA Integrity Number (RIN) greater than 7.0. Approximately 10 μg of total RNA representing a specific adipose type was used to isolate poly(A) mRNA with poly-T oligo-attached magnetic beads (Invitrogen). Following purification, both poly(A)- and poly(A)+ RNA fractions were fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were then reverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNA-Seq sample preparation kit (Illumina, San Diego, USA). The average insert size for the paired-end libraries was 300 bp (50 bp). We then performed paired-end sequencing on an Illumina NovaSeq 6000 at lc-bio (China) following the vendor's recommended protocol.

last: zhang


title: Transcriptomics revealed changes in the skin of HTRA1 knockout mice

summary: RNA sequencing (RNA-seq) analysis identified alterations in gene expression within the skin of HTRA1 knockout mice compared to wild-type mice.

overall_design: Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The quantity and purity of the total RNA were analyzed using a Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA), with an RNA Integrity Number (RIN) greater than 7.0. Approximately 10 μg of total RNA representing a specific adipose type was used to isolate poly(A) mRNA with poly-T oligo-attached magnetic beads (Invitrogen). Following purification, both poly(A)- and poly(A)+ RNA fractions were fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were then reverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNA-Seq sample preparation kit (Illumina, San Diego, USA). The average insert size for the paired-end libraries was 300 bp (50 bp). We then performed paired-end sequencing on an Illumina NovaSeq X Plus at lc-bio (China) following the vendor's recommended protocol.


title: Phospho-proteome profiling in human neurons reveals targets of TBK1 in ALS/FTD-Phospho-proteome profiling in human neurons reveals targets of TBK1 in ALS/FTD-associated autophagy networks

overall_design: RNA-seq profiling of  control, TBK1 KO, OPTN KO, TBK1 E696KSNV/WT and TBK1 E696KSNV/SNV hiPSCs

last: Lu


title: Ribosomal protein control of hematopoietic stem cell transformation through direct, non-canonical regulation of metabolism

city: Philadelphia

first: David

last: Wiest

address: 333 Cottman Ave

institute: Fox Chase Cancer Center

summary: We report here that expression of the ribosomal protein, RPL22, is frequently reduced in human myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML); reduced RPL22 expression is associated with worse outcomes. Mice null for Rpl22 display characteristics of an MDS-like syndrome and develop leukemia at an accelerated rate. Rpl22-deficient mice also display enhanced hematopoietic stem cell (HSC) self-renewal and obstructed differentiation potential, which arises not from reduced protein synthesis but from increased expression of the Rpl22 target, ALOX12, an upstream regulator of fatty acid oxidation (FAO). The increased FAO mediated by Rpl22-deficiency also persists in leukemia cells and promotes their survival. Altogether, these findings reveal that Rpl22 insufficiency enhances the leukemia potential of HSC via non-canonical de-repression of its target, ALOX12, which enhances FAO, a process that may serve as a therapeutic vulnerability of Rpl22 low MDS and AML leukemia cells.

overall_design: Splenocytes were isolated from genetically engineered Mll-AF9 mice that are either Rpl22 proficient or Rpl22 deficient. After sorting for CD11b+Gr1+ leukemic blasts, RNA was isolated, and library preparation was performed using Illumina mRNA stranded kit. Libraries were pooled and sequenced on the NextSeq 2000 platform

first: Truong

last: Billy

first: Price

last: Rachael

first: Rhodes

last: Michele

first: Wiest

last: David


title: Pneumococcal H₂O₂ Reshapes Mitochondrial Function and Reprograms Host Cell Metabolism

city: Jackson

first: Jorge

middle: Eugenio

last: Vidal

state: MS

address: 2500 North State Street, Jackson MS 39216

department: Cell and Molecular Biology

institute: University of Mississippi Medical Center

summary: Streptococcus pneumoniae (Spn), a primary cause of pneumonia, induces acute lung parenchymal damage through a unique metabolic pathway generating hydrogen peroxide (H2O2) as a byproduct. This study demonstrates that Spn-derived H2O2, primarily produced by pyruvate oxidase (SpxB), inhibits key tricarboxylic acid (TCA) cycle enzymes (aconitase, glutamate dehydrogenase, and α-ketoglutarate dehydrogenase) in lung epithelial cells, leading to citrate accumulation and diminished NADH production for oxidative phosphorylation. RNA sequencing reveals SpxBdependent upregulation of glycolytic genes (HIF1A, IER3, HK2, PFKP), restricting pyruvate entry into the TCA cycle and increasing glucose consumption and lactate/acetate production, indicative of a Warburg-like metabolic shift that enhances bacterial survival. Notably, mitochondrial membrane potential remains largely preserved, with minimal apoptosis despite Spn-induced stress. These findings uncover a novel mechanism of Spn-driven host metabolic reprogramming, highlighting potential therapeutic targets for pneumococcal diseases.

overall_design: Comparative transcriptomic analysis of RNA-seq data from human Calu-3 bronchial epithelial cells, either mock-infected or infected with Streptococcus pneumoniae strain TIGR4 and its isogenic mutant derivatives.


title: Retinal polyunsaturated fatty acid supplementation reverses aging-related vision loss in mice

city: Irvine

first: Emily

last: Tom

address: 847 Health Sciences Quad, FLF Lab #6131A

institute: University of California, Irvine

summary: The retina is uniquely enriched in polyunsaturated fatty acids (PUFAs), which are primarily localized in cell membranes, where they govern membrane biophysical properties such as diffusion, permeability, domain formation, and curvature generation. During aging, alterations in lipid metabolism lead to reduced content of very long-chain PUFAs (VLC-PUFAs) in the retina, and this decline is associated with normal age-related visual decline and pathological age-related macular degeneration (AMD). ELOVL2 (Elongation of very-long-chain fatty acids-like 2) encodes a transmembrane protein that produces precursors to docosahexaenoic acid (DHA) and VLC-PUFAs, and methylation level  of its promoter is currently one of the best predictors of chronological age. Here, we show that mice lacking ELOVL2-specific enzymatic activity (Elovl2C234W) have impaired contrast sensitivity and slower rod response recovery following bright light exposure. Short-term and long-term intravitreal supplementation with the direct product of ELOVL2, 24:5n-3, in aged animals improved visual function and reduced accumulation of ApoE and C3d in sub-RPE deposits. At the molecular level, the gene expression pattern observed in retinas supplemented with 24:5n-3 exhibited a partial rejuvenation profile, including decreased expression of aging-related genes and a transcriptomic signature similar to younger retinas. Finally, we present  human genetic data showing significan t association of two variants in the ELOVL2 locus with the onset of intermediate AMD, underlining the translational importance of our findings. In sum, our study identifies potential therapeutic opportunities and defines ELOVL2 as a promising target for interventions aimed at preventing age-related vision loss.

overall_design: RNA-seq profiling of retinas from 12-month-old wildtype and Elovl2^C234W and 18-month-old wildtype C57BL6/J mice; vehicle- and 24:5-supplemented retinas from 18-month-old mice 5 days after intravitreal injection

first: Fangyuan

last: Gao

first: Cezary

last: Rydz

first: William

last: Cho

first: Dorota

last: Skowronska-Krawczyk


title: Transcriptional and functional architecture of the whole neutrophil compartment [bulkRNAseq_human_donors]

city: New Haven

first: Andrés

last: Hidalgo

address: 10 Amistad Street

institute: Yale University

summary: In this study, we present NeuMAP, a comprehensive single-cell analysis of neutrophils spanning over 40 anatomical, physiological, and pathological contexts in mice. NeuMAP confirms and expands previous models of neutrophil diversity, revealing the organization of neutrophils into distinct functional hubs under both normal and pathological conditions. Furthermore, we delineate prototypical trajectories as key organizers of granulopoiesis and examine neutrophil dynamics along these trajectories during acute inflammation and cancer. Integrating insights from fate mapping, mutant mouse models, and in vitro experiments, we identified immunological signals guiding neutrophil through these typical trajectories of granulopoiesis, that balance immune protection, tissue homeostasis and repair. Specifically, we identified IFNB, GMCSF, and TGFB as drivers of pro-inflammatory, cancer-associated, and mature neutrophil states, respectively and found that the transcription factor JUNB drives the angiogenic and immunosuppressive function of neutrophils. Additionally, we uncover conserved transcriptional signatures for human neutrophil states, validate their prognostic significance in cancer patients, and introduce a proof-of-concept strategy for exploring the diagnostic potential of neutrophil functional diversity across various diseases using single-cell transcriptomics of blood neutrophils. Our study provides a model that delineates the global architecture of the neutrophil compartment in mammals, and stablishes a robust framework for further exploration of neutrophil biology.

overall_design: Bone marrow samples were obtained from healthy donors under informed consent approved by the ethics committee of the University Hospital Tübingen. CD34⁺ HSPCs were isolated through Ficoll gradient centrifugation followed by magnetic bead-based separation using the EasySep™ Human CD34⁺ Cell Selection Kit II (Stem Cell Technologies, #17856). CD34⁺ cells (n=4; purity 95.4 ± 1.9%) were cultured at a density of 5 × 10⁵ cells/ml in StemSpan™ SFEM II hematopoietic stem cell medium (Stem Cell Technologies, #09655), supplemented with 1% penicillin/streptomycin (P/S), 20 ng/ml IL-3, 20 ng/ml IL-6, 20 ng/ml TPO, 50 ng/ml SCF, and 50 ng/ml FLT-3L (all cytokines purchased from R&D Systems). Cells were cultured under standard conditions (37°C, 5% CO₂) and frozen for future use. For granulocytic differentiation in vitro, cells were seeded at a density of 2 × 10⁵ cells/ml. During the first 8 days of differentiation (days 0–7), cells were maintained in a myeloid cell expansion medium - RPMI 1640 supplemented with 10% FCS, 1% penicillin/streptomycin (P/S), 5 ng/ml SCF, 5 ng/ml IL-3, and 1 ng/ml G-CSF. The medium was changed every two days. On day 8 of culture, the medium was replaced with a granulocytic cell differentiation medium (RPMI 1640 supplemented with 10% FCS, 1% P/S, and 1 ng/ml G-CSF). The medium was changed every two days until day 14. On day 13 of differentiation, cells were collected and counted. 800,000 cells were lysed for RNA isolation, 50,000 cells for FACS, and 40,000 cells for cytospins. The remaining cells were resuspended in fresh granulocytic differentiation medium at a seed density of 2 × 10⁵ cells/ml and divided into four groups. Group one was maintained in granulocytic differentiation medium, group two was treated with 10 ng/ml TGF-β, group three with 10 ng/ml IFNβ (refreshed after 24h), and group 4 with 10 ng/ml GM-CSF. RNA-seq analyses were performed 48 hours after stimulation.

first: Daniela

last: Cerezo-Wallis

first: Ivan

last: Ballesteros

first: Andrea

last: Rubio


title: Architecture of the neutrophil compartment in mice [scRNA-seq rewilded]

overall_design: 8 weeks old Male BALB/C and C57Bl6 mice were sacrificed via CO2, and blood, spleen, bone marrow, and lung were collected. 1ml of blood was collected in BD Microtainer tubes containing EDTA (365974, BD), and red blood cell were lysed in 10ml of 1X ACK buffer for 10min on ice. The spleen, lung and tumors  were enzymatically digested to generate cell suspensions that were  washed, resuspended in 1ml of 1X ACK buffer and incubated for 5min on ice to get rid of the red blood cells. The cell suspension was washed, resuspended in cold staining buffer, and kept on ice. The bone marrow was harvested by flushing the femur’s shaft with cold staining buffer and filtered to obtain a single cell suspension. The cells were washed and red blood cell were lysed using 1ml of 1X ACK buffer (5min on ice). After lysis, the cells were further washed, resuspended in cold staining buffer, and kept on ice. Cell suspensions  were incubated with Fc block (TruStain FcX PLUS – 156603 – Biolegend) for 10min on ice, and for another 30min with an antibody cocktail containing: anti-Ly6G_PE (127608 - Biolegend), anti-CD11b_APC (101211 - Biolegend). Before acquisition and sorting, single cell suspensions were incubated for 15min with Dapi (NBP2311561, Novus Biologicals). Live-CD11b+-Ly6G+ neutrophils were sorted out. After sorting, the  neutrophil suspensions were Taged (BD Multiplexing Kit)  and pooled in equal proportion. For the generation of single-cell whole-transcriptomes we used a BD Rhapsody system according to the manufacturer instructions. Briefly, cell suspensions from each condition were incubated with Sample Tags (BD) for 20 minutes at RT. Cells were then washed three times and pooled in a single tube. Viability and cell concentration using the Countess III cell counter (Thermofisher). 60,000 cells were loaded into a Rhapsody Single Cell Analysis System cartridge. Cell capture and cDNA synthesis were performed according to manufacturer’s instructions; cells were isolated into nanowells by gravity, then cells lysed and mRNAs together with sample tags oligos were released and captured by beads present in the nanowells. Each bead contained a unique oligo named “cell label” to identify each individual bead. All beads present in the cartridge were collected and cDNA synthesis took place in a single reaction. At this point each cDNA and Sample Tag oligo were attached to its corresponding “cell label” oligo. Two separated indexed libraries were prepared for whole transcriptome analysis and Sample Tag demultiplexing following manufacturer’s instructions. The average size of the libraries was calculated using the 2100 Bioanalyzer (Agilent) and the concentration was determined using the Qubit fluorometer (Thermofisher). Finally, libraries were combined and sequenced together in a paired-end run (60x42) using a NextSeq 2000 system (Illumina) and a P2 flow cell. Output files were processed with NextSeq 1000/2000 Control Software Suite v1.4.1. FastQ files for each sample were obtained using BCL Convert v3.6.3 software (Illumina).

first: Cerezo-Wallis

last: Daniela

first: Rubio-Ponce

last: Andrea

first: Ballesteros

last: Iván

first: Hidalgo

last: Andrés


title: Modelling interactions between somites and neural tube with human Trunk-like Structures (hTLS)

first: Naomi

last: Moris

address: 1 Midland Way

institute: Francis Crick Institute

summary: Human stem cell-based embryo-like models have opened new avenues of research into the principles of embryonic development using experimentally amenable in vitro systems. One of the benefits of embryo-like models over organoids is their multilineage differentiation, which allows the co-development of different populations and interaction between tissues. Here, we develop a method of generating somite- and neural tube-containing models, called human Trunk-like Structures (hTLS). These hTLS contain SOX2+ TBXT+ neuromesodermal progenitors, presomitic mesoderm, and somitic epithelial tissues, alongside a neural tube structure. As well as the expression of Wnt/FGF ligands posteriorly and Retinoic Acid (RA) pathway components anteriorly, we observe oscillatory HES7 expression, and show that increasing exposure to a Sonic Hedgehog signalling agonist (SAG) increasingly ventralises tissue identity. Additionally, we show that RA and noncanonical Wnt interactions likely occur between the somites and neural tube. In particular, we observe medially-localised ALDH1A2 expression in the somites - at the interface with the neural tube – which we validated in mouse embryos, whereas in somite-only structures, ALDH1A2 is anteroposteriorly polarised. This suggests that interactions between the neural tube and the somites might be responsible for mediolateral somitic organisation, both in vitro and in vivo, and highlights the value of embryo models to reveal insights into mammalian tissue co-development.

overall_design: Human trunk-like structures were sampled at 3 timepoints (72, 96, 120h), dissociated to single cells, and sequenced using the 10x transcriptomic platform.


title: ZNF514 promotes esophageal cancer progression by enhancing cell proliferation, migration and invasion

city: JiNan

first: Lin

last: Lv

state: Student

address: Department of Thoracic Surgery, Shandong University, Jinan, 250013, Shandong, P.R.China;

institute: Department of Thoracic Surgery, Jinan Central Hospital, Shandong University

summary: Esophageal cancer , a malignant tumor occurring in the upper gastrointestinal tract, is the seventh most common cancer worldwide. Zinc finger proteins, the most abundant family of transcription factors in humans, play a crucial role in the initiation and progression of various malignant tumors. However, its function in esophageal cancer remains unclear. The present study aimed to elucidate the role of ZNF514 in the development and progression of esophageal cancer, and to investigate its underlying mechanism. RNA expression and protein levels of ZNF514 were assessed using reverse transcription-quantitative PCR and western blotting . To assess functional roles, multiple cellular assays were performed, including: 5-ethynyl-2’-deoxyuridine incorporation ; Cell Counting Kit-8 ; wound healing assays ; colony formation assays ; Transwell assays. Subsequently, Gene Ontology, Gene Set Enrichment Analysis, Kyoto Encyclopedia of Genes and Genomes analysis and Ingenuity Pathway Analysis were performed through transcriptomic analysis. In EC, ZNF514 exhibited high expression at both the mRNA and protein levels. Additionally, ZNF514 influenced the migration, invasion and proliferation of EC cells. Gene enrichment analyses and IPA demonstrated that ZNF514 knockdown significantly affected multiple signaling pathways, such as Fcγ receptors, the complement system,  G-protein coupled receptors-related receptors, the Ribosomal S6 kinase pathway, RAS/MEK, PI3K/AKT, STAT3, Nucleotide Binding Oligomerization Domain Containing , and NF-κB pathways. The anti-cancer mechanisms induced by ZNF514 knockdown may be related to the enhancement of Fcγ receptor and complement system activation, as well as the inhibition of GPCR, RSK, RAS/MEK, PI3K/AKT, STAT3, NOD1/2 and NF-κB pathways.

overall_design: RNA sequence analysis of wild-type KYSE-150 cells and their knockout ZNF514 (siZNF514)

first: Liangming

last: Zhu

first: Xiaoming

last: Sun


title: Combinatorial BMP4 and Activin direct choice between alternate routes to endoderm during human gastrulation [scRNA-seq]

first: Silvia

last: Santos

summary: Cellular differentiation requires the proper interpretation of multiple signalling cues which vary in concentration. How exactly the combination and history of signals a cell is exposed to influences a fate decision remains poorly understood. In this study we use hESCs as a tractable model system to explore how combinations of cues guide state transitions during gastrulation. Using single-cell transcriptomics and live-cell imaging of engineered hESCs expressing endogenous cell state reporters, we reconstructed developmental lineages and obtained single-cell measurements of fate specification dynamics during gastrulation. We found that definitive endoderm, one of the three germ layers, arises from two distinct developmental trajectories: a direct route from pluripotency, and an indirect route via a mesoderm progenitor state. Furthermore, by modulating the signalling input we found that the relative concentration of Activin and BMP4 controls the choice between alternate trajectories to endoderm. These findings reveal a lineage convergence event during human gastrulation with multiple routes existing to definitive endoderm dictated by the combination of signalling cues presented. This work shows that the combination cues a cell is exposed to not only directs the final fate outcome it assumes, but the developmental route taken.

overall_design: scRNA-Seq was performed on cultured hESC line H1 WT  cells treated with Activin 100 ng/mL and BMP4 10 ng/mL or BMP4 50 ng/mL in a E5 basal media and switched between cues at different points in time. The aim of this work was to characterise what cell fates are aquired when hESCs are treated with these signals for different durations and orders.


title: snoRNA Snord3 promotes rheumatoid arthritis by epigenetic regulation of ESM1 in fibroblast-like synoviocytes in mice

city: Hong Kong

first: chunhao

last: cao

address: Hong Kong

department: School of Chinese Medicine

country: Hong Kong

institute: Hong Kong Baptist University

summary: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by systemic inflammation, synovial hyperplasia, and the destruction of bone and cartilage. Despite the use of various immunosuppressive disease-modifying antirheumatic drugs (DMARDs), a considerable proportion of RA patients remain symptomatic. As RA progresses, fibroblast-like synoviocytes (FLSs) undergo a phenotypic transition to an aggressive state, making them attractive non-immune cellular targets for RA treatment. However, there are currently no clinically available therapies that selectively ablate RA-FLSs due to the lack of specific molecular targets.  Our research focuses on the knockdown and overexpression of the SNORD3 gene in RA-FLSs. SNORD3 is expressed in the cell  of RA-FLSs and plays a crucial role in their aggressive transition. By specific knockout or overexpression of NCL, we observed significant aggressive transformations of RA-FLS.

overall_design: To investigate whether SNORD3 plays a critical role in the tumor-like transformation and overactivation of RA-FLSs, we established two RA-FLSs stable line in which the  target gene was silenced  down or expressed.


title: Transcriptional and functional architecture of the whole neutrophil compartment [RNA-seq Hoxb8]

overall_design: HoxB8-immortalized myeloid progenitors were routinely tested for mycoplasma contamination and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 30 μM β-mercaptoethanol (Thermo Fisher Scientific), 1% supernatant from SCF-producing CHO cells, and 1 μM β-estradiol (Sigma-Aldrich) to maintain the progenitor state. Neutrophil differentiation was initiated by β-estradiol withdrawal and continued culture in medium supplemented with 1% SCF-containing supernatant. Differentiation into neutrophils was achieved by culturing cells in RPMI 1640 medium containing 10% FCS, 30 μM β-mercaptoethanol, 4% SCF supernatant, and 20 ng ml⁻¹ granulocyte colony-stimulating factor (G-CSF) under standard tissue culture conditions (37 °C, 5% CO₂) To generate .JunB Overexpressing HoxB8 cells we used Lentiviral vectors for Junb overexpression. Cells were co-transfected with (i) a transfer plasmid containing the Junb cDNA under the control of the human PGK promoter, (ii) packaging plasmid psPax2, and (iii) envelope plasmid pMD2.G encoding VSV-G.

last: Rubio-Ponce

first: Jon

last: Sicilia


title: Disrupting the CBP/ β-catenin Axis: A Path to Suppress Survivin and Induce Apoptosis in Melanoma

city: Lodz

first: Michal

last: Wozniak

address: Mazowiecka 6/8

country: Poland

institute: Medical University of Lodz

summary: Survivin, the member of the inhibitors of apoptosis (IAP) protein family is overexpressed in various cancer types while remaining nearly undetectable in normal cells. Survivin overexpression is associated with increased chemotherapy resistance, tumor recurrence, and shorter patient survival. Bioinformatics analysis supported the observation that increased expression of survivin may be a biomarker of poorer patients’ survival and may contribute to resistance to standard targeted therapy of melanoma. The lack of identifiable active sites and enzymatic activity make targeting survivin challenging. Therefore, indirect targeting of survivin may represent a feasible therapeutic approach. To downregulate survivin, we used PRI-724, an inhibitor of CBP/β-catenin signaling axis.  While PRI-724 has demonstrated efficacy in preclinical models and clinical trials in different cancer types, its potential in melanoma had not been previously assessed. Treatment of drug-naïve, trametinib- and vemurafenib-resistant melanoma cells with PRI-724 was sufficient to downregulate survivin and other CBP/β-catenin target proteins, inhibit proliferation, reduce invasiveness and induce apoptosis. These findings indicated that PRI-724 exhibited significant antitumor activity in both drug-naïve melanoma cells and their drug-resistant counterparts. Among the resistant populations, trametinib-resistant melanoma cells showed the greatest sensitivity to PRI-724 treatment, whereas vemurafenib-resistant melanoma cells, which exhibited the higher expression of survivin, were the less responsive to PRI-724 treatment.

overall_design: RNA-seq data was prepared from DMBC21 patient-derived melanoma cell lines and their drug-resistant counterparts: cells with acquired resistance to vemurafenib (DMBC21PLXR) and trametinib (DMBC21TRAR)

first: Anna

last: Gajos-Michniewicz

first: Katarzyna

last: Kluszczynska

first: Malgorzata

last: Czyz


title: Selective RNA sequestration in biomolecular condensates directs cell fate transitions [smallRNAseq]

city: Boulder

first: Mika

last: Nevo

state: Colorado

address: 2315 Walnut St

institute: CU Boulder

type: Non-coding RNA profiling by high throughput sequencing

summary: Post-transcriptional regulation is increasingly recognized as pivotal in guiding the gene expression programs that instruct cell fate decisions. Recent studies have highlighted the significance of RNA condensates in modulating gene expression through RNA processing and translational control. Yet, the functional roles and composition of RNA condensates in the context of cell fate specification remain unclear. In this study, we leveraged Fluorescence-Activated Particle Sorting to profile the coding and non-coding transcriptome within cytoplasmic condensates known as P-bodies across diverse developmental contexts and vertebrate species. Our analyses revealed the highly conserved, cell type-specific sequestration of fate-instructive RNAs, which are translationally suppressed and stored within P-bodies. Using degron systems in both human and mouse pluripotent cells, we demonstrate that the acute dissolution of P-bodies facilitates the translation of mRNAs otherwise suppressed in condensates, including genes associated with zygotic genome activation and totipotency. Consistently, loss of RNA sequestration awakens the totipotency transcriptional program in human and mouse pluripotent cells. Mechanistically, we provide evidence that miRNAs play a pivotal role in orchestrating the sequestration of specific RNAs into P-bodies in a context-dependent manner. Accordingly, perturbing AGO2, alternative polyadenylation, and miRNA function profoundly reshapes the sequestration of RNAs into P-bodies and alters cell fate. We further identify mir-300/381 as a key repressor of the totipotent transcriptional state. Collectively, our findings connect RNA processing within condensates to cell fate decisions across vertebrate species.

overall_design: The hESC and mESC were differentiated into each of the cell types below, and then pbody contents were sorted and sequence, and cytoplasmic fractions were sequenced seperately. Two replicates were sequenced for each sample. In treatment or knockout conditions, each treatment condition was sequenced alongside a control cell line, two replicates of each for pbody and cytoplasm.