title: Phosphate starvation induces root cell-type specific transcriptional responses and alternative splicing

city: Pozuelo de Alracon

first: Juan

middle: Carlos

last: del Pozo

state: Madrid

address: Campus de Montegancedo, UPM

department: Biotechnology

institute: Centro de Biotecnología y Genómica de Plantas

summary: Optimal plant nutrition is crucial for plant growth, development, and production. Phosphorus (P) is a vital macronutrient for plants, but its availability in soil is often limited. Despite high levels of P in natural soils, much of it is inaccessible to plants. Plants acquire phosphorus as inorganic phosphate (Pi) from the rhizosphere, and Pi starvation triggers adaptive responses to enhance Pi acquisition. These responses include changes in root development and gene transcriptional reprogramming among others. Although several root transcriptomic analyses have identified key genes involved in Pi starvation responses, almost all of them have been conducted with illuminated roots, a condition that masks molecular responses to Pi starvation. In this work, a comparative transcriptomic analysis of specific root cell types identified over 7,000 genes with differential expression in response to Pi deficiency. Additionally, this study highlights the role of alternative splicing in regulating Pi starvation responses and demonstrates that both transcriptional and posttranscriptional responses are cell type specific. Overall, this work provides new insights into the cell-specific responses to Pi starvation, enhancing our understanding of plant adaptation to phosphorus deficiency.

overall_design: To identify novel genes involved in phosphate starvation responses, we carried out a transcriptomic analyses using RNA from  specific cell types of seedlings grown in the D-Root device. RNAseq analyses of polyA mRNAs isolated from roots of GFP/YFP markers  seedlings grown at 22ºC in half strength MS medium with high or low phsophate for 6 days. Afterwards, different cell types were shorted by fluorescence-activated cell sorting (FACS)

first: J

first: M

last: P. Gonzalez-Garcia

last: Lanza

last: Moreno-Risueño

first: E

last: Caro

first: S

last: Gonzalez-Bodi

first: V

last: Baca-Gonzalez

first: L

last: Serrano

last: Bustillo-Avendaño

last: Garcia-Mina

first: A

last: Zamarreño

last: Garnica-Ochoa


title: Single-Cell Chromatin Accessibility Landscape of Developing Perinatal Mouse Skin: A Multi-Omics Approach to Lineage Specification

status: Public on Mar 12 2026

city: Seoul

first: Hanjae

last: Lee

address: 103, Daehak-ro, Jongno-gu

country: South Korea

institute: Seoul National University

summary: This study presents a time-series scATAC analysis of developing mouse skin. Motif and footprint analyses at the chromatin level, as well as integrated analysis with single-cell and spatial transcriptomic data, enable an in-depth identification of key transcription factors and driver genes involved in fibroblast differentiation and hair follicle formation. This study demonstrates the utility of scATAC analysis combined with multi-omics approaches in providing a comprehensive understanding of skin development. It serves as a valuable reference for future research on skin development and regeneration, supported by an interactive online portal.

overall_design: Formalin-fixed paraffin-embedded (FFPE) tissue sections (5 μm thick) were prepared and subsequently analyzed using the Xenium Analyzer. After deparaffinization and permeabilization, mRNAs were targeted with a custom 50-gene panel of skin markers and driver genes, supplemented by the 10x mouse brain panel (247 genes; https://www.10xgenomics.com/products/xenium-panels). Post-analysis H&E staining was performed, and data visualization was carried out using Xenium Explorer (v3.0). The initial Xenium Analyzer data were reprocessed with the updated Xenium Ranger (v2.0), adjusting nuclei boundary expansion 2 μm.


title: The effect of intrauterine growth restriction on the developing pancreatic immune system

city: Philadelphia

first: James

last: Garifallou

state: Pennsylvania

address: 3615 Civic Center Boulevard, ARC1014

department: Neonatology

country: USA

institute: Children's Hospital of Philadelphia

type: Expression profiling by high throughput sequencing

summary: Immune cells in the pancreas are known to participate in organ development and homeostasis. However, the resident pancreatic immune system has yet to be fully defined.  Immune cells also play a role in pathology and are implicated in diseases such as diabetes induced by intrauterine growth restriction (IUGR).  We hypothesized that the resident immune system is established during neonatal development and disrupted by IUGR.  Using single cell RNAseq and flow cytometry we identified many immune cell populations in the near term fetus (at embryologic day 22) and neonatal (postnatal day 1, 7, &14) rat islets  and non-endocrine pancreas and in the spleen.  Using flow cytometry, we observed the resident immune system is established during neonatal development in the pancreas and spleen.  We identified 9 distinct immune populations in the endocrine pancreas and 8 distinct immune populations in the spleen by single cell RNAseq.  There were no sex-specific differences in the relative proportion of immune cells in the pancreas or spleen.  Finally, using the bilateral uterine artery ligation model, we tested if IUGR disrupted the neonatal immune system.  We found significant changes to the percentage of CD11B+ HIS48- and CD8+ T cells in the islets and non-endocrine pancreas and in the spleen.  IUGR-induced alterations were influenced by the tissue environment and the sex of the offspring.  Interestingly, it that took longer for immune cells to reach percentages similar to controls in male than female IUGR offspring.   In the female spleen, but not male, IUGR altered the percentage of several immune populations at e22, but the percentage was similar to controls post birth.  Future research to define the role of these immune cells in pancreatic development may identify disrupted pathways that contribute to the development of diabetes following IUGR.

overall_design: CD45+ cells from the pancreas and spleen were isolated from postnatal day 1 rats. Cell populations and transcriptional signatures were analyzes using single cell RNA-sequencing.

first: Thea

last: Golden

middle: P

first: Colin

middle: C

last: Conine

first: George

middle: S

last: Worthen

first: Rebecca

middle: A

last: Simmons


title: RNA G-quadruplex dynamics control gene expression and genome stability through DHX36-mediated regulation

city: Guangzhou

first: Tiegang

last: Meng

address: Guangzhou Key Laboratory of Metabolic Diseases and Reproductive Health

country: China

institute: Guangdong Second Provincial General Hospital

type: Genome binding/occupancy profiling by high throughput sequencing

summary: RNA G-quadruplexes (rG4s) serve as critical regulators of gene expression, yet their comprehensive mapping and dynamic regulation in physiological contexts remain technically challenging. Here we develop Ultra-low-input rG4-seq (ULI-rG4-seq), enabling precise rG4 detection in samples as small as 100 cells. This technological advance, combined with acute protein depletion strategies, reveals three fundamental principles of RNA structure regulation: First, we uncover a temporal dimension of rG4 control, where acute and chronic loss of the helicase DHX36 leads to opposing effects on rG4 levels. This observation extends beyond the traditional view of helicases as unwinding enzymes and suggests sophisticated cellular mechanisms maintaining RNA structural homeostasis. Second, through integrated analysis of rG4 landscapes and DHX36 binding profiles, we demonstrate coordination between cytoplasmic rG4 regulation and nuclear gene expression, revealing how RNA structure dynamics orchestrate genome-wide transcriptional programs. This mechanism provides insights into how modulation of a single RNA helicase can broadly impact cellular state. Third, using mouse oocytes as a model system, we establish rG4 regulation as essential for development and female fertility. DHX36 deficiency results in transcriptional defects, genome instability, and premature ovarian failure, demonstrating the physiological importance of proper RNA structure maintenance. These findings advance our understanding of RNA-based regulation and suggest therapeutic strategies for treating disorders of RNA metabolism. The technological and conceptual advances presented here provide new approaches for investigating RNA structure biology in development and disease.

overall_design: Single cell RNA-Seq, ULI-rG4-seq, LACE-seq, and CUT&Tag analyse the RNA G-quadruplex regulome in gene expression and genome integrity.

first: Tie-Gang

first: Wei

last: Yue

first: Chao

last: Li

first: Di

last: Gao

first: Shi-Ming

last: Luo

first: Ruibao

last: Su

first: Xiang-Hong

last: Ou

first: Qing-Yuan

last: Sun


title: Transcriptome-wide profiling of m6A modifications in the mouse embryonic fibroblasts (MEFs) replicative senescence

city: Dongguan

first: Yuting Li

middle: Yuting

address: Xin Cheng Avenue 1#, Songshan Lake,

institute: Guangdong Medical University

summary: N6-Methyladenosine (m6A), an abundant and dynamically chemical modification, has recently emerged as a critical posttranscriptional regulator of gene expression, which affect diverse eukaryotic biological processes. Nonetheless, the roles of m6A modification in aging research was still rarely reported. Herein, we uncovered that the m6A modification level and the methyltransferase Mettl3 expression level were significantly enhanced in aging process, including physiological aging mice model, cellular replicative senescence model. Furthermore, silencing of Mettl3 staved off senescent phenotype of MEF cells, as evidenced by down-regulation of p16, decreased β-galactosidase activity and enhanced cell proliferative capacity, while Mettl3 overexpression accelerated the cellular senescence. Results of in vivo experiments showed that Mettl3 overexpression mice exhibited more pronounced senescence phenotype accompanied with shortened lifespan. To deepen into the understanding of the molecular mechanism of m6A and Mettl3 on aging process, the high throughput MeRIP sequencing was performed in the young or senescent MEFs

overall_design: The Methylated RNA Immunoprecipitation sequencing(MeRIP-seq) in the young or senescent mouse embryonic fibroblasts (MEFs)


title: 2′-O-methylation-dependent installation of N2-methylguanosine in the U6 internal stem loop facilitates efficient spliceosome assembly

city: Göttingen

first: Katherine

last: Bohnsack

address: Humboldtallee 23

country: Germany

institute: University Medical Centre Göttingen

summary: The internal stem loop (ISL) of the human U6 snRNA, which catalyzes pre-mRNA splicing, contains LARP7-dependent, snoRNA-guided 2′-O-methylations and an N2-methylguanosine (m2G) that is required for splicing of weak splice sites. Here, we show that installation of m2G72 by the THUMPD2-TRMT112 methyltransferase complex is one of the last maturation events during U6 snRNP biogenesis. We dissect features of THUMPD2 required for association with U6 and present an experimentally validated model of the THUMPD2-TRMT112-U6 complex. Using in vitro methylation assays as well as a newly developed m2G-sensitive deoxyribozyme to monitor U6-m2G72 levels in cellular RNAs, we reveal that 2′-O-methylations within the U6 ISL enhance methylation of G72. We show that m2G72 and the 2′-O-methylations in U6 independently and interdependently influence alternative splicing. Furthermore, our data demonstrate that 2′-O-methylations in the ISL are required for incorporation of U6 into snRNPs whereas m2G72 influences the progression of the U6 snRNP into larger assemblies, highlighting distinct roles of these modifications during spliceosome assembly.

overall_design: Flp-In T-Rex HEK293 cell lines expressing THUMPD2-His-FLAG or the His-FLAG tag alone were crosslinked using UV and analyzed by CRAC.

first: Nicole

last: Kleiber

first: Chairini

last: Thomé

first: Philipp

last: Hackert


overall_design: Flp-In T-Rex HEK293 cell lines expressing THUMPD2-His-FLAG or the His-FLAG tag alone were crosslinked using UV and anti-FLAG IPs were performed. Co-purified RNAs were analysed by 3' RACE.


title: Parkinson's disease (PD) genetic asymmetry in neuronal nuclei from paired hemisphere samples of prefrontal cortex

city: Grand Rapids

first: Gerry

last: Coetzee

state: MI

address: 333 Bostwick Ave NE

department: CNS

institute: Van Andel Research Institute

summary: We measured gene expression in neuronal nuclei from cortex derived from PD (18 male, 10 female) and healthy control (5 male, 6 female) brains.  Each brain sample consisted of cortex from both right and left hemispheres. Th side of PD onset, age, duration of disease, as well as technical variables were used to define linear models of expression for each gene. Significant differences in expression due to age, disease, sex, hemisphere, side of PD onset, or combinations were determined

overall_design: Frozen prefrontal cortex was prepared and sorted to isolate neuronal nuclei. RNA was preparred for bulk RNA-sequencing. Results were analyzed using generalized linear modeling after controling for unwanted variation.

first: Gerhard

first: Steven

middle: E

last: Pierce

first: J

last: vanderSchans

first: Elizabeth

last: Ensink


title: TERT expression attenuates metabolic disorders in obese mice by promoting adipocyte formation and remodeling macrophage landscape [snRNA-Seq]

city: Marseille

first: Laura

last: BRAUD

address: 27 boulevard Leï Roure

country: France

institute: Institut Paoli Calmettes (IPC)

summary: Obesity is linked to limited adipose tissue (AT) remodeling capacity, leading to hypertrophic adipocytes, senescence, and inflammation. We used a mouse model expressing mTert, or its catalytically inactive form mTertCi, from the Cdkn1a locus to investigate the role of mTERT in obesity-induced metabolic disorders in mice on a high-fat-diet (HFD). We found that mTERT expression attenuates p21 expression, telomere attrition, senescence, and inflammation in the AT of HFD-induced obese mice, thereby reducing associated metabolic disorders. In vivo and in vitro data reveal that mTERT promotes differentiation of adipose stem and progenitor cells into adipocytes in obese mice. Strikingly, single nucleus RNA-seq data show that both mTERT and mTERTCi remodels the landscape of macrophages in AT of obese mice thereby reducing the immune response. These results emphasize involvement of the canonical and non-canonical mTERT functions in ameliorating metabolic disorders and suggest conditional TERT expression as a potential therapeutic option for obesity.

overall_design: To assess the effects of p21 promoter-driven mTert and mTertCi expression on global transcription, we performed RNA-seq experiments on the SVF. We analyzed gene expression in the SVF of the p21+/-, p21+/Tert, and p21+/TertCi mice fed HFD and p21+/mCherry fed SD (Dataset 1).

last: Braud

first: Manuel

last: Bernabe

first: Julien

last: Vernerey

first: Dmitri

last: Churikov

first: Manon

last: Richaud

first: Antonio

middle: M

last: Miranda

first: Liam

last: Mc  Allan

first: Christophe

last: Lachaud

first: Jesus

last: Gil

first: William

last: Scott

first: Vincent

last: Geli


title: Unfolded protein response is activated in leukocyte RNA from patients with severe heat stroke

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

country: Japan

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

summary: Compared to normal subjects, patients with severe heat stroke showed altered expression of RNAs, especially those involved in the endoplasmic reticulum stress response.

overall_design: Prospective cohort study.

first: Hisatake

last: Matsumoto

last: Okuzaki


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

status: Public on Mar 11 2026

city: Huntsville

first: Jesse

middle: Nicholas

last: Cochran

state: AL

address: 601 Genome Way

institute: HudsonAlpha Institute for Biotechnology

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

overall_design: LentiMPRA in KOLF2.1h-NGN2 Neurons (transduced on day 14, collected on day 18) and HEK293FT cells using 270bp oligos as test sequences

first: Rebecca

last: Hauser

first: Nicholas


title: An integrated landscape of mRNA and protein isoforms [PacBio]

city: Shenzhen

first: Wei

last: Chen

state: Guangdong

address: No. 1088 Xueyuan Avenue, Nanshan District

department: School of Life Sciences

institute: Southern University of Science and Technology

summary: Current proteomic methods are not well suited to detect protein isoforms. On the one hand, standard shotgun (that is, bottom-up) proteomics involves digestion of proteins into peptides. While this approach identifies many proteins, it results in a loss of isoform information. On the other hand, mass spectrometric analysis of intact proteins (that is, top-down proteomics) distinguishes protein isoforms but only covers a small subset of the proteome. We developed peptide correlation profiling (PepCP) as a method to obtain protein-level information from peptide-centric (that is, bottom-up proteomic) data: First, proteins are fractionated by SDS-PAGE to polypeptides of different length. Second, individual protein fractions are digested into peptides. Third, peptides are identified and quantified in all fractions using quantitative mass spectrometry-based proteomics. Finally, peptide abundance profiles across fractions are analysed to obtain protein-level information.

overall_design:
To construct the full-length transcriptome of RPE-1 cell line, we extracted RNA and made PacBio library in RPE-1 cell line, without replicates.
After full-length RNA sequencing, we corrected the reads with next generation sequencing data, and constructed full-length RNA isoforms

first: Mengran

last: Wang

first: Henrik

last: Zauber

first: Amir

last: Kedan

first: Qionghua

last: Zhu

first: Matthias

last: Selbach


title: Immunological mechanisms underlying the clinical heterogeneity of Kikuchi disease: The potential role of atypical memory B cells

city: Taipei

first: Shan-Chi

last: Yu

address: No 7 Chung Shan S Rd

country: Taiwan

institute: National Taiwan University Hospital

summary: Building on our previous research, which classified Kikuchi disease into three subtypes based on predominant symptoms and fever status—febrile type, febrile lymphadenopathy (FebLAP), and afebrile lymphadenopathy (aLAP)—we further investigated the underlying mechanisms contributing to their distinct clinical differences.

overall_design: Using NanoString nCounter technology, we analyzed the gene expression profiles of 35 Kikuchi disease FFPE lymph node specimens and compared them across the subtypes.

first: Tseng-Cheng

first: Chun-Nan

first: Tsung-Lin

last: Yang


title: DMSO-induced human embryo model reveals V-ATPase requirement in cavitation

city: Beijing

first: Wenfeng

last: Xie

state: Beijing

address: 49 North Garden Road

department: Department of Obstetrics and Gynecology

institute: Peking University Third Hospital

summary: This study focuses on the key regulatory mechanisms of lysosomal acidification in early human embryonic development. By employing single-cell RNA sequencing technology (Smart-seq2), we conducted a systematic analysis of human morulae derived from three pronuclei, embryos arrested in development after treatment with 1 nM Concanamycin A, and control blastocysts. Through sequence alignment and quantitative analysis based on the human reference genome, the study successfully revealed the significant biological role of lysosomal acidification in the differentiation process of the trophectoderm (TE). This finding provides a new theoretical perspective for a deeper understanding of the molecular mechanisms underlying embryonic developmental arrest.

overall_design: This study employed single-cell RNA sequencing (Smart-seq2) to analyze morulae derived from three pronuclei, embryos arrested in development by 1 nM Concanamycin A treatment, and control blastocysts, aiming to investigate the regulatory mechanisms of lysosomal acidification in early human embryonic development.

first: Yang


title: TERT expression attenuates metabolic disorders in obese mice by promoting adipocyte formation and remodeling macrophage landscape [RNA-Seq]

first: Laura

last: BRAUD

last: Braud

first: Manuel

last: Bernabe

first: Julien

last: Vernerey

first: Dmitri

last: Churikov

first: Manon

last: Richaud

first: Antonio

last: Miranda

first: Liam

last: Mc Allan

first: Christophe

last: Lachaud

first: Jesus

last: Gil

first: William

first: Vincent


title: Igh nuclear speckle association promotes V(D)J recombination

city: Chicago

first: Hammad

last: Farooq

state: IL

address: 835 S Wolcot

department: Microbiology and Immunology

institute: University of Illinois Chicago, College of Medicine

summary: Hi-C analyses of pro-B lymphocytes

overall_design: We report the application of in situ Hi-C for analysis of progenitor (pro)-B cells. We generated genome-wide Hi-C heatmaps for pro-B cells from Rag2-/- and Rag2-/-SiteI.3Pr-/- mice.

first: Amy

middle: L

last: Kenter

first: Khalid

last: Hussain Bhat

first: Sarah

last: Naiyer

first: Jie

last: Liang

first: Saurabh

last: Priyadarshi


title: An integrated landscape of mRNA and protein isoforms [NGS]

first: Wei

last: Chen

overall_design:
To construct the full-length transcriptome of RPE-1 cell line, we performed next generation sequencing to assist the construction and quantification of full-length transcript.
Two replicates of RPE-1 cell line were used, after RNA extraction and library construction, mRNA-seq was performed. Next, the reads were used for correcting full-length reads from third generation sequencing, the splice-junction information was used to construct full-length transcriptome, and expression profiling analysis was performed to assist the quantification of full-length transcript.

first: Mengran

last: Wang

first: Henrik

last: Zauber

first: Amir

last: Kedan

first: Qionghua

last: Zhu

first: Matthias

last: Selbach


title: Whole Transcriptome Profiling of Lesional Psoriatic and Adjacent Normal Skin Tissues

city: Kolkata

first: Raghunath

last: Chatterjee

state: WB

address: 203 BT Road, HGU, Indian Statistical Institute

department: Human Genetics Unit

country: India

institute: Indian Statistical institute

summary:
Purpose: To identify the differentially expressed genes in lesional skin of psoriasis patients compared to adjacent normal skin tissue by Next  Generation Sequencing.
Methods: Total RNA was isolated from 24 paired lesional and adjacent normal skin tissue samples and Next Generation Sequencing was performed. The sequences reads were trimmed of adapter sequences and quality trimmed using CutAdapt, followed by alignment to the human genome (hg19/GRCh37) by 'STAR  (v2.7.10a)'. The aligned files were sorted based on chromosomal location. Trancript assembly was performed using StringTie (v1.3.3b) and python script prepDE.py was used to obtain the raw read counts for all annotated genes. Differnetial expression analysis was performed with the raw reads using R-package 'edgeR'.  Gene ontology with the deregulated genes was performed uign DAVID GO. The differentially expressed genes were integrated with differentially expressed miRNAs (GSE183547) and differentially methylated CpGs (GSE115797) to identify the genes regulated by miRNA and DNA metylation respectively.
Results:  We identified 1,541  differentially expressed protein-coding genes with at least 2-fold deregulation that were statistically significant (FDR ≤ 0.05). The deregulated genes were found to be associated with biological processes including inflammatory response, cytokine mediated signaling, keratinization, neutrophil chemotaxis and defense response to virus  among the top enriched processes. Inetgrative analysis with small RNA-seq and DNA methylation profiling using Illumina Infinum 450K BeadArray identified 487 and 321 genes to be regulated by miRNA and DNA methylation respectively.
Conclusions: Our multi-omics integrative analysis identfied the biological processes regulated exclusively as well as combinatorially by miRNAs and DNA methylation in the disease pathogenesis of psoriasis.

overall_design: Whole transcriptome profiling of 24 paired lesional psoriatic skin and adjacent normal skin

first: Sayantan

last: Laha

first: Swapan

last: Senapati

first: Gobinda


title: Whole-Protein Screening and Multi-Modal Profiling of Antigen-Specific CD4+ T Cells at Single-Cell Resolution

city: Seattle

first: Rongyu

last: Zhang

state: WA

address: 401 Terry Ave N

institute: ISB

summary: Systematic whole-protein screening and comprehensive profiling of antigen-specific CD4+ T cells are crucial for advancing vaccine design and cancer immunotherapies, yet remain technically challenging. Here, we present a high-throughput platform that utilizes large-scale class II single-chain trimer libraries to detect antigen-specific CD4+ T cells, while simultaneously profiling their antigen specificity, TCRα/β sequences, MHC restriction, whole transcriptomes, and patient/timepoint origins at single-cell resolution. Upon rigorous platform validation, we screened the full SARS-CoV-2 spike receptor binding domain in a longitudinal cohort of 22 participants, identifying 2,188 antigen-specific CD4+ T cells and revealing key metrics defining the immunogenicity of class II-restricted viral antigens. We further extended the platform to whole-protein screening of HPV-16 E6/E7 in a cohort of precancerous patients, uncovering HPV-specific CD4 TCRs that, upon extensive characterization, demonstrated strong therapeutic potential. By integrating high-throughput antigen screening with high-dimensional, multi-modal cellular characterization, our approach provides an unprecedented window into CD4+ T cell immunity, potentially guiding vaccine design and next-generation TCR-based cancer immunotherapies.

overall_design: RNA-seq profiling of antigen-specific CD4+ T cells identified through single-chain trimer (SCT) dextramers to characterize the immunological states of cells in response to SARS-CoV-2 and HPV-16 infections at various timepoints.


title: A single-cell atlas revealing cellular heterogeneity across healthy and diseased human thymus

city: Vienna

first: Martin

last: Direder

state: Vienna

address: Lazarettgasse 14

department: Orthopedic and Trauma Surgery

country: Austria

institute: Medical University Vienna

summary: Transcriptional data of B-type thymoma, true thymic hyperplasia and thymic carcinoma

overall_design: Single cell RNA-sequencing gene expression of true thymic hyperplasia, B-type thymoma and thymic carcinoma. Single-cell suspension from all samples were prepared using MACS Miltenyi Tumor Dissociation Kit (Miltenyi). Libraries were generated using Chromium Next GEM Single Cell 5´GEM, Library & Gel Bead Kit v5.2, Chromium Next GEM Chip K Single Cell Kit and Single Index Kit TT Set A (all 10x Genomics, Pleasanton, CA, USA).

first: Maria

last: Laggner

first: Dragan

last: Copic

first: Katharina

last: Klas

first: Daniel

last: Bormann

first: Marie-Therese

last: Lingitz

first: Leonhard

last: Müllauer

first: Ana-Iris

last: Schiefer

first: Michael

last: Mildner

first: Hendrik

middle: J

last: Ankersmit

first: Bernhard

last: Moser

last: Wielscher

first: Melanie

last: Salek

first: Bahar

last: Golabi

first: Hannes

last: Kühtreiber

first: Wolfgang

last: Weninger

first: Clemens

last: Aigner


title: RNA seqeuncing of Fusobacterium polymorphum strains from patients with leukoplakia

first: Nezar

last: Al-hebshi

state: PA

address: 3223 N Broad Street, Office L213

institute: Temple University

summary: Fusobacterium polymorphum has been implicated in oral squamous cell carcinoma, yet its ubiquitous presence in healthy individuals complicates its role as a disease driver and raises the possibility that some clinical isolates may possess greater pathogenic potential than others. To explore potential functional differences among isolates, we performed transcriptomic profiling of F. polymorphum strains obtained from across the oral epithelial disease spectrum. Despite being genetically indistinguishable, isolates derived from dysplastic lesions exhibited distinct transcriptional signatures, including significant upregulation of genes involved in heme uptake compared with isolates from healthy sites. These findings suggest that transcriptional heterogeneity among clinical isolates may contribute to functional differences that could influence their pathogenic potential.

overall_design: RNA sequencing of strains of F. polymorphum strains isolated from mucosa of patients with varying levels of oral dysplasia and from healthy controls.

last: Al-Hebshi

first: Gary

last: Moran

first: Doaa

last: Elhadedy


title: An integrated landscape of mRNA and protein isoforms

first: Wei

last: Chen

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

overall_design: Refer to individual Series


title: putzig safeguards genome integrity by contributing to the Piwi-mediated repression of transposon activity in the female germline of Drosophila in a two-tiered fashion

city: Stuttgart

first: Lea

last: Hüttinger

address: Garbenstr. 30

department: Molecular Genetics (190g)

institute: University of Hohenheim

summary: Genome integrity in the germline is jeopardized by the activity of transposable elements (TEs). Transposon activity is kept in check by the Piwi-piRNA pathway, which silences TEs post-transcriptionally in the cytoplasm as well as co-transcriptionally in the nucleus. piRNAs derive from long precursor transcripts originating at piRNA-loci by non-conventional transcription. They are processed in the cytoplasm and loaded into Piwi-piRNA complexes that then enter the nucleus to bind to the target RNA and direct local heterochromatin formation at TE loci, involving several downstream effectors. In this work, we have analyzed the role of Putzig (Pzg) in Piwi-mediated TE silencing in the female germline. Pzg has multi-facetted roles during Drosophila development and is important for DNA integrity, germ cell differentiation and survival. Here, we provide evidence for a two-tier activity of Pzg by serving as a hub for various Piwi-pathway members. Firstly, Pzg assists transcription initiation at piRNA clusters by coupling the Trf2-Moonshiner transcription initiation complex to the Rhino-Deadlock-Cutoff complex, thereby promoting formation of piRNA-precursor transcripts. Secondly, in the context of co-transcriptional gene silencing, Pzg licences heterochromatin formation by linking the Piwi-piRNA silencing machinery and the histone demethylase Lsd1, involved in promoter inactivation.

overall_design: ChIP experiments followed by sequencing (ChIP-seq) were used to identify the genomic distribution of Pzg in Drosophila ovaries.

first: Ludmilla

last: Kober

first: Mirjam

last: Zimmermann

first: Sebastian

last: Schlensog

first: Marc

last: Engelhart

first: Anja

last: Nagel


title: Pheromone circuits and transcriptional cascades modulating transcriptional and chromatin states in the Drosophila central brain with social experience (CUT&RUN)

city: Durham

first: Chengcheng

last: Du

state: North Carolina

address: French Family Science Center,, 124 Science Dr.

institute: Duke University

summary: Social experience significantly influences the behavioral and physiological responses of animals, including humans. In many animals, social isolation increases aggression, courtship, locomotion, and feeding while disrupting sleep. This occurs when peripheral neurons detect social signals, such as pheromones, which activate decision-making circuits in the brain. However, the molecular and circuit mechanisms of how chronic social isolation or enrichment alter gene expression and affect neuronal function and behavior remain unclear. In this study, we examined how transcription patterns and chromatin marks in male Drosophila brains change in response to social experience, and the effect of pheromone circuits and transcription factors involved in social circuit function. We focused on pheromone receptors Or47b and Or67d, as well as transcription factors FruM and DsxM. Our findings suggest that social experience affects multiple genes in the central brain. Disrupting Or47b, Or67d, FruM, and DsxM function moderated the transcriptional responses through antagonistic interactions. Specifically, Or47b circuits showed a primary association with transcriptional responses to social isolation in conjunction with DsxM function, while Or67d and FruM were linked to the regulation of responses to group housing. Notably, mutants of fruM and dsxM exhibited more extensive transcriptional changes in the brain than Or mutants, especially for FruM/DsxM target genes. While social experience did not lead to detectable alterations in the overall chromatin profile in the whole brain, mutants of the four genes resulted in significant changes in the enrichment of H3K4me3 and RNA polymerase II (RNAPolII) compared to wild type. Furthermore, mutants in fruM and dsxM generally eliminated social experience-dependent changes in sleep and locomotion behaviors, whereas Or mutants exhibited more modest disruptions. Overall, our results uncover the pheromone circuits and transcriptional cascades in regulating molecular and behavioral responses to social experience.

overall_design: CUT&RUN followed by sequencing to map genome-wide landscapes of H3K4me3 and RNA polymerase II in Drosophila wild-type (CS), and Or47b, Or67d, fru, and dsx mutant male brains under group-housed (GH) and single-housed (SH) conditions.

first: Pelin

last: Volkan


title: A STAT5B-driven mouse model of hepatosplenic γδ T cell lymphoma reveals therapeutic efficacy of JAK inhibitors

first: Thomas

last: Eder

address: Veterinaerplatz 1

department: Department for Biological Sciences and Pathobiology

institute: University of Veterinary Medicine, Vienna

summary: Hepatosplenic T cell lymphoma (HSTL) is a rare, aggressive disease primarily affecting young adults. The disease progresses rapidly, with patients displaying low survival rates and poor responses to standard chemotherapy-based treatments. Currently no targeted therapies are available for HSTL, and pre-clinical models to test new treatment options have not been established. Hence, new models recapitulating HSTL are needed. The JAK-STAT signalling pathway is a key pathway dysregulated in HSTL and STAT5B N642H is the most frequent oncogenic mutation. Using a transgenic mouse model, we have previously validated STAT5B N642H as a strong driver of γδ T cell lymphoma. Here, we have established clonal, murine γδ T cell lymphoma cell lines driven by oncogenic STAT5B, which resemble key features of human HSTL. The IL-2 dependent cell line, C15, displays immunophenotypic features as well as commonly dysregulated gene expression profiles shared with patient-derived HSTL cell lines. C15 cells can be engrafted intravenously into immunocompetent mice to generate an aggressive HSTL-like disease. Recipient mice display an expansion of malignant γδ T cells causing hepatosplenomegaly and elevated alanine transaminase (ALT) and aspartate transaminase (AST) levels. Using these models, we tested the potential of JAK inhibition as a targeted treatment strategy for HSTL, with the clinically-approved JAK inhibitor upadacitinib displaying significant anti-tumor efficacy against HSTL cells in vitro and in vivo. Overall, we have developed a robust, accessible and faithful preclinical model of HSTL with the advantage of immune-competence, which can facilitate the study of physiological disease mechanisms and new (immuno)therapy strategies.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: We generated clonal mouse γδ T cell lymphoma cell lines harboring a STAT5B N642H mutation transgene. To compare their transcriptional profiles to human HSTL cells, we performed RNA-seq analysis using our mouse cell lines as well as two human HSTL cell lines that also harbor the STAT5B N642H mutation, DERL-2 and DERL-7. Differential gene expression analysis was performed for the mouse and human cell lines using primary murine γδ T cells and primary human CD3+ T cells as controls, respectively.

first: Neubauer

last: Heidi

first: Herling

last: Marco

first: Müller

last: Tony

first: Jungherz

last: Dennis

first: Eder

last: Thomas


title: A dual role for PGLYRP1 in host defense and immune regulation during B. pertussis infection

city: Baltimore

first: Ciaran

last: Skerry

state: MD

address: 685 W. Baltimore Street

institute: University of Maryland School of Medicine

summary: Bordetella pertussis, the etiologic agent of whooping cough, remains a serious public health concern despite widespread vaccination. Improved therapeutics and vaccines are urgently needed to treat and prevent pertussis disease. Host recognition of bacterial peptidoglycan (PGN), including B. pertussis extracellular PGN fragment tracheal cytotoxin (TCT), shapes the immune response to infection. Peptidoglycan recognition proteins (PGLYRPs) are a conserved family of innate immune molecules which bind bacterial PGN. While they function as immune signaling receptors in arthropods, PGLYRPs in mammals have thus far been primarily recognized for their bactericidal activity. Previously thought to function only as antimicrobial peptides in mammals, the immune modulatory roles of this family of peptidoglycan recognition proteins are beginning to gain greater appreciation. Peptidoglycan recognition protein 1 (PGLYRP1) is a secreted antimicrobial protein. However, its role in mammalian host defenses and immune signaling during infection with Gram-negative pathogens, such as B. pertussis, remain largely unknown. Here, we identify a dual role for PGLYRP1 in modulating host immune responses to B. pertussis. Using knockout mice, single-cell and bulk transcriptomics and functional assays, we show that PGLYRP1 has bactericidal activity against B. pertussis in vitro and promotes early bacterial control in vivo. PGLYRP1 also dampens inflammatory responses and impedes bacterial killing later in infection. Mechanistically, PGLYRP1 enhances nucleotide oligomerization domain (NOD)-1 signaling in response to TCT while suppressing NOD2- and triggering receptor expressed on myeloid cells-1 (TREM-1)-mediated inflammatory pathways. TCT-bound PGLYRP1 selectively impairs TREM-1 activation compared to PGNs from other bacteria, revealing a novel bacterial immune evasion strategy. These findings demonstrate that B. pertussis co-opts PGLYRP1 to temper inflammation and alter immune signaling, revealing a novel immune evasion mechanism of manipulating the availability and structure of their exogenous peptidoglycan, revealing implications for host-pathogen evolution, vaccine design and host-directed therapeutics.

overall_design: Mice challenged intranasally with 50 uLs of PBS-sham or B. pertussis (2x10^6 CFU). One hour before challenge with B. pertussis, mice in the AALR group were treated with S1P agonist, AALR. Four days after infection, mice were euthanized, perfused, and lungs were collected.

first: David

last: Rickert

first: Sasha

last: Cardozo

first: Nicholas

middle: H

last: Carbonetti

first: William

last: Goldman

first: Karen

last: Scanlon


title: Peroxisomes orchestrate metabolic flexibility and longevity via an interorganelle cascade

city: Boston

middle: B.

last: Mair

state: MA

address: 677 Huntington Ave, Boston,

institute: Harvard School of Public Health

summary: Aging impairs coordinated organelle dynamics essential for lipid metabolism, causing a decline in intracellular metabolic flexibility. However, the drivers of organelle collapse and their temporal order remain unclear. Here, we identify peroxisomal function as a critical regulator of metabolic flexibility during youth and low-energy states. Using Caenorhabditis elegans, we show that fasting robustly induces peroxisomal function in youth, while this response is blunted during aging. Loss of peroxisomal import via PRX-5 declines over age, causing pathological lipid droplet expansion, dysfunctional mitochondrial bioenergetics, and metabolic inflexibility. While targeted PRX-5 degradation recapitulates metabolic aging, its overexpression preserves lipid dynamics and mitochondrial integrity. Notably, dietary restriction maintains peroxisomal pathways and organelle coordination into late life, and peroxisomal function causally underpins dietary restriction-mediated longevity. Our findings highlight peroxisomes as central, upstream regulators of a dynamic inter-organelle cascade driving metabolic plasticity and highlight peroxisomal maintenance as a key determinant of metabolic flexibility during aging.

overall_design: Expression profiling by high throughput RNA sequencing. Wild-type N2 C. elegans were subjected to RNA expression profiling at ages young adult (YA, young) and day 8 (D8, adult) under fed and 6 hour fasted conditions to specifically identify genes either induced/repressed with fasting in young but not aged animals.

first: Arpit

last: Sharma

first: Aditi

last: Prabhakar

first: Miriam

last: Valera-Alberni

first: Jamie

middle: D

last: Kraft

first: Abigail

last: Ellis

first: Ryan

last: Sheldon

first: Meeta

last: Mistry

first: Pallas

last: Yao

first: Yanshan

last: Liang

first: Sheng

last: Hui

middle: B


title: SbmC limits the mutation rate of Escherichia coli

first: Monica

last: Guo

address: 750 Republican St

department: Microbiology

institute: University of Washington

summary: DNA damage induces the mutations that drive bacterial adaption, evolution, and antibiotic escape. Both mutagenic and non-mutagenic DNA damage repair is coordinated by the SOS response, but despite extensive work, the functions of some SOS-induced genes remain obscure. Here, we clarify the function of Escherichia coli SbmC (GyrI). Despite its proposed function as a gyrase inhibitor, cells either lacking or overexpressing SbmC instead exhibit phenotypes consistent with a role in limiting DNA damage and cellular variation. Importantly, SbmC levels inversely correlate with E. coli mutation rate. Excess SbmC limits mutation whereas loss of SbmC increases mutation, possibly because ∆sbmC cells variably induce the SOS response, including mutagenic DNA Pol V. We additionally show that SbmC is dispensable for survival in the presence of double-strand break inducing drugs but is required to limit their mutational effects. Finally, evolutionary analysis indicates that bacterial SbmC homologs maintain their small molecule-binding domain but not the gyrase interacting residues identified in E. coli. Together, our findings suggest that SbmC-like proteins may bind to a yet-unknown cofactor to limit DNA damage and organismal evolution.

overall_design: RNA-seq data from Escherichia coli MG1655 wild-type and ∆sbmC cells in exponential phase harveted at OD600 0.5.

first: Katie

last: Leung


title: Base editing and nanoparticle transfection of airway cell types essential for treatment of cystic fibrosis

first: Garry

middle: R

last: Cutting

address: 733 N Broadway

department: Genetic Medicine

institute: Johns Hopkins School of Medicine

summary: Cystic Fibrosis (CF) is a life-limiting genetic disorder caused by deleterious variants in the CFTR gene that results in altered mucous impairing the airway epithelia. Durable correction of these variants in airway cells remain a therapeutic challenge for ~10% of individuals unresponsive to CFTR modulators. A common disease-causing CFTR splice site variant 3120+1G>A was corrected in primary CF airway cells using base editor RNAs. Single-cell RNA sequencing revealed a remarkable increase in detectable CFTR transcript in most CF airway epithelial cell types resulting in notable enrichment of CFTR-expressing ionocytes and secretory goblet cells. Progenitor basal cell subtypes were edited but they decreased as a fraction of total cells and CFTR expressing cells compared to unedited cells.

overall_design: Primary human nasal epithelial cells were isolated and cultured from a CF individual bearing one copy of the 3120+1G>A variant. Following electroporation of ABE8e mRNA/sgRNA4long or sg5long or no sgRNA (mock) to targeting the 3120+1G>A variant, cells were allowed to differentiate for 21 days on air-liquid-interface before undergoing processing for scRNASeq analysis.

first: Erin

middle: W

last: Kavanagh

first: Anya

middle: T

last: Joynt

first: Audrey

last: Pion

first: Alice

last: Eastman

first: Alianna

middle: I

last: Parr

first: Katherine

last: Starego

first: Manav

last: Jain

first: Sydney

last: Shannon

first: Edwin

last: Yoo

first: Gregory

last: Newby

first: Stephany

middle: Y

last: Tzeng

first: Neeraj

first: Jordan

last: Green


title: Mechanical Compression Causes Lung Hypoplasia in Congenital Diaphragmatic Hernia with GATA4 Genetic Variants

city: La Jolla

first: David

last: McCulley

state: CA

address: 9300 Campus Point Dr, MC 7774

institute: University of California, San Diego

summary: Congenital diaphragmatic hernia (CDH) is a common and severe structural malformation in which the high rate of morbidity and mortality is caused by lung hypoplasia and pulmonary hypertension. Genetic variants have been identified in 30% of CDH patients and are associated with increased morbidity and mortality but it is unclear how these variants impact lung and pulmonary vascular defect severity. Deletions of 8p23.1 account for 3-5% of cases and encompass GATA4, a transcription factor that directs gene expression throughout the developing embryo. CDH patients with GATA4 haploinsufficiency have high mortality and severe lung hypoplasia and pulmonary hypertension. Given this information, our aim was to characterize the role of GATA4 during lung and pulmonary vascular development. We generated mice with lung-specific deletion of Gata4 and found that GATA4 is not required during lung or pulmonary vascular development. However, mice with diaphragm-specific inactivation of Gata4 (Gata4 D-CKO) die after birth with abnormal diaphragm formation and lung hypoplasia thus we focused on the latest stage of embryonic development. We obtained whole lung RNA at embryonic day 18 (E18) at the latest stage prior to delivery due to early postnatal death of Gata4 D-CKO mice. We performed bulk RNA sequencing on control embryos (termed Gata4 D-control, Prx1Cre;Gata4F/+) and embryos with diaphragm-specific inactivation of Gata4 (termed Gata4 D-CKO, Prx1Cre;Gata4F/D). In this bulk RNA sequencing dataset are expression data from Gata4 D-control and D-CKO whole lungs at E18 implicating abnormal expression of genes involved in cell cycling and motor protein pathways. Mechanical compression of the embryonic lungs was also associated with increased phosphorylation of mechanosensory protein YAP1 resulting in decreased cell cycling. Our data suggest that the lung and pulmonary vascular phenotype of patients with CDH and GATA4 haploinsufficiency is due to mechanical compression.

overall_design: We performed bulk RNA sequencing on whole lung RNA collected at embryonic day 18 (E18) from 3 controls (Prx1Cre;Gata4F/+) and 3 Gata4 CKO (Prx1Cre;Gata4F/D) embryos. The diaphragm and lungs of Gata4 controls (Prx1Cre;Gata4F/+) develop normally. In contrast, Gata4 CKO (Prx1Cre;Gata4F/D) embryos lack Gata4 expression in the lateral plate mesoderm leading to deletion of Gata4 in the developing diaphragm and resulting in defects during diaphragm formation, lung hypoplasia, and decreased lung cell proliferation. This dataset was used to compare the gene expression in E18 Prx1Cre;Gata4F/+ control lungs with the expression in E18 Prx1Cre;Gata4F/D Gata4 CKO lungs and to investigate the mechanisms responsible for lung hypoplasia in Gata4 CKO embryos.

first: Betty

last: Pham

first: Gidsela

last: Luna


title: Transcrptomic analysis of dysplastic oral keratinocytes infected with Fusobacterium polymorphum strains from patients with leukoplakia

city: Philadelphia

first: Nezar

last: Al-hebshi

summary: Fusobacterium polymorphum has been implicated in oral squamous cell carcinoma, yet its widespread presence in the oral cavity raises questions about whether different clinical isolates exert distinct effects on host cells. To explore host transcriptional responses, we performed transcriptomic profiling of dysplastic oral keratinocytes (DOK) following exposure to F. polymorphum isolates derived from across the oral epithelial disease spectrum. The host transcriptomic response revealed a broadly shared pro-inflammatory signature across all isolates, regardless of their clinical origin. However, expression of genes and pathways associated with cell proliferation and related signaling processes varied among isolates and correlated with the dysplasia severity of the lesions from which the strains were derived. These findings suggest that while F. polymorphum isolates elicit a common inflammatory response in epithelial cells, variation in host transcriptional programs linked to proliferation may reflect differences in their biological impact.

overall_design: RNA sequencing of dysplastic oral keratinocytes (DOK) following infection with Fusobacterium polymorphum strains isolated from the oral mucosa of patients with varying degrees of epithelial dysplasia and from healthy controls to characterize host transcriptional responses. Uninfected controls were included

last: Al-Hebshi

first: Gary

last: Moran

first: Doaa

last: Elhadedy


title: Pheromone circuits and transcriptional cascades modulating transcriptional and chromatin states in the Drosophila central brain with social experience (RNAseq)

first: Chengcheng

last: Du

overall_design: Transcriptomic profiling (RNA-seq) of Drosophila wild-type (CS), and Or47b, Or67d, fru, and dsx mutant male brains under group-housed (GH) and single-housed (SH) conditions.

first: Pelin

last: Volkan


title: SEPHS2 deficiency reprograms glucose metabolism to activate the pentose phosphate pathway by stabilizing PCK1 through NAD+

city: shenzhen

first: yihuizhi

last: zhang

state: guangdong

address: 1088 Xueyuan Avenue, Nanshan District

department: School of Medicine

summary: SEPHS2, a key enzyme in synthesizing selenoproteins, which converts hydrogen selenide into selenophosphate. We found that SEPHS2 knockout inhibits oxidative phosphorylation (OXPHOS), but has no effect on glycolysis, as indicated by an seahorse assay. SEPHS2 loss redirects more glucose to the pentose phosphate pathway (PPP). Mechanistically, SEPHS2 knockout elevates NAD+ levels, which are essential for Sirtuin activity. The increase in the activity of SIRT2 leads to deacetylation of PCK1, a posttranslational modification required to prevent PCK1’s ubiquitination and subsequent degradation. Thus, SEPHS2 knockout stabilizes PCK1 by reducing its ubiquitination. Since PCK1 is a key regulator of gluconeogenesis, SEPHS2 knockout promotes gluconeogenesis while reducing cellularglucose uptake, down-regulating oxidative phosphorylation and enhancing the PPP.

overall_design: Differential gene expression caused by A-375 control and SEPHS2 knockout cells were determined by RNA-seq

first: Siyuan

last: Xia

first: Yihuizhi

last: Zhang


title: Single-cell RNA-sequencing of Immortalized Alveolar type 2 Cell Lines from Healthy Lungs

city: Los Angeles

first: Anas

last: Rabata

address: 127 S. San Vicente Blvd

department: Medicine

institute: Cedars-Sinai Medical Center

summary: Using the 10x Genomics platform, we conducted single-cell RNA sequencing to profile our newly immortalized alveolar type 2 (AT2) cell lines. Since primary human AT2 cells are notoriously difficult to propagate long-term, passage repeatedly, and modify genetically, we developed several stable, immortalized lines derived from healthy AT2 cells.

overall_design: To characterize and functionally study immortalized AT2 cell lines, we generated stable immortalized AT2 lines as follows. EpCAM+ cells were magnetically enriched from healthy human lung samples using CD326 (EpCAM) MicroBeads. The enriched cells were resuspended in a 3:1 mixture of DMEM/F12 and DMEM, supplemented with 5% FBS, 0.4 mg/mL hydrocortisone, 5 μg/mL insulin, 8.4 ng/mL cholera toxin, 10 ng/mL recombinant human EGF, Antibiotic-Antimycotic, and 10 μM Y-27632. Cells were allowed to adhere, with medium refreshed every 2–3 days thereafter. At passages 3–4, cells were transduced with lentivirus expressing SV40 Large T antigen to induce immortalization, followed by selection with G418. immortalized AT2 cells were isolated by FACS as EpCAM+ HTII-280+ CD31− CD45− cells from the total epithelial population. Then cells cultured in 2D, after that cells were collected and performed single cell analysis using the 10x Genomics Chromium platform.

first: Dianhua

last: Jiang


title: IL-17RC signaling connects intestinal microbiota and neuroimmune interactions in atherosclerosis

first: Ekaterina

last: Koltsova

address: 8700 Beverly Blvd

summary: While dysbiosis and inflammation were previously implicated in cardiovascular diseases, the circuits of how microbiota drives distant perivascular innervation, neuroinflammation, and atherosclerosis remains unknown. Here, we report that IL-17RC signaling in the intestine protects from atherosclerosis controlling intestinal barrier and microbiota, and loss of IL-17RC in intestinal epithelial cells alters microbiota, enhances perivascular innervation and aortic inflammation, augmenting the disease. Neuronal outgrowth is functionally dependent on microbiota and is essential for neuroinflammation and augmentation of atherosclerosis as chemical denervation or chemogenetic inhibition reduces inflammation, macrophage activation and disease progression. Microbiota-dependent IL-17A producing gd T cells accumulate in the aorta to promote neuronal outgrowth and activation that can be reversed by gd T cell blockade. Perivascular neuron activation is further dependent on cell-autonomous IL-17 signaling as IL-17RC ablation in sympathetic neurons protected mice from microbiota-driven atherosclerosis. Together, our data illuminate how intestinal cytokine signaling distantly restrains neuroimmune interactions in aorta and uncovers a novel link between IL-17 signaling, microbiota, perivascular innervation and neuroimmune pro-inflammatory crosstalk instrumental for atherosclerosis progression.

overall_design: RNA-seq profiling of tissues from mice with intestinal epithelial cell-specific knockout of IL17RC and their littermate controls, all on an Ldlr-/- background. Tissues analyzed include ileum and aorta from separately housed mice, aortas from antibiotic-treated mice, and aortas from co-housed mice.

first: A

last: Mazitova

first: E

middle: K


title: HTRA1 Mutation and Expression Regulation in Cerebral Small Vessel Disease

city: Shanxi

first: Shina

last: Song

address: Jiefangnan 85 Road

institute: The First Hospital of Shanxi Medical University

summary: Cerebral small vessel disease (CSVD) refers to a series of clinical, radiological, and pathological syndromes caused by various etiologies affecting small arteries, arterioles, venules, capillaries, and small veins in the brain. It can lead to cognitive impairment, stroke, gait abnormalities, and other neurological symptoms and signs. Globally, approximately 25% to 30% of strokes are caused by CSVD. The underlying mechanisms of CSVD are multifaceted, involving endothelial dysfunction, blood-brain barrier (BBB) inflammation, neuronal apoptosis, chronic cerebral hypoperfusion, and their complex interactions. Current treatments often fail to achieve satisfactory outcomes. Therefore, understanding the pathogenic mechanisms of CSVD is crucial for developing effective therapeutic strategies to mitigate its detrimental effects. The high-temperature requirement protease A-1 (HTRA1) mutations can cause hereditary CSVD. CSVD associated with HTRA1 mutations is referred to as HTRA1-associated CSVD. Homozygous HTRA1 mutations cause Cerebral Autosomal Recessive Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CARASIL), a rare hereditary CSVD that is inherited in an autosomal recessive manner. Heterozygous HTRA1 mutations can lead to Cerebral Autosomal-Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy type 2 (CADASIL 2), also known as heterozygous HTRA1 mutation carriers. This condition is characterized by clinical manifestations such as stroke, cognitive impairment, gait abnormalities, alopecia, and spondylosis. To date, 35 pathogenic mutations in the HTRA1 gene have been reported, primarily exhibiting autosomal dominant inheritance. Current research mainly focuses on case reports and the potential pathogenic mechanisms associated with different mutation sites. HTRA1 mutations can lead to downregulation of HTRA1 mRNA and protein expression, thereby reducing HTRA1 protease activity. However, the specific morphological and functional changes associated with CSVD, especially those in endothelial cells, remain unclear. This study aims to explore the mutation spectrum and clinical phenotypes of heterozygous HTRA1 mutation carriers. By comparing whole-blood RNA sequencing (RNA-seq) analysis between heterozygous HTRA1 mutation carriers and healthy controls, we identify differentially expressed genes. Based on the RNA-seq results, we further investigate the effects of abnormal HtrA1 expression on the biological functions of mouse brain microvascular endothelial cells and mouse cognitive behavior. This study elucidates the role of HTRA1 in CSVD, providing insights into the pathogenesis and new therapeutic targets for patients with heterozygous HTRA1 mutations.

overall_design: This submission includes peripheral blood samples from heterozygous HTRA1 mutation carriers (HTRA1mc) and healthy controls (HC). A total of eight samples were analyzed, including four HTRA1mc patients from two independent families and four unrelated healthy controls. The experimental variable under investigation is the presence of heterozygous HTRA1 mutations. HTRA1 mutation carriers represent the experimental group, while healthy controls serve as the reference group. All samples are independent biological replicates.


title: Role of microRNAs in flunarizine-mediated neuroprotection of motoneuron disease spinal muscular atrophy

city: 91400 Orsay

first: Florent

last: Dumont

state: Ile de France

address: 17 avenue des sciences

institute: Université Paris-Saclay

type: Non-coding RNA profiling by high throughput sequencing

summary: Spinal muscular atrophy (SMA) is caused by mutations in the Survival   Motor Neuron 1 (SMN1) gene, leading to reduced SMN protein levels and   widespread disruption in RNA metabolism. Despite the availability of   disease-modifying therapies, clinical outcomes remain variable.   Dysregulation of small non-coding RNAs, including microRNAs, has been   reported in SMA, but their contribution to the disease remains poorly   understood. We previously showed that flunarizine could improve the   phenotype of an SMA mouse model. Here, the microRNA profile of   flunarizine-treated SMA patient fibroblasts is determined by small-RNA   sequencing, a subset of 30 microRNAs is validated by RT-qPCR and the   effect of the differentially expressed microRNAs is analyzed using miR   mimics and inhibitors in murine motor neuron-like NSC34 cells. The   miR-128-3p, miR-149-5p, miR-191-5p, and miR-320a-3p are identified as   novel SMA-dysregulated microRNAs. The transfection of the miR-128-3p   inhibitor in NSC34 cells reduces the flunarizine-induced neurite   outgrowth, a morphological hallmark of neurons. The homeodomain   interacting protein kinase 2 (HIPK2) is identified as a miR-128-3p   target among the differentially expressed genes by flunarizine. The   transfection of miR-128-3p mimic reduces the Hipk2 mRNA levels whereas   miR-128-3p inhibitor increases them. Moreover, Hipk2 expression is   regulated in a genotype- and sex-dependent manner by flunarizine at   early and late stages of disease in an SMA mouse model. Flunarizine   changes the microRNA profile in NSC34 cells and attenuates the   dysregulation of microRNAs in the SMA mouse model. We propose that an   early accumulation of microRNAs can contribute to molecular dysfunctions   and may represent a key-initiating event in SMA pathogenesis.

first: Suzie

last: Lefebvre


title: Brain-Engrafted Monocyte-derived Macrophages from Blood and Skull-Bone Marrow Exhibit Distinct Properties [scRNA_DR3X]

city: St. Louis

first: Jonathan

last: Kipnis

state: MO

address: 4515 McKinley Ave

institute: Washington University in St. Louis

summary: Microglia arise from yolk sac progenitors and are thought to persist throughout life with minimal input from adult hematopoiesis. However, whether brain-engrafted monocyte-derived macrophages (MDM) exist at homeostasis and during turnover, and how they function relative to yolk sac-derived microglia (YSM) remain unsettled. Here, we combine lineage tracing, pharmacological microglia depletion, and multi-omics profiling to define the ontogeny, identity, and function of brain parenchymal macrophages. Despite sharing the parenchymal milieu, MDM display transcriptional and epigenetic landscapes distinct from YSM. Fate-mapping reveals that brain-engrafted MDM transiently express CD206, echoing a developmental stage of microglial precursors. MDM engraftment and polarization are modulated by IL-34 and CCR2. Furthermore, parabiosis and skull‑flap transplantation reveal that both blood and skull marrow supply the niche, yielding origin‑biased MDM states. Functionally, MDM engraftment enhanced cuprizone-mediated demyelination. Together, our study defines the origins, molecular features, and context-dependent roles of brain parenchymal macrophages across homeostasis, turnover, and CNS pathology.

overall_design: To investigate the presence of brain-engrafted MDM during microglia turnover, we fed mice with PLX5622 (PLX), a pharmacological inhibitor of colony-stimulating factor-1 (CSF1R), to deplete microglia. We performed either one round (DR-1x)35 or three rounds of depletion-repopulation (DR-3x). Brains were analyzed immediately after the final PLX cycle, prior to repopulation (CTR-0). To assess whether MDM engraftment also occurs in aged animals and whether age influences the transcriptional programs of engrafted MDM, we included an additional cohort of 15-month-old mice in this analysis. For brain, sample preparation was executed on ice to preserve tissue integrity. Mice were administered a lethal dose of Euthasol (10% v/v) intraperitoneally, followed by transcardial perfusion with PBS containing 0.025% heparin. Post-decapitation, brains were excised and immediately submerged in cold RPMI (Gibco). For FACS, the choroid plexus was carefully extracted and discarded. Brain tissue was then dissociated using a dounce homogenizer, filtered through a 70 μm cell strainer into a 50 ml conical tube, and centrifuged for 10 mins/450xg at 4°C. The resulting brain cell pellet was resuspended in a 30% isotonic percoll solution (GE Healthcare) to remove myelin, and myelin fraction was depleted by centrifugation for 15 mins/850xg at 4°C (acceleration 6, break 1). The clean cell pellets were then resuspended in a FACS buffer containing 1% BSA (Equitech-Bio), 1mM EDTA (Corning), and 10mM HEPES (Corning) in preparation for staining. For flow cytometry, brain parenchymal macrophages were defined as live singlets, CD19⁻, CD45⁺ CD11b⁺, Ly6G⁻, Ly6C⁻, MERTK⁺, and CD11c- and CD206low unless otherwise specified.

first: Siling

first: Feiya

last: Ou

first: Antoine

last: Drieu

first: Eric

last: Xu

first: Yumeng

last: Cheng

first: Steffen

last: Storck

first: Tornike

last: Mamuladze

first: Jay

last: Cao

first: Nora

last: Abduljawad

first: Bishan

last: Bhattarai

first: Justin

last: Rustenhoven

first: Niall

last: Mortimer

first: Simone

last: Brioschi

first: Khai

last: Nguyen

first: Patrick

middle: F

last: Rodrigues

first: Igor

last: Smirnov

first: Daniel

last: Gibson

first: J

last: White

first: Wandy

last: Beatty

first: David

last: DeNardo

first: Qingyun

last: Li

first: Michael

last: Meers

first: Claudia

middle: Z

last: Han

last: Sun

last: Ginhoux

first: Marina

last: Cella

first: Marco

last: Colonna


title: Wollemia nobilis methylation sequencing

city: Cold Spring Harbor

first: W. Richard

last: McCombie

state: NY

address: 1 Bungtown Road

institute: Cold Spring Harbor Laboratory

type: Methylation profiling by high throughput sequencing

summary: We performed methylation profiling analysis from Bisulfite-seq of 2 different reproductive tissues: ovule and pollen

overall_design: Methylation profiling analysis of Bisulfite-seq data for ovule and pollen tissues

first: Srividya

last: Ramakrishnan

first: Cristiane

last: de Santis Alves Rosa

first: Lais

last: Araujo Coelho

first: Melissa

last: Kramer

first: Sara

last: Goodwin

first: Olivia

last: Mendivil Ramos

first: Gil

last: Eshel

first: Veronica

last: Sondervan

first: Samantha

last: Frangos

first: Cecilia

last: Zumajo-Cardona

first: Katharine

last: Jenike

first: Shujun

last: Ou

first: Xiaojin

last: Wang

first: Yin

last: Peng Lee

first: Stella

last: Loke

first: Maurizio

last: Rossetto

first: Hannah

last: McPherson

first: Sebastiano

last: Nigris

first: Silvia

last: Moschin

first: Damon

middle: P

last: Little

first: Manpreet

middle: S

last: Katari

first: Kranthi

last: Varala

first: Sergios-Orestis

last: Kolokotronis

first: Barbara

middle: A

last: Ambrose

first: Larry

last: Croft

first: Gloria

last: Coruzzi

first: Dennis

last: Stevenson

middle: C

last: Schatz

first: Robert

last: Martienssen


title: FOXM1 is prevalent in lung cancer and targetable via T cell receptor engineering

city: Houston

first: Peixin

last: Jiang

state: TX

address: 1515 Holcombe Blvd

institute: UT MD Anderson Cancer Center

summary: We identified epitopes derived from FOXM1 which were immunogenic on HLA-A*02:01, HLA-A*24:02, and HLA-A*23:01, endogenously-processed and presented, and resulted in T cell activation and cytotoxic T cell responses. Following the generation of TCR-T cells, sensitivity and specificity were confirmed by peptide dose-response and X-scan, respectively. Most importantly, adoptive transfer of TCR engineered T cells led to a significant reduction in tumor growth, as well as significantly prolonged survival in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) tumor-bearing murine model. Our studies confirm the immunogenicity of FOXM1 and feasibility of targeting this antigen using TCR-engineering.

overall_design: Expanded, reactive antigen-specific T cells were analyzed using scRNAseq for identifying and synthsis of CDR3 α/β sequences assembly.

first: Minying

last: Zhang

first: Emily

last: Bontekoe

first: Alexandre

last: Reuben


title: Single-cell chromatin profiling reveals dynamic regulatory logic and enhancer elements in brain and retina development

first: Bushra

last: Raj

address: 421 Curie Boulevard

institute: University of Pennsylvania

summary: Cell type identity in the nervous system is encoded within cis-regulatory landscapes that integrate transcription factor activity with chromatin accessibility. However, how these regulatory programs are organized and remodeled during post-embryonic neural development remains poorly understood. We generate a temporally resolved single-cell chromatin accessibility atlas of ~95,000 zebrafish brain and retina nuclei spanning larval, juvenile, and adult stages. We define 212 discrete chromatin states and uncover widespread, cell type-specific chromatin reorganization across development. By integrating with transcriptomic data, we link motif accessibility to transcription factor expression and identify regulatory programs that are either maintained or reconfigured during post-embryonic development of each cell type. Leveraging this atlas, we systematically identify and functionally validate candidate enhancers in vivo. Focusing on the slc1a3b locus in radial glia, we define evolutionarily conserved, compact enhancer modules that act combinatorially to drive gene expression. Together, these findings provide a systems-level framework for decoding neural regulatory logic and enable functional dissection of conserved cis-regulatory programs in the vertebrate nervous system.

overall_design: scATAC-seq profiling of wildtype, TL/AB zebrafish brain and eyes.  Dissection of zebrafish whole heads at 3 dpf and isolated zebrafish brain and eyes at 21 dpf and adult (5mpf). Lens and cornea were removed during dissection of adult eyes to enrich for retinal populations.

first: Jessie

middle: E

last: Greenslade

first: Hemagowri

last: Veeravenkatasubramanian

first: Marisa

last: Reed


city: Rochester

first: Zhiquan

last: Wang

state: MN

address: 200 1st ST SW

institute: Mayo Clinic

summary: Defining the acute BTK-loss transcriptional program in CLL using a BTK degrader by RNA-seq

overall_design: Primary CLL B cells received various treatments and the gene expression was determined by RNA-seq


title: Brain-Engrafted Monocyte-derived Macrophages from Blood and Skull-Bone Marrow Exhibit Distinct Properties [scRNA_FIRE-ctrl-blood-skull]

first: Jonathan

last: Kipnis

overall_design: For parabiosis, UBC-GFP mice, together with sex and age matched WT mice were rendered fully muscle-relaxed via intraperitoneal injection of a xylazine (10 mg/kg) and ketamine (100 mg/kg) mixture. The respective lateral regions were first shaved, then applied with betadine and alcohol in three alternating cycles. The surgery was performed using aseptic technique. Next, a cut extending from the olecranon to the knee joint was made, allowing for the separation of the subcutaneous fascia to yield a 0.5 cm margin of free skin. The olecranon and knee joints were then conjoined with 5-0 Vicryl absorbable sutures. The dermis of the parabiotic pairs was approximated, deliberately excluding epidermal layers at the junction, and sutured closed. Postoperative care included Buprenorphine SR (1.0 mg/kg) and antibiotic administration in drinking water. For skull transplant, mice were anesthetized using a xylazine (10 mg/kg) and ketamine (100 mg/kg) mixture. Throughout the surgical procedure, body temperature was maintained at 37°C using a heating pad. For the initial preparation, the heads of WT mice were shaved, and a midline incision was made to expose the skull. A cranial window (4-mm-by-6-mm) was drilled into the parietal and interparietal bones of the skull using an electric drill. To prevent thermal damage, the drilling site was intermittently cooled with 0.9% sodium chloride solution. After the cranial window was detached, careful craniotomy was conducted without damaging the underlying dura mater, brain, or sinus structures. Simultaneously, a sex-matched UBC-GFP mouse was used as a skull BM donor. After euthanization and decapitation, a piece of the donor’s calvarium, matching the host’s window size, was harvested. The periosteum and pericranium were left intact on the bone flap to enhance survival post-transplant. The meninges were removed from the donor skull piece containing the bone marrow, which was then placed within the surgical window of the host. The donor skull was affixed to the host site using cyanoacrylate glue and secured with a 10-0 nylon suture. Post-surgery, the incision was closed, and mice were kept on a heating pad until recovery. Postoperative care included Buprenorphine SR (1.0 mg/kg) and antibiotic administration in drinking water for the first five days to mitigate inflammation and prevent infection. Experimental animals were collected in two independent batches. Within each batch, individual mice were labeled using cell hashing, and scRNA-seq was performed on sorted immune cells.

first: Siling

last: Du

first: Feiya

last: Ou

first: Antoine

last: Drieu

first: Eric

last: Xu

first: Yumeng

last: Cheng

first: Steffen

last: Storck

first: Tornike

last: Mamuladze

first: Jay

last: Cao

first: Nora

last: Abduljawad

first: Bishan

last: Bhattarai

first: Justin

last: Rustenhoven

first: Niall

last: Mortimer

first: Simone

last: Brioschi

first: Khai

last: Nguyen

first: Patrick

last: Rodrigues

first: Igor

last: Smirnov

first: Daniel

last: Gibson

first: J

middle: M

last: White

first: Wandy

last: Beatty

first: David

last: DeNardo

first: Qingyun

last: Li

first: Michael

last: Meers

first: Claudia

last: Han

last: Sun

first: Florent

last: Ginhoux

first: Marina

last: Cella

first: Marco

last: Colonna


title: MERTK inhibition cooperates with immunomodulatory cyclophosphamide to induce CXCL9-positive monocyte-macrophage programming and durable anti-tumor immunity in triple negative breast cancer

first: Xiang

last: Zhang

address: One Baylor Plaza

institute: Baylor College of Medicine

summary: Triple negative breast cancer (TNBC) has high rates of recurrence despite chemotherapy and immune checkpoint blockade (ICB). Tumor associated macrophages (TAMs) can either suppress or support anti-tumor immunity, but the mechanisms governing these states and their therapeutic targeting remain unclear. Here, by integrating public single-cell datasets with TNBC patient cohorts, we identify a prognostic myeloid signature defined by CXCL9-high and C1Q-low TAM programs that is associated with improved survival and increased lymphocyte activation pathways. Using immunocompetent p53-null syngeneic TNBC models spanning basal-like (2153L) and claudin-low (T12) subtypes, we show that immunomodulatory cyclophosphamide (CTX) reprograms hematopoiesis toward the monocytic lineage and induces an interferon conditioned tumor microenvironment that supports CXCL9-positive monocyte-derived macrophages in basal-like disease. Combining CTX with the next generation MERTK-selective inhibitor ULC2371 (MRX-2843) drives complete tumor remissions in both models; however, durable long-term responses occur selectively in the basal-like subtype. Responding tumors exhibit expansion of antigen-presenting CXCL9-positive monocyte-derived macrophages and a reduction of C1q-positive phagocytic TAMs. Mechanistically, MERTK inhibition relieves MAPK and SOCS1 mediated restraint of interferon signaling, driving a positive feedback loop involving IRF7, STAT1, and IRF1 that promotes sustained CXCL9 induction. Functionally, tumor control requires CXCL9-CXCR3 dependent CD4 T cell recruitment, accumulation of stem-like memory CD4 T cells, and germinal center-like immune organization in tumor draining lymph nodes. PD-1 blockade further enhances response durability, preventing recurrence in most treated basal-like tumors.Together, these findings define an interferon-licensed, MERTK-regulated myeloid checkpoint that can be therapeutically targeted to convert immunosuppressive TNBC microenvironments into durable adaptive immunity, supporting clinical translation of CTX and MRX-2843 based combination therapies in basal-like TNBC.

overall_design: Day 7 mice were treated 3 hours before euthanasia and harvesting. Tumors were processed, digested, and diluted as mentioned above. Spleens were harvested as mentioned above. Anti-CD16/32 antibody was used to block FcR binding, then incubated with anti-CD45 (BioLegend, 103128 1:250) for 45 min. Cells were then stained in collection buffer (PBS + 20%FBS) with Dapi. Minimally, 150,000 cells were sorted for Dapi-CD45+ on a Sony MA900 in the Cytometry and Cell Sorting Core at Baylor College of Medicine. After sorting, cells were spun, resuspended in PBS, and submitted to the Single Cell Genomics Core at Baylor College of Medicine.

first: Alex

last: Smith

first: Jeffrey

last: Rosen


title: Single-cell transcriptomic analysis of microglia and brain cells after ischemic stroke

city: Yushima, Bunkyo-ku

first: Jun

last: Tsuyama

state: Tokyo

department: Medical Research Laboratory

institute: Institute of Science Tokyo

summary: To investigate microglial heterogeneity and the effects of Zfp384 perturbation on brain cell populations after ischemic stroke, we performed single-cell RNA-seq of microglia and total brain cells from mouse brains at multiple conditions after middle cerebral artery occlusion (MCAO).

overall_design: Single-cell RNA-seq of FACS-sorted microglia from sham-operated, post-ischemic day 14 (Igf1-CreER;Ai14), day 28 (Zfp384flox/flox vs. Cx3cr1-CreER;Zfp384flox/flox; ASO-control vs. ASO-Zfp384), and day 56 (ASO-control vs. ASO-Zfp384) mouse brains; and total brain cells from sham-operated, post-ischemic day 14, and day 28 (ASO-control vs. ASO-Zfp384) mouse brains.

first: Seiichiro

last: Sakai

first: Yuichiro

last: Hara

first: Hideya

last: Kawaji

first: Takashi

last: Shichita


title: Chromatin accessibility profiling of mouse microglia after ischemic stroke

summary: To investigate time-dependent changes in the epigenetic chromatin state in microglia after ischemic stroke, we performed assay for transposase-accessible chromatin with sequencing (ATAC-seq) of microglia from mouse brains at multiple time points after middle cerebral artery occlusion (MCAO).

overall_design: ATAC-seq of microglia collected from sham-operated and post-ischemic day 1, 6, 14, and 28 mouse brains (wild-type), and from Igf1-CreER;Ai14 mice on day 28 post-MCAO (tdTomato+ dysfunctional vs. tdTomato– homeostatic microglia).


title: Brain-Engrafted Monocyte-derived Macrophages from Blood and Skull-Bone Marrow Exhibit Distinct Properties [Cut-n-Tag]

first: Jonathan

last: Kipnis

overall_design: CUT&Tag was performed on freshly isolated brain macrophages using a low-input protocol adapted from Kaya-Okur et al.115. Briefly, brain parenchymal macrophages were FACS-sorted, and approximately 100,000 cells per reaction (plus ~20% excess) were collected in low-retention tubes. Cells were pelleted at 600 × g for 3 min at room temperature, the supernatant was removed, and pellets were resuspended in 100 µL cold Nuclei Extraction Buffer (Epicypher, Pre-Nuclei Extraction Buffer, 21-1026). Cells were incubated on ice for 10 min to lyse the plasma membrane and release nuclei. Nuclei were collected by centrifugation at 600 x g for 5 min at 4°C, and the supernatant was carefully removed. A total of 10,000-30,000 nuclei were allocated for each CUT&Tag reaction. Nuclei were bound to Concanavalin A-coated magnetic beads that had been pre-washed and activated in binding buffer. Bead–nuclei suspensions were rotated at room temperature for 10 min and transferred to 0.6-mL tubes. Bead-bound nuclei were resuspended in 50 µL antibody buffer (wash buffer supplemented with 2 mM EDTA) containing anti-H3K27ac antibody (Abcam, ab4729; 0.5 µg per reaction) or anti-H3K4me3 antibody (Epicypher, 13-0041; 0.5 µg per reaction) and incubated overnight at 4°C with gentle rotation. The following morning, samples were washed with wash buffer and incubated for 1 h at room temperature with a secondary anti-rabbit IgG antibody (1:100 in wash buffer). After two washes with wash buffer, nuclei-bead complexes were incubated with pAG-Tn5 transposase pre-loaded with sequencing adapters (2.5 µL per reaction, diluted to a final volume of 100 µL in 300-wash buffer) for 1 h at room temperature with rotation. Excess pAG-Tn5 was removed by two washes in 300-wash buffer, and bead-bound nuclei were resuspended in 50 µL CUT&Tag tagmentation buffer (300-wash buffer supplemented with 10 mM MgCl2). Tagmentation was carried out at 37°C for 1 h. Beads were then washed once with 10 mM TAPS (pH 8.5), and tagmented chromatin was released by adding 10 µL of 1% SDS in 10 mM TAPS and incubating at 58°C for 1 h with a heated lid. After release, 11 µL 8.75% Triton X-100 was added to neutralize SDS, followed by 2 µL each of 10 µM i5 and i7 indexing primers and 25 µL NEBNext High-Fidelity 2× PCR Master Mix (New England Biolabs). Libraries were amplified using a 16-cycle PCR program (initial gap-filling at 58°C and 72°C, followed by 15 amplification cycles at 98°C/60°C). Libraries were purified with SPRI beads (1.1x ratio), washed twice in 80% ethanol, and eluted in 16 µL 10 mM Tris-HCl (pH 8.0). Library quality was assessed on an Agilent TapeStation to confirm the expected CUT&Tag fragment-size distribution. Libraries were quantified by Qubit, pooled equimolarly, and sequenced on an Illumina NovaSeq platform using paired-end 50-bp reads, targeting 10 million uniquely aligned reads per sample.

first: Siling

last: Du

first: Feiya

last: Ou

first: Antoine

last: Drieu

first: Eric

last: Xu

first: Yumeng

last: Cheng

first: Steffen

last: Storck

first: Tornike

last: Mamuladze

first: Jay

last: Cao

first: Nora

last: Abduljawad

first: Bishan

last: Bhattarai

first: Justin

last: Rustenhoven

first: Niall

last: Mortimer

first: Simone

last: Brioschi

first: Khai

last: Nguyen

first: Patrick

last: Rodrigues

first: Igor

last: Smirnov

first: Daniel

last: Gibson

first: J

last: White

last: Beatty


title: Celastrol induces autophagy and apoptosis of human osteosarcoma cells via downregulation PI3K/Akt/mTOR signaling pathway

city: Nanchang

first: fengfen

last: guo

address: No.17 Yongwaizheng Street, Donghu District

department: Department of Orthopedics

institute: The First Affiliated Hospital of Nanchang University

summary: Celastrol, a terpenoid compound isolated from the plant Tripterygium wilfordii (Thunder God Vine), has been shown to have powerful anti-tumor effects against various developing cancers. Osteosarcoma is the most common primary bone malignancy in children and adolescents, and its long-term survival has remained stagnant over the past few decades.

overall_design: Here, we aim to investigate the inhibitory effect of celastrol on osteosarcoma and its molecular mechanism. Our study confirmed that celastrol can inhibit the proliferation, migration and invasion of osteosarcoma cells, and induce apoptosis through endogenous and exogenous pathways. Further studies confirmed that celastrol can induce autophagy and promote apoptosis via downregulation PI3K/Akt/mTOR signaling pathway. Furthermore, autophagy inhibitors and PI3K pathway activators significantly reduced celastrol-induced autophagy and apoptosis. Finally, celastrol inhibited the growth of osteosarcoma and lung metastasis in mice at safe doses. Taken together, our results suggest that celastrol can induce autophagy and apoptosis in human osteosarcoma cells via downregulation the PI3K/Akt/mTOR signaling pathway, and inhibit cell proliferation, migration, invasion and lung metastasis.

first: Fengfen

last: Guo


title: Xenopus gene ag1 influence on the skin would healing in mice

city: Moscow

first: Andrey

last: Zaraisky

address: Miklukho-Maklaya str. 16/10

country: Russia

institute: Institute of Bioorganic Chemistry (RAS)

summary: Here, we investigated whether the ag1 gene, which plays a key role in regeneration in fish and amphibians and was lost in amniotes, could enhance skin wound healing in mice. We generated transgenic mice with inducible expression of the Xenopus laevis ag1 gene in the skin and compared wound healing dynamics between induced and non-induced groups. Induction of ag1 resulted in approximately 20% faster wound closure. Transcriptomic analysis revealed enhanced activation of multpathways involved in wound repair

overall_design: Two biological replicates were analyzed for 2 conditions (wound tissues of wild-type adult mice vs similar tissues of adult transgenic mice ubiquitously expressing the Xenopus laevis ag1)

first: Anastasiya

middle: V

last: Kosykh

first: Nadya

middle: G

last: Gurskaya

first: Anastasiia

last: Ivanova

first: Helen

last: Makarenkova


title: Transcriptomic profiling of mouse microglia after ischemic stroke

first: Jun

last: Tsuyama

summary: To investigate time-dependent changes in gene expression in microglia after ischemic stroke, we performed RNA-seq of FACS-sorted  microglia from mouse brains at multiple time points after middle cerebral artery occlusion (MCAO).

overall_design: RNA-seq of microglia collected from sham-operated and post-ischemic day 1, 6, 14, and 28 mouse brains (wild-type); Igf1-Egfp transgenic mice on day 6 and day 14 (EGFP+ vs. EGFP– microglia); and Cx3cr1-CreER;Yy1flox/flox vs. Yy1flox/flox mice on day 6 (3 biological replicates each).

first: Tsuyama

last: Jun

first: Shichita

last: Takashi


title: Association between elevated expression of GDF15/GFRAL and sarcopenia risk

city: hefei

last: Wu

state: Anhui

address: Meishan Road 81

institute: Anhui medical university

summary: Glial cell line-derived neurotrophic factor family receptor α-like (GFRAL) is the exclusive receptor for growth differentiation factor 15 (GDF15) and plays distinct roles in various diseases. However, its impact on sarcopenia remains poorly understood. This study, comprising participants of White British and Chinese descent, demonstrated that elevated serum levels of GDF15 and its receptor GFRAL are associated with an increased risk of sarcopenia, particularly among individuals with low physical performance. While GDF15 exerts effects across the whole population, GFRAL appears to be more detrimental in women. Complementary single-cell RNA sequencing and immunohistochemical analyses in animal models further support the involvement of the GDF15–GFRAL axis in mediating skeletal muscle damage. These findings highlight the clinical and preventive relevance of the GDF15–GFRAL axis and underscore the need for future mechanistic studies to elucidate its role in sarcopenia.

overall_design: The tibialis anterior muscles from 26-month-old female C57BL/6 mice underwent enzymatic digestion, followed by filtration through a 30–70 µm cell sieve. Erythrocytes were removed, and the sample was further filtered using MS Columns to eliminate reagents and dead cells. The resultant cell suspension was prepared in 100 µl of 1×PBS containing 0.04% BSA. Cell counting was conducted using a hemocytometer or the Countess II Automated Cell Counter, resulting in a concentration of 700–1200 cells/µl. The single-cell suspension was processed using a 10× Chromium chip in accordance with the 10X Genomics Chromium Single-Cell 3′ kit (V4) protocol. cDNA amplification and library construction were performed following standard procedures. Considering that sarcopenia underlies frailty and exhibits similar changes in skeletal muscles, frail mice were designated as the case group based on the 31-item frailty index (FI) evaluation, with an FI score ≥ 0.18, while mice with FI scores < 0.18 were classified as controls. Following data quality filtration, two frail mice and three control mice were included in the single-cell sequencing analysis.


title: Starve-feed cycles regulate gene expression to control quiescence to proliferation transitions in Drosophila Follicle Stem Cells

city: 3601 Spruce Street

first: Yan

last: Zhou

address: 333 Cottman Ave

department: Bioinformatics

institute: Fox Chase Cancer Center

summary: we used sequential screening to uncover feeding-dependent transcriptional regulators of Q->P transitions in FSCs. Our first step was to endogenously label nascent transcripts induced within FSCs during the 6 hour Q->P transition timecourse using Thiouracil Tagging (TU-Tagging)40, 41. We then purified and sequenced transcripts that were differentially enriched in FSCs and early follicle cells whose expression changed during the 6 hour Q->P transition. Next, we evaluated expression of prioritized candidate genes in the germarium using the RNA in situ hybridization approach, RNAScope42, pinpointing genes enriched in FSCs, specifically. Finally, we performed functional analysis of genes isolated via our primary and secondary gene expression screens to identify novel regulators of Q->P. We present evidence that feeding-induced, cell autonomous Q->P regulators are found in predictable pathways, including cholesterol trafficking and feeding regulation, and also in unexpected pathways that regulate neuronal and glial signaling. This work highlights the value of conducting unbiased, forward genetic screening for identification of dynamic new factors critical for stem cell regulation.

overall_design: RNAs from 3 separate RNA pools (biological triplicates) derived from >100 ovary pairs each were used. Transcriptome profile between TU-tagged libraries and Input RNA were compared and differentially expressed genes were identified.

first: Eric

middle: H

last: Lee

first: Jacqueline

last: Simonet

first: Alana

last: O'Reilly


title: H3K27ac HiChIP of mouse microglia after ischemic stroke

summary: To detect chromatin loops maintaining genomic interactions among enhancer and promoter regions in microglia after ischemic stroke, we performed H3K27ac HiChIP of FACS-sorted CD45intCD11bint microglia from sham-operated and post-ischemic day 6 mouse brains.

overall_design: H3K27ac HiChIP of CD45intCD11bint microglia collected from sham-operated and post-ischemic day 6 mouse brains.

first: Takashi

last: Shichita


title: Amino acid supplementation enhances in vivo efficacy of lipid nanoparticle-mediated mRNA delivery in preclinical models

city: Chicago

first: Zongjie

last: Wang

state: Illinois

address: 400 N Aberdeen St., STE800

department: Biohub Chicago

institute: Biohub

summary: Lipid nanoparticles (LNPs) play a critical role in the delivery of therapeutic mRNA. Despite extensive efforts to optimize lipid formulations for in vivo delivery, efficacy of mRNA by LNPs remains suboptimal in many organs. Here, we demonstrate that LNP delivery efficacy is influenced by cellular metabolism, with the physiologic metabolome imposing constraints on mRNA expression from LNPs. Using an in vitro system, we found that simulated physiologic metabolic conditions led to the downregulation of certain amino acid metabolic programs. Supplementation of an optimized formulation of methionine, arginine, and serine as an amino acid supplement (AAS)  which enhanced the uptake of LNPs and the expression of delivered mRNA cargo in epithelial cells in vitro. Co-administration of AAS with LNPs led to a 5- to 20-fold improvement in mRNA expression across various cell types and lipid formulations in vitro . In vivo delivery of mRNA by LNPs co-administered with AAS  by multiple routes enhanced in vivo mRNA expression. Delivery of  growth hormone encoding mRNA by LNPs with co-administration of AAS improved the liver GH expression and the therapeutic outcomes in a model of inflammatory liver damage. Delivery of gene editing materials by LNP and AAS through intratracheal route increased lung-targeted in vivo gene editing efficiency compared with LNP alone. The addition of an optimized amino acid supplement as a co-delivered agent with LNPs may provide a simple strategy to broadly improve the efficacy of mRNA-based cell and gene therapies.

overall_design: RNA-seq profiling of H441 and HT29 cell cultured in RPMI-1640 and HPLM medium for 7 days.

first: Kangfu

last: Chen

first: Wenhan


title: Stable Bioreactor Control Reveals Acidic pH–Driven Metabolic Reprogramming and Mitochondrial Dysfunction in Human Lymphoblastoid Cells

city: Thuwal

first: Mo

last: Li

address: King Abdullah University Of Science And Technology

country: Saudi Arabia

summary: Human cells require precise pH regulation to maintain optimal physiological functions, yet the molecular impacts of acidic environments remain underexplored due to the lack of technologies capable of precisely controlling the cell culture environment. Here, we employed a gas-only bioreactor to stably control pH, dissolved oxygen, and temperature during the culture of human B lymphoblastoid cells. Integrated transcriptomic, epigenomic, and metabolomic analysis revealed that acidic pH (6.8) induces a metabolic shift towards glycolysis, suppresses energy-intensive processes such as cell proliferation, and triggers intracellular accumulation of lactate and oncometabolites and mitochondrial dysfunction. This environment also elevates reactive oxygen species (ROS) and upregulates inflammatory and immune pathways, leading to heteroplasmic shifts in a pathogenic mitochondrial mutation. Mechanistically, acidic pH caused marked depletion of intracellular NAD⁺ driven in part by PARP1 activation, and restoration of NAD⁺ levels via nicotinamide mononucleotide supplementation or PARP1 inhibition partially rescued cellular proliferation, mitochondrial genomic integrity, and stress-associated transcriptional programs. These findings highlight the critical role of pH homeostasis in cellular metabolism and immune response, offering insights into the cellular adaptations to acidic stress, which may inform therapeutic strategies for conditions like cancer and metabolic disorders.

overall_design: GM12878 cells were thawed from liquid nitrogen stocks and expanded in batch culture until entering logarithmic growth phase. Cells were then transferred to bioreactor culture systems equilibrated to the indicated pH conditions (pH 7.4 or pH 6.8), with dissolved oxygen and pH continuously monitored to ensure stability throughout the experiment. Cells were seeded at a density of approximately 2.5 × 10⁵ cells/mL and cultured for 72 hours. For NAD⁺ precursor supplementation, nicotinamide mononucleotide (NMN, Abcam; Cat# ab223879) was added to the culture medium at a final concentration of 0.5 mM at the start of the bioreactor culture. For inhibition of NAD⁺ consumption, a PARP1 inhibitor (Rucaparib, Sigma; Cat# AMBH2D6FA67F) was added at a final concentration of 5 μM. NMN-treated and PARP1 inhibitor–treated cultures were performed in parallel with untreated controls under both pH conditions. No medium changes were performed during the intervention period to maintain consistent metabolic conditions. At the end of the 72-hour treatment period, cells and conditioned media were harvested for downstream analyses. All intervention conditions were processed in parallel to minimize batch effects. Total RNA was extracted using Qiagen RNeasy mini kit (cat#74106), and the quality was assessed with the Bioanalyzer 2100. The RNA samples with RIN > 7.0 are outsourced to X Genomics for directional library preparation and sequencing.

first: Samhan

last: Alsolami

first: Yiqing

last: Jin

first: Yingzi

last: Zhang


title: Glycine max JmjC domain-containing proteins JMJ19/20 exhibit endopeptidase activity and interact with LUXs to mediate flowering time

city: Shanghai

first: Chenjiang

last: You

state: Shanghai

address: 2005 Songhu Rd

institute: Fudan University

summary: Histone demethylases serve essential functions in plant growth and development across various species. However, their roles in Glycine max remain largely unexplored. This study identified GmJMJ19 and GmJMJ20, encoding JmjC domain-containing proteins. They exhibit circadian rhythmic expression patterns and interact with LUX ARRHYTHMO 2 (GmLUX2) both in vitro and in vivo. Although GmJMJ19/20 bind to histones, they lack conventional histone demethylase activity. Rather, GmJMJ19/20 function as endopeptidases that specifically cleave histone H3 peptides at unmethylated lysine 27 residues, in a manner independent of Fe²⁺ and α-ketoglutarate (α-KG), the cofactors required for typical JmjC enzymes. Structural modeling supports the occlusion of the catalytic pocket that prevents access to methylated substrates. The simultaneous knockout of GmJMJ19/20 significantly delays flowering time. Comparative transcriptomic analyses reveal that the GmJM19/20-GmLUX2 module enhances GmFULc expression via independent of the canonical evening complex. Haplotype analysis suggests that GmJMJ19 underwent selection during domestication, potentially contributing to soybean geographical adaptation.

overall_design: Genomic occupancy of GmJMJ20 in soybean leaves at ZT12 in W82 plants

first: Yapeng

last: Han

first: Yingxiang