title: Insulin resistance is associated with mammary mitochondrial dysfunction at the onset of human lactation

city: Oxford

first: Fadil

last: Hannan

state: Oxfordshire

address: Level 3, Women's Centre, John Radcliffe Hospital

department: Nuffield Department of Women's and Reproductive Health

country: United Kingdom

institute: University of Oxford

summary: Insulin resistance (IR) has emerged as a risk factor for lactation insufficiency and delays the onset of milk secretion after childbirth, termed secretory activation (SA). This may cause inadequate infant weight gain and early breastfeeding cessation. However, the mechanisms underlying delayed SA in insulin resistant women are unknown. To investigate this, we characterized the mammary transcriptomes and IR-related hormones of 75 breastfeeding women with healthy term infants during postpartum days 1-5. Participants were divided into IR tertiles based on plasma leptin-to-adiponectin ratio measurements. Those in the highest tertile had later SA onset with greater neonatal weight loss during postpartum days 1-5. Transcriptomic analysis on postpartum day 2 (n=4 high IR vs. n=8 low IR participants) showed transient suppression of mammary insulin and prolactin signaling genes, increased pro-inflammatory gene expression and altered expression of >200 mammary mitochondrial genes. These alterations were absent on postpartum days 3-5. Cultured mammary epithelial cells (MECs) treated with insulin showed upregulation of prolactin signaling and oxidative phosphorylation (OXPHOS) genes, with imaging and bioenergetic studies demonstrating that insulin promotes mitochondrial biogenesis and OXPHOS. Thus, our findings delineate roles for insulin in mammary bioenergetics and highlight mitochondrial dysfunction as a mechanism for delayed SA in insulin resistant women.

overall_design: RNA-seq profiling of three datasets generated as part of the same study: (1) differentiated HC11 cells treated with 0nM (control) or 100nM insulin for 8 hours; (2) differentiated WT MCF-12A cells treated with 0, 10, or 100 nM insulin, and Akt knockdown cells treated with 0 or 100 nM insulin; (3) milk fat globule (MFG) RNA from breastfeeding women with varying degrees of insulin resistance.

first: Xin

last: Meng

first: Taha

last: Elajnaf

first: Hussam

last: Rostom

first: Michelle

last: Ma

first: Annalee

last: Furst

first: Bryony

last: Davies

first: Isabella

last: Honess

first: Gaurav

last: Pandey

first: Isadora

last: Furigo

first: Craig

middle: L

last: Doig

first: Jayne

middle: F

last: Martin Carli

first: Rajesh

last: Thakker

first: Lars

last: Bode

first: Kelsey

last: Johnson


title: Store-operated calcium signaling modulators improve a dendritic cell-based vaccine against melanoma

status: Public on Aug 07 2026

city: Geneva

first: María

last: Cruz Cobo

address: Rue Michel-Servet 1, CMU

department: Pathology and Immunology

country: Switzerland

institute: University of Geneva

type: Expression profiling by high throughput sequencing

summary: Dendritic cells (DCs) are crucial for eliciting cytotoxic CD8+ T cell responses against intracellular pathogens, viruses and cancer. DC-based vaccines can stimulate both cytotoxic and humoral immunity, as well as memory against cancer. However, efficacy has remained low, leaving room for improvement. Our previous work using conditional Stim1 knock-out mice highlighted the importance of store-operated calcium entry (SOCE) in promoting DC migration, phagosome maturation and DC_x0002_driven CD8+ T cell responses. Yet its potential as a target to improve DC-mediated immune stimulation remains incompletely understood. We hypothesized that transient pharmacological modulation of SOCE signaling could boost DC-mediated T cell activity. Here, a targeted screen of SOCE modulators identified two compounds, thapsigargin (Tg) and para-bromo-2-aminoethyl diphenylborinate (pBr), that improved T cell proliferation, IL-2, and IFNG secretion in mouse and human DC:T cell co-cultures. In a B16 melanoma model, DC vaccines boosted with either compound reduced tumor growth by 50% and doubled the median survival benefit, increasing infiltration of activated CD8+ T cells, CD4+ T, natural killer, CD80+ B cells, and M1 macrophages. Neither compound affected the surface expression of MHC-I-peptide complexes, MHC-II, CD86, nor DC migration, although thapsigargin promoted antigen retention. Bulk RNA sequencing revealed instead broad changes in pathways regulating secretion, where cytokine array analysis confirmed both compounds boosted secretion of several immunomodulatory factors. Together, this work identifies SOCE as a druggable axis that may be exploited to boost DC-mediated T cell activation and improve DC-based anticancer therapies.

overall_design: RNAseq profiling of human monocyte-derived dendritic cells from 3 healthy donors, matured overnight with a cytokine cocktail (IL-1b, IL-6 and TNFa), loaded with melanoma-associated peptide (Melan-A long), and either untreated or concomitantly treated with SOCE modulators thapsigargin (Tg) and para-bromo-2-aminoethyl diplenylborinate (pBr)

first: Laure

last: Garnier

first: Marion

last: Mandavit

first: Maral

last: Azam

first: Isabelle

last: Dentand Quadri

first: Julien

last: Angelillo

first: Dale

last: Brighouse

first: Mathilde

last: Bausart

first: Valérie

last: Dutoit

first: Achille

last: Schild

first: Valeria

middle: M

last: Oliva

first: Martin

last: Lochner

first: Bénédicte

last: Manoury

first: Stéphanie

last: Hugues

first: Paula

last: Nunes-Hasler


title: Methylation array of cultured GBM models to create surrogate CNV profiles

city: Amsterdam

first: Bart A.

last: Westerman

address: van der Boechorststraat 6b

department: Human Genetics

country: Netherlands

institute: Amsterdam UMC

type: Methylation profiling by genome tiling array

summary: Background: Epidermal growth factor receptor (EGFR) amplification occurs in ~50% of IDH-wildtype glioblastoma (GBM) cases, frequently accompanied by expression of the oncogenic EGFRvIII variant. Although EGFR represents an attractive therapeutic target, EGFR-directed therapies have shown limited clinical efficacy in GBM. Resistance to kinase inhibitors is frequently attributed to activation of compensatory signaling pathways (“kinome rewiring”). We therefore investigated whether EGFR inhibition in GBM induces broad adaptive kinase responses that could be co-targeted to overcome resistance.  Methods: We molecularly profiled 29 patient-derived GBM cell lines for EGFR status and selected five representative models spanning distinct EGFR amplification states for functional analyses. Cells were treated with EGFR inhibitors and responses were assessed using 72-hour viability assays, time-resolved immunoblotting, and phosphoproteomics (LC-MS/MS) with kinase activity inference.  Results: EGFR inhibitors preferentially impaired viability in EGFR-driven models and transiently reduced EGFR phosphorylation during the initial response. However, partial restoration of EGFR phosphorylation and downstream signaling occurred after 24 hours of continuous inhibitor exposure. Phosphoproteomics revealed no evidence of broad kinome rewiring within this timeframe but instead identified increased EGFR protein levels associated with restored EGFR pathway activity. The phosphorylated-to-total EGFR ratio remained relatively stable, consistent with sustained target engagement despite signaling rebound.  Conclusions: Early adaptive responses to EGFR inhibition in GBM were not characterized by broad kinome rewiring but by restoration of EGFR signaling associated with increased EGFR abundance. These findings suggest that adaptive signaling remains largely EGFR-dependent despite continued inhibitor exposure, identifying regulation of EGFR abundance as a potential contributor to therapeutic resistance.

overall_design: Single specimen per cell culture model

first: Bart

first: Yoran

last: Broersma

first: Megan

last: Houweling


title: Multi-platform reassessment of human mitochondrial DNA methylation reveals signals consistent with technical artifacts

city: Toronto

first: Sagi

last: Abelson

state: ON

address: 661 University Avenue

country: Canada

institute: Ontario Institute for Cancer Research

type: Methylation profiling by high throughput sequencing

summary: The existence and functional relevance of mitochondrial DNA methylation remain controversial. Here, we systematically profiled cytosine methylation and hydroxymethylation across human brain and blood tissues spanning healthy and malignant states using orthogonal sequencing approaches that avoid chemical conversion during library preparation. While nuclear DNA exhibited canonical methylation patterns, mitochondrial DNA consistently showed negligible signal, indistinguishable from background technical noise. By mapping cytosine-guanine sites between mitochondrial DNA and nuclear-embedded mitochondrial sequences, we demonstrate the potential of these nuclear counterparts to confound not only cytosine methylation but also hydroxymethylation measurements, corroborating and extending prior findings implicating nuclear contamination as a potential source of apparent mitochondrial epigenetic signals. Additional technical factors that inflate apparent mtDNA methylation signals were identified, including sequence context biases, flow cell chemistries, and coverage-dependent discrepancies between the heavy and light strands. Collectively, these results provide convergent evidence against the presence of biologically meaningful cytosine methylation or hydroxymethylation in mitochondrial DNA. These findings caution against interpreting apparent mtDNA methylation signals in human adult tissues as meaningful without rigorous orthogonal validation and comprehensive consideration of technical and analytical confounding factors.

overall_design: Blood and brain samples were assayed using the Biomodal enzymatic sequencing platform to measure 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels of mitochondrial DNA.


title: Comparison of NK cell transcriptomes between C57BL6 and NOD mouse strains

city: Winnipeg

first: Peter

last: Thompson

state: MB

address: 715 McDermot ave

department: Physiology & Pathophysiology

institute: University of Manitoba

summary: Natural Killer (NK) cells are essential for surveillance of stressed, infected and malignant cells in the body. The autoimmune diabetes prone nonobese diabetic (NOD) mouse has a deficiency and dysfunction of NK cells compared to other strains but the molecular basis remains unclear. Here we performed bulk mRNA-seq on bead enriched splenic NK cells from early disease prediabetic 8 week-old female NOD mice as compared with age-matched C57BL6 female mice as controls.

overall_design: Splenocytes were isolated from 8 week-old female NOD/ShiLtJ and C57BL6/J mice and subjected to NK cell purification/enrichment using a bead-based negative selection (StemCell Technologies NK cell kit). NK cell enrichment was confirmed by flow cytometry for NKp46 and CD49b(DX5) along with CD3-negativity. Cells were lysed in Trizol for total RNA isolation and submitted for library preparation and next-gen sequencing using the Illumina Hi-Seq platform.

middle: J

first: Jasmine

last: Pipella


title: Senescent Fibroblast-derived SASP Drives Chemotherapy-Induced Peripheral Neuropathy

city: Saint Louis

first: Sheila

last: Stewart

address: 660 South Euclid Avenue

country: USA

institute: Washington University School of Medicine

summary: Hindpaw skin from mice treated with vehicle solution or PTX followed by normal or MK2i compounded chow were digested with 2mg/mL Collagenase A, 1mg/mL hyaluronidase and 2U/mL Dnase I.

overall_design: Mice were treated with vehicle or PTX (50mg/kg) via tail vein injection for 2 days. Following Vehicle or PTX treatment, mice were given normal or MK2i compounded chow for the following 7 days. On day 9, skin from the planar surface of the hindpaw was then digested and CD45- cells were isolated by FACS and analyzed using scRNA-seq. Samples from 5 mice were pooled for each sample.

first: Taylor

last: Malachowski

middle: A


title: Acute viral infection with influenza A virus and SARS-CoV-2 accelerates the progression of ALS in SOD1G93A mice.

city: Hamilton

first: Imran

middle: Syed

last: Ahmed

state: Ontario

address: 1280 Main Street West

institute: McMaster University

summary: While several viral infections have been associated with amyotrophic lateral sclerosis (ALS), the mechanism(s) through which they promote disease has remained almost entirely elusive. This study investigated the impact of common, acute viral infections prior to disease onset on ALS progression in the SOD1G93A mouse model. A single sublethal infection prior to onset of ALS clinical signs was associated with markedly accelerated ALS disease progression characterized by rapid loss of hindlimb function. Prior infection resulted in gliosis in the lumbar spine and upregulation of transcriptional pathways involved in inflammatory responses, metabolic dysregulation, and muscular dysfunction. Therapeutic suppression of gliosis with an anti-inflammatory small molecule, or administration of a direct-acting antiviral, was associated with significantly improved ALS clinical signs, akin to what was observed in uninfected animals. This study provides causal and mechanistic evidence that the immune response elicited by acute viral infections may be an important etiological factor that alters ALS disease trajectory and provides insight into novel therapeutic and preventative strategies for ALS.

overall_design: Bulk RNA-Seq profiling of transcripts in the lumbar spinal cord of SOD1G93A infected and uninfected mice  30 days post infection with a sublethal dose of IAV.

first: Anna

middle: D

last: Gheva


title: Targeting NCAM1/FGFR1 signaling to alleviate sleep disturbance-induced neuroinflammation in the hypothalamus

city: Dalian

first: Yanxiang

last: Qu

address: No.9 West Section, Lvshun South Road, Dalian, Liaoning Province

country: China

institute: Dalian Medical University

summary: Sleep disturbance is a prevalent public health concern linked to cognitive deficits and emotional dysregulation, yet the underlying mechanisms by which SD leads to hypothalamic dysfunction remain poorly understood. We demonstrated that two-week SD induced significant neuroinflammation in the hypothalamus, characterized by elevated expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, as well as the chemokine Ccl2, alongside progressive neuronal loss, as evidenced by reduced nissl bodies and NeuN+ cells. Single-nucleus RNA sequencing revealed that inhibitory neurons were the most responsive hypothalamic cell cluster to SD, exhibiting enriched pro-inflammatory pathways (e.g., TNF and IL-17 signaling) and neurodegenerative transcriptional programs. Furthermore, SD triggered microglial activation and a phenotypic shift toward a disease-associated state, with enhanced antigen-presenting and pro-inflammatory capacity. Notably, intercellular communication analysis identified the neural cell adhesion molecule 1 (NCAM1)/fibroblast growth factor receptor 1 (FGFR1) signaling axis as a key mediator of crosstalk between microglia and other cell types. In vitro experiments in BV2 cells revealed that NCAM1, via FGFR1, suppressed lipopolysaccharide-induced M1 polarization and the subsequent release of pro-inflammatory cytokines. Collectively, our findings elucidate a critical protective role for the NCAM1/FGFR1 axis in mitigating SD-induced hypothalamic neuroinflammation and neuronal injury, highlighting a potential therapeutic target for sleep-related neurological disorders.

overall_design: For both the control and sleep deprivation (SD) groups, a single biological sample was prepared by pooling hypothalamic tissues from three rats, resulting in one sample per group (n = 1 per group).

first: Bo

last: Li

first: Shixuan

last: Yan

first: Chuanjie

last: Zhang

first: Guohua

last: Ji

first: Kai

first: Yujie

last: Zhao

first: Zuoyang

last: Wang

last: Song

first: Xiaopeng

first: Lina


title: CAR-NKT cells induce low cytokine release syndrome by targeting hyperinflammatory macrophages with mitigation from GM-SCF inhibition.

city: Los Angeles

first: Yichen

last: Zhu

state: California

address: 610 Charles E Young Drive S

institute: University of California, Los Angeles

summary: Chimeric antigen receptor (CAR)-T cell therapy has demonstrated remarkable efficacy in cancer treatment but is frequently limited by severe toxicities, including cytokine release syndrome (CRS), which is largely driven by host myeloid cell activation. CAR-engineered invariant natural killer T (CAR-NKT) cells have emerged as a promising alternative with comparable antitumor activity and reduced toxicity; however, the mechanisms underlying their improved safety profile remain poorly defined. Here, we systematically evaluate CRS induced by human CAR-NKT and CAR-T cells using a heavily tumor-bearing xenograft mouse model that recapitulates clinically relevant CRS. Compared with conventional CAR-T cells, CAR-NKT cells induce significantly lower CRS severity across multiple clinical and biochemical parameters. To elucidate the underlying mechanisms, we perform comprehensive single-cell transcriptomic profiling of mouse immune cells, including myeloid and granulocytic populations, across six anatomical compartments: tumor, blood, liver, lung, spleen, and bone marrow. This integrative analysis reveals dynamic immune remodeling during tumor progression, therapeutic intervention, and CRS development. Mechanistically, CAR-NKT cells preferentially target CD1d⁺ hyperinflammatory macrophages, a key driver of CRS, thereby limiting myeloid-mediated inflammatory responses. Furthermore, pharmacological inhibition of GM-CSF further attenuate CRS, supporting a combinatorial strategy to enhance the safety of CAR-NKT cell therapy. Collectively, these findings provide mechanistic insight into the reduced toxicity of CAR-NKT cells and support their development as a safer and effective platform for cancer immunotherapy.

overall_design: Gene expression profiling of mouse immune cells extracted from in vivo CRS models.

first: Annabel

first: Yan-Ruide

first: Lili

last: Yang


title: Diseaase context dictates the cellular targets of IL-17 in inflammatory skin disease [2]

city: San Diego

first: Kellen

last: Cavagnero

address: 9500 Gilman Dr

institute: Univeristy of California, San Diego

summary: Therapeutic blockade of IL-17 and TNF can effectively treat inflammatory skin diseases such as hidradenitis suppurativa and psoriasis, yet the relative importance of the different cell types that respond to IL-17 and TNF remains unresolved. Keratinocytes are viewed as the dominant effector cells, whereas fibroblasts have recently emerged as important contributors. In mice, topical imiquimod induces IL-17- and TNF-dependent skin inflammation and is frequently used to model psoriasis. Here, we demonstrate that intradermal injection of recombinant IL-17 and TNF elicits skin inflammation with features of hidradenitis suppurativa, including a gene expression program that is distinct from psoriasis and imiquimod-induced inflammation. Single-cell transcriptomic network analysis identified dermal fibroblasts as the dominant cell communication hub in hidradenitis suppurativa and in mice injected with IL-17 and TNF. In contrast, fibroblasts and keratinocytes both show strong network involvement in psoriasis and in mice challenged with imiquimod. Cell-type-specific deletion of IL-17 receptor A in mice revealed that imiquimod-induced inflammation depends equally on IL-17 signaling in fibroblasts and keratinocytes, whereas inflammation induced by intradermal IL-17 and TNF only requires fibroblasts to recognize IL-17 and is independent of keratinocyte IL-17 sensing. Single-cell transcriptomic analysis of these conditional knockout mice further demonstrated that keratinocytes and fibroblasts activate divergent and disease-dependent transcriptional programs following activation by IL-17. Together, these findings introduce a new conceptual framework wherein IL-17 signaling is routed through distinct cellular and molecular pathways depending on disease context and establish complementary experimental systems for interrogating type 17 skin inflammation.

overall_design: scRNA-seq of control and conditional knockout mouse skin + IMQ or IL-17A/TNF

first: Johann

middle: E

last: Gudjonsson

first: Lam

middle: C

last: Tsoi


title: Transgenerational inheritance of altered H3K27me3 in wildtype Caenorhabditis elegans

city: Genève

first: François-Xavier

last: Stubbe

state: Genève

address: 30, quai Ernest-Ansermet

department: MOCEL

summary: Epigenetic inheritance provides mutation-free mechanisms for phenotypic adaptation, including modifications to DNA or DNA-associated proteins that create patterns of heritable gene regulation. However, the limits and regulation of this inheritance remain incompletely understood. Here, we developed a C. elegans system to study the transgenerational epigenetic inheritance of H3K27me3, a conserved histone posttranslational modification associated with gene repression. We find that induced alterations of the genome-wide H3K27me3 landscape and the associated fertility defects persist for many generations in genetically wildtype descendants under selective pressure. We uncover that the inheritance of the altered H3K27me3 landscape involves an initiation phase that relies on SET-32/H3K23me3 and a maintenance phase that requires MES-4/H3K36me3. Our results demonstrate that epigenetic inheritance can act as a mutation-independent, heritable mechanism of adaptation.

overall_design: For the RNA-seq analysis we collected germ lines from P0wt (wildtype control; n=3), subfertile individuals from P0array (n=2), subfertile individuals from F3subfertile (n=2), and fertile individuals from F3subfertile (n=2). We estimated whether individual animals were fertile or subfertile by letting them lay eggs until the second day of adulthood, and then dissected germ lines for RNA extraction.

first: Florian

last: Steiner


title: B-cell depletion improves therapeutic index of combination checkpoint blockade in patients with advanced melanoma

city: Seattle

first: Kavita

last: Dhodapkar

state: Washington

address: 1100 Fairview Ave N

institute: Fred Hutchinson Cancer Center

summary: Combined checkpoint blockade (CCB) of programmed-death-1 (PD-1) and cytotoxic-T-lymphocyte-associated protein-4 (CTLA-4) is highly active in melanoma but limited by significant morbidity from immune-related adverse events (irAEs). Effective strategies toprevent CCB-mediated irAEs are lacking. Patients with advanced melanoma were randomly assigned to receive standard of care ipilimumab and nivolumab alone (Arm-A: ipi/nivo, n=7) or with one cycle of rituximab (Arm-B; ipi/nivo+rituximab, n=7). Patients receiving ipi/nivo+rituximab experienced lower rates of >grade-3(G3) irAEs (14% versus 57%) and superior G3-irAE-free survival compared to those in ipi/nivo arm (2-year G3- irAE-free survival 86% versus 29% (p=0.01), without adverse impact on tumor regression or survival. Rituximab depleted pre-therapy activated naïve B cells linked to autoimmunity and enhanced CCB-mediated induction of myeloid inflammation and CXCL13+ICOS+ CD4 T cells. B-cell depletion favorably modulates CCB-mediated immune activation and reduces irAE risk.

overall_design: CITE-seq of PBMCs from human subjects in a clinical trial. The study includes baseline and post-tratment samples

first: Antonio

last: Matera

first: Alyssa

last: Duffy

first: Azmain

last: Taz

first: Renee Julia

last: Manalo

first: Melinda

last: Yushak

first: Ragini

last: Kuchadkar

first: David

middle: H

last: Lawson

first: Madhav

middle: V


title: Intersection of transient cell states with stable cell types in hippocampus

city: La Jolla

first: Jack

last: Olmstead

state: CA

institute: UC San Diego

summary: The transcriptome of a brain cell encodes both its stable identity and its dynamic responses to environmental stimuli. While significant progress has been made in categorizing cell types within the brain, deciphering to what extent transcriptional identity and transcriptional state are related remains a major technical and conceptual challenge. Here, we present a single-nucleus RNA-sequencing atlas of the mouse hippocampus spanning physiological and pathological stimuli and multiple circadian phases, enabling unified analysis of activity-, circadian-, and cell-type-dependent transcriptional programs. Taxonomically assigned cell types are largely stable despite the induction of different activity states, with a notable exception in the dentate gyrus. Activity and circadian rhythm each drive robust, largely nonoverlapping transcriptional responses, with convergent regulation on genes involved in specific pathways, including endocannabinoid signaling, excitability, and chromatin remodeling. These results underscore the necessity of integrating cell-type taxonomy with transcriptional state to capture how diverse cell types respond to experience.

overall_design: Wild-type C57/BL6J mice were exposed to various behavioral conditions such as enriched environment (EE), kainic-acid-induced (KA) seizures, or home cage (negative control). These exposures occurred at various time points within the 24h circadian cycle (denoted by ZT0, ZT4, ZT16, ZT20) and for durations of 30 minutes or 6 hours in the case of EE and KA exposures. Hippocampi from exposed mice were radpily dissected and flash frozen following exposures. Thawing and snRNA-seq library preparation occurred in parallel from 48 different biological replicates (i.e., mice) using the SPLiT-Seq protocol (Parse Biosciences).

first: Lauren

last: King

first: Brenda

last: Bloodgood


title: m6Am-seq profiling of macrophage-specific Pcif1 knockout mice during experimental colitis

city: BeiJing

first: Kai

last: Li

address: Yi He Yuan Street No.5

department: School of Life Science

institute: Peking University

summary: Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation accompanied by alterations in immune cell function and enteric nervous system homeostasis. To investigate the role of PCIF1-mediated N6,2′-O-dimethyladenosine (m6Am) RNA methylation in macrophages during colitis, we generated macrophage-specific Pcif1 knockout mice and performed transcriptome-wide RNA sequencing (RNA-seq) and m6Am profiling (m6Am-seq). These datasets were generated to characterize m6Am-regulated gene expression programs and identify molecular pathways associated with extracellular matrix remodeling, macrophage function, and enteric neuronal regulation during intestinal inflammation.

overall_design: Wild-type (WT) bone marrow-derived macrophages (BMDMs) were used for m6Am-seq to identify transcriptome-wide m6Am-modified genes in macrophages. RNA fragments (>200 nt) were fragmented and subjected to sequential immunoprecipitation: cap-m7G IP using an anti-m7G antibody, followed by FTO demethylase treatment (FTO+ or FTO-) and m6A IP using an anti-m6A antibody. Four libraries were generated: total RNA Input, m7G-IP, FTO(+)/m6A-IP, and FTO(-)/m6A-IP. The Input library serves as the RNA-seq reference for normalization and gene expression analysis.

first: Ye

last: Xiao


title: Tissue nanotransfection-mediated induction of neurogenic programs promotes myoprotective responses in denervated skeletal muscle

city: Columbus

first: Ana

middle: Isabel

last: Salazar Puerta

state: OH

address: 140 W 19th Ave Building 151

institute: The Ohio State University

summary: Peripheral nerve injuries often result in prolonged skeletal muscle denervation, leading to progressive atrophy, fibrosis, neuromuscular instability, and loss of regenerative capacity before axons can reinnervate distal targets. Here, we developed a non-viral strategy using tissue nanotransfection (TNT) to deliver the neurogenic transcription factor cocktail Ascl1, Brn2, and Myt1l (ABM) directly to denervated skeletal muscle. In vitro, ABM-transfected myoblasts sustained expression of the reprogramming factors, acquired neuron-like morphologies, and upregulated neuronal markers including Tuj1, Map2, and Syp. RNA sequencing confirmed broad activation of neurogenic transcriptional programs, with enrichment of pathways associated with neuronal fate commitment, neuron differentiation, axon guidance, synaptogenesis, and developmental signaling. In a mouse model of sciatic nerve resection, TNT enabled localized ABM expression in denervated gastrocnemius muscle. ABM-TNT treatment accelerated resolution of denervation-associated fibrillation potentials and showed trends toward improved twitch and tetanic torque, compound muscle action potential amplitudes, and muscle mass preservation. Transcriptomic profiling of treated muscles 5 weeks after injury revealed distinct gene expression programs enriched for muscle regeneration, neuromuscular organization, trophic support, extracellular matrix remodeling, angiogenesis, myogenesis, and metabolic adaptation. Network analyses further identified activation of neurogenic regulators, neurotrophic signaling, and vascular-support pathways. These findings establish TNT-mediated ABM delivery as a non-viral platform for inducing neurogenic and myoprotective programs in denervated muscle and suggest a strategy to preserve muscle viability during the prolonged interval required for peripheral nerve regeneration.

overall_design: RNAseq profiling of gastrocnemious muscle tissue electrotransfected with TFs Ascl1, Brn2 and Myt1l using TNT. The muscle was subjected to TNT after the transection of the nerve to separate both ends. Muscle tissue was collected after 5 weeks and processed for RNAseq.

middle: I

last: Salazar-Puerta

first: Sara

last: Kheirkhah

first: Jordan

middle: T

last: Moore

first: Carlos

last: Vasquez-Martinez

first: Carolina

last: Velasquez-Quintero

first: Hallie

last: Harris

first: Manami

last: Fukuda

first: Megumi

first: Jonathan

middle: P

last: Stranan

first: Kavya

last: Dathathreya

first: Jared

last: Albert

first: Prameela

last: Bobbili

first: Fangli

last: Zhao

first: Charles

last: Wendt

last: Winograd

first: Ian

last: Valerio

first: Candice

last: Askwith

first: Amy

first: William

last: Arnold

first: Daniel

last: Gallego-Perez


title: Temporal changes in metabolism guide oligodendrocyte precursor cell dynamics in aging and multiple sclerosis [BulkRNAseq]

city: Palo Alto

first: Erin

last: Gibson

address: 3165 Porter Drive

department: Psychiatry and Behavioral Sciences

institute: Stanford University

summary: Impaired oligodendrocyte precursor cell (OPC) differentiation limits myelin renewal in aging and contributes to multiple sclerosis (MS) progression. How aging drives OPC deficits remains incompletely understood. We find dysregulation of genes associated with the circadian clock, including Bmal1, and metabolism in aged compared with young OPCs. Targeted loss of Bmal1 in OPCs drives metabolic dysfunction, leading to cellular senescence and impaired dynamics. OPC proliferation and differentiation occur at different rates throughout the day in young adult mice and become disrupted with aging. Chronotherapeutic targeting of BMAL1-controlled sirtuin signaling restores Bmal1-disrupted OPC dynamics after demyelination via sirtuin 2 (Sirt2)-dependent mechanisms. Induced pluripotent stem cell (iPSC)-derived OPCs from MS patients and MS lesion oligodendroglia recapitulate BMAL1 and SIRT2 disruptions. These findings establish BMAL1 as a key regulator of OPC energy metabolism, sirtuin homeostasis, and senescence. We anticipate that this work will provide a foundation for future studies investigating the interconnected roles of aging, circadian disruption, and myelin biology.

overall_design: Following hiPSCs differentiation into OPCs, we purified PDGFRα+ cells, synchronized them, and collected samples 24 hours later at four circadian timepoints (CT0, 6, 12, 18) for bulk RNA sequencing.

first: Tess

last: Dierckx

first: Sarah

last: Wilson

first: Rebecca

last: Buchanan

first: Themistoklis

last: Tsarouchas

first: Yuhsiang

last: Cheng

first: Maria

last: Sacconi Nunez

first: Kelly

last: Strickland

first: Dena

last: Kumsa

first: Yohan

last: Auguste

first: Sam

last: Vanherle

first: Sander

last: Bouwman

first: Daniela

last: Rojo

first: Louisa

last: Dal Cengio

first: Celia

last: Lerma Martin

first: Yassene

last: Mohammed

first: Lucas

last: Schirmer

first: Jeroen

last: Bogie

first: Bart

last: Eggen


title: Compound heterozygous TDRD9 variants are associated with postmeiotic spermatid arrest and impaired LINE-1 suppression in a man with non-obstructive azoospermia

status: Public on Aug 13 2026

city: hangzhou

first: Huimei

last: Zheng

address: yuhangtang roda866

institute: ZheJiang Univerisity

summary: Background: Biallelic variants in TDRD9, a component of the germline piRNA pathway, have been associated with male infertility, but the stage-specific consequences of partial TDRD9 dysfunction in humans remain unclear. Methods: We studied a 22-year-old man with idiopathic non-obstructive azoospermia and his unaffected parents. Exome sequencing and segregation analysis were integrated with minigene splicing and protein-stability assays, testicular histology, acrosomal staining, meiotic chromosome spreads, exploratory bulk RNA sequencing, transposable-element profiling, and LINE-1 open reading frame 1 protein immunofluorescence. Obstructive azoospermia samples with preserved spermatogenesis served as controls where indicated. Results: The proband carried a paternal splice-acceptor variant, c.1714-1G>C, and a maternal missense variant, c.2402A>C (p.Gln801Pro), in TDRD9. The splice variant caused exon 16 skipping and was predicted to generate p.Val572ArgfsTer5; p.Gln801Pro accelerated protein decay and was associated with reduced testicular TDRD9 abundance. Histology and acrosomal staining showed round-spermatid arrest, abnormal acrosome formation, and rare late spermatids. In the spermatocytes examined, chromosome-axis formation, synapsis, and sex-body formation showed no overt abnormalities. Exploratory transcriptomic findings were concordant with depletion of late spermiogenic programs. Evolutionarily young LINE-1 features were enriched among upregulated transposable-element features, and LINE-1 open reading frame 1 protein accumulated in spermatids. Conclusions: This deeply phenotyped case supports an association between compound heterozygous TDRD9 variants, postmeiotic spermatogenic failure, and impaired LINE-1 suppression. The data are compatible with residual TDRD9 activity permitting meiotic progression in the cells examined, but larger genotype-phenotype cohorts and direct piRNA analyses are required to test this model and assess clinical utility.

overall_design: RNA-seq profiling of the seminiferous tubules from three obstructive azoospermia patients and the proband with Compound heterozygous TDRD9 variants

first: Yunbo

last: Wang

first: Cheng

last: Wu

first: Jianhua

last: Chen

first: Xiaohang

last: Yang

first: Yongmei

last: Xi

first: Jingping

last: Li


title: CCR10-Regulated Mucosal Immune Responses Support Restoration of Lung Tissue Homeostasis after SARS-CoV-2 infection.

city: San Antonio

last: Xiong

state: TX

address: 7703 Floyd Curl

department: MIMG

institute: UT Health Science Center at San Antonio

summary: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. While most patients can clear the infection and recover, some develop severe symptoms associated with dysregulated immune activation that leads to injury of lungs. Clinical studies found that severities of COVID-19 patients correlated with levels of mucosa-homing CCR10+ immunoglobulin A (IgA) antibody-secreting cells (IgA-ASCs) while recovery was linked to expansion of CCR10+ type 2 innate lymphoid cells (ILC2s). However, exact roles of CCR10-regulated mucosal immune responses in SARS-CoV-2 infection and pathology are not clear.  Here, we addressed these questions using CCR10-knockout (KO) mice infected with a mouse-adapted strain of SARS-CoV-2. SARS-CoV-2-infected CCR10-KO mice had reduced IgA-ASCs but increased ILCs and T cells in lungs compared to control wild-type (WT) mice. However, CCR10-KO mice cleared SARS-CoV-2 as efficiently as WT mice, suggesting that CCR10-regulated mucosal immune responses are not critical for viral clearance. On the other hand, CCR10-KO mice, particularly males, were impaired in recovery from SARS-CoV-2-induced lung damages due to the immune dysregulation. Intranasal immunization overcame defective mucosal immune responses and prevented SARS-CoV-2-induced lung pathology in CCR10-KO mice. Our results revealed an important role of CCR10-regulated mucosal immune responses in restoration of lung homeostasis after SARS-CoV-2 infection.

overall_design: RNA-seq profiling of lung tissues from SARS-CoV-2 MA10 Intranasally infected CCR10 heterozygous mice and CCR10-KO mice at Day 14.

first: Juan

last: Garcia

first: Austin

middle: W

last: Todd

first: Alexei

last: Tumanov

first: Yong

last: Ge

first: Yan

last: Xiang


title: Transplantation of endothelial progenitor cells attenuates pulmonary fibrosis via IFITM3-containing exosomes

city: Phoenix

first: Ying-Wei

last: Lan

address: 475 N. 5th Street

department: Child Health

institute: The University of Arizona College of Medicine-Phoenix

summary: Pulmonary endothelial progenitor cells contribute to vascular regeneration, but their molecular characteristics in the adult lung remain incompletely defined. In this study, single-cell RNA sequencing was performed on endothelial cells isolated from adult wild-type mouse lungs. KIT-positive endothelial cells were stratified into Foxf1-positive capillary endothelial progenitor cells (capEPCs) and Foxf1-negative endothelial cells. Differential gene expression analysis identified Ifitm3 as highly enriched in capEPCs, supporting a role for the FOXF1–IFITM3 pathway in endothelial regeneration and pulmonary fibrosis.

overall_design: A single-cell suspension was prepared from adult wild-type mouse lungs, and lung endothelial cells were isolated for single-cell RNA sequencing. One sequencing library, WT_Lung, was generated. Single-cell transcriptomes were analyzed using Seurat. KIT-positive endothelial cells were stratified into Foxf1-positive capEPCs and Foxf1-negative endothelial cells, followed by differential gene expression analysis


title: GAR1, a DNA Binding Gibberellin Receptor, Directly Activates Gene Expression to Regulate Rice Height and Fertility

city: China

first: Yue

last: Jin

address: 100 Guilin Road,Xuhui DistrictC

institute: Shanghai Normal University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Gibberellins (GAs) regulate multiple aspects of growth and development in flowering plants, primarily through the canonical GID1-DELLA signaling module. Contrary to the prevailing paradigm that all GA responses require GID1, we demonstrated that a rice GID1-like 2 protein (designated GA Receptor 1, GAR1) is a GA receptor that functions independently of GID1. Isothermal titration calorimetry revealed that GAR1 can bind bioactive GAs, and multiple assays confirmed that GAR1 physically interacts with the promoter of GAMYB, a central regulator of GA responses, whereas GID1 does not. Using a transgenic rice line containing the GA biosensor GIBBERELLIN PERCEPTION SENSOR 1 (GPS1), we demonstrated that the subcellular distributions of GAR1 and its homologs GAR3 and GAR4 overlapped with strong GA signals in specific cells of rice leaves, anthers, and filaments. gar1 gar2 double mutants exhibited a semi-dwarf phenotype with slightly shorter anthers, whereas gar3 gar4 mutants displayed normal plant height with elongated filaments; both double mutants showed slightly reduced male fertility. ChIP-seq analyses revealed enrichment of GAR1 at the promoters of MYBs and other GA-responsive genes associated with tiller height and anther development, and RT-qPCR confirmed that height-related genes were downregulated in gar1 gar2 double mutants. Our findings reveal a non-canonical GA signaling pathway mediated by GAR1, providing new mechanistic insights into GA perception and novel targets for biotechnological manipulation.

overall_design: Leaves and inflorescences separately from GAR1-eGFP transgenic plants were subjected to cross-linking and then used for ChIP assays.


title: Multiplexed single cell transcriptomics optimizes mesodermal patterning and hemogenic endothelial output from murine embryonic stem cells

first: Brandon

last: Hadland

state: WA

address: 1100 Fairview Ave N, Mailstop D2-100

department: Translational Science and Therapeutics

summary: Background: Early patterning of mesodermal precursor populations is a key step of hematopoietic development in the embryo. To better understand this process, we employed sci-Plex, a high-throughput method of measuring multiplexed perturbations at the single-cell level, to evaluate the transcriptional response of mouse embryonic stem cells subjected to a gradient of two key morphogens in early mesoderm/hematopoietic development, Activin and BMP4. Results: sci-Plex revealed varying combinations of Activin and BMP4 temporally influenced mesoderm patterning in vitro and subsequent production of cell types reflecting their in vivo counterparts. We leveraged sci-Plex data to further optimize the generation of intraembryonic-like hemogenic endothelial cells that serve as the precursors of definitive hematopoietic lineages, including hematopoietic stem cells. Conclusions: This study highlights the utility of sci-Plex to dissect how dose and temporal integration of interacting signal pathways determines cell fates and serves as a resource to analyze cell fate choices in early mesoderm patterning at single cell resolution.

overall_design: Multiplexed single cell RNA-seq libraries were generated for mouse embryos aged E6.5-7 or E8.5-9, and mouse embryonic stem cell (mESC)-derived embryoid bodies treated with a combinatorial gradient of Activin and bone morphogenic protein 4 (BMP4) harvested on days 4, 5 and 6 of differentiation. These single cell RNA-seq libraries were generated using sci-Plex, a multiplexing methodology previously described in Srivatsan et al. 2020 Science 367(6473) p.45-51.

first: Barbara

last: Varnum-Finney

first: Adam

last: Heck

first: Sanjay

middle: R

last: Srivatsan

first: Stacey

last: Dozono

first: Rachel

last: Wellington

first: Alisa

middle: B

last: Chankhunthod

first: Cynthia

last: Nourigat-McKay

first: Tessa

last: Dignum

first: Cole

last: Trapnell


title: D3 dopamine receptors implicate a striatal neuronal subtype in the aversive effects of quetiapine

city: Princeton

first: Jessie

last: Muir

state: NJ

address: 40 Woodlands Wy

department: Princeton Neuroscience Institute

institute: Princeton

summary: Second generation antipsychotics (SGAs) are widely used clinical tools; yet, they often cause negative side effects and take weeks to become effective, leading to poor patient compliance. The effect/side effect profile of individual SGAs is highly variable, and the mechanisms that underlie this variability are not well understood. Here, we identify a role of type 3 dopamine receptor (D3R) neurons in the Nucleus Accumbens (NAc) that mimics the aversive effects of quetiapine. Using single-nucleus RNA sequencing, we show that D3R is expressed in a subpopulation of D1R neurons and defines a distinct NAc cell type. We found that both clozapine and quetiapine cause acute conditioned place aversion in mice, but aversion subsides only after 21 days of treatment with quetiapine, a drug characterized as an arrestin-biased agonist at D3R. We provide evidence at both the cellular and population level that quetiapine inhibits D3R-expressing neurons in the lateral shell (LatSh) of the NAc. We also demonstrate that local injection of quetiapine into LatSh NAc is sufficient to cause place aversion. Selective optogenetic inhibition of D3R-neurons in the LatSh produces real time place aversion in mice, correlating this cell type to the aversive effects of quetiapine. These findings suggest a cell-type-specific mechanism underlying quetiapine-induced aversion and its potential attenuation with chronic treatment, offering insight into the cell types and circuitry that shape the quetiapine side effect profile.

overall_design: single nucleus RNA seq profiling of Nuceus Accumbens (NAc) of D3-cre::Ai14 mice

first: Elinor

last: Lewis

first: Jennifer

middle: L

last: Whistler

first: Christina

middle: K

last: Kim


title: Ultrasound-Triggered piRNA Delivery Using Biomimetic Phase-Changeable Nanoparticles for Theranostics of Myocardial Infarction

city: Qingdao

first: yajun

last: zhang

address: No. 16 Jiangsu Road, Shinan District

department: Department of Cardiac Ultrasound

institute: The Affiliated Hospital of Qingdao University

summary: Myocardial infarction (MI) remains a leading cause of acute cardiovascular morbidity and is among the most difficult to diagnose. Early treatment options fail to delay necroptosis of cardiomyocytes, restrict the fibrosis of the heart, and prevent the adverse remodeling of the myocardium.

overall_design: Here, we present the first evidence that piR-1873 is significantly down-regulated in MI patients, mouse models, and H2O2-treated cardiomyocytes. Serum piR-1873 levels inversely correlated with established MI biomarkers, and ROC analysis revealed good diagnostic accuracy (AUC=0.8389). Mechanistically, piR-1873 protected cardiomyocytes by directly targeting Usp2 and suppressing the RIP1/RIP3/MLKL-mediated necroptosis pathway. To enable efficient in vivo delivery, we engineered ultrasound (US)-responsive piRNA nanoparticles (piR-NPs) consisting of a phase-change perfluorohexane (PFH) core loaded with piR-1873 and encapsulated within a cardiomyocyte membrane shell. In vitro, piR-NPs were efficiently taken up by H2O2-treated cardiomyocytes, restored piR-1873 levels, and inhibited necroptosis, with US exposure further enhancing these effects. In MI mice, piR-NPs preferentially accumulated in the infarcted myocardium, and US prolonged their retention, leading to improved cardiac structure and function, reduced fibrosis, and decreased necroptosis, without detectable systemic toxicity. Collectively, this study demonstrates the diagnostic and cardioprotective potential of piR-1873 and introduces a biomimetic, US-triggered piRNA delivery platform with high translational promise for MI therapy.

first: Yajun

last: Zhang


title: Methionine-Driven N6-Methyladenosine Modification of PPARG in Macrophages Promotes Immunosuppression in Glioblastoma

city: Harbin

first: Hong

last: Hu

state: 黑龙江

address: youzheng street

institute: Harbin medical university

summary: Glioblastoma (GBM) is characterized by a methionine-enriched metabolic microenvironment and extensive infiltration of immunosuppressive macrophages. Although tumor metabolism is closely associated with immune suppression, the mechanisms by which elevated methionine availability affects macrophage function remain incompletely understood. In this study, we investigated whether a high-methionine environment alters N6-methyladenosine (m6A) RNA modification in macrophages and thereby regulates macrophage anti-tumor immunity. THP-1 cells were differentiated into macrophage-like cells using phorbol 12-myristate 13-acetate (PMA) and subsequently cultured under normal methionine conditions or treated with 0.8 mM methionine for 48 h. Arraystar Human m6A-mRNA&lncRNA Epitranscriptomic Microarray analysis was performed to characterize transcriptome-wide changes in m6A modification and to identify candidate regulatory factors associated with high-methionine-induced alterations in macrophage anti-tumor immune function. These data were generated as part of a study investigating the methionine/m6A regulatory axis in macrophage-mediated immunosuppression in glioblastoma.

overall_design: THP-1 cells were differentiated into macrophage-like cells by PMA treatment and divided into two experimental groups. Four biological replicates were cultured under normal methionine conditions as controls, and four biological replicates were cultured in medium containing 0.8 mM methionine for 48 h. Total RNA was collected from the eight samples and subjected to m6A immunoprecipitation. The immunoprecipitated m6A-enriched RNA fraction (IP) and the corresponding supernatant fraction (Sup) were analyzed using the Arraystar Human m6A-mRNA&lncRNA Epitranscriptomic Microarray. IP and Sup RNAs were labeled with Cy5 and Cy3, respectively, and hybridized on two-color arrays. The experiment was designed to identify changes in m6A RNA methylation associated with high-methionine exposure in PMA-differentiated THP-1 macrophage-like cells.

first: Yishuai

last: Huang

first: Jiafeng

last: Li

first: Honglei

first: Sha

last: Luan

first: Jiaqi

last: Liu

first: Ruijie

first: Meng

last: Cai

first: Hao

last: Chen

first: Ning

last: Wang

first: Weichen

first: Cong

first: Zhenfeng

last: Jiang

first: Kaikai

first: Lei

last: Teng

first: Huailei


title: Gene expression + ATAC profiling of trisomic hippocampal neurons upon SAHA treatment [RNA-seq]

city: Lausanne

first: Cesar

last: Sierra

address: SV 2513 (Bâtiment SV)

department: SV

institute: EPFL

summary: Down syndrome (DS) is associated with widespread molecular alterations, including aberrant gene expression, which not only affects chromosome 21 genes but perturbs the whole transcriptome. The genome-wide nature of this alteration suggests that it might be epigenetically mediated, which may also offer therapeutic opportunities given the reversibility of epigenetic modifications. Here, we show that decreased global acetylation level in the hippocampus of the trisomic mouse model Ts65Dn is associated with decreased chromatin accessibility at gene promoters in hippocampal neurons. Strikingly, pharmacological restoration of histone acetylation using the clinically approved histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) normalized transcriptomic, epigenetic and cortico-hippocampal memory deficits in trisomic mice. This rescue was mediated by an unexpected heterochromatization of the supernumerary chromosome. Altogether, our results identify an unanticipated epigenetic mechanism linking histone acetylation dynamics to chromosomal dosage homeostasis.

overall_design: Hippocampal neuronal nuclei from 4 month old WT, Ts65Dn andTs65Dn+HDACi mice were isolated by Fluorescence-activated nuclear sorting (FANS). 50000 nuclei were processed for ATAC-seq and 200000 for RNA-seq.

first: René

last: Crans

first: Mara

last: Dierssen


title: Gene expression + ATAC profiling of trisomic hippocampal neurons upon SAHA treatment [ATAC-seq]


title: Immunological Differences in Atopic Dermatitis Across Age Groups: Insights from Single-Cell Multi-Omics

city: San Francisco

first: Gian Carlo

middle: L.

last: Baldonado

address: 2340 Sutter Street

department: Dermatology

institute: University of California San Francisco

summary: Background: Atopic dermatitis (AD) occurs across all ages but presents distinct clinical and immunologic features between children, adults, and older adults. The molecular programs underlying these age-specific immune differences remain poorly understood.  Methods: We performed single-cell multi-omics profiling of peripheral blood mononuclear cells (PBMCs) from 29 AD patients and 29 matched healthy controls (HC), spanning pediatric (0–17 years), adult (18–59 years), and geriatric (≥60 years) groups. Using Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq), we simultaneously quantified transcriptomic (RNA) and surface proteomic (ADT) profiles across ~280,000 immune cells. Integrated analyses identified 30 immune subsets for cell-type proportion and differential expression analyses. Machine-learning classifiers were trained on significant gene and protein features to distinguish AD subgroups by age.  Results: Compared with HC, AD blood showed enrichment of CD14+ monocytes, plasmacytoid dendritic cells, and CD4+ proliferating T cells, and differential gene expression analysis of AD vs HC revealed downstream Th2-associated signatures shared across all age groups. Within AD, pediatric patients had increased γδ T cells, naïve CD4+, and naïve CD8+ T cells, while geriatric patients exhibited more CD4+ cytotoxic and CD8+ central memory T cells, indicating a shift from naive to effector predominance with aging. Transcriptomic and proteomic analyses revealed distinct programs: pediatric AD was enriched for IL-10 and cytokine–cytokine receptor signaling; adult AD demonstrated activation of metabolic and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)/Th1/Th17 pathways; and geriatric AD exhibited reduced adaptive immune activity but increased innate signaling. Machine-learning models based on differentially expressed genes and proteins accurately classified AD age groups (transcript-based F1 = 0.70, AUC = 0.79), identifying stable markers such as *IRF2*, *PDK4*, *ZFP90*, CD21, CD94, and CD122.  Conclusions: Single-cell multi-omics profiling revealed immune differences across the AD lifespan, transitioning from developmental tolerance in children to inflammatory and metabolic activation in adults to enhanced innate signaling in geriatric individuals. These findings highlight molecular signatures that could support age-group subtyping and therapeutic strategies for AD across the lifespan.

overall_design: We have collected PBMCs from atopic dermatitis patients across all age groups (pediatric, adult, and geriatric) with age-matched healthy controls. Peripheral blood was collected in Acid Citrate Dextrose (ACD) Vacutainer tubes and processed within 4 hours of phlebotomy. PBMCs were isolated by Ficoll gradient centrifugation, washed, and cryopreserved in 90% fetal bovine serum (FBS) + 10% dimethyl sulfoxide (DMSO) until use. For CITE-seq, 1×10⁶ cryopreserved PBMCs per subject were thawed, counted on a Vi-CELL XR (Beckman Coulter), and pooled in batches of ten subjects balanced by condition and age group. Following Fc blocking (Human TruStain FcX, BioLegend), cells were stained with a 140-plex TotalSeq-B antibody panel (Supplementary Table 2, Additional File 2) for 30 min at 4°C, washed three times with Cell Staining Buffer, and filtered through 40 µm strainers. Cell viability exceeded 85% in all preparations. Libraries were sequenced on an Illumina NovaSeq 6000 (S4 flow cell) with a target depth of ~20,000–40,000 reads/cell (RNA) and 5,000–10,000 reads/cell (ADT). Subjects were subsequently demultiplexed using Demuxlet (Popscle suite), leveraging imputed SNP genotypes to assign each droplet to its donor and remove inter-individual doublets. Remaining intra-sample doublets were removed using DoubletFinder (v2.0).

first: Baldonado

last: Gian Carlo L.

first: Kumar

last: Sugandh

first: Jin

last: Joy

first: Fang

last: Xiaohui

first: Ildardashty

last: Alexander

first: Braun

last: Mitchell

first: Neuhaus

last: Isaac M.

first: Mathes

last: Erin

first: Bhutani

last: Tina

first: Liao

last: Wilson


title: Single-cell sequencing of E10.5 mouse cardiac outflow tract

city: Nanjing

first: haiyue

last: Feng

state: Jiangsu

address: No.22 Hankou Road,Nanjing City

institute: Nanjing University

summary: E10.5 mouse cardiac outflow tract cells were isolated and subjected to single cell sequencing.  We used this approach to characterize the transcriptomic profile of the  intercalated valve swelling located in the inferior midline OFT (imICVS).

overall_design: The cardiac outflow tract was dissected from WT mice at E10.5 to identify imICVS cells and characterize their transcriptomic profiles.

first: Haiyue

first: Yuke

last: Ji

first: Zixuan

last: Li

first: Jie

last: Yao

first: Jing

last: Cui

first: Lijun

last: Zhang

first: Xiaodong

last: Wang

first: Wenli

last: Fan

first: Xianfa

last: Yang

first: Naihe

last: Jing

first: Zhongzhou


title: ppGpp regulates transcription elongation via direct and indirect inputs to RNA polymerase

city: Madison

first: Michael

last: Wolfe

state: WI

address: 1300 University Ave

institute: University of Wisconsin-Madison

summary: The signaling molecules guanosine 5′-tri/diphosphate 3′-diphosphate, (p)ppGpp, control bacterial protein synthesis rates and cell growth by targeting transcription, translation, NTP synthesis, and other functions. In lineages like E. coli, (p)ppGpp produced in response to charged-tRNA deficiency targets transcribing RNAP polymerase (RNAP) to match its pace to the pioneering ribosome on the nascent RNA (transcription–translation coupling). However, the mechanism by which (p)ppGpp slows RNAP is poorly defined. (p)ppGpp may stimulate RNAP pausing, inhibit catalysis, or promote backtracking allosterically, may inhibit GTP synthesis, may compete for GTP binding, or may uncouple transcription–translation by inhibiting ribosomes. Using a combination of cryo-EM, biochemical assays, quantitative nascent elongating transcript sequencing (qNET-seq), and quantitative ChIP-seq (qChIP-seq), we establish that (p)ppGpp allosterically regulates pausing and nucleotide addition via distinct motions of the RNAP swivel module and both competes with and lowers GTP. (p)ppGpp stimulates swiveling at pause sites to delay escape but may also inhibit counter-swiveling required in every round of nucleotide addition.

overall_design:
ChIP-seq of RNAP in E coli cells in the presence and absence of increased ppGpp. ppGpp is increased through overexpression of RelA* and compared to overexpression of a truncated inactive RelA. Comparisons were made in strains with WT RNAP and strains with RNAP site1 deleted. All samples have a corresponding input sample.
NET-seq of RNAP in E coli cells in the presence and absence of increased ppGpp. ppGpp is increased through overexpression of RelA* and compared to overexpression of a truncated inactive RelA. Comparisons were made in strains with WT RNAP and strains with RNAP site1 deleted.

first: Yu

last: Bao

last: Engstrom

first: Rachel

middle: A

last: Mooney

first: Robert

last: Landick


title: CAR-NKT cells induce low cytokine release syndrome by targeting hyperinflammatory macrophages with mitigation from GM-SCF inhibition

city: Los Angeles

first: Yichen

last: Zhu

address: 610 Charles E Young Drive S

institute: University of California, Los Angeles

summary: Chimeric antigen receptor (CAR)-T cell therapy has demonstrated remarkable efficacy in cancer treatment but is frequently limited by severe toxicities, including cytokine release syndrome (CRS), which is largely driven by host myeloid cell activation. CAR-engineered invariant natural killer T (CAR-NKT) cells have emerged as a promising alternative with comparable antitumor activity and reduced toxicity; however, the mechanisms underlying their improved safety profile remain poorly defined. Here, we systematically evaluate CRS induced by human CAR-NKT and CAR-T cells using a heavily tumor-bearing xenograft mouse model that recapitulates clinically relevant CRS. Compared with conventional CAR-T cells, CAR-NKT cells induce significantly lower CRS severity across multiple clinical and biochemical parameters. To elucidate the underlying mechanisms, we perform comprehensive single-cell transcriptomic profiling of mouse immune cells, including myeloid and granulocytic populations, across six anatomical compartments: tumor, blood, liver, lung, spleen, and bone marrow. This integrative analysis reveals dynamic immune remodeling during tumor progression, therapeutic intervention, and CRS development. Mechanistically, CAR-NKT cells preferentially target CD1d⁺ hyperinflammatory macrophages, a key driver of CRS, thereby limiting myeloid-mediated inflammatory responses. Furthermore, pharmacological inhibition of GM-CSF further attenuate CRS, supporting a combinatorial strategy to enhance the safety of CAR-NKT cell therapy. Collectively, these findings provide mechanistic insight into the reduced toxicity of CAR-NKT cells and support their development as a safer and effective platform for cancer immunotherapy.

overall_design: Gene expression profiling of therapeutic cells extracted from in vivo CRS models, including CD19 targeting CAR-T, and CAR-NKT cells

first: Annabel

last: Zhao

first: Yan-Ruide

first: Lili


title: H3K27M co-opts ASCL1 to maintain progenitor states and drive gliomagenesis

city: Oxford

first: Alan

last: Jiao

state: MA

address: Old Road Campus Research Building

institute: Ludwig Institute for Cancer Research, Oxford University

summary: Phenotypic plasticity, a hallmark of cancer facilitated by epigenomic reprogramming, represents a significant challenge but also therapeutic opportunity for cancer treatment. How plasticity is regulated in diffuse midline gliomas (DMGs), incurable pediatric brain tumors characterized by oncogenic H3K27M histone mutations, is not understood. Here we demonstrate that H3K27M-driven plasticity requires the lineage-specific, pioneer transcription factor (TF) ASCL1. Prolonged differentiation reveals clinically relevant plasticity in DMGs, which segregate into ASCL1-high, neural progenitor cell (NPC)-like gliomaspheres and ASCL1-low, mesenchymal (MES)-like adherent subpopulations. Direct transcriptional activation by ASCL1 enables super enhancers to sustain NPC programs de-repressed by H3K27M. H3K27M loss induces an irreversible MES-like state, suppresses plasticity by abolishing the ASCL1-high state, and is largely phenocopied by loss of ASCL1. Knocking out ASCL1 in DMG lines robustly inhibited tumor growth and significantly extended survival of recipient mice. Our results suggest targeting epigenetic plasticity to suppress master TF-driven progenitor states as a general therapeutic opportunity in cancer.

overall_design: To investigate how ASCL1 regulates transcriptional heterogeneity in diffuse midline gliomas, we knocked out ASCL1 in the BT245 cell line. Control and ASCL1-KO cells were treated with differentiation medium containing 10% FBS for 6 days. Nuclei were isolated and joint snRNA- and scATAC libraries were prepared using the 10X Genomics platform.

first: Yang

last: Shi


title: Mitochondrial tRNA-derived fragments link mtDNA variation to metastatic burden [lung]

city: Kansas City

first: Emily

last: Schueddig

state: KS

address: 2901 Rainbow Boulevard

department: Department of Biostatistics & Data Science

institute: University of Kansas Medical Center

type: Non-coding RNA profiling by high throughput sequencing

summary: How mitochondrial DNA (mtDNA) polymorphisms influence complex phenotypes remains poorly understood. Using Mitochondrial-Nuclear eXchange (MNX) mice, we previously showed that mtDNA single nucleotide polymorphisms (SNP) modify metastasis, cardiovascular disease, and epigenetic marks independently of metabolic differences, suggesting a role for non-protein-coding loci. The only SNP correlating with these phenotypes resides in the gene encoding mitochondrial tRNA-Arginine (mt-tRNAArg (UCG), mt-TR). Here we identify and preliminarily characterize previously undescribed tRNA-derived fragments (tRF) generated from mt-TR. Northern blotting revealed distinct tRF that are differentially expressed between lung and liver and between sexes. Small RNA sequencing failed to capture these tRF unless samples were pre-treated to enzymatically remove non-canonical RNA termini and base methylation. Focusing on fragments with complete homology to mt-TR, the two most abundant tRF align uniquely to the mitochondrial genome and share conserved cleavage sites. Together, these findings uncover mitochondrial-derived tRF as a previously unrecognized small RNA metastasis modifiers and expand the functional output of the mitochondrial genome, supporting a model in which mtDNA-encoded tRF contribute to phenotype modification.

overall_design: Small RNA sequencing (RealSeq, Santa Cruz, CA) of liver tissue from wild-type mice (CC, HH, FF) and MNX mice (HC, CH, and CF) (C=C57BL/6J, H=C3H/HeN, F=FVB/NJ).

first: Katy

last: Swancutt

first: R

middle: M

last: Walsh

first: Sydney

last: Quigano

first: Devin

last: Koestler

first: Adam

last: Scheid

first: Tony

last: Bush

first: Yi

first: Isidore

last: Rigoutsos

first: Danny

last: Welch


title: A novel sorting method uncovers metabolic heterogeneity between mononucleated and binucleated tetraploid hepatocytes

city: Hongo

first: Yusuke

last: Watanabe

state: Bunkyo, Tokyo

department: Bioengineering

country: Japan

institute: University of Tokyo

summary: Hepatocytes display notable ploidy diversity, varying both in the number of genomic copies and in the number of nuclei. In adult mice, more than 50% of hepatocytes are tetraploid (4n), which can exist as either mononucleated (1x4n) or binucleated (2x2n) cells. Despite this distinction, these two cell types have traditionally been grouped and studied as a single population. One likely reason for this is that conventional ploidy-sorting methods classify hepatocytes based solely on total DNA content, without distinguishing between mononucleated and binucleated states. Consequently, it remains unclear whether 1x4n and 2x2n hepatocytes are functionally equivalent. In this study, we developed a novel FACS strategy to distinguish and isolate 1x4n and 2x2n hepatocytes. Our approach leverages Hoechst-area to assess total ploidy and Hoechst-height to differentiate mononucleated and binucleated hepatocytes. Transcriptome analysis comparing these two populations revealed that 1x4n hepatocytes exhibit a broader and more metabolically active gene expression profile. Importantly, these metabolic gene expression programs were independent of liver zonation, a well-known driver of metabolic heterogeneity in hepatocytes. Our findings uncover a previously underappreciated layer of functional diversity in the liver and provide a new framework for studying the physiological and pathological roles of nuclear configuration in hepatocytes.

overall_design: RNA-seq profile of WT adult mouce hepatocytes sorted by flow cytometry based on Hoechst fluorescence.

first: Jonathan

last: Sussman

first: Tomoya

last: Anami

first: Moena

last: Hattori

first: Saaya

last: Yamane

first: Masaki

last: Nishikawa

first: Yasuyuki

last: Sakai

first: Takeshi

last: Katsuda


title: Mitochondrial tRNA-derived fragments link mtDNA variation to metastatic burden

first: Emily

last: Schueddig

overall_design: Small RNA sequencing of liver tissue from wild-type mice (CC, HH, FF) and MNX mice (HC, CH, and CF) (C=C57BL/6J, H=C3H/HeN, F=FVB/NJ).

first: Katy

middle: D

last: Jing


title: A single-cycle, recombinant VSV platform Nipah vaccine cross-protects against Hendra virus in nonhuman primates

city: Galveston

first: Jacquelyn

last: Turcinovic

address: Galveston National Laboratory, 301 University Blvd.

department: Microbiology and Immunology

institute: University of Texas Medical Branch

summary: Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic paramyxoviruses that produce severe, often fatal disease in humans and animals. Zoonotic spillover of these henipaviruses from the Pteropus bat natural reservoir occurs near annually in densely populated regions of the world, particularly for NiV. Outbreaks of NiV frequently exceed case fatality rates of 75%, and NiV can be transmitted person-to-person. HeV is less transmissible between humans; however, infection is nearly always fatal. Approaches to human vaccine development have largely focused on NiV given the larger case burden, and immunogen selection has centered on display of the NiV attachment (G) or fusion (F) surface glycoproteins. However, experimental vaccines displaying these NiV antigens have failed to uniformly cross-protect against HeV disease in preclinical models. The HeV (G) antigen was shown to cross-protect against both HeV and NiV when delivered in a protein subunit form; however, attempts to utilize mRNA or canarypox vectors failed to achieve equivalent protection. We previously developed and evaluated two experimental vaccines to NiV and HeV using a single-cycle, recombinant vesicular stomatitis virus platform (G*rVSV∆G) displaying either the (G) glycoprotein of HeV (G*rVSV∆G-HeV-G) or NiV (G*rVSV∆G-NiVB-G). The G*rVSV∆G-NiVB-G vaccine demonstrated ideal characteristics of rapid and durable protection in nonhuman primates, and the HeV-G vaccine was capable of cross protecting against both NiV and HeV in a lethal rodent model. In the present work, we show that the G*rVSV∆G-NiVB-G vaccine cross-protects against lethal HeV challenge, with the protective response driven by a balance of both cell-mediated and humoral compartments.

overall_design: Time-series of circulating blood in control or vaccinated NHPs (rVSV-NiV) after challenge with HeV

first: Declan

last: Pigeaud

first: Robert

last: Cross

first: Thomas

last: Geisbert


title: Viral entry defines the hepatitis E virus species barrier in murine hepatocytes

city: Bochum

first: Leyla

last: Sirkinti

address: Universitätsstraße 150

department: Translational and Computational Infection Research

country: Germany

institute: Ruhr University

summary: The Paslahepevirus balayani hepatitis E virus (HEV) and the distantly related Rocahepevirus ratti rat HEV pose a risk for zoonotic transmission to humans. However, the molecular determinants for HEV transmission between species remain unknown. Despite the broad host range including its ability to infect certain rodent species, infections of animals of the genus Mus within the subfamily Murinae are rarely documented. To dissect the molecular mechanisms underlying species barriers to HEV infection, this study aimed to investigate the virus replication cycle and immune-related determinants responsible for restricted HEV infection in murine hepatocytes. Murine hepatic cell lines supported moderate levels of zoonotic HEV replication and infectious virion production upon transfection of in vitro transcribed viral RNA. Notably, viral replication was not restricted by innate immune responses or presence of dominant restriction factors but was limited by absence of host-specific dependency factors. While successful attachment to murine hepatic cells was detected, mechanisms of viral entry differed between human and murine hepatocytes, correlating with murine cell lines and primary murine hepatocytes being refractory to HEV infection. In summary, the murine barrier to HEV infection is defined at the viral entry stage, specifically by a block post attachment and before viral replication is initiated. These findings shed new light on the fundamental role of viral entry mechanisms in defining HEV species tropism.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Primary Maus hepatocytes were analyzed for basal gene expression or differentially expressed genes upon inoculation with cell-culture derived HEV-3.  Murine and human hepatic cell lines were analyzed for basal gene expression levels as well as differentially expressed genes upon transfection of full-length in vitro transcribed HEV-3 RNA and interferon alpha treatment.

first: Nicola

last: Frericks

first: Olinda Pinto

last: Veiga

first: Tina

last: Sliwinski

first: Hongbo

last: Guo

first: Yannick

last: Brüggemann

last: Burkard

first: Rainer G

last: Ulrich

first: Richard J.P.

last: Brown

first: Tran

last: Tuoc

first: Huu Phuc

last: Nguyen

first: Wenshi

last: Wang

first: Daniel

last: Todt

first: Volker

last: Kinast

first: Eike

last: Steinmann


title: Spatial biology reveals macrophage dysfunction in immunosuppressed non-melanoma skin cancer [scTCR-Seq]

city: Houston

first: Moran

last: Amit

address: 1515 Holcombe Blvd

department: Head & Neck Surgery

institute: University of Texas - MD Anderson Cancer Center

summary: The rising incidence of non-melanoma skin cancer (NMSC), including basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC), particularly among immunosuppressed individuals, underscores the need for a deeper understanding of the tumor microenvironment (TME) to develop effective and safe immunotherapies. This study employed single-cell multi-omic analyses to investigate the spatial distribution and transcriptional profiles of immune cells in NMSC from both immunocompetent (IC) and immunosuppressed (IS) patients. Our results challenge the prevailing notion that immunosuppression is primarily driven by reduced immune cell abundance or lymphocytic dysfunction. We found that IS tumors retain all requisite immune cell populations but display functional suppression and disrupted spatial organization. Our data revealed a limited number of distinct programs, particularly in macrophages and their spatially organized niches, that are linked to restricted T-cell repertoire diversity and impaired clonal expansion, providing a mechanistic bridge between innate dysfunction and adaptive immune failure. These perturbations were further confirmed by a novel spatial epigenomic atlas of the NMSC TME that revealed immune status–specific regulatory programs, including MAF- and ATF3-driven macrophages and CUX1- enriched fibroblast niches, that shape the epigenetic landscape of immunosuppressed tumors. These findings highlight the central role of innate immunity in shaping anti-tumor responses and offer potential therapeutic targets to enhance immunotherapy in this vulnerable patient population.

overall_design: 34 samples of tumor tissue (21 immunosuppressed and 13 immunocompetent) obtained from 29 untreated patients with basal cell carcinom or cutaneous squamous cell carcinoma. FACS was used to enrich for CD45+ cells and then single cell TCR sequencing was performed.

first: Shorook

last: Naara

first: Veena

last: Kochat

first: Xiayu

last: Rao

first: Emre

last: Arslan

last: Saddawi-Konefka

first: Suresh

last: Satpai

first: Jennifer

last: Garbarino

last: Anderson

first: Frederico

middle: O

last: Gleber-Netto

first: Priyadharsini

last: Nagarajan

first: Travis

last: Kerr

first: Shamima

last: Akhter

first: Sophie

last: Li

first: Ray

last: Fodor

first: Shlomo

last: Koyfman

first: Dan

last: Yaniv

first: Tongxin

last: Xie

first: Mica

last: Glaun

first: Michael

last: Bobian

first: William

last: Pardon

first: Colin

last: Ng

first: Preethi

last: Gunaratne

first: Shiyanth

last: Thevasagayampillai

last: Migden

first: Hussein

last: Abbas

first: Patrick

last: Reville

first: Keneth

middle: Y

last: Tsai

last: McGrail

first: Jing

first: Jeffrey

middle: N

last: Myers

first: Neil

last: Gross

first: Kunal

last: Rai


title: Olfactomedin 4 marks luminal progenitor cells that give rise to a secretory cell lineage in the mouse cervix

city: Dallas

first: Mala

last: Mahendroo

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

summary: Development of the female reproductive tract in mice occurs in early postnatal life. The current model identifies Trp63 as the master regulator that initiates differentiation of simple columnar Keratin 8+ epithelium in the cervix and vagina into a stratified squamous epithelium. Thereafter Trp63+ basal progenitors maintain cervicovaginal epithelial cell homeostasis and in the adult serve as the progenitor for hormone-regulated shifts in stratified squamous and secretory luminal cells. This model differs from the human in which two progenitors, one columnar and the other basal gives rise to secretory cells in the endocervix and stratified squamous epithelia in the ectocervix and vagina respectively. In the current study, we identify a population of Krt8+, Tp63- epithelial cells that are retained in the cervicovaginal epithelium during the postnatal developmental period and into adulthood. Single cell datasets from the cervices of adult mice, identify Olfactomedin 4 (Olfm4), as a unique marker of the Krt8+Trp63- population. Adult lineage tracing and reassessment of gene markers during postnatal development support a revised model in which two progenitors are delineated in the mouse cervix and vagina by PND15. Olfactomedin 4+ progenitors give rise to specialized secretory goblet cells, while Trp63+ basal progenitors give rise to stratified squamous luminal cells in the cervix and vagina of nonpregnant and pregnant mice. Consistent with the expansion of goblet cells in pregnancy, the Olfm4+ progenitor is highly proliferative in early pregnancy and progesterone regulates increased goblet cell differentiation.  These findings reveal a previously unrecognized species similarity between mice and humans in which goblet cell and squamous keratinized cell subtypes are derived from two progenitor populations respectively.

overall_design: Transcriptome profiles were obtained from two biological groups consisting of ovariectomized (OVX) control mice and OVX mice treated with progesterone, with n = 9 mice pooled per group. Libraries were prepared using the 10x Genomics Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (Dual Index) on the 10x Genomics Chromium Controller and sequenced on an Illumina NextSeq 2000 (P2 flow cell) using the 28‑bp + 90‑bp + 10‑bp + 10‑bp configuration, producing a 90‑bp single‑end cDNA read for transcript quantification. All libraries were constructed following high‑level guidance from the 10x Genomics protocol CG000389, rev C.

first: ShanmugaPriyaa

last: Madhukaran

first: Govindkumar

last: Balagannavar

first: Lei

last: Wang

first: Gary

last: Hon


title: Identifying mediators of cytosolic myddosome assembly

city: Boston

first: Daniel

last: Fisch

address: 1 Blackfan Circle

institute: Boston Children's Hospital

summary: Toll-like receptor (TLR) activation induces the formation of highly dynamic myddosomes in the cytosol of macrophages. However, not all cells respond, macrophage populations consistently contain bystanders whose frequency tracks with ligand concentration and activated TLR. We established a flow cytometry–based assay to separate responder and bystander cells via SSC changes (SSClo vs SSChi) reflecting cytosolic myddosomes in immortalized bone marrow-derived macrophages (iBMDM) with endogenously-tagged MyD88  (MyD88-AGF). We performed bulk RNA-Seq to compare responder and bystander cells to untreated cells and LPS-treated but unsorted pools. Clustering and individual comparisons confirmed high similarity between responders and the LPS pool and a distinct bystander signature. Comparing bystanders to responders and untreated to LPS-treated pools showed reduced induction of TLR primary and secondary response genes (PRGs/SRGs) in bystanders, while interferon-stimulated genes (ISGs) induced via paracrine signaling remained intact. Thus, myddosome-containing cells drive the LPS-induced transcriptional program, whereas bystanders only retain IFN responsiveness.

overall_design: RNA-Seq profiling of LPS-treated iBMDMs treated with 10 ng/mL LPS for 6 hours with subsequent sorting into responder and bystander populations.

first: Jonathan

middle: C

last: Kagan


title: Characterization of TLR signaling in Ticam2-/- macrophages

summary: Early Toll-like receptor (TLR) signaling termination in TRAM (Ticam2) KO macrophages selectively impairs activation of secondary response genes (SRG) and interferon-stimulated genes (ISGs), whereas primary response gene (PRG) induction remains largely intact. We performed bulk RNA-Seq on LTA- or LPS-stimulated primary WT and Ticam2-/- BMDMs after 2 h and 6 h of PAMP-treatment to analyze this phenomenom.

overall_design: RNA-Seq profiling of LPS- and LTA-treated primary BMDMs treated with 10 ng/mL LPS or 100 ng/mL LTA for 2 or 6 hours. Each replicate represents cells from one animal.


title: CAD-C: An engineered nuclease enables repair-free in situ proximity ligation and nucleosome-resolution chromosome walks in human cells II

city: New York

first: Viviana

last: Risca

state: NY

address: 1230 York Ave

institute: The Rockefeller University

summary: Chromosome conformation capture (3C)-derived methods have become an indispensable tool in the study of gene regulation. The three-dimensional contacts can probe depend strongly on the properties of the enzyme used to fragment chromatin prior to proximity-driven ligation. Micrococcal nuclease (MNase), used in Micro-C, increases resolution at the expense of low ligation efficiency and the need for extensive enzyme titration. To overcome these limitations, we engineered a highly active, TEV protease-activatable caspase-activated DNase (CAD) to enable an efficient, low-sequence-bias, and high-resolution proximity ligation assay we call CAD-C. CAD-C was successful on the first attempt for each human cell line tested and the resulting datasets capture loops, TADs, compartments, and stripes similarly to Micro-C. However, compared to Micro-C and Hi-C, CAD-C shows enhanced sensitivity for promoter-enhancer loops. Leveraging the ligation-competent DNA ends produced by CAD cleavage, we show that CAD-C is compatible with a highly streamlined, repair-free protocol and produces multi-step CADwalks, consecutive ligations between nucleosomal or sub-nucleosomal fragments. With these walks, we probe local chromatin fiber folding contacts, nucleosomal and sub-nucleosomal footprints, and long-range nuclear organization regimes in human cell lines. CAD-C is an efficient, robust chromatin structure assay that can span sub-nucleosomal to chromosomal length scales in a single experiment.

overall_design: CADwalk to wild type GM12878 cells.


title: CAD-C: An engineered nuclease enables repair-free in situ proximity ligation and nucleosome-resolution chromosome walks in human cells [Classic, Express and Direct CAD–C]

overall_design: Classic, Express and Direct CAD–C to different cell types.


title: CAD-C: An engineered nuclease enables repair-free in situ proximity ligation and nucleosome-resolution chromosome walks in human cells [Cut & Tag]

overall_design: CUT&Tag assays targeting Pol II Ser5 and H3K4me1 were carried out in hTERT-RPE1 cells.


title: CAD-C: An engineered nuclease enables repair-free in situ proximity ligation and nucleosome-resolution chromosome walks in human cells [CAD-Seq]

overall_design: CAD-seq and digestion control assays in wild type hTERT-RPE1, GM12878 and K562 cells.


title: CAD-C: An engineered nuclease enables repair-free in situ proximity ligation and nucleosome-resolution chromosome walks in human cells [Micro-C]

overall_design: Micro‑C performed on wild‑type hTERT‑RPE1 cells


title: Targeting CDK12/CYCLIN K induces a gene activation program which is mediated by P-TEFb [Cut&RUN]

city: Beer Sheva

first: Praveenkumar

last: Murugavelu

address: David Ben Gurion Blvd

country: Israel

institute: Ben Gurion University

summary: Regulation of transcription in human cells is a multistep process controlled by coordinated actions of host transcription factors and their associated kinases. Despite major advances, fundamental gaps still remain in understanding how transcriptional programs are dynamically regulated. Gene expression of Human immunodeficiency virus (HIV) is tightly controlled by the cellular transcription machinery, providing a powerful model for studying transcription regulation. Such gene expression regulation is clinically significant, as HIV persists in long-lived reservoirs even under antiretroviral therapy. Here, we show that selective targeting of CDK12/CYCLIN K (CCNK) induces a global gene activation program, which is mediated by positive transcription elongation factor b (P-TEFb). CDK12/CCNK targeting reshapes promoter-proximal and gene bodies landscape at chromatin, and promotes release of P-TEFb from its inhibitory 7SK snRNP complex. Additional transcriptomic profiling reveals preferential upregulation of P-TEFb-target genes, accompanied by induction of relatively short genes and repression of CDK12-regulated DNA damage response pathways. In HIV- infected cells, targeting CDK12/CCNK activates a robust viral gene expression, accompanied with latency reversal and importantly, synergistic reactivation effects when combined with latency- reversing agents. Together, these results uncover a previously unrecognized compensatory interplay between transcriptional kinases that rewires the cellular gene expression program with implications for HIV latency reversal and cancer biology.

overall_design: Cut&RUN of the histone modification (H3K4me3 and H3K27ac), RNA Pol II CTD, Ser2 and Ser5 in SR4835 treated 2D10 cells

first: Jagrity

last: Rani

first: Heli

last: Glebko

first: Nili

last: Tickotsky

first: Liron

last: Levin

first: Alona

last: Kuzmina

first: Ran

last: Taube


title: Characterization of Circulating Neoantigen-Specific T Cell Responses and Their Public TCR Repertoires in Lynch Syndrome Carriers (scTCRseq)

first: Eduardo

last: Vilar-Sanchez

institute: The University of Texas MD Anderson Cancer Center

summary: Lynch syndrome (LS), a common hereditary cancer syndrome, provides a unique model to study immune surveillance at the earliest stages of tumorigenesis. A hallmark of LS carcinogenesis is the generation of highly immunogenic mutated neoantigens derived from insertion deletions, yet the transcriptomic states of the neoantigen-specific T cells that recognize them remain poorly characterized. Here, we characterized neoantigen-specific T cells from LS carriers using functional assays, single-cell RNA/TCR sequencing, and repertoire integration with existing data sets. Recurrent neoantigens elicited strong immune responses, with neoantigen-specific T cells mediating cytotoxicity against patient-derived tumor organoids. Single-cell analysis revealed oligoclonal expansions spanning effector and memory states with LS cancer survivors showing enrichment for metabolically exhausted subsets and previvor LS carriers retaining polyfunctional effectors. Cross-cohort analyses identified public clonotypes overlapping with tumor-infiltrating repertoire. These findings define the architecture of circulating neoantigen-specific immunity in LS and identify public TCRs as biomarkers for immune monitoring and immunoprevention.

overall_design: To define the phenotypic and transcriptional states of circulating neoAg-specific T cells, we performed single-cell multi-omics on six LS carriers, including three previvors (P1, P5 and P7) and 3 survivors (S2, S8 and S10) with >1% dextramer positivity.


title: CAD-C: An engineered nuclease enables repair-free in situ proximity ligation and nucleosome-resolution chromosome walks in human cells

overall_design: ATAC-seq assays in wild type hTERT-RPE1 cells.


title: Massively parallel characterization of adolescent idiopathic scoliosis risk variants [RNA-Seq]

city: Gainesville

first: Darius

last: Ramkhalawan

address: 1200 Newell Drive

institute: University of Florida

summary:
Adolescent idiopathic scoliosis (AIS) is a common pediatric musculoskeletal disorder characterized by lateral spinal curvature, often leading to chronic pain and deformity. While a significant genetic component to AIS is recognized, the functional impact of most associated genetic variants, particularly those in non-coding regions, remains largely unknown.
Using massively parallel reporter assays, we characterize 1,664 variant positions in linkage disequilibrium with 26 AIS lead variants identified by genome-wide association studies (GWAS) in chondrocytes, a major cell type implicated in AIS pathogenesis. Using a library of 7,173 candidate regulatory sequences we compare the 1,664 reference alleles against 4,708 alternate alleles in two human chondrocyte cell lines (TC28a2 and SW1353). Our analysis identifies 92 variants that exhibit significant differential regulatory activity between their reference and alternate alleles, 79 of which are predicted to disrupt transcription factor binding sites, often correlating with their observed regulatory effect.

overall_design: RNA sequencing of untreated TC28a2 human chondrocytes

first: Justin

last: Koesterich

first: Anat

last: Kreimer

first: Nadja

last: Makki


title: Clonal Hematopoiesis Drives Fibrotic Remodeling of the Bone Marrow Microenvironment [dataset 2]

city: Toronto

first: Soheil

last: Jahangiri

address: 610 University Ave

department: Princess Margaret Cancer Centre

institute: University Health Network

summary: Clonal hematopoiesis of indeterminate potential (CHIP) is associated with systemic inflammation and increased malignant risk, yet how it reshapes the bone marrow (BM) niche remains poorly understood. Using single-cell transcriptomic profiling of freshly isolated BM, we identify fibro-inflammatory remodeling as an early hallmark of CHIP. Beyond the established myeloid-biased inflammatory state, CHIP BM displayed coordinated TNFα activation spanning hematopoietic progenitors, monocytes, and stromal cells. Stromal populations showed early fibrotic remodeling, with mesenchymal and vascular cells upregulating collagen and extracellular matrix gene programs. Fibroblasts were markedly expanded regardless of mutational subtype and acquired cancer-associated fibroblast (CAF)-like transcriptional states. Histological analysis confirmed the presence of pre-fibrotic stromal remodeling in CHIP BM, representing an early and potentially disease-defining pathological change. Spatial transcriptomics further revealed discrete fiber-enriched regions populated by active and CXCL12⁺ fibroblasts, macrophages, and lymphoid cells, establishing fibroblast-immune crosstalk as a defining architectural feature of the CHIP niche. Strikingly, these regions were characterized by significant enrichment of pro-inflammatory pathways, including TGF-β and TNF signaling, identifying them as spatially confined inflammatory hubs within the BM microenvironment. Together, these findings establish fibro-inflammatory BM remodeling as a conserved early consequence of clonal expansion, linking CHIP to prefibrotic stromal changes, immune dysregulation, and a niche architecture that may prime the transition to overt malignancy.

overall_design: We performed Xenium in situ spatial transcriptomics on bone marrow tissue from patients with and without CHIP.

first: Aguilar-Navarro

last: Alicia G.

first: Edun

last: Gibran

first: Jahangiri

last: Soheil


title: Targeting CDK12/CYCLIN K induces a gene activation program which is mediated by P-TEFb [ChIP-seq]

overall_design: ChIP-seq of the CDK9 in HIV infected Jurkat cells treated with SR4835.


title: Engineering antigenic breadth against SARS-CoV-2 by pairing divergent RBDs within a single mRNA immunogen

city: Wellington

first: Lisa

last: Connor

address: Kelburn Parade

country: New Zealand

institute: Malaghan Institute of Medical Research

summary: Vaccines capable of eliciting broadly neutralising antibodies (bnAbs)are a major goal for pandemic preparedness. A persistent challengeacross vaccine fields is how to deliberately recruit the rare B cellclones that recognise conserved epitopes shared across diverse viralvariants.  BnAbs have been known to frequently emerge throughextensive somatic hypermutation during affinity maturation, here wedescribe an alternative, structure-driven mechanism for bnAbselection. We designed an mRNA vaccine in which two antigenicallydistinct SARS-CoV-2 variant’s (Omicron and Delta; O-Δ) receptorbinding domains (RBDs) are physically fused on a singlepolypeptide. This design is predicted to favour B cell antigenreceptors capable of engaging conserved epitopes on both RBDs withenhanced avidity. A matched non-divergent tandem RBD (Delta-Delta;Δ-Δ) served as a control. The divergent (O-Δ) immunogen was robustlyexpressed and retained high-affinity ACE2 binding. In mice,immunisation elicited potent antibody responses and increased thefrequency of antigen-specific cross-reactive B cells, recognisingDelta, Omicron, and the 2002 pandemic strain SARS-CoV RBDs. Usingmulticolour RBD tetramers and single-cell B cell receptor sequencing,we show that breadth arises via two distinct pathways.  The divergentvaccine preferentially enriches clonally distinct cross-reactive Bcells (not present within non-cross-reactive B cell pools) with lowlevels of somatic hypermutation (SHM), consistent with selection ofgermline-biased precursors. In contrast, the matched control vaccineyields cross-reactivity primarily within existing clonal lineages(clonal overlap between cross- reactive and non-cross-reactive cells)and at higher mutational burdens, consistent with affinity-maturation-driven acquisition of breadth. Together, these findingsdemonstrate that antigen structure can bias B cell selection towardscross-reactive specificities without requiring extensive SHM. Thiswork establishes a simple, modular antigen-design principle in whichjuxtaposing appropriately divergent antigens on a single scaffoldpromotes the enrichment of bnAb-prone B cells, providing a scalablestrategy for vaccine development against rapidly evolving pathogens.

overall_design: Cohorts (n = 12 per vaccine) received two doses of either matched (Δ-Δ) tRBD-NTD or divergent (Δ-O) tRBD-NTD vaccine. Lymph nodes were processed individually, indexed with sample tags and then pooled for cell sorting. Antigen-specific B cells were sorted by multicolour baits into three tiers of polyreactivity: (I) single-binding (Delta only), (ii) mid cross-reactive (Delta+ Wuhan+ or Delta+CoV-1+), and (iii) multi-binding (Delta+Wuhan+CoV-1+) (Fig. 5a). To ensure that cross-reactivity metrics were not artificially driven by inclusion of Omicron in the divergent (Δ-O) tRBD-NTD vaccine, Omicron probes were excluded from the sequencing sorts. Sorted cells were stained with oligo-tagged surrogate antibodies (CD19 and B220) to retain tier identity before loading onto BD Rhapsody single-cell separation cartridge. Whole transcriptomic single-cell sequencing, V(D)J sequencing, clone calling and somatic hypermutation quantification were performed on a single cell level.

first: Isabelle

last: Montgomerie

first: Rebecca

middle: E

last: McKenzie

first: Olga

last: Palmer

first: Ngarangi

last: Mason

first: Joanna

last: Kuang

first: Theresa

last: Pankhurst

first: Sarah

last: Draper

first: Sventja

last: von Daake

first: David

last: Eccles

first: Thomas

last: Bird

first: Abby

last: Martin

first: Isaac

last: Green

first: Lydia

last: White

first: Jordan

middle: J

last: Minnell

first: Sam

last: Small

first: Ian

middle: F

last: Hermans

first: Gavin

last: Painter

first: James

last: Ussher

first: Miguel

last: Quiñones-Mateu

first: Wayne

last: Patrick

first: Davide

last: Comoletti


title: Targeting CDK12/CYCLIN K induces a gene activation program which is mediated by P-TEFb

first: Praveenkumar

last: Murugavelu

overall_design: 2D10 cells were treated with SR4835 drug for reactivation for 6hrs and subjected to RNA-seq

first: Jagrity

last: Rani

first: Heli

last: Glebko

first: Nili

last: Tickotsky

first: Liron

last: Levin

first: Alona

last: Kuzmina

first: Ran

last: Taube


title: Pooled CRISPR screens identify genes and non-coding genomic regions that regulate red blood cell density

city: Montreal

first: Guillaume

last: Lettre

state: Quebec

address: 5000 Belanger

institute: Montreal Heart Institute

summary: Genome-wide association studies have identified >1000 loci associated with clinically important red blood cell (RBC) traits, such as hemoglobin content and cell volume. However, few of these associations have been characterized at the molecular level such that most causal genes and variants remain elusive. Here, we performed pooled CRISPR screens in an erythroid cell line to identify genes and regulatory non-coding sequences that control RBC density. We perturbed 556 candidate genes and genomic sequences near 2114 GWAS variants. We used a density gradient to quantify the impact of these CRISPR perturbations on cell density. After validation, we found 17 genes and 13 regions near GWAS variants that regulate cell density. Some of these genes have previously been implicated in RBC biology (e.g. ATP2B4, CCND3, EPOR) although many are novel (e.g. CHTF8, CTU2, DNASE2). We confirmed that deletions in the osmotic stress response kinase gene OXSR1 increase cell density, and a phosphoproteome analysis in OXSR1-depleted cells indicated that this phenotype is accompanied with a dephosphorylation of the upstream kinase WNK1 and the downstream target KCC3 (SLC12A6). We also combined CRISPR perturbations and RNA-sequencing to show how a non-coding genomic sequence near rs13255015 regulates the expression of the transcription factor ZFAT in cis and SLC4A1 in trans. SLC4A1 encodes Band3, a known regulator of RBC hydration and volume. Our results suggest experimental strategies to characterize GWAS findings and provide new molecular insights into the regulation of complex RBC traits.

overall_design: This study includes 3 sequencing experiments. The first one is a discovery CRISPR/Cas9 screen (DS) using 17219 gRNA. The second is a validation CRISPR/Cas9 screen (VS) using 190 gRNA. Both screens were performed in HUDEP-2 cells stably expressing either Cas9, dCas9-KRAB, or dCas9-VP64. The pHKO plasmid was used in Cas9 and dCas9-KRAB expressing cells and pMS2 plasmid was used in dCas9_VP64 expressing cells. After transduction of the gRNA librairies, the HUDEP-2 cells were placed on a 4 layers Percoll gradient. After centrifugation, the cells were collected at each interface as well as the palet, making 5 fractions for each gradient. After extraction of gDNA, the gRNA sequences were amplified by PCR. These PCR products were sequenced using different index for each Percoll gradient fraction. The abundance of each gRNA in each fraction was determined to establsih the effect of each gRNA on HUDEP-2 cell density. The first screen with 17219 gRNA was performed in 4 replicates and the validation screen, with 190 gRNA was performed in 3 replicates. The third experiement is an RNA-seq (RNAseq) experiment in which 6 different gRNA were individually transduced in HUDEP-2 cells expressing Cas9 as well as a safe gRNA as control. We assessde the effect of each of these gRNA on the transcriptome.

first: Nicolas

last: Brosseau

first: Thomas

last: Pincez

first: Ken Sin

last: Lo

first: Chao-Sheng

first: Mélissa

last: Beaudoin