title: NOD2/NF-κB/NLRP2 signaling axis potentially induces early spontaneous abortion following IVF/ICSI-ET via promoting pyroptosis in extravillous trophoblast cells

city: Zhanjiang City

first: Xiao

last: Wang

state: Guangdong Province

address: No. 57 South Renmin Avenue

institute: Affiliated Hospital of Guangdong Medical University

summary: Early spontaneous abortion following in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) remains a significant clinical challenge. The underlying molecular mechanisms, particularly involving trophoblast dysfunction, are not fully elucidated.

overall_design: Transcriptomic profiling (RNA-seq) was performed on chorionic villi from patients with early spontaneous abortion following IVF/ICSI-ET and controls with elective termination. Bioinformatics analysis identified differentially expressed genes and associated pathways. Functional validation was conducted using the human extravillous trophoblast cell line HTR-8/SVneo, with overexpression of NOD1 and NOD2 via plasmid transfection. Pyroptosis and inflammatory responses were assessed by RT-qCR, Western blot, flow cytometry, ELISA, and immunofluorescence. For in vivo validation, a pregnant mouse model was established by intrauterine injection of NOD2 overexpressing lentivirus (oeNOD2), with or without the NF κB inhibitor BAY 11-7082, to determine whether this signaling axis contributes to early pregnancy loss.


title: Somatic mutations reveal the ontogeny of microglia in human aging [snATAC-seq]

status: Public on Jul 13 2026

city: Stanford

first: Julia

last: Belk

state: California

address: 450 Serra Mall

country: USA

institute: Stanford University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Microglia are the resident macrophages of the central nervous system. In mice, microglia seed the brain during embryogenesis and can be maintained throughout life with minimal input from adult hematopoiesis. The origins of human microglia are less clear, but recent evidence suggests that marrow-derived cells contribute to the human microglial pool in certain individuals. Here, to investigate the ontogeny of human microglia, we develop a method that uses the collection of accumulated somatic mutations which uniquely labels each clone of cells to track the infiltration of marrow-derived cells into the human brain. Applying this method to 20 aged individuals, we find evidence of an influx of marrow-derived cells into the brain in all examined individuals. Single cell analysis, including single cell lineage tracing using mitochondrial DNA variants, demonstrates that these infiltrating cells are similar to microglia and can comprise a large fraction of the microglial pool. Analysis of large-scale sequencing cohorts demonstrates a protective association between most types of clonal hematopoiesis and Alzheimer’s disease. In sum, this work uncovers a widespread influx of myeloid cells into the healthy human brain which contributes to the pool of human microglia and becomes common with aging.

overall_design: snATAC-seq was performed on archival tissue specimens from the UW BRaIN Lab. Individuals were identified who harbored marrow-derived cells in their brain (assessed via passenger-assisted clone tracking), and snATAC-seq enabled assessment of the immune cell phenotypes present in each individual.

middle: A

first: Howard

middle: Y

last: Chang

first: Siddhartha

last: Jaiswal


title: Antigen presentation requirements for effective cDC1-based cancer immunotherapy

city: Houston

first: LI

last: SHEN

state: TX

address: 1515 Holcombe Blvd.

institute: MD Anderson Cancer Center

type: Expression profiling by high throughput sequencing

summary: Type 1 conventional dendritic cells (cDC1s) are important for generating and sustaining antitumor immunity. Accordingly, the abundance of cDC1s in human tumors correlates with improved outcomes in cancer. Capitalizing on this role, we previously demonstrated that vaccination with murine cDC1s, generated in culture from bone marrow cells (termed here “in vitro–derived cDC1s”), elicits durable tumor control in multiple preclinical models; however, the immunological mechanisms underlying the efficacy of cDC1 vaccination remain unclear. Here, we examined whether in vitro–derived cDC1s resemble tumor-infiltrating DC populations and whether MHC-I and MHC-II antigen presentation contribute to cDC1-mediated tumor control following vaccination in melanoma. As expected, MHC-I or MHC-II deficiency had minimal impact on the transcriptional state of cDC1s in homeostasis or following stimulation with the adjuvant poly dI:dC. Moreover, in vitro–derived cDC1s cultured under steady-state conditions closely resembled tumor-infiltrating cDC1s, whereas their poly dI:dC–stimulated counterparts resembled CCR7+ tumor-infiltrating DC populations, also referred to as mregDCs or LAMP3+ DCs. Our data further show that both MHC-I and MHC-II contribute to tumor control upon cDC1 vaccination and that coexpression of MHC-I and MHC-II on the same cDC1 is necessary for a robust vaccine response. We also identified an important function for host cDC1s in supporting the efficacy of vaccination with in vitro–derived cDC1s, as judged by impaired tumor control in Irf8 + 32−/− mice, which lack endogenous cDC1s. Overall, these results indicate that effective antitumor responses depend on MHC-I and MHC-II antigen presentation by vaccine-delivered cDC1s, with additional contributions from host cDC1s.

overall_design: RNA-seq profiling of in vitro-derived cDC1s from wild-type C57BL/6J mice and their knockdown derivatives (MHC-I KO and MHC-II KO) under steady-state conditions or following stimulation with poly dI:dC, either alone or in combination with an agonistic anti-CD40 antibody (αCD40).

first: Josué

middle: E

last: Pineda

first: Tomoyuki

last: Minowa

first: Li

last: Shen

first: Stephanie

middle: S

last: Watowich

first: Matthew

middle: M

last: Gubin


title: mTOR Signaling is a Key Modifier and Therapeutic Target in a Mouse Model of X-linked Alport Syndrome.

city: Bar Harbor

first: Courtney

last: Willey

state: Maine

address: 600 Main Street

institute: The Jackson Laboratory

summary:
X-linked Alport syndrome (XLAS) is a hereditary kidney disease with substantial phenotypic variability despite well-defined causal mutations in type IV collagen. To identify modifier pathways influencing disease severity, we previously generated and analyzed a population of ~200 genetically diverse mice with XLAS and identified two candidate genes associated with albuminuria (Dgke and Pik3r1) both involved in the PI3K-AKT-mTOR pathway.

To investigate the involvement of the PI3K-AKT-mTOR pathway in XLAS, we inhibited mTOR with rapamycin and found that treatment extended time to renal failure by 44%, whereas partial mTORC2 reduction via Rictor haploinsufficiency did not alter disease. A multi-drug intervention showed that rapamycin, meclizine, and fisetin significantly reduced albumin-to-creatinine ratio (ACR) without improving glomerular filtration rate (GFR), indicating protection from kidney injury rather than restoration of filtration. Together, these findings highlight mTORC1 signaling as a key modifier of XLAS.

overall_design: Male mice with XLAS either treated with control diet or diet containing 500ppm of fisetin (all mice are on the B6 background). Bulk RNA-seq of kidneys collected at 30-weeks of age.

first: Ron

last: Korstanje


title: PGK1 acts as a non-catalytic gatekeeper of ferroptosis by stabilizing SLC7A11 [RNA-Seq]

city: ChongQing

first: yuanze

last: zhou

address: No.30, Gaotanyan Main Street, Shapingba District

country: China

institute: The First Hospital Affiliated to Army Medical University (Southwest Hospital)

summary: Ferroptosis is restrained by layered antioxidant defenses, yet whether glycolytic enzymes directly constitute ferroptosis-defense machinery remains unclear. Here we identify phosphoglycerate kinase 1 (PGK1) as a non-canonical suppressor of ferroptosis that functions independently of its glycolytic kinase activity. Across pan-cancer bulk and single-cell transcriptomes, glycolysis inversely associates with ferroptosis programs, with PGK1 emerging as a prognostic hub. PGK1 loss in multiple tumor models elicits hallmark ferroptotic features, elevates lipid peroxidation and labile iron, and suppresses xenograft growth, all of which are reversed by liproxstatin-1. Mechanistically, PGK1 physically binds the C-terminal tail of SLC7A11 (aa 471-501) and shields it from ubiquitin-proteasome degradation, thereby sustaining cystine-driven glutathione synthesis and redox homeostasis. This scaffolding role is preserved in a kinase-dead PGK1 mutant, establishing a strict separation between metabolic and ferroptosis-regulatory functions. Restoring SLC7A11 rescues PGK1-deficient tumor growth, whereas a rationally designed competing peptide SLC7A11471-501 (FP-01) disrupts the PGK1-SLC7A11 interface, depletes SLC7A11/GSH, triggers ferroptosis, and suppresses tumor progression in vivo. Collectively, the PGK1-SLC7A11 interaction represents a druggable ferroptosis vulnerability that enables selective ferroptosis induction while sparing essential glycolysis.

overall_design: To investigate the function of PGK1 in HT1080 cell, we established stable PGK1 overexpression in HT1080 cell and performed RNA-seq.

first: Guixian

last: Wu

first: Yuanze

last: Zhou

first: Chengxin

last: Luo

first: Yaqun

last: Ding

first: Xi

last: Li

first: Hui

first: Shuangnian

last: Xu


title: Loss of TSPOAP1 exacerbates mitochondrial dysfunction and neuronal apoptosis after intracerebral hemorrhage

city: Chongqing

first: yuhao

last: duan

address: No. 136 Zhongshan 2nd Road, Yuzhong District

department: School of Pediatrics

institute: Children's Hospital of Chongqing Medical University

summary: Intracerebral hemorrhage (ICH) induces severe secondary neuronal injury, in which mitochondrial dysfunction and apoptosis play central roles. Accumulating evidence suggests that mitochondrial failure represents a critical convergence point linking diverse injurious stimuli to neuronal apoptosis after ICH. However, the molecular mechanisms that connect mitochondrial dysfunction to irreversible apoptotic commitment remain incompletely understood. In particular, the upstream regulators governing mitochondrial permeability transition pore (mPTP) opening during ICH have yet to be fully defined.

overall_design: Transcriptomic analyses of perihematomal tissue from patients with ICH and mouse models were integrated with functional studies using an HT22 neuronal cell line deficient in 18-kDa translocator protein (TSPO)-associated protein 1 (TSPOAP1), together with pharmacological intervention using FG-7142. Mitochondrial integrity, mPTP opening, apoptotic signaling, and protein–protein interactions were evaluated in vitro and in vivo.

first: Yuhao

last: Duan


title: Small RNA sequencing of whole blood from a rat model of chronic primary pain with adrenergic receptor blockade

city: Durham

first: Nathaniel

middle: P

last: Hernandez

state: NC

address: 3 Genome Ct

department: Anesthesiology

institute: Duke University

type: Non-coding RNA profiling by high throughput sequencing

summary: The sympathetic nervous system has been implicated in the development of chronic primary pain conditions (CPPCs), yet the downstream molecular mechanisms linking sustained catecholamine signaling to chronic pain remain poorly understood. Here, we profiled circulating small RNAs in a rat model of CPPC induced by sustained catecholamine elevation through catechol-O-methyltransferase (COMT) inhibition. Female Sprague-Dawley rats (N = 5/group) were assigned to four treatment groups: Vehicle + Vehicle (pain-free control), OR486 + Vehicle (primary pain condition), Vehicle + ICI-118,551/SR59230A, or OR486 + ICI-118,551/SR59230A. OR486 and vehicle were delivered continuously for 14 days by subcutaneous osmotic minipump. Separate minipumps delivered the ADRB2-selective antagonist ICI-118,551 and the ADRB3-selective antagonist SR59230A or vehicle. Whole blood was collected on Day 14 for small RNA isolation and Illumina small RNA sequencing. Differential expression analysis identified a discrete set of circulating miRNAs altered during primary pain, including significant downregulation of miR-133a-3p and upregulation of multiple inflammation-associated miRNAs. Pharmacologic blockade of ADRB2/3 prevented miR-133a-3p downregulation, identifying miR-133a-3p as an adrenergic receptor-dependent regulator associated with chronic primary pain.

overall_design: Small RNA-seq profiling of whole blood from female CPPC rats treated with ADRB2 and ADRB3 antagonists and their respective controls (Vehicle+Vehicle "VV", OR486+Vehicle "OV", Vehicle + ICI118551/SR59230A "VI", or OR468 + ICI118551/SR59230A "OI")

first: Brittney

last: Ciszek

first: Matt

last: Kanke

first: Praveen

last: Sethupathy

first: Andrea

middle: G

last: Nackley


title: Engineering hematopoietic stem and progenitor cells to generate red blood cells as viral traps against HIV-1

first: Matthew

last: Porteus

address: 265 Campus Drive

department: Pediatrics

summary: Canonical HIV-1 entry into target cells depends on binding to CD4 as a primary receptor. Because of this, use of the CD4 receptor as a viral trap (a decoy receptor used to prevent infection of target cells) is a promising strategy for the treatment of HIV-1. One challenge in using CD4 viral traps is maintaining enough of the decoy receptor in circulation to remain effective. Here we present a strategy to produce cell-based CD4 viral traps by engineering hematopoietic stem and progenitor cells (HSPCs) to express the CD4 receptor in red blood cell (RBC) progeny. This takes advantage of the ability of the HSPC to repopulate the blood system for a lifetime, while leveraging the fact that RBCs greatly outnumber any cell targeted for infection. CRISPR/Cas9-engineered HSPCs efficiently express CD4 on their cell surface after differentiation to the RBC lineage in vitro. Fusion of CD4 to glycophorin A (GPA) and introduction of a truncated erythropoietin receptor (tEPOR) leads to increased CD4 expression and enrichment of edited cells (CD4-GPA-tEPOR) to levels capable of neutralizing HIV-1 pseudovirus in vitro. In sum, this work presents a potential strategy for the one-time delivery of CD4-RBC viral traps through autologous transplantation of engineered HSPCs.

overall_design: Bulk RNA-seq profiling of day 14 differentiated erythroid cells derived from human hematopoietic stem and progenitor cells (HSPCs) engineered to express the following transgenes: GFP integrated at the CCR5 locus, or CD4-GPA or CD4-GPA-tEPOR integrated at the HBA1 locus. Experiment includes n=3 mock control, CCR5/GFP, HBA1/CD4-GPA, and HBA1/CD4-GPA-tEPOR samples.

first: Sofia

last: Luna

first: Willaim

middle: N

last: Feist

first: Kaya

middle: R

last: Ben-Efraim

first: Freja

middle: K

last: Ekman

middle: H


title: Protocol for low-input whole-genome bisulfite sequencing in plants

city: München

first: Jie

last: Yang

state: Bavaria

address: Karlsfelder Str. 38a

country: Germany

institute: Technical University of Munich (TUM)

type: Methylation profiling by high throughput sequencing

summary: DNA methylation profiling of rare plant samples is limited by the scarcity of input DNA. This protocol uses Arabidopsis leaf to describe tissue cryo-milling, proteinase K lysis, direct bisulfite conversion, post-bisulfite adapter tagging library construction with random priming and adapter ligation, and MethylStar-based alignment and methylation calling. DNA inputs as low as 50 pg generate methylomes comparable to conventional 50-ng WGBS.

overall_design: Whole-genome bisulfite sequencing (WGBS) libraries were generated from a serial dilution of Arabidopsis thaliana leaf genomic DNA (5 pg to 50 ng) using a low-input post-bisulfite adapter tagging (PBAT) protocol, and benchmarked against standard bulk WGBS libraries prepared from 50 ng input (samples S45_1-3). 2-3 biological replicates were sequenced per DNA-input concentration group.

first: Binh

middle: T

last: Vo

first: Frank

last: Johannes


title: tracheal mucosal injury caused by hyperinflation cuff pressure

city: Nanjing

first: Xuan

last: Wang

address: Guangzhou Road

institute: Nanjing medical university

summary: RNA seq of tracheal mucosa between normal rabbits and tracheal injury rabbits

overall_design: Two groups were seted and a tracheal mucosal injury model was established by inserting a 3.0 mm tracheal tube to a depth of 13 cm and maintaining the cuff pressure at 35 cmH2O for 2 h in New Zealand rabbits (3 months old), n = 3 per group


title: Loss of cellular RNA homeostasis contributes to MDA5 activation during virus infection

city: Melbourne

first: Jamie

last: Gearing

address: 27-31 Wright St

country: Australia

institute: Hudson Institute of Medical Research

summary: MDA5 is an innate immune RNA sensor that senses infection with a range of viruses and other pathogens. MDA5’s RNA agonists are not well defined. We used single-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) to study its ligands. Surprisingly, upon infection with SARS-CoV-2 or encephalomyocarditis virus, MDA5 bound overwhelmingly to cellular RNAs. Many binding sites were intronic and proximal to Alu elements and to potentially base-paired structures. Concomitantly, cytoplasmic levels of aberrant transcripts and intron-containing unspliced transcripts increased in infected cells and displayed enrichment of MDA5 iCLIP peaks. Moreover, overexpression of the splicing factor SRSF3 reduced aberrant transcription and abrogated MDA5 activation. Taken together, we propose that MDA5 surveys RNA processing fidelity and can detect infections by sensing perturbations of posttranscriptional events such as splicing.

overall_design: LIM1215 cells overexpressing either GFP or SRSF3-GFP were left uninfected, infected with EMCV (MOI = 0.01) for 48 hr, or treated with IFN for 6 hr. RNA was extracted from the cytoplasmic fractions, rRNA-depleted and sequenced. 4 replicates were performed.

first: Natalia

last: Sampaio

first: Linden

middle: J

first: Alexandra

middle: L

last: McAllan


title: NRAS knockdown reverses monocytic cell state and resensitizes resistant FLT3-AML cells to gilteritinib

city: Wilsonville

first: Srivathsan

middle: V

last: Ranganathan

state: OR

address: 30519 SW Ruth St

department: DOS

institute: OHSU

summary: While the FLT3 inhibitor gilteritinib is initially effective in patients with FLT3-mutated acute myeloid leukemia (AML), patients invariably relapse within months of treatment. Gilteritinib resistance is commonly driven by the emergence of NRAS mutations and a shift towards a more monocytic cell state. We hypothesized that directly targeting and depleting NRAS protein would reverse these adaptive differentiation changes and restore therapeutic sensitivity. To test this, we utilized a mutation-agnostic antisense oligonucleotide (ASO) to selectively knock down NRAS expression across gilteritinib-resistant AML cell lines, in vivo cell-line-derived xenografts, and primary patient samples. NRAS ASO successfully resensitized gilteritinib resistant cells with multiple distinct NRAS mutations, displaying superior efficacy compared to downstream MEK inhibition. This therapeutic efficacy was independent of NRAS mutant variant allele frequency, suggesting that wild-type NRAS may also contribute to resistance. Comprehensive multi-omic profiling (transcriptomics, proteomics, phosphoproteomics) revealed that NRAS knockdown consistently reversed monocytic phenotype, shifting cells back toward a more primitive cell state. Monocytic differentiation and NRAS mutations are established drivers of resistance to diverse targeted regimens in AML, including FLT3, IDH, and BCL2 inhibitors, so we also tested venetoclax resistant primary cells with NRAS mutations. Resistant cells were resensitized to venetoclax after NRAS knockdown, suggesting that NRAS knockdown may be more broadly applicable in overcoming monocytic cell state and drug resistance in AML.

overall_design: RNAseq of gilteritinib-resistant cell lines (Joshi et al Cancer Cell 2021; N=8) and parental MOLM-14 cells. No ASO refers to media controls (no added ASO). Control=5 uM non-targeting ASO control. NRAS=5 uM NRAS ASO. Cells were then treated with gilteritinib for 0, 4, and 24 hours.

first: Sunil

last: Joshi

first: Hadi

last: Maazi

first: Janét

last: Pittsenbarger

first: Belinda

middle: B

last: Garana

first: Andy

last: Kaempf

first: Ariane

last: Huang

first: Marina

last: Gritsenko

first: Chelsea

last: Hutchinson

first: Karl

last: Weitz

first: Rosalie

last: Chu

first: Sara

last: Gosline

first: Paul

middle: D

last: Piehowski

first: Ann-Kathrin

last: Eisfeld

first: Martin

last: Carrol

first: Alexander

last: Perl

first: Karin

last: Rodland

first: Bill

last: Chang

first: Alexey

last: Revenko

first: Jeffrey

middle: W

last: Tyner

first: Brian

last: Druker

first: Elie

last: Traer


title: Neural cell state modulation by PARK2 and dopaminergic neuroprotection by small molecule Parkin agonism [scRNA-seq]

city: Cleveland

first: Tyler

last: Alban

state: OH

address: 9500 Euclid Ave

institute: Cleveland Clinic

summary: Parkin, an E3 ubiquitin ligase encoded by PARK2, plays a key role in both hereditary and sporadic Parkinson’s disease (PD), yet there are no therapies currently available that can target this important pathway. Here, we show that Parkin is critical for successful neuronal differentiation and survival, and we develop small-molecule Parkin agonists that can protect dopaminergic neurons. Upon differentiation of neural progenitor cells, loss of Parkin results in a reduced capacity to maintain neuronal cell state, dopaminergic neuronal phenotypes, and stress resistance. Moreover, Parkin loss disrupted cell morphology and the stability of neurites. Transcriptional and single-cell analyses reveal that Parkin controls critical pathways regulating stem-like cell transitions and is needed for stable neuronal maturation. We also examined the effects of FB231, a small molecule enhancer of Parkin E3 ligase activity, in models of PD. FB231 reduced pathological α-synuclein and enhanced cell survival in human iPSC-derived dopaminergic neurons treated with α-synuclein preformed fibrils. Furthermore, FB231 attenuated α-synuclein pathology and dopaminergic neurodegeneration in a gut α-synuclein murine model of PD. Our findings support that Parkin plays a crucial role in maintaining neuronal homeostasis and that pharmacologic activation of Parkin may be a promising strategy to attenuate neurodegeneration in PD.

overall_design: scRNAseq profiling of Wildtype nPC and their Parkin KO cells

first: Yongxing

last: Gong

first: Armin

last: Bayati

first: Prerana

last: B Parthasarathy

first: Fang

last: Zhou

first: Vladimir

last: Makarov

first: Yuhang

last: Zhao

first: Chienwen

last: Su

first: Jackson

last: Schumacher

first: Vijay

last: Singh

first: Laura

middle: A

last: Volpicelli-Daley

first: Wen

last: Luo

first: Thomas

last: Durcan

first: Shovan

last: Dutta

first: Michael

last: Schwarzschild

first: Jennifer

last: Johnston

first: Timothy

last: Chan

first: Xiqun

last: Chen


title: Neural cell state modulation by PARK2 and dopaminergic neuroprotection by small molecule Parkin agonism [RNA-Seq]

first: Tyler

last: Alban

overall_design: RNAseq profilign of wild type NPC cells and their PARKIN KO

first: Yongxing

last: Gong


title: Transcriptomic profiling of liver tissues from DDC-treated and control mice

city: Harbin

first: Wenjing

last: Sun

address: Baojian road

institute: Harbin Medical University

summary: This study aimed to characterize transcriptomic alterations in mouse liver tissues during DDC-induced cholestatic liver injury and biliary fibrosis. Bulk RNA sequencing was performed on liver tissues from control and DDC-treated mice. Differential gene expression analysis was conducted to identify pathways associated with extracellular matrix remodeling, TGF-beta signaling and fibrosis.

overall_design: Bulk RNA-seq was performed on liver tissues from control mice and DDC-treated mice. Biological replicates were included for each group. Total RNA was extracted from liver tissues, libraries were constructed and sequenced using an Illumina platform. Processed gene-level count matrices and normalized expression tables are provided.

first: Yujuan

last: Wa

first: Songbin

last: Fu

first: Qingbo

last: Cui

first: Hao

last: Wang


title: CYP3A4-mediated metabolic variability drives resistance and systemic toxicities under targeted therapies for non–small cell lung cancer

city: Tampa

first: Pragya

last: Kumar

state: FL

address: 4502 Montego Bay Court

institute: University of South Florida

summary: Targeted therapies, including small molecule inhibitors of mutant ALK, EGFR, and RAS in lung cancers, can produce strong and durable clinical responses, yet a subset of tumors fail to respond, and even strong responders eventually relapse. Further, although targeted therapies are typically well tolerated, a subset of patients displays dose-limiting adverse effects. Therapy resistance is generally considered to be a tumor-intrinsic phenomenon, mediated by cell-intrinsic or microenvironmental mechanisms. We found that hyperactivation of CYP3A4, a P450 family enzyme, can lead to tumor-extrinsic resistance by altering pharmacokinetics and reducing systemic and tumor drug exposure, whereas reduced CYP3A4 activity is expected to increase systemic toxicities. Retrospective analysis of clinical cohorts suggests that variability in CYP3A4 activity might be a major contributor to variability in clinical outcomes. To understand the consequences of altered drug metabolism, we developed a mathematical model linking variability in CYP3A4 activity with temporal variability in systemic drug concentrations. These in silico studies indicate that the impact of altered CYP3A4 activity can be mitigated by altering drug dosing and intervals. Further, we demonstrate that tumor sensitivity can be restored by pharmacological inhibition of CYP3A4. These findings establish systemic metabolic variability as a clinically actionable driver of both resistance and toxicity and provide a framework for personalized dosing to improve safety and efficacy of targeted therapy.  The Bulk RNA sequencing data is from mouse livers, either untreated or treated with candidate drug JIB-04 to assess the change in gene expression landscape.

overall_design: RNA seq profiling of livers from NSG mice. 2 samples (C1 and C2) represent livers from 2 untreated / control mice and 2 samples (J1 and J2) represent livers from 2 mice treated with 55mg/kg JIB-04 twice weekly, for 5 doses before collection.

first: Julia

last: Pelesko

first: Malgorzata

last: Weh

first: Menkara

middle: C

last: Henry

first: Bina

last: Desai

first: Ramu

last: Kakumanu

first: Min

last: Liu

first: Natalia

last: Siqueira

first: Robert

last: Vander Velde

first: Viktoriya

last: Marusyk

first: Jyllica

last: Aurelio

first: Eric

last: Haura

first: Jhanelle

last: Gray

first: Bruna

last: Pellini

first: David

last: Basanta

first: Andriy


title: Fertility Gene Introns Harbor Transposable Elements that Shape Y-Loop Architecture

city: Farmington

first: Mayu

last: Inaba

state: CT

address: 263 Farmington Ave

institute: UConn Health

summary: Transposable elements (TEs) are powerful drivers of genome evolution, yet how they persist under selection and become incorporated into host regulatory networks remains poorly understood. In the Drosophila male germline, TEs are highly expressed during the spermatocyte stage, coinciding with activation of giant fertility genes on the Y chromosome. These genes contain megabase-scale introns enriched for repetitive DNA, and three of these genes form prominent nuclear structures known as Y-loops, providing a unique system to investigate gene regulation. Here, we show that multiple TEs expressed in spermatocytes are transcribed from the introns of Y-linked fertility genes. RNA fluorescence in situ hybridization (FISH) targeting several TEs, including accord2, Juan, and HMS Beagle, illuminates distinct nuclear regions corresponding to kl-2, kl-3, and kl-5, respectively. Genetic perturbation of these fertility gene loci or disruption of RNA-processing factors eliminates these TE transcripts, demonstrating that these TE sequences are embedded within Y chromosome-associated nascent transcripts rather than being independently transcribed. The identity and expression patterns of Y-loop-associated TEs vary extensively among closely related Drosophila species, consistent with the previously documented rapid evolution of Y-linked loci and suggesting that TEs may contribute to the genetic diversification of these giant fertility genes. We propose that continual turnover of repetitive elements within Y-linked introns provides a mechanism by which rapidly evolving repetitive DNA influences germline gene regulation, male fertility, and speciation.

overall_design: RNA-seq of sorted spermatocytes from control (blanks-gfp) and blanks-gfp, bamGal4>U2af38 RNAi Drosophila testes

last: Beard

first: Jeffrey

last: Gamer

first: Amelie

last: Raz


title: Decitabine induces innate immune gene expression in rare melanoma models

city: Aurora

first: Kasey

middle: L.

last: Couts

state: CO

institute: University of Colorado Anschutz Medical Campus

summary: Rare melanoma subtypes, including mucosal melanoma, can exhibit reduced tumor-intrinsic innate immune and interferon-associated gene expression, which may contribute to immune evasion and limited responses to immune checkpoint inhibitors. We investigated whether decitabine, a DNA methyltransferase inhibitor known to induce viral mimicry and immune-associated transcriptional programs, could restore innate immune and interferon-associated signaling in rare melanomas. Here, we report RNA-seq profiling of the MB2141 anorectal mucosal melanoma model following decitabine treatment in vitro and in vivo. Decitabine treatment resulted in robust induction of innate immune pathogen sensing, type I interferon signaling, antigen presentation, and other immune-associated gene expression programs. These data provide a transcriptional framework for understanding how DNA hypomethylating agents modulate immune gene expression in mucosal melanoma and support further investigation of decitabine as an immune-priming strategy for rare melanoma subtypes.

overall_design: RNA-seq profiling was performed on MB2141 anorectal mucosal melanoma cells treated with decitabine in vitro and MB2141 xenograft tumors treated with decitabine in vivo, each compared with matched untreated or vehicle-treated controls.


title: Resilience to bioprinting-induced cellular stress by heat shock preconditioning

city: Heidelberg

first: Ralph

last: Grand

address: Im Neunheimer feld 345

institute: Heidelberg University

summary: Bioprinting holds the promise to revolutionize therapeutic development and healthcare; however, we still lack an in-depth understanding of how cells respond to stress experienced during the bioprinting process. Here, we assess at the molecular and cellular level the effects of bioprinting-induced stress on cell morphology, metabolism, and behavior using human umbilical vein endothelial cells. We demonstrate that mild preconditioning of the stress response in cells using heat shock before bioprinting helps cells to rapidly respond to bioprinting-associated stress, mitigating negative effects. We hypothesize this is in part mediated by the small heat shock protein 27 (Hsp27) which is known to play a role in stabilizing actin in the cytoskeleton. This results in restoration of cellular viability, morphology, metabolism, and function, including vascularization. Our study outlines an easy-to-implement strategy to improve cell viability following bioprinting, facilitating the real-life application of bioprinting.

overall_design: Cells were bioprinted with and with out mild heat shock pre-conditioning. RNA was isolated at 1 hour and 14 hours post-printing.

first: Erin

last: Spiller

first: Martina

last: Capriati

first: Jule

last: Zimmermann

middle: S

first: Daniela

middle: F

last: Duarte Campos


title: RNA-seq profiling of control and treatment breast cancer cell-line samples

city: Hefei

first: Jun

last: Xu

state: Anhui

address: 17 Lujiang Road

institute: The First Affiliated Hospital of USTC, Anhui Provincial Hospital

summary: Bulk RNA sequencing was performed on human breast cancer cell-line samples to compare transcriptomic profiles between control and treatment conditions.

overall_design: Six cell-line RNA-seq libraries were analyzed, including three control biological replicates and three treatment biological replicates. Gene expression values were provided as a processed FPKM matrix.


title: RNA-seq profiling of treatment-sensitive and treatment-resistant breast cancer cell-line samples

summary: Bulk RNA sequencing was performed on human breast cancer cell-line samples to compare transcriptomic profiles between treatment-sensitive and treatment-resistant conditions.

overall_design: Six cell-line RNA-seq libraries were analyzed, including three treatment-sensitive biological replicates and three treatment-resistant biological replicates. Gene expression values were provided as a processed FPKM matrix.


title: RNA-seq profiling of breast cancer cell-line samples with LRIG1 and EGFR L747A manipulation

summary: Bulk RNA sequencing was performed on human breast cancer cell-line samples to compare transcriptomic profiles among control, LRIG1-minus, LRIG1, and LRIG1 plus EGFR L747A conditions.

overall_design: Twelve cell-line RNA-seq libraries were analyzed, including control, LRIG1-minus, LRIG1, and LRIG1 plus EGFR L747A groups with three biological replicates per group. Gene expression values were provided as a processed FPKM matrix.


title: RNA-seq profiling of FFPE samples from treatment-sensitive and treatment-resistant breast cancer cases

summary: Bulk RNA sequencing was performed on formalin-fixed paraffin-embedded (FFPE) tumor samples to compare transcriptomic profiles between treatment-sensitive and treatment-resistant breast cancer cases.

overall_design: Ten FFPE tumor samples were analyzed by bulk RNA-seq, including five treatment-sensitive samples and five treatment-resistant samples. Gene-level raw counts were provided as processed data.


title: The Small Non-coding RNA MicC is a Critical Regulator of the Stress Resistance and Virulence of Extraintestinal Pathogenic Escherichia coli

city: Salt Lake City

first: Matthew

last: Mulvey

state: Utah

address: 1390 Presidents' Circle

department: Biology

institute: University of Utah

summary: Small non-coding RNA (sRNA) molecules can modulate diverse bacterial functions ranging from carbon metabolism to virulence gene expression. Here, we used a series of deletion strains to probe a panel of seven conserved sRNAs (DsrA, RprA, OxyS, RyhB, MicF, MicC, and Spot 42) of Extraintestinal Pathogenic Escherichia coli (ExPEC) isolate UTI89 for effects on the response to oxidative stress. ExPEC strains are the primary etiologic agents of urinary tract infections, which consistently rank among the most common infectious diseases and are a leading cause of sepsis. In broth culture, each sRNA deletion knockout grew like the wild-type strain, whereas the spot42 (spf) and micC knockout strains were both markedly impaired in their ability to deal with oxidative stress generated by methyl viologen. UTI89ΔmicC had several additional in vitro and in vivo phenotypes important for pathogenesis. in vitro, the micC knockout had impaired motility, reduced association with and survival within bladder epithelial cells (BECs), and reduced resistance to human serum. in vivo, the micC knockout was defective in its ability to colonize and persist in the mouse urinary tract, gastrointestinal tract, and bloodstream. RNA sequencing of UTI89ΔmicC as well as micC overexpressed using an inducible plasmid revealed differential expression of many genes, especially within metabolism and motility pathways. Notably, expression of the maltose transport operon malEFG was significantly reduced in UTI89ΔmicC, as was expression of the structural flagellar genes flgC and flgE. ompC, the only known target of MicC, did not have increased expression in the deletion, but did have significantly decreased expression in the micC overexpression strain. Here, we demonstrate that the conserved ExPEC sRNA MicC serves as a critical regulator of both stress resistance and virulence in a mammalian host, extending further the enigmatic functions of this bacterial sRNA.

overall_design: Overnight cultures of UTI89/pRRK1, UTI89ΔmicC/pRRK1, and UTI89ΔmicC/pMicC were back diluted 1:100 into 5 mL LB and grown for 3 hours 15 min at 37°C, shaking 225 rpm. At the two-hour growth mark IPTG was added to each culture to a final concentration of 0.1 mM. 1.7 mL of each culture was pelleted, and RNA was extracted using a PureLinkTM RNA Mini Kit (Invitrogen) and TRIzol (Life Technologies). RNA sequencing was performed through the University of Colorado Anschutz Genomics and Microarray Core. Sequences were processed using BBduk v37.10 to trim adapters. Reads were then mapped to UTI89 reference strain GCF_000013265 using bowtie2 v2.5.3 and the very-sensitive argument. Samtools v1.20 was used to read features. Differential gene expression was analyzed using the DESeq2 package in R v.4.4.0.

first: O'Connor

last: Matthews

first: Brittany

last: Fleming

first: Kevin

last: Nelson

first: Alejandra

last: Mendez

first: Alan

last: Stenquist

first: Sam

last: Bonkowsky

first: Richard

last: Kulesus

first: William

last: Brazelton

last: Blango


title: Inflammation-triggered sensing and miRNA immunotherapy with a wireless battery-free hydrogel bioelectronic patch

city: Hangzhou

first: Yi

address: Qiutao Street 166

institute: Zhejiang University

summary: Early diagnosis and timely intervention are crucial for the management of periodontal diseases driven by immune dysregulation. However, in situ sensing and drug delivery in the oral cavity remain challenging due to biological disparities between electronic devices and oral tissues. Here, we report a flexible, battery-free, and hydrogel-based bioelectronics that integrates biological functions and wireless signal transmission for periodontal inflammation monitoring and localized microRNA (miRNA) immunomodulation. The hydrogel, synthesized from gelatin with bioactive peptide sequences cleavable by matrix metalloproteinases (MMPs), responds specifically to protease activity associated with periodontal disease progression. Its enzymatic degradation is wirelessly transduced into quantitative radio-frequency signals of local immune activity, while simultaneously triggering the release of embedded miRNAs as critical immune regulators. In vitro and animal validations demonstrated diagnostic accuracy and therapeutic efficacy, leading to suppression of the inflammatory response. The combination of biosensing, wireless feedback, and miRNA-based immunotherapy provides a closed-loop, adaptable platform for personalized management of periodontal and other inflammation-related disorders.

overall_design: To investigate the effects of let-7c-5p transfection into RAW264.7 cells on macrophage polarization and corresponding phenotypic functions

first: Zhenghan

last: Shi

first: Lingkai

last: Su


title: Smart-seq2 analysis of tdTomato-positive neurons from LH+ NR and LH+ Run mice

city: Guangzhou

first: Can-Yuan

last: Zhang

state: Guangdong

address: No.1023-1063 South Shatai Road, Baiyun District, Guangzhou City, Guangdong Province, P.R.China

department: Department of Neurobiology, School of Basic Medical Sciences

institute: Southern Medical University

summary: Newborn neurons in the adult hippocampal dentate gyrus (DG) are hyperexcitable, competitive cells involved in behavioral responses to stress and antidepressants. The role and the mechanisms underlying the competitiveness of the newborn neurons in moderating behavioral responses to stress have not yet been comprehensively inves-tigated. Using FosTRAP2 transgenic mice in the Learned Helplessness (LH) paradigm, we report that voluntary running, a guideline-supported intervention for depression, induces neurogenesis, and that the newly generated neurons compete with LH-TRAPed neurons, thereby reducing stress-induced helpless behavior. Mechanistically, we found that increasing neurogenesis is sufficient to reduce spine density in LH-TRAPed neurons via p21-activated kinase 4 (Pak4) and to attenuate Ca2+ activity in these neurons. Taken together, our findings suggest that highly excitable newborn neurons compete with LH-TRAPed neurons, attenuating helpless behaviors and providing therapeutic value for depressed individuals, who typically show enhanced memory for negative events. This GEO submission contains Smart-seq2 transcriptomic data from FACS-isolated tdTomato-positive neurons from LH+ NR and LH+ Run mice.

overall_design: After the learned helplessness paradigm and 28 days of voluntary running, Fos-iCreERT2; Rosa26-LSL-tdTomato mice were assigned to the LH+ NR and LH+ Run groups. LH-TRAPed tdTomato-positive neurons were isolated by fluorescence-activated cell sorting and subjected to full-length single-cell RNA sequencing using the Smart-seq2 protocol.

first: CanYuan

first: SuFen

last: Wei

first: LiangYu

last: Chen

first: WeiZhong

last: Cai


title: Postoperative delirium in hip fracture patients linked to epigenetic alterations in inflammatory and immune pathways: a genome-wide DNA methylation study

city: Palo Alto

first: Shota

last: Nishitani

state: CA

address: 3165 Porter Dr.

department: Department of Psychiatry and Behavioral Sciences

institute: Stanford University

type: Methylation profiling by genome tiling array

summary: Delirium is a common yet underdiagnosed condition in elderly hospitalized patients. The lack of effective diagnostic and therapeutic methods can be attributed to the limited understanding of its pathophysiology. Delirium has recently been reported to be linked to neuroinflammation and epigenetic changes. The aim of this study was to validate the pathways in a larger cohort with a uniform type of surgery, while rigorously adjusting for potential covariates. This study primarily investigated DNA methylation (DNAm) changes before and after surgery in postoperative delirium (POD) among older adults having undergone femoral fracture surgery. After propensity analysis, 65 subjects were divided into 2 subgroups; one consisted of subjects who had blood samples collected preoperatively and on postoperative day 0, and the other consisted of those who had samples collected preoperatively and on postoperative day 3. We performed differential methylation analysis and enrichment analysis on each subgroup. Enrichment analysis using CpGs that exhibited substantial DNAm changes between pre- and postoperative day 0 samples in POD cases showed inflammation- and immunity-related pathways such as "leukocyte mediated immunity" and "NF-kappa B signaling pathway." Inflammation- and immunity-related pathways became less noticeable between pre- and postoperative day 3 samples. Inflammation- and immunity-related pathways showed in this study align with previous studies across diverse populations, reinforcing the role of inflammation- and immunity-related epigenetic mechanisms in delirium including POD. Notably, these DNAm changes were potentially transient, corresponding to the typical onset of delirium, suggesting their potential as biomarkers for early diagnosis.

overall_design: Bisulfite-converted whole blood DNA were hybridized into the Illumina Infinium Methylation EPIC Kit array version 2

first: Gen

last: Shinozaki


title: EHMT1/KMT1D drives neointima formation via epigenetic repression of the PPARγ–PDIA6 axis

city: Wuhan

first: Ding-Sheng

last: Jiang

state: None Selected

address: 1095 Jiefang Ave., Wuhan 430030, China

institute: Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology

summary: Neointima formation underlies restenosis and other occlusive vascular diseases, yet the epigenetic regulators that drive this process remain poorly understood. We aimed to define the role of the histone methyltransferase EHMT1 in neointima formation and to delineate its downstream mechanisms. Here, we found that the EHMT1 expression and its histone marks H3K9me1/2 were markedly upregulated in proliferating VSMCs and in injured arteries exhibiting neointima formation. Deletion of EHMT1 in VSMCs suppressed phenotypic switching and significantly reduced neointimal hyperplasia in vivo. In vitro, EHMT1 knockdown inhibited, whereas EHMT1 overexpression promoted, VSMC proliferation, migration, and acquisition of a synthetic phenotype. Unbiased transcriptome sequencing revealed significant enrichment of peroxisome proliferator-activated receptor gamma (PPARγ) target genes following EHMT1 knockdown. Mechanistically, we demonstrated that EHMT1 directly repressed PPARγ expression via H3K9 dimethylation, and pharmacological activation of PPARγ with rosiglitazone reversed the proliferative, migratory, and synthetic effects induced by EHMT1 overexpression. Further analysis identified protein disulfide isomerase A6 (PDIA6) as a direct PPARγ target: PPARγ bound to the PDIA6 promoter (-567 to -385 bp) to suppress its expression. Overexpression of PDIA6 rescued the inhibitory effects of EHMT1 knockdown on VSMC proliferation and migration, whereas PDIA6 silencing abrogated the pro-proliferative and pro-migratory effects of EHMT1 overexpression. EHMT1 acts as a critical epigenetic regulator of neointima formation by controlling VSMC phenotypic switching through a novel EHMT1–PPARγ–PDIA6 axis. These findings provide mechanistic insight into epigenetic regulation of vascular remodeling and identify EHMT1–PPARγ–PDIA6 as a promising target for therapeutic intervention in restenosis.

overall_design: RNA-seq profiling of human aortic smooth muscle cells (HASMCs) transfected with lentiviral-shRNA or shEHMT1.

first: Hanshen

last: Luo

first: Jingjie

first: Yanxin

last: Huang

first: Bo

last: Huo

first: Xian

last: Guo

first: Xingliang

last: Wu

first: Liyuan

last: Liu

first: Wenxin

last: Lin

first: Xin

last: Feng

first: Ze-Min

last: Fang

last: Yi


title: Methodological Considerations for Studying HOXA10, HOXA11 and LIF Expression in Unexplained Infertility: A Pilot Study in Colombian Women

city: Bogota

first: Maria

middle: Camila

last: Rincon

state: Bogota

department: Biomedical Engineering

country: Colombia

institute: Universidad de los Andes

summary: This study investigates the transcriptomic profile of endometrial tissue from patients with idiopathic infertility, focusing on the expression of implantation- and inflammation-related genes, including HOXA10, HOXA11, and LIF, PKHD1L1, HS6ST1, LTF, CCDC1441. This study also focuses on identifying potential molecular markers that may be associated with unexplained infertility. RNA sequencing (RNA-seq) was performed to evaluate gene expression patterns and their potential association with impaired endometrial receptivity. These data provide valuable insights into the molecular mechanisms underlying idiopathic infertility and represent a useful resource for further studies aimed at improving diagnosis and therapeutic strategies in reproductive medicine.

overall_design: Gene expression profiling analysis of RNA-seq data from endometrium biopsies of controls and patients with unexplained infertility.

last: Rincon-Garcia

first: Veronica

last: Almeida Toro

first: Fernando

last: Gomez-Corredor

first: Daniela

last: Rubio

first: Johanna

last: Stepanian

first: Juan

last: Cruz

first: Jorge

last: Duitama

first: Carolina

last: Muñoz-Camargo


title: Transcriptional signatures indicating RB function may predict response to CDK4/6 inhibition in endometrial cancer [WES]

city: Boston

first: Sarah

middle: J.

last: Hill

state: MA

address: 450 Brookline Ave., Dana Building, Room 1410C

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary: RNA sequencing was performed on RNA samples from endometrial cancers from patients who participated in two different abemaciclib combination clinical trials. For a subset of patients, we also generated genomic DNA from the tumor samples and performed whole exome sequencing. The goal was to determine if the tumors had functional or dysfunctional RB using different transcriptional signatures and then to determine if the RB functional status of the tumor could predict patient response to the CDK4/6 inhibitor in the clinical trial.

overall_design:
Genomic DNA was prepared from formalin fixed paraffin embedded endometrial tumors scraped from slides. DNA underwent whole exome sequencing and analysis. Methods are provided with each patient sample directly from the linked manuscript. For additional details, please see the linked manuscript.

***************************************************************
Due to privacy concerns, we are only providing processed data.
***************************************************************

first: Zelei

last: Yang

first: Elizabeth

last: Partan

first: Shannan

last: Ho Sui


title: Transcriptional signatures indicating RB function may predict response to CDK4/6 inhibition in endometrial cancer [RNA-Seq]

overall_design:
Formalin fixed paraffin embedded endometrial tumor tissue was scraped from slides, and RNA was prepared from this tissue. RNA underwent RNA sequencing and subsequent analysis. Methods are provided with each patient sample directly from the linked manuscript. For additional details, please see the linked manuscript.

***************************************************************
Due to privacy concerns, we are only providing processed data.
***************************************************************


title: Recipient Endothelial IRF1 mediates IFNgamma-driven tissue tolerance in acute Graft-versus-Host Disease

city: Columbus

first: Katherine

middle: E

last: Miller

address: 700 Children's Dr.

department: Institute for Genomic Medicine

institute: Nationwide Children's Hospital

summary: Acute graft-versus-host disease (GVHD) arises from dysregulated donor T cell mediated immune interactions against the host, following allogeneic hematopoietic cell transplantation (allo-HCT).  However, mechanisms via which non-hematopoietic host tissues actively shape post-transplant outcomes remains incompletely defined. Here, we identify endothelial cell (EC)-intrinsic interferon-g (IFNg)—IRF1 signaling as a key pathway controlling vascular remodeling and immune tolerance after allo-HCT. Using single cell RNA sequencing of liver ECs from syngeneic and allogeneic transplant recipients, we reveal that liver ECs undergo rapid, subset-specific transcriptional reprogramming after allo-HCT, marked by strong induction of IFNg-responsive pathways. We show IRF1 is a key upstream regulator, linking IFNg signaling to endothelial activation and functional remodeling. Both in vitro and in vivo studies reveal that endothelial intrinsic IFNg—IRF1 signaling is dispensable for Th1 differentiation but required for endothelial-driven Treg expansion. Loss of endothelial IRF1 in vivo uncouples vascular integrity from endothelial-mediated tolerance, reducing endothelial activation and apoptosis but exacerbating GVHD due to impaired Treg induction. Our findings suggest that therapeutic strategies that preserve endothelial IRF1-dependent tolerogenic programs may reduce GVHD severity while maintaining protective alloimmune responses.

overall_design: Single cell RNA-sequencing (scRNA-seq) was performed to characterize transcriptional changes in endothelial cells from the liver of mice who received either a syngeneic or allogeneic hematopoietic stem cell transplant. Endothelial cells were collected 4 days or 14 days after transplant and isolated from the liver via flow staining and sorting for Cd45- Cd11b+ Cd31+. Up to 10,000 cells per condition were targeted for scRNA-seq via the 10x Genomics Flex singleplex kit (BC001) following the manufacturer’s protocol. Libraries were sequenced on an Illumina NovaSeq6000. Raw BCL files were converted to FASTQ format using Cell Ranger v8.0.0 mkfastq, and alignment to the mm10 mouse probe set was performed using Cell Ranger v8.0.0 multi with default settings, including filtering, barcode counting, and UMI quantification. Downstream analysis was conducted in R v4.3.3 using Seurat v5 [PMCID: PMC10928517].

first: Lotus

last: Neidemire-Colley

first: Rathan

last: Kumar

first: Malith

last: Karunasiri

first: Elizabeth A

last: Garfinkle

first: Adithe

last: Rivaldi

first: Katherine E

first: Parvathi

last: Ranganathan


title: A Phase 1 clinical trial and single-cell correlates of motixafortide, cemiplimab, gemcitabine and nab-paclitaxel for metastatic treatment-naïve metastatic pancreatic ductal adenocarcinoma.

city: New York

first: Edridge

middle: Kevin

last: D'Souza

state: NY

address: 1130 St Nicholas Ave

department: Hematology/Oncology

institute: Columbia University

summary: The C-X-C motif chemokine receptor 4 (CXCR4)/C-X-C motif chemokine ligand 12 (CXCL12) axis is a well-established contributor to the immunosuppressive and immune-excluded TME in pancreatic adenocarcinoma (PDA). Building on pre-clinical data demonstrating a survival benefit with the addition of gemcitabine to CXCR4 and PD1 inhibition in the KPC mouse model, we conducted an open-label, single-arm phase 1 clinical trial combining CXCR4 inhibition (motixafortide), PD-1 blockade (cemiplimab), and chemotherapy (gemcitabine/nab-paclitaxel; MCGN) in treatment-naïve patients with metastatic PDA (n=11). MCGN was safe and tolerable, achieving a 64% partial response (PR) by iRECIST, a 55% confirmed partial response (cPR), and a 91% disease control rate (DCR). The median PFS and OS were 9.7 months (95% confidence interval [CI]: 5.9-not reached [NR]) and 10.1 months (95% CI: 9.3-NR), respectively. One patient achieved a pathological complete response within the primary tumor and the hepatic metastasis after undergoing a pancreatoduodenectomy and hepatectomy and has remained free of disease for 18 months, suggesting that durable responses are possible with MCGN treatment. Single-nucleus RNA-sequencing of serial tissue biopsies from all trial participants revealed a reduction in transcriptional heterogeneity and depletion of cells expressing epithelial-to-mesenchymal transition states, while treatment-resistant tumors maintained tumor heterogeneity. The presence of CXCL12+ proaxogenic Cancer Associated Fibroblasts (pCAFs) were predictive of MCGN efficacy and depleted in resistant samples, suggesting that they represent the substrate for treatment response. MCGN also induced a highly inflamed tumor-microenvironment, which we confirmed with serial tissue staining, and rescue of T cell dysfunction. Based on these promising data and biomarkers, we have launched a multicenter randomized phase 2 trial comparing MGCN to GN in patients with treatment-naïve metastatic PDA (NCT04543071), which is ongoing.

overall_design:
We loaded 0.9-1.5e3 nuclei in ST buffer without RNAse inhibitor using a Chromium controller and 5’ chemistry v2 capture (10X Genomics, #1000263). We prepared cDNA libraries according to manufacturer instructions for reverse transcription and cleanup, and performed an extra cycle of cDNA amplification to compensate for the reduced total RNA content when analyzing single nuclei compared to single cells. We prepared final sequencing libraries with the 10X Library Construction Kit (#1000190) and Dual Index Kit TT Set A (#1000215) and quantified them using D1000 and D5000 reagents (Agilent, #5067-5583 and #5067-5589) on an Agilent 2200 TapeStation system. We then mixed samples and sequenced to a target depth of at least 20,000 reads per droplet using NovaSeq S4 (Illumina, San Diego, CA) with at least 2x100bp read length.

***************************************************************
Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access.
***************************************************************

first: Alexander

last: Raufi

middle: K

first: Michael

last: May

last: Sedycias

first: Ilenia

last: Pelliciotta

first: Samuel

last: Pan

first: Shikun

last: Wang

first: Carmine

last: Palermo

first: Steven

last: Sastra

first: Naomi

last: Sender

first: Isabelle

last: Ross

first: Li

last: Li

first: Karan

last: Luthria

first: Parin

last: Shah

first: Somnath

last: Tagore

first: Zachary

last: Walsh

first: Andrew

last: Turk

first: Sema

last: Bircan

first: Yashaswi

last: Manneru

first: Alina

last: Iuga

first: Sun

last: Dajiang

last: Kluger

first: Beth

last: Schrope

first: Dane

last: Brown

first: Tito

last: Fojo

first: Juanma

last: Schvartzman

first: Yoanna

last: Pumpalova

first: Ryan

last: H Moy

first: Sysan

last: Bates

first: Linda

last: Wu

first: Hanina

last: Hibshoosh

first: Aik

last: Tan

first: John

last: Chabot

first: Kenneth

middle: P

last: Olive

first: Benjamin

last: Izar

first: Gulam

last: Manji


title: Identifying potential biomarkers for mild COVID-19 in saliva by miRNA profiling using NGS and ddPCR

city: San Cristobal de La Laguna

first: Malena

last: Gajate Arenas

address: Astrofisico Sanchez Avenue

country: Spain

institute: Universidad de La Laguna

summary: miRNAs represent promising biomarkers for several diseases, including viral respiratory infections. Saliva screening for miRNA expression was performed using NGS, followed by a validation step through RT-qPCR and ddPCR. Our data revealed that miRNAs are found to be dysregulated in early stages of SARS-CoV-2 infection.

overall_design:
RNA-seq profiling in saliva samples from COVID-19 cases and healthy control. The most dysregulated miRNAs from NGS are analyzed by RT-qPCR and ddPCR.

***************************************************************
We have had a problem with the hard drive used to store the raw sequencing data, and so far, we haven't been able to recover them
***************************************************************

first: Gajate-Arenas

last: Malena

first: Córdoba-Lanús

last: Elizabeth


title: Lymphoid structures in the skull bone marrow control homeostatic and anti-tumor immune responses in the CNS [dura/skull/sternum]

city: St. Louis

first: Jonathan

last: Kipnis

state: MO

address: 4515 McKinley Ave

institute: Washington University in St. Louis

summary: Accumulating evidence demonstrates that the central nervous system (CNS) is not disconnected from the peripheral immune system; however, precisely how the adaptive immune system surveils the CNS remains a critical question. Recent findings reveal that channels between the dura mater and the skull facilitate the exchange of cerebrospinal fluid and immune cells between the CNS and skull bone marrow (BM) under both homeostatic and disease conditions. Skull BM serves as a source of innate immune cells for the CNS, yet its role in adaptive immune responses remains insufficiently characterized. Here, we identify lymphoid structures within the skull BM, featuring germinal center-like formations and harboring a distinct population of follicular helper-like T cells that promote B cell activation and humoral immunity through CD40L, IL-21, and IFN-γ signaling. Adaptive immune cells within these skull BM lymphoid structures surveil and respond to CNS-derived antigens, which we demonstrate to be critical for optimal antitumor immunity in murine brain cancer models. Our discovery of distinct anatomical sites in the skull BM that enable adaptive immunosurveillance of the CNS highlights promising avenues for immunotherapies targeting neurological diseases, including brain cancers.

overall_design: 10X genomics was used to measure single cell RNA sequencing (scRNA-seq) to characterize T cells in the skull, sternum bone marrow, and dura. Live CD45+B220-CD11b-Thy1.2+CD4+CD8+ cells were sorted with FACS Aria II.

first: Jang Hyun

last: Park

first: Daviti

last: Abramishvili

first: Ruben

last: Silva

first: Gustavo

middle: G

last: Davanzo

first: Xingxing

last: Gu

first: Siling

last: Du

first: Daniel

last: Lee

first: Bernd

middle: H

last: Zinselmeyer

first: Jackson

last: Turner

first: Gwendalyn

middle: J

last: Randolph

first: Igor

last: Smirnov


title: Low physiological pH drives P300-mediated acetylation of PARP1 and promotes PARP inhibitor resistance [RNA-seq]

city: Houston

first: Yuan

last: Qi

state: TX

address: 1400 Pressler St

department: Bioinformatics & Computational Biology

institute: University of Texas M.D. Anderson Cancer Center

summary: Resistance to PARP inhibitors (PARPi) remains a major clinical obstacle in epithelial ovarian carcinoma (EOC), ultimately limiting therapeutic efficacy and contributing to patient mortality. Here, we demonstrate that the acidic tumor microenvironment drives a novel mechanism of PARPi resistance. Decreasing the pH is sufficient to reduce PARPi anti-tumor efficacy in multiple in vitro and in vivo EOC models. Through three independent, epigenetically focused CRISPR/Cas9 screens conducted under low pH conditions, we identified p300 as a druggable target for overcoming pH-induced PARPi resistance. Mechanistically, in an unbiased functional proteomic evaluation we uncovered an ERK1/2–p300–PARP1 signaling axis activated under low pH, which alleviates PARPi-induced PARP1 trapping by promoting PARP1 K505 acetylation and thereby reducing replication fork collapse and associated DNA damage and driving PARPi resistance. Notably, in multiple in vivo patient-derived and syngeneic EOC models, two novel p300 bromodomain inhibitors, IACS-16559 and TT125-802, restore sensitivity to PARPi. Together, our findings establish p300 as a promising therapeutic target for overcoming pH-driven PARPi resistance in EOC.

overall_design: RMG1 cells cultured at the pH 7.4 or pH 6.5 medium were treated with vehicle (DMSO) or P300 inhibitors, CCS-1477 and IACS-16559 for 48h, then the cells were harvested and performed RNA-seq.

first: Rugang

last: Zhang

first: Liping

last: Liao

first: Hao

last: Nie


title: Mining phage-genomes for regulatory elements to develop a SynBio toolbox for non-model Pseudomonads

city: Heverlee

first: Rob

last: Lavigne

address: Kasteelpark Arenberg 20

department: Biosystems

country: Belgium

institute: KU Leuven

summary: Bacteriophages represent a largely untapped reservoir of regulatory elements that can expand the genetic toolbox for engineering non‑model hosts. Using ONT‑cappable‑sequencing, we mapped full‑length primary transcripts across five Pseudomonas phages and identified 232 transcription start sites (TSSs) and 176 transcription termination sites (TTSs), including 38 intrinsic factor‑independent terminators. Motif discovery revealed 59 s70-like promoters in KIL5, 37 in KIL3b, and unique phage‑encoded RNA polymerase (RNAP) promoter motifs for KNP, ϕ2, and pphageB21, enabling discrimination between host‑ and phage‑dependent transcriptional control. To verify these predictions, a subset of ten promoters and five terminators was quantitatively characterized in vivo in P. fluorescens strain GL-S-306, demonstrating promoter activities spanning a large dynamic range and terminator efficiencies allowing complete transcriptional termination. These data establish the first experimentally validated, phage‑derived library of promoters and terminators for P. fluorescens and P. syringae, providing chassis‑specific regulatory parts that enable more finetuned gene‑expression than current cross‑species SynBio tools. Our results position phage transcriptomics as a scalable strategy for mining regulatory elements tailored to non‑model bacterial hosts and accelerate the development of new synthetic biology platforms.

sample_organism:
Pseudomonas fluorescens
Pseudomonas savastanoi pv. phaseolicola
Pseudomonas coronafaciens pv. porri
Pseudomonas phage phi2
Pseudomonas phage vB_PsyM_KIL3b
Pseudomonas phage vB_PsyM_KIL5
Pseudomonas phage KNP
Pseudomonas phage pphageB21

overall_design: ONT-cappable-sequencing of 5 Pseudomonas phages (KIL3b, KIL5, pphage B21, f2 and KNP) and their bacterial hosts

first: Jorien

last: Poppeliers

first: Maarten

last: Boon

first: Alison

last: Kerremans


title: Low physiological pH drives P300-mediated acetylation of PARP1 and promotes PARP inhibitor resistance [Cut & Run]

overall_design: RMG1 cells were treated with vehicle (DMSO) or P300 inhibitors, CCS-1477 and IACS-16559 for 48h, then the cells were harvest and perform CUT&Run seq, using IgG and an anti-P300 antibody

first: Chen

last: Wang


title: A Bivalent Molecular Glue Linking Lysine Acetyltransferases to Oncogene-directed Cell Death [CUT&RUN]

city: Stanford

first: Sai

last: Gourisankar

address: 240 Pasteur Dr, Rm 4700

department: Chemical Systems Biology

institute: Stanford

summary: Lysine acetyltransferases (KATs) such as p300 and CBP regulate gene networks in healthy and malignant cells by scaffolding transcriptional machinery and by acetylating chromatin and transcription factors (TFs). In certain cancers including diffuse large B cell lymphomas, they contribute to tumor suppression and are therapeutic vulnerabilities when deregulated. Inhibitors and degraders of KATs are limited by potential toxicity associated with targeting these essential proteins. We introduce KAT-TCIPs (lysine acetyltransferase transcriptional/epigenetic chemical inducers of proximity), bivalent molecules that redirect the KAT and co-activating activity of p300 and CBP towards programmed cell death signaling repressed by the oncogenic TF BCL6. The lead KAT-TCIP molecule kills lymphoma cells at ~0.8 nM by redistributing chromatin acetylation, activating the pro-apoptotic protein PUMA, and repressing c-MYC. Analysis of the ternary co-crystal structure containing p300 and BCL6 reveals fortuitous interactions that drive the potency and specificity of KAT-TCIPs and sets a precedent for KAT-targeting induced proximity therapeutics.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone acetylation (H3K27ac and H2BK20ac) after TCIP3 treatment in SUDHL5 cells

first: Meredith

middle: N

last: Nix

first: Roman

last: Sarott

first: Brendan

last: Dwyer

first: Sabin

last: Nettles

first: Michael

middle: M

last: Martinez

first: Hind

last: Abuzaid

first: Yanlan

first: Haopeng

last: Yang

first: Juste

last: Simanauskaite

first: Bryan

last: Romero

first: Hannah

last: Jones

first: Andrey

last: Krokhotin

first: Tara

last: Loewinson

first: Lei

last: Chen

first: Mark

last: Davis

last: Fernandez

first: Tinghu

middle: R

last: Green

first: Stephen

last: Hinshaw

first: Nathanael

last: Gray

first: Gerald

last: Crabtree


title: R-loop maintains the deposition of H3K36me3 to prevent invasion of H3K4me3 during SN-oocyte development [4]

first: Ying

middle: liang

last: Sheng

address: No. 9 XingDaoHuanBei Road, Guangzhou International Bio Island

institute: Guangzhou National Laboratory

summary: Establishment of epigenetic modifications in an orderly manner is crucial for oocyte maturation and embryonic development. R-loop (DNA/RNA hybrids) plays an important role in regulating genome stability, epigenetic modifications and gene expression. However, the specific functions and regulatory mechanisms of R-loop on the crosstalk between histone modifications during oocyte development remain unclear. Here, we find that loss of R-loop by overexpressing Rnaseh1 significantly slow down oocyte development. R-loop loss in germinal vesicle (GV) oocyte leads to downregulation of H3K36me3 at key oocyte developmental gene bodies, aberrant invasion of H3K4me3 into H3K36me3-marked regions, resulting in dysregulated gene expression and ultimately hindering oocyte development. Importantly, this dysregulated gene expression can be rescued by histone H3K36me3 methyltransferase SETD2 targeting on specific R-loop-dependent H3K36me3-marked regions. Together, this finding reveals that R-loop acts as a crucial regulator in controlling epigenetic crosstalk of H3K36me3 and H3K4me3 in maternal genome, ensuring normal oocyte development.

overall_design: RNA-seq after overexpression of Rnaseh1 and mCherry in NSN and SN oocytes.

first: Yingliang

first: Tianyan

last: Guo

first: Hongjie

last: Yao


title: Phage-encoded sRNA counteracts xenogeneic silencing in pathogenic E. coli

city: Sydney

first: Jai

middle: Justin

last: Tree

state: NSW

address: Rm s110 Bldg F25, UNSW, Gate 11 Botany St

department: School of Biotechnology and Biomolecular Sciences

institute: University of New South Wales

summary: Horizontal gene transfer introduces foreign DNA that can disrupt cellular processes and is therefore subject to xenogeneic silencing by nucleoid-associated proteins such as H-NS and Hha. In Enterohaemorrhagic Escherichia coli (EHEC), prophages make up a large fraction of the accessory genome and encode many virulence factors, yet their expression must overcome this silencing. We identify a prophage-encoded small RNA (sRNA), HnrS, that functions as an anti-silencing factor by targeting the H-NS paralogue Hha. HnrS is a short (66-nt) sRNA present in up to nine copies in EHEC and EPEC genomes and is enriched in LEE⁺ E. coli strains. We show that HnrS directly base-pairs with the ribosome-binding site of hha mRNA, repressing its translation and reduces Hha-enhanced silencing. This leads to de-repression of the locus of enterocyte effacement (LEE) type III secretion system and repression of motility. Transcriptomic profiling further revealed that HnrS activates genes involved in nitrate/nitrite respiration and nitric oxide resistance, metabolic pathways the contribute to survival in the inflamed gastrointestinal tract. Deletion of hnrS reduced expression of nitrate reductase genes and impaired actin pedestal formation on host epithelial cells. Our results indicate that multiple hnrS copies encoded on prophages provide a counter-silencing mechanism that reduces Hha–H-NS repression at specific virulence loci. This likely enables expression of horizontally acquired genes without broadly disrupting the global H-NS regulon. HnrS illustrates how mobile genetic elements can deploy sRNAs to counteract xenogeneic silencing and promote virulence gene expression, enhancing colonisation of the host.

overall_design: Total RNA-seq on wild type EHEC str. Sakai, an isogenic triple deletion of hnrS (EcOnc10, EcOnc11, EcOnc12), and the complemented deletion strain (pZE12::hnrS)

first: Pranita

last: Poudyal

first: Daniel

last: Mediati


title: Single-cell mapping of chromosome breaks identifies multiple fragile site classes with distinct DNA replication timing landscapes

city: Kobe

first: Jothivanan

last: Elumalai

state: Hyogo

address: 2-2-3, Minatojima Minamimachi, Chuo-ku

department: Biosystems Dynamics Research (BDR)

country: Japan

institute: RIKEN

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series

first: Ichiro

last: Hiratani


title: Lymphoid structures in the skull bone marrow control homeostatic and anti-tumor immune responses in the CNS [skull/sternum]

first: Jonathan

last: Kipnis

overall_design: 10X genomics was used to measure single cell RNA sequencing (scRNA-seq) and TCR or BCR sequencing to characterize T cells and B cells in the skull, sternum bone marrow. Live CD45+CD3e-CD138-CD19+ B cells and CD45+IgD-CD3e-CD138+TACI+ antibody-secreting cells (ASCs) were sorted with FACS Aria II.

first: Jang Hyun

last: Park

first: Daviti

last: Abramishvili

first: Ruben

last: Silva

first: Gustavo

last: Davanzo

first: Xingxing

last: Gu

first: Siling

last: Du

middle: D

last: Lee

first: Bernd

last: Zinselmeyer

first: Jackson

last: Turner

first: Gwendalyn

last: Randolph

first: Igor

last: Smirnov


title: Mapping intasome integration sites and nucleosome dyads in chromatinized yeast DNA

city: London

first: Peter

last: Cherepanov

address: 1 Midland Road

country: United Kingdom

institute: The Francis Crick Institute

summary: HIV-1 and other lentiviruses hijack the cellular chromatin-binding protein LEDGF/p75 to facilitate integration into active transcription units. However, the mechanism of chromatin engagement by lentiviral intasomes and the structural role of LEDGF/p75 in this process remain poorly understood. To address these gaps, we studied the activities of in vitro-assembled retroviral intasomes in the presence of chromatinized target DNA. While LEDGF/p75 was both essential and minimally sufficient to enhance lentiviral integration into chromatin containing histone H3 trimethylated on Lys36, it unexpectedly facilitated integration outside of the nucleosome core particles. LEDGF/p75 additionally inhibited integration into unmodified chromatin in a dose-dependent manner, promoting integration into naked DNA. The results establish the preference of lentiviral intasomes for linker DNA regions within H3K36Me3-enriched chromatin.

overall_design: These experiments addressed interactions between retroviral intasomes and near-by chromatin structure through mapping both integration sites and the nucleosome dyads simultaneously. To this end, yeast genomic was chromatinized using human histones including H4(S47C)-phenanthroline conjugate and H3 with or without trimethylation of Lys36. Following incubation with HIV-1, MVV, or PFV intasomes, the strand transfer products were fragmented using Fenton reaction, end-repaired, amplified by linker-mediated PCR and sequenced using Oxford Nanopore technology. The reads were mapped on yeast genome to determine the linked integration and Fenton fragmentation sites.

first: Nicola

last: Cook

first: Joshua

last: Hope


title: Comprehensive transcriptomic profiling reveals impaired polyamine metabolism as a contributor to age-related muscle decline.

city: Rome

first: Andrea

last: Bracaglia

address: Via Antonio Scarpa 16

country: Italy

institute: University of Rome La Sapienza

summary: Sarcopenia, the age-associated decline in skeletal muscle mass and function, remains mechanistically poorly understood, particularly at the spatial and fiber type-specific levels. Here, we present a spatial transcriptomic atlas of skeletal muscle from young and aged mice, resolving age-associated transcriptional reprogramming across distinct myofiber types and tissue compartments. Our analyses uncover coordinated alterations in sarcomeric organization, excitation-contraction coupling, and oxidative stress responses, together with fiber type-specific metabolic rewiring toward glycolytic or oxidative programs. We also delineate conserved molecular signatures across muscles and species, highlighting Car3 as a potential pan-muscle biomarker of sarcopenia. In this context, we identify selective downregulation of key enzymes in the polyamine biosynthetic pathway, including Amd1 and Smox, resulting in reduced spermidine levels in aged muscle. This decline in polyamine availability also impacts muscle-resident cell populations. Indeed, pharmacological inhibition of Amd1 in fibro-adipogenic progenitors (FAPs) recapitulates aging-associated phenotypes, including myofibroblast differentiation, extracellular matrix dysregulation, and impaired support for satellite cell-mediated myogenesis. Collectively, our work reveals spatially organized, fiber type-specific, and polyamine-linked mechanisms of muscle aging and highlights the polyamine pathway as a promising therapeutic target to counteract sarcopenia.

overall_design: Spatial transcriptomics of tibialis anterior cryosections derived from young (5 months-old) and old (24 months-old) C57BL/6 mice.

first: Veronica

last: Ruggieri

first: Luca

last: Madaro


title: Mutation-Agnostic Base Editing of the Progerin Farnesylation Site Rescues Hutchinson-Gilford Progeria Syndrome Phenotypes in Neuromuscular Organoids

city: Seoul

first: Eun-Ji

last: Kwon

address: 1, Gwanak-ro

country: South Korea

institute: Seoul National University

summary: Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare, fatal premature aging disorder caused by a de novo mutation in the LMNA gene that leads to the production of progerin, a farnesylated, pathogenic form of lamin A. Treatment with farnesyltransferase inhibitors achieves significant yet limited life extension, highlighting progerin farnesylation as a key pathogenic driver of HGPS. In this study, rather than correcting the single pathogenic point mutation, we introduce Farnesylation Amino acid Targeted Editing (FATE), a novel, mutation-agnostic precision genome editing strategy that selectively disrupts the farnesylation site of LMNA. Next-generation sequencing confirmed that FATE exclusively edits the LMNA locus without inducing off-target mutations or affecting other genes encoding farnesylated proteins. Using neuromuscular organoids (NMOs) derived from two isogenic pairs of human pluripotent stem cells (hPSCs) carrying the HGPS mutation (HGPS-hPSCs), we found perinuclear progerin accumulation exclusive to the muscular compartment to be associated with defective formation of DNA damage foci and loss of perinuclear heterochromatin. Notably, applying FATE to HGPS-hPSCs successfully abolished these muscle-specific pathologies in subsequently-derived NMOs. Direct delivery of FATE mRNA into HGPS-NMOs likewise effectively inhibited perinuclear accumulation of progerin and rescued the formation of DNA damage repair foci. These findings demonstrate FATE as a broadly applicable, mutation-agnostic editing approach that targets a fundamental pathogenic mechanism in HGPS and therefore has feasible utility in clinical application.

overall_design: Following differentiation, Neuromuscular organoids (NMOs) derived from HGPS iPSCs, genome-edited iPSCs, and FATE-treated iPSCs were analyzed using scRNA-seq.

first: Hyuk-Jin

last: Cha


title: Lymphoid structures in the skull bone marrow control homeostatic and anti-tumor immune responses in the CNS [skull/brain]

first: Jonathan

last: Kipnis

overall_design: 8-wks-old mice were intracranially injected with 1x10^5 cells of CT2A. After 14 days of injection, tissues were harvested. 10X genomics was used to measure single cell RNA sequencing (scRNA-seq) and BCR sequencing to characterize B cells in CT2A brain tumor tissue (14 days post injection) from C57BL/6 mice. PNA+ cells were magnetically sorted from tissues. After that, DAPI-negative live cells were sorted with FACS Aria III

first: Jang Hyun

last: Park

first: Daviti

last: Abramishvili

last: Silva

first: Daniel


title: Transcription factor Satb1 modulates the functionality of TILs (scRNA-seq)

city: Kyoto

first: Wooseok

last: Seo

address: Yoshida-Konoe-cho, Sakyo-ku

institute: Kyoto University Graduate School of Medicine

summary: Transcription factor Satb1 plays important roles in the development of various immune cells. By utilizing genetic tools such as conditional knockout in mice have provided valuable information about its roles in T cell development. For example, CD4-cre transgenic mouse has been extensively used to delete Satb1 gene from T cells. While CD4-cre is useful to knockout a target gene floxed by LoxP sites from T cells, its expression lacks tissue specificity. To decipher the specific roles of Satb1 in T cells, we generated Satb1 conditional knockout mice with ThPOK-cre and E8I-cre to delete Satb1 from peripheral CD4+ and CD8+ T cells, respectively. Subsequently, total RNA-seq was performed to examine changes in transcriptome in the absence of Satb1 from peripheral T cells.

overall_design: scRNA-seq analysis of TILs from WT, Satb1 F/F; ThPOK-cre and Satb1 F/F; E8I-cre mice.

first: Ichiro

last: Taniuchi


title: Extracellular vesicles mediate stem cell signaling and systemic RNAi in planarians

city: Kansas City

first: Alejandro

last: Sanchez Alvarado

address: 1000 East 50th Street

institute: Stowers Institute for Medical Research

summary: Planarian flatworms are known for their remarkable regenerative capacity; however, the precise intercellular communication mechanisms underlying this process remain unsolved. Here, we report the discovery and characterization of abundant extracellular vesicles (EVs) in planarians. Using imaging and molecular analysis, we show conservation of biogenesis, morphology, and protein composition of planarian EVs. Environmental stressors significantly elevate EV release, indicating that planarians dynamically regulate vesicle production. Functionally, planarian EVs mediate intercellular communication by transferring regulatory signals: We find that they shuttle small RNAs that effect systemic RNA interference (RNAi) throughout the organism. Notably, gene knockdown experiments reveal a crucial role for AGO-3, a member of the Argonaute family of proteins, in modulating the association of small interfering RNAs with EVs, linking the intracellular RNAi machinery to EV-based signaling. These findings highlight EVs as pivotal mediators of cell-cell communication in planarians, with broad implications for understanding the coordination of gene regulation and tissue regeneration in animals.

overall_design: sRNA profiling fo RNA collected from EVs in both wildtype and RNAi conditions

first: Vidyanand

last: Sasidharan

first: Laura

last: Ancellotti

first: Viraj

last: Doddihal

first: Carolyn

last: Brewster

first: Frederick

last: Mann

first: Mary Cathleen

last: McKinney

first: Joseph

last: Varberg

first: Eric

last: Ross

first: Fengyan

last: Deng

first: Yi

last: Kexi

last: Sánchez Alvarado


title: Regulation of mRNA polyadenylation governs body plan formation in gastruloids

city: Schlieren

first: Ataman

last: Sendoel

address: Wagistrasse 12

department: Institute of Regenerative Medicine

country: Switzerland

institute: University of Zurich

summary: Gastrulation marks a landmark event in early embryogenesis, laying the foundation for the three germ layers and body plan formation. While transcriptional and epigenetic regulation during gastrulation has been extensively studied, post-transcriptional mechanisms remain underexplored, partly due to technical constraints and limited access to mammalian embryos. Here, we combine the scalability of gastruloids, an in vivo model of early embryonic development, with a single-cell CRISPR screening platform to systematically dissect post-transcriptional regulators of germ layer differentiation. Our screen uncovers previously unrecognized regulators of mesodermal and endodermal fate and enables cell-type-specific functional characterization with single-cell transcriptomic readout. Focusing on the de-adenylase Cnot8, we demonstrate that Cnot8-mediated poly(A) tail regulation is essential for mesodermal differentiation and proper body axis formation, underscoring the critical role of post-transcriptional control in early mammalian development. Together, our findings support a critical role of poly(A) tail regulation in the patterning of the mammalian embryo and establish gastruloid based single cell CRISPR screens as tool to uncover novel regulators and mechanisms of embryogenesis.

overall_design: We use mouse gastruloids as a model to systematically identify novel regulators of germ layer differentiation using a single cell CRISPR screen. We employ lentiviral libraries 669 sgRNAs targeting with 201 mRNA regulators and including non-binding, depletion and germ layer controls to infect Cas9 expressing mouse embryonic stem cells (mESCs), We then generate gastruloids and use a CRISPR CROP-seq strategy to document cell-type specific differentiation defects. We validate one candidate using ribsoome profiling, nanopre direct RNA sequencing, RNA degradation assays and single cell sequencing. Together our results reveal Cnot8 as crucial regulator of mesodermal patterning, differentiation and the mammalian body plan.

first: David

last: Taborsky

first: Fabiola

last: Valdivia-Francia

first: Clara

last: Duré

first: Umesh

last: Ghoshdastider

last: Llop-Grau

first: Neguin

last: Ranjbar

first: Peter

last: Renz

first: Merve

last: Yigit

first: Ramona

last: Weber

first: Aleksei

last: Mironov

first: Katie

last: Hyams

first: Stefano

last: Vianello

first: Matthias

last: Lütolf


title: Single-cell transcriptomics revealing LRFC specification dynamics

city: Shanghai

first: Haotian

last: Shi

state: Shanghai

address: 800 Dongchuan RD. Minhang District

institute: Shanghai Jiaotong University

summary: A conserved GA–ROS–GASA1 regulatory module specifies and commits plant lateral root founder cells by orchestrating nuclear migration, asymmetric division, and ROS homeostasis.

overall_design: The transcriptional landscape of xylem pole pericycle cells (XPPCs) transitioning to lateral root founder cell (LRFC) at single-cell resolution, capturing the earliest molecular events underlying LRFC commitment. XPPCs were isolated at 0, 1, 3, and 6 hours post-NAA treatment using protoplasting and fluorescence-activated cell sorting (FACS). The isolated cells were subjected to scRNA-seq.


title: Transcription factor Satb1 modulates the functionality of TILs (RNA-seq)

first: Wooseok

last: Seo

first: Ichiro

last: Taniuchi


title: Molecular signature of human endometrial stem/progenitor cells at the single cell level

city: Herston

first: Sally

last: Mortlock

state: Queensland

address: AWaGHR Centre, School of Public Health, The University of Queensland

department: School of Public Health

institute: University of Queensland

summary: Human endometrium sheds and regenerates each month during the menstrual cycle. N-cadherin+ (CDH2) glandular epithelial progenitors and SUSD2+ MSC and their niches have been identified but their signaling interactions remain unknown. SSEA1+ epithelial cells resurface the endometrium generating a new luminal epithelium each cycle. Based on these markers, we characterised the gene expression of human endometrial stem/progenitor cell-enriched populations derived from FACSorted hysterectomy endometrium by scRNAseq. Two of 10 epithelial clusters contained CDH2+SOX9+ cells with high TRH and IHH expression. N-cadherin and IHH, and SSEA1 and Hedgehog coreceptor BOC immunocolocalised in the basal layer of endometrial glands from which the new functional layer glands regenerate each menstrual cycle. Two of six mesenchymal clusters contained SUSD2+ MSC, one with high MUSTN1 expression. Epithelial progenitors and endometrial MSC transitioned to their respective progeny. We provide new insights into human endometrial stem/progenitor cell signaling pathways and niche interactions regulating their function.

overall_design: Isolated primary human endometrial cells enriched with stem/progenitor cell subpopulations

first: Harriet

middle: C

last: Fitzgerald

first: Caitlin

last: Filby

first: Fiona

middle: L

last: Cousins

first: Elizabeth

last: Marquez-Garcia

first: Katherine

last: Wyatt

first: Brett

last: McKinnon

first: Luk

last: Rombauts

first: Jim

last: Tsaltas

first: Grant

middle: W

last: Montgomery

first: Caroline

last: Gargett


title: Heparan sulfate Sulfatases are essential for the patterning of stem cell–derived midbrain dopaminergic neurons

city: Stockholm

first: Carmen

last: Abaurre

address: Solnavägen 9

department: MBB

country: Sweden

institute: Karolinska Institutet

summary: Midbrain dopaminergic (mDA) neuron loss underlies Parkinson’s disease (PD), making it critical to understand how mDA identity is established during development for cell replacement therapy. While intrinsic transcriptional programs and morphogen gradients driving mDA are known, the contribution of extracellular modulators remains less explored. Here, we show that the heparan sulfate (HS)–modifying enzymes SULF1 and SULF2 are essential for proper mDA neuron differentiation in vitro. Using CRISPR/Cas9-engineered human iPSCs, we demonstrate that SULF1/2 double knockout (DKO) cells fail to acquire ventral midbrain identity and instead adopt dorso-caudal fates, marked by ectopic expression of CDX2, HOX genes, neural crest, and Schwann cell markers. Mechanistically, SULF1/2 loss leads to increased 6-O-sulfation of HS chains, enhancing FGF signaling. FGF inhibition in SULF1/2 DKO cells suppresses CDX2+ axial progenitors and caudal derivatives, but does not completely restore ventral midbrain identity. Instead, it redirects cells toward dorsal lineages, including the emergence of ependymal-like cells. Our findings identify SULF1/2 as key modulators of anterior-posterior and dorsal-ventral patterning during mDA neuron development, possibly acting through a tight control of signaling molecules balance, and offer insights to refine differentiation protocols for PD cell therapy.

overall_design:
hiPSC cells were differentiated following a mDA neuron differentiation protocol in three samples: wild type (WT), SULF1/2 DKO and FGF-inhibited (PD/SU treated) SULF1/2 DKO. At day 16, nuclei were isolated from each sample and analyzed with snRNA-seq.

***************************************************************
Raw files for human/patient samples are being made available in EGA (https://www.ebi.ac.uk/ega/) for controlled access to the personally identifiable sequence data: EGAS50000001153.
***************************************************************

first: Chiara

last: Tremolanti

first: Eneritz

last: Agirre

first: Ulrika

last: Marklund

first: Gonçalo

last: Castelo-Branco


title: Reassessment of the roles of coronin proteins as actin effectors and in signalling

city: Basel

first: Jean

last: Pieters

address: Spitalstrasse 61

department: Biozentrum

institute: University of Basel

summary: Coronin proteins are present in all known non-plant eukaryotes, and are involved in key biological processes including cytoskeletal dynamics and the regulation of cell population sizes in amoeba and mammals. How, exactly, coronin proteins exert their function is debated. On the one hand, coronins are widely considered as F-actin-binding and regulatory proteins. On the other hand, coronin proteins were also shown to act as regulators of cAMP/Ca2+ signalling. Here, we demonstrate that endogenously expressed mammalian coronin 1, 2 and 3 did not depend on F-actin for their subcellular localisation and did not impact actin-related processes that were previously reported to be coronin-dependent. We also show that the fusion of GFP or FLAG-tags to coronin proteins resulted in their increased colocalisation with actin and phenocopied loss-of-function mutation in mice. Together these results suggest that inclusion of tags on coronin proteins causes increased colocalisation with F-actin and can disrupt their in vivo function. Furthermore, we show that the ubiquitously expressed coronin 2 and coronin 3 regulated cAMP production regardless of the presence of F-actin. The analysis presented here may contribute to a re-evaluation of the function of coronin proteins in signalling, independently of actin modulation.

overall_design: RNA-seq profiling of wildtype Jurkat T-cell cell line and its two drivatives with knocked out either  Coronin 1 (CORO1A)  or Coronin 3 (CORO1C) gene. Three biological replicates of each sample have been sequenced.

first: Tohnyui

last: Ndinyanka Fabrice

first: Urszula

last: Brykczynska Kunzmann


title: AMPK suppresses multiple cell death pathways to sustain tumor-associated macrophage proportion and promotes tumor progression

city: Beijing

first: Huan

last: Wang

address: 38 Life Science Park Road

institute: National Center for Protein Sciences, Beijing

summary: Tumor-associated macrophages (TAMs) are a major immune cell population in the tumor microenvironment (TME). This part depends on their capacity to resist cell death by adapting to the harsh tumor microenvironment, characterized by nutrient deprivation and the accumulation of metabolic waste, through metabolic reprogramming. This enables TAMs to not only utilize excess metabolites and metabolic waste in the tumor microenvironment as energy sources, but also to cope with metabolic stress by upregulating cysteine uptake and activating hypoxia-inducible factors, thereby maintaining their survival and function. However, the core genes that suppress TAM death in response to the metabolic dynamics of the tumor microenvironment during tumor progression remain unknown. In this study, we used proteomics analysis combined with experimental validation found that the degree of TAM apoptosis decreased while the level of AMPK activation increased during tumor progression. Notably, myeloid-specific knockout of AMPK significantly inhibits tumor growth, reduces TAM proportion in the tumor, and weakens its pro-tumor phenotype. Bulk RNA sequencing data analysis further revealed that specific knockout of AMPK in TAMs triggers multiple forms of cell death, including apoptosis, ferroptosis, and necroptosis. Further experimental validation confirmed that AMPK knockout not only induces apoptosis and ferroptosis in TAMs but may also trigger disulfidptosis. Mechanistic research suggested that AMPK might regulate cysteine uptake and the pentose phosphate pathway to inhibit ferroptosis and disulfidptosis in TAMs. In conclusion, this study identifies AMPK as a key regulator that responds to the metabolic dynamics of the TME and inhibits various forms of cell death in TAMs, providing new theoretical insights for TAM-targeted metabolic therapies.

overall_design:
To investigate how AMPK regulate the function of TAMs, we performed bulk RNA-seq analysis of TAMs from the tumor of AMPKfl/fl and AMPKfl/flLyz2cre/cre mice.
To investigate how  TAMs resist cell death during tumor progression, we performed bulk RNA-seq analysis of TAMs from the tumor of early and late stage.

first: Ruixuan

first: Di

last: Liu

first: Li

last: Tang


title: Transcriptional rewiring by enhancer methylation in CBFA2T3-GLIS2–driven pediatric acute megakaryoblastic leukemia [RRBS]

city: Little Rock

first: Samrat

last: Roy Choudhury

state: AR

address: 13 Children's Way, Arkansas Children's Research Institute

institute: University of Arkansas for Medical Sciences

summary: Chemotherapy resistance and relapse remain major challenges in pediatric acute myeloid leukemia (pAML), particularly in acute megakaryoblastic leukemia (AMKL) driven by the CBFA2T3-GLIS2 (C/G) fusion. To investigate the epigenetic mechanisms contributing to these challenges, we employed multi-epigenomic approaches to identify oncogenic enhancers at cis-regulatory elements (CREs) that drive aberrant gene expression.

overall_design: We generated a CRISPR-based DNMT3B knockout in M07e cells and compared its DNA methylation to the control M07e cell line. Total genomic DNA was extracted using the AllPrep DNA/RNA/Protein Kit, bilsulfite converted and subjected to library preparation for Reduced Representation Bisulfite Sequencing (RRBS) to identify differentially methylated regions.


title: Transcriptional rewiring by enhancer methylation in CBFA2T3-GLIS2–driven pediatric acute megakaryoblastic leukemia

overall_design: This study analyzed mRNA sequencing profiles in C/G+ AML cell lines M07e, compared to cord blood-derived CD34+ hematopoietic stem cells (HSCs) and CD41+ differentiated megakaryocyte progenitors. Additionally, we generated a CRISPR-based DNMT3B knockout in M07e cells and compared its transcriptome to the control M07e cell line. Total RNA was extracted using the AllPrep DNA/RNA/Protein Kit and subjected to library preparation for RNA sequencing


title: Transcriptional rewiring by enhancer methylation in CBFA2T3-GLIS2–driven pediatric acute megakaryoblastic leukemia [Hi-C]

summary: Chemotherapy resistance and relapse remain major challenges in pediatric acute myeloid leukemia (pAML), particularly in acute megakaryoblastic leukemia (AMKL) driven by the CBFA2T3-GLIS2 (C/G) fusion. To investigate the epigenetic mechanisms contributing to these challenges, we employed multi-epigenomic approaches to identify oncogenic enhancers at cis-regulatory elements (CREs) that drive aberrant gene expression.In this study, Promoter capture Hi-C (PCHi-C) was used to analyze long-range chromosomal interactions between promoters and distal regulatory regions in C/G+ M07e cells, compared to Hematopoietic Stem Cells (HSC)

overall_design: For Promoter capture Hi-C (PCHi-C) libraries were prepared using the Dovetail® Pan Promoter™ Assay (Catalog #35013E, Cantata Bio), following the manufacturer’s protocol.


title: Hyodeoxycholic Acid Suppresses Hepatosteatosis via GLP-1/iNKT Cell Crosstalk and PPARα Activation (scRNAseq_primary hepatocytes)

city: Chiba

first: Eunyoung

last: Lee

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

department: Department of Medical Physiology

institute: Chiba University, Graduate School of Medicine

summary: Hepatosteatosis, a common liver condition, involves excessive lipid accumulation and can lead to severe complications. Its molecular mechanisms are poorly understood, highlighting the need for therapies.  The molecular mechanisms are poorly understood, emphasizing the need for effective therapies.  In this study, we identified hyodeoxycholic acid (HDCA) as a potent anti-steatotic agent capable of preventing diet-induced fatty liver.  Mechanistically, HDCA treatment enhanced the hepatic expression of numerous PPARα target genes while also promoting the secretion of the gut hormone GLP-1.  The significance of PPARα signalling was emphasised by the fact that the anti-steatotic effects of HDCA were completely abolished in PPARα-knockout mice.  Our findings further reveal a novel mechanism involving invariant natural killer T (iNKT) cells.  HDCA treatment increased both the number and proliferation of iNKT cells in vivo, a phenomenon recapitulated in vitro using a GLP-1 agonist.  This indicates that the anti-steatotic action of HDCA requires the coordinated activity of PPARα signalling and iNKT cells.  These insights offer a promising avenue for developing targeted therapies for MASLD.

overall_design: Mice were treated with different diets, normal diet (ND), 60% high fat diet (HFD), or 60% HFD plus 0.25% hyodeoxycholic acid (HDCA), respectiviely. After 12 weeks feeding, primary hepatocytes were isolated from the mice treated with different diets. Primary hepatocyte were isolated with a two step-digestion method. Gene expression was evaluted using single cell RNA-seq.

first: Ryo

last: Hatano

first: Masanori

last: Fujimoto

first: Tomoaki

last: Tanaka

first: Takashi

last: Miki


title: Hyodeoxycholic Acid Suppresses Hepatosteatosis via GLP-1/iNKT Cell Crosstalk and PPARα Activation (Bulk RNA-seq)

overall_design: For bulk RNA-seq of RNA, liver was isolated from mice fed normal diet (ND), 60% high fat diet (HFD), or 60% HFD plus 0.25% hyodeoxycholic acid (HDCA)  for 12 weeks starting at 8 weeks of age.