title: A Transcriptional Hub for Endochondral ossification adn Fracture Repair.

city: Pamplona

first: Asier

last: Ullate-Agote

state: Navarra

address: Av. de Pío XII, 55

department: Hemato-Oncology

institute: CIMA

summary: After injury, bone tissue initiates a reparative response that will recover its structure and function, failure to initiate or delay this response results in fracture nonunion. The molecular mechanisms underlying fracture nonunion appearance are not yet stablished. We proposed that the signaling pathways derived from hypoxia and mediated by reactive oxygen species (ROS) homeostasis control Bmp2 expression and fracture healing initiation, having potential to induce fracture nonunion when excessive ROS develops into oxidative stress. Here, we silenced Apex1, the final transducer of ROS signaling and responsible of the activation of key transcription factors by oxidoreduction of cysteines, evaluating its role during endochondral ossification and fracture repair. Silencing Apex1 in limb bud mesenchyme results in transient metaphyseal dysplasia derived of impaired chondrocyte differentiation. During bone regeneration, silencing Apex1 generates a fracture nonunion phenotype characterized by delayed fracture healing initiation with impaired periosteal response and reduced chondrocyte and osteoblasts differentiation. In addition, compromised chondrocyte differentiation hampers fracture healing progression and callus vascularization. Our data supports a molecular mechanism of fracture healing initiation dependent of hypoxia derived ROS signaling in mesenchymal progenitors.

overall_design: Bulk RNA-seq for fracture calluses of wild-type and Apex1 KO mice at different days post fracture(-dpf)  3-, 6-, 10- and 14-dpf)

first: Froilán

last: Granero-Moltó

first: Emma

last: Muiños-López

first: José

last: Valdés-Fernández


title: CDK9 is a Dependency in GATA-3 driven and MCL-1 independent T-cell Lymphomas

status: Public on Nov 24 2025

city: Ann Arbor

first: Chenguang

last: Wang

state: Michigan

address: 1500E Medical Center Dr, Room 4411

department: Department of Internal Medicine

country: USA

institute: University of Michigan

type: Expression profiling by high throughput sequencing

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: HIF1α gates tendon response to overload and drives tendinopathy independently from vascular recruitment

city: Zurich

first: Jess

middle: Gerrit

last: Snedeker

address: Lengghalde 5

department: D-HEST

country: Switzerland

institute: ETH Zurich

summary: To investigate the role for HIF1α stabilization in tendon pathology, we generated mice with a tendon cell-targeted knockout of von Hippel-Lindau (VHL) protein by intercrossing ScxCre mice, in which Cre recombinase is under the Scleraxis promoter, with mice carrying the loxP -flanked Vhl alleles. VHL acts as a ubiquitin ligase that marks HIFs for proteolytic degradation under normoxic conditions, thereby silencing the HIF dependent transcriptional program. Hence, Vhl knockout results in stabilization of HIFs. We then performed gene expression analysis of tail tendon fascicles to confirm the activation of a HIF-dependent angiogenic and metabolic transcriptional program in the knockout mice compared to the wild types.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for wild type and ScxCre;Vhlf/f tail tendon fascicles (TTFs)

first: Greta

last: Moschini

first: Archana

middle: G

last: Mohanan

first: Izabella

middle: S

last: Niewczas

first: Diane

middle: E

last: Taylor

first: Patrick

middle: K

last: Jaeger

first: Amro

middle: A

last: Hussien

first: Stefania

middle: L

last: Wunderli

first: Olivia

last: Baumberger

first: Maja

last: Wolleb

first: Barbara

last: Niederoest

last: Bollhalder

first: Raphaela

last: Ardicoglu

first: Guillermo

last: Turiel

first: Evi

last: Masschelein

first: Sarah

last: Morice

first: Santiago

last: Ardiles

first: Lieke

last: Mous

first: Matthew

middle: R

last: Aronoff

first: Monika

last: Hilbe

first: Farah

last: Selman

first: Karl

last: Wieser

first: Sandro

middle: F

last: Fucentese

first: Fabian

last: Passini

first: Ulrich

last: Blache

first: Didier

last: Surdez

first: Helma

last: Wennemers

first: Dirk

last: Elewaut

first: Jonathan

last: Clark

first: Katrien

last: De Bock


title: Transcriptomic profiles of the maternal-fetal interface in wild-type and C1qtnf6-KO mice

city: Yangzhou

first: Yuchen

last: Tang

state: Jiangsu

address: Wenhui Road Yangzhou Citr No.48

country: China

institute: College of Veterinary Medicine Yangzhou University

summary: To understand the function of CTRP6 protein during gestation, we performed RNA-seq experiments for the maternal-fetal interface in wild-type and C1qtnf6-KO mice.

overall_design: We collected the maternal-fetal interface samples from wild-type and C1qtnf6-KO mice at four different gestation stages, includinggd10.5, gd14.5, gd16.5 and gd19.5. Three replicates per sample were used for RNA-seq experiments.

first: Hairui

last: Fan

first: Xiaolei

last: Chen

first: Cui

last: Du

first: Ming-an

last: Sun


title: VEGFA Stop-gained Variant Deteriorates Cardiac Remodeling in Myocardial Infarction [scRNA-seq]

status: Public on Nov 23 2025

city: shanghai

first: Zhongxiang

state: Shanghai

address: 浦东新区东方路1678号上海儿童医学中心

institute: shanghai children's medical center

summary: A sustained dosage of Vascular Endothelial Growth Factor A (VEGFA) is crucial for angiogenesis in both homeostasis and cardiovascular diseases. CUG-initiated alternative translation is a conserved mechanism for producing mature VEGFA. Genetic surveys have identified stop-gained variants predicted to prematurely terminate CUG-initiated translation without affecting ATG-initiated translation. However, the impacts of these variants on the vasculature in steady-state and disease conditions remain unknown. Here, we identified that homozygous mice carrying the stop-gained variant were viable. VEGFA dosage reduced to 70% in the Q150X homeostatic heart, with no significant alteration in cardiac function or vasculature. In the MI model, VEGFA dosage in Q150X was reduced to about 40% within the first-week post-infarction, leading to functional deterioration in the post-MI hearts. Significant changes in cellular composition were observed three days post-MI. In particular, endothelial cells in Q150X diverged into a state that showed higher level of hypoxia stress, an elevated inflammatory response, and increased extracellular matrix secretion. Additionally, we observed an increase of Nppb+ stressed cardiomyocytes in both 3 days post-MI and homeostasis. Finally, pro-inflammatory macrophages, neutrophils, and Cd8+T cells were enriched in the ischemic zone of Q150X hearts. CUG-initiated translation contributes significantly to the production of mature VEGFA in ischemic hearts. VEGFA dosage is critical in determining the cellular microenvironment during ischemic injury.

overall_design: Using CRISPR/Cas9 genome editing, we established VegfaQ150X/Q150 allele (Q150X), a mouse genetic model that mimics the human VEGFA stop-gained variant. The effects of this variant were tested in both adult homeostatic conditions and the acute myocardial infarction (MI) model. We then performed gene expression profiling analysis using data obtained from bulk RNAseq of whole hearts, endothelial cells(EC) and cardiomyocytes(CM) and and single-nulceus RNAseq of whole hearts from WT and Q150X(HO)  at homeostasis(homeo) and different time points following MI.

first: Min

last: Zhang


title: An integrated multi-omics and network analysis of neutrophil differentiation from early- to late-stage[RNA-Seq]

city: Beijing

first: Cheng

last: Li

state: Beijing

address: No.5 Yiheyuan Road, Haidian District

department: School of Life Sciences

institute: Peking University

summary: The goal of this study is to study the gene regulation mechanism during ATRA-induced HL-60 cell neutrophil differentiation especially in the early time-point. We demonstrated the striking early changes event that trigger the whole differentiation process. We also identified the pioneer TFs (Transcription factors) that ignite the differentiation process with the corresponding TF correlation networks.

overall_design: Genome-wide mapping of transcriptomic, chromatin accessibility, and chromatin interaction in differenti time-points during neutrophil differentiation.

first: Wei

first: Minglei

last: Shi

first: Qing


title: An integrated multi-omics and network analysis of neutrophil differentiation from early- to late-stage [ATAC-Seq]


title: Piezo1-dependent gene expression in mouse colon cancer cell line CMT-93

city: Tsukuba

first: Yutaro

last: Kumagai

state: Ibaraki

address: Central 5-41, Higashi 1-1-1

department: BRD

country: Japan

institute: AIST

summary: A wild-type or Piezo1-deficient colon cancer cell line CMT-93 was stimulated with single-strand RNA (ssRNA) or Yoda-1, and gene expression was compared by using microarray.

overall_design: A wild-type or Piezo1-deficient colon cancer cell line CMT-93 was stimulated with single-strand RNA (ssRNA) or Yoda-1 for 24 hours and total RNA was subjected to microarray analysis.

first: Kenta

last: Maruyama


title: An integrated multi-omics and network analysis of neutrophil differentiation from early- to late-stage [OCEAN-C]


title: DOT1L provides transcriptional memory through PRC1.1 antagonism [ChIP-Seq]

city: Melbourne

first: Daniel

last: Neville

state: Victoria

address: 99 Commercial Road Level 6, Alfred Centre

department: Australian Centre for Blood Diseases

country: Australia

institute: Monash University

summary: DOT1L and Menin are essential cofactors for the oncogenic activity of MLL-fusion proteins (MLL-FPs) in leukaemia. However, the mechanisms underpinning the therapeutic effects of their inhibitors remain unclear. Here, we identify a critical role for the non-canonical Polycomb repressive complex 1.1 (PRC1.1) in mediating the cellular responses to DOT1L and Menin inhibitors. Menin inhibition induces PRC1.1-dependent deposition of H2AK119ub to silence a subset of MLL-FP targets, whereas DOT1L inhibition results in a genome-wide increase in H2AK119ub. We show that enhanced PRC1.1 activity arises specifically from the progressive loss of DOT1L-mediated H3K79 methylation, independent of MLL-FP displacement or transcriptional repression. This regulatory crosstalk is conserved across cell types and is driven by direct biochemical antagonism between H3K79 methylation and PRC1.1 activity. Together, our findings establish DOT1L as a component of transcriptional memory co-opted in leukaemia and suggest it serves as the missing link balancing the opposing forces of the MLL-Polycomb system.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: ChIP sequencing of AML cell lines (murine FLAG-MLLAF9 and MOLM13) and K562 cells with and without DOT1L or Menin inhibitor treatment. ChIP-sequencing of HuDEP-2 (human erythroid progenitors) and murine embryonic stem cells with and without DOT1L inhibitor treatment.

first: Omer

last: Gilan


title: A single-nucleus dataset sampling subcutaneous white adipose from living donors to study familial partial lipodystrophy 2

status: Public on Nov 22 2025

first: Bioinformatics

last: Core

state: MI

address: BRCF Bioinformatics Core, 2800 Plymouth Rd

summary: Familial partial lipodystrophy 2 (FPLD2) is a rare disease characterized by adipose tissue loss and redistribution, and metabolic dysfunction. FPLD2 is caused by pathogenic variants in the LMNA gene, encoding nuclear lamins A/C, structural proteins that control nuclear function and gene expression. However, the mechanisms driving adipocyte loss in FPLD2 remain poorly defined. In this study, we recruited eight families with developing or established FPLD2 and performed clinical, histological, and transcriptomic analyses of subcutaneous adipose tissue biopsies. Bulk and single-nuclei RNA-sequencing revealed suppression of lipid metabolism and mitochondrial pathways, alongside increased inflammation. These signatures were mirrored in tamoxifen-inducible adipocyte-specific Lmna knockout mice, in which lamin A/C-deficient adipocytes shrank and disappeared. Lmna-deficient fibroblasts shared similar gene expression changes, linked to altered chromatin accessibility, underscoring lamin A/C’s potential regulatory role in lipid metabolism and inflammatory programs. By directly comparing atrophic and hypertrophic adipose depots in FPLD2, and integrating human, mouse, and in vitro models, this study provides new insights into disease progression and potential therapeutic targets.

overall_design: Single nuclei RNA-seq of human subcutaneous white adipose tissue surgical biopsies from patients with developing FPLD2, developed FPLD2, or control unaffected patients, either from the abdomen or dorsocervical region (upper neck)

first: Jessica

middle: N

last: Maung

first: Akira

last: Nishii

first: Rebecca

last: Schill

first: Ormond

last: MacDougald


title: A novel interferon stimulated gene GALNT2 is required to restrict respiratory virus infections

city: Guangzhou

first: fenghua

last: chen

address: 195 Dongfengxi Road, Yuexiu District

institute: Guangzhou Medical University

summary: Respiratory viruses, particularly coronaviruses (CoVs) and influenza A viruses (IAVs), have driven global pandemics, with zoonotic spillover posing ongoing threats to public health. SARS-CoV-2, the cause of COVID-19, emerged in 2019, devastating global health and claiming over 7 million lives, while the 2009 influenza pandemic caused at least 200,000 deaths. The innate immune system serves as the first line of defense, where type I interferons (IFNs) and interferon-stimulated genes (ISGs) play pivotal antiviral roles. Impaired IFN-I responses are linked to severe COVID-19 and influenza, underscoring their importance: life-threatening COVID-19 cases often involve genetic defects in IFN pathways or neutralizing autoantibodies that dampen IFN activity, whereas children’s resilience to severe infection correlates with robust IFN responses. Recombinant IFNs show therapeutic potential, though timing is critical. Key ISGs like OAS1 and OASL further illustrate IFN-mediated protection, highlighting the need to dissect how individual ISGs aid viral clearance and mitigate disease. In this context, we identified GALNT2, an O-GalNAc transferase, as a novel ISG that enhances viral clearance and reduces susceptibility and severity in both SARS-CoV-2 and IAV infections.

overall_design: RNA sequencing was performed on A549 cell lines with or without GALNT2 overexpression, with three biological replicates for each experimental condition


title: The highly localized interaction between Neurofascin-186 and Gliomedin promotes subcellular innervation by the chandelier cell

city: Columbus

first: Hiroki

last: Taniguchi

state: OH

address: 333 W 10th Ave

department: Department of Neuroscience

institute: Ohio State University

summary: A variety of cortical inhibitory interneuron (IN) subtypes differentially but coordinately operate in local circuits to process neuronal signals. The subcellular arrangement of IN synaptic outputs contributes to subtype-specific functional specialization. However, it remains poorly understood what intercellular molecular interactions enable the matched synapse formation between IN subtypes and specific subcellular domains. Using mice of both sexes, we demonstrate that Neurofascin-186 (NF186), a cell adhesion molecule specifically expressed in the axon initial segments (AISs) of pyramidal neurons, is necessary for chandelier cells (ChCs) to develop a string of synaptic boutons along the AIS (an axon cartridge). Furthermore, we discovered that Gliomedin, a known receptor for NF186 in the nodes of Ranvier, is preferentially expressed in ChCs and mediates ChC axon cartridge development by acting as a major receptor for NF186. Thus, the intercellular interaction through subcellularly-restricted ligands and cell type-specific receptors ensures a high degree of IN subcellular synapse specificity.

overall_design: RNAseq profiling of cortical interneuron subtypes extracted from postnatal day 5 VIP-Cre;Ai47, SOM-Cre;Ai47, Nkx2.1-CreER;Ai47 mice

first: Yugo

last: Ishino

first: Yasufumi

last: Hayano

first: Gogce

middle: C

last: Crynen


title: NBL1 Associates with Renal Phenotypes in Mice, but Partial Nbl1 Reduction Does Not Ameliorate Kidney Disease: Male Mice with XLAS either HET or WT for Nbl1

city: Bar Harbor

first: Courtney

last: Willey

state: Maine

address: 600 Main Street

institute: The Jackson Laboratory

summary: Background: Increased concentrations of neuroblastoma suppressor of tumorgenicity 1 (NBL1) in the blood have been associated with disease progression in diabetic kidney disease and IgA nephropathy. However, it is unclear whether NBL1 is a causal factor for kidney disease and what is driving these increased concentrations in the blood. Methods: We tested causality of NBL1 by knocking out Nbl1 in two different mouse models of kidney disease (X-linked Alport Syndrome (XLAS) and low-dose cisplatin treatment) and performed a genetic analysis for the drivers of NBL1 concentrations in two independent cohorts of genetically diverse (DO) mice with X-linked Alport Syndrome (DO-XLAS). Results: Significant correlations were found between NBL1 concentrations in the blood and both GFR and ACR in DO-XLAS mice. However, XLAS mice with only one functional Nbl1 allele and reduced NBL1 concentrations had similar GFR and ACR compared to Nbl1 wildtype animals. Similarly, there was no difference between animals with one or two functional Nbl1 alleles after chronic low-dose cisplatin treatment. Genetic analysis of NBL1 concentrations in our DO-XLAS cohorts identified associations with loci on Chromosomes 4 and 17, with Clcnka, and MHC2 as potential candidates. Conclusion: Increased NBL1 in blood is a marker for different forms of kidney disease but is not a causal factor.

overall_design: Male mice with XLAS either HET or WT for Nbl1 (all mice are on the B6 background). Bulk RNA-seq of kidneys collected at 15-weeks of age.

first: Rei

last: Bufi

first: Harsphpreet

last: Chandok

first: Ron

last: Korstanje


title: DOT1L provides transcriptional memory through PRC1.1 antagonism [RNA-seq]

overall_design:
3'RNA sequencing of MOLM13, MV4;11, murine MLLAF9, K562, mESC, HuDEP-2 cells with and without DOT1L and/or Menin inhibitor treatment in control or PCGF1, BCOR KO. RNA-seq experiments were conducted in triplicate.

RNA sequencing of AML cell lines with and without DOT1L or Menin inhibitor treatment. RNA-seq experiments were conducted in triplicate.


title: UHRF1 Deficiency Exacerbates Intestinal Inflammation by Epigenetic Modulation of NPY1R Gene Methylation [gene expression]

city: Xi'an

first: Xiaodi

last: Zhao

address: No.127 Changle West Road

department: Xijing Hospital of Digestive Diseases

institute: Xijing Hospital of Fourth Military Medical University

summary: Epigenetic modifications play a crucial role in the pathogenesis of inflammatory bowel disease (IBD) by mediating gene–environment interactions. We previously showed that UHRF1, a central regulator of DNA methylation, contributes to cancer progression; however, its function in IBD remains poorly understood. Here, we revealed that UHRF1 is frequently reduced in inflamed tissues of IBD patients and that its deficiency exacerbates intestinal epithelial cell (IEC) damage. Through a multi-level approach incorporating human cell models and an intestinal epithelial-specific Uhrf1 knockout mouse model, we established UHRF1 as a key mitigator of IBD progression. Mechanistically, UHRF1 bound to the NPY1R promoter, promoting its methylation and leading to transcriptional suppression. The NPY1R upregulation resulting from UHRF1 deficiency attenuated cAMP–PKA–CREB signaling in IECs, thereby enhancing NF-κB activation and subsequent pro-inflammatory responses, which compromised intestinal epithelial barrier integrity. Furthermore, we identified miR-141 as a negative regulator of NPY1R, highlighting its potential as a therapeutic agent. Collectively, our results identified the UHRF1–NPY1R regulatory axis as a critical epigenetic mechanism in intestinal inflammation and underscored its dual promise for IBD diagnostics and therapy.

overall_design: gene expression in NCM460-shCtrl and NCM460-shUHRF1 cells was measured

first: Han

last: Yanan

first: Bai

last: Xue


title: Multi-drug tolerance in Leishmania persister-like cells

city: Antwerp

first: Pieter

last: Monsieurs

address: Nationalestraat 155

country: Belgium

institute: Institute of Tropical Medicine

summary: Leishmaniasis is caused by parasitic protozoa of the genus Leishmania and is found widely across the tropics and sub-tropics, afflicting hundreds of thousands of people. The disease is notoriously difficult to treat. Here, we present evidence of the existence of persister-like cells in cultured Leishmania populations, induced upon exposure to normally lethal doses of antimony, a widely used anti-leishmanial.  Persisters are a small fraction of non-proliferative cells with reduced metabolism that are adapted to withstand a variety of environmental assaults, including lethal doses of antimicrobials. We show that Leishmania persister-like cells survive lethal doses of antimonials by adopting a quiescence phenotype characterised by reduced proliferation, constrained metabolism, and diminished mitochondrial membrane potential. What is more, these cells demonstrate cross-tolerance to other anti-leishmanial drugs. Wild-type persister-like cells reverted to similar levels of drug susceptibility once the antimony-induced pressure was removed. Surprisingly, cells which had previously been selected for genetic changes causing resistance to antimony acquired a level of hyper-resistance after transient passage through the quiescent state, without further genetic change. Our results demonstrate the extreme versatility of this eukaryotic pathogen in adaptation to drug pressure and highlight the need for the development of new anti-leishmanials targeting non-proliferative forms.

overall_design:
bulk RNA-seq analysis of PAT exposed Leishmania donovani cells. RNA is extracted at Day 0, Day2, Day 4 and after PAT pressure, for both strains BPK026 and BPK275.

UPDATE: [Feb-04-2026] Samples GSM9291748-GSM9291751 were removed because they did not pass quality control.

first: Allison

last: Aroni-Soto

first: Odin

last: Goovaerts

first: Kaoutar

last: Choukri

first: Basudha

last: Khanal

first: Wim

last: Adriaensen

first: Jean-Claude

last: Dujardin

first: Michael P

last: Barrett

first: Malgorzata A

last: Domagalska


title: Acute kidney injury following fatty liver ischemia-reperfusion injury: indirect protection by hepatic ferroptosis inhibition

status: Public on Nov 21 2025

city: Indianapolis

first: Wenjun

last: Zhang

state: IN

address: 1044 W. Walnut Street, R4 W116

institute: Indiana University School of Medicine

summary: To understand the roles of ferroptosis in subsequent acute kidney injury (AKI) following hepatic ischemia-reperfusion injury (hIRI) in steatosis livers. The hIRI was induced in mice fed either a high-fat, high-sucrose diet (HFD) or a normal diet (ND)  to mimic the AKI commonly observed clinically after fatty liver transplantation. RNA sequencing was conducted to investigate the underlying mechanisms.  We found that apoptosis and inflammation are the prominent kidney injury mechanisms following fatty liver IRI. Although ferroptosis may not be directly involved in the renal injury, anti-ferroptosis intervention mitigates AKI, supporting the concept that ferroptosis-mediated liver injury may serve as the primary upstream trigger in this context.

overall_design: The 10-week-old C57BL/6 mice (n=3 in each group) were fed with either a control diet (ND) or a high-fat diet (HFD) for 12 weeks before being subjected to 1hr hepatic ischemia-reperfusion injury (IRI) or a sham procedure.  Total 4 groups: HFD_IRI, HFD_sham, ND_IRI, and ND_sham. The mouse liver and kidney tissue were harvested in 6 hrs after ischemia injury.

first: Zachary

last: Rokop

first: Chandan

last: Sen

first: Chandrashekhar

last: Kubal

first: Hongyu

last: Gao


title: Early life microbiota skew long-term gene expression and epigenome of bone marrow hematopoietic precursors

city: Toronto

first: Wilder

last: Scott

state: ON

address: 2075 Bayview Ave

country: Canada

institute: SRI

summary: Asthma is the most prevalent chronic childhood disease in developed nations, yet its etiology remains incompletely understood. Emerging evidence suggest a crucial role for the gut microbiota in shaping immune development and future susceptibility to atopic disease. We previously demonstrated that short-chain fatty acids (SCFAs) as fermentation products of normal gut bacteria, regulate an innate type 2 immune axis that serves to dampen susceptibility to allergic lung inflammation. Here, we show that neonatal depletion of SCFA-producing gut bacteria via antibiotics robustly alters Lin⁻Sca-1⁺c-Kit⁺ (LSK) hematopoietic stem and progenitor cell (HSPC) output, resulting in a transplantable atopic immune phenotype:  bone marrow (BM) transplants from these mice generates recipients with increased lung B cell IgE expression, elevated serum levels of IgE, dowstream increased IgE bound to basophils and exacerbated allergic lung inflammation following papain challenge. Notably, neonatal depletion of SCFA bacteria also enhanced susceptibility to chemotherapy (5-fluorocuracil) -induced myelosuppression, marked by impaired neutrophil recovery and increased DNA damage in long-term HSCs. Importantly, these antibiotic effects were reversed by supplementation of antibiotic-treated mice with exogenous SCFA. Single-cell RNA sequencing (scRNA-seq) of LSK HSPCs revealed distinct shifts in gene expression following vancomycin treatment, including increased proliferation-associated signatures with stress-adaptive features. Epigenetic profiling further revealed differential histone acetylation in HSPCs, consistent with an SCFA-mediated epigenetic regulatory mechanism. Collectively, these findings establish an epigenetic link between gut microbiota composition, hematopoiesis, and long-term immune function, providing a mechanistic basis for microbiota-driven atopic disease susceptibility and hematopoietic dysfunction.

overall_design: Approximately 1 x 10^4 viable HSPCs (live-gatedCD45+Lin-Sca1+cKit+) were sorted (FACSAria II, BD Biosciences, San Jose, CA) from BM of 8-week-old male mice (3 mice per group) reared on vancomycin, vancomycin with BAP or regular drinking water. Sorted cells were encapsulated into droplets, and transcript libraries were prepared using Single Cell 3′ reagent kits (10x Genomics) and then sequenced using a 75 cycle NextSeq500. Samples were aligned using the mm10 reference transcriptome and the standard Cell Ranger count pipeline. Data analysis and visualization was performed using the R package Seurat

first: Ahmed

last: Kabil

first: Alissa

last: Cait

first: Michael

last: Hughes

first: Kelly

middle: M

last: McNagny


title: Extracellular vesicles secreted from human myotubes of T2D and NGT donors

city: Oslo

first: Stian

last: Christiansen

address: Folke Bernadottes Vei 13D

country: Norway

institute: OsloMet

summary: Type 2 diabetes (T2D) is associated with increased morbidity, mortality, and substantial healthcare costs. Peripheral insulin resistance, involving interconnected dysregulation of multiple organs, is considered a major driver of T2D. Extracellular vesicles (EVs) are suggested as mediators of this dysregulation based on their properties in intercellular communication. Given the role of skeletal muscle in glucose metabolism, the content of skeletal muscle-derived EVs may provide insights into mechanisms of T2D.  To examine this, myotubes from severely obese female T2D donors and matched women with normal glucose tolerance (NGT) were cultured. Small EVs (sEVs) were isolated by differential centrifugation and filter columns and characterized by nanoparticle tracking analysis, flow cytometry, and transmission electron microscopy. The micro-RNA (miR) content of sEV was analyzed via Affymetrix GeneChip miRNA 4.0, while proteins were detected by LC-MS/MS.

overall_design: Small extracellular vesicles (sEVs) were isolated from primary human skeletal muscle cell cultures established from biopsies of 12 female donors (6 with type 2 diabetes, 6 with normal glucose tolerance). Cultures were differentiated into myotubes, conditioned media collected, and sEVs isolated and characterized by nanoparticle tracking analysis, transmission electron microscopy, and flow cytometry. sEV protein cargo was analyzed by nanoLC-MS/MS (Q Exactive, Thermo), and data were processed in MaxQuant with LFQ normalization. In parallel, sEV RNA was isolated and profiled using Affymetrix GeneChip miRNA 4.0 arrays, followed by RMA normalization and log2 transformation. One sample in the NGT group was excluded after quality control, yielding n=5 (NGT) and n=6 (T2D) for miRNA profiling, and n=5 per group for proteomic analysis. Differential expression was assessed with t-tests and multiple testing correction.

first: Kari

middle: B

last: Haug

first: Misbah

last: Hussain

first: Abdille

last: Hussein

first: Berit

last: Brusletto

first: Reidun

last: Øvstebø

first: Oliwia

last: Witczak

first: Vigdis

last: Aas


title: Bulk RNA-seq time-course of human iPSc differentiation to thymic epithelial progenitors

city: Nantes

first: Matthieu

last: Giraud

address: 30 bd Jean Monnet

department: CRTI

country: France

institute: INSERM _ Université de Nantes

summary: Bulk RNA-seq was performed across a time-course of human iPSc differentiation toward thymic epithelial progenitors (TEPs). Twenty-nine experimental libraries spanning eleven time-points (days 0, 1, 2, 3, 7, 9, 10, 14, 15, 17, 22) plus four primary thymic epithelial cell (TEC) controls revealed progressive down-regulation of pluripotency genes and stepwise activation of transcriptional modules corresponding to anterior endoderm, third pharyngeal pouch endoderm, and thymic epithelial fate.

overall_design: Human induced pluripotent stem cells (iPSc; line LON71) were differentiated toward thymic epithelial progenitors (TEPs) using our defined iPSc-to-TEP protocol. RNA was extracted with the RNeasy Micro Kit (Qiagen) at eleven time-points: D0 (n = 3), D1 (n = 1), D2 (n = 1), D3 (n = 4), D7 (n = 5), D9 (n = 6), D10 (n = 4), D14 (n = 2), D15 (n = 1), D17 (n = 1), D22 (n = 1). Four primary pediatric thymic epithelial cell (TEC) samples served as references. All samples were sequenced using 3′ RNA-seq (DGE-seq).

first: Nathan

last: Provin


title: Rb1 deficiency induces synthetic lethality with ATR and PKMYT1 co-inhibition in breast cancer

city: Houston

first: Khandan

last: Keyomarsi

state: Texas

address: 1515 Holcombe Blvd

department: Experimental Radiation Onc

institute: University of Texas - MD Anderson Cancer Center

summary: Targeting cell cycle checkpoints has emerged as a promising strategy in cancer therapy, yet single-agent inhibitors often fail due to compensatory mechanisms. Here, we demonstrate that co-inhibition of ATR (RP-3500) and PKMYT1 (RP-6306) induces synthetic lethality in Rb1-deficient breast cancers by disrupting both S/G2 and G2/M checkpoints, resulting in replication stress, premature mitotic entry, and DNA damage accumulation. In vitro, Rb1-deficient breast cancer cell lines exhibited marked apoptosis and loss of clonogenic potential, whereas Rb1-proficient models remained largely resistant to combination treatment. Genetic manipulation of Rb1 confirmed this dependency: Rb1 knockdown sensitized resistant models, and overexpression conferred protection. In vivo, patient-derived xenograft (PDX) models recapitulated these findings. Rb1-deficient tumors underwent complete regression, while Rb1-proficient tumors showed limited response with RP-3500/RP-6306 treatment. Combination therapy was well tolerated in vivo, without significant toxicity or weight loss. Biomarker analysis revealed increased γH2AX and reduced Ki67 staining exclusively in Rb1-deficient PDX models, underscoring the specificity of this response. Mechanistically, Rb1 loss impaired double-strand DNA repair by attenuating homologous recombination and non-homologous end joining, leading to replication fork collapse, chromosomal instability, and mitotic catastrophe. Proteogenomic analysis identified JNK/p38 stress response pathway activation as a key driver of apoptosis following ATR/PKMYT1 inhibition in Rb1-deficient cells. Clinically, analysis of stage IV breast cancer patient datasets revealed that Rb1-low tumors display reduced DNA repair pathway activity and are enriched in triple-negative and CDK4/6 inhibitor-resistant luminal breast cancers. These findings establish Rb1 loss as a functional biomarker for ATR/PKMYT1-targeted therapy, offering a precision treatment strategy for advanced breast cancers.

overall_design: Breast cancer cell lines MDA-MB-231, BT-549, MCF-7, MCF-7 PalbociclibResistant (MCF7PR) were collect at either baseline (DMSO) or under treatment 30 nM RP6306 (MYTi), 10 nM RP3500 (ATRi) or in combination (COMB) for 48 hours and harvest for total RNA and RNA sequencing. Each group havs at least three biological replicates.

first: Xiao-Ting

last: Jiang

first: Mei-Kuang

last: Chen

first: Jiwang

last: Lee

first: Xiayu

last: Rao

first: Jing

last: Wang


title: iPSc-to-thymic epithelial progenitor differentiation under multifactorial DoE optimization and transcriptomic profiling

summary: This study investigates the differentiation of human induced pluripotent stem cells (iPSc) into thymic epithelial progenitors (TEPs) through a combinatorial experimental design (Design of Experiments, DoE). Bulk RNA-seq was performed across 96 differentiation conditions covering key developmental transitions: definitive endoderm (DE), anterior foregut endoderm (AFE), third pharyngeal pouch endoderm (3PPE), and thymic epithelial progenitors (TEP). The multifactorial screen leveraged Plackett-Burman designs and transcriptomic similarity to in vivo pharyngeal development atlases to identify optimal differentiation cues.

overall_design: RNA was extracted at the AFE, 3PPE and TEP stages of directed differentiation from iPSc across 96 conditions generated from three Plackett-Burman designs (36+36+24 runs), corresponding respectively to the DE_to_AFE, the AFE_to_3PPE and the 3PPE_to_TEP transitions. Each condition combined low/high concentrations of pathway modulators (BMP, WNT, FGF, TGFβ, SHH, etc.) administered at defined time windows. Samples were sequenced using 3’ RNA-seq (DGE-seq) and analyzed via bulk transcriptomics to evaluate proximity to gene signatures from developmental single-cell atlases.


title: Eosinophils enhance granuloma-mediated control of persistent Salmonella infection

city: Stanford

first: Daniel

last: Arve-Butler

state: CA

address: 299 Campus Drive, FAIRCHILD BUILDING D33

institute: Stanford University

summary: Salmonella enterica can persist asymptomatically within tissues for extended periods. This is achieved through intricate host-pathogen interactions in immune cell aggregates called granulomas, wherein Salmonella establish favorable cellular niches to exploit while the host limits its expansion and tissue dissemination. Here, using a mouse model of persistent Salmonella infection, we identify a host-protective role for eosinophils in control of Salmonella Typhimurium (STm) infection within the mesenteric lymph nodes (MLN), the main lymphoid tissue of STm persistence. Combining spatial transcriptomics and experimental manipulations, we found that monocytes and macrophages responding to STm infection recruited eosinophils in a C-C motif chemokine ligand 11 (CCL11)-dependent manner and enhanced their activation. The protein major basic protein, primarily expressed by eosinophils, was associated with altered macrophage polarization and bacterial control. Thus, eosinophils play a vital role in restraining Salmonella exploitation of granuloma macrophages at a key site of bacterial persistence.

overall_design: Sixteen Xenium in situ spatial transcriptomics samples from mouse mesenteric lymph node (MLN): Uninfected controls (UI1–UI2), WT infected at 4 wpi (WT1–WT8), and DblGATA1 infected at 4 wpi (GATA1–GATA6). Raw Xenium outputs (cell_feature_matrix.zarr.zip, cells.csv.gz, transcripts.parquet) deposited per sample. Processed outputs include an integrated Seurat object (RDS), per-cell cluster table (CSV), and marker tables (CSV).

last: Butler

first: Blanda

last: Di Luccia

first: Jose

last: Vilches-Moure

first: Denise

last: Monack


title: Selective elimination of donor bacteria enables global profiling of plasmid gene expression at early stages of conjugation

city: Gif sur Yvette

first: Elena

last: ESPINOSA

address: 1, Avenue de la Terrasse

institute: CNRS

summary: Conjugative plasmids are a major driving force for the dissemination of antimicrobial resistance. During conjugation, plasmid DNA is transferred from the donor cell as a single-stranded (ss) linear molecule. Recent researches highlighted intriguing DNA reactions at the early stage of conjugation that are important for plasmid establishment in the recipient cell, including prompt expression of anti-defense genes. However, genomics-based approaches to investigate plasmid establishment have been challenging, because identical, fully established plasmids in donor cells mask transconjugant-specific signals. To overcome this limitation, we developed a new EDTA method which exploits a donor mutant hypersensitive to hypoosmotic shock. It allows unprecedently quick and efficient Elimination of Donor population for Transconjugant Accumulation. RNAseq analysis revealed highly selective and robust induction of plasmid genes during early stage of conjugation. We experimentally identified +1 sites of 6 possible operons that are matched with ssDNA promoter predictions. We also showed gene expression program is altered in different recipient cells in which plasmid establishment is perturbed. As the EDTA method is straightforward and broadly applicable, it will further understanding of the plasmid establishment processes in new host cell, not only gene expression but also ss to double-stranded DNA conversion and plasmid circularization.

sample_organism:
Sinorhizobium meliloti
Escherichia coli

platform_organism:
Sinorhizobium meliloti
Escherichia coli

overall_design: RNA-seq of recipient cells after 30 minutes or 60 minutes conjugation of pESBL and F plasmid

first: Meng

last: Wen

last: Fraikin

first: Emma

last: Mettouchi

first: Christian

last: Lesterlin

last: Espinosa

first: Yoshiharu

last: Yamaichi


title: VEGFA Stop-gained Variant Deteriorates Cardiac Remodeling in Myocardial Infarction [RNA-seq]

first: Zhongxiang

address: 浦东新区东方路1678号上海儿童医学中心

summary: A sustained dosage of Vascular Endothelial Growth Factor A (VEGFA) is crucial for angiogenesis in both homeostasis and cardiovascular diseases. CUG-initiated alternative translation is a conserved mechanism for producing mature VEGFA. Genetic surveys have identified stop-gained variants predicted to prematurely terminate CUG-initiated translation without affecting ATG-initiated translation. However, the impacts of these variants on the vasculature in steady-state and disease conditions remain unknown. Here, we identified that homozygous mice carrying the stop-gained variant were viable. VEGFA dosage reduced to 70% in the Q150X homeostatic heart, with no significant alteration in cardiac function or vasculature. In the MI model, VEGFA dosage in Q150X was reduced to about 40% within the first-week post-infarction, leading to functional deterioration in the post-MI hearts. Significant changes in cellular composition were observed three days post-MI. In particular, endothelial cells in Q150X diverged into a state that showed higher level of hypoxia stress, an elevated inflammatory response, and increased extracellular matrix secretion. Additionally, we observed an increase of Nppb+ stressed cardiomyocytes in both 3 days post-MI and homeostasis. Finally, pro-inflammatory macrophages, neutrophils, and Cd8+T cells were enriched in the ischemic zone of Q150X hearts. CUG-initiated translation contributes significantly to the production of mature VEGFA in ischemic hearts. VEGFA dosage is critical in determining the cellular microenvironment during ischemic injury.

overall_design: Using CRISPR/Cas9 genome editing, we established VegfaQ150X/Q150 allele (Q150X), a mouse genetic model that mimics the human VEGFA stop-gained variant. The effects of this variant were tested in both adult homeostatic conditions and the acute myocardial infarction (MI) model. We then performed gene expression profiling analysis using data obtained from bulk RNAseq of whole hearts, endothelial cells(EC) and cardiomyocytes(CM) and and single-nulceus RNAseq of whole hearts from WT and Q150X(HO)  at homeostasis(homeo) and different time points following MI.

first: Min

last: Zhang


title: Ectopic expression of MUC5B in the respiratory bronchiole initiates ER stress in the IPF lung

city: Denver

first: Rachel

last: Blumhagen

state: CO

address: 1400 Jackson St.

department: Center for Genes, Environment and Health

institute: National Jewish Health

summary: Subcellular spatial transcriptomics on lung tissue from idiopathic pulmonary fibrosis (IPF) and unaffected control subjects to further understand the pathogenesis of MUC5B-driven lung fibrosis.

overall_design: CosMxTM technology [CosMxTM Human Universal Cell Characterization RNA Panel (1000-plex) + 24 custom genes; Nanostring, USA] was applied to lung tissues from 15 IPF patients and 10 unaffected controls. 2 tissue microarrays were constructed from 25 FFPE tissue blocks, resulting 43 fields of view (FoVs). The following cell surface markers were used for morphology visualization: B2M/CD298, PanCK, CD45, CD3 antibodies and DAPI.

first: Ivana

last: Yang


title: Detaching cells from human iPSc-derived thymic organoids (hTOs) seeded with primary early thymic progenitors (ETPs) at late phase of maturation (D28)

first: Matthieu

last: Giraud

summary: We have employed a single-cell sequencing approach using 10x Genomics scRNA-seq to study hematopoietic cell generation from hTOs (at the late phase of differentiation/maturation at D28) obtained through reaggreagation of iPSc-derived thymic epithelial progenitors (TEPs) with primary ETPs.

overall_design: At D28 of organoid formation, cells were harvested by soft mechanical dissociation to obtain the CD45+ cell fraction and analyzed using scRNA-seq

first: Nathan

last: Provin


title: Isolated TEC and DC from human iPSc-derived thymic organoids (hTOs) seeded with primary early thymic progenitors (ETPs) at late phase of maturation (D28)

summary: We employed an ultra-sensitive bulk RNA sequencing approach to study thymic epithelial cells (TECs), sorted by FACS based on their HLA-DR expression levels, and dendritic cells (DCs), both isolated from hTOs at the late stage of differentiation (day 28). These hTOs were generated by reaggregating iPSc-derived thymic epithelial progenitors (TEPs) with primary ETPs.

overall_design: At D28 of organoid formation, cells were isolated to obtain the CD45-EPCAM+ HLA-DRhigh and HLA-DRlow fractions (TEChi and TEClo), as well as the CD45+EPCAMlo fraction (DC), analyzed using SMARTER bulk RNA-seq


title: Human iPSc-derived thymic organoids (hTOs) seeded with primary early thymic progenitors (ETPs) at early phase of maturation (D7)

summary: We have employed a single-cell sequencing approach to using 10x Genomics scRNA-seq to study the differentation/maturation of thymic epithalial cell progenitors (TEPs) obtained via our iPSc differentitation protocol based on design of experiment (DoE) in the early phase of hTO formation at D7after reaggregation with primary ETPs.

overall_design: At D7 of organoid formation, hTOs were dissociated and EPCAM+CD45- cells isolated by FACS sorting and analyzed using scRNA-seq


title: CD205+ iPSc-derived thymic epithelial progenitor cells

summary: We employed an ultra-sensitive bulk RNA sequencing approach to study thymic epithelial progenitor cells (TEPs) derived from iPSc differentiation and sorted by FACS based on their CD205 expression levels.

overall_design: iPSc were differentiated using our DoE based protocol. The CD205+ fraction of the resulting TEPs was analyzed using SMARTER bulk RNA-seq


title: Maternal acute SARS-CoV-2 infection impairs preimplantation embryo development and reprograms early offspring hematopoietic system

city: Shanghai

first: Yuxuan

last: Zheng

address: 825 Zhangheng Rd.

institute: Fudan University

type: Methylation profiling by high throughput sequencing

summary: The COVID-19 pandemic has raised significant concerns regarding its impact on reproductive health, particularly in the context of assisted reproductive technology (ART). This study investigates the effects of maternal acute SARS-CoV-2 infection on preimplantation embryo development and early offspring hematopoietic system. We analyzed embryo development and neonatal immune responses in women undergoing ART during acute SARS-CoV-2 infection. Our findings reveal that maternal SARS-CoV-2 infection significantly impairs blastocyst formation and quality, with molecular evidence of developmental delay in embryos. Single-cell RNA sequencing (scRNA-seq) of blastocysts and umbilical cord blood (UCB) cells demonstrated dysregulated mitochondrial function, impaired mTORC1 signaling, and altered immune responses in neonates from infected mothers. Additionally, DNA methylation analysis revealed hypermethylation of immune-related genes in embryos from infected mothers. These results highlight the potential long-term impacts of maternal SARS-CoV-2 infection on embryonic development and offspring immune health, emphasizing the need for further research to balance patient safety and reproductive outcomes during the pandemic.

overall_design:
From December 17, 2022, to January 6, 2023, a total of 123 patients underwent ovarian stimulation cycles in our ART clinic. Among these, 28 patients were assigned to the SARS-CoV-2-positive (SARS-CoV-2 infected, IS) group, defined by a positive nucleic acid test or antigen test for SARS-CoV-2 within 24 hours prior to oocyte retrieval and a body temperature below 37°C during the procedure. The remaining 95 patients were initially classified as non-infected; however, 9 were excluded due to unsuccessful oocytes retrieval or cryopreservation of oocytes, resulting in 86 patients in the SARS-CoV-2 negative group (non-infected, NI). All participants in IS and NI groups underwent either intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Good-quality blastocysts were transferred back into the maternal uterus to achieve pregnancy, while additional blastocysts were cryopreserved for future use. With the patients’ informed consent, the cryopreserved blastocysts and neonatal umbilical cord blood (UCB) were collected for experimental purposes in this study.

***************************************************************
The raw sequence data reported in this study have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA010475) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human.
***************************************************************

first: Meiling

first: Di

last: Liu

first: Songmao

last: Li

first: Jiansheng

first: Fanghao

last: Guo

first: Haibin

last: Zhu

first: Li

last: Sun

first: Yu

last: Yan

first: Yanquan

first: Rui

last: Qiao

first: Haixia

last: Ding

first: Qing

first: Mengxi

first: Yongjian

last: Ma

first: Zhiwei

first: Wen


title: UHRF1 Deficiency Exacerbates Intestinal Inflammation by Epigenetic Modulation of NPY1R Gene Methylation

first: Xiaodi

last: Zhao

summary: Epigenetic modifications play a crucial role in the pathogenesis of inflammatory bowel disease (IBD) by mediating gene–environment interactions. We previously showed that UHRF1, a central regulator of DNA methylation, contributes to cancer progression; however, its function in IBD remains poorly understood. Here, we revealed that UHRF1 is frequently reduced in inflamed tissues of IBD patients and that its deficiency exacerbates intestinal epithelial cell (IEC) damage. Through a multi-level approach incorporating human cell models and an intestinal epithelial-specific Uhrf1 knockout mouse model, we established UHRF1 as a key mitigator of IBD progression. Mechanistically, UHRF1 bound to the NPY1R promoter, promoting its methylation and leading to transcriptional suppression. The NPY1R upregulation resulting from UHRF1 deficiency attenuated cAMP–PKA–CREB signaling in IECs, thereby enhancing NF-κB activation and subsequent pro-inflammatory responses, which compromised intestinal epithelial barrier integrity. Furthermore, we identified miR-141 as a negative regulator of NPY1R, highlighting its potential as a therapeutic agent. Collectively, our results identified the UHRF1–NPY1R regulatory axis as a critical epigenetic mechanism in intestinal inflammation and underscored its dual promise for IBD diagnostics and therapy.

overall_design: Genomic DNA was isolated from NCM460-shCtrl and NCM460-shUHRF1 cells using the QIAamp DNA Mini Kit (Qiagen). DNA purity and concentration were measured with a Nanodrop 2000 spectrophotometer (Thermo Scientific). Approximately 500 ng of DNA from each sample was subjected to sodium bisulfite conversion using the EZ DNA Methylation-Gold Kit (Zymo Research) according to the manufacturer’s protocol. Genome-wide DNA methylation was assessed using the Illumina Infinium HumanMethylation850K BeadChip (Illumina) following the manufacturer’s instructions. Array data (IDAT files) were analyzed with the ChAMP package in R to determine methylation levels. The methylation status of each probe was represented as a β value, defined as the ratio of methylated probe intensity to total probe intensity (sum of methylated and unmethylated intensities plus a constant α, α = 100). CpG sites with |Δβ| ≥ 0.20 (test vs. control) and an adjusted P value ≤ 0.05 were considered differentially methylated. A CpG site was classified as hypermethylated if Δβ ≥ 0.20 or hypomethylated if Δβ ≤ −0.20.

first: Han

last: Yanan


title: Injured epithelial cell states impact kidney allograft survival after T-cell-mediated rejection

city: Hannover

first: Christian

last: Hinze

address: Carl-Neuberg-Str. 1

department: Department of Nephrology and Hypertension

country: Germany

institute: Hannover Medical School

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

overall_design: Refer to individual Series


summary: Background: T-cell mediated rejection (TCMR) remains a significant challenge after kidney transplantation and is associated with reduced allograft outcome. Previous research highlighted the critical role of TCMR-induced renal epithelial injury. Yet, the detailed cellular origin of these injury responses and the associated clinical implications remain poorly understood.  Methods: To induce acute TCMR, we used mouse models of allogeneic (C57BL/6 to BALB/c and BALB/c to C57BL/6) kidney transplantation and syngeneic controls (C57BL/6 to C57BL/6 and BALB/c to BALB/c). Molecular changes were analyzed 7 days post-transplant using single-nucleus RNA sequencing and spatial transcriptomics. Results were compared with snRNA-seq data from three human TCMR biopsies and three stable allografts without rejection. The clinical impact of TCMR-induced epithelial injury was evaluated using marker gene sets on bulk transcriptomic data from 1292 kidney allografts, including 95 TCMR samples, with allograft outcome.  Results: Mouse kidneys from allogeneic transplants exhibited all hallmark histological features of TCMR. Single-nucleus RNA sequencing revealed TCMR-induced injured cell states and significant gene expression changes particularly in proximal tubules (PT) and thick ascending limbs (TAL). Spatial transcriptomics showed a heterogeneous spatial distribution of these injured cell states and proximity to leukocytes. Cross-species analysis confirmed similar injured PT and TAL cell states in human TCMR. Signatures of a severely injured TCMR-induced TAL cell state correlated strongly with allograft survival in a large kidney transplant cohort specifically after TCMR.  Conclusion: This is the first study showing kidney allograft outcome with distinct injured epithelial cell states. We found a particularly strong correlation between kidney allograft survival and TAL injury in TCMR, which occurred with reduced proximity to leukocytes, suggesting potential epithelial injury driven by non-immune mechanisms.

overall_design: Xenium spatial transcriptomics data from 7 mouse kidney transplantations including 2 syngeneic controls and 5 allogeneic transplantations with acute rejection.

first: Anna

last: Pfefferkorn

first: Lorenz

last: Jahn

first: Patrick

last: Gauthier

first: Janna

last: Leiz

first: Sadia

last: Safraz

first: Vera

last: Kuhlow

first: Izabela

last: Plumbom

first: Svjetlana

last: Lovric

first: Jessica

last: Schmitz

first: Jan

last: Bräsen

first: Irina

last: Scheffner

first: Michael

last: Fähling

first: Igor

last: Sauer

first: Felix

last: Aigner

first: Janine

last: Altmüller

first: Thomas

last: Conrad

first: Kai

last: Schmidt-Ott

first: Wilfried

last: Gwinner

first: Philipp

last: Halloran

first: Muhammad

last: Ashraf


city: Boston

first: Nikolaos

last: Tsopoulidis

state: MA

address: 185 Cambridge Street, CPZN 4100

institute: MGH Molecular Biology

type: Non-coding RNA profiling by high throughput sequencing

summary: Stem cells continually self-renew and differentiate to sustain tissue homeostasis, yet the role of post-transcriptional mechanisms in guiding these processes remains incompletely understood. Here, we demonstrate that the regulation of 3’UTR length via alternative mRNA polyadenylation (APA) is essential for stem cell function across diverse tissues. Modulating the APA regulator Nudt21 reveals that stem cell self-renewal and differentiation depend on distinct dosage thresholds and thus can be uncoupled. Specifically, moderate Nudt21 suppression  elicits a maturation arrest of stem cells due to 3’UTR-shortening of differentiation-associated mRNAs that escape miRNA regulation and perturb ceRNA networks. By contrast, complete Nudt21 suppression additionally shortens the 3’UTRs of mRNAs encoding essential multiprotein complexes, including the nuclear pore, leading to complex destabilization, proteotoxic stress, DNA damage, and cell cycle arrest. Critically, deletion of the alternative 3’UTRs of individual nucleoporins recapitulates defects observed with Nudt21 loss. We further demonstrate that the co-translational assembly of dozens of protein complexes is impaired in Nudt21-deficient cells, providing a mechanistic framework for compromised complex integrity. Collectively, our results show that APA plays distinct, dose-dependent roles in stem cell homeostasis by fine-tuning the expression of differentiation-associated genes and coordinating the biogenesis of multiprotein complexes essential for cell cycle progression.

overall_design:
RNA-seq timecourse for Nudt21 KO and Nudt21 Hypomorph esophageal basal cells:
RNA-seq was performed for Nudt21 KO (d0, d2, d4, d6) and Nudt21 Hypomorph (d4, d6) in triplicate. Day 0 are cells that were treated with ETOH instead of 4OHT, thus they retain the floxed Nudt21 allel

PAS-seq for Nudt21 KO and Nudt21 Hypomorph esophageal basal cells:
PAS-seq was performed for Control, Nudt21 KO (d4) and Nudt21 Hypomorph (d4) in triplicate. Control samples  were treated with ETOH instead of 4OHT, thus they retain the floxed Nudt21 allel

Small-RNA-seq for Control and Nudt21 KO esophageal basal cells:
small-RNA-seq was performed for Control and Nudt21 KO (d4) in triplicate. Control samples  were treated with ETOH instead of 4OHT, thus they retain the floxed Nudt21 allel

first: Konrad

last: Hochedlinger


title: GPR25 promotes the formation of lung and liver tissue-resident memory CD8+ T cells

city: LA JOLLA, SAN DIEGO

first: Pandurangan

last: Vijayanand

state: CALIFORNIA

address: 9420 ATHENA CIRCLE

department: Vaccine Discovery

institute: La Jolla Institute for Immunology

summary: Tissue-resident memory (TRM) CD8+ T cells are key players that orchestrate protective anti-viral and anti-tumor immune responses. The molecules that support their development and function are not fully defined. Here, we report on the regulation and function of an orphan G-protein coupled receptor, GPR25 that is expressed at higher levels in TRM cells. TGF-b, a key cytokine involved in the development to TRM cells, induces the expression of GPR25 in CD8+ T cells, and TRM-associated cis-regulatory elements in the GPR25 locus show binding of SMAD1, key transcription factors downstream of TGF-b signaling. By using Gpr25-deficient T cells in an LCMV infection model, we show that Gpr25 acts in a cell-intrinsic manner to promote the development of both primary and secondary TRM cells in the liver. Gpr25 deficiency in T cells also impairs their capacity to develop into CD69+CD103+ TRM cells in the lungs as well as to control lung metastasis in a tumor challenge model. Single-cell transcriptomic analysis of Gpr25-deficient memory T cells and TRM cells showed potential defects in restraining the ZEB2-S1PR5 pathway that drives tissue egress and in supporting a stem-like memory program as opposed to effector differentiation. Our findings support the concept that modulating Gpr25 function may be an attractive therapeutic option to boost the magnitude and quality of TRM responses generated in the context of infection and cancer.

overall_design: CD45.1.2 Gpr25 WT and CD45.2 Gpr25 KO OT-I cells were mixed in a 1:1 ratio and adoptively transferred at 1 x 10^6 total cells per recipient CD45.1 mouse. Mice were then infected with LCMV-OVA. Thirty days after infection, liver and lung tissues were detached and CD8+ T cells (or CD69+ CD8+ T cells) were isolated by FACS sorting.

first: Sungjun

last: Park

first: Han

last: Feng

first: Jae

last: Woo Shin

first: Francisco

last: Castañeda-Castro

first: Benjamin

last: Schmiedel

first: Changlu

last: Liu

last: Jackson

first: Christian

middle: H

last: Ottensmeier


title: Dual promoter–enhancer activities reflect a unified regulatory logic

status: Public on Nov 20 2025

city: Ithaca

first: Haiyuan

last: Yu

state: New York

address: 237 Tower Road

department: Department of Computational Biology, Weill Institute for Cell and Molecular Biology

institute: Cornell University

summary: Accumulating evidence indicates that both putative promoters and enhancers can exert dual promoter and enhancer functions. However, how these activities are related within individual elements and what determines this dual functionality remain poorly understood. We developed a massively parallel dual reporter assay that enables simultaneous assessment of the intrinsic promoter and enhancer potential exerted by the same sequence. Parallel quantification for thousands of elements reveals that canonical human promoters and enhancers can act as both promoters and enhancers under the same contexts, and that promoter activity may be necessary but not sufficient for enhancer function. Perturbations to element transcription factor binding motifs lead to disruptions in both activities, implicating a shared syntax for the two regulatory functions. Combinations of elements with different minimal promoters reveal reciprocal activity modulation, which, together with a strong correlation between promoter and enhancer functions, imply a bidirectional feedback loop to sustain environments of high transcriptional activity. Finally, we validate this reciprocity and correlation in situ using CRISPR activation at the human β-globin locus. Our approach reveals that the functional convergence between promoters and enhancers arises from a shared regulatory logic and sequence syntax, advancing a unified model for regulatory element biology.

overall_design: Quantitative Unifying Assay for Simultaneously Active Regulatory Regions by sequencing (QUASARR-seq), a massively parallel dual-reporter assay that simultaneously measures the intrinsic promoter and enhancer activities exerted by the same DNA sequence.

first: Mauricio

middle: I

last: Paramo

first: John

middle: T

last: Lis


title: Spatial transcriptomics to dissect molecular signature of cardiac fibroblasts in experimental autoimmune myocarditis

city: Kraków

first: Monika

middle: Marta

last: Stefanska

state: malopolska

address: Medical College, Wielicka 265

department: Department of Clinical Immunology

country: Poland

institute: Jagiellonian University

summary: Myocarditis is a severe inflammatory cardiac disease that can progress to dilated cardiomyopathy and heart failure. Experimental autoimmune myocarditis (EAM) represents an animal model of acute myocarditis, which is followed by the development of post-inflammatory cardiac fibrosis and systolic dysfunction. In this model, cardiac inflammation is characterized by the massive influx of myeloid infiltrates and is paralleled by the activation of extracellular matrix-producing cardiac fibroblasts. 6–8-week-old BALB/c mice were immunized with αMyHC peptide together with Complete Freunds’ Adjuvant to induce EAM. Heart tissues were isolated, embedded in OTC, sectioned into 10 µm-thick fragments, and placed on a 10x Genomics Visium Gene Expression Slide. Single-cell spatial libraries were prepared using 10x Genomics Visium protocols, and the sequencing was performed using Illumina Chemistry. Single-cell RNA sequencing data was analyzed using RStudio software. In our analysis, we included three biological time points: a healthy heart (d0), acute myocarditis (d19). Following sequencing, we analyzed 3789 spots under the tissue. We identified the main cell populations present in the mouse heart, including fibroblasts, immune cells, endothelial cells, smooth muscle cells, and cardiomyocytes. Subset analysis on cardiac fibroblasts resulted in the presence of four different subsets (FB1–FB6). Subset FB3 has a myofibroblastic signature defined by the expression of, among others, Acta2, Postn, Vim, Col1a1 and Col3a1. This subset is transiently present at d19 of EAM. Spatial analysis of infiltrating immune cells characterized by the expression of, e.g., Ly6c1 and Lyz2 resulted in the presence of five subsets (INF1–INF5). The INF4 subset is present only at acute myocarditis phase (d19) and is spatiotemporally colocalized with the myofibroblastic subset(FB3). Our data indicate that myofibroblasts and infiltrating immune cells colocalize within the tissue during acute phase of myocarditis. This may indicate active molecular interplay between inflammatory cells and cardiac fibroblasts.

overall_design: Spatial transcriptomic data of mouse heart at different stages of experimental autoimmune myocarditis.  6-8-week-old BALB/c mice were immunized with αMyHC peptide together with Complete Freunds’ Adjuvant to induce EAM. Heart tissues were isolated, embedded in OTC, sectioned into 10 µm-thick fragments, and placed on a 10x Genomics Visium Gene Expression Slide. Single-cell spatial libraries were prepared using 10x Genomics Visium protocols, and the sequencing was performed using Illumina Chemistry. Single-cell RNA sequencing data was analyzed using RStudio software.

first: Katarzyna

last: Sarad

first: Marta

last: Kot

first: Martyna

last: Strzelec

first: Daria

last: Krzysztofik


title: The conserved SEN1 DNA/RNA helicase has multiple functions during yeast meiosis

city: Stony Brook

first: Ed

last: Luk

state: NY

address: 100 Nicolls Rd, Life Sciences Building

department: Biochemistry and Cell Biology

institute: Stony Brook University

summary: DNA:RNA hybrids are unusual structures found throughout the genomes of many species, including yeast and mammals. While DNA:RNA hybrids may promote various cellular functions, persistent hybrids lead to double strand breaks, resulting in genomic instability. DNA:RNA hybrid formation and removal are therefore highly regulated, including by enzymes that either degrade or unwind RNA from the hybrid. Meiosis is the specialized cell division that creates haploid gametes for sexual reproduction. Previous work in yeast and mammals showed that elimination of DNA:RNA hybrids by RNase H facilitates meiotic recombination. This work demonstrates that the conserved Sen1 DNA/RNA helicase functions during three temporally distinct processes during yeast meiosis. First, SEN1 allows meiosis-specific genes to be expressed at the proper time to allow entry into meiosis. Second, SEN1 prevents the accumulation of hybrids during premeiotic DNA replication. Third, SEN1 promotes the repair of programmed meiotic double strand breaks that are necessary to form crossovers between homologous chromosomes to allow their proper segregation at the first meiotic division. Given the evolutionary conservation of Sen1 with its mammalian counterpart, Senataxin, studies of Sen1 function in yeast are likely to be informative about the regulation of DNA:RNA hybrids during humans as well.

overall_design: To investigate how SEN1 contributes to the regulation of IME1, nanopore direct RNA sequencing (dRNA-seq) was performed on coding and non-coding transcripts isolated from SEN1 and sen1-md cells in Spo medium. dRNA-seq requires that each transcript has a 3’ poly(A) tail to which a sequencing adapter and motor protein that threads the RNA through a pore can be ligated. Non-coding RNAs—particularly those that fail to terminate properly in the absence of the Nrd1-Nab3-Sen1 (NNS) complex lack poly(A) tails and therefore would be underrepresented. To capture these transcripts, poly(A) tails are added in vitro. However the high abundance of the four ribosomal RNAs (rRNAs) (25S, 18S, 5.8S and 5S) in total RNA preparations would swamp out this reaction and so must be removed prior to polyadenylation. The four rRNAs were depleted from total RNA isolated from SEN1 and sen1-md cells by hybridization to the combined biotinylated anti-sense transcripts for each rRNA, followed by streptavidin-based removal. The resulting rRNA-depleted RNA was then polyadenylated in vitro using E. coli poly(A) polymerase prior to nanopore library preparation. Two biological replicates were performed for SEN1 and sen1-md strains after two hours in Spo medium, yielding ~6-7 million uniquely aligned reads per replicate to the S. cerevisiae SK1 genome.

first: Robert

last: Gaglione

first: Leonidas

last: Pierrakeas

first: Lihong

last: Wan

first: Jonathan

last: Caradonna

first: Amy

middle: J

last: MacQueen

first: Nancy

last: Hollingsworth


title: Clonal T cell responses in human xenograft rejection

city: New York

first: Ramin

last: Herati

address: 430 E. 29th Street, 1st floor, room 135A-C

institute: NYU School of Medicine

summary: Despite the life-saving successes of solid organ transplantation, the number of individuals needing organ transplant far exceeds the number of organs available for use each year. Porcine xenotransplantation, or the use of pig organs for transplantation in people, holds substantial promise but xenograft rejection in humans is poorly understood. T cell rejection by the host immune system is a major challenge for human allografts and may limit the longevity of porcine xenografts. To study the xenograft rejection, we evaluated T cell responses and repertoire dynamics across tissues following porcine xenograft transplantation in a decedent model over 61 days after bilateral native kidney nephrectomy. Despite induction with anti-thymocyte globulin and ongoing  immune suppression consisting of rituximab, corticosteroids, calcineurin inhibition, and proliferation inhibition, human T cell infiltration of the xenograft was observed and was associated with xenograft dysfunction. Longitudinal analysis of T cell clonotypes in biopsies of the xenokidney revealed accumulation of clonal human CD4 and CD8 T cell responses. Moreover, circulating activated T cells, including circulating T follicular helper (cTfh), were xeno-reactive and increased in frequency around rejection events. A single CD8 clonotype dominated in the circulation leading up to the acute cellular rejection event. Following re-treatment with anti-thymocyte globulin and intensification of corticosteroids, the xeno-reactive T cell clonotypes were dramatically diminished in frequency in lymph nodes, though not eliminated. Together, these data demonstrate T cell repertoire dynamics across tissues in the setting of xenograft rejection and highlight opportunities for early surveillance and potential intervention.

overall_design: Multimodal single-cell RNA sequencing was used to compare T cells over time following porcine xenotransplantion in a decendent model.

first: Ekaterina

last: Novikova

first: Elizabeth

last: Severa

first: Fiorella

last: Chacon

first: Han

last: Chen

first: Farshid

last: Fathi

first: Nathan

last: Suek

first: Benjamin

last: Vermette

first: Robin

last: Bombardi

first: Eloi

last: Schmauch

first: Simon

last: Williams

first: Imad

last: Aljabban

first: Ian

last: Jaffe

first: Karen

last: Khalil

first: Jacqueline

last: Kim

first: Elaina

middle: P

last: Weldon

first: Anoma

last: Nellore

first: Megan

last: Sykes

first: Adam

middle: D

last: Griesemer

first: Brendan

last: Keating

first: Jeffrey

last: Stern

last: Montgomery


title: Asymmetric envelope surface disposition of secreted protein YjbI controls bimodal antibiotic susceptibilities in C. crescentus

city: Würzburg

first: Manuel

last: Velasco Gomariz

address: Josef-Schneider-Straße 2/D15

institute: Helmholtz Institute for RNA-based Infection Research (HIRI)

summary: Cytoplasmic pentapeptide repeat proteins (PRPs) protect bacterial DNA gyrase from quinolone antibiotics. While some secreted PRPs are essential upon quinolone exposure, their role in the regulation of antibiotic resistance remains to be fully characterized. We show that a YjbI-type secreted PRP regulates antibiotic sensitivity, bimodally for small or large molecules, via modulation of the Caulobacter crescentus outer membrane (OM). YjbI silences two converging envelope-stress pathways that globally reprogram the OM proteome via TonB-dependent receptors (TBDRs), periplasmic proteases, and AcrAB-NodT, a multidrug efflux pump whose induction by small molecules and antibiotics is lethal to yjbI mutant cells. Loss of YjbI also confers sensitivity to vancomycin and bacitracin, two large peptidoglycan-targeting and zinc-binding antibiotics that permeate the outer membrane via the previously uncharacterized TBDR BugA and its orthologs. Zinc stress triggers rapid proteolytic removal of Yjbl, activates expression of TBDRs, including BugA, and ultimately leads to replenishment of YjbI. Molecular dynamics simulations and reactive thiol probing imply an asymmetric surface disposition of YjbI, explaining the differential accessibility of its conserved cysteine pairs that flank the quadrilateral β-helix. Taken together, our findings identify a role of YjbI as a cell surface-regulator of outer membrane composition and antibiotic sensitivity in a Gram-negative bacterium.

overall_design: Transcriptomic of Caulobacter NA100 wild-type (KFS-6) & Caulobacter NA100 Δyjbl (PV20792) in the absence/presence on 80 µM ZnSo4. Cells were growing in PYE medium at 30°C until they reached OD660=0.4. 80 µM of ZnSO4 or mock was added to the cells and after 30´ samples were collected for RNAprep

first: Kathrin

middle: S

last: Fröhlich

first: J

last: Costrafolaz

first: P

last: Viollier


title: NBL1 Associates with Renal Phenotypes in Mice, but Partial Nbl1 Reduction Does Not Ameliorate Kidney Disease: Male Nbl1 HET or WT Mice Treated with Cisplatin or PBS

city: Bar Harbor

first: Courtney

last: Willey

address: 600 Main Street

institute: The Jackson Laboratory

summary: Background: Increased concentrations of neuroblastoma suppressor of tumorgenicity 1 (NBL1) in the blood have been associated with disease progression in diabetic kidney disease and IgA nephropathy. However, it is unclear whether NBL1 is a causal factor for kidney disease and what is driving these increased concentrations in the blood. Methods: We tested causality of NBL1 by knocking out Nbl1 in two different mouse models of kidney disease (X-linked Alport Syndrome (XLAS) and low-dose cisplatin treatment) and performed a genetic analysis for the drivers of NBL1 concentrations in two independent cohorts of genetically diverse (DO) mice with X-linked Alport Syndrome (DO-XLAS). Results: Significant correlations were found between NBL1 concentrations in the blood and both GFR and ACR in DO-XLAS mice. However, XLAS mice with only one functional Nbl1 allele and reduced NBL1 concentrations had similar GFR and ACR compared to Nbl1 wildtype animals. Similarly, there was no difference between animals with one or two functional Nbl1 alleles after chronic low-dose cisplatin treatment. Genetic analysis of NBL1 concentrations in our DO-XLAS cohorts identified associations with loci on Chromosomes 4 and 17, with Clcnka, and MHC2 as potential candidates. Conclusion: Increased NBL1 in blood is a marker for different forms of kidney disease but is not a causal factor.

overall_design: Male Nbl1 HET or WT mice treated with Cisplatin or PBS (Control) for 4 consecutive weeks starting at 8 weeks of age (all mice are on the B6 background). Bulk RNA-seq of kidneys collected 3 days post treatment at 11-weeks of age.

first: Rei

last: Bufi

first: Harsphpreet

last: Chandok

first: Ron

last: Korstanje


title: PNPLA3-I148M genetic variant rewires lipid metabolism to drive programmed cell death in human hepatocytes

city: Pittsburgh

first: Soto

middle: Gutierrez

last: Alejandro

state: PA

address: 200 Lothrop St.

department: Department of Pathology

institute: University of Pittsburgh

summary: Genetic variants in lipid metabolism influence the risk of developing metabolic dysfunction-associated steatotic liver disease (MASLD), cirrhosis, and end-stage liver disease (ESLD). The mechanisms by which these variants drive disease are poorly understood. Because of the PNPLA3-I148M variant's strong correlation with all stages of the MASLD spectrum and the lack of tractable therapeutic targets, we sought to understand its impact on cellular function and liver metabolism. Primary human hepatocytes (HAH) and iPSC-derived hepatocytes (iHeps) from healthy individuals possessing the PNPLA3-I148M mutation were characterized for changes in lipid metabolism, cellular stress, and survival. Using lipidomics, metabolomics, stable isotope tracing, and flux propensity analysis, we created a comprehensive metabolic profile of the changes associated with the PNPLA3-I148M variant. Functional analysis showed that the presence of the PNPLA3-I148M variant increased endoplasmic reticulum stress, mitochondrial dysfunction, and peroxisomal β-oxidation, ultimately leading to cell death via ferroptosis. Nutritional interventions, ferroptosis-specific inhibitors, and genetic approaches modulating GPX4 activity in PNPLA3-I148M HAH and iHeps decreased programmed cell death. Our findings indicate that therapies targeting ferroptosis in patients carrying the PNPLA3-I148M variant could affect the development of MASLD and ESLD and highlight the utility of iPSC-based models for the study of genetic contributions to hepatic disorders.

overall_design: RNA-Seq profiling of isolated primary human hepatocytes carrying the SNP PNPLA3-I148M as compared to the WT

first: Rodrigo

last: Florentino

first: Olamide

last: Animasahun

first: Nils

last: Haep

first: Minal

last: Nenwani

first: Kehinde

middle: O

last: Omoloja

first: Leyla

last: Altay

first: Abhinav

last: Achreja

first: Kazutoyo

last: Morita

first: Takashi

last: Motomura

first: Ricardo

last: Diaz-Aragon

first: Lanuza

last: Faccioli

first: Yiyue

last: Sun

first: Zhenghao

last: Liu

first: Zhiping

last: Hu

first: Bo

last: Yang

first: Fulei

last: Wuchu

first: Ajay

last: Shankaran

first: Miya

last: Paserba

first: Annalisa

last: Baratta

first: Shohrat

last: Arazov

first: Zehra

last: Kocas-Kilicarslan

first: Noah

last: Meurs

first: Jaideep

last: Behari

first: Edgar

last: Tafaleng

first: Jonathan

last: Franks

first: Alina

last: Ostrowska

first: Takahiro

last: Tomiyama

first: Kyohei

last: Yugawa

first: Akinari

last: Morinaga

first: Zi

last: Wang

first: Kazuki

last: Takeishi

first: Dillon

last: Gavlock

first: Mark

last: Miedel

first: D.

last: Taylor

first: Ira

last: Fox

first: Tomoharu

last: Yoshizumi

first: Deepak

last: Nagrath

first: Alejandro

last: Soto-Gutierrez


title: ClpC1-targeting peptide natural products differentially dysregulate the proteome of Mycobacterium tuberculosis.

city: Camperdown

first: Daniel

last: Hesselson

state: NSW

address: Bldg 93, RPA Hospital, Missenden Rd

institute: Centenary Institute

summary: Targeting the protein quality control system in Mycobacterium tuberculosis offers a powerful and largely untapped opportunity for antibiotic development. The ClpC1:ClpP1P2 protease is an essential component of the system and is involved in both regulatory and stress-related protein degradation. Several non-ribosomal peptide natural product families have been discovered, including ecumicin, ilamycins (also called rufomycins) and cyclomarins, which bind to the ClpC1 chaperone component of the system and possess potent antimycobacterial activity. This has led to significant interest in these molecules, and the ClpC1:ClpP1P2 system more generally, as a bona fide target for the development of new tuberculosis drugs. In this study, we apply a systematic and multi-layered approach combining quantitative proteomics, bioinformatics, transcriptomic profiling, CRISPRi gene knockdown, and targeted biochemical and biophysical assays to dissect the mechanisms of ecumicin, ilamycin, and cyclomarin in clinically relevant Mycobacterium tuberculosis. Strikingly, despite exhibiting similar binding modes to ClpC1, each natural product elicits distinct substrate-specific effects on protein-degradation. We further show that derivatives of ilamycin and ecumicin engage the stress-response chaperones ClpC2 and Hsp20 respectively, albeit with varied sequestration capacity. Together our data reveal that these natural products dysregulate ClpC1 substrate recognition and perturb stress response chaperones in distinct ways, highlighting opportunities to target multiple nodes of protein quality control for the development of next generation antimycobacterials.

overall_design: Exponentially growing Mtb H37Rv cultures were treated with MIC90 Ecu* (1) and incubated for a further 48 h at 37 C. The Mtb cells were then centrifuged at 3725 g for 10 min to pellet bacteria. Pellets were resuspended with 1 mL TRIzol and transferred to a 2 mL O-ring tube with acid-washed glass beads. To disrupt the mycobacterial cell envelope, cells were subjected to six cycles of bead beating (20 s at 4600 rpm and 40 s on ice) with a (Biopec products) mini beadbeater. Samples were then clarified at 18000 g for 1 min. After a short room temp incubation chloroform was added to each sample and samples were inverted by hand for 15 seconds and incubated at room temperature for 3 min. Samples were then centrifuged at 12000 g for 15 min at 4 C. The upper aqueous phase containing RNA was collected. RNA was purified from the aqueous phase with the Isolate II RNA micro kit. Libraries were prepared using the (Illumina) Total RNA library kit with Ribo-Zero Plus chemistry for rRNA depletion and sequenced on an (Illumina) NovaSeq 6000 platform to generate 150 bp paired-end reads (>65 million pairs per sample).

first: Isabel

middle: K

last: Barter

first: Max

last: Bedding

first: Julia

last: Leodolter

first: Joshua

middle: W

last: Maxwell

first: Paige

last: Hawkins

first: Maxwell

last: Stevens

first: Matthew

last: McNeil

first: William

last: Jowsey

first: Trixie

first: Diana

last: Quan

first: Sabryna

last: Junker

first: Manuela

last: Flórido

first: Gregory

last: Cook

first: Tim

last: Clausen

first: Warwick

last: Britton

last: Larance

first: Richard

last: Payne


title: A gene expression atlas of a juvenile nervous system.

city: Nashville

first: David

last: Miller III

state: Tennessee

address: 465 21st Avenue South

department: Cell and Developmental Biology

institute: Vanderbilt University

summary: Although the fundamental architecture of metazoan nervous systems is typically generated in the embryo, substantial numbers of neurons are added during post-natal development as existing neurons also expand in size and undergo additional differentiation. To reveal the underlying molecular determinants of post-embryonic neurogenesis and maturation, we have produced gene expression profiles of all neuron types and their progenitors in the first larval stage (L1) of C. elegans. Comparisons of the L1 profile to the embryo and to the later L4 larval stage identified thousands of differentially expressed genes across individual neurons throughout the nervous system. Key neuropeptide signaling networks, for example, are remodeled during larval development. Gene regulatory network analysis revealed potential transcription factors driving the temporal changes in gene expression across the nervous system, including a likely broad role of the heterochronic gene lin-14. We utilized available connectomic data of juvenile animals in combination with our neuron-specific atlas to identify potential molecular determinants of membrane contact and synaptic connectivity. These expression data are available through a user-friendly interface at CeNGEN.org for independent investigations of the maturation, connectivity and function of a developing nervous system.

overall_design: Neurons were isolated from L1 stage hermaphrodite Caenorhabditis elegans using Fluroescence-activated cell sorting (FACS) and analyzed using scRNA-seq.

first: Seth

middle: R

last: Taylor

first: Claire

last: Olson

first: Lidia

last: Ripoll-Sanchez

first: Giulio

last: Valperga

first: Rebecca

last: McWhirter

first: S. Talmage

last: Barney

first: Alexander

last: Atkinson

first: Sidharth

last: Goel

first: Alexis

last: Weinreb

first: Andrew

last: Hardin

last: Rolfson

first: Jacob

last: Pattee

first: G. Robert

last: Aguilar

first: Daniel

last: Merritt

last: Eroglu

first: Maryam

last: Majeed

first: Ethan

last: Grungvig

last: Child

last: Beets

first: Petra

last: Vertes

last: Schafer

first: Erdem

last: Varol

first: Marc

last: Hammarlund

first: Oliver

last: Hobert


title: Non-reciprocal callosal projections and input gradients underlie interhemispheric communication in binocular visual cortex

first: Elisabetta

last: Furlanis

address: Longwood avenue

institute: Harvard Medical School

summary: How the brain combines information received independently by the two hemispheres is not fully understood. Here we describe a non-reciprocal circuit for interhemispheric communication in mouse binocular visual cortex via anatomically segregated neuronal populations. Callosal projecting neurons (CPNs) received only weak or no callosal input, whereas callosal receiving neurons (CRNs) made only weak or no callosal projections. Both populations received direct input from the thalamus. At the cellular level, CRNs had reduced excitability compared to non-CRNs (putative CPNs) due to elevated Kv1 potassium channel expression (encoded by the KCNA2 gene), with excitability of CRNs correlated with the magnitude of callosal input. Functionally, CRNs were predominately binocular, with binocularity correlated with callosal input, whereas non-CRNs (putative CPNs) were predominately monocular. In summary, we find that non-reciprocal callosal projections between CPNs and CRNs together with differences in excitability shaped by callosal input underlie interhemispheric communication in binocular visual cortex.

overall_design: RNA-seq profiling of virally labeled and FAC-sorted callosal projecting (CPN) and callosal receiving (CRN) neurons from the mouse binocula visula cortex


title: Oncogenic KRAS/ERK/JUNB signaling suppresses differentiation regulator GATA6 in pancreatic cancer

city: Singapore

first: David

last: Virshup

address: 8 College Road

country: Singapore

institute: Duke-NUS Medical School

summary: GATA6 is a master regulator of differentiation in the pancreas and its expression levels determine the two main molecular subtypes of pancreatic cancer. High GATA6 contributes to the “classical” pancreatic cancer subtype, which is associated with a higher degree of tumor differentiation and better disease prognosis. However, why GATA6 expression varies across pancreatic cancers and what regulate GATA6 expression remain elusive. Here we report that the oncogenic KRAS-activated ERK signaling suppresses GATA6 transcription in pancreatic cancers. GATA6 mRNA levels inversely correlated with KRAS/ERK activity in pancreatic tumors. A genome-wide CRISPR screen in a GATA6-EGFP reporter knockin cell line identified JUNB as the ERK-regulated transcriptional repressor for GATA6. Active ERK stabilizes JUNB protein while KRAS/ERK inhibition led to ubiquitin-independent proteasomal degradation of JUNB and increased transcription of GATA6. Up-regulation of GATA6 enhanced chemosensitivity of pancreatic cancer cells and KRAS/ERK inhibitors synergized with chemotherapy in a GATA6-dependent manner. Our study identifies how oncogenic KRAS/ERK signaling suppresses GATA6 to cause dedifferentiation in pancreatic cancer. Combining KRAS/ERK inhibitors with standard-of-care chemotherapies could be a promising therapeutic strategy for treating pancreatic cancers.

overall_design: To explore the genome occupancy of JUNB in pancreatic cancer and how MEK/ERK inhibition affects JUNB’s genome occupancy, we performed Cut&Run assays with anti-JUNB antibody or IgG control in HPAF-II cells treated with or without the MEK inhibitor trametinib, followed by next-generation sequencing (Cut&Run-seq).

first: Zheng

last: Zhong


title: Lineage-specific enhancer insertions regulate Prdm14 to drive the rapid transition from naïve to formative pluripotency in rodents

city: Sanda

first: Yoshiyuki

last: Seki

state: hyogo

address: 1, Gakuen Uegahara

department: School of Biological and Environmental Sciences

institute: Kwansei Gakuin University

summary: The network of transcription factors is dynamically reorganized during the transition from naïve to formative pluripotency. In mice, Prdm14 is expressed in naïve pluripotent cells but rapidly downregulated upon exit from the naïve state. In contrast, PRDM14 expression persists throughout pluripotency transitions in non-rodent mammals, including pigs and humans. Here, we investigate the molecular mechanisms underlying the rodent-specific expression of Prdm14. Using CRISPR/Cas9-mediated deletions, we demonstrated that OCT4 and TFCP2L1 recognition sequences within Muroidea-specific cis-regulatory elements located downstream of Prdm14 are essential for its transcriptional upregulation required for the upregulation of Prdm14 in naive embryonic stem cells (ESCs). Loss of these enhancers suppresses Pramel7 induction and impairs degradation of UHRF1, thereby attenuating global DNA demethylation under 2iL conditions. Moreover, deletion of PRDM14-binding motifs in Muroidea-specific enhancers disrupts its negative feedback loop, resulting in delayed transition from the naïve to formative pluripotent state.

overall_design: RNA-seq experiments of WT, Cis KO of Prdm14, Prdm14-overexpressing embryonic stem cells


title: Lamin A/C-regulated cysteine catabolic flux modulates stem cell fate through epigenome reprogramming [ES_RNA_seq_G609G_LA_EXP2]

city: Mannheim

first: Gergana

last: Dobreva

state: Baden Württemberg

address: Ludolf-Krehl-Str. 7-11

department: Cardiovascular Genomics and Epigenomics

institute: Medical Faculty Mannheim/University of Heidelberg

summary: Spatiotemporal changes in the nuclear lamina and cell metabolism shape cell fate, yet their interplay is poorly understood. Here, we identify lamin A/C as a key regulator of cysteine catabolic flux essential for proper cell fate and longevity. Its loss in naïve mouse pluripotent stem cells leads to upregulation of the cysteine generating and catabolizing enzymes, cystathionine γ-lyase (CTH) and cystathionine β-synthase (CBS), thereby promoting de novo cysteine synthesis. Increased cysteine flux into acetyl-CoA fosters histone H3K9 and H3K27 acetylation, triggering a transition from naïve to primed pluripotency and abnormal cell fate and function. Conversely, the toxic gain-of-function mutation of Lmna, encoding lamin A/C and associated with premature aging, reduces CTH and CBS levels. This reroutes cysteine catabolic flux and alters the balance between H3K9 acetylation and methylation, crucially impacting germ layer formation and genome stability. Importantly, modulation of Cth and Cbs rescues the abnormal cell fate and function, restores the DNA damage repair capacity, and alleviates the senescent phenotype caused by lamin A/C mutations, highlighting the potential of modulating cell metabolism to mitigate epigenetic diseases.

overall_design: RNA-seq of Lmna+/+, LmnaG609G/+,, LmnaG609G/G609G as well as CBS-OE-LmnaG609G/G609G embryonic stem cells

first: Yinuo

last: Wang

first: Haoije

last: Shi

first: Julio

last: Cordero


title: Lamin A/C-regulated cysteine catabolic flux modulates stem cell fate through epigenome reprogramming [ES_RNA_seq_G609G_LA_EXP1]


title: AAV Assembled Capsids Are Produced in Cells Blocked fromFrom Cell Cycle Progression

city: Ottawa

first: Ziying

last: Liu

address: 1200 Montreal Road

institute: National Research Council, Canada

summary:
Adeno-associated virus (AAV) is a promising delivery system for gene therapy. However, current
manufacturing of AAV suffers from very low yields compared to other biotherapeutics. The AAV
dose per patient ranges between 1011and 1015 viral genomes (vg), requiring an average of 10 to 30 L
production/dose. As a consequence, production costs are prohibitive for most indications. Our
recent studies revealed that only 10% of the HEK293 cells that have received the AAV encoding DNA
produce assembled AAV capsids. This observation prompts the question: Why would cells that have
been successfully transfected, be unable to produce AAV. To answer this question, we undertook a
detailed study to characterize the two sub-populations from the same transfection, the cells that
were making assembled capsids and those that were not. We found that the two populations had
distinct cell cycle profiles, with a block in cell cycle progression characterizing the producer
population. RNA-seq analysis of the two populations reveals differences in the molecular pathways
impacted and provides a basis for making changes to improve productivity.

overall_design: 11 RNA samples were used for RNA sequencing. All RNA extractions were performed on paraformaldehyde-fixed and antibody-stained cells. 2 RNA samples were isolated from un-transfected, fixed and stained 293SF cells, 3 from mock-transfected, fixed and stained cells and 6 from cells that were sorted into 2 populations after transfection, fixing and staining,  one producing virus and the other not, after transfection, fixing and staining.

first: Alaka

last: Mullick

first: Audrey

last: Morasse

first: Melanie

last: Leclerc


title: Zymomonas mobilis oxidative stress transcriptomics

city: Madison

first: Kevin

last: Myers

state: WI

address: 5127 WEI, 1552 University Ave

department: Great Lakes Bioenergy Research Center

institute: University of Wisconsin - Madison

summary: Zymomonas mobilis is an important bioenergy organism that has potential to produce biofuels, including ethanol, in high volumes. Here we examined the response of Zymomonas mobilis to various oxidative stresses using genome-scale transcriptomics data. We first examined the transcrpit abundance in WT aerobic growth compared to aerobic grown in paraquat, which forms superoxide. Under anaerobic growth conditions we compared WT Zymomonas mobilis with strains grown in media lacking iron as well as strains lacking iron that were treated with the iron chelator DIP before collection. Finally we examined transcript abundance in cells lacking ZMO_0422 (Rrf2 family transcription factor homolog) and ZMO_1411 (Fur homolog) grown under anaerobic conditions.

sample_organism: Zymomonas mobilis subsp. mobilis ZM4 = ATCC 31821

overall_design: Transcriptomic analysis of WT, a deletion of ZMO_0422, and a deletion of ZMO_1411 in Zymomonas mobilis ZM4 under aerobic and anaerobic growth conditions along with various oxidative stresses:  Paraquate addition, No Iron, and hydrogen peroxide addition.

first: Isabel

last: Askenasy

first: Kevin S

first: Patricia J

last: Kiley


title: Diabetes exacerbates destructive inflammation by activating the CD137L-CD137 axis in dendritic and gd T-cells

city: Philadelphia

first: Michael

last: Gonzalez

state: Pennsylvania

address: 3535 Market St

department: Translational Medicine and Human Genetics

institute: University of Pennsylvania

summary: Periodontal disease, a bacterial infection affecting a large percentage of the world's population, is an important risk factor for several systemic diseases and is significantly worsened by diabetes. To investigate how diabetes exacerbates the inflammatory response to bacteria in this disease, we combined insights from murine and human studies. Through single-cell RNA sequencing, we identified a compelling hyperglycemia-driven molecular pathway: the upregulation of CD137L in dendritic cells and increased expression of its receptor, CD137, in γδ T-cells. The CD137L-CD137 axis emerged as a pivotal mediator of diabetes-induced inflammatory tissue destruction. Antibody-mediated inhibition of CD137L markedly reduced the diabetes-driven bone loss, neutrophil recruitment, expansion of γδ T-cells, and excessive infiltration by IL17A+ cells.

overall_design:
Wild-type mice were CD11CCre-/- mice, which are on a mixed C57B6 - FVB background. Type-I diabetes mellitus was induced in mice using streptozotocin. Bacterial induced inflammation for two weeks, waiting period was two weeks. Cells were isolated from the gingiva, pooled from 3 mice per group with negative selection of dead cells (DAPI+), endothelial cells (CD31+), RBC (Ter-119+)and positive selection of CD45+ cells with 20% enrichment of CD11c+CD45+ cells by FACS sorting. 10,000 FACS sorted cells were loaded per lane of a 10x Genomics Chromium instrument for scRNA-seq analysis

***************************************************************
Submitter states that missing raw files are due to file loss.
***************************************************************

first: Xin

last: Huang

first: Min

middle: V

first: Rahul

last: Debnath

first: Hamideh

last: Afzali

first: Yongwon

last: Choi

first: Su

middle: A

last: Kim

first: Kang

last: Ko

first: Dana

last: Graves


title: Sequential DC-intrinsic Lymphotoxin Beta Receptor and Retinoic Acid Receptor signals regulate cDC2 fates

first: Chrysothemis

last: Brown

department: Immuno-Oncology Program

institute: Memorial Sloan Kettering Cancer Center

summary: Type II conventional dendritic cells (cDC2) are functionally and phenotypically heterogenous. Previous work identified two major cDC2 subsets (cDC2A and cDC2B) and a transcriptionally related monocytic DC3 subset; however, the underlying microenvironmental cues governing their distinct differentiation pathways remain unclear. Here we delineate cDC2 lineage relationships, and the sequential signals required for cDC2A specification and maintenance. We show that T-bet+ cDC2A, arising from a Clec9a+ DC progenitor, express the retinol receptor RBPR2, require cell-intrinsic retinoic acid (RA) signaling for their maintenance in the spleen and mesenteric lymph nodes, and depend on intrinsic RA receptor signaling to sustain downstream Notch signals. Prior to their expression of T-bet, splenic cDC require cell-intrinsic Lymphotoxin beta receptor (LTbR) which is needed to reduce F-actin content and prevents the escape of cDC2 to the blood. We propose a novel mechanism whereby splenic cDC2-intrinsic LTbR signaling anchors cDC2 within the spleen so that they may access macromolecules such as retinol to promote cDC2A development.

overall_design: scRNA-seq (gene expression and HTOs) of YFP+ and YFP– CD45+Lin–MHCII+ cells from spleen and mLN of Tbx21RFP-creERT2_Clec9a-cre/cre_R26-YFP (Clec9a fate-mapping) mice, pooled from 4 biological replicates.

first: Logan

last: Fisher

first: Anushka

last: Yadav