title: Cellular heterogeneity and therapeutic response profiling of human IDH+ glioma stem cell cultures

city: Seattle

first: Bahar

last: Tercan

state: WA

address: 401 Terry Ave. N.

institute: Institute for Systems Biology

summary: Glioblastoma stem cell (GSC) cultures are initiated from glioblastoma (GBM) surgical resection tissue. They can capture and propagate key GBM primary tumor molecular and cellular features. We have deeply characterized four IDH-expressing (IDH+ or ‘wildtype’) GSC cultures from unrelated adults to serve as cellular models for the majority of adult primary GBM. We demonstrate that GSC cultures can be continuously propagated in defined, serum-free media and 5% oxygen without requiring specialized growth substrates; have well-defined genomic and mtDNA variants and gene/protein expression profiles; and highly reproducible dose-survival curves when treated with the GBM standard-of-care therapies of ionizing radiation (IR) and temozolomide (TMZ). We also illustrate how expressed lentiviral barcodes, mtDNA variants and single cell gene expression profiling can be used to define and track cellular heterogeneity over 40 days after IR treatment. These well-characterized IDH+ GSC cultures can support many high throughput in vitro assay formats, including xenograft, organoid and other GBM disease modeling protocols. They should prove a useful resource to better understand GBM biology, and to identify new and more effective GBM therapies and treatment regimens.

overall_design: scRNAseq data from IDH-wildtype Glioma Stem Cell Cultures  from unrelated adults.

first: Nyasha

last: Chambwe

first: Scott

last: Kennedy

first: Brendan

middle: F

last: Kohrn

first: Pavlo

last: Lazarchuk

first: Mario

last: Leuter

first: Guangrong

last: Qin

first: Monica

last: Sanchez-Contreras

first: Weiliang

last: Tang

first: Jerome

middle: J

last: Graber

first: Patrick

last: Paddison

first: Judit

last: Villén

first: Ilya

last: Shmulevich

first: Raymond

last: Monnat


title: Transcriptomes of trichlorobenzenes-respiring Dehalococcoides mccartyi strain CBDB1

status: Public on Jan 28 2026

city: Würzburg

first: Tom

last: Gräfenhan

address: Josef-Schneider-Str. 2, Bau D15

department: Core Unit SysMed

country: Germany

institute: University of Wuerzburg

type: Expression profiling by high throughput sequencing

summary: Dehalococcoides mccartyi obligately depends on organohalide respiration for energy conservation and growth. The genome of strain CBDB1 encodes 32 reductive dehalogenases, which enable the reductive dehalogenation of a broad range of halogenated compounds. It is one of the few strains able to respire chlorinated benzenes. The differential transcriptional response of the dehalogenase-encoding and –associated genes to halogenated aromatic compounds has so far not been studied on a genome-wide level. To understand the global transcriptional response to specific halogenated aromatic compounds, we analyzed and compared the transcriptomes during growth with 1,2,3- and 1,2,4-trichlorobenzene (TCB).

overall_design: Strain CBDB1 was grown with either 1,2,3- or 1,2,4-TCB as electron acceptor over several transfers. Three replicate samples each were then harvested during the transition from the exponential to the stationary growth phase. RNA was isolated and subjected to RNA-seq and the different expression profiles were compared.

first: Ute

last: Lechner

first: Franziska

last: Greiner-Haas

first: Tobias

last: Goris

first: Gary

last: Sawers

first: Thorsten

last: Bischler

first: Konrad

middle: U

last: Förstner

first: Cynthia

middle: M

last: Sharma


title: Clonal memory of colitis accumulates and promotes tumor growth

city: Cambridge

first: Surya

last: Nagaraja

state: MA

address: 7 Divinity Ave, Bauer 2nd Floor 208

department: HSCRB

country: USA

institute: Harvard University

type: Genome binding/occupancy profiling by high throughput sequencing

type: Methylation profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Single-cell RNA-seq of femoral bone cells reveals cellular heterogeneity and molecular mechanisms of chronic intermittent hypoxia-induced bone growth impairment

city: Shanghai

first: Peilin

last: Zhang

address: 600 Yishan Road

country: China

institute: Shanghai Sixth People's Hospital, Shanghai Jiao Tong University

summary: We employed single-cell RNA sequencing to analyze cells isolated from the spongiosa region of the femoral bone in young female mice exposed to chronic intermittent hypoxia (CIH) or normoxia for 4 weeks. This study provides novel insights into cellular heterogeneity and molecular mechanisms involved in CIH-induced bone growth impairment.

overall_design: Following 4-week CIH or normoxia exposure, cells from the spongiosa region of the femoral bone in young female mice were isolated and analyzed using scRNA-seq.


title: Neoadjuvant Immune Checkpoint Blockade Before Chemoradiation for Patients with FIGO Stage IB3–IVA Cervical Squamous Carcinoma: GINECO Window-Of-Opportunity COLIBRI Study

city: Lyon

first: PIERRE

last: SAINTIGNY

address: 28 Rue Laennec

country: France

institute: Centre Léon Bérard

summary: Combining immune checkpoint blockade (ICB) with chemoradiation is effective in locally advanced cervical cancer. However, the impact of dual ICB before any therapy on immune modulation and treatment response is poorly understood.

overall_design: Targeted gene expression profiling of 2549 genes was performed using HTG sequencing at three timepoints

first: Isabelle

last: Ray-Coquard

first: Marie-Christine

last: Kaminsky-Forrett

first: Ryotaro

last: Ohkuma

first: Aymeric

last: de Montfort

first: Florence

last: Joly

last: Treilleux

first: Sarah

last: Ghamry-Barrin

first: Diana

last: Bello-Roufai

first: Pierre

last: Saintigny

first: Antoine

last: Angelergues

first: Lucas

last: Michon

first: Anne-Claire

last: Hardy-Bessard

first: Valéry

last: Attignon

first: Jessie

last: Auclair

first: Gabriel

last: Chemin

first: Alexandra

last: Lainé

first: Hélène

last: Pere

first: David

last: Veyer

first: Aude-Marie

last: Savoye

first: Justine

last: Berthet

first: Christophe

last: Caux

first: Fabrice

last: Lecuru

first: Bertrand

last: Dubois

last: Bétrian


title: Genetic background and transient prenatal disruption of vitamin A signaling determine susceptibility to airway hyperresponsiveness in mice [scMultiome]

city: College Station

first: Masako

last: Suzuki

state: TX

address: 498 Olsen Blvd, Norman E. Borlaug Building 202C

department: Nutrition

institute: Texas A&M University

summary: Airway structural changes and hyperresponsiveness (AHR), hallmarks of asthma, are crucially influenced by genetic variations and adverse exposures. While intrauterine environmental perturbations leading to dysfunctional lung development have been linked to adult pulmonary disease, still little is known about the developmental events leading to these postnatal abnormalities. Here, we provide evidence of genetic background playing a key role in this process. Using A/J and C57BL/6J mice known for their distinct susceptibility to AHR, we show that A/J but not C57BL/6J develop an aberrant airway smooth muscle (SM) program and AHR in adulthood when exposed transiently to a vitamin A/retinoic acid (RA)-disrupted intrauterine environment in vivo by a maternal BMS493 administration. Single nuclei multiomics analysis identified a subpopulation of mesenchymal cells that overactivated TGFβ targets in response to BMS selectively in A/J, but not C57BL/6J, embryonic lungs. These cells, localized to sites of airway SM initiation, exhibited robust BMS-mediated upregulation of SMAD2/3 targets, including regulators of SM program Pdgfra and Tnc, and showed stable cell proportions despite the marked transcriptional rewiring following RA disruption. These findings identify TGFβ-activating mesenchymal cells as a critical niche responsive to RA signaling and reveal how genetic background determines developmental susceptibility to micronutrient perturbations with long-term impact on airway function.

overall_design: We assessed gene expression and chromatin accessibility at single-cell resolution using the 10XGenomic single-cell Multiome platform on E14.5 male mouse lungs of control and BMS493 treatment groups in A/J and B6 strains.

first: Takehiro

last: Otoshi

first: Ayyappa

last: Kumar Sista Kameshwar

first: Benjamin

middle: D

last: Kotton

first: Yoshinori

last: Seki

first: Zachary

last: Cardell

first: Ke

last: Xiangyi

first: Yuta

last: Matsuno

first: Pooja

last: Rajaram

first: Youn-Kyung

last: Kim

first: Loredana

last: Quadro

first: Wellington

middle: V

last: Cardoso


title: Mature Polarized Hepatocyte Organoids Derived from Human Embryonic Stem Cells for Modeling the Pathogenesis and Progression of Metabolic Dysfunction-Associated Steatotic Liver Disease and Therapeutic Drug Screening

city: Guangzhou

first: Changlu

last: Qin

address: No. 382, Outer Ring Road, Higher Education Mega Center, Xiaoguwei Subdistrict, Panyu District, Guangzhou City, Guangdong Province

institute: South China University of Technology

summary: Metabolic dysfunction-associated steatotic liver disease (MASLD), the most prevalent chronic liver disorder worldwide, exhibits complex pathogenesis and lacks effective targeted therapeutics. Existing animal models are limited by prolonged induction periods and interspecies discrepancies, while conventional monolayer hepatocyte cultures fail to recapitulate disease pathology due to inadequate polarization and functional immaturity. To overcome these constraints, this study established an in vitro MASLD model using human-derived mature polarized hepatocyte organoids (P-hep-orgs). P-hep-orgs were generated through directed differentiation of human embryonic stem cells (hESCs), with their structural and functional maturity validated by sequential expression analysis of stage-specific markers (OCT4, SOX17, ALB) and polarized localization of tight junction protein ZO-1 and multidrug resistance protein MDR1. Subsequent treatment with 450 μM free fatty acids (FFAs) induced key pathological features including lipid accumulation, oxidative stress, and disruption of polarized architecture. Dynamic disease progression was modeled through time-dependent interventions: short-term exposure (1-2 days) elicited early-stage phenotypes (e.g., extensive lipid droplet deposition, elevated triglycerides and supernatant glucose, increased reactive oxygen species, upregulation of pro-apoptotic genes), whereas prolonged treatment (≥10 days) recapitulated advanced manifestations (cholesterol accumulation, enhanced apoptosis, loss of polarization, and ductular reaction). Transcriptomic profiling demonstrated high similarity between this model and metabolic dysfunction-associated steatohepatitis (MASH) patient livers, revealing conserved mechanisms of glucolipid metabolic dysregulation, inflammation, and fibrotic pathway activation. Pharmacological validation confirmed that vitamin E, ursodeoxycholic acid, and empagliflozin significantly ameliorated lipid deposition, suppressed ROS generation, and restored albumin secretion. The model enables disease induction and drug evaluation within two weeks, substantially shortening the timeline compared to animal models, while its human origin eliminates species-specific drug response biases. This P-heporg-based MASLD platform provides an efficient and physiologically relevant foundation for investigating disease mechanisms and accelerating anti-MASLD drug discovery. Future integration of non-parenchymal cells will further enhance microenvironmental fidelity.

overall_design: RNA-seq profiling of P-hep-orgs treated with FFAs for 2 days and 10 days, along with their respective negative control groups untreated with FFAs.

first: Yuyou

last: Duan

first: Haibin

last: Wu


title: Cellular heterogeneity and therapeutic response profiling of human IDH-wildtype glioma stem cell cultures

city: Manhasset

first: Nyasha

last: Chambwe

state: NY

address: 350 Community Drive

department: Institute of Molecular Medicine

institute: Feinstein Institutes for Medical Research

summary: Glioblastoma stem cell cultures (GSCs) are initiated from glioblastoma (GBM) surgical resection tissue, and can capture and propagate key GBM molecular and cellular features. We have deeply characterized four IDH-wildtype GSCs from unrelated adults as cellular models of a majority of adult primary GBMs. These GSC cultures could be continuously propagated in defined, serum-free media and 5% oxygen without requiring specialized growth substrates. They had well-defined genomic and mtDNA variants and gene and protein expression profiles. All four had highly reproducible dose response curves to the GBM standard-of-care therapies of ionizing radiation (IR) and temozolomide (TMZ). Expressed lentiviral barcodes, mtDNA variants and single cell gene expression profiling were used to define cellular heterogeneity, and to track individual cell trajectories and their gene expression programs over 40 days after IR treatment. These well-characterized GSC cultures can support many high throughput in vitro assay formats, as well as xenograft, organoid and other GBM disease modeling approaches. Their use should help to better understand GBM biology, and to identify new and more effective GBM therapies and treatment regimens.

overall_design: 18 samples

first: Scott

middle: R

last: Kennedy

first: Brendan

last: Kohrn

first: Mario

last: Leutert

first: Guangrong

first: Bahar

last: Tercan

first: Monica

last: Sanchez-Contreras

first: Weiliang

last: Tang

first: Jerome

last: Graber

first: Patrick

last: Paddison

first: Judit

last: Villén

first: Ilya

last: Shmulevich

first: Raymond

last: Monnat Jr


title: Inhibition of SPI1 by ADAP regulates S100A8/A9 signaling in macrophages to control the development of colitis

city: Suzhou

first: Xinyue

last: Lv

address: 199 Ren'ai Road

institute: Soochow university

summary: Although the immune adapter protein ADAP (adhesion and degranulation protein adaptor protein) plays a critical role in regulating the inflammatory responses of macrophages, the impact of this regulation on intestinal inflammation remains elusive. This study reveals that mice with ADAP deficiency had an increased susceptibility to dextran sulfate sodium (DSS)-induced colitis. Furthermore, the absence of ADAP leads to increased expression of S100A8/A9 (also known as MRP8 and MRP14, respectively) both in vivo and in vitro, and increased susceptibility to intestinal inflammation. Mechanistically, we demonstrate that ADAP upregulates macrophage E3 ubiquitin ligase FBXW7 (F-box and WD repeat domain-containing 7), promoting proteasomal degradation of the transcription factor SPI1(SPI-1 proto-oncogene) and mediates this effect in macrophages during colitis. Whereas ADAP inhibits SPI1 expression in macrophages, ADAP deficiency promotes SPI1 expression and increases the binding of SPI1 to the S100A8/A9 promoter region. Blockade of SPI1 effectively prevents colitis-induced S100A8/A9 upregulation in macrophages. Thus, our findings highlight the potential link between ADAP and intestinal inflammation and pave the way for therapeutic interventions targeting the ADAP-SPI1-S100A8/A9 signaling axis in inflammatory colitis.

overall_design: The mice were randomly grouped. The mice in the control group were given normal drinking water, whereas those in the DSS group were given fresh 3% DSS (Yeasen, 60316ES60, China) in their drinking-water. The DSS mixture was changed every two days and administered to the mice for seven consecutive days to induce ulcerative colitis. Each mouse was treated with 200 μL of clodronate liposomes (Yeasen, 40337ES08, China) via intraperitoneal injection on the first, second, and fifth day of DSS treatment. In the control group, 200 μL of liposomes were treated with control liposomes.After establishing the mouse model, the colon tissue was homogenized, and total RNA was extracted using TRIzol reagent (Sigma-Aldrich, T9424, USA).

first: Yanqi

last: Wang

first: Xiao

last: Li

last: lv

first: Pengchao

last: Zhang

first: Hebin

last: Liu


last: Qin

address: 401 Terry Ave N

overall_design: Four GSC cultures were treated either without IR or with IR treatment. The cells were barcoded using Cell Tags. Following a 40-days time course, we detected the abundance of  Cell Tags at each time points to monitor the cell enrichment or depletion.

first: Nyasha

last: Chambwe

first: Scott

last: Kennedy

first: Pavlo

last: Lazarchuk

last: Monnat Jr.


title: Brief Pulses of High-Level Fluid Shear Stress Enhance Metastatic Potential and Rapidly Alter the Metabolism of Cancer Cells

city: Norfolk

first: Michael

last: Henry

state: Virgina

address: 700 W Olney Rd, PO Box 1980

department: Department of Biomedical and Translational Sciences

institute: Eastern Virginia Medical School at Old Dominion University

summary: Circulating tumor cells (CTCs) face challenges to their survival including mechanical and oxidative stresses that are different from cancer cells in solid primary and metastatic tumors. The impact of adaptations to the fluid microenvironment of the circulation on the outcome of the metastatic cascade are not well understood. Here we find that cancer cells (PC-3, MDA-MB-231, Myc-CaP) exposed to brief pulses of high-level FSS exhibit enhanced invasiveness and anchorage-independent proliferation in vitro and enhanced metastatic colonization/tumor formation in vivo. Cancer cells exposed to FSS rapidly alter their metabolism in a manner that promotes survival by providing energy for cytoskeletal remodeling and contractility as well as reducing equivalents to counter oxidative stress associated with cell detachment. Thus, exposure to FSS may provide CTCs an unexpected survival benefit that promotes metastatic colonization.

overall_design: RNAsequeince of PC-3  that were either briefly exposed to fluid shear stress (sheared) or not (static) and then held in suspension for 3, 12, or 24 hours after exposure. Experiment was performed such that sheared and static samples for an individual biological replicate within a timepoint can be paired as they are from an original pool of cells. Data from between timepoints are biologically independent

first: Devon

last: Moose


title: Developing precision medicine for remote liver cancer patients

city: Townsville

first: Lionel

last: Hebbard

state: QLD

address: 1 James Cook Dr, Douglas QLD 4811

country: Australia

institute: James Cook University

summary: Background and Aim Hepatocellular carcinoma (HCC) is a growing burden in Australia’s rural, regional and remote areas and knowledge of its molecular mechanisms and hence potential therapies are limited. To address this, we investigated HCC genetics from patients treated at a regional hospital and used a novel computational model for low-cost therapeutic prognostication. Methods We profiled paired tumour and adjacent non-tumour liver biopsies from 19 patients admitted to the Townsville University Hospital, based in North Queensland (NQ). RNA-seq was used to characterize transcriptomic and mutational features and to test a transcriptome-only adaptation of the TARGET-SL pipeline for drug target prediction. Results Differential expression analysis identified 923 genes altered in our cohort, of which 64% overlapped with The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC), and the cohort-mean gene expression correlated strongly (Spearman Rho = 0.96). Somatic variant calling from RNA highlighted mutational heterogeneity, with CTNNB1 (47%) and TP53 (21%) the most frequently mutated genes, consistent with TCGA findings. Copy number inference detected recurrent deletions on 8p, 6q, and 17p, congruous with known HCC patterns. We ran TARGET-SL on RNA-Seq to identify personalised driver genes in these patients and identified drug candidates in 63% of patients. Conclusions Our results demonstrate that NQ HCC shares core molecular features with larger TCGA cohorts, and that a transcriptome-based approach can feasibly support precision oncology in resource-limited regional settings.

overall_design: RNAseq profiling of paired human hepatocellular carcinoma and adjacent normal tissue biopsies.

first: Rhys

last: Gillman

first: Miriam

last: Wankell

first: Eun

last: Sun

first: Matan

last: Ben David

first: Rozemary

last: Karamatic

first: Pranavan

last: Palamuthusingam

first: Matt

middle: A

last: Field

first: Ulf

last: Schmitz


title: TTBK2-Driven Ciliogenesis Is Required for Intrinsic Neuronal Regeneration after Spinal Cord Injury

city: changchun

first: Zhang

last: Renfeng

state: jilin

address: 218 Ziqiang Street

institute: Jilin University Second Hospital: The Second Hospital of Jilin University

summary:
Primary cilia (PCs) are microtubule-based organelles that function as critical signaling hubs in both embryonic neurodevelopment and mature neurons. Tau tubulin kinase 2 (TTBK2) is a key initiator of ciliogenesis, but its role in neuronal regeneration following spinal cord injury (SCI) remains unclear. Here, we investigated whether TTBK2 regulates axonal regeneration and circuit remodeling after SCI via a cilia-dependent mechanism.
Using both in vitro spinal neuron cultures and an in vivo conditional knockout (TTBK2^fl/fl-Cre ERT^+/-) mouse model of T10 spinal hemisection, we demonstrate that TTBK2 deletion impairs PC formation, downregulates Sonic Hedgehog (SHH) signaling, reduces MAP2 expression, and disrupts axonal elongation. Overexpression of TTBK2 rescued these deficits, while loss of ciliary function via KIF3A knockdown replicated the phenotypes. Transcriptomic and proteomic profiling revealed that TTBK2 deficiency suppresses axonogenesis-related pathways while enhancing synaptic excitability markers. Notably, activation of SHH signaling by Smoothened agonist partially restored MAP2 expression and axonal growth in TTBK2-deficient neurons.
In vivo, TTBK2 knockout mice displayed impaired motor recovery, increased glial scarring, disrupted axonal projection, and elevated expression of axon growth inhibitors (NogoA/NogoR). Confocal imaging and electron microscopy confirmed the loss of PCs and morphological signs of demyelination and aberrant synaptogenesis. Furthermore, endogenous neural stem cells in TTBK2-deficient mice failed to differentiate into mature neurons, correlating with reduced SHH activity.
Collectively, our findings identify a TTBK2–PC–SHH–MAP2 regulatory axis that governs axonal regeneration and circuit reassembly after SCI. This study expands the functional relevance of primary cilia beyond neurodevelopment and highlights TTBK2 as a potential therapeutic target for promoting neural repair in the adult CNS.

overall_design:
Neuronal cells were isolated from the spinal cord of fetal mice at 15 days of gestation. Cells were cultured at a density of 1.5*10^6/well (six-well plate) for immunofluorescence experiments Neuronal cells were grown in u-Plate 24 Well Black (ibidi, Germany). Neuronal growing media included DMEM (Gibco, USA) and FBS (Gibco, USA), and neuronal media included Neurobasal medium (Gibco, USA), 2% B27neural supplement (Gibco-Invitrogen, USA), 1 mM L- glutamine solution (Sigma-Aldrich, G7513, USA) and 100 ng/ml penicillin-streptomycin (Gibco-Invitrogen, USA). Neuronal cells were changed every 2 days and a half, and subsequent experiments were performed on day 5 to identify neuronal cells by immunofluorescence assay. The experiment was divided into five groups, including: NC, shKIF3A, shTTBK2, OETTBK2, shKIF3A+OETTBK2. Cells were seeded on day 5 of the plate and infected using adenovirus, using the 1/2-volume infection method, half of the medium in the original well plate was removed, and the corresponding viral solution was added according to the MOI=100. Adv5-MIR30 (shTTBK2, NM_001024856), Adv5-MIR30(shKIF3A, NM_001290805), pDC316-mCMV(mTTBK2 , NM_001024856) The sequences of the target genes are shown in Table S1, and silencing of KIF3A, TTBK2, overexpression of TTBK2 and silencing of KIF3A+ overexpression of TTBK2, and negative plasmids were added to the control group. After 8h of infection, the virus-shouting medium was discarded and replaced with neuronal medium for further cultivation, and the infection efficiency was identified by qRT-PCR.
National Natural Science Foundation of China, NO.82171388

first: Yang

last: Xiaoyu

first: Qi

last: Zhiping


title: Transcriptomic evaluation of ovine intervertebral disc cells as an in vitro model to explore degenerative processes

city: Nantes

first: Romain

last: Guiho

address: 1 place Alexis Ricordeau

institute: Inserm UMRS 1229-RMeS Regenerative Medicine & Skeleton

summary: Intervertebral disc degeneration (IVDD) is a leading cause of chronic back pain and disability. Understanding the cellular and molecular mechanisms underlying disc degeneration is crucial for developing effective therapies. This study investigates the relevance of a cellular model using annulus fibrosus (AF) and nucleus pulposus (NP) cells isolated from lamb and aged sheep discs. Histological analysis and MRI were used to confirm moderate degeneration in 7–8-year-old sheep discs. High-throughput RNA sequencing (RNAseq) was performed on four samples per condition: AF lamb, NP lamb, AF sheep, and NP sheep. Transcriptomic analysis using Gene Set Enrichment Analysis (GSEA) and Weighted Gene Co-Expression Network Analysis (WGCNA) revealed significant differences, including a higher propensity for cellular senescence and altered metabolism in sheep samples. Further in vitro functional studies elucidated these differences, providing deeper insights into the mechanisms of IVDD.

overall_design: Intervertebral discs (IVDs) were collected from 4 female 6-month-old lambs and 4 female sheep between 7-8 years old (Vendée breed, GAEC HEAS, Les Rabelais, Ligné, France) in the accredited Centre of Research and Pre-Clinical Investigations (ONIRIS, National Veterinary School of Nantes). IVDs were dissected from the vertebral bodies, the AF and NP were separated from each other and diced into pieces. The tissue fragments obtained were rinsed three times in PBS with 2% penicillin/streptomycin (P/S) for 2 minutes. The extracellular matrix was digested with 0.05% hyaluronidase H4272 in PBS at 37 °C for 15 minutes. After two rinses with PBS, 15–20 mL 0.2% trypsin T9935 in PBS were added and incubated at 37 °C for 30 minutes. After two more rinses with PBS, 15–20 mL collagenase C2674 in complete medium were added and the mixtures were incubated at 37 °C overnight. The recovered suspension was filtered through a 70-μm-pore filter and centrifuged for 5 minutes at 300 g. The cells were counted and seeded in T75 flasks. AF and NP cells were grown at 37 °C and 5% CO2 in T75 flasks in complete culture medium (DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S)). Total RNA was extracted from AF and NP cells at passage 1 (P1) of lamb and sheep was extracted using NucleoSpin RNA XS kit (Macherey-Nagel, 740902). Library preparation was performed with Illumina® Stranded mRNA Prep kit. Sequencing was then performed on an Illumina HiSeq with NovaSeq 6000 SP Reagent Kit (200 cycles) v1.5. Sequencing data was then aligned with STAR using Ovis_aries_rambouillet.ARS-UI_Ramb_v2.0.111 genome.

first: Paul

last: Humbert


title: eIF3g binding to GUCG boxes spread in mRNA coding regions mediates translational heat shock response

city: Kumamoto

first: Shingo

last: Usuki

address: 2-2-1 Honjo, Chuo-ku

department: Institute of Molecular Embryology and Genetics (IMEG)

country: Japan

institute: Kumamoto University

summary: Heat shock response (HSR) is crucial for life’s adaptation to heat. Here, we discovered a novel mechanism of HSR controlled at the translational level, which depends on the multi-subunit translation initiation factor, eIF3. The cis-element for this regulation is called GUCG box and occurs degenerately within protein-coding regions of regulated mRNAs. Being positioned at ribosome leading-edge through -propeller subunit eIF3i, the RNA-binding subunit eIF3g binds the motif and stabilizes mRNA anchoring during translation under mild heat. The eIF3g/i-regulated HSR consists of 64 genes including heat shock protein genes, engaging a quarter of the cellular ribosomes. The 5’-terminal coding regions of the HSR mRNAs are enriched with more GUCG boxes, some of which are masked by weak secondary structures.  This arrangement enables eIF3g/i-dependent enhancement in part through melting inhibitory secondary structures. GUCG boxes spread evenly across the entire protein-coding regions of strongly translated mRNAs ~20 nt. apart, as if to prevent ribosome collision. Thus, eIF3 not only stabilizes initiating ribosomes at its leading-edge but may contribute to anchoring elongating ribosomes throughout coding regions under heat insult.

overall_design: SELEX was performed for 12 rounds in each experiment, as described  in 5, with 18 base-long randomized ribonucleotide sequence flanked by primer binding sites designed for cDNA synthesis, PCR and RNA synthesis by T7 RNA polymerase. For these experiments we used His6-tagged eIF3g/Tif35p or its complex with (untagged) eIF3i/Tif34p. The DNA fragments encoding them were amplified by PCR using budding yeast Saccharomyces cerevisiae genomic DNA and then cloned into pET28b (with hexa-histidine tag fused to its N-terminus, in the case of eIF3g) and were co-expressed in E. coli strain BL21-CodonPlus (DE3)-RILP (Stratagene).  The expressed protein(s) was purified by Ni-NTA (Qiagen) and Q-sepharose (Cytiva).

first: Hiroaki

last: Kato

first: Akihiro

last: Oguro

first: Yuanhui

last: Mao

first: Kinsley

last: Ochsner

first: Hailey

last: Casey

first: Grace

last: Sakai

first: Ji

last: Wan

first: Leiming

last: Tang

first: Brandon

last: Rider

first: Mizuki

last: Asano

first: Nirmala

last: Bardiya

first: Kade

last: Kaufman

first: Haana

last: Yamada

first: Susumu

last: Ishiguro

first: Naoki

last: Tani

first: Kazuyuki

last: Kumagai

first: Akira

last: Nakamura

first: Chingakham Ranjit

last: Singh

first: Taiichi

last: Sakamoto

first: Eiji

last: Obayashi

first: Shu-Bing

last: Qian

first: Katsura


title: DNA Damage-Induced Nuclear STING Translocation Orchestrates Innate Immune Activation and Chromatin Remodeling [RNA-Seq]

first: Yihao

last: Wang

state: State

address: Zhizaoju Road 833, Shanghai

department: Department of Ophthalmology

institute: Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine

summary: To define STING’s role in the DNA damage response, we collected STING wild-type (WT) and STING knockout (KO) HeLa cells following 24 hours of BMN673 treatment, as well as non-treated cells. We then performed gene expression profiling analysis using data obtained from RNA-seq dataset across four conditions: STING WT and STING KO cells, with or without 24 hours of BMN673 treatment.

overall_design:
Comparative gene expression profiling analysis of RNA-seq data for STING WT and STING KO HeLa cells after 24 hours of BMN673 treatment or in the non-treated condition.
Comparative gene expression profiling analysis of RNA-seq data for STING WT and STING KO HeLa cells after 24 hours of BMN673 treatment or in the non-treated condition.

first: Jianfeng

last: Shen

first: Lingjie

last: Li

first: Zan


title: Targeting ATR offers multifaceted treatment strategies involving RAD51-mediated compensatory DNA repair in bladder cancer

city: Freiburg

first: Geoffroy

last: Andrieux

address: Breisacherstr 153

institute: University clinics Freiburg

summary: Background: Muscle-invasive bladder cancer treatment depends on histological and molecular subtypes. While urothelial carcinoma (UC) benefits from diverse therapies, options beyond radical cystectomy for rare subtypes such as squamous cell carcinoma (SCC) remain limited. We previously demonstrated that ATR inhibitor (ATRi) Ceralasertib, enhanced UC and SCC treatment efficacy in vitro. Thus, we investigated the therapeutic impact of ATRi, its downstream effects and compensatory pathways bypassing ATR dysfunction in patient-derived ex vivo models.  Results: Patient-derived ex vivo ATRi-adapted models (p-SCCATRi) were generated through long-term ATRi treatment (Ceralasertib) and characterized via RNA-seq profiling. In p-SCCATRi, ATRi adaptation led to decreased sensitivity (up to 3.3-fold IC50 increase), with compensatory upregulation of DNA repair, particularly homologous recombination (HR) genes like BRCA1 and RAD51, plus chromatin reorganization and immune downregulation. HR upregulation was targetd with application of RAD51 inhibitor (B02).  Conclusion: Our results propose ATR as a promising target in bladder cancer by (1) enhancing radiosensitivity through classical ATR inhibition, and (2) exploiting ATRi-adaptation as a vulnerability by targeting compensatory HR activation through RAD51 inhibition. These findings offer novel strategies for improving bladder cancer treatment.

overall_design: For patient-derived ex vivo models, cells were isolated from fresh SCC cystectomy tissue and cultured in supplemented MEBM medium with 2% matrigel. Dysfunctional ATR models in p-SCCs (p-SCCATRi) were generated by long-term treatment (>6 months) with the ATR inhibitor Ceralasertib (AZD6738), with the concentration gradually increased from 0.05 µM to 0.60 µM over time. Parallel control cultures were treated with DMSO. Two ATRi-adapted models (p-SCC1ATRi and p-SCC2ATRi) and their respective DMSO-treated controls (p-SCC1DMSO and p-SCC2DMSO) were profiled for transcriptomic changes using RNA-seq.

first: Melanie

last: Boerries

first: Michael

last: Rose

first: Nadine

middle: T

last: Gaisa

first: Julia

last: Pannhausen


title: Dietary antigens drive the generation of functional cytotoxic intraepithelial lymphocytes for early defense against foodborne pathogens

city: Suwon

first: Jaeu

last: Yi

state: Gyeonggi-do

address: 206, World cup-ro

department: Department of Biological Science

country: South Korea

institute: Ajou University

summary: Intraepithelial lymphocytes (IELs) constitute the largest lymphocyte population in the body and exhibit direct cytotoxic effector functions. How IELs are generated and maintained as effector T cells, however, is poorly understood. While significant attention has been given to the role of commensal microbiota in the intestine, the predominance of dietary components in the small intestine suggests that dietary antigens may regulate IEL generation and homeostasis. Here, we show that conventional TCRab + CD4 + and CD8 + IEL populations are present in normal numbers in germ-free (GF) mice which lack microbiota.However, these IELs are severely depleted in antigen-free (AF) mice, GF mice fed an amino acid diet devoid of proteins. Moreover, the few remaining IELs in AF mice lack effector function. Notably, while TCRab + CD8ab + IELs in adult GF mice can persist for prolonged periods, they lose their effector function when fed an AF diet. IL-12 presumably produced by intestinal dendritic cells plays a critical role in the maintenance of TCRab + CD8ab + IELs and their effector functions. Importantly, mice lacking functional dietary antigen-induced TCRab + CD8ab + IELs exhibit a defect in early protection against a foodborne pathogen, Listeria monocytogenes. Collectively, these findings support that dietary antigen exposure drives the generation of innate-like cytotoxic IELs thatprovide rapid and local protection against foodborne-pathogens.

overall_design: RNA seq profiling of Small intestinal DCs isolated from SPF, GF, or AF mice

first: Jisun

last: Jung

first: Kwang Soon

last: Kim


title: A STAT1-GBP3-STING positive feedback loop governs inflammation, oxidative stress, and DNA damage to trigger acute aortic dissection [scRNA-Seq]

city: Shijiazhuang

first: siming

last: Bu

state: CHINA

address: Zhongshan East Road

institute: Hebei Medical university

summary: Acute aortic dissection (AAD) is a degenerative aortic remodeling disease characterized by exceedingly high mortality without effective pharmacologic therapies. Although oxidative stress, DNA damage, and inflammation are associated with AAD, the precise interplay among these responses has remained unclear. In this study, aortas from mouse Control and AD models were subjected to integrative ATAC-seq, RNA-seq and scRNA-seq analysis.

overall_design: scRNA-seq of Control and AAD aortic tissues. ScRNA-seq  was generated from  from 3-4 weeks old C57BL/6 mice with β-Aminopropionitrile drinking freely for 2 weeks and then intraperitoneal injection of angiotensin II (BAPN/AngII administration) 3 days.

first: Siming

first: Wenli

first: Zhixue

last: Song

first: Dong

last: Ma


title: Ex vivo spatiotemporal characterization of spermatogenesis in mouse testicular organoids

city: nanjing

first: yutong

last: li

state: jiangsu

address: No. 818, Tianyuan East Road

institute: Nanjing medical university

summary: Testicular organoids (TOs) offer an opportunity to preserve fertility, but current TO protocols are limited by suboptimal maintenance of the organoid structure, meiotic defects, and incomplete in vitro spermatogenesis (IVS). Here, we developed a strategy for self-reconstitution of single-cell suspensions of neonatal mouse testes into TOs containing the major testicular cell types and generation of lumen-like structures and haploids. Morphological and lineage-specific marker analyses revealed that these TOs met the criteria for organoids, including germ cell differentiation and recapitulation of key events in meiosis (e.g., chromosome recombination and synapsis). Notably, the spatiotemporal characteristics of spermatogenesis in the TOs were comparable to those in testes, and their derived haploids resembled step-4/5-like round spermatids in vivo. Further scRNA-seq analysis confirmed that haploids accounted for approximately 2.43% of the total germ cells and that undifferentiated germ cells were able to develop into haploids even when chimeric TOs were reconstructed via testicular somatic cells (Sertoli cells or Leydig cells) from mice with different genetic backgrounds. Collectively, our TO-based findings provide a promising platform for studies on testicular microenvironment construction, IVS and fertility preservation.

overall_design: Cells were isolated through enzymatic dissociation from both 28-day-old testicular organoids (TOs) and 28-day-old mouse testes.

first: Jiachen

last: Sun

first: Lifa

last: Zhu

first: Yutong

first: Xin

last: Wu


title: Transcriptomic profiling of tumor-infiltrating and lymph node-derived Treg and B cells in Foxp3-Cre-YFP and Foxp3-Cre-YFP Appl2f/f melanoma-bearing Mice

first: Shunyan

last: Kan

address: No.601, West Huangpu Avenue

country: Afghanistan

institute: Jinan University

summary: Tregs are a critical target for developing anti-tumor immunotherapy, however, they are also critical for maintaining systemic immune tolerance and homeostasis. It remains a formidable challenge to specifically disable Tregs in the tumor microenvironment without compromising systemic immune tolerance. An ideal target is that barely expressed or dispensable in peripheral Tregs but significantly increased and critical for the immunosuppressive activity of tumor infiltrated Tregs (TIL-Tregs). In an attempt to search for specific targets that mediate TIL-Tregs fitness in the tumor microenvironment, we performed RNA-seq of TIL-Tregs and Tregs from the draining lymph nodes (dLN-Tregs) of mice bearing B16-F0 melanoma.

overall_design: For bulk RNA-sequencing, Treg cells or B cells in the tumor infiltrated lymphocytes (TILs) or draining lymph nodes (dLNs) were sorted from Foxp3-Cre or Foxp3-Cre Appl2f/f mice bearing B16-F0 melanoma. RNA was extracted and used for RNA-Sequencing analysis.


title: Transcriptomics effects of ALDH1A3 inhibitor,CLM296, in MDA-MB-231 Triple negative breast cancer cells

city: Halifax

first: Paola

last: Marcato

state: NS

address: 5850 College street

country: Canada

institute: Dalhousie University

summary: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer associated with poor survival outcomes. Aldehyde dehydrogenase 1A3 (ALDH1A3) promotes tumor growth, metastasis, and chemoresistance across multiple cancer types including TNBC, glioblastoma, melanoma lung, and colon cancers. Currently, there are no ALDH1A3 inhibitors in clinic and developing targeted ALDH1A3 inhibitors is novel precision medicine approach to treat ALDH1A3+ tumors. For this purpose, we have developed a novel ALDH1A3 specific inhibitor,CLM296

overall_design: MDA-MB-231 breast cancer cells, overpressing ALDH1A3 or vector control, were treated with or without 100nM CLM296 for 24 hours. The RNA was collected and sequencing was performed on Illumina NovaseqX platform. Three replicates per sample were used in this study.

first: Maya

last: MacLean

first: Bianca

middle: L

last: Bernardoni

first: Wasundara

last: Fernando

first: Giovanni

last: Petrarolo

first: Cheryl

last: Dean

first: Ilaria

last: D’Agostino

first: Christopher

last: Hughes

first: Jaganathan

last: Venkatesh

first: Geetha

last: Marivel

first: Olivia

last: Walker

first: RajPranap

last: Arun

first: Hannah

last: Cahill

first: Kerry

last: Goralski

first: Concettina

last: LaMotta


title: RNA-seq of RAW264.7, PBMC of patients with sepsis and nonseptic controls

first: wenchen

last: luo

address: Dongan Road

institute: Fudan University

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Effects of different diets on autophagy control and unfolded protein response in the aging heart

city: Baton Rouge

first: Jaroslaw

last: Staszkiewicz

state: LA

address: 6400 Perkins Rd

department: Genomics Core Facility

institute: Pennington Biomedical Research Center

summary: We reported a decrease in mitochondrial function in the hearts of aging mice undergone a low protein diet, but an increased lifespan. For that, we hypothesized that the increased autophagy and unfolded protein response plays an important role in this phenomenon. For that, we want to identify changes in the heart transcriptome profiling (RNA-seq) that could explain this lifespan increased in aging mice.

overall_design: Heart samples from aging mice were undergone to normal protein, low protein, high- fat diet and high-fat diet plus low protein.

last: Axelrod

first: Richard

middle: P

last: Carmouche

first: Gangarao

last: Davuluri

first: John

last: Kirwan

first: Wagner

middle: S

last: Dantas


title: DNA Damage-Induced Nuclear STING Translocation Orchestrates Innate Immune Activation and Chromatin Remodeling [ChIP-Seq]

city: Shanghai

first: Yihao

last: Wang

summary: To investigate the function of nuclear STING and its regulatory mechanisms, we treated wild-type (WT) HeLa cells with BMN673 for 24 hours to promote nuclear STING translocation.  ChIP-seq was performed using HeLa cells treated with BMN673 for 24 hours.

overall_design: ChIP-seq analysis of WT HeLa cells after 24 hours of BMN673 treatment to promote nuclear STING translocation.

first: Jianfeng

last: Shen

first: Lingjie

last: Li

first: Zan


title: Transcriptomic profiling of tumor-infiltrating Treg cells in Foxp3-Cre-YFP melanoma-bearing mice

first: Shunyan

last: Kan

summary: Tregs restrain anti-tumor immunity and facilitate the evasion of tumor cells from immune surveillance, thus has long been considered as a promising target for anti-tumor therapy. Recent studies found that excessive type 1 immune responses (mainly mediated by IFN-γ and IL-12 et al.) in the tumor microenvironment drive Treg fragility that downregulates FOXP3 but upregulates IFN-γ, hereby compromises its suppressive function.The expression level of FOXP3 in Treg cells regulates its immunosuppressive function. Therefore, we sorted FOXP3-high and FOXP3-low Treg cells and strived to find their differentially expressed genes and their functional regulatory mechanisms.

overall_design: For bulk RNA-sequencing, FOXP3-high and FOXP3-low Treg cells in the tumor infiltrated lymphocytes (TILs) were sorted from Foxp3-Cre-YFP  mice bearing B16-F0 melanoma. RNA was extracted and used for RNA-Sequencing analysis.


title: Interleukin-27 is antiviral at the maternal-fetal interface

city: Philadelphia

first: Kellie

last: Jurado

state: Pennsylvania

address: 3400 Civic Center Blvd, Building 421

department: Microbiology

institute: University of Pennsylvania

summary: Congenital viral infections can have severe consequences for pregnancy and fetal outcomes. Remarkably, the fetal-derived placenta serves as a robust barrier to infection through meticulous regulation by immune effectors and a diverse repertoire of cytokines. Yet, the regulatory roles of many cytokines remain undefined at the maternal-fetal interface. Interleukin 27 (IL-27) is a highly expressed cytokine in the placenta whose functional consequence during congenital infection is unknown. Here, we utilized trophoblast organoids (TO) derived from primary human placentas and a mouse model of congenital viral infection to uncover the functional role of IL-27 signaling during pregnancy. We show that TOs constitutively express IL-27 and its receptor, IL27RA, and demonstrate that IL-27 signaling restricts Zika virus (ZIKV) infection of TOs. Through bulk RNA-sequencing of TOs in the absence and presence of IL-27 signaling, we demonstrate IL-27-mediated upregulation of antiviral genes. Finally, we show that IL-27 signaling is critical within the context of congenital murine ZIKV infection, as IL-27 restricts placental ZIKV burdens and protects against pathologic fetal outcomes early in gestation. These findings collectively demonstrate a novel role for IL-27 in the placenta and establish IL-27 as an innate antiviral defense at the maternal-fetal interface during congenital viral infection.

overall_design: RNA sequencing of trophoblast organoids (TO) derived from primary human placental tissue.

first: Merlino

last: Madeline S

first: Barksdale

last: Briah

first: Negatu

last: Seble G

first: Clements

last: Rebecca L

first: Miller-Ensminger

last: Taylor

first: Lopez

last: Alexandra H

first: Mainigi

last: Monica N

first: Hunter

last: Christopher A

first: Jurado

last: Kellie A


title: Single-cell RNA sequencing (scRNA-Seq) analysis of tumor-infiltrating lymphocytes (TILs) from B16-F0 melanoma in mice

summary: To comprehensively analyze the influences on the tumor infiltrated immune cells after deficiency of APPL2 specifically in Tregs, we employed single-cell RNA sequencing (scRNAseq) to profile CD45+ TILs from both Foxp3YFP-Cre Appl2f/f and Foxp3YFP-Cre control mice. To our surprise, tumor infiltrated B cells were dramatically decreased in Foxp3YFP-Cre Appl2f/f mice. Hereby, there was a trend of increase for most of the other immune subsets. These data indicated that the infiltrated B cells, as a whole, were tumor supportive and restricting protumoral B cell infiltration was critical for Appl2-KO Tregs in suppressing tumor growth.

overall_design: For scRNA-Seq analysis, TILs from mice bearing B16-F0 melanoma were suspended in RPMI-1640 medium containing 2% FBS and DAPI solution ( BD, Cat: 564907, 1:3000). CD45+ DAPI- cells were sorted and prepared as approximately 1 × 106 cells/ml, cell viability was above 97%. Cell suspensions were loaded onto a 10×Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 according to the manufacturer’s instructions.


title: Mutant p53 negatively regulates ER⍺ activity to drive resistance to endocrine therapy [RNA-seq]

first: Priyankara

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: High-grade serous ovarian cancer (HGSOC) is a highly lethal gynecologic malignancy in women.  Women diagnosed with HGSOC initially respond to chemotherapy but there is a greater than 80% rate of relapse.  There is thus a significant unmet need for new therapeutic targets for HGSOC.  Estrogen receptor alpha (ER⍺) is a particularly attractive candidate, as 70-80% of HGSOC tumors stain positively for ER⍺ and there are approved inhibitors that show limited toxicity.  Unlike the case for breast cancer however, endocrine therapy for HGSOC has not shown consistently promising results.  In this work we show that mutant forms of p53, which occur in 70% of HGSOC, inhibits ER⍺ function and confer resistance to fulvestrant and elacestrant.  Mechanistically we show that mutant p53 only inhibits one arm of the ER⍺ pathway: the transactivation of jointly regulated ER⍺-SP1 target genes, like DEPTOR, which encodes an mTOR inhibitor.  We show that silencing mutant p53 restores the ability of ER⍺ to transactivate ER⍺-SP1 target genes, and renders HGSOC sensitive to endocrine therapy.  This work resolves part of the underlying reason for endocrine resistance of HGSOC, and reveals one mechanism to reverse such: refold or ablate mutant p53.

overall_design: RNA-seq analysis of control and p53 knockdown PEO1 cells at 48 hours after 10 μM Fulvestrant treatment.

first: Chunlei

last: Shao

first: Maureen

last: Murphy


title: Mutant p53 negatively regulates ER⍺ activity to drive resistance to endocrine therapy

overall_design: RNA-seq analysis of PEO1 cells transfected with siControl or sip53 and then treated with 10 nM E2 for 2 or 8 hours.


title: The two-step purification method ViREn identifies a single NSUN6-mediated 5-methylcytosine modification promoting dengue virus RNA genome turnover.

city: Heidelberg

first: Alessia

last: Ruggieri

address: Im Neuenheimer Feld 344

department: Dpt. of Infectious Diseases, Molecular Virology

institute: Heidelberg University

summary: Chemical modifications on cellular and viral RNAs are new layers of post-transcriptional regulation of cellular processes including RNA stability and translation. Although advances in analytical methods have improved detection of RNA modifications, the precision of mapping at single-base resolution remains challenging. Requirements for sensitivity and purity limit accuracy and reproducibility especially for low abundant viral RNAs extracted from infected cells. Here we report the two-step method, ViREn, for the enrichment of the genomic RNA (gRNA) of dengue virus (DENV), a positive-sense single-stranded RNA virus. This approach enabled the preparation of gRNA with significantly increased purity and led to the identification of a high-confidence 5-methylcytosine (m5C) site in DENV gRNA. This finding was orthogonally validated by Illumina-based bisulfite sequencing and direct RNA sequencing by Nanopore Oxford Technologies. Strikingly, this m5C modification was detected exclusively in gRNA extracted from infected cells but not in gRNA extracted from viral particles. We identified NSUN6 as the host methyltransferase catalyzing this modification and demonstrated a role for m5C in regulating DENV gRNA turnover. ViREn thus enables the mapping of m5C on low abundance viral gRNA with unprecedented precision and sensitivity and facilitates future mechanistic studies into the role of RNA modification in virus replication.

overall_design: RNA-seq quantifying the RNA sample composition before and after antisense affinity capture of ViREn. WT Huh7 cells were infected with DENV2 NGC for 48h. Total RNA was extracted following ViREn protocol.

first: Chia Ching

last: Wu

first: Johanna

last: Schott

first: Doris

last: Lindner

first: Ruggieri

last: Alessia


title: PRPF40B regulates NTRK2 pre-mRNA splicing to control its dominant-negative receptor, preventing BDNF signaling inhibition in neurons

city: Paris

first: Pierre

last: de la Grange

address: 29, rue du Faubourg Saint-Jacques

department: Paris Biotech Santé

institute: GenoSplice technology

summary: BDNF signaling through its receptor TRKB plays a critical role in brain development, neuroplasticity, and maintaining homeostasis. The TRKB gene, NTRK2, encodes both the full-length receptor (TRKB-FL) and a truncated isoform (TRKB-T1) generated by alternative splicing, which acts as a dominant-negative mutant, inhibiting TRKB-FL signaling. Dysregulation of BDNF-TRKB signaling, including TRKB-T1 upregulation, has been observed in neurodegenerative diseases, psychiatric disorders, and cognitive impairments. Here, we show that PRPF40B, a splicing factor associated with neuronal dysfunctions, regulates the TRKB-FL/TRKB-T1 balance during neuronal differentiation by modulating splicing. Silencing PRPF40B increases the TRKB-T1 expression, impairing neuronal differentiation and synaptic plasticity. Our data identify PRPF40B as a key regulator of the TRKB receptor balance, crucial for fine-tuning neuronal responses and preventing neuroplasticity or survival impairments.

overall_design: RNA-seq profiling of SH-SY5Y neuroblastoma wild-type (WT) and their knockdown derivatives (Scramble and G2).

first: Carles

last: Suñé


title: 16S rRNA sequencing analysis of bacteiral community composition in antibiotic-treated mouse fecal sample

city: Nam-gu, Pohang-si

first: Haeun

last: Ko

state: Gyeongsangbuk-do

address: 77, Cheongam-ro

department: Life Science

institute: POSTECH

summary: Purpose: This study aims to compare and analyze the differences in bacterial community composition in fecal samples from mice treated with Control(DW), Vancomycin (VAN), Ampicillin (AMP), Neomycin (NEO), Metronidazole (MET), and a combination of all antibiotics (ALL, VANM) using 16S rRNA sequencing. Methods: Each antibiotics treated mice's fecal samples were collected and stored -80'c until analyzation. DNA was extracted using the NucleoSpin DNA Stool Kit (MACHEREY-NAGEL) following the manufacturer’s protocol. Metagenomic sequencing was performed on an Illumina MiSeq platform (Illumina), targeting the V3 and V4 regions of the 16S rRNA gene according to the manufacturer's instructions. PCR products were purified using AMPure XP beads, and sequencing adapters were added using the Nextera XT Index Kit (Illumina). The library was further purified with AMPure XP beads and quantified using automated electrophoresis with the TapeStation System (Agilent). Sequencing was performed using the MiSeq v3 reagent kit (Illumina), following the manufacturer’s protocol. Results: QIIME2 (v2023.02) was used to process and analyze 16S rRNA gene amplicon sequencing data, from sequence preprocessing to taxonomic classification. Paired-end sequences were merged and quality-filtered using Deblur. The resulting amplicon sequence variants (ASVs) were used for downstream analyses. Conclusions: Our study presents a comparative analysis of bacterial community composition in fecal samples from antibiotic-treated mice. We observed that microbiota composition varied distinctly depending on the type of antibiotic administered.

overall_design: 16S rRNA profiles of fecal sample of antibiotic treated mice. Six replicates for each group were anaylzed.

first: Sin-Hyeog

last: Im


title: Mutant p53 negatively regulates ER⍺ activity to drive resistance to endocrine therapy [ChIP-seq]

overall_design: ChIP-seq analysis of ER⍺ and SP1 in control and sip53 PEO1 cells, with or without E2 treatment.


title: Obesity-associated TRIM15 promotes the proliferation of esophageal adenocarcinoma through the YY2/FOXRED1 axis

city: Changsha

first: Huaiyuan

last: Liang

address: 183 Renmin Middle Road

institute: Institute of Urological Cancer, Central South University

summary: Obesity has been identified as an independent risk factor for gastroesophageal reflux disease (GERD) and esophageal adenocarcinoma (EAC). Oxidative stress and inflammation driven by chronic GERD are the main causes of the tumorigenesis of EAC, but the underlying mechanism remains elusive. Here, we identified the inflammation-upregulated E3 ligase, tripartite motif 15 (TRIM15) as a key driver of obesity-associated EAC. We demonstrated that TRIM15 promotes the degradation of YY2 through the ubiquitin-proteasome system, which in turn dysregulates the lipid metabolism and enhances the proliferation of EAC cells. Furthermore, we showed that YY2 transcriptionally increases FOXRED1 expression. We then found that FOXRED1 is one of the essential effectors for the TRIM15-induced dysregulation of lipid and energy metabolism in EAC cells. Thus, we identified a novel obesity-associated TRIM15/YY2/FOXRED1 axis that contributes to the proliferation of EAC. Given that lipid metabolism regulates ferroptosis by controlling cellular processes associated with phospholipid peroxidation. We show that the TRIM15/YY2/FOXRED1 axis regulates EAC sensitivity to ferroptosis-inducing compounds by modulating the levels of SLC3A2 and GPX4, providing a therapeutic strategy for EAC.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for these: (1) Subcutaneous xenograft tumor tissues from nude mice on a high-fat diet (HFD) or a normal diet (ND) ; (2) OE33 cells transfected with OE-TRIM15 or empty vector (EV); (3) OE33 cells transfected with shYY2 or shControl; (4) OE33 cells transfected with shFOXRED1 or shControl;

first: Xin

last: Jin

first: Haohui

last: Wang


title: Cardiac Radiotherapy-Induced Epigenetic Memory Underlies Electrophysiologic and Metabolic Reprogramming [CUT&Tag]

city: Saint Louis

first: Shuhua

last: Fu

state: MO

address: 660 S. Euclid Avenue Campus Box 8103

institute: Washington University in St. Louis

summary: Stereotactic arrhythmia radiotherapy (STAR) is emerging as a highly effective treatment for ventricular tachycardia (VT). Growing evidence indicates that STAR favorably reprograms the electrical substrate via modulating ion channel expression, though the mechanisms whereby single-fraction radiation mediates durable changes in gene expression are incompletely understood. The DNA damage response (DDR) recruits epigenetic regulators during the repair process after irradiation (IR). Here, we demonstrate acute changes in the cardiomyocyte epigenome and transcriptome after IR in vivo and in vitro. A subset of changes persist as late as 6 weeks post-IR in mice, including increased Scn5a expression and chromatin accessibility (encoding the alpha subunit of the sodium channel, NaV1.5), demonstrating a role for epigenetic memory in conduction velocity increases observed after STAR. Transcriptomic and epigenetic sequencing further identify dynamic changes to gene expression and regulatory regions involved in cellular repolarization, calcium handling, and metabolism after IR. Gene expression changes are mirrored by dose-dependent and cell-autonomous changes in repolarization, calcium flux, and mitochondrial respiration after IR, highlighting important cellular processes which may mediate both therapeutic and toxic effects of STAR. Overall, we find that cardiomyocytes exposed to a single fraction of high-dose IR exhibit epigenetic reprogramming that mediates broad and dynamic physiologic responses.

overall_design: CUT&Tag profiliing was performed in control and 25 Gy irradiated hiPSC-cardiomyocytes (commercially available iCell Cardiomyocytes^2, FujiFilm, female). CUT&Tag was performed 7-14 days after treatment, probing for H3K27me3, H3K27ac, H3K4me3, H3K4me1, or IgG isotype control.

first: Samuel

last: Jordan

first: Abigail

last: Fulkerson

first: Khadija

last: Khan

first: Donghua

last: Hu

first: Sherwin

last: Ng

first: David

last: Zhang

first: Sneha

last: Manikandan

first: Jeff

last: Szymanski

first: Anish

last: Bedi

first: James

last: Tabor

first: Lauren

last: Boggs

first: Lori

last: Strong

first: Stephanie

last: Hicks

first: Lavanya

last: Aryan

first: Nishanth

last: Gabriel

first: Kuo-Chan

last: Weng

first: Nathaniel

last: Huebsch

first: Julie

middle: K

last: Schwarz

first: Bo

first: Stacey

last: Rentschler


title: RNA sequencing analysis of gene expresssion profiles in spleen and Peyer's patches T follicular helper cells

first: Haeun

last: Ko

summary: Purpose: The goal of this study is to compare and examine the transcriptional profiles in spleen(SP) Tfh cells versus Peyer's patches(PP) Tfh cells by mRNA sequencing.  Methods: Tfh cells (CD4+CXCR5+PD1+GITR-CD44+) were sorted by a MoFlo-Astrios (Beckman Coulter). Total RNA was extracted using TRIzol Reagent (GeneAll), according to the manufacturer’s instructions. A library for mRNA sequencing was prepared using the SMART-Seq mRNA Kit/NEXtera XT DNA Library Preparation Kit (Illumina) according to the manufacturer’s instructions. Sequencing was performed with Novaseq 6000 (Illumina). Results: For alignment and transcript quantification of raw RNA sequencing data, STAR (v2.7.10b) and RSEM(v1.3.3) were used. To normalize the expected read counts while accounting for differences in sequencing depth and gene length, GeTMM method was applied.  Differentially expressed genes (DEGs) analysis was conducted using DESeq2 R package (DESeq2 version 1.42.1; R version 4.1.2) (Log2 fold-change > 1 and P < 0.01).  Conclusions: Our study presents comparative gene expression analysis of Tfh cells from SP and PP in wild type SPF mice. We concluded that PP-Tfh cells express higher levels of Tfh related genes. The data reported here also provide supplementary files of read counts and GeTMM

overall_design: mRNA profiles of Tfh cells from the spleen and Peyer's patches of C57BL/6 mice. Three replicates for each genotypes were anaylzed.

first: Sin-Hyeog

last: Im


title: Cardiac Radiotherapy-Induced Epigenetic Memory Underlies Electrophysiologic and Metabolic Reprogramming [RNA-seq]

first: Shuhua

last: Fu

summary: Stereotactic arrhythmia radiotherapy (STAR) is emerging as a highly effective treatment for ventricular tachycardia (VT). Growing evidence indicates that STAR favorably reprograms the electrical substrate via modulating ion channel expression, though the mechanisms whereby single-fraction radiation mediates durable changes in gene expression are incompletely understood. The DNA damage response (DDR) recruits epigenetic regulators during the repair process after irradiation (IR). Here, we demonstrate acute changes in the cardiomyocyte epigenome and transcriptome after IR in vivo and in vitro. A subset of changes persist as late as 6 weeks post-IR in mice, including increased Scn5a expression and chromatin accessibility (encoding the alpha subunit of the sodium channel, NaV1.5), demonstrating a role for epigenetic memory in conduction velocity increases observed after STAR. Transcriptomic and epigenetic sequencing further identify dynamic changes to gene expression and regulatory regions involved in cellular repolarization, calcium handling, and metabolism after IR. Gene expression changes are mirrored by dose-dependent and cell-autonomous changes in repolarization, calcium flux, and mitochondrial respiration after IR, highlighting important cellular processes which may mediate both therapeutic and toxic effects of STAR. Cardiomyocyte-specific knockout of p53 failed to blut conduction velocity changes post-IR, and improved ROS scavenging via SOD2 overexpression had minimal impact on the post-IR transcriptome. Overall, we find that cardiomyocytes exposed to a single fraction of high-dose IR exhibit epigenetic reprogramming that mediates broad and dynamic physiologic responses.

overall_design:
RNAseq profiliing was performed in control and 25 Gy irradiated hiPSC-cardiomyocytes from two independent sources : (1) commercially available iCell Cardiomyocytes^2 (female) and (2) WTC11-AAV-CAG-GCaMP6 (GCaMP6) differentiated via small-molecule Wnt modulation (male). RNA was prepared at 1 day, 7 days, 14 days, or 28 days (GCaMP6 only) after treatment.  RNAseq profiling was also performed in mice after whole-heart irradiation with 25 Gy or sham treatment. Left ventricle tissue was dissociated, nuclei were isolated, and flow cytometry was used to select PCM1+ cardiomyocyte nuclei prior to sequencing. Samples were collected from sham mice and 6 weeks post-IR.
Myosin Heavy Chain6-Superoxide dismutase (Myh6-SOD) mice overexpresses SOD2 in cardiomyocytes, resulting in enhanced ROS scavenging. After exposuring Myh6-SOD and wild-type FVB mice to 25 Gy whole-heart IR or sham conditions, we performed bulk RNA sequencing at 2 days and 6 weeks post-IR.

first: Samuel

last: Jordan

first: Abigail

last: Fulkerson

first: Khadija

last: Khan

first: Donghua

last: Hu

first: Sherwin

last: Ng

first: David


title: Cardiac Radiotherapy-Induced Epigenetic Memory Underlies Electrophysiologic and Metabolic Reprogramming [ATAC-seq]

overall_design: ATACseq profiliing was performed in control and 25 Gy irradiated hiPSC-cardiomyocytes from two independent sources : (1) commercially available iCell Cardiomyocytes^2 (FujiFilm, female) and (2) WTC11-AAV-CAG-GCaMP6 (GCaMP6, male) differentiated via small-molecule Wnt modulation. ATACseq was performed at 1 day, 7 days, 14 days, or 28 days (GCaMP6 only) after treatment.  ATACseq profiling was also performed in mice after whole-heart irradiation with 25 Gy or sham treatment. Left ventricle tissue was dissociated, nuclei were isolated, and flow cytometry was used to select PCM1+ cardiomyocyte nuclei prior to sequencing. Samples were collected from sham mice and 2 days or 6 weeks post-IR.

last: Zhang

first: Sneha

last: Manikandan

first: Jeff

last: Szymanski

first: Anish

last: Bedi

first: James

last: Tabor

first: Lauren

last: Boggs

first: Lori

first: Stephanie


title: Next-generation sequencing analysis of METRNL+/+(WT) and METRNL-/- (METRNL KO) macrophages treated with candida albicans

city: ChongQing

first: Jiayu

last: Liu

address: No. 1 Yi xue yuan Road, Yuzhong  District，ChongQing

department: School of Medical Laboratory

institute: Chongqing Medical University

summary: To investigate the gene expression profilie of METRNL knock out  macrophages , we designed RNA-Seq analysis of   METRNL+/+ and METRNL-/-  macrophages treated with candida albicans.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for METRNL+/+ and METRNL-/- macrophages treated with Candida albicans.

first: Wang

last: Tan

first: Ju

last: cao


title: Next-generation sequencing analysis of vehicle and rmMETRNL mouse macrophages challenged with Candida albicans.

summary: To investigate the effect of METRNL on macrophages phagocytosis and killing of Candida albicans, we designed RNA-Seq analysis of macrophages pretreated with vehicle or rmMETRNL challenged with Candida albicans for 6 hours in vitro.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for macrophages pretreated with vehicle or rmMETRNL challenged with Candida albicans for 6 hours.

last: Cao


title: Next-generation sequencing analysis of peripheral blood mononuclear cells (Control mice vs systemic Candida albicans-infected  mice)

summary: To investigate the gene expression profilie of systemic Candida albicans infection(invasive candidiasis), we designed RNA-Seq analysis of  peripheral blood mononuclear cells for Control mice(uinfected)  and  systemic Candida albicans-infected  mice(infected for 7days).

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Control mice and  systemic Candida albicans-infected  mice for 7 days.


title: Long-term evaluation of human iPSC-derived cartilage for repairing chondral defects

city: Pittsburgh

first: Shuchang

address: 203 Lothrop Street

institute: University of Pittsburgh

summary: Previously, induced pluripotent stem cells (iPSCs)-derived cartilage have demonstrated superior capacity to regenerate hyaline cartilage compared to mesenchymal stromal cells (MSCs). Since many patients requiring chondral repair are young, the regenerated cartilage must maintain a hyaline cartilage phenotype and function for decades. However, most animal studies testing iPSC chondrogenesis have only conducted short-term assessments. In this study, we first performed a long-term (8 weeks) in vitro chondrogenesis of human iPSC-derived multipotent cells (iMPCs) and human MSCs, and examined cell phenotypes at various time points. The results showed that the expression levels of chondrogenic genes in MSCs peaked earlier than iMPCs. The expression levels of hypertrophy-related genes increased over time in both groups, which however were lower in the iMPC group than the MSC group. Furthermore, iMPC-derived chondrocytes exhibited significantly lower apoptosis levels than MSC-derived chondrocytes. We then implanted the in vitro-generated cartilage into osteochondral defects in normal rats up to 48 weeks (equivalent to ~30 years in humans). At both short and long-term time points (12 and 48 weeks), the cartilage tissue in the defect sites from the iMPC group maintained more cartilage matrix with less collagen type I and osteocalcin deposition than the MSC group. Importantly, at 48 weeks, the native cartilage surrounding the defect areas from the MSC group showed continuous degradation in some rats, which was not observed in the iMPC group. In conclusion, iMPCs represent a safe and effective cell source for long-term hyaline cartilage repair.

overall_design: In this study, we first optimized the culture conditions and evaluated in vitro chondrogenesis at different time points, extending to 8 weeks. We then implanted these in vitro-generated cartilage tissues into osteochondral defects in rats. Histology and immunostaining techniques were employed to assess the phenotype of the regenerated tissues. Lastly, we examined the changes in the host cartilage to evaluate the effect of the implants. Our studies provide crucial information regarding the long-term efficacy and safety of both human MSCs and iPSC-derived cartilage, which is vital for informing future clinical applications.

first: Yangfan

last: Lu

first: Elizabeth

last: Kats

first: Sophie

last: Hines

first: Jia-jun

first: Silvia

first: MaCalus

last: Hogan

first: Hang

last: Lin


title: ClpX inhibition promotes ferroptosis through mitochondrial dysfunction to suppress pancreatic ductal adenocarcinoma progression

first: Cai-guang

last: Yang

address: 555 Zuchongzhi Rd

institute: Shanghai Institute of Materia Medica

summary: Caseinolytic protease X (ClpX) is an unfoldase that forms the ClpXP complex with caseinolytic protease P (ClpP), playing a critical role in maintaining mitochondrial protein homeostasis. While targeting ClpP has emerged as a promising cancer intervention strategy, the biological function of ClpX in cancer remains largely unexplored. In this study, we identify elevated expression of CLPX in pancreatic ductal adenocarcinoma (PDAC), which correlates with poor patient prognosis. CLPX knockdown significantly inhibits cell proliferation and disrupts mitochondrial protein homeostasis in PDAC cells. This knockdown also induces mitochondrial oxidative stress, impairs oxidative phosphorylation, and activates the unfolded protein response. CLPX knockdown increases reactive oxygen species (ROS) levels, ferrous ion accumulation, lipid peroxidation, and elevates malonaldehyde content, thus promoting ferroptosis. Furthermore, screening of reported ATPase inhibitors reveals that MSC1094308 binds to ClpX, inhibiting ClpXP-mediated substrate degradation. MSC1094308 induces unfolded protein stress and ferroptosis in PDAC cells. Altogether, our findings suggest that ClpX inhibition represents a potent therapeutic strategy for combating PDAC.

overall_design: RNA-seq profiling of MIA PaCa-2 cells and their knockdown derivatives (shCLPX-1) of global genes expression differentiation.

first: Pengyu

last: Wang

first: Ranran

first: Yihui

last: Pan

first: Yanling

last: Zhao

first: Chao

last: Yan

first: Wei

last: Wu

first: Tao

first: Cai

middle: G


title: Ultra-resolution, multiscale live imaging reveals TADs as domains of cohesin-accelerated search

city: Stanford

first: Joo

last: Lee

state: CA

address: 279 Campus Dr

department: Developmental Biology

institute: Stanford University

summary: In the last decade, sequencing methods like Hi-C have made it clear the genome is intricately folded, (e.g. in  TADs, stripes and dots), and that this organization contributes significantly to the control of gene expression and thence cell fate and behavior. Single-cell DNA tracing microscopy and polymer physics-based simulations of genome folding, have proposed these population-scale patterns arise from motor-driven, heterogeneous movement, rather than stable 3D genomic architecture, implying  that motion, rather than structure, is key to understanding genome function. However tools to directly observe this motion in vivo have been limited in coverage and resolution. Here, we introduce TRansposon Assisted Chromatin Kinetic Imaging Technology (TRACK-IT), which combines a suite of imaging and labeling improvements to achieve ultra-resolution in space and time, with self-mapping transposons to distribute labels across the chromosome. We find, in the presence of cohesin, that elements at submegabase separation exhibit search times as fast as a few to few tens of seconds - faster than the sampling interval of most recent studies. Acute degradation of cohesin leads to longer search times and a stronger dependence of search time on the linear separation at submegabase scales, but minimal change at multimegabase scales. Loci separated by a TAD border exhibited increased search times compared to distance matched controls. Finally, cohesin containing cells exhibited rare, processive movements, not seen in cohesin depleted cells. These processive trajectories exhibit extrusion rates of four kb/s across three distinct genomic intervals, four fold faster than recent in vitro measurements.   Taken together, these results reveal a genome in constant motion on the seconds timescale, where motor dependent extrusion produces TADs that manifest as kinetic domains of accelerated local search.

overall_design: RNA-seq of T7 RNAP in situ transcripts (isT-seq) originating from T7 promoter positioned within sleeping beauty transposon, which is then used to identify ITR-genome junction to efficiently map transposon insertion sites. 4 bulk samples (ctrl: no transposon, pre: TRACK-IT parent cell line before transposon remobilization, post: TRACK-IT labels remobilized following sleeping beauty expression, rnd: positive control where TRACK-IT labels were remobilized from multi copy transfected plasmids) and 9 single clonal cell line (5kb, 20kb, 55kb, 70kb, 134kb, 260kb, 2Mb, 12Mb, 73Mb) were used as samples.

first: Liang-Fu

last: Chen

first: Simon

last: Gaudin

first: Kavyaa

last: Gupta

first: Andrew

last: Spakowitz

first: Alistair

last: Boettiger

first: Ana

last: Novacic


title: Branched Chain Amino Acids (BCAA) Modulate Metabolic Inflammation

first: Bokai

last: Zhu

address: Bridgeside Point 1, 5th Floor, 100 Technology Drive

institute: Aging Institute of UPMC/University of Pittsburgh School of Medicine

summary: H3K27ac ChIP-seq using Abcam ab4729 antibody was performed on the liver of mice with PBS or BCAA injection to determine epigenetic effects of BCAA

overall_design: ChIP-Seq of liver samples harvested from mice injected with BCAA or PBS (control) for five days

first: Tristan

last: Pesaresi

first: William

last: Dion

first: Silvia

last: Liu

first: Matthew

last: Steinhauser


title: Parathyroidectomy Restores Skeletal Muscle Strength and Transcriptome in Individuals with Primary Hyperparathyroidism

city: Stockholm

first: Carolina

last: Nylén

address: Karolinska University Hospital

country: Sweden

institute: Karolinska Institutet

summary:
Background: Primary hyperparathyroidism (pHPT) is a common endocrine disorder leading to hypercalcemia and skeletal muscle dysfunction. Muscle weakness is associated with increased risk of morbidity and mortality but is overlooked in surgical guidelines for parathyroidectomy. While parathyroidectomy is the only curative treatment, its effects on skeletal muscle strength and molecular remodelling remain underexplored.
Methods: We conducted a prospective observational cohort study involving 21 postmenopausal women with pHPT, of whom 15 completed the full protocol. Clinical assessments, magnetic resonance imaging (MRI), and skeletal muscle biopsies were performed before and three months post-parathyroidectomy. Muscle strength was evaluated using the timed stand test (TST) and isokinetic dynamometry, while RNA sequencing characterized transcriptomic changes in muscle biopsies.
Findings: Parathyroidectomy normalized calcium and PTH levels, accompanied by significant improvements in muscle strength and composition. MRI revealed an increase in muscle volume and a reduction in fat fraction, without changes in physical activity levels. Transcriptomic analysis identified 981 differentially expressed genes post-surgery, enriched in pathways related to extracellular matrix remodelling, angiogenesis, and mitochondrial metabolism. Notably, transcriptional changes mirrored exercise-induced adaptations.

overall_design: RNA-seq profiling of skeletal muscle tissue from women pre- and post-parathyroidectomy

first: Sofia

middle: B

last: Öberg

first: Nicolas

last: Pillon


title: GALNT3 is a novel target driving lymphomagenesis via O-glycosylation of FGFR2

first: sun

last: Ruixin

address: Building 7, 277 South Chongqing Road

department: Biochemistry

institute: Shanghai Jiao Tong University, School of Medicine

summary: Despite the dual roles of GALNT3 in various cellular processes related to tumorigenesis and solid tumors, the clinical and biological significance of GALNT3 in lymphomagenesis remains largely unknown. Herein, our bioinformatics analysis uncovers that GALNT3 dependent glucose metabolism was significantly elevated in DLBCL. Our DLBCL cohort study revealed that GALNT3 positivity is significantly associated with worse prognosis and clinical outcomes in DLBCL pathogenesis. Biologically, GALNT3 overexpression were found to promote DLBCL cell proliferation and cell cycle progression, while silencing GALNT3 inhibited lymphomagenesis. RNA-seq uncovers that GALNT3 upregulated FGFR2-MAPK signaling pathway in DLBCL. Moreover, protein mass spectrometry identified two potential O-glycosylation sites of FGFR2 (Thr319 and Ser299). Further point mutation of glycosylation sites, conﬁrmed that GALNT3 O-glycosylates FGFR2 through Thr319. Besides, in vitro and in vivo rescue experiments demonstrated that Thr319 is dispensable for GALNT3-FGFR2-MAPK induced lymphomagenesis. In vitro pharmacological inhibition of FGFR2 with a selective inhibitor Futibatinib further demonstrated that Futibatinib inhibits DLBCL cell growth, cell proliferation, induced cell cycle arrest, promoted cell apoptosis. Further in vivo study also indicated that combination of Futibatinib with chemotherapy demonstrated better anti-tumor activity relative to single drug therapy in DLBCL treatment. Collectively, our data highlight the importance of considering the GALNT3-FGFR2-MAPK signaling axis as an attractive therapeutic target for lymphomagenesis.

overall_design: RNA-seq profiling of DLBCL cells and their GALNT3 overexpressing derivatives (ctrl,GALNT3-OE)

first: Ma

last: Jiao

first: Ruixin

last: Sun

first: Zizhen

last: Xu


title: Tachykinin signaling defines distinct populations of glia in the enteric nervous system [Bulk RNA-seq]

city: Boston

first: Meenakshi

last: Rao

state: MA

address: 300 Longwood Ave

department: Pediatrics-Division of Gastroenterology

institute: Boston Children's Hospital

summary: One of the largest populations of glia outside the brain is in the gut. These enteric glia are involved in many functions from intestinal peristalsis to immunity, yet it is unclear if there are defined types with distinct roles in homeostasis. Comparing glia from divergent microenvironments in the mouse intestine, we found that mucosal glia resembled microglia and macrophages, while muscularis glia resembled satellite glia. Tacr3, encoding the receptor for neuropeptide Neurokinin B (NKB), was enriched within muscularis glia associated with neuronal soma and was undetectable in extraintestinal glia. Genetic or pharmacological manipulation of NKB-TACR3 signaling disrupted establishment of enteric glial populations during postnatal development, and dynamically modulated intestinal motor behaviors in adult mice. Collectively, we delineate spatially, transcriptionally and functionally distinct populations of enteric glia, identify one as an unanticipated target of TACR3 antagonists in clinical use, and establish this pathway as necessary for enteric glial diversification and function.

overall_design:
To determine how muscularis and mucosal enteric glia might differ in their transcriptional and functional properties,  GFP+ and GFP- cells from mucosa and muscle from the distal small intestine (ileum) of male and female 8–12-week-old Plp1eGFP mice were sorted for bulk RNA-sequencing.
We then performed gene expression profiling analysis of four different sample types: GFP+ mucosal, GFP+ muscularis glia, GFP- mucosal cells, GFP- muscularis cells

first: Anoohya

middle: N

last: Muppirala

first: Perry

middle: E

last: Mitchell

first: Neha

last: Mani

first: Maryam

last: Dadabhoy

first: Sushmita

last: Debnath

first: Maria

last: Paraskevopoulou

first: Thomas

last: Gould


title: Latent cytomegalovirus disrupts NK cell responses to P. falciparum and impairs parasite control

city: Brisbane

first: Zuleima

last: Pava Imitola

state: Queensland

address: 300 Herston road

institute: QIMR Berghofer

summary:
NK cells are major innate and adaptive responders to malaria, with multiple roles in protection. Function of NK cells is heterogeneous, underpinned by expression of a diversity of receptors.  One driver of NK cell heterogeneity is latent CMV infection, which drives the expansion of memory-like NK cells. We have recently reported that latent CMV infection can negatively impact the adaptive immune response to malaria, but whether CMV-mediated changes to the NK cell compartment also impact responses to malaria is unknown.

Methods: We investigated the impact of latent CMV infection on NK cell response to the malaria parasite Plasmodium falciparum in vitro, and in CMV seronegative and seropositive individuals during controlled human malaria infection. We analysed NK cell activation, cytotoxicity and NK cell receptor expression. Additionally, we investigated the impact of CMV serostatus on cytokine production in response to TLR stimulation in the myeloid cell compartment. The impact of CMV and NK cell responses on malaria symptoms and parasite control and symptoms of malaria was investigated.

Results: NK cells from CMV seropositive individuals had reduced responsiveness to P. falciparum parasites in vitro and had reduced activation during controlled human infection. Reduced activation was not restricted to NK subsets modulated by CMV but occurred across the entire NK cell compartment. Consistent with global NK cell attenuation, IL-12 production from myeloid cells, a response that supports NK cell activation on exposure to P. falciparum parasites, was lower in CMV infected individuals. Linking NK cell activation to clinical outcomes, NK cells expressing perforin were strongly associated with parasite control in CMV seronegative individuals.

Conclusion: CMV infection modulates NK cell responses during malaria by disruption of IL-12, leading to reduced parasite control.

overall_design: PBMCs were co-cultured at 37 °C, 5% CO2 in a 96-well U-bottom plate for 4 hours at a 1:1 cell ratio with 1x106 mature trophozoite stage pRBCs. After incubation NK cells subsets were FACS sorted using the BD FACSAria™ III Cell Sorter. RNA was extracted from isolated cell population lysates using the QIAGEN PicoPureTM RNA isolation kit (Applied Biosystems™, KIT0204), and RNA quality confirmed with the 2200 TapeStation system (G2964AA) by High Sensitivity RNA ScreenTape (5067- 5579). RNA sequencing libraries were constructed using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (E6420S) and NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs) (E6440S). The libraries were sequenced using a NetSeq 550 system

first: Reena

last: Mukhiya

first: Jessica

last: Loughland

first: Nick

last: Dooley

last: Pava

first: Damian

last: Oyong

first: Dean

middle: W

last: Andrew

first: Julian

last: Hamelink

first: Kiana

last: Berry

first: James

last: McCarthy

first: Bridget

last: Barber

first: J. Alejandro

last: Lopez

first: Christian

last: Engwerda

first: Michelle

last: Boyle


title: Whole transcriptome analysis reveals T cell signaling activation in septic patients with thrombocytopenia

city: Taichung City

first: thh

last: lab

address: 1650 Taiwan Boulevard Sect. 4, Taichung, Taiwan 40705, ROC

country: Taiwan

institute: TCVGH

summary: Sepsis, a life-threatening condition triggered by a dysregulated immune response to infection, remains one of the leading causes of mortality worldwide. Thrombocytopenia, a common sepsis complication, is linked to poor outcomes and reflects disease severity and specific immunological changes affecting patient outcomes. This study aims to elucidate this relationship using whole-transcriptome RNA sequencing (RNA-Seq) in intensive care unit (ICU) patients with sepsis.

overall_design: We conducted a prospective study on 52 patients who were admitted to ICU for sepsis, including 22 with thrombocytopenia and 30 without. Blood samples were collected on both day-1 and -8, and RNA-Seq was performed to analyze transcriptomic profiles. Differentially expressed genes (DEGs) were identified, and functional enrichment analyses were conducted to explore biological pathways and processes. T-cell receptor (TCR) diversity was assessed using MiXCR and VDJTools software.

first: I-Chieh

last: Chen

first: Yu-Han

last: Jiang

first: Tzu-Hung

last: Hsiao

first: Wen-Chen

last: Chao


title: Deletion of the mouse homolog of human FHR1 (FHRE) protects ApoE-/- mice from atherosclerosis

city: Jena

first: Sascha

last: Schäuble

address: Beutenbergstraße 11a

institute: Leibniz Institute for Natural Product Research and Infection Biology

summary: Atherosclerosis is the leading cause of heart attack and stroke worldwide. The key characteristic of atherosclerosis is accumulation of LDL cholesterol in artery walls, subsequent infiltration by monocytes/macrophages, and development of inflammation. Macrophage-driven inflammation causes oxidative stress, thereby enhancing endothelial cell activation, immune cell recruitment, and foam cell formation. Lowering LDL plasma levels, as well as inhibiting inflammation, reduces the risk of cardiovascular events. Recently, we reported that plasma protein complement factor H-related 1 (FHR1) binds to the necrotic surfaces of cardiovascular plaques and induces inflammation driven by monocytes/macrophages and neutrophilic granulocytes. Moreover, the concentration of FHR1 is higher, whereas CFHR1 gene deletion frequency is significantly lower, in patients with atherosclerosis than in healthy controls. To investigate the link between FHR1 and atherosclerosis, we generated FHRE-/- (the murine homolog of FHR1) knockout mice and then crossed them with ApoE-/- knockout mice (a model of human atherosclerosis). Notably, deletion of FHRE enhanced lipid degradation in liver as seen by RNAseq analysis, resulting in normalized cholesterol levels, reduced inflammation and no plaque formation in FHRE-/-ApoE-/- mice than in ApoE-/- mice expressing FHRE. These data indicate that FHRE directs uptake of oxLDL by macrophages, and supports foam cell formation, plaque development, and inflammation in dislipidemic mice. As human FHR1 correlates with non-HDL cholesterol concentrations and inflammation markers in patients with atherosclerosis-associated cardiovascular disease (ACVD) we assume that FHR1/FHRE plays a key role in development of atherosclerosis and subsequent events such as stroke and heart attack.

overall_design: We generated FHRE-/- (the murine homolog of FHR1) knockout mice and then crossed them with ApoE-/- knockout mice (a model of human atherosclerosis).

first: Luce

last: Perie

first: Sarah

last: Herr

first: Tomas

last: Ghebreslassie

first: Sonia

last: Wulf

first: Ina

last: Löschmann

first: Andjela

last: Sekulic

first: Anna-Karina

last: Maier-Wenzel

first: Berit

last: Jungnickel

first: Gianni

last: Panagiotou

first: Olaf

last: Strauß

first: Thorsten

last: Wiech

first: Peter

middle: F

last: Zipfel

first: Svante

first: Christine

last: Skerka


title: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) of glioma was conducted in 3 paired glioma and normal tissues

city: Jinan

first: Shun

last: Wang

address: BEIYUAN road 247

institute: The Second Hospital of Shandong University

summary: MeRIP was performed using a Magna Methylated RNA Immunoprecipitation Kit m6A Kit (Merck Millipore, USA). All the experimental procedures were carried out in accordance with the manufacturer's instructions.

overall_design: Surgical samples were obtained from glioma patients and patients with traumatic head injury (normal tissues). To assess the difference in the m6A-modified transcriptome between glioma tissues and normal tissues, m6A-meRIP followed by high-throughput sequencing of 3 paired glioma and normal tissues was performed. MeRIP-seq and data analysis were performed by Shanghai Genechem Co., Ltd. We reveal abnormalities in the m6A epitranscriptome in glioma via MeRIP-seq, and our results emphasize that ALKBH5 mediates the upregulation of SH3RF3-AS1 for its stabilization and that SH3RF3-AS1 further exacerbates the malignant biological behavior of glioma cells through upregulation of SH3RF3.

last: wang


title: Combined targeting poly (ADP-ribose) polymerase and receptor tyrosine kinase inhibits ovarian clear cell carcinoma progression through disrupted ribosome biogenesis

city: Guishan, Taoyuan

first: Chiao-Yun

last: Lin

address: 5 Fu-Shin St

institute: Department of Obstetrics and Gynecology

summary: OCCC has extremely poor prognosis. ARID1A mutation-related chromatin remodeling errors are key molecular features of OCCC. Dysregulation of receptor tyrosine kinases-related signaling pathways is common in OCCC. Here we show that combination of niraparib and lenvatinib exhibits significant synergistic inhibitory effects against platinum–resistant OCCC cells in vitro, in vivo xenografts and prolonged survival in the platinum-refractory patient-derived tumor xenograft (PDX) model. RNA-sequencing revealed the most differently expressed genes in PDX treated with a combination of niraparib and lenvatinib versus control, were in structural ribosomal components. Selected differently expressed ribosomal proteins (RPs: RPS2, RPS5, RPS9 and RPL3) was validated using quantitative polymerase chain reaction. Nucleophosmin (NPM1) expression, which is involved in ribosome biogenesis, was inhibited by niraparib and lenvatinib. Our findings imply that combined niraparib and lenvatinib reduces platinum-resistant OCCC progression by attenuating Src phosphorylation, NPM1 expression and ribosome biogenesis. These results highlight the necessity for continued exploration of this promising treatment strategy, particularly in future clinical trials for platinum-resistant or platinum-refractory OCCC.

first: An-Shine

last: Chao

first: Angel

first: Ren-Chin

last: Wu

first: Alice

last: Yu

first: Chiung-Hui

last: Fu

first: Yu-Chuan

last: Teng

first: Ruby

middle: Y

last: Huang

first: Chyong-Huey

last: Lai


title: Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA binding proteins with prion-like domains

first: Lin

last: Guo

address: 1020 Locust Street

institute: Thomas Jefferson University

summary: RNA-binding proteins with prion-like domains, such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs (24-48 nucleotides) that prevent FUS fibrillization by promoting liquid phases, and distinct short RNAs that prevent and, remarkably, reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. Importantly, we define a short RNA that dissolves aberrant cytoplasmic FUS condensates, restores nuclear FUS, and mitigates FUS proteotoxicity in optogenetic models and ALS-patient derived motor neurons. Another short RNA dissolves aberrant cytoplasmic TDP-43 condensates, restores nuclear TDP-43, and mitigates TDP-43 proteotoxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have therapeutic utility for ALS/FTD and related disorders.

overall_design: A common feature of ALS/FTD is the cytoplasmic mislocalization and aggregation of nuclear RNA-binding proteins (RBPs) with prion-like domains (PrLDs), such as TDP-43 or FUS, in degenerating neurons. A key therapeutic innovation for ALS/FTD would be to develop agents that reverse the aberrant cytoplasmic aggregation of TDP-43 and FUS, and return these proteins to native form and nuclear function. We aimed to identify short specific RNAs (24-48nts) that antagonize aberrant FUS assembly. While extracting recombinant GST-FUS from E. coli, we found that purified GST-FUS was bound to RNA. We sought to identify enriched RNA motifs in this FUS-bound RNA population as they might antagonize aberrant FUS assembly. RNA extraction followed by electrophoresis revealed a population of RNAs with a size range of ~50-100nts. From this RNA population, a cDNA library was constructed and sequenced. We identified 42 enriched motifs between 8-12nts in our library.

first: Jacob

last: Mann

first: Jocelyn

middle: C

last: Mauna

first: Katie

last: Copley

first: Hejia

first: Jack

last: Rubien

first: Hana

middle: M

last: Odeh

first: Jiabei

first: BoLim

last: Lee

first: Laura

last: Ganser

first: Emma

last: Robinson

first: Kevin

last: Kim

first: Anastasia

last: Murthy

first: Tapas

last: Paul

first: Bede

last: Portz

first: Amanda

last: Gleixner

first: Zamia

last: Diaz

first: Jenny

last: Carey

first: Ashleigh

last: Smirnov

first: George

last: Padilla

first: Ellen

last: Lavorando

first: Carolann

last: Espy

first: Yulei

last: Shang

first: Eric

middle: J

first: Alessandra

last: Chesi

first: Nicolas

last: Fawzi

first: Sua

last: Myong

first: Christopher

last: Donnelly

first: James

last: Shorter


title: CD99 Promotes Self-renewal in Hematopoietic Stem Cells and Leukemia Stem Cells by Regulating Protein Synthesis

city: Dallas

first: Stephen

middle: Shiu-wah

last: Chung

state: TX

address: 6001 Forest Park Road

department: Internal Medicine

institute: University of Texas Southwetsern

summary: Blood production is sustained by hematopoietic stem cells (HSCs), which are typically the only blood cells capable of long-term self-renewal. HSCs exhibit and depend on low levels of protein synthesis to self-renew. However, the mechanisms by which HSCs regulate protein synthesis to maintain their self-renewal capacity during proliferative stress and leukemogenesis remain unknown. Here we show CD99, a protein upregulated in leukemia stem cells (LSCs) in acute myeloid leukemia (AML), is required for the self-renewal of proliferating HSCs and LSCs. We found that loss of CD99 in HSCs and LSCs leads to increased protein synthesis and that their self-renewal capacity can be restored by translation inhibition. These data demonstrate a functional role for CD99 in constraining protein synthesis, which may promote the clonal expansion of HSCs and LSCs that leads to AML. Furthermore, these studies demonstrate that similar to HSCs, LSCs depend on maintenance of tightly regulated protein synthesis rates.

overall_design: To investigate the molecular consequences of loss of CD99 in hematopoietic stem and progenitor cells, hematopoietic stem cells (HSCs) and granulocyte macrophage progenitors (GMPs) were purified from the bone marrow of 10-12 week old CD99 KO mice for RNA-sequencing.

first: Liang

last: Guo

first: Wenhuo

last: Hu

first: Benjamin

last: Kroger


title: IL-10 from tumoral B cells modulates the microenvironment of diffuse large B-cell lymphoma and influences immunotherapy responses

city: Pamplona

first: Francisco

last: Novo

address: Irunlarrea 1

department: Biochemistry and Genetics

country: Spain

institute: University of Navarra

summary: The contribution of IL-10 secreted by tumoral B cells to the progression and shaping of the microenvironment in diffuse large B-cell lymphoma (DLBCL) with activated B-cell (ABC) phenotype is not yet completely understood. To shed light on this issue, we generated an immunocompetent mouse model of ABC-DLBCL with conditional knock-out of IL-10 specifically in malignant B cells. Unexpectedly, these mice had significantly worse overall survival when left untreated, but experienced increased sensitivity to conventional anti-CD20 immunotherapy or regulatory T cell depletion. We identified various immunomodulatory mechanisms involved in this behavior. In particular, we show that IL-10-deficient lymphomas acquire a highly immunosuppressed and T-cell exhausted microenvironment with increased angiogenesis that results in a more aggressive phenotype, refractory to PD-1 immune checkpoint blockade. However, the response of IL-10-deficient mice to anti-CD20 immunotherapy was greatly enhanced by upregulation of calcium channels in B cells. In general, IL-10 autocrine signaling promotes survival of malignant B cells, while the paracrine action of B cell-derived IL-10 maintains an immunoreactive microenvironment that influences the efficacy of emerging immunotherapy strategies aimed at the lymphoma microenvironment. Furthermore, IL-10-associated transcriptional signatures derived from our studies may correctly predict clinical outcomes of DLBCL patients treated with R-CHOP. Thus, our work provides important functional and mechanistic insights into the role of B cell-derived IL-10 in the biology of ABC-DLBCL.

overall_design: Single-cell RNA-seq analysis of one pBIC mouse and one pBIC10 mouse. For each, a gene expression library and a TCR-enriched library were sequenced.

first: Sergio

last: Roa