title: Examining preharvest genetic and morphological factors contributing to lettuce (Lactuca sativa L.) shelf life

city: GAINESVILLE

first: FAHONG

last: YU

state: FL

address: 2033 Morway RD

department: ICBR

institute: UNIVERSITY OF FLORIDA

summary: Lettuce (Lactuca sativa L.) is a highly perishable horticultural crop with a relatively short shelf life due to leaf senescence that limits its commercial value and contributes to food waste. Postharvest senescence varies with influences of both environmental and genetic factors. Preharvest genetic factors can be indicative of postharvest quality. Discovery of additional preharvest markers to assess lettuce shelf life is an important step towards increasing the efficiency of lettuce breeding efforts for improved shelf life. We selected and evaluated three romaine lettuces with variable shelf lives with the aim of identifying preharvest markers of lettuce postharvest shelf life. We evaluated leaf morphological characteristics for each of the three cultivars. To assess molecular indicators of shelf life, we used an RNA sequencing approach to construct transcriptomic profiles of two of the cultivars, a short shelf life (SSL) breeding line 60184 and a long shelf life (LSL) cultivar ‘Okeechobee’ at maturity. We identified 552 upregulated and 315 downregulated differentially expressed (DE) genes between the genotypes. We found that 27 % of the DE lettuce genes had an Arabidopsis thaliana ortholog characterized as senescence-associated, indicating that variable expression of senescence-associated genes (SAGs) could serve as a tool for preharvest markers of postharvest shelf life. Notably, we identified several SAGs and functional groupings with highly differential expression between the cultivars. This includes several jasmonate ZIM-domain (JAZ), jasmonic acid (JA) signaling genes, chlorophyll a-b binding (CAB) chloroplast-associated genes, and cell wall modification genes including pectate lyases (PL) and expansins (EXP). This study presented an innovative approach for identifying molecular markers for preharvest factors linked to postharvest traits for prolonged shelf. These genes could potentially be developed further as preharvest predictors of shelf life for lettuce breeding

overall_design: To select and evaluate romaine lettuces with variable shelf lives with the aim of identifying preharvest markers of lettuce postharvest shelf life

first: Kathryn

last: Chase

first: Catherine

last: Belisle

first: Yogesh

last: Ahlawat

first: Fahong

first: Steven

last: Sargent

first: German

last: Sandoya

first: Kevin

last: Begcy

first: Tie

last: Liu


title: Reactivation of memory-encoding dentate gyrus neurons during memory consolidation is associated with subregion-specific, learning- and sleep-mediated biosynthetic changes.

status: Public on Feb 27 2024

city: Ann Arbor

first: Bioinformatics

last: Core

state: MI

address: BRCF Bioinformatics Core, 2800 Plymouth Rd

country: USA

institute: University of Michigan

type: Expression profiling by high throughput sequencing

summary: This superseries contain NanoString GeoMx spatial profiling datasets that measured RNA and protein expression levels from ROIs of mice that received fear-conditioning and/or sleep deprivation. ROIs were collected from both hemispheres of the brain, including 5 different subregions of the hippocampus (CA1, CA3, DG-superior blade, DG-inferior blade, and DG-hilus).

sample_organism:
Mus musculus
blank sample

platform_organism:
Mus musculus
blank sample

overall_design: Refer to individual Series


title: Gene expression profile of tumor infiltrating cells in MC38 tumor that grown on C57/Bl6 mice subjected to total body irradiation (TBI) with ABT263 treatment,

status: Public on Feb 26 2024

city: Montreal

first: Christian

last: Beauséjour

state: QC

address: 3175, chemin de la Côte-Sainte-Catherine, 6.17.315.6

country: Canada

institute: CHUSJ

summary: The goal of this scRNA_seq was to interogate the immunosuppresive profile of infiltrating tumor immune cells in mice previously exposed to TBI and treated or not with ABT263 to kill senescent cells

overall_design: Live, tumor infiltrating, bone marrow or splenic cells were sorted by flow cytometry after sacrifice of the animal and tumor resection.

first: Damien

last: Maggiorani

first: Véronique

last: Lisi

first: Vincent

middle: P

last: Lavallée


title: Iterative assay for transposase-accessible chromatin by sequencing to home in on functionally relevant neuronal subtypes

status: Public on Feb 28 2024

city: Salt Lake City

first: Adrian

last: Rothenfluh

state: UT

institute: University of Utah

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The Drosophila brain contains tens of thousands of distinct cell types. Numerous different transgenic lines reproducibly target specific neuron subsets, yet most still express in several distinct cell types. Furthermore, most lines were developed without a priori knowledge of where the transgenes would be expressed. To aid in the development of cell type-specific tools for neuronal identification and manipulation, we used an iterative assay for transposase-accessible chromatin by sequencing (ATAC-seq) approach. Open chromatin regions (OCRs) that were enriched in neurons compared to whole bodies drove transgene expression preferentially in subsets of neurons. A second round of ATAC-seq from these specific neuron subsets revealed additional enriched OCRs that further restricted transgene expression within the chosen neuron subset. This approach allows for continued refinement of transgene expression and can be used to identify neurons relevant for sleep behavior. Furthermore, this approach is widely applicable to other cell types and to other organisms.

overall_design: GFP-positive nuclei from whole flies, neurons in the head, and neuronal subtypes were isolated by fluorescence-activated nuclei sorting and were used to generate ATAC-seq libraries.

first: Collin

middle: B

last: Merrill

first: Iris

last: Titos

first: Miguel

middle: A

last: Pabon

first: Austin

last: Montgomery

first: Aylin

middle: R

last: Rodan


title: scRNA-Seq: First Atlas and Cellular Landscape of Lacrimal Sac: Implications in Primary Acquired Nasolacrimal Duct Obstruction Pathogenesis.

city: Shanghai

first: Caiwen

last: Xiao

state: -- Select State/Province --

address: Zhizaoju Road

country: China

institute: Shanghai Ninth People's Hospital

summary:
Purpose:  The aim of the study is to describe the transcriptional changes of individual cellular components in the lacrimal sac in patients with primary acquired nasolacrimal duct obstruction  (PANDO) and attempt to construct the first lacrimal sac cellular atlas to elucidate the potential mechanisms that may drive the disease pathogenesis.
Methods: Lacrimal sac samples were obtained intraoperatively during the endoscopic dacryocystorhinostomy (EnDCR) procedure from five patients. Single-cell RNA sequencing was performed to analyze each individual cell population including epithelial and immune cells during the early-inflammatory and late-inflammatory phases of the disease.
Results: Eleven cell types were identified among 25,791 cells. T cells and B cells were the cell populations with the greatest variation in cell numbers between the two phases and were, involved in immune response and epithelium migration-related pathways. The present study showed that epithelial cells highly expressed the genes of senescence-associated secretory phenotype (SASP) and were involved in influencing the inflammation, neutrophil chemotaxis, and migration during the late-inflammatory stage. Enhanced activity of CXCLs-CXCRs between the epithelial cells and neutrophils was noted by the cell-cell communication analysis and is suspected to play a role in inflammation by recruiting more neutrophils.
Conclusions: The study presents a comprehensive single-cell landscape of the lacrimal sac cells in different phases of PANDO. The contribution of T, B cells and epithelial cells to the inflammatory response, and construction of the intercellular signaling networks between the cells within the lacrimal sac has further enhanced the present understanding of the PANDO pathogenesis.

overall_design: using BD Rhapsody system in March 2023

first: Wenyue

last: Zhang

first: Huan

last: Huang

first: Xueru

first: Leilei

first: Lunhao

last: Li

first: Yi

last: Ding

first: Yichuan

first: Mohammad

middle: J

last: Ali

first: Hao

last: Sun


title: Select EZH2 inhibitors enhance the viral mimicry effects of DNMT inhibition through a mechanism involving Calcium-Calcineurin-NFAT signaling [EM-seq]

city: Grand Rapids

first: Scott

last: Rothbart

address: 333 Bostwick Avenue NE

institute: Van Andel Research Institute

type: Methylation profiling by high throughput sequencing

summary: DNA methyltransferase inhibitors (DNMTi) are FDA-approved for various hematological malignancies but have limited efficacy in solid tumors. DNA hypomethylation with these drugs is associated with elevated lysine 27 tri-methylation on histone H3 (H3K27me3). We hypothesized that this EZH2-dependent repressive mark limits the full potential of DNMTi. Here, we show in cell line and tumoroid models of colorectal cancer, that low-dose DNMTi sensitizes cells to selective EZH2 inhibitors (EZH2i) that have limited single agent toxicity, and that EZH2i enhances DNMTi-driven molecular and therapeutic effects. Through integrative epigenomic analyses, we reveal that DNMTi induces H3K27me3 accumulation at genomic regions poised with EZH2. Unexpectedly, combined treatment alters the epigenome landscape to promote transcriptional upregulation of the Calcium-Calcineurin-NFAT signaling pathway. Blocking this pathway limits the transcriptional activating effects of the drug combination, including expression of transposable elements and innate immune response genes within a viral defense pathway. Consistently, we demonstrate positive correlations between DNMTi- and viral infection-associated transcription profiles and Calcium signal activation in colon cancer patient samples. Collectively, our study demonstrates that compensatory EZH2 activity following DNA hypomethylation presents a barrier to the therapeutic action of DNMTi in colon cancer, reveals a new application of EZH2i beyond cancers associated with PRC2 hyperactivity, and links Calcium-Calcineurin-NFAT signaling to epigenetic therapy-induced viral mimicry.

overall_design: HCT116 cells were treated with two different doses of DAC (30 nM, 300 nM) and DMSO for the Vehicle control in biological duplicate for 72 hours. Cells were then fixed and prepped for chromatin immunoprecipitation of H3K27me3. Purified DNA fragments from the H3K27me3 ChIP assay were then processed through Enzymatic Methyl-seq (EM-seq) to query the DNA methylation status of the H3K27me3 purified fragments from each drug treatment.

first: Alison

last: Chomiak

first: Rochelle

middle: L

last: Tiedemann

first: Yanqing

last: Liu

first: Xiangqian

last: Kong

first: Ying

last: Cui

first: Kate

last: Thurlow

first: Evan

middle: M

last: Cornett

first: Michael

last: Topper

first: Stephen

last: Baylin


title: Chitin-mediated blockade of chitinase-like proteins reduces tumor immunosuppression, inhibits lymphatic metastasis and enhances anti-PD-1 efficacy in complementary TNBC models.

status: Public on Feb 25 2024

city: Merelbeke

first: Jonas

last: Steenbrugge

address: Salisburylaan 133

department: Veterinary and Biosciences

country: Belgium

institute: Ghent University

summary:
Purpose: Evaluate the anti-tumorigenic efficacy and immunomodulatory effects of chitin as general blocker of immunosuppressive chitinase-like proteins (CLPs) in combination with and without anti-PD-1 immune checkpoint blockade (ICB) in an immunocompetent 4T1- and 66cl4-based intraductal model for triple-negative breast cancer (TNBC).
Methods: 4T1 and 66cl4 mammary tumor-bearing female BALB/c mice were either left untreated or were treated with anti-PD-1, chitin or chitin + anti-PD-1 for 2 weeks (w) between 3 and 5 w post-inoculation (p.i.) of the mammary tumor cells. After the 2-w treatment, 4T1 and 66cl4 primary tumors were resected and RNA was isolated from the tissue using in-house developed protocols.
Results: Chitin-mediated reduction in innate immunosuppression and increased anti-tumor T-cell immunity in primary tumors of both TNBC models was clearly presented at the genomic level based on RNA-sequencing analysis. More specifically, a selection of 68 and 55 innate immunity-related genes in respectively 4T1 and 66cl4 primary tumors were downregulated upon chitin treatment in combination with or without anti-PD-1. Several of the selected innate immunity genes could be linked to immunosuppressive myeloid cell types, including Ccl2, Cxcl2, Csf1r and Itgam. Genes related to T-cell exhaustion, including Pdcd1, Cd274, Havcr2 and Lag3, were downregulated upon chitin and chitin + anti-PD-1 treatment in 4T1 tumors. Chitin- and chitin + anti-PD-1-treated 66cl4 tumors showed upregulation of genes associated with enhanced T-cell activity, including Cd8a, Cd8b1, Ifng, Il12b and Il18r1. In line with an overall reduced inflammation upon chitin treatment at the protein level, predominantly through myeloid cell reduction, a selected set of 68 and 48 inflammation-related genes were downregulated in chitin- and chitin + anti-PD-1-treated primary tumors of respectively the 4T1- and the 66cl4-based model. Importantly, genes that have been associated with the signaling and pro-tumorigenic activity of a prominent CLP family member, i.e. CHI3L1, were also downregulated in primary tumors following chitin treatment either with or without anti-PD-1, including Usf1 and Rab37 in the 4T1-based model and Lgals3 in the 66cl4-based model.
Conclusion: Our findings highlight that chitin, as a general CLP blocker, reduces cancer-associated immunosuppression and enhances anti-tumor immunity as well as ICB responses in complementary ICB-resistant TNBC models, supporting its potential clinical relevance in immunosuppressed TNBC patients.

overall_design: RNA was isolated from 4T1 and 66cl4 primary tumors at 5 w p.i. using the Rneasy Mini Kit (QiAgen, Valencia, CA, USA). All analyses were done on 5 samples per treatment group from both TNBC models, yielding a total of 40 samples. Only 4T1 tumor sample 3 from the untreated group showed RNA degradation and was therefore excluded from the analyses. RNA sequencing was done in collaboration with Azenta Life Sciences and performed on an Illumina Novaseq system (paired end, read length of 150 bp), aiming for 10 million reads per sample.

first: Robbe

last: Salembier

first: Caro

last: De Haes

first: Julie

last: Bellemans

first: Kristel

last: Demeyere

first: Wim

last: Van Den Broeck

first: Niek

middle: N

last: Sanders

first: Steven

last: Van Laere

first: Traci

last: Lyons

first: Evelyne

last: Meyer


title: Select EZH2 inhibitors enhance the viral mimicry effects of DNMT inhibition through a mechanism involving Calcium-Calcineurin-NFAT signaling

first: Scott

last: Rothbart

type: Methylation profiling by genome tiling array

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


title: Bacterial vitamin B6 is required for post-embryonic development in C. elegans

status: Public on Feb 23 2024

city: college station

first: Min

last: Feng

state: Texas

address: 200 Spence street

department: Chemical Engineering

institute: Texas A&M University

summary: Nutritional intake influences animal growth, reproductive capacity, and survival of animals. Under nutrition deficiency, animal developmental arrest occurs as an adaptive strategy to survive. However, the nutritional basis and the underlying nutrient sensing mechanism essential for animal regrowth after developmental arrest remain to be explored. In Caenorhabditis elegans, larvae undergo early developmental arrest are stress resistant, and they require certain nutrients to recover postembryonic development. Here, we investigated the developmental arrest in C. elegans feeding on Lactiplantibacillus plantarum, and the rescue of the diapause state with trace supplementation of Escherichia coli. We performed a genome-wide screen using 3983 individual gene deletion E. coli mutants and identified E. coli genes that are indispensable for C. elegans larval growth on originally not nutritionally sufficient bacteria L. plantarum. Among these crucial genes, we confirmed E. coli pdxH, and the downstream metabolite pyridoxal 5-P (PLP, Vitamin B6) as important nutritional factors for C. elegans postembryonic development. Transcriptome results suggest that bacterial pdxH affects host development by coordinating host metabolic processes and PLP binding. Additionally, the developmental arrest induced by the L. plantarum diet in worm does not depend on the activation of FoxO/DAF-16. Altogether, these results highlight the role of microbial metabolite PLP as a crucial cofactor to restore postembryonic development in C. elegans.

overall_design: To investigate the signaling mechanism that leads to developmental arrest of C. elegans fed on L. plantarum due to the absence of pdxH in E. coli, we carried out RNA sequencing and expression analysis to identify the host pathways regulated by E. coli pdxH activity. Specifically, we compared the RNA-seq results of worms fed a L. plantarum diet with either a trace amount of E. coli pdxH mutant or wild type E. coli BW25113.

first: L

last: Garcia

first: Qing

last: Sun


title: Trimethylamine N-oxide impairs β-cell function and glucose tolerance

city: Beijing

first: lijuan

last: kong

state: 北京

address: 1 Xian Nong Tan Street

institute: Institute of Materia Medica, Chinese Academy of Medical Sciences

summary: β-Cell dysfunction, manifested as impaired glucose-stimulated insulin secretion (GSIS), and β-cell loss, which presents as dedifferentiation, inhibited transcriptional identity and death, are the hallmarks of type 2 diabetes. Trimethylamine N-oxide (TMAO), a gut microbiota metabolite, has been shown to play a role in cardiovascular disease. Here, we found that plasma TMAO levels are elevated in both diabetic mice and human subjects and that TMAO at a similar concentration to that found in diabetes could directly decrease β-cell GSIS in both MIN6 cells and primary islets from mice or humans. Elevation of TMAO levels through choline diet feeding impairs GSIS, the β-cell proportion, and glucose tolerance. TMAO inhibits calcium transients through NLRP3 inflammasome-related inflammatory cytokines and induced Serca2 loss, and a Serca2 agonist reversed the effect of TMAO on β-cell function in vitro and in vivo. Additionally, long-term TMAO exposure promotes β-cell ER stress, dedifferentiation, and apoptosis and inhibits β-cell transcriptional identity. Inhibition of TMAO production through either genetic knockdown or antisense oligomers of Fmo3, the TMAO-producing enzyme, improves β-cell GSIS, the β-cell proportion, and glucose tolerance in both db/db and choline diet-fed mice. These observations elucidate a novel role for TMAO in β-cell dysfunction and maintenance, and inhibition of TMAO could be a new approach for the treatment of type 2 diabetes.

overall_design:
To further explore the mechanism, we performed RNAseq (RNA sequencing) of MIN6 cells treated with TMAO (100 nM, 18 h)/vehicle with low or high glucose. Each group includes 3 biological replicates.
NOTE FROM SUBMITTER: We can't provide the raw data files, because the company that did RNAseq for us said that it has been deleted.

first: Lijuan

last: Kong

first: Pingping

last: Li


title: Systematic analyses of RNA-binding proteins uncover therapeutically promising vulnerabilities in osteosarcoma

city: Heidelberg

first: Thileepan

last: Sekaran

address: Mayerhoffstarsse

country: Germany

institute: EMBL

summary: Osteosarcoma (OS) is the most common primary malignant bone tumor with a strong tendency to metastasize, limiting the prognosis of affected patients. Genomic, epigenomic and transcriptomic analyses have demonstrated the exquisite molecular complexity of this tumor, but haven't sufficiently defined the underlying mechanisms or identified promising therapeutic targets. To systematically explore RNA-protein interactions relevant to OS, we defined the RNA interactomes together with the full proteome and the transcriptome of cells from five malignant bone tumors (four osteosarcomata and one malignant giant cell tumor of the bone) and from normal mesenchymal stem cells and osteoblasts. These analyses uncovered both systematic changes of RNA-binding activity common to all osteosarcomata and individual alterations that were observed in only a subset of tumors. Functional analyses especially revealed a vulnerability of these tumors to translation inhibition and a positive feedback circuit involving the RNA-binding protein IGF2BP3 and the transcription factor Myc which affected cellular translation and OS cell viability. Our results provide novel and potentially clinically relevant insights into a previously uncharted molecular landscape of osteosarcoma.

overall_design:
To investigate the RNA-protein interactome in osteosarcoma, four patient-derived osteosarcoma (OS) cell lines and one giant cell tumor of bone (GCTB) were used for enhanced RNA interactome capture (eRIC) and full proteome analysis in comparison with normal osteoblasts (OB) and bone mesenchymal cells (MSC).
Total RNA was also isolated from the seven cell types and used for RNAseq. Differential gene expression analysis was done for the OS and GCTB cells in comparison with the OB and MSC in order to determine upregulated and downregulated mgenes in the OS and GCTB cells

first: Zhou

last: Yang

first: Ray

last: Partho Sarothi

first: Hentze

last: Matthias W.

first: Kulozik

last: Andreas E.


title: Select EZH2 inhibitors enhance the viral mimicry effects of DNMT inhibition through a mechanism involving Calcium-Calcineurin-NFAT signaling [EPIC]

overall_design: Genomic DNA was isolated from HCT116 cells treated with Vehicle (Veh-DMSO), 5'aza-2'deoxycytidine (30 nM), 5'aza-2'deoxycytidine (300 nM), Tazemetostat (1uM), or 5'aza-2'deoxycytidine (30 nM) + Tazemetostat (1 uM) in biological duplicate.

first: Alison

last: Chomiak

first: Rochelle

last: Tiedemann

first: Yanqing

last: Liu

first: Xiangqian

first: Ying

last: Cui

first: Kate

last: Thurlow

first: Evan

last: Cornett

first: Bradley

last: Dickson

first: Michael

last: Topper

first: Stephen

last: Baylin


title: Clinical and genomic characterization of chemoradiation resistant HPV-positive squamous cell carcinoma

city: La Jolla

first: Theresa

last: Guo

state: California

address: 3855 Health Sciences Dr, MC 0987

institute: University of California, San Diego

summary: This study performed genomic sequencing on a rare phenotype of HPV-positive oropharyngeal squamous cell carcinoma which was resistant to standard of care platinum based chemoradiation treatment.  Tissue was collected from archival FFPE when available from pretreatment and post treamtent samples from four patients and these were batch corrected to be compared to known HPV-positive tumors without recurrent disease

overall_design:
Tumor tissue from patients with chemoradiation resistant HPV positive oropharyngeal cancer tumors
Authors declare that raw files are planned to be submitted to dbGAP.  In the interim, they are available upon request from corresponding author Theresa Guo (twguo@health.ucsd.edu)

***************************************************************
Submitter states that missing raw data are being made available for controlled access in dbGaP.
***************************************************************

first: Fernando

last: Zamuner

first: Daria

last: Gaykalova

first: Ranee

last: Mehra


title: Tgfbr1 controls developmental plasticity between the hindlimb and external genitalia by remodeling their regulatory landscape

city: Oeiras

first: Anastasiia

last: Lozovska

address: Rua da Quinta Grande 6

country: Portugal

institute: Instituto Gulbenkian de Ciência

summary: The hindlimb and external genitalia of present-day tetrapods are thought to derive from an ancestral common primordium that evolved to generate a wide diversity of structures adapted for efficient locomotion and mating in the ecological niche conquered by the species. We show that despite long evolutionary distance from the ancestral condition, the early primordium of the mouse external genitalia preserved the capacity to take hindlimb fates. In the absence of Tgfbr1, the pericloacal mesoderm generates an extra pair of hindlimbs at the expense of the external genitalia. It has been shown that the hindlimb and the genital primordia share many of their key regulatory factors. Tgfbr1 controls the response to those factors acting in a pioneer-like mode to modulate the accessibility status of regulatory elements that control the gene regulatory networks leading to the formation of genital or hindlimb structures. Our work uncovers a remarkable tissue plasticity with potential implications in the evolution of the hindlimb/genital area of tetrapods, and identifies a novel mechanism for Tgfbr1 activity that might also contribute to the control of other physiological or pathological processes.

overall_design: Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) performed on control hindlimb and genital tubercle (GT) and on Tgfbr1 conditional knock out (cKO) extra hindlimb and GT.

first: Artemis

last: Korovesi

first: André

last: Dias

first: Donald

last: Fowler

first: Gabriel

middle: G

last: Martins

first: Moisés

last: Mallo


title: Select EZH2 inhibitors enhance the viral mimicry effects of DNMT inhibition through a mechanism involving Calcium-Calcineurin-NFAT signaling [RNA-seq]

first: Scott

last: Rothbart

overall_design: HCT116 cells were treated with single agent DAC (30 nM), TAZ (1 uM), or DAC + TAZ (Combo) in biological triplicate for 3 (dosed once) and 6 days (dosed twice) to be compared back to the Vehicle control (DMSO). RNA was purified and submitted for sequencing. HCT116 cells were also treated with single agent Cyclosporin A (CsA, 5 uM) and CsA + Combo (DAC + TAZ) in biological triplicate to be compared to Vehicle controls (DMSO) for 72 hours.

first: Alison

last: Chomiak

first: Rochelle

last: Tiedemann

first: Yanqing

last: Liu

first: Xiangqian

last: Kong


title: A proof-of-concept phase 2 study of the CHK1 inhibitor prexasertib with translational study outcomes in platinum-resistant recurrent, BRCA wild-type  high-grade serous ovarian carcinoma

status: Public on Feb 22 2024

city: Bethesda

first: Jayakumar

last: Nair

state: MD

address: 10 center drive

department: Women malignancies Branch

institute: National Cancer Institute

summary: This proof-of-concept phase 2 trial (NCT02203513) evaluated the clinical activity of the CHK1 inhibitor (CHK1i), prexasertib, in platinum-resistant (PR) recurrent high-grade serous ovarian carcinoma (HGSOC) patients without BRCA mutation. The primary endpoint was objective response rate (ORR). Secondary outcomes were safety and progression-free survival (PFS). Translational research was an exploratory endpoint. Potential biomarkers were investigated using pre-treatment fresh biopsies and serial blood samples. 49 heavily-pretreated patients were enrolled. Among the 39 RECISTv1.1-evaluable patients, ORR was 30.7%, the median PFS was 5.8 months. Toxicity was manageable. Transcriptomic analysis revealed high levels of DNA replication-related genes (POLA1, POLE, GINS3, MCM7) associated with lack of clinical benefit. Subsequent preclinical experiments demonstrated synthetic lethality of POLA1 inhibition in combination with CHK1i in PR-HGSOC cell line models. Therefore, POLA1 expression may be predictive for CHK1i resistance, and the concurrent POLA1 inhibition may improve the efficacy of CHK1i monotherapy in this hard-to-treat population, deserving further investigation.

overall_design: RNAseq was performed using a HiSeq4000 sequencing system (Illumina) at the CCR Sequencing Facility/NCI on pre-treatment biopsy samples from 17 patients.. NovaSeq S1 using Illumina TruSeq Stranded Total RNA Library Prep and paired-end sequencing was used to perform RNAseq using a TruSeq stranded library. Raw RNAseq counts were used as input. STAR v2.7.0f was used to align raw reads in 2-pass mode. RNAseq by expectation-maximization (RSEM) v1.3.1 was used for quantification. Principal component analysis was constructed from the batch-normalized data after centering. The first two PCs were plotted across all data with labels for batch and tumor/normal status. Two samples did not match with ovarian cancer tissue profiles obtained from GTEx database, since they were more likely normal or necrotic tissues. 15 biopsies were selected for further in silico analysis. Quartile normalization and log-transformation prior to analysis were performed on datasets.

first: Elena

last: Giudice

first: Grant

last: Zurcher

first: Tzu-Ting

last: Huang

first: Darryl

last: Nousome

first: Marc

last: Radke

first: Jung-Min

last: Lee


title: The genetic regulation of the gastric transcriptome is associated with metabolic and obesity-related traits and diseases

city: Marburg

first: Laura

last: Koebbe

address: Baldingerstraße

institute: University of Marburg

summary: Tissue-specific gene expression and gene regulation lead to a better understanding of tissue-specific physiology and pathophysiology. We analyzed the transcriptome and genetic regulatory profiles of two distinct gastric sites, corpus and antrum, to identify tissue-specific gene expression and its regulation. The transcriptome data from corpus and antral mucosa highlights the heterogeneity of gene expression in the stomach. We identified enriched pathways revealing distinct and common physiological processes in gastric corpus and antrum. Physiological differences are mostly related to digestion and epithelial protection. Furthermore, we found an enrichment of the single nucleotide polymorphism (SNP)-based heritability of metabolic, obesity-related, and cardiovascular traits and diseases by considering corpus- and antrum-specifically expressed genes. In particular, we could prioritize gastric-specific candidate genes for multiple metabolic traits, like NQO1 which is involved in glucose metabolism, MUC1 which contributes to purine and protein metabolism or RAB27B being a regulator of weight and body composition.

overall_design: Gastric corpus and antrum mucosa biopsies were collected during routine gastroscopies from up to 431 healthy individuals. We performed gene expression profiling of the two major gastric regions, corpus and antrum, using data obtained from RNA-seq.

first: Timo

last: Hess

first: Ann-Sophie

last: Giel

first: Jessica

last: Bigge

first: Jan

last: Gehlen

first: Vitalia

last: Schueller

first: Michael

last: Geppert

first: Franz

last: Dumoulin

first: Joerg

last: Heller

last: Schepke

first: Dominik

last: Plaßmann

last: Vieth

first: Marino

last: Venerito

first: Johannes

last: Schumacher

first: Carlo

last: Maj


title: RNA-Sequencing of CD4, CD8 and CD19 splenocytes from mouse models of SLE

city: Syracuse

first: Frank

last: Middleton

state: NY

address: 505 Irving Avenue

department: Neuroscience and Physiology

institute: SUNY Upstate Medical Univ

summary: Systemic Lupus Erythematosus (SLE) is an autoimmune disease that affects 20-150 out of 100,000 people globally. Reactive oxygen species (ROS) generation from mitochondrial dysfunction contributes to risk for SLE and autoimmune states and are potential targets for the treatment of autoimmunity. HRES-1/Rab4 (Rab4A) is a GTPase linked to mitochondrial oxidative stress and activation of the mechanistic target of rapamycin (mTOR), and has been linked to SLE pathogenesis. Here, RNA-sequencing (RNA-seq) was used to examine the expression of different classes of splenocytes (CD4+, CD8+, CD19+) from the lupus-prone  SLE1.2.3. triple-congenic (B6.TC) mouse, as well as Rab4AQ72L mutant mice (which exhibits constitutive overexpression of Rab4a), and mice with CD4 T cell-specific deletion of Rab4A (KO).

overall_design: Stranded paired-end RNA-sequencing of purified CD4, CD8 and CD19 splenocytes obtained from adult mice with different genetic backgrounds related to lupus

first: Thomas

last: Winans

first: Andras

last: Perl


title: Cancer cell genetics shaping of the tumor microenvironmentreveals myeloid cell-centric exploitable vulnerabilities in hepatocellular carcinoma [WES 2]

city: Amsterdam

first: Masami

last: Ando-Kuri

address: Plesmanlaan 121

country: Netherlands

institute: Netherlands Cancer Institute (NKI)

summary: Hepatocellular carcinoma (HCC) is a highly heterogenous disease associated with an equally dynamic tumor microenvironment (TME).  We generated somatic HCC mouse models bearing clinically-relevant oncogenic driver combinations that faithfully recapitulated different human HCC subclasses. Using WES data, we explored the tumor mutation frequencies between models and compared them with human datasets.

overall_design: WES profiles of bulk tumors from genetically-distinct HCC mouse models. 3 biological replicates for each genotype and 3 for control livers.

first: Christel

middle: F

last: Ramirez

first: Serena

last: Vegna

first: Roelof

last: Kluin


title: Dbf4-dependent kinase promotes cell-cycle controlled resection of DNA double strand breaks and repair by homologous recombination

city: Planegg-Martinsried

first: Martina

last: Peritore

state: Bayern

address: Am Klopferspitz 18

institute: MPIB

summary: DNA double-strand breaks (DSBs) can be repaired by several pathways. In eukaryotes, repair pathway choice – the cellular decision making underlying DSB repair – occurs at the level of DSB resection and is controlled by the cell cycle. Upon cell cycle-dependent activation, cyclin-dependent kinases (CDKs) phosphorylate resection proteins and thereby stimulate DSB resection and repair by homologous recombination (HR). Here, we identify Dbf4-dependent kinase (DDK) as a second major cell cycle regulator of DNA end resection. Using inducible genetic and chemical inhibition of DDK in budding yeast and human cells, we show that DNA resection and HR require activation by DDK. Mechanistically, DDK catalyzes phosphorylation of at least two resection nucleases. Via phosphorylation of the Mre11 activator Sae2 it promotes activation of resection initiation, via phosphorylation of the Dna2 nuclease it promotes long-range resection. Notably, synthetic activiation of DDK allows limited resection and HR in G1 cells, suggesting that DDK is a key component of DSB repair decision making.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for Replication Protein A (RPA) in different Saccharomyces cerevisiae strains: WT, dbf4-3AID, sae2-S267E, GAL-DDK, sae2-S267E GAL-DDK. N ≥ 2 replicates

first: Lorenzo

last: Galanti

first: Robert

last: Gnügge

first: Elda

last: Cannavó

first: Maria

last: Palumbieri

first: Barbara

last: Steigenberger

first: Lorraine

middle: S

last: Symington

first: Petr

last: Cejka

first: Boris

last: Pfander


title: A fast-acting lipid checkpoint in G1 prevents mitotic defects

city: Stanford

first: Peter

middle: K

last: Jackson

state: CA

address: 279 Campus Dr., CCSR 3230

institute: Stanford University

summary: Lipid synthesis must increase during the cell cycle to double membrane mass, but how insufficient synthesis restricts cell-cycle entry is not understood. Here, we identify a lipid checkpoint in G1 phase of the mammalian cell cycle by using live single-cell imaging, lipidome, and transcriptome analysis in non-transformed cells. We show that synthesis of fatty acids in G1 not only increases lipid mass but extensively shifts the lipid composition to unsaturated phospholipids and neutral lipids. Strikingly, acute lowering of lipid synthesis rapidly activates the PERK/ATF4 endoplasmic reticulum (ER) stress pathway that blocks cell-cycle entry by increasing p21 levels, decreasing Cyclin D levels, and suppressing Retinoblastoma protein phosphorylation. Together, our study identifies a rapid anticipatory ER lipid checkpoint in G1 that prevents cells from starting the cell cycle as long as lipid synthesis is low, thereby preventing mitotic failure and cell death, which are triggered by low lipid synthesis much later in mitosis.

overall_design:
To investigate the effect of fatty acid metabolism inhibition on cell cycle progression, we treated MCF-10A cells that were previously synchronized through starvation and released into the cell cycle with epidermal growth factor (EGF) with compounds inhibiting fatty acid metabolism.
We performed gene expression profiling on MCF-10A cells released with EGF for 9, 12, and 15 hours. Cells were treated with fatty acid synthase inhibitors C75 (30µM), or GSK2194069 (50µM), or stearoyl-CoA desaturase (SCD) inhibitor (32µM) or DMSO (control) for 3 or 6 hours after 9 hours of EGF release.
Comparative gene expression profiling analysis of RNA-Seq data was performed between inhibitor treatment and DMSO control.

first: Marielle

last: Köberlin

first: Yilin

last: Fan

first: Tobias

last: Meyer


title: Accelerated DNA replication fork speed due to Loss of R-Loops in Myelodysplastic Syndromes with SF3B1 Mutation [RNAseq_erythroHuman]

city: Paris

first: David

last: Rombaut

address: 22 rue Méchain

country: France

institute: Institut Cochin

summary: Myelodysplastic syndromes (MDS) with mutated SF3B1 gene have many features including a favorable outcome that are distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases of these divergences are poorly understood. Here we show that SF3B1-mutated MDS are characterized by a dramatically reduced R-loop formation predominating in gene bodies, which associates with intron retention reduction specifically found in SF3B1-mutated, but not in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibited augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restored Rloop formation,slowed down DNA replication forks and improved SF3B1-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress is a hallmark of SF3B1-mutated MDS ineffective erythropoiesis, which could be used as a new therapeutic target.

overall_design: Comparative gene and splicing expression analysis of RNA-seq data for bone marrow-derived basophilic and polychromatophilic erythroblasts, SF3B1 MUT vs WT.

first: Carine

last: Lefèvre

first: Tony

last: Rached

first: Sabrina

last: Bondu

first: Anne

last: Letessier

first: Raphael

last: Mangione

first: Batoul

last: Farhat

first: Auriane

last: Lesieur-Pasquier

first: Daisy

last: Castillo-Guzman

first: Ismael

last: Boussaid

first: Chloé

last: Friedrich

first: Aurore

last: Tourville

first: Magali

last: de Carvalho

first: Françoise

last: Levavasseur

first: Marjorie

last: Leduc

first: Morgane

last: Le Gall

first: Sarah

last: Battault

first: Marie

last: Temple

first: Alexandre

last: Houy

first: Didier

last: Bouscary

first: Lise

last: Willems

first: Sophie

last: Park

last: Raynaud

first: Thomas

last: Cluzeau

first: Emmanuelle

last: Clappier

first: Pierre

last: Fenaux

first: Lionel

last: Adès

last: Margueron

first: Michel

last: Wassef

first: Samar

last: Alsafadi

first: Nicolas

last: Chapuis

first: Olivier

last: Kosmider

first: Eric

last: Solary

first: Angelos

last: Constantinou

first: Marc-Henri

last: Stern

first: Nathalie

last: Droin

first: Benoit

last: Palancade

last: Miotto

first: Frédéric

last: Chédin

first: Michaela

last: Fontenay


title: Accelerated DNA replication fork speed due to Loss of R-Loops in Myelodysplastic Syndromes with SF3B1 Mutation [RNAseq_CMNhuman]

first: David

last: Rombaut

overall_design: Comparative gene and splicing expression analysis of RNA-seq data for bone marrow mononuclear cells, SF3B1 MUT vs WT.

first: Carine

last: Lefèvre

first: Tony

last: Rached

first: Sabrina

last: Bondu

first: Anne

last: Letessier

first: Raphael

last: Mangione

first: Batoul

last: Farhat

first: Auriane

last: Lesieur-Pasquier

first: Daisy

last: Castillo-Guzman

first: Ismael

last: Boussaid

first: Chloé

last: Friedrich

first: Aurore

last: Tourville

first: Magali

last: de Carvalho

first: Françoise

last: Levavasseur

first: Marjorie

last: Leduc

first: Morgane

last: Le Gall

first: Sarah

last: Battault

first: Marie

last: Temple

first: Alexandre

last: Houy

first: Didier

last: Bouscary

first: Lise

last: Willems

last: Park

last: Raynaud

last: Cluzeau


title: Accelerated DNA replication fork speed due to Loss of R-Loops in Myelodysplastic Syndromes with SF3B1 Mutation

first: David

last: Rombaut

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Accelerated DNA replication fork speed due to Loss of R-Loops in Myelodysplastic Syndromes with SF3B1 Mutation [RNAseq_G1EER]

overall_design: Comparative gene and splicing expression analysis of RNA-seq data for G1E-ER4 cells, SF3B1 MUT (K700E, CRISPR-Cas9 genererated) vs WT, estradiol treated or not.

first: Carine

last: Lefèvre

first: Tony

last: Rached

first: Sabrina

last: Bondu

first: Anne

last: Letessier

first: Raphael

last: Mangione

last: Farhat

last: Lesieur-Pasquier

last: Castillo-Guzman

first: Sophie

first: Thomas

first: Emmanuelle

last: Clappier

first: Pierre

last: Fenaux

first: Lionel

last: Adès

last: Margueron

first: Michel

last: Wassef

first: Samar

last: Alsafadi

first: Nicolas

last: Chapuis

first: Olivier

last: Kosmider

first: Eric

last: Solary

first: Angelos

last: Constantinou

first: Marc-Henri

last: Stern

first: Nathalie

last: Droin

first: Benoit

last: Palancade

last: Miotto

first: Frédéric

last: Chédin

first: Michaela

last: Fontenay


title: Accelerated DNA replication fork speed due to Loss of R-Loops in Myelodysplastic Syndromes with SF3B1 Mutation [DRIP-seq]

first: David

last: Rombaut

summary: Myelodysplastic syndromes (MDS) with mutated SF3B1 gene have many features including a favorable outcome that are distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases of these divergences are poorly understood. Here we show that SF3B1-mutated MDS are characterized by a dramatically reduced R-loop formation predominating in gene bodies, which tightly associates with reduced retention of introns specifically found in SF3B1-mutated, but not in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibited augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation, which were recapitulated in murine Sf3b1K700E/+ proerythroblasts. Importantly, R-loop formation was restored by histone deacetylase inhibition using SAHA/vorinostat, which improved Sf3b1K700E/+ erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress is a hallmark of SF3B1- mutated MDS ineffective erythropoiesis, which could be used as a new therapeutic target.

overall_design: Comparative Rloop analysis with DRIP-seq data for CD34+-derived proE/basoE cells, with or without HDACi treatment. Please note that not all samples have been treated with HDACi. Therefore, in fig3A, all SF3B1 WT and all SF3B1 MUT samples have been used, whereas in fig6A, samples labelled WT4, 5 and 6, and MUT3, 4 and 5 were used.

first: Carine

last: Lefèvre

first: Tony

last: Rached

first: Sabrina

last: Bondu

first: Anne

last: Letessier

first: Raphael

last: Mangione

first: Batoul

last: Farhat

first: Auriane

last: Lesieur-Pasquier

first: Daisy

last: Castillo-Guzman

first: Ismael

last: Boussaid

first: Chloé

last: Friedrich

first: Aurore

last: Tourville

first: Magali

last: de Carvalho

first: Françoise

last: Levavasseur

first: Marjorie

last: Leduc

first: Morgane

last: Le Gall

first: Sarah

last: Battault

first: Marie

last: Temple

first: Alexandre

last: Houy

first: Didier

last: Bouscary

first: Lise

last: Willems

last: Park

last: Raynaud

last: Cluzeau


title: Cancer cell genetics shaping of the tumor microenvironment reveals myeloid cell-centric exploitable vulnerabilities in hepatocellular carcinoma.

first: Masami

last: Ando-Kuri


title: Effect of replacing soybean meal with Hermetia illucens meal on cecal microbiome, liver transcriptome and plasma metabolome of broilers

status: Public on Feb 21 2024

city: Gießen

first: Robert

last: Ringseis

address: Heinrich-Buff-Ring 26-32

department: Institute of Animal Nutrition and Nutrition Physiology

institute: JLU Gießen

summary: Despite the existence of a number of studies investigating the effect of insect meal on the growth performance of broilers, knowledge about the metabolic effects of insect meal in broilers is still scarce. Thus, the present study investigated the effect of partial replacement of soybean meal with Hermetia illucens (HI) larvae meal on the liver transcriptome, the plasma metabolome and the cecal microbiome in broilers. For the study, 72 male one-day-old Cobb 500 broilers were divided into three groups (n = 12) and fed three different diets with either 0% (HI0), 7.5% (HI7.5) or 15% (HI15) defatted HI meal for 35 d. While body weight (BW) gain, feed intake, and feed:gain ratio did not differ between groups, breast muscle weight, carcass yield and apparent ileal digestibility (AID) of 12 amino acids were higher in group HI15 than in group HI0 (P > 0.05). Indicators of α-diversity (Chao1 and Observed) in the cecal digesta were higher in groups HI15 and HI7.5 than in group HI0 (P < 0.05). The abundance of 5 families and 18 genera, all of which belonged to the Firmicutes phylum, in the cecal digesta differed among groups (P < 0.05). Concentrations of butyric acid, valeric acid and isobutyric acid in the cecal digesta were lower in group HI15 than in the other two groups (P > 0.05), whereas those of total and other short-chain fatty acids were not different between groups. Liver transcriptomics revealed a total of 70 and 61 differentially expressed transcripts between groups HI15 vs. HI0 and between groups HI7.5 vs HI0, respectively, (P > 0.05). Targeted metabolomics identified 138 metabolites, most of which were triglyceride species, being different between the three groups (FDR < 0.05). According to this study, dietary inclusion of HI larvae meal has no detrimental impact but increases breast muscle weight and carcass weight in broilers suggesting that HI larvae meal can be recommended as a sustainable alternative protein source for broilers.

first: Simone

last: Beller

first: Klaus

last: Eder


title: Cancer cell genetics shaping of the tumor microenvironment reveals myeloid cell-centric exploitable vulnerabilities in hepatocellular carcinoma [scRNA-Seq]

summary: Hepatocellular carcinoma (HCC) is a highly heterogenous disease associated with an equally dynamic tumor microenvironment (TME).  We generated somatic HCC mouse models bearing clinically-relevant oncogenic driver combinations that faithfully recapitulated different human HCC subclasses. Using scRNA-seq data, we explored changes in the TME to characterize phenotypic differences between immune populations.

overall_design: scRNA-seq data from a NrasG12DPtenKO HCC and a control liver. 1 biological replicate for each sample.

first: Christel

last: Ramirez

first: Daniel

last: Taranto

first: Marnix

middle: H

last: de Groot

first: Serena

last: Vegna


title: Exploration of Mechanisms of DrugResistance in a Microfluidic Device andPatient Tissues

status: Public on Feb 24 2024

city: Suwon

first: Inwoo

last: Hwang

address: 2066, SEOBU-RO, JANGAN-GU

department: School of Medicine

country: South Korea

institute: Sungkyunkwan University

summary: Chemoresistance is a major cause of treatment failure in many cancers. However, the lifecycle of cancer cells as they respond to and survive environmental and therapeutic stress isunderstudied. In this study, we utilized a microfluidic device to induce the development ofdoxorubicin-resistant (DOXR) cells from triple negative breast cancer (TNBC) cells within 11days by generating gradients of DOX and medium. In vivo chemoresistant xenograft models,an unbiased genome-wide transcriptome analysis, and a patient data/tissue analysis allshowed that chemoresistance arose from failed epigenetic control of the nuclear protein-1(NUPR1)/histone deacetylase 11 (HDAC11) axis, and high Nupr1 expression correlated withpoor clinical outcomes. These results suggest that the chip can rapidly induce resistant cellsthat increase tumor heterogeneity and chemoresistance, highlighting the need for furtherstudies on the epigenetic control of the NUPR1/HDAC11 axis in TNBC.

overall_design: A total of 1 ×10^5 cells/10 μL was suspended in culture medium, and 1 μL of the solution was gently added to thechip using a pipette with a tip through the cell seeding hole. The hole was plugged with a sterilizedstainless pin, and the chip was incubated at 37 ℃ with 5% CO2 overnight. The next day, 250 μL ofculture medium and culture medium containing 1.5 μM DOX were added to two of the diagonalreservoirs, and 50 μL of culture medium was added to the rest of the diagonal reservoirs. Thefresh culture medium and drug were replaced every day. After 11 days, trypsin (Gibco) was addedto the chip, which was incubated at 37 ℃ for 5 min. The detached cells were flushed out of thechip and collected from the reservoirs by injecting 1 mL of culture medium through the seedinghole with a needle-free syringe. To remove non-resistant cells, the collected cells were grown inmedium containing 0.05 μM DOX for one week

first: Wanyoung

last: Lim

first: Jiande

last: Zhang

first: Zhenzhong

last: Chen

first: Jeonghun

last: Han

first: Jaehyung

last: Jeon

first: Bon-Kyoung

last: Koo

first: Sangmin

last: Kim

first: JeongEon

last: Lee

first: Kenneth

last: Pienta

last: Amend

last: Austin

first: Jee-Yin

last: Ahn

first: Sungsu


title: Rational derivation of midbrain dopamine neuron progenitors from hPSCs using a selective FZD5 antibody agonist

city: Toronto

first: De Hua Andy

last: Yang

state: Ontario

address: 160 College St

department: CCBR

institute: University of Toronto

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


title: Rational derivation of midbrain dopamine neuron progenitors from hPSCs using a selective FZD5 antibody agonist [RNA-Seq]

summary: Current ventral midbrain dopaminergic progenitor differentiation protocols utilize small molecule inhibitors targeting Glycogen synthase kinase-3 (GSK3) to activate Wnt signaling, a step required for the anterior-posterior patterning of the nervous system and acquisition of the midbrain fate. However, GSK3a and GSK3β are pleiotropic kinases involved in multiple signaling pathways and their inhibition is a known trigger of neurogenesis. We predicted that direct activation of specific Wnt receptors naturally involved during neural patterning would allow for a more precise spatiotemporal control of Wnt/β-catenin signaling and mimic endogenous cell-cell communication mechanisms to improve hPSC differentiation. Here, we characterized the expression of FZD receptors at the surface of neural progenitor cells with different regional identity. Our data shows that FZD5 expression is uniquely upregulated in anterior neural progenitors and is rapidly downregulated as cells adopt a posterior fate. This spatial regulation of Frizzled cell surface expression constitutes a novel regulatory mechanism adjusting the levels of β-catenin signaling along the anterior-posterior axis and possibly contribute to midbrain-hindbrain boundary formation. Using a tetravalent antibody that selectively triggers FZD5 and LRP6 clustering to activate Wnt/β-catenin signaling, we show that the resulting midbrain-patterned neural progenitors exhibit a gene expression program more closely aligned with the anatomical origin of dopaminergic neurons and could hence represent a more efficient source for cell transplant therapies.

overall_design: Bulk RNA-Seq was performed in differentiated VM progenitors from hPSC patterned with F5L6.13 and CHIR99021 treatments at day 16, 3 replicates each, and compared to the control H1 hESC, 2 replicates

first: Andy

first: Stephane

last: Angers


title: Rational derivation of midbrain dopamine neuron progenitors from hPSCs using a selective FZD5 antibody agonist [scRNA-Seq]

overall_design: Differentiated VM progenitors with CHIR or F5L6.13 from hPSC were subjected to single cell RNA sequencing using 10X CellPlex at day 11 and at day 30.


title: Gene expression profile at single cell level of human neural stem cells (hNSCs) at 35degC and 37degC treatment.

status: Public on Feb 20 2024

city: Shanghai

first: Yuxiao

last: Ma

address: No. 1630 Pujian Road

institute: Renji Hospital, School of Medicine, Shanghai Jiao Tong University

summary: In order to elucidate why mild hypothermia of 35degC could promote neurogenesis of cultured hNSCs, we performed single-cell RNA sequencing (scRNA-seq) to reveal the molecular mechanism.

overall_design: hNSCs cultured under differentiation condition at 35degC and 37degC were collected to perform scRNA sequence.

first: Zhenghui

last: He

first: Jiangchang

last: Wang

first: Ping

last: Zheng

first: Zixuan

first: Qian

last: Liang

first: Qiao

first: Xiongfei

last: Zhao

first: Jialin

last: Huang

first: Weiji

last: Weng

first: Jiyao

last: Jiang

first: Junfeng

last: Feng


title: Dissecting the spatiotemporal diversity of adult neural stem cells

city: Berlin

first: Nina

last: Mitic

address: Hannoversche Strasse 28

department: Berlin Institute for Medical Systems Biology

institute: Max-Delbrück-Center for Molecular Medicine

summary: Adult stem cells are important for tissue turnover and regeneration. However, in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here, we dissected the diversity of neural stem cells in the adult zebrafish brain, an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single-cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells, with some subtypes being restricted to a single brain region, while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation, with different states being involved in proliferative and non-proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for functional manipulation of neural stem cell subtypes.

overall_design: Single cell RNA-seq of adult zebrafish brain performed using 10x Genomics Gene Expression protocols to explore transcriptomic diversity of adult cell types. The samples were prepared either from whole brain, FACS-sorted cells (GFP positive cells from gfap:GFP line) or a dissected single brain region, as indicated in the sample title. CRISPR-based lineage tracing was performed for a subset of samples to dissect developmental lineage relationships between cells. ScSLAM-seq was performed in combination with Notch pathway inhibition to assess response of stem cells to perturbation.


title: MRNAs bound to RBM3 of hNSCs under differentiation conditions at 35℃ and 37℃.

summary: To isolate RNA molecules bound to RBM3, we performed CLIP-seq analysis using differentiated hNSCs cultured in neural differentiation medium at 35℃ and 37℃.

overall_design: hNSCs cultured under differentiation conditions at 35℃and 37℃ were collected to perform CLIP-seq. 37℃ was designed as control. No replicates were included

last: Zhang


title: Distinct responses of patient-derived GBM cell lines to hypoxia

city: New York

first: Aarthi

last: Ramakrishnan

state: New York

address: 1425 Madison Avenue

department: Neuroscience

institute: Icahn School of Medicine at Mount Sinai

summary: Glioblastoma (GBM) is the most common primary malignant cancer of the central nervous system. Hypoxia, low oxygen, has been linked to GBM infiltration and aggressive tumor progression leading to poor patient outcome. Hypoxia-induced pathways are activated by the stabilization of HIF1-α, which upregulates genes related to reprogramming metabolism, angiogenesis, cell trafficking, and cell survival. However, underlying mechanisms of HIF-1α involvement with GBM cell specific pathways is not fully understood. Here, we show that hypoxia can induce specific genes dependent on GBM subtype. In a in vitro migration assay, we screened eight patient-derived GBM stem cell lines. Two lines, UCA and UNB, were found to induce an increased migration ability in response to hypoxia. By RNA bulk sequencing, we identified key specific genes that were upregulated in each cell line that were not shared between them. Furthermore, we found that this data set correlates to previous single-cell RNA sequencing findings of GBM having differentiated transcriptional subtypes. Our results demonstrate how hypoxia induces a specific response in tumor cells by activating genes for survival and tumor progression. Our study forms the basis for future approaches to characterize top candidate genes and elucidate their role in tumor survival in response to hypoxia. Inhibition of these top candidate genes can be a strategy used to dampen hypoxia pro-tumor properties and may lead to better outcomes for GBM patients.

overall_design: 3 replicates of UCA vs. 3 replicates of UNB exposed to either normoxia (UCA_NORM and UNB_NORM) or hypoxia (UCA_HYPO and UNB_HYPO) for 72h before extracting RNA for RNA bulk.

first: Valerie

last: Marallano

first: Rut

last: Tejero

first: Rohana

last: Ramalingam

first: Lauren

last: Stalbow

first: Anirudh

last: Sattiraju

first: Yong

first: Shen

last: Li

first: Mary

middle: E

last: Ughetta

first: Alexander

last: Tsankov

first: Santosh

last: Kesari

first: Alexandre

last: Wojcinski

first: Hongyan

last: Zou

first: Roland

last: Friedel


title: SOX17/ETV2 Improves the Direct Reprogramming of Adult Fibroblasts to Endothelial Cells

city: Boston

last: Grath

state: MA

address: 360 Huntington Ave

department: Bioengineering

institute: Northeastern University

summary: In this study, we directly reprogram adult human dermal fibroblasts (NHDF) into reprogrammed ECs (rECs) by overexpressing SOX17 in conjunction with ETV2. The rECs are capable of emulating in vitro and in vivo EC functions better than cells reprogrammed with ETV2 alone, such as improved reprogramming efficiency, enriched in more EC genes, and form large blood vessels carrying blood from the host, and most importantly, start to express eNOS in vivo. From these results, we present an improved method to reprogram adult fibroblasts into functional ECs and posit ideas for the future that could potentially further improve the reprogramming process.

overall_design: NHDF were transduced with an ETV2 lentivirus (ETV2-ECs) or both the ETV2 and SOX17 lentivirus (rECs) to observe the effects of SOX17 on the fibroblast to EC transdifferentiation process. Each sample has biological triplicates. NHDF served as a negative control, while HUVEC served as a positive control.

first: Guohao

last: Dai


title: Dupilumab-associated head and neck dermatitis shows a pronounced type 22 immune signature mediated by oligoclonally expanded T cells

city: Vienna

first: Natalia

last: Alkon

address: Währinger Gürtel 18-20

department: Department of Dermatology

country: Austria

institute: Medical University of Vienna

summary:
Background: Dupilumab, an IL4R-blocking antibody, has shown clinical efficacy for atopic dermatitis (AD) treatment. In addition to conjunctivitis/blepharitis, the _de novo_ appearance of head/neck dermatitis is now recognized as a distinct side effect, occurring in up to 10% of patients. Histopathological features distinct from AD suggest a drug effect, but exact underlying mechanisms remain unknown.
Methods: We profiled punch biopsies from dupilumab-associated head and neck dermatitis (DAHND) by using single-cell RNA sequencing and compared data with untreated AD and healthy control skin.
Results: We show that dupilumab treatment was accompanied by normalization of IL-4/IL-13 downstream activity markers such as _CCL13, CCL17_, _CCL18 _and_ CCL26_. By contrast, we found strong increases in type 22-associated markers (_IL22, AHR_) especially in oligoclonally expanded T cells, accompanied by enhanced keratinocyte activation and IL-22 receptor upregulation.
Conclusions: Taken together, we demonstrate that dupilumab effectively dampens conventional type 2 inflammation in DAHND lesions, with concomitant hyperactivation of _IL22_-associated responses.

overall_design:
Comparison of skin cells obtained from DAHND, Head/neck AD, Trunk AD and HC donors.
Submitter declares "Due to patient privacy concerns raw data is not provided."

first: Christine

last: Bangert*

last: Alkon*

first: Sumanth

last: Chennareddy

first: Tamara

last: Arnoldner

first: Jasmine

last: Levine

first: Magdalena

last: Pilz

first: Marco

last: Medjimorec

first: John

last: Ruggiero

first: E

last: Cohenour

first: Constanze

last: Jonak

first: William

last: Damsky

first: Johannes

last: Griss

first: Patrick

last: Brunner


title: Effect of constitutive AHR activation in skeletal muscle [AHRmKO_RNAseq]

city: Gainesville

first: Terence

middle: E.

last: Ryan

state: Florida

address: 1864 Stadium Rd

department: Applied Physiology and Kinesiology

institute: University of Florida

summary: To investigate the RNA differences in skeletal muscle in muscle-specific AHR (aryl hydrocarbon receptor) knockout mice and their AHR-floxed littermate controls (wildtype) following a 16-week chronic cigarette smoke exposure intervention

overall_design: Male AHR-floxed (wildtype) and muscle-specific AHR knockout (AHRmKO) mice were subjected to daily cigarette smoke exposures for 16 weeks. RNA was harvested from the gastrocnemius muscle 72 hours following the final smoke exposure. RNA sequencing was performed to explored transcriptional changes.

first: Ryan

last: Terence


title: Tissue-specific responses to DNA damage limit function and regulate ageing in progeroid mice

city: Cambridge

first: Alastair

last: Crisp

address: Francis Crick Avenue

country: United Kingdom

institute: MRC Laboratory of Molecular Biology

summary: DNA repair deficiency can lead to segmental phenotypes in humans and mice, in which certain tissues lose homeostasis while others remain seemingly unaffected. This may be due to different tissues facing varying levels of damage or having different reliance on specific DNA repair pathways. However, we find that the cellular response to DNA damage determines different tissue-specific outcomes. Here, we use a mouse model of the human XPF-ERCC1 progeroid syndrome (XFE) caused by loss of DNA repair. We find that p53, a central regulator of the cellular response to DNA damage, regulates tissue dysfunction in Ercc1-/- mice in different ways. We show that ablation of p53 rescues the loss of hematopoietic stem cells, and has no effect on kidney, germ cell or brain dysfunction, but exacerbates liver pathology and polyploidisation. Mechanistically, we find that p53 ablation led to the loss of cell-cycle regulation in the liver, with reduced p21 expression. Eventually, p16/Cdkn2a expression is induced, serving as a fail-safe brake to proliferation in the absence of the p53-p21 axis. Taken together, our data show that distinct and tissue-specific functions of p53, in response to DNA damage, play a crucial role in regulating tissue-specific phenotypes.

overall_design: RNAseq analysis on HSCs, PGCs and hepatocytes from C57BL/6 x 129S4S6/Sv F1 background mice with knockouts for p53, ERCC1, p53+ERCC1 and Fanca

first: Ross

last: Hill

first: Nazereno

last: Bona

first: Gerry

last: Crossan


title: Activation of endogenous retroviruses and induction of viral mimicry by MEK1/2 inhibition in pancreatic cancer

status: Public on Feb 19 2024

city: Milan

first: Serena

last: Ghisletti

address: Via Adamello 16

department: Department of Experimental Oncology

country: Italy

institute: European Institute of Oncology


title: Activation of endogenous retroviruses and induction of viral mimicry by MEK1/2 inhibition in pancreatic cancer [ChIP-seq]

summary: While pancreatic ductal adenocarcinomas (PDAC) are addicted to KRAS-activating mutations, inhibitors of downstream effectors in the KRAS pathway, such as the clinically approved MEK1/2 kinases inhibitor Trametinib, are devoid of significant therapeutic effects. Nevertheless, the extensive rewiring of regulatory circuits driven by the attenuation of the KRAS pathway can induce novel functional states and vulnerabilities of potential therapeutic relevance. An in-depth molecular analysis of the transcriptional and epigenomic alterations occurring in PDAC cells in the initial hours after MEK1/2 inhibition by Trametinib, unveiled a massive induction of a large number of retroelements, in particular endogenous retroviruses (ERVs), that in these cells escaped epigenetic silencing. In turn, the increased abundance of ERV-derived double stranded RNAs induced a strong Interferon (IFN) response. Pathway deconvolution allowed us to track ERV activation to the early induction of the transcription factor ELF3, which extensively bound and activated non-silenced retroelements and synergized with IRF1 (Interferon regulatory factor 1) in the activation of interferons and IFN-stimulated genes. Trametinib-induced viral mimicry in PDAC may be exploited in the design of combination therapies in immuno-oncology.

overall_design:
To determine the enrichment of retrotransposon sequences in differentially acetylated regions, we generated H3K27ac ChIP-seq data sets in two low-grade (CAPAN2 and CFPAC1) and two high-grade (MiaPaca2 and PANC1) PDAC cell lines that were sampled in the untreated condition (UT) and at multiple time points (3h, 12h and 24h) after Trametinib treatment.
To quantify the genomic locus-specific levels of H3K9me3 after Trametinib treatment, we generated H3K9me3 ChIP-seq data sets in CFPAC1 cells that were sampled in the untreated condition (UT) and at multiple time points (3h, 12h, 24h and 48h) after Trametinib treatment.
To assess whether ELF3 binds ERVs and other retroelements induced by Trametinib, we generated ELF3 ChIP-seq data sets in untreated and Trametinib-treated (24h) CFPAC1 cells.

first: Alice

last: Cortesi

first: Francesco

last: Gandolfi

first: Fabiana

last: Arco

first: Emanuele

last: Valli

first: Sara

last: Polletti

first: Roberta

last: Noberini

last: Gualdrini

first: Sergio

last: Attanasio

first: Francesca

last: Citron

first: Simona

last: Rodighiero

first: Nico

last: Mitro

first: Tiziana

last: Bonaldi

first: Andrea

last: Viale

first: Giuseppe

last: Diaferia

first: Gioacchino

last: Natoli


title: Single cell RNA-seq analysis for spleen tissue under steady state, phenohydrazine (PHZ) and bleeding treatment.

city: Singapore

first: Rui

last: Yokomori

address: 14 Medical Drive

country: Singapore

institute: Cancer Science Institute of Singapore, National University of Singapore

summary:
Single cell RNA-seq analysis for spleen tissue isolated from mice under steady state, and subjected to phenohydrazine (PHZ) and bleeding treatment respectively.
Single cell RNA-seq analysis for bone marrow cells isolated from mice under steady state.

overall_design:
Mice were subjected to PHZ and bleeding induced acute anemic stresses, live cells from spleen under each condition were isolated for scRNA-seq.
Live cells isolated from fresh murine bone marrow were isolated for scRNA-seq.

first: Yang

last: Chong

first: Takaomi

last: Sanda

first: Toshio

last: Suda


title: Cancer cell genetics shaping of the tumor microenvironment reveals myeloid cell-centric exploitable vulnerabilities in hepatocellular carcinoma [RNA-Seq]

first: Masami

last: Ando-Kuri

summary: Hepatocellular carcinoma (HCC) is a highly heterogenous disease associated with an equally dynamic tumor microenvironment (TME).  We generated somatic HCC mouse models bearing clinically-relevant oncogenic driver combinations that faithfully recapitulated different human HCC subclasses. Using RNA-seq data, we explored changes in the TME and dissected cancer cell intrinsic shapers of the TME.

overall_design: mRNA profiles of bulk tumors, CD45+ cells and cancer cell lines from  genetically-distinct HCC mouse models. 3-4 biological replicates.

first: Christel

last: Ramirez

first: Daniel

last: Taranto

first: Marnix

last: de Groot

first: Serena

last: Vegna


title: Primate-expressed EPIREGULIN promotes basal progenitor proliferation in the developing neocortex

city: Dresden

first: Mareike

last: Albert

address: Fetscherstr. 105

department: Center for Regenerative Therapies TU Dresden

institute: TUD Dresden University of Technology

summary: Neocortex expansion during evolution is linked to higher numbers of neurons thought to result from increased proliferative capacity and neurogenic potential of basal progenitors during development. Here, we show that EREG, encoding the growth factor EPIREGULIN, is expressed in the human developing neocortex, but not in the mouse neocortex. Addition of EPIREGULIN to the mouse neocortex increases proliferation of both major basal progenitor types, intermediate basal progenitors and basal radial glia, whereas ablation of EPIREGULIN in human cortical organoids reduces basal progenitor proliferation. Here, we analyzed gene expression changes upon addition of EPIREGULIN to the mouse neocortex for 24 hours using hemisphere rotation culture. We performed fluorescent activated cell sorting to isolate radial glia (RG), intermediate progenitor (IP) cells and neurons (N) based on the nuclear markers Sox2 and Tbr2, and expression of GFP in neurons isolated from a Tubb3::GFP mouse reporter line.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for mouse neocortex treated without (Ctl) or with 50 ng/mL of EPIREGULIN (Epi) for 24 hours followed by isolation of radial glia (RG), intermediate progenitor (IP) cells and neurons (N).

first: Paula

last: Cubillos

first: Theresa

last: Schütze


title: PD-1 restrains thymic IL-2 production and regulatory T cell development

city: Portland

first: Amy

last: Moran

state: Oregon

address: 3181 SW Sam Jackson Park Rd, Mail code KR-CDCB

department: Department of Cell, Developmental & Cancer Biology

institute: Oregon Health & Science University

summary: Inhibitory proteins, such as programmed cell death protein 1 (PD-1), have been extensively studied in peripheral T cell responses to foreign, self, and neoantigens. Notably, these proteins are first expressed during T cell development in the thymus. Reports suggest that PD-1 limits regulatory T cell (Treg) development, but the mechanism by which PD-1 exerts this function remains unknown. The present study expands the evaluation of PD-1 and its ligands in the thymus, demonstrating that some of the highest expressers of PD-1 and PD-L1 are agonist selected cells. Surprisingly, we reveal a selective role for PD-1 in regulating the developmental niche only for Tregs as other agonist selected cell populations, such as natural killer T cells, remain unchanged. We also ruled out PD-1 as a regulator of proliferation or cell death of agonist selected Tregs and further demonstrated that PD-1 deficient Tregs have reduced TCR signaling. Unexpectedly, the data suggests that PD-1 deficient thymocytes produce elevated levels of IL-2, a Treg niche limiting cytokine. Collectively, these data suggest a novel role for PD-1 in regulating IL-2 production and the concurrent agonist selection of thymic Tregs. This observation has implications for the use of checkpoint blockade in the context of cancer and infection.

overall_design: Thymus from 3-week-old WT or PD-1 knockout animals (C57BL/6J) were processed for flourescence-activated cell sorting (FACS) to sort CD4+ HSA+ CD73- CD25- (CD4 single positive thymocytes) and CD4+ HSA+ CD73- CD25+ (regulatory T cells and CD25+ regulatory T cell progenitors). We sought to examine differences between the WT and PD-1 knockout cells using scRNAseq.

first: Breanna

last: Caruso

first: Benjamin

last: Weeder

first: Reid

last: Thompson


title: Activation of endogenous retroviruses and induction of viral mimicry by MEK1/2 inhibition in pancreatic cancer [RNA-seq]

last: Ghisletti

overall_design:
To explore the transcriptional impact of Trametinib in PDAC, we generated RNA-seq data sets in two low-grade (CAPAN2 and CFPAC1) and two high-grade (MiaPaca2 and PANC1) PDAC cell lines that were sampled in the untreated condition (UT) and at multiple time points (3h, 12h and 24h) after Trametinib treatment.
To investigate the role of ELF3 induction by Trametinib in the transcriptional activation of retroelements and in the activation of the IFN response, we generated RNA-seq data set of ELF3-Knockout and Wild-Type CFPAC1 cells in both untreated and treated condition (24h) after Trametinib treatment.

first: Alice

last: Cortesi

first: Francesco

last: Gandolfi

first: Fabiana

last: Arco

first: Emanuele

last: Valli

first: Sara

last: Polletti

first: Roberta

last: Noberini

last: Gualdrini

first: Sergio

last: Attanasio

first: Francesca

last: Citron

first: Simona

last: Rodighiero

first: Nico

last: Mitro

first: Tiziana

last: Bonaldi

first: Andrea

last: Viale

first: Giuseppe

last: Diaferia

first: Gioacchino

last: Natoli


title: Cooperative super-enhancer inactivation caused by heterozygous loss of CREBBP and KMT2D skews B-cell fate decisions and accelerates development of T-cell depleted lymphomas (scRNA-Seq)

city: New York City

first: Christopher

middle: Russell

last: Chin

address: 413 E 69th Street, Belfer Building, BB-1462

institute: Weill Cornell

summary: Mutations in chromatin modifiers are a hallmark of many tumors, especially lymphomas arising from germinal center (GC) B cells. Given that these lymphoma mutations all induce aberrant gene repression, it is surprising that they often co-occur in individual patients. The most common pairing are mutations affecting CREBBP and KMT2D. Both impair enhancer activity in overlapping pathways to facilitate lymphomagenesis, hence their co-occurrence is especially puzzling. Herein, we report that combined haploinsufficiency of CREBBP and KMT2D (C+K) do indeed accelerate lymphomagenesis (vs either allele alone) and confer a more malignant phenotype.  Single cell RNA-seq analysis of GC reaction showed that C+K haploinsufficiency induced aberrant GC hyperplasia by altering cell fate decisions, skewing B cells away from memory B and plasma cell differentiation and favored instead expansion of centroblasts.  Integrative epigenomic studies in murine and human B cells showed that C+K deficiency particularly impairs enhancer activation for immune synapse genes involved in exiting the GC reaction. This effect was especially severe at super-enhancers for genes governing cell fate decisions induced by T cell help, pointing to a particular dependency for both co-activators at these specialized regulatory elements. Mechanistically, CREBBP and KMT2D formed a complex, were highly co-localized on chromatin, and were required for each-other’s stable recruitment to enhancers.  Given the impaired expression of immune synapse genes, it was notable that C+K lymphomas in mice and humans manifested significantly reduced CD8+ T cell abundance. This suggests that deficiency of the two chromatin modifiers cooperatively induced an immune evasive phenotype due to failure to activate key immune synapse super-enhancers, associated with altered immune cell fate decisions. These findings point to the potential need for epigenetic adjuvant therapy to augment reactivity with immunotherapy approaches in patients with C+K deficiency.

overall_design: Single-cell RNA-seq profiling of mouse B220+IgD- splenic cells sorted from sheep red blood cell (SRBC)-immunized genetically engineered mouse models (GEMMs), which include Cg1Cre+/- (WT); Cg1Cre+/-, Crebbp fl/+ (C); Cg1Cre+/-, Kmt2d fl/+ (K); Cg1Cre+/-, Crebbp fl/+, Kmt2d fl/+ (CK); n = 1 mouse for each genotype.

first: Jie

last: Li

first: Wendy

last: Béguelin

first: Ari

last: Melnick


title: Full-length Sequencing of Non-capped RNAs Reveals the Heterogeneity of RNA Processing and Novel Classes of Noncoding RNAs

city: Guangzhou

first: Jian-Hua

last: Yang

address: No. 135, Xingang Xi Road

department: School of Life Sciences

institute: Sun Yat-sen University

summary:
Purpose:Up to 80% of the human genome gives rise to “dark matter” RNAs that mainly composes of non-capped RNAs (napRNAs). However, determining the functional impacts and metabolisms of the napRNAs requires identification of their full-length sequences.
Method:We developed an approach to globally profile the full-length sequences of napRNAs at single-nucleotide resolution
Results:We discovered, for the first time, the stable expressed linear intron RNA (sliRNA), a novel class of snoRNA-intron (snotron) RNAs, a new class of RNA embedded in miRNA spacers (misRNA) and thousands of new exceptional structured ncRNAs and repeat-derived ncRNAs (repeatRNA) in humans and mouse. Importantly, these new napRNAs are dynamic changes in response to various stimuli and biological processes, suggesting that they have potential regulatory functions. Furthermore, we show that functional napRNA FS4, discovered by NAP-seq firstly, promotes HepG2 cell proliferation by interacting with DKC1 to maintain the protein stability.
Conclusion:Collectively, our approach establishes a paradigm for mapping and annotating novel class of ncRNAs of many living species.

overall_design: Next generation sequencing of NAP-seq  was applicated in 6 human cell types, including U87, HEK293T, HepG2 (including 4 conditions: non-treated, poly(I:C)-treated, Adriamycin-treated, CoCl2-treated), and in  4 differentiation stages of mouse skeletal muscle cell C2C12, including 0 hour, 72 hours, 96 hours, 144 hours after differentiation. Third generation sequencing  of NAP-seq and normal RNA-seq was applicated in non-treated HepG2 and C2C12. NAP-seq combined with SHAPE-Map was applicated in non-treated HepG2. Each sample has three biological replicates (except for CoCl2-treated HepG2, which has two).

first: Jianhua

first: Shurong

last: Liu


title: Cooperative super-enhancer inactivation caused by heterozygous loss of CREBBP and KMT2D skews B-cell fate decisions and accelerates development of T-cell depleted lymphomas [ATAC-seq]

overall_design: ATAC-seq-based chromatin accessibility profiling for mouse GC B cells sorted from sheep red blood cell (SRBC)-immunized genetically engineered mouse models (GEMMs), which include Cg1Cre+/- (WT); Cg1Cre+/-, Crebbp fl/+ (C); Cg1Cre+/-, Kmt2d fl/+ (K); Cg1Cre+/-, Crebbp fl/+, Kmt2d fl/+ (CK); n = 4-5 mice for each genotype.


title: Cooperative super-enhancer inactivation caused by heterozygous loss of CREBBP and KMT2D skews B-cell fate decisions and accelerates development of T-cell depleted lymphomas (Cut&Run)

overall_design: Cleavage Under Targets & Release Using Nuclease (CUT&RUN) profiling of H3K4me1, H3K27ac, and H3K4me3 in isogenic human GCB-DLBCL OCI-Ly7 cell lines, which include WT, CREBBP-R1446C (C, HAT inactivating point mutation), KMT2D-KO (K), and CREBBP-R1446C + KMT2D-KO (CK); n = 2 biological replicates for each genotype.


title: Cooperative super-enhancer inactivation caused by heterozygous loss of CREBBP and KMT2D skews B-cell fate decisions and accelerates development of T-cell depleted lymphomas.

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

overall_design: Refer to individual Series


title: Cooperative super-enhancer inactivation caused by heterozygous loss of CREBBP and KMT2D skews B-cell fate decisions and accelerates development of T-cell depleted lymphomas [RNA-Seq]

overall_design: Comparative gene expression profiling analysis of RNA-seq data for 1) mouse centroblasts (CB) and centrocytes (CC) sorted from sheep red blood cell (SRBC)-immunized genetically engineered mouse models (GEMMs), which include Cg1Cre+/- (WT); Cg1Cre+/-, Crebbp fl/+ (C); Cg1Cre+/-, Kmt2d fl/+ (K); Cg1Cre+/-, Crebbp fl/+, Kmt2d fl/+ (CK); n = 3-4 mice for each genotype. 2) isogenic human GCB-DLBCL OCI-Ly7 cell lines, which include WT, CREBBP-R1446C (C, HAT inactivating point mutation), KMT2D-KO (K), and CREBBP-R1446C + KMT2D-KO (CK); n = 2 biological replicates for each genotype. 3) mouse GC B cells sorted from lymphomagenesis mice at day 235 post-BMT, which include VavP-BCL2+/- (BCL2); VavP-BCL2+/-, Cg1Cre+/-, Crebbp fl/+ (BCL2+C); VavP-BCL2+/-, Cg1Cre+/-, Kmt2d fl/+ (BCL2+K); VavP-BCL2+/-, Cg1Cre+/-, Crebbp fl/+, Kmt2d fl/+ (BCL2+CK); n = 4 mice for each genotype.


title: Transcriptomic landscape of the anterior thalamic nuclei of the mouse [Xenium]

status: Public on Feb 17 2024

city: Vancouver

first: Mark

middle: Steven

last: Cembrowski

state: BC

address: 2350 Health Sciences Mall

department: Cellular and Physiological Sciences

institute: University of British Columbia

summary: Here, we examined diversity in the cell-type composition of the mouse anterior thalamic nuclei (ATN) using single-cell RNA-seq and single-cell spatial transcriptomics.

overall_design: Examination of anterior thalamic nuclei neuron transcriptomes.


title: Real-time quantitative PCR analysis of ccRCC model rats III

city: Kashihara

first: Takuto

last: Shimizu

state: Nara

address: 840 Shijo-cho

department: Department of Urology

country: Japan

institute: Nara Medical University

summary: Carcinogen-induced ccRCC rat models of mRNA expression arrays were used to explore genes potentially associated with the destruction of pseudo capsule (PC).

overall_design: The mRNA was compared between the i-Cap 1 (contorol) group (n = 3) and i-Cap 2-3 (PC destruction) group (n = 3)

first: Kota

last: Iida

first: Makito

last: Miyake


title: Maternal dietary fat during lactation shapes single nucleus transcriptomic profile of postnatal offspring hypothalamus in a sexually dimorphic manner in mice

status: Public on Feb 18 2024

city: Shenzhen

first: John

middle: R.

last: Speakman

state: Guangdong

address: 1068 Xueyuan Avenue, Shenzhen University Town

institute: Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences

summary: Maternal overnutrition during pregnancy/or lactation predisposes offspring obesity and related metabolic syndromes in later life. Hypothalamus is the highest heterogeneous brain region located at the base of the brain that is critical to the regulation of the energy homeostasis.We investigated how the maternal programming shapes the single-nucleus transcriptomic landscapes in postnatal day15 offspring hypothalamus in a sex-specific manner.

overall_design: Hypothalamus of male and female postnatal day 15 offspring raised by mothers fed high fat and low fat diets during lacatation were dissected and processed with 10X snRNAseq

first: Yi

last: Huang

first: Guanlin

last: Wang


title: NAP-seq Reveals Novel Classes of Structured Noncoding RNAs with Regulatory Functions

summary:
Purpose:Up to 80% of the human genome produces “dark matter” RNAs, most of which are noncapped RNAs (napRNAs). However, determining the functional impacts and metabolism of napRNAs requires the identification of their full-length sequences.
Method:Here, by developing a novel method, NAP-seq, to globally profile the full-length sequences of napRNAs at single-nucleotide resolution, we revealed several novel classes of exceptionally structured noncoding RNAs (ncRNAs).
Results:We discovered stably expressed linear intron RNAs (sliRNAs), a novel class of snoRNA-intron RNAs (snotrons), a new class of RNAs embedded in miRNA spacers (misRNAs) and thousands of new structured ncRNAs in humans and mice. These new napRNAs undergo dynamic changes in response to various stimuli and differentiation stages. Importantly, we showed that the novel napRNA DINAP interacts with dyskerin pseudouridine synthase 1 (DKC1) to promote cell proliferation by maintaining DKC1 protein stability.
Conclusion:Our approach establishes a paradigm for discovering novel classes of ncRNAs with regulatory potency.

overall_design: Next generation sequencing was applicated in 6 human cell types, including U87, HEK293T, HepG2 (including 4 conditions: non-treated, poly(I:C)-treated, Adriamycin-treated, CoCl2-treated), and in  4 differentiation stages of mouse skeletal muscle cell C2C12, including 0 hour, 72 hours, 96 hours, 144 hours after differentiation. Third generation sequencing was applicated in non-treated HepG2. Each sample has three biological replicates (except for CoCl2-treated HepG2, which has two).


title: Real-time quantitative PCR analysis of ccRCC model rats


title: The expression of trefoil factor family member 2 in increased at an acidic pH

status: Public on Feb 16 2024

city: SASEBO

first: KYOKO

last: FUJIMOTO

state: NAGASAKI

address: 2825-7 HUIS TEN BOSCH

department: PHARMACY

institute: NAGASAKI INTERNATIONAL UNIVERSITY

summary: Trefoil factor family member 2 (Tff2) is significantly involved in intestinal tumor growth in ApcMin/+ mice, which can be used as a human colon cancer model. TFF2, which encodes TFF2 (spasmolytic protein 1) is highly expressed in human cancer tissues, including the pancreas, colon and bile ducts, as well as in normal gastric and duodenum tissues. By contrast, TFF2 exhibits low expression levels in other normal tissues, including the small and large intestine. Furthermore, TFF2 expression has not been detected in DLD-1 cells, a cell line derived from human colon cancer. The trigger of TFF2 expression in normal and tumor cells remains unknown. Highly malignant tumor tissues are characterized by higher temperatures and lower pH (6.2-6.9) than in normal tissues, where the normal pH ranges from 7.2 to 7.4. This microenvironment exacerbates malignancy by promoting the acquisition of cell death resistance, drug resistance and immune escape. Therefore, the present study examined how TFF2 expression is affected in cultured cells that imitate the tumor tissue microenvironment. The incubation temperature was increased from 37 to 40˚C, but no expression of TFF2 was induced. Subsequently, a culture solution with an acidic pH was prepared to simulate the Warburg effect in tumors. TFF2 expression was increased by 42.8- and 5.8-fold in cells cultured in acidic medium at pH 6.5 and 6.8 compared with at pH 7.4, respectively, as determined using the relative quantification method following quantitative polymerase chain reaction. The present study also analyzed fluctuations in the expression levels of genes other than TFF2, under acidic conditions. Acidic conditions upregulated the expression of genes related to cell membranes and glycoproteins, based on the Database for Annotation, Visualization, and Integrated Discovery. In conclusion, TFF2 was highly expressed under acidic conditions, implying that it may have an important function in protecting the plasma membrane from acidic environments in both normal and cancer cells. These findings warrant further investigation of TFF2 as a target of cancer therapy and diagnosis.

overall_design: The cells were incubated at 37℃ in a humidified atmosphere with 5 % CO2. DLD-1 was incubated for 48 hours with different pH of the culture supernatant as follows: pH 7.4 as control, pH 6.8 and pH 6.5. Opti-MEM medium was used to reduce protein denaturation, and HEPES was added for the acidic condition.

first: Yui

last: Masumoto

first: Suzuka

last: Matsuo

first: Natsuno

last: Kinjou

first: Yuka

last: Narieda

first: Morimasa

last: Wada

first: Kyoko

last: Fujimoto


title: In vitro aging alters the gene expression and secretome composition of canine adipose-derived mesenchymal stem cells

status: Public on Feb 15 2024

city: Zagreb

first: Marina

last: Prišlin

address: Savska cesta 148

department: Virology Department

country: Croatia

institute: Croatian Veterinary Institute

summary: Canine adipose-derived mesenchymal stem cells (cAD-MSCs) show therapeutic promise for their regenerative potential, particularly in their secretome. However, concerns arise regarding the impact of in vitro cultivation need for storing therapeutic doses, prompting this study to comprehensively explore the impact of in vitro aging on gene expression and secretome composition. The study involved collecting abdominal adipose tissue samples from nine healthy female dogs, from which cAD-MSCs were extracted and cultured. Stem cells were validated through trilineage differentiation assays and flow cytometry immunophenotyping. Gene expression profiling, using RT-qPCR array and cAD-MSCs secretome LC-MS/MS analysis, were conducted at passages 3 and 6 to reveal gene expression and protein composition alterations during in vitro culture. Gene expression profiling of in vitro aged cAD-MSCs revealed  expression alterations, while significant downregulation was observed in two MSC-associated genes. Proteomic analysis revealed 10% distinctively expressed proteins, and several up- and downregulations. Grouping these proteins with biologically significant ones, Gene Ontology Panther Pathway analysis revealed that P3 proteins were significantly associated with cytoskeletal regulation, nicotinic acetylcholine receptor signaling, inflammation mediated by chemokine and cytokine signaling, Wnt signaling, and CCKR signaling map. In contrast, P6 proteins were linked to the xanthine and guanine salvage pathway, adenine and hypoxanthine salvage pathway, and blood coagulation pathway. To the best of our knowledge, this study presents the first original perspective on the changes in secretome composition that occur when cAD-MSCs age in vitro. Our findings highlight significant changes that, in conclusion, indicate the regenerative potential of cAD-MSCs and that their secretome may be compromised due to in vitro aging. Consequently, our study suggests a preference for earlier passages when considering these cells for therapeutic applications.

overall_design: Nine biological replicates are studied in two different cell conditions, passage 3 and passage 6.

first: Dragan

last: Brnić


title: Real-time quantitative PCR analysis of ccRCC model rats II


title: Characterisation of  ILCs isolated from Mtb infected mice lungs

city: Chicago

first: Shabaana

last: Khader

state: Illinois

address: 920 East 58th Street

institute: The University of Chicago

summary: ILCs were sorted from the lungs of Mtb infected mice at 5 and 14 days post infection and subjected to sc-RNA sequencing to determine  gene signature profile

overall_design: 10 mice were pooled per sample per time point. Total 2 samples for 2 time points.

first: Shibali

last: Das


title: Ex vivo activation of the GCN2 pathway metabolically reprograms T cells leading to enhanced adoptive cell therapy

city: Toronto

first: Pamela

last: Ohashi

state: ON

address: 610 University Ave

department: Tumor Immunotherapy Program

institute: University Health Network

summary: The manipulation of T cell metabolism to enhance anti-tumor activity is an area of active investigation. Here, we report that activating the amino acid starvation response in effector CD8+ T cells ex vivo using the General Control Non-depressible 2 (GCN2) agonist halofuginone (halo) enhances oxidative metabolism and effector function. Further characterization revealed that halo-treated CD8+ T cells increased expression of the large neutral amino acid (LNAA) transporter CD98 as well as the co-stimulatory marker 4-1BB. Mechanistically, we identified autophagy coupled with the CD98-mTOR axis as key downstream mediators of the phenotype induced by halo treatment. The adoptive transfer of halo-treated CD8+ T cells into mice bearing well-established tumors led to robust tumor control and curative responses. Halo-treated T cells also synergized in vivo with a 4-1BB agonistic antibody to control tumor growth in a mouse model resistant to immunotherapy.  Importantly, treatment of human CD8+ T cells with halo resulted in similar metabolic and functional reprogramming. These findings demonstrate that activating the amino acid starvation response with the GCN2 agonist halofuginone can enhance T cell metabolism, effector function and anti-tumor activity, with the potential to augment existing clinical immunotherapeutic approaches.

overall_design: 12 CD8+ T-cell Samples profiled by RIBO-Seq: 3 Halofuginone (HALO)-treated samples, Ribosome-Protected RNA Fragments (RPF) profiled; 3 Control samples, RPF-profiled; 3 Halo-treated samples, total RNA profiled; 3 control samples, total RNA profiled.

first: St-Paul

last: Michael

first: Saibil

last: Samuel

first: Ohashi

last: Pamela

first: Sayad

last: Azin


title: Multimodal stimulation screens reveal unique and shared regulators limiting T-cell fitness [IntenseAcuteChronic_screen]

city: Amsterdam

first: Chun-Pu

last: Lin

address: Plesmanlaan 121

department: Molecular Oncology and Immunology

institute: NKI-AvL

summary: It has not been systematically studied whether T-cell fitness regulators uniquely or broadly contribute to specific traits limiting antitumor activity. We performed genome-scale CRISPR/Cas9 knockout screens in primary CD8 T-cells to uncover genes negatively impacting on their fitness upon three modes of stimulation: (1) intense stimulation, causing activation-induced cell death (AICD); (2) acute stimulation, triggering T-cell expansion; (3) chronic stimulation, resulting in dysfunction. Besides established regulators, we uncovered several genes controlling T-cell fitness either specifically or commonly under various stimulation modalities. Ablation of Dap5, ranking highly in all three screens, increased translation while enhancing tumor killing. Loss of Icam1-mediated homotypic T-cell clustering amplified T-cell expansion and effector functions after both acute and intense stimulation. Finally, inactivation of Ctbp1 induced T-cell persistence exclusively upon chronic stimulation. Our results functionally annotate T-cell fitness regulators based on their unique or shared contribution to traits limiting T-cell antitumor activity.

overall_design: Naive OT-I/Cas9 T-cells were primed using CD3 and CD28 antibody and transduced with the genome-wide Brie sgRNA library. Day 1 after puromycin selection we harvested a library reference sample (t0). After 6d puro-selection, we harvested a Pre-reactivation reference sample. For different stimulation screens, T-cells were stimulated under three different conditions: (1) Intense: T-cells were stimulated with plate-bound CD3 antibody for two times in two days. (2) Acute: T-cells were stimulated once with CD3 antibody for 24h. Cells were then removed from the plates and cultured for another 3d before harvesting. (3) Chronic: T-cells were co-cultured daily with fresh D4M.OVA tumor cells for 11d (11 times). For the resting condition, cells were refreshed daily without adding tumor cells. Both stimulated and resting cells were harvested for analysis after 11 days (11 times) tumor stimulation. Genomic DNA was isolated, sgRNAs were amplified using a one-step barcoding PCR.

first: Pierre

last: Levy

first: Georgi

last: Apriamashvili

first: Joleen

last: Traets

first: Austin

last: George

first: Daniel

last: Peeper


title: B-cell Intrinsic Regulation of Antibody Mediated Immunity by Histone H2A Deubiquitinase BAP1 [RNA-seq]

city: Montreal

first: Anastasia

last: Nijnik

state: Quebec

address: Rm 368 - 3649 Promenade Sir William Osler

department: Physiology

institute: McGill University

summary: BAP1 is a deubiquitinase (DUB) of the Ubiquitin C-terminal Hydrolase (UCH) family that regulates gene expression and other cellular processes, through its direct catalytic activity on the repressive epigenetic mark histone H2AK119ub, as well as several other substrates. BAP1 is also a highly important tumor suppressor, expressed and functional across many cell types and tissues. In recent work, we demonstrated a cell intrinsic role of BAP1 in the B cell lineage development in murine bone marrow, however the role of BAP1 in the regulation of B cell mediated humoral immune response has not been previously explored. In the current study, we demonstrate that a B-cell intrinsic loss of BAP1 function in activated B cells in the Bap1fl/fl Cγ1-cre murine model results in a severe defect in antibody production, with altered dynamics of germinal centre B cell, memory B cell, and plasma cell numbers. At the cellular and molecular level, BAP1 was dispensable for B cell immunoglobulin class switching but resulted in an impaired proliferation of activated B cells, with genome-wide dysregulation in histone H2AK119ub levels and gene expression. In summary, our study establishes the B-cell intrinsic role of BAP1 in antibody mediated immune response and indicates its central role in the regulation of the genome-wide landscapes of histone H2AK119ub and downstream transcriptional programs of B cell activation and humoral immunity.

overall_design: To further analyze the role of BAP1 in the transcriptional and epigenetic regulation of B cell activation, RNA-Seq gene expression analysis was conducted on germinal centre B cells isolated from the spleen of Bap1fl/fl Cγ1-cre and control Bap1+/+ Cγ1-cre mice at day 11-14 post-SRBC immunization, with the cells gated for FACS-sort.

first: Yue

last: Liang

first: Hanchen

last: Wang

first: Noé

last: Seija

first: Yun

first: Lin

middle: T

last: Tung

first: Javier

last: Di Noia

first: David

last: Langlais


title: B-cell Intrinsic Regulation of Antibody Mediated Immunity by Histone H2A Deubiquitinase BAP1 [ChIP-seq]

overall_design: To identify the genomic regions directly regulated by BAP1 in activated B cells, we conducted ChIP-seq to map the BAP1 genome-wide DNA-binding sites in CH12F3 cells, firstly using wild type cells with an anti-BAP1 antibody, repeating the analyses with both untreated cells and cells at 72 hours of stimulation with TGF-β, IL-4, and anti-CD40, corresponding to the standard protocol for CH12F3 cell activation. ChIP-seq analysis was further repeated using the CH12F3 cells stably expressing 3xFLAG-tagged BAP1 with an antibody against the FLAG epitope.