title: Comparative transcriptome of normal and cancer-associated fibroblasts

status: Public on Sep 26 2024

city: Bengaluru

first: Prathibha

last: Ranganathan

state: Karnataka

address: Electronic City Phase I

institute: Centre for Human Genetics

summary: Background The characteristics of a tumor are largely determined by its interaction with the surrounding micro-environment (TME). TME consists of both cellular and non-cellular components. Cancer-associated fibroblasts (CAFs) are a major component of the TME. They are a source of many secreted factors that influence the survival and progression of tumors as well as their response to drugs. Identification of markers either overexpressed in CAFs or unique to CAFs would pave the way for novel therapeutic strategies that in combination with conventional chemotherapy are likely to have  better patient outcome.  Methods  Fibroblasts have been derived from Benign Prostatic Hyperplasia (BPH) and prostate cancer. RNA from these has been used  to perform a transcriptome analysis in order to get a comparative profile of normal and cancer-associated fibroblasts.  Results  The study has identified 818 differentially expressed mRNAs and 17 lincRNAs between normal and cancer-associated fibroblasts.   Also, 15 potential lincRNA-miRNA-mRNA combinations have been identified which may be potential biomarkers.  Conclusions Identified differentially expressed markers between normal and cancer-associated fibroblasts would help in CAFs/derived factors targeted therapy in combination with conventional therapy. However, this would in future need more experimental validation.

overall_design: In order to get a comparison of the transcriptoome from normal and cancer associated fibroblasts biopsy samples from control (benign prostatic hyperplasia/BPH) and prostate cancer were obtained. Fibroblasts were derived from these tissues. Total RNA was isolated from the fibroblasts and subjected to RNA-seq using Illumina 2000 platform. Differentially expressed mRNAs and long non-coding RNAs were identified between the two groups.

first: Apoorva

last: Abikar

first: Mohammad

last: Mehaboob Subhani Mustafa

first: Radhika

last: Rajiv Athalye

first: Namratha

last: Nadig

first: Ninad

last: Tamboli

first: Vinod

last: Babu

first: Ramaiah

last: Keshavamurthy


title: Loss of PI5P4Kα slows the progression of a Pten mutant basal cell model of prostate cancer

status: Public on Sep 24 2024

city: Bern

first: Andrej

last: Benjak

address: Murtenstrasse 24-28

department: Department for BioMedical Research

country: Switzerland

institute: University of Bern

type: Expression profiling by high throughput sequencing

summary: We use in vivo mouse models to characterize the outcome of deleting PI5P4Kα in prostate basal cells.  Single cell RNA-Seq analysis reveals the molecular consequence of altering PI5P4Kα and links impaired lipid metabolism with delayed mPIN development.

overall_design: We used a flow cytometry-based selection of eYFP+ cells from our luminal and basal in vivo systems. Two animals per group and 1500 individual cells were sequenced following FACs selection using the SORT-seq RNA-Seq platform, a partially robotized method based on the CEL-Seq2 protocol.

first: Joanna

last: Triscott

first: Marika

last: Lehner

first: Mark

middle: A

last: Rubin


title: Primary Human articular chondrocyte isolation and miR-199a-5p or miR-199b-5p level manipulation

status: Public on Oct 01 2024

city: NEWCASTLE UPON TYNE

first: David

last: Young

address: Centre For Life

country: United Kingdom

institute: Newcastle University

summary: Objective  Animal models of post-traumatic osteoarthritis (PTOA) recapitulate the pathological changes observed in human PTOA. Here we aimed to compare the cartilage transcriptome responses of a non-surgical, mechanically induced rupture of the anterior cruciate ligament (ACL) model and the surgical destabilisation of the medial meniscus (DMM) model.   Methods  Skeletally mature male C57Bl6 mice were subjected to either the non-surgical, mechanical ACL rupture or surgical DMM models and transcriptome profiling performed on micro-dissected cartilage at day 7 and 42 post-procedure, respectively; in general, naïve animals served as controls. MicroRNA profiling was also performed on the ACL rupture model. Expression levels of a miRNA of interest, miR-199-5p, were inhibited in primary human articular chondrocytes (HAC) with RNA-seq and 3’UTR assays used to identify and valid potential target genes.    Results  The number of differentially expressed genes between the two models were comparable and highly correlative (Spearman R =0.8, P<2.2E-16).  Gene ontology enrichment analysis identified similarly enriched pathways, containing anabolic terms including ‘extracellular matrix organisation’ enriched for the upregulated genes. Within the ACL rupture miRNA transcriptome, miR-199-5p family members were amongst the most abundantly, and differentially expressed, which was replicated in the DMM cartilage by qRT-PCR. Inhibition of miR-199-5p in HAC led to a comparable transcriptome response to that observed in both human OA damaged vs intact cartilage and murine DMM cartilage datasets. Several genes, including GIT1, NCEH1, SOS2 and ECE1 were all experimentally verified as targets.  Conclusion  For the first time, we have characterised both the mRNA and miRNA articular cartilage signature in the ACL rupture model and demonstrated highly correlative responses with the DMM PTOA model. These data support the use of the ACL rupture model as a non-invasive alternative to DMM.

overall_design: Human articular chondrocyte (HAC) were isolated from knee cartilage by enzymatic digestion (briefly, macroscopically normal cartilage was removed from the subchondral bone and dissected into ~1mm pieces using scalpel and forceps. Enzymatic digestion was performed using hyaluronidase, trypsin and then collagenase overnight at 37oC).  Tissue was donated by four patients (CIMA55, CIMA56, CIMA64, CIMA67) with diagnosed osteoarthritis and undergoing joint replacement surgery (age 59-85; 3 female, 1 male) with informed consent and ethics committee approval (REC 19/LO/0389). Briefly, macroscopically normal cartilage was removed from the subchondral bone and dissected into ~1mm pieces using scalpel and forceps. Enzymatic digestion was performed using hyaluronidase, trypsin and then collagenase overnight at 37oC (28).  For modulation of miR-199 levels in HAC, Dharmacon miRIDIAN hairpin inhibitor against miR-199a-5p or miR-199b-5p or Dharmacon miRIDIAN miRNA hairpin inhibitor nontargeting Control #2 (IN-002005-01) were transfected into 40%–50% confluent HAC using Dharmafect 1 lipid reagent (Horizon Discovery) at 100 nM final concentration.  24 hours later RNA was isolated following the miRVana protocol, quality assessed, and cDNA libraries generated using the Illumina TruSeq Stranded mRNA protocol and sequenced on an Illumina NextSeq500 instrument. Kallisto, Tximport and DeSeq2 were used for analysis as described above

first: Matt

middle: J

last: Barter

first: Jamie

last: Soul


title: Insights into Molecular Mechanisms and Therapeutic Potential of Arborvitae Essential Oil (Thuja plicata) on Cervical Cancer Cells

status: Public on Sep 30 2024

city: Guadalajara

first: Adriana

middle: Del Carmen

last: Aguilar Lemarroy

state: Jalisco

address: Sierra Mojada 800, Independencia Oriente, 44340 Guadalajara, Jal.

department: División de Inmunología

country: Mexico

institute: Centro de Investigación Biomédica de Occidente

summary: This analysis aimed to assess the effect of arborvitae (Thuja plicata) essential oil (AEO) in an in vitro model of cell lines derived from cervical cancer, namely HeLa and SiHa, by examining its influence on gene expression modulation. The working cell lines were HeLa, and SiHa. Total RNA sequencing was performed on the Illumina NovaSeq 6000 platform by Novogene Bioinformatics Technology Co., Ltd, Beijing, China, with two independent replicate sequences for each cell model. Methodology: The execution of the bioinformatics pipeline analysis was carried out as follow: R (ver. 4.3.1), RStudio (ver. 2023.09.1 +494) and Galaxy (https://www.usegalaxy.orgaccessed on 02 February 2024) open-source platforms were used to analyze the Illumina raw data. The Quality Check was conducted through the FastQC tool (v0.74+galaxy0). Afterward, all the reads were processed by the Rsubread package (v2.14.2); at that point, the reads were trimmed and aligned to the Human Genome Reference (GRCh38.p14 v44). The obtained output was the BAM files, of which the number of reads was counted by the FeatureCounts tool (v2.0.3); at the final step, the Differential Expression Analysis was settled by the DESeq2 package for R (ver. 1.42. 0). The differential gene expression measurements were normalized by DESeq2's median of ratios (median of ratio of gene counts relative to geometric mean per gene) method. All genes with an adjusted p-value (p-adj) minus or equal to 0.05 and fold change (Log2FC) up to 2 or less than minus 2 were selected as differentially expressed genes (DEGs). Conclusions: Our study found that AEO regulates genes related with cell cycle regulation, cell growth, differentiation, and cell death in both cell lines, mainly through downregulation; however, its effect appears to be mediated by different pathways in each cell line.

overall_design: Cervical cancer-derived cell lines underwent total  RNA extraction (SiHa and HeLa) after 4 hrs AEO or DMSO (vehicle control) treatment; this process was addressed in duplicate. After that, the replicates were compared as a single end of each cell line vs. the control for the development of the differential expression analysis.

first: Ruben

last: Piña-Cruz

first: Andrea

last: Molina-Pineda

first: Marco

last: Aguila-Estrada

first: María

middle: M

last: Villaseñor-García

first: Georgina

last: Hernández-Flores

first: Luis

middle: F

last: Jave-Suárez

last: Aguilar-Lemarroy


title: Transcriptomic analysis of fetal epaxial muscle fibers from wildtype and dyW/dyW mice at embryonic day 17.5

status: Public on Sep 23 2024

city: Lisboa

first: Ana Rita

last: Carlos

address: FCUL, Campo Grande

country: Portugal

institute: Faculty of Sciences, University of Lisbon

summary: LAMA2-congenital muscular dystrophy (LAMA2-CMD) is the most common congenital muscular dystrophy and is triggered by mutations in LAMA2, coding for laminin α2 chain. Several phenotypes have been associated with LAMA2-CMD, including inflammation, fibrosis and increased oxidative stress. However, it is not yet known what mechanisms are faulty, right at disease onset, which in the mouse model of LAMA2-CMD dyW/dyW has been previously established to occur between embryonic days (E) 17.5 and E18.5. This transcriptomic analysis of fetal muscle fibers perfomed at E17.5 provides critical information regarding the genes and pathways that are altered in LAMA2-CMD right at the onset of the disease.

overall_design: This analysis was performed via the comparison of 4 samples each obtained from a different wildtype mouse and 3 samples each obtained from a different dyW/dyW mouse

first: Susana

middle: G

last: Martins

first: Vanessa

last: Ribeiro

first: Pedro

last: dos Santos

first: Dario

last: Antonini

first: Rita

last: Zilhão

first: Sólveig

last: Thorsteinsdóttir

first: Ana

middle: R


title: HIF1α-glycolysis engages activation-induced cell death to drive IFN-γ induction in hypoxic T cells

city: Birmingham

first: Hongxing

last: Shen

state: AL

address: 1700 6TH AVE S, HSROC 2222N

department: Department of Radiation Oncology

country: USA

institute: University of Alabama at Birmingham

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The role of HIF1a-glycolysis in regulating IFN-g induction in hypoxic T cells is unknown. Given that hypoxia is a common feature in a wide array of pathophysiological contexts such as tumor and that IFN-g is instrumental for protective immunity, it is of great significance to gain a clear idea on this. Combining pharmacological and genetic gain-of-function and loss-of-function approaches, we find that HIF1a-glycolysis controls IFN-g induction in both human and mouse T cells activated under hypoxia. Specific deletion of HIF1a in T cells (HIF1a–/–) and glycolytic inhibition significantly abrogate IFN-g induction. Conversely, HIF1a stabilization in T cells by hypoxia and VHL deletion (VHL–/–) promotes IFN-g production. Mechanistically, reduced IFN-g production in hypoxic HIF1a–/– T cells is due to attenuated activation-induced cell death but not proliferative defect. We further show that depletion of intracellular acetyl-CoA  is a key metabolic underlying mechanism. Hypoxic HIF1a–/– T cells are less able to kill tumor cells, and HIF1a–/– tumor-bearing mice are not responsive to immune checkpoint blockade (ICB) therapy, indicating loss of HIF1a in T cells is a major mechanism of therapeutic resistance to ICBs. Importantly, acetate supplementation restores IFN-g production in hypoxic HIF1a–/– T cells and re-sensitizes HIF1a–/– tumor-bearing mice to ICBs, providing an effective strategy to overcome ICB resistance. Taken together, our results highlight T cell HIF1a-anaerobic glycolysis as a principal mediator of IFN-g induction and anti-tumor immunity. Considering that acetate supplementation (i.e., glycerol triacetate (GTA)) is approved to treat infants with Canavan disease, we envision a rapid translation of our findings, justifying further testing of GTA as a repurposed medicine for ICB resistance, a pressing unmet medical need

overall_design:
We conducted whole transcriptome analysis (RNA-Seq) using total RNAs isolated from WT and HIF1a–/– T cells that were activated, under normoxia and hypoxia for 48h.

cleavage under targets and release using nuclease(Cut&Run) data for the histone H3 modification H3K9Acetate

first: Oluwagbemiga

last: Ojo

first: Chuan

last: Xing

first: Lewis

middle: Z

last: Shi


title: The effect of G-MDSCs depletion on the gene expression profile of hepatocytes at different times following 70% hepatectomy

status: Public on Sep 29 2024

city: Ramat Gan

first: Niv

last: Pencovich

address: 2 Derech Sheba

department: Surgery and transplantation

country: Israel

institute: Sheba medical center

summary: Liver regeneration following resection is a complex process relying on coordinated pathways and cell types in the remnant organ. Myeloid-Derived Suppressor Cells (MDSCs) have a role in liver regeneration-related angiogenesis but their influence on hepatocyte proliferation and immune modulation during liver regeneration is unclear. We examined the transcriptional response of regenerating liver hepatocytes after major resection in mice with CD11b+Ly6G+ MDSCs (G-MDSCs) depletion using RNA sequencing. Global gene expression profiling of regenerating hepatocytes upon G-MDSC depletion revealed disrupted transcriptional progression from day one to day two after major liver resection. Key genes and pathways related to hepatocyte proliferation and immune response were differentially expressed upon MDSC depletion. This study provides evidence that MDSCs contribute to early liver regeneration by promoting hepatocyte proliferation and modulating the intra-liver immune response. These findings illuminate the multifaceted role of MDSCs in liver regeneration.

overall_design: To investigate the effect of G-MDSCs on regenerating hepatocytes, we performed RNA-seq on mouse hepatocytes isolated at post-operative day (POD) 1 or 2 following 70% hepatectomy, after injection of either an anti mouse Gr-1 antibody (causing G-MDSC depletion), or an isotype control. 3 replicates per group were used.

first: Nachmani

last: Ido

first: Shir

last: Nevo

first: Sarit

last: Edelheit

first: Gilgi

last: Friedlander

first: Avital

last: Sarusi-Portuguez

first: Oran

last: Yakubovsky


title: RABIF promotes hepatocellular carcinoma progression through regulation of mitophagy and glycolysis

status: Public on Sep 28 2024

city: Xuzhou

first: Nianli

last: Liu

address: West Huaihai Road

country: China

institute: Xuzhou Medical University

summary: The RAB interacting factor (RABIF) is a putative guanine nucleotide exchange factor that also functions as a RAB-stabilizing holdase chaperone. It has been implicated in pathogenesis of several cancers. However, the functional role and molecular mechanism of RABIF in hepatocellular carcinoma (HCC) are not entirely known. Here, we demonstrate an upregulation of RABIF in patients with HCC, correlating with a poor prognosis. RABIF inhibition results in decreased HCC cell growth both in vitro and in vivo. Our study reveals that depleting RABIF attenuates the STOML2-PARL-PGAM5 axis-mediated mitophagy. Consequently, this reduction in mitophagy results in diminished mitochondrial reactive oxygen species (mitoROS) production, thereby alleviating the HIF1α-mediated downregulation of glycolytic genes HK1, HKDC1, and LDHB. Additionally, we illustrate that RABIF regulates glucose uptake by controlling RAB10 expression. Importantly, the knockout of RABIF or blockade of mitophagy sensitizes HCC cells to sorafenib. This study uncovers a novel role of RABIF crucial for HCC growth and identifies it as a potential therapeutic target.

overall_design: RNA sequencing was performed in RABIF knock out Huh7 cells using CRISPR/Cas 9.  Wide typel Huh7 cells were used as control.

first: Ning

last: Feng

first: Rui

last: Zhang


title: Spatial multi-omics in skeletal muscle unravel complex myofiber architecture

status: Public on Sep 19 2024

city: Leiden

first: Clara

last: Martinez Mir

address: Einthovenweg 20

department: Anatomy and Embryology

country: Netherlands

institute: Leiden University Medical Center

summary: Skeletal muscles are complex organs consisting of multiple tissues, including connective tissue, blood vessels, nerves and multinucleated myofibers. Single-nucleus transcriptomics analyses have revealed distinctive myonuclear populations related to myofiber type and to specialized regions, including neuromuscular and myotendinous junctions. However, the use of tissue dissociation methods prevented access to global spatial organization. Here we applied RNA tomography (TOMOseq), a spatial transcriptomics approach, to explore gene expression differences along murine tibialis anterior muscles.

overall_design: RNA sequencing data from tissue sections isolated from murine tibialis anterior muscles

first: Paola

last: Pisterzi

first: Isabel

last: De Poorter

first: Maria

last: Rilou

first: Melissa

last: van Kranenburg

first: Bram

last: Heijs

first: Anna

last: Alemany

first: Fanny

last: Sage

first: Niels

last: Geijsen


title: H3K9ac-marked three-dimensional genome dynamics in rice under drought stress [RNA-Seq]

city: Wuhan

first: Yu

last: Chang

state: Hubei

address: No.1, Shizishan Street, Hongshan District

department: National Key Laboratory of Crop Genetic Improvement

institute: Huazhong Agricultural University

summary: Drought stress response involves vigorous reprogramming of transcriptome while the mechanism modulating this process remains elusive. The role of 3D-genome in the regulation of rice development has recently been unveiled in rice, which exhibited characteristics distinct from that in mammals and other plants. However, the relationship between spatial chromosome organization and drought responsive gene reprogramming is still poorly understood. In this study, we identified H3K9ac as an efficient histone mark that response dynamically to drought stress in rice and re-constructed high-resolution 3D genome contact maps based on these sites under the normal, drought, and recovery conditions. We discovered significant decondensation of chromatin contacts with over 10000 chromatin loops lost upon drought stress treatment while the recovery of 3D genome was limited after 4-day’s re-watering. We identified dominate promoter-promoter interacting (PPI) loops under each condition and identified their significant correlations to altered gene expressions in response to the corresponding condition. Based on the relative contact intensities of the PPI connections, we characterized super-promoter regions that integrate closer connections of genes with more vigorous inductions to condition shifts. Especially, about 75% of the drought stress-dominate PPI loops were associated to the binding by a well-defined drought stress-responsive transcription factor, OsbZIP23. The mutation of OsbZIP23 led to the diffuse of over 80% DS-dominate PPI loops and impaired the expression of the connected genes. As a case study, we showed how OsbZIP23 binding to a super-promoter region can integrate the PPI loop formation and transcriptional activation of four function-vital dehydrin genes upon drought stress. Our results shed light on the mechanisms of 3D genome dynamic in response to water supply variations in rice and highlight the role of OsbZIP23 in the regulation of chromatin loop formation under drought stress.

overall_design: This study involves 8 ChIA-PET samples, 8 RNA-seq samples, and 13 ChIP-seq samples. All experiments in this study were carried out using two biological replicates. Each replicate involved at least 30 rice seedlings. All buckets were randomly placed in the growth chamber during the treatments.

first: Jiahan

first: Minrong

last: Guo

first: Xingwang

last: Li

first: Lizhong

last: Xiong


title: H3K9ac-marked three-dimensional genome dynamics in rice under drought stress

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

overall_design: Refer to individual Series


city: Changchun

first: Xiaorui

last: Yang

address: Xincheng Street

institute: Jilin Agricultural University

summary: Exploring the effect of m6A methylation modification on the progression of bovine hypothalamic tissue before and after the onset of puberty

first: Ziming

last: Wang

first: Wenfa

last: Lu


title: Comparison of iPSC-derived human intestinal epithelial cells with Caco-2 cells and human in vivo data after exposure to Lactiplantibacillus plantarum WCFS1

status: Public on Sep 18 2024

city: Wageningen

first: Benthe

last: van der Lugt

address: Akkermaalsbos 2

institute: Wageningen University & Research

summary: To investigate intestinal health and its potential disruptors in vitro, representative models are required. Human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cells (IECs) more closely resemble the in vivo intestinal tissue than conventional in vitro models like human colonic adenocarcinoma Caco-2 cells. However, the potential of IECs to study immune-related responses upon external stimuli has not been investigated in detail yet. The aim of the current study was to evaluate immune-related effects of IECs by challenging them with a pro-inflammatory cytokine cocktail. Subsequently, the effects of Lactiplantibacillus plantarum WCFS1 were investigated in unchallenged and challenged IECs. All exposures were compared to Caco-2 cells and in vivo data where possible. Upon the inflammatory challenge, IECs and Caco-2 cells induced a pro-inflammatory response which was strongest in IECs. Heat-killed L. plantarum exerted the strongest effect on immune parameters in the IEC model, while L. plantarum in the stationary growth phase had most pronounced effects on immune-related gene expression in Caco-2 cells. Unfortunately, comparison to in vivo transcriptomics data showed limited similarities, which could be explained by essential differences in the study setups. Altogether, hiPSC-derived IECs show a high potential as a model to study immune-related responses in the intestinal epithelium in vitro.

overall_design: To investigate the effects of L. plantarum on hiPSC-derived IECs and Caco-2 cell models, both cell models were exposed to heat-killed and stationary L. plantarum for 96 hours. After 24h of L. plantarum exposure, the pro-inflammatory stimulus was intitated (i.e., 2 ng/mL IFN-γ for 24 hours, followed by exposure to 1 ng/mL IL-1β and 10 ng/mL TNFα for another 48 hours). After exposure, RNAseq analysis was performed on three samples per experimental condition, i.e., healthy Caco-2 and IECs (unchallenged cells without L. plantarum (control) and exposed to stationary or heat-killed L. plantarum), and challenged Caco-2 and IECs (challenged cells without L. plantarum exposure (control) and exposed to stationary or heat-killed L. plantarum.

first: Aafke

middle: W

last: Janssen

first: Loes

middle: P

last: Duivenvoorde

first: Arjan

last: Vos

first: Shanna

last: Bastiaan-Net

first: Monic

last: Tomassen

first: Janine

last: Verbokkem

first: Emmie

last: Blok-Heimerikx

first: Guido

last: Hooiveld

first: Peter

last: van Baarlen

first: Laurent

last: Ferrier

first: Meike

last: van der Zande


title: Systematic perturbations of SETD2, NSD1, NSD2, NSD3 and ASH1L reveal their distinct contributions to H3K36 methylation

status: Public on Sep 25 2024

city: Montreal

first: Jacek

last: Majewski

state: Quebec

address: 740 Dr. Penfield

department: Human Genetics

country: Canada

institute: McGill University

summary: Methylation of histone 3 lysine 36 (H3K36me) has emerged as an essential epigenetic component for the faithful regulation of gene expression. Despite its importance in development, disease, and cancer, how the molecular agents collectively shape the genome-wide deposition of H3K36me is unclear. Here, we use mouse mesenchymal stem cells to comprehensively perturb the components of the H3K36me deposition machinery and infer the activities of the five most prominent players: SETD2, NSD1, NSD2, NSD3, and ASH1L. We find that H3K36me2 is the most abundant of the three methylation states and that it is predominantly deposited at intergenic regions by NSD1, and in part by NSD2. In contrast, H3K36me1/3 are most abundant within exons and are positively correlated with gene expression. We demonstrate that while SETD2 is responsible for depositing most H3K36me3, it also deposits H3K36me2 within transcribed genes. Additionally, loss of SETD2 results in an increase of exonic H3K36me1, suggesting that other H3K36 methyltransferases may prime gene bodies with lower methylation states ahead of transcription. Through a reductive approach, we uncover the distribution patterns of NSD3- and ASH1L-catalyzed H3K36me2. While NSD1/2 establish broad intergenic H3K36me2 domains, NSD3 deposits H3K36me2 peaks centered on active promoter and enhancer regions. Meanwhile, the activity of ASH1L is restricted to regulatory elements of developmentally relevant genes, and our analyses implicate PBX2 as a potential recruitment factor. Overall, our study provides new insights into the regulation of H3K36me and helps to consolidate the wealth of previous observations in the context of structured analyses.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-Seq) for histone modifications H3K36me1, H3K36me2 and H3K36me3 as well as transposase-accessible chromatin with sequencing (ATAC-Seq) in C3H10T1/2 mouse mesenchymal stem cells.

first: Gerald

last: Shipman

first: Reinnier

last: Padilla

first: Cynthia

last: Horth

first: Bo

last: Hu

first: Eric

last: Bareke

first: Francisca

last: Vitorino

first: Joanna

last: Gongora

first: Benjamin

last: Garcia

first: Chao


title: H3K9ac-marked three-dimensional genome dynamics in rice under drought stress [ChIP-Seq]

first: Yu

last: Chang

first: Jiahan

last: Liu

first: Minrong

last: Guo

first: Xingwang

last: Li

first: Lizhong

last: Xiong


title: Aptamer-Drug Conjugates-loaded Bacteria for Pancreatic Cancer Synergistic Therapy

city: Shanghai

last: Xiao

address: No. 160 Pujian Road, Pudong New Area, Shanghai, China

institute: Shanghai Jiao Tong University School of Medicine

summary: Pancreatic cancer is one of the most malignant tumors with the highest mortality rates, and it currently lacks effective drugs. Aptamer-drug conjugates (ApDC), as a form of nucleic acid drug, show great potential in cancer therapy. However, the instability of nucleic acid-based drugs in vivo and the avascularity of pancreatic cancer with dense stroma have limited their application. Fortunately, VNP20009, a genetically modified strain of Salmonella typhimurium, which has a preference for anaerobic environments, but is toxic and lacks specificity, can potentially serve as a delivery vehicle for ApDC. Here, we propose an approach to synergistic therapy that utilizes the penetrative capability of bacteria and the targeting and toxic effects of ApDC by conjugating ApDC to VNP20009 via straightforward, one-step click chemistry. With this strategy, bacteria specifically target pancreatic cancer through anaerobic chemotaxis and subsequently adhere to tumor cells driven by the aptamer's specific binding. Results indicate that this method prolongs the serum stability of ApDC up to 48 hours and resulted in increased drug concentration at tumor sites compared to the free drugs group. Moreover, the aptamer's targeted binding to cancer cells tripled bacterial colonization at the tumor site, leading to increased death of tumor cells and T cell infiltration. Notably, by integrating chemotherapy and immunotherapy, the effectiveness of the treatment is significantly enhanced, showing consistent results across various animal models. Overall, this strategy takes advantage of bacteria and ApDC and thus presents an effective synergistic strategy for pancreatic cancer treatment.

overall_design: First, bacteria act as microbial stealth bombers aimed at the tumor microenvironment (TME) through the aptamer’s guidance system to deliver a potent drug payload, but also as protective shields for ApDC, effectively mitigating the risk of renal clearance and nuclease degradation. Second, owing to the bacterial predilection for the hypoxic TME and their innate tissue-penetrating capabilities, ApDC can navigate the otherwise formidable tumor stroma, ensuring precise drug delivery to the innermost tumor regions and achieving efficacious tumor cell killing. Moreover, leveraging the binding affinity of the nucleic acid aptamer Sgc8c to pancreatic cancer cells, these drug-loaded bacteria can proficiently transport the medication to the tumor site, thereby enhancing the therapeutic effect. Simultaneously, the proliferation of bacteria acts as a catalyst for an immune response, thereby facilitating a targeted combination therapy against tumors. This strategy effectively leverages the strengths of both ApDC-targeted therapy and bacterial therapy, presenting a promising strategy for the treatment of pancreatic cancer.

first: Yang

last: Sun

first: Weihong

last: Tan

first: Haiyan

last: Xu


title: Chromodomain protein CDYL confers forebrain identity to human cortical organoids by inhibiting neuronatin [ChIP-seq]

city: Beijing

first: Liang-Yu

last: Ni

state: Beijing

address: Peking university Health Science center

institute: Peking University

summary: Fate determination of neural stem cells is crucial for cerebral cortex development and is closely linked to neurodevelopmental disorders when gene expression networks are disrupted. The transcriptional corepressor chromodomain Y-like (CDYL) is widely expressed across diverse cell populations within the human embryonic cortex, though its precise role in this complex developmental process remains unclear. Here, we show that CDYL is critical for human cortical neurogenesis. CDYL deficiency leads to a substantial increase in gamma-aminobutyric acid (GABA)-ergic neurons in human cortical organoids, instead of the expected generation of typical cortical neurons. Subsequently, neuronatin (NNAT) is identified as a significant target of CDYL in this process, and its abnormal expression obviously influences the proliferation and fate commitment of cortical neural stem cells (NSCs) within human cortical organoids. Cross-species comparisons of CDYL targets unravel a distinct developmental trajectory between human cortical organoids and mouse cortex at an analogous stage. Collectively, our data provide insight into the evolutionary roles of CDYL in human cortex development, emphasizing the critical function of CDYL in maintaining the fate of human cortical NSCs.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: cortical organoids generated from CDYL +/+ and CDYL -/- human ESCs are collected at day 30; mouse cortex at E15.5 were collected

first: Yaming

last: Yang

first: Bai-Rong

last: Chen

first: Xi-Yin

last: Zhang

first: Yun

last: Wang


title: Chromodomain protein CDYL confers forebrain identity to human cortical organoids by inhibiting neuronatin [RNA-seq]

summary: Epigenetic regulation is essential for the normal development of human cerebral cortex, and its disruptions would lead to diverse neurodevelopmental disorders. The epigenetic co-repressor CDYL exhibits widespread expression across various cell clusters within the human embryonic cortex, yet its roles in this intricate process have remained elusive. Here, we show that CDYL is critical for cortical neurogenesis, and CDYL deficiency led to an augmentation in the generation of GABAergic neurons instead of cortical progenitors and neurons in the cortical organoid model. Combining analysis of bulk RNA-seq and ChIP seq, we identified NNAT as a significant CDYL target by H3K27me3 modification, crucial for the fate determination of neural stem cells within human cortical organoids, distinctly diverging from the murine cortex at a similar developmental stage. Collectively, our study sheds light on the critical function of CDYL in the maintenance of cortical neural stem cell fate commitment during human corticogenesis through the inhibition of NNAT expression.

overall_design: cortical organoids generated from CDYL +/+ and CDYL -/- human ESCs are collected at day 30 and day 80; mouse cortex at E15.5 from control and Emx1-cre-Cdyl-loxp group were collected


title: Transcriptomic profiling of adipose tissues across estrous cycles in mice.

city: shanghai

first: baiyier

middle: yier

last: bai

state: shanghai

address: shanghai huangpuqu

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

summary: To investigate the impact of the reproductive cycle on adipose tissue, we analyzed transcriptome profiling across the entire estrous cycle in the adipose tissue of female mice.

overall_design:
Twenty 8-week-old female C57BL/6 mice were used for the study. The stages of their estrous cycle (proestrus, estrus, metestrus, diestrus) were determined through vaginal smears and hormone level assessments. Inguinal subcutaneous fat was then collected for RNA extraction, library construction, and sequencing. The data presented show FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values from transcriptome profiling of adipose tissues across different stages of the estrous cycle in these mice.

***************************************************************
The RNA-seq raw data have been deposited in the Genome Sequence Archive (GSA: CRA014866) under BioProject PRJCA020845 that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.
***************************************************************

first: Hongjie

last: Zheng

first: Yier

last: Bai

first: Shan

last: Wu

first: Zhixuan

last: Jiang

first: Qing

last: Pei

first: Min

last: Yao


title: DNA demethylation triggers cell free DNA release in colorectal cancer cells

city: Candiolo

first: Federica

last: Di Nicolantonio

state: Torino

address: Candiolo Cancer Institute FPO-IRCCS, SP142, km 3.95

department: Department of Oncology

country: Italy

institute: University of Torino

summary: Background: Liquid biopsy based on cell free DNA (cfDNA) analysis holds significant promise as a minimally invasive approach for the diagnosis, genotyping and monitoring of solid malignancies. Human tumors release cfDNA in the bloodstream through a combination of events, including cell death, active and passive release. However, the precise mechanisms leading to cfDNA shedding remain to be characterized. Addressing this question in patients is confounded by several factors, such as tumor burden extent, anatomical and vasculature barriers, release of nucleic acids from normal cells. In this work, we exploited cancer models to dissect basic mechanisms of DNA release.  Methods: We measured cell loss ratio, doubling time and cfDNA release in the supernatant of a colorectal cancer (CRC) cell line collection (N=76) representative of the molecular subtypes previously identified in cancer patients. Association analyses between quantitative parameters of cfDNA release, cell proliferation and molecular features were evaluated. Functional experiments were performed to test the impact of modulating DNA methylation on cfDNA release.  Results: Higher levels of supernatant cfDNA were significantly associated with slower cell cycling and increased cellular crashes. In addition, a higher cfDNA shedding was found in non-CpG Island Methylator Phenotype (CIMP) models. These results indicate a positive correlation between lower methylation and increased cfDNA levels. To explore this further, we exploited methylation microarrays to identify a subset of probes significantly associated with cfDNA shedding and derive a methylation signature capable of discriminating high from low cfDNA releasers. We applied this signature to an independent set of 176 CRC cell lines and patient derived organoids to select 14 models predicted to be low or high releasers. The methylation profile successfully predicted the amount of cfDNA released in the supernatant. At the functional level, genetic ablation of DNA methyl-transferases increased chromatin accessibility and DNA fragmentation, leading to increased cfDNA release in isogenic CRC cell lines. Furthermore, in vitro treatment of five low releaser CRC cells with a demethylating agent was able to induce a significant increase in cfDNA shedding.  Conclusion: Methylation status of cancer cell lines contributes to the variability of cfDNA shedding in vitro. Changes in methylation pattern are associated with cfDNA release levels and might be exploited to increase sensitivity of liquid biopsy assays.

overall_design: DNA methylation profiles were obtained for 12 CRC patient derived organoids grown in vitro without any treatment

first: Elisa

last: Mariella

first: Ludovic

last: Barault

first: Giulia

last: Gionfriddo


title: Long non-coding RNA GRASLND affects melanoma phenotypic switch and modulates immunogenicity by inhibition of the IFNγ signaling pathway (GRASLND KD).

city: Dortmund

first: Kim

middle: Denise

last: Fischer

address: Otto-Hahn-Straße 15

country: Germany

institute: Max Planck Institute for molecular Physiology

summary: Melanoma is a highly malignant tumor, that stands as the most lethal form of skin cancer and is characterized by notable phenotypic plasticity and intratumoral heterogeneity. Melanoma plasticity is involved in tumor growth, metastasis and therapy resistance. Long non-coding RNAs (lncRNAs) could influence plasticity due to their regulatory function. However, their role and mode of action are poorly studied. Here, we show a relevance of lncRNA GRASLND in melanoma phenotypic switch, IFNγ signaling and immunogenicity. GRASLND knockdown revealed switching towards a dedifferentiated, slow-proliferating and highly-invasive cell state. Interestingly, GRASLND is overexpressed in differentiated melanomas and associated with poor prognosis. Accordingly, we found GRASLND expressed in immunological “cold” tumors and it negatively correlates with gene signatures of immune response activation. In line, silencing of GRASLND under IFNγ enhanced the expression of IFNγ-stimulated genes, including HLA-I antigen presentation, demonstrating suppressive activity of GRASLND on IFNγ signaling. Based on our findings, we hypothesize an adaptive resistance mechanism of melanoma cells evading the immune system via inhibition of IFNγ signaling by GRASLND overexpression and highlight its value as a negative prognostic factor for melanoma.

overall_design: To investigate effects of GRASLND knockdown on 501Mel melanoma cells, we generated dox-inducible shRNA knockdown cell lines with one control shRNA (lacZ) and 2 GRASLND-targeting shRNAs (sh4 and sh8)


title: SATB1 prevents immune cell infiltration by regulating chromatin organization and gene expression of a chemokine gene cluster in T cells

first: bao

last: wang

address: jin zun road

institute: shanghai jiao tong university school of medicine

summary: The precise regulation of T lymphocyte development and functions is crucial for preventing the initiation and progression of autoimmunity. SATB1, a key regulator of T cell development, governs lineage-specific transcriptional programs. However, its role in autoimmunity remains unclear. Here we show that conditional knockout of Satb1 in CD4+ T cells in mice led to T cell hyperactivation and inflammatory cell infiltration in the lungs and stomach. By performing transcriptome profiling, we show that the loss of SATB1 led to the aberrant upregulation of chemokines, particularly the ones belonging to the CC subfamily. Treating Satb1 conditional knockout mice with the inhibitor of the CC chemokine receptor alleviated inflammatory cell infiltration and autoimmune phenotypes. Intriguingly, SATB1's transcriptional regulation of chemokine genes could not be attributed to its direct binding to chemokine promoters or modulation of H3K27Ac. Instead, SATB1 exerted its regulatory effects through the control of higher-order chromatin organization at the CC chemokine locus. The loss of SATB1 led to the emergence of a new chromatin domain encompassing the Ccl3, Ccl4, Ccl5, Ccl6, and Ccl9 genes and a distal enhancer, resulting in increased contacts between the enhancer and all five chemokine genes, thus inducing their upregulation. Collectively, these results demonstrate that SATB1 safeguards organisms against autoimmunity by ensuring proper chemokine expression and preventing inflammatory cell infiltration, providing valuable insights into the understanding of autoimmune diseases.


title: Long non-coding RNA GRASLND affects melanoma phenotypic switch and modulates immunogenicity by inhibition of the IFNγ signaling pathway (IFNg treatment and GRASLND KD).

overall_design: To investigate a potential effect of GRASLND on the transcription of IFNγ-stimulated genes, doxycycline-inducible GRASLND knockdown melanoma cells (501Mel) were generated, cell lines with control shRNA (lacZ) and 2 GRASLND-targeting shRNAs (sh4 and sh8). These GRASLND knockdown or lacZ control cells were treated with IFNγ and were either uninduced or induced with doxycycline.


title: Spatial transcriptomics revealed that S1pr2 deletion in keratinocytes increases the MyD88/NF-kB pathway and psoriasis-related cytokine expression in the epidermis

status: Public on Sep 20 2024

city: La Jolla

first: Kana

last: Kuroki

state: CA

address: 9500 Gilman Drive

department: Dermatology

institute: University of California San Diego

summary: The importance of the cross-talk between keratinocytes and immune cells in the pathogenesis of psoriasis has recently been reaffirmed. Recent studies have found that several S1PR functional antagonists, other than S1PR2, are effective in improving psoriasis. We performed spatial transcriptomics to map the immune cell landscape and its association with metabolic pathways in an imiquimod (IMQ)-induced psoriasis-like inflammation in S1pr2fl/fl K14-Cre mice that could not sense sphingosine-1-phosphate (S1P) in the epidermis through the S1PR2 receptor. Our analysis suggests that S1PR2 in keratinocytes plays a major role in psoriasis-like inflammation compared to other S1PRs. It acts as a down-regulator, inhibiting the recruitment of Th17 cells into the skin.

overall_design: Mice at 8 weeks old of S1pr2fl/fl and S1pr2fl/fl K14-Cre were depilated on the back skin 1 day before the treatment, then received a daily topical dose of 62.5 mg of 5% IMQ cream for 7 days. Skin samples were collected, formalin-fixed, and paraffin-embedded. Skin samples from S1pr2fl/fl mouse treated with IMQ (S1pr2wtIMQ) and S1pr2fl/fl K14-Cre mouse treated with IMQ (S1pr2K14IMQ) were analyzed using spatial transcriptomics.

first: Shahrzad

last: Alimohammadi

first: Samantha

last: Lowry

first: Anna

last: DiNardo


title: Probe-based spatial host-pathogen genes expression to study bacterial pathogenesis and the regulation of bacterial virulence factors in tissue.

city: Milan

first: Federico

last: Di Marco

state: Outside U.S./Canada

address: Via Olgettina 60

institute: IRCCS San Raffaele Scientific Institute

summary: Co-localization of host and pathogen spatial transcriptome information can improve our understanding on how inflammatory environments modulate pathogen virulence in tissues and vice versa. Here, we demonstrate for the first time that bacterial probes can be used to simultaneously identify host-pathogen mutual interactions in formalin-fixed, paraffin-embedded (FFPE) tissues using spatial transcriptomics technology. With properly designed probes targeting pathogens of interest, our approach could provide an improved definition of infection processes by detecting pathogen and host genes expression simultaneously, spatially distributed, and unbiased.

overall_design: We collected lung tissue section of mice displaying chronic infection by Mycobacterium abscessus (ATCC 19977 strain). Spatial Gene Expression assay was performed on lung tissue section of mouse chronically infected with M. abscessus (14 days post bacterial challenge) and of sham infected mice as negative control

first: Nicola

middle: I

last: Lorè

first: Francesca

last: Nicola

first: Stefano

last: de Pretis


title: MUTATIONS IN FIBRONECTIN IMPAIR CHONDROGENESIS IN CORNER FRACTURE TYPE SPONDYLOMETAPHYSEAL DYSPLASIA

first: Neha

last: Dinesh

address: 3640 Rue University

department: Anatomy and Cell Biology

summary: The study aims to investigate  molecular consequences of mutations in  matrix glycoprotein fibronectin (FN) in  pathological condition corner fracture type  spondylometaphyseal dysplasia (SMDCF).  SMDCF is a rare group of skeletal dysplasia  wherein the patients are characterized by  severe skeletal anomalies such as short  stature, scoliosis , coxa vara, genu varum and more.  To investigate the molecular mechanism underlying the pathogenesis of SMDCF, we analyzed  the cellular transcriptome of SMDCF patient using next generation RNA sequencing on  iPSC derived mesenchymal stem cells and  chondrocytes.

overall_design: We determined the differentially expressed genes in control versus firbonectin mutant cells at mesenchymal stem cell stage and after differentiation into chondrocytes

middle: E

first: Philippe

last: Campeau

first: Dieter

last: Reinhardt


title: 10X single-cell RNA-seq profiling of ccRCC model tumors and normal kidney tissue

city: Boston

first: Kevin

last: Bi

state: MA

address: 360 Longwood Ave

institute: Dana-Farber Cancer Institute

summary: To achieve a robust immunocompetent mouse model of human ccRCC, we directly injected adenovirus-associated viruses (AAVs) encoding sgRNAs against VHL and other known/suspected ccRCC tumor suppressor genes into the kidneys of C57BL/6 mice under conditions where Cas9 was under the control of the Pax8 promoter to induce kidney tumors. An AAV targeting Vhl, Pbrm1, Keap1 and Tsc1 reproducibly caused macroscopic ccRCCs that partially resembled human ccRCC tumors with respect to transcriptome and cell of origin and responded to a ccRCC standard of care agent, axitinib.

overall_design: LSL-Cas9/+ mice were crossed with Pax8 Cre mice to produce LSL-Cas9/+; Pax8 Cre/+ mice and orthotopically injected in the kidney with AAV expressing sgRNAs for Keap1, Pbrm1, Vhl and Tsc1. Four model tumors and four normal kidney samples were collected from mice, dissociated, and subjected to single cell RNA-sequencing.

first: Laura

last: Stransky

first: Wenhua

last: Gao

middle: S

last: Schmidt


title: NanoString nCounter Cancer CN panel assay CNV detection of high-grade serous carcinoma samples

city: Moscow

first: Pavel

last: Grebnev

email: grepavel@gmail.com, pgrebnev@cspfmba.ru

state: --- Select One ---

address: Pogodinskaya Street, 10, bld. 1, 119121 Moscow, Russia

country: Russia

institute: CSP

type: Genome variation profiling by genome tiling array

summary:
CNVs detection using 13 samples’ pairs of (12 samples’ pairs of histologically confirmed diagnosis of high-grade serous carcinoma and one patient with endometrioid carcinoma and matching normal tissues. Samples have IHC status for p53, WT1, Ki-67, PAX-8, and TNM classification according to WHO Classification of Tumors.

Please cite the original atricle:
Grebnev, P.A.; Meshkov, I.O.; Ershov, P.V.; Makhotenko, A.V.; Azarian, V.B.; Erokhina, M.V.; Galeta, A.A.; Zakubanskiy, A.V.; Shingalieva, O.S.; Tregubova, A.V.; et al. Benchmarking of Approaches for Gene Copy-Number Variation Analysis and Its Utility for Genetic Aberration Detection in High-Grade Serous Ovarian Carcinomas. Cancers 2024, 16, 3252. https://doi.org/10.3390/cancers16193252

overall_design: 13 samples’ pairs, 12 samples’ pairs of histologically confirmed diagnosis of high-grade serous carcinoma and one patient with endometrioid carcinoma and matching normal tissues

first: P

first: M

middle: V

last: Erokhina

first: A

last: Galeta

last: Zakubanskiy

first: O

last: Shingalieva

last: Tregubova

last: Asaturova

first: E

last: Snigir


title: Scaled and efficient derivation of loss-of-function alleles in risk genes for neurodevelopmental and psychiatric disorders in human iPSCs

status: Public on Sep 17 2024

city: Evanston

first: Jubao

last: Duan

state: IL

address: 1001 University Place

department: Center for Psychiatric Genetics

institute: NorthShore University HealthSystem

summary: Translating genetic findings for neurodevelopmental and psychiatric disorders (NPD) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, here we developed a pipeline for clonal loss-of-function (LoF) allele mutagenesis in human induced pluripotent stem cells (hiPSCs) by introducing premature stop-codons (iSTOP) that lead to mRNA nonsense-mediated-decay (NMD) or protein truncation. We tested the pipeline for 23 NPD genes on 3 hiPSC lines and achieved highly reproducible, efficient iSTOP editing in 22 NPD genes. Using RNAseq, we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Interestingly, for three schizophrenia risk genes (SETD1A, TRIO, CUL1), despite the high efficiency of base editing, we only obtained heterozygous LoF alleles, suggesting their essential roles for cell growth. We replicated the reported neural phenotypes of SHANK3-haploinsufficiency and found CUL1-LoF reduced neurite branches and synaptic puncta density. This iSTOP pipeline enables a scaled and efficient LoF mutagenesis of NPD genes, yielding an invaluable shareable resource.

overall_design: As part of the SSPsyGene Consortium, our Assay and Data Generation Center (ADGC) for the Model of iPSC-derived Neurons for NPD (MiNND) aims to employ the CBE editor-based iSTOP approach to generate isogenic hiPSC lines carrying LoF alleles for about 150-200 NPD genes on multiple donor genetic backgrounds.  Here, leveraging an improved reporter gene editing enrichment system that can substantially increase the CBE iSTOP editing efficiency in hiPSC, we established a semi-automated pipeline for parallel and efficient clonal LoF mutagenesis of a large number of genes. We tested the workflow on 23 NPD genes with 3 donor hiPSC lines (KOLF2.2J, CW20107, MGS_CD14). We obtained high and reproducible iSTOP editing efficiency across all three hiPSC lines. We systematically characterized the engineered isogenic iSTOP hiPSC lines for pluripotency, karyotyping, neuron differentiation capacity, and the expected NMD and LoF.

first: Siwei

last: Zhang

first: Zhiping

last: Pang


title: The five homologous CiaR-controlled sRNAs modulate Streptococcus pneumoniae Zn-resistance

city: Houston

first: Nicholas

last: De Lay

state: Texas

address: 6431 Fannin St

department: Microbiology and Molecular Genetics

institute: University of Texas Health Science Center

summary: Zn and Mn are essential micronutrients for many bacteria including Streptococcus pneumoniae. While Zn performs vital structural or catalytic roles in certain proteins, in excess, Zn can inhibit Mn uptake by S. pneumoniae and displace, but not functionally replace Mn from key enzymes including superoxide dismutase A (SodA). Here, we show that the Ccn small regulatory RNAs promote S. pneumoniae Mn uptake and resistance to the oxidative stress. Furthermore, we demonstrate that these small regulatory RNAs modulate the ability of S. pneumoniae to cause invasive pneumonia. Altogether, these findings reveal a new layer of regulation of S. pneumoniae Zn and Mn homeostasis and suggest that there are factors in addition to known transporters that modulate intracellular Mn levels.

sample_organism:
Streptococcus pneumoniae TIGR4
Streptococcus pneumoniae D39

overall_design: Characterization of the five homologous Ccn sRNAs in Streptococcus pneumoniae D39 using RNA-seq. Gene expression of  S. pneumoniae strain D39, TIGR4,  and its derived ccnABCDE mutants grown in BHI broth alone or supplemented with 0.2 mM ZnSO4 was determined by RNA-seq using biological triplicates.


title: The lifespan and kinetics of human Dendritic Cell subsets and their precursors in health and inflammation

status: Public on Sep 15 2024

city: Jerusalem

first: Ruth

last: Lubin

address: Ein-Kerem

department: The Institute of Biomedical and Oral Research

institute: Hebrew university

summary: Dendritic cells (DC) are specialised mononuclear phagocytes connecting innate and adaptive immunity. They comprise two principal subsets: plasmacytoid DC and conventional DC. Here, single cell RNA sequencing (scRNA-seq) was performed to analyze the expression levels of different genes in circulating human DC subsets.

overall_design: Following myeloid HLA+ cell enrichment of whole blood according to the method outlined (Lubin et al., 2023). The cells were subsequently stained and  sorted using a Fluoresecence-avtivated cell soritng (FACS) to isolate DC. Sorted cells were  separated into droplet emulsion using Chromium X (10X Genomics) and analyzed using scRNAseq.

first: Amit

last: Patel

first: Jonas

last: Mackerodt

first: Yan

first: Rotem

last: Gvili

last: Mulder

first: Charles-Antoine

last: Dutertre

first: Parinaaz

last: Jalali

first: Jame

last: Glanville

first: Idit

last: Hazan

first: Nikhila

last: Sridharan

first: Leonid

last: Kandel

first: Gurion

last: Rivkin

first: Ayse

last: Akarca

first: Derek

last: Gilroy

first: Teresa

last: Marafioti

first: Alexander

last: Mildner

first: Asaf

last: Wilensky

first: Becca

last: Asquith

first: Florent

last: Ginhoux

last: Macallan

first: Abed

last: Nasereddin

last: Shiff

first: Simon

last: Yona


title: H3K9ac-marked three-dimensional genome dynamics in rice under drought stress [ChIA-PET]

first: Yu

last: Chang

address: No.1, Shizishan Street, Hongshan District

institute: Huazhong Agricultural University

summary: Drought stress response involves vigorous reprogramming of transcriptome while the mechanism modulating this process remains elusive. The role of 3D-genome in the regulation of rice development has recently been unveiled in rice, which exhibited characteristics distinct from that in mammals and other plants. However, the relationship between spatial chromosome organization and drought responsive gene reprogramming is still poorly understood. In this study, we identified H3K9ac as an efficient histone mark that response dynamically to drought stress in rice and re-constructed high-resolution 3D genome contact maps based on these sites under the normal, drought, and recovery conditions. We discovered significant decondensation of chromatin contacts with over 10000 chromatin loops lost upon drought stress treatment while the recovery of 3D genome was limited after 4-day’s re-watering. We identified dominate promoter-promoter interacting (PPI) loops under each condition and identified their significant correlations to altered gene expressions in response to the corresponding condition. Based on the relative contact intensities of the PPI connections, we characterized super-promoter regions that integrate closer connections of genes with more vigorous inductions to condition shifts. Especially, about 75% of the drought stress-dominate PPI loops were associated to the binding by a well-defined drought stress-responsive transcription factor, OsbZIP23. The mutation of OsbZIP23 led to the diffuse of over 80% DS-dominate PPI loops and impaired the expression of the connected genes. As a case study, we showed how OsbZIP23 binding to a super-promoter region can integrate the PPI loop formation and transcriptional activation of four function-vital dehydrin genes upon drought stress. Our results shed light on the mechanisms of 3D genome dynamic in response to water supply variations in rice and highlight the role of OsbZIP23 in the regulation of chromatin loop formation under drought stress.

overall_design: This study involves 8 ChIA-PET samples, 8 RNA-seq samples, and 13 ChIP-seq samples. All experiments in this study were carried out using two biological replicates. Each replicate involved at least 30 rice seedlings. All buckets were randomly placed in the growth chamber during the treatments.

first: Jiahan

last: Liu

first: Minrong

last: Guo

first: Xingwang

last: Li

first: Lizhong

last: Xiong


title: Neuroblastoma heterogeneity and plasticity over disease progression are rooted in the dynamics of an early sympathetic transcriptional trajectory

status: Public on Sep 13 2024

city: Utrecht

first: Arjan

last: Boltjes

address: Heidelberglaan 25

institute: Princess Máxima Center

summary:
This GEO series contains human scRNA-seq data of Neuroblastoma patients, used in two manuscripts.
n=7 paired samples, primary tumour vs. bone marrow metastases
n=2 non-paired samples, bone marrow metastases

Titles:
Article 1 PUBLISHED: Neuroblastoma plasticity during metastatic progression stems from the dynamics of an early sympathetic transcriptomic trajectory
See Citation and PubMed ID below.

Article 2 UNDER REVIEW: Single-cell analyses of paired primary neuroblastoma tumor and bone marrow metastases reveals intra-patient heterogeneity and therapy-resistant subpopulations

Abstract of article 1:
Despite their indisputable importance in neuroblastoma (NB) pathology, knowledge of
the bases of NB plasticity and heterogeneity remains incomplete. They may be rooted
in developmental trajectories of their lineage of origin, the sympatho-adrenal neural
crest. We found that implanting human NB cells in the neural crest of the avian embryo
allows recapitulating the metastatic sequence until bone marrow involvement. Using
deep single cell RNA sequencing, we characterized transcriptome states of NB cells
and their dynamics over time and space, and compared them to those of fetal
sympatho-adrenal tissues and patient tumors and bone marrow samples. Here we
report remarkable transcriptomic proximities restricted to an early sympathetic
neuroblast branch that co-exist with phenotypical adaptations over disease
progression and recapitulate intratumor and interpatient heterogeneity. Combining
avian and patient datasets, we identified a list of genes upregulated upon bone marrow
involvement and associated with growth dependency, validating the relevance of our
multimodal approach.

Despite being key pathological features, Neuroblastoma (NB) plasticity and Heterogeneity remain largely misunderstood. They may be rooted in complex developmental trajectories of their embryonic lineage of origin, the sympatho-adrenal neural crest. We found that implanting human NB cells in the neural crest of the avian embryo allows recapitulating the metastatic sequence until bone marrow involvement. Using deep single cell RNA sequencing, we characterized transcriptional states of NB cells and their dynamics over time and space, and compared them to those of fetal sympatho-adrenal tissues and patient tumors, including matched primary tumor and bone marrow samples. We found remarkable transcriptomic proximities restricted to an early sympathetic neuroblast branch that co-exist with phenotypical adaptations over disease progression and recapitulate intratumor and interpatient heterogeneity. Combining avian model and patient datasets, we identified a list of candidate genes upon bone marrow involvement associated with NB growth dependency, validating the relevance of our multimodal approach.

overall_design:
We isolated tumour cells and associated cells from tissue - both adrenal gland and bone marrow – , sorted these on a single cell basis into 384 well plates, and performed scRNA-seq.

GSM7837988-GSM7838055: raw data available via "SRA Run Selector" link at the foot of this record.
GSM8299123-GSM8299133: no raw data available for now due to research participant privacy/consent.

first: Caroline

last: Hochheuser

first: Kaylee

last: Keller

first: Ilse

last: Timmerman

first: Lieve

last: Tytgat

first: Jan

last: Molenaar


title: LPS-induced systemic inflammation is suppressed by the PDZ motif peptide of ZO-1 via regulation of macrophage M1/M2 polarization

city: Busan

first: Kyoung Seob

last: Song

address: Gamcheon-ro, Seo-gu, Busan 49267

department: College of Medicine

country: South Korea

institute: Kosin University

summary: The gram-negative bacterium lipopolysaccharide (LPS) is frequently administered to generate models of systemic inflammation. In particular, both kidney and lung are more sensitive to acute injury caused by LPS-induced systemic inflammation. However, there are several side effects and no effective treatment for LPS-induced systemic inflammation. PEGylated PDZ peptide was based on the first PDZ domain of the zonula occludens-1 (ZO-1) protein. PEGylated PDZ peptide was analyzed for effects on systemic inflammation induced by LPS. PDZ peptide administration led to restoration of tissue injuries (kidney, liver, and lung) and prevented alterations in biochemical plasma markers. The production of pro-inflammatory cytokines was significantly decreased in the plasma and lung BALF in the PDZ-administered mice. Flow cytometry analysis revealed the PDZ peptide significantly inhibited inflammation, mainly by decreasing the population of M1 macrophages, neutrophils (immature and mature), and increasing M2 macrophages. Using RNA sequencing analysis, the expression levels of the NF-κB-related proteins were lower in PDZ-treated cells than in LPS-treated cells. In addition, wild-type PDZ peptide significantly increased mitochondrial membrane integrity and decreased LPS-induced mitochondria fission. Interestingly, PDZ peptide dramatically could reduce LPS-induced NF-κB signaling, ROS production, and the expression of M1 macrophage marker proteins, but increased the expression of M2 macrophage marker proteins. These results indicated that PEGylated PDZ peptide inhibits LPS-induced systemic inflammation, reducing tissue injuries and reestablishing homeostasis and may be a therapeutic candidate against systemic inflammation.

overall_design: BEAS-2B cells were treated with wild-type PDZ or mutant PDZ peptide for 24 h and then incubated with LPS for 2 h, after which RNA sequencing analysis was performed.

first: Hyun-Chae

last: Lee

first: Sun-Hee

last: Park

first: Hye

last: Jeong

first: Kyoung


title: RNA-seq analysis after STXBP5 overexpression or STXBP5 knockdown  in HA-progerin HEK293 cells

status: Public on Sep 14 2024

city: Tianjin

first: Jinchao

address: Tongyan Road

institute: Nankai University

summary: Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by a mutant LMNA called progerin. To determine the mechanism of STXBP5 on progerin, we over expressed STXBP5 or knocked down STXBP5 in HA-progerin HEK293 cells, then analyzed the effect on the expression of coding genes. In this study, we identified STXBP5 as an influencing factor for HA-progerin HEK293 cells. Lowering the expression of STXBP5 may be a new therapeutic strategy for treating age-related phenotypes in HGPS.

overall_design: In order to gain insight into the function of the identified related STXBP5 and progerin, HA-progerin HEK293 cells with over vector-FLAG (Control-OE), over vector-FLAG-STXBP5 (STXBP5), gAAVS1, gSTXBP5 respectively were used to performed four groups of RNA-sequencing (RNA-seq) gene expression profiling of HA-progerin HEK293 cells.

first: Hongqian

last: Qi

first: Yingying

last: Wu


title: Long non-coding RNA GRASLND links melanoma differentiation and interferon-gamma response

first: Kim

last: Fischer

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

overall_design: Refer to individual Series


title: Single-cell landscape of bronchoalveolar immune cells in patients with immune checkpoint inhibitor-related pneumonitis

city: beijing

first: lei

last: zhang

address: xueyuan road 38th

department: heath science center

institute: peking university

summary: We conducted single-cell and T-cell receptor transcriptomic sequencing on the bronchoalveolar lavage fluid from five patients with grade ≥2 immune checkpoint inhibitor-related pneumonitis. Our analyses revealed a prominent enrichment of T cells in the bronchoalveolar lavage fluid of patients with immune checkpoint inhibitor-related pneumonitis.

overall_design: All enrolled patients in the prospective cohort underwent standard bronchoscopy under general anesthesia to collect BALF samples. Generally, the bronchoscope was guided to the ICP-affected lung lobe or segment, and preheated sterile saline was injected into the airway multiple times. A total of 30ml of lavage fluid was retrieved from each patient, with 5-10ml reserved for routine clinical tests and the remainder used for scRNA-seq.

first: Zhening

last: Zhang

first: Lei

first: Keqiang

last: Wang

first: Tong

last: Xie

first: Xiaotian

first: Wenyi

last: Yu

first: Yanyan

first: Lin

last: Shen

first: Ran

first: Zhi

last: Peng


title: Parallel genome-scale CRISPR-Cas9 screens uncouple human pluripotent stem cell identity versus fitness [CRISPR]

status: Public on Sep 12 2024

city: New York

first: Hyein

middle: Sophia

last: Cho

state: NY

address: 430 E 67th St

department: Developmental Biology

institute: Memorial Sloan Kettering Cancer Center

summary: Pluripotent stem cells are defined by their self-renewal capacity, which is the ability of the stem cells to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into any somatic cell lineage. However, understanding the mechanisms that control stem cell fitness versus the pluripotent cell identity is challenging. To investigate the interplay between these two aspects of pluripotency, we performed four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSC self-renewal conditions, and the dissolution of the primed pluripotency identity during early differentiation. Comparative analyses led to the discovery of genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further discovered a core set of factors that control both stem cell fitness and pluripotent identity, including a network of chromatin factors that safeguard pluripotency. Our unbiased and systematic screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide rich datasets for exploring pluripotent cell identity versus cell fitness, and offer a valuable model for categorizing gene function in broad biological contexts.

overall_design: CRISPR screen of stem cells at conditions of definitive endoderm and neuroectoderm differentiation and maintenance of pluripotency. GFP+ and GFP- provided. 2 replicates each.

first: Bess

last: Rosen

first: Qing

last: Li

first: Danwei

last: Huangfu


title: DNA methylation profiling identifies TBKBP1 as potent amplifier of cytotoxic activity in CMV-specific human CD8+ T cells

city: Saarbrücken

first: Fabian

last: Müller

address: Campus A 2.4

institute: Saarland University

type: Methylation profiling by high throughput sequencing

summary: Epigenetic mechanisms stabilize gene expression patterns during CD8+ T cell differentiation. Although adoptive transfer of virus-specific T cells is clinically applied to reduce the risk of virus infection or reactivation in immunocompromised individuals, the DNA methylation pattern of virus-specific CD8+ T cells is largely unknown. Hence, we here performed whole-genome bisulfite sequencing of cytomegalovirus-specific human CD8+ T cells and found that they display a unique DNA methylation pattern consisting of 79 differentially methylated regions (DMRs) when compared to memory CD8+ T cells. Among the top demethylated DMRs in cytomegalovirus-specific CD8+ T cells was TBKBP1, coding for TBK-binding protein 1 that can interact with TANK-binding kinase 1 (TBK1) and mediate pro-inflammatory responses in innate immune cells downstream of intracellular virus sensing. Since TBKBP1 has not yet been reported in T cells, we aimed to unravel its role in virus-specific CD8+ T cells. TBKBP1 demethylation in terminal effector CD8+ T cells correlated with higher TBKBP1 expression at both mRNA and protein level, independent of alternative splicing of TBKBP1 transcripts. Notably, the distinct DNA methylation patterns in CD8+ T cell subsets was stable upon long-term in vitro culture. TBKBP1 overexpression resulted in enhanced TBK1 phosphorylation upon stimulation of CD8+ T cells and significantly improved their virus neutralization capacity. Collectively, our data demonstrate that TBKBP1 modulates virus-specific CD8+ T cell responses and could be exploited as therapeutic target to improve adoptive T cell therapies.

overall_design: Peripheral blood mononuclear cells (PBMCs) from CMV-seropositive donors were were isolated, stimulated with CMV-pp65 overlapping peptide pool and subjected to flow cytometric sorting. Genomic DNA was isolated from sorted naive CD8+ T cells, interferon gamma-negative memory CD8+ T cells and interferon gamma-positive CD8+ T cells, and used for Bisulfite-Seq. Differentially methylated regions were identified by pairwise comparison of the samples with the software BSmooth.

first: Zheng

first: Varun

last: Sasidharan-Nair

first: Agnes

last: Bonifacius

first: Jana

last: Niemz

first: Britta

last: Eiz-Vesper

first: Stefan

last: Floess

first: Jochen

last: Huehn


title: Parallel genome-scale CRISPR-Cas9 screens uncouple human pluripotent stem cell identity versus fitness [bulk RNA-seq]

overall_design: Bulk RNA-seq of E8 stem cells at WT and TADA2B KO. 3 replicates each.


title: Dosage-sensitive maternal siRNAs determine hybridization success in Capsella

city: Uppsala

first: Juan

middle: C

last: Santos-González

state: Uppsala

address: Almas Allé 5

department: Department of Plant Biology and Forest Genetics

country: Sweden

institute: SLU

type: Non-coding RNA profiling by high throughput sequencing

summary: Hybrid seed failure arising from wide crosses between plant species is a recurring obstacle in plant breeding, impeding the transfer of desirable traits. This postzygotic reproductive barrier primarily occurs in the endosperm, a tissue that nourishes the embryo and functions similarly to the placenta in mammals. We found that incompatible seeds show a loss of DNA methylation and chromatin condensation in the endosperm, similar to seeds lacking maternal RNA polymerase IV activity. This similarity is linked to a decrease in small interfering RNAs in the endosperm (sirenRNAs), maternal RNA polymerase IV-dependent short interfering RNAs that regulate DNA methylation. Several AGAMOUS-like MADS-box transcription factor genes (AGLs), key regulators of endosperm development, are targeted by sirenRNAs in cis and in trans. This finding aligns with the enrichment of AGL target genes among deregulated genes. We propose that hybrid seed failure results from reduced maternal sirenRNAs combined with increased AGL expression, leading to abnormal gene regulation in the endosperm.

sample_organism: Capsella bursa-pastoris x Capsella grandiflora

overall_design: Each RNA-Seq library was prepared from seeds taken out of 10-15 siliques. Three biological replicates were used for each genotype. The BS-Seq and sRNA-SEq libraries were prepared from manually dissected endosperm of around 600 seeds. Two biological replicates were used for each genotype.

first: Katarzyna

last: Dziasek

first: Kai

first: Yichun

last: Qiu

first: Jiali

last: Zhu

first: Diana

last: Rigola

first: Koen

last: Nijbroek

first: Claudia

last: Köhler


title: RTP801 INTERACTS WITH THE tRNA LIGASE COMPLEX AND DYSREGULATES ITS RNA LIGASE ACTIVITY IN ALZHEIMER’S DISEASE

city: Barcelona

first: Genís

last: Campoy Campos

state: Barcelona

address: C. Casanova 143 3rd Floor North Wing, Biochemistry Unit

country: Spain

institute: Universitat De Barcelona

summary: RTP801/REDD1 is a stress-responsive protein overexpressed in neurodegenerative diseases such as Alzheimer’s disease (AD) that contributes to cognitive deficits and neuroinflammation. Here we found that RTP801 interacts with HSPC117, DDX1, and CGI-99, three members of the tRNA ligase complex (tRNA-LC), which ligates the excised exons of intron-containing tRNAs and the mRNA exons of the transcription factor XBP1 during the unfolded protein response (UPR). We also found that RTP801 modulates the mRNA ligase activity of the complex in vitro, since RTP801 knockdown promoted XBP1 splicing and the expression of its transcriptional target, SEC24D. On the contrary, RTP801 overexpression inhibited the splicing of XBP1. In this line, in human AD postmortem hippocampal samples, where RTP801 protein levels are upregulated, we found that XBP1 splicing dramatically decreased. In the 5xFAD mouse model of AD, silencing RTP801 expression in hippocampal neurons promoted Xbp1 splicing and prevented the accumulation of intron-containing pre-tRNAs. Finally, the tRNA-enriched fraction obtained from 5xFAD mice promoted abnormal dendritic arborization in cultured hippocampal neurons, and RTP801 silencing in the source neurons prevented this phenotype. Altogether, these results show that elevated RTP801 impairs RNA processing in vitro and in vivo, in the context of AD and suggest that RTP801 inhibition could be a promising therapeutic approach.

overall_design: To study the effect of RTP801 on tRNA processing, we specifically silenced RTP801 in the hippocampal neurons of 6-month-old wild type (WT) and 5xFAD mice using shRNA (shRTP801, or shControl (shCT)) and analyzed the pool of hippocampal tRNAs by Hydro-tRNA-seq

last: Campoy-Campos

first: Julia

last: Solana-Balaguer

first: Anna

last: Guisado-Corcoll

first: Almudena

last: Chicote-González

first: Pol

last: Garcia-Segura

first: Leticia

last: Pérez-Sisqués

first: Adrian

last: Gabriel Torres

first: Mercè

last: Canal

first: Laura

last: Molina-Porcel

first: Joaquín

last: Fernández-Irigoyen

first: Enrique

last: Santamaria

first: Lluis

last: Ribas de Pouplana

first: Jordi

last: Alberch

first: Eulàlia

last: Martí

first: Albert

last: Giralt

first: Esther

last: Pérez-Navarro

first: Cristina

last: Malagelada


title: Force-mediated recruitment and reprogramming of healthy endothelial cells in cerebral cavernous malformations

city: Leuven

first: Apeksha

last: Shapeti

address: Celestijnelaan 300C

department: Mechanical Engineering

country: Belgium

institute: KU Leuven

summary: Cerebral cavernous malformations (CCMs) are vascular abnormalities in the brain characterized by clusters of leaky, tumor-like vessels that often lead to cerebral hemorrhage. Disruptions in signaling pathways involved in endothelial mechanotransduction and angiogenesis due to loss-of-function mutations in CCM genes are known to contribute to lesion formation. Recent studies have highlighted the critical role of wild-type cell recruitment from the surrounding endothelium by CCM mutants in lesion progression. However, the specific mechanisms underlying this recruitment process remain poorly understood. We report here that hyper-angiogenic CCM2-deficient endothelial cells enhance angiogenic invasion of neighboring wild-type cells. Mechanically hyperactive CCM2-deficient tips generate pulling forces on wild-type stalk cells and leave degraded paths in the matrix as cues to promote invasion. We wanted to further answer if mutant cells are capable of modifying the transcriptomic signature of neighbouring healthy endothelial changes in addition to the phenotypic modifications. We used  single-cell RNA-sequencing (10x Genomics Chromium) to identifiy endothelial cell clusters. We enriched the endothelial cells and then used 10X Genomics Chromium technology to capture 1434 cells. The analysis was then performed using Seurat to identify each cell cluster. The results revealed that mutant cells were able to reprogram wild-type cells into stalk cells, with the activation of matrisome and DNA replication programs, eventually leading to cell proliferation. These findings unveil a novel mechanism by which proliferative wild-type cells contribute to CCM lesion growth and identify dysregulated cell mechanics as a “third hit” in CCM pathogenesis.

overall_design: Wild type (WT) human umbilical vein endothelial cells (HUVECs) were labeled with Lifeact-RFP, while control (CT) or CCM2 siRNA silenced HUVECs were labeled with Lifeact-GFP. Red wild type HUVECs were combined in a 1:1 ratio with either siCT or siCCM2 HUVECs and allowed to invade overnight as mosaics into a PEG based hydrogel using our 3D-in vitro model of angiogenic sprouting. Hydrogels were then dissolved and the cells collected. WT cells were separated from the siRNA silenced HUVECs using Fluorescence activated cell sorting  (FACs) according to the presence of lifeact-GFP or lifeact-RFP signal. The 3 samples collected for single cell RNA sequencing were WT cells invading in mosaics with siCT cells termed "WTinCT", WT cells invading in mosaics with siCCM2 cells termed "WTinCCM2", and siCCM2 cells from the mosaics termed "CCM2". The cells were collected from 4 independent experiments and in triplicates of hydrogels per trial. All collected and sorted cells were then pooled such that each sample (WTinCT, WTinCCM2, CCM2) consisted of the respective cell type from all trials and replicates.

first: Jorge

last: Barrasa-Fano

first: Abdel

last: Abdel Fattah

first: Janne

last: de Jong

first: José

middle: A

last: Sanz-Herrera

first: Mylène

last: Pezet

first: Said

last: Assou

first: Emilie

last: de Vet

first: Seyed

last: Ali Elahi

last: Ranga

first: Eva

last: Faurobert

first: Hans

last: Van Oosterwyck


title: Detect IGF1R function in postnatal bone marrow mesenchymal stem cell

status: Public on Sep 11 2024

city: Guangzhou

first: Qi

last: Lou

address: 74 Zhongshan 2nd road

institute: Sun Yat Sen University

summary: 1. IGF1R signaling pathway is highly enriched in P0.5 BMMSC compared to adult BMMSC; 2. Niche factors are highly expressed in PDGFRa+Sca1+ (PaS) cells. 3. Knockout IGF1R in Prx1+ BMMSC significantly impaired niche factors expression in P0.5 BMMSC.

overall_design: Flow sorted adult PDGFRa+ adult BMSCs, perinatal PDGFRa+Sca1+ BMSCs, perinatal PDGFRa+Sca1- cells, and PDGFRa+Sca1+ perinatal BMSCs with or without IGF1R deletion for RNAseq.


title: Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34+ cell therapy to induce fetal hemoglobin for sickle cell disease [CHANGE-seq]

city: Memphis

first: Shengdar

last: Tsai

state: TN

address: 262 Danny Thomas Pl

department: Department of Hematology

institute: St Jude Children's Research Hospital

summary: To define the genome-wide activity of Cas9-sgRNA directed at the gamma-globin promoter genes, we performed CHANGE-seq and identified 678 off-target sites from plerixafor-mobilized normal donors of African American ancestry (pre-GMP engineering runs, n = 3). Most of these off-targets are in intergenic and intronic regions of the human genome, as expected based on their relative genomic proportions. We selected the 277 CHANGE-seq candidate sites reproducibly identified in 2 or more biological donors and in silico predicted sites for multiplexed targeted amplicon sequencing (rhAmp-seq, IDT).

overall_design: We applied CHANGE-seq, a highly-sensitive in vitro based biochemical assay to detect potential off-target sites from plerixafor-mobilized normal donors of African American ancestry (pre-GMP engineering runs, n = 3).

first: Varun

last: Katta

first: Garret

last: Manquen

first: Yichao

last: Li


title: Immune niches in brain metastases contain TCF1+ stem-like T cells, are associated with disease control and are modulated by preoperative SRS

city: Atlanta

first: Haydn

last: Kissick

state: Georgia

address: 1462 Clifton Rd

institute: Emory University

summary: We analyzed immune infiltration in 67 brain metastasis (BrM) and found high frequencies of PD1+TCF1+stem-like CD8+T-cells and TCF1-effector-like cells.

overall_design: CD45+ TILS and CD8+ TILS were mixed at a 1:1 ratio from patients with irradiated and unirradiated brain metastases were FACS-purified for every sample. Single-cell suspensions of FACS-purified cells were loaded onto the 10X Genomics Chromium Controller. Library construction was performed using the Chromium Single Cell 5’ Library Construction Kit followed by sequencing on a HiSeq3000.

first: Zach

last: Buchwald

first: Meghana

last: Pagadala


title: 2’3’-cGAMP binds and activates Rab18 to promote cell migration

city: Chapel Hill

first: Austin

middle: J

last: Hepperla

state: NC

address: 7018B Mary Ellen Jones Building

department: Genetics

institute: University of North Carolina at Chapel Hill

summary: Cyclic dinucleotides serve as bacterial secondary messengers regulating sporulation, motility, biofilm formation and virulence. A nonsymmetric c-di-GAMP is firstly identified in bacteria to promote colonization, while mammalian 2’3’-cGAMP is synthesized by cGAS through binding and activating STING to trigger innate immune activation. However, pathophysiological function of 2’3’-cGAMP beyond innate immunity remains elusive. Here, we report 2’3’-cGAMP facilitates cell migration independent of STING and its innate immune function. 2’3’-cGAMP interactome analysis with a targeted shRNA-screen identifies the GTPase Rab18 as a direct 2’3’-cGAMP binding partner and effector in cell migration control. Mechanistically, 2’3’-cGAMP binds Rab18-S17, E36 and R92 residues to facilitate Rab18 activation and subsequently promotes FosB transcription in promoting cell migration. Low-dose doxorubicin induces 2’3’-cGAMP synthesis and facilitates cell migration in vitro and in vivo. Interestingly, lovastatin induces Rab18 phosphorylation abolishes 2’3’-cGAMP recognition to antagonize 2’3’-cGAMP induced cell migration.  Together, our study reveals a novel 2’3’-cGAMP function in cell migration control beyond innate immunity via binding Rab18 that provides new insights into clinical applications of 2’3’-cGAMP.

overall_design: RNA-seq comparing THP-1 cells that are untreated or treated with 2'3'-cGAMP

first: Yu

last: Deng

first: Zhichuan

last: Zhu

first: Yanqiong

last: Zhang

first: Brenda

last: Temple

first: Jeremy

last: Simon

first: Charlene

last: Santos

first: Laura

last: Herring

first: Albert

last: Baldwin

first: Pengda

last: Liu


title: MDM2 induces pro-inflammatory and glycolytic responses in M1 macrophages by integrating iNOS-nitric oxide and HIF-1α pathways in mice

status: Public on Sep 16 2024

city: Hung Hom

first: Kelvin

middle: Ka-lok

last: Wu

state: Kowloon

address: Y938, Lee Shau Kee Building, The Hong Kong Polytechnic University

country: Hong Kong

institute: The Hong Kong Polytechnic University

summary: M1 macrophages induce protective immunity against infection, but also contribute to metabolic and inflammatory diseases. Here we show that he E3 ubiquitin ligase, MDM2, promotes the glycolytic and inflammatory activities of M1 macrophage by increasing the production of IL-1β, MCP-1 and nitric oxide (NO). Mechanistically, MDM2 triggers the ubiquitination and degradation of E3 ligase, SPSB2, to stabilize iNOS and increases production of NO, which s-nitrosylates and activates HIF-1α for triggering the glycolytic and pro-inflammatory programs in M1 macrophages. Myeloid-specific haplo-deletion of MDM2 in mice not only blunts LPS-induced endotoxemia and NO production, but also alleviates obesity-induced adipose tissue-resident macrophage inflammation. By contrast, MDM2 haplodeletion induces higher mortality, tissue damage and bacterial burden, and also suppresses M1 macrophage response, in the cecal ligation and puncture-induced sepsis mouse model. Our findings thus identify MDM2 as an activator of glycolytic and inflammatory responses in M1 macrophages by connecting the iNOS-NO and HIF-1α pathways.

overall_design: To investigate the role of MDM2 in M1 macrophages, we first isolated bone marrow cells from C57BL/6J mice and differentiated them into bone marrow-derived macrophages (BMDM) using L929-conditioned medium. BMDM were then transfected with siRNA targeting MDM2 (siMdm2) or a scramble control (siScramble) respectively, followed by stimulation with 100 ng/ml lipopolysaccharide (LPS) and 100 ng/ml interferon-gamma (IFNg) for 20 hours.

first: Kelvin K

first: Xiaofan

last: Xu

first: Manyin

first: Xiaomu

first: Moinul

last: Hoque

first: Gloria

middle: Y

first: Qizhou

last: Lian

first: Kekao

last: Long

first: Tongxi

last: Zhou

first: Hailong

last: Piao

first: Aimin

first: Hannah

middle: X

last: Hui

first: Kenneth K

last: Cheng


title: Evaluation of breast cancer PDX tumor heterogeneity at single cell resolution II

city: Richmond

first: Wright Center for Clinical and Translational Research

last: Bioinformatics Core

state: VA

address: 203 E Cary St

institute: Virginia Commonwealth University

summary: Breast cancer is the most commonly diagnosed cancer among women. PDXs (patient-derived xenografts) are similar to cancer cell lines but differ in that they are maintained in a physiological setting as soon as they are isolated from the patient and for subsequent passages. These models are valuable for preclinical trials because PDX models have been shown to closely match their patient counterparts, both in genomic profile and response to treatment. One challenge to treatment development is tumor heterogeneity. In this study, we profiled ER+ and triple negative breast cancer PDX models using single-cell RNA-sequencing. This data may help identify populations of cells which are susceptible to certain treatments in order to improve clinical outcomes for breast cancer patients.

overall_design:
ER+, HER2 amplified, and triple-negative mammary tumors were subjected to single-cell RNA sequencing with the goal of identifying and profiling sub-populations within models to improve outcomes of targeted treatments. This dataset contains a total of 117 samples that includes new and reanalyzed samples with and without raw data present in the SRA.

This GEO record is organized as follows: the “Samples” section below contains 23 new samples with raw data submitted to the SRA; the “Relations” section contains links to the remaining 94 reanalyzed sample GSM records (see the “GSE276609_sample_file_association_for_reanalysis.txt.gz” file for mapping to reanalyzed data and sample names); of the 94 reanalyzed samples 25 are from GSE235168 that have raw data previously submitted to the SRA and are listed in the “TABLE OF REANALYZED/REUSED DATATABLE OF REANALYZED/REUSED DATA”; the remaining 69 samples are from GEO Series GSE161529 and do not have raw data archived in the SRA. These 69 samples from GSE161529 were re-analyzed by the original authors on our request by aligning the raw data using 10X Genomics CellRanger v6 software and then sharing the read count matrices with our team who incorporated them into the same pipeline as the rest of the data in this submission. 10X formatted read count matrices and additional process data for the 69 samples from GSE161529 can be found in the tar file below named “GSE276609_GSE161529_reanalysis.tar.gz”.  Processed data, including 10X formatted read count matrices, for the 25 reanalyzed samples from GSE235168 can be found in the tar file below named “GSE276609_GSE235168_reanalysis.tar.gz”. Likewise, the read counts and other processed data files for the 23 new samples that are part of this submission can be found in the “GSE276609_RAW.tar” tar file below. For all data, the processed files with “CancerCellsOnly” in the file name contains a list of the cell barcodes that we identified as cancer cells using the inferCNV tool as described in the manuscript.  In addition to read counts and cancer cell barcode lists, we provide 5 Loupe Cell Browser files that contain the merged dataset as presented in the paper (.cloupe files).  To view layouts consistent with the manuscript, you need to change the projection to the imported “UMAP_Seurat” or the imported “tSNE_Seurat” options as the defaults are not consistent with the publication figures.  Loupe file contents are as follows: MasterMerge_AllSamples.cloupe.gz contains all 117 samples with human cells only; CancerOnlyDataset.cloupe contains the subset of identified malignant cells from the Master Merge file with all normal samples removed (the CancerCellsOnly processed data files contain a list of barcodes for each sample that are in this dataset);   CancerOnly_TNBC-Samples.cloupe is a subset of the CancerOnlyDataset file contain only TNBC samples; CancerOnly_ERpositive-Samples.cloupe is a subset of the CancerOnlyDataset file that contains only ER+ samples; WHIM30_PDX_PDXO_SamplesOnly.cloupe contains only the WHIM30 PDX and organoid samples.

*********************************************************************
The table below lists GEO accessions reused/reanalyzed for this study where fastq files are available at SRA.
*********************************************************************

first: J

last: Harrell

first: A

middle: L

last: Olex

first: M

last: Dozmorov

last: Altman

first: D

last: Boyd

first: E

last: Zboril


title: mRNA deadenylation modeling at permissive and stress conditions reveals complex relations to decay

city: Warsaw

first: Andrzej

last: Dziembowski

address: 4 Ks. Trojdena Street

country: Poland

institute: International Institute Of Molecular And Cell Biology In Warsaw

summary: The prevailing model postulates that complete cytoplasmic polyadenosine tail (pA-tail) deadenylation is essential for initiating mRNA decapping and subsequent degradation. To investigate this, we conducted direct RNA sequencing of yeast mRNAs derived from steady-state and stress condition chase experiments. Subsequently, we developed a numerical model based on a modified gamma distribution function, which estimated the transcriptomic deadenylation rate at 10 A/min. A simplified independent method, based on the delineation of quantile pA-tail values, showed a correlation between the decay and deadenylation rates of individual mRNA, which appeared consistent within functional transcript groups and associated with to codon optimality. Notably, these rates varied during the stress response. Detailed analysis of ribosomal protein-coding mRNAs (RPG mRNAs), constituting 40 % of the transcriptome, singled out this transcript group. While RPG mRNA deadenylation and decay accelerated under heat stress, their degradation could proceed even when deadenylation was blocked, depending entirely on ongoing nuclear export. Our findings support the general primary function of deadenylation in dictating decapping onset, while also demonstrating complex relations between these processes.

overall_design: We implemented Nanopore Direct RNA Sequencing to we experimentally model RNA deadenylation and decay rates under steady-state and stress conditions using: (1) the Anchor-Away system, to deplete the main cellular export factor, Mex67, thus rapidly inducing massive nuclear degradation of newly-synthesized mRNAs, (2) depletion of the decapping enzyme Dcp2, Xrn1 5'-3' exonuclease, or Pab1 poly(A)-binidng protein using the auxin-inducible degron (AID) system, (3) a heat stress chase, (4) a thiolutin chase.

first: Agnieszka

last: Czarnocka-Cieciura

last: Tudek

first: Paweł

last: Krawczyk

first: Seweryn

last: Mroczek


title: Unbiased screening of regulatory insulators

status: Public on Sep 09 2024

city: Lausanne

first: Maria Cristina

last: Gambetta

address: Genopode Building

department: Center for Integrative Genomics

country: Switzerland

institute: University of Lausanne

summary: Long-range transcriptional activation of gene promoters by abundant enhancers in animal genomes raises the need for mechanisms to limit inappropriate gene regulation. DNA elements known as insulators provide one such mechanism by shielding promoters from an enhancer when placed in between these two elements. Whereas promoters and enhancers have been extensively characterized in animal genomes, insulators have not because of the lack of a high-throughput screening assay. In the absence of unbiased insulator screening in a genome, basic questions remain about the identity, density and diversity of genomic insulators. Here, we establish “insulator-seq” as a plasmid-based massively parallel reporter assay in Drosophila tissue culture cells to perform the first unbiased screen for insulators in any genome. By screening developmental gene loci, we found that not all characterized insulator protein binding sites are able to block enhancer-promoter communication. By dissecting the sequence determinants of functional insulators, we found that an unexpectedly broad sequence context around the central insulator protein binding motif is required for functionality. The ability to screen millions of DNA sequences in parallel without any positional effect has enabled the first functional mapping of insulators and provided further insights into the determinants of functional insulators.

overall_design: For insulator-seq, test fragments were cloned into a barcoded insulator reporter acceptor plasmid library, then transfected into Drosophila S2R+ tissue culture cells. 72 hours post-transfection, total RNA was isolated and PCR amplicon libraries of two reporter genes (eGFP and mCherry) with a shared unique transcribed barcode were generated. In parallel, DNA-sequencing libraries were generated to link a transcribed barcode in a reporter gene to a cloned test fragment. Three screens were performed to test fragments from the abdominal HOX gene locus (BX-C), an extended region around the even-skipped (eve) gene, or synthetic CTCF-dependent insulators and systematically mutated variants thereof. Also, wildtype or BEAF-320 mutant Drosophila melanogaster pupal heads were subjected to Hi-C.

first: Anastasiia

last: Semenova

first: Pascal

last: Cousin

first: Aleksander

last: Jankowski

first: Bihan

last: Wang

first: Julien

last: Dorier

first: Jonas

last: Barraud

first: Sanyami

last: Zunjarrao

first: Maria


title: Non-homogenous intratumor ionizing radiation doses synergize with PD1 and CXCR2 blockade

status: Public on Sep 06 2024

city: Fontenay-Aux-Roses

first: Fabien

last: Milliat

address: 31 avenue de la division Leclerc

country: France

institute: IRSN

summary: Partial tumor irradiation combining low dose radiation with high dose radiation within the same tumor mass would prompt an antitumor effect when combined with immunomodulators. Partial irradiations (50%/50%) of subcutaneous MC38 tumors with millimetric precision to deliver low dose radiation (2Gy) and high dose radiation (16Gy) within the same tumor was performed and single-cell RNA sequencing of CD45+ cells analysis was done in both the two part of the tumor to identify tumor-infiltrating immune cells.

overall_design: scRNA-seq datas of CD45+ cells from subcutaneous MC38 tumors from control mice (Ctrl, 4 replicates, total tumor irradiation (TI) at 2 Gy (TI2, 2 replicates) or 16 Gy (TI16, 2 replicates) or partial irradiations (PI) (50%/50%) either 16Gy/0Gy (PI16/0, 2 replicates) or 16Gy/2Gy (PI16/2, 2 replicates). A total of 16 samples is included in this study.

first: Michele

last: Mondini

first: Paul

last: Bergeron

first: Amine

last: Benadjaoud


title: Cardiomyocyte-specific knockout of ADAM17 alleviates doxorubicin-induced cardiomyopathy via inhibiting TNFα-TRAF3-TAK1-MAPK axis

status: Public on Sep 10 2024

city: Jinan, Shandong

first: Lin

last: Xie

address: No.107, Wen Hua Xi Road

institute: Qilu Hospital of Shandong University

summary: Doxorubicin as a commonly used anthracycline has become the cornerstone of chemotherapy in a wide range of cancers owing to its high efficacy. However, clinical applications of doxorubicin are limited mainly due to its toxic effects on myocardium but the pathogenic mechanism of doxorubicin-induced cardiomyopathy are poorly understood. ADAM17 is known as tumor necrosis factor α converting enzyme (TACE), and the cleavage of TNF-α by ADAM17 is a prerequisite for pro-inflammatory TNF-α activity, which raises a possibility that inhibition of ADAM17 may exert a beneficial effect on disease processes where TNF-α plays an essential role. Our previous research has shown that cardiomyocyte specific knockout of ADAM17 improves diabetic cardiomyopathy by modulating cardiomyocyte apoptosis. However, the relationship between ADAM17 and doxorubicin-induced cardiomyopathy is unclear.Through RNA sequencing analysis, we observed significant changes in the TNF signaling pathway genes in the heart tissue of mice with or without cardiomyocyte ADAM17 knockout.

overall_design: The gene expression of 3 samples from ADAM17flox/flox+NS  was compared to 3 samples from ADAM17flox/flox+DOX by RNA-sequencing. And the gene expression of 3 samples from ADAM17flox/flox+DOX was compared to 3 samples from ADAM17α-MHCKO+DOX were compared by RNA sequencing.

first: L

first: F

last: Xue

first: J

middle: M

last: Yang

first: C

last: Zhang

first: Y