title: Single nucleus RNA sequencing analysis of vagal sensory neurons

city: bronx

first: Young-Hwan

last: Jo

state: NY

address: 1300 Morris Park ave, Forchheimer 505B

institute: Albert Einstein college of Medicine

summary: We report that molecularly distinct populations of vagal sensory neurons would play a role in causing differences in metabolic homeostasis between the sexes.

overall_design: Examination of the molecular identity of vagal sensory neurons in female and male mice


title: Combined bioinformatic and clinical assay uncovers resistance and susceptibility mechanisms of human glioblastomas to temozolomide and identifies new survival biomarkers outperforming MGMT promoter methylation

status: Public on Apr 17 2024

city: Moscow

first: Alexander

last: Modestov

address: 8-2 Trubetskaya str.

country: Russia

institute: I.M. Sechenov First Moscow State Medical University

type: Expression profiling by high throughput sequencing

summary: Glioblastoma (GBM) is the most aggressive and lethal CNS tumor with only 14 months median overall survival after diagnosis and ∼5% 5-years survival rate. The treatment strategy is mainly surgery and/or radiation therapy, both combined with adjuvant temozolomide (TMZ) chemotherapy. TMZ treatment results in methylation of DNA purine bases, where the primary cytotoxic lesion is O6-methylguanine that can be removed by DNA repair enzyme methylguanine methyltransferase (MGMT) when tumors express this protein. Historically, methylation of MGMT gene promoter is used as the major biomarker predicting individual tumor response to TMZ, but there are also additional biomarkers. However, most of them were developed using TCGA project collection of molecular profiles which were unproperly diagnosed as GBM and now need to be reclassified according to the most recent WHO CNS5 tumor classification. Here we for the first time compared biomarker potentials of MGMT methylation, expression levels of 361 DNA repair genes, and activation levels of 38 DNA repair pathways on updated TCGA and other samplings and validated the results on our experimental multicenter GBM patient cohort (n=50). We found that expression/activation levels of 7 and 6 emerging gene/pathway biomarkers served as high-quality positive (HR<0.61) and negative (HR>1.63), respectively, patient survival biomarkers, all performed significantly better than MGMT methylation. Positive survival biomarkers were enriched in the processes of ATM-dependent checkpoint activation and cell cycle arrest whereas negative – in excision DNA repair. We also built and characterized gene signature which was informative for GBM patient survival following TMZ administration (HR 0.27-0.44, p<0,0004; AUC 0.69-0.9).

overall_design: This study was designed to investigate whether the metrics connected to TMZ-related DNA alkylation repair and alternative to MGMT promoter methylation may be related with response on TMZ and survival of GBM patients. The group of 50 experimental clinically annotated GBM biosamples was obtained during a multi-center study of University of Mainz (Germany), Ljubljana University (Slovenia), and Vitamed Clinic (Russia) for patients undergoing Oncobox molecular testing. Among them, 4 biosamples and the respective RNA sequencing profiles were obtained in Vitamed Clinic (Russia).

first: Maria

last: Suntsova

first: Anton

last: Buzdin


title: Maternal and zygotic factors sequentially shape the tissue regionalization of chromatin landscapes in early vertebrate embryos

status: Public on Apr 19 2024

city: Irvine

first: Kitt

middle: D.

last: Paraiso

state: CA

address: 4410 Nat Sci II, University of California, Irvine

department: Department of Developmental and Cell Biology

country: USA

institute: University of California, Irvine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: ChIP-seq and RNA-seq analyses to understand the establishment of the cis-regulatory mark H3K4me1 during early embryogenesis.

overall_design: We examined the spatial regionalization of H3K4me1 marking across the early Xenopus tropicalis embryo. We performed H3K4me1 ChIP-seq in ectoderm, mesoderm and endoderm samples [note that the endoderm samples were previously released in GSE118024]. Subsequently, we performed H3K4me1 ChIP-seq alpha-amanitin treated embryos (ectoderm, mesoderm and endoderm) and Otx1/Vegt/Foxh1 knockdown embryos (endoderm). We also performed single embryo RNA-seq on 15 individual embryos from 3 clutches to compare the expression variation genes with or without H3K4me1 marking.

middle: D

first: Ken

middle: W

last: Cho


title: Spatial Transcriptomic Analysis of Superficial Peritoneal Endometriotic Lesions

status: Public on Apr 16 2024

city: Grand Rapids

first: Gregory

last: Burns

state: MI

address: 400 Monroe Ave NW

department: Obstetrics, Gynecology and Reproductive Biology

institute: Michigan State University

summary: The mechanisms underlying the pathophysiology of endometriosis, characterized by the presence of endometrium-like tissue outside the uterus, remain poorly understood. This study aimed to identify cell type-specific gene expression changes in superficial peritoneal endometriotic lesions and elucidate the crosstalk among the stroma, epithelium, and macrophages compared to patient-matched eutopic endometrium. Surprisingly, comparison between lesions and eutopic endometrium revealed transcriptional similarities, indicating minimal alterations in the sub-epithelial stroma and epithelium of lesions. Spatial transcriptomics (NanoString GeoMx) highlighted increased signaling between the lesion epithelium and macrophages, emphasizing the role of the epithelium in driving lesion inflammation. We propose that the superficial endometriotic lesion epithelium orchestrates inflammatory signaling and promotes a pro-repair phenotype in macrophages, providing a new role for Complement 3 in lesion pathobiology. This study underscores the significance of considering spatial context and cellular interactions in uncovering mechanisms governing disease in endometriotic lesions.

overall_design: Superficial peritoneal lesions and eutopic uterine tissue were collected from five women in the secretory phase of the menstrual cycle. Matched lesion and eutopic tissues (n = 10) were selected. Duplicate regions of interest were selected for each tissue and segmented based on fluorescence intensity for macrophages (marker: CD68+), epithelium (marker: pan-cytokeratin+), and stroma (marker: ACTA2-, CD68-, pan-cytokeratin-), resulting in 60 segments/AOI (area of illumination).

first: Zhen

last: Fu

first: Erin

middle: L

last: Vegter

first: Zachary

middle: B

last: Madaj

last: Greaves

first: Idhaliz

last: Flores

first: Asgerally

middle: T

last: Fazleabas


title: Age-dependent regulation of axoglial interactions and behavior by oligodendrocyte AnkyrinG

status: Public on Apr 12 2024

city: Houston

first: Matthew

middle: Neil

last: Rasband

state: Texas

address: 1 Baylor Plaza

department: Neuroscience

institute: Baylor College of Medicine

summary: The bipolar disorder (BD) risk gene ANK3 encodes the scaffolding protein AnkyrinG(AnkG). In neurons, AnkG regulates polarity and ion channel clustering at axon initial segments and nodes of Ranvier. Disruption of neuronal AnkG causes BD-like phenotypes in mice. During development, AnkG is also expressed at comparable levels in oligodendrocytes and facilitates the efficient assembly of paranodal junctions. However, the physiological roles of glial AnkG in the mature nervous system, and its contributions to BD-like phenotypes, remain unexplored. Here, we generated oligodendroglia-specific AnkG conditional knockout mice and observed the destabilization of axoglial interactions in aged but not young adult mice. In addition, these mice exhibited profound histological, electrophysiological, and behavioral pathophysiologies. Unbiased translatomic profiling revealed potential compensatory machineries. These results highlight the critical functions of glial AnkG in maintaining proper axoglial interactions throughout aging and suggests a previously unrecognized contribution of glial AnkG to neuropsychiatric disorders.

overall_design: We performed unbiased translatomic profiling of oligodendrocytes from the whole brain homogenate of NG2CreERTM;Ank3F/F;Rpl22HA/+ (AnkG icKO RiboTag) versus NG2CreERTM;Rpl22HA/+ (control RiboTag) mice

first: Xiaoyun

last: Ding

first: Emily

last: Ricco

first: Victoria

last: Rodriguez


title: Stroma-derived Dickkopf-1 suppresses NK cell cytotoxicity in breast cancer

status: Public on Apr 11 2024

city: SAINT LOUIS

first: Seunghyun

last: Lee

state: MO

address: 660 S. Euclid Ave., Box 8228

institute: Washington University in St.Louis

summary: Effects of DKK1 on tumor cells and NK cells

overall_design: Tumor cells: GFP-H2B-mApple-Thy1.1+ PyMT-BO1 cells from orthotopic breast tumors 15 days after tumor inoculation in IgG or mDKN01-treated WT mice were sorted, after exclusion of Ter119+ erythrocytes and CD45+ immune cells and extracted RNA for sequencing. NK cells: NK cells sorted from the spleen of poly I:C treated C57BL/6 mice using negative selection beads and stimulated ex vivo with rDKK1 for 4 hours. Then, further sorted cells based on CD45+CD3-NK1.1+ markers  and extracted RNA for sequencing

first: Roberta

last: Faccio


title: RNAseq for WT and osino80

status: Public on Apr 10 2024

city: Shanghai

first: Wenhao

last: Xie

state: Shanghai

address: No. 2005, Songhu Road

department: School of Life Science

country: China

institute: Fudan University

summary: To investigate the effect of the OsINO80, we analysed differentially expressed genes of osino80 mutant compared to WT. We then performed gene expression profiling analysis using data obtained from RNA-seq of WT and osino80 mutant.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for WT and osino80 plants.

first: Aiwu

last: Dong

first: Jiachen

last: Wang

first: Xiang

last: Li

first: Liufan

last: Yin

first: Kangxi

last: Du

first: Jiabing

last: Wu


summary: To investigate the effect of the OsINO80, we analysed OsINO80 binding regions and analysed genome-wide H3, H2A, H2A.Z, H2Aub, H3K9me2, H3K4me2, H3K4me3, H3K27me3, and H3K36me3 in the mutant and WT by ChIP-seq.

overall_design: ChIP-seq for OsINO80 location using YFP antibody, and ChIP-seq for WT and osino80 using H3, H2A, H2A.Z, H2Aub, H3K9me2, H3K4me2, H3K4me3, H3K27me3, and H3K36me3 antibodies.


title: ChIP-seq, RNA-seq and BS-seq for WT and osino80

type: Methylation profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Anchor-Enhanced Bead Design for Reduced Oligonucleotide Synthesis Errors in Single-cell sequencing

status: Public on Apr 09 2024

city: Oxford

first: Adam

last: Cribbs

address: Windmill Road

department: NDORMS

country: United Kingdom

institute: University of Oxford

summary: Single-cell transcriptomics, reliant on the incorporation of barcodes and unique molecular identifiers (UMIs) into captured polyA+ mRNA, faces a significant challenge due to synthesis errors in oligonucleotide capture sequences. These inaccuracies, which are especially problematic in long-read sequencing, impair the precise identification of sequences and result in inaccuracies in UMI deduplication. To mitigate this issue, we have modified the oligonucleotide capture design, which integrates an interposed anchor between the barcode and UMI, and a 'V' base anchor adjacent to the polyA capture region. This configuration is devised to ensure compatibility with both short and long-read sequencing technologies, facilitating improved UMI recovery and enhanced feature detection, thereby improving the efficacy of droplet-based sequencing methods.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: In order to evaluate the accuracy of our spacer UMI assignment we captured mixed mouse and human cells (at ratio of 50:50) and then performed single-cell RNA sequencing.

middle: P


title: BSseq for WT and osino80

summary: To investigate the effect of the OsINO80, we analysed DNA methylation of osino80 mutant compared to WT.

overall_design: Comparative DNA methylation analysis of BS-seq data for WT and osino80 in rice.


title: Single-cell Transcriptomic Analysis Reveals Landscape of Epithelial-Mesenchymal Transition Molecular Heterogeneity in Esophageal Squamous Cell Carcinoma

city: Jinan

first: Dianhao

last: Guo

address: 6699 Qingdao Road

department: Institute of Basic Medicine

institute: shandong first medical university

summary: We performed gene expression profiling analysis using data obtained from RNA-seq of 2 ESCC cell line and 1 normal esophageal

overall_design: Comparative gene expression profiling analysis of RNA-seq data for kyse30/150 cells and heec cell line.

first: Xinxing

last: Lyu

first: Yang

last: Jia


title: Role of hypothalamic microglial activation on ventricular arrhythmias in stress cardiomyopathy

status: Public on Apr 01 2024

city: Harbin

first: Bin

state: Heilongjiang

address: No. 23, Postal Street, Nangang District

department: Department of Cardiology

institute: First Affiliated Hospital of Harbin Medical University

summary:
Background: Current stress cardiomyopathy (SCM) has a high incidence in older adults, and the theories revolve mostly around "catecholamine myocardial toxicity" and "sympathetic hyperactivation". However, the role of the central nervous system (CNS) in the pathogenesis of stress cardiomyopathy remains unknown.
Methods: We investigated the role of microglia activation in the paraventricular hypothalamic nucleus (PVN) in the development of SCM. To create a SCM model, male Sprague-Dawley (SD) rats were immobilized for 6 hours every day for a week. Electrocardiogram, cardiac electrophysiology, and echocardiography were measured to verify the changes in cardiac structure and function in rats with stress cardiomyopathy. RNA sequencing was measured to explore the changes in the hypothalamus of stress cardiomyopathy. In addition, brain and heart tissues were extracted to detect microglial activation and sympathetic activity.
Results: (1) Immobilization stress successfully induced SCM in SD rats. (2) Microglia were significantly activated in the hypothalamus, as evidenced by cytosol thickened and an increased in the number of branches and specifically enriched at PVN. (3) In SCM, microglia in the PVN increased central and peripheral cardiac sympathetic activity by increasing the expression of neuroinflammatory factors. (4) It is possible that inhibiting microglia activation could suppress central and heart sympathetic activity and increase the cardiac electrical stability in SCM rats.
Conclusions: Immobilization stress induced SCM in SD rats can activate hypothalamic microglia, and the activated microglia are specifically enriched at PVN, which can increase the central and peripheral sympathetic nerve activity by regulating the expression of neuro-inflammatory factor, mediate left heart dysfunction and increase the susceptibility to ventricular fibrillation.

overall_design: To investigate the role of PVN microglial cell activation in stress cardiomyopathy, we established a rat model of stress cardiomyopathy using immobilized stress, and microglial cells can be inhibited by minocycline. We collected PVN (the experiment was divided into three groups: model, minocycline, and control) for RNA- seq, comparing changes in gene expression in the RNA-seq data

first: Pengqi

last: Lin

first: Quanwei

last: Pei

first: Jiemei

last: Yang

first: Lina

last: Zou

first: Dezhan

last: Su

first: Junpei

last: Zhang

first: Hongpeng

first: Mbabazi

last: Nadine

first: Junjie

first: Zhaolei

last: Jiang

first: Jingjie

first: Dechun


title: Integrative transcriptome and proteome profiling of insulin-resistant kidney cell models and patient biopsies reveals common and cell-type-specific mechanisms underpinning Diabetic Kidney Disease

status: Public on Apr 02 2024

city: Lausanne

first: Richard

last: Coward

address: Dorothy Hodgkin Building, Whitson Street, Bristol

department: Bristol Medical School

institute: Bristol Royal hospital for Sick Children & University of Bristol

summary: Diabetic kidney disease (DKD) is the leading cause of end stage kidney failure worldwide. It is now clear that cellular insulin resistance is a major driver of this disease. Using established human conditionally immortalised podocytes (Pods), glomerular endothelial cells (GECs), mesangial cells (MCs), and proximal tubular cells (PTCs), we modelled both insulin sensitivity and insulin resistance and performed simultaneous transcriptomics and proteomics for integrated analysis. Our data was further compared with bulk- and single-cell transcriptomic kidney biopsy data from early- and advanced-stage DKD patient cohorts. We identified several consistent changes (individual genes, proteins, and molecular pathways) occurring across all insulin-resistant kidney cell types, which were replicated in human early- and/or advanced-stage DKD biopsies. These included the genes CTSS, NRBF2, C3, CXCL1, TFPI2 and PFKFB3, and pathways related to the inflammatory response, ER stress and glycoprotein metabolism. We further identified several cell-line-specific molecular changes occurring in response to insulin resistance, which were replicated in single-cell sequencing data from DKD, together with a selective reduction in mitochondrial function in Pods, MCs and PTC, but not GECs. This study provides a rich data resource to direct future studies in elucidating underlying kidney signalling pathways and potential therapeutic targets in DKD.

overall_design: Conditionally immortalised human glomerular endothelial cells (GECs), podocytes (Pods), mesangial cells (MCs) and proximal tubular cells (PTCs) were studied in vitro in a basal and insulin resistant environment (consisting of 1ng/ml TNFα, 1ng/ml IL-6, 25mM glucose and 100nmol/L insulin). Insulin-sensitive cell lines were established via stable overexpression of the human insulin receptor.


title: Robust single nucleus RNA sequencing reveals depot-specific cell population dynamics in adipose tissue remodeling during obesity

city: Indianapolis

first: Jisun

last: So

state: IN

address: 635 Barnhill Drive

department: Biochemistry and Molecular Biology

institute: Indiana University School of Medicine

summary: Single nucleus RNA sequencing (snRNA-seq), an alternative to single cell RNA sequencing (scRNA-seq), encounters technical challenges in obtaining high-quality nuclei and RNA, persistently hindering its applications. Here, we present a robust technique for isolating nuclei across various tissue types, remarkably enhancing snRNA-seq data quality. Employing this approach, we comprehensively characterize the depot-dependent cellular dynamics of various cell types underlying adipose tissue remodeling during obesity. By integrating nuclear RNA-seq data from adipocyte nuclei of varying sizes, we identify distinct adipocyte subpopulations categorized by size and functionality. Specifically, we characterize dysfunctional hypertrophic adipocytes prevalent in visceral adipose tissues during obesity, exhibiting cellular stress, inflammation and impaired metabolic gene expression. Obesity-induced changes in gene expression profiles of adipocyte subpopulations reveal their distinct contributions to adipose tissue pathophysiology. Our study establishes a robust snRNA-seq method, providing novel insights into the mechanisms orchestrating adipose tissue remodeling during obesity, with broader applicability across diverse biological systems.

overall_design: We conducted a comparative gene expression profiling analysis of snRNA-seq data from epididymal and inguinal white adipose tissues of lean and obese mice. Additionally, to define nuclear gene expression signatures associated with nucleus size and potentially corresponding to distinct adipocyte subpopulations, we performed RNA-seq on adipocyte nuclei isolated from inguinal white adipose tissues (iWAT) of Adipoq-NuTRAP mice fed a high-fat diet (HFD), sorted by size using flow cytometry. Adipocyte nuclei from iWAT of mice fed a normal chow diet were also included as controls.

first: Hyun Cheol

last: Roh


title: Modulation of apple leaf trancriptome by Erwinia amylovora infection in Evereste (resistant) and MM106 (susceptible) genotypes

city: Beaucouzé

first: Matthieu

last: Gaucher

address: 42 rue Georges Morel

department: SPE

country: France

institute: INRAE

summary: Investigation of whole genome gene expression level changes in leaves of Evereste and MM106 genotypes 6 and 24 hours after infiltration (hpi) by Erwinia amylovora (Ea) versus mock  (water + 0,01 % Silwet). This analysis include transcritome comaprison between the two apple genotypes just before inoculation (T0).

overall_design: A total of 30 samples, corresponding to 3 biological replicates (from 3 independent experiments) for each treatment, were used for differential gene expression analysis : Evereste (T0), MM106 (T0), Evereste (mock 6 hpi), Evereste (Ea 6 hpi), MM106 (mock 6 hpi), MM106 (Ea 6 hpi), Evereste (mock 24 hpi), Evereste (Ea 24 hpi), MM106 (mock 24 hpi), MM106 (Ea 24 hpi).

first: Sébastien

last: Aubourg

first: Sandra

last: Pelletier

first: Marie-Noëlle

last: Brisset


title: Sonic hedgehog medulloblastoma cells in co-culture with cerebellar organoids converge towards in vivo malignant cell states

first: John

last: Jacob

address: Headley Way

department: NDCN

summary: In the malignant brain tumour, sonic hedgehog medulloblastoma the properties of cancer cells are influenced by their microenvironment, but the nature of those effects and the phenotypic consequences for the tumour are poorly understood. The aim of this study was to identify phenotypic properties of SHH-MB cells that were driven by the non-malignant tumour microenvironment. Human iPSC were differentiated to cerebellar organoids to simulate the non-malignant tumour microenvironment. Tumour spheroids were generated from two distinct, long-established SHH-MB cell lines which were co-cultured with cerebellar organoids. We profiled the cellular transcriptomes of malignant and non-malignant cells by performing droplet-based single cell RNA-seq of thousands of cells. The transcriptional profiles of tumour cells in co-culture were compared with those of malignant cells cultured in isolation and with public SHH-MB datasets of patient tumours and PDX models. Heterogeneity of cell states was observed for SHH-MB cells cultured in isolation and in co-culture. Tumour cells in co-culture activated a key marker of differentiating granule cells, NEUROD1 that was not observed in control tumour cells cultured in isolation. In malignant cells in co-culture, cancer stem cell (CSC)-like states emerged that were not observed in control cultures and closely similar or equivalent cell states could be identified in patient tumours. For both SHH-MB cell lines grown in co-culture, there was a convergence of malignant cell states towards in vivo cell states observed in SHH-MB patient tumours and PDX models that were non-cell autonomously induced by the non-malignant microenvironment.

overall_design:
We profiled the cellular transcriptomes of malignant medulloblastoma and non-malignant cerebellar organoid cells by performing droplet-based single cell RNA-seq of thousands of cells.

Grant award numbers: MR/V037730/1 and C2195/A28699
Grant title: Evaluation of a novel in vitro model of sonic hedgehog medulloblastoma by single cell transcriptomics
Funding source: Medical Research Council (UK) to JJ and Cancer Research UK (CRUK) Oxford Centre Clinical Research Training Fellowship to MvE

first: Max

middle: J

last: van Essen

first: Alina

last: Nicheperovich

first: Benjamin

last: Schuster-Böckler

first: Esther

last: Becker


title: Modulation of apple leaf trancriptome by Acibenzolar-S-methyl (ASM)

summary: Investigation of whole genome gene expression level changes in leaves of apple seedlings (Golden delicious) 3 days after treatment by Bion 50WG (ASM) versus water.

overall_design: A total of 6 samples, corresponding to 3 biological replicates (from 3 independent experiments) for each treatment, were used for differential gene expression analysis


title: Castrate Resistant Prostate Cancer Response to Taxane is Determined by an HNF1-dependent Apoptosis Resistance Circuit (RNA-Seq)

city: Bethesda

first: Adam

middle: G.

last: Sowalsky

state: MD

address: 37 Convent Drive Building 37 Room 1062B

department: Genitourinary Malignancies Branch

institute: National Cancer Institute

summary: Metastatic castrate-resistant prostate cancer (mCRPC) is a genetically and phenotypically heterogeneous cancer where advancements are needed in biomarker discovery and targeted therapy. A critical and often effective component of treatment includes taxanes.  We performed one of the first high throughput screens across a cohort of 30 diverse patient-derived CRPC organoids to a library of 78 drugs.   Combining quantitative response measures with transcriptomic analyses demonstrated that HNF1 Homeobox A (HNF1A) drives a transcriptional program of taxane resistance, dependent upon cellular inhibitor of apoptosis protein 2 (cIAP2).  Monotherapy with cIAP2 inhibitor LCL161 was sufficient to treat HNF1A+ models of mCRPC previously resistant to docetaxel.

overall_design:
RNA-seq for LuCaP 23.1 organoids with and without HNF1A expression
***************************************************************
Submitter states that missing raw data are being made available for controlled access in dbGaP under accession ID: phs001587.v3.p1
***************************************************************

first: Ilya

middle: S

last: Senatorov

first: Joel

last: Bowman

first: Brian

last: Capaldo

first: Kathleen

last: Kelly


title: Dynamic co-evolution of transposable elements and the piRNA pathway in East African cichlid fishes [smallRNA-seq]

city: Cambridge

first: Miguel

middle: Vasconcelos

last: Almeida

address: Tennis Court Road

department: Department of Biochemistry

institute: University of Cambridge

type: Non-coding RNA profiling by high throughput sequencing

summary: East African cichlid fishes have diversified in an explosive fashion, but the (epi)genetic basis of the phenotypic diversity of these fishes remains largely unknown. Although transposable elements (TEs) have been associated with phenotypic variation in cichlids, little is known about their transcriptional activity and epigenetic silencing. Here, we describe dynamic patterns of TE expression in African cichlid gonads and during early development. Orthology inference revealed an expansion of piwil1 genes in Lake Malawi cichlids, likely driven by PiggyBac TEs. The expanded piwil1 copies have signatures of positive selection and retain amino acid residues essential for catalytic activity. Furthermore, the gonads of African cichlids express a Piwi-interacting RNA (piRNA) pathway that target TEs. We define the genomic sites of piRNA production in African cichlids and find divergence in closely related species, in line with fast evolution of piRNA-producing loci. Our findings suggest dynamic co-evolution of TEs and host silencing pathways in the African cichlid radiations. We propose that this co-evolution has contributed to cichlid genomic diversity.

sample_organism:
Oreochromis niloticus
Haplochromis burtoni
Astatotilapia calliptera
Maylandia zebra
Tropheops sp. 'mauve'
Pundamilia nyererei

overall_design: To profile small RNAs and identify Piwi-interacting RNAs (piRNAs) in the East African cichlid radiations, we have sequenced small RNAs from ovaries and testes of cichlid species representative of the three East African Great Lakes, as well as of Nile Tilapia (Orechromis niloticus) an outgroup to radiating cichlids. We also sequenced muscle, a somatic tissue which is not expected to have prominent piRNA populations.

middle: V

first: Jonathan

last: Price

first: Eric

middle: A

last: Miska


title: Altered expression of PBMC miRNA profile following the third dose of inactivated COVID-19 vaccine

city: shaoxing

first: guanguan

last: qiu

state: zhejiang

address: Yanan Road123

institute: shaoxing second hospital

summary: COVID-19 vaccination is the most effective approach to prevent severe disease and death. Inactivated vaccines are the most accessible type of COVID-19 vaccines in the developing countries. Several studies, including work from our group, have demonstrated that the third (booster) dose of inactivated COVID-19 vaccine induces robust humoral and cellular immune responses. The aim of this study was to examine miRNA expression profile in participants received a homogenous third dose of the CoronaVac vaccine.

overall_design: The discovery and validation cohorts included 6 volunteers.Samples of peripheral blood mononuclear cells (PBMCs) were harvested from healthcare volunteers before and 1-2 weeks after the third dose of CoronaVac.

first: Guanguan

last: Qiu


title: Castrate Resistant Prostate Cancer Response to Taxane is Determined by an HNF1-dependent Apoptosis Resistance Circuit (ChIP-Seq)

overall_design:
Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for H3K27ac and HFN1a in LuCaP 23.1, 170.2 and 170.3 prostate cancer organoids

***************************************************************
Submitter states that missing raw data are being made available for controlled access in dbGaP under accession ID: phs001587.v3.p1
***************************************************************


title: Single-cell RNA sequencing reveals the cellular and molecular heterogeneity of treatment-naïve primary osteosarcoma in dogs

status: Public on Mar 28 2024

city: Fort Collins

first: Dylan

last: Ammons

state: CO

address: 200 W Lake St

department: Clinical Sciences

institute: Colorado State University

summary: We used single-cell RNA sequencing to characterize the heterogeneity of canine osteosarcoma (OS) tumors.

overall_design: The complete dataset consists of 150bp paired end sequnecing data obtained using a 10x genomics single-cell RNA sequencing platform. The final dataset consists of data from tumor biopsies of 6 dogs diagnosed with osteosarcoma. (Note the raw data pairs tumor_no_tx_1_2 + tumor_no_tx_1_2 and tumor_no_tx_2_1 + tumor_no_tx_2_2 are pairs technical replicates, so the four raw data samples represent 2 biological replicates).

first: Steven

last: Dow


title: Electrolyte and Metabolite Composition of Cystic Fluid from a rat model of ARPKD

status: Public on Mar 30 2024

city: Tampa

first: Alexander

last: Starushchenko

state: FL

address: 560 Channelside Dr

department: Molecular Pharmacology & Physiology

institute: University of South Florida, College of medicine

summary: Polycystic Kidney Disease (PKD) is a devastating inherited kidney disease that is a major cause of kidney failure. Large fluid-filled cysts develop in the kidney and continually expand over time, damaging the surrounding tissue until renal replacement therapy is required. Because the development of cysts is the major feature of this disease, researchers have long sought to determine the composition of cystic fluid. To the best of our knowledge, no one has previously published the ion concentrations of cystic fluid from any PKD rodent models. The overall goal of this study was to characterize the cyst fluid composition of a rat model of ARPKD, the PCK rat. This model developed spontaneously from the Charles River Japan cesarean derived Sprague Dawley (Crj:CD/SD) strain and features a mutation in Pkhd1, the ortholog to the causative ARPKD gene in humans. In addition to analyzing cystic fluid, we complemented our metabolomics approach with transcriptomic analysis of male and female PCK rat kidney cortex tissue.

overall_design: We used the PCK rats, a model of ARPKD (male and female rats, 12-14 wks) and performed RNAseq on kidney tissue to assess sex differences in this PKD rat model

last: Starusachenko

first: Christine

last: Klemens


title: Growth regulated co-occupancy of Mediator and Lsm3 at intronic ribosomal protein genes [RNA-seq]

status: Public on Mar 26 2024

city: Umea

first: ALEXANDER

last: VERGARA

address: Historiegrand 9, 1002

department: Physics

country: Sweden

institute: Umea University

summary: Mediator is a well-known transcriptional co-regulator and serves as an adaptor between gene-specific regulatory proteins and RNA polymerase II. Studies on the chromatin-bound form of Mediator revealed interactions with additional protein complexes involved in various transcription-related processes, such as the Lsm2-8 complex which is part of the spliceosomal U6 snRNP complex. Here we employ ChIP-seq of chromatin associated with the Lsm3 protein and the Med1 or Med15 Mediator subunits. We identify 86 genes co-occupied by both Lsm3 and Mediator of which 73 were intron-containing ribosomal protein genes. In logarithmically growing cells, Mediator primarily binds to their promoter regions but also shows a second, less pronounced occupancy at their 3´-exons. During late exponential phase, we observe a near complete transition of Mediator from these promoters to a position in their 3´-ends, overlapping the Lsm3 binding sites approximately 250 bp downstream of their last intron-exon boundaries. Using an unbiased RNA-seq approach we show that transition of Mediator from promoters to the last exon of these genes correlates to reduction of both their mRNA levels and splicing ratios, indicating that the Mediator and Lsm complexes co-operate to control growth-regulated expression of intron-containing ribosomal protein genes at the levels of transcription initiation and splicing.

overall_design: By employing ChIP-seq of chromatin associated with the Lsm3 subunit of the spliceosomal U6 snRNP complex and Med1 or Med15, subunits of the middle and tail modules of Mediator, respectively, we identified 86 genes co-occupied

first: Wael

middle: R

last: Abdel-Fattah

first: Mattias

last: Carlsson

first: Guo-Zhen

last: Hu

first: Ajeet

last: Singh

last: Vergara

first: Hans

last: Ronne

first: Stefan

last: Björklund

first: Rameen

last: Aslam


title: Growth regulated co-occupancy of Mediator and Lsm3 at intronic ribosomal protein genes [ChIP-seq]

overall_design: By employing ChIP-seq of chromatin associated with the Lsm3 subunit of the spliceosomal U6 snRNP complex and Med1 or Med15, subunits of the middle and tail modules of Mediator, respectively, we identified 86 genes co-occupied by both Lsm3 and Mediator


title: Two distinct Trypanosoma eIF4F complexes co-exist, bind different mRNAs and are regulated during nutritional stress

status: Public on Apr 14 2024

city: Würzburg

first: Bernardo

last: Papini Gabiatti

address: Sanderring 2

country: Germany

institute: University of Würzburg

summary: Many eIF4F subunits and PABP paralogues are found in trypanosomes: six eIF4E, five eIF4G, one eIF4A and two PABPs. They are expressed simultaneously and assemble into different complexes, contrasting the situation in metazoans that use distinct complexes in different cell types or developmental stages. Each eIF4F complex has its own proteins, mRNAs and, consequently, a distinct function. We set out to study the function and regulation of the two major eIF4F complexes of Trypanosoma cruzi. We identified the associated proteins and mRNAs of eIF4E3 and eIF4E4 in cells in exponential growth and in nutritional stress. Upon stress, eIF4G/eIF4A and PABP remain associated to the eIF4E, but the association with other 43S pre-initiation factors decreases, indicating that ribosome attachment is impaired. Most eIF4E3-associated mRNAs encode for metabolic proteins, while eIF4E4 associate to mRNAs encoding ribosomal proteins. Interestingly, for both eIF4E3/4, more mRNAs were associated in stressed cells than in non-stressed cells, even though stress causes ribosome disassembly. In summary, trypanosomes have two co-existing eIF4F complexes involved in translation of distinct mRNA cohorts important for growth. Under stress conditions, both complexes exit translation but remain bound to their mRNA targets.

overall_design: We analysed RNAs associated to Trypanosoma cruzi eIF4E3 and eIF4E4 using RIP-seq in exponential growth and nutritional stress. We also analysed polyA total RNA and sequenced polysomal RNA (Ribo-seq). in these two conditions.

first: Eden

last: Ribeiro Freire

first: Jimena

last: Ferreira da Costa

first: Mariana

last: Galvão Ferrarini

first: Tatiana

last: Reichert Assunção de Matos

first: Henrique

last: Preti

first: Isadora

last: Munhoz da Rocha

first: Beatriz

last: Gomes Guimarães

first: Susanne

last: Kramer

first: Nilson

last: Ivo Tonin Zanchin

first: Fabíola

last: Barbieri Holetz


title: Patient-derived response estimates from zero-passage organoids of luminal breast cancer

status: Public on Mar 22 2024

city: Charlottesville

first: Kevin

middle: A.

last: Janes

state: VA

address: Box 800759 Health System

department: Department of Biomedical Engineering

institute: University of Virginia

summary: Background: Primary luminal breast cancers lose hormone receptors rapidly in standard tissue culture.  Breast cancer organoids are thought to retain tumor characteristics better, but long-term viability of luminal-subtype cases is a persistent challenge.   Methods: We freshly isolated patient-derived cells from luminal tumor scrapes, miniaturized the organoid format into 5 µl replicates to increase throughput, and set an endpoint of 14 days to minimize drift. Therapeutic hormone targeting was mimicked in these “zero–passage” organoids by withdrawing β-estradiol and adding tamoxifen. We tested sulforaphane as an electrophilic stress and commercial neutraceutical. Genetic perturbations were introduced by lentiviral transduction of two complementary sgRNAs and Cas9 stabilization for the first week of organoid culture. Transcriptional changes were measured by RT-qPCR or RNA sequencing; organoid phenotypes were quantified by serial brightfield imaging, digital image segmentation, and regression modeling of cellular doubling times.   Results: We achieved >50% success in initiating luminal breast cancer organoids from tumor scrapes and maintaining them to the 14-day zero-passage endpoint. Few clinical parameters reliably impacted success. Abundance of ESR1 and PGR in zero-passage organoids consistently remained within the range of patient variability at the endpoint. However, responsiveness to hormone withdrawal and blockade was highly variable among luminal breast cancer cases. Combining sulforaphane with knockout of NQO1 (a phase II enzyme) also yielded a breadth of growth phenotypes, including growth inhibition with sulforaphane, growth promotion with NQO1 knockout, and growth antagonism when combined.   Conclusions: Zero-passage organoids are a rapid and scalable way to interrogate properties of luminal breast cancer cells from patient-derived material.

overall_design: RNA-seq was performed on paired tumor scrapes, organoids, and 2D cultured cells from 6 different patients (considered as batches). 6 different luminal A breast cancer cell lines were used as additional controls. For 5 of the patient-derived organoid cultures, we also treated with 0.1% DMSO or 200 nM or 3 µM 4-hydroxytamoxifen from day 5 to day 14 of culture.

first: Roza

middle: K

last: Przanowska

first: Piotr

last: Przanowski


title: Temporal constraints on enhancer usage and enhancer-promoter connectivity shape the regulation of limb gene transcription

city: Geneva

first: Guillaume

last: Andrey

address: Rue Michel-Servet 1

department: Department of Genetic Medicine and Development

country: Switzerland

institute: University of Geneva

summary: Repertoires of transcriptional enhancers orchestrate gene expression during embryonic development, thereby shaping the forms and functions of organs. Within these repertoires individual enhancers display spatially distinct or overlapping activities that collectively build up the expression domain of cognate genes. However, the temporal specificity of these enhancers - how their activities change over developmental time to dynamically influence gene expression - remains uncharacterized. Here, we observed that temporally restricted enhancer repertoires are embedded at numerous loci associated with mouse limb development. To monitor how such enhancer repertoires govern gene transcription in vivo across extensive developmental periods, we introduce the regulatory trajectory framework. This paradigm conceptually involves transcriptional initiation, marking the beginning of gene expression, followed by its maintenance over time, and ultimately decommissioning, leading to gene repression. To track and sort cells undergoing these distinct phases, we devised a transgenic recorder approach at the Shox2 model locus. Through this method, we discovered that cells maintaining Shox2 transcription in early and late limb development relies on distinct, temporally restricted enhancer repertoires. We demonstrate that eliminating early- or late-acting enhancers only transiently affects Shox2 expression indicating that these enhancer repertoires function independently. Additionally, we found that changes in the 3D topology of the locus associate with enhancer activities and that a rapid loss of enhancer-promoter contacts occurs during decommissioning. Finally, we show that the decommissioning of the Shox2 locus can be actively driven by Hoxd13, a gene which expression is known to antagonize Shox2. Overall, our work uncovers the dependency of developmental genes on enhancers with temporally restricted activities to generate complex expression patterns over time and shed light on the dynamics of enhancer-promoter interactions.

overall_design: Refer to individual Series


title: Temporal constraints on enhancer usage and enhancer-promoter connectivity shape the regulation of limb gene transcription [scRNA-seq]

overall_design: We devised a dual-reporter system to monitor Shox2 regulatory trajectories and sort cells undergoing these distinct phases. First, we inserted in mouse embryonic stem cells (mESCs), 1kb upstream of the Shox2 transcriptional start site, a regulatory sensor cassette constituted by a minimal β-globin promoter, a mCherry reporter gene followed by a destabilized PEST sequence, a P2A self-cleavage sequence and a CRE recombinase gene. These cells cells were then retargeted to integrate, at the Rosa26 locus, a cassette with a splice acceptor followed by a floxed 3x SV40pA STOP signal and the EYFP gene open reading frame. We studied the transcriptional identity of these cells by scRNA-seq. We the profile the identity of sorted cells by RNA-seq and their regulatory profile by CHiC and ChIP-seq

first: Raquel

last: Rouco


title: Temporal constraints on enhancer usage and enhancer-promoter connectivity shape the regulation of limb gene transcription [Caupture-HiC]

summary: The repertoire of enhancers orchestrating gene expression during embryonic development shapes the forms and functions of organs. Within regulatory landscapes, enhancers control their target genes through spatially distinct or overlapping activities. However, the temporal specificity of enhancers repertoires—how their activities change over time to dynamically influence gene expression—is less well characterized. In this study, we introduce the regulatory trajectory framework to monitor how enhancer landscapes govern gene transcription in vivo across extensive developmental periods. This paradigm conceptually involves transcriptional initiation, marking the beginning of gene expression, followed by its maintenance over time, and ultimately decommissioning, leading to gene repression. To track and sort cells undergoing these distinct phases, we devised a transgenic recorder approach at the Shox2 model locus. Through this method, we discovered that cells maintaining Shox2 transcription in early and late limb development relies on distinct, temporally restricted enhancer repertoires. We demonstrate that eliminating early- or late-acting enhancers only transiently affects Shox2 expression indicating that these enhancer repertoires function independently. Additionally, we found that changes in the locus' 3D topology associate with enhancer activities and that a rapid loss of enhancer-promoter contacts occurs during decommissioning. Finally, we show that the decommissioning of the Shox2 locus can be actively driven by Hoxd13, a gene which expression is known to antagonize Shox2. Overall, our work uncovers the dependency of developmental genes on enhancers with temporally restricted activities to generate complex expression patterns over time and shed light on the dynamics of enhancer-promoter interactions.


title: Genome-wide analysis of DNA replication by TrAEL-seq in mouse embryonic stem cells

status: Public on Mar 15 2024

first: Sarah

last: Inglesfield

address: Babraham Research Campus

department: Bioinformatics

institute: The Babraham Institute

summary: We have applied the TrAEL-seq method to profile DNA replication in wild type and  Snf2h knockoutmouse embryonic stem cells.

overall_design: 129S6/SvEvTac cells (wt and Snf2h KO) were cultured in duplicate and profiled by the standard TrAEL-seq protocol

first: Abdulkadir

last: Abakir

first: Neesha

last: Kara

first: Jonathan

last: Houseley


title: Dynamic co-evolution of transposable elements and the piRNA pathway in East African cichlid fishes

first: Miguel

last: Almeida

platform_organism:
Oreochromis niloticus
Haplochromis burtoni
Astatotilapia calliptera
Maylandia zebra
Tropheops sp. 'mauve'
Pundamilia nyererei

overall_design: To profile transposable element expression in the East African cichlid radiation, we have sequenced mRNA from ovaries, testes, and brains of cichlid species representative of the three East African Great Lakes, as well as of Nile Tilapia (Orechromis niloticus) an outgroup to radiating cichlids.

last: Price

first: Eric

last: Miska


title: Transcriptomic response of ginsenoside Rc (gRc) in C2C12 myotube under dexamethasone (DEX) treatment

status: Public on Mar 20 2024

city: Daejeon

first: Musun

last: Park

address: 1672 Yuseong-daero, Yuseong-gu

country: South Korea

institute: Korean Institute of Oriental Medicine

summary: This study aimed to observe the effect of gRc on C2C12 myotubes under resting state and DEX treatment to indentify the transcript expression induced by gRc in the C2C12 myotubes.

overall_design: To investigate the influence of gRc on DEX stimulation, C2C12 myotubes were pre-treated with gRc or vehicle (0.1% DMSO) for 12 hours, and then treated with DEX for 24 hours.

first: Aeyung

last: Kim

first: Seongwon

last: Cha


title: Temporal constraints on enhancer usage and enhancer-promoter connectivity shape the regulation of limb gene transcription [RNA-seq]

overall_design:
We devised a dual-reporter system to monitor Shox2 regulatory trajectories and sort cells undergoing these distinct phases. First, we inserted in mouse embryonic stem cells (mESCs), 1kb upstream of the Shox2 transcriptional start site, a regulatory sensor cassette constituted by a minimal β-globin promoter, a mCherry reporter gene followed by a destabilized PEST sequence, a P2A self-cleavage sequence and a CRE recombinase gene. These cells cells were then retargeted to integrate, at the Rosa26 locus, a cassette with a splice acceptor followed by a floxed 3x SV40pA STOP signal and the EYFP gene open reading frame. We studied the transcriptional identity of these cells by scRNA-seq. We the profile the identity of sorted cells by RNA-seq and their regulatory profile by CHiC and ChIP-seq

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The table below lists GEO accessions reused/reanalyzed for this study.
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title: Inflammatory transcriptomic signatures and cell type compositions in inflamed and non-inflamed colonic mucosa of ulcerative colitis

status: Public on Mar 19 2024

city: Dongjak-gu

first: Junil

state: Seoul

address: 360 Songdo-ro

department: Department of Bioinformatics

institute: Soongsil University

summary: Ulcerative colitis is a chronic inflammatory disorder in the lower part of the digestive system. Despite of many functional studies, the hidden mechanisms of this complex disease limit complete remission. Here, we investigated transcriptomic signature of ulcerative colitis using RNA sequencing obtained from 15 active (inflamed), 15 inactive (non-inflamed) samples of ulcerative colitis, and 15 healthy controls. The transcriptomics profiling revealed that inflammatory transcriptomic signature was highly enriched in active samples compared to inactive or healthy control samples. However, this bulk RNAseq cannot provide the cell type information, which play a key role in such a chronic inflammation. To overcome this limitation, we borrowed the power of single cell RNAseq. Deconvolution of bulk RNAseq based on the single cell expression estimated active UC enriched cell types including inflammatory fibroblast and inflammatory monocytes. This integrative approach using bulk and single cell transcriptomics provide not only target genes but also target cell types of ulcerative colitis for therapeutic purpose.

overall_design: We performed RNAseq on total RNA from both areas of the colonic mucosa with active inflammation and without inflammation during the colonoscopy.

first: Zehra

last: Ramsha

first: Jahanzeb

last: Saqib


title: Pan-cancer bioinformatics analysis of TNFRSF12A and functional study in colorectal cancer

status: Public on Mar 13 2024

city: Guangzhou

first: Chuyue

last: Wang

address: Tianhe District, Guangzhou, Guangdong Province

institute: Sun Yat-Sen University

summary: Cancer has become a major public health problem in the world, however challenges such as drug resistance, metastasis and cancer recurrence continue to be unresolved issues. Therefore, it is important to find new cancer markers or therapeutic targets. Tn this study, we found TNF receptor superfamily member 12A (TNFRSF12A) upregulated in cancer and associated with a poor prognosis. TNFRSF12A knockdown inhibited growth, colony formation and migration of colorectal cancer cells, and overexpression TNFRSF12A promoted these behaviors of colorectal cancer cells. RNA-seq results showed that TNFRSF12A overexpression caused increased NF-κB signaling, transcriptional misregulation, and significant upregulation of BIRC3 gene, which may be the transcription target of NF-κB member RELA. Through BIRC3 knockdown in overexpressed TNFRSF12A cells, we proved BIRC3 is a key downstream of TNFRSF12A. Therefore, we speculated the existance of TNFRSF12A/RELA/BIRC3 regulatory axis in colorectal cancer.

overall_design: Comparative gene expression profiling of RNA-seq data from doxycycline-induced TNFRSF12A overexpression DLD-1 cells (oeT) and corresponding control group (oeCtl).


title: Castrate Resistant Prostate Cancer Response to Taxane is Determined by an HNF1-dependent Apoptosis Resistance Circuit

first: Adam

last: Sowalsky

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


title: Arresting the Bad Seed: HDAC3 Regulates Proliferation of Different Microglia after Ischemic Stroke [RNA-seq]

city: Shanghai

first: Yue

last: Zhang

address: 131, Dong'an Road, Xuhui

department: Institute of Brain Science

institute: Fudan University

summary: The rapid accumulation of self-renewed polarized microglia in the penumbra is the critical neuroinflammatory process after the onset of ischemic stroke, leading to secondary demyelination and neuronal loss. HDAC3 has been reported to regulate cell proliferation of tumour cells and modulate neuroinflammation. However, the mechanism by which HDAC3 regulates microgliosis and microglial polarization remains ambiguous. Herein, we demonstrated that microglia-specific ablation of HDAC3 (HDAC3-miKO) ameliorated poststroke long-term functional and histological outcomes. Starting with unbiased RNA seq of microglia, we identified mitosis as the most significant process reversed by loss of HDAC3. Notably, HDAC3-miKO specifically inhibited the proliferation of M1-like microglia but not M2-like microglia, resulting in microglial transition to an M1-like state. Moreover, ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) revealed that HDAC3 deletion induced drastic closing of accessible regions enriched with motifs for PU.1. Taken together, we uncovered for the first time that HDAC3/PU.1-mediated differential proliferation-related reprogramming in different microglia populations drives poststroke inflammatory state transition of microglia and thereby contributes to the pathophysiology of ischemic stroke.

overall_design: Expression profile of RNA-seq data for  WT-Sham, miKO-Sham, WT-tFCI, miKO-Tfci.

first: Jiaying

last: Li

first: Yichen

last: Huang

first: Ziyu

last: Shi

first: Hailian

first: Hui

last: Cao

last: Tan

first: Chenran

first: Yana

first: Di

last: Chen

first: Shuning

first: Yongfang

last: Zhao

first: Yangfan

first: Yueyan

last: Zhu

first: Yan

last: Jiang

first: Ye

last: Gong

first: Yanqin

last: Gao


title: Gene expression dynamics of branching morphogenesis in pancreatic cancer organoids (2)

status: Public on Mar 08 2024

city: Munich

first: Hans

middle: Carlo

last: Maurer

state: Bavaria

address: Ismaninger Str. 22

department: II. Medizinische Klinik

institute: Klinikum rechts der Isar

summary:
The transcriptional dynamics occurring at the self-organizing glandular epithelial during branching morphogenesis are pivotal for deciphering various processes ranging from normal tissue growth to cancer formation.
For the past decade matrigel© has been widely used as extracellular matrix for the propagation of organoids, here we report that only in the presence of a higher than 70% collagen matrix symmetry breaking can be observed. Comparing the transcriptomic profile of collagen and matrigel grown-organoids we identified that matrigel organoids are more basal-like, have a higher EMT signature (GSEA) compared to the more classical collagen grown organoids.
In this study we used single primary murine pancreatic ductal adenocarcinoma cells (PDAC) cells embedded in floating collagen gels and adapted the media supplementation, thus allowing organoids to self-organize into highly complex branched structures, replicating the in vivo tumour architecture. During organoid development we identified four distinct morphogenetic phases, each characterized by a unique pattern of cell proliferation, invasion, matrix deformation and protein expression. In our search for novel molecular drivers of branching morphogenesis we compared the transcriptome of two distinct phases, an early development (day 7) vs a late mature time point (day 13). We compared these two distinct developmental time points in using PCA, GSEA and respective heat maps of major processes illustrating the overexpression of genes associated with: proliferation, ECM interaction, signalling by Rho GTPases and EMT in day-7 organoids and genes associated with ion channel transport at day-13 organoids.
An important aspect raised during our investigation was the effect of different genotypes (Ptf1aCre/+; KrasG12D/+, Pdx1Cre/+; KrasG12D/+; TP53fl/fl) on the branch formation of organoids. To this end we generated organoids derived from KC and KPC tumours, which interestingly gave rise to strikingly similar branching organoids. Although the morphology was not significantly altered the transcriptional profiling showed higher proliferation (Myc targets, E2F targets), EMT score for the KPC organoids.
We propose that the spatiotemporal synchronized processes of cell proliferation, matrix remodeling, contraction and ion channel flux are key-events of the different morphogenic phases and lead to the formation of these complex structures. Importantly, these dynamic processes are accompanied by strong transcriptional profile changes, with the organoids undergoing de-differentiation during branch elongation and later on activating an epithelial gene expression program upon maturation. Taken together, these results illustrate the ability of tumour cells to self-organize in multicellular complex structures and provide with a novel system to study branching morphogenesis and tumour biology in vitro.
In the updated submission we include new data sets from our recent publication. More specifically we focus on how we can capture inter- and intratumoral heterogeneity using our previously published organoid system (Randriamanantsoa, Papargyriou et al. 2022) of pancreatic cancer and how these morphologically diverse organoids respond to targeted therapy.  We aim to understand the following four biological questions: First, we aim to address intertumoral heterogeneity by culturing classical and basal-like PDAC cells in 2D culture conditions and 3D floating collagen gels. Indeed, in both cases we observe a clear separation between mesenchymal and epithelial organoids and describe pivotal pathways (f.e. TGF pathway) that are differentially regulated between the two culture conditions (2D vs 3D). In detail, we culture 3 epithelial (ID: 8442, 9591, 53631) and 3 mesenchymal (ID: 8028, 9091, 16992) murine pancreatic cancer lines in established 2D conditions and in 3D floating collagen gels. Next, we focus on understanding the transcriptional background of distinct organoid morphologies derived from the same parental cell lines of either epithelial or mesenchymal origin. In more detail we transcriptionally analyze specific epithelial (line ID 9591) as a bulk population and their subclones: TEBBO, cystic branched, thick-branched and tree-like and mesenchymal pancreatic cancer line (line ID 16992) as a bulk population and their subclones: branched mesenchymal, firework and star-like. After performing a 102-drug screening on these clones we identified potent target therapy drugs acting in specific phenotypes. To validate the effects on the transcriptional level we treated all the epithelial organoid phenotypes with the combination of AZD5153 (BET/BRD4 inhibitor)+Poziotinib (pan-HER inhibitor) and the mesenchymal phenotypes with monotreatments of the following drugs: Birinapant (SMAC antagonist) Poziotinib (pan-HER inhibitor), Saracatinib (Src inhibitor) and RO5126766 (RAF/MEK inhibitor). Furthermore, to translate our findings in a human setting we developed a new media composition which enables the formation of branching organoids in pancreatic cancer patient-derived organoids (PDOs). We examined the effect of our newly developed media, the Basal Branching PDO Media on the transcriptome of 3 PDOs (PDO line ID: B211, B250, B320) compared to previously established media conditions such as the Full PDO Media.

overall_design: Study 1. The batch 332AP contains: bulk 2D cell lines vs. 3D organoids grown in collagen (for the line 9591 use the previously submitted samples: S40-S45 & S49-S56). Study 2. The batch 432AP: Understanding intra-cell line heterogeneity in 3D Organoids (note that for the 9591 bulk population samples were included in the first submission. Study 3. The batch 448AP: PDO lines comparison grown in different Media conditions (Full PDO Media vs Basal Branching PDO Media). Study 4. The batch 536APm: Mesenchymal organoids comparison control vs. targeted therapy treated. Study 5. The batch 536APe: Epithelial organoids comparison control vs. combinatory targeted therapy treated organoids.

first: Aristeidis

last: Papargyriou

first: H. Carlo

first: Rupert

last: Öllinger

first: Roland

last: Rad

first: Maximilian

last: Reichert


title: Altered DNA methylation underlies monocyte dysregulation and immune exhaustion memory in sepsis

status: Public on Mar 05 2024

city: Blacksburg

first: Blake

middle: Alexander

last: Caldwell

address: 149 Life Sciences I Building, 970 Washington Street SW

department: Biological Sciences

institute: Virginia Tech


title: Growth regulated co-occupancy of Mediator and Lsm3 at intronic ribosomal protein genes

first: ALEXANDER

last: VERGARA


title: Altered DNA methylation underlies monocyte dysregulation and immune exhaustion memory in sepsis [Array]

summary: Innate immune memory is the process by which pathogen exposure elicits cell-intrinsic states to alter the strength of future immune challenges. Such altered memory states drive monocyte dysregulation during sepsis, promoting pathogenic behavior characterized by pro-inflammatory, immunosuppressive gene expression and emergency hematopoiesis. Epigenetic changes, notably via histone modifications, have been shown to underlie innate immune memory, but the contribution of DNA methylation remains poorly understood. Using an ex vivo sepsis model, we discovered broad changes in DNA methylation throughout the genome of exhausted monocytes, including at several genes implicated in immune dysregulation during sepsis and Covid-19 infection (e.g. Plac8). Methylome reprogramming is driven in part by Wnt signaling inhibition in exhausted monocytes, and can be reversed with DNA methyltransferase inhibitors, Wnt agonists, or immune training molecules. These changes are recapitulated in septic mice following cecal slurry injection, supporting the involvement of DNA methylation in acute and long-term monocyte dysregulation during sepsis.

overall_design: Genome-wide DNA methylation profiling was performed using Infinium Mouse Methylation BeadChip arrays. Based on variability observed in previous studies, a minimum of 4 biological replicates was determined to be sufficient for most BMMC culture experiments. To account for greater mouse-to-mouse variability in response to in vivo cecal slurry injections, a minimum of 6 biological replicates was collected for control and experimental day 7 mice. From discussions with the sesame analysis pipeline author, it was determined that n = 3 and n = 4 was sufficiently powered to detect differences in 5mC as low as 5% for BMMC culture and in vivo bone marrow monocytes, respectively. All experiments were performed on at least two separate days and with mice from at least 3 separate litters. For in vivo cecal slurry experiments, mice were randomly split between cecal slurry injection and PBS control injection groups by researchers uninvolved with downstream analyses.

first: Yajun

last: Wu

first: Jing

last: Wang

first: Liwu

last: Li


title: RPA and Rad27 limit templated and inverted insertions at DNA breaks

status: Public on Mar 07 2024

city: Boston

first: Xin

state: MA

address: 300 Longwood Ave,

institute: Boston children's hospital

summary: Formation of templated insertions at DNA double-strand breaks (DSBs) is very common in cancer cells. The mechanisms and enzymes regulating these events are largely unknown. Here, we investigated templated insertions in yeast at DSBs using amplicon sequencing across a repaired locus. We document very short (most ~5-34 bp), templated inverted duplications at DSBs. They are generated through a foldback mechanism that utilizes microhomologies adjacent to the DSB. Enzymatic requirements suggest a hybrid mechanism wherein one end requires Polδ-mediated synthesis while the other end is captured by nonhomologous end joining (NHEJ) or rarely by alternative end joining (Alt-EJ). This process is exacerbated in mutants with low levels or mutated RPA (rtt105Δ; rfa1-t33) or extensive resection mutant (sgs1Δ exo1Δ). Templated insertions from various distant genomic locations also increase in these mutants as well as in rad27 and originate from fragile regions of the genome. Among complex insertions, common events are insertions of two sequences, originating from the same locus and with inverted orientation. We propose that these inversions are also formed by microhomology-mediated template switching. Taken together, we propose that a shortage of RPA typical in cancer cells is one possible factor stimulating the formation of templated insertions.

overall_design: To study insertion at DSBs in yeast, we used a high-throughput amplicon sequencing based method called Break-Ins (Break Insertions) which allows screening of hundreds of thousands of NHEJ products simultaneously. We used yeast cells carrying a galactose-inducible HO endonuclease that generates a single double-strand break (DSB) per genome at the MATa locus. This DSB can't be repaired by homologous recombination; only the cells that repaired the break imprecisely, altering the HO recognition site, can survive continuous HO induction and form colonies.Yeast cells were grown overnight in YPD (1% yeast extract, 2% peptone, 2% dextrose), washed twice with YP-raffinose (1% yeast extract, 2% peptone, 2% raffinose) media, inoculated into 10 mL YP-raffinose media, and incubated at 30 °C overnight. When the cell density was about 2x107/mL, 500 µL cells were spread onto YP-galactose (1% yeast extract, 2% peptone, 2% galactose, 2.5% agar) plates to induce HO expression and the DSB at MATa locus, and incubated at 30 °C for 5 days. Each sample was spread on 5-7 150x15 mm YP-galactose plates. The number of colonies was counted. All colonies on YP-galactose plates were collected, combined, and mixed vigorously. Approximately 80 μL cells were spun down for isolating genomic DNA. Standard glass beads-phenol/chloroform/isoamyl alcohol (25:24:1) method. The DNA was dissolved in water and adjusted to a concentration of 10 ng/μL. The construction of the amplicon sequencing library was adapted from Illumina’s protocol #15044223 Rev. B. Briefly, the sequencing libraries were constructed with two rounds of PCR using KAPA HiFi HotStart ReadyMix polymerase (Roche, 7958927001). For the first round of PCR, 3.2 μL genomic DNA and 0.5 μL 10 μM primers were used for a total volume of 25 μL. The forward and reverse primers targeting the MATa locus are located 11 bp upstream or 18 bp downstream of the HO cleavage site respectively, containing Illumina adapter sequence and 3 bases unique home index as described (7). The thermocycling conditions were: 95 °C for 5 min; 22 cycles of 98 °C for 20 s, 65 °C for 30 s, 72 °C for 3 min; 72 °C for 10 min. After the first round of PCR, 18μL PCR product was purified with an equal volume of AMPure XP beads (Beckman Coulter, A63880). The DNA was eluted with 52.5 μL of 10 mM Tris (pH 8.5) and used as the templates for the second round of PCR. For the second round of PCR, 5 μl template DNA, 5 μL index primer N7xx and 5 μL index primer S5xx from Nextera XT V2 Index kit (Illumina, FC-131-2001) were used for the total volume of 50 μL. The following conditions were used for the second round of PCR: 95 °C for 5 min; 8 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 3 min; 72 °C for 10 min. Then, the PCR product was purified with 1.2x volume of AMPure XP beads and eluted with 10 mM Tris pH 8.5. The average size of each sample was measured with TapeStation and the DNA concentration was measured with Qubit dsDNA BR Assay Kit (ThermoFisher, Q32850). Then, the DNA was diluted to a concentration of 4 nM. An equal volume of DNA from ~20 samples was pooled into the library. The 0.2 M NaOH was used to denature the pooled library and PhiX library (Illumina, FC‐110‐3001). After diluting the libraries with pre-chilled HT1 to 12 pM, pooled library and PhiX library were mixed with the ratio 9:1, further denatured by incubating at 96 °C for 2 min and immediately put in an ice-water bath for 5 min. The MiSeq and Reagent Kit v3 (600 cycles) (Illumina, MS-102-3003) were used for sequencing. The cluster density for all libraries was ~1100 K/mm2.

first: Yang

last: Yu

first: Kaifu

first: Grzegorz

last: Ira


title: Arresting the Bad Seed: HDAC3 Regulates Proliferation of Different Microglia after Ischemic Stroke

first: Yue

last: Zhang


title: Proteogenomic analysis reveals adaptive strategies for alleviating the consequences of aneuploidy in cancer

status: Public on Mar 04 2024

city: Kaiserslautern

first: Jan-Eric

last: Boekenkamp

address: Paul-Ehrlich-Strasse 24

department: Molecular Genetics

institute: University of Kaiserslautern-Landau

type: Genome variation profiling by genome tiling array

summary: Aneuploidy is prevalent in cancer, conferring fitness advantage, multidrug resistance, and poor prognosis. In contrast, experimentally induced aneuploidy often results in adverse effects and impaired proliferation. This paradox underscores the necessity of cancer cells to adapt to abnormal chromosome numbers. To identify molecular mechanisms of adaptation to aneuploidy, we initiated in vitro evolution of cells with extra chromosomes added via microcell-mediated chromosome transfer. To this end, we cultured cells in a nutrient-rich medium for 50 passages or plated the cells at a low density and selectively collected the largest colonies originating from a single cell (colony selection). The anaylsis of somatic copy number variations in the evolved cell lines was then carried out based on low-coverage whole genome sequencing (WGS) and array-based comparative genomic hybridization (aCGH).

overall_design: Detection of copy number variations using genome-wide chromosomal genomic hybridization (CGH) analysis with aneuploid cell lines derived from HCT116 and RPE-1 cell lines before (passsage 0) and after in-vitro evolution (along their near-diploid parental counterparts) and with commercially available gDNAs as references.

last: Bökenkamp

first: Kristina

last: Keuper

first: Zuzana

last: Storchova

first: Silvia

last: Stingele


title: Temporal constraints on enhancer usage and enhancer-promoter connectivity shape the regulation of limb gene transcription [ChIP-seq]

first: Guillaume

last: Andrey

first: Raquel

last: Rouco


title: Dynamic change of polarity is involved in the establishment of metastasis by Spread Through Air Spaces

status: Public on Mar 01 2024

city: Kyoto

first: Masahiro

last: Inoue

address: Shimoadachi-cho 46, Sakyou-ku

country: Japan

institute: Kyoto University, Graduate School of Medicine

summary:
Spread through the airspace (STAS) is a histological finding of lung tumors where tumor cells exist within the airspace of the lung parenchyma beyond the margin of the main tumor. Although STAS is an important prognostic factor, the etiology of STAS is largely unknown. In this study, histopathological analysis revealed the presence of an apical membrane outside the STAS surrounding colorectal cancer (CRC) lung metastases and lung adenocarcinomas. When CRC cells were intratracheally administered to mice, cancer cell clusters had greater metastatic potential than single cells. An in vitro model of STAS co-cultured with CRC organoids and 2D cultured mouse airway epithelial organoids (2D-MAO) was established. Adhesion of CRC clusters to 2D-MAO was much less than to type I collagen or endothelial cells, suggesting a protective role of the airway epithelium against adhesion of cancer cell clusters. Loss of the apical membrane of CRC clusters at the contact surface with 2D-MAO after adhesion was responsible for establishing adhesion. In the co-culture model, airway epithelium stimulated by TGF-β1 promoted adhesion of CRC organoids to the airway epithelium. Among TGF-β1-induced genes in airway epithelium, FSTL1 promoted CRC organoid adhesion, suggesting that FSTL1 may promote metastasis by STAS in the inflammatory microenvironment. Elucidating the mechanism of STAS will contribute to the development of therapies to prevent STAS and its associated metastases.
To examine the downstream of TGF-β1 in 2D-MAO, gene expression profiles were analyzed and compared between TGF-β1-treated with non-treated 2D-MAO. The number of differentially expressed genes that showed statistically more than 2-fold or greater changes was 709, with 571 upregulated and 138 downregulated.

overall_design: 2D-MAO was plated in 6-well plates, and treated with or without TGF-β1 in medium without noggin and A83-01 on day 3. The cells were collected at day 7. Total RNA was extracted. Microarray analysis, from three biological replicates, was performed.

first: Yoshiaki

last: Matsuura

first: Roberto

last: Coppo


title: iPSC-derived human sensory neurons reveal a subset of TRPV1 antagonists as anti-pruritic compounds

status: Public on Feb 27 2024

city: Singapore

first: Jeremy

last: Pang

address: 61 Biopolis Drive

country: Singapore

institute: A*STAR

summary: Given the inaccessibility of human sensory neurons (SNs), it is yet to be established whether the signalling pathway between histamine 1 receptor (H1R) and transient receptor potential cation channel subfamily V member 1 (TRPV1) is conserved between humans and mammalian models. Accessible human SNs are vital for identifying TRPV1 antagonists with higher potential for success in clinical trials targeting histaminergic itch, especially given TRPV1's species specificity concerns. Hence to build a humanized histamine-dependent itch model, we derived peripheral sensory neurons from human pluripotent stem cells (hiPSC-SNs) and validated channel functionality using immunostaining, calcium imaging and multielectrode array (MEA) recordings. Here, we demonstrated that a subset of hiPSC-SNs exhibits co-expression of H1R and TRPV1, responding to both histamine and capsaicin agonists. We found that inhibiting TRPV1 prevented histamine activation. Moreover, we show that silencing histamine-sensitive neurons reduces capsaicin response, and silencing capsaicin-sensitive neurons diminishes histamine response. To assess the ability of hiPSC-SNs in TRPV1 antagonist drug screening, we evaluated two well-established hyperthermic and three thermal-neutral TRPV1 antagonists. Our finding identifies SB366791, a thermal-neutral antagonist, as a potent inhibitor of H1R activation. The use of hiPSC-SNs may therefore provide a physiologically relevant means to perform large-scale screening to discover anti-pruritic agents predictive of actual efficacy in human clinical trials.

overall_design: 46-day old hiPSC-SNs were dissociated using papain and analyzed using scRNA-seq.

first: Shermaine

middle: H

last: Tay

first: Winanto

last: Ng

first: Chong

middle: Y

first: Zi

last: Khong

first: Zheng-Shan

last: Chong

first: Shi-Yan


title: Molecular characterizations of cell types in the adult sea lamprey retina

city: Los Angeles

first: Yi-Rong

last: Peng

address: 100 Stein Plaza Driveway, B-133

department: Department of Ophthalmology

institute: University of California, Los Angeles

summary: As an ancient jawless vertebrate species, the lamprey offers an important model to probe the evolutionary history of retinal cells. In this study, we generated a cell atlas of the adult sea lamprey retina using single-cell RNA sequencing

overall_design: Retinal cells were dissociated from adult lamprey retina and analyzed using scRNAseq

first: Lin

first: Junqiang

last: Wang

first: Alapakkam

last: Sampath


title: Expression profiling of microRNAs in mouse livers with deletion of paternally-expressed, imprinted Mir483 [miRNA-seq]

status: Public on Feb 26 2024

city: Cambridge

first: Brian

middle: Yee Hong

last: Lam

address: Box 289 Addenbookes Hospital, Hills Road

department: Metabolic Research Laboratories

institute: University of Cambridge

summary: In this study we generated mice with constitutive deletion of microRNA Mir483, which is imprinted and expressed from the paternal allele only. Global analysis of microRNA expression was performed by miRNA-seq in livers collected from E18.5 fetuses (mutants with deletion of paternal allele - Mir483Pat-KO - and wild-type controls).

overall_design: We performed miRNA-seq analysis in E18.5 mouse livers of female fetuses with deletion of paternally-expressed microRNA Mir483 (n=7 biological replicates) and wild-type littermate controls (n=5 biological replicates)

first: Ionel

last: Sandovici

first: Miguel

last: Constancia


title: Expression profiling of whole E10.5 embryos expressing 5 copies of Mir483 at the endogenous locus

summary: In this study we generated mice with over-expression of microRNA Mir483, by insertion of four extra-copies at the endogenous locus. Whole transcriptome analysis was performed by RNA-seq in in total lysates of E10.5 embryos (mutants with five copies - 5C - and wild-type controls).

overall_design: We performed RNA-seq analysis in E10.5 mouse embryos with five copies of microRNA Mir483 (n=3 biological replicates) and wild-type littermate controls (n=3 biological replicates)

first: Wendy

last: Cooper


title: Expression profiling of mouse livers with inducible over-expression of microRNA Mir483

summary: In this study we generated mice with inducible over-expression of microRNA Mir483, using a transgene inserted at the Rosa26 locus. Whole transcriptome analysis was performed by RNA-seq in livers collected from 15-week old adult mice (over-expressors and controls).

overall_design: We performed RNA-seq analysis in 15-week old mouse livers of adult females with inducible over-expression of microRNA Mir483 (n=6 biological replicates) and littermate controls (n=6 biological replicates)


title: Expression profiling of mouse livers with deletion of paternally-expressed, imprinted Mir483 [RNA-seq]

summary: In this study we generated mice with constitutive deletion of microRNA Mir483, which is imprinted and expressed from the paternal allele only. Whole transcriptome analysis was performed by RNA-seq in livers collected from E18.5 fetuses (mutants with deletion of paternal allele - Mir483Pat-KO - and wild-type controls).

overall_design: We performed RNA-seq analysis in E18.5 mouse livers of female fetuses with deletion of paternally-expressed microRNA Mir483 (n=5 biological replicates) and wild-type littermate controls (n=5 biological replicates)


title: The role of residue chirality of polypeptide hydrogels in regulating local immune microenvironment for anti-tumor immune responses

status: Public on Mar 14 2024

city: changchun

first: Junfeng

last: Ding

state: Jilin

address: No. 5625 Renmin Rd.

institute: Changchun Institute of Applied Chemistry, Chinese Academy of Sciences

summary: The impact of chirality on immune response has attracted great interest in cancer vaccine research recently. However, the study of chiral synthetic polypeptide hydrogels as cancer vaccines as well as of the impact of biomaterials itself for antitumor immunotherapy has rarely been reported. Herein, we demonstrated the key role of residue chirality of polypeptides hydrogels in antitumor immunity and local immune microenvironment regulation. Compared to poly(γ-ethyl-L-glutamate)-based hydrogels (L-Gel), poly(γ-ethyl-D-glutamate)-based hydrogels (D-Gel) induced greater immune cell infiltration. Unexpectedly, D-Gel caused higher levels of suppressive markers on antigen-presenting cells and even induced stronger T cells exhaustion than L-Gel. Finally, D-Gel established a local chronic inflamed and immunosuppressive microenvironment and showed insufficient anti-tumor effects. Conversely, the milder host immune responses induced by L-Gel led to more effective tumor inhibition. This study provides new insights on the role of residue chirality in the regulation of local immune microenvironment and affecting antitumor immune response.

overall_design: To further characterize the heterogeneity of the local immune microenvironment following injection with the different hydrogels, bulk RNA sequencing (RNA-seq) was performed with the Illumina sequencing platform. We then performed gene expression profiling analysis using data obtained from RNA-seq of three hydrogel tissues of each group. Comparative gene expression profiling analysis of RNA-seq data for L-Gel and D-Gel.

first: Yan

last: Rong

first: Chaoliang

last: He


title: Tumor suppressive function of IGF2BP1 in gastric cancer through decreasing MYC

city: Hangzhou

first: Ning

state: Zhejiang

address: Yuhangtang Road

institute: Zhejiang University School of Medicine

summary: Gastric cancer is one of the most common causes of cancer-related death worldwide. The N6-methyladenosine (m6A) reader IGF2BP1 (insulin-like growth factor-2 mRNA binding protein 1) has been reported to promote cancer progression by stabilizing oncogenic mRNAs through its m6A-binding activity in some tumors. However, the role of IGF2BP1 in gastric carcinogenesis remains unclear. In this study, we find that IGF2BP1 is significantly downregulated in tumor tissues from patients with gastric cancer. Lower expression of IGF2BP1 is associated with poor prognosis. IGF2BP1 suppresses gastric cancer cell proliferation in an m6A-dependent manner. Additionally, IGF2BP1 is able to significantly attenuate tumor growth of gastric cancer cells. Further m6A sequencing and m6A-RIP (RNA immunoprecipitation) assays show that MYC (c-myc proto-oncogene) mRNA is a target transcript of IGF2BP1 in gastric cancer cells. IGF2BP1 inhibits gastric cancer cell proliferation by reducing the mRNA and protein expression of MYC. Mechanistically, IGF2BP1 promotes the degradation of MYC mRNA and inhibits its translation efficiency. Taken together, these data suggest that IGF2BP1 plays a tumor-suppressive role in gastric carcinogenesis by downregulating MYC in an m6A-dependent manner, thereby making the IGF2BP1-MYC axis a potential target for gastric cancer treatment.

overall_design: To explore the potential target transcript mediating the role of IGF2BP1 in gastric cancer, we performed m6A-RIP seq and RNA-seq in IGF2BP1-depleted MKN45 cells.

first: Wenwen

last: Chen


title: Altered DNA methylation underlies monocyte dysregulation and immune exhaustion memory in sepsis [CUT&RUN]

first: Blake

last: Caldwell

overall_design: CUT&RUN sequencing for enhancer (H3K4me1) and promoter (H3K4me3) histone modifications in cultured mouse bone marrow monocytes under PBS control or repetitive LPS stimulation

first: Yajun

last: Wu

first: Jing

first: Liwu

last: Li


title: Dual inhibition of BET and EP300 has antitumor activity in undifferentiated pleomorphic sarcomas and synergizes with ferroptosis induction [CRISPR‒Cas9 knockout screen]

status: Public on Feb 20 2024

city: Bordeaux

first: Amandine

last: Crombe

address: 229 cours de l'Argonne

institute: Institut Bergonié

summary:
Purpose: Patients with UPS exhibit a poor prognosis and have few therapeutic options. Epigenetic modifiers has been extensively investigated in recent years leading to the development of novel therapeutic agents. Dual BET/EP300 inhibitors have shown synergistic antitumor activity and have recently entered clinical development. To date, no data related to potential of BET/EP300 inhibition as a treatment in UPS have been reported.
Experimental design: To investigate the therapeutic potential of BET/EP300 inhibition, we first evaluated the prognostic value of BRD4 expression in 2 cohorts of sarcomas. wWe then investigated evaluated the antitumor activity of three compounds in vitro via MTT, apoptosis and cell cycle assays. The most potent inhibitor was evaluated in vivo in two animal models and the mechanisms of action were investigated by RNA sequencing, Western blotting and immunofluorescence staining. A CRISPR knockout screen was performed to identify resistance mechanisms.
Results: High BRD4 expression were associated with worsened metastasis-free survival of sarcoma patients. Among the three compounds tested, the dual inhibitor NEO2734 was the most potent, decreased the viability of UPS cells in vitro through a regulation of E2F targets and cell cycle and decreased the tumor growth in vivo. Moreover, we identified GPX4 as a gene involved in resistance and showed synergy between BET inhibition and ferroptosis induction.
Conclusion: The present study demonstrated that dual BET/EP300 inhibitors have a relevant anti-tumor activity in a subgroup of UPS characterized by expression of MYC-targets pathway and identified a potent combination therapeutic strategy that deserves further investigation in the clinical setting.

overall_design: To decipher the mechanism of resistance to BET inhibition in UPS cell lines, We performed a genome-wide pooled lentiviral CRISPR‒Cas9 knockout screen in KN473 cells stably infected with the cas9 nuclease. After GeCKO library infection and 72 h of NEO2734 treatment at 1 µM, a dose that normally does not kill KN473 cells, genomic DNA was extracted and sequenced by NGS to identify the genes whose knockout causes cell death and hypersensitivity to NEO2734

first: Stephanie

last: Verbeke

first: Aurélien

last: Bourdon

first: Mathilde

last: Lafon

first: Vanessa

last: Chaire

first: Frederic

last: Bertolo

first: Amina

last: Naït Eldjoudi

first: Marie-Alix

last: Derieppe

first: Francis

last: Giles

first: Antoine

last: Italiano


title: Dual inhibition of BET and EP300 has antitumor activity in undifferentiated pleomorphic sarcomas and synergizes with ferroptosis induction [RNA-seq]

overall_design: To decipher the mechanism of action of BET inhibition in STS cell lines, we explored the transcriptomic changes in 4 UPS cells after 3 days of treatment by RNA sequencing. UPS cells were untreated or treated with NEO2734 at the IC50  for 3 days in triplicate. RNA was then extracted and sequenced on a NovaSeq6000 S2 platform.


title: Novel synthetic ecteinascidins exhibit potent anti-melanoma activity by targeting super-enhancer-driven oncogenic transcription

status: Public on Feb 23 2024

city: Illkirch-Graffenstaden

first: Frédéric

last: Coin

address: 1 Rue Laurent Fries

institute: IGBMC

summary: This SuperSeries is composed of the SubSeries listed below. Note that Comp = PM54.

overall_design: Refer to individual Series


title: Novel synthetic ecteinascidins exhibit potent anti-melanoma activity by targeting super-enhancer-driven oncogenic transcription [RNA-Seq]

summary: Melanoma cells exhibit a dynamic capacity for transitioning through various cellular states. This intricate biological trait poses significant challenges to current therapeutics approaches, emphasizing the urgent need for innovative treatment strategies. Here, we initially demonstrated the efficacy of Lurbinectedin, a synthetic ecteinascidin derived from marine sources, against melanoma cells, irrespective of their driver mutations or phenotypic manifestations. Subsequently, we designed and evaluated two derivatives of Lurbinectedin, termed Ecubectedin and PM54(Comp IA), both of which fully recapitulate the cytotoxic effects of Lurbinectedin in vitro and might present benefits versus the original compound. These derivatives also exert a noteworthy in vivo impact on tumor growth and animal survival, inducing apoptosis in melanoma cells. Mechanistically, our investigations revealed that ecteinascidins strongly inhibit oncogenic super-enhancer-mediated gene expression by a three-pronged mechanism. Firstly, the binding to the promoters of lineage-specific master transcription factors and the disruption of their associated auto-regulatory circuits, secondly, the binding and inhibition of genes encoding ubiquitous transcription factors/coactivators, heavily enriched at oncogenic SEs, and thirdly, the direct drug-binding to these regulatory elements, seemingly hampering the assembly of transcriptional condensates, all likely work in tandem to disrupt oncogene expression. These findings underscore the potential of ecteinascidins as promising therapeutics for melanoma and potentially other transcriptionally-addicted cancers.

overall_design: Differentiated melanoma cells MM074 and 501mel, undifferentiated melanoma cells MM029, and SCLC DMS53 cells have been treated with DMSO or 10xIC50s of either the transcriptional inhibitors Lurbinectedin, Ecubectedin or PM54(Comp IA) for 8h, to analyze gene expression changes caused by the drugs versus DMSO control. Each condition in biological duplicate.

first: Max

last: CIGRANG


title: Effects of Ginseng Berries Concentrate and Ginsenoside Re on gene expression profiles.

status: Public on Mar 03 2024

city: Gwangju

first: Dongryeol

last: Ryu

address: 123 Cheomdangwagi-ro

department: Biomedical science and engineering

institute: Gwangju Institute of Science and Technology

summary: C2C12 myocytes were treated with Ginseng Berries Concentrate and Ginsenoside Re to identify gene expression changes.

overall_design: Total RNA from C2C12 myocytes treated with Ginseng Berries Concentrate and Ginsenoside Re.

first: Yunju

last: Jo

first: Eun-ju

last: Jin

first: Minjeong

last: Kim


title: Novel synthetic ecteinascidins exhibit potent anti-melanoma activity by targeting super-enhancer-driven oncogenic transcription [Cut & Tag, ATAC-Seq]

overall_design: In order to analyze the genomic binding sites of Lurbinectedin and PM54(Comp IA), biotinylated versions of these drugs were used to perform Chem-Mapping on differentiated 501mel and undifferentiated MM029 melanoma cells. To assess transcriptionally active and open chromatin regions, Cut&Tag experiments targeting H3K27ac, RNAPII and BRD4 were also performed, in addition to ATAC-Seq. Biological triplicates for Chem-Mapping and biological duplicates for Cut&Tag and ATAC-Seq.

first: Julian

last: OBID


title: Genetic code expansion reveals site-specific lactylation in living cells reshapes protein function

status: Public on Feb 12 2024

city: Nanjing

first: Qi

last: Yuan

address: school of pharmacy

institute: China Pharmaceutical University

summary: Still in its infancy, the functions of lactylation remain elusive. To address this, we established a comprehensive workflow for lactylation studies that integrates the discovery of lactylation sites with proteomics, the expression of site-specifically lactylated proteins in living cells via genetic code expansion (GCE), and the evaluation of the resulting biological consequences. Specifically, we developed a wet-and-dry-lab combined proteomics strategy, and identified highly conserved lactylation at ALDOA-K147. Driven by its potential biological significance, we site-specifically expressed this lactylated ALDOA in mammalian cells and interrogated the biological changes. We discovered that it not only inhibited enzyme activity but also elicited gain-of-function effects——it dramatically reshaped the functionality of ALDOA by improving stability, enhancing nuclear translocation and affecting gene expression. Further, we demonstrated broad applicability of this workflow to study distinct histone lactylation sites. Together, we anticipate its wide uses in elucidating causative links between site-specific lactylation and target-centric or cell-wide changes.

overall_design: We performed RNA sequencing to understandwe performed RNA-sequencing (RNA-seq) analysis on HEK293T cells that site-specifically encode ALDOA-K147 (147TAG+Klac) through co-transfection of KlaRS1/tRNA pair and ALDOA-147TAG plasmids and cultured with Klac, and used cells that were transfected in a similar manner but cultured without Klac as the control group (147TAG)

first: Chang

last: Shao

first: Shuo

last: Tang

first: Siqin

last: Yu

first: Chenguang

last: Liu


title: MftG is crucial for alcohol metabolism of mycobacteria by linking mycofactocin oxidation to respiration

city: Kulmbach

first: Gerald

last: Lackner

address: Fritz-Hornschuch-Str. 9

department: Chair of Biochemistry of Microorganisms

institute: University of Bayreuth

summary: Mycofactocin is a redox cofactor essential for the alcohol metabolism of Mycobacteria. While the biosynthesis of mycofactocin is well established, the mftG gene, which encodes an oxidoreductase of the glucose-methanol-choline superfamily remained functionally uncharacterized. Here, we show that MftG enzymes strictly require mft biosynthetic genes, and are found in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the ∆mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin re-oxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.

overall_design: In order to access the impact of ethanol and glucose on the mutant strain lacking the mftG gene on Mycolicibacterium smegmatis genome MC2 155, transcriptomic data of the WT strain and mutant growing until exponential phase in each carbon source  was obtained. Biological triplicates of M. smegmatis WT and ∆mftG strains were incubated in the two different conditions (glucose and ethanol).  The wt grown on each condition (carbon source) was defined as control condition.

first: Ana

last: Graca

first: Tilman

last: Klassert

first: Malte

last: Siemers


title: RNA-seq profiling of Methylotuvimicrobium alcaliphilum 20ZR grown with or without La-supplementation

status: Public on Feb 02 2024

city: «Sirius» Federal Territory

first: Semyon

last: Kolmykov

state: Krasnodar region

address: Olympic Ave., 1

institute: Sirius University of Science and Technology

summary:
In this study we investigated the steady-state growth of Methylotuvimicrobium alcaliphilum 20ZR in media containing calcium (Ca) or lanthanum (La, a REE element). RNA-seq profiling of  Methylomicrobium alcaliphilum strain 20ZR  in bioreactor on methane. Sample cultures, La-optimum, La-CH4 limited, Ca-optimum and Ca-CH4 limited, were collected and immediately transferred into tubes containing 5 ml of the stop solution (5% water-equilibrated phenol in ethanol). It was found, that cells supplemented with La show a higher growth rate compared to Ca-cultures; however, the efficiency of carbon conversion, estimated as biomass yield, is higher in cells grown with Ca.

The study was financially supported by DOE under FOA DE-FOA-0001085 and by NSF-CBET award 1605031

overall_design: CaBR1: P3% CH4 +Ca +W +Cu CaBR2: P3% CH4 +Ca +W +Cu CaOLBR1: P3% low CH4 (methane limitation) +Ca +W +Cu CaOLBR2: P3% low CH4 (methane limitation) +Ca +W +Cu LaBR1: P(La)3% CH4 +La +W +Cu LaBR2: P(La)3% CH4 +La +W +Cu LaOLBR1: P(La)3% low CH4 (methane limitation) +La +W +Cu LaOLBR2: P(La)3% low CH4 (methane limitation) +La +W +Cu

first: Richard

last: Hamilton

first: Marina

middle: G

last: Kalyuzhnaya

first: Tatiana

last: Sokolova


title: Adam8Mutant vs DBA intervertebral disc injury

status: Public on Feb 13 2024

city: Philadelphia

first: Ken

last: Chen

state: Pennsylvania

address: 3701 Chestnut Street, Apt 1136

institute: UNIVERSITY OF PENNSYLVANIA

summary: To examine the alteration of biological pathways in response to ADAM8-inactivation, an unbiased approach comparing the transcriptome of IVDs in the mutant and WT mice was used.

overall_design: Messenger RNA was isolated from the Adam8EQ and wild type mouse IVDs, and bulk RNAseq was performed followed by transcriptomics data analysis.


title: Effect of CYP1B1 inhibition and estrogens on pulmonary vascular smooth muscle cells

status: Public on Feb 01 2024

city: Nashville

first: James

last: West

state: TN

address: 4200 E 9TH Ave, Box B133

department: Medicine

institute: Vanderbilt University

summary: PMVEC treated with scrambled siRNA, or siRNA to CYP1B1 are treated with vehicle, 1uM E2, or 1uM 16aOHE

overall_design: Examination of effect of E2 or 16aOHE in PMVEC with and without a cytochrome that metabolizes E2

middle: D

first: Xinping

first: Jingyuan

first: Eric

last: Austin