title: MiR-146a-5p deficiency in extracellular vesicles of glioma-associated macrophages promotes epithelial-mesenchymal transition through the NF-κB signaling pathway

city: Chongqing

first: Chao

last: Xu

address: 118 Xingguang Boulevard, Liang Jiang New Area

institute: Chongqing General Hospital

summary: Background: Glioma-associated macrophages (GAMs) are one of the most important cellular components in the tumor immune microenvironment (TIME). GAMs display a mostly M2-like phenotype with anti-inflammatory features, which is closely related to the malignancy and progression of cancers. Extracellular vesicles derived from GAMs (GAM-EVs), which are important components of the TIME, contain a variety of microRNAs (miRNAs). These miRNAs play a crucial role in communication between tumor cells and GAMs. In this study, we aimed to explore the effect of GAM-EVs on the malignancy and progression of glioblastoma multiforme (GBM) through miRNA-mediated communication and its possible signaling pathway.  Methods: The GBM and LGG data from The Cancer Genome Atlas (TCGA) were analyzed, and the immunological features of 12 clinical glioma samples, including GBM and low-grade glioma (LGG) samples, were estimated by immunohistochemistry. To evaluate the changes induced by M2-EVs compared to M1-EVs, the human GBM cell lines U87MG and A172 were cocultured with M2-EVs and M1-EVs, and their epithelial-mesenchymal transition (EMT) behaviors were investigated. The contents of M2-EVs and M1-EVs were determined by miRNA sequencing, and the differences were analyzed. Then, the EMT signatures were evaluated after a miRNA mimic was transfected into GBM cells. Six databases were used to predict the targets of the miRNA. Human GBM cell lines with interference and overexpression of the most valuable candidate miRNA targets were established. The related signaling pathways were further explored. Finally, an orthotopic homograft mouse model was employed to verify the results of the in vitro experiments. Results: The immunosuppressive GAM genes were upregulated in GBM compared with LGG, showing that the TIME of GBM is more immunosuppressive. To explore the effect of the immunosuppressive GAMs and TIME on the progression of GBM, we established immunostimulatory (M1-like) and immunosuppressive (M2-like) GAMs and compared the differences in their EVs. The migration and invasion of U87MG and A172 cells were enhanced under coculture with M2-EVs. MiR-146a-5p was deficient in M2-EVs compared with M1-EVs. Upon treatment with the miR-146a-5p mimic, the migration and invasion abilities of GBM cells were weakened, and the EMT signature was correspondingly decreased. According to the database prediction, tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin 1 receptor-associated kinase 1 (IRAK1) are the targets of miR-146a-5p. TRAF6 is an E3 ubiquitin ligase, and IRAK1 is the substrate. Bimolecular fluorescence complementation (BiFC) and coimmunoprecipitation assays confirmed interactions between TRAF6 and IRAK1. Interference with both TRAF6 and IRAK1 expression weakened but overexpression of both TRAF6 and IRAK1 promoted the EMT behaviors of GBM cells. The TRAF6-Glioma-associated macrophages (GAMs) are pivotal chains in the tumor immune microenvironment (TIME). GAMs mostly display M2-like phenotypes with anti-inflammatory features related to the malignancy and progression of cancers. Extracellular vesicles derived from immunosuppressive GAMs (M2-EVs), the essential components of the TIME, greatly impact the malignant behavior of GBM cells. M1- or M2-EVs were isolated in vitro, and human GBM cell invasion and migration were reinforced under M2-EV treatment. Signatures of the epithelial-mesenchymal transition (EMT) were also enhanced by M2-EVs. Compared with M1-EVs, miR-146a-5p, considered the key factor in TIME regulation, was deficient in M2-EVs according to miRNA-sequencing. When the miR-146a-5p mimic was added, EMT signatures and the invasive and migratory abilities of GBM cells were correspondingly weakened. Public databases predicted the miRNA binding targets and interleukin 1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were screened as miR-146a-5p binding genes. Bimolecular fluorescent complementation and coimmunoprecipitation confirmed interactions between TRAF6 and IRAK1. The correlation between TRAF6 and IRAK1 was evaluated with immunofluorescence (IF)-stained clinical glioma samples. The TRAF6-IRAK1 complex is the switch and the brake that modulates IKK complex phosphorylation and NF-κB pathway activation, as well as the EMT behaviors of GBM cells. Furthermore, a homograft nude mouse model was explored and mice transplanted with TRAF6/IRAK1-overexpressing glioma cells had shorter survival times while mice transplanted with glioma cells with miR-146a-5p overexpression or TRAF6/IRAK1 knockdown lived longer. This work indicated that in the TIME of GBM, the deficiency of miR-146a-5p in M2-EVs enhances tumor EMT through disinhibition of the TRAF6-IRAK1 complex and IKK-dependent NF-κB signaling pathway providing a novel therapeutic strategy targeting the TIME of GBM.

overall_design: Comparative micoRNA expression profiling analysis of RNA-seq data for EVs derived from THP-1 cells that stimulated GM-CSF and IL-4/IL-13 followed PMA treatment.

first: Pan

last: Wang

first: Haiyan

last: Guo

first: Chuan

last: Shao

first: Bin

last: Liao

first: Sheng

last: Gong

first: Yanghao

last: Zhou

first: Bingjie

last: Yang

first: Haotian

last: Jiang

first: Gang

last: Zhang

last: Wu


title: DNA selection by the master transcription factor PU.1

status: Public on Jun 28 2023

city: Bronx

first: Samuel

middle: John

last: Taylor

state: NY

address: 1300 Morris Park Ave, Chanin building 601

country: USA

institute: Albert Einstein College of Medicine

summary: This study aims to understand the binding preferences of WT vs Q226E mutant versions of PU.1 throughout the genome.

overall_design: HEK293T cells were transduced with WT PU.1 or Q226E expression vectors, and after 48hrs PU.1 genomic localization was examined via CUT&Tag

first: Gregory

last: Poon

first: Ulrich

last: Steidl


title: Single-cell multiomics of human fetal hematopoiesis defines a  developmental specific population and a fetal signature

city: Lund

first: Charlotta

last: Böiers

state: Lund

address: BMC B12

country: Sweden

institute: Lund University,

type: Expression profiling by high throughput sequencing

summary:
Knowledge of human fetal blood development and how it differs from adult is highly relevant to our understanding of congenital blood and immune disorders and childhood leukemia, of which the latter can originate in utero. Blood formation occurs in waves that overlap in time and space adding to heterogeneity, which necessitates single-cell approaches. Here, a combined single cell immunophenotypic and transcriptional map of first trimester primitive blood development is presented. Using CITE-seq (Cellular-Indexing-of-Transcriptomes-and-Epitopes-by-Sequencing) the molecular profile of established immunophenotypic gated progenitors was analyzed in the fetal liver (FL). Classical markers for hematopoietic stem cells (HSCs) such as CD90 and CD49F were largely preserved, whereas CD135 (FLT3) and CD123 (IL3R) had a ubiquitous expression pattern capturing heterogenous populations. Direct molecular comparison with an adult bone marrow (BM) dataset revealed that the HSC state was less frequent in FL, whereas cells with lympho-myeloid signature were more abundant. An erythro-myeloid primed multipotent progenitor cluster was identified, potentially representing a transient, fetal-specific population. Furthermore, differentially expressed genes between fetal and adult counterparts were specifically analyzed, and a fetal core signature identified. The core gene set could separate subgroups of Acute Lymphoblastic Leukemia (ALL) by age, suggesting that a fetal program may be partially retained in specific sub-groups of pediatric leukemia.
Our detailed single-cell map presented herein emphasizes molecular and immunophenotypic differences between fetal and adult blood cells, of significance for future studies of pediatric leukemia and blood development in general.

overall_design:
CITE--seq

>>>Submitter states: the data being deposited derives from living EU citizens and their sequence data is protected by the GDPR.  Therefore, raw data are not provided for these samples.<<<

first: Mikael

middle: N

last: Sommarin

first: Rasmus

last: Olofzon

first: Sara

last: Palo

first: Parashar

last: Dhapola

first: Shamit

last: Soneji

first: Göran

last: Karlsson


title: Spatial transcriptomics (Visium, 10x Genomics) data of Duchenne mouse models

city: Leiden

first: Pietro

last: Spitali

state: Zuid Holland

address: Postzone S4-P, Albinusdreef 2

department: Human Genetics

country: Netherlands

institute: Leiden University Medical Center

summary: Quadricep sections of 10µm thick were placed on Visium spatial transcriptomics slide to obtain spatial datasets for these skeletal muscle sections

overall_design: healthy controls (C57BL10 and DBA/2J) and DMD affected mouse models (mdx and D2-mdx samples) all n=1

first: Laura

last: Heezen

first: Tamim

last: Abdelaal

first: Maaike

last: van Putten

first: Annemieke

last: Aartsma-Rus

first: Ahmed

last: Mahfouz


title: 'Mitogen-Induced Defective Mitosis Transforms Neural Progenitor Cells

city: Calgary

last: Cairncross

state: AB

address: 3330 Hospital Dr NW

department: Clinical Neurosciences

country: Canada

institute: University of Calgary

summary: To investigate the cause(s) of the phenotype induced by PDGFA versus EGF/FGF (control) in p53-null neural progenitor cells, we performed a transcriptomic analysis in 3 independent cell lines exposed to those different growth factor conditions for 4 days.

overall_design: Comparative gene expression profiling analysis of RNA-Seq data from p53-null Neural Progenitor cells in EGF/FGF (control, n=3) and PDGFA (experimental, n=3)

first: Mathieu

last: Meode

first: J

middle: G


title: Expression data of Tubb3+ cells from E4 chicken neural tube

status: Public on Jun 27 2023

city: Barcelona

first: Antonio

last: Herrera

address: Baldiri i Reixac

country: Spain

institute: IBMB-CSIC

summary:
The specification of the different neuronal subtypes is associated with a profound morphological transformation, involving processes common to most neurons and others characteristic of each neuronal subtype. To study the Tcf-dependent genes that were active during commissural neuron differentiation, we compared the genes in differentiating neurons (Tubb3+ cells) after the repression of Tcf-dependet transcription in chichen neural tube from E3 to E4, period when mostly commissural neurons are differentiating.
We used Affymetrix GeneChip arrays to study TCF regulated genes during this process.

overall_design: We transfected E3 chick embryos for 24 h to produce either Tubb3::CAG:TcfEnR (suppression of Tcf-dependent transcription in neurons) or Tubb3::CAG:control (controls), dissected the NTs and cell-sorted the GFP+ neural populations. Both vectors are bicistronic and contain GFP CDS after IRES.

first: A

first: S

last: Pons


title: High-throughput functional characterization of combinations of transcriptional activators and repressors II

city: Stanford

first: Adi

middle: Xiyal

last: Mukund

state: CA

address: 443 via Ortega, Shriram 036

institute: Stanford University

summary: Despite growing knowledge of the functions of individual human transcriptional effector domains, much less is understood about how multiple effector domains within the same protein combine to regulate gene expression. Here, we measure transcriptional activity for 8,400 effector domain combinations by recruiting them to reporter genes in human cells. In our assay, weak and moderate activation domains synergize to drive strong gene expression, while combining strong activators often results in weaker activation. In contrast, repressors combine linearly and produce full gene silencing, and repressor domains often overpower activation domains. We use this information to build a synthetic transcription factor whose function can be tuned between repression and activation independent of recruitment to target genes by using a small molecule drug. Altogether, we outline the basic principles of how effector domains combine to regulate gene expression and demonstrate their value in building precise and flexible synthetic biology tools.

overall_design: Methods reproduced from Tycko et al. 2020:

first: Mukund

last: Adi Xiyal

first: Tycko

last: Josh

first: Allen

last: Sage

first: Robinson

last: Stephanie A

first: Andrews

last: Cecelia

first: Sinha

last: Joydeb

first: Ludwig

last: Connor H

first: Spees

last: Kaitlyn

first: Bassik

last: Michael C

first: Bintu

last: Lacramioara


title: Malignant Transformation Drives DNA Methylation Loss and Transcriptional Activation of Transposable Element Loci

city: Washington

first: Katherine

last: Chiappinelli

state: MA

address: 800 22nd Street, NW

department: Molecular Immunology and Tropical Medicine

institute: George Washington University

type: Methylation profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Effect of cyclic dinucleotides produced by cGAS-like receptors on gene expression in Crassostrea virginica

city: Boston

first: Yao

last: Li

address: 1 Jimmy Fund Way, SM512

institute: Dana-Farber Cancer Institute

summary: We performed transcriptime analysis (RNA-seq) in the stony coral Crassostrea virginica treated with different nucleotide messengers produced by cGLRs.

overall_design: Several spicemens from two colonies of Crassostrea virginica were incubated with 250 µM of 3'3'-cUA or 2'3'-cGAMP in seawater or seawater control for 18h and subjected to RNA-seq analysis.

first: Anamaria

last: Elk

first: Shani

last: Levy

first: Tali

last: Mass

first: Arnau

last: Sebe-Pedros

first: Philip

middle: J

last: Kranzusch


title: Hi-C of GAF-depleted Drosophila melanogaster embryos

city: Madison

first: Melissa

middle: M

last: Harrison

email: mharrison3@wisc.edu, ykaur@wisc.edu, torreszelada@wisc.edu, mmfreund2@wisc.edu

state: WI

address: 420 Henry Mall

department: Biomolecular Chemistry

institute: University of Wisconsin Madison

overall_design: Hi-C

first: Michael

last: Stadler


title: Gene expression profiles of NR1D1 knockout MDA-MB-231 cells treated with H2O2

city: Seoul

first: Na-Lee

last: Ka

address: 1 Gwanak-ro, Gwanak-gu

country: South Korea

institute: Seoul National University

summary: To understand the role of NR1D1 in the progression of breast cancer, NR1D1 knockout (KO) MDA-MB-231 cells, established using the CRISPR/Cas9 system, were treated with 200 μM H2O2 for 72 h. Gene expression patterns were analyzed by microarray.

overall_design: Control or NR1D1 KO MDA-MB-231 cells were treated with 200 μM H2O2 for 72 h. RNA was extracted and subjected to microarray analysis.

first: Mi-Ock

last: Lee

first: Ho

first: Mi

middle: K

last: Park


title: Gene expression profiles of the mammary gland tumors from Nr1d1+/+;PyMT and Nr1d1-/-;PyMT mice

city: Goyang

first: Mi Kyung

address: 323, Ilsan-ro, Ilsandong-gu

institute: National Cancer Center

summary: To investigate the role of NR1D1 in the progression of breast cancer, mammary gland tumor tissues were obtained from 14 weeks old FVB Nr1d1+/+;PyMT and Nr1d1-/-;PyMT mice and the gene expression patterns were analyzed by RNA-seq.

overall_design: Gene expression profiling analysis of RNA-seq data for mammary gland tumors from Nr1d1+/+;PyMT and Nr1d1-/-;PyMT mice.

first: MiKyung


title: Effect of cyclic dinucleotides produced by cGAS-like receptors on gene expression in Stylophora pistillata

summary: We performed transcriptime analysis (RNA-seq) in the stony coral Stylophora pistillata treated with different nucleotide messengers produced by cGLRs.

overall_design: Several spicemens from two colonies of adult coral S. pistillata were incubated with 250 µM of 3'3'-cUA or 2'3'-cGAMP in seawater or seawater control for 18h and subjected to RNA-seq analysis.


title: In vivo structure probing of RNA in Archaea: Novel insights into the ribosome structure of Methanosarcina acetivorans

city: University Park

first: Elizabeth

last: Jolley

state: PA

address: 239 Chemistry Bldg, Pennsylvania State University

institute: Pennsylvania State University

summary: Structure probing combined with next-generation sequencing (NGS) has provided novel insights into RNA structure-function relationships. To date such studies have focused largely on bacteria and eukaryotes, with little attention given to the third domain of life, archaea. Furthermore, functional RNAs have not been extensively studied in archaea, leaving open questions about RNA structure and function within this domain of life. With archaeal species being diverse and having many similarities to both bacteria and eukaryotes, the archaea domain has the potential to be an evolutionary bridge. In this study, we introduce a method for probing RNA structure in vivo in the archaea domain of life. We investigated the structure of ribosomal RNA (rRNA) from Methanosarcina acetivorans, a well-studied anaerobic archaeal species, grown with either methanol or acetate. After probing the RNA in vivo with dimethyl sulfate (DMS), Structure-seq2 libraries were generated, sequenced, and analyzed. We mapped the reactivity of DMS onto the secondary structure of the ribosome, which we determined independently with comparative analysis, and confirmed the accuracy of DMS probing in M. acetivorans. Accessibility of the rRNA to DMS in the two carbon sources was found to be quite similar, although some differences were found. Overall, this study establishes the Structure-seq2 pipeline in the archaea domain of life and informs about ribosomal structure within M. acetivorans.

overall_design: Structure-seq2 library construction for 3 biological replicates of M. acetivorans grown with acetate or methanol resulting in 3 control acetate grown samples, 3 DMS treated acetate grown samples, 3 control methanol samples, and 3 DMS treated methanol samples.

first: Allison

last: Williams

middle: A

first: Michel

last: Santiago-Martinez

first: Cheong

middle: X

last: Chan

first: Robin

middle: R

last: Gutell

first: James

last: Ferry

middle: C

last: Bevilacqua


title: MAPK-related gene signature predicting MAPKi sensitivity in pLGG

city: Jena

first: Romain

last: Sigaud

address: Am Klinikum 1

department: Pediatrics and Adolescent Medicine

country: Germany

institute: UKJ


title: Pyruvate Anaplerosis is a Targetable Vulnerability in Persistent Leukaemic Stem Cells

city: Glasgow

first: Kevin

last: Rattigan

address: Institute of Cancer Sciences, Wolfson Wohl Cancer Research Centre, Garscube Estate, Glasgow, Bearsden

country: United Kingdom

institute: University of Glasgow

summary: Deregulated oxidative metabolism is a hallmark of leukaemia. While use of tyrosine kinase inhibitors (TKIs) such as imatinib has led to increased survival of chronic myeloid leukaemia (CML) patients, it fails to eradicate disease initiating leukemic stem cells (LSCs). Whether TKI-treated CML LSCs remain metabolically deregulated is unknown. Using clinically and physiologically relevant assays we generated multi-omics datasets that offer unprecedented insight into nutrient fate in patient derived CML LSCs. We demonstrate that LSCs have increased glucose anaplerosis when compared to normal hematopoietic stem cells. While imatinib treatment reverses BCR-ABL-mediated LSC metabolic reprogramming, stable isotope-assisted metabolomics revealed that deregulated glucose anaplerosis is unaffected by imatinib. Encouragingly, genetic ablation of glucose anaplerosis sensitises CML cells to imatinib. Finally, we demonstrate that the clinical oral-available inhibitor MSDC-0160 targets glucose anaplerosis in robust pre-clinical CML models. Collectively these results highlight glucose anaplerosis as a persistent and therapeutically targetable vulnerability in imatinib-treated CML patient samples.

overall_design: To investigate the effect of TKI treatment on the metabolic functions of primary CML LSCs, CD34+ cells obtained from four patients were cultured in +/- 2µm Imatinib for 48 hours.

first: Zuzana

last: Brabcova

first: Daniele

last: Sarnello

first: Martha

last: Zarou

first: Kiron

last: Roy

first: Lucie

last: de Beauchamp

first: Amy

last: Dawson

first: Angela

last: Ianniciello

first: Eric

last: Kalkman

first: Mary

last: Scott

first: Karen

last: Dunn

first: David

last: Sumpton

first: Alison

last: Michie

first: Mhairi

last: Copland

first: Saverio

last: Tardito

first: Eyal

last: Gottlieb

first: Vignir

last: Helgason


title: Localization of the pioneer factor GAF to subnuclear foci is driven by DNA binding and required to silence satellite repeat expression [ChIP-Seq]

first: Melissa

last: Harrison

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The eukaryotic genome is organized to enable the precise regulation of gene expression required for development. This organization is established during early development when the embryo transitions from a fertilized germ cell to the totipotent zygote. To understand the factors and processes that drive genomic organization, we focused on the pioneer factor GAGA factor (GAF) that is required for early embryonic development in Drosophila. GAF transcriptionally activates the zygotic genome and is localized to subnuclear foci. We show that this non-uniform distribution is driven by binding to the highly abundant GA-satellite repeats. At GA-repeats, GAF is necessary to form heterochromatin and silence transcription. Thus, GAF is required to establish both active and silent regions. We propose that the foci formation enables GAF to have opposing transcriptional roles within a single nucleus. Our data support a model in which modulation of the subnuclear concentration of transcription factors acts to organize the nucleus into functionally distinct domains that are essential for the robust regulation of gene expression.

overall_design: ChIP-seq was performed using the anti-GFP antibody (Abcam #abcam290) on 2-3 hr AEL embryos from mothers expressing endogenously tagged sfGFP-GAF (sfGFP-GAF/+) or endogenously tagged sfGFP-GAF with the polyQ region of the short isoform deleted (sfGFP-GAFS-delta-PQ/sfGFP-GAFS-delta-PQ). DBD-sfGFP ChIP-seq was performed using the anti-GFP antibody on 2-2.5 hr AEL embryos laid by mothers expressing a truncated version of GAF from a transgene that contained only the DNA-binding domain (310-391aa) tagged with sfGFP. Anti-H3K9me3 ChIP-seq was performed using the anti-H3K9me3 antibody (Active motif #39161) on 2-2.5 hr AEL sfGFP-GAF homozygous controls and GAFdeGradFP embryos that have GAF knocked down. All experiments were done in duplicate, and sequencing was performed on immunoprecipitated samples as well as input controls.

first: M

last: Gaskill

first: I

middle: V

last: Soluri

first: A

middle: E

last: Branks

middle: P

last: Boka

last: Stadler

first: K

last: Vietor

first: H

middle: S

last: Huang

first: T

last: Gibson

last: Mir

first: S

last: Blythe


title: GATA2 mitotic bookmarking is required for definitive hematopoiesis [scRNA-seq]

first: Ilia

last: Kurochkin

state: Skane

address: BMC A12, Sölvegatan 17

institute: Lund University

summary: In mitosis, most transcription factors detach from chromatin, but some are retained and bookmark genomic sites. Mitotic bookmarking has been implicated in lineage inheritance, pluripotency and reprogramming. However, the biological significance of this mechanism in vivo remains unclear. Here, we address mitotic retention of the hemogenic factors GATA2, GFI1B and FOS during haematopoietic specification. We show that GATA2 remains bound to chromatin throughout mitosis, in contrast to GFI1B and FOS, via C-terminal zinc finger-mediated DNA binding. GATA2 bookmarks a subset of its interphase targets that are co-enriched for RUNX1 and other regulators of definitive haematopoiesis. Remarkably, homozygous mice harbouring the cyclin B1 mitosis degradation domain upstream Gata2 partially phenocopy knockout mice. Degradation of GATA2 at mitotic exit abolishes definitive haematopoiesis at aorta-gonad-mesonephros, placenta and foetal liver, but does not impair yolk sac haematopoiesis. Our findings implicate GATA2-mediated mitotic bookmarking as critical for definitive haematopoiesis and highlight a dependency on bookmarkers for lineage commitment.

overall_design: RNAseq profiling on single cells, generated after induction of hemogenic reprogramming in human dermal fibroblasts (HDFs, 2 donors), day4 and day6 cells after induction in HDFs with MD-GATA2 or MDmut-GATA2, in combination with GFI1B and FOS. Single cell RNAseq profiling of HDFs (2 donors) was used as controls.

first: Rita

last: Silvério-Alves

first: Carlos-Filipe

last: Pereira


title: RNA sequencing of metastatic breast patient derived organoids (PDOs) treated with control and CAF-conditioned media, in the presence and absence of Fulvestrant

first: Laura

last: Selfors

address: 240 Longwood Avenue

institute: Harvard Medical School

summary: To gain insight into the gene expression profile of breast patient derived organoids (PDOs) under endocrine therapy, and how cancer associated fibroblasts (CAFs) might influence gene expression and stimulate resistance, we performed RNA sequencing on three of the metastatic PDOs treated with control media and CAF-conditioned media, in the presence and absence of Fulvestrant (4-day treatment). These data revealed that Fulvestrant significantly suppressed the ER signature in the PDOs, suggesting that the ER-pathway is still active in these tumors although the patients had relapsed on ER-targeting agents (Tamoxifen, Letrozole).

overall_design: Four replicates of patient #8 and #10 PDOs and 2 replicates of patient #26 PDOs grown in organoid media or CAF-CM spiked with 500 nM Fulvestrant.  For PDO#8 and #10 samples were prepared in two batches.

first: Jenny

last: Hogstrom

first: Taru

last: Muranen


title: Mating activates transcriptional programs and represses microRNAs in the Drosophila melanogaster female reproductive tract

city: New York

first: Sofie

last: Delbare

address: 435 East 30th Street

institute: NYU Langone

type: Non-coding RNA profiling by high throughput sequencing


title: Targeting lysine-specific demethylase 1 (KDM1A/LSD1) impairs colorectal cancer tumorigenesis by affecting cancer cells stemness, motility and differentiation

city: Novara

first: Francesco

last: Favero

state: NO

address: Corso Trieste 15/A

department: Translational Medicine - CAAD (Center for Translational Research on Autoimmune and Allergic Diseases)

country: Italy

institute: University of Eastern Piedmont

summary: We explored the potential role of KDM1A targeting in CRC by characterizing the effect of KDM1A silencing in differentiated Colorectal Cancer Stem Cells human (CRC-SCs).

overall_design: Total RNA Seq of KDM1A silenced (sh71) or control (shNT) Colorectal Cancer Stem Cells human (CRC-SCs)

first: Annamaria

last: Antona

first: Stefania

last: Faletti

first: Giuliana

last: Pelicci

first: Davide

last: Corà

first: Daniela

last: Capello


title: Mating activates transcriptional programs and represses microRNAs in the Drosophila melanogaster female reproductive tract [miRNA-seq]

summary: The female’s reproductive tract is exposed directly to the male’s ejaculate, making it a hotspot for mating-induced responses shortly after mating. In Drosophila melanogaster, changes in the reproductive tract are essential to optimize fertilization. To detect the earliest gene regulatory events that underlie these changes, we measured transcript abundances using RNA-seq and microRNA-seq of reproductive tracts of unmated females and females collected within 10-15 minutes after the end of mating, either to a wildtype male or to a male with defective BMP signaling in secondary cells of the accessory gland, which influences the composition of the male’s ejaculate. We observed transcript abundance changes for genes with roles in tissue morphogenesis, wound healing, the immune response and metabolism. Strikingly, predicted targets of microRNAs that respond to mating are enriched for overlapping functions, suggesting that mating-induced changes are in part regulated by microRNAs. Most of the differentially expressed RNAs are upregulated in response to mating, while most of the differentially expressed microRNAs are downregulated. This pattern suggests a response of activation and de-repression of gene programs that switch the reproductive tract to a “mated” state, rather than a repression of virgin-specific programs. Male genotype did not influence transcript levels, indicating that the earliest transcriptomic responses in the reproductive tract are not dependent on ejaculate components that require BMP signaling in secondary cells. Our results shed light on the molecular changes that accompany very early responses to mating and present candidate genes and microRNAs that can be further examined for their participation in alterations of the reproductive tract microenvironment in response to signals from the male.

overall_design: RNA-seq and microRNA-seq was done on the reproductive tracts of unmated D. melanogaster females, and on females mated to control males or mated to males with inhibited BMP signaling in the secondary cells of their accessory glands. Females were collected for reproductive tract dissections within 10-15 minutes after the end of mating. 3 biological replicates were collected for each treatment.

middle: Y

first: Andrew

last: Clark

first: Mariana

middle: F

last: Wolfner


title: Mating activates transcriptional programs and represses microRNAs in the Drosophila melanogaster female reproductive tract [mRNA-seq]


status: Public on Jun 26 2023

first: Hui

last: Pan

address: 1 Joslin Place

department: Bioinformatics and Biostatistics Core

institute: Joslin Diabetes Center

summary: To evaluate the global transcriptomic changes induced by Neuronal Regeneration Related Protein (NREP) downregulation, we employed RNA-sequencing in human primary hepatocytes lacking NREP.

overall_design: We used pure, highly viable, and plateable primary human hepatocytes from 5 pooled donors (ThermoFisher, #HMCPP5). Briefly, hepatocytes were seeded on 6-well collagen-treated plates containing William E media supplemented with primary hepatocyte supplements (ThermoFisher) and HepExtend supplement (ThermoFisher).  Hepatocytes were allowed to attach for 6h. Media was exchanged to eliminate non-viable/attached cells that were forward transfected with Lipofectamine RNAiMAX (ThermoFisher) and 100nM of siGENOME Non-Targeting siRNA Pool (Dharmacon, # D-001206-13-05), or siGENOME human NREP siRNA (Dharmacon, # M-019848-00-0005) in complete media. Media was then changed at 6h post-transfection with complete media. At 48h post-transfection cells were starved for 16h in William E media containing 0.1% BSA, washed three times with ice-cold DPBS, and snap-frozen and pelleted for further analyses.

first: Dario

last: DeJesus

first: Rohit

last: Kulkarni


title: Single-cell transcriptome and TCR repertoire sequencing of cholinergic CD4+ T cells in liver cancer

city: Toronto

first: Tak

last: Mak

state: ON

address: 610 University Ave

department: The Campbell Family Institute for Breast Cancer Research

institute: Princess Margaret Cancer Centre, University Health Network

summary: ACh was originally isolated from spleen back in 1929, however, its contribution to immune regulation has only recently been appreciated. Subsets of both T and B lymphocytes have been found to express choline acetyltransferase (Chat) and produce ACh. To date, Chat-expressing T cells have been described as relaying neural signals in the spleen to modulate immune responses; regulating blood pressure; and promoting anti-viral immune responses. In a murine multi-hit model of hepatocellular carcinoma, we observed activation of the adaptive immune response and induction of Chat-expressing CD4+ T cells. These cholinergic T cells curtail the development of liver cancer by supporting anti-tumor immune responses. We used single-cell RNA sequencing to investigate the transcriptome and TCR repertoire of the Chat-expressing CD4+ T cells in healthy and HCC-bearing livers.

overall_design: HCC was induced in Chat-GFP mice by Myc overexpression and combined Trp53 and Pten ablation. HCC livers (from two male and two female mice) and control livers (from sex-matched littermates) were collected for hepatic mononuclear cell (MNC) isolation. Hepatic MNCs were stained with antibodies for CD4, CD8, CD19, TCR-β, NK1.1, CD45 and CD1d tetramer, as well as with barcode antibodies (TotalSeq-C0304, C0305, C0306, or C0307) to hashtag cells from individual mice of the HCC or control group. CD45+DAPI-NK1.1-CD1dTetramer-CD19-TCR-β+CD4+CD8-GFP+ (Chat-GFP+ CD4 T cells) and CD45+DAPI-NK1.1-CD1dTetramer-CD19-TCR-β+CD4+CD8-GFP- (Chat-GFP- CD4 T cells) populations were sorted. The two CD4 T cell compartments were individually sorted from each mouse. The same CD4 T compartments from mice under the same treatment were pooled into 4 samples: control Chat-GFP+, control Chat-GFP-, HCC Chat-GFP+ and HCC Chat-GFP-. After sorting and pooling, the samples were immediately submitted to the Princess Margaret Genomics Centre for downstream processing.

first: Chunxing

last: Zheng

middle: W


title: Characterized functions of hypothetical genes identified by Tn-seq important for pathgoenesis of Staphylococcus aureus pneumonia

city: Newark

first: Dane

last: Parker

state: NJ

address: 205 South Organge Ave, G-1208

department: Pathology, Immunology and Laboratory Medicine

institute: Rutgers New Jersey Medical School

summary: To identify new genetic elements required for S.aureus pathogenesis, we performed a transposon sequencing in the context of acute murine penumonia. We screened conditionally essential genes identified by Tn-seq and confirmed 6 previously uncharacterized genes were important during infection. As further study, we performed RNA-seq to identify uncharacterized genes.

overall_design: 6 hypothetical mutants (SAUSA300_1720, SAUSA300_1902, SAUSA300_1650, SAUSA300_2068, SAUSA300_0903 and SAUSA300_0917 ) were grown in LB broth up to 1.0 (OD600nm). Bacterial RNA was extracted by the EZRNA isolation kit (Omega biotech) and then RNA-seq performed

first: Gyu-Lee

last: Kim


title: GATA2 mitotic bookmarking is required for definitive haematopoiesis [ChIP-seq]

city: Lund

first: Ilia

last: Kurochkin

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for GATA2 in asynchronous and mitotic K562 cells.

first: R

last: Silvério-Alves

first: I

first: F

last: Pereira


title: TMPRSS2-ERG and gain-of-function p53 mutants co-dictate pyrimidine synthesis and prostate cancer fitness [ERG and p53 ChIP-seq]

city: Rochester

first: LIGUO

last: WANG

state: MN

address: 200 1st St SW

department: Division of Computational Biology

institute: Mayo Clinic

summary: TMPRSS2-ERG fusion is the most common genetic alteration in prostate cancer (PCa) and TP53 is the most frequently mutated gene in human cancers. However, their precise roles in PCa pathogenesis remain elusive. Here we showed that TMPRSS2-ERG fusion co-occurred with TP53 deletion/mutation in PCa patient specimens. ERG overexpression and Trp53 knockout/R172H mutant knockin induced pyrimidine synthesis gene (PSG) expression and prostate tumorigenesis in mice. Gain-of-function p53 mutants bound to the CTNNB1 promoter and upregulated β-Catenin. Overexpressed ERG and β-Catenin co-occupied PSG loci and mediated PSG expression, and high PSG expression associated with increased β-Catenin level and poor overall survival of PCa patients. β-Catenin inhibition by proteolysis-targeting chimeras (PROTACs) of its co-activator CBP and partner proteins LEF1/TCFs blocked ERG/p53-mutant PCa growth. Our study identifies CTNNB1 as a transcriptional target of p53 GOF-mutants, and reveals a druggable dependency on β-Catenin and pyrimidine synthesis in p53-mutated cancers with or without TMPRSS2-ERG fusion.

overall_design: To investigate the genes occupied by ERG or p53 in VCaP cells

first: Haojie

last: Huang

first: Liguo

last: Wang

first: Alexandra

last: Blee

first: Donglin

last: Ding


title: Antibody-microarrays in human primary hepatocytes lacking NREP

summary: To evaluate the global signaling changes induced by Neuronal Regeneration Related Protein (NREP) downregulation, we employed antibody-microarrays in human primary hepatocytes lacking NREP.


title: Norepinephrine modulates calcium dynamics in cortical oligodendrocyte precursor cells promoting proliferation during arousal in mice

city: Los Angeles

first: Riki

last: Kawaguchi

address: 760 Westwood Plaza, Room 37-420

department: Department of Psychiatry and Neurology Department of Psychiatry

institute: University of California, Los Angeles

summary:
Oligodendrocyte precusor cells (OPCs) exhibit near membrane calcium transients that can be enhacned by norepinephrine-mediated activation of alpha1A adrenergic receptors in vivo.
In order to determine if norepinephrine directly modulates OPC behaviors, we performed an unbiased, transcriptomic-based assessment of biological processes altered in OPCs after activation of alpha1 adrenergic receptors

overall_design: Comparative gene expression profiling analysis of RNA-seq data for primary mouse OPCs (isolated from P4-P7 CD-1 pups, equal sexes) treated with and without phenylephrine (PE, 20 uM for 1 hr)

first: Tsai-Yi

last: Lu

first: Dwight

last: Bergles


title: Gene expression analysis comparing rat neonatal cardiomyocytes and human iPSC-cardiomyocytes

city: Montreal

first: Terence

last: Hébert

state: Quebec

address: 3655 Promenade Sir William Osler

department: Pharmacology and Therapeutics

institute: McGill University

summary: Bulk RNA sequencing of rat neonatal cardiomyocytes and hiPSC-cardiomyocytes treated with vehicle or GPCR agonists for 60 minutes.

sample_organism:
Homo sapiens
Rattus norvegicus

platform_organism:
Homo sapiens
Rattus norvegicus

first: Kyla

last: Bourque


title: Identification of N6-methyladenosine (m6A) modified RNA in activated ILC3

first: Yuefeng

address: 701 West 168th Street

department: Microbiology & Immunology

institute: Columbia University

summary: Innate lymphoid cells (ILCs) are able to directly respond to alarmin signals and produce an array of effector molecules for immune protection and tissue homeostasis. However, how posttranscriptional machinery in ILCs execute extracellular stimuli towards robust gene expression is yet to understand. Here, we reported a cell type-specific role of N6-methyladenosine (m6A) RNA methylation in ILCs. Inducible deletion of m6A methyltransferase METTL3 had little impact on ILC maintenance in the steady state or cytokine-induced ILC1 or ILC3 activation, but dramatically diminished IL-25-triggered ILC2 response. Specific deletion of Mettl3 in ILC2 significantly attenuated cell expansion, cytokine production, inter-organ migration, and anti-helminth immunity. To investigate the molecular mechanism by which m6A modification regulates ILC response, we subjected IL-1 plus IL-23-activated ILC3 to a m6A-tagged mRNA immunoprecipitation sequencing (meRIP-seq) to identify the m6A modified mRNA.

overall_design: We sorted IL-1 plus IL-23-induced ILC3 that were activated ILC3 in small intestine of C57BL/6 mice and isolated the total RNA from the ILC3. Then we performed methylated RNA immunoprecipitation (meRIP) using anti-m6A antibody to obtain the unbound and pellet fractions. The unbound fraction is non-m6A modified RNA fraction while the pellet fraction is m6A modified RNA fraction. We next constructed the libraries for unbound and pellet fractions and performed the sequencing.

first: Yingyu

last: Zhang

first: Wanwei

first: Jingyao

last: Zhao

first: Takamasa

last: Ito

first: Jiacheng

last: Jin

first: Alexis

last: Aparicio

first: Junsong

last: Zhou

first: Vincent

last: Guichard

first: Joseph

last: Urban Jr

first: Jacob

middle: H

last: Hanna

first: Sankar

last: Ghosh

first: Xuebing

last: Wu

first: Lei

first: Uttiya

last: Basu


title: Identification of N6-methyladenosine (m6A) modified RNA in ILC2 of WT and Mettl3 Klrg1Cre mice

last: Huang

summary: Innate lymphoid cells (ILCs) are able to directly respond to alarmin signals and produce an array of effector molecules for immune protection and tissue homeostasis. However, how posttranscriptional machinery in ILCs execute extracellular stimuli towards robust gene expression is yet to understand. Here, we reported a cell type-specific role of N6-methyladenosine (m6A) RNA methylation in ILCs. Inducible deletion of m6A methyltransferase METTL3 had little impact on ILC maintenance in the steady state or cytokine-induced ILC1 or ILC3 activation, but dramatically diminished IL-25-triggered ILC2 response. Specific deletion of Mettl3 in ILC2 significantly attenuated cell expansion, cytokine production, inter-organ migration, and anti-helminth immunity. To investigate the molecular mechanism by which m6A modification regulates ILC2 response, we subjected ILC2 of WT and Mettl3 Klrg1Cre mice to a m6A-tagged mRNA immunoprecipitation sequencing (meRIP-seq) to identify the m6A modified mRNA.

overall_design: We sorted ILC2 from small intestine of WT and Melttl3 Klrg1Cre mice and isolated the total RNA from the ILC2. Then we performed methylated RNA immunoprecipitation (meRIP) using anti-m6A antibody to obtain the pellet fractions which are m6A modified RNA fractions. We next constructed the libraries for pellet fractions and performed the sequencing.

last: Ding


title: The single-cell transcriptome and chromatin accessibility datasets of peripheral blood mononuclear cells in Chinese Holstein cattle

city: Kongens Lyngby

first: Xiao

last: Wang

address: Bagsværdvej 103, ST

country: Denmark

institute: Konge Larsen Aps

summary: This study is performed in the frame of a bigger study dedicated to the integrated analysis of the single-cell transcriptome and chromatin accessibility datasets of peripheral blood mononuclear cells (PBMCs) with a large-scale GWAS of 45 complex traits in Chinese Holstein cattle. In dairy cattle, lipopolysaccharide (LPS) is a crucial mediator of chronic inflammation to modulate immune responses. PBMCs include primary T and B cells, natural killer (NK) cells, monocytes (Mono), and dendritic cells (DC). It still remains unknown how LPS stimulated PBMCs at the single-cell level in dairy cattle. Therefore, the single-cell transcriptome and chromatin accessibility datasets in this study enable the further understanding and application of the cell types and functions of PBMCs and their responses to LPS stimulation in vitro in other studies.

overall_design: Four whole blood samples were collected from four 2-year old Holstein female lactating cattle through the tail vein. Afterwards, they were mixed in one pool and evenly divided into four replicates. Four whole-blood treatments for each replicate included one control (no LPS) treatment and three case treatments (2 h, 4 h, and 8 h LPS), where 2 µg/ml LPS (Product Number: L2880, Sigma-Aldrich, Saint Louis, MO, USA) was used at 37 °C.

first: Jianbin

last: Li


title: Identification of N6-methyladenosine (m6A) modified RNA in activated ILC1, activated ILC3 and ILC2 of WT and Mettl3 Klrg1Cre mice

first: Yuefeng

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

overall_design: Refer to individual Series


title: Identification of N6-methyladenosine (m6A) modified RNA in activated ILC1

summary: Innate lymphoid cells (ILCs) are able to directly respond to alarmin signals and produce an array of effector molecules for immune protection and tissue homeostasis. However, how posttranscriptional machinery in ILCs execute extracellular stimuli towards robust gene expression is yet to understand. Here, we reported a cell type-specific role of N6-methyladenosine (m6A) RNA methylation in ILCs. Inducible deletion of m6A methyltransferase METTL3 had little impact on ILC maintenance in the steady state or cytokine-induced ILC1 or ILC3 activation, but dramatically diminished IL-25-triggered ILC2 response. Specific deletion of Mettl3 in ILC2 significantly attenuated cell expansion, cytokine production, inter-organ migration, and anti-helminth immunity. To investigate the molecular mechanism by which m6A modification regulates ILC response, we subjected IL-12 plus IL-18-activated ILC1 to a m6A-tagged mRNA immunoprecipitation sequencing (meRIP-seq) to identify the m6A modified mRNA.

overall_design: We sorted IL-12 plus IL-18-induced ILC1 that were activated ILC1 in liver of C57BL/6 mice and isolated the total RNA from the ILC1. Then we performed methylated RNA immunoprecipitation (meRIP) using anti-m6A antibody to obtain the unbound and pellet fractions. The unbound fraction is non-m6A modified RNA fraction while the pellet fraction is m6A modified RNA fraction. We next constructed the libraries for unbound and pellet fractions and performed the sequencing.

first: Yingyu


title: miRNA effectors of estrogen’s bone anabolic activity in a murine model of post-menopausal osteoporosis

first: Michael

middle: Justin

last: Emch

state: Minnesota

address: 200 1st Street SW

department: Biochemistry and Molecular Biology

summary: Estrogen signaling is critical for the development and maintenance of healthy bone and age-related declines in estrogen levels leads to the development of post-menopausal osteoporosis. The large majority of bones consist of a dense outer cortical shell and an internal mesh-like network of trabecular bone which respond differently to external cues such as hormonal signaling. To date, no study has assessed the transcriptomic differences that occur specifically in cortical and trabecular bone compartments in response to hormonal changes. To investigate this, we employed a mouse model of post-menopausal osteoporosis (ovariectomy (OVX)) and estrogen replacement therapy (ERT).  mRNA and miR sequencing revealed highly distinct transcriptomic profiles between cortical and trabecular bone in the setting of OVX and ERT. Through extensive bioinformatic analyses, 7 miRs were identified as most likely to contribute to the observed estrogen-mediated gene expression changes.  Of these, 4 miRNAs were prioritized for further study and were shown to be capable of decreasing the expression of predicted target genes in bone cells, to be estrogen regulated, to be able to enhance the expression of osteoblast differentiation genes, and to confer alterations to the mineralization capacity of primary calvarial osteoblasts. As such, candidate miRs, and miR mimics, may have the capacity to mimic the bone beneficial responses of ERT without the unwanted side effects and therefore represent a novel class of therapeutic approaches to combat diseases related to bone loss.

overall_design: miRNA sequencing in cortical and trabecular bone compartments in a murine model of post-menopausal osteoporosis (sham operated, ovariecomized mice, and ovariecomized + estrogen replacement therapy)

first: John

last: Hawse


title: miRNA effectors of estrogen’s bone anabolic activity in a murine model of post-menopausal osteoporosis [mRNA]

overall_design: mRNA sequencing in cortical and trabecular bone compartments in a murine model of post-menopausal osteoporosis (sham operated, ovariecomized mice, and ovariecomized + estrogen replacement therapy)


title: CRISPR/Cas9 screen of T-ALL cells (DND-41) in the presence of DMSO, CB-103 or GSI (DAPT).

city: Lausanne

first: Nadine

last: Fournier

state: Vaud

address: Agora Cancer Research Center, Bugon 25A

country: Switzerland

institute: Agora Cancer Research Center & Swiss Institute of Bioinformatics

summary: The efficacy of targeted therapies for treatment of cancer patients is often limited by development of drug resistance. Potential resistance mechanisms to pharmacological Notch1 inhibition mediated by GSI or CB-103 in T-ALL are currently unclear. Thus, we performed a genome-wide loss-of-function (LoF) CRISPR/Cas9 screen to identify genes responsible for resistance to Notch inhibition and novel combination therapies for efficient treatment of human T-ALL. We used a Notch-dependent human T-ALL cell line, DND-41, which responds moderately to both GSI and CB-103 treatment in vitro. DND-41 cells stably expressing Cas9 were infected with human GeCKO v2 CRIPSR libraries, containing 123,411 sgRNAs targeting 19,050 genes and treated with either vehicle, GSI or CB-103 for 21 days enabling both positive and negative selection of sgRNAs.

overall_design: In total, 10 samples (one sample at time zero and triplicates of DMSO, CB-103, and GSI treated samples) were sequenced.( Time zero is for initial library quality check. ) The other 9 samples are to identify genes responsible for resistance to Notch inhibition and novel combination therapies for efficient treatment of human T-ALL.

first: Gustavo

last: Ruiz Buendia

first: Linlin

last: Cao

first: Freddy

last: Radtke


title: Expression profile of PIK3R1 knockout and non-targeting control cells in response to Notch inhibition

summary: To gain insights how loss of PIK3R1 confers resistance to pharmacological Notch inhibition in T-ALL cells, we performed gene expression analysis. Libraries for mRNA-seq were prepared with the Stranded mRNA Ligation method (Illumina) starting from 1µg RNA, according to manufacturer’s instructions. Libraries, all bearing unique dual indices, were subsequently loaded at 250pM in a HiSeq 4000 instrument (Illumina) and sequenced according to manufacturer’s instructions, yielding paired-end reads of 75 nucleotides. Reads were trimmed of their adapters with bcl2fastq v2.20 (Illumina) and quality-controlled with FastQC v0.11.9. PIK3R1 deficiency leads to up-regulation of cell cycle and pro-survival genes in response to Notch inhibition.

overall_design: Expression profile comparing PIK3R1 knockout and non-targeting control RPMI-8402 cells in response to Notch inhibitor CB-103 or GSI (LY3039278) for 24hrs. Experiments are set up in triplicates.


title: BiTE-secreting T cells effectively target ovarian cancer and prolong antitumor immunity by persisting in the extratumoral peritoneal cavity

city: Buffalo

first: Mark

middle: D

last: Long

address: Elm & Carlton Streets

department: Bioinformatics & Biostatistics

institute: Roswell Park Comprehensive Cancer Center

summary: Cancer immunotherapies can produce complete therapeutic responses, however outcomes in ovarian cancer (OC) are modest and while adoptive transfer of engineered T cells (ACT) has been evaluated in OC, durable effects are rarely observed. T cells lacking tumor specificity (bystander T cells) readily accumulate in tumors and leveraging bystander T cell activity represents a promising opportunity to enhance antitumor immunity following ACT. To this end, we have generated T cells that stably secrete a FRα-directed bispecific T cell engager (FR-B T cells), which robustly targeted FRα+ tumor cells in OC patient specimens and effectively engaged patient T cells present in the tumor microenvironment (TME). Therapeutic testing of FR-B T cells using an immunocompetent OC model demonstrated robust activity, with response duration dependent on both endogenous T cells and FR-B T cell persistence. Cytokine preconditioning prior to infusion produced less differentiated FR-B T cells and improved therapeutic efficacy, with mechanistic studies revealing preferential accumulation of TCF-1+ CD69-CD39- stem-like CD8+ FR-B T cells in the peritoneal cavity over solid tumors. These findings highlight the potential of FR-B T cell therapy in OC and suggest the peritoneal TME permits FR-B T cells to persist in the extratumoral space while actively directing antitumor immunity.

overall_design: RNAseq analysis of sorted CD8+ FR-B TALs pre-conditioned with  IL-2/IL-7 or IL-2/IL-15 was conducted using the Takara Bio USA, Inc. SMART-Seq® v4 PLUS Kit

first: Robert

last: McGray


title: Localization of the Drosophila pioneer factor GAF to subnuclear foci is driven by DNA binding and required to silence satellite repeat expression

city: Madison

first: Melissa

last: Harrison

address: 420 Henry Mall

institute: University of Wisconsin Madison


title: Convergence of distinct RNA-silencing pathways on GW182/Tnrc6 [sgRNA screen]

city: Basel

first: Helge

last: Grosshans

address: Maulbeerstrasse 66

institute: Friedriche Miescher Institute for Biomedical Research (FMI)

summary: sgRNA screens for factors influencing Trim71 silencing activity.

overall_design: Screen1: Genome-wide screen: Cells were transduced with two separate lentiviral sgRNA libraries (library 1: 52,000 unique sgRNAs (~5 per gene); library 2: 39,000 unique sgRNAs (~5 per gene); (Zeng et al., 2018). Cells were transduced at a multiplicity of infection (MOI) of 0.31 using 10 µg/ml polybrene at 700x coverage/sgRNA. Screen2: validation screen: Cells were transduced at an MOI of 0.1 using 10 µg/ml polybrene with a 500x coverage/sgRNA.

first: Thomas

last: Welte

first: Alison

last: Goulois

middle: B

last: Stadler

first: Daniel

last: Hess

first: Charlotte

last: Soneson

first: Anca

last: Neagu

first: Chiara

last: Azzi

first: Marlena

last: Lübke

first: Jan

last: Seebacher

first: Isabel

last: Schmidt

first: David

last: Estoppey

first: Florian

last: Nigsch

last: Reece-Hoyes

first: Dominic

last: Hoepfner

last: Großhans


title: Single cell transcriptomic analysis of cortical cell type composition underlying diabetes-associated cognitive decline

city: Belfast

first: Aditi

last: Singh

state: Please Select State

address: 97 Lisburn Road, Belfast

department: The Wellcome-Wolfson Institute for Experimental Medicine, SMDBS

institute: Queen's University Belfast

summary: Type 2 diabetes (T2D) is associated with increased risk of cognitive decline although the precise underlying pathogenesis remains obscure. Based on the single-cell RNA sequencing approach applied to the cerebral cortex of a T2D mouse model (db/db), we identified novel regulatory mechanisms underlying diabetes-associated cognitive deficits. Our data adds detail to the highly complex neurovascular pathology associated with T2D and highlights key cell-specific deficits in mitochondrial bioenergetics linked to neuroinflammation, which underlie T2D-linked cognitive impairment.

overall_design: For scRNA-seq, mouse were sacrificed at 16 weeks of age. The cortex was dissected and dissociated using the papain dissociation system (Worthington, Lakewood, NJ) as per the manufacturer’s instructions.

first: Karis

last: Little

first: Alan

last: Stitt

first: Vijay

last: Tiwari


title: Co-transcriptional pseudouridylation of mRNAs by the H/ACA complex controls translational efficiency.

city: Solna

first: Federico

last: Agostini

state: Stockholm

address: Tomtebodavägen 23a

institute: Science for Life Laboratories


title: R-loop associated G-quadruplexes reinforce CTCF interactions

city: Philadelphia

first: Phillip

last: Wulfridge

state: Pennsylvania

address: 3601 Spruce St

institute: The Wistar Institute

summary: We report that three-stranded R-loop structures facilitate CTCF binding genome-wide through the formation of G-quadruplex (G4) structures. Genome-wide assays of CTCF and R-loop formation under various treatment conditions reveal that R-loop removal and G4 stabilization can both alter CTCF occupancy, and these changes are associated with alterations in gene expression and chromatin organization.

overall_design: CUT&RUN, MapR, RNA-Seq, ChIP, and Hi-C was performed on E14 and EN52.9.1 mouse embryonic stem cells. Cells were untreated, treated with 150 U Rnase H for 1 hour during CUT&RUN and MapR protocol, or treated with auxin, pyridostatin, PhenDC3, and/or actinomycin D prior to genomics experiments.

first: Qingqing

last: Yan

first: Kavitha

last: Sarma


title: Co-transcriptional pseudouridylation of mRNAs by the H/ACA complex controls translational efficiency [ChIP-seq]

summary: Post-transcriptional modifications of mRNA have emerged as novel regulators of gene expression. Pseudouridylation is the most abundant and widespread type of RNA modification in living organisms; however, the biological role of pseudouridine in mRNAs remains poorly understood. Here, we show that the pseudouridine synthase dyskerin associates with RNA polymerase II and is enriched at RNA polymerase II-transcribed genes genome-wide. As part of the H/ACA complex, dyskerin binds to thousands of mRNAs and is responsible for their pseudouridylation, an action that occurs in chromatin and does not appear to require a fully complementary guide RNA. In cells lacking dyskerin, pseudouridylation of mRNAs is reduced, while at the same time, de novo protein production is enhanced, indicating that pseudouridylation of mRNAs by dyskerin interferes with translation. Accordingly, pseudouridylation of an mRNA by dyskerin in vitro results in reduced translation of that mRNA. Moreover, in patients with dyskeratosis congenita caused by inherited mutations in the DKC1 gene, binding between mutated dyskerin and mRNAs is altered and pseudouridylation of mRNAs severely reduced. Our findings reveal a new critical function of the H/ACA complex in directing translation control with important implications for development and disease.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for RNAPII and dyskerin in U2OS and HCT116 cells.

last: Pederiva


title: Identification of N6-methyladenosine (m6A) modified RNA in activated ILC2

first: Yuefeng

last: Huang

state: NY

summary: Innate lymphoid cells (ILCs) are able to directly respond to alarmin signals and produce an array of effector molecules for immune protection and tissue homeostasis. However, how posttranscriptional machinery in ILCs execute extracellular stimuli towards robust gene expression is yet to understand. Here, we reported a cell type-specific role of N6-methyladenosine (m6A) RNA methylation in ILCs. Inducible deletion of m6A methyltransferase METTL3 had little impact on ILC maintenance in the steady state or cytokine-induced ILC1 or ILC3 activation, but dramatically diminished IL-25-triggered ILC2 response. Specific deletion of Mettl3 in ILC2 significantly attenuated cell expansion, cytokine production, inter-organ migration, and anti-helminth immunity. To investigate the molecular mechanism by which m6A modification regulates ILC2 response, we subjected IL-25-activated iILC2 to a m6A-tagged mRNA immunoprecipitation sequencing (meRIP-seq) to identify the m6A modified mRNA.

overall_design: We sorted IL-25-induced iILC2 that were activated ILC2 in MLN of C57BL/6 mice and isolated the total RNA from the iILC2 which was the input fraction. Then we performed methylated RNA immunoprecipitation (meRIP) using anti-m6A antibody to obtain the unbound and pellet fractions. The unbound fraction is non-m6A modified RNA fraction while the pellet fraction is m6A modified RNA fraction. We next constructed the libraries for input, unbound and pellet fractions and performed the sequencing.

first: Yingyu

last: Zhang

first: Wanwei

first: Jingyao

last: Zhao

first: Takamasa

last: Ito

first: Jiacheng

last: Jin

first: Alexis

last: Aparicio

first: Joseph

last: Urban Jr

first: Jacob

last: Hanna

first: Xuebing

last: Wu

first: Lei

last: Ding

first: Uttiya

last: Basu


title: Convergence of distinct RNA-silencing pathways on GW182/Tnrc6 [RIP-seq]

first: Helge

last: Grosshans

summary: RIP-sequencing in mouse embryonic stem cells. Experiment 1 (Samples 1-18): Base cell line (untagged control cells): GFP_5xT71BS (mES [129S6/SvEvTac], yGlob [GFP_5xT71BS], stable transfection FLAG-Cas9::T2A::Blasticidin). HA-Tnrc6a: endogenously HA-AVI-tagged Tnrc6a. HA-Tnrc6a_Trim71KO: HA-AVI-tagged Tnrc6a, Trim71-/-. Experiment 2 (Samples 19-42): Ctrl: base cell line GFP_5xT71BS. Tnrc6a: endogenously HA-AVI-tagged Tnrc6a. Tnrc6a_Trim71KO: HA-AVI-tagged Tnrc6a, Trim71-/-. Tnrc6a_Ago2KO: HA-AVI-tagged Tnrc6a, Ago2-/-.

overall_design: Cells were grown in triplicates to confluency in 10 cm dishes. Cells were washed 1x with PBS and lysed with lysis buffer (25 mM Tris-HCL at pH 7.5, 100 mM KCl, 0.5% [v/v] NP-40, 1 mM EDTA, EDTA-free cOmplete miniprotease inhibitor [Roche], PhosSTOP [Roche]. RNAse inhibitors [SUPERase•In™ RNase Inhibitor, Invitrogen, AM2694] was added to all buffers. Lysates were cleared by centrifugation. Equal amounts of protein were incubated with 20 µL of Pierce™ HA magnetic beads (Thermo Scientific, 88836) for 2 h at 4°C. Beads were washed three times with 200 µL of washing buffer (50 mM Tris-HCl at pH 7.5, 150 mM KCl, 5 mM MgCl2, 0.05% NP-40, EDTA-free cOmplete miniprotease inhibitor [Roche, 11836153001], PhosSTOP [Roche]). RNA was isolated using the Norgen single-cell RNA purification kit (Norgen, 51800) with on-column DNase digestion (Norgen, 25710).

first: Thomas

last: Welte

first: Alison

last: Goulois

first: Michael

last: Stadler

first: Daniel

last: Hess

first: Charlotte

last: Soneson

first: Anca

last: Neagu

first: Chiara

last: Azzi

first: Marlena

last: Lübke

first: Jan

last: Seebacher

first: Isabel

last: Schmidt

first: David

last: Estoppey

first: Florian

last: Nigsch

first: John

last: Reece-Hoyes

first: Dominic

last: Hoepfner

last: Großhans


title: Convergence of distinct RNA-silencing pathways on GW182/Tnrc6 [RNA-seq]

summary: RNA-sequencing in mouse embryonic stem cells. Experiment 1 (Samples 1-9): Base cell line (WT): GFP_5xT71BS (mES [129S6/SvEvTac], yGlob [GFP_5xT71BS], stable transfection FLAG-Cas9::T2A::Blasticidin). Trim71 KO: GFP_5xT71BS Trim71-/-.  Ago2 KO: GFP_5xT71BS Ago2-/-. Experiment 2 (Samples 10-21): parental cell line: GFP_5xT71BS. T6B_Wt: Overexpression of T6B_WT, T6B_Mut: Overexpression of T6B_MUT. Trim71_KO: GFP_5xT71BS, Trim71-/- transfected with empty plasmid. Ago2_KO: GFP_5xT71BS, Ago2-/- transfected with empty plasmid.

overall_design: RNA-seq of all cell lines was done with 3x biological replicates.


title: Co-transcriptional pseudouridylation of mRNAs by the H/ACA complex controls translational efficiency [RNA-seq]

first: Federico

last: Agostini

overall_design: Gene expression profiling through RNAseq of U2OS

last: Pederiva


title: Gene expression profiling of tumor associated macrophages following perfluoro-crown-ether nanoparticles administration in a mouse model of glioma.

first: Nadine

last: Fournier

summary: High-grade gliomas, the most common and aggressive primary brain tumors, are characterized by a complex and multifaceted tumor microenvironment (TME). Among the non-malignant cells of the glioma TME, tumor-associated microglia and macrophages (TAMs) constitute the major compartment. In patients, a greater abundance of TAMs is associated with high-grade and more aggressive disease. In a range of glioma mouse models, we have observed specific alterations in TAM dynamics and functions during tumor development and following different therapeutic interventions, including irradiation. The underlying TAM abundance may also modulate the subsequent response to TAM-targeted therapy, including the anti-CSF1R inhibitor BLZ945, which results in a pronounced tumor regression in different preclinical models of brain cancers, as our lab has recently shown.  These observations indicate that it is crucial to evaluate TAM abundance and dynamics in gliomas. Current techniques to quantify TAMs in patients rely mainly on histological staining of tumor biopsies. While informative, these techniques require an invasive procedure to harvest the tissue sample and typically only result in a snapshot of a small region at a single point in time. Fluorine isotope 19 MRI (19F MRI) has been shown to be a powerful tool to non-invasively and longitudinally monitor myeloid cells in other pathological conditions by injecting perfluorocarbon-containing nanoparticles (PFC-NP). In this study, we demonstrated the feasibility and power of 19F MRI in preclinical glioma models and brain metastasis models and showed that the major cellular source of 19F signal consists of TAMs. Moreover, PFC-NP accumulation occurred predominantly in proximity to dysmorphic vessels, thereby enabling the identification of a TAM subpopulation with a specific oxidative metabolism. We also evaluated this imaging modality in the context of irradiation (IR) treatment and found that the 19F signal enables the tracking of TAM dynamics over time following IR. Moreover, multispectral 19F MRI with two different PFC-NP allowed us to identify spatially- and temporally-distinct TAM niches in IR-recurrent tumors. Together, we have been able to image TAMs non-invasively and longitudinally with a powerful integrated cellular, spatial and temporal resolution, while revealing new biological insights into the many functions of TAMs, including in disease recurrence.

overall_design: This study aimed to compare the expression profile of PFCE-NP uptaking versus non-uptaking TAMs. Glioma-bearing mice (n=6) were intravenously injected with fluorescently-labelled Rhodamine-PFCE-NP. 24 hours later, the tumor was harvested and processed for fluorescence-activated cell sorting (FACS). Paired Rhodamine+ TAMs and Rhodamine- TAMs (>2000 cells, gating stategy based on CD45+/Cd11b+/Ly6C-/Ly6C-) were sorted directly into TRIzol LS (Thermo Fischer Scientific) and immediately snap frozen in liquid nitrogen.

first: Davide

last: Croci

last: Fournier Zangger

first: Joahanna

last: Joyce


title: Insights into energy balance dysregulation from a mouse model of methylmalonic aciduria

city: Neuherberg

first: Johannes

last: Beckers

address: Ingolstaedter Landstr. 1

department: Institute of Experimental Genetics

institute: Helmholtz Zentrum Muenchen

summary: Maintenance of energetic homeostasis during periods of limited supply requires metabolic adaptation. Inherited disorders of mitochondrial metabolism, including isolated methylmalonic aciduria (MMAuria), present unique challenges to this homeostasis by disrupting energetic pathways. To better understand the systemic and cellular responses to long-term energy shortage, we investigated a hemizygous mouse model of MMAuria that has a knock-in (ki) missense allele combined with a knock-out (ko) allele (Mmut-ko/ki) and shows typical clinical features including reduced growth. We found that Mmut-ko/ki (mutant) mice, compared to their Mmut-ki/wt (control) littermates, have reduced body mass along with a relative reduction in lean mass but increase in fat mass. White adipose tissue appears histologically normal, but brown adipose tissue show a process of whitening, in line with their lower body surface temperature and lesser ability to cope with cold challenge. Ad libitum fed mice show constitutive hypometabolism: they have reduced food intake, reduced energy expenditure, and reduced plasma glucose, but elevated lactate and fibroblast growth factor 21 (Fgf21). Together, these changes culminate in a loss of metabolic flexibility in mutant mice. This is manifested in lesser ability to regulate energy sources when switching from the fed to fasted state, and in a delayed glucose clearance in response to exogenous glucose and insulin. Liver investigations find multi-metabolite accumulation and suggest upregulation of peroxisome proliferator-activated receptor (Ppar)- and Fgf21-controlled metabolic pathways, including beta-oxidation, and downregulation of oxidative phosphorylation, as molecular consequences. Altogether, this study identifies a multi-faceted metabolic response to chronic energy shortage in MMAuria and suggests hypometabolism combined with dysregulated adaptive capability to be underlying features.

overall_design: We performed gene expression microarray analysis of liver from mice hemizygous for Mmut and control animals

first: Marie

last: Lucienne

first: Raffaele

last: Gerlini

first: Birgit

last: Rathkolb

first: Julia

last: Calzada-Wack

first: Patrick

last: Forny

first: Stephan

last: Wueest

first: Andres

last: Kaech

last: Traversi

first: Merima

first: Céline

last: Bürer

first: Antonio

last: Aguilar-Pimentel

first: Martin

last: Irmler

first: Sven

last: Sauer

first: Stefan

last: Kölker

first: Joseph

last: Dewulf

first: Guido

middle: T

last: Bommer

last: Hoces

first: Valerie

last: Gailus-Durner

first: Helmut

last: Fuchs

last: Rozman

first: D

last: Froese

first: Matthias

last: Baumgartner

last: Hrabě de Angelis


title: Co-transcriptional pseudouridylation of mRNAs by the H/ACA complex controls translational efficiency [iCLIP]

first: Federico

last: Agostini

overall_design: Mapping of dyskerin-RNA and GAR1-RNA interactions through iCLIP in U2OS cells.

first: Chiara

last: Pederiva


title: A designer peptide against the EAG2-Kvβ2 potassium channel targets the interaction of cancer cells and neurons to treat glioblastoma

status: Public on Jun 25 2023

city: Changsha

first: Siyi

last: Wanggou

state: Hunan

address: Xiangya Road, No.87

department: Departmeng of Neurosurgery

country: China

institute: Xiangya Hospital, Central South University

summary: This dataset includes the raw single-cell RNAseq data from Xenograft GBM tumor (G411)-bearing NSG mice model treated with either control peptide K59-78TAT or designer peptide K90-114TAT and raw bulk RNAseq data from GBM cell (G411)-neuron coculture model treated with either control peptide K59-78TAT or designer peptide K90-114TAT. In this project, we focused on transcriptomic alteration caused by designer peptide in tumor cells.

overall_design: Single-Cell RNAseq from Xenograft GBM tumor (G411)-bearing NSG mice model treated with either control peptide K59-78TAT or designer peptide K90-114TAT. Bulk RNAseq data from GBM cell (G411)-neuron coculture model treated with either control peptide K59-78TAT or designer peptide K90-114TAT.

first: Weifan

last: Dong

first: Hongwei

last: Liu

first: Xi

last: Huang


title: Mechanical Confinement Promotes Heat Resistance of Hepatocellular Carcinoma via SP1/IL4I1/AHR Axis

city: Guangzhou

first: Guopei

last: Zhang

address: 58 Zhongshan Road 2

institute: Sun Yat-sen University

summary: Mechanical stress can modulate the fate of cells in both physiological and extreme conditions. Recurrence of tumors after thermal ablation, a radical therapy for many cancers, indicates that some tumor cells can endure temperatures far beyond physiological ones. This unusual heat resistance with unknown mechanisms remains a key obstacle to fully realizing the clinical potential of thermal ablation. By developing a 3D bioprinting-based thermal ablation system, we demonstrate that hepatocellular carcinoma (HCC) cells in this 3D model exhibit enhanced heat resistance as compared with cells on plates. Mechanistically, the activation of transcription factor SP1 under mechanical confinement enhances the transcription of Interleukin-4-Induced-1, which catalyzes tryptophan metabolites to activate the aryl hydrocarbon receptor (AHR), leading to heat resistance. Encouragingly, the AHR inhibitor prevents HCC recurrence after thermal ablation. These findings reveal a previously unknown role of mechanical confinement in heat resistance and provide a rationale for AHR inhibitors as neoadjuvant therapy.

overall_design: To investigate the specific factors endowed by a bona fide 3D environment and reveal the mechanism of heat resistance in the 3D model, we performed RNA sequencing to delineate the transcriptional states of HCC cells in the 2D, 3D, and animal models. To interrogate the key factor among 11 metabolic genes that altered tumor metabolism to induce heat resistance, we performed an additional RNA-sequencing to explore the transcriptional state of HCC cells in the 3D model after heat stress compared with untreated control.

first: Ming

last: Kuang


title: Immunometabolic actions of trabectedin and lurbinectedin on human macrophages: Relevance for their antitumor activity

city: Madrid

first: Lisardo

last: Bosca

state: Madrid

address: Arturo Duperier 4

department: Metabolism and cellular Signaling

institute: Instituto Investigaciones Biomedicas Alberto Sols

summary: In recent years, it has been recognized the central role of cell bioenergetics in regulating immune cell function and fate giving rise to the interest in immunometabolism, an area of research focused on the interaction between metabolic regulation and immune function. Immunometabolism has been studied in the modulation of macrophage polarization.  Thus, early metabolic changes associated with the polarisation of macrophages into pro-inflammatory or pro-resolving cells under different stimuli have been characterized. Tumour-associated macrophages are among the most abundant cells in the tumour microenvironment; however, it exists an unmet need to study the effect of chemotherapeutics on macrophage immunometabolism. Here, a systems biology approach that integrates transcriptomics and metabolomics unveils the immunometabolic effects of trabectedin (TRB) and lurbinectedin (LUR), two intercalating DNA agents with proved antitumor activity in the low nanomolar range. Our results show that TRB and LUR activate human macrophages towards a pro-inflammatory functional phenotype by inducing a specific metabolic rewiring program that includes ROS production and changes in the mitochondrial inner membrane potential, increased pentose phosphate pathway, TCA cycle serine and methylglyoxal pathways in human macrophages. glutamine, aspartate, histidine, and proline consumption are increased whereas 50 nM TRB increases lactate release and oxygen consumption is depressed . The observed immunometabolic rewiring could explain additional antitumor activities of these compounds and open new avenues to design therapeutic interventions that specifically target the immunometabolic landscape in the treatment of cancer.

overall_design: mRNA profiles of primary cultures of human monocyte-derived macrophages treated with TRB or LUR 100nM for 6h in RPMI+2%FBS or vehicle (controls RPMI+2%FBS)

last: Boscá

first: Antonio

last: Castrillo

first: Adrián

last: Povo-retana


title: Coordinated single cell tumor microenvironment dynamics reinforce pancreatic cancer subtype

city: Stony Brook

first: Scott

last: Powers

address: 101 Nicolls Rd

department: Pathology

institute: Stony Brook University School of Medicine -  - Stony Brook, NY

summary: Bulk analyses of pancreatic ductal adenocarcinoma (PDAC) samples are complicated by the tumor microenvironment (TME), i.e. signals from fibroblasts, endocrine, exocrine, and immune cells. Despite this, we and others have established tumor and stroma subtypes with prognostic significance. However, the interaction of underlying signals driving distinct immune and stromal landscapes is still unclear. Here we integrate 92 single cell RNA-seq samples from seven independent studies to build a reproducible PDAC atlas with a focus on tumor-TME interdependence. Patients with activated stroma are synonymous with higher myofibroblastic and immunogenic fibroblasts, and furthermore show increased M2-like macrophages and regulatory T-cells. Contrastingly, patients with ‘normal’ stroma showed M1 recruitment, elevated effector and exhausted T-cells. To aid interoperability of future studies, we provide a pretrained cell type classifier and an atlas of subtype-based signaling factors that we also validate in mouse data. Ultimately, this work leverages the heterogeneity among single-cell studies to create a comprehensive view of the orchestra of signaling interactions governing PDAC.

overall_design: Single-cell analysis of human derived pancreatic tumor tissue. Dissociated PDAC tissue of 2 patients undergoing Whipple procedure.

first: Ki

last: Oh

first: Manisha

last: Rao

first: Georgakis

last: Georgios

first: Aaron

last: Sasson

first: Mei

last: Gao

first: Richard

last: Moffitt


title: Convergence of distinct RNA-silencing pathways on GW182/Tnrc6

first: Helge

last: Grosshans

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

overall_design: Refer to individual Series


title: Single cell analysis of transcriptome and open chromatin reveals the dynamics of hair follicle stem cell aging

city: Chicago

first: Rui

last: Yi

state: IL

address: 303 E. Chicago Avenue

institute: Northwestern University


title: Effect of cholesterol  overload on gene expression of RAW264 macrophages

city: Nagoya

first: Michiko

last: Itoh

state: Aichi

address: Furo-cho, Chikusa-ku

department: Research Institute of Environmental Medicine

country: Japan

institute: Nagoya University

summary: Hepatocyte death plays a critical role in the disease progression from simple steatosis to non-alcoholic steatohepatitis (NASH). We have previously reported that hepatocyte death locally induces phenotypic changes in the macrophages surrounding the corpse and remnant lipids, thereby promoting liver fibrosis in a murine model of NASH. Here, we demonstrated free cholesterol was accumulated in macrophages around dead hepatocytes containing cholesterol crystals. In vitro experiments revealed that cholesterol-induced lysosomal stress triggered profibrotic activation in macrophages. This study provides evidence that dysregulated cholesterol metabolism in macrophages would be a novel mechanism of NASH.

overall_design: RAW264 macrophages were treated with cholesterol crystals (500 μg/ml) with or without β-cyclodextrin polyrotaxane for 24 hours. RNA sequencing analysis was performed to investigate the lysosomal stress response induced by cholesterol crystals.

first: Yasuyoshi

last: Oka

first: Tomoo

last: Ogi

first: Takayoshi

last: Suganami


title: Single cell analysis of transcriptome and open chromatin reveals the dynamics of hair follicle stem cell aging [scATAC-Seq]

summary: As the biggest organ of human body, skin and its appendages offer excellent experimental systems for aging studies. Skin wrinkling, hair greying and hair loss are among the most striking and observable traits of aging. However, the chronic changes and cell-to-cell variation accumulated during aging in different hair follicle lineages remain unexplored. Here we present a detailed analysis of scRNAseq and scATACseq data from young and aging animals. We found distinct cellular state changes of individual hair follicle populations.

overall_design:
scATAC-Seq P28, 12 months
Single cells were isolated from 24 month wildtype mouse back skin and then used for scATACseq

first: Chi

last: Zhang

first: Dongmei

last: Wang

first: Robin

last: Dowell


title: Spatial transcriptomics reveals distinct and conserved tumor core and edge architectures that predict survival and targeted therapy response

city: Calgary

first: Pinaki

last: Bose

state: AB

address: HMRB 354, 3330 Hospital Dr. NW

institute: University of Calgary

summary: We performed the first integrative single-cell and spatial transcriptomic analysis on HPV-negative oral squamous cell carcinoma (OSCC) to comprehensively characterize tumor core (TC) and leading edge (LE) transcriptional architectures. We show that the TC and LE are characterized by unique transcriptional profiles, cellular compositions, and ligand-receptor interactions. We demonstrate that LE regions are conserved across multiple cancers while TC states are more tissue specific, highlighting the existence of common mechanisms underlying tumor progression and invasion. Additionally, we found our LE gene signature is associated with worse clinical outcomes while the TC gene signature is associated with improved prognosis across multiple cancer types. Finally, using an in silico modeling approach, we describe spatially-regulated patterns of cell development in OSCC that are predictably associated with drug response. Our work provides pan-cancer insights into TC and LE biology and a platform for data exploration (http://www.pboselab.ca/spatial_OSCC/) that can be foundational for developing novel targeted therapies.

overall_design:
Overall Design

Fresh-frozen OSCC tissue was obtained from the Alberta Cancer Research Biobank under the approval of the Health Research Ethics Board of Alberta – Cancer Committee (reference number: HREBA.CC-16-0644). The BioBank OSCC tumor samples were collected at the time of the surgery, embedded in optimal cutting temperature (OCT) compound, and stored frozen at -80C until retrieval for the study. The study samples were sectioned in a cryostat at 10 um, stained with hematoxylin and eosin (H&E) and used for ST. Tissue optimization was performed to determine the optimal permeabilization time for OSCC tissue for the downstream gene expression protocol. Spatial transcriptomics was performed on OSCC cryosections using the Visium platform according to the manufacturer’s protocol (10x Genomics, Pleasanton, CA, USA). Briefly, OCT-embedded 10 micrometer-thick cryosections of OSCC samples were placed on the Visium spatial slide. Sections were enzymatically permeabilized for 24 min. cDNA was obtained from mRNA bound to capture oligos printed on the slide. cDNA quantification was performed using Agilent Bioanalyzer High Sensitivity Kit on an Agilent Bioanalyzer 2100 (Agilent Technologies, CA, USA). cDNA libraries were sequenced on an Illumina NovaSeq 6000 sequencer using the SP flowcell (200 cycles) at the Centre for Health Genomics and Informatics (CHGI, University of Calgary, Alberta, Canada). Sequencing reads were aligned using the 10s Genomics Space Ranger 1.3.1 pipeline to the standard GRCh38 reference genome. 12 samples passing alignment QC were aggregated together using the 10x Genomics Space Ranger aggr function to normalize for read depth between samples. Aggregated samples (n = 12) recovered a total number of 24,876 spots containing tissue sequenced to 43,648 post-normalization mean reads per spot.

Extract protocol

Fresh-frozen OSCC tissue was obtained from the Alberta Cancer Research Biobank under the approval of the Health Research Ethics Board of Alberta – Cancer Committee (reference number: HREBA.CC-16-0644). The BioBank OSCC tumor samples were collected at the time of the surgery, embedded in optimal cutting temperature (OCT) compound, and stored frozen at -80C until retrieval for the study. The study samples were sectioned in a cryostat at 10 um, stained with hematoxylin and eosin (H&E) and used for ST.

first: Rohit

last: Arora

first: Christian

last: Cao

first: Mehul

last: Kumar

first: Sarthak

last: Sinha

first: Ayan

last: Chanda

first: Divya

last: Samuel

first: Reid

last: McNeil

first: T

last: Matthews

first: Samir

last: Chandarana

first: Robert

last: Hart

first: Joseph

last: Dort

first: Jeff

last: Biernaskie

first: Paola

last: Neri

first: Martin

last: Hyrcza


title: Single cell analysis of transcriptome and open chromatin reveals the dynamics of hair follicle stem cell aging [scRNA-Seq]

first: Rui

last: Yi

overall_design: scRNA-Seq, 3 samples

first: Chi

last: Zhang

first: Dongmei

last: Wang


title: Datasets for the effects of RUNX2 silencing on transcriptomic and metabolomic profiles in SJSA-1 osteosarcoma cells

first: Jianlin

last: Zhou

address: 36 Lushan road

department: College of Life Science

institute: Hunan Normal University

summary: Osteosarcoma is the most common primary malignant bone tumor with a high risk of metastasis and recurrence. Metabolic reprogramming is a hallmark of osteosarcoma and other cancers and is associated with genetic and epigenetic alterations. RUNX2 is an important transcription factor for osteoblastic differentiation, and aberrant expression of the gene contributes to the development and progression of osteosarcoma. To identify the effects of RUNX2 silencing on transcriptomic and metabolomic profiles in osteosarcomas, we generated SJSA-1 osteosarcoma cells stably expressing RUNX2 shRNA and SJSA-1 cells stably expressing scramble shRNA and analyzed transcriptome and metabolome profiles in the two cell types using Illumina NovaSeq 6000 and ultrahigh-performance liquid chromatography coupled with time-of-flight mass spectrometry, respectively. The datasets can be used by researchers to identify novel targets of RUNX2 and elucidate the role and underlying mechanism of RUNX2 in osteosarcoma pathogenesis and metabolic reprogramming.

overall_design: Comparing the mRNA profilings of SJSA-1 cells stably expressing RUNX2 shRNA or scramble shRNA.

first: Mai Nhu Uyen

last: Le

first: Ruiqi

last: Chen

first: Liang-e

last: Xiang

first: Yichong

last: Ning


title: Ancient vertebrate dermal armor evolved from trunk neural crest

status: Public on Jun 24 2023

city: Pasadena

first: Jan

last: Stundl

state: California

address: 1200 E. California Blvd

institute: California Institute of Technology

summary: Bone is an evolutionary novelty of vertebrates, likely to have first emerged as part of ancestral dermal armor that consisted of osteogenic and odontogenic components. Whether these early vertebrate structures arose from mesoderm or neural crest cells has been a matter of considerable debate. To examine the developmental origin of the bony part of the dermal armor, we have performed in vivo lineage tracing in the sterlet sturgeon, a representative of non-teleost ray-finned fish that has retained an extensive postcranial dermal skeleton. The results definitively show that sterlet trunk neural crest cells give rise to osteoblasts of the scutes. Transcriptional profiling further reveals neural crest gene signature in sterlet scutes as well as bichir scales. Finally, histological and microCT analysis of ray-finned fish dermal armor show that their scales and scutes are formed by bone, dentin and hypermineralized covering tissues, in various combinations, that resemble those of the first armored vertebrates. Taken together, our results support a primitive skeletogenic role for the neural crest along the entire body axis, that was later progressively restricted to the cranial region during vertebrate evolution. Thus, the neural crest was a crucial evolutionary innovation driving the origin and diversification of dermal armor along the entire body axis.

sample_organism:
Acipenser ruthenus
Danio rerio
Polypterus senegalus

overall_design: To investigate the expression profiles of the dermal armor, we performed bulk RNA sequencing of sterlet sturgeon scutes, scales of the Senegal bichir, and zebrafish scales.

first: Megan

middle: L

last: Martik

first: Ayyappa

last: Desingu

first: Marianne

last: Bronner


title: A novel tumor suppressing role for RUNX3 is shown where a direct interaction with MYC leads to disruption of MYC-MAX heterodimers and consequent degradation of the MYC oncoprotein

city: Singapore

first: Vincent

last: oei

address: 14 Medical Dr, #12-01 Centre for Translational Medicine, Singapore

country: Singapore

institute: Cancer Science Institute of Singapore

summary: Developmental regulator RUNX3 targets MYC protein for rapid degradation through the glycogen synthase kinase-3 beta-box/WD repeat-containing protein 7 (GSK3β-FBXW7) proteolytic pathway. We therefore uncover  a previously unknown mode of MYC destabilization by RUNX3 and provide an explanation as to why RUNX3 inhibits early-stage cancer development in gastrointestinal and lung mouse cancer models.

overall_design: Transcriptional profiling of HeLa-RUNX3 and MKN28-RUNX3 doxycycline inducible cell lines comparing non-treated samples with doxycycline-treatment (RUNX3 induction) samples.  The aim of this experiment was to evaluate the functions and effects of RUNX3 induction in these cell lines in vitro. There are three replicates for each condition.

last: Oei

first: Chuang

last: Huey

first: Junichi

last: Matsuo

first: Supriya

last: Srivastava

first: Ming

last: Teh

first: Yoshiaki

last: Ito


title: BRG1-dependent regulation of Wnt signaling pathway during skeletal muscle differentiation

city: Worcester

first: Tapan

last: Sharma

state: MA

address: 368 Plantation Street

department: Gene Therapy Centre

institute: University of Massachusetts Medical School

summary: RNA-seq analysis for C2C12 myoblasts knocked down for either only-BRG1 or only-BRM or both BRG1 and BRM. Cells were treated with an siRNA SMARTpools of four oligos each targeting either BRG1 (siGENOME mouse Smarca4 pool #M-041135-01-0020) or BRM (siGENOME mouse Smarca2 pool #M-056591-00-0020) or both. Control samples were treated with non-targeting scrambled oligos (siRNA SMARTpool ON-TARGETplus scrambled non-targeting pool # D-001810-10-20). siRNA pools were purchased from Dharmacon, Horizon Discovery Ltd., USA.

overall_design: RNA-seq data was generated from undifferentiated and differentiated C2C12 cells. Cells were treated with siRNA oligos targeting either BRG1 or BRM or both BRG1 and BRM. Samples were harvested at proliferative stage (GM) and 48h post induction of differentiation (DM 48h). RNA from biological duplicates (for each treatment and timepoint) was extracted. Library preparation, sequencing and downstream analysis was performed by BGI Americas.

first: Anthony

middle: N

last: Imbalzano


title: Vertical and horizontal gene transfer shaped plant colonization and biomass degradation in the fungal genus Armillaria

status: Public on Jun 23 2023

city: Sopron

first: Boris

last: Indic

address: Bajcsy-Zsilinszky u. 4

department: Functional Genomics and Bioinformatics Group

country: Hungary

institute: Institute of Forest and Natural Resource Management, University of Sopron

summary: Members of the fungal genus Armillaria are necrotrophic pathogens with efficient plant biomass-degrading strategies. Armillaria species are some of the largest terrestrial organisms on Earth that cause tremendous losses in diverse ecosystems. Despite their global importance, how Armillaria evolved pathogenicity in a clade of dominantly non-pathogenic wood-degraders (Agaricales) remains elusive. Here, using new genomic data, we show that Armillaria species, in addition to widespread gene duplications and de novo gene origins, appear to have acquired at least 1025 genes via 124 horizontal gene transfer (HGT) events, primarily from Ascomycota donors. Functional and expression data suggest that HGT might have affected plant biomass-degrading and virulence abilities of Armillaria, two pivotal traits in their lifestyle. HGT provides an explanation for their soft-rot like biomass degrading strategy too, which is markedly different from the primarily white rot decay mechanism of related species. Combined multi-species expression data revealed putative virulence factors, extensive regulation of horizontally acquired and wood-decay related genes as well as novel noserved pathogenicity-induced small secreted proteins (PiSSPs). Two PiSSPs induced necrosis in live plants, suggesting they are potential virulence effectors conserved across Armillaria. Overall, this study details how evolution knitted together horizontally and vertically inherited genes in complex adaptive traits, such as plant biomass degradation and pathogenicityin one of the most influential fungal pathogens of temperate forest ecosystems.

overall_design: In vitro invasion assays using freshly cut conifer stems were set up to explore potential virulence factors in Armillaria analyzing the gene expression in highly and less-virulent isolates of the conifer-specific Armillaria ostoyae.

first: Simang

last: Champramary

first: György

last: Sipos

first: László

middle: G

last: Nagy


title: Transcriptome and chromatin accessibility profiles of mouse liver following iron overload

city: Rehovot

first: Ido

last: Goldstein

address: POB 12

department: Institute of Biochemistry, Food Science and Nutrition

country: Israel

institute: The Hebrew University of Jerusalem

summary: Iron overload disorders are an important factor contributing to various pathologies. However, the effects of iron overload on liver transcriptional regulation are largely unknown. To examine the effects of iron overload on liver transcriptome and chromatin accessibility, we fed mice a high-iron diet (HID) for 2 weeks. Then, livers were collected and profiled by RNA-seq and ATAC-seq.

overall_design: 8 weeks old C57BL/6J male mice were fed chow (standard) diet (2018-Teklad Global 18% Protein Rodent Diet) and 2% carbonyl iron diet (TD 09521-2% carbonyl iron diet, Teklad) for 2 weeks (chronic treatment) and for 48 hours (acute treatment). Each group has 3 replicates.

first: Talia

last: Radushkevitz-Frishman


title: Platelet factor 4 (PF4) is induced by klotho and rejuvenates cognition and its molecular signature in the aging hippocampus

city: South San Francisco

first: Oliver

last: Hahn

state: CA

address: 1170 Veterans Blvd

institute: Calico Life Sciences, LLC

summary: Platelet factors regulate wound healing and also signal from the blood to the brain. However, whether platelet factors modulate cognition, a highly valued and central manifestation of brain function, is unknown. Here, we show that systemic platelet factor 4 (PF4) modulates cognition and its molecular signature. Klotho, a longevity and cognition-enhancing protein, acutely activated platelets and increased circulating platelet factors, most robustly platelet factor 4 (PF4). To directly test PF4 effects on the brain, we treated mice with vehicle or systemic PF4. In young mice, PF4 enhanced synaptic plasticity and cognition. In aging mice, PF4 restored cognitive deficits and rejuvenated a molecular signature of cognition in the aging hippocampus. Augmenting platelet factors such as PF4, a possible messenger of klotho, may enhance cognition in the young brain and rejuvenate cognitive deficits in the aging brain.

overall_design: mRNA-seq data of hippocampus extracted from 3-5 months (young) or 17-20 months (old), vehicle- (Veh) or PF4-treated (PF4) mice (C57BL/6J) were generated by deep sequencing using Illumina sequencing.

first: Cana

last: Park

first: Shweta

last: Gupta

first: J

middle: A

last: Moreno

first: Dan

last: Wang

first: Tony

last: Wyss-Coray

first: Dena

last: Dubal


sample_organism:
Armillaria borealis
Armillaria ostoyae

platform_organism:
Armillaria borealis
Armillaria ostoyae


title: Expression data from MSCs of healthy donors vs. CML patients

city: Aachen

first: Mirle

last: Schemionek

state: NRW

address: Pauwelsstr. 30

department: Hematology, Oncology, Hemostaseology and SCT

institute: University Hospital RWTH Aachen

summary:
The management of patients with chronic myeloid leukemia (CML) has been revolutionized by the introduction of tyrosine kinase inhibitors (TKIs), which induce deep molecular responses so that treatment can eventually be discontinued, leading to treatment-free remission (TFR) in a subset of patients. Unfortunately, leukemic stem cells (LSCs) often persist and a fraction of these can again expand in about half of patients that attempt TKI discontinuation. In this study, we show that presence of myelofibrosis (MF) at the time of diagnosis is a factor that strongly associates with TFR failure. Fibrotic transformation is governed by the action of several cytokines, and interestingly, some of them have also been described to support LSC persistence. At the cellular level, these could be produced by both malignant cells and by components of the bone marrow (BM) niche, including megakaryocytes (MKs) and mesenchymal stromal cells (MSCs). In our cohort of 57 patients, around 40% presented with MF at diagnosis and the number of blasts in the peripheral blood and BM was significantly elevated in patients with higher grade of MF. Employing a CML transgenic mouse model, we could observe higher levels of alpha-smooth muscle actin (α-SMA) in the BM when compared to control mice. Short-term treatment with the TKI nilotinib, efficiently reduced spleen weight and BCR-ABL1 mRNA levels, while α-SMA expression was only partially reduced. Interestingly, the number of MKs was increased in the spleen of CML mice and elevated in both BM and spleen upon nilotinib treatment. Microarray analysis of human CML- vs healthy donor (HD)-derived MSCs showed an altered expression of gene signatures reflecting fibrosis as well as hematopoietic support, thus suggesting MSCs as a potential player in these two processes. Finally, in our cohort, 12 patients qualified for TKI discontinuation, and here we observed that all patients who failed TFR had BM fibrosis at diagnosis, further linking fibrosis to LSC persistence. Although we have not investigated whether MF is directly responsible for LSC persistence or whether it is merely an epiphenomenon occurring alongside the development of an LSC-supportive niche, we strongly believe that this is a mechanism that merits further investigation
We used microarrays to compare MSCs expression profiles of HD vs. CML patients.

overall_design: MSCs from 4 healthy donors (CML replicates) and 4 CML patients (CML replicates) were isolated from the bone and separated by plastic adherence. RNA was isolated and hybridized on Human Clariom D Assay gene chip.

first: Henrike

last: Jacobi

first: Margherita

last: Vieri

first: Marlena

last: Bütow

first: Carolina

last: Namasu

first: Laura

last: Flüter

first: Ivan

last: Costa

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