title: RNA Sequencing Shows Activation of Type I Interferon Pathway in Zymosan-Induced Mast Cells

status: Public on Jan 27 2021

city: Frankfurt am Main

first: Klaus

last: Scholich

address: Theodor Stern Kai 7

department: Institute of Clinical Pharmacology

institute: University Hospital Goethe-University Frankfurt

summary: Next to their role in IgE-mediated allergic diseases and in promoting inflammation, mast cells also have antiinflammatory functions. They release pro- as well as antiinflammatory mediators, depending on the biological setting. Here we aimed to better understand the role of mast cells during the resolution phase of a local inflammation induced with the Tolllike receptor (TLR)-2 agonist zymosan. Multiple sequential immunohistology combined with a statistical neighborhood analysis showed that mast cells are located in a predominantly antiinflammatory microenvironment during resolution of inflammation and that mast cell-deficiency causes decreased efferocytosis in the resolution phase. Accordingly, FACS analysis showed decreased phagocytosis of zymosan and neutrophils by macrophages in mast cell-deficient mice. mRNA sequencing using zymosan-induced bone marrow-derived mast cells (BMMC) revealed a strong type I interferon (IFN) response, which is known to enhance phagocytosis by macrophages. Both, zymosan and lipopolysaccharides (LPS) induced IFN-b synthesis in BMMCs in similar amounts as in bone marrow derived macrophages. IFN-b was expressed by mast cells in paws from naïve mice and during zymosan-induced inflammation. As described for macrophages the release of type I IFNs from mast cells depended on TLR internalization and endosome acidification. In conclusion, mast cells are able to produce several mediators including IFN-b, which are alone or in combination with each other able to regulate the phagocytotic activity of macrophages during resolution of inflammation.

overall_design: mRNA sequencing  of bone marrow-derived mast cells treated with 10 μg/ml zymosan for 24 h or 48 h or left untreated (control, 0 h), 4 samples per condition.

first: Lisa

last: Kornstädt

first: Tobias

last: Schmid

first: Stefanie

last: Ebersberger

first: Ingo


title: Genome-wide analysis of PGC-1α-binding in microglia

city: Tianjin

first: Bin

last: Han

address: 154 Anshan Road, Heping District

country: China

institute: Tianjin Medical University General Hospital

type: Genome binding/occupancy profiling by high throughput sequencing

summary: PGC-1α in microglia protects against ischemia-induced brain damage in mice. The data suggest that microglia-specific PGC-1α play a key role in limiting ischemia-induced brain damage and potently participates in regulating microglial function. To further clarify the mechanism of PGC-1α, we conducted chromatin immunoprecipitation-sequencing (ChIP-Seq) analysis to identify the targets of PGC-1α in microglia from mPGC-1α mice at 24 h after ischemic stroke. KEGG pathway analysis of these genes identified the mitophagy signaling pathway as one of the most highly enriched pathways. Finally, we demonstrated that PGC-1α induces mitophagy by regulating ULK1 expression in an ERRα-dependent manner, thereby reducing neuroinflammatory reactions.

overall_design: Primary microglia were isolated from mPGC-1α mice at 24 h after ischemic stroke using the MojoSortTM mouse P2RY12 selection kit (Biolegend, San Diego, CA, USA) according to the manufacturer’s protocol. Then the samples were processed to perform ChIP-Seq analysis.

first: Wei

last: Jiang

first: Junwei

last: Hao


title: The Xa7 Resistance Gene Guards the Susceptibility Gene SWEET14 of Rice Against Exploitation by Bacterial Blight Pathogen

city: Tempe

first: R. Taylor

last: Raborn

state: AZ

address: 727 E Tyler St

department: Center for Mechanisms of Evolution

country: USA

institute: Arizona State University

summary: TAL effectors of bacteria hijack host gene expression to condition disease susceptibility, while host plants counteract TAL effectors with resistance genes to thwart pathogen infections. Here we report the cloning of Xa7, TAL effector-specific expression by the essential virulence TAL effectors AvrXa7 and PthXo3, and the prevalence of Xa7 in indica rice cultivars.

overall_design: RAMPAGE in Xoo treated- and untreated O. sativa IRBB7.

first: R

middle: T

first: Volker

middle: P

last: Brendel

first: Bing

last: Yang


title: RNA-Seq analysis comparing transcriptome of MYB117 overexpression transgenic and wild hybrid poplar grown in the greenhouse

city: Victoria

first: C. Peter

last: Constabel

state: British Columbia

address: 3800 Finnerty Rd

department: Biology

country: Canada

institute: University of Victoria

type: Expression profiling by high throughput sequencing

summary: We compared the transcriptiome of MYB117-overexpressing and wild type hybrid poplars and try to identify the differentially expressed genes.

overall_design: Three wild type and three MYB117-overexpressing transgenic poplars were grown in greenhouse (University of Victoria) for two months. Leaves number 7-10 (LPI7-10) were harvested and analyzed.

first: Peter

middle: C

first: Dawei

last: Ma


title: RALYL increases hepatocellular carcinoma stemness by sustaining the mRNA stability of TGF-β2

city: Hong Kong

first: XIA

last: WANG

address: 21 Sassoon Rd, Pokfulam

department: Clinical Oncology

country: Hong Kong

institute: The University of Hong Kong

summary: To investigate the role of RALYL in HCC development and stemness, RALYL was overexpressed in PLC-8024 HCC cell line. Transcriptome sequencing was performed to investigate subsequent target and signaling pathway of RALYL.

overall_design: Examination of gene expression of RALYL-overexpressing and control cells.

first: Xia

last: Wang

first: Lingxi

first: Xin-Yuan

last: Guan


title: Genetic deletion of ZNF91 in human embryonic stem cells leads to ectopic activation of SVAs and collective upregulation of KRAB zinc finger gene clusters

status: Public on Jan 28 2021

city: Amsterdam

first: Frank

last: Jacobs

address: Science Park 904

department: Sammerdam Institute for Life Sciences

country: Netherlands

institute: University of Amsterdam

summary: Transposable element (TE) invasions have shaped vertebrate genomes over the course of evolution. They have contributed an extra layer of species-specific gene regulation by providing novel transcription factor binding sites. In humans, SVA elements are one of three still active TE families, and approximately 2800 SVA insertions exist in the human genome, half of which are human-specific. TEs are often silenced by KRAB zinc finger (KZNF) proteins recruiting co-repressor proteins that establish a repressive chromatin state. A number of KZNFs have been reported to bind SVAs, but their individual contribution to repressing SVAs and their roles in suppressing SVA-mediated gene-regulatory effects remains elusive. We analyzed the genome-wide binding profile for ZNF91 in human cells and found that ZNF91 interacts with the VNTR region of SVAs. Through CRISPR-Cas9 mediated deletion of ZNF91 in human embryonic stem cells we established that loss of ZNF91 results in increased transcriptional activity of SVAs. In contrast, SVA activation was not observed upon genetic deletion of the ZNF611 gene encoding another strong SVA-interactor. Epigenetic profiling confirmed the loss of SVA repression in the absence of ZNF91 and revealed that mainly evolutionary young SVAs gain gene activation-associated epigenetic modifications. Genes close to activated SVAs showed a mild upregulation, indicating SVAs adopt properties of cis-regulatory elements in the absence of repression. Intriguingly, genome-wide de-repression of SVAs elicited the communal upregulation of KZNFs that reside in KZNF clusters. This phenomenon may provide new insights into the potential mechanisms utilized by the host genome to sense and counteract TE invasions.

overall_design: Identification of ZNF91 binding sites in HEK293 cells using ZNF91-GFP overexpression followed by ChIP-seq. RNA seq and ChIP seq analysis of CRISPR/Cas9 ZNF91 wild type and knockout human embryonic stem cell lines

first: Nina

middle: L

last: Haring

first: Elisabeth

middle: J

last: van Bree

first: Whitney

middle: S

last: Jordaan

first: Judith

middle: R

last: Roels

first: Gonzalo

last: Congrains Sotomayor

first: Tiziana

middle: M

last: Hey

first: Fred

last: White

first: Marc

middle: D

last: Galland

first: Marten

last: Smidt


title: Transcriptional landscape of  CD8 T cell subsets

city: Stockholm

first: Marcus

last: Buggert

state: PA

address: C2:94; Karolinska Universitetssjukhuset Huddinge

department: Microbiology

country: Sweden

institute: Karolinska Institutet

summary: Transcriptional study of sorted CD8 Tcell subsets from LymphNode and Tumor Draining Lymph Node

overall_design: Three or more biological replicates were analyzed for each of the sorted CD8 T cell subsets

first: Laura

last: Vella

first: E

last: Wherry


title: Epigenetic landscape of CD8 T cell subsets

summary: Open chromatin accessibility analysis of sorted CD8 Tcell subsets

overall_design: 2-3 Biological replicates were analyzed for each of the sorted T cell subsets from lymph node and tumor draining lymph node


title: The identity of human tissue-emigrant CD8+ T cells

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

overall_design: Refer to individual Series


title: Single cell RNA-seq of relapsed and refractory multiple myeloma patients defines new resistance pathways and targeted treatments

city: Rehovot

first: Ido

last: Amit

address: 234 Herzl st.

department: Immunology

country: Israel

institute: Weizmann Institute of Science

summary: Multiple myeloma (MM) is a neoplastic plasma cell (PC) disorder, characterized by clonal proliferation of malignant PC. Despite extensive research, disease heterogeneity within and between treatment resistant patients is poorly characterized. Here, we conduct a prospective multi-center single-arm clinical trial (NCT04065789), combined with longitudinal single cell RNA-seq to study the molecular dynamics of MM resistance mechanisms. Forty-one newly diagnosed MM patients who either failed to respond or experienced early relapse after a bortezomib-containing induction regimen, were enrolled to evaluate the safety and efficacy of a daratumumab/carfilzomib/lenalidomide/dexamethasone combination. Primary clinical endpoint was safety and tolerability. Secondary endpoints included overall response rate; progression free survival, and overall survival. Treatment was safe and well-tolerated, deep and durable responses were achieved. In prespecified exploratory analyses, comparison of 41 primary-refractory and early-relapsed patients with 11 healthy subjects and 15 newly diagnosed MM patients revealed new MM molecular pathways of resistance, including hypoxia tolerance, protein folding, and mitochondria respiration, that generalized to larger clinical cohorts (CoMMpass). We found peptidylprolyl isomerase A (PPIA), a central gene in the protein-folding response pathway, as a potential new target for resistant MM. CRISPR/Cas9 deletion of PPIA or inhibition of PPIA with cyclosporin A significantly sensitizes MM tumor cells to proteasome inhibitors. Together, our study defines a roadmap for integrating scRNA-seq in clinical trials, identifies a signature of highly resistant MM patients and discovers PPIA as a potent therapeutic target for these tumors.

overall_design: To better understand early resistance mechanisms of PRMM patients, we designed an open-label single arm prospective clinical and translational trial combined with comprehensive scRNA-seq. We recruited patients with MM who received a bortezomib-based induction and either failed to achieve a timely response (<4 months/ cohort A) or progressed early (<18 months/ cohort B). The designed therapeutic treatment includes a quadruple regimen comprised of Daratumumab, Carfilzomib, Lenalidomide and Dexamethasone (DARA-KRD) (Methods; KYDAR Clinical Trial). We sequenced bone marrow PC from a total of 41 KYDAR patients: 34 baseline, 27 cycle-4 (3 months -marked post), and 10 cycle-10 (9 months -marked post2). In addition, we also characterized BM PC using MARS-seq from 15 newly diagnosed MM (NDMM) patients and 11 healthy aged control group undergoing hip replacement surgery as previously described.

first: Yael

last: Cohen

first: Mor

last: Zada

first: Shuang-Yin

first: Assaf

last: Weiner


title: The identity of human tissue-emigrant CD8+ T cells (scRNAseq, scTCRseq, bulk RNA-seq)

summary: Lymphocyte migration is essential for adaptive immune surveillance. However, ourcurrent understanding of this process is rudimentary, because most human studieshave been restricted to immunological analyses of blood and various tissues. Toaddress this, we used an integrated approach to characterize tissue-emigrant immunecells in thoracic duct lymph (TDL). The most prevalent immune cells in human andnon-human primate efferent lymph were T cells. Cytolytic CD8+ T cell subsets witheffector-like epigenetic and transcriptional signatures were clonotypically skewed andselectively confined to the intravascular circulation, whereas non-cytolytic CD8+ T cellsubsets with stem-like epigenetic and transcriptional signatures predominated intissues and TDL. Moreover, these anatomically distinct gene expression profiles wererecapitulated within individual antigen-specific clonotypes, suggesting paralleldifferentiation programs independent of the expressed antigen receptor. Our collectivedataset provides a framework of the migratory immune system and defines the natureof tissue-emigrant CD8+ T cells that recirculate via TDL.

overall_design:
scRNAseq and scTCRseq of 1) HIV-specific or CMV-specific CD8+ T-cells and 2) memory CD8+ T-cells between TDL and blood

bulk RNA-seq of FACS sorted CD8+ Tcell memory subsets from human peripheral blood (samples re-used from GSE148234).

first: Vincent

middle: H

last: Wu

first: Son

middle: N

last: Nguyen


title: Defective metabolic programming impairs early neuronal morphogenesis in neural cultures and an organoid model of Leigh syndrome [rRNA-depleted RNA-seq]

status: Public on Jan 29 2021

city: Berlin

first: Petar

last: Glazar

address: Robert Rössle Str. 10 (H. 87)

department: Systems Biology of Gene Regulatory Elements

country: Germany

institute: Max Delbrück Center for Molecular Medicine

summary: Mutations in the mitochondrial complex IV assembly factor SURF1 represent a major cause of Leigh syndrome (LS), a fatal neurological disorder. SURF1-deficient animals failed to recapitulate the neuronal failure of LS, which is considered an early-onset neurodegeneration. Using patient induced pluripotent stem cells (iPSCs) and genetically corrected cells, we discovered aberrant bioenergetics initiating in neural progenitor cells (NPCs) leading to impaired neuronal differentiation and maturation. iPSC-derived cerebral organoids recapitulated the neurogenesis defects and appeared smaller with reduced cortical thickness. We suggest defective neurogenesis as a central mechanism in LS pathogenesis and propose NPC function as an interventional target, which allowed us to identify SURF1 gene augmentation as a strategy for restoring neural function in this fatal disease.

overall_design: polyA-selected RNA sequencing of neuronal cultures enriched for dopaminergic neurons, derived from two control and two patient-derived hESC/iPSC cell lines, each in three replicates at two time points (4 and 8 weeks of differentiation)

first: Gizem

last: Inak

first: Agnieszka

last: Rybak-Wolf

first: Pawel

last: Lisowski

last: Glažar

first: Nikolaus

last: Rajewsky

first: Markus

last: Schuelke

first: Alessandro

last: Prigione


title: Mouse B cells responding to Plasmodium yoelii infection

city: Iowa City

first: Noah

last: Butler

state: IL

address: 51 Newton Road

department: Microbiology and Immunology

institute: University of Iowa

summary: This study was designed to capture single cell transcriptomic and BCR repertoire information among subsets of B cells activated during rodent malaria

overall_design: Naïve B cells (CD19+CD138-IgD+), plasmablasts (CD19+CD138hiIgDneg), and GC B cells (CD19+GL7+Fas+) from P. yoelii-infected mice

first: Jenna

last: Guthmiller

first: Linda

middle: Y

last: Lan


title: Inflammatory B cells correlate with failure to checkpoint blockade in melanoma patients

city: Epalinges

first: Daniel

middle: E

last: Speiser

state: VD

address: Route de la Corniche 9A

department: Department of Oncology

country: Switzerland

institute: University of Lausanne

summary: We report the RNA sequencing of ex vivo sorted B cells isolated from melanoma patients within the tumoral tissue or the blood. Sorted B cells from healthy donor blood were included as control.

overall_design: Transcriptome analysis of B cells in patients' blood and tumor, compared to healthy donors' blood.

first: Kaat

last: de Jonge

first: Sina

last: Nassiri

first: Daneil


title: Role of regorafenib in macrophage and in antitumor immunity in hepatocellular carcinoma

city: Taipei

first: Chia-Lang

last: Hsu

address: No. 7, Zhongshan S. Rd.

department: Department of Medical Research

country: Taiwan

institute: National Taiwan University Hospital


title: High-throughput Transcriptomic Analysis of Testosterone-Manipulated epididymis

city: Da’an Dist

first: Tse-en

last: Wang

state: Taipei City

address: No. 1, Sec. 4, Roosevelt Rd., Da’an Dist., Taipei City 106, Taiwan (R.O.C.)

department: Veterinary medicine

institute: National Taiwan University

summary: Androgenic modulation of the structural integrity and functional maintenance of the epididymis have been demonstrated. Androgen receptor is a typical receptor to modulate classical testosterone signaling pathway, while accumulated studies suggested number of membranous receptors or intermediated molecules also involved in testosterone pathway. In RNAseq analysis of castration mouse model, profile of testosterone responsive genes was identified. Comibining with our in-vitro studies, an novel testosterone action which incoperated with glutamine and glutamate transportation has been proposed in epididymal epithlium cells.

overall_design: To establish a loss- and restore-of-function animal model, 13 adult mice were randomly allocated into control (n=3) and castration (n=10) group. Castration was performed by bilateral removal of testes. Control animals underwent the same surgical procedures except for removing their testes (sham). Fourteen days post-surgery, five castrated mice were subcutaneously given testosterone propionate (5 mg/kg body weight, dissolved in corn oil) for 10 consecutive days (Cas+T); the other five castrated animals received corn oil served as vehicle controls (Cas+CO).

first: Pei-shiue

last: Tsai


title: Transcriptome analysis of the differentiated C2C12 cells in 3 different substrate, Tissue culture plate, Methacarylated gelatin and 15% Methacarylated gelatin mixed with 1% PSS (poly sodium 4-styrenesulfonate)

status: Public on Jan 26 2021

city: cheonan

first: ji hoon

last: jeong

address: cheonan

country: South Korea

institute: Soonchunhyang Institute of Medi-bio Science

summary: Purpose : To determine transcriptome profile of the diffferentiated C2C12 cells in 3 different cell culture substrate, Tissue culture plate, Methacarylated gelatin and 15% Methacarylated gelatin mixed with 1% PSS (poly sodium 4-styrenesulfonate)  Method : RNA smples prepared from differentiated C2C12 cells in 3 different cell culture substrate, Tissue culture plate, Methacarylated gelatin and 15% Methacarylated gelatin mixed with 1% PSS (poly sodium 4-styrenesulfonate)  Results : Paired-ends 101bp reads, we mapped about 72 million reads - 145 million reads per sample to the mouse genome (build mm10) and assembled with cufflinks program (value : FPKM)

overall_design:
RNA seq on the samples isolated from the 3 different cell culture substrates (Tissue culture plate, Methacarylated gelatin, 15% Methacarylated gelatin mixed with 1% PSS (poly sodium 4-styrenesulfonate))
-------------------------------------
Authors state "we want submit our data without the raw data, because we can not locate raw data", and "our original raw fastq files also defective and company already deleted our data so we couldn't get raw files".

first: Yongsung

last: Hwang

first: Jeong

middle: K

last: Yoon


title: Inceptor counteracts insulin signalling in β-cells to control glycaemia

city: Neuherberg

first: Johannes

last: Beckers

address: Ingolstaedter Landstr. 1

department: Institute of Experimental Genetics

institute: Helmholtz Zentrum Muenchen

summary: Resistance to insulin and insulin-like growth factor 1 (IGF1) in pancreatic β-cells causes overt diabetes in mice; thus, therapies that sensitize β-cells to insulin may protect patients with diabetes against β-cell failure. Here we identify an inhibitor of insulin receptor (INSR) and IGF1 receptor (IGF1R) signalling in mouse β-cells, which we name the insulin inhibitory receptor (inceptor; encoded by the gene Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to INSR and IGF1R4, and a mannose 6-phosphate domain that is also found in the IGF2 receptor (IGF2R)5. Knockout mice that lack inceptor (Iir-/-) exhibit signs of hyperinsulinaemia and hypoglycaemia, and die within a few hours of birth. Molecular and cellular analyses of embryonic and postnatal pancreases from Iir-/- mice showed an increase in the activation of INSR–IGF1R in Iir-/- pancreatic tissue, resulting in an increase in the proliferation and mass of β-cells. Similarly, inducible β-cell-specific Iir-/- knockout in adult mice and in ex vivo islets led to an increase in the activation of INSR–IGF1R and increased proliferation of β-cells, resulting in improved glucose tolerance in vivo. Mechanistically, inceptor interacts with INSR–IGF1R to facilitate clathrin-mediated endocytosis for receptor desensitization. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of inceptor resulted in the retention of inceptor and INSR at the plasma membrane to sustain the activation of INSR–IGF1R in β-cells. Together, our findings show that inceptor shields insulinproducing β-cells from constitutive pathway activation, and identify inceptor as a potential molecular target for INSR–IGF1R sensitization and diabetes therapy.

overall_design: We performed gene expression microarray analysis on pancreas from inceptor-deficient (Iir-/-) and control mice at E18.5 and P0

last: Ansarullah

first: Chirag

last: Jain

first: Fataneh

last: Fathi Far

first: Katharina

last: Wißmiller

first: Sarah

last: Homberg

first: Felizitas

last: Gräfin von Hahn

first: Aurelia

last: Raducanu

first: Silvia

last: Schirge

first: Michael

last: Sterr

first: Sara

last: Bilekova

first: Johanna

last: Siehler

first: Julius

last: Wiener

first: Lena

last: Oppenländer

first: Amir

last: Morshedi

first: Aimée

last: Bastidas-Ponce

first: Gustav

last: Collden

first: Martin

last: Irmler

first: Annette

last: Feuchtinger

first: Michal

last: Grzybek

first: Christin

last: Ahlbrecht

first: Regina

last: Feederle

first: Oliver

last: Plettenburg

first: Timo

last: Müller

first: Matthias

last: Meier

last: Tschöp

first: Ünal

last: Coskun

first: Heiko

last: Lickert


title: HOXBLINC is aberrantly expressed in acute myeloid leukemia and functions as a potent oncogenic long non-coding RNA in leukemogenesis

city: Hangzhou

first: Huacheng

last: Luo

state: Zhejiang

address: No. 150, Fucheng Road, Qiantang District

institute: Hangzhou Institute of Medicine (HIM), The Chinese Academy of Sciences

summary: Dysregulation of HOXA/B genes is a dominant mechanism of leukemic transformation.HOXB locus-associated long non-coding RNA (lncRNAs), HOXBLINC, regulates transcription of the anterior HOXB genes and plays a critical role in hematopoiesis development. Here, we show that HOXBLINC lncRNA is up-regulated in over 60% of patients with AML. Interestingly, AML patients with high HOXBLINC expression have a significantly shortened survival as compared to low HOXBLINC expressing patients. Transgenic expression of HoxBlinc in mice leads to dramatically increased pools of long-term (LT)- and short-term (ST)-hematopoietic stem cells (HSCs) and development of AML. Mechanistically, HoxBlinc overexpression alters the expression of genes (including HoxB1-6, Met, Meis1, Cdx2,HoxA9,Runx1, Irf8 and Wnt5a) critical for HSC regulation and/or leukemogenesis,and enhances enhancer/promoter chromatin accessibility in these loci. Importantly, knockdown of HOXBLINC suppresses OCI-AML3 leukemic cell proliferation both in vitro and in vivo through abrogating the aberrant expression of HOXB and other leukemogenic genes. Our study identifies HOXBLINC as a potent oncogenic lncRNA in leukemogenesis, which coordinate to maintain an oncogenic transcription program for leukemic transformation. Our study also provides novel insights into the HSC regulation by lncRNAs.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: RNA-SEQ, ATAC-seq, ChIP-seq and 4C-seq were carried out with WT and Hoxblinc transgenic mice cells.LSK cells (Lin- Sca1+ Kit+) were sorted by FACS from the bone marrow of WT and HoxBlinc transgenic mice for RNA-seq and ATAC-seq assay, and similarly, LK cells  (Lin- Kit+) were sorted for CHIP-seq and 4C assay.

first: Suming

last: Huang

first: Mingjiang

last: Xu

first: Ganqian

last: Zhu


title: Role of regorafenib in macrophage

first: Chia-Lang

last: Hsu

summary: Regorafenib increased M1/M2 ratio of BMDMs polarization and proliferation/activation of co-cultured T cells in vitro, indicating angiogenesis-independent immunomodulatory effects. Suppression of p38 kinase phosphorylation and downstream CREB-KLF4 activity in BMDMs by regorafenib reversed M2 polarization. Regorafenib enhanced antitumor efficacy of adoptively transferred antigen-specific T cells, whereas macrophage deletion negated regorafenib’s antitumor effects. Synergistic antitumor efficacy between low-dose regorafenib and anti-PD1 was associated with multiple immune-related pathways in the tumor microenvironment.

overall_design: Mouse bone marrow-derived macrophages were polarized with IFN-gamma + LPS or IL4 for 24 h to induce M1 or M2 phenotypes, respectively.

first: D

last: Ou

first: C


title: Role of regorafenib in antitumor immunity in hepatocellular carcinoma

summary: Low-dose regorafenib (5 mg/kg/day, corresponding to about half of human clinical dosage) inhibited tumor growth and angiogenesis in vivo similarly to DC-101 (anti-VEGFR antibody) but produced higher T cell activation and M1 macrophage polarization, Regorafenib increased M1/M2 ratio of BMDMs polarization and proliferation/activation of co-cultured T cells in vitro, indicating angiogenesis-independent immunomodulatory effects. Suppression of p38 kinase phosphorylation and downstream CREB-KLF4 activity in BMDMs by regorafenib reversed M2 polarization. Regorafenib enhanced antitumor efficacy of adoptively transferred antigen-specific T cells, whereas macrophage deletion negated regorafenib’s antitumor effects. Synergistic antitumor efficacy between low-dose regorafenib and anti-PD1 was associated with multiple immune-related pathways in the tumor microenvironment.

overall_design: BNL-MEA tumor-bearing Balb/c mice treated with vehicle, regorafenib, DC-101, and anti-PD1


title: Slc20a1b is essential for hematopoietic stem/progenitor cell expansion in zebrafish

city: Guangzhou

first: Gaofei

last: Li

address: Guangzhou Higher Education Mega Centre, Panyu District

institute: South China University of Technology

summary:
Hematopoietic stem and progenitor cells (HSPCs) are able to self-renew and can give rise to all blood lineages throughout their lifetime, yet the mechanisms regulating HSPC development have yet to be discovered.
In this study, we characterized a hematopoiesis defective zebrafish mutant line named smu07, which was obtained from our previous forward genetic screening, and found the HSPC expansion deficiency in the mutant.
Positional cloning identified that slc20a1b, which encodes a sodium phosphate cotransporter, contributed to the smu07 blood phenotype.
Further analysis demonstrated that mutation of slc20a1b affects HSPC expansion through cell cycle arrest at G2/M phases in a cell-autonomous manner.
Our study shows that slc20a1b is a vital regulator for HSPC proliferation in zebrafish early hematopoiesis and provides valuable insights into HSPC development.

overall_design: A mini-bulk RNA-Seq analysis to compare the expression profiles of wild-type HSPCs and smu07 mutant HSPCs

first: Jiakui

last: Chen

first: Yiyue

last: Zhang


title: T-cell CX3CR1 expression as a dynamic blood-based biomarker of response to immune checkpoint inhibitors

city: Buffalo

first: Sebastiano

last: Battaglia

state: NY

address: Elm and Carlton st

department: Department of Immunology

institute: Roswell Park Comprehensive Cancer Center

summary:
We investigated the frequency of CX3CR1+CD8+ T cells in peripheral blood (PB) before and during immune checkpoint inhibitor (ICI) therapy, and delineated the TCR repertoire in peripheral CX3CR1+CD8+ T-cell subsets and CD8+ tumor-infiltrating lymphocytes (TILs) using preclinical models. To understand the clinical utility of CX3CR1 as a circulating T-cell biomarker, we analyzed longitudinal PB samples from non-small cell lung cancer (NSCLC) patients undergoing anti-PD-1 therapy, and evaluated predictive and prognostic value of changes in the frequency of PB CX3CR1+ CD8+ T cells

Methods:
Female C57BL/6 mice were inoculated subcutaneously with 5 x 10^5 MC38 tumor cells per mouse on the right flank on day 0. When tumor volume reached approximately 50 mm^3, 200 µg of anti-PD-L1 Ab (clone 10F.9G2, BioXcell) and 100 µg of anti-CTLA-4 Ab (clone 9H10, BioXcell) were administered intraperitoneally every 3 days and every other day, respectively.  MC38 tumor-bearing mice were treated for 14 days, and spleens and tumors were harvested.
[Tumor cells] Tumors were cut into small pieces of 2-4 mm. Single-cell suspensions were obtained by mechanical dispersion consisting of two 30-min incubations at 37°C, 5% CO2 in 5 ml RPMI 1640 (Gibco) and tumor dissociation kit (Miltenyi Biotec) in C Tubes (Miltenyi Biotec) interspersed with three mechanical dispersions on a GentleMACS dissociator (Miltenyi Biotec). The tumor cell suspensions were then filtered through a cell strainer (70 μm; BD Biosciences). An EasySep Mouse CD8a Positive Selection Kit II (STEMCELL Technologies) was used to isolate murine CD8+ tumor-infiltrating lymphocytes (TILs) for TCR sequencing.
[Splenocytes] Spleens were homogenized by forcing the tissue through a cell strainer (70 μm; BD Biosciences). Red blood cells in blood and spleen were lysed using ACK Lysis Buffer (Gibco). For TCR sequencing of murine splenic CD8+ T cells, single cell suspensions from freshly isolated splenocytes were stained. CD45+ CD3+ CD8+ T cells were gated, and CD27lo CX3CR1-, CD27hi CX3CR1-, and CX3CR1+ CD8+ T cells were sorted using BD FACSAria II Cell Sorter.
DNA from flow-isolated murine splenic CD8+ T cells and CD8+ TILs, and PB CD8+ T cells was extracted using QIAamp DNA Micro Kit (QIAGEN). DNA was quantified using Qubit dsDNA BR Assay (Invitrogen). Amplification and sequencing of TCRβ CDR3 regions was performed using ImmunoSEQ immune profiling system at the survey level (Adaptive Biotechnologies). Sequencing was performed on an Illumina NextSeq system using 150 cycle mid-output kit (Illumina Inc.). Processed data were uploaded to the ImmunoSEQ Platform (Adaptive Biotechnologies) for bioinformatics analysis. Processed data were downloaded and frequencies/counts for TCR clonotypes and diversity were examined by nucleotide sequences after non-productive reads were filtered out.

Results:
Clonally expanded TCR repertoires of CD8+ TILs are enriched in the peripheral CX3CR1+ subset during ICI therapy.
ICI therapy induces high degree of TCR sequence similarity and clonality between CD8+ TILs and the peripheral CX3CR1+ CD8+ T cells.

overall_design:
12 samples, splenic CD27loCX3CR1lo, CD27hiCX3CR1lo,  and CX3CR1hi CD8 T cells, and CD8 TILs

*** Comment from submitter: "Files are from Adaptive Biotech ImmunoSeq" ***

first: Ito

last: Fumito

first: Battaglia

last: Sebastiano

first: Jain

last: Vaibhav


title: Transcriptome analysis of ER+/PR+, ER-/PR+ and triple negative breast tumors

city: Dijon

first: Romain

last: Boidot

address: 1, rue Pr Marion

country: France

institute: CGFL

summary: The goal of this study is to analyse whether ER-/PR+ breast tumors could be transcriptionally different from ER+/PR+ and/or from triple negative breast tumors

overall_design: Comparison of ER-/PR+ breast tumors with ER+/PR+ and triple negative breast tumors

first: Françoise

last: Beltjens

first: Laurent

last: Arnould

first: Corentin

last: Richard


title: MeRIP, RIP and RNA Sequencing of the H1299 stable cell lines which the endogenous YTHDF2 was silenced by a short hairpin RNA targeting YTHDF2 3’UTR (shYTHDF2) and then re-expressed Flag-tagged YTHDF2-WT orYTHDF2-K571R

city: Shanghai

first: Jianxiu

last: Yu

address: 280 South Chongqing Road, Shanghai 200025, China

department: Department of Biochemistry and Molecular Cell Biology

institute: shanghai jiaotong university school of medicine

summary: SUMOylation affects many aspects of target proteins such as activity, stability, localization and protein-protein interactions. We have found that SUMOylation of YTHDF2 increased its binding activity with m6A-RNAs by using different experimental approaches. To confiremd this conclusion,the analysis of RIP-seq, MeRIP-seq and RNA-seq in H1299-shYTHDF2 cells re-expressing YTHDF2-WT and YTHDF2-K571R was performed. MeRIP+RIP targets showed lower binding affinities in the mutant YTHDF2-K571R when compared with YTHDF2-WT. Compared to the control group, the binding capacities of YTHDF2 to RIP targets in treated group with either 2-D08 or GA were decreased, especially to MeRIP+RIP targets. Moreover, SUMOylated YTHDF2 promoted  m6A-RNAs degradation. Combined analysis of RNA-seq, RIP-seq and MeRIP-seq showed that the mRNA levels were up-regulated in shYTHDF2 stable cells re-expressing YTHDF2-K571R compared with those in re-expressing YTHDF2-WT.

overall_design:
Examination of the binding affinity of YTHDF2 and mRNA profiles  between H1299-shYTHD2-WT and H1299-shYTHDF2-K571R cells
Examination of the binding affinity of YTHDF2 in H1299-shYTHD2-WT cells under Ginkgolic acid or 2-D08 treatment

first: Guofang

last: Hou


title: Transcriptome and Chromatin Landscape of iNKT cells are Shaped by Subset Differentiation and Antigen Exposure [ATAC-seq]

city: San Diego

first: Mitchell

last: Kronenberg

state: CA

address: 9420 Athena Circle

institute: La Jolla Institute for Immunology

summary: Invariant natural killer T cells (iNKT cells) differentiate into thymic NKT1, NKT2 and NKT17 subsets that are also peripheral.  We determined if the gene programs associated with these thymic subsets were maintained in peripheral sites, the influence of tissue location, and if there were large-scale changes after antigen exposure.  RNA-seq and ATAC-seq analyses showed that iNKT cells in any subset were similar, regardless of tissue location.  Lung iNKT cell subsets shared the most distinct location-specific features, shared with other innate lymphocytes in the lung, possibly consistent with increased activation. After antigenic stimulation, iNKT cells underwent chromatin and transcription changes leading to two populations: one similar to follicular helper T cells and the other like NK cells. Phenotypic analysis indicated these changes were observed long-term, suggesting that iNKT cells gene programs are not fixed, but they are capable of chromatin remodeling after antigen to give rise to several new subsets.

overall_design: Transcriptome and epigenome analysis of NKT cell subsets isolated from thymus, spleen, lung and liver from unchallenged C57Bl/6J mice, as well as splenic NKT cells isolated from mice 6d post-challenge with the strong NKT agonist alpha-galactosyl-ceramide.

first: Mallory

last: Murray

first: Isaac

last: Engel

first: Gregory

last: Seumois

first: Sara

last: Herrera de la Mata

first: Sandy

last: Rosales

first: Ashu

last: Sethi

first: Ashmitaa

last: Premlal

first: Jason

last: Greenbaum

first: Anjana

last: Rao

first: Pandurangan

last: Vijayanand

first: James

last: Browne


title: Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes [ChIP-seq]

city: Xiamen

first: Zhenkui

last: Li

state: Fujian

address: Xiangan south road

institute: Xiamen University

summary: Male and female gametocytes are sexual precursor cells essential for transmission of malaria parasite in the mosquitoes. Differentiation of gametocytes to fertile gametes (gametogenesis) relies on the gender-specific transcriptome. However, how the parasites establish distinct repertoire of gene transcription in the male and female gametocytes remains largely unknown. Here, we report that an Apetala2 (AP2) family transcription factor (TF) AP2-O3 operates as a transcription repressor regulating female gametocyte transcriptome. AP2-O3 is specifically localized in the nucleus of the female gametocytes. AP2-O3-deficient parasites produce apparently normal female gametocytes, which fail to differentiate to fully fertile female gametes, leading to developmental arrest in fertilization and early development post-fertilization. AP2-O3 disruption causes massive up-regulation of transcriptionally dormant male genes and simultaneously down-regulation of highly transcribed female genes in female gametocytes. ChIP-seq and EMSA analysis establish AP2-O3 as a transcription repressor that targets a significant proportion of the upregulated male genes by recognizing an eight-base DNA motif in the promoters. In addition, the maternal AP2-O3 is removed after fertilization, which is required for the zygote to ookinete development. These results demonstrate that global transcriptional repression of male genes in the female gametocytes is required for safeguarding female-specific transcriptome and essential for the mosquito transmission of Plasmodium.

overall_design: We report genome-wide binding profile of Plasmodium transcription factor AP2-O3. Two independent experiments were peformed.


title: Malaria parasite transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes

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

overall_design: Refer to individual Series


title: Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes [RNA-seq]

overall_design: We performed RNA-Seq to gain insights into the genome-wide gene expression changes due to the loss of AP2-O3 in female gametocytes. To obtain female gametocytes of high purity, we used a double fluorescence reporters strain P. yoelii DFsc7, in which GFP and mCherry are specifically expressed in the male and female gametocytes respectively. We deleted the endogenous ap2-o3 in the DFsc7 background and generated the mutant DFsc7;Δap2-o3.Using fluorescence-activated cell sorting (FACS), the mCherry+ female gametocytes from both DFsc7 and DFsc7;Δap2-o3 strains were collected with more than 99% purity for RNA-seq.


title: Transcriptome and Chromatin Landscape of iNKT Cells are Shaped by Subset Differentiation and Antigen Exposure [RNA-seq]

summary: Invariant natural killer T cells (iNKT cells) differentiate into thymic NKT1, NKT2 and NKT17 subsets that are also peripheral. We determined if the gene programs associated with these thymic subsets were maintained in peripheral sites, the influence of tissue location, and if there were large-scale changes after antigen exposure. RNA-seq and ATAC-seq analyses showed that iNKT cells in any subset were similar, regardless of tissue location. Lung iNKT cell subsets shared the most distinct location-specific features, shared with other innate lymphocytes in the lung, possibly consistent with increased activation. After antigenic stimulation, iNKT cells underwent chromatin and transcription changes leading to two populations: one similar to follicular helper T cells and the other like NK cells. Phenotypic analysis indicated these changes were observed long-term, suggesting that iNKT cells gene programs are not fixed, but they are capable of chromatin remodeling after antigen to give rise to several new subsets.

last: Logandha Ramamoorthy Premlal


title: Epigenetic and Transcriptional profiling of Influenza-specific memory B cells

status: Public on Jan 25 2021

city: Atlanta

first: Chris

last: Scharer

state: GA

address: 1510 Clifton Rd, Suite 3086A

institute: Emory University

summary:
This study aimed to characterize the chromatin accessibility and transcriptional differences between influenza-specific murine memory B cells and naive B cells.  Memory B cells were subdivided into those that expressed either an IgM or IgG B cell receptor (BCR).  Using B cell tetramers, we FACS isolated influenza-specific cells 35 days post-PR8 infection and performed ATAC-seq and RNA-seq.

This SuperSeries is composed of the SubSeries listed below.


title: The Aim2 inflammasome and Gasdermin D promote atherosclerotic plaque necrosis in Jak2V617F clonal hematopoiesis

city: New York

first: Chenyi

last: Xue

institute: Columbia University

summary: scRNA-Sequencing of CD45+ cells harvested from aortas of Ldlr-/- mice following a 12 week western type diet.

overall_design: CD45+ cells were isolated from aortas of Ldlr-/- mice with bone marrow from mice with MX1-Cre driving Jak2V617F mutations, Mx1-Jak2V617F mutations lacking Gsdmd, and littermate control.

first: Trevor

last: Fidler

first: Alan

last: Tall

last: Wang


title: Influenza-specific Memory B cell ATAC seq

summary: To determine the differences in chromatin accessibility between antigen-experienced memory B cells and naïve B cells, we isolated influenza-specific memory B cells expressing either IgG or IgM BCR and compared the chromatin accessibility landscape to naive B cells using ATAC-seq.

overall_design: ATAC-seq of IgG and IgM memory B cells compared to naïve B cells.

first: C

first: M

last: Price

first: J

last: Boss


title: Influenza-specific Memory B cell RNA seq

summary: To determine the differences in gene expression between antigen-experienced memory B cells and naïve B cells, we isolated influenza-specific memory B cells expressing either IgG or IgM BCR and compared their transcriptomes to naive B cells using RNA-seq.

overall_design: RNA-seq of IgG and IgM memory B cells compared to naïve B cells.


title: RNA-seq of miR-204-5p overexpressing 4T1 murine breast cancer cells

city: Seoul

first: Boksil

last: Hong

address: 101, Daehak-ro, Jongno-gu

institute: Seoul National University of Hopsital

summary: We established two distinct stable clones which ectopically overexpress miR-204-5p to different levels, then performed transcriptome profiling of miR-204-5p overexpressing cells compared to control 4T1 cells to understand the molecular mechanisms of the miR-204-5p’s effect on cancer cells.

overall_design: Triplicate of control 4T1 and two distinct clones of miR-204-5p overexpression

first: Hyeong-Gon

last: Moon

first: Bok


title: Dimerisation of the PICTS complex via LC8/Cut-up drives co-transcriptional transposon silencing in Drosophila [RNA-Seq]

city: Cambridge

first: Susanne

last: Bornelöv

address: Robinson Way

department: Cancer Research UK - Cambridge Institute

country: United Kingdom

institute: University of Cambridge

summary: In animal gonads, the PIWI-interacting RNA (piRNA) pathway guards genome integrity in part through the co-transcriptional gene silencing of transposon insertions. In Drosophila ovaries, piRNA-loaded Piwi detects nascent transposon transcripts and instructs heterochromatin formation through the Panoramix-induced co-transcriptional silencing (PICTS) complex, containing Panoramix, Nxf2 and Nxt1. Here, we report that the highly conserved dynein light chain LC8/Cut-up (Ctp) is an essential component of the PICTS complex. Loss of Ctp results in transposon de-repression and a reduction in repressive chromatin marks specifically at transposon loci. In turn, Ctp can enforce transcriptional silencing when artificially recruited to RNA and DNA reporters. We show that Ctp drives dimerisation of the PICTS complex through its interaction with conserved motifs within Panoramix. Artificial dimerisation of Panoramix bypasses the necessity for its interaction with Ctp, demonstrating that conscription of a protein from a ubiquitous cellular machinery has fulfilled a fundamental requirement for a transposon silencing complex.

overall_design: RNA sequencing from Drosophila melanogaster ovarian somatic cells (OSCs) treated with either Ctp or control (GFP) knockdowns, and from Drosophila melanogaster ovaries with either Ctp, Panx or control (white) germline knockdowns. Four replicates per condition.

first: Evelyn

last: Eastwood

first: Kayla

middle: A

last: Jara

first: Marzia

last: Munafò

first: Vasileios

last: Frantzis

first: Emma

last: Kneuss

first: Elisar

last: Barbar

first: Benjamin

last: Czech

first: Gregory

last: Hannon


title: SLAMF5 blocking of sorted Bregs CD19+IL10+ (GFP+)

city: Rehovot

first: Idit

last: Shachar

address: Herzl 234

department: immunology

institute: Weizmann Institute of Science

summary:
Purpose: Our results show that in the absence of SLAMF5 ( by KO or blocking ab), there is an accumulation of Bregs (CD19+IL10+) and a specific increase of their survival. Here, using RNAseq, we wished to identify genes that take place in the regulation of Breg by SLAMF5 blocking.
Method: Total RNA was extracted from 3x10e5 sorted CD19+GFP+cells from 2-3 Vert-x mice (induced with EAE) incubated with SLAMF5 blocking Ab or control IgG using the Direct-zol RNA kit (ZymoResearch) according to the manufacturer’s instructions. RNA-seq was performed using the MARS-seq protocol. Showing here 2 biological replicates of each group.

overall_design: RNA of CD19+IL10+ sorted cells (il10 GFP reporter mice) derived of EAE sick mice,  incubated with SLAMF5 blocking Ab or control IgG.

first: Lihi

last: Radomir


title: Dimerisation of the PICTS complex via LC8/Cut-up drives co-transcriptional transposon silencing in Drosophila [sRNA-Seq]

overall_design: Small RNA (sRNA) sequencing from Drosophila melanogaster ovarian somatic cells (OSCs) treated with either Ctp or control (GFP) knockdowns. Two replicates per condition.


title: Dimerisation of the PICTS complex via LC8/Cut-up drives co-transcriptional transposon silencing in Drosophila

type: Non-coding RNA profiling by high throughput sequencing


title: Multiplexed single-cell transcriptomic analysis of normal and impaired lung development in the mouse

city: Ottawa

first: David

last: Cook

state: ON

address: 501 Smyth Rd

department: Cancer Therapeutics Program

institute: Ottawa Hospital Research Institute

summary: During late lung development, alveolar and microvascular development is finalized to enable sufficient gas exchange. Impaired late lung development manifests as bronchopulmonary dysplasia (BPD) in preterm infants. Single-cell RNA sequencing (scRNA-seq) allows for assessment of complex cellular dynamics during biological processes, such as development. Here, we use MULTI-seq to generate scRNA-seq profiles of over 66,000 cells from 36 mice during normal or impaired lung development secondary to hyperoxia. We observed dynamic populations of cells, including several rare cell types and putative progenitors. Hyperoxia exposure, which mimics the BPD phenotype, alters the composition of all cellular compartments, particularly alveolar epithelium, capillary endothelium and macrophage populations. Pathway analysis and predicted dynamic cellular crosstalk suggests inflammatory signalling as the main driver of hyperoxia-induced changes. Our data provides a novel single-cell view of cellular changes associated with late lung development in health and in disease.

overall_design: scRNA-seq profiles of 36 mice during normal or impaired lung development secondary to hyperoxia.

first: Ivana

last: Mižíková

first: Maria

last: Hurskainen

first: Bernard

last: Thébaud


title: Dimerisation of the PICTS complex via LC8/Cut-up drives co-transcriptional transposon silencing in Drosophila [ChIP-Seq]

overall_design: ChIP-seq for H3K4me2 and H3K9me3 from Drosophila melanogaster ovarian somatic cells (OSCs) and Schneider 2 (S2) cells. OSCs were treated with either Ctp or control (GFP) knockdown. S2 cells harbouring a stable integration of the DNA sensor plasmid were transfected with either Lacl-Egg, Lacl-Panx or Lacl-Renilla (Control). Two replicates per condition.


title: Defective viral genomes determine respiratory syncytial virus disease severity in adult and children cohorts

city: St Louis

first: Carolina

middle: B

last: Lopez

state: MO

address: 425 S Euclid Ave

institute: Washington University in St. Louis

summary: Defective viral genomes of the copy-back type (cbDVGs) are generated during RSV infectin and are suggested to impact the clinical outcome. Here in this study we selected the nasal secretions from 13 hospitalized pediatric pateints, including both cnDVG+ and cbDVG-, to perform RNA-seq to understand the impact of cbDVGs on patient transcriptome.

overall_design: 6 cbDVG- samples and 7 cbDVG+ samples

first: Yan

last: Sun

first: Sebastien

last: Felt

last: López

first: Emna

last: Achouri


title: Functional analysis of TMEM53, the disease gene for a novel type of sclerosing bone disorder

city: Tokyo

first: Long

last: Guo

address: 4-6-1 Shirokanedai, Minato-ku

country: Japan

institute: RIKEN

summary: Purpose: The goal of this study is to explore the molecular mechanism of the phenotypes found in the TMEM53 mutant human and mouse. Methods: Calvarial mRNA profiles of 3-day-old Tmem53+/- and Tmem53-/- mice were generated by sequencing, in triplicate, using NovaSeq 6000 sequencer. The reads from the RNA-seq were aligned to the mouse genome (NCBI build37.2) using Tophat. Aligned reads were quantified using Cufflinks. Differential gene expression analysis was performed with Cuffdiff. Gene ontology (GO) term enrichment analysis was performed by DAVID 6.8. Results:The gene expression profiling displayed a total of 14,836 genes, including 1,278 differentially expressed genes (DEGs). These DEGs were enriched in some GO terms, and the most enriched term is “ossification”, which is considered to be closely associated with the thickened calvaria in the Tmem53-/- mice. The known marker genes of osteogenic precursors, osteoblasts and osteocytes (Runx2, Sp7, Alpl, Bglap and Ibsp) were up-regulated in  Tmem53-/- mice according to the RNA-seq dataset. The DEGs were also enriched in the reported target gene set of Bmp-Smad pathway. The representative downstream transcriptional targets of Bmp signaling, Id1, Dlx3 and Dlx5, were up-regulated in the calvaria of the Tmem53-/- mice. Conclusions: Our results shows enhanced osteoblastogenesis in the calvaria of the Tmem53-/- mice, which would be attributed to the over-activated Bmp-Smad pathway caused by the Tmem53 deficiency.

overall_design: Calvarial mRNA profiles of 3-day-old Tmem53+/- (n=3) and Tmem53-/- mice (n=3)

first: Juan

last: Wang

first: Katta

last: Girisha

first: Shiro

last: Ikegawa


title: IL-36 Amplifies Neutrophilic Lung Inflammation

city: Biberach

first: Christian

middle: Thaddaeus

last: Wohnhaas

address: Birkendorfer Straße 65

institute: Boehringer Ingelheim

summary: IL-36, which belongs to the IL-1 superfamily, is increasingly linked to neutrophilic inflammation. Here, we performed single-cell RNA-seq on an acute LPS mouse model of lung inflammation to provide insights into the intercellular signaling pathways and mechanisms through which IL-36 promotes lung inflammation. We identified neutrophils as a source of IL-36 which provides a rationale for targeting IL-36 to improve treatment of a variety of neutrophilic lung diseases.

overall_design: Mice were exposed to aerolized LPS for 4h; for scRNA seq BALF of 8 animals were equally pooled to obtain a single BAL pool that contained the same cell count per animal.

first: Carolin

last: Koss

first: Jonathan

last: Baker

first: Cornelia

last: Tilp

first: Michèl

last: Przibilla

first: Carmen

last: Lerner

first: Silvia

last: Frey

first: Martina

last: Keck

first: Cara

last: Williams

first: Daniel

last: Peter

first: Meera

last: Ramanujam

first: Jay

last: Fine

first: Florian

last: Gantner

first: Matthew

last: Thomas

first: Peter

last: Barnes

first: Louise

last: Donnelly

first: Karim

last: El Kasmi


title: The effect of COX2 inhibition on RAS-induced senescence

first: Guy

last: Slater

institute: Cancer Research UK Cambridge Institute

summary: ER:RAS-G12V expressing IMR90 cells were treated with either 100nM 4-OHT or 40uM Celecoxib or both for 6 days leading to RAS-induced senescence (RIS) with or without COX2 inhibition.

overall_design: IMR90 cells expressing a 4-hydroxytamoxifen (4-OHT) inducible estrogen receptor (ER)-coupled RAS-G12V (ER:RAS-G12V) were treated with either 100nM 4-OHT or 40uM Celecoxib or both for 6 days leading to RAS-induced senescence (RIS) with or without COX2 inhibition. Oligo-dT captured RNA was then used for library preparation and then analysed using mRNA-sequencing. There were 8 (eight) biological replicatesfor each of the experimental conditions. Untreated ER:RAS IMR90 cells were the control condition

first: Matt

last: Hoare


title: Transcriptome and Chromatin Landscape of iNKT Cells are Shaped by Subset Differentiation and Antigen Exposure

first: Mitchell

last: Kronenberg


title: RNA sequencing reveals that methionine supply during late-gestation alters neonatal Holstein heifer calf liver transcriptome profiles

status: Public on Jan 24 2021

city: Campobasso

first: Valentino

last: Palombo

address: via Francesco De Sanctis

department: Dipartimento Agricoltura Ambiente e Alimenti

country: Italy

institute: Università degli Studi del Molise

summary: Maternal methionine supply during late pregnancy can affect offspring growth and development via nutritional programming. In the present study, we investigated if increasing the supply of methionine during late-pregnancy in Holstein cows affects transcriptome profiles in the offspring.

overall_design: Liver biopsies were harvested at 4 days of age from 6 calves per treatment born to cows fed a control diet or the control plus ethyl-cellulose rumen-protected methionine for the last 28 days prepartum, and were fed and managed similarly after birth

first: Abdulrahman

last: Alharthi

first: Fernanda

last: Batistel

first: Mariasilvia

last: D'Andrea

first: Claudia

last: Parys

first: Jessie

last: Guyader

first: Juan

last: Loor


title: Generation of blastocyst-like structures from human embryonic stem cells III [scRNA-seq]

city: Dallas

first: Gary

last: Hon

address: 5323 Harry Hines Blvd.

department: Department of Obstetrics and Gynecology

institute: UT Southwestern

summary: We established a method that enables the generation of blastocyst-like structures from human pluripotent stem cells.

overall_design: To model human pre-/peri-implantation development, we reported a novel strategy to generate blastocyst-like structures from human pluripotent stem cells (hPSCs) in vitro, which resemble blastocysts in morphology and cell-lineage allocation.

first: Jun

last: Wu

first: Jialei

last: Duan


title: Generation of blastocyst-like structures from human embryonic stem cells II [scRNA-seq]


title: Effect of 9 days of inhibited GDNF signaling on the transcriptome of the mouse testis

city: Ithaca

first: Jennifer

last: Grenier

address: 526 Campus Rd

department: Biomedical Sciences

institute: Cornell University

summary: In the normal adult testis GDNF specifically targets spermatogonial stem cells and early progenitor spermatogonia. Inhibition of GDNF signaling for 9 days alters only 171 of the 15,000 transcripts expressed in the mouse testis. Many of these transcripts are known to be expressed by the spermatogonial stem cells. One transcript that is affected is Kif26A, a known suppressor of GDNF signaling.

overall_design: We used a chemical-genetic approach to inhibit GDNF signaling in vivo. Mice were treated for 9 days with inhibitor or vehicle (n=3/group) and testes collect on day 14 of the experiment. Total testis RNA was isolated, polyA+ libraries were generated with the NEBNext Ultra Directional RNA Library kit. Libraries were sequenced using an Illumina NextSeq500 instrument, to minimum 30 million single end 81nt reads per library.

first: William

last: Wright


title: A Multiplexed, Next-Generation Sequencing Platform for High-Throughput Detection of SARS-CoV-2 [PoC cohort]

city: Toronto

first: Jeff

last: Wrana

address: 600 Unv Ave

institute: Lunenfeld Tanenbaum Research Institute

summary: Population scale sweeps of viral pathogens, such as SARS-CoV-2, require high intensity testing for effective management. However, reliable systems affording parallel testing of thousands of patients for pathogen infection have not yet been routinely employed. Here we describe “Systematic Parallel Analysis of RNA coupled to Sequencing for Covid-19 screening” (C19-SPAR-Seq), a multiplexed, readily automated platform for SARS-CoV-2 detection capable of analyzing tens of thousands of patient samples in a single instrument run. To address strict requirements for control of assay parameters and output demanded by clinical diagnostics, we employed a control-based Precision-Recall and Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of several hundred patients performed with a specificity of 100% and sensitivity of 91% on samples with low viral loads. Our study thus establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.

sample_organism:
Homo sapiens
synthetic RNA

platform_organism:
Homo sapiens
synthetic RNA

overall_design: Highly parallelized RT-PCR amplification of SARS-CoV-2 sequences from patient swabs coupled to next-generation sequencing (NGS) for multiplexed samples.

first: Marie

last: Aynaud

first: Javier

last: Hernandez

first: Seda

last: Barutcu

first: Ulrich

last: Braunschweig

first: Kin

last: Chan

first: Joel

last: Pearson

first: Daniel

last: Trcka

first: Hong

last: Han

first: Suzanna

last: Prosser

first: Jaeyoun

last: Kim

first: Miriam

last: Barrios-Rodiles

first: Mark

last: Jen

first: Siyuan

last: Song

first: Jess

last: Shen

first: Christine

last: Bruce

first: Bryn

last: Hazlett

first: Susan

last: Poutanen

first: Liliana

last: Attisano

first: Rod

last: Bremner

first: Tony

last: Mazzulli

first: Benjamin

last: Blencowe

first: Laurence

last: Pelletier

first: Jeffrey


title: The deubiquitinase Usp9x regulates PRC2-mediated chromatin reprogramming during mouse development

first: Miguel

last: Ramalho-Santos

address: 25 Orde Street

institute: Lunenfeld-Tanenbaum Research Institute

summary: Pluripotent cells of the mammalian embryo undergo extensive chromatin rewiring to prepare for lineage commitment after implantation. Repressive H3K27me3, deposited by Polycomb Repressive Complex 2 (PRC2), is reallocated from large blankets in pre-implantation embryos to mark promoters of developmental genes. The regulation of the global redistribution of H3K27me3 is poorly understood. Here we report a post-translational mechanism that destabilizes PRC2 to constrict H3K27me3 during lineage commitment. Using an auxin-inducible degron system, we show that the deubiquitinase Usp9x is required for mouse embryonic stem (ES) cell self-renewal. Usp9x-high ES cells have high PRC2 levels and bear a chromatin and transcriptional signature of the pre-implantation embryo, whereas Usp9x-low ES cells resemble the post-implantation, gastrulating epiblast. We show that Usp9x interacts with, deubiquitinates and stabilizes PRC2. Deletion of Usp9x in post-implantation embryos results in the derepression of genes that normally gain H3K27me3 after gastrulation, followed by the appearance of morphological abnormalities at E9.5, pointing to a recurrent link between Usp9x and PRC2 during development. Usp9x is a marker of “stemness” and is mutated in various neurological disorders and cancers. Our results unveil a Usp9x-PRC2 regulatory axis that is critical at peri-implantation and may be redeployed in other stem cell fate transitions and disease states.

overall_design:
Spike-in normalized transcriptional profiling of mouse ES cells carrying an auxin-inducible degron system for depletion of Usp9x. Cells were treated with auxin for 8 hours, sorted based on Usp9x status (Usp9x-high or Usp9x-low), and harvested or allowed to recover without auxin for 48 hours. Vehicle-treated and auxin-resistant (Flag-tagged Usp9x) cells serve as controls.

Spike-in normalized H3K27me3 ChIP-seq profiles of mouse ES cells carrying an auxin-inducible degron system for depletion of Usp9x. Cells were sorted based on Usp9x status (Usp9x-high or Usp9x-low) following 8 hours of auxin treatment.

Transcriptional profiling of single E8.5 male embryos either wild-type or mutant for Usp9x. Usp9x-fl/fl females were crossed to Sox2-Cre males to delete Usp9x in epiblast derivatives in offspring. Litter-matched Cre-negative (Usp9x-fl/Y) embryos serve as controls.

first: Trisha

last: Macrae


title: A Multiplexed, Next-Generation Sequencing Platform for High-Throughput Detection of SARS-CoV-2 [extended cohort]

first: Jeff

last: Wrana

sample_organism:
Homo sapiens
synthetic construct
synthetic RNA

first: Marie

last: Aynaud

first: J

last: Hernandez

first: Seda

last: Barutcu

first: Ulrich

last: Braunschweig

first: Kin

last: Chan

first: Joel

last: Pearson

first: Daniel

last: Trcka

first: Hong

last: Han

first: Suzanna


title: A Multiplexed, Next-Generation Sequencing Platform for High-Throughput Detection of SARS-CoV-2 [pilot cohort]

platform_organism:
Homo sapiens
synthetic construct
synthetic RNA

last: Prosser

first: Jaeyoun

last: Kim

first: Miriam

last: Barrios-Rodiles

first: Mark

last: Jen

first: Siyuan

last: Song

first: Jess

last: Shen

first: Christine

last: Bruce

first: Bryn

last: Hazlett

first: Susan

last: Poutanen

first: Liliana

first: Rod


title: A Multiplexed, Next-Generation Sequencing Platform for High-Throughput Detection of SARS-CoV-2 [test cohort]

first: Jeff

last: Wrana

first: Marie

last: Aynaud

first: J

first: Seda

last: Attisano

last: Bremner

first: Tony

last: Mazzulli

first: Benjamin

last: Blencowe

first: Laurence

last: Pelletier

first: Jeffrey


title: High throughput sequncing for YTHDC1 binding RNAs [4sUDRB-seq]

city: GuangZhou

first: Jiangping

last: He

address: Kai yuan avenue 190

institute: Guangzhou Institutes of Biomedicine and Health (GIBH), CAS

summary: N6-methyladenoisne (m6A) plays critical roles in many biological processes. However, the function of m6A at the early phase of mammalian development remains poorly understood. Here we show that m6A reader Ythdc1 is required for the proliferation of mouse embryonic stem cells (mESCs) in an m6A-dependent manner and its deletion resets mESCs back to a 2C-like state.

overall_design: We analyzed the transcriptome expression and epigenetic  profiles after YTHDC1 Knock out in mouse embyronic stem cells


title: High throughput sequncing for YTHDC1 binding RNAs [ChIRP-seq]

overall_design: We analyzed the transcriptome expression and epigenetic  profiles after YTHDC1 Knock out treated only with DMSO in mouse embyronic stem cells.


title: Generation of blastocyst-like structures from human embryonic stem cells

first: Gary

last: Hon

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

overall_design: Refer to individual Series


title: High throughput sequencing for YTHDC1 binding RNAs [RNA-seq]


title: High throughput sequencing for YTHDC1 binding RNAs [ChIP-seq]


title: Proteomics profiling of arginine methylation mediated by PRMT family reveals a critical role of arginine methylation in RNA splicing and cancer cell growth

status: Public on Jan 23 2021

first: Guosheng

last: Hu

address: Xiang'an South Road, Xiang'an District

department: School of Pharmaceutical Sciences

institute: xiamen University

summary: Protein arginine methylation, catalyzed by the protein arginine methyltransferase (PRMT) family, is recognized as a widespread post-translational modification (PTM) with implications in a plethora of biological processes in eukaryotes. PRMT proteins were classified into three types, type I, II and III, according to the final methyl-arginine products generated. Despite that thousands of substrates have been identified for Type I and II PRMTs, a full scope of arginine methylation catalyzed by the only type III PRMT, PRMT7, as well as its connection with that of type I and II PRMTs remain unknown, limiting our understanding of the network of arginine methylation and its functions in cells. In this study, global profiling of PRMT4 (type I), PRMT5 (type II) and PRMT7 (type III) substrates (also referred as methylome) revealed that PRMT7 methylated a GAR (glycine and arginine) motif similar as PRMT5, while PRMT4 uniquely methylated a motif in which proline was highly enriched. PRMT4, 5 and 7-methylome were all enriched with proteins functioning in mRNA splicing, and knockdown of PRMT4, 5 or 7 led to a global change of alternative splicing events, among which a cohort with implications in cancer development was co-regulated by all three PRMTs. PRMT4, 5 and 7-mediated arginine methylation on splicing factors such as hnRNPA1, though at different status, were shown to enhance RNA binding in general and participate in the regulation of PRMT4, 5 and 7 co-regulated alternative splicing events.The clinical significance of PRMT4, 5 and 7-mediated arginine methylation was underscored by the fact that these three PRMTs as well as hnRNPA1 arginine methylation were over-represented in multiple types of cancers, which were associated with aberrant gene alternative splicing. Consistently, silencing or pharmacological inhibition of PRMT4, 5 and 7 altered gene alternative splicing and suppressed cancer cell growth, and co-treatment exhibited synergistic effects. Taken together, our study provided an integrative view of substrates for the three types of PRMTs, revealing the important function of arginine methylation in RNA splicing and cancer.

overall_design: RNA-seq performed in this study was designed to reveal the gene splicing events regulated by PRMT4, 5 or 7

first: Wen

last: Liu

first: Wen-juan

last: Li

first: Guo-sheng


title: Genome-wide maps of  m5C methylation in neuron of mouse.

status: Public on Jan 22 2021

city: Tianjin

first: Huan

last: Jian

address: NO.22 Qixiangtai Road

institute: Tianjin medical university

type: Methylation profiling by high throughput sequencing

summary: We report the application of RNA bisulfite sequencing(RNA-BS-seq) technology for high-throughput profiling of mouse neuron. Here, we successfully constructed a neuronal oxygen-glucose deprivation/reoxygenation (OGD/R) model,1.5 hours and 3 hours respectively, and obtained an overview of the transcriptome-wide m5C profiles using RNA-BS-seq. We discovered that the distribution of neuronal m5C modifications was highly conserved, significantly enriched in CG-rich regions and concentrated in the mRNA translation initiation regions. After OGD/R, modification level of m5C increased, whereas the number of methylated mRNA genes decreased. The amount of overlap of m5C sites with the binding sites of most RBPs increased significantly, except for that of the RBM3-binding protein. Moreover, hypermethylated genes in neurons were significantly enriched in pathological processes, and the hub hypermethylated genes RPL8 and RPS9 identified by the protein-protein interaction (PPI) network were significantly related to cerebral injury. This study identified novel m5C mRNAs associated with ischemia-reperfusion in neurons, providing valuable perspectives for future studies on the role of the RNA methylation in cerebral ischemia-reperfusion injury.

overall_design:
Examing 3 conditions, each with 3 replicates

*m5c sample: RNA was bisulfite-converted;  *mock samples: RNA was not bisulfite-converted
Each bed file was generated from *m5c and *mock raw data, and is linked to the corresponding *m5c sample records.


title: Generation of blastocyst-like structures from human embryonic stem cells I [scRNA-seq]

first: Jun

last: Wu

first: Jialei

last: Duan


title: A Multiplexed, Next-Generation Sequencing Platform for High-Throughput Detection of SARS-CoV-2

first: Jeff


title: The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity

summary:
The RNA modification N6-methyladenosine (m6A) has critical roles in many biological processes. However, the function of m6A in the early phase of mammalian development remains poorly understood. Here we show that the m6A reader YT521-B homology-domain-containing protein 1 (YTHDC1) is required for the maintenance of mouse embryonic stem (ES) cells in an m6A-dependent manner, and that its deletion initiates cellular reprogramming to a 2C-like state. Mechanistically, YTHDC1 binds to the transcripts of retrotransposons (such as intracisternal A particles, ERVK and LINE1) in mouse ES cells and its depletion results in the reactivation of these silenced retrotransposons, accompanied by a global decrease in SETDB1-mediated trimethylation at lysine 9 of histone H3 (H3K9me3). We further demonstrate that YTHDC1 and its target m6A RNAs act upstream of SETDB1 to repress retrotransposons and Dux, the master inducer of the two-cell stage (2C)-like program. This study reveals an essential role for m6A RNA and YTHDC1 in chromatin modification and retrotransposon repression.

This SuperSeries is composed of the SubSeries listed below.


title: The comparative methylome and transcriptome after change of direction compared to straight line running exercise in human skeletal muscle

status: Public on Jan 21 2021

city: Oslo

first: Adam

last: Sharples

address: 220 Sognsveien

department: Institute for Physical Performance

country: Norway

institute: Norwegian School of Sport Sciences

summary: The methylome and transcriptome signatures following exercise that are physiologically and metabolically relevant to sporting contexts such as team sports or health prescription scenarios (e.g. high intensity interval training/HIIT) has not been investigated. To explore this, we performed two different sport/exercise relevant high-intensity running protocols in 5 male sport team members using a repeated measures design of: 1) Change of direction (COD) versus; 2) straight line (ST) exercise with a wash-out period of at least 2 weeks between trials. Skeletal muscle biopsies collected from the vastus lateralis 30 minutes and 24 hours post exercise, were assayed using 850K methylation arrays and a comparative analysis with recent (subject-unmatched) sprint and acute aerobic exercise meta-analysis transcriptomes was performed. Despite COD and ST exercise being matched for classically defined intensity measures (speed x distance and number of accelerations/decelerations), COD exercise elicited greater movement (GPS-Playerload), physiological (HR), metabolic (lactate) as well as central and peripheral (differential RPE) measures compared with ST exercise, suggesting COD exercise evoked a higher exercise intensity. The exercise response alone across both conditions evoked extensive alterations in the methylome 30 mins and 24 hrs post exercise, particularly in MAPK, AMPK and axon guidance pathways. COD evoked a considerably greater hypomethylated signature across the genome compared with ST exercise, particularly at 30 minutes post exercise, enriched in: Protein binding, MAPK, AMPK, insulin, and axon guidance pathways. Comparative methylome analysis with sprint running transcriptomes identified considerable overlap, with 49% of genes that were altered at the expression level also differentially methylated after COD exercise. After differential methylated region analysis, we observed that VEGFA and its downstream nuclear transcription factor, NR4A1 had enriched hypomethylation within their promoter regions. VEGFA and NR4A1 were also significantly upregulated in the sprint transcriptome and meta-analysis of exercise transcriptomes. We confirmed increased gene expression of VEGFA, and considerably larger increases in the expression of canonical metabolic genes, PPARGC1A (that encodes PGC1-α) and NR4A3 in COD vs. ST exercise. Overall, we demonstrate that increased physiological/metabolic load via change of direction exercise in human skeletal muscle evokes considerable epigenetic modifications that are associated with changes in expression of genes responsible for adaptation to exercise.

overall_design: A within-subject design conducted in young adult male humans. Each participant (1-3) underwent two running trials separated by a minimum of 2 weeks. Trial 1) Change of direction (COD) versus; Trial 2) straight line (ST) running exercise. Skeletal muscle biopsies were collected from the vastus lateralis muscle at baseline (at rest pre-exercise under overnight fasted conditions) as well as after 30 minutes and 24 hours post exercise in both trials. These samples were assayed using 850K Illumina EPIC methylation arrays and a comparative analysis with recent (subject-unmatched) sprint and acute aerobic exercise meta-analysis transcriptomes. Despite COD and ST exercise being matched for classically defined intensity measures (speed x distance and number of accelerations/decelerations), COD exercise elicited greater movement (GPS-Playerload), physiological (HR), metabolic (lactate) as well as central and peripheral (differential RPE) measures compared with ST exercise, suggesting COD exercise evoked a higher exercise intensity or ‘player-loading’. Therefore, the samples provided an opportunity to assess differences in DNA methylation across the genome following running exercise that evoked differential exercise intensities/playload in a model that was physiologically and metabolically relevant to sporting contexts such as team sports or health prescription scenarios (e.g. high intensity interval training/HIIT). Note - Participant 1 had their methylome profiled at baseline (pre-exercise under rested conditions) prior to both trials (COD & ST) separated by at least two weeks. These 2 baseline biopsies in the same participant were taken under the same conditions (baseline/rest/overnight fast) before each trial and demonstrated little variation in methylome profiles. Therefore, for the remaining participants (2 & 3) due to the reason above and because this was a within-subject trial, the baseline (pre-exercise/rested) sample for the first trial undertaken only (COD trial) was used as the baseline sample in downstream analysis. Hence why participants 1-3 have baseline, 30 minute and 24 hr samples for their COD trial and participants 2 & 3 only have 30 minute and 24 hr samples designated for their ST trial.


title: High throughput sequencing for YTHDC1 binding RNAs [RIP-seq]


title: Short telomeres trigger epithelial to mesenchymal transition programmes and sensitize to folic acid-induced kidney fibrosis in mice [young vs old]

city: Madrid

first: Osvaldo

last: Graña

state: Comunidad de Madrid

address: C/ Melchor Fernández Almagro, 3

department: Structural Biology

country: Spain

institute: CNIO

summary: Gene expression profiles of Tert+/+ and generation 3 (G3) Tert-/- male and female mice.

overall_design: 7 and 47 weeks old Tert+/+ and generation 3 (G3) Tert-/- male mice were used in the study.

first: Sarita

last: Saraswati

last: Graña-Castro

first: Paula

last: Martínez

first: María

last: Blasco


title: Short telomeres trigger epithelial to mesenchymal transition programmes and sensitize to folic acid-induced kidney fibrosis in mice


title: A surge of DNA damage links transcriptional reprogramming and hematopoietic deficit in Fanconi anemia

city: Houston

first: Haoqiang

last: Ying

state: Texas

address: 1515 Holcombe Blvd

institute: M. D. Anderson Cancer Center

summary: Impaired DNA crosslink repair leads to Fanconi anemia (FA), characterized by a unique manifestation of bone marrow failure and pancytopenia among diseases caused by DNA damage response defects. As a germline disorder, why the hematopoietic hierarchy is specifically affected is not fully understood. We find that reprogramming transcription during hematopoietic differentiation results in an overload of genotoxic stress, which causes aborted differentiation and depletion of FA mutant progenitor cells. The onset of DNA damage most likely arises from formaldehyde, an obligate by-product of oxidative protein demethylation during transcription regulation. Our results demonstrate that rapid and extensive transcription reprogramming associated with hematopoietic differentiation poses a major threat to genome stability and cell viability in the absence of the FA pathway. The connection between differentiation and DNA damage accumulation reveals a novel mechanism of genome scarring and is critical to exploring therapies to counteract the aplastic anemia for the treatment of FA patients.

overall_design: Examine the effect of FANCL knock-out in DNA repair and hematopieotic cell differentiation

first: Xi

last: Shen

first: Rui

last: Wang

first: Moon

last: Kim

first: Qianghua

first: Chih-Chao

last: Hsu

last: Yao

first: Naeh

last: Klages-Mundt

first: Yanyan

last: Tian

first: Erica

last: Lynn

first: Thomas

middle: F

last: Brewer

first: Yilei

last: Zhang

first: Banu

last: Arun

first: Boyi

last: Gan

first: Michael

last: Andreeff

first: Shunichi

last: Takeda

first: Junjie

last: Chen

first: Jae-il

last: Park

first: Xiaobing

last: Shi

first: Christopher

last: Chang

first: Sung

last: Jung

last: Qin

first: Lei


title: Disturbed intramitochondrial phosphatidic acid transport impairs cellular stress signaling

city: Cologne

first: Franziska

last: Metge

address: Joseph-Stelzmann-Str 9b

department: Bioinformatics Corefacility

institute: Max Planck Institute for Biology of Ageing

summary: Lipid transfer proteins of the Ups1/PRELID1 family facilitate the transport of phospholipids across the intermembrane space of mitochondria in a lipid-specific manner. Heterodimeric complexes of yeast Ups1/Mdm35 or human PRELID1/TRIAP1 shuttle phosphatidic acid (PA) synthesized in the endoplasmic reticulum (ER) to the inner membrane, where it is converted to cardiolipin (CL), the signature phospholipid of mitochondria. Loss of Ups1/PRELID1 proteins impairs the accumulation of CL and broadly affects mitochondrial structure and function. Unexpectedly and unlike yeast cells lacking the cardiolipin synthase Crd1, Ups1 deficient yeast cells exhibit glycolytic growth defects, pointing to functions of Ups1-mediated PA transfer beyond CL synthesis. Here, we show that the disturbed intramitochondrial transport of PA in ups1D cells leads to altered phospholipid composition of the ER membrane, independent of disturbances in CL synthesis. The impaired flux of PA into mitochondria is associated with the increased synthesis of phosphatidylcholine (PC) and a reduced phosphatidylethanolamine (PE)/PC ratio in the ER of ups1D cells which suppresses the unfolded protein response (UPR). Moreover, we observed inhibition of TORC1 signaling in these cells. Activation of either UPR by ER protein stress or of TORC1 signaling by disruption of its negative regulator, the SEACIT complex, increased cytosolic protein synthesis and restored glycolytic growth of ups1D cells. These results demonstrate that PA influx into mitochondria is required to preserve ER membrane homeostasis and that its disturbance is associated with impaired glycolytic growth and cellular stress signaling.

overall_design: Transcript profiles of WT and ups1-null yeast cells grown under glycolytic conditions or treated with tunicamycin for 3 h. The dataset includes biological triplicates for each condition (12 samples in total).

first: Akinori

last: Eiyama

last: Langer

first: Janine

last: Altmüller


title: Cryopreservation of microglia enables single-cell RNA sequencing with minimal effects on disease-related gene expression patterns

city: OMAHA

first: Meng

last: Niu

state: NE

address: 42nd and Emile

department: Department of Neurological sciences

institute: University of Nebraska Medical Center

summary: We report the effect of cryopreservation on isolated microglia and macrophages from the brains of rhesus monkeys. At the time of necropsy, cells were isolated from the brain and either used fresh or cryopreserved. For single cell sequencing, isolates were purified for CD11b expression and captured and processed through the 10xGenomics platform following by sequencing. We find that cryopreservation altered the expression of approximately 2% of the genes. These were not enriched for any known microglia/macrophage set of differentially regulated genes, but was enriched in immediate early genes and the acute phase pathway. Assessment of the fresh and cryopreseved cells in comparison to a prior dataset from animals with encephalitis revelaed equal ability to detect differentially expresed genes. Thus, given the practical difficulty of freshly processing microglia from human and non-human primate brains for isolation and scRNAseq processing the same day, and the ability to archive sampoles for potential use in scRNAseq experiments, cryopreservation is an optimal choice for future analysis of these important brainn cells, and likely other cell types.

overall_design: Rhesus macaques were infected with SIVmac251.  Five weeks post infection, one animal was treated with a two-drug cART consisting of 8mg/ml dolutegravier (DTG), 80 mg/ml emtricitabine (FTC) or a four-drug cART, consisting of 4 mg/ml DTG, 1mg/ml tenofovir alafenamide fumerate (TAF), 40 mg/ml FTC, 12 mg/ml maraviroc (MVC). Animals remained on drug regime for 6 months post latency (<50 SIV copies/ml plasma) and were sacrificed as per experimental design. MIcroglia were isolated and either further purified and subjected to capture and library production, or cryopreserved followed by thawing, purification, capute and library preparation. LIbraries were then subjected to sequencing.

first: Brenda

last: Morsey

first: Howard

last: Fox


title: Role of socs3 in the exon  expression in thymic epithelial cells.

city: Solna

first: Martin

last: Rottenberg

address: Solna vägen 9

institute: Karolinska Institutet

summary: Analysis of RNA from sorted thymic epithelial cells (EpCAM+ CD45 neg) from 3 wild type socs3fl/fl  and 3  socs3fl/fl act creER mice 7 days after tamoxifen treatment

overall_design: Sorted thymic epithelial cells (EpCAM+ CD45 neg) from 3 wild type socs3fl/fl  and 3  socs3fl/fl act creER mice 7 days after tamoxifen treatment

first: M


title: A neural m6A/YTHDF pathway is required for learning and memory in Drosophila

city: New York

first: Brian

last: Joseph

state: New York

address: 430 E 67th St

department: Department of Developmental Biology

institute: Sloan-Kettering Institute

summary: The roles of epitranscriptomic modifications in mRNA regulation have recently received substantial attention, with appreciation growing for their phenotypically selective impacts within the animal. We adopted Drosophila melanogaster as a model system to study m6A, the most abundant internal modification of mRNA. Here, we report proteomic and functional analyses of fly m6A-binding proteins, confirming nuclear (YTHDC) and cytoplasmic (YTHDF) YTH domain proteins as the major m6A binders. Since all core m6A pathway mutants are viable, we assessed in vivo requirements of the m6A pathway in cognitive processes. Assays of short term memory revealed an age-dependent requirement of m6A writers working via YTHDF, but not YTHDC, comprising the first phenotypes assigned to Drosophila mutants of the cytoplasmic m6A reader. These factors promote memory via neural-autonomous activities, and are required in the mushroom body, the center for associative learning. To inform their basis, we mapped m6A from wild-type and mettl3 null mutant heads, allowing robust discrimination of Mettl3-dependent m6A sites. In contrast to mammalian m6A, which is predominant in 3' UTRs, Drosophila m6A is highly enriched in 5' UTRs and occurs in an adenosine-rich context. Genomic analyses demonstrate that Drosophila m6A does not directionally affect RNA stability, but is preferentially deposited on genes with low translational efficiency. However, functional tests indicate a role for m6A in translational activation, since we observe reduced nascent protein synthesis in mettl3-KO cells. Finally, we show that ectopic YTHDF can increase m6A target reporter output in an m6A-binding dependent manner, and that this activity is required for in vivo neural function of YTHDF in memory. Altogether, we provide the first tissue-specific m6A maps in this model organism and reveal selective behavioral and translational defects for m6A/YTHDF mutants.

overall_design: miCLIP and RNA sequencing libraries prepared from fly head RNA samples

first: Lijuan

last: Kan

first: Stanislav

last: Ott

first: Eun-Sil

first: Crystal

last: Dai

first: Ralph

last: Kleiner

first: Adam

last: Claridge-Chang

first: Eric

last: Lai


title: CRISPR-TRAPSeq identifies the QKI RNA binding protein as important for astrocytic maturation and control of thalamocortical synapses

first: Joseph

last: Dougherty

state: Missouri

address: 4566 Scott Ave, box 8232

department: Genetics and Psychiatry

institute: Washington University


title: Tumorigenic effects of TLX over-expression in HEK 293T cells

city: Gothenburg

first: Toshima

middle: Z.

last: Parris

address: Medicinaregatan 1G, 6th floor

department: Department of Oncology

institute: University of Gothenburg

summary: Transcriptomic profiling to investigate the effect of TLX overexpression with an inducible vector system in the HEK 293T cell line

overall_design: Evaluation of common fusion transcripts, genetic variants, and gene expression patterns in TLX-overexpressing HEK 293T cells

first: T

middle: Z

first: D

last: Vizlin-Hodzic

first: S

last: Salmela

first: K

last: Funa


title: Identification of pannexin 1-regulated genes, interactome, and pathways in rhabdomyosarcoma and its tumor inhibitory interaction with AHNAK

city: Ottawa

first: Xiao

last: Xiang

address: 451 Smyth Road

institute: University of Ottawa

summary:
Purpose: Our laboratory has recently reported novel tumor-suppressive roles of PANX1 in both embryonal (eRMS) and alveolar (aRMS) rhabdomyosarcoma, but the underlying molecular mechanism remains to be elucidated.
Methods: PANX1-induced differential gene expression profile in alveolar rhabdomyosarcoma (RMS) cell line, Rh30, was generated by deep sequencing using Illumina HiSeq2000. The sequence reads that passed quality filters were analyzed by BOWTIE/TOPHAT pipeline with accessary programs SAMTOOLS (v1.0) and CUTADAPT (v1.7.1) followed by Cufflinks (v2.2.1). The protein expression of the differentially regulated genes was validated by Western blotting.
Results: Using RNA-seq and DAVID analysis, we showed that the Gene Ontology (GO) terms such as apoptosis and migration, and KEGG pathways such as MAPK and Rap1 Signaling pathways were amongst the most highly enriched processes in Rh30 (aRMS) cells with ectopic PANX1 expression. We found that both GJA1 and its encoded protein connexin 43 (Cx43) were significantly increased in PANX1 expressing Rh30 (aRMS) cells.
Conclusion: PANX1 transcriptome and proteome databases hold great potential for discovery of novel PANX1 tumor-suppressive mechanisms and therapeutic targets for RMS.

first: Kyle

last: Cowan


title: FTO-mediated cytoplasmic m6Am demethylation adjusts stem-like properties in colorectal cancer cell

status: Public on Jan 20 2021

city: MONTPELLIER

first: Julie

last: Ripoll

state: OCCITANIE

address: 860 rue de St Priest

department: Informatics

institute: LIRMM

summary: Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications dynamic in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its m6Am (N6,2'-O-dimethyladenosine) demethylase activity. While m6Am is strategically located next to the m7G-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates m6Am level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear m6Am methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of m6Am modification in stem-like properties acquisition. FTO-mediated regulation of m6Am marking constitutes a novel, reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the m6Am modification and its potential adverse consequences for colorectal cancer management.

overall_design: Colorectal cancer cells with depletion of FTO (sh-FTO) and without (sh-CTL) comparison at transcriptome and translatome scales

first: Sébastien

last: Relier

first: Hélène

last: Guillorit

first: Amandine

last: Amalric

first: Cyrinne

last: Achour

first: Florence

last: Boissière

first: Jérôme

last: Vialaret

first: Aurore

last: Attina

first: Françoise

last: Debart

first: Armelle

last: Choquet

last: Macari

first: Virginie

last: Marchand

first: Iouri

last: Motorin

first: Emmanuelle

last: Samalin

first: Jean-Jacques

last: Vasseur

last: Pannequin

first: Francesca

last: Aguilo

first: Evelyne

last: Crapez

first: Christophe

last: Hirtz

last: Rivals

last: Bastide

first: Alexandre

last: David


title: Impact of cyclophosphamide pre-conditioning on solid tumor microenvironemt and impacts on CAR T cell therapy

city: Duarte

first: John

middle: P.

last: Murad

state: California

address: 1500 E. Duarte Rd

institute: City of Hope

summary: Tumor bearing mice treated with or without cyclophosphamide (100mg/kg; i.p.) and mock T cells or CAR T cells. Tumors harvested at early timepoint following therapy and RNA extracted from whole tumors was used for bulk RNA sequencing analysis.

overall_design: Tumor engraftment Day 0 (1x10^6 RM9 ; s.c.). Cyclophosphamideor vehicle (pbs) treatment (100mg/kg ; i.p.) on day 6 post engraftment. Mock or CAR T cell treatment (i.v.) on day 7 post engraftment. Tumor harvest, lysate, and RNA extraction on day 10 post engraftment. RNA submitted for bulk RNA sequencing.

first: Saul

last: Priceman


city: Shanghai

first: Ding

last: Yu

address: Number 953 Jianchuan Road

country: China

institute: Newcore biotechnology

summary:
Purpose:In recent years, increasing evidence has confirmed that exosomal circular RNAs (circRNAs) play a crucial role in the prognostic prediction and diagnosis of liver cancer （LC）. In this study, we first compared the expression patterns of exosomal circRNAs during transarterial chemoembolization (TACE).
Methods:CircRNA-seq identified 390 differentially expressed circRNAs between the prior TACE and TACE1 groups and 489 differentially expressed circRNAs between the prior TACE and TACE2 groups. GO analysis of the differentially expressed circRNAs showed that they were associated with fatty acid metabolism, receptor binding and membrane protein complexes. KEGG pathway analysis predicted that protein digestion and absorption pathways were activated after TACE. And we screened out a novel gene , hsa_circRNA_G004213 (circ_G004213) was significantly upregulated after TACE (fold change >10, P<0.01). Further analysis found circ_G004213 significantly increased the cisplatin sensitivity of HepG2 cells and positively correlated with the prognosis of tumor-bearing mice. Based on the potential downstream miRNAs and mRNAs, the circRNA‐miRNA-mRNA network was constructed.
Results: We demonstrated that circ_G004213 regulates cisplatin resistance via the miR-513b-5p/PRPF39 axis. Finally, our study confirmed that circ_G004213 was positively correlated with the prognosis of LC patients after TACE. Therefore, circ_G004213 may be used as an indicator for predicting the efficacy of TACE.
Conclusions: Hsa_circRNA_G004213 Promotes Cisplatin Sensitivity by Regulating miR-513b-5p/PRPF39 in Liver Cancer.

overall_design: We collected hepatic artery blood from 5 LC patients at three time points: prior to TACE treatment (prior TACE), after the first TACE operation (TACE1) and after the second TACE operation (TACE2). Then, the exosomes were extracted from the blood, and the alterations of the circRNAs in exosomes were further analyzed.

first: Jun

last: Li

first: Lin

last: Qin


title: CRISPR-TRAPSeq identifies the QKI RNA binding protein as important for astrocytic maturation and control of thalamocortical synapses (CLIP-Seq dataset)

city: St Louis

first: Joseph

last: Dougherty

country: USA

summary: We immunoprecipitated QK-6 from P21 mouse brains to identify which mRNAs QK-6 binds to in vivo.

overall_design: P21 mouse brains were flash frozen, powderized and UV crosslinked prior to homogenization and QK-6 IP, or IgG control IP. mRNA bound to QK-6 was extracted after size selection on SDS-PAGE. Sequencing libraries were made from these samples as well as total crosslinked mRNA from homogenized brain (Input).  After RNA-Seq, reads were aligned to mouse genome.  Using QK-6 IP samples, candidate locations of binding (peaks) were discovered using the Piranha peak calling algorithm in a gene agnostic fashion.  These candidate peaks were then tested for enrichment by statistically comparing read depth of QK-6 IP vs. control (Igg IP and Input)  at each peak using EdgeR.  Final data file was annotated with gene coordinate information for those peaks overlaping genes based on mm2.

first: Kristina

last: Sakers


title: CRISPR-TRAPSeq identifies the QKI RNA binding protein as important for astrocytic maturation and control of thalamocortical synapses (TRAP-Seq dataset)

summary: Quaking RNA binding protein(QKI) is essential for oligodendrocyte development as myelination requires MBP mRNA regulation and localization to distal processes by its cytoplasmic isoforms(e.g. QKI-6). QKI-6 is also highly expressed in astrocytes, which we and others recently demonstrated have regulated mRNA localization. Here, we show via CLIPseq that QKI-6 binds 3’UTRs of a subset of astrocytic mRNAs, including many enriched in peripheral processes. Binding is enriched near stop codons, which is mediated partially by QKI binding motifs(QBMs) yet spreads to adjacent sequences. We developed CRISPR TRAPseq: a viral approach for mosaic, cell-type specific gene mutation with simultaneous translational profiling. This enabled study of QKI-6 CLIP targets in QKI-deleted astrocytes in an otherwise normal brain. Astrocyte-targeted QKI deletion altered translation and maturation, while also increasing synaptic density within the astrocyte's territory. Overall, our data indicate QKI is required for astrocyte maturation and demonstrate an approach for a highly targeted translational assessment of gene knockout in specific cell-types in vivo.

overall_design: Six Cas9 positive and six Cas9 negative samples from mouse cortex


title: Myofibroblast-specific 5-HT2B Mediates Adverse Remodeling Following Myocardial Infarction

city: Nashville

middle: Caleb

last: Snider

state: TN

address: 9445 MRB4, 2213 Garland Ave

department: Biomedical Engineering

institute: Vanderbilt University

summary: We report that ablation of the serotonin 2B receptor in periostin-expressing cells improves cardiac structure and function following myocardial infarction. We isolated PDGFRa expressing cells during the fibrotic healing phase after injury, and show that these cells have alterations in the expression of genes controlling cell cycle progression as well as a few markers of cell adhesion and migration.

overall_design: 2 Htr2bfl/fl controls and 4 Htr2bfl/flPostnMCM/+ experimental animals. All mice were given tamoxifen to activate Cre. All injuries were conducted at the same time, and tissue was harvested 7 days after injury.

first: J

middle: C

first: Lance

last: Riley

first: W

last: Merryman


title: Short telomeres trigger epithelial to mesenchymal transition programmes and sensitize to folic acid-induced kidney fibrosis in mice  [folic acid]

first: Osvaldo

last: Graña

summary: Gene expression profiles of Tert+/+ and generation 3 (G3) Tert-/- male an female mice were  untretaed or tretaed  witth Folic acid.

overall_design: 6-8 weeks of Tert+/+ and generation 3 (G3) Tert-/- male and female mice were  administered a single intraperitoneal injection of FA with low dose of 125 mg/kg body weight or 0.3 M NaHCO3 (200ul).  Mice were sacrificed at day 14 and the kidneys were ice cold PBS perfused and harvested.

first: Sarita

last: Saraswati

last: Graña-Castro

first: Paula

last: Martínez

first: María

last: Blasco