title: Cytotoxic Granzyme C Expressing ILC1s Contribute to Antitumor Immunity and Neonatal Autoimmunity

first: Ming

last: Li

address: 200, East 89th Street

department: Immunology Program

institute: Sloan Kettering Institute

summary: Innate lymphocytes are integral components of the cellular immune system that coordinates host defense against a multitude of challenges and can trigger immunopathology when dysregulated. Natural killer (NK) cells and innate lymphoid cells (ILCs) are innate immune effectors postulated to functionally mirror conventional cytotoxic T lymphocytes and helper T cells, respectively. Here, we show that the cytolytic molecule granzyme C was surprisingly expressed in cells with the phenotype of type 1 ILCs (ILC1s) in mouse liver and salivary gland. Cell fate-mapping and transfer studies revealed that granzyme C-expressing innate lymphocytes could be derived from ILC progenitors and did not interconvert with NK cells, ILC2s, or ILC3s. Granzyme C defined a maturation state of ILC1s, which required the transcription factor T-bet and to a lesser extent Eomes specifically in the salivary gland for their maintenance. Furthermore, transforming growth factor-b (TGF-b) signaling promoted maintenance of granzyme C-expressing ILC1s in the salivary gland and in the tumor of a transgenic breast cancer model, and their depletion caused accelerated tumor growth. ILC1s gained granzyme C expression following interleukin-15 (IL-15) stimulation, which enabled perforin-mediated cytotoxicity. Strikingly, constitutive activation of the IL-15-regulated transcription factor Stat5 in granzyme C-fate-mapped ILC1s triggered lethal perforin-dependent autoimmunity in neonatal mice. Thus, granzyme C marks a cytotoxic effector state of ILC1s, broadening their function beyond ‘helper-like’ lymphocytes.

overall_design: ATAC-seq and bulk RNA-seq data sets analyzed in Nixon, et al. 2022. Murine cell populations profiled by ATAC-seq include two populations with an NK cell phenotype (NK1.1+NKp46+CD49a-CD49b+) from the liver and spleen, and two populations with an ILC1 phenotype (NK1.1+NKp46+CD49a+) from the liver and salivary gland (Figures 1 and S1, Tables S1 and S2). Murine cell populations profiled by bulk RNA-seq from the murine liver include S1pr5eGFP+ NK cells as well as three different ILC1 populations: ILC1s with current granzyme C expression (GzmC+GzmcFM+, double-positive [DP]), past but not current granzyme C expression (GzmC-GzmcFM+, fate mapped single-positive [FMSP]), and no history of expression (GzmC-GzmcFM-, double-negative [DN]) (Figures 6, S9, and S10; Tables S3 and S4). Four DP, four FMSP, three DN, and four NK cell samples were processed from a total of six mice.

first: Briana

last: Nixon

middle: O

first: Chirag

last: Krishna

first: Christina

last: Leslie

first: Andrew

middle: E

last: Cornish


title: Expression data from MCF7 cells treated with hits from screening cytotoxic michael acceptors

status: Public on Feb 17 2022

city: Linköping

first: Johannes

middle: Artiaga

last: Gubat

address: Ing. 68 Campus US. Linköpings Universitet

department: Biomedical and Clinical Sciences

country: Sweden

institute: Linköping university

summary:
We used a chemical library to screen for cytotoxic compounds with Michael acceptor groups that induce a cellular response characteristic of proteasome inhibition. We then determined the differentially expressed genes in MCF7 cells following treatment with each of the identified hits
This dataset includes expression data from MCF7 cells treated with either DMSO or one of the treatment compounds

overall_design: A total of 36 samples were analyzed. There were 12 treatment conditions (11 treatment compounds + 1 DMSO/Control) and 3 replicates. All treatments were done for 6 hours at 37C. For the respective treatments: Samples 113_1-3 were treated with CB113 10µM; Samples 383_1&2 were treated with CB383 2.5µM; Samples 686_1&2 were treated with CB686 10µM; Samples 729_1-3 were treated with CB729 10µM; Samples 742_1-3 were treated with CB742 5µM; Samples 826_1-3 were treated with CB826 2.5µM; Samples 916_1-3 were treated with CB916 5µM; Samples 997_1-3 were treated with CB997 10µM; Samples 360_1-3 were treated with CB360 10µM; Samples bAP15_1-3 were treated with b-AP15 1µM; Samples 688_1-3 were treated with CB688 2.5µM; samples control_1-3 were exposed to 0.5% DMSO. Pairwise comparisons of the treatment compounds and the control were done to obtain the differentially expressed genes. 2 outliers were excluded from the analysis.


title: Pulmonary Neuroendocrine Cells Regulate Gas Exchange Through Controlling Lung Fluid Balance

status: Public on Feb 18 2022

city: San Diego

first: Xin

last: Sun

state: CA

address: 9500 Gilman Dr., CMME, Room 1055

department: Department of Pediatrics, Department of Biological Sciences

country: USA

institute: UNIVERSITY OF CALIFORNIA AT SAN DIEGO

type: Expression profiling by high throughput sequencing

summary: Limiting fluid in lung is critical for efficient gas exchange. Here we discovered a mechanism of neuropeptidergic control of lung fluid balance by pulmonary neuroendocrine cells (PNECs), potent sensors of chemical and mechanical cues. We studied the first animal model of neuroendocrine cell hyperplasia of infancy (NEHI), which faithfully recapitulated patient phenotypes including PNEC hyperplasia and impaired gas exchange. Double mutants showed that increased PNECs and excess PNEC products such as CGRP are responsible for poor gas exchange, acting through downregulating endothelial junctions, increasing vessel leakage and fluid accumulation. Endothelium-specific inactivation of CGRP receptor, or treatment with CGRP receptor antagonist reduced fluid and improved gas exchange. In lungs with acute respiratory distress syndrome (ARDS), including those caused by COVID-19, there was a striking increase of CGRP-expressing PNECs. These findings raise the possibility that increased neuropeptides would contribute to excess extravascular lung fluid and antagonizing their function may improve gas exchange.

overall_design: Single-cell suspensions were prepared from 3 whole mouse lungs (gender mixed) for each indicated genotype. Live epithelial and endothelial cells were FACS-sorted and re-proportionated with 2:1 ratio before loading. Single-cell barcoded droplets were produced using 10X Single Cell 3’ v3 chemistry following user guide provided by 10X Genomics. Libraries generated were sequenced using Illumina Novaseq instrument. Reads were aligned and gene level unique molecular identifier (UMI) counts were obtained using the Cell Ranger pipeline. Cell clustering and gene expression profile in different cell type and conditions are performed by Seurat (version 3.0) package.

first: Le

last: Xu

first: Jinhao


title: A herpesvirus microRNA targets host microRNA processing to promote virus reactivation from latency [dRNA-seq]

city: Würzburg

first: Florian

last: Erhard

address: Versbacher Str. 7

department: Institut für Virologie und Immunbiologie

country: Germany

institute: Julius-Maximilians-Universität Würzburg

summary: In a major study, we found that miR-aU14, a potential miRNA expressed by human herpesvirus 6A (HHV-6A), acts to selectively inhibit the processing of members of the human miR-30 family through direct RNA:RNA interaction and causes changes in mitochondrial arcchitecture through p53-Drp1 axis. In order to characterize miR-aU14, we carried out differentia lRNA-seq (dRNA-seq) from HHV-6A lytic infected HSB2 cells.

overall_design: HSB-2 cells were obtained from HHV-6 Foundation, USA and were maintained in RPMI 1640 media with 200 units of Penicilin and Streptomycin. HSB2 cells were infected with HHV-6A at an MOI of 5-10. 3 days post infection, total RNA was prepared using TRI-reagent (SIGMA). Two biological replicates were prepared on two different days. Transcription start site profiling dataset using dRNA-seq was prepared according to the published protocol (Sharma and Vogel, 2014) with some modifications by the Core Unit Systems Medicine (Würzburg). In brief, total RNA was extracted from HHV-6A and mock infected HSB-2 cells.

first: Bhupesh

middle: K

last: Prusty

first: Lars

last: Dölken


title: A herpesvirus microRNA targets host microRNA processing to promote virus reactivation from latency [Lytic infection]

type: Non-coding RNA profiling by high throughput sequencing

summary: In a major study, we found that miR-aU14, a potential miRNA expressed by human herpesvirus 6A (HHV-6A), acts to selectively inhibit the processing of members of the human miR-30 family through direct RNA:RNA interaction and causes changes in mitochondrial arcchitecture through p53-Drp1 axis. In order to characterize miR-aU14 during virus reactivation, we carried out small RNA-seq from U2-OS cells carrying latent HHV-6A. Virus reactivation was carried out using 80 ng/ml of Trichostatin-A (TSA).

overall_design: HSB-2 cells were infected with HHV-6A.  To study Argonaut bound miRNAs, Ago2 immunoprecipitation was carried out.

first: Archana

middle: B


title: Preparation of chimeric recombinant SARS-CoV-2

status: Public on Feb 16 2022

city: Minato-ku

first: Kei

last: Sato

state: Tokyo

address: 4-6-1 Shiroganedai

department: Department of Microbiology and Immunology

country: Japan

institute: The Institute of Medical Science, The University of Tokyo

summary: To investigate the virological properties of a SARS-CoV-2 variant, Omicron BA.2, we generated chimeric recombinant viruses that express GFP and encodes the S gene of B.1.1 (ancestral D614G-bearing virus), Delta, BA.1 and BA.2. To verify the genome sequence of the working viruses, we performed viral RNA-sequencing of the viral stock.

sample_organism: Severe acute respiratory syndrome coronavirus 2

overall_design:
We prepared the seed recombinant viruses by CPER technique. We then amplified the recombinant viruses using VeroE6/TMPRSS2 cells. At 3 days postinfection, the culture medium was harvested and centrifuged, and the supernatant was collected as the working viruses. The genome sequences of the working viruses were verified using a viral RNA-sequencing analysis. Viral RNA was extracted using QIAamp viral RNA mini kit (Qiagen, Cat# 52906). The sequencing library for total RNA-sequencing was prepared using NEB Next Ultra RNA Library Prep Kit for Illumina (New England Biolabs, MA, Cat# E7530). Paired-end, 76-bp sequencing was performed using MiSeq (Illumina, San Diego, CA) with MiSeq Reagent Kit v3 (Illumina, Cat# MS-102-3001).

Contributor: The Genotype to Phenotype Japan (G2P-Japan) Consortium

first: Mai

last: Suganami

first: Jumpei

last: Ito


title: Chronic Stress Induces Coordinated Cortical Microcircuit Cell-Type Transcriptomic Changes Consistent With Altered Information Processing

city: Toronto

first: Etienne

last: Sibille

state: Ontario

address: RM,133, 250 college street

department: Neuroscience of Aging and Depression

country: Canada

institute: Center for Addiction and Mental Health

summary: Major depressive disorder (MDD) is a severe psychiatric disorder, characterized by deficits in GABAergic and glutamatergic signaling in cortical brain regions. Unpredictable chronic mild stress (UCMS), a murine stress model, induces behavioural and neurobiological changes reminiscent of MDD. Despite increasing knowledge of the cellular complexity of the cortical microcircuitry, our understanding of how distinct cell-types are impacted by UCMS is extremely limited. We thus endeavoured to determine the effect of UCMS on Pyramidal (PYR) cells and three classes of interneurons: Somatostatin (SST), Parvalbumin (PV), and Vasoactive intestinal peptide-expressing (VIP) cells in the medial prefrontal cortex. C57Bl/6 mice, exposed to UCMS or control housing for five weeks, were assessed with a battery of anxiety- and depressive-like behavioural tests. Laser-capture microdissection was used to isolate samples of microcircuit cell-types, which underwent RNA-sequencing. UCMS-exposed mice showed significantly elevated emotionality, and isolate cell-types showed robust specificity. Cell-types exhibited unique transcriptomic profiles after UCMS. Briefly, PYR-cells showed decreased signaling and post-synaptic receptor expression, SST-cells showed increased activation of the unfolded protein response, PV-cells showed increased axonal microtubule expression, and VIP-cells showed increased apoptotic signaling and altered cytoskeletal gene expression. Co-expression analyses revealed that these changes were largely correlated across cell-types and that UCMS increased transcriptome-wide co-expression between PYR and both PV and SST cells. Overall, these findings suggest decreased PYR, SST, and VIP-cell function, elevated PV-cell function, and identify the dysregulation of PYR-cells as a central locus of UCMS-induced transcriptomic change. Further studies interrogating potential antidepressant-like activity of compounds reversing these cell-specific changes are warranted.

overall_design: Examination of gene expression in mice exposed to chronic stress, versus controls, in four neuronal cell-types isolated from the medial prefrontal cortex.

first: Dwight

middle: F

last: Newton

middle: L


title: RNA sequencing of pancreatic islets from WT, Mfn1-/-, Mfn2-/- and Mfn1/2-/- mice

city: Ann Arbor

first: Scott

last: Soleimanpour

state: Michigan

address: 1000 Wall St,

department: Internal Medicine

institute: University Of Michigan

summary: The dynamin-like GTPases Mitofusin 1 and 2 (Mfn1 and Mfn2) are essential for mitochondrial function, which has been principally attributed to their regulation of fission/fusion dynamics. Here, we report that Mfn1 and 2 are critical for glucose-stimulated insulin secretion (GSIS) primarily through control of mtDNA content. Whereas Mfn1 and Mfn2 individually were dispensable for glucose homeostasis, combined Mfn1/2 deletion in β-cells reduced mtDNA content, induced impaired mitochondrial morphology and networking fragmentation, and impaired decreased respiratory function, ultimately resulting in severe glucose intolerance. Importantly, gene dosage studies unexpectedly revealed that Mfn1/2 control of glucose homeostasis was dependent on maintenance of mtDNA content, rather than mitochondrial structure. Indeed, pharmacologic mitofusin agonists rescued islet mtDNA depletion due to mitofusin deficiency independent of changes on mitochondrial structure. Mfn1/2 maintain mtDNA content by regulating the expression of the crucial mitochondrial transcription factor Tfam, as Tfam overexpression ameliorated the reduction in mtDNA content and GSIS in Mfn1/2-deficient β-cells. Thus, the primary physiologic role of Mfn1 and 2 in β-cells is coupled to preservation of mtDNA content rather than mitochondrial architecture, and Mfn1 and 2 may be promising targets to overcome mitochondrial dysfunction and restore glucose control in diabetes.

overall_design: RNA libraries from WT, Mfn1-/-, Mfn2-/- and Mfn1/2-/- mouse islets were prepared by standard Illumina protocols and sequenced using the NovaSeq-6000 platform (Illumina).

first: Vaibhav

last: Sidarala

first: Jie

last: Zhu

first: Elena

last: Levi-D’Ancona

first: Gemma

last: Pearson

first: Emma

middle: C

last: Reck

first: Emily

middle: M

last: Walker

first: Brett

middle: A

last: Kaufman


title: Bulk RNA-seq on NOD mice prostate tissues treated with etanercept

city: West Lafayette

first: Nadia

middle: Marie

last: Atallah

state: IN

address: 201 S. University St

department: Center for Cancer Research

institute: Purdue University

summary: Immunohistochemistry studies indicated that etanercept treatment reduced inflammation, epithlial proliferation, and epithelial NFkB activity in NOD prostate tissues compared to control treated mice. A bulk RNA-seq study was conducted on whole mouse prostate tissues from NOD mice treated with PBS control or etanercept for 5 weeks to identify putative pathways involved in these processes. These analyses determined differentially expressed genes and numerous enriched pathways yielding a greater understanding of pathways involved in prostate regression and inhibition of inflammation.

overall_design: whole prostate tissues from 4 control or 4 etanercept-treated mice used for RNA isolation, followed by bulk RNA-seq

first: Renee

last: Vickman

first: Simon

last: Hayward

last: Lanman


title: Inhibition of Mitochondrial Biogenesis and Tumor Bioenergetics for Glioma [A172 and U87MG]

city: Durham

first: Gao

last: Zhang

state: NC

address: 203 Research Dr | MSRB1, Rm 179

department: The Preston Robert Tisch Brain Tumor Center

institute: Duke University School of Medicine

summary: The obejctive of this study is to To establish the therapeutic role of Gamitrinib for gliomas in vitro and in vivo and to elucidate the anti-tumor mechanism of Gamitrinib in glioma cells. And the ultimate goal of this proposal is to identify a novel Gamitrinib-based combination therapy for primary and recurrent gliomas.

overall_design: RNAseq of Glioma Cells treated with Gamitrinib or DMSO

first: Delong

last: Yin


title: A bleomycin induced murine model of scleroderma in GSDMD-/- versus wild type

city: Guangzhou

first: Huan

last: Yang

state: Guangdong

address: No. 2 Lujing Road

department: Dermatology Department

country: China

institute: Dermatology Hospital of Southern Medical University

summary: The NLRP3 inflammasome and IL-1β are essential for scleroderma pathogenesis. Nevertheless, the role of pyroptosis executor gasdermin D(GSDMD), which is a downstream molecule of NLRP3 and is required for IL-1β release in some situations, has not yet been well elucidated in scleroderma. The purpose of this study was to explore the differences in the degree of skin fibrosis between GSDMD-/- and wild type mice in a bleomycin-induced scleroderma model, and the molecular pathways that may be involved.Total RNA from skin biopsies from GSDMD-/- and wild type mice after subcutaneous injection of BLM to induce skin fibrosis was examined by nextGen RNA sequencing

overall_design: GSDMD-/- and WT female mice were subcutaneously injected with BLM every other day for a total 28 days. 1x0.5cm biopsies were obtained from the skin lesions. RNA was extracted using TRIzol Reagent and quantified and qualified by Agilent 2100/2200 Bioanalyzer (Agilent  Technologies, Palo Alto, CA, USA). cDNA libraries were prepared according to standard protocol and loaded on Novaseq instrument(Illumina).Sequencing was carried  out using a 2x150 paired-end (PE) configuration.image analysis and base calling were conducted by the NovaSeq Control Software (NCS) + OLB + GAPipeline1.6 (Illumina) on the NovaSeq instrument.

first: Yanqiang

last: Shi

first: Huiting

last: Liu

first: Feiyan

last: Lin

first: Biying

last: Qiu

first: Qinglan

last: Feng

first: Yu

last: Wang

first: Bin


title: Whole-genome profiling of DNA methylation and hydroxymethylation identify distinct regulatory programs among innate lymphocytes

city: St. Louis

first: Vincent

last: Peng

state: MO

address: 660 S. Euclid Ave.

institute: Washington University School of Medicine in St. Louis

type: Methylation profiling by high throughput sequencing

summary: Innate lymphocytes encompass a diverse array of phenotypic identities with specialized effector functions. DNA methylation and hydroxymethylation are essential mechanisms for epigenetic fidelity and fate commitment. The landscapes of these modifications are unknown in innate lymphocytes. Here, we characterized the whole-genome distribution of methyl-CpG and 5-hydroxymethylcytosine (5hmC) in mouse ILC3, ILC2, and NK cells. We identified differentially methylated and hydroxymethylated DNA regions between ILC-NK subsets and correlated them with signature transcriptional modules. We associated lineage-determining transcription factors with demethylation and demonstrated unique patterns of DNA methylation/hydroxymethylation in relationship to open chromatin regions, histone modifications, and transcription factor binding sites. We also discover a novel association between 5hmC and NK cell super-enhancers. Using Tet2-deficient mice, we showed that the DNA hydroxymethylase Tet2 is required for optimal production of hallmark cytokines by ILC3 and production of IL-17A by inflammatory ILC2. These findings provide a powerful resource for studying immune epigenetic regulation and further decode the regulatory logic governing innate lymphocyte identity.

overall_design: Splenic NK cells, intestinal ILC2, and intestinal ILC3 were sorted from mice and MeDIP-seq, MRE-seq, and hmC-seal libraries were generated. Two biological replicates were used for each cell type.

first: Marco

last: Colonna

first: Ting


title: Transcriptome analysis of human colonic organoids infected SARS-CoV-2

city: New York

first: Kyungku

last: Jang

state: NY

address: 430 East 29th Street

department: Microbiology

institute: New York University

summary:
Purpose: To identify the diferentially expressed genes in SARS-CoV-2 susceptible and resistant organoids during the ifnection.
Method: We selected 3 susceptible (C8, C9, and C10)- and 3 restant (C1, C2, and C7)-organoids lines and infected SARS-CoV-2 at multiplicity of infection (MOI) of 4 for 24 and 72 hrs. The RNAs were collected and then sequenced by CEL-seq2. Sequencing was performed on Illumina NovaSeq 6000.
Results: Longitudinal transcriptome analyses identified robust yet late transcriptional changes induced by SARS-CoV-2, the magnitude of which corresponded to the levels of viral infection.

overall_design: 3 SARS-CoV-2 resistant and 3 SARS-CoV-2 susceptible colonic organoids lines; technical duplicates or triplicates; 0 h control, 24 h infection, 72 h control, and 72 h infection

first: Kyung-Ku

first: Simone

last: Dallari

first: Ken

last: Cadwell


title: The effect of two lysines in PML/RARa on mouse haematopoietic stem/progenitor cells

city: Hangzhou

first: Xuejing

last: Shao

address: 866, Yuhangtang Road

department: Zhejiang University

institute: College of Pharmaceutical Sciences

summary: Analysis of mHSPCs infected with PML/RARa(WT) or PML/RARa(2KR: K227R and K360R in RARa moiety). We used RNA-sequencing to detail the global programme of gene expression in PML/RARa(2KR)-overexpressing mHSPCs. Results provide insight into the reduced clonogenicity caused by PML/RARa(2KR)

overall_design: Bone marrow cells were first obtained from the femurs of 5-fluorouracil-treated C57BL/6 mice, and then mHSPCs were selected from bone marrow cells using the Easysep Mouse Hematopoietic Progenitor Cell Isolation Kit (Stemcell Technologies). mHSPCs were cultured in a six-well plate and exposed to concentrated lentivirus (pMSCV, PML/RARa(WT) or PML/RARa(2KR)) in the presence of 6 µg/ml of polybrene. Infection was obtained by centrifugation of the cells in viral supernatant at 1,800 rpm for 40 min. The cells were cultured for 24 h at 37 °C. The following day, the medium was removed and replaced with fresh culture medium. Transduced mHSPCs were sorted and collected for RNA extraction and RNA-sequencng.

first: Meidan

last: Ying


title: RNA-Seq transcriptome profiling of human iPS cell-derived cardiomyocytes (hiPSC-CMs) following overexpression (OE) of estrogen-related receptor gamma (ERRg)

city: Philadelphia

first: Tomoya

last: Sakamoto

state: PA

address: Smilow Center for Translational Research 11th FL (Room 11-172) 3400 Civic Center Blvd Bldg 421

department: Cardiovascular Institute

institute: University of Pennsylvania

summary: Transcriptional regulatory circuits that drive cardiomyocyte maturation are poorly understood. Human iPS cell-derived cardiomyocytes (hiPSC-CMs) have been shown to have fetal cardiomyocyte features in terms of metabolic gene expression profiles. Here we found that in hiPSC-CMs, overexpression of estrogen-related receptor gamma (ERRg) is sufficient to drive cardiomyocyte metabolic maturation programs including the induction of a number of oxidative mitochondrial metabolic genes.

overall_design: Examination of how gene expression levels are changed by OE ERRg compared to OE GFP in hiPSC-CMs in triplicate.

first: Kirill

last: Batmanov

first: Daniel

middle: P

last: Kelly


title: Whole genome bisulfite sequencing of WT and Dnmt3a KO HSCs following chronic infection

city: Houston

first: Katherine

middle: Yudeh

last: King

state: Texas

address: 1102 Bates Ave., Ste.1150

department: Pediatrics and Infectious diseases

institute: Baylor College of Medicine

summary:
Since DNMT3A is a de novo DNA methyltransferase, transcriptional differences in WT versus Dnmt3a-/- HSCs upon infection suggest a possible role for epigenetic regulation in the HSC transcriptional response to inflammation. Therefore, we wanted to investigate if differences in stress and presence of Dnmt3a affect the methylome overall, and specific IFNg responses genes.
100000 LT-HSCs (LK CD150+ CD48- were sorted into lysis buffer from the pools of naive or 1-month (M. avium) infected WT/ Dnmt3-/-_x0001_ mice (n = 7-8 per group). Around 300 ng of DNA per group was extracted with AllPrep DNA/RNA Mini Kit. NEBNext Ultra II DNA library prep kit was used for library preparation. Bisulfite conversion step was added after ‘‘methylated’’ adaptor ligation and USER excision. Then, methylated adaptor ligated DNA fragment was treated with bisulfite conversion using EZ DNA methylationlightning kit. These modified DNA fragments were then amplified with index primers with Kapa HiFi urasil+ specialized polymerase for bisulfite converted DNA. For WGBS library sequencing, 150-high output kit from illumine was used. Samples (400 million reads/ sample (15-20X coverage)) were run in NovaSeq S1 FC (2, lanes 1,600 Million reads).
Global methylation of Dnmt3a-/- cells was more divergent upon infection compared to WT. There were vastly more hyper- and hypomethylated regions in infected versus naive Dnmt3a-/- HSCs compared to WT. Among the altered regions, both WT and Dnmt3a-/- HSCs showed slightly more hypomethylated than hypermethylated regions, although changes in both directions were present. In the WT background, regions of hyper- and hypomethylation were mostly restricted to CpG islands. By contrast, highly significant differences in hypomethylation were found in the Dnmt3a-/- HSCs in CpG islands, shores, enhancers, and promoters
DNMT3A is highly active in HSCs during infection and that the loss of DNMT3A function results in significant global changes in methylation, including both hyper- and hypomethylation, in genome regions that likely affect gene transcription. we examined the methylation of Batf2, Jun, and Fos. All 3 of these prodifferentiation genes showed increased methylation in the promoter region in the Dnmt3a-/- background as compared to the WT background. Hypermethylation of these promoter regions is consistent with their transcriptional repression.

overall_design: Comparison of the methylome between HSCs that are either WT or Dnmt3a KO exposed to 1 month M. avium infection

first: Roman

last: Jaksik

first: Bailee

last: Kain

first: Dan

last: Hormaechea-Agulla

first: Duy

middle: T

last: Le

middle: Y


title: Inhibition of Mitochondrial Biogenesis and Tumor Bioenergetics for Glioma

first: Gao

last: Zhang

first: Delong

last: Yin


title: The Nuclear Receptor ERR  Cooperates with the Cardiogenic Factor GATA4 to Orchestrate Transcriptional Control of Cardiac Maturation

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Assay for transposase-accessible chromatin sequencing (ATAC-seq) on estrogen-related receptor alpha and gamma (ERRa/g) knock out (KO) human iPS cell-derived cardiomyocytes (hiPSC-CMs)

summary: To determine the role of estrogen-related receptor alpha and gamma (ERRa/g) on chromatin accessibility, we performed ATAC-seq studies with WT control and ERRa/g KO hiPSC-CMs. We found that ERRa/g activates cardiac chromatin accessibilities in hiPSC-CMs. A subset of the decreased ATAC signals by loss of ERRa/g overlapped ERRg peaks (GSE113760) in hiPSC-CMs, suggesting that ERRg might be directly involved in the activation of cardiac chromatin accessibilities. The motif analysis with decreased ATAC signal by loss of ERRa/g revealed that cardiac-enriched transcription factors such as MEF2 and GATA could cooperate with ERR.

overall_design: Examination of chromatin accessibility with ATAC-seq in wild type control or ERRa/g KO hiPSC-CMs from two different lines with CRISPR/Cas9


title: RNA-Seq transcriptome profiling of estrogen-related receptor alpha and gamma (ERRa/g) knock out (KO) human iPS cell-derived cardiomyocytes (hiPSC-CMs)

summary: Transcriptional regulatory circuits that drive cardiomyocyte maturation are poorly understood. Here we found that in ERRa/g KO hiPSC-CMs, cardiac energy metabolic and cardiac structural transcriptional programs are dysregulated.

overall_design: Examination of gene expression levels in wild type control or ERRa/g KO hiPSC-CMs from two different lines with CRISPR/Cas9


title: Dynamic transcriptional activity and chromatin remodeling of regulatory T cells after varied duration of IL-2R signaling

city: Miami

first: Thomas

last: Malek

state: Florida

address: 1600 N.W. 10th Avenue

institute: University of Miami Miller School of Medicine

summary: Regulatory T cells (Tregs) are necessary for the maintenance of immune self-tolerance and homeostasis. They have a heightened sensitivity to IL-2, which may create a therapeutic window to promote immune regulation by their selective stimulation. While IL-2 responsive genes are well characterized, yet remain unknown genetic, chromatin and metabolic programs during sustained IL-2R signaling. Longitudinal transcriptional and chromatin accessibility profiling and metabolic studies were performed on peripheral Tregs in response to long-lived mIL-2/CD25 fusion protein. We found an initially increased STAT5 dependent gene modulation enabled by enhanced chromatin accessibility, whereas a STAT5-independent genome-wide chromatin closing to inhibit transcription and cell signaling occurred later. Interestingly, IL-2-dependent genes were induced upon re-stimulation. Mitochondrial metabolism was reprogrammed to fulfill the bioenergetics demand for the burst of proliferation. We propose that a shift from euchromatin to heterochromatin represents a mechanism to restrain, but not abrogate, persistent IL-2R signaling to maintain Treg homeostasis.

overall_design: Mouse spleen Tregs DNA accessibility of mIL-2, mIL-2/CD25 (FP) or control PBS injected mice were generated 0.75 hr post injection by deep sequencing in triplicate using NextSeq 500 with a High Output Kit 150-cycle flow cell (Illumina). Reads from ATAC-seq were mapped to the Mus musculus genome GRCm38 using bwa-mem and MACS was used to identify the peak regions. The Irreproducible Discovery Rate (IDR) method was used to identify reproducible peaks between two pseudo-replicates that were generated for each sample. Only reproducible peaks (IDR > 0.1) were retained for downstream analyses.  Differential expressed genes in Tregs between the mIL-2 or mIL-2/CD25 and PBS injected mice were identified using DESeq2, and determined according to threshold of p <0.05

first: Alejandro

last: Moro

first: Zhen

last: Gao

first: Lily

last: Wang

first: Aixin

last: Yu

first: Yuguang

last: Ban

first: Aimin

last: Yan

first: Corneliu

last: Sologon

first: Xi

last: Chen

middle: R


title: Genome-wide acetylation of histone 3 at lysine residuce (H3K27ac) deposition in human iPS cell-derived cardiomyocytes (hiPSC-CMs)

first: Tomoya

last: Sakamoto

summary: To determine the role of estrogen-related receptor gamma (ERRg) in cardiac myocyte enhancers, we performed ChIP-seq studies with hiPSC-CMs using antibody directed against acetylated histone 3 at lysine residue 27 (H3K27ac), a known active enhancer mark. We found that knocking out ERRg downregulated H3K27ac depositions on several adult cardiac contractile protein and mitochondrial oxidative metabolic genes. These results establish significant roles of ERR to activate cardiac enhancer and promoter activities.

overall_design: With ChIP-seq, examination of H3K27ac deposition on the human genome in wild type hiPSC-CMs and ERRg KO hiPSC-CMs.

first: Kirill

last: Batmanov

first: Daniel

last: Kelly


title: Improved patient-derived tumor models in pancreatic ductal adenocarcinoma employing orthotopic implantation

city: Tokyo

first: Masayuki

last: Komatsu

address: 5-1-1, Tsukiji, Chuo-ku

institute: National Cancer Center Research Institute

summary:
Pancreatic ductal adenocarcinoma has a very poor prognosis, and new therapies and preclinical models are urgently needed. We developed patient-derived xenografts (PDXs), established PDX-derived cell lines (PDCLs), and generated cell line-derived xenografts (CDXs), and integrated these to create 13 matched trios, as systematic models for this cancer. Orthotopic implantation (OI) of PDCLs showed tumorigenesis and metastases to the liver and peritoneum. Morphological comparisons of OI-CDX and OI-PDX with passaged tumors showed that histopathological features of the original tumor were maintained in both models. Molecular alterations in PDX tumors (including those to KRAS, TP53, SMAD4, and CDKN2A) were similar to those in the respective PDCLs and CDX tumors. Comparing gene expression in PDCLs, ectopic tumors, and OI tumors, CXCR4 and CXCL12 genes were specifically upregulated in OI tumors, whose immunohistochemical profiles suggested epithelial-mesenchymal transition and adeno-squamous trans-differentiation. These patient-derived tumor models provide useful tools for preclinical research into pancreatic ductal adenocarcinoma.
We performed comprehensive gene expression profiling of 13 pancreatic cancer cell lines, 14 CDX and 14 PDX tumors by Affymetrix Gene Chip HG-U133Plus2.0.

overall_design: Forty one RNA samples from cell lines, CDXs, and PDXs of human pancreactic cancer.

first: Hiroki

last: Sasaki

first: Hazuyoshi

last: Yanagihara


title: Secretion of interleukin-1 by dedifferentiated melanoma cells inhibits JAK1-STAT3-driven actomyosin contractility of lymph node fibroblastic reticular cells

city: Nice

first: Virginie

last: Prod'homme

address: 151, route de Ginestère

country: France

institute: Inserm

summary: Fibroblastic reticular cells (FRC) are immunologically specialized myofibroblasts that control the elasticity of the lymph node (LN), in part through their contractile properties. Swelling of tumor-draining LN is a hallmark of lymphophilic cancers such as cutaneous melanoma. Melanoma displays high intratumoral heterogeneity with the coexistence of melanoma cells with variable differentiation phenotypes, from melanocytic to dedifferentiated states. Factors secreted by melanoma cells promote pre-metastatic LN reprograming and tumor spreading. Elucidating the impact of the melanoma secretome on FRC could help identify approaches to prevent metastasis. Here we show that melanocytic and dedifferentiated melanoma cells differentially impact the FRC contractile phenotype. Factors secreted by dedifferentiated cells, but not by melanocytic cells, strongly inhibited actomyosin-dependent contractile forces of FRC by decreasing the activity of the RHOA-ROCK pathway and the mechano-responsive transcriptional co-activator YAP. Transcriptional profiling and biochemical analyses indicated that actomyosin cytoskeleton relaxation in FRC is driven by inhibition of the JAK1-STAT3 pathway. This FRC relaxation was associated with increased FRC proliferation and activation and with elevated tumor invasion in vitro. The secretome of dedifferentiated melanoma cells also modulated the biomechanical properties of distant LN in pre-metastatic mouse models. Lastly, interleukin-1 produced by dedifferentiated cells was involved in the inhibition of FRC contractility. These data highlight the role of the JAK1-STAT3 and YAP pathways in spontaneous contractility of resting FRC. They also suggest that dedifferentiated melanoma cells specifically target FRC biomechanical properties to favor tumor spreading in the pre-metastatic LN niche. Targeting this remote communication could be an effective strategy to prevent metastatic spread of the disease.

overall_design: Gene expression was measured in human lymph node fibroblasts exposed for 48 hours to conditioned medium prepared from the 1205Lu human melanoma cell line, or maintained in control culture conditions. Four independent experiments were performed.

first: Sophie

last: Tartare-Deckert


title: Lack of Nhe6 associated with increased endothelial permeability

status: Public on Feb 15 2022

city: Basel

last: Bodmer

address: Hebelstrasse 20

department: Department of Biomedicine

country: Switzerland

institute: University Hospital Basel

summary: Our earlier study showed that hearing loss in Nhe6 KO animals was due to cochlear damage and that NHE6 is important for normal hearing function. These results were based on the data extracted from experiments involving hearing/sensory structures in organ of Corti. Considering the importance of blood-labyrinth barrier (BLB) in transport of substances between the blood and intrastrial space, and the fact that the physiology and overall properties of the BLB are not completely elucidated so far, we have isolated and subcultured endothelial cells from mouse BLB and by RNAseq we want to compare overall gene expression between Nhe6 KO and WT mice in order to discover further differences between the two phenotypes.

overall_design: Examining differences between the Nhe6 KO and WT mice in the overall gene expression from primary endothelial cells isolated from stria vascularis capillaries.

first: Marijana

last: Sekulic

first: Vesna

last: Petkovic


title: Protease dysregulation during response and resistance to immune checkpoint blockade therapy (RNA-seq)

city: Atlanta

first: Gabriel

last: Kwong

state: Georgia

address: 345 Ferst Dr NW

department: Biomedical Engineering

institute: Georgia Institute of Technology

summary: Immune checkpoint blockade (ICB) therapy does not benefit the majority of treated patients, and those who respond to the therapy can become resistant to it. Here we report the design and performance of systemically administered activity sensors conjugated to anti-programmed cell death protein 1 (αPD1) antibodies for the monitoring of antitumour responses to ICB therapy. RNA sequencing analysis revealed that differential expression of tumour and immune proteases underpins ICB response and resistance. We then designed a library of mass-barcoded sensors that, when cleaved by proteases, are released into urine, where they can be detected by mass spectrometry. By using syngeneic mouse models of colorectal cancer, we show that random-forest classification trained on mass-spectrometry signatures can be used to detect early antitumour responses and to discriminate resistance to ICB therapy driven by loss-of-function mutations in either the B2m or Jak1 genes. Our data supports the use of activity-based biomarkers that leverage protease dysregulation for response assessment and classification of refractory tumours.

overall_design: 6- to 8-week-old female C57BL/6 mice were subcutaneously inoculated with MC38 tumour cells, including wild type cells and cells harbouring loss-of-function mutations in B2m or Jak1. Mice underwent 3 doses of immune checkpoint therapy with aPD1 antibody or IgG1 isotype control, after which mRNA was isolated from tumors (30 samples, 5 per group) for RNA seq.

first: Quoc

middle: D

last: Mac

first: Anirudh

last: Sivakumar

first: Congmin

last: Xu

first: Peng

last: Qiu


title: NANOG prion-like assembly mediates DNA bridging

city: HOUSTON

first: chuangye

last: Qi

address: One plaza

department: Huffington Center

summary: NANOG is a key stem cell pluripotency factor. NANOG’s unique ability to form prion-like assemblies provides a cooperative and concerted DNA bridging mechanism essential for chromatin reorganization and dose-sensitive activation of ground state pluripotency. We take advantage of Hi-C and ChIP-seq experiments to directly address whether NANOG can bridge DNA elements together in human cells, and our results support this.

overall_design: NANOG function in DNA bridging in human cells.

first: Josephine

last: Ferreon

first: Wenbo

last: Li


title: Dynamic transcriptional activity and chromatin remodeling of regulatory T cells after varied duration of IL-2R signaling [RNA-seq]

first: Thomas

last: Malek

overall_design: Splenic murine Tregs mRNA profiles of mIL-2, mIL-2/CD25 (FP) or control PBS injected mice were generated 1.5 hr post injection by deep sequencing in quadruplicate using NextSeq 500 with a High Output Kit 150-cycle flow cell (Illumina). Reads from RNA-seq were mapped to the Mus musculus genome GRCm38 using STAR (ver.2.5.0) aligner. Raw counts were generated on Ensembl gene (GENCODE M13) with featureCounts (ver.1.5.0). Differential expressed genes in Tregs between the mIL-2 or mIL-2/CD25 and PBS injected mice were identified using DESeq2, and determined according to threshold of false discovery rate (FDR) <0.01

first: Alejandro

last: Moro

first: Zhen

last: Gao

first: Lily

last: Wang

first: Aixin

last: Yu

first: Yuguang

last: Ban

first: Aimin

last: Yan

first: Corneliu

last: Sologon

first: Xi

last: Chen


title: Microarray of Fab072+ and Fab072- epithelial cells from P18T-derived xenografts

city: Barcelona

first: Adria

last: Caballe

address: C/ Baldiri Reixac 10

department: biostatistics

country: Spain

institute: IRB

summary: Lgr5+ and Lgr5- cells from the epithelial tumor compartment of P18T-derived xenografts were analyzed for their transcriptomic expression profile.

overall_design: P18T organoids were injected subcutaneously into Swiss-nude mice. When tumors were grown above 100mm3, animals were sacrified and xenografts were dissagregated and stained for Epcam+ and Fab072 (Lgr5). Within the alive Epcam+ compartment, 1000 cells of the 10% most positive Fab072 cells vs. Fab072- were FACS sorted in picoprofile lysis buffer. Two replicas were collected from two different xenografts. RNA was extracted and amplified with the picoprofile technique and retrotranscrived to cDNA. samples were hybridized to Affimetrix Microarray Chips.

first: Carme

last: Cortina


title: RNA sequencing of human CRC PDOs treated with Cetuximab versus MCLA-158

first: Camille

last: Stephan Otto Attolini

state: Select a State or Province

address: Baldiri Reixac 10

department: Biostatistics and bioinformatics Unit

institute: IRB Barcelona

summary: We profiled 2 PDOs (P18T and C55T) in control medium, Cetuximab or MCLA-158 treatment during 48 hours at 2 concentrations

overall_design: 2 PDOs, 3 antibody treatments, 2 concentrations, 2 replicated

first: Cañellas

last: Adrià

first: Batlle

last: Eduard


title: Single-cell RNA sequencing data for 1-month human spinal cord organoids derived from human pluripotent stem cells

city: SEOUL

first: Ju-Hyun

last: Lee

address: 73 Inchon-ro

country: South Korea

institute: Korea University College of Medicine

summary: We performed single-cell RNA seqeucing to examine whether human spinal cord organoids reflect the characteristics of spinal cord tissue.

overall_design: Multiple organoids pooled together were used for single-cell RNA sequencing.

first: Woong

last: Sun

first: Subin

last: Cho


title: Topical estrogen application promotes cutaneous wound healing in db/db female mice with type 2 diabetes

city: Kanazawa

first: Shin-ichi

last: Horike

state: Ishikawa

address: 13-1 Takaramachi

department: Research Center for Experimental Modeling of Human Disease

institute: Kanazawa University

summary: Female sex hormones are beneficial effects for wound healing. However, till date, whether topical estrogen application can promote cutaneous wound healing in diabetes remains unclear. Therefore, the present study aimed to validate the effect of topical estrogen application on cutaneous wound healing in a type 2 diabetes db/db mice model. In total, 22 db/db female mice with type 2 diabetes and eight C57BL/6J female mice were subjected to two full-thickness wound injuries. The mice were divided into the db/db, db/db + estrogen, db/db + vehicle, and wild type (WT) groups. Wound healing was assessed until day 14. The db/db group had a significantly high wound area ratio (wound area/initial wound area) on days 3–14 and a significantly low re-epithelialization ratio on days 7 and 14. Moreover, their angiogenesis ratio was significantly low on day 7 and high on day 14. In contrast, compared with the db/db group, the db/db + estrogen group had a significantly lower wound area ratio on days 1–14 and angiogenesis ratio on day 14, thereby indicating early withdrawal of new blood vessels, as well as a significantly higher re-epithelialization ratio on days 7 and 14 and Ym1+ M2 macrophage/macrophage ratio on day 7. Moreover, microarray analysis showed that the top 10 upregulated or downregulated genes in the db/db group were reversed by estrogen treatment, particularly on day 14, in comparison with the WT group. Thus, topical estrogen application reduced the wound area, promoted re-epithelialization and angiogenesis, and increased the number of M2 macrophages in mice with type 2 diabetes. Furthermore, it improved the differential regulation of genes in db/db mice. Therefore, such treatment can enhance cutaneous wound healing in female mice with type 2 diabetes.

overall_design: Comparison in eight groups: diabetic group (db/db), a topical estrogen wound treatment diabetic group (db/db+E), vehicle wound treatment diabetic group (db/db+vehicle) and wild-type group (WT) on Day7 and Day14 after wound, respectively.There are two biological replicates for each conditions.

first: Kanae

last: Mukai

first: Makiko

last: Meguro-Horike

first: Yukari

last: Nakajima

first: Arya

last: Iswara

first: Toshio

last: Nakatani


title: Coordinate regulation of ribosomal protein gene transcription mediates embryonic secretory cell growth

city: Duluth

first: Matthew

last: Slattery

state: MN

address: 1035 University Drive, SMed 255

department: Biomedical Sciences

institute: University of Minnesota Medical School

summary: Cell growth is well defined for the late (post-embryonic) stages of development, but evidence for early (embryonic) cell growth during post-mitotic morphogenesis is quite limited. Here, we identify early cell growth as a key characteristic of Drosophila embryonic salivary gland (SG) tubulogenesis. A BTB/POZ domain nuclear factor, Ribbon (Rib), mediates early cell growth without affecting endoreplication. Rib binds the transcription start site of nearly every SG-expressed ribosomal protein gene (RPG), and is required for full expression of all RPGs tested. Rib binding to RPG enhancers is, however, weak and not sequence-specific, suggesting that specificity is achieved through co-factor interactions. We demonstrate Rib’s ability to physically interact with each of the three known activators of RPG transcription. Surprisingly, Rib-dependent early cell growth in another tubular organ—the embryonic trachea—is not mediated by direct RPG transcription. Our results support a model for tissue-specific transcriptional regulation in early cell growth programs.

overall_design: ChIP-seq of Drosophila melanogaster embryos

first: Rajprasad

last: Loganathan

first: Daniel

last: Levings

first: Deborah

middle: J

last: Andrew


title: CmLHP1 proteins play a key role in plant development and maintenance of monoecy in Cucumis melo (RNA-Seq)

city: Paris

first: Jing

last: An

address: 630 Rue Noetzlin

institute: Universite Paris Saclay

summary:
Purpose: We conducted a reverse genetic approach in order to elucidate the role of H3K27me3 writers and erasers in the process of sex determination and expression in melon.
Methods: We used TILLING to find loss-of function mutants of cmLHP1 proteins in melon. Selected alleles were phenotyped at the developmental and molecular levels.  We generated the cmlhp1ab mutant, for which we observed a pleiotropic phenotype.  For this double mutant, we integrated RNA-seq (leaves), ChIP-seq (leaves) and RT-PCR (flowers) for elucidating the impact of cmLHP1 loss of function at the molecular level.
Results: cmlhp1ab displayed a pleiotropic phenotype which correlated with genome-wide changes of H3K27me3 (moore oftenly hypomethylation) and deregulation of genes involved in flower development and hormone responses.

overall_design: 3 biological replicates of RNA-seq from leaves of WT and Cmlhp1ab double mutants.

first: Natalia

last: Rodriguez

first: Abdel

last: Bendahmane

first: Moussa

last: Benhamed

first: Sanchari

last: Sincar

first: Rim

last: Brik-Chauche


title: CmLHP1 proteins play a key role in plant development and maintenance of monoecy in Cucumis melo (ChIP-Seq)

overall_design:
2 biological replicates of H3K27me3 ChIP-seq from leaves of WT and Cmlhp1ab double mutants. One Input library was produced for each genotype

1 biological replicate of Melon PolII ChIP-seq from leaves


title: CmLHP1 proteins play a key role in plant development and maintenance of monoecy in Cucumis melo

overall_design: Refer to individual Series


title: A herpesvirus microRNA targets host microRNA processing to promote virus reactivation from latency [Latent reactivation]

city: Würzburg

first: Florian

last: Erhard

address: Versbacher Str. 7

department: Institut für Virologie und Immunbiologie

institute: Julius-Maximilians-Universität Würzburg

summary: In a major study, we found that miR-aU14, a potential miRNA expressed by human herpesvirus 6A (HHV-6A), acts to selectively inhibit the processing of members of the human miR-30 family through direct RNA:RNA interaction and causes changes in mitochondrial arcchitecture through p53-Drp1 axis. In order to characterize miR-aU14 during virus reactivation, we carried out small RNA-seq from U2-OS cells carrying latent HHV-6A. Virus reactivation was carried out using 80 ng/ml of Trichostatin-A (TSA).

overall_design: U2-OS cells were purchased from ATCC and were cultured in McCoy5A medium supplemented with 10% (v/v) FBS and 200 units/ml penicillin-streptomycin. U2-OS cells carrying latent HHV-6A were reactivated by treating the cells with 80 ng/ml of TSA for 48 h.

first: Bhupesh

last: Prusty

first: Lars

last: Dölken


title: Transcriptional profilng of CCNE1 overexpressing hTERT-immortalized p53-R175H fallopian tube cell lines (FT282)

city: Toronto

first: David

middle: WILLIAM

last: Gallo

state: ON

address: 600 University Ave

institute: Lunenfeld-Tanenbaum research instituite

summary: In this study we used RNA-seq to profile transcriptional changes in two clones of FT282-hTERT p53-R175H cells engineered to overexpress CCNE1. We compared the differential gene expression (DGE) of CCNE1 OE to their wild type counterparts and identified that CCNE1 OE increases expression of genes involved in G2/M progression. Subsequent gene set enrichment analysis (GSEA) revealed significant enrichment of genes co-regulated by MYBL2 and FOXM1 transcription factors.

overall_design: mRNA profiles (3x replicates) of FT282-hTERT p53-R175H WT and CCNE1 OE clone 3 and 4

last: Durocher


title: RNA LEVER mediates long-range regulation of ε-globin by keeping PRC2 in check

city: Singapore

first: Chan-Shuo

last: Wu

address: 14 Medical Drive, #12-01

department: Cancer Science Institute

country: Singapore

institute: National University of Singapore

summary: EZH2, a core component of polycomb repressive complex 2 (PRC2), is a histone H3 methyltransferase required for gene silencing during embryonic development. The regulatory function of promiscuous binding of RNA to EZH2 is not known, although evidences suggest that binding of RNA inactivates EZH2 in vitro. Here we present a seesaw mechanism for the regulation of ε-globin through inactivating EZH2 by an upstream RNA (LEVER) in K562 cells. We show that LEVER, identified by RNA immune precipitation sequencing (RIP-seq) and NANOPORE sequencing, binds EZH2 and thereby preventing the accumulation of H3K27 methylation along the axis where LEVER emerges. The opened chromatin within LEVER in turn competes for the DNA looping between the locus control region (LCR) and the promoter of ε-globin. Hence, LEVER downregulates ε-globin by inhibiting EZH2.

overall_design: EZH2 associated RNA in wild type K562 cells is profiled by RIP-seq. Subcellular distribution of EZH2 associated RNA is characterized through profiling K562 cytoplasmic, nuclear, nuclear poly A+ or nuclear polyA- RNA by RNA-seq. LEVER RNA structure is determined by NANOPORE sequencing on K562 nuclear RNA or total polyA+ RNA. LEVER is either knockout or knockdown in K562 cells by CRISPR-Cas9 or -dCas9 strategy. H3K27 histones marks in K562 NT-gRNA (control) or LEVER gRNA KD cells are profiled by ChIP-seq. Intra-chromatin interaction at ε-globin promoter or locus control region (LCR) in NT-gRNA (control) or LEVER KO cells is analysed through 4C-seq.

first: Wei

middle: W

last: Teo

first: Xinang

last: Cao

first: Chan

middle: S

first: Hong

last: Tan

first: Qiling

last: Zhou

first: Omer

first: Henry

last: Yang

middle: G

last: Tenen


title: Identification of human tumor-unique signaling networks between antigen-presenting cells and T cells using multi-omic single cell analysis

city: Seattle

first: Martin

last: Prlic

state: WA

address: 1100 Fairview Ave, Mail stop E5-110

institute: Fred Hutch

summary: Tissue residence and prolonged exposure to inflammation profoundly affect lymphocyte and myeloid cell function. Immune cells derived solid tumors are thought to undergo additional adaptation, and the identification of such tumor-driven unique immune cell alterations might allow for more efficient and precise tumor therapies. We used a multi-omic single-cell analysis approach to define the immune landscape of human oral squamous cell carcinomas (OSCC) and inflamed non-malignant oral tissues to identify tumor-unique immune cell interactions.   The immune infiltrate in non-malignant, inflamed tissues showed substantial phenotypic congruence with immune phenotypes that are typically tumor-associated.  Yet, computational and machine learning analyses of single-cell data allowed identification of putative tumor-unique subsets and interactions of regulatory T cells (Tregs) and antigen-presenting cells (APCs). Subsequent experimental validation confirmed tumor-unique chemokine and cytokine signaling axes between APCs and Tregs. We identified ICOS and IL-1R1 as uniquely co-expressed biomarkers on tumor Tregs that allow for specific therapeutic targeting.

overall_design: Immune cells were isolated from inflamed oral mucosa, oral squamous cell carcinomas, or peripheral blood. These cells were then process for transcriptional profiling using both targeted and whole transcriptome approaches (Rhapsody and 10x, respectivly)

first: Jami

last: Erickson

last: Mair


title: Transcriptomics of small RNAs upon HHV-6A reactivation

summary: In order to understand the effect of HHV-6A reactivation on host cell, U2-OS bone osteosarcoma cells were generated carrying latent HHV-6A genome. These cells were either treated with DMSO (solvent control) or Trichostatin-A (TSA) for viral reactivation. As a control cells carrying no HHV-6A were used and treated similarly. Two biological replicates of each sample were processed for small RNA transcriptomics (small RNAseq). Furthermore, HeLa (cervical epithelial cells) were generated carrying lentiviral insertions of one of the small non-coding RNA from HHV-6A (sncRNA-U14). These cells could be transiently induced for sncRNA-U14 transcription using Doxycycline. HeLa cells having a mock lentiviral backbone was used as a control and was treated similarly. Two biological replicates of each sample were processed for small RNA transcriptomics (small RNAseq).

overall_design: Small RNAseq was performed in 48 hr treated cells. Biological replicates were generated on a completely separate day using a different batch of cells.

middle: K


title: RNA-sequencing of bone marrow and splenic megakaryocytes during homeostasis and sepsis

status: Public on Feb 14 2022

city: San Francisco

first: Colin

last: Valet

state: California

address: 513 Parnassus avenue

institute: UCSF

summary: Platelets have a wide range of functions including critical roles in hemostasis, thrombosis, and immunity. We hypothesized that during acute inflammation, such as in life-threatening sepsis, there are fundamental changes in the sites of platelet production and phenotypes of resultant platelets. Here, we showed during sepsis that the spleen is a major site of megakaryopoiesis and platelet production. Sepsis provoked an adrenergic-dependent mobilization of megakaryocyte-erythrocyte progenitors (MEPs) from the bone marrow to the spleen where interleukin-3 (IL-3) induced their differentiation into megakaryocytes. In the spleen, immune-skewed megakaryocytes produced a CD40L-high platelet population with potent immunomodulatory functions. Transfusions of post-sepsis platelets enriched from splenic production enhanced immune responses and reduced overall mortality in sepsis-challenged animals. These findings identify a spleen-derived protective platelet population that may be broadly immunomodulatory in acute inflammatory states such as sepsis.

overall_design: Bulk RNA sequencing of Bone marrow and Spleen Megakaryocytes from C57BL/6 mice 5 days after sepsis or not.

first: Mark

last: Looney

first: Michael

last: Adkisson

first: Andrea

last: Barczak

first: Andrew

last: Schroeder


title: Intron retention analysis using isogrowth profiling

city: Köln

last: Lukačišin

state: Deutschland

address: Zülpicher Straße 47a

department: Institute for Biological Physics

institute: Universität zu Köln

summary: We present an analysis of intron retention under stress from two different drugs and their combinations in yeast Saccharomyces cerevisiae. We previously established isogrowth profiling, a method to abstract the non-specific effects of growth rate inhibition from the specific effect of perturbation by a small molecule: two drugs are used at varied ratios, but at fixed overall growth inhibition. Here, cycloheximide and LiCl were used at seven different ratios along the 50% growth inhibition isobole and the total ribodepleted RNA was sequenced. This allowed us to gauge the changes in intron retention due to the used drugs, while ensuring that the effects are not caused by growth inhibition. We found a prominent increase in intron retention under LiCl treatment that preferentially affects introns contained in the transcripts of ribosomal proteins.

overall_design: Saccharomyces cerevisiae strain BY4741 was grown in 7 conditions of varying ratios of LiCl and cycloheximide, ensuring 50% growth inhibition, and in YPD containing no drug. Before sequencing, the extracted RNA was ribodepleted rather than polyA-enriched.

first: Tobias

last: Bollenbach


title: Reactive human iPSC astrocytes suppress oligodendrocyte precursor cell differentiation

city: Baltimore

first: Matthew

last: Smith

state: MD

address: 600 N Wolfe Street

department: Neurology

institute: Johns Hopkins University

summary: Astrocytes are instrumental in both maintaining CNS homeostasis and responding to tissue injury. While astrocyte activation may be a beneficial response to acute pathologies, prolonged reactive gliosis is thought to be injurious in neurodegenerative diseases, including multiple sclerosis (MS). A major limitation of studying neurodegenerative diseases is lack of human pathological specimens obtained during the acute stages, thereby relegating research to post mortem specimens often obtained years after the initiation of pathology. Rodent reactive astrocytes have been shown to be cytotoxic to neurons and oligodendrocytes but may differ from human cells, especially in diseases with known genetic susceptibility. Herein, we purified human CD49f+ astrocytes from differentiated induced pluripotent stem cells derived from individual patient and control peripheral white blood cell samples. We compared TNF and IL1a stimulated human reactive astrocytes from 7 persons with MS and 6 non-MS controls and show their specific astrocyte transcriptomic profiles are remarkably similar to those described in rodents. The functional effect of astrocyte conditioned media (ACM) was examined in a human oligodendrocyte precursor cell (OPC) line differentiation assay using a transgenic secreted reporter of myelin basic protein (MBP) expression. ACM was not cytotoxic to the OPCs but robustly inhibited the MBP reporter. No differences were seen between MS and control stimulated astrocytes at either the transcript level or in functional OPC suppression assays. We next used RNAseq to interrogate differentially expressed genes in the OPC lines that had suppressed differentiation from the human ACM. Remarkably, not only was OPC differentiation and myelin gene expression suppressed, but we observed induction of several immune pathways and Nf-κB signaling in OPCs exposed to the ACM. These data support the notion that reactive astrocytes can inhibit OPC differentiation thereby limiting their remyelination capacity, and that OPCs take on an immune profile in the context of inflammatory cues.

overall_design: hiPSC Astrocytes: 7 Persons with MS, 6 Non-MS Controls, 2 conditions per person (Stimulated and Unstimulated), 26 samples. hOPC Reporters: 2 media only controls, 4 astrocyte conditioned media from PwMS, 4 astrocyte conditioned media from NMSC, 2 conditions per person (Stimulated and Unstimulated), 20 samples

first: Xitiz

last: Chamling

first: Hector

last: Martinez

first: Weifeng

last: Li

first: Donald

last: Zack

first: Valentina

last: Fossati

first: Peter

last: Calabresi


title: A herpesvirus microRNA targets host microRNA processing to promote virus reactivation from latency

first: Florian

last: Erhard

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


title: scRNAseq of established brain metastasis

city: Madrid

first: Osvaldo

last: Graña

state: Comunidad de Madrid

address: C/ Melchor Fernández Almagro, 3

department: Structural Biology

institute: CNIO

overall_design: H2030-BrM cell line (lung adenocarcinoma, in vivo selected to increase its brain tropism) was injected intracardilly in three mice and when metastases were established as assesed by bioluminescence brains were microdissected to obtain GFP+ metastases. Metastases from the three mice were pooled and cell sorted for GFP. A pure GFP+ population was loaded onto a 10x Chromium Single Cell controller chip B (10x Genomics). The intention with the experiment is to evaluate intra-metastasis heterogeneity.

first: Neibla

last: Priego

first: Lauritz

last: Miarka

last: Graña-Castro

first: Manuel

last: Valiente


title: CUT&Tag2for1: a modified method for simultaneous profiling of the accessible and silenced regulome in single cells

first: Jorja

last: Henikoff

address: 1100 Fairview AV N, A1-162

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

summary: Cleavage Under Targets & Tagmentation (CUT&Tag) is an antibody-directed transposase-tethering chromatin profiling strategy for small samples and single cells. Previously, we showed that activation of tethered Tn5 transposase under low-salt conditions using antibodies that target promoters and enhancers produces high-resolution genome-wide chromatin accessibility maps. Here we show that low-salt CUT&Tag using a mixture of an antibody to the initiation form of RNA Polymerase II (Pol2 Serine-5 phosphate) and an antibody to repressive Polycomb domains (H3K27me3) followed by computational signal deconvolution produces efficient high-resolution maps of both the active and repressive regulomes. We have extended this CUT&Tag2for1 method to single cells using a novel deconvolution approach, thus producing high-quality multifactorial single-cell chromatin maps with a workflow that is identical to that for standard single-cell CUT&Tag. The ability to seamlessly map both the active and repressive regulatory elements in single cells provides a complete regulome profiling strategy suitable for high-throughput single-cell platforms.

overall_design: We used Cleavage under targets and Tagmentation (Cut-and-Tag), a chromatin profiling strategy in which antibody-targeted controlled integration of DNA sequencing adapters produces libraries for paired-end DNA sequencing.

first: Derek

middle: H

last: Janssens

first: Dominik

last: Otto

last: Meers

first: Manu

last: Setty

first: Kami

last: Ahmad

first: Steven


title: ETV2 regulates PARP-1 binding protein to induce ER stress-mediated cell death in tuberin-deficient cells

city: Boston

first: Boston University

last: Microarray and Sequencing Resource

state: MA

address: 72 East Concord Street, E631

department: Microarray and Sequencing Resource

institute: Boston University

summary:
Syk inhibitor demonstrates antiproliferative effect on Tsc2-deficient cells and LAM lung nodules similar to rapamycin. A feedback loop between Syk inhibition and mTORC1 inhibition pathways is also present in these Tsc2-deficient cells.
We used microarrays to discern the potential difference in Syk inhibition and mTORC1-inhibition pathways. The goal was to investigate regulatory pathways or targets of Syk inhibition to induce cytocidal response in Tsc2-deficient cells, independent of its crosstalk with mTORC1 signaling.

overall_design: Tsc2-deficient ELT3V cells were plated in 10 cm culture dish per condition in DMEM culture media supplemented with 10% FBS. Twenty-four hours after plating cells were starved overnight (16 hours). The cells were then treated with DMSO, Syk-inhibitor or rapamycin for 24 hours. Cells were harvested by trypsinization for total RNA isolation and microarray processing.

first: Shikshya

last: Shrestha

first: Souheil

last: El-Chemaly


title: Extrinsic KRAS signaling shapes the pancreatic microenvironment through fibroblast reprogramming

city: Ann Arbor

first: Marina

last: Pasca di Magliano

state: MI

address: 1500 E Medical Center Dr

institute: University of Michigan

summary: We utilized the inducible and reversible iKras* genetically engineered mouse model to intiate oncogenic Kras (Kras*) expression in the pancreatic epithelium by providing mice with doxycycline water, and then switched mice to regular water thereby turning expression of Kras* off for 3 days, 3 weeks post-induction of acute pancreatitis. Single cell RNA sequencing revealed that fibroblasts are reprogrammed by signals originating from epithelial Kras*-expressing cells to shape the immune microenvironment during pancreatic tumorigenesis.

overall_design: This 10X Genomics single cell RNA sequencing repository includes raw and processed data files from iKras* mice expressing oncogenic Kras (Kras*) for 3 weeks following induction of acute pancreatitis, and then without Kras* expression for an additional 3 days prior to harvest (n=3 mice pooled prior to submission). Single cell cDNA libraries were prepared and sequenced at the University of Michigan Advanced Genomics Core using the 10x Genomics platform. The sample was run using paired-end 50 cycle reads on HiSeq 400 (Illumina) to a depth of 100,000 reads. Alignment was performed by the University of Michigan Advanced Genomics Core. Parameters include Cell Ranger version 3.0.0 (default settings), Single Cell 3' v3 chemistry, mm10 transcriptome, initial expected cell count of 10,000.

first: Ashley

last: Velez-Delgado

first: Katelyn

last: Donahue


title: A strategy to personalize the use of radiation in patients with brain metastasis based on S100A9-mediated resistance

state: MADRID

address: Calle de Melchor Fernández Almagro, 3

institute: Centro Nacional de Investigaciones Oncológicas

summary: We report a strategy to personalize the use of radiotherapy on brain metastasis from lung cancer and breast cancer compatible with liquid biopsy and the use of a radiosensitizer.

overall_design: We perform 4 types of in vitro cultures using the lung adenoicarcinoma cell line H2030-BrM. These four culture conditions include regular adherent cultures (Adh), oncospheres (Onco), co-cultures between the cancer cell line and the glia using inserts (Insert) (the glia in the upper chamber; cancer cells in ower chamber) or mixed in the plate (Cocult). An additional condition included the administration of irradiation (10Gy) to all these four conditions. Irradiated and non-irradiated cultures (every culture in duplicate) were processed for RNAseq (Co-cultures plated in the same well underwent a sorting process based on the expression of GFP by cancer cells) 24 hours from irradiation.

first: Cátia

last: Monteiro

first: Coral

last: Fustero-Torre


title: Transcriptome profiling of DSS-induced colonic and anal inflammation and injury in mice

city: Chicago

first: Cambrian

middle: Yangshao

last: Liu

state: IL

address: 5841 S Maryland Ave

department: Department of Medicine

institute: The University of Chicago

summary:
Oral administration of dextran sulfate sodium (DSS) to mice induces ulceration and inflammation in the colons of mice. These changes are somewhat similar to those seen in human inflammatory bowel disease.
Here we provide a full transcriptome (RNA-Seq) analysis of different gastrointestinal tissues in the regions affected by the DSS chemical, at different timepoints during the injury-inflammation-recovery cycle. The profiled regions include the distal colon (rectum), where the histological changes are most pronounced, the mid-colon, the anus, which is relatively resistant to damage, and the squamous neo-epithelium of the colon, a skin-like tissue that is found in the colon and is believed to be associated with wound healing.
We find that administration of DSS is associated with upregulation of cytokines and inflammatory markers. Moreover, stem cell markers of the homeostatic colonic epithelium are depleted, consistent with reprogramming of epithelia to adopt a wound-healing phenotype. Thus, this dataset faithfully recovers broad changes in gene expression in a mouse model of inflammatory bowel disease-like damage.

overall_design:
Wildtype C57Bl/6 mice were exposed to the DSS colitis model for 6 d, beginning on experimental day (exp d) 0. They were dissected and their anorectal junctions analyzed via single-cell RNA-Seq at experimental day (exp d) 0 (unchallenged mice), exp d 7 (highly colitic mice), and exp d 42 (recovered mice).
On either exp d 0 (uninjured controls), 3, 6, 9, 14, or 20, mice were dissected and their distal gastrointestinal tracts isolated.
The tissue was divided into mid-colonic ("prox"), rectal ("dist"), anal ("anal"), and squamous neo-epithelial ("meta") sections for transcriptome profiling using RNA-Seq. In each tissue section, the muscle layer was dissected out and discarded prior to RNA isolation.

first: D

middle: B

last: Polk


title: Single cell characterization of the cellular landscape of acral melanoma identifies novel targets for immunotherapy

city: Tampa

first: Keiran

last: Smalley

state: FL

address: 12902 Magnolia Dr

institute: Moffitt Cancer Center

summary: Acral melanoma is a rare subtype of melanoma that arises on the non-hair-bearing skin of the palms, soles and nail beds. In this study, we used single cell RNA-seq (scRNA-seq) to map the transcriptional landscape of acral melanoma and identify novel immunotherapeutic targets.

overall_design: Single cell rnaseq of immune microenvironment of acral melanoma

first: Jiannong

last: Li

first: Inna

first: Zhihua

last: Chen

first: Jheng-Yu

last: Wu

first: Manali

last: Phadke

first: Yian

middle: A


title: Single-cell RNA-Seq profiling of the mouse anorectal junction before, during, and after acute colitis

summary:
We have identified a squamous epithelium that repairs the distal colon of mice after injury and colitis. Lineage tracing studies demonstrate that this squamous epithelium is derived from a small zone of transitional cells located at the squamocolumnar junction of the rectum and anus.
To characterize the molecular properties of this junctional epithelium in homeostasis, colitis, and regeneration, we profiled the distal colon and anus of mice using single-cell RNA-Seq.
We found that junctional cells in this transition zone express high levels of Krt7 and, at baseline, co-express markers of both colonic epithelium and squamous epithelium. These cells thus represent a specialized reserve epithelial population capable of striking plasticity in their generation of a hybrid tissue type to mediate colonic repair.

overall_design: Wildtype C57Bl/6 mice were exposed to the DSS colitis model for 6 d. They were dissected and their anorectal junctions analyzed via single-cell RNA-Seq at experimental day (exp d) 0 (unchallenged mice), exp d 7 (highly colitic mice), and exp d 42 (recovered mice).


title: Integrating lncRNAs and mRNAs expression profiles in penicillin-induced persistent chlamydial infection in HeLa cells

city: Guangzhou

first: Xiaobao

last: Huang

state: Guangdong

address: No. 58 Zhongshan Second Road, Guangzhou, Guangdong, China

department: Department of dermatology

institute: The First Affiliated Hospital, Sun Yat-sen University

summary: Chlamydia trachomatis (C. trachomatis) is a major etiological agent of sexually transmitted infection. Some stressing conditions can result in persistent chlamydial infection, which is thought to associate with severe complications such as ectopic pregnancy and tubal factor infertility. Long noncoding RNAs (lncRNAs) have been identified as key modulators in many biological processes. However, the role of lncRNAs in persistent chlamydial infection is still unclear. In this study, we used lncRNA and mRNA microarray to identify the global lncRNAs and mRNAs expression in penicillin-induced persistent chlamydial infection in HeLa cells as well as the control group (HeLa cells without C. trachomatis infection). Among 1005 differentially expressed lncRNAs, 585 lncRNAs were upregulated and 420 downregulated in persistent chlamydial infection, while 410 mRNAs were identified to express differentially, of which 113 mRNAs were upregulated and 297 downregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with differentially expressed genes were performed. We then constructed the lncRNA-miRNA-mRNA competing endogenous RNAs (ceRNAs) network. Four mRNAs were validated to be changed by quantitative real-time PCR which were correlated with the microarray result. Integration of protein-protein interaction (PPI) network was constructed and hub genes were identified. These findings provide a new perspective on the molecular mechanism of penicillin-induced persistent chlamydial infection.

overall_design: Group: INF(infected), CTL(control), with three samples in each group

first: Qian

last: Liufu

first: Rui

last: Xu

first: Xiaohong

first: Mingna

first: Jiande

last: Han

first: Hongyu

last: Guan

first: Chunguang

last: Ma


title: Silk scaffolding drives self-assembly of functional and mature human brain organoids [scRNA-seq]

status: Public on Feb 12 2022

city: Basel

first: Edoardo

last: Sozzi

address: Klingelbergstrasse 48

institute: ETH Zurich

summary: Human pluripotent stem cells (hPSCs) are intrinsically able to self-organize into cerebral organoids that mimic features of developing human brain tissue. These three-dimensional (3D) structures provide a unique opportunity to generate cytoarchitecture and cell-cell interactions reminiscent of human brain complexity in a dish. However, current in vitro brain organoid methodologies often result in intra-organoid variability, limiting their use in recapitulating later developmental stages as well as in disease modeling and drug discovery. In addition, cell stress and hypoxia resulting from long-term culture lead to incomplete maturation and cell death within the inner core. Here, we used a recombinant silk microfiber network as a scaffold to drive human PSCs to self-arrange into engineered cerebral organoids. Silk scaffolding promoted neuroectoderm formation and reduced heterogeneity of cellular organization within individual organoids. Bulk and single cell transcriptomics confirmed that silk cerebral organoids display more homogeneous and functionally mature neuronal properties than organoids grown in the absence of silk fibers. Furthermore, oxygen sensing analysis showed that silk scaffolds create more favorable growth and differentiation conditions by facilitating the delivery of oxygen and nutrients. Silk-engineering platform appears to reduce intra-organoid variability and enhances functional maturation during spontaneous self-patterning in human brain organoid differentiation.

overall_design:
Single-cell RNA-seq of silk and non-silk organoids

**RAW data not provided due to patient privacy concerns**

first: Petter

last: Storm

first: Alessandro

last: Fiorenzano

first: Malin

last: Parmar


title: The integrated single cell RNAseq analysis of lung cells in Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV) mouse model

status: Public on Feb 13 2022

city: Cincinnati

first: Guolun

last: Wang

state: OH

address: 3333 Burnet Avenue

institute: Cincinnati Children's Hospital Medical Center

summary: we report the application of single cell RNAseq analysis of lung from Foxf1 S52F heterozygous ACDMPV model, and healthy lung from littermate used as control.

overall_design: The mutant sample and control samples were prepared from E18.5 heterozygous mouse embryo. Three embryos were pooled together for both healthy control and mutant sample. The samples were dissociated into small pieces and digested into single cell suspension. The CD45+ cells were depleted by MACS column and CD45- cells used to prepare single cell RNA 3' v3 library for sequencing with 10X Genomics platform.

first: Vladimir

last: Kalinichenko


title: Real-time quantitative PCR analysis of human CD14+ monocytes during the confinement within the Eskiz experiment

status: Public on Feb 11 2022

city: Moscow

first: Anastasia

last: Sadova

state: Russian Federation

address: Khoroshevskoe Shosse 76a

country: Russia

institute: Institute of Biomedical Problems of RAS

summary: Human CD14 positive monocytes were purified from healthy volunteers' blood

overall_design: qPCR gene expression profiling.  Monocytes from six donors were extracted at four different timepoints during the confinement in a sealed chamber within the Eskiz experiment. Equal amount total RNA from each donor was pooled prior to gene expression analysis.

first: Daria

last: Vlasova

first: Nickolay

last: Germanov

first: Veronica

last: Galina

first: Vyacheslav

last: Shmarov

first: Olga

middle: V

last: Kutko

first: Sophia

middle: M

last: Shulgina

first: Evgenia

middle: N

last: Antropova

first: Marina

last: Rykova

first: Ksenia

last: Orlova

first: Sergey

last: Ponomarev


title: Single Cell RNA in RA Synovium

city: Rochester

first: Andrew

last: McDavid

state: NY

address: 265 Crittenden Boulevard, CU 420630

institute: University of Rochester

summary: Ectopic lymphoid structures (ELS) can develop in rheumatoid arthritis (RA) synovial tissue, but the precise pathways of B cell activation and selection are not well understood. Here, we identified a unique B cell population in the synovium characterized by co-expression of a family of orphan nuclear receptors, NR4A1 (also known as NUR77), NR4A2 (NURR1) and NR4A3 (NOR1), that is highly enriched at both early and late stages of RA. See doi:10.1101/2021.05.14.443150 for details.

overall_design: B cells from Synovium (4 donors) were disaggregated by combination of an enzymatic digestion and mechanical disruption.  For one matched donor, B cells were sorted from peripheral blood. B cells were gated as CD45+CD3-CD14-CD19+.

first: Nida

last: Meednu

first: Javier

last: Rangel-Moreno

first: Fan

last: Zhang

first: Katherine

last: Escalera-Rivera

first: Elisa

last: Corsiero

first: Edoardo

last: Prediletto

first: Edward

last: DiCarlo

first: Susan

last: Goodman

first: Laura

middle: T

last: Donlin

first: Soumya

last: Raychauduri

first: Michele

last: Bombardieri

first: Costantino

last: Pitzalis

first: Dana

middle: E

last: Orange

first: Jennifer

last: Anolik


title: TRIM71R595H/R595H mutations and TRIM71-KO in mESC lead to similar transcriptomic changes, which indicate a poised state toward neural differentiation

city: Bonn

first: Joachim

last: Schultze

state: NRW

address: Carl-Troll-Strasse 31

department: Genomics and Immunoregulation

institute: LIMES (Life and Medical Sciences Center Genomics and Immunoregulation)

summary: Mutations in TRIM71’s RNA-binding domain leads to congenital hydrocephalus in human patients and a TRIM71 mutant mouse model (Trim71R595H/+). Additionally, homozygous  Trim71R595H/R595H mice phenocopy the neural tube defects and premature neural differentiation observed in TRIM71-KO mice. As RNA-Seq analyses revealed that TRIM71-KO mESC are poised for neural differentiation, we aimed to also examine the transcriptomes of TRIM71R595H/R595H and TRIM71R595H/+ mESC. Both TRIM71-KO and TRIM71R595H/R595H mutations in mESC result in transcriptomic changes with similar patterns, when compared to control mESC. Similar to TRIM71-KO, the transcriptome of Trim71R595H/R595H mESC indicates a poised state of mutated mESC towards neural differentiation.

overall_design: bulk-mRNA sequencing of Trim71-cond, -KO, and WT, Trim71-R595H/R595H, -R595H/+ mESC

first: Stefan

middle: C

last: Weise

first: Phan

middle: Q

last: Duy

first: Thomas

last: Ulas

first: Kristian

last: Händler

first: Elena

last: De Domenico

first: Matthias

last: Becker

last: Schulze

first: Kristopher

last: Kahle

first: Waldemar

last: Kolanus


title: Melanoma-secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis

city: New York

first: Kelly

last: Ruggles

address: 227 East 30th Street

institute: NYU Grossman School of Medicine

summary: Brain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. Our unbiased proteomics analysis of melanoma short-term cultures revealed that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared to those derived from extracranial metastases. We showed that melanoma cells require amyloid beta (Ab) for growth and survival in the brain parenchyma. Melanoma-secreted Ab activates surrounding astrocytes to a prometastatic, anti-inflammatory phenotype and prevents phagocytosis of melanoma by microglia. Finally, we demonstrate that pharmacological inhibition of Ab decreases brain metastatic burden.

overall_design: Assess how astrocytes (with and without amyloid beta) are impacted by melanoma co-culture

first: Kevin

last: Kleffman

first: Eva

last: Hernando


title: miRNAs associated with the pathogenesis of bovine rotavirus

status: Public on Feb 10 2022

city: Wuhan

first: Gehad

last: Elkady

state: Hubei

address: Huazhong Agricultural University, No.1,Shizishan Street, Hongshan District,Wuhan

department: Virology

institute: HZAU

summary: Bovine Rotavirus (BRV) causes massive economic losses in the livestock industry worldwide. MA-104 cell line has become a convenient tool for discover BRV–host interactions. Notwithstanding, the role of miRNAs in MA-104 cells during BRV infection is still ambiguous. In our study, we performed Illumina RNA sequencing analysis of miRNA libraries of BRV-infected and mock-infected MA-104 cells (at different time points 0 hpi (just after adsorption time (90 min)), 6 hpi, 12 hpi, 24 hpi, 36 hpi and 48 hpi), and the total clean reads 74701041 and 74184124 were obtained from BRV-infected and uninfected cells, respectively. From these findings, 579 categorized as known miRNAs and 144 as novel miRNAs, 160 differentially expressed (DE) miRNAs in BRV-infected in comparison to uninfected MA-104 cells were successfully investigated, 95 were upregulated and 65 exhibited downregulation. The target mRNAs of DE miRNAs were expected by bioinformatics. The functional annotation of the target genes by GO and KEGG suggested that they were mainly contributed to biological pathways, endocytosis, apoptotic process, trans-Golgi membrane and lysosome, besides, the mTOR, NF-κB , Rap 1, cAMP, TNF, Ras, pathways of cancer and MAPK signaling pathways. What is more, real-time quantitative reverse transcription-polymerase chain reaction verified the expression pictures of ten selected DE miRNAs, which were consistent with the results of the deep sequencing (from 0 hpi up to 48 hpi), and 20 from their corresponding mRNA targets mainly of regulatory pathways of the cellular machinery and immune importance. Our study is the first novel approach which uncover the impact of BRV infection on miRNA expression of MA-104 cells, and it offers clues for identifying potential candidates for antiviral or vaccine strategies.

first: Aizhen

last: Guo


title: Transcriptomic Characterization of Pancreatic Cancer-Associated Macrophage Polarization

first: Brian

last: Magnuson

address: 1500 East Medical Center Drive

summary: The pancreatic ductal adenocarcinoma (PDA) microenvironment is composed of a variety of cell types and marked by extensive fibrosis and inflammation. Tumor-associated macrophages (TAM) are abundant, and they are important mediators of disease progression and invasion. TAMs are polarized in situ to a tumor promoting and immunosuppressive phenotype via cytokine signaling and metabolic crosstalk from malignant epithelial cells and other components of the tumor microenvironment (TME). However, the specific distinguishing features and functions of TAMs remain poorly defined. Here, we generated tumor-educated macrophages (TEM) in vitro andanalyzed the transcriptome.

overall_design: Three replicates each of six conditions were subjected to polyA+ RNA-seq.  Bone marrow-derived macrophages (BMDMs) were treated with M-CSF, LPS, IL4, or conditioned media from Kras-Off ("noDOX", 3 days or 5 days) or Kras-On ("plusDOX") pacreatic adenocarcinoma (PDA) cells.

first: Seth

last: Boyer

first: Ho-Joon

last: Lee

first: Christopher

middle: J

last: Halbrook

first: Costas

last: Lyssiotis


title: Enhanced lipogenesis through Pparγ helps cavefish adapt to food scarcity

city: Kansas City

first: Shiyuan

middle: (Cynthia)

last: Chen

state: MO

department: Computational Biology

institute: Stowers Institute for Medical Research

summary: To test wheather cavefish have higher lipogenesis capability than surface fish, we used liver sample from fasted and refed fish to do RNA-seq to compare their transcriptome responding to feeding in surface fish and Pachón cavefish. Moreover, we did the Pparγ ChIP-seq with liver from fed surface fish and Pachón cavefish. We amied to test whether Pparγ in Pachón cavefish have more biding sites and higher binding peaks than surface fish.

overall_design: For RNA-seq study, we 2 feeding regimes(starved and refed) and 2 fish populations (surface and Pachón cavefish). We had 3 replicates for each fish group. For ChIP-seq, we had one input control and two replicates for each fish population (surface and Pachón cavefish).

first: Shaolei

last: Xiong

first: Nicolas

last: Rohner


title: TLR7 gain-of-function genetic variation causes human lupus

city: Canberra

first: Carola

last: Vinuesa

state: ACT

address: 131 Garran Rd, Acton

country: Australia

institute: The John Curtin School of Medical Research

summary: While circumstantial evidence supports enhanced TLR7 signaling as a mechanism of human systemic autoimmune disease, we have lacked the proof afforded by lupus-causing TLR7 gene variants. Here we describe human systemic lupus erythematosus (SLE) caused by TLR7 gain-of-function (GoF). We identified a de novo, novel, missense TLR7 Y264H variant in a child with severe lupus. The de novo TLR7 Y264H variant selectively increased sensing of guanosine and was sufficient to cause lupus when introduced in mice (kika mice). We performed RNA-seq of kika and wild type B cells cultured for 20 hours with anti-IgM and found a decreased tendency to apoptosis in kika B cells, including decreased expression of active Caspase 3 and also a small decrease in proliferation. Overall, these results suggest that hypersensitive TLR7 signaling allows the survival of B cells that are binding self-antigen through their surface BCR.

overall_design: Spleens were obtained from wild type or Tlr7Y264H mice (kika mice). Total B cells were purified and then stimulated with anti-mouse IgM (10 µg/mL) for 20 hours.

first: Grant

last: Brown

first: Hao

last: Wang


title: Reconstruction of intercellular communication and signaling impacting de novo HSC emergence using CellComm platform

first: Edroaldo

last: Lummertz da Rocha

address: 1 Blackfan Circle

department: Hematology/Oncology

institute: Boston Children's Hospital

summary: Cellular transitions are shaped by cross-talk between cell-intrinsic and extrinsic factors. Signals derived from the cell’s microenvironment are integrated and processed by the cell to modulate gene expression programs to instruct cell function and fate. Recent advances in single-cell technologies and analytics have provided an unprecedented resolution to study intercellular communication and differentiation trajectories. Here, we developed CellComm, a systems biology platform to infer intercellular communication networks and transcriptional regulators downstream of cell surface receptors predicted to mediate cellular interactions. We applied CellComm to investigate how the unique cellular niche of the aorta-gonad-mesonephros (AGM) region is involved in hematopoietic stem cell emergence and identified key cell types and microenvironmental signals implicated in hematopoietic development. We extensively validated CellComm’s predictions in three different species - zebrafish, mouse and human - and found both known and novel genes involved in developmental hematopoiesis, such as Stat3, Nr0b2, Ybx1 and, surprisingly, App, which is often reported in neurodegenerative disorders. Strikingly, CellComm revealed extensive cross-talk of signaling pathways feeding into the same transcriptional regulators, indicating a tightly regulated developmental program ensuring dynamic adaptation to changes in the embryonic environment. Taken together, our work contributes to a better understanding on how the embryonic niche is involved in developmental hematopoiesis as well as provides a new algorithm and data resource for the scientific community.

overall_design: Single-cell RNA-sequecing of random sampling of the aorta-gonad-mesonephros region (AGM) complemented with sorted endothelial cells and immunophenotypic T1-preHSCs

middle: L

last: da Rocha


title: Spatial-CUT&Tag: Spatially resolved chromatin modification profiling at the cellular level

city: New Haven

first: Yanxiang

last: Deng

state: CT

address: 55 Prospect Street

department: Biomedical Engineering

institute: Yale University

summary: Spatial omics emerged as a new frontier of biological and biomedical research. Here, we present spatial-CUT&Tag for spatially resolved genome-wide profiling of histone modifications by combining in situ CUT&Tag chemistry, microfluidic deterministic barcoding, and next-generation sequencing. Spatially resolved chromatin states in mouse embryos revealed tissue-type-specific epigenetic regulations in concordance with ENCODE references and provide spatial information at tissue scale. Spatial-CUT&Tag revealed epigenetic control of the cortical layer development and spatial patterning of cell types determined by histone modification in mouse brain. Single-cell epigenomes can be derived in situ by identifying 20-micrometer pixels containing only one nucleus using immunofluorescence imaging. Spatial chromatin modification profiling in tissue may offer new opportunities to study epigenetic regulation, cell function, and fate decision in normal physiology and pathogenesis.

overall_design:
Spatially resolved genome-wide profiling of histone modifications (e.g., H3K27me3, H3K4me3, and H3K27ac) in mouse embryos, olfactory bulbs, and mouse brains.

Please note that the *spatial.tar.gz files (containing images and coordinates needed for downstream analysis) were added on Feb 17, 2022.

first: Rong

last: Fan


title: CDK4/6 inhibition suppresses p73 phosphorylation and activates DR5 to potentiate chemotherapy and immune checkpoint blockade

status: Public on Feb 09 2022

city: Pittsburgh

first: Lin

last: Zhang

state: Pennsylvania

address: 5117 Centre Ave, Hillman Cancer Ctr Rm2.42a, 5117 Centre Ave

department: Pharmacology and Chemical Biology

institute: University of Pittsburgh

summary: Targeting cyclin-dependent kinases 4 and 6 (CDK4/6) is a successful therapeutic approach against breast and other solid tumors. Inhibition of CDK4/6 halts cell cycle progression and promotes antitumor immunity. However, the mechanisms underlying the antitumor activity of CDK4/6 inhibitors are not fully understood. We found that CDK4/6 bind and phosphorylate the p53 family member p73 at threonine 86, which sequesters p73 in the cytoplasm. Inhibition of CDK4/6 led to dephosphorylation and nuclear translocation of p73, which transcriptionally activated death receptor 5 (DR5), a cytokine receptor and key component of the extrinsic apoptotic pathway. p73-mediated induction of DR5 by CDK4/6 inhibitors promoted immunogenic cell death (ICD) of cancer cells. Deletion of DR5 in cancer cells in vitro and in vivo abrogated the potentiating effects of CDK4/6 inhibitors on immune cytokine TNF-related apoptosis-inducing ligand (TRAIL), 5-fluorouracil (5-FU) chemotherapy, and anti-PD-1 immunotherapy. Together, these results reveal a previously unrecognized consequence of CDK4/6 inhibition, which may be critical for potentiating the killing and immunogenic effects on cancer cells.

overall_design: mRNA profiles of HCT116 cells, HCT116 cells treated with palbociclib for 24 hours, HCT116 cells transfected with siRNA against CDK4 for 24 hours.

first: Jingshan

last: Tong

first: Xiao

last: Tan

first: Xiangping

last: Song

first: Man

last: Gao

first: Denise

last: Risnik

first: Suisui

last: Hao

first: Kaylee

last: Ermine

first: Peng

first: Hua

last: Li

first: Yi

last: Huang

first: Jian

last: Yu


title: Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity

city: Memphis

first: Jeremy

middle: Chase

last: Crawford

state: TN

address: MS 221, 262 Danny Thomas Place

department: Department of Host-Microbe Interactions

institute: St. Jude Children's Research Hospital

summary: Infection and vaccination repeatedly expose individuals to antigens that are conserved between influenza virus subtypes. Nevertheless, antibodies recognizing conserved influenza epitopes greatly outnumber antibodies reactive against variable epitopes. Elucidating factors contributing to the paucity of broadly reactive influenza antibodies remains a major obstacle for developing a universal influenza vaccine. Here, we report that inducing broadly reactive influenza antibodies increases autoreactive antibodies in humans and mice and exacerbates disease in four distinct models of autoimmune disease. Importantly, transferring broadly reactive influenza antibodies augments disease in the presence of inflammation or autoimmune susceptibility. Further, broadly reactive influenza antibodies spontaneously arise in mice with defects in B cell tolerance. Together, these data suggest that self-tolerance mechanisms limit the prevalence of broadly reactive influenza antibodies, which can exacerbate disease in the context of additional risk factors.

overall_design:
Autoantigen arrays were performed at Microarray and Immune Phenotyping Core Facility of University of Texas Southwestern Medical Center (UTSW) and carried out as described previously (Li et al., 2007). Briefly, serum samples from HKx31-infected mice, treated with rapamycin or PBS for 14 days, were frozen and sent to UTSW. The sera were pre-treated with DNase I and incubated on autoantigen arrays, and the bound antibodies were detected using a Cy3 labeled anti-mouse IgG (532nm) or Cy5 labeled anti-mouse IgM (635nm) to generate Tiff images.

012993_Slide1: Autoantigen Microarray Super Panel (128 antigen panel)
https://microarray.swmed.edu/products/product/autoantigen-microarray-super-panel-128-antigen-pan/

012995_Slide2: Autoantigen Microarray Panel IV
https://microarray.swmed.edu/products/product/autoantigen-microarray-panel-iv/

first: Maureen

last: McGargill


title: Quantitative Analysis of Transcriptomes of Hamster Liver Treated with or without DC371739

city: Shanghai

first: He

address: 555 Zuchongzhi Rd

institute: Shanghai Institute of Materia Medica

summary:
Purpose: The goal of this study is to reveal the transcriptional dynamics of hamster liver upon DC371739 treatment
Methods: Total RNAs were extracted from the livers of HFD-hamsters control and DC371739-treated (30 mg/kg for 21 days) hamsters. Seven biological replicates were performed for each studied condition. The libraries were sequenced using NovaSeq 6000 (pair-end 150 bp). All reads were mapped to the reference genome of Mesocricetus auratus using HISAT2 v.2.2.1 as instructed. RNA counts were generated from feature Counts.
Results: Ccompared with the vehicle group, the transcription levels of Pcsk9 and Angptl3 of the DC371739 treated group have been decreased by 41.3% (P = 0.016) and 15.7% (P = 0.031), respectively, while the transcription levels of Hnf1α and Ldlr have not shown the statistically significant difference after DC371739 treatment.
Conclusions: We conclude that DC371739 negatively modulates the transcription of two crucial therapeutic targets PCSK9 and ANGPTL3 for hypercholesterolemia treatment.

overall_design: mRNA profiles of hamster liver with or without DC371739 treatment

first: Hong

last: Liu