title: cIAP1/2 antagonism induces antigen-specific T cell dependent immunity

city: Boston

first: Stephanie

last: Dougan

address: 1 Jimmy Fund Way

department: Cancer Immunology & Virology

institute: Dana-Farber Cancer Institute

summary: Checkpoint blockade immunotherapy has failed in pancreatic cancer and other poorly responsive tumor types in part due to inadequate T cell priming. Naïve T cells can receive co-stimulation not only via CD28 but also through TNF superfamily receptors that signal via NF-κB. Antagonists of the ubiquitin ligases cIAP1/2, also called SMAC mimetics, induce degradation of cIAP1/2 proteins, allowing for the accumulation of NIK and constitutive, ligand-independent activation of alternate NF-κB signaling that mimics co-stimulation in T cells. In tumor cells, cIAP1/2 antagonists can increase TNF production and TNF-mediated apoptosis; however, pancreatic cancer cells are resistant to cytokine-mediated apoptosis, even in the presence of cIAP1/2 antagonism. Here, we confirm our previous findings that even poorly immunogenic tumors with a paucity of T cells can experience T cell-dependent anti-tumor immunity, and we provide transcriptional clues into how these rare T cells coordinate downstream immune responses.

overall_design: TCRα-/- mice were implanted orthotopically with M8 live passage organoids and treated with vehicle or LCL-161 (75mg/kg) by oral gavage every 3 days starting on day 4. Tumors and spleens were harvested on day 12 after 3 doses of LCL-161 and single cell suspensions prepared. CD4 and CD8 T cells were isolated by FACS and subjected to limited bulk transcriptional profiling. N=3 per group.

first: Katherine

last: Ventre

first: Kevin

last: Roehle

first: Elisa

last: Bello

first: Aladdin

last: Bhuiyan

first: Tamara

last: Biary

last: Crowley

first: Patrick

middle: T

last: Bruck

first: Max

last: Heckler

last: Lenehan

first: Lestat

middle: R

last: Ali

first: Courtney

last: Stump

first: Victoria

last: Lippert

first: Eleanor

last: Clancy-Thompson

first: Megan

last: Hoffman

first: Li

last: Qiang

first: Marc

last: Pelletier

first: James

last: Akin

first: Michael


title: Expression profiling by bulk RNA sequencing

status: Public on Jan 25 2023

city: Isehara-shi

first: Atsushi

last: Fukuda

country: Japan

institute: Tokai University

type: Expression profiling by high throughput sequencing

summary: We report that the effect of Nanosheet coating on human pluripotent stem cell  by bulk RNA-sequencing.

overall_design: Transcriptome sequencing of 8 total RNA samples from human stem cells.

first: Yosuke

last: Okamura

first: Yuka

last: Sakata

first: Hong

last: Zhang


title: Histone variant H3.3 facilitates open chromatin at promoters [ATAC-seq]

city: Dallas

first: Laura

last: Banaszynski

state: TX

address: 5323 Harry Hines Blvd

department: Green Center for Reproductive Biology Sciences

country: USA

institute: UT Southwestern Medical Center

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We analyze the contribution of H3.3 to chromatin accessibility, post-translational modification states, and transcription in mouse embryonic stem cells (mESC).

overall_design: ATAC-seq analysis in mouse embryonic stem cell lines and embryoid bodies (Control, H33KO).

first: Sara

last: Martire

middle: A


title: MLL3/MLL4 methyltransferase activities control early embryonic development and ESC differentiation in a lineage-selective manner

city: Bethesda

first: Kai

last: Ge

state: MD

address: 50 South Dr Rm 4154

department: NIDDK

institute: NIH

summary: H3K4me1 methyltransferases MLL3 (KMT2C) and MLL4 (KMT2D) are critical for enhancer activation, cell differentiation and development. However, roles of MLL3/4 enzymatic activities and MLL3/4-mediated enhancer H3K4me1 in these processes remain unclear. Here, we report that constitutive elimination of both MLL3 and MLL4 enzymatic activities prevents initiation of gastrulation and leads to early embryonic lethality in mice. However, selective elimination of MLL3/4 enzymatic activities in embryonic, but not extraembryonic, lineages leaves gastrulation largely intact. Consistently, embryonic stem cells (ESCs) lacking MLL3/4 enzymatic activities can differentiate towards the three embryonic germ layers but show aberrant differentiation to extraembryonic endoderm and trophectoderm. The failure in extraembryonic endoderm differentiation can be attributed to markedly reduced enhancer-binding of the lineage-determining transcription factor GATA6. Furthermore, we show that MLL3/4-catalyzed H3K4me1 is largely dispensable for enhancer activation during ESC differentiation. Together, our findings suggest a lineage-selective, but enhancer activation-independent, role of MLL3/4 methyltransferase activities in early embryonic development and ESC differentiation.

overall_design: Expression profiling by RNA-Seq in ESCs (D0), at D4/D10 of EB differentiation and at D8/D16 of neural differentiation, and ChIP-Seq profiling of MLL4 as well as active enhancer marks H3K4me1 and H3K27ac in ESCs (D0), at D4 of EB differentiation and at D8 of neural differentiation, profiling of chromatin accessibility by ATAC-Seq in ESCs (D0) and at D4 of EB differentiation

first: Xie

last: Guojia

first: Lee

last: Ji-Eun

first: Park

last: Young-Kwon

first: Peng

last: Weiqun

first: Ge

last: Kai


title: Histone variant H3.3 facilitates open chromatin at promoters [GRO-seq]

overall_design: GRO-seq analysis in mouse embryonic stem cell lines (Control, H33KO).


title: Linking chromatin acylation mark-defined proteome and genome in living cells

status: Public on Jan 27 2023

city: Beijing

first: Xiong

last: Ji

address: Yiheyuan Road

department: School of life science

country: China

institute: Peking University

summary: A generalizable strategy with programmable site-specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a Single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications, and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and β-hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation, and established linkages for chromatin acylation mark-defined proteome, genome and functions. This led to the identification of GLYR1 as a distinct interacting protein in modulating H3K56cr’s gene body localization as well as the discovery of an elevated super-enhancer repertoire underlying bhb-mediated chromatin modulations. SiTomics offers a platform technology for elucidating the “metabolites-modification-regulation” axis, which is widely applicable for multi-omics profiling and functional dissection of modifications beyond acylations and proteins beyond histones.

overall_design: ChIP-Seq was performed to explore the site-specific histone acylations in the metabolites regulated gene expression

first: Fangfei

last: Qin

first: Boyuan

last: Li


title: Genomic profiling of HIV-1 integration in microglia cells links viral integration to the topologically associated domains

status: Public on Jan 24 2023

city: Cleveland

first: Konstantin

last: Leskov

state: OHIO

address: 2109 Adelbert Rd, Wood W200

department: Molecular Biology and Microbiology

institute: Case Western Reserve University School of Medicine

summary: HIV-1 encounters the hierarchically organized host chromatin to stably integrate and persist in anatomically distinct latent reservoirs. The contribution of genome organization in HIV-1 infection has been largely understudied across different HIV-1 targets. Here we determine HIV-1 integration sites (IS), associate them to chromatin and expression signatures at different genomic scales in a microglia cell model and profile them together with the primary T cell reservoir. HIV-1 insertions into introns of actively transcribed genes with IS hotspots in genic- and super-enhancers, characteristic of blood cells, are maintained in the microglia cell model. Genome organization analysis reveals dynamic CCCTC-binding factor (CTCF) clusters in cells with active and repressed HIV-1 transcription, while CTCF removal impairs viral integration. We identify CTCF-enriched topologically associated domain (TAD) boundaries with signatures of transcriptionally active chromatin as HIV-1 integration determinant in microglia and CD4+ T cells, highlighting the importance of the host genome organization in HIV-1 infection.

overall_design: For single cell RNA-Seq (scRNA-Seq), iPSC-derived microglia cells (~0.5 million cells) were pre-treated with Human Fc block (BD, Catalog # 564219) for 10 min at room temperature, then incubated on ice for 40 min with a cocktail of oligo-indexed antibodies to 41 surface marker proteins. The cocktail included 30 antibodies from the BD ABseq Immune Discovery panel (BD,Catalog # 625970) supplemented with 11 antibodies to microglia-specific antigens as prescribed by the manufacturer (see table below). After a total of three washes with BD Stain buffer (Catalog # 554656), cells were applied to the cartridge of the BD Rhapsody system for single-cells RNAseq/ABseq. After single cell capturing with barcoded beads, we performed reverse transcription and generated Whole Transcriptome and ABseq libraries. Sequencing was performed at Medgenome, Inc. using the Illumina HiSeq X system, PE150.

first: Marina

last: Lusic


title: Single nuclei sequencing to understand mechanism of action of the melanocortin 1 receptor agonist Pl8177 in Dextran sodium sulfate (DSS)-induced colitis in rats

city: Cranbury

first: Paul

last: Kayne

state: New Jersey

address: 4-B Cedar Brook Drive

institute: Palatin Technologies

summary: PL8177 is a potent and selective agonist of the melanocortin 1 receptor (MC1R). PL8177 has shown efficacy in reversing intestinal inflammation in a cannulated rat ulcerative colitis model. To facilitate oral delivery, a novel, polymer-encapsulated formulation of PL8177 was developed and tested in dextran sulfate sodium induced rat ulcerative colitis model. Rats treated with 50 µg oral PL8177 demonstrated significantly lower macroscopic colon damage scores and improvement in colon weight, stool consistency, and fecal occult blood vs the vehicle without active drug. We used single nuclei RNA sequencing of colon tissues  to characterize the mechanism of action and identify relative cell population and key gene expression changes between treated, healthy and vehicle.

overall_design: Nuclei were isolated from 9 flash-frozen rat colon tissue samples (n=3 for each treatment group: PL8177-50ug, Sham and placebo) and were used to construct 3’ single cell gene expression libraries (Next GEM v3.1) using the 10x Genomics Chromium system (10x Genomics, Pleasanton, CA). Libraries were sequenced with ~150 million read pairs (PE150) per sample on an Illumina NovaSeq sequencing system (Illumina, San Diego, CA). After sequencing, gene counts were generated by Cell Ranger v6.0.1 (10x Genomics) using the rat reference genome Rattus norvegicus 6.0.97. Introns were included in the analysis. CellBender was applied to eliminate empty droplets and technical artifacts. Only genes detected in a minimum of 3 cells, cells with at least 200 genes, and cells with <5% of mitochondrial reads were retained

first: Priyanka

last: Dhingra

first: Alison

last: Obr

first: John

last: Dodd

first: Carl

last: Spana


title: Comparative transcriptomic response analysis of Escherichia coli K-12 MG1655 to nine antibiotics

city: Qingdao

first: Mingyu

last: Wang

address: 72 Haibin Rd.

department: State Key Laboratory of Microbial Technology

institute: Shandong University

summary: To investigate and compare transcriptomic changes of Escherichia coli K-12 MG1655, the bacterium was exposed to nine antibiotics (tetracycline, mitomycin C ,imipenem, ceftazidime, kanamycin, ciprofloxacin, polymyxin E, erythromycin, and chloramphenicol) , and RNA-Seq was performed to determine comparative transcriptomic changes.

overall_design: Escherichia coli K-12 MG1655 was grown in MHB medium supplemented with antibiotics at concentrations that inhibit 50% growth (tetracycline 1.8 mg/L, mitomycin C 1.4 mg/L, imipenum 0.06 mg/L, ceftazidime 0.12 mg/L, kanamycin 1.6 mg/L, ciprofloxacin 0.012 mg/L, polymyxin E 0.4 mg/L, chloramphenicol 1.1 mg/L, erythromycin 7 mg/L). Cells were grown to mid-log phase, havested, and subject to RNA-Seq analysis. Controls were performed (ethanol for erythromycin and chrloamphenicol as they are dissolved in ethanol, and water for every other antibiotic). Triplicates were performed.

first: Luyao

last: Bie

first: Mengge

last: Zhang

first: Juan

first: Meng

last: Fang

first: Ling

first: Hai

last: Xu


title: Characterizing of Breast Cancer translational PEARL clinical trial

status: Public on Jan 26 2023

city: Valencia

first: Antonio

last: Fernandez-Serra

address: Beltraan Baguena, 8-11

department: Molecular Biology Lab

country: Spain

institute: Fundacion Instituto Valenciano de Oncologia

summary:
Introduction
In HR+/HER2- metastatic breast cancer (MBC) is imperative to identify patients who respond poorly to CDK4/6i and to discover therapeutic targets to reverse this resistance. Non-luminal breast cancer subtype and high levels of CCNE1 are candidate biomarkers in this setting but further validation is needed.
Methods
We performed mRNA gene expression profiling and correlation with progression-free-survival (PFS) on 455 tumor samples included in the phase III PEARL study, that assigned HR+/HER2- MBC patients to receive palbociclib+ET vs capecitabine. ER+/HER2- breast cancer cell lines were used to generate and characterize resistance to palbociclib+ET.
Results
Non-luminal subtype was more prevalent in metastatic (14%) than in primary tumor samples (4%). Patients with non-luminal tumors had median PFS of 2.4months (m) with palbociclib+ET and 9.3m with capecitabine; HR:4.16, adjusted p-value<0.0001. Tumors with high CCNE1 expression (above median) had also worse median PFS with palbociclib+ET (6.2m) than with capecitabine (9.3m); HR:1.55, adjusted p-value=0.0036. In patients refractory to palbociclib+ET (PFS in the lower quartile) we found higher levels of Polo Like Kinase 1 (PLK1). In an independent data set (PALOMA3), tumors with high PLK1 show worse median PFS than those with low PLK1 expression under palbociclib+ET treatment. In ER+/HER2- cell line models we show that PLK1 inhibition reverses resistance to palbociclib+ET.
Conclusion
We confirm the association of non-luminal subtype and CCNE1 with resistance to CDK4/6i+ET in HR+ MBC. High levels of PLK1 mRNA identify patients with poor response to palbociclib, suggesting PLK1 could also play a role in the setting of resistance to CDK4/6i.

overall_design: Breast Cancer samples were included

first: Miguel

last: Martín


title: Hedgehog signaling affects platelet-derived growth factor signaling mediated alveolar septum formation during postnatal lung development [scRNA-seq]

city: New York City

first: Alireza

last: Khodadadi-Jamayran

state: NY

address: 550 1st Ave, MSB 304

department: Division of Advanced Research Technologies (DART)

institute: New York University, NYU Langone Medical Center

summary: HH inhibition reproduces the lung phenotype of PDGF pathway knockout lungs and results in profound effects on differentiation and gene expression of Pdgfra-expressing alveolar mesenchymal cells, in particular myofibroblasts.

overall_design: Whole lung single cell suspensions of Gli1creERT2/+;R26RtdTom/+ reporter lungs (P6) from mice, labelled with tamoxifen at P1 followed by treatment with anti-HH antibody 5E1 or control IgG at P3 were loaded onto a GEM code instrument (10xGenomics).

first: Matthias

middle: C

last: Kugler


title: Single cell RNA sequencing-based comparison of mouse splenic pDC, pDC-like cells, tDC, cDC1 and cDC2 at steady state and during a viral infection

city: marseille

first: Thien-Phong

last: Vu Manh

address: 163 avenue de Luminy

country: France

institute: CIML

summary: Plasmacytoid dendritic cells (pDC) are the main source of type I interferon (IFN) during viral infections. Their other functions are debated, due to a lack of tools to identify and target them in vivo without affecting pDC-like cells and transitional DC (tDC), which harbor overlapping phenotypes and transcriptomes but a higher efficacy for T cell activation. To overcome this bottleneck, we designed, generated and validated a pDC-Tomato reporter mouse. We bred  pDC-Tomato with Zbtb46 GFP mice to yield the he ZeST mouse strain that enabled transcriptomic profiling of all splenic DC types, by single cell RNA sequencing, both at steady state and during the course of the infection with mouse cytomegalovirus (MCMV). Analyses of the transcriptomic dataset unraveled diverging activation of pDC-like cells vs tDC during the infection. This dataset and the associated specific gene modules will be useful to delineate the physiological functions of pDC versus other DC types.

overall_design: pDC, pDC-like cells, tDC, cDC1 and cDC2 were sorted from ZeST reporter mice at 0, 36 or 48 hours after MCMV infection. Single-cells were FACS-sorted into fourteen 96-well plates (4 plates for 0 hours, 5 plates for each of the two other time points). All plate maps were identical, encompassing at the time of sorting: 14 individual cells for each of the following populations, pDC expressing Tomato, pDC negative for Tomato, pDC-like cells, tDC, and cells positive for both Ly6D and Zbtb46; 12 cDC1, 12 cDC2 and 2 empty control wells. Gene expression was profiled by 5'-end single-cell RNAseq following the FB5P-seq protocol.

first: Michael

last: Valente

first: Laurine

last: Gil

first: Pierre

last: Milpied

first: Elena

last: Tomasello

first: Marc

last: Dalod


first: Xiong

last: Ji

overall_design: ATAC-Seq was performed to explore the nucleosome accessibility caused by site-specific histone acylations in the metabolites regulated gene expression

first: Fangfei

last: Qin

first: Boyuan

last: Li


title: miRNA profiles of EVs released from menstrual blood-derived stem cells

city: Xinxiang

first: JIE

last: LIAN

state: Henan

address: Jinsui Road

department: Henan Joint International Research Laboratory of Stem Cell Medicine

institute: Xinxiang Medical University

summary: To identify specific miRNAs carried by MenSC-EVs, and explore their crucial roles in MenSC-EV-based improvement of inflammatory diseases.

overall_design: miRNAs expression in MenSC-EVs derived from two different donors (25-35 years old, female). EVs were isolated from the cell-free conditioned medium of MenSCs culture over 48 hr by differential ultracentrifugation.

first: Juntang

last: Lin

first: Jie

last: Lian

first: Xinxing

last: Zhu

first: Rui

last: Guo


title: Impact of Genomic and Clinical Factors on Outcome of Children ≥18 Months of Age with INSS Stage 3 Neuroblastoma with Unfavorable Histology without MYCN Amplification

city: Los angeles

first: Xiangming

last: Ding

state: California

address: 4650 Sunset Blvd

institute: CHLA

summary: Patients with INSS stage 3, MYCN–non-amplified, unfavorable histology neuroblastoma represent a rare subset of children with outcomes that are inferior to those of most patients with advanced locoregional disease, and superior to those of most high-risk patients. We evaluated the impact of clinical variables on outcome and used available tumor samples from subjects within this subgroup to correlate whole exome sequencing (WES) and RNA sequencing (RNASeq) profiles with outcome. We found that subjects with tumors harboring chromosome 11q losses had inferior outcomes and describe an expression profile with universally favorable outcomes. These findings provide a framework for a refined risk stratification where patients with 11q loss could be included with traditional high-risk groups and patients with a favorable expression profile could be considered to have non-high-risk disease.

overall_design: Patients enrolled on Children’s Oncology Group (COG) protocols A3973 (n=34) and ANBL0532 (n=27), and/or biology protocol ANBL00B1 (n=72) were analyzed. Tumors with available DNA (n=65) and RNA (n=42) were subjected to whole exome sequencing (WES) and RNAseq. WES analyses and gene expression profiling were evaluated for their impact on survival. Multivariate analyses of EFS and OS using significant factors from univariate analyses were performed.

first: Shahab

last: Asgharzadeh


title: Analyses of regulatory network and discovery of potential biomarkers for Korean rockfish (Sebastes schlegelii) in responses to starvation stress through transcriptome and metabolome

city: Shanghai

first: Xiaozhao

last: Han

address: 999 Huchengring Road

institute: Shanghai Ocean University

summary: Whether in aquaculture or in nature, starvation stress limits the growth of fish. The purpose of the study was to clarify the detailed molecular mechanisms underlying starvation stress in Korean rockfish (Sebastes schlegelii) through liver transcriptome and metabolome analysis. Transcriptome results showed that liver genes associated with cell cycle and fatty acid synthesis were down-regulated, whereas those related to fatty acid decomposition were up-regulated in the experimental group (EG; starved for 72 days) compared to the control group (CG; feeding). Metabolomic results showed that there were significant differences in the levels of metabolites related to nucleotide metabolism and energy metabolism, such as purine metabolism, histidine metabolism and oxidative phosphorylation. Five fatty acids (C22:6n-3; C22:5n-3; C20:5n-3; C20:4n-3; C18:3n-6) were selected as possible biomarkers of starvation stress from the differential metabolites of metabolome. Subsequently, correlation between these differential genes of lipid metabolism and cell cycle and differential metabolites were analyzed, and observed that these five fatty acids were significantly correlated with the differential genes. These results provide new clues for understanding the role of fatty acid metabolism and cell cycle in fish under starvation stress. It also provides a reference for promoting the biomarker identification of starvation stress and stress tolerance breeding research.

overall_design: We performed gene expression profiling analysis using data obtained from RNA-seq of livers in Korean rockfish.

first: Jiying

last: Wang

first: Baoshan

first: Zhidong

last: Song

first: Peiyu

first: Bingshan

last: Huang

first: Chengqiang

first: Yongzhi

last: Sun

first: Xiaoyan

first: Tiantian

last: Hao


title: IL-13 signaling via epithelial cell-expressed type II IL-4 receptor mediates eosinophilic esophagitis

city: Tel Aviv

first: Guy

last: Shapira

address: Chaim Levanon 30

country: Israel

institute: Tel Aviv University

summary:
Purpose: We aimed to define the molecular pathways that are regulated by IL13Ra1 in our experimental murine EoE model
Methods: 1cm piece of the middle part from the esophagus of OXA or Vehicle treated WT or Il13ra1 KO mice were obtained for RNA extraction using TRIZOL. Thereafter, RNA samples were prepared using the CEL-Seq2 protocol, as published by Hashimshony et al. Genome Biology (2016) 17:77, with minor changes. Instead of single-cells as input, 2 ng purified RNA was taken as input for library preparation. The CEL-Seq library was run on an Illumina NextSeq550 instrument. The number of reads ranged from 6,819,051 to 34,217,489 per sample. The reads were mapped to the Mus musculus, GRCm38 genome using Tophat2 version 2.1.0, with up to 2 mismatches allowed per read, the minimum and maximum intron sizes were set to 50 and 100,000, respectively, and an annotation file was provided to the mapper. The percentage of uniquely mapped reads ranged from 86.64% to 90.05% per sample. Only uniquely mapped reads were counted to genes, using 'HTSeq-count' package version 0.6.1 with ‘union’ mode. Normalization and differential expression analyses were conducted using DESeq2 R package version 1.28.0.. Sample preparation, sequencing, quality control, and differential expression analyses were conducted by the "Technion Genome Center," Life Science and EngineeringPurpose: We aimed to define the molecular pathways that are regulated by IL13Ra1 in our experimental murine EoE model.
Results: IL13ra1 regulates transcriptome signature in EoE murine model.
Conclusion: We demonstrate that IL13ra1 results EoE related pathways.

overall_design: RNAseq of esophagus  from EoE (OXA) or control (Vehicle) WT or Il13ra1 KO mice

first: Shmuel

last: Avlas

first: Ariel

last: Munitz


title: Inhibition of Fap Promotes Cardiac Repair by Stabilizing BNP

first: Dandan

last: Cao

address: 1239 Siping Road, Yangpu District

institute: Tongji University

summary: Inhibition of fibroblast activation protein promotes cardiac repair by stabilizing brain natriuretic peptide after myocardial infarction

overall_design:
Bulk RNA-seq data of mouse cardiac fibroblasts (CFs) cultured alone in 3D GelMA hydrogel vs mouse CFs co-cultured with cardiac and lung endothelial cells in a ratio of 2:1 in 3D GelMA hydrogel.
RNA-seq libraries from 3 biological replicates of mouse CFs cultured alone in 3D GelMA hydrogel vs mouse CFs co-cultured with cardiac and lung endothelial cells in a ratio of 2:1 in 3D GelMA hydrogel. Samples were subjected to paired-end sequencing by HiSeq XTen systems (Illumina, 150 bp reads).

This study aims to test whether Fap is a critical regulator of cardiac repair after MI, and to uncover the underlying cellular and molecular mechanisms for better treatment of Myocardial infarction (MI).
We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different tissues from sham, MI, FAPI (MI), FapKO (MI).
Comparative gene expression profiling analysis of RNA-seq data for border zones of sham, MI, FAPI (MI), FapKO (MI).

last: Yue

first: Dachun

last: Xu

first: Li

last: Zhang

first: Yuxi

first: Mengqiu

last: Ma


title: Strain differences in conjunctival gene expression profiles

city: La Jolla

first: Tomoaki

last: Ando

address: 9420 Athena Circle

institute: La Jolla Institute for Allergy and Immunology

summary: Gel-forming mucins secreted by conjunctival goblet cells have been implicated in the clearance of allergens, pathogens, and debris. Here, we found that B6J mouse conjunctival goblet cell mucins are Alcian blue-positive due to sialylation in the steady state. However, Balb/c mouse strain was found to have Alcian blue-negative goblet cells in the conjunctiva. To understand the mechanisms underlying this difference, we performed microarray analysis.

overall_design: Conjunctival tissue was collected from naïve female B6J and Balb/c mice. Total RNA samples from 3 mice were pooled for each strain.

first: Moe

last: Matsuzawa

first: Mutusko

last: Hara

first: Jiro

last: Kitaura


title: IL-17A expression by both T cells and non-T cells contribute to HSV-IL-2-induced CNS demyelination

city: Los Angeles

first: Shaohui

last: Wang

state: CA

address: 8700 Beverly BLVD

department: Surgery

institute: Cedars Sinai Medical Center

summary: Adoptive transfer of T cells from wild-type (WT) mice to IL-17A-/- mice or T cells from IL-17A-/- mice to Rag-/- mice induced CNS demyelination in infected mice. Adoptive T cell experiments suggest that both T cells and non-T cells expressing IL-17A contribute to HSV-IL-2-induced CNS demyelination with no difference in the severity of demyelination between the two groups of IL-17A producing cells. IL-6, IL-10, or TGFβ did not contribute to CNS demyelination in infected mice. Transcriptome analysis between IL-17A-/- brain and spinal cord of infected mice with and without T cell transfer from WT mice revealed that “neuron projection extension involved in neuron projection guidance” and “ensheathment of neurons” pathways were associated with CNS demyelination. Collectively, the results indicate the importance of IL-17A in CNS demyelination and the possible involvement of more than three of IL-17 receptors in CNS demyelination

overall_design: Comparative gene expression profiling analysis of RNA-seq data for two types of tissue (brain and spinal cord) from three groups( IL17 KO

first: Satoshi

last: Hirose

first: Ujjaldeep

last: Jaggi

first: Harry

middle: H

last: Matundan

first: Mihoko

last: Kato

first: Xue-Ying

first: Sara

middle: J

last: Molesworth-Kenyon

first: Robert

middle: N

last: Lausch

first: Homayon

last: Ghiasi


title: Kabuki syndrome DNA methylation data

city: Glostrup

first: Mathis

last: Hildonen

state: Denmark

address: Gamle Landevej 7

department: Department of Clinical Genetics

country: Denmark

institute: Rigshospitalet

type: Methylation profiling by genome tiling array

summary: Disease-specific DNA methylation patterns (DNAm signatures) have been established for an increasing number of genetic disorders and represent a valuable tool for classification of genetic variants of uncertain significance (VUS). Sample size and batch effects are critical issues for establishing DNAm signatures, but their impact on the sensitivity and specificity of an already established DNAm signature as a predictive tool of variant pathogenicity has not previously been tested. Here, we assessed whether publicly available DNAm data can be employed to generate a binary machine learning classifier for VUS classification, and used variants in KMT2D, the gene associated with Kabuki syndrome, together with an existing DNAm signature as proof-of-concept. Using publicly available data for training, a classifier for KMT2D variants that correctly discriminated DNA samples from individuals with molecularly confirmed Kabuki syndrome and unaffected individuals was generated. These data document the clinical utility of a robust DNAm signature even with small numbers of patients to be tested. This study further underlines the importance of data sharing in the field of rare genetic disorders.

overall_design: Bisulphite converted DNA samples were hybridized to the Illumina Infinium Human MethylationEPIC Beadchip.

first: Marco

last: Ferilli

first: Tina

middle: D

last: Hjortshøj

first: Morten

last: Dunø

first: Lotte

last: Risom

first: Mads

last: Bak

first: Jakob

last: Ek

first: Rikke

middle: S

last: Møller

first: Andrea

last: Ciolfi

last: Tartaglia

first: Zeynep

last: Tümer


title: AP2XII-2 binding sites in Toxoplasma gondii

city: Indianapolis

first: Michael

middle: James

last: Holmes

state: IN

address: 635 Barnhill Drive

department: Pharmacology & Toxicology

institute: Indiana University School of Medicine

summary: Developmental regulation of the intracellular parasite Toxoplasma gondii is an understudied topic despite being central for a bid to control its dissemination around the globe. Of particular neglect are the factors that contribute to its sexual development. While it has previously been shown that generalized transcriptional repression machinery plays an important role in silencing spurious gene expression of sexually-committed parasites, the specific factors that target the generalized machinery to genetic loci remains unexplored. Here, we uncover that a member of the AP2 transcription factor family, AP2XII-2, is targeted to genomic loci that are associated with sexually-committed parasites along with the generalized regulators of transcriptional silencing, HDAC3 and MORC. Despite widespread association with gene promoters, AP2XII-2 is required for silencing of relatively few genes. We place two genes associated with sexual development downstream of AP2XII-2 control, transcription factor AP2X-10 and the amino acid hydroxylase AAH1. Dissecting gene regulatory pathways of Toxoplasma sexual development will likely be essential for controlling Toxoplasma dissemination in the future.

overall_design: Toxoplasma gondii with AP2XII-2 tagged with (HA-AID) were collected and processed by CUT&Tag protocol to determine AP2XII-2 binding sites genome-wide in tachyzoites

first: William

last: Sullivan Jr


title: Improved Efficacy of MSCs stably expressing CXCR4 and IL-10 in a xenogeneic Graft versus Host Disease Mouse Model

city: Madrid

first: Cristian

last: Suarez Cabrera

state: Madrid

address: Av. Complutense 40

institute: CIEMAT

summary: Previous clinical trials have shown that mesenchymal stromal cells (MSCs) can modulate graft versus host disease (GvHD) after allogeneic hematopoietic transplantation, although with variable efficacy. To improve the anti-GvHD effect of these cells, adipose  tissue derived-human MSCs (Ad-MSCs) were transduced with a lentiviral vector conferring stable expression of CXCR4, a molecule involved in cell migration to inflamed sites, and IL-10, a cytokine with potent anti-inflammatory properties. In vitro experiments showed that the expression of these molecules in Ad-MSCs (named CXCR4-IL10-MSCs) efficiently enhanced their migration towards SDF‐1α and also improved their immunomodulatory properties compared to unmodified Ad‐MSCs (WT‐MSCs). Moreover, using a humanized GvHD mouse model, CXCR4-IL10-MSCs showed improved therapeutic effects, which were confirmed by histopathologic analysis in the target organs. Additionally, compared to WT-MSCs, CXCR4-IL10-MSCs induced a more marked reduction in the number of pro-inflammatory Th1 and Th17 cells, a higher polarization towards an anti-inflammatory T cell profile (CD3+-IL10+ cells), and increased the number of regulatory T and B cells. Our in vitro and in vivo studies strongly suggest that CXCR4-IL10-MSCs should constitute an important new generation of MSCs for the treatment of GvHD in patients transplanted with allogeneic hematopoietic grafts.

overall_design: Analysis of hCD45 cells from spleen of NSG mice in animals without treatment (GvHD model), treated with WT-MSCs and modified-MSCs (CXCR4-IL10-MSCs). 3 animales per group were analyzed.

first: Rosario

last: Hervás-Salcedo

first: Maria

last: Fernandez-Garcia

first: Miriam

last: Hernando-Rodriguez

last: Suarez-Cabrera

first: Juan

last: Bueren

first: Rosa

middle: M

last: Yanez


title: nCounter PanCancer mouse immune profiling in CT26-bearing mouse tumor treated with DSP-0509 combined with anti PD-1 antibody

city: Osaka

first: Yosuke

last: Ota

state: Osaka

address: 3-1-98 Kasugade-naka konohanaku

department: Cancer Research Unit

institute: Sumitomo Pharma

summary: Combinatio of DSP-0509, small molecule TLR7 agonist, with anti PD-1 antibody enhanced anti-tumor inflammatory tumor microenvironment in CT26 tumor.

overall_design: This study consist of  4 treatment groups including vehicle, DSP-0509, anti PD-1 antibody and comibnation. Each group have three mice. Total RNA derived from each tumor were analyzed by mouse PanCancer immune profiling panel including 770 genes offered by NanoString.


title: Integrated analysis of extrachromosomal DNA elements in SCLC [scCNV]

city: Bethesda

first: Lorinc

middle: Sandor

last: Pongor

state: Maryland

address: 37 Convent drive

department: NCI

institute: NIH

summary: Integrated analysis of whole-genome sequencing, long-range optical mapping, single-cell DNA sequencing, and fluorescence in situ hybridization to find extrachromosomal DNA (ecDNA) as the primary source of MYC amplifications and driver fusions in SCLC. ecDNAs bring to proximity enhancer elements and oncogenes through circularization, creating transcription-amplifying units, driving heterogeneity of MYC gene dosage and expression of SCLC lineage-defining transcription factors.

overall_design: scCNV analysis of targeted regions

first: Parth

last: Desai

first: Anish

last: Thomas


title: Histone variant H3.3 facilitates open chromatin at promoters [CUT&TAG]

city: Dallas

first: Laura

last: Banaszynski

address: 5323 Harry Hines Blvd

institute: UT Southwestern Medical Center

summary: We analyze the contribution of H3.3 to chromatin accessibility, post-translational modification states, transcription factor binding and transcription in mouse embryonic stem cells (mESC).

overall_design: Transcription factor CUT&Tag analysis in mouse embryonic stem cell lines (H33WT, H33KO).

first: Ryan

last: O'Hara


title: IL-13 signaling via epithelial cell-expressed type II IL-4 receptor mediates eosinophilic esophagitis  II

first: Guy

last: Shapira

summary:
Purpose: We aimed to define the molecular pathways and the transcriptome signature of experimental EoE treated WT mice
Methods: 1cm piece of the middle part from the esophagus of OXA or Vehicle treated WT mice was obtained for RNA extraction using TRIZOL . Thereafter, RNA samples were prepared for sequencing using the CEL-Seq2 protocol, with minor modifications. Briefly, purified RNA (2 ng) was taken as input for library preparation. Thereafter, the CEL-Seq library was run on an Illumina NextSeq550 instrument. The number of reads ranged from 6,819,051 to 34,217,489 per sample. The reads were mapped to the Mus musculus, GRCm38 genome using Tophat2 version 2.1.0, with up to 2 mismatches allowed per read, the minimum and maximum intron sizes were set to 50 and 100,000, respectively, and an annotation file was provided to the mapper. The percentage of uniquely mapped reads ranged from 86.64% to 90.05% per sample. Only uniquely mapped reads were counted to genes, using 'HTSeq-count' package version 0.6.1 with ‘union’ mode. Normalization and differential expression analyses were conducted using DESeq2 R package version 1.28.0. Sample preparation, sequencing, quality control, and differential expression analyses were conducted by the "Technion Genome Center," Life Science and Engineering Interdisciplinary Research Center, Technion, Haifa, Israel.
Results: Experimental EoE is characterized by predominant inflammation response, proliferation, and epithelial changes.
Conclusion:We demonstrate that our new experimental EoE model results with an inflammation signature associated with type 2 immunity

overall_design: RNAseq of esophagus  from EoE (OXA) or control (Vehicle) WT mice

first: Shmuel

last: Avlas

first: Ariel

last: Munitz


title: Ageing-associated changes in transcription and splicing efficiency influence longevity

city: Köln

first: Antonios

last: Papadakis

address: Fürvelser Str. 15

country: Germany

institute: CECAD

summary: Ageing alters cellular homeostasis thereby compromising multiple cellular processes such as transcription and splicing. However, both the extent of these changes and the molecular mechanisms are not well understood so far. In order to address this question, we analyzed transcription-coupled processes on a genomic scale in five animal species (C. elegans, D. melanogaster, M. musculus, R. norvegicus, H. sapiens) at different adult life stages. Using total RNA profiling, we quantified alterations of transcriptional elongation speed, splicing efficiency, and splicing precision. We consistently observed across all analyzed species, that the genome-average speed of RNA polymerase II (Pol-II) increased with age. These effects were reverted under lifespan-extending conditions, such as dietary restriction or modulation of insulin signaling. Reducing the speed of Pol-II in worms and flies improved splicing efficiency and increased lifespan. We further observed reduced precision in nucleosome positioning in senescent compared to proliferating cells, which correlated with reduced splicing efficiency. Thus, an age-associated increase of transcriptional speed results in reduced splicing efficiency and splicing quality. These findings substantially extend our understanding about the molecular mechanisms driving animal aging, and suggest new mechanisms underlying lifespan-extending interventions.

sample_organism:
Caenorhabditis elegans
Drosophila melanogaster
Homo sapiens
Mus musculus

overall_design: We used total RNA-seq to quantify changes in transcription speed on a genomic scale in five animal species: the worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, the mouse Mus musculus, the rat Rattus norvegicus, and humans Homo sapiens, at different adult life-stages. To address whether the lifespan-extending interventions dietary restriction (DR) and inhibition of insulin/insulin-like growth factor signaling (IIS) affect transcriptional elongation speeds, we sequenced samples from IIS mutants, using daf-2 mutant worms at day 14 and fly heads from dilp2-3,5 mutants at day 30 and day 50, and hypothalamus from aged wild type and IRS1-/- null mice. Further, we sequenced samples from kidney and liver of dietary restricted (DR) and ad libitum fed mice.

first: Cédric

last: Debès

first: Özlem

last: Karalay

first: Sebastian

last: Grönke

first: Luke

last: Tain

first: Athanasia

last: Mizi

first: Shuhei

last: Nakamura

first: Anne

last: Zirkel

first: Isabell

last: Brusius

first: Konstantinos

last: Sofiadis

first: Mantha

last: Lambrousi

first: Yu-Xuan

last: Lu

first: Wenming

last: Huang

first: Reza

last: Esmaillie

first: Torsten

last: Kubacki

first: Martin

last: Späth

first: Bernhard

last: Schermer

first: Thomas

last: Benzing

first: Roman-Ulrich

last: Müller

first: Adam

last: Antebi

first: Linda

last: Partridge

first: Argyris

last: Papantonis

first: Andreas

last: Beyer


title: DNA methylome analysis of primary prostate cancer and pathologic lymph node metastases in patients with low and high Gleason score

status: Public on Jan 23 2023

city: Munich

first: Kristian

last: Unger

address: Marchioninistr. 15

department: Department of radiation oncology

institute: LMU University Hospital

summary: DNA-methylation profiles of primary tumors (PT) and paired synchronous lymph node metastases (LN) from Gleason Score (GS)-6/7a (n=20 LN-positive, n=20 LN-negative) and GS-9/10 patients (LN-positive n=20) after prostatectomy and lymphonodectomy were analyzed.

overall_design: 5 groups were compared: GS-6/7a N0 primary tumors,  GS-6/7a N1 primary tumors,  GS-6/7a N1 lymph node metastasis, GS-9/10 N1 primary tumors,  GS-9/10 N1 lymph node metastasis

first: Claus

last: Belka


title: Characterization of MdMYB68, a suberin master regulator in russeted apples.

city: Belvaux

first: Sylvain

last: Legay

address: 41, rue du Brill

department: ERIN department

country: Luxembourg

institute: Luxembourg Institute of Science and Technology

summary: Apple russeting is mainly due to the accumulation of suberin in the cell wall in response of defects and damages in the cuticle layer. Over the last decades, massive efforts have been done to better understand the complex interplay between pathways involved in the suberization process in model plants. However, the regulation mechanisms which orchestrate this complex process are still under investigation. Our previous studies highlighted number of transcription factor candidates from the Myeloblastosis (MYB) transcription factor family, which might regulate the suberization in the russeted or suberized apple fruit skin. Among these, we identified MdMYB68, which was co-expressed with number of well-known key suberin biosynthesis genes. Using transient expression in Nicotiana benthamiana and whole gene expression profiling analysis, we showed that MdMYB68 was able to trigger the expression of the globality of the already defined suberin biosynthesis pathway. The lipid content analysis further confirmed that MdMYB68 regulates the deposition of suberin in cell. Furthermore, we also investigate the alteration of the cell wall components and showed that MdMYB68 triggered a massive modification of the hemicellulose and pectins as suggested in our previous work. In summary, we here describe MdMYB68 as a new master regulator of the suberin deposition in apple fruit, and also provide, for the first time, new insights related to the fate of the cell wall during the suberization process.

overall_design: In order to draw a consistent picture of the MdMYB68 function, we compared  4 biological replicates of Nicotiana benthamiana leaves infiltrated with Pearleygate103 35S::MdMYB68/pBINP19 and 4 biological replicates of control plants infiltrated with P103empty vector /pBINP19.

first: Xuan

last: Xu

first: Gea

last: Guerriero

first: Frederic

last: Domergue

first: Emmanuelle

last: Cocco

first: Roberto

last: Berni

first: Olga

last: Beine-Golovchuk

first: Kjell

last: Sergeant

first: Jean-Francois

last: Hausman


title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells [scRNA-seq II]

city: Turin

first: Andrea

last: Lauria

address: Via Accademia Albertina, 13

department: Life Sciences and Systems Biology

country: Italy

institute: University of Turin

summary: The correct establishment of DNA methylation patterns during mouse early development is essential for cell fate specification. However, the molecular targets as well as the mechanisms that determine the specificity of the de novo methylation machinery during differentiation are not completely elucidated. Here we show that the DNMT3B-dependent DNA methylation in epiblasts of key developmental regulatory regions provides an epigenetic priming that ensures flawless commitment at later stages. Using in vitro stem cell differentiation and loss of function experiments combined with high-throughput genome-wide bisulfite-, bulk-, and single cell RNA-sequencing, we dissected the specific role of DNMT3B in cell specification, we identified the DNMT3B-dependent regulatory elements on the genome which, in Dnmt3b knockout (3BKO), impair the differentiation into meso-endodermal (ME) progenitors and redirect epiblast-like cells (EpiLCs) towards the neuro-ectodermal lineages. Moreover, ectopic expression of DNMT3B in 3BKO re-establishes the DNA methylation of the master regulator Sox2 super-enhancer, down-modulates its expression, and restores the expression of ME markers. Taken together, our data reveal that DNMT3B-dependent methylation at the epiblast stage is essential for the priming of the meso-endodermal lineages and provide functional characterization of the de novo DNMTs during EpiLCs lineage determination.

overall_design: Single cell RNA-seq transcriptome profiling of wild-type (WT) wild-type (WT), DNMT3A -/- (3AKO) and DNMT3B -/- (3BKO) mouse Embryonic Stem Cells (ESC) differentiating in Embryoid Bodies. 965 cells (related to Supplementary Fig. 1).

first: Guohua

last: Meng

first: Valentina

last: Proserpio

first: Stefania

last: Rapelli

first: Mara

last: Maldotti

first: Isabelle

middle: L

last: Polignano

first: Francesca

last: Anselmi

first: Danny

last: Incarnato

first: Anna

last: Krepleova

first: Chiara

last: Levron

first: Daniela

last: Donna

first: Giacomo

last: Donati

first: Ivan

last: Molineris

first: Francesco

last: Neri

first: Salvatore

last: Oliviero


title: Endothelial cell-derived oxysterol ablation attenuates experimental autoimmune encephalomyelitis

city: Epalinges

first: Caroline

last: Pot

address: Chemin des Boveresses 155

department: Department of Clinical Neurosciences

country: Switzerland

institute: Lausanne University Hospital

summary: The vasculature is a key regulator of leukocyte trafficking into the central nervous system (CNS) during inflammatory diseases including multiple sclerosis. However, the impact of endothelial-derived factors on other aspects of CNS immune responses remains unknown. Bioactive lipids, in particular oxysterols downstream Cholesterol-25-hydroxylase (Ch25h) promote neuroinflammation but their functions in the CNS remain unclear. Using a floxed-reporter Ch25h knock-in mice, we traced Ch25h expression to CNS endothelial cells (ECs) and myeloid cells and demonstrated that Ch25h-specific ablation in ECs attenuates experimental autoimmune encephalomyelitis. Mechanistically, inflamed Ch25h-deficient CNS ECs displayed altered lipid metabolism favoring polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) expansion relative to other leukocyte subsets that suppress encephalitogenic T lymphocytes proliferation. Finally, endothelial Ch25h-deficiency combined with mobilization of immature neutrophils into circulation resulted in nearly complete EAE protection. Our findings reveal a central role for CNS endothelial-derived Ch25h in promoting neuroinflammation by regulating expansion of immunosuppressive myeloid cell populations.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for primary brain microvascular endothelial cells isolated from Ch25h-sufficient (Ch25hfl/fl x VE-Cadherin-Cre-) or mice with endothelial-specific Ch25h deletion (Ch25hfl/fl x VE-Cadherin-Cre+), treated or not with IL-1beta

first: Florian

last: Ruiz

first: Solenne

last: Vigne

first: Benjamin

last: Peter

first: Jessica

last: Rebeaud

first: Tania

last: Wyss

first: Yannick

last: Yersin

first: Valentine

last: Bressoud

first: Martin

last: Roumain

first: Camille

last: Kowalski

first: Gael

last: Boivin

first: Leonard

last: Roth

first: Markus

last: Schwaninger

first: Giulio

middle: G

last: Muccioli

first: Stephanie

last: Hugues

first: Tatiana

middle: V

last: Petrova


title: NAD+ supplementation limits triple-negative breast cancer metastasis via SIRT1-P66Shc signaling

city: Beijing

first: Yi

last: Jiang

address: Hai Dian

department: School of Life Sciences

institute: Tsinghua University

summary: NAD+ levels decline with age and in certain disease conditions. NAD+ precursors have been shown to stimulate NAD+ biosynthesis and ameliorate various age-associated diseases in mouse models. However, NAD+ metabolism is complicated in cancer and its role in triple-negative breast cancer (TNBC) remains elusive. Here, we show that NAD+ supplement suppresses tumor metastasis in a TNBC orthotopic patient-derived xenograft (PDX) model. Sirtuin1 lysine deacetylase (SIRT1) is required for the effects since SIRT1 knockdown blocks NAD+-suppressed tumor metastasis. Overexpression of SIRT1 effectively impairs the metastatic potential of TNBC. Importantly, the interaction between SIRT1 and p66Shc causes the deacetylation and functional inactivation of p66Shc, which inhibits epithelial-mesenchymal transition (EMT). Overall, we demonstrate that NAD+ supplementation executes its anti-tumor function via activating the SIRT1-p66Shc axis, which highlights the preventive and therapeutic potential of SIRT1 activators as effective interventions for TNBC.

overall_design: RNA sequencing (RNA-seq) was performed on tumors from mice treated with either NAD+ precursor NMN (500 mg/kg/day) or vehicle in the HCC1937-orthotopic model.

first: Zongrui

last: Luo

first: Yuanchao

last: Gong

first: Yan

last: Fu

first: Yongzhang


title: B cells and peritoneal macrophages RNA-seq

city: Bunkyo-ku

first: Masashi

last: Kanayama

state: Tokyo

address: 1-5-45 Yushima

department: Department of Biodefense Research

institute: Medical Research Laboratory, Institute of Science Tokyo

summary: We found that CD11b-expressing B cells were increased in the BM of LPS-treatmed mice. To examine the differences in gene expression, we isolated CD11b-negative and CD11b-positive B cells were isolated and compared the gene expression profiles with peritoneal macrophages obtained from naive mice by RNA-sequence.

overall_design: We intraperitoneally injected lipopolysaccharide (LPS, 5 mg/kg) to WT C57BL/6 mice and isolated CD11b-negative and CD11b-positive B cells from the bone marrow 48 h after LPS-treatment. Peritoneal macrophages were isolated from peritoneal cavity of naïve WT mice. Comparative gene expression profiling analysis with RNA-seq  was performed for those cells.

first: Toshiaki

last: Ohteki


title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells [BSAS]

first: Andrea

last: Lauria

type: Methylation profiling by high throughput sequencing

summary: The correct establishment of DNA methylation patterns during mouse early development is essential for cell fate specification. However, the molecular targets as well as the mechanisms that determine the specificity of the de novo methylation machinery during differentiation are not completely elucidated. Here we show that the DNMT3B-dependent DNA methylation in epiblasts of key developmental regulatory regions provides an epigenetic priming that ensures flawless commitment at later stages. Using in vitro stem cell differentiation and loss of function experiments combined with high-throughput genome-wide bisulfite-, bulk-, and single cell RNA-sequencing, we dissected the specific role of DNMT3B in cell specification, we identified the DNMT3B-dependent regulatory elements on the genome which, in Dnmt3b knockout (3BKO), impair the differentiation into meso-endodermal (ME) progenitors and redirect epiblast-like cells (EpiLCs) towards the neuro-ectodermal lineages. Moreover, ectopic expression of DNMT3B in 3BKO re-establishes the DNA methylation of the master regulator Sox2 super-enhancer, down-modulates its expression, and restores the expression of ME markers.  Taken together, our data reveal that DNMT3B-dependent methylation at the epiblast stage is essential for the priming of the meso-endodermal lineages and provide functional characterization of the de novo DNMTs during EpiLCs lineage determination.

overall_design: 1) Targeted bisulfite amplicon sequencing (BSAS) analysis of DNA methylation in Sox2 locus of WT, 3BKO and 3BKO ectopically expressing Dnmt3b (Rescued) mouse ES-derived EpiLCs. 8 samples, 2 independent 3BKO clones (B77,B126).  2) Targeted bisulfite amplicon sequencing (BSAS) analysis of DNA methylation in Sox2 locus of 3BKO and 3BKO ESC transfected with CRISPRoff targeting 4 enhancers (E1-E4). 6 samples, 2 independent 3BKO clones (B77,B126).

first: Guohua

last: Meng

first: Valentina

last: Proserpio

first: Stefania

last: Rapelli

first: Mara

last: Maldotti

first: Isabelle

last: Polignano

first: Francesca

last: Anselmi

first: Danny

last: Incarnato

first: Anna

last: Krepleova

first: Chiara

last: Levra Levron

first: Daniela

last: Donna

first: Giacomo

last: Donati

first: Ivan

last: Molineris

first: Francesco

last: Neri

first: Salvatore

last: Oliviero


title: Hippocampus and visceral adipose tissues cellular landscape profiling to characterize the effects of obesity-amyloidosis comorbidity

city: Jerusalem

first: Naomi

last: Habib

address: Safra Campus

department: Center for brain sciences

institute: Hebrew University of Jerusalem

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Integrated analysis of extrachromosomal DNA elements in SCLC

first: Lorinc

last: Pongor


title: Hippocampus and visceral adipose tissues cellular landscape profiling to characterize the effects of obesity-amyloidosis comorbidity [scRNA-seq]

summary: To explore the link between Alzheimer's disease (AD) and obisity, we profiled the transcriptional changes of the co-morbidites as well as indivitual morbidities on brain, immune and adipose tissue, at the single cell level. We observed effects of both morbidities, including AD-associated alterations in microglia and astrocytes in AD mice, and HFD-associated transcriptional changes in teh brain and the adipose. See details in: doi: https://doi.org/10.1101/2022.02.05.479219

overall_design: We preforemed single nuceli RNA-seq sequencing of the hippocampus and adipose tissue of WT and AD-model (5xFAD) mice maintained on control or high fat diet, in 7-9 months old mice.

last: Greka

first: Michal

last: Schwartz


title: Angiopoietin-Tie2 signaling protects the kidney from ischemia-reperfusion injury in mice

city: Chicago

first: Yalu

last: Zhou

state: IL

address: 303 E. Superior St,

department: Department of Biochemistry and Molecular Genetics

institute: Northwestern University

summary:
Single cell RNAseq analysis of kidneys from Hepta-ANG1-treated and vehicle-treated mice identified endothelial-specific gene signatures and emergence of a new glomerular endothelial subpopulation associated with improved kidney function.

Total RNA of whole kidneys was sequenced to compare the transcriptional response of control and VE-PTPiKO kidneys after 5 hours of reperfusion. We saw fewer genes altered  in the VE-PTPiKO kidney after IR injury compared to control.

overall_design:
Kidneys from vehicle or Hepta-ANG1 treated male C57BL/6 mice were harvested 24 h after IR injury with 20min ischemia time.

bulk RNA sequencing of total RNA isolated from whole kidney 5 hour after IR induced acute kidney injury and sham surgery

first: Yanyang

last: Li

first: Pan

last: Liu

first: Hiroshi

last: Maekawa

first: Benjamin

last: Thomson

first: Jing

last: Jin

first: Susan

middle: E

last: Quaggin


title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells [RNA-seq]

first: Andrea

last: Lauria

overall_design: 1) Time course RNA-seq transcriptome profiling of wild-type (WT) and Dnmt3b -/- (3BKO) mouse Embryonic Stem Cells (ESCs) differentiating in mouse ES-derived Epiblast-Like Stem Cells (EpiLCs) and meso-endodermal (ME) progenitors. 24 samples, 2 biological replicates for each stage/condition, 2 independent 3BKO clones (B77,B126). 2) Time course RNA-seq transcriptome profiling of 3BKO and 3BKO ectopically expressing Dnmt3b (Rescued) mouse ES-derived EpiLCs and ME progenitors. 24 samples, 2 biological replicates for each stage/condition, 2 independent 3BKO clones (B77,B126).

first: Guohua

last: Meng

first: Valentina

last: Proserpio

first: Stefania

last: Rapelli

first: Mara

last: Maldotti

first: Isabelle

last: Polignano

first: Francesca

last: Anselmi

first: Danny

last: Incarnato

first: Anna

last: Krepleova

last: Levra Levron


title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells [WGBS]

overall_design: WGBS profiling of wild-type (WT) and DNMT3B -/- (3BKO) mouse ESCs, Epiblast-Like Cells (EpiLCs) and meso-endoderm (ME) cells. 16 samples, 2 biological replicates for each stage/condition, 2 independent Dnmt3b -/- clones (B77,B126).

first: Chiara

first: Daniela

last: Donna

first: Giacomo

last: Donati

first: Ivan

last: Molineris

first: Francesco

last: Neri

first: Salvatore

last: Oliviero


title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells [scRNA-seq]

overall_design: Single cell RNA-seq transcriptome profiling of wild-type (WT) wild-type (WT), DNMT3A -/- (3AKO) and DNMT3B -/- (3BKO) mouse Embryonic Stem Cells (ESC) differentiating in Embryoid Bodies. 550 cells.


title: Transcriptome profile of drug resistant metaplastic breast cancer cell lines

city: London

first: Ernesto

last: Yague

address: Du Cane Road

department: Division of Cancer

country: United Kingdom

institute: Imperial College London

summary: Two metaplastic breast cancer cell lines were used: a widely available, HS578T, and a novel line isolated from a metaplastic breast cancer tumor, BAS. Doxorubicin and paclitaxel resistant derivatives of these lines were generated and transcriptome profiling performed.

overall_design: Cells were treated with increasing concentrations of drug until they were able to survive and proliferate after a pulse of cytotoxic drug.  HS-TX: HS578T cells, 100 nM paclitaxel, 5 days pulse; HS-DOX: HS-578T cells, 100 nM doxorubicin, 3 days pulse; BAS-TX: BAS cells, 20 nM paclitaxe, 3 days pulse; BAS-DOX, BAS cells, 160 nM doxorubicin, 3 days pulse. For the array experiment, RNA isolated from three (drug resistant cells) or four (parental naive cells) independent cultures were used for each condition.

first: E

first: U

last: Kumar


title: AP2XII-2-dependent gene regulation in Toxoplasma gondii tachyzoites

first: Michael

last: Holmes

summary: Developmental regulation of the intracellular parasite Toxoplasma gondii is an understudied topic despite being central for a bid to control its dissemination around the globe. Of particular neglect are the factors that contribute to its sexual development. While it has previously been shown that generalized transcriptional repression machinery plays an important role in silencing spurious gene expression of sexually-committed parasites, the specific factors that target the generalized machinery to genetic loci remains unexplored. Here, we uncover that a member of the AP2 transcription factor family, AP2XII-2, is targeted to genomic loci that are associated with sexually-committed parasites along with the generalized regulators of transcriptional silencing, HDAC3 and MORC. Despite widespread association with gene promoters, AP2XII-2 is required for silencing of relatively few genes. We place two genes associated with sexual development downstream of AP2XII-2 control, transcription factor AP2X-10 and the amino acid hydroxylase AAH1. Dissecting gene regulatory pathways of Toxoplasma sexual development will likely be essential for controlling Toxoplasma dissemination in the future.

overall_design: Toxoplasma gondii with AP2XII-2 tagged with (HA-AID) were treated with vehicle or IAA and processed polyA-enriched RNAseq in tachyzoites

first: William

last: Sullivan Jr


title: Targeting prolyl-tRNA synthetase via a series of ATP-mimetics to accelerate drug discovery against toxoplasmosis

status: Public on Jan 22 2023

city: La Tronche

first: Alexandre

last: Bougdour

state: Isère

address: Domaine de la Merci, Campus santé

department: Prevention and Therapy of Chronic diseases

institute: Centre de Recherche Inserm U1209 / CNRS UMR5309 / UGA

summary: To shed light on the parasiticidal mechanisms of L35, we have adapted a workflow which combines a forward genetic approach based on transcriptome sequencing, computational mutation discovery, and CRISPR/Cas9 genome editing in Toxoplasma gondii. Drug-resistant parasites were generated by chemical random mutagenesis. Multiple independent resistant lines were isolated. Single nucleotide variations (SNVs) were identified based on NGS transcriptomic analysis. By focusing on mutations present in coding sequences, we identified a single gene, TgPRS, that harbored SNVs leading to amino acid substitutions in the 6 drug-resistant lines obtained that were not present in the parental strain. Finally, using CRISPR/Cas9 genome editing we confirmed that the mutations identified confer resistance against L35.

sample_organism:
Homo sapiens
Toxoplasma gondii RH

platform_organism:
Homo sapiens
Toxoplasma gondii RH

overall_design: Methods: RNA profiles were generated from HFF cells infected by RH wild-type or L35-resistant T. gondii strains. Total RNAs were extracted and purified using TRIzol (Invitrogen, Carlsbad, CA, USA) and RNeasy Plus Mini Kit (Qiagen). RNA-sequencing was performed by GENEWIZ (South Plainfield, NJ, USA). The RNA quality was assessed using the Qubit Fluorometer and the Fragment Analyzer system with the PROSize 3.0 software (Agilent Technologies, Palo Alto, California, USA). Illumina TruSEQ RNA library prep of selected polyA RNAs and sequencing reagents were used following the manufacturer's recommendations (Illumina, San Diego, CA, USA). The samples were paired-end multiplex sequenced (2 × 150 bp) on the Illumina Hiseq platform and generated at least 20 million reads for each sample.

first: Manickam

last: Yogavel

first: Nipun

last: Malhotra

first: Jyoti

last: Chhibber-Goel

first: Siddhartha

last: Mishra

first: Valeria

last: Bellini

first: Karl

last: Harlos

first: Benoît

last: Laleu

first: Mohamed-Ali

last: Hakimi

first: Amit

last: Sharma


title: Human CD4 cytotoxic T lymphocytes mediate potent tumor control via tumor cell HLA 2 class II expression in a humanized immune system mouse model

city: Tarrytown

first: Gabrielle

last: Choonoo

address: 777 Old Saw Mill River Rd

institute: Regeneron Pharmaceuticals, Inc

summary: Efficacy of immune checkpoint inhibitors in cancer can be limited by dysfunction of CD8 T cells or down-regulation of HLA class I. Tumor control mechanisms independent of the CD8/HLA-I axis would bypass these limitations. CD4 cytotoxic T lymphocytes (CTLs) have been detected in diverse human cancers. However, their independent roles in tumor immunity are underexplored. Here, we report CD4 T cell-dependent spontaneous tumor regression and subsequent memory responses in a humanized immune system (HIS) mouse model. HT-29 tumors, which upregulate HLA class II expression in response to IFN-γ, regressed or were eliminated in a subset of tumor implanted HIS mice. Mice with regressing tumors showed increased cytotoxic CD4 T cells in blood and tumors and enhanced HLA-II expression on tumor cells compared to mice with progressing tumors. The intratumoral CD4 T cell subset associated with tumor regression expressed multiple cytotoxic markers and exhibited clonal expansion. Notably, tumor control was abrogated by depletion of CD4 but not CD8 T cells. CD4 T cells derived from tumor-regressing mice exhibited HLA-II-dependent and tumor cell-specific killing ex vivo. Taken together, our study demonstrates a critical role of human CD4 CTLs in mediating potent tumor control independent of CD8 T cells and provides a novel platform to study human CD4 CTL-mediated anti-tumor immunity.

overall_design: Data includes untreated same donor-engrafted StRG47 HIS mice implanted with HT-29 colon tumor. Tumor samples were collected after 1 month and sorted human CD45+ and mouse CD45+ for sequencing. There are 11 samples in total, 5 StRG47 HIS regressors and 6 StRG47 HIS progressors. The single cell data is aligned to both the human and mouse genome and the TCR data is aligned to human.

first: Wen

last: Lin

first: Varan

last: Singh

first: Raynel

last: Springer

first: Namita

last: Gupta

first: Aditi

last: Khatri

first: Davor

last: Frleta

first: Jun

last: Zhong

first: Tomasz

last: Owczarek

first: Lynn

last: Macdonald

first: Andrew

last: Murphy

first: Gavin

last: Thurston

first: Markus

last: Mohrs

first: Ella

last: Ioffe

first: Yi-Fen

last: Lu


title: RNAseq analysis of Hep3B cells upon overexpression of LINE1 retrotransposon encoded protein L1orf1p

status: Public on Jan 21 2023

city: Newcastle upon Tyne

first: Ruchi

last: Shukla

address: Ellison A Building

department: Applied Biosciences

institute: Northumbria University

summary: L1 retrotransposons are known to get activated during hepatocarcinogenesis. We have developed inducible (TET-ON) overexpression of L1orf1 using piggybac vector in Hep3B cell line and a control TET-ON-empty cell line was also generated. Influence of L1orf1 overexpression on overall cellular transcritome was then analysed by RNAseq followed by pathway analysis. Overall, a number of key oncogeneic pathways are dysregulated upon L1orf1 overexpression is observed.

overall_design: RNAseq was carried out for 2 cell lines derived from Hep3B cells (TET-ON-L1orf1 and TET-On-empty control) 72 hrs after doxycycline (1ug/ml) induction (RNA isolated from 3 independent cell pellets).


title: Integrated analysis of extrachromosomal DNA elements in SCLC [DNA-seq]

first: Lorinc

last: Pongor

summary: Integrated analysis of whole-genome sequencing, long-range optical mapping, single-cell DNA sequencing, and fluorescence in situ hybridization to find extrachromosomal DNA (ecDNA) as the primary source of MYC amplifications and driver fusions in SCLC. ecDNAs bring to proximity enhancer elements and oncogenes through circularization, creating transcription-amplifying units, driving heterogeneity of MYC gene dosage and expression of SCLC lineage-defining transcription factors.

overall_design: DNA sequencing of NCI-H524 adherent and suspension cells

first: Parth

last: Desai

first: Anish

last: Thomas


title: RNAseq analysis of Huh7 cells upon stable-knockdown of L1 elements

summary: L1 elements are repeat elements known to get activated during hepatocarcinogenesis. We have knocked-down expression of L1 elements using shRNA targeting L1-ORF1 in Huh7 cell line and a control non-targeting cell line was also generated. Influence of L1 knockdown on overall cellular transcritome was then analysed by RNAseq followed by pathway analysis. Overall, a crosstalk between L1 elements and TGFbeta signaling pathway is observed.

overall_design: RNAseq was carried out for 2 cell lines derived from Huh7 cells (L1-knockdown and non-targeting control) in triplicate (RNA isolated from 3 independent cell pellets).


title: Liver lineage confers sensitivity of cancer cells to sulfonation-dependent alkylators

city: Chapel Hill

first: William

last: Kim

state: NC

address: 450 West Dr

department: Lineberger Comprehensive Cancer Center

institute: UNC Chapel Hill

summary: Adult liver malignancies, including intrahepatic cholangiocarcinoma and hepatocellular carcinoma, are the 2nd leading cause of cancer related death worldwide. The majority of patients are currently treated with either combination chemotherapy or immunotherapy, respectively, without specific biomarkers for patient selection. Here, using high-throughput screens, proteomics, and in vitro resistance models, we identify the small molecule, YC-1, as selectively active against a defined subset of cell lines derived from both major liver cancer types. We demonstrate that selectivity in vitro and in vivo is determined by expression of a human cytosolic sulfotransferase – the liver resident enzyme SULT1A1, which sulfonates YC-1. Sulfonation stimulates covalent binding of YC-1 to lysine residues in protein targets, enriching for RNA binding factors. Using computational analysis as well as structural modeling, we also define a wider group of structurally related SULT1A1-activated small molecules with distinct target profiles, which together constitute an untapped small molecule class. These studies provide a foundation for pre-clinical development of these agents and point to the broader potential of exploiting the liver lineage enzyme SULT1A1 for selective targeting strategies.

overall_design: Two SULT1A1 expressing, IDH1 mutant human cholangiocarcinoma cell lines RBE and SNU1079 were used to study YC-1 induced transcriptomic alterations. Specific experimental design includes YC-1 and DMSO vehicle treated conditions at early (6 hour) and late (16 hour) time points, with three biological replicates for each condition. In total, 24 samples were collected for RNA extraction and subsequently RNA sequencing.

first: Lei

last: Shi

first: Mi

last: Zhou

first: Ujjawal

last: Manocha

middle: Y

first: Nabeel

last: Bardeesy


title: Integrated analysis of extrachromosomal DNA elements in SCLC [HiChIP]

overall_design: H3K27ac HiChIP samples.


title: miRNA deregulation and relationship with metabolic parameters after Mediterrran ary intervention in BRCA-mutated women

city: Bari

first: Simona

last: De Summa

state: (Click to Select US State)

address: Via Orazio Flacco, 65

institute: IRCCS-Istituto Tumori "Giovanni Paolo II"

summary: Breast cancer could be related to BRCA1/2 alterations genetically shared in familial context. However, genetic predisposition is not the only factor determining tumor onset. It is well-known the importance of genetics-environment interaction. Indeed, the influence of insulin, glucose and estradiol have been reported. The aim of the present study was to uncover the relationships between Mediterranean ary intervention, miRNA expression and metabolic parameters.

overall_design: Plasma samples of BRCA-alterated subjects have been collected at the baseline and after six months from the beginning of  regimen. Moreover, subjects have been randomized in two groups: ary intervention and placebo. miRNA profiling and subsequent ddPCR validation have been performed in all the subjects and in both time points.

first: Stefania

last: Tommasi


title: Characterization of human erythropoiesis using ex vivo differentiation of CD34 HSC (NG Capture-C)

city: Headington

first: Damien

last: Downes

state: Oxfordshire

address: John Radcliffe Hospital

department: MRC Weatherall Institute of Medicine

institute: The University of Oxford

summary: Genome Wides Association Studies (GWAS) have identified tens of thousands of associations between human genetic variation and common disease. Despite the abundance of GWAS associations, functional identification and characterization of causative variants and effector genes remains a challenging prospect. Human erythropoiesis provides a highly tractable model system for the development of tools for GWAS analysis. We have developed a three-phase protocol for differentiation of peripheral CD34+ Haematopoietic Stem Cells (HSC) into mature enucleating erythrocytes. This protocol has been characterized for it’s the changes observed morphologically, immunologically (by FACS) and epigenetically (by ATAC-seq, ChIP-seq, and RNA-seq). We have used this protocol to study the effects of common GWAS variants affecting red blood cell traits, and more severe mutations causing  Type-1 Congenital Dyserythropoietic Anemia (CDA-I).

overall_design: To identify regulatory interactions of variants associated with red blood cell traits we performed high resolution chromatin conformation capture (3C) using NG CaptureC (Davies 2016). NG CaptureC was performed in triplicate for Day 10 differentiating erythroid (Ery) cells from three donors, human Embryonic Stem Cells (H1 hESC; WiCell), and Human Umbilical Vein Endothelial Cells (HUVEC; Lonza/Gibco/PromoCell). Capture was performed with biotinylated oligonucleotides targeting sequences adjacent to DpnII sites in two separate designs. Probes were divided into pools targeting SNPs more than (enhancer) or less than (promoter) 5 kb from known transcription start sites.

first: Caroline

last: Scott

first: Lea

last: Nussbaum

first: Stephanie

last: Hill

first: Jim

last: Hughes


title: NF-κB-Inducing Kinase (NIK) Governs the Mitochondrial Respiratory Capacity, Differentiation, and Inflammatory Status of Innate Immune Cells

status: Public on Jan 20 2023

city: Bryan

first: Raquel

last: Sitcheran

state: TX

address: 8447 Riverside parkway

institute: Texas A&M University School of Medicine

summary: NF-κB-Inducing Kinase (NIK), which is essential for the activation of the noncanonical NF-κB pathway, regulates diverse processes in immunity, development, and disease.  While recent studies have elucidated important functions of NIK in adaptive immune cells and cancer cell metabolism, the role of NIK in metabolic-driven inflammatory responses in innate immune cells remains unclear. Here we demonstrate that NIK-deficient bone marrow-derived macrophages exhibit defects in mitochondrial-dependent metabolism and oxidative phosphorylation (OXPHOS) functions that impair acquisition of a pro-repair, anti-inflammatory phenotype. Subsequently, NIK-deficient mice exhibit skewing of myeloid cells characterized by aberrant eosinophil, monocyte, and macrophage cell populations in the blood, bone marrow, and adipose tissue. Furthermore, NIK-deficient blood monocytes display hyperresponsiveness to bacterial lipopolysaccharide and elevated TNFα production ex vivo, indicative of systemic inflammation. These findings suggest that NIK is required for the metabolic rewiring of mitochondrial respiration that is critical for balancing pro- and anti-inflammatory myeloid immune cell functions.  Overall, our work highlights a previously unrecognized role for NIK as a molecular rheostat that fine-tunes immunometabolism in innate immunity and suggests that metabolic dysfunction may be an important driver of inflammatory diseases caused by aberrant NIK expression or activity.

overall_design: 1 million WT and NIK KO bone marrow derrived macrophages from 2 mice per genotype and treatment were treated with PBS or 20ng/mL IL-4 for 6 hours

first: Justin

last: Keeney

first: Ashley

last: Winters

first: A

middle: P

last: West


title: Immune landscape in ER+ breast cancer subtypes identifies a differential role for macrophages

status: Public on Jan 29 2023

city: Pittsburgh

first: Anthony

middle: Richard

last: Cillo

state: PA

address: The Assembly, Room 4070C Bay 10, 5051 Centre Ave

department: Immunology

institute: University of Pittsburgh

summary: Current immunotherapies are ineffective for estrogen receptor positive (ER+) breast cancers, which account for nearly 70% of all breast cancers. Amongst the ER+ breast cancers, the major subtypes are invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC). We utilized droplet-based single-cell RNAseq (10X Genomics) to analyzed the immune landscape of both peripheral blood and tumor infiltrating immune populations in both IDC and ILC. These analyses demonstrated substantial heterogeneity of macrophage within the tumor microenvironment.

overall_design: Single-cell RNAseq was performed on paired peripheral blood samples and tissue samples from patients with ER+ breast cancers to assess the transcriptional immune landscape. Live immune cells were sorted from peripheral blood and single cell suspensions generated from tumor specimens. Both 3'-based and 5'-based single-cell RNAseq protocols from 10X Genomics were utilized. Samples were collected from a total of 15 patients (6 IDC and 9 ILC).

middle: R

first: Sayali

last: Onkar

first: Jian

last: Cui

first: Steffi

last: Oesterreich

first: Dario

last: Vignali


title: Characterization of human erythropoiesis using ex vivo differentiation of CD34 HSC (ChIP-seq)

overall_design: To characterise the epigenetic landscape and identify chromatin modifications associated with nucleosome depleted regions, ChIP-seq was carried out on differentiating erythroid cells at Day 10. CD34+ cells from normal leukocyte cones (NCO) were differentiated simultaneously to Day 10 and ChIP was performed for enhancer (H3K4me1), promoter (H3K4me3) and active transcription (H3K27ac) marks, as well as for the boundary protein CTCF. All ChIPs were performed on the same material simultaneously and a 5% input was stored prior to immunoprecipitation as a control.

first: Jelena

last: Telenius


title: T cell egress via lymphatic vessels limits the intratumoral T cell repertoire in melanoma

city: New York

first: Amanda

last: Lund

state: New York

address: 522 First Ave

institute: NYU Langone Health

summary: Antigen-specific CD8+ T cell accumulation in tumors is a prerequisite for effective immunotherapy, and yet, the mechanisms of lymphocyte transit remain poorly defined. We find that tumor-associated lymphatic vessels control T cell exit from tumors via the chemokine CXCL12, and intratumoral antigen encounter tunes CXCR4 expression on effector CD8+ T cells. Only high affinity antigen downregulates CXCR4 and upregulates the CXCL12 decoy receptor, ACKR3, thereby reducing CXCL12 sensitivity and promoting T cell retention. A diverse repertoire of functional tumor-specific CD8+ T cells exit the tumor, thereby limiting tumor control. CXCR4 inhibition and loss of lymphatic-specific CXCL12 boosts T cell retention and enhances tumor control. Strategies that limit T cell egress, therefore, provide a new tool to boost the response to immunotherapy.

overall_design: YUMMER1.7 murine melanoma cells were intradermally implanted into 3 C57Bl/6 mice. At day 21, tumors were harvested and digested into cell suspensions. Cell suspensions were immunostained with CD45-APC antibodies and then enriched for Miltenyi APC microbeads and the QuadroMACs Magnetic Seperator and LS Columns. Enriched CD45+ cell suspensions were stained with DAPI and then sorted on SY3200 HAPS sorter to improve purity of live CD45+ cells. 2.5e5 cells were sorted from each tumor and then pooled. The data was output to fastq format, converted to 10X format, then into a Seurat object. The Seurat object was dimensionally reduced and clustered, with CD3d/CD3E+ cells identified, then dimensionally reduced and clustered again, with CD8+ cells identified.

middle: W

first: Maria

last: Steele

first: Sunny

last: Son


title: Transcriptomic differences in livers from pubertal mice fed the AIN93G standard diet or a high-fat diet

city: Grand Forks

first: Lin

last: Yan

state: North Dakota

address: 2420 2nd Ave N

institute: USDA- Grand Forks Human Nutrition Research Center

summary: The analyses show that consumption of a high-fat diet, compared to a standard control diet, alters hepatic transcriptome in pubertal mice.

overall_design: Weanling three-week-old female C57BL/6 mice were randomly assigned into two groups and fed the AIN93G standard diet (providing 16% of energy from soybean oil) or a high-fat diet (a modified AIN93 formulation providing 45% of energy from soybean oil) for three weeks. At termination, mice were euthanized every four hours followed by exsanguination for a period of 48 hours. Livers were collected and stored at -80°C for designed analyses. Only samples from the first 24-hour cycle were used for the RNA-seq analysis.

first: Sneha

last: Sundaram

first: Bret

last: Rust

first: Huawei

last: Zeng


title: UV irradiation remodels the specificity landscape of transcription factors [84702_PhotoProduct_Antibody]

city: Durham

first: Raluca

last: Gordan

address: 101 Science Dr, CIEMAS 2179

department: Center for Genomic and Computational Biology

institute: Duke University

summary: Somatic mutations are highly enriched at transcription factor (TF) binding sites, with the strongest trend being observed for ultraviolet light (UV)-induced mutations in melanomas. One of the main mechanisms proposed for this hyper-mutation pattern is the inefficient repair of UV lesions within TF-binding sites, caused by competition between TFs bound to these lesions and the DNA repair proteins that must recognize the lesions to initiate repair. However, TF binding to UV-irradiated DNA is poorly characterized, and it is unclear whether TFs maintain specificity for their DNA sites after UV exposure. We developed UV-Bind, a high-throughput approach to investigate the impact of UV irradiation on protein-DNA binding specificity. We applied UV-Bind to ten TFs from eight structural families, and found that UV lesions significantly altered the DNA-binding preferences of all TFs tested. The main effect was a decrease in binding specificity, but the precise effects and their magnitude differ across factors. Importantly, we found that despite the overall reduction in DNA-binding specificity in the presence of UV lesions, TFs can still compete with repair proteins for lesion recognition, in a manner consistent with their specificity for UV-irradiated DNA. In addition, for a subset of TFs we identified a surprising but reproducible effect at certain non-consensus DNA sequences, where UV irradiation leads to a high increase in the level of TF binding. These changes in DNA-binding specificity after UV irradiation, at both consensus and non-consensus sites, have important implications for the regulatory and mutagenic roles of TFs in the cell.

overall_design: The UV-Bind assay was performed for CPD and 6-4PP antibodies for UV-irradiated arrays. A universal design that allows for the measurement of k-mers for CPD and 6-4PP antibody signal. A detailed description is provided in the manuscript.

first: Zachery

last: Mielko


title: MYTHO: a novel regulator of skeletal muscle autophagy and integrity

city: Montreal

first: Gilles

last: Gouspillou

state: Québec

address: 141 Avenue du Président Kennedy

department: Département des sciences de l'activité physique

country: Canada

institute: UQAM

summary: Autophagy is a critical process in the regulation of muscle mass, function and integrity. The molecular mechanisms regulating autophagy are complex and still partly understood. Here, we identify and characterize a novel FoxO-dependent gene, d230025d16rik which we named Mytho (Macroautophagy and YouTH Optimizer), as a regulator of autophagy and skeletal muscle integrity in vivo. Mytho is significantly up-regulated in various mouse models of skeletal muscle atrophy. Short term depletion of MYTHO in mice attenuates muscle atrophy caused by fasting, denervation, cancer cachexia and sepsis. While MYTHO overexpression is sufficient to trigger muscle atrophy, MYTHO knockdown results in a progressive increase in muscle mass associated with a sustained activation of the mTORC1 signaling pathway. Prolonged MYTHO knockdown is associated with severe myopathic features, including impaired autophagy, muscle weakness, myofiber degeneration, and extensive ultrastructural defects, such as accumulation of autophagic vacuoles and tubular aggregates. Inhibition of the mTORC1 signaling pathway in mice using rapamycin treatment attenuates the myopathic phenotype triggered by MYTHO knockdown. Skeletal muscles from human patients diagnosed with myotonic dystrophy type 1 (DM1) display reduced Mytho expression, activation of the mTORC1 signaling pathway and impaired autophagy, raising the possibility that low Mytho expression might contribute to the progression of the disease. We conclude that MYTHO is a key regulator of muscle autophagy and integrity.

overall_design: Refer to individual Series


title: Gene expression profile of primary ovarian tumors and matched metastasis

city: Hangzhou

first: Kelie

last: Chen

address: #886 Yuhangtang Road

institute: Zhejiang University

summary: Ovarian cancer is the most frequent cause of cancer death in women and the leading cause of death related to gynecological cancer, accounting for 5% of estimated cancer deaths. Due to its asymptomatic nature at early stages, most ovarian cancers were diagnosed at an advanced stage, with distant metastasis in the abdominal cavity. Cancer metastasis is the primary cause of morbidity, and contributes to 95% of cancer-related deaths. Metastatic ovarian cancer is closely related with recurrence and drug resistance, rendering metastasis as the major challenge in the course of ovarian cancer treatment. Thorough understanding of ovarian cancer metastasis is believed to contribute to improve cancer cure rates, while it is still not well elucidated. One important aspect of ovarian cancer metastasis research is the identification of driver molecular. To identify proteins required for ovarian cancer metastasis, we carried out unbiased high-throughput screening by comparing the expression profile of ovarian cancer primary and metastasis tissues.

overall_design: RNA-seq was performed of 5 pairs of matched primary ovarian tumors and metastasis from ovarian cancer patients. RNA samples were sequenced on BGISEQ platform (BGI, China). Raw reads were mapped to the reference genome (Homo sapiens, ensemble, GRCh38) using Bowtie2 with the default settings. Numbers of reads assigned to genes were counted by featureCounts. DEGs were retrieved by R package DESeq2.

first: Shengchao

last: Wang

first: Yihua

last: Wu

first: Weiguo

last: Lu


title: Mature B cells and Mesenchymal Stem Cells control emergency myelopoiesis

city: Seoul

first: Jungmin

last: Choi

state: Seoul

address: 73, Goryeodae-ro, Seongbuk-gu

department: Department of Biomedical Sciences

country: South Korea

institute: Korea University College of Medicine

summary: Systemic inflammation halts lymphopoiesis and prioritizes myeloid cell production. How blood cell production switches from homeostasis to emergency myelopoiesis is incompletely understood. Here we show that Lymphotoxin-b receptor (LTbR) signaling in combination with TNF and IL1 receptor signaling in mesenchymal stem cells (MSCs) downregulates Il7 expression to shutdown lymphopoiesis during systemic inflammation. LTbR signaling in MSCs also promoted CCL2 production to enable inflammatory monocyte egress from the bone marrow. Furthermore, pharmacological or genetic blocking of Il7 downregulation in MSCs impaired myeloid cell production and egress, which reduced survival against systemic bacterial and viral infections. Interestingly, lymphotoxin a1b2 delivered by B-lineage cells, and specifically by mature B cells, contributed to promote Il7 downregulation and reduce MSC lymphopoietic activity. Our studies revealed an unexpected role for LTbR signaling in MSCs and identified mature B cells as an important regulator of emergency myelopoiesis.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Mesenchymal Stem Cells and its treatment derivatives.

first: Vivian

last: Lim

first: Joao

last: Pereira


title: A single cell host/pathogen transcriptome atlas of the HPV16 infected epidermis identifies a novel differentiation-dissonant keratinocyte population implicated in cancer

city: Cincinnati

first: Mike

last: Adam

state: Ohio

address: 240 Albert Sabin Way

department: CBDI

institute: Cincinnati Children's Hospital Research Foundation

summary: We report the first single cell transcriptome atlas of HPV16-infected epithelium, describe relevant cell populations and molecular networks as a foundation for antiviral target discovery, and identify a novel differentiation-dissonant keratinocyte population that is implicated in HPV-associated cancer types

overall_design: Human HPV16 infected keratinocytes were analyzed using 10X single-cell rna-seq

first: Susanne

last: Wells


title: Hippocampus and visceral adipose tissues cellular landscape profiling to characterize the effects of obesity-amyloidosis comorbidity [Bulk RNA-seq]

city: Jerusalem

first: Naomi

last: Habib

address: Safra Campus

institute: Hebrew University of Jerusalem

summary: Treatment of human cultured T cells with NANA to evaluate the transcriptional changes induced by NANA

overall_design: T cells were isolated form PBMCs of 4 individuals. The cells were cultured with and without NANA for 120hr. RNA was extracted from the cells and used for preparation of RNA-seq libraries

first: Anna

last: Greka

first: Michal

last: Schwartz


title: Evaluation of a camptothetin analog, Topotecan, as an HIV "block and lock" agent

city: La Jolla

first: Nadejda

middle: S.

last: Beliakova-Bethell

state: CA

address: 9500 Gilman Dr.

department: Medicine

institute: University of California, San Diego

summary:
The goal of this study was to investigate transcriptome remodeling induced by treatment with a camptothetin analog, Topotecan (TPT), of a primary T cell model of HIV latency. The study aimed to determine whether TPT has a global inhibitory effect on gene expression in primary CD4+ T cells, and identify mechanisms of action of this drug as an HIV “block and lock” agent.
Methods: CD4+ T cells were isolated from blood of HIV seronegative study participants (N=3) and utilized to generate an in vitro model of latent HIV infection (model developed in the Spina laboratory and previously described by Spina et al., 2013 and Soto et al., 2022). Following generation of the model, cells were treated with 10 uM Topotecan or its solvent dimethyl sulfoxide (DMSO) for 24 hours. Cells were lyzed with RLT buffer containing beta-mercaptoethanol from a RNeasy micro kit (Qiagen, Inc. cat # 74004). ERCC spikes (Thermo Fisher Scientific, Inc.) were added to cell lysates based on cell number in each sample (10 ul of 1:800 dilution per million cells). RNA was extracted and subjected to deep sequencing at the Institute for Genomics Medicine (IGM) Genomics Center at the University of California San Diego. Filtering low quality reads and removal of the 3’ adapter sequences were performed using the Trim Galore tool. Reads were mapped to the human genome (build hg38) using HISAT2, and to HIV and ERCC references using bowtie. Mapped human reads were counted against the human GENCODE annotation (v37) using HT-Seq. ERCC counts were used to determine whether TPT had global inhibitory effect on transcription in primary CD4+ T cells. Because no such effect was observed, differential gene expression analysis was performed using library EdgeR in Bioconductor R without correction of expression based on ERCC spikes. Genes with false discovery rate (FDR)-corrected p-value less than 0.05 and an absolute fold change in TPT-treated samples compared to DMSO controls greater than 2, were considered significantly modulated by TPT. Pathway and gene ontology (GO) term enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v2022q3. Pathways and GO terms with FDR-corrected p-value less than 0.05 were considered significantly enriched for differentially expressed genes.
Results: An average of 19.7 million reads per sample were acquired and mapped to the human genome (build hg38). After applying filtering criteria, 9,678 human genes were identified with the HISAT2 and HTSeq workflow. Differential expression analysis was performed between TPT- and DMSO-treated samples using EdgeR. A total of 1,749 differentially expressed genes (DEGs) were identified with FDR p-value <0.05 and an absolute fold change greater than 2; of which 522 were up- and 1227 downregulated by TPT. Pathway and GO term enrichment analysis revealed that TPT interferes with gene transcription and cell signaling pathways (e.g. T cell receptor signaling, positive regulation of JNK cascade, regulation of actin cytoskeleton and positive regulation of GTPase activity were affected by TPT treatment).
Conclusion: The study demonstrated that TPT induces multiple effects on transcriptome in primary CD4+ T cells. Some of these changes may represent direct and indirect mechanisms of action of TPT as an HIV “block and lock” agent.

overall_design: Transcriptomic profiling of the primary CD4+ T cell model of HIV latency treated with a camptothetin analog, Topotecan (TPT), or its solvent DMSO, in three replicate experiments that used cells from different blood donors.

first: Amey

last: Mukim

first: Davey

last: Smith


title: Syngeneic model of carcinogen-induced tumor mimics basal/squamous, stromal-rich, and neuroendocrine molecular and immunological features of muscle-invasive bladder cancer

city: Buffalo

first: Mark

last: Long

state: NY

address: Elm & Carlton Streets

department: Bioinformatics & Biostatistics

institute: Roswell Park Comprehensive Cancer Center

summary: Invasive bladder tumors were induced in C57BL/6 mice when continuously exposed to N-butyl-N-(4-hydroxbutyl nitrosamine) (BBN) in the drinking water. Tumors were excised and serially passed by subcutaneous implantation into sex-matched syngeneic C57BL/6 hosts. Eight lines were named BBN-induced Urothelium Roswell Park (BURP) tumor lines. BURP lines were characterized by applying consensus molecular classification to RNA expression.

overall_design: RNAseq analysis of BURP cell lines

first: Barbara

last: Foster

first: Wendy

last: Huss

first: Craig

last: Brackett


title: ASPSCR1-TFE3 orchestrates the angiogenic program of alveolar soft part sarcoma 2

city: Tokyo

first: Takuro

last: Nakamura

address: 3-8-31 Ariake, Koto-ku

department: The Cancer Institute

institute: Japanese Foundation for Cancer Research

summary:
Alveolar soft part sarcoma (ASPS) is a rare soft part malignancy affecting adolescents and young adult. ASPS is characterized by its alveolar structure consisting of tumor cells and highly integrated vascular network, and its high metastatic potential indicates the importance of the prominent angiogenic activity of ASPS. Here we find that the expression of ASPSCR1-TFE3, the fusion transcription factor causatively associated with ASPS, is dispensable for in vitro tumor maintenance but required for in vivo tumor development via angiogenesis. ASPSCR1-TFE3 frequently associates with active enhancers including super-enhancers (SE) upon its DNA binding, and the loss of its expression induces dynamic modification of SE distribution related to genes belonging to the angiogenesis pathway. Using epigenomic CRISPR/dCas9 screening, we identify Pdgfb, Rab27a, Sytl2, and Vwf as critical target genes associated with reduced enhancer activities due to the ASPSCR1-TFE3 loss. ASPSCR1-TFE3 thus orchestrates higher ordered angiogenesis via enhanced intracellular trafficking of angiogenic factors.
We used microarrays to detail the global program of gene expression in mouse and human ASPS cells.

overall_design: Murine and human ASPS cell lines expressing ASPSCR1-TFE3 were examined.

first: Miwa

last: Tanaka


title: ASPSCR1-TFE3 orchestrates the angiogenic program of alveolar soft part sarcoma 3

summary: Alveolar soft part sarcoma (ASPS) is a rare soft part malignancy affecting adolescents and young adult. ASPS is characterized by its alveolar structure consisting of tumor cells and highly integrated vascular network, and its high metastatic potential indicates the importance of the prominent angiogenic activity of ASPS. Here we find that the expression of ASPSCR1-TFE3, the fusion transcription factor causatively associated with ASPS, is dispensable for in vitro tumor maintenance but required for in vivo tumor development via angiogenesis. ASPSCR1-TFE3 frequently associates with active enhancers including super-enhancers (SE) upon its DNA binding, and the loss of its expression induces dynamic modification of SE distribution related to genes belonging to the angiogenesis pathway. Using epigenomic CRISPR/dCas9 screening, we identify Pdgfb, Rab27a, Sytl2, and Vwf as critical target genes associated with reduced enhancer activities due to the ASPSCR1-TFE3 loss. ASPSCR1-TFE3 thus orchestrates higher ordered angiogenesis via enhanced intracellular trafficking of angiogenic factors.

overall_design: Mouse ASPS was induced by expressing ASPSCR1-TFE3 in embryonic mesenchymal cells followed by subcutaneous transplantation into nude mice. Subcutaneous tumors were subjected to cell culture. Human ASPS cell lines were used for gene knockdown experiments.


title: Uncovering the transcriptional changes in egressed vs intratumoral effector CD8+ T cells

first: Amanda

last: Lund

summary: Comparative gene expression profiling of RNA-seq data for tumor-retained, tumor-egressed, and circulating effector CD44+ CD8+ T cells.

overall_design: YUMMER1.7 tumors were implanted into Kaede-Tg mice. On day 11, tumors were photoconverted, and on day 12, YUMMER1.7 tumors, draining lymph nodes, and spleens were harvested. Kaede red+ CD44+ CD8+ T cells were sorted from YUMMER1.7 tumors (tumor-retained) and draining lymph nodes (tumor-egressed) along with CD44+ CD8+ T cells from the spleens (circulating effector CD8+ T cells). Tissues were harvested and sorted from 4 replicate mice. MedGenome Inc performed RNA extraction, library prep, and sequencing.

first: Maria

last: Steele

first: Ian

last: Dryg