title: Developmental signals control chromosome segregation fidelity during early lineage specification and neurogenesis by modulating replicative stress

city: Utrecht

first: Jeroen

last: van den Berg

state: Utrecht

address: Uppsalalaan 8

institute: Hubrecht Institute

summary: Human development and homeostasis rely on the correct replication, maintenance and segregation of our genetic blueprints. How these intracellular processes are monitored across different human cellular lineages, and why the spatio-temporal distribution of mosaicism varies during development  remain unknown. Using human and mouse pluripotent stem cells, we identify that several lineage specification signals –including WNT, BMP and FGF– converge into the modulation of DNA  replication stress and damage during S-phase, which in turn controls spindle dynamics and  chromosome segregation fidelity in mitosis. We show that patterning signals associated with  anteriorisation during mammalian gastrulation increase chromosome missegregation, while the  posteriorising signals WNT and BMP protect pluripotent stem cells from excessive origin firing,  DNA damage and chromosome missegregation derived from stalled forks. Through epistasis  experiments, we find that WNT, BMP and FGF have distinct roles during DNA replication, and  demonstrate that WNT/GSK3 signalling sits at the helm of this regulatory cascade. Cell signalling  control of chromosome segregation declines after pluripotency exit and specification into the three human germ layers, but re-emerges in differentiating neural progenitors. Through ex vivo and in vivo analyses, we also show that FGF and WNT signalling display opposite roles in chromosome segregation fidelity in mouse neural progenitors during the onset of neurogenesis (E14.5), but not during their expansion (E12.5). In particular, we find that the neurogenic factor FGF2 induces DNA replication stress-mediated chromosome missegregation, which could provide a rationale for the elevated chromosomal mosaicism of the developing brain. Our results highlight a role for patterning signals and cellular identity in genome maintenance that contributes to somatic mosaicism during mouse and human early lineage specification and neurogenesis.

overall_design: hiPSC were treated with DMSO, Aphidicolin (200nM) or DKK1 (250ng/ml) for 3hrs prior to double pulse EdU labeling, single cells were sorted and subjected to scEdU-seq (van den Berg, et al., Nature Methods,2024

first: Anchel

last: De Jaime-Soguero

first: Sergio

last: Acebrón


title: Mechanism for controlled assembly of transcriptional condensates by Aire [5'-ethynyl uridine RNA-seq]

status: Public on Jul 08 2024

city: Boston

first: Qianxia

last: Zhang

state: MA

address: 3 Blackfan Circle, RM 3117.16

country: USA

institute: Boston Children's Hospital

summary: Transcriptional condensates play a crucial role in gene expression and regulation, yet their assembly mechanisms remain poorly understood. We here report a multi-layered mechanism for condensate assembly by Autoimmune regulator (Aire), an essential transcriptional regulator (TR) that orchestrates gene expression reprogramming for central T-cell tolerance. Aire condensates assemble on enhancers, stimulating local transcriptional activities and connecting disparate inter-chromosomal loci. This functional condensate formation hinges upon the coordination between three Aire domains: polymerization domain CARD, histone binding domain PHD1 and C-terminal tail (CTT). Specifically, CTT binds coactivators CBP/p300, recruiting Aire to CBP/p300-rich enhancers and promoting CARD-mediated condensate assembly. Conversely, PHD1 binds to the ubiquitous histone mark H3K4me0, keeping Aire dispersed throughout the genome until Aire nucleates on enhancers. Our findings showed that the balance between PHD1-mediated suppression and CTT-mediated stimulation of Aire polymerization is crucial to form transcriptionally active condensates at target sites, providing new insights into controlled polymerization of TRs.

overall_design: AIRE is known to be dynamically expressed in a small subset of mTECs, which has made mechanistic studies using mTECs challenging. We thus utilized the human thymic epithelial cell line 4D6, in which AIRE expression was temporally controlled through a doxycycline (Dox)-inducible promoter, to investigate the mechanism of AIRE polymerization in the nucleus. We examined whether ectopic expression of AIRE in 4D6 cells could recapitulate AIRE behaviours in mTECs including broad transcriptomic changes post AIRE expression.

first: Sun

last: Hur

first: Yu-San

last: Huoh


title: Mechanism for controlled assembly of transcriptional condensates by Aire [ATAC-seq]

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: To investigate the impact of AIRE.CTT–CBP/p300 interaction on AIRE's target site selection, we compared ChIP-seq of WT AIRE to deltaCTT.R3 mutan, which is deficient in CBP/p300 binding. To investigate the exent to which a polymerization defect contributes to AIRE targeting, we compared ChIP-seq of WT AIRE to deltaCARD mutant, which is deficient in polymerization. We also examine what histone marks including H3K27ac, H3K4me1, H3K27me3 are associated with AIRE-enriched sites, and whether p300 enrichment is correlated with AIRE enrichment in the genome. Next, we asked whether p300 inhibitors would affect AIRE targeting.


title: Targeting adenocarcinoma and enzalutamide-resistant prostate cancer using the novel anti-androgen inhibitor ADA-308

city: Vancouver

first: Amina

last: Zoubeidi

state: BC

address: 2660 Oak Street

country: Canada

institute: Vancouver Prostate Centre

type: Expression profiling by high throughput sequencing

summary: Prostate cancer is a leading cause of cancer-related death among men globally. It often develops resistance to standard androgen deprivation therapy and androgen receptor (AR) pathway inhibitors such as enzalutamide. This resistance highlights the urgent need for novel therapeutic strategies. ADA-308 emerges as a promising candidate, demonstrating potent inhibition of both AR-sensitive adenocarcinoma as well as enzalutamide-resistant prostate cancer cell lines. Our studies reveal that ADA-308 effectively blocks AR activity, including nuclear localization, and inhibits cell proliferation in vitro. Furthermore, ADA-308 demonstrates remarkable efficacy in vivo, showcasing a robust antitumor response in enzalutamide-resistant models. These findings establish ADA-308’s role as a potent AR inhibitor that overcomes resistance to AR-targeted therapies and highlight its potential as a novel therapeutic approach in advanced prostate cancer management.

overall_design: LNCaP cell lines were cultured in RPMI-1640 media plus 5% FBS treated with10µmol/L enzalutamide or ADA-308 for 72 hours. Cells were collected for RNA sequencing.

first: Shaghayegh

last: Nouruzi

first: Fraser

last: Johnson

first: Sahil

last: Kumar

first: Milla

last: Koistinaho

first: Anu

last: Muona


title: HMGA1 causes a global shift in chromatin architecture linked to transcriptional changes [scRNA-Seq]

city: Cambridge

first: Ioana

last: Olan

state: Cambridgeshire

address: Robinson Way

department: Cancer Research UK Cambridge Institute

country: United Kingdom

institute: University of Cambridge

summary: High Mobility Group A1 (HMGA1) is an architectural chromatin-binding protein that regulates gene expression in various biological contexts, including differentiation and cancer. While previous studies show that HMGA1 acts by binding specific gene promoters or by contributing to the enhanceosome, its global effect on transcription remains unclear. HMGA1 is also critical for the formation of senescence-associated heterochromatic foci (SAHF) in oncogene-induced senescence (OIS), a stable form of cell cycle arrest with a pro-inflammatory phenotype. This link between the two roles of HMGA1, gene regulation and chromatin organisation, has not been fully explained. Here, we used graph theory and data integration from high-throughput assays to investigate how HMGA1 binding impacts the chromatin interactions network. Our results demonstrate that HMGA1 profoundly remodels the global chromatin network during OIS and contributes to stronger compartmentalization. This remodelling goes beyond SAHF, leading to substantial gene repositioning and differential gene expression, highlighting a novel role for HMGA1 in altering chromatin environments. The HMGA1 effects of gene expression go beyond senescence and appear to be relevant in other contexts, such as lung cancer.

overall_design: Hi-C, ChIP-seq, RNA-seq and scRNA-seq was performed in IMR90 cells in the Grow and RAS-induced senescence conditions, with and without shHMGA1. ChIP-seq and RNA-seq were also performed in the H1299 cell line.


title: HMGA1 causes a global shift in chromatin architecture linked to transcriptional changes [Hi-C]


title: HMGA1 causes a global shift in chromatin architecture linked to transcriptional changes [ChIP-Seq]


title: HMGA1 causes a global shift in chromatin architecture linked to transcriptional changes [RNA-Seq]


title: Mechanism for controlled assembly of transcriptional condensates by Aire [ChIP-seq]


title: HMGA1 causes a global shift in chromatin architecture linked to transcriptional changes

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

overall_design: Refer to individual Series


title: Mechanism for controlled assembly of transcriptional condensates by Aire [CRISPR]

overall_design: To understand the molecular mechanisms by which AIRE C-terminal tail (AIRE.CTT) contributes to the TAD-like activity, we investigated genetic interaction partners of AIRE.CTT. A genome-wide CRISPR screen was performed using a modified reporter system, wherein the screening reporter mKate2 was induced upon expression of Gal4DBD-CTT fused with the expression reporter GFP via a self-cleavable peptide 2A.


title: Mechanism for controlled assembly of transcriptional condensates by Aire [RNA-seq]


title: Mechanism for controlled assembly of transcriptional condensates by Aire

summary:
Transcriptional condensates play a crucial role in gene expression and regulation, yet their assembly mechanisms remain poorly understood. We here report a multi-layered mechanism for condensate assembly by Autoimmune regulator (Aire), an essential transcriptional regulator (TR) that orchestrates gene expression reprogramming for central T-cell tolerance. Aire condensates assemble on enhancers, stimulating local transcriptional activities and connecting disparate inter-chromosomal loci. This functional condensate formation hinges upon the coordination between three Aire domains: polymerization domain CARD, histone binding domain PHD1 and C-terminal tail (CTT). Specifically, CTT binds coactivators CBP/p300, recruiting Aire to CBP/p300-rich enhancers and promoting CARD-mediated condensate assembly. Conversely, PHD1 binds to the ubiquitous histone mark H3K4me0, keeping Aire dispersed throughout the genome until Aire nucleates on enhancers. Our findings showed that the balance between PHD1-mediated suppression and CTT-mediated stimulation of Aire polymerization is crucial to form transcriptionally active condensates at target sites, providing new insights into controlled polymerization of TRs.
This SuperSeries is composed of the SubSeries listed below.


title: Hippocampal differential expression underlying the neuroprotective effect of delta-9-tetrahydrocannabinol (THC) microdose on old mice

status: Public on Jul 06 2024

city: Tel Aviv

first: Guy

last: Shapira

address: Chaim Levanon 30

country: Israel

institute: Tel Aviv University

summary: Delta-9-tetrahydrocannabinol (THC) is the primary psychoactive compound of the cannabis plant and an exogenous ligand of the endocannabinoid system. In previous studies, we demonstrated that a single microdose of THC (0.002mg/kg, 3–4 orders of magnitude lower than the standard dose for rodents) exerts distinct, long-term neuroprotection in model mice subjected to acute neurological insults. When administered to old, healthy mice, the THC microdose induced remarkable long-lasting (weeks) improvement in a wide range of cognitive functions, including significant morphological and biochemical brain alterations. To elucidate the mechanisms underlying these effects, we analyzed the gene expression of hippocampal samples from the model mice. Samples taken 5 days after THC treatment showed significant differential expression of genes associated with neurogenesis and brain development. In samples taken 5 weeks after treatment, the transcriptional signature was shifted to that of neuronal differentiation and survival. This study demonstrated the use of hippocampal transcriptome profiling in uncovering the molecular basis of the atypical, anti- aging effects of THC microdose treatment in old mice.

overall_design: Experiments were performed on old (24 months old) female and young (2 months old) male mice of the Institute of Cancer Research. Each age-group was treated with a THC microdose (0.002mg/kg), or vehicle solution. Hippocampal samples were obtained from old and young mice 5 weeks after administration, an extra group of treated old mice were sampled 5 days after treatment. RNA was extracted from the samples and sequenced.

first: Ifat

last: Israel-Elgali

first: Noam

last: Shomron

first: Yosef

last: Sarne


title: Variants in tubule epithelial regulatory elements mediate most heritable differences in human kidney function

status: Public on Jul 05 2024

city: San Francisco

first: Gabriel

last: Loeb

state: CA

address: 555 Mission Bay Blvd South, Rm 382H

department: Medicine

institute: UCSF

summary: We used bulk and single-cell ATAC-seq to measure chromatin accessibilty in human kidneys. This allowed us to define candidate cis-regulatory elements of each human kidney cell type and identify the role of podocyte and tubule regulatory elements in human kidney function.

overall_design:
Single cell ATAC-sequencing of 3 human kidneys. Single cell ATAC-sequencing was performed on cortex from all 3 kidneys and medulla from two of the kidneys.
Bulk ATAC-sequencing from the cortex of one human kidney was also performed.

**Please note that the records have been updated with additional data on Aug 8, 2024.****

middle: B

first: Jeremy

middle: F

last: Reiter


title: Single cell gene expression profiling of the SW620 colorectal cell line

status: Public on Jul 16 2024

city: Rotterdam

first: Mathijs

last: Verhagen

address: Doctor Molewaterplein 40

country: Netherlands

institute: Erasmus Medical Center

summary: Intra-tumor heterogeneity, i.e. the presence of diverse cell types and subpopulations within tumors, presents a significant obstacle in cancer treatment due to its negative consequences for resistance to therapy and disease recurrence. However, the mechanisms which underlie intra-tumor heterogeneity and result in the plethora of different cancer cells within a single lesion remain poorly understood. Here, we leverage the SW480 and SW620 cell lines as a model system to investigate the molecular and functional diversity of colon cancer cells. Through a combination of fluorescence activated cell sorting (FACS) analysis and transcriptomic profiling, we identify three distinct subpopulations, namely resident cancer stem cells (rCSCs), migratory CSCs (mCSCs), and high-relapse cells (HRCs). These subpopulations show varying Wnt signaling levels and gene expression profiles mirroring their stem-like properties. Examination of publicly available spatial transcriptomic data confirms the presence of these subpopulations in patient-derived cancers and reveals their distinct spatial distribution relative to the tumor microenvironment.

overall_design: Cell lines were cultured in DMEM medium with FCS. After FACS sorting of the bulk, live population, single cell gene expression profiling was performed according to droplet based sequencing (10X Genomics)

first: M

middle: P

first: A

last: Sacchetti

first: R

last: Fodde


title: Gene expression of alveolar macrophage in mice with AT2 cell-specific Cpt1a deletion

city: Taipei

first: Chia-Lang

last: Hsu

address: No. 7, Zhongshan S. Rd.

department: Department of Medical Research

country: Taiwan

institute: National Taiwan University Hospital

summary: Carnitine palmitoyltransferase 1a (CPT1a) is the key regulator of mitochondrial long-chain fatty acid beta-oxidation (LCFAO). However, the functional significance of AT2 cell-specific LCFAO at baseline and during acute lung injury (ALI) is not fully understood. In this study, Murine models of AT2 cell-specific Cpt1a deletion were generated for investigating the role of CPT1a in regulating AT2 cell function and the severity of lipopolysaccharide-induced murine ALI models.

overall_design: To investigate whether impaired LCFAO in AT2 cells regulates the responses of alveolar macrophages, we also isolated alveolar macrophages from Cpt1a-KO and control mice for trancriptomic analysis.

first: Kuei-Pin

last: Chung


title: Interferon subverts an AHR-JUN axis to promote CXCL13+ T cells in lupus

city: Chicago

first: Calvin

last: Law

state: IL

address: 303 E Superior St Lurie 5-220

department: Dermatology

institute: Northwestern University

summary: SLE is prototypical autoimmune disease driven by pathologic T cell-B cell interactions. Expansion of B cell-helper T cells including T follicular helper (Tfh) and T peripheral helper (Tph) cells is a prominent feature of systemic lupus erythematosus (SLE). Human Tfh and Tph cells characteristically produce high levels of the B cell chemoattractant CXCL13, yet regulation of T cell CXCL13 production and the relationship between CXCL13+ T cells and other T cell states remains unclear. Here, we identify an imbalance in CD4 T cell phenotypes in SLE patients, with expansion of PD-1+/ICOS+ CXCL13+ T cells and reduction of CD96hi IL-22+ T cells. Using CRISPR screens, we identify the aryl hydrocarbon receptor (AHR) as a potent negative regulator of CXCL13 production by human CD4 T cells. Transcriptomic, epigenetic, and functional studies demonstrate that AHR coordinates with AP-1 family member JUN to prevent CXCL13+ Tph/Tfh cell differentiation and promote an IL-22+ phenotype. Type I interferon (IFN), a pathogenic driver of SLE, opposes AHR and JUN to promote T cell production of CXCL13. These results place CXCL13+ Tph/Tfh cells on a polarization axis opposite from Th22 cells and reveal AHR, JUN, and IFN as key regulators of these divergent T cell states.


title: Transcriptome Analysis of the Mouse Cerebral Cortex in Brain ischemia Injury Model

city: Shanghai

first: Qimin

last: Zheng

address: 131 Dongan Road,Xuhui District

department: Institutes of Brain Science

country: China

institute: Fudan University

summary: Ischemic stroke caused a high level of death and disability in China. However, the underlying mechanism still remains largely unknown. Here, we examined changes in the transciptomic profiles in the brain of mice with ischemic stroke using RNA sequencing (RNA-seq)technology. And we introduced a new kind of Nano-particle material of Cu9S8-SNAP to investigate the effect in ischemic stroke

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 different group of brain tissue from mice, including Operation group, Cu9S8-SNAP treated group after tMCAO (Temporary Middle Cerebral Artery Occlusion) operation. Combining with naïve mice, at 3 days after operation.

first: Qing

last: Ye


title: CBX2 overexpression in the OVCAR4 high grade serous cell line

city: Denver

first: Benjamin

last: Bitler

state: CO

department: OB-GYN

institute: The University of Colorado

summary: CBX2 regulates the tumor immune microenvironment of high grade serous carcinoma. Through genetic knockdown and overexpression, we discovered that CBX2 regulates immune regulated genes, promotes macrophage polarization, and phagocytosis.

overall_design: CBX2 was cloned from full-length OVCAR4 cDNA into the pCDH-CMV-MCS-EF1-Puro vector. OVCAR4 cells were transduced with pCDH-CMV-MCS-EF1-Puro or pCDH-CMV-MCS-EF1-Puro-CBX2 using lentiviral particles.  The gene expression profiles of OVCAR4 vector-control and a CBX2 overexpression cell lines were compared using RNAseq with two replicates for each.

first: Lindsay

last: Brubaker

first: Lily

last: Nguyen

first: Meher

last: Boorgula

first: Thomas

last: Danhorn


title: Endocytosed dsRNAs induce lysosomal membrane permeabilization that allows cytosolic dsRNA translocation for Drosophila RNAi response

status: Public on Jul 07 2024

city: Kumamoto

first: Shingo

last: Usuki

address: 2-2-1 Honjo, Chuo-ku

department: Institute of Molecular Embryology and Genetics (IMEG)

country: Japan

institute: Kumamoto University

type: Non-coding RNA profiling by high throughput sequencing

summary: RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. Herein, we show that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl−/H+ antiporter ClC-b/DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells.

overall_design: Ago2 complex was immunopurified and copurified RNAs are deep-sequenced. Samples 1-4 were control saline injected w (1), and DCV injected w (2), clc-b_KO (3), and ostm1_KO (4) flies, and whole fly lysates were used for immunoprecipitation. Samples 5-10 were control S2 (5, 6), clc-b_KO (7, 8), and dmostm1_KO (9, 10) cell lines clutured in without (5, 7, 9) or with (6, 8, 10) dsRNA for firefly luciferase CDS in the medium.

first: Tsubasa

last: Tanaka

first: Tamaki

last: Yano

first: Kazuko

last: Hanyu-Nakamura

first: Akira

last: Nakamura


title: CBX2 knockdown in the PEO1 high grade serous cell line

overall_design: Small hairpin RNA used to knockdown CBX2 in PEO1 cells.  The gene expression profiles of PEO1 parental and a CBX2 knockdown cell lines were compared using RNAseq with two replicates for each.


title: CBX2 Knockout in the OVCAR4 high grade serous cell line

overall_design: CRISPR/Cas9 was used to knockout CBX2 in OVCAR4 cells and two clonal populations were isolated. The gene expression profiles of OVCAR4 parental and a CBX2 knockout cell lines were compared using RNAseq with two replicates for each.


title: Alveolar epithelial cells mitigate neutrophilic inflammation in acute lung injury through regulating mitochondrial fatty acid oxidation

address: No 7, Chung-Shan South Rd

department: Laboratory Medicine

overall_design: AT2 cells were isolated from the lungs from mice with AT2 cell-specific Cpt1a deletion (Cpt1a-KO) and the control (Sftpc-CreERt2 +/-) at baseline and 24 hours after intra-tracheal LPS administration. To investigate whether impaired LCFAO in AT2 cells regulates the responses of alveolar macrophages in ALI, we also isolated alveolar macrophages from Cpt1a-KO and control mice at 4 hours after intra-tracheal LPS administration, and alveolar macrophages from control mice at baseline. Alveolar macrophages from 2 mice were pooled to make one sample for trancriptomic analysis.

first: Chong-Jen

last: Yu


title: Replicative RNA enables directed evolution and Darwinian adaptation in mammalian cells

city: Beijing

first: Yihan

last: Lin

address: No.5 Yiheyuan Road, Haidian

institute: Peking University


title: Gene expression changes in SHI-1 cells following exposure to disulfiram/copper, niclosamde or combined drugs

status: Public on Jul 03 2024

city: London

first: Owen

last: Williams

address: 30 Guilford Street

department: Cancer section

institute: UCL GOS ICH

summary: Disulfiram and niclosamide were identified as drugs that induced depletion of an MLL-AF9-luciferase fusion protein in THP-1 AML cells, in a bioluminescence sreen. MLL-fusion protein depletion was confirmed in AML and ALL cell lines expressing different MLL-fusion proteins. Combination of disulfiram with niclosamide was found to enhance depletion of MLL-fusion proteins. To investigate whether this enhanced depletion resulted in increased suppression of downstream target genes. MLL-AF6 expressing SHI-1 cells were exposed to disulfiram/copper, nciclosamide or combined drugs. SHI-1 cells were treated for 16 hours with 0.3uM disulfiram / 1uM copper, 5uM niclosamide or combined drugs and RNAseq performed on isolated RNA.

overall_design: Total cellular RNA was purified from SHI-1 cells treated with DMSO, 0.3uM disulfiram / 1uM copper, 5uM niclosamide or combined drugs for 16 hours, from three independent experiments

first: Jasper

last: de Boer

first: Noelia

last: Che

first: Sandra

last: Cantilena


title: The receptor protein tyrosine phosphatase PTPRK promotes intestinal repair and catalysis-independent tumor suppression

status: Public on Jul 02 2024

first: Simon

middle: Richard

last: Andrews

state: Cambs

address: Babraham Research Campus

department: Science Services

institute: The Babraham Institute

summary: PTPRK is a receptor tyrosine phosphatase linked to the regulation of growth factor signalling and tumor suppression. It is stabilized at the plasma membrane by trans homophilic interactions upon cell-cell contact. It regulates cell-cell adhesion, but is also reported to regulate numerous cancer-associated signalling pathways. However, its signalling mechanism remains to be determined. Here, we find that PTPRK regulates cell adhesion signalling, suppresses invasion and promotes collective, directed migration in colorectal cancer cells. In vivo, PTPRK supports recovery from inflammation-induced colitis. In addition, we confirm that PTPRK functions as a tumor suppressor in the mouse colon and in colorectal cancer xenografts. PTPRK regulates growth factor and adhesion signalling, and suppresses epithelial to mesenchymal transition (EMT). Contrary to the prevailing notion that PTPRK directly dephosphorylates EGFR, we find that PTPRK regulation of both EGFR and EMT is independent of its catalytic function. This suggests that additional adaptor and scaffold functions are important features of PTPRK signalling.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Reintroduction of doxycycline inducible PTPRK and mutants into PTPRK knockout colorectal cancer cells grown as xenografts

first: Katherine

last: Young

first: Hayley

last: Sharpe


title: Disease-associated microglia signature genes are up-regulated during photoreceptor degeneration

first: Wenchuan

last: Zhou

address: No. 1665, Kongjiang Road

institute: Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine

summary: Retinal degeneration is a collection of devastating conditions with progressive loss of vision which often lead to blindness. Research on retinal microglial cells offers great therapeutic potential in deterring the progression of the condition. This study explored the mechanisms underlying the TREM2-mediated protective function of activated microglial cells during retinal degeneration. A significant upregulation of disease-associated microglia signature genes was observed during photoreceptor degeneration.

overall_design: RNA-Seq on whole-retina samples from rd10 and wild-type  mouse retina. Two biological replicates of each.

first: Jincan

last: He

first: Jing

last: Li


title: Fc-engineered antibodies leverage neutrophils to drive control of Mycobacterium tuberculosis

status: Public on Jul 01 2024

first: Charles

middle: Ara

last: Demurjian

address: 31 Ames Street

institute: BioMicro Center at MIT

summary: Novel vaccination and therapeutic strategies are urgently needed to mitigate the tuberculosis (TB) epidemic. While extensive efforts have focused on potentiating cell-mediated immunity to control Mycobacterium tuberculosis (Mtb) infection, less effort has been invested in exploiting the humoral immune system to combat Mtb. Emerging data point to a role for antibodies in microbial control of Mtb, however the precise mechanism(s) of this control remain incompletely understood. Here we took an antibody Fc-engineering approach to determine whether Fc-modifications could improve the ability of antibodies to restrict Mtb, and to define Fc-mediated mechanism(s) antibodies leverage for this restriction. Using an antibody specific to the capsular polysaccharide α-glucan, we engineer a panel of Fc variants to augment or dampen select antibody effector functions, rationally building antibodies with enhanced capacity to promote Mtb restriction in a human whole blood model of infection. Surprisingly, restrictive Fc-engineered antibodies drive Mtb control in a neutrophil, not monocyte, dependent manner. Using single cell RNA sequencing, we show that restrictive antibodies promote neutrophil survival and expression of cell intrinsic antimicrobial programs. These data provide a roadmap for exploiting Fc-engineered antibodies as a novel class of TB therapeutics able to harness the protective functions of neutrophils to achieve disease control.

overall_design:
Whole-blood Mtb infection with antibody was placed through a 10X Genomics NextGEM Single Cell 3’ kit and processed using CellRanger v6.1.2 to generate gene expression count matrices and deMULTIplex to generate LMO barcode count matrices.

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Submitter states that missing raw files are due to patient privacy concerns.
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first: Edward

last: Irvine

first: Angel

last: Nikolov

first: Mehak

middle: Z

last: Khan

first: Sarah

middle: M

last: Fortune


title: Sculpting the tumour microenvironment by combining radiotherapy and ATR inhibition for curative-intent adjuvant immunotherapy

status: Public on Jul 04 2024

first: Anton

last: Patrikeev

state: London

address: Chester Beatty Laboratories, 237 Fulham Rd.

department: Targeted Therapy

institute: The Institute of Cancer Research

summary: Results of combining radiotherapy/chemoradiotherapy and immune checkpoint blockade have been disappointing in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). For such a potentially radiocurable disease, there remains an imperative to explore novel combination approaches. Here, we show that combining ATR inhibition with radiotherapy (ATRi/RT) increases the frequency of activated NKG2A/PD-1 double-positive T cells in animal models of HNSCC. Addition of dual anti-NKG2A/-PD-L1 blockade to ATRi/RT in the adjuvant, post-radiotherapy setting induces a robust antitumour response. Efficacy of the combination relies on CD40/CD40L costimulatory-mediated infiltration of activated/proliferative/memory CD8 and CD4 T cells with persistent or new T cell receptor (TCR) signalling, respectively. In this favourable therapeutic context, we reveal increased richness of the TCR repertoire and the emergence of numerous and large TCR clusters that share antigen specificity in response to combination therapy. Collectively, our data point towards promising combination approaches for future clinical testing in HNSCC.

overall_design: To investigate therapeutic effects of combination of ATR inhibition with radiotherapy (ATRi/RT) and dual anti-NKG2A and anti-PD-1/-PD-L1 antibodies, we used 8- to 14-week-old females C57BL/6 WT mice, inoculated with  head and neck cancer cells from several cell lines. We used models of murine immunocompetent HPV-negative and HPV-positive head and neck cancer, MOC1/MOC2 and mEER, respectively. Mice were inoculated subcutaneously (ectopic) on the right flank with 4 × 10^6 cells for the MOC1 or 10^6 cells for the MOC2 and mEER models. Mice were further treated with combination of the ATR inhibitor AZD6738 and radiotherapy OR with anti-NKG2A/anti-PD-L1 antibodies OR with both. Additionally, vehicle-treated mice were added as a control group. Each treatment group consisted of four biological replicates. We then performed gene expression profiling analysis using data obtained from RNA-seq of tumour cells from the treated mice.


title: Deciphering the initial steps of SARS-CoV2 infection in whole human lungs identifies macrophages as primary targets and reveals subset-specific responses.

city: marseille

first: Thien-Phong

last: Vu Manh

address: 163 avenue de Luminy

country: France

institute: CIML

summary: Several in vitro models have been established to mimic the initial interaction between lung cell types and SARS-CoV-2, mainly using explant-based techniques that generated contrasted results about the cell types that were infected. These contrasting results could be due to biases in the various tissue-culture conditions. Furthermore, all these systems do not respect the spatial tissue architecture and the anatomic constraints that shape cell type interactions with viruses.  In this study, we used whole human lungs maintained alive ex vivo for 10 h, based on a technique used in lung transplantation (ex vivo lung perfusion or EVLP), to analyze the first steps of SARS-CoV-2 infection in the lung. Five human donor lungs declined for transplantation were perfused and ventilated at 37°C and three of them were infected with SARS-CoV-2 using nebulization of the Wuhan lineage and D614G variants. Single-cell RNA-sequencing (scRNA-seq) using 10x Genomics 3' (v3 Chemistry) has been performed on these samples.

overall_design:
Donor lungs used in this study were from donation after brain death and were determined to be unsuitable for transplantation. Donor lung retrieval was carried out according to current clinical practice. After transportation at 4°C, lungs were processed to EVLP, with or without virus nebulization. Lung biopsies were taken just before EVLP (0 h) and after 10 h EVLP, and analyzed using scRNAseq.

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Submitter states that missing raw files are due to patient privacy concerns.
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first: Isabelle

last: Schwartz

first: Marc

last: Dalod

first: Edouard

last: Sage


title: NAPRT silencing in FH-deficient renal cell carcinoma confers therapeutic vulnerabilities via NAD+ depletion

city: New Haven

first: Ranjit

last: Bindra

state: CT

address: 15 York Street

department: Therapeutic Radiology

institute: Yale University

summary: Genome wide DNA methylation profiling of normal kidney and FH-deficient renal cell carcinoma cell lines, as well as wild type and IDH1 mutant glioma cell lines to understand patterns of DNA methylation in oncometabolite-producing cancer cell lines. The Illumina Human EPIC Bead Array (850k) platform was used to obtain methylation beta values of over 850k CpG sites throughout the genome across all cell lines.

overall_design: 1 microgram of genomic DNA was bisulfite-converted and analyzed for genome-wide methylation patterns using the Illumina Human EPIC Bead Array (850k) platform at the Yale Center for Genome Analysis for all samples.

middle: S

first: Katelyn

middle: J

last: Noronha

first: Sam

last: Friedman


title: Fasting induces a metabolic switch and a spatial redistribution of tanycyte functions on lipid processing and neuronal interactions.

city: Lausanne

first: Federico

last: Santoni

address: Chemin du Bugnon 27

country: Switzerland

institute: CHUV

summary: The ependyma lining the third ventricle (3V) in the mediobasal hypothalamus is recognized as a critical player in controlling energy balance and glucose homeostasis. Its molecularly distinct cell types, namely diverse tanycyte subpopulations and typical ependymal cells, confer a high functional heterogeneity and plasticity to this complex ependymal system, but the molecular mechanisms governing its features, especially in the context of metabolic regulation, are still not fully understood.Here, 5481 hypothalamic ependymocytes were cataloged using FACS-assisted single-cell RNA sequencing from fed, 12h-fasted, and 24h-fasted adult male mice. We first observed the limitations of using standard clustering analysis to characterize the different ependymal cell subpopulations lining the 3V. Indeed, while typical ependymal cells and β2-tanycytes are sharply defined at the molecular level using this approach, other subpopulations (i.e., β1- and α-tanycytes) display fuzzy boundaries and very few specific markers. Moreover, 12h- and 24h-fasting dynamically modulate gene expression, increasing tanycyte subgroup heterogeneity. To better analyze the dynamic changes in tanycytes’ gene expression profile, we implemented a pseudospatial analysis based on 3V neuroanatomical distribution, which we validated using in-situ hybridization, immunohistochemistry, and histologic staining. This approach improved the characterization of tanycyte markers, refining our knowledge of specific versus shared features and the segregation of subgroup-specific versus general tanycyte functions. Importantly, we showed that fasting dynamically shifts patterns in gene expression and transcription activity along the 3V, inducing metabolic and functional switches in tanycyte subpopulations, particularly regarding lipid metabolism. Additionally, numerous functions related to tanycyte-neuron and tanycyte-synapse interactions, notably through Nrxn1 expression, present a functional dorso→ventral shift towards the arcuate nucleus during fasting. Altogether, this data shows that changes in energy status lead to a spatial redistribution of distinct cell type-specific responses along the 3V and gives new insights into molecular and functional diversity and plasticity within the tanycyte population.

overall_design: Single-cell RNA sequencing data of mouse in different metabolic conditions; normally fed (FED), fasting for 12 hours (FAST12) and fasting for 24 hours (FAST24). Cells were FACS sorted based on tdTomato expression

first: Fanny

last: Langlet

first: Maxime

last: Brunner

first: David

last: Lopez-Rodriguez

first: Judith

last: Estrada-Meza

first: Rafik

last: Dali

first: Antoine

last: Rorhbach

first: Tamara

last: Deglise

first: Andrea

last: Messina

first: Bernard

last: Thorens


title: Blockade of IL-1b and PD-1 with combination chemotherapy reduces systemic myeloid suppression in metastatic pancreatic cancer with heterogeneous effects in the tumor

city: San Diego

first: Diane

last: Simeone

address: 3855 Health Sciences Drive

institute: UCSD Health

summary: Clinical and translational results from a phase Ib trial testing whether IL-1β blockade in human pancreatic cancer would alleviate myeloid immunosuppression and reveal anti-tumor T cell responses to PD-1 blockade.

overall_design: Single-cell RNA-seq analysis of pre- and post-treatment samples from the phase Ib trial

first: Paul

middle: E

last: Oberstein

first: Andressa

last: Dias Costa

first: Emily

middle: A

last: Kawaler

first: Victoire

last: Cardot-Ruffino

first: Jonathan

last: Nowak

first: Stephanie

middle: K

last: Dougan


title: TULIPs decorate the three-dimensional genome of PFA ependymoma

city: Houston

first: Marco

last: Gallo

state: TX

address: 1102 Bates Avenue

department: Pediatrics

institute: Baylor College of Medicine

summary: Hi-C and RNA-seq for a large cohort of pediatric brain tumors including ependymoma (PFA, PFB, Ste, spinal), medulloblastoma (G3, G4, SHH), high grade glioma (H3K27 and H3-WT), pilocytic astrocytoma, and more.

overall_design:
Assessing 3D genome conformation by Hi-C for many types of pediatric brain tumors.

>>>Submitter states that raw data have been deposited at EGA under accession EGAS00001005476<<<

first: Michael

middle: D

last: Taylor

first: Nada

last: Jabado


title: Molecular gatekeepers: how peptide maturation molecules coordinate cell-cell communication in Streptococcus pneumoniae

city: Pittsburgh

first: N.

middle: Luisa

last: Hiller

state: PA

address: 4400 Fifth Ave

department: Biological Sciences

institute: Carnegie Mellon University

summary: Cell-cell communication systems enable populations to regulate behavior via modifications in gene expression. In Gram-positive bacteria, these systems are often composed of secreted, signaling peptides that disperse across the population and interact with cognate transcriptional regulators. While cells generally possess multiple such systems, there is a gap in knowledge regarding molecular mechanisms that coordinate these systems to allow for optimization of phenotypic responses. The human pathogen Streptococcus pneumoniae (Spn) encodes several cell-cell communication systems, notably multiple members of the Rgg/SHP and the Tpr/Phr family. Previous studies have characterized their individual regulatory networks and established their importance in Spn virulence and until now, these two signaling families were thought to work independently. Our study provides the molecular link between Rgg/SHP and TprA/PhrA systems by demonstrating that conserved peptide maturation molecules are integral to the coordination of both systems in Spn. We reveal that the ABC transporter PptAB and the transmembrane enzyme Eep display a dual mode of action, simultaneously activating the Rgg/SHP systems and repressing the TprA/PhrA system. Specifically, they regulate the respective precursor peptides (SHP and PhrA) before these leave the cell. This dual function leads to temporal coordination of these systems producing an overlap between their respective regulons during host cell infection. Thus, we have identified a single molecular mechanism that targets diverse cell-cell communication systems. Moreover, the signaling systems and peptide maturation molecules are also encoded by closely related species and other Gram-positive bacteria suggesting this coordination mechanism may have applications across species boundaries.

overall_design: We employed RNA-seq to compare gene expression between WT Spn and a deletion of the pptAB transporter.  Deletion of the phrA signaling peptide was used as control for lack of signaling. A double deletion of pptAB and of the peptide importer amiA-F was used to capture a condition where extracellular PhrA is not imported.

first: Karina

last: Mueller Brown

first: Derek

last: Wang

first: Qidong

last: Jia

first: Jason

last: Rosch

first: N

middle: L


title: Reduced WNT4 expression in normal skin fibroblasts leads to ‘Dupuytren-like’ changes in the transcriptome

city: Crawley

first: Andrew

middle: William

last: Stevenson

state: Western Australia

address: Room 112b, Curnow Building, Hackett Drive Carpark Entrance 2, UWA

department: School of Biomedical Sciences

country: Australia

institute: University of Western Australia

summary: Background: Dupuytren’s disease (DD) is a fibro-proliferative disorder of unknown aetiology. Previous studies have implicated multiple WNT signalling genes/proteins in Dupuytren pathology, including WNT4. However, it is not yet clear whether WNT signalling dysregulation plays an important role in the initiation of the disease or progression. The aim of this study was to determine if loss of WNT4 expression triggered ‘Dupuytren-like’ changes in the transcriptome of healthy skin fibroblasts. Methods: Fibroblasts were isolated from the wrists of healthy adult males and from the wrists and disease cord tissue from males in a family positive for Dupuytren’s disease.  Normal skin fibroblasts from healthy controls were treated with WNT4 siRNA and scrambled controls. RNASeq was used to analyse the transcriptomes of disease and non-disease fibroblasts from patients with Dupuytren’s as well as in siRNA treated and non-treated control fibroblasts.  Results: Analysis of the transcriptomes from DD patient and normal skin fibroblasts showed significant differences, including in WNT4 expression. Downregulation of WNT4 in normal skin fibroblasts using siRNA led to ‘DD-like’ changes in the transcriptome. Conclusion: In people susceptible to DD WNT4 is downregulated even in non-fibrotic fibroblasts. Knockdown of WNT4 in normal fibroblasts led to changes that made cells ‘DD-like’. This study shows that WNT4 is down regulated in ‘non-disease’ cells, and that downregulating WNT4 in normal skin fibroblasts leads to widespread ‘DD like’ changes in the transcriptome, suggesting WNT4 downregulation is a key driver of DD.

overall_design: Cells cultured from the diseased cord tissue of patients with dupuytrens disease (DD), as well as unaffected wrist skin from these same patients (DDC), were compared to cultured cells from wrist skin of unaffected controls (NS). Knockdown of WNT4 using siRNA was carried on the DDC cells, and compared to the existing RNAseq for the DD, NS and DDC cells.

first: Mark

middle: W

last: Fear

first: Aoife

last: O'Brien

first: Amira

last: Allahham


title: p53R172H and p53R245W Mutants Drive Distinct Transcriptomes in Mouse Mammary Tumors Through a Convergent Transcriptional Mediator

first: Xiaoping

last: Su

state: Texas

address: 7300 Brompton St, Apt 5633

institute: MD Anderson Cancer Center

summary: Aggressive breast cancers harbor TP53 missense mutations. Tumor cells with TP53 missense mutations exhibit enhanced growth and survival through transcriptional rewiring. To delineate how TP53 mutations in breast cancer contribute to tumorigenesis and progression in vivo, we created a somatic mouse model driven by mammary epithelial cell-specific expression of Trp53 mutations. Mice developed primary mammary tumors reflecting the human molecular subtypes of Luminal A, Luminal B, HER2-enriched, and Triple Negative Breast Cancer with metastases. Transcriptomic analyses comparing MaPR172H/- or MaPR245W/- mammary tumors to MaP-/-tumors revealed (1) differences in cancer associated pathways activated in bothp53 mutants and (2) Nr5a2 as a novel transcriptional mediator of distinct pathways in p53 mutants. Meta-analyses of human breast tumors corroborated these results. In vitro assays demonstrate mutant p53 upregulates specific target genes that are enriched for Nr5a2 response elements in their promoters. Co-immunoprecipitation studies revealed p53R172H and p53R245W interact with Nr5a2. These findings implicate NR5A2 as a novel mediator of mutant p53 transcriptional activity in breast cancer.

overall_design:
Paired end RNA Seq of mammary tumors harvested from  MaPR172H/-, MaPR245W/-, and MaP-/- mice to undertand how mutant p53 contributes to breast cancer.
T1 is the tumor tissue extracted from left side of mouse sample, and T2 is the tumor tissue extracted from right side of mouse sample.

first: Joy

last: McDaniel

first: Guillermina

last: Lozano


title: Genome wide mapping of IKAROS binding (ChIP-Seq) in U-2932 cell line.

city: Summit

first: Matthew

last: Stokes

state: NJ

address: 556 Morris Ave

institute: Bristol Myers Squibb

summary: We here report the genome-wide binding of IKAROS in diffuse large B-cell lymphoma (DLBCL) cell line U-2932.

overall_design: One IKAROS ChIP-Seq for U-2932 cell line; One input control.

first: Junfei

last: Zhao

first: Chih-Chao

last: Hsu

first: Patrick

last: Hagner


title: Aberrant DNA methylation occurs in RUNX1 heterozygous mutations harboring hematopoietic progenitor cells [CUT&RUN]

city: Yokohama

first: Yuki

last: Tanaka

state: Kanagawa

address: 1-7-22 Suehiro-cho, Tsurumi-ku

department: Center for Integrative Medical Sciences

institute: RIKEN

summary:
Background: Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by a heterozygous germline mutation inRUNX1. The patients with FPDMM show not only thrombocytopenia with platelet dysfunction, but also a high level of developing hematological malignancies, strongly suggesting that FPDMM is in a precancerous state. However, the DNA methylation status of FPDMM has not yet been elucidated due to no animal models for FPDMM and the difficulty in obtaining the patient-derived samples.
Results: We found that differentiation efficiencies into HPCs and megakaryocytes was reduced in the FPDMM-mimicking cells, which were established by genome editing for human iPS cells as a FPDMM-model, compared with those of wild-type cells. The FPDMM-mimicking HPCs were subjected to DNA methylation analysis, and the HPCs showed the distinct DNA methylation patterns compared to wild-type HPCs. Furthermore, we identified FLI1 as a putative causative transcription factor of the differential DNA hypermethylation involved in both promoting the binding site-directed DNA demethylation and regulating megakaryopoiesis.

overall_design:
To establish the human iPS cell lines with FPDMM-mimicking RUNX1 heterozygous mutations, we performed CRISP−Cas9-mediated genome editing of RUNX1, followed by single-cell cloning of the resulting genome-edited cells.
We performed cleavage under targets and release using nuclease (CUT&RUN) sequencing for FLI1 to confirm its potential binding in the vicinity of hypermethylated differentially methylated cytosine-guanine dinucleotides (DMCs) in FPDMM-mimicking HPCs.

first: Yuri

last: Nakanishi

first: Erina

last: Furuhata

first: Ken-ichi

last: Nakada

first: Rino

last: Maruyama

first: Harukazu

last: Suzuki

first: Takahiro


title: Transcriptome profiles of wild-type, IRF8KO microglia

city: Bethesda

first: Keita

last: Saeki

state: Maryland

address: 6 Center Drive

institute: NIH/NICHD

summary: The lineage-determining transcription factor IRF8's function in microglia is unclear. Our study aims to shed light on this by analyzing the genome-wide, temporal transcriptome profiles of wild-type and IRF8KO microglia. We used deep-sequencing and differential analysis to identify the effects of IRF8 on transcription programs in microglia, such as microglia identity gene expression. This study provides a new understanding of the transcriptional programs in microglia.

overall_design: Transcriptome profiles of WT and IRF8KO microglia were analyzed.

first: Keiko

last: Ozato


title: IRF8 defines the epigenetic landscape in postnatal microglia, thereby directing their transcriptome programs - additional datasets

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

overall_design: Refer to individual Series


title: Diurnal Retinal and Choroidal Gene Expression Patterns Distinguish Myopia Progression from Myopia Onset

city: Boston

first: Debora

last: Nickla

address: 424 Beacon St

department: Dept. of Biosciences and Disease

institute: New England College of Optometry

summary: Following laboratory and clinical findings implicating circadian biology in the pathogenesis of myopia (nearsightedness), we examined gene expression in two crucial tissues controlling post-natal refractive development, the retina and choroid. Inducing unilateral visual form deprivation myopia in young chicks, a widely studied and validated model, we isolated retinal and choroidal tissues every 4 hours over a single day from myopic and contralateral control eyes during a period time when myopia progresses rapidly.

overall_design: After applying RNA-Seq to each tissue extract, we used various bioinformatics and gene expression profiling analyses to assess the retinas and choroids at each time point. We extended these findings with Gene Set Enrichment Analysis and with comparison to candidate genes for human myopia.

first: Richard

last: Stone

first: John

last: Tobias

first: P

last: Iuvone


title: Single-cell transcriptome profiles of wild-type and IRF8KO microglia

summary: IRF8 is a transcription factor that is crucial in determining the lineage of microglia. We previously showed that IRF8 is robustly bound during the early postnatal period. To better understand IRF8's postnatal functions, we analyzed mice in which IRF8 was removed after P16. By using single-cell nuclei sequencing, we analyzed cells in which IRF8 was definitely knocked out and investigated both the transcriptome and chromatin accessibility that IRF8 regulates. The differential analysis showed the importance of IRF8 in regulating postnatal transcriptional programs and epigenetic landscapes that are related to microglial identity and disease-associated genes. This study provides a new perspective on how IRF8-mediated transcriptional programs affect microglia.

overall_design: Transcriptome profiles and chromatin landscapes of adult WT and IRF8 conditional knockout (IRF8cKO) microglia were analyzed simultaneously at a single-cell level using 10xGenomics.


title: Attenuated effector T cells are linked to control of chronic HBV infection

city: Freiburg

first: Sagar

address: Hugstetter Straße 55

department: Department of Internal Medicine II

country: Germany

institute: University Medical Center Freiburg

summary: Hepatitis B virus (HBV)-specific CD8+ T cells play a dominant role during acute-resolving HBV infection but are functionally impaired during chronic HBV infection in humans. These functional deficits have been linked with metabolic and phenotypic heterogeneity, but it has remained unclear to what extent different subsets of HBV-specific CD8+ T cells still suppress viral replication. We addressed this issue by deep profiling, functional testing and perturbation of HBV-specific CD8+ T cells during different phases of chronic HBV infection. Our data revealed a mechanism of effector CD8+ T cell attenuation that emerges alongside classical CD8+ T cell exhaustion. Attenuated HBV-specific CD8+ T cells were characterized by cytotoxic properties and a dampened effector differentiation program, determined by antigen recognition and TGFβ signaling, and were associated with viral control during chronic HBV infection. These observations identify a distinct subset of CD8+ T cells linked with immune efficacy in the context of a chronic human viral infection with immunotherapeutic potential.

overall_design:
Live HBV epitope-specific CD8+ T cells were sorted in 1,5mL microcentrifuge tubes (Eppendorf, Germany) containing 20µl PBS using BD Aria Fusion (BD, Germany). Naive, CD45RA+CCR7+, T cells were excluded. Sorted cells were processed through the 10x Genomics single-cell workflow with feature barcoding technology to multiplex cells from different donors so that they could be loaded on one well to reduce costs and minimize technical variability. Hashtag oligos were obtained as purified and already oligo-conjugated in TotalSeq-C (5’ chemistry) format from BioLegend.

**Raw data are not provided due to privacy concerns**

last: Sagar

first: Kathrin

last: Heim

first: Robert

last: Thimme

first: Maike

last: Hofmann


title: Genome-wide open chromatin status in WT and IRF8KO microglia

summary: The role of a transcription factor, Interferon Regulatory Factor 8 (IRF8), in microglia is yet to be fully understood. We conducted a study to examine the chromatin status of postnatal and adult wild-type (WT) and IRF8-knockout (IRF8KO) microglia through ATAC-Seq. We also added 5xFAD Alzheimer's disease microglia to observe any chromatin events that may be similar in disease-associated microglia. After deep-sequencing and differential analysis, we discovered that chromatin accessibility in microglia is dependent on IRF8. In the absence of IRF8, there was aberrant chromatin accessibility that potentially resulted in mRNA transcription of genes that were upregulated in IRF8KO microglia, which was distinct from 5xFAD microglia. This study contributes to a better understanding of epigenetic regulation in microglia.

overall_design: Examination of chromatin accessibility in microglia.


title: ParBF DNA binding profiles over various supercoiling level and on linear DNA

city: Toulouse

first: Jean-Yves

last: Bouet

address: 169 avenue Marianne Grunberg-Manago

institute: CNRS

summary: We report the genome-wide analysis from chromatin immunoprecipitated DNA (ChIP-sequencing) of the DNA binding pattern of ParBF (SopB) of plasmid F. This study, performed in E. coli and Salmonella typhimurium, investigates the impact of DNA supercoiling on ParBF DNA binding profiles in vivo. We found that variation in DNA supercoiling does not significantly affect the ParB DNA binding profiles even on linear DNA.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2

overall_design: Chromatin immunoprecipitation DNA-sequencing for ParB of plasmid F (ParBF) in bacterial strains exhibiting various DNA supercoiling level, and on linearized plasmid F at several positions relative to the centromere parSF. Biological replicates are labelled IP1 and IP2. Anti ParBF antibody is affinity purified before used.

first: Hicham

last: Sekkouri Alaoui

first: Jerome

last: Rech

first: Delphine

last: Labourdette


title: Tumor intrinsic activity of Chromobox 2 (CBX2) remodels the tumor microenvironment in high grade serous carcinoma

city: Denver

first: Benjamin

last: Bitler

institute: The University of Colorado


title: Iron-Dependent KDM4D Activity Controls the Quiescence-Activity Balance of MSCs via the PI3K-Akt-Foxo1 Pathway [CUT&Tag]

city: Shenzhen

first: Yunshu

last: Che

address: NO.3025 shennan road

institute: Sun Yat-sen University

summary: Iron deficiency is a common nutritional deficit that can lead to organ damage or dysfunction. Research is increasingly linking iron deficiency to dysfunction of bone metabolism, although the exact mechanisms remain unclear. Some studies suggest that iron-dependent methylation-erasing enzyme activity regulates cell proliferation and differentiation under physiological or pathological conditions. Whether iron deficiency inhibits the activation of quiescent mesenchymal stem cells (MSCs) by affecting histone demethylase activity is unclear. In our study, we discovered that KDM4D plays a pivotal role in the activation of quiescent MSCs. Under conditions of iron deficiency, the H3K9me3 demethylase activity of KDM4D significantly decreased. This change led to increased heterochromatin with H3K9me3 near the PIK3R3 promoter, hindering the expression of PIK3R3. Subsequently, the activation of quiescent MSCs was inhibited via the PI3K-Akt-Foxo1 pathway. Iron-deficient mice exhibited significantly inhibited bone marrow MSC activation and reduced bone mass compared to normal mice. Modulating the PI3K-Akt-Foxo1 pathway could reverse iron deficiency-induced bone loss.

overall_design: CUT&Tag-seq for histone modification H3K9me3 in MSCs

first: Huiyong

last: Shen

first: Zhongyu

last: Xie


title: Iron-Dependent KDM4D Activity Controls the Quiescence-Activity Balance of MSCs via the PI3K-Akt-Foxo1 Pathway [RNA-Seq]

overall_design: Examination of the mRNA profiles in MSCs under DFO treatment or KDM4D knockdown during quiescent MSC mobilization


title: Iron-Dependent KDM4D Activity Controls the Quiescence-Activity Balance of MSCs via the PI3K-Akt-Foxo1 Pathway


title: A Th17 cell-intrinsic glutathione/mitochondrial-IL-22 axis protects against intestinal inflammation

city: Esch-sur-Alzette

first: Joseph

last: Longworth

state: Luxembourg

address: 29, rue Henri Koch

department: Department of Infection and Immunity

country: Luxembourg

institute: LIH

summary: The intestinal tract generates significant reactive oxygen species (ROS), but the role of T cell antioxidant mechanisms in maintaining intestinal homeostasis is poorly understood. We used T cell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), which impaired glutathione (GSH) production, crucially reducing IL-22 production by Th17 cells in the lamina propria, which is critical for gut protection. Under steady-state conditions, Gclc deficiency did not alter cytokine secretion; however, C. rodentium infection induced increased ROS and disrupted mitochondrial function and TFAM-driven mitochondrial gene expression, resulting in decreased cellular ATP. These changes impaired the PI3K/AKT/mTOR pathway, reducing phosphorylation of 4E-BP1 and consequently limiting IL-22 translation. The resultant low IL-22 levels led to poor bacterial clearance, severe intestinal damage, and high mortality. Our findings highlight a previously unrecognized, essential role of Th17 cell-intrinsic GSH in promoting mitochondrial function and cellular signaling for IL-22 protein synthesis, which is critical for intestinal integrity and defense against gastrointestinal infections.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Th17 cells with and without Gclc knockout using Cd4Cre Gclc fl/fl mice. Cd4Cre Gclc fl/fl mice investigated with and without NAC (N-Acetyl Cysteine) treatment. Each condition with 3 Biological replicates included.

first: Dirk

last: Brenner

first: Lynn

last: Bonetti

first: Veronika

last: Horkova


title: CRISPR activation screens identify the SWI-SNF ATPases as suppressors of ferroptosis [NGS4182_ATAC]

city: South San Francisco

first: Marc

last: Hafner

address: Building 45-1, 1 DNA Way

department: Oncology Bioinformatics

institute: Genentech

summary: Ferroptosis is an iron-dependent cell death mechanism characterized by an accumulation of toxic lipid peroxides and membrane rupture. The glutathione dependent enzyme, GPX4 (glutathione peroxidase 4), prevents ferroptosis by reducing these lipid peroxides into non-toxic lipid alcohols. Ferroptosis induction by GPX4 inhibition has emerged as a vulnerability of cancer cells, thus highlighting the need to identify ferroptosis regulators that may be exploited therapeutically. Through genome-wide screens and a series of genetic, genomic, and quantitative imaging approaches, we identify the SWI-SNF ATPases BRM and BRG1 as ferroptosis suppressors. Mechanistically, they directly bind to and catalytically increase chromatin accessibility at NRF2 target loci, thus boosting NRF2 transcriptional output. This primes cells to counter lipid peroxidation and confers resistance to GPX4 inhibition and ferroptosis. Importantly, we demonstrate that the BRM/BRG1-ferroptosis connection can be leveraged to enhance the paralog dependency of BRG1-mutant lung cancer cells on BRM, especially in lines that are less sensitive to BRM inhibition or degradation. Our data reveal ferroptosis induction as a potential avenue for broadening the efficacy of BRM degraders/inhibitors and define a specific genetic context for exploiting GPX4 dependency.

overall_design: Bulk ATAC seq

first: Kamakoti

last: Bhat

first: Xin

last: Ye

first: Musaddeque

last: Ahmed

first: Michal

last: Kabza

first: Jinchu

last: Vijay


title: CRISPR activation screens identify the SWI-SNF ATPases as suppressors of ferroptosis [NGS4183_ATAC]


title: CRISPR activation screens identify the SWI-SNF ATPases as suppressors of ferroptosis [NGS3994_ATAC]

last: hafner


title: CRISPR activation screens identify the SWI-SNF ATPases as suppressors of ferroptosis [NGS3991_RNA]

overall_design: Bulk RNA-Seq


title: CRISPR activation screens identify the SWI-SNF ATPases as suppressors of ferroptosis


title: CRISPR activation screens identify the SWI-SNF ATPases as suppressors of ferroptosis [NGS3855_barcodes_gDNA]

overall_design: Sequencing of TraCe-seq barcodes from gDNA of BRM-inducible KP4 cells treated with or without doxycycline to induce BRM overexpression, followed by ML210 treatment and a 3-day recovery period.

first: Jun

last: Zou

first: Caroline

last: Vilas

first: Steffen

last: Durinck


title: Expression data derived from adenocarcinoma or neuroendocrine prostate cancer of TRAMP and SPARC deficient TRAMP mice.

city: Milan

first: Annamaria

last: Piva

address: via Amadeo, 42

country: Italy

institute: Fondazione IRCCS Istituto Nazionale dei Tumori

summary: In this dataset we show transcriptional profiles derived from adenocarcinoma or incipient neuroendocrine prostate cancer (NEPC) derived from TRAMP or SPARC-deficient TRAMP mice respectively. Genetic ablation of SPARC rendered prostate cancer cells to be prone to NEPC differentiation.

overall_design: Total RNA was obtained from prostate tumors from 18 and 30-week-old with SPARC-deleted TRAMP mice with adenocarcinoma and NEPC (n=7 for each time point and tumor type). Then, we performed transcription array with Clariom S assay.

first: Roberta

last: Sulsenti

first: Giuseppina

middle: B

last: Scialpi

first: Barbara

last: Frossi

first: Laura

last: Botti

first: Renata

last: Ferri

first: Irene

last: Tripodi

first: Sabina

last: Sangaletti

first: Valeria

last: Cancila

first: Claudia

last: Chiodoni

first: Daniele

last: Lecis

first: Francesca

last: Bianchi

last: Fischetti

last: Enriquez

first: Filippo

last: Crivelli

first: Marco

last: Bregni

first: Giuseppe

last: Renne

first: Claudio

last: Tripodo

first: Carlo

last: Pucillo

first: Mario

last: Colombo

first: Elena

last: Jachetti


title: An engineered trafficking biosensor reveals a role for DNAJC13 in DOR downregulation

city: Portland

first: Braden

last: Lobingier

state: Oregon

address: 3181 SW Sam Jackson Park Road, Mail Code L-334

department: Chemical Physiology and Biochemistry

institute: Oregon Health and Sciences University

summary: Trafficking of G protein-coupled receptors (GPCRs) through the endo-lysosomal pathway is critical to homeostatic regulation of GPCRs following activation with agonist. Identifying the genes involved in GPCR trafficking is challenging due to the complexity of sorting operations and low affinity protein-receptor interactions. Here we show that the engineered enzyme APEX2 is a highly sensitive biosensor for GPCR trafficking to the lysosome, and this trafficking can be monitored through APEX-based activation of fluorogenic substrates such as Amplex UltraRed (AUR). We used GPCR-APEX2/AUR to perform a genome-wide CRISPR interference screen focused on the delta type opioid receptor (DOR), a GPCR which modulates anxiety and pain. We provide a genetic map of components in the endo-lysosomal pathway necessary for DOR trafficking to the lysosome and subsequent downregulation. Using our GPCR-APEX2 pipeline, we demonstrate that a gene identified in our screen, DNAJC13, controls trafficking of DOR to the lysosome by regulating the endosomal proteome and endosomal homeostasis.

overall_design: Single guide RNA (sgRNA) enrichment within sorted cell populations based on fluorogenic reaction readout for opioid receptor abundance after receptor agonist stimulation.

first: Brandon

last: Novy

first: Aleksandra

last: Dagunts

first: Tatum

last: Weishaar

first: Emily

last: Holland

first: Hayden

last: Adoff

last: Hutchinson

first: Monica

last: De Maria

first: Martin

last: Kampmann

first: Nikoleta

middle: G

last: Tsvetanova

middle: T


title: CRISPR activation screens identify the SWI-SNF ATPases as suppressors of ferroptosis [NGS3789_snRNA]

first: Marc

last: Hafner

overall_design: Single cell multiome profiling of BRM-inducible KP4 cells treated with or without doxycycline to induce BRM overexpression (snRNA-seq).

first: Kamakoti

middle: P

last: Bhat

first: Xin

last: Ye

first: Caroline

last: Vilas

first: Steffen

last: Durinck


title: CRISPR activation screens identify the SWI-SNF ATPases as suppressors of ferroptosis [NGS3790_scATAC]

overall_design: Single cell multiome profiling of BRM-inducible KP4 cells treated with or without doxycycline to induce BRM overexpression (scATAC-seq).


title: Effect of Molidustat treatment on hepatocellular carcinoma cells Huh7

city: Lyon

first: Olivier

last: Diaz

address: Avenue Tony Garnier

department: Centre International de Recherche en Infectiologie (CIRI)

institute: INSERM U1111 – CNRS UMR5308 Université Lyon 1, ENS de LyonINSERM U1111 – CNRS UMR5308 Université Lyon 1, ENS de Lyon

summary: Molidustat is an inhibitor of prolyl hydroxylase that activates the hypoxia response pathway. We analyzed the transcriptome of liver cells treated with this drug and the consequences on metabolic reprogramming.

overall_design: Triplicate RNA extractions were analyzed for each cell line

first: Pierre-Olivier

last: Vidalain

first: Laure

last: Perrin-Cocon

first: Vincent

last: Lotteau

first: Clémence

last: Jaquemin


title: Direct in vivo cardiac reprogramming improves cardiac function and ventricular remodeling in chronic MI.

city: Shinjuku

first: Taketaro

last: Sadahiro

state: Tokyo

address: 35 Shinanomachi

institute: Keio University School of Medicine

summary:
Heart failure (HF) is a leading cause of morbidity and mortality. As adult cardiomyocytes (CMs) have little regenerative capacity, after myocardial infarction (MI), resident cardiac fibroblasts (CFs) synthesize extracellular matrix to form scar tissues, resulting in myocardial remodeling and HF. Thus, both cardiac regeneration and fibrosis are therapeutic targets for chronic MI.
We previously reported that fibroblasts were directly reprogrammed into induced CMs (iCMs) by overexpression of cardiogenic transcription factors in acute and chronic MI. Here we show that in vivo cardiac reprogramming improved cardiac function, and reversed cardiac remodeling in chronic MI using a novel transgenic mouse system. Transcriptome analysis revealed that in vivo cardiac reprogramming suppressed signs of fibrosis and inflammation. Thus, in vivo cardiac reprogramming may be a promising approach for chronic HF.

overall_design: Hearts were harvested from myocardial infarction at 2 or 3 months (8W-Ctrl, 12W-Ctrl and 12W-TTg) for microarray analysis (n = 2 independent biological replicates) (Clariom S Array, Mouse, Affymetrix).

first: Hidenori

last: Tani

first: Yuto

last: Abe

first: Masaki

last: Ieda


title: A non-canonical role for a small nucleolar RNA in ribosome biogenesis and senescence (Ribosome profiling)

city: Dallas

first: Joshua

last: Mendell

address: 6000 Harry Hines Blvd., NA6.200A

department: Molecular Biology

institute: UT Southwestern Medical Center

summary: Oncogene-induced senescence is an important tumor suppressor mechanism that limits proliferation of cells harboring oncogenic mutations. Through a genome-wide screen, we discovered a conserved snoRNA, SNORA13, that is required for this pathway. Although SNORA13 guides the pseudouridylation of a highly conserved nucleotide in the ribosomal decoding center, loss of this snoRNA minimally impacts translation. Instead, we found that SNORA13 negatively regulates ribosome biogenesis. Oncogenic stress perturbs ribosome biogenesis, resulting in the accumulation of free ribosomal proteins (RPs) that trigger p53 activation. We showed that SNORA13 interacts directly with RPL23, decreasing its incorporation into maturing 60S subunits and, consequently, increasing the pool of free RPs, thereby promoting the p53-mediated senescence program. Thus, SNORA13 regulates ribosome biogenesis and the p53 pathway through a non-canonical mechanism distinct from its role in guiding RNA modification. These findings expand our understanding of snoRNA functions and the roles of these abundant noncoding RNAs in cellular signaling.

overall_design: Ribosome profiling was performed in WT and SNORA13 KO BJ cells. Two independent WT clones and two independent KO clones were used.  Samples were collected without (4-OHT negative) and with 7 days (Z)-4-Hydroxytamoxifen treatment (4-OHT positive).

first: Yujing

last: Cheng

first: Siwen

last: Wang

first: He

last: Zhang


title: Biomimetic Cell Stimulation with Graphene Oxide Antigen Presenting Platform (GO-APP) for Developing T Cell-Based Therapies

city: Los Angeles

first: Lili

last: Yang

state: California

address: 610 Charles E. Young Dr. East, TLSB 3045

institute: UCLA Lili Yang Lab

overall_design: Refer to individual Series


title: Biomimetic Cell Stimulation with Graphene Oxide Antigen Presenting Platform (GO-APP) for Developing T Cell-Based Therapies [RNA-Seq]

summary: Graphene Oxide Antigen Presenting Platform was designed for human T cell activation superior to commercial beads. We used deep RNA sequencing to study the mechanism of the T cell activation from PBMCs.

overall_design: T cells were activated and expanded from PBMCs using Beads3/28 (αCD3 and αCD28 on commercial beads) and GO-APP3/28 (αCD3 and αCD28 on graphene oxide antigen presenting platform) respectively. Activated T cells were analyzed using deep RNA sequencing.

first: Zhu

last: Enbo

first: Yu

last: Jiaji

first: Li

last: Yan-Ruide

first: Ma

last: Feiyang

first: Liu

last: Miao

first: Kim

last: Yu Jeong

first: Hanh

last: Zoe

first: Zhou

first: Brown

last: James

first: Zhang

last: Yuchong

first: Pelegrini

last: Matteo

first: Hsiai

last: Tzung

first: Yang

last: Lili

first: Huang

last: Yu


title: A non-canonical role for a small nucleolar RNA in ribosome biogenesis and senescence (RNA-sequencing)

first: Joshua

last: Mendell

overall_design: RNA-sequencing was performed in WT and SNORA13 KO BJ cells. Two independent WT clones and two independent KO clones were used.  Samples were collected without (4-OHT negative) and with 7 days (Z)-4-Hydroxytamoxifen treatment (4-OHT positive).

first: Yujing

last: Cheng

first: Siwen


title: Biomimetic Cell Stimulation with Graphene Oxide Antigen Presenting Platform (GO-APP) for Developing T Cell-Based Therapies [scRNA-Seq]

first: Lili

summary: Graphene Oxide Antigen Presenting Platform was designed for human T cell activation superior to commercial beads. We used single cell RNA sequencing (scRNA-seq) to study the genome-wide transcriptome profiling of the activated T cells from PBMCs.

overall_design: T cells were activated and expanded from PBMCs using Beads3/28 (αCD3 and αCD28 on commercial beads) and GO-APP3/28 (αCD3 and αCD28 on graphene oxide antigen presenting platform) respectively. Activated T cells were analyzed using single cell RNA sequencing (scRNA-seq).

first: Zhu


title: A non-canonical role for a small nucleolar RNA in ribosome biogenesis and senescence (CRISPRi screen)

overall_design: A genome-wide CRISPRi screen was performed in BJ cells expressing tamoxifen-inducible HRAS(G12V) and dCas9. Two independent clones were used for the screen. Samples were collected before (D0, control samples) and 21 days after (Z)-4-Hydroxytamoxifen treatment (D21).

last: Wang

first: He

last: Zhang


title: Deciphering metastatic route-specific signals and their microenvironment interactions in peritoneal metastasis of gastric cancer

city: Seoul

first: Kitae

last: Kim

country: South Korea

institute: Seoul National University

summary: Gastric cancer (GC) constitutes a significant cause of cancer-related mortality worldwide, with metastatic patterns including hematogenous, peritoneal, and ovarian routes. Although GC gene expression patterns have been extensively researched, the metastasis-specific gene expression landscape remains largely unexplored. This study undertook a whole transcriptome sequencing analysis of 66 paired primary and metastatic (hematogenous, peritoneal, or ovarian) GC tumors from 14 patients, leading to the identification of 122 unique metastasis-specific epithelial-mesenchymal transition (msEMT) genes. These genes demonstrated varying expression patterns depending on the metastatic route, suggesting route-specific molecular mechanisms in GC metastasis. High expression of msEMT genes in primary tumors was associated with more frequent CDH1 mutations, the genomically stable subtype, and poor prognosis in The Cancer Genome Atlas GC cohort. This association was further corroborated by poor prognosis and high predictive performance for peritoneal or ovarian recurrence in two independent cohorts (GSE66229; n=300, GSE84437; n=433). Single-cell RNA sequencing analysis of primary tumors (GSE167297) and four independent ascites samples from GC patients revealed that msEMT genes were predominantly expressed in diverse fibroblast sub-populations, rather than cancer cells. This study illuminates the route-specific mechanisms and underlines the significance of msEMT genes and cancer-associated fibroblasts in GC metastasis, highlighting potential directions for future research.

overall_design: Gastric cancer (GC) with metastatic including hematogenous, peritoneal, and ovarian.


title: Transcriptomic profiling of dormancy developmental cycle in DHA-treated P. falciparum

city: Singapore

first: Zbynek

last: Bozdech

address: 60 Nanyang Drive

department: School of Biological Sciences

country: Singapore

institute: Nanyang Technological University

summary: Transcriptome of DHA-treated Plasmodium falciparum 3D7 parasite strain was generated by collecting RNA samples from 500 sorted cells from Day 1 till Day 12 after the drug treatment

overall_design: A total of 37 transcriptomes collected from Day 1 till Day 12 after the drug treatment, sequenced on Illumina NovaSeq6000 platform

first: Jaishree

last: Tripathi

first: Michal

last: Stoklasa

first: Sourav

last: Nayak


title: Impaired repopulating ability of Uhrf2-/- hematopoietic progenitor cells in mice. [RNA-Seq]

city: Fukushima

first: Kazuhiko

last: Ikeda

state: Fukushima

address: 1 Hikariga-oka

department: Blood Transfusion and Transplantation Immunology

institute: Fukushima Medical University

summary: We generated Uhrf2-/- mice to clarify the role of deletion of UHRF2 in hematopoiesis.  In competitive repopulation assay, the proportions of Uhrf2-/- cells were decreased relative to Uhrf2+/+ cells in all lineages.

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of Uhrf2-KO and WT LSK cells.

first: Koki

last: Ueda

first: Keiji

last: Minakawa


title: Exosomes from VZV-infected neurons

city: Burlington

first: Seth

last: Frietze

state: VT

address: 149 Beaumont Avenue

department: Biomedical and Health Sciences

institute: University of Vermont

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


title: A non-canonical role for a small nucleolar RNA in ribosome biogenesis and senescence


title: Comprehensive analysis of the secretome and transcriptome stability from WJ-Mesenchymal Stromal cells throughout GMP-Manufacturing

status: Public on Jun 30 2024

city: Bogota

first: Cristian

middle: Camilo

last: Galindo

state: Bogota DC

address: Cra. 32 #12 - 81 Puente Aranda

country: Colombia

institute: Instituto Distrital de Ciencia, Biotecnología e Innovación en Salud IDCBIS

summary: Mesenchymal stromal cells (MSCs) hold promise for cell-based therapies due to their ability to stimulate tissue repair and modulate immune responses.  Umbilical cord-derived MSC from Wharton’s jelly (WJ), offer advantages such as low immunogenicity and potent immune modulatory effects. However, ensuring consistent quality and safety throughout their manufacturing process remains critical. RNA sequencing (RNA-seq) emerges as a crucial tool for assessing genetic stability and expression dynamics. This study examines the secretome and transcriptome signatures throughout WJ-MSCs Good Manufacturing Practice (GMP) production, focusing in the role of Total RNA or Massive Analysis of cDNA Ends (MACE-Seq)-RNA sequencing. Through comprehensive transcriptomic analysis, we demonstrated the stability of WJ-MSC transcriptome across manufacturing stages. Notably, MACE-Seq enhanced the identification of key expression patterns related to senescence and immunomodulation. These findings highlight the potential of MACE-Seq as a routine quality assessment tool for WJ-MSC-based therapies, ensuring their efficacy and safety in clinical applications. Importantly, MACE-Seq proves valuable for characterizing WJ-MSC products, providing insights unattainable through traditional assays.

overall_design: Using conventional RNA-Seq and MACE-seq, we compared the transcriptome of WJ-MSCs at different stages of GMP production.  MSCs from a master cell bank (MCB) were seeded at two different cell densities (high and low). Cells at high density reached passage 3, while cells at low density reached passage 2. RNA was extracted from cells of the MCB and from pasasges 2 and 3. The same RNA sample was used for both RNA sequencing techniques. Comparative gene expression analysis was performed, focusing on the effect of the two seeding densities and the unique gene expression signatures identified by each sequencing approach

first: Mariana

last: Cañas

last: Camilo Galindo

first: Monica

last: Cruz Barrera

first: Katherine

last: Herrera

first: Karl

last: Beltrán

first: Alejandro

last: Rodríguez

first: Björn

last: Rotter

first: Bernardo

last: Camacho

first: Gustavo

last: Salguero


title: MSTR-1::FLAG and N2

city: Toronto

first: Matthew

last: Eroglu

state: ON

address: 686 Bay St

department: DSCB

institute: Hospital for Sick Children


title: CLIP-seq of MSTR-1::FLAG and N2

summary: Worms expressing 3xFLAG-tagged MSTR-1 (F22D6.2) were grown to young adult stage and CLIP-seq was performed to identify binding regions on target RNAs

overall_design: Two biological replicates of MSTR-1::FLAG expressing worms, one biological replicate of WT (control) worms not expressing FLAG, and one size-matched input experiment

first: W

last: Derry


title: Large field of view and spatial region of interest transcriptomics in frozen and FFPE tissue

city: Qingdao

first: Xiaonan

last: Cui

address: 238 Songling Road

institute: The Ocean University of China

summary: Expression profiling in spatially defined regions is crucial for systematically understanding tissue complexity. Here, we report a method of photo-irradiation for in-situ barcoding hybridization and ligation sequencing, named PBHL-seq, which allows targeted expression profiling from the photo-irradiated region of interest in intact fresh frozen and FFPE tissue samples. PBHL-seq uses photo-caged oligodeoxynucleotides for in situ reverse transcription followed by spatially targeted barcoding of cDNAs to create spatially indexed transcriptomes of photo-illuminated regions. We recover thousands of differentially enriched transcripts from different regions by applying PBHL-seq to OCT-embedded tissue (E14.5 mouse embryo and mouse brain) and FFPE mouse embryo (E15.5). We also apply PBHL-seq to the subcellular microstructures (cytoplasm and nucleus, respectively) and detect thousands of differential expression genes. Thus, PBHL-seq provides an accessible workflow for expression profiles from the region of interest in frozen and FFPE tissue at subcellular resolution with areas expandable to centimeter scale, while preserving the sample intact for downstream analysis.

overall_design: The new approach (PBHL-seq) is applied to frozen mouse embryo (E14.5), mouse brain, FFPE mouse embryo (E15.5)  and subcelluar of Hela for ROIs expression analysis.

first: Weiyang

last: Shi

first: Xue

last: Dong

first: Mengzhu

last: Hu

first: Wenjian

last: Zhou


title: Macrophage-mediated myelin recycling fuels brain cancer malignancy (VISIUM)

city: Amsterdam

first: Daan

middle: Juri

last: Kloosterman

address: Plesmanlaan 121

department: Tumor Biology & Immunology

institute: The Netherlands Cancer

summary: Tumors growing in metabolically-challenged environments, such as glioblastoma in the brain, are particularly reliant on cross-talk with their tumor microenvironment (TME) to satisfy their high energetic needs. However, the intricacies of this metabolic interplay and the consequences on immune cell subset diversity and function remain largely unexplored. We interrogated the heterogeneity of the glioblastoma TME using single cell multi-omics analyses in preclinical glioblastoma mouse models and patient samples, and identified metabolically-rewired tumor-associated macrophage (TAM) subpopulations that fuel glioblastoma malignancy. These TAM subsets, termed lipid-laden macrophages (LLMs) to reflect their increased lipid metabolism activity and cholesterol storage, are epigenetically rewired, display immunosuppressive features and are enriched in the aggressive mesenchymal glioblastoma subtype. In response to TME-derived cues triggering liver-X-receptor (LXR) expression, macrophages increase engulfment of cholesterol-rich myelin debris and acquire an LLM phenotype. Subsequently, LLMs directly transfer myelin-derived lipids to cancer cells in an LXR/Abca1-dependent manner, thereby fueling the heightened metabolic demands of mesenchymal glioblastoma. Furthermore, LLM content predicts clinical outcomes and immune checkpoint blockade response in glioblastoma patients and other cancer types. Our work provides an in-depth understanding of the immune-metabolic interplay during glioblastoma progression in a subtype- and microanatomical niche-dependent manner, thereby laying a framework for the discovery of targetable metabolic vulnerabilities in glioblastoma.

overall_design: To reveal the reciprocal interactions between macrophages and tumor cells that fuel glioblastoma malignancy, we investigated the dynamic contexture of the glioblastoma TME at the single cell and spatial transcriptomic levels, in two genetically-engineered mouse models (GEMMs) that recapitulate key characteristics of human gliomagenesis and therapeutic response. Both GEMMs involve enforced expression of platelet-derived growth factor-b in Nestin-positive progenitor cells, either in an Ink4a/ArfKO background (herein termed PDG-Ink4a), or in combination with a short-hairpin-mediated knockdown of p53 in tumor cells (herein termed PDG-p53). Orthogonally, publicly available transcriptional datasets of glioblastoma patients enabled the validation of our murine models’ clinical relevance. We explored the dynamic heterogeneity of macrophage subsets in relation to their local microenvironment, employing multi-omics approaches and a wide range of functional ex vivo and in vitro assays to unveil novel pro-tumorigenic interactions underlying macrophage and tumor cell co-evolution in the glioblastoma TME.

first: Johanna

last: Erbani

first: Menno

last: Boon

first: Martina

last: Farber

first: Shanna

last: Handgraaf

first: Masami

last: Ando-Kuri

first: Bauke

last: Fontein

first: Elena

last: Sánchez-López

first: Marjolijn

last: Mertz

first: Marja

last: Nieuwland

first: Ning

middle: Q

last: Liu

first: Gabriel

last: Forn-Cuni

first: Nicole

middle: N

last: van der Wel

first: Anita

last: Grootemaat

first: Luuk

last: Reinalda

first: Sander

middle: I

last: van Kasteren

first: Elzo

last: de Wit

first: Brian

last: Ruffel

first: Ewa

last: Snaar-Jagalska

first: Kevin

last: Petrecca

first: Dieta

last: Brandsma

first: Alexander

last: Kros

first: Martin

last: Giera

first: Leila

last: Akkari


title: Macrophage-mediated myelin recycling fuels brain cancer malignancy (scRNA-Seq)

first: Daan

last: Kloosterman

middle: J

first: Johanna

last: Erbani

first: Menno

last: Boon

first: Martina

last: Farber

first: Shanna

middle: M

last: Handgraaf

first: Masami

last: Ando-Kuri

first: Bauke

last: Fontein

first: Elena

last: Sánchez-López

middle: E


title: Macrophage-mediated myelin recycling fuels brain cancer malignancy (RNA-Seq ex vivo)

first: Marjolijn

last: Mertz

first: Marja

last: Nieuwland

first: Ning

last: Liu

first: Gabriel

last: Forn-Cuni

first: Nicole

last: van der Wel

first: Anita

last: Grootemaat

first: Luuk

last: Reinalda

first: Sander

last: van Kasteren


title: Cellular dynamics of pig-to-human kdiney xenotransplantation [single cell RNA-seq]

city: New York

first: Weimin

last: Zhang

state: NY

address: 435 E 30th street

department: Institute for Systems Genetics

country: USA

institute: NYU Langone Health

summary: We performed comprehensive single-cell RNA sequencing (scRNA-seq) analyses of the two porcine xenografts and their contralateral untransplanted kidneys to dissect xenotransplantation-associated cellular dynamics and xenograft-recipient interactions. We additionally performed longitudinal bulk RNA-seq on core biopsies to dissect the time-resolved physiological changes of the xenografts and longitudinal scRNA-seq of peripheral blood mononuclear cells (PBMCs) to detect recipient’s immune responses across time.

sample_organism:
Homo sapiens
Sus scrofa

platform_organism:
Homo sapiens
Sus scrofa

overall_design: Xenografts and contralateral control kidneys went through the same procedure for preparation of transplantation. Kidney tissues from xenograft and control kidneys in both cases were collected at the end of the study (54 hours). PBMC samples were collected from the recipient in case 2 at the following timepoints: pre-transplant (0 h) and then at 6h, 12h, 24h, 48h, and 53h (right before termination).

first: Wanqing

last: Pan

first: Binghan

last: Zheng

first: Brendan

last: Camellato

first: Jeffrey

last: Stern

first: Ziyan

last: Lin

first: Alireza

last: Khodadadi-Jamayran

first: Jacqueline

last: Kim

first: Philip

last: Sommer

first: Karen

last: Khalil

first: Elaina

last: Weldon

first: Jiangshan

last: Bai

first: Yinan

last: Zhu

first: Peter

last: Meyn

first: Adriana

last: Heguy

first: Massimo

last: Mangiola

first: Adam

last: Griesemer

last: Keating

first: Robert

middle: A

last: Montgomery

first: Bo

last: Xia

first: Jef

middle: D

last: Boeke


title: Cellular dynamics of pig-to-human kdiney xenotransplantation [RNA-seq]

first: Weimin

last: Zhang

overall_design: Longitudinal biopsy samples were collected in xenotransplantation case 2 at 0h from untransplanted control kidney, and then at 12h, 24h, and 48h from the xenograft.

first: Wanqing

last: Pan

first: Binghan

last: Zheng

first: Brendan

last: Camellato

last: Stern


title: Transcriptional Remodeling in Human Cardiac Fibroblasts and Myocytes Induced by Oxygen-Dependent Crosstalk

first: Megan

middle: L

last: McCain

state: CA

address: 1024 Downey Way

institute: USC

summary: Myocardial infarctions are caused by coronary occlusions that deprive downstream myocardial tissue of oxygenated blood, leading to localized necrosis of cardiac myocytes. Hypoxic injury drives a remodeling process in which cardiac fibroblasts activate and synthesize matrix proteins to form a scar. During this process, cardiac cells are exposed to an oxygen gradient at the infarct border zone that transitions from 0% oxygen at the site of injury to 10% oxygen in healthy myocardium. However, the impact of any crosstalk between hypoxic cardiac fibroblasts and neighboring, normoxic (10% oxygen) cardiac cells is unexplored because conventional research tools cannot re-create spatially heterogeneous oxygen landscapes. Here, we used a microfluidic device to co-culture hypoxic cardiac fibroblasts with (1) normoxic cardiac fibroblasts or (2) normoxic human induced pluripotent stem cell (hiPSC)-derived cardiac myocytes. We then compared their transcriptome to the same cells cultured in uniform hypoxia or normoxia. These data contribute to more advanced understanding of how oxygen-dependent crosstalk between cardiac cells impacts the wound healing process post-myocardial infarction.

overall_design: Primary human adult cardiac fibroblasts or human induced pluripotent stem cell (hiPSC)-derived cardiac myocytes were cultured on fibronectin-coated PDMS microfluidic devices designed to expose cells to 0% oxygen, 10% oxygen, or adjacent co-culture regions of 0% and 10% oxygen. After 24h of oxygen modulation, cells from each half of the co-culture region were immediately separated with a razor blade and all conditions (0% control, 0% in co-culture, 10% in co-culture, and 10% control) were lysed. RNA was isolated and sequenced, and gene expression was analyzed using Partek Flow Genomics Software.

first: Natalie

last: Khalil

first: Divya

last: Gupta

first: Sarah

last: Parker


title: Temporally resolved proteomics identifies nidogen-2 as a co-target in pancreatic cancer that modulates fibrosis and therapeutic efficacy

city: Darlinghurst

first: Leonard

last: Goldstein

address: 384 Victoria St

institute: Garvan Institute of Medical Research

summary: Pancreatic ductal adenocarcinoma (PDAC) is characterized by increasing fibrosis, which can enhance tumor progression and spread. Here, we undertook an unbiased temporal assessment of the matrisome of the highly-metastatic KPC and poorly-metastatic KPflC genetically engineered mouse models of pancreatic cancer using mass spectrometry proteomics. Our assessment at early-, mid- and late-stage disease reveals an increased abundance of nidogen-2 (NID2) in the KPC model compared to KPflC, with further validation showing that NID2 is primarily expressed by cancer-associated fibroblasts (CAFs). Using biomechanical assessments, second harmonic generation multiphoton imaging and Picrosirius Red staining with birefringence analysis, we show that NID2 reduction by CRISPR interference (CRISPRi) in CAFs reduces stiffness and fibrosis, leading to impaired cancer cell invasion. Intravital imaging with Quantum Dots exhibited improved vascular patency in live NID2-depleted tumors, with enhanced response to gemcitabine/Abraxane (nab-paclitaxel) chemotherapy. In orthotopic models, mice bearing NID2 CRISPRi tumors had significantly reduced liver metastasis and increased survival, highlighting NID2 as a potential new target in PDAC.

overall_design: KPC CAF cells (mt-CAF; Vennin et al 2019 Nature Communications DOI: 10.1038/s41467-019-10968-6) were genetically modified via CRISPR inactivation (CRISPRi). There are three cell lines: 1) KPC CAF GFP-1 KRAB, with gRNA targeting GFP-1 as non-targeting control, 2) KPC CAF B500 NID2 KRAB, with gRNA targeting the NID2 gene, 3) KPC CAF C500 NID2 KRAB, w gRNA targeting NID2 gene.  RNA sequencing was performed on these 3 different cells in triplicate.

first: Brooke

last: Pereira

first: Shona

last: Ritchie

first: Marjan

last: Naeini

first: Thomas

middle: R

last: Cox

first: Paul

last: Timpson


title: Aging affects ciliated cells development in human endometrial epithelium

city: Maastricht

first: Darina

last: Obukhova

state: Limburg

address: P. Debyelaan 25

department: Department of Clinical Genetics

institute: Maastricht University Medical Center +

summary: The twenties are typically considered the prime reproductive years for women. However, in today’s modern world many women are choosing to delay family planning, resulting in an increase of females in their forties seeking fertility treatment. Although in vitro fertilization (IVF) with donated oocytes and preimplantation genetic testing may help to address the impact of maternal age, the success rate for IVF treatment in this age group is still significantly lower. While endometrial changes, such as abnormal endometrial thickness, inflammatory background, and altered hormone response signaling, are associated with aging, little is known about molecular features of endometrial aging and their impact on the ability to support embryo implantation. To better understand age-specific changes, we performed endometrial transcriptome profiling of young and advanced age females, undergoing hormonal replacement therapy (HRT) before frozen embryo transfer, followed by immunohistology analysis and single-cell-based deconvolution. Here, we identified 491 differentially expressed genes pointing to the effect of aging on decidualization, cell signaling, inflammation and endometrial receptivity. Our results indicate that p16INK4a   may be involved in cellular senescence and the suppression of metabolic and inflammatory processes essential for endometrial preparation for embryo implantation. We have also shown that the proportion of ciliated cells along with ciliary processes is affected by endometrial aging. These findings have important implications for future strategies aimed at improving infertility treatment in women of advanced reproductive age.

overall_design: To investigate the differences in endometrial tissue between advanced maternal age and young maternal age groups, we obtained endometrial biopsies on the day 5 of progesterone administration and analyzed them using bulk RNA-seq.

first: Marina

last: Loid

first: Keiu

last: Kask

first: Alvin

last: Meltsov

first: Kasper

last: Derks

first: Signe

last: Altmäe

first: Merli

last: Saare

first: Maire

last: Peters

first: Ave

last: Minajeva

first: Priit

last: Adler

first: Kaarel

last: Krjutškov

first: Masoud

last: Zamani Esteki

first: Andres

last: Salumets


title: Single-cell gene expression profiling of early tumorigenesis in genetically engineered mouse models of breast carcinoma

city: Boston

first: Judy

last: Lieberman

state: MA

address: 200 Longwood Ave

department: PCMM

institute: Boston Children's Hospital

summary: During early tumorigenesis tumors edit their gene expression to evade immune surveillance. Because of difficulty capturing early tumors, studies of gene editing have to date mostly investigated changes in expression of candidate genes involved in activating adaptive immunity, such as those needed for antigen presentation and processing. Here we took advantage of an aggressive genetically engineered mouse model (GEMM) of breast cancer that expresses inducible oncogenic HER2 to identify tumor edited genes without bias.

overall_design: Eight-week-old ErbB2ΔEx16+/-MTB+/- female mice were fed with 2 mg/ml doxycycline in the drinking water to induce multifocal breast tumor. scRNA-seq libraries were prepared from tumors harvested 5-10 days (early) or 30-35 days (late) (n=4 samples/group) after oncogene induction.

first: Ying

last: Zhang

first: Pourya

last: Naderi Yeganeh

first: Winston

last: Hide


title: In vivo CRISPR screens for Toxoplasma hyperLOPIT-unassigened proteins_1

city: Suita

first: Yuta

last: Tachibana

state: Osaka

address: 3-1, Yamadaoka

department: Research Institute for Microbial Diseases (RIMD)

institute: Osaka University

summary: We performed in vivo CRISPR screens of Toxoplasma targeting genes with hyperLOPIT-unassigened subcellular localisation during mouse infection.

overall_design: This Unassigned_1 library targeting 295 genes which consisted 2944 gRNAs targeting hyperLOPIT-unassigened genes together with dispensable and essential control genes.

first: Masahiro

last: Yamamoto


title: Genomewide methylation analysis of B16F10 and 4T1 after Decitabine treatment with Reduced Representative Bisulfite Sequencing [RRBS]

first: Eric

last: Greer

address: 320 Longwood Avenue

department: Newborn Medicine/Department of Pediatrics

institute: Boston Children's Hospital/Harvard Medical School

type: Methylation profiling by high throughput sequencing

summary: We report genomewide methylation levels on CpG sites and segments before and after Decitabine treatment

overall_design: Bisulfite treated DNA was built for deep sequencing, in triplicate, using NovaSeq S4

first: Simon

middle: Y

last: Wang