title: Tumour-intrinsic endomembrane trafficking by ARF6 shapes an immunosuppressive microenvironment that drives melanomagenesis and response to checkpoint blockade therapy [scRNA-seq]

city: Salt Lake City

first: Allie

last: Grossmann

state: Utah

address: 2000 Circle of Hope

department: Pathology

institute: University of Utah

summary: Tumour-host immune interactions lead to complex changes in the tumour microenvironment (TME), impacting progression, metastasis and response to therapy. While it is clear that cancer cells can have the capacity to alter immune landscapes, our understanding of this process is incomplete. Herein we show that endocytic trafficking at the plasma membrane, mediated by the small GTPase ARF6, enables melanoma cells to impose an immunosuppressive TME that accelerates tumour development. This ARF6-dependent TME is vulnerable to immune checkpoint blockade therapy (ICB) but in murine melanoma, loss of Arf6 causes resistance to ICB. Likewise, downregulation of ARF6 in patient tumours correlates with inferior overall survival after ICB. Mechanistically, these phenotypes are at least partially explained by ARF6-dependent recycling, which controls plasma membrane density of the Interferon-gamma receptor. Collectively, our findings reveal the importance of endomembrane trafficking in outfitting tumour cells with the ability to shape their immune microenvironment and respond to immunotherapy.

overall_design: scRNA-seq data from cd45+ sorted cells from 3 Arf6null and matched 3 Arf6wt dissociated tumors

first: Yinshen

last: Wee

first: Junhua

last: Wang

first: Emily

last: Wilson

first: Coulson

middle: P

last: Rich

first: Aaron

last: Rogers

first: Chris

last: Stubben

first: Roger

middle: K

last: Wolff


title: Tumour-intrinsic endomembrane trafficking by ARF6 shapes an immunosuppressive microenvironment that drives melanomagenesis and response to checkpoint blockade therapy [bulk RNA-seq]

status: Public on Jun 27 2024

country: USA

type: Expression profiling by high throughput sequencing

overall_design: RNASeq based comparion of gene expression between 6 Arf6null and 6 matched Arf6wt tumors

middle: H


title: Tumour-intrinsic endomembrane trafficking by ARF6 shapes an immunosuppressive microenvironment that drives melanomagenesis and response to checkpoint blockade therapy.

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

overall_design: Refer to individual Series


title: SAMD1 suppresses epithelial-mesenchymal transition pathways in pancreatic ductal adenocarcinoma [RNA-Seq]

city: Marburg

first: Robert

last: Liefke

state: Hessen

address: Hans-Meerwein-Str. 2

department: Institute of Molecular Biology and Tumor Research (IMT)

country: Germany

institute: Philipps University of Marburg

summary: Pancreatic ductal adenocarcinoma (PDAC) poses a significant threat due to its tendency to evade early detection, frequent metastasis, and the subsequent challenges in devising effective treatments. Processes that govern epithelial-mesenchymal transition (EMT) in PDAC hold promise for advancing novel therapeutic strategies. SAMD1 (SAM domain-containing protein 1) is a CpG island-binding protein that plays a pivotal role in the repression of its target genes. Here, we revealed that SAMD1 acts as a repressor of genes associated with epithelial-mesenchymal transition (EMT). Upon deletion of SAMD1 in PDAC cells, we observed significantly increased migration rates. SAMD1 exerts its effects by binding to specific genomic targets, including CDH2, encoding N-cadherin, which emerged as a driver of enhanced migration upon SAMD1 knockout. Furthermore, we discovered the FBXO11-containing E3 ubiquitin ligase complex as an interactor and negative regulator of SAMD1, which inhibits SAMD1 chromatin binding genome-wide. High FBXO11 expression in PDAC is associated with poor prognosis and increased expression of EMT-related genes, underlining an antagonistic relationship between SAMD1 and FBXO11. In summary, our findings provide insights into the regulation of EMT-related genes in PDAC, shedding light on the intricate role of SAMD1 and its interplay with FBXO11 in this cancer type.

overall_design: ChIP-Seq of SAMD1 in Control and SAMD1 KO cells (Experiment 1), and Control and FBXO11 KO cells (Experiment 2).

first: Clara

last: Simon

first: Bastian

last: Stielow

first: Andrea

last: Nist


title: SAMD1 suppresses epithelial-mesenchymal transition pathways in pancreatic ductal adenocarcinoma

type: Genome binding/occupancy profiling by high throughput sequencing


title: SAMD1 suppresses epithelial-mesenchymal transition pathways in pancreatic ductal adenocarcinoma [ChIP-Seq]

overall_design: ChIP-Seq of SAMD1 in Control and SAMD1 KO cells (Experiment 1), Control and FBXO11 KO cells (Experiment 2), and second replicate of Control and FBXO11 KO cells (Experiment 3).


title: O-GlcNAcase Regulates Pluripotency States of Human Embryonic Stem Cells

status: Public on Jun 26 2024

city: Hangzhou

first: Qianyu

last: Liu

address: YuhangTang Road

country: China

institute: Zhejiang University

summary: In order to study which part, if any, of the EP300 phenotype is related to OGA regulation.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for siNC, siOGA, siEP300, siEP300+OGA naïve  hESCs.

first: Wen

last: Yi


title: Multi-organ transcriptomic profiles and gene-regulation network underlying vibriosis resistance in tongue sole

city: Qingdao

first: Quanchao

last: Chen

state: Shandong

address: Yushan Campus, No. 5 Yushan Road,Qingdao

institute: Ocean university of china

summary: Vibrio spp. are major pathogens responsible for mortality and disease in various marine aquaculture organisms. Effective disease control and genetic breeding strategies rely heavily on understanding host vibriosis resistance mechanisms. The Chinese tongue sole (Cynoglossus semilaevis) is economically vital but suffers from substantial mortalities due to vibriosis. Through continuous selective breeding, we have successfully obtained vibriosis-resistant families of this species. In this study, we conducted RNA-seq analysis on three organs, including liver, spleen and intestine from selected resistant and susceptible tongue soles. Additionally, we integrated these data with our previously published RNA-seq datasets of skin and gill, enabling the construction of organ-specific transcriptional profiles and a comprehensive gene co-expression network elucidating the differences in vibriosis resistance. Furthermore, we identified 12 modules with organ-specific functional implications. Overall, our findings provide a valuable resource for investigating the molecular basis of vibriosis resistance in fish, offering insights into target genes and pathways essential for molecular selection and genetic manipulation to enhance vibriosis resistance in fish breeding programs.

overall_design: To characterize and compare the gene expression patterns between the Vibrio resistant and susceptible fish from multi-generation selective breeding, we collected gill, skin, liver, spleen, and intestine tissues from both groups, with each group including three biological replicates.

first: Xinran

last: Ma

first: Jie

first: Meng

last: Shi

first: Songlin

first: Qian

last: Zhou


title: Loss of ZNF408 Is Linked to Immune Escape in Breast Carcinogenesis [CUT&Tag]

city: Beijing

first: Chunyu

last: Yu

address: Xueyuan Road 38#

institute: Peking University

summary: Understanding cancer immune escape is important to the comprehension of cancer development and to the development of cancer immunotherapy. We report here that ZNF408, encoded by a gene linked to familial exudative vitreoretinopathy (FEVR) and autosomal recessive retinitis pigmentosa (RP), is physically associated with the chromatin remodeler SETD1A/COMPASS complex in breast cancer cells. The ZNF408/SETD1A/COMPASS complex orchestrates the transcriptional activation of a cohort of genes via histone H3K4 trimethylation, particularly STING1 that are critically involved in innate immune response, leading to the release of multiple cytokines and activation of cytotoxic lymphocyte cells in tumor microenvironment. ZNF408 enhances STING expression and promotes anti-tumor immune responses both in vitro and in vivo. Importantly, the expression of ZNF408 is downregulated in breast carcinomas, and its level of expression is positively correlated with that of STING1 and negatively correlated with the histological grade of breast cancer. High ZNF408 is also correlated with high CD8+ T cell infiltration and favorable prognosis of breast cancer patients. Our study uncovers an important role for ZNF408 in innate immune response, supporting further investigations of the ZNF408-SETD1A/COMPASS-STING1 axis in cancer immune escape as well as in other ZNF408-associated diseases including FEVR and RP.

overall_design: The study comprises ATAC-seq samples from MCF-7 cells with 2 replicates, RNA-seq samples with 3 replicates each for both wild-type and ZNF408 knockdown conditions, and CUT&Tag samples for SETD1A with 2 replicates. There are no replicates for CUT&Tag samples targeting ZNF408 and H3K4me3. This setup includes control samples for RNA-seq and comparative analysis across conditions to explore the effects of ZNF408 knockdown on gene expression and chromatin state.

first: Jing

last: Liang

first: Xiao

last: Cheng

first: Yan

last: Zhang


title: Loss of ZNF408 Is Linked to Immune Escape in Breast Carcinogenesis [RNA-seq]


title: Loss of ZNF408 Is Linked to Immune Escape in Breast Carcinogenesis [ATAC-seq]


title: Loss of ZNF408 Is Linked to Immune Escape in Breast Carcinogenesis.


title: Deciphering the heterogeneity of differentiating hPSC-derived corneal limbal stem cells through single-cell RNA-sequencing

city: Nijmegen

first: Jo Huiqing

address: Geert Grooteplein 26/28

country: Netherlands

institute: Radboud University


title: Deciphering the heterogeneity of differentiating hPSC-derived corneal limbal stem cells through single-cell RNA-sequencing [scRNA-seq]

summary: A comprehensive understanding of the human pluripotent stem cell (hPSC) differentiation process stands as a prerequisite for the development hPSC-based therapeutics. In this study, single-cell RNA-sequencing (scRNA-seq) was performed to decipher the heterogeneity during differentiation of three hPSC lines towards corneal limbal stem cells (LSCs). The scRNA-seq data revealed the presence of nine clusters, among which five clusters followed the anticipated differentiation path of LSCs. The remaining four clusters were linked to previously undescribed cell states that were annotated as either mesodermal or undifferentiated subpopulations, and their prevalence was hPSC line-dependent. Distinct cluster-specific marker genes identified in this study were confirmed by immunofluorescence analysis and employed to purify hPSC-derived LSCs, which effectively minimized the variation in the line-dependent differentiation efficiency. In summary, scRNA-seq offered molecular insights into the heterogeneity of hPSC-LSC differentiation, allowing a data-driven strategy to be adopted for consistent and robust generation of LSCs, essential for future advancement toward clinical translation.

overall_design: hESC, hiPSC1, and hiPSC2 at different time points

first: Meri

last: Vattulainen

first: Jos

middle: G

last: Smits

first: Dulce

last: Lima Cunha

first: Tanja

last: Ilmarinen

first: Heli

last: Skottman

first: Huiqing


title: Deciphering the heterogeneity of differentiating hPSC-derived corneal limbal stem cells through single-cell RNA-sequencing [Bulk-seq]

overall_design: iPSC1 (n=2) and hESC1 (n=2) were differentiated into induced limbal stem cells, cells were FACs selected on AREG6+, ITGA6+ and PODXL- and cultured for an additional 15 days.  Cells were subjected to bulkRNA sequencing to quantify  marker gene expression in the induced limbal stem cells originating from iPSCs and hESC.


title: IL-13 decreases suceptibility to airway epithelial SARS-CoV-2 infection but increases disease severity in vivo [Bulk RNA-seq]

city: Iowa city

first: Alejandro

middle: Pezzulo

last: Colmenares

state: Iowa

address: 169 Newton Road

department: Internal medicine

institute: University of Iowa

summary: Human airway epithelia (HAE) undergo inflammation-induced remodeling in chronic lung diseases such as asthma and chronic bronchitis. The role of type 2 inflammation-induced epithelial remodeling in SARS-CoV-2 infection and the course of COVID-19 is unclear, moreover, there is discrepancy in the literature regarding the potential benefit of treatments that modulate type 2 inflammation. We investigated the role of IL-13-induced inflammation on SARS-CoV-2 binding/entry, replication, and host response in primary HAE cells in vitro and in a model of mouse-adapted SARS-CoV-2 in vivo. IL-13 protected airway epithelial cells from SARS-CoV-2 infection in vitro by decreasing the abundance of ACE2- expressing ciliated cells rather than by neutralization in the airway surface liquid or by interferon-mediated antiviral effects. In contrast, IL-13 worsened the severity of disease in mice in vivo; the effects were mediated by eicosanoid signaling and were abolished in mice deficient in the phospholipase A2 enzyme PLA2G2D. We conclude that IL-13-induced inflammation affects multiple steps of SARS-CoV-2-induced disease pathogenesis. Whereas IL-13-induced inflammation may be protective against initial infection at the airway epithelium, it enhances disease severity once infection progresses in vivo; blockade of IL-13 and/or eicosanoid signaling may be protective against progression to severe lung disease.

overall_design: To investigate the role of IL-13 induced inflammation on SARS-CoV-2 in vivo, we intranasally infected 4 mice by 1,000PFU of SARS2-N501YMA30 after a 4-day pretreatment of 2.5 μg/day intranasal IL-13 or vehicle. We harvested lungs  on the fifth day and then performed bulk RNA-seq.

first: Shreya

last: Ghimire

first: Biyun

last: Xue

first: Kun

last: Li

first: Ryan

middle: M

last: Gannon

first: Christine

middle: L

last: Wolford-Lenane

first: Andrew

last: Thurman

first: Huiyu

last: Gong

first: Grace

middle: C

last: Necker

first: Jian

last: Zheng

first: David

last: Meyerholz

first: Stanley

last: Perlman

first: Paul

middle: B

last: McCray

middle: A

last: Pezzulo


title: In-depth analysis of transcriptomes in ovarian cortical follicles from children and adults reveals interfollicular heterogeneity [RNA-Seq 2]

city: Tallinn

first: Ilmatar

last: Rooda

address: Akadeemia tee 15

department: Chemistry and Biotechnology

country: Estonia

institute: Tallinn University of Technology

summary: The ovarian cortical reserve of follicles is vital for fertility. Some medical treatments are toxic to follicles, leading to premature ovarian insufficiency. Ovarian tissue cryopreservation is an established method to preserve fertility in adults and even applied in prepuberty despite unproven efficacy. Here, we analyze transcriptomes of 120 cortical follicles from children and adults for detailed comparison. We discover heterogeneity with two main types of follicles in both age groups: one with expected oocyte-granulosa profiles and another with predicted role in signaling. Transcriptional changes during growth to the secondary stage are similar overall in children and adults, but variations related to extracellular matrix, theca cells, and miRNA profiles are found. Notably, cyclophosphamide dose correlates with interferon signaling in child follicles. Additionally, morphology alone is insufficient for follicle categorization suggesting a need for additional markers. Marker genes for early follicle activation are determined. These findings will help refine follicular classification and fertility preservation techniques across critical ages.

overall_design: Parallel analysis of the RNA content, including both long RNAs containing a poly-A-tail and small RNAs, was performed on the same individual follicles. To reveal differences between prepubertal and adult follicle gene expression, two groups of patients were included. Ovarian tissue was collected from adult gender reassignment patients (GRP) and from children undergoing fertility preservation.  Ovarian cortical samples were collected and cryopreserved from both patient groups. The cryopreserved cortical pieces were then thawed, and enzymatically digested to collect ovarian follicles. The quality of follicles was assessed by neutral red staining, and only those follicles that stained positively were collected. Each follicle was individually photographed, lysed, and frozen at -80°C until RNA library preparation.

first: Jasmin

last: Hassan

first: Jie

last: Hao

first: Timo

last: Tuuri

first: Kirsi

last: Jahnukainen

first: Marjut

last: Otala

first: Katri

last: Knuus

first: Kiriaki

last: Papaikonomou

first: Sebastian

last: Gidlöf

first: Kersti

last: Jääger

first: Andres

last: Saluments

first: Agne

last: Velthut-Meikas

first: Pauliina

last: Damdimopoulou


title: In-depth analysis of transcriptomes in ovarian cortical follicles from children and adults reveals interfollicular heterogeneity [miRNA-Seq]

type: Non-coding RNA profiling by high throughput sequencing


title: In-depth analysis of transcriptomes in ovarian cortical follicles from children and adults reveals interfollicular heterogeneity


title: West Nile Virus-induced expression of senescent gene Lgals3bpregulates microglial phenotype within cerebral cortex

status: Public on Jun 25 2024

city: London

first: Robyn

middle: S.

last: Klein

state: ON

address: 100 Perth Dr

country: Canada

institute: University of Western Ontario

summary: Microglia, the resident macrophages of the central nervous system, exhibit altered gene expression in response to various neurological conditions. This study investigates the relationship between West Nile Virus infection, and microglial senescence, focusing on the role of LGALS3BP, a protein implicated in both antiviral responses and aging. Using spatial transcriptomics, RNA sequencing and flow cytometry, we characterized changes in microglial gene signatures in adult and aged mice following recovery from WNV encephalitis. Additionally, we analyzed Lgals3bp expression and generated Lgals3bp-deficient mice to assess the impact on neuroinflammation and microglial phenotypes. Our results show that WNV-activated microglia share transcriptional signatures with aged microglia, including upregulation of genes involved in interferon response and inflammation. Lgals3bp was broadly expressed in the CNS and robustly upregulated during WNV infection and aging. Lgals3bp-deficient mice exhibited reduced neuroinflammation, increased homeostatic microglial numbers, and altered T cell populations without differences in virologic control or survival. This data indicates that LGALS3BP has a role in regulating neuroinflammation and microglial activation and suggest that targeting LGALS3BP might provide a potential route for mitigating neuroinflammation-related cognitive decline in aging and post-viral infections.

overall_design: CD11b+ cells extracted from Murine forebrain 30 days after mock- or West Nile Virus E218A infection of 16 or 85 week old C57BL6/J mice

first: Artem

last: Arutyunov


title: Resolving the three-dimensional interactome of Human Accelerated Regions during human and chimpanzee neurodevelopment

city: New Haven

first: Atreyo

last: Pal

state: CT

address: 333 Cedar St

department: Genetics

institute: Yale University

summary: Human Accelerated Regions (HARs) are highly conserved across species but exhibit a significant excess of human-specific sequence changes, suggesting they may have gained novel functions in human evolution. HARs include gene regulatory elements with human-specific activity and have been implicated in the evolution of the human brain. However, our understanding of how HARs contributed to uniquely human features of the brain is hindered by a lack of insight into the genes and pathways that HARs regulate. It is unknown whether HARs acted by altering the expression of gene targets conserved between HARs and their chimpanzee orthologs or by gaining new gene targets in human, a mechanism termed enhancer hijacking. We generated a high-resolution map of chromatin interactions for 1,590 HARs and their orthologs in human and chimpanzee neural stem cells (NSCs) to comprehensively identify gene targets in both species. HARs and their chimpanzee orthologs targeted a conserved set of 2,963 genes enriched for neurodevelopmental processes including neurogenesis and synaptic transmission. Changes in HAR enhancer activity were correlated with changes in conserved gene target expression. Conserved targets were enriched among genes differentially expressed between human and chimpanzee NSCs or between human and non-human primate developing and adult brain. Species-specific HAR gene targets did not converge on known biological functions and were not significantly enriched among differentially expressed genes, suggesting that most HARs did not alter gene expression via enhancer hijacking. HAR gene targets, including differentially expressed targets, also showed cell type-specific expression patterns in the developing human brain, notably in outer radial glia, which are hypothesized to contribute to human cortical expansion. Our findings support that HARs influenced human brain evolution by altering the expression of a conserved set of gene targets and provide the means to functionally link HARs with novel human brain features.

sample_organism:
Pan troglodytes
Homo sapiens

platform_organism:
Pan troglodytes
Homo sapiens

overall_design: Capture HiC (CHiC) for Human Accelerated Regions (HARs) and differentially active Human Gained Enhancers (HGEs) from Uebbing et al 2021 performed in human and chimpanzee Neural Stem Cells (hNSCs and cNSCs) and human neurons. Cut & Run (H3K27ac, H3K27me3, CTCF, RAD21 and POLII) and RNA-Seq were performed on two replicates each of hNSCs, cNSCs and human neurons. Single cell RNA-Seq was performed in a multiplexed sample of hNSCs and cNSCs to validate homogeneity at the NSC stage. The number of replicates for each assay are as follows:Three replicates each for CHiC in hNSCs and cNSCs, two replicates for CHiC in human neurons, two replicates for each chromatin mark in Cut & Run for hNSC, cNSC, human neuron, four replicates each for RNA-Seq in hNSCs and cNSCs and RNA-seq in human neuron, and a single multiplexed scRNA-Seq replicate for hNSCs and cNSCs.

first: James

last: Noonan


title: Super-enhancer-driven PPP1R15B/CReP, regulated by C/EBPβ and YY1, is oncogenic in multiple myeloma [HiChIP II]

city: Singapore

first: Tze King

last: Tan

address: 14 Medical Drive

department: Cancer Science Institute

country: Singapore

institute: National University of Singapore

summary: H3K27ac HiChIP analysis was performed in multiple myeloma cell lines and primary sample to analyze active chromatin-chromatin interactions in myeloma.

overall_design: MM cells were crosslinked with formaldehyde for 10 min.  H3K27ac HiChIP libraries were generated using HiChIP MNase Kit from Dovetail. HiChIP was performed using an antibody against H3K27ac Antibody (Cell Signaling Technology, #8173).

first: Xiong

last: Sinan

first: Zhou

last: Jianbiao

first: Tze


title: In-depth analysis of transcriptomes in ovarian cortical follicles from children and adults reveals interfollicular heterogeneity [RNA-Seq 1]

first: Ilmatar

last: Rooda

first: Jasmin

last: Hassan

first: Jie

last: Hao

first: Timo

last: Tuuri

first: Kirsi


title: Massively Parallel Binding Assay (MPBA) reveals limited transcription factor cooperation, challenging models of specificity

city: Rehovot

first: Naama

last: Barkai

address: Herztel 234

department: Molecular genetics

country: Israel

institute: Weizmann institute of science

summary: We provide the data collected using the method MPBA, allowing parallel measurement of TF binding to thousands of DNA sequence-variants within cells. The method was applied to quantify binding of dozens of transcription factors (TFs) to libraries of mutated regulatory regions, systematically screening all motif combinations.

overall_design:
Massively Parallel Binding Assay (MPBA), a novel method allowing parallel measurement of transcription factor (TF) binding to thousands of DNA sequence-variants within cells. In this project we apply it to quantify binding of dozens of TFs to libraries of mutated regulatory regions, systematically screening all motif combinations. In brief, we integrate a library of DNA variants into a designed plasmid and transform it into cells carrying an endogenous TF of interest fused to a Micrococcal Nuclease (MNase), which allows triggering the cleavage of TF-bound DNA upon a short calcium pulse. Sample titles are convention:
TranscriptionFactor_libraryNumber_experimentNumber_biologicalRepeat_TimePoint_TechnicalRepeat.
Note that each fastq file is muliplexed in two levels:
(1) sample (TF,experimentNum,biologicalRepeat,TimePoint,TechnicalRepeat).
(2) libraries (LibaryNum).

ATAC-seq: profiles for the wild-type strain BY4741 and for a strain with double deletion of Msn2/4 genes (2 repeats for each of the tested strains).

first: Tamar

last: Jana Lang

first: Sagie

last: Brodsky

first: Wajd

last: Manadre

first: Gili

last: Valinsky

first: Vladimir

last: Mindel

first: Felix

last: Jonas


title: Super-enhancer-driven PPP1R15B/CReP, regulated by C/EBPβ and YY1, is oncogenic in multiple myeloma


title: Functional Transition of CD11b+CD33+HLA-DR-/lowCD14- G-MDSC: Immunostimulation to Immunosuppression Post Allo-HSCT

city: Gui Yang

first: MING

last: NI

address: 28 Guiyi St., Yunyan District

department: Department of Hematology

institute: Affiliated Hospital of Guizhou Medical University,

summary: The prediction of acute graft-versus-host disease (aGvHD) post allogeneic hematopoietic stem cell transplantation (allo-HSCT) is critical for treatment decisions. The granulocytic myeloid-derived suppressor cells (G-MDSCs) show a fast recovery post-transplantation and constituted the major part of peripheral blood in the early phase after allo-HSCT. These cells were previously believed to exhibit immunosuppressive properties. However, they also promote inflammation under specific conditions. Thus, we conducted a comprehensive study of G-MDSCs from 82 patients after allo-HSCT within 90 days to elucidate the role of them in post-transplantation immunity. Strikingly, our results showed that G-MDSCs promoted inflammation in the early-stage, by facilitating cytokine secretion and proliferation of T cells, as well as their differentiation into pro-inflammatory T helper subsets. At day 28, patients with a higher number of G-MDSCs exhibited an increased risk of developing aGvHD. Besides, adoptive transfer of G-MDSCs from patients into humanized mice exacerbated aGvHD. However, at day 90, G-MDSCs showed immunosuppressive features, characterized by upregulated expression of indoleamine 2,3-dioxygenase gene and interleukin-10 secretion, coupled with the inhibition of T cell proliferation. Furthermore, transcriptional analysis of G-MDSCs at day 28 and day 90 revealed that a total of 1445 genes were differentially expressed. Genes linked to the endoplasmic reticulum stress were upregulated in patients without aGvHD. After the genetic overlap analysis, DERL1 may be the hub-gene. Our findings elucidate the alteration in the immune characteristics of G-MDSCs within the first 90 days post allo-HSCT. Moreover, the quantity of G-MDSCs at day 28 could serve as a predictive indicator for aGvHD.

overall_design: G-MDSCs from the patients at day 28 and day 90 under follow-up in outpatient department were collected and sorted using FACS sorting

first: Ming

first: Jing

last: Cui

first: Xin

last: Yang

first: Yuntian

last: Ding

first: Peng

last: Zhao

first: Tianzhen

last: Hu

first: Yun

last: Zhan

first: Qian

last: Kang

first: Xiuying

first: Jiangyuan

first: Yao

last: Xu

first: Lu

last: Chen

first: Min

last: Liu

first: Mei

first: Fengqi

last: Zhang

first: Shisi

last: Huang

first: Ya

last: Li

first: Xueying

first: Luxin

first: Tianzhuo

first: Bo

last: Deng

first: Bing

first: Deqin

last: Lu

first: Jishi

last: Wang


title: ChAHP and ChAHP2 control diverse retrotransposons by complementary activities [RNA-seq]

city: Basel

first: Fabio

last: Mohn

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

summary: Retrotransposon control in mammals is an intricate process that is effectuated by a broad network of chromatin regulatory pathways. We previously discovered ChAHP, a protein complex with repressive activity against SINE retrotransposons, composed of the transcription factor ADNP, chromatin remodeler CHD4, and HP1 proteins. Here we identify ChAHP2, a protein complex homologous to ChAHP, wherein ADNP is replaced by ADNP2. ChAHP2 is predominantly targeted to ERVs and LINEs, via HP1b-mediated binding of H3K9 trimethylated histones. We further demonstrate that ChAHP also binds these elements in a mechanistically equivalent manner to ChAHP2, and distinct from DNA sequence-specific recruitment at SINEs. Genetic ablation of ADNP2 alleviates ERV and LINE1 repression, which is synthetically exacerbated by additional depletion of ADNP. Together, our results reveal that the ChAHP and ChAHP2 complexes function to control both non-autonomous and autonomous retrotransposons by complementary activities, further adding to the complexity of mammalian transposon control.

overall_design: All experiments were performed with 2-4 biological replicates in mouse ES cells grown in media containing serum, LIF, GSK3b inhibitor and MEK inhibitor (2i). ChIP samples were produced under a variety of conditions (wild type controls, point mutations in ADNP or ADNP2, SETDB1 depletion)  to assay the levels and distribution of ADNP2, ADNP and H3K9me3. Total RNAseq was performed upon individual and combined removal of ADNP and ADNP2 (genetic KO for ADNP2, protein depletion for ADNP) to test function in transcription regulation.

first: Josip

last: Ahel

first: Aparna

last: Pandey

first: Michaela

last: Schwaiger

first: Marc

last: Bühler


title: ChAHP and ChAHP2 control diverse retrotransposons by complementary activities


title: ChAHP and ChAHP2 control diverse retrotransposons by complementary activities [ATAC-seq]


title: ChAHP and ChAHP2 control diverse retrotransposons by complementary activities [ChIP-seq]


title: Peach flower bud development

city: Legnaro

first: Giovanni Battista

last: Tornielli

state: Padova

address: Viale dell'Università, 16

department: DAFNAE

country: Italy

institute: University of Padova

summary: Thinning is indispensable practice in peach cultivation aiming to reduce fruit number per plant, promoting sink-source balance and reducing competition among fruit, which results in bigger fruit and the improvement of other fruit-quality parameters. Inhibition of floral induction by GAs has been largely demonstrated and commercial products based on GAs have been used to this aim. We tested a product GA4/7 based in different moments after full bloom in peach to reduce the number of flowers in the following season. Return to bloom and transcriptome analysis were performed to identify the best moment for the treatment, increasing the product efficacy and understanding the product action at genetic level.

overall_design: The main field trial has been carried out in 2019 on the flat-nectarine cultivar Platinet 1 (Prunus persica var. platycarpa). This cultivar is harvested approximately 22 days before the reference cv RedHaven. The experiment was conducted at Vivai Zanzi’s farm in Ferrara, Italy (44°46’56.355”, 11°39’55.419”). A suitable number of homogeneous trees with a suitable fruit load, all protected by anti-hail nets, were selected and marked among 40 trees in total in order to build a single block experiment with 5 trees per block. Blocks were separated by two untreated trees to avoid drift effects. The experimental formulation FAL900 (10 g/L GA7) was sprayed at 100 ppm by using a shoulder sprayer until run-off, at 45 (T1), 51 (T2), 58 (T3), 65 (T4), and 79 (T5) days after full bloom (DAFB). Before spraying, a suitable number of shoots were collected in three biological replicates and the buds from the top and the bottom halves of the shoot excised separately, frozen in liquid nitrogen and stored at -80°C for following analyses.

first: Alessandro

last: Botton

first: Monica

last: Canton

first: Francesco

last: Girardi

first: Francesca

last: Populin


title: TempO-Seq sequencing of Dimethylamine

city: Hannover

first: Matthias

last: Wehr

address: Nikolai-Fuchs-Str. 1

institute: Fraunhofer ITEM

summary: This study compares the dose dependent transcriptome data obtained for the volatile compound dimethylamine, which has been exposed in an in vitro system via air-liquid-interface (ALI) exposure to a pulmonary cell line (A549 cells).

overall_design: A549 cells were exposed to dimethylamine under ALI-conditions in 12-well plates (P.R.I.T.® ExpoCube®, Ritter et al. 2018 ). Exposure took place for 60 minutes by applying exposure flows of 3 ml/min per 1cm² ALI culture. The experimental design was based on 3 groups including 4 cultures / plate being exposed to the test substance, 4 cultures / plate being exposed to clean air as exposure control and the remaining wells / plate as non-exposure controls (no application of exposure flow). Vapor concentrations were set up by controlled in-line evaporation of the liquids using an impinger and dilution with clean air. Concentrations were monitored online using quantitative FT-IR analysis (Gasmet DX4000). High dose (HD) = 81.7 ppm, mid dose (MD) = 41.1 ppm, low dose (LD) = 14.9 ppm.  Exposure for one hour was followed by a 23hr recovery period before the next exposure started. Within a 72hr interval a total of 3 exposure and recovery periods were accomplished without medium change in between. For each exposure condition 3 biological replicates where tested, each was based on 2 pooled wells.


title: Qiagen RT-PCR array Dimethylamin validation experiment

overall_design: A549 cells were exposed to dimethylamine under ALI-conditions in 12-well plates (P.R.I.T.® ExpoCube®, Ritter et al. 2018 ). Exposure took place for 60 minutes by applying exposure flows of 3 ml/min per 1cm² ALI culture. The experimental design was based on 3 groups including 4 cultures / plate being exposed to the test substance, 4 cultures / plate being exposed to clean air as exposure control and the remaining wells / plate as non-exposure controls (no application of exposure flow). Vapor concentrations were set up by controlled in-line evaporation of the liquids using an impinger and dilution with clean air. Concentrations were monitored online using quantitative FT-IR analysis (Gasmet DX4000). High dose (HD) = 81.7 ppm, mid dose (MD) = 41.1 ppm, low dose (LD) = 14.9 ppm.   Exposure for one hour was followed by a 23hr recovery period before the next exposure started. Within a 72hr interval a total of 3 exposure and recovery periods were accomplished without medium change in between. For each exposure condition 3 biological replicates where tested, each was based on 2 pooled wells.

first: Monika

last: Niehof


title: A549 in vitro cell culture exposed to 3 concentrations of Dimethylamin in an Air-liquid Interface.

type:
Expression profiling by array
Expression profiling by RT-PCR

overall_design: Refer to individual Series


title: M3.3 Clariom D Arrays Dimethylamin

first: Stella

last: Reamon-Buettner


title: The primitive endoderm supports lineage plasticity to enable regulative development

city: Copenhagen

first: Josh

last: Brickman

address: 3B Blegdamsvej

department: reNEW

country: Denmark

institute: Copenhagen University


first: Tuan Zea

last: Tan

address: 14 Medical Drive, #11-01, Singapore

department: Cancer Science Institute Singapore

summary: We identified a novel super-enhance-driven oncogene PPP1R15B overexpressed in multiple myeloma as compared to normal plasma cells and B lymphoma cells. We used PPP1R15B specific shRNAs to study its function.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for H929 cells and its KD derivatives (shPPP1R15B).

first: Jianbiao

last: Zhou

first: Sinan

last: Xiong


title: The primitive endoderm supports lineage plasticity to enable regulative development [RNA-seq]

summary: Mammalian blastocyst formation involves the sequential specification of trophectoderm and the differentiation of inner cell mass into either epiblast or primitive endoderm. During this time, the embryo maintains a window of plasticity and able to redirect its cellular fate when challenged experimentally. In this context, we found that the primitive endoderm alone was capable of regenerating a complete blastocyst and continue normal postimplantation development. We identify an in vitro population similar to the early primitive endoderm in vivo that exhibits plasticity, forms three dimensional embryoid structures and exhibits multilineage competence in chimera assays. Here, we find OCT4 as the main player in collaborating with endodermal transcription factors to maintain pluripotent enhancers in a state that is primed for activation mediated by JAK/STAT signalling. Our observations support the notion that transcription factor persistence underlies plasticity in regulative development and highlights the importance of primitive endoderm in perturbed development.

overall_design: scRNA-seq of mouse 2iLIF ESCs, naïve extra-embryonic endoderm (nEnd), 3D nEnd and nEnd differentiated is trophoblast stem cell medium (TSC)

first: Madeleine

last: Linneberg-Agerholm

first: Alba

last: Redó-Riveiro

first: Annika

last: Sell

first: Martin

last: Proks

first: Teresa

middle: E

last: Knudsen

first: Marta

last: Perera

first: Joshua


title: Super-enhancer-driven PPP1R15B/CReP, regulated by C/EBPβ and YY1, is oncogenic in multiple myeloma [HiChIP I]

first: Tze King

summary: H3K27ac HiChIP analysis was performed in two multiple myeloma cell lines to analyze active chromatin-chromatin interactions in myeloma.

first: S

first: J


title: Mapping of flumioxazin tolerance in a snap bean diversity panel leads to the discovery of a master genomic region controlling multiple stress resistance genes

status: Public on Jun 24 2024

city: Urbana

first: Matthew

middle: D.

last: Brooks

state: Illinois

address: 1201 W Gregory Drive

department: GCPRU

institute: USDA

summary: Effective weed management tools are crucial for maintaining the profitable production of snap bean (Phaseolus vulgaris [L.]). Preemergence herbicides help the crop to gain a size advantage over the weeds, but the few preemergence herbicides registered in snap bean have poor waterhemp (Amaranthus tuberculatus) control, a major pest in snap bean production. Waterhemp and other difficult-to-control weeds can be managed by flumioxazin, an herbicide that inhibits protoporphyrinogen oxidase (PPO). However, there is limited knowledge about crop tolerance to this herbicide. We aimed to quantify the degree of snap bean tolerance to flumioxazin and explore the underlying mechanisms. A genome-wide association mapping approach was employed, utilizing field-collected data from a snap bean diversity panel. The response to a preemergence application of flumioxazin was measured by assessing plant population density and shoot biomass variables. Snap bean tolerance to flumioxazin is associated with a single genomic location in chromosome 02. Tolerance is influenced by several factors, including those that are indirectly affected by seed size/weight and those that directly impact the herbicide's metabolism and protect the cell from reactive oxygen species induced damage. Transcriptional  profiling and co-expression network analysis identified biological pathways likely involved in flumioxazin tolerance, including oxidoreductase processes. Upregulation of genes involved in those processes is possibly orchestrated by a transcription regulator located in the region identified in the GWAS analysis. Several entries belonging to the Romano class, including Bush Romano 350, Roma II, and Romano Purpiat presented high levels of tolerance in this study. The alleles identified in the diversity panel that condition snap bean tolerance to flumioxazin shed light on a novel mechanism of herbicide tolerance and can be used in crop improvement.

overall_design: We aimed to identify the biological pathways likely involved in flumioxazin tolerance. Snap bean seedlings of 12 cultivars shown to have contrasting phenotypic response to the application (6 sensitive and 6 tolerant) and carrying contrasting versions of a genomic region identified by GWAS, were germinated and harvested at emergence. Herbicide was not applied, with the assumption that differential gene expression contributing to tolerance would be constitutive

first: Ana

last: Saballos

last: Williams II


title: The primitive endoderm supports lineage plasticity to enable regulative development [CUT&Tag]

overall_design: Cleavage Under Targets & Tagmentation (CUT&Tag) of naïve extra-embryonic endoderm (nEnd) subpopulations


title: PKM2 aggregation drives metabolism reprograming during aging process

first: Juntao

last: Bie

address: Xueyuan road 38

institute: Peking university

summary: Mice was administrated with doxorubicin (DOX) to induce premature aging. Also these mice were administered intragastrically with vehicle or K35 or K27 compounds. After 20 days, these mice were sacrificed for liver tissue which were subjected to RNA sequencing to find the differentially expressed genes between control and treated groups.

overall_design: The liver of C57BL/6J was treated   with Dox and K35/K27 were sacrificed for liver tissue which were subjected to RNA sequencing to find the diferentially expressed genes.


title: Rational cell fate engineering through chromatin dynamics [RNA-Seq II]

first: Chengchen

last: Zhao

state: Zhejiang

address: Dunyu Road No.600, Xihu District

institute: Westlake University

summary: Mechanism controlling cell fate remains elusive. Chromatin remodeling complex interacts with transcription factors to colocalize across the genome and regulate region-specific epigenetic environment. Here, we propose an engineering approach for controlling cell fate through chromatin closing and opening. We utilize chromatin remodeling complex, BAF, known to activate gene expression by opening chromatin loci. By grafting BAF interacting motifs onto Nanog, we show that engineering factors could promote somatic cell reprogramming with Oct4. Furthermore, mutation on the interacting motifs render iPSC generation. The syntactic factors facilitate cell fate transition by recruiting BAF complex to modulate chromatin accessibility and reorganize cell state specific enhancers. Our findings reveal alternative methods to control cell fate by manipulating chromatin accessibility.

overall_design: We analyzed the gene expression profiles of NanogBiD or OKS mediated  iPSCs  derived from mouse tail tip fibroblasts and mouse neonatal fibroblasts by RNA-seq. We also sequenced  MNF and TTF.

first: Tao

last: HUANG

first: Dong

last: LIU

last: ZHAO

first: Bo

last: WANG

first: Duanqing

last: PEI


title: Rational cell fate engineering through chromatin dynamics [CUT&TAG II]

overall_design: CUT&Tag analysis was performed in reprogramming intermediates of day 3 to map Nanog, Nr5a2, Sox2 and Oct4 binding sites.


title: Rational cell fate engineering through chromatin dynamics [ATAC-Seq]

overall_design: We analyzed the chromatin accessibility profiles of NanogN70 or Nanog mediated somatic cell reprogramming at different reprogramming time points (Days 0,1,3,5,7,8,10,12) by ATAC-seq.


title: Rational cell fate engineering through chromatin dynamics [RNA-Seq]

overall_design: We analyzed the gene expression profiles of NanogN70 or Nanog mediated somatic cell reprogramming at different reprogramming time points (Days 0,1,3,5,7,8,10,12) by RNA-seq. In addition, we also sequenced mouse ES cells, MEF, and iPSCs reprogrammed from mouse fibroblasts using  NanogN70 or Nanog combinated with Oct4.


title: MARS-seq on SU-DIPG13 cells treated Sulfopin/Vorinostat/Sulfopin+Vorinostat or DMSO

first: Danielle

last: Algranati

address: 234 Herzl Street

department: Department of Immunology & Regenerative Biology

institute: Weizmann Institute of Science

summary: Diffuse midline gliomas (DMG) are aggressive pediatric tumors of the central nervous system that are highly resistant to treatments and are inevitably fatal. Lysine to methionine substitution of residue 27 on histone H3 (H3-K27M) is a driver mutation in DMGs, reshaping the epigenetic landscape of these cells to promote tumorigenesis. H3-K27M gliomas are characterized by deregulation of histone acetylation and methylation pathways, as well as the oncogenic MYC pathway. In search of effective treatment, we examined the therapeutic potential of dual targeting of histone deacetylases (HDACs) and MYC in these tumors. Treatment of H3-K27M patient-derived cells with Sulfopin, an inhibitor shown to block MYC-driven tumors in vivo, in combination with the HDAC inhibitor Vorinostat, resulted in substantial decrease in cell viability. Moreover, transcriptome and epigenome profiling revealed synergistic effect of this drug combination in downregulation of prominent oncogenic pathways such as mTOR. Finally, in vivo studies of patient-derived orthotopic xenograft models showed significant tumor size reduction in mice treated with the drug combination. These results highlight the combined treatment of Sulfopin and Vorinostat as a promising therapeutic approach for these aggressive tumors.

overall_design: MARS-seq analysis performed on SU-DIPG 13 cells treated with Sulfopin (12h/8d)/Vorinostat/Sulfopin+Vorinostat or DMSO

first: Bareket

last: Dassa

first: Efrat

last: Shema


title: Cut&Run SU-DIPG13 cells treated Sulfopin/Vorinostat/Sulfopin+Vorinostat or DMSO

overall_design: Cut and Run analysis performed on SU-DIPG 13 cells treated with Sulfopin/Vorinostat/Sulfopin+Vorinostat or DMSO, using the following primary antibodies: anti-histone H3K27me3 and anti-histone H3K27ac. Secondary antibody anti-rabbit HRP was used as a negative control.


title: Cut&Run and MARS-seq on SU-DIPG13 cells treated Sulfopin/Vorinostat/Sulfopin+Vorinostat or DMSO

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


title: Rational cell fate engineering through chromatin dynamics


title: Oligoclonal CD4+CXCR5+ T cells with a cytotoxic phenotype in tonsils and blood

city: Würzburg 97074

first: Chunguang

last: Liang

state: Bayern

address: Bioinformatik, Biozentrum, Uni Würzburg, Am Hubland

department: Bioinformatics

institute: Biozentrum

summary: In human studies, mononuclear cells from peripheral blood (PBMC) often serve as a source of clinical study markers. Therefore, it is essential to understand to what extent phenotypes of PBMCs reflect their tissue-resident equivalents. In order to determine the heterogeneity of T-follicular helper (TFH) cells in peripheral blood versus tonsils, CD3+CD4+CD45RA–CXCR5+ cells of both origins were sorted and transcriptomes, TCR repertoires and cell-surface protein expression were analysed by single-cell RNA sequencing, flow cytometry and immunohistochemistry. Four subsets of TFH cells with classical T-follicular gene expression patterns were mostly found in tonsils. Interestingly, however, all circulating CD3+CD4+CXCR5+ T-cell subpopulations also appeared in tonsils. Still, when circulating, cells of the same cluster rather displayed markers of proliferation and migration, whereas their tonsillar counterparts exhibited known TFH characteristics resembling bona fide germinal center-typical TFH cell subtypes. Surprisingly, one distinct and oligoclonal CD4+CXCR5+ subpopulation displayed pronounced cytotoxic properties. Those ‘killer TFH (TFK) cells’ were found among PBMCs as well as tonsillar cells but were located outside of germinal centers. Accordingly, they were the only CD4+CXCR5+ T-cell subtype in tonsils featuring transcripts for CXCR3 and PSLG1. They appeared terminally differentiated and could be distinguished from all other TFH subsets by expression of NKG7 (TIA-1), granzymes, perforin, CCL5, CCR5, EOMES, CRTAM and CX3CR1. This hitherto undescribed subpopulation is indicative for chronic infection and inflammation and therefore is a valuable candidate to be included in any CD4+CXCR5+ TFH cell assessment.

overall_design: Tonsil samples taken from three donors, Cyclosporin A treatment (during/after) samples from two patients, booster vaccination (before and after) samples from two healthy donors are measured by scRNA-seq, CITE-seq, TCR-seq

first: Silvia

last: Spoerl

first: Yin

last: Xiao

first: Katharina

last: Habenicht

first: Diana

last: Dudziak

first: Andreas

last: Rosenwald

first: Matthias

last: Edinger

first: Michael

last: Rehli

first: Petra

last: Hoffmann

first: Thomas

last: Winkler

first: Friederike

last: Berberich-Siebelt


title: Function of ARID1A during osteoclastogenesis (scRNA-Seq)

status: Public on Jun 23 2024

city: Shanghai

first: Jiahui

last: Du

address: Zhizaoju Road 639

institute: Shanghai Jiao Tong University

summary: We show that ARID1A promotes osteoclastogenesis

overall_design: We detected the function of ARID1A in bone marrow-derived monocytes (BMDMs) during osteoclastic differentiation with M-CSF and RANKL.


title: Function of ARID1A during osteoclastogenesis (RNA-Seq)


title: Rational cell fate engineering through chromatin dynamics [CUT&TAG]

city: Hangzhou

first: Chengchen

last: Zhao

overall_design: CUT&Tag analysis was performed in reprogramming intermediates of day 5 to map Nanog, Smarca4, H3K27ac and H3K4me binding sites.

first: Tao

last: HUANG

first: Dong

last: LIU

last: ZHAO

first: Bo

last: WANG


title: Cell-specific crosstalk proteomics reveals cathepsin B signaling as a driver of glioblastoma malignancy near the subventricular zone

city: Jacksonville

first: Hugo

last: Guerrero-Cazares

state: Florida

address: 4500 San Pablo Rd., Griffin Building 111

department: Neurosurgery

institute: Mayo Clinic

summary: Glioblastoma (GBM) is the most prevalent and aggressive malignant primary brain tumor. GBM proximal to the lateral ventricles (LVs) is more aggressive, potentially due to subventricular zone (SVZ) contact. Despite this, crosstalk between GBM and neural stem/progenitor cells (NSC/NPCs) is not well understood. Using cell-specific proteomics, we show that LV-proximal GBM prevents neuronal maturation of NSCs through induction of senescence. Additionally, GBM brain tumor initiating cells (BTICs) increase expression of CTSB upon interaction with NPCs. Lentiviral knockdown and recombinant protein experiments reveal both cell-intrinsic and soluble CTSB promote malignancy-associated phenotypes in BTICs. Soluble CTSB stalls neuronal maturation in NPCs while promoting senescence, providing a link between LV-tumor proximity and neurogenesis disruption. Finally, we show LV-proximal CTSB upregulation in patients, showing the relevance of this crosstalk in human GBM biology. These results demonstrate the value of proteomic analysis in tumor microenvironment research and provide direction for new therapeutic strategies in GBM.

overall_design: To investigate how CTSB contributes to gene expression in glioblastoma brain tumor initiating cells, we performed RNA sequencing on three technical repliactes of primary line GBM1A transduced with empty vector (EV) or shRNA against CTSB (KD 1). We then performed gene expression comparisons to identify differentially expressed genes.

first: Emily

middle: S

last: Norton

first: Lauren

last: Whaley

first: Vanessa

last: Jones

first: Mieu

last: Brooks

first: Marissa

middle: N

last: Russo

first: Dmytro

last: Morderer

first: Erik

last: Jessen

first: Paula

last: Schiapparelli

first: Andres

last: Ramos-Fresnedo

first: Natanael

last: Zarco

first: Anna

last: Carrano

first: Wilfried

last: Rossoll

first: Yan

middle: W

last: Asmann

first: TuKiet

middle: T

last: Lam

first: Kaisorn

last: Chaichana

first: Panos

middle: Z

last: Anastasiadis

first: Alfredo

last: Quiñones-Hinojosa

last: Guerrero-Cázares


title: Single cell RNA-seq of Oncorhynchus mykiss PBLs from infected and healthy fish

status: Public on Jun 21 2024

city: Valdeolmos

first: Pablo

last: Jiménez-Barrios

state: Madrid

address: Carretera de Algete a El Casar, Km 8

department: Animal Health Research Centre

country: Spain

institute: CISA-INIA (CSIC)

summary: Single cell RNA-Seq data generated on a 10x Chromium genomics platform from infected and healthy PBLs from Oncorhynchus mykiss.

overall_design: Two rainbow trout were killed by benzocaine overdose. Blood was extracted with a heparinized needle from the caudal vein and diluted 10 times with Leibovitz medium. PBLs were obtained by centrifugation of diluted blood on 51% continuous Percoll density gradients. IPNV (Sp strain, ATCC VR 1318) was propagated in the RTG-2 (Rainbow Trout Gonad-2) cell line. To produce viral stocks, complete culture media was removed from 80% confluent cell cultures in 75 cm2 culture flasks and IPNV inoculated. For the in vitro infection of rainbow trout PBLs, cells resuspended in L-15 containing antibiotics and 5% FCS were disposed in 24-well plates. Cell suspensions (1,000 cells/µl ) from each sample were loaded into the chips of the Chromium™ Single Cell 5′ Gel Beads Kit (10x Genomics) and subjected to the Chromium Controller instrument to generate single cell Gel Bead-In Emulsions (GEMs). GEMs were subjected to library construction using the Chromium™ Single Cell 5′ Library Kit v1 (10x Genomics). Samples were sequenced using a NextSeq instrument (Illumina) with 150PE chemistry.

first: Pedro

last: Perdiguero

first: Esther

last: Morel

first: Beatriz

last: Abós

first: Patricia

last: Díaz-Rosales

first: Carolina

last: Tafalla


title: Single-Mitochondrion Sequencing Uncovers Distinct Mutational Patterns and Heteroplasmy Landscape in Mouse Astrocytes and Neurons

city: Philadelphia

first: Junhyong

last: Kim

state: PA

address: 433 S University Avenue

department: Biology

institute: University of Pennsylvania

summary:
BACKGROUND: Mitochondrial (mt) heteroplasmy can cause adverse biological consequences when deleterious mtDNA mutations accumulate disrupting ‘normal’ mt-driven processes and cellular functions. To investigate the heteroplasmy of such mtDNA changes we developed a moderate throughput mt isolation procedure to quantify the mt single-nucleotide variant (SNV) landscape in individual mouse neurons and astrocytes In this study we amplified mt-genomes from 1,645 single mitochondria (mts) isolated from mouse single astrocytes and neurons to 1. determine the distribution and proportion of mt-SNVs as well as mutation pattern in specific target regions across the mt-genome,2. assess differences in mtDNA SNVs between neurons and astrocytes, and 3. Study cosegregation of variants in the mouse mtDNA.
RESULTS: 1. The data show that specific sites of the mt-genome are permissive to SNV presentation while others appear to be under stringent purifying selection. Nested hierarchical analysis at the levels of mitochondrion, cell, and mouse reveals distinct patterns of inter- and intra-cellular variation for mt-SNVs at different sites. 2. Further, differences in the SNV incidence were observed between mouse neurons and astrocytes for two mt-SNV 9027:G>A and 9419:C>T showing variation in the mutational propensity between these cell types. Purifying selection was observed in neurons as shown by the Ka/Ks statistic, suggesting that neurons are under stronger evolutionary constraint as compared to astrocytes. 3. Intriguingly, these data show strong linkage between the SNV sites at nucleotide positions 9027 and 9461.
CONCLUSION: This study suggests that segregation as well as clonal expansion of mt-SNVs is specific to individual genomic loci, which is important foundational data in understanding of heteroplasmy and disease thresholds for mutation of pathogenic variants.

overall_design: From 13 mice, 59 neuron cells and 43 astrocytes were harvested by micropipetting. On avarage, from a single neuron or astrocyte 16 mitochondria were sampled, and in total 1645 single mitochondria were collected. In parallel, 73 control samples were made on various conditions: (1) no PCR primers, (2) no input mtDNA, (3) both (1) and (2), (4) with PCR primers and with input mtDNA but pooling multiple mitochondria. Condition (1-3) serves as negative control while (4) serves as positive control. In total, there are 179 libraries and 1717 samples including controls.

first: Parnika

last: Kadam

first: Zijian

last: Yang

first: Youtao

last: Lu

first: Hua

last: Zhu

first: Yasemin

last: Atiyas

first: Nishal

last: Shah

first: Stephen

last: Fisher

first: Erik

last: Nordgren

first: David

last: Issadore

first: James

last: Eberwine


title: IL-1R signaling drives enteric glia-macrophage interaction in colorectal cancer progression [scRNA-Seq]

city: Leuven

first: Gianluca

last: Matteoli

address: Herestraat 49

country: Belgium

institute: KU Leuven

summary: To characterize the immune landscape of colorectal tumors, we analysed the immune tumor microenvironment cells from mice bearing orthotopic tumors using single cell transcriptomics.

overall_design: scRNA-seq data from BL6 mice bearing orthotopic colon tumors, 21 days after tumor induction (n = 3).

first: Lies

last: van Baarle

first: Veronica

last: De Simone

first: Linda

last: Schneider

first: Sneha

last: Santhosh

first: Saeed

last: Abdurahiman

first: Francesca

last: Biscu

first: Reiner

first: Lisa

last: Zanoletti

first: Sara

last: Verbandt

first: Zedong

last: Hu

first: Michelle

last: Stakenborg

first: Bo-Jun

last: Ke

first: Balbina

last: García-Reyes

first: Jonas

last: Henn

first: Marieta

last: Toma

first: Maxime

last: Vanmechelen

first: Frederik

last: De Smet

first: Sales

last: Ibiza

first: Sabine

last: Tejpar

first: Sven

last: Wehner


title: Changes in the transcriptomic profile of cumulus cells under the influence of pre-incubation of human oocytes in patients treated in ART cycles

status: Public on Jun 22 2024

city: Tehran

first: Naser

last: Elmi Ghiasi

address: Bioinformatics Deparment, Qods Street, Enghelab Street

country: Iran

institute: University of Tehran

summary: In this study, we tried to find out if the pre-incubation of the cumulus-oocyte complex can lead to better cumulus cell (CCs) function and higher oocyte quality by changing the transcriptomic profile of CCs. For this purpose, 140 cumulus cell samples were isolated from 12 participants and divided into two groups based on pre-incubation time. In the T0 group, the cumulus-oocyte complex was immediately dissected to separate the CCs from around the oocytes. In the T2 group, dissection and preparation of CCs were done after 2 hours of incubation. Then, the transcriptomic profile of the CCs of the non-pre-incubation group (T0) was compared to the 2-hour pre-incubation group (T2). 70 samples were placed in each group. 40 samples from each group were used for RNA-Sequencing and the remaining 30 samples of each group were used to confirm RNA-Sequencing results via qRT‑PCR. The CCs transcriptome analysis showed 17 genes were downregulated and 22 genes upregulated in the T2 group compared to the T0 group. Also, the pathways related to ATP production (oxidative phosphorylation, electron transport chain, and Mitochondrial complex I assembly model OXPHOS system), TNF-alpha signaling pathway, and glucocorticoid receptor pathway enriched in the T2 group compared to the T0 group. Also, the TGF-β pathway was decreased in the T2 group compared to the T0 group. This study showed that 2 hours’ pre-incubation leads to changes in important pathways in CCs, which has a positive effect on oocyte quality. 2 hours’ pre-incubation may lead to a better support of the CCs to the oocyte and thus to a more complete maturation of the oocyte.

overall_design: There is a difference of opinion about the time of pre-incubation, and because the direct examination of the human oocyte is not ethical, in this study, the changes in the transcriptomic profile of cumulus cells (CCs) with zero and T2 pre-incubation were investigated. 140 cumulus cell samples were isolated from 12 participants and divided into two groups based on pre-incubation time. In the T0 group, cumulus-oocyte complex was immediately dissected to separate the CCs from around the oocytes. In the T2 group, dissection and preparation of CCs were done after 2 hours of incubation. 70 samples were placed in each group. 40 samples from each group were used for RNA-Sequencing and the remaining 30 samples of each group were used for confirmation of RNA-Sequencing results via qRT‑PCR.

first: Mehdi

last: Mehdizadeh

first: Azam

last: Govahi

first: Sahar

last: Eghbali

first: Zahra

last: Zandieh

first: Marziyeh

last: Ajdary

first: Rana


title: Differentiation of monocyte-derived Langerhans cells in the presence of anandamide affects the immunomodulatory action of the cells

city: Debrecen

first: Attila

middle: Gábor

last: Szöllősi

address: Egyetem tér 1.

department: Department of Immunology

country: Hungary

institute: University of Debrecen

summary: The endocannabinoid system (ECS), named after the chemical compounds found in the cannabis plant, is a regulatory network of neurotransmitters, receptors, and enzymes that plays crucial roles in skin health and disease. Endogenous ligands of the ECS, called endocannabinoids, have proven to be important regulators of immune responses. One of the most prevalent endocannabinoids, arachidonoylethanolamide (also known as anandamide), is known for its anti-inflammatory effects. It reduces the production of pro-inflammatory cytokines in various immune cells, weakens both humoral and cellular immune responses, and promotes cell migration. Langerhans cells (LCs) are the sole antigen-presenting cells present in the human epidermis. They serve as the first line of defense against invading pathogens and are essential for the skin's specific immune responses and play a critical role in maintaining tissue homeostasis; however, little is known about the effect of endocannabinoids, specifically anandamide, on the biological functions of these cells. Our research aimed to provide the connection between monocyte-derived Langerhans cells (moLCs) and the ECS, shedding light on their collaborative roles in immune homeostasis and inflammation. Anandamide did not significantly affect the viability of moLCs up to 10 µM. When applied during the differentiation process it had only a negligible effect on CD207 expression, the prototypic marker of LCs; however, there was an observed reduction in CD1a expression by moLCs. Anandamide had no significant effects on the maturation status of moLCs, nor did it affect the maturation induced by TLR3 and TLR7/8 agonists. moLCs differentiated in the presence of anandamide did however show decreased production of CXCL8, IL-6, IL-10 and IL-12 cytokines induced by TLR3 and TLR7/8 activation. Anandamide-treated moLCs showed an increased capability to activate naïve T cells; however, not to the level seen with combined TLR agonism. RNAseq analysis of moLCs differentiated with anandamide showed modest changes to the transcriptome of the cells but did reveal an inhibitory effect on oxidative phosphorylation specifically in activated moLCs. Anandamide also promoted the polarization of naïve T cells towards a Th1 phenotype. Our results show that anandamide has nuanced effects on the differentiation, maturation, cytokine secretion, metabolism and function of activated moLCs. Among these changes the decrease in CD1a expression on moLCs holds promise to selectively dampen inflammation induced by CD1a restricted T cells, which have been implicated as drivers of inflammation in common inflammatory skin conditions such as psoriasis, atopic dermatitis and contact dermatitis.

overall_design: To determine the effect of anandamide (arachidonoylethanolamide) on the dfferentiation of monocyte-derived Langerhans cells we cultured monocytes in a cytokine mixutre that induces their differentiation into Langerhans cells with and without anandamide. Some the samples were also activated on day 4 of the differentiation protocol with TLR agonists (polyinosinic:polycytidylic acid and CL075). Samples were collected on day 5 of the differentiaion protocol.

last: Szollosi

first: Zsofia

last: Penzes


title: Synthetic essentiality of thymine DNA glycosylase in p53-deficient cancer [ChIP-seq]

city: shanghai

first: Zhenyu

last: Shao

state: --- Select a state ---

address: Yueyang Road 320

department: Center for Excellence in Molecular Cell Science

institute: Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

summary: Thymine DNA Glycosylase (TDG) is a versatile protein involved in DNA mismatch repair, DNA demethylation, and transcriptional regulation. Here, we identify TDG as a synthetic essential effector in p53-deficient tumours. We found that deletion of TDG inhibits cell proliferation specifically in p53-deficient cancer cells. Using a genetically engineered mouse model of lung adenocarcinoma (LUAD), we demonstrate that depletion of TDG suppresses tumour growth in the p53-deficient context. Notably, A novel and selective inhibitor developed to disrupt the DNA binding activity of TDG exhibits therapeutic efficacy against p53-deficient tumours in both cellular and xenograft models. Mechanistically, TDG coordinates with p53 to orchestrate the transcription of RNA helicase DHX9, which functions to eliminate dsRNA. Depletion of TDG in p53-deficient cells results in the downregulation of DHX9 and aberrant cytoplasmic double-strand (dsRNA) accumulation derived from Alu elements, subsequently activating the RIG-I/MDA5-MAVS mediated antiviral response. Moreover, single-cell RNA-sequencing analysis revealed the depletion of TDG in p53-deficient tumours facilitates the recruitment of tumour-infiltrating lymphocytes. We observed that TDG inhibition combined with immune checkpoint blockade (ICB) achieved a strong synergistic anti-tumour effect in p53-deficient tumours. This study highlights the function of TDG in maintaining p53-deficient tumour growth and provides an alternative therapeutic target for p53-deficient cancers.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for TDG and p53  as well as CUT&Tag for P300 and the histone modifications H3K9me3, H3K4me3 in B16 cells.

first: Jiaxin

last: Zhou


title: Function of ARID1A during osteoclastogenesis (ChIP-Seq)

first: Jiahui

last: Du


title: IL-1R signaling drives enteric glia-macrophage interaction in colorectal cancer progression [bulkRNA-Seq]

first: Gianluca

last: Matteoli

summary: To examine the mechanisms by which EGCs affect the immune CRC compartment with particular regard to the TAMs, we first investigated their transcriptional adaptations upon CRC onset. To this end, we established an in vitro setup to study EGC-CRC interactions able to mimic the response of EGCs to the factors secreted by the TME. These EGCs, from now onwards defined as TME-conditioned medium-treated EGCs (TME-CM EGCs), were generated by stimulating primary EGCs with the CM of digested murine MC38 orthotopic tumor tissues (Figure 2A). Bulk RNA sequencing (RNA-seq) was performed to determine transcriptional differences in TME-CM EGCs, compared to unstimulated and healthy conditioned medium-treated (H-CM) primary EGCs, at different time points.

overall_design: Transcriptome analysis of in vitro primary neurosphere-derived embryonic EGCs alone or stimulated with healthy conditioned medium (H-CM) or tumor microenvironment conditioned medium (TME-CM) at different time points (6h, 12h, and 24h, n = 4).

first: Lies

last: van Baarle

first: Veronica

last: De Simone

first: Linda

last: Schneider

first: Sneha

last: Santhosh

first: Saeed

last: Abdurahiman

first: Francesca

last: Biscu

first: Reiner

first: Lisa

last: Zanoletti

first: Sara

last: Verbandt

first: Zedong

last: Hu

first: Michelle

last: Stakenborg

first: Bo-Jun

last: Ke

first: Balbina

last: García-Reyes

first: Jonas

last: Henn

first: Marieta

last: Toma

first: Maxime

last: Vanmechelen


title: Specific silencing of pathogenic mRNA by a novel compact RNA-targeting tool TaqTth-hpRNA [RNA-seq]

status: Public on Jun 20 2024

city: Nanjing

first: Shu

last: Xu

state: Jiangsu Province

address: China Pharmaceutical University

institute: China Pharmaceutical University

summary: Pathogenic allele silencing is a promising treatment for genetic hereditary diseases. However, the concern about the specificity of present RNA-knockdown strategies has limited their in vivo applications. Here a TaqTth-hpRNA system consisting of a small, chimeric protein (TaqTth) and hairpin-RNA probe (hpRNA) is provided. The TaqTth-hpRNA showed a high-specific knockdown against targeted mRNA with minimal flanking sequence-motif requirement and less cell viability damage, then was applied to mutant APPswe mRNA silencing without altering the wild-type APP mRNA in Alzheimer’s disease. Notably, the combination of the TaqTth and human apolipoprotein E 2 (APOE2) overexpression encoded in a single AAV vector is available due to the compact size of TaqTth, and showed stronger inhibition of pathologies in the mouse model. Altogether, we provided the basis for a small RNA-targeting tool with high selectivity, minimal flanking sequence-motif requirement, less cell viability damage and potentially being an alternative in therapeutic applications.

overall_design: RNA sequencing. Total RNA was extracted using the Total RNA Extractor (Trizol) kit (B511311, Sangon, China) according to the manufacturer’s protocol, and treated with RNase-free DNase I to remove genomic DNA contamination. RNA integrity was evaluated with a 1.0% agarose gel. Thereafter, the quality and quantity of RNA were assessed using a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) and a Qubit® 2.0 Flurometer (Invitrogen). The high-quality RNA samples were subsequently submitted to the Sangon Biotech (Shanghai) Co., Ltd. for library preparation and sequencing. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using VAHTSTM mRNA-seq V2 Library Prep Kit for Illumina® following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in VAHTSTM First Strand Synthesis Reaction Buffer. First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second-strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. Remaining overhangs was converted into blunt ends via exonuclease/polymerase activities. After the adenylation of 3’ ends of DNA fragments, the adaptor was ligated to prepare for the library. To select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3-μL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The libraries were then quantified and pooled. Paired-end sequencing of the library was performed on the NovaSeq sequencers (Illumina, San Diego, CA) Gene expression values of the transcripts were computed by StringTie (version 1.3.3b). Principal Component Analysis (PCA) and Principal co-ordinates analysis (PCoA) were performed to reflect the distance and difference between samples. The TPM (Transcripts Per Million), eliminates the influence of gene lengths and sequencing discrepancies to enable direct comparison of gene expression between samples.


title: Dimethyl Fumarate Mediates Sustained VSMC Remodeling in a Mouse Model of Cerebral Aneurysm

city: New Orleans

first: Alejandra

last: Martinez Lege

state: United States

address: 1430 Tulane Ave

institute: Tulane University

summary: Cerebral aneurysms (CA) are a type of vascular disease that causes significant morbidity and mortality with rupture. Dysfunction of the vascular smooth muscle cells (VSMCs) from circle of Willis (CoW) vessels mediates CA formation as they are the major cell type of the arterial wall and play a role in maintaining vessel integrity. Dimethyl fumarate (DMF), a first-line oral treatment for relapsing-remitting multiple sclerosis, has been shown to inhibit VSMC proliferation and reduce CA formation in a mouse model. Potential unwanted side effects of DMF on VSMC function have not been investigated yet. The present study characterizes the impact of DMF on VSMC using scRNA-seq in CoW vessels following CA induction and further explores its role in mitochondrial function using in vitro VSMC cultures. Two weeks of DMF treatment following CA induction impaired the transcription of the glutathione redox system and downregulated mitochondrial respiration genes in VSMCs. In vitro, DMF treatment increased lactate formation and enhanced the mitochondrial production of reactive oxygen species (ROS). These effects rendered VSMCs vul-nerable to oxidative stress and led to mitochondrial dysfunction and enhancement of apoptosis. Taken together, our data support the concept that the DMF-mediated antiproliferative effect on VSMCs is linked to disturbed antioxidative functions resulting in altered mitochondrial metabo-lism. This negative impact of DMF treatment on VSMCs may be linked to preexisting alterations of cerebrovascular function due to renal hypertension. Therefore, before severe adverse effects emerge, it would be clinically relevant to develop indices or biomarkers linked to this disturbed antioxidative function to monitor patients undergoing DMF treatment.

overall_design: Single-cell suspension from surgically isolated circle of Willis arteries from sham, animals that formed cerebral aneurysms and animals that had ruptured

first: A

last: Martinez

first: G

last: Tortelote

last: Dumont


title: Uremic toxin indoxyl sulfate induces trained immunity via the AhR-dependent arachidonic acid pathway in end-stage renal disease [RNAseq]

city: Seoul

first: Hee Young

last: Kim

address: 103 Daehak-ro, Jongno-gu,

department: Department of Microbiology and Immunology

country: South Korea

institute: Seoul National University College of Medicine

summary: Trained immunity is the long-term functional reprogramming of innate immune cells, which results in altered responses toward a secondary challenge. Despite indoxyl sulfate (IS) being a potent stimulus associated with chronic kidney disease (CKD)-related inflammation, its impact on trained immunity has not been explored. Here, we demonstrate that IS induces trained immunity in monocytes via epigenetic and metabolic reprogramming, resulting in augmented cytokine production. Mechanistically, the aryl hydrocarbon receptor (AhR) contributes to IS-trained immunity by enhancing the expression of arachidonic acid (AA) metabolism-related genes such as Arachidonate 5-Lipoxygenase (ALOX5) and ALOX5 Activating Protein (ALOX5AP). Inhibition of AhR during IS training suppresses the induction of IS-trained immunity. Monocytes from end-stage renal disease (ESRD) patients have increased ALOX5 expression and after 6-day training, they exhibit enhanced TNF-α and IL-6 production to LPS. Furthermore, healthy control-derived monocytes trained with uremic sera from ESRD patients exhibit increased production of TNF-α and IL-6. Consistently, IS-trained mice and their splenic myeloid cells had increased production of TNF-α after in vivo and ex vivo LPS stimulation compared to that of control mice. These results provide insight into the role of IS in the induction of trained immunity, which is critical during inflammatory immune responses in CKD patients.

overall_design: To investigate the mechanism involved in IS-induced trained immunity, we performed RNA-seq  using IS-trained cells and non-trained cells at 6 days. Differentially expressed genes (DEGs) and  gene oncology (GO) pathways were analyzed

first: Hee-Young

first: Su-Jeong

last: Lee

first: Won-Woo


title: Uremic toxin indoxyl sulfate induces trained immunity via the AhR-dependent arachidonic acid pathway in end-stage renal disease [ChIPseq]

overall_design: To investigate the involvement of epigenetic reprogramming in IS-induced trained immunity, we performed ChIP-seq for histone 3 K4 trimethylation (H3K4me3)  in IS-trained immunity at 6 days.


title: Study on the functional mechanism of NOP2 in regulating lung cancer development

city: Zhengzhou

first: Junhu

last: Wan

address: No. 1 Jianshe East Road

institute: The First Affiliated Hospital of Zhengzhou University

summary: NOP2 is a member of the NOL1/NOP2/SUN structural domain (NSUN) family and is responsible for catalyzing post-transcriptional modification of RNA via 5-methylcytosine (m5C). m5C modification dysregulation has been implicated in the pathogenesis of a variety of malignancies. Here, we investigated the expression of NOP2 in lung cancer tissues and cells and found that its expression was significantly upregulated. This is consistent with previous reports. To further investigate the mechanism by which NOP2 affects lung cancer development, RNA sequence analysis was performed. In conclusion, our study emphasizes the important role of NOP2 in lung cancer, and its promise as a potential target for lung cancer therapy.

overall_design: To investigate the mechanism by which NOP2 affects lung cancer development, RNA sequence analysis was performed.


title: Specific silencing of pathogenic mRNA by a novel compact RNA-targeting tool TaqTth-hpRNA [Amplicon sequencing]

overall_design: Sequence preference detection. An oligo sequence, with two degeneracy bases at the 5' end, a target in the middle, and four degeneracy bases at the 3' end, was designed and inserted into the ampicillin start codon to construct a plasmid library. The ampicillin-resistant plasmid library, the chloramphenicol-resistant TaqTth protein expression plasmid and the spectinomycin-resistant probe plasmid were transferred into the electrical natural competence by bio-red electroporation. The expression of TaqTth was induced by the Tet inducer. Then 16 hours later, cells were scraped off plates and extracted plasmid DNA. The unrelated probe NT was used as the control group. To assess sequence preference, the nucleotide portion of the original library containing the target sequence was extracted and PCR-specific amplification was performed. The specific PCR product was sequenced by Sangon Biotech (Shanghai, China).


title: DSB profiles in human spermatozoa highlight the role of TMEJ in the male germline

status: Public on Jun 19 2024

city: Mainz

first: Maurice

last: Scheuren

address: Anselm-Franz-von-Bentzel-Weg 7

department: Division of Anthropology, Faculty of Biology, Institute of Organismic and Molecular Evolution

institute: Johannes Gutenberg-University Mainz

summary: The male mammalian germline is characterized by two rounds of significant chromatin remodeling of the genome, first by the histone-to-protamine transition during spermatogenesis, followed by the reversal to nucleo-histones in the male pronucleus of the zygote. Both transitions are associated with the extensive formation of DNA double-strand breaks (DSBs), requiring an estimated 5 to 10 million transient DSBs per spermatozoa. Additionally, the high transcription rate in early stages of spermatogenesis leads to transcription-coupled damage preceding meiotic homologous recombination, potentially further contributing to the DSB landscape in mature spermatozoa. Once meiosis is completed, spermatozoa remain haploid and therefore cannot rely on error-free homologous recombination, but instead depend on error-prone classical non-homologous end joining (cNHEJ). This DNA damage/repair-scenario is proposed to be one of the main causes of the observed paternal mutation propensity in human evolution. Recent studies have shown that DSBs in the male pronucleus are repaired by maternally provided Polθ in C. elegans through Polθ-mediated end joining (TMEJ). Additionally, population genetic datasets have revealed a preponderance of TMEJ signatures associated with human variation. Since these signatures are the result of the combined effect of TMEJ and DSB formation in spermatozoa and male pronuclei, we used a BLISS-based protocol to analyze recurrent DSBs in mature human sperm heads as a proxy of the male pronucleus before zygotic chromatin remodeling. The DSBs were found to be enriched in (YR)n short tandem repeats and in evolutionarily young SINEs, reminiscent to patterns observed in murine spermatids, indicating evolutionary hotspots of recurrent DSB formation in mammalian spermatozoa. Additionally, we detected a similar DSB pattern in diploid human IMR90 cells when cNHEJ was selectively inhibited, indicating the significant impact of absent cNHEJ on the sperm DSB landscape. Strikingly, regions associated with most retained histones, and therefore less condensed chromatin, were not strongly enriched with recurrent DSBs. In contrast, the fraction of retained H3K27me3 in the mature spermatozoa displayed a strong association with recurrent DSBs. DSBs in H3K27me3 are associated with a preference for TMEJ over cNHEJ during repair. We hypothesize that the retained H3K27me3 may trigger transgenerational DNA repair by priming maternal Polθ to these regions.

overall_design: Genomic DNA double-strand break profiling by high troughpout sequencing using BLISS-like protocol of human spermatozoa and somatic human cells.

first: Hans

last: Zischler


title: Therapeutic potential of co-signaling receptor modulation in hepatitis B [RNA-Seq]

city: Milano

first: Chiara

last: Laura

address: Via Olgettina 58

institute: San Raffaele Scientific Institute

summary: Reversing CD8+ T cell dysfunction is crucial in therapeutic approaches to eliminate chronic hepatitis B virus (HBV) infection. However, the specific molecular targets for achieving this goal are not fully understood. In this study, we conducted a comprehensive analysis of the co-signaling receptors induced during hepatocellular priming and examined the trajectory and long-term fate of dysfunctional HBV-specific CD8+ T cells. Our findings demonstrate that early during intrahepatic priming, HBV-specific CD8+ T cells selectively upregulate co-inhibitory receptors PD-1, CTLA-4, and LAG-3, as well as co-stimulatory receptors OX40, 4-1BB, and ICOS. While blocking the co-inhibitory receptors had no significant impact, activating 4-1BB and OX40, but not ICOS, transformed these cells into potent antiviral effectors. Prolonged antigenic stimulation of intrahepatically-primed dysfunctional T cells led to the development of a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile that only partially overlaps with other forms of T cell hyporesponsiveness. This population consists of a dysfunctional progenitor or stem-like (TSL) population and two distinct dysfunctional tissue-resident memory (TRM) cell populations, which are hierarchically related. While 4-1BB expression is maintained across all populations, OX40 expression is limited to TSL cells. At later stages, only stimulation of 4-1BB, not OX40, conferred antiviral activity to HBV-specific CD8+ T cells. These findings highlight the therapeutic potential of co-signaling receptor modulation for chronic HBV infection and suggest that targeting all dysfunctional tissue-resident T cells, rather than solely the stem-like precursors, represents a promising strategy, distinguishing it from other conditions characterized by chronic antigenic stimulation.

overall_design: Bulk RNA sequencing was performed on Cor93 T cells adoptively transferred in HBV-Tg mice. These cells were challenged 24 hours later with agonistic mAbs targeting either OX40 or 4-1BB. After 5 days, intrahepatic Cor93 were FACS-sorted and processed for bulk RNAseq.

first: Francesco

last: Andreata

first: Micol

last: Ravà

first: Caroline

last: Krüger

first: Xenia

last: Ficht

first: Xeigo

last: Kawashima

first: Christian B.

last: Beccaria

first: Matteo

last: Iannacone


title: GTPBP8 is essential for mitoribosome formation in human mitochondria

city: Nashville

first: James

last: Marks

state: Tennessee

address: 638A Robinson Research Building

institute: NIAMS/NIH

summary: Mitochondrial gene expression relies on mitoribosomes to translate mitochondrial mRNAs. The biogenesis of mitoribosomes is an intricate process involving multiple assembly factors. Among these factors, GTP-binding proteins (GTPBPs) play crucial roles. In bacterial systems, numerous GTPBPs are required for ribosome subunit maturation, with EngB being a GTPBP involved in the ribosomal large subunit assembly. In this study, we focused on exploring the function of GTPBP8, the human homolog of EngB. We found that ablation of GTPBP8 leads to the inhibition of mitochondrial translation, resulting in significant impairment of oxidative phosphorylation. Structural analysis of mitoribosomes from GTPBP8 knock-out cells showed the accumulation of mitoribosomal large subunit assembly intermediates that are incapable of forming functional monosomes. Furthermore, fPAR-CLIP analysis revealed that GTPBP8 is an RNA-binding protein that interacts specifically with the mitochondrial ribosome large subunit16S rRNA. Our study highlights the crucial role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitochondrial large subunit maturation.

overall_design: We performed Flag-GTPBP8 and V5-AUH fPAR-CLIP to assess protein occupancy on RNA targets in doxacycline incudcible HEK293 cells from whole cell lysates

first: Miriam

last: Cipullo

first: Genís

last: Valentín Gesé

first: Shreekara

last: Gopalakrishna

first: Annika

last: Krueger

first: Vivian

last: Lobo

first: Maria

last: Pirozhkova

first: Petra

last: Páleníková

first: Dmitrii

last: Shiriaev

first: Yong

last: Liu

first: Jelena

last: Misic

first: Yu

last: Cai

first: Minh

last: Nguyen

first: Abubakar

last: Abdelbagi

first: Xinping

last: Li

first: Michal

last: Minczuk

first: Markus

last: Hafner

first: Daniel

last: Benhalevy

first: Aishe

last: Sarshad

first: Ilian

last: Attanasov

first: Martin

last: Hällberg

first: Joanna

last: Rorbach


title: Chromatin conformation and histone modification profiling across human kidney anatomic regions [Hi-C]

city: New Haven

first: Haikuo

address: 55 Prospect St

institute: Washington University in St. Louis

summary: Here, we leveraged our recently described high-yield nuclei extraction method and performed Hi-C and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing of histone modifications (H3K4me3 and H3K27me3) in parallel on the kidney cortex, medulla and papilla samples dissected from the same donor. This site describes the Hi-C data.

overall_design: Hi-C was performed with the Arima-HiC kit (A510008, Arima Genomics). Hi-C library preparation was performed with the Arima Library Prep Kit v2 (A303011, Arima Genomics). CUT&RUN was performed with the CUTANA™ ChIC / CUT&RUN Kit v3. CUT&RUN library preparation was performed with the NEBNext® Ultra™ II DNA Library Prep Kit. All 6 dual-indexed Hi-C libraries and 18 dual-indexed CUT&RUN libraries were sequenced on a 300-cycle NovaSeq X Plus platform (Illumina).

first: Benjamin

last: Humphreys


title: Immunological memory diversity in the human upper airway

city: La Jolla

first: Shane

last: Crotty

state: CA

address: 9420 Athena Circle

institute: La Jolla Institute for Immunology

summary: Limited CITE-seq antibody panel and 5' gene expression profiling of human nasopharyngeal cells collected with flocked swabs, paired with TCR and BCR sequencing of NP lymphocytes. FACS-sorted SARS-CoV-2 antigen-specific memory B and T cells with 5' gene expression profiling and scBCR and scTCRseq.

overall_design: 10x Genomics 5' immune profiling using BioLegend TotalSeq-C TBNK, hashtag antibodies, and SARS-CoV-2 labeled probes.

first: Sydney

middle: I

last: Ramirez

first: Farhoud

last: Faraji


title: Chromatin conformation and histone modification profiling across human kidney anatomic regions [CUT&RUN]

summary: Here, we leveraged our recently described high-yield nuclei extraction method and performed Hi-C and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing of histone modifications (H3K4me3 and H3K27me3) in parallel on the kidney cortex, medulla and papilla samples dissected from the same donor. This site describes the CUT&RUN data.

overall_design: CUT&RUN was performed with the CUTANA™ ChIC / CUT&RUN Kit v3. CUT&RUN library preparation was performed with the NEBNext® Ultra™ II DNA Library Prep Kit. All 6 dual-indexed Hi-C libraries and 18 dual-indexed CUT&RUN libraries were sequenced on a 300-cycle NovaSeq X Plus platform (Illumina).


title: IL-1R signaling drives enteric glia-macrophage interactions in colorectal cancer

first: Gianluca

last: Matteoli

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

overall_design: Refer to individual Series


title: Therapeutic potential of co-signaling receptor modulation in hepatitis B [scRNA-Seq]

first: Chiara

last: Laura

overall_design: Single-cell RNA sequencing was performed on Cor93 T cells 28 days after adoptive transfer into HBV Tg mice. These hepatocellularly (HC)-primed dysfunctional cells were compared to control Cor93 T cells injected into wild-type (WT) mice transduced with a replication-defective recombinant lymphocytic choriomeningitis-based vector (rLCMV-core) that targets the same antigen, a non-secretable version of the HBV core protein, to Kupffer cells (KC) and dendritic cells that are not naturally infected by HBV.

first: Francesco

last: Andreata

first: Micol

last: Ravà

first: Caroline

last: Krüger

first: Xenia

last: Ficht

first: Xeigo

last: Kawashima

first: Cristian G.

last: Beccaria

first: Matteo

last: Iannacone


title: PRC2-AgeIndex: a universal biomarker of aging and rejuvenation [WGBS]

city: Stanford

first: Vittorio

last: Sebastiano

state: California

address: 265 Campus Drive

department: OB-GYN/Reproductive, Perinatal and Stem Cell Biology Research

institute: Stanford University

type: Methylation profiling by high throughput sequencing

summary: DNA methylation (DNAm) is one of the most reliable biomarkers of aging across many mammalian tissues. While the age-dependent global loss of DNAm has been well characterized, age-dependent DNAm gain is less specified. Multiple studies have demonstrated that polycomb repressive complex 2 (PRC2) targets are enriched among the CpG sites which gain methylation with age. However, systematic whole-genome examination of all PRC2 targets in the context of aging methylome as well as determination of the pan-tissue or tissue-specific nature of these associations is lacking. Here, by analyzing DNAm data from different assays and from multiple young and old human and mouse tissues, we found that low-methylated regions (LMRs) which are highly bound by PRC2 in embryonic stem cells (PRC2 LMRs) gain methylation with age in all examined somatic mitotic cells. We also estimated that this epigenetic change represents around 90% of the age-dependent DNAm gain genome-wide. Therefore, we propose the “PRC2-AgeIndex,” defined as the average DNAm in PRC2 LMRs, as a universal biomarker of cellular aging in somatic cells. In addition, we demonstrate the application of this biomarker in the evaluation of different anti-aging interventions, including dietary restriction and partial epigenetic reprogramming.

overall_design: WGBS data of neonatal and old fibroblasts from three different donors each.

first: Madhi

last: Moqri

first: Andrea

last: Cipriano

first: Daniel

middle: J

last: Simpson

first: Sajede

last: Rasouli

first: Tineke

last: de Jong


title: CD47 blockade synergizes with doxorubicin in enhancing macrophage phagocytosis of Ewing sarcoma cells and deceasing xenograft tumor growth and metastasis in an orthotopic model of Ewing sarcoma

city: Columbus

first: Katherine

last: Miller

state: OH

address: 700 Children's Dr.

department: Institute for Genomic Medicine

institute: Nationwide Children's Hospital

summary: Macrophages are immune cells whose major functions include phagocytosing tumor cells. However, tumors escape macrophage phagocytosis through the expression of anti-phagocytic signals, most commonly CD47. Moreover, in Ewing sarcoma (ES), we found that tumor cells utilize dual mechanisms to evade macrophage clearance by simultaneously over-expressing CD47 and down-regulating cell surface calreticulin (csCRT), the pro-phagocytic signal. Here we utilize a CD47 blockade (magrolimab, MAG) to inhibit the anti-phagocytic signal and a chemotherapy regimen (doxorubicin, DOX) to enhance the pro-phagocytic signal and evaluated the effects of MAG or DOX alone and in combination on macrophage phagocytosis of ES cells in vitro and tumor formation and development in vivo. Furthermore, we performed RNA-Seq to identify potential mechanisms of resistance to the CD47 blockade combinatorial therapy.

overall_design: Doxorubicin was utilized to enhance csCRT on ES cells. Magrolimab, a humanized CD47 monoclonal antibody was used to block CD47 signal. An ES orthotopic mouse model was utilized to assess the effect of doxorubicin and magrolimab on ES tumor growth and metastasis and animal survival. Tumors were harvested at the end of the study. Total RNA was extracted from the tumors and bulk RNA-Seq was carried out on NovaSeq 6000.

first: Wen

last: Luo

first: Mitchell

last: Cairo


title: PRC2-AgeIndex: a universal biomarker of aging and rejuvenation


title: PRC2-AgeIndex: a universal biomarker of aging and rejuvenation [ChIP-seq]

overall_design: ChIP data of neonatal fibroblasts, old fibroblasts and CD4 T-cells, each from three different donors.


title: RNA sequencing of bone marrow

city: Chongqing

first: Mengjia

last: Hu

address: Gaotanyan Street 30

institute: Third Military Medical University


title: RNA sequencing of eGFPlo and eGFPhi LT-HSCs in the bone marrow from Trim47+/eGFP mice

summary: eGFPlo and eGFPhi LT-HSCs in the bone marrow from Trim47+/eGFP mice were isolated to explore the differentially expressed genes by RNA-seq.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for eGFPlo and eGFPhi LT-HSCs in the bone marrow rrom Trim47+/eGFP mice

first: Fang

last: Chen


title: RNA sequencing of LT-HSCs in the bone marrow from WT and Trim47-/-  mice untreated or treated with 5-FU for 10 days

summary: LT-HSCs in the bone marrow of WT and Trim47-/-  mice untreated or treated with 5-FU for 10 days were isolated to explore the differentially expressed genes by RNA-seq.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for LT-HSCs in the bone marrow from WT and Trim47-/- mice


title: BET activity plays an essential role in control of stem cell attributes in Xenopus

city: Evanston

first: Paul

middle: Benjamin

last: Huber

address: 2205 Tech Drive

department: Molecular Biosciences

institute: Northwestern University

summary:
Here we examine the role that epigenetic readers of the BET (Bromodomain and Extra Terminal)
family play in controlling the potential of pluripotent blastula and neural crest cells.
We find that inhibiting BET activity leads to loss of pluripotency and inability
to respond to lineage instructing cues at blastula stages, and a loss of neural crest at neurula stages.

overall_design:
We compare the effects of HDAC (an eraser of acetylation marks) and BET (a reader of acetylation) inhibition
and find that they lead to similar cellular outcomes through distinct effects on the transcriptome.
TSA and IBET or vehicle treated animal pole explants (epidermal or neural crest-reprogrammed)
were collected at stages 9,13, and 17, and total RNA was collected
from those samples and submitted for high throughout sequencing.

first: Anjali

last: Rao

first: Carole

last: LaBonne