title: EWS::FLI cooperatively binds at GGAA microsatellites via DBD-a4 helix in TTC-466 cells [FLAG_CnT]

city: Columbus

first: Ariunaa

last: Bayanjargal

state: OH

address: 700 Children's Dr

institute: Nationwide Childrens Hospital

summary: Multi-omics study of DBD-a4 helix of EWS::FLI to understand the underlying mechanism of transcriptional regulation in Ewing sarcoma cell line: TTC466

overall_design: In TTC-466 cells, we knock-down endogenous EWSR1::ERG with shRNA  (KD) and rescued with FLAG tagged DBD and DBD+ constructs. Cells are then harvested for RNA-Seq (3 bio reps), CUT&Tag (3 bio reps for CTCF, 4 bio reps for H3K27ac, 4 bio reps for FLAG), and Micro-C libraries (2 bio reps).

first: Julia

last: Selich-Anderson

first: Emily

last: Theisen

first: Jesse

last: Crow


title: The transcription factor ZNF469 regulates collagen production in liver fibrosis [RNAseq_HSC]

status: Public on Feb 27 2025

city: NASHVILLE

first: Marisol

last: Ramirez

state: TENNESSEE

address: 2220 Pierce Avenue, 571 Preston Research Building

department: Center for Quantitative Sciences, Department of Biostatisticss

country: USA

institute: Vanderbilt University Medical Center

type: Expression profiling by high throughput sequencing

summary: Non-alcoholic fatty liver disease (NAFLD)—characterized by excess accumulation of fat in the liver—now affects one third of the world’s population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis.

overall_design: HSCs were electroporated with ribonucleoproteins (RNPs) targeting individual transcription factors vs a non-targeting gRNA control. N=3 per CRISPR group.

first: Quanhu

last: Sheng

first: Jonathan

last: Brown

first: Sebastian

last: Steinhauser

last: Ramirez-Solano


title: The transcription factor ZNF469 regulates collagen production in liver fibrosis [MouseMCD]

overall_design: Male and Female C57BL/6J animals were fed a methionine-choline deficient diet ("MCD") or control diet ("MCD Control") (n=6 animals per diet per sex) for 20 weeks.


title: The transcription factor ZNF469 regulates collagen production in liver fibrosis [LX2_CUTRUN_ZNF]

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: Human immortalized stellate cel lline (LX2) were grown in standard conditions.   Cells were transfected with a piggybac transposon vector expressing full length human ZNF469 gene or  human ZNF469 with deleted zinc finger (dZF).  3 days after transfection, stable lines were selected with puromycin at 1 microgram/mL.  After selection, cells were treated with doxycycline (1 microgram/mL) to induce expression of ZNF469 or dz-ZNF469 for 24 hours.   Samples were then processed for CUT&RUN.


title: The transcription factor ZNF469 regulates collagen production in liver fibrosis [MicroC]

overall_design: Primary human hepatic stellate cell line cultured in standard growth media without stimulation.


title: The transcription factor ZNF469 regulates collagen production in liver fibrosis [RNA-Seq liver]

overall_design: Human Liver biopsies from patients referred for bariatric surgery.  Both males and females are included.


title: The transcription factor ZNF469 regulates collagen production in liver fibrosis [MouseFFD]

overall_design: Male and Female C57BL/6J animals were fed a high fat diet ("FFD") or control diet (n=6 per diet per sex) for 20 weeks.


title: The transcription factor ZNF469 regulates collagen production in liver fibrosis [Cohort_CUTRUN_H3K27ac]


title: EWS::FLI cooperatively binds at GGAA microsatellites via DBD-a4 helix in TTC-466 [CTCF_CnT]

summary: Multi-omics study of DBD-a4 helix of EWS::FLI to understand the underlying mechanism of transcriptional regulation in Ewing sarcoma cell line: TTC-466

middle: R

middle: C


title: EWS::FLI cooperatively binds at GGAA microsatellites via DBD-a4 helix in TTC-466 [RNA-seq]


title: Sox9 and Nfi transcription factors regulate the timing of neurogenesis and ependymal maturation in dopamine progenitors [scRNA]

city: Stockholm

first: Behzad

last: Yaghmaeian Salmani

state: Solna, Stockholm

address: Department of Cell and Molecular Biology (CMB) Karolinska Institute,, Biomedicum, Solnavägen 9, Solna, Stockholm, P.O. Box 285 SE-171 77 Stockholm, Sweden

department: CMB

country: Sweden

institute: Karolinska Institute

summary: Correct timing of neurogenesis is critical for both generating the correct number and subtypes of glia and neurons in the embryo, as well as preventing tumours and the depletion of stem cell pools in the adults. Here we analyse how the midbrain dopamine neuron (mDA) progenitors transition into cell cycle arrest (G0) and begin to mature into ependymal cells. The comparison of mDA progenitors from different embryonic stages revealed the upregulation of Nfi and Sox9 transcription factors during development. Their conditional inactivation in the early embryonic midbrain leads to delayed G0 entry and ependymal maturation, reduced gliogenesis, and increased generation of neurons, including mDA neurons. In contrast, their inactivation in late embryogenesis does not result in mitotic re-entry, suggesting that these factors are necessary for the G0 induction, but not for its maintenance. Our characterisation of mDA-progenitor-derived adult ependymal cells by single-cell RNA sequencing and histology show that they both retain several progenitor features but also secrete neuropeptides and contact neighbouring cells and blood vessels, indicating that these cells may form a novel part of the circumventricular organ system.

overall_design: Ependymal and glial cells for scRNA sequencing were prepared from freshly dissected brains. Ependymal tissue was microdissected from the lateral ventricles, midbrain aqueduct, third ventricle, and spinal cord from 3-month-old FoxJ1CreERT2 R26TdT animals (n=10; 5 females & 5 males). Midbrain tissue was also microdissected from 3-month-old Nkx2-2Cre R26TrapC mice (n=7, all females).

first: Laura

last: Lahti

first: Nikolaos

last: Volakakis

first: Linda

last: Gillberg

last: Yaghmaeian-Salmani

first: Katarína

last: Tiklová

first: Nigel

last: Kee

first: Hilda

last: Lundén-Miguel

first: Maarten

last: Werkman

first: Michael

last: Piper

first: Richard

last: Gronostajski

first: Thomas

last: Perlmann


title: The transcription factor ZNF469 regulates collagen production in liver fibrosis [HSC_CUTRUN_ZNF469AB]

last: Ramirez

overall_design: Primary human HSCs were cultured in standard conditions.  Cells were electroporated with RNPs (ribonucleoproteins) containing spCas9 with non-targeting gRNA (i.e. control) or gRNAs targeting ZNF469 (target gene).  Following cell recovery and expansion, CUT&RUN was performed with anti-ZNF469 antibody.  N=3 replicates per CRISPR condition.


title: Immunosuppressive crosstalk between tumour cells, myeloid cells and T cells in an anti-PD-1 plus anti-CTLA-4 therapy resistant mouse ccRCC model [RNA-seq]

city: Freiburg

first: Patrick

last: Metzger

address: Breisacher Str. 153

department: Institute for Medical Bioinformatics and Systems Medicine

country: Germany

institute: University Hospital Freiburg

summary: Many patients respond poorly or develop resistance to immune checkpoint therapies for metastatic clear cell renal cell carcinoma (ccRCC). Here we demonstrate that ccRCCs in the Vhl/Trp53/Rb1 mutant autochthonous mouse model are resistant to anti-PD-1 plus anti-CTLA-4 therapy, providing a model of checkpoint therapy resistance. In vivo checkpoint therapy allowed isolated T cells to recognise cultured ccRCC cells, implicating the tumour microenvironment in suppression of T cell activation. Single cell RNA-seq identified putative immunosuppressive PMN-MDSC, M-MDSC and TAM populations that show similar features to myeloid cells in the microenvironment of human ccRCC. Conditioned medium of ccRCC cell lines attracted bone marrow-derived macrophages in a MIF-dependent manner and induced the mis-differentiation of bone marrow cells to M-MDSC and TAM2-like phenotypes. These cells suppressed the viability and proliferation of activated CD8+ T cells. These analyses identify new candidate cellular and molecular barriers to effective immune checkpoint therapy. We validate this concept by showing that GALECTIN-3 is the predominant checkpoint ligand expressed by ccRCC cells and myeloid cells and that anti-LAG-3 treatment increases T cell infiltration in this model.

overall_design: RNA-seq of conditioned medium-differentiated bone marrow cultures compared to freshly-isolated bone marrow

first: Asin

last: Peighambari

first: Hsin

last: Huang

first: Mojca

last: Adlesic

first: Kyra

last: Zodel

first: Rouven

last: Hoefflin

first: Silvia

last: Schäfer

first: Philipp

last: Seidel

first: Lukas

last: Braun

first: Jan

last: Hülsdünker

first: Wolfgang

last: Melchinger

first: Marie

last: Follo

first: Manching

last: Ku

first: Stefan

last: Haug

first: Yong

last: Li

first: Anna

last: Köttgen

first: Christoph

last: Schell

first: Dominik

last: von Elverfeldt

first: Wilfried

last: Reichardt

first: Robert

last: Zeiser

first: Mathias

last: Heikenwalder

first: Melanie

last: Boerries

first: Ian

middle: J

last: Frew


title: Heterogeneous Cardiac- and Neural Crest-Derived Aortic Smooth Muscle Cells have Similar Transcriptional Changes after TGFβ Signaling Disruption [scRNA-seq-of-human-aortic-media-vascular-cells]

city: New Haven

first: George

last: Tellides

state: CT

address: 10 Amistad st, 337B

department: Surgery

institute: Yale University

summary: We report single-cell RNA-seq data of different segments from human thoracic aortas

overall_design:
Root/Asc/Arch/Desc aortas were collected from human donors in operation room, then digested in collagenase and elastase, stained with CD45, CD235a and viability dye, then subjected to FACS and scRNA-seq

***************************************************************
Submitter states that missing raw data are being made available for controlled access in dbGaP.
***************************************************************

first: Pengwei

last: Ren


title: The transcription factor ZNF469 regulates collagen production in liver fibrosis [MouseOBOB]

first: Marisol

last: Ramirez

overall_design: Animals were housed in standard mouse cages with standard chow diet ("OBOB_FLO") or standard chow diet along with addition of 0.165% DL Ethionine ("OBOB_Fibrotic") in the drinking water for 2 weeks.  Only Male mice n=6 per group) were used.

first: Quanhu

last: Sheng

first: Jonathan

last: Brown

first: Sebastian

last: Steinhauser

last: Ramirez-Solano


title: The endogenous antigen-specific CD8+ T cell repertoire is composed of unbiased and biased clonotypes with differential fate commitment.

city: Lebanon

first: Leena

last: Abdullah

state: NH

address: 1 Medical Center Dr

institute: Dartmouth College

summary: Generating balanced populations of CD8+ effector and memory T cells is necessary for immediate and durable immunity to infections and cancer. Yet, a definitive understanding of how a diverse CD8+ T cell repertoire differentiates remains unclear. We identified several hundred TCR clonotypes that constitute the polyclonal response against a single antigen and found that a majority of TCR clonotypes were highly biased towards memory or effector fate. TCR-intrinsic biases were not stochastic and were dominant over environmental cues. Differential gene expression analysis of memory- or effector-biased TCR clonotypes showed bifurcation of differential fates at the early effector stage. Additionally, phylogenetic analysis revealed that memory-biased clonotypes retain their fate preferences in subclonal populations but effector-biased subclones can switch to memory fate. Our study highlights that the polyclonal CD8+ T cell response is a composite of unbiased and biased clonotypes with varying capacity to incorporate environmental cues in their cell fate decisions.

overall_design: scRNA-seq, scRAGE-seq (scTCR-seq) and scCARLIN barcode sequencing were performed on splenic CD8+ T cells isolated from CARLIN mice infected with VSV-OVA acute viral infection. scRNA-seq for transcriptional profiling of the cells and the scCARLIN barcode sequencing to uncover the edits in the CARLIN array were performed using Illumina sequencing. scTCR-seq was performed using Nanopore technology as done in the original RAGE-seq paper (https://www.nature.com/articles/s41467-019-11049-4).

first: Francesco

middle: E

last: Emiliani

first: Chinmay

middle: M

last: Vaidya

first: Hannah

last: Stuart

first: Shawn

last: Musial

first: Fred

middle: W

last: Kolling

first: Joshua

last: Obar

first: Pamela

last: Rosato

first: Margaret

last: Ackerman

first: Li

last: Song

first: Aaron

last: McKenna

first: Yina

middle: H

last: Huang


title: The effects of ALKBH5 on liver function [RNA-seq]

city: Harbin

first: Zheng

last: Chen

state: Heilongjiang

address: 92 Xidazhijie  Harbin Institute of Technology

country: China

institute: Harbin Institute of Technology

summary: The goal of this study is to investigate how ALKBH5 regulates liver function.

overall_design:
mRNA profiles in livers of Alkbh5flox/flox and Alkbh5-HKO mice were generated by deep sequencing using Illumina Novaseq 6000 platform (n=3 for each group).
Methods: Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from livers of Alkbh5flox/flox and Alkbh5-HKO mice at 9 weeks old. Three independent biological replicates for each group were used for RNA-seq. RNA-seq was performed by deep sequencing using an Illumina Novaseq 6000 platform. Paired-end clean reads were aligned to the mouse reference genome(GRCm38.p6) with Hisat2 v2.0.5, and the aligned reads were used to quantify mRNA expression by using featureCounts v1.5.0-p3.
Our study represents the first detailed analysis of liver transcriptomes from  Alkbh5flox/flox and Alkbh5-HKO mice, generated by RNA-seq technology. The RNA-seq analysis showed that 1107 genes were downregulated and 1248 genes were upregulated in the livers of Alkbh5-HKO mice.

first: Kaixin

last: Ding


title: Heterogeneous Cardiac- and Neural Crest-Derived Aortic Smooth Muscle Cells have Similar Transcriptional Changes after TGFβ Signaling Disruption [scRNA-seq-of-Nkx2-5-Cre-mTmG]

first: George

last: Tellides

summary: We report single-cell RNA-seq data of Nkx2-5 lineage marked, integrin alpha 8 labeled smooth muscle cells (SMCs) from murine aortas at 12 weeks of age

overall_design: Root/Ascending/Arch/Descending aortic segments were procured from 12-week-old  Nkx2-5-Cre.mT/mG mice, ITGA8+ SMCs were isolated after cryophilic enzyme digestion, and single-cell RNA-seq analysis was performed

first: Pengwei

last: Ren


title: Heterogeneous Cardiac- and Neural Crest-Derived Aortic Smooth Muscle Cells have Similar Transcriptional Changes after TGFβ Signaling Disruption [Bulk-RNA-seq-of-Human-aortas]

summary: We report bulk RNA-seq data of different segments of human thoracic aortas

overall_design:
Human aortic segments were collected in operation room and kept in -80 . Total RNA was isolated using a RNeasy Mini Kit (74104, Qiagen), and sent for bulk RNA-seq with rRNA depletion.

***************************************************************
Submitter states that missing raw data are being made available for controlled access in dbGaP.
***************************************************************


title: Heterogeneous Cardiac- and Neural Crest-Derived Aortic Smooth Muscle Cells have Similar Transcriptional Changes after TGFβ Signaling Disruption [scRNA-seq-of-human-aortic-cells]

overall_design:
Root/Asc/Arch aortas were collected from human donors in operation room, then digested in collagenase and elastase, subjected to FACS and scRNA-seq

***************************************************************
Submitter states that missing raw data are being made available for controlled access in dbGaP.
***************************************************************


title: The transcription factor ZNF469 regulates collagen production in liver fibrosis

first: Marisol

last: Ramirez

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Heterogeneous Cardiac- and Neural Crest-Derived Aortic Smooth Muscle Cells have Similar Transcriptional Changes after TGFβ Signaling Disruption


title: Cannabinoid receptor 2 selective agonist alleviates systemic sclerosis by inhibiting Th2 differentiation through JAK/SOCS3 signaling

city: Shanghai

last: Tian

address: South Chongqing Road

institute: Shanghai Jiao Tong University

summary: Systemic sclerosis (SSc) poses a significant challenge in autoimmunology, characterized by the development of debilitating fibrosis of skin and internal organs. The pivotal role of dysregulated T cells, notably the skewed polarization toward Th2 cells, has been implicated in the vascular damage and progressive fibrosis observed in SSc. In this study, we explored the underlying mechanisms by which cannabinoid receptor 2 (CB2) highly selective agonist HU-308 restores the imbalance of T cells to alleviate SSc. Using a bleomycin-induced SSc (BLM-SSc) mouse model, we demonstrated that HU-308 effectively attenuates skin and lung fibrosis by specifically activating CB2 on CD4+ T cells to inhibit the polarization of Th2 cells in BLM-SSc mice, which was validated by Cnr2-specific-deficient mice. Different from classical signaling downstream of G protein-coupled receptors (GPCRs), HU-308 facilitates the expression of SOCS3 protein and subsequently impedes the IL2/STAT5 signaling pathway during Th2 differentiation. The deficiency of SOCS3 partially mitigated the impact of HU-308. Analysis of a cohort comprising 62 SSc patients and 82 healthy controls revealed an abnormal elevation in the Th2/Th1 ratio in SSc patients. Administration of HU-308 to PBMCs and peripheral CD4+ T cells from SSc patients led to the upregulation of SOCS3, which effectively suppressed the aberrantly activated STAT5 signaling pathway and the proportion of CD4+IL4+ T cells. In conclusion, our findings unveil a novel mechanism by which the CB2 agonist HU-308 ameliorates fibrosis in SSc by targeting and reducing Th2 responses. These insights provide a foundation for future therapeutic approaches in SSc by modulating Th2 responses.

overall_design: Naive CD4+ T cells were isolated and cultured under Th2 polarization conditions with or without the addition of HU308. On day 4, cells were re-stimulated using anti-CD3/CD28 beads and harvested after 12h. Total RNA was extracted by TRIzol reagent. Subsequently, library preparation and sequencing were performed by Novogene Corporation (Beijing, China).

first: Hao

last: Cheng

first: Yu

last: Du

first: Yi

last: Lei


title: CCR7 Expression Distinguishes Functionally Distinct pTfh1 Subsets with Roles in Malaria-Specific Immunity

city: Brisbane

first: Zuleima

last: Pava Imitola

state: Queensland

address: 300 Herston road

country: Australia

institute: QIMR Berghofer

summary: T-follicular CD4 T (Tfh) cells play essential roles in antibody induction during infection and following vaccination. In humans, peripheral Tfh (pTfh) cells are commonly analysed based on expression of CXCR3 and CCR6, with different subsets of pTfh (pTfh1, pTfh2, pTfh17) associated with antibody induction in a context-dependent manner. In malaria, the specific roles of pTfh subsets in antibody development is not clear. Several studies in human malaria infection and vaccination have identified an important role of pTfh2 cells, which associate with antibody development while pTfh1 cells do not. However, in vitro studies and animal models highlight that pTfh1 cells are key drivers of cytophilic antibody development, which are protective. To dissect these contradictions, we mapped the heterogeneity of pTfh cells in healthy individuals and individuals with controlled human malaria infection using scRNAseq. We identified two, previously unidentified, pTfh1-like subsets with functional relevance, which can be defined based on CCR7 expression. CCR7pos pTfh1 cells have increased capacity to produce IL-21, whereas CCR7neg pTfh1 cells express markers of cytotoxicity. In controlled human malaria infection, we show that both CCR7pos and CCR7neg pTfh1 cells, along with Tfh2 cells, clonally expand, are transcriptionally and phenotypically activated, and are malaria specific. However, only CCR7pos pTfh1 and pTfh2 cells associated with antibody responses to infection. Our data advance our knowledge of Tfh cell diversity, and may inform the development of vaccines and therapeutics that target these key CD4 T cell responses.

overall_design: In brief, healthy malaria naive individuals underwent Controlled Human Malaria Infection with 2800 viable P. falciparum parasitized RBCs, and peripheral parasitaemia was measured at least daily by qPCR. Participants were treated with antimalarial drugs at day 8 of infection when parasitaemia reaches approximately 20,000 parasites/ml. Blood samples (from 5 studies across 6 independent cohorts) were collected prior to infection (day 0), at peak infection (day 8) and 14 or 15 and 27-36 days (end of study, EOS) after inoculation (in analyses these time points are grouped as 0, 8, 14/15 and EOS). PBMCs were isolated by Ficoll-Paque (Sigma, USA) density gradient centrifugation, isolated PBMCs were cryopreserved in 10% DMSO/FBS. Participants were healthy malaria naïve adults with no prior exposure to malaria or residence in malaria-endemic regions.  Samples used here were from samples opportunistically collected from volunteers who consented to donate blood for immunological studies within the parent clinical trial. As such, no sample size estimation was performed for this immunology study.

first: M

middle: S

last: Soon

first: D

middle: A

last: Oyong

first: N

last: Dooley

first: R

last: Mukhiya

first: Z

last: Pava

last: Andrew

first: J

last: Loughland

last: McCarthy

last: Chan

middle: G

last: Beeson

first: C

last: Engwerda

first: A

last: Haque

last: Boyle


title: EWS::FLI cooperatively binds at GGAA microsatellites via DBD-a4 helix in TTC-466 [H3K27ac_CnT]

first: Ariunaa

last: Bayanjargal

overall_design: In TTC-466 cells, we knock-down endogenous EWSR1::ERG with shRNA  (KD) and rescued with FLAG tagged DBD and DBD+ constructs. Cells are then harvested for RNA-Seq (3 bio reps), CUT&Tag (3 bio reps for CTCF, 4 bio reps for H3K27ac, 4 bio reps for FLAG), and Micro-C libraries (2 bio reps).

first: Emily

last: Theisen

first: Julia

last: Selich-Anderson

first: Jesse

last: Crow


title: The transcription factor ZNF469 regulates collagen production in liver fibrosis [HSC_CUTRUN_H3K27ac]

first: Marisol

last: Ramirez

overall_design: Primary human HSCs were cultured in standard conditions.  Cells were electroporated with RNPs (ribonucleoproteins) containing spCas9 with non-targeting gRNA (i.e. control) or gRNAs targeting ZNF469 (target knockdown).  Following cell recovery and expansion, CUT&RUN was performed with anti-H3K27ac antibody.  N=3 replicates per CRISPR condition.

first: Quanhu

last: Sheng

first: Jonathan

last: Brown

first: Sebastian

last: Steinhauser

last: Ramirez-Solano


title: Genome binding/occupancy profiling of ALKBH5 by high throughput sequencing  [Cut&Tag]

first: Zheng

last: Chen

summary: Alkylation repair homolog protein 5 (ALKBH5) binds to RNA and mediates RNA m6A demethylation. However, whether ALKBH5 binds to DNA is largely unknown. In this study, our CUT&Tag assay identified 12333 peaks in ALKBH5-overexpressing Hepa1-6 cells, indicating that ALKBH5 can bind to DNA.

overall_design: To investigate whether ALKBH5 regulates gene expression by binding to DNA, primary hepatocytes were infected with Ad-FLAG-ALKBH5 adenovirus, and Cut&Tag assay was performed.

first: Kaixin

last: Ding


title: Heterogeneous Cardiac- and Neural Crest-Derived Aortic Smooth Muscle Cells have Similar Transcriptional Changes after TGFβ Signaling Disruption [scRNA-seq-of-Wnt1-Cre2-mTmG]

first: George

last: Tellides

summary: We report single-cell RNA-seq data of Wnt1-Cre2 lineage marked, integrin alpha 8 labeled smooth muscle cells (SMCs) from murine aortas at 12 weeks of age

overall_design: Root/Ascending/Arch segments were procured from 12-week-old  Wnt1-Cre2.mT/mG mice, ITGA8+ SMCs were isolated after cryophilic enzyme digestion, and single-cell RNA-seq analysis was performed

first: Pengwei

last: Ren


title: The effects of ALKBH5 on m6A mRNA modification in the liver [MeRIP-seq]

summary: Through  m6A mRNA-profiling, we aim to characterize the  m6A mRNA changes in the liver between Alkbh5flox/flox and Alkbh5-HKO mice.

overall_design:
To map the mRNA m6A modification caused by ALKBH5 in the liver, MeRIP-seq was performed in the livers of Alkbh5flox/flox and Alkbh5-HKO mice.
Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from livers of Alkbh5flox/flox and Alkbh5-HKO mice at 9 weeks old. A total of 300 μg RNAs were pooled from three mice.Fragmented mRNA
(~100 nt) was incubated for 2 hr at 4oC with anti-m6A polyclonal antibody (Synaptic Systems) in the immunoprecipitation experiment. Then, immunoprecipitated mRNAs or Input was used for library construction with NEBNext ultra RNA library prepare kit for Illumina (New England Biolabs). The library preparations were sequenced on an Illumina Novaseq6000 platform with a paired-end read length of 150 bp according to the standard protocols. After mapping reads to the reference genome, exomePeak R package (version 2.16.0) was used for the m6A peak identification in each anti-m6A immunoprecipitation group with the corresponding input samples serving as a control, and q-value threshold of enrichment of 0.05 was used for all data sets. The m6A-enriched motifs of each group were identified by HOMER (version 4.9.1).
The liver mRNA m6A profiles in Alkbh5flox/flox and Alkbh5-HKO mice were characterized.


title: Transforming Growth Factor-β (TGFβ) Prevents Hypertension-Induced Aortic Dissection in Adult Mice via Extracellular Matrix Production (Single cells KO vs. WT SMC)

summary: We report single-cell RNA-seq data of Myh11 lineage marked smooth muscle cells (SMCs) from murine aortas at 1 week after conditional disruption of TGFβ signaling versus controls

overall_design: Ascending aorta/aortic arch segments were procured from tamoxifen-treated, 12-week-old  Myh11-CreERT2.mT/mG (designated as GFPiSMC or simply WT) or Tgfbr1f/f.Tgfbr2f/f.Myh11-CreERT2.mT/mG (designated as Tgfbr1/2iSMCKO or simply KO) mice, GFP+ SMCs were isolated after cryophilic enzyme digestion, and single-cell RNA-seq analysis was performed

first: Bo

last: Jiang

first: Dejian

last: Zhao


title: Mapping the Molecular Signature of Progressive Multiple Sclerosis [197191]

status: Public on Feb 26 2025

city: London

first: Carolina

last: Barcellos Machado

address: 4-8 Maple St

country: United Kingdom

institute: BenevolentAI

summary: Multiple Sclerosis is a complex neurological disorder characterised by inflammation, demyelination, and neurodegeneration of the central nervous system. While treatments exist for relapsing-remitting multiple sclerosis, therapeutic options for the progressive forms remain limited, highlighting the need to understand their distinct pathological mechanisms. To identify subtype specific molecular signatures in patients with multiple sclerosis, bulk RNA sequencing was performed in 90 brain tissue samples from donors with relapsing-remitting, secondary progressive, and primary progressive multiple sclerosis, as well as controls without the disease. This study provides open access to transcriptomic data from multiple sclerosis tissue samples, enabling other researchers to perform independent analyses, validate findings, and generate new hypotheses.

overall_design: RNA sequencing profiling of brain tissue from donors with relapsing remitting, secondary progressive and primary progressive multiple sclerosis. Control samples obtained from donors without MS.

first: Janet

last: Small

first: Yogesh

last: Kamra

first: Tara

last: Herbert

first: Peri

last: Tate

first: Elloise

last: Matthews

first: Alison

last: McGarvey

first: Jingwen

last: Wang

first: Camilo

last: Albornoz Carranza

first: Manuela

last: Salvucci

first: Pijika

last: Watcharapichat

first: Meltem

last: Gurel


title: Mapping the Molecular Signature of Progressive Multiple Sclerosis


title: Mapping the Molecular Signature of Progressive Multiple Sclerosis [194017]


title: Leaf maturation transcriptional dynamics are plastic in response to stress (hormone data)

city: La Jolla

first: Joseph

last: Ecker

state: CA

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

institute: HHMI-Salk-Institute

summary: Leaf development is dynamic in response to environmental stress. Although such developmental plasticity in response to stress is well described at the physiological level, the underlying transcriptional changes that adjust leaf development and produce adaptive phenotypes remain poorly understood. Here, using single-nucleus transcriptome sequencing, we demonstrate that that drought-induced changes in leaf development involve promoting transcriptional responses linked to leaf maturation across leaves of all ages. Notably, we find that these transcriptional changes scale with stress intensity, ensuring the resulting tissue size is dose-responsive with environmental change.

overall_design: Using 10X transcriptomic approaches, we analyzed the transcriptional changes of Arabidopsis leaf tissue to different hormone treatments

last: Swift

first: Xuelin

last: Wu

first: Tanvi

last: Jain

first: Jiaying

last: Xu

first: Natanella

last: Illouz-Eliaz

last: Nery

first: Joanne

last: Chory


title: Aged and BRCA mutated stromal cells drive epithelial cell transformation

city: Pittsburgh

first: Lan

last: Coffman

state: PA

address: 5051 Centre Ave

institute: UPMC Hillman Cancer Center

summary: The fundamental steps in high-grade serous ovarian cancer (HGSOC) initiation are unclear presenting critical barriers in prevention and early detection of this deadly disease. Current models propose that fallopian tube epithelial (FTE) cells transform into serous tubal intraepithelial carcinoma (STIC) precursor lesions and subsequently HGSOC.  Here we report that an epigenetically altered mesenchymal stem cell niche, termed high risk MSC (hrMSC), exists prior to STIC lesion formation. hrMSCs are enriched in STIC stroma and contribute to a stromal ‘field effect’ extending beyond the borders of STIC lesion. hrMSCs promote DNA damage in FTE cells while also fostering FTE cell survival. hrMSCs induce malignant transformation of FTE resulting in metastatic cancer in vivo, indicating hrMSCs promote cancer initiation.  hrMSCs are significantly enriched in BRCA1/2 mutation carriers and increase with age. Combined, these findings indicate that hrMSCs can incite ovarian cancer initiation and have important implications for ovarian cancer detection and prevention.

overall_design: FFPE embedded fallopian tube samples were sectioned onto slides. Samples were stained with CD105, CD90, CD73, p53, CD45, and PanCk and processed through the Nanostring GeoMx Digital Spatial Profiler. Using morphology stains, ROI categories were called according to staining combinations. The (v1.0) Human NGS Whole Transcriptome Atlas RNA was used for all samples.

first: Geyon

middle: L

last: Garcia

first: Taylor

last: Orellana

first: Grace

last: Gorecki

first: Leonard

last: Frisbie

first: Roja

last: Baruwal

first: Swathi

last: Suresh

first: Ester

last: Goldfield

first: Ian

last: Beddows

middle: P

last: MacFawn

first: Ananya

middle: K

last: Britt

first: Macy

last: Hale

first: Amal

middle: T

last: Elhaw

first: Brian

last: Isett

first: Nadine

last: Hempel

first: Riyue

last: Bao

first: Hui

last: Shen

first: Ronald

last: Buckanovich

first: Toren

last: Finkel

first: Ronny

last: Drapkin

first: T

last: Soong

first: Tullia

last: Bruno

first: Huda

middle: I

last: Atiya


title: scRNA-seq data of spleen immune cells from Pb-infection and Un-infection mice

city: Beijing

first: Weixuan

last: Wang

address: No. 38 Xueyuan Road, Haidian District

institute: Peking University

summary: To explore the immune microenvironment of spleen in the state of Plasmodium infection, we employed single-cell RNA sequencing (scRNA-seq)..

overall_design: Mice were intraperitoneally injected with 1 × 10⁴ iRBCs, and on day 5 post-infection, spleens from both uninfected and Pb-infected mice were collected to isolate lymphocytes. CD45+ cells were then sorted by flow cytometry for subsequent single-cell sequencing analysis.

first: Sun

last: Xin

first: Dan

last: Lu


title: THE INTEGRATIVE GENOMIC AND FUNCTIONAL IMMUNOLOGICAL ANALYSES OF COLORECTAL CANCER INITIATING CELLS TO MODULATE STEMNESS PROPERTIES AND THE SUSCEPTIBILIY TO IMMUNE RESPONSES [miRNA]

city: Doha

first: Cristina

last: Maccalli

address: Doha

country: Qatar

institute: Sidra Medicine

summary: The nCounter technology (Nanostring Technology) was used for the characterization of the repertoire of miRNAs expressed in CICs (initiating cells) and FBS (differentiated tumor cell) colorectal cancer cell lines. The Nanostring miRNA panel V3 (including ~800 targets) was run on all samples. The class comparison of the profile of miRNAs in CRC-CIC vs. FBS tumor cells led to the identification of N=57 differentially expressed miRNAs (p≤0.05). Most of the miRNAs were found to be upregulated in CICs vs. FBS cell lines, including the N=11 top scored differentially expressed (p<0.02, miRNAs -299, -15a, -210, -196a, -362, -378d, -3144, -4461, -4488 and let-7d). Pathway Analysis performed on target genes for the miRNAs, showing their involvement in tumorigenic functions, such as cell cycle, migration, development and proliferation and the regulation of EMT.

overall_design: miRNA profiling of 20 CICs primary cell lines or Colorectal cancer initiating cells (CICs) cell lines and 3 FBS (Colorectal cancer differentiated tumor cell lines). We evaluated A differential gene expression profile of 800 miRNAs detected in CICs vs. FBS tumor cells using the nCounter technology (Nanostring Tech) we used the Nanostring miRNA panel V3 (including ~800 targets).

first: Salim

last: Bougarn


title: THE INTEGRATIVE GENOMIC AND FUNCTIONAL IMMUNOLOGICAL ANALYSES OF COLORECTAL CANCER INITIATING CELLS TO MODULATE STEMNESS PROPERTIES AND THE SUSCEPTIBILIY TO IMMUNE RESPONSES

type: Methylation profiling by genome tiling array

summary: We conducted a genome wide DNA methylation profiling of primary CICs-CRC and differentiated tumor cell lines-CRC, including autologous pairs. The Illumina Infinium Methylation EPIC v1.0 BeadChip Array was used to obtain DNA methylation profiles.  Idat files generated by Infinium MethylationV.1 BeadChip were analyzed using the RnBeads R package.  In total, 782939 probes were retained for further differential DNA methylation analysis. Differential methylation analysis between CIC and FBS cell lines was conducted using limmaR package at the CpG sites and region levels, defined as tiling (5 kb), genes, promoters, and CpG islands. For differential methylation measures, a combined rank a total of 3529 differentially methylated sites were identified. A combined rank among the 500 best ranking regions was applied to identify differentially methylated genes (n=79) and promoters (n=31) in CRC-CICs vs. FBS cell lines. N=48 and 31 genes were hypomethylated and hypermethylated, respectively while, at the promoter levels, N=9 and 22 were hypomethylated and hypermethylated, respectively in CRC-CIC as compared to FBS cell lines (p-value ≤0.11). CICs exhibited distinct methylation patterns, compared to differentiated tumor cells (p<0.05 or 0.01). Following the data quality control and normalization,

overall_design: DNA extraction and purification from CICs and FBS cell lines were performed using the AllPrep DNA/RNA Mini Kit. DNA methylation has been performed, starting from a minimum of 250 ng of utilizing the Infinium Methylation EPIC V.1 BeadChip, upon bisulfite conversion of DNA through the EZ kit, followed by library construction using the Infinium Epic Bead Chip kit. The prepared library has been sequenced with sequencing depths of 50M paired end reads with the HiSeq 3000 instrument. DNA methylation profiling was performed using the Illumina EPIC array V.1.

first: Alex

last: Tout


title: Chromatin disruptions in pathophysiologically relevant gene promoters underlie unique SARS-Cov-2 nucleocapsid-mediated gene regulation

city: Tallahassee

first: Jonathan

last: Dennis

state: FL

address: 319 Stadium Drive

department: Biological Science

institute: Florida State University

summary: SARS-CoV-2, the causative agent of COVID-19, is a positive-sense, single-stranded RNA virus that causes a spectrum of disease severity, from asymptomatic infection to severe illness to long-term sequelae. Similar to other human coronaviruses, SARS-CoV-2 proteins modulate host genomic responses through epigenomic modifications, facilitating viral replication and immune evasion. While the nucleocapsid protein is well known for its role in RNA stability and immune modulation, its impact on host chromatin organization remains unclear. To investigate this, we generated stable human alveolar cell lines expressing nucleocapsid proteins from endemic and pandemic human coronaviruses. Our analysis revealed that nucleocapsid proteins from all tested coronaviruses induced changes in nucleosome positioning and occupancy at specific gene promoters involved in coagulation pathways, hormone signaling, and innate immune responses. Additionally, SARS-CoV-2-specific alterations were identified in genes dysregulated in severe infections, such as  suggesting a direct role for epigenomic modifications in disease pathophysiology. We also observed extensive changes in nucleosome susceptibility to nuclease digestion in SARS-CoV and SARS-CoV-2 samples that were not observed in common cold cell lines. Promoters with altered sensitivity and resistance to nuclease were linked to innate immune, metabolic, olfactory, and signaling pathways known to be dysregulated in severe COVID-19 and post-acute sequelae (PASC). These findings demonstrate that nucleocapsid protein expression alters chromatin structure at specific loci, implicating viral proteins in host genome dysregulation. Furthermore, we identified both shared and unique chromatin targets of SARS-CoV-2 and common cold coronaviruses, highlighting pathways for further investigation and potential therapeutic intervention.

overall_design: To determine the role of CoV nucleocapsid proteins on nucleosome distribution and susceptibility to enzymatic digestion within promoters, we generated stable A549 human lung epithelial cell lines expressing the full length nucleocapsid proteins of seven human CoVs. Replicate cell lines were generated for each of the following nucleocapsids: HCoV-HKU1, HCoV-NL63, HCoVOC43, HCoV-229E, SARS-CoV, MERS-CoV, and SARS-CoV-2 which were compared to cells expressing an empty vector construct. The distribution of nucleosomes across promoters was determined by digesting crosslinked chromatin with Micrococcal nuclease (MNase), an enzyme which selectively cleaves linker DNA leaving nucleosomal DNA intact for high throughput sequencing (Shivaswamy et al. 2008; Schones et al. 2008). In addition to mapping the location of individual nucleosomes, a titration of MNase was used to determine the susceptibility of a given nucleosome to enzymatic digestion (Vera et al. 2014; Turpin et al. 2018; Cole and Dennis 2020; Cole et al. 2021; Mieczkowski et al. 2016; Brahma and Henikoff 2019).  We chose to focus our analysis on promoter regions as nucleosomes within promoter regions have distinct roles in gene regulation, are highly occupied, and are especially well positioned compared to the rest of the genome.

first: Jane

last: Benoit


title: THE INTEGRATIVE GENOMIC AND FUNCTIONAL IMMUNOLOGICAL ANALYSES OF COLORECTAL CANCER INITIATING CELLS TO MODULATE STEMNESS PROPERTIES AND THE SUSCEPTIBILIY TO IMMUNE RESPONSES [RNA-Seq]

summary: We conducted a the transcriptomic profile of CICs or FBS cell lines, upon treatment or not with immunomodulatory or epigenetic drugs or with miRNA modulators, using the QuantSeq 3’mRNA-Seq Library Prep Kit-FWD (Lexogen). Differentially expressed genes (DEGs) were assessed in CICs and FBS cell line pairs (#1076 and 1247) upon treatment with either miRNA-15a or -196a modulators (inhibitors or mimics) or immunomodulatory or epigenetic agents (HDACi, butyrate or IFN-γ + butyrate). Numerous DEGs were detected among the comparisons of the different treatments. In CICs, N= 176, 238, 131, 168, 1155, 1237 and 1368 DEGs were significantly (p<0.05) detected following their treatment with miR-15a mimic or inhibitor, miR-196a mimic or inhibitor, butyrate, HDACi or IFN-γ + butyrate, respectively. The results corroborated the role of miRNA-15a and miRNA-196a mimics and of epigenetic drugs as beneficial treatments to improve CRC-specific immune responses through mechanisms including TGF-β signaling downmodulation, HLA and APM upregulation and negative regulation of tumorigenic pathways.

overall_design: CICs and FBS cell line pairs (#1076 and 1247) were treateed with either miRNA-15a or -196a modulators (inhibitors or mimics) or immunomodulatory or epigenetic agents (HDACi, butyrate or IFN-γ + butyrate).


title: Epigenetic priming promotes acquisition of TKI resistance and oncogene amplification in human lung cancer [CUT&RUN2]

first: Eric

last: Sun

address: 10 Amistad St

department: Genetics

summary: In mammalian cells, gene copy number is tightly controlled to maintain gene expression and genome stability. However, a near-universal molecular feature across cancer types is oncogene amplification, which promotes cancer progression by dramatically increasing the copy number and expression of tumor-promoting genes. For example, in tyrosine kinase inhibitor (TKI)-resistant lung adenocarcinoma (LUAD), oncogene amplification occurs in over 40% of patients’ tumors. Despite the prevalence of oncogene amplification in TKI-resistant tumors, the mechanisms facilitating oncogene amplification are not fully understood. Here, we find that LUAD tumors exhibit a unique chromatin signature demarcated by strong CTCF and cohesin deposition in drug-naïve tumors, which correlates with the boundaries of oncogene amplicons in TKI-resistant LUAD cells. Throughout the acquisition of TKI resistance, we identified a global chromatin priming effect, marked by a dynamic increase of H3K27Ac, cohesin loading, and inter-TAD interactions, which occurs before the onset of oncogene amplification. Furthermore, we have identified that the protein METTL7A, which was previously reported to localize to the ER and inner nuclear membrane, has a novel chromatin regulatory function in binding to amplified oncogenes and regulating cohesin recruitment and inter-TAD interactions. Surprisingly, we discovered that METTL7A remodels the chromatin landscape prior to any noticeable oncogene copy number gains. Furthermore, while METTL7A depletion has little effect on drug-naïve cells, METTL7A depletion prevents the formation of TKI resistant-clones, highlighting the specific role of METTL7A as cells are acquiring resistance. In summary, we discovered an unexpected mechanism required for the acquisition of TKI resistance regulated by a largely uncharacterized factor, METTL7A. This discovery sheds new light into maintenance of oncogene copy number and paves the way to the development of new therapeutics for preventing TKI resistance in LUAD.

overall_design: CUT&RUN: CUT&RUN was performed in PC9 wild-type and PC9 METTL7A KO (PC9_M7A_KO) against H2A and TOP2B.

first: Rebecca

last: Starble

first: Rana

last: Gybli

last: Radda

first: Jiuwei

first: Tyler

middle: B

last: Jensen

first: Ning

first: Shuai

last: Zhao

first: Nitin

last: Roper

first: Gang

first: Jikui

last: Song

first: Katerina

last: Politi

first: Siyuan

first: Andrew

middle: Z

last: Xiao


title: Alpha-ketoglutarate -mediated carnitine synthesis promotes homologous recombination via histone acetylation

first: Katherine

last: Aird

address: 5051 Centre Ave.

institute: University of Pittsburgh School of Medicine

summary: Homologous recombination (HR) deficiency enhances sensitivity to DNA damaging agents commonly used to treat cancer. In HR-proficient cancers, metabolic mechanisms driving response or resistance to DNA damaging agents remain unclear. Here we identified that depletion of alpha-ketoglutarate (αKG) sensitizes HR-proficient cells to DNA damaging agents by metabolic regulation of histone acetylation. αKG is required for the activity of αKG-dependent dioxygenases (αKGDDs), and prior work has shown that changes in αKGDD affect demethylases. Using a targeted CRISPR knockout library consisting of 64 αKGDDs, we discovered that Trimethyllysine Hydroxylase Epsilon (TMLHE), the first and rate-limiting enzyme in de novo carnitine synthesis, is necessary for proliferation of HR-proficient cells in the presence of DNA damaging agents. Unexpectedly, αKG-mediated TMLHE-dependent carnitine synthesis was required for histone acetylation, while histone methylation was affected but dispensable. The increase in histone acetylation via αKG-dependent carnitine synthesis promoted HR-mediated DNA repair through site- and substrate-specific histone acetylation. These data demonstrate for the first time that HR-proficiency is mediated through αKG directly influencing histone acetylation via carnitine synthesis and provide a metabolic avenue to induce HR-deficiency and sensitivity to DNA damaging agents.

overall_design: We used Ovcar8 high grade serous cancer cells with CCNE1 overexpression and knocked down TMLHE using 2 independent shRNAs. All samples were done in triplicate. Comparative gene expression profiling analysis for CCNE1 overexpression vs. control and shTMLHE vs. CCNE1 overexpression.


title: Coronavirus and influenza virus triggers antiviral RNAi immunity [AIV-miRNA-seq]

city: Nanjing

first: Yaotang

last: Wu

address: Nanjing Agricultural University, Wei gang 1

institute: Nanjing Agricultural University

type: Non-coding RNA profiling by high throughput sequencing

summary: RNA interference (RNAi) is a key antiviral immune mechanism in eukaryotes. However, in vertebrates such as birds and mammals, antiviral RNAi has only been observed in cells with poor interferon systems (stem cells and oocytes) or in viral suppressors of RNAi (VSR) deficiency virus infections. Our research originally discovered that infecting macrophages with wild-type coronavirus (Infectious bronchitis virus, IBV) and influenza viruses (Avian influenza virus, AIV) can trigger RNAi antiviral immunity and produce a certain amount of virus-derived siRNA (vsiRNA). These vsiRNAs have an inhibitory effect on the virus and carry out targeted silencing along the Dicer-Ago2-vsiRNA axis. Notably, these vsiRNAs are distributed throughout of the virus’s entire genome, with a predilection for A/U at the 5’ and 3’ termini of vsiRNA. In addition, Dicer cleavage produces vsiRNA based on the RWM motif, where R represents A/G, W represents A/C, and M represents A/U. Additionally, we discovered that avian LGP2 and MDA5 proteins positively impact the expression of the Dicer protein and the Dicer subtype “DicerM”, which exhibits a potent antiviral activity compared to Dicer itself. Most importantly, the psilencer4.1-plasmid constructed based on vsiRNA combined with nanomaterial polyetherimide (PEI) showed excellent anti-virus activity in specific-pathogen-free (SPF) chickens. These findings show that RNA viruses trigger the production of the vsiRNA in avian somatic cells, which is of great significance for the application of therapeutic vaccines in poultry.

overall_design: For HD11 cell experiments, the infection level of AIV-H9N2 is 1 MOI. They were incubated at 37℃ with 5% CO2 for 2 h and then cleaned using RPMI 1640. The non-infected virus cells for the Blank group. The RNA samples were collected according to the indicated time (24 h). The groups are divided inot Blank group and AIV-H9N2 infection group.

first: Jian

last: Lin

first: Qian

last: Yang


title: Hepatic sphingomyelin phosphodiesterase 3 promotes steatohepatitis by disrupting membrane sphingolipid metabolism.

first: Cen

last: Xie

address: 501 Haike Road

institute: Shanghai Institute of Materia Medica

summary: Metabolic dysfunction-associated steatohepatitis (MASH) remains a significant health challenge. Herein, we identify sphingomyelin phosphodiesterase 3 (SMPD3) as a key driver of hepatic ceramide accumulation through increasing sphingomyelin hydrolysis at the cell membrane. Hepatocyte-specific Smpd3 gene disruption and pharmacological inhibition of SMPD3 alleviate MASH, whereas reintroducing SMPD3 reverses the resolution of MASH. While healthy livers express low-level SMPD3, lipotoxicity-induced DNA damage suppresses SIRT1, triggering an upregulation of SMPD3 during MASH. This disrupts membrane sphingomyelin-ceramide balance and promotes MASH progression by enhancing caveolae-dependent lipid uptake in hepatocytes and extracellular vesicle secretion from steatotic hepatocytes to worsen inflammation and fibrosis. Thus, SMPD3 acts as a central hub integrating key MASH hallmarks. Notably, we discovered a bifunctional agent that simultaneously activates SIRT1 and inhibits SMPD3, which shows significant therapeutic potential in MASH treatment. These findings suggest that inhibition of hepatic SMPD3 restores membrane sphingolipid balance and holds great promise for developing novel MASH therapies.

overall_design: Male C57BL/6J mice at the age of 6-8 weeks. Mice were subjected to GAN diet for 12 weeks and then those mice were randomly grouped for 15-week intragastric administration of vehicle and DPTIP. Saline solution containing 1% DMSO and 2% Tween-80 serves as vehicle, and the daily dose of DPTIP was 15 mg/kg.

first: Jie

last: Jiang

first: Yuqing

last: Gao

first: Jiang

last: Wang

first: Yan

last: Huang

first: Rong


title: Transcriptome sequencing of pancreatic cancer cells transfected with MMP28 overexpressing lentivirus

city: Lanzhou

first: Shi

last: Dong

address: No.222, Tianshui South Road, Chengguan District, Lanzhou City

institute: Lanzhou University

summary: The mechanism by which MMP28 regulates cytokine secretion remains unclear. In our previous study, we found that MMP28 can regulate the expression and secretion of IL-8 and VEGFA in pancreatic cancer cells, and in order to further explore the specific mechanisms involved, we performed transcriptome sequencing.

overall_design: The control group was treated with null virus transfection and the experimental group was treated with MMP28 overexpressing lentivirus


title: Effect of IL-8 and VEGFA on polarization of tumor-associated macrophages

summary: We demonstrated in a previous study that IL-8 and VEGFA have a role in recruiting and polarizing macrophages. To fully confirm the specific mechanisms by which IL-8 and VEGFA secreted by pancreatic cancer cells affect macrophage polarization, we performed transcriptome sequencing on groups of macrophages after treatment of the co-culture system with anti-IL-8 and anti-VEGFA neutralizing antibodies.

overall_design: The experiment was divided into four groups, one of which was the control group, another was the MMP28 overexpression co-culture group, and the remaining two groups were the anti-IL-8 and anti-VEGFA neutralizing antibody co-culture groups, respectively, which were added on the basis of MMP28 overexpression.


title: Staphylococcus aureus-specific TIGIT+ Treg are present in the blood of healthy subjects – a hurdle for vaccination?

city: Siena

first: Elisabetta

last: Soldaini

address: via Fiorentina, 1

country: Italy

institute: GSK

summary: Staphylococcus aureus poses an enormous burden of morbidity and mortality worldwide. Making an efficacious vaccine has however proven extremely challenging. Due to colonizing interactions, pre-existing S. aureus-specific CD4+ T cells are often found in the human population and yet a detailed characterization of their phenotypes and how they might in turn impact vaccine efficacy are thus far unknown. Using an activation induced marker assay to sort for S. aureus-specific CD4+ T cells in an effector function-independent manner, single cell transcriptomic analysis was conducted. Remarkably, S. aureus-specific CD4+ T cells consisted not only of a broader spectrum of conventional T cells than previously described but also of Treg. As compared to polyclonally-activated CD4+ T cells, S. aureus-specific Tcon were enriched for the expression of the Th17-type cytokine genes IL17A, IL22 and IL26, while higher percentages of S. aureus-specific Treg expressed the T Cell Immunoreceptor with Ig and ITIM domains (TIGIT), a pleiotropic immune checkpoint. Notably, the antagonistic anti-TIGIT mAb Tiragolumab increased IL-1b production in response to S. aureus in vitro. Therefore, these results uncover the presence of S. aureus-specific TIGIT+ Treg in the blood of healthy subjects that could blunt responses to vaccination and indicate TIGIT as a potential targetable biomarker to overcome pre-exposure-induced immunosuppression.

sample_organism: Staphylococcus aureus subsp. aureus USA300

overall_design: scRNA-seq of CD4+ T cells from PBMCs of healthy subects activated in vitro with heat-killed S. aureus (HKSA stimulation) or anti-CD3/CD28 antibodies (Polyclonal stimulation) for 24hs. Activated CD4+ T cells from each of the 6 donors analyzed (01, 02, 04, 05, 09 and 15) were identified as OX40+CD137+ and sorted in single in two 384-well precast plates from Single Cell Discoveries. A library was generated from each plate (1 and 2).

first: Jonah

last: Clegg

first: Małgorzata

last: Mnich

first: Alberto

last: Carignano

first: Giovanni

last: Cova

first: Simona

last: Tavarini

first: Chiara

last: Sammicheli

first: Bruna

last: Clemente

first: Megan

last: Smith

first: Emilio

last: Siena

first: Monia

last: Bardelli

first: Michela

last: Brazzoli

first: Fabio

last: Bagnoli

first: Rachel

last: McLoughlin


title: An in vitro pharmacogenomic approach reveals subtype-specific therapeutic vulnerabilities in atypical teratoid/rhabdoid tumors (AT/RT) [Methylation]

city: Homburg (Saar)

first: Marc

last: Remke

state: Saarland

address: Kirrberger Straße 100, Gebäude 9

department: Pediatric Translational Oncology Lab

institute: Universitätsklinikum des Saarlandes

summary: Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant embryonal brain tumor driven by genetic alterations inactivating the SMARCB1 or, less commonly, the SMARCA4 gene. Large-scale molecular profiling studies have identified distinct molecular subtypes termed AT/RT-TYR, -SHH and -MYC. Despite the increasing knowledge of AT/RT biology, curative treatment options are still lacking for certain risk groups and outcomes of these patients remain poor. We performed an in vitro high-throughput drug screen of 768 small molecule drugs covering conventional chemotherapeutic agents and late-stage developmental drugs in 13 AT/RT cell lines and determined intra- and inter-entity differential responses to unravel specific vulnerabilities. Our data demonstrated in vitro preferential activity of mitogen-activated protein kinase kinase (MEK) and mouse double minute 2 homolog (MDM2) inhibitors in AT/RT cell lines compared to other high-grade brain tumor cell lines including medulloblastoma and malignant glioma models. Moreover, we were able to link distinct drug response patterns to AT/RT molecular subtypes through integration of drug response data with large-scale DNA methylation and RNASeq-based expression profiles. Subtype-dependent drug response profiles demonstrated sensitivity of AT/RT-SHH cell lines to B-cell lymphoma 2 (BCL2) and heat shock protein 90 (HSP90) inhibitors, and increased activity of microtubule inhibitors, kinesin spindle protein (KSP) inhibitors, and the eukaryotic translation initiation factor 4E (eIF4E) inhibitor briciclib in a subset of AT/RT-MYC cell lines. In summary, our in vitro pharmacogenomic approach revealed preclinical evidence of tumor type- and subtype-specific therapeutic vulnerabilities in AT/RT cell lines that may inform future in vivo and clinical evaluations of novel pharmacological strategies.

overall_design: We aimed to profile the methylome of AT/RT cell lines grown in serum-free media. DNA was analyzed using the Infinium MethylationEPIC array in a cohort of 13 cell lines.

first: Daniel

last: Picard

first: David

last: Pauck

first: Guido

last: Reifenberger


title: Epigenetic priming promotes acquisition of TKI resistance and oncogene amplification in human lung cancer [ChIP-seq 2]

first: Eric

last: Sun

institute: Yale University

overall_design: ChIP-seq: ChIP-seq was performed against IgG or MYC-tag in Wild-Type or METTL7A-MYC-tag-FLAG expressing cells.

first: Rebecca

last: Starble

first: Rana

last: Gybli

first: Jonathan

last: Radda

first: Jiuwei

last: Lu

first: Tyler

last: Jensen

first: Ning

first: Shuai

last: Zhao

first: Nitin

last: Roper

first: Gang

last: Wang

first: Jikui

last: Song

first: Katerina

last: Politi

first: Siyuan

first: Andrew

last: Xiao


title: Coronavirus and influenza virus triggers antiviral RNAi immunity [IBV_miRNA-seq]

first: Yaotang

last: Wu

overall_design: For HD11 cell experiments, the infection level of IBV-QX is 5 MOI. They were incubated at 37℃ with 5% CO2 for 2 h and then cleaned using RPMI 1640. The non-infected virus cells for the Blank group. The RNA samples were collected according to the indicated time (24 h). The group contain the NO1, NO2, NO3, NO4, NO5, and NO6.

first: Jian

last: Lin

first: Qian

last: Yang


title: Receptor Allostery Promotes Sonic Hedgehog Signaling During Embryonic Development

city: Memphis

first: Yong-Dong

state: TN

address: 262 Danny Thomas Pl, MS1135

institute: St. Jude Children's Research Hospital

summary: Sonic Hedgehog (SHH) signaling functions in temporal and context-dependent manners to pattern diverse tissues during embryogenesis. The signal transducer Smoothened (SMO) is regulated by sterols, oxysterols, and arachidonic acid (AA) through binding pockets in its extracellular cysteine rich domain (CRD) and 7-transmembrane (7TM) bundle. In vitro analyses suggest SMO signaling is allosterically enhanced by combinatorial ligand binding to the CRD and 7TM pockets but in vivo evidence of SMO allostery is lacking. Herein, we demonstrate that disruption of AA binding to a pocket at the top of the 7TM attenuates SHH and sterol-stimulated SMO activity. We generated a knockin mouse model of compromised AA binding and show that homozygous mutants are cyanotic with high perinatal lethality. Surviving animals fail to thrive due to cardiopulmonary mispatterning. Neurodevelopment is unaltered in these mice, suggesting context-dependent allosteric regulation of SMO signaling allows for precise tuning of pathway activity for heart and lung development.

overall_design: Mature SmoK399A/+ females were crossed with SmoK399A/+ males to generate litters of E9.5 Smo+/+ and SmoK399A/K399A embryos.  Multiple litters were harvested, and expression profiles of wild type and mutant were comparied.

first: Shariq

last: Ansar

first: Miriam

last: Dillard

first: Stacey

last: Ogden


title: Single-cell single-molecule spatial transcriptomics using CosMx on colectomy specimens collected during Pouch surgery in IBD patients.

city: Chicago

first: David

last: Zemmour

state: Illinois

address: 924 E 57th St

department: Pathology

institute: University of Chicago

summary: Total abdominal colectomy (TAC) with a staged ileal pouch-anal anastomosis (IPAA) is a common surgical treatment for ulcerative colitis (UC). However, a significant percentage of patients experience pouch failure, leading to considerable morbidity. This retrospective case-control study aimed to identify histopathological features of the TAC specimen associated with subsequent pouch failure and to investigate the underlying molecular mechanisms of this susceptibility using single-cell spatial transcriptomics.

overall_design: Four J-pouch failure vs. four J-pouch non-failure colectomy specimens collected during IPAA surgery in IBD patients prior to pouch formation.

first: Paul Chak Mou

last: Ngai

first: Emily

last: Schahrer


title: An in vitro pharmacogenomic approach reveals subtype-specific therapeutic vulnerabilities in atypical teratoid/rhabdoid tumors (AT/RT) [RNASeq]

first: Marc

last: Remke

overall_design: We aimed to profile the transcriptome of AT/RT cell lines grown in serum-free media. RNA was analyzed using paired-end RNA sequencing in a cohort of 13 cell lines.

first: Daniel

last: Picard

last: Pauck

first: Guido

last: Rifenberger


title: Inhibition of PRC2 enables self-renewal of naïve pluripotent stem cells from chimpanzee [bisulfite-seq]

city: Minato-ku

first: Hideki

last: Masaki

state: Tokyo

address: 4-6-1 Shirokanedai

department: Institute of Medical Science

country: Japan

institute: University of Tokyo

type: Methylation profiling by high throughput sequencing

summary: Naïve pluripotent stem cells (nPSCs) correspond to nascent epiblast in the pre-implantation embryo. nPSCs from mouse and human differ in self-renewal requirements and potency for trophectoderm generation. Here we investigated chimpanzee (Pan troglodytes) nPSCs. Naïve type colonies emerged after resetting or reprogramming but failed to expand. We found that the block to self-renewal is overcome by inhibition of EZH2, the enzymatic component of Polycomb repressor group 2 (PRC2). Chimpanzee nPSCs are euploid, produce teratomas, and can be capacitated for somatic lineage differentiation in vitro. They show transcriptome relatedness to human nPSCs and early epiblast, with shared expression of a subset of pluripotency transcription factors. Chimpanzee nPSCs differentiate to trophectoderm and form tri-lineage blastoids. We confirmed that PRC2 suppresses self-renewal by genetic deletions. Furthermore, we demonstrate that EZH2 inhibition facilitates feeder-free propagation of human nPSCs. In summary, chimpanzee nPSCs expand the repertoire of systems for studying primate pluripotency and early embryogenesis.

overall_design: Whole genome bisulfite sequencing was conducted on primed, naïve, and capacitated chimpanzee iPSC lines to determine if there are changes in methylation states across different pluripotent states.

first: Tao

last: Huang

first: Ayaka

last: Yanagida

first: Arthur

last: Radley

first: Zhili

last: Ren

first: Francesca

last: Carlisle

first: Paul

last: O’Neill

first: Hiroo

last: Imai

first: Hiromitsu

last: Nakauchi

first: Ge

last: Guo

first: Austin

last: Smith

first: Somayyeh

last: Tahajjodi

first: James

last: Clarke

first: Madeline

last: Lancaster

first: Zoe

last: Heckhausen

first: Rosie

last: Bamford

first: Aaron

last: Jeffries

first: Jonathon

last: Mill

first: Petra

last: Hajkova

first: Ferdinand

last: von Meyenn

first: Joao Pedro Agostinho

last: de Sousa


title: REG3A promotes the development of pancreatic cancer by activating EGFR pathway

first: Kunxin

last: Xie

state: China

address: 101Longmian Avenue, Jiangning District, Nanjing

institute: Nanjing Medical University

summary: REG3A is highly expressed in pancreatic tissues, but its significance is unclear. We found that REG3A overexpressed in paracancerous acinar cells interacted with EGFR on tumor cells to stimulate downstream tumor growth signals and promote the occurrence and development of tumors

overall_design: Wild-type PANC1 cells and OE-REG3a-transfected PANC1 cells were used for 3v3 RNA-seq

first: Peng

last: Sun


title: Embryonic heat conditioning induces paternal heredity of immunological cross- tolerance: coordinative role of CpG DNA methylation and miR-200a regulation [RNA-seq]

city: Rishon Lezion

first: Padma Malini

last: Ravi

address: Hamacabim

country: Israel

institute: ARO

summary: The first-generation embryos underwent inovo heat conditioning (EHC) and the offspring were subjected to an intracranial lipopolysaccharide (LPS) challenge, demonstrating immune resilience in second generation which is mediated through the paternal DNA methylation pattern. DNA was extracted from the sperm and reduced representation bisulfite sequencing (RRBS) was performed

overall_design: RNA was isolated from PO/AH and mRNA profile of 3 day old Naive (t0) and 6h hours into miR-200a challenge, by bulk MARS-seq method. 15ng of purified RNA was used for library preparation with the bulk MARS-seq method, involving barcoding of samples by reverse transcription using an oligo dT primer, pooling of samples, and subsequent molecular reactions for linear amplification and preparation for Illumina sequencing. 100bp single reads were sequenced on a Novaseq 6000 sequencing system. The output was ~16 million reads per sample. Poly-A/T stretches and Illumina adapters were trimmed from the reads using Cutadapt (28). The resulting reads shorter than 30bp were discarded. The remaining reads were mapped onto 3′ UTR regions (1000 bases) of the Gallus genome (galGal5.0, UCSD) according to Refseq annotations, using STAR with EndToEnd option and out Filter Mismatch Nover Lmax was set to 0.05. Deduplication was carried out by flagging all reads that were mapped to the same gene and had the same UMI. Read counts per gene were quantified using HTSeq-count (30). UMI counts were corrected for saturation by considering the expected number of unique elements when sampling without replacement. Differentially expressed genes were identified using DESeq2 (31) with the betaPrior, cooksCutoff and independent Filtering parameters set to False. Raw P values were adjusted for multiple testing using the procedure of Benjamini and Hochberg. Pipeline was run using snakemake.

first: PadmaMalini

first: Tatiana

last: Kisliouk

first: Noam

last: Meiri


title: Embryonic heat conditioning induces paternal heredity of immunological cross- tolerance: coordinative role of CpG DNA methylation and miR-200a regulation [BiSulfite-seq]

overall_design: DNA was isolated from the sperm sample using Genomic Mini AX Swab & Semen Spin (A&A Biotechnology, Gdansk, Poland). 70-140ng of DNA was used for RRBS library preparation using Zymo-Seq RRBS Library Kit (Zymo Research, Irvine, CA). The paired end sequencing was done using a SP 100 cycle kit on Nova seq 6000 instrument (Illumina). The quality trimming and trimming of Illumina adapters was performed with Trim galore and the reads were mapped to the chicken genome (galGal 5.0) using Bismark v. 0-22-3 bowtie mode. The methylation calls were extracted with Bismark, methylation extractor mode. Edge R was used for the extraction and analysis of differential methylation on single CpGs (DMS) with the cutoff of 8 reads in every sample and FDR <0.05. DMRs are extracted and analysed by metilene v. 0.2-8. PO/AH mRNA profile of 3 day old Naive (t0) and 6h hours into miR-200a challenge, by bulk MARS-seq method


title: Coronavirus and influenza virus triggers antiviral RNAi immunity [AIVKO_miRNA-seq]

first: Yaotang

last: Wu

overall_design: For HD11 knockout cell experiments, the infection level of AIV-H9N2 is 1 MOI. They were incubated at 37℃ with 5% CO2 for 2 hours and then cleaned using RPMI 1640. The non-infected virus cells for the Blank group. The RNA samples were collected according to the indicated time (24 h). The group divided into Group1 "HD11 cells", Group2 "HD11 Dicer KO cells", Group3 "HD11 DicerM KO cells", Group4 "HD11 Ago2 KO cells", Group5 "HD11 cells and AIV infection", Group6 "HD11 Dicer KO cells and AIV infection", Group7 "HD11 DicerM KO cells and AIV", and Group8 "HD11 Ago2 KO cells and AIV infection".

first: Jian

last: Lin

first: Qian

last: Yang


title: Th1-mediated immune programming is protective during craniotomy infection

city: White/Caucasian

first: Tammy

last: Kielian

state: NE

address: 985900 Nebraska Medical Center

institute: UNMC

summary: A craniotomy is a neurosurgical procedure performed to access the intracranial space. In 3-5% of cases, infection can develop, most commonly caused by Staphylococcus aureus biofilm formation on the surface of the skull. Medical management of this infection is difficult, as biofilm properties confer immune- and antimicrobial recalcitrance to the infection and necessitate additional surgical procedures. Still, treatment failure rates can be appreciably high. These factors, in conjunction with rapidly expanding rates of antibacterial resistance, necessitate the development of alternative treatment strategies targeting and reversing the dysfunctional immune response that develops during biofilm infection. Our recent work has identified Th1 and Th17 CD4+ T cells as potent regulators of innate immune antimicrobial capacity during craniotomy infection, where enhanced signatures of IFN-γ production and signaling were observed at the site of infection. Here, we follow up on these findings to determine the specific role of IFN-γ in programing the immune response to biofilm infection using global and cell-type-restricted IFN-γR deficient mice. We show exaggerated bacterial outgrowth in the absence of IFN-γR, despite few changes in immune cell recruitment relative to WT mice. Single-cell transcriptomic analysis identified candidate explanations for this phenotype as alterations in cell death pathways, innate immune cell activation, MHC-II expression, and CD4+ T cell skewing in IFN-γR KO mice. While PMN pyroptosis in vitro and macrophage/microglia MHC-II levels in vivo displayed sensitivity to IFN-γ signaling, granulocyte- and macrophage/microglia-specific conditional IFN-γR KO experiments isolated these factors from observed increases in bacterial burden. Instead, we uncovered evidence of a decreased Th1/Th17 ratio which was corroborated by elevated IL-17 levels during IFN-γR deficiency and correlated with dysfunctional cellular communication with the innate immune system. Further, Th17 cells were less effective than Th1 cells in stimulating macrophage and microglia bacterial killing.  In sum, this work identifies Th1 cells as uniquely protective during craniotomy infection, where they likely enhance macrophage and microglial antibacterial abilities in an IFN-γ-independent manner. Therefore, augmentation of CD4+ T cell differentiation towards a Th1 bias may represent a novel therapeutic strategy for treating craniotomy infection in the future.

overall_design: To better understand the role of IFN-gamma signaling during craniotomy infection, we collected immune cells from a mouse model of craniotomy infection to perform scRNA-seq. CD45+ immune cells were sorted from brain and galea homogenates of wildtype (WT; C57BL/6) or IFNgR KO (KO) mice subjected to craniotomy infection at day 14 post-craniotomy infection.

first: Zachary

last: Van Roy

first: Gunjan

last: Kak

first: Rachel

last: Fallet


title: The effect of Zinc defficiency on the mouse paneth cell transcriptome in TNF context

city: Gent

first: Steven

last: Timmermans

address: Technologiepark 71

country: Belgium

institute: VIB-UGent

summary: We investigated the effect of zinc depletion on the mouse intestine, and specifically on paneth cells. Mice were placed on a zinc depleted diet for 7 weeks after which they were given TNF for 3 hours and the the effects of dietary zinc shortage on paneth cells in TNF context were investigated. A zinc is an essential element for correct paneth cell function and zinc defficiency is a human and animal health problem investigating the PC transcriptom my provide valuable insights into the exact mechanism.

overall_design: 2 goups with 4 biological replicates each were used: one group was given the standerd chow and one group was given zinc depleted food for 7 weeks, both were injected with TNF 3 hours before sampling

first: Charlotte

last: Wallaeys

first: Natalia

last: Garcia-Gonzalez

first: Claude

last: Libert


title: Paneth cell trancriptome: effect of DEX on TNF induced inflammatory response

summary: We aimed to investiagte the protective anti-inflammatory effect of glucocorticoids (dexamethsone, DEX) on paneth cells from mice treated with TNF. TNF treatment causes leaky gut syndrome and paneth cell disfunction. Using pre-treatment with DEX we aim to protect paneth cell against TNF effects

overall_design: 4 groups with 4 biological replicates each for a total of 16 samples: PBS+PB treated, DEX+PBS treated, PBS+TNF treated and DEX+TNF treated


title: The effect of Dinc defficiency on the mouse paneth cell transcriptome

summary: We invstigated the effect of zinc depletion on the mouse intestine, and specifically on paneth cells. Mice ere placed on a zinc depleted diet for 7 weeks after which the the effects of dietary zinc shortage on paneth cells were investigated. A zinc is an essential element for correct paneth cell function and zinc defficiency is a human and animal health problem investigating the PC transcriptom my provide valuable insights into the exact mechanism.

overall_design: 2 goups with 4 biological replicates each were used: one group was given the standerd chow and one group was given zinc depleted food for 7 weeks


title: Paneth cell gene expression profiles after zinc suplementation in TNF context

summary: Mice were given zinc as a salt (ZnSO4; 25 mM) in the drinking water for 7 days, then mice were injected with TNF and sampling was done 3 hours after injection. Expression profiling with RNA-seq was performed on purified paneth cellq to investigate the genome wide effects of zinc supplemenation on the paneth cell gene expression profile in the conext of TNF

overall_design: 2 groups of 4 mice each were given zinc in ultrapure water or just ultrapure water for 7 days. Afterard the were injected with TNF and paneth cells were isolated for sequencing 3 hours later


title: Expression profiling of purified cells after (mature) paneth cell depletion.

summary: We used transgenic mice expressing diphtheria toxin in a paneth cell specific manner to eablate paneth cells, but found that we could still detect and also purify (paneth) cells in the transgenic mice. We purified this population and a control WT population of paneth cells for expression profiling, with the goal to check if the diphtheria toxin expression system had low penetrance or if our PC population in TG mice was immature , and thus did not yet express the active diphtheria toxin.

overall_design: 1 group of wild type mice and one group of transgenic mice withe paneth cells expressing diphtheria toxin, with 4 replictes per group


title: Paneth cell gene expression profiles after zinc suplementation - exp1

summary: Mice were given zinc as a salt (ZnSO4; 25 mM) in the drinking water for 7 days. Expression profiling with RNA-seq was performed on purified paneth cell to investigate the genome wide effects of zinc supplemenation on the paneth cell gene expression profile

overall_design: 2 groups of 4 mice each were given zinc in ultrapure water or just ultrapure water for 7 days, afterwars paneth cells were isolated for sequencing


title: Effect of TNF of gene expression in mouse paneth cells after 3 hours

summary: The study of how gene expression in paneth cells changes 3 hours after TNF admistration. We used deep paired-end sequencing to also be able to investigate alternative splicing and non-coding RNA

overall_design: To investigate the effects of TNF on paneth cell gene expression we use 2 goups of fou biological repliactaes each: one PBS control group and one TNF group


title: Paneth cell gene expression with and wihout zinc in different microbiome contexts (antibiotics, germ-free, normal)

summary: We investigated the differences in paneth cell gene expression in different intestinal microbiome contexts and with and without zinc supplementation. We used mice housed in an SPF animal house that wre untreated, mice housed in ans SPF animal house that were treated with antobiotics for 3 weeks and germ-free mice to investigated the effect of the microbiome on paneth cell gene expression. Zinc is an essential element for paneth cell fuction. Mice of each group were further divide in zinc supplemented and non-zinc supplemented drinking water subgroups. This allowed us to check if zinc would enhance the  differences between SPF, atibioicts and germ-free mice or reduce them.

overall_design: In total there are six groups of interest: SPF housed mice that recieved Milli-Q water or zinc supplemented water (3 biological replicates per group). SPF housed mice that recieved Milli-Q water or zinc supplemented water ans had their microbiome depleded with broad spectrum antibioics 3 wees before sampling (4 biological replicates per group). germ-free mice that recived Milli-Q water (3 biological replictes) or zinc supplemented water (4 biological replictes)


title: Investigation of genes involoved in Corydalis incisa (Thunb.) Pers. (CIP)-induced differentiation in acute myeloid leukemia (AML).

city: Busan

first: Ji-Eun

last: Lee

address: 2, Busandaehak-ro 63beon-gil

country: South Korea

institute: Pusan national university

summary: Acute myeloid leukemia (AML) is the most common form of leukemia in adults. Differentiation therapy targets the key pathologic features of AML, the blockade of differentiation in myeloid progenitor cells. This study screened 100 plant extracts native to South Korea to search for those with differentiation-inducing activity in AML. Of these, Corydalis incisa (Thunb.) Pers. (CIP) exhibited the highest efficacy.CIP induced myeloid differentiation, decreased cell viability, increased cell apoptosis, and cell cycle arrest in multiple AML cell lines. We performed Quantseq 3' mRNA seqencing to understand CIP-induced differentiation and identify potential targets of CIP using CIP-resistant cell line.

overall_design:
To identify genes involoved in Corydalis incisa (Thunb.) Pers. (CIP)-induced AML differentiation, we generated CIP-resistant HL-60 cells (HL-60-CIP).
We then performed Quantseq 3' mRNA sequencing using HL-60 DMSO and HL-60 CIP cells.
Comparative gene expression analysis of Quantseq 3' mRNA seq data for HL-60 DMSO and HL-60 CIP cells

first: Byeol-Eun

last: Jeon

first: Chan-seong

last: Kwon

first: Hyeon-Young

last: Kim

first: Tae-Jin

first: Young-Seob

last: Seo

first: Sang Hun

first: Ho-Jin

last: Shin

first: Sang-Woo


title: Inhibition of PRC2 enables self-renewal of naïve pluripotent stem cells from chimpanzee

first: Hideki

last: Masaki

overall_design: To elucidate the developmental potential of chimpanzee naïve pluripotent stem cells (PSCs), we dissociated induced blastoids into single cells and performed single-cell RNA sequencing (scRNAseq). We then compared each single-cell transcriptome with human embryo scRNAseq datasets to identify the corresponding lineages.

first: Tao

last: Huang

first: Ayaka

last: Yanagida

first: Arthur

last: Radley

first: Zhili

last: Ren

first: Francesca

last: Carlisle

first: Paul

last: O’Neill

first: Hiroo

last: Imai

first: Hiromitsu

last: Nakauchi

first: Ge

last: Guo

first: Austin

last: Smith

first: Somayyeh

last: Tahajjodi

first: James

last: Clarke

first: Madeline

last: Lancaster

first: Zoe

last: Heckhausen

first: Rosie

last: Bamford

first: Aaron

last: Jeffries

first: Jonathon

last: Mill

first: Petra

last: Hajkova


title: Inhibition of Atg7 in intestinal epithelial cells drives resistance against Citrobacter rodentium

city: PARIS

first: Béatrice

last: ROMAGNOLO

address: 24 rue du Fb St Jacques 75014

department: CANCER

country: France

institute: INSERM-Institut Cochin

summary: Autophagy, a cytoprotective mechanism in intestinal epithelial cells, plays a crucial role in maintaining intestinal homeostasis. Beyond its cell-autonomous effects, the significance of autophagy in these cells is increasingly acknowledged in the dynamic interplay between the microbiota and the immune response. In the context of colon cancer, intestinal epithelium disruption of autophagy has been identified as a critical factor influencing tumor development. This disruption modulates the composition of the gut microbiota, eliciting an anti-tumoral immune response. Here, we report that Atg7 deficiency in intestinal epithelial cells shapes the intestinal microbiota leading to an associated limitation of colitis induced by Citrobacter rodentium infection. Mice with an inducible, intestinal epithelial-cell-specific deletion of the autophagy gene, Atg7, exhibited enhanced clearance of C. rodentium, mitigated hyperplasia, and reduced pathogen-induced goblet cell loss. This protective effect is associated with the downregulation of pro-inflammatory pathways and an increase in Th17 and Treg responses—known protective immune responses against C. rodentium infection that are influenced by specific gut microbiota. Fecal microbiota transplantation and antibiotic treatment approaches revealed that the Atg7-deficiency-shapped microbiota, especially Gram-positive bacteria, plays a central role in driving resistance to C. rodentium infection. In summary, our findings highlight that inhibiting autophagy in intestinal epithelial cells contributes to maintaining homeostasis and preventing detrimental intestinal inflammation through microbiota-mediated colonization resistance against C. rodentium. This underscores the central role played by autophagy in shaping the microbiota in promoting immune-mediated resistance against enteropathogens

overall_design: we conducted a comprehensive analysis of the colonic gene expression profile in response to C. rodentium infection using RNA sequencing. Total RNA was extracted from the colons of infected WT and Atg7ΔIEC mice at day 8 and day 18 post-infection (p.i). A cohort of uninfected WT and Atg7ΔIEC mice were also analyzed as controls.

first: David

last: Cune

first: Caterina

last: Pitasi

first: Alessia

last: Rubiola

first: Trinath

last: Jamma

first: Luca

last: Simula

first: Camille

last: Boucher

first: Apolline

last: Fortun

first: Lucie

last: Adoux

first: Franck

last: Letourneur

first: Benjamin

last: Saintpierre

first: Emmanuel

last: Donnadieu

first: Benoît

last: Terris

first: Pascale

last: Bossard

first: Benoit

last: Chassaing

last: Romagnolo


title: Glutamatergic argonaute2 promotes the formation of the neurovascular unit in mice

city: Schwerzenbach

first: Adhideb

last: Ghosh

address: Schorenstrasse 16

department: Health Sciences and Technology

country: Switzerland

institute: ETH Zurich

summary: Proper formation of the complex neurovascular unit (NVU) along with the blood-brain barrier is critical for building and sustaining a healthy, functioning central nervous system. The RNA binding protein argonaute2 (Ago2) mediates microRNA (miRNA)–mediated gene silencing, which is critical for many facets of brain development, including NVU development. Here, we found that Ago2 in glutamatergic neurons was critical for NVU formation in the developing cortices of mice. Glutamatergic neuron–specific loss of Ago2 diminished synaptic formation, neuronal-to-endothelial cell contacts, and morphogenesis of the brain vasculature, ultimately compromising the integrity of the blood-brain barrier. Ago2 facilitated miRNA targeting of phosphatase and tensin homolog (Pten) mRNA, which encodes a phosphatase that modulates reelin-dependent phosphatidylinositol 3-kinase (PI3K)–Akt signaling within the glutamatergic subpopulation. Conditionally deleting Pten in Ago2-deficient neurons restored Akt2 phosphorylation as well as postnatal development and survival. Several mutations in AGO2 impair small RNA silencing and are associated with Lessel-Kreienkamp syndrome, a neurodevelopmental disorder. When expressed in a neuronal cell line, these human AGO2 loss-of-function variants failed to suppress PTEN, resulting in attenuated PI3K-Akt signaling, further indicating that dysregulation of Ago2 function may contribute to both impaired development and neurological disorders. Together, these results identify Ago2 as central to the engagement of neurons with blood vessels in the developing brain.

overall_design: To understand the role of endogenous Ago2 in the CNS, we generate a mutant mouse line where Ago2 expression was deleted in Vglut2-expressing excitatory neurons (by crossing the Slc17a6-Cre mouse line with an Ago2 conditional allele, Slc17a6-Cre, Ago2flox/flox mice). The cortical cells will be isolated from mice brain at P14 for scRNAseq analysis

first: Chandan

last: Sona

first: Xin

last: Yan

first: Laura

last: Hinte

first: Ferdinand

last: von Meyenn

first: Matthew

middle: N

last: Poy


title: Differences of tsRNA expression profiles efficiently discriminate monozygotic twins in peripheral blood

city: Shenyang

first: Meihui

last: Tian

address: No.77, Puhe Road

department: School of Forensic Medicine

institute: China Medical University

summary: Since monozygotic twins (MZTs) share extremely similar genomic DNA sequences, they cannot be distinguished by conventional forensic STR genotyping. The utilization of epigenetic factors in forensic application has gradually risen over the last few years. Epigenetic factors are characterized by dynamic changes and can be either inherited or accumulated as a result of acquired environmental influences. In this study, we analyzed the tsRNAs expression profiles in peripheral blood from four pairs of adult MZTs using PANDORA-sequencing (PANDORA-seq). Subsequently, the differential expressed tsRNAs (DEtsRNAs) were screened and confirmed by RT-qPCR and droplet digital PCR (ddPCR) in both adult and newborn MZTs. Additionally, the characteristics of the DEtsRNAs including longitudinal temporal stability, suitability for aged bloodstains and multigelation were evaluated.

overall_design: To explore the potential of using epigenetic factors to distinguish monozygotic twins, we used PANDORA-seq to investigate the differential expression of tRNA-derived small RNA(tsRNA) in human peripheral blood of monozygotic twins.

first: Dawei

last: Guan

first: Jun

last: Yao

first: Xiangnian

last: Liu

first: Danyang

last: Wang

first: Yuxi

first: Siwen

first: Jiayi

last: Wei


title: Lung-delivered IL-10 Mitigates Lung Inflammation Induced by Repeated Endotoxin Exposures

city: Omaha

first: Aaron

middle: D

last: Schwab

address: 42nd and Emile St.

institute: University of Nebraska Medical Center

summary: Background: Therapeutic options capable of resolving inflammatory lung disease resulting from high-consequence occupational and environmental hazards are lacking. Objective: This study seeks to determine the therapeutic potential of direct lung-delivered interleukin (IL)-10 following repeated high concentration lipopolysaccharide exposures. Methods: C57BL/6 mice were intratracheally instilled with LPS (10μg) and then IL-10 (1μg) or vehicle (saline) control 5-hours later. This LPS exposure and treatment paradigm was then repeated daily for the next 2 days. Serum and lung tissues were collected and assessed for proinflammatory and profibrotic mediators. Lung cell infiltrates were enumerated by flow cytometry. Lung sections were stained for myeloperoxidase (MPO), CCR2, vimentin and post-translational protein citrullination (CIT) and malondialdehyde-acetaldehyde (MAA) modifications. Lung function testing and longitudinal in vivo micro-CT imaging were performed. Whole lungs were profiled using bulk RNA sequencing. Results: Repeated IL-10 therapy significantly (p<0.05) mitigated several LPS-induced adverse effects. IL-10 treatment reduced LPS-induced weight loss and serum pentraxin-2 and IL-6 levels. LPS-induced proinflammatory/profibrotic mediators (i.e., TNF-α, IL-6, CXCL1, CCL2, MMP-8, MMP-9, TIMP-1, fibronectin) were decreased with IL-10 treatment. IL-10 reduced LPS-induced influx of lung neutrophils, CD8+ T cells, NK cells, recruited monocyte-macrophages, and monocytes, as well as tissue expression of CCR2+ monocytes/macrophages, MPO+ neutrophils, vimentin, CIT, and MAA. IL-10 treatment reversed LPS-induced airway hyperresponsiveness. Micro-CT imaging confirmed reduction in LPS-induced lung density by IL-10 treatment. Lung-delivered IL-10 therapy administered after daily repeated endotoxin exposures strikingly reduces lung inflammatory and profibrotic processes and airway dysfunction to hasten lung recovery and resolution. Short-term, lung-localized IL-10 therapy following high-consequence environmental exposure events may represent a novel therapy to prevent chronic lung disease development.

overall_design: C57BL/6 male mice (between 6 and 8 weeks of age) were purchased from The Jackson Laboratory (Bar Harbor, ME), randomized upon arrival, and allowed to acclimate for 1 week prior to initiation of experiments. 6 mice were intratracheally instilled with LPS (10μg). Half of these mice were then intratracheally instilled with IL-10 (1μg) and the other half was intratracheally instilled with vehicle (saline) control 5-hours later. This exposure paradigm was then repeated  for the next 2 days so that each mouse received 3 total doses of LPS in the morning and 3 total doses of either IL-10 or the vehicle control 5 hours post-LPS. Animals were euthanized 24 hours following the final LPS exposure. Whole lungs were harvested and profiled using bulk RNA sequencing.

first: Jill

last: Poole


title: Long-read sequencing for the assessment of therapeutic CRISPR/Cas9 strategy for the removal of aberrant DM1-causing CTG repeats in DMPK gene locus

city: Milan

first: Fabio

last: Martelli

address: Via Olgettina 58

institute: IRCCS Policlinico San Donato

summary: The genetic cause of Myotonic Dystrophy Type 1 (DM1) is the expansion of unstable CTG-repeats in the 3´ untranslated region of the DMPK gene. Therapeutic gene editing for treatment of monogenic diseases could in principle remove definitively the disease-causing genetic defect. Here we describe the application of the CRISPR/Cas9 strategy to remove the CTG-expansion in the DMPK gene in myogenic cells derived from a DM1 patient that is heterozygous for the expansion. The efficacy of on-target genomic events was assessed by performing long-read DNA sequencing (Oxford Nanopore Technologies) after applying Cas9-mediated target enrichment of the Regions Of Interest (ROI), enabling the simultaneous analysis (a) of cuts by both sgRNAs resulting in the effective excision of the CTG expansion, (b) of cuts induced only by a single sgRNA, (c) of the potential occurrence of aberrant events such as inversions, and (d) the existence of non-edited sequences, within the same sample.

overall_design: New guide RNA pairs were designed and assessed by PCR. The most effective pair (sgC3/sg384) was utilized to perform editing in proliferating (Growth Medium, GM) and differentiated (Differentiation Medium, DM) myogenic cells. For target enrichment, CRISPR RNAs (crRNAs) were designed to cut the ROI four times, i.e., twice upstream on the plus strand and twice downstream on the minus strand, to avoid incomplete cleavage. crRNA pools against two ROI were used: one targeting the full-length DMPK gene (~14kbp, ROI1) to screen for potential long deletions, the other flanking the sgC3/sg384 editing sites within the 3’UTR region (~4kbp, ROI2). Five libraries were prepared and sequenced from edited DM1 myogenic cell populations in both DM (1 ROI1 and 2 ROI2 samples) and GM (2 ROI2 samples), yielding an overall sequencing coverage ≥ 140X for both cell states.

first: Christine

last: Voellenkle

first: Claudia

last: Provenzano

first: Svetlana

last: Frolova

first: Spyros

last: Tastsoglou

first: Beatrice

last: Cardinali

first: Germana

last: Falcone


title: m6A-mediated epi-transcriptomic dysregulation underlies synaptic dysfunction in fragile X syndrome [MeRIP-seq]

city: Cincinnati

first: Ziyuan

last: Guo

address: 3333 Burnet Ave

institute: CCHMC

summary: N6-methyladenosine (m6A) is a prevalent mRNA modification essential for post-transcriptional regulation. Fragile X syndrome (FXS), the leading genetic cause of intellectual disability, arises from FMR1 gene silencing and loss of FMRP, which has been shown to antagonize m6A readers. However, the impact of FMRP loss on transcriptome-wide m6A modifications in human FXS remains unknown. Using cortical neurons derived from healthy subjects and FXS patient-induced pluripotent stem cells, we found that FXS neurons exhibited synaptic and neuronal network defects, mirroring clinical EEG data from the same FXS patients. FMRP deficiency led to increased translation of m6A writers, resulting in transcriptome-wide hypermethylation, primarily affecting synapse-associated transcripts and increasing mRNA decay. Treatment with an m6A writer inhibitor rescued synaptic defects in FXS neurons, highlighting its therapeutic potential. Our findings uncover an epi-transcriptomic mechanism by which FMRP deficiency disrupts m6A modifications in FXS, contributing to FXS pathogenesis, and suggest a promising avenue for m6A-targeted therapies.

overall_design: To unravel the transcriptome-wide m6A landscape in FXS, we employed methylated RNA immunoprecipitation sequencing (MeRIP-Seq) on FXS neurons derived from six FXS patients and compared the results with seven control samples. Additionally, MeRIP-seq was performed on postmortem cortex tissues obtained from three FXS patients and healthy control subjects.

first: Lu

last: Lu


title: Genome-wide DNA methylation markers associated with metabolic liver cancer

city: Jacksonville

first: Samuel

middle: O

last: Antwi

address: 4500 San Pablo Road South, 756N Stabile Building

department: Quantitative Health Sciences

institute: Mayo Clinic

summary:
Background and Aims: Metabolic liver disease is the fastest rising cause of hepatocellular carcinoma (HCC) worldwide, but the underlying molecular processes that drive HCC development in the setting of metabolic perturbations are unclear. We investigated the role of aberrant DNA methylation in metabolic HCC development in a multicenter international study.
Results: We enrolled 272 metabolic HCC patients and 316 control patients with metabolic liver disease from six sites. Fifty-five differentially methylated CpGs were identified; 33 hypermethylated and 22 hypomethylated in cases versus controls. The panel of 55 CpGs discriminated between cases and controls with AUC=0.79 (95%CI=0.71-0.87), sensitivity=0.77 (95%CI=0.66-0.89), and specificity=0.74 (95%CI=0.64-0.85). The 55-CpG classifier panel performed better than a base model that comprised age, sex, race, and diabetes mellitus (AUC=0.65, 95%CI=0.55-0.75, sensitivity=0.62 (95%CI=0.49-0.75) and specificity=0.64 (95%CI=0.52-0.75). A multifactorial model that combined the 55 CpGs with age, sex, race, and diabetes, yielded AUC=0.78 (95%CI=0.70-0.86), sensitivity=0.81 (95%CI=0.71-0.92), and specificity=0.67 (95%CI=0.55-0.78).
Conclusions: A panel of 55 blood leukocyte DNA methylation markers differentiates patients with metabolic HCC from control patients with benign metabolic liver disease, with a slightly higher sensitivity when combined with demographic and clinical information.

overall_design:
We used a case-control design, frequency-matched on age, sex, and study site. Genome-wide profiling of peripheral blood leukocyte DNA was performed using the 850k EPIC array. Cell type proportions were estimated from the methylation data. The study samples were split 80% and 20% for training and validation. Differential methylation analysis was performed with adjustment for cell type, and we generated area under the receiver-operating curves (ROC-AUC).
***NOTE FROM SUBMITTER: The data cannot be shared for 107 subjects due to European Union (EU) law (derived from EU patients that are protected by General Data Protection Regulation).***